Reproductive biology of Paysandisia archon

Transcription

MARCHE POLYTECHNIC UNIVERSITY
Department of Agricultural, Food and Environmental Sciences
Scientific Field: AGR 11 - General and Applied Entomology
PhD School 2010-2012
Curriculum: Crop Production and Environment
11th Cycle new series
Reproductive biology of Paysandisia archon
(Burmeister 1880): behavioural and
morphological investigations
PhD Thesis of:
Dr. Elisa Verdolini
Tutor:
Cotutor:
Coordinator:
Dr. Paola Riolo
Prof. Nunzio Isidoro
Prof. Bruno Mezzetti
Contents
Abstract
1
Introduction
The Castniidae family
Paysandisia archon (Burmesister 1880), the castniid palm moth
Pest management with semiochemicals
Intraspecific communication in Lepidoptera
References
3
4
5
7
8
9
Objective
15
Chapter 1
Materials and methods
Results
Discussion
References
17
17
21
37
41
Chapter 2
Results
Discussion
References
45
46
47
57
60
Concluding remarks and future works
65
Acknowledgements
67
Abstract
Paysandisia archon (Burmeister 1880) has been accidentally introduced to
Europe from Argentina in the mid nineties, becoming an invasive species in
Italy, Spain and France, where it has been the cause of high palm mortalities,
in both nurseries and urban areas.
The aim of this study was to provide a qualitative and quantitative
description of P. archon courtship and copulatory behaviour in semifield
condition. Then, to assess the role of olfaction, visual and mechanosensory
stimuli, bioassays with antennectomized adults and dummies, were
performed. Moreover morphological investigations of ovipositor and
antennae were carried out.
A fairly fixed pattern of courtship and copulatory sequence emerged,
consisting of 10 steps. In P. archon courtship behaviour, we showed that
visual cues are involved deeply in mate location. P. archon females first
approached the perching male who then pursued activated mainly by the
movement of flying female. We hypothesized that at close range, during
female first approach or/and pair flight, olfactory cues could be released by
the female stimulating the copulatory behaviour.
Our morphological investigations confirmed that there is no evidence of
glandular tissues in the ovipositor, commonly found in female moths.
Moreover we observed that P. archon antennae are thin and clubbed without
any evident sexual dimorphism and reduced sensillar surface as in day-flying
butterflies. Six types of sensilla were found: chaetica, likely mechanogustatory chemoreceptors; trichoidea, basiconica and auricillica multiporous
olfactory chemoreceptors; coeloconica with olfactory or a double olfactorythermoreceptory function and ampullacea likely thermo-hygroreceptors.
All these results have pointed out behavioural and morphological similarities
of this castniid palm moth with butterflies rather than moths, providing the
necessary background knowledge on several aspects of P. archon
reproductive behaviour of practical importance in Integrated Pest
Management strategies.
Paysandisia archon (Burmeister, 1880) è stata accidentalmente introdotta in
Europa dall’Argentina nella metà degli anni ’90, diventando una specie
dannosa in Francia, Italia e Spagna causando un’elevata mortalità di palme,
sia in vivaio che in ambiente urbano.
Lo scopo di questo studio è stato di fornire una descrizione qualitativa e
quantitativa del comportamento di corteggiamento e accoppiamento in P.
archon. Inoltre, per approfondire il ruolo degli stimoli olfattivi, visivi e
1
meccanosensoriali, sono stati condotti dei biosaggi con adulti aventi antenne
amputate e zimbelli. Infine sono state effettuate indagini di morfologia
funzionale dell’ovopositore e delle antenne.
E’ stato osservato che la sequenza di corteggiamento e accoppiamento in P.
archon è composta da 10 fasi. È emerso che la vista è coinvolta nella
localizzazione del partner. Le femmine si sono avvicinate per prime al
maschio che poi le ha inseguite stimolato principalmente dal movimento
della femmina in volo. Abbiamo ipotizzato che a breve raggio, durante
l’avvicinamento della femmina e/o il volo di coppia, stimoli olfattivi
potrebbero essere emessi dalla femmina stimolando l’accoppiamento.
Le indagini morfologiche hanno confermato che, in P. archon, non c’è
evidenza di tessuti ghiandolari nell’ovopositore, generalmente presenti nei
lepidotteri notturni. Inoltre, le antenne sono clavate senza alcun evidente
dimorfismo sessuale e con una ridotta superficie sensillare come nei
lepidotteri diurni. Infine 6 tipi di sensilli sono stati identificati: chetici,
probabili meccano-chemiocettori gustativi; tricoidei, basiconici e auricillici
chemiorecettori olfattivi; celoconici olfattivi o chemiorecettori olfattivi e
termorecettori e ampullacei probabili termo-igrorecettori.
Questi risultati hanno evidenziato in P. archon analogie comportamentali e
morfologiche con i lepidotteri diurni piuttosto che con i lepidotteri notturni,
fornendo conoscenze su diversi aspetti del comportamento riproduttivo di
questa specie di importanza applicativa nelle strategie di lotta integrata.
2
Introduction
The worldwide intensive trade of plants and goods and an increase in tourist
traffic have caused the incidental introduction of exotic phytophagous
insects throughout Europe and in Italy, becoming a quite common event.
Further, due to Mediterranean climate, several alien subtropical species have
recently become established (Pellizzari et al., 2005).
Two alien pests of palm trees have been accidentally introduced into Europe
through the import of infested plants: Paysandisia archon (Burmeister 1880)
(Lepidoptera, Castniidae), the castniid palm moth, and Rhynchophorus
ferrugineus (Olivier 1790) (Coleoptera, Curculionidae), the red palm weevil.
Both species, endophagous borers, have become invasive in many European
countries, representing a serious threat to palms. They are listed in the EPPO
(European and Mediterranean Plant Protection Organization) A2 List of
“Pest recommended for regulation as quarantine pests”, respectively n. 338
and n. 332 (EPPO, 2008 a and b).
The potential risk and impact on the palm tree populations and landscape in
Southern Europe can be severe.
Palms are present in very large number as amenity trees along streets, in
public and private gardens, golf courses, hotel grounds and collections of
historical value, becoming over the years an essential element of urban
landscape.
Native palms are threatened: Phoenix canariensis (Chabaud) in Canary
Islands, Phoenix theophrasti (Greuter) in Crete and Chamerops humilis
(Linneus) endemic all over the Mediterranean basin and also in Italy.
Tourism in the Elche palm oasis in Spain, UNESCO site, and in the French
and Italian Riviera can be negatively affected.
Furthermore, numerous nurseries specialized in palm growing of great
economic value are present throughout the Mediterranean basin and date
palm production is important from North Africa to the Persian Gulf (CABI,
2012).
Also in the Marche region, Central-Eastern Italy, ornamental palm species
are not only an extremely important part of the landscape, the Adriatic Palm
Riviera, but also of high economic importance for tourism and nurseries
specialized in their commercial cultivation (Riolo et al., 2004).
3
The Castniidae family
Currently the Castniidae family (Lepidoptera) is placed into the Sesioideae
superfamily with Brachodidae (little bear moths) and Sesiidae (clear-wing
moths) (Minet, 1991). Some authors have suggested the Castniidae family to
be allied with Cossidae (wood moths) basing on some characteristics such as
the egg shape, the endophagous habit of the larvae and the presence of dorsal
abdominal spines on pupae (Mosher, 1916; Forbes, 1923; Miller, 1986).
The family includes two subfamilies. The Castniinae subfamily contains the
Neotropical species, tribes Castniini and Gazerini, and the Australian
species, tribe Synemonini (Miller, 1995; Edwards et al., 1998). Castniini and
Gazerini are found in Central and South America containing 134 species
listed by Miller (1995) and reduced to 81 assigned in 32 genera following
the revision of Lamas (1995). The Synemonini are found in mainland
Australia containing 44 species, of which only 24 described and placed in
the genus Synemon (Doubleday) (www.environment.gov.au). The
Tascininae is a small subfamily with a single genus Tascina (Westwood)
with five species occurring in South East Asia, Indo-Malayan region
(Fukuda, 2000).
There is a paucity of information concerning life history, ecology and
distribution because of the endophagous habit of the larvae, the diversity of
larval foodplants and the scarce economic importance of most castniid
species. Few castniid species infest crops such as banana and sugar cane
(Miller, 1986).
Neotropical castniid larvae are stem and root borers associated with
monocotyledonous plants of the tropical moist forest vegetation, such as
Arecaceae, Bromeliaceae, Cyperaceae, Marantaceae, Orchidaceae, Poaceae,
Ecdeiocoleaceae and Lomandraceae (Scoble, 1992; Edwards et al., 1998).
Castniid adults are medium-large sized moths with diurnal flight habit and
cryptic forewings but brightly coloured hindwings, characteristics leading
rather to call them “butterfly-moths” or “sun moth” (Tindale, 1980).
As they are day-flying moths, some reports exist about their day flight
period. Seitz (1913) has reported they start to fly when the tropical sun is
strong, after 10.00 hour, confirmed by Miller (1986) who has observed a
flight period from 9.30 to 15.00 hours.
Regarding mating behaviour, copulations of Neotropical castniids have been
rarely observed lasting for an average of 30 minutes in Castniomera
atymnius (Dalman) and 1-3 hours, even more, in Eupalamides cyparissias
(Fabricius), crepuscular species (Lara, 1964; Korytkowski and Ruiz, 1980).
4
Introduction
Paysandisia archon (Burmesister 1880), the castniid palm moth
Paysandisia archon (Burmeister 1880) (Lepidoptera: Castniidae) is a
Neotropical castniid moth indigenous to South America, restricted to
northern Argentina, South East Brazil, Western Uruguay, and Paraguay
(Miller, 1986; Lamas, 1995; Sarto I Monteys, 2002).
The moth is the only species of the Castniidae family present in Europe. It
has been accidentally introduced to Europe from Argentina in the mid
nineties, hiding as larvae in imported palm trees, mostly Butia yatay
(Martius) Beccari and Trithrinax campestris (Burmeister) Drude and
Grisebach. At present, the moth has been reported from Bulgaria, Cyprus
Island, Czech Republic, Denmark, France, Greece, Italy, Slovenia, Spain,
Switzerland and United Kingdom. In France, Italy and Spain P. archon is
considered an invasive species (EPPO Reporting Service, 2010/145; CABI,
2012). In Italy the first record dates back to November 2002. The moth has
first been detected in the coastal area of Campania, province of Salerno,
approximately one year later in Marche, Ascoli Piceno province, and then in
Tuscany, Sicily, Abruzzo, Puglia, Liguria, Emilia-Romagna, Veneto, Friuli
Venezia Giulia, Lazio and Lombardy (EPPO Reporting Service, 2010/054,
2010/207; CABI, 2012).
In Europe it appears to have a large host range, including C. humilis (EPPO,
2008 b). In the Marche region, Central-Eastern Italy, attacks by P. archon in
ornamental palm nurseries have been resulted in up to 90% loss of
production. In the Marche region, this moth bas been detected on: C.
humilis, P. canariensis, Trachycarpus fortunei (Hooker) Wendland and
Washingtonia filifera (Lindley) Wendland (Riolo et al., 2004, 2005) as well
as in France and Spain. Long time ago Bourquin (1933) have reported that
this castniid palm moth had the potential to become a serious threat of
palms.
Larvae, internal specialized feeders, are not easily to detect and symptoms
differ among palm species, shown at different levels such as leaves, rachis
and stipe. Heavy larval attack may kill the palm tree, but infested plants can
be asymptomatic (Drescher and Dufay, 2001; Riolo et al., 2004; Sarto I
Monteys and Aguilar, 2005).
Adults are powerful day-flying moths. Their males are very territorial
showing patrolling behaviour in the wild (Sarto I Monteys et al., 2012) as
reported for other Neotropical castniids (Seitz, 1913, Salt, 1929; Lara, 1964;
Miller, 1986; Romero, 1998).
Regarding mating behaviour, very few copulations have been observed
lasting an average of about 38 minutes (Sarto I Monteys et al., 2012).
