POSTCOPULATORY SEXUAL SELECTION IN THE
SOLDIER FLY MEROSARGUS CINGULATUS
A dissertation presented to
The Faculty of the Graduate School
University of Missouri
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
by
FLAVIA BARBOSA
Dr. Reginald Cocroft and Dr. H. Carl Gerhardt, Dissertation Supervisors
DECEMBER 2011
The undersigned, appointed by the dean of the Graduate School,
have examined the dissertation entitled
POSTCOPULATORY SEXUAL SELECTION IN THE SOLDIER FLY MEROSARGUS CINGULATUS
Presented by Flavia Araújo Barbosa
A candidate for the degree of
Doctor of Philosophy
And hereby certify that, in their opinion, it is worthy of acceptance.
Reginald Cocroft, Ph.D.
H. Carl Gerhardt, Ph.D.
Lori Eggert, , Ph.D.
Robert Sites, Ph.D.
William Eberhard, Ph.D. ACKNOWLEDGEMENTS
This work could not have been accomplished without the support of many, many people. I am thankful for all the advice and support of my supervisors, Rex Cocroft and
Carl Gerhardt. They did an amazing job fostering my development as an independent researcher. Not only have they taught me so many necessary skills for being a successful scientist, but they also set an inspiring example. I am also thankful to my dissertation committee, Lori Eggert, Bob Sites, and especially Bill Eberhard. From the beginning of my graduate studies, Bill played a major role in my career, and I owe my success to his advice and encouragement. The days I spent conducting experiments on his compost pile at his backyard in Gamboa are one of my fondest memories of graduate school.
I have loved science from an early age, and was lucky to have had wonderful mentors who guided me towards this career. I am grateful to Airton Carrião, for teaching me how to be a scientist. Élida Rabelo, my first research mentor, was extremely influential in my life. I will be forever grateful for all the opportunities and guidance she gave me, as well as for her friendship. I thank Andrea Aspbury, Ray
Engeszer and Mike Ryan for their encouragement and help. I am also deeply thankful for
Júlio Fontenelle, my undergraduate thesis advisor. Júlio introduced me to my study system, the soldier flies, but he did so much more than that. He taught me invaluable skills and instilled me with a passion for animal behavior. I will be forever grateful to him.
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I started my graduate studies at the University of Texas, and I am grateful to
Mike Ryan for being my mentor. I was also lucky to have the opportunity to work alongside a number of talented graduate students, undergraduate researchers and postdocs. I am thankful to all the members of the Ryan lab at the University of Texas, the Gerhardt lab and the Cocroft lab, as well as several fellow graduate students who offered me their support and friendship. Karin Akre, Natalia Biani, Carlos Guarnizo, Katy
Klymus, Ashley Groves, Peter Marting, Jeremy Gibson and many, many others, thank you for all the help and all the fun moments. I am especially thankful to Abbie Green,
Ximena Bernal and Jen Hamel.
The Division of Biological Sciences has so many people who also contributed to my degree. I thank all the wonderful staff for all their help and support, but especially
Shannon Dennis, Nila Emerich, Alan Marshall, Josh Hartley and Tyeece Little. I am also grateful to Richard Daniel for the opportunity to teach Bio 1500 for several semesters and for all the fun at the Herpetology field trips. I thank the staff at the Smithsonian
Tropical Research Institute, the Organization for Tropical Studies, and Guido Berguido.
The governments of Panama and Costa Rica provided research and export permits for this work, and a number of organizations provided financial support.
This work could not have been made without the love and support of my family and friends. Para minha mãe, obrigada por uma vida inteira de amor incondicional. Por toda a minha vida, você fez o possível e o impossível para me ajudar a conquistar meus sonhos, e eu não estaria aqui se não fosse por você. Eu também sou gratas às minhas
iii irmãs maravilhosas. Joyce, obrigada por uma vida inteira de amizade, e pelas conversas quase diárias durante todo o meu doutorado. Janaina e Vitória, obrigada por fazer minha vida mais feliz. I am thankful for all my friends, especially Rafaela, Leticia and
Aninha. I am also immensely grateful for my American family. They have generously welcomed me into their family, and thanks to them, I feel like this country is also my home. Thank you Mary Lou, Walt, Rebecca, Elizabeth, Grandma Gerstle, Grandma and
Grandpa Reichert.
Finally and most importantly, I am fortunate that my graduate career led me to finding the person with whom I share my life. To Michael, there are no words to thank you for being part of everything I do. Thank you for the love, the encouragement, the constant companionship. Thank you for revising countless versions of the same manuscript and for all the statistical consulting. You had an immense influence in this dissertation in so many ways. Thank you for being my partner in life.
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TABLE OF CONTENTS
Acknowledgements ...... ii
List of figures ...... vii
List of tables ...... viii
Abstract ...... ix
CHAPTERS
1. Introduction ..…..…….………………………………………………….……………………………………..01 Sperm competition……………………………………………………………………………………………02 Cryptic female choice……………………………………………………………………………………….05 Cryptic male choice………………………………………………………………………………………….07 Study system…………………………………………………………………………………….………………08 Figures………………………………………………………………………………………………………………11 References……………………………………………………………………………………………….……….13
2. Cryptic female choice by female control of oviposition timing in a soldier fly .... 21 Introduction…………………………………………………………………………………………..…………22 Methods………………………………………………………………………………………………………..…24 Results…………………………………………………………………………………………………………..…29 Discussion……………………………………………………………….…………………………………….…30 Tables………………………………………………………………………………..………………………….…34 Figures…………………………………………………………………………………………………………..…36 References………………………………..…………………………………………………………………..…37
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3. Copulation duration in the soldier fly: the roles of cryptic male choice and sperm competition risk ………………………………….………………………………………………….……….……40 Introduction………………………………………………………………………………………..……………41 Methods…………………………………………………………………………………………..………………45 Results……………………………………………………………………………………………..………………47 Discussion……………………………………………………………….……………………….………………48 Figures………………………………………………………………………………………….….………………52 References………………………………..……………………………………………………..………………56
4. Males responding to sperm competition cues have higher fertilization success in a soldier fly …………………….……………………………………………………………………….……….….…61
Introduction………………………………………………………………………………………..……………62 Methods……………………………………………………………………………………………..……………65 Results…………………………………………………………………………………………………………..…69 Discussion……………………………………………………………….…………………………………….…71 Tables………………………………………………………………………………..………………………….…75 Figures…………………………………………………………………………………………………………..…77 References………………………………..…………………………………………………………………..…80
5. Discussion ...... 85 Summary of results……………………………………………………………………………………..……85 Future directions …………………………………………………………………………………………..…87 References………………………………..………………………………………………………………………90
Vita ...... 93
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LIST OF FIGURES
CHAPTER 1
Figure 1…………………………………………………………………………………………………………….11
Figure 2…………………………………………………………………………………………………………….12
CHAPTER 2
Figure 1…………………………………………………………………………………………………………….36
CHAPTER 3
Figure 1…………………………………………………………………………………………………………….52
Figure 2……………………………………………………………………………………………..……………..53
Figure 3…………………………………………………………………………………..………………………..54
Figure 4…………………………………………………………………………………………………………….55
CHAPTER 4
Figure 1…………………………………………………………………………………………………………….77
Figure 2…………………………………………………………………………………………………………….78
Figure 3……………………………………………………………………………………..……………………..79
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LIST OF TABLES
CHAPTER 2
Table 1…………….……………………………………………………………………………………………..34
CHAPTER 4
Table 1…………….……………………………………………………………………………………………….75
Table 2…………….……………………………………………………………………………………………….76
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ABSTRACT
My dissertation research focuses on the largely understudied field of postcopulatory (or “cryptic”) mate choice. For the past few decades, it has become increasingly clear that both males and females can perform mate choice during and after copulation, but the implications of this for sexual selection and evolution have been little explored. As part of my dissertation research, I have developed a novel model system for the study of postcopulatory mate choice, the soldier fly Merosargus cingulatus (Diptera: Stratiomyidae). The soldier flies have proven to be an excellent study organism, since they are extremely tractable for field experiments, which I conducted at the Smithsonian Tropical Research Institute field station in Gamboa,
Panama, and at the Organization for Tropical Studies field station in La Selva, Costa Rica.
