INFLUENCE of a JUVENILE HORMONE ANALOG and DIETARY PROTEIN on MALE CARIBBEAN FRUIT FLY, Anastrepha Suspensa (DIPTERA: TEPHRITIDAE), SEXUAL BEHAVIOR

INFLUENCE OF A JUVENILE HORMONE ANALOG AND DIETARY PROTEIN ON MALE CARIBBEAN FRUIT FLY, Anastrepha suspensa (DIPTERA: TEPHRITIDAE), SEXUAL BEHAVIOR

RUI MANUEL CARDOSO PEREIRA

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2005

ACKNOWLEDGMENTS

I thank my advisor, John Sivinski, for his involvement, support, and

encouragement. I have benefited immensely from his critical and creative thinking, and also been greatly inspired by his enthusiasm for science. I am grateful to my committee,

Jane Brockmann, Jorge Hendrichs, Heather McAuslane, Gary Steck, and Peter Teal, for their unstinting help. The diverse perspectives they provided enriched my work.

I would like to thank the Centro de Ciência e Tecnologia da Madeira for financial

support through the Ph.D. grant BD I/2002-004.

Heidi Burnside and Nancy Lowman maintained the Caribbean fruit fly colony from

which I obtained the flies for my experiments, 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 work and Meghan Brennan for the advice in the statistical survival analyses performed in Chapter 5.

Finally, I thank Hoa Hguyen for the indispensable technical assistance in most of

the experimental activities performed.

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 JUVENILE HORMONE ANALOG AND DIETARY PROTEIN ON MALE SEXUAL SUCCESS ...... 14

Introduction...... 14 Material and Methods...... 15 Results...... 20 Discussion...... 25

3 INFLUENCE OF A JUVENILE 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 JUVENILE HORMONE ANALOG AND DIETARY PROTEIN ON MALE ATTRACTIVENESS TO FEMALES...... 43

Introduction...... 43 Material and Methods...... 44 Results...... 51 Discussion...... 54

iii 5 EFFECTS OF SEXUAL INTERACTIONS ON MALE LONGEVITY ...... 58

Introduction...... 58 Material and Methods...... 60 Results...... 63 Discussion...... 66

6 INFLUENCE OF A JUVENILE HORMONE ANALOG AND DIETARY PROTEIN ON MALE BODY LIPID 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

LIST OF TABLES

Table page

1-1 List of tephritids that form leks...... 6

3-1 Number of male Caribbean fruit flies that occupied the various 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 parameters 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 treatments for different ages...... 78

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 Caribbean fruit flies from day 4 to the end of experiment (day 35)...... 22

2-3 Percentage of male Caribbean fruit flies that mated more than once...... 23

2-4 Number of matings obtained per treatment 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 Caribbean fruit flies that mated 1, 2, and 3 consecutive times...... 26

3-1 Lek parameters and sexual success 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 fruit 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 5-1 Cumulative survival probability of male 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

Abstract of Dissertation Presented 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 HORMONE ANALOG AND DIETARY PROTEIN ON MALE CARIBBEAN FRUIT FLY, Anastrepha suspensa (DIPTERA: TEPHRITIDAE), SEXUAL BEHAVIOR

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) directed towards this pest requires the release of males that can compete with wild males and attract wild females. Mass-reared males must form leks, engage in male-male agonistic interactions, 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 application and protein supply

+ + + - (M PP ); methoprene application and no protein supply (M PP ); no methoprene application

- + - - and protein supply (M PP ); and no application of methoprene or protein supply (M PP ).

Laboratory, field cage, and greenhouse experiments compared male sexual performance and other reproductive parameters on a lifetime and daily basis. Numbers of copulations, lek initiation, lek participation, pheromone signaling (calling), and female attraction were

viii + + all significantly greater in M PP males. Simultaneously, methoprene caused earlier male maturation. Sexual interactions, both intrasexual and intersexual (resource 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 application have the potential to produce more efficacious sterile males for SIT programs.

ix CHAPTER 1 GENERAL INTRODUCTION

The sterile insect technique (SIT) is the principal non-chemical component of pest management of tephritid (Diptera: Tephritidae) flies (Hendrichs et al. 2002).

Improvements in production costs and insect quality influence the efficacy of the technique and even the practicality of its use under certain circumstances (Robinson et al.

2002). In this study I examine the influence of juvenile hormone analog, methoprene and dietary protein as a tool to improve SIT.

Control of insects with SIT technique is based 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 majority of females in the target populations

(Hendrichs et al. 2002). Thus for SIT to succeed, 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 possible. Understanding the evolution 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 accelerates sexual maturation and increases pheromone production (Teal et al. 2000). However, there might be sexual-

1 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 selection is typically divided into competition

for mates (intrasexual selection) and mate choice (intersexual selection). It is responsible

for most adaptations of reproductive biology, including attributes 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 offspring’s chance of surviving at the cost of the parent being able to produce other offspring. 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 ability to produce her eggs, and multiple mating does not result in more offspring. However, ejaculates 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 access to mates, and this often results in aggression and/or increased searching or mobility.

Intersexual selection may result from differences among males in their capacity to

provide “direct benefits”, from “runaway sexual selection”, from variance in male genetic

quality (“good genes”) and through manipulation of female perceptions (“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 males and so produce more fit offspring, benefit

from the choice of good genes (Kirkpatrick and Revigné 2002). Males that are able to

influence female behavior through non-sexual signals acquire mates through sensory bias

(Fuller et al. 2005).

Both intrasexual and intersexual selection can act simultaneously on the mating

behavior of a species and it may, therefore, be difficult to differentiate 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 further subdivided to describe 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 males to defend locations where females occur create both different arenas and different intensities of sexual selection. When many

males fail to mate then there is high variance 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 species, 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 formation). 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 predictable 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 specialist 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 male mating aggregations (Aluja et al. 1983,

Hendrichs 1986, Robacker et al. 1991, Aluja 1994, Aluja et al. 2000, Sivinski et al.

2000).

There are revealing exceptions to the rule. In apparently 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 particular fruit at an acceptable rate and thus selection will not

favor fruit guarding. Alternatively, predation on fruit may be higher in the tropics and this

has forced males to give up fruit-based territories 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 sexual selection, and are non-resource based (Bradbury 1981,

Höglund and Alatalo 1995).

Lek mating systems are found in diverse taxa, including insects (Shelly and

Whittier 1997), fish (McKaye et al. 1990), birds (Höglund 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 family Tephritidae form leks (Shelly and

Whittier 1997). Male tephritids often aggregate and hold individual territories from which they emit chemical, acoustic and visual signals (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 leks and associated behaviors are presented in Table 1-1. In most cases males use chemical 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.

Table 1-1. List of tephritids that form leks (updated from Shelly and Whittier 1997). Species Lek Male Mc1 Mm2 Ms3 Source size signals Anastrepha fraterculus 2-8 Chemical y y ? Morgante 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/ y y y Nation 1972, Burk 1983, Acoustic 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”), and site-quality (“hotspot”) (Westcott 1994).

The female preference hypothesis proposes that females prefer mating in leks because of the advantages they gain compared to mating at solitary-male sites. Among these advantages is reduced risk of predation (Burk 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 from fruit (Hendrichs and Hendrichs 1998).