Recently, Delle Vedove et al. (2012) have provided basic information on P.
5
archon reproductive biology: the diurnal flight activity has a peak from
11.00 to 16.00 hours with sexual activity mainly occurred from 11.00 to
15.00 on the emergence day, proving that the adults are sexually mature few
hours after emergence. Moreover females are generally monandrous, laying
eggs from 0 to 4 days after mating.
Current Integrated Pest Management (IPM). Because P. archon is not a pest
in its native country due to its lack of economic importance, there is little
information and no specific control measures have been developed
(Bourquin, 1933; Drescher and Dufay, 2002; EPPO, 2008 b; Sarto I Monteys
and Aguilar, 2005).
In Europe prevention by plant trade regulation is necessary to prevent further
spread. After inclusion in the EPPO A2 List as quarantine pest, since 2009
the moth have been included in European Legislation, annexes II, IV and V
of Commission Directive 2000/29/EC amended by Commission Directive
2009/7/EC, on protective measures against the introduction into the
Community of organisms harmful to plants or plant products and against
their spread (EU, 2009).
Because of the concealed nature of their larvae management by chemical
insecticides appears difficult. Frequent applications have been required
against first instar larvae and have proved to be not effective in reaching
hiding larvae and cocoons (Sarto I Monteys and Aguilar, 2005; Nardi et al.,
2009). Moreover, the EU Directive 91/414 has restricted the available
insecticides authorized (EU, 1991) mainly in urban areas.
Biological control may represent an interesting and viable alternative.
In laboratory trials conducted in France with a strain of the
entomopathogenic fungus Beauveria bassiana (Balsamo Crivelli) Vuillemin,
good results against P. archon have been obtained (Millet et al., 2007;
Besse-Millet et al., 2008). Field trials have been carried out in Italy and
Spain to evaluate the efficacy of the entomopathogenic nematode
Steinernema carpocapsae (Weiser) (Martinez de Altube and Martinez Peña,
2009; Nardi et al., 2009). Recently, several natural toxins such as arthropodderived substances and entomopathogenic fungi-derived toxins have been
tested towards pest species of different orders (Mazet et al., 1994, QuesadaMoraga and Vey, 2004; Fitches et al., 2009).
In Europe there is no evidence of P. archon natural enemies, except
observations of perforated eggs, suggesting parasitoid attack, and eaten
adults, suggesting predation by birds. However natural enemies have been
occasionally reported in other Neotropical castniids (Sarto I Monteys and
Aguilar, 2005 and references therein).
6
Introduction
Until now, no castniid pheromone is known but hexane ovipositor extracts of
Castnia licus (Drury), the giant sugarcane borer, have elicited positive
responses in males and the main compounds have been identified by gaschromatography and mass-spectrometry (Reboucas et al., 1999). Therefore,
no detecting methods or strategies for monitoring and control are available
through the use of pheromones.
Pest management with semiochemicals
Semiochemicals are molecules that mediate communication between
organisms. Pheromones act between individuals of the same species.
Allelochemicals affect individuals of different species distinguishing
kairomones, benefiting the receiver, allomones favourable to the emitter and
synomones advantageous for both (Nordlund and Lewis, 1976).
Semiochemicals are chemicals nontoxic to vertebrates and beneficial insects
and therefore have been widely used in IPM programs, particularly sex
pheromones.
Sex pheromones mediate behaviours bringing the sexes together and
increasing mating success (Birch and Haynes, 1982). Butenandt et al. (1959)
have identified the first sex pheromone of the silk moth Bombyx mori
(Linneus). The existence of sex pheromones has been demonstrated in many
insect orders as well as in Lepidoptera, where more than 1500 have been
identified (www.pherobase.com). They are largely used in traps for detecting
the arrival of migrating pests in a new area and for population relative
abundance that can suggest timing of control measures. Sex pheromones are
also used in mass trapping, one of the oldest approaches, that consists of
removing the greatest number of reproductively active population through a
large number of traps and communication disruptions between sexes,
preventing or reducing female fertilization.
Other semiochemicals are used in IPM for example plant-derived chemicals
are known to improve attraction to pheromone lures and non-host volatiles
used in push and pull techniques (Witzgall et al., 2010 and references
therein).
Whatever way they are used, the integration of semiochemicals in pest
management needs a complete understanding of insect behaviour and
ecology.
7
Intraspecific communication in Lepidoptera
Insect communication have shown a great variety of chemical, visual, tactile
and acoustical signals to realize a broad spectrum of activities: selection of
food plant, choice of oviposition site, location of prey, defense and offence,
mate selection, courtship, organization of social activities and many other
behaviours (Birch and Haynes, 1982; Virant-Doberlet and Cokl, 2004).
Mating behaviour involves a) long-range mate location, consisting in
orientation and approach, and b) close-range courtship, consisting in close
interactions that can lead to copulation (Thornhill and Alcock, 1983).
Long-range sex pheromones are means of mate location, released by females
and involved in the attraction of mates, flying towards the source over long
distances (Hartlieb and Anderson, 1999). In females, sex pheromone glands
are consistent in their location and histological features. They are located at
the level of the last abdominal segments as modified intersegmental
membrane between the 8th and 9th abdominal segments (Percy-Cunningham
and MacDonald, 1987). They are also sporadically found between the 7th and
8th urite (Sreng and Sreng, 1988) or close to the opening of the ostium bursae
(Chow et al., 1976). Glandular cells are hypertrophied epidermal cells in
direct contact with overlying surface cuticle provided frequently with
projections (Percy-Cunningham and MacDonald, 1987). These glands are
often eversible and when everted they are exposed releasing pheromones in a
behaviour named “calling” (Birch and Haynes, 1982).
Close-range sex pheromones, both in males and females, are involved in
courtship behaviour (Scott, 1972; Hartlieb and Anderson, 1999; Costanzo
and Monteiro, 2007). Male sex pheromones can provide information about
the mate fitness, such as quality and quantity of nuptial gifts (Dussord et al.,
1991), can indicate the presence of other conspecific males (Iyengar et al.,
2001), can increase the acceptance of males by females (Grant and Brady,
1975; Scott, 1972) and can facilitate reproductive isolation in related species
(Phelan and Baker, 1987; Löfstedt et al., 1991). Male sex pheromones are
associated with morphological structures called androconia by Scudder
(1877). Androconia are found on almost any parts of the body such as wings,
thorax, abdomen and legs, varying in form from simple scales, brushes and
hair-pencils to eversible structures like coremata, inflatable tube organs.
Histologically, these scent organs consist of hypertrophied epidermal cells
and the associates scales or hairs (Birch et al., 1990; Hall and Harvey, 2002).
Female sex pheromones acting at close-range evoke the male pursuing
response (Scott, 1972).
Sex pheromones are detected trough antennae, whose primary function is to
be multimodal sensory organs bearing sensilla through which the insects
8
Introduction
monitor the external environment (exteroceptors) and their body parts
(proprioceptors) (Zacharuk, 1985).
In moths, having nocturnal or crepuscular habits, mate location at long-range
is due to males detecting the sex pheromone of conspecific females, highly
species-specific. After approaching, the male moths can release close-range
sex pheromones accompanied by visual, mechanosensory and acoustical
cues when courting females (Grant, 1987; Nakano et al., 2008, 2009).
On the other hand, in day-flying butterflies the long-range mate-locating
behaviour depend largely on visual cues. Visual cues appear to be weakly
specific and less differentiated, more important in early courthip (Scott,
1972; Scott, 1986). Butterfly species sharing mimicry and even behaviour
and habitat, have problem to distinguish one from another by sight alone.
Close range sex pheromone, acting in later courtship stages both in males
and females, release courtship behaviours (Siberglied, 1984;Vane-Wrigth
and Bopprè, 1993; Fordyce et al., 2002; Scott, 1972); these chemical cues
are not alternative to visual cues but additional. In some cases tactile,
contact-chemoreception or acoustical signals can be involved (Vane-Wrigth
and Bopprè, 1993).
In butterflies, searching individuals show typical strategies such as
patrolling, perching or hill topping (Rutowski, 1991).
The use of several cues in mate choice, displayed through complex sexual
behaviours, has received increasing attention. Recent studies have suggested
that the use of multiple cues decreases the cost reducing the number of mates
inspected, time and energy consuming. Therefore the evolution should prefer
the use of multiple cues explaining the fact this is so common (Candolin,
2003).
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Miller J.Y. (1995). Castniidae. In: Checklist Part 2. Hyblaeoidea - Pyraloidea Tortricoidea. Atlas of Neotropical Lepidoptera. Heppner J.B. Eds. Association
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13
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14
Objective
Paysandisia archon has caused serious damages and plant mortalities,
mainly in ornamental palm nurseries, in France, Italy and Spain. By now the
moth is considered an invasive species (Riolo et al., 2004; Sarto I Monteys
and Aguilar, 2005). There is a paucity of information on P. archon and in
general on Neotropical castniid life history, ecology and distribution in the
native habitat due to the lack of economic importance and the endophagous
habit of the larvae (Miller, 1986).
Recently Sarto I Monteys et al. (2012) and Delle-Vedove et al. (2012) have
pointed out some characteristics of reproductive behaviour and biology in P.
archon, providing background knowledge, but several aspects still need to
be detailed.
The knowledge of the mating behaviour at different levels (behavioural,
sensorial, chemical) is an important point to develop new strategies for
phytophagous control (detection, monitoring, mass trapping, communication
disruptions, etc) (Witzgall et al., 2010).
The aims of this research were:
a) to study the courtship and copulatory behaviour of the castniid palm
moth, P. archon, providing a qualitative and quantitative description and
investigating on possible cues (chemical, visual, mechanosensory) involved;
b) to carry out morphological and ultrastructural investigations of secretory
and sensory structures (ovipositor and antennae), by traditional light
microscopy, scanning and transmission electron microscopy, to fully
characterize the intraspecific communication in P. archon mating behaviour.
15
16
Chapter 1: Behavioural investigations
The understanding of the courtship and copulatory behaviour of an insect, is
essential to develop and optimize practical applications. Preliminary
observations into the wild and subsequently semifield and laboratory
experiments which test a series of hypotheses are needed and can lead to
insights of great practical potential.
Many studies have focused on lepidopterans because of their economic
importance as agricultural pest worldwide. It is well know that in moths
chemical cues are involved in mate location over long distances; on the
contrary butterflies depend largely on visual cues (Hartlieb and Anderson,
1999). During interactions over close range chemical, visual,
mechanosensory and acoustical cues are involved both in moths and
butterflies (Scott, 1972; Silberglied, 1984; Grant, 1987; Vane-Wrigth and
Bopprè, 1993; Fordyce et al., 2002; Nakano et al., 2008, 2009).
Paysandisia archon (Burmeister 1880), “butterfly-moth” of the Castniidae
family, has become invasive in many European countries, representing a
serious threat to palms, especially in nursery production (Riolo et al., 2004,
2005).
In literature are present scarce informations on courtship and copulatory
behaviour in castniid species. Sarto I Monteys et al. (2012) have observed
courtship behaviour in 4 P. archon pairs in the wild.
The aim of this study was to provide a qualitative and quantitative
description of P. archon courtship and copulatory behaviour in semifield
condition. Moreover further bioassays were carried out to investigate the
possible cues (chemical, visual, mechanosensory) involved during courtship
and copulatory behaviour.
Insects
P. archon specimens were obtained from potted 2-3 year old infested plants
of Chamerops humilis (Linneus) (Figure 1.1), from nurseries located in the
municipality of Grottammare (42°59'22”N; 13°51'56"E), province of Ascoli
Piceno, Marche region, Central-Eastern Italy. The infested palms (about
400) were placed in a net tunnel, inside a greenhouse (Department of
Agricultural, Food and Environmental Sciences), in Ancona (43°35'11"N;
13°30'50"E), Marche region.
