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Influence of a Juvenile Hormone Analog and Dietary Protein on Male Caribbean Fruit Fly, Anastrepha suspensa (Diptera: Tephritidae), Sexual Behavior

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Title:
Influence of a Juvenile Hormone Analog and Dietary Protein on Male Caribbean Fruit Fly, Anastrepha suspensa (Diptera: Tephritidae), Sexual Behavior
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PEREIRA, RUI MANUEL CARDOSO ( Author, Primary )
Copyright Date:
2008

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Aggregation ( jstor )
Female animals ( jstor )
Fruit flies ( jstor )
Insects ( jstor )
Juvenile hormones ( jstor )
Lipids ( jstor )
Mating behavior ( jstor )
Pheromones ( jstor )
Signals ( jstor )
Table sugars ( jstor )

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University of Florida
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University of Florida
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Copyright Rui Manuel Cardoso Pereira. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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12/31/2006
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496175030 ( OCLC )

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INFLUENCE OF A JUVENILE HORM ONE ANALOG AND DIETARY PROTEIN ON MALE CARIBBEAN FRUIT FLY, Anastrepha suspensa (DIPTERA: TEPHRITIDAE), SEXUAL BEHAVIOR By RUI MANUEL CARDOSO PEREIRA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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ACKNOWLEDGMENTS I thank my advisor, John Sivinski , for his involvement, support, and encouragement. I have benefited immensely from his critical a nd creative thinking, and also been greatly inspired by his enthusiasm fo r science. I am grateful to my committee, Jane Brockmann, Jorge Hendrichs, Heather Mc Auslane, Gary Steck, and Peter Teal, for their unstinting help. The diverse perspec tives they provided enriched my work. I would like to thank the Centro de Cincia e Tecnologi a da Madeira for financial support through the Ph.D. grant BD I/2002-004. Heidi Burnside and Nancy Lowman maintain ed the Caribbean fruit fly colony from which I obtained the flies for my experime nts, Barbara Dueben helped prepare the methoprene solutions and Jeff Shapiro had the patience to teach me protein and lipid analytical techniques. I also thank Paul Shirk and Jeffrey Lotz for the beautiful Caribbean fruit fly pictures used in presentations of my wo rk and Meghan Brennan for the advice in the statistical survival analyses performed in Chapter 5. Finally, I thank Hoa Hguyen for the indispen sable technical assistance in most of the experimental activities performed. ii

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ..................................................................................................ii LIST OF TABLES ...............................................................................................................v LIST OF FIGURES ...........................................................................................................vi ABSTRACT .....................................................................................................................viii CHAPTER 1 GENERAL INTRODUCTION....................................................................................1 2 INFLUENCE OF A JUVENI LE HORMONE ANALOG AND DIETARY PROTEIN ON MALE SEXUAL SUCCESS.............................................................14 Introduction .................................................................................................................14 Material and Methods .................................................................................................15 Results .........................................................................................................................20 Discussion ...................................................................................................................25 3 INFLUENCE OF A JUVENI LE HORMONE ANALOG AND DIETARY PROTEIN ON MALE SEXUAL BEHAVIORS WITHIN MATING AGGREGATIONS.....................................................................................................29 Introduction .................................................................................................................29 Material and Methods .................................................................................................31 Results .........................................................................................................................35 Discussion ...................................................................................................................38 4 INFLUENCE OF A JUVENI LE HORMONE ANALOG AND DIETARY PROTEIN ON MALE ATTRACTI VENESS TO FEMALES...................................43 Introduction .................................................................................................................43 Material and Methods .................................................................................................44 Results .........................................................................................................................51 Discussion ...................................................................................................................54 iii

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5 EFFECTS OF SEXUAL INTERACTIONS ON MALE LONGEVITY...................58 Introduction .................................................................................................................58 Material and Methods .................................................................................................60 Results .........................................................................................................................63 Discussion ...................................................................................................................66 6 INFLUENCE OF A JUVENI LE HORMONE ANALOG AND DIETARY PROTEIN ON MALE BODY LIPI D AND PROTEIN CONTENTS.......................70 Introduction .................................................................................................................70 Material and Methods .................................................................................................72 Results .........................................................................................................................76 Discussion ...................................................................................................................81 7 GENERAL DISCUSSION.........................................................................................84 LIST OF REFERENCES ...................................................................................................89 BIOGRAPHICAL SKETCH ...........................................................................................102 iv

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LIST OF TABLES Table page 1-1 List of tephritids that form leks. .................................................................................6 3-1 Number of male Caribbean fruit flies th at occupied the vari ous positions within leks. ..........................................................................................................................38 3-2 Percentages of resident male Caribbean fruit flies (by treatment) in leks that won contests against intruders. ........................................................................................38 3-3 Summary of sexual success para meters in Caribbean fruit fly. ...............................40 5-1 Results of the survival analysis. ...............................................................................65 6-1 Analysis of variance (ANOVA) for male Caribbean fruit fly weight, total lipids, and total proteins among different ages in six different treatments. ........................78 6-2 Analysis of variance (ANOVA) for male Caribbean fruit fly weight, total lipids, and total proteins among trea tments for different ages. ...........................................78 v

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LIST OF FIGURES Figure page 2-1 Percentage of matings per treatment of male Caribbean fruit flies in laboratory and field cage experiments. ......................................................................................21 2-2 Percentage of matings of male Caribb ean fruit flies from day 4 to the end of experiment (day 35). ................................................................................................22 2-3 Percentage of male Caribbean fr uit flies that mated more than once. ......................23 2-4 Number of matings obtained per treatm ent at various ages by male Caribbean fruit flies. ..................................................................................................................24 2-5 Average number of male Caribbean fruit flies that mated twice, or three times on the same day. ............................................................................................................25 2-6 Mean copulation duration of male Ca ribbean fruit flies that mated 1, 2, and 3 consecutive times. ....................................................................................................26 3-1 Lek parameters and sexual succ ess of male Caribbean fruit flies. ...........................36 3-2 Female Caribbean fruit fly acceptance index. ..........................................................39 4-1 Arrangement of the experiment (attractiveness in laboratory). ................................47 4-2 Arrangement of the experiment (attractiveness in greenhouse). ..............................48 4-3 Artificial leks used in the greenhouse experiment. ..................................................50 4-4 Time spent by male Caribbean frui t flies calling and time spend by female visiting the males. .....................................................................................................52 4-5 Percentage of male Caribbean fruit flies in a greenhouse calling and female approaches to males. ................................................................................................53 4-6 Correlation between the number of male Caribbean fruit flies calling within an artificial lek and the number of female visiting in the greenhouse environment. ....54 vi

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5-1 Cumulative survival probability of ma le Caribbean fruit flies in the three experimental groups. ................................................................................................64 5-2 Cumulative survival probability of male Caribbean fruit flies in the four treatments. ................................................................................................................66 6-1 Average adult weight of male Caribbean fruit flies. ................................................77 6-2 Average total lipid contents of male Caribbean fruit flies. ......................................79 6-3 Average total protein contents of male Caribbean fruit flies. ..................................80 vii

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INFLUENCE OF A JUVENILE HORM ONE ANALOG AND DIETARY PROTEIN ON MALE CARIBBEAN FRUIT FLY, Anastrepha suspensa (DIPTERA: TEPHRITIDAE), SEXUAL BEHAVIOR By Rui Manuel Cardoso Pereira December 2005 Chair: John Sivinski Major Department: Entomology and Nematology The Caribbean fruit fly, Anastrepha suspensa (Loew), like many polyphagous tephritids (Diptera: Tephritidae), adopts a lek polygyny mating system. The success of sterile insect technique (SIT) di rected towards this pest requires the release of males that can compete with wild males and attract w ild females. Mass-reared males must form leks, engage in male-male agonistic interac tions, court females, and mate to transfer sterile sperm. The effects of application of a juvenile hormone analog, methoprene, and dietary protein on male Caribbean fruit fly sexual success were evaluated in the four possible combinations of the two factors (methoprene app lication and protein supply (M + P P + ); methoprene applicati on and no protein supply (M + PP ); no methoprene application and protein supply (M P P + ); and no application of me thoprene or protein supply (M PP ). Laboratory, field cage, and greenhouse experiments compared male sexual performance and other reproductive parameters on a lifetim e and daily basis. Numbers of copulations, lek initiation, lek par ticipation, pheromone signaling (calling), and female attraction were viii

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all significantly greater in M + P P + males. Simultaneously, methoprene caused earlier male maturation. Sexual interactions, both intrase xual and intersexual (r esource expenditure), and protein consumption (resource availability ) resulted in significant reductions and increases, respectively, in male longevity. No impact of methoprene on survival was observed. A protein-rich diet increased the weight, total lipid content and total protein content of males during the first 35 days of adult life. No impact of methoprene on nutritional status was observed. The substantial improvement in male sexual performance due to the methoprene application, protein supply, and interaction of methoprene and protein, and an earlier sexual maturation due to methoprene applicati on have the potential to produce more efficacious ster ile males for SIT programs. ix

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CHAPTER 1 GENERAL INTRODUCTION The sterile insect technique (SIT) is the principal non-chemical component of pest management of tephritid (Diptera: Tephr itidae) flies (Hendrichs et al. 2002). Improvements in production costs and insect quality influence the efficacy of the technique and even the practicality of its us e under certain circumstances (Robinson et al. 2002). In this study I examine the influence of juvenile hormone analog, methoprene and dietary protein as a t ool to improve SIT. Control of insects with SIT technique is ba sed on the release of sterile males of the target species that mate with wild females who then produce unviable offspring (Knipling 1955). Sterile males are typically mass-reared in facilities having the capacity to produce up to hundreds of millions of males per week (Hendrichs et al. 1995). However, to be effective the released sterile males have to successfully transfer their sperm carrying dominant lethal mutations to a large major ity of females in th e target populations (Hendrichs et al. 2002). Thus for SIT to su cceed, males must be able to participate in courtship, compete in male-male interactions, attract wild females, copulate and inhibit females from remating for as long as possibl e. Understanding the e volution of sexual behavior and the physiological consequences of reproductive adaptations is ultimately critical to control using SIT methods. One way to improve SIT in the Caribbean fruit fly, Anastrepha suspensa (Loew), is to apply the juvenile hormone analog, methoprene, which ac celerates sexual maturation and increases pheromone production (Teal et al. 2000). However, there might be sexual1

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2 quality costs to artificially inducing early maturation, and treated flies may not acquire sufficient nutritional resources to court for extended periods or they may suffer from early mortality. Any extra nutritional requirements , when methoprene is applied , need to be investigated. Sexual selection. Sexual selection was proposed by Darwin to account for sexual differences (secondary sexual characters) that were difficult to explain in terms of differential survival (Darwin 1876). Sexual selec tion is typically divided into competition for mates (intrasexual selection) and mate choi ce (intersexual selectio n). It is responsible for most adaptations of reproductive biology, including attrib utes related to courtship, copulation and fertilization, operational sex ratios, mating systems and parental care (Thornhill 1979, Kokko et al. 2002) Ultimately, both intersexual and intrasexual selection occur because of differences in parental investment between the sexes (Trivers 1972), with parental investment defined as that which increases an offspri ng’s chance of surviving at the cost of the parent being able to produce other offspri ng. There is a fundamental sexual dimorphism in parental investment because of the female ’s investment in large, resource rich eggs, and the male’s in small, inexpensive sperm. If all other things are equal, a female’s capacity to reproduce is limited by her abi lity to produce her eggs, and multiple mating does not result in more offspring. However, ej aculates are inexpensive and there is little inherent cost to insemination in most species. As a result, selection favors males that successfully compete with one another for acce ss to mates, and this often results in aggression and/or increased searching or mobility.

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3 Intersexual selection may result from diffe rences among males in their capacity to provide “direct benefits”, from “runaway sexua l selection”, from variance in male genetic quality (“good genes”) and thr ough manipulation of female pe rceptions (“sensory bias”). Females that acquire a resource or access to a resource through mate choice derive a direct benefit (Kirkpatrick and Ryan 1991). Female preference for an arbitrary male attribute that is genetically correlated with the capacity of males to produce the attribute results in runaway sexual selection (Ryan 1997). Females that recognize and prefer heritable differences in fitness among male s and so produce more fit offspring, benefit from the choice of good genes (Kirkpatrick and Revign 200 2). Males that are able to influence female behavior through n on-sexual signals acquire mates through sensory bias (Fuller et al. 2005). Both intrasexual and intersexual selec tion can act simultaneously on the mating behavior of a species and it ma y, therefore, be difficult to di fferentiate the role of each. For example, male displays that are used in agonistic actions against rival males also attract females and influence their choice (Andersson 1994). Mating systems. There are three major types of mating system, monogamous, polygynous, and polyandrous, that can be furthe r subdivided to desc ribe a variety of male-male and male-female interactions (Thornhill and Alcock 1983). Emlen and Oring (1977) argue that mating systems evolve through the interactions of two major environmental factors: resource distribution and resource size. The resulting degrees of female predictability and capacities of male s to defend locations where females occur create both different arenas and different intensities of sexual selection. When many

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4 males fail to mate then there is high vari ance in male reproductive success which ensures that selection in the male sex will be stronger (Shuster and Wade 2003). Some tephritids have been objects of intensive mating behavior studies (Prokopy and Hendrichs 1979, Burk 1983, Whittier et al. 1993, Lance et al. 2000) because they are important fruit pests. Among these sp ecies, resource defense polygyny and mating aggregations (leks) are two commonly encountered mating systems (Prokopy 1980, Burk 1981). Monophagous species, often temperate in distribution, mate where the female oviposits (fruit or site of gall formati on). Polyphagous species, often tropical or subtropical, usually mate in aggregations on the foliage of a host plant, but not necessarily in the immediate vicinity of the oviposition resource. Males of monophagous species may be able to defend fruits that will predictably attract females, whereas females of polyphagous species may be less predictabl e due to the breadth of their host range (Sivinski et al. 2000). For example, in the predominantly tropical genus Anastrepha Schiner, males of only a single sp ecialist species guard fruits ( Anastrepha bistrigata Bezzi) (Morgante et al. 1983), whereas other, polyphagous, species sexually signal from host plant leaves, and many of these form ma le mating aggregations (Aluja et al. 1983, Hendrichs 1986, Robacker et al. 1991, Aluj a 1994, Aluja et al. 2000, Sivinski et al. 2000). There are revealing exceptions to the rule. In apparent ly monophagous but tropical species, such as Anastrepha hamata (Loew), males call from host plant leaves rather than from fruits (Sivinski et al. 2000). Typical fruit densities suggest that the fruits on these trees are so abundant relative to females that a fruit-guarding male can no longer expect females to arrive at any part icular fruit at an acceptable ra te and thus selection will not

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5 favor fruit guarding. Alternatively, predation on fruit may be higher in the tropics and this has forced males to give up fruit-based terr itories and signal from nearby foliage (Burk 1982, Hendrichs and Hendrichs 1998). A lek is defined as a communal display area where males congregate and to which females come for mating (Wilson 1975). Leks are characterized by the absence of parental care, intense sexua l selection, and are non-re source based (Bradbury 1981, Hglund and Alatalo 1995). Lek mating systems are found in divers e taxa, including in sects (Shelly and Whittier 1997), fish (McKaye et al. 1 990), birds (Hglund and Lundberg 1987) and mammals (Appolonio et al. 1989, Isvaran and Jhala 2000). Among insects, the majority of mating aggregations occur in the order Diptera. Other insect orders in which lekking is reported in more than a few species are the Lepidoptera and Hymenoptera, and occasional species of Homoptera, Hemiptera, Coleoptera, and Orthoptera also lek (Shelly and Whittier 1997). A substantial number of the dipteran fa mily Tephritidae form leks (Shelly and Whittier 1997). Male tephritids often aggregate and hold individual territories from which they emit chemical, acoustic and visual signal s (Burk 1981). Females arrive at these leks and choose mates and the variance in male reproductive success is typically high (Burk 1983). The list of the species known to form le ks and associated behaviors are presented in Table 1-1. In most cases males use chemi cal signals to attract mates to the leks. In almost all species males have been observed to engage in aggression and female mate choice has been reported.