Females may also prefer leks because of the opportunity to compare adjacent males. By such a preference they reduce mate search costs and 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 fighting (Robacker et al. 1991).

The hotshot hypotheses suggest that leks form as a result of high variance in male

attractiveness. Males that are less attractive cluster around those that are successful where

they might adopt alternative, cryptic, or satellite strategies for obtaining females within

the communal display areas rather than avoiding 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 calling 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 encountered (Droney 1994). Males may use female-

emitted 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 particular parts of host trees (Sivinski 1989).

These same locations tend to be the sites occupied by sexually active males.

Males may also prefer to hold 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 long as the following day (Sivinski et al. 1994). In addition, leaf

integrity is important. Males tend to select undamaged, symmetrical leaves as lek sites.

Thus, the choice of location in a lek is not random and is probably composed of a combination of factors such as leaf size, texture and integrity, wind, light intensity, and

direct sunlight (Kaspi and Yuval 1999). It may be that there are locations on host trees

where fruits are not abundant, but which have high female densities because of favorable

microenvironments (Field et al. 2002).

Signaling. Three types of selection pressure occur in leks which all tend to increase

the elaborateness and complexity of male sexual signals: the necessity 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 (Alcock and Pyle 1979, Thornhill

and Alcock 1983, Sivinski and Burk 1989). However, species sometimes appear to

partition the signaling environment. For example, sympatric Anastrepha species call at

different times of the day (Aluja et al. 2000).

The courtship behavior of tephritids are wonderfully varied. They range in complexity from males that couple after little preliminary courtship signaling to those that produce an elaborate repertoire 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-arching body movements, and extensions of the mouthparts (Sivinski et al. 1984, Sivinski and Burk 1989). Burk (1983) and

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 (Sivinski and Wing 2004).

These signals include postures that may communicate size, and movements such as wing waving that communicate the physiological state of the signaler. However, there is no evidence that wing color-patterns function in sexual communication and sexual dimorphism in tephritid wing patterns are rare (Sivinski and Pereira 2005).

Males of sexually active lekking tephritids produce medium to long distance chemical attractants that are dispersed from pleural abdominal pouches and anal membranes that distend (Nation 1974, Fletcher 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 abdomen to the bottom surface of leaves to deposit the sex pheromone and increase the effective surface area of pheromone evaporation (Nation 1972, Sivinski et al. 1994).

Acoustical signals of tephritids may be attractive to the opposite sex and the same sex, and may be important to successful courtship 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, but 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 (precopulatory song). It is very energetic and its sound intensity was

shown experimentally to be an important factor in determining whether females would

allow singing males to copulate (Sivinski et al. 1984, Eberhard 1996). Song structure can

vary according to the situation. For example, pulse trains (episodes of wing beating)

increase in duration in the presence of males, and the interval between pulse trains

decreases when females are nearby (Sivinski and Webb 1986). While pheromone dispersal may have been the original function of wing fanning, the sounds produced by such movements, in at least some instances, have taken on a signaling significance of their own (Sivinski et al. 2000).

Nutritional status. Both male and female tephritids 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 spending his early life feeding and acquiring

energy reserves before entering the competition (Thornhill and Alcock 1983). These

sexual expenditures could justify the relatively 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 tephritids indicate that providing protein to

males in the days following eclosion advances 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).

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 and achieve significantly fewer matings compared with protein-fed males.

Juvenile hormone. In addition to nutritional 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 that 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 addition to methoprene affecting the rate of

maturation, males treated with methoprene have greater sexual performance following

maturation (Teal et al. 2000). Teal and Gomez-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 Florida since 1965 (Nation 1972). It infests over 100

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 above, it is a polyphagous species that forms leks. Behavioral

aspects of its mating system, have been described in detail (Nation 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 contribute 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 particularly important when hormones titers are

manipulated. Additionally, methoprene applications and the availability of dietary protein

may result in different nutritional conditions in A. suspensa males.

The effects of methoprene application, adult 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

13 performance on a daily and lifetime basis (Chapter 2), lek tenure, as influenced by male- male interactions (Chapter 3), male sexual attractiveness 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 methoprene application and dietary protein supply on male nutritional status (Chapter 6).

CHAPTER 2 INFLUENCE OF A JUVENILE HORMONE ANALOG AND DIETARY PROTEIN ON MALE SEXUAL SUCCESS

Introduction

Polyphagous tephritid fruit flies often have complex mating systems in which

aggregated males occupy individual territories from which they emit chemical, acoustic

and visual signals (Prokopy 1980, Burk 1981, Sivinski 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 obtain the majority of copulations (Shelly and

Whittier 1997). Differences among males in terms 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 characteristic, but more rapid

14 15

maturation can lead to cost reductions at fly 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 territorial 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 maturity 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 the effects of a juvenile hormone analog

(methoprene) application, of protein in the adult diet and their interactions on the sexual

performance of male Anastrepha suspensa (Loew), specifically male competitiveness in agonistic interactions with competing mates, attractiveness to females, and mating success. Experiments were conducted in laboratory 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 study had been in a laboratory colony at

the Center for Medical, Agricultural and 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

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 obtained 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 (Burk 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 (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) with light intensity of 550±50 lux, temperature of

25±1°C 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 PP ) + - • methoprene application and sugar as adult food (M PP ) - + • no methoprene application and sugar and hydrolyzed yeast as adult food (M PP ) - - • no methoprene application and sugar as adult food (M PP )

The methoprene was applied topically in the first 24 hours after adult emergence at

a rate of 5 μg in 1 μl acetone solution per male. In M- treatments, 1 μl 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

through the net onto the dorsal surface of the thorax, No anesthesia was used to

immobilize 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 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 (Jácome 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 screen 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 replications (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). These 13-16 days old, virgin sexually mature

males were previously marked (on the day before the experiment) with a dot of water-

based paint (different color per treatment and rotated among treatments) on the dorsal

surface of thorax (FAO/IAEA/USDA 2003). At 17:00, a 20-23 days old, sexually mature

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 males 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 male compatibility and sexual performance in tephritids

(FAO/IAEA/USDA 2003). Cages are cylindrical, 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 for 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, 20-23 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 individual vials and mating duration 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).

The abiotic conditions during the 6 days of the field cage experiment were similar.

The temperature at 17:00 was similar on the six days of experiments, 28ºC to 30ºC, and varied from 24ºC to 30ºC throughout the 2 hour period of the study (17:00 to 19:00).

Daily fluctuations were on the order of 2ºC to 4ºC. RH varied from 48% to 79%. Light intensity dropped from 1,112±135 lux at 17:00 to 291±71 lux at 19:00. The maximum light intensity was registered on October 12th at 17:00 (1,345 lux). Sunset occurred at

19:11 on October 4th (1st day of experiment) and at 19:00 on October 13th (last day of

experiment).