The vast majority of studies of postcopulatory sexual selection have been conducted in the laboratory, and as a consequence, little is known about the effects of postcopulatory sexual selection on natural populations. By performing field experiments, I was able to obtain results that are more relevant to natural populations than those of lab experiments.
I have demonstrated that cryptic female choice occurs in M. cingulatus through a novel mechanism: female control of oviposition timing. In this study, I experimentally manipulated males’ ability to perform copulatory courtship. I showed that females are less likely to oviposit immediately after mating when a male does not perform
ix copulatory courtship than when he does. I also developed AFLP profiles to perform paternity analysis in this species. Through parentage analysis, I determined that last male sperm precedence occurs in this species. These results show that failure to oviposit before remating by the female decreases the previous male’s reproductive success. This study was the first demonstration of cryptic female choice in the field in any species.
In another study, I tested different hypotheses that can explain variation in copulation duration in M. cingulatus . Different factors can affect copulation duration, and one of them is the social environment: males are expected to prolong copulation duration in situations where the risk of sperm competition is high. I demonstrated that this occurs in M. cingulatus . In addition, I demonstrated that cryptic male choice occurs in this species as well. While the prevalent view is that in most animal species, females choose among competing males, male choice is expected to occur whenever there is sufficient variation in female quality and male mating effort is high. Postcopulatory or
“cryptic” male choice is defined as variation in the amount of resources males allocate to females of varying quality. If resources such as time, energy or sperm are limited to males, they are expected to selectively allocate them to females that will maximize their reproductive success. In M. cingulatus , males selectively allocate resources, investing more in copulations with larger females.
Since I found that males increase copulation duration when the risk of sperm competition is high and when they mate with larger females, I also tested the hypothesis that longer copulations result in higher reproductive success for males. I
x found no effect of copulation duration on the number of eggs laid by females. However,
I performed parentage analysis using AFLP profiles, and found that fertilization success is influenced by copulation duration: males that mated for longer fertilized a higher percentage of the clutch than those with shorter copulations. This is only the second study to show a direct effect of sperm competition risk on reproductive success, and the first to do so in a field population. These results have been submitted for publication.
My dissertation work is an unusually complete study of postcopulatory sexual selection, since I have examined cryptic female choice mechanisms, selected traits, and their interactions with cryptic male choice in a single species.
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1. Introduction
When Darwin defined sexual selection as the process that leads to variation in reproductive success between individuals due to variation in their ability to obtain mates, he assumed that females were monogamous. (Darwin 1871). Because of that, early studies of sexual selection focused only on processes happening prior to copulation. However, more recent studies have accumulated a vast amount of evidence that polyandry is extremely common, and taxonomically widespread (Birkhead and
Moller 1995, Jennions and Petrie 2000, Zeh and Zeh 2001, Arnqvist and Nilsson 2000). If females mate multiply, the number of mates a male obtains does not necessarily translate into reproductive success; thus, it soon became apparent that processes happening during or after copulation could play an important role in sexual selection.
These processes are deemed postcopulatory sexual selection (although this term encompasses processes that occur both during and after copulations) (Birkhead and
Moller 1998, Eberhard 1996).
Sexual selection has two main components, intrasexual selection, or male-male competition, and intersexual selection, or mate choice. Similarly, postcopulatory sexual selection has an intrasexual and an intersexual component: sperm competition is the postcopulatory equivalent of male-male competition, while cryptic mate choice corresponds to mate choice (figure 1). Sperm competition theory was first proposed in
1970 by Geoffrey Parker and was quickly accepted as an important process shaping male physiology, morphology and behavior, as well as an almost ubiquitous process
1
among animals (Parker 1970, Birkhead 1998, Simmons 2001). Cryptic female choice, on the other hand, did not receive as much attention at first, and the role it plays in sexual selection was controversial until recently (Birkhead 1998, Birkhead 2000, Kempenaers et al. 2000). Research on cryptic female choice is considerably less advanced than research on sperm competition, but its importance for sexual selection has finally been acknowledged, thanks largely to an extensive review by William Eberhard (1996). Even more recently, it was suggested that males also perform postcopulatory choice by allocating more resources to females that will result in higher reproductive success
(Bonduriansky 2001). This cryptic male choice has since been demonstrated to also play an important role in sexual selection. Here I will discuss these three different aspects of postcopulatory sexual selection.
SPERM COMPETITION
Sperm competition was defined by Parker (1970) as competition between the sperm of multiple males in the reproductive tract of a single female for the fertilization of the ova. It is analogous to precopulatory male-male competition and since it was proposed, its importance for sexual selection has been strongly acknowledged.
Mechanisms through which sperm competition can occur and its role in shaping male behavior, morphology and physiology have been demonstrated in many taxa (e.g.
Simmons 2001, Birkhead and Moller 1998).
Males use different mechanisms in sperm competition, but these mechanisms mainly fall into two categories: offensive mechanisms have to do with males
2
outcompeting males the female has previously mated with, while defensive mechanisms are aimed at keeping the female from remating (Parker and Pizzari 2010). Sperm competition models seek to find evolutionary stable strategies (ESS) of sperm use. A common offense strategy is to increase ejaculate size: for raffle models, where sperm from different males is thoroughly mixed inside the female genital tract and each sperm has an equal chance of fertilizing the ova, the ESS is to increase ejaculate expenditure when the risk of sperm competition increases (Parker et al. 1997, reviewed by Wedell et al. 2002, Parker and Pizzari 2010). These models assume that females will either mate once or twice, and the risk of sperm competition is the probability that a given female will remate (i.e., the probability that sperm competition will occur). This prediction is strongly supported by empirical data, both across and within species. For example, comparative studies show that in vertebrates, testes size correlates with the female level of polyandry (Harcourt et al. 1981). Within species, males have been shown to increase ejaculate size when male density is high at the time of copulation, which males presumably perceived as an increased risk of sperm competition (Gage and Barnard
1996, Gage 1991, Ramm and Stockley 2007, Wedell and Cook 1999).
In some sperm competition situations, however, males are not expected to increase ejaculate sizes. Intensity models relate to species with high levels of sperm competition, often external fertilizers that are group spawners. Sperm competition intensity refers to the number of ejaculates from different males competing within a female’s genital tract. The ESS in this case is to transfer the least sperm when there is no sperm competition, to transfer the most sperm when only two males are competing,
3
and then decrease ejaculate size as sperm competition intensity increases, since in these situations males will obtain diminished returns from their investment in ejaculates
(Parker 1996). These models have also found strong empirical support (Fuller 1998,
Simmons and Kvarnemo 1997, Wedell and Cook 1999).