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6 Table 1-1. List of tephritids that form leks (updated from Shelly and Whittier 1997). Species Lek size Male signals Mc 1 Mm 2 Ms 3 Source Anastrepha fraterculus 2-8 Chemical y y ? Mo rgante et al. 1983, Malavasi et al. 1983 Anastrepha ludens 2-10 Chemical y y y Aluja et al. 1983, Robacker et al.1991 Anastrepha obliqua 2-8 Chemical y y y Aluja et al. 1983, Burk 1991 Anastrepha pseudoparallela 2-5 Chemical y y y Burk 1991 Anastrepha serpentina 2-5 Chemical y y y Burk 1991 Anastrepha sororcula 2-5 Chemical y y y Burk 1991 Anastrepha suspensa 2-13 Chemical/ Acoustic y y y Nation 1972, Burk 1983, Sivinski 1984 Bactrocera cucurbitae 2-20 Chemical y y y Kuba and Koyama 1985, Iwahashi and Majima 1986 Bactrocera dorsalis 2-11 Chemical n y ? Shelly and Kaneshiro 1991, Shelly 2001 Bactrocera tryoni ? Chemical y ? y Tychsen 1977 Ceratitis capitata 2-20 Chemical y y y Prokopy and Hendrichs 1979, Arita and Kaneshiro 1989, Shelly 2001 Ceratitis rosa 2-10 Chemical y y ? Quilici et al. 2002 Paramyolia nigricornis 2-10 ? n y y Steck and Sutton 2006 Procecidochares sp . 37-51 ? y y y Dodson 1986 1 Male courtship present or absent (y/n) 2 Male-male aggression prior to female arrival present or absent (y/n) 3 Mate sampling by females present or absent (y/n) (?) No information available Three general hypotheses have been used to explain the evolution of leks, female preference, male attractiveness (“hotshot”), a nd site-quality (“hots pot”) (Westcott 1994). The female preference hypothesis proposes that females pr efer mating in leks because of the advantages they gain compared to ma ting at solitary-male sites. Among these advantages is reduced risk of predation (B urk 1982). An individual in a group is able to hide more effectively among its companions, reducing the risk that it will be one of the unfortunate ones consumed during a period of signaling (Thornhill and Alcock 1983). As noted above, predation pressure on tephritids at host fruit may have driven flies to form mating aggregations on foliage and away fr om fruit (Hendrichs and Hendrichs 1998).

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7 Females may also prefer leks because of the opportunity to compare adjacent males. By such a preference th ey reduce mate search costs a nd are allowed to make more efficient comparisons (Thornhill and Alcock 1983). By searching for mates on contested leaves, females may also have a good chance of finding a more fit male that has won or held its leaf during recent fi ghting (Robacker et al. 1991). The hotshot hypotheses suggest that leks form as a result of high variance in male attractiveness. Males that ar e less attractive cluster around th ose that are successful where they might adopt alternative, cryptic, or sate llite strategies for obtaining females within the communal display areas rather than avoi ding male aggregations altogether (Emlen and Oring 1977). This would be the case if the chance of a less-attractive male being chosen by error or default were larger than being chosen when calli ng alone (Field et al. 2002). The hotspot hypothesis suggests that patterns of female movement and dispersion determine where males settle. Leks should form where female densities are highest or where females are most likely to be enc ountered (Droney 1994). Males may use femaleemitted cues to locate a site where they have some chance of attracting and intercepting receptive females (Thornhill and Alcock 1983). In A. suspensa , sexually inactive females and non-signaling males accumulate in partic ular parts of host trees (Sivinski 1989). These same locations tend to be the sites occupied by sexually active males. Males may also prefer to hol d territories on sites where other males, or themselves, had called previously (Burk 1983). This may be because of pheromones deposited by residents that can last as l ong as the following day (Sivinski et al. 1994). In addition, leaf integrity is important. Males tend to select undamaged, symmet rical leaves as lek sites.

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8 Thus, the choice of location in a lek is not random and is probably composed of a combination of factors such as leaf size, te xture and integrity, wi nd, light intensity, and direct sunlight (Kaspi and Yuval 1999). It ma y be that there are lo cations on host trees where fruits are not abundant, but which have high female de nsities because of favorable microenvironments (Field et al. 2002). Signaling. Three types of selection pressure occu r in leks which all tend to increase the elaborateness and complexity of male sexual signals: the nece ssity of attracting females, competition with neighboring males, and the demands of female choice (Burk 1981, Thornhill and Alcock 1983). Courtship patterns are often species specific, however, species isolation may not be the context in which they have differentiated. Signals might help females pick the fittest males and as a result males of other species are rejected (Alcoc k and Pyle 1979, Thornhill and Alcock 1983, Sivinski a nd Burk 1989). However, species sometimes appear to partition the signaling environmen t. For example, sympatric Anastrepha species call at different times of the day (Aluja et al. 2000). The courtship behavior of tephritids ar e wonderfully varied. They range in complexity from males that couple after litt le preliminary courtship signaling to those that produce an elaborate re pertoire of signals (Burk 1981). In general, the known signaling systems of male Anastrepha species often include pheromone emissions from pleural glands and evaginated anal membranes, pheromone depositions on leaves, wing fanning-acoustic signals (songs) produced both prior to and during coupling, wing motions accompanied by graceful sideways-a rching body movements, and extensions of the mouthparts (Sivinski et al. 1984, Sivinski and Burk 1989). Burk (1983) and

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9 Hendrichs (1986) described the sequence of A. suspensa signaling. Visual, chemical and acoustical signals play a major role in A. suspensa sexual interaction. Tephritid fruit fly visual signals may be used in agonistic encounters between competing males or as courtship signals between the sexes (S ivinski and Wing 2004). These signals include postures that may co mmunicate size, and movements such as wing waving that communicate the phys iological state of the signa ler. However, there is no evidence that wing color-patterns func tion in sexual communication and sexual dimorphism in tephritid wing patterns are rare (Sivinski and Pereira 2005). Males of sexually active lekking tephriti ds produce medium to long distance chemical attractants that are dispersed from pleural abdominal pouches and anal membranes that distend (Nation 1974, Fletch er and Kitching 1995). Males, adopting their pheromone-calling or puffing posture, wave their wings, fanning them rapidly, spin around, and frequently touch the tip of the a bdomen to the bottom surface of leaves to deposit the sex pheromone and increase th e effective surface area of pheromone evaporation (Nation 1972, Sivinski et al. 1994). Acoustical signals of tephritids may be at tractive to the opposite sex and the same sex, and may be important to successful cour tship and in agonistic encounters (Fletcher and Kitching 1995, Sivinski et al. 1984). In A. suspensa , there are at least two forms of acoustic signals. One takes place when the male expands his pleural glands and dabs pheromone unto the leaf territory surface (calling song). These calling songs elicit responses from virgin females and males, bu t not from mated females (Burk and Webb 1983, Sivinski 1993). The other occurs when the male mounts the female and attempts to engage her genitalia (precopulat ory song). It is very energeti c and its sound intensity was

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10 shown experimentally to be an important f actor in determining whether females would allow singing males to copulate (Sivinski et al. 1984, Eber hard 1996). Song structure can vary according to the situation. For example, pulse trains (episodes of wing beating) increase in duration in the presence of male s, and the interval between pulse trains decreases when females are nearby (Siv inski and Webb 1986). While pheromone dispersal may have been the original f unction of wing fanning, the sounds produced by such movements, in at least some instan ces, have taken on a signaling significance of their own (Sivinski et al. 2000). Nutritional status. Both male and female tephritid s are anautogenous, i.e. they emerge as adults with gonads that are immature, and rely on feeding during adult life to provide the nutrients needed for sexual development (Yuval et al. 2002). The energy spent signaling is obtained in part from food that the adult ingests during the precopulatory period (Landolt and Sivinski 1992). If fighting among males for mates is severe and costly, a male may gain by sp ending his early life feeding and acquiring energy reserves before entering the comp etition (Thornhill and Alcock 1983). These sexual expenditures could justify the relativel y long precopulatory periods in some adult tephritids, up to 20 days in Anastrepha interrupta Stone (Pereira et al. 2006a). Recent studies on several species of tephr itids indicate that providing protein to males in the days following eclosion adva nces pheromone emission and stimulates greater volume (Papadopoulos et al. 1998, Yuval et al. 1998, Teal et al. 2000), which enhances male reproductive success (Warburg and Yuval 1997). In addition to protein, carbohydrates must be frequently ingested to fuel metabolic activities (Landolt and Sivinski 1992, Teal et al. 2004).

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11 Males of the medfly, Ceratitis capitata (Wied.) that participate in leks in the field are significantly heavier than resting males and this difference corresponds to higher nutrient content (Yuval et al. 1998). Similarly, Shelly et al . (2002) and Blay and Yuval (1997) in independent studies conducted in field cages found that protein-deprived wild C. capitata males have lower attractiveness a nd achieve significantly fewer matings compared with protein-fed males. Juvenile hormone. In addition to nutriti onal status there are some indications that physiological aspects like juvenile hormone titers can play an important role in the sexual maturation and signaling capacity of male tephritids (Teal et al . 2000). Research on a number of tropical Anastrepha species ( Anastrepha ludens (Loew), A. suspensa and Anastrepha obliqua (Macquart)) has shown that juvenile hormone is critical to regulating sexual maturity and sexual signaling and th at methoprene (a juvenile hormone analog) has a similar effect (Teal and Gomez-Simuta 2002a). In A. suspensa , sexual signaling in males and responses by females to pheromones are correlated with sexual maturity (Nation 1972). Thus, neither males nor females engage in sexual behavior until they have achieved sexual maturity (Nation 1974). In a ddition to methoprene affecting the rate of maturation, males treated with methoprene ha ve greater sexual performance following maturation (Teal et al. 2000). Teal and Go mez-Simuta (2002b), found that enhancement of sexual signaling, pheromone release and mating was induced by juvenile hormones analogs. The Caribbean fruit fly . The Caribbean fruit fly, A. suspensa , is indigenous to the West Indies, but was first detected in Key West in 1930 (Weems 1966), and has been established in central and southern Florid a since 1965 (Nation 1972). It infests over 100

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12 fruits (Swanson and Baranowski 1972), including common guava, Surinam cherry, peach, loquat, rose apple, and tropical almond (Weems 1966, Nguyen et al. 1992). While it typically infests only overripe commercial citrus, it represents a threat to the Florida citrus industry. Citrus growers have spent millions of dollars monitoring and managing this pest, primarily to comply with requirements for exporting grapefruit to Japan (Nigg et al. 2004). As discussed a bove, it is a polyphagous species that forms leks. Behavioral aspects of its mating system, have been described in detail (N ation 1972, Perdomo 1974, Perdomo et al. 1976, Mazomenos et al. 1977, Dodson 1982, Burk 1983, Sivinski 1984, Hendrichs 1986, Sivinski 1989, Sivinski et al. 1994). Study objectives. I examined the effect of a number of interacting factors that may influence the effectiveness of SIT employed against A. suspensa . The manipulation of juvenile hormones titers can c ontribute to an increase in male pheromone production and under certain conditions, sexual competitiveness. At the same time, it leads to earlier sexual maturation which can reduce the cost of the sterile insect technique (SIT), particularly in fruit fly species with a long precopulatory periods like A. suspensa, due to space savings at fly handling facilities. However, accelerated maturity may have nutritional consequences since there is less time for flies to acquire reserves. Thus the addition of protein rich adult diet may be pa rticularly important when hormones titers are manipulated. Additionally, methopr ene applications and the avai lability of dietary protein may result in different nutritional conditions in A. suspensa males. The effects of methoprene application, adu lt protein diet and their interactions on the sexual performance of Caribbean fruit fly males are the main goals of this project. Major components of sexual success to be studied in detail include: male sexual

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13 performance on a daily and lifetime basis (Chapt er 2), lek tenure, as influenced by malemale interactions (Chapter 3), male sexual a ttractiveness to females (Chapter 4), and male survival (Chapter 5). Additionally, total lipid and protein contents at different ages were determined to detect the influence of met hoprene application and di etary protein supply on male nutritional status (Chapter 6).

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CHAPTER 2 INFLUENCE OF A JUVENILE HORM ONE ANALOG AND DIETARY PROTEIN ON MALE SEXUAL SUCCESS Introduction Polyphagous tephritid fruit flies often have complex mating systems in which aggregated males occupy individual territori es from which they emit chemical, acoustic and visual signals (Prokopy 1980, Burk 1981, Sivi nski and Burk 1989). Females arrive at these leks in order to choose mates and the variance in male reproductive success is typically high, i.e., relatively few males obtai n the majority of copulations (Shelly and Whittier 1997). Differences among males in term s of competitiveness and attractiveness play major roles in generating this variance . Since the success of the sterile insect technique (SIT) requires the release of males that can compete in these arenas (Knipling 1955), it is important to fully understand the target-pest’s mating behavior and to incorporate the best possible sexual qualities into the mass-reared insects (Hendrichs et al. 2002). Exposure to juvenile hormones, analogs of juvenile hormones and increased protein consumption during the adult pre-sexual maturation period accelerate male tephritid development and may lead to greater sexual success through increased pheromone production (Teal et al. 2000, Teal and Gomez-Simuta 2002a). Similar phenomena have been reported in other insects, such as the German cockroach, Blatella germanica (L.) (Schal et al. 1994), and the social wasp, Polybia occidentalis (Olivier) (O’Donnell and Jeanne 1993). Greater sexual ability is obviously a valuable characteri stic, but more rapid 14

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15 maturation can lead to cost reductions at fl y handling facilities in SIT programs due to space savings. In fruit fly species with long adult precopulatory periods, like Anastrepha species (Aluja 1994), early maturation is particularly important. Obtaining nutritional resources prior to engaging in territo rial or courtship behavior is essential for reproductive success in many tephritids. Blay and Yuval (1997) report the improvement of sexual performance of medfly, Ceratitis capitata (Wied.), due to additional protein in diets. Accelerated matu rity due to hormone manipulation may have particularly serious nutritional consequences since there is less time for flies to acquire reserves. Thus, the addition of a protein rich adult diet could be critical to methods employed to accelerate male development by delivering exogenous hormone. For example, in the mountain spiny lizard, Sceloporus jarrovi , males with testosterone implants required additional food in order to survive at rates similar to untreated males (Marler and Moore 1991). The goal of this study was to determine th e effects of a juve nile hormone analog (methoprene) application, of protein in the a dult diet and their inte ractions on the sexual performance of male Anastrepha suspensa (Loew), specifically male competitiveness in agonistic interactions with competing mates, attractiven ess to females, and mating success. Experiments were conducted in labo ratory and field cages, and on daily and lifetime bases. The implications of the results for SIT are discussed. Material and Methods Insects. Caribbean fruit flies used in the st udy had been in a laboratory colony at the Center for Medical, Agricultural a nd Veterinary Entomology (CMAVE) USDA-ARS, at Gainesville, FL, for less than 2 years and were reared as described elsewhere (FDACS 1995). The flies were maintained under low stress condition (~ 100 flies in 20 by 20 by

PAGE 25

16 20 cm adult cages and one larvae per 4 g of diet ), which results in low selection pressure for characteristics associated with do mestication (Liedo et al. 2002, Mangan 2003). Flies to be used in experiments were obt ained from pupae sorted into size classes with a sorting machine (FAO/IAEA/USDA 2003). This was done to eliminate any impact of size on male competitiveness (Bur k and Webb 1983, Burk 1984, Webb et al. 1984, Sivinski and Dodson 1992, Sivinski 1993). Males used in experiments came from a size class whose average weight was 10.7 0.9 mg (n=40). Females were obtained from the next larger class size, whose average weight was 11.9 0.9 mg (n=30). In the field, males are typically 80% of the female’s size (S ivinski and Calkins 1990, Sivinski 1993). These pupal weights were in the middle range of A. suspensa pupae collected from infested fruits in nature (Hendrichs 1986). After emergence the flies were maintained in a laboratory room with a photoperiod of 13L:11D (light from 7:00 to 20:00) with light intensity of 550 50 lux, temperature of 25 1C and relative humidity of 55 5%. All laboratory experiments were conducted in this same room under the same environmental conditions. Treatments. The study compared sexual performance of male A. suspensa subjected to the following four treatments: application of juvenile hormone analog, methoprene (M), and sugar and hydrolyzed yeast (protein source) as adult food (M + P P + ) methoprene application and sugar as adult food (M + P P ) no methoprene application and sugar a nd hydrolyzed yeast as adult food (M P P + ) no methoprene application and sugar as adult food (M P P ) The methoprene was applied topically in the first 24 hours after adult emergence at a rate of 5 g in 1 l acetone solution per male. In M treatments, 1 l of acetone was applied, to serve as a control. Males were immobilized in a net bag (as in standard

PAGE 26

17 marking techniques, FAO/IAEA/USDA (2003 )) and the solution applied via pipette through the net onto the dorsal surface of th e thorax, No anesthesia was used to immobilize the flies. Two different net ba gs and pipettes were used (one for M + treatments and other for M treatments) to prevent methoprene contaminations. Males from each treatment were maintained in independent 30 cm by 30 cm by 30 cm screen cages with a maximum male density of 200 f lies/cage and with the type of food assigned for each treatment. In P treatments only water and sugar ad libitum were supplied to the flies. In P treatments only water and sugar ad libitum were supplied to the flies. In the P + treatments hydrolyzed protein was added to the sugar diet in a proportion of three parts of sugar and one part of hydrolyzed yeast, and water were supplied ad libitum . This mixture is considered a high quality diet for Anastrepha species (Jcome et al. 1995, Aluja et al. 2001). Females used in the experiments were sexed on the first day of adult life and maintained in 20 cm by 20 cm by 20 cm scr een cages without exposure to males. They were provided with a P + diet, i.e., sugar plus hydrolyzed yeast (3:1) and water ad libitum . Sexual success in laboratory. The experiment was conducted in 20 cm by 20 cm by 20 cm screen cages. There were 12 replicat ions (different days) with 15 cages per replication for a total of 180 cages. In each cage four males (one per treatment) were released in the mid afternoon (15:00). Th ese 13-16 days old, virgin sexually mature males were previously marked (on the day be fore the experiment) with a dot of waterbased paint (different color per treatmen t and rotated among treatments) on the dorsal surface of thorax (FAO/IAEA/USDA 2003). At 17:00, a 20-23 days old, sexually mature

PAGE 27

18 virgin female was released into each cage and observed until 19: 00. This afternoon period coincides with the peak of sexual activity in A. suspensa (Dodson 1982, Burk 1983, Hendrichs 1986, Landolt and Sivinski 1992). When mating occurred, the pair was removed and all the flies, including the 3 male s that did not mate, were killed in a freezer. Male wing lengths (right wing of each fly) were measured at the end of the experiments to quantify male size (Landolt and Sivinski 1992, Yuval et al. 1998). Sexual success in field cages. The experiment was conducted in a standard field cage used for the study of ma le compatibility and sexual performance in tephritids (FAO/IAEA/USDA 2003). Cages are cylindric al, with flat floor and ceiling, 2.9 m diameter and 2.0 m high (Calkins and Webb 1983). In this experiment, 2 cages were observed per day for 6 days (4, 5, 6, 10, 12, and 13 October, 2004) for a total of 12 replications. In each cage a 1.8 m high potted guava plant ( Psidium guajava L.), a preferred host of Caribbean fruit fly (Dodson 1982, Hendrichs 1986, Landolt and Sivinski 1992, Sivinski 1989), served as a substrate fo r calling males and sexual interactions. Sixty virgin males (15 per treatment), 13-16 days old and color marked as above, were released at 16:50. Ten minutes later 30 virgin females, 2023 days old, were added. The experiment ran until 19:00 to coincide with A. suspensa ’s sexual activity peak (Dodson 1982, Burk 1983, Hendrichs 1986, Landolt and Sivinski 1992). During these 2 hours, temperature, relative humidity and light intensity were measured every 30 minutes. Mating pairs were removed to 10 ml indivi dual vials and mating dur ation was recorded, as was position inside the cage (cage or tree), plant part, elevation within the tree canopy (high, middle or low), and side of leaves on which mating occurred (under or over).