Sexual performance over life. This experiment was conducted in the laboratory in

individual cylindrical cages (10 cm high and 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 period of 35 days. Virgin males in cages were

provided with the adult food appropriate 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 (sugar plus hydrolyzed yeast in 3:1 proportion)

and water ad libitum. Mating and copulation duration were recorded. If mating occurred,

females were removed at the end of copulation (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

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 copula (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 interactions between methoprene and protein. These analyses

were followed by an ANOVA to detect differences 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 experiment a two-way ANOVA was performed to detect treatment and age effects. The significance value used in tests was 95% (α=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 significant effects of methoprene application

(F1,44=122.89, p<0.05), protein supply (F1,44=85.81, p<0.05), and the interaction of the methoprene application and protein supply (F1,44=16.81, p<0.05) on mating.

60 aA

50 Field cage Laboratory 40 30 Matings (%) B b 20 b B

10 cC 0 M+P+ M+P- M-P+ M-P- Treatment

Figure 2-1. Percentage of matings per treatment 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 replications in both in laboratory and field cage experiments). Different lowercase and capital letters represent significant differences among treatments, for field cage and laboratory tests respectively (Tukey’s test, α=0.05).

+ + Of the total matings, 55% were performed by M PP males, which were significantly

- - higher compared with males from all the other treatments (F3,44=75.17, p<0.05). M PP males had significantly fewer matings (5%) than other treatments in this competitive arena. Wing lengths of males that did and did 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 (F1,44=109.17, p<0.05), protein supply (F1,44=125.32, p<0.05), and interaction

of the methoprene application and protein supply (F1,44=20.04, p<0.05) on mating.

+ + The M PP males with 61 matings (59%) had significantly higher mating success

- - (F3,44=84.84, p<0.001) compared to other treatments (Figure 2-1). M PP males obtained

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 the 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

(F1,124=15.51, p<0.05), protein supply (F1,124=31.25, p<0.05), and interaction of the

+ + methoprene and protein (F1,124=5.98, p=0.016) was found. M PP males obtained

significantly more copulations (F3,124=15.57, p<0.001) than males with no methoprene

application (Figure 2-2).

60 a 50 a,b 40 b 30 b Matings (%) Matings 20

0 M+P+ M+P- M-P+ M-P- Tre a tme nt

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, α=0.05).

The earliest mating occurred on day 4 for M+P+ males, on day 5 for M+P- males

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).

25 M+P+ M+P- 20 M-P+

15 M-P-

Males (%) 10

0 23>3 Number of consecutive matings

Figure 2-3. Percentage of male Caribbean fruit flies that mated more than once over the 35 day duration of the experiment when raised 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 (F3,18=49.56, p<0.05) and adult age (F6,18=12.63, p<0.05) on mating in a daily

+ + basis. At each of the examined ages M PP 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 PP 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 PP , 1 M PP , and none of the M PP males were able to mate so often (Figure 2-5).

M+P+ 40 M+P- M-P+ 30 M-P-

10 Number of matings of Number

0 5 101520253035 Adult age (days)

Figure 2-4. Number of matings obtained per treatment at various 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±8.6 minutes and there were no significant differences among treatments

(F3,100=0.176, p=0.912). Similarly there were no differences in duration of copulation

among treatments (F3,577=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 (F3,577=0.854, p=0.465). The total of 581 matings observed in the lifetime

experiment averaged 26.9 ± 9.8 minutes.

On a daily basis, no differences in mating duration were found among treatments

(F3,415=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 (F3,415=40.30, p<0.001) (Figure

2-6).

7 6 a 5 M+P+ 4 M+P- M-P+ 3 b aaM-P- 2 a,b a,b

Number of males of Number c 1 c b bbb 0 1+1 2 3 Number of matings

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 significantly different (Tukey’s test, α=0.05). ANOVA values: 1+1 matings (F3,24=3.56, p=0.038); 2 matings (F3,24=15.74, p<0.001); 3 matings (F3,24=9.74, p<0.001).

Discussion

Both laboratory and field cage tests found a clear increase in male sexual

performance due to methoprene application, the addition of protein in the diet and the interaction of methoprene and 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%). This 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 field cages might have given females more

opportunities to exercise mate choice and more easily escape the attentions of unwanted

suitors.

a 30 a a

20 b

10 Copulation duration (min) duration Copulation n=303 n=42 n=58 n=16 0 11+12 3 Number of matings

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, α=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 Mangan (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-fed males, which reduces subsequent female remating. In our

study, protein diets improved sexual performance. This was true whether methoprene was

applied or not, although the addition of methoprene to protein resulted 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

that win territorial contests may obtain more suitable and attractive locations within leks

(Burk 1983, Thornhill and Alcock 1983, Hendrichs 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 provided separately. This

+ + effect was consistent in all the tests performed. For example, basically only M PP males were able to perform the sexual feat of mating 3 consecutive times in a 3 hour window. It seems likely that insects with an optimal diet would have the energetic capacity to exploit the physiological effects of additional methoprene.

Average mating duration was similar in field cage (coincident with Hendrichs

(1986) data under a similar protocol) and in laboratory studies (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 final copulation was significantly shorter.

Aluja et al. (2001) found no differences in the durations of copulations performed

by males fed on different diets in A. ludens, A. obliqua, and A. serpentina. On the other

hand Pérez-Staples and Aluja (2004), found that protein-fed males of A. striata have

significantly shorter copulation durations, but these males are able to mate more often.

Inconsistent results were found for diet effects on copulation duration in C. capitata.

Protein-deprived, mass reared males had longer 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 Kennelly 2002). The causes of this sort of variability are presently not understood but could be due to either male capacity or female

28 perception of male quality. In either case, Fritz (2004) found that copulation duration was positively correlated with the quantity of sperm stored by the female A. suspensa.

The improved sexual performance of males and their earlier maturation due to methoprene application could contribute enormously 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 Gomez-Simuta 2002b), appear to lead to both significantly greater daily and life-time sexual success (Teal et al. 2000).

Additionally, earlier maturation can significantly 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 untreated 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-diet and methoprene resulted in greater sexual success than either treatment alone, and for this reason 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.

CHAPTER 3 INFLUENCE OF A JUVENILE HORMONE 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 individual territories from which they emit chemical, acoustic and visual signals (Prokopy 1980, Burk 1981). Females visit these male aggregations, or

“leks”, for mating (Emlen and Oring 1977, Höglund and Alatalo 1995, Field 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, Shelly and Whittier 1997). This high variance could be due to differences among males in their attractiveness to females or their success in male-male agonistic interactions or both. 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 aggregations some of which bear only partial resemblance to classic vertebrate leks (Höglund and Alatalo 1995). This variety includes aerial swarms whose male participants typically do not engage in elaborate signals

(Sivinski and Petersson 1997), and substrate aggregations that are incompletely independent of female-required resources. Polyphagous tephritid fruit flies, such as the

Caribbean fruit fly, Anastrepha suspensa (Loew), are examples of the latter, with leks

29 30

usually forming on the foliage or in the vicinity of fruiting host trees (Sivinski et al.

2000).

