Vol 85 Num 3 Septiembre 2013

Transcription

Anais da Academia Brasileira de Ciências
versão impressa ISSN 0001-3765
Sumário
An. Acad. Bras. Ciênc. vol.85 no.3 Rio de Janeiro set. 2013
Nota Editorial
• A closer look at the Impact Factor (JCR 2012): problems, concerns and
actions needed
KELLNER, ALEXANDER W.A.; AZEVEDO, RICARDO A.
• texto em Inglês
• pdf em Inglês
Ciências Matemáticas
• A decoding method of an n length binary BCH code through (n + 1)n
length binary cyclic code
SHAH, TARIQ; KHAN, MUBASHAR; ANDRADE, ANTONIO A. DE
• resumo em Português | Inglês
• pdf em Inglês
Ciências Químicas
• Chemical constituents of Distictella elongata (Vahl) Urb.
(Bignoniaceae)
SIMOES, LEANDRO R.; MACIEL, GLAUBER M.; BRANDAO, GERALDO C.; FILHO, JOSE D.
S.; OLIVEIRA, ALAIDE B.; CASTILHO, RACHEL O.
• pdf em Inglês
• Flavonoids from leaves of Derris urucu: assessment of potential
effects on seed germination and development of weeds
SILVA, EWERTON A.S. DA; LOBO, LIVIA T.; SILVA, GEILSON A. DA; SOUZA FILHO,
ANTONIO PEDRO DA S.; SILVA, MILTON N. DA; ARRUDA, ALBERTO C.; GUILHON,
GISELLE M.S.P.; SANTOS, LOURIVALDO S.; ARRUDA, MARA S.P.
• pdf em Inglês
• Multivariate optimization of an ultrasound-assisted extraction
procedure for Cu, Mn, Ni and Zn determination in ration to chickens
BARROS, JOELIA M.; BEZERRA, MARCOS A.; VALASQUES, GISSELI S.; NASCIMENTO
JUNIOR, BARAQUIZIO B. DO; SOUZA, ANDERSON S.; ARAGAO, NADIA M. DE
• pdf em Inglês
• In vitro trypanocidal activity of solamargine and extracts from
Solanum palinacanthum and Solanum lycocarpum of brazilian cerrado
MOREIRA, RAQUEL R.D.; MARTINS, GILMARCIO Z.; MAGALHAES, NATHALIA O.;
ALMEIDA, ADELIA E.; PIETRO, ROSEMEIRE C.L.R.; SILVA, FLAVIA A. J.; CICARELLI,
REGINA M.B.
• pdf em Inglês
• Active polyketides isolated from Penicillium herquei
MARINHO, ANDREY M.R.; MARINHO, PATRICIA S.B.; SANTOS, LOURIVALDO S.;
RODRIGUES FILHO, EDSON; FERREIRA, IZABEL C.P.
• pdf em Inglês
Ciências da Terra
• A study of structural lineaments in Pantanal (Brazil) using remote
sensing data
PARANHOS FILHO, ANTONIO C.; NUMMER, ALEXIS R.; ALBREZ, EDILCE A.; RIBEIRO,
ALISSON A.; MACHADO, ROMULO
• pdf em Inglês
Ciências Biológicas
• The Brazilian research contribution to knowledge of the plant
communities from Antarctic ice free areas
PEREIRA, ANTONIO B.; PUTZKE, JAIR
• pdf em Inglês
• Evidence of separate karyotype evolutionary pathway in Euglossa
orchid bees by cytogenetic analyses
FERNANDES, ANDERSON; WERNECK, HUGO A.; POMPOLO, SILVIA G.; LOPES, DENILCE
M.
• pdf em Inglês
• Evaluation of wound healing and antimicrobial properties of aqueous
extract from Bowdichia virgilioides stem barks in mice
AGRA, ISABELA K.R.; PIRES, LUANA L.S.; CARVALHO, PAULO S.M.; SILVA-FILHO,
EURIPEDES A.; SMANIOTTO, SALETE; BARRETO, EMILIANO
• pdf em Inglês
• Assessment of the cytotoxic, genotoxic, and antigenotoxic activities of
Celtis iguanaea (Jacq.) in mice
BORGES, FLAVIO F.V.; MACHADO, THIAGO C.; CUNHA, KENYA S.; PEREIRA, KARLA C.;
COSTA, ELSON A.; PAULA, JOSE R. DE; CHEN-CHEN, LEE
• pdf em Inglês
• Expression of manganese peroxidase by Lentinula edodes and
Lentinula boryana in solid state and submerged system fermentation
HERMANN, KATIA L.; COSTA, ALESSANDRA; HELM, CRISTIANE V.; LIMA, EDSON A. DE;
TAVARES, LORENA B.B.
• pdf em Inglês
• Chironomidae (Insecta: Diptera) of different habitats and
microhabitats of the Vacacaí-Mirim River microbasin, Southern Brazil
KONIG, RODRIGO; SANTOS, SANDRO
• pdf em Inglês
• Evaluation of analgesic and anti-inflammatory activities of
Hydrocotyle umbellata L., Araliaceae (acariçoba) in mice
FLORENTINO, IZIARA F.; NASCIMENTO, MARCUS VINICIUS M.; GALDINO, PABLINNY M.;
BRITO, ADRIANE F. DE; ROCHA, FABIO F. DA; TONUSSI, CARLOS R.; LIMA, THEREZA
CHRISTINA M. DE; PAULA, JOSE R. DE; COSTA, ELSON A.
• pdf em Inglês
• Treadmill exercise does not change gene expression of adrenal
catecholamine biosynthetic enzymes in chronically stressed rats
GAVRILOVIC, LJUBICA; STOJILJKOVIC, VESNA; KASAPOVIC, JELENA; POPOVIC,
NATASA; PAJOVIC, SNEZANA B.; DRONJAK, SLADJANA
• pdf em Inglês
• Impacts of hydroelectric dams on alluvial riparian plant communities
in eastern Brazilian Amazonian
FERREIRA, LEANDRO VALLE; CUNHA, DENISE A.; CHAVES, PRISCILLA P.; MATOS,
DARLEY C.L.; PAROLIN, PIA
• pdf em Inglês
• Germination responses of the invasive Calotropis procera (Ait.) R. Br.
(Apocynaceae): comparisons with seeds from two ecosystems in
northeastern Brazil
LEAL, LAURA C.; MEIADO, MARCOS V.; LOPES, ARIADNA V.; LEAL, INARA R.
• pdf em Inglês
• Are leaf physiological traits related to leaf water isotopic enrichment
in restinga woody species?
ROSADO, BRUNO H.P.; MATTOS, EDUARDO A. DE; STERNBERG, LEONEL DA S.L.
• pdf em Inglês
• Daily and seasonal activity patterns of free range South-American
rattlesnake (Crotalus durissus)
TOZETTI, ALEXANDRO M.; MARTINS, MARCIO
• pdf em Inglês
• Regional movements of the tiger shark, Galeocerdo cuvier, off
northeastern Brazil: inferences regarding shark attack hazard
HAZIN, FABIO H.V.; AFONSO, ANDRE S.; CASTILHO, PEDRO C. DE; FERREIRA, LUCIANA
C.; ROCHA, BRUNO C.L.M.
• pdf em Inglês
• Nuclear entropy, angular second moment, variance and texture
correlation of thymus cortical and medullar lymphocytes: Grey level cooccurrence matrix analysis
PANTIC, IGOR; PANTIC, SENKA; PAUNOVIC, JOVANA; PEROVIC, MILAN
• pdf em Inglês
• Chemical, enzymatic and cellular antioxidant activity studies of
Agaricus blazei Murrill
HAKIME-SILVA, RICARDO A.; VELLOSA, JOSE C.R.; KHALIL, NAJEH M.; KHALIL, OMAR A.
K.; BRUNETTI, IGUATEMY L.; OLIVEIRA, OLGA M.M.F.
• pdf em Inglês
• Cryopreservation of mutton snapper ( Lutjanus analis) sperm
SANCHES, EDUARDO G.; OLIVEIRA, IDILI R.; SERRALHEIRO, PEDRO C. DA SILVA;
CERQUEIRA, VINICIUS R.
• pdf em Inglês
• Anurans in a forest remnant in the transition zone between cerrado
and atlantic rain forest domains in southeastern Brazil
PIRANI, RENATA M.; NASCIMENTO, LUCIANA B.; FEIO, RENATO N.
• pdf em Inglês
• The role of phytophysiognomies and seasonality on the structure of
ground-dwelling anuran (Amphibia) in the Pampa biome, southern Brazil
MARAGNO, FRANCIELE P.; SANTOS, TIAGO G.; CECHIN, SONIA Z.
• pdf em Inglês
• Ecological features of titica vine (Heteropsis flexuosa (Kunth) GS
Bunting) in Rondônia State, Northwest Brazilian Amazon
GAMA, MICHELLINY M. BENTES; VIEIRA, ABADIO H.; ROCHA, RODRIGO B.
• pdf em Inglês
• Efficiency of snake sampling methods in the Brazilian semiarid region
Mesquita, Paulo C.M.D.; Passos, Daniel C.; Cechin, Sonia Z.
• pdf em Inglês
• Pouteria ramiflora extract inhibits salivary amylolytic activity and
decreases glycemic level in mice
GOUVEIA, NEIRE M. DE; ALBUQUERQUE, CIBELE L. DE; ESPINDOLA, LAILA S.;
ESPINDOLA, FOUED S.
• pdf em Inglês
• Morphological and hematological studies of Trypanosoma spp.
infecting ornamental armored catfish from Guamá River-PA, Brazil
Fujimoto, Rodrigo Y.; Neves, Mikaelle S.; Santos, Ruda F.B.; Souza, Natalino C.; Couto,
Marcia V.S. do; Lopes, Josiane N.S.; Diniz, Daniel G.; Eiras, Jorge C.
• pdf em Inglês
• The role of cyclooxygenase-2 on endurance exercise training in female
LDL-receptor knockout ovariectomized mice
OLIVEIRA, FLAVIA DE; MAIFRINO, LAURA B.M.; JESUS, GUSTAVO P.P. DE; CARVALHO,
JULIANA G.; MARCHON, CLAUDIA; RIBEIRO, DANIEL A.
• pdf em Inglês
• Effect of l-glutamine on myenteric neuron and of the mucous of the
ileum of diabetic rats
TRONCHINI, ELEANDRO A.; TREVIZAN, ALINE R.; TASHIMA, CRISTIANO M.; FREITAS,
PRISCILA DE; BAZOTTE, ROBERTO B.; PEREIRA, MARLI A.S.; ZANONI, JACQUELINE N.
• pdf em Inglês
• Hawkmoth fauna (Sphingidae, Lepidoptera) in a semi-deciduous
rainforest remnant: composition, temporal fluctuations, and new records
for northeastern Brazil
PRIMO, LUIS M.; DUARTE, JOSE A.; MACHADO, ISABEL C.
• pdf em Inglês
• Handling sticky Resin by Stingless Bees: Adhesive Properties of
Surface Structures
GASTAUER, MARKUS; CAMPOS, LUCIO A.O.; WITTMANN, DIETER
Ciências Biomédicas
• pdf em Inglês
• Predictive factors of subjective sleep quality and insomnia complaint
in patients with stroke: implications for clinical practice
ROCHA, PATRICIA C. DA; BARROSO, MARINA T.M.; DANTAS, ANA AMALIA T.S.G.; MELO,
LUCIANA P.; CAMPOS, TANIA F.
• pdf em Inglês
• Biochemical and hematological analysis in acute intermittent
porphyria (AIP): a case report
SANTOS, ANNA R.R. DOS; ALBUQUERQUE, RAFAELA R. DE; DORIQUI, MARIA J.R.;
COSTA, GRACIOMAR C.; SANTOS, ANA PAULA S.A. DOS
Academia Brasileira de Ciências
R. Anfilofio de Carvalho, 29, 3. and.
20030-060 Rio de Janeiro RJ Brazil
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
• pdf em Inglês
http://dx.doi.org/10.1590/S0001-37652013000300001
Editorial Note
A closer look at the Impact Factor (JCR 2012): problems, concerns and
actions needed
ALEXANDER W.A. KELLNER1, RICARDO A. AZEVEDO2
1
Laboratório de Sistemática e Tafonomia de Répteis Fósseis, Departamento de Geologia e
Paleontologia, Museu Nacional/UFRJ, Quinta da Boa Vista, s/n, Bairro Imperial de São Cristóvão,
20940-040 Rio de Janeiro, RJ, Brasil
2 Departamento de Genética, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São
Paulo, 13418-900 Piracicaba, SP, Brasil
The present editorial is a little bit different from the ones regularly published in the Anais da Academia
Brasileira de Ciências (AABC). Instead of focusing on contributions of the current issue as we normally
Kellner 2011
do (e.g.,
), here we report a situation that occurred with the AABC in 2012 and that might be
of general interest for editors and anyone else that uses bibliometric parameters to evaluate scientific
performance (as CNPq, CAPES and Graduate Programs do in Brazil), since it is directly related to the
Impact Factor (IF), Journal Citation Reports® (JCR) by Thomson Reuters JCR and the Web of
KnowledgeSM (WoS). We do not intend to discuss the JCR or the importance of the IF and all the useful
data published about each journal indexed in the JCR. However, in the light of the present case, we
would like to point out that problems with the JCR do exist, that they are serious and that new actions
and measures should be taken in order to mitigate the resulting complications.
For quite some time now, June has become the month of the year in which the anxiety of journal's
Editors reaches the highest level because of the release of the Journal Citation Reports® (JCR) by
Thomson Reuters. The AABC is the sole multidisciplinary journal produced in Brazil, publishing mainly
original research in distinct areas such as Mathematical Sciences (e.g.,
g.,
Jiang and Wang 2011
), Biomedical Studies (e.g.,
Kwakkel et al. 2011
), Paleontology (e.
Cruz et al. 2012
), Agriculture (e.g., Monteiro et al. 2012),
Rocha et al. 2012
Social Sciences (e.g.,
) and everything in-between. The four issues per year have gradually
increased from about 15 to 35 articles per issue since 2007. The year 2012 was not different and the
volume 84 was published with four issues. However, by the end of July 2012, we noticed that the issue
84(2) had not yet appeared in the WoS. A few months later the issue 84(3) was included and all papers
published made available in the WoS, but still no sign of issue 84(2). When issue 84(3) was made
available in the WoS, AABC contacted Thomson Reuters and submitted several requests alerting about
the missing issue, most only resulting in their standard e-mail response. With the help of the Thomson
Reuters office in Brazil and the organization Scientific Library On Line (SciELO, based in São Paulo), it
was only in May, 1st 2013, that the issue 84(2) finally appeared in the WoS. Problem solved and 7 weeks
later the JCR 2012 was published (mid-June) with the new IFs, which in the case of the AABC, was
down from 1.094 to 0.851. A revision of AABC's IF was requested but Thomson Reuters informed
AABC that there is nothing they could do about this at this point, a quite disappointing notice. AABC
was not expecting any significant changes in its IF, but we were aware that at least 3 valid citations
(articles published in 2010-2011) from papers published in the 84(2) issue were not used in the IF
calculation because of the delay in having the issue included in the WoS. If they had been counted the
AABC's 2012 IF would only change to 0.865, a modest 1.64% increase over the published IF. The
current IF actually reveals that the variation is within the normal AABC's IF trend over the last 5 years:
0.881, 1.074, 0.925, 1.094 and 0.851 for JCR 2008, 2009, 2010, 2011 and 2012, respectively (data from
the JCR).
What was interesting and somehow worrying in reviewing all 25 articles published in the AABC 84(2)
issue is the fact that from over 700 listed references, 55 of them concerned papers published in 20102011 in 45 different journals (Rev Palaeobot Palynol, Remote Sens Environ, Brazil J Geod, Sedim Geol,
J Geol, Geology, J South Am Earth Sci, Mineral Mag, Environ Sci Pollut Res, J Soils Sedim, Mar
Environm Res, Mar Pollut Bull, Conserv Genet Resour, PLoS One, Neurosci Lett, Blood, Nature, J Biol
Chem, Int J Parasitol, Biochim Biophys Acta, J Pineal Res, Antioxid Redox Signal, Thromb Haemost,
Purinergic Signaling, Cell Microbiol, Arthritis Rheum, Science, Eukaryot Cell, Malar J, PLoS Pathog,
Exp Parasitol, Parasitology, J Agr Food Chem, Plant Physiol Biochem, Plant Methods, Planta, Environ
Exp Bot, Process Biochem, Mol Breed, Plant Growth Regul, Ann Appl Biol, New Phytol, Plant Sci
2010, Plant Biol, Analyst). All of them would have counted to these journal's IFs published in the JCR
2012 if this issue would have been processed. Once again, we suspect that those extra citations would
not have resulted in significant changes to these journals' IFs although the actual impact might vary. For
instance, in an extreme case, Nature received 3 valid citations, which is insignificant compared to the
65,731 citations the journal has received in 2012. Yet, it is amazing to see the number of citations that
were not used for IF calculation due to the delay in adding the AABC 84(2) issue to the WoS database.
A true “bibliometric domino effect”!
The present case raises the question if such incidents are rare or if they occur more regularly with
volumes/issues from other periodicals as well. If similar “missing issues” or “late issue additions”
regarding this prestigious database are more common and not corrected, than the credibility of the
system is at stake. Perhaps it might be interesting that someone would look more deeply into this
question and perform a fully evaluation of what can be called the “missing issue” or “late issue addition
factors” in the IF of periodicals with distinct IFs (e.g., IF higher than 3, from 3 to 1, and lower than 1).
Curiously, after the JCR data were published in June 2013, the section “2012 JCR Data Update” of the
JCR Notices (http://admin- apps. webofknowledge.com/JCR/static_html/notices/notices.htm) has been
updated weekly with additions or adjustments to the JCR data. This section is set to be closed in
September 2013. By checking the list changes carried out to the JCR 2012 data, 98 updates were
performed and for 58 (59.2%) of them the “reason for update” was “missing issues”. So, adjustments
and corrections are possible, but, as already mentioned, an update of the AABC's IF due to the missing
issue 84(2) was not granted so far.
One should note that the editorial board of several periodicals produced in the so-called ‘developing
world’ are working very hard to get their journals as part of the international mainstream journals, what
necessarily passes through the IF. Efforts vary from inviting special papers on hot topics (e.g.,
Almeida 2011
) and organizing complete volumes on relevant subjects for a particular field (e.g.,
Tomida 2011
Verjovski-
Kellner and
), to showing directions in a specific area where the journal would like to be heading (e.g.,
Azevedo 2012
). In what can be perhaps regarded a more extreme case, some journals even changed their
titles into English, the lingua franca of science, hoping to make the journal more attractive to both,
authors and readers. Furthermore, there is the growing trend to evaluate and compare science
achievement of authors, departments, institutions and even countries. Despite the complexity and the
inevitable controversy associated with this issue (e.g.,
Pinto and Andrade 1999 Hirsch 2005 Jin et al. 2007 Ball
,
,
,
2007 Mugnaini et al. 2008 Kellner and Ponciano 2008
,
,
), scientific publications have become paramount in this
matter. For all this to work properly, there must be a reliable database where the presented IF of journals
can be trusted.
One of the key concerns of the AABC case reported here is the fact that the 84(2) 2012 issue entered the
WoS some 8-10 months later (1st of May 2013) than it should have normally entered, which means that
the 24 articles and 1 editorial published were not made available to the users of this important database
for a much longer period than expected. As a consequence this specific issue of AABC most likely has
lost the chance of being cited more often in 2013 papers since its visibility to the users might have been
reduced. Consequently, we can assume that the 84(2) issue may also contribute with fewer citations than
normally expected and possibly with a smaller impact on the journal's 2013 IF to be published in June
2014. Still part of the “bibliometric domino effect”.
Finally, we have also noticed that 17 citations granted to papers published in 2010-2011 of WoS 2012
were incorrectly cited. Among the mistakes are spelling errors of author names, volume numbers, issue
numbers, page numbers and the year of publication. As a matter of fact, these gross errors by authors are
a major problem. Because of these errors, the 17 citations that would have counted for the 2012 IF were
not used in the calculation. The good news is that the WoS offers the possibility to suggest corrections to
their database, improving the quality of the data for the readers and statistics generated by the system.
Therefore, it is important that editors and their staff check regularly the citations received by the journal
and make sure that any corrections needed are sent to the Thomson Reuters as soon as possible so all
citations are counted. If the 17 errors would have been included in the IF calculation, plus at least the 3
citations from the 84(2) issue, the IF would have been 0.947, a 11.3% increase in the 2012 IF.
As a consequence, the editorial office of the AABC is now considering to establish a routine check of
every citation granted to the papers published in the journal of the preceding year, as well as working
more closely with authors of accepted papers to ensure that the cited references will be free of errors.
Perhaps other periodicals that are not already doing so might also follow this initiative to ensure more
accuracy regarding citations. Just another task for the editorial board of scientific journals in what is
becoming an increasingly complex scientific world where an expressive number of researchers
(particularly the younger generation) are being driven by what has been defined as the “bakery effect” (=
the need for constantly producing new and “fresh” publications, preferably in journals with a high IF;
see Kellner and Ponciano 2007). This reality arguable leads to the production of “salami-slicesCastiel and Sanz-Valero 2007
science” (e.g.,
), not to speak of the mounting plagiarism and self-plagiarism.
Furthermore, editors have to deal with the high importance given to indexes and other bibliometric
parameters, to which the evaluation of databases' reliability has just been now added.
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This is an Open Access article distributed under the terms of the Creative Commons Attribution NonCommercial License, which permits unrestricted non-commercial use, distribution, and reproduction in
any medium, provided the original work is properly cited.
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Mathematical Sciences
A decoding method of an n length binary BCH code through (n + 1)n length binary cyclic code
TARIQ SHAH1, MUBASHAR KHAN1, ANTONIO A. DE ANDRADE2
1
2
Department of Mathematics, Quaid-i-Azam University, 45320, Islamabad, Pakistan
Departamento de Matemática, IBILCE, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Rua Cristóvão Colombo, 2265, Bairro Jardim Nazareth, 15054-000 São José do Rio Preto, SP, Brasil
ABSTRACT
For a given binary BCH code Cn of length n = 2 s - 1 generated by a polynomial
of degree r there is no binary BCH code of length (n + 1)n generated by a generalized polynomial
of degree 2r. However, it does exist a binary cyclic code C (n+1)n of length (n + 1)n such that the binary BCH code Cn is embedded in C (n+1)n . Accordingly a high code rate is attained through a binary cyclic code C (n+1)n for a binary BCH code Cn . Furthermore, an algorithm proposed facilitates in a decoding of a binary BCH code Cn through the decoding of a binary cyclic code C (n+1)n , while the codes Cn and C (n+1)n have the same minimum hamming distance.
Keywords: BCH code; binary cyclic code; binary Hamming code; decoding algorithm
RESUMO
Para um determinado código binário BCH Cn de comprimento n = 2 s −1 gerado por um polinômio
de grau r não existe um código BCH binário de comprimento (n + 1)n gerado por um polinômio generalizado
de grau 2r. No entanto, não existe um código cíclico binário C(n+1)n de comprimento (n + 1)n de tal modo que o código BCH binário C n é imerso em C(n+1)n . Assim, um código de taxa elevada é alcançado através de um código cíclico binário C (n+1)n para um código BCH binário C n . Além disso, propomos um algoritmo que facilita na decodificação de um código BCH binário C n através da decodificação de um código cíclico binário C (n+1)n , ao passo que os códigos C n e C(n+1)n possuem a mesma distância de Hamming mínima.
Palavras-Chave: Código BCH; código cíclico binário; código de Hamming binário; algoritmo de decodificação
INTRODUCTION
The applications of finite commutative rings, particularly finite local rings, have great importance due to their principal ideals. In the design of communication systems and high rate digital computers, encoding and decoding have an importance for error control. The main component of the conventional error-correcting codes are ideals in a finite commutative principal ideal ring.
In
Cazaran and Kelarev 1997
authors introduce the necessary and sufficient conditions for the ideal to be a principal ideal and describe all finite principal ideal rings
constructed through a polynomial ring in one indeterminate with a finite coefficient ring. Shah in
, where I is generated by univariate polynomials. Moreover, in
Shah et al. 2011a b
, , instead of a polynomial ring, the construction methodology of cyclic, BCH, alternant, Goppa, and Srivastava codes over a finite ring is used through a semigroup ring, where the results of
, respectively. However, in (
The existence of a binary cyclic (
Andrade et al. 2010
Andrade and Palazzo 2005
Cazaran and Kelarev 1999
, they obtained conditions for certain rings to be finite commutative principal ideal rings. However, the extension of a BCH code embedded in a semigroup ring
are improved in such a way that in the place of cancellative torsion free additive monoid
of non negative integers, the cancellative torsion free additive monoids
, where S is a finite semigroup, is introduced by
and
) authors describe the decoding principle based on modified Berlekamp-Massey algorithm for BCH, alternant and Goppa codes constructed through monoid rings
We were provoked by Shah et al. 2012 and initiated the inquiry in support to binary BCH codes alike binary cyclic codes. However, we observed that for an n length binary BCH code with n = 2 s − 1 generated by the polynomial
as ideals. A numerous information related with several ring constructions and concerning polynomial codes was given by
Kelarev 2002
. Whereas, in
Kelarev 2007 2008
,
, Kelarev discusses the concerning extensions of BCH codes in several ring constructions, where the results can also be considered as particular cases of semigroup rings of particular nature.
Shah et al. 2011a b
, , R is considered as a finite unitary commutative ring for the quotient rings
Andrade and Palazzo 2005
elaborated the cyclic, BCH, alternant, Goppa and Srivastava codes over finite rings, which are in real meanings
and
.
. Furthermore, in Shah et al. 2012 a decoding procedure for binary cyclic (n, n − r) code by the binary cyclic (
,
) code is also given, which improve the code rate and error corrections capabilities.
of degree 2r. However, in this study, we established that corresponding to a binary BCH code C n (n, n − r) there is a binary cyclic code C(n+1)n ((n+1)n, (n+1)n−2r) such that C n is embedded in C(n+1)n . Furthermore, we propose an algorithm that enables decoding a binary BCH code of length n through the decoding of (n + 1)n length binary cyclic code.
of degree r it is not possible to construct a binary BCH code of length (n+1)n generated by the generalized polynomial
This paper is formulated as follows. In Section 2, first we investigate that, for a positive integer n = 2 s − 1, where s is a positive integer, such that if a polynomial
which we can obtain the decoding of a binary BCH code (n, n−r). Concluding remarks are given in Section 5.
, where an algorithm was given for computing the weights of extensions for codes embedded in
are taken, respectively. Consequently, this new structure gives a construction of a finite quotient ring of a polynomial ring into a finite quotient ring of monoid rings of particular nature. In
) code, where k is a positive integer, corresponding to a binary cyclic (n, n − r) code is established in Shah et al. 2012 by monoid ring
,
Cazaran et al. 2006
of degree r divides xn − 1, then a generalized polynomial
of degree 2r divides
in
. In Section 3, we discuss the non existence and existence of a binary BCH code of length (n+1)n and a cyclic code of length (n+1)n against a binary BCH code of length n, respectively. Consequently, a link of a BCH code (n, n−r) and a cyclic code ((n+1)n, (n+1)n−2r) is developed. However, in Section 4, we present the decoding procedure for a binary cyclic code ((n + 1)n, (n + 1)n − 2r) by
. Second, we discuss cyclic codes of length (n+1)n generated by
CYCLIC CODE OF LENGTH (n + 1)n CONSTRUCTED THROUGH
A semigroup ring R[x; S] is the set of all finitely nonzero functions from a semigroup (S,*) into an associative ring (R,+, ·) in which binary operations addition and multiplication are given by (f + g)(s) = f(s) + g(s) and
, where the
is an ordered monoid, it follows that we can define the degree of an element in
We initiate this study by an observation that the indeterminate of generalized polynomials in a semigroup ring
Given any generalized polynomial
Proposition 1 Let
Proof. Clearly
, where fi 6 ≠ 0 and si 6 ≠ sj for i ≠ j. If S is
shows that the sum is taken over all pairs (t, u) of elements of S such that t * u = s, otherwise (fg)(s) = 0. If S is a monoid, then R[x; S] is called monoid ring. A nonzero element f of R[x; S] has unique representation
and R is an associative ring, particularly the binary field
, then the semigroup ring R[x; S] is simply the polynomial ring R[x]. Clearly, it follows that
.
is given by
and it behaves like an indeterminate x in
, we can construct the factor ring
be a polynomial of degree r. If n = 2 s − 1, where s is a positive integer, then the generalized polynomial
. For instance, for a torsion free cancellative monoid S, it follows that the monoid ring
, where
is a Euclidean domain if
is a principal ideal in
is a field and
or
generated by
(
Gilmer and Parker 1974
). Of course, here
. The elements of the factor ring are the cosets of the ideal
in
. The factor ring is a field if, and only if,
.
is a torsion free cancellative and isomorphic to
is irreducible over
.
.
. Since
divides
Now onward, if
where ζ denotes the coset
. If
, then
, then
is given by
divides
.
.
Thus, f(ζ) = 0, where ζ satisfies the relation ζ n (n+1)−1 = 0. Let us now make a change in notation and write
in place of ζ. Thus, the ring
The multiplication * in the ring
becomes
in wich the relation
is modulo
holds, that is
.
. So, given
, we write
, then
. Otherwise,
for some generalized polynomial
. Practically, to obtain
to denote their product in the ring
is the remainder left on dividing
, we simply compute the ordinary product
and
by
and then put
to denote their product in the ring
. In other words, if
. If deg
, then
,
and so on. Now, consider
, then
and it would be
That is
Particularly, we can take the product
in
by following lema.
Lemma 2 The
is isomorphic to
Proof. It follows that
, where
.
. In fact, we deal the coefficients of generalized polynomials
of
, there is a n(n + 1)-tuppled vector (
We observed that, multiplication by
in the ring
elements of the code C are now referred as codewords or code generalized polynomials.
By use of the techniques of (Shah et al. 2012), the following results can easily be established for a positive integer n(n + 1) instead of
Theorem 3 (Shah et al. 2012) Let C be a linear code over
Theorem 4 (Shah et al. 2012) A subset C of
It is noticed that
. Then C is cyclic if, and only if,
corresponds to cyclic shift σ in
in
. Thus, there is an isomorphism between the vector space
, that is
. A subspace C of
and
defined by
is a linear code. From Lemma 2, identifying every vector c in
. So
is a cyclic code if, and only if, C is an ideal of the ring
with the polynomial
in
, it follows that
.
.
, where
represents the principal ideal generated by
. Corresponding to
.
.
for every
has n(n + 1) terms and hence the coefficients in
in the ring
.
. The
Theorem 5 (Shah et al. 2012) If C is a nonzero ideal in the ring,
then,
1. there exists a unique monic polynomial
2.
of least degree in C;
divides divides
in
3. for all
;
divides
4.
in
; and
.
Conversely, if C is an ideal generated by
, then
From Theorem 5, it follows that the only ideals in the ring
is a generalized polynomial of least degree in C if, and only if,
are linear codes which are generated by the factors of
divides
. Thus, we can obtain all cyclic codes of length n(n + 1) over
in
if we find all factors of
.
. In the case of trivial factors, we get trivial codes. If
, then
. Whereas
implies
.
Remark 6 If
does not divide
, then
Definition 7 Let C be a nonzero ideal in
cannot be of least degree in the ideal
. If
Note that if
is the ideal generated by
.
is a unique monic generalized polynomial of least degree in C, then
, then
is the generator generalized polynomial of C if, and only if,
is called the generator generalized polynomial of the cyclic code C.
is monic and divides
.
A LINK OF A BCH CODE (n, n−r) AND A CYCLIC CODE ((n+1)n, (n+1)n−2r)
In this section, we develop a link between a binary BCH code (n, n−r) and a binary cyclic code ((n+1)n, (n+1)n − 2r). For this, let Cn be a binary BCH code based on the positive integers c, δ 1, q = 2 and n such that 2 ≤ δ 1 ≤ n with gcd(n, 2) = 1 and n = 2 s − 1, where
. So, there is a cyclic code C (n+1)n generated by
. Consequently, the binary BCH code Cn has generator polynomial of degree r given by g(x) = lcm{mi (x) : i = c, c + 1, ... , c +δ 1−2}, where mi (x) are minimal polynomials of ζ i for i = c, c + 1, ... , c + 1−2. Whereas ζ is the primitive nth root of unity in
in
. Since
divides
. Since mi (x) divides xn − 1 for each i, it follows that g(x) divides xn − 1. This implies Cn = (g(x)) is a principal ideal in the factor ring
in
. From Proposition 1, it follows that the generalized polynomial
, it follows that
in
. Now, by third isomorphism theorem for rings, it follows that
. Thus, Cn is embedded in C (n+1)n and the monomorphism ϕ : Cn → C (n+1)n is defined as
, where a(x) ∈ Cn . The above discussion shape the following theorem.
Theorem 8 Let s be a positive integer. If Cn is a binary BCH code of length n = 2 s − 1 generated a polynomial of degree r given by
, then
1. there exist a binary cyclic code C (n+1)n of length (n+1)n generated by a generalized polynomial of degree 2r given by
; and
2. the binary BCH code Cn is embedded in the binary cyclic code C (n+1)n .
For a binary BCH code Cn with generator polynomial g(x) it is not possible to construct a binary BCH code C (n+1)n with generator polynomial
. Hence,
. Indeed, as we know that generator polynomial of a binary BCH code is the least common multiple of irreducible polynomials over
. For instance, if
is the generator polynomial of the binary BCH code Cn , then
is not the least common multiple of irreducible polynomials in
is not qualify for a generator of a binary BCH code.
Example 9 Let s = 2, n = 2 s − 1 = 22 − 1 = 3, δ = 3, c = 1 and p(x) = x 2 + x + 1 a primitive polynomial of degree 2. Thus,
, where ζ satisfies the relation ζ2 + ζ + 1 = 0. Using this relation, it follows that {0, ζ1 = ζ , ζ2 = 1 + ζ , ζ3 = 1}. Let mi (x) be the minimal polynomial of ζ i , where i = c, c + 1, ... , c, c + 1, ... , c + δ − 2. Thus, m 1(x) = x 2 + x + 1, and hence, g(x) = lcm{mi (x) : i = c, c + 1, ... , c + δ − 2} = x 2 + x + 1. Also,
in
, it follows that the corresponding cyclic code C 12(12, 8) is generated by
is a binary BCH code based on the positive integers c = 1, δ = 3, q = 2 and n = 3 such that 2 ≤ δ ≤ n with gcd(n, 2) = 1. Since
divides
.
GENERAL DECODING PRINCIPLE
Berlekamp et al. 1978
demonstrated that the maximum-likelihood decoding is a NP-hard problem for general linear codes. Whereas by the principle of maximum-likelihood decoding we obtain a code after decoding which is closest to the received vector when the errors are corrected. We use the decoding procedure which follows the same principle.
In the following we interpret the decoding terminology for a binary cyclic code C (n+1)n with length (n+1)n and having parity-check matrix H. If the vector b is received, then we obtain the syndrome vector of b given by S(b) = bHT . In this way, we calculate a table of syndromes which is useful in determining the error vector e such that S(b) = S(e). So the decoding of received vector b has done as the transmitted vector a = b − e.
The general principle of decoding is to pick the codeword nearest to the received vector. For this purpose, we prepare a look-up table that gives the nearest codeword for every possible received vector. The algebraic structure of a linear code as a subspace provides a convenient method for preparing such a table. If C (n+1)n is a subspace of
Let y be any vector in
, and supose
Theorem 12 Let C (n+1)n be an ((n+1)n, (n+1)n−2r) code over
. Recall that for every
is the codeword nearest to y. Now, x lies in the coset
Theorem 10 Let
Definition 11 Let C (n+1)n be a linear code in
, then C (n+1)n is a subgroup of the additive group
be a linear code. Given a vector
. For all
, the codeword x nearest to y is given by x = y − e, where e is the vector of least weight in the coset containing y. If the coset containing y has more than one vector of least weight, then there are more than one codewords nearest to y.
. The coset leader of a given coset of C (n+1)n is defined to be the vector with the least weight in the coset.
. If H is a parity-check matrix of C (n+1)n , then
, the set
.
is called a coset of C (n+1)n and the set of these cosets form a partition of the set
, it follows that d(y,x) ≤ d(y,c), i.e. w(y − x) ≤ w(y − c). Hence, y − x is the vector of least weight in the coset containing y. Writing e = y − x, it follows that x = y − e. Thus, the following theorem is obtained.
. Hence,
is the disjoint union of distinct cosets of C (n+1)n .
From Theorem 12, it follows that S(y) = 0 if, and only if,
where N = qn–k ,
. Thus, S(y) = S(y′) holds if, and only if, (y − y′)HT = 0, that is,
. Let y such that
. Hence, two vectors have the same syndrome if, and only if, they lie in the same coset of C (n+1)n . Thus, there is a one-to-one correspondence between the cosets of C (n+1)n and the syndromes. A table with two columns showing the coset leader ei and the corresponding syndromes S(ei ) is called the syndrome table. To decode a received vector y, we compute its syndrome S(y) and then look at the table to find the coset leader e for which S(e) = S(y). Then y is decoded as x = y − e. The syndrome table is given by
and S(ei ) = eiHT for 1 ≤ i ≤ N.
Now, consider a binary BCH code Cn based on the positive integers c, δ, q = 2 and n such that 2 ≤ δ ≤ n with n = 2 s − 1, where s is a positive integer. Let ζ be a primitive nth root of unity in
Assume that C (n+1)n is the corresponding binary cyclic code of length (n + 1)n with minimum distance d and with generator generalized polynomial
Of course,
is the parity-check matrix de order (2r × (n + 1)n for binary cyclic code C (n+1)n of dimension k = (n + 1)n − 2r. Syndrome of the vector
denote the minimal polynomial of ζ i . Let g(x) be the product of distinct polynomials among mi (x), for i = c, c + 1, ... , c + δ − 2, that is, g(x) = lcm{mi (x) : i = c, c + 1, ... , c + δ − 2}.
. Let
which has the check generalized polynomial
. Thus, the matrix H is given by
is denoted as S(a) = aHT . For the vector a given by
, it follows that the generalized polynomial is given by
in
Now, assume that the codeword v ∈ C is transmitted and the received vector is given by a = v + e, where
Therefor, S(e) = S(a). Now, the syndrome table for the binary cyclic code C (n+1)n is
is the error vector which has the polynomial form
So, S(a) = aHT , where
where N = 2(n+1)n–k , k = (n+1)n − 2r and S(ei ) = eiHT for 1 ≤ i ≤ N.
DECODING ALGORITHM
We establish a decoding method of a binary BCH code of length n through binary cyclic code of length (n+1)n. Though, here in the following we sum up the procedure which indicates the steps in decoding a received word of the cyclic code of length (n + 1)n and explain the technique obtaining the wrapped codeword of the BCH code of length n.
Step 1: Evaluate the check generalized polynomial
of binary ciclic code C (n+1)n .
Step 2: Construct the syndrome table for the binary cyclic code C (n+1)n .
Step 3: Calculate the received generalized polynomial
corresponding to received polynomial
.
Step 4: Calculate the syndrome vector for the vector
corresponding to the received generalized polynomial
Step 5: By looking at syndrome table (step 2), find the coset leader e for which S(b′) = S(e).
Step 6: Decode b′ as b′ − e = a′.
Step 7: The corresponding corrected codeword polynomial a(x) in binary BCH code Cn is obtained.
Example 13 Let s = 2, n = 22 − 1 = 3 and C 3 be the BCH code with positive integers c, δ, gcd(n, 2) = 1 and generated by
and parity-check matrix with check polynomial
Syndrome table is given by
. In this case,
is given by
is the generator polynomial of the corresponding binary cyclic code C (3+1)3 = C 12(12, 8, d). The generator matrix of C 12 is given by
Let
be the received vector of binary BCH code C 3 . Then, its polynomial representation is given by b(x) = 1 + x in
corrected codeword in binary BCH code C 3 is a(x) = 1 + x + x 2 in
and the corresponding received polynomial in the cyclic code C 12 is given by
in
, that is a = 111.
AN APPLICATION TO COGNITIVE RADIO
Cognitive radio is a most recent technology in wireless communication by which the spectrum is vigorously used when the primary user, the approved possessor of the spectrum, is not consumed. The scheme of cognitive radio is initiated in
The fundamental map in
By (
Mitola 2000
Zhao and Sadler 2007
Mitola 2000
is to issue license spectrum to secondary users and hurdle the interference observed by primary users. To guard the primary user from the interference activated by the secondary user during transmission, (
. Rendering this notion, the cognitive radio has the competence to judge the radio environs and step up the decision according to the transmission parameters such as code rate, modulation scheme, power, carrier frequency and bandwidth.
Srinivasa and Jafar 2006
) offered an organization of transmission models as interweave, underlay and overlay.
), in the interweave model the secondary user has opportunistic accesses to the spectrum slum while the primary user is not in and pull out when the primary user wants to in once more. For cognitive radio transformation under the interweave model we may get spectrum corresponding to the binary cyclic code C (n+1)n for data transfer of the primary user. Now, the setup only allow the secondary user having binary BCH code Cn for its data transfer. Accordingly the secondary user obtain high speed data transfer as compare to its own scheme of the BCH code Cn .
CONCLUSION
This paper addresses the following aspects:
of degree 2r corresponding to a binary BCH code of length n with n = 2 s − 1 generated by a polynomial
1. There does not exist a binary BCH code of length (n+1)n generated by a generalized polynomial
of degree 2r corresponding to a binary BCH code of length n with n = 2 s − 1 generated by a polynomial
2. There does exist a binary cyclic code of length (n + 1)n generated by a generalized polynomial
3. An algorithm is given which enables in decoding of a given binary BCH code Cn of length n through the decoding of a binary cyclic code C (n+1)n of length (n + 1)n. Consequently, we have the advantage that, if n − r message transmitted under the cover of binary cyclic code C (n+1)n , then we obtain high speed data transfer as compare to the BCH code Cn . Whereas the codes Cn and C (n+1)n have same minimum hamming distance.
4. By the interweave model for cognitive radio, the secondary user transfers its data through the binary BCH code Cn and has opportunistic accesses to the spectrum of primary user which uses binary cyclic code C (n+1)n for its data transfer. As a result the secondary user achieve high data transfer rate as compare to its own scheme based on the BCH code Cn .
Acknowledgements
The authors are very grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) by financial support, 2007/56052-8 and 2011/03441-2.
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Kelarev AV. 2007. Algorithms for computing parameters of graph-based extensions of BCH-codes. J Discrete Algorithms 5: 553-563. [ Links ]
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Mitola J. 2000. Cognitive Radio: An Integrated Agent Architecture for Software Defined Radio. Ph.D. Dissertation, KTH, Stockholm, Sweden, 304 p. (Unpublished). [ Links ]
Shah T, Khan A and Andrade AA. 2011a. Encoding through generalized polynomial codes. Comput Appl Math 30(2): 349-366. [ Links ]
Shah T, Khan A and Andrade AA. 2011b. Constructions of codes through semigroup ring
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Received: April30, , 2012; Accepted: April29, , 2013
Correspondence to: Antonio Aparecido de Andrade E-mail: [email protected]
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Tel: +55 21 3907-8100
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of degree r such that Cn is embedded in C (n+1)n .
of degree r such that Cn is embedded in C (n+1)n .
, and its vector representation is
. Also, S(b′) = b′*(H′) T = 0010 = S(e 5), hence the corrected codeword in C 12 is a′ = b′ + e 5 = 101010000000 and its polynomial representation is a′ (x) = 1+x2+x4 in
. Hence, the corresponding
http://dx.doi.org/10.1590/S0001-37652013000300003
Chemical Sciences
Chemical constituents of Distictella elongata (Vahl) Urb. (Bignoniaceae)
LEANDRO R. SIMÕES1, GLAUBER M. MACIEL1, GERALDO C. BRANDÃO1, JOSÉ D.S. FILHO2, ALAÍDE B. OLIVEIRA1, RACHEL O. CASTILHO1
1
Faculdade de Farmácia, Departamento de Produtos Farmacêuticos, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Campus Pampulha, 31270901 Belo Horizonte, MG, Brasil
2 Departamento de Química, ICEX, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Campus Pampulha, 31270-901 Belo Horizonte, MG, Brasil
ABSTRACT
Pectolinarin, a flavone heteroside, was isolated from Distictella elongata (Vahl) Urb. leaves ethanol extract, along with a mixture of ursolic, pomolic and oleanolic acids,
besides β-sitosterol. Their structures were established on the basis of spectral analysis (1H and 13C NMR, 1D and 2D) and they were compared with literature. This is the
first report on the occurrence of this flavonoid in a species of the Bignoniaceae family.
Keywords: Bignoniaceae; Distictella elongata; pectolinarin; triterpenes
RESUMO
Pectolinarina, uma flavona heterosídica, foi isolada do extrato etanólico das folhas de Distictella elongata (Vahl) Urb., além de uma mistura de ácidos ursólico, pomólico
e oleanólico, além de β-sitosterol. Suas estruturas foram estabelecidas com base em análise espectral (RMN de 1H e 13C 1D e 2D) em comparação com a literatura. Esta
é a primeira vez em que se relata a ocorrência deste flavonoide em uma espécies da família Bignoniaceae.
Palavras-Chave: Bignoniaceae; Distictella elongata; pectolinarina; triterpenos
INTRODUCTION
Bignonieae is a large and diverse clade of neotropical lianas. The group is widely distributed in the neotropics, occurring in Central America, Amazonia, the
Atlantic forests of eastern Brazil, and the open dry forests and savannahs of Argentina, Bolivia, Brazil, and Paraguay. Bignonieae contains all the lianas of the
Brazilian Bignoniaceae and most of the species (approximately 250 of the 350) are in the four large genera, Arrabidaea, Adenocalymma, Anemopaegma and
Lohmann 2006
Pool 2009
Memora (
). Distictella Kuntze is a genus of 18 species in this tribe. The species are lianas or, less frequently, shrubs (
). Distictella elongata
(Vahl) Urb. appears as Distictis elongata Bureau & K. Schum. in the FLORA BRASILIENSIS and its actual name is Amphilophium elongatum (Vahl) L.G.
Lohmann (
Cipriani et al. 2007 Bedir et al. 2009 Lohmann 2010
,
,
).
Bignoniaceae is characterized by the presence of terpenoids, flavonoids, alkaloids, and special aromatic derivatives of the shikimic acid pathway (
A β-lapachone derivative was also previously obtained from the roots of D. elongata (
Cipriani et al. 2007
).
Bedir et al. 2009
).
In the present study, leaves ethanol extract (LEE) from D. elongata were phytochemically investigated affording the flavonoid di-O-glycoside pectolinarin (1) along
with a mixture of the triterpenoids: ursolic, pomolic and oleanolic acids and β-sitosterol.
MATERIALS AND METHODS
General Experimental Procedures
Optical rotation was measured on a Bellingham + Stanley Ltda ADP 220 polarimeter, whereas Infrared (IR) spectra were recorded on a Perkin-Elmer Spectrum One
spectrophotometer. Melting point was determined on an electrothermal digital apparatus (model MQAPF-30; Microquímica, Brazil), without correction. UV spectra
were measured in a UV-2900 UV-VIS recording spectrophotometer (HITACHI, Japan). NMR spectra were obtained in DMSO-d6 with TMS as internal standard
and they were recorded on a Bruker Avance DRX-400 equipment, (DQ/ICEX/UFMG). The MS system used was a quadrupole time-of-flight instrument (UltrOTOFQ, Bruker Daltonics, Billerica, MA, U.S.A) equipped with an ESI positive and negative ion source. The analyses were performed with the mass spectrometer in
positive mode. The following settings were applied throughout the analyses: capillary voltage 4500 V; dry gas temperature 150 °C; dry gas flow 4 L/min.; nebulizer
gas nitrogen.
Plant Material
The leaves of Distictella elongata (Vahl) Urb. were collected in 2008 in the UFMG Campus Pampulha, Belo Horizonte, Minas Gerais State, Brazil. The species was
identified by Dr. Lúcia Lohmann, Universidade de São Paula (USP), São Paulo, SP, and a voucher sample is deposited at the BHCB, UFMG, Belo Horizonte,
Brazil, under the number 21.862.
Extraction and Isolation
Dried, powdered leaves (481 g) of D. elongata were extracted by percolation with EtOH 95%. After removing the solvent by evaporation under reduced pressure at
40°C, the leaves ethanol extract (LEE) (130.6 g; 27.2%) was obtained. Fractionation of LEE (7.4 g) was carried out by column chromatography (CC) on silica gel
(740 g, 70-320 mesh - Merck®) eluting with n-hexane, n-hexane-CH2Cl2 (1:1), CH2Cl2, CH2Cl2-EtOAc (1:1), EtOAc, EtOAc-MeOH (1:1), MeOH and H2O
successively. The EtOAc fraction (500 mg) was further chromatographed over a flash silica gel column chromatography (15 g, 200-300 mesh - Merck®) eluted with
n-hexane, n-hexane-EtOAc (1.7:0.3; 7:3; 1:1), and EtOAc. The n-hexane-EtOAc 7:3 fraction 21-35 (26 mg) was identified by TLC, 1H and 13C NMR as β-sitosterol
Castilho and Kaplan 2008
in comparison with literature (
). The n-hexane-EtOAc 7:3 fraction 51-73 (99 mg) was submitted to preparative TLC using CH2Cl2-EtOAc (3:2)
as eluent affording a mixture (17 mg) of ursolic, pomolic and oleanolic acids that were identified by 1H and 13C NMR. A portion of the EtOAc:MeOH (1:1) fraction
(230 mg) was further purified by preparative reverse phase HPLC separation to give the flavone pectolinarin (17 mg).
Pectolinarin (1). Pale yellow amorphous powder; mp 248-351 °C; [α]D 22,4 (MeOH, 0.5 mg/mL): -0.06; UV λmax nm (MeOH): 273.5, 328.5, λmax nm (MeOH +
NaOMe): 295, 370.5; λmax nm (MeOH + NaOAc): 273.5, (321-sh); λmax nm (MeOH + AlCl3): 349, 298, 281.5; (MeOH + AlCl3 + HCl): 348, 298, 281.5; IR v max
(cm–1): 3353 (OH), 1656 (C=O), 1608, 1583 (aromatic C=C), 1068 (C-O); ESI-MS m / : 623 [M + H]+, 645 [M + Na]+, 661 [M + K]+.
z
Preparative HPLC
Pectolinarin was purified on a Shimadzu HPLC system (Japan) composed of pump LC-8A, UV-Vis detector SPD-GAV, controller system SCL-8A and integrator CR4A. An ODS column (250 × 20 mm I.D., 10 mm; Shimadzu, Japan) was employed at room temperature, at a flow rate of 4.0 mL/min and UV220 detection. The
mobile phase was consisted of H2O (solvent A) and MeOH (solvent B). The following segmented gradient was used: A-B (95:5, v/v) to A-B (40:60, v/v), in 2 min;
A-B (40:60, v/v) to A-B (0:100, v/v), in 40 min; A-B (0:100, v/v) to A-B (95:5, v/v) in 20 min. HPLC grade solvents (Tédia, Brazil) were used and were degassed
by sonication before use. Samples were dissolved in MeOH, in an ultrasonic bath for 20 min (25 mg/mL). After filtration through 0.45 µm membrane syringe filter,
the sample solutions (200 µL) were injected into the apparatus.
RP-HPLC-DAD Profile
RP-HPLC-DAD analyses were carried out on a Waters alliance 2695 HPLC system composed of a quaternary pump, an auto sampler, a photodiode array detector
(DAD) 2996 and a Waters Empower pro data handling system (Waters Corporation, Milford, USA). An ODS column (125 × 4.0 mm i.d., 5 mm; Merck, Darmstadt,
Germany) was employed for the analysis. The profiles were performed employing a linear gradient of H2O (A) and MeOH (B), A-B (95:5, v/v) to A-B (5:95, v/v)
in 60 min; followed by 5 min of isocratic elution of A-B (5:95, v/v), at a temperature of 40°C and flow rate of 1.0 mL/min. The injection volume was 20 µL. The
chromatograms were obtained at λ 220 nm UV spectra from λ 220 to 400 nm were recorded online. Samples were dissolved in MeOH, in ultrasonic bath for 20
min, and then filtrated at 0.45 µm membrane syringe filter, giving a final concentration of 10 mg/mL for extract and fractions, and 4 mg/mL for the isolated
substance.
TLC Analyses
TLC plates (Merck, Silica gel 60 F254 – 0.25 mm); plates spots were detected under UV light (λ 365 nm), after spraying with anisaldehyde-H2SO4 reagent
(general), Liebermann-Burchard reagent (terpenoids and steroids) or aluminium chloride (flavonoids) followed by heating.
Hydrolysis
Hertog et al. 1992
The isolated flavone (2 mg) was hydrolysed in 2 M HCl in 20% aqueous methanol, by heating in a water bath at 80°C for 4 h (
). The final solution
was extracted with CH2Cl2. The aqueous layer was used for identification of the sugars by comparison with standards (β-D-glucose, α-L-rhamnose, β-D-galactose
and α-L-arabinose; Sigma-Aldrich) in TLC with CHCl3-MeOH-H2O (70:30:4) as mobile phase and anisaldehyde-H2SO4 reagent as spraying reagent.
RESULTS AND DISCUSSION
The RP-HPLC profile of the LEE from D. elongata has shown the major peaks in the range of 19.0 to 31.0 min. The UV spectra registered online are characteristic
of cinnamoyl derivatives: peaks with Rt 19.6 to 20.5 min; and flavones: peaks with Rt 26.5 to 31.0 min (
Mabry et al. 1970
). TLC phytochemical screening carried out
Wagner et al. 1984
for LEE indicated the presence of phenols, including flavonoids, terpenoids and/or steroids (
).
Fractionation of LEE by column chromatography (CC) on silica gel yielded eight fractions. TLC analysis of the EtOAc fraction has shown mainly the presence of
terpenoids and/or steroids. It was further chromatographed over a flash silica gel chromatography column to give 17 mg of a mixture of triterpenoids characterized
by 1H and 13C NMR spectroscopy.
The 1H NMR spectrum showed many signals at region δ 0.66-2.00 ppm corresponding to methyl and methylene hydrogens. The signal at δ 5.13 was attributed to
the olefin hydrogen bonded to C-12 of ursane/oleanane triterpenoids (
Castilho and Kaplan 2008
). 13C NMR spectra have shown the chemical shift of the carbinol and
olefin carbons that led to the identification of the three terpenoids: ursolic, pomolic and oleanolic acids (
Mahato and Kundu 1994
).
Wagner et al. 1984
TLC analysis of the EtOAc:MeOH (1:1) fraction has shown that it contained phenols only (
). RP-HPLC-DAD analyses of the EtOAc and EtOAc:
Meoh (1:1) fractions have shown a peak with retention time of approximately 31.0 min. The substance corresponding to this peak was isolated from the fraction
EtOAc:MeOH (1:1), by preparative RP-HPLC, and was identified by spectrometric analyses.
The UV spectrum of the isolated substance was typical of a flavone (λmax 273.5 and 328.5 nm). The presence of a free hydroxy group at C-5 and the absence of free
hydroxy at C-4′ and C-7 was indicated by the effects of AlCl3 and NaOAc in the UV curve. The difference in band I in the methanol spectra (λ 328.5 nm) and after
addition of NaOMe (λ 370.5 nm) is 42 nm with a decrease in intensity, indicating the absence of free OH at C-7 and C-4′. No modification of the UV curve,
relatively to the AlCl3 curve, after addition of HCl, indicated the presence of a free 5-hydroxyl group (
Mabry et al. 1970 Markham 1982
,
).
The 1D 1H NMR spectrum showed an AA′XX′ system of spins at δ 8.04 and 7.17 ppm (2H each, J = 8.0 Hz, 2′ and 6′; 3′ and 5′, respectively) due to hydrogens in a
4′-oxygenated B ring. The doublets at 4.57 ppm (J = 1.1 Hz, H-1′″) and δ 5.12 ppm (J = 4.00 Hz, H-1″) are typical of anomeric hydrogens. The singlet at δ 12.96
ppm for the hydroxy proton indicate the formation of a hydrogen bond with the neighboring oxygen. H-8 and H-3 appeared as singlets at δ 6.94 and 6.93 ppm,
respectively. The doublet at δ 1.06 (3H, J = 4.0 Hz, H-6′″) was related to a rhamnose methyl group. Two methoxy singlets at δ 3.78 (4′-OCH3) and 3.97 ppm (6-
OCH3) were observed and they were confirmed by the 1D 13C NMR signals at δ 55.6 (4′-OCH3) and δ 60.3 ppm (6-OCH3). Nine signals were observed in the 13C
NMR in the range of δ 66.0-76.5 ppm, corresponding to two sugar units: D-glucose and L-rhamnose as confirmed by 2D HMBC and HSQC-TOCSY experiments.
The methyl group of rhamnose gave a signal at δ 17.8 ppm in the 13C spectrum. Acid hydrolysis of the isolated substance yielded an aglycone, pectolinarigenin,
besides D-glucose and L-rhamnose which were confirmed by TLC. According to the coupling constant observed in the 1H NMR spectrum, the configurations at the
anomeric carbons of D-glucose and L-rhamnose were determined as β- (J = 1.1 Hz) and α-linkages (J = 4.00 Hz), respectively.
The chemical shifts of the carbon signals for D-glucose and L-rhamnose, indicated L-rhamnose as the terminal sugar. The C-6 glucose signal at δ 66.0 ppm
Moccelini et al. 2009
indicated that the disaccharide should be rutinose (
). The DEPT experiment confirmed this signal for a methylene group. The linkage of the
rutinosyl moiety to the oxygen atom attached to C-7 was confirmed by the HMBC correlation between the glucosyl H-1 (δ 5.12) and C-7 (δ 157.3). The 13C NMR
data also supported the attachment of the rutinosyl moiety to the oxygen at the 7-position of a flavone (Table I and Figure 1).
Fig. 1 Structure of pectolinarin.
TABLE I 13C and 1H NMR data for pectolinarin in DMSO-d6.
C
CH
C
C
C
C
CH
C
C
-
δC ppm
164.3
103.5
182.9
152.7
133.2
157.3
94.3
152.1
105.4
-
δH ppm (M, J)
6.93 (s)
6.94 (s)
12.96 (s, 5-OH)
1′
C
122.8
-
2′
CH
128.4
8.04 (d, J = 8.0 Hz)
3′
CH
114.8
7.17 (d, J = 8.0 Hz)
4′
C
162.8
-
5′
CH
114.8
7.17 (d, J = 8.0 Hz)
6′
6-OCH3
CH
128.4
8.04 (d, J = 8.0 Hz)
CH3
60.3
3.87 (s)
4′-OCH3
CH3
55.6
3.78 (s)
1″
CH
100.4
5.12 (d, J = 4.0 Hz)
2″
CH
73.2
3.33*
3″
CH
76.5
3.35*
4″
CH
69.5
3.18*
5″
CH
75.8
3.63*
6″
CH2
66.0
3.89; 3.48 (dd)
1′″
CH
100.4
4.57 (d, J = 1.1 Hz)
2′″
CH
70.8
3.46*
3′″
CH
70.4
3.67*
4′″
CH
72.0
3.16*
5′″
CH
68.3
3.41*
Position
Type
2
3
4
5
6
7
8
9
10
-
6′″
CH3
17.8
1.06 (d, J = 4.0 HZ)
M: Multiplicity, J: coupling constant
*
Approximate shifts obtained by HSQC experiment.
Direct correlations observed in the 2D HMBC maps confirmed the identification of this compound as pectolinarin (Figure 2). H-3 signal (δ 6.93 ppm) is recognized
by 2 J coupling with C-2 and C-4 (δ 164.3 and 182.9 ppm, respectively) and by 3 J coupling with C-10 and C-1′ (δ 105.4 and 122.8 ppm, respectively). H-8 (δ 6.94
ppm) is determined by 2 J coupling with C-7 and C-9 (δ 157.3, 152.9 ppm, respectively), 3 J with C-6 (δ 133.2 ppm) and 4 J with C-5 (δ 152.7) while H-6′ H-2′ (δ
8.04 ppm) showed 3 J coupling with C-2 and C-4′ (δ164.3, 162.8, respectively). Similarly, H-3′H-5′ (δ 7.17 ppm) showed 2 J coupling with C-4′ (δ 162.8) and 3 J
with C-1′ (δ 122.8). The direct coupling between H-2′H-6′ and H-3′H-5′ was not observed in the HMBC spectrum. The direct correlation between the OCH3
hydrogens (δ 3.78 ppm) and C-4′ (δ 162.8 ppm) confirmed the position of this methoxyl group at C-4′. Similar correlations were observed between the methoxyl
group signal at 3.87 ppm and C-6 (down field at δ 133.2 ppm) (Figure 1) (
Silverstein et al. 2007 Yim et al. 2003
,
).
Fig. 2 Main 1H →13C correlations inferred from HMBC pectolinarin spectra.
The ESI-MS spectrum showed an ion peak at m/z 623 [M + H]+ and ion peaks at 645 [M + Na]+ and 661 [M + K]+, which are coherent with the molecular formula
C29H34O15 for the flavone, pectolinarin.
This is the first report on the occurrence of pectolinarin, a flavone, in the Bignoniaceae family. Earlier studies indicated some bioactivities of this flavone.
The antioxidant potential of seven Korean thistles rich in pectolinarin was evaluated via the peroxynitrite. The DPPH free radical assays exhibiting strong activity
(
Jeong et al. 2008
). Pectolinarin isolated of the leaves of Cirsium setidens (Compositae), demonstrated hepatoprotective efficacy in a rat model of hepatic injury caused
by D-galactosamine. It was suggested that the activity occurs mainly via SOD (superoxide dismutase) antioxidant mechanism (
Yoo et al. 2008
).
However, in the present study, pectolinarin has not presented any radical scavenging activity on the DPPH assay, although the LEE and the EtOAc:MeOH (1:1)
fraction presented antioxidant activity by the DPPH assay (data not show). These results might be indicative of the presence of other phenols in LEE and EtOAc:
MeOH (1:1) fraction which would be responsible for their antioxidant activity.
In vivo studies have demonstrated that oral administration of pectolinarin and a fraction rich in pectolinarigenin isolated from aerial parts of Cirsium chanroenicum
at 20-100 mg/kg in several animal models resulted in inhibitory activities of inflammation/allergy: arachidonic acid-induced mouse ear edema, carrageenan-induced
mouse paw edema and passive cutaneous anaphylaxis. All of these results suggest that pectolinarigenin and pectolinarin possess anti-inflammatory activity and that
they may inhibit eicosanoid formation in inflammatory lesions. These activities certainly contribute to the anti-inflammatory effects of C. chanroenicum (
Lim et al.
2008
).
The promising application of pectolinarin in the osteogenesis imperfecta (OI) type I pharmacological therapy was shown by in vitro tests. The flavonoid normalized
collagen synthesis in OI cells. It was suggested that it exerts its effects through β1-integrin-mediated signaling (
Galicka and Nazarruk 2007
).
In vitro studies have demonstrated that leaves ethanol extract of D. elongate and pectolinarin isolated from the same species have antiviral activity against vaccinia
virus Western Reserve (VACV-WR) and dengue virus 2 (DENV-2) (
Simões et al. 2011
).
Although no ethnomedical use is reported for D. elongata, it might be considered useful as a source of pectolinarin for its disclosed antiviral, anti-inflamatory and
collagen inducing synthesis effects.
Acknowledgements
To Dr. Lúcia Lohmann, USP, São Paulo, SP, Brazil, for taxonomical identification of the plant species. To Dr. Norberto P. Lopes, USP, Ribeirão Preto, SP, Brazil,
for MS spectrum. To Conselho Nacional de Desenvolvimento Cietífico e Tecnológico (CNPq) (IA: A. B. O.) and Fundação de Amparo à Pesquisa do Estado de
Minas Gerais (FAPEMIG) for financial support and fellowship (BIC: G. M. M.).
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(Rutaceae). Quim Nova 32: 131-133. [ Links ]
Pool A. 2009. A Review of the Genus Distictella (Bignoniaceae). Ann Miss Bot Garden 96: 286-323. [ Links ]
Silverstein RM, Webster FX and Kiemle DJ. 2007. Identificação espectrométrica de compostos orgânicos. Rio de Janeiro: LTC, 490 p. [ Links ]
Simões LR, Maciel GM, Brandão GC, Kroon EG, Castilho RO and Oliveira AB. 2011. Antiviral activity of Distictella elongata (Vahl) Urb. (Bignoniaceae), a
potentially useful source of anti-dengue drugs from the state of Minas Gerais, Brazil. Lett Appl Microbiol 53: 602-607. [ Links ]
Wagner H, Bladt S and Zgainski EM. 1984. Plant drug analysis: a thin layer chromatography atlas. Berlim: Springer Verlag, 320 p. [ Links ]
Yim SH, Kim HJ and Lee IS. 2003. A polyacetylene and flavonoids from Cirsium rhinoceros. Arch Pharmacal Res 26: 128-131. [ Links ]
Yoo Y, Nam J, Kim M, Choi J and Park H. 2008. Pectolinarin and Pectolinarigenin of Cirsium setidens Prevent the Hepatic Injury in Rats Caused by DGalactosamine via an Antioxidant Mechanism. Biol Pharm Bull 31: 760-764. [ Links ]
Received: June22, , 2011; Accepted: May8, , 2012
Correspondence to: Rachel Oliveira Castilho E-mail: [email protected]
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http://dx.doi.org/10.1590/S0001-37652013000300004
Chemical Sciences
Flavonoids from leaves of Derris urucu: assessment of potential effects on seed germination and development of weeds
EWERTON A.S. DA SILVA1, LÍVIA T. LÔBO1, GEILSON A. DA SILVA1, ANTONIO PEDRO DA S. SOUZA FILHO2, MILTON N. DA SILVA1, ALBERTO C. ARRUDA1, GISELLE M.S.P. GUILHON1, LOURIVALDO S. SANTOS1, MARA S.P. ARRUDA1
1
2
Programa de Pós-Graduação em Química, Instituto de Ciências Exatas e Naturais, Universidade Federal do Pará, Cidade Universitária Professor Jose da Silveira Netto, 66075-970 Belém, PA, Brasil
Laboratório de Agroindústria, Embrapa Amazônia Oriental, Trav. Dr. Enéas Pinheiro, s/n, 66095-100 Belém, PA, Brasil
ABSTRACT
In some previous studies, we described the isolation of nine compounds from leaves of Derris urucu, a species found widely in the Amazon rainforest, identified as five stilbenes and four dihydroflavonols. In this work, three of these dihydroflavonols [urucuol A (1), urucuol B (2) and isotirumalin (3)] were evaluated to identify their potential as
allelochemicals, and we are also reporting the isolation and structural determination of a new flavonoid [5,3′-dihydroxy-4′-methoxy-(7,6:5″,6″)-2″,2″-dimethylpyranoflavanone (4)]. We investigated the effects of the dihydroflavonols 1-3 on seed germination and radicle and hypocotyl growth of the weed Mimosa pudica, using solutions at 150 mg.L–1.
Urucuol B, alone, was the substance with the greatest potential to inhibit seed germination (26%), while isotirumalin showed greater ability to reduce the development of the hypocotyl (25%), but none of the three substances showed the potential to inhibit radicle. When combined in pairs, the substances showed synergism for the development of root and
hypocotyl and effects on seed germination that could be attributed to antagonism. When tested separately, the trend has become more intense effects on seed germination, while for the substances tested in pairs, the intensity of the effect was greater on development of weed.
Keywords: Derris urucu; flavonoids; allelopathy; development of weeds; seed germination
RESUMO
Em estudos anteriores descrevemos o isolamento de nove substâncias, a partir das folhas de Derris urucu, uma espécie encontrada amplamente na floresta Amazônica, as quais foram identificadas como cinco estilbenos e quatro diidroflavonóis. Neste trabalho, três desses diidroflavonóis [urucuol A (1), urucuol B (2) e isotirumalina (3)] foram avaliados
para identificar seus potenciais como aleloquímicos. Estamos relatando também, o isolamento e a determinação estrutural de um novo flavonóide [5,3′-diidroxi-4′-metoxi-(7,6:5″,6″)-2″,2″-dimetilpiranoflavanona (4)]. Investigamos os efeitos dos diidroflavonóis 1-3 sobre a germinação de sementes e desenvolvimento da radícula e do hipocótilo da planta
daninha Mimosa pudica, usando soluções a 150 mg.L–1. Urucuol B, isoladamente, foi quem apresentou o maior potencial para inibir a germinação de sementes (26%), por sua vez, isotirulamina exibiu maior capacidade para reduzir o desenvolvimento do hipocótilo (25%), porém nenhuma das três substâncias mostrou potencial para inibir o
desenvolvimento da radícula. Quando combinadas aos pares, as substâncias mostraram sinergismo ao desenvolvimento da radícula e do hipocótilo e efeitos, na germinação de sementes, que poderiam ser atribuídos a antagonismo. Quando testadas separadamente, as substâncias apresentaram maior tendência para inibir a germinação de sementes, enquanto
que, quando testadas aos pares, observou-se aumento no efeito de inibição do desenvolvimento da planta daninha.
Palavras-Chave: Derris urucu; flavonóides; alelopatia; desenvolvimento da planta; germinação de semente
INTRODUCTION
Over the past few decades, in order to meet a growing demand for food, the so-called modern agriculture has been increasingly dependent on agrochemicals, such as fertilizers, and specially, agricultural defensives. In tropical regions, such as the Amazon, those needs are more present mainly due to acid soil conditions and low natural fertility,
and because environmental conditions are extremely favorable to endemic development. The use of these products has produced discontent of social order, mainly because they pollute natural resources, and jeopardize the quality of food of human diet, and for being associated with health problems (
Moreira et al. 2010
resistant to commercial herbicides are more frequent in agricultural systems (
Chemical substances produced by the plants themselves have been one of the many viable alternatives to face such issues. Like herbicides many of these substances act in the plant metabolism, thus a potential replacement to synthetic products available in the market (
Duke and Abbas 1995
action between natural products and synthetic products (
Anaya 1999
). Besides, varieties of weeds
). Such information shows that novel strategies are required for controlling weeds.
). Additionally, natural molecules have specificities which present low risk to natural resources and to the interests of society (
Cutler 1988
Duke et al. 2002
). Another favorable topic is the similarity of molecular sites of
).
The Amazon forest known for its wealth and biological diversity of species, may offer opportunities for discoveries of innovative and efficient chemical molecules which may be used in large scale in most agricultural activities, including management of both agricultural pests, of economic importance, and weed control. As a result, during the
last few years several studies have been conducted with native plants from the Amazon which have helped to identify powerful allelochemicals for possible agricultural use (
Arruda et al. 2005
, Souza Filho et al. 2005, 2006a, 2009,
Santos et al. 2007 Lôbo et al. 2008 Vilhena et al. 2009
,
,
).
Among the many species and varieties of native plants occurring in the Amazon forest, with chemical molecules properties with potential use in agriculture are Derris (Lonchocarpus/Deguelia) genera. As a result, Derris urucu, known as timbó vermelho, is the species which has raised our interest for its pesticide properties (
Hien et al. 2003
), and
Lôbo et al. 2009 2010
has recently been studied by us, resulting in the isolation of substances belonging to the dihydroflavonol and stilbene classes (
,
). Following up on this study, a hypothesis was raised that the dihydroflavonols isolated from the leaves may present potential activity for use in weed management. Therefore, the urucuol A (1),
urucuol B (2) and isotirumalin (3) substances (Fig. 1) were evaluated to characterize, isolated or in pairs, the potential allelopathic activity of these substances. We also describe the isolation and structural determination of a new substance, the flavanone 4 (Fig. 1), also isolated from the leaves of this plant.
Fig. 1 Chemical structure of flavonoids 1-4 isolated from Derris urucu leaves.
General Experimental Procedure
NMR spectra, including 1H-1H COSY, HMQC, HMBC experiments, were recorded on a Varian Mercury-300 spectrometer, operating at 300 MHz at 1H and 75 MHz at 13C, using d-chloroform as solvent and internal standard. Mass spectral analyses were performed at high resolution analyses on micro TOF II-ESI-TOF (Brucker, Daltonics
Billerica MA, USA) only at the cationazed ion region. HPLC was carried out in a preparative LC-8A Shimadzu system with SPD-10AV Shimadzu UV detector (Tokyo, Japan); using a Phenomenex Gemini C18 column (250 x10 mm, 5µ), an isocratic system of water/acetonitrile (38:62 for DU-3 fraction and 30:70 for DU-4 fraction) and a flow
rate of 4.7 mL per mim. Detection was performed at 280 and 320 nm. All solvents were filtered through a 0.45 µm membrane filter prior to analysis. Absorbance measurements were recorded on a Spectrum UV SP-220® spectrophotometer.
Plant Material
The plants were collected at the Experimental Field of Embrapa Amazônia Oriental, located in Belém, Pará, Brazil when the plants were flowering. A fertile sample was obtained and stored at the the Botanical Laboratory, and deposited as a dried and pressed specimen under the IAN-179599 register. Approximately 2.0 kg Derris urucu (Killip
& A.C.Sm.) green leaves were collected (Killip & A.C.Sm.) J. F. Macbr. (Leguminosae – Pap.) – being its synonym Lonchocarpus urucu (Killip & A.C.Sm) and Deguelia rufescens var. urucu (Killip & A.C.Sm.) J.F. Macbr. (A.M.G. Azevedo Tozzi, unpublished data), - which were dried in a forced air greenhouse, at 40 °C, until constant
weight was obtained. Next, trituration was performed with a knife mill.
Extraction and Isolation Procedure
Lôbo et al. 2009
The dried and powdered leaves of Derris urucu (700 g) were extracted with ethanol at room temperature. Part of the crude extract obtained (30 g) was treated following the methodology described (
), giving rise, among others, to fractions DU-3 (5.17 g) and DU-4 (5.36 g). Fraction DU-3 (1.0 g) was purified on a HPLC system
using a Phenomenex Gemini C18 column (250×10 mm, 5µ), an isocratic system of water: acetonitrile (38:62) and a flow rate of 4.7 mL min–1, yielding five fractions. Fraction four showed chromatographic peak with retention time 19.24 and was identified as a flavanone 4 (11 mg). The fraction DU-4 (1.0 g) was purified on the same HPLC
column using an isocratic system of water: acetonitrile (46:54) and a flow rate of 4.7 mL min–1, yielding the compounds 1 (80 mg), 2 (28 mg) and 3 (20 mg), which showed chromatographic peaks with retention times 9.10, 7.84 and 16.27, respectively.
Bioassay Evaluation of Allelopathic Activity
The germination bioassay was developed in a controlled temperature chamber at 25 °C, for 12 hours of photoperiod. Germination was checked every day for 10 days, with daily counting and the elimination of the germinated seeds. Seeds were considered germinated when they reached 2.0 cm or more in length of radicle. Each 9.0 cm diameter
Petri dish covered with qualitative filter received 25 seeds.
The bioassays of the radicle and hypocotyl development were developed similar to the seed germination, except for the photoperiod which was now 24 hours. Each Petri dish covered with qualitative filter received three pre germinated seeds, with approximately two days of germination. After 10 days of growth, the radicle and hypocotyl length
was measured.
Other Experimental Procedures
The substances were tested separately and in pairs, under 150 mg.L–1 concentrations. Each Petri dish received 3.0 mL of test solution. Specifically for those bioassays involving tests of substances in pairs, 50% of the volume was used for each substance. Following the eluent evaporation, the same volume of distilled water was added, thus,
maintaining the original concentration. The solutions were added just once, at the beginning of the tests, and then, whenever necessary, only distilled water was added.
Malicia (Mimosa pudica) as a common weed in the Amazon, was chosen to evaluate the allelopathic potential of the substances investigated in this work. The seeds of that species were collected in cultivated pasture areas, in the Municipality of Terra Alta, State of Pará, and eventually cleaned and treated to break the dormancy, by immersing
them into sulfuric acid for 20 minutes, as specified by Souza Filho et al. (1998).
Experimental Delineation and Statistical Analysis
The experimental delineation was entirely randomized with four repetitions using distilled water as control treatment. The data were transformed to arc. sine √x, to follow normal distribution. The values obtained were submitted to variance analysis, using F-test, and when treatment effects presented significant differences (p< 0.05), the means
were compared using the Tukey test. We used the Statistical Analysis System (1990) to perform the data analysis.
RESULTS
Phytochemical Investigation
Lôbo et al. 2009 2010
Fractionation of ethanol extract obtained from the leaves of D. urucu, allowed the isolation and structural identification of nine compounds (
,
). From the same extract, a new substance (4) was isolated as a white powder. The HRESIMS of 4 displayed a pseudomolecular ion [M+Na]+ at m/z 391.1327, consistent with the
molecular formula C21H20O6Na, further corroborated by the NMR data of 4. The 1H and 13C NMR spectra of 4 (Table I) showed characteristic sets of signals at δH 5.30 (dd, J=12.6 and 2.7 Hz, H-2), 3.05 (dd, J=17.4 and 12.6 Hz, H-3β) and 2.76 (dd, J=17.4 and 2.7 Hz, H-3α) and at δC 78.8 (CH, C-2) and 43.1 (CH2, C-3) of a flavanone
Jang et al. 2002
skeleton (
). A lowfield singlet at δH 12.29 indicated a C-5 OH group hydrogen-bonded to a carbonyl at C-4. Aromatic proton signals at δH 7.03 (d, J= 1.8 Hz, H-2′), 6.92 (dd, J= 8.1 and 1.8 Hz, H-6′) and 6.87 (d, J= 8.1 Hz, H-5′) could be assigned as 1,3,4-trisubstituted aromatic ring B protons, as evident from the HMBC
correlations from H-2′ and H-6′ to C-2 (Table I). The 3 J correlations of H-2′, H-6′, and a singlet at δH 3.91 (3H) to C-4′ (δC 145.8) indicated the attachment of a OCH3 group at C-4′. The correlations of H-2′ to both C-3′ (δC 146.9) and C-4′, and the presence of only one signal of OMe (δH 3.91) showed a OH group linked to C-3′. The 1H NMR
signals at δH 5.49 (d, J= 9.9 Hz, H-3″), 6.61 (d, J= 9.9 Hz, H-4″) and 1.43 (s, 6H) showed 1 J correlations with the 13C NMR signals at δC 126.2, 115.2 and 28.4 (2Me), respectively, and were assigned to a dimethylchromene group. The key HMBC correlations between OH-5/C-5 (δC 158.3) and H-4″/C-5 required the placement of a chromene
Jang et al. 2002
ring at C-6 and C-7. The configuration at C-2 was assigned as S based on a vicinal coupling constant of 12.6 Hz, in comparison to those of previously reported flavanones (
TABLE I
1H
). Compound 4 was thus identified as 5,3′-dihydroxy-4′-methoxy-(7,6:5″,6″)-2″,2″-dimethylpyranoflavanone and was given the trivial name urucunone.
(300 MHz) and 13C (75 MHz) NMR spectral data for 4, including heteronuclear 2D shift-correlated obtained by 1 H and 13 C-COSY- n J CH (n=1, HETCOR, n=2 and 3, HMBC) experiments, in CDCl3 as solvent, chemical shifts (δ, ppm) and coupling constants (J , Hz, in parenthesis).a
1H-13C-COSY- 1
Position
1H-13C-COSY- n
J CH
δH
δC
5.30 (dd, 12.6 and 2.7)
78.8
3α
2.76 (dd, 17.4 and 2.7)
43.1
3β
4
5
3.05 (dd, 17.4 and 12.6)
2
195.4
158.3
2
J CH
H-3β/C-2
102.9
7
162.0
H-8/C-7
96.2
162.3
103.1
H-8/C-9
131.5
H-6′/C-1′
5.95 (s)
1′
2′
7.03 (d, 1.8)
J CH
OH-5, H-8, H-3″/C-6
H-4″/C-7
H-8, OH-5/C-10
H-5′/C-1′
112.6
3′
146.9
H-2′, OH-3′/C-3′
4′
145.8
H-5/C-4′
5′
6.87 (d, 8.1)
110.5
6′
6.92 (dd, 8.1 and 1.8)
118.1
78.2
2″
3
H-2′/C-2
OH-5/C-5
6
8
9
10
J CH
3″
5.49 (d, 9.9)
126.2
4″
6.61 (d, 9.9)
115.2
2Me-2″
1.43 (s)
28.4/28.3
OMe-4′
3.91 (s)
56.0
H-2′, H-6′/C-4′
H-2′/C-6′
H-3″, 2Me-2″/C-2″
2Me-2″/C-3″
OH-5
12,29 (s)
OH-3′
5,72 (s)
a
Number of hydrogens bound to carbon atoms deduced by comparative analysis of DEPT-13C NMR spectra. 1H-1H-COSY spectrum was also used in these assignments.
Phytotoxicity of Dihydroflavonols Isolated
The potential phytotoxic promoted over seed germination (Fig. 2), of malicia as a receptor plant varied significantly (p<0.05) between the substances tested (1-3). Substance 2 caused the most intense inhibiting factors, always significantly higher (p<0.05), than those of 1 and 3. However, the magnitudes of these effects were always lower than
30%. No significant difference was observed (p>0.05) between the inhibitory potential of substances 1 and 3. When analyzed in pairs, the intensity of inhibitions were always lower (p<0.05) when compared to effects caused by each substance alone, what might indicate the existence of antagonism between both 1 and 2, as well as between 1 and
3, and 2 and 3.
Fig. 2 Effects of dihydroflavonols 1, 2 and 3, separately, and in pairs, over seed germination of malícia weed seedling. Data is shown in percentages of inhibition in relation to control treatment: distilled water. Letters show significant differences by the Tukey test (5%).
Figure 3 shows the effects over radicle development. The data show that there were no significant differences (p>0.05) between the three substances, when tested separately, with effect intensity always below 20%. Comparatively to the inhibitory effects over seed germination, we observed that when the substances are administered separately,
they presented more phytotoxic potential over seed germination, although this difference was not so significant. Under all conditions, when the substances were tested, inhibition was significantly (p<0.05) higher than those promoted by the substances separately. In some combination, such as in 1+3, the increase in relation to the two separated
substances was 200%. For combination 1+2, it was observed an increase in the phytotoxic capacity by 150%. These data sets show that there was a synergy between the three substances when administered in combination. The data differ from those observed over seed germination, where antagonistic effects might be involved.
Fig. 3 Effects of dihydroflavonols 1, 2 and 3, separately, and in pairs, over root development of malícia weed seedling. Data is shown in percentages of inhibition in relation to control treatment distilled water. Letters show significant differences by the Tukey test (5%).
For effects over the hypocotyl development (Figure 4), no significant differences were observed (p>0.05) between substances 1 and 2. The inhibitions promoted by these two substances, when tested separately, were below 10%. When tested separately, substance 3 presented greater potential (p<0.05) to inhibit radicle development than 1 and 2,
promoting, however, inhibition below 30%. When combined in pairs, the substances always promoted inhibitions of greater magnitude when compared to the effects achieved by the substances separately, indicating the existence of synergic effects among the three substances. This result replicates that observed on the radicle development
(Figure 3) and is different from the effects promoted over seed germination (Figure 2), indicating possibility of antagonism. In general, the tendency observed was that the substances, when tested separately, promoted more intense effects over seed germination, and when in pairs, they inhibited the radicle and the hypocotyl in a greater scale.
Separately, substance 1 was more effective in inhibition of seed germination of malicia weed, while 3 showed that it was more effective on inhibition of hypocotyl development. No significant differences were observed (p>0.05) on inhibitions promoted by allelochemicals 1 and 2, on both the radicle and the hypocotyl development.
Fig. 4 Effects of dihydroflavonols 1, 2 e 3, separately, and in pairs, over the hypocotyl development of malícia weed seedling. Data is shown in percentages of inhibition in relation to control treatment: distilled water. Letters show significant differences by the Tukey test (5%).
DISCUSSION
The rich Amazon plant biodiversity offers new and revealing opportunities to discover chemical molecules with potential use in weed management. Timbó vermelho (Derris urucu) is a species found in South America and Asia, with large dispersion in the Amazon Region. Several chemical prospecting studies of this plant have been more
restricted to its roots, being identified the presence of rotenoids rotenone, deguelin, tefrosin and rotenolone as major metabolites and minor compounds belonging to the flavanone, isoflavanone, chalcone, flavonol, pterocarpan, stilbene and saponin classes (Braz Filho et al. 1975,
studies, including this one, involving the leaves of D. urucu, have shown the absence of rotenoids. Apparently, the occurrence of these substances is restricted to the roots and barks of this species.
Flavonoids form an important group with different biological activities. In terms of allelopathy, some information is found in the literature indicating this activity for different compounds of flavonoids (
Anaya et al. 2003 Tseng et al. 2003 Perry et al. 2007 Simões et al. 2008
,
,
,
Parente and Mors 1980 Fang and Casida 1999 Pereira et al. 2000
). Recent studies (
,
,
Cipollini et al. 2008 Simões et al. 2008
,
). Recent
) show the importance of
flavonoids as allelophatic agents. There are no references concerning the allelopathic activity of isolated dihydroflavonoids in this study. Thus, the results found for other compounds of the group may serve as point of reference, facilitating assessment the potential of the three substances isolated and tested in this study. For instance,
(2008)
Lôbo et al.
Arruda et al. (2005)
e
obtained, for lower concentration, effects much greater than those observed in this study, for the same weed specie indicating a wide variation in allelopathic activity of flavonoids. In addition allelopathic inhibition obtained for chemical substances from the leaves of timbó vermelho (Derris urucu) constitutes a factor that
values the native flora of the Amazon rainforest and must be taken into consideration when speculating on the importance forest preservation in the Amazon as an alternative source of chemical molecules with potential use in weed management.
Reynolds (1987)
Bitencourt et al. (2007)
Attempts to relate the allelopathic activity to the structure of a molecule are rare in the literature, as seen in a study by
is a good example. Small differences in the chemical structure of certain substances may be favorable to increase in allelopathic activity (Souza Filho et al. 2006b). According to
, the presence or
absence of certain functional groups in a molecule may represent the increase or decrease of allelopathic activity. Of the three dihydroflavonoids tested in this study (1-3), Urucuol A (1) and Urucuol B (2) present chemical structures extremely similar, with differences only in the group connected to C-5, OMe in 2 and OH in 1. This difference
produced effects more significant to Urucuol B (2), especially in relation to inhibition of seed germination. The Isotirumalin structure (3) differs from the other two substances due to the absence of ring 2.2-dimethylchromene linked to ring A, and its effects were more intense over the hypocotyl development. Apparently, the Urucuol B structure
was more compatible to reduce weed germination while the Isotirumalin structure was more effective to reduce hypocotyl development, however, it is difficult to determine if only one functional group is operative and determinant of the molecule allelopathic activity, and it has not been clear yet, which specific factors act in each case.
In plant communities, the effects which may be attributed to the allelopathy phenomenon result not only from the action of a single allelochemical, but from various components acting simultaneously in the target plant. As a result, one may assume that allelopathic activity of a mixture of allelochemicals will be determined not only for its
Kubo et al. 1992 Vokou et al. 2003
concentration, but also, by the positive or negative interaction which may exist among the substances. There is information in the literature suggesting the existence of synergism as a result from the combination of different allelochemicals (
,
). In most studies where this hypothesis has been tested, the combination
among the allelochemicals involve fixed concentration and the interferences are based in the increase of inhibitory activity in relation to effects promoted by each substances alone (leading us to conclude the existence of synergism) and the reduction in inhibitory activity (suggesting the occurrence of antagonism). The results observed for
evaluation of the three dihydroflavonoids indicate the existence of synergic effects, as well as possible antagonist effects. In the seed germination bioassay, the predominant tendency was suggestive of antagonism, while synergic effects were observed in radicle and hypocotyl development. Apparently, the manifestation of one or the other of
these two attributes depends not only in the capacity that the substances are thought to be able to augment each other's activity, but also the factor of the plant under analysis. It is known, for example, that in general, seed germination is less sensitive to allelochemical effects than the growth of seedlings (
extent, may explain the variations observed in this study.
Reigosa and Malvido 2007
), which, to some
The set of results obtained once more indicate the Amazon forest as a source of chemical molecules with properties to be used in weed management, thus contributing to the aggregation of economic value of the two species and consequently for its preservation.
Acknowledgements
The authors are grateful to Prof. Norberto Peporine for HRMS measurements. We gratefully acknowledge Fundação de Amparo à Pesquisa do Estado do Pará-Brasil (Fapespa) for financial support. We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico do Ministério de Ciência e Tecnologia – Brasil (CNPq) for a grant.
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Received: December5, , 2011; Accepted: September4, , 2012
Correspondence to: Mara Silvia Pinheiro Arruda E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
http://dx.doi.org/10.1590/S0001-37652013000300005
Chemical Sciences
Multivariate optimization of an ultrasound-assisted extraction procedure for Cu, Mn, Ni and Zn determination in ration to chickens
JOÉLIA M. BARROS1, MARCOS A. BEZERRA1, GISSELI S. VALASQUES1, BARAQUÍZIO B. DO NASCIMENTO JÚNIOR1, ANDERSON S. SOUZA2, NÁDIA M. DE ARAGÃO1
1
2
Laboratório de Química Analítica, Departamento de Química e Exatas, Universidade Estadual do Sudoeste da Bahia, Av. José Moreira Sobrinho, s/n, Bairro Jequiezinho, 45200-190 Jequié, BA, Brasil
Núcleo Multidisciplinar em Saúde, Universidade Federal da Bahia, Rua Rio de Contas, 58, Bairro Candeias, 45028-100 Vitória da Conquista, BA, Brasil
ABSTRACT
In this work, multivariate optimization techniques were used to develop a method based on the ultrasound-assisted extraction for copper, manganese, nickel and zinc determination from rations for chicken nutrition using flame atomic absorption spectrometry. The proportions of extracting components (2.0 mol.L–1 nitric, hydrochloric and acetic acid
solutions) were optimized using centroid-simplex mixture design. The optimum proportions of this mixture taken as percentage of each component were respectively 20%, 37% and 43%. Variables of method (sample mass, sonication time and final acid concentration) were optimized using Doehlert design. The optimum values found for these variables
were respectively 0.24 g, 18s and 3.6 mol.L–1. The developed method allows copper, manganese, nickel and zinc determination with quantification limits of 2.82; 4.52; 10.7; e 9.69 µg.g–1, and precision expressed as relative standard deviation (%RSD, 25 µg.g–1, N = 5) of 5.30; 2.13; 0.88; and 0.83%, respectively. This method was applied in the analytes
determination from chicken rations collected from specialized commerce in Jequié city (Bahia State/Brazil). Application of paired t-test at the obtained results, in a confidence level of 95%, does not show significant difference between the proposed method and the microwave-assisted digestion.
Keywords: rations to chicken; ultrasound-assisted extraction; mixture design; Doehlert design; metals
RESUMO
Neste trabalho, técnicas de otimização multivariada foram usadas no desenvolvimento de um método com base na extração assistida por ultrassom para determinação de cobre, manganês, níquel e zinco em rações para nutrição de frangos usando espectrometria de absorção atômica com chama. As proporções dos componentes extratores (soluções 2.0 mol.
L–1 dos ácidos nítrico, clorídrico e acético) foram otimizadas usando-se um planejamento de misturas centróide simplex. As proporções ótimas desta misturas representadas em percentagem de cada componente foram, respectivamente, 20, 37 e 43%. As variáveis de método (massa da amostra, tempo de sonicação e concentração final do ácido) foram
otimizadas usando-se planejamento Doehlert. Os valores ótimos encontrados para estas três variáveis foram respectivamente 0,24g; 18s e 3,6 mol.L–1. O método desenvolvido permite a determinação de cobre, manganês, níquel e zinco com limites de quantificação de 2,82; 4,52; 10,7 e 9,69 µg.g–1, e precisão expressa como desvio padrão relativo (%
SRD, 25 µg.g–1, N = 5) de 5,30; 2,13; 0,88 e 0,83%, respectivamente. Este método foi aplicado na determinação de analitos em rações de frango coletadas no comércio especializado da cidade de Jequié (Estado da Bahia/Brasil). A aplicação do test t pareado aos resultados obtidos, no nível de confiança de 95%, mostrou não haver diferença significativa
entre o método proposto e a digestão assistida por microondas.
Palavras-Chave: rações para frango; extração assistida por ultrassom; planejamento de misturas; planejamento Doehlert; metais
INTRODUCTION
Nowadays, Brazil is a major producer and exporter of chicken meat, and in 2007 produced 10.3 million tons and exported 3.2 million for foreign market. In the last decade, the domestic consumption of chicken meat in the country exceeded the consumption of other meats, such as beef and pork. Therefore, assessing factors such as adequate
food for birds is very important to improve the quality of the produced meat (
Roppa 2008
).
The use of rations for nutrition of chickens destined for meats and eggs production is an economical, efficient and rational way of providing all the nutrients in a balanced way to these animals. Macro (Ca, Mg, P, K, Na, Cl and S) and micro nutrients (Cu, Co, Zn, Fe, Ni, Mn, I, Mo, Se and Cr) must be present in rations in adequate amounts,
avoiding risks of malnutrition, which would be difficult to control using other types of food. Determining mineral content in rations for animal nutrition is of great importance in assessing its quality, mainly due to the necessity of verifying the quantities of mineral nutrients supplied by these rations and evaluating the possibility of
contamination by toxic metals (such as Pb, Hg and Cd) that can often be present in rations (Edwards and Veltman 1983).
The search for new procedures that are quick, easy to perform and that require smaller quantities of reagents has been a major driver of research in the field of Analytical Chemistry in the recent years. Within this context, the energy present in the ultrasonic waves has been used for developing methodologies for extraction of chemical species in
different types of samples (
Mason and Lorimer 2002
).
Ultrasounds are mechanical waves that propagate in material media, with a speed that can vary from hundreds of meters per second in the air up to thousands of meters per second in solids. That occurs in consecutive cycles of compression and rarefaction at frequencies higher than the audible frequencies for humans, which is above 16 KHz
(
Korn et al. 2003
).
Ultrasound-assisted leaching is considered by many authors an efficient way to extract metal species present in different types of samples and constitute an environmentally friendly technique (
Nascentes et al. 2001
Ca, Mg, Mn and Zn in plant tissue samples (
); Ca, Mg, Mn and Zn in samples of fish ration (
Collasiol et al. 2004
One can also mention the Hg determination in soil, river and sea sediments (
Hristozov et al. 2004
Pb and Zn in sewage sludge (
) and metals in edible seaweed (
Neves et al. 2009
); electrolytic manganese in slag (
); Cd and Pb present in plant leaves (
Gonzalez et al. 2005
Ruiz-Jimenez et al. 2003
Li et al. 2008
Luque-Garcia and Castro 2003 Magalhães et al. 2009
,
); Mg, Mn and Zn in plant tissue samples (
Filgueiras et al. 2000
). One can mention the following works that use the ultrasound energy for extracting metals: determining of
); cadmium and lead in mussel samples (
Biurrun et al. 2005
). In these papers FAAS was used as detection technique.
) using graphite furnace atomizer in AAS; trace elements in hair samples by inductively coupled plasma mass spectrometry (ICP-MS) (
Batista et al. 2009
); determination of Cd, Co, Cr, Cu, Mn, Ni,
) using inductively coupled plasma optical emission spectrometry (ICP OES).
In this paper, it was applied multivariate optimization techniques (mixture and Doehlert designs) to develop an analytical method, using ultrasound-assisted extraction of Cu, Mn, Ni and Zn in rations used for chicken nutrition and determination using flame atomic absorption spectroscopy (FAAS).
Instrumentation
A Perkin Elmer (Norwalk, CT, USA) Analyst model 200 flame atomic absorption spectrometer equipped with a deuterium lamp for background correction was used for measuring absorbance. Hollow cathode lamps for copper, manganese, nickel and zinc were used in accordance with the recommendations of the manufacturer (0.7, 0.2, 0.2,
and 0.7, for bandwidth of the slit, 324.8, 279.5, 232.0, and 213.9 nm for wavelengths to Cu, Mn, Ni and Zn, respectively). The burner height (13.5 mm) was also used with conventional values. Flame was consisted of acetylene (flow rate 2.0 L.min–1) and air (flow rate 13.5 L.min–1). Flow rate used for the nebulizer was 5.0 mL.min–1.
A Unique (São Paulo, Brazil) ultrasonic bath, Maxiclean 1450 model was used to promote the extraction of target elements. The ultrasound bath equipment was filled with 2.8 L of water and one-by-time glass centrifuge tube was placed, with the aid of a base, in the center of the bath to perform the ultrasound assisted extraction.
A QUIMIS T 222 QM centrifuge (São Paulo, Brazil) with 50 mL centrifuge tubes was used to separate solid from liquid-phase. A Sartorius (model BL D105) analytical balance was used to establish the sample mass. Digestion of rations samples was carried out in a Parr 4749 (Moline, IL, USA) digestion bomb enclosing a chemically inert
Teflon sample cup of 23 mL.
Reagents and Solutions
All chemicals and reagents used in this study were of analytical-reagent grade. Ultrapure water (Milli-Q®, Millipore, USA) with resistivity of 18.2 mΩ cm was used throughout. The laboratory glassware was kept in diluted nitric acid at least overnight, and subsequently washed with deionized water. Solutions of 10% (m/v) Triton X-100
surfactant (Sigma-Aldrich, Milwaukee, USA) were prepared in high purity deionized water. One thousand milligrams per liter stock standard solutions of Cu, Mn, Ni and Zn were purchased from Merck (Darmstadt, Germany). Nitric and hydrochloric acid solutions were prepared by direct dilution with deionized water from the concentrated
solutions (Merck, Darmstadt,Germany).