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19 The abiotic conditions during the 6 days of the field cage experiment were similar. The temperature at 17:00 was similar on the si x days of experiments, 28C to 30C, and varied from 24C to 30C throughout the 2 hour period of the study (17:00 to 19:00). Daily fluctuations were on the order of 2C to 4C. RH va ried from 48% to 79%. Light intensity dropped from 1,112 lux at 17:00 to 291 lux at 19:00. The maximum light intensity was registered on October 12 th at 17:00 (1,345 lux). Sunset occurred at 19:11 on October 4 th (1 st day of experiment) and at 19:00 on October 13 th (last day of experiment). Sexual performance over life. This experiment was conducted in the laboratory in individual cylindrical cages (10 cm high a nd 7 cm diameter). Cages were placed over a container that supplied water ad libitum to the flies through a cotton wick. Eighty cages (20 per treatment) were examined over a peri od of 35 days. Virgin males in cages were provided with the adult food appropria te to each treatment (sugar in P treatments and sugar and hydrolyzed yeast (3:1) in P + treatments). Daily, at 17:00, one virgin female (20-23 days old) was released in to each cage and observed until 19:00. The adult females were maintained with optimal adult diet (s ugar plus hydrolyzed y east in 3:1 proportion) and water ad libitum . Mating and copulation duration we re recorded. If mating occurred, females were removed at the end of copulat ion (copulations lasted about 30 minutes on average). All females that had not mated were removed at 19:00. The ones that were still in copula were removed as soon as they finished. The same procedures were repeated daily until males were 35 days of age. Sexual performance on a daily basis. This experiment was conducted in the laboratory in individual cylindrical cages (10 cm high and 7 cm diameter) as described

PAGE 29

20 above. Eighty cages (20 per treatment) were examined with males of 5, 10, 15, 20, 25, 30, and 35 days of age. Males were maintained prior to transfer in a 30 cm by 30 cm by 30 cm cage (one cage per treatment). At each of the above mentioned ages, males were transferred to individual cages at 15:00. One 20-23 days old virgin female per cage was released at 16:00. After a 1 hour interval (at 17:00) the resident female was replaced by another female whether or not the original female had mated. If a mating was still in progress the female was replaced immediately after the pair separated and another female was introduced. The procedure was repeated at 18:00. The experiment finished at 19:00, with the exception of the pairs still in c opula (continued until pair separated). Mating and copulation duration were recorded. Statistical analyses. The data were analyzed using a two-way analysis of variance (ANOVA) to detect the inte ractions between methoprene and protein. These analyses were followed by an ANOVA to detect differenc es between means in the treatments. If differences in means were detected through ANOVA, complementary multiple comparisons of means (Tukey’s test) were performed (Ott and Longnecker 2001). In the sexual performance on a daily basis experi ment a two-way ANOVA was performed to detect treatment and age effects. The si gnificance value used in tests was 95% ( =0.05). Statistical analyses were performed using R software (version 2.1.0, www.r-project.org). Results Sexual success in laboratory. From a total of 180 cages (12 replications with 15 cages each), 131 successful matings were recorded in the laboratory (Figure 2-1) so that 73% of all females mated. There were signi ficant effects of me thoprene application (F 1,44 =122.89, p<0.05), protein supply (F 1,44 =85.81, p<0.05), and the interaction of the methoprene application and protein supply (F 1,44 =16.81, p<0.05) on mating.

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21 0 10 20 30 40 50 60 70 80 M+P+ M+PM-P+ M-PTreatmentMatings (%) Field cage LaboratoryaA b B B b cC 0 10 20 30 40 50 60 70 80 M+P+ M+PM-P+ M-PTreatmentMatings (%) Field cage LaboratoryaA b B B b cC Figure 2-1. Percentage of matings per treat ment of male Caribbean fruit flies in laboratory and field cage experiments, when treated or not with methoprene (M + /M ) and fed or not with protein (P + /P) (mean plus standard deviation). Data were obtained from the 12 replic ations in both in laboratory and field cage experiments). Different lowercase a nd capital letters represent significant differences among treatments, for field cage and laboratory tests respectively (Tukey’s test, =0.05). Of the total matings, 55% were performed by M + P P + males, which were significantly higher compared with males from all the other treatments (F =75.17, p<0.05). M 3,44 PP males had significantly fewer matings (5%) th an other treatments in this competitive arena. Wing lengths of males that did and di d not mate were not significantly different (mated-average= 4.7 0.3 mm (n=131); average unmated= 4.7 0.2 mm (n=393)). Sexual success in field cages. A total of 104 matings (29% of females) were recorded in the 12 field cage replicates. There were significant effects of methoprene application (F 1,44 =109.17, p<0.05), protein supply (F 1,44 =125.32, p<0.05), and interaction of the methoprene applica tion and protein supply (F 1,44 =20.04, p<0.05) on mating. The M + P P + males with 61 matings (59%) had significantly higher mating success (F =84.84, p<0.001) compared to other treatments (Figure 2-1). M 3,44 PP males obtained

PAGE 31

22 only 3 matings (3%) and had a significantly lower male competitive ability when compared to the other treatments. Of the 102 matings that occurred on th e tree, all were on leaves and 94 (92%) occurred on the undersides of leaves. Seventy five matings (73%) occurred in the highest part of the canopy, 22 (22%) in the middle part and only 5 (5%) in the lower part. Sexual performance over life. A significant effect of methoprene application (F 1,124 =15.51, p<0.05), protein supply (F 1,124 =31.25, p<0.05), and interaction of the methoprene and protein (F 1,124 =5.98, p=0.016) was found. M + P P + males obtained significantly more copulations (F =15.57, p<0.001) than males with no methoprene application (Figure 2-2). 3,124 0 10 20 30 40 50 60 70 M+P+ M+PM-P+ M-PTreatmentMatings (%)a,b a b b 0 10 20 30 40 50 60 70 M+P+ M+PM-P+ M-PTreatmentMatings (%)a,b a b b Figure 2-2. Percentage of matings of male Caribbean fruit flies from day 4 to the end of experiment (day 35), when treated or not with methoprene (M + /M ) and fed or not with protein (P + / P ) (mean plus standard deviation). Bars with the same letter were not significantly different (Tukey’s test, =0.05).

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23 The earliest mating occurred on day 4 for M+P+ males, on day 5 for M+Pmales and on day 6 for males that did not receive methoprene. In addition, higher percentages of M+P+ males mated consecutively over 3 or more days than males in other treatments (Figure 2-3). 0 5 10 15 20 25 30 23> 3 Number of consecutive matingsMales (%) M+P+ M+PM-P+ M-PFigure 2-3. Percentage of male Caribbean fruit flies that mated more than once over the 35 day duration of the experiment when ra ised under four treatments: with or without methoprene (M + /M ) and fed or not with protein (P + /P ). Sexual performance on a daily basis. There was a significant effect of both treatment (F 3,18 =49.56, p<0.05) and adult age (F 6,18 =12.63, p<0.05) on mating in a daily basis. At each of the examined ages M + P P + males obtained more copulations (Figure 2-4). As in the previous experiment, the M + males began to mate earlier (see adult age 5 in Figure 2-4). At 5 days of age, only one M male mated. M + P+ P males were not only more likely to mate, but 10% (14 males) were able to mate 3 times on the same day. Only 1 M + P P , 1 M P+ P , and none of the M P P males were able to mate so often (Figure 2-5).

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24 0 10 20 30 40 5101520253035Adult age (days)Number of matings M+P+ M+PM-P+ M-PFigure 2-4. Number of matings obtained per treatment at vari ous ages by male Caribbean fruit flies when treated or not with methoprene (M + /M ) and fed or not with protein (P + /P ). Copulation duration. The 104 matings performed in the field cages averaged 30.8.6 minutes and there were no significant differences among treatments (F 3,100 =0.176, p=0.912). Similarly there were no di fferences in duration of copulation among treatments (F 3,577 =1.69, p=0.304) on a lifetime basis or in the durations of matings by number of matings during their lifetimes (1, 2, 3, and more than 3) within each treatment (F 3,577 =0.854, p=0.465). The total of 581 mati ngs observed in the lifetime experiment averaged 26.9 9.8 minutes. On a daily basis, no differences in ma ting duration were found among treatments (F 3,415 =1.001, p=0.392). However, in males that mated for a third time within a single day the duration of the third mating was significantly shorter (F 3,415 =40.30, p<0.001) (Figure 2-6).

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25 0 1 2 3 4 5 6 7 1+1 2 3 Number of matingsNumber of males M+P+ M+PM-P+ M-P-aa a a,b b bbb a,b b c c 0 1 2 3 4 5 6 7 1+1 2 3 Number of matingsNumber of males M+P+ M+PM-P+ M-P-aa a a,b b bbb a,b b c c Figure 2-5. Average number of male Caribbean fruit flies that mated twice (2 consecutive or 1+1 within at least a 1 hour period), or three times on the same day (mean plus standard deviation) . Data derived from a total of 140 males (20 per treatment of 7 different ages, when treated or not with methoprene (M + /M ) and fed or not with protein (P + /P ). Bars with the same letter for each number of matings were not signifi cantly different (Tukey’s test, =0.05). ANOVA values: 1+1 matings (F 3,24 =3.56, p=0.038); 2 matings (F 3,24 =15.74, p<0.001); 3 matings (F 3,24 =9.74, p<0.001). Discussion Both laboratory and field cage tests f ound a clear increase in male sexual performance due to methoprene application, th e addition of protein in the diet and the interaction of methoprene a nd protein. The proportions of females simultaneously exposed to males of different treatments and that subsequently copulated differed in the laboratory and in field cages (73% vs 29%). Th is may be due to the greater ratio of males to females in the laboratory tests (4:1 in laboratory versus 2:1 in field cages). However, the larger space available inside the fiel d cages might have given females more opportunities to exercise mate choice and more easily escape the attentions of unwanted suitors.

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26 0 10 20 30 40 11 + 123 Number of matingsCopulation duration (min)a b a a n=303 n=16 n=58 n=42 0 10 20 30 40 11 + 123 Number of matingsCopulation duration (min)a b a a 0 10 20 30 40 11 + 123 Number of matingsCopulation duration (min)a b a a n=303 n=16 n=58 n=42 Figure 2-6. Mean (plus standard deviation) copulation duration of male Caribbean fruit flies that mated 1, 2 (2 consecutive or 1+1 with 1 hour interval) and 3 consecutive times on the same day. Bars with the same letter were not significantly different (Tukey’s test, =0.05). Sugar is a basic nutritional requirement for sexual activities of male A. suspensa (Landolt and Sivinski 1992, Teal et al. 2004). Incorporation of protein in adult diet improves male sexual success in tephritids such as C. capitata (Blay and Yuval 1997, Yuval et al. 1998, Kaspi et al. 2000), Anastrepha obliqua (Macquart), Anastrepha serpentina (Wied.), Anastrepha striata Schiner (Aluja et al. 2001), and A. suspensa (Landolt and Sivinski 1992). Aluja et al. (2001) and Manga n (2003) found no effect of additional protein on male Anastrepha ludens (Loew) sexual success. Taylor and Yuval (1999) reported that female C. capitata store more sperm when they copulate with protein-fe d males, which reduces subsequent female remating. In our study, protein diets improved sexual performan ce. This was true whether methoprene was applied or not, although the addition of met hoprene to protein resu lted in the highest levels of male sexual success. Protein diet might also influence success in agonistic encounters, a frequent occurrence in lekking A. suspensa (Burk 1984, Chapter 3). Males

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27 that win territorial contests may obtain more suitable and at tractive locations within leks (Burk 1983, Thornhill and Alcock 1983, He ndrichs 1986, Sivinski and Heath 1988, Sivinski 1989). The combination of methoprene application with a supply of protein-enriched food resulted in greater male sexual success than when either was provi ded separately. This effect was consistent in all the tests performed. For example, basically only M + P P + males were able to perform the sexua l feat of mating 3 consecutive times in a 3 hour window. It seems likely that insects with an optimal diet would have the energe tic capacity to exploit the physiological effects of additional methoprene. Average mating duration was similar in field cage (coinciden t with Hendrichs (1986) data under a similar prot ocol) and in laboratory studi es (coincident with Fritz (2004) data). No effect on duration was found among treatments. However, in males that performed a third mating on the same day the fi nal copulation was significantly shorter. Aluja et al. (2001) found no differences in the durations of co pulations performed by males fed on different diets in A. ludens, A. obliqua , and A. serpentina. On the other hand Prez-Staples and Aluja (2004), found that protein-fed males of A. striata have significantly shorter copulation durations, but th ese males are able to mate more often. Inconsistent results were found for di et effects on copulation duration in C. capitata . Protein-deprived, mass reared males had longe r copulations than protein-fed males (Blay and Yuval 1997, Field and Yuval 1999), however no such differences were found in wild males (Taylor et al. 2000, Shelly and Kenne lly 2002). The causes of this sort of variability are presently not unde rstood but could be due to either male capacity or female

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28 perception of male quality. In either case, Fritz (2004) found that copulation duration was positively correlated with the quantit y of sperm stored by the female A. suspensa. The improved sexual performance of ma les and their earlier maturation due to methoprene application could contribute enor mously to the success of SIT. Ultimately, this technique is based on release in the field of competitive sterile males (Knipling 1955), and increased male signaling and pheromone production, while not physiologically well understood (Teal and Gome z-Simuta 2002b), appear to lead to both significantly greater daily and life-time sexual succe ss (Teal et al. 2000). Additionally, earlier maturation can signifi cantly reduce the costs of fly handling operations. This is particularly the case in tephritids with long adult precopulatory periods like the Anastrepha species (Aluja 1994), but less so for species like C. capitata with shorter precopulatory periods (Liedo et al. 2002). Sterile A. suspensa flies treated with methoprene might be ready to release 2-3 days earlier than untre ated males and this means that space for storage of adults could be considerably reduced. Even medflies could be released 1 day earlier. With the number of ongoing medfly programs (Hendrichs et al. 2002), the release of sexually mature males even a day earlier could represent a significant increase in SIT efficacy. The combination of protein-di et and methoprene resulted in greater sexual success than either treatment alone, and for this reas on the incorporation of protein in adult diets in SIT programs together with methoprene is highly recommended. In addition, the release of well fed flies would allow sterile males to immediately forage for mates rather than food.