In addition to strategic sperm allocation, many other sperm competition strategies have evolved. For example, some males can displace or even actively remove the sperm of rivals from the female genital tract. For example, males of the damselfly
Calopteryx maculata use structures in their penises to “scoop out” sperm from the females’ spermathecae, before depositing their own ejaculate (Waage 1979). Sperm morphology is also affected by sperm competition, favoring faster designs: comparative studies in mammals and birds have shown that sperm cells are longer in species where sperm competition is higher; longer sperm cells are faster (Gomendio and Roldan 1991,
Briskie et al. 1997).
Sperm competition defense mechanisms include mate guarding, when the male remains with the female after copulation to keep her from mating with additional males
(Alcock 1994). Males may also produce a mating plug, which blocks the female genital tract (Thornhill and Alcock 1983, Wedell 2007). Finally, they may transfer ejaculates containing anti-aphrodisiac compounds to the female, which decrease female receptivity (Wedell 2005, Wolfner 1997).
4
CRYPTIC FEMALE CHOICE
The idea that females could influence offspring paternity after mating was first suggested by Thornhill (1983) and is referred to as cryptic female choice. It is defined as a female-controlled, postcopulatory process that biases paternity towards males that have a preferred trait over males that lack that trait, when the female has mated with both (Eberhard 1996). The word “cryptic” here refers to the fact that, originally, female choice was believed to have ended when copulation begins. Female choice happening during or after copulation is then “invisible” from that point of view. It does not mean that choice is “cryptic” because it occurs inside the female’s reproductive tract: in fact, cryptic female choice often happens outside of the female body and is often readily observable (Eberhard 1996).
Relative to sperm competition, cryptic female choice has received less attention, since its role in sexual selection was initially seen as controversial (Birkhead 1998,
Birkhead 2000, Kempenaers et al. 2000). One of the reasons for this is that cryptic female choice is more difficult to study and demonstrate than sperm competition. It is even more subtle than precopulatory female choice, since sometimes it is a process happening inside the female’s genital tract. It is also difficult to separate it from sperm competition effects. Another problem is that many times cryptic female choice studies require knowledge on the mechanisms of insemination, sperm storage in females and fertilization, which are not available for many species.
Eberhard (1996) extensively reviewed various mechanisms through which cryptic female choice can occur. Although indirect evidence suggests there may be over 20
5
mechanisms, there is experimental evidence for only a few. Other potential mechanisms have not been demonstrated, but are likely to be widespread. Prefertilization mechanisms include selective sperm discharge, selective interruption of copula
(resulting in decreased sperm transfer), biasing movement of sperm inside the genital tract, storage of sperm in locations where it is more likely to reach the egg or less likely to be removed by other males, selective failure to ovulate and selective remating.
Postfertilization mechanisms include selective production of less offspring, selective abortion and biased offspring investments. There is evidence that these mechanisms are widespread in many different taxa, which is consistent with the occurrence of cryptic female choice. However, there are few studies where a direct link between these mechanisms and female choice was made (see chapter 2). This is partly due to the difficulty of disentangling the role of males and females in controlling fertilization. In addition, in studies of cryptic female choice, it is often difficult to determine what male features the female is assessing and selecting.
Cryptic female choice has important evolutionary consequences for both males and females. As in precopulatory female choice, it plays a role in shaping behavior and morphology of both males and females. Perhaps one of the best examples of a trait that probably evolved as a result of cryptic female choice is copulatory courtship, or courtship that occurs during copulation. This behavior might seem paradoxical at first, since it occurs after a male has already obtained his mate. However, studies have shown that copulatory courtship affects several postcopulatory female processes, such as sperm usage, oviposition timing, sperm dumping and remating (Eberhard 1991, Tallamy
6
et al. 2002, Barbosa 2009, Edvardsson and Arnqvist 2000, Peretti and Eberhard 2010).
Copulatory courtship is a widespread behavior in arthropods: a survey of 131 species found that it occurs in 81% of them. In addition, in over 80% of the species where copulatory courtship was observed, the male abandoned the female after mating, suggesting that the behavior was not aimed at having the female remate with that male, and supporting the idea that copulatory courtship is aimed at cryptic female choice
(Eberhard 1994).
CRYPTIC MALE CHOICE
The cost of sperm production is not as trivial for males as it was originally thought:
while single sperm might be cheap for males to produce, large amounts of it are not
necessarily so (Dewsbury 1982, Tang-Martinez and Ryder 2005). Although male
investment in reproduction is lower than that of females, reproduction is still costly for
them: in addition to producing sperm, males often produce large ejaculates containing
nutrients and other seminal fluids (Sakaluk 1985, Oberhauser 1988, Fox et al. 1995,
Wagner 2005), spend time and energy in courtship and copulation (Vehrencamp et al.
1989, Prestwich 1994), and provide females with direct benefits such as territory or
food (Vahed 1998), and paternal care (Gross and Sargent 1985, Tallamy 2000).
If a male’s number of copulations is limited by resources allocated for
reproduction rather than the availability of potential mates, it can maximize its
reproductive success by having mating preferences. It will benefit from selectively
allocating resources to higher quality females. Male choice can be precopulatory, when
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males refuse to mate with low quality females, and postcopulatory, which is referred to as cryptic male choice (Bonduriansky 2001).
When cryptic male choice occurs, males are expected to selectively allocate more resources to more fecund females. These resources could include ejaculate, time and energy spent mating, and paternal care. Often, larger females are more fecund, and in several species males transfer more sperm, copulate for longer, or provide larger nuptial gifts when mating with large females (Gage and Barnard 1996, Gage 1998, Parker et al.
1999, Engqvist and Sauer 2001, Reinhold et al. 2002, Cornwallis and O’Connor 2009, Xu and Wang 2009).
Cryptic male choice also includes male responses that relate to sperm competition: female age and mating status can influence male reproductive success, and males should prefer to mate with virgin females, to avoid sperm competition. If only mated females are available, however, then sperm competition is inevitable and males should transfer more sperm (Parker 1998). In natural populations where females mate multiply, age and mating history are likely to be correlated: the older a female, the more times she is likely to have mated. This means that males may detect female mating status by assessing it directly, or by assessing female age (Wedell and Ritchie 2004).
STUDY SYSTEM
Arthropods constitute excellent model organisms for studies of postcopulatory sexual selection. Postcopulatory sexual selection in arthropods has been widely described (Eberhard 1996, Simmons and Siva-Jothy 1998) and it is known to play an
8
important role in shaping both morphology (Burger et al. 2003, King Sirot 2003) and behavior (Eberhard 1994) in many species. The soldier fly Merosargus cingulatus
(Diptera: Stratiomyidae) is especially tractable for answering questions in this area. They are easy to manipulate and study in the field, which allows studies that address their behavior in nature. This is particularly important, since few studies in postcopulatory sexual selection have been conducted in the field.
Merosargus is a neotropical genus which contains over one hundred species
(James and McFadden 1971). They are morphologically and ecologically diverse, being commonly found in a variety of habitats, such as the rainforest, suburban residential areas and in city parks (Woodley 2001). Even though this is a diverse and abundant genus, very little is known about its biology and natural history. The larvae are detritivores, and feed on a variety of decomposing plant matter. Several species develop in flower parts of Heliconia plants (Seifert & Seifert 1976; Seifert & Seifert 1979), others are commonly found in a variety of decomposing fruits, recently cut grass, and fallen stems of succulents; a few species develop in animal fecal matter (Woodley 2001).
Adults aggregate around this larval substrate, where females oviposit and males often defend territories. Very little is known about the foraging habits of the adults.