Collection of Samples
Samples of chicken ration used were collected in the trade of Jequié city, Bahia, Brazil. Samples were stored on their own sale packaging and transported to the laboratory, where they were subjected to preliminary treatments. Rations were collected for three different purposes (promote growth, fattening, and laying eggs).
Preliminary Treatments of Samples
Samples were taken to oven and dried at a temperature of 70°C until all moisture was removed. After drying, samples were left in order to reach room temperature to be crushed in a knives mill. Comminuted samples were sieved through 300 µm sieves and later 100 µm and then stored in polyethylene bottles within dissectors. The remaining
fractions were comminuted and sieved again, until those samples do not present some segregation.
Total Digestion of Chicken Ration Samples in Microwave Oven
Ration samples (0.1 g) were digested by adding 2.0 mL of 1:1 (v/v) nitric acid solution to the sample. A Teflon cup was placed in a PARR 4781 digestion bomb and heated in a microwave system for 1 min at 264.9 W. Blank samples were prepared in the same way. Subsequently, samples were cooled to room temperature, transferred to a
volumetric flask and diluted to a final volume of 10 mL with deionized water. Metals were determined in the final solution by flame atomic absorption spectrometry (FAAS). Assays of metals found in the samples using the microwave radiation-assisted acid digestion method were taken as reference values (
percentage of extracted metals in relation to the method of ultrasound-assisted extraction.
Santos et al. 2009
) in establishing the
Optimization of Ultrasound-Assisted Extraction Procedure
To optimize the ultrasound-assisted extraction method of metals (Cu, Mn, Ni and Zn) in ration samples for chicken nutrition, we used two types of experimental design: a simplex centroid mixture design (
2007
Bruns et al. 2006 Massart et al. 1997
,
) to optimize the ratio of extracting solutions (HCl, HNO3 and H3COOH), and a Doehlert design (
Ferreira et al.
) for optimization of method variables (final concentration of acid, sample mass, and sonication time).
To allow the simultaneous optimization of the four obtained responses (percentage of metal extraction), a mathematical approach developed by Derringer and Suich was used (
Derringer and Suich 1980
). It is based on desirability functions applied in optimizing multi-response experiments. The approach consists of first converting each response yi
into an individual desirability function di , which varies within the range 0 ≤ di ≤ 1. If the response is the desired, di = 1, and if the response is outside the acceptable region, d = 0. Thus, the factor levels are chosen to maximize the overall desirability given by the expression:
(Eq. 1)
Where m is the number of response variables. For example: if the target value T is a maximum , there will be:
(Eq. 2)
where L is the lowest acceptable value for the response, and s is the weight (when equal to 1 = linear desirability function). In this work, L values were the lowest recovery found in a set of experiments for each metal and T values were the highest recovery also for each metal.
Use of Mixture Design in the Optimization of Extracting Acid Solutions Proportions
The following procedure was followed: about 0.3 g of chicken ration sample was weighed directly into a 50 mL dry centrifuge tube. Later, different volumes of each extracting solution were added in order to always have a final volume of 6.0 mL. The proportion of each acid solution in each experiment is established by the mixture design
matrix used (centroid simplex design). In this step, variables of the method such as acid concentration, sample mass and sonication time were fixed at 2.0 mol.L–1, 0.3 g and 15 minutes, respectively.
Use of Doehlert Design in the Optimization of Method Variables
Variables of the method to be optimized in this stage were: acid concentration (AC) in mol.L–1, sample mass (SM) in grams, and sonication time (ST) in minutes. Due to the characteristics of the experimental design matrix used in this study, AC and SM were studied in five levels and ST in three levels. The acid proportions considered as
optimal in the previous mixture design were used.
The following procedure was followed: 0.1 to 0.5 g of chicken ration sample was weighed directly into a 50 mL dry centrifuge tube. Later, volumes considered as optimal from each of the extracting solutions were added. The concentration of acids, sample mass, and sonication time corresponding to each experiment are set by the design
matrix.
The development of fast and accurate methods to allow determining amounts of micro nutrients such as Cu, Mn, Ni and Zn in rations for animal nutrition is of concern due the necessity of monitoring their levels and to ensure that metals are offered in adequate concentration to the chickens. Low concentrations of these metals cause
malnutrition but, on the other hand, high levels can be toxic and affect the bird health decreasing the performance of the creation. Thus, quality control protocols based on reliable analytical methods must be implemented to guarantee the food security of population.
Simplex Centroid Mixture Design
Simplex centroid design is very useful in establishing optimal proportions among the various components of a mixture. In ultrasound-assisted extraction, acid solutions are usually used as extracting agents due to their good efficiency. This efficiency can be improved by using mixtures of these solutions. However, it is necessary to use an
appropriate proportion of these extractors to achieve a good extraction and improve the analytical characteristics of the method.
The experimental simplex centroid design used, the recoveries for the studied metals, and the overall desirability are shown in Table I. All recoveries, and consequently all the overall desirability, were obtained in duplicate for each experiment in order to estimate the experimental error.
TABLE I Mixtures design matrix and responses (recovery percentage of metals) to optimize the proportions of extracting solutions of nitric acid (HNO3), hydrochloric acid (HCl) and acetic acid (HAc).
Variables
Extraction (%)
HNO3
HCl
HAc
Cu
Mn
Ni
Zn
6 (1)
0 (0)
0 (0)
3 (1/2)
3 (1/2)
0 (0)
2 (1/3)
4 (4/6)
1(1/6)
1(1/6)
0 (0)
6 (1)
0 (0)
3 (1/2)
0 (0)
3 (1/2)
2 (1/3)
1(1/6)
4 (4/6)
1(1/6)
0 (0)
0 (0)
6 (1)
0 (0)
3 (1/2)
3 (1/2)
2 (1/3)
1(1/6)
1(1/6)
4 (4/6)
81.01 / 86.00
74.34 / 60.07
62.63 / 62.98
73.44 / 76.62
97.27 / 89.42
80.62 / 78.43
86.73 / 84.43
84.46 / 84.54
82.32 / 78.43
85.44 / 89.00
83.21 / 88.77
81.51 / 71.38
90.33 / 88.35
82.79 / 82.05
97.91 / 97.68
90.83 / 94.11
91.64 / 98.84
92.57 / 96.71
88.76 / 89.07
96.59 / 100.76
46.23 / 51.85
78.34 / 78.34
39.50 / 45.16
45.63 / 65.12
52.69 / 58.45
85.44 / 97.91
78.10 / 78.10
58.39 / 58.45
105.2 / 97.92
59.07 / 58.17
99.54 / 97.11
85.04 / 81.98
87.74 / 88.22
95.14 / 92.01
101.3 / 97.05
95.07 / 92.48
100.2 / 97.17
99.22 / 92.94
97.35 / 94.34
97.15 / 98.27
Overall Desirability
0.3809 / 0.4962
0.2820 / 0.0000
0.0000 / 0.1882
0.3067 / 0.4251
0.6523 / 0.6312
0.6449 / 0.6552
0.6544 / 0.7827
0.5900 / 0.5117
0.7282 / 0.6410
0.6080 / 0.6569
Note: The coded values are given in brackets.
Linear, quadratic, special cubic, and full cubic mathematical models were fitted to the data generated for overall desirability to obtain the best description of the experimental region. To assess the quality of the model, the lack of fit was verified. If the mathematical model is well fitted to the experimental data, the mean square of lack of fit
(MSlof) should reflect only the random errors inherent to the system. Additionally, mean square of pure error (MSpe) should also be an estimative of these errors and it is assumed that these two values are not statistically different. Thus, it is possible to use the F distribution to assess whether there is any statistical difference between these two
means.
According to Analysis of Variance (Anova) presented in Table II, the linear model showed lack of fit (Fcalculated > Ftabled) and left large residues. Therefore, it presents low predictive capability. The quadratic model did not show lack of fit (Fcalculated <Ftabled), left smaller residues and, therefore, has a good predictive capability. The special
cubic and full cubic models also left low residues, but the small improvement in predictive capability is not a rationale for the use of a more complex model. Thus, it was decided to use the quadratic model to the description of the studied experimental region.
TABLE II Analysis of variance (ANOVA) for linear, quadratic, special cubic, and full cubic models, fitted to overall desirability, with a 95% confidence level.
Linear Model
Source of Variation
SS
df
MS
Fcalculated
Ftabled
Model
Total error
Lack of fit
Pure error
0.0363
0.970
0.884
0.085
2
17
7
10
0.0181
0.0571
0.126
0.008
0.318
3.59
14.79
3.14
Quadratic Model
Source of Variation
SS
df
MS
Fcalculated
Ftabled
Model
Total error
Lack of fit
Pure error
0.860
0.146
0.054
0.085
5
14
4
10
0.172
0.010
0.014
0.008
16.48
2.96
1.59
3.48
Special Cubic Model
Source of Variation
SS
df
MS
Fcalculated
Ftabled
Model
Total error
Lack of fit
Pure error
0.870
0.137
0.045
0.085
6
13
3
10
0.145
0.0105
0.015
0.008
13.8
2.92
1.72
3.71
Full Cubic Model
Source of Variation
SS
df
MS
Fcalculated
Ftabled
Model
Total error
Lack of fit
Pure error
0.913
0.0932
0.007
0.085
8
11
1
10
0.114
0.00848
0.007
0.008
13.5
2.95
0.87
4.96
SS, sum of square; df, degree of freedom; MS, mean of square.
The following quadratic equation illustrates the relation among the three component volumes of the studied mixtures and overall desirability (OD):
(Eq. 3)
In equation 3, terms in bold are not significant. They were not eliminated from the equation to allow calculation of the critical point. In Figure 1a and 1b, response surface and the contour plot described by equation 1 are presented and show the behavior of response with the level variation of the studied factors.
Figure 1 (a) Response surface and (b) contour graph for optimization of extraction proportions in an ultrasound extraction procedure using centroid mixture design.
Figure 2 shows the individual desirability profiles (related to each metal) and the overall desirability profile indicating the optimum proportions of each component. The fourth column of graphics, presented in Figure 2, shows how the profiles vary from individual desirability for each response according to the limits established for L , T and s
in equation 1. Since L was the lowest value found for each set of responses, T was the highest of them and s was equal to 1, the graphics present linear profiles.
Figure 2 Profiles for predictive values, individual and overall desirability in optimizing the proportions of the mixture variables.
Using overall desirability as response, it possible to see that there is a maximum as critical point. The coordinates of these points are the proportions of components that generate the highest response for the studied system. Thus, analyzing the desirability profile graph, the optimal values found to performing simultaneous extractions of studied
metals were: 1.2 mL (20%) of HNO3, 2.2 (37%) of HCl, and 2.6 (43%) of HAc.
Doehlert Design
The performance of ultrasound-assisted extraction depends on proportions of acid solutions used to extract analytes, as well as some method variables such as particle size, acid concentration, sample mass, sonication time, extractor volume, among others.
The experimental Doehlert design used, recoveries for the studied metals, and the overall desirability are shown in Table III. For description of the experimental area, the overall desirability was once again used as response, in order to obtain a simultaneous optimization of the four metals studied. According to the ANOVA Table (Table IV),
both linear and quadratic model showed lack of fit (Fcalculated > Ftabled). In spite of the quadratic model presents lack of fit, it leaves lower residue values than the linear and it was used to indicate the optimum experimental conditions. Experiments were performed using these conditions and it was verified that there were quantitative recoveries
for all studied metals. The quadratic equation shown below was found:
(Eq. 4)
Again, in equation 4, terms in bold are not significant, but they were not eliminated from the equation to allow calculation of the critical point.
TABLE III Doehlert design and responses (recovery percentage of copper, manganese, nickel, and zinc) for optimization of variables method: acid concentration (AC), sample mass (SM), and sonication time (ST).
AC
3 (0)
2 (-0.5)
2 (-0.5)
4 (0.5)
4 (0.5)
1 (-1)
3 (0)
5 (1)
2 (-0.5)
2 (-0.5)
4 (0.5)
4 (0.5)
3 (0)
Variables
SM
0,5 (1)
0,4 (0.5)
0,4 (0.5)
0,4 (0.5)
0,4 (0.5)
0,3 (0)
0,3 (0)
0,3 (0)
0,2 (-0.5)
0,2 (-0.5)
0,2 (-0.5)
0,2 (-0.5)
0,1 (-1)
Extraction (%)
ST
15 (0)
5 (-0.707)
25 (0.707)
5 (-0.707)
25 (0.707)
15 (0)
15 (0)
15 (0)
5 (-0.707)
25 (0.707)
5 (-0.707)
25 (0.707)
15 (0)
Cu
71.08 / 64.44
48.33 / 51.79
66.56 / 62.74
70.49 / 63.70
100.8 / 100.75
86.27 / 90.84
82.10 / 72.69
101.9 / 100.79
75.76 / 68.66
79.55 / 86.79
76.40 / 69.85
88.20 / 84.61
79.47 / 81.06
Mn
79.60 / 76.98
77.65 / 76.43
90.63 / 80.87
73.28 / 75.39
79.16 / 75.91
91.67 / 91.65
98.19 / 98.76
82.12 / 83.63
91.32 / 86.89
93.88 / 89.41
83.32 / 83.86
89.43 / 82.68
101.6 / 91.31
Ni
44.33 / 53.05
60.78 / 54.67
69.83 / 61.09
44.21 / 43.70
64.83 / 66.35
66.58 / 58.65
94.47 / 88.65
95.63 / 109.18
65.65 / 66.50
83.15 / 62.13
62.89 / 63.88
88.01 / 99.50
43.62 / 40.44
Overall Desirability
Zn
61.95 / 62.75
73.58 / 75.76
75.38 / 78.03
68.10 / 61.79
61.28 / 64.41
78.92 / 81.54
98.37 / 92.22
82.89 / 94.90
79.90 / 84.50
94.91 / 93.79
76.85 / 83.36
88.54 / 96.68
89.04 / 90.54
0.0991 / 0.1299
0.0000 / 0.1554
0.4290 / 0.3145
0.0035 / 0.0612
0.0000 / 0.2318
0.5369 / 0.5223
0.8124 / 0.6993
0.6179 / 0.7546
0.4949 / 0.4561
0.6989 / 0.5799
0.3994 / 0.4178
0.6815 / 0.6552
0.3765 / 0.3450
Note: The coded values are given in brackets.
TABLE IV Analysis of variance (ANOVA) for linear and quadratic models fitted to overall desirability, with a 95% confidence level.
Linear Model
Source of Variation
SS
df
MS
Fcalculated
Ftabled
Regression
Residual
Lack of fit
Pure error
Total
0.705
0.895
0.823
0.0723
1.60
3
22
9
13
25
0.235
0.0407
0.0915
0.00556
5.77
3.05
16.4
2.71
Source of Variation
SS
df
MS
Fcalculated
Ftabled
Regression
Residual
Lack of fit
Pure error
Total
1.30
0.301
0.228
0.0723
1.60
9
16
3
13
25
0.144
0.0188
0.0761
0.00556
7.68
2.54
13.7
3.41
Quadratic Model
SS, sum of square; df, degree of freedom; MS, mean of square.
Figure 3 shows the individual and the overall desirability profiles. As it was carried out to calculate the individual desirability for mixture, L was the lowest value found for each set of responses, T was the highest of these set and s was equal to 1. Analyzing this graph, indicating that higher responses are obtained with an acid concentration of
3.2 mol.L–1, sample mass of 0.24 g, and sonication time of 18 min.
Figure 3 Profiles for predictive values, individual and overall desirability in optimizing the values of the method variables.
Analytical Features of the Procedure
The limit of detection (LOD) was calculated based on three times (3 s) of standard deviation of blank signal by 10 replicate measurements. The limit of quantification (LOQ) is the concentration that gives a response equivalent to 10 times the standard deviation of blank signal (n = 10), and it defines the lower limit of the range. For the
proposed procedure, LODs were 0.85, 1.28, 3.50, and 2.90 µg.g–1, and LOQs were 2.82, 4.52, 10.7, and 9.69 µg.g–1, and precision expressed as relative standard deviation (%RSD, N = 5) of 8.30, 2.13, 0.88, and 0.83% for copper, manganese, nickel and zinc, respectively. Relative standard deviation was determinate with data originated after
perform five extractions from a sample that was spiked to a final concentration of 25 µg.g–1. Additionally, it was verified that Triton X-100 surfactant solutions contribute to increase the blank signal and, consequently, the limit of detection for zinc.
Application and Accuracy
After defining the optimal conditions for ultrasound-assisted extraction of the elements studies from rations for chicken nutrition, the developed method was applied to the analysis of nine samples collected in the specialized market of Jequié city, at Bahia State.
The accuracy of the developed procedure was verified by comparing the generated results with those of microwave-assisted digestion for three samples. The results of this analysis are presented in Table V.
TABLE V Concentrations (µg.g–1) of copper, manganese, nickel and zinc found in samples of rations analyzed using the Ultrasound-Assisted Extraction (UAE) and Microwave-Assisted Digestion (MAD).
Kind of ration
Growth
Fattening
Posture
Sample
1
2
3
1
2
3
1
2
3
Cu
UAE
96 ±3
24±1
51±6
36±3
26±1
3,9±0,4
53±2
5,3±0,3
4,7±0,6
Mn
MAD
93±2
36±1
54±1
-
UAE
111 ±7
98±5
78±1
158±1
126±3
71±3
110±5
96±1
61±3
Ni
MAD
115±3
159±2
109±1
-
UAE
49 ±5
21±2
12±2
51±2
73±2
38±3
47±3
27±3
14±2
Zn
MAD
47±3
56±6
46±3
-
UAE
64 ±3
65±3
62±2
71±3
72±4
58±2
62±5
61±3
55±3
MAD
65±3
72±2
65±2
-
UAE, Ultrasound-assisted extraction; MAD, Microwave-assisted digestion.
Three results from each sample for growth, fattening and posture using the proposed method were compared with the results of microwave-assisted digestion. Paired t-test was used to compare them with a 95% confidence level. Through this test, it was not noticed significant difference between data generated by the two methodologies. The t
values found respectively for Cu, Mn, Ni and Zn were 0.555, 0.918, 0.305, and 2.50, all values below the tabled value (4.30) indicating that, at this confidence level, results found for both methods are not statistically different.
CONCLUSIONS
Application of mixture and Doehlert designs allowed the development of an analytical procedure for determination of copper, manganese, nickel and zinc from ration for chicken nutrition using flame atomic absorption spectrometry (FAAS), after ultrasonic-assisted extraction. Since FAAS is low cost and a relatively simple analytical
technique, its association with efficient extraction procedures can make it more adequate for quality control in industries for chicken rations production.
The described method offers a quick, easy and efficient sample preparation method for direct determination of these metals from these samples. The use of ultrasonic energy allowed leaching of the target analytes in a shorter time than required by the conventional wet acid digestion. The ultrasonic-assisted extraction is a quick, inexpensive,
easy, and precise technique.
Acknowledgements
Authors acknowledge the financial support of the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Financiadora de Estudos e Projetos (FINEP).
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Received: August30, , 2011; Accepted: September5, , 2012
Correspondence to: Marcos de Almeida Bezerra E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013000300006
Chemical Sciences
In vitro trypanocidal activity of solamargine and extracts from Solanum palinacanthum and Solanum lycocarpum
of brazilian cerrado
RAQUEL R.D. MOREIRA1, GILMARCIO Z. MARTINS1, NATHÁLIA O. MAGALHÃES1, ADÉLIA E. ALMEIDA2, ROSEMEIRE C.L.R. PIETRO2,
FLÁVIA A. J. SILVA, REGINA M.B. CICARELLI3
1
Departamento de Princípios Ativos Naturais e Toxicologia, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista/UNESP, Rodovia Araraquara
– Jaú, Km 1, s/n, 14801-502 Araraquara, SP, Brasil
2 Departamento de Fármacos e Medicamentos, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista/UNESP, Rodovia Araraquara – Jaú, Km 1, s/
n, 14801-502 Araraquara, SP, Brasil
3 Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista/UNESP, Rodovia Araraquara – Jaú, Km 1, s/n,
14801-502 Araraquara, SP, Brasil
ABSTRACT
The present investigation was to evaluate the potential trypanocidal activity of crude ethanolic extract of the fruits of Solanum palinacanthum, Solanum lycocarpum and
the glycoalcaloid, solamargine. S. palinacanthum and S. lycocarpum fruit powders were submitted to exhaustively extraction with 96% ethanol and solamargine were
isolated from the extract of S. palinacanthum. Both extracts and solamargine were analysed for trypanocidal activity by using MTT colorimetric assay. Extracts of S.
palinacanthum showed to be more active (IC50 = 175.9 µg.ml–1) than S. lycocarpum (IC50 = 194.7 µg.ml–1). Solamargine presented a strong activity (IC50 = 15.3 µg.ml–
1),
which can explain the better activity of the both extracts. Benznidazol (IC50 = 9.0 µg.ml–1) is the only drug used to treat Chagas' disease. These findings demonstrate
for the first time that ethanol extracts obtained from both fruits of S. palinacanthum and S. lycocarpum and also solamargine have a potential anti-trypanosomal activity.
Keywords: Solanum palinacanthum; Solanum lycocarpum; solamargine; epimastigote; antitrypanosomal activity; Trypanosoma cruzi
RESUMO
O objetivo deste estudo foi avaliar a potencial atividade tripanocida do extrato bruto etanólico dos frutos de Solanum palinacanthum, Solanum lycocarpum e do
glicoalcalóide solamargina. Pó do fruto seco de S. palinacanthum e S. lycocarpum foram submetidos a extracção por refluxo com etanol a 96% e solamargina foi
isolada a partir do extrato bruto de S. palinacanthum. Foram determinadas de ambos os extratos e a solamargina a atividade tripanocida utilizando o ensaio
colorimétrico MTT. O Extrato de S. palinacanthum mostrou-se mais ativo (IC50 = 175,9 µg.ml–1) de que o extrato de S. lycocarpum (IC50 = 194,7 µg.ml–1). A
solamargina apresentou forte atividade tripanocida (IC50 = 15,3 µg.ml–1), o que pode explicar a melhor atividade de ambos os extratos. O benzonidazol (IC50 = 9,0 µg.
ml–1) é a única droga utilizada para o tratamento da doença de Chagas. Estes resultados demonstram pela primeira vez que os extratos etanólicos obtidos a partir de
frutos de S. palinacanthum e S. lycocarpum, além da solamargina apresentam uma atividade tripanocida potencial.
Palavras-Chave: Solanum palinacanthum; Solanum lycocarpum; solamargina; epimastigota; atividade tripanocida; Trypanosoma cruzi
INTRODUCTION
Solanum palinacanthum Dunal. and Solanum lycocarpum St. Hill are members of the Solanaceae family, found in tropical and temperate zones, including the
Brazilian Cerrado. They have great importance for food and medicinal use (
Blankemeyer et al. 1998 Almeida et al. 2010
,
Balasubramanian et al. 2007
inflammatory and other properties of these plants have been intensively studied (
not been given for other healing properties.
). The antiviral, diuretic, anti-spasmodic, anti-
; as cited by
Soares-Mota et al. 2009
). However, attention has
Phytochemical studies revealed that polyphenols and glycoalcaloids, the most common elements obtained from some species of Solanum, which are secondary
synthesized metabolites, seek protection against animals and insects because of their toxicity (
Hall et al. 2006
). Glycoalkaloids solamargine and solasonine were
Hall et al. 2006 Sun et al. 2010
identified in various species of Solanaceae family, which have extensive anti-viral function (
,
). However, the trypanocidal effects of the
glycoalkaloids and plants extracts have never been reported before. The aim of the present investigation is to evaluate the potential trypanocidal activity of 95%
crude ethanol extracts of the fruits of S. palinacanthum, S. lycocarpum and solamargine isolated from S. palinacanthum, the major constituent of these plants.
Plant Material and Extraction
The study was performed with fruits from a Solanum palinacanthum Dunal. and Solanum lycocarpum St. Hill collected in Itápolis and Barretos, respectively, cities
within districts of São Paulo State, Brazil. The voucher specimen of S. palinacanthum Dunal. has been deposited at the Herbário do Instituto de Biociências of the
Universidade Estadual Paulista (UNESP), Rio Claro, São Paulo State, Brazil; S. lycocarpum St. Hill has been deposited at the Herbário do Departamento de
Biologia of the Faculdade de Filosofia Ciências e Letras de Ribeirão Preto of the Universidade de São Paulo (SPFR 11.308), Ribeirão Preto, São Paulo, Brazil. All
fruits were dried in hot air flow at 60 °C and then crushed in a blade grinder, to facilitate extraction of the chemical constituents.
Fruits powder (500 g) of S. palinacanthum were submitted to exhaustively extraction with 96% ethanol (EtOH) by four times at temperature (4 oC). The solvent
proportion corresponded to five times the plant material. Subsequently, exhaustive extraction with hot EtOH was done and evaporation of the solvent under
reduced pressure afforded a crude extract (100.2 g). The crude extract was redissolved in 5% aqueous acetic acid (v/v) and after 24 hours, a preliminary
purification involving defatting was done by successive benzene extraction. The final solution was changed to pH 10.0 with ammonium hydroxide. After 24 hours
at room temperature the precipitated was obtained, filtered and dried (24.2 g).
Fruits powder (35 g) of Solanum lycocarpum was submitted to exhaustively extraction with 250 mL of 96% ethanol by 4 hours at boiling temperature, followed by
filtration under reduced pressure. These processes were repeated with 200 mL of 96% ethanol. Were blended the extracts and concentrated under reduced pressure
until syrupy consistency and then dried to room temperature by air suction (8.12 g) (Almeida et al. 1995).
Isolation and Identification of Solamargine
The glycoalkaloid mixture of Solanum palinacanthum was subjected to chromatographic column (aluminum oxide, neutral) and after elution with 40% aqueous
ethanol (v/v) yielded solamargine (0.92 g) as showed in the Fig. 1. The separation was monitored by TLC in solvent system (n-butanol:acetic acid glacial:water
(6:3:1), nebulized and developed with 10% aqueous sulfuric acid (v/v) and then heated at 150 °C for 5 min. Solamargine was obtained and rechromatographed on
aluminum for purification and repeatedly recrystallised with methanol, and its identity ascertained by comparing physical and spectral data (mp, IV) (Almeida et
al. 1995).
Figure 1 Chemical structures of solamargine
Trypanocidal Activity-MTT Assay
An in vitro assay was performed measuring the viability of Trypanosoma cruzi (Y strain) epimastigote forms growing in axenic culture using MTT assay
Cotinguiba et al. 2009
(
). The parasites were grown axenically at 28 °C in liver-infusion Tryptose (LIT) medium supplemented with 10% fetal calf serum and harvested
during the exponential phase of growth (7 day-old culture forms). Extracts and compounds were dissolved in dimethylsulfoxide (DMSO) and further added to a 96well tissue culture plate (TPP) in different final concentrations. T. cruzi (1 x 107 parasites.mL–1) were added into each well and the same quantity of LIT medium
was added into the control wells. These plates were maintained at 28 °C for 72 h, 10 µL of a 2.5 mg.mL–1 MTT-PMS solution was added to each well and the
plates were incubated for 75 min in the dark at 28 °C. A solution of 10% (100 µL) of sodium dodecyl sulfate (SDS) was added to the anterior solution and
maintained at room temperature and in the dark at 30 min. The absorbance of the samples had read at 595 nm. The 50% inhibitory concentration (IC50) values
were determined by linear regression analysis after a 72 h incubation period. All the tests were done in triplicates and the IC50 values of samples and benznidazole
Muelas-Serrano et al. 2000
(positive control) were determined. For the statistical analysis, probit's method was employed (
).
Chagas' disease or American trypanosomiasis caused by the protozoan flagellate T. cruzi, is an important public health problem in Latin America, affecting
Sanchez-Burgos et al. 2003
millions of people annually (
). The most common treatment for this disease involves two drugs, benznidazole (A) and nifurtimox (B) (Fig.
2), which are active only during the acute and short-term chronic phases. Beznidazole (Rochagan®/Brazil and Radanil®/Argentina) is now the only drug still
available since the production of nifurtimox was discontinued. Besides developing severe side effects, narrow therapeutic windows, and variable drug
Coura and Castro 2002
susceptibilities among T. cruzi strains, result in low clinical efficacies for these 2-nitroimidazole (
).
Figure 2 Chemical structures of benznidazole (A) and nifurtimox (B)
In many Solanum species, the glycoalkaloids are presented as pairs of structurally related compounds which share a common aglycone with either solasodine and
differ only in their carbohydrate moiety (
Chataing et al. 1998
). The literature contains several reports on the biological activity of nitrogen-containing steroids of the
Chataing et al. 1998 Hall et al. 2006
species Solanum (
,
).
In this work, we presented the inhibitory activity the ethanol extracts from fruits of S. palinacanthum and S. lycocarpum, as well as the purified solamargine,
comparing with benznidazole (as positive control). The results are shown in Table I.
TABLE I In vitro trypanocidal activity of ethanolic extracts of S. palinacanthum and S. lycocarpum and solamargine.
Compounds
IC50 (µg.mL–1)
extract of S. palinacanthum
175.9
extract of S. lycocarpum
solamargine
benznidazole*
194.7
15.3
9.0
*
positive control.
The solamargine is a dominant steroidal glycoalkaloid extract from plants belonging to the genus Solanum and showed an IC50 very close to the benznidazole.
Chataing et al. 1998 Hall et al. 2006
Similar results were showed by other authors in the past (
,
, Valaderesa et al. 2009).
There was a light difference in the response between the two extracts: IC50 = 175.9 µg.ml–1 for extract of S. palinacanthum and IC50 = 194.7 µg.ml–1 for S.
lycocarpum.
Solamargine isolated from S. palinacanthum showed an IC50 = 15.3 µg.ml–1, which was very interesting by explaining the better activity of the both extracts.
Solamargine had also a pronounced effect on T. cruzi, closely similar to benznidazole (IC50 = 9.0 µg.ml–1). However, other methodologies should be used to
rightly understand the mechanism of action.
Our results were very similar to those described by
Hall et al. (2006)
, but using different approach. They have speculated that the positioning of the terminal sugars in
α-solamargine is sterically more favorable to bind with the parasites mucin-rich cell surface than those of α-solasonine (
Hall et al. 2006
concentrations, the alkaloid's lipid moiety would induce cytolysis through hydrophobic interactions with membrane sterols (
). Once bound in sufficient
Hall et al. 2006
).
Since the alkaloid used in this study was derived from ripe fruits of a Solanum species. There have been numerous hypotheses put forth to explain the allocation of
secondary metabolites in ripe fleshy fruits (
Hall et al. 2006 Li et al. 2007
,
), including the retention of high levels of glycoalkaloids in fruits of some Solanum species
Kuo et al. 2000 Hall et al. 2006
(
,
).
The 96% ethanolic extracts from the fruits of S. palinacanthum and S. lycocarpum exhibited the most potent in vitro trypanocidal activity. These species are an
interesting group for more studies and search of novel therapeutic plants against Chagas' disease (Almeida et al. 1995;
Soares-Mota et al. 2009
).
CONCLUSION
It can be concluded from this study that 96% ethanolic extracts from the fruits of S. palinacanthum and S. lycocarpum presented a potential anti-trypanosomal
activity based on toxic effect against epimastigote form of T. cruzi Y strain. The glycoalkaloid solamargine has been isolated from S. palinacanthum and
demonstrated to be active in the trypanocidal effect and would be a candidate to new therapeutic substance.
In view of these findings, further chemical and pharmacological investigations to identify others secondary metabolites and to evaluate the potential of these
Solanum species as antichagasic agents in vivo are recommended.
REFERENCES
Almeida AE, Cardoso CRP, Almeida DV, Moreira RRD, Silva M and Varanda EA. 2010. Mutagenic Activity of Glycoallkaloids from Solanum palinacanthum
Dunal (Solanaceae) found in the Brazilian cerrado. Lat Am J Pharm 29: 122-126. [ Links ]
Almeida AE and Rocca MA. 1995. Glicoalcalóides dos frutos de Solanum flaccidum Vell. Rev Cienc Farm 16: 111-118. [ Links ]
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shrimp. Aquaculture 23: 15-19. [ Links ]
Blankemeyer JT, McWillians ML, Rayburn JR, Weissenberg M and Friedman M. 1998. Developmental Toxicology of Solamargine and Solasonine
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Chataing B, Concepción JL, Lobatón R and Usubillaga A. 1998. Inhibition of Trypanosoma cruzi Growth in vitro by Solanum alkaloids: A comparison with
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anti-trypanosomal agents. Med Chem Res. Doi: 10.1007/s00044-008-9161-9. [ Links ]
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Hall CA, Hobby T and Cipollini M. 2006. Efficacy and mechanisms of α-solasonine and α-solamargine-induced cytolysis on two strains of Trypanosoma cruzi. J
Chemical Ecol 32: 2405-2416. [ Links ]
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solamargine: Triggering apoptosis in human hepatoma cells. Biochem Pharmacol 60: 1865-1873. [ Links ]
Li S, He DJ, Zang X, Ni WH, Zhou YF and Zhang LP. 2007. Modification of Sugar Chains in Glycoalkaloids and Variation of Anticancer Activity. Chem Res
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Sanchez-Burgos G, Mezquita-Veja RG, Escobedo- Ortegon J, Ramirez-Sierra MJ, Arjona-Torres A, Ouaissi A, Rodrigues MM and Dumonteil E. 2003.
Comparative evaluation of therapeutic DNA vaccines against Trypanosoma cruzi in mice. FEMS Immunol Med Microbiol 50: 333-341. [ Links ]
Soares-Mota MR, Schwars A, Bernardi MM, Maiorka PC and Spinosa HS. 2009. Toxicological evaluation of 10 % Solanum lycocarpum St. Hill fruit
consumption in the diet of growing rats: Hematologica, biochemical and histophatological effects. Exp Toxicol Pathol. Doi: 10.1016/j.etp.2009.07.006. [ Links ]
Sun F, Li S, He D, Cao G, Ni X, Tai G, Zhou Y and Wang D. 2010. Effects of glycoalkaloids from Solanum plants on cucumber root growth. Phytochemistry.
doi:10.1016/j.phytochem.2010.06.002. [ Links ]
Valadaresa YM, Brandão GC, Kroonb EG, Souza Filho JD, Oliveira AB and Braga FC. 2009. Antiviral Activity of Solanum paniculatum Extract and
Constituents. Z Naturforsch 64: 813-818. [ Links ]
Received: May28, , 2012; Accepted: September28, , 2012
Correspondence to: Raquel Regina Duarte Moreira E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
An. Acad. Bras. Ciênc. vol.85 no.3 Rio de Janeiro set. 2013 Epub 20-Ago-2013
http://dx.doi.org/10.1590/S0001-37652013005000048
Chemical Sciences
Active polyketides isolated from Penicillium herquei
ANDREY M.R. MARINHO1, PATRÍCIA S.B. MARINHO1, LOURIVALDO S. SANTOS1, EDSON RODRIGUES FILHO2, IZABEL C.P. FERREIRA3
1
Faculdade de Química, Universidade Federal do Pará, Rua Augusto Corrêa, 01, 66075-110 Belém, PA, Brasil
Departamento de Química, Universidade Federal de São Carlos, Rod. Washington Luiz Km 235, 13565-905 São Carlos, SP, Brasil
3 Departamento de Análises Clínicas, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR, Brasil
2
ABSTRACT
In this work we are reporting the isolation by classical methods of chromatography of six polyketides from Penicillium herquei. The compounds citreorosein ( 1) , emodin ( 2) , janthinone ( 3) , citrinin ( 4) , citrinin H1
( 5) and dicitrinol ( 6) were identified by spectral methods of 1D and 2D NMR and MS. Compounds 1, 2 and 3 were tested against promastigotes forms of Leishmania brasiliensis and 1 and 2 were also assayed against
Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis and showed good activity.
Keywords: polyketides; biological assay; P. herquei
RESUMO
Neste trabalho estamos relatando o isolamento por métodos clássicos de cromatografia de seis policetídeos de Penicillium herquei. Os compostos citreoseine ( 1) , emodina ( 2) , janthinona ( 3) , citrinina ( 4) , citrinina H1
( 5) e dicitrinol ( 6) foram identificados por métodos espectrais de RMN 1D e 2D e EM. Os compostos 1, 2 e 3 foram testados contra formas promastigotas de Leishmania brasiliensis e 1 e 2 também foram ensaiados
contra a Escherichia coli, Pseudomonas aeruginosa e Bacillus subtilis e mostraram boa atividade.
Palavras-Chave: policetídeos; ensaio biológico; P. herquei
INTRODUCTION
The search for substances with useful biological activities to man is one of the most studied fields in science as a whole. There is always the need to renew the arsenal of active compounds due to the parasites
acquire resistance to drugs already on the market, as well as the emergence of new diseases.
Fungi are good producers of secondary metabolites, many with useful biological activity (
Petrini et al. 1992 Jarvis and Miller 1996 Li et al. 1998 Stierle et al. 1995
,
the host plant to fight infestations by other fungi, bacteria and viruses, even assisting in its development (
great importance to study the biological activities of substances produced by endophytic fungi.
,
Shittu et al. 2009
,
) . Endophytic fungi often produce substances that can help
, Hamayun et al. 2009) . Since such substances can also be useful for people, it is therefore of
In this paper we report the isolation of six polyketides citreorosein ( 1) , emodin ( 2) , janthinone ( 3) , citrinin ( 4) , citrinin H1 ( 5) and dicitrinol ( 6) ( Figure 1) . Compounds 1, 2 and 3 were tested against
promastigote forms of Leishmania brasiliensis and 1 and 2 were also assayed for antimicrobial activities.
Figure 1 polyketides isolated from P. herquei.
General Procedures
The IR spectrum was measured in BOMEN MB-102 spectrophotometer in KBr pellet. APCIMS data were acquired in negative mode using a MICROMASS QUATTRO-LC instrument ( Waters, Manchester –
United Kingdom) equipped with an ESI/APCI ion source “Z-spray” type. 1H and 13C NMR experiments were obtained in a BRUKER DRX-400 spectrometer ( Bruker Daltonics, Germany) in CDCl3 with TMS as
internal standard.
Microorganism
P. herquei belong to the collection of the Laboratório de Bioquímica Micromolecular de Microorganismos, Departamento de Química - Universidade Federal de São Carlos and it is identified by the number
LaBioMi 019. This collection contains isolates from Melia azedarach (
Santos et al. 2003
).
Culture of P. herqueii in Rice and Polyketides Isolation
Forty-five Erlenmeyer flasks ( 500 mL) containing 90 g rice ( “Uncle's Been's®”) and 75 mL distilled water per flask were autoclaved for 45 min at 121°C. Small cubes of PDA medium containing mycelium of P.
herquei were added in 42 Erlenmeyer flasks under sterile condition. Three flasks were used as control. After 20 days of growth at 25°C the biomass obtained was macerated with dichloromethane, ethyl acetate and
methanol. The dichloromethane solution was evaporated under reduced pressure, producing a yellowish residue ( 24.2 g) . Part of this residue ( 10.0 g) was subjected to a low-pressure silica gel CC eluted with nhexane, ethyl acetate and methanol gradient. The medium polarity fractions eluted with ethyl acetate were repeatedly chromatographed on silica gel CC eluted with n-hexane, acetone and methanol gradient.
Finally, they provide the polyketides citreorosein ( 1) , emodin ( 2) , janthinone ( 3) , citrinin ( 4) , citrinin H1 ( 5) and dicitrinol ( 6) .
Leishmanicidal Assay
Promastigote forms of Leishmania viannia braziliensis MHOM/BR1987/M11272 were grown at 25°C in Schneider's Drosophila medium supplemented with 10% fetal calf serum ( FCS) . Cells were collected at
logarithmic phase, resuspended in fresh medium, counted in Neubauer chamber and the concentration adjusted to 4x106 cells/mL. The test was conducted in vitro. Substances were added at 320 µg/mL to 0.125 µg/
mL solubilized in DMSO and incubated at 25°C for 24 h. After this period, surviving parasites were counted in Neubauer chamber and compared with controls. Pentamidine isethionate ( Eurofarma®) was used as
positive control drug and DMSO as negative control. The LD50/24 was determined by linear regression analysis of the inhibition percentage with 10% statistical error.
Antibacterial Bioassay
Microorganisms' susceptibity to the polyketides test were determined by microbroth dilution assay as recommended by the Subcommittee on Antifungal Susceptibility Testing of the US National Committee for
Clinical Laboratory Standards ( NCCLS 1997) .
Polyketides Identification
Compounds 1, 2 and 3 were identified by 1D and 2D NMR and MS spectral analyses in comparison with the literature and showed total similarity to the polyketides citreorosein ( 1) , emodin ( 2) and janthinone
( 3) (
Fujimoto et al. 2004 Cohen and Towers 1995 Marinho et al. 2005
janthinellum (
,
,
Marinho and Rodrigues Filho 2011
) . Substances 4, 5 and 6 1D and 2D NMR and MS data are consistent with citrinin, citrinin H1 and dicitrinol, previously isolated by our team from P.
).
Leishmanicidal Test
The substance 2 inhibited 50% of parasites at 320 µg/mL showing a promising result. However, substances 1 and 3 inhibited about 20% and 22% of parasites at 320 µg/mL, respectively. It seems that the methyl
hydroxylation in the substance 1 reduced the activity in more than 50%. Results obtained in leishmanicidal tests with substances 1, 2 and 3 suggest that positions C-3 and C-6 must be reduced and oxidized,
respectively, for there being activity, since when both positions are oxidized or reduced there is significant decrease in the activity presented.
Antimicrobial Activity
The antibacterial activity of citreorosein ( 1) was examined in the presence of E. coli, P. aeruginosa and B. subtilis. Results were compared with those obtained for emodin ( 2) under the same conditions ( Table I) .
In general, citreorosein is less active than emodin, except against E. coli, which stops growing in a medium containing 31.25 µg/mL of 1.
TABLE I Bacteria growth behavior in the presence of compounds 1 and 2 at different concentrations.
Concentration c [µg/mL] of test compounds
500
250
125
62.50
31.25
15.63
7.81
= bactericidal effect, - bacteriostatic effect, + no active.
E. coli
1
=
=
=
=
=
+
+
P. aeruginosa
2
=
+
+
+
+
+
+
1
=
=
=
=
+
+
+
B. subtilis
2
=
=
=
=
-
1
=
+
+
+
+
+
2
=
=
+
+
Acknowledgements
The authors thank the Fundação Amazônica Paraense de Amparo à Pesquisa ( FAPESPA) , Fundação de Amparo à Pesquisa do Estado de São Paulo ( FAPESP) , Conselho Nacional de Desenvolvimento Científico
e Tecnológico ( CNPq) , Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior ( CAPES) and Ministério da Saúde ( MS) for financial support.
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Received: September15, , 2011; Accepted: January10, , 2013
Correspondence to: Andrey M.R. Marinho E-mail: [email protected]
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any
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Tel: +55 21 3907-8100
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http://dx.doi.org/10.1590/S0001-37652013000300007
Earth Sciences
A study of structural lineaments in Pantanal (Brazil) using remote sensing data
ANTONIO C. PARANHOS FILHO12*, ALEXIS R. NUMMER23**, EDILCE A. ALBREZ1, ALISSON A. RIBEIRO1, RÔMULO MACHADO2
1
2
3
Centro de Ciências Exatas e Tecnologia, Universidade Federal de Mato Grosso do Sul, Campus Universitário, s/nº, Caixa Postal 549, 79070-900 Campo Grande, MS, Brasil
Instituto de Geociências, Universidade de São Paulo, Rua do Lago, 562, 05508-080 São Paulo, SP, Brasil
Departamento de Geociências, Instituto de Agronomia, Universidade Federal Rural do Rio de Janeiro, Rod. BR 465, Km 7, 23890-000 Seropédica, RJ, Brasil
ABSTRACT
This paper presents a study of the structural lineaments of the Pantanal extracted visually from satellite images (CBERS-2B satellite, Wide Field Imager sensor, a free image available in INTERNET) and a comparison with the structural lineaments of Precambrian and Paleozoic rocks surrounding the Cenozoic Pantanal Basin. Using a free software for
satellite image analysis, the photointerpretation showed that the NS, NE and NW directions observed on the Pantanal satellite images are the same recorded in the older rocks surrounding the basin, suggesting reactivation of these basement structural directions during the Quaternary. So the Pantanal Basin has an active tectonics and its evolution seems to
be linked to changes that occurred during the Andean subduction.
Keywords: free remote sensing software; free satellite images; structural geology; lineaments; photointerpretation; Pantanal
RESUMO
Este trabalho apresenta um estudo dos lineamentos estruturais extraídos manualmente a partir de imagens de satélite (CBERS-2B, sensor WFI, uma imagem gratuita disponível na internet) da região do Pantanal e faz uma comparação com os lineamentos estruturais de rochas Pré-Cambrianas e Paleozóicas que circundam a bacia Cenozóica do Pantanal.
Usando software livre para análise de imagens de satélite, a fotointerpretação mostrou que as direções NS, NE e NW observadas no Pantanal são as mesmas registradas nas rochas mais antigas que circundam a bacia, sugerindo reativação destas mesmas direções estruturais durante o Quaternário. Logo, a Bacia do Pantanal possui uma tectônica ativa e sua
evolução parece estar ligada às mudanças relacionadas com a subducção Andina.
Palavras-Chave: programa livre para sensoriamento remoto; imagem de satélite gratuita; geologia estrutural; lineamentos; fotointerpretação; Pantanal
INTRODUCTION
The main purpose of this paper is to study the structural lineaments of the Pantanal extracted from CBERS-2B satellite images (Wide Field Imager sensor – WFI) combined with field information, and to investigate possible relationships between the structural directions thus obtained and the reactivation of the basement structures.
Besides the identification of geological structures, another purpose of this study is the utilization of free satellite images and a free software in order to evaluate their potential as structural analysis tools.
In the present work we identified three main lineament directions: NE, around N-S, and E-W. The first two structures are older and related to reactivations of basement structures. The youngest E-W developed from the end of the Cenozoic to the Quaternary.
In this paper the term lineament is used according to the definition of
O'Leary et al. (1976)
, “mappable linear surface features which differ distinctly from the patterns of adjacent features and presumably reflect subsurface phenomena”.
The study area encompasses not only the Pantanal but also the surrounding Precambrian rocks and the northwestern margin of the Phanerozoic Paraná Basin.
Pantanal is a plain humid area situated in the middle of the South America Plate. Due to its biodiversity, which is one of the largest in the world, it is considered part of the human patrimony. The origin of the Pantanal Basin is still not well understood and if on one hand it is possible to attest a tectonic origin for the Pantanal basin, on the other
hand, the main structural systems have not been made available in the scientific literature. Thus, the aim of this work is to present the main Pantanal structural systems compiled from photointerpretation of remote sensing images checked against field data.
Geological Setting
In his pioneer work on the Brazilian and South American tectonics,
Almeida (1967)
describes two regions that were the sites of sedimentation on the South American Platform during the Quaternary: the Amazon and the Pantanal basins.
The Pantanal is nowadays a region of intense sedimentation, even in human terms (
Ab'Saber 1988 2006
,
). Despite the alleged stability during the Cenozoic, the Pantanal Basin shows several evidences of recent and current tectonic activities (
Assine 2010 Assine and Soares 2004 Hasui 1990 2010 Assumpção and Suárez 1988 Assumpção et al. 2009a b Facincani et al.
,
,
,
,
,
, ,
2011 Zani and Assine 2011
,
).
Riccomini and Assumpção (1999)
explain that the concept of tectonic stability has long prevailed for most of the South American Platform. Therefore, the investigation on the Quaternary tectonics in Brazil has begun only in the 1990's.
Assine (2010)
considers the Pantanal as a tectonically active sedimentary basin, with many of its morphological features resultant from a paleogeographic evolution conditioned by climatic and tectonic changes that have occurred since the late Pleistocene to the present day. The author points out to the presence of active faults within the Pantanal
Basin and considers that many of them are related to the Transbrasiliano lineament.
Pantanal has been considered a seismic region at least since the beginning of the twentieth century (
Zani and Assine (2011)
Branner 1912
). Even being inside the South American plate neotectonic activities have been documented, even including several earthquakes records (
Assumpção et al. 2009a Facincani et al. 2011
,
).
present the results of the systematic mapping of the Taquari River megafan paleochannels using remote sensing images and conclude that there are areas under the influence of recent tectonic movements.
Ab'Saber 1988
Ruellan (1952, apud
) is the first to identify the physiographic feature containing the Pantanal depression and called it an inlier: a geomorphological term used to describe an inversion of the relief. It is represented by a wide arcuate shield (called “aboboda”) formed by Precambrian terrains excavated by erosional processes, which in
this case ended up forming the depression occupied by Pantanal Basin.
The relief of this region originated in the late Cretaceous, when there was no Paraguay depression. Instead, there was an elevated region between the Andean region and the northwestern portion of the Paraná sedimentary basin (
place, affecting the Precambrian rocks. The Upper Paraguay hydrographic basin contains the Pantanal Plain and its former rivers.
Almeida (1965)
Almeida 1965
). In the region that nowadays corresponds to the Upper Paraguay hydrographic basin major faulting took
identifies a set of NNE-SSW structures affecting the Gran Chaco in Bolivia and Paraguay and the main core of the Brazilian Upper Paraguay river basin, representing two separated tectonic compartments, limited by the hills along the border between Brazil and Bolivia. So the Post-Cretaceous and Pre-Pliocene tectonics may have
been wider and more complex than the residual tectonics that has been responsible for the generation of the Pleistocenic Pantanal Basin.
According to
Ab'Saber (1988 2006)
,
, the Pantanal formed in the Pleistocene was the result of a regional system of faults forming a graben system inside the boutinière.
The regional context is represented in Figure 1 (Companhia de Pesquisa de Recursos Minerais (CPRM) – Schobbenhausa and Bellizzia 2001). It is possible to observe the main structures around the Pantanal Basin, as follows:
●
●
●
●
- the Paraguay belt to the north, with Precambrian metamorphic structures following a NNE/SSW trend;
- the EW/EEW-trending Proterozoic rocks of the Tucavaca belt to the west, surrounded by Quaternary/Cenozoic sediments from Bolivia and several hills aligned NNE/SSW on the Brazil - Bolivia border;
- the NS-trending Bodoquena Hills to the south constituted by Precambrian carbonates and high-grade metamorphic rocks;
- the Pantanal eastern border with the Paraná Sedimentary Basin, constituted by an almost NS-trending tectonic structure.
Figure 1 The geological context of Pantanal in relation to regional tectonic units (simplified from Schobbenhaus and Bellizzia 2001): (1) Precambrian metamorphic rocks; (2) Gondwana sequence with Paleozoic sedimentary rocks; (3) Tertiary sedimentary rocks, and (4) Cenozoic sequences including Pantanal.
It is important to observe the structural context with the metamorphic rocks of Paraguay Belt to the north; Bodoquena Hills to the south, composed by carbonates and other metamorphites; the Paraná Basin to the east, and Precambrian rocks of the Tucavaca Belt and Bolivian Cenozoic sediments to the west.
Over a false color composition of a CBERS-2B Satellite image, WFI sensor (Instituto Nacional de Pesquisas Espaciais (INPE), 2008a, b), the main lineaments were identified and vectorized on screen by means of the Quantum GIS Software (OSGeo 2011).
The Quantum GIS Project, supported by the Open Source Geospatial Foundation (OSGeo), is currently developing a user friendly Open Source Geographic Information System (GIS) licensed under the GNU General Public License. It is a free software and can be customized freely. It runs on several operating systems and supports numerous
vector, raster, and database formats and functionalities.
The WFI (wide field imager) images, available for free download from the INPE website, have 260m spatial resolution and two channels in the red and near infrared spectral regions. The third band used to create the false color composition is the NDVI (Normalized Difference Vegetation Index), derived from the red and infrared channels.
One interesting characteristic is that WFI sensor with its 890 km wide scanning range allows to view large portions of viewed land cover at once, allowing large portions of land cover to be viewed so that the whole Pantanal is sampled in only two scenes of the same date. Other sensors, like Landsat TM or ETM+, for example, would capture
the same land area in at least 48 days. The resulting mosaic would show differences in the vegetation cover due the humidity and phenology, whereas this bias is negligible in the WFI images obtained on the same day.
The dry season, due to hydric stress, causes a decrease of chlorophyll in vegetation and consequent changes in the spectral response of the land cover in the satellite images. The rainy station also causes variations in the land cover, mainly through flood pulses. Images from rainy and dry seasons were compared in order to depict possible
differences due to seasonality and define the correct location of the photointerpreted lineaments.
The images were used in compliance with PROBIO (official limits established by the Brazilian Ministry of Environment for the Pantanal, published in
Silva and Abdon 1998
) and also the official limits of the Upper Paraguay river hydrographic basin.
The Brazilian Geological Survey (CPRM – Schobbenhaus and Bellizzia 2001) has also geological maps for the Pantanal surrounding region focusing on prospecting, ruling out the Pantanal Basin, which is of Cenozoic age. However, the map of lineaments generated by CPRM for the surrounding area was used in the analysis of the
photointerpreted structures. There are about a dozen CPRM maps of that cover the Cenozoic Plain of Pantanal (Figure 2).
Figure 2 Map of regional lineaments surrounding the Pantanal (structural data from CPRM – Schobbenhaus and Bellizzia 2001). The Upper Paraguay basin and Pantanal limits are from PROBIO (
Silva and Abdon 1998
).
Field control was carried out in two field trips, the first in July, 2011 and the second in August, 2011, in order to check the structural data compiled from the images. The southern and eastern borders of Pantanal were covered during these trips, totaling more than 300 ground control points.
During the field works the physiography of the terrain (breaklines) was analyzed and the directions of the rock bedding were obtained by means of the geological compass. The vectors of the structural lineaments were checked against a road map to avoid misinterpretations.
The structures compiled by means of photointerpretation of the CBERS-2B satellite image are presented in Figure 3. Three main systems are distinguished:
Figure 3 The lineaments obtained through photointerpretation of satellite images. The main systems are NNE/SSW, EW, NE/SW and NW/SE (the last two being associated sub-systems).
1. - An almost NS, NNE/SSW-trending system that encompasses the eastern edge of the Pantanal Basin, coinciding with the western boundary of the Paraná Sedimentary Basin. This direction also controls the Paraguay River on the western borderline of the Pantanal Basin.
2. - Two lineament systems, a main NE-SW and a secondary NW-SE compose the second system.
3. - The third system trending EW is situated on the southern edge of the Pantanal Basin, being well marked between the alignments of the Negro River and the Tucavaca lineament. It is also visible on the middle of Pantanal.
NE and NW lineaments are long (over 50 km), continuous, and extremely straight, penetrating the Pantanal region and the adjacent regional tectonic units. Lineaments with these same directions, but with smaller length, occur on the eastern edge of the Pantanal Basin.
The lineaments following NS structures are also persistently continuous in the images. Nonetheless, they are more targeted and their trace is restricted to “corridors”, as the eastern edge of the Pantanal Basin, where such structures are coincident with the edge of the Paraná Basin and with the outcrops of Precambrian rocks visited during the
fieldtrips. The Paraguay River flows in a corridor with this orientation on the west border of Pantanal.
The largest EW lineaments, with length exceeding 50 km, generally occur in the central part of the Pantanal.
The comparison between the data for the Pantanal Basin and the surrounding area, taking into account the correspondence with the direction and length of the lineaments, is shown in Figure 4. There is a good correlation between these diagrams for each area. There is a meaningful difference between the two areas. The directions in the area of
the Pantanal Basin are concentrated around ENE and WNW, whereas there is a great dispersion of surrounding data in the rose diagrams, which show a main concentration around NE and another around NS.
Figure 4 Rose diagrams with the lineaments from CPRM (A) and from the photointerpretation of the satellite image (B). No stronger trend is depicted in “A”, except for a suggestion of NE and NS structures. The rose diagrams with length-proportional frequency show the importance of the NE direction, which is
associated with the Transbrasiliano Lineament. There are two important systems marked on B: one trending ENE and the other trending almost EW.
The NNE/SSW lineaments found in this work could be associated with the initial phases of the opening of the Pantanal Basin before the Pleistocenic deposition, according to the model proposed by
In the central region of Brazil the directions associated with the N45E directions according to
(1998) Assine and Soares (2004)
,
and
Hasui (1990)
Almeida (1965)
for the Pantanal Basin, corroborated by
Ab'Saber (1988 2006)
,
may be associated with neotectonic activities and to the Transbrasiliano Lineament (Schobbenhaus et al. 1975), a Neoproterozoic brittle structure with reactivation phases in the Paleozoic, Mesozoic and Cenozoic. According to
Facincani et al. (2011)
, these structures played a significant rule on the evolution of the Pantanal Basin.
These structures are visible not only in the Pantanal Basin, but also crossing its limits towards the Precambrian, Paleozoic and Mesozoic terrains arround the basin. This means that there has been reactivation of these structures in different geological periods.
Riccomini (1989 1995a b)
,
,
.
proposed a stress field with σ1 oriented to EW during the Cenozoic. For some authors this same stress field persisted during the Quaternary and continued until the present time (
Assumpção 1992 Lima and Nascimento 1994 Riccomini 1995a
,
,
).
Soares et al.
It is important to observe that the field control showed that the structures observed in the images match with positive and negative physiographic breaklines on the terrain (Figure 5)
Figure 5 A series of hills aligned according to the NS direction. The picture was taken from the Bodoquena Belt looking northwards.
The presence of structures with the magnitude of those found in the Pantanal Basin is associated with active tectonics or reactivation as this basin is filled with recent and immature sediments that can be easily remobilized by the basin fluvial systems, thus masking the structural system. Some sort of structural control must exist to keep these
lineaments visible and controlling the drainage under an intense depositional regime.
CONCLUSIONS
The results of this study are similar to the data obtained by the Geological Survey of Brazil in the area surrounding the Pantanal Basin. They only differ in the smaller dispersion of the data and the concentration of the main lineament directions. In the area of the Pantanal, directions are concentrated around ENE and WNW, whereas in its
surroundings they are concentrated around NE and NS.
The NE, NW and NS tectonic directions characterized in the Pantanal region have been reactivated since the late Pleistocene to the present day and they correspond to the general structural pattern present in basement rocks. Special exception for EW structures, which are newer than the others, as well as the sedimentary sequence of the
Pantanal Basin. Although many authors consider the NE structural directions of the Pantanal Basin to be related to the reactivation of the Transbrasiliano Lineament, the EW stress field produced in the South American Plate during the late Pleistocene to Quaternary may also be responsible for such reactivation.
The Pantanal corresponds to a tectonic basin elongated in the NS direction, whose evolution and tectono-sedimentary deformation are probably related to the Andean convergence, including the EW structures, younger than the stratigraphic succession of the Pantanal Basin. The structural directions NE, NW and EW are responsible for
controlling the main rivers that currently drain the Pantanal plain.
Concerning the methodological aspects of this work, the application of a free software and free images is recommended for further environmental studies.
Acknowledgements
To Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (process number 151762/2010-4) for ACPF Post-Doctor Senior scholarship and to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (process number 2010/52614-4) for supporting part of the field work.
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Assumpção M and Suárez G. 1988. Source mechanisms of moderate size earthquakes and stress orientation in mid-plate South America. Geophys J 92: 253-267. [ Links ]
Branner JC. 1912. Earthquakes in Brazil. Bull Seismic Soc Am 2(2): 105-117. [ Links ]
Facincani EM, Assumpção MS, Assine ML and França GLSA. 2011. Sismicidade da Bacia do Pantanal Mato-Grossense. In: Anais do XIII Simpósio Nacional de Estudos Tectônicos (XIII SNET). Campinas, SP 1: 314-317. [ Links ]
Hasui Y. 1990. Neotectônica aspectos fundamentais da tectônica ressurgente no Brasil. In: Worshop sobre a tectônica e sedimentação Cenozóica continental no sudeste brasileiro, 1. 1990, Belo Horizonte. Anais... Belo Horizonte: SBG-MG, p. 1-31. (Boletim 11). [ Links ]
Hasui Y. 2010. A grande colisão pré-cambriana do sudeste brasileiro e a estruturação regional. Universidade Estadual Paulista “Julio de Mesquita Filho”. Geociências 29(2): 141-169. [ Links ]
INPE – Instituto Nacional de Pesquisas Espaciais. Imagem de Satélite. 2008a. CBERS 2B – Sensor WFI. Canais 1 e 2. São José dos Campos. Órbita 165 ponto 116. De 24 de Agosto de 2008 (a). Disponível em: http://www.dgi.inpe.br/CDSR/. Last access on: feb, 8/2011. [ Links ]
INPE – Instituto Nacional de Pesquisas Espaciais. Imagem de Satélite. 2008b. CBERS 2B – Sensor WFI. Canais 1 e 2. São José dos Campos. Órbita 165 ponto 124. De 24 de Agosto de 2008 (b). Disponível em: http://www.dgi.inpe.br/CDSR/. Last access on: feb, 8/2011. [ Links ]
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Riccomini C. 1995a. Tectonismo Gerador e Deformador dos Depósitos Sedimentares Pós-Gondwânicos da Porção Centro-Oriental do Estado de São Paulo e Áreas Vizinhas. Tese de Livre-Docência, Universidade de São Paulo, Brasil, 100 p. (restricted circulation). [ Links ]
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Soares PC, Assine ML and Rabelo L. 1998. The Pantanal Basin: Recent Tectonics, Relationships to the Transbrasiliano Lineament In: SIMPÓSIO BRASILEIRO DE SENSORIAMENTO REMOTO, 9. (SBSR), 1998, Santos. Anais... São José dos Campos: INPE, p. 459-469. CD-ROM. ISBN 85-17-00015-3. (INPE-6909-PRE/2875).
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Received: April24, , 2012; Accepted: February27, , 2013
Correspondence to: Alisson André Ribeiro E-mail: [email protected]
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http://dx.doi.org/10.1590/S0001-37652013000300008
Biological Sciences
The Brazilian research contribution to knowledge of the plant communities from Antarctic ice free areas
ANTONIO B. PEREIRA1, JAIR PUTZKE2
1
2
Instituto Nacional de Ciência e Tecnologia Antártico de Pesquisas Ambientais/INCT-APA, Universidade Federal do Pampa/UNIPAMPA , Campus São Gabriel, Av. Antonio Trilha, 1847, 93000-000 São Gabriel, RS, Brasil
Instituto Nacional de Ciência e Tecnologia Antártico de Pesquisas Ambientais/INCT-APA, Universidade de Santa Cruz do Sul/UNISC, Av. Independência, 2293, Bairro Marechal Rondon, 96815-900 Santa Cruz do Sul, RS, Brasil
ABSTRACT
This work aims to summarize the results of research carried out by Brazilian researchers on the plant communities of Antarctic ice free areas during the last twenty five years. Since 1988 field work has been carried out in Elephant Island, King George Island, Nelson Island and Deception Island. During this period six papers were published on the
chemistry of lichens, seven papers on plant taxonomy, five papers on plant biology, two studies on UVB photoprotection, three studies about the relationships between plant communities and bird colonies and eleven papers on plant communities from ice free areas. At the present, Brazilian botanists are researching the plant communities of
Antarctic ice free areas in order to understand their relationships to soil microbial communities, the biodiversity, the distribution of the plants populations and their relationship with birds colonies. In addition to these activities, a group of Brazilian researchers are undertaking studies related to Antarctic plant genetic diversity, plant chemistry and
their biotechnological applications.
Keywords: Scientific publication; Botany; South Shetlands Island; Antarctica
RESUMO
Este trabalho tem como objetivo resumir os resultados das pesquisas realizadas por brasileiros nas comunidades vegetais de áreas livres de gelo da Antártida, durante os últimos vinte e cinco anos. Tais investigações iniciaram em 1988, sendo realizados nas ilhas Elefante, Rei George, Nelson de Deception. Durante este período foram publicados seis
trabalhos sobre a química de líquens, sete sobre taxonomia vegetal, cinco sobre a biologia de plantas, dois sobre fotoproteção UVB, três sobre as relações entre as comunidades vegetais e colônias de aves e onze sobre as comunidades de plantas de áreas de degelo, abordando biogeografia e fitossociologia. Atualmente, os botânicos brasileiros estão
pesquisando as comunidades vegetais de áreas de degelo da Antártica, buscando entender suas relações com as comunidades microbianas do solo, a biodiversidade, a distribuição das populações de plantas e sua relação com as colónias de aves. Além dessas atividades, um grupo de pesquisadores está investigando a diversidade genética vegetal
através de estudos de genética de populações e a bioquímica das plantas.
Palavras-Chave: Produção científica; Botânica; Shetlands do Sul; Antarctica
INTRODUCTION
The importance of the study of plant species found in the ice-free areas of Antarctic were related with the environmental monitoring, as a source for to evaluate the global changes. Climate changes is expected to have a major impact on the terrestrial biota of the Antarctic. Studies suggested that increasing temperatures and greater water
Turner and Marshall 2011
availability could extend the active season, increase development rates and reduce the life cycle leading to altered the species distribution (
).
One of the first expeditions to the South Pole was carried out by the French explorer Lozier Bouvet in 1739. His discovery was named as Bouvet Island, but he had no scientific interest on the region. The first systematic botanic studies were carried out by J. Torrey in 1823, when he describes the Usnea fasciata. J. Eights was the first
Putzke and Pereira 2001
scientist to collect lichens, mosses, sea algae and grasses, between 1829-1830 (
).
The Brazilian introduction in the Antarctica began in 1882, when the astronomer Luiz Antonio Cruls, on request of the French Academy of Science, held several observation about the Venus passage across the sun. Hundred years after, in 1982, starts the effective Brazilian participation through the first Brazilian Antarctic expedition on
Souza 2008
board of the oceanographic support ship Barão de Teffé (
).
Olech 1996
The plant communities in the Antarctic ice free areas are comprised of populations of three flowering plants (of which two are natives): Deschampsia antarctica Desv. (Poaceae) and Colobanthus quitensis (Kunth) Bartl (Caryophyllaceae) and Poa annua L. (Poaceae), accidently introduced in the 1980s (
). The latter is native to
Europe and was found growing in the vicinity of the Polish Arctowski Station, Admiralty Bay, King George Island, South Shetlands. For bryophytes 110 mosses species and 22 liverworts species are cited. More than 360 species of lichens are recognized (Øvstedal and Lewis Smith 2004). There are two species of macroscopic algae
growing in and at the surroundings of bird colonies (
Pereira 2004
).
The Antarctic flora has been studied continuously since its discovery, but only a few efforts have been made to really explore the species richness and relationship in different ice-free areas. The South Shetland Archipelago has 540 km of islands distributed over a SW to NE line, located in the northern part of the Antarctic Peninsula (ca.
160 km at north). King George Island is the largest one and the main area used for research bases in Antarctica. Argentina, Brazil, Chile, China, Poland, Russia, South Korea and Uruguay have permanently keep open bases on this island. Other countries have seasonal summer stations on different parts of this island, which demonstrates
that human impact are strong on King George Island relative to other areas in the Maritime and Continental Antarctic.
This work aims to present the results of research carried out by Brazilian researchers on the plant communities of Antarctic ice free areas at the last twenty years in the South Shetland islands.
Since 1988 field work has been carried out in different areas of the South Shetland Islands: Elephant Island (Stinker Point), King George Island (Admiralty Bay, Fildes Peninsula, Turret Point and Lions Rump), Nelson Island (Rip Point), Deception Island (Figures 1A-B, Figure 2). Phytosociological approaches were used to study the
plant succession cover in the four islands.
Figure 1 Maps of Islands visited by the group: Elephant island (upper part), King George and Nelson Islands (middle) and Deception Island (down). 1 – Penguin Island. 2 – 11 King George Island: 2- Turret Point; 3- Hennequin point; 4-Ullman Point and Stenhouse Bluff; 5- Keller Peninsula; 6- Region from
Thomas Point to Telefone Point; 7- Dufaiel Island; 8- Potter Peninsula; 9- Barton Peninsula; 10- Ardley Island; 11- Fildes Peninsula; 12 – Nelson Island (Rip Point).
Figure 2 Deception Island map, where all ice free areas were visited.
Species identification were carried out with help of specialized literature, such Øvstedal and Lewis Smith (2001, 2004, 2009),
Putzke and Pereira (2001)
and
Ochyra et al. (2008)
. The mosses names was up to date using the plant names database available in W3Tropicos web server (www.tropicos.org). For Bryaceae species was use the recent
Spence 2005 2007
nomenclature following (
,
). The lichen names was revised using the index fungorum database (www.indexfungorum.org) and most recent literature for antarctic lichens (Øvstedal and Lewis Smith 2001, 2004, 2009). The exsiccates of the specimens collected in Antarctica were deposited principally in the HCB Herbarium,
ULBRA Herbarium, Botanical Garden of Rio de Janeiro Herbarium (RB) and HBEI (UNIPAMPA).
The Brazilian contribution to the study of plant communities in Antarctic ice free areas, began as the foundation of the Brazilian Antarctic Program that performed the first Scientific Antarctic Expedition in Austral-Summer 1982/1983. In the first and second expeditions only oceanology studies were carried out, including cartographic
and marine biology survey. Research on the land ecosystems was made possible after the inauguration of the Brazilian Antarctic Station in 1984, named Comandante Ferraz. Research on terrestrial plants began in the Austral Summer 1985 and 1986, with the study of lichens chemistry published by
Czeczuga and Xavier-Filho (1987 1988)
,
Xavier-Filho (1986 1988 1989 1990)
,
Since 1988 the Brazilian Antarctic Program botanists initiated the study of plant communities in melting areas. Early works are published on the plant taxonomy, including first moss species list for King George Island (
taxonomical studies (
Putzke and Pereira 1996 1998a
,
out studies on plant biology as
by
Pereira et al. (1990a b)
,
and
,
,
and
, describing beta-carotenoid, natural products and the antimicrobial activity of several lichen species.
) (see plant checklist in the present paper). More recently the first book on Antarctic Mosses (
Elster et al. 2008 Gonçalves et al. 2008 Kovácik et al. 2004 Martins et al. 2004
,
Sander et al. (2004)
,
,
and
Kovácik and Pereira 2001
Pereira 1990
), as summarize in Table I, as well as the new records for Usnea trachycarpa (
Sramkova et al. 2007
), was published a contribution on diatoms of Deception Island (
Putzke et al. 2004
) and the first record of a myxomycete species in Antarctica (
), and other
). Were also carried
detail information on the Mastodia tesselata (Hook. f. & Harv.) Hook. f. & Harv. lichenization processes. In addition, studies regarding the relationships between plant communities and bird colonies were published
. The photoprotective effect of extracts of Deschampsia. antarctica, Colobanthus quitensis, and Polytrichum juniperinum Hedw., against UVB. They attributed their photoprotective properties to several molecules, such as flavonoids and carotenoids, that absorb UV, act as antioxidants, and
Kappel Pereira et al. 2004 2009
stimulate DNA-repair processes (
,
). Several plant community studies were carried out, such as
useful for environmental impact evaluations in the ice-free areas by the Brazilian Antarctic Program in the future.
Pereira (2004)
(the first phytosociological survey for Antarctica),
Pereira and Putzke (1994) Pereira et al. (2007 2008 2009) Putzke and Pereira (1998b) Victoria et al. (2004 2006 2009a b)
,
,
,
,
,
,
,
,
and
Victoria and Pereira (2007)
. These studies will be
TABLE I Moss species collected in the four South Shetland Islands by Brazilian botanist.
MOSSES
Elephant
King George
Nelson
Deception
AMBLYSTEGIACEAE
Cratoneuropsis relaxa ssp. minor (Hook. f. & Wilson) Ochyra [=Orthotheciella varia (Hedw.) Ochyra]
Sanionia uncinata (Hedw.) Loeske
Sanionia georgicouncinata (Müll Hal.) Ochyra & Hedenas
Warnstorfia fontinaliopsis (Müll. Hal.) Ochyra
Warnstorfia sarmentosa (Wahlenb.) Hedenas
ANDREAEACEAE
Andreaea depressinervis Cardot
Andreaea gainii Cardot
Andreaea regularis Müll. Hal.
BARTRAMIACEAE
Bartramia patens Brid.
Conostomum magellanicum Sull.
Philonotis polymorpha (Müll. Hal.) Kindb. (=Bartramia polymorpha Müll. Hal.)
BRACHYTHECIACEAE
Brachythecium austrosalebrosum (Müll. Hal.) Paris
Brachythecium austroglareosum (Müll. Hal.) Paris
Brachythecium subpilosum (Hook. f. & Wilson) A. Jaeger
BRACHYTHECIACEAE
Sciuro-hypnum glaciale (Schimp.) Ignatov & Huttunen (=Brachythecium glaciale Schimp)
BRYACEAE
Bryum archangelicum Bruch & Schimp (=Bryum amblyodon Müll. Hal.)
Bryum argenteum Hedw.
Gemmabryum dichotomum (Hedw.) J. R. Spence & H. P. Ramsay (=Bryum dichotomum Hedw.)
Bryum orbiculatifolium Cardot & Broth.
Bryum pallescens Schleich. ex Schwaegr.
Ptychostomum pseudotriquetrum (Hedw.) J. R. Spence & H. P. Ramsay [=Bryum pseudotriquetrum (Hedw.) P. Gaertn., B. Mey. & Scherb.]
Pohlia cruda (Hedw.) Lindb.
Pohlia drummondii (Müll. Hal.) A. L. Andrews
Pohlia nutans (Hedw.) Lindb.
Pohlia wahlenbergii (F. Weber & D. Mohr) A.L. Andrews
DICRANACEAE
Dicranella campylophylla (Taylor) A. Jaeger [=Anisothecium cardotii (R. Br. bis.) Ochyra]
Chorisodontium aciphyllum (Hook. f. & Wills.) Broth.
Kiaeria pumila (Mitt.) Ochyra
DITRICHACEAE
Ceratodon purpureus (Hedw.) Brid.
Distichium capillaceum (Hedw.) Bruch & Schimp
Ditrichum hyalinum (Mitt.) Kuntze
Ditrichum hyalinocuspidatum Cardot (=Ditrichum lewis-smithii Ochyra)
ENCALYPTACEAE
Encalypta rhaptocarpa Schwaegr.
GRIMMIACEAE
Orthogrimmia sessitana (De Not.) Ochyra & Zarnowiec
Bucklandiella sudetica (Funck) Bednarek-Ochyra & Ochyra
Schistidium amblyophyllum (Müll. Hal.) Ochyra & Hertel
Schistidium antarctici (Cardot) L. I. Savicz & Smirnova
Schistidium cupulare (Müll. Hal.) Ochyra
Schistidium falcatum (Hook. f. & Wils.) B. Bremer
Schistidium halinae Ochyra
Schistidium andinum (Mitt.) Herzog [=Schistidium occultum (Müll. Hal.) Ochyra & Matteri]
Schistidium rivulare (Brid.) Pobp.
Schistidium steerei Ochyra
Schistidium urnulaceum (Müll. Hal.) B. G. Bell.
HYPNACEAE
Hypnum revolutum (Mitt.) Lindb.
Platydictya jungermannioides (Brid.) H. A.Crum
MEESIACEAE
Meesia uliginosa Hedw.
ORTHOTRICHACEAE
Muelleriella crassifolia (Hook. f. & Wils.) Dusén
POLYTRICHACEAE
Notoligotrichum trichodon (Hook. & Wilson) G. L. Sm.
Polytrichastrum alpinum (Hedw.) G. L. Smith
Polytrichum strictum Menzies ex Brid.
Polytrichum juniperinum Hedw.
Polytrichum piliferum Hedw.
POTTIACEAE
Didymodon brachyphyllus (Sull.) R. H. Zander
Hennediella antarctica (Angström) Ochyra & Matteri
Hennediella heimii (Hedw.) R. H. Zander.
Stegonia latifolia (Schwaegr.) Vent ex Broth.
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Syntrichia filaris (Müll. Hal.) R. H. Zander
Syntrichia magellanica (Mont.) R.H. Zander [=Syntrichia princeps (De Not.) Mitt.]
Syntrichia saxicola (Cardot) R. H. Zander
SELIGERACEAE
Hymenoloma crispulum (Hedw.) Ochyra
Hymenoloma grimmiaceum (Müll. Hal.) Ochyra
Hymenoloma antarcticum (Müll. Hal.) Ochyra
Holodontium strictum (Hook f. & Wilson) Ochyra
Syntrichia saxicola (Cardot) R. H. Zander
SELIGERACEAE
Holodontium strictum (Hook f. & Wilson) Ochyra
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Pereira and Putzke 1994
Comparing the data on the vegetal communities of different areas, we can see that Stinker Point has the greater Bryum argenteum and Chorisodontium acyphyllum formations, which are reduced in the other areas (
Putzke and Pereira 1998a b
Pereira et al. 2007
the lakes rich in submerged communities (
, ). The biggest lake in Elephant Island also has a submerged community that is, not observed on the Keller Peninsula (
are found. For description of the main plants communities see
Pereira et al. 2008
and
Victoria et al. 2009b
). At Rip Point the mosses are dispersed over all the area, in small patches or cushions, rarely carpets, and
). The vegetation of Deception Island (Figure 2) is still recovering the last volcanic eruption, but some fast growing patches of D. antarctica
.
Pereira and Putzke 1994
On Elephant and Nelson Islands human impact are minimal since the only buildings present are small refuges that are only sporadically occupied (
, Puztke and Pereira 1998b). On the first island, the Brazilian refugee Wiltgen was completely removed, contributing to the moss regeneration at Stinker Point.
On Deception Island the touristic activity has been affecting the flora, but new rules are being followed and the impact is being reduced.
Pereira et al. 2007 2008
Keller Peninsula studies over the years have shown that human activity impacts the moss formations in a negative manner, but in some areas the plant communities increase faster (
,
).
Pereira et al. 1990a b Sander et al. 2004
The use of land plants for nest building was studied in Catharacta spp, in L. dominicanus and in Phalacrocorax atriceps. In the three cases studied land plant were the principal material in the nest, but lots of garbage are being used (
Plant checklist from the areas studied, collected and indentified by Brazilian researchers and preserve in herbarium
ANGIOSPERMAE
POACEAE
Deschampsia antarctica Desv.
CARYOPHYLLACEAE
Colobanthus quitensis (Kunth) Bartl.
LIVERWOTS
Cephaloziella varians (Gottsche) Stephani
Lophozia excisa (Dicks.) Dumort.
Marchantia berteroana Lehm. & Lindenb.
MOSSES
AMBLYSTEGIACEAE
Cratoneuropsis relaxa ssp. minor
Sanionia uncinata (Hedw.) Loeske
Sanionia georgico-uncinata (Müll Hal.) Ochyra & Hedenas
Warnstorfia laculosa (Müll. Hal.) Ochyra & Matteri
Warnstorfia sarmentosa (Wahlenb.) Hedenas
ANDREAEACEAE
Andreaea depressinervis Cardot
Andreaea gainii Cardot
Andreaea regularis Müll. Hal.
BARTRAMIACEAE
Bartramia patens Brid.
Conostomum magellanicum Sull.
BRACHYTHECIACEAE
Brachythecium austrosalebrosum (Müll. Hal.) Kindb.
, ,
).
Sciuro-hypnum glaciale (Schimp.) Ignatov & Huttunen
BRYACEAE
Bryum archangelicum Bruch & Schimp
Bryum argenteum Hedw.
Bryum orbiculatifolium Cardot & Broth.
Bryum pallescens Schleich. ex Schwaegr.
Ptychostomum pseudotriquetrum (Hedw.) J. R. Spence & H. P.
Pohlia cruda (Hedw.) Lindb.
Pohlia drummondii (Müll. Hal.) A. L. Andrews in Grout
Pohlia nutans (Hedw.) Lindb.
Pohlia wahlenbergii (F. Weber & D. Mohr) A.L. Andrews
DICRANACEAE
Chorisodontium aciphyllum (Hook. f. & Wills.) Broth.
Dicranella campylophylla (Taylor) A. Jaeger
Dicranella varia (Hedw.) Schimp
Kiaeria pumila (Mitt. & Hook. f.) Ochyra - very rare.
DITRICHACEAE
Ceratodon purpureus (Hedw.) Brid.
Distichium capillaceum (Hedw.) Bruch & Schimp
Ditrichum hyalinum (Mitt.) Kuntze
Ditrichum hyalinocuspidatum Cardot
ENCALYPTACEAE
Encalypta rhaptocarpa Schwaegr.
GRIMMIACEAE
Grimmia reflexidens Müll. Hal.
Bucklandiella sudetica (Funck) Bednarek-Ochyra & Ochyra
Schistidium amblyophyllum (Müll. Hal.) Ochyra & Hertel
Schistidium antactici (Cardot) L. I. Savicz & Smirnova
Schistidium cupulare (Müll. Hal.) Ochyra
Schistidium falcatum (Hook. f. & Wils.) B. Bremer
Schistidium halinae Ochyra
Schistidium andinum (Mitt.) Herzog Schistidium rivulare (Brid.) Pobp.
Schistidium steerei Ochyra
Schistidium urnulaceum (Müll. Hal.) B. G. Bell.
HYPNACEAE
Hypnum revolutum (Mitt.) Lindb.
Platydictya jungermannioides (Brid.) Crum
MEESIACEAE
Meesia uliginosa Hedw.
ORTHOTRICHACEAE
Muelleriella crassifolia (Hook. f. & Wils.) Dusén
POLYTRICHACEAE
Polytrichastrum alpinum (Hedw.) G. L. Smith
Polytrichum strictum Brid.
Polytrichum juniperinum Hedw.
Polytrichum piliferum Hedw.
POTTIACEAE
Didymodon brachyphyllus (Sull.) R. H. Zander
Hennediella antarctica (Angstr.) Ochyra & Matteri
Hennediella heimii (Hedw.) Zand.
Stegonia latifolia (Schwaegr.) Vent ex Broth.
Syntrichia filaris (Müll. Hal.) Zand.
Syntrichia magellanica (Mont.) R.H. Zander
Syntrichia saxicola (Cardot) Zand.
SELIGERACEAE
ALGAE
MACROSCOPIC CONTINENTAL ALGAE
Prasiola crispa (Lightfoot) Menegh
MACROSCOPIC FUNGI
Lamprospora miniatopsis Spooner
Omphalina antarctica Sing.
Galerina moelleri Bas.
Galerina perara Sing.
LICHENIZED AND LICHENICOLOUS FUNGI
Acarospora macrocyclos Vain.
Amandinea coniops (Wahlenb.) M. Choisy ex Scheid. & H. Mayrhofer. (=Buellia coniops)
Amandinea petermanii (Hue) Matzer, H. Mayrhofer & Scheid. [=Rinodina petermanii (Hue) Darb.]
Arthopyrenia maritima Øvstedal
Arthrorhaphis citrinella (Ach.) Poelt
Austrolecia antarctica Hertel
Bacidia stipata Lamb
Biatorella antarctica B. J. Murray
Bryonora castanea (Hepp) Poelt
Bryoria chalybeiformis (L.) Brodo et D. Hawksw.
Buellia anisomera Vain.
Buellia augusta Vain.
Buellia cladocarpiza Lamb
Buellia granulosa (Darb.) Dodge
Buellia latemarginata Darb.
Buellia nelsonii Darb.
Buellia papillata (Sommerf.) Tuck.
Buellia perlata (Hue) Darb.
Buellia pycnogonoides Darb.
Buellia russa (Hue) Darb.
Buellia subpedicillata (Hue) Darb.
Caloplaca amniospila (Ach.) H. Olivier
Caloplaca athallina Darb.
Caloplaca buelliae Olech & Søchting
Caloplaca cirrochrooides (Vain.) Zahlbr.
Caloplaca citrina (Hoffm.) Th. Fr.
Caloplaca iomma Olech & Søchting
Caloplaca millegrana (Müll. Arg.) Zahlbr.
Caloplaca psoromatis Olech & Søchting
Caloplaca regalis (Vain.) Zahlbr.
Caloplaca siphonospora Olech & Søchting
Caloplaca sublobulata (Vain.) Zahlbr.
Caloplaca tetraspora (Nyl.) H. Oliv.
Caloplaca tiroliensis Zahlbr.
Candelaria murrayi (Dodge) Poelt
Candelariella hallettensis (Murray) Øvstedal
Candelariella vitellina (Hoffm.) Müll. Arg.
Carbonea assetiens (Nyl.) Hertel
Carbonea vorticosa (Flörke) Hertel
Catapyrenium daedaleum (Kremp.) Stein
Catapyrenium lachneum (Ach.) R. Sant.
Catillaria corymbosa (Hue) Lamb
Cetraria aculeata (Schreb.) Fr. [=Coelocaulon aculeatum (Schreber) Link]
Cladonia cariosa (Ach.) Spreng.
Cladonia chlorophaea (Flörke ex Sommerf.) Spreng.
Cladonia furcata (Huds.) Schrader
Cladonia gracilis (L.) Willd.
Cladonia metacorallifera Asah.
Cladonia phyllophora Ehrh. ex Hoffm.
Cladonia pyxidata (L.) Hoffm.
Cladonia rangiferina (L.) Weber
Coleopogon epiphorellus (Nyl.) Brusse & Kärnefelt [=Coelocaulon epiphorellum (Nyl) Kärnef.]
Cystocoleus ebeneus (Dillwyn) Thwaites
Dermatocarpon intestiniforme (Körb.) Hasse
Haematomma erythroma (Nyl.) Zahlbr.
Himantormia lugubris (Hue) Lamb
Huea cerussata (Hue) C.W. Dodge & G.E. Baker
Huea coralligera (Hue) C.W. Dodge & G.E. Baker
Hypogymnia lugubris (Pers.) Krog
Hypogymnia lububris var. compactior (Zahlbr.) D. C. Linds.
Japewia tornoensis (Nyl.) Tønsberg
Lecania brialmontii (Vain.) Zahlbr.
Lecania gerlachei (Vain.) Zahlbr.
Lecanora dispersa (Pers.) Sommerf.
Lecanora expectans Darb.
Lecanora mons-nivis Darb.
Lecanora physciella (Darb.) Hertel
Lecanora polytropa (Hoffm.) Rabenh.
Lecidea atrobrunnea (Ramond ex Lam. et DC.) Schaer.
Lecidea lapicida (Ach.) Ach.
Lecidea sarcogynoides Körb.
Lecidea sciatrapha Hue
Lecidella stigmatea (Ach.) Hertel and Leuckert
Lecidella wulfenii (Hepp) Körb.
Leptogium menziesii Mont.
Leptogium puberulum Hue
Massalongia carnosa (Dicks.) Körb.
Mastodia tesselata (Hook.f. & Harv.) Hook.f. & Harvey
Megaspora verrucosa (Ach.) Hafellner
Micarea assimilata (Nyl.) Coppins (=Lecidea assimilata Nyl.)
Microglaena antarctica Lamb
Ochrolechia frigida (Sw.) Lynge
Ochrolechia parella (L.) A. Massal.
Pannaria hookeri (Borrer ex Sm.) Nyl.
Parmelia saxatilis (L.) Ach.
Physcia caesia (Hoffm.) Fürnr.
Physcia dubia (Hoffm.) Lettau
Physconia muscigena (Ach.) Poelt
Placopsis contortuplicata Lamb
Poeltidea perusta (Nyl.) Hertel et Hafellner
Polyblastia gothica Th. Fr.
Porpidia albocaerulescens (Wulfen) Hertel et Knoph
Porpidia crustulata (Ach.) Hertel et knoph
Pseudephebe minuscula (Nyl. ex Arnold) Brodo et D. Hawksw. (=Alectoria minuscula Lindsay)
Pseudephebe pubescens (L.) Choisy
Psoroma hypnorum (Vahl) Gray
Ramalina terebrata Hook et Tayl.
Rhizocarpon geminatum Körb.
Rhizocarpon geographicum (L.) DC.
Rhizocarpon polycarpon (Hepp) Th. Fr.
Rhizoplaca aspidophora (Vain.) Redón
Rhizoplaca melanophthalma (DC. in Lam. et DC.) Leuck. et Poelt
Rinodina deceptionis Lamb
Rinodina mniaraea (Ach.) Körb.
Rinodina turfacea (Wahlenb.) Körb.
Sphaeorophorus fragilis (L.) Pers.
Sphaeorophorus globosus (Hudson) Vain.
Sphaeorophorus melanocarpus (Sw.) DC.
Staurothele gelida (Hook & Tayl.) Lamb
Stereocaulon alpinum Laurer ex Funck
Stereocaulon glabrum (Müll. Arg.) Vain.
Tephromela atra (Hudson) Hafellner
Thelocarpon cyaneum Olech et Alstrup
Tremolecia atrata (Ach.) Hertel
Umbilicaria aprina Nyl.
Umbilicaria decussata (Vill.) Zahlbr.
Umbilicaria propagulifera (Vain.) Llano
Umbilicaria rufidula (Hue) Filson
Usnea acromelana Stirton
Usnea antarctica Du Rietz
Usnea aurantiacoatra (Jacq.) Bory
Usnea trachycarpa (Stirt.) Mull. Arg.
Verrucaria ceuthocarpa Wahlenb.
Verrucaria cylindrophora Vain.
Verrucaria dispartita Vain.
Verrucaria elaeoplaca Vain.
Verrucaria halizoa Leight.
Verrucaria psycrophila Lamb
Verrucaria racovitzae Vain.
Verrucaria tesselatula Nyl.
Xanthoria candelaria (L.) Th. Fr.
Xanthoria elegans (Link.) Th. Fr.
Actually, the main concern of the Brazilian botanists in Antarctica is to describe and map the plant and microbial community in order to better understand the processes that drive in the Antarctic ice-free areas, and to relate these to human impacts or natural phenomena. Global changes are leading to the loss of ice cover in Antarctica, and
with it, potentially dramatic changes in plant and microbial community composition. There is an urgent need to establish georeferenced baseline maps of plant and soil microbial distribution in order to evaluate future compositional changes through the years. Brazilian researchers have already began this important work, contributing to
Pereira and Putzke 1994 Pereira et al. 2007 2008 2009 Putzke and Pereira 1998b Victoria et al. 2004 2006 2009a b Victoria and Pereira 2007
the management and monitoring studies in the Antarctic ice free areas (
,
,
,
,
,
,
,
, ,
).
CONCLUSIONS
In twenty-five years of work by botanists from the Brazilian Antarctic Program investigating the plant communities in Antarctic ice free areas, six papers on the chemistry of lichens, six papers on plant taxonomy, five papers on plant biology, two works on UVB photoprotection and eleven papers on plant communities were published.
At the present, Brazilian botanists are studying the plant communities in Antarctic ice free areas in order to understand their relationship to soil microbial communities, their biodiversity, the distribution of the plants populations and their relationship with birds colonies. Baseline maps of plant communities and their distribution are being
made in order to arguments studies of their development and environmental impacts in the future. In addition to these activities, a group of Brazilian researchers are undertaking studies related to Antarctic plant genetic diversity, plant chemistry and the biotechnological applications of these. It is believed that knowledge of the
biotechnological potentials of Antarctic plants, in addition to research on plant/communities biology and evolving processes is essential to the valuation and therefore the preservation of these natural resources.
Acknowledgements
This work was supported by the Brazilian Antarctic Program through the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq process no. 574018/2008), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ process E-26/170.023/2008), Ministry of Environment – MMA, Ministry of Science,
Technology and Innovation – MCTI and CIRM.
REFERENCES
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Czeczuga B and Xavier-Filho L. 1988. Investigations on caratenoids in Embryophyta X. I. The presence of torulene in mosses from the Antarctica. Ser Cient INACH 37: 111-116. [ Links ]
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http://dx.doi.org/10.1590/S0001-37652013005000050
Biological Sciences
Evidence of separate karyotype evolutionary pathway in Euglossa orchid bees by cytogenetic analyses
ANDERSON FERNANDES1, HUGO A. WERNECK2, SILVIA G. POMPOLO2, DENILCE M. LOPES2
1
2
Departamento de Ciências Biológicas, Universidade do Estado de Mato Grosso, MT Rodovia MT, 358, Km 07, Jardim Aeroporto, 78300-000 Tangará da Serra, MT, Brasil
Departamento de Biologia Geral, Universidade Federal de Viçosa, Av. P.H. Rolfs, s/n, 36570-000 Viçosa, MG, Brasil
ABSTRACT
Euglossini are solitary bees considered important pollinators of many orchid species. Information regarding chromosome organization is available for only a small number of species in this group. In the present work, the species Euglossa townsendi and E. carolina were analyzed by cytogenetic techniques to
collect information that may aid the understanding of their evolution and chromosomal organization. The chromosome number found was n = 21 for males and 2n = 42 for females in the two species. The distribution and amount of heterochromatin regions differed in the two species analyzed, where they were
classified as “high” or “low” heterochromatin content, similarly to what has already been performed in social bee species of the genus Melipona. Banding patterns found in this study suggest that other mechanisms may have occurred in the karyotype evolution of this group, unlike those suggested for social bees
and ants. Karyotype evolution of solitary bees appears to have occurred as an event separate from other hymenopterans and did not involve chromosome fissions and heterochromatin amplification.
Keywords: Cytogenetics; Euglossini; heterochromatin; karyotypic evolution
RESUMO
Os Euglossini são abelhas solitárias consideradas importantes polinizadores de muitas espécies de orquídeas. Somente um pequeno número de espécies desse grupo possui informações sobre sua organização cromossômica. Neste trabalho as espécies Euglossa townsendi e E. carolina foram analisadas por
técnicas citogenéticas a fim de obter informações que auxiliem no entendimento de sua evolução e organização cromossômica. O número cromossômico encontrado foi n = 21 para os machos e 2n = 42 para as fêmeas das duas espécies. A distribuição e a quantidade de heterocromatina diferem nas duas espécies
analizadas e podem ser classificadas como alta e baixa quantidade de heterocromatina, similarmente como já foi feito anteriormente para espécies de abelhas sociais do gênero Melipona. O padrão de bandeamento encontrado no presente trabalho sugere que outros mecanismos podem estar envolvidos na
evolução cariotípica do grupo, diferente daqueles sugeridos para as abelhas sociais e formigas. A evolução cariotípica de abelhas solitárias parece ter acontecido por eventos distintos que não envolveram fissões cromossômicas e amplificação da heterocromatina.
Palavras-Chave: Citogenética; Euglossini; heterocromatina; evolução cariotípica
INTRODUCTION
Among insects, the Hymenoptera order attracts great interest from the field of cytogenetics. Different bee ( review in
Rocha et al. 2003
) and ant species (
Lorite and Palomeque 2010
) have already been investigated. However, there is almost no information on genome organization at the chromosomal level of
some groups. One example includes the bee Euglossini. These bees exhibit solitary behavior and have received attention in recent years in particular because they are effective pollinators of nearly 700 orchid species (
Among the five genera of Euglossini, Euglossa Latreille ( 1802) is the most diverse, composed of six subgenera with about 122 species (
due to the large number of morphological similarities.
Cytogenetics of the Euglossini group is currently restricted to few species.
Eltz et al. ( 1997)
Nemésio 2009
Ramírez 2005
) . For this reason they are also known as “orchid bees”.
) . This number may be much higher as there are constant revisions and faunal surveys needed, as well as the group taxonomy is extremely complicated
determined chromosome numbers of the Euglossa cyanaspis and E. hyacinthina species ( n = 21 and n = 20, respectively) , and also confirmed that chromosome morphology was submetacentric in both species. In
Kerr and Laidlaw ( 1956)
Gomes et al. 1998
the same study, the researchers cited that
determined the chromosome number for the Eufriesea violacea species as n = 16. This species was further studied with other techniques (
) . In this study, the authors used the techniques of C and G banding to obtained greater
detail on heterochromatin distribution and a more secure pairing between homologues. They also established the chromosome number for the species as 2n = 30 in all submetacentric chromosomes. Another study was performed with a species of Euglossa ( unidentified) in which the authors observed n
= 21 for males, and reported marking by the Ag-NOR technique ( silver impregnation - Nucleolar Organizer Region) in five chromosomes (
Maffei et al. 2001
).
Nemésio 2009
Considering the ecological importance of these bees and the lack of cytogenetic studies with Euglossini species, the present work aimed to expand the cytogenetic information by analyzing two species of the Euglossa genera, E. ( Euglossa) carolina
( 1904) , using conventional staining, C band, restriction enzymes and fluorochromes for better understanding of the karyotype evolution of these bees. restriction enzymes and fluorochromes for better understanding of the karyotype evolution of these bees.
and E. ( Euglossa) tonwsendi Cockerell
Individuals captured in bamboo tube nest traps were used in the present study ( as describle by
Camillo et al. 1995
) . Euglossa carolina was collected in Cataguases, MG, Brazil, ( three nest obtaining a total of 12 individuals) and E. tonwsendi in the city of Viçosa, MG, Brazil ( two nest obtaining a total of
9 individuals) . The cerebral ganglia of larvae in the final defecation stage were removed and used for chromosomic preparations. Metaphase chromosomes were obtained according to the methodology proposed by
Imai et al. ( 1988)
.
