CARYOLOGIA Vol. 56, no. 4

Vol. 56, no. 4: 405-411, 2003
CARYOLOGIA
Cytogenetic characterization of hybrids offspring
between Colossoma macropomum (Cuvier, 1818)
and Piaractus brachypomus (Cuvier, 1817)
from Caicara del Orinoco, Venezuela
M. NIRCHIO1, A.S. FENOCCHIO2, A.C. SWARÇA3, J.E. PÉREZ4, A. GRANADO5, A. ESTRADA5 and E. RON1
1
Escuela de Ciencias Aplicadas del Mar. Universidad de Oriente, Isla de Margarita, Venezuela. Apartado Postal 630-Porlamar.
Universidade Federal do Paraná, Departamento de Genética, Centro Politécnico, Curitiba, Brazil; e-mail: afenocch@
fceqyn.unam.edu.ar.
3 Universidade Estadual de Londrina, Departamento de Biologia Geral, Caixa Postal 6001, CEP 86051-990, Londrina, PR,
Brasil; e-mail: [email protected].
4 Instituto Oceanográfico de Venezuela, Universidad de Oriente, Apartado 243, Cumaná, Venezuela; e-mail: jperez@
telcel.net.ve.
5 Instituto Limnológico, Universidad de Oriente, Caicara del Orinoco, Estado Bolívar, Venezuela; e-mail: [email protected].
2
Abstract - Conventional karyotype and nucleolar organizer regions (NORs) of
C. macropomum x P. brachypomus hybrids and parental species are reported.
A modal diploid number of 54 chromosomes and a fundamental number (NF) of
108 were found for C. macropomum, P. brachypomus and their hybrids. P. brachypomus shows a pair of silver stained chromosomes, while cells with three and four
Ag-NOR bearing chromosomes were observed in C. macropomum. The hybrids
consistently presented cells with a single metacentric Ag-NOR bearing chromosome and cells with three Ag-NOR bearing chromosomes. The FISH technique
was employed to localize 18S rDNA in the chromosomes of the parentals and the
hybrids. In P. brachypomus the FITC signals appeared in the SM pair as when
stained with silver salts. In C. macropomum the signals were evidenced in six
chromosomes. In the hybrids, as expected, the FITC dots were observed in four
chromosomes. All the techniques employed in the present work represent good
tools to identify the parentals and distinguish them of the hybrids.
Key words: Fisheries; Interspecific hybrids; Colossoma macropomum; Piaractus
brachypomus; Cytogenetic Monitoring; NORs; in situ hybridization.
INTRODUCTION
The production of interspecific offsprings to
obtain individuals in which the best characteristics of the parental species could converge has
been one of the goals of aquaculturists (DUNHAM
1987; CHEVASSUS and DORSON 1990).
In Latin America hybridization between different species of the Family Characidae has been
* Corresponding author: e-mail: [email protected]
achieved with success in several Research Institutes. Particularly Colossoma macropomum and
Piaractus brachypomus have been object of attention because of their high production yields, easiness for cultivation and massive production of
alevins under controlled reproduction conditions
(ALMEIDA-TOLEDO et al. 1988; KOSSOSWKI et al.
1983; MARTINO 1994).
The analysis of genome structure of hybrid
fishes is necessary to achieve an adequate genetic evaluation in order to get a basic understanding that allows to distinguish the types of genet-
406
NIRCHIO, FENOCCHIO, SWARÇA, PÉREZ, GRANADO, ESTRADA
ic products resulting from a successful hybridization process (A LMEIDA-T OLEDO FILHO et al.
1994) and to assess the potential biological hazards of the hybrid to the genetic integrity of the
parental species, wild or cultivated (PÉREZ and
RYLANDER 1998). Cytogenetics provides some
techniques that could help to understand better
the behavior and structure of the chromosomes
that was integrate in the hybrids. So, the analysis
of the karyotype and principally of the nucleolus
organizer regions (NORs) can be appropriate
and sensitive methods to verify the ploidy levels
as well as the origin of the chromosomes in fishes (ALMEIDA-TOLEDO FILHO et al. 1994; ZHANG
and TIERSCH 1997).
Locations of the NORs are easily detected by
silver impregnation but this technique has the
limitation imposed by their necessary activity in
the previous interphase.
DNA-DNA in situ hybridization is increasingly important for understanding the physical
position of all classes of DNA sequences along
chromosomes of both plants and animals (HESLOP-HARRISON 1991). The fluorescence in situ
hybridization (FISH) with rDNA probes is a useful tool to reveal precisely all the chromosomes
involved in nucleolar organization as well as the
physical location and copy number of the ribosomal genes.