17
In early summer, during 2011 and 2012, the infested palms were cut to
sample P. archon specimens (larvae and pupae). Specimens were separated
by gender according to the morphology of pupae (Riolo, unpublished data),
placed in separate cages (Bugdorm-1, Mega View Science, Taichung,
Taiwan) and stored in a climatic chamber (Binder KBWF 240, Tuttlingen,
Germany) (15±1°C, 24D, 60% RH). A stock of pupae was periodically
removed from the chamber and left under natural photoperiod, temperature
and humidity conditions to allow adult emergence.
Figure 1.1 – Paysandisia archon infested palms: a) dust; b) leaf perforations
arranged in a semicircle.
Bioassays
Courtship and copulatory behavioural sequence. After emergence, adults
were kept at 25±1°C, under natural photoperiod and humidity conditions,
and placed individually in a net cage (Bugdorm-1) until bioassays were
carried out. Males and females were kept in separate rooms to prevent them
exposing to each other.
Bioassays were performed in a mesh cage (1.0 x 3.0 x 1.3 m) containing 4
uninfested potted palms (Figure 1.2), in a net tunnel from 9.00 to 17.30
hours, between 26th July and 7th September 2011. Virgin 0-4-day-old P.
archon adults were used. Adults were placed individually in net cages 30
minutes (min) before testing. P. archon pairs were observed. Each pair was
observed for 2 hours and for more time until the copulation end.
Temperature, humidity (Humidity/Temp Monitor 800016, Sper Scientific,
Scottsdale, USA) and light intensity (Light Meter HD 2302.0, Delta OHM,
Padova, Italy) were measured before and after each bioassay. For each pair
18
Chapter 1
Behavioural investigations
were recorded: a) time from bioassay beginning to first female flight
approaching the male, b) time from first female flight to first pair flight, c)
time from first pair flight to first copulation attempt, d) time from first to last
copulation attempt, e) time from last copulation attempt to copulation, f)
copulation duration and g) copulation time.
Moreover, the frequencies of ovipositor extrusion and antennal cleaning
were recorded. The ovipositor extrusion was distinguished in: a) pre- and
post-copulation extrusion, b) partial or total extrusion. Furthermore, the
distance of the male from the female during its ovipositor extrusion was
measured (more and less than 50 cm). The frequency of antennal cleaning
was observed distinguishing 5 phases during bioassays: 1) before copulation
when male and female were distant (more than 50 cm from each other) 2)
before copulation when male and female were close (less than 50 cm from
each other) 3) during copulation, 4) after copulation when male and female
were distant and 5) after copulation when male and female were close.
Figure 1.2 - Mesh cage containing 4 uninfested potted palms, where bioassays
were performed.
19
Investigation on cues involved in courtship and copulatory behaviour.
Antennectomized adults. To assess the role of olfaction in P. archon
courtship and copulation, bioassays were performed with antennectomized
males or females.
After emergence, adults were kept individually in net cages (9 ᴓ x 12 cm),
under natural conditions. Males and females were kept in separate rooms to
prevent them exposing to each other.
Bioassays were performed in a mesh cage (60 x 60 x 90 cm) (Figure 1.3 a)
placed in a greenhouse room, from 9.00 to 16.30 hours, between 19th July
and 30th August 2012. Virgin 0-4-day-old P. archon adults were used.
Adults were anaesthetized with CO2 and antennae were cut with a razor
blade, leaving the first 4-7 antennomeres. Normal adults were anaesthetized
using CO2 as well. Treated insects were allowed to recover for one day
before being tested. Adults were placed in the greenhouse room 30 min
before bioassays. One female and one male were observed for 2 hours and
for more time until the copulation end. Normal females were tested with
antennectomized males (n=7) and vice versa (n=6).
Cages were cleaned with soapy water and absolute ethanol after each
replicate. Temperature, humidity and light intensity were measured before
and after each bioassay. For each pair were recorded: act) activation time
(when insects left the release cage), a) time from bioassay beginning to first
female flight approaching the male, b) time from first female flight to first
pair flight, c) time from first pair flight to first copulation attempt, d) time
from first to last copulation attempt, e) time from last copulation attempt to
copulation, f) copulation duration and g) copulation time. During bioassays
were observed the “main steps” of the courtship and copulatory behavioural
sequence and contact and male head dipping as “secondary steps”.
Dummy adults. To assess the role of visual and mechanosensory stimuli in
P. archon courtship and copulation, bioassays were performed with dummy
males and females.
Trials were performed as described for antennectomized insects. Normal
females were tested with male dummies (n=7) and vice versa (n=6).
The body of the P. archon dummy consisted of an amber glass vial (8.2 ᴓ x
40 mm) (Sigma-Aldrich, Germany), while the wings were printed on a paper
(female wingspan was of 8 cm; male wingspan was of 6.5 cm) (Figure 1.3
b). The wings were rinsed with hexane and fixed to amber glass by double
sided tape. The dummy was positioned facing up on the sunny side of the
mesh cage, using a piece of wire. Glass vials and wires were cleaned with
absolute ethanol and baked overnight at 200°C after each replicate, while
paper wings were used only one time.
20
Chapter 1
Temperature, humidity and light intensity were measured before and after
each bioassay. For each bioassays were recorded: act) activation time (when
insects left the release cage) and a) time from bioassay beginning to first
adult flight approaching the dummy. During bioassays were observed the
perching, female flight, alighting and male abdomen curling as “main steps”
of the courtship and copulatory behavioural sequence and contact and male
head dipping as “secondary steps”.
Data analysis. Cocoon sex ratio was tested with Chi-square (Zar, 1999).
Antennal cleaning data and times were analyzed by one-way ANOVA,
followed by Tukey’s test for mean separation. All statistical analysis was
performed at P<0.05, using Systat 11 (Systat Software Inc.).
Figure 1.3 – a) Mesh cage positioned inside the greenhouse room, where
bioassays with antennectomized and dummy adults were performed and detail
of dummy adult in ventral view inside the red square; b) dorsal view of dummy
adult.
Results
Insects
From 27th June to 30th August 2011, a total of 197 cocoons were collected
from about 400 infested palms. Cocoon sex ratio was 0.80:1 male:female
(χ2=2.198; df=1; P>0.05). Only 98 adults (51 males and 47 females)
emerged from 1st July to 30th October 2011, corresponding to the 49.8 % of
total specimens collected.
A total of 142 cocoons were collected from infested palms from 24th May to
30th July 2012. Cocoon sex ratio was 0.8:1 male:female (χ2=1.667; df=1;
P>0.05). Only 59 adults (27 males and 32 females) emerged from 11th June
21
to 5th September 2012, corresponding to the 41.6 % of total collected
specimens.
Bioassays
Courtship and copulatory behavioural sequence.
1.Successful courtships. Courtship behaviour performed by P. archon pairs,
until copulation attempt or copulation, was defined successful. Of the 33
pairs observed, 12 successful courtships and 10 copulations occurred (30.3%
of mating rate). All mated adults were 0-2-day-old, except one 4-day-old
female. A fairly fixed pattern of courtship and copulatory sequence emerged.
Ten steps were reported: 6 “main steps”, that occurred in all successful
courtships, and 4 “secondary steps”, that did not always occur. The
following step were observed (Figure 1.4):
Step 1 - Perching (100.0% of occurrence): the male and female await
stationary in a perching site (a palm leaf or the mesh) distant (more than 50
cm) from each other.
Step 2 - Female flight (100.0% of occurrence): the female approaches the
perching male by flying less than 50 cm from the male.
Step 3 - Pair flight (100.0% of occurrence): the male chases the female and
male and female fly together.
Step 4 - Alighting (100.0% of occurrence): male and female stop flying and
alight facing up at less than 10 cm from each other. No male flickering was
observed after alighting (Figure 1.5 a).
Step 5 - Ovipositor extrusion before copulation (66.7% of occurrence): the
female alights less than 10 cm from the male and extrudes the ovipositor
once or repeatedly: more than 10 consecutive extrusions were observed. The
extrusions were partial and/or total: 58.3% of the females performed only
partial ovipositor extrusions; 8.3% both total and partial consecutively.
Step 6 - Contact (25.0% of occurrence): the male approaches the female
from behind and touches the female wings with antennae or forelegs (Figure
1.5 b).
Step 7 - Male head dipping (16.7% of occurrence): the male dips its head
under the female abdomen or wings before attempting copulation (Figure 1.5
c).
Step 8 - Male abdomen curling (copulation attempt) (100.0% of occurrence):
the male curls its abdomen and shows the claspers, trying to grasp the female
copulatory orifice (Figure 1.5 d). In the 41.7% of the cases only one attempt
was sufficient for the male to grasp the female. In the remaining cases 2-10
attempts were necessary.
Step 9 - Copulation (83.3% of occurrence): during copulation the moths are
motionless, facing up, next to each other, the male with curled abdomen on
22
Chapter 1
the female left or right side (Figure 1.5 e). After copulation, in the 70.0% of
cases the male flew away before the female.
Step 10 - Ovipositor extrusion after copulation (75.0% of occurrence): the
female extrudes the ovipositor once or repeatedly (even more than 90 times
were observed). The 41.7% of the females performed total and partial
ovipositor extrusions at different times, the 25.0% partial and the 8.3% total
(Figure 1.5 f).
Figure 1.4 - Occurrence (%) of main (black bars) and secondary (white bars)
steps during successful courtship sequences (n=12 pairs); tp: ovipositor
extruded totally and partially; p: ovipositor extruded partially (tip or half); t:
ovipositor extruded totally.
Courtship steps n° 1, 2, 3, 4, 5, 6, 7 and 8 occurred more than once during
the successful courtship sequences (Table 1.1).
23
Figure 1.5 – Ground courtship and copulatory steps: a) Step 4 - Alighting:
adults facing up next to each other; b) Step 6 - Contact: male touches female
wings with its legs; c) Step 7 - Male head dipping under female wings; d) Step 8
- Male abdomen curling: male shows its claspers, trying to grasp the female
copulatory orifice; e) Step 9 - Copulation: adults facing up, male on the left side
with curled abdomen; f) Step 10 - Female ovipositor tip partial extrusion after
copulation.
24
Chapter 1
Time (mean±SE) from bioassays beginning to first female flight approaching
the male (a) was 30.09±7.26 min. Time from first female flight to first pair
flight (b) was 0.46±0.08 min (Table 1.1). The mean duration of the interval
from first pair flight to first copulation attempt (c) was 14.39±6.85. Time
from first to last copulation attempt (d) was 15.10±4.52 min. The mean
duration of the interval from last copulation attempt to copulation (e) was
0.95±0.26 min. Copulations lasted on average 54.42±5.72 min (f) (Table
1.1). Copulations took place on the cage lateral mesh except one pair that
mated on a palm leaf. During all the ground courtship and copulatory steps,
adults were always facing up, and their wings were closed in the typical
moth position (over the body or pressed flat on either side of the body). All
copulations took place in the morning, around 9.30 to 11.30 hours, except
two pairs, that mated after 14.00 hours (g) (Table 1.1).
Mean environmental parameters recorded during bioassays were
28.7±0.7°C, 51±2% RH and 69376±3310 lux.
The 50% of the perching females performed ovipositor extrusions from 2 to
45 times: the 25.0% of the females performed only partial ovipositor
extrusions and the 25.0% both total and partial.
Antennal cleaning frequency was higher in females than in males (F=14.991;
P<0.001; df=1) (Table 1.2). In particular, females had a higher frequency of
antennal cleaning when they were distant (more than 50 cm) from the males
before copulation (male vs female comparison: F=13.190; P<0.01; df=1;
comparison among females: F=6.977; P<0.001; df=4). No significant
differences emerged among males that cleaned their antennae with the same
frequency during all the bioassay duration (F=1.756; P>0.05; df=4) (Table
1.2).
2.Unsuccessful courtships. Courtships that did not lead to any copulation
attempt (Step 8) were defined unsuccessful. Of the 33 pairs observed, 21
were unsuccessful. Courtships stopped at maximum at step 3. The following
steps were observed (Figure 1.6; Table 1.3):
Step 1 - Perching (100.0% of occurrence).
Step 2 - Female flight (52.4% of occurrence): 33.3% of the females flew less
than 50 cm from the male more than once; 19.1% only once. In the rest of
the cases the female flew more than 50 cm from the male (19.0%) or didn’t
fly at all (28.6%).