After obtaining metaphase chromosomes, the slides were subjected to staining techniques. For the conventional staining, a solution of 4% Giemsa in Sörensen buffer, pH 6.8, was used for 15 minutes. The C banding ( BSG method: Bariumhydroxide/Saline/Giemsa) was performed as suggested by
and Pompolo ( 1998)
. Sequential staining with the fluorochromes chromomycin A3 ( CMA3) and DAPI ( 4′-6- diamino-2-phenylindole) was carried out according to
was carried according to
Seabright ( 1971)
and restriction banding with DraI ( AAA/TTT) and HaeIII ( CC/GG) as reported by
Gosálvez et al. ( 1987)
Schweizer ( 1980)
with the modifications proposed by
Rocha et al. ( 2002)
Rocha
. Quinacrine mustard was used according to Schmid ( 1980) . G banding
.
The slides were then analyzed under an Olympus BX60 light/fluorescence microscope with immersion objective, seeking to observe an average of 10 metaphases per slide. For karyotype mounting, the chromosomes were arranged according to the classification proposed by
Levan et al. ( 1964)
.
RESULTS
Euglossa Tonwsendi
Euglossa townsendii presented submetacentric chromosomes, 2n = 42 for females and n = 21 for males ( Fig. 1) , with heterochromatin blocks on the pericentromeric region of the long arm, and in some chromosomes on the short arm ( Fig. 1B) . An interstitial and a telomeric block were also observed
in three chromosomes ( Fig 1B) .
Figure 1 Karyotypes of Euglossa townsendi, n = 21 ( male) and 2n = 42 ( female) . Conventional staining ( A) . C-banding ( B) . DAPI fluorochrome ( C) . CMA3 fluorochrome ( D) . The asterisk indicates sites of chromosome overlap. Scale bar = 5 µm.
When treated with the fluorochromes DAPI and CMA3 the pericentromeric heterochromatin showed to be rich in AT bases ( markings DAPI+ and CMA3−, Fig. 1C and D, respectively) , except for the telomeric region of the long arm of chromosomes 2, 3, and 7 which indicated minimal markings by
CMA3 ( Fig. 1D) .
Positive G bands were observed in both euchromatin and heterochromatin regions ( Fig 2A) . Some positive G bands were also QM+, especially in the pericentromeric and telomeric regions, indicating that they are rich in AT sequences ( Fig 2A and 2B) .
Figure 2 Metaphases of Euglossa townsendi. G-band ( A) . Quinacrine mustard used sequentially to G-band ( B) . Restriction enzyme HaeIII with conventional staining ( C) and DAPI ( D) . Restriction enzyme DraI with conventional staining ( E) and DAPI ( F) . Arrows
indicate site of intense markings. Scale bar = 5 µm.
By using the restriction enzyme HaeIII ( target site CC/GG) followed by conventional staining, it was possible to visualize enzyme activity at the end of one arm of the 14 chromosomes of the haploid set ( Fig. 2C) . However, when the enzyme was used sequentially with DAPI, a new marking pattern
was encountered ( Fig. 2D) . Remarkable were the markings on the pericentromeric and terminal regions of the arms of most chromosomes. Two chromosomes of the haploid set were highlighted because they have very intense markings at the terminal region of one arm. Results of the treatment with
the enzyme DraI ( recognition site AAA/TTT) ( Fig. 2E and F) were very similar to those found for HaeIII. Four chromosomes of the diploid set showed strong markings at the end of one arm.
Euglossa Carolina
The chromosome number of this species was 2n = 42 for females and n = 21 for males, and all chromosomes present submetacentric morphology ( Fig. 3A and B) . In some chromosomes, it was possible to visualize the presence of secondary constrictions. Chromosomes were almost entirely
heterochromatic with exception of teloremic regions ( Figure 3B) .
Figure 3 Karyotypes of Euglossa carolina, n = 21 and 2n = 42. Conventional staining of female ( A) . C-banding ( B) . DAPI fluorochrome ( C) . CMA3 fluorochrome ( D) . The asterisk indicates sites of chromosome overlap. Scale bar = 5 µm.
The fluorochromes showed a centromeric region with composition rich in AT ( DAPI+) , while GC-rich regions were verified throughout the chromosome where it was possible to observe several bands ( Fig. 3C and D) . Heterochromatin therefore appears to be heterogeneous with blocks rich in AT
and others in GC.
DISCUSSION
Chromosomal Organization
Eltz et al. 1997
Only two Euglossa species have been studied cytogenetically, E. cyanaspis and E. hyacinthina (
) , and chromosome numbers presented by these species are n = 21 and n = 20, respectively. The chromosome numbers of the two species studied in the present work were n = 21. Conservation
of the chromosome number ( or little change) in bees appears to be a feature commonly found in these species.
Secondary constrictions indicated by conventional staining in E. carolina can be associated with the Nucleolar Organizer Region ( NOR) . The same pattern has been previously observed in Euglossa sp. by
confirmation should be obtained by specific techniques such as Ag-NOR or in situ hybridization. However, good results with Ag-NOR in bees are restricted to only few species (
Maffei et al. ( 2001)
Maffei et al. 2001 Duarte et al. 2009
,
, who matched positive CMA3 marks on five chromosomes. Further
) and in situ hybridization is not yet widely used in this group.
Variations regarding heterochromatin quantity and location were observed between the E. tonwsendi and E. carolina species. While E. tonwsendi presented very little heterochromatin, a large quantity of heterocromatin was observed in E. carolina located in the interstitial region, with euchromatin
restricted to chromosome ends. A pattern similar to E. carolina was found in Eufriesea violacea (
Gomes et al. 1998
) , another Euglossini bee.
Rocha and Pompolo 1998
In the genus Melipona the pattern of heterochromatin distribution allowed division of these bees into two groups: one showing a high heterochromatin content and the other a low content (
) . Compared to the Euglossini bees studied so far, E. tonwsendi could be classified as
belonging to the group of low heterochromatin content with pericentromeric distribution, while E. carolina to the group of high heterochromatin content with interstitial distribution throughout the chromosome.
Fluorochrome staining showed that positive C bands were predominantly DAPI+ with the exception of a few CMA3+ heterochromatic blocks in E. carolina chromosomes. This demonstrates that in E. carolina the heterochromatin is not homogeneous, which may be related to distinct sequences that
make up the heterochromatin and are not highlighted by C banding. Usually these few C+/CMA3+ regions of the genome are associated with NORs (
Maffei et al. 2001 Rocha et al. 2002 2003 Brito et al. 2005
,
,
,
).
The G-banding technique indicated rich patterns of bands in E. tonwsendi in both euchromatic and heterochromatic regions of all chromosomes ( Figure 2A) . When followed by QM, some G+ bands were also shown as QM+ ( rich in AT bases) , especially those located in pericentromeric regions and
at the end of one arm ( Figure 2B) .
Despite the contrasting recognition sites, both restriction enzymes HaeIII ( CC/GG) and DraI ( AAA/TTT) cleaved DNA at the chromosomes ends of E. townsendi. ( Fig. 2D and F) .
This result seems contradictory since fluorochromes bind to DNA and restriction enzymes ( RE) initially function by removing DNA. However, a different action mechanism of some REs should be mentioned which would result in chromatin decondensation (
This decondensation may increase accessibility to DNA providing a greater number of binding sites between fluorochromes (
Verma and Babu 1995
).
Mezzanote and Ferrucci 1984 Miller et al. 1984
,
).
Heterochromatin Content and Karyotype Evolution
Imai et al. ( 1994)
One mechanism proposed to explain karyotype evolution in the Hymenoptera order is centric fission. This mechanism is supported by the Minimum Interaction Theory proposed by
. It predicts that the karyotype evolved to minimize DNA damage due to interactions between the
chromosomes by decreasing the size of chromosomes resultant from centric fission, which would increase the chromosome number. To regain stability in the fission region, there would have occurred the in tandem growth of heterochromatin in this region, leading to chromosomes with a
heterochromatic arm (
Imai et al. 1988
) . In Meliponini, the karyotype analyses of 70 species ( review
however, it appears that heterochromatin was added without being preceded by fission (
Rocha et al. 2003
Rocha et al. 2003
) appeared to indicate that for most species the karyotypic changes in these organisms are in accordance with events predicted in the Theory of Minimal Interaction. In the Melipona genus
).
In Euglossa other mechanisms may have caused karyotype evolution as individuals have high chromosomes numbers, higher than other bees species. The chromosomes are large and present submetacentric morphology which is contrary to the characteristics predicted in the Theory of Minimal
Interaction. Moreover, different patterns of heterochromatin distribution are encountered, similar to that seen in Melipona.
Therefore, karyotype evolution of the Hymenoptera order ( or at least in solitary bees) seems to have occurred in separate events involving fission followed by heterochromatin amplification. Therefore, this study provides important data on solitary bees. However due to the small number of species
studied these questions cannot yet be answered with certainty but as the number of Euglossini species analyzed increases the processes involved in the karyotype evolution may be better understood.
Acknowledgements
The authors thank Fundação de Amparo à Pesquisa do Estado de Minas Gerais ( FAPEMIG) , Fundação de Amparo à Pesquisa do Estado de Mato Grosso ( FAPEMAT) and Conselho Nacional de Desenvolvimento Científico e Tecnológico ( CNPq) for the financial support.
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Received: February12, , 2011; Accepted: September26, , 2011
Correspondence to: Denilce Meneses Lopes E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013005000049
Biological Sciences
Evaluation of wound healing and antimicrobial properties of aqueous extract from Bowdichia virgilioides stem barks in mice
ISABELA K.R. AGRA1, LUANA L.S. PIRES2, PAULO S.M. CARVALHO1, EURÍPEDES A. SILVA-FILHO2, SALETE SMANIOTTO1, EMILIANO BARRETO1
1
Laboratório de Biologia Celular, Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Alagoas, Campus A.C. Simões, Avenida Lourival Melo Mota, s/n, Tabuleiro dos Martins, 57072-970 Maceió, Al, Brasil
Laboratório de Genética de Microorganismos, Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Alagoas, Campus A.C. Simões, Avenida Lourival Melo Mota, s/n, Tabuleiro dos Martins, 57072-970 Maceió,
Al, Brasil
2
ABSTRACT
The decoction of the stem barks from Bowdichia virgilioides KUNTH is a folk remedy used to treat inflammatory disorders in Latin American and Brazil. In the present study, the wound healing activity of aqueous extract of the stem
bark from B. virgilioides, called AEBv, was evaluated by the rate of healing by wound contraction and period of epithelization at different days post-wound using the wound excisional model. On day 9, the AEBv-treated animals
exhibited significative reduction in the wound area when compared with controls. In wound infected with S. aureus, the AEBv significantly improved the wound contraction when compared to the saline-treated mice. The histological
analysis showed that AEBv induced a collagen deposition, increase in the fibroblast count and few inflammatory cells than compared to saline-treated group. The expression of collagen type I was increased in the group treated with AEBv
as indicated by immunohistochemical staining. In vitro, the AEBv was effective only against S. aureus but not against P. aeruginosa. Together, the results of this study demonstrate, for the first time, the healing and antimicrobiological
effects of aqueous extract of the stem bark from B. virgilioides in the therapy of skin wounds.
Keywords: wound healing; antimicrobial effect; Bowdichia virgilioides ; medicinal plant
RESUMO
A decocção das cascas do caule de Bowdichia virgilioides Kunth é um medicamento popular usado para tratar doenças inflamatórias na América Latina e no Brasil. Neste estudo, a atividade de cicatrização de feridas do extrato aquoso da
casca do caule de B. virgilioides, chamado AEBv, foi avaliada pela contração da ferida e pelo período de epitelização em diferentes dias pós-ferida usando o modelo ferida excisional. No nono dia, os animais tratados com AEBv
apresentaram uma redução significativa na área da ferida, quando comparados com os controles. Nas feridas infectadas com S. aureus, o AEBv melhorou significativamente a contração da ferida quando comparado com os camundongos
tratados com solução salina. A análise histológica mostrou que AEBv induziu uma deposição de colágeno, aumento na contagem de fibroblastos e poucas células inflamatórias do que em relação ao grupo tratado com solução salina. A
expressão de colágeno tipo I mostrou-se aumentada no grupo tratado com AEBv como indicado pela coloração imuno histoquímica. In vitro, o AEBv foi eficaz apenas contra S. aureus, mas não contra P. aeruginosa. Juntos, os resultados
deste estudo demonstram, pela primeira vez, a cura e os efeitos antimicrobianos do extrato aquoso da casca do caule de B. virgilioides na terapia de feridas cutâneas.
Palavras-Chave: Cicatrização de feridas; efeito antimicrobiano; Bowdichia virgilioides ; planta medicinal
INTRODUCTION
Bowdichia virgilioides Kunth ( Fabaceae) is a plant that grows commonly in several South American countries such as Venezuela, Guiana and Brazil (
Brazilian traditional medicine for treatment of diseases. The bark is used for wound healing, as anti-ulcer and anti-diabetic (
treat rheumatism, arthritis, and skin diseases (
Flores and Rodrigues 2010
) . Various parts of B. virgilioides are used in the
Bacchi 1986 Oliveira and Saito 1989 Braga 1953
,
,
) . Other plant parts such as seeds are used in folk medicine to
Cruz 1965
).
The stem bark preparations of the B. virgilioides are reported to have antimalarial (
chemical substances such as lupeol, lupeol acetate, sitosterol and stigmasterol (
virgilioides has been attributed to its essential oil of seeds (
virgilioides.
Deharo et al. 2001
) , analgesic and anti-inflammatory activities (
Melo et al. 2001
Silva et al. 2010 Thomazzi et al. 2010 Barros et al. 2010
,
) . Others include alkaloid named acosmine, ormosanine and podopetaline (
,
) . The stem bark contains several
Barbosa-Filho et al. 2004
) . The antimicrobial property from B.
Almeida et al. 2006
) and leaves ( J.G.R. Feitosa et al., unpublished data) . However, there are no published reports on the antimicrobial activity of the stem barks from B.
Open wounds are particularly prone to infection, especially by bacteria, and also provide an entry point for systemic infections. Infected wounds heal less rapidly and also often result in the formation of unpleasant exudates and
toxins that will be produced with concomitant killing of regenerating cells. Consequently, there is a need to stimulate healing and restore the normal functions of the affected part of the body to ease the discomfort and pain
associated with wounds, preventing infection, and activating tissue repair processes. Antibacterial and healing compounds in a traditional remedy can induce this occurrence and may be beneficial in treating wounds (
2008
Reddy et al.
) . In spite of recorded uses of the B. virgilioides, there is no scientific evidence that confirms the healing effect and antibacterial activity of stem bark of B. virgilioides. Thus, this work was undertaken to explore the
antimicrobial and wound healing effects of B. virgilioides stem bark extract.
Plant Material and Preparations of Aqueous Extract
Stem bark from B. virgilioides Kunth ( Family Fabaceae) was collected in Maceió, Alagoas State, Brazil ( 9°33′12″S and 35°46′9″W) . The plant was identified by Prof. Rosângela Lemos, Instituto do Meio Ambiente, Maceió,
Brazil, and the voucher specimen ( number MAC29914) has been deposited at the Herbarium MAC of the Instituto do Meio Ambiente.
After collection, the stem barks were dried at ambient temperature and triturated. The aqueous extract of B. virgilioides, called AEBv, was prepared by infusing 50 g of powdered plant material for 20 minutes using 300 mL of
boiling water. The extract was filtered and lyophilized. The yield of the infusion was 17.2% ( wt=wt) . At the time of use, extract was reconstituted in water ( sterile endotoxin-free) at the required concentration ( 10 mg.kg−1)
according as previous results ( J.P. Silva, unpublished data) .
Animals
Swiss mice of either sex weighing 18–22 g were obtained from the Universidade Federal de Alagoas ( UFAL) breeding unit. The animals were maintained with free access to food and water and kept at 22-28°C with a controlled
12-hour light/dark cycle at the Instituto de Ciências Biológicas e da Saúde, UFAL. Experiments were performed during the light phase of the cycle. The animals were allowed to adapt to the laboratory for at least 2 hours before
testing and were used only once. All experiments were carried out in accordance with institutional guidelines and ethics ( License Number 23065.12614/2006-89) .
Excision Wound Model
The animals were anesthetized with anesthetic ether and shaved at the predetermined site before wounding. A circular wound was inflicted by cutting away approximately 1.6 cm of diameter of the predetermined area on the
anterior-dorsal side of each mice using sterile surgical blade (
left open to the environment.
Morton and Malone 1972
) . The animals were then placed in separate cages to avoid any disturbance. The bedding was changed daily. After skin excision, the wound was
In other set of experiments, the wound was inoculated ( 10 µL) with Staphylococcus aureus ( ATCC 25923) at 108 Colony Forming Unit ( CFU) . All animals received topical application ( 200 µL) of solutions containing saline
( NaCl, 0.9%) or AEBv ( 10 mg.kg−1) for once a day for 9 consecutive days starting from the day of wounding. As standard treatment was used fibrinolysin ( Fibrase SA®) on non-infected wounds or 1% silver sulfadiazine
( Dermazine®) on infected wounds. The animals of the Fibrase SA® or Dermazine ® groups were topically treated once a day with 0.5 g of each ointment. Wound contraction was calculated as percentage reduction in wound area.
The progressive changes in wound area were monitored by a camera ( Sony Cyber Shot, Dsc w80) on wounding day, followed by measurements on 3th, 6th and 9th day. Later on, wound area was evaluated by using ImageJ program
(
Nicoli et al. 2008
) . A specimen sample of tissue was isolated from the healed skin of each group of mice for the histopathological examination.
Wound Healing Rate
The wound area of each animal was measured on days 3, 6, and 9 post-surgery. The wound size measurements taken at the time of surgery and at the time of biopsy were used to calculate the percent wound contraction, using
equation:
[( A0 – At) /A0] × 100 = % of wound closure
where A0 is the original wound area, and At is the area of wound at the time of biopsy.
Antimicrobial Sensitivity Test
Antimicrobial activities of extract was evaluated against Staphylococcus aureus ( ATCC 25923) , Pseudomonas aeruginosa ( ATCC 27853) and Staphylococcus aureus ( MRSA) which was supplied by Dr. Euripedes A. SilvaFillho, UFAL, Maceió-AL, Brazil. The MRSA-strain was based on their resistance to methicillin ( DMPPC) and oxacillin ( MPIPC) according to the guidelines of the
National Committee for Clinical Laboratory Standards ( 2003)
. A modified
Joung et al. 2010
diffusion test was used to determine the antimicrobial activity (
) . The colonies were taken directly from the plate and were suspended in 5 mL of sterile 0.85% saline. The turbidity of the initial suspension was
adjusted by comparing with 0.5 McFarland's standard. When adjusted to the turbidity of the 0.5 McFarland's standard, the bacteria suspension contains about 108 colony forming unites ( CFU) . mL−1. In different petridish the
Murray et al. 1995
bacterial strains were grown to exponential phase in Mueller-Hinton broth at 37 °C for 18 h and adjusted to a final density of 108 CFU/ml by diluting fresh cultures and comparing with McFarland density (
) . The
blank control was performed with distillated water. Chloramphenicol was used as the positive control. In each petridish were made bores ( 4 mm) where each bore was loaded with 40 µL of water, AEBv ( 1, 2 and 4 mg.mL−1) or
chloramphenicol ( 1 mg.mL−1) . After incubation at 37°C for 24 h the inhibition zones around the bores were measured. The tests were performed in triplicate and the results were expressed in mm as the arithmetic media of
diameters of the inhibition zones. After incubation, the result of antimicrobial activity test was reported as the average diameter of the inhibition zone surrounding the wells containing the test solution.
Histopathological Analysis
Tumen et al. ( 2012)
The skin specimens from each group were collected at 9 days after beginning of the experiment to evaluate the histopathological alterations in accordance with
, being the analysis performed blindly by a
pathologist. Samples were fixed in 10% buffered formalin, processed and blocked with paraffin. Then, sample were sectioned into 5 µm-thick sections and stained with hematoxylin and eosin ( HE) and Masson's trichrome ( MT) .
The tissues were examined by light microscope ( Olympus BX51 attached DP70 Digital Camera System) and graded subjectively as mild ( +) , moderate ( ++) and severe ( +++) for epidermal or dermal remodeling,
reepithelization; fibroblast proliferation, mononuclear and/or polymorphonuclear cells and collagen depositions in dermis were analyzed to score the epidermal or dermal remodeling.
Immunohistochemical Staining
Six µm thick skin cryostat sections were used for detecting type I collagen expression by immunoperoxidase staining. The sections were then treated with 0.3% hydrogen peroxide ( H2O2) in phosphate buffered saline ( PBS) for 10
min to quench any endogenous peroxidase activity within the tissue. The nonspecific binding sites were blocked with 0.5% bovine serum albumin ( BSA) for 10 min at room temperature. After washing with PBS, specimens were
incubated with purified rabbit anti-mouse type I collagen antibody ( Novotec - lot 338i) diluted 1:80 in PBS for 1 h at room temperature. After washing, the specimens were incubated with peroxidase-conjugated goat anti-rabbit
IgG second antibody ( Sigma) diluted 1:200 in PBS for 45 min at room temperature. Immunoreactivity was visualized with a diaminobenzidine ( DAB) ( Sigma) containing 0.02% H2O2 for 10 min. The control sections were
incubated directly with the secondary antibody in the absence of the primary antibody and processed as above. The specimens were observed using light microscope ( Nikon Eclipse 50i) .
Statistical Analysis
Data are mean ± SEM values. The statistical analysis involving two groups was done using Student's t test. Analysis of variance followed by the Student-Neuman-Keuls test was used to compare three or more groups. Values of P <
0.05 were considered as indicative of significance.
RESULTS
Wound Healing Activity
The wound healing activity of the aqueous extract prepared from the stem barks of Bowdichia virgilioides was evaluated on mice in the excision wound models to confirm the folkloric usage of the plant. The histopathological
changes induced by this extract and its antimicrobial activity in vitro were also assessed. The area of the wound was measurement on the days 3, 6 and 9 days post surgery in all groups. The measurements of the progress of wound
healing induced by the extract, reference drug and saline treated-groups in the excision of non-infected wounds are shown in Table I.
TABLE I Effect of aqueous extract of stem barks from Bowdichia virgilioides on non-infected wounds.
Parameter
Treatment
Saline
3
1.67 ± 0.15
Wound area ( cm2) on day
6
1.48 ± 0.11
9
1.11 ± 0.08
Period of epithelization ( days)
13.00 ± 0.00
Fibrase®
1.60 ± 0.06
1.40 ± 0.09
0.52 ± 0.08 ***
10.75 ± 0.5 **
AEBv
1.50 ± 0.12c
0.75 ± 0.06 *** a
0.18 ± 0.03 *** b
9.60 ± 0.33 *** c
Values represent the mean ± S.E.M., ( n=4) . Statistical differences were determined by ANOVA followed Student-Newman-Keuls test.
***
P<0.001,
**
P<0.01,
*
P<0.05 as compared to respective saline treatment.
a
P<0.001,
b
P<0.01,
c
P<0.05 as compared to Fibrase-treated animals.
Table I shows the measured values of the closure progression of non-infected wound in different groups. After application of AEBv topically onto non-infected wounds the area of wound reduced 25% of their original size ( 2 cm2)
on day 3, 62.5% on day 6 and 91% on day 9, and complete closure around day 10. In saline-treated animals, the area was reduced to 16.5% ( day 3) , 26% cm2 ( day 6) and 44.5% ( day 9) . The wound closure in animals treated
with reference drug, Fibrase, were 20% ( day 3) , 30% and 74% ( day 9) ( Table I) . Treatment with AEBv in non-infected wounds was able to reduce to 9 days the period of epithelialization when compared with saline-treated
group and Fibrase group, which were, respectively, 13 and 10 days.
Table II shows the measured values of the closure progression of infected wound in different groups. After application of AEBv topically onto infected wounds the area of wound reduced 43% of their original size ( 2 cm2) on day
3, 84% on day 6 and 95.5% on day 9, and complete closure on day 10 ( Table II) . On the other hand, in saline-treated animals, the area was reduced to 16.8% ( day 3) , 26.3% ( day 6) and 44.7% ( day 9) . The wound closure in
animals treated with reference drug, Dermazine®, were 23.8% ( day 3) , 41.2% and 64.1% ( day 9) ( Table II) . Treatment with AEBv in infected wounds was able to reduce to 10 days the period of epithelialization when compared
with saline-treated group and Dermazine® group, which were, respectively, 17.5 and 15 days.
TABLE II Effect of aqueous extract of stem barks from Bowdichia virgilioides on infected wounds.
Parameter
Treatment
Saline
Dermazine®
AEBv
3
1.53 ± 0.03
Wound area ( cm2) on day
6
1.25 ± 0.03
9
1.04 ± 0.03
Period of epithelization ( days)
17.50 ± 0.50
1.53 ± 0.05
1.18 ± 0.04
0.72 ± 0.04 ***
15.00 ± 0.00 **
1.14 ± 0.12 **
0.32 ± 0.08 *** a
0.09 ± 0.04 *** a
10.00 ± 0.00 *** a
***
P<0.001,
**
a
P<0.001 as compared to Dermazine-treated animals.
histopathological analysis
In order to confirm the experimental results, histopathological analysis was also performed. Figure 1 shows the histology of saline, AEBv and Fibrase-treated groups at 9 days of analysis in non-infected wound. The AEBv and
Fibrase-treated groups shows faster wound healing processes if compared with saline-treated animals. There was attenuation in the infiltration of inflammatory cells and enhanced proliferation of fibroblasts as a result of treatment
with our extract and the reference drug. There was full thickness reepithelialization, in which epidermis was thin and well organized, comparable to the normal adjacent skin which was not involved in the wound generation and
healing process. AEBv-treated wounds were associated with enhanced formation of epidermis and deposition of connective tissue when compared to that of control group animals. Less epithelialization and less collagen formation
in saline-treated animals indicated incomplete healing.
Figure 1 Histopathological view of epidermal/dermal remodeling in non-infected wounds. In A, B and C show skin sections stained with hematoxylin and eosin. In D, E and F show skin sections stained with
Masson's trichrome. In G, H and I show immunohistological staining to expression of collagen type I. The original magnification was 100x. Data are representative of 4 animals per group. A, D and G Salinetreated group ( 9-day-old wound tissue treated with only saline) ; B, E and H AEBv group ( 9-day-old wound tissue treated with B. virgilioides extract) ; C, F and I Fibrase-treated group ( 9-day-old wound tissue
treated with Fibrase) . Arrows pointing events during wound healing: RE: reepithelization; C: collagen; PMN: polymorphonuclear cells.
The expression of type I collagen was detected by the immunohistochemistry method ( Figure 1) . In contrast to the group treated with saline, a considerable expression of collagen type I was detected in the tissue after 9 days of
treatment with AEBv ( Figure 1G - 1H) . This increase in collagen type I expression was the most effective in AEBv-treated group than compared to Fibrase-treated group ( Figure 1H - 1I) .
Figure 2 shows the histology of saline, AEBv and Dermazine-treated groups at 9 days of analysis in infected wound. The saline-treated group demonstrated delayed wound healing processes compared to the other groups. The
epidermis in infected wounds was thick and disorganized, especially when compared with the adjacent normal skin. Clumps of degenerating tissue, necrotic changes, and the persistence of inflammatory exudates in the upper
dermis with loss of epidermis were observed in infected wounds on day 9. AEBv and Dermazine-treated mice showed marked epithelialization and moderate amount of connective tissue synthesis. Following histopathological
examination to both infected and non-infected wound, the scored results were combined, summarized and presented in Table III.
Figure 2 Histopathological view of epidermal/dermal remodeling in infected wounds. In A, B and C show skin sections stained with hematoxylin and eosin. In D, E and F show skin sections stained with
Masson's trichrome. In G, H and I show immunohistological staining to expression of collagen type I. The original magnification was 100x. Data are representative of 4 animals per group. A, D and G Salinetreated group ( 9-day-old wound tissue treated with only saline) ; B, E and H AEBv group ( 9-day-old wound tissue treated with B. virgilioides extract) ; C, F and I Dermazine-treated group ( 9-day-old wound
tissue treated with Dermazine) . Arrows pointing events during wound healing: RE: reepithelization; C: collagen; PMN: polymorphonuclear cells.
TABLE III Wound healing processes of aqueous extract of stem barks from Bowdichia virgilioides.
Groups
Wound healing processes
Saline
+/++
+
+
++
RE
FP
CD
PMN
Non-infected wounds
Fibrase
++
+++
+/++
-/+
AEBv
+++
+++
+++
-/+
Saline
-/+
+
-/+
++
Infected wounds
Dermazine
++
+/++
-/+
+
AEBv
+++
++
++/+++
-/+
***
P<0.001,
**
aP<0.001 as compared to Dermazine-treated animals.
The expression of collagen type I was detected in 9 days after injury in infected wounds ( Figure 2) . The AEBv-treated group demonstrated clusters of slight increase in expression of collagen type I compared with the salinetreated group ( Figure 2G - 2H) . Dermazine treatment had less effect on the collagen type I expression in comparison with the AEBv-treated group ( Figure 2H- 2I) .
Antimicrobial Sensitivity Test
Table IV shows the antibacterial activity of aqueous extracts of the stem bark of Bowdichia virgilioides against two bacterial strains, S. aureus and P. aeruginosa. The AEBv showed the highest antibacterial activity against S.
aureus while had no effect against P. aeruginosa. Chloramphenicol, a standard antibiotic, showed a significantly anti-bacterial activity against the test organisms.
TABLE IV Antibacterial activity of the aqueous stem bark extracts of B. virgilioides.
Treatment
Dose ( mg.mL−1)
S. aureus ( ATCC 25923)
Zone of inhibition ( mm)
P. aeruginosa ( ATCC 27853)
1
2
4
1
EABv
Chloramphenicol
15.0 ± 0.0
16.0 ± 0.0
18.0 ± 0.0
20.0 ± 0.0
NA
NA
NA
10.0 ± 0.0
NA, not active. Values represent the mean ± S.E.M.
Due to the high antimicrobial activity of the AEBv against S. aureus ATCC 25923, the antimicrobial activity against S. aureus MRSA was assessed. Results demonstrated that the AEBv ( 4 mg.mL−1) induced an inhibition zone of
18 mm for strain MRSA, value similar to that presented by the standard strain.
DISCUSSION
In the present paper we report the wound healing potential of the aqueous extract of the stem bark of Bowdichia virgilioides, AEBv, applied on infected and non-infected wounds in mice. The extract of Bowdichia virgilioides
Almeida et al. 2006
Silva et al. 2010 Thomazzi et al. 2010 Barros et al. 2010
showed antimicrobial activity ( J.G.R. Feitosa et al., unpublished data,
) , analgesic and anti-inflammatory effects (
,
,
analgesic and anti-inflammatory activities together, it can be supposed that this material also may help to promote wound healing and contribute skin regeneration.
) . So, if any plant material presents antimicrobial,
We observed that the topical application of AEBv enhances cutaneous healing, which appeared completed in 9 days. The histological findings showed that the original tissue regeneration is much greater in skin wounds treated with
the extract than in wounds saline-treated. The wound contraction is mediated by specialized myofibroblasts found in the granulated tissue (
result of the enhanced activity of fibroblasts.
Moulin et al. 2000
) . So, the increase in wound contraction in AEBv-treated mice might be a
Indeed, the response to injury involves the migration and proliferation of cells such as fibroblasts, endothelial and epithelial cells, and deposition of connective tissue and contraction of the wound. Collagen not only confers
strength and integrity to the tissue matrix but also plays an important role in homeostasis and in epithelialization at the later phase of healing (
the deposition of the bands of collagen, a phenomenon that appears to contribute with the increase in wound contraction.
Clark 1996
) . Here, our finding revealed that treatment with AEBv caused an increased in
Collagen type I is the most common protein in animals and provides the tensile strength of healing in wounds. Besides contributing to the skin strength, collagen type I is also important to guide keratinocytes and dermal fibroblasts
migration in the wounded area (
Bennett and Schultz 1993
) . Considering this, our results suggest that topical treatment with AEBv could be beneficial to wounds skin repair in both conditions infected and non-infected.
Skin wound healing starts immediately after injury and consists of three phases: inflammation, proliferation, and maturation. The first response is inflammation, acting as a defense mechanism of the tissue, able to provide a
resistance to the microbial contaminations (
Kondo 2007
) . But, a long duration in the inflammatory phase causes a delay in healing process. Anti-inflammatory activity is necessary for shorten the healing period (
Therefore, the significant wound healing activity of AEBv may be related to its remarkable anti-inflammatory effect as presented in previous reports (
Shimizu et al. 2000
).
Silva et al. 2010 Thomazzi et al. 2010
,
).
Antimicrobial activity is important for the wound healing period, because the wound exposed to external environment is more prone to microbial attacks, which usually results in a delay in the healing process. So, risk factors such
Arora and Kaur 2007
as infections may compromise the repair process. S. aureus and P. aeruginosa are the most common pathogens responsible for infection in skin wounds (
) . Topical applications of drugs are effective both as
microbicide and increasing wound healing rate because of its greater availability at the infected wound site. In this study, the slow rate of wound closure in control mice may be attributable to the presence of microorganisms and
their metabolites, which inhibits wound contraction and impair healing. In this study, even in infected wounds where the period of epithelialization is greater, when the animals were treated with AEBv there was a better wound
healing if compared to animals treated with saline.
In vitro analysis of the antimicrobial effect of AEBv showed a potential inhibitory effect against Gram-positive bacteria S. aureus, but not against Gram-negative bacteria such as P. aeruginosa. In line with this observation,
previous results from J.G.R. Feitosa et al. ( unpublished data) showed that essential oil of seeds from B. virgilioides possess an antimicrobial activity against Gram-positive B. subtilis, B. vulgaris, E. faecalis and S. aureus and had
low activity in vitro against Gram-negative P. aeruginosa, S. enteritidis and E. coli. This antibacterial effectiveness may be attributed to the fact that cell wall in Gram-positive bacteria consists of a single layer, whereas Gramnegative cell wall is a multilayered structure bounded by an outer cell membrane ( Mahomoodally et al. 2010) . Moreover, findings from the present study showed that AEBv was effective against a methicillin-resistant strain of S.
aureus ( MRSA) . These MRSA are difficult to treat because they are also multiresistant and up to now there are no satisfactory antimicrobial drugs (
virgilioides seem to be a potential tool to combat the problem of MRSA.
Joung et al. 2010
) . Therefore, regarding to the present result, extract from B.
The results of our study indicate, for the first time, that B. virgilioides may be a potential candidate for dermal wound healing because of its positive influence on phases of the healing process and particularly effective in view of it
antimicrobial properties. Therefore, there is, the need for further studies into the stability of the extract to ensure an efficacious formulation of products for wound healing.
Acknowledgements
This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico ( CNPq) , the Programa de Cooperação Acadêmica/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
( PROCAD/CAPES) and the Fundação de Amparo à Pesquisa do Estado de Alagoas ( FAPEAL) ( Brazil) .
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Received: September2, , 2011; Accepted: October19, , 2011
Correspondence to Emiliano Barreto E-mail: [email protected]
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided
the original work is properly cited.
Tel: +55 21 3907-8100
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An. Acad. Bras. Ciênc. vol.85 no.3 Rio de Janeiro set. 2013 Epub 28-Ago2013
http://dx.doi.org/10.1590/S0001-37652013005000054
Biological Sciences
Assessment of the cytotoxic, genotoxic, and antigenotoxic activities of
Celtis iguanaea (Jacq.) in mice
FLAVIO F.V. BORGES1, THIAGO C. MACHADO1, KÊNYA S. CUNHA2, KARLA C. PEREIRA2,
ELSON A. COSTA3, JOSÉ R. DE PAULA4, LEE CHEN-CHEN1
1
Departamento de Biologia Geral, Instituto de Ciências Biológicas (ICB), Universidade Federal de
Goiás (UFG), Campus-II, 74001-970 Goiânia, GO, Brasil
2 Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas (ICB),
Universidade Federal de Goiás (UFG), Campus-II, 74001-970 Goiânia, GO, Brasil
3 Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas (ICB), Universidade Federal
de Goiás (UFG), Campus-II, 74001-970 Goiânia, GO, Brasil
4 Departamento de Tecnologia Farmacêutica, Faculdade de Farmácia, Universidade Federal de Goiás
(UFG), Campus-I, 74605-220 Goiânia, GO, Brasil
ABSTRACT
Ethnobotanical surveys of Cerrado native plants show that leaves of Celtis iguanaea (Jacq.) Sargent
(Cannabaceae), popularly known in Brazil as “esporão de galo”, are used in folk medicine for body pain,
asthma, cramps, poor digestion, urinary infection, kidney dysfunctions, as well as a stimulant and diuretic.
This work aimed at evaluating possible C. iguanaea aqueous leaf extract (CALE) cytotoxicity,
genotoxicity, and antigenotoxicity using the mouse bone marrow micronucleous test. To assess CALE
genotoxicity, Swiss mice were orally treated with three different extract concentrations (100, 300, and 500
mgkg−1). To evaluate its antigenotoxicity, the same doses were used simultaneously with a single i.p. dose
of mitomycin C (MMC, 4mg.kg−1). The frequencies of micronucleated polychromatic erythrocytes
(MNPCE) were evaluated 24 h and 48 h after administration except for the negative control (24 h).
Genotoxicity was evaluated using the frequency of micronucleated polychromatic erythrocytes (MNPCE),
whereas cytotoxicity was assessed by the polychromatic and normochromatic erythrocytes ratio (PCE/
NCE). The results showed that CALE did not exhibit a significant reduction in the PCE/NCE ratio, neither
a considerable increase in the frequency of MNPCE. Nonetheless, CALE reduced bone marrow toxicity
(increased PCE/NCE ratio) and decreased the micronuclei frequency induced by MMC. We can conclude
that CALE presented no cytotoxic and genotoxic effects, but showed antigenotoxic and anticytotoxic
actions under the experimental conditions applied in this study.
Keywords: Cytotoxicity; genotoxicity; mice; medicinal plant
RESUMO
Levantamentos etnobotânicos de plantas nativas do cerrado evidenciam que as folhas de Celtis iguanaea
(Jacq.) Sargent (Cannabaceae), popularmente conhecidas como “esporão-de-galo” são usadas na medicina
popular para dores no corpo, asma, cólicas, má-digestão, infecções urinárias, disfunções renais, como
estimulante ou como diurético. O presente trabalho teve como objetivo avaliar as possíveis atividades
citotóxica, genotóxica e antigenotóxica do extrato aquoso das folhas de C. Iguanaea (CALE) através do
teste do micronúcleo em medula óssea de camundongos. Para avaliar a genotoxicidade de CALE, os
camundongos foram tratados por via oral com três diferentes concentrações de extrato (100, 300 e 500 mg.
kg−1). Para a investigação da atividade antigenotóxica, foram utilizadas as mesmas doses
concomitantemente a uma única dose i.p. de Mitomicina C (MMC, 4mg.kg−1). Com exceção do controle
negativo (apenas 24h), todas as outras doses tiveram as frequências de eritrócitos policromáticos
micronucleados (EPCMN) avaliadas em 24 e 48 horas após o tratamento. A Genotoxicidade foi avaliada
por meio da frequência de eritrócitos policromáticos micronucleados (MNPCE), enquanto que a
citotoxicidade foi mensurada pela razão entre eritrócitos policromáticos e normocromáticos (PCE / NCE).
Os resultados mostraram que CALE não apresentou uma redução significativa na razão PCE/NCE, nem um
aumento considerável na frequência de EPCMN. No entanto, CALE reduziu a toxicidade na medula óssea
(aumento na razão PCE/NCE) e diminuiu a frequência de micronúcleos induzidos pela MMC. Nos
resultados obtidos, CALE não apresentou efeitos citotóxicos e genotóxicos, contudo apresentou ações
antigenotóxicas e anticitotóxicas nas condições experimentais aplicadas neste estudo.
Palavras-Chave: Citotoxicidade; genotoxicidade; camundongos; planta medicinal
INTRODUCTION
The use of plant products for the treatment, cure, and prevention of general disorders is one of the
Halberstein 2005
earliest forms of medical practice, and probably almost as old as the human species (
). It is
estimated that natural products and herbal preparations are responsible for 25% of the medical
prescriptions in developed countries and about 80% in developing countries. The number of bioactive
plant compounds exceeds 100,000, but the actual number must be much higher because only 20 - 30%
Wink 2009
of plants have been investigated so far (
). Also, a significant number of synthetic drugs was
obtained from natural precursors (Raskin et al. 2002,
Rates 2001
).
The Brazilian Cerrado is the richest savanna formation in the world, responsible for about 5% of global
Myers et al. 2000
biodiversity, and considered one of the world's hotspots. (
). It has an endemic level of 44%
for vascular plants and 70% for herbaceous plants, representing a valuable spectrum of organic
Machado et al. 2008
molecules with medical and therapeutic potential (
).
Safety and toxicity studies of plants used in therapy are vital, due to its considerable range of
applications and its widespread use in folk medicine, which, according to
the only therapy of many communities and ethnical groups.
Maciel et al. 2002
, often represent
In the Brazilian Cerrado traditional medicine, the leaf infusion of Celtis iguanaea (Jacq.) Sargent,
Souza and Lorenzi 2005
popularly known as “esporão-de-galo” (
), is used in the treatment of body pains,
asthma, colic, poor digestion, urinary infection, kidney dysfunctions, as well as a stimulant and diuretic
Carneiro 2009 Silva and Proença 2008 Piliackas et al. 2001
(
,
,
). According to ethnobotanical surveys of the Cerrado
Silva and Proença 2008
native plants, this species showed a considerable consensus of popular usage (
),
which may indicate potential medical properties and strengthen the need of proper pharmacological and
Vendruscolo and Mentz 2006
toxicological studies (
). However, despite the wide use of this plant, a rather
scarce literature is dedicated to the species and little information regarding the biological effects of its
constituents can be found, especially when it comes to their potential toxicological properties.
Several studies on phytotherapic substances have already reported that many medicinal plant compounds
present undesirable properties, such as mutagenicity, carcinogenicity and toxicity, limiting their use as
therapeutic agents (
Déciga-Campos et al. 2007 Marques et al. 2003
,
). On the other hand, a number of studies have
Aruoma
also shown that phytotherapic compounds may possess antigenotoxic/anticarcinogenic effects (
2003 Gupta et al. 2001 Waters et al. 1996
,
,
). Therefore, the investigation of traditionally used medicinal plants is
valuable both as a source of potential chemotherapeutic drugs, and as a measure of safety for the
Verschaeve et al. 2004
continuous use by the population (
).
Short-term assays have been used for more than 30 years to identify chemical, physical, and biological
genotoxic agents, as well as to assess their carcinogenic potential. Although the genetic toxicity is not a
direct measure of carcinogenicity, it is often used as an indicator for cancer, since genotoxicity tests
Fearon and Volgelstein 1990
measure an initial or intermediary event in tumorigenesis (
).
Among the methods for in vivo genotoxicity investigation, the micronucleus test has been widely
Mateuca et al. 2006 Choy 2001
accepted by regulatory agencies and governmental institutions (
,
). This assay
Schmid 1975
was initially developed in mouse bone marrow erythrocytes (
assess the genotoxic potential of physical and chemical agents (
). Since then, it has been used to
Ding et al. 2003 Chung et al. 2002
,
),
Bolognesi et al. 2004 Majer et al. 2001
biomonitor human populations occupationally exposed to mutagens (
in the search for carcinogenesis inhibiting compounds (
ecotoxicological studies (
,
),
Roy et al. 2003 Izzotti et al. 2001
,
), and in
Llorente et al. 2002 Gauthier et al. 1999
,
).
The micronucleus test detects genetic alterations arising from chromosomal damage and/or damage to
the mitotic apparatus caused by clastogenic or aneugenic agents, respectively. As micronuclei (MN) are
Azevedo et al. 2003
indicative of irreversible DNA loss, their frequency may be used as a mutation index (
It is already known that there is a positive correlation between increased frequency of MN and the
appearance of tumors in rodents and humans (Clare et al. 2006,
).
Azevedo et al. 2003
).
Thus, considering the widespread use of this plant by the Brazilian population, the present work aimed
at evaluating the cytotoxic, genotoxic, and antigenotoxic activities of Celtis iguanaea aqueous leaf
extract (CALE) using the in vivo mouse bone marrow micronucleus test.
Plant Material: Celtis iguanaea Extract
Celtis iguanaea (Jacq.) Sargent (“esporão de galo”) leaves were collected in a riparian forest located in
the municipality of Campestre (16°45′44&″ S; 49°41′40&″ W; altitude = 651 m), in the state of Goiás,
Midwestern Region of Brazil. The botanical material was identified and a voucher specimen deposited
in the Herbarium of the Universidade Federal de Goiás, Goiânia, GO, under the number 40110/UFG.
Paula, 2009
The plant material was prepared according to
: The leaves were dried in an oven at 40°C with
forced ventilation and then ground into a powder. CALE was obtained by infusion of the powder at 3%
at 80°C for 30 min, with agitation every 10 min. After vacuum filtration, the filtrate was concentrated
under reduced pressure at 45°C. The yield of the extract was determined by the dry weight method
(20%), and the final concentration was 60 mg.mL−1. At this concentration, CALE showed pH of 7.22,
was odorless, had a greenish aspect and presented mild viscosity, probably due to the presence of
mucilage. CALE solutions were always prepared with distilled water (extract concentrations of 100,
300, and 500 mg.kg−1) immediately before their use in the experiments.
Animals
This study was approved by the Human and Animal Research Ethics Commitee of the Universidade
Federal de Goiás (CEPMHA/HC/UFG n° 014/09). Healthy young male adults (8–12 weeks) outbred
mice (Mus musculus, Swiss Webster), weighing 30–40 g, obtained from the Central Animal Facility of
Universidade Federal de Goiás (Goiás, Brazil) were randomly allocated to treated groups. All animals
were brought to the laboratory 7 days before the experiments and housed in polyethylene cages (40 cm x
30 cm x 16 cm), in groups of five animals, lined with wood shavings, in air-conditioned rooms at 25 ± 2°
C and 50 ± 10% relative humidity, with a 12-h light/dark natural cycle. Food (appropriate commercial
rodent diet Labina, Ecibra Ltda.) and water were given ad libitum.
Experimental Procedure
To evaluate the genotoxicity of the extract, five animal groups were orally treated with three different
doses (100, 300, and 500 mg.kg−1 body weight) of CALE. A positive control group (4 mg.kg−1 i.p.
mitomycin C, C15H18N4O5, MMC, Bristol-Myers Squibb) and another negative (sterile distilled water)
control group were included. In order to assess antigenotoxicity, the same CALE doses were
administered simultaneously with a single i.p. dose of MMC (4 mg.kg−1). All treatments were evaluated
24 h and 48 h after administration except for the negative control (24 h). The animals were euthanized
by cervical dislocation, femurs were dissected, opened, and the bone marrow was gently flushed out
using fetal calf serum (Soralli). After homogenization of the bone marrow in serum, it was centrifuged at
1,000 rpm for 5 min. The bone marrow cells were smeared on glass slides, coded for blind analysis, airdried, and fixed with absolute methanol (CH4O, LabSynth) for 5 min. The smears were stained with
Giemsa (Doles), dibasic sodium phosphate (Na2HPO412H2O, Sigma-Aldrich Chemical Co.), and
monobasic sodium phosphate (NaH2PO4H2O, Sigma-Aldrich Chemical Co.) to detect micronucleated
polychromatic erythrocytes (MNPCE). For each mouse, three slides were prepared and a minimum of
2,000 polychromatic erythrocytes (PCE) were counted to determine the frequency of MNPCE. To
evaluate CIE cytotoxicity, 1,000 normocromatic erythrocytes (NCE) were counted, as well as the
frequency of polycromatic erythrocytes (PCE) within the same microscope fields, and the PCE/NCE
ratio was then calculated to measure bone marrow toxicity. The slides were analyzed by microscopy
(Olympus BH-2 10x100). The micronucleus test and MNPCE scoring were carried out according to
Schmid (1975)
.
To evaluate the genotoxic activity of CALE, the frequency of MNPCE in the treated groups was
compared to the results of the negative control group (genotoxicity assessment) or to the results from the
positive control group (antigenotoxicity evaluation), using one-way analysis of variance (ANOVA),
followed by the multiple comparison test (Tukey). P values lower than 0.05 (p < 0.05) were considered
indicative of statistical significance. In order to assess CALE cytotoxicity, the polychromatic/
normochromatic erythrocytes ratio (PCE/NCE) of all treated groups was compared to the result of the
negative control group (cytotoxicity assessment) or to the result of the positive control group
(anticytotoxicity evaluation), using qui-square test (χ 2). A value of p < 0.05 was taken as the criterion of
statistical significance.
RESULTS
Table I summarizes the frequencies of MNPCE and PCE/NCE ratio in mouse bone marrow cells
treated with CALE.
TABLE I MNPCE frequencies and PCE/NCE ratio in mouse bone marrow cells treated with
Celtis iguanaea aqueous extract (CALE) at different doses and times.
Treatment (mg.kg−1)
Time (h)
Negative control 1
24
24
48
Positive control 2
MN/2000 PCE
Individual data
Mean ± SD4
5 4 6 4 4 4.60 ± 0.89a
36 29 34 31 33 32.60 ± 2.70b
14 12 11 11 15 12.60 ± 1.81c
CALE 3
PCE/NCE ratio 4
1.13 ± 0.10d
0.51 ± 0.04e
0.39 ± 0.03f
100 mg.kg−1
24
4
7
3
4
5
4.60 ± 1.51a
1.22 ± 0.08d
300 mg.kg−1
24
5
5
6
4
5
5.00 ± 0.70a
1.08 ± 0.04d
500 mg.kg−1
24
7
4
4
6
6
5.40 ± 1.34a
1.18 ± 0.07d
100 mg.kg−1
48
3
6
4
5
6
4.80 ± 1.30a
1.13 ± 0.05d
300 mg.kg−1
48
6
7
4
7
3
5.40 ± 1.81a
1.16 ± 0.07d
500 mg.kg−1
48
5
3
4
5
7
4.80 ± 1.48a
1.27 ± 0.15d
1
Sterile distilled water.
2
Mitomycin C (4 mg.kg−1).
3
CALE doses are compared to their respective positive controls at the same exposure times.
4
Same symbols in the same column – p > 0.05; different symbols in the same column – p < 0.05.
The results obtained showed no significant increase in MNPCE frequency either 24 h (4.6, 5.0, 5.4) or
48 h (4.8, 5.4, 4.8) after the administration of CALE at any tested dose (100, 300, 500 mg.kg−1) when
compared to the negative control (p > 0.05). There was significant increase of MNPCE frequency in the
positive control group compared to the negative control group (p < 0.05). This result was already
Kang et al. 2006
expected, since MMC is described as a highly genotoxic and mutagenic agent (
).
Regarding cytotoxicity, no significant decrease in the PCE/NCE ratio was observed when comparing
mice treated with CALE with the negative control group for all tested doses and different times of
evaluation (p > 0.05). As expected, the PCE/NCE value of MMC was much lower compared to the
Estrem and Vanleeuwen 2000
negative control or CALE doses, confirming its well-known cytotoxic activity (
Kraut and Drnovsek-Olup 1996
,
).
Table II summarizes the frequency of MNPCE and PCE/NCE ratio in mice bone marrow cells treated
simultaneously with different doses of CALE and 4.0 mg.kg−1 of MMC.
TABLE II MNPCE frequencies and PCE/NCE ratio after simultaneous treatment with Celtis
iguanaea extract (CALE) and mitomycin C (MMC).
Treatment (mg.kg−1)
Time (h)
Negative control 1
24
24
48
Positive control 2
MN/2000 PCE
Individual data
Mean ± SD4
5 4 6 4 4 4.60 ± 0.89a
36 29 34 31 33 32.60 ± 2.70b
14 12 11 11 15 12.60 ± 1.81c
CALE 3
PCE/NCE ratio 4
1.13 ± 0.10d
0.51 ± 0.04b
0.39 ± 0.03c
100 mg.kg−1 + MMC
24
24 26 27 21 22
24.0 ± 2.54d
0.64 ± 0.07a
300 mg.kg−1 + MMC
24
21 21 25 24 20
22.2 ± 2.16d
0.71 ± 0.09a
500 mg.kg−1 + MMC
24
23 20 19 20 18
20.0 ± 1.87d
0.69 ± 0.06a
100 mg.kg−1 + MMC
48
10 8
9 11 10
9.60 ± 1.14e
0.58 ± 0.07f
300 mg.kg−1 + MMC
48
9
8
8
9
8
8.40 ± 0.54e
0.55 ± 0.06f
500 mg.kg−1 + MMC
48
8
7 10 7
9
8.20 ± 1.30e
0.62 ± 0.04f
1
Sterile distilled water.
2
Mitomycin C (4 mg.kg−1).
3
CALE doses are compared to their respective positive controls at the same exposure times.
4
Same symbols in the same column – p > 0.05; different symbols in the same column – p < 0.05.
The results of the antigenotoxicity evaluation showed that CALE significantly decreased MNPCE
frequency either 24 h (24.0, 22.2, 20.0) or 48 h (9.60, 8.40, 8.20) after the administration of any of the
three tested doses (100, 300, 500 mg.kg−1 g co-treated with MMC) compared with the positive control
(p < 0.05).
In relation to the anticytotoxic assessment of CALE, we observed an attenuation of the cytotoxic action
provoked by MMC at all tested doses, either 24 h or 48 h after exposure when compared with the
respective positive control (p < 0.05).
DISCUSSION
In the present study, we aimed to evaluate the cytotoxic, genotoxic, and antigenotoxic activities of
CALE using the mouse bone marrow micronucleus test. This short-term assay is recommended by
regulatory agencies all over the world as the first in vivo test to be conducted in chemical safety analyses
(
Khrishna and Hayashi 2000
). It has proven to be a reliable method to investigate the genotoxic (clastogenic
Hayashi et al. 1990 Schmid 1976
and/or aneugenic) effects of chemical and physical agents (
,
).
Fenech 2000
MN appear in erythrocytes due to damage induced in parental cells (
). These small masses of
chromatin are originated from acentric fragments or lagging chromosomes that fail to incorporate into
one of the daughter nuclei during telophase of the mitotic cells. MN frequency in polychromatic
Suzuki et al. 2008
erythrocytes (PCE) of mouse bone marrow is a very sensitive index of damage (
), being
induced by oxidative stress, exposure to clastogenic or aneugenic agents, genetic defects in cell cycle
checkpoints, and/or DNA repair genes, and also by the deficiency of nutrients required as co-factors in
DNA metabolism and chromosome segregation machinery (Bonassi et al. 2007). All of these events that
cause MN formation are associated with the chromosomal instability commonly observed in cancer
(
Rajagopalan et al. 2004 Fenech 2002
,
).
The results of the present work demonstrate that CALE did not provoke a significant increase in
MNPCE frequency when compared with the negative control at all tested doses and times of exposure,
indicating that this extract did not exhibit genotoxic effects in PCE of mouse bone marrow.
The micronucleus assay also detects cytotoxic effects by the PCE/NCE ratio. When normal proliferation
of bone marrow cells is affected by a toxic agent, there is a decrease in the number of immature
erythrocytes (PCE) in relation to the number of mature erythrocytes (NCE), reflecting bone marrow
toxicity and cell depression (
Shahrim et al. 2006
).
Our results showed no significant reduction of the PCE/NCE ratio at any CALE doses and times of
exposure compared with the negative control. Therefore, these results indicate that CALE did not
present cytotoxic action.
The results of the antigenotoxicity evaluation (CALE + MMC) showed that CALE significantly reduced
the frequency of MMC-induced MNPCE at all concentrations tested and times of exposure, attenuating
the genotoxic activity of the alkylating agent.
In relation to the anticytotoxic activity of CIE, we observed an attenuation of MMC cytotoxic action at
all CALE doses and times of exposure tested.
The phytochemical analysis of Celtis iguanaea leaves revealed the presence of coumarins, mucilage, and
Paula 2009
flavonoids (
).
Coumarins present a wide variety of bioactivities, including anti-inflammatory, anticoagulant,
antimicrobial, vasodilating, anthelmintic, sedative, hypnotic, analgesic, hypothermic, and antitumorGarcía-Argáez et al. 2000 Fujioka et al. 1999 O'Kennedy and Thornes 1997 Mizuno et al. 1994
promoting activity (
,
,
,
). The
majority of tests for assessing mutagenic and genotoxic potential suggest that coumarins are not
genotoxic agents, and exposure to coumarins from food, medicines, and/or cosmetic products poses no
Lake 1999
health risks to humans (
). However, possible phototoxic effects of coumarin furanoderivatives
Edwards et al. 1994
should be kept in mind (
).
Mucilage is used in medicine mainly as an emollient and a demulcent, but some mucilaginous plants
have other applications, such as Aloe vera gel, which has been used since ancient times to treat burns
Choy
and other wounds due to its ability to enhance the healing rate and to reduce the risk of infection (
and Chung 2003 Capasso et al. 1998
,
).
Flavonoids have been recognized to possess anti-inflammatory, analgesic, antiallergic, hepatoprotective,
Havsteen 2002 Hodek et al. 2002
anti-bacterial, antiviral, and anticarcinogenic activities (
,
). Although the
mechanisms of flavonoid protection against DNA damage and potential protection against
carcinogenesis are largely unknown, it is suggested that they may act as antioxidant, free radical
scavengers, inhibitors of tumor cell growth, inducers of apoptosis, modulators of DNA repair, or
carcinogen inactivators (
Lee et al. 2003 Duthie and Dobson 1999
,
).
Antioxidant agents are broadly known to significantly reduce cytotoxicity and genotoxicity of
Borek 2005 Halliwell 2002 Marnett 2000
compounds that generate free radicals (
,
,
). It is also known that the
hazardous effect of MMC is related to its ability to alkylate DNA and produce reactive free radicals,
Kang et al. 2006
causing different types of cellular damage, including DNA breaks (
). Consequently, the
antigenotoxic and anticytotoxic activities of CALE detected in our experiments can be associated, at
least partially, to the presence of flavonoids exerting protective effects by scavenging reactive oxygen,
reducing alkylation and/or other antioxidant mechanisms. However, the complexity of plant extracts
should not be overlooked, as the final response of a treatment using them is likely to be the result of
synergistic, antagonistic, and other interactive effects among their biologically active components.
Concluding, our results in the present work indicate that CALE did not exhibit genotoxic or cytotoxic
effects in mouse bone marrow micronucleus test. Nonetheless, this plant extract showed antigenotoxic
and anticytotoxic effects under the experimental conditions tested.
Acknowledgements
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Apoio à
Pesquisa (FUNAPE – UFG) and Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG).
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Author Disclosure Statement
No competing financial interests exist.
Received: September2, , 2011; Accepted: October28, , 2011
Correspondence to: Lee Chen-Chen E-mail: [email protected]
This is an Open Access article distributed under the terms of the Creative Commons Attribution NonCommercial License, which permits unrestricted non-commercial use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Tel: +55 21 3907-8100
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[email protected]
http://dx.doi.org/10.1590/S0001-37652013000300009
Biological Sciences
Expression of manganese peroxidase by Lentinula edodes and Lentinula boryana in solid state and submerged system fermentation
KATIA L. HERMANN1, ALESSANDRA COSTA2, CRISTIANE V. HELM3, EDSON A. DE LIMA3, LORENA B.B. TAVARES2
1
Pós-graduação em Engenharia Ambiental, Universidade Regional de Blumenau/FURB, Rua São Paulo, 3250, 89030-000 Blumenau, SC, Brasil
Laboratório de Engenharia Bioquímica, Departamento de Engenharia Química, Universidade Regional de Blumenau/FURB, Rua São Paulo, 3250, 89030-000 Blumenau, SC, Brasil
3 Pesquisador da Embrapa Florestas. Estrada da Ribeira, Km 111, Caixa Postal 319, 83411-000 Colombo, PR, Brasil
2
ABSTRACT
The production of ethanol from lignocellulosic biomass is referred as a second generation biofuel, whose processing is one of the most promising technologies under development. There are few available studies on the use of enzymes produced by fungi as active for the
biodegradation of lignocellulosic biomass. However, the manganese peroxidase (MnP) enzyme presents high potential to degrade lignin and the basidiomycetes are the major producers of this oxidase. Thus, this study aimed at evaluating the ability of fungi Lentinula edodes
and Lentinula boryana to produce this enzyme when cultivated in submerged fermentation system (SS) and also in solid-state fermentation system (SSF) containing Eucalyptus benthamii sawdust with or without corn cob meal. In the SS the greatest MnP expression occurred
on the 25th day, being of 70 UI.L–1 for L. boryana and of 20 UI.L–1 for L. edodes. In the SSF, the best results were obtained on the 10th day for L. edodes, while for L. boryana it happened between the 20th and the 25th days, despite both species presented values close to 110
UI.L–1. Therefore, the results indicated that the studied fungi express the enzyme of interest and that its production is enhanced when cultivated in solid system.
Keywords: basidiomycetes; Eucalyptus benthamii; enzyme; ethanol
RESUMO
A produção de etanol a partir de biomassa lignocelulósica é referida como uma segunda geração de biocombustíveis, sendo que este processo é uma das mais promissoras tecnologias em fase de desenvolvimento. Poucas são as pesquisas disponíveis sobre o uso de enzimas
produzidas por fungos como ativos para biodegradação de biomassa lignocelulósica. No entanto, a enzima manganês peroxidase (MnP) apresenta alto potencial para degradar a lignina e os fungos basidiomicetos são os principais produtores desta oxidase. Portanto, esse estudo
teve por objetivo avaliar a capacidade dos fungos Lentinula edodes e Lentinula boryana em produzir essa enzima quando cultivados em fermentação em sistema submerso (FSS) e também em fermentação em sistema sólido (FES) contendo serragem de Eucalyptus benthamii
suplementada ou não com farelo de sabugo de milho. Na FSS, a maior expressão de MnP ocorreu no 25° dia de cultivo, sendo 70 UI.L–1 para L. boryana e 20 UI.L–1 para L. edodes. Na FES, os melhores resultados foram obtidos no 10° dia para L. edodes, enquanto que para L.
boryana foram entre o 20° e o 25° dia, apesar de ambas as espécies terem apresentado valores próximos a 110 UI.L–1. Portanto, os resultados indicaram que os fungos estudados expressam a enzima de interesse e que a sua produção é otimizada quando cultivados em sistema
sólido.
Palavras-Chave: basidiomicetos; Eucalyptus benthamii; enzima; etanol
INTRODUCTION
The increase in ecological awareness starting in the late 20th century made it clear that the great challenge facing humanity for the coming decades is balancing the production of goods and services, also economic growth, social equity and environmental sustainability.
Among the issues that have been studied lately, lignocellulosic biomass has received increasing attention from the Brazilian government and researchers who have dedicated time and thought to study the transformation of this material, often an environmental problem,
into a value-added product, such as the second generation ethanol, also known as cellulosic biomass ethanol or bioethanol.
The lignocellulosic material from wood biomass has been viewed as a promising energy source due to its renewable capacity and the amout of carbohydrates (
Sun and Cheng 2002
hydrolysis of pretreated biomass has as its main technical and economic barriers the difficulty to establish an efficient and appropriate pretreatment of raw material (
). However, the use of lignocellulosic biomass to produce ethanol from the enzymatic
McMillan 1994
).
The basidiomycetes are highlighted in the degradation of lignocellulosic materials due to the fact that they produce enzymes that hydrolyze cellulose, such as: avicelase, carboxymethyl-cellulase and glucosidase (cellulase), which act on the cellulosic portion;
xylanases, mannanases, glucanases and galactanases (hemicellulases), acting on the hemicellulosic portion and oxidative enzymes, as lignin peroxidase, manganese peroxidase and laccase, defined as phenoloxidases. They have the ability of acting on phenolic
compounds as lignin (
peroxidase (MnP).
Coelho 2007
). These enzymes degrade lignocellulosic materials to produce fermentable sugars for biofuels as an example, the second generation ethanol, making it possible through the bioconversion carried out by enzymes like manganese
MnP is one of the lignolytic enzymes expressed by white rot basidiomycetes and has been widely studied as active in the decolorization of synthetic dyes used in textile industries (
of biodegradation of lignin, polycyclic aromatic hydrocarbons, humic acids and chlorinated pollutants (
Coelho 2007 Hamid and Rehmana 2009
,
Palma 2003
,
,
). Furthermore, it has been used in studies
). Therefore, the remarkable degradation potential of MnP makes it an attractive enzyme for biotechnological applications. The
production of commercial enzymes is usually performed by biological processes in liquid medium - submerged fermentation system (SS) - in mixed reactors (
solid state fermentation (SSF) promote higher enzymatic activity, despite the difficult control performance (
Boer et al. 2004 Mohorcic et al. 2006 Park et al. 2007
Dalsenter and Tavares 1999
). However, several studies have shown that the biological processes carried out in
).
Throughout the production of enzymes by basidiomycetes, the SSF system seems quite appropriate given the characteristics of the substrate involved in the process, which consists essentially of lignocellulosic material (
better process conditions, the addition of lignocellulosic supplements such as cob corn meal has been proposed to stimulate the expression of lignolytic enzymes, in particular MnP (
suitable for the development of basidiomycetes are nutritionally adequate for mycelial growth (Valadares Filho et al. 2002).
Boer et al. 2004
Wisniewski et al. 2010
). Thus, as the SS presents
). Therefore, the corn cob and the presence of lignocellulosic material
Accordingly, this study aims to evaluate the expression of MnP by the basidiomycetes Lentinula edodes and Lentinula boryana in SSF, cultivated in lignocellulosic biomass of Eucalyptus benthamii, and in SS, seeking knowledge on the differences between the two
systems in order to define the best conditions for enzyme express.
Fungi and Cultivation Conditions
L. edodes (CNPF 21) and L. boryana (CNPF 24) were both maintained on Potato Dextrose Agar medium (PDA - prepared according to manufacturer's instructions) at 4 °C (called as primary matrix). For the production of secondary matrix, 7 mm plugs of the primary
matrix were placed separately in Petri dishes containing PDA medium. These plates were incubated in a B.O.D chamber, at 20 °C for L. boryana (
of the fungus on the plate. After that, the matrix was used as inoculum for SS and SSF.
For SS, a modified SOCAREAN medium was prepared (
Couri and Farias 1995
Faria et al. 2007
) and at 25 °C for L. edodes (
Regina and Broetto 2005
), in the absence of light, until the complete outspreading
), containing 3.0 g.L–1 of sodium nitrate p.a. (NaNO3), 0.5 g.L–1 of magnesium sulphate p.a. (MgSO4), 0.5 g.L–1 of potassium chloride p.a. (KCl), 0.01 g.L–1 of ferrous sulfate heptahydrate p.a.
(FeSO4.7H2O), 1.0 g.L–1 of dibasic potassium phosphate p.a. (K2HPO4) and 12.0 g.L–1 of corn cob meal (obtained from Embrapa Florestas). The medium (50 mL) was placed into 125 mL Erlenmeyer flasks and sterilized in an autoclave at 121 °C for 15 min. The
flasks were inoculated with 6 plugs of the secondary matrix and incubated for 30 days in a B.O.D chamber at 20 °C for L. boryana and at 25 °C for L. edodes, in the absence of light. Four flasks were randomly taken every five days for kinetic analysis of cellular
growth, glucose residual value, pH and MnP activity.
For SSF, two treatments (culture medium) were used and both were prepared with sawdust (≤ 3 mm) of Eucalyptus benthamii (obtained from Embrapa Florestas). T1 treatment consisted of 95% sawdust and 5% soybean meal. T2 treatment consisted of 75% sawdust,
5% soybean meal, 10% cassava bagasse and 10% corn cob meal. Forty grams (40 g) of the treatments were transferred to 500 mL flasks and 50 mL of distilled water were added to each of them. The flasks were sterilized in an autoclave at 121 °C for 1 hour. The two
media were inoculated with 5 plugs of the secondary matrix and incubated for 30 days in a B.O.D chamber at 20 °C for L. boryana and at 25 °C for L. edodes, in the absence of light. Three flasks were randomly taken every five days for kinetic analysis of the moisture
content, aw, pH and MnP activity.
Kinetic Analysis of Submerged System
The fungi mycelium was separated from the culture medium through vacuum filtration and, in order to obtain the enzymatic extract, the medium was centrifuged (Hermle refrigerated centrifuge) at 2,990.65 × g and 4 °C during 15 min. The supernatant (extract) was
collected for MnP quantification.
To estimate the biomass, the gravimetric method of dry weight was used. The mycelium and corn cob meal retained on the filter paper, after vacuum filtration, were placed in a laboratory oven at 70 °C until constant weight was achieved. Mycelium mass was
calculated by the difference between total mass and corn cob meal mass added to the flask. Therefore, cell concentration (Cc) was calculated as the ratio between mycelium mass and the medium's volume.
The average growth rate (Gr) was calculated as the ratio between the variation of cell concentration and cultivation time. Maximum specific growth rate (µmax ) was determined using a linear regression of natural logarithm of cell concentration against cultivation time.
Pirt 1967
The slope obtained during logarithm phase represented µmax (
).
The residual glucose concentration in the culture medium was determined by spectrophotometry (in Shimadzu UV-1650 spectrophotometer) using the enzymatic method of glucose oxidase (GOD), performed as described by the manufacturer of the enzymatic glucose
test kit (Doles). The pH was directly measured in all samples using the potentiometric method (Tecnal pH meter).
Kinetic Analysis of Solid State Fermentation
Humidity (H) was determined by drying the substrate samples in a laboratory oven at 70 °C until reaching constant weight. The difference between wet mass (mW ) and dry mass (mD ) was calculated as shown in Equation 1.
(1)
Water activity (aw = Pm / Ps) of the medium depends on its moisture content and composition as it concerns the ratio between the vapor pressure of the culture medium (Pm) and the vapor pressure of pure water (Ps) at the same temperature (
sample of the SSF, aw was determined by direct measurement of substrate samples in an
Aqualab®
Wenqing et al. 2003
). In each
device (Decagon).
To obtain the enzymatic extract from the solid medium, it was placed on Erlenmeyer flasks, in a ratio of 6 mL of distilled water/gram of substrate, and shaken for 3 hours at 20 °C. After that, the mixture was vacuum filtered and the liquid portion was centrifuged at
2,990.65 × g at 4 °C during 15 min. The supernatant (extract) was collected for MnP quantification and pH direct measurement.
Quantification of MNP in the Extracts
The MnP activity in the extracts obtained from SS and SSF was determined through spectrophotometry, using the modified method of
Wariishi et. al (1992)
. A reaction mixture containing 0.4 mL of extract, 0.4 mL of maganese sulfate p.a. (MnSO4) 10 mM, 2.8 mL of
sodium malonate buffer 50 mM, pH 4.5, and 0.4 mL of hydrogen peroxide p.a. (H2O2) 0.5 mM was prepared. The oxidation of MnSO4 by H2O2 results in the formation of the complex Mn(III)-malonate which was monitored at 30 °C, during 5 min, at 270 nm
(ε270=11,590 M-1.cm-1).
The enzymatic activity (EA) expressed in International Unit per liter (UI.L–1), defined as the amount of enzyme required to oxidize 1 µM of substrate per minute per liter of extract was calculated by:
(2)
where ∆A/t is the graph slope of absorbance variation (∆A) over reaction time (t), d is the path length, ε is the molar extinction coefficient, VR is the total reaction volume and VE is the extract volume.
All analyses were performed in triplicate. The results of biomass production, MnP activity, pH, moisture and aw were examined by analysis of variance (ANOVA). Means with p-values under 0.05 were considered statistically different, and, in these cases the Tukey's
test was also applied.
Fermentation in Submerged System
Biomass production (expressed by cell concentration) and MnP activity analyzed during the SS of L. edodes and L. boryana are shown in Figure 1. It is possible to notice that there were significant differences between the cell concentration means of the two species
only on the 5th and 25th day of cultivation, indicating that they grow in a similar way, although the Gr of L. edodes (0.14 g.L–1.day-1) was greater than that of L. boryana (0.08 g.L–1.day–1).
Fig. 1 Cell concentration and MnP activity during the SS of L. boryana (LB) and L. edodes (LE). (The vertical bars represent standard deviation).
Due to the fact that the cultivation was performed in multiple flasks for sample collection, the occurrence of oscillations during growth is acceptable, such as the peak cell concentration of L. edodes on the 15th day followed by a decay of biomass, which is not usual in
reality. For this reason it was not possible to precisely define the stages of fungal growth. The use of natural logarithm made possible to verify the absence of a lag phase. To calculate the µmax, the L. boryana logarithmic's phase was defined between 0 and 10 days
(reaching µmax of 0.05.day–1) and also L. edodes' between 0 and 5 days ( µmax of 0.10.day–1) (Figure 2). These values confirm what was observed visually during the experiment, that L. edodes presented higher mycelial density in a shorter cultivation time.
Fig. 2 Natural logarithm of cell concentration against cultivation time to obtain the maximum.
After the period of maximum growth the system got into a phase of reduction in the rate of biomass production (characterized as stationary phase), in which secondary metabolism is activated, resulting in the production of bioactive compounds such as phenol
Ferreira et al. (2010)
oxidative enzymes.
states that nutritional stress may be one of the responsible factors for MnP synthesis, explaining why this enzyme expression was observed only after the 15th day (Figure 1). Maximum levels of enzyme activity were obtained at 25
days of cultivation for both species. For L. boryana this value was much higher (70 UI.L–1). The values obtained at 30 days for both species. The one obtained at 25 days for L. edodes showed no statistical difference.
Regina and Broetto (2005)
MnP activity expressed by L. edodes in this study was lower than the one found in the literature for the same fungus.
obtained maximum values of MnP activity around 1,400 UI.L–1 for L. edodes grown in liquid media composed by an infusion of
cassava bagasse and dextrose. However, these authors also observed the influence of substrate on enzyme expression, as in an infusion of sugar cane bagasse and dextrose, the maximum value obtained was of 400 UI.L–1.
The SS medium used in this study contained corn cob meal which has more than 60% of polysaccharides with high concentration of xylan, followed by the monosaccharides xylose and glucose (R.F.M. Silveira, unpublished data). However, the presence of residual
glucose during the cultivations was not detected, showing that this lignocellulosic substrate may not have been used by the fungi during its development. Another hypothesis is that the low enzymatic activity promoted a reduced biodegradation, influencing the low
production of sugars for cellular metabolism and thus influencing global cellular metabolism.
The medium presented an initial pH near 5.0 (Table I), but a statistically significant decrease in this value after the beginning of both species of Lentinula's growth was observed.
TABLE I Values of pH obtained during SS for L. edodes and L. boryana during cultivation time.
Fungi
L. boryana
L. edodes
0
5.06±0.13Aa
5.06±0.013Aa
5
4.57±0.13Ab
4.39±0.20Ab
10
4.65±0.08Ab
4.56±0.11Ab
Cultivation time (days)
15
4.56±0.07Ab
4.23±0.16Ab
20
4.26±0.05Ac
3.77±0.04Bc
25
4.10±0.07Ac
3.59±0.16 Bc
30
4.50±0.01Ab
4.16±0.05Bb
Means ± standard deviation followed by the same lowercase letters in the line and by the same uppercase letters in the row do not differ by Tukey's test (p ≤ 0.05).
The assays for quantification of MnP use buffer with pH 4.5, and then it seems that values of pH near this one help in the enzyme stability. Although pH has fluctuated throughout the growth of fungi, it has remained between 3.6 and 4.6. It was noted that for both
species, the remarkable lower values occurred at 20, and especially at 25 days; periods at which a greater expression of the enzyme was observed. It indicates that the biosynthesis of metabolites of this species could significantly change the pH in the case of SS, a fact
that should be better studied.
Analyzing the obtained data, it seems that SS presents suitable characteristics for MnP production and it is feasible to study its application in the development of second generation ethanol. However, considering what was observed in this study, the time required for
the occurrence of enzyme expression in this system becomes a barrier for its application in an industrial process.
Solid State Fermentation
Values of aw, pH and moisture concerning SSF are presented in Table II. Despite the samplings being performed in periods of 5 days, the values mentioned in Table II only correspond to the beginning, middle and end of cultivation time. Unlike submerged culture, in
SSF there was no statistical difference in the values of pH throughout the cultivation of both species neither between the species for the same day.
TABLE II Values of pH, moisture content (%) and aw obtained during SSF.
Treatment
Fungi
T1
T2
pH
L. boryana
L. edodes
0 day
4.64 ± 0.05Aa
4.64 ± 0.05Aa
15 days
4.54 ± 0.04Aa
4.57 ± 0.09Aa
L. boryana
L. edodes
53.01 ± 0.67Aa
53.01 ± 0.67Aa
52.95 ± 3.10Aa
55.52 ± 2.44Aa
L. boryana
L. edodes
0.999±0.001Aa
0.999±0.001Aa
0.997±0.003Aa
0.997±0.002Aa
30 days
4.99 ± 0.13Aa
4.87 ± 0.10Aa
Moisture content (%)
39.65 ± 2.65Aa
54.73 ± 5.91Aa
aw
0.988±0.006Aa
1.002±0.003Aa
0 day
4.60 ± 0.02Aa
4.60 ± 0.02Aa
15 days
4.46 ± 0.30Aa
4.44 ± 0.03Aa
30 days
5.12 ± 0.04Aa
4.77 ± 0.09Aa
53.37 ± 0.58Aa
53.37 ± 0.58Aa
52.05 ± 3.9Aa
56.13 ± 2.55Aa
44.29 ± 3.97Aa
47.03 ± 3.10Aa
1.000±0.003Aa
1.000±0.003Aa
0.997±0.002Aa
0.999±0.002Aa
0.991±0.007Aa
0.997±0.003Aa
Means ± standard deviation followed by the same lowercase letters in the line and by the same uppercase letters in the row do not differ by Tukey's test (p ≤ 0.05).
V.M.C.S Santos (unpublished data) reports that some species of basidiomycetes have the characteristic of self-regulating pH, tending to stabilize its value in the optimum pH for growth, regardless the initial pH value. This fact was also observed by
study with Polyporus tricoloma for antibiotic production. The initial pH value does not seem to influence the lengthening of hyphae in Macrocybe titans as well.
titans in PDA medium. According to the author, there was no influence on mycelial growth, which can be explained by self-regulation.
Wisniewski et al. (2010)
Vieira et al. (2008)
in a
evaluated the effect of initial pH values (5, 6, 7 and 8) in radial mycelial growth of M.
Regarding humidity, there were no statistical differences between fungi neither throughout cultivation time (Table II). The values remained constant until the 15th day for the L. boryana's culture, being reduced after that by over 15%. For the cultivation of L. edodes, in
T1 treatment values remained constant throughout the whole cultivation, unlike the values of T2 treatment, which have decreased. Concerning the humidity achieved during SSF, the aw values showed no statistically significant differences (Table II).
However, analyzing the data from a physiological point of view, it is known that an aw of 0.988 may be different from 1.0, since it is possible to have aw values close to 1.0 with moisture contents between 20 and 100%, which can lead to the verification of different
consequences for cell growth and enzymes expression.
MnP activity values obtained throughout the SSF of fungi is shown in Figure 3. It was realized that although the maximum value of fungi's enzymatic activity was very close, around 110 UI.L–1 (equivalent to 0.66 UI.g–1), the enzyme expression occurred at different
times. For L. edodes the maximum value was obtained at 10 days of cultivation, while for L. boryana the maximum value was observed between 20 and 25 days. It was also noted that the supplementation of culture medium with cassava bagasse or corn cob (which
made T1 different from T2) showed no significant difference for the species of Lentinula. Consequently, the combined addition of these carbon sources, in the studied concentrations, did not contribute to increase the production of the enzyme MnP.
Fig. 3 MnP activity during the SSF of L. edodes (LE) and L. boryana (LB) in the different treatments (T1 and T2).
MnP activity showed a decrease in both treatments in SSF after reaching the highest activity level. This was also reported by
Regina (2004)
, who found a significant decreasing in MnP activity after the 8th day of incubation of different L. edodes' strains. According to
Kadimaliev (2003)
, changes in lignolytic activity and in peroxidase biosynthesis activity by fungi during solid state cultivation, depend on the type of substrate used. The author states that the amount of substances that restrict the mycelium growth (such as resins) and the
existence of a more intricate supramolecular structure may be linked to this outcome.
According to
Silva (2004)
, many basidiomycetes are developed in a simple media which presents availability of assimilable carbon, nitrogen and phosphorus sources and essential mineral salts. The ability of the fungus to grow in lignocellulosic substrates is related to the
vigor of the inoculated mycelium and to the capacity to activate physiological mechanisms necessary to use the nutrients from the culture medium (
Mata et al. 2001
). Thus, the nutritional needs for a satisfactory mycelium growth, especially at the initial stage of
Donini et al. 2006
cultivation, can be achieved depending on the type of material used in culture medium supplementation. The addition of certain meals, for example, provides nitrogen sources and can stimulate the enzymatic action of mycelium (
).
However, J.H.A. Betini (unpublished data) proved that the fungus Aspergillus niveus produced a 30% higher concentration of xylanase when grown in a medium containing only wheat bran as carbon source, when compared to the medium which also contained
corncob. This demonstrates that supplementation with cob meal does not seem to respond satisfactorily to the fungal enzyme production, although
cultivated in medium supplemented with high concentration of corncob.
Kadowaki et al. (1997)
, in their studies, have obtained maximum production of xylanase by Aspergillus tamarii when it was
CONCLUSION
When considering the two cultivation systems, it seems clear that the process developed in liquid medium, apart from presenting a lower enzymatic activity, reduced the kinetics of MnP's production. On the other hand, lignocellulosic materials, such as eucalyptus
wood, proved to be promising for enzymes production by basidiomycetes, for their stimulation capacity of MnP expression. Thus, considering the importance of eucalyptus in the energy matrix to obtain bioethanol, studies on how to optimize the production processes
of enzymes able to degrade lignocellulolytic compounds should be encouraged.
Acknowledgements
The authors are thankful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina
(FAPESC) for financial support through research grants and to Embrapa Florestas for financial support and supply of the Lentinula strains and the E. benthamii sawdust.
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Regina M. 2004. Atividade de enzimas lignocelulolíticas no crescimento de Lentinula edodes em subprodutos energéticos. Dissertation (Doutorado). Faculdade de Ciências Agronômicas, Universidade Estadual Paulista, 107 p. (Unpublished). [ Links ]
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Received: October19, , 2011; Accepted: January2, , 2012
Correspondence to: Katia Luiza Hermann E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013000300010
Biological Sciences
Chironomidae (Insecta: Diptera) of different habitats and microhabitats of the Vacacaí-Mirim River microbasin, Southern Brazil
RODRIGO KÖNIG1, SANDRO SANTOS2
1
2
Departamento de Ciências Biológicas, Instituto Federal Farroupilha, Campus Júlio de Castilhos, Rodovia RS 527, s/n, 98130-000 Júlio de Castilhos, RS, Brasil
PPG Biodiversidade Animal, Departamento de Biologia, Universidade Federal de Santa Maria, Av. Roraima, 1000, Bairro Camobi, 97105-900 Santa Maria, RS, Brasil
ABSTRACT
The Chironomidae family is one of the main groups of aquatic insects present in streams. This work aimed to investigate the influences of anthropogenic pressures and substrate types on chironomid community composition and structure. Chironomid larvae were collected during the summer of 2007 at four sites along the Vacacaí-Mirim River
microbasin, chosen based on the different available benthic substrates. The organisms were identified in the laboratory at the genus level, and the density, the rarefied taxonomic richness and the Shannon diversity index were calculated. The faunal structure was subjected to an ANOVA to compare the metrics among sites and substrates. The community
composition of the sites was subjected to a multivariate statistical analysis. Differences in the composition, richness, density, and diversity were observed among the sites due to differences in the levels of nutrients and solids generated by the presence of crops close to water bodies. Samples collected from sandy substrates exhibited a lower density and
taxonomic richness because sand is a poorer substrate than the others that were sampled. In organic or mixed substrates with higher energy availability and better shelter conditions, the observed densities were higher. The physical and chemical conditions and the morphometric characteristics of the sites were more influential than the substrates.
Keywords: aquatic insects; benthic substrates; anthropogenic pressures; faunal structure
RESUMO
A família Chironomidae é um dos principais grupos de insetos aquáticos presentes em riachos. Este trabalho teve como objetivo investigar a influência de pressões antrópicas e do tipo de substrato sobre a estrutura e composição da comunidade de Chironomidae. Larvas de Chironomidae foram coletadas durante o verão de 2007 em quatro locais da
microbacia do rio Vacacaí-Mirim, considerando os diferentes substratos bentônicos disponíveis. Os organismos foram identificados em laboratório até o nível de gênero e foi calculada a densidade, a riqueza taxonômica rarefeita e a diversidade de Shannon. A estrutura da fauna foi submetida à ANOVA para comparação das métricas entre locais e
substratos. A composição da comunidade dos locais foi submetida à estatística multivariada. Diferenças na composição, riqueza, densidade e diversidade foram observadas nos locais, devido a diferentes níveis de nutrientes e sólidos gerados pela presença de culturas próximas aos corpos d'água. Amostras de substrato arenoso apresentaram menor
densidade e riqueza por este ser um substrato mais pobre em relação aos demais amostrados. Em substratos orgânicos ou mistos, que tem maior disponibilidade de energia e melhores condições de abrigo, as densidades observadas foram maiores. Fatores físicos, químicos e morfométricos dos locais foram mais influentes do que as características dos
substratos.
Palavras-Chave: insetos aquáticos; substratos bentônicos; pressões antrópicas; estrutura da fauna
INTRODUCTION
Fesl 2002
The patterns of diversity for organisms in aquatic environments have received considerable attention from researchers in recent years because, along with abiotic environmental factors, the data on these patterns can indicate the conservation status of a river, stream or other water body (
may affect the structure (density, abundance, and diversity) of distinct biological communities, such as the benthic macroinvertebrates.
). Any environmental disturbance to a water body
Armitage et al. 1995
Among the macroinvertebrates, Chironomidae (Diptera) larvae are predominant in freshwater environments (
, Brito Jr et al. 2005). Immature stages of Chironomidae generally live on or in the sediment, feeding on organic detritus and associated microfauna and flora. This group occupies an important position in aquatic
ecosystem trophodynamics, recycling nutrients in the sediment and modifying the composition of particulate organic matter (
Sankarperumal and Pandian 1992
).
Henriques-Oliveira et al. 2003
The composition of a Chironomidae community changes based on alterations in trophic, physical and chemical conditions. Anthropogenic impacts frequently affect those conditions. Natural features may also shape the patterns of diversity in aquatic environments, with the substrate being one decisive factor (
Substrates containing more complex structures, such as leaves and wood, usually shelter a greater diversity of taxa compared with simple substrates, such as sand or rocks (
).
Vinson and Hawkins 1998
). Studies of the distribution of Chironomidae taxa may generate additional information that would be of use in the assessment of aquatic systems, e.
Kleine and Trivinho-Strixino 2005
g., data that indicate the influence of disturbances to the substrate, on hydrological variables and on edge vegetation, among others (
).
Spies and Reis 1996
The number of chironomid species in the Neotropical zone has been underestimated, taking into consideration the high diversity of the group (
understanding of the group (Pannata et al. 2006).
). Therefore, in this zone, in addition to the challenges posed by the interpretation of Chironomidae diversity patterns, there is also a need to further develop the taxonomic
In Southern Brazil, some segments of the Vacacaí-Mirim River microbasin are located in urban zones and in areas of agricultural influence, whereas some of its affluents have springs located on the edges of Serra Geral, a conservation area with good water quality. Such characteristics make this basin ideal for studies associating the
characteristics of macro and micro environments with the structure of the Chironomidae community. The aim of this study was to investigate the influences of anthropogenic pressures and of substrate types on chironomid community composition and structure, as well as correlate the results with the environmental characteristics of the study
areas and identify the factors that most influence the local fauna.
The area studied is part of the Vacacaí-Mirim River's hydrographic microbasin, which drains a total area of 11,077 km2 (SEMA 2010). Together with the Vacacaí River microbasin, this constitutes one of the biggest hydrographic basins of the State of Rio Grande do Sul, Brazil. The climate is subtropical with an annual average temperature of
19.1°C and an annual average rainfall of 1,712 mm (
Sutili et al. 2009 Heldwein et al. 2009
,
).
Four sampling sites were selected within the Vacacaí-Mirim River microbasin in the Santa Maria municipality. The segments varied from first-order to third-order. Sites A and B displayed environmental characteristics with less human influence, whereas sites C and D were influenced by areas of rice cultivation located close to the water
body.
Data Collection
Baumart and Santos 2010
Chironomidae fauna were collected during the summer of 2007, at the time when agricultural activities in the region reach their maximum (
). Samples were collected with a Surber sampler with a 0.25 mm mesh net and a collection area of 0.1 m2. A distinct number of samples were collected from each site to represent all
of the substrates present in each area. The types of substrates varied according to site; however, with the exception of site D, all sites included areas of rocks, leaves, and mixed substrate (organic and inorganic). A total of 26 samples were collected:
Site A (29°40′08″ S, 53°45′26″ W): rocks, leaves, mixed (rocks/leaves).
Site B (29°40′27″ S, 53°45′05″ W): rocks, sand, leaves, mixed (rocks/leaves).
Site C (29°40′58″ S, 53°43′46″ W): rocks, leaves, mixed (rocks/leaves).
Site D (29°41′44″ S, 53°41′07″ W): rocks; sand.
At least two samples representing each substrate were collected from each site. The collected sediment was stored in plastic bags and fixed with a formaldehyde solution at 5%. At the laboratory, the material was sorted by size using a series of sieves. The residue was examined under a stereomicroscope. Semi-permanent slides were
manufactured according to Trivinho-Strixino and Strixino's guide (1995), which was used to identify specimens along with the Epler guide (2001).
For every site and substrate, several abiotic variables were determined:
Martinelli and Krusche (2004)
1. - In situ: water current velocity was measured with the use of a floater, according to
; pH was determined with an Ohaus pH meter; electrical conductivity was determined using a Marte MB-11P conductivimeter; and dissolved oxygen was measured with a Diginet oxymeter;
2. - In the laboratory: the total solids present in water samples were measured via gravimetry; the total nitrate and phosphate contents of water samples were determined using the spectrophotometric method; and the organic matter content of sediment samples was analyzed based on ash-free dry mass.
All analyses were performed according to procedures described in the Standard Methods for the Examination of Water and Wastewater (APHA 1998).
Data Analysis
Mcalecee et al. 1997
The average density (number of individuals per m2) of chironomid taxa was determined for each site and substrate. Using the Biodiversity Pro program (
), the rarefied taxonomic richness and the Shannon diversity index were also established, following
Magurran (1988)
.
The UPGMA method and the Bray-Curtis distance were used to produce a dendrogram of the samples with the use of the biological matrix. In this way, we were able to verify whether the classifications were made based on site or substrate. Four types of substrates were considered: rocks, sand, organic (leaves), and mixed substrate (organic
and inorganic).
To determine whether there were differences in the community structure (richness, density, and diversity), an analysis of variance (two-way ANOVA) was applied, including site and substrate factors. A multivariate analysis of variance (MANOVA) was applied to identify any differences in the community compositions of the samples.
The relative importance of site versus substrate was assessed based on a partial canonical correspondence analysis (pCCA). This analysis used the taxa density matrix to partition the results into independent components, revealing the percentage of the results that is explained by the spatial component (site), the percentage explained by the
substrate type, the fraction that is shared (attributable to both site and substrate), and the fraction that is undetermined. Two different canonical correspondence analyses (CCA) were processed separately to assess the association between taxa and sites, and between taxa and substrates. Taxa with an average density representing below 0.2% of
Oksanen et al. 2010
the total fauna were discarded for the CCA. The “vegan” package (
R Development Core Team 2010
) of the R program (
) was used for the above analyses.
RESULTS
A total of 6,577 chironomid larvae were identified. Members of the Chironominae, Orthocladiinae and Tanypodinae subfamilies were collected from all sites, but a greater density of these subfamilies was present at site C. The average proportions of the total larvae were 48% Chironomidae, 32% Orthocladiinae, and 20% Tanypodinae. A
total of 29 Chironomidae taxa were collected (Table I). Rheotanytarsus, Cricotopus, Thienemannimyia, and Polypedilum were the most abundant taxa.
TABLE I The absolute average density (number of larvae m–2) of Chironomidae genera in the sampling sites and substrates of the Vacacaí-Mirim River microbasin, Santa Maria, Brazil.
Site
Substrate
Apedilum
Caladomyia
Chironomus
Cryptochironomus
Dicrotendipes
Endotribelos
Harnischia
Kiefferulus (?)
Parachironomus
Paratendipes
Polypedilum
Rheotanytarsus
Stenochironomus
Tanytarsus
Tanytarsini type I
R
A
O
B
M
Chironominae
S
Ablabesmyia
Labrundinia
Larsia
Pentaneura
Thienemannimyia
Zavrelimyia
Pentaneurini type I
O
M
O
O
O
O
O
O
O
R
C
O
D
M
R
S
O
O
X
X
O
X
X
□
□
X
□
O
□
X
X
X
X
X
X
□
X
□
X
□
□
O
∆
□
∆
□
□
□
O
□
□
□
O
□
□
□
X
Orthocladiinae
Corynoneura
Cricotopus
Lopescladius
Nanocladius
Parametriocnemus
Rheocricotopus
Thienemanniella
R
X
X
X
□
□
□
O
X
□
X
X
X
X
X
X
□
O
□
X
X
□
□
O
∆
X
X
Tanypodinae
□
□
□
∆
∆
∆
O
∆
∆
□
∆
∆
∆
X
X
O
X
X
X
O
O
O
X
□
X
O
X
O
□
X
□
X
X
X
□
X
X
□
□
□
X
□
O
□
□
X
X
X
□
X
X
X
□
□
O
O
R: Rocky; O: Organic; M: Mixed; S: Sandy. O: 0-20; X: 21-100; □:101-500; ∆: >500.
The dendrogram generated from the biological matrix exhibited a strong tendency to structure the communities based on the site (Figure 1), although there was a tendency for identical substrates to be grouped together within the same site.
□
∆
∆
X
X
X
X
∆
∆
∆
□
X
Figure 1 Dendrogram displaying the sampling sites and substrates of the Vacacaí-Mirim River microbasin, Santa Maria, Brazil, created using cluster analysis with the UPGMA method and Bray-Curtis distance. Each branch in dendrogram corresponds to one replicate. The first letter indicates the sampling site
and the second letter the substrate type: Organic (O), Mixed (M), Rocky (R), Sandy (S); numbers define substrate replicates (e.g., AO1 = sample number 1 of the organic substrate at site A).
Chironomid densities were higher at site C. At the sites where organic and mixed substrates were found, was observed a higher density of organisms compared to the other substrates. The rocky substrate exhibited a slightly lower density than the mixed and organic substrates. The sandy substrate indicated the lowest average chironomid
density in the study overall (Figure 2). The ANOVA test revealed significant differences in chironomid density among sites (p < 0.01, F = 455.241), among substrates (p < 0.01, F = 49.85), and for the site/substrate interaction (p < 0.01, F = 26.57).
Figure 2 Chironomidae density (ind. m–2) in the sampling sites and substrates of the Vacacaí-Mirim River microbasin, Santa Maria, Brazil. Horizontal bars indicate standard errors.
Taxonomic richness estimated using the technique of rarefaction revealed higher values for sites A and B (Figure 3). The same pattern occurred in the Shannon diversity index values (Figure 4). No patterns of richness or diversity were found within sites A, B or C for the rocky, organic or mixed substrates. Sand had the lowest levels of
richness and diversity in the sites where it was present. According to the ANOVA test, richness differed among sites (p < 0.01, F = 32.24) and for the site/substrate interaction (p < 0.01, F = 2.13), but not among substrates. The same pattern was observed for diversity (p < 0.01, F = 42.74 for site; p < 0.01, F = 2.97 for site/substrate).
Figure 3 Rarefied richness (number of taxa) in the sampling sites and substrates of the Vacacaí-Mirim River microbasin, Santa Maria, Brazil. Horizontal bars indicate standard errors.
Figure 4 Diversity (Shannon Index) of the Chironomidae fauna in the sampling sites and substrates of the Vacacaí-Mirim River microbasin, Santa Maria, Brazil. Horizontal bars indicate standard errors.
The multivariate analysis used to compare faunal composition revealed differences among sites (p < 0.01, F = 25.53), substrates (p < 0.01, F = 2.90), and site/substrate (p < 0.05; F = 2.39).
According to the pCCA, the site explains 24.1% of the variation found among samples in the study, whereas the substrate explains only 4.3%. The value of clarification shared by the two factors was 2.3%.
The first two axes of the CCA based on site explain 79.2% of the total variance in the data (Figure 5). Axis 1 explains 50.5% of the variance, and it was positively correlated with high values for nutrient content (phosphate and nitrate), electrical conductivity, total solids, organic matter, and water current velocity, all of which were
characteristics of sites C and D. Parachironomus, Dicrotendipes, and Nanocladius were the taxa associated with this axis and with site D. Chironomus was associated with site C.
Figure 5 Ordination diagram of the CCA with the abiotic variables and the most representative Chironomidae genera found in the sampling sites of Vacacaí-Mirim River microbasin, Santa Maria, Brazil. Abl (Ablabesmyia); Cri (Cricotopus); Nan (Nanocladius); Pac (Parachironomus); Dic (Dicrotendipes); Chi
(Chironomus); Rhe (Rheotanytarsus); Thia (Thienemannimyia); Thla (Thienemanniella); Lar (Larsia); Pol (Polypedilum); Pat (Paratendipes); Pam (Parametriocnemus); Pen (Pentaneura); Rhc (Rheocricotopus); Lop (Lopescladius); Lab (Labrundinia); Har (Harnischia); Cor (Corynoneura); Tat (Tanytarsus). P
(Total Phosphate); T.S. (Total Solids); Cond. (Electrical Conductivity); NO3 (nitrate); C.V. (Current Velocity); O.M. (Organic Matter); D.O.(Dissolved Oxygen).