This paper reports the karyotypic formulaes
and organizer nucleolar regions (NORs) of Colossoma macropomum, Piaractus brachypomus and
their hybrids by means of conventional and AgNOR staining and in situ hybridization with rDNA
biotinilated probes.
MATERIALS AND METHODS
Five individuals of each parental species (Colossoma macropomum and Piaractus brachypomus) and 25
specimens of the resulting offspring obtained by in vitro fecundation of ovocites of P. brachypomus (female
N° 2) with sperm of C. macropomum (males N° 2 and
N° 63) were analyzed.
Each specimen was injected intraperitonially with
a yeast suspension (1cc/100 g of weight) to stimulate
the cellular proliferation in the hematopoietic organ
(kidney) (LOZANO et al. 1988). After a 18–hour period each fish was injected with colchicine solution
(0.5%; 1 ml/100g body weight), and maintained in a
well aerated aquarium for 4-6 hours. The preparations for the visualization of the chromosomes, were
and RON
obtained following the technique described by NIRCHIO and CEQUEA (1998).
For the determination of the conventional karyotype, the preparations were stained during 20 minutes with Giemsa (10%) diluted in buffer phosphate,
pH 6.8. The detection of the nucleolar organizer
regions (NORs) was performed following the methodology described by HOWELL and BLACK (1980). The
18S rDNA segment containing 1700pb of the fish
Oreochromis niloticus was used for fluorescence in
situ hybridization (FISH) and labeled with biotin-14dATP by nick translation according to the manufacturer’s instructions (Gibco cat Nº 18247-015). The
hybridization technique, post-hybridization washes
and visualization were performed according to
SWARÇA et al. (2001).
The mitotic figures were photographed using
green filter and ASA 50 film. The resulting photographs were then scanned (1,200 dpi) and stored as
*.tif images
Long arm (L), short arm (S) and total length were
measured from each chromosome to the nearest 0.01
mm using the measuring tool of ADOBE PHOTOSHOP Software v. 5.0. Chromosomes were classified
as described by LEVAN et al. (1964) and ordered in
decreasing size.
RESULTS AND DISCUSSION
The diploid numbers determined for Colossoma macropomum, Piaractus brachypomus and their
hybrids were 54 chromosomes (all bi-armed) and
the fundamental number (FN) were 108 (Fig. 1).
The chromosome groups were arranged according to the proportion L arm/S arm. The karyotypes of parental species showed a complement
constituted by 14 pairs of M and 13 pairs of SM
chromosomes, however a clear differentiation
among the karyotypes of the species studied here
have been observed: the first pair of SM chromosomes of C. macropomum is around 30% longer
than the second instead, in P. brachypomus were
evidenced a gradual size decrease of all the chromosomes of the complement (Fig. 1a, b).
In the hybrid 28 M and 26 SM chromosomes
were distinguished (Fig. 1c). In this case these
elements were not paired because in some cases
they not constitute homologues and in other they
would be homeologue chromosomes. This feature is very evident in the group of SM when the
first chromosome has not any paireable partner
(Fig. 1c).
These results are partially in agreement with
previous reports that indicate a numerical com-
CYTOGENETIC CHARACTERIZATION OF HYBRIDS BETWEEN COLOSSOMA MACROPOMUM AND PIARACTUS BRACHYPOMUS
407
a
b
c
Fig. 1 – Conventional karyotype (Giemsa) of Colossoma macropomum (a), Piaractus brachymomus (b) and the hybrid (c).
M: metacentric; SM: submetacentric.
408
plement 2n=54 (NF= 108) for C. macropomum
and P. brachypomus, but differs respect to the
number of M and SM elements. In the case of C.
macropomum previous reports indicate 18M +
36 SM (KOSSOWSKI et al. 1983); 20M + 34 SM
(ALMEIDA TOLEDO 1987) and 26 M + 28 SM
(NAKAYAMA et al. 1990), while for P. brachypomus
a complement constituted by 34M + 20 SM
(NAKAYAMA et al. 1990) has been described.
These differences could be attributed to technical
features, as chromosomal condensation.
ALMEIDA TOLEDO et al. (1988) found that
conventional staining with Giemsa would not
allow to differentiate the hybrids from the
parental species in the cross C. macropomum x P.
mesopotamicus.
NIRCHIO, FENOCCHIO, SWARÇA, PÉREZ, GRANADO, ESTRADA
and RON
In the case of the cross C. macropomum () x
Mylossoma duriventris () the karyotype of the
parental species are numerically equal, but there is
an acrocentric marker in M. duriventris which is
detected in the hybrids (KOSSOWSKI et al. 1983).
P. brachypomus studied in the present work
have a pair of SM NOR bearing chromosomes
when silver stained. These regions are located at
the ends of the long arm (Fig. 2a, b).