Step 3 - Pair flight (38.1% of occurrence): in 28.6% of the cases more than
one pair flight was observed, while in 9.5% of the cases only one pair flight
occurred. Male and female alighted more than 50 cm from each other, after
chasing, and the courtship behaviour stopped.
25
Table 1.1 - Steps observed during successful courtship sequences (n= 12 pairs) and times recorded: a) from bioassay
beginning to first female flight approaching the male; b) from first female flight to first pair flight; c) from first pair flight to
first copulation attempt; d) from first to last copulation attempt; e) from last attempt to copulation; f) copulation duration
and g) copulation time.
26
Pair (n)
1
2
3
4
5
6
7
8
9
10
11
12
Step sequence
1-2-3-4-8-8-1-2-3-1-2-3-4-5-7-8-9-10
1-2-3-4-5-8-8-1-2-3-4-8-8-5-8-5-8-6-8-5-6-8-1-2-3-4-5-8-8
1-2-3-1-2-3-4-8-5-1-2-3-4-8-5-1-2-3-1-2-3-1-2-3
1-2-3-1-2-3-4-8-9-10
1-2-3-1-2-3-1-2-3-1-2-3-4-5-6-8-9-10
1-2-3-1-2-3-4-8-5-1-2-3-4-8-9-10
1-2-3-4-8-9-10
1-2-3-1-2-3-4-5-8-9-10
1-2-3-4-5-8-1-2-3-1-2-3-4-8-9-10
1-2-3-4-5-6-8-8-8-9-10
1-2-3-4-1-2-3-1-2-3-4-1-2-3-4-8-9-10
1-2-3-4-7-8-8-7-8-7-8-1-2-3-4-8-9
mean
SE
*Steps not performed
26
a
0.00
41.10
57.33
37.25
0.75
27.17
36.67
47.37
0.42
1.25
34.75
77.00
30.09
±7.26
b
0.08
0.50
0.67
0.58
0.33
0.85
0.25
1.00
0.13
0.47
0.30
0.40
0.46
±0.08
c
0.17
0.73
2.75
17.53
32.98
19.55
0.42
12.58
0.20
3.18
81.95
0.60
14.39
±6.85
Times (min)
d
e
5.28
0.47
34.67
-*
24.37
-*
-*
1.14
-*
1.43
19.93
0.50
-*
0.32
-*
1.00
14.32
0.95
4.10
0.50
-*
3.00
3.00
0.20
15.10
0.95
±4.52
±0.26
f
94.58
-*
-*
76.50
54.33
50.00
54.35
51.45
45.98
41.67
34.58
40.80
54.42
±5.72
g
10.16
14.04
10.37
11.26
14.14
10.57
10.56
9.39
11.35
11.06
Chapter 1
Table 1.2 - Antennal cleaning frequency of males and females in successful
courtship sequences. Data are means±SE. Asterisks indicate significant
difference in male vs female comparisons (*P<0.05; **P<0.01; ***P<0.001; oneway ANOVA). Identical letters within the same column indicate no statistical
difference in comparison between males or females (Tukey’s test, P<0.05).
Antennal cleaning frequency
Males
Females
Before copulation, distant
0.3±0.1 a
2.9±0.7 ** a
Before copulation, close
0.1±0.1 a
0.4±0.3
b
During copulation
0.2±0.2 a
0.1±0.1
b
After copulation, distant
0.6±0.2 a
1.1±0.4
b
After copulation, close
0.7±0.3 a
0.7±0.3
b
Overall means ±SE
1.6±0.3
4.9±0.8 ***
“Distant”: male and female more than 50 cm from each other; “Close”:
female less than 50 cm from each other
n
12
12
10
10
10
12
male and
Figure 1.6 - Occurrence (%) of main steps (secondary steps did not occur)
during unsuccessful courtship sequences (n=21 pairs).
During unsuccessful courtships, time (mean±SE) from bioassays beginning
to first female flight approaching the male (a) was 35.76±9.66 min; time
27
from first female flight to first pair flight (b) was 0.39±0.07 min (Table 1.3).
28.8±0.7°C, 46±2% RH and 72071±2369 lux.
Ovipositor extrusions (from 1 to 31 times) were performed by the 71.4% of
the females. The 42.9% of the females performed only partial ovipositor
extrusions; the 28.6% both total and partial.
During unsuccessful courtships, antennal cleaning occurred only when the
adults were more than 50 cm from each other. The antennal cleaning
frequency of the females was greater than that of males (F=20.550; P<0.001;
df=1) (Table 1.4).
Comparing successful and unsuccessful courtships, time from bioassays
beginning to first female flight approaching male (a) and time from first
female flight to first pair flight (b) did not result significantly different
(F=0.225; P>0.05; df=1; F=0.376; P>0.05; df=1).
No significant differences emerged in antennal cleaning frequency among
successful and unsuccessful males (F=1.105; P>0.05; df=1) or females
(F=0.724; P>0.05; df=1) (Table 1.4).
28
Chapter 1
Table 1.3 - Steps observed during unsuccessful courtship sequences (n=21
pairs) and times recorded: a) from bioassay beginning to first female flight
approaching the male; b) from first female flight to first pair flight.
Pair (n)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Step sequence
1
1
1
1-2-1-2-1-2-1
1
1-2-3-1-2-3-1-2-3-1-2-3-1
1-2-3-1-2-3-1
1
1-2-3-1-2-1-2-1-2-3-1-2-1-2-1
1
1-2-3-1
1
1-2-3-1
1
1-2-3-1-2-3-1-2-3-1
1-2-3-1-2-3-1-2-3-1
1
1
1-2-1-2-1-2-1-2-1
1-2-1
1-2-3-1
mean
SE
* Steps not performed
Times (min)
a
b
-*
-*
-*
-*
-*
-*
104.00
-*
-*
-*
2.43
0.72
45.60
0.20
-*
-*
22.00
0.17
-*
-*
8.83
0.55
-*
-*
0.00
0.23
-*
-*
55.08
0.30
52.83
0.58
-*
-*
-*
-*
6.62
-*
33.98
-*
62.00
0.40
35.76
0.39
±9.66
±0.07
Table 1.4 - Antennal cleaning frequency in males and females in successful and
unsuccessful courtship sequences. Data are means±SE. Asterisks indicate
significant difference in male vs female comparisons (*P<0.05; **P<0.01;
***P<0.001; one-way ANOVA). Identical letters within the same column
indicate no statistical difference between males or females (one-way ANOVA,
P<0.05).
Successful courtship sequence
Unsuccessful courtship sequence
Overall means ±SE
Antennal cleaning frequency
Males
Females
1.6±0.3 a
4.9±0.8 *** a
1.0±0.4 a
4.1±0.6 *** a
1.2±0.3
4.4±0.5 ***
29
n
12
21
33
Investigation on cues involved during courtship and copulatory behaviour.
Courtship behaviour performed by P. archon adults, until copulation attempt
or copulation, was defined successful.
1.Normal female and antennectomized male. Of the 7 pairs observed, 1
successful courtship occurred (14.3% of occurrence), but male antennal
amputation affected the copulation success (0.0% of mating rate). All
successful adults were 1-2-day-old. The following steps were observed
(Figure 1.7; Table 1.5):
Step 2 - Female flight (100.0% of occurrence): 85.7% of the females flew
more than once approaching the male, 14.3% only once.
Step 3 - Pair flight (85.7% of occurrence): all the pairs flew more than once
together.
Step 4 - Alighting (100.0% of occurrence).
Step 6 - Contact (28.5% of occurrence): males touched female wings with
the forelegs.
Step 8 - Male abdomen curling (14.3% of occurrence): one male made 18
copulation attempts.
Activation time (mean±SE) was 20.69±12.30 min in normal females and
17.56±7.55 min in antennectomized males. Time from bioassay beginning to
first female flight approaching the male (a) was 21.83±12.08 min. Time
from first female flight to first pair flight (b) was 2.85±2.73 min; times from
first pair flight to first copulation attempt (c) and from first to last copulation
attempt d) were recorded only for the pair that displayed copulation attempts
(25.07 min and 88.45 min, respectively) (Table 1.5).
31.5±0.6°C, 46±4% RH and 18078±5100 lux.
2.Normal male and antennectomized female. Of the 6 pairs observed, 3
successful courtships occurred (50.0% of occurrence) and the copulation
success was of 33.3%. All successful adults were 1-3-day-old. The following
steps were observed (Figure 1.8; Table 1.6):
Step 2 - Female flight (50.0% of occurrence): 33.3% of the females flew
more than once approaching the male, 16.7% only once.
Step 3 - Pair flight (50.0% of occurrence): 33.3% of the pairs flew together
only once, 16.7% more than once.
Step 8 - Male abdomen curling (50.0% of occurrence): each male made 2
copulation attempts .
Step 9 - Copulation (33.3% of occurrence).
30
Chapter 1
Figure 1.7 - Occurrence (%) of courtship and copulatory steps in normal
females tested with antennectomized males (n=7 pairs).
Activation time (mean±SE) was 59.67±20.80 min in antennectomized
females and 9.02±3.68 min in normal males. Time from bioassay beginning
to first female flight approaching the male (a) was 50.99±25.32 min. Time
from first female flight to first pair flight (b) was 0.88±0.87 min. The
duration of the interval from first pair flight to first copulation attempt (c)
was 8.75±8.02 min. Time from first to last copulation attempt (d) was
6.44±4.54 min. The duration of the interval from last copulation attempt to
copulation (e) was 0.07±0.04 min. The two copulations observed lasted
36.28 min and 40.43 min, respectively, and took place in the late morning on
the cage lateral mesh (Table 1.6).
30.3±0.5°C, 44±1% RH and 16383±4533 lux.
31
Table 1.5 – Steps observed and times recorded during bioassays with normal females vs antennectomized males (n=7 pairs):
act) activation time; a) from bioassay beginning to first female flight approaching the male; b) from first female flight to
first pair flight; c) from first pair flight to first copulation attempt; d) from first to last copulation attempt.
Pair (n)
32
Step sequence
1-2-3-4-1-2-1-2-1-2-1-2-3-1-2-3-4-1-2-3-1-2-3-1-2-1-2-3-1-21
1-2-3-1-2-3-1-2-3-1-2-3-1-2-1-2-3-1-2-3-1-2-1-2-3-1-2-1-2-31-2-1-2-3-1-2-1-2-3-1-2-1-2-1-2-1-2-4
1-2-3-1-2-3-4-8-8-8-8-8-8-8-8-8-8-8-8-1-2-3-1-2-3-4-8-8-8-82
8-8
3
1-2--1-2-3-1-2--1-2-3-4-1-2-3-1-2-3-1-2-3-1-2-3-1-2-3-4
4
1-2-3-1-2-3-1-2-3-1-2-3-1-2-1-2-1-2-1-2-4
5
1-2-3-1-2-3-1-2-3-1-2-4-6
6
1-2-4
7
1-2-1-2-3-1-2-3-4-6-1-2-3-1-2-3-1-2-3
Mean
SE
32
act ♀
act ♂
Times (min)
a
b
c
d
2.67
0.02
2.67
0.02
-*
-*
0.08
0.18
0.08
0.10
25.07
88.45
29.05
6.27
90.72
15.18
0.85
20.69
±12.30
29.45
6.98
42.20
43.03
1.07
17.56
±7.55
29.05
7.17
90.93
15.18
7.75
21.83
±12.08
0.43
0.02
0.03
-*
16.48
2.85
±2.73
-*
-*
-*
-*
-*
-*
-*
-*
-*
-*
Chapter 1
Figure 1.8 - Occurrence (%) of courtship and copulatory steps in normal males
tested with antennectomized females (n=6 pairs).
3.Normal female and dummy male. In presence of dummy males, normal
females performed the following courtship steps (Figure 1.9; Table 1.7):
Step 2 - Female flight (100.0% of occurrence): all the females flew more
than once approaching the dummy male.
Step 6 - Contact (28.6% of occurrence): females touched male dummies with
the legs; no contact by antennae on male paper wings was observed.