Exposure to the juvenile hormone analog, methoprene at emergence accelerates

male development (Teal et al. 2000) and may lead to greater sexual success through

increased pheromone production (Teal and Gomez-Simuta 2002a). Protein consumption

during the adult stage can contribute to male gonadal and accessory glands development

and influence sexual success (Yuval et al. 1998). The greater sexual success of protein-

fed 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, Aluja et al. (2001) and Mangan (2003) found no

sexual effect of additional protein in Anastrepha ludens (Loew). The combination of

methoprene and a protein-rich diet has an additive or synergistic effect on sexual success

in A. suspensa (Chapter 2). Males appear to need adequate nutrition 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 treated with different methoprene and protein regimens. Initiation and participation in leks, male pheromone-calling, male position in the lek, male-male and male-female interactions, 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), requires the release of males that can form leks, engage in agonistic and sexual interactions, and attract wild females. I discuss whether or

31 not materials, such as methoprene and protein, that substantially improve male sexual success can be economic additions 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) USDA-

ARS, 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 obtained 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 (Burk 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 (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±1°C and a relative humidity of 55±5%.

Treatments. The study compared sexual performance of male A. suspensa

subjected to the following four treatments:

Methoprene was applied topically in the first 24 hours after adult emergence at a

rate of 5 μg in 1 μl acetone solution per male in M+ treatments. In M- treatments, 1 μl 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 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

cage 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 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 (Jácome 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 screen cages without exposure to males. They

were provided with a P+ diet, i.e., sugar plus hydrolyzed yeast (3:1) and water ad libitum.

Lek tenure in field cages. The experiment was conducted in a standard field cage used for the study of compatibility and male sexual performance 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 previous day (Sivinski et al. 1994).

In each cage 40 sexually mature, 13-16 day 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 later, 20 sexually mature virgin females, 20-

23 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 hours, temperature, relative humidity and

light intensity were measured every 30 minutes.

During the 12 days of the field cage experiment, the temperature ranged from 24ºC

to 32ºC, with a daily variation of 1ºC to 6ºC. 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. Sunsets occurred between 20:28 and 20:32.

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 first male to create an aggregation, calling or

not), male calling (indicated by the expansion 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. Mating pairs were removed to 10 ml individual vials and copulation duration was recorded.

Males that landed within 20 cm of another calling male were considered participants in a lek (Sivinski 1989). Positions in leks were divided into three tiers: central- the male is located on the middle leaves of the aggregation; surrounding- males 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 satellites- males that are adjacent and peripheral to the surrounding males. Male-male interactions normally took place when 2 males occupied the same leaf (the resident occupied the leaf and the intruder arrived and attempted to displace the resident). Typically this resulted in an agonistic 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:

mate toattempts male ofNumber ofNumber male toattempts mate index acceptance Female acceptance index = matings successful ofNumber ofNumber successful matings

Statistical analyses. Lek initiation, lek participation, males calling, and matings

were analyzed using a two-way analysis of variance (ANOVA) to detect methoprene

application effect, protein supply effect, and the interactions between methoprene and

protein. These analyses were followed by an ANOVA to detect differences between

means in the treatments. Other data was analyzed by ANOVA. If differences in means

were detected, complementary multiple comparisons of means (Tukey’s test) were performed (Ott and Longnecker 2001). The significance value used in the tests was 95%

(α=0.05). Statistical analyses were performed using R software (version 2.1.0, www.r- project.org).

Results

Initiation and participation in the aggregation. The number of leks per

replication averaged 3.4±0.8 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±1.1

males per aggregation.

Significant effects of methoprene application (F1,44=63.14, p<0.05), protein supply

(F1,44=72.84, p<0.05), and the interaction of the methoprene application and protein

supply (F1,44=19.49, p<0.05) were found in lek initiation. The percentage of males, by

treatment, initiating aggregations is presented in Figure 3-1. Of a total of 41 leks

+ + observed, 28 (68%) were initiated by M PP males (2.3±0.7 per replication), which was

- - significantly higher compared with all the other treatments (F3,44=51.8, p<0.05). M PP males initiated significantly fewer (none).

Significant effects of methoprene application (F1,44=137.84, p<0.05), protein supply

(F1,44=199.99, p<0.05), and interaction of the methoprene application and protein supply

(F1,44=31.40, p<0.05) were found in lek participation. Significant effects of methoprene

application (F1,44=64.57, p<0.05), protein supply (F1,44=84.34, p<0.05), and interaction of

the methoprene application and protein supply (F1,44=8.23, p=0.006) were found for male

calling as well.

90 80 Lek initiation Lek participation 70 a a a Males calling 60 a Mating 50 40

Males (%) b bbb 30 bb b b 20 c c 10 c c 0 M+P+ M+P- M-P+ M-P- Treatments

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, α=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, participating 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±2.6 per replication), 53% (112) were

+ + M PP males, which was significantly higher than all the other treatments (F3,44=123.1,

- - p<0.05). M 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 PP males, which was significantly higher than all the other treatments

- - (F3,44=52.4, p<0.05). M PP males had significantly fewer males (4%) calling. Calling

duration of those males that called was not significantly different among treatments

(F3,174=1.397, p=0.245). On average males spent 18.8±9.1 minutes calling in an

aggregation.

Matings. Significant effects for methoprene application (F1,44=65.32, p<0.05),

protein supply (F1,44=83.90, p<0.05), and interaction of the methoprene application and

protein supply (F1,44=25.52, p<0.05) were found. Of a total of 73 matings observed in the

+ + 12 replicates (6.3±1.0 per replication), 67% were performed by M PP males, which was

- - significantly higher than all other treatments (F3,44=57.6, p<0.05). M PP males had

significantly fewer copulations (only 1%) than all other treatments (Figure 3-1). On

average there were 6.1±1.0 matings per replication with a range of 5 to 8. All matings

were by males that were participating in aggregations. Copulation duration was not

different among treatments (F3,69=0.351, p=0.789), and averaged 27.4±5.7 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 correlated with treatment (Table 3-1). Fifty

+ + seven of the 94 center males (60.6%) were M PP , 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 PP resident males that lost 42% of their

contests.

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 copulated 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, unsuccessful mating attempts were common; 224

male-female interactions without copulation were observed. On average and across all

treatments, males attempted 3.1±0.8 matings per copulation. However, when female

+ + acceptance index were analyzed by treatment (Figure 3-2), M PP males made

significantly fewer unsuccessful attempts (2.5±0.4) than males from other treatments

(F3,24=26.561, p<0.001).

Table 3-2. Percentages of resident male Caribbean 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, α = 0.05). Intruders Residents Total M+P+ M+P- M-P+ M-P- 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, dietary protein, and the interaction of methoprene and protein significantly improved most parameters of lekking and male sexual success

(Table 3-3). From the males that initiated a lek, 78% went on to copulate, which represents a high probability of success. Lek 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

methoprene or protein resulted in higher rates of lek initiation and ultimately greater

sexual success.

6 b 5 b

4 b

3 a

1 Female acceptance index 0 M+P+ M+P- M-P+ M-P- Treatment

Figure 3-2. Female Caribbean fruit fly acceptance 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, α = 0.05).

What are the physiological and/or behavioral consequences of methoprene and

protein that resulted in this 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 PP males that participated and called in leks was significantly higher than in other treatments, however calling duration was not significantly different among treatments. That is, although other-treatment males were less likely to participate in leks

+ + those that did so spent as much time calling. On the other hand, M PP 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 PP males in agonistic encounters, particularly in

the role of resident, might allow them to spend more time in the aggregation, and all other

things being equal, be available longer to visiting females. In addition, certain territories

may be particularly valuable within the lek either because they serve as a superior

signaling platform or because females prefer them 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 PP M PP M PP M PP 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 (α=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 PP males. For one thing, M PP males had a lower female acceptance index, evidence

that they required fewer encounters on average 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 important component of the variance in male

reproductive success.