Axis 2 explains 28.7% of the total variance, and it was correlated positively with pH and phosphate content. High oxygen concentrations were negatively projected on axes 1 and 2, especially associated with site A. Paratendipes was strongly associated with site A, whereas Tanytarsus and Corynoneura were associated with site B. To a lesser
extent, Parametriocnemus, Pentaneura, Rheocricotopus, Lopescladius, Labrundinia, and Harnischia were also associated with these sites and/or conditions.
The first and second axes of the CCA generated for substrates (Figure 6) explain 80.7% and 17.3% of the total variance, respectively. Larsia, Lopescladius, Rheocricotopus, Labrundinia, and Pentaneura were correlated with high values of current velocity and organic matter content. These variables and taxa were negatively associated with
sandy substrates. Tanytarsus was negatively associated with rocky substrates. Harnischia and Paratendipes were strongly associated with mixed and organic substrates, whereas Nanocladius was associated with sand.
Figure 6 Ordination diagram of the CCA with the abiotic variables and the most representative Chironomidae genera found in the sampling substrates of Vacacaí-Mirim River microbasin, Santa Maria, Brazil. Abl (Ablabesmyia); Cri (Cricotopus); Nan (Nanocladius); Pac (Parachironomus); Dic (Dicrotendipes);
Chi (Chironomus); Rhe (Rheotanytarsus); Thia (Thienemannimyia); Thla (Thienemanniella); Lar (Larsia); Pol (Polypedilum); Pat (Paratendipes); Pam (Parametriocnemus); Pen (Pentaneura); Rhc (Rheocricotopus); Lop (Lopescladius); Lab (Labrundinia); Har (Harnischia); Cor (Corynoneura); Tat (Tanytarsus). P
(Total Phosphate); T.S. (Total Solids); Cond. (Electrical Conductivity); NO3 (nitrate); C.V. (Current Velocity); O.M. (Organic Matter); D.O.(Dissolved Oxygen).
DISCUSSION
Kleine and Trivinho-Strixino (2005)
The present study comprises the first survey of Chironomidae fauna performed in the central region of Rio Grande do Sul. The total taxonomic richness found in this study area is similar to results found by other studies conducted in Brazil.
collected 33 Chironomidae taxa from three sampling sites in a river in
São Paulo State (Brazil), 24 of which were collected in an environment located in a preservation area. It presented similar results to those of site B in the present study. Community structure can be influenced by environmental characteristics at different scales, such as those related to the site and/or microhabitats. In this study, we could
observe that the general characteristics of the sites exerted a great influence over faunal distribution, with distinct types of substrates within the same environment ending up with more similarities than the same substrates in different environments.
The spatial distribution of aquatic insects is directly related to river current velocity, water temperature, dissolved oxygen, pH, size of substrate particles, food availability, and other variables. Physical, chemical, and morphometric characteristics define the macro-distribution, whereas substrates and food availability tend to influence microFesl 2002
distribution (
). Physical and chemical factors were determinant for Chironomidae fauna, and they can better explain the community distribution than substrate type, as revealed in the classification analysis and pCCA results.
Primack and Rodrigues 2001
Sites A and B exhibited the highest taxonomic diversity and richness. At these sites, the river is protected from impacts by the presence of riparian vegetation (
). Similar morphometric properties, such as low values for total solids, percentage of organic materials, electrical conductivity, and nutrients as well as a high
level of dissolved oxygen, characterize sites A and B as the least-modified environments regarding physical and chemical characteristics. These attributes characterize well-preserved environments and low-order rivers. Some of the taxonomic groups found in these sites are considered sensitive to environmental disturbances, such as
Ruse and Wilson 1995
Paratendipes and Rheocricotopus (
).
However, although sites A and B share similar physical and chemical attributes, they display unique characteristics in terms of taxonomic richness, diversity and faunal composition. According to
site B may be a result of the greater heterogeneity of habitats found in this environment.
Coffman (1995)
, highly heterogeneous rivers are usually associated with a greater abundance of species. Therefore, the variety of fauna observed at
Richness and diversity were significantly different at sites C and D compared to the other environments. Several site characteristics contributed to this dissimilarity, particularly the high levels of nutrients (nitrogen at site C and phosphate at site D) and organic matter, as well as the high-velocity water current. The recorded data for sites C and
D regarding content of nutrients and organic matter characterize altered environments with low richness, because only highly tolerant groups can remain in such conditions. Tolerant taxa, such as Thienemannyimia, Cricotopus and Rheotanytarsus, sustained large populations, especially at site C. According to
Amorim et al. (2004)
, environmental
Merritt and Cummins 1996
disturbances tend to promote an increase in biomass and a decrease in richness. Rheotanytarsus individuals were present in great numbers on the macrophyte plants at site C. Those plants provide shelter and allow the taxon to exploit the fast-moving water of the site. Rheotanytarsus are collector-filterers (
of getting food is favored from a high-velocity current (
can influence a community.
), and their way
Sanseverino and Nessimian 2001 Kikuchi and Uieda 2005
,
). The biological contrasts between the two groups of environments (sites C-D versus sites A-B) can easily be perceived. However, the sites also differ among themselves, as evidenced by faunal composition, another signal that spatiality
Some taxa were characteristic of less-disturbed sites, whereas others characterized more impacted ones.
Roque et al. (2000)
cited Corynoneura, Lopescladius, Harnischia, and Labrundinia among the taxa found in environmental conditions characterizing less-impacted areas. That trend was confirmed in this study, because those taxa were
Rae 1989
associated with sites A and/or B. However, other taxa were also related to less-disturbed environments, such as Parametriocnemus, Pentaneura, Rheocricotopus, and Tanytarsus. Dicrotendipes characterizes agricultural areas (
) and it was found only at site D (influenced by rice cultivation). Parachironomus and Nanocladius were also
Marques et al. 1999
typical of site D, indicating a possible link with intermediate-quality water. Chironomus was associated with site C, corroborating existing information about the relationship of this taxon with impacted water quality (
Polypedium can tolerate a wide range of environmental disturbances. Rheotanytarsus larvae were numerous at both of the sites that were enriched by nutrients (C and D), possibly due to the proximity of rice cultivation.
). Confirming another tendency in our data,
Roque et al. (2000)
suggested that the larvae of Rheotanytarsus and
Fend and Carter 1995
Even with the site exerting a greater influence on community structure than the substrate in the present study, it is well-established that the distribution of several groups of aquatic insects is defined according to the types of substrate available; this factor often determines the presence and abundance of various populations (
).
Baptista et al. (2001)
There are a number of notable tendencies in this study. Substrates primarily influenced the density and composition of the community. Substrates containing at least a fraction of organic matter generally exhibited greater chironomid density, corroborating the conclusions of
that such organic components, especially leaves,
may be favorable because they provide better feeding and sheltering conditions in addition to having a richer periphytic flora. As the presence of leaves is typical in low-order rivers and Chironomidae are numerous in aquatic environments, the idea that a great number of organisms use leaves as a source of energy is plausible (Callisto and
Gonçalves Jr 2007).
Aburaya and Callil 2007
Among the recorded taxa, Polypedilum exhibited high densities across all substrates due to its cosmopolitan characteristics (
), but the values were higher for vegetation or mixed substrates. Harnischia and Paratendipes also revealed a preference for these substrates, possibly because of the better food conditions offered.
Sand was the least inviting substrate for the development of Chironomidae fauna, presenting a lower amount of organic matter and a greater accumulation of solids than other substrates. It is likely that substrate instability linked to the low availability of organic matter in sandy substrates hindered taxonomic richness and organism density.
Tikkanen et al. 1994
Substrate stability is proportional to particle size; thus, smaller particles, such as those found in mostly sandy substrates, can be moved more easily (
).
Henriques-Oliveira et al. (2003)
also reported lower taxonomic richness in sites characterized by greater quantities of sandy substrate.
It is also important to highlight the negative correlation between some taxa and sandy substrates. Larsia displayed this tendency more pronouncedly, and it was recorded in all substrates except sand. Labrundinia and Pentaneura densities declined sharply in sand. This trend does not corroborate
Wiederholm's (1983)
findings, which indicates a
Wetzel 1993
tendency for the subfamily Tanypodinae to be present in higher numbers in sandy substrates. One explanation may be the predation habit of the group (
), which includes the intake of other chironomids. The decreased density in sandy substrates of groups that serve as food may have contributed to the lower abundance of these three
Tanypodinae taxa in that substrate. Other taxa may also have been influenced by the conditions of the sandy substrate, such as Lopescladius and Rheocricotopus. Nanocladius was the only taxon that indicated a preference for sandy substrates.
The rocky substrate also supported a lower average Chironomidae density regarding organic substrates, but no meaningful difference concerning faunal richness was observed. Benthic communities are more stable in rocky areas than sandy areas. Communities in rocky substrates are controlled by biological interactions, whereas in sand,
Kikuchi and Uieda 2005
community structures are physically determined and therefore more susceptible to environment variations (
). Neither of the groups exhibited a strong relationship with the rocky substrate, but the absence of Tanytarsus in this substrate, despite its high density in organic substrates, confirms the results of
Nessimian (1998)
Epler 2001
, indicating that the larvae of this taxon primarily inhabit deposited litter. The presence of Stenochironomus in rocky substrates is unusual and most likely occurred accidentally, because this taxon is a miner of submerged leaves and wood fragments (
mixture of plant debris colonized by Stenochironomus larvae in the rock sample, or these organisms may have been washed onto the rocky substrate by the current.
Future applied studies could confirm the potential utility of Chironomidae taxa as indicators of different anthropogenic disturbances and types of substrates, considering that in this study some groups occurred only in particular sites and microhabitats.
Acknowledgements
This work was supported by grants provided by Pró-Publicações Internacionais/PRPGP/UFSM and by a MSc scholarship grant provided by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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Received: October10, , 2011; Accepted: March12, , 2012
Correspondence to: Rodrigo König E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013000300011
Biological Sciences
Evaluation of analgesic and anti-inflammatory activities of Hydrocotyle umbellata L., Araliaceae (acariçoba) in
mice
IZIARA F. FLORENTINO1, MARCUS VINÍCIUS M. NASCIMENTO1, PABLINNY M. GALDINO1, ADRIANE F. DE BRITO1, FABIO F. DA ROCHA3,
CARLOS R. TONUSSI4, THEREZA CHRISTINA M. DE LIMA4, JOSÉ R. DE PAULA2, ELSON A. COSTA1
1
Laboratório de Farmacologia de Produtos Naturais, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade Federal de Goiás,
Campus II, Km 13, 74001-970 Goiânia, GO, Brasil
2 Laboratório de Pesquisa de Produtos Naturais, Faculdade de Farmácia, Universidade Federal de Goiás, Praça Universitária, 1166, Setor Universitário, 74605-220
Goiânia, GO, Brasil
3 Departamento de Ciências Fisiológicas, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro, BR 465, Km 07, 23890-000 Seropédica, RJ, Brasil
4 Departamento de Farmacologia, Universidade Federal de Santa Catarina, Campus Universitário, 88049-900 Florianópolis, SC, Brasil
ABSTRACT
The Hydrocotyle umbellata L. is a specimen of the Araliaceae family popularly known as acariçoba. Its indications in folk medicine include treatment of skin ulcers,
and rheumatism. The aim of this study was to evaluate the antinociceptive and anti-inflammatory activities of the ethanolic extract from acariçoba's underground parts
(EEA). EEA reduced the nociceptive response of the animals as evaluated in the acetic acid-induced writhing test and in both phases of formalin test. EEA also
presented a supraspinal analgesic activity by increasing the pain latency in the hot plate test. Moreover, EEA reduced the leukocytes migration and plasma extravasation
to pleural cavity in the carrageenan-induced pleurisy, besides reducing the edema induced by carrageenan until the second hour and also the edema induced by dextran.
In conclusion our results showed that EEA of H. umbellata L. presents analgesic and anti-inflammatory activities, and that a blockade of activity or reduction in the
release of different mediators, such as histamine and serotonin, could be involved in these pharmacologic effects.
Keywords: Acariçoba; anti-inflammatory; antinociceptive; Biomedical Sciences; Hydrocotyle umbellata L
RESUMO
A Hydrocotyle umbellata L., é uma espécie pertence à família Araliaceae, popularmente conhecida como acariçoba. Suas indicações na medicina popular incluem
tratamento de úlceras cutâneas, eczemas e reumatismo. O objetivo deste trabalho é avaliar as atividades antinociceptiva e/ou anti-inflamatória do extrato etanólico das
partes subterrâneas da acariçoba (EEA). O EEA reduziu a resposta nociceptiva do animais no teste das contorções abdominais induzidas por ácido acético e em ambas as
fases do teste da formalina. O EEA também mostrou uma atividade analgésica supra-espinhal ao aumentar o limiar nociceptivo no método da placa quente. O EEA
reduziu tanto o número de leucócitos migrados quanto o extravasamento plasmático para a cavidade pleural na pleurisia induzida por carragenina. Além de reduzir o
edema de pata induzido por carragenina até a segunda hora e também o edema de pata induzido por dextrana. Em conclusão os nossos resultados mostram que o EEA de
H. umbellata L. possui atividades analgésica e anti-inflamatória, e que um bloqueio da atividade ou redução da liberação de diferentes mediadores tais como histamina e
serotonina, podem estar envolvidos nestes efeitos farmacológicos.
Palavras-Chave: Acariçoba; anti-inflamatório; antinociceptivo; Ciências Biomédicas; Hydrocotyle umbellata L
INTRODUCTION
The Hydrocotyle umbellata L., popularly known as acariçoba, is a creeping specimen that belongs to the Araliaceae family. It species is native in Argentina, Cuba,
India and Brazil, where this plant can be found in MidSouth states of São Paulo, Paraná, Santa Catarina, Rio Grande do Sul and Goiás (Corrêa 1984,
Fischer et al.
1994
).
Plant species as Hydrocotyle umbellata L. and Hydrocotyle asiatica (L.) have great interest in folk phytotherapy and in the Ayurvedic medicine (Indian) because of
Reis et al. 1992
its potential anxiolytic, memory stimulant effects and its use in the cosmetic industry (
). Ethnobotanical studies suggest that these plants are used as
true panaceas, being employed as healing, diuretic, anti-hypertensive and to treat skin ulcers, eczema, dermatitis, psoriasis, erysipelas, rheumatism, tuberculosis,
and spleen, liver and intestinal disorders. H. umbellata L. leaves in high doses produce emetic effects and are potentially toxic (Corrêa 1984,
al. 1994
Reis et al. 1992 Fischer et
,
Rocha et al. 2011
). Our previous study found that the ethanolic extract from H. umbellata L. showed anxiolytic-like and sedative effects in mice (
).
Acariçoba is a plant with long and petiolate leaves, white small and numerous flowers and a small fruit-shaped capsule flattened. In the Brazilian folk medicine a
decoction of its leaves (fresh or dried) is used as baths, and orally for the treatment of inflammatory processes (Pharmacopeia dos Estados Unidos do Brasil, 1926 –
Brazilian Pharmacopeia). Inflammation is the body's immediate response the tissue aggressions caused by pathogens, toxic substances or physical harm. The
inflammatory process is characterized by the release of several mediators and the development of the classic signs of inflammation such as swelling, warmth,
redness and pain (Queiroz et al. 2010).
Adams et al. 1989
Phytochemical studies showed the presence of flavonoids, essential oils, saponins, tannins and absence of alkaloids and anthracene derivatives (
Fischer et al. 1994
). Thus, the aim of this study was to evaluate the pharmacological activity of ethanolic extract from Hydrocotyle umbellata L. (acariçoba)
underground parts in pain and inflammation models seeking to scientifically give support the use of this plant by the Brazilian population.
,
Botanical Material and Extraction
Hydrocotyle umbellata L. underground parts were authenticated by Prof. Heleno Dias Ferreira (Institute of Biological Sciences/Federal University of Goiás/ICB/
UFG) and collected in the Medicinal Plants Garden of Faculty of Pharmacy from the Universidade Federal de Goiás (UFG). A voucher specimen has been
deposited in the Herbarium of UFG under the number UFG/22.394.
To prepare the ethanolic extract from acariçoba underground parts (EEA) the vegetal material was dried and milled and the powder was then macerated in ethanol
(96° GL) for three days, followed by filtration. After that, the filtrated was concentrated to dryness under reduced pressure in rotavapor (at 40 °C). The extract was
obtained by this extractive process with a yield of 3.0 %.
Animals
Male Swiss albino mice weighing approximately 30 g from the Central Animal House of UFG were used in this study. The animals received food and water ad
libitum and were maintained in a room with light and temperature regulation. All experimental protocol was developed in accordance with the principles of ethics
and animal welfare designated by the SBCAL/COBEA, as well as approved by the Ethics Committee in Research of UFG (number 104/08).
Drugs
Acetone (Isofar, Brazil); carrageenan (Sigma, USA); cyproheptadine (Sigma, USA); crotonoil (Sigma, USA); dexamethasone - decadron (Ache, Brazil); dextran
(Sigma, USA); ethanol 95% P.A. (Synth, Brazil); formaldehyde (Synth, Brazil); glacial acetic acid (Vetec, Brazil); Heparin (Hipolabor, Brazil); indomethacin
(Prodome, Brazil); morphine - Dimorf® (Cristalia, Brazil); NaCl (Belga); Türk solution (Bioshop, Brazil).
Acute Oral Toxicity Study
Acute oral toxicity assay was performed in adult male albino Swiss mice (30-35 g) divided into different groups following the OECD guidelines-423 (OECD,
2001). Two groups of mice n=3 albino Swiss were treated with EEA (4,000 mg/kg), orally. The vehicle group received distilled water at the same volume and route.
Analgesic Activity
The analgesic activity of the ethanolic extract from underground parts acariçoba was investigated using the following models.
Acetic acid-induced abdominal writhing
Experimental groups of mice (n = 9) were treated (p.o.) with vehicle (10 mL/kg), EEA (250, 500 or 1,000 mg/kg) or indomethacin (10 mg/kg) 60 min before the
administration of 1.2% acetic acid solution (10 mL/kg, i.p.). The number of writhing produced in each group for the following 30 min was counted and the results
Koster et al. 1959 Vacher et al. 1964
were expressed as mean ± standard error of mean (SEM) in percentage of control group (
,
).
Formalin-induced pain
Experimental groups of mice (n = 9) were treated with vehicle (10 mL/kg, p.o.), EEA (1,000 mg/kg, p.o.), indomethacin (10 mg/kg, p.o.) or morphine (5 mg/kg s.
c.), 60 min after the p.o. treatments or 30 min after s.c. treatment. The animals were treated with formalin 3% (20 µL) in the right hindpaw. Following injection of
the phlogistic agent, the mouse was placed into an acrylic box, with a mirror placed under the box at 45° to facilitate the observation of the formalin-injected paw
Hunskaar
for 30 min. The pain reaction time (time for licking the paw) was observed in two periods, 0-5 min (neurogenic pain) and 15-30 min (inflammatory pain) (
and Hole 1987
).
Hot plate test
The latency (in seconds) to reaction of the mice, expressed as licking, shaking or lifting the hind paws, on hot plate at 55.5 + 0.5°C was analyzed according to
Woolfe and MacDonald (1944)
. The animals were divided into five experimental groups (n = 9) consisting of animals treated with vehicle (10 mL/kg, p.o.), EEA (250,
500, or 1,000 mg/kg, p.o.) or morphine (10 mg/kg, s.c.). The latency to pain reaction was measured at -60, -30, 0, 30, 60, 90 and 120 min of the treatment.
Anti-Inflammatory Activity
The anti-inflammatory activity of the ethanolic extract from acariçoba underground parts was investigated using the following models.
Carrageenan-induced pleurisy
Each animal received, intravenously, 200 µL of 2.5% Evan's blue in normal saline, two hours after the animals were being treated with vehicle (10 mL/kg, p.o.),
EEA (250, 500, or 1,000 mg/kg, p.o.) or dexamethasone (2 mg/kg, p.o.). One hour after the treatments, the animals received an injection of 100 µL of 1%
carrageenan into pleural cavity. Four hours after phlogistic agent administration the pleural exudate was collected with 1 mL of heparinized PBS. One aliquot was
used to count the number of total leukocytes, using Türk solution, in a Neubauer's chamber. Other aliquot was used to determine the Evan's blue concentration on a
Saleh et al. 1999 Vinegar et al. 1973
spectrophotometer at 600 nm (
,
).
Croton oil-induced ear edema
Animals were treated (p.o.) with vehicle (10 mL/kg, p.o.), dexamethasone (2 mg/kg, p.o.), or EEA (250, 500, or 1,000 mg/kg, p.o.). One hour after the treatments,
the inflammation was induced by the application of 2.5 % (v/v) croton oil solution in acetone (2.5%) on the inner surface of the right ears. The same volume of
Zanini et al. 1992
acetone was applied to the left ear (
). After 4 hours, the mice were sacrificed and segments of both ears were removed. The inflammatory was
measured by the difference between the weights of the segments.
Carrageenan-induced paw edema
Groups of mice (n =9) were treated orally with vehicle (10 mL/kg), EEA (250, 500 or 1,000 mg/kg) or indomethacin (10 mg/kg) 1 h before the injection of 50 µL
of 1% carrageenan in the right paw. The left paw was used as control and received the same volume of 0.9% NaCl solution. Then, the edema was measured by the
Passos 2007
difference in the volume between the paws using a plethysmometer (Ugo Basile Co. - Italy) at several time-points after injection of phlogistc agent (
).
Dextran-induced paw edema
Experimental groups of mice were treated orally with vehicle (10 mL/kg), EEA (1,000 mg/kg) or cyproheptadine (5 mg/kg) one hour before the injection of 50 µL
of 1% dextran in the right paw. The left paw was used as control and received the same volume of 0.9% NaCl solution. After the injections the edema was
measured in time intervals of 0, 30, 60, 90 and 120 min using a plethysmometer (Ugo Basile Co.- Italy). The formation of edema was assessed by the difference
between the paws volume.
Results were expressed as means ± S.E.M. Differences between two means were detected using the Student's t test. Differences between more than 2 means were
Sokal and
detected using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls' test. The results were considered significant when p < 0.05 (
Rohlf 1981
).
RESULTS
Acute Oral Toxicity
In the acute oral toxicity test doses up to 4,000 mg/kg of EEA did not cause any death in mice during 14-days of observation. Mice did not show any signs of
toxicity or change in general behavior or other physiological activities.
Acetic Acid-Induced Abdominal Writhing
The EEA (250, 500 or 1,000 mg/kg) produced a significant reduction (24, 32 and 41%, respectively) in the number of acetic acid-induced writhes when compared
with control values (vehicle 10 mL/kg; 113.0 ± 6.4). The indomethacin reduced the abdominal writhes by 40% (Figure 1).
Figure 1 Effect of the ethanolic extract from acariçoba underground parts (EEA) (250, 500 and 1,000 mg/kg p.o.) in the number of acetic acidinduced writhes in mice. Indomethacin (10 mg/kg p.o.) was used as positive control. Vertical bars represent mean ± SEM of cumulated writhings in
30 min for each experimental group. *p < 0.05; ***p < 0.001. According ANOVA followed by Student-Newman-Keuls' test.
Formalin-Induced Pain
Treatment with EEA 1,000 mg/kg p.o. or morphine 10 mg/kg s.c. reduced by 41 and 99%, respectively, the licking time of hind paw after the intraplantar injection
of formalin in the neurogenic phase (0-5 min) from control values (vehicle 10 mL/kg; 62.6 ± 4.8 s). In the inflammatory phase (15-30 min), treatment with EEA
1,000 mg/kg p.o., morphine or indomethacin reduced the licking time by 48, 99 and 43% respectively, from a control value of 131.0 ± 17.1 s (Figure 2).
Figure 2 Effect of the ethanolic extract from acariçoba underground parts (EEA) (1,000 mg/kg p.o.), indomethacin (10 mg/kg p.o.) and morphine
(10 mg/kg s.c.) on the licking time of Formalin-induced pain, in mice, in the first phase (0-5 min) and the second phase (15-30 min). Vertical bars
represent mean ± SEM of reaction time pain, in seconds. *** p < 0.001 vs first phase control; ## p < 0.01 and ### p < 0.001 vs second phase control.
According ANOVA followed by Student-Newman-Keuls' test.
Hot Plate Test
Treatment with EEA increased by 60% the latency to reaction only with the highest dose (1,000 mg/kg) 1 h after the treatment from a control value (vehicle 10 mL/
kg) of 10.6 ± 0.82. The positive control morphine (10 mg/kg s.c.) increased the latency to reaction to 90 min after the treatment (Figure 3).
Figure 3 Effect of the ethanolic extract from acariçoba underground parts (EEA) (250, 500 and 1,000 mg/kg p.o.) or morphine (10 mg/kg s.c.) on
the nociceptive response of mice in the hot-plate test. Values are expressed as mean ± SEM of the latency for the nociceptive behavior. * p < 0.05;
*** p < 0.001. According ANOVA followed by Student-Newman-Keuls' test.
Carrageenan- Induced Pleurisy
The EEA (500 and 1,000 mg/kg) or dexamethasone (2.0 mg/kg) reduced by 40, 44 and 50 % the number of migrated leukocytes/ mL to the pleural cavity from a
control value of 4.8 ± 0.46 × 106 leukocytes/ mL (vehicle 10 mL/kg), respectively (Figure 4). Treatment with EEA (250, 500 and 1,000 mg/kg) reduced by 33, 38.8
and 50% the Evan's blue concentration in the pleural exudate from a control value (vehicle 10 mL/kg) by 1.8 ± 0.17 µg/mL, respectively. Dexamethasone reduced
this value by 50% (Figure 5).
Figure 4 Effect of the ethanolic extract from acariçoba underground parts (EEA) (250, 500 and 1,000 mg/kg p.o.), in carrageenan-induced pleurisy
in mice. Dexamethasone (2.0 mg/kg p.o.) was used as positive control. The bars represent the means ± SEM of number of migrated leukocytes /mL
(× 106) migrated to pleural cavity after carrageenan injection relative to control group. *** p < 0.001. According ANOVA followed by Student-NewmanKeuls' test.
Figure 5 Effect of the ethanolic extract from acariçoba underground parts (EEA) (250, 500 and 1,000 mg/kg p.o.), in carrageenan-induced pleurisy
in mice. Dexamethasone (2.0 mg/kg p.o.) was used as positive control. The bars represent the means ± SEM of Evan's blue concentration (µg/mL)
in the pleural exudate after carrageenan injection. ** p < 0.01 and *** p < 0.001. According ANOVA followed by Student-Newman-Keuls' test.
Croton Oil-Induced Ear Edema
EEA (250, 500 and 1,000 mg/kg) reduced the edema by 20, 25 and 38%, in a dose-dependent manner, from a control value (vehicle 10 mL/kg) of 17.8 ± 0.77 mg,
respectively. Dexamethasone (2.0 mg/kg) reduced the edema by 78% (Figure 6).
Figure 6 Effect of the ethanolic extract from acariçoba underground parts (EEA) (250, 500 and 1,000 mg/kg p.o.), in croton oil-induced ear edema
in mice. Dexamethasone (2.0 mg/kg p.o.) was used as positive control. Vertical bars represent mean ± SEM of the difference between left and right
ear plugs, in milligrams. * p < 0.05; ** p < 0.01; *** p < 0.001. According ANOVA followed by Student-Newman-Keuls' test.
Carrageenan-Induced Paw Edema
In this model, treatment with EEA (250, 500 and 1,000 mg/kg) or indomethacin reduced the edema (µL) at the first and second hour. At the first hour, it was
observed a reduction of 27, 44, 57 and 21% from control value (vehicle 10 mL/kg) of 55.0 ± 4.3, respectively. At the second hour, from control value of 57.66 ±
3.51 to 39.33 ± 2.54; EEA reduced the edema by 32, 59 and 47%, respectively. The indomethacin reduced the edema by 45%. After the second hour of edema, only
indomethacin was able to reduce the edema formation (Figure 7).
Figure 7 Effect of the ethanolic extract from acariçoba underground parts (EEA) (250, 500 and 1,000 mg/kg p.o.) or indomethacin (10 mg/kg p.o.)
on the carrageenan-induced paw edema. The edema was measured 1, 2, 3 and 4 h after the injection of the phogistic agent. Vertical bars represent
mean ± SEM of the difference between the volumes of the paws. *p < 0.05; **p < 0.01; ***p < 0.001. According ANOVA followed by Student-Newman-
Keuls' test.
Dextran-Induced Paw Edema
In the model of dextran-induced paw edema, treatment with EEA (1,000 mg/kg) or cyproheptadine (5.0 mg/kg) reduced the edema (µL) of 36 and 37%,
respectively, at 30 min after the injection of dextran, from a control value (vehicle 10 mL/kg) of 87.8 ± 7.4, as well as, at 60 min after the treatment, there was a
reduction of 41 and 45%, from control value (60.0 ± 7.3), respectively (Figure 8).
Figure 8 Effect of the ethanolic extract from acariçoba underground parts (EEA) (1,000 g/kg p.o.) or cyproheptadine (5.0 mg/kg p.o.) on the
dextran-induced paw edema. The edema was measured 0, 30, 60, 90 and 120 min after the injection of dextran. Vertical bars represent mean ±
SEM of the difference between the volumes of the paws. * p < 0.05 and ** p < 0.01. According ANOVA followed by Student-Newman-Keuls' test.
DISCUSSION
In the present study the analgesic and anti-inflammatory effects of the ethanolic extract (EEA) from acariçoba's underground parts was evaluated, since this plant
Santana 2001
has the popular reputation of these effects. The results obtained with this extract in the acute oral toxicity evaluation indicated no toxic effects (
).
The acetic acid induce nociception by stimulates nociceptive fibers directly, besides promote the release of endogenous mediators involved in pain modulation,
Whittle 1964 Berkenkopf and Weichman 1988 Chau 1989
among which is bradykinin, serotonin, histamine and prostaglandins (
,
,
). Treatment with EEA decreased the number
of acetic acid-induced writhing. However, this test is considered as having high sensitivity but low selectivity, thus false-positive results may occur with different
groups of drugs such as antihistaminics, central nervous system stimulants, monoamine oxidase inhibitors (MAOIs), serotonin antagonists, muscle relaxants and
Ikeda et al. 2001
neuroleptics (
, Le Bars et al. 2001).
The formalin test, on the other hand, allows the study of two types of pain. The first phase (0-5 min) corresponds to a neurogenic pain with the involvement of
Dubuisson and Dennis 1977
mediators such as substance P, bradykinin, histamine and serotonin, and the evidence of direct stimulation of nociceptors by formalin (
,
Hunskaar and Hole 1987 Shibata et al. 1989 Corrêa and Calixto 1993 Munron 2007
,
,
,
). The second phase (15-30 min) is associated with the production and release of various pro-
inflammatory mediators such as histamine, bradykinin, serotonin, prostaglandins, tachykinins and glutamate by cells activated by formalin (
and Calixto 1997 Cao et al. 1998 Omote et al. 1998
,
,
and
Fujimaki et al. 1992 Santos
,
Beirith et al. 2002
). The formalin also activates primary afferent sensory neurons through a specific and direct action on
TRPA1, which is a member of the Transient Receptor Potential family of cation channels that is highly expressed by a subset of C-fiber nociceptores (
McNamara et al.
2007
).
Considering the fact that formalin-induced pain test is more specific for pain study than abdominal contortions induced by acetic acid, with the expectation to
confirm the true analgesic activity of this EEA, we decided to work with the higher dose.
The EEA reduced the pain reactivity in both phases of the formalin test. This effect may be a TRPA1 inhibition, seen that TRPA1 antagonist receptor eliminated
McNamara et al. 2007
pain-related flinching in both phases of the formalin response in vivo (
). But also, this action in the both phases of formalin test does not allow us
to separate an anti-inflammatory activity independent of a central analgesic, making necessary to evaluate the extract in specific models of inflammation. The
inflammation is a complex process, thus it is necessary to use various inflammation models, involving the analysis of several parameters, to evaluate an antiinflammatory activity of a given substance (
Chiabrando et al. 1989
).
The highest dose of EEA also increased the nociceptive threshold to thermal stimulation 1 h after the treatment. This effect in the hot plate test suggests a central
Yaksh and Rudy 1977
analgesic activity (
).
In the carrageenan-induced pleurisy test, it is possible to evaluate an important inflammation parameter, the leukocyte migration. Carrageenan is a phlogistic agent
that when injected in the pleural cavity promotes a severe inflammation, in which various inflammatory mediators are released, such as histamine, serotonin,
cytokines and eicosanoids, resulting in an intense leukocyte migration, and an pleural exudate formation caused by protein extravasation, being this extravasation
Steele and Wilhelm 1966
indirectly quantified by Evan's blue concentration in the pleural exudate (
).
Treatment with EEA at the doses of 500 and 1,000 mg/kg reduced the number of leukocytes migration into the pleural cavity. These effects suggest a reduction in
Henriques et al. 1987
the level of mediators responsible for the cell migration, such as cytokines (IL-1 and TNF-α), nitric oxide, prostanoids, serotonin and histamine (
,
Hopkins 2003
). A similar reduction was observed in Evan's blue concentration in the pleural exudate, where the EEA promoted a reduction up to 50%, suggesting a
Saleh et al. 1997 Vianna and Calixto 1998
reduction of protein extravasation to pleural cavity (
,
).
Swingle et al. 1981
The croton oil causes leukocyte migration, increases the vascular permeability and the plasma exudation (Lapa 2003,
). The anti-edematogenic
effect observed in this method suggests an anti-inflammatory action. Nevertheless, this model does not clearly show in which stage of the inflammatory process
EEA acts.
Winter et al. 1962
The carrageenan-induced paw edema is a model of acute inflammation which involves a gradual and complex response (
). The edema evolution is
marked by the release of various inflammatory mediators such as histamine, bradykinin, serotonin and prostaglandins. These mediators are released at different
times. In the first two hours, histamine and serotonin are the primary mediators; in the third hour, prostaglandins are predominant in the acute edema, while the
complement system operates throughout the process (Di Rosa et al. 1971). In this test EEA reduced the edema in the first two hours.
Similar results were observed with extracts of leaf and stem bark of Lafoensia pacari, a Cerrado's species. These extracts showed antinociceptive activity in
Nascimento et al.
writhing test. The two phases of the formalin test reduced the ear edema and the cell migration, suggesting an analgesic and anti-inflammatory effect (
2011 Guimarães et al. 2010
Nascimento et al. (2011)
,
).
is not blocked by naloxone.
showed that this analgesic activity may be independent of anti-inflammatory action of ellagic acid and that this effect
The Spiranthera odoratissima, other Cerrado's specie popularly kwon by manacá, has only anti-inflammatory activity by reducing the writhing test, the second
phase of formalin test, the ear edema, the second and third hours of paw edema, leukocyte migration, protein extravasation and inhibition of inflammation
Barbosa et al. 2012 Nascimento et al. 2012
mediators such as phospholipase A2 and TNF-α, without analgesic effect in the first phase of the formalin and hot plate tests (
,
).
The inhibition in the edema caused by EEA in the first two hours suggests that the extract interferes with the action or release of serotonin or histamine, because
Santos and Rao 2000
these mediators are predominant in the initial phase of carrageenan-induced edema (
). The effect observed with EEA in the dextran-induced edema
model corroborates with this possibility. The dextran is a phlogistic agent known to induce edema mediated by 5-hydroxytryptamine and histamine (
Lo et al. 1982
,
Katz et al. 1984
).
Kumar et al. 2005
Histamine is a vasoactive amine, which is among the preformed mediators released during the inflammatory process (
). This amine causes
Sherwood
vasodilation and increases the vascular permeability through the action over specific receptors (H1 and H2), together with other inflammatory mediators (
and Toliver-Kinsky 2004
).
Eide and Hole 1993
In this regard, serotonin (5-hydroxtryptamine [5-HT]) has long been associated with pain processing and modulation (
spinal analgesic action of 5-HT released from brainstem structures (
). Some studies have shown a
Yaksh and Wilson 1979
). However, the peripheral action of serotonin is different from its central
Dray 1995
actions. Serotonin is considered an inflammatory mediator in the periphery, being released from platelets and mast cells after tissue injury (
).
Moreover, our results with cyproheptadine pretreatment in the edema induced by dextran suggest a blocker action on both histaminergic H1 and serotonergic 5HT2
receptors (
Rang et al. 2007
).
CONCLUSIONS
Results showed that the ethanolic extract (EEA) from acariçoba (Hydrocotyle umbellata) presents analgesic and anti-inflammatory activity in different
experimental models, suggesting an anti-migration and anti-edematous action that can be due to the blockade or inhibition in the release of histamine or serotonin.
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors are grateful to Dra. Ekaterina A. F. B. Rivera and Jackson Nascimento de Lima for technical assistance. This study was financially supported by
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Apoio à Pesquisa (FUNAPE/UFG), Fundação de Amparo à Pesquisa do
Estado de Goiás (FAPEG) e Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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Received: September20, , 2011; Accepted: April16, , 2012
Correspondence to: Elson Alves Costa E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
An. Acad. Bras. Ciênc. vol.85 no.3 Rio de Janeiro set. 2013 Epub 22-Jul-2013
http://dx.doi.org/10.1590/S0001-37652013005000041
Biological Sciences
Treadmill exercise does not change gene expression of adrenal catecholamine biosynthetic enzymes in chronically stressed rats
LJUBICA GAVRILOVI•, VESNA STOJILJKOVI•, JELENA KASAPOVI•, NATAŠA POPOVI•, SNEŽANA B. PAJOVI•, SLADJANA DRONJAK
Institute of Nuclear Sciences “Vin•a”, Laboratory of Molecular Biology and Endocrinology, University of Belgrade, 11001, Belgrade, Serbia
ABSTRACT
ABSTRACT
Chronic isolation of adult animals represents a form of psychological stress that produces sympatho-adrenomedullar activation. Exercise training acts as an important modulator of sympatho-adrenomedullary system. This study aimed to investigate physical exercise-related changes in gene
expression of catecholamine biosynthetic enzymes (tyrosine hydroxylase, dopamine-ß-hydroxylase and phenylethanolamine N-methyltransferase) and cyclic adenosine monophosphate response element-binding (CREB) in the adrenal medulla, concentrations of catecholamines and
corticosterone (CORT) in the plasma and the weight of adrenal glands of chronically psychosocially stressed adult rats exposed daily to 20 min treadmill running for 12 weeks. Also, we examined how additional acute immobilization stress changes the mentioned parameters.
Treadmill running did not result in modulation of gene expression of catecholamine synthesizing enzymes and it decreased the level of CREB mRNA in the adrenal medulla of chronically psychosocially stressed adult rats. The potentially negative physiological adaptations after treadmill
running were recorded as increased concentrations of catecholamines and decreased morning CORT concentration in the plasma, as well as the adrenal gland hypertrophy of chronically psychosocially stressed rats. The additional acute immobilization stress increases gene expression of
catecholamine biosynthetic enzymes in the adrenal medulla, as well as catecholamines and CORT levels in the plasma.
Treadmill exercise does not change the activity of sympatho-adrenomedullary system of chronically psychosocially stressed rats.
Keywords: adrenal medulla; acute immobilization stress; catecholamine; chronic social isolation; gene expression; treadmill exercise
RESUMO
RESUMO
O isolamento crônico de animais adultos representa uma forma de estresse psicológico que produz ativação do sistema simpático adrenomedular. O treinamento físico atua como um importante modulador do sistema simpático adrenomedular. Este estudo tem como objetivo investigar mudanças
relacionadas ao exercício físico na expressão dos genes de enzimas envolvidas na biossíntese de catecolaminas (tirosina hidroxilase, dopamina-B-hidroxilase e feniletanolamina N-metiltransferase) e da proteína de ligação ao elemento de resposta ao monofosfato cíclico de adenosina (CREB) na
medula adrenal, concentrações de catecolaminas e corticosterona (CORT) no plasma e o peso das glândulas adrenais de ratos adultos psicologicamente estressados por exposição diária a 20 min de corrida em esteira durante 12 semanas. Além disso, examinamos como o estresse adicional por
imobilização aguda altera os parâmetros mencionados. O treinamento em esteira não resultou na modulação da expressão dos genes de enzimas que sintetizam catecolaminas, mas houve uma diminuição dos níveis de RNAm de CREB na medula adrenal de ratos adultos com estresse
psicossocial crônico. As adaptações psicológicas potencialmente negativas após corrida na esteira foram registradas como um aumento na concentração de catecolaminas e diminuição matinal nas concentrações de CORT no plasma, bem como na hipertrofia da glândula adrenal de ratos
psicossocialmente estressados. O estresse adicional por imobilização aguda aumenta a expressão dos genes de enzimas da via de biossíntese de catecolaminas na medula adrenal, bem como os níveis de catecolaminas e CORT no plasma. O exercício em esteira não altera a atividade do sistema
simpático adrenomedular de ratos com estresse psicossocial crônico.
Palavras-Chave: medula adrenal; estresse por imobilização aguda; catecolaminas; isolamento social crônico; expressão gênica; exercício em esteira
INTRODUCTION
A number of diseases and pathological conditions are related to the long-term adaptive response to stress, particularly under conditions of chronic stress when allostasis can shift from a healthy toward a pathological state. Chronic individual housing of rats, frequently termed “isolation
Bartolomucci et al. 2003 Võikar et al. 2005
stress”, represents a very strong psychosocial stress (
,
stress. In humans, regular exercise has a beneficial impact on depression (
Dunn et al. 2005
Ishida et al. 2003
). It is known that social isolation is a risk factor for human depression (
). The literature data indicate that exercise training reduces the risk of developing diseases related to chronic
). Changes in catecholamine biosynthesis represent one of the mechanisms by which exercise training may diminish circulating catecholamine levels. The exercise training lowers plasma
Seals et al. 1994
Fleshner (2000)
noradrenaline (NA) concentration in elderly patients with elevated baseline levels (
).
found that 4 weeks of freewheel running attenuates the elevation of plasma noradrenaline produced by acute exposure to inescapable tail shock stress. Although a number of
markers are frequently used to assess the involvement of the sympatho-adrenal response (plasma and tissue noradrenaline and adrenaline levels), it is important to examine more specific variables such as gene expression of key enzymes involved in catecholamine biosynthesis. Tyrosine
Kvetnansky et al. 2004
hydroxylase (TH), as the “rate limiting” enzyme in the biosynthesis of catecholamines, is localized in all cells that produce catecholamines (
NA. During repeated immobilization stress DBH may become “rate limiting” when dopamine formation is markedly accelerated (
ethanolamine N-methyltransferase (PNMT) is considered as the “rate limiting” enzyme for the synthesis of adrenaline (A) (
). Dopamine-ß-hydroxylase (DBH) is another important catecholamine biosynthetic enzyme that converts dopamine (DA) into
). In addition,
Scatton et al. (1984)
showed that under conditions of neural activation DBH becomes “rate limiting” enzyme. Phenyl
).
Gavrilovi• et al. 2008
Our earlier studies showed that long-term social isolation (12 weeks) of adult rat males produced a decreased gene expression of catecholamine biosynthetic enzymes in the adrenal medulla (
Gavrilovi• et al.
), and increased concentrations of catecholamines in the plasma (
2010
). In this study we wanted to investigate whether treadmill exercise changed the activity of sympatho-adrenomedullary system of chronically psychosocially stressed rats. We applied a combined model of chronic social isolation and treadmill running (CSITR) in rats. One of the key
questions in stress research is how the same stressor can elicit a variant or altered response depending on prior experience with the current or different stressor. Immobilization is frequently used as an additional acute stressor and considered as one of the most intensive stressors that
significantly changes gene expression (
2008
Gavrilovi• et al.
). Our previous data showed that exposure of socially isolated rats to additional immobilization produced exaggerated responses in gene expression of catecholamine biosynthetic enzymes in the adrenal medulla (
). The response to novel additional acute immobilization stress might reveal the detailed mechanisms underlying the gene expression of catecholamine biosynthetic enzymes in the adrenal medulla in specific stress conditions.
This study aimed to investigate physical exercise-related changes in gene expression of catecholamine biosynthetic enzymes tyrosine (TH, DBH and PNMT) and expression of cAMP response element-binding protein (CREB) in the adrenal medulla, concentrations of catecholamines
and morning corticosterone (CORT) in the plasma and the weight of adrenal glands of chronically psychosocially stressed adult rats exposed daily to 20 min treadmill exercise for 12 weeks. In addition, we examined the impact of the additional acute immobilization stress (CSITR
+IMM) on the mRNA and protein levels of catecholamine biosynthetic enzymes in the adrenal medulla, as well as concentrations of NA, A and CORT in the plasma of rats. Levels of CORT were measured in the morning because previous findings had shown that light is capable of
eliciting a rapid CORT response in rats (
Mohawk et al. 2007
), humans (
Leproult et al. 2001
) and mice (
Ishida et al. 2005
).
Detecting regulatory physiological mechanism for catecholamine synthesis in the adrenal medulla in conditions provoked by prolonged social isolation stress and physical activity is extremely important in the prevention of diseases caused by chronic stress in sports medicine and
pathophysiology.
Animals and Stress Models
In this study Wistar male rats 11-week-old were used. Animals were under standard laboratory conditions with water and food ad libitum and kept three to four per cage. The care was taken to minimize the pain and discomfort of the animals according to the recommendations of the
Ethical Committee of the Vin•a Institute of Nuclear Sciences, Belgrade, Serbia, which are in accordance with the Guide for Care and Use of Laboratory Animals of the National Institute of Health, Bethesda, MD, U.S.A. The experiment is complied with the current laws of the Republic
of Serbia.
Animals were divided into four groups. The control group (n = 10) was not exposed to any treatment. CSITR group (n = 10) consisted of animals exposed to treatment of chronic combined social isolation and treadmill running. CSITR treatment was achieved by exposing the
individually housed rats to the daily treadmill running for a period of 12 weeks. The duration and speed of running was gradually increased from week to week, from the initial 10 minutes-10m/min up to 20 minutes-20m/min at 00 incline. The treadmill training protocol used in this
study involves a gradual increase in running intensity and is commonly used in the similar studies (
Kvetnansky and Mikulaj (1970)
Tümer et al. 2001 Erdem et al. 2002
,
). IMM group (n = 40) consisted of animals exposed to acute stress immobilization, for a period of 2 hours. Immobilization stress was provoked as
described by
. CSITR+IMM group (n = 40) consisted of animals exposed to CSITR treatment for a period of 12 weeks and, after chronic treatment, these animals were exposed to additional acute IMM stress for a period of 2 hours. Groups IMM and CSITR
+IMM contained 40 animals. These groups were divided into four subgroups (n = 10), as we examined changes in catecholamine biosynthetic enzymes in four different time periods after the cessation of immobilization. The animals were sacrificed after CSITR treatment, immediately
Kvetnansky et al. 2009 Wong and Tank 2007
after the cessation of acute immobilization and 3, 6 and 22 hours after the acute immobilization. Literature data show that in these periods, changes in gene expression of catecholamine biosynthetic enzymes in the adrenal medulla are expected (
,
).
Fliedner et al. 2010
Samples of blood were collected and both adrenal glands were isolated and weighed. Separation of cortex and medulla was performed as previously described (
). The tissue was kept frozen on dry ice at all times. The adrenals were cut centrally, perpendicular to the
longest axis. The surface cut was visually inspected for the presence of adrenal medulla. The border between the cortex and medulla was easily identified by the red/brown color of the cortical zona reticularis. The tissue was again cut perpendicular to the longest axis at a distance of 1
cm from the first cut, until an area with a wider section of medulla was found. The tissues were examined under the dissection microscope, and the gray/pink medulla was carefully cut out of the surrounding cortex with size 11 scalpel blades. The adrenal medullas were stored in liquid
nitrogen until analyzed. The morning CORT was measured to determine whether CSITR affects the diurnal rhythm of CORT in this experiment.
RNA Isolation and cDNA Synthesis
Total RNAs were isolated using TRIZOL reagent (Invitrogen, USA). After the isolation of mRNA, DNA-ase treatment was applied with DNAse I (Fermentas, Lithuania). Concentration of total mRNA was measured in triplicates on a spectrophotometer. Quality of mRNA was checked
on agarose gel. Reverse transcription was performed using Ready-To-Go You-Prime First-Strand Bead (Amersham Biosciences, UK) and pd (N)6 Random Hexamer (Amersham Biosciences, UK) primer according to the manufacturer's protocol.
Real-Time RT-PCR
TaqMan PCR assays were carried out using Assay-on-Demand Gene Expression Products (Applied Biosystems,USA) for TH (Rn00562500_m1), DBH (Rn00565819_m1), PNMT (Rn01495589_g1) and CREB (Rn01441386_g1). The gene expression assays contained primers for
amplification of the target gene and the TaqMan MGB (Minor Groove Binder) probe 6-FAM dye-labeled for the quantification. Reactions were performed in a 25 µL reaction mixture containing 1× TaqMan Universal Master Mix with AmpErase UNG, 1× Assay Mix (Applied
Biosystems, USA) and cDNA template (10 ng of RNA converted to cDNA). PCR was carried out in the ABI Prism 7000 Sequence Detection System at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. The experimental threshold was
calculated based on the mean baseline fluorescence signal from cycle 3 to 15 plus 10 standard deviations. Each sample was operated in triplicates and the mean value of each Ct triplicate was used for further calculations. The reference gene (endogenous control) was included in each
analysis to correct for the differences in the inter-assay amplification efficiency and all transcripts were normalized to cyclophyline A (Rn00690933_m1) expression. The reaction mixture for endogenous control gene amplification consisted of 1x TaqMan Universal Master Mix with
AmpErase UNG (Applied Biosystems, USA), 1x Assay (6-FAM dye-labeled MGB probes) and cDNA (10 ng of RNA converted to cDNA). The levels of expression of cyclophyline A in samples under different treatments were checked by additional experiments that confirmed that the
chosen reference gene was not regulated. Before quantification, validation experiments were performed to determine the similar amplification efficiency of endogenous control and each target gene. We tested cyclophyline A and demonstrated that its efficiency of amplification was
approximately equal to all assays used for target genes. Briefly, serial dilutions of cDNA were prepared and amplified by real-time PCR using specific primers and fluorogenic probes for target and endogenous control gene.
Quantification was done using the 2−∆∆Ct method according to
. The results obtained were analyzed by the RQ Study Add On software for 7000 v 1.1 SDS instrument (ABI Prism Sequence Detection System, Applied Biosystems, USA) with a confidence level of
95% (P < 0.05). The relative expression of the target gene was normalized to cyclophyline A and expressed in relation to the calibrator, i.e. the control sample. Due to individual differences among animals, one sample from control group with the expression value closest to the mean of
all samples in this group and with the lowest measurement error was chosen as a calibrator. The results are reported as a fold change relative to the calibrator and normalized to cyclophyline A using the equation: Nsample = 2−∆∆Ct.
Livak and Schmittgen (2001)
Western Blot Analysis
Stich (1990)
Laemmli (1970)
The adrenal medullas were homogenized in 0.05 M sodium phosphate buffer (pH 6.65). Subsequently, the protein concentration was determined using BCA method (Pierce, USA), described by
. The samples were boiled in denaturing buffer according to
, for 5
min at 95°C. Fifteen micrograms of protein extract from adrenal medulla were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to a supported nitrocellulose membrane (Hybond™ C Extra, Amersham Biosciences, UK). The membrane was blocked in 5%
™
non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST). All following washes and antibody incubations were also carried out in TBST at room temperature on a shaker. The antibody incubation time was 1 hour. Protein molecular mass standards (PageRuler Plus
Prestained Protein Ladder, Fermentas) were used for calibration. Antibodies used for quantification of specific proteins were as follows: for TH the monoclonal primary antibody against mouse TH (monoclonal antibody against TH from mouse-mouse hybrid cells, clone 2/40/15,
dilution 1:5000, Chemicon International, USA); for DBH the anti-dopamine-ß hydroxylase (N-terminal) antibody, sheep (dilution 1:5000, Sigma, USA); for PNMT the polyclonal ant-PNMT primary antibody, rabbit (dilution 1:1000, Protos Biotech Corporation, USA); and for ß-actin
the rabbit polyclonal anti-ß-actin (ab8227, dilution 1:5000, Abcam, USA). After washing, the membranes were incubated in the secondary anti-mouse, anti-rabbit (dilution 1:5000, Amersham ECLTM Western Blotting Analysis System, UK) and anti-sheep (dilution 1:5000,
Calbiochem, Germany) antibodies conjugated to horseradish peroxidase. A secondary antibody was then visualized by the Western blotting enhanced chemiluminiscent detection system (ECL, Amersham Biosciences, UK). The membranes were exposed to ECL film (Amersham
Biosciences, UK). Densitometry of protein bands on ECL film was performed by Image J analysis PC software. The result was expressed in arbitrary units normalized in relation to ß actin.
Catecholamine and Cort Measurements
Peuler and Johnson (1977)
Plasma catecholamines were measured by a standard radioenzymatic assay described previously by
and the values were expressed as pg/mL plasma. Catecholamines present in the plasma aliquots were converted to their labeled O-methylated derivatives by S-(3H)
adenosylmethionine (Lacomed, Czech Republic) and the lyophilized catechol-O-methyl transferase isolated from the rat liver. The O-methylated derivatives of the amines were then extracted along with unlabeled carrier compounds.
Plasma CORT was measured upon prior extraction directly, using RIA commercial kits (MP Biomedicals, Germany) and the values were expressed as ng CORT/mL plasma.
DATA ANALYSIS
The data are presented as means ± S.E.M. Differences of gene expression (mRNA and protein levels) of catecholamine biosynthetic enzymes (TH, DBH, PNMT) and level of CREB mRNA in the adrenal medulla and concentration of NA, A and CORT in the plasma, as well as weight
of adrenal gland were analyzed by One-way ANOVA. The effects of chronic social isolation and treadmill running (CSITR) and acute immobilization stress (IMM) compared to control, as well as the effects of additional acute immobilization stress after chronic social isolation and
treadmill running (CSITR+IMM) compared to chronic social isolation and treadmill running (CSITR), were tested by Tukey post-hoc test.
The correlation between PNMT protein level and A level of animals exposed to CSITR was analyzed by the Spearman test, using the Sigma Plot v10.0 (with SigmaStat integration).
Statistical significance (p) was set to 0.05, statistical power (1-ß) exceeded 82%. Statistical power confirms that the number of animals (n = 10) was sufficient for this experiment. Reliability test was designed so we did three repeated measurements of the level of gene expression of TH,
DBH, PNMT and CREB. The calculated value of the ICCR test of >0.80 was considered to be satisfactory and it proves the reliability of the applied methods. Statistical analysis was carried out using the SPSS.
RESULTS
One-way ANOVA analysis revealed significant changes of TH (F = 10.19; p < 0.01), DBH (F = 7.22; p < 0.05), PNMT (F = 11.78; p < 0.05), CREB (F = 12.29; p < 0.01) mRNA levels, and TH (F = 8.18; p < 0.01), DBH (F = 7.12; p < 0.05), PNMT (F = 16.20; p < 0.01) protein levels
in the adrenal medulla, as well as NA (F = 19.28; p < 0.01), A (F = 21.7; p < 0.01) and CORT (F = 24.7; p < 0.01) plasma concentrations and the weight of adrenal glands (F = 11.07; p < 0.05) under examined treatments.
Changes in the Plasma Concentrations of NA, A, Cort and the Weight of Adrenal Glands
CSITR treatment
CSITR treatment significantly increased the plasma concentrations of NA by 78% (p < 0.01, Tukey test, Fig. 1a) and A by 87% (p < 0.01, Tukey test, Fig. 1b), decreased CORT concentration by 73% (p < 0.01, Tukey test, Fig. 1c) and increased weight of adrenal glands by 26% (p <
0.05, Tukey test, Fig. 1d), compared with control animals.
Figure 1 Effects of chronic social isolation and treadmill running and additional acute immobilization stress on the concentration of noradrenaline (NA) [a], adrenaline (A) [b] and corticosterone (CORT) [c] in
the plasma and the weight of adrenal gland [d]. The values are means ± S.E.M. of 10 rats. Statistical significance: +p < 0.05, ++p < 0.01 animals exposed to chronic social isolation and treadmill running vs. control animals (Tukey test); *p < 0.05, **p
< 0.01 animals exposed to acute 2h immobilization vs. control animals (Tukey test); #p < 0.05, ##p < 0.01 animals exposed to additional acute 2h-immobilization stress after chronic social isolation and treadmill running vs. animals exposed to chronic social
isolation and treadmill running (Tukey test).
IMM and CSITR+IMM treatments
The exposure of the control animals to acute immobilization stress significantly increased NA concentration by 185% (p < 0.01, Tukey test, Fig. 1a) and A concentration by 112% (p < 0.01, Tukey test, Fig. 1b), while the additional acute immobilization of CSITR animals increased NA
concentration by 20% (p < 0.05, Tukey test, Fig. 1a) and A concentration by 20% (p < 0.05, Tukey test Fig. 1b) 3 hours after the cessation of immobilization. After 22 hours, the plasma NA in the control group was reduced by 40% when comparing to 3 hours. However, after 22 hours
the NA plasma level in CSITR group was higher by 26% when compared to 3 hours. Also, we found that acute IMM increased CORT concentration by 66% (p < 0.05, Tukey test, Fig. 1c), while the exposure of CSITR animals to additional acute immobilization stress led to increased
CORT concentration by 212% (p < 0.01, Tukey test, Fig. 1c) in the plasma immediately after the cessation of immobilization. After 3 hours, the plasma CORT in the control group returned to the basal value, while in CSITR group was reduced by 40% when comparing to the value
immediately after the cessation of immobilization.
Changes of the TH, DBH, PNMT and Creb mRNA Levels in the Adrenal Medulla
CSITR treatment
The animals exposed to CSITR showed a decreased levels of TH mRNA by 10% (p < 0.05, Tukey test, Fig. 2a), DBH mRNA by 11% (p < 0.05, Tukey test, Fig. 2b), PNMT mRNA by 20% (p < 0.05, Tukey test, Fig. 2c) and CREB mRNA by 50% (p < 0.01, Tukey test, Fig. 2d),
compared with control animals.
Figure 2 Effects of chronic social isolation and treadmill running and additional acute immobilization stress on tyrosine hydroxylase (TH) [a], dopamine-ß-hydroxylase (DBH) [b], phenylethanolamine Nmethyltransferase (PNMT) [c] and cAMP response element-binding (CREB) [d] mRNA levels in the adrenal medulla. The values are means ± S.E.M. of 10 rats. Statistical significance: +p < 0.05, ++p < 0.01 animals exposed to chronic
social isolation and treadmill running vs. control animals (Tukey test); **p < 0.01 animals exposed to acute 2h immobilization vs. control animals (Tukey test); #p < 0.05, ##p < 0.01 animals exposed to additional acute 2h immobilization stress after chronic social
isolation and treadmill running vs. animals exposed to chronic social isolation and treadmill running (Tukey test). The final result was expressed as fold change relative to the calibrator and normalized to cyclophyline A.
IMM treatment significantly increased the level of TH mRNA by 110% (p < 0.01, Tukey test) 3 hours after the cessation of immobilization and by 100% (p < 0.01, Tukey test) 6 hours after the cessation of immobilization, compared to the control group (Fig. 2a). The additional acute
immobilization of CSITR animals increased mRNA levels of TH by 111% (p < 0.01, Tukey test, Fig. 2a), DBH by 46% (p < 0.05, Tukey test, Fig. 2b) 3 hours after the cessation of immobilization and PNMT by 14% (p < 0.05, Tukey test, Fig. 2c) 6 hours after the cessation of
immobilization, compared to the CSITR group.
Changes of the TH, DBH and PNMT Protein Levels in the Adrenal Medulla
CSITR treatment
CSITR provoked a decreased protein levels of TH by 64% (p < 0.01, Tukey test, Fig. 3a) and of DBH by 20% (p < 0.05, Tukey test, Fig. 3b); however, it induced the increase of PNMT protein level by 18% (p < 0.05, Tukey test, Fig. 3c), compared with the controls. The significant
positive correlation was found between the levels of PNMT protein in the adrenal medulla and A concentration in the plasma of animals exposed to CSITR (Spearman ρ = 0.904, P < 0.0005, Fig. 4).
Figure 3 Effects of chronic social isolation and treadmill running and additional acute immobilization stress on tyrosine hydroxylase (TH) [a], dopamine-ß-hydroxylase (DBH) [b] and phenylethanolamine Nmethyltransferase (PNMT) [c] protein levels in the adrenal medulla. The values are means ±S.E.M. of 10 rats. Statistical significance: +p < 0.05, ++p < 0.01 animals exposed to chronic social isolation and treadmill running vs. control animals;
(Tukey test); *p < 0.05 animals exposed to acute 2h immobilization vs. control animals (Tukey test); #p < 0.05, ##p < 0.01 animals exposed to additional acute 2h immobilization stress after chronic social isolation and treadmill running vs. animals exposed to
chronic social isolation and treadmill running (Tukey test). The result was expressed in arbitrary units normalized in relation to ß actin. (A) Distribution of TH, DBH, PNMT and ß-actin proteins in the adrenal medulla of control animals [I], animals exposed to
CSITR [II], animals exposed to IMM (3h after immobilization) [III], animals exposed to CSITR+IMM (3h after immobilization) [IV], animals exposed to IMM (22h after immobilization) [V] and animals exposed to CSITR+IMM (22h after immobilization) [VI].
Figure 4 The correlation between PNMT protein level in the adrenal medulla and A level in the plasma of animals exposed to chronic social isolation and treadmill running (Spearman).
Acute IMM increased the protein levels of TH by 10% (p < 0.05, Tukey test, Fig. 3a) and PNMT by 42% (p < 0.05, Tukey test, Fig. 3c) 22 hours after the cessation of immobilization, while the additional exposure of CSITR animals to acute immobilization stress led to increased
protein levels of TH by 100% (p < 0.05, Tukey test, Fig. 3a), and PNMT by 133% (p < 0.01, Tukey test Fig. 3c) 3 hours after the cessation of immobilization and DBH by 33% (p < 0.05, Tukey test, Fig. 3b) 22 hours after the cessation of immobilization.
DISCUSSION
Changes in the Plasma Concentrations of NA, A and Cort
Micutkova et al. 2003
When the sympathoadrenal system is activated repeatedly over a long period of time, the response is not only adaptive, but also maladaptive (
). It is known that catecholamine hyperactivity and glucocorticoid disregulation are the biological consequences of chronic
stress. In our previous study (
), we found that long-term isolation stress caused the increased concentration of catecholamines in the plasma. Although we expected positive adaptations in this study, we observed that CSITR produced adaptive physiological changes,
indicating chronic stress. The potentially negative physiological adaptations after CSITR were recorded as increased concentration of catecholamines and decreased morning CORT concentration in the plasma, as well as the adrenal gland hypertrophy. Treadmill running does not
change the activity of sympatho-adrenomedullary system of chronically psychosocially stressed rats. According to the literature data, treadmill running is forced exercise and it is a combination of hard physical and psychological stressors.
Moraska et al. (2000)
found that the treadmill
Pagliari and Peyrin 1995
training for a period of 8 weeks may cause physiological adaptations indicative of chronic stress. Also, the treadmill running stimulated concomitantly peripheral catecholamines secretion and central noradrenergic activity, i.e. NA turnover and release (
study, we observed that CSITR provoked a decreased morning CORT concentration in the plasma.
Zarkovi• et al. (2003)
). In this
showed that chronic stress is associated with a transient suppression of the HPA axis, manifested by the lower morning cortisol and the reduced adrenal cortisol
response to ACTH stimulation. This HPA response pattern is manifested by decreased morning plasma or urinary cortisol, especially in the subjects with post-traumatic stress disorder (PTSD) (
Yehuda et al. 2002
). It is interesting to note that reduced concentration of CORT in the plasma
Malkesman et al. 2006
was recorded in depressed conditions (
evidence of depressive conditions (
). Adrenal hypertrophy found in our experiments may be interpreted as the consequence of stress conditions. The weight of the adrenal gland may be a reliable criterion of the experienced chronic stress and may serve as the
Westenbroek et al. 2003
Nemeroff et al. 1992
). Hypertrophy of the adrenal glands has also been found in depressed patients (
), indicating that adrenal size provides a good measure of the stress perception over periods of time.
Response to the acute IMM stress is characterized by the activation of the sympatho-adrenomedullary system and the HPA axis. Our results show that the acute IMM stressor triggers an exaggerated elevation of the plasma catecholamines. However, heterotypic novel additional acute
IMM stressor does elevate the plasma catecholamines but not excessively in the animals previously exposed to CSITR. This finding might be explained by the quality and especially intensity of the stressor used. Immobilization is considered as one of the most intensive stressors. The
novel stressors elicit exaggerated responses in prestressed animals, when the novel stressor is of equal or greater intensity or duration and/or it is repeated (
). In this work, animals exposed to CSITR are already prepared to manage the new situation evoked by a novel
stressor and the exaggerated response is not necessary. Our results, together with above mentioned data, show that the CSITR induces adaptations that are indicative of chronic stress.
Changes of the TH, DBH and PNMT Gene Expression in the Adrenal Medulla
In our previous study we showed that long-term isolation had induced a reduction gene expression of catecholamine biosynthetic enzymes in the adrenal medulla (
). Also, in the present study we have found that the CSITR has induced a reduction of TH and DBH
mRNAs and protein levels in the adrenal medulla. Treadmill exercise did not result in further modulation of gene expression of catecholamine synthesizing enzymes in the adrenal medulla of chronically psychosocially stressed adult rats. In the adrenal medulla, the DNA-binding
Erdös et al. 2007
activities of AP-1 and CREB play a major role in regulating the expression of TH and DBH genes during forced exercise (
). Transcription factor CREB may be important in establishing the stress-induced patterns of gene expression. For these reasons, we analyzed the
transcription factor involved in the down regulation of TH and DBH gene expression. Our results demonstrate that reduced level of CREB mRNA coincide with the reduced TH and DBH mRNA levels. Many authors have confirmed that chronic stress is associated with the reduction of
Trentani et al. (2002)
Wang et al. (2006)
phospho-CREB expression.
showed that in male rats chronically exposed to a mild electrostimulation, phospho-CREB expression was reduced, especially in the subcortical and cortical region.
observed that chronic stress significantly reduces the
expression of cAMP dependent kinase A (PKA) and phospho-CREB in the hippocampus of rats. However, in stressed rats treated with fluoxetine, the expression of phospho-CREB was significantly increased, indicating that chronic stress can affect the PKA and phospho-CREB
expression and that the antidepressant is an antagonist. In addition to that, the decreased TH and DBH gene expression may be the consequence of the adrenomedular cell desensitization. The excessive stimulation by chronic stress could provoke the adrenomedular cell desensitization
and depress the catecholamine synthesis pathway. It is known that the glucocorticoids regulate the expression and enzyme activity of TH and DBH genes (
the plasma, which coincides with the decrease of TH and DBH mRNA levels in the adrenal medulla.
Núñez et al. 2009 Hwang and Joh 1993 McMahon and Sabban 1992
,
.
). We found that CSITR induces the decrease of CORT concentration in
A significant result in this work is that CSITR does not affect de novo synthesis of TH enzyme, but increases the concentration of NA in the plasma. Many factors can affect the activity of TH enzyme without changing its expression. One of the answers may be in the intracellular level
Baruchin et al. 1990
of tetrahydrobiopterin, which can be altered by stress and sympathetic nervous activity and thus, it may affect the activity of TH without changing the level of the enzyme (
). Many studies have shown that noncholinergic neurotransmitters affect the biosynthesis of
Bobrovskaya et al. (2007)
catecholamines.
observed that the amount of TH enzyme is regulated by pituitary adenylate cyclase-activating peptide (PACAP). Specifically, prolonged activation of TH enzyme resulting from phosphorylation of TH at Ser 40 can maintain the synthesis of
catecholamine without synthesis of TH enzyme. Although expression of TH and DBH gene is decreased in the adrenal medulla after CSITR treatment regimes, this treatment may lead to continuous increased biosynthesis of NA as well as increased releasing of NA in plasma, which
might represent an adaptation on applied stress regime. Also, it is important that the sympathetic nervous system (stellate ganglia) may be a source of NA in the circulation after chronic stress. In our previous work (
TH and DBH gene expressions in stellate ganglia. Increased synthesis of TH and DBH enzymes in stellate ganglia causes the increase in NA plasma levels, which is in accordance with the reports of
Sabban et al. (2004)
), we found that chronic stress causes the increase of
.
Glucocorticoids are important regulators of PNMT gene expression. In this work, we observed that CSITR induces the decrease of CORT concentration in the plasma, coinciding with the decrease of PNMT mRNA level in the adrenal medulla. Studies on the hypophysectomised rats
Evinger et al. 1992 Wong et al. 1995 Krizanova et al. 2001
have shown that reduced amounts of corticosteroids cause the reduction of level of PNMT mRNA level (
,
,
). A significant result in this study is that CSITR induces the increase of PNMT protein level in the adrenal medulla, with the
consequent increase of A level in the plasma. We found a significant positive correlation between the levels of PNMT protein in the adrenal medulla and A in the plasma of the animals exposed to CSITR. It is interesting to note that although PNMT mRNA is decreased after CSITR
treatment regimes, this treatment may lead to continuous accumulation of its proteins as an adaptation on applied stress regime. This adaptive response is necessary to maintain the A biosynthetic capacity in the adrenal medulla during periods of sustained A secretion. Long term stress
induces appropriate translational mechanisms (
Xu et al. 2007
). During CSITR treatment the increased synthesis of PNMT protein affects the sustained increase of A secretion. Our results confirm that the CSITR shows adaptations that are indicative of chronic stress.
In this study, we showed that acute immobilization stress induces the increase of TH and PNMT protein levels in the adrenal medulla and levels of cateholamines in the plasma. However, acute immobilization does not change the levels of DBH and PNMT mRNAs, as well as the
protein levels of DBH.
Nankova et al. (1999)
found that the DBH gene expression changes are caused by prolonged or repeated stress. A significant result in this work is that chronically stressed animals (CSITR) have statistically more significant expression of TH, DBH and PNMT genes
after additional acute immobilization stress compared with the animals exposed to acute immobilization stress.
Wong et al. (2002)
reported that PNMT protein and enzymatic activity change require additional time of approximately 18–20 hours to reach maximum stimulated levels.
Kvetnansky et al. (2006)
showed that in rats exposed repeated immobilization stress (7 days), 3 hours after a termination of immobilization stimulus PNMT level in the adrenal medulla was significantly increased. We found significantly elevated levels of TH and PNMT proteins 3 hours
after, and DBH proteins 22 hours after the cessation IMM in chronically stressed animals. This data suggest the possibility of increased catecholamine synthesis in the adrenal medulla of chronically stressed animals after novel immobilization stress. This could mean that prior
experience may condition physiological systems to “expect” a problem and therefore, be more ready to respond to a novel additional acute stressor. The readiness of the organism prolongly exposed to homotypic stressor to respond to a heterotypic stressor by an exaggerated expression
of catecholamine biosynthetic enzymes that is considered to be an important adaptive phenomenon of the sympatho-adrenomedullary system in rats (
).
Our results confirm that the CSITR shows adaptations that are indicative of chronic stress. We found that treadmill running did not result in modulation of gene expression of catecholamine synthesizing enzymes in the adrenal medulla of chronically psychosocially stressed adult rats. A
significant result in this study is that during CSITR treatment the increased synthesis of PNMT protein in the adrenal medulla affects the sustained increase of A secretion. The potentially negative physiological adaptations after CSITR were recorded as increased concentrations of
catecholamines in the plasma. It is known that increased level of catecholamines in the plasma after chronic stress is the allostatic load that may provoke disease indicative of sports medicine and pathophysiology. The results presented here confirm that the chronic treadmill running in
rats is forced exercise. An alternative to forced exercise paradigms would be to allow experimental animals free access to run wheels and to allow voluntary exercising for extended periods of time. Using this method, the stressor effects of forced training schedules could be avoided.
Acknowledgements
This work was supported by the Ministry of Education and Science of the Republic of Serbia, Contract No. III 41027, III 41022 and OI 173044. The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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Received: October18, , 2011; Accepted: April27, , 2012
Correspondence to: Ljubica Gavrilovi• E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013000300012
Biological Sciences
Impacts of hydroelectric dams on alluvial riparian plant communities in eastern Brazilian Amazonian
LEANDRO VALLE FERREIRA1, DENISE A. CUNHA1, PRISCILLA P. CHAVES1, DARLEY C.L. MATOS1, PIA PAROLIN2
1
2
Museu Paraense Emílio Goeldi, Coordenação de Botânica, Avenida Perimetral, 1901, Bairro Terra Firme, 66077-530 Belém, PA, Brasil
Biodiversität, Evolution und Ökologie der Pflanzen/BEE, Biozentrum Klein Flottbek und Botanischer Garten, Universität Hamburg, Ohnhorststr., 18, 22609 Hamburg, Deutschland
ABSTRACT
The major rivers of the Amazon River basin and their biota are threatened by the planned construction of large hydroelectric dams that are expected to have strong impacts on floodplain plant communities. The present study presents forest inventories from three floodplain sites colonized by alluvial riparian vegetation in
the Tapajós, Xingu and Tocantins River basins in eastern Amazonian. Results indicate that tree species of the highly specialized alluvial riparian vegetation are clearly distinct among the three river basins, although they are not very distinct from each other and environmental constraints are very similar. With only 6 of 74
species occurring in all three inventories, most tree and shrub species are restricted to only one of the rivers, indicating a high degree of local distribution. Different species occupy similar environmental niches, making these fragile riparian formations highly valuable. Conservation plans must consider species
complementarily when decisions are made on where to place floodplain forest conservation units to avoid the irreversible loss of unique alluvial riparian vegetation biodiversity.
Keywords: Amazon River basin; biodiversity loss; floodplain forest; hydroelectric dams
RESUMO
Os principais rios da bacia Amazônia e sua biota estão ameaçados pelos planos de construção de grandes usinas hidrelétricas que poderá causar fortes impactos nas comunidades vegetais das áreas alagadas. O objetivo desse estudo é apresentar os inventários botânicos em três áreas de vegetações inundadas dos rios
Tocantins, Xingu e Tapajós no leste da Amazônia. Os resultados do estudo mostram uma flora altamente especializada das vegetações inundadas, claramente distintas entre as três bacias, embora os ambientes entre as bacias dos rios não sejam distintos entre si e as condições ambientais muito semelhantes. Somente 6 das
74 espécies ocorreram nos três inventários, sendo a maioria das arvores e arbustos restritos a um dos rios, indicando um alto grau de distribuição local. Espécies diferentes ocupam nichos ambientais semelhantes, mostrando alto valor de conservação dessas formações vegetais. Os planos de conservação devem considerar a
complementaridade das espécies nas tomadas de decisões de onde colocar as unidades de conservação nesses nas florestas inundadas a fim de evitar de forma irreversível a perda da biodiversidade.
Palavras-Chave: Amazônia; perda de biodiversidade; florestas inundáveis; usinas hidrelétricas
INTRODUCTION
Fearnside 1995
In the Brazilian Amazon River basin, two huge hydroelectric dams are currently in operation: Balbina, in Amazonas state, and Tucuruí, Pará state. Their construction caused the flooding of thousands of square kilometers of forested area (
). At the moment, two large hydroelectric dams are under
construction: Jirau and Santo Antônio on the Madeira River, in Rondônia, and the construction of the Belo Monte hydroelectric dam on the Xingu River, in Pará state, began in the second semester of 2011. The planning of six new hydroelectric dams is now in progress. These are for the Tapajós and Jamanxin
Tundisi 2007
Rivers in Pará state, totaling 2,600 km2 of flooded area and affecting directly many protected conservation areas and indigenous lands (
).
The hydroelectric dams planned for the Xingu, Tapajós and Jamanxim Rivers are the focus of an intensive controversy due to the vast areas they will flood and the magnitude of their expected impacts.
Junk 1989
In their natural state, large tracts of floodplain forests along these rivers are subject to periodic and predictable periods of annual flooding caused by cyclical fluctuations in water levels linked to precipitation throughout the Amazon basin (
manifold adaptations against waterlogging and submergence (
). The organisms colonizing Amazonian floodplain forests exhibit
Junk 1989 Adis and Messner 1997 Parolin et al. 2004
,
,
), including morphological, physiological, and behavioral adaptations. Efficient mechanisms of fixation to substrates allow some organisms to withstand the mechanical strains brought about by flooding and
Junk 1989 Parolin 2001
high water velocity mainly in the draining period (
,
). Other organisms exhibit vertical migration on trees.
Salomão et al. 2007
There are two main types of floodplain vegetation in the Tapajós, Xingu and Tocantins River basins in eastern Amazonian, dense ombrophilous flood forest (
islands in the river beds (
Veloso et al. 1991
) and alluvial riparian vegetation (
). These vegetations colonize mainly sandy and rocky islands along river margins or on
Veloso et al. 1991
).
Silva et al. 1974
The particularity of these vegetation formations is that they occur on nutrient-poor rocky formations that belong to the oldest of the world, dating back to more than a billion years (
inundation and strong water currents during the high water period, and they are sensitive to habitat alterations (
). These formations are highly specialized climax communities adapted to a lack of soil nutrients; long periods of
Cunha and Ferreira 2012
).
Wittmann et al. 2004 2010 Salomão et al. 2007
In the alluvial riparian vegetation, the forest structure is low, formed mostly by shrubs and small trees that do not form a close canopy. Emergent species reach only 15 m in height. Species richness is very low compared to other types of Amazonian floodplain forests (
,
,
).
Alho 2011
The construction of hydroelectric dams in Amazonian will cause the flooding of extensive areas due to the formation of reservoirs, resulting in huge losses of forested areas that are the habitats of many species of plants and animals (
alluvial riparian vegetation can be expected to influence growth, survival and species composition of these forests and have a major negative impact on their functions and conservation (
). Artificial alterations of the hydrological cycle and the drowning of
Ferreira et al. 2010
). This outcome can already be observed in the reservoir of the Tucuruí Dam on the lower Tocantins River
Veloso et al. 1991
which is responsible for a great loss of habitat of natural vegetation cover with significant decrease in species richness, and changes in species composition (
).
In order to formulate management and conservation plans, as well as environmental compensation measures, local alluvial riparian vegetation in areas to be flooded should be inventoried and their status determined. The objective of this study is to assess the and compare species richness, alpha and beta diversity,
density and composition of the riparian plant vegetation of the Tapajós, Xingu and Tocantins River basins in areas that will be heavily influenced by projected hydroelectric dams in Pará state in the Brazilian Amazon, as well as to suggest mitigatory measures for environmental compensation. The key focus is on
a comparison of riparian forest in three adjacent river systems with similar environmental constraints to the vegetation, namely poor soils, rocky substrates, and periodic recurring flooding. We analyzed species composition and complementarily over short distances between the river basins.
Study Area
Forest inventories were carried out in the areas near the existing Tucuruí hydroelectric dam, on the Tocantins River, and two projected hydroelectric plants, Belo Monte on the Xingu River and São Luiz do Tapajós on the Tapajós River, all three in Pará state (Figure 1). Sampled sites are 200 to 400 km apart, in
the three river basins.
Figure 1 Satellite image of the Tapajós, Xingu, and Tocantins Rivers (from west to east), showing the locations of the Tucuruí dam on the Tocantins River and future dams sites on the Xingu and Tapajós Rivers.
Data Collection
Veloso et al. 1991
The vegetation was inventoried on 130 islands of different sizes, ranging from 0.1 to 10.0 hectares. All were colonized by alluvial riparian vegetation (
(Figure 2).
). Surveys were carried out between September and November, 2010, during the low water period when this type of vegetation is not flooded
Figure 2 Typical alluvial riparian vegetation on the Xingu River (A) covering whole islands e showing rocky substrate during low water (B), with an indication of flood period water level (C). (Photographs by Leandro Ferreira, 2010).
In three sampled river basins 130 plots of 5 × 20 meters were established on the islands, varying from 30 plots on the Tocantins River to 50 plots on the Tapajós and Xingu Rivers. In each plot all trees and shrubs with DBH (diameter at breast height) greater than or equal to 1 cm were measured and the
individuals determined to species level. Height of trees was estimated. Fertile botanic material was collected for all species and deposited in the João Murça Pires Herbarium of the Museu Paraense Emílio Goeldi (MPEG), in Belém, Brazil.
Data Analysis
Cientec 2006
Floristic and phytosociological data were analyzed with the Mata Nativa program (
Cain 1956
). To test the sufficiency of plots the collector's curve was used (
).
Zar 2010
Differences in species richness and diversity (dependent variables) among the three rivers (factors) were tested using simple analysis of variance and the Tukey's test, in order to detect differences in the dependent variables in relation to the factors (
similarity index (
Krebs 1999
Mccune and Brace 2002
), through an analysis of ordinance (non-metric multidimensional scheduling, NMS), using Euclidean distance as measure for species similarity and next neighbor to test for links between the plots (
RESULTS
Sampling Efficiency
The collector's curves reached a stable level in the three inventoried sites, indicating that sampling effort for each area was sufficient to determine local species richness (Figure 3).
).
). Species similarity was analyzed with the Sørensen
Figure 3 Collector's curves for the three sample sites of alluvial riparian vegetation on the Tapajós, Xingu, and Tocantins Rivers.
Species Richness
A total of 4,823 individuals of shrubs and trees were sampled, belonging to 39 species on the Tapajós River, 42 species on the Xingu River, and 25 species on the Tocantins River. These were clear differences in species richness and diversity among the three sites, these being significantly higher in plots on the
Xingu River compared to those on the Tapajós and Tocantins Rivers which were not significantly different from one another (Figure 4).
Figure 4 Species richness and diversity in the three sites with alluvial riparian vegetation on the Tapajós, Xingu, and Tocantins Rivers (different letters indicate significant differences).
Species Similarity and Species Composition
Ordinance analysis showed a clear separation of species composition among the alluvial riparian vegetation of the three rivers (Figure 5). The plant communities of the three river basins showed a geographic pattern in a west to east direction, of complementarities in species composition.
Figure 5 Multidimensional scaling analysis, showing grouping of plots based on speciescomposition in the three sample sites from west to east of alluvial riparian vegetation on the Tapajós (ta), Xingu (xi) and Tocantins (to) Rivers.
Only 6 species (12.2%) of the total of 74 species were common to all three sampling sites (Table I). Only a few species showed the highest relative densities of the sampled individuals on the Tapajós, Xingu, and Tocantins Rivers (Table I). This indicates the dominance of a group of few species in the three rivers
sampled, showing that the alluvial riparian vegetation is characterized by low species richness associated with a great number of rare species and a low frequency of species common to all three rivers.
TABLE I Vegetation (shrubs and trees) of alluvial riparian vegetation on the Tapajós, Xingu, and Tocantins Rivers.
Scientific name
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Acosmium nitens
Aeschynomene brevifolia
Alchornea fluviatilis
Amajoua guianensis
Anisomeres preslii
Astrocaryum jauari
Basanacantha armata
Buchenavia grandis
Buchenavia oxycarpa
Calophyllum brasiliense
Campsiandra comosa
Clitoria amazonum
Clitoria racemosa
Coccoloba acuminata
Coccoloba pichuna
Couepia cataractae
Couepia paraensis
Crataeva benthamii
Dalbergia inundata
Eugenia biflora
Eugenia brachipoda
Eugenia flavescens
Eugenia inundata
Eugenia patens
Gaya scopulorum
Genipa spruceana
Hidrochoria corimbosa
Licania apetala
Licania leptostachya
Mabea caudata
Machaerium aristulatum
Macrolobium sp.
Malouetia tamaquarina
Maripa reticulata
Mascagnia benthamiana
Maytenus ebenifolia
Mimosa sensitiva
Myrcia inundata
RDo
12.8
Tapajós River
RD
2
RF
4.4
0.2
0.5
0.3
1
0.3
3.4
0
0.1
0.3
0
0.1
0.3
0.4
0.3
1.4
1.2
1.4
3.7
0
14.2
2
0.1
24.9
1.3
0.3
13.3
2.4
0
0.2
3.5
0.2
3.4
0.3
0.4
0.2
0.7
5.5
5.2
5.4
0.1
Xingu River
RD
2.9
RF
6.4
0.2
0.1
0.3
0.1
6.2
1.4
9.6
0
0.6
5.4
0.2
8.5
0.1
0.8
6.2
0.3
8.6
0.3
0
0.2
0.3
3.1
10.3
9.4
0.3
0.1
0
1.2
0.9
0.1
1.6
1.1
0.3
RDo
Tocantins River
RD
RF
1.5
6.9
4.1
4.2
3.6
5.5
5.7
0.5
1.2
1.6
0.9
1.2
2.2
2.8
2.9
1.1
6.1
1.2
0.2
0.5
1.2
0.9
3.2
2.4
3.2
12.5
11.5
0
10
0.1
1.3
0.6
1.2
0.5
1
1.8
0.1
0.5
4.2
0.7
0.4
0.5
0.1
RDo
20
0
0.8
0.1
1.3
0.1
3.9
1
0.6
0.3
3.5
0.8
1.9
0
7.4
0
1.2
0.3
0.1
2.6
0.2
0.3
0.1
0.3
5.4
0.3
0.8
0.3
0
0
0.1
0.2
0.3
0.3
0.3
0
0.3
1.4
0
0.3
0.3
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
Myrcia phalax
Myrcia sp
Myrciaria floribunda
Ouratea racemiformis
Ouratea salicifolia
Palicourea quadrifolia
Passiflora nitida
Piranhea trifoliata
Pouteria procera
Psidium acuminatum
Psidium guianense
Psidium paraense
Psidium sessilis
Psidium verrucosum
Pterocarpus amazonicus
Retiniphyllum schomburgkii
Robinia sp.
Rotula pohlii
Rudgea cornifolia
Ruprechtia brachysepala
Ryania angustifolia
Sapium duckei
Sarcaulus brasiliensis
Simaba guianensis
Simaba polyphylla
Stachyarrhenia spicata
Swartzia cf. laurifolia
Swartzia leptopetala
Symmeria paniculata
Tocoyena brasiliensis
Trichilia singularis
Vasivaea alchorneoides
Vasivaea loranthoides
Vitex cf. duckei
Vitex cymosa
Zygia cauliflora
0.7
0.9
23.1
0.8
0.1
0.7
0.3
30.7
2
0.4
2
0.7
16.7
2.4
1.4
0
14.4
0.1
1.3
0.3
4.4
0.1
0.2
0.3
0.2
0.4
1.4
0.2
0.7
2
0
0.2
0.7
0.1
0.3
0.3
0.4
0.3
1
1.9
0.8
3.5
0.2
4.4
0.7
0
0
0.1
0
0.2
0.1
0.4
0.2
0.7
0.3
1.7
0.7
5.6
12.4
0.7
15.9
2.4
12.6
6
22.1
10
0
0.1
0.3
5.9
0.3
1.5
0.2
2.4
0.3
0
2.1
0.1
5.9
0.3
3.7
0
1.1
0
0.1
0
0.3
0.8
0.1
0.3
0.1
0.5
1.6
0.3
1.3
0.3
1.2
2.1
4
0
0
1.2
0.1
0.1
0.3
0.3
0.3
1.3
22.8
0.3
9.4
2.1
7
4
6.5
12.8
9.6
0.5
2.3
3.2
7.9
12.6
10.3
33.8
23.2
15.2
0
1.3
8.3
0.1
1.1
4
0.6
3.6
4.2
0.4
3.2
0.5
20.7
2.4
11.5
0.3
0.6
1.8
7.4
2.6
5.5
0.5
0.5
1.2
0
0.1
0.6
1.3
1.5
1.2
RDo: relative dominance; RD: relative density and RF: relative frequency; Bold: species occurring in all three sampling sites.
DISCUSSION
Most plant species found in the alluvial riparian formations of this study are rare at local level and clearly restricted to one of the three sampled river basins. Only six species were common to the plots in all three river systems. Even among the most dominant species, only two were common to all three sampling
sites. It is important to emphasize that in sites with high environmental stresses, such as rocky substrates or periodically waterlogged or submerged sites, the dominance of few species is typical. Not so many species are expected to be adapted to such harsh environmental constraints. Limiting environmental
Salomão et al. 2007 Cunha and Ferreira 2012
factors of the pioneering formations include periodical fluctuations of river levels and low nutrient availability on the rocky substrates of the islands, as well as mechanical stresses related to river currents during flooding (
as data from central Amazonian floodplain forests (
,
). However, as suggested by these data, suggest as well
Ferreira 1997 Parolin et al. 2004
,
), a range of species are adapted and can fill interchangeable niches within these plant communities. This leads to a high level of endemism with both high species richness and high local variability.
Observed differences in species richness, diversity and composition in alluvial riparian vegetation on the Tapajós, Xingu and Tocantins Rivers demonstrate that most species are restricted to only one of the rivers, an indication of high local rarity. Although the environmental constraints are almost identical, only
6 of 74 species occurred in all three inventoried sites. This indicates the filling of this niche with many different species having similar functional traits. Therefore, it is important for conservation actions to preserve these locally distributed species. Environmental compensation programs for hydroelectric dams
should protect the riparian areas to which these plants are adopted.
Whether already implanted or in the future, all planning for hydroelectric dams has to consider that many plant species occurring on Amazonian river systems are both rare and to only one river basin. There is a high complementarily of the total set of species, with alternative designs of equal fitness occurring in
similar environments, which make these fragile ecosystems of pioneering formations so valuable. As is typical for tropical forests in Amazonian, the majority of species are rare and thus the forests are characterized by high species richness associated with low abundances.
Lyons 2005
Many floristic studies carried out in the Amazon basin characterize a specific type of vegetation, its structure and species abundance, but these data are not generally compared with the total set of species of the community (
). By comparing three similar ecosystems, all within just 200 to 400 km, we
show that the assayed plant communities can differ in species composition, although they are composed of similar functional types adapted to rocky substrate and comprising typical pioneering formations. Attention must be given to the serious problem of conservation of the regional flora, with focus on the
preservation of plant communities.
The construction of hydroelectric dams with flooding and destruction of huge areas will thus most likely result in the strong reduction of species diversity and richness of the alluvial riparian vegetation in the Tapajós, Xingu and Tocantins River basins, as well as in other Amazonian localities where other
Junk 1987
hydroelectric dams are planned. This knowledge is not new (
), but little has been done to avoid foreseen impacts.
Junk 1989
Ferreira and Parolin (2007)
Several studies have demonstrated that one of the basic factors maintaining ecosystem functions and biotic diversity in Amazonian floodplains is the periodic fluctuation of river levels, causing a natural alternation of flooded and un-flooded periods (
).
demonstrated that tree
reproduction and establishment in central Amazonian floodplains are associated with river level fluctuations and largely depend upon them. Other studies showed that fish species also depend on the connectivity among water bodies and on annual flooding periodicity for their great migrations and reproductive
cycles (
Barthem et al. 1991 Barthem and Goulding 1997
,
Alho 2011
). River seasonal hydrological flux is also important for freshwater turtles, wild mammals both terrestrial and aquatic (
).
Large Amazonian dams, now in various phases of building, will cause environmental impacts on plant communities. Such as the loss of vegetation types, disappearance of specific habitats, and loss or migration of important faunal elements that have key functions for pollination and plant dispersal. Besides of the
loss of single species with unknown potentials, changes in vegetation cover may modify the hydrologic patterns, the aeration of the rhizosphere and microclimatic patterns. Increased tree mortality will be caused by new flooding patterns imposed by water level regulation of the dams, and to a large extent by the
elimination of an un-flooded period on which the vegetation depends for growth and seedling establishment.
The reservoir of the Tucuruí dam already caused extensive habitat loss for alluvial riparian vegetation of the Tocantins River, reflected in lower species richness and diversity in comparison with still pristine formations on the Xingu and Tapajós Rivers that are yet unaffected by the implantation of the planned
Cunha and Ferreira 2012
hydroelectric dams. Some 90% of the riparian habitats for pioneer formations on the Xingu River will be affected by construction of the Belo Monte hydroelectric dam (
projected hydroelectric dams.
). The same will occur with alluvial riparian vegetation on the Tapajós and Jamanxin Rivers with the construction of five
This study emphasizes that, in the analyzed areas, rare species will be threatened by the construction of hydroelectric dams. Political decisions concerning measures for environmental compensation of the hydroelectric enterprises must be specific to each of the hydrographic basins. Alluvial riparian vegetation of
Vane-
Amazonian river systems will soon suffer the impacts of new giant hydroelectric dams. In order to protect these plant communities, conservation units must consider the principle of complementarily which aims at increasing the species diversity in reserves while minimizing redundancy or species repetition (
Wright et al. 1991 Pressey et al. 1993
Anacleto et al. 2005
,
). The principle of complementarily states that areas with different attributes (species, habitats and landscapes) should be chosen for reserves and not areas that duplicate the same attributes (
). In the alluvial riparian vegetation of Amazonian rivers, where most
species are restricted to a single hydrographic basin, the most important attribute for conservation is complementarily of species composition. If this is not considered as a conservation strategy, original biodiversity will be lost irreversibly, without the possibility for compensation at other locations.
Acknowledgements
The Brazilian Ministry of Science, Technology and Innovation helped finance this study through the Network for Amazon Environmental Modeling (Geoma), the Museu Paraense Emílio Goeldi provided support, Mr. Luiz Carlos Batista Lobato assisted in the collection and identification of plants. Dr. William
Overal and Patrick Meir kindly reviewed the manuscript.
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McCune B and Brace JB. 2002. Analysis of ecological communities. MjM Software Design, Gleneden Beach, Oregon, USA. [ Links ]
Parolin P. 2001. Morphological and physiological adjustments to waterlogging and drought in seedlings of Amazonian floodplain trees. Oecol 128: 326-335. [ Links ]
Parolin P, De Simone O, Haake K, Waldhoff D, Rottenberger S, Kuhn U, Kesselmeier J, Schmit W, Piedade MTF and Junk WJ. 2004. Central Amazon floodplain forests: tree survival in a pulsing system. The Bot Rev 70: 357-380. [ Links ]
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Silva GG, Lima MIC, Andrade ARF, Issler RS and Guimarães G. 1974. Geologia das folhas SB.22 Araguaia e Parte de SC.22 Tocantins. In: Programa de Integração Nacional, Levantamento de Recursos Naturais. Vol. 4, Rio de Janeiro: DNPM, Projeto RADAM, p. 91-143. [ Links ]
Tundisi JG. 2007. Exploração do potencial hidrelétrico da Amazônia. Estud av 21(59): 109-117. [ Links ]
Vane-Wright RI, Humphries CJ and Williams PH. 1991. What to protect? Systematics and the agony of choice. Biol Conser 55: 235-254. [ Links ]
Veloso HP, Rangel FAL and Lima JCA. 1991. Classificação da vegetação brasileira adaptada ao sistema universal, Rio de Janeiro: IBGE, 124 p. [ Links ]
Wittmann F, Junk WJ and Piedade MTF. 2004. The várzea forests in Amazonia: Flooding and the highly dynamic geomorphology interact with natural forest succession. Forest Ecol Manag 196: 199-212. [ Links ]
Wittmann F, Junk WJ and Schongart J. 2010. Phytogeography, species diversity, community structure and dynamics of central Amazonian floodplain forests. In: Junk WJ et al. (Eds), Central Amazonian Floodplain Forests: Ecophysiology, Biodiversity and Sustainable Management, Ecological Studies, Springer
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Received: October31, , 2011; Accepted: May15, , 2012
Correspondence to: Leandro Valle Ferreira E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013000300013
Biological Sciences
Germination responses of the invasive Calotropis procera (Ait.) R. Br. (Apocynaceae): comparisons with seeds from two ecosystems in northeastern Brazil
LAURA C. LEAL1, MARCOS V. MEIADO12, ARIADNA V. LOPES3, INARA R. LEAL3
1
Programa de Pós-Graduação em Biologia Vegetal, Universidade Federal de Pernambuco, Av. Professor Moraes Rego, s/n, Cidade Universitária, 50670-901 Recife, PE, Brasil
Departamento de Biociências, Universidade Federal de Sergipe, Av. Vereador Olímpio Grande, s/n, Centro, 40500-000 Itabaiana, SE, Brasil
3 Departamento de Botânica, Universidade Federal de Pernambuco, Av. Professor Moraes Rego, s/n, Cidade Universitária, 50670-901 Recife, PE, Brasil
2
ABSTRACT
Life history traits are considered key indicators of plant invasibility. Among them, the germination behavior of seeds is of major relevance because it is influenced by environmental factors of invaded ecosystem. Here, we investigated how seed traits and seed tolerance to environmental
factors on seed germination of Calotropis procera vary depending on the invaded ecosystems in northeastern Brazil. We have tested seeds from two vegetation types – Caatinga and Restinga – to different levels of light intensity, salinity, and water stress. Previous to those experiments,
seed-set and morphometric analysis were carried out for both studied populations. We have observed a higher seed-set in Caatinga. Seeds produced in this ecosystem had lower seed moisture content. Seeds from Restinga showed lower germination time when light intensity decreased. We
observed a reduction in both the germinability and the synchronization index with decreasing osmotic potential and increasing salinity. Nevertheless, both populations exhibited changes in photoblastism when seeds were submitted to water and saline stress. In conclusion, C. procera
seeds are tolerant to environmental factors assessed. That characteristic ensures the colonization success and wide distribution of this plant species in the studied ecosystems.