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CHAPTER 3 INFLUENCE OF A JUVENILE HORM ONE ANALOG AND DIETARY PROTEIN ON MALE SEXUAL BEHAVIORS WITHIN MATING AGGREGATIONS Introduction Polyphagous tephritid fruit flies often have complex mating systems in which aggregated males defend indi vidual territories from which they emit chemical, acoustic and visual signals (Prokopy 1980, Burk 1981). Fe males visit these male aggregations, or “leks”, for mating (Emlen and Oring 1977, H glund and Alatalo 1995, Fi eld et al. 2002). As in the leks of other species, the variance in male reproductive success on tephritid leks is typically high; i.e., relatively few males obtain the majority of copulations (Thornhill and Alcock 1983, Sivinski and Burk 1989, She lly and Whittier 1997). This high variance could be due to differences among males in th eir attractiveness to females or their success in male-male agonistic interactions or bot h. Selection for long-distance attraction of females, effective courtship, and male competition all occur in the context of lekking and affect the intensity and complexity of male sexual signals. Insects show a variety of mating aggregat ions some of which bear only partial resemblance to classic vertebrate leks (H glund and Alatalo 1995). This variety includes aerial swarms whose male participants typi cally do not engage in elaborate signals (Sivinski and Petersson 1997), and substrat e aggregations that are incompletely independent of female-required resources. Polyph agous tephritid fruit flies, such as the Caribbean fruit fly, Anastrepha suspensa (Loew), are examples of the latter, with leks 29

PAGE 39

30 usually forming on the foliage or in the vici nity of fruiting host trees (Sivinski et al. 2000). Exposure to the juvenile hormone anal og, methoprene at emergence accelerates male development (Teal et al. 2000) and ma y lead to greater sexual success through increased pheromone production (Teal and Go mez-Simuta 2002a). Protein consumption during the adult stage can cont ribute to male gonadal and accessory glands development and influence sexual success (Y uval et al. 1998). The greater sexual success of proteinfed males has been documented for Anastrepha obliqua (Macquart), Anastrepha serpentina (Wied.), Anastrepha striata Schiner (Aluja et al. 2001), and A. suspensa (Landolt and Sivinski 1992). However, Al uja et al. (2001) a nd Mangan (2003) found no sexual effect of additional protein in Anastrepha ludens (Loew). The combination of methoprene and a protein-rich diet has an a dditive or synergistic e ffect on sexual success in A. suspensa (Chapter 2). Males appear to need adequate nutr ition to produce large quantities of pheromone. Evaluation of methoprene application, adult protein diet and their interactions on male A. suspensa performance within leks is the main goal of this study. Experiments in field cages were conducted with males trea ted with different me thoprene and protein regimens. Initiation and partic ipation in leks, male pheromone-calling, male position in the lek, male-male and male-female inter actions, and sexual success were observed. Success of the sterile insect technique (SIT) (Knipling 1955) , a commonly used means of tephritid control (Hendrichs et al. 2002), require s the release of males that can form leks, engage in agonistic and sexual interactions, a nd attract wild females. I discuss whether or

PAGE 40

31 not materials, such as methoprene and protein, that substantially improve male sexual success can be economic addi tions to SIT programs. Material and Methods Insects. The Caribbean fruit flies used in the study had been in a laboratory colony at the Center for Medical, Agricultural and Veterinary Entomology (CMAVE) USDAARS, at Gainesville, FL, for less than 3 years and were produced according to a specific mass rearing protocol (FDACS 1995). The flies were maintained under low stress condition (~ 100 flies in 20 by 20 by 20 cm adult cages and one larvae per 4 g of diet), which results in low selection pressure for characteristics associated with domestication (Liedo et al. 2002, Mangan 2003). Flies to be used in experiments were obt ained from pupae sorted into size classes with a sorting machine (FAO/IAEA/USDA 2003). This was done to eliminate any impact of size on male competitiveness (Bur k and Webb 1983, Burk 1984, Webb et al. 1984, Sivinski and Dodson 1992, Sivinski 1993). Males for the experiment came from the size class whose average weight was 10.8 0.8 mg (n=30). Females were obtained from the next larger class size, with an average weight of 11.9 0.9 mg (n=30). In the field, males are typically 80% of the female size (Sivin ski and Calkins 1990, Sivinski 1993). These pupal weights were in the middle range of A. suspensa pupae collected from infested fruits in nature (Hendrichs 1986). After emergence the flies were maintained in a laboratory room with a photoperiod of 13L:11D (light from 7:00 to 20:00), a light intensity of 550 50 lux, a temperature of 25 1C and a relative humidity of 55 5%.

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32 Treatments. The study compared sexual performance of male A. suspensa subjected to the following four treatments: application of juvenile hormone analog, methoprene (M), and sugar and hydrolyzed yeast (protein source) as adult food (M + P P + ) methoprene application and sugar as adult food (M + P P ) no methoprene application and sugar a nd hydrolyzed yeast as adult food (M P P + ) no methoprene application and sugar as adult food (M P P ) Methoprene was applied topically in the fi rst 24 hours after ad ult emergence at a rate of 5 g in 1 l acetone solution per male in M + treatments. In M treatments, 1 l of acetone was applied, to serve as control. Males were immobilized in a net bag (as used in standard marking techniques, FAO/IAEA/USDA (2003)) and the solution applied via pipette through the net onto the dorsal surface of the thorax. No anesthesia was used to immobilize the flies. Two different net ba gs and pipettes were used (one for M + treatments and other for M treatments) to prevent methoprene contaminations. Males from each treatment were maintained in independent 30 cm by 30 cm by 30 cm screen cage with a maximum male density of 200 flie s/cage and with the type of food assigned for each treatment. In the P treatments only water and sugar ad libitum were supplied to the flies. In the P + treatments hydrolyzed yeast was added to the adult diet as protein source (mixed with sugar in a proportion of three parts of s ugar and one part of hydrolyzed yeast). This mixture is considered a high quality diet for Anastrepha species (Jcome et al. 1995, Aluja et al. 2001). Females used in the experiments were sexed on the first day of adult life and maintained in 20 cm by 20 cm by 20 cm scr een cages without exposure to males. They were provided with a P + diet, i.e., sugar plus hydrolyzed yeast (3:1) and water ad libitum .

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33 Lek tenure in field cages. The experiment was conducted in a standard field cage used for the study of compa tibility and male sexual pe rformance in tephritids (FAO/IAEA/USDA 2003). These screen cages are cylindrical, 2.9 m diameter and 2.0 m high with a flat floor and ceiling (Calkins and Webb 1983). Twelve replications were run (one per day) from 7-19 June, 2005 (except on 12 June). In each cage a potted 1.8 m high guava ( Psidium guajava L.) was moved inside to serve as a substrate for sexual interactions. Guava is considered a key host of A. suspensa and a common substrate for lekking (Dodson 1982, Hendrichs 1986, Landolt and Sivinski 1992, Sivinski 1989). A different potted guava was used every day to prevent the influence of male pheromones deposited on leaves the previ ous day (Sivinski et al. 1994). In each cage 40 sexually mature, 13-16 da y old, virgin males (10 per treatment) were released at 16:50. They were previously marked with a dot of water-based paint on the dorsal surface of the thorax to identify the males from each treatment. The colors were rotated among treatments. Ten minutes la ter, 20 sexually mature virgin females, 2023 days old, were released inside the cage. The experiment was run until 19:00 to coincide with the sexual activity peak (Dodson 1982, Burk 1983, Hendrichs 1986, Landolt and Sivinski 1992). During these two ho urs, temperature, relative humidity and light intensity were measured every 30 minutes. During the 12 days of the field cage expe riment, the temperature ranged from 24C to 32C, with a daily variation of 1C to 6 C. Relative humidity varied from 40% to 94%. Light intensity varied from 3,540 lux to 12,090 lux. Rapidly developing clouds contributed to these variations. Suns ets occurred between 20:28 and 20:32.

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34 Marked males were observed as they moved about inside the cage . Initiation (first male that started to emit pheromone in a certain area of the plant canopy) and participation in leks (males that join the fi rst male to create an aggregation, calling or not), male calling (indicated by the expansi on of pleural regions of the abdomen and eversion of glistening rectal tissue), male position on the lek, male-male and male-female interactions, and matings were observed. Mati ng pairs were removed to 10 ml individual vials and copulation duration was recorded. Males that landed within 20 cm of a nother calling male were considered participants in a lek (Sivinski 1989). Positions in leks were divided into three tiers: centralthe male is located on the middle leaves of the aggregation; surroundingmales that land on the leaves adjacent to the center (within a 20 cm radius of the edge of the center area of the aggregation); and satellit esmales that are adjacent and peripheral to the surrounding males. Male-male interactions normally took place when 2 males occupied the same leaf (the resident occ upied the leaf and the intruder arrived and attempted to displace the resident). Typically th is resulted in an agoni stic interaction with wing waving that lasted for several seconds although physical contact was rare. Losers left and the winners stayed on the leaf. Male -female interactions occurred when males attempted to mate. Males could succeed or be rejected when females flew away or moved to the upper side of the leaf. A female acceptance index was calculated according the following formula: matings successful ofNumber mate toattempts male ofNumber index acceptance Female Statistical analyses. Lek initiation, lek participat ion, males calling, and matings were analyzed using a two-way analysis of variance (ANOVA) to detect methoprene

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35 application effect, protein supply effect, a nd the interactions between methoprene and protein. These analyses were followed by an ANOVA to detect differences between means in the treatments. Other data was an alyzed by ANOVA. If differences in means were detected, complementary multiple co mparisons of means (Tukey’s test) were performed (Ott and Longnecker 2001). The sign ificance value used in the tests was 95% ( =0.05). Statistical analyses were performed using R software (version 2.1.0, www.rproject.org). Results Initiation and participation in the aggregation. The number of leks per replication averaged 3.4.8 and varied between 2 and 5. The number of males simultaneously aggregating (lek size) varied between 2 and 6 with an average of 3.6.1 males per aggregation. Significant effects of me thoprene application (F 1,44 =63.14, p<0.05), protein supply (F 1,44 =72.84, p<0.05), and the interaction of th e methoprene application and protein supply (F 1,44 =19.49, p<0.05) were found in lek initiation. The percentage of males, by treatment, initiating aggregations is pres ented in Figure 3-1. Of a total of 41 leks observed, 28 (68%) were initiated by M + P P + males (2.3.7 per replication), which was significantly higher compared w ith all the other treatments (F =51.8, p<0.05). M 3,44 PP males initiated significantly fewer (none). Significant effects of me thoprene application (F 1,44 =137.84, p<0.05), protein supply (F 1,44 =199.99, p<0.05), and interaction of the met hoprene application and protein supply (F 1,44 =31.40, p<0.05) were found in lek participat ion. Significant eff ects of methoprene application (F 1,44 =64.57, p<0.05), protein supply (F 1,44 =84.34, p<0.05), and interaction of

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36 the methoprene application and protein supply (F 1,44 =8.23, p=0.006) were found for male calling as well. 0 10 20 30 40 50 60 70 80 90 M+P+ M+PM-P+ M-PTreatmentsMales (%) Lek initiation Lek participation Males calling Matinga a a a bb b b c b bbb c c c 0 10 20 30 40 50 60 70 80 90 M+P+ M+PM-P+ M-PTreatmentsMales (%) Lek initiation Lek participation Males calling Matinga a a a bb b b c b bbb c c c Figure 3-1. Lek parameters and sexual success of male Caribbean fruit flies presented as percentages of males (mean plus standard deviation), when treated or not with methoprene (M + /M ) and fed or not with protein (P + /P ). Bars with the same letter for each parameter were not significantly different (Tukey’s test, =0.05). Observations based on 41 leks (lek initiation), where 212 males participatd, 178 called, and 73 matings occurred. The percentages of males, by treatment, particip ating in aggregations and calling, are presented on Figure 3-1. The data show a similar pattern for both participation and calling. Of a total of 212 males participating (17.7.6 per replication), 53% (112) were M + P P + males, which was significantly higher than all the other treatments (F =123.1, p<0.05). M 3,44 PP males had significantly fewer males (7 %) participating in aggregations. One hundred and seventy eight males called (84% of total participating in leks). Of these, 55% were M + P P + males, which was significantly higher than all the other treatments (F =52.4, p<0.05). M 3,44 PP males had significantly fewer males (4%) calling. Calling duration of those males that called was not significantly different among treatments

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37 (F 3,174 =1.397, p=0.245). On average males spent 18.8.1 minutes calling in an aggregation. Matings. Significant effects for methoprene application (F 1,44 =65.32, p<0.05), protein supply (F 1,44 =83.90, p<0.05), and interaction of the methoprene application and protein supply (F 1,44 =25.52, p<0.05) were found. Of a tota l of 73 matings observed in the 12 replicates (6.3.0 per replica tion), 67% were performed by M + P P + males, which was significantly higher than all other treatments (F =57.6, p<0.05). M 3,44 PP males had significantly fewer copulations (only 1%) than all other tr eatments (Figure 3-1). On average there were 6.1.0 matings per replication with a ra nge of 5 to 8. All matings were by males that were participating in aggregations. Copulat ion duration was not different among treatments (F 3,69 =0.351, p=0.789), and averaged 27.4.7 minutes (range of 15 to 40 minutes). Among all treatments a high percentage of males (78%) that initiated the aggregation subsequently copulated. Position in the lek. Sixty four percent of matings were obtained by males in the center of the aggregation, 36% by surrounding males, and none by the satellites. Numbers of males in each category differed so that of the 94 center males 50.0% copulated, while 24.8% of the 105 males on surrounding territories mated (Table 3-1). None of the 13 satellite males copulated. Sexual success was co rrelated with treatment (Table 3-1). Fifty seven of the 94 center males (60.6%) were M + P P + , and of those, more than half copulated (54.4%). Male-male and male-female interaction. In general, residents had an advantage over intruders (Table 3-2). The exception was M P P resident males that lost 42% of their contests.

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38 Table 3-1. Number of male Caribbean fruit flies that occupied the various positions within leks, when treated or not with methoprene (M + /M ) and fed or not with protein (P + /P ). Percentages of males that c opulated are inside parenthesis. Position on the lek M + P + M + P M P + M P Total per position Central 57 (54.4%) 14 (42.9%) 21 (47.6%) 2 (0.0%) 94 (50.0%) Surrounding 49 (36.7%) 22 (18.2%) 22 (13.6%) 12 (8.3%) 105 (24.8%) Satellites 6 (0.0%) 3 (0.0%) 3 (0.0%) 1 (0.0%) 13 (0.0%) Total per treatment 112(43.8%) 39 (25.6%) 46 (28.3%) 15 (6.7%) 212 (34.4%) Among male-female interactions, unsuccessf ul mating attempts were common; 224 male-female interactions without copulati on were observed. On average and across all treatments , males attempted 3.1.8 matings per copulation. However, when female acceptance index were analyzed by treatment (Figure 3-2), M + P P + males made significantly fewer unsuccessful attempts (2.5.4) than males from other treatments (F =26.561, p<0.001). 3,24 Table 3-2. Percentages of resident male Caribbe an fruit flies (by treatment) in leks that won contests against intruders, when treated or not with methoprene (M + /M ) and fed or not with protein (P + /P ). Number of interactions is in parenthesis. Totals with different letter are significantly different (Tukey’s test, = 0.05). Intruders Residents M + P + M + P M P + M P Total M + P + 94.4 (n=36) 88.2 (n=17) 92.0 (n= 25) 100.0 (n=22) 94.0 (n=100) a M + P 92.9 (n=14) 100.0 (n=5) 100.0 (n=9) 100.0 (n=2) 96.7 (n=30) a M P + 72.2 (n=18) 100.0 (n=5) 100.0 (n=2) 100.0 (n=7) 84.4 (n=32) a M P 50.0 (n=6) 66.7 (n=3) 66.7 (n=3) (-) (n=0) 58.3 (n=12) b Discussion Methoprene application, diet ary protein, and the intera ction of methoprene and protein significantly improved most parame ters of lekking and male sexual success (Table 3-3). From the males that initia ted a lek, 78% went on to copulate, which represents a high probability of success. Le k initiation in various tephritids can be dependent on the nutritional status of the male (Yuval et al. 1998) or perhaps by the male’s hormonal state (Teal et al. 2000). In the case of the Caribbean fruit fly, either

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39 methoprene or protein resulted in higher rate s of lek initiation a nd ultimately greater sexual success. 0 1 2 3 4 5 6 7 M+P+ M+PM-P+ M-PTreatmentFemale acceptance indexa b b b 0 1 2 3 4 5 6 7 M+P+ M+PM-P+ M-PTreatmentFemale acceptance indexa b b b Figure 3-2. Female Caribbean fruit fly accep tance index when treated or not with methoprene (M + /M ) and fed or not with protein (P + /P ). Bars with the same letter were not significantly different (Tukey’s test, = 0.05). What are the physiological and/or behavi oral consequences of methoprene and protein that resulted in th is improved sexual performance? Some possibilities include longer calling durations, more pheromone per unit of calling time, and more time spent in leks (or in better locations within leks) due to favorable outcomes in agonistic encounters. The proportion of M + P P + males that participated and calle d in leks was significantly higher than in other treatments, however calling duration was not significan tly different among treatments. That is, although other-treatment male s were less likely to participate in leks those that did so spent as much time calling. On the other hand, M + P+ P males are known to produce more pheromone per unit time of calling (Teal et al. 2000) and this could produce a more powerful signal that might attract more females to their positions within aggregations. The greater success of M + P P + males in agonistic encounters, particularly in the role of resident, might allow them to sp end more time in the aggregation, and all other

PAGE 49

40 things being equal, be available longer to vi siting females. In addition, certain territories may be particularly valuable within the le k either because they serve as a superior signaling platform or because females prefer th em due to their safety from predation or because they indicate male quality (Field et al. 2002). Males may fight to establish calling stations in favorable locations or to keep other males at a distance (Dodson 1982). In the Mexican fruit fly, A. ludens, male mating success is influenced by the propensity to engage in fights with other males, and fighting ability (Robacker et al. 1991). Table 3-3. Summary of sexual success parameters in Caribbean fruit fly when treated or not with methoprene (M + /M ) and fed or not with protein (P + /P ). Parameters M + P P + M + P P + M + P P + M + P P + Initiation of aggregation a b b c Male participation a b b c Males calling a b b c Calling duration ns ns ns ns Matings a b b c Copula duration ns ns ns ns Male-male interaction a a a b Male-female interaction a b b b ns-non significant differences a > b > c significant different ( =0.05) While it is difficult to separate some of these possibilities it does seem unlikely that increased female encounter rates alone are sufficient to explain the sexual success of M + P P + males. For one thing, M + P+ P males had a lower female acceptance index, evidence that they required fewer encounters on aver age to successfully initiate copulation. Thus female preference, either for a more powerful signal(s) or for the male’s position within the aggregation, seems to be an import ant component of th e variance in male reproductive success. Can the relative importance of signal and re sidence quality to female mate choice be determined? Hendrichs (1986) ex amined the sexual behavior of A. suspensa in a field