In Merosargus cingulatus , copulations occur at oviposition sites such as piles of rotting fruit or recently cut grass, in which the larvae develop, and where males patrol and defend a territory. Males will defend their territory from any insect that approaches, including heterospecifics (personal observation). They do so by chasing the intruder; if the intruder is a conspecific, the interaction often escalates. First, the males
9
will collide against each other; then, one of the males may use his legs to capture the other one and carry him away from the territory. He then releases that male and returns
(personal observation).
Oviposition sites attract large numbers of males and females. Male territories are relatively small, with neighboring males typically about ten to fifteen centimeters apart. The male attempts to grab and copulate with any female that flies near his territory. Females do not appear to have any opportunity to choose a mate, and do not seem to resist mating once they are grasped by a male. It is unclear if females come to the territories primarily for mating or oviposition. Males perform copulatory courtship throughout the copulation. Copulatory courtship involves two distinct behaviors: males tap the female’s abdomen with their hind legs, and wave their legs in the air. It lasts for the entire duration of copulation, and although there is considerable variation in copulation duration, there is very little variation in the rate of tapping and waving behaviors (Barbosa 2009).
After mating, females usually lay eggs deep in the rotting vegetable matter in or close to the male’s territory. When females leave a male’s territory without ovipositing, they will likely mate again before they have the chance to oviposit elsewhere (personal observation). Male density at the oviposition sites is high and if a female is detected by a male, he will likely grab her and mate (personal observation). Observations of marked animals show that both males and females mate multiply in the field (personal observation).
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FIGURES
Precopulatory Postcopulatory sexual selection sexual selection
Male-male Mate Sperm Cryptic mate competition choice competition choice
Figure 1 – The intrasexual and intersexual components of sexual selection before and after copulation.
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(a)
(b)
Figure 2 – Merosargus cingulatus larva (a) and adult (b).
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2. Cryptic female choice by female control of
oviposition timing in a soldier fly
ABSTRACT: There is substantial evidence that cryptic female choice (CFC) is present in
numerous taxa. Several mechanisms have been proposed for CFC; however, we only
have experimental evidence for a few of them. Female control of oviposition timing is a
potentially widespread mechanism of CFC, but it has never been experimentally
demonstrated. The aims of this study are to test two critical predictions of the
hypothesis that CFC through control of oviposition timing occurs in the soldier fly
Merosargus cingulatus : (1) to determine if M. cingulatus females are less likely to
oviposit immediately after mating when the male does not perform copulatory
courtship than when he does; and (2) to determine if failure to immediately oviposit by
the female results in lower reproductive success for the male she just mated with. To
answer the first question, I compared the oviposition behavior of females that mated
with control males versus females that mated with manipulated males that could not
perform copulatory courtship. I showed that M. cingulatus females fail to oviposit immediately following copulation when males do not perform copulatory courtship. To answer the second question, I showed that there is last male sperm precedence in M.
cingulatus . Since the last male to mate fertilizes most of the female’s eggs, a male will
benefit when females oviposit immediately after mating with him and before remating
with another male.
21
KEY WORDS: copulatory courtship; cryptic female choice; oviposition timing;
postcopulatory sexual selection; soldier fly; Stratiomyidae.
INTRODUCTION
Postcopulatory sexual selection is potentially important in polygamous species,
where the number of copulations by males does not necessarily predict their
reproductive success. There are two primary mechanisms: sperm competition and
cryptic female choice (CFC). CFC is a female-controlled, postcopulatory process that
biases paternity towards males that have a preferred trait over males that lack that trait,
when the female has mated with both (Eberhard 1996). Whereas there is abundant
evidence for sperm competition (Birkhead and Møller 1998, Simmons 2001), there are
far fewer studies that provide direct evidence for CFC (see table 1). However, there is
substantial indirect evidence for CFC in numerous taxa (Eberhard 1996).
Several potential mechanisms have been proposed for CFC (Eberhard 1996).
Although indirect evidence suggests there may be over 20 mechanisms, there is
experimental evidence for only a few: female adjustment of the number of eggs laid,
female control of copulation duration and sperm transfer, and internal sperm
manipulation by the female (see table 1). Other potential mechanisms have not been
demonstrated, but are likely to be widespread. One such mechanism is female control
of oviposition timing. A female can bias paternity towards a male by ovipositing soon
after mating with him and before she mates with another male, if last male sperm
precedence occurs (i.e., the last male to mate with a female fertilizes most of her eggs).
22
This means that if a female mates with a second male before ovipositing, she will greatly decrease the first male’s reproductive success. Female control of oviposition timing is a potentially important mechanism of CFC, since last male sperm precedence is widespread among insects (Thornhill and Alcock 1983). However, there are no known examples of CFC through oviposition timing. The goal of this study is to experimentally test the hypothesis that CFC by female control of oviposition timing occurs in the soldier fly Merosargus cingulatus (Diptera: Stratiomyidae).
In M. cingulatus , copulations occur at oviposition sites such as piles of rotting fruit or recently cut grass, in which the larvae develop, and where males patrol and defend a territory. Oviposition sites attract large numbers of males and females. Male territories are relatively small, with neighboring males typically about ten to fifteen centimeters apart (personal observation). The male attempts to grab and copulate with any female that flies near his territory. Females do not appear to have any opportunity to choose a mate, and do not seem to resist mating once they are grasped by a male. It is unclear if females come to the territories primarily for mating or oviposition. Males perform copulatory courtship throughout the copulation. Copulatory courtship involves two distinct behaviors: males tap the female’s abdomen with their hind legs, and wave their legs in the air.
After mating, females usually lay eggs deep in the rotting vegetable matter in or close to the male’s territory. When females leave a male’s territory without ovipositing, they will likely mate again before they have the chance to oviposit elsewhere. Male density at the oviposition sites is high and if a female is detected by a male, he will likely
23
grab her and mate (personal observation). Observations of marked animals show that both males and females mate multiply in the field (personal observation).
The aims of this study are to test two critical predictions of the hypothesis that
CFC through control of oviposition timing occurs in M. cingulatus : (1) to determine if
females are less likely to oviposit immediately after mating when the male does not
perform copulatory courtship than when he does; and (2) to determine if failure to
immediately oviposit by the female results in lower reproductive success for the male
she just mated with. To answer the first question, I conducted a field experiment where
I compared the oviposition behavior of females that mated with control males versus
females that mated with manipulated males that could not perform copulatory
courtship. I showed that M. cingulatus females fail to oviposit immediately after
copulation when males do not perform copulatory courtship. To answer the second
question, I tested for last-male sperm precedence using AFLP markers. I showed that the
last male to mate with a female fertilizes most of her eggs. This means that a male will
benefit when females oviposit immediately after mating with him and before remating
with another male.
METHODS
Copulatory courtship
All field observations and experiments were conducted at the Smithsonian
Tropical Research Institute field station in Gamboa, Panama, in August 2006 and in July
2007. I set up piles of decomposing fruit peels in open areas by small patches of forest in
24
a residential area. Both male and female M. cingulatus were attracted to the fruit. I captured a total of 62 males. Half of them were treated so that they could not perform copulatory courtship by applying black acrylic paint to their hind legs. This treatment made the legs stiff and prevented normal movement during copulatory courtship; I did not observe any other change in behavior caused by this treatment, and they obtained mates as readily as untreated males. The other half of the males received the control treatment: they were caught and handled like the manipulated males, but their hind legs were not painted, allowing for normal movement during copulatory courtship. All males were uniquely marked on the thorax with acrylic paint to allow individual identification and were released immediately after being processed. When males returned to their territories, I observed them until they mated. Not all of the marked males returned to their territories and mated; I only collected data from the ones that did so. I filmed the copulation and the female behavior until she left the oviposition site, and then collected the male. I allowed each male to mate only once.