Can the relative importance of signal and residence quality to female mate choice

be determined? Hendrichs (1986) examined the sexual behavior of A. suspensa in a field

cage and found that males compete for leaves in the centers of aggregations and females

usually mate in the center as well, which was substantiated by this study. Territory

location within leks figures in two prominent theories of lek evolution; the “hotspot” and

“hotshot” models (Höglund and Alatalo 1995), both of which are consistent with the

results of this study. In the first, females choose 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 certain locations provide more protection from

predators during periods of concentration and awkward positioning.

In the second model (“hotshot”), satellite 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 model 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 compounds persist for at least 24 hours (Sivinski

et al. 1994). Thus occupation could make a territory 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 being 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 quickly surrounded by less capable satellites but

still manages to copulate. However, lek sites can be consistent over time (Sivinski 1989)

+ + and it could be that M PP 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 Caribbean fruit fly leks, it is abundantly clear

+ + from the present study that M PP males, and to a lesser extent those that receive either

methoprene or protein are more prone to initiate leks, more likely to occupy lek-centers,

better able to defend their territories and ultimately 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 the associated cost of hydrolyzed yeast or other protein sources inhibits their use in SIT programs. However, data such as presented

here would suggest that avoiding 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-reared 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 experiments 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.

CHAPTER 4 INFLUENCE OF A JUVENILE HORMONE 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 aggregations (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). Due, presumably, to variance in these

elaborate signals and behaviors male sexual 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 males, visual and acoustic signals

probably act at close range (Sivinski et al. 1984, Sivinski and Pereira 2005), but

pheromones may have both short and long-range 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 locate 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 analog, methoprene, and protein consumption

during the adult pre-sexual maturation period accelerates male development and may lead

to greater sexual success through increased pheromone production (Teal et al. 2000).

However, accelerated maturity and increased pheromone production may have nutritional consequences since there is less time for young flies to acquire reserves and these

43 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 mass-

reared flies (Kaspi and Yuval 2000). However, Shelly and McInnis (2003) found no

influence of protein in adult diet on mating success of sterile males. Thus the addition of a protein rich adult diet on sexual behaviors needs to be investigated for A. suspensa and it may have particularly important consequences 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 methoprene and protein adult 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 relative rates of female encounter experienced by

variously treated males in both the laboratory 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) USDA-

ARS, 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 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 obtained 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 (Burk and Webb 1983, Burk 1984, Webb et al. 1984,

Sivinski and Dodson 1992, Sivinski 1993). Males 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 class 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±1°C and a relative humidity of 55±5%.

Treatments. The study compared sexual performance of male A. suspensa

subjected to the following four treatments:

Methoprene, a synthetic juvenile hormone analog, was applied topically in the first

24 hours after adult emergence at a rate of 5 μg in 1 μl acetone solution per male in M+ treatments. On M- treatments, 1 μl of acetone was applied, to serve as a 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 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 was supplied to the flies. In the

+ PP 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 (Jácome 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 cylindrical 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 cages were observed until 19:00. No food or

water was supplied in the outer cage in order not to complicate female responses and no

food (only water) was supplied to the male inner cages so as not to interfere with male

attractiveness. Male pheromone signaling (calling) 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 ♀

♂♂

Figure 4-1. Arrangement of the experiment (attractiveness in laboratory). Four males in inner cylindrical cages (one per treatment, 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 the number of female visitations (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

that differential male pheromone deposition on the previous day might influence female

response (Sivinski et al. 1994). Laboratory conditions were the same as those used to maintain flies in the laboratory before the experiments (light intensity of 550±50 lux,

temperature of 25±1°C 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 greenhouse 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).

8.6 m

6.0 m

Figure 4-2. Arrangement of the experiment (attractiveness in greenhouse). Open circles represent tree canopy and filled circles represents the local where the six-male artificial leks were located (1 artificial lek per treatment).

Four artificial leks (see below) were hung within particular tree canopies each test

day. These were placed on the second and fifth tree of the second line (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 greenhouse 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 interaction 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 confound female responses. At 16:50, 100 virgin 20-

23 day old females were evenly distributed in the greenhouse (four females in each tree

canopy plus 4 on the center). Ten minutes later, 13-16 day old males were hung in their

lek-cages. The experiment was conducted over 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 depositions 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. Temperature, relative humidity and light intensity were measured every 30 minutes during the experiment

(from 17:00 to 19:00). The numbers of males calling and the number of females in the

immediate vicinity of each lek (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. Temperature, relative humidity and light intensity were measured every 30 minutes during the experiment

(from 17:00 to 19:00). The numbers of males calling and the number of females in the

immediate vicinity of each lek (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 temperature, relative humidity and light intensity.

Inside the greenhouse, the temperature ranged from 25.0ºC to 35.6ºC, with a daily

variation of 1ºC to 3ºC over the observation period. Relative 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 female visitation, both in laboratory and

greenhouse experiments, were analyzed using a two-way analysis of variance (ANOVA)

to detect interactions between methoprene and protein. These analyses were followed by

an ANOVA to detect differences between means in the treatments. 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 visitation in the greenhouse experiment. The

significance value used in tests was 95% (α=0.05). Statistical analyses were performed

using R software (version 2.1.0, www.r-project.org).

Results

Male attractiveness in the laboratory. Significant effects of methoprene

application (F1,44=72.00, p<0.05), protein supply (F1,44=81.31, p<0.05), and the

interaction of the methoprene application and protein supply (F1,44=7.51, p=0.009) on

male calling duration were found. In terms of female visitation, there were also

significant effects of methoprene application (F1,44=120.53, p<0.05), protein supply

(F1,44=104.30, p<0.05), and interaction of the methoprene application and protein supply

+ + (F1,44=28.75, p<0.05). M PP 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: F3,44=53.7, p<0.05; and female visitation: F3,44=84.5, p<0.05).

Significant effects of methoprene application (F1,44=120.14, p<0.05), protein supply

(F1,44=106.23, p<0.05), and interaction of the methoprene application and protein supply

+ + (F1,44=22.51, p<0.05) on female visitation were also found. M PP males received a

significantly higher number of female visits (1.71±0.28) when compared with the other

- - treatments (F3,44=83.0, p<0.05). M PP males had significantly fewer female visits

+ - + - (0.35±0.17) than males of the other treatments (M PP =0.75±0.24; M PP =0.71±0.18).

50 Males calling a Femele visitation 40

30 A bb 20 Duration (min) Duration

10 B c B C 0 M+P+ M+P- M-P+ M-P- Treatment

Figure 4-4. Time spent by male Caribbean fruit 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 capital for female visitation) are not significantly different (Tukey’s test, α=0.05).

Male attractiveness in the greenhouse. Significant effects of methoprene

application (F1,44=106.76, p<0.05) and protein supply (F1,44=93.59, p<0.05) were found

on males calling. However, no interaction was found between methoprene application

and protein supply (F1,44=3.13, p=0.084). In terms of female visitation, there were

significant effects of methoprene application (F1,44=88.11, p<0.05), protein supply

(F1,44=85.97, p<0.05), and the interaction of methoprene application and protein supply

(F1,44=12.59, p<0.05).

