Reproductive biology of Paysandisia archon
Transcription
Reproductive biology of Paysandisia archon
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 Materials and methods 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). References Besse-Millet S., Bonhomme A., Panchaud K. (2008). Efficacy of a Beauveria bassiana (Vuillemin) strain against the Castniid palm borer Paysandisia archon (Burmeister, 1880) under laboratory and natural conditions. IOBC/wprs Bulletin 31, 216-219. Birch C., Poppy G. M., Baker T. C.(1990). Scent and eversible scent structures of male moths. Annual Review of Entomology 35, 25-58. Birch M.C., Haynes K.F. (1982). Insect pheromones. E. Arnold, London. Bourquin F. (1933). 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[Paysandisia 12 Introduction archon (Lepidoptera, Castniidae): first report of damages of the dangerous palm borer on the adriatic coast.] Informatore Fitopatologico 10, 28-31. Riolo P., Nardi S., Isidoro N. (2005). Indagini biologiche su un lepidottero minatore delle palme di recente introduzione in Italia. XX Congresso Nazionale Italiano di Entomologia, 262. Romero F. (1998). Comportamiento, oviposición y distribución de Duboisvalia simulans (Boisduval) en Venezuela (Lepidoptera:Castniidae). Boletin de Entomologia Venezolana N.S. 13 (1), 91-92. Rutowski R.L. (1991). The evolution of male mate-locating behavior in butterflies. American Naturalist 138 (5), 1121-1139. Salt G. (1929). Castniomera humboldti (Boisduval), a pest of bananas. Bulletin of Entomological Research 20 (2), 187-193. Sarto I Monteys V. (2002). The discovery, description and taxonomy of Paysandisia archon (Burmeister, 1880), a castniid species recently found in south-western Europe (Castniidae). Nota Lepidopterologica 25(1), 3-15. Sarto I Monteys V., Acín P., Rosell G., Quero C., Jiménez M.A., Guerrero A. (2012) Moths behaving like butterflies. Evolutionary loss of long range attractant pheromones in Castniid moths: a Paysandisia archon model. PLoS ONE 7(1), e29282. doi:10.1371/journal.pone.0029282. Sarto I Monteys V., Aguilar L. (2005). The Castniid Palm Borer, Paysandisia archon (Burmeister, 1880) (Lep. Castniidae), in Europe: comparative biology, pest status and possible control methods. Nachrichten des Entomologischen Vereins Apollo N.F 26 (1-2), 61-94. Scoble M.J. (1992). The Lepidoptera form, function and diversity. Oxford, Oxford University Press. Scott (1972). Mating of butterflies. The Journal of Research on the Lepidoptera 11, 99-127. Scott J.A. (1986). The Butterflies of North America: a natural history and field guide. Stanford University Press, Stanford, California Scudder S.H. (1877). Antigeny, or sexual dimorphism in butterflies. Proceedings of the American Academy of Arts and Sciences 12, 150-158. Seitz A., Strand E. (1913). Famille Castniidae. In: Les Macrolépidoptères du globe. Bombyx et Sphinx américains. Seitz A. Eds. Paris (Cabinet EntomologiqueE. Le Moult).. 6, 5-19. Silberglied R.E. (1984). Visual communication and sexual selection among butteflies. In: The biology of butterflies. Vane-Wright R.I., Ackery P.R Eds. London, UK: Academic, 207-233. Sreng I., Sreng L. (1988). Fine structure of the female sex pheromone-producing glands in Sesamia nonagrioides Lef. (Lepidoptera: Noctuidae). International Journal Insect Morphology and Embryology 17, 345-357. Thornhill R., Alcock J. (1983). The evolution of insect mating systems. Harvard University Press, Cambridge, Massachusetts. Tindale N.B. (1980). Origin of the Lepidoptera with description of a new mid Triassic species and notes on the origin of the butterfly stem. Journal of Lepidoptera Society 34 (3), 263 -285. 13 Vane-Wright R.I., Bopprè M. (1993). Visual and chemical signalling in butterflies: functional and phylogenetic perspectives. Philosophical Transactions of the Royal Society: Biological Sciences 340 (1292), 197-205. Virant-Doberlet M., Cokl A. (2004). Vibrational communication in insects. Neotropical Entomology 33 (2), 121-134. Witzgall P., Kirsch P., Cork A. (2010). Sex Pheromones and Their Impact on Pest Management. Journal of Chemical Ecology 36, 80-100. www.environment.gov.au www.pherobase.com Zacharuk R.Y. (1985). Antennae and sensilla. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut G.A., Gilbert L.I. Eds. Pergamon, Oxford 6, 1-69. 