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41 cage and found that males compete for leaves in the centers of aggregations and females usually mate in the center as well, whic h was substantiated by this study. Territory location within leks figures in two prominent theories of lek evolution; the “hotspot” and “hotshot” models (Hglund and Alatalo 1995), both of which are consistent with the results of this study. In the first, females c hoose males on the basis of location within the aggregation either because male-male competition for a particular site acts as a “filter” that guarantees male quality, or because ce rtain locations provide more protection from predators during periods of concen tration and awkward positioning. In the second model (“hotshot”), sa tellite males accumulate around unusually attractive males attempting to intercept females as they try to obtain access to the “hotshot”. By surrounding the “hotshot” these satellite males place him in their center. There is an unusual permutation of this m odel in the case of many tephritids. Calling males deposit pheromones on leaf-surfaces while calling, probably to enlarge the surface area for evaporation, and some of these compou nds persist for at least 24 hours (Sivinski et al. 1994). Thus occupation could make a te rritory increasingly valuable as a signaling site and turn residents into relative “hotshots”. With the present data it is difficult to completely eliminate one or the other explanation. Certainly the chain of events that leads from males that initiate leks to be more likely to occupy the lek-center, to bei ng better able to defend their territories, and then to subsequently mate more often would seem to favor the “hotshot” model; i.e., an attractive (high-output signaling) male is qui ckly surrounded by less cap able satellites but still manages to copulate. However, lek site s can be consistent ove r time (Sivinski 1989)

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42 and it could be that M + P P + and other successful males are more competent at locating “hotspots” and thus being the first in to signal from incipient leks. Regardless of the selective history of Cari bbean fruit fly leks, it is abundantly clear from the present study that M + P P + males, and to a lesser extent those that receive either methoprene or protein are more prone to initi ate leks, more likely to occupy lek-centers, better able to defend their territories and ul timately enjoy greater sexual success. Since capacity of mass-reared males to compete with wild males is the foundation of SIT these findings have important implications for fruit fly area-wide control. Protein rich adult diets have been previously shown to increase male sexual success in the medfly (Kaspi and Yuval 2000), but th e associated cost of hydrolyzed yeast or other protein sources inhibits their use in SIT programs. Howe ver, data such as presented here would suggest that avoi ding the costs of protein (or methoprene) might seriously undercut the potential of SIT. Caribbean fruit fly males with neither methoprene nor protein, i.e., those most resembling mass-r eared males at present, were sexually incompetent in comparison to those receiving either food or hormone additives. Two further studies are immediately suggested by this work. The first is to identify the most economic source of protein for adult mass-reared flies. Incorporation of bacteria in diets to optimize the microbial symbiont flora (Lauzon et al. 2000) might offer one avenue. The second is to repeat these experime nts using radiation-sterilized flies. It is ultimately sterilized flies that must compete in the field, and sterilization often results in decreased sexual performance (Heath et al. 1994, Lux et al. 2002, Barry et al. 2003). Methoprene and protein may prove to be even more critical given the expected loss of vigor.

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CHAPTER 4 INFLUENCE OF A JUVENILE HORM ONE ANALOG AND DIETARY PROTEIN ON MALE ATTRACTIVENESS TO FEMALES Introduction Polyphagous tephritid fruit flies, such as Anastrepha suspensa (Loew), the Caribbean fruit fly, often form mating aggrega tions (leks) (Sivinski et al. 2000). Males employ chemical, acoustic and visual signals in intra-sexual interactions, and to attract and court females (Prokopy 1980, Burk 1981). Du e, presumably, to variance in these elaborate signals and behaviors male se xual success normally is skewed, and a relatively few males obtain the majority of the copulations (Thornhill and Alcock 1983). Among the signal-channels employed by ma les, visual and acoustic signals probably act at close range (Sivinski et al. 1984, Sivinski an d Pereira 2005), but pheromones may have both short and long-ra nge effects (Nation 1972, Webb et al. 1983, Sivinski et al. 1994). In addition to attracting females, male A. suspensa respond to pheromones as well, presumably to help lo cate lekking sites (Burk 1983). However, in another tephritid, medfly, Ceratitis capitata (Wied.) Shelly (2000) found no response of males to other lekking males emitting pheromone. Exposure to the juvenile hormone anal og, methoprene, and protein consumption during the adult pre-sexual maturation period accelerates male development and may lead to greater sexual success th rough increased pheromone pr oduction (Teal et al. 2000). However, accelerated maturity and increased pheromone production may have nutritional consequences since there is less time fo r young flies to acquire reserves and these 43

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44 nutrients may be used at a higher rate. In the medfly, protein-fed males call and mate more frequently than protein-deprived males in both wild (Kaspi et al. 2000) and in massreared flies (Kaspi and Yuval 2000). Howe ver, Shelly and McInnis (2003) found no influence of protein in adult diet on mating su ccess of sterile males. Thus the addition of a protein rich adult diet on sexual beha viors needs to be investigated for A. suspensa and it may have particularly important conse quences when juvenile hormones titers are manipulated. In the previous chapters, methoprene application and a protein-rich adult diet improved male sexual success (Chapter 2) and performance within leks (Chapter 3). This effect was more pronounced when both me thoprene and protein ad ult diet were used together. The major goal of this study was to determine the effects of methoprene application, a protein-rich adult diet and their interactions on A. suspensa male attractiveness to females, i.e., the relativ e rates of female encounter experienced by variously treated males in both the laborat ory and under semi-natural conditions in a greenhouse. Material and Methods Insects. The Caribbean fruit flies used in the study had been in a laboratory colony at the Center for Medical, Agricultural and Veterinary Entomology (CMAVE) USDAARS, at Gainesville, FL, for less than 3 years and were produced according to their mass rearing protocol (FDACS 1995). The flies were maintained under low stress condition (~ 100 flies in 20 by 20 by 20 cm adult cages and on e larvae per 4 g of diet), which results in low selection pressure for characteristics associated with domestication (Liedo et al. 2002, Mangan 2003).

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45 Flies to be used in experiments were obt ained from pupae sorted into size classes with a sorting machine (FAO/IAEA/USDA 2003). This was done to eliminate any impact of size on male competitiveness (Bur k and Webb 1983, Burk 1984, Webb et al. 1984, Sivinski and Dodson 1992, Sivinski 1993). Male s used for the experiment came from a size class with an average weight of 10.8 0.9 mg (n=30). Females were obtained from the next larger cla ss size, with an average weight of 11.7 0.8 mg (n=30). In the field, males are typically 80% of the female size (Sivinski and Calkins 1990, Sivinski 1993). These pupal weights were in the middle range of A. suspensa pupae collected from infested fruits in nature (Hendrichs 1986). After emergence , the flies were maintained in a laboratory room with a photoperiod of 13L:11D (light from 7:00 to 20:00), a light intensity of 550 50 lux, a temperature of 25 1C and a relative humidity of 55 5%. Treatments. The study compared sexual performance of male A. suspensa subjected to the following four treatments: application of juvenile hormone analog, methoprene (M), and sugar and hydrolyzed yeast (protein source) as adult food (M + P P + ) methoprene application and sugar as adult food (M + P P ) no methoprene application and sugar a nd hydrolyzed yeast as adult food (M P P + ) no methoprene application and sugar as adult food (M P P ) Methoprene, a synthetic juven ile hormone analog, was applied topically in the first 24 hours after adult emergence at a rate of 5 g in 1 l acetone solution per male in M + treatments. On M treatments, 1 l of acetone was applied, to serve as a control. Males were immobilized in a net bag (as us ed in standard marking techniques, FAO/IAEA/USDA (2003)) and the solution applied via pipette thr ough the net onto the dorsal surface of the thorax. No anesthesia was used to immobilize the flies. Two

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46 different net bags and pipe ttes were used (one for M + treatments and other for M treatments) to prevent methoprene contaminations. Males from each treatment were maintained in independent 30 cm by 30 cm by 30 cm screen cages with a maximum male density of 200 flies/cage and with the type of food assigned for each treatment. In the P treatments only water and sugar ad libitum was supplied to the flies. In the P P + treatments hydrolyzed yeast was added to the adult diet as protein source (mixed with sugar in a proportion of three parts of sugar and one part of hydrolyzed yeast). This mixture is considered a high quality diet for Anastrepha species (Jcome et al. 1995, Aluja et al. 2001). Females used in the experiments were sexed on the first day of adult life and maintained in absence of males with the P + diet, i.e., sugar plus hydrolyzed yeast (3:1) and water ad libitum . Male attractiveness in laboratory. Thirteen to 16 day old males were transferred to individual cylindrical screen cages (10 cm high and 7 cm diameter) with water supplied by a cotton wick situated in a separate cup of water and penetrating the cage bottom. Each replicate included one male from each of the four treatment groups. Four individual cages (each containing one male from a treatment) were placed inside a larger 30 cm by 30 cm by 30 cm screen cage (Figure 4-1). A single male was released into each of the four individual cylindric al cages at 16:00. A 20-23 day old fully mature female was then released into the outer, larger cage one hour later (17:00), and her movements and approaches to the male-containing inner cag es were observed until 19:00. No food or water was supplied in the outer cage in order not to compli cate female responses and no food (only water) was supplied to the male inner cages so as not to interfere with male

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47 attractiveness. Male pheromone signaling (cal ling) was identified by the distention of lateral pouches on their abdomens and the extruded anal membranes, moistened with pheromone, which appeared as a droplet (Burk 1983). 30 cm 30 cm 30 cm 30 cm 30 cm 30 cm Figure 4-1. Arrangement of the experiment (att ractiveness in laboratory). Four males in inner cylindrical cages (one per treatmen t, 7 cm diameter) attract the released female in the outer cage. The time spent by males calling when females were in the outer cage was recorded (from 17:00 to 19:00), as was th e number of female visita tions (landing on cylindrical male cages) and the time they spent on the male-cages. The position of each male cage (treatment) was rotated inside the outer screen cage between replicates. Four large screen cages were run daily during a total of 12 days so that a total of 48 females were observed. Each day new individual cylindrical screen cages were used to eliminate the possibility

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48 that differential male pheromone deposition on the previous day might influence female response (Sivinski et al. 1994). Laboratory condi tions were the same as those used to maintain flies in the laboratory before the experiments (li ght intensity of 550 50 lux, temperature of 25 1C and relative humidity of 55 5%). Male attractiveness under semi-natural conditions. The experiment was conducted in a 8.6 m by 6.0 m gree nhouse containing 24 potted guavas ( Psidium guajava L.; 1.8 m to 2.0 m high). Guavas are a key host fruit and are commonly used by A. suspensa as lek sites in the wild (Dodson 1982, Hendrichs 1986, Landolt and Sivinski 1992, Sivinski 1989). The potted trees were distributed in four rows of six trees each (Figure 4-2). 6.0 m 8.6 m 6.0 m 8.6 m Figure 4-2. Arrangement of the experiment (a ttractiveness in gree nhouse). Open circles represent tree canopy and filled circles represents the local where the six-male artificial leks were located (1 artificial lek per treatment).

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49 Four artificial leks (see below) were hung within particular tree canopies each test day. These were placed on the second and fift h tree of the second lin e (that part of the canopy facing line one) and on the second and fifth tree of the third line (part of canopy facing line four) at 2.0 m from the greenhous e sidewalls and 2.2 m from the end walls. Males from each treatment occupied one of the four positions and treatments were rotated every day. Each artificial lek consisted of six males, one male in each of the six (4 cm high and 2 cm diameter) cylindrical screen cages, which were bound together in a bundle (Figure 4-3). The cage ends were closed with cotton wicks. Males were caged individually to prevent intense male-male in teraction when in a confined area and to permit pheromones to attract females to a particular emitter. No food or water was supplied to the males so as to not to c onfound female responses. At 16:50, 100 virgin 2023 day old females were evenly distributed in the greenhouse (four females in each tree canopy plus 4 on the center). Ten minutes late r, 13-16 day old males were hung in their lek-cages. The experiment was conducted ove r 12 days (28 and 30 June 2005, and 2 to 9 and 11 and 13 July 2005) with new males in new individual cages on each day (replicate) in order to prevent previous pheromone depos itions from influencing female behavior (Sivinski et al. 1994). Females from the previous replicate were removed from the greenhouse the following morning and new ones released at 16 :50 on the test day. Te mperature, relative humidity and light intensity were measur ed every 30 minutes dur ing the experiment (from 17:00 to 19:00). The numbers of males calling and the number of females in the

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50 immediate vicinity of each le k (within a radius of 25 cm) were recorded every 10 minutes and values were averaged across one replicate. Figure 4-3. Artificial leks used in the greenhouse experiment. Females from the previous replicate were removed from the greenhouse the following morning and new ones released at 16 :50 on the test day. Te mperature, relative humidity and light intensity were measur ed every 30 minutes dur ing the experiment (from 17:00 to 19:00). The numbers of males calling and the number of females in the immediate vicinity of each le k (within a radius of 25 cm) were recorded every 10 minutes and values were averaged across one replicate. The abiotic conditions during the 12 days of the greenhouse experiment (17:00 to 19:00) varied according to outside temperatur e, relative humidity and light intensity.

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51 Inside the greenhouse, the temperature ra nged from 25.0C to 35.6C, with a daily variation of 1C to 3C over the observation period. Re lative humidity ranged from 46% to 99%, and light intensities from 656 lux to 13,700 lux. Sunset occurred between 20:31 and 20:33 over the course of the experiment. Statistical analyses. Male calling and fema le visitation, both in laboratory and greenhouse experiments, were analyzed usi ng a two-way analysis of variance (ANOVA) to detect interactions betw een methoprene and protein. These analyses were followed by an ANOVA to detect differences between means in the trea tments. If differences in means were detected through ANOVA, a complementary multiple comparisons of means (Tukey’s test) was performed (Ott and Longnecker 2001). Linear regression was used to correlate calling males with female vi sitation in the greenhouse experiment. The significance value used in tests was 95% ( =0.05). Statistical anal yses were performed using R software (version 2.1.0, www.r-project.org). Results Male attractiveness in the laboratory. S ignificant effects of methoprene application (F 1,44 =72.00, p<0.05), protein supply (F 1,44 =81.31, p<0.05), and the interaction of the methoprene application and protein supply (F 1,44 =7.51, p=0.009) on male calling duration were found. In terms of female visitation, there were also significant effects of me thoprene application (F 1,44 =120.53, p<0.05), protein supply (F 1,44 =104.30, p<0.05), and interaction of the met hoprene application and protein supply (F 1,44 =28.75, p<0.05). M + P P + males spent more time calling and were approached by females more often than males in any of the other treatments. M PP males called significantly less often and had fewer females approach than males of other treatments (Figure 4-4; males calling: F 3,44 =53.7, p<0.05; and female visitation: F 3,44 =84.5, p<0.05).

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52 Significant effects of me thoprene application (F 1,44 =120.14, p<0.05), protein supply (F 1,44 =106.23, p<0.05), and interaction of the met hoprene application and protein supply (F 1,44 =22.51, p<0.05) on female visitation were also found. M + P P + males received a significantly higher number of female visits (1.71.28) when compared with the other treatments (F =83.0, p<0.05). M 3,44 PP males had significantly fewer female visits (0.35.17) than males of the other treatments (M + P P =0.75.24; M + PP =0.71.18). 0 10 20 30 40 50 60 M+P+ M+PM-P+ M-PTreatmentDuration (min) Males calling Femele visitationa bb c A B B C 0 10 20 30 40 50 60 M+P+ M+PM-P+ M-PTreatmentDuration (min) Males calling Femele visitationa bb c A B B C a bb c A B B C Figure 4-4. Time spent by male Caribbean fr uit flies calling and time spend by female visiting the males (mean plus standard deviation), when treated or not with methoprene (M + /M ) and fed or not with protein (P + / P ). Bars with the same letter (lowercase for males calling and cap ital for female visitation) are not significantly different (Tukey’s test, =0.05). Male attractiveness in the greenhouse. Significant effects of methoprene application (F 1,44 =106.76, p<0.05) and protein supply (F 1,44 =93.59, p<0.05) were found on males calling. However, no interaction was found between methoprene application and protein supply (F 1,44 =3.13, p=0.084). In terms of female visitation, there were

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53 significant effects of me thoprene application (F 1,44 =88.11, p<0.05), protein supply (F 1,44 =85.97, p<0.05), and the interaction of meth oprene application and protein supply (F 1,44 =12.59, p<0.05). In the greenhouse, M + P P + males called more often and we re approached by females more frequently than males in other tr eatments (Figure 4-5; males calling: F =67.1, p<0.001; and females attracted: F=62.2, p<0.001). M 3,44 3,44 PP males had significantly fewer males calling and attracted fewer females. 0 10 20 30 40 50 60 70 M+P+ M+PM-P+ M-PTreatment% Calling males Female visitationa bb c A BB C 0 10 20 30 40 50 60 70 M+P+ M+PM-P+ M-PTreatment% Calling males Female visitationa bb c A BB C Figure 4-5. Percentage of male Caribbean fruit flies in a greenhouse calling and female approaches to males (mean plus standard deviation), when treated or not with methoprene (M + /M ) and fed or not with protein (P + /P ). Bars with the same letter (lowercase for males calling and cap ital for female visitation) are not significantly different (Tukey’s test, =0.05). Male calling frequency, regardless of tr eatment, was correlated with female visitation (Figure 4-6).