+ + In the greenhouse, M PP males called more often and were approached by females

more frequently than males in other treatments (Figure 4-5; males calling: F3,44=67.1,

- - p<0.001; and females attracted: F3,44 =62.2, p<0.001). M PP males had significantly fewer

males calling and attracted fewer females.

60 Calling males A Female visitation 50 a 40 % 30 bbBB 20 c 10 C

0 M+P+ M+P- M-P+ M-P- Treatment

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 capital for female visitation) are not significantly different (Tukey’s test, α=0.05).

Male calling frequency, regardless of treatment, was correlated with female

visitation (Figure 4-6).

2.5

1.5

1 y = 0.6082x - 0.2157 0.5 R2 = 0.7753 Female visitation

0 00.511.522.533.54 -0.5 Calling males

Figure 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.

Discussion

Laboratory and greenhouse experiments, using individual and aggregated males

+ + respectively, obtained similar results. M PP 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 treatments 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, Teal et al. 2004). Yuval et al. (1998) in field studies of

the medfly found that only males with adequate energy reserves could engage in

reproductive activities, including pheromone calling. In addition, medfly males that

called more frequently had a higher mating success (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, Aluja et al. (2001) and Mangan (2003) found no

sexual effect of additional protein for Anastrepha ludens (Loew).

While male calling is significantly higher 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 protein alone enhances calling, but without the

physiological effects of methoprene this increase 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 calling 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 the behaviors associated with pheromone signaling may not accurately predict the 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), female attraction was a direct

reflection of male signal output. In both situations 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 18- and 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 present 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 proteins as enhancers of

male qualities for use in sterile insect technique (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, Pereira et al. 2006a), and

under these conditions methoprene treatment has an additional advantage for mass- rearing. 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 and 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

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.

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 directed toward females and, occasionally, copulations with visiting females (Burk 1983, Thornhill and Alcock 1983). These forms of reproductive competition presumably have energetic costs, deplete resources and can result in physical damage such as frayed wings. Such costs, in addition to hazards like sexually-transmitted diseases and predation (Cade 1975, Tuttle and Ryan 1981, Burk

1982, Hendrichs and Hendrichs 1998), may be critical 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, Cordts 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 decrease 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) lived as long as virgin males kept alone.

58 59

Other nutritional and physiological factors are known to affect longevity of A. suspensa. Landolt and Sivinski (1992) showed that a consistent supply of sucrose was necessary to maintain male pheromone production and calling, 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 nutrition, 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 sources of nutrition following release and that this leads to greater longevity. Other influences on the longevity of mass-reared male tephritids are sterilizing-irradiation dose (Heath et al. 1994, Lux et al. 2002, Barry et al.

2003) and strain selection within colonies (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, likelihood 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 intersexual 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 with no interactions; those with daily male- male 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

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 destined 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) USDA-

ARS, 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 classes with a sorting machine (FAO/IAEA/USDA 2003). This was done to eliminate any impact of size on male competitiveness (Burk 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 10.9±0.7 mg (n=30).

Females were obtained from the next larger 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 fruits in nature (Hendrichs 1986).

After emergence and during experiments, 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±1°C and relative humidity of 55±5%.

Treatments to manipulate the intensity of sexual interactions and the availability of resources. In order to more fully reveal the roles of resource expenditure

(intensity of sexual interactions) and resource availability (presence of protein) on male survival, methoprene levels and dietary protein were manipulated. Both of these treatments are known to increase male propensity 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:

Methoprene, a synthetic juvenile hormone analog, was applied topically in the first

24 hours after adult emergence at a rate of 5 μg in 1 μl acetone solution per male in M+ treatments. In M- treatments, 1 μl 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 through the net onto the dorsal surface of the thorax, No anesthesia was used to immobilize 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 the adult diet as a 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 (Jácome et al. 1995,

Aluja et al. 2001).

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 experimental 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 and 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 laboratory in small cylindrical cages (10 cm high and 7 cm diameter) with the same abiotic conditions as described before (550±50 lux, temperature of 25±1°C 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-based paint on the upper part of the thorax to distinguish them from the focal males. Marked males were removed at 19:00. The same

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, individual 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 copula 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 given the four treatments in all three of the interaction–type group). The same was true when protein was withheld. However, for the

+ - PP treatment, survival was significantly higher than P in all interaction groups (Table 5-

1). No effect of methoprene application was observed in any of the experimental groups.

Reproductive behaviors, both with potential mates and sexual rivals were a significant source of mortality. Longevity in the three interaction groups (Figure 5-2) showed that within each treatment, males maintained alone survived significantly longer than those interacting with males or females (Table 5-1).

0.8

0.6 M+P+ 0.4 M+P- M-P+ 0.2 M-P- A

0 1234567891011121314151617181920212223242526272829303132333435 y 1

0.8 B 0.6

0.4 M+P+ M+P- 0.2 M-P+ M-P- 0 Comulative male sulvival probabilit 1234567891011121314151617181920212223242526272829303132333435

0.8 C

0.6

0.4 M+P+ M+P- 0.2 M-P+ M-P- 0 1234567891011121314151617181920212223242526272829303132333435 Adult age (days)

Figure 5-1. Cumulative survival probability of male Caribbean fruit flies in the three experimental groups (A- males alone-no interaction; B- male-male interaction; C- male-female interaction), when treated or not with methoprene (M+/M-) and + - fed or not with protein (P /PP ).

65 - P -

M p<0.05 p<0.05 z=-7.50 z=-4.89 p<0.05 z=1.30 p=0.19 ns z=-6.67 p<0.05 - z=4.53 z=4.53 value + P -

- z=1.81 z=1.81 p<0.05 z=3.19 z=3.19 z=-6.72 p=0.0014 p<0.05

z=7.86 z=7.86 p=0.071 ns - 15 P +

M - -non-significant differences p<0.05 z=-7.48 z=1.33 p=0.18 ns p<0.05 z=- z=-

Male-female interaction interaction Male-female the table; z value negative + P + ntly different if p<0.05 (z

- 6. p<0.05 z=-6.16 z=2.86 p=0.004 - P -

p<0.05 z=-6.51 p<0.05 z=-1.95 p=0.051 ns z=-7.91 p<0.05 - z=-11.60 z=-11.60 on the top of table). ns + P -

- M

z=-6.72 p<0.05 z=1.51 p=0.13 ns p<0.05 z=6.09 z=6.09

eatment located on the top of located on the top eatment - P + Figure 5.2). Pairs are significa z=-6.89 p<0.05

p<0.05 M

z=-4.64 z=-4.64 Male-male interaction interaction Male-male

+ P +

z=-8.88 p<0.05 al group/treatment located al group/treatment - P - M -

z=-3.53 p<0.05 z=1.33 p=0.18 ns z=-2.71 p=0.007 is (data on Figure 5-1 and + P -

z=-0.81 p=0.42 ns p<0.05 - z=4.03 z=4.03 - P No interaction interaction No +

M -

p<0.05 - z=-4.85 z=-4.85 + P + M - between the experimental group/treatment compared. group/treatment between the experimental represent lower survival in the experiment survival lower represent positive represent higher survival in the experimental group/tr survival in the experimental higher represent positive + - + - + - + - + - + - P P P P P P P P P P P P + + - - + + - - + + - -

M M M M M M M M M M M M

interaction interaction interaction interaction

No interaction interaction No Male-male Male-female of the survival analys Table 5-1. Results Cohorts 66

M+P+ M+P- 1 1

a 0.8 0.8 a 0.6 b 0.6

0.4 c 0.4 b No interaction b 0.2 Male-male interaction 0.2

Cumulative male survival Cumulative male Male-female interaction Cumulative survival male 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Adult age (days) Adult age (days)

- + - - 1 M P 1 M P a 0.8 0.8 a

0.6 0.6 b 0.4 0.4 b 0.2 c 0.2

Cumulative male survival Cumulative male survival c

0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Adult age (days) Adult age (days)

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 different (Cox proportional hazard model, α=0.05).