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. Materials and methods 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 Behavioural investigations 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 Behavioural investigations 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 Behavioural investigations 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 Behavioural investigations 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). Mean environmental parameters recorded during bioassays were 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 Behavioural investigations 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 1 - Perching (100.0% of occurrence). 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). Mean environmental parameters recorded during bioassays were 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 1 - Perching (100.0% of occurrence). 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 4 - Alighting (66.7% of occurrence). 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 Behavioural investigations 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). Mean environmental parameters recorded during bioassays were 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 *Steps not performed 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 Behavioural investigations 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 1 - Perching (100.0% of occurrence). Step 2 - Female flight (100.0% of occurrence): all the females flew more than once approaching the dummy male. Step 4 - Alighting (71.4% of occurrence). 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). Mean environmental parameters recorded during bioassays were 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 *Steps not performed 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 Behavioural investigations 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 1 - Perching (100.0% of occurrence). 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 Behavioural investigations 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 *Steps not performed 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 Behavioural investigations 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 Behavioural investigations 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 Andersson M. (1994). Sexual selection. Princeton, New Jersey: Princeton University Press. 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, 845-851 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 of the National Academy of Sciences USA 88, 9224-9227. Fordyce J.A., Nice C.C., Forister M.L., Shapiro A.M. (2002). The significance of wing pattern diversity in the Lycaenidae: mate discrimination by two recently 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 pheromone and associated stimuli. Annals of the Entomological Society of America 80, 78-88. 41 Grant G.G., Brady U.E. (1975). Courtship behavior of Phycitid moths. Comparison of Plodia interpunctella and Cadra cautella and the role of male scent glands. Canadian Journal of Zoology 53, 813-826. Hartlieb E., Anderson P. (1999). Olfactory-released behaviours. In Insect olfaction. Hanson B.S. Eds.. Berlin, Germany: Springer, 315-349. Korytkowski C.A., Ruiz E.R. (1980). El barreno de los racimos de la palma aceitera, Castnia daedalus (Cramer), Lepidopt.:Castniidae, en la plantación de Tocache, Perú. Revista Peruana de Entomología 22, 49-62. Lara F. (1964). The banana stalk borer Castniomera humboldti (Boisduval) in La Estrella Valley, Costa Rica. II. Bionomics. Turrialba 14 (4), 188-195. McDonald A.K., Nijhout H.F. (2000). The effect of environmental conditions on mating activity of the buckeye butterfly, Proecis coenia. Journal of Research on the Lepidoptera 35, 22-28. Nakano R., Skals N., Takanashi T., Surlykke A., Koike T., Yoshida K., Maruyama H., Tatsuki S., Ishikawa, Y. (2008). Moths produce extremely quiet ultrasonic courtship songs by rubbing specialized scales. Proceedings of the National Academy of Sciences USA 105, 11812-11817. Nakano R., Takanashi T., Fujii T., Skals N., Surlykke A., Ishikawa Y. (2009). Moths are not silent, but whisper ultrasonic courtship songs. The Journal of Expimental Biology 212, 4072-4078. Nieberding C.M. , de Vos H., Schneider M.V., Lassance J.M., Estramil N., Andersson J., Bang J., Hedenstrom E., Löfstedt C., Brakefield P.M. (2008) The male sex pheromone of the butterfly Bicyclus