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54 y = 0.6082x 0.2157 R2 = 0.7753 -0.5 0 0.5 1 1.5 2 2.5 00.511.522.533.54 Calling malesFemale visitation Figure 4-6. Correlation between th e number of male Caribbean fruit flies calling within an artificial lek and the number of female visiting in the greenhouse environment. Discussion Laboratory and greenhouse e xperiments, using individua l and aggregated males respectively, obtained similar results. M + P P + males spent significantly more time calling than males from other treatments and attracted more females, and M PP males called significantly less than males from other tr eatments and attracted significantly fewer females. Overall, the immediate effects of both methoprene and protein were to increase male signaling and enhance sexual success, and the interaction of these two factors was additive. Greater pheromone emission could be due to either more males calling or because they produce more pheromone per unit of calling time. The importance of nutrition to male signaling and sexual attractiveness has been documented in a number of tephritids, including the role of sugars in the Caribbean fruit fly (Landolt and Sivinski 1992, T eal et al. 2004). Yuval et al . (1998) in field studies of the medfly found that only males with ad equate energy reserves could engage in

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55 reproductive activities, including pheromone calling. In addition, medfly males that called more frequently had a higher mating su ccess (Shelly 2000) and those that attended leks contained higher nutritive reserves (sugar and proteins) than males not participating in leks (Yuval et al. 1998). Among Anastrepha species, incorporation of protein in adult diet improves male sexual success in Anastrepha obliqua (Macquart), Anastrepha serpentina (Wied.), Anastrepha striata Schiner (Aluja et al. 2001), and A. suspensa (Landolt and Sivinski 1992). However, Al uja et al. (2001) a nd Mangan (2003) found no sexual effect of additional protein for Anastrepha ludens (Loew). While male calling is significantly highe r when males received only methoprene, an absence of protein may limit the potential amount of calling possible with a superior diet. Alternatively, the addition of protei n alone enhances calling, but without the physiological effects of methoprene this in crease does not reach the highest levels. Increased calling is linearly related to female visitation, however the slope of this relationship is less than 1; i.e., doubling the number of cal ling males did not double the number of female visits. Certain theories of lek evolution argue that females prefer to compare males in close proximity and thus are disproportionately attracted to male aggregations (Field et al. 2002). While our data did not support this argument there are methodological difficulties in periodically counting the numbers of males. The observations of these behaviors were not continuous and th e behaviors associated with pheromone signaling may not accurately predict th e intensity of the signal itself. I suggest that the response of A. suspensa females to different numbers of males be explored with formulated pheromones released at different rates. In medfly (Shelly 2000) and Oriental fruit fly ( Bactrocera dorsalis (Hendel)) (Shelly 2001), fema le attraction was a direct

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56 reflection of male signal output. In both s ituations the relationship between female observations and calling activity among leks suggest that a difference in signal production by itself accounted for inter-lek variation in female visitation. However , in later studies Shelly (2001) found that while female medfly sightings per calling male were similar between the 18and 36-male leks , the ratios observed for these larger leks were significantly greater than those noted for the six-male leks. However, unlike the artificial leks in the presen t study, those artificial aggregations were larger than those found in the field (Prokopy and Hendrichs 1979, Arita and Kaneshiro 1989). This study shows the potential for use of hormones and pr oteins as enhancers of male qualities for use in sterile insect t echnique (SIT) programs. The use of hormones can increase the likelihood of male sexual signaling and consequently attract more females, which is a crucial factor for mating success. Male Anastrepha species have an extended, often weeks long, precopulatory period (Aluja 1994, Pereir a et al. 2006a), and under these conditions methoprene treatmen t has an additional advantage for massrearing. Application of juvenile hormone analog accelerates sexual maturation and as a consequence space is saved in fly handling facilities and costs are reduced. In addition, reducing the precopulatory period means males can be released already sexual mature or more likely to survive to sexual maturity . Protein rich adult diets have been shown to be of crucial importance in increasing male sexual success of medfly (Kaspi a nd Yuval 2000). However, the expense of hydrolyzed yeast or other protein sources has deterred its adoption by SIT programs. Detailed studies involving effectiveness of protein-fed compared to protein-deprived

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57 males, particularly sterile males, need to be done on a larger scale to support the addition of protein in adult diet and its concomitant costs.

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CHAPTER 5 EFFECTS OF SEXUAL INTERACTIONS ON MALE LONGEVITY Introduction Male Caribbean fruit fly, Anastrepha suspensa (Loew), forms lek mating aggregations in which there are intense defense of leaf territories, male acoustic displays, pheromone emissions and visual signals di rected toward females and, occasionally, copulations with visiting females (Burk 1983, Thornhill and Alcock 1983). These forms of reproductive competition presumably have energetic costs, depl ete resources and can result in physical damage such as frayed wi ngs. Such costs, in addition to hazards like sexually-transmitted diseases and preda tion (Cade 1975, Tuttle and Ryan 1981, Burk 1982, Hendrichs and Hendrichs 1998), may be cr itical in limiting an individual male’s sexual success and ultimately their survival. In Drosophila melanogaster L. exposure to virgin females reduces male longevity (Partridge and Farquhar 1981, Partridge and Andrews 1985, Co rdts and Partridge 1996). In the tephritid Ceratitis capitata (Wied.) male survival is reduced as male density increases in caged flies (Gaskin et al. 2002). This is attributed to the deleterious effects of increasing male-male behavioral interactions . In less territorial Diptera, such as D. melanogaster, male-male interactions actually de crease with density (Hoffmann and Cacoyianni, 1990). Little information exists on the costs of male and female interactions and the effect of reproduction on survival in male A. suspensa . Sivinski (1993) found that males caged with females (not replaced daily) li ved as long as virgin males kept alone. 58

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59 Other nutritional and physiological factor s are known to aff ect longevity of A. suspensa . Landolt and Sivinski (1992) showed th at a consistent supply of sucrose was necessary to maintain male pheromone production and cal ling, and Teal et al. (2004) demonstrated that males require carbohydrates in the adult diet to survive. There have been several studies of the impact of adult nu trition, principally of protein, on survival in other tephritids. Male C. capitata longevity is reduced when starved following protein feeding (Kaspy and Yuval 2000, Levy et al. 2005 ) which may be due to an irreversible metabolic cascade. In contrast, Maor et al . (2004) concluded that protein-fed males are better able to exploit naturally occurring s ources of nutrition following release and that this leads to greater longevit y. Other influences on the longevity of mass-reared male tephritids are sterilizing-irradi ation dose (Heath et al. 1994, L ux et al. 2002, Barry et al. 2003) and strain selection within co lonies (McInnis et al. 2002). Juvenile hormone titers influence behavior and may ultimately affect life span. In A. suspensa application of the juvenile hormone analog, methoprene, increases male calling, participation in leks, like lihood of copulation and success in male-male agonistic interactions (Chapter 3). The physiological expenses and physical hazards of these changes might also increase mortality. To measure the impact of both interse xual and intrasexual activities on male survival in A. suspensa and the interacting effects of reproductive behavior, diet and methoprene levels, three experimental groups of individually-caged flies were monitored for a period of 35 days: males caged alone w ith no interactions; those with daily malemale interactions; and those with a daily male -female interaction. In the last two cases the “interacting” fly was presented to the “focal ” fly for the two hours that coincided with peak time for sexual activity (Dodson 1982, Burk 1983, Hendrichs 1986, Landolt and

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60 Sivinski 1992). The intensity of the sexual interactions was manipulated within each experimental group by the addition of a methoprene application and/or dietary protein to various subgroups of males. The consequences of methoprene and protein treatments on longevity of mass-reared male tephritids destin ed for sterile insect technique (SIT) control programs are discussed. Material and Methods Insects. The Caribbean fruit flies used in the study had been in a laboratory colony at the Center for Medical, Agricultural and Veterinary Entomology (CMAVE) USDAARS, at Gainesville, FL, for less than 3 years and were produced according to their mass rearing protocol (FDACS 1995). Flies to be used in experiments were obtained from pupae sorted into size classe s with a sorting machine (F AO/IAEA/USDA 2003). This was done to eliminate any imp act of size on male competitiveness (Burk and Webb 1983, Burk 1984, Webb et al. 1984, Sivinski and D odson 1992, Sivinski 1993). Males used in experiments came from a size clas s whose average weight was 10.9 0.7 mg (n=30). Females were obtained from the next larg er class size, whose average weight was 11.9 0.8 mg (n=30). In the field, males are typically 80% of the female size (Sivinski and Calkins 1990, Sivinski 1993). These pupal weights were in the middle range of A. suspensa pupae collected from infested frui ts in nature (Hendrichs 1986). After emergence and during experiments, th e flies were maintained in a laboratory room with a photoperiod of 13L: 11D (light from 7:00 to 20:00 ), with light intensity of 550 50 lux, temperature of 25 1C and relative humidity of 55 5%. Treatments to manipulate the intensit y of sexual interactions and the availability of resources. In order to more fully reveal the roles of resource expenditure

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61 (intensity of sexual interactions) and resour ce availability (presence of protein) on male survival, methoprene levels and dietary pr otein were manipulated. Both of these treatments are known to increase male propens ity to emit pheromone, participate in leks, win agonistic encounters and copulate with females. The following treatments were compared within the experimental groups of males described in the section below: application of juvenile hormone analog, methoprene (M), and sugar and hydrolyzed yeast (protein source) as adult food (M + P P + ) methoprene application and sugar as adult food (M + P P ) no methoprene application and sugar a nd hydrolyzed yeast as adult food (M P P + ) no methoprene application and sugar as adult food (M P P ) Methoprene, a synthetic juven ile hormone analog, was applied topically in the first 24 hours after adult emergence at a rate of 5 g in 1 l acetone solution per male in M + treatments. In M treatments, 1 l of acetone was applied, to serve as a control. Males were immobilized in a net bag (as in standard marking techniques, FAO/IAEA/USDA (2003)) and the solution applied vi a pipette through the net onto the dorsal surface of the thorax, No anesthesia was used to immob ilize the flies. Two different net bags and pipettes were used (one for M + treatments and other for M treatments) to prevent methoprene contaminations. Males from each treatment were maintained in independent 30 cm by 30 cm by 30 cm screen cages with a maximum male density of 200 flies/cage and with the type of food assigned for each treatment. In the P treatments only water and sugar ad libitum were supplied to the flies. In the P + treatments hydrolyzed yeast was added to th e adult diet as a protein source (mixed with sugar in a proportion of three parts of s ugar and one part of hydrolyzed yeast). This mixture is considered a high quality diet for Anastrepha species (Jcome et al. 1995, Aluja et al. 2001).

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62 Females used in the experiment were sexed on the first day of adult life and maintained virgin (in absence of males) with the P + diet, i.e., sugar plus hydrolyzed yeast (3:1) and water ad libitum . Male survival. Survival in the three experiment al groups of flies was monitored daily for 35 days. These groups consisted of: 1) males alone (no interaction); 2) males with the daily addition of a 13-16 day old a nd sexually mature male from 17:00 to 19:00 (male-male interaction); and 3) males with the daily addition of a 20-23 day old sexually mature virgin female between 17:00 to 19:00 (male-female interaction). The experiments were conducted in the labor atory in small cylindrical cages (10 cm high and 7 cm diameter) with the same ab iotic conditions as described before (550 50 lux, temperature of 25 1C and relative humidity of 55 5%). The cages were placed over a water container that supplied water ad libitum to the flies through a cotton wick. For each of the three experimental groups 80 caged males (20 per treatment) were observed from just after emergence to 35 days of age. Males in cages were provided with the adult food appropriate to each treatment (sugar in P treatments and sugar and hydrolyzed protein (3:1) in P + treatments). In the males alone group, mortality was observed daily at 17:00. In the male-male interaction group, one sexually mature 13-16 day old male was released into the company of each focal male at 17:00. These “interacting” males had been maintained with optimal adult diet (sugar plus hydrolyzed yeast in 3:1 proportion) and water ad libitum until that age. They were marked with a dot of water-b ased paint on the upper part of the thorax to distinguish them from the focal males. Ma rked males were removed at 19:00. The same

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63 procedures were repeated daily until the test males reached 35 days of age. Mortality was observed daily at 17:00. For the male-female interaction group, i ndividual 20-23 day old virgin females, previously maintained with the P + diet (sugar plus hydrolyzed yeast in 3:1 proportion) and water ad libitum until that age , were placed in each male cage at 17:00. If mating occurred, females were removed at the end of copula. All females that had not mated were removed at 19:00. Those still in c opula at 19:00 were removed as soon they finished. The same procedures were repeated daily until 35 days of age. Mortality was observed daily at 17:00. Statistical analyses. The data were analyzed using survival analysis and the Cox proportional hazard model (Everitt and Pickles 2004). Statistical analyses were performed using R software (version 2.1.0, www.r-project.org). Results For all combinations of experimental groups and methoprene application there were no significant differences in longevity when protein was provided (Figure 5-1 shows the cumulative survival probability of males give n the four treatments in all three of the interaction–type group). The same was true when protein was withheld. However, for the P P + treatment, survival was si gnificantly higher than P in all interaction groups (Table 51). No effect of methoprene application was observed in any of the experimental groups. Reproductive behaviors, both with potent ial mates and sexual rivals were a significant source of mortality. Longevity in the three interaction groups (Figure 5-2) showed that within each trea tment, males maintained alone survived significantly longer than those interacting with ma les or females (Table 5-1).

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64 Figure 5-1. Cumulative survival probability of male Caribbean fruit flies in the three experimental groups (Amales alone-no interaction; Bmale-male interaction; Cmale-female interac tion), when treated or not with methoprene (M + /M ) and fed or not with protein (P + /P 0 0.2 0.4 0.6 0.8 11234567891011121314151617181920212223242526272829303132333435Comulative male sulvival probabilit y M+P+ M+PM-P+ M-P0 0.2 0.4 0.6 0.8 11234567891011121314151617181920212223242526272829303132333435Adult age (days) M+P+ M+PM-P+ M-P0 0.2 0.4 0.6 0.8 11234567891011121314151617181920212223242526272829303132333435 M+P+ M+PM-P+ M-PA B C 0 0.2 0.4 0.6 0.8 11234567891011121314151617181920212223242526272829303132333435Comulative male sulvival probabilit y M+P+ M+PM-P+ M-P0 0.2 0.4 0.6 0.8 11234567891011121314151617181920212223242526272829303132333435Adult age (days) M+P+ M+PM-P+ M-P0 0.2 0.4 0.6 0.8 11234567891011121314151617181920212223242526272829303132333435 M-PM-P+ M+PM+P+ P ). B C A

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65 Table 5-1. Results of the survival analys is (data on Figure 5-1 and Figure 5.2). Pairs are significa ntly different if p<0.05 (z value positive represent higher survival in the experimental group/tr eatment located on the top of the table; z value negative represent lower survival in the experiment al group/treatment located on the top of the table). ns -non-significant differences between the experimental group/treatment compared. No interaction Male-male interaction Male-female interaction Cohorts M + P + M + P M P + M P M + P + M + P M P + M P M + P + M + P M P + M P M + P + z=-4.85 p<0.05 z=-0.81 p=0.42 ns z=-3.53 p<0.05 z=-8.88 p<0.05 z=-6.16 p<0.05 M + P z=4.03 p<0.05 z=1.33 p=0.18 ns z=-6.89 p<0.05 z=-7.48 p<0.05 M P + z=-2.71 p=0.007 z=-6.72 p<0.05 z=-6.72 p<0.05 No interaction M P z=-11.60 p<0.05 z=-7.50 p<0.05 M + P + z=-4.64 p<0.05 z=1.51 p=0.13 ns z=-6.51 p<0.05 z=2.86 p=0.004 M + P z=6.09 p<0.05 z=-1.95 p=0.051 ns z=1.33 p=0.18 ns M P + -6. z=-7.91 p<0.05 z=3.19 p=0.0014 Male-male interaction M P z=4.53 p<0.05 M + P + z=15 z=1.81 p<0.05 p=0.071 ns z=-4.89 p<0.05 M + P z=7.86 p<0.05 z=1.30 p=0.19 ns M P + z=-6.67 p<0.05 Male-female interaction M P