Male-male interaction effects on survival were more costly than male-female

+ - interactions, with the exception of the M PP 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 maturation) (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 interaction was almost always greater (except for

+ - M PP males where no significant differences were found). Regardless of social

interactions, protein-fed males lived longer than those that were protein-deprived. There

was no effect of methoprene on survivorship 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 wing-

produced acoustic signals. These in combination with body and wing movements

performed during close range courtships appear 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, Teal 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 amplifies 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 available during the period before

calling and the biochemical mechanisms are developed 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 courtship and mating that were not taken into

account in the present study. The considerable damage 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

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 territoriality 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 interact at an earlier age with other 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 interaction but not in the earliest days of male- female interaction.

The importance of certain nutrients is clearly 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, pheromone 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. Thus there are few non-fiscal drawbacks and

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

CHAPTER 6 INFLUENCE OF A JUVENILE HORMONE 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 addition, exposure to methoprene at emergence

accelerates male development (Teal et al. 2000) and may lead to greater sexual success

through increased pheromone production (Teal and Gomez-Simuta 2002a). In the related

medfly, Ceratitis capitata (Wied.), protein consumption during the adult stage can

contribute to male gonadal and accessory gland development and also influence sexual

success (Yuval et al. 2002).

Lipid and protein content of the body represent energy reserves and “building-

blocks” and may be related to the ability of males to sustain participation in leks, sexual

calling, male-male agonistic interactions and ultimately mating (Yuval et al. 1998). It

may also affect lifespan. The relationship between nutritional status and mass-reared fly

quality, flight-ability and survival, and sexual success has been investigated to some

extent in C. capitata. These are all key aspects in success of sterile insect technique (SIT)

programs (Fisher and Cáceres 2000, Hendrichs et al. 2002).

Adult tephritids feed on a variety 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 tephritid nutritional studies concentrated on the

70 71

utility of various diets in mass-rearing programs, 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 evaluations of the impact of larval diets on adult biology, including reproductive capacity and behavior (Kaspi et al. 2002). The impacts of adult diet and energy reserves on sexual behavior and relative reproductive 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 earlier 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 effects on survival and mating

success (Shelly el al. 2005).

Among Anastrepha species, Aluja et al. (2001) evaluated the effects of different adult nutrients, including protein 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 potential. 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 metabolites and found a lower

attraction for protein-fed males. The influence of adult diet and age on lipid reserves of A.

serpentina was quantified by Jácome et al. (1995).

Relatively little nutritional work has been done with A. suspensa. However, some

nutritional studies were conducted to investigate consumption 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 adult diets and methoprene application on lipid and protein body contents.

In this study I examine the impact of the application of the juvenile hormone

analog, methoprene, protein supply, 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 information may provide insights into the

physiological conditions that underlie male sexual performance (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) USDA-

ARS, at Gainesville, FL, for less than 3 years and were produced according 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

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 considered 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±1°C 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 PP ) + - • methoprene application in acetone solution, and sugar as adult food (M PP ) • no methoprene application, but acetone, and sugar and hydrolyzed yeast as adult - + food (M PP ) - - • no methoprene application, but acetone, and sugar as adult food (M PP ) • no methoprene or acetone application, and sugar and hydrolyzed yeast as adult food (P+) • no methoprene or acetone application 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 juvenile hormone analog, was applied topically in the first

24 hours after adult emergence at a rate of 5 μg in 1 μl acetone solution per male in M+ treatments. In M- treatments, 1 μl 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 μg in 1 μl acetone solution per male. In M- treatments, 1 μl 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

through the net onto the dorsal surface of the thorax, No anesthesia was used to

immobilize 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 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 (Jácome 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 killed in the freezer and stored at -84ºC.

Five newly emerged flies, were also collected 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 homogenization 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 Na2HPO4 in 1 l of water). The homogenate was then brought up to 4.0 ml with PBS. Lipid was extracted (Bligh and

Dyer 1959) by adding 40 mg of Na2SO4 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 the 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). Vanillin reagent (27 mg of vanillin in 4.5 ml of pure

water and 18 ml of H3PO4) 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 μl) were dried under N2 and 500 μl

of H2SO4 was added to the dried samples and standards, which were then heated for 10

min at 90ºC. A190 μl quantity of vanillin reagent was added to 10 μl of sample, standard

or blank. Lipid content was colorimetrically 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 μl were removed (1/8 of the homogenized insect),

and 100 μl of sodium deoxychoate reagent (0.15 w/v) and 100 μl of 72% (w/v) tricloroacetic acid (TCA) were added to the 500 μl 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

the supernatant was discarded. Proteins formed a pellet at the bottom of the tube, to

which 50 μl of 5% (w/v) sodium dodecyl sulfate (SDS) and 1 ml of Pierce micro BCATM

protein assay reagent were added to the pellet (Pierce 1999). After incubation in a water

bath at 37ºC for 30 min, proteins in samples 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 interactions between ages and treatments for the parameters

studied (adult weight, lipid content and protein content). These 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) were performed (Ott and Longnecker 2001).

Statistical analysis among ages includes values obtained at emergence (day zero, before treatments were applied). The significance value used in tests was 95% (α=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 (F5,192=24.46, p<0.05). However, age had

no significant effect (F7,192=1.59, p=0.140) nor did the interaction of treatment and adult

age (F35,192=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 various 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 regardless of whether or not protein was supplied.

6 Adult weight (mg) weight Adult 4

M+P+ M+P- 2 M-P+ M-P- P+ P- 0 0 5 10 15 20 25 30 35 Adult age (days)

Figure 6-1. Average (±sd) adult weight (n=5) of male Caribbean fruit flies at different ages among the six treatments featuring methoprene (M) and protein (P) and their various combinations.

Lipid contents. Significant effects of treatment (F5,192=131.37, p<0.05), adult age

(F7,192=83.14, p<0.05), and the interaction of adult age and treatment (F35,192=6.34,

p<0.05) were found. Male lipid content per treatment 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 dropped precipitously between emergence and day five (after treatment application). A significant drop in protein-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.

Protein-fed treatments at emergence, day five and day 10 were significantly higher than all the other ages.