Activation time (mean±SE) was 8.54±3.68 min. Time from bioassay
beginning to first female flight approaching the dummy (a) was 26.35±13.15
min (Table 1.7).
31.5±0.8°C, 38±3% RH and 14214±4409 lux.
33
Table 1.6 – Steps observed and times recorded during bioassays with normal males vs antennectomized females (n=6 pairs):
act) activation time; a) from bioassay beginning to first female flight approaching the male; b) from first female flight to
first pair flight; c) from first pair flight to first copulation attempt; d) from first to last copulation attempt; e) from last
attempt to copulation; f) copulation duration and g) copulation time.
34
Pair (n)
Step sequence
1
1-4-1-2-1-2-1-2-1-2-3-1-2-1-2-4-1-2-4-1-24
2
1-2-3-4-1-2-3-1-2-3-1-2-3-1-2-3-4-8-8
3
1-2-3-4-8-8-9
4
1
5
1-4-8-8-9
6
1
mean
SE
Times (min)
c
act ♀
act ♂
A
b
17.20
24.10
43.40
2.62
92.45
10.82
117.88
60.00
-*
59.67
±20.80
12.95
0.00
6.00
10.47
0.62
9.02
±3.68
98.15
11.42
-*
-*
-*
50.99
±25.32
0.02
0.02
-*
-*
-*
0.88
±0.87
34
d
e
f
g
-*
-*
-*
-*
-
16.77
0.73
-*
-*
-*
8.75
±8.02
3.57
0.43
-*
15.33
-*
6.44
±4.54
-*
0.12
-*
0.03
-*
0.07
±0.04
-*
40.43
-*
36.28
-*
38.36
±2.07
11.47
11.30
-
Chapter 1
4.Normal male and dummy female. In presence of dummy females, normal
males performed the following courtship steps (Figure 1.9; Table 1.8):
Step 4 - Alighting (83.3% of occurrence): males approached the dummy
female flying towards it.
Step 6 - Contact (33.3% of occurrence): males touched dummy female wings
with the forelegs; no contact by male antennae on female paper wings was
observed.
Step 8 - Male abdomen curling (16.7% of occurrence): only one copulation
attempt was observed, at a distance of 50 cm from the female dummy.
Activation time (mean±SE) was 5.49±3.42 min and time from bioassay
beginning to first male flight approaching the dummy (a) was 9.34±5.33 min
(Table 1.8). Mean environmental parameters recorded during bioassays were
31.6±0.4°C, 45±2% RH and 8600±1635 lux.
Figure 1.9 - Occurrence (%) of courtship and copulatory steps in normal
females tested with dummy males (n=7; red bars) and vice versa (n=6; blue
bars).
35
Table 1.7 – Steps observed and times recorded during bioassays with normal females tested with dummy males (n=7): act)
activation time; a) from bioassay beginning to first female flight approaching the dummy.
Pair (n)
1
36
2
3
4
5
6
7
Step sequence
1-2-1-2-1-2-1-2-1-2
1-2-1-2-1-2-1-2-1-2-1-2-4-1-2-1-2-1-2-1-2-4-1-2-4-1-2-4-1-2-4-1-2-1-2-1-2-1-2-1-2-4-12-4-1-2
1-2-1-2-1-2-1-2-1-2-1-2
1-2-4-1-2
1-2-1-2-1-2-4-1-2-1-2-1-2-4-1-2-4
1-4-6-1-4-1-2-1-2-1-2-4
1-2-1-2-4-1-2-4-1-2-1-2-1-2-1-2-4-1-2-4-1-2-1-2-1-2-4-1-2-4-1-2-1-2-1-2-4-6-1-2-4
mean
SE
36
Times (min)
a
act ♀
0.00
3.47
0.22
5.38
5.03
3.25
27.42
10.22
13.65
8.54
±3.68
15.17
4.62
27.42
101.88
26.50
26.35
±13.15
Chapter 1
Table 1.8 – Steps observed and times recorded during bioassays with normal
males tested with dummy females (n=6): act) activation time; a) from bioassay
beginning to first male flight approaching the dummy.
Pair (n)
1
2
3
4
5
6
Step sequence
1
1-4-1-4-1-4-6-1-4
1-4
1-4-1-4-1-4-8-1-4-1-4-1-4-1-4-1-4-1-4
1-4
1-4-6
mean
SE
Times (min)
act ♂
0.47
20.18
11.08
1.17
0.00
0.03
5.49
±3.42
a
0.47
26.50
-*
17.25
1.62
0.85
9.34
±5.33
Considering the bioassays with antennectomized and dummy adults to
investigate the cues involved during courtship and copulatory behaviour,
antennal amputation did not affect male activation times (normal vs
antennectomized: F=1.289; P>0.05; df=2), while antennectomized females
tended to be slower (normal vs antennectomized: F=4.106; P<0.05; df=2)
(Table 1.9).
Table 1.9 – Activation time in males and females. Data are means±SE. Identical
letters within the same column indicate no statistical difference between males
or females (Tukey’s test, P<0.05).
Normal female and antennectomized male
Normal male and antennectomized female
Normal female and dummy male
Normal male and dummy female
Activation time (min) (mean±SE)
Females
Males
20.69±12.30 ab 17.56±7.55 a
59.67±20.80 a
9.02±3.68 a
8.54±3.68
b
5.49±3.42 a
n
7
6
7
6
Discussion
Butterflies use two different strategies for mate location. In perching
behaviour, male rests in advantageous place at certain time of the day to find
mates. Female flies near the perching male who then pursues attracted to
moving objects. In patrolling behaviour males search for females by flying
37
and are often attracted by motionless objects of the same color of their
females. Males use movement and color wing in finding females, then
pheromones are involved in close-range courtship. Studies have shown that
the color of the female is the most attractive to the male of the same species,
especially colored strips, bands and spots on the wings (Scott, 1986).
Reports of few species adopting both strategies are present (Courtney and
Parker, 1985; Rutowski et al., 1988). The male behaviour can be influenced
by many factors (Courtney and Anderson, 1986), for example males prefer
perching to patrolling when temperatures are low, light weak and wind speed
high (Wickman, 1988).
During our bioassays the courtship and copulatory behaviour of P. archon
revealed a fairly fixed sequence of 10 steps, of which 6 main steps and 4
secondary steps.
The perching behaviour of P. archon males was observed. Inside the mesh
cage, the perching sites were generally represented by the higher part of the
cage in sunny side. In the wild P. archon males have been observed to patrol
(Sarto I Monteys et al., 2012), so this species could adopt both mate location
behaviours.
In our bioassays, was always the P. archon female that first approached the
perching male in these advantageous sites, who then pursued, attracted
probably to moving objects. The hypothesis is that female localizes the
perching male by sight and the male is activated by the movement of flying
female as reported in many butterflies (Scott, 1986). Moreover this
behaviour suggests the lack of female sex pheromone active over long-range,
as in butterflies. This is also supported by the fact that ovipositor extrusions
were performed by P. archon females before and after copulation. In P.
archon, ovipositor extrusions could be not related with calling behaviour and
long-range sex pheromone emission as in moths (Birch and Haynes, 1982),
but rather with egg-laying probing activity or with other functional aspects
of reproductive biology. This hypothesis was supported by the fact that also
during unsuccessful courtships the females performed ovipositor extrusions.
In P. archon courtship behaviour, the pair flight seems to be very important
in communication between sexes. During pursuit the wing beats could
stimulate the partner as mechanosensory or anemotactile cue. Moreover
olfactory cues, active at close range, could be released by the female and/or
male and received by the opposite sex stimulating the pursuing response and
then the alighting close to each other. The alighting at close-range after
pursuit, could represent the acceptance of male by female that has been
reported one of the functions of close-range pheromones both in moths and
butterflies (Grant and Brady, 1975; Brower et al., 1965). The close-range
volatile diffusion during pair flight could replace the male wing clapping or
38
Chapter 1
flickering observed in moths, Grapholita molesta (Busck), Plodia
interpunctella (Hübner), Cadra cautella (Walker), and butterflies, Bicyclus
anynana (Butler), Agraulis vanilla (Linneus) and likely involved in transfer
of chemical cues, pheromones, and anemotactile cues as well (Grant e
Brady, 1975; Baker and Cardè, 1979; Rutowski and Schaefer, 1984;
Nieberding et al., 2008). During our bioassays, no hairpencil, brushes or
coremata extrusions were observed in P. archon males. However,
androconia could be present in this castniid species as simply modified
scales on wings or abdomen. Moreover Sarto I Monteys et al. (2012) have
suggested the presence of chemical cues on P. archon male wings.
The role of the scales have been reported to be widespread in lepidopterans
(Sanders and Lucuik, 1992; Trematerra 1992 and references therein) and
have shown to be not species-specific (Shimizu and Tamaki, 1980; Sanders,
1979). For example, the role of the scales has been assessed in
Choristoneura fumiferana (Clemens), in which males did not attempt to
copulate with models without wings attached (Sanders and Lucuik, 1992).
Trematerra (1992) have stated that likely, in addition to tactile
(mechanosensory) cues due to scale shape, a chemical cue active at closerange could be emitted by the scales as well.
During our bioassays the contact of the male forelegs with the female wings
was sporadically observed. In Choristoneura fumiferana (Clemens) the
contact has been reported to be important in releasing copulatory behaviour
(Sanders, 1979; Grant, 1987; Sanders and Lucuik, 1992).
In our bioassays the antennal cleaning frequency resulted always higher in
females, likely preparing themselves to odor reception (plant host odors for
search of oviposition site or close-range pheromone reception).
In P. archon copulatory behaviour the male head dipping was observed in
very low percentage. This behaviour has been reported in the butterfly
Precis coenia (Hubner) to serve to reach the female abdomen into a position
that the male can easily couple with (McDonald and Nijhout, 2000).
As reported for some Nymphalidae butterfly species, Danaus plexippus
(Linneus), D. gilippus (Cramer), and D. chrysippus (Linneus) (Brower et al.,
1965; Pliske, 1974), P. archon pairs engaged multiple repetitions of
courtship steps before copulation occurred, so the courtship and copulatory
sequences resulted highly variable in duration. Sexual selection has been
reported to be important in butterflies, at least in males; in fact sometimes
females, depending on their receptivity, can copulate after prolongated
courtships by the males (Scott, 1972).
In our observation, P. archon copulation lasted on average 54 min
(maximum time of 95 min). Sarto I Monteys et al. (2012) and Delle Vedove
et al. (2012) have reported, for the same castniid species, an average
39
copulation time of 38 min and 35 min respectively. Reports of other
Neotropical castniids have recorded copulation for an average of 30 min in
Castniomera atymnius (Dalman) and 1-3 hours, even more, in Eupalamides
cyparissias (Fabricius) (Lara, 1964; Korytkowski and Ruiz, 1980).
During our bioassays a low mating rate occurred, about 30%. This may be
due to duration of each bioassay (2 hours). In recent experiments Delle
Vedove et al. (2012) have reported higher mating rate for a longer
observation time (10 hours).
P. archon specimens used in bioassays emerged from 2-3 year-old potted
palms and this could have affected the specimen quality and nuptial gift
quality. Size plays an important role in mate quality assessment so in insects
as in other organisms (Andersson, 1994 and references therein). Close-range
pheromones have proved to provide information about the mate fitness; titer
and content of these molecules involved in mate quality assessment have
shown to be correlated with insect size (Conner et al., 1990; Dussord et al.,
1991).
Furthermore environmental conditions could not have been optimal. In fact
temperature and light level have shown to be critical for courtship activity in
the butterfly P. coenia (McDonald and Nijhout, 2002) and also in P. archon
(personal observations).
In our bioassays with antennectomized adults, more than 80% of the
antennectomized males were stimulated by female flight and pair flight was
performed even if only one copulation attempt occurred. This observations
confirmed that P. archon perching males are first attracted to fluttering
females as reported in many butterflies (Scott, 1986).