Sperm precedence
I collected mated pairs in the field and raised their offspring for paternity analysis in order to assess last male sperm precedence. Since all individuals were field caught, their previous mating history is unknown. I observed copulations and afterwards watched the female as she oviposited. I then collected the male and female and the piece of fruit on which she had oviposited. Since the fruit peels came from fresh fruit and I allowed them to decompose in sealed enclosures, the only eggs present were
25
those laid by the female that I observed mating. I allowed the eggs to develop to three week old larvae and preserved the adults and these larvae in 100% ethanol. I collected a total of nine “families” (the mated pair and the larvae resulting from that clutch), which had an average of 22.77 offspring per family. Twenty offspring per family were randomly picked for the paternity analysis for the families that had more than 20 offspring; all offspring were analyzed in four families with fewer than 20 (number of larvae = 7,8,19, and 19).
DNA extraction
I extracted DNA from the tissue from flight muscles and legs of adults. For the larvae, I removed the head and used the rest of the body. I froze the tissues with liquid nitrogen and macerated them with a lysis buffer (250 mM tris pH 7.5, 2M NaCl, 100mM
EDTA, 2.5% SDS and 2% proteinase K), incubating for two hours at 58°C. The DNA was purified with a 10% CTAB solution, a 24:1 chloroform: isoamyl alcohol solution, and 5M ammonium acetate. Samples were then treated with a 100 ug/mL solution of RNAse A for 30 minutes at 37°C. DNA was precipitated with ethanol and resuspended in low TE
(0.5M EDTA, 1M Tris-HCl). DNA samples were quantified with a NanoDrop N-1000 spectrophotometer and diluted to a final concentration of 50 ng/uL.
AFLP
Generating AFLP markers involved four steps. First, I digested the DNA with two restriction endonucleases, EcoR1 and Mse1. The fragments generated were then ligated
26
to adaptors, which are small DNA fragments with sticky ends complementary to the enzyme’s cutting sites. These two steps were combined in a single reaction. The fragments were then submitted to two PCR reactions: the preselective and the selective amplifications, where the adaptor sequence is used to build the primers. The purpose of the PCR reactions is to amplify only a subset of the fragments by adding one to three arbitrary bases to the primer sequence. The primers used in the preselective PCR have one base pair added, and the selective PCR primer can have up to three bases added to its sequence.
The protocol used was adapted from Vos et al. 1995. First, 50 ng of DNA were used in a restriction-ligation reaction (1x T4 DNA ligase buffer with ATP, 0.05M NaCl,
5ug/mL bovine serum albumin, 5 units Eco RI, 1 unit MseI, 1U T4 ligase, 50 uM MseI adaptor, 5uM EcoRI adaptor), and incubated at 37°C for two hours. The adaptors were synthesized by Integrated DNA Technologies. The sequence of the adaptor was, for
EcoR1 forward: 5’-CTC GTA GAC TGC GTA CC-3’; reverse: 5’- AAT TGG TAC GCA GTC TAC
-3’; and for MseI forward: 5’-GAC GAT GAG TCC TGA-3’; and reverse: 5’-TAC TCA GGA
CTC AT-3’. The second step was a preselective amplification. The product from the reaction above was diluted 1:10 with low TE and 2.5ul of it was combined with 1.5U of
Taq DNA polymerase, 20ul of 10x Taq DNA polymerase buffer, 1uL of the preselective amplification primer mix and 0.5ul of 10mM dNTP solution, and this solution was brought to 20ul with water. The PCR began with a cycle of 72°C for two minutes, followed by 30 cycles of 94°C for 30 seconds, 56°C for 30 seconds and 72°C for two minutes, and a final cycle of 60°C for 10 minutes. The structure of the preselective
27
primers used were 5’-GAC TGC GTA CCA ATT CT-3’ for the EcoR1 primer, and 5’-GAT
GAG TCC TGA GTA AC-3’ for the Mse1 primer, and they were both synthesized by
Integrated DNA Technologies. The amplification product was diluted 1:5 with low TE and used in a selective PCR. The selective PCR used 1uL of 10x Taq DNA polymerase buffer,
0.75U of Taq DNA polymerase, 2pmols of the Mse1 selective primer, 0.4 pmols of the
EcoR1 selective primer, 1.5uL of the diluted reaction and 0.25ul of 10mM dNTP solution.
This mixture was brought to a volume of 10ul with water. One primer pair combination was identified from a set of eight primer pairs as the most polymorphic, and therefore most informative. The Mse1 primer had three selective nucleotides added. It was synthesized by Integrated DNA Technologies and its structure is 5’-GAT GAG TCC TGA
GTA ACA T-3’. The EcoR1 primer had two selective nucleotides added and was fluorescently labeled with 6FAM dye. Its sequence is 5’-GAC TGC GTA CCA ATT CTG-3’ and it was synthesized by Applied Biosystems. Fragments were separated by gel electrophoresis and detected by an ABI 3730 DNA Analyzer, which uses an automated fluorescence-based detection system. Fragment size was accurately detected by the addition of an internal lane size standard that was also fluorescent marked (Genescan
600 LIZ).
AFLP Analysis
Profiles were visualized and scored for presence/absence of fragments ranging from 60 to 400 bp using Softgenetics GeneMarker software. For the paternity analysis, I used Famoz software (Gerber et al. 2003) to calculate the average exclusion
28
probabilities and to determine paternity of each offspring through categorical allocation. The LOD score (logarithm of the likelihood ratio) of each father-offspring pair was calculated and used to determine if the males analyzed were the fathers of the offspring of the females they mated with. The exclusion probability equations for dominant markers can be found at Gerber et al. 2000.
RESULTS
Copulatory courtship
None of the ten manipulated males performed copulatory courtship. Of the ten females that mated with manipulated males, none oviposited after mating; all flew away soon after copulation ended. All of the 13 control males performed copulatory courtship and all 13 females that mated with controls oviposited (difference in oviposition behavior of females with control vs. manipulated males: Fisher’s exact test, p<0.005). There was no significant difference in the duration of copulation between manipulated and control males (Control group: 95.93 ± 43.97 seconds (x±SD); manipulated group: 121.10 ± 59.49. Two-tailed t-test, p=0.245). Samples were checked for normal distribution (Shapiro-Wilk, p control = 0.558; p manipulated = 0.745).
Sperm precedence
The AFLP selective primer pair used generated 127 polymorphic loci, with an average exclusion probability of 0.996. In the nine families analyzed, average p 2, or
percentage of offspring sired by the last male, was 0.839 (range 0.55 to 1) (Fig.1). Since
29
the females used in the analysis were field caught and their mating history is unknown, it is possible that in the three cases where p 2 was 1, those females only mated once. The average p 2 when these females are excluded from the analysis is 0.759 (one-sample t-
test, null hypothesis: p 2=0.5, t=4.317, p=0.008). In either case, these results show that there is last male sperm precedence in M. cingulatus .
DISCUSSION
Cryptic female choice is a female-controlled process that biases paternity towards males that have a preferred trait, after having mated with males with and without the trait. In M. cingulatus , male copulatory courtship influences female oviposition behavior: females oviposited immediately after copulating with males that performed copulatory courtship, but not with males that performed no copulatory courtship. There is last male sperm precedence in this species, so failure of the female to oviposit before remating will decrease the first male’s reproductive success. Because oviposition is under female control, the behavior of female M. cingulatus is an expression of CFC. To my knowledge, this study is the first to demonstrate CFC through female control of oviposition timing.