anynana: towards an evolutionary analysis. PLoS ONE 3(7): e2751. doi:10.1371/journal.pone.0002751 Riolo P., Nardi S., Carboni M., Riga F., Piunti A., Ferracini C., Alma A., Isidoro N. (2004). Paysandisia archon (Lepidoptera, Castniidae): prima segnalazione di danni del pericoloso minatore delle palme sulla riviera adriatica. [Paysandisia archon (Lepidoptera, Castniidae): first report of damages of the dangerous palm borer on the adriatic coast.] Informatore Fitopatologico 10, 28-31. Riolo P., Nardi S., Isidoro N. (2005). Indagini biologiche su un lepidottero minatore delle palme di recente introduzione in Italia. XX Congresso Nazionale Italiano di Entomologia, 262. Pliske, T.E. (1974). Courtship behaviour of the monarch butterfly, Danaus plexippus. Annals of the Entomological Society of America 68, 143-151. Rutowski R.L., Gilchrist G.W., Terkanian B. (1988). Mate locating behavior in Euphydryas chalcedona (Lepidoptera: Nymphalidae) related to pupation site preferences. Journal of insect behavior 1, 277-289. Rutowski R.L., Schaefer J. (1984). Courtship behavior of the gulf fritillary, Agraulis vanilla, (Nymphalidae). Journal of the Lepidopterists Society 38, 23-31. Sanders C.J. (1979). Mate location and mate in eastern spruce budworm. Department of the Environment, Canadian Forest Service, Ottawa. Ontario Biman. Research Notes 35, 2-3. Sanders C.J., Lucuick G.S.M. (1992). Mating behavior of the spruce budworm moths, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). The Canadian Entomologist 124, 273-286. 42 Chapter 1 Behavioural investigations Sarto I Monteys V., Acín P., Rosell G., Quero C., Jiménez M.A., Guerrero A. (2012) Moths behaving like butterflies. Evolutionary loss of long range attractant pheromones in Castniid moths: a Paysandisia archon model. PLoS ONE 7(1), e29282. doi:10.1371/journal.pone.0029282 Scott (1972). Mating of butterflies. The Journal of Research on the Lepidoptera 11, 99-127. Scott J.A. (1986). The Butterflies of North America: a natural history and field guide. Stanford University Press, Stanford, California. Shimizu K., Tamaki Y. (1980). Releasers of male copulatory attempt in the smaller tea tortrix moth (Lepidoptera: Tortricidae). Applied Entomology and Zoology 15, 140-150. Silberglied R.E. (1984). Visual communication and sexual selection among butteflies. In: The biology of butterflies. Vane-Wright R.I.,. Ackery P.R. Eds. London, UK: Academic, 207-233. Trematerra P. (1992). Su alcuni meccanismi di richiamo sessuale adottati da Corcyra cephalonica (Stainton) (Lepidoptera Galleriidae). Redia LXXV, 109121. Wickman P.O. (1988). Dynamics of mate searching behavior in hilltopping butterfly, Lasiommata megera (L.): the effects of weather on male density. Zoological Journal of the Linnean Society 93, 357-377. Vane-Wright R.I., Bopprè M. (1993). Visual and chemical signalling in butterflies: functional and phylogenetic perspectives. Philosophical Transactions of the Royal Society: Biological Sciences 340 (1292), 197-205. Zar J.H. (1999). Biostatistical analysis. Upper Saddle River, New Jersey:Prentice Hall. 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 Materials and methods 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 Morphological investigations 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 Morphological investigations 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 Morphological investigations 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 Morphological investigations 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 Morphological investigations 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 Morphological investigations 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. References Altner H , Sass H , Altner I. (1977) Relationship between structure and function of antennal chemoreceptive, hygroreceptive, and thermoreceptive sensilla in Periplaneta americana. Cell and Tissue Research 176, 389-405. Altner H. (1977). Insect sensillum specificity and structure: an approach to a new tipology. Olfaction taste 6, 295-303. Altner H., Prillinger L. (1980). Ultrastructure of invertebrate chemo, thermo and hygroreceptors and its functional significance. 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Pergamon, Oxford 6, 1-69. 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
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