On the other hand, P. archon antennectomized females reduce their
performance to approach perching males: only the 50% performed female
flight approaching the male compared to the 100% of normal females. In this
case, 2 copulations occurred suggesting that female close range pheromones
could be used by P. archon males during courtship. Scott (1972 and
references therein) have reported that female close-range pheromones
probably occur in most butterfly species and their existence is suspected
from behavioural evidence.
Antennectomized females tended to be slower in activation time then normal
females while the antennal amputation did not affect the activation time in
males. P. archon females are likely more sensitive than males with respect to
the antennae, as way of relating to the environment for example for choice of
oviposition site. In our bioassays in which we used dummy adults, males
flew approaching the dummy female and alighted close to it (80 % of
occurrence); also one copulation attempt was observed. On the other hand
females approached dummy males (100% of occurrence) and alighted close
40
Chapter 1
to it (70 % of occurrence). Laboratory experiments on B. anynana have
suggested that when multiple cues are involved in mate location and
courtship, the presence of at least one correct stimulus (visual or olfactory) is
sufficient to lead to copulation, even if multiple cues are favored (Costanzo
and Monteiro, 2007).
Anyway further bioassays are necessary to deepen the mechanisms of
intraspecific communication in P. archon mating behaviour.
References
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Baker T.C., Carde R.T. (1979). Courtship behavior of the oriental fruit moth
(Grapholitha molesta): experimental analysis and consideration of the role of
sexual selection in the evolution of courtship pheromones in the Lepidoptera.
Annals of the Entomological Society of America 72, 173-188.
Birch M.C., Haynes K.F. (1982). Insect pheromones. E. Arnold, London.
Brower L.P., Brower J.V.Z., Cranston F.P. (1965). Courtship behavior of the queen
butterfly, Danaus gilippus berenice. Zoologica (New York) 50, 1-39.
Conner W.E., Roach B., Benedict E., Meinwald J., Eisner T. (1990). Courtship
pheromone production and body size as correlates of larval diet in males of the
arctiid moth, Utetheisa ornatrix. Journal of Chemical Ecology 16, 543-552.
Costanzo K., Monteiro A. (2007). The use of chemical and visual cues in female
choice in the butterfly Bicyclus anynana. Proceedings: Biological Sciences 274,
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Courtney S.P., Anderson K. (1986). Behaviour around encounter sites. Behavioural
Ecology and Sociobiology 19-241-248.
Courtney S.P., Parker G.A. (1985). Mating behavior of the tiger blue butterfly
(Tarucus theophrastus): competitive mate searching when not all females are
captured Behavioural Ecology and Sociobiology 17, 213-221.
Delle Vedove R., Beaudoin Ollivier L., Hossaert McKey M., Frérot B. (2012).
Reproductive biology of the palm borer, Paysandisia archon (Lepidoptera:
Castniidae). European Journal of Entomology, 109 (2), 289-292.
Dussord D. E., Harvis C.A., Meinwald J., Eisner T.(1991). Pheromonal
advertisement of a nuptial gift by a male moth (Utetheisa ornatrix). Proceedings
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Fordyce J.A., Nice C.C., Forister M.L., Shapiro A.M. (2002). The significance of
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diverged species. Journal of Evolutionary Biology 15, 871-879.
Grant G.G. (1987). Copulatory behaviour of spruce budworm, Choristoneura
funiferana (Lepidoptera: Tortricidae): experimental analysis of the role of sex
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Grant G.G., Brady U.E. (1975). Courtship behavior of Phycitid moths. Comparison
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Hartlieb E., Anderson P. (1999). Olfactory-released behaviours. In Insect olfaction.
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Korytkowski C.A., Ruiz E.R. (1980). El barreno de los racimos de la palma aceitera,
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43
44
Chapter 2: Morphological investigations
Fine morphological studies of the pheromone secretory systems and sensory
structures are important tasks to fully characterize the mechanisms of
intraspecific communication in mating behaviour. Moreover these
investigations are important starting points and complementary for
physiological and behavioural studies, providing invaluable information of
practical importance in Integrated Pest Management (IPM) strategies.
In female moths sex pheromone glands are commonly found as modified
intersegmental membrane between the 8th and the 9th abdominal segments
(Percy and Weatherson, 1974).
The antennae are the primary sensory organs bearing small structures known
as sensilla through which insects can perceive tactile, chemo, hygro and
thermo stimuli by antennal feeling (Zacharuck, 1985). Historically, the
classification of insect sensilla was based on the shape of the cuticular parts
by light microscopy (Snodgrass, 1926, 1935; Schneider, 1964). The
development of electron microscopy techniques has led to new classification
based on ultrastructural characteristics (Altner, 1977; Altner and Prillinger,
1980). Only electrophysiological recordings are finally conclusive in order
to assess a function to sensilla.
At present, in Paysandisia archon (Burmeister 1880) the casniid palm moth,
has been hyphotesized the lack of female pheromone glands associated with
ovipositor, basing only on external morphological features (Sarto I Monteys
et al., 2012).
In many ditrysian Lepidoptera, as P. archon, the antennal sensillar types are
fairly fixed, comprising olfactory sensilla (trichodea, basiconica, auricillica,
coeloconica), gustatory sensilla (chaetica) and thermo-hygroreceptory
sensilla (styloconica) (Hallberg and Hansson, 1999).
So far, P. archon antennae have been investigated by steremicroscopy and
scanning electron microscopy reporting 4 types of sensilla (Sarto I Monteys
et al., 2012).
The aim of this study was to provide for the first time ultrastructural
characterization of the P. archon ovipositor, as possible site of sex
pheromone gland, and of the antennal sensory equipment.
45
Insects
P. archon adults for morphological investigations were obtained as
described in Chapter 1.
Light microscopy
Ovipositor. Ten newly emerged virgin females were used for the
observations. After anaesthetization by freezing, ovipositors were excised
and immersed in a 10% aqueous solution of caustic potash (KOH) at 100°C
for 4 hours. After digestion, the ovipositors were observed under a stereo
microscope Leica MZ 125 (Wetzlar, Germany) in order to identify the
anatomical parts.
For ultrastructural investigations excised ovipositors were immediately
immersed for 5 h in the fixing Carnoy solution (60 ml absolute ethanol, 30
ml chloroform, 10 ml glacial acetic acid) at room temperature. Dehydration
in absolute ethanol was followed by embedding in paraffin with xylene as
bridging solvent. Sections (20 µm) were taken on a microtome Olympus Cut
4060E (Wetzlar, Germany) and mounted on glass slides. Finally, the sections
were investigated with Zeiss microscope 0312 (Oberkochen, Germany), after
staining with hematoxylin-eosin. Digital pictures were obtained using a high
resolution digital camera Color View 12 (Soft Imaging System GmbH,
Münster, Germany).
Antennae. In order to count the number of antennomeres per antenna, both in
males and females, antennae were detached from anaesthetized adults and
were observed under a stereo microscope Leica MZ 125 (Wetzlar,
Germany). Ten males and ten females were used.
Scanning electron microscopy (SEM)
Ovipositor. The ovipositors of five anaesthetized females were excised and
dehydrated in a series of graded ethanol, from 50% to 99%. After
dehydration, specimens were mounted on aluminum stubs and goldsputtered using a Balzers Union SCD 040 unit (Balzers, Liechtenstein). The
observations were carried out using a scanning electron microscope Zeiss
Supra 40 (Oberkochen, Germany).
Antennae. Ten adults of both sexes were anaesthetized and dissected
removing the antennae from the head capsule. In some cases the whole head
with the antennae in their natural position was detached from the rest of the
body. Specimens were dehydrated in a series of graded ethanol, from 50% to
99%. After dehydration, specimens were mounted on aluminum stubs,
taking care to place them with different orientations in order to obtain a view
of the ventral, dorsal and both lateral sides. Mounted specimens were goldsputtered using a Balzers Union SCD 040 unit (Balzers Liechtenstein).
46
Chapter 2
Morphological investigations
The observations were carried out using a scanning electron microscope
Zeiss Supra 40 (Oberkochen, Germany).
Sensilla mapping. In order to count the number of sensilla ten adults of both
sexes were used. Each antennal club was divided in 4 equal sections from
the tip to the base of the club. In each section an area unit (50x50 µm) was
randomly chosen and used to count the number of sensilla.
Data analysis. Sensilla lengths and densities were analyzed by one-way
ANOVA, followed by Tukey’s test for mean separation. The statistical
analysis was performed at the P<0.05, using Systat 11 (Systat Software Inc.).
Transmission electron microscopy (TEM)
Antennae. Ten adults of both sexes were used for ultrastructural
investigations. The clubs of the antennae were isolated and immediately
immersed in a solution of glutaraldehyde and paraformaldehyde 2.5% in 0.1
M cacodylate buffer 5% sucrose, pH 7.2–7.3 (Karnovsky fixing solution).
The clubs were cut in small fragments (2-3 flagellomeres) to aid fixative
penetration and left at 4°C for 2 h. After rinsing overnight in 0.1 M
cacodylate buffer, the club fragments were post fixed in 1% OsO4 (osmium
tetroxide) for 1 h at 4°C and rinsed in the same buffer. Dehydration in a
graded ethanol series was followed by embedding in Epon-Araldite with
propylene oxide as bridging solvent. Thin sections (100 nm) were taken with
a diamond knife (Diatome, Biel/Bienne, Switzerland) on Leica Ultracut R
ultramicrotome (Wetzlar, Germany) and mounted on formvar coated 50
mesh grids. Finally, the sections were investigated with a Philips EM 208
miscroscope (Eindhoven, The Netherlands) after staining with uranyl acetate
(15 min, room temperature) and lead citrate (5 min, room temperature).
Digital pictures were obtained using a high resolution digital camera
MegaView III (Soft Imaging System GmbH, Münster, Germany) connected
to the TEM.
Results
Ovipositor. In P. archon the ovipositor is a telescopic one consisting of the
last 3 abdominal segments: 8th uromere forming the ovipositor base and
9th+10th uromeres fused together, making up the true ovipositor, connected to
the base by an intersegmental membrane, long about as much as the two
uromeres together (Figure 2.1 a). Two apodemes are present throughout the
ovipositor giving reinforcement and support to it. They are connected to the
cuticle of the 9th and 10th uromeres, while they are no longer connected to the
cuticle into the intersegmental membrane and the 8th abdominal segment
47
(Figure 2.1 a). The ovipositor in the resting position lies completely retracted
beneath the abdomen.
The outer surface of the intersegmental membrane at SEM observation
displays parallel series of longitudinal grooves and smooth surface, devoid
of any aperture or projection (Figure 2.1 b).
Paraffin cross sections of intersegmental membrane investigated by light
microscope show the integument consisting of a thick cuticle over a single
layer of atrophied epidermal cells resting on a thin basement membrane
(Figure 2.2 c and d). The apodemes are surrounded by a well developed
musculature, regulating ovipositor movement. The intestine is surrounded by
thick layers of muscles also. Moreover numerous tracheae are visible (Figure
2.2 a-d).
Figure 2.1 - Ovipositor pictures: a) light microscope dorsal view showing the
8th uromere, the intersegmental membrane (IM), the 9th + 10th uromeres and
the apomedes (Ap); b) SEM detail of the IM. Bar scale: b=2 µm
Antennae. P. archon presents segmented clubbed antennae composed of:
scape, pedicel and flagellum. Scape is the first basal antennal segment
articulated with the head through the torulus, connected by an elastic joint
membrane. Pedicel, the second segment, is articulated proximally with the
scape and distally with the rest of the antenna, that is the flagellum.
Flagellum, composed of most flagellomeres, is divided into club, the
extended distal portion, and apiculus. Apiculus, consisting of the last 5-10
segments, is tapered, recurved slightly upwards; numerous long setae are
present on the tip (Figure 2.3).
The number of antennomeres varied, but not significantly (F=0.925; P>0.05;
df=1), in dependence of sex: males 58.70±1.11 (mean±SE) and females
57.40±0.78 antennomeres (Table 2.1).