In C. macropomum were observed cells with
four (Fig. 2c) and three (Fig. 2d and 2e) metacentric Ag-NOR bearing chromosomes. The silver salts produce evident dark blocks on terminal
regions of these chromosomes (Fig 2f).
The hybrids consistently presented cells with
a single Ag-NOR bearing chromosome and cells
Fig. 2 – Chromosomic complement of Colossoma macropomum stained with Silver Nitrate. The arrows show the NOR-bearing chromosomes. In the insets, amplification of silver stained chromosomes with the nucleolar organizer region. Piaractus
brachypomus (a, b); Colossoma macropomum (c, d, e, f); hybrid (g, h, i, j, k).
CYTOGENETIC CHARACTERIZATION OF HYBRIDS BETWEEN COLOSSOMA MACROPOMUM AND PIARACTUS BRACHYPOMUS
409
Fig. 3 – Fluorescence in situ hybridization with biotin-labeled 18S rDNA probe in Colossoma macropomum (a, b),
Piaractus brachypomus (c, d) and the hybrid (e, f). Arrows indicate rDNA FISH.
410
with three Ag-NOR bearing chromosome (Fig.
2g, i, respectively). In the case of the cells with a
single silver stained chromosome, it was a SM
element like the one of P. brachypomus (Fig. 2h).
When three Ag-NOR bearing chromosomes were
observed, the conspicuous black dots always was
located at the terminal ends of the long arm of an
element SM and less evident blocks on the terminal region of two metacentrics (Fig. 2j), like in
the parental C. macropomum.
The silver staining of the NORs only reveal
the position of the genes for the ribosomal RNA
in chromosomes that were active in a preceding
interphase (HSU et al. 1975; MILLER et al. 1976).
An alternative to detect all the chromosomes
involved in nucleolar organization as well as the
exact location of the ribosomal genes, is the application of the fluorescence in situ hybridization
(FISH) with rDNA probes.
In this work the FISH technique was successfully employed to localize 18S rDNA on the chromosomes of Colossoma macropomum, Piaractus
brachypomus and their hybrids. In P. brachypomus
the FITC signals appeared on the long arm of the
submetacentric pair in all analyzed metaphases
(Fig. 3c,d), like when the chromosomes preparation were stained with silver salts.
Furthermore, in C. macropomum the signals
were evidenced on six chromosomes, at the short
arm of two metacentric pairs and on the long
arm of one submetacentric pair (Fig. 3a, b), but
when was applied the Ag-NO3 were evidenced
only three or four NOR-bearing metacentric
chromosomes.
According to PENDÁS et al. (1993) the silver
staining is not recommended to localize rRNA
genes, but is the best approach to study NOR
expression. In accordance with these authors, in C.
macropomum the FISH technique have evidenced
two chromosomes that were not detectable by
AgNO3 staining (probably inactives or silent during the previous interphase). This inactivation may
be due to the presence of some type of gene interaction, like a partial inactivation of the genes.
In the hybrids, when was applied the AgNO3
staining were evidenced cells with a single metacentric chromosome marked and cells with three
chromosomes marked, one SM and two M chromosomes. However, in this case, FISH allows to
identify the positive FITC signals on four chromosomes, on the short arm of two metacentric
pairs and on the long arm of two submetacentric
(Fig 3e, f), as expected.
NIRCHIO, FENOCCHIO, SWARÇA, PÉREZ, GRANADO, ESTRADA
and RON
On the basis of shape and size, was inferred
that one of these two submetacentrics correspond
to the Ag-NOR bearing chromosomes of Piaractus brachypomus, and the others (one SM and
two M) would correspond to the NOR bearing
chromosomes of C. macropomum.
This results, in the same way that in C. macropomum confirm that the silver staining only shows
NORs that were active in the preceding interphase, once the AgNO3 react with acid proteins
associated with rDNA (JORDAN 1987).
In this work, conventional coloration helps to
identify the chromosome types and allow to distinguish clearly the karyotype of the offspring
resulting from the cross between C. macropomum
and P. brachypomus, the hybrids also were perfectly differentiated by the number and location
of nucleolar genes.
Thus, both, conventional karyotype (Giemsa
stained) and NOR phenotype (Ag and FISH),
can be used as valid methods to identificate
parental species and to verify the origin of the
hybrids.
Acknowledgments – The authors are thankful to
Dra. Ana Lúcia Dias and Dra. Lúcia Giuliano-Caetano from the University Estadual de Londrina
(Brazil) for providing the rDNA 18S probe for
hybridization experiments and for their continuous cooperation. Swarça, A.C. was supported by
fellowship from CAPES.
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Received December 19, 2002; accepted April 8, 2003