It is important to note that there is an alternative explanation for the observed results that cannot be ruled out with the present data. It is possible that females fail to oviposit in the absence of courtship because sperm transfer (and therefore, fertilization) does not happen in that case. Copulatory courtship has been demonstrated to be critical for males to achieve complete penetration (and spermatophore transfer) in a
30
chrysomelid beetle (Tallamy et al. 2002). To distinguish between the two scenarios, it
would be necessary to determine whether manipulated males that do not perform
courtship transfer sperm to the female. However, it should also be noted that females
can oviposit even if the last male they mated with does not transfer sperm, since they
are likely to have sperm stored from previous matings. Therefore, it seems unlikely that
females fail to oviposit due to lack of sperm transfer by manipulated males. Another
possibility is that manipulated males transfer less sperm than the control males. In that
case, the observed female behavior is still an example of CFC by female control of
oviposition timing.
Last male sperm precedence is a widespread pattern among insects (Thornhill
and Alcock 1983), which suggests that control of oviposition timing is likely to be a
common mechanism of CFC. The possibility that females in other species can influence
paternity of their offspring by changing oviposition timing has been overlooked and
deserves to be explored.
Copulatory courtship is a male trait that is likely under selection by CFC, such
that males performing more or superior courtship benefit from increased paternity.
Since copulatory courtship occurs only after copulation has begun, it does not play a
role in attracting the courted female. In most species it also does not seem to function
in obtaining a second mating with the female being courted, as males leave after a
single copulation (Eberhard 1991, 1994). Copulatory courtship is therefore likely to be a
trait that influences paternity after mating. The widespread occurrence of copulatory
courtship in arthropods (Eberhard 1991, 1994) suggests that CFC is also widespread. A
31
relationship between copulatory courtship and male reproductive success has been found in two other species (see table 1). However, this is the first study to show CFC of copulatory courtship through an external and easily observable female behavior. This discovery increases the possibilities for future studies, since such female behaviors can be easily scored.
This is the first study of CFC conducted in the field, rather than in the laboratory.
Using field individuals entails some limitations, the main one being that the mating history of those individuals is unknown. However, field experiments provide results that are relevant to natural populations. Female choice can play out very differently in the field and in the laboratory: factors such as predation risk and the costs of sampling males can make female preferences more costly, which may result in preferences not being expressed as strongly, if at all. In a laboratory setting, these factors are often removed, which in some species is known to lead to a level of female choosiness that would not be expressed in the field (Jennions and Petrie 1997). Thus, field studies are fundamental to assess the relevance of CFC: only by showing that these female preferences are expressed in natural populations can we confirm that CFC is an important agent of sexual selection.
FUNDING
This work was supported by the Smithsonian Tropical Research Institute, The Dorothea
Bennett Memorial Graduate Research Fund and the Animal Behavior Society.
32
ACKNOWLEDGMENTS
William Eberhard shared invaluable ideas for the execution of this work. Ulrich Mueller,
Robert Snyder and Lori Eggert provided information on the AFLP technique and analysis.
Michael Reichert, Rex Cocroft, William Eberhard, Carl Gerhardt and two anonymous reviewers made helpful comments on drafts of this manuscript. I would also like to thank the staff at Smithsonian Tropical Research Institute for their assistance, and the
Autoridad Nacional del Ambiente (ANAM) at the Republic of Panama for issuing research permits.
33
TABLES
Table 1 – Studies of cryptic female choice where the postcopulatory mechanism could be attributed to the female, and was shown to affect male reproductive success
Organism Female mechanism Male trait favored by References female Cucumber beetle, Controlling male access to Intensity of Tallamy et al.
Diabrotica bursa copulatrix and copulatory courtship 2002
undecimpunctata spermatophore transfer
howardi
Water strider, Clutch size adjustment Larger body size Arnqvist and
Gerris lateralis Danielsson 1999
Scorpionfly, Clutch size adjustment Large body size and Thornhill 1983
Harpobittacus large nuptial gift prey
nigriceps item (which are
correlated)
Guppy, Poecilia Controlling amount of Relatively colorful Pilastro et al.
reticulate retained sperm males 2004
Water frog, Rana Clutch size adjustment Not hybrid males Reyer et al . 1999
lessonae-esculenta
Yellow dung fly, Unknown pgm genotype Ward 2007
Scathophaga (preference varies Ward 2000
stercoraria with environment) Ward 1998
Spider, Argiope Controlling copulat ion Small body size Elgar et al. 2000
34
keyserlingi duration by mate
cannibalism
Flour beetle, Controlling sperm transfer Intensity of Bloch -Qazi 2003
Tribolium and storage copulatory courtship Edvardsson and castaneum Arnqvist 2000
Fly, Dryomyza anilis Transferring s perm to Number of Otronen and
singlet spermatheca, which postcopulatory Siva-Jothy 1991
is primarily used when genital taps
ovipositing
Field cricket, Clutch size adjustment Higher dominance Bretman et al.
Gryllus bimaculatus status 2006
Decorated cricke t, Controlling duration of Large Sakaluk and
Gryllodes sigillatus spermatophore spermatophylax size Eggert 1996
attachment, and therefore Sakaluk 1997
of sperm transfer
35
FIGURES
Figure 1 – P2, or percentage of offspring assigned to the last male to mate with each
female. The dotted line shows the p 2 value of 0.5.
36
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39
3. Copulation duration in the soldier fly: the roles of
cryptic male choice and sperm competition risk
ABSTRACT: In most animal species, females invest more in reproduction than males do and are therefore expected to be the choosier sex. However, male choice is also expected to occur whenever there is sufficient variation in female quality and male mating effort is high. Postcopulatory or ‘‘cryptic’’ male choice is defined as variation in the amount of resources males allocate to females of varying quality. The soldier fly
Merosargus cingulatus exhibits significant variation in copulation duration. This variation in mating effort may be a consequence of cryptic male choice, although other processes such as sperm competition may also play a role. In this study, I manipulated factors associated with the risk of sperm competition (male density) and varying mate quality (female size) to determine whether these factors affected male mating effort. I found direct evidence that male M. cingulatus can control copulation duration.
Copulations were longer when male density was high, which support the hypothesis that males increase copulation duration when there is a high risk of sperm competition.
In addition, copulation duration was longer when males mated with larger more fecund females, which supports the hypothesis of cryptic male choice. Key words: copulation duration, cryptic male choice, postcopulatory sexual selection, soldier fly, sperm competition.
40
KEY WORDS: copulation duration; cryptic male choice; sperm competition;
postcopulatory sexual selection; soldier fly.
INTRODUCTION
The theory of sexual selection explains differences in sex roles as a result of
differential parental investment: The sex that invests relatively little in reproduction is
expected to compete for mates, whereas the one with higher investment often has
mating preferences. In most animal species, females invest more in reproduction than
males, hence the classical view of females as the ‘‘choosy’’ sex. This view stems in great
part from the idea that gamete expenditure is much lower for males than for females
(Trivers 1972). However, while single sperm might be cheap for males to produce, large
amounts of it are not necessarily so (Dewsbury 1982; Tang-Martinez andRyder 2005),
which means that even if males contribute nothing but sperm for reproduction,
copulations come at a cost. In addition, males often provide more than just sperm,
investing in large ejaculates containing nutrients and other seminal fluids (Sakaluk 1985;
Oberhauser 1988; Fox et al. 1995; Wagner 2005), time and energy spent in courtship
and copulation (Vehrencamp et al. 1989; Prestwich 1994), direct benefits such as
territory or food (Vahed 1998), and paternal care (Gross and Sargent 1985; Tallamy
2000).