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66 0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival 0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival 0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival 0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival No interaction Male-male interaction Male-female interactionM+P+M-P-M-P+M+P-c a a a a b b b b b c c 0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival 0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival 0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival 0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival No interaction Male-male interaction Male-female interactionM+P+M-P-M-P+M+P0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival 0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival 0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival 0 0.2 0.4 0.6 0.8 105101520253035Adult age (days)Cumulative male survival No interaction Male-male interaction Male-female interactionM+P+M-P-M-P+M+P-c a a a a b b b b b c c Figure 5-2. Cumulative survival probability of male Caribbean fruit flies in the four treatments, when treated or not with methoprene (M + /M ) and fed or not with protein (P + /P ), for the three experimental groups (no interaction; male-male interaction; and male-female interaction). Lines with the same letter for each treatment were not significantly diff erent (Cox proportional hazard model, =0.05). Male-male interaction effects on survival were more costly than male-female interactions, with the exception of the M + P P treatment where mortality in the context of male-male interactions was not significantly different from male-female interactions. In part this costly male-male interaction was due to the higher mortality in the first week of adult life (coincident with male ma turation) (Figure 5-1 (B) and (C)). Discussion Mortality increased significantly when males interacted with either females or other males. However the cost of male-male intera ction was almost always greater (except for M + P P males where no significant difference s were found). Regardless of social

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67 interactions, protein-fed males lived longer th an those that were pr otein-deprived. There was no effect of methoprene on survivorsh ip in any of the experimental groups. Polyphagous male tephritids in general, and A. suspensa in particular, synthesize relatively large quantities of sexual pheromone which are emitted in concert with wingproduced acoustic signals. These in co mbination with body and wing movements performed during close range courtships appe ar to be energetically expensive. The intensity of these daily, hours-long performances is reflected in the need for a continuous supply of sucrose (Landolt and Sivinski 1992, Te al et al. 2004). The increased mortality of males presented daily with a virgin female is perhaps due to the exhaustion of vital nutrients such as lipids (see Chapter 6). However, if this were the sole cause then it is surprising that while methoprene amplifie s pheromone production and other factors involved with sexual success, it has no effect on longevity. However, pheromone production is presumably de novo and thus, if sugar is avai lable during the period before calling and the biochemical mechanisms are de veloped then the synthesis would proceed as usual. No extra protein is needed because it may be more likely that males who mate must replenish accessory gland secretions which require significant amounts of protein and lipid biosynthesis. Male-female interaction is costly for males, but less so than male-male interactions. Perhaps there are other physical costs to c ourtship and mating that were not taken into account in the present study. The considerable da mage to wings in males presented with a daily rival may be one reflection of this physical cost. The more abrupt decline in longevity in males continually confronted with males in three of four treatments, than with potential mates, would suggest that the physical costs of competition are larger than

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68 the physiological costs of sex. In some other Diptera, e.g., D. melanogaster , costs associated with survival are mainly due to sexual activity with females (Partridge and Andrews 1985). However, there is little male te rritoriality in this species (Hoffmann and Cacoyianni 1990). The relative shapes of the relationships curves between age and survivorship in male and female interaction groups reflected some differences in the timing of mortality factors. Males appear to inter act at an earlier age with ot her males, prior to complete sexual maturity, when they begin to interact with females. As a result, some mortality occurs in the first days of male-male intera ction but not in the ear liest days of malefemale interaction. The importance of certain nutrients is clea rly reflected in the relative longevity of protein-fed males regardless of the type of social interaction or application of methoprene. In addition to longer life spans protein-fed males are more likely to win agonistic encounters with other males, pheromo ne signal, initiate leks and copulate with females than protein-deprived males (Chapter 3). Apparently, both sugars and proteins are required to maintain sexual competitiveness. The results of this study clearly indicate an increase in survival when protein is supplied to the males. This can have practical implications in sterile insect technique (SIT) programs, since release-recapture studies, at least with sugar-only fed C. capitata , have found a very low percentage of recaptures after four days in the field (Hendrichs et al. 1993, Barbosa et al. 2000). In addition, methoprene application improved sexual performance but had no effect on survival. T hus there are few non-fi scal drawbacks and

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69 many advantages to the addition of both of these treatments to fruit fly mass-rearing protocols.

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CHAPTER 6 INFLUENCE OF A JUVENILE HORM ONE ANALOG AND DIETARY PROTEIN ON MALE BODY LIPID AND PROTEIN CONTENTS Introduction Both the application of the juvenile hormone analog, methoprene and the addition of protein to the adult diet of Caribbean fruit fly, Anastrepha suspensa (Loew) increased male sexual success (Chapter 2). In additi on, exposure to methoprene at emergence accelerates male development (Teal et al. 2000 ) and may lead to greater sexual success through increased pheromone pr oduction (Teal and Gomez-Simu ta 2002a). In the related medfly, Ceratitis capitata (Wied.), protein consumption during the adult stage can contribute to male gonadal and accessory gl and development and also influence sexual success (Yuval et al. 2002). Lipid and protein content of the body re present energy reserves and “buildingblocks” and may be related to the ability of males to sustain participation in leks, sexual calling, male-male agonistic interactions a nd ultimately mating (Yuval et al. 1998). It may also affect lifespan. The relationship be tween nutritional status and mass-reared fly quality, flight-ability and su rvival, and sexual success has be en investigated to some extent in C. capitata. These are all key aspects in success of sterile insect technique (SIT) programs (Fisher and Cceres 2000, Hendrichs et al. 2002). Adult tephritids feed on a va riety of carbohydrates and proteins derived from fruit juices, honeydew and bird feces (Hendrichs et al. 1991, Warburg and Yuval 1997, Yuval and Hendrichs 2000). Historically, most tephri tid nutritional studies concentrated on the 70

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71 utility of various diets in mass-rearing pr ograms, with emphasis on diet selection by larvae (Zuculoto 1987, Economopoulos et al. 1990) , and larval development (Chang et al. 2001). Additionally, there have been few evaluati ons of the impact of larval diets on adult biology, including reproductive capacity and behavi or (Kaspi et al. 2002). The impacts of adult diet and energy reserves on sexual be havior and relative re productive performance have also been studied in C. capitata (Warburg and Yuval 1997, Kaspi et al. 2000, Shelly and Kennelly 2002, Shelly et al. 2002, Yuval et al. 2002, Shelly and McInnis 2003). Protein-fed males start to call earlie r in life (Papadopoulos et al. 1998). Also, the impact of adult diet on postcopulatory sexual selection has been examined (Taylor and Yuval 1999). In some cases, the influence of different diets on lipid reserves, both for adults (Nestel et al. 1985, Warburg and Yuval 1996) and late instar pupae and emerging adults (Nestel et al. 2004), has been investigated, as have levels of lipids, carbohydrates and proteins during metamorphosis (Nestel et al. 2003). In Bactrocera dorsalis (Hendel), incorporation of protein into adult diet has significant e ffects on survival and mating success (Shelly el al. 2005). Among Anastrepha species, Aluja et al. (2001) evaluated the effects of different adult nutrients, including protei n and sugar, on male sexual performance in adults of four species (Anastrepha ludens (Loew), Anastrepha obliqua (Macquart), Anastrepha serpentina (Wied.), Anastrepha striata Schiner), while Mangan (2003) determined adult nutritional effects on A. ludens female reproductive potenti al. Protein-fed males were more sexually successful than protein-deprived, except for A. ludens where no differences were found. Robacker and Moreno (1995) studied the influence of protein diets on the attraction of A. ludens to volatile bacterial meta bolites and found a lower

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72 attraction for protein-fed males. The influence of adult diet and age on lipid reserves of A. serpentina was quantified by Jcome et al. (1995). Relatively little nutritional work has been done with A. suspensa . However, some nutritional studies were conducted to investigate consump tion of carbohydrates, proteins and amino acids by adults (Sharp and Chambers 1984, Landolt and Davis-Hernandez 1993), food availability and quality on male pheromone production (Epsky and Heath 1993, Teal et al. 2000, Teal and Gomez-Simuta 2002a), and the influence of sucrose on male calling (Landolt and Sivinski 1992), and survival (Teal et al. 2004). However, no work has been done on the impact of protein incorporation in adu lt diets and methoprene application on lipid and protein body contents. In this study I examine the impact of the application of th e juvenile hormone analog, methoprene, protein supp ly, and their interactions on A. suspensa male body lipid and protein content during the adult stage (from emergence until day 35). Male weight was followed during the same period. Such in formation may provide insights into the physiological conditions that underlie male sexual performa nce (previous chapters) and ultimately improve the quality of released males in SIT programs. Material and Methods Insects. The Caribbean fruit flies used in the study had been in a laboratory colony at the Center for Medical, Agricultural and Veterinary Entomology (CMAVE) USDAARS, at Gainesville, FL, for less than 3 year s and were produced ac cording to their mass rearing protocol (FDACS 1995). Pupae were sorted by class size with a pupal sorting machine (FAO/IAEA/USDA 2003) which was used to obtain males for the experiment with low variation in weight (Burk and Webb 1983, Burk 1984, Webb et al . 1984, Sivinski and Dodson 1992, Sivinski

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73 1993). The flies used in this experiment were from a size class that averaged 10.9 0.7 mg (n=30) in weight. This is consider ed a mid size pupa for field collected A. suspensa males (Hendrichs 1986). After emergence and during the experiment, the flies were maintained in a laboratory room with a photoperiod of 13L:11D (light from 7:00 to 20:00), a light intensity of 550 50 lux, a temperature of 25 1C and a relative humidity of 55 5%. Treatments. The study compared male A. suspensa weight, lipid and protein contents from emergence until day 35 following one of six treatments: application of juvenile hormone analog, methoprene (M) in acetone solution, and sugar and hydrolyzed yeast as adult food (M + P P + ) methoprene application in acetone so lution, and sugar as adult food (M + P P ) no methoprene application, but acetone, a nd sugar and hydrolyzed yeast as adult food (M P P + ) no methoprene application, but acet one, and sugar as adult food (M P P ) no methoprene or acetone application, and sugar and hydrolyzed yeast as adult food (P + ) no methoprene or acetone applicat ion and sugar as adult food (P ). I included two additional treatments (P + ) and (P ) in addition to those considered in previous chapters to provide information on the effect of acetone on nutritional status. Methoprene, a synthetic juven ile hormone analog, was applied topically in the first 24 hours after adult emergence at a rate of 5 g in 1 l acetone solution per male in M + treatments. In M treatments, 1 l of acetone was applied. Treatments (P + ) and (P ) did not receive methoprene or acetone. The methoprene was applied topically in the first 24 hours after adult emergence at a rate of 5 g in 1 l acetone solution per male. In M treatments, 1 l of acetone was applied, to serve as a control. Males were immobilized in a net bag (as in standard marking techniques, FAO/IAEA/USDA (2003 )) and the solution applied via pipette

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74 through the net onto the dorsal surface of th e thorax, No anesthesia was used to immobilize the flies. Two different net ba gs and pipettes were used (one for M + treatments and other for M treatments) to prevent methoprene contaminations. Males from each treatment were maintained in independent 30 cm by 30 cm by 30 cm screen cages with a maximum male density of 200 f lies/cage and with the type of food assigned for each treatment. In the P treatments, only water and sugar ad libitum were supplied to the flies. In the P + treatments hydrolyzed yeast was added to the adult diet as protein source (mixed with sugar in a proportion of three parts of s ugar and one part of hydrolyzed yeast). This mixture is considered a high quality diet for Anastrepha species (Jcome et al. 1995, Aluja et al. 2001). Males were maintained in the laboratory in independent 30 cm by 30 cm by 30 cm cages per treatment until 5, 10, 15, 20, 25, 30, and 35 days of age. A sample of five flies from each treatment group and at each age was k illed in the freezer and stored at -84C. Five newly emerged flies, were also collect ed and stored just prior to the treatment application. In C. capitata , lipid contents have been found to vary according to the time of the day, due to the different activities in which males were engaged (Warburg and Yuval 1997). For this reason, I always sampled males at the same time of day (16:30, at the beginning of the calling period). Prior to homogeniza tion for lipid and protein determination flies were weighed individually. Lipid contents. Individuals were homogenized in a solution of PBS buffer at pH 7.25 (8.77 g of 0.15 M NaCl and 7.1 g of 50 mM Na 2 HPO 4 in 1 l of water). The homogenate was then brought up to 4.0 ml w ith PBS. Lipid was extracted (Bligh and

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75 Dyer 1959) by adding 40 mg of Na 2 SO 4 to 2.0 ml of the homogenate sample (1/2 of the insect), and 3.75 ml of chloroform: methanol (1:2). Later 1.25 ml of chloroform was added and sample was vortexed to obtain two separate phases. After being centrifuged for 4 min at 4,000 rpm, the lower chloroform phase was collected. A second addition of chloroform (1.875 ml) served to extract th e remaining lipids. The lower phase was collected and added to the first collection. The solution was dried in a Speed Vac device (Thermo Savant, San Jose, CA). Lipid contents were determined through the vanillin reagent method (Van Handel 1985, Warburg and Yuval 1996). Van illin reagent (27 mg of vanillin in 4.5 ml of pure water and 18 ml of H 3 PO 4 ) was mixed on a stir plate for use in colorimetric determination. Standards were prepared with triolein weighed and diluted 1.0 mg/ml with chloroform. The standards (500, 200, 100, 50, and 25 l) were dried under N 2 and 500 l of H 2 SO 4 was added to the dried samples and sta ndards, which were then heated for 10 min at 90C. A190 l quantity of vanillin reagent was added to 10 l of sample, standard or blank. Lipid content was colorimetr ically determined at 530 nm in a spectrophotometer plate reader (Bio -tek Instruments, Winooski, VT). Protein contents. Protein determination was done according the Pierce BCA protein assay (Pierce, Rockford, IL). Previously obtained insect homogenate (1.0 ml) was centrifuged 1 min at 14,000 rpm, 500 l were removed (1/8 of the homogenized insect), and 100 l of sodium deoxychoate r eagent (0.15 w/v) and 100 l of 72% (w/v) tricloroacetic acid (TCA) were added to the 500 l samples and to the standard solutions previously prepared with Pierce BCA standard stock, to precipitate the proteins. After incubation at room temperature for 10 min and centrifugation for 10 min at 14,000 rpm

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76 the supernatant was discarded. Proteins form ed a pellet at the bottom of the tube, to which 50 l of 5% (w/v) sodium dod ecyl sulfate (SDS) and 1 ml of Pierce micro BCA TM protein assay reagent were added to the pell et (Pierce 1999). After incubation in a water bath at 37C for 30 min, proteins in samp les and standards were colorimetrically determined at 562 nm in the plate reader. Statistical analyses. The data were analyzed using a two-way analysis of variance (ANOVA) to detect the inter actions between ages and treatments for the parameters studied (adult weight, lipid c ontent and protein content). Th ese analyses were followed by an ANOVA to detect differences between means in the treatments and ages. If differences in means were detected through ANOVA, complementary multiple comparisons of means (Tukey’s test) we re performed (Ott and Longnecker 2001). Statistical analysis among ages includes valu es obtained at emergence (day zero, before treatments were applied). The signif icance value used in tests was 95% ( =0.05). Statistical analyses were performed using R software (version 2.1.0, www.r-project.org). Results Male weight. Average adult weights varied between 5.8 mg and 11.6 mg (Figure 6-1). There was a significant effect of treatment (F 5,192 =24.46, p<0.05). However, age had no significant effect (F 7,192 =1.59, p=0.140) nor did the intera ction of treatment and adult age (F 35,192 =1.16, p=0.256). No differences in weight were found among males of different ages within any of the treatments (F values in Table 6-1). Significant differences were found for all ages among va rious treatments (Table 6-2) . No significant differences were found among the three protein-fed treatments, nor were there any among the protein-deprived treatments. This means that there was no significant impact of methoprene or acetone on male weight regard less of whether or not protein was supplied.

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77 0 2 4 6 8 10 12 14 05101520253035 Adult age (days)Adult weight (mg) M+P+ M+PM-P+ M-PP+ PFigure 6-1. Average (sd) adult weight (n=5) of male Caribbean fruit flies at different ages among the six treatments featuri ng methoprene (M) and protein (P) and their various combinations. Lipid contents. S ignificant effects of treatment (F 5,192 =131.37, p<0.05), adult age (F 7,192 =83.14, p<0.05), and the interaction of adult age and treatment (F 35,192 =6.34, p<0.05) were found. Male lipid content per trea tment per age (Figure 6-2) differed both among ages and for different treatments (Table 6-1) and among treatments for different ages (Table 6-2). Male total lipid contents declined during adult life for all treatments studied. Protein-deprived treatments droppe d precipitously between emergence and day five (after treatment applic ation). A significant drop in pr otein-fed treatments occurred between day 10 and day 15 (after males reached sexual maturity). Lipid contents of protein-deprived treatments were significantly higher at emergence than at the other ages.