Keywords: biological invasions; Caatinga; environmental factors; Restinga; seed germination; seed-set
RESUMO
Características da história de vida são consideradas indicadoras chave do potencial invasivo de vegetais. Dentre essas características, o comportamento germinativo de sementes é de grande relevância, pois é influenciado por fatores ambientais do ecossistema invadido. Aqui, investigamos
como características das sementes de Calotropis procera e sua tolerância a fatores ambientais na germinação varia dependendo do ecossistema invadido no Nordeste do Brasil. Nós testamos o comportamento germinativo de sementes de duas populações - Caatinga e Restinga - sob
diferentes níveis de luminosidade, salinidade e déficit hídrico. Nós também quantificamos as sementes produzidas por fruto em cada população e realizamos análise morfométrica das sementes. Nós observamos maior número de sementes em frutos de Caatinga e as sementes produzidas
nesse ecossistema tiveram menor teor de umidade. Com o decréscimo da intensidade luminosa, sementes de Restinga apresentaram menor tempo médio de germinação. Nós também observamos redução na germinabilidade e na sincronização da germinação com o descréscimo do
potencial osmótico e com o aumento da salinidade. Ambas populações exibiram mudanças no fotoblastismo quando sementes foram submetidas a estresse salino e hídrico. Dessa forma, concluímos que sementes de C. procera são tolerantes aos fatores ambientais testados e que essa
característica pode ser responsável pelo sucesso de colonização da espécie nos ecossistemas estudados.
Palavras-Chave: invasões biológicas; Caatinga; fatores ambientais; Restinga; germinação de sementes; produção de sementes
INTRODUCTION
To understand how environmental factors affect seed germination responses it is essential to comprehend processes as establishment, succession and natural regeneration of vegetation (
and seedling establishment are affected by environmental factors such as light intensity, temperature, water availability, and soil salinity (
Labouriau 1983 Baskin and Baskin 1998 Donohue et al. 2010
seed germination and, consequently, the distribution of many plant species (
(Delesalle and Blum 1994).
,
,
Baskin and Baskin 1988
, Vázquez-Yanes and Orozco-Segovia 1993). In natural habitats, seed germination
). In addition, seed germination may also be affected by environmental conditions to which mother plants were exposed during seed development
Studies on seed traits and germination behavior have been useful in the investigation of environmental factors affecting geographic distribution of native species (
Smith and Knapp 2001
understanding colonization abilities and geographical distribution of exotic plants introduced in new habitats (
Bazzaz 1979
). Thus, it is possible to assert that the range of environmental conditions may be pointed out as one of the factors affecting
Abreu and Garcia 2005 Donohue et al. 2010 Silveira et al. 2011
,
,
). Furthermore, such investigations are important tools for
). When species with high dispersal capacity are removed from their native habitat and introduced into an exotic one, they may find favorable
Richardson et al. 2000
microclimatic and ecological conditions (e.g. predators and competitors absence) to the establishment of viable populations without human assistance (
). Species that have this ability may become a serious threat to native species, and identifying factors that
Smith and Knapp 2001
affect or favors the establishment of exotic species is important to guide strategies for prevention and control of invasive alien plant species worldwide (
).
Thus, the aim of this study was to evaluate if seed traits and germination responses to environmental factors of an invasive species vary depending on the invaded ecosystem. For that we studied populations of Calotropis procera (Ait.) R. Br. (Apocynaceae), an invasive plant of
two Brazilian northeastern ecosystems – Caatinga and Restinga. Our discussion contributes to the characterization of invasive species traits, focusing on how environmental factors of the invaded ecosystem can influence the establishment of invasive alien plant species.
Study Area and Species
The study was carried out with seeds of Calotropis procera collected in two Brazilian northeastern ecosystems - Caatinga and Restinga. The Caatinga is a mosaic of seasonally tropical dry forest (sensu
of 730,000
Sampaio 1995
km2 (
).
between June and December (
Mooney et al. 1995 Pennington et al. 2000
,
) and scrub vegetation that covers an area
Seeds were collected in a population located at Serra Talhada (7°59′S, 38°19′W), Pernambuco State, northeast Brazil. Annual rainfall in the region is ∼ 803 mm, the average temperature is 26°C and there is a pronounced dry season, that occurs
Machado et al. 1997
). The predominant soils are Cambisoils and Litosoils with marked salinity due to salt accumulation resulting from fast water evaporation and poor soil drainage. Vegetation is dominated by shrubs of 3 to 4 m tall (
Machado et al. 1997
The herbaceous stratum has low density and is composed mainly by annual species that are present only during the rainy season (
Machado et al. 1997
In Restinga, seeds of C. procera were collected from a population located a few meters away from the sea, in Caucaia (03°44′S, 38°39′W), Ceará State, northeast Brazil. The average temperature in the region varies between 25 and 27°C, with maximum amplitude of 5°C (
et al. 2003
). The annual rainfall is ∼ 1,255 mm, with the dry season occurring between July and December (
the fixation of vegetation in some places which attenuates the wind dynamics (
Holanda et al. 2003
Holanda et al. 2003
).
).
Holanda
). The soil is formed by non-consolidated sand and clay sedimentary particles, with the formation of mobile dunes characterized by the absence of plants or
).
Calotropis procera is popularly known in Brazil as “algodão-de-seda”, “algodão brabo”, “algodão-de-praia” or “leiteiro” because of the long white fibers attached to its seeds as an adaptation to wind dispersal. Calotropis procera has a wide geographical distribution, mainly in
semiarid areas (
Melo et al. 2001
). The species is native from Africa, India, Persia, and Afghanistan. It has been naturalized in Australia, Mexico, Central America, South America, Caribbean and Pacific Islands (
Rahman and Wilcock 1991
commonly found in invasive plants such as continuous flowering and autogamy in invaded areas, high seed production efficiently dispersed by wind, and fast growth after establishment (G.M. Tabatinga, unpublished data,
affect seed germination, establishment and distribution of this species have not been studied in detail.
In Brazil, C. procera was probably introduced as an ornamental in the early 20th century (
Caatinga and Restinga and is considered a threat to the native vegetation (
Kissmann and Groth 1992
Souza and Lorenzi 2005
). Later, it became invasive of pasture areas, due to the high dissemination of its seeds by the wind (
Corrêa 1939
). Calotropis procera has some attributes
Sobrinho et al. 2013
). However, environmental factors that
). Currently, the species can be easily found in areas of
Melo et al. 2001
). It is a common species in pasture areas, road margins and cultivated areas, forming dense populations that are difficult to eradicate (
).
Seed-Set, Morphometry and Germination
During May (wet season at Caatinga study site) and June (wet season at Restinga study site) of 2007, we collected 30 fruits per ecosystem, one fruit per individual (randomly selected in both populations/ecosystems). All the fruits were completely developed but still closed, thus
we could verify the seed-set. We measured the length and width of seeds (mm), dry biomass (mg), and the seed moisture content (%). All the morphometric parameters followed the Brazilian Norms for Seeds Analysis (
sample per individual) to check the dry biomass, seed moisture content, length and width of the seeds.
Brasil 2009
). We used 30 samples of 10 seeds each (one
We assessed the effect of light intensity, salinity and water stress on seed germination of both populations. Values of light intensity, water and salinity stress used in these experiments were calculated in order to simulate the range of abiotic conditions to which Calotropis procera
can be submitted in both ecosystems. We used five Petri dishes with 50 seeds each per treatment watered with the test solutions. In each dish, we used seeds from one individual of each population randomly selected. The different levels of light intensity (0, 30, 50, 70 and 100%,
which represented the full light treatment with a light intensity of 20 µmol.m–2.s–1) were simulated with the use of black polypropylene shade cloth. We used a commercial solution of sodium chloride (
Braccini et al. 1996
) and polyethylene glycol 6000 (
Villela et al. 1991
salinity and water stress, respectively. In both experiments, we evaluated the effect of osmotic potentials 0.0, -0.2, -0.4, -0.6, -0.8 and -1.0 MPa on seed germination. The dishes were placed in ideal conditions for the C. procera seed germination, according to
(1976)
) to simulate the
Labouriau and Valadares
(i.e., constant temperature of 30°C, under light or continuous darkness, simulated with the use of black plastic bags). The radicle protrusion was the criterion for germination seeds. The evaluations were made daily for a period of 35 days.
Data Analysis
We compared seed-set and morphometric parameters between the two populations with t tests. For each seed germination treatment, we calculated: 1) germinability (%); 2) mean germination time [t = Σni.ti/Σni, where ti is the time from the start of the experiment to the ith
observation (day), and ni is the number of seeds germinated in the time i (not the accumulated number, but the number correspondent to the ith observation)]; and 3) the synchronization index of germination [E = -Σfi.log2.fi, where fi is the relative frequency of germination (i.e.,
Ranal and Santana (2006)
. The germinability data were arcsine square-root transformed in order to adjust count data to the normal distribution and apply parametric tests. For the evaluation of light intensity effects we
the proportion of germinated seeds in an interval)] according to
used a Two Way ANOVA (ecosystem and light intensity). To verify the influence of the water stress between the populations and to check for the existence of a photoblastic change in the seeds submitted to the treatments, we compared the germination of all treatments using a
Three Way ANOVA (ecosystem, photoblastism and water availability). The same analysis was conducted with germination data of seeds submitted to saline stress. We verified the normality of the residuals and homogeneity of the variances through Shapiro-Wilk and Levene
tests, respectively. All statistical analyses are described in
Zar (1999)
and were assessed with STATISTICA 7.0 with a significance level equal to 0.05.
RESULTS
The number of seeds per fruit of Calotropis procera ranged from 213 to 391 in Caatinga and 162 to 403 in Restinga, which resulted in a seed production in Caatinga 11.85% higher than in Restinga (t = 3.01, df = 58, P = 0.004, Table I). For the morphometric parameters, we
found no significant differences in seed length (t = -0.49, df = 58, P = 0.630), seed width (t = -0.57, df = 58, P = 0.571), or dry biomass (t = 0.75, df = 58, P = 0.480, Table I) between ecosystems. For seed moisture, however, we found a water content about 60% lower in the
Caatinga (t = -5.60, df = 58, P < 0.001, Table I) compared to the Restinga population.
TABLE I Number of seeds per fruit unit and dimensions, dry biomass and moisture content of Calotropis procera (Ait.) R. Br. (Apocynaceae) seeds collected in the same developmental stage in Caatinga and Restinga areas of northeastern Brazil.
Different letters in the same column indicate significant differences between ecosystems. Data are expressed as mean ± SE.
Seeds per Fruit
329 ± 43 a
290 ± 57 b
Caatinga
Restinga
Seed Length (mm)
6.3 ± 0.3 a
6.3 ± 0.3 a
Seed Width (mm)
4.2 ± 0.2 a
4.2 ± 0.2 a
Dry Biomass (mg)
56.5 ± 8.1 a
56.3 ± 7.8 a
Seed Moisture Content (%)
9.3 ± 0.9 b
15.4 ± 5.9 a
Seeds of C. procera showed a positive response to a wide range of environmental factors. The germinability was not influenced by the variation in light intensity (F = 0.40, df = 4, P = 0.770), with seeds of both populations having a final germination superior to 95% (Table II).
Similarly, the synchronization index of seed germination was not influenced by the different levels of light intensity (F = 1.10, df = 4, P = 0.380, Table II). On the other hand, the mean germination time was affected by the ecosystem (F = 27.42, df = 4, P < 0.001) and light
intensity (F = 8.61, df = 4; P < 0.001). Seeds from Restinga germinated more rapidly when exposed to lower light intensities (Table II).
TABLE II Germination parameters of Calotropis procera (Ait.) R. Br. (Apocynaceae) seeds from two ecosystems of northeastern Brazil (Caatinga and Restinga) subjected to light treatments at 30°C after 35 days. Different capital and small letters
indicate significant differences between ecosystem type and light treatments, respectively. Data are expressed as mean ± SE.
Light Intensity (%)
0
30
50
70
100
Germinability (%)
Caatinga
Restinga
97.6 ± 5.37 Aa
100 ± 0.0 Aa
98.4 ± 2.19 Aa
98.8 ± 1.79 Aa
99.2 ± 1.79 Aa
100 ± 0.0 Aa
99.6 ± 0.89 Aa
100 ± 0.0 Aa
97.2 ± 6.26 Aa
100 ± 0.0 Aa
Mean Germination Time (day)
Caatinga
Restinga
2.03 ± 0.09 Ab
0.97 ± 0.04 Ab
2.91 ± 1.24 Aab
2.20 ± 0.43 Aab
3.27 ± 0.65 Aab
2.28 ± 0.37 Aab
3.36 ± 1.08 Aab
2.51 ± 0.32 Aa
4.05 ± 1.01 Aa
2.38 ± 0.44 Bab
Synchronization Index
Caatinga
Restinga
0.12 ± 0.20 Aa
0.16 ± 0.22 Aa
0.37 ± 0.27 Aa
0.03 ± 0.06 Aa
0.42 ± 0.76 Aa
0.58 ± 0.40 Aa
0.36 ± 0.13 Aa
0.67 ± 0.22 Aa
0.80 ± 0.72Aa
0.54 ± 0.48 Aa
The salinity stress negatively influenced the germinability of C. procera (F = 107.29, df = 5, P < 0.001), completely inhibiting it in both population when seeds were submitted to a concentration equal or higher than -0.8 MPa (Fig. 1A). With increase in salinity concentration, we
found changes in the photoblastic response of both populations, with a higher germinability in the darkness (F = 50.12, df = 1, P < 0.001). We did not observe statistical significant differences in the germinability between the populations subjected to different salinity
concentrations (F = 0.21, df = 1, P = 0.956). However, the synchronization index of seed germination under the influence of salinity stress was higher in the absence of light (F = 8.83, df = 1, P < 0.001). Moreover, there was no significant difference in the mean germination time
between ecosystems (F = 0.23, df = 1, P = 0.630, Table III).
Fig. 1 Germinability (%) of Calotropis procera (Ait.) R. Br. (Apocynaceae) seeds from Caatinga (C) and Restinga (R), northeastern Brazil, subjected to saline (A) and water stress (B) in light and darkness at 30°C after 35 days. Bars indicate ± SE.
TABLE III Germination parameters of Calotropis procera (Ait.) R. Br. (Apocynaceae) seeds from two ecosystems of northeastern Brazil (Caatinga and Restinga) and subjected to salinity stress in light and darkness at 30°C after 35 days. Greek, capital
and small letters indicate statistic results of comparisons between photoblastism, ecosystem type and salinity treatments, respectively. Data are expressed as mean ± SE.
Light Regime
Salinity (MPa)
0.0
Light
Darkness
Caatinga
Restinga
Caatinga
Restinga
4.05 ± 1.01 αAa
2.38 ± 0.51 αAa
0.80 ± 0.71 αAa
0.54 ± 0.47 αAa
0.2
4.97 ± 3.51 αAa
3.52 ± 2.50 αAa
0.98 ± 0.84 αAa
0.35 ± 0.47 αAa
0.4
7.60 ± 11.26 αAa
5.55b ± 8.07 αAa
0.20 ± 0.44 αAa
0.00 ± 0.00 αAa
0.6
-
0.8
1.0
2.20 ± 3.03 αAa
-
-
0.00 ± 0.00 αAa
-
0.00 ± 0.00 αAa
-
0.0
2.03 ± 0.11 αAa
0.97 ± 0.04 αAa
0.12 ± 0.20 αAa
0.16 ± 0.22 αAb
0.2
2.59 ± 0.44 αAa
1.96 ± 0.08 αAa
0.38 ± 0.30αAa
0.14 ± 0.30 αAb
0.4
3.56 ± 0.34 αAa
3.67 ± 0.87 αAa
0.82 ± 0.46αAa
1.44 ± 0.40 αAa
0.6
6.54 ± 2.29 αAa
-
5.48 ± 3.15 αAa
-
0.82 ± 0.64 αAa
-
0.39 ± 0.90 αAa
-
0.8
1.0
- No seed germination.
As observed with the saline stress, the populations exhibited changes in the photoblastism when subjected to water stress (F = 238.69, df = 4, P < 0.001). The reduction in the availability of water also negatively affected the synchronization index (F = 5.80, df = 4, P < 0.001) and
germinability (F = 10.28, df = 4, P < 0.001, Fig. 1B), with seeds from Caatinga germinating at concentration of -0.8 MPa, while the germination was inhibited at concentrations of less than -0.4 MPa in seeds from Restinga. With reduction of water availability, we also found a
significantly increase in mean germination time (F = 7.48, df = 4, P < 0.001, Table IV).
TABLE IV Germination of Calotropis procera (Ait.) R. Br. (Apocynaceae) seeds from two ecosystems of northeastern Brazil (Caatinga and Restinga) and subjected to water stress in light and darkness at 30°C after 35 days. Greek, capital and small
letters indicate significant differences between photoblastism, ecosystem type and water stress treatments, respectively. Data are expressed as mean ± SE.
Light regimes
Caatinga
Restinga
Treatments (MPa)
Darkness
Restinga
4.05 ± 1.01 αAb
2.38 ± 0.51 αAa
0.80 ± 0.71 αAa
0.54 ± 0.47 αAa
0.2
12.42 ± 6.92 αAa
2.80 ± 3.83 αBa
0.66 ± 0.46 αAa
0.00 ± 0.00 αAa
0.4
0.6
0.8
1.0
10.37 ± 10.83 αAa
-
0.00 ± 0.00 αBa
-
0.25 ± 0.56 αAa
-
0.00 ± 0.00 βAa
-
0.0
2.03 ± 0.09 αAa
0.97 ± 0.04 αAa
0.12 ± 0.20 αAd
0.16 ± 0.22 αAab
0.2
2.71 ± 0.31 βAa
2.00 ± 0.00 αAa
0.46 ± 0.38 αAcd
0.00 ± 0.00 αAb
0.4
4.26 ± 0.50 αAa
3.50 ± 0.37 αAa
1.25 ± 0.55 αAb
0.87 ± 0.50 αAa
0.6
8.03 ± 1.93 αAa
0.00 ± 0.00 αAa
1.82 ± 0.45 αAa
0.00 ± 0.00 αBb
0.8
10.50 ± 9.63 αAa
-
0.00 ± 0.00 αAa
-
0.20 ± 0.45 αAc
-
0.00 ± 0.00 αAb
-
0.0
Light
Caatinga
1.0
- No seed germination.
DISCUSSION
Roach and Wullf 1987
Plant phenotype variation might be affected not only by the genotype, but also by the environment where plants live as well as by the conditions to which the parental plants are growing (Delesalle and Blum 1994,
). Thus, we expected that individuals from
different populations would differ in morphological or physiological adaptations, which allow them to establish in several habitats. Seed-set is one of the main phases of plants life cycle. This phase can be affected by environmental pressures suffered by parental plants in the site
Roach and Wullf 1987
where they are established (
). We observed significant differences in seed-set between populations, which indicated that C. procera can produce different number of seeds depending on the invaded environment. Although seed-set may varies between years, the
dry season preceding fruit collection was typical in both ecosystems. This fact leads us to believe that the differences between populations are not result of atypical climatic variations during the production of the fruits used in these experiments.
Seeds of Calotropis procera from both populations/ecosystems are aphotoblastic (sensu
) and had the same germination behavior in the presence or absence of light, with a mean germination time less than 5 days. The mean germination time represents an
Abreu and Garcia 2005
important parameter in seed germination studies, especially for alien plant species, as it is a useful parameter for the evaluation of niche occupation speed and efficiency (
). Reduction in light intensity caused a reduction in the mean germination time, with the
higher average found in seeds in a constant darkness. Thus, the germinability and the efficiency of space occupation of this species are positively favored by the absence of light. Moreover, under the influence of water and salinity stress the seeds showed a type of photoblastism,
behaving as “relative negative photoblastic” (sensu
), indicating, once more, that the success of establishment is higher when seeds are in darkness.
Many alien plants show an increase in their germination rates when exposed to high light conditions, which favors their performance in disturbed areas (
Drewitz and DiTomaso 2004
Harradine 1991
). Seeds of Cortaderia jubata (Lemoine) Stapf (Poaceae), an invasive species of coastal California, for example,
have 3.3 times higher germinability in high light than in dark (
). However, the ability to germinate under different degrees of light intensity, like observed here, can increase the invasion ability. Thus, these alien plants can colonize many habitats with a
wide spectrum of light conditions. Other alien species show photoblastic germination behavior similar to that found in C. procera, such as Cortaderia selloana (Schultes & Schultes fil.) Asch. & Graebner (Poaceae) seeds. It is an European alien plant, which presents high
germination rate under different light conditions, but are favored by light intensity below 100% (
Domènech and Vilà 2008
).
The ecosystems where the two studied populations were located are marked by a high soil salinity, either by fluvial-marine influence, such as in the Restinga (
Fassbender and Bornemisza 1987 Mello et al. 1983
Caatinga soils (
,
Gutterman 1993 Kigel 1995
ecosystems usually exhibit high tolerance to salinity (
Sugiyama 1998
), or by the high water evaporation and deficient drainage of soil, resulting in the accumulation of salt in the
). Thus, due to the high occurrence of individuals in those environments, we expected a similar seed germination response to salinity for both populations, which was confirmed in our results. In fact, plant species of arid
,
). On the other hand, water availability differs between ecosystems, with Restinga areas showing higher pluviometric rates and air relative humidity than Caatinga. However, part of the water in this
Miller and Donahue 1990
Miller and Donahue 1990
environment might not be available for the plants due to the high concentration of salt in water (
). Thus, in Restinga, plants have several strategies to promote water absorption, most of them involving energetic expenses (
). Based on these
statements, we expected that seeds from Restinga had higher seed moisture content, as these seeds lose less water during the seed development. In fact, this pattern was observed in our studies. Seeds collected in the Restinga had higher levels of water content than those collected
in Caatinga. According to
Castro et al. (2004)
, higher levels of moisture make seeds more sensitive to water stress during the germination period, and this was observed in our results.
In conclusion, although we found some differences in seed attributes and germination response of Calotropis procera between Caatinga and Restinga, both populations showed high germination success. They showed a reduction in the synchronization index of germination and an
increase in the mean germination time. Further, seeds from both ecosystems showed positive responses to a wide range of environmental factors. Thus, these factors do not act as barriers to the invasive capacity of C. procera, ensuring its colonization success and wide
distribution in both ecosystems. Based on its seed-set, seed size, dispersal mode and germination behavior, C. procera seems to be an effective invasive species in Caatinga and Restinga areas, and its potential to invade other ecosystems in the world should be considered.
Acknowledgements
The study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, PROCAD Grant number 0166057). The Centro de Pesquisas Ambientais do Nordeste (CEPAN) and The Conservação Internacional do Brasil (CI-Brasil) provided logistic
support during field work. We are also grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), which provided an undergraduate fellowship to L. C. Leal and research grants to A. V. Lopes and I. R. Leal.
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Received: April28, , 2011; Accepted: June6, , 2012
Correspondence to: Inara Roberta Leal E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013005000051
Biological Sciences
Are leaf physiological traits related to leaf water isotopic enrichment in restinga woody species?
BRUNO H.P. ROSADO13, EDUARDO A. DE MATTOS1, LEONEL DA S.L. STERNBERG2
1
2
3
Departamento de Ecologia, IB, CCS, Universidade Federal do Rio de Janeiro, Av. Brigadeiro Trompowski, s/n, C.P. 68020, 21941-970 Rio de Janeiro, RJ, Brasil
Department of Biology, University of Miami, Coral Gables, FL 33124, USA
Departamento de Ecologia, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, Rua São Francisco Xavier, 524, Maracanã, 20550-019 Rio de Janeiro, RJ, Brasil
ABSTRACT
During plant-transpiration, water molecules having the lighter stable isotopes of oxygen and hydrogen evaporate and diffuse at a faster rate through the stomata than molecules having the heavier isotopes, which cause isotopic enrichment of leaf water. Although previous models have assumed that leaf water is well-mixed and isotopically uniform, non-uniform stomatal closure, promoting different enrichments between cells, and different pools of water within leaves, due to
morpho-physiological traits, might lead to inaccuracies in isotopic models predicting leaf water enrichment. We evaluate the role of leaf morpho-physiological traits on leaf water isotopic enrichment in woody species occurring in a coastal vegetation of Brazil known as restinga. Hydrogen and oxygen stable isotope values of soil, plant stem and leaf water and leaf traits were measured in six species from restinga vegetation during a drought and a wet period. Leaf water isotopic
enrichment relative to stem water was more homogeneous among species during the drought in contrast to the wet period suggesting convergent responses to deal to temporal heterogeneity in water availability. Average leaf water isotopic enrichment relative to stem water during the drought period was highly correlated with relative apoplastic water content. We discuss this observation in the context of current models of leaf water isotopic enrichment as a function of the Péclet
effect. We suggest that future studies should include relative apoplastic water content in isotopic models.
Keywords: leaf water isotopic enrichment; Péclet effect; restinga; leaf morpho-physiological traits
RESUMO
Durante a transpiração das plantas, moléculas de água com isótopos mais leves de oxigênio e hidrogênio evaporam e se difundem mais rapidamente através dos estômatos do que moléculas com isótopos mais pesados, o que causa enriquecimento isotópico de água em folhas. Embora modelos prévios assumam que a água da folha é bem misturada e isotopicamente uniforme, o fechamento não uniforme dos estômatos, promovendo enriquecimentos diferentes entre as células e
diferentes compartimentos de água dentro de folhas, devido às características morfo-fisiológicas, podem levar a imprecisões dos modelos isotópicos na previsão de enriquecimento da água foliar. Nós avaliamos o papel de atributos morfo-fisiológicos foliares sobre o enriquecimento isotópico da água foliar em espécies arbóreas que ocorrem em planícies arenosas costeiras no Brasil, conhecidas como restingas. Isótopos estáveis de hidrogênio e oxigênio em amostras de solo,
ramos e folhas e características foliares foram medidos em seis espécies arbóreas de restinga durante períodos seco e chuvoso. O enriquecimento isotópico de água foliar relativo à água dos ramos foi mais homogêneo entre as espécies durante a seca, em contraste com o período chuvoso, sugerindo respostas convergentes para lidar com a heterogeneidade temporal na disponibilidade de água. A média de enriquecimento isotópico de água foliar relativo à água dos ramos durante
o período de seca foi altamente correlacionada com o conteúdo relativo de água apoplasmática. Nós discutimos estes resultados no contexto de modelos atuais de enriquecimento isotópico de água foliar como uma função do efeito Péclet. Sugerimos que estudos futuros devem incluir o conteúdo relativo de água apoplasmático em modelos isotópicos.
Palavras-Chave: enriquecimento isotópico foliar; efeito Péclet; restinga; atributos morfofisiológicos
INTRODUCTION
Flanagan 1993 Roden and Ehleringer 1999
Water molecules having the lighter isotopes of hydrogen or oxygen (16O and 1H) will evaporate and diffuse at a faster rate through the stomata than those having the heavier isotopes (18O and 2H), which will cause isotopic enrichment of leaf water. Many studies have indicated that this isotopic enrichment can be indicative of the relative humidity during the transpiration flux (
in plant cellulose for environmental reconstructions via tree-ring cellulose (
The model developed by
Craig and Gordon (1965)
Roden and Ehleringer 2000
Yakir and Sternberg 2000 Lai et al. 2008
). Additionally, the understanding of the causes of leaf water isotopic enrichment is an useful tool to parameterize models, based on plant responses, i.e., to predict the gas exchanges between biosphere-atmosphere in different scales (
,
Flanagan et al. 1991 Dawson et al. 2002
predicts that isotopic enrichment is mainly driven by climate and that there are two isotope effects during evaporation: an equilibrium effect that occurs during isotope exchange reactions converting water from liquid to vapor; and a kinetic effect promoted by distinct mass-dependent diffusion rates (
results for soil water undergoing evaporation (
Mathieu and Bariac 1996 Clark and Fritz 1999
,
,
). It can also be used to identify isotope composition
).
,
). A good fit has been observed between the Craig-Gordon model and experimental
).
Previous models have assumed that leaf water is well-mixed and isotopically uniform. The enrichment of leaf water above stem water (∆e) during transpiration was therefore described by an equation using the same principles developed by
Craig and Gordon (1965)
:
where ε* is the proportional depression of water vapour pressure over heavy isotopes compared with the vapour pressure over light isotopes; εk is the kinetic fractionation as water vapour moves through the stomata and leaf boundary layer; and ∆v is the isotope composition of atmospheric water vapour relative to plant stem water. The parameter e is the water vapor pressure and subscripts a and i refer to bulk air and intercellular air spaces, respectively. It can be
seen that the vapor pressure gradient (e a /e i ), which is directly related to relative humidity, is an important component dictating leaf water isotopic enrichment.
Yakir et al. 1990 Flanagan and Ehleringer 1991 Flanagan et al. 1991 Yakir et al. 1993 Yakir and Sternberg 2000
However, many studies have reported that the observed isotopic composition of bulk leaf water is usually less enriched than that predicted by the above equation (
,
,
,
,
Lai et al. 2008
). The reasons for these discrepancies are especially affected by life form and plant hydraulic properties (
) which are related to (i) non-uniform stomatal closure that promote different enrichments
Flanagan et al. 1991 Luo and Sternberg 1992
between cells adjacent to open and closed stomata (
,
reflects the water absorbed from roots (
Farquhar and Lloyd (1993)
Flanagan and Ehleringer 1991 Luo and Sternberg 1992
,
Yakir et al. 1989 1990 1993 Flanagan et al. 1991 Yakir and Sternberg 2000
) and (ii) different pools of water within leaves (apoplastic and symplastic water as well as water in veins) some of which may not be exposed directly to evaporative conditions (
composition influenced by environmental conditions, showing diurnal isotope ratio fluctuations (
Yakir et al. 1989 1990
,
Yakir and Sternberg 2000
). This pool of water does not completely mix with bulk leaf water (
,
,
,
,
). The cell wall surfaces, which are exposed to evaporation, correspond to part of the apoplastic water fraction that have its isotopic
). The symplastic water is not completely exposed to evaporation but may be affected by the isotopically enriched apoplastic water (
). The third pool consists of the water in the veins showing a high proportion of unfractionated water that
).
modeled leaf water isotopic composition by invoking the Péclet effect, which explained the above discrepancies and other observed characteristics of leaf water isotopic enrichment. The Péclet effect is quantified as ρ and is the result of the mixing between the convective flux of unfractionated water and the back diffusion of isotopically enriched water from the evaporating sites into the leaf, represented by the equation:
The molar concentration of water (5.56x104 mol.m−3) is represented by C, D is the diffusivity of DHO and H2 18O through water, T is the rate of transpiration rate and L is the effective length in the leaf that water has to travel from the xylem to the evaporative site. This parameter is a product of the constant of proportionality, k, and the distance from the evaporating surfaces to the leaf veins, L (
Barbour et al. 2000 Barbour and Farquhar 2003
,
). The isotopic enrichment of
bulk leaf water relative to stem water (∆L) is integrated over this mixing gradient and given by:
Thus, according to this model, the greater the Péclet number, the less enriched the leaf water will be relative to stem water. Likewise, leaf water enrichment approaches the values of ∆e predicted by equation 1 as the Péclet number decreases. The Péclet number in turn is a function of different leaf morpho-physiological characteristics which might affect the two components of the Péclet values: transpiration rate and the path length. Although the contribution of
Lai et al. 2008
Kahmen et al. 2008 Kahmen et al. 2009
), for path length, however, few studies have investigated to what degree leaf morpho-physiological traits could affect leaf water isotopic enrichment (
day- and nighttime transpiration to equilibrium between leaf water and atmospheric water vapor have been described (
study indicates, to some extent, the role of leaf morpho-physiological traits in leaf water isotopic enrichment, which may be relevant in the improvement of models describing leaf isotopic enrichment.
,
Ferrio et al. 2009
). In fact, it has been observed that leaf hydraulic properties related to mesophyll features are directly related to changes in L (
). This later
Here, we evaluate in which way leaf morpho-physiological traits could affect leaf water isotopic enrichment in six restinga woody species. For this purpose, we choose leaf traits commonly used in ecophysiological studies, such as leaf mass per area, leaf thickness, leaf density and leaf physiological traits obtained from pressure-volume curves that describe performance of species in terms of water use (Rosado and de Mattos 2010). We collected these data in a
coastal vegetation of Brazil known as restinga, where the substrate is formed by coastal sandy barriers from marine sources; characterized by low water retention and poor nutrients and organic matter. Although restinga species possess functional traits to deal with low water and nutrient availability, variations in leaf morphological traits among species (Rosado and de Mattos 2010) might lead to distinct species responses to fluctuations in resources' availability
and consequently in the isotopic identity of leaf water.
Study Area
Araujo et al. 1998
Measurements were done in the Restinga of Jurubatiba National Park covering 14,140 hectares and located north of the state of Rio de Janeiro, Brazil (22° and 22°23′S and 41°15′ and 41°35′W). The mean annual precipitation, between 1,100 to 1,300 mm, is seasonal with the month average minimum in winter of 41 mm and month average maximum in summer of 189 mm. The mean annual temperature is 22.6° C (
Jurubatiba is derived from the Atlantic rain forest and has eleven different vegetation assemblages with different species and life forms (
photosynthetic pathway with the exception of Clusia hilariana (Clusiaceae). The most common species found in these thickets (
Araujo et al. 2004
Araujo et al. 2004
Araujo et al. 2004
). The plant assemblage known as the open Clusia scrub is the most common assemblage. It represents 45% of park area (
). The flora of the Restinga of
) and is the focus of this study. The Clusia scrub assemblage is characterized by areas of bare sand and vegetation thickets having woody shrubs with broad leaves and the C3
) and the ones studied here are: Protium icicariba (DC.) Marchand (Burseraceae), Byrsonima sericea DC. (Malpighiaceae), Myrsine parvifolia A.DC (Myristicaceae), Erythroxylum ovalifolium Peyr. (Erythroxylaceae), Eugenia umbelliflora Berg (Myrtaceae) and Maytenus obtusifolia Mart. (Celastraceae).
Measurement of Leaf Mass Per Area, Succulence, Thickness and Density
We used the mean values from the dataset obtained by Rosado and de Mattos (2007) from nine measurements made from February of 2003 to August of 2005 (three per year) for the above species at this site. To measure the above parameters, ten leaves from separate individuals of each species were collected on the north side of their respective canopies. A leaf disc from each leaf was taken with a cork borer for the determination of leaf attributes. Each disc was
immersed in water for a period of 24 hours for the measurement of saturated mass (FMsat, g). After leaf discs were fully hydrated, each disc had their thickness (TH, mm) measured with a digital caliper and was then oven-dried for 72 hours for determination of dry mass (DM, g). Leaf mass per area (LMA, g.m−2) was calculated by dividing disc mass by its area (A) while the capacity of water storage in the leaf known as the succulence index (SUC, g.m−2) was
measured as: (Fmsat – DM)/A. The foliar density (DEN, mm.mm−3) was estimated following the formula: LMA = TH * DEN (
Witkowski and Lamont 1991
).
Relative Water Content of Apoplast (RWCAPOP)
During the wet season of 2005, one sun-exposed branch from each of seven individuals with mature leaves was cut from each species and used to measure the pressure-volume parameters. The base of each branch was cut under water in the laboratory and placed in a water recipient, covered with a humidified plastic bag and equilibrated in a dark room for about 12 hours. After initial measurements of water potential (PMS Instruments, Corvallis, OR, USA) and
Tyree and Hammel 1972
fresh mass, branches were dry on the bench and water potential and branch mass were periodically measured during dehydration (
Boyer 1995
). The RWCapop was obtained from the pressure-volume curves of each species based on approximately 20 measurements. P-V curves have a non-linear and a linear part (
). We determined the slope and intercept of a linear regression on the linear portion of the inverse balance pressure vs. fresh weight of
the branch plot. The RWCapop was obtained by dividing the intercept by the slope of the linear regression.
Stable Isotopes Analysis
Oxygen and Hydrogen Isotope Analyses
Lai et al. 2008
We collected samples from four individuals of each species on November 2005 and January 2006. Despite the observed variation in isotopic composition in leaves along the day (
), we collected the samples during the later morning for all species where we expect the leaves were transpiring and probably reaching a steady state. Although these months are considered as “wet” season, the January 2006 samples were collected at the end of a three week
rainless period (dry spell). Dry spells on the restingas are common, which may decrease the water availability even during the wet season due to the low water retention of sandy soils (Cavalin and de Mattos 2007). For each individual we sampled a well suberized stem (up to 4 cm in length and up to 1 cm in diameter) and one of the sun exposed north facing leaves, which was subsequently deveined. Eight soil samples were also collected 10 cm from the soil
Moreira et al. 2000
surface in November 2005 and January 2006. Soil, stems and deveined leaves were sealed in Vacutainer© tubes, further sealed with Parafilm© and kept frozen until water distillation (
Oxygen and hydrogen isotope ratios are reported here as δ18O, and δD respectively and calculated as:
). After distillation, oxygen and hydrogen isotope ratios of water were measured by equilibration on an Isoprime© Isotope Ratio Mass Spectrometer connected to a Multiflow© system (Elementar, Hanau, Germany) with the respective precision of ±0.1‰ and ±2.0‰.
in which Rsample and RSMOW represent the heavy (to light isotope ratio of the sample and the standard respectively. The standard for water isotope ratios used here is Vienna standard mean ocean water (vSMOW).
We calculated the isotopic leaf water enrichment relative to stem water (∆L) for both hydrogen and oxygen isotopes according to the following equation:
in which δL and δS are either the δ18O or δD values of leaf and stem water respectively.
RESULTS
The δD and δ18O values of stem water for November 2005 and January 2006 were close to the Global Meteoric Water Line (GMWL) compared with those of soil water (Fig. 1). However, the isotopic values of the January samples were more depleted than those of November (Fig. 1 and Table I). When stem and leaf water δD values are plotted against the respective δ18O values a highly significant linear relationship was described with a slope lower than that of
the GMWL (a slope=2.62, R2=0.79 for November and a slope=3.53, R2=0.91 for January). November and January Isotopic ratios of soil water were between those of leaf and stem water values with November samples being more enriched than those of January (Fig. 1).
Figure 1 Hydrogen isotope ratios of leaf, stem and soil water versus the respective oxygen isotope ratios of each species and soil samples for November of 2005 (y = 2.62 – 1.576x; R2=0.79) and January of 2006 (y = 3.53 – 19.03x; R2=0.91) compared to the meteoric relationship between δ18O and δ 2H of precipitation (δ2H=8.13 δ18O + 10.8).
TABLE I Leaf water oxygen and hydrogen isotopic enrichment (∆L) and hydrogen and oxygen isotope ratios of plant stem water (δ) for each species for November of 2005 and January of 2006. Different letters for each month in each variable indicating significant differences among species (p<0.05) and asterisks indicating significant differences in each species for each variable between months (p<0.05).
Byrsonima sericea
Erythroxylum ovalifolium
∆LD
∆L 18O
Species
δD Stem
δ18O Stem
Jan
Nov
Jan
Nov
Jan
Nov
Jan
17.66±3.25 a*
5.84±0.24ab
68.56±21.79a
17.99±2.06a
-4.83±2.06a
0.92±0.22b
-47.70±15.03 a*
-0.86±0.99a
a*
11.52±12.04a
a*
a*
-1.80±1.02a
a*
Nov
5.22±1.79a
52.86±2.93
0.71±0.30b
-24.66±1.74
Eugenia umbelliflora
16.04±1.52 a*
10.94±0.80c
56.81±9.81 a*
29.29±1.48a
-3.76±1.48a
-0.76±0.19a
-32.18±9.39 a*
-6.12±0.90a
Maytenus obtusifolia
14.40±0.71 a*
10.51±0.57bc
45.25±0.91 a*
25.28±2.62a
-3.66±0.25 a*
-1.23±0.18a
-25.72±1.90 a*
-2.09±1.72a
Myrsine parvifolia
15.50±0.84
a*
4.66±3.17abc
57.17±3.40
a*
5.10±20.73a
-4.10±0.25
a*
-1.21±0.34a
-30.32±0.88
a*
-5.41±3.28a
Protium icicariba
15.85±0.70 a*
12.24±0.99c
64.09±10.09 a*
25.33±4.62a
-2.45±0.53 a*
-0.74±0.22a
-26.76±1.06 a*
-1.28±1.50a
15.58±1.32
-1.75±0.33
Erythroxilum ovalifolium and Byrsonima sericea had significantly higher δ18O values of stem water compared to those of the other species during the wet November collection (Table I). No significant difference in the isotopic ratios of stem water between species during the dry period sampling in January was observed. The range of dry season stem water isotopic values, however, was greater than those observed during the wet sampling in November. Leaf
water oxygen and hydrogen isotopic enrichment (∆L) of Protium icicariba, Eugenia umbelliflora and Maytenus obtusifolia was higher than the other species during the wet November sampling (Table I), but no significant difference was observed for the dry January samples.
RWCapop varied from 0.203 to 0.590 among the six species studied (Fig. 2) and it was not related to any of the other measured morphological traits (Table II). The January average ∆L 18O and ∆LD values were negatively correlated only with RWCapop at a highly significant level (R2=0.84, P<0.01 and R2=0.86, P<0.01 respectively, Fig. 2; Table II) and the relationship remained significant even when Byrsonima (the outlier value) was removed (R2=0.72; P<0.04
and R2=0.68; P=0.05, respectively). The leaf water isotopic enrichment (∆L 18O and ∆LD) for the November sample did not correlate with any of the leaf traits measured here.
Figure 2 Negative relationship between Relative Apoplastic Water Content (RWCapop) and ∆L 18OJan and ∆LDJan (y = 93.80 + 127.59x; R2 = 0.86 and y = 20.47 + 16.25x; R2 = 0.84, respectively). Without Byrsonima (∆L 18OJan R2=0.72; P<0.04; y=18.89 + 11.35x); ∆LDJan (R2=0.68; p=0.05; y=91.53 + 120.51x).
TABLE II Correlation matrix between isotopic ratios of stem water and leaf water isotopic enrichment versus different leaf traits. Bold values are statistically significant (p<0.05).
Leaf Traits
δD Stem Jan
δD Stem Nov
δ18O Stem Jan
δ18O Stem Nov
∆LD Jan
∆LD Nov
∆L 18O Jan
∆L 18O Nov
Leaf Mass per Area
Leaf Succulence
Leaf Thickness
Leaf Density
Relative Apoplastic Water Content
-0.16
0.0
-0.09
-0.11
0.66
-0.58
-0.76
-0.76
0.54
-0.31
-0.66
-0.59
-0.64
0.11
0.09
-0.5
-0.6
-0.52
0.1
-0.72
-0.41
-0.48
-0.4
0.25
-0.93
0.33
-0.09
-0.16
0.85
0.2
-0.23
-0.35
-0.26
0.18
-0.92
0.19
-0.11
-0.2
0.78
0.32
DISCUSSION
Clark and Fritz 1999 Yakir and Sternberg 2000
According to the Craig-Gordon model soil water becomes isotopically enriched during periods of intense evaporation (
,
). In both months, the isotopic ratios of soil water were disproportionally enriched in 18O relative to the GMWL indicating evaporative effects near the soil surface. The isotopic composition of stem water which were closer to the meteoric water line are probably indicative of water use deeper in the soil
profile and similar to the average isotopic composition of rain water at the time of sampling. If so, then the average δ18O and δD value of rainfall during the month of November was greater than that occurring during January. It was not possible, however, to determine at which depth of the soil profile the species absorbed water, because we did not collect soil samples at different depth for isotopic analyses of soil water
The range of average δ18O values of stem water for each species during January (-4.83 to -1.75) was greater than that observed for the November samples (-1.23 to +0.92). This may be caused not only by a greater variability in water source between species, but also between individuals of the same species during drought periods. More variable water sourcing by plants in a coastal sand dune community under salt water impact was previously observed by
and Sternberg (2006)
Greaver
, where plants in the seaward side of sandy coastal plains did not show significantly different water source relative to landward plants based on isotopic composition of stem water. Seaward plants, however, showed greater variance in the isotopic composition of stem water than landward plants, indicative of a more variable water source for seaward coastal dune plants.
Despite the fact that the January water source was isotopically lighter than those of November, factors such as lower relative humidity (RH) or a different transpiration regime could have caused the isotopic enrichment of leaf water of the January samples to be greater than those of November (
). Our results at the species level showed absence of significant differences in leaf water isotopic enrichment among species over the long dry spell of
January, but not during favorable water availability in November. Recently, a trait-based approach in restinga indicated that temporal variation in water availability may predispose plants to water shortage during rainless days what may explain the convergence of leaf water isotopic fractionation during the dry spell (Rosado and de Mattos 2010). Restinga plants show high values of leaf water potential at the turgor loss point, less negative osmotic pressure and
most species have indications of chronic photoinhibition during the dry season. In addition, some species have a lower amount of leaf area available for transpiration during the dry season (Rosado and de Mattos 2010). In terms of leaf morphological traits, Rosado and de Mattos (2007) also reported in the same restinga species, a trend of highest values of LMA, SUC, TH and DEN in dry months during three years of study suggesting convergent responses to deal
with temporal heterogeneity of water availability. Additionally, in a broad scale, the study of Huxman et al. (2004) also observed that despite differences across biomes in annual precipitation, physiognomy and climatic history, rain use efficiency (i.e., the ratio of aboveground net primary production to precipitation) converged during years when water was the most liming factor. Thus, in spite of differential ability of plants to cope with water shortage, during
periods of strong water limitation, plants may show convergence of responses, which might be more related to a universal limit of plant survival under stressful conditions.
We observed that the greater the RWCapop, the lower the average ∆L 18O and ∆LD of leaf water during the dry spell. According to
Steudle et al. (1993)
, the water movement within leaves can occur simultaneously in three different pathways: (i) apoplastic flow through cell walls or cell to cell movement; (ii) via aquaporins; and (iii) plasmodesmata.
Barbour et al. (2000)
pointed out the importance of the increases in the effective length (L) to produce significant Péclet
Barbour and Farquhar (2003)
using wheat leaf as a model, estimated L for apoplastic pathway, between 9 and 15 mm, and for plasmodesmata and aquaporin pathway, between 121 and 201 mm and 9 and 15 mm, respectively. The authors observed that the three pathways of water movement presented different contributions to the enrichment between the
effect because of increases in the tortuosity in the water movement from the vein to the leaf evaporative surface.
vein and the evaporation sites and consequently could create a significant Péclet effect. We suggest that a best test for an important control of the Péclet effect would be the parameter 1-De/Dl, where De is Craig-Gordon enrichment and Dl is observed steady-state leaf water enrichment, against the parameter that is presumed to be controlling the Péclet number, which would the RWCapop.
Ferrio et al. 2012
Although we did not measure leaf transpiration, our results open new perspectives about the leaf traits that could affect ∆L. Interestingly, despite recent findings demonstrating that traits related to leaf hydraulic conductance are positively related to L (
conductance might be negatively related to such leaf traits like LMA and TH (
Aasamaa et al. 2005 Flexas et al. 2008
,
), our non-significant relationships between stem and leaf water isotopic enrichment and leaf morphological traits are contrary to these results since leaf hydraulic conductance and mesophyll
Ferrio et al. 2009 Zhou et al. 2011
). In fact, in cases where water flow is reduced due to tortuous apoplastic pathway, increases in effective L has been observed (
,
). As mentioned by
Lai et al (2008)
, L has never been directly measured and the effect of the uncertainties of measurement of L on ∆L is unknown. Therefore, we suggest that RWCapop, as proxy for L,
may perform a conspicuous role, in the movement of water and the tortuosity of the pathway from leaf vein to the evaporative surface.
As discussed previously, bulk leaf water isotopic enrichment is in part a function of the Péclet number, which in turn is a function of transpiration (T) and the effective diffusive length of the leaf (L). Here, we propose that during the relatively wet month of November, transpiration typical of each species is a source of distinction in the leaf water isotopic enrichment. During the dry spell, however, transpiration rates may decrease to a consistently low value
Lai et al. 2008
between restinga species (L.B. Lignani et al., unpublished data), raising the importance of the effective diffusive length as of great source of variability for the bulk leaf water enrichment among species. Contrary to other studies performed in tropical forests where plants are subjected to high environmental heterogeneity (
), the RH and isotope ratios of water vapor cannot explain the variability observed here since during sample collection all plants
were exposed to the same RH and vapor pressure deficit (B.H.P. Rosado et al., unpublished data). Supporting this, we observed that during the wet season average leaf water enrichment did not correlate with RWCapop. Hence, future studies performed in drought prone habitats where transpiration rate is low, should consider the role of RWCapop as a proxy for the effective diffusive length of the leaf (L) in isotopic models of leaf water enrichment.
In conclusion, we raised important aspects that will deepen our understanding regarding the mechanisms affecting leaf water enrichment and the diversity of ecophysiological behaviors in restinga plants. We demonstrated that despite differences in isotopic signatures during periods of higher water availability, restinga plants may converge to a similar foliar response during a long dry spell amidst the rainy season. Moreover, during a drought period the average
leaf water isotopic enrichment may be determined by anatomical properties of leaves, which are reflected in the RWCapop. Therefore, we suggest that RWCapop may be an important functional trait to be used as a proxy L in isotopic models explaining leaf water isotopic enrichment.
Acknowledgements
We are grateful to P. Cavalin for his help in field work and to G. Cardinot for help us with P-V curves. Sincere thanks go to Maria C. Pinzon, Amartya Saha and Patrick Ellsworth for great help in laboratory and useful discussions. We thank the staff from NUPEM-UFRJ and National Park for logistic support. This study was supported with grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do
Estado do Rio de Janeiro (FAPERJ) and the Brazilian Long Term Ecological Research Program (PELD-CNPq) for B.H.P.R. and E.A. de M. and NSF #0322051 and #0420553 for L. da S.L.S.
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Yakir D, DeNiro MJ and Rundel P. 1989. Isotopic inhomogeneity of leaf water: Evidence and implications for the use of isotopic signals transduced by plants. Geochim Cosmochim Ac 53: 2769-2773. [ Links ]
Yakir D and Sternberg LSL. 2000. The use of stable isotopes to study ecosystem gas exchange. Oecologia 123: 297-311. [ Links ]
Witkowski ETF and Lamont BB. 1991. Leaf specific mass confounds leaf density and thickness. Oecologia 88: 486-493. [ Links ]
Zhou Y, Grice K, Chikaraishi Y, Stuart-Williams H, Farquhar GD and Ohkouchi N. 2011. Temperature effect on leaf water deuterium enrichment and isotopic fractionation during leaf lipid biosynthesis: results from controlled growth of C3 and C4 land plants. Phytochemistry 72: 207-213. [ Links ]
Received: March23, , 2012; Accepted: August23, , 2012
Correspondence to: Bruno Henrique Pimentel Rosado E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013005000043
Biological Sciences
Daily and seasonal activity patterns of free range South-American rattlesnake (Crotalus durissus)
ALEXANDRO M. TOZETTI1, MARCIO MARTINS2
1Laboratório
de Ecologia de Vertebrados Terrestres, Universidade do Vale do Rio dos Sinos/UNISINOS, São Leopoldo, RS, Brasil, Laboratório de Ecologia de Vertebrados Terrestres,
Universidade do Vale do Rio dos Sinos/ UNISINOS, Avenida Unisinos, 950, 93022-000 São Leopoldo, RS, Brasil
2 Departamento de Ecologia, Instituto de Biociências, Universidade de São Paulo, Caixa Postal 11461, 05422-970 São Paulo, SP, Brasil
ABSTRACT
This study aimed at describing daily and seasonal variation in the activity of a population of South-American rattlesnakes (Crotalus durissus) in a savanna like habitat (Cerrado) in Southeastern Brazil.
Seasonal and daily activities of snakes were evaluated by the number of captures of snakes during road surveys, accidental encounters, and relocations by radio-tracking. Our results show that climatic
variables such as air temperature and rainfall have little influence on the activity pattern of rattlesnakes. Our findings indicate that rattlesnakes spend most of the day resting and most of the night in
ambush posture. The South-American rattlesnake is active throughout the year with a discrete peak in activity of males during the matting season. The possibility of maintaining activity levels even
during the coldest and driest season can facilitate the colonization of several habitats in South America. This possibility currently facilitates the colonization of deforested areas by rattlesnakes.
Keywords: Cerrado; ethology; squamata; radio-track
RESUMO
O estudo teve como objetivo descrever as variações na atividade sazonal e diária de uma população de cascavéis (Crotalus durissus) no Cerrado do sudeste brasileiro. As atividades sazonais e diárias
das cobras foram avaliadas por meio de capturas em amostragens em rodovias, encontros ocasionais e rádio rastreamento. Nossos resultados indicam que variáveis ambientais como a temperatura do
ar e a pluviosidade têm pouca influencia na atividade das cascavéis. Nossos dados indicam que elas passam a maior parte do dia em repouso e a maior parte da noite forrageando. A cascavel da
América do Sul se mostra ativa o ano todo com um pico moderado na atividade de machos durante a estação reprodutiva. A possibilidade de manter níveis de atividade até mesmo durante a estação
mais fria e seca do ano pode facilitar a colonização de vários habitats na América do Sul favorecendo a colonização de cascavéis em áreas desflorestadas.
Palavras-Chave: Cerrado; etologia; squamata; radio-telemetria
INTRODUCTION
Although the concept of activity is very broad, herpetological studies frequently associate activity with movement patterns of snakes, and consequently to their capture rates (
1987 Oliveira et al. 2013
,
Gibbons and Semlitsch
). These movements might be associated with migration, dispersion of newborns, escape from predators, search for shelters for daily thermoregulation, food, or mates for
reproduction (
Gibbons and Semlitsch 1987
). As a result of its association with movements, activity may vary depending on gender, age, reproductive condition, or molting (
Duvall et al. 1985 Macartney et
,
al. 1988
). Unlike in temperate areas, in most tropical regions, the winter, even the most harsh, does not seem to prevent the activity of most ectotherms, which tend to remain active throughout the
year (
Marques et al. 2000 Tozetti et al. 2010
,
).
In in tropical areas of South America, snake assemblages have shown activity peaks during the warmer, rainy season (mid-spring to early autumn;
Marques et al. 2000 Maciel et al. 2003
,
). The
rattlesnake Crotalus durissus, however, exhibits a peak of activity in the dry/colder season (autumn) (
). The peak activity of rattlesnakes in the coldest season of the year may
Huey et al. 1989
seem unexpected, especially when considering that the activity of ectotherms is directly dependent on the thermal regime of the environment (
). However this pattern may reflect the
Salomão and Almeida-Santos 2002
fact that during this season males search for females to mate (April to June) (
). Activity can also be subdivided into specific activities, such as foraging, reproduction
or thermoregulation. The difficulty of examining all behaviors that compose the activity of snakes might be the reason why most studies have presented activity from a general point of view,
Salomão et al. 1995 Dorcas and Willson 2009
based on encounter rates of snakes found during visual surveys, captured in traps or based on records of specimens in collections (
,
). However, the
importance of each one of these factors on snake activity remains poorly understood and data from field studies on South American rattlesnakes are extremely rare in the literature. Here we
describe the daily and seasonal activity patterns of C. durissus based on the encounter rate and radio tracking of a population of this species inhabiting an open Cerrado remnant, a savanna like
habitat in Southeastern Brazil.
Field work was conducted at the Itirapina Ecological Station (IES: 2.300 ha; 22°13′24′ S; 47°54′03′W; 700 m of elevation), municipality of Itirapina, State of São Paulo, Southeastern Brazil.
Regular sampling was conducted from December 2003 to December 2004. The main vegetation types in the reserve are grasslands and shrubby grasslands. The climate is mesothermic, with two
well-defined seasons, a dry/cold between April and August (average monthly rainfall from 18-42 mm) and a wet/warm season between September and March (88-368 mm). During the dry/cold
season in the study period, the minimum air temperatures ranged from 0 to 5° C and maximum from 31 to 35° C. At the same period in the rainy/warm season the air temperature variations were
7 to 15° C and 36 to 39°; C respectively.
Snake's activity was evaluated by: (1) captures during car surveys along unpaved roads and firebreaks that cut the study area, (2) accidental encounters, and (3) relocation of animals equipped
with radio-transmitters. Field work consisted of regular sampling during five consecutive days, every 20 days. In each sampling period, unpaved roads and firebreaks at IES were run daily
between 6 am and 24 pm, in a systematic manner with a vehicle at a speed below 35 km/h. Captured animals were weighed, measured, and implanted with a passive integrated transponder.
Snakes were also examined by abdominal palpation in order to detect gravid females. Animals with body mass above 250 g were equipped with radio-transmitters (model SI-2; 9 g, 33 mm × 11
mm; Holohil Systems Ltd., Ontario, Canada) externally attached with adhesive tape. The device never accounted for more than 5% of the animal's body mass. The transmitter was externally
attached to the dorsal region, at the posterior third of the snake's body using duct tape. The detachment of transmitters occurred during shedding. Animals were observed moving through dense
vegetation, basking, foraging, and even capturing and ingesting a prey offered by us. This suggests that the apparatus generate a low level of interference on snake's behavior (
Tozetti and Martins
2007 2008
,
). Sequential relocations of a radio-tagged individual snake were conducted with an interval of minimum 12 hours. Radio-tracking provided visual contact with almost all snakes that
were relocated both by day and at night to maximize the observation of different behaviors. In each snake re-location, air temperature, relative humidity, and substrate temperature were
recorded. Climate data were obtained from a meteorological station near the study area. We have estimates both, seasonal and diel snake's activity. Seasonal activity was assessed exclusively
based on the numbers of captured snakes during car surveys. We considered snake capture rates as an indicator of snake activity. Capture rates were estimated by dividing the total number of
captures by the sampling effort of each month (e. g., in January the sampling effort was 53.7 km/day and we captured two snakes; thus, the capture rate was 2 / 53.7 = 0.037 snakes/km/day).
Diel activity was assessed using the records of capture of snakes by car surveys, accidental encounters, and relocations from radio-tracking. Diel activity was classified as: coiled alert, coiled
hunting, moving, coiled resting, and basking. Each observation (capture or re-location) was considered as a record. Records of animals that were disturbed when they were found were not
Oliveira and Martins (2001)
considered. The activity of non-moving animals was determined based on the method proposed by
, as follow: (a) coiled hunting when the body was coiled, the neck was
forming an S-coil and the head was lying over the body coils forming an angle > 20o in relation to the ground ; (b) coiled alert when the body was coiled, the neck was forming an S-coil and the
head was lying over the body coils forming and angle < 20o in relation to the ground; (c) basking when the snake was found during daytime with the body extended or partially extended; and (d)
coiled resting when the body was loosely coiled, the neck was held straight and the head formed an angle < 20° in relation to the ground.
Considering the null hypothesis that rattlesnakes have the same probability of being observed performing each of the five types of activity, the expected number of monthly records for each
activity was defined as one fifth of the total number of observations in each month. We classified as daytime activities all records from 6:00 am to 5:59 pm and nighttime as those made between
6:00 to 12:00 pm. We used a Mann-Whitney U-test to test for differences in activity between sexes and seasons. The relationships between activity and environmental variables were compared
using Spearman Rank Correlation tests (rs ). In all cases, differences were considered significant when P < 0.05 (
Zar 1999
).
RESULTS
During the study, 38 rattlesnakes were captured (16 = car surveys; 22 = accidental encounters). Twelve were females (mean body size = 896.2 mm; range = 537-1,335 mm) and twenty-six were
males (mean body size = 977.7 mm; range = 637-1256 mm). A total of 12,000.36 km were covered with a vehicle in 224 sampling days. Throughout the study, the capture rate of rattlesnakes
based exclusively on the captures obtained with the road survey (n = 16) was 0.30 snakes/km/day. Capture rates significantly increased as air humidity increased (rs = 0.58; P < 0.05; n = 12). On
the other hand, no significant correlation was found between capture rate and daily maximum air temperatures (rs = 0.45; P = 0.14; n = 12), minimum air temperatures (rs = 0.51; P = 0.09; n =
12) or rainfall (rs = 0.30; P = 0.32; n = 12). Capture rates were not significantly different when the dry and rainy seasons were compared (0.9 and 0.20 snakes/km/day, respectively; U = 17; P =
0.93; n = 12). Capture rates were not different between sexes (0.17 snakes/km/day for males and 0.13 snakes/km/day for females; U = 63; P = 0.58; n = 24). Capture rates of females were not
different between the rainy and dry seasons (0.11 snakes/km/day and 0.02 snakes/km/day, respectively; U = 10; P = 0.22; n = 12; Figure 1). Similarly, capture rates of males were not different
between the rainy and dry seasons (0.09 snakes/km/day and 0.07 snakes/km/day, respectively; U = 17; P = 0.93; n = 12), despite a discrete peak in activity of males during the matting season
(Figure 1).
Figure 1 Seasonal activity (snakes/km/day) of Crotalus durissus based on captures during the road survey in Southeastern Brazil. Black columns = males; white columns =
females; solid line = mean of relative air humidity; dashed lines = maximum and minimum air temperatures; MMF = male-male fighting. Reproductive data obtained from:
Salomão et al. (1995) Salomão and Almeida-Santos (2002) Almeida-Santos et al. (2004)
,
,
.
Only 20 rattlesnakes (13 males and 7 females) were able to be equipped with radio-transmitters (exhibit minimal body mass). Snakes were radio-tracked during a mean of 64.9 days (9.5
relocations). During the day, resting was observed in a higher frequency than expected (46.1% of samples; χ2 = 30.3; GL = 10; P < 0.001; Table I). The number of observations of the remaining
activities was not significantly different from expected (alert: χ2 = 4.4; GL = 8; P = 0.82; hunting: χ2 = 3.35; GL = 5; P = 0.65; movement: χ2 = 1.2; GL = 2; P = 0.56; basking: χ2 = 8.5; GL = 8;
P = 0.38; Table I). At night, the activities observed did not vary significantly in relation to expected values (alert: χ2 = 1.97; GL = 7; P = 0.96; hunting: χ2 = 23.25; GL = 18; P = 0.18;
movement: χ2 = 1.87; GL = 6; P = 0.93; resting: χ2 = 3.07; GL = 15; P = 0.99; Table I).
TABLE I Number of records for each activity performed by rattlesnakes (Crotalus durissus) at the moment they were found during road surveys, accidental encounters, and
radio-telemetry at the IES. The percentage of a given activity in a given period of the day is shown between parentheses.
Activity
Coiled alert
Coiled hunting
Moving
Coiled resting
Basking
Totals
Day
16 (15.4%)
10 (9.6%)
6 (5.8%)
48 (46.1%)*
24 (23.1%)
104 (100%)
Night
17 (23.3%)
35 (47.9%)
9 (12.3%)
12 (16.4%)
73 (100%)
*
= observed value significantly higher than expected.
DISCUSSION
Our data show that C. durissus remains active throughout the year with a discrete peak in activity of males during the matting season. This may reflect the fact that during this season males
search for females to mate (April to June;
Salomão and Almeida-Santos 2002
). This variation may not be significant due to the small number of males captured. For females, the lack of changes in the
Sazima 1988
activity between seasons may be due by their sedentary lifestyle, as observed for females of Bothrops jararaca (
captured, supporting the hypothesis that gravid females are less mobile (
) and B. atrox (
Oliveira and Martins 2001
). No females with embryos were
Gibbons and Semlitsch 1987
). Also, gravid females represent a small part of the female population due to the biannual
Almeida-Santos and Salomão 1997 Almeida-Santos et al. 2004
reproductive cycle of rattlesnakes (
,
).
Previous data of activity of C. durissus was obtained from preserved specimens in herpetological collections and reveal that most of the seasonal variation in rattlesnakes activity is due to
dispersal of juveniles (
). On the other hand, our findings indicate that adult rattlesnakes have a constant activity pattern throughout the year, with slight drops during colder
months. Moreover, changes in activity could be detected in future studies that investigate intraday variation of the activity. During the colder months, for example, individuals of C. atrox, C.
Secor 1994 Beck 1995
cerastes, C. molossus and C. tigris were more active at midday while in the cooler were predominantly nocturnal or crepuscular (
pattern for the genus (
,
). We hypothesized that this might be a
Tozetti et al. 2009
).
Concerning dial activity, resting is the predominant activity during the day and movements are not very frequent, as also observed for Bothrops jararaca, B. atrox and other species of Crotalus
(
Duvall et al. 1985 Sazima 1988 Beck 1995 Oliveira and Martins 2001
,
et al. 1999
,
,
). Movements probably increase the risk of predation and exposure to adverse environmental conditions (
Gibbons and Semlitsch 1987 Bonnet
,
), which may be avoided during the day. From this point of view, the selection of a foraging site that could also provide the diurnal thermoregulatory requirements is essential, reducing
Huey et al. 1989
the need of moving. Despite the importance of basking to control the metabolic activity of ectotherms (
), the number of records of animals performing this activity was relatively
low. Perhaps the vegetation structure, associated with the relatively warm winter, keep the average daily temperatures from oscillating much among seasons, decreasing the need of prominent
changes in activity associated with thermoregulatory needs. Hunting was typically a nocturnal activity, the period that coincides with the peak of prey activity (rodents; see
In addition, the selection of hunting sites is not random, but rather based on chemical cues from prey, maximizing the probability of finding them even during the day (
Emmons and Feer 1997
).
Reinert et al. 1984
).
Our findings support some of the hypothesis on the daily and seasonal activities of South American rattlesnakes previously described in studies on specimens from collections, on captive
animals, or from opportunistic sightings in the field. Compared to the North-American rattlesnakes, C. durissus is active throughout the year what may be allowed both by favorable weather
conditions. These features clearly facilitated the colonization by C. durissus of several open habitats in South America during its spread in the continent (
invasion of deforested areas throughout its distribution (e. g.,
WÜster et al. 2005
), as well as facilitated its
Bastos et al. 2005
).
Acknowledgements
This study was developed with field assistance of several people, especially Victor Vetorazzo. We also thank Denise Zancheta (Inst. Florestal – SP) for permissions, Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the grants received.
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Received: June13, , 2011; Accepted: September4, , 2012
Correspondence to: Alexandro Marques Tozetti E-mail: [email protected]
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013005000055
Biological Sciences
Regional movements of the tiger shark, Galeocerdo cuvier, off northeastern Brazil: inferences regarding shark attack hazard
FÁBIO H.V. HAZIN1, ANDRÉ S. AFONSO12, PEDRO C. DE CASTILHO1, LUCIANA C. FERREIRA1, BRUNO C.L.M. ROCHA1
1
Departamento de Pesca e Aquicultura, Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros, s/n, 52171-030 Recife, PE, Brasil
de Ciências e Tecnologia, Universidade do Algarve, Campus de Gambelas, Faro, Portugal, Faculdade de Ciências e Tecnologia, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
2Faculdade
ABSTRACT
An abnormally high shark attack rate verified off Recife could be related to migratory behavior of tiger sharks. This situation started after the construction of the Suape port to the south of Recife. A previous study suggested that attacking
sharks could be following northward currents and that they were being attracted shoreward by approaching vessels. In this scenario, such northward movement pattern could imply a higher probability of sharks accessing the littoral area
of Recife after leaving Suape. Pop-up satellite archival tags were deployed on five tiger sharks caught off Recife to assess their movement patterns off northeastern Brazil. All tags transmitted from northward latitudes after 7-74 days of
freedom. The shorter, soak distance between deployment and pop-up locations ranged between 33-209 km and implied minimum average speeds of 0.02-0.98 km.h−1. Both pop-up locations and depth data suggest that tiger shark
movements were conducted mostly over the continental shelf. The smaller sharks moved to deeper waters within 24 hours after releasing, but they assumed a shallower (< 50 m) vertical distribution for most of the monitoring period.
While presenting the first data on tiger shark movements in the South Atlantic, this study also adds new information for the reasoning of the high shark attack rate verified in this region.
Keywords: migration; Recife; satellite telemetry; shark attack; Suape
RESUMO
A elevada taxa de ataques de tubarão verificada em Recife poderá estar relacionada com o comportamento migratório do tubarão tigre. O início desta situação coincidiu com a construção do porto de Suape localizado a Sul de Recife. Um
estudo anterior sugeriu que algumas das espécies responsáveis pelos ataques poderiam estar seguindo as correntes costeiras para Norte e que o trânsito marítimo estaria atraindo as mesmas para junto da costa. Neste cenário, a
movimentação dos tubarões para Norte implicaria uma maior probabilidade de estes acessarem o litoral de Recife após sairem de Suape. Para averiguar os padrões de movimentação do tubarão tigre no nordeste brasileiro, foram aplicados
transmissores via satélite a 5 espécimes capturados ao largo de Recife. Todas as marcas transmitiram de latitudes a Norte após 7-74 dias em liberdade. A menor distância entre os locais de captura e de primeira transmissão foi de 33-209
km, correspondendo a velocidades médias mínimas entre 0.02-0.98 km.h−1. As localizações da primeira transmissão e os dados de profundidade sugerem que os movimentos dos animais foram realizados principalmente sobre a
plataforma continental. Os tubarões menores deslocaram-se para águas profundas em até 24 horas após a liberação, mas assumiram uma distribuição mais superficial (< 50 m) durante a maior parte do tempo. Este estudo apresenta os
primeiros resultados das movimentações de tubarões tigre no Atlântico Sul e adiciona novas informações para a compreensão dos motivos que levaram à problemática de ataques de tubarão verificada nesta região.
Palavras-Chave: migração; Recife; telemetria via satélite; ataque de tubarão; Suape
INTRODUCTION
Understanding the factors that elicit repeated shark attack events on humans in a local scale is essential in every sort of aspects. Since 1992, the metropolitan region of Recife, Brazil, exhibits one the highest shark attack rates per
Fischer et al. 2009
Gadig and Sazima 2003 Hazin et al. 2008
unit of area in the world, accounting for 53 attacks which resulted in 20 fatalities. Relative abundances assessed off Recife (
), together with forensic analysis (
,
) indicated the tiger
shark, Galeocerdo cuvier, and the bull shark, Carcharhinus leucas, to be responsible for most of the attacks. Both species are considered potentially aggressive and have been frequently implicated in attacks on humans worldwide
(International Shark Attack File, http://www.flmnh.ufl.edu/fish/Sharks/ISAF/ISAF.htm. Accessed January 26, 2011).
Hazin et al. (2008)
suggested that the construction of a port complex in Suape, about 20 km to the south of Recife, was the main factor responsible for the attack outbreak due not only to strong environmental degradation, resulting
from construction activities, but also to increased attraction of sharks into this area in association with higher maritime traffic. Sharks are known to follow ships (Baldridge 1974,
(
Coppleson 1958 McCord and Lamberth 2009 Meyer et al. 2009
,
,
Myrberg 2001
). Attraction of sharks by low-frequency sounds (
Schultz 1975
) and to visit port areas and harbors
Averson and Vendittis 2000
) emitted by transiting vessels (
) could partially explain such behavior, as well as the common
Hazin et al. (2008)
habit of ship crews to throw garbage overboard. Thus, it is likely that the Suape port may have favored the approximation of sharks to these shores.
suggested that potentially aggressive sharks occurring off Recife
could be moving downstream following northward coastal currents, which would lead them from the coast of Suape directly to the beaches of Recife where most attacks occurred. This would be most applicable in relation to tiger
Meyer et al. 2009
Heithaus et al. 2007 Kohler et al. 1998
sharks, which have wide home ranges (
) and have been reported to perform long-distance movements in a short amount of time (
,
). If tiger sharks off northeastern Brazil are moving
northward then, under the assumption that sharks are being attracted shoreward towards Suape by incoming vessels, one might predict that they would subsequently visit the littoral area of Recife. This note investigates the regional
movements of tiger sharks caught off Recife and addresses the implications of such movements regarding local attack hazard.
Recife is located in the northeastern Brazilian coast (8°03′S, 34°53′W; Fig. 1), about 20 km to the north of the Suape port. The Suape port was built in a large, mangrove-bordered estuarine system which used to be relatively
pristine until construction activities inflicted intense habitat degradation, including the partial barring of two of the four rivers previously discharging in this system. In the metropolitan region of Recife, the area where most of the
Souza 2007
attacks occurred corresponds to a 20 km stretch of densely populated beaches, which includes the Barra de Jangadas estuary. The continental margin of northeastern Brazil consists of a narrow, 63 km width shelf (
bordered by one of the longest, consistently steep (4 to 20°) slopes in the world (
et al. 2005
).
Fainstein and Milliman 1979
)
Stramma 1991 Stramma et al. 1995 Bittencourt
). In this region, coastal currents assume a northward direction almost all year-round (
,
,
Figure 1 Deployment and pop-up location of Pop-up Satellite Archival Tags (PSAT) fitted on five tiger sharks caught off Recife, Brazil. On the map, locations are represented by: Cross = T1, T2, T4, and T5
tag deployments; plus sign = T3 tag deployment; solid square = T1 tag pop-up; empty circle = T2 tag first transmission; solid circle = T2 tag pop-up location estimate; solid pentagon = T3 tag pop-up; hash
mark = T5 tag first transmission. The darkness gradient in the oceanic region is directly proportional to water depth relatively to the continental shelf, which is represented in white.
Pop-up satellite archival (PSAT) tags (model mk10; Wildlife Computers, Washington) were deployed on 5 tiger sharks (T1-T5; Table I) caught off Recife between 2008 and 2010 during the winter season (June-August), since
Hazin et al. 2008
winter presented higher shark attack rates (
). All sharks were caught within 2.0 km from shore between 8-12 m isobaths, using a bottom longline equipped with 10 m length branch lines and 18/0, 0% offset circle
hooks, except for T3 which was caught 17 km from shore at about 30 m depth. All sharks were carefully brought onboard, eye-covered, and restrained on deck (T1 and T2) or in a wooden tank filled with sea water (T3, T4 and
T5), which was readily assembled after the shark was first sighted. Sharks were then quickly transported offshore in order to remove them away from the area of risk, and released at isobaths between 20-40 m, depending on
oceanographic conditions and the health status of the shark. Before releasing, sharks were measured, sexed, and tagged with both a conventional, stainless steel, dart tag, and a PSAT-tag. The conventional tag was fitted to the
dorsal musculature just below the first dorsal fin, while the PSAT-tag was attached to the proximal, anterior region of the first dorsal fin by passing a coated, 2.0 mm polyamide monofilament through a hole pierced with a 3.0 mm
gauge needle and tightly adjusting the length of the monofilament to prevent the tag from crossing over the dorsal fin towards its anterior region while being towed by the shark. Every procedure carried out with sharks was
conducted in accordance to the recommendations at the Regiment of the Commission of Ethics on the Usage of Animals from the Universidade Federal Rural de Pernambuco. This study was ethically approved by the license
number 041/2009 (protocol number 23082.009679/2009 D18). All tiger sharks were juveniles, measuring 128-193 cm TL, and were released in good health, generally swimming away from the boat immediately after being
returned to the water except for T4, which had to be assisted during approximately 15 min before demonstrating enough strength for being released.
TABLE I Summary of PAT-tag deployments on tiger sharks off Recife, Brazil between 2008-2010. Tagging location was about [8.1 S; 34.8 W] for all sharks except for T3 [8.25 S; 34.77 W]. Note that T4 tag
uplinked to the satellite insufficient times and so pop-up location and tracking data are not available (n.a.).
Tag Sex TL (cm) Tag date Prog. span Track span Pop-up location
T1
T2
T3
T4
T5
M
M
F
F
M
130
193
128
154
150
28-Jun-08
25-Jul-09
1-Jun-10
1-Aug-10
7-Aug-10
30 d
75 d
73 d
50 d
99 d
30 d
4d
74 d
42 d
72 d
6.32S;34.79W
7.34S;34.65W*
7.97S;34.67W
n.a.
7.08S;34.85W**
Linear distance (km)
209
94*
33
n.a.
125**
Km from shore Max. depth (m)
23
16
15
n.a.
inland**
248
56
200
n.a.
304
Min. Avg. speed (km.h−1)
0.29
0.98
0.01
n.a.
n.a.
∆Temp. (°C) Hours. Bin-1
13.6 – 27.0
25.0 – 27.6
15.0 – 29.0
n.a.
13.0 – 28.0
24
3
3
2
4
*
estimated from tag drift;
**
relative to first transmission.
PSAT-tags were programmed to release between 30-99 days after deployment (Table I). Water depth and temperature were recorded every second and further summarized into bins of between 2-24 hours for transmitting during
satellite uplinks, which occurs once the tag pops off the shark and floats to the surface. Depth and temperature data were binned in 14 strata that were set before deployment. The time spent at each stratum was continuously
monitored. Disregarding minor variations, depth strata were generally arranged by classes < 0, 0-5, 5-10, 10-20, 20-40, 40-60, 60-80, 80-100, 100-125, 125-150, 150-200, 200-250, 250-300, and > 300 m. Temperature strata were
arranged by classes < 12, 12-14, 14-16, 16-18, 18-20, 20-22, 22-24, 24-25, 25-26, 26-27, 27-28, 28-29, 29-30, and > 30 °C. Heterogeneous strata sizes required temporal data to be standardized by either depth- or temperature-unit
24
before assessing tiger shark environmental preferences. Data analysis was performed with R 2.12.2 ( ) and IGOR Pro 6.1®. In one circumstance, the pop-up position was derived from surface current direction and speed which
Hays et al. 2001
were estimated based on tag drift during the first 24 hours of satellite-linked transmissions using only messages with location quality LC ≥ 1 (Argos-based geolocation error < 1 km for LQ ≥ 1) (
). Luminosity-based
geolocation estimates, which provide a proxy of the horizontal movements performed by the tagged individual during the tracking period, were not considered because position estimates errors at tropical latitudes are yet too great
for assessing movements conducted in small spatial scales and thus are most effective for studies conducted in the oceanic realm (Musyl et al. 2011).
RESULTS
Following pop-up, all satellite tags transmitted to the north of Recife at linear distances varying between 33 and 209 km from the deployment location (Fig. 1), except for T4 tag. Although the T4 tag transmitted a few messages by
the programmed pop-up date, the amount of consecutive transmissions were insufficient for generating a geolocation estimate, what could be ascribed to a technical failure such as the antennae being damaged during the
deployment. Due to some premature releases, tracking duration varied between 4 and 74 days (Table I). T2 tag transmitted 7 days after deployment, about 152 km to the north of Recife (Fig. 1); however, data analysis showed that
the tag had been drifting at the surface for about 3 days before transmitting to the satellite (Fig. 2B). Estimated pop-up position derived from surface current direction (about 10° NW) and speed (0.25 m.s−1) corresponded to 94 km
northward from the tagging location (7.34° S; 34.65° W). The first transmission of 3 out of the 4 successfully deployed tags occurred at 15-23 km from the coastline. T5 tag also prematurely released but it was washed up to the
beach before transmitting (3 days after releasing) and so the estimate of pop-up position was impossible to assess. Assuming the shorter, soak course between deployment and pop-up locations, calculated minimum average speeds
were low for both T1 and T3 (0.29 and 0.02 km.h−1, respectively), but higher for T2 (mean speed of 0.98 km.h−1, equaling to 0.13 body length per second).
Figure 2 Depth-and-temperature profiles of the vertical movements performed by tiger shark (A) T1, (B) T2, (C) T3, and (D) T5 off Northeastern Brazil. Data illustrates the depth range and associated water
temperature per each time-unit successfully sampled and blanks correspond to data which was not successfully up-linked to ARGOS satellites. Note that both tags T2 and T5 prematurely detached off the
animals and drifted on the surface for 3 days before transmitting. Data pertains to years 2008 (T1), 2009 (T2), and 2010 (T3 and T5).
The vertical depth-and-temperature profiles were relatively consistent among all tracks. T1, T3 and T5 showed a clear tendency for occupying deeper water layers during the first ~13 days of tracking, frequently performing dives
between 150-300 m deep (Fig. 2). T5 performed the deepest dive observed (= 304 m). The thermal gradients of the dives of these three sharks went up to 13.4 °C, indicating deep penetration into the thermocline layer. After that
first period, the sharks assumed a more superficial behavior for the remainder of the tracking period, generally never exceeding the 50 m isobaths, except for T1 which repeated the deep-diving pattern soon after for a couple of
days (Fig. 2A). Despite being tracked for only 4 days, T2 movements were conducted exclusively in shallow water (< 56 m) in an environment with very little thermal variation (Fig. 2B), resulting in a quite homogeneous depthand-temperature profile which was similar to the profiles of the other three sharks when their movements were restricted to more superficial waters. All the individuals moved to the surface during just about every temporal unit of
the respective track.
Overall, all the individuals showed a strong preference for shallower waters, spending on average 52.5% (SD = 6.5%) of the tracking time at depths < 10 m and 88.0% (SD = 7.5%) at depths < 40 m (Fig. 3A). T5 spent the least
amount of time (44.5%) at depths < 10 m, while T2 spent most of the time (59.7%) at those same depths. T3 spent the least amount of time (1.6%) at depths > 40 m. Standardization of depth strata further reduced the time per unit
of depth spent by all sharks at deeper waters and it increased the time per unit of depth spent at isobaths < 10 m (average = 81.6%, SD = 7.8%). Generally, all sharks exhibited a decreasing preference for water layers deeper than
10 m, except for T3, which spent twice as much time per unit of depth at depths between 20-40 m than between 10-20 m (Fig. 3B).
Figure 3 Proportion of time spent by tiger sharks T1, T2, T3 and T4 at each depth strata for the entire tracking period considering (A) the raw time data as provided by the tag manufacturer, and (B) the depthstandardized time data for each depth bin as the relative time per unit of depth. The diameter of each circle is directly proportional to the cubic root of relative time in order to reduce differences between circle
sizes of different depth strata. Plot produced in R version 2.12.2 with balloonplot function from
Wickham (2009)
.
All sharks spent most of the tracking time (average = 94.6%, SD = 6.8%) between 24-28°C. T1, T2 and T3 spent considerable time (average = 84.3%, SD = 9.9%) at warmer waters, between 26-28°C, while T5 spent 74.8% of the
tracking time between 24-26°C and only 20.0% between 26-28°C (Fig. 4).
Figure 4 Proportion of time spent by tiger sharks T1, T2, T3 and T4 at each temperature strata for the entire tracking period. The diameter of each circle is directly proportional to the cubic root of relative time
in order to reduce differences between circle sizes of different temperature strata. Plot produced in R version 2.12.2 with balloonplot function from
Wickham (2009)
.
DISCUSSION
The present results sustain the hypothesis that tiger sharks occurring off Recife perform regional northward movements, following the direction of coastal currents. Long-distance movements of tiger sharks in both coastal and
Heithaus et al. 2007 Kohler et al. 1998
oceanic realms have been evidenced in previous studies (
,
Heithaus 2001 Wirsing et al. 2006
), and seasonal migrations were suggested to occur in some regions (
move throughout wide-ranging, coastal habitats (15-109 km) which they patrol during a period of time before leaving to a different area (
,
). In Hawaii, tiger sharks were noted to
Meyer et al. 2009
). Unfortunately, the poor quality of luminosity-based geolocation in tropical
Musyl et al. 2001
waters, most especially regarding latitude estimates (
), did not allow the assessment of directional shifts of latitudinal displacement which could indicate patrolling behavior. Nevertheless, the close proximity of T3
tag to the original deployment location after a long period of time (74 days) suggests that such behavior may have occurred. It contrasted with T2 horizontal movement which produced a considerable displacement in little time,
thus indicating a relatively well oriented course northwardly after being tagged off Recife. In any case, if patrolling behavior occurs in tiger sharks off northeastern Brazil, the overall net displacement appears to be directional,
aiming to the north, since no tag transmitted to the south of Recife.
Tagged individuals were all juveniles measuring < 200 cm TL because the majority of the tiger sharks caught off Recife belong to that size-class, exceeding the number of larger specimens by 10-fold. They were by far the most
Hazin et al. 2000
prevailing component amongst the catch composition of potentially aggressive species (
). In spite of considerably low relative abundances, juvenile tiger sharks have been noted to occur off Recife in temporal
clusters, with two or more individuals being caught in the same week or even in the same fishing set (F.H.V. Hazin et al., unpublished data). Even though the role of these smaller individuals in the shark attack problematic is not
hitherto clear, it seems plausible that they may have been responsible for some of the incidents.
All tags popped-up over the continental shelf of northward regions, suggesting that juvenile tiger sharks preferentially move in the neritic zone off northeastern Brazil. This was sustained by the depth-temperature profiles
registered by electronic tags, which evidenced a clear preference for superficial waters. Indeed, the observed vertical distribution may reflect a depth preference as much as a bathymetric constraint. The continental shelf off
Manso et al. 2003
northeastern Brazil is relatively monotonous, slanting gently from the shoreline until a depth of about 50-60 m (
), where the slope abruptly starts. General maximum dive depths of 40-60 m observed in the greatest
extent of all tracks suggest that juvenile tiger sharks in this region move mostly over the continental shelf, at least during the winter season when they appear to be more abundant off Recife (F.H.V. Hazin et al., unpublished data).
The tagged individuals stayed most of the time at the mixed layer and so they were mainly exposed to a thermal niche of 24-28°C throughout the tracking period, as verified by temperature data. The lower temperature associated
Hazin et al. 2000
with T5 movements should be attributed not only to deep-diving behavior but also to seasonal variation in the water temperature, which usually tends to be minimum in August (
). Interestingly, deeper dives into
depths > 100 m were performed by all the three smaller individuals almost exclusively within the first ~13 days after tagging, with all sharks adopting a shallower vertical distribution after that period. The similarity of such pattern
between different individuals suggests that it could be ascribed to post-release, stress-mediated behavior. It also suggests that tagged individuals seek refuge in offshore, deeper waters after being released. In fact, three of the sharks
were clearly off the continental shelf within a 24 h period after being released. The duration of deep-diving pattern suggests that the behavior of caught-and-released juvenile tiger sharks could be altered during a couple of weeks
after releasing, eventually implicating some behavioral biases during the beginning of telemetry studies. T2 was the only individual who did not exhibited such a propensity for escaping to deeper waters, which could be related to
its larger size compared to the other specimens. The vertical distribution evidenced by these 4 tiger sharks is comparable with results obtained in Hawaii (
Holland et al. 1999 2001
,
).
Observed minimum average speeds, measured by considering the shorter, soak distance between deployment and pop-up locations, were greatly different. T2 was the largest shark and appeared to move faster. However, the small
duration of its track probably reduced the amount of biases imposed by both longitudinal and vertical movements, which would tend to diminish average speeds in longer tracks. In accordance, T2 minimum average speed was
roughly comparable to tiger shark speeds assessed in previous studies. In Hawaii, an average speed of 0.29 body length per second (BL.s−1) was measured for six acoustically tracked, mostly > 300 cm TL tiger sharks (
Holland et al.
BL.s−1
1999
m.s−1,
km.h−1
m.s−1).
). Assuming 0.29
as a validated speed, the expected speed for T2 would be 0.57
which is within the same order of magnitude of the measured speed (0.98
= 0.27
Such compatibility suggests T2 to
have moved in a relatively well oriented latitudinal course, as opposed to longitudinal and/or vertical displacements, since T2 would already be expected to swim slower than > 300 cm TL individuals due to its smaller size. The
pop-up latitudes of T1- and T3-tags were distinct, as well as the duration of their tracks. In spite of T3 having the longest track span (74 days), its location was the closest to the tagging site by the end of the tracking period,
resulting in an unreasonable estimate of minimum average speed. On the other hand, T1 exhibited the highest latitudinal displacement by the end of its track (30 days), indicating that juvenile tiger sharks also utilize wide-ranging
habitats off the continental shelf of northeastern Brazil. Despite the long distance achieved, the relatively low minimum average speed of T1 could be attributed to the more frequent longitudinal and vertical displacements when
compared to the T2 shark. Tiger shark diel rhythmicity has been recorded in previous studies (
Heithaus et al. 2006 2007 Lowe et al. 1996 2006
Tricas et al. 1981
), usually comprising predatory excursions to shallow habitats with high prey density to forage and subsequent returns to
Meyer et al. (2009)
deeper waters (
,
,
,
), although
found no evidence of rhythmic patterns of behavior in a more recent study. Off the Brazilian coast northward of Recife, such behavior would
imply longitudinal movements which would necessarily reduce the rate of latitudinal displacement.
Tiger sharks visiting the littoral area of Recife during winter season appear to be performing northward movements at least on a regional scale. Whether tiger sharks approach Recife coming from eastern oceanic waters, following
the South Equatorial Current, or coming from south, following the Brazilian coast, is a question that further satellite-tagging should clarify. In any case, if tiger sharks are being attracted shoreward by vessels approaching the Suape
port, they might be expected to further move north into the littoral area of Recife. The probability of a tiger shark accessing into the risk area would then be a function of both bioecological features determining temporal variability
of tiger shark abundance, and the intensity of maritime traffic to Suape, which is variable at week- and year-level. Significant statistical correlations between periods of higher maritime traffic into Suape and shark attack events off
Hazin et al. 2008
Recife have been previously evidenced (
), which further supports this hypothesis. Based on the assumption that the port of Suape is attracting sharks shoreward, the pattern of tiger shark regional movements suggested
by the present study may possibly emerge as a significant factor contributing for the shark attack outbreak verified at Recife.
Acknowledgements
The authors are thankful to Rafael Muniz, José Pacheco, Diogo Nunes, Lucas Flores, Renato Santiago, Sidney Silva, Juliana Viana, Wesley Oliveira, Gabriel Crema, Camilo Rosa, Camila Araújo, Anderson Silva, and the crew of
R.V. Sinuelo and R.V. Pedrinho for assisting with field work. Precious guidance regarding data analysis was kindly supplied by B. Block, S. Jorgensen and M. Castleton, to whom we are deeply appreciated. The authors would like
to acknowledge the State Government of Pernambuco and Petrobrás (Brazil) and Fundacão para a Ciência e Tecnologia (Portugal) for funding this research and providing a PhD grant to Afonso AS (MCTES/FCT/SFRH/
BD/37065/2007).
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Received: November7, , 2011; Accepted: October5, , 2012
Correspondence to: André Sucena Afonso E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
●
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http://dx.doi.org/10.1590/S0001-37652013005000045
Biological Sciences
Nuclear entropy, angular second moment, variance and texture correlation of thymus cortical and medullar lymphocytes: Grey level co-occurrence matrix analysis
IGOR PANTIC1, SENKA PANTIC2, JOVANA PAUNOVIC2, MILAN PEROVIC3
1
2
3
Laboratory for Cellular Physiology, Institute of Medical Physiology, School of Medicine, University of Belgrade, Visegradska, 26/II, 11129 Belgrade, Serbia
Institute of Histology and Embryology, School of Medicine, University of Belgrade, Visegradska, 26/II, 11129 Belgrade, Serbia
Hospital Center “Narodni Front”, School of Medicine, University of Belgrade, Kraljice Natalije, 62, 11000 Belgrade, Serbia
ABSTRACT
Grey level co-occurrence matrix analysis (GLCM) is a well-known mathematical method for quantification of cell and tissue textural properties, such as homogeneity, complexity and level of disorder. Recently, it was demonstrated that this method is capable of evaluating fine structural changes in nuclear structure that otherwise are undetectable during standard microscopy analysis. In this article, we present the results indicating
that entropy, angular second moment, variance, and texture correlation of lymphocyte nuclear structure determined by GLCM method are different in thymus cortex when compared to medulla. A total of 300 thymus lymphocyte nuclei from 10 one-month-old mice were analyzed: 150 nuclei from cortex and 150 nuclei from medullar regions of thymus. Nuclear GLCM analysis was carried out using National Institutes of Health
ImageJ software. For each nucleus, entropy, angular second moment, variance and texture correlation were determined. Cortical lymphocytes had significantly higher chromatin angular second moment (p < 0.001) and texture correlation (p < 0.05) compared to medullar lymphocytes. Nuclear GLCM entropy and variance of cortical lymphocytes were on the other hand significantly lower than in medullar lymphocytes (p < 0.001).
These results suggest that GLCM as a method might have a certain potential in detecting discrete changes in nuclear structure associated with lymphocyte migration and maturation in thymus.
Keywords: texture analysis; GLCM matrix; CD4; CD8; gene
RESUMO
Análise da matriz de co-ocorrência de níveis de cinza (GLCM) é um método matemático bem conhecido para a quantificação das propriedades estruturais das células e tecidos, incluindo homogeneidade, complexidade e nível de desordem. Recentemente foi demonstrado que esse método é capaz de avaliar as mudanças estruturais finas do núcleo que, de outra forma não são detectáveis pela análise microscópica habitual. Nesse
artigo, apresentamos resultados indicando que a entropia, segundo momento angular, variância, e correlação de textura da estrutura nuclear de linfócitos, determinada pelo método de GLCM, diferem na córtex do timo em comparação a medula. Um total de 300 núcleos de linfócitos do timo de 10 camundongos de um mês de idade foram analisados: 150 núcleos da córtex e 150 núcleos da medula do timo. Análise de GLCM nuclear
foi realizada usando o software ImageJ do National Institutes of Health (NIH). Para cada núcleo, entropia, segundo momento angular, variância e correlação de textura foram determinadas. Linfócitos corticais continham significativamente maiores os parâmetros segundo momento angular da cromatina (p<0.001) e correlação de textura (p<0.05) comparados a linfócitos medulares. A entropia GLCM nuclear e variância de linfócitos
corticais eram por outro lado significativamente menores que nos linfócitos medulares (p<0.001). Esses resultados sugerem que GLCM como método tenha um potencial de detector mudanças discretas na estrutura nuclear associadas a migração e maturação de linfócitos no timo.
Palavras-Chave: análise de textura; matriz GLCM; CD4; CD8; gene
INTRODUCTION
During the past several decades, there have been numerous research efforts to design and implement a simple, accurate, and affordable computational biology method for quantification and evaluation of nuclear architecture. Many of the developed techniques include image analysis of cell cultures and stained tissue micrographs, and they combine standard morphometric measurements with second and higher order
statistical parameters. One of the recently applied techniques that include second order statistical texture analysis is the so-called grey level co-occurrence matrix method (GLCM). GLCM is a rapid mathematical method for assessing image structural properties, such as homogeneity, complexity and level of disorder. It was introduced in the work of
on pixel pair grey level occurrences within a grey scale image. In time, GLCM was successfully applied in nuclear magnetic resonance imaging (
Li et al. 2009
suggested that in cell cultures, GLCM parameters of nuclear structure may be important for evaluation of chromatin structural changes during apoptosis (
Huber et al. 2011
), computed tomography (
Haralick et al. (1973)
, who have established a total of 14 parameters based
Shamir et al. 2009
) and other clinical research areas. In fundamental medical and biology research, Shamir et al. demonstrated that tissue GLCM entropy is an indicator of tissue age-related structural degradation (
). Losa et al.
Losa and Castelli 2005
).
Shamir et al. 2009 Pantic and Pantic 2012 Pantic et al. 2012a Losa and Castelli 2005 Li et al. 2009 Alvarenga et al. 2010
Today, of all GLCM parameters, entropy, angular second moment (ASM), variance and texture correlation are perhaps most commonly used in experimental and clinical medicine (
,
,
,
,
,
). In recent research carried out by our laboratory, we have suggested that GLCM entropy and angular second moment can be successfully
used to evaluate structural properties of a secondary lymphoid organ. In immunized guinea-pigs, entropy of spleen germinal centers was negatively correlated with humoral immune response, indicating that entropy may be a good predictor of structural deterioration and degradation that occurs in spleen germinal center tissue (
(where the process of B-lymphocyte selection takes place), entropy and angular second moment of lymphocyte nuclear structures are related to the nuclear size (
Pantic and Pantic 2012
). Also, in another study, we found that in germinal center light zones
Pantic et al. 2012a
). These results suggested that nuclear GLCM parameters may be good indicators of structural changes that follow the shrinkage of nuclei during early stages of programmed cell death (apoptosis) in spleen white pulp.
Unlike secondary lymphoid organs, there are virtually no data regarding primary lymphoid organ nuclear GLCM properties. In this article, we present the results indicating that lymphocytes located in thymus cortex have significantly different nuclear entropy, ASM and correlation, when compared to the lymphocytes located in thymus medulla. These findings suggest that GLCM as a method might be capable of
detecting fine structural changes in cell nuclei associated with migration and maturation of thymus lymphocytes.
Thymus tissue was obtained from 10 male one-month-old Swiss white albino outbred mice. All animals were previously bread and kept under same laboratory and environmental conditions (standard laboratory diet, ambient temperature of 22°C, natural light cycle). The research followed the rules presented in the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institute of Health
(NIH Publication No. 85 – 23, revised 1985). The principal investigator (IP) had an authorization from the Ethical Commission of the University of Belgrade, School of Medicine, and the Serbian Ministry of agriculture for the experimental work with the laboratory animals (Approval No. 323-07-03985/2012-05/1).
Thymus tissue was fixated in Carnoy solution, mounted on glass slides (5 micrometer thickness), and stained with hematoxylin/eosin (H/E). An Olympus C-5060 Wide Zoom digital camera instrument with an oil immersion objective (×1,000 magnification) of Olympus BX41 microscope (1 pixel equaled 0.048 µm) was used for micrograph acquisition similarly to our previous study (
thymus lymphocyte nuclei (30 randomly selected non-overlapping lymphocyte nuclei from each animal) were analyzed: 150 from cortex (15 per animal) and 150 from medullar regions (15 per animal) of thymus (figure 1).