Table 6-1. Analysis of variance (ANOVA) for male Caribbean fruit fly weight, total lipids, and total proteins among different ages in six different treatments featuring methoprene (M) and protein (P) and their various combinations (ns, non significant differences, p>0.05; * 0.01

No significant differences were found among protein-fed treatments for any of studied ages. The same was true among protein-deprived treatments. There was no effect of methoprene or acetone on total lipid contents. This implies that the two protein-type diet suites (P+ types and P- types) yielded two significantly different relationship clusters,

- + - except for age 20 when treatment M PP was not different from treatment P .

Table 6-2. Analysis of variance (ANOVA) for male Caribbean fruit fly weight, total lipids, and total proteins among treatments for different ages featuring juvenile hormone (JH) and protein (P) and their various combinations (* 0.01

350

300

250

200

150 Lipids (ug/insect) 100 M+P+ M+P- M-P+ M-P- 50 P+ P-

0 0 5 10 15 20 25 30 35 Adult age (days)

Figure 6-2. Average (±sd) total lipid contents of male Caribbean fruit flies at different adult ages among the 6 treatments featuring methoprene (M) and protein (P) and their various combinations.

Protein contents. Significant effects of treatment (F5,192=44.63, p<0.05), adult age

(F7,192=15.00, p<0.05), and the interaction of adult age and treatment (F35,192=4.77,

p<0.05) were found. Significant differences were found for each treatment among

- + different ages (Figure 6-3) (except for treatment M PP ) (Table 6-1), and among treatments

at all ages (Table 6-2).

As with lipids, P+ type and P- type diets yielded two relationship clusters. The

inclusion of protein in the diet had no effect on the protein levels of males in the M- P+ treatment at various ages, but dietary protein did have significant effect on protein levels of males at various ages in the other two protein-fed treatments (Table 6-1). All protein-

deprived males had significant age-related differences within the treatments (Table 6-1).

Different responses over time were observed. In protein-fed treatments there was an

increase in total protein after day 15 of adult life. In protein-deprived treatments there

was a significant decrease until day 10 with a subsequent increase.

900

800

700

600

500

400

Protein (ug/insect) 300

200 M+P+ M+P- M-P+ M-P- 100 P+ P-

0 0 5 10 15 20 25 30 35 Adult age (days)

Figure 6-3. Average (±sd) total protein contents of male Caribbean fruit flies at different adult ages among the 6 treatments featuring methoprene (M) and protein (P) and their various combinations.

At different ages there were always significant differences in protein content among the treatments (Table 6-2). However, no significant differences were found among

+ - + - PP and P treatments for each of the studied ages. As with lipids, P and P type diets

yielded two clusters of protein content relationships. There was no effect of methoprene

or acetone on total protein contents in either protein-deprived or protein-fed males.

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 contents, 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 occur 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 contained significantly more

protein and sugar than resting males. There were no differences in lipids (note that the

analysis calculated lipids per milligram of insect, 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 are 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 activities, 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 trust fund, whereas carbohydrates are comparable

to a readily accessible cash account.

In male A. suspensa, at least in the first 10 days of adult life of protein-fed males,

there are indications of lipogenesis. The same phenomenon occurs in C. capitata

(Warburg and Yuval 1996) and A. serpentina (Jácome et al. 1995). However, total lipid

contents decline following male Caribbean fruit fly maturation. Sharp decreases indicate

that males start to utilize their 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 (Jácome et al. 1995). This decline 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 production 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 protein-fed or protein-

deprived males when methoprene was applied. Thus lipid reserves in this species may not

play a significant role in pheromone synthesis or release.

The differences in total protein content between protein-fed and protein-deprived males may be influenced by ingested protein in the gut. However, the gradual increase in total protein contents over time in both protein-deprived and protein-fed males is both difficult to explain and inconsistent with gut protein alone accounting for the difference.

Tephritids are known to feed on feces (Prokopy et al. 1993, Epsky et al. 1997) and perhaps the consumption of their own bacteria-containing fecal materials inadvertently provided them with a protein source.

The findings of this study can have direct implications for SIT programs. The

incorporation of dietary protein has an effect on adult weight which can directly influence

male-male interactions (Sivinski 1993). The effects of lipid and protein 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.

CHAPTER 7 GENERAL DISCUSSION

Male sexual performance, in terms of lek tenure, attractiveness to females, winning

in agonistic encounters and number of copulations obtained, was significantly improved

through both applications of the juvenile hormone 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 understood in many insects and the costs and

benefits of foraging for food have been examined 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 effective 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 positive impact on longevity (Chapter 5) which is

correlated with a potential increase in offspring production.

Less well understood is the role of methoprene, 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 apparently are (i.e., as high as they are after

84 85

treatment), or perhaps more heuristically, what limits male hormone production to levels

below those that would yield more copulations? In general, there may be limits on the availability of juvenile hormone components, or there may be physiological costs to juvenile hormone production (in the present study additional juvenile hormone analog is

“free” to males), or there may be expensive physiological consequences to higher

juvenile hormone titers.

Little is known about the costs of juvenile hormone production 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 physiological consequences, these experiments

have directly addressed only a single characteristic, 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 methoprene 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 maturation. Not only are such males more sexually successful on a daily basis, they also begin their reproductive lives earlier. 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

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 anauthogeneous 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 mature sooner. Pereira et al. (2006b) saw this clearly when they found that the time under domestication affected the female pre- maturation period (mass-reared flies mature earlier 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 carbohydrates 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 chemical, visual and acoustic signals.

Regardless of the unanswered evolutionary questions posed by these experiments,

there are clear pragmatic applications for the sterile insect technique (SIT). SIT is a

principal non-chemical component of pest tephritid management (Hendrichs et al. 2002),

and improvements in production costs and insect quality influence the efficacy of the

technique and even the practicality of its use 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, prevention or suppression) should influence the strategies

adopted.

SIT is based on the release of sterile insects 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 sperm carrying dominant lethal mutations to a

large majority of females of the target populations (Hendrichs et al. 2002). For SIT to

succeed, these males must be able to join the leks 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 significantly 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 means that early sexual maturation results in more flies and in lower costs, due to space savings at fly handling facilities.

Incorporation of these experimental results into SIT programs requires that certain

technical problems be addressed. Alternative sources of less expensive protein need to be

88 identified and suitable means of their incorporation 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 topical application such as spraying 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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BIOGRAPHICAL SKETCH

Rui Manuel Cardoso Pereira received his B.Sc. Degree in Agricultural Engineering

from Lisbon Technical University (Universidade Técnica de Lisboa, Instituto Superior de

Agronomia), in Lisbon, Portugal, in 1991. He then pursued graduate 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 trapping systems in monitoring 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) control program against

Mediterranean fruit fly in the Madeira Islands, Portugal. As part of his activities at

Madeira-Med, he received training in Guatemala (1995), Hawaii and California, USA

(1996).

Since 1996 he served as chief scientific investigator of four Food and Agriculture

Organization / International Atomic Energy Agency (FAO/IAEA) coordinated research

programs: Development of female medfly attractants 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 sterile male performance in fruit fly SIT

programmes.

He entered the Ph.D. program at Entomology and Nematology Department,

University of Florida, in January 2003. His doctoral research focused on the use of a

102 103 juvenile hormone analog and dietary protein to improve male sexual performance in

Caribbean fruit fly. He received a Ph.D. in Entomology in December 2005.