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78 Protein-fed treatments at emer gence, day five and day 10 were significantly higher than all the other ages. Table 6-1. Analysis of variance (ANOVA) fo r male Caribbean fruit fly weight, total lipids, and total proteins among differen t ages in six different treatments featuring methoprene (M) and protein (P) and their various combinations (ns, non significant differences, p>0.05; * 0.01
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79 0 50 100 150 200 250 300 350 05101520253035 Adult age (days)Lipids (ug/insect) M+P+ M+PM-P+ M-PP+ PFigure 6-2. Average (sd) total lipid contents of male Caribbean fruit flies at different adult ages among the 6 treatments feat uring methoprene (M) and protein (P) and their various combinations. Protein contents. S ignificant effects of treatment (F 5,192 =44.63, p<0.05), adult age (F 7,192 =15.00, p<0.05), and the interaction of adult age and treatment (F 35,192 =4.77, p<0.05) were found. Significant difference s were found for each treatment among different ages (Figure 6-3) (except for treatment M P P + ) (Table 6-1), and among treatments at all ages (Table 6-2). As with lipids, P + type and P type diets yielded two re lationship clusters. The inclusion of protein in the diet had no eff ect on the protein levels of males in the M P + treatment at various ages, but dietary protein did ha ve significant effect on protein levels of males at various ages in the other two protein-fed treatments (Table 6-1). All protein

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80 deprived males had significant age-related differences within the treatments (Table 6-1). Different responses over time were observed. In protein-fed treat ments there was an increase in total protein afte r day 15 of adult life. In prot ein-deprived treatments there was a significant decrease until day 10 with a subsequent increase. 0 100 200 300 400 500 600 700 800 900 05101520253035 Adult age (days)Protein (ug/insect) M+P+ M+PM-P+ M-PP+ PFigure 6-3. Average (sd) total protein contents of male Caribbean fruit flies at different adult ages among the 6 treatments feat uring methoprene (M) and protein (P) and their various combinations. At different ages there we re always significant differe nces in protein content among the treatments (Table 6-2). However, no significant differences were found among P P + and P treatments for each of the studied ages. As with lipids, P + and P type diets yielded two clusters of protein content rela tionships. There was no effect of methoprene or acetone on total protein contents in e ither protein-deprived or protein-fed males.

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81 Discussion There is a clear effect of protein added to the diet on the weight, total lipids and total protein contents during the first 35 days of adult life in male Caribbean fruit flies. In contrast, there is no impact of methoprene or acetone application on these same variables. Weight and total protein conten ts, regardless of diet type, were relatively stable during adult life. However, total lipid content steadily decreased with age in flies fed a diet without protein, although this loss did not occu r until after 10 days of age for protein-fed flies. Male weight was significantly higher when protein was supplied in the adult diet. In C. capitata Yuval et al. (1998) found no differences in the sizes of lekking and resting males in nature. However, lekking males were heavier and containe d significantly more protein and sugar than resting males. There were no differences in lipids (note that the analysis calculated lipids per milligram of in sect, not total lipid contents). In another study with the medfly, Kaspi et al. (2000) showed a lower content of lipids in protein-fed males than in protein-deprived males. These data are inconsistent with the present study of A. suspensa. The lipids most important to insects are fatty acids, phospholipids, and sterols. Many kinds of fatty acids and phospholipids ar e synthesized by insects, but all insects require sterols in their diet (Chapman 1998). Reduction of total lipid content during life can be the result of somatic ac tivities, since lipids represent stored energy, even if some restoration of lipid reserves occurs by lipogenesis (Warburg and Yuval 1996). In a study of energy use by male mosquitoes, Yuval et al. (1994) postulated that lipids metaphorically represent an energetic trus t fund, whereas carbohydrates are comparable to a readily accessible cash account.

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82 In male A. suspensa, at least in the first 10 days of adult life of protein-fed males, there are indications of lipogenesi s. The same phenomenon occurs in C. capitata (Warburg and Yuval 1996) and A. serpentina (Jcome et al. 1995). However, total lipid contents decline following male Caribbean fr uit fly maturation. Sharp decreases indicate that males start to utilize th eir metabolic reserves, and this seems likely to correspond to the energetic requirements of pheromone production and engagement in male-male agonistic interactions. In A. serpentina, lipid reserves increased (day 4 to day 16 of adult life) when sugar and protein were supplied as adult food, but decreased when only sugar was provided (Jcome et al. 1995). This declin e was consistent with the present results for A. suspensa. Nestel et al. (1986) suggested that lipid reserves in male C. capitata may play an important role in the regulation and produc tion of sex pheromone. For male Caribbean fruit fly, pheromone production increases when methoprene is applied (Teal et al. 2000). However, this study shows that lipids did not increase in either pr otein-fed or proteindeprived males when methoprene was applied. T hus lipid reserves in this species may not play a significant role in phero mone synthesis or release. The differences in total protein content between protein-fed a nd protein-deprived males may be influenced by ingested protein in the gut. However, the gradual increase in total protein contents over tim e in both protein-deprived a nd protein-fed males is both difficult to explain and inconsis tent with gut protein alone accounting for the difference. Tephritids are known to feed on feces (P rokopy et al. 1993, Epsky et al. 1997) and perhaps the consumption of their own bacter ia-containing fecal materials inadvertently provided them with a protein source.

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83 The findings of this study can have dire ct implications for SIT programs. The incorporation of dietary protei n has an effect on adult weight which can directly influence male-male interactions (Sivinski 1993). The eff ects of lipid and protei n contents are also important since they play a role in male sexual performance (Yuval et al. 1998). Due these effects, the incorporation of protein in adult diet for SIT programs is recommended. This can contribute to more nutritionally stable males, and consequently increased effectiveness of sterile males.

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CHAPTER 7 GENERAL DISCUSSION Male sexual performance, in terms of lek tenu re, attractiveness to females, winning in agonistic encounters and nu mber of copulations obtained, was significantly improved through both applications of the juvenile ho rmone analog, methoprene and the addition of protein to adult diet. Used together they had an additive effect. Protein by itself had a positive impact on male longevity and on balanced nutritional status. The advantages of good diet are well unders tood in many insects and the costs and benefits of foraging for food have been exam ined in a number of studies (DeClerk and DeLoof 1983, Nestel et al. 1985, Cohen et al. 1987, Wheeler and Slansky 1991, Stockhoof 1993, Yuval et al. 2002). Differences in male sexual success can be in part due to differences in their diets. In addition to a well-nourished male being capable of greater activity, females may choose males on the basis of their perceived or inferred nutritional status. By mating with males that are able to maintain their position in the lek, females are mating with males that have been eff ective in acquiring food, and consequently may provide genes that affect offspring viability and male fitness (Field et al. 2002). Another advantage of a protein diet is a clear positiv e impact on longevity (Chapter 5) which is correlated with a potential increase in offspring production. Less well understood is the role of met hoprene, i.e., juvenile hormone, titer in sexual behavior and selection. For example, given the great sexual advantages available to males that have higher juvenile hormone titers, the question arises as to why these levels are not generally higher than they appa rently are (i.e., as high as they are after 84

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85 treatment), or perhaps more heuristically, what limits male hormone production to levels below those that would yield more copulati ons? In general, there may be limits on the availability of juvenile hormone component s, or there may be physiological costs to juvenile hormone production (in the present study additional j uvenile hormone analog is “free” to males), or there may be expens ive physiological consequences to higher juvenile hormone titers. Little is known about the co sts of juvenile hormone produc tion other than that it is not stored but must be produced in the corpora allata and then released into the hemolymph (Chapman 1998). In terms of phys iological consequences, these experiments have directly addressed only a single charac teristic, adult life span. While additional protein had a clear and positive effect on male survival there was no effect of methoprene on longevity. In sexual terms, neither the effect of methopr ene itself, nor the greater sexual activity it engendered, lessened the time males had to forage for mates. However, there may have been an effect later in life. While 35 days is believed to be a long life for a released tephritid (Hendrichs et al. 1993, Barbosa et al. 2000), Anastrepha suspensa (Loew) can live up to 1 year (Sivinski 1993) , and the effects of juvenile hormones on middle-aged and elderly flies is unknown. Methoprene also causes accelerated matu ration. Not only are such males more sexually successful on a daily basis, they al so begin their reproduc tive lives ear lier. All other things being equal, males that mature sooner have more sexual opportunities. So why then do they not mature as quickly as they could (as quickly as in these experiments) in nature? Tephritids often have extended, pre-reproductive periods in both males and females, although these tend to be longer in females. Male pre-reproductive periods can

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86 vary from about 5 days in Ceratitis capitata (Wied.) (Liedo et al. 2002) to 20 days in Anastrepha interrupta Stone (Pereira et al. 2006a). Male maturation in A. suspensa (Dodson 1982, Teal et al. 2000) and Anastrepha ludens (Loew) (Robacker et al. 1991) occurs in 5 to 7 days. This is due to the anau thogeneous nature of tephritids, i.e., adults emerge with unmatured gonads (Yuval et al . 2002). It is known that the timing of maturation is flexible. Environmental factors, such as presence of the male pheromone, can be used as cues for timing facultative A. suspensa female maturation (Pereira et al. 2006b). However, changes in pre-reproductive periods under mass-rearing suggest the parameters of maturation are set by nutritional requirements, i.e., that flies given a predictable laboratory diet are selected to ma ture sooner. Pereira et al. (2006b) saw this clearly when they found that the time under domestication affected the female prematuration period (mass-reared flies mature ear lier than recently domesticated flies and wild flies took longer still). The same phenomenon occurs in male medfly (Liedo et al. 2002). As adults in the field, tephritids feed on a variety of carbohydr ates and proteins derived from fruits juices, honeydew and bird feces (Hendrichs. et al 1991, Prokopy et al. 1993, Epsky et al. 1997, Yuval and Hendrichs 2000) . Given the nature of their diets, it may typically take an extended period of time to gather sufficient resources for both eggs and the rigors of producing expensive ch emical, visual and acoustic signals. Regardless of the unanswered evolutionary questions posed by these experiments, there are clear pragmatic applications for th e sterile insect technique (SIT). SIT is a principal non-chemical component of pest te phritid management (Hendrichs et al. 2002), and improvements in production costs and inse ct quality influence the efficacy of the

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87 technique and even the practicality of its us e under certain circumstances (Robinson et al. 2002). Economic support for these area-wide programs is based on ecological, commercial, and regulatory aspects (Munford 2004). However, the final objective of the technique (eradication, preven tion or suppression) should influence the strategies adopted. SIT is based on the release of sterile insect s of the target species to compete with wild males for the female (Knipling 1955). These insects normally are produced in mass rearing facilities with production capacities of up to hundreds of millions of insects per week (Hendrichs et al. 1995). However, to be effective the released sterile mass-reared males have to successfully transfer their sp erm carrying dominant lethal mutations to a large majority of females of the target popul ations (Hendrichs et al. 2002). For SIT to succeed, these males must be able to join the le ks of wild males or establish leks of their own, participate in male-male interactions, attract wild females, court, copulate and inseminate them, and inhibit them from remating for as long is possible. This study has demonstrated that methoprene and protein are capable of signifi cantly improving these qualities in mass-reared males. The delay between adult emergence and sexual maturity poses a significant problem for SIT programs because males must be held for a long period of time prior to release, or have to be released before becoming sexually mature, resulting in fewer surviving to maturity and copulation. This m eans that early sexual maturation results in more flies and in lower costs, due to space savings at fly handling facilities. Incorporation of these experi mental results into SIT programs requires that certain technical problems be addressed. Alternative sources of less e xpensive protein need to be

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88 identified and suitable means of their in corporation into agar diets developed. Additionally, inoculation with bacteria to restore the gut fauna after irradiation could further enhance optimal diets (Lauzon et al. 2000). The effect of methoprene when provided in a diet rather than being topically applied needs to be determined, since the physiological effect on A. suspensa could be different. Should this be the case , perhaps an alternative technique of topi cal application such as spra ying pupae, would be efficacious. And finally, these experiments need to be repeated with mass-reared and sterilized flies and transferred to other SIT target species like A. ludens.

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LIST OF REFERENCES Alcock J., and D. W. Pyle. 1979. The complex courtship behavior of Physiphora demandata (F.). Zeit. Treip. 49: 532-562. Aluja, M. 1994. Bionomics and management of Anastrepha. Annu. Rev. Entomol. 39: 155-178. Aluja, M., J. Hendrichs, and M. Cabrera. 1983. Behavior and interactions between Anastrepha ludens (Loew) and A. obliqua (Macquart) on a field caged mango tree. 1-Lekking behavior and male territoriality, pp. 122-133. In : R. Cavalloro (ed.), Fruit Flies of Economic Importance. Balkema, Athens, Greece. Aluja, M., J. Piero, I. Jcome, F. Daz-Fleischer, and J. Sivinski. 2000. Behavior of flies in the genus Anastrepha (Trypetinae: Toxotrypanini), pp. 375-406. In M. Aluja and A. L. Norrbom (eds.), Fruit f lies (Tephritidae): p hylogeny and evolution of behavior. CRC, Boca Raton, FL. Aluja, M., I. Jcome, and R. Macas-Ordez. 2001. Effect of adult nutrition on male sexual performance in four neotropi cal fruit fly species of the genus Anastrepha. (Diptera: Tephritidae). J. Insect Behav. 14: 759-775. Andersson, M. 1994. Sexual selection. Princeton Un iversity Press, Princeton, NJ. Appolonio, M., M. Festa-Bianchet, and F. Mari. 1989. Correlates of copulatory success in a fallow deer lek. Beha v. Ecol. Sociobiol. 25: 89-97. Arita, L. H., and K. Y. Kaneshiro. 1989. Sexual selection and lek behavior in the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae). Pac. Sc. 43: 135143. Barbosa, S., A. Mexia, and R. Pereira. 2000. Field study of the dispersal and survival of released Mediterranean fruit fly sterile “tsl” males, pp. 527-533. In T. K. Hong (ed.), Area-wide control of fruit flies and other insect pests. Universiti Sains Malaysia Press, Penang, Malaysia. Barry, J. D., D. O. McInnis, D. Gates, and J. G. Morse. 2003. Effects of irradiation on Mediterranean fruit flies (Diptera: Tephri tidae): emergence, survivorship, lure attraction, and mating competiti on. J. Econ. Entomol. 96: 615-622. Blay, S., and B. Yuval. 1997. Nutritional correlates of reproductive success of male Mediterranean fruit flies (Diptera: Tephritidae). Anim. Behav. 54: 59-66. 89

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101 Webb, J. C., J. Sivinski, and C. Litzkow. 1984. Acoustical behavior and sexual success in the Caribbean fruit fly, Anastrepha suspensa (Loew) (Diptera: Tephritidae). Environ. Entomol. 13: 650-656. Weems, H. V. 1966. The Caribbean fruit fly in Florida. Proc. Fla. State Hortic. Soc. 79: 401-405. Westcott, D. A. 1994. Leks of leks: a role for hotspots in lek evolution. Proc. R. Soc. Lond. B. 258: 281-286. Wheeler, G. S., and F. Slansky. 1991. Compensatory responses of the fall armyworm ( Spodoptera frugiperda ) when fed waterand cellulos e-diluted diets. Physiol. Entomol. 16: 361-374. Whittier, T. S., F. Y. Nam, T. E. Shelly, and K. Y. Kaneshiro. 1993. Male courtship success and female discrimination in the Mediterranean fruit fly (Diptera: Tephritidae). J. Insect. Behav. 7: 159-170 Wilson, E. O. 1975. Sociobiology: the new synthesis. Harvard University Press, Cambridge, MA. Yuval, B., and J. Hendrichs. 2000. Behavior of flies in the genus Ceratitis , pp. 429-456. In M. Aluja and A. L. Norrbom (eds.), Fruit flies (Tephritidae): phylogeny and evolution of behavior. CRC, Boca Raton, FL. Yuval, B., M. Holliday-Hanso n, and R. K. Washino. 1994. Energy budget of swarming male mosquitoes. Ecol. Entomol. 19: 74-78. Yuval, B., R. Kaspi, S. Sholmit, and M. S. Warburg. 1998. Nutritional reserves regulates male participation in Mediterrane an fruit fly leks. Ecol. Entomol. 23: 211215. Yuval, B., R. Kaspi, S. A. Field, S. Blay, and P. Taylor. 2002 . Effects of post-ternal nutrition on reproductive success of male Mediterranean fruit flies (Diptera: Tephritidae). Fla. Entomol. 85: 165-170. Zucoloto, F. S. 1987. Feeding habits of Ceratitis capitata (Diptera: Tephritidae): can larvae recognize a nutritionally effectiv e diet? J. Insect Physiol. 33: 349-353.

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BIOGRAPHICAL SKETCH Rui Manuel Cardoso Pereira received his B.Sc. Degree in Agricultural Engineering from Lisbon Technical University (Universidad e Tcnica de Lisboa, Instituto Superior de Agronomia), in Lisbon, Portugal, in 1991. He then pursued gra duate studies in Integrated Pest Management at the same University, and received a M.Sc. Degree in 1996. For his M.Sc. Degree, he studied the use of tra pping systems in monito ring and control of Mediterranean fruit fly. He worked as head of field activities (1994-1997) and later as program director (1997-2002) of Madeira-Med, a sterile insect technique (SIT) cont rol program against Mediterranean fruit fly in the Madeira Islands, Portugal. As part of his activities at Madeira-Med, he received training in Gu atemala (1995), Hawaii and California, USA (1996). Since 1996 he served as chief scientific i nvestigator of four Food and Agriculture Organization / International Atomic Energy Agency (FAO/IAEA) coordinated research programs : Development of female medfly attractan ts systems for trapping and sterility assessment; Development of improved attractants and their integration in to SIT fruit fly management programmes; Quality assurance in mass-reared and released fruit flies for use in SIT programmes; and Improving ster ile male performance in fruit fly SIT programmes. He entered the Ph.D. program at En tomology and Nematology Department, University of Florida, in January 2003. Hi s doctoral research focused on the use of a 102

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103 juvenile hormone analog and dietary protei n to improve male sexual performance in Caribbean fruit fly. He received a Ph.D. in Entomology in December 2005.