Pantic et al. 2012a
). A total of 300
Figure 1 Digital micrographs (1,000x magnification) of thymus cortex (A) and medulla (B).
Nuclear image acquisition was done after creating an automatic threshold of the nuclear boundaries in 8-bit format using National Institutes of Health ImageJ (version 1.44p) software. The following protocol was used for each nucleus. The digital micrographs of thymus cortex/medulla in tiff format were converted to 8-bit (ImageJ>Type>8-bit). The converted 8-bit micrographs were then processed using the function
ImageJ>Adjust>Threshold. During the visualization of the nuclei in ImageJ software, two independent histology experts determined the values of the upper and lower threshold level settings, which in their opinion, provided the most accurate quality of the segmentation. Upper threshold level was set to 110 while lower threshold level was 0. Although this visual evaluation of automatic image processing is subjective in
nature, we assume that it has not significantly influenced the final results, as the values of GLCM parameters depend much more on textural patterns of the interior of the nuclei, than on precision of nuclear boundary determination. The sample of non-overlapping lymphocyte nuclei was selected by [Wand tracing tool] (Mode: Legacy, Tolerance:0.0) and cropped (ImageJ>Image>Crop). After the cropping process,
individual nuclei images were analyzed by GLCM method.
GLCM method was first introduced in 1973, by Haralick et al. who developed a method able to quantify spatial relationship between grey tones (pixel brightness values) in an image. This method uses [second order] statistics, which means that it estimates the relationship between groups of two resolution cells (pixels) separated by a defined distance d. GLCM analysis in our study was carried out by ImageJ and its
texture analysis plugins developed by Julio E. Cabrera, and Toby C. Cornish.
Grey level co-occurrence matrix entropy, angular second moment, variance and correlation for each nucleus were calculated based on the following formulas:
Haralick et al. 1973
where i and j are coordinates of the co-occurrence matrix, whereas σ and µ represent means and standard deviations of px and py for a selected distance d and angle θ (
).
GLCM parameters were determined separately for direction angles θ equal to 0°, 45°, 90° and 135°, as well as distances d = 1, d = 2 and d = 3, after which the average values were calculated similarly to the work of
this subject (
Pantic and Pantic 2012 Pantic et al. 2012a
,
), the work of
Losa and Castelli (2005)
. This way, a total of 3,600 individual GLCM measurements were calculated. For details regarding the mathematical protocol of the study, the reader is referred to our previous articles on
Haralick et al. (1973)
, as well as technical data of National Institutes of Health ImageJ software.
Statistical analysis was done using GraphPad Prism software (La Jolla, CA, USA) and SPSS statistical package (v. 10; SPSS Inc., Chicago, IL, USA). Student's t-test was used to determine possible statistically significant difference between cortical and medullar lymphocytes.
RESULTS
Average values of GLCM entropy, angular second moment and correlation for different GLCM distances d are presented in table I. All parameters presented statistically significant difference.
TABLE I Average values of chromatin entropy, angular second moment, variance and correlation for different GLCM distances (d).
GLCM feature
Entropy
Angular second moment
Variance
Correlation
Cortex
Medulla
Cortex
Medulla
Cortex
Medulla
Cortex
Medulla
d=1
6.389±0.253
6.510±0.280***
0.0021±0.0006
0.0019±0.0007***
174.3±102.8
216.4± 108.5***
0.0065±0.0030
0.0055±0.0038*
d=2
6.537±0.264
6.675±0.294***
0.0018±0.0005
0.0016±0.0006***
170.8±98.1
212.1±104.3***
0.0058±0.0025
0.0050±0.0033*
d=3
6.611±0.270
6.756±0.301***
0.0016±0.0005
0.0014±0.0006***
170.8±96.4
211.4±102.9***
0.0048±0.0019
0.0043±0.0027*
*
p<0.05,
**
p<0.01,
***
p<0.001.
For distance d = 1, mean entropy was 6.389 ± 0.253 for cortical lymphocytes and 6.510 ± 0.280 for medullar lymphocytes. The difference between the two lymphocyte populations was statistically highly significant (p < 0.001, figure 2). Average angular second moment for cortical lymphocytes was 0.0021 ± 0.0006, and it was significantly higher than the ASM of medullar lymphocytes (0.0019 ± 0.0007, p < 0.001,
figure 2). Mean GLCM variance in cortical and medullar lymphocytes were 174.3 ± 102.8 and 216.4 ± 108.5, respectively (p < 0.001). Mean texture correlation of cortical lymphocytes was significantly higher than correlation of medullar lymphocytes (0.0065 ± 0.0030 versus 0.0055 ± 0.0038, p < 0.05, figure 2).
Figure 2 GLCM entropy (a), angular second moment (b), correlation (c) and variance (d) of thymus cortical and medullar lymphocyte nuclei.
For distance d = 2, mean entropy was 6.537 ± 0.264 for cortical lymphocytes and 6.675 ± 0.294 for medullar lymphocytes. The entropy of medullar lymphocytes was significantly higher (p < 0.001, figure 2). Similar results were found for textural variance (p < 0.001; 170.8 ± 98.1 versus 212.1 ± 104.3). Cortical lymphocyte population had higher ASM (0.0018 ± 0.0005 versus 0.0016 ± 0.0006, p < 0.001, figure 2) and
texture correlation (0.0058 ± 0.0025 versus 0.0050 ± 0.0033, p < 0.05, figure 2).
For distance d = 3, mean ASM was 0.0016 ± 0.0005 for cortical lymphocytes and 0.0014 ± 0.0006 for medullar lymphocytes. The difference between the two lymphocyte populations was statistically highly significant (p < 0.001, figure 2). Cortical lymphocyte population had lower entropy (6.611 ± 0.270 versus 6.756 ± 0.301, p < 0.001, figure 2) and variance (170.8 ± 96.4 versus 211.4 ± 102.9) as well as higher
texture correlation (0.0048 ± 0.0019 versus 0.0043 ± 0.0027, p < 0.05, figure 2).
Of all determined parameters, entropy in both thymus regions showed the highest degree of homogeneity with inter and intra-individual variability lower than 10%. There was no significant difference in GLCM parameters between individual animals (ANOVA, p > 0.05).
For each GLCM parameter a receiver operating characteristic (ROC) curve was constructed to illustrate the performance of the parameter as a binary classifier of lymphocyte nuclei in terms of their location in thymus. As presented in figure 3, the areas under the ROC curves were 0.652 for entropy, 0.649 for angular second moment, 0.626 for correlation and 0.637 for variance.
Figure 3 Receiver operating characteristic (ROC) curves for GLCM parameters.
DISCUSSION
The results of our study suggest that lymphocytes of thymus medulla might have higher nuclear GLCM entropy and variance than lymphocytes in thymus cortex. Angular second moment and texture correlation are on the other hand lower in medullar lymphocytes. In biology research, these GLCM parameters are generally seen as indicators of cell/tissue structural complexity and heterogeneity. High entropy and
Shamir et al. 2009 Haralick et al. 1973
variance usually implies elevated level of disorder and disorganization, whereas angular second moment is a measure of homogeneity (
,
quantification of cell/tissue texture, and for detection of fine cell/tissue structural changes that are otherwise undetectable during standard microscopy analysis.
Haralick et al. 1973
). Texture correlation is a measure of grey level linear dependencies in a micrograph (
) and can have values from 0 (uncorrelated micrograph) to +1 and -1 (perfectly correlated). GLCM parameters are today commonly used for
Thymus is a primary lymphoid organ with various biological functions regarding immune system development and its ability to respond to infections. Cortex and medulla are two major thymus histological compartments. Both compartments consist primarily of T-lymphocytes (T-cells), cells which are essential for adaptive immune responses. One of the main events that take place in thymus environment is selection
Sleckman 2005 Nitta et al. 2008 Krangel 2009
and maturation of T-cells. In cortex, these cells undergo the so-called [T-cell receptor gene rearrangement], a process involving random joining of gene segments in nucleus in order to form a genetic sequence responsible for creation of cell surface T-cell receptor (TCR), which is an essential property of a functional T-lymphocyte antigen recognition ability (
After this TCR gene rearrangement, the T-cell expresses two surface TCR co-receptor CD molecules: CD4 and CD8 (double positive CD4+CD8+ immature T-cell).
Another process that takes place in thymus cortex is positive selection of immature T-cells (Teng et al. 2011,
fail to receive this signal as their TCR is unable to interact with MHC molecule (
al. 2011, Kurobe et al. 2006,
,
,
, Seitan et al. 2011).
Takahama et al. 2010 Klein et al. 2009 Gommeaux et al. 2009
,
,
). Lymphocytes that have T-cell receptor capable of binding to a major histocompatibility complex (MHC) molecule, receive pro-survival signal. It is thought that during positive selection approximately 90% of immature T-cells (double positive CD4+CD8+)
Clarke et al. 2009
). As a result, these cells go through programmed cell death (apoptosis). The remaining, surviving T-cells become either CD4+CD8- or CD4-CD8+ (single positive) through the process of downregulation and upregulation. This lineage commitment is followed by migration of single positive T-cells to thymus medulla (Teng et
Choi et al. 2008
).
In thymus medulla, the single positive T-cells that show affinity to self-antigens presented on MHC molecules receive pro-apoptotic signal (Le Borgne et al. 2009,
cells in cortex, it is much more pronounced and efficient in medulla.
Qiu et al. 2010 Atibalentja et al. 2009 McCaughtry et al. 2008
,
,
). This elimination of auto-reactive T-cells is called negative selection, and its main purpose is to prevent the development of autoimmunity. Although negative selection can also occur in double positive T-
Therefore, cortical and medullar lymphocytes, although all being T-cells, have some major differences in their functional and maturational status. First, these two populations have different expression of TCR co-receptor CD molecules: cortical lymphocytes are predominantly double negative (CD4-CD8-) or double positive (CD4+CD8+), while medullar, more mature lymphocytes are mostly single positive (CD4+CD8Nitta et al. 2008 Benz et al. 2004 Ma et al. 2013
or CD4-CD8+). Second, the microenvironment of thymus cortex is substantially different than in medulla; this refers to different interleukin levels, presence or absence of certain growth factors, as well as differences in epithelial reticular cell functions (
,
,
). All these factors can have an impact on nuclear structural organization of the two lymphocyte populations.
Losa and Castelli (2005)
Our results indicate that GLCM as a method may be capable of detecting discrete nuclear structural changes that are associated with thymus lymphocyte migration and maturation. This is in accordance with the study of
who suggested that some GLCM parameters (i.e. textural correlation) may be very efficient in identifying chromatin organizational changes in cell cultures, in some cases with
greater sensitivity than conventional molecular biology methods such as TdT-mediated dUTP nick end labeling assay, FACScan Sub G0/G1 peak, or Annexin-V labelling. In our study, of analyzed GLCM parameters, changes in entropy, variance and angular second moment were more pronounced than in correlation. In our study, in addition to the standard statistical difference tests, the ability of the GLCM parameters
to discriminate the two lymphocyte populations was tested by ROC analysis. In clinical sciences, ROC analysis is essential for evaluating potential value of the studied diagnostic test. ROC curve is constructed based on the fraction of true positives out of the positives (sensitivity) and the fraction of false positives out of the negatives (1-specificity), taking into account different thresholds. In our work, in SPSS software
[state variable]=1 was assigned to the cortical lymphocytes, while [state variable]=0 was assigned to the medullar lymphocytes. Therefore, cortical location would be equivalent to [positive] diagnosis in a clinical diagnostic test.
Zweig and Campbell 1993 Sandelowsky et al.
Potential discriminatory performance of the test is evaluated by analyzing the area under the ROC curve. A test with no diagnostic value would have the area under the ROC curve approximately higher than 0.5 and lower than 0.6. Area between 0.6 and 0.7 indicates [poor] performance, area between 0.7 and 0.8 [fair] performance, and area between 0.8 and 0.9 [good] performance (
,
2011
). Tests that belong to the category [excellent] usually have the ROC areas higher than 0.9.
In this study, all four tested GLCM parameters had ROC areas between 0.6 and 0.7, with entropy having the highest accuracy and correlation the lowest. Although these values show relatively low discriminatory performance of GLCM parameters, it should be noted that at present there is no known image analysis method that would represent a gold standard for nuclear structure classification in thymus. Also, we cannot
exclude the possibility that in some future study, a scoring system combining various GLCM parameters could be designed and would have much higher performance than individual GLCM parameters taken separately. Alternatively, one could combine GLCM method with other known image complexity analysis techniques, such as fractal analysis, particularly having in mind that some computational biology programs
Pantic et al. 2012b
(i.e. ImageJ) can be easily upgraded to apply both techniques on same digital micrographs (
).
Pantic et al. 2012a
The presented findings regarding entropy and angular second moment are also in accordance with the results of our previous study concerning GLCM analysis of B-lymphocytes (B-cells) in spleen germinal center (
). We suggest that nuclear structural entropy and ASM of spleen germinal center light zone B-cell nuclei are correlated with nuclear structural reorganization during apoptosis. This
reorganization may have included chromatin condensation, marginalization and fluctuations in euchromatin / heterochromatin ratio. The chromatin changes that occur during lymphocyte maturation in thymus (and migration from cortex to medulla) are probably more discrete than the ones taking place in germinal center, which makes GLCM as a method potentially even more sensitive than previously thought.
Calder et al. 2011 Gruver et al. 2007
Our study had several potential limitations. First, all 300 analyzed lymphocyte nuclei originated from thymus tissues of one-month-old mice. It is well known that with age, thymus as a primary lymphoid organ begins to atrophy, followed by its decreased function (
,
). It remains unclear whether GLCM method would detect the same differences between cortical and medullar lymphocyte
chromatin in older tissues. Perhaps, in the future, it would be of interest to conduct a similar, but larger study which would investigate possible effects of aging on T-cell chromatin GLCM parameters. Second, if we consider the total number of T-cells that are present in thymus at a given time, the sample of 300 nuclei is somewhat limited. Nevertheless, we should have in mind that GLCM as a mathematical and
computational biology method is relatively exact and when combined with NIH ImageJ software, it significantly lowers possible measurement variations that would otherwise be the result of subjective interpretation, researcher bias, or small sample size.
In conclusion, our findings suggest that grey level co-occurrence matrix entropy, angular second moment, variance and correlation might be useful in detection of discrete structural changes of lymphocyte nuclei in thymus. Although discriminatory performance of the calculated GLCM parameters is relatively poor, we assume that in the future, a more sensitive scoring system based on a combination of individual
GLCM features could be designed. Along with results of other studies, it would represent a basis for further research regarding applicability of GLCM method in medical and biological sciences.
Acknowledgements
The authors are grateful to The Ministry of Education and Science, Republic of Serbia, Research Projects oi-175059 and iii-41027, as well as the staff at The Institute of Physiology Dragisa Nikitovic and Dragana Pavlovic for their help in obtaining the laboratory animals.
The authors report no conflict of interests regarding this manuscript.
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Received: April3, , 2012; Accepted: October11, , 2012
Correspondence to: Igor Pantic E-mail: [email protected] / [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013005000044
Biological Sciences
Chemical, enzymatic and cellular antioxidant activity studies of Agaricus blazei Murrill
RICARDO A. HAKIME-SILVA1, JOSÉ C.R. VELLOSA2, NAJEH M. KHALIL3, OMAR A.K. KHALIL4, IGUATEMY L. BRUNETTI5, OLGA M.M.F. OLIVEIRA1
1
Universidade Estadual Paulista “Júlio de Mesquita Filho”, Departamento de Bioquímica e Tecnologia Química, Instituto de Química, 14800-900 Araraquara, SP, Brasil
Universidade Estadual de Ponta Grossa/UEPG, Programa de Pós-graduação em Ciências Farmacêuticas, Campus Uvaranas, 84030-900 Ponta Grossa, PR, Brasil
Estadual do Centro-Oeste, Departamento de Farmácia, Guarapuava, PR, Brasil, Universidade Estadual do Centro-Oeste/UNICENTRO, Departamento de Farmácia, Campus CEDETEG, 85040-080 Guarapuava, PR, Brasil
4Instituto Federal de Goiás, Formosa, GO, Brasil, Instituto Federal de Goiás, Campus Formosa, 73813-816 Formosa, GO, Brasil
5 Universidade Estadual Paulista “Júlio de Mesquita Filho”, Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, 14801-902 Araraquara, SP, Brasil
2
3Universidade
ABSTRACT
Mushrooms possess nutritional and medicinal properties that have long been used for human health preservation and that have been considered by researchers as possible sources of free radical scavengers. In this work, the antioxidant properties of water extracts from Agaricus blazei Murill, produced by maceration and decoction, are demonstrated in vitro. Resistance to oxidation is demonstrated through three mechanisms: i) inhibition of enzymatic oxidative process, with 100% inhibition of HRP
(horseradish peroxidase) and MPO (myeloperoxidase); ii) inhibition of cellular oxidative stress, with 80% inhibition of the oxidative burst of polymorphonuclear neutrophils (PMNs); and iii) direct action over reactive species, with 62% and 87% suppression of HOCl and superoxide anion radical (O2• –), respectively. From the data, it was concluded that the aqueous extract of A. blazei has significant antioxidant activity, indicating its possible application for nutraceutical and medicinal purposes.
Keywords: Agaricus blazei Murrill; antioxidant; oxidant species; chemiluminescence; polymorphonuclear neutrophils
RESUMO
Cogumelos apresentam propriedades nutricionais e medicinais bastante empregadas na manutenção da saúde humana e têm sido considerado por pesquisadores como possíveis fontes de seqÜestradores de radicais livres. Neste trabalho, as propriedades antioxidantes de extratos aquosos de Agaricus blazei Murill, produzidos por maceração e decocção, são demonstrados in vitro. A resistência à oxidação é demonstrada através de três mecanismos: i) inibição de processos oxidativos enzimáticos, with 100% de
inibição da HRP (horseradish peroxidase) e MPO (myeloperoxidase); ii) inibição do estresse oxidativo celular, com 80% de inibição do oxidative burst de neutrófilos polimorfonucleares (PMN) e iii) ação direta sobre espécies reativas, com 62% e 87% de supressão do HOCl e ânion superóxido (O2• –), respectivamente. A partir destes dados, concluise que o extrato aquoso deste cogumelo tem significativa capacidade antioxidante, indicando sua possível aplicação para propósitos nutracêuticos e medicinais.
Palavras-Chave: Agaricus blazei Murril; antioxidantes; espécies oxidantes; quimiluminescência; neutrófilos polimorfonucleares
INTRODUCTION
Oxidations are essential reactions in the biological processes of many organisms. However, reactive oxygen species (ROS), which are continuously produced in vivo, are known to promote cell death and tissue damage (
There is an increasing emphasis on research aimed to identify and utilise antioxidants from natural sources (
Ramarathnam et al. 1995
Calabrese et al. 2007 Halliwell and Gutteridge 1999
,
). Antioxidants are of great interest because of their possible role in protecting the organism against damage by ROS (
). Aging and diseases, including atherosclerosis, diabetes, cancer and cirrhosis, have been linked to oxidative damage (
Halliwell et al. 1995b
) such as superoxide anion radical (O2• –), hydrogen peroxide (H2O2) and hydroxyl radical
(HO•).
Vellosa et al. 2007a Morton et al 2000 Eastwood 1999 Vinson et al. 1995 Halliwell et al. 1995a
,
,
,
,
).
These species are by-products of normal metabolism that can attack biological molecules such as lipids, proteins, DNA and RNA,
Jung et al. 1999
leading to cell or tissue injury associated with degenerative diseases (
).
Agaricus blazei Murrill (Agaricaceae), an edible mushroom, is native to the small Brazilian town of Piedade (
Mizuno 1995
, Heinemann et al. 1993). Today, A. blazei is consumed globally as food or in tea on account of its putative medicinal properties (
Mshandete and Cuff 2007 Kaneno et al. 2004
,
). Previous studies with isolated fractions of the A. blazei fruit bodies indicated that some samples exhibited antimutagenic, anticarcinogenic and immunostimulative activities (
Mizuno et al. 1998 Kawagishi et al.
,
1990 Itoh et al. 1994 Osaki et al. 1994 Mizuno et al. 1990
,
,
,
).
Natural products with antioxidant activities are used to aid the endogenous immune system. As a result, increasing interest has been expressed in the antioxidative roles of nutraceutical products (
Pietta et al. 1998
). Polyphenols and flavonoids are plant constituents with a probable effect on the organism's ability to scavenge free radicals (
Vellosa et al. 2006
). Studies by
Barros et al. (2007) Ferreira et al. (2007)
,
and others (
Turkoglu et al. 2007 Oliveira et al. 2007 Cheung et al. 2003 Cheung and Cheung 2005 Lo and Cheung
,
,
,
,
2005 Yang et al. 2002
,
) indicate a correlation between the mushrooms' antioxidant activity and their phenolic content.
The aim of this study is to evaluate the antioxidant properties of aqueous extracts of A. blazei, particularly the reduction in activity of oxidoredutase enzymes such as HRP, the scavenging effects on radicals such as superoxide radical and HOCl and the inhibition of the oxidative burst of activity of polymorphonuclear neutrophils (PMNs).
The enzyme horseradish peroxidise (HRP) type VI, phorbol-12-myristate-13-acetate (PMA), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 5-thio-2-nitrobenzoic acid (TNB), phenazine methosulphate (PMS), nicotinamide adenine dinucleotide reduced (NADH), nitrobluetetrazolium (NBT), luminol and 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) were purchased from Sigma-Aldrich Chemical Company, (St Louis, MO, USA). Hydrogen peroxide (30% solution) was purchased
from Peroxides, Brazil (São Paulo, SP, Brazil). All of the reagents used for buffer preparation were analytical grade.
Apparatus
All chemiluminescence measurements were carried out using a luminescence photometer Luminometer 1251 (BioOrbit model, Finland) and were monitored on a computer workstation running the program Multiuse v 2.0. All assays were conducted on an HP 8453 Diode Array Spectrophotometer.
Mushroom Material
Agaricus blazei Murrill (A. blazei) fruiting bodies were collected at Valemar Ranch (São José do Rio Preto, SP, Brazil) and identified by Dr. Arailde Fontes Urben (EMBRAPA researcher).
Two types of A. blazei aqueous extracts were prepared. First, a DECOCTION extract was obtained by decocting 1 g of powdered mushroom in 100 mL of distilled water until the volume was reduced by half. Next, a MACERATION extract was obtained by macerating 1 g of powdered mushroom in 50 mL of distilled water. Material was refrigerated at 4°C for 60 minutes and then filtered and fractionated. The extract was used within 4 hours.
HRP/H2O2/Guaiacol Kinetics Activity Measurements
Both macerate and decoction extracts were subjected to the peroxidase colorimetric assay. All horseradish peroxidase activity assays were conducted by measuring the oxidation of guaiacol as substrate. H2O2 and HRP solutions were prepared in MilliQ water and their concentrations were determined spectrophotometrically using the molar absorption coefficients of •230 nm = 80 M−1cm−1 (
mixtures contained 10−9 M HRP enzyme, 50 mM phosphate buffer, pH 7.0 and 25 mM guaiacol, at 25°C. The reaction was started by the addition of H2O2 and product formation was followed spectrophotometrically at 470 nm (
) and • 403 nm = 1.02 × 105 M−1cm−1 (
Brestel 1985
Ohlsson and Paul 1976
), respectively. Typical reaction
Makinen and Tenovuo 1982 Gazarian and Lagrimini 1996
,
). The initial reaction rate (vo) was determined by the angular coefficient of the plot of absorbance at 470 nm versus time (seconds), extrapolated to time zero. All reactions were conducted in triplicate (Desser et al. 1972).
Chemiluminescence Assay of HRP Activity
Inhibition of HRP activity by A. blazei was measured by chemiluminescence. Luminol solutions were prepared in MilliQ water and their concentrations were determined spectrophotometrically using the molar absorption coefficient • 347 nm = 7,636 M−1cm−1 (
). Typical reaction mixtures contained 3×10−8 M HRP enzyme, 50 mM phosphate buffer, pH 7.0 and 5×10−6 M luminol, at 37 °C (
Allen and Loose 1976
Brestel 1985
). All reactions were conducted in triplicate. The net reaction is illustrated in
the following scheme:
where HRP, HRP-I and HRP-II are the ferric peroxidase and intermediate compounds of horseradish peroxidase; and L and L• are luminol and its radical product of one electron oxidation, respectively. The radical product of luminol oxidation is then converted into 3-aminophthalate, resulting in light emission (
Dodeigne et al. 2000
). The integrated light emission was taken as the analytical readout.
MPO/H2O2/TNB Kinetic Activity Measurements
All myeloperoxidase (MPO) activity assays were conducted by measuring the oxidation of 5-thio-2-nitrobenzoic acid (TNB). TNB was prepared according to
. The concentration of TNB was determined by measuring the absorbance at 412 nm using • = 13,600 M−1 cm−1.
Ching et al. (1994)
Typical reaction mixtures contained 0.04 U/mg MPO enzyme, 50 mM phosphate buffer, pH 7.0, 0.07 mM TNB, 0.1 mM taurine, 50 µL of A. blazei, and 0.1 mM H2O2, at 25°C. The reaction was started by the addition of H2O2 and the TNB consumption was monitored spectrophotometrically at 412 nm. The initial reaction rate (vo) was determined by the angular coefficient of the plot of the absorbance at 412 nm versus time (in seconds), extrapolated to time zero. All reactions were
conducted in triplicate.
The antioxidant activity of the extract from macerated A. blazei was determined by comparing the results of three reactions:
The antioxidant activity was calculated as the percent inhibition of TNB oxidation to DTNB by the following equation:
where A, B and C correspond to the absorbance change observed for reactions A, B and C, respectively.
Chemiluminescence Assay Using PMNS
Primary polymorphonuclear neutrophils (PMNs) were obtained according to the following protocol. A 5% oyster glycogen solution (dissolved in 0.85% NaCl) was injected into the peritoneum of anesthetized rats. This work was submitted to the Ethics Committee of FCFAR / UNESP and authorized by the protocol CEP n° 65/2009. The animals were kept with food and liquid ad libitum and sacrificed 12 h after injection. Calcium-free Dulbecco's phosphate-buffered saline (PBS-D, 20 mL),
containing 10 IU heparin per mL, was injected into the peritoneal cavity (
Babior and Cohen 1981 Paino et al. 2005
,
). The discharge was collected and centrifuged for 3 min at 2,000 g. The cell pellet was layered on Ficoll-Hypaque 1077 and centrifuged for 25 min at 2,000 g. PMN cells were collected, washed and kept in ice-cold PBS-D until required.
The effect of mushroom macerate on PMN activity was assayed by chemiluminescence (
). PMNs (1 × 106 cells/mL), suspended in PBS-D buffer, pH 7.2, were stimulated by the addition of phorbol-12-myristate-13-acetate (PMA, 1×10−5 mM) in the presence of luminol (10−5 M). The chemiluminescence emission was measured in millivolts (mV) at 37°C in 1-s intervals for 1 hour. The ideal dose was determined by the addition of different volumes of A. blazei extracts. PMN
activity was expressed as a percent reduction in the maximum chemiluminescence response of PMNs to PMA in a positive control reaction. Negative controls were performed by omitting PMA. The integrated light emission was taken as the analytical readout.
Parij et al. 1998
Assay of Superoxide Anion Radical (O2• –) Suppression
Kakkar et al. 1984
Superoxide radicals, produced by reduced nicotinamide adenine dinucleotide (NADH) and phenazine methosulphate (PMS), reduce nitrotetrazolium blue chloride (NBT) to produce a formazan compound. The intensity of the colour is inversely proportional to the antioxidant concentration (Vellosa et al. 2007B,
). The assay was carried out in sodium pyrophosphate buffer (25 mM, pH 8.3) and the mixture contained 25 µL of 0.372 mM PMS, 75 µL of 0.6 mM NBT, 50 µL of
1.56 mM NADH, macerate mushroom extract (several volumes) and buffer to a final volume of 1 mL. Reactions were started by the addition of NADH. After a 90 seconds incubation at 25°C, 100 µL of glacial acetic acid and 900 µL of sodium pyrophosphate buffer were added. After vigorous homogenisation, the colour intensity of the mixture was measured at 560 nm. All reactions were conducted in triplicate.
Data are reported as the mean ± SD. The Student t-test was used to determine the difference between test and control preparations, with the level of significance set at p < 0.05.
The goal of this study was to evaluate the aqueous extract from A. blazei as a source of pharmacological agents against oxidative stress. The aqueous extracts used for most experiments were obtained by maceration at 25°C, as it was observed that extracts obtained by decoction had low activity (Figure 1). A possible explanation for this observation is that the high temperature (∼100°C) of the extraction induces polymerization of lower molar mass phenols, leading to a lower antioxidant
capacity.
Figure 1 HRP/guaiacol/H2O2 assay in aqueous extract, macerated and decoction of A. blazei. The incubation mixture contained HRP/ 3×10−5 mM; H2O2 /3.0×10−1 mM; Guaiacol /5.0×10−4 mM, (Mean ± SD; n=3).
Kinetics Enzymatic HRP/Guaiacol/H2O2 Assay
Job and Dunford (1976)
have studied the oxidation of several phenols and aromatic amines through horseradish peroxidase (HRP). The one-electron oxidation of organic compounds (AH) by horseradish peroxidase may be represented as follows:
The HRP activity was studied by spectrophotometrically monitoring guaiacol oxidation, which generates a chromophore with an absorbance at 470 nm within 1 minute. Figure 1 presents the inhibitory effects of aqueous extracts of A. blazei, prepared by either maceration or decoction, on the HRP/guaiacol/H2O2 assay. HRP activity was completely inhibited in the presence of the macerate. The mechanism by which the extracts exert their antioxidant properties cannot be determined by this
method alone; however, it is likely that the macerate extract provides phenolic compounds able to act on the enzyme system or to reduce the oxidised product.
Chemiluminescence Assay Using HRP and Agaricus Blazei Murill
The luminol-dependent chemiluminescent assay lacks specificity regarding the ROS generated upon neutrophil activation (
Dodeigne et al. 2000
); therefore, experiments were performed to determine the effect of A. blazei extracts on the activity of the HRP/H2O2 enzymatic system. Peroxidases catalyse the oxidation of luminol by hydrogen peroxide.
Figure 2 presents the inhibitory effects of aqueous macerated extracts from A. blazei on the HRP-catalysed, luminol-dependent chemiluminescence assay. Note that in the control reaction of luminol, HRP and H2O2, the light intensity is 2,000 mV; the addition of 100 µL of aqueous macerated extract reduces the light intensity to 18.5 mV and, hence, reduces oxidation by HRP.
Figure 2 Effect of the mushroom macerate extract over chemiluminescence intensity during luminol oxidation by hydrogen peroxide catalyzed by HRP. The incubation mixture contained 50 mM of Phosphate buffer, pH 7.0, 3×10−8 mM HRP, and 5×10−6 mM luminol. Chemiluminescence intensity was registered 5 min, by 37°C, after the start of the reaction.
Antioxidant Activity of Agaricus blazei murill by the MPO/H2O2/TNB System
The neutrophils' oxidative burst is the result of the assembly of the multi-enzyme NADPH-oxidase system that promotes the one-electron reduction of oxygen to superoxide anion radical (
proposed to be the primary agent responsible for the antimicrobial action of PMNs. Figure 3 represents the antioxidant activity of aqueous extracts of A. blazei in the following reactions:
Babior 2000
). Next, this species is reduced to hydrogen peroxide by superoxide dismutase. Finally, hydrogen peroxide is used by myeloperoxidase (MPO) to oxidise chloride to hypochlorous acid (HOCl) (
This assay is based on the ability of a substance (scavenger) to inhibit the oxidation of 5-thio-2-nitrobenzoic acid (TNB) to 5,5′-dithio-2-nitrobenzoic acid (DTNB) in the presence of the oxidants HOCl and chloramine-taurine (Tau-Cl, a species also formed in vivo), (
The antioxidant activity, calculated as the percent inhibition of TNB oxidation, was 62%.
Ching et al. 1994
Hampton et al. 1998 Podrez et al. 2000 Lapenna and Cuccurullo 1996
,
) generated in vitro by MPO and H2O2. The antioxidant activity of the macerated extract of A. blazei is expressed by the following reaction:
,
). This highly oxidising molecule has been
Figure 3 Antioxidant activity of aqueous macerate extract of A. blazei on the system MPO/Taurine/H2O2/Cl−. (Mean ± SD; n=3). (Mean ± SD; n=3). The incubation mixture contained: MPO/0.04 U/mg, H2O2/2×10−5 mM, TNB/0.074 mM, Tau/0.1 mM, PBS-D buffer by Cl−, pH 7.4. Columns: A, MPO/Taurine/Catalase/TNB, by absorbance of 1.130; B, MPO/Taurine/H2O2/Catalase/TNB, by absorbance of 0.130; and C, MPO/Taurine/H2O2/Catalase/
TNB/A. blazei, by absorbance of 0.749.
Luminol-Dependent Chemiluminescence Assay Using Polymorphonuclear Neutrophils (PMNS): Oxidative Burst Inhibition by Agaricus Blazei Murill
Luminol-dependent chemiluminescence (LDCL), commonly believed to result from the production of O2• –, HOCl and H2O2, is observed during the oxidative burst of PMNs (Dahlgren and Karlsson 1987, Allen 2000).
The oxidative burst of PMNs was monitored using the soluble stimulant phormol-12-myristate-13-acetate (PMA). Studies by Dahlgren and Karlsson (1987) showed that LDCL produced by PMNs depends largely on the generation of HOCl by the MPO/H2O2/Cl− system. Thus, the oxidation of luminol can occur either by the peroxidase reaction or by the direct reaction of luminol with HOCl.
PMN assays conducted in the presence of the A. blazei aqueous extracts demonstrate reduced LDCL on PMA-stimulated PMNs (Figure 4). This effect may be linked to the action of compounds in the A. blazei extract on the oxidative burst enzymes or to a direct reaction with HOCl and possibly other reactive oxygen species such as superoxide anion.
Figure 4 Effect of the mushroom macerate extract over luminol-dependent chemiluminescence assay using PMN. The incubation mixture contained PMN/1×106 cells/mL stimulators by PMA/1×10−5 mM the absence end presence of 50µL of A. blazei.
Suppression of superoxide anion radical (O2• –)
Kakkar et al. 1984
We have also evaluated the potential of macerated extracts of A. blazei to suppress superoxide anion formation using a non-enzymatic superoxide generation method (
). The radical is generated by reacting phenazine methosulphate with NADH, resulting in NBT reduction. We used this reaction to evaluate if the extract (Figure 5) was able to suppress superoxide anion formation in vitro. Note that in this reaction, NBT must be present in excess to evaluate the real potency of
samples in suppressing superoxide anion (Vellosa et al. 2007B). The ability of A. blazei extracts to scavenge superoxide anion radical (O2• –) (% inhibition = 51.8 ± 2.1%) may be expected to contribute to its possible anti-radical action.
Figure 5 Suppression of superoxide anion radical (O2• –) by different volumes (µL) of A. blazei macerate extract. The incubation mixture contained: Phenazine Methosulphate – 168×10−6 mM; NBT – 30×10−6 mM; NADH – 78×10−6 mM; Sodium pyrophosfate buffer – 25 mM. (Mean ± SD; n=3).
CONCLUSION
The results indicate that A. blazei aqueous macerated extracts are capable of diminishing ROS levels by interfering with enzymatic ROS generators or by suppressing different reactive species. Evidence in support of these conclusions came from the inhibited formation of superoxide anion, the lessened extent of HRP-dependent luminol-dependent chemiluminescence, the inhibition of the oxidative-burst in neutrophils and the inhibition of HRP and MPO enzymatic oxidative kinetics. From
these observations, it may be suggested that the A. blazei mushroom can be used as a possible pharmacological agent against oxidative stress and as a nutritional and pharmaceutical source of new therapeutic compounds, due to the mushroom's excellent antioxidant properties.
Acknowledgements
The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support. The authors thank Regina H. C. Garcia and Edalcy G. Cerrano for providing most of the A. blazei used in this study.
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Received: August29, , 2011; Accepted: October22, , 2012
Correspondence to: José Carlos Rebuglio Vellosa E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013005000047
Biological Sciences
Cryopreservation of mutton snapper ( Lutjanus analis) sperm
EDUARDO G. SANCHES1, IDILI R. OLIVEIRA1, PEDRO C. DA SILVA SERRALHEIRO1, VINICIUS R. CERQUEIRA2
1
2
Núcleo de Pesquisa e Desenvolvimento do Litoral Norte, Instituto de Pesca/APTA/SAA, Rua Joaquim Lauro Monte Claro Neto, 2275, 11680-000 Ubatuba, SP, Brasil
Laboratório Piscicultura Marinha, Departamento de Aquicultura, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Rod. Ademar Gonzaga, 1346, 88040-900 Florianópolis, SC, Brasil
ABSTRACT
This study aimed to develop a protocol of semen cryopreservation of the mutton snapper Lutjanus analis. The interaction between three extenders ( pH 6.1; 7.8 and 8.2) , two concentrations of dimethyl sulfoxide ( DMSO, 5 and 10%)
and three cooling rates ( -90; -60 and -30°C.min−1) on the sperm motility rate and motility time were analyzed by a factorial experiment. A sample of 30 fishes ( 1,261 ± 449 g) collected in the nature was kept in floating net cages. The
semen was frozen by using cryogenic straws, in nitrogen vapour and transferred, later, to liquid nitrogen. Fertilization test was accomplished to evaluate the viability of the cryopreserved sperm. The highest sperm motility rate and
motility time ( P < 0.05) was achieved by combining extender C ( pH 8.2) with DMSO ( 10%) and cooling rate of -60°C.min−1 ( P < 0.05) . The use of cryopreserved sperm presented fertilization rates higher than 59% validating the
present protocol for mutton snapper.
Keywords: cryoprotector; extender; Lutjanus analis ; reproduction; sperm
RESUMO
Este trabalho foi realizado com a finalidade de desenvolver um protocolo de crioconservação do sêmen da cioba Lutjanus analis. Em um experimento fatorial foram analisados os efeitos de três diluentes ( pH 6,1; 7,8 e 8,2) , duas
concentrações de dimetilsulfóxido ( DMSO, 5 e 10%) e três velocidades de congelamento ( -90, -60 e -30°C.min−1) sobre a taxa de motilidade e tempo de motilidade espermáticas do sêmen crioconservado. Uma amostra de 30
exemplares com peso médio de 1.261,11 ± 449,0 g, oriunda da natureza, foi mantida em tanques-rede. O sêmen obtido foi congelado empregando-se palhetas criogênicas em vapor de nitrogênio e, posteriormente, transferido para
nitrogênio líquido. Posteriormente um teste de fertilização foi realizado para avaliar a viabilidade do sêmen crioconservado. A combinação que propiciou maior taxa de motilidade e tempo de motilidade espermáticas ( P < 0,05) foi
proporcionada pelo emprego do diluente de pH 8,2 com 10% de DMSO e uma velocidade de congelamento de -60°C.min−1 ( P < 0,05) . O sêmen crioconservado apresentou taxa de fertilização superior a 59% validando o presente
protocolo para a cioba.
Palavras-Chave: crioprotetor; diluidores; Lutjanus analis ; reprodução; sêmen
INTRODUCTION
Interest in the culture of snappers ( Lutjanidae family) has been developed throughout the world because of declines in wild stocks combined with a consistent high demand and market value (
2002,
(
Watanabe et al. 1998
, Benetti et al.
Garcia-Ortega 2009
) . Some lutjanid species, such as Lutjanus argentimaculatus, Lutjanus johnii, Lutjanus russelli and Lutjanus sebae, are currently farmed in Pakistan, China, Singapore, Malaysia, Thailand and Philippines
Hong and Zhang 2002
).
The mutton snapper Lutjanus analis ( Cuvier 1828) is distributed along the West Atlantic coast. This species is important as commercial food fish due to the quality of meat, high market value and because it is listed as vulnerable
by the International Union for Conservation of Nature and Natural Resources ( IUCN) . Experimental farms in the Caribbean, Colombia and Brazil have shown the potential of this species for marine aquaculture ( Benetti et al.
2002,
Botero and Ospina 2003 Sanches 2011
,
).
One of the major hurdles to the development of marine fish farming is the breeding of larval stages in species with a potential for commercial culture. The sperm cryopreservation is an important tool for optimizing reproduction
procedures. Utilization of cryopreserved sperm in spawning of mutton snapper allows efforts to be focused on maintaining female broodstock, monitoring ovarian development and increasing efficiency during the strip-spawning
process. When properly collected and frozen and after genetic characterization, cryopreserved sperm can be used for restoration programs of endangered species or specific genetic recovering programs to increase growth rates
and resistance to diseases or to develop specific desired functional properties (
Cabrita et al. 2009
).
In Brazil, the sperm cryopreservation has been used in few marine species, including dusky grouper Epinephelus marginatus (
Centropomus parallelus (
Tiba et al. 2009
Sanches et al. 2008
) , Brazilian flounder Paralichthys orbignyanus (
Lanes et al. 2008
) and fat snook
) . However, few studies have focused on the sperm freezing technique of lutjanids. To date, cryopreservation protocols have been developed for only three species: red snapper Lutjanus
campechanus, grey snapper Lutjanus griseus and mangrove red snapper Lutjanus argentimaculatus (
Riley et al. 2004 2008 Vuthiphandchai et al. 2009
,
,
).
The development of an effective sperm cryopreservation protocol depends on knowledge of the factors affecting the process, such as extender composition, sperm:extender ratio, cryoprotectant type and concentration, cooling
and thawing rates and equilibrium time. Fertilization tests comparing cryopreserved and fresh sperm are also important. The successful cryopreservation of mutton snapper sperm would expand the sperm bank of endangered
marine species (
Sanches et al. 2008
) and represent a vital tool for the conservation and commercial farming of the species.
This study aimed to develop a species-specific cryopreservation protocol for mutton snapper ( Lutjanus analis) sperm to improve its reproduction in captivity.
Fish
The mutton snapper individuals ( n = 30) , which were originally collected from the coast of Ubatuba/SP, Brazil, by line and hook, during October and November 2007, were kept in 2 m × 2 m × 2 m ( 8 m3) net cages at a density
of 2 individuals.m−3. The net cages were set up in the coastal area at Itaguá Beach, Ubatuba/SP and fish were fed daily with a commercial feed for marine fish ( 45% crude protein and 12% ethereal extract) .
Sperm Collection
In the early spawning season ( January 2008) , 30 individuals, fasted for 24 h, were anesthetized with benzocaine ( 0.1 g.L−1) , and their length ( cm) and weight ( g) were measured. The sperm was collected ( without hormonal
induction) in plastic graduated syringes ( 1 mL) that were placed on the urogenital papilla while applying gentle abdominal pressure until the first sign of blood. The syringes were wrapped in foil paper to avoid exposure to light
and the volumes were registered.
Sperm Characterization
The motility of each sperm sample was estimated by recording the percentage of sperm actively moving forward in the microscope field. The duration of the motility was timed from the initiation of motility until the end. The
analyses of sperm motility rate and motility time were performed simultaneously in the same preparation by a single technician on a single, randomly chosen focal field. The sperm density was determined by counting the sperm
cells under a microscope at 200-X magnification in a sperm sample that was previously diluted with 5% buffered formalin and prepared in a Neubauer hematimetric chamber ( 1 mm3) . The spermatocrit technique was used for
the determination of the sperm density. The sperm cells were transferred into microhematocrit capillaries, with one tip sealed with plastiline, and centrifuged for 15 min in a microcentrifuge at 7,000 rpm ( 18,000 g) . These
settings were optimized in a previous experiment. After centrifugation, the cell mass was determined with a graduated ruler, and the values are expressed in parcentages. The correlation between the spermatocrit values and sperm
density was determined.
Sperm Cryopreservation
Experiment I – Cryopreservation protocol
The effect of different extenders, cooling rates and dimethyl sulfoxide ( DMSO) concentrations on the motility rate and motility time of cryopreserved sperm was analyzed. A factorial design was used in the experiment, with
three extender solutions ( A, B, C) , two cryoprotectant concentrations ( DMSO at 5% and 10%) and three cooling rates ( -90, -60 and -30°C.min−1) , with three replicates for each treatment.
Three extenders that were previously shown to be successful in the cryopreservation of marine fish sperm were used as follows:
Extender A ( g.L−1) : NaCl, 7.89; KCl, 1.19; CaCl2, 0.2; MgCl2, 0.4266; pH 6.1; 158 mOsm (
Extender B ( g.L−1) : NaCl, 6.5; KCl, 3.0; CaCl2, 0.3; NaHCO3, 0.2; pH 7.8; 157 mOsm (
Chao et al. 1975
);
Peleteiro et al. 1996
);
Extender C ( g.L−1) : NaCl, 7.89; KCl, 1.19; CaCl2, 0.22; MgCl2, 0.72531; NaH2PO4, 0.0805; NaHCO3, 0.84; pH 8.2; 172 mOsm (
Sanches et al. 2008
).
To achieve different cooling rates, straws were manually constructed from cryogenic plastic tubes with an internal diameter of 4 mm to contain final volumes of 0.25, 0.50 and 1.00 mL. The tubes were cut to different lengths to
store the different volumes of diluted sperm so that the cooling rates selected in this study could be achieved during cooling. Cooling rates were previously determined in sperm samples with extenders in test straws, using thermo
electrical pair ( Ethics Scientific Equipment, 521-200) . The temperatures evaluated ranged from 26 °C to -196 °C.
Only sperm with a motility higher than 90% were used in the cryopreservation procedure. The sperm cells from 10 individuals were collected, mixed in equal volumes and placed into opaque plastic flasks for use with the
respective extenders. The extenders with previously added DMSO at 5% and 10% were slowly added to the sperm up to the desired dilution of 1:4 ( v/v) . The equilibration time between the initial sperm dilution and initial
cooling was 60 seconds. A cryogenic container with nitrogen steam at -196°C ( CP 100 Taylor-Wharton - Harsco Corp., Theodore, AL, USA) was used for cooling the sperm samples. After 24 h, the straws were transferred to a
storage container ( Cryometal, model DS-34) with liquid nitrogen.
After 180 days, the straws were thawed in water at 26°C by two minutes for the determination of the sperm motility rate and motility time.
Experiment II – Evaluation of fertilization
Fertility tests were conducted simultaneously with fresh and cryopreserved sperm from the 2008 and 2009 spawning seasons through insemination of oocytes from the same female. LH-RHa 50 µg.kg−1 ( SIGMA, USA) was used
to induce the female to spawn. The sperm used in this experiment were cryopreserved with extender C ( pH 8.2) and 10% DMSO at a cooling rate of -60°C.min−1, with an equilibrium time of 1 min and a dilution of 1:3 ( v/v) .
The release of mature oocytes began approximately 36 h after induction, and the extrusion for the dry fertilization tests was performed. The oocytes were collected in plastic trays and separated into 30 aliquots, with
approximately 1,000 oocytes each. The oocytes were placed into 50-mL plastic containers for the simultaneous fertilization of 10 aliquots of fresh sperm and 10 aliquots of each of cryopreserved sperm from the 2008 and 2009
spawning seasons. Before being mixed, the fresh sperm was previously diluted in the same extender at the same sperm:extender ratio that had been used for the cryopreserved sperm.
After the mixture of sperm and oocytes using 0.05 mL of sperm per 1,000 oocytes for a sperm:oocyte ratio of 200,000:1, 20 mL of seawater ( 35 ppt) were added to activate the sperm and initiate fertilization. After 5 min, each
aliquot was placed in an individual incubator ( 1 L) and kept in a tank with a continuous circulation of seawater at 28°C. The fertilization rates, based on the relationship between the number of fertilized eggs and total number of
eggs, were calculated 4 h after the fertilization.
Statistical Analyses
Data were analyzed using two-way ANOVA. Tukey's multiple comparison test was applied to estimate differences among groups. The significant level was set at P < 0.05 unless otherwise noted. Results are presented as mean ±
SE. Percentage data were arcsine transformed before analysis. SAS software ( Statistical Analyses System, SAS/STAT 6.11) ( Sas Institute Inc. 1990) was used.
RESULTS
Sperm Characterization
Running milt was available from all individuals ( n = 30) . The average volume of sperm collected from each fish was 0.90 ± 0.52 mL. Mean density of spermatozoa was 2.6 ± 0.3×109 cells.mL −1 ( Table I) . There was a positive
correlation between the sperm density and spermatocrit value ( Fig. 1) . The regression equation y = 0.0428x + 0.7887 ( y = sperm density, x = spermatocrit; r2 = 0.90, P < 0.05) was determined from the sperm density and
spermatocrit values. Therefore, the sperm density of mutton snapper can be estimated from spermatocrit values.
Fig. 1 Correlation between spermatocrit ( %) and spermatozoa density ( cells.mL−1) for mutton snapper sperm ( n = 30 males, adjusted R2 = 0.90, P < 0.05) .
TABLE I Morphometric and semen characterization parameters of mutton snapper ( n = 30) .
Parameter
Standart length ( cm)
Body wet weigth ( g)
Mean ± SE
43.7 ± 4.3
1,261.11 ± 449.0
Sperm density ( x 109 cells/mL)
Collection volume ( mL)
Initial sperm motility ( %)
Sperm motility time ( s)
Spermatocrit ( %)
0.90 ± 0.52
100 ± 0
148 ± 29
78.2 ± 4.6
2.6 ± 0.3
Sperm Cryopreservation
Experiment I – Cryopreservation protocol
There was a significant interaction among the factors influencing sperm motility rate ( interaction extender*cooling rate*DMSO, p = 0.018) . A Tukey test was conducted to improve the fit among the three factors. The use of
extender C ( pH 8.2) with 10% DMSO at a cooling rate of -60°C.min−1 resulted in the highest motility ( 90.1%) ( Table II) .
TABLE II Sperm motility rate ( %, mean ± SE) of mutton snapper sperm ( n = 10 males) with different extenders, cooling rates and DMSO concentrations interaction.
Extender
Cooling rate
-90°C.min−1
A ( pH = 6.1) ( 158 mOsm)
-60°C.min−1
-30°C.min−1
-90°C.min−1
B ( pH = 7.8) ( 157 mOsm)
-60°C.min−1
-30°C.min−1
-90°C.min−1
C ( pH = 8.2) ( 172 mOsm)
-60°C.min−1
DMSO
Mean ± SE
5%
39.3 ± 6.1c
10%
12.0 ± 0.3d
5%
61.4 ± 3.2b
10%
30.1 ± 8.6cd
5%
72.3 ± 4.2b
10%
22.0 ± 0.8cd
5%
29.1 ± 4.1cd
10%
12.0 ± 0.0d
5%
29.1 ± 4.1cd
10%
12.8 ± 0.0d
5%
34.3 ± 3.3cd
10%
16.0 ± 0.6d
5%
40.1 ± 5.4c
10%
40.1 ± 5.4c
5%
50.0 ± 0.0c
-30°C.min−1
10%
90.1 ± 0.6a
5%
60.2 ± 3.1bc
10%
75.2 ± 3.3ab
a-d
Data are represented as mean ± SE. Values sharing different letters in each colune indicate a significant difference among treatments ( P < 0.05) .
Data on the sperm motility time after cryopreservation were submitted to ANOVA. The interaction between the extender solution, DMSO concentration and cooling rate was not significant ( p = 0.176) . However, the interaction
between each two of the factors was statistically significant ( P < 0.05) .
The sperm motility time was the longest with the use of extender C ( pH 8.2) at a cooling rate of -60°C.min−1 ( Table III) . When the extender and DMSO concentration are considered together, the motility time was higher with
the use of extender C ( pH 8.2) and 10% DMSO ( Table IV) . For the combination of cooling rate and DMSO concentration, the sperm motility time was higher at a cooling rate of -60°C.min−1 with 10% DMSO ( Table V) .
TABLE III Sperm motility time ( seconds, mean ± SE) of mutton snapper sperm ( n = 10 males) in three extenders at different cooling rates.
Cooling rate
-90°C.min−1
20c
-60°C.min−1
-30°C.min−1
21b
295 ± 19b
Extender A
210 ±
Extender B
140 ± 13d
220 ± 18c
170 ± 12d
Extender C
290 ± 18b
420 ± 26a
330 ± 28b
300 ±
a-d
The means followed by different superscripts are statistically different ( P < 0.05) .
TABLE IV Sperm motility time ( seconds, mean ± SE) of mutton snapper sperm ( n = 10 males) in three extenders and two DMSO concentrations.
Extender A
Extender B
Extender C
5% DMSO
210 ± 12cd
160 ± 22d
360 ± 15b
10% DMSO
20c
12d
410 ± 28a
290 ±
190 ±
a-d
TABLE V Sperm motility time ( seconds, mean ± standard deviation) of mutton snapper sperm ( n = 10 males) at different cooling rates in two DMSO concentrations.
Cooling rate
-90°C.min−1
5% DMSO
10% DMSO
180 ±
52d
275 ±
20cd
a-d
-60°C.min−1
-30°C.min−1
295 ±
31c
192 ± 35d
425 ±
29a
338 ± 29b
Experiment II – Fertilization tests
The fertilization rate for fresh sperm ( 81%) was significantly higher ( P < 0.05) than that of the cryopreserved sperm. There were no significant differences between the fertilization rates for cryopreserved sperm from the 2008
( 59%) and 2009 ( 66%) spawning seasons ( Fig. 2) .
Fig. 2 Fertilization rates ( mean ± SE.) with fresh sperm and cryopreserved sperm of eggs from one mutton snapper female spawned in 2009. Bars sharing different letters indicate significant difference
among treatments ( P < 0.05) .
DISCUSSION
The sperm density for mutton snapper are higher than those observed for red snapper ( 1.0 × 109 cells. mL−1) (
comparable to those of mangrove red snapper, which ranged from 1.3 to 2.8 ×
1010
cells.
in the geographic range or by sampling at different periods during the spawning season (
mL−1
(
) and lane snapper Lutjanus synagris ( 2.2 × 109 cells.mL−1) ( Sanches and Cerqueira 2010) but they are
Riley et al. 2004
Vuthiphandchai et al. 2009
) . The variation in sperm density between different species of the same genus may be caused by differences
Lanes et al. 2010
).
The best result among the different ionic compositions and pH values tested in this study was achieved in the extender solution at 172 mOsm and pH 8.2. The extender HBSS with an osmolality of 200 mOsm was used for the
cryopreservation of red snapper sperm and it was close to the values used in this study, but it was lower than the value of 315 mOsm used for mangrove red snapper sperm (
Wayman and Tiersch ( 1998)
) . The cryopreservation of sperm
has also been successful in other marine species using extender solutions with osmolalities close to 200 mOsm. For instance,
, studying extender solutions at different osmolalities ( 200, 300 and 400
mOsm) , found that 200 mOsm was the most suitable for the preservation of sperm quality in red drum Sciaenops ocellatus. Moreover, an extender with an osmolality of 200 mOsm was successfully used for the cryopreservation
of common snook Centropomus undecimalis sperm (
Tiersch et al. 2004
).
Several authors have reported the effect of pH on sperm motility rate. In fact, extenders with different pH values may affect sperm motility (
accumulation of sperm metabolites, during the cooling process, from changing the sperm pH and damaging the sperm cells.
Chen et al. ( 2004)
Cosson 2004 Alavi and Cosson 2005
,
) . Buffered extenders are used to prevent the
, using three extenders ( 202, 335 and 363 mOsm) at different pH values ( 6.7, 8.2 and
6.5 respectively) , had the best results for the cryopreservation of turbot Scophthalmus maximus sperm at pH 8.2. The same pH value was successfully used for the cryopreservation of dusky grouper (
flounder (
Lanes et al. 2008
) and fat snook sperm (
Tiba et al. 2009
Sanches et al. 2008
) , Brazilian
).
The sperm motility time was more positively influenced when extender C ( pH 8.2 and 172 mOsm) was used. The average duration of fresh sperm motility was 174 s, and the motility time was 400 s after cryopreservation in
Sanches et al. 2008
extender C; a significant increase even when compared to the semen cryopreserved in other extender solutions. A similar result was achieved with dusky grouper sperm cryopreserved in the same extender (
) . The
increase in sperm movement, which may result in increased sperm viability, is probably associated with particular features of the extender solution, such as the alkaline pH, higher NaHCO3 concentration and osmolality, that
create a new microhabitat for the sperm that is different from that in seminal plasma. Apparently, these constituents were able to interact with the chemical components of the internal structures of the flagella responsible for the
initiation of sperm motility (
Inaba 2003
) , changing the pattern of the motility time that was preserved after the freezing process.
Different cooling rates can be obtained in the same freezing straws depending on its maintenance, stabilization time and use frequency. Cooling rates of -45°C.min−1 (
) and -35.6°C.min−1 (
) were
recorded for the same container model ( CP300) . Moreover, different cooling rates can be achieved more economically than by using controlled-rate programmable freezers by placing straws of different volumes in the same
Carolsfeld et al. 2003
freezing compartment; although controlled-rate programmable freezers are more efficient, they are also high-maintenance and expensive (
considered, the best cooling rate for mutton snapper sperm was
C.min−1;
)
Epinephelus moara (
-60°C.min−1.
Maria et al. 2006
Richardson et al. 1999 Yasui et al. 2008
,
) . When the sperm motility rate and motility time were
This value is higher than that used for the cryopreservation of sperm from red snapper ( -16°C.min−1;
Riley et al. 2004
) and mangrove red snapper ( -10°
. Nevertheless, the cooling rate with the best result in this study is similar to the cooling rates used for the cryopreservation of grouper Epinephelus malabaricus (
Miyaki et al. 2005
) , fat snook (
Tiba et al. 2009
Chao et al. 1992
) , kelp grouper
He et al. 2011
) and red spotted grouper Epinephelus akaara sperm (
).
DMSO has been considered to be the most efficient cryoprotectant for use in the cryopreservation of marine fish sperm because of its low toxicity and its protection of sperm during cooling due to its capacity for reducing ice
formation by lowering the freezing point of intracellular fluid (
cryopreservation of red snapper (
cobia Rachycentron canadum (
Peleteiro et al. 1996
) . In this study, a solution with 10% DMSO was the most efficient cryoprotectant, and a similar concentration was suggested for the
Riley et al. 2004
) and mangrove red snapper sperm (
Caylor et al. 1994
) , common snook (
) . In other commercial marine species, satisfactory results were achieved with 10% DMSO for the cryopreservation of
Tiersch et al. 2004
) and fat snook sperm (
Tiba et al. 2009
) . The beneficial effect with rapid cooling may also rely on the use of DMSO as the cryoprotectant because
DMSO can penetrate sperm cells rapidly and prolonged equilibration during slow cooling can exert more cytotoxicity of DMSO on sperm cells (
He et al. 2011
)
Although sperm motility rate is the key parameter in sperm quality, the cryopreservation process should also be evaluated based on the fertilization capacity of oocytes. However, the sperm:oocyte ratio and contact time may
affect fertilization rates, hindering comparisons between different studies (
(
Rurangwa et al. 2004
) . To ensure maximum fertilization, sperm cells in excess are usually used for the fertilization of fish oocytes with cryopreserved sperm
Viveiros et al. 2009
) . However, this procedure may mislead the evaluation of the cryopreservation process. Therefore, to avoid controversy, a sperm:oocyte ratio of 200,000:1 was used in this study. The same ratio was used for
cryopreserved sperm in mangrove red snapper (
) , although
Riley et al. ( 2004)
used a ratio of 430,000:1 in the same species.
The fertilization rates observed in this study ( 81% for fresh sperm and 59% and 66% for cryopreserved sperm) show that a cryopreservation protocol for mutton snapper has been successfully developed. However, the ideal
sperm:oocyte ratio for mutton snapper was not determined and should be addressed in future studies. The use of the ideal ratio may result in fertilization rates for cryopreserved sperm that are similar to those for fresh sperm. Red
snapper cryopreserved sperm yielded fertilization rates ranging from 11% to 85%, and this variation was attributed to differences in oocyte quality (
Riley et al. 2004
) . Moreover, high fertilization rates ( 90%) were observed in
mangrove red snapper using cryopreserved sperm (
) . Sperm refrigerated for 48 h yielded fertilization rates greater than 50% in lane snapper ( Sanches and Cerqueira 2010) . Although cryopreservation
protocols are designed to yield similar fertilization rates, differences between fresh and cryopreserved sperm are common. For instance, no significant differences in fertilization rates between fresh and cryopreserved sperm
( 54% and 41%, respectively) were observed in common snook (
Tiersch et al. 2004
) . Nevertheless, fertilization rates in fat snook were higher for fresh ( 84%) than for cryopreserved ( 74%) sperm (
Tiba et al. 2009
).
Therefore, the cryopreservation of mutton snapper sperm was the most successful when using an extender solution with pH 8.2, 172 mOsm and 10% DMSO at a cooling rate of -60°C.min−1.
This is the first report on successful cryopreservation of mutton snapper sperm, this procedure should improve broodstock management techniques for this species and consequently augment the potential for its culture.
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Viveiros ATM, Oliveira AV, Maria AN, Orfão LH and Souza JC. 2009. Sensibilidade dos espermatozóides de dourado ( Salminus brasiliensis) a diferentes soluções crioprotetoras. Arq Bras Med Vet Zootec 61: 883-889.
[ Links ]
Vuthiphandchai V, Chomphuthawach S and Nimrat S. 2009. Cryopreservation of red snapper Lutjanus argentimaculatus sperm: effect of cryoprotectants and cooling rates on sperm motility, sperm viability and fertilization
capacity. Theriogenology 72: 129-138. [ Links ]
Watanabe WO, Ellis EP, Ellis SC, Chaves J, Manfredi C, Hagood RW, Sparsis M and Arneson S. 1998. Artificial propagation of mutton snapper Lutjanus analis, a new candidate marine fish species for aquaculture. J World
Aquac Soc 29: 37-43. [ Links ]
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Received: April25, , 2012; Accepted: November12, , 2012
Correspondence to: Eduardo Gomes Sanches E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013000300014
Biological Sciences
Anurans in a forest remnant in the transition zone between cerrado and atlantic rain forest domains in southeastern Brazil
RENATA M. PIRANI1, LUCIANA B. NASCIMENTO2, RENATO N. FEIO1
1
2
Departamento de Biologia Animal, Museu de Zoologia João Moojen, Vila Gianetti, 32, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brasil
Programa de Pós-graduação em Zoologia de Vertebrados, Museu de Ciências Naturais, Pontifícia Universidade Católica de Minas Gerais, 30535-610 Belo Horizonte, MG, Brasil
ABSTRACT
This study presents the species richness, temporal distribution and reproductive activity of anurans from the Uaimií State Forest (Floresta Estadual do Uaimií – FLOE Uaimií), situated in the Quadrilátero Ferrífero region, municipality of Ouro Preto, Minas Gerais state, Brazil. Field activities were performed monthly from September 2009 to August 2010.
We recorded 36 anurans species, distributed in 10 families. The greatest richness of the sampled sites corresponds to a permanent rivulet in a secondary forest. The majority of anuran species presented seasonal vocalization activity pattern, mainly in the rainy season. The anuran species composition of FLOE Uaimií is similar to others studied areas from
the Quadrilátero Ferrífero region.
Keywords: amphibians; Espinhaço Mountain Range; inventory; reproductive activity
RESUMO
O estudo apresenta a riqueza de espécies, distribuição temporal e atividade reprodutiva dos anfíbios anuros da Floresta Estadual do Uaimií situado na região do Quadrilátero Ferrífero, município de Ouro Preto, estado de Minas Gerais, Brasil. Foram realizadas campanhas mensais no período de Setembro de 2009 a Agosto de 2010. Foram registradas 36
espécies de anuros, distribuídas entre 10 famílias. Maior riqueza de espécies foi verificada em uma área de riacho permanente em mata secundária. A maioria das espécies de anuros apresentou um padrão de atividade de vocalização sazonal, principalmente relacionado à estação chuvosa. A composição de espécies de anuros da FLOE Uaimií é similar a de
outras áreas estudadas na região do Quadrilátero Ferrífero.
Palavras-Chave: anfíbios; Serra do Espinhaço; inventário; atividade reprodutiva
INTRODUCTION
Communities can be seen as units determined by biotic interactions (
Cardoso et al. 1989
calling sites (
Pianka 1973
), preferences for activities in different seasons (
). In anurans communities, several strategies are used allowing many species to coexist within the same environment (
Cardoso and Haddad 1992 Eterovick and Sazima 2000 Conte and Rossa-Feres 2006
,
,
), or for activities at different times of the day (
Cardoso and Haddad 1992
Cardoso and Haddad 1992
). Among these strategies, there are differences in the use of breeding (
Rossa-Feres and Jim 1994 Conte and Rossa-Feres 2007
,
). These differences can be interpreted as mechanisms of pre-zygotic reproductive isolation (
Wells 1977
) and
).
Brazil presents a great diversity of geographical reliefs, among them are the mountain ranges of Espinhaço and Mantiqueira, in eastern Brazil. The Espinhaço Mountain Range (hereafter called EMR) corresponds to a transition between two domains, the Cerrado and the Atlantic Forest, and is the largest rocky meadow formation in Brazil,
considered by some authors as another domain (
Gontijo 2009
). The EMR covers an area of approximately three million hectares and its geographical range beginning in the south in the municipality of Ouro Preto, Minas Gerais state, extending northwards to the municipality of Jacobina, in Bahia state (
Mantiqueira Mountain Range (hereafter called MMR) connects with the plateaus and hills of the EMR through an elevated region, which joins Serra do Ibitipoca (situated in MMR) to the meridional portion of the Espinhaço, making it difficult to establish the precise limits between them (
The Quadrilátero Ferrífero region, at Minas Gerais state, is included in south portion of in EMR (
modify the natural landscape (
Segalla 2005
Silvano and Segalla 2005
Gontijo 2009
) and comprises the mountain ranges of Caraça, Curral, Piedade, Itacolomi, Itabirito, Ouro Branco, and Moeda (
). The north portion of the
).
). The region is known for the iron ore richness and, consequently, for intense mining activities that
). Besides, the Quadilátero Ferrífero region is considered a priority area for amphibian conservation in Minas Gerais state. The main current threat to amphibian populations at this region is the natural habitat destruction by deforestation, due to urban, agricultural and mining activities (
). Faced with these threats, one of the recommendations to protect this region is scientific research (
Among the studies conducted in Minas Gerais state involving reproductive activity and/or frogs communities,
Moura et al. (2012)
Viana and Filgueiras 2009
Gontijo 2009
Cruz and Feio 2007
Drummond et al. 2005
Silvano and
).
Nascimento et al. (1994) Eterovick and Sazima (2000) Pedralli et al. (2001) Eterovick (2003) Eterovick and Sazima (2004) Nascimento et al. (2005) Canelas and Bertoluci (2007)
,
,
,
,
in the MMR can be highlighted. EMR and MMR have been showing a high endemism rate which needs the development of long-term studies for understanding their biogeographical patterns (
,
Cruz and Feio 2007
,
, São Pedro and Feio (2010, 2011) in the EMR;
Cruz et al. (2009) Lacerda et al. (2009)
,
, and
).
According to the context given above, the present study shows data on the species richness and temporal distribution of an anuran community in Uaimií State Forest (Floresta Estadual do Uaimií - FLOE Uaimií), a conservation unit in the municipality of Ouro Preto, at Quadrilátero Ferrifero region, at Minas Gerais state, with comments on the
biogeography of southeastern region of Brazil. Additionally, it describes the annual reproductive activity patterns, based mainly on vocalization activity.
Study Area
Alvarenga et al. 1997
The FLOE Uaimií (20°29′66″S, 43°57′47″W, Figure 1) comprises an area of approximately 4,400 ha. located in the municipality of Ouro Preto, Minas Gerais state, at Quadrilátero Ferrífero region, in the southern portion of the Espinhaço mountain range (
, IEF 2008). The conservation unit has typical vegetation of the transition
between the Cerrado and Atlantic Forest morphoclimatic domains, with the occurrence of rocky meadows (campos rupestres) at the highest elevations, with altitudes ranging from 900 to 1,400 metres. It is the main source water source of the Rio das Velhas, the largest tributary of the São Francisco River Basin (IEF 2008).
Figure 1 Map of the sampled sites of the study showing the temporal distribution at FLOE Uaimií (Ouro Preto, MG): Riacho do Alojamento (R1); Cachoeira de São Bartolomeu (R2); Brejo da Estrada (P1); Brejo do Campo Rupestre (P2).
The climate class of the study region is Cwa (sensu
per month (Sá Junior et al. 2011).
Köppen 1918
) comprising a rainy season that occurs from October to March, with a monthly average of more than 110 mm, accounting for approximately 81.5% of the total annual average at 1,373 mm. The dry season is from April through September, during which the average rainfall is 42 mm
Field Activities
Expeditions to study the anurans temporal distribution were performed monthly, lasting five days from September 2009 to August 2010. The temporal distribution study was conducted at four sites: two permanent rivulets (named Riacho do Alojamento – R1 - and the Cachoeira de São Bartolomeu –R2), one permanent pond (Brejo da Estrada –
P1) and one temporary pond (Brejo do Campo Rupestre – P2). The areas were chosen based on their potential to host anuran species and also for easy access (Table I).
TABLE I Sample sites for the study of temporal distribution characterization of the anuran communities in the FLOE Uaimií (Ouro Preto, MG) from September 2009 to August 2010.
Sampled sites
Coordinates
Height
Description
R1 - Riacho do alojamento
20° 17′ 39″ S43° 34′ 46″ W
1,077 m
100 m transect on a permanent rivulet in an area of secondary forest, surrounded by trees and shrubs on the banks
R2- Cachoeira de São Bartolomeu
20° 17′ 39″ S43° 33′ 71″ W
1,080 m
50 m transect on a permanent rivulet with waterfalls and a sequent pond in secondary forest, surrounded by trees and shrubs on the banks
P1 - Brejo da estrada
20° 29′ 71″ S43° 56′ 05″ W
1,015 m
100 m transect on a permanent enlarged water body in a secondary forest, surrounded by grasses and shrubs on the edge
P2 – Brejo do Campo Rupestre
20° 14′ 40″S43° 35′ 17″ W
1,147 m
An approximate 200 m transect in a temporary swamp rocky meadow, with sand and quartzitic rocks, surrounded by shrubs and grasses on the banks
Direct visualisations of individuals and/or zoophony were used for recording the species. Reproductive activity evidence was recorded considering the following parameters: a) couples in amplexus; b) mature females identified by visualization of oocytes through abdominal transparency; c) clutches; d) tadpoles and e) froglets.
The vocalization patterns of anuran species at FLOE Uaimií were defined as: (1) continuous: for species that vocalize, or for more than nine months of the study; (2) rainy season: for species with calling activity associated with the rainy months; (3) dry season: vocalization associated with the dry months; (4) opportunistic: for species that
vocalized only after rains; and (5) not defined: species for which it was not possible to define a pattern (adapted from
Bertoluci 1998 Bertoluci and Rodrigues 2002 Canelas and Bertoluci 2007
,
,
, São Pedro and Feio 2010).
For the species inventory, data collected at other sampling sites in FLOE Uaimií and from other campaigns in the area were used. The data for accumulated rainfall in the region were provided by Área de Controle Ambiental from the company Novelis do Brasil LTDA, located in the municipality of Ouro Preto, Minas Gerais state, around 13 km
from FLOE Uaimií. The air temperature at each site was measured at the beginning and the end of each night during observation activities. Its average was used as the month's temperature.
Data Analysis
The taxonomic nomenclature used in the present study is according to
The Sorensen index (
Pedralli et al. 2001
(
2009
Krebs 1999
Pyron and Wiens (2011)
.
) was used to verify the similarity in anurans species composition at FLOE Uaimií to the following mountain regions of south-central and south-eastern Minas Gerais state: Serra do Ouro Branco (São Pedro and Feio 2011), Parque Estadual do Itacolomi and Estação Ecológica do Tripuí (PE Itacolomi/EE Tripuí)
), Parque Nacional Serra do Cipó (PARNA Cipó) (
Eterovick and Sazima 2004
), Reserva Particular do Patrimônio Nacional da Serra do Caraça (RPPN Serra do Caraça) (
Canelas and Bertoluci 2007
), and Parque Nacional da Serra do Caparaó (PARNA Caparaó) (V.K. Verdade et al. unpublished data), in the MMR. A cluster analysis by the unweighted average method (UPGMA) (
similarity. For this analysis we considered only taxa with confirmed specific identity.
Krebs, 1999
), in the EMR; and Parque Estadual da Serra do Brigadeiro (PESB) (
Moura et al. 2012
), Parque Estadual do Ibitipoca (PE Ibitipoca) (
Cruz et al.
) was performed to determine the existence of locations with similar species composition. The clusters were defined at a level with at least 40%
Voucher specimens, collected under the licenses IBAMA n° 20724-1 and IEF n° 055/09, were deposited in the collection of Amphibians in the Museu de Zoologia João Moojen, Universidade Federal de Viçosa (MZUFV), Viçosa, Minas Gerais state, Brazil.
RESULTS
We recorded 36 anurans species in FLOE Uaimií, distributed in 10 families: Brachycephalidae (2 species), Bufonidae (2), Centrolenidae (1), Craugastoridae (1), Odontophrynidae (2), Hylidae (19), Hylodidae (1), Leiuperidae (2), Leptodactylidae (5) and Microhylidae (1). The sample site P1 presented the highest richness (14 species), while the
P2 and R1 sites had the lowest, with only seven species in each one (Figure 2).
Figure 2 List of amphibians species and the habitat where they occurred: Riacho do Alojamento (R1); Cachoeira do Bartlomeu (R2); Brejo da Estrada (P1); Brejo do Campo Rupestre (P2). * Species recorded outside the monitored environments, in FLOE Uaimií (Ouro Preto, MG).
The family Hylidae was the most representative, with a total of 19 species (51%), and two that were unidentified, Bokermannohyla sp. (gr. circumdata) and Scinax sp. (aff. perereca). The last one corresponds to a species that is in the description process (L.B. Nascimento unpublished et al., data).
The Figure 3 presents the vocalization patterns displayed by the recorded anuran species. They were associated with the following patterns: 1) continuous: Aplastodiscus arildae and Hypsiboas polytaenius; 2) rainy season: Ischnocnema izecksohni, I. juipoca, Vitreorana uranoscopa, Aplastodiscus cavicola, Phyllomedusa burmeisteri, Scinax sp.
(aff. perereca), Physalaemus cuvieri, Pseudopaludicola serrana, Leptodactylus cunicularius, L. furnarius, and L. fuscus; 3) dry season: Scinax luizotavioi; 4) opportunistic: Proceratophrys boiei; and 5) not defined: Rhinella pombali, R. rubescens, Haddadus binotatus, Bokermannohyla sp. (gr. circumdata), Dendropsophus minutus, Hypsiboas
albopunctatus, and H. faber. The species Phasmahyla jandaia and Elachistocleis cesarii could not be placed in any of the defined patterns since they were not observed vocalizing. The species Phasmahyla jandaia was recorded only in February and a young of E. cesarii was recorded only in October. Although Bokermannohyla sp. (gr.
circumdata) was placed in the not defined vocalization pattern, mature females were observed in October and January, a male was observed vocalizing in February and soon after (May and June), tadpoles and froglets were observed. In spite of the species Haddadus binotatus being assigned to the not defined pattern, a male was observed
vocalizing in March.
Figure 3 Monthly rainfall (bars), average monthly temperature (line) and annual pattern of reproductive activity of 24 anuran species sampled in FLOE Uaimií (Ouro Preto, MG), from September 2009 to August 2010.
The species composition of FLOE Uaimií was most similar to other conservation units in the municipality of Ouro Preto (PE Itacolomi and EE Tripuí) which were also similar to Serra do Ouro Branco. The group formed by them has showed greater similarity with the RPPN Serra do Caraça. All these locations are in the Quadrilátero Ferrífero
region, forming a separate group in the similarity dendrogram (Figure 4A). Two other mountainous regions in the MMR, the PESB and PARNA Caparaó, are similar regarding the anurans composition and they form a group with the Quadrilátero Ferrífero studied areas. The PE Ibitipoca, also in the MMR, formed an isolated branch, indicating
that its species composition is different from that found in the other locations. The PARNA Cipó, in the EMR, was the location with the lowest similarity to the study area (Figures 4A and 4B).
Figure 4 (A) Similarity between localities and (B) Map of the dendrogram in overlapping areas of central-south and southeastern Minas Gerais state, Brazil: FLOE Uaimií (present study), Serra do Ouro Branco (São Pedro and Feio 2011), Parque Estadual do Itacolomi and Estação Ecológica do Tripuí (PE
Itacolomi/EE Tripuí) (
Pedralli et al. 2001
Cruz et al. 2009
Ibitipoca) (
), Parque Nacional da Serra do Cipó (PARNA Cipó) (
Eterovick and Sazima 2004
), Reserva Particular do Patrimônio Natural Serra do Caraça (RPPN Caraça) (
), Parque Estadual Serra do Brigadeiro (PESB) (
Moura et al. 2012
), Parque Estadual do Ibitipoca (PE
) and Parque Nacional da Serra do Caparaó (PARNA Caparaó) (V.K. Verdade et al., unpublished data). Cophenetic correlation coefficient = 0.9213
DISCUSSION
The 36 species recorded in FLOE Uaimií correspond to 50% of the anurofauna known in the Quadrilátero Ferrífero region, situated in EMR, Minas Gerais state (
43 species (
). The recording of the species Hylodes babax represents the first record for the Quadrilátero Ferrífero region (
Pirani et al. 2010
Leite et al. 2008
). The species richness of this conservation unit is only surpassed by Serra do Ouro Branco, with 47 species (São Pedro and Feio 2011), and RPPN Serra do Caraça, with
).
The great richness of amphibian species associated with the EMR is due not only to its large area and high longitudinal and altitudinal amplitude, but certainly to the existence of a complex and intricate relationship between this historical and ecological formation and its adjacent biomes, the Cerrado and the Atlantic Forest (
Pedralli et al. 2001 Canelas and Bertoluci 2007
EMR has a large environmental heterogeneity, presenting a mosaic of habitats from open areas to forests, providing a high resources variety, which has also been shown in other studies (
pattern of great richness is observed for the flora of the rocky meadows of the EMR which also presents a high endemism rate.
,
, São Pedro and Feio 2010, São Pedro and Feio 2011). According to
Rapini et al. (2008)
and
Leite et al. 2008
Versieux et al. (2008)
). The
, the same
Cardoso et al. (1989)
Conte and Rossa-Feres (2006)
Among the study sites, the Brejo da Estrada (P1) was the richest environment, housing 14 species.
and
highlighted spatial heterogeneity as a determinant of the environment richness. The amount of microhabitats with substrates for male calling activity available in each environment can be a
determining factor for differentiating the richest site. Although we have not quantified, the P1 seems to be the sampled environment that presented the largest number of microhabitats.
The Cachoeira de São Bartolomeu rivulet (R2) presented lower richness, with only eight species, which may be related to the high flow of people in the area compared to the others study sites, due to the presence of a waterfall and an oratory dedicated to Saint Bartholomew. The Riacho do Alojamento (R1) and Brejo do Campo Rupestre (P2)
presented the lowest richness, with seven species. Other studies show that temporary environments attract more species due to the reduced amount or absence of tadpoles predators (
Alford 1999 Bertoluci and Rodrigues 2002
,
). This is not observed at P2 in FLOE Uaimií.
The majority of the FLOE Uaimií anurans followed a seasonal pattern of calling activity in which the species vocalized during the warmer and wetter months of the year. This same pattern was noted in studies conducted in other Brazil regions (
1999 Vasconcelos and Rossa-Feres 2005 Canelas and Bertoluci 2007
,
,
, São Pedro and Feio 2010). In tropical regions, the rain seems to be the main factor regulating the anurans reproductive activities (
Gottsberger and Gruber 2004
duration of their reproductive sites (
Aichinger 1987 Heyer 1973
,
,
, Pombal Jr 1997,
Bertoluci 1998 Bernarde and Anjos 1999 Bernarde et al.
,
,
).
Some species like Aplastodiscus arildae and Hypsiboas polytaenius had a continuous vocalization period. The same pattern was observed for the A. arildae in Parque das Mangabeiras, in the municipality of Belo Horizonte (
Serra do Ouro Branco (
Heyer et al. 1990 Rossa-Feres and Jim 1994
), not only because of the vulnerability of these animals to desiccation, but also because it is the rain that determines the viability and
Carvalho et al. 2006
) and at RPPN Serra do Caraça (
) and for H. polytaenius at this last locality and at
, São Pedro and Feio 2010, respectively).
It was not possible to define the vocalization pattern for some species, such as D. minutus, H. albopunctatus, and H. faber, because they were registered only once during the study period at FLOE Uaimií. However, the three species are very abundant in other studied areas within the Quadrilátero Ferrífero region and were registered almost all
the year, presented a higher density of calling males during the rainy season (
Other anuran species can present explosive reproductive patterns (sensu
Bertoluci 1998
of São Paulo (
) and RPPN Serra do Caraça (
Grandinetti and Jacobi 2005 Canelas and Bertoluci 2007
,
Wells 1977
, São Pedro and Feio 2010).
), so they call for only a short period. Field activities did not coincide with the nights when they vocalized (
Toledo et al. 2003
). This may be the case for the species Elachistocleis cesarii, which demonstrated explosive reproductive patterns in Fazenda Intervales, state
).
Heyer 1978
Three species of the genus Leptodactylus, from the L. fuscus species group (sensu
), occurring in P2, had overlapping vocalization periods in the rainy season, as observed in other studies (
edge. Leptodactylus fuscus was found on the soil, L. furnarius within the grasses, and L. cunicularius among the rocks. However, the indication of a spatial partitioning could be speculative in this case.
, São Pedro and Feio 2010). Apparently, they demonstrated a different spatial distribution of calling sites on the body water
Leite et al. (2008)
The studied communities at localities that are included at the Quadrilátero Ferrífero region (FLOE Uaimií, PE Itacolomi/EE Tripuí, Serra do Ouro Branco, and RPPN Serra do Caraça) form a cluster in the similarity dendrogram. According to
, and within what is currently known, it is possible to recognise three major relief units
along the EMR, based on the endemic anurans species distribution, and one of which is at the Quadrilátero Ferrífero. The present study confirms this proposal, because the most similar anuran communities are from this unit. The dissimilarity verified in this study among the PARNA Cipó and others studied areas from the EMR could be also
explained by the proposal of
Leite et al. (2008)
. The second proposed unit begins at Serra do Cipó and continues to mountains in the north of the Minas Gerais state. One endemic species of the EMR could exemplify this fact, Bokermannohyla martinsi, whose distribution is related only to the Quadrilátero Ferrífero unit (
Madeira et al. 2008
is important to point out that most studies conducted at PARNA Cipó are related to the rocky meadows complex and on the western slopes of the mountain range bordering the São Francisco river basin (
groups, including amphibians.
The Cerrado and Atlantic Forest biomes influence on the EMR is an important factor for understanding the current observed distribution of these amphibian species (
Leite et al. 2008
southeast) and it acts as a barrier for many species typical of these different phytogeographic formations (
Leite et al. 2008
). According to
Madeira et al. (2008)
). The EMR not only separates the coastal hydrographic basins from the inland basins, but also the Cerrado (in the west) and Atlantic Forest biomes (in the east/
Leite et al. 2008
).
Species with restricted distributions, related physiologically, occupying the same environmental physiognomy in different mountain ranges (Espinhaço and Mantiqueira), suggest biogeographic homologies and raise speculations about the influence of the geomorphologic evolution in the anurans speciation patterns (
). Species with distribution in both the EMR and MMR (e.g. Hylodes babax Quadrilátero Ferrífero clade.
Pirani et al. 2010
The PE Ibitipoca is composed of quartzite, forming a prominent elevated lowland hill (
; and Scinax luizotavioi –
Cruz et al. 2009
). Although, it
). Thus, the western EMR acts as the occurrence limit of species or even genera (e.g. Phasmahyla, Thoropa, Crossodactylus, Hylodes and Cycloramphus) that have the centre of their occurrence areas in the Atlantic Forest. The
same can be observed for species typical from Cerrado biome (e.g. Ameerega flavopicta and Physalaemus centralis), whose geographic distribution does not extend further than the western slope of the EMR (
Feio 2007
Leite et al. 2008
, the Atlantic Forest portion located in Serra do Cipó is poorly sampled for various species
Moura et al. 2012
; Physalaemus maxinus and Dendropsophus giesleri – São Pedro and Feio 2011; Ischnocnema izecksohni -
Taucce et al. 2012
). Its vegetation is composed mainly of montane meadows at higher altitudes (from 1,050 to 1,784 m), patches of rocky meadows, and forest formations (
between the EMR and MMR, differences in the geological formations and relationships to the biogeographic domains provide PE Ibitipoca with some environmental peculiarities, making it less similar to FLOE Uaimií (
Cruz et al. 2009
Nascimento et al. 2005 Cruz and
,
) could explain an anuran fauna similarity of PESB and PARNA Caparaó with the
Cruz et al. 2009
). Even though the continuous altimetry provides some form of connection
).
Although the most part of the amphibians species registered in this study have showed wide geographical distribution, the FLOE Uaimií anuran community include species from both mountain complex, the EMR and MMR. Endemic distribution of anurans species emphasizes the needing of strategies to conserve the FLOE Uaimií and also the
EMR.
Acknowledgements
We would like to thank everyone who helped us during the fieldwork. We are grateful to Paulo C. A. Garcia and Emanuel Texeira da Silva (ETS) for valuable suggestions on a previous version of the manuscript. ETS for his help in the statistical analysis. Mário Moura and Diego Santana for construction of the map. All residents of São
Bartolomeu for their logistical support. RMP thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação Artur Bernardes (FUNARBE) for financial support. LBN thanks Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq- Process 479457/2012-03), and Fundação de Amparo à Pesquisa
do Estado de Minas Gerais (FAPEMIG, Process APQ-00683-12). RNF thanks for Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the productivity fellowship conceded.
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Received: June25, , 2012; Accepted: November13, , 2012
Correspondence to: Renata Magalhães Pirani E-mail: [email protected]
Tel: +55 21 3907-8100
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http://dx.doi.org/10.1590/S0001-37652013005000042
Biological Sciences
The role of phytophysiognomies and seasonality on the structure of ground-dwelling anuran (Amphibia) in the Pampa biome, southern Brazil
FRANCIÉLE P. MARAGNO1, TIAGO G. SANTOS2, SONIA Z. CECHIN3
1
Programa de Pós-Graduação em Biodiversidade Animal, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brasil
Universidade Federal do Pampa, Campus São Gabriel, 97300-000 São Gabriel, RS, Brasil
3Departamento de Biologia, Programa de Pós-Graduação em Biodiversidade Animal, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brasil
2
ABSTRACT
ABSTRACT
Considering that habitat use by amphibians is related both with climate and environmental features, we tested the hypothesis that anuran assemblages found in different phytophysiognomies and in different seasons vary in structure.
Additionally, we searched for species which can be indicators of habitat and seasons. The study was conducted in the Pampa biome, southern Brazil. Sampling was done through pitfall traps placed in three phytophysiognomies: grassland,
ecotone grassland/forest; and forest. The seasonality factor was created by grouping months in warn and cold seasons. Sixteen species were found and the assemblages were influenced both by phytophysiognomies and climatic
seasonality. In a paired comparison, the three phytophysiognomies differed in structure of assemblage from each other. Physalaemus henselii, P. riograndensis, Pseudopaludicola falcipes and Pseudis minuta were indicators of ecotone.
Leptodactylus gracilis and Physalaemus biligonigerus were indicators of grassland. None species was indicator of forest. Most of the species were indicators of warm season: Elachistocleis bicolor, Leptodactylus fuscus, L. gracilis, L.
latinasus, L. latrans, L. mystacinus, Physalaemus biligonigerus, P. cuvieri and Pseudis minuta. None species was indicator of cold season. We found that even for species of open areas, as Pampa, heterogeneous phytophysiognomies are
important for maintaining abundance and constancy of populations of anuran.
Keywords: amphibian; community; ecotone; pitfalls traps; seasons
RESUMO
RESUMO
Considerando que o uso do habitat pelos anfíbios está relacionado tanto com clima quanto com características ambientais, testamos as hipóteses de que taxocenoses de anuros encontradas em diferentes fitofisionomias e estações diferem
em estrutura. Adicionalmente, buscamos espécies que podem ser indicadoras de habitat e estações. O estudo foi desenvolvido no bioma Pampa, sul do Brasil. A amostragem foi realizada através de armadilhas de interceptação e queda
instaladas em três fitofisionomias: campo, ecótono campo/mata e mata. O fator sazonalidade climática foi criado agrupando os meses em estações quente e fria. Dezesseis espécies foram encontradas e as taxocenoses foram influenciadas
pelas fitofisionomias e sazonalidade climática. Em uma comparação pareada, as três fitofisionomias diferiram entre si em estrutura da taxocenose. Physalaemus henselii, P. riograndensis, Pseudopaludicola falcipes e Pseudis minuta
foram indicadoras de ecótono. Leptodactylus gracilis e Physalaemus biligonigerus foram indicadoras de campo. Nenhuma espécie foi indicadora de mata. A maioria das espécies foi indicadora da estação quente: Elachistocleis bicolor,
Leptodactylus fuscus, L. gracilis, L. latinasus, L. latrans, L. mystacinus, Physalaemus biligonigerus, P. cuvieri e Pseudis minuta. Nenhuma espécie foi indicadora da estação fria. Observamos que mesmo para espécies de áreas abertas,
como o Pampa, fitofisionomias heterogêneas são importantes para manutenção da abundância e constância das populações de anuros.
Palavras-Chave: anfíbios; comunidade; ecótono; armadilhas pitfalls ; estações
INTRODUCTION
The factors influencing organization of anuran assemblage are related with availability, sharing and partitioning of resources like prey, shelter against predators, vocalization and egg deposition sites, maintaining of body
temperature and moisture, interactions with other species, and historical constraints (
association of all these factors some patterns can emerge.
Lillywhite et al. 1973 Toft 1985 Barbault 1991 Wellborn et al. 1996 Lima et al. 2000
,
,
,
,
, Wente and Philips 2005,
Prado et al. 2005 Tomé et al. 2010
,
). From the
The temporal pattern of activity of amphibians is highly influenced by climatic conditions, mainly due to their reproductive characteristics. In this sense, the constancy and intensity of precipitation throughout the year are related to
a continuous reproductive activity, as in tropical forests, or to a seasonal reproductive activity, as in regions with markedly dry and rainy seasons (
Gottsberger and Gruber 2004 Prado et al. 2005 Vasconcelos and Rossa-Feres 2005 Bernarde 2007
,
,
,
Sanchez et al. 2007 Both et al. 2008 Canavero et al. 2009 Canavero and Arim 2009
A seasonal reproductive pattern also occurs where temperature and photoperiod are seasonal, but rainfall is constant, as in subtropical climate (
,
,
increase in the activity of anurans is expected during the breeding season, mainly associated with higher rates of movements in search of breeding sites and recruitment of juveniles.
,
). In this sense, an
On a local scale, environmental features such as microclimate provided by vegetation, density of bushes, density of canopy cover, and depth of leaf-litter are some of the important issues regulating amphibian assemblages in
forests (
Krishnamurthy 2003 Paris 2004
,
).
, Van Sluys et al. 2007). In areas of open vegetation, the presence and abundance of a species may be associated with availability of favorable sites for reproduction, as well as the structural
features of the reproductive sites (
Prado et al. 2005 Vasconcelos et al. 2009 Silva and Rossa-Feres 2011 Silva et al. 2012
,
,
,
). A distinct pattern is found in the ecotones, where there are resources belonging to both types of vegetation, with greater
Ries et al. 2004
Connel 1978
environmental complexity and richness of organisms (
). Moreover, according to the intermediate disturbance hypothesis (
), landscape changes in intermediary intensity and frequency, as observed in ecotones,
produce a greater diversity of organisms as there is a constant turnover of species in these regions. Temperature, density of shrubs, canopy and leaf-litter cover, and parasites have been related to composition of herpetofauna and
Schlaepfer and Gavin 2001 Urbina-Cardona et al. 2006
distance from the edge (
,
). Differences between a gradient from open to forestall phytophysiognomies have being portrayed in studies concerning habitat fragmentation (
Urbina-Cardona et al. 2006 Vasconcelos et al. 2010
,
). However, few studies discuss habitat use in natural mosaics of vegetation (e.g.
Ries et al. 2004
,
Julião et al. 2004
).
The Pampa biome is composed by natural grasslands that cover south of Brazil, Uruguay and central region of Argentina. The typical ecosystem on the south of Brazil is a natural mosaic of Seasonal Forests from Atlantic Domain
and grassland (IBGE 2004,
Oliveira-Filho et al. 2006
). Currently, 50% of the natural vegetation of Brazilian Pampa was converted in pastures, crops and forestry (
regions of Brazilian Pampa are listed as priorities for biodiversity conservation, only 2.58% are designed to conservation units (
Cordeiro and Hasenack 2009 Santos and Trevisan 2009
,
). Indeed, although several
Brandão et al. 2007
).
Moreira et al. 2007 Both et al. 2008
Moreira et al. 2010
In southern Brazil, studies that address the range of amphibian assemblages along environmental and seasonal gradients are recent (e.g.
,
, Santos et al. 2008,
). Such studies were
developed manly at breeding sites and did not evaluate the habitat use as a whole. Studies beyond breeding sites identify the relationship of species and the matrix surrounding these reproductive places, complementing the
knowledge generated from studies in breeding sites. As habitat use by amphibians is related both with climate and environmental features, we tested the hypothesis that anuran assemblages found in different phytophysiognomies
and in different seasons vary in structure. Moreover, considering that the species requirements (e.g. feeding, reproductions and thermic shelter) may vary according to seasonal climatic changes and that phytophysiognomies may
vary on the availability of resources according to its characteristics (e.g. heterogeneity), we also test an integrated effect of the factors “habitat” and “season”. Additionally, we searched for species which can be indicators of habitat
and seasons.
The study was conducted in Santa Maria, central portion of Rio Grande do Sul, southern Brazil. The region belongs to Pampa biome (IBGE 2004), near the southern boundary of the Semideciduous Forest. The climate is
subtropical humid (STUM), with annual precipitation of 1,708 mm and no dry season (
Maluf 2000
). The climate is characterized by seasonal variation of photoperiod and temperature, varying from zero or few negatives in the winter
Pereira et al. 1989
to 40°C in the summer, with the average annual temperature of 19.2°C (
). The photoperiod varies from 10 (May-August) to 14 hours (December-January) along the year (Observatório Nacional Brasileiro 2012).
The sample site is located at Campo de Instrução do Exército de Santa Maria (Army Field Instruction, CISM), (29°43′44′S and 53°42′44′W; Figure 1). The CISM has an area of 5,786 ha, of which about 30% are composed of
Cechin and Martins 2000
clumps of native forest and riparian vegetation, with the remainder covered by native grassland partially intended for livestock. Sampling was performed using pitfall traps with drift fences (
). Pitfalls were
placed in three distinct phytophysiognomies: 1) grassland; 2) ecotone grassland/forest; and 3) forest. In each phytophysiognomie we placed ten barrels of 200 l in line, spaced 15 m apart and connected by fences of 1 m high. The
line of traps in the grassland was spaced 2 km apart from the line of traps in the ecotone and 4 km from the traps in the forest. In the ecotone, the line of traps was placed exactly at the edge between the grassland and a fragment of
riparian forest. The traps in the forest were placed in the interior of the fragment and approximately 1 km from the forest edge (Figure 1). The traps were open continuously from January 1996 to March 1998, totalizing 27 months
of sampling, and they were reviewed every two days. All individuals were collected and deposited at the “Coleção de Herpetologia da Universidade Federal de Santa Maria” (ZUFSM). For analysis, we included only species with
total abundance greater than 10 individuals considering all sampling sites and time to avoid punctual occurrences.
Figure 1 Study site localization. Army Field Instructions of Santa Maria, Rio Grande do Sul, Brazil. Numbers indicate the position of the lines of pitfall traps in the three phytophysiognomies (grassland,
ecotone, and forest) to sampling of anurans.
To investigate the variation in the structure of assemblage (in the sense of composition and abundance simultaneously) between the phytophysiognomies and between the seasons we used a PERMANOVA for repeating measures
Anderson et al. 2008
(
), considering two factors: i) phytophysiognomies (grassland, ecotone, and forest) and ii) seasonality (spring/summer and autumn/winter). To compose the seasonality factor, months were grouped in two seasons
(warm and cold) since the higher activity of anurans in the southern Brazil occurs during hotter and longer photoperiods (
Both et al. 2008
, Santos et al. 2008).
The PERMANOVA allows to explore the effect of each factor individually (phytophysiognomies and seasonality), as well as the interaction between factors. The modality “repeated measures” was developed to deal with sampling
Clark and Gorley 2006
designs containing measures repeated in time (temporal pseudo-replication). The PERMANOVA was based at the indices of binomial dissimilarity deviation (
), using species abundance transformed by square
root and 999 permutations. Additionally, the dissimilarity matrix was represented by a Non- Metric Multidimensional Scaling (NMDS) to exhibit the dispersion of samples (months) on the two factors analyzed
(phytophysiognomies and seasonality). Information loss during the ordination was measured by stress (S) according to
Clark and Warwick (2001)
. Analyses were conduct on package PERMANOVA+ add on 1.01 (
Anderson et al. 2008
),
available in PRIMER-E 6.1.11 (Clarke and Gorley 2006).