If copulation is costly for males and if the number of mates is limited by resources allocated for reproduction rather than the availability of potential mates, males can maximize their reproductive success by having mating preferences. They will
41
benefit from selectively allocating resources to higher quality females. Male mate choice, therefore, is expected to occur even in species where female choice is present as well (reviewed in Bonduriansky 2001; Clutton-Brock 2009). Male choice can be precopulatory, when males refuse to mate with low-quality females, and postcopulatory, which is referred to as cryptic male choice (sensu Bonduriansky 2001).
When cryptic male choice occurs, males are expected to selectively allocate more resources to more fecund females. These resources could include ejaculate, time and energy spent mating, and paternal care. Often, larger females are more fecund, and in several species, males transfer more sperm and copulate for longer when mating with large females (Gage and Barnard 1996; Gage 1998; Parker et al. 1999; Engqvist and
Sauer 2001, Reinhold et al. 2002; Cornwallis and O’Connor 2009; Xu and Wang 2009).
Variation in copulation duration can be a result of cryptic male choice. There are, however, other processes that can explain this variation. Males are expected to increase copulation duration in situations where the risk of sperm competition is high because they may attempt to increase sperm transfer, add seminal fluids to affect female future receptivity, or prevent the female from remating by mate guarding (Simmons 2001).
Cryptic female choice can also be at play, if females prefer males that mate for longer than other males. These hypotheses all assume that males can control copulation duration, but females, as well as males, can benefit by controlling copulation duration.
Long copulations can be costly for females if they increase predation risks or reduce foraging opportunities. Long copulations can also be beneficial when females obtain resources from mating (Arnqvist 1989; Rowe 1992; Crudgington and Siva-Jothy 2000;
42
Schneider et al. 2006). In either case, optimal copulation duration can be different for males and females, a discrepancy that can lead to sexual conflict for the control of copulation duration (Arnqvist and Rowe 2005). Because internal as well as external events may be involved, determining which sex is controlling copulation duration is difficult and requires teasing apart the roles of each sex during copulation. Previous studies have tried to circumvent this difficulty by manipulating one sex and then attributing to that sex the observed changes in copulation duration (Carazo et al. 2007;
Wilder and Rypstra 2007; Holwell 2008; Sakaluk and Mu¨ ller 2008; Bretman et al. 2009;
Carlos and Seiji 2010; Herberstein et al. 2010; Morse 2010). This approach fails to rule out the role of the other sex, which can also perceive and respond to such manipulations. For example, some studies have tested if males prolong copulations when the risk of sperm competition is high by comparing the copulation durations of isolated mating pairs with those in the presence of other males or their chemical cues.
(Carazo et al. 2007; Sakaluk and Mueller 2008; Bretman et al. 2009). Observed differences in copulation duration cannot be attributed, however, to one sex or the other because the extra males can be perceived by and affect the behavior of both males and females.
I will test 2 different hypotheses that may explain variation in copulation duration in the soldier fly Merosargus cingulatus . This species was especially well suited for this study because there is great variation in copulation duration (personal observation). Males seem to be in control of copulation duration because copulation in this species ends when males release the females. Females also do not perform any
43
externally visible behavior to signal the end of copulation. I will address the question of whether males can influence copulation duration by affecting female behavior rather than or in addition to controlling it by directly restraining and then releasing the female.
More specifically, I will test the hypothesis that males of M. cingulatus influence copulation duration by affecting female behavior through copulatory courtship.
Merosargus cingulatus mates at its oviposition site, in decomposing vegetation, where males defend territories. The decomposing vegetation attracts a large number of males and females, and the mating frequency of both sexes is high (personal observation). Variation in female size and a high female encounter rate suggest that cryptic male choice could happen in this species. Males attempt to grab and copulate with any approaching females, whereas females do not appear to resist mating once they are grasped by a male. After mating, females oviposit in or around a male’s territory. Females lay eggs multiple times in their lifetime. For the entire duration of copulation, males perform copulatory courtship, which consists of an alternation of bouts of 2 male behaviors: 1) tapping the female’s abdomen with their hind legs and 2) waving their legs in the air (Barbosa 2009). There is significant variation in copulation duration and producing ejaculates and copulatory courtship are likely to be energetically expensive to males.
The risk of sperm competition predicts that longer copulations occur when male
density is high. Another explanation, cryptic male choice, predicts longer copulations
with more fecund females. Here, I present the results of tests of these 2 non-mutually
exclusive hypotheses. To test the first hypothesis, I compared copulation durations
44
when males—but not females—were exposed to different male densities prior to copulation. Because females remain receptive after mating, a higher male density at the oviposition site increases the risk of sperm competition. To test the second hypothesis, I compared copulation duration when males mated with females of different sizes. I will show that copulation duration is longer both in high densities and with larger females, indicating that both cryptic male choice and sperm competition are at play in this species. These are not the only possible explanations for the variation in mating duration observed, and I will discuss some testable alternatives.
METHODS
Experiments were conducted at the Smithsonian Tropical Research Institute field
station in Gamboa, Panama, between August and October 2008. A 1.8 x 1.8 x 1.8 m
mesh enclosure was built in an open area adjacent to small patches of forest in a
residential area of Gamboa. In the enclosure, I covered an area of 80 x 40 cm with fruit
peels to create suitable oviposition sites. All individuals used were field caught.
The effect of male density on copulation duration
To test the hypothesis that increased risk of sperm competition results in longer
copulations, I made sure that any changes seen in copulation duration were attributable
to the males, not the females. The experiment consisted of 2 treatments: low density (n
= 20), in which a male was introduced into the enclosure alone and high density (n = 20),
in which a male was introduced with 4 other males. For both treatments, males had an
45
acclimation period of 1 h, after which the 4 extra males in the high-density treatment were removed. For both treatments, I then introduced a female into the enclosure with an entomological net. Because the additional males in the high-density treatment had already been removed, the females were not exposed to different male densities. I allowed the pair to mate, video-recorded the copulations, and measured the copulation duration from the video recording.
It is possible that males can influence copulation duration not by restraining or releasing the female but by affecting female behavior through copulatory courtship. I tested this hypothesis by measuring different components of copulatory courtship through video analysis and comparing them between the 2 treatment groups. I measured the rate and the average duration of bouts of both taps and leg waves.
The effect of female size on copulation duration
Before testing the hypothesis that mating with preferred females results in
longer copulations, I made measurements to confirm my assumption that large females
would be preferred because they are more fecund than small females. I attracted
females with fruit peels and collected 46 individuals. I weighed these females and
dissected them to count the number of mature eggs in their reproductive tract. I used
linear regression to evaluate the relationship between female size and fecundity.
Having shown that larger females have higher fecundity (see Results), I then tested whether female size influenced copulation duration. I allowed males to mate with either a small (46.0 mg, n = 13), medium (46.0–67.2 mg, n = 14) or large (67.2 mg, n
46
= 12) female. I then weighed field-caught females and assigned them to 1 of the 3 size groups based on previous measures of the body weight of 54 females; the groups corresponded to the bottom, middle, and top thirds of the size distribution. Males were introduced individually to the mesh enclosure containing an oviposition area. After 30 min of acclimation, I introduced a female from 1 of the 3 groups. I allowed pairs to mate and measured the copulation duration from video recordings of the copulations.