The 80% of the antenna is covered by enlarged and distally dentate scales;
the 20% appears without scales but bearing numerous sensilla. The sensillar
area starts as a non-continuous ventral strip at the level of 10th-15th
48
Chapter 2
Figure 2.2 - Paraffin cross sections of the ovipositor: a-b) cross section of 9th
and 10th uromeres at the positions A and B as reported in Figure 2.1 a; c-d)
cross section of IM at the positions C and D as reported in Figure 2.1 a; Ap:
apodemes; In: integument; Int: intestine; Mu: muscles; Tr: tracheae. Bar scale:
a-d=200 µm.
flagellomer, becoming uniform for about 10 flagellomeres and then
enlarging progressively. The 85% of the total sensilla are placed on the
ventral side of the club (Figure 2.3).
Club length was bigger in females (6.58±0.17 mm) than in males (5.52±0.16
mm) (F=19.927; P<0.001; df=1); the opposite was observed for the club
maximum diameter (males: 658.2±4.7 µm; females 618.1±12.3 µm)
(F=9.335; P<0.01; df=1). Significant difference did not result in the club
basal diameter between sexes (males: 330.7±7.4 µm; females 336.1±7.3 µm)
(F=0.279; P>0.05; df=1) (Table 2.1).
49
SEM and TEM investigations of P. archon flagellum revealed the presence
of six types of sensilla: sensilla chaetica, trichodea, basiconica, auricillica,
coeloconica and sensilla ampullacea.
Figure 2.3 - Schematic drawing of antenna in ventral-lateral view. Sc: scape,
first antennal segment; Pe: pedicel, second antennal segment; Fl: flagellum; Cl:
club, expanded distal portion of the flagellum; Ap: apiculus, last 5-10 segments.
Table 2.1 - Number of antennomeres and club measures in males and females
antennae (n=10). Data are means ± SE. Asterisks indicate significant difference
in male vs female comparisons (ns=not significant; *P <0.05; **P <0.01; ***P
<0.001; one-way ANOVA test).
Number of antennomeres
Club length (mm)
Club maximum diameter (µm)
Club basal diameter (µm)
Males
58.70±1.11
5.52±0.16
658.2±4.7
330.7±7.4
Females
57.40±0.78
6.58±0.17 ***
618.1±12.3 **
336.1±7.3
Sensilla chaetica. Sensilla chaetica appear as an elongated cuticular shaft
with a flexible well-defined basal socket characterized by a joint membrane
with developed suspension fibers (Figure 2.4 a and b). The hair shaft
diameter decreases toward the rounded tip pierced by few pores. The sensilla
are about 20 µm length and possess a thick cuticular wall.
TEM investigations show that the cellular components consist of 5 sensory
neurons with 3 accessory cells. Four sensory neurons enter the peg lumen as
outer dendritic segments enclosed in a common dendritic sheath and reach
unbranched the tip of the shaft. The outer dendritic segment of the fifth
sensory neuron ends in a tubular body attached to the joint membrane
(Figure 2.4 b and c).
50
Chapter 2
Figure 2.4 – Sensilla chaetica: a) SEM detail of a single sensillum; b) TEM
cross section at the socket level showing the joint membrane with suspension
fibers (JM); c) detail of the tubular body (TB white asterisk) and 4 outer
dendritic segments (ODS black asterisks). Bar scale: a, b, c=2 µm.
Sensilla trichoidea. They are characterized by an elongated cuticular shaft
decreasing in diameter toward the apex. The cuticular shaft is about 40 µm
long (Figure 2.5 a). The sensilla length did not result significantly different
between sexes (males: 40.56±1.25 µm; females: 41.02±0.81 µm; n=20)
(F=0.098; P>0.05; df=1). Moreover, the sensilla length did not change
between the prossimal and distal area of the club in both sexes (males:
F=0.330; P>0.05; df=1;females: F=0.031; P>0.05; df=1).
Numerous pores are present dorsally along herringbone grooves (Figure 2.5
b).
TEM investigations show a thick wall sensillum innervated by 2 or 3 sensory
neurons with 3 accessory cells. The outer dendritic segments of the sensory
neurons are enclosed in a common dendritic sheath; they, after entering the
peg lumen, reach unbranched or lightly branched the tip of the shaft (Figure
2.5 c and d).
Sensilla basiconica. They are characterized by a thin elongated cuticular
shaft, about 20 µm long, in which numerous pores are present (Figure 2.6 a
and b). They were longer in males (20.85±0.45 µm, n=20) than in females
(19.12±0.49 µm) (F=6.644; P<0.05; df=1). The sensilla length did not
change between the prossimal or distal area of the club in both sexes (males:
F=0.529; P>0.05; df=1; females: F=0.340; P>0.05; df=1).
51
TEM investigations show a thin wall sensillum with 2 or 3 sensory neurons
with 3 accessory cells. The outer dendritic segments of the sensory neurons
are enclosed in a common dendritic sheath and after entering the peg lumen,
reach branched the tip of the shaft (Figure 2.6 c).
Figure 2.5 – Sensilla trichoidea: a) SEM pictures showing sensilla trichoidea; b)
detail of sensillum trichodeum showing herringbone grooves and pores (Po); c)
TEM cross section of the shaft showing thick wall cuticle, pores (Po) and 3
outer dendritic segments (ODS); d) Three ODS (black asterisks) enclosed in a
common dendritic sheath. Bar scale: a=10 µm; b=1 µm; c=500 nm; d=2 µm.
Sensilla auricillica. They are characterized by an elongated and flattened
cuticular shaft about 12 µm long (Figure 2.7 a). The cuticular wall is
uniformly covered by numerous porous (Figure 2.7 a-c).
They resulted as long in males as in females (males: 11.89±0.31 µm;
females: 12.02±0.25 µm; n=20) (F=0.111; P>0.05; df=1). The sensilla length
did not change between the prossimal or distal area of the club in both sexes
(males: F=1.532; P>0.05; df=1; females: F=1.620; P>0.05; df=1).
TEM investigations show a thin wall sensillum innervated by 2 sensory
neurons with 3 accessory cells. The sensory neurons enter the peg lumen as
outer dendritic segments enclosed in a common dendritic sheath and reach
branched the tip of the shaft (Figure 2.7 c and d).
52
Chapter 2
Figure 2.6 – Sensilla basiconica: a) SEM picture showing sensillum
basiconicum; b) detail of sensillum basiconicum showing numerous pores (Po);
c) TEM cross section at the level of the peg showing dendritic branches (DB)
and pores (Po); d) TEM oblique section showing ciliary constriction (CC). Bar
scale: a, d=2 µm; b=200 nm; c=1 µm.
Figure 2.7 - Sensilla auricillica: a) SEM picture showing sensillum auricillicum;
b) detail of sensillum auricillicum showing pores (Po); c) TEM cross section
showing thin cuticular wall, pores (Po) and dendritic branches (DB); d) TEM
longitudinal section at the base of the peg showing 2 outer dendritic segments
(ODS). Bar scale: a, c=1 µm; b= 200 nm; d=2 µm.
53
Sensilla coeloconica. Cuticular parts of sensilla coeloconica appear as a
small and clavate peg with wall proximally smooth and distally grooved (16
grooves). Many pores are present along the grooves except on the tip. The
pores are set in round.
The peg, about 5 μm long, is completely embedded within the antennal wall,
in an ellipsoidal shaped cavity of amplitude 6,5 μm. The cavity is shallow
and wider at the bottom than at its opening (Figure 2.8 a and b).
TEM investigations show a double walled sensillum innervated by 3 sensory
neurons with 3 accessory cells. The outer dendritic segments of the sensory
neurons are enclosed in a common dendritic sheath and reach unbranched
the tip of the peg (Figure 2.8 c).
Figure 2.8 - Sensilla coeloconica. a) SEM picture showing sensillum
coeloconicum; b) TEM section showing grooves (Gr), pores (Po) and outer
dendritic segments (ODS); c) ODS (black asterisks) enclosed in a common
dendritic sheath . Bar scale a, b, c=2 µm.
Sensilla ampullacea. Sensilla ampullacea appear as a dome-shaped peg
about 3 µm long, completely embedded within a cuticular cavity which
diameter is about 3.5 µm. Pores are not present on the cuticular wall (Figure
2.9).
Sensilla mapping. Sensilla chaetica, coelonica and ampullacea are not
widespread over the sensillar area, but present in low number. Sensilla
chaetica are located at the base of antennomeres and approximately on all
sensory antennomeres in variable number: from 1 into the last 5
antennomeres to a maximum of 5 into the others. Sensilla coeloconica and
sensilla ampullacea are more present in the lateral sensillar areas near the
scales and their distribution did not seem to follow a fixed pattern, being not
uniform between individuals.
54
Chapter 2
Figure 2.9 – Sensilla ampullacea: a) SEM picture showing the sensillar area of
the antenna and a sensillum ampullaceum inside the white square; b) sensillum
ampullaceum. Bar scale a=10 µm; b=1 µm .
Particularly, sensilla coeloconica are found up to 8 in some antennomeres.
Sensilla ampullacea are present only on few antennomeres in number of 1 or
2 per antennomer.
Sensilla trichoidea, basiconica and auricillica are widespread over the entire
sensillar area of the antenna. Sensilla trichoidea were the most abundant both
in males (F=379.481; P<0.001; df=2) and females (F=46.153; P<0.001;
df=2), but their density was higher in males (9.98±0.36 sensilla/area unit)
than in females (8.05± 0.46 sensilla/area unit) (F=10.930; P<0.01; df=1).
Sensilla basiconica were fewer than trichoidea in both sexes, but their
density was higher in females (5.18±0.36 sensilla/area unit) than in males
(2.45±0.15 sensilla/area unit) (F=47.932; P<0.001; df=1). Sensilla auricillica
resulted fewer in number than trichoidea and basiconica. Their density was
higher in females (3.15±0.22 sensilla/area unit) than in males (1.48±0.15
sensilla/area unit) (F=39.404; P<0.001; df=1) (Table 2.2).
Considering the distribution on different sections of the club, in both males
and females the density of sensilla trichodea (males: F=30.436; P<0.001;
df=3; females: F=17.502; P<0.001; df=3) and basiconica (males: F=4.810;
P<0.01; df=3; females: F=12.278; P<0.001; df=3) decreased from the tip to
the base of the club as detailed in Table 2.2. The density of sensilla
auricillica in males was higher towards the base of the club (F=5.913;
P<0.01; df=3) and uniform in females (F=1.613; P>0.05; df=3) (Table 2.2).
55
Table 2.2 - Sensilla trichoidea, basiconica and auricillica densities in different sections of the antennal club in males and
females. Asterisks indicate significant difference in male vs female comparisons (*P <0.05; **P <0.01; ***P <0.001; one-way
ANOVA test). Identical letters within the same column indicate no statistical difference (Tukey’s test, P <0.05). C1= distal
section of the club; C2-C3= intermediate sections of the club; C4= prossimal section of the club.
56
C1
C2
C3
C4
Overall means
Trichoidea
Males
Females
12.60±0.52 a
11.60±0.67 a
10.50±0.31 b
8.10±0.60 b
9.40±0.31 b
6.80±0.65 bc
7.40±0.40 c
5.70±0.52 c
9.98±0.36
8.05±0.46 **
Sensilla density (n° sensilla/area unit)
Basiconica
Auricillica
Males
Females
Males
Females
2.20±0.25 a
7.90±0.57 a
1.20±0.13 a
3.30±0.26 a
3.30±0.26 b
4.30±0.47 b
1.00±0.15 a
3.80±0.49 a
2.10±0.23 a
3.70±0.30 b
2.40±0.34 b
3.00±0.58 a
2.20±0.29 a
4.80±0.71 b
1.30±0.33 a
2.50±0.31 a
2.45±0.15
5.18±0.36 ***
1.48±0.15
3.15±0.22 ***
56
n
10
10
10
10
40
Chapter 2
Discussion
Ovipositor. Sex pheromone glands are often associated with genital region of
body. Such glands in female moths are consistent in their location, highly
conserved, and histological features, more than in other insect orders. Glands
are commonly found as modified intersegmental membrane between the 8th
and the 9th abdominal segments (Percy and Weatherson, 1974; Tamaki,
1985).