Previous to PERMANOVA, the homogeneity of variance (sphericity) of samples was tested through Levene's test. When the test is significant, the homogeneity must be rejected. Variances were homogeneous for samples from the
three phytophysiognomies (F = 2.77; p > 0.05).
Dufrêne and Legendre 1997
Additionally, we used an Indicator Species Analysis (ISA) (
) to identify the species that contributed most to the segregation between the three phytophysiognomies and two seasons. In this analysis, we define
groups a priori and test which species represent the groups. For this, each species gain an indicator value (IV) for each group. This value range from zero (none indication) to 100 (perfect indication) and is calculated based on
Dufrêne and Legendre 1997
species abundance and frequency in the sampling unities (phytophysiognomies and seasons). Therefore, a good indicative species must be frequent and abundant in the samples of its group (
hypothesis of this analysis considers the maximum indicator value is not greater than expected by chance. The significance of groups IV was tested through Monte Carlo permutation (5000).
). The null
RESULTS
We found 16 species of ground-dwelling anuran, belonging to six families. Species richness varied from 13 to 15 in the phytophysiognomies (Figure 2). The greater number of individuals was found in the ecotone grassland/forest
(62.8%), followed by grassland (23.4%) and forest (13.8%). In overall, Pseudopaludicola falcipes, Leptodactylus latrans and Physalaemus cuvieri were the most abundant. Three species were rare at study site: Leptodactylus
chaquensis, Rhinella achavali and R. henselli (Table I). Physalaemus cuvieri (66.7%), P. biligonigerus (59%) and Leptodactylus gracilis (59%) were the most frequent species of grassland. In the forest, Physalaemus cuvieri
(59%), Leptodactylus latrans (55.5%) and L. fuscus (37%) were the most frequent; while in the ecotone, the most frequent were Physalaemus cuvieri (81.5%), P. riograndensis (77.8%) and Pseudopaludicola falcipes (74%) (Table
I).
Figure 2 Venn's diagram representing the richness of ground-dwelling anurans in three phytophysiognomies (grassland, ecotone, and forest), and richness shared among phytophysiognomies in an
assemblage of Pampa biome, Brazil, from January 1996 to March 1998.
TABLE I Abundance of ground-dwelling anurans in three phytophysiognomies (grassland, ecotone, and forest) and two seasons (warm and cold) of Pampa biome, Brazil, from January 1996 to March 1998. %
O = Percentage of Occurrence (percentage of months in which a species was found in relation to total months of the study (27).
Species
Grassland
Rhinella achavali
Rhinella henselli
1
0
Odontophrynus americanus
6
Pseudis minuta
1
Physalaemus biligonigerus
Physalaemus cuvieri
Physalaemus henselii
Physalaemus riograndensis
Pseudopaludicola falcipes
82
102
0
29
11
Leptodactylus chaquensis
Leptodactylus fuscus
Leptodactylus gracilis
Leptodactylus latinasus
Leptodactylus latrans
Leptodactylus mystacinus
0
109
72
2
34
10
Elachistocleis bicolor
Total abundance
Richness
19
479
13
Phytophysiognomies
Forest
%O
Bufonidae
3.7
0
0
0
5
18
Cycloramphidae
18
3
11
Hylidae
3.7
9
18
Leiuperidae
59
3
11
67
164
59
0
0
0
3.7
1
3.7
26
18
33
Leptodactylidae
0
1
3.7
41
40
37
59
1
3.7
3.7
3
11
48
26
55
18
3
11
Microhylidae
59
7
0.19
283
14
%O
Seasons
%O
Cold
%O
2
1
13
6.7
0
4
0
17
15
5
27
8
42
145
52
148
87
8
33
10
220
32
158
259
19
81
48
78
74
82
400
22
157
196
80
100
60
80
87
13
86
10
31
92
50
75
33
75
75
1
95
22
7
225
24
3.7
63
48
22
55
30
0
232
83
12
248
28
0
87
87
53
87
53
2
12
12
0
37
6
17
33
42
0
50
25
85
1287
15
48
103
1720
15
87
8
327
14
50
Ecotone
%O
Warm
1
0
3.7
0
4
The anuran assemblage was significantly influenced by phytophysiognomies and seasons individually (Pseudo-F = 25.83, p < 0.01; Pseudo-F = 31.74, p < 0.01, respectively). The interaction between these two factors did not affect
the assemblage (Pseudo-F = 1.08 and p = 0.39). In a paired comparison, we found that the three phytophysiognomies differ in structure of assemblage from each other (T = 3.56 and p < 0.01 (comparing grassland with forest); T =
5.33 and p < 0.01 (comparing grassland with ecotone); T = 5.56 and p < 0.01 (comparing forest with ecotone). The greatest dissimilarity was among samples from ecotone, while the lowest dissimilarity was among samples from
the forest. In addition, grassland and ecotone were the most dissimilar to each other, while grassland and forest were the least dissimilar (Figure 3; Table II).
Figure 3 Non-Metric Multidimensional Scaling (NMDS) representing the dispersion of samples in relation to two factors: phytophysiognomies (grassland, ecotone, and forest) and seasons (warm and cold) for
an assemblage of ground-dwelling anurans of Pampa biome, Brazil, from January 1996 to March 1998.
TABLE II Average dissimilarity between and within the factor phytophysiognomies (grassland, ecotone, and forest) for an assemblage of ground-dwelling anuran of Pampa biome, Brazil, from January 1996
to March 1998.
Grassland
Forest
Ecotone
Grassland
5.89
6.27
10.48
Forest
4.01
9.54
Ecotone
8.97
According to the Indicator Species Analysis (ISA), six species (46% of total) had occurrence and abundance significantly further than expected by chance in some of the phytophysiognomies (p < 0.05). Physalaemus henselii, P.
riograndensis, Pseudopaludicola falcipes and Pseudis minuta were indicative of ecotone. Two species were indicative of grassland: Leptodactylus gracilis and Physalaemus biligonigerus. None species was indicative of forest
(Table III). Regarding seasonality, nine species (56% of total) were indicative of warm season: Elachistocleis bicolor, Leptodactylus fuscus, L. gracilis, L. latinasus, L. latrans, L. mystacinus, Physalaemus biligonigerus, P. cuvieri
and Pseudis minuta. None species was indicative of cold season (Table IV).
TABLE III Indicator value (IV) resulting from an Indicator Species Analysis (ISA) for an assemblage of ground-dwelling anuran in three phytophysiognomies (grassland, ecotone, and forest) in the Pampa
biome, Brazil, from January 1996 to March 1998. * for p ≤ 0.05 and *** for p ≤ 0.001.
Species
P. falcipes
P. riograndensis
P. biligonigerus
P. henselii
L. gracilis
P. minutus
L. latrans
E. bicolor
P. cuvieri
L. fuscus
L. latinasus
L. mistacinus
O. americanus
Phytophysiognomies
Ecotone
Ecotone
Grassland
Ecotone
Grassland
Ecotone
Ecotone
Ecotone
Ecotone
Ecotone
Ecotone
Ecotone
Grassland
IV
90.6***
75.8***
66.2***
65***
61.1***
51.6***
43.6
42.2*
37.3
27.4
18.1
16
10.9
TABLE IV Indicator value (IV) resulting from an Indicator Species Analysis (ISA) for an assemblage of ground-dwelling anuran during two seasons (warm and cold) in the Pampa biome, Brazil, from January
1996 to March 1998. * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001.
Species
P. cuvieri
L. fuscus
L. latrans
L. gracilis
E. bicolor
P. biligonigerus
P. riograndensis
P. falcipes
P. minutus
L. latinasus
L. mistacinus
O. americanus
P. henselii
Seasons
Warm
Warm
Warm
Warm
Warm
Warm
Warm
Warm
Warm
Warm
Warm
Cold
Warm
IV
81.1***
75***
67.6***
51***
49.3**
40.5*
39.9
37.6
34.7*
28.6**
28.2*
16.7
16.4
DISCUSSION
Phytophysiognomies influenced the ground-dwelling anuran so that each vegetation type had distinct structure of assemblage. It is known that characteristic of habitat can influence the presence of amphibians at the environment.
In fact, studies of Neotropical amphibian assemblage observed a distinct structure than expected by chance, for both adults and tadpoles (
Vasconcelos et al. 2009 Both et al. 2011
,
, Moreira and Maltichik 2012).
In this study, the differences in structure of assemblage were mainly due variation in abundance and frequency of occurrence of each species in each phytophysiognomie, since the composition of species was similar between
phytophysiognomies (see Table I). In the grassland, the greatest abundance and frequency of occurrence of some species, mainly Leptodactylus fuscus, L. gracilis and Physalaemus biligonigerus, distinguished the assemblage
structure from the other phytophysiognomies. In fact, this two last species were indicator of grassland. These species are considered abundant along their geographic distribution and are very plastic in habitat use: being found in
grasslands, wetland, dunes, forest, growing areas of soybean and rice, and urban areas (
Peltzer et al. 2003 Attademo et al. 2005 Loebmann and Vieira 2005 Lucas and Fortes 2008
,
,
,
, Santos et al. 2008). Leptodactylus gracilis belongs to
Leptodactyus fuscus group, whose species deposit eggs in cavities in the ground that are flooded, releasing the tadpoles to complete their development in ponds closer (
Heyer 1978
). This reproductive mode confers higher protection
against dehydration and predation, and possibilities to a great versatility in habitat use. Physalaemus biligonigerus is able of reproducing both in ponds within agricultural lands and in areas of pristine vegetation (
Peltzer et al. 2006
),
Zarancho et al. 2004
using shallow, temporary ponds, and semi-covered by grasses and sedges (
). Thus, these species present a set of characteristics which allow them to successfully use open areas.
Grassland is the reproductive habitat of pampean species and a high abundance and frequency of occurrence would be expected in this phytophysiognomy (
Cei 1980 Manzano et al. 2004 Núñez et al. 2004 Both et al. 2008
,
,
,
Inger and Colwell 1977 Silvano et al. 2003 Urbina-Cardona et al. 2006
However, some species were more abundant in other phytophysiognomies. Open areas are environments of large variation in climatic conditions (
species less tolerant to these variations search for shelter in phytophysiognomies as forests or ecotone, returning to the grassland only for breeding.
,
,
) and it is possible that
In the forest occurred the lowest anuran abundance, with dominance of Physalaemus cuvieri. Furthermore, most species found in the forest had low frequency throughout the study, as well as anuran samples of this vegetation type
were the most similar to each other, revealing great homogeneity. In forests, the richness and composition of vegetation, elevation, structure and depth of leaf-litter, and proximity to ponds are some factors that may influence
richness, abundance, and composition of anuran assemblages (
Paris and McCarthy 1999 Willians and Hero 2001 Peltzer et al. 2003
,
,
, Van Sluys et al. 2007). However, in the present study, the structure found in the forest seems to be a
consequence of an opportunist occurrence to foraging and maintenance of temperature and body moisture, as observed for anurans in fragments of Semideciduous Forest (
In a community, species may be affected in a lesser or greater degree by the environment, depending on how they use this environment (
Silva and Rossa-Feres 2007
).
Urbina-Cardona et al. 2006
). Anurans of Pampa use mainly lentic ponds localized in grassland or
Both et al. 2008
associated with wet lands as reproduction sites (
, Santos et al. 2008). In the present study, there were no lentic ponds in the forest and the sampling method excludes species that uses the vertical stratification of
vegetation. These two cues may explain the absence of indicator species in this phytophysiognomie.
The structure of the ground-dwelling anuran assemblage in the ecotone was distinct from the two others phytophysiognomies and most species (56%) were found in 48% or more of the months during the study. In addition, anuran
samples from ecotone were the least similar to each other, demonstrating the heterogeneity of samples. In this study, the ecotone emerges from the natural proximity of grassland and riparian forest. Considering the high ecological
flow, the access to resources from two distinct phytophysiognomies, and the ecological interactions in the ecotones, it is expected that communities in ecotones differs from those which created them (
Ries et al. 2004
). In this study,
the assemblages from the grassland and from the forest are more similar to each other than the samples from ecotone among themselves. This reinforces the idea of heterogeneity of communities in transition areas (
the same study site, highest richness of snakes was also found in ecotone (S.Z. Cechin, unpublished data).
Schlaepfer and Gavin 2001
Connel 1978
). For
Pineda and Halffter 2004
Environmental characteristics may cause a differential effect in each population (
, Willian and Hero 2001,
) and in this study, Physalaemus henselii, P. riograndensis, Pseudopaludicola
falcipes and Pseudis minuta were associated to the ecotone. This is a consequence of a high abundance and frequency of these species in this phytophysiognomie along the months, with P. henselii found only in the ecotone. All
Peltzer et al. 2003 Attademo et al. 2005 Peltzer et al. 2006
these species may be found in open and disturbed habitats (
,
,
, Santos et al. 2008). However, some studies which evaluated their reproductive sites occupancy recorded them in ponds
Peltzer et al. 2003 2006
close to forest areas or inside no disturbed riparian forest (
,
). In this study, the riparian forest was periodically waterlogged providing a moister substrate that amphibians may use as shelter against dehydration,
Antonini et al. 2003
especially small species such as Physalaemus riograndensis and Pseudopaludicola falcipes. In addition, forests edges may concentrate a high number of insects (
) that are important food resources for amphibians,
and they also offer a variety of shelters against predation created by the heterogeneity of the vegetation. Thus, it is possible that species indicators of ecotone are more sensitive to the availability of all this resources. Leptodactylus
fuscus, L. latrans and Physalaemus cuvieri also occurred in high frequency and abundance in the ecotone, but these species were not identified as indicator of this phytophysiognomy because they were also abundant and frequent
in the others two phytophysiognomies.
The structure of the assemblage had a seasonal pattern, with the most of the species indicators of warm season. This result can be understood as an overall pattern of activity of the assemblage. The association between the annual
cycle of activity of amphibians with climatic factors have been widely reported (e.g.
Gottsberger and Gruber 2004 Bernarde 2007 Canelas and Bertoluci 2007 Sanchez et al. 2007
,
,
,
). In subtropical temperate regions, temperature and photoperiod are
Both et al. 2008 Canavero and Arim 2009
considered the most important factors on the regulation of reproductive activity of anurans (
,
), which is more intense in warmer and with longer photoperiod months (
Both et al. 2008
, Santos et al.
Both et al. 2008
2008). A higher frequency of capture in the warm season was then expected since the most species of amphibians are in reproductive activity during this period in the southern Brazil (
From conservation point of view, removal of native vegetation with consequent emergence of an edge affects negatively species with restrict requirements and that tolerate little variation in environmental conditions (
2003
). However, Pampa is a very complex biome naturally composed by native mosaics of grassland and forests (
Silvano et al.
Boldrini 2009
) and whose resulting ecotones are keys to ensure the diversity of species, serving as shelter, feeding areas
Bencke 2009
and corridors (
). The present study reinforces the importance of theses natural mosaics and ecotones of Pampa since we demonstrated that each phytophysiognomie was responsible for the maintenance of distinct
assemblage of ground-dwelling anuran. In this sense, the increase of removal of riparian forest, patches of forest, and wetlands stimulated by the changes proposed by the new Brazilian Forest Code may have a negative impact also
on the amphibian fauna.
Acknowledgements
We are grateful to CISM for allowing access to the study site. We also thank Cynthia P.A. Prado, Elaine Lucas, Nilton Caceres and Vanessa Fortes for suggestions made on the manuscript. FPM is grateful to Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the doctorate's degree fellowships granted, and SZC is grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a research fellowship
(Process No. 303359/2009–9).
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Received: January18, , 2012; Accepted: December6, , 2012
Correspondence to: Franciéle Pereira Maragno E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
http://dx.doi.org/10.1590/S0001-37652013000300015
Biological Sciences
Ecological features of titica vine (Heteropsis flexuosa (Kunth) GS Bunting) in Rondônia State, Northwest Brazilian Amazon
MICHELLINY M. BENTES GAMA, ABADIO H. VIEIRA, RODRIGO B. ROCHA
EMBRAPA, Centro de Pesquisa Agroflorestal de Rondônia, BR 364, Km 5,5, Caixa Postal 127, 76815-800 Porto Velho, RO, Brasil
ABSTRACT
The aim of this study was to characterize the forest structure with H. flexuosa in Rondônia State, as a first step in developing sustainable harvest and conservation guidelines for the species. Crown attributes, total height (m) and diameter at breast height ≥ 10 (cm) of trees and palms with H. flexuosa were evaluated in three permanent plots (100 m x 150 m each) randomized in 219 ha. A total of 22 botanical families hosted H. flexuosa. It was not observed
any specific preference for the species standing up itself. Nevertheless it was frequently associated to Burseraceae, Leguminosae, Sapotaceae, Annonaceae, Chrysobalanaceae, Apocynaceae, Moraceae and Myristicaceae and species with thick bark like Schweilera coriacea, Protium sp. and Licania membranaceae. The species was less frequent with taller and broader trees and tended to develop in understory light condition. Trunks or branches were the
main position for the attaching of H. flexuosa (90.83%) and a low frequency of other lianas was observed on the same host trees. These highlight the need to promote appropriate management practices for root harvesting and species conservation in order to maintain the species in open ombrophylus forest habitat.
Keywords: non-timber forest product; ecology; plant fibers; habitat preference; multiple-use forest management
RESUMO
O objetivo deste estudo foi caracterizar a estrutura florestal com H. flexuosa no Estado de Rondônia, como base para a coleta sustentável e diretrizes de conservação da espécie. Os atributos de copa, altura total (m) e o diâmetro a altura do peito ≥ 10 (cm) de árvores e palmeiras com H. flexuosa foram avaliados em três parcelas permanentes (100 m x 150 m cada), aleatorizadas em 219 ha. Um total de 22 famílias botânicas foram hospedeiras de H. flexuosa.
Não foi observada preferencia específica para desenvolver sua capacidade de sustentar a si mesma. Entretanto, a espécie esteve frequentemente associada às famílias Burseraceae, Leguminosae, Sapotaceae, Annonaceae, Chrysobalanaceae e espécies que apresentam a casca espessa, como Schweilera coriacea, Protium sp. e Licania membranaceae. A espécie ocorreu com menos frequência conforme o aumento das classes de altura e diâmetro e tendeu a
desenvolver-se sob a condição de luminosidade de sub-bosque. Os troncos ou galhos foram o principal local de fixação de H. flexuosa (90.83%) e uma baixa frequência de outras espécies de cipós foi observada nas mesmas árvores hospedeiras. Estes resultados indicam a necessidade de promover práticas de manejo apropriadas para a coleta de raízes e a conservação da espécie em habitat de Floresta Ombrófila Aberta.
Palavras-Chave: produto florestal não-madeireiro; ecologia; fibras vegetais; preferência de habitat; manejo florestal de uso-múltiplo
INTRODUCTION
The magnitude and importance of the natural resources in the Amazon tropics have not been properly appreciated to date. Misinformation and disinformation on many patterns and processes involving the diversity and distribution of Amazonian organisms are main factors that cause threats to the wilderness, biodiversity and local people. The open ombrophylus forest in Rondonia State, which encompasses a main biological repository in the
Northwestern Brazilian Amazon, covering up to 50% of upland forest ecosystems in the region, is a current area under threat due to the accelerated development since the 1980's.
Among the ecosystem functions and products that many species and their populations of tropical biodiversity may offer, vegetal fibers may constitute a real opportunity for new markets and incomes for rural communities who rely on fiber extraction and processing. It may in turn contribute to the promotion and development of sustainable non-wood-based regional economies (
Some plant fibers such as carauá (Ananas erectifolius), babaçu (Orbignya spp.), buriti (Mauritia flexuosa), carnauba (Copernicia prunifera), and piaçava (Attalea funifera) feature in the non-timber economy of many Brazilian rural communities (
the Amazon, little scientific information on major species that could high-value agricultural products is available to develop technical guidelines for sustainable management.
Multi-use approaches to tropical forests invite a great number of possibilities to generate forest goods and services (
Fearnside 2008
IBGE 2005
). In addition to that, recent studies have demonstrated the potential of these non-wood species for numerous applications (
). Nevertheless non-timber forest products (NTFPs) are still far from having clear guidelines on sustainable multiple-use management (
Beyond encouraging the extraction of the raw material itself in order to expand the use of vegetable fibers in the Amazon, it is necessary to investigate the natural species behavior (Balcázar-Vargas and van Andel 2005,
Balcázar-Vargas et al. 2011
Shanley et al. 2002
).
Monteiro et al. 2006a b Marques et al. 2007 Zambrana et al. 2007
, ,
,
). Still, in
Summers et al. 2004 Guariguata et al. 2010
,
).
) to allow improvement in the current harvesting method of non-wood species (
Leoni and Marques 2008
), for which there are few protective laws or regulations in most of the Brazilian Amazon states.
From a biophysical standpoint, it is important to know more about the mechanisms and patterns by which these species establish themselves in their natural environment. It will contribute in filling the lack of scientific information to support management recommendations regarding the conservation of species of important biological and economic value.
The main concerns regarding non-wood forest species management relates to inadequate removal practices, which can compromise the mechanisms of resilience through the continued removal of their populations. This is the case of titica vine (Heteropsis flexuosa (HBK) GS Bunting), one of the eight natural fibers used in the regional markets of natural products in Rondônia State (Diário... 2010).
Extraction of the species is regulated only in the Brazilian states of Amapá and Amazonas (
Amapá 2001 Amazonas 2008
,
); while in the other states 100% of Heteropsis spp. roots are removed, causing severe damages or death to the mother plant of the vine. Amapá State, actually, is the main producer of Heteropsis sp. in the Amazon Region, with an average monthly production of 45 tons of raw fiber-followed by the states of Pará, Amazonas and
IBGE 2005
Rondônia (
).
Morais 2008
Plowden et al. (2003)
The genus Heteropsis encompasses 17 species (
) and is described by
as a hemi-epiphytic root of natural occurrence in most of the Amazon Basin forests. In Rondônia State, at least two species of titica vine are commonly found in the main forest type formation (Heteropsis flexuosa (Kunth) GS Bunting and Heteropsis spruceana Schott). Although the economic use of the fiber may be low in contrast to agricultural
income, Heteropsis spp. roots are widely appreciated by their resistance for baskets, furniture and other manual handcrafts, therefore a source of additional income for local communities.
The objective of this study was to characterize the forest structure with H. flexuosa in open ombrophylus “terra firme” forest in Rondônia State as a basis for future sustainable harvest and conservation guidelines of the species.
Study Species
Titica vine (Heteropsis spp.), Araceae, is found in a variety of Central American (Costa Rica, Nicaragua) and particularly South American (Colombia, Venezuela, Ecuador, Guyana, Perú, Brazil) habitats. In Brazil it ranges from the Amazon to the Atlantic forest (
Morais 2008
).
The species differs from true epiphytes by germinating in soil. After germination, it commonly uses adventitious root to climb a plant, usually trees, and reach a point where it can growth and receive light and nutrients for development. The trees that stand the liana are known as host trees. The vine reaches the juvenile phase when it produces its first large leaf and a vegetative elongation, forming what is commonly called as the vine's
“mother plant”. To survive, the mother plant sends down an absorbent root, allowing a connection to the ground to transport water and nutrients (
Plowden et al. 2003
).
The vine usually grows attached to the trunk of the host tree and it is possible to find inflorescences of H. flexuosa from 7 m upwards, with most inflorescences at heights of > 10 m (
Balcázar-Vargas et al. 2011
). In addition, when it develops a root with downward growth it may be susceptible to damage by herbivores. When the meristem is damaged, a node can be observed together with the appearance of new roots which will keep on growing
Plowden et al. 2003 Morais 2008
until they reach the soil (
,
). For commercial purposes, good quality roots should show few nodes and no sign of weevil infestation (Balcázar-Vargas and van Andel 2005). In rural Brazilian communities, the absence of nodes is a main indicator for resistance and durability to the local production (
Almeida 2010
).
Study Site, Sample Design and Field Measurements
Fieldwork was conducted during the dry season, in October 2006, at the research station of Embrapa in the municipality of Machadinho do Oeste, Northwestern of Rondônia Sate (61°47′ and 63°00′ WGr, 9°19′ and 10°00′ S). The climate in this area is Am – topical monsoon according to Köppen Climatic Classification and it presents an average annual precipitation of nearly 2,400 mm, with dry season from June to August and rainy season
from December to March, where peaks of 1,300 mm are registered. The annual temperature average is 26.2°C and the relative humidity annual mean is 85%. The topography of the study area, with low hills, reaches at maximum 180 m above sea level with predominance of Yellow Latosol (Table I).
TABLE I Edafoclimatic conditions of site with naturally occurrence of H. flexuosa in Rondônia State, Brazilian Amazon (Source: Rondônia 2005,
- Localization:
Miranda et al. 2002
).
- Rainfall (mm):
2,390 - annual mean
61°47′ and 63°00′ WGr9°19′ and 10°00′ S
219 ha - primary forest
A m – tropical monsoon
- Dry season:
June to August
- Climate1:
- Rainy season:
December to March (1300mm)
- Temperature:
- Relative humidity:
26.2 °C - annual mean
85% - annual mean
- Soil:
- Altitude/Relief:
Yellow Latosol
180 m above sea level/Smooth
- Study area:
1
Köppen Climatic Classification
Open ombrophylus forest is the dominant vegetation type in the region (Projeto... 1978). The survey was carried out in undisturbed upland forest locally known as “terra firme” in three permanent vegetation plots, randomized in an area of 219 ha, each measuring 1,5 ha. Each plot was 100 m x 150 m, subdivided into 30 subplots of 20 m x 25 m (0.05 ha). Total height (m) and diameter at breast height (dbh) ≥ 10 cm of trees and palms that
hosted H. flexuosa were evaluated, accordingly to the Amazon non-timber forest products network of EMBRAPA (Kamukaia).
The life-cycle stage of the individuals in this study was categorized as adults, following
Balcázar-Vargas et al. (2011)
, as having upper height ≥ 10 m. All the adult plants of H. flexuosa were identified in the entire plot and tagged for future management test purposes. The trees of the entire forest community had already been tagged for monitoring growth (
For each H. flexuosa host tree the upper height was taken with a measuring tape and climbing techniques were used to reach the plant when needed. The dbh ≥ 10 cm of host trees was also taken. Furthermore, the crown attributes of each host tree were evaluated following
presence of adult H. flexuosa in the stem, crown, or crown and stem, and the current stage of infestation by other lianas (without liana, presence up to 25%, up to 75%, above 75%) were also considered.
Synnott (1979)
Vieira et al. 2002
).
for crown form (perfect, good, tolerable, poor, and very poor) with adaptions for crown position (dominant, co-dominant, intermediate, and suppressed). The
The botanical identification of the species in this study was done in a first moment based on the experience of a field assistant (“mateiro”), and later by comparison with exsiccates deposited at the herbarium of Embrapa Amazônia Oriental (CPATU), in Pará State.
Descriptive statistics (
Brower and Zar 1984
) with significance level at 99% were used to test differences among the assessed variables. The biodiversity measure among botanical families that host H. flexuosa was obtained following
Odum (1983)
:
1. Shannon-Weaver Diversity Index, which takes into account the number and the relative abundance of the species,
2. Uniformity of Pielou Index, which measures the uniformity defined by the distribution of the species,
An extensive inventory of the entire forest community was carried out in 2000 at the research station used in the current study (Embrapa in Machadinho do Oeste). The object of the survey was to study the phytosociology, to assess marketable species, and to monitor forest dynamics (
Vieira et al. 2002
). For comparison of the diversity and uniformity between host trees and the entire forest community the Hutchinson t-test (
Zar 1996
) was used:
The estimator for the variance (a) and the degrees of freedom (b) of the Hutchison t-test is defined by:
where N 1 and N 2 are the number of observations used to obtain the estimative of N′1 and N′2.
As for comparison of tree size (dbh) means between host trees and the entire forest community in the survey, F-test (
where
RESULTS
Callegari-Jacques 2003
is the sample mean, u is the population mean, and σ is the standard deviation of the population mean.
) was used:
Based on the survey of the three permanent vegetation plots, 403 trees were found (268.66 individuals.ha–1) distributed into 24 botanical families hosting H. flexuosa alive. Established, adults of H. flexuosa were mostly found on the trunk, branches or canopies of natural endemic families such as Lecythidaceae, Burseraceae, Leguminosae, Sapotaceae, Annonaceae, Chrysobalanaceae, Apocynaceae, Moraceae, Myristicaceae, and Myrtaceae.
Lecythidaceae and Leguminosae constituted 38.05% of all botanical families surveyed in the study area (Table II). The species belonging to the main botanical families were: Schweilera coriacea, Protium sp., Licania membranacea, Rollinia exsucca and Pouteria pachycarpa.
TABLE II Frequency of the host botanical families of H. flexuosa in open ombrophylus forest in Machadinho do Oeste, Rondônia.
#
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
...
Botanical Family
Lecythidaceae
Burseraceae
Leguminosae
Sapotaceae
Annonaceae
Chrysobalanaceae
Apocynaceae
Moraceae
Myristicaceae
Myrtaceae
Others
HTC (n)
115
61
45
34
27
27
21
18
13
9
33
403
22
FC (n)
515
217
507
241
255
94
199
223
93
71
271
2,686
24
H′
e
2.34ns
2.41ns
0.757
0.760
r(HTC/FC)
0.88
Total of individuals (n)
Botanical family (n)/tree community
HTC (%)
28.54
15.14
11.17
8.44
6.70
6.70
5.21
4.47
3.23
2.23
8.19
100
FC (%)
19.17
8.08
18.88
8.97
9.49
3.50
7.41
8.30
3.46
2.64
10.08
100
ns
– not significant by Hutchinson t- test of Shannon-Weaver estimative (p < 0.001). HTC – host tree community; FC – entire forest community; n – number of individuals.
H′– Shannon-Weaver Diversity Index; e – Pielou Equability Index; and r – Simple correlation coefficient.
The mean height of hosts was 20.40 m (SD ± 6.4 m) and only 29.78% of H. flexuosa were found above the mean height; still, in this forest type the vines were found on a wide range of heights (0.8 to 33.0 m height), with 7.2 m of mean height, independently of the maturity of plants. The canopy in this stratum performs light attenuation to the forest soil. The diameter distribution of host trees of H. flexuosa did not differ from that of the entire
forest community > 10 cm dbh (z-test0.01 = 1.96), with mean dbh of 19.55 cm ± 13.64 cm, with 53% of the adults of H. flexuosa climbing up the smallest diameter category of hosts (10.19 to 91.35 cm dbh), and in both the research sample and the forest community the size distribution showed an inverse J shape (Fig. 1).
Figure 1 Tree size (dbh) of open ombrophylus forest with H. flexuosa in Machadinho do Oeste, Rondônia. Common letters indicate means that are not significantly different from one another by Z Test (p < 0.001). Source: research data.
Over all crown categories, only 26.05% of host tree crowns were considered as perfect and good crown (complete or incomplete circle); and tolerable and poor crowns (half-crown or less than half-crown) together were over 63% amongst the host tree community, while few host trees showed crowns with any degree due natural damages (Table III).
TABLE III Crown position and form of host tree community (HTC) of H. flexuosa in open ombrophylus forest in Machadinho do Oeste, Rondônia.
Crow position
1
2
3
4
Individuals (n)
19.00 b
12.33 b
49.00 a
53.33 a
Crown form
1
2
3
4
5
Individuals (n)
8.66 b
26.00 ab
45.66 a
39.66 a
13.66 b
Common letters indicate means that are not significantly different from one another by Tukey Test (p < 0.001).
Crow position (illumination): 1 – dominant, 2 – co-dominant, 3 – intermediate and 4 – suppressed. Crown form: 1 – perfect, 2 – good, 3 – tolerable, 4 - poor, and 5 – very poor.
Regarding position of establishment, 90.82% of H. flexuosa were observed attached to trunks or branches of the hosts. H. flexuosa was less observed only in the trunk (6.15%) or only in the canopy positions (3.03%) and the majority of the vines were growing at a mean height of 10.2 m of the host trees. Only 23.57% of crowns were found dominant or co-dominant amongst the host tree community; thus, 41.44% of hosts presented suppressed
crowns; followed by intermediate crowns (36.72%). Moreover, low frequency of other lianas species (21.87%) was found in the same host trees of H. flexuosa (Table III).
Estimates of host tree family diversity and uniformity by information content are given in Table III. These estimates refer to the sample of 22 families and 403 trees and they were close to that found in the entire forest community of 24 families and 2,686 trees, corroborating that host trees of H. flexuosa are important species in the forest type studied.
DISCUSSION
The majority of H. flexuosa adults had climbed up small to medium size host trees in the forest studied, and no specific preference to specific botanical families was found across all the observed forest area. However, the vines were frequently found on the trunk, branches or canopies of natural endemic families such as Lecythidaceae, Burseraceae, Leguminosae, Sapotaceae, Annonaceae, Chrysobalanaceae, Apocynaceae, Moraceae,
Myristicaceae, and Myrtaceae. This finding is consistent with the results of other studies on host tree characteristics of Heteropsis genus along the areas of East and Central Amazon (
ombrophylus forests, mainly those trees with thick and deep longitudinal fissured barks (
Plowden et al. 2003 Morais 2008
,
) that showed Lecythidaceae, Chrysobalanaceae and Burseraceae as the main families associated with Heteropsis spp. The authors suggested that this fact may be explained as Heteropsis spp. could colonize a variety of tree species in
Morais 2008
), as occurred in Machadinho do Oeste.
Most of the adults of H. flexuosa in the area tended to be less frequent in taller host trees. In fact, adult H. flexuosa in Machadinho do Oeste occurred on a lower range of hosts between 20.0 - 95.0 cm dbh and on a higher range of trees and palms with 10.0 - 20.0 cm dbh. These results are similar to those of
Vargas et al. (2011)
and
Knab-Vispo et al. (2003)
that found Heteropsis spp. in large trees with dbh > 20 cm in “terra firme” forest in Colombia and Venezuela, respectively. However, indicates, as in
The tree size (dbh) did not influence H. flexuosa colonization in the forest community and just like in
, which observed most H. flexuosa in host trees from 10.5 m and above in Brazil; and they differ from those in
Balcázar-
, that the species has a preference for abiotic characteristics of the forest type rather than the density of preferred host tree species or sizes.
Balcázar-Vargas et al. (2011)
Crown form and position, which reflect the light conditions prevailing at a particular moment in the forest canopy (
forest of Amapá State (
Plowden et al. (2003)
, the diameter distributions showed clear inverse J shapes.
Synnott 1979
), or the tree ability to access light resources (Kainer et al. 2007), are important to explain several ecological processes including variations in fruit production and photosynthesis. The findings for host tree attributes are consistent with the results of the study on growth and management of H. flexuosa in “terra firme”
Pereira and Guedes 2008
), which have climbed host trees from 11.8 m ± 0.8 m up to 14.3 m ± 0.8 m of mean height.
Concerning the ability to compete for light, nutrients and water, the scarcity of other lianas species in the same host trees as H. flexuosa (21.87%) could indicate a minimum competitive niche, or that those other lianas were not adapted to the abiotic traits at the mean height colonized by H. flexuosa (Table III). The diversity indexes followed the expected range in the region (
has a random preference pattern for its host trees, or, as in
Fotopoulos 2006 Silva and Bentes-Gama 2008
,
), suggesting that H. flexuosa
, that the simply distribution of preferred host tree sizes could not explain its distribution and density. These results highlight the importance of identifying which habitat type is suitable to support H. flexuosa populations, to recommend future sustainable harvest guidelines for the species in open ombrophylus forest in the Amazon.
CONCLUSION
●
●
●
●
●
●
- H. flexuosa did not present specific host preferences for developing the capacity to hold itself upright, but was frequently associated with botanical families (Burseraceae, Leguminosae, Sapotaceae, Annonaceae, Chrysobalanaceae, Apocynaceae, Moraceae and Myristicaceae) and species with thick bark (Schweilera coriacea, Protium sp. and Licania membranaceae) of common frequency in the study area.
- H. flexuosa was less frequent with taller and broader trees in the study community.
- Crown form and position were important explanatory variables in this study and indicated the species preference for developing under understory strata light condition.
- Trunks or branches of host trees were the prefered position for the attaching of H. flexuosa (90.83%).
- The low frequency of other lianas (21.87%) observed on host trees indicated that they could not be adapted to the abiotic characteristics at the mean height colonized by H. flexuosa.
- These results highlight the importance of identifying which habitat type is suitable to support H. flexuosa populations, to recommend future sustainable harvest guidelines for the species in open ombrophylus forest in the Amazon.
Acknowledgements
The authors thank members and partners of the Research Network for non-wood forest products of Embrapa (Kamukaia) for all the efforts to make it possible to research this theme. This research was also supported by scholarships of EMBRAPA/Ministério da Ciência e Tecnologia-Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCT-CNPq) from 2006 to 2008.
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Received: May6, , 2011; Accepted: January10, , 2013
Correspondence to: Michelliny de Matos Bentes Gama E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013005000040
Biological Sciences
Efficiency of snake sampling methods in the Brazilian semiarid region
Paulo C.M.D. Mesquita1, Daniel C. Passos2, Sonia Z. Cechin1
1
2
Laboratório de Herpetologia, Departamento de Biologia, Universidade Federal de Santa Maria, Avenida Roraima, 1000, 97105-900 Santa Maria, RS, Brasil
Núcleo Regional de Ofiologia/NUROF, Universidade Federal do Ceará/UFC, Campus do Pici, Centro de Ciências, Bloco 905, 60455-760 Fortaleza, CE, Brasil
ABSTRACT
The choice of sampling methods is a crucial step in every field survey in herpetology. In countries where time and financial support are limited, the choice of the methods is critical. The methods used to sample snakes often lack objective criteria, and the traditional methods have apparently been more important when making the choice. Consequently researches using not-standardized methods are frequently found in
the literature. We have compared four commonly used methods for sampling snake assemblages in a semiarid area in Brazil. We compared the efficacy of each method based on the cost-benefit regarding the number of individuals and species captured, time, and financial investment. We found that pitfall traps were the less effective method in all aspects that were evaluated and it was not complementary to the other
methods in terms of abundance of species and assemblage structure. We conclude that methods can only be considered complementary if they are standardized to the objectives of the study. The use of pitfall traps in short-term surveys of the snake fauna in areas with shrubby vegetation and stony soil is not recommended.
Keywords: Caatinga; comparison; pitfall traps; Squamata; techniques
RESUMO
A escolha de métodos de amostragem é um passo crucial em todas as pesquisas de campo em herpetologia. Em países onde há frequentemente limitações de tempo e apoio financeiro a escolha dos métodos é crítica. Os métodos utilizados para amostrar as serpentes por vezes carecem de critérios objetivos e aparentemente a tradição tem sido mais importante para a escolha. Consequentemente, pesquisas usando
métodos não padronizados são frequentemente encontradas na literatura. Nós comparamos quatro métodos comumente utilizados para a amostragem de taxocenoses de serpentes em uma área semiárida no Brasil. Comparamos a eficácia de cada método baseado no custo-benefício em relação ao número de indivíduos e espécies capturadas, investimento de tempo e financeiro. Constatamos que armadilhas de "pitfall"
foi o método menos eficiente em todos os aspectos avaliados além de não ter sido complementar aos outros métodos, em relação à abundância das espécies e estrutura da assembleia. Concluímos que os métodos podem ser considerados complementares apenas se padronizados para os objetivos do estudo. O uso de armadilhas de queda em pesquisas de curto prazo da fauna de serpentes em áreas arbustivas e de solo
pedregoso não é recomendado.
Palavras-Chave: Caatinga; comparação; armadilhas pitfall; Squamata; técnicas
INTRODUCTION
The choice of sampling methods of biodiversity should be the most cost-effective as possible in attending to the requirements of the research. In this perspective, several factors must be considered: the surveyed taxon, research objectives, environment studied, time available for the development of the field work, and availability of financial resources (
al. 2010)
Aurichio and Salomão 2002 Silveira et al. 2010 Spence-Bailey et
,
,
.
Sampling methods for herpetofauna can be applied in the presence or absence of the researcher in the field (
Heyer et al. 1994 Franco et al. 2002 Willson and Dorcas 2004 Silveira et al. 2010
,
,
,
). The most used methods to capture snakes that require the presence of researchers in the field are: time-constrained search (TCS), transects and occasional encounters by the research staff (OE) (
see Martins and Oliveira 1998 Bernarde and Abe 2006 Zanella and Cechin 2006 Winck et al. 2007 Sawaya et al. 2008 Hartmann et al. 2009a b Araujo et al. 2010
often criticized because it is hard to standardize, as it is not subject to strict planning. Nevertheless, this sampling technique has been useful and used by a large number of researchers in different neotropical biomes (
,
,
,
,
,
, ,
Franco et al. 2002
). The OE method is
).
Among the methods that do not require the constant presence of the researcher in the field, the most used methods are traps and occasional encounters by thirds (OET). Among the traps, the pitfall traps (PIT) are the most used in neotropical environments, while other techniques, such as "funnel traps", "glue traps", and artificial shelters are still rarely used possibly because of limited funding (
Martins 2000 Auricchio and Salomão 2002 Sawaya et al. 2008 Silveira et al. 2010
,
,
,
).
Cechin and
The literature on the methods of sampling snakes indicates a different efficiency for each method in different environments, and treats the snake fauna included in the studies of herpetofauna in general (
et al. 2011
Cechin and Martins 2000 Ribeiro-Júnior et al. 2008
,
). Not specifying the methods for the group often leads to the snakes being subsampled (
Greenberg et al. 1994 Parris 1999 Gardner et al. 2007 Garden et al. 2007 Ribeiro-Júnior
,
,
,
,
).
Unlike studies on many animal groups, where specific methods for the studied groups are deve loped, the methods for sampling snakes usually follow traditional methods for collecting general herpetofauna, rather than targeting the group itself (
Freilich et al. 2005 Willson and Dorcas 2004
,
), despite the knowledge of the wide variety of shapes and habits that snakes may have (
Cadle and Greene 1993
Silva 2010
of the simultaneous use of several methods is the difficulty in standardizing the samples of each method, making it difficult to compare the efficiency between these and adding sampling biases to the final results on the research (
Shine and Bonnet (2000)
e.g. Vogt and Hine 1982 Jacobson 1984 Stubbs et al. 1984 Coyne 1994 Gascon 1994 Jones and Hayes-Odum 1994 McDiarmid and Altig 1999 Auricchio and Salomão 2002 Franco et al. 2002
,
,
,
,
,
,
). Studies that focus on snakes often rely on multiple methods to sample snakes in order to capture the highest number of individuals and thus obtaining a good representation of the assemblage richness (
,
,
e.g. Bernarde and Abe 2006 Sawaya et al. 2008
,
,
). The major problem
).
indicate that snakes are good model organisms for many ecological questions due to the natural history plasticity of the group. The authors showed that there is a growing trend in using snakes to test ecological theories. Nevertheless, the majority of studies on tropical snakes address species inventories and characteristics of the natural history of some species, rarely seeking the
understanding of patterns and processes at the community level (
but see Vitt and Vangilder 1983 Luiselli et al. 1998 Brown and Shine 2002 Luiselli 2006 Akani et al. 2007 Brooks et al. 2009
,
,
,
,
,
). This is an issue that deserves more attention as it is known that most species of snakes and the richest assemblages occur in tropical environments (
Vitt 1987 Greene 1997 Martins and Oliveira 1998
,
,
).
Herein we present a comparison between four of the most commonly used methods to sample snakes (TCS, OET, OE and PIT) in relation to the main limiting factors: capture rate over time; financial cost for each snake collected and environment while discussing the relationship and applicability in studies of species inventories and community ecology.
STUDY AREA
The study area was the experimental farm Vale do Curu (main entrance: 03°49'06.1''S / 39°20'14.8''W, GPS Datum: WGS 84) in Pentecoste Municipality, State of Ceará, Northeast Brazil. It is a semiarid region with an annual mean temperature of 26.8°C, 73% relative humidity, and 723.3 mm of rainfall with a remarkable seasonality in the rainfall regime with a wet period from February to July and a dry
period for the rest of the year (
Barros et al. 2002 Leão et al. 2004
,
).
Between January 2008 and June 2010, we made monthly visits to the study area, in which each visit lasted approximately 90 hours. During the first six months we performed a pilot study to delineate the sampling methods and to train a local worker.
We considered each method as follows: time-constrained search (TCS), occasional encounters by the staff (OE), occasional encounters by third (OET) and pitfall traps with drift-fences (PIT).
TCS
Consisted of the search for snakes on foot or by car (maximum speed of 25 km/h) carried out by three people at a slow pace in trails and roads within the area and surroundings. Each month we carried out a total of 24 hours of TCS, with 22 hours of TCS by foot and two hours by car. For logistical issues, we carried out 16 hours of diurnal TCS and eight hours of nocturnal TCS each month. For this
method the initial investment was approximately $170.00 (USD) (flashlight + hook for handling snakes + sampling bags + batteries). We did not include the expenses for the car fuel because the search by car occurred exclusively during the traveling from and to the original city in a vicinal road and a road within the study area.
OE
Consisted of the encounter of snakes during the field campaign when TCS was not being applied. We estimate the time that the staff was subject to OE in 46 hours per month (90 hours total, less 24 hours of TCS = 66 hours; less 20 hours of estimated period of inactivity of the researchers). There was no specific cost for the application of this method.
PIT
Consisted of three sets of traps installed in three different vegetation types (arboreal Caatinga, shrub Caatinga and river floodplain). Each set of traps was composed of 20 plastic containers (50 liters) buried in the soil in radial conformation interconnected by five meter long and 60 cm high drift fences (
Mengak and Guynn-Jr 1987
). A total of 60 containers were installed.
Cechin and Martins (2000)
Within each bucket, we placed litter and a sheet of Styrofoam to serve as shelter against the weather conditions for the captured animals. We chose not to follow the suggestion of
, of a linear conformation of the traps and 100 L buckets, because the stony soil and the shrubby vegetation make this alternative unfeasible. Each container was kept open for 60 hours. Considering the
three sets of traps (60 buckets), we totalized 3,600 hours of PIT per month. The traps were checked every 20 hours during the field campaigns. For this method, the initial investment was approximately $1,260.00 (USD) (buckets + drift-fences (including replacements) + payment for local workers that helped digging the soil for the installation of the traps). We did not include the costs of durable
equipment that was easily obtained in rural areas such as shovels, crowbars, and pickaxes because most of them were provided by the local workers.
OET
To fit the legal requirements and to standardize the effort of OET, a single local worker was trained during the first six months of the pilot study for the techniques of handling and capturing snakes (training time consisted of 72 hours) and we requested him to capture only the snakes he encountered occasionally during his daily activities. The native collector was not paid to avoid the active sampling of
animals. As snake encounters by this method are merely incidental, we considered the effort of OET as 540 monthly hours (720 h reduced by 180 h of the estimated inactivity of the collector). The cost of OET was nearly $20.00 (USD) (a handcrafted hook + plastic containers to store the snakes).
Expenses with accommodation and transport for the study area were not included because these costs are not inherent to any particular method.
ANALYSIS
We compared the observed and estimated species richness (Sobs and Sest, respectively) using the first order Jackniffe estimator for each method using the software EstimateS 8.0 (
Colwell 2005, USA
). For a better visual comparison, we have built sample-based rarefaction curves for the richness estimated for each method and for all the methods together (
Gotelli and Colwell 2001
).
We measured the snout-vent length (SVL) of captured snakes and compared it by each method through the analysis of variance (ANOVA) and post hoc Tukey's pairwise test to verify the differences in the size classes of the snakes captured. All analyses were performed with an alpha set at 0.05.
The capture rate of the snakes was evaluated as a function of the monetary investment (in USD invested/snake captured) and the time spent (in hours of effort/snake captured). We also observed the presence or absence of untargeted species captured by each method.
In order to access the suitability of using the combination of these methods in ecological studies, we compared the structures of the assemblage (composition, richness, and abundance) indicated by each method by building diagrams of the distribution of abundances for each method and by a cluster analysis UPGMA (Unweighted Pair Group method with Mean Arithmetic) using Euclidean distances as a
measure of similarity between the structures resulted from each method.
RESULTS
NUMBER OF INDIVIDUALS AND SPECIES RICHNESS
After 24 months of samplings, we recorded 620 snakes through the four evaluated methods. TCS responded for 55.8% (n = 346) of the captures, followed by OET with 37.1% (n = 230), PIT (n = 23), and OE (n = 21) contributed with less than 4% each (Table I).
TABLE I Number of individuals of the species captured by each method at Pentecoste municipality, Northeast Brazil.
Species
TCS OET OE PIT
Apostolepis cearensis Gomes, 1915
0
2 0 0
Boa constrictor Linnaeus, 1758
4 13 1 0
Boiruna sertaneja Zaher, 1996
5
2 0 0
Crotalus durissus (Linnaeus, 1758)
1
0 0 0
Epicrates assisi Machado, 1945
6
4 0 0
Leptodeira annulata (Linnaeus, 1758)
16 15 1 1
Leptophis ahaetulla (Linnaeus, 1758)
37 13 0 1
Liophis dilepis (Cope, 1862)
10 7 2 0
Liophis mossoroensis Hoge & Lima-Verde, 1972
0
1 0 0
Liophis poecilogyrus Wied, 1825
17 29 1 0
Liophis viridis Günther 1862
7
9 3 14
Mastigodryas bifossatus (Raddi, 1820)
0
1 0 0
Micrurus ibiboboca (Merrem, 1820)
8 18 1 2
Oxybelis aeneus (Wagler, 1824)
87 36 4 0
Oxyrhopus trigeminus Duméril, Bibron & Duméril 1854 28 16 3 2
Philodryas nattereri Steindachner, 1870
44 28 3 1
Philodryas olfersii (Lichtenstein, 1823)
52 9 0 2
Pseudoboa nigra (Duméril, Bibron & Duméril, 1854)
Psomophis joberti (Sauvage, 1884)
Tantilla melanocephala (Linnaeus, 1758)
Thamnodynastes cf. strigilis (Mikan, 1828)
Xenodon merremii (Wagler, 1824)
15
5
1
1
2
9
7
5
0
6
2
0
0
0
0
0
0
0
0
0
All methods together captured 22 species of snakes. OET was the method that captured the highest number of species (n = 20), followed by TCS (n = 19), OE (n = 10) and PIT (n = 7).
The first order Jackniffe estimator of richness predicted an assemblage composed of 22 (Sobs) to 26 species (Sest = 25.83) based on all the methods together. The same estimator predicts a richness of 23 species for OET (estimated value = 22.88), 22 species for TCS (estimated value = 21.88), 18 species for OE (estimated value = 17.67) and 11 species for PIT (estimated value = 10.83).
Observing the rarefaction curves, the methods of OE and PIT were far from reaching the plateau, in spite of the relatively high effort (especially for PIT). TCS and OET showed similar patterns in their curves, after a fast growth in the early samples both maintained a slow and steady growth until the end of the study, while OE and PIT showed a slow and continuous growth since the first samples (Figure
1).
Figure 1 Sample-based rarefaction curves for all sampling methods. Confidence interval is shown only for all methods together. TCS: time-constrained survey, OET: occasional encounters by thirds, PIT: pitfall traps, OE: occasional encounters by the field staff.
DIFFERENCES IN THE SIZES OF SNAKES CAPTURED
TCS captured snakes between 98 and 1,210 mm SVL (mean = 623.22 ± SD 243.27), OET captured snakes between 125 and 2,300 mm (mean = 575.40 ± SD 313.90), OE between 169 and 1,250 mm (mean = 531.05 ± SD 267.14) and PIT captured individuals between 186 and 580 mm (mean = 322.35 ± SD 83.41).
The results from the ANOVA (F = 8.19; p = 0.0001) and the Tukey's pair-wise comparison indi cated that only snakes captured by PIT presented the shorter amplitude of sizes and were significantly smaller than those captured by TCS (q = 6.76; p < 0.01) and OET (q = 5.60; p < 0.01) (Figure 2).
Figure 2 Non-outlier relation between size and sampling method for snakes in the study area. TCS: time-constrained survey, OET: occasional encounters by thirds, PIT: pitfall traps, OE: occasional encounters by the field staff.
FINANCIAL COSTS
One snake was captured for every $ 0.49 USD invested in TCS, $ 0.09 USD in OET, no specific cost was estimated for OE, and one snake for every $ 54.78 USD invested in PIT.
TIME COSTS
TCS was the method with the highest rate of capture at nearly 0.6 snakes per hour, or approximately one snake every 1 hour and 40 minutes of effort. The methods of OET and OE showed similar encounter rates of 0.018 snake/hour (OET) and 0.019 snake/hour (OE), or approximately one snake every 55 hours and 30 minutes of effort in OET and one snake every 52 hours and 40 minutes in OE. PIT
presented the lowest capture rate with 0.0053 snakes/hour (considering one set of traps, i.e. 20 buckets) or one snake every 187 hours and 40 minutes. If we consider the three sets of traps, the success rate falls to 0.0018 snakes/hour and considering each bucket individually the capture rate is 0.00026 snakes/hour, or one snake for every 3,756 hours and 30 minutes of effort in PIT.
ASSEMBLAGE STRUCTURE
Three species were registered exclusively by OET (Apostolepis cearensis [n = 2], Liophis mossoroensis [n = 1] and Mastigodryas bifossatus [n = 1]), two only by TCS (Crotalus durissus [n=1] and Thamnodynastes cf. strigilis [n=1]) and no exclusive species were captured by OE or PIT (Figure 3).
Figure 3 Diagrams of distributions of abundances of the species registered by each method. Ace (Apostolepis cearensis), Bco (Boa constrictor), Bse (Boiruna sertaneja), Cdu (Crotalus durissus) Eas (Epicrates assisi), Lah (Leptophis ahaetulla), Lan (Leptodeira annulata), Ldi (Liophis dilepis), Lmo (Liophis mossoroensis), Lpo (Liophis poecilogyrus), Lvi (Liophis
viridis), Mbi (Mastigodryas bifossatus), Mib (Micrurus ibiboboca), Oan (Oxybelis aeneus), Otr (Oxyrhopus trigeminus), Pjo (Psomophis joberti), Pna (Philodryas nattereri), Pni (Pseudoboa nigra), Pol (Philodryas olfersii), Tme (Tantilla melanocephala), Tst (Thamnodynastes cf. strigilis), Xme (Xenodon merremii).
The diagrams of distribution of abundances represent the importance of each component of the assemblage indicated by each method; we observed that PIT is the only method with a strong trend toward a single species (Figure 3).
Regarding the assemblage structure (species composition, richness, and abundance) that is indicated by each method, the UPGMA formed a group including TCS, OET, and OE, whereas PIT did not cluster (Figure 4).
Figure 4 UPGMA dendrogram representing the similarities among the assemblage structure obtained by each sampling methods (r = 0.99).
DISCUSSION
TCS and OET were the most efficient methods in terms of representation of the richness of the area. The rarefaction curves for those methods were within the range of 95% of the sample-based rarefaction curves of all methods together. Although the rarefaction curves of TCS and OET nearly reached the plateau at the end of the samplings, we observed that the sample-based curves did not reach a steady
plateau for any of the methods, which was expected as it is virtually impossible to sample all the species from any biological community (
Gotelli and Colwell 2001
).
TCS was the method with the highest rate of captures, and it resulted in a good representation of the richness as its estimated richness was the same as the total number of species known for the area observed, including all the methods. Overall, the financial costs of TCS are low, but the results depend on the experience of the researchers involved in the work (
al. 2007
Silveira et al. 2010 Ribeiro-Júnior et al. 2008 Garden et
,
,
). TCS has the disadvantage of being very physically demanding and requires prior training and good fitness of the field staff. It has the advantage of being the most efficient method regarding the capture rate of snakes over time, presenting a relatively low maintenance cost (basically the replacement of batteries) and a reasonable rate of catch per monetary unit invested ($ 0.49 USD per snake).
Another advantage of TCS is that there is no mortality or capture of untargeted species. In addition to that, because it is a method specifically directed to the capture of snakes, it allows field observations of the activity and behavior of the snakes, providing valuable information on ecology and natural history of many species. Our TCS results support the option made by
exclusively in their study on ecological patterns in a tropical snake assemblage.
Brown and Shine (2002)
that used TCS
OET was a good representative of the snake assemblage in the area; the estimated richness by this method was only one species higher than what is known to the area. Although there is a slow addition of individuals per hour, this addition is constant throughout the study period, which increases the probability of success of the method. The three species captured exclusively by this method clearly show
the importance of OET in studies with snakes in semi-arid environments. OET has a disadvantage because it may be biased by the day to day work areas and schedule of the local collector, for example if the collector works in crops with seasonal harvest and, therefore, information about the seasonal activity of the captured snakes should be discarded because of the periods that the collector stays longer
in field.
Sometimes it is argued that one cannot rely on the information given by locals, but this problem can be mitigated by decreasing the number of collectors and training them properly, thus valuing the quality of the information rather than the quantity (
Akani et al. 2007
rarely the authority responsible for environmental permits authorizes its use, because it allegedly encourages the killing of snakes (
Dorcas and Willson 2009
). Another disadvantage of this method is the time required for training the collectors. OET is a method recognized as controversial and
). During our study, no snakes were killed by the trained collector, although he did occasionally collect some animals that were roadkill or killed by other locals.
OET is advantageous because it requires almost no maintenance and - at least initially - local people from the study area tend to see more animals than recently arrived researchers. It also enables the researcher to collect information throughout the study period, even without the presence of researchers in the field (
local helpers in field research that requires snake sampling, as long as they are not paid and receive previous training in all aspects of this method (e.g. human and animal safety).
OE was not efficient regarding the number of individuals in this study because of the relatively short time of possible encounters by OE. The method presents a slow rate of addition of new species, but the capture rate in hours of application is very similar to that of OET.
Because, similarly to OE, every snake encounter by OET is merely incidental and it should obviously have the same probability of occurrence in the absence of observer bias. Another advantage of OE is that there are no specific financial costs involved.
Zanella and Cechin (2006)
FitzGibbon and Jones 2006 Franco et al. 2002
,
). Those are important reasons for the participation of
intuitively named this method as "Occasional Encounters by Thirds", which is consistent with our results.
Gibbons and Semlitsch (1982)
PIT was the least efficient method in all aspects evaluated in this study. It is the least representative of the observed richness, did not capture any exclusive species, has the lowest capture rate, and the highest financial investment by snake captured (more than 100 times higher than TCS).
suggest that PIT presents problems due to the variable efficiency in sampling different
species. From 23 PIT-trapped individuals, 60.86% (n=14) belonged to a single small-sized species (Liophis viridis) and it was the only method with a significant difference in the size class of the sampled snakes, tending to catch smaller snakes and in a smaller size range. There is evidence that the size of the containers influences the capture rate and size of the snakes caught, so the constraint observed in
Cechin and Martins 2000 Todd et al. 2007
our studies may be due to the size of the buckets we used (
,
). However, with the impossibility of using larger containers in stony soils as those of many areas of the Brazilian Caatinga, this method is not advantageous in this type of environment. Considering the financial expenses of PIT, it is recommended in studies where it is expected that a large number of terrestrial,
Enge 2001 Rueda-Almonacid et al. 2006
fossorial, and cryptozoic snakes, in long-term studies and at sites that are relatively plain and with sandy soils (
,
).
Among the methods evaluated, PIT is also the method that requires the greatest time and financial investment in maintenance due to damage to the drift-fences caused by intense solar radiation, low humidity, and animal or human action. Furthermore, the installation period is physically demanding because of the stony ground and high temperatures, requiring a large number of field-assistants (in this
study, ten assistants) resulting in higher financial expenses.
Ribeiro-Júnior et al. (2008)
Ribeiro-Júnior et al. 2011
containers than those used in our study (
Garden et al. (2007)
As found by
has not been quantified.
and
Enge (2001)
suggest that PIT is a cost-effective method for sampling lizards, amphibians, and small mammals. In this study, we demonstrate that the same cannot be said about snakes in the semi-arid regions in Brazil. This is consistent with the results in other areas that also captured a low number of snakes, even using larger
).
, the number of untargeted groups, such as invertebrates, frogs, lizards, and small mammals, captured by PIT is high, making the method advantageous for general faunistic inventories, but not to studies that target snakes specifically. Moreover, it was the only method in which the mortality of the untargeted species was observed even though the mortality rate
Cechin and Martins 2000
It is difficult to compare the eficiency of PIT in different biomes, because, each study use slightly different delineation to apply this method. Nevertheless the capture rate of snakes per container per month was much lower than the rates for other regions of Brazil, even considering studies that used smaller containers (
). Although pragmatically, this relationship is not the most
appropriate because containers connected by drift-fences should not be considered as independent units, but we chose to discuss this in order to be able to compare the available information. The most obvious advantages of PIT is that, after installation and proper maintenance, the sampling is passive and does not require a constant presence in the field for long periods, it is not biased by the researcher
experience, and it allows some extent of quantification of prey availability.
Our results are consistent with
Silveira et al. (2010) Garden et al. (2007)
,
, and
Enge (2001)
that PIT sample small and medium-sized animals or juveniles of larger animals, with terrestrial or fossorial habits. While active methods are usually more general, and record a wide variety of species.
e.g. Zanella and Cechin 2006 Sawaya et al. 2008
for snakes in this semi-arid area, but it has been demonstrated for other tropical biomes (
,
Campbell and Christman (1982)
suggest that PIT capture animals rarely found by visual encounters, which was not corroborated
).
We showed that TCS, OET, and OE are grouped and could be compared regarding the structure of the assemblage while the results obtained by PIT are inappropriate for comparisons in studies that are based on abundances or assemblage structure at least in semiarid regions. PIT resulted in a very different assemblage structure compared to the other methods due to the strong tendency to capture only a
small group of species, being unable to capture even some of the most abundant species.
The study area should also be considered for the methods to be cost-effective. Our results indicate that in semi-arid areas with stony soil, the most cost-effective set of sampling methods tested are TCS, OET, and OE. We suggest the non-application of PIT under the given conditions, and research of tropical areas should also consider other sampling methods not tested here, such as box and funnel traps
that have been successfully used in many extratropical environments (
Kjoss and Litvaitis 2001
).
The choice of methods should be made carefully in relation to the study objectives and methods should be adapted to the reality of each location (
Silveira et al. 2010
). When the objective is to identify the species richness in a particular area or to investigate aspects of natural history and the autoecology of certain species of snakes it is accepted and recommended to use as many methods as possible, because
Magurran 2004 Prudente et al. 2010 Silva 2010
some methods may capture species that are difficult to be registered by other methods and it may increase the number of individuals sampled from the target group. In these cases, the use of multiple methods can be considered as complementary (
,
,
). However, if the objective of the study is related to patterns of population dynamics, abundance, or assemblage
structure, we suggest great care in selecting the sampling methods. In these cases, some methods cannot be regarded as complementary, as they may add overlapping information that may distort rather than reveal natural patterns. This approach was already considered in research on more easily sampled taxa. In those studies the sampling delineation is directed to obtain very consistent data, even
see Heyer et al. 1994 Huang and Hou 2004 Gardner et al. 2007b Dixo and Martins 2008 Siqueira et al. 2009
reducing (and often excluding) the number of records of some species that could be captured by some different methods (
,
,
,
,
).
In conclusion, although it is one of the most efficient methods to sample other groups of herpetofauna, and therefore extremely useful in faunistic inventories, PIT was ineffective in catching snakes in the Brazilian semiarid region. Considering the difficult to sample snakes in the field (
Reinert 1993
), the limited time and funding, we suggest that the time and funds usually invested in PIT should be invested
Ribeiro-Júnior et al. 2011
in other methods that are more cost-effective and that are truly complementary, especially in shrubby environments with stony soils. Furthermore, we suggest that when there are the simultaneous uses of different methods, the sampling effort should be standardized (in hours of effort, for example) to allow comparisons between different methods and studies (
al. (2010)
that the methods should be used, focusing neither on convenience nor tradition, but rather fundamentally to obtain quality data that provide results that fulfill the objectives of the study (
Measey 2006 Garden et al. 2007 Ribeiro-Junior et al. 2008 Dorcas and Willson 2009 Spence-Bailey et al. 2010
,
,
,
,
). We agree with
Silveira et
).
Acknowledgements
We would like to thank Dra. Teresa Cristina Sauer Ávila-Pires, Dra. Noeli Zanella, Dra. Maria da Graça Salomão, and Dr. Everton Behr for their valuable suggestions on the first versions of the manuscript. Dra. Diva Maria Borges-Nojosa and the staff of the Núcleo Regional de Ofiologia/Universidade Federal do Ceará for material and logistic support. ICMbio for the license code: SISBIO/18596-1.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support, and SZC thanks Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the research funding (protocol 303359/2009-9).
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Received: May28, , 2012; Accepted: February27, , 2013
Correspondence to: Paulo Cesar Mattos Dourado Mesquita E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013000300016
Biological Sciences
Pouteria ramiflora extract inhibits salivary amylolytic activity and decreases glycemic level in mice
NEIRE M. DE GOUVEIA1, CIBELE L. DE ALBUQUERQUE1, LAILA S. ESPINDOLA2, FOUED S. ESPINDOLA1
1
2
Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, Laboratório de Bioquímica e Biologia Molecular, Campus Umuarama, Bloco 2E, Sala 235, 38400-902 Uberlândia, MG, Brasil
Universidade de Brasília, Faculdade de Ciências da Saúde, Laboratório de Farmacognosia, Campus Universitário Darcy Ribeiro, Asa Norte, 70910-900 Brasilia, DF, Brasil
ABSTRACT
In this study, extracts of plant species from the Cerrado biome were assessed in order to find potential inhibitors of human salivary alpha-amylase. The plants were collected and extracts were obtained from leaves, bark, and roots. We
performed a preliminary phytochemical analysis and a screening for salivar alpha-amylase inhibitory activity. Only three botanical families (Sapotaceae, Sapindaceae and Flacourtiaceae) and 16 extracts showed a substantial inhibition (>75%)
of alpha-amylase. The ethanolic extracts of Pouteria ramiflora obtained from stem barks and root barks decreased amylolytic activity above 95% at a final concentration of 20 µg/mL. Thus, adult male Swiss mice were treated orally with P.
ramiflora in acute toxicity and glycemic control studies. Daily administration with 25, 50 and 100 mg/kg of aqueous extract of P. ramiflora for eight days can reduce significantly body weight and blood glucose level in mice. These data
suggest that the crude polar extract of P. ramiflora decreases salivary amylolytic activity while lowering the blood levels of glucose.
Keywords: alpha-amylase inhibition; hypoglycemia; Pouteria ramiflora; Sapotaceae
RESUMO
Neste estudo, extratos de espécies de plantas do bioma Cerrado foram avaliados em busca de um potencial inibidor da enzima alfa-amilase salivar humana. As plantas foram coletadas e os extratos obtidos das folhas, da casca e da raiz. Nós
realizamos uma análise fitoquímica preliminar e uma triagem destas plantas utilizando um ensaio de inibição da alfa-amilase salivar. Inicialmente, somente três famílias botânicas (Sapotaceae, Sapindaceae e Flacourtiaceae) e 16 extratos
apresentaram inibição da alfa-amilase maior que 75% sendo que os extratos etanólicos da casca do caule e da raiz de Pouteria ramiflora tiveram mais de 95% de inibição para a concentração final de 20 µg/mL. Assim, camundongos Swiss
adultos machos foram oralmente tratados com extrato aquoso de P. ramiflora para avaliar a toxicidade aguda e o controle glicêmico. A administração diária do extrato de P. ramiflora nas doses de 25, 50 e 100 mg/kg, por oito dias, foi capaz
de reduzir o peso corporal e o nível glicêmico nos animais. Esses dados sugerem que o extrato polar de P. ramiflora diminui a atividade amilolítica da saliva, além disso, reduz os níveis de glicose sanguínea.
Palavras-Chave: inibição da alfa-amilase; hipoglicemiante; Pouteria ramiflora; Sapotaceae
INTRODUCTION
Recently, several studies of alpha-amylase inhibition have been conducted with the aim of discovering new potential drugs capable of reducing postprandial hyperglycemia that could be used in the treatment of diabetes mellitus type 2
and obesity. Inhibitors of this enzyme reduce postprandial hyperglycemia by delaying carbohydrate digestion and decrease intestinal glucose absorption (
Ali et al. 2006 Funke and Melzig 2006 Gad et al. 2006 Kim et al. 2005 Shu et al. 2009
,
,
,
,
). Plants
Marles and Farnsworth 1994 Oliveira et al. 2005
continue to play an important role in the treatment of diabetes, particularly in developing countries (
(de Mesquita et al. 2009, Flausino et al. 2009,
,
). Medicinal plants from the Cerrado biome has been previously investigated by bioprospection
Mesquita et al. 2005 Napolitano et al. 2005 Rodrigues et al. 2006
,
,
). However, few studies have investigated the biological activity of such medicinal plants extracts or their isolated components on the
Silva et al. 2007 2009
activity of alpha-amylase (
,
).
Inhibition studies of alpha-amylase activity by natural products have been performed in vitro using human pancreatic alpha-amylase (HPA) (
Ali et al. 2006 Conforti et al. 2005 Gad et al. 2006
,
,
). Alternatively, as human salivary alpha-amylase
Brayer et al. 1995 Ramasubbu et al. 2003
(α-1,4-D-glucan-4-glucanohydrolases, EC 3.2.1.1, HSA) is highly homologous to HPA, and both proteins adopt very similar structures (
,
), the use of HSA as an experimental model for in vitro
inhibition studies with natural products has the advantage of saliva being ease and not invasive to collect. Previous studies have shown similar in vitro inhibition of HPA and HSA activities (
Kim et al. 2005 McDougall et al. 2005
,
).
The aims of this study are (1) to screen selected plants extracts from the Brazilian Cerrado for possible HSA inhibitory activity, and (2) to carry out a biological assay in mice to investigate any effects on glycemic and toxicity of the
stem bark extract from Pouteria ramiflora (Mart.) Radlk.
Collection and Preparation of the Plants for Screening
The plants were collected in the Cerrado biome, in the outskirts of Brasilia, Distrito Federal, Brazil, and were identified at the Department of Vegetable Anatomy, Institute of Biology, Universidade de Brasília (UnB). The voucher
specimens were deposited in the Herbarium of UnB. Extracts were obtained from leaves, wood bark, stem bark and roots of the selected plants. Air-dried and powdered plant material were successively extracted with hexane and
ethanol 95% by maceration. Crude extracts were obtained after evaporation of the solvents under reduced pressure at 40°C. Extracts were dissolved in dimethylsulfoxide (DMSO) (Sigma-Aldrich, Saint Louis, USA) at a concentration
of 10 mg/mL. Stock solutions of each extract were prepared before experiments. Samples were stored at -20°C until analysis. Plant species investigated, part used, solvent and voucher numbers are presented in Table I.
TABLE I Family, plant species investigated, part used/solvent and voucher number.
Family
Apocynaceae
Asteraceae
Bignoniaceae
Flacourtiaceae
Sapindaceae
Sapotaceae
Species
Aspidosperma macrocarpa Woodson
Himatantus obovatus (M. Arg) Woodson
Hancornia pubescens (Nees & Mart.) M. Arg.
Piptocarpha rotundifolia (Less.) Baker
Tabebuia caraiba (Silva Manso) Benth. & Hook.f ex S. Moore
Anemopaegma arvense (Vell.) Stellf.
Cybistax antisyphilitica (Mart.) Mart.
Casearia sylvestris SW. var. lingua (Camb.) Eichl.
Piptocarpha macropoda (DC.) Baker
Cupania vernalis Cambess
Matayba guianensis Aubl.
Serjania lethalis A.St.Hil.
Magonia pubescens A.St.Hill.
Chrysophyllum soboliferum Rizzini
Pouteria ramiflora (Mart.) Radlk
Pouteria gardnerii (Mart. & Miq.) Baehni
Pouteria torta Radlk.
Part used/sovent
SW (E, H); SB (E, H); RW (E); RB (E, H); L (E)
RB (H);L (H)
L (H); RW (H); RB (H)
RW (E, H); L (E, H); RB (E, H); SB (H); SW (E)
RB (E); SB (H); L (E, H); SW (E, H); SB (E); RB (H); RW (E)
L (E, H); S (H); R (E, H); FS (E, H); S (E)
WS (E, H); L (E, H); SW (E, H); SB (E, H);
SW (E, H); RB (E, H); SB (E, H); RW (H); R (E); L (E, H)
SB (H); L (H)
SB (E, H); RB (E, H); RW (H); SW (E, H); L (E, H); R (E)
SB (E, H);SW (E, H); RB (E, H)
L (E, H); SW (E, H); SB (E, H); RB (E, H)
L (H); SB (H)
L (E, H)
L (E, H);RB (E, H); SW (E, H); RW (E); SB (E, H)
L (E, H)
L (E, H)
Voucher
(UB) 3692
(UB) 3678
(UB) 3677
(UB) 3676
(UB) 3701
(UB) 3691
(UB) 3696
(UB) 3693
(UB) 3708
(UB) 3695
(UB) 3697
(UB) 3702
(UB) 3733
(UB) 3671
(UB) 3672)
(UB) 3674
Part used : L, leaf; SW, stem wood; SB, stem bark; RW, root wood; RB, root bark; R, root; FS, fruit+seed; S, stem (bark+wood); WS, wood+stem bark. Extraction solvent: H, hexane; E, ethanol.
Collection and Preparation of the Pouteria ramiflora Extract for in vivo Assay
Stem barks of P. ramiflora were collected in the Cerrado biome in the outskirts of Uberlândia, Minas Gerais, Brazil. The plant was botanically identified at the Institute of Biology, Universidade Federal de Uberlândia, and placed in the
herbarium of the same institution (voucher specimen, HUFU, 45,535). The vegetables were dried at 37°C and then grounded in an electric mill. Aqueous solution of P. ramiflora (PrSBAE) was used for the extraction process that lasted
eight days. After extraction, the solution was filtered and lyophilized. Extract was then solubilized in water for in vitro studies.
Preliminary TLC and HPLC Phytochemical Analysis
The TLC analysis was carried out using 60G silica gel plates (10 × 10 cm; 0.25 mm; Aldrich), eluted with BAW (n-butanol:acetic acid:water, 4:1:5, upper phase) or CHCl3:MeOH (8:2) and stained with NP/PEG (natural products polyethylene glycol reagent) and vanillin-sulphuric acid reagent in separated plates. All extracts were also analyzed in analytical HPLC (Shimadzu, Kyoto, Japan) with SupelcosilTM LC-18 column (250 × 4.6 mm; Supelco) and PDA
detector. The mobile phase consisted of 0.1% aqueous acetic acid and methanol with a flow rate of 1 mL/min. The mobile phase composition began with 10% methanol, followed by linear increase of 66% in 32 min and returned to the
initial condition in 35 min for the next run.
Screening for Alpha-Amylase Inhibitiory Activity
Alpha-amylase inhibition was performed using a commercial kit (Kit Analisa - Belo Horizonte, Minas Gerais, Brazil) based on starch-iodine color changes (
Caraway 1959
). Saliva was collected from nine individuals using a modified
Navazesh 1993
method (
). The pooled saliva was centrifuged at 14,000xg for 15 min at 4°C. The supernatant was diluted (500×) in phosphate buffered saline, pH 7.4. Extracts (10 mg/mL) were pre-incubated with the diluted saliva
supernatant for 4 min 35 sec at 37°C. The final concentration of the plant extract in the incubation was 200 µg/mL. The reaction was started by addition of 10 µL of the supernatant solution in 250 µL of starch solution. The tubes were
incubated at 37°C for 7 min 30 sec. The reaction was stopped by the addition of iodine. This assay was also carried out with acarbose (10 mg/mL) (provided by EMS S/A, São Paulo, Brazil) - a drug therapeutically used as alphaglucosidase and alpha-amylase inhibitor (positive control). The final concentration of acarbose was 200 µg/mL. DMSO was used as a negative control (10 µL). The final concentration of the negative control in the incubation was 0.022
µg/mL.
The inhibition of the activity of HSA was expressed as a percentage in which alpha-amylase activity of the non-incubated saliva supernatant was considered 100%. Extracts that presented the highest inhibition rates were selected for a
subsequent inhibitory assay at lower concentrations. For the secondary assay, the same method described before was used with increasing final concentration of the extract (20, 50 and 100 µg/mL) incubated with the diluted saliva
supernatant.
Animals
Healthy adult male Swiss mice aged 7-8 weeks (weigh: 25-30g) were used for this study. Animals were housed under standard conditions (25°C, 12h light and 12h dark cycle) and fed with rat chow and water ad libitum. All procedures
for handling, use and euthanasia of these animals followed carefully the resolutions proposed by the Brazilian Society of Science in Laboratory Animals and were approved by the Ethics Committee in Animal Research of the
Universidade Federal de Uberlândia, Brazil (CEUA/UFU 060/10).
Acute Toxicity Study
Brito 1994
Experiments were carried out as previously described (
). In the acute toxicity study, animals were treated orally and by intraperitoneal injection with PrSBAE extract. Mice were given PrSBAE at doses of 50, 500 and 5,000 mg/
kg daily for a period of 18 days (n=5). Animals were observed for 2h continuously and then hourly for 8h, and finally after every 24h up to 18 days for any physical signs of toxicity such as writhing, hypnosis, dyspnea or mortality.
Effects of the PrSBAE Extract on the Levels of Blood Glycaemia
Twenty mice were divided randomly in four groups and treated orally for eight days in the following manner: Group 1 (water, control), 2 (25 mg/kg of PrSBAE), 3 (50 mg/kg of PrSBAE), and 4 (100 mg/kg of PrSBAE). Treatments
were administrated in the same time, once a day, in alternate days. After the eighth day, mice were fasted overnight (for 8h) and then were weighed and sacrificed. Glycemic level in blood serum was measured by a colorimetric
enzymatic method (Labtest, Minas Gerais, Brazil).
Statistical comparisons were made using one-way ANOVA followed by the Tukey test. p-values lower than 0.05 were considered statistically significant.
One hundred and nine crude extracts were tested, of which 47 were ethanolic and 52 hexanic. In the initial screening, all extracts were prepared as hexanic and ethanolic crude extracts from different parts of 17 plant species belonging
to six botanical families (Flacourtiaceae, Sapindaceae, Sapotaceae, Bignoniaceae, Asteraceae and Apocynaceae) (Table I). The percentages of inhibition of the HSA by the most effective extracts are shown in Table II. Only 16 extracts
from six species belonging to the botanical families, Sapotaceae, Flacourtiaceae and Sapindaceae, were found to have an inhibitory effect on HSA activity (greater than 75%), including four ethanolic extracts obtained from root bark,
root wood and stem bark of P. ramiflora. The extracts were more effective to inhibit the activity of alpha-amylase in vitro than acarbose in the same concentration (200 µg/mL). We observed that ethanolic extracts presented more
effective HSA inhibition activity than hexanic extracts. This result was also reported in other studies of HSA inhibition activity with plant extracts of different families from the Cerrado biome (de Souza et al. 2012,
TABLE II Plant species investigated, part used/solvent, yields in terms of dry starting material and percentage of inhibitory activity of crude extracts on human salivary alpha-amylase.
Species
Cupania vernalis Cambess
Matayba guianensis Aubl.
Serjania lethalis A.St.Hil.
Casearia sylvestris SW. var. lingua (Camb.) Eichl.
Part used/sovent
L (H)
SW (E)
RB (E)
SB (E)
R (E)
SB (E)
RB (E)
SB (H)
SB (E)
SW (E)
RB (H)
RB (E)
RB (H)
RB (E)
SB (E)
SW (E)
L (H)
Yield (%)
7.82
3.78
6.15
0.34
3.20
9.74
0.46
2.56
2.25
4.23
14.97
11.02
-
Inhibition (%)
57±50.1
63±55.3
94±5.6
82±16.0
81±21.9
79±0.0
86±9.1
92±6.8
98±1.5
92±3.5
66±14.0
89±1.4
58±50.9
68±31.3
90±5.8
87±5.2
36±27.1
Silva et al. 2009
).
L (E)
RB (E)
RW (E)
SB (E)
SW (H)
L (E)
-
Pouteria ramiflora (Mart.) Radlk
Pouteria torta Radlk.
acarbose (positive control)
DMSO (negative control)
4.84
8.65
3.62
5.98
5.08
-
61±42.5
96±4.9
97±4.6
77±19.9
83±15.3
77±6.7
58±2.8
0±4.7
Part used : L, leaf; SW, stem wood; SB, stem bark; RW, root wood; RB, root bark. Extraction solvent : H, hexane; E, ethanol. Results are represented as means of replicates±S.D.
Preliminary qualitative phytochemical analysis of the active extracts was performed to determine the probable type of compounds present in the extracts causing HSA inhibition. We visualize by TLC the components of only few
extracts. Flavonoids were found in the ethanolic extract of the P. torta leaves and hexane extract of the P. ramiflora stem bark. Tannins and saponins were found in the ethanolic extract of the Serjania lethalis stem bark and root bark,
and ethanolic extracts of the P. ramiflora root wood. Tannins were found in the ethanolic extracts of the P. ramiflora root bark. As we observed, previous studies have reported that the capacity of alpha-amylase inhibition by vegetal
extracts is normally associated with polar compounds, as phenolic, tannins and triterpenoids compounds (
Ali et al. 2006 Gad et al. 2006 Kandra et al. 2004
,
,
).
The extracts that showed the highest inhibition in the screening test at a concentration of 200 µg/mL were subsequently investigated at concentrations of 100, 50 and 20 µg/mL. The S. lethalis stem wood ethanolic extract and stem bark
ethanolic extract (20 µg/mL) inhibited almost 40% of the HSA activity. The ethanolic extracts (20 µg/mL) of Matayba guianenses and S. lethalis root bark and P. torta leaves inhibited 70-80% of the HSA activity. Ethanolic extract of
the P. ramiflora root bark and hexane extract of the P. ramiflora stem bark (20 µg/mL) showed a 95-100% of inhibition effect, respectively. Studies from the bark of P. caimito reveal the presence of the triterpenes (
Ardon and Nakano 1973
Pellicciari et al. 1972
). Seven polyphenolic compounds, gallic acid, (+)-gallocatechin, (+)-catechin, (-)-epicatechin, dihydromyricetin, (+)-catechin-3-O-gallate, and myricitrin, were isolated and identified from the fresh fruits of P.
Ma et al. 2004
campechiana, P. sapota and P. viridis (
). These reports indicate that the capacity of alpha-amylase inhibition by genus Pouteria might be associated with such compounds.
We selected the stem bark aqueous extract of P. ramiflora (PrSBAE) for in vivo studies because it inhibited 92% of the HSA activity when it was diluted 1/10. In the acute toxicity test, mice treated with PrSBAE extract in doses higher
than 500 mg/kg showed toxicity signals as hypnoses, dyspnea, writhing. Two animals died. In mice that received 5,000 mg/kg of the extract we observed signals such as hypnosis, dyspnea. One animal died. When mice were treated
orally and intraperitoneal with 100 mg/kg of the PrSBAE dose, they did not show any toxicity effect.
Weight and blood glucose level in mice treated with 25, 50 and 100 mg/kg of the PrSBAE extract are shown in figures 1 and 2. All administrated doses have shown a significant effect on weight loss when compared to the control
group after eight days of treatment. Animals that received 50 and 100 mg/kg of extracts showed a significant reduction of the levels of blood glucose when compared to the control group. Mice treated with acarbose, a competitive
Santeusanio and Compagnucci 1994
inhibitor of intestinal alpha-glucosidases that slows the breakdown of sucrose and starch (
after 30 min of treatment (
Mai and Chuyen 2007
). A previous study of our laboratory (
Heo et al. (2009)
), were not effective to reduce blood glucose level when compared with polyphenolic enriched crude plant extract
Deconte et al. 2011
) conducted in rats treated by 20 days with acarbose did not reveal difference of weight gain and glycemic level of non-diabetic and
diabetic groups when compared with the respective controls.
showed that diphlorethohydroxycarmalol, isolated from Ishige okamurae, had inhibitory effects on alpha-glucosidase and alpha-amylase activities higher than
those of acarbose. Therefore, our results showed that the P. ramiflora extracts were more effective to inhibit the activity of alpha-amylase in vitro than acarbose. The in vivo results suggest that PrSBAE reduction of glucose level of
treated mice may be due to inhibition of carbohydrate-hydrolyzing enzymes.
,
Figure 1 The effect of administration of stem bark aqueous extract from P. ramiflora (PrSBAE) for eigth days on body weight loss in mice. Each column represents mean±S.E.M for five mice. * P < 0.05, ** P <
0.01, *** P < 0.001 different from control group.
Figure 2 The effect of administration of stem bark aqueous extract from P. ramiflora (PrSBAE) for eigth days on blood glucose level in mice. Each column represents mean±S.E.M for five mice. * P < 0.05, ** P <
0.01, *** P < 0.001 different from control group.
Several plant extracts have high concentrations of tannins, that are compounds that may exert an anti-nutritional effect by interfering with gut function (
Gin et al. 1999
Carbonaro et al. 2001
) and reduce the glycemic response to carbohydrate foods in
Oliveira et al. 2005
humans (
). In another study, it was investigated whether blood glucose reduction was due to reduced food intake in mice treated with the Syzygium cumini ethanol extract and tannic acid (
). In the study by
Oliveira et al., it was hypothesized that polyphenolic compounds contribute to the reduction of food intake, body weight and the levels of blood glucose. Hence, we cannot exclude the possibility that the P. ramiflora extract may
contain phenolic compounds and that its effects on weight reduction and the decrease in blood glucose are related to the dietary restriction associated with the inhibition of alpha-amylase.
In conclusion, the present study showed that M. guianensis, S. lethalis, P. torta and P. ramiflora inhibit alpha-amylase activity in vitro even at low concentrations. In addition, P. ramiflora bears positive effects on body weight and
blood glucose levels.
Acknowledgements
Grants to FSE from Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, MG, Brasil). Fellowships to NMG from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasil). We also thank Dr.
Jose Elias de Paula (Botanical Garden of UnB) and Dra. Ana Angelica Almeida Barbosa (Botanical Garden of UFU) for approving the authenticity of the plants used in the research, and Dr. Paulo S. Pereira and Dra. Suzelei C. França
(Department of Biotechnologyt, Ribeirao Preto University, UNAERP, Brazil) for their provision of TLC and HPLC preliminary phytochemical analysis.
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Received: June27, , 2012; Accepted: February27, , 2013
Correspondence to: Foued Salmen Espindola E-mail: [email protected]
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Tel: +55 21 3907-8100
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http://dx.doi.org/10.1590/S0001-37652013005000039
Biological Sciences
Morphological and hematological studies of Trypanosoma spp. infecting ornamental armored catfish from Guamá River-PA, Brazil
Rodrigo Y. Fujimoto1, Mikaelle S. Neves2, Rudã F.B. Santos2, Natalino C. Souza2, Márcia V.S. do Couto2, Josiane N.S. Lopes2, Daniel G. Diniz3, Jorge C. Eiras45
1
Embrapa Tabuleiros Costeiros, Avenida Beira Mar, 3250, Caixa Postal 44, 49025-040 Aracaju, SE, Brasil
Universidade Federal do Pará, Instituto de Estudos Costeiros, Rua Leandro Ribeiro, s/n, Aldeia, 68370-000 Bragança, PA, Brasil
Universidade Federal do Pará, Instituto de Ciências Biológicas, Laboratório de Investigações em Neurodegeneração e Infecção, Hospital Universitário João de Barros Barreto, Rua dos Mundurucus, 4487, Guamá, 66073-005 Belém, PA, Brasil
4 Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal
5 CIIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental/CIMAR, Universidade do Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal
2
3
ABSTRACT
A total of 281 specimens of freshwater armored ornamental fish species (Leporacanthicus galaxias, Lasiancistrus saetiger, Cochliodon sp., Hypostomus sp., Pseudacanthicus spinosus, Ancistrus sp. and Rineloricaria cf. lanceolata) were captured at the hydrological basin of Guamá River, Pará, Brazil. The infection by Trypanosoma spp. was inspected. The morphological and morphometric characterization of the parasites and the
hematological parameters were determined. Leporacanthicus galaxias and Pseudacanthicus spinosus presented 100% infection prevalence, and the other species showed a variable prevalence of infection. The parasites showed clearly different morphotypes and dimensions, and probably belong to different species. The hematological response to the infection varied with the host. Cochliodon sp. showed no differences between
infected and not infected fish. In other species several modifications on some hematological parameters were found, but apparently without causing disease. It is emphasized the possibility of introduction of the parasites in new environments due to the artificial movements of these ornamental fish.
Keywords: freshwater fish; hematological parameters; infection; Trypanosoma spp
RESUMO
Um total de 281 espécimes de peixes ornamentais de água doce - das espécies Leporacanthicus galaxias, Lasiancistrus saetiger, Cochliodon sp., Hypostomus sp., Pseudacanthicus spinosus, Ancistrus sp. e Rineloricaria cf. lanceolata - foram capturados na bacia hidrográfica do rio Guamá, Pará, Brasil. A infecção por Trypanosoma spp. foi inspecionada. A caracterização morfológica e morfométrica dos parasitas e os parâmetros
hematológicos foram determinados. Todas as espécies foram infectadas e todos os espécimes de Leporacanthicus galaxias e Pseudacanthicus spinosus estavam parasitados. As outras espécies mostraram uma prevalência variável da infecção. Os parasitas mostraram claramente morfotipos e dimensões diferentes, e provavelmente, pertencem a espécies diferentes. A resposta hematológica à infecção variou de acordo com o
hospedeiro. Em Cochliodon sp. não houve diferença entre peixes infectados e não infectados. Em outras espécies diversas modificações em alguns parâmetros hematológicos foram encontrados, mas aparentemente sem causar doença. Ressalta-se a possibilidade de introdução de parasitas em novos ambientes devido aos movimentos artificiais destes peixes ornamentais.
Palavras-Chave: peixes de água doce; parâmetros hematológicos; infecção; Trypanosoma spp
INTRODUCTION
The northeast of Pará State, in Brazil, especially the hydrographic basin of Guamá River, is an important area for the fishery of ornamental freshwater fish. This is due to the facility of reaching the fishing grounds and to the proximity of Belém, from where the fish are exported to other countries. According to
Most of these fish are exported, but a sanitary inspection of the specimens is not performed. Therefore, the dissemination risk of parasites and diseases is high.
There are some observations on the parasites of ornamental fish in Brazil, mostly about ectoparasites (
Garcia et al. 2009 Piazza et al. 2006 Prang 2007 Tavares-Dias et al. 2010
,
hosts (over 105 specimens /mm3), like Cyprinus carpio and Carassius auratus, may cause anemia, anorexia and ascites (
11 Eiras et al. (2010, 2012)
,
,
), and some studies deal with the infection by Trypanosoma spp. These parasites may not harmful to the fish hosts (
Torres (2007)
, the capture of ornamental armored fish represents up to 55% of the monthly income to fishermen.
Untergasser 1989
), or cause anemia, damage of the hematopoietic tissues and, finally, the death of the fish (
Noga 1996
). Massive infections in some
Paperna 1996
).
According to
, there are at least 62 nominal species of trypanosomes infecting freshwater fish in Brazil, a number of them parasitizing armored fish like Hypostomus spp. and Pterodoras spp. (
assuming specificity in parasitization.
D'Agosto et al. 1985 Lopes et al. 1991 Bara et al. 1985 Fróes et al. 1979
,
,
,
). It must be stressed out that a number of these parasites were identified only with basis on morphology and morphometry, and in most of the times
In this paper we report the infection of 7 different species of ornamental armored freshwater fish from the Guamá River by Trypanosoma spp. The morphology and morphometry of each form is described, and the prevalence, abundance and mean intensity of infection is reported. Furthermore, the hematological characteristics of the hosts are referred to.
A total of 281 armored ornamental specimens were captured at Guamá River, including 41 Leporacanthicus galaxias (Isbrüker and Nijssen 1989) (common name: acari pinima), 48 Lasiancistrus saetiger (Armbruster 2005) (acari canoa), 35 Cochliodon sp. (acari pleco), 10 Pseudacanthicus spinosus (Castelnau 1855) (acari assacu), 57 Rineloricaia cf. lanceolata (acari loricaria), 42 Hypostomus sp. (acari picoto), and 48
Ancistrus sp. (acari ancistrus).
Immediately after capture, a blood sample was taken from the caudal vein with syringes coated with 10% EDTA. After blood sampling the fish were measured (total and standard length) and inspected for ectoparasites, and integument or gill lesions. The following blood parameters were determined: glucose (mg/dL) using the automatic meter Prestige IQ 50, hematocrit (Ht, at 13,000 rpm during 3 minutes), total plasma
protein (g/dL) using a Quimis refractometer, total hemoglobin (HB, g/dL) using a Celmi 500 and Celmi 550 meter, and number of erythrocytes per mm3 counted in Neubauer chamber.
Blood smears were air dried and stained with May Grunwald Giemsa modified by
Rosenfeld (1947)
. The smears were used for leukocyte differential counting. The determination of mean corpuscular volume (MCV = Ht / Er x 10, fentoliter), mean corpuscular hemoglobin (MCH = Hb / Er x 10, picograms), and mean corpuscular hemoglobin concentration (MCHC= Hb / Ht x 100, g/dL) were recorded according to
Vallada
(1999)
.
Ranzani-Paiva 1995
Blood smears and hematocrit were also used for determining the presence of trypanosomes. In the positive samples the mean intensity of infection was determined indirectly relating the number of parasites with the amount of 1,000 erythrocytes (modified from
method of differential counting of white blood cells). The parasites were photographed using a digital camera, and the photographs were used
latter (employing the software Motic Images Advanced 3.0) to determine the cell characteristics: total length (TL), maximum width of the body (W), nucleus length (NL) and width (NW), distance between the middle of nucleus and anterior (DNA) and posterior (DNP) extremities, length of the free flagellum (FF) and number of folds of the undulating membrane (UM). With the data obtained, the nuclear index (IN)
according to
D'Agosto and Serra-Freire (1993)
and
Gu et al. (2006)
was calculated.
All the hematological results, as well as the morphometric ones were submitted to analysis of variance (BioEstt 4.0 programme). For F significant values, it was employed the Tukey test (5% of probability) to compare the means values. For the analysis of the variance of folds of the undulating membrane, it was employed the Kruskal-Wallis method for non-parametric data. It was also used the normality test with basis
in the deviations values to verify the existence of outliers which were eliminated.
RESULTS
The species Pseudacanthius spinosus and Leporacanthicus galaxias presented 100% of prevalence of infection, the other hosts showed a prevalence value varying between 22.6% (Cochliodon sp.) and 58.3% (Lasanciastrus sp.) (Table I). Therefore, most of the fishes were infected, and a high proportion of them were also infected by unidentified leeches. However, some of the specimens infected with leeches were not
parasitized by trypanosomes.
TABLE I Total weight (W), standard (SL) and total length (TL) of fish, prevalence of infection (P), mean intensity of infection (MI), abundance of infection (AB), and hematological parameters in infected and uninfected fish. Figures highlighted in ictalicized bold indicate significant differences between infected and uninfected fish; Ns: not significant.
W
SL
TL
P
MI
AB
GLUC
HT
TPP
HB
ER
MCV
MCH
Cochliodon sp.
Not infected
Infected
22.0±5.73ns
21.8±12.03ns
9.2±1.90ns
9.8±1.77ns
12.2±1.02ns
12.9±1.99ns
_
26.66
_
1
_
0.26
92.4±10.80ns
42.7±11.74ns
20.6±10.76ns
21.8±15.56ns
7.8±2.45ns
8.4±3.16ns
6.8±4.20ns
5.2.54ns
0.3±0.46ns
0.2±0.18ns
1097.9±286.90ns
568.9±250.54ns
270.8±175.63ns
211.9±108.40ns
CMCH
38.6±29.07ns
35.7±2.94ns
LYM
NEU
MON
56.1±20.65ns
39.5±21.16ns
4.2±1.48ns
Leporacanthicus
galaxias
Infected
28.4±14.6
10.1±2.0
12.9±2.6
100
1.1
1.1
60.2±27.8
31.6±13.2
8.6±3.4
10.9±3.8
0.4±0.3
843.7±661.4
353.6±348.3
37.4±17.7
27.3±7.8
68.9±8.5
3.7±1.9
47.2±20.12ns
48.2±21.29ns
4.5±2.51ns
Pseudacanthicus
spinosus
Infected
25.2±27.9
9.5±2.7
12.6±2.7
100
1.2
1.2
27.6±21.6
10.1±5.9
6.3±2.6
5.7±3.2
0.2±0.2
1887.0±180.6
1541.2±145.1
62.7±17.2
62.8±8.7
28.8±6.9
8.4±2.6
W
SL
TL
P
MI
AB
GLIC
HT
TPP
HB
ER
MCV
MCH
CMCH
LYM
NEU
MON
Ancistrus sp.
Lasiancistrus saetiger.
Not infected
Infected
Not infected
Infected
39.2±13.98ns
26.6±5.46ns
28.4±15.68ns
32.7±15.23ns
11.2±1.43ns
11.4±0.30ns
10.1±1.76ns
9.8±1.99ns
13.9±2.07ns
14.1±0.81ns
11.4±2.30ns
11.4±3.50ns
_
20
_
58.3
_
1
_
1
_
0.2
_
0.58
72.4±28.72ns
95±13.85ns
55.2±18.37ns
44.1±8.75ns
20.4±10.46ns
14.3±8.96ns
17.8±7.49 b
28.0±11.69 a
4.6±1.69ns
3.0±1.17ns
8.9±2.30 a
6.5±2.53b
8.9±5.54ns
9.3±5.95ns
9.23±4.52ns
9.7±4.97ns
0.6±16.99ns
0.6±0.26ns
0.3±0.32 b
1.3±0.06 a
373.7±156.62ns 289.2±264.15ns 845.8±786.60ns
230.9±99.29ns
170.5±107.62ns 162.3±87.09ns 1106.4±1917.10ns 71.6±37.18ns
155.4±216.18
41.3±17.67 b
63.4±39.44 a
29.4±10.97 b
a
75.6±19.56ns
81.3±2.51ns
94.4±13.23
64.4±15.33
19.9±16.89ns
11.3±1.52ns
4.0±9.6
28.0±13.8
3.2±3.24ns
7.3±3.21ns
1.2±0.8
7.5±4.4
Hypostomus sp.
Rineloricaria cf. lanceolata
Not infected
Infected
Not infected
Infected
21.4±8.76ns
23.9±6.05ns
19.4±6.88ns
13.0±5.38ns
8.7±1.33ns
9.1±0.96ns
15.3±2.10ns
15.5±2.53ns
11.0±1.53ns
11.±1.51ns
18.0±2.80ns
17±4.69ns
_
20
_
46.6
_
1.3
_
0.5
_
0.2
_
0.2
62.3±15.29ns
48.6±8.5ns
102.4±30.04a
59.5±22.12b
16.9±7.01ns
21±7.54ns
16.0±6.76ns
17.5±3.93ns
8.2±2.18ns
10.3±0.91ns
9.0±2.00ns
9.0±1.85ns
9.9±5.09ns
9.7±0.92ns
9.5±4.73ns
4.9±2.86ns
0.6±0.30ns
0.4±0.20ns
0.6±0.34ns
0.2±0.18ns
693.7±740.41ns 490.9±167.64ns 557.4±848.57ns 1070.4±1073.02ns
342.1±354.24ns 246.2±108.77ns 199.5±1066.66ns 216.5±110.35ns
58.8±24.71ns 50.4±18.28ns
58.3±47.05a
26.4±11.84b
29.4±5.85a
14.66±9.23b
80.2±7.71a
64.7±9.46b
67.5±5.61ns
76±15.09ns
14.6±7.16b
25±5.29a
3±1.75ns
9.3±7.57ns
4.3±3.25b
10.2±4.27a
Abbreviations: GLUC, glucose (mg/dl), HT, hematocrit, TPP, total plasma proteins g/dl, HB, hemoglobin g/dl, ER, number of erythrocytes (number of cells x 106/mm3); MCV, mean corpuscular volume (fentoliter); MCH, median corpuscular hemoglobin (pg); CMCH, concentration of the median corpuscular hemoglobin in g/dl; LYM, total number of lymphocytes; NEUT, neutrophils (%); MON, monocytes (%).
Hypostomus sp. presented the highest intensity of infection (1.3 parasites by 1,000 erythrocytes), and Rineloricaria cf. lanceolata showed the lowest one (0.5). The other species presented intermediate values (Table I). Pseudacanthicus spinosus had the highest abundance level, and Rineloricaria cf. lanceolata the smallest one, while the other species presented intermediate values (Table I).
The morphology (Fig. 1) of the parasites varied. In general, they had a rounded anterior extremity and a tapered posterior one. In some cases both the extremities were slightly tapered. The nuclei were most of the times oval-shaped, sometimes almost circular, in some cells occupying all the cell width. The kinetoplast was mostly rounded, in most of cases having a sub-terminal location. Usually the small part of the cell
located before the kinetoplast was difficult to observe clearly due to poor staining of this part of the body.
Figure 1 Types of Trypanosomes found in some host species (A , Cochliodon sp.; B, Lasiancistrus saetiger; C, Leporacanthicus galaxia; D, Pseudacanthicus spinosus; E, Cochliodon sp.). Note the very different morphotypes especially concerning the width of the body and the length of free flagellum. Magnification: 1,000.
The undulating membrane was well defined, developing all over the body length, or about half of the length. In some cases it was observed only near the extremity, presenting only two folds in the specimens with smaller values of body width. The undulating membrane was especially evident in Cochliodon sp., presenting in this host more folds. The cytoplasm varied from basophilic to eosinophilic. The free flagellum
was sometimes hard to distinguish because it was not so intensely stained, and its length varied between short and long.
There was a great morphometrical variation in the several characteristics as it can be seen in Table II, and several features presented a great variation depending from the host species.
TABLE II Morphometric characteristics (average plus standard deviation, figures in micrometers) of Trypanosoma spp. infecting armored fish.
TL
W
NL
NW
DNA
DNP
UM
FF
NI
Leporacanthicus
galaxias
52.1±3.6 a
5.2±1.1 a
5.9±1.1 a
4.6±1.2 a
26.1±3.2 a
24.8±4.7 a
7.9±1.9 ab
5.6±1.4 a
0.9±0.1 a
Cochliodon
sp.
58.0±9.4 a
3.9±0.6 b
6.0±1.4 a
3.5±0.6 b
32.0±2.5 ab
26.3±5.4 a
8.7±1.5 a
20.9±13.0 ab
0.8±0.1 b
Pseudacanthicus
spinosus
38.1±4.3 ab
3.4±0.3 bc
4.8±0.9 a
3.2b±0.4 bc
21.2ab±4.6 ab
15.5b±4.0 ab
3.5cd±0.9 abc
4.0±1.5 bc
0.6±0.2 b
Hypostomus
sp.
47.4±13.0 abc
4.9±1.3 bcd
3.9±0.7 ab
4.4±1.4 bc
27.1±11.3 ab
20.3±4.3 ab
6±1.4 abcd
6.9±2.1 bc
0.8±0.3 b
Lasiancistrus
saetiger
44.9±5.7 abc
5.3±0.7 bcd
5.4±1.1 ab
5.0±0.7 bc
25.8±5.0 ab
19.3±5.4 abc
5.7±2.4 abcde
2.3±0.6 bc
0.7±0.2 b
Ancistrus
sp.
38.1±7.7 c
3.5±0.9 d
4.2±0.8 bd
3.3±0.9 c
23.2±2.6 b
15.1±5.7 c
3.6±1.3 e
13.5±9.2 c
0.7±0.3 b
Rineloricaria cf.
lanceolata
47.5±4.3 c
3.8±1.1 d
5.4±0.9 d
3.7±1.0 c
22.4±3.5 b
24.6±2.8 c
2.8±1.1 e
2.1±0.8 c
1.1±0.1 b
Abbreviations: TL, total length; W, width; NL, nucleus length; NW, nucleus width; DNA, distance from the nucleus till the anterior extremity; DNP, distance from the nucleus till the posterior extremity; UM, number of folds of the undulating membrane; FF, length of the free flagellum; NI, nuclear index. Values followed by different letters in the same line indicate significant differences (5% probability in Tukey test).
The hematological study showed the infection caused varied effects on the hosts. Interestingly, in Cochliodon sp. no hematological alterations were found between infected and not infected specimens. In Ancistrus sp. and Hypostomus sp. the repercussions were minimal - the first specimens showed only a pronounced increase of the concentration of mean corpuscular hemoglobin, and the second revealed increase in the
percentage of lymphocytes. In Rineloricaria cf. lanceolata the infection caused decrease of mean corpuscular hemoglobin concentration and glucose, and modifications in the white blood cells (decrease of lymphocytes and neutrophils and increase in monocytes). Finally, Lasiancistrus saetiger showed increase in the hematocrit and erythrocytes, and decrease of total plasma proteins and of mean corpuscular hemoglobin.
DISCUSSION
The first conclusion to be drawn from our results is that the prevalence of the infection varied considerably with the host species, in two of them (L. galaxias and P. spinosus) reaching a prevalence of 100%. It is known that these parasites are transmitted by the bite of leeches. Therefore, the facility of infection by leeches promotes the parasitization by trypanosomes, and the behavior of the fish may contribute to a
higher or lesser probability of leech infection. The armored fish have a benthic behavior that facilitates the infection by leeches, and high values of infection by trypanosomes are not uncommon.
D'Agosto and Serra-Freire (1990)
reported 100% of prevalence for Trypanosoma chagasi and T. guaiabensis infecting the armored Hypostomus punctatus from lake Açú at Rio de Janeiro. Other reports on infections in several species
Fróes et al. 1978 1979 Lopes et al. 1989 Ribeiro et al. 1989 Eiras et al. 1989 1990
of armored fish showed a high variability on the prevalence and intensity of infection values (
,
,
,
,
,
There are in Brazil at least 62 species of trypanosomes described from freshwater fish, and at least 28 from those were described from armored fish (
review the Brazilian fish trypanosomes as it was done with trypanosomes from Africa performed by
). Considering these facts, and the fact that apparently the probability of leech infection in the fishes from our sample was the same for all the host species, it is possible to conclud that the resistance of the fish to the infection varies with the fish species.
Eiras et al. 2010
). Most of the descriptions were done assuming a strict specificity of infection, and a form observed in a new host was considered a new species (
Baker (1960)
, resulting in a substantial reduction of the number of blood flagellate species. Besides, one confusing factor is the variability in length during infection and the existence of pleomorphic species (
Thatcher 2006
). Today it is recognized that strict specificity may be an exception but not a role, and it is urgent to
Gibson et al. 2005
).
Our data do not allow the identification of the parasite species and, for the reasons described above, a comparison with the Brazilian species of trypanosomes would be useless. The identification based solely on morphological features is usually not possible, and the absence of specific infections, at least in most of the cases, do not allow a positive identification without the aid of molecular tools, and characterization of
the development of the parasite within the vector, which were not considered in the present research. However, it is highly probable that we face different species due to the so pronounced differences in morphology and morphometry of the parasites, as depicted in Figure 1 and Table II. It is the authors' intent to pursue this study in the future in order to elucidate this question.
According to the hematological data obtained, it seems that some host species (Lasiancistrus saetiger and Rineloricaria cf. lanceolata) were more affected than others (Ancistrus sp. and Hypostomus sp.), while Cochliodon sp. apparently had the hematological parameters not altered by the infection. Therefore, it can be concluded that some hosts adapted better than others to the infection.
Some results of other authors for different freshwater hosts species show results sometimes similar to ours: anemia in Carassius auratus infected with Trypanosoma danilewskyi (
parasites and hosts, and
Aguilar et al. (2005)
consider that T. granulosum has only minor effects in the host Anguilla anguilla. According to
Dyková and Lom 1979
), in Barilius blendelisis parasitized by Trypanosoma sp. (
Rauthan et al. 1995
), and in Cyprinus carpio infected with T. borreli (
Lom (1979)
, "it seems, on the evidence obtained from observations of natural infections, that species of this genus live in a more or less balanced state with their host".
In summary, we conclude that the armored ornamental freshwater fish species studied are highly infected by trypanosomes (representing most probably different species) and react differently to the infection as showed by the hematological observations.
Furthermore, it is important to emphasize the risk of dissemination of these parasites, due to the artificial movements of the hosts, in spite of the need of a vector to transmit the parasites to uninfected fish. This problem is especially important because the infection is not detectable by visual inspection of the fish.
Acknowledgements
Paticipation of J.C. Eiras on this research was partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through FCT - Fundação para a Ciência e a Tecnologia, under the project "PEst-C/MAR/LA0015/2011
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Received: October30, , 2012; Accepted: February27, , 2013
Clauss et al. 2008
). Other authors reported minimal changes in blood parameters for different species of
Correspondence to: Daniel Guerreiro Diniz E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013005000046
Biological Sciences
The role of cyclooxygenase-2 on endurance exercise training in female LDL-receptor knockout ovariectomized mice
FLAVIA DE OLIVEIRA1, LAURA B.M. MAIFRINO23, GUSTAVO P.P. DE JESUS1, JULIANA G. CARVALHO1, CLÁUDIA MARCHON2, DANIEL A. RIBEIRO1
1
Departamento de Biociências, Universidade Federal de São Paulo, Campus Baixada Santista, Avenida Ana Costa, 95, Vila Mathias, 11060-001 Santos, SP, Brasil
Laboratório de Análises Morfoquantitativa e Imunohistoquímica, Universidade São Judas Tadeu, Rua Taquari, 546, Móoca, 03166-000 São Paulo, SP, Brasil
3 Instituto Dante Pazzanese de Cardiologia, Av. Dr. Dante Pazzanese, 500, Vila Mariana, 04012-180 São Paulo, SP, Brasil
2
ABSTRACT
Estrogen deprivation in postmenopausal women increases cardiovascular risk. Cardiovascular risk as a result of atherosclerosis is able to induce an inflammatory disease as far as cyclooxygenase-2 ( COX-2) expression. The purpose of
the study was to investigate the role of COX-2 on exercise training in female mice low-density lipoprotein receptor knockout ( LDL-KO) with or without ovariectomy. A total of 15 female C57BL/6 mice and 15 female LDL-KO mice
were distributed into 6 groups: sedentary control, sedentary control ovariectomized, trained control ovariectomized, LDL-KO sedentary, LDL-KO sedentary ovariectomized and LDL-KO trained ovariectomized. The ascending part of the
aorta was stained with H&E and COX-2 expression was assessed by immunohistochemistry. Results revealed that ovariectomy as well as exercise training were not able to induce histopathological changes in mouse aorta for all groups
investigated. LDL-KO mice demonstrated plaque containing cholesterol clefts, foamy histiocytes and mild inflammatory process for all groups indistinctly. Ovariectomy induced a strong immunoexpression in atherosclerosis lesion of
LDL-KO mice. Nevertheless, a down-regulation of COX-2 expression was detected in LDL-KO trained ovariectomized when compared to LDL-KO sedentary. Our results are consistent with the notion that exercise training is able to
modulate COX-2 expression in LDL-KO mice as a result of COX-2 down-regulation.
Keywords: atherosclerosis; Ciclooxygenase-2; exercise; menopause
RESUMO
A diminuição de estrogênio em mulheres pós-menopausa aumenta o risco de doenças cardiovasculares. Esse risco, como resultado da aterosclerose, pode induzir o processo inflamatório e a expressão da Ciclooxigenase-2 ( COX-2) . O
objetivo deste estudo foi investigar a expressão da COX-2 em camundongos fêmeas knockout para o receptor de lipoproteína de baixa densidade ( LDL-KO) submetidas ao exercício com ou sem ovariectomia. Um total de 15 fêmeas
C57BL/6 e 15 fêmeas LDL-KO foram divididas em 6 grupos: controle sedentário, controle ovariectomizado sedentário, controle ovariectomizado treinado, LDL-KO sedentário, LDL-KO ovariectomizado sedentário e LDL-KO
ovariectomizado treinado. A parte ascendente da aorta foi analisada com H&E e com imunohistoquímica para COX-2. Os resultados revelaram que a ovariectomia bem como o exercício não induziram alterações histopatológicas da aorta
dos camundongos em todos os grupos investigados. Camundongos LDL-KO demonstraram placas de ateroma, histiócitos espumosos e processo inflamatório moderado em todos os grupos indistintamente. A ovariectomia induziu a forte
imunoexpressão da COX-2 na lesão aterosclerótica dos camundongos LDL-KO. No entanto, foi detectada uma diminuição da expressão da COX-2 no LDL-KO ovariectomizado treinado quando comparado com o LDL-KO sedentário.
Nossos dados estão de acordo com a noção de que o exercício pode modular a expressão da COX-2 nos camundongos LDL-KO como resultado da diminuição da expressão da COX-2.
Palavras-Chave: aterosclerose; Ciclooxigenase-2; exercício; menopausa
INTRODUCTION
Menopause is a critical period in a woman's life, characterized by decreased ovarian hormone production due to age (
increasing the susceptibility to obesity and its associated comorbidities, as far as inflammation (
ischemic stroke as a result of an inflammatory disease (
Ludgero-Correia et al. 2011
) . Removal of ovarian hormones in mice by ovariectomy mimics women in menopause,
Hong et al. 2009 Vieira et al. 2007
,
) . Atherosclerosis has particular interest, as it may be the underlying cause of myocardial infarction and
Ross 1999
).
Cyclooxygenase ( COX) plays a key role in the conversion of arachidonic acid to prostaglandins in inflammation. COX has 2 isoforms: COX-1, expressed by most tissues and mediates normal physiological functions; and COX-2,
rapidly induced at sites of inflammation (
Burleigh et al. 2002
) . According to studies with human atherosclerotic lesions, COX-2 is expressed by endothelial cells, smooth cells and macrophages (
Schonbeck et al. 1999 Baker et al. 1999
,
Accumulating evidence suggests the benefits of exercise on body weight, bone constitution, muscle strength and endurance, flexibility, oxygen consumption, blood pressure, and metabolic control after menopause (
2004
Petersen and Pedersen 2005
).
Asikainen et al.
) . In addition, regular exercise offers protection against mortality, primarily by protection against cardiovascular disease (
) . However, it would be interesting to know if, and to what extent, exercise
training is able to modulate COX-2 expression in mice suffering atherosclerosis and menopause, particularly because there are no previous reports. Therefore, the purpose of the present study was to investigate if exercise training
associated or not with menopause is able to modulate the inflammatory process as a result of COX-2 expression in mice suffering atherosclerosis.
Animals and Groups
All experiments were performed on 15 female C57BL/6 mice and 15 female low-density lipoprotein receptor knockout mice LDL-KO ( 20-25 g) from the Animal Shelter of Universidade São Judas Tadeu, São Paulo, Brazil. The
mice received standard laboratory chow and water ad libitum. The animals were housed in cages in a temperature-controlled room ( 22°C) with a 12-h dark-light cycle. All mice were treated similarly in terms of daily
manipulation. All surgical procedures and protocols were approved by the Experimental Animal Use Committee of Universidade São Judas Tadeu ( 058/2007) . The mice were randomly assigned to one of six groups ( n = 5 per
group) : sedentary control ( G1) , sedentary control ovariectomized ( G2) , trained control ovariectomized ( G3) , LDL-KO sedentary ( G4) , LDL-KO sedentary ovariectomized ( G5) and LDL-KO trained ovariectomized ( G6) .
Ovariectomy
At nine months of age, animals were anesthetized ( Ketamine 120 mg/Kg + Xylazin 20 mg/Kg) , and a small abdominal incision was made. The ovaries were then located, and a silk thread was tightly tied around the oviduct,
including the ovarian blood vessels. The oviduct was sectioned and the ovary removed. The skin and muscle wall were then sutured with silk thread (
Marsh et al. 1999 Irigoyen et al. 2005
,
).
Exercise Training
All animals from trained groups ( G3 and G6) were adapted to the treadmill ( 10 min/day; 0.3 Km/h) for 3 days prior to beginning the exercise training protocol. A maximal treadmill test (
Irigoyen et al. 2005 Souza et al. 2007 Rodrigues et
,
,
al. 2007
) was performed in trained groups a week after ovariectomy: first at the beginning of the experiment; in a second time in the fourth weeks of the training protocol. The purpose was to determine aerobic capacity and exercise
training intensity. Exercise training was performed on a motor treadmill at low-moderate intensity ( 50-60% maximal running speed) for one hour a day, 5 days a week for 4 weeks, with a gradual increase in speed from 0.3 to 1.2
Km/h.
The ascending part of aorta were taken and kept in 10% formalin buffered for 24h. The specimens were routinely embedded in paraffin blocks and cut in transversal sections ( 3 µm) . The slides were stained with hematoxylin and
eosin. The specimens were analyzed with light microscope ( Axio Observer.D1 Zeiss®, Germany) .
Immunohistochemistry
Paraffin was removed with xylene from serial sections of 3 µm and the sections were rehydrated in graded ethanol. Then, they were pretreated in a microwave with 0.01M citric acid buffer ( pH 6) for three cycles of 5 minutes each
at 850W for antigen retrieval. The material was pre-incubated with 0.3% hydrogen peroxide in phosphate-buffered saline ( PBS) solution for 5 minutes to inactive the endogenous peroxidase and then blocked with 5% normal goat
serum in PBS solution for 10 minutes. The specimens were then incubated with anti-COX-2 polyclonal primary antibody ( Santa Cruz Biotechnology, Santa Cruz, CA) at a concentration of 1:200. Incubations were carried out
overnight at 4°C in a refrigerator. This was followed by two washes in PBS for 10 minutes. The sections were then incubated with biotin-conjugated secondary antibody anti-rabbit IgG ( Vector Laboratories, Burlingame, CA) at a
concentration of 1:200 in PBS for 1 hour. The sections were washed twice with PBS followed by the application of performed avidin biotin complex conjugated to peroxidase ( Vector Laboratories) for 45 minutes. The bound
complexes were visualized by the application of a 0.05% solution of 3-3 – diaminobenzidine solution and counterstained with Harris hematoxylin. On control studies of the antibodies, the serial sections were treated with rabbit IgG
( Vector Laboratories) at a concentration of 1:200 in place of the primary antibody. Additionally, internal positive controls were performed with each staining bath.
Quantification of Immunohistochemistry
Sections stained using immunohistochemistry were analyzed for the percentages of immuno-positive cells in control and “hot spot” areas. A total of 1,000 cells were evaluated in 3 to 5 fields at 400X magnification. These values
were used as labeling indices.
Statistical Methods
Statistical analysis was perfomed by Kruskal-Wallis non-parametric test followed by the Dunn's test using SPSS software pack ( version 1.0) . “p” value < 0.05 was considered for statistic significance.
RESULTS
The histopathological evaluation revealed that there were no remarkable changes in the sedentary control group ( G1) ( Figure 1A) as well as in control mice submitted to ovariectomy ( G2) ( Figure 1B) and/or trained
ovariectomized ( G3) ( Figure 1C) . However, LDL-KO sedentary mice revealed histopathological changes such as the presence of plaque with cholesterol clefts, foamy histiocytes, and mild inflammatory process ( G4) ( Figure
2A) . The ovariectomy ( G5) or ovariectomy associated with exercise training ( G6) did not interfere with the presence of atherosclerosis in LDL-KO mice ( Figures 2B and 2C, respectively) .
Figure 1 Histopathological evaluation of groups G1, G2 and G3. In 1A, sedentary control ( G1) ; In 1B, sedentary control and ovariectomized group ( G2) and in 1C trained control ovariectomized group
( G3) . H&E staining. Scale Bar = 100 µm.
Figure 2 Histopathological evaluation of groups G4, G5 and G6. In 2A, LDL-KO sedentary group ( G4) ; In 2B LDL-KO and ovariectomy group ( G5) and in 2C LDL-KO trained ovariectomized group ( G6) .
Asterisk indicates atherosclerosis. H&E staining. Scale Bar = 100 µm.
Immunohistochemical Data
Immunohistochemical pattern was considered cytoplasmic for COX-2 immunomarker investi-gated in this setting.
In the sedentary control group ( G1) , COX-2 immunoexpression was weak in the aorta wall ( Figure 3A) . In the same way, experimental sedentary control ovariectomized ( G2) or trained control ovariectomized ( G3) was
considered negative indistincly for COX-2 immunoexpression ( Figures 3B and 3C, respectively) .
Figure 3 Immunoexpression of COX-2 ( arrow) in G1, G2 and G3 groups. In 3A, sedentary control ( G1) ; In 3B, sedentary control and ovariectomized group ( G2) and in 3C trained control ovariectomized
group ( G3) . Immunohistochemistry staining. Scale Bar = 100 µm.
Regarding LDL-KO sedentary group ( G4) , a moderate immunoexpression was detected ( Figure 4A) . However, the association with LDL-KO and ovariectomy ( G5) induced a strong pattern of COX-2 immunoexpression in
some cells of atherosclerosis lesion ( Figure 4B) . The LDL-KO ovariectomized and trained ( G6) was able to decrease COX-2 expresssion when compared to G4 ( Figure 4C) . Such findings are summarized in the graph showed in
Figure 5.
Figure 4 Immunoexpression of COX-2 ( arrow) in G4, G5 and G6 groups. In 4A, LDL-KO sedentary group ( G4) ; In 4B LDL-KO and ovariectomy group ( G5) and in 4C LDL-KO trained ovariectomized group
( G6) . Immunohistochemistry staining. Scale Bar = 100 µm.
Figure 5 Graph showing COX-2 labeling index in the groups sedentary control ( G1) ; sedentary control ovariectomized ( G2) ; trained control ovariectomized ( G3) ; LDL-KO sedentary ( G4) ; LDL-KO
ovariectomy ( G5) and LDL-KO trained ovariectomized ( G6) . Values were expressed as means ± SD. ( *) p < 0.05.
DISCUSSION
The aim of this study was to evaluate the protective effects of exercise training on inflammatory process induced by atherosclerosis in ovariectomized mice. The investigation was conducted using the immunohistochemistry for
COX-2. To the best of our knowledge, the approach has not been addressed before.
Histopathological analysis revealed that the groups exposed to exercise training or ovariectomy did not exhibit any morphological changes in this setting. However, LDL-KO mice revealed histopathological changes such as plaque
containing cholesterol clefts, foamy histiocytes, and mild inflammatory process. The association of ovariectomy or exercise training did not change the microscopic findings. Taken as whole, the outcomes exerted by menopause or
exercise training are not able to induce histopa-thological changes in aortic tissue.
Accumulating evidence suggests that prosta-glandin may also play key roles in growth and differentiation control of the several tissues (
Cansell et al. 2007
) . In prostaglandin biosynthesis, cyclooxygenases catalyze the key reactions
Feng et al. 1993
(
) . As an important member of the cyclooxygenase isoenzymes, COX-2 was usually found to be expressed in a variety of tumors and inflammatory reactions. To further elucidate the role of COX-2 in dislipidemic
mice suffering atherosclerosis, we evaluated the expression pattern of COX-2 in the aortic cells to explore the possible outcomes of certain therapeutic modalities in the normal structure and function of the aorta. Our results
demonstrated that COX-2 immunoexpression was weakly detected in mice from the control group ( sedentary) as well as in those subjected to ovariectomy or exercise training. However, a strong expression was detected in the
atherosclerosis lesion of LDL-KO mice exposed to experimental menopause. In fact, some authors have postulated that COX-2 promotes early atherosclerotic lesion formation in LDL Receptor-Deficient Mice in vivo (
2002
Burleigh et al.
) . By comparison, some studies have provided evidence that selective inhibition of the COX-2 enzyme with celecoxib prevented the development of atherosclerotic lesions in the proximal aortas from apo E-/- mice (
Jacob et al.
2008
) . One of the possible mechanisms is reduction in endothelial expression of the cell intercellular adhesion molecule and vascular cell adhesion molecule, which plays a key role in the recruitment of inflammatory cells during
the early stages of atherogenesis. Moreover, some authors demonstrated that selective inhibition of COX-2 and elimination of COX-2 from macrophages significantly reduces early atherosclerotic lesion formation in apoE-deficient
Burleigh et al. 2005
and C57BL/6 mice (
) . Deletion of the prostacyclin receptor removed the atheroprotective effect of estrogen in ovariectomized female mice (
important role in a proatherogenic mechanism, and support the potential of anti-inflammatory therapeutic approaches for atherosclerosis.
Egan et al. 2004
) . Therefore, it seems that COX-2 expression plays an
Nowadays, there is little information regarding whether exercise training above a certain intensity or duration could have beneficial effects. Accumulating evidence suggests that exercise training enhances oxygen consumption, in
an intensity dependent fashion, which is associated with augmented generation of immediate oxidative by-products: reactive oxygen species (
Davies et al. 1982
) . Exercise-induced increases in oxygen radical species production
Ascensao et al. 2005
stimulates enzymatic and nonenzymatic antioxidant responses, especially in cases of recurrent efforts (
) . Our results demonstrated that the exercise training associated with dislipidemia was able to reduce COX-2
immunoexpression in aortic cells suferring atheroscherosis. The findings are new, and therefore, difficult to discuss. It has been established that selective inhibition of COX-1 ( aspirin) or COX-2 ( rofecoxib) does not affect
Weber et al. 2007
Leem et al. 2011
systemic prostacyclin synthesis after physical exercise in trained healthy volunteers (
) . Conversely, chronic exercise ameliorates the neuroinflammation in mice (
) . Some authors have assumed that
inhibition of COX-2 in cardiomyocytes may contribute to heart failure in patients receiving nonsteroidal anti-inflammatory drugs specific for inhibition of COX-2 ( Wang et al. 2009) . This requires further study.
Taken together, our results are consistent with the notion that exercise training is able to modulate COX-2 expression in LDL-KO mice as a result of COX-2 down-regulation. As extrapolation from animal to human research
requires considerable caution, further studies are warranted to provide additional evidence of the long-term consequences of menopause or exercise training.
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Received: October9, , 2012; Accepted: March12, , 2013
Correspondence to: Flavia de Oliveira E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013005000052
Biological Sciences
Effect of l-glutamine on myenteric neuron and of the mucous of the ileum of diabetic rats
ELEANDRO A. TRONCHINI1, ALINE R. TREVIZAN1, CRISTIANO M. TASHIMA1, PRISCILA DE FREITAS1, ROBERTO B. BAZOTTE2, MARLI A.S. PEREIRA1, JACQUELINE N. ZANONI1
1
2
Departamento de Ciências Morfológicas, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR, Brasil
Departamento de Farmácia e Farmacologia, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR, Brasil
ABSTRACT
The objective of this work was to investigate the effect of the L-glutamine supplementation to prevent - diabetes induced changes in myenteric neurons and also to verify the effect on the mucosa of the ileum of Wistar rats. The animals
were divided in five groups (n = 5): untreated normoglycaemic (UN), normoglycaemic treated with L-glutamine (NG), untreated diabetics (UD), diabetics treated with L-glutamine, starting on the 4th (DG4) or 45th day following diabetes
induction (DG45). The amino acid was added to the diet at 1%. The density and size of neurons, the metaphasic index in the crypt, the height of the villus, the depth of the crypt and the number of globet cells were determined. There was
no difference in the neuronal density and in the cellular body area of the myosin-stained myenteric neurons of groups DG4 and DG45 when compared to group D. The metaphase index and the number of goblet cells showed no significant
differences when all groups were compared (P > 0.05). The villi height of groups DG4 and DG45 were 45.5% (P < 0.05) and 32.4% (P > 0.05) higher than those in group UD, respectively. The analyzed crypts showed similar depth for all
studied groups.
Keywords: diabetes mellitus; L-glutamine; myenteric plexus; myosin-V; rats
RESUMO
O objetivo deste estudo foi investigar o efeito da suplementação com L-glutamina para prevenir as alterações induzidas pelo diabetes sobre os neurônios mientéricos e também verificar o efeito sobre a mucosa do íleo de ratos Wistar. Os
animais foram divididos em cinco grupos (n = 5): normoglicêmico não tratado (UN), normoglicêmico tratado com L-glutamina (NG), diabético não tratado (UD), diabético tratado com L-glutamina, iniciando no 4° (DG4) ou 45° (DG45)
dias após a indução do diabetes. O aminoácido foi adicionado à dieta a 1%. A densidade e o tamanho dos neurônios, o índice metafásico nas criptas, a altura das vilosidades, a profundidade das criptas e o número de células caliciformes
foram determinados. Não houve diferença na densidade neuronal e na área do corpo celular dos neurónios mientéricos miosina-V imunomarcados dos grupos DG4 e DG45 quando comparados com o grupo D. O índice metafásico e o
número de células caliciformes não apresentaram diferenças significativas quando todos os grupos foram comparados (P > 0,05). A altura das vilosidades dos grupos DG4 e DG45 foi 45,5% (P < 0,05) e 32,4% (P > 0,05) superior que no
grupo UD, respectivamente. As criptas analisadas apresentaram profundidade semelhante para todos os grupos estudados.
Palavras-Chave: diabetes mellitus; L-glutamina; plexo mioentérico; miosina-V; ratos
INTRODUCTION
Diabetes mellitus is a heterogeneous clinical syndrome characterized by endocrine-metabolic abnormalities that alter homeostasis. The neurological manifestations are probably the most common among the chronic complications
caused by diabetes mellitus. Diabetic neuropathy is a heterogeneous group of disorders, with an extensive range of abnormalities (
Vinik and Mehrabyan 2004
), that can affect the peripheral nervous system harming the quality of life of
Vinik 1999 Afzaal and Saleem 2002
individuals (
,
).
Many studies have demonstrated alterations in the myenteric plexus of several intestinal segments of diabetic rats, not just regarding the reduction on the number, but also on the size of neurons (
1990 Zanoni et al. 1997 2003
,
,
). An important factor related to the diabetic neuropathy is oxidative stress (
Kuyvenhoven and Meinders 1999
) and/or a reduction on the efficiency of the endogenous antioxidants (
Vinson et al. 1989 Baynes 1991
,
Belai et al. 1988 1991 Belai and Burnstock
,
), which happens when there is an increase of reactive oxygen species (ROS) (
,
Parthiban et al. 1995
,
Baynes 1991 Kuyvenhoven and Meinders 1999
,
).
Another factor that provokes neuronal degeneration is the intracellular increase of sorbitol and fructose, due to the increase of the aldose reductase enzyme activity in the polyol metabolic pathway as a consequence of
Vinik 1999 Giugliano et al. 1996 Afzaal and Saleem 2002
Giugliano et al. 1996
hyperglycemia (
,
,
). As the glucose conversion by aldose reductase into sorbitol consumes NADPH, there is a reduction on the ratio NADPH:NADP+ (
). That reduction
can impair the reactions that NADPH need, especially the oxidized glutathione conversion to reduced glutathione by the glutathione reductase. Consequently, reduced levels of glutathione is observed in diabetes mellitus, which
Vincent et al. 2004
increases the susceptibility of cells to oxidative stress (
). Hence, measures that increase the availability of antioxidants may be relevant in the treatment of the chronic complications associated with diabetes mellitus.
Within this context, the L-glutamine supplementation is likely to present some neuroprotective effects, since it is a substrate for the production of glutathione (
Amores-Sánchez and Medina 1999
).
L-glutamine is a non essential amino acid, stored mainly in the skeletal muscle (75%) and in the liver (25%). Among its multiple functions, L-glutamine serves as primary carrier of nitrogen among the tissues and it is the main
Vahdat 2001
source of energy for the speed proliferation of cells, such as the intestinal epithelium (
). L-glutamine is metabolized through the phosphate-L-glutaminase, resulting in glutamate and ammonia. Glutamate is transported to
the cytosol and it can be used in the synthesis of glutathione (Amores-Sánches and Medina 1999), thus, maintaining the glutathione concentration in the cells. The absence of L-glutamine compromises nitrogen transport, causes a
Wolfgand et al. 2003
depletion of energy and a reduction on the glutathione levels (
may help to keep the integrity of the intestinal mucosa in diabetes mellitus.
). According to
Watford et al. (1984)
, there is a reduction of the plasmatic L-glutamine during diabetes mellitus. Therefore, it is possible that glutamine
The objective of this work was to evaluate the effect of the L-glutamine supplementation on myosin-V-stained myenteric neurons and on the mucosa in the ileum of streptozotocin-induced diabetic rats.
Procedure with the Animals
All of the experiments described in this work were supervised and approved by the Committee of Ethics on Animal Experimentation of the Universidade Estadual de Maringá. Twenty-five male Wistar rats (Rattus norvegicus), 90
days of age were used. Diabetes was induced through the intravenous administration of 35 mg.kg−1 of body weight of streptozotocin (Sigma, St. Louis, USA), dissolved in a buffer solution of 10 mmol.L−1 (pH 4.5), prior to a 14Bergmeyer and Bernet 1974
hour-fast. The injection with streptozotocin resulted in the diabetic syndrome with polyuria, polyphagia and polydipsia. The glycaemia (
) of each animal was assessed on the 4th day after the diabetes onset,
−1
with a mean value of 486.9 ± 14.25 mg.dL . The rats were divided in five groups: untreated normoglycaemic (UN), normoglycaemic treated with L-glutamine (NG), untreated diabetics (UD), diabetics treated with L-glutamine,
starting on the 4th (DG4) or 45th day following the onset of diabetes (DG45). The amount of food intake, water consumption and eliminated urine were assessed for five days of the last week of every month, with use of metabolic
cages.
The animals were kept in individual cages, receiving water and food (Nuvital® lab) ad libitum, with a photoperiod (6 am - 6 pm) and controlled room temperature (24°C±2°C). Some animals were fed chow supplemented with Lglutamine (1%) (Ajinomoto, Tokyo, Japan), which was prepared weekly.
On the 210th day, the animals were anesthetized with sodium thiopental (40 mg.kg−1 of body weight) and their blood collected by heart puncture to measure the glucose (
Bergmeyer and Bernet 1974
).
Neuronal Counting and Morphometric Analysis of Myenteric Neurons
The rats were perfused with 250 mL of fixing solution containing sodium periodate (10 mM), lysine (75 mM), paraformaldehyde (1%) in phosphate buffer (PB), pH 7.4 (37 mM) followed by a 1.1% saline solution. After perfusion,
each ileum was removed, rinsed with saline solution, opened and immersed for 1 hour in the same fixing solution. Subsequently, the segments were dehydrated in ethanol (50, 70, 80, 90, and 100%), remaining in each solution for
10 min, cleared in xylol (10 min), rehydrated with 100, 95, 90, 80, 70% ethanol, and stored in 70% ethanol. Afterwards, the segments were dissected under a stereomicroscope with transillumination, to remove the mucous and
Drengk et al. 2000
submucous layer, obtaining whole mount external muscle layers preparations. The neurons were stained for immunoreactivity to myosin V (
) as described previously using an antibody developed in the laboratories
of Cellular and Molecular Biology Department, Universidade de São Paulo (USP), Ribeirão Preto. These preparations were rinsed twice in phosphate-buffered saline (PBS) 0.1 M, pH 7.4 and blocked with PBS, bovine serum
albumin (BSA) (2%) (Sigma, St Louis, MO, USA), goat serum (2%), Triton-X-100 (0.5%) (Sigma, St Louis, MO, USA) at room temperature (RT). Then, the segments were incubated in a solution containing a 1:750 diluition of
the rabbit serum containing the anti-myosin-V antibody in PBS, BSA (2%), Triton-X-100 (0.1%) and normal goat serum (2%) at RT for 24 h. After incubation, the tissues were rinsed twice in PBS Triton-X-100 (0.1%) and twice
in the solution of PBS + Tween 0.05% (Sigma, St Louis, MO, USA). Segments were incubated in secondary antibodies (anti rabbit) conjugated with peroxidase 1:1,000 (Pierce, Rockford, USA) at RT, by shaking for 24 h. They
were then rinsed 4 x 15 min, in PBS with 0.05% Tween-20. The tissues were developed with diaminobenzidine (DAB) (Sigma, St Louis, MO, USA) and mounted in glycerol gel. The negative control was performed with the lack
of primary antibody.
Enteric neurons were counted using a BX 40 Olympus microscope with a 40x objective. Quantitative analysis was performed at the intermediate region (60° - 120°; 240° - 300°, with 0° as the mesenteric insertion) (
Sixty microscope fields were randomly counted for each preparation. The area of each microscopic field was 0.229 mm2. The results were shown in cells.cm2.
Zanoni et al. 2005
For morphometric analysis of the myenteric neurons, images were taken with a high-resolution camera and sent to a microcomputer. The cell profile (µm2) of 100 cell bodies for each animal was measured through the imageanalyses software Image-Pro-Plus 4, in a total of 500 neurons for each studied group. Neurons were classified by size into 100 µm2 bins and the percentage of each group was calculated for each bin.
Histological Procedure
Metaphasic index
Two hours prior the sacrifice, the animals were injected with 1 mg.Kg−1 vincristine sulfate (Oncovin®, Eli Lilly, Brazil), a blocking agent of the mitotic spindle. The injections were always given at the same time of day to avoid
circadian variations. Soon afterwards, the ileum was collected and carefully washed to remove the feces. The segments were opened along the mesenteric border, fixed with pins into a paper card with the mucosa surface upward.
They were then immersed in buffered formalin for 6 hours.
After fixation, the ileum was dehydrated and embedded in 2-hydroxyethyl-metacrilate (Leica Historesine - Embedding Kit, Germany). Two micrometer semi-serial sections were made in a Leica RM 2145 microtome, with glass
razor. The sectioned resins were stained by hematoxylin-eosin (HE).
The metaphasic index was calculated by counting the interphasic and metaphasic epithelial nuclei in the ileum crypts. Approximately 2,500 cells per animal were counted on BX 40 Olympus microscope under a 40 X lens. The
metaphasic index was expressed as the percentage of nuclei in metaphase divided by the overall number of counted nuclei.
).
Goblet cells in the villus
Resins sectioned were stained using the pricro-indigo-carmin/periodic acid Schiff (PAS) histochemical technique to stain goblet cells. The number of stained goblet cells in one side of the villus and the picro-indigo-carmim/
periodic acid-Schiff (PAS) method in one side of the villus and the overall number of cells on the same side of the villus were counted in all rats of each group. Approximately 2,500 cells per animal were counted - including
positive or negative PAS - in longitudinal sections of intact villus (
divided by the overall number of counted cells.
Hernandes et al. 2003
) using BX 40 Olympus microscope under a 40 X lens. The number of goblet cells was expressed as the percentage of PAS-stained goblet cells
Morphometry of villus and crypts
The height of the villus and the depth of the crypts were measured in well-guided longitudinal sections. The crypts depth measured as the distance between the junction crypt-villi and the crypt base. The length of the villus is
measured as the distance between the crypt-villus junction and the villus apex. The morphometric assessment was carried out on 30 villus and 30 crypts per animal (150 measurements per group) using a 10x objective and an ocular
with Zeiss micrometer grade.
Statistical analysis
The data were analyzed by the minimum squares method, through the Variance Analysis (ANOVA), followed by Tukey test, which was used as a post-test to compare the means. Since the areas of the cell bodies did not present a
similar distribution, we employed the Variance Analysis and t-test to compare the means. The analyses were carried out with the software Prisma 3.0. The results are shown as means ± standard error (M ± SE).
RESULTS
Procedures Animals
We verified in this study that the diabetic rats (groups UD, DG4 and DG45) did not gain weight in the same proportion as the animals from the normoglycaemic groups (UN and NG) (P < 0.05) (Table I). The L-glutamine
supplementation did not alter the final body weight of rats from groups DG4 and DG45 when compared to the animals from group UD (P > 0.05) (Table I). The diabetic condition was kept throughout the studied period, since
glycaemia levels were high in all of the investigated diabetic groups. The diabetic syndrome was also verified by the polyphagia, polyuria, polydipsia and the L-glutamine supplementation did not alter these parameters (Table I).
TABLE I Glycaemia (GI), initial and final body weight (IBW and FBW), daily water consumption (DWC), daily food intake (DFI) and daily urine excretion (DUE) for animals belonging to the following groups:
untreated normoglycaemic (UN), glutamine-treated normoglycaemic (NG), untreated diabetic (UD), glutamine-treated diabetic (DG4), glutamine-treated diabetic (DG45). All results are shown as means ± SE. n
= 5 rats per group.
Parameters
UN
NG
UD
DG4
DG45
GI mg.dl−1
158.1 ± 9.3a
143.2 ± 7.1a
649.6 ± 36.5b
524.7 ± 44.8c
500.5 ± 28.4c
IBW.g−1
330.6 ± 8.5a
300.3 ± 9.3a
314.8 ± 9.2a
328.0 ± 8.2a
312.4 ± 9.0a
FBW.g−1
480.3 ±
10.7a
406.4 ±
13.4b
292.1 ±
10.9c
286.7 ± 18.5c
54.35 ±
2.6a
49.2 ±
1.35a
213.3 ±
7.0b
160.6 ± 5.0b
54.82 ± 1.2b
48.3 ± 2.5b
DWC.ml−1
DFI.g−1
DUE.ml−1
309. 3 ±
17.7c
185.7 ±
11.7b
32.1 ± 0.75a
28.77 ± 1.1a
50.8 ± 2.1b
1.7a
1.6a
5.5b
12.82 ±
10.7 ±
101.7 ±
127 ±
7.0c
88.7 ± 3.8b
Means followed by different letter on the same line are different by the test of Tukey (P < 0.05).
Myenteric Neurons
The myosin-V technique allowed us to observe the three components of the myenteric plexus (Fig. 1). The primary plexus was well evidenced, with the presence of ganglia and interganglionic nerve fibers. Isolated neurons were
observed in the path of the nerve fibers of the primary plexus. The secondary and tertiary plexuses were also quite evident. The nerve fibers of the secondary plexus are parallel to the muscular cells of the circular layer of the
muscular tunica. The tertiary plexus was formed by fine nerve fibers, situated between the spaces left by the primary plexus (Fig. 1). It was observed a different intensity in the neurons staining in the myenteric ganglions (Fig. 2).
Figure 1 Micrographs showing the components of the myenteric plexus of the ileum of rats stained by the myosin-V technique. Figure A e B: 1 – primary plexus; 2 – secondary plexus; 3 – tertiary plexus; 4 –
isolated neurons. Calibration bar: (A) 50µm and (B) 30µm.
Figure 2 Micrographs of the myenteric plexus of the ileum of rats showing myenteric ganglia stained by myosin-V belonging to animals from the groups: untreated normoglycaemic (A), L-glutamine-treated
normoglycaemic (B), untreated diabetic (C), L-glutamine-treated diabetic (D), L-glutamine-treated diabetic (E). Calibration bar: 20µm
There was a 36.72% and 36.33% reduction in the neuronal density in the ileum of animals of group UD when compared to the untreated normoglycaemic (UN) and the normoglycaemic treated with L-glutamine (NG) respectively
(P < 0.05). The neuronal density of animals of groups DG4 and DG45 was similar to that observed in the untreated diabetics (UD) (P > 0.05) ( Fig. 3).
Figure 3 Density (Neurons/cm2) of myenteric neurons stained by myosin-V in the ileum of rats. Groups: untreated normoglycaemic (UN), glutamine-treated normoglycaemic (NG), untreated diabetic (UD),
glutamine-treated diabetic (DG4), glutamine-treated diabetic (DG45). All results were shown as means ± standard error. n = 5 rats per group. *P < 0.05 when compared to group UN and NG.
The cell profile area of the myosin-V stained-neurons of animals of group UD was 4.6% larger when compared to the animals of group UN (P > 0.05) (Table II). The L-glutamine supplementation (group DG4 and DG45) did not
change these results, with means similar to those of animals to group UD (P > 0.05). Most neurons had cell profiles that varied from 101 to 400 µm2. The proportion of neurons in this range for groups UN, NG, UD, DG4 and
DG45 was of 82.2%, 89.6%, 84.4%, 92.2% and 88.4% respectively (Fig. 4).
Figure 4 Size distribution of myosin-V stained myenteric neurons. They were classified in classes of 100 µm2 for the animals in the groups: untreated normoglycaemic (UN), glutamine-treated
normoglycaemic (NG), untreated diabetic (UD), glutamine-treated diabetic (DG4), glutamine-treated diabetic (DG45). n = 5 rats per group.
TABLE II Means and standard errors of the cell body areas of myosin-V stained neurons in groups: untreated normoglycaemic (UN), glutamine-treated normoglycaemic (NG), untreated diabetic (UD),
glutamine-treated diabetic (DG4), glutamine-treated diabetic (DG45). n = 5 rats per group.
Group
UN
NG
UD
DG4
DG45
myosin-V
273.5 ± 17.17
255.4 ± 5.268
286.6 ± 11.59
285.5 ± 21.16
274 ± 12.91
Means did not have significant differences by the t- test (P > 0.05).
Metaphasic Index and Estimation of Goblet Cells in the Villus
The data related to the metaphasic index (Table III). The methafasic index of group UD was 23.2% higher when compared to group UN (P > 0.05). The treatment with L-glutamine (DG4 group) promotes a increase of 6.92% in
metaphasic index in relation to the UD group (P > 0.05). The number of goblet cells of animals of groups UD, DG4 and DG45 was similar to those of animals of group UN and NG (P > 0.05) (Table III).
TABLE III Frequency of Goblet cells and metaphasic indexes of crypts of the ileum of rats belonging to groups in non-treated normoglycaemic rats (UN), L-glutamine-treated normoglycaemic (NG), nontreated diabetic (UD), L-glutamine-treated diabetic (DG4) and (DG45). All results were shown as means ± standard error. n = 5 rats per group.
Grupo
UN
NG
UD
DG4
Goblet Cells (%)
15.63 ± 0.441
14.53 ± 1.273
15.54 ± 0.955
15.79 ± 0.798
Index Metaphasic (%)
6.792 ± 0.4207
8.326 ± 0.7619
8.856 ± 0.8805
8.843 ± 0.5702
DG45
14.87 ± 0.733
7.586 ± 0.8120
There were no significant differences when the groups were compared by the test of Tukey (P > 0.05).
Morphometry of Villus and Crypts
Villus height in rats from groups DG4 and DG45 were 45.5% (P < 0.05) and 32.4% (P > 0.05) higher than those in group UD, respectively. The crypts of animals of group UD had a depth similar to those evidenced in the animals
of group UN (P > 0.05). The crypt depth in DG4 and DG45 were 27.63% and 29.17% larger than in UD (P > 0.05), respectively. The means of the villi height (µm) and crypt depth (µm) (Fig. 5).
Figure 5 Villi height (µm) and crypts depth (µm) in the ileum mucosa of untreated normoglycaemic rats (UN), L-glutamine-treated normoglycaemic (NG), untreated diabetic (UD), L-glutamine-treated diabetic
(DG4) and (DG45). All results were shown as means ± standard error. n = 5 rats per group. * P < 0.05 when compared to group UD.
DISCUSSION
Our experimental model of diabetes was effective because there was an increase on the daily water consumption, food intake and glycaemia. The animals of groups UD, DG4 and DG45 had a smaller body weight when compared
to the normoglycaemic animals (groups UN and NG).
Myenteric neurons: The myosin-V immunohistochemical technique was used to stain the myenteric neurons in the muscular layer whole mounts (
Drengk et al. 2000
). Several authors have used this technique to stain myenteric
Buttow et al. 2003 2004 Zanoni et al. 2003 2005 Schoffen et al. 2005
neurons in different areas of the gastrintestinal tract (
,
,
,
,
Langford 2002
axon (
). Myosin-V is a versatile motor protein and it is involved in the fast transport of vesicles from the dendrites to the
). This technique is used as marker of the overall neural population because the myosin-V is present in cell bodies and nerve fibers (
Drengk et al. 2000
the myenteric plexus. We verified the presence of the three components that compose this plexus (primary, secondary and tertiary) similar to previous studies
myenteric neurons inside the ganglia was observed in all studied groups. According to
Drengk et al. (2000)
). With this technique, we are able to observe the organization of
Furness and Costa (1980)
. The variable intestinity of imunoreactivity in the
heterogeneity of intensity may indicate different levels of neuronal activity.
We observed a 36.72% decrease on density of myenteric neurons stained by the myosin-V in group UD when compared to the normoglycaemic (UN). This result was similar to the one found by
Zanoni et al. (2003)
and
Alves et al. (2006)
who verified a reduction in the neuron density in the ileum of diabetic rats. There are several factors that contribute to the reduction on the number of myenteric neurons. Some of them are: a) the increase of the aldose reductase
activity, associated to an increase in the sorbitol and fructose levels (
Ferraz et al. 1997
); b) an increase in oxidative stress (
Baynes 1991 Vincent et al. 2004
,
levels of endogenous antioxidants. The glutathione levels are reduced during the hyperglycaemia leading to an increase in ROS (
) that causes an increment in the production of free radicals; c) a decrease in the
Parthiban et al. 1995 Greene et al. 1999 Vincent et al. 2004
,
,
). Glutathione is an important antioxidant in most
Vincent et al. 2004
cells of mammals (
). This substance serves as an essential cofactor for the glutathione peroxidase enzyme, which removes hydroperoxidase, and the formation of oxidized glutathione (GSSG). The reduced
glutathione (GSH) is regenerated by the enzyme glutathione reductase using NADPH (
ROS (
Parthiban et al. 1995 Vincent et al. 2004
,
). Therefore, the hyperactivity of the polyol pathway reduces the glutathione levels, causing an increase in the
Nakamura 2002
). This reduction is related to the increase of the aldose reductase activity that consumes NADPH, which is also necessary for the formation of the reduced glutathione (
Tesfamariam 1994 Giugliano et al. 1996
,
). All of
Srinivasan et al. 2000 Barber 2003
these events involve an increase in oxidative stress and are related to the activation of a cascade of events that generate the apoptosis (
,
).
We hypothesized that L-glutamine supplementation in the DG4 group of animals would prevent the development of diabetic neuropathy. Furthermore, the objective of L-glutamine supplementation of the DG45 group of animals
was to work as a treatment for diabetic. However, the L-glutamine supplementation was not efficient either in preventing or in treating this pathology at the dose used. L-glutamine is a very important energy substratum for cells,
Vincent et al. 2004
Roth et al. (2002)
being also a precursor for nucleotides, glutamate and, especially, the GSH synthesis. This amino acid has its concentration reduced during the catabolic stress (
). Studies carried out by
showed that rats
fed with L-glutamine had an increase in their cellular content of GSH. We observed that L-glutamine supplementation (DG4 and DG45) did not avoid the decrease on the number of myenteric neurons. This might be due to the fact
Vincent et al. 2004
that several mechanisms were producing ROS. Among them are: the polyol pathway, the autoxidative glucosilation, the reduction of antioxidants and the reduction of the efficiency of the enzymatic system (
).
Zanoni et al. 2003
Therefore, a combined treatment of substances already studied individually (as the ascorbic acid [
] or acetil-l-carnitine [Miranda-Neto et al. 2005]) with L-glutamine could be effective, because ROS would be
neutralized by different mechanisms. We speculate that another factor to be questioned would be in relation to the concentration of used L-glutamine. In this experiment a concentration of 1% was used and could have been too
small to produce the wanted biological effect. We worked with this concentration, because there is evidence in the literature that higher concentrations (4%) is harmful the intestinal mucous membrane (
Shinozaki et al. 1997 Ameho et al.
,
1997
). It is possible that an intermediate concentration could be beneficial to the neurons without damage to the intestinal mucous membrane.
There were no significant differences regarding the neuronal profile area when the five groups were compared. When comparing group UD with group UN, we observed that the immunoreactive neurons to myosin-V of animals
belonging to UD were 4.6% larger than the animals from group UN. We have demonstrated previously
Zanoni et al. (2003)
that the cellular profile of myosin-V myenteric neurons of the ileum of diabetic rats was unaltered by
Zanoni et al. (2002 2003 )
treatment with ascorbic acid. On the other hand, we have also demonstrated that VIP-ergic and nitrergic neurons
,
in the ileum had an increase in their area in diabetic rats and further, that ascorbic acid
supplementation prevented this increase. We can infer that diabetes mellitus affects in a different way the subpopulations of myenteric neurons, since we have noticed no changes in the neuronal area when analyzing the neurons
overall population and the suplementation with L-glutamine did not alter these results. When we analyze the distribution of the neuronal population in random classes at intervals of 100 µm2, we verified that despite the size
variability observed in the area of the neuronal profile (47.301 to 855.102 µm2), most neurons in both groups had a profile cell area ranging from 101 to 400 µm2. This shows the uniformity in the neuronal distribution between the
groups.
Mucosa of the ileum: Our results did not show significant differences in the cell proliferation, crypts depth and villus height of the ileum mucosa at the end of 120th experiment day in UD when compared to UN and NG. Several
Miller et al. 1977 Zhao et al. 2003
studies have already shown changes in the small intestine of non-supplemented diabetic rats such as hyperplasia, hypertrophy of the mucosa (
,
), increase on the crypts depth and villus heights after 4 days
Zhao et al. 2003
of diabetes in the duodenum and 7 days of diabetes in jejunum and ileum (
). These results differ due to their use of a model of acute diabetes mellitus while our experimental model is of chronic diabetes mellitus. It is
likely that the intestinal mucosa underwent an adaptation to the chronic pathogenesis of the diabetes mellitus. Data observed by
intestine of rats after 84 days of the diabetes mellitus onset.
Zoubi et al. (1995)
did not find morphometric changes in the crypts depth and villus height in the small
The intestinal mucous membrane of the animals of the group DG4 presented an increase in the cellular proliferation in the proportion of 6.92% and an increase in 45.5% in the height of the villus when compared to the group UD.
The villus in groups DG45 was 32.4% higher than those in group UD. The crypt depth in groups DG4 and DG45 were larger when compared to group UD (27.63% and 29.17% respectively). These results can be related the
functional activity of L-glutamine which is considered the main energy source for the enterocytes (
Fleming et al. 1997
) to promote proliferation and cellular migration of the intestinal mucosa cells (
Ruemmele et al. 1999
). L-glutamine
Palanch 2000
links to receptors at the luminal or basolateral surface of enterocytes and causes its own absorption (
). It is also possible that the villus suffered a delay in its cell death process, the L-glutamine may have prevented the
villus apoptosis from happening because glutamine is a precursor of glutathione, which is reduced in intestinal mucosa due to the diabetes mellitus (
Bhor et al. 2004
). Previous studies have demonstrated that rats supplemented with L-
Klimberg et al. 1990
glutamine (3%), for a period of 8 days, had an increased on the height of villus and number of mitotic cells per crypt after treatment with chemotherapeutics (
).
The number of goblet cells counts was similar among groups UD, UN, NG. A morphological study of the enteric epithelial mucosa of streptozotocin-induced diabetic rats, with 14 days of age has demonstrated similar number of
goblet cells in the jejunum, ileum and duodenum Etharh and Carr (1997) or just ileum
did not alter the number of goblet cells counts in the villus.
Mantle et al. (1989)
when comparing the diabetic and control groups. In the present study, L-glutamine supplementation (groups DG4 and DG45)
Summing up, the present work showed that the L-glutamine supplementation (1%) was not effective in altering the diabetes-induced loss of myenteric neurons, but promoted an increase of the size of the villus and depth of the
crypts in the animals of the group DG4 and DG45. These results allow us to suggest that it is necessary new researches to prove the effectiveness of the suplementation with L-glutamine on the innervation enteric of the ileum of
diabetic rats.
Acknowledgements
The authors would like to thank the support of Maria Euride do Carmo Cancino, Maria dos Anjos Fortunato, Valdir Trombeli and José Antonio de Souza for their excellent technical support.
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Received: May17, , 2012; Accepted: March15, , 2013
Correspondence to: Jacqueline Nelisis Zanoni E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013000300017
Biological Sciences
Hawkmoth fauna (Sphingidae, Lepidoptera) in a semi-deciduous rainforest remnant: composition, temporal fluctuations, and new records for northeastern Brazil
LUIS M. PRIMO1, JOSÉ A. DUARTE2, ISABEL C. MACHADO3
1
Programa de Pós-Graduação em Biologia Vegetal, Universidade Federal de Pernambuco, Departamento de Botânica, Av. Prof. Moraes Rego, s/n, Cidade Universitária, 50372-970 Recife, PE, Brasil
Núcleo de Pesquisa Educacionais do Rio Grande do Norte/NUPERN, BR 101, Km 0, Centro Administrativo, Lagoa Nova, 59064-901 Natal, RN, Brasil
3 Departamento de Botânica, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, s/n, Cidade Universitária, 50372-970 Recife, PE, Brasil
2
ABSTRACT
We carried out a qualitative and quantitative inventory of the hawkmoth fauna (Sphingidae) of an area of semi-deciduous seasonal rainforest in the state of Pernambuco (Tapacurá Ecological Station), northeastern Brazil. Hawkmoths were sampled monthly from October 2004 to February 2007 (27 months). We recorded 31 species from 16 genera, three
tribes, and three families. Macroglossinae was the most abundant subfamily and represented ca. 71% of all species. Out of the 277 individuals collected, 88.4% were males. Five new records were made for northeastern Brazil: Enyo gorgon (Cramer, 1777), Perigonia stulta (Herrich-Schäffer, [1854]), Eupyrrhoglossum sagra (Poey, 1832), Nyceryx
coffaeae (Walker, 1856) and Xylophanes chiron (Drury, 1773). Eight further species were recorded for the first time for the Pernambuco Endemism Center, showing the important role played by Tapacurá Station in preserving the biodiversity of this insect group. Species richness and abundance were directly related to rainfall: about 70% of all
individuals were captured during the rainy season. Changes in Sphingidae populations may, however, be caused by other factors that directly affect either larvae and adults of those insects, such as matrix effect and forest fragment size, which influence migration processes and the presence of predators.
Keywords: dry forest; Pernambuco Endemism Center; phenology; seasonality; Sphingidae abundance and richness; sphingophily
RESUMO
Nós analisamos o inventário qualitativo e quantitativo da fauna de mariposas esfingídeas (Sphingidae) de uma área de floresta úmida semi-decidual no estado de Pernambuco (Estação Ecológica de Tapacurá), Nordeste do Brasil. Os esfingídeos foram amostrados mensalmente de Outubro de 2004 a Fevereiro de 2007 (27 meses). Nós registramos 31
espécies distribuídas em 16 gêneros, três tribos e três famílias. Macroglossinae foi a sub-família mais abundante, representada por ca. 71% de todas as espécies. Dos 277 indivíduos coletados, 88.4% foram machos. Cinco novos registros foram obtidos para o Nordeste do Brasil: Enyo gorgon (Cramer, 1777), Perigonia stulta (Herrich-Schäffer, [1854]),
Eupyrrhoglossum sagra (Poey, 1832), Nyceryx coffaeae (Walker, 1856) and Xylophanes chiron (Drury, 1773). Outras oito species foram registradas pela primeira vez para o Centro de Endemismo Pernambuco, mostrando o importante papel desempenhado pela Estação de Tapacurá na preservação da biodiversidade desse grupo de insetos. Riqueza e
abundância das espécies apresentaram relação direta com a precipitação: cerca de 70% de todos os indivíduos foram capturados durante a estação chuvosa. Variações nas populações de Sphingidae, entretanto, podem ser causadas por outros fatores que afetam diretamente as larvas e adultos daqueles insetos, como o efeito da matriz e do tamanho do
fragmento florestal, que influenciam os processos de migração e a presença de predadores.
Palavras-Chave: floresta seca; Centro de Endemismo Pernambuco; fenologia; sazonalidade; abundância e riqueza de Sphingidae; esfingofilia
INTRODUCTION
Kitching and Cadiou 2000
Sphingidae (Lepidoptera, Bombycoidea) have a pantropical distribution with approximately 1,200 species (
Haber 1983
), from which 75% occur in tropical regions (
Duarte et al.
). In Brazil, this family is represented by 29 genera and 210 species. However, this number is probably higher as the available data are still poor (
2008
).
As hawkmoths, like all lepidopterans, are holometabolous insects, their life cycle is very uniform. The larvae are generally oligophagous. However they may exist in the same subfamily or tribe representatives both oligo and polyphagous. In general, Sphingidae feeding on young leaves low in tannin, although many species of Smerinthini
Pittaway 1993
(Smerinthinae), as the Saturniidae, feed on leaves rich in tannin (
). Except Smerinthini and Sphingulini, adults, in turn, are polyphagous-nectarivorous generalists, which forage on flowers of many species (Kieslev et al. 1972,
Haber and Frankie 1989 Darrault and Schlindwein 2002
,
), and may be responsible for 5 to 10% of the pollination of
Bawa et al. 1985 Haber and Frankie 1989 Oliveira et al. 2004 Primo 2008
the tropical flora (
,
,
,
).
The Atlantic Forest located to the north of São Francisco River is known as the Pernambuco Endemism Center (sensu
areas, but none in dry forest areas (sensu
Prance 1987
); faunal inventories in its remnants are of high biogeographic interest. However, there are only two studies on the sphingid fauna in this ecosystem:
Gentry 1995
Duarte et al. 2001 Duarte and Schlindwein 2005b Gusmão and Creão-Duarte 2004
). Five other inventories have been carried out in northeastern Brazil in other ecosystems, such as Caatinga (
,
,
Duarte and Schlindwein (2005a)
and
Lopes et al. (2006)
, both in rainforest
Gusmão and Creão-Duarte 2004
), Ombrophilous Montane Forest - “Brejo de Altitude” (
Darrault and
) and Tabuleiro (
Schlindwein 2002
).
Considering that hawkmoths play a vital role in the stability of ecosystems, acting on the pollination and reproduction of plant species, the main purpose of our study was to improve the basic knowledge on these lepidopterans by answering the following questions: What is the composition and abundance of sphingids in the area? Is the
phenology of this group seasonal? We also aimed to test whether there was a direct relationship between richness and abundance of sphingids and rainfall in the dry forest (sensu
Gentry 1995
), as it has been observed in other ecosystems.
Janzen 1988a b 1997
This information is essential not only to improve the biogeographic research on this insect group, but also for the management and conservation of natural environments, mainly dry forests, which are fast becoming degraded, as they are located in sites of flat relief that are eminently suitable for agriculture (
,,
).
Study Site
The present study was carried out in Tapacurá Ecological Station (Estação Ecológica do Tapacurá), São Lourenço da Mata municipality, 50 km from the coast of Pernambuco State (08°01′S, 35°11′W), northeastern Brazil. The region's annual rainfall is ca. 1,300 mm and the average temperature is 25.5 °C. There are marked seasons: the
rainy season occurs from March to August and the dry season between September and February (ITEP-LAMEPE) (Fig. 1).
Fig. 1 Multiannual rainfall and temperature at the Tapacurá Ecological Station, Pernambuco, Brazil. Source: ITEP (Instituto de Tecnologia de Pernambuco), São Lourenço da Mata (barragem Tapacurá) station (historical average of 30 years).
Andrade-Lima
The forest remnant area is approximately 400 ha and it is surrounded by a matrix of sugar cane plantations and pastures. The vegetation is classified as semi-deciduous seasonal rainforest, as the variation between dry (six months with rainfall below 100 mm) and rainy seasons imposes seasonality on the dominant tree species (
1960 Veloso et al. 1991
,
Rodal et al. 2005
). Although there is a floristic similarity with the rainforest near the Atlantic coast, the semi-deciduous forest shows a relatively less diverse and structurally simpler community (
).
Sphingid Surveys
Sampling was carried out at each new moon over two consecutive nights, between 5:30 pm and 2:30 am, for 27 months, from October 2004 to February 2007 (only June 2005 and January 2006 were not sampled), for a total of 486 sampling hours.
To sample the moths, we used a light trap composed of a mercury vapor lamp (250 watts) in a vertical position two meters above the ground in front of a white cloth of 3 × 2 m. We always set the trap in the same site: an open area that allowed the light to shine across a large forest area.
Sphingids were collected by hand, using an entomological net, killed with an injection of ethyl acetate ventrally between the thorax and the abdomen, conserved in individual wax-paper envelopes, and later dry-pinned, labeled and deposited in the collection of the Laboratory of Floral and Reproductive Biology (Laboratório de Biologia
Floral e Reprodutiva), at the Botanical Department of Universidade Federal de Pernambuco, Recife, Brazil. Species were identified using
bristle in the male) (
Kitching and Cadiou 2000
).
Kitching and Cadiou (2000)
D'Abrera 1986 Moré et al. 2005
and additional literature (
,
). We determined the sex of each individual by the morphology of the frenulum (a brush of bristles in the female, a single strong
Ayres et al. 2007
To test for differences in species richness and abundance between dry and rainy seasons, as well as the relationship between population size of the most abundant species and annual rainfall, we used Pearson's correlation. Calculations were made using the package BioStat 5.0 (
calculated as a percentage of number of individual collected monthly in relation to the total of captured individuals. The normality of the data was checked using the Liliefors' test (
). The relative abundance of hawkmoth species was
Sokal and Rohlf 1995
).
RESULTS
Composition of the Sphingid Fauna
We captured 277 sphingids of 16 genera and 31 species (Table I). The subfamily Macroglossinae was most abundant (22 species, 70.96%) followed by Sphinginae (eight species, 25.8%) and Smerinthinae (one species, 3.22%). Eight species were represented by only one individual (Table I). The most abundant species were Callionima parce
(Fabricius, 1775), Manduca lefeburii (Guérin-Méneville, 1844), Protambulyx strigilis (Linnaeus, 1771) and Manduca hannibal (Cramer, 1779) (Fig. 2).
Fig. 2 Individuals number of Hawkmoth (Lepidoptera: Sphingidae) and relative abundance recorded at the Tapacurá Ecological Station, Pernambuco, Brazil, from October 2004 to February 2007.
TABLE I Hawkmoth (Lepidoptera: Sphingidae) species recorded at the Tapacurá Ecological Station, Pernambuco, Brazil, from October 2004 to February 2007.
Species
Smerinthinae Ambulycini Protambulyx strigilis (Linnaeus, 1771)
Sphinginae Sphingini Cocytius antaeus (Drury, 1773)
Manduca diffissa tropicalis (Rothschild. & Jordan, 1903)
Manduca hannibal hannibal (Cramer, 1779)
Manduca lefeburii lefeburii (Guérin-Méneville, [1844])
Manduca rustica rustica (Fabricius, 1775)
Manduca sexta paphus (Cramer, 1779)
Neogene dynaeus (Hübner, [1827]-[1831])
Acherontiini Agrius cingulata (Fabricius, 1775)
Macroglossinae Dilophonotini Callionima parce (Fabricius, 1775)
Enyo gorgon (Cramer, 1777)
Enyo ocypete (Linnaeus, 1758)
Erinnyis alope alope (Drury, 1773)
Erinnyis crameri (Schaus, 1898)
Erinnyis ello ello (Linnaeus, 1758)
Eupyrrhoglossum sagra (Poey, 1832)
Hemeroplanes triptolemus (Cramer, 1779)
Nyceryx coffaeae (Walker, 1856)
Pachylia ficus (Linnaeus, 1758)
Perigonia lusca lusca (Fabricius, 1777)
Perigonia pallida Rothschild & Jordan, 1903
Perigonia stulta Herrich-Schäffer, [1854]
Pseudosphinx tetrio (Linnaeus, 1771)
Philampelini Eumorpha anchemolus (Cramer, 1779)
Eumorpha fasciatus fasciatus (Sulzer, 1876)
Eumorpha vitis vitis (Linnaeus, 1758)
Macroglossini Xylophanes chiron nechus (Cramer, 1777)
Xylophanes libya (Druce, 1878)
Xylophanes loelia (Druce, 1878)
Xylophanes pluto (Fabricius, 1777)
Xylophanes tersa tersa (Linnaeus, 1771)
Total
Male
35
4
1
20
42
4
8
7
1
58
3
1
2
6
7
2
1
3
5
3
1
3
1
6
2
2
1
1
1
2
12
245
Female
1
1
0
1
4
1
1
1
1
1
1
0
4
7
5
0
0
0
0
0
0
0
0
1
1
0
0
0
0
1
0
32
Total
36
5
1
21
46
5
9
8
2
59
4
1
6
13
12
2
1
3
5
3
1
3
1
7
3
2
1
1
1
3
12
277
Proboscis length ± SD (mm)
28.48 ±1.85
104.18 ±22.95
61
76.23 ±5.84
54.75 ±4.33
125.56 ±8.77
80.72 ±9.61
21.52 ±5.38
99.15 ±19.02
15.9 ±1.4
24.8 ±1.66
25.3
40.95 ±2.28
34.05 ±2
33.44 ±2.48
18.65 ±0.49
18.6
12.6 ±0.56
49.89 ±5.02
17.15 ±2.19
13.1
19.1 ±1.17
48
45.06 ±5.77
43.73 ±1.1
40.8 ±2.54
40.04
54
35.6
31.11
31.15 ±1.37
In addition to the species captured with the light trap, we also recorded, though not systematically, the diurnal species Aellopus titan (Cramer 1777) (Macroglossinae, Dilophonotini) visiting flowers of Caesalpinia echinata Lam. (Caesalpinoideae), Psychotria capitata Ruiz & Pavon (Rubiaceae) and Casearia hirsuta Sw. (Flacourtiaceae).
The sex ratio was 7.5:1, male to female (239 males and 32 females). The distribution of individuals and species by tribes, as well as their variations between dry and rainy seasons, are expressed in Figure 3. Proboscis length varied from 13.1 mm (Perigonia pallida) to 125.56 mm (Manduca rustica) (Table I).
Fig. 3 Relative proportions in the number of individuals (A) and species (B) collected per tribes at the Tapacurá Ecological Station, Pernambuco, Brazil, from October 2004 to February 2007, and distributed in each season: individuals number per tribes in dry season (C) and in rain season (D), species
number in the dry season (E) and rain season (F).
The species accumulation curve (Fig. 4) showed an apparent stabilization at twenty species, between December 2005 and February 2006, but then there was an increment of 11 species at the end of the sampling.
Fig. 4 Species accumulation curve of hawkmoths (Lepidoptera: Sphingidae) during the study, at the Tapacurá Ecological Station, Pernambuco, Brazil.
Seasonality and Abundance
We collected 69.7% of the specimens during the rainy season (March-August), which indicates a positive relationship with annual rainfall (richness: r = 0.65, abundance: r = 0.67).
May 2006 had the highest abundance (36 specimens) and richness (14 species) of hawkmoths, whereas in October 2005 and November 2006 (dry months) no sphingids were collected (Fig. 5). Twelve species were captured only during the rainy season and four only in the dry season, whereas the other 15 species occurred in both seasons
(Table II).
Fig. 5 Seasonal variability of abundance and richness of Hawkmoth (Lepidoptera: Sphingidae) and precipitation during the study period at the Tapacurá Ecological Station, Pernambuco, Brazil (June 2005 and January 2006 were not sampled).
TABLE II Seasonality of hawkmoth (Lepidoptera: Sphingidae) monthly collected with a light trap at the Tapacurá Ecological Station, Pernambuco, Brazil, during 27 months from October 2004 to February 2007. The moths were organized from the collections in January (hatched months correspond to the rainy
season).
Species
Manduca lefeburii lefeburii
Erinnyis ello ello
Hemeroplanes triptolemus
Perigonia lusca lusca
Manduca hannibal hannibal
Callionima parce
Nyceryx coffaeae
Enyo gorgon
Manduca rustica rustica
Manduca diffissa tropicalis
Neogene dynaeus
Erinnyis alope alope
Erinnyis crameri
Pachylia fícus
Eumorpha anchemolus
Eumorpha fasciatus fasciatus
Perigonia pallida
Protambulyx strigilis
Manduca sexta paphus
Xylophanes tersa tersa
Eumorpha vitis vitis
Agrius cingulata
Perigonia stulta
Xylophanes chiron nechus
Xylophanes libya
Cocytius antaeus
Enyo ocypete
Eupyrrhoglossum sagra
J
3
1
1
1
F
19
3
7
1
1
M
14
9
7
1
1
1
2
1
1
1
2
1
1
A
4
4
M
6
2
2
4
3
10
1
1
2
1
1
1
3
J
J
A
1
O
N
1
12
1
4
1
4
1
1
D
3
1
1
8
1
1
3
2
1
2
2
1
4
1
7
1
1
3
2
1
1
1
6
4
4
1
2
3
1
1
2
3
1
7
1
2
1
1
2
2
1
1
6
1
3
1
S
10
1
2
1
1
1
1
Total moths/
5
6
1
3
8
10
3
3
4
1
4
4
6
4
3
3
1
9
4
6
2
1
1
1
1
3
1
2
Individuals/ especie
46
12
1
3
21
59
3
4
5
1
8
6
13
5
7
3
1
36
9
12
2
2
3
1
1
5
1
2
Xylophanes loelia
Xylophanes pluto
Pseudosphinx tetrio
Total species/moths
Total individuals
1
3
4
5
13
14
17
8
6
10
5
8
6
1
7
1
1
1
1
3
1
277
The seven most abundant species were collected in both seasons, whereas the eighth most frequent species, Manduca sexta, was collected only in the rainy season. When analyzing each species separately, the cyclic behavior in relation to rainfall varied (Fig. 6). Only three species exhibited a positive correlation with rainfall: Callionima
parce (r = 0.62), Erinnyis crameri (r = 1) and M. sexta (r = 0.81).
Fig 6 Seasonal distribution of the eight most abundant species and its relationship with rainfall at the Tapacurá Ecological Station, Pernambuco, Brazil, in 27 months of samplig, from October 2004 to February 2007.
DISCUSSION
Although the present study has recorded the largest number of sphingid species for any study so far in northeastern Brazil, the distribution of individuals among the subfamilies corroborates
belong to the tribes Dilophonotini and Macroglossini.
Duarte et al. (2008)
, who noted that in all sphingid fauna inventories in Brazil, Macroglossinae has the highest number of individuals, and that over 50%
In the present study we made several new records for northeastern Brazil, which represent species that occur in low abundance: Enyo gorgon, Perigonia stulta, Eupyrrhoglossum sagra, Nyceryx coffaeae and Xylophanes chiron. Apart from these, eight other species were recorded for the first time for the Pernambuco Endemism Center:
Callionima parce, Erinnyis crameri, Xylophanes pluto, Agrius cingulata, Eumorpha vitis, Perigonia lusca, Perigonia pallida and the subspecies Manduca diffissa tropicalis. This result shows the importance of Tapacurá Station, an example of a dry forest, as a refuge for those insects.
Furthermore, some species that are rarely collected in northeastern Brazil were abundant in Tapacurá Ecological Station, such as Manduca lefeburii (the second most frequent species, with 46 individuals). This species had previously only been known from a single record of an individual collected in the open Atlantic rainforest of the state of
Lopes et al. 2006
Alagoas (
Duarte and Schlindwein 2005a
). Likewise, Manduca hannibal, very abundant in Tapacurá (21 individuals), has been recorded only once in the Atlantic Forest of the state of Pernambuco, where three individuals were collected (
“Tabuleiros” (Savanna areas) of the state of Paraíba (
). Finally, E. crameri, which had only one record with two individuals in the
Darrault and Schlindwein 2002
), was the fifth most abundant in Tapacurá, where 12 individuals were recorded. Again, this demonstrates the important role played by the Tapacurá Ecological Station to preserve and to maintain this insect group.
However, when comparing species abundance and richness among sites, it must be acknowledged that sampling methods have not been standardized in all inventories in northeastern Brazil, either in terms of number and time of sampling, total of sampling hours or the type of light trap used.
Despite the major difference in the time established to end captures (in most studies captures were carried out until 5 or 6 am, whereas in the present study captures ended at 2 am), we have obtained in Tapacurá Station the highest number of sphingid species for northeastern Brazil. Maybe this was a consequence of greater sampling effort in
the present study: 486 h (27 sampling sessions). Other studies had sampling efforts of 308 h (14 sessions) (
(
), 288 h (12 sessions) (
Duarte and Schlindwein 2005b
), 264 h (12 sessions) (
), 144 h (12 sessions) (
Duarte et al. 2001
), 72 h (six sessions) (
) and 16 h (four sessions)
Lopes et al. 2006
). The apparent stabilization of the species accumulation curve evident in the middle of the present sampling period may also have happened in other studies, leading to an interruption of sampling. Probably, an increase in hours and in number of sampling sessions could have raised species richness, as in Tapacurá.
In spite of the large sampling effort, species richness in Tapacurá may be even higher than recorded in the present study, given that there was an increment of one species one month before the last month of sampling (even after five sampling sessions without any new inclusion).
In tropical ecosystems, the change between well-marked dry and rainy seasons imposes cycles of leaf availability of host plants to the larvae of hawkmoths, which, according to
caterpillars, which hardly occurs in the dry season.
Haber and Frankie (1989)
, then determines the seasonal occurrence of adults. In a similar way,
Janzen (1988b)
found seasonality in the leaf-cutting activity of lepidopteran
Haber and Frankie 1989
As observed in Tapacurá, a positive relationship between the rainy season and the maximum richness and abundance of Sphingidae has been recorded in several ecosystems, such as dry forests of Costa Rica (
Laroca and Mielke 1975
) and the Atlantic Forest of the state of Paraná, Southern Brazil (
).
Inventories carried out in ecosystems of northeastern Brazil partially corroborate this hypothesis of a correlation between richness and abundance, and rainfall. There were significant correlations in a Caatinga area in the state of Paraíba (
in the Tabuleiros of the state of Paraíba (
). However, this correlations were not observed in humid swamp areas of the state of Paraíba (
Duarte and Schlindwein 2005b
) and in the state of Rio Grande do Norte (
), as well as
) or in the humid Atlantic Forest of the state of Pernambuco, Brazil (
).
The seasonality analysis of each species reveals cyclic patterns that remain unnoticed when the phenology of the sphingid community is analyzed as a whole. In only two species, Erinnyis crameri and Manduca sexta, the temporal distribution seems to follow the annual rainfall cycle. An exception can be noted for Callionima parce, which
exhibits a positive correlation with rainfall, but shows regularity in its activities, since it was captured in almost all months. In turn, Manduca lefeburii and M. hannibal, which have no relationship with rainfall, exhibited evident regularity; there was a demographic increment three or four months before rainfall peaks. Possibly the marked
population cycle of these two species is related to the phenology of the larvae's host plants in the study area or its surroundings. According to
Moré et al. (2005)
Rodal et al. 2005
, the host plants of M. lefeburii belong to the genus Casearia of the Salicaceae, a family that has not been sampled in our study area (
). There are some records of larval
Moss 1920
hosts for M. hannibal mostly in Solanaceae (eg. Solanum campaniforme Roem. & Schult.) and Verbenaceae, as in Clerodendrum fragrans (Vent.) Willd. (
; see Daniel Janzen's website for Costa Rican Lepidoptera).
No period seems to be more or less favorable for Protambulyx strigilis, Xylophanes tersa and Erinnyis ello. Seasonality analysis for the other species was not possible because of the low number of individuals collected.
It is premature to propose a comprehensive hypothesis regarding the cause of Sphingidae seasonality in Tapacurá Ecological Station, as the host plant community of the larvae and its phenology are unknown. However, the correlation between rainfall and occurrence of adult sphingids offers at least a plausible partial explanation for the
seasonal phenology of this insect group, and suggests that many other variables are involved.
Haber and Frankie (1989)
and
Cruz-Neto et al. (2011)
observed a relationship between seasonality of sphingid abundance and flowering phenology of plants pollinated by them. Similarly,
Lopes et al. (2006)
proposed a hypothesis that the reduction or
Primo 2008
disappearance of sphingid populations in small fragments of Atlantic Forest may occur due to the decrease of sphingophilous plants. However, in Estação Ecológica do Tapacurá there seems to be no positive relationship between sphingid abundance and number of flowering sphingophilous plants (
).
Apart from food availability for larvae (host plants) and adults (sphingophilous plants), variations in sphingid populations can be influenced by several other factors acting together. Among these factors might be, for example, the effect of the matrix, the size of the forest fragment, migration, and the presence of predators of larvae or adults.
Therefore, more studies are necessary to determine the complex causes of hawkmoth seasonality.
Acknowledgements
To Mr. Paulo Martins (Universidade Federal Rural de Pernambuco) for logistical support and permission for the development of the field work at Tapacurá Ecological Station; to Edinaldo Mendes da Silva, Airton Torres Carvalho and Eduardo Oliveira Barros for help with some of the samples. To Ivano Bonamici and Tarcila Nadia for their
help with the graphics. Financial support was provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, PhD fellowship to LMP - Graduate Student Program Agreement - PEC/PG/CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Research Grant to ICM).
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Received: July16, , 2012; Accepted: March15, , 2013
Correspondence to: Isabel Cristina Machado E-mail: [email protected]
Tel: +55 21 3907-8100
Fax: +55 21 3907-8101
[email protected]
http://dx.doi.org/10.1590/S0001-37652013000300018
Biological Sciences
Handling sticky Resin by Stingless Bees: Adhesive Properties of Surface Structures
MARKUS GASTAUER1*, LUCIO A.O. CAMPOS2, DIETER WITTMANN1
1
2
Institut für Nutzpflanzenwissenschaften und Ressourcenschutz (INRES), Fachbereich Ökologie der Kulturlandschaft: Tierökologie, Melbweg 42, 53127 Bonn, Germany
Universidade Federal de Viçosa, Departamento de Biologia Geral, Campus Universitário, s/n, 36570-000 Viçosa, MG, Brasil
ABSTRACT
Many Stingless Bees (Hymenoptera: Meliponini) like Tetragonisca angustula collect resin to defend their nests against intruders like ants or Robber Bees. Small portions of resin are attached to intruders bodies and extremities causing their immobilization. It has been observed that resin is removed easily from the bee's mandible but adheres strongly to
the intruder's cuticle. We tested the hypothesis that resin sticks lesser to the mandibles of Stingless Bees than to the surface of intruders due to special surface structures or adhesive properties of these structures. The surface structures of the mandible of T. angustula and the trochanter of Camponotus sericeiventris were studied by scanning electron
microscopy. To measure adhesion properties, selected surfaces were fixed on a fine glass pin and withdrawn from a glass tip covered with resin. The deformation of the glass pin indicates adhesion forces operating between the resin and the selective surface. The absolute value of the forces is computed from the glass pin's stiffness. It has been shown that
resin sticks more to the smooth mandible of the bee than to the structured trochanter of the ant. A new hypothesis to be tested says that the bees might lubricate their mandibles with nectar or honey to reduce the resin's adhesion temporarily.
Keywords: measuring adhesion forces; scanning electron microscopy; surface properties; cuticle; ultrastructure
RESUMO
Muitas espécies das abelhas-sem-ferrão (Hymenoptera: Meliponini) como Tetragonisca angustula coletam resina para a defesa dos seus ninhos contra invasores como formigas ou abelhas cleptobióticas. Pequenas porções de resina são aderidas nos corpos e extremidades dos invasores causando a sua imobilização. Observa-se que a resina é removida com
facilidade das mandíbulas das abelhas, mas cola fortemente na cutícula dos invasores. Foi testada a hipótese de que a resina adere menos nas mandíbulas das abelhas-sem-ferrão do que na superfície dos invasores devido a estruturas especiais de superfície ou a propriedades adesivas dessas estruturas. As superfícies da mandíbula de T. angustula e do
trocanter de Camponotus sericeiventris foram estudadas por microscopia eletrônica de varredura. Para medir suas propriedades de adesão, superfícies selecionadas foram montadas em uma alfinete de vidro fino e retiradas de uma ponta de vidro coberta com resina. A deformação do alfinete de vidro indica forças de adesão operando entre a resina e a
superfície selecionada. O valor absoluto da força é calculado através da rigidez do alfinete. Foi observado que a resina adere mais na mandíbula da abelha com superfície lisa do que no trocanter estruturado da formiga. Uma nova hipótese a ser testada diz que as abelhas lubrificam as mandíbulas com néctar ou mel para reduzir a adesão da resina
temporariamente.
Palavras-Chave: medir forças de adesão; microscopia eletrônica de varredura; propriedades de superfície; cutícula; ultraestrutura
INTRODUCTION
Armbruster 1984 Marsaioli et al. 1999
The Stingless Bee (Hymenoptera: Meliponini) Tetragonisca angustula (Latreille, 1811) defends its nests with resins collected from different plants (
,
Roubik 1989 Nogueira-Neto 1997
) which are stored in deposits within their nests (
Formicinae) enter the nest, worker bees form small pebbles of resin from these deposits and stick them onto the intruder's extremities until legs and antennas are tied up causing immobilization (
Radtke 1994
,
). When intruders like the ant Camponotus sericeiventris (Guérin, 1834, Hymenoptera:
Gastauer et al. 2011
). These pebbles are formed and transported by the bee's mandibles (
).
Gastauer et al. 2011
It has been observed that the resin, once attached on the intruder's extremities, clings very tight to its cuticle, while it is removed easily and without residues from the bee's mandible (
). Resin seems to stick lesser to the bee's mandible than to other insect's surfaces.
The aim of this paper is to test the hypothesis that resin sticks lesser to the bee's mandible than to the extremities of typical intruders because of specialized surface structure or composition. The mandible of T. angustula and the trochanter of the hind leg of C. sericeiventris have been selected as representative surfaces, on which structure and
adhesive properties were analyzed.
Scanning Electron Microscopy (SEM)
Mandibles of T. angustula as well as the trochanter of the hind leg of C. sericeiventris have been examined with the scanning electron microscope Hitachi S-2460, Japan, at voltages varying between 20 and 25 kV.
Individuals of both species had been killed by means of acetic ether. The body parts used for analysis had been dissected and immediately cleaned in a solution of dish washing liquid, kept in alcohol 70% for five days, then dehydrated in an ascending alcohol series. Afterwards the samples were transferred into acetone and subsequently cleaned
for 10 minutes in a bath of ultrasound (Bandolin Sonorex). The samples were mounted on pin stubs and sputtered with a 40 nm layer of gold.
Vouchers of both species have been incorporated in the collections of the Entomological Museum of the Universidade Federal de Viçosa (UFV-B).
Adhesion Force
To measure the adhesive properties of the cuticles, these were mounted to fine, pulled-out glass pins (P1) being withdrawn from a similar glass pin P2 covered with resin (Figure 1). The adhesive properties were measured as forces necessary to provoke adhesive failure. These forces are called adhesive forces in the following.
Figure 1 Instrument to measure adhesion forces. Left – Overall view, right – details of the working range. The adjustments (D) of the micromanipulator (MM) are used to position the cuticles fixed on the glassy pin P1 horizontally; the vertical positioning is executed with the coarse (C) and the fine (F)
adjustment. MC: Microscope, P2 : Glass pin covered with resin, s: distance describing the deformation of P1 between point of contact (shown as upper pin1) and position of separation (lower pin1).
Production of Glass Pins
The pins P1 and P2 were produced from glass bar fragments (diameter 1 mm, length 20 cm). With the Puller P-97 from Sutter Instruments Co the midpoint of the bars was heated up to 560°C. With an acceleration of 110 m/s2, the pins were stretched; the resulting diameters of the pins' tips ranged from 1 and 10 µm. For pin types P1, diameters
of 3 to 4 µm were selected, types P2 of 10 µm.
Fixation of the Cuticle Sample on P1
For analyzing the adhesive properties of the cuticles of the trochanter of C. sericeiventris and the mandible of T. angustula, the specimen were captured, knocked out with CO2 and dissected immediately.
All samples were fixed to the tip of P1 with warmed dental wax (65°C). P1 was mounted on the micromanipulator (MM in Figure 1) and resin was installed on P2 immediately.
Installation of Resin on P2
The dichloromethane cleaned P2 was dipped 2 - 3 mm into a mass of 2 g resin collected from a deposit of a colony of T. angustula. When the tip of P2 was covered completely by a drop of resin (length: 20-50 µm, width: 10-20 µm, see Figure 2), it was clipped perpendicular to P1 on the object table of the microscope MC (Figure 1). The
behavior of the attached resin is described in Figure 2.
Figure 2 Behavior of resin after the attachment on P2. Left - Resin from the bees hive immediately after the attachment on P2. Middle - Once being attached to the glass pin it crawls upwards towards the base of P2 (dashed arrow). For a short moment just the tip of the pin is covered with resin (white arrow).
This is the moment to measure the adhesive properties on the cuticle samples. Right - About 60 seconds later the cover on the tip of P2 is vanishing (white arrow) and the resin has to be reinstalled to continue measurement.
Measurement
All treatments and measurements with resin were conducted at 19°C.
The adhesion forces were measured at the moment, when P2 was covered with only a thin, but still complete layer of resin (Figure 2, middle). P1 was slowly moved upwards to P2 until the cuticle sample touched the resin on P2. The two glass pins were kept in this position for 5 seconds so that resin and cuticle contacted each other. Then P1 was
slowly withdrawn from P2 with a constant speed of 62.5 µm/s (corresponding to one turn of the micromanipulator's fine adjustment), until resin and cuticle disconnected. The distance s (Figure 1) between position of contact and position of separation describes the deformation of P1 and depends on the stiffness of P1 as well as the adhesion
forces between resin and cuticle.
Calibration of P1
The adhesion forces were calculated from the stiffness of P1. Each P1 that was used for measurement was carefully pressed with the micromanipulator onto a digital balance in steps of 62.5 µm. Step by step the weight indicated by the balance was recorded.
Calculation of the Adhesive Properties
The adhesion forces FA is given by the product of mean distance s between position of contact and position of separation and the stiffness c of P1.
Statistical Evaluation
To proof the results statistically, the non-parametric Mann-Whitney Test has been used. Probability values lesser than 1% were considered as highly significant.
RESULTS
The cutting edge of the mandible of workers of T. angustula is composed of a large blade and a pointed tooth. The surface of these structures are smooth (Figure 3) without microstructures. On several mandibles we found distinct traces of use like scratches or broken edges. On the other hand, the trochanter of the ant C. sericeiventris is scaled
(Figure 4).
Figure 3 Left – Smooth surface of the ventral side of the tip of the mandible of Tetragonisca angustula, right – mandible's tip with distinct traces of use.
Figure 4 Surface structures on the trochanter of Camponotus sericeiventris.
Calibration trials of all P1 show linear relation (R2 ≥ 0.998) between deformation in µm of P1 and weight in µg indicated by the balance (Figure 5), so that adhesion forces had been interpolated by linear regression.
Figure 5 Calibration trial of glass pins P1 used to measure adhesion failure between resin and the surface of the mandible of Tetragonisca angustula and the trochanter of Camponotus sericeiventris.
During measurement of the adhesion forces, hydrophobic interaction has been observed between resin and the tested surfaces. The resin attached on P2 attracts the tested cuticles and the cover slip on P1 with a jerk as soon as it reaches a critical distance somewhere between 1.5 µm and 1 µm.
Figure 6 shows that adhesion of resin on the structured cuticle of the trochanter of C. sericeiventris is six times smaller than on the smooth cuticle of the tip of the mandible of T. angustula. These differences are highly significant using the Mann-Whitney-Test for independent samples.
Figure 6 Adhesion of resin on the surface of the trochanter of Camponotus sericeiventris and the mandible of Tetragonisca angustula in µg with electron-microscopical photos of the examined surfaces. The circles on the photographs show the contact area of the resin-covered glass pin P2 . Data include
standard deviation and number of repetitions.
DISCUSSION
The results of the adhesion force measurement refute our hypothesis that resin sticks lesser to the bees' mandible than to other insects' surfaces – even if the contact area between resinous and insect's surface in experiment is smaller than in reality.
Kendall 2001
Adhesion on rough surfaces like the scaled trochanter of C. sericeiventris is lower than on smooth ones like the mandible of T. angstula (
should stick more on the bee's mandible than on the trochanter of the ant when contact area is increased.
). Major contact areas covering hairs and various scales on the ant's trochanter surface, will reduce adhesion further. As such adhesion reducing structures are lacking on the bee's mandible, resin
However, these findings do not explain why resin, once attached to the mandible of T. angustula, is removed easily and without leaving residues behind. To manage that, the bee should possess mechanisms to reduce adhesion of resin on the mandible. T. angustula might change the adhesive properties temporarily by lubricating their mandibles
Voigt and Gorb 2008
with adhesion reducing substances. Lubricating substances seem to be common within insects. The body surface of the mirid bug Pameridea roridulae (Reuter 1907) is covered by a lipid layer avoiding the bug getting stuck in the resinous surface of its host plant (
secretions of the mandibular, salivary or other cephalic glands as proposed by
distributed, it reduces the adhesion of resin on the complete mandible.
Santos et al. (2009)
). In stingless bees such lubricating substances might be
. As production of secretions is a complex and energy consuming processes, worker bees might even regurgitate nectar from the nectar crop. The liquid could run down the ventral side of the mandible via a small groove as illustrated in Figure 7. Once
Figure 7 Electron-microscopical overall-view of the posture of Stingless Bees' mandibles, here from Plebeia lucii (Moure, 2004, magnification 200x, 25 kV). White arrows mark the hypothetical path of secretions or regurgitated nectar or honey moisten the complete ventral side of the mandible.
This groove on the base of the ventral side of the mandible, also observed in other stingless bee species (Figure 8,
order to form, cut and knead resins without sticking to them.
Stort et al. 1986
) and the honey bee Apis mellifera, is discussed by
Goodman (2003)
as a feature to channel liquids to the tips of the mandibles. The groove might be an adaptation of fsecbees to alter adhesive properties of the mandibles in
Figure 8 Electron-microscopical photos showing the similarity of the mandible's ventral side of some Stingless Bees that collect resin. Left – right mandible of T. angustula, right – left mandible of Frieseomelitta varia (Lepeletier, 1836).
Further investigations have to test, if adhesion of resin is reduced on mandibles greased with nectar or honey. To prove our hypothesis, chemical research activities have to attest the existence of residues of the adhesion reducing substances like honey, nectar or sugar in resins treated by Stingless Bees.
Acknowledgements
Thanks to Bernd Hoffmann and Wolfgang Rubner from the Institute of Bio- and Nanosystems, Institute 4 (Biological Layers), Research Center Jülich, Germany, for their help in developing a transportable apparatus to measure adhesion forces. We are grateful to Karin Ulmen from the Museum König in Bonn, Germany, to grant access to the
SEM. Some parts of this research were supported by the DFG and the DAAD.
REFERENCES
Armbruster WS. 1984. The role of resin in angiosperm pollination: Ecological and chemical considerations. Am J Bot 71: 1149-1160. [ Links ]
Gastauer MM, Campos LAO and Wittmann D. 2011. Handling sticky resin by Stingless Bees. Rev Bras Ent 55: 234-240. [ Links ]
Goodman J. 2003. Form and function in the Honey Bee. Cardiff: International Bee Research Association, 220 p. [ Links ]
Kendall K. 2001. Molecular Adhesion and Its Applications: The Sticky Universe. New York, Boston, Dordrecht, London, Moscow: Kluwer Academic, 440 p. [ Links ]
Marsaioli AJ, Porto ALM, Gonçalves RAC, Oliveira CMA, Manfio GP and Bittrich V. 1999. The Ecosystem of Microorganisms, Bees, and Clusia Floral Res

Vol 85 Num 3 Septiembre 2013

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