Investigations of gland surface have reported to be common the presence of
protuberances, although with some structural variations (spikes, spines or
microspines) (Weatherson and Percy 1970; Conner et al., 1980; Aubrey et
al., 1983), in Lepidoptera and not only. These projections are supposed to be
involved in secretion storage and evaporation regulation during gland
eversion by calling females (Percy-Cunningham and McDonald, 1987;
Solinas and Isidoro 1991; Isidoro et al., 1992).
In P. archon ovipositor, SEM investigations showed a smooth cuticle
surface of the intersegmental membrane, devoid of any protuberance or
aperture. Moreover, ultrastructural investigations revealed the epidermal
cells of the intersegmental membrane did not show typical features of
glandular cells observed in many pheromone glands such as hypertrophy,
large nucleus and cytoplasm features typical of intense secretory activity
(many vacuoles, extensive smooth endoplasmic reticulum, abundant
ribosomes, well developed Golgi apparatus etc.) (Percy and Weatherson,,
1974; Percy-Cunningham and McDonald, 1987). Hence, in P. archon there
is no evidence of pheromone gland in the intersegmental membrane of the
ovipositor.
Sarto I Monteys et al. (2012) has come to the same conclusion supported by
SEM observation and EAG responses of ovipositor extracts in male antennae
suggesting an evolutionary loss of long-range female pheromone in P.
archon.
So far, no castniid pheromone is known but another castniid species, Castnia
licus (Drury), the giant sugarcane borer, has been investigated to identify the
female sex pheromone. In fact, hexane extracts of ovipositors have been
analyzed by gas chromatography and mass spectrometry and have been
found to elicit male responses in behavioural bioassays by olfactometer
(Reboucas et al., 1999).
Other diurnal moths (families Sesiidae, Zygaenidae and Arctiidae) have been
reported to use female sex pheromone for male attraction as predominant
cue, despite to their diurnal habit (Naka et al., 2007; Subchev et al., 2009;
Kondo et al., 2012).
57
Antennae. P. archon antennae are thin and club shaped without any evident
sexual dimorphism. The surface of the antenna covered by sensilla is
extremely reduced, housed mainly on the ventral side of the club as in dayflying butterflies that depend more on visual stimuli than on olfactory cues
(Silberglied, 1984; Vane-Wright and Bopprè, 1993; Sarto I Monteys et al.,
2012).
On the contrary in moth species, using female long-distance sex pheromones
for mate location, the antennae are often sexually dimorphic. The female
antennae are filiform, while the male antennae range from being filiform in
some species, to being extremely developed, representing a surface
amplification, with a larger number of sensilla and a greater length of these
sensilla. (Steinbrecht, 1987).
Six different types of sensilla were found on the antennae of both sexes of P.
archon. As reported by Hallberg and Hansson (1999) all these sensilla types
are fairly costant in “higher” ditrysian Lepidoptera. Until now, P. archon
antennae have been investigated by stereomicroscopy and SEM reporting 4
types of sensilla: chaetica, trichoidea, basiconica and auricillica (Sarto I
Monteys et al., 2012).
Our SEM and TEM data are consistent with a double mechanochemosensory gustatory function for P. archon sensilla chaetica. They are
characterized by the presence of few apical pores, thick wall and a set of 5
sensory neurons whose outer dendritic segments reach unbranched the tip.
One outer dendritic segment ends in a tubular body attached to the joint
membrane at the base of the shaft. Moreover these sensilla are present lateroventrally in antennae of both sexes, as noted most often on insect
appendages used by contact (Zacharuk, 1980; 1985).
In P. archon, sensilla trichoidea, basiconica and auricillica have a porous
hair shaft, indicative of an olfactory chemosensory function. Sensilla
trichoidea are characterized by the presence of numerous pores on the
cuticular wall along herringbone grooves, single thick wall and a set of 2 or
3 neurons with outer dendritic segments unbranched or lightly branched in
the hair lumen. Sensilla trichoidea have been largely investigated in
Lepidoptera. In male moth antennae, long sensilla trichoidea have been
reported to be female sex pheromone receptors (Kaissling, 1979; Faucheux,
1990; Hansson, 1995; Castrejon-Gomez et al., 1999, 2003). Shorter sensilla
trichoidea have been reported in females to be involved in the detection of
volatile compounds released by the host plant (Anderson et al., 1995;
Calatayud et al., 2006). In P. archon their length did not vary with sex
contrary to what reported by Sarto I Monteys et al. (2012) and as generally
observed in moths (Hallberg et al., 2003). Although their density was higher
in males, the club size suggests a compensation in number of sensilla
58
Chapter 2
trichoidea in females. For these reasons and because there is no evidence of
pheromone gland in the intersegmental membrane of the ovipositor, sensilla
trichoidea in P. archon males appear not to be involved in long range female
pheromone detection, as suggested by Sarto I Monteys et al. (2012).
In P. archon sensilla basiconica and auricillica are characterized by
numerous pores on the cuticular wall without grooves, a single thin wall and
a set of 2 or 3 neurons with outer dendritic segments branched up to the tip
of the sensillum. Sensilla basiconica have been shown to be involved both in
host-odour and pheromone detection (Yamazaki, 1966; Grant et al., 1989;
Kafka, 1987). In the Arctiid moth, Utetheisa ornatrix (Linneus), basiconica
sensilla in females are unexpectedly sensitive to male produced pheromone,
since receptor neurons responsive to insect pheromones in male moths
appear, as a rule, to be associated with sensilla trichoidea (Grant et al., 1989;
Cuperus, 1985). In P. archon, sensilla basiconica, fewer than trichoidea,
showed to be longer in males, contrary to what generally observed in moths
(Hallberg et al., 2003), and more abundant in females. Sensilla auricillica
have been reported to be receptor for plant volatiles (Den Otter et al., 1978;
Mochizuki et al.,1992; Anderson et al., 2000) and more recently for sex
pheromone (Ansebo, 2004). Their length in P. archon did not vary with sex
as generally observed in moths (Hallberg et al., 2003). They showed to be
fewer than basiconica and more abundant in P. archon females.
Our data on sensilla density confirm most of recent investigations on P.
archon antennae, except that sensilla basiconica have been reported to be
fewer than auricillica by Sarto I Montes et al. (2012). Although no sexual
dimorphism was observed in sensillar types and features, differences
between sexes were found in densities of sensilla supposed to be olfactory
(trichoidea, basiconica and auricillica), suggesting they could respond to
different volatiles.
In Bombyx mori (Linneus) long sensilla trichoidea with 2 neurons have been
found to be the most abundant in both sexes. Those of the male are
specifically responsive to female sex pheromone but those of the female are
not (Boeckh et al., 1965; Kaissling et al., 1978). Sensilla trichoidea in B.
mori females respond to a set of compounds proved to be plant volatiles and
components of male-produced pheromones in other Noctuid species (Birch
et al., 1990 and references therein; Heath et al., 1992; Anderson et al.,
2009).
Hence, function is not associate with a morphological sensillum type but
rather to the specificity of the neuron receptors (Steinbrecht, 1999 and
references therein).
59
Moreover, a certain overlap has been observed in the specificity of receptor
cells housed in morphologically distinct sensillum types so that more sensilla
can contribute to plant odour reception (Pophof, 1997; Pophof et al., 2005).
In P. archon, sensilla coeloconica appear as short double walled sensilla set
in a pit with 3 sensory neurons and could be referred to MPG “Multiporous
Grooved Peg” found in many insect orders (Altner and Prillinger, 1980; Keil
and Steinbrecht, 1984; Steinbrecht, 1997; Zacharuk, 1980). They are found
scattered in low number neighboring the surface covered by scales. They
have been often reported as grouped in small patches in specific antennal
areas (Altner et al., 1981; Hunger and Steinbrecht, 1998; Ruchty et al.,
2009). Functionally, MGP, are associated with olfactory function or with a
double olfactory-thermoreceptory function (Altner et al., 1977; Altner and
Prillinger, 1980; Zacharuk, 1985; Diehl et al., 2003; Pophof et al., 2005).
In B. mori, as in other species, double walled sensilla coeloconica have been
reported to be sensitive to host plant odors and not to humidity or
temperature (Pophof, 1997; Pophof et al., 2005).
Finally our data are consistent with a possible role of sensilla ampullacea as
thermo-hygroreceptors in P. archon. There is no evidence of TEM data
because of their very low number, but they are distributed on both lateral
side of the antennomeres, embedded within the antennal wall, closely related
to no pore sensilla (Altner, 1977) described in many insect orders (Zacharuk,
1985).
Further work by elettroantennography and single cell recording has to be
done to validate these functional hypotheses.
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64
Concluding remarks and future works
In this thesis the courtship and copulatory behaviour of the castniid palm
moth, Paysandisia archon (Burmeister 1880) have been investigated,
focusing on the better understanding of the mechanisms involved in the steps
leading to mating. Morphological investigations of the ovipositor, as
possible site of sex pheromone gland, and of the antennal sensory equipment
have provided a complementary support to fully characterize the
communication between sexes. The knowledge of the reproductive
behaviour at different levels (behavioural, sensorial, chemical) is an
important point to develop new strategies for phytophagous control.
Especially for quarantine pests as P. archon, primary and principal tasks in
Integrated Pest Management (IPM) are to monitor the pest population and
early detect the infestation, to forecast the expansion and to evaluate the
success of eradication and control efforts in new areas of introduction. This
is mostly crucial for those pests that are concealed borers.
P. archon courtship and copulatory behavioural sequence have been
described, providing both qualitative and quantitative data for the first time.
In P. archon courtship behaviour, we have shown that visual cues are
involved deeply in mate location. P. archon females first approached the
perching male, who then pursued, mainly activated by the movement of
flying female. These findings have suggested the lack of female sex
pheromone active over long-range, confirmed by ultrastructural
investigations of the ovipositor.
In P. archon courtship behaviour the pair flight seems to be very important
in communication between sexes at close range. We have hypothesized that
anemotactile/mechanosensory cues could stimulate male and female each
other during the pair flight and visual cues are involved in close-range
interactions as well. Moreover, at close range, during female first approach
or/and pair flight olfactory cues could be released by the female and received
by male stimulating the next copulatory behaviour. Further bioassays are
needed to confirm these hypotheses.
These are the first ultrastructural investigations of the P. archon antennae
and ovipositor. No pheromone gland has been found in the intersegmental
membrane of the P. archon ovipositor. The P. archon antennae have shown
a clubbed shape, reduced sensillar surface without evident sexual
dimorphism. Six different types of sensilla have been found on the antennae
of both sexes of P. archon: sensilla chaetica, trichoidea, basiconica,
auricillica, coeloconica and ampullacea. Sensilla coeloconica and
65
ampullacea have never been found so far. Hypotheses on function of these
sensilla have to be assessed by further electrophysilogical investigations.
All these results have pointed out behavioural and morphological similarities
of this castniid palm moth with butterflies rather than moths. In fact
Neotropical castniids, according to its diurnal habit and the bright
colorations of hind wings, are called “butterfly-moths” or “sun moth”.
This thesis has effects on practical applications. At the moment, it is not
possible to use pheromone traps for P. archon monitoring, or direct control
methods (mass trapping, communication disruption etc.), as usually in many
moths. Other devices exploiting visual stimuli have to be developed and
tested. Therefore future dedicated work of the vision assessing various
aspects including visual range and attractive colors are also needed to fully
characterize the P. archon adult behaviour.
66
Acknowledgements
I am very grateful to Dr. S. Ruschioni and Dr. R. Minux for their
cooperation and support in the realization of this thesis.
Thanks to Dr. V. M. Rossi Stacconi and Mr. C. Dentini, Perugia University,
for cooperation and help in morphological investigations.
Thanks are also due to the technical staff of the Department of Materials,
Environmental Sciences and Urban Planning, Marche Polytechnic
University, and of the University Electron Microscopy Centre (CUME),
Perugia University.
I would also like to thank Dr. A. Piunti and the staff of the experimental
farm “P. Rosati”, Marche Polytechnic University.
67

Reproductive biology of Paysandisia archon

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