RESULTS
The effect of male density on copulation duration
Copulations were longer when male density was higher at the oviposition sites
(high density: 128.21 ± 86.09 s, low density: 68.45 ± 54.05 s, mean ± standard deviation
[SD]; n = 20 for each group, Mann–Whitney test, p = 0.012; Figure 1). There were no differences between the 2 treatment groups for the courtship behaviors analyzed
(Mann–Whitney test, all p values > 0.5, Figure 2).
The effect of female size on fecundity
As expected, larger females had more eggs in their reproductive tract (R 2 = 0.76,
P < 0.001, Figure 3).
The effect of female size on copulation duration
Copulation duration was strongly influenced by female size, with males copulating 4 times longer, on average, with the largest than with the smallest females.
47
Copulation duration was significantly different between the 3 female size groups (small females: 30.08 ± 11.48 s [mean ± SD]; medium: 71.53 ± 55.49; large: 125.88 ± 79.03;
Kruskal–Wallis, P < 0.0005, Figure 4). When pairwise comparisons were performed between the groups, copulation duration was significantly different after correction for multiple comparisons with both sequential Bonferroni and Sidak adjustment methods between small versus medium and small versus large females (Mann–Whitney test, P =
0.013, P < 0.0005, respectively) and marginally significant between medium versus large females (Mann–Whitney test, P = 0.057). These results support the hypothesis that there is cryptic male choice in M. cingulatus : males invest more in copulations with
larger females by increasing copulation duration.
DISCUSSION
The results above show that copulation duration in M. cingulatus is influenced by
sperm competition and suggest that cryptic male choice also occurs. Experiment 1
provides direct evidence that males can influence copulation duration: in this
experiment, the only difference between treatments was pre-mating male density, a
cue available only to males. The difference in copulation duration must therefore be due
to a change in male behavior.
Males can control copulation duration directly by restraining or releasing the
female; they could also influence it by affecting female behavior. For example, in the
scorpionfly Panorpa cognata , males can prolong copulations by offering the female
larger nuptial gifts of salivary masses (Engqvist and Sauer 2001). One way M. cingulatus
48
males could influence female behavior is through copulatory courtship, but the results presented here suggest that this is not the case. The analysis of copulatory courtship behavior of males from the 2 density treatments showed no significant differences in the behavior of the 2 groups. It is conceivable that there could have been other differences between the groups that were not explored and that those differences were perceived by the female. However, any differences between groups would still have resulted from the different treatments, which means that the changes in copulation duration observed have to be attributed to the male. In addition, a previous study where M. cingulatus males were manipulated so they could not perform copulatory
courtship showed no difference in copulation duration between these males and the
controls, which adds further support to the idea that copulation duration is not affected
by copulatory courtship. Male control of copulation duration in M. cingulatus probably
occurs by male restraint of the female.
Longer copulations in higher densities suggest sperm competition is at play.
However, these results do not allow a distinction between different sperm competition
mechanisms: Both mate guarding and increasing amount of sperm transferred predict
longer copulations when the risk of sperm competition is higher. In M. cingulatus , mate
guarding seems unlikely because even the longest copulations are relatively short when
compared with other species where in-copula mate guarding occurs (Alcock 1982;
Cordero 1990; Sakaluk 1991). Further studies are necessary to verify if males are
prolonging copulation duration to increase sperm transfer. The amount of sperm
transferred is probably a function of copulation duration in this species, but the nature
49
of this function is unknown. These results also provided an important experimental tool to manipulate copulation duration: manipulating male density. This is a much less disruptive technique than interrupting copulations, making it less likely to confound results or change the individuals’ behaviors.
The results from experiment 2 support the hypothesis of cryptic male choice in
M. cingulatus : males mate longer with larger more fecund females. Long copulations are
probably costly to males in terms of time and energy, so males should benefit from
selectively allocating their efforts to females that give them the highest reproductive
success. An alternative explanation is that larger females mate longer because they can
store more sperm. This hypothesis assumes that sperm transfer is a function of
copulation duration in this species, which is unknown. Although the results from
experiment 1 demonstrate that males can control copulation duration, females might
still be able to exert some control as well, and as discussed below, there is evidence for
postmating cryptic choice.
There are different possible ways males benefit from longer copulations with
larger females. If sperm transfer is indeed a function of copulation duration, longer
copulations would allow males to fare better in sperm competition. It is also possible
that females prefer longer copulations. A previous study demonstrated that cryptic
female choice for copulatory courtship occurs in this species: Females that mated with
males that did not perform copulatory courtship left the males’ territories without
ovipositing (Barbosa 2009). It is possible that females exhibit preferences for long
copulations as well and that males that mate for longer father a larger percentage of
50
a female’s offspring through sperm selection. Further studies are necessary to tease apart these different hypotheses.
FUNDING
American Philosophical Society Lewis and Clark Fund and Sigma Xi.
ACKNOWLEDGEMENTS
I thank William Eberhard and Rex Cocroft for sharing invaluable ideas on the experiments and data analysis and for comments on the manuscript. I also thank
Michael Reichert, Carl Gerhardt, and 2 anonymous reviewers for comments on the manuscript, the staff at Smithsonian Tropical Research Institute for their assistance, and the Autoridad Nacional del Ambiente (ANAM) at the Republic of Panama for issuing research permits.
51
FIGURES
Figure 1 - Mean copulation duration in high- and low-density oviposition sites.
*indicates a P value < 0.05. Error bars represent standard errors.
52
Figure 2 - Copulatory courtship behavior of males of 2 treatment groups, high density
(black) and low density (white): rate of taps (a), mean duration of tap bouts (b), rate of waves (c), and mean duration of wave bouts (d). Error bars represent standard errors.
53
Figure 3 - Number of eggs present in female reproductive tract as a function of her weight.
54
Figure 4 - Mean copulation duration with females of different size ranges. *indicates a P value < 0.05, **indicates a P value < 0.01. Error bars represent standard errors.
55
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4. Males responding to sperm competition cues have
higher fertilization success in a soldier fly
ABSTRACT - Sperm competition has been demonstrated to be an important force shaping male behavior in a number of species. For example, males may prolong copulation duration when they perceive sperm competition to be high. Although male behavioral responses to sperm competition have been shown in several species, their effects on reproductive success have rarely been demonstrated. In the soldier fly
Merosargus cingulatus , males prolong copulations when sperm competition is high and when mating with more fecund females. Here, I tested the hypothesis that this behavioral response results in higher reproductive success for males. I exposed males to different simulated levels of sperm competition (high or low male density at the oviposition site), then introduced a female. I allowed the pair to mate and the female to oviposit. I determined the percentage of offspring sired by the male using AFLP profiles.
Sperm competition did not affect clutch size, but it did affect fertilization success: males under higher simulated sperm competition increased copulation duration and fertilized a higher percentage of a female’s egg clutch than did males under lower sperm competition.
KEY WORDS: Sperm competition; postcopulatory sexual selection; fertilization success; clutch size; soldier fly.
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INTRODUCTION
Sperm competition plays an important role in shaping male morphology and behavior (Simmons 2001). Whenever sperm is a limited resource, males are expected to selectively allocate sperm in a way that can increase their reproductive success (Gage
1991, Cook and Gage 1995, Gage and Barnard 1996, Marconato and Shapiro 1996,
Simmons and Kvarnemo 1997, Fuller 1998, Parker et al. 1999). Sperm competition
theory makes different predictions about male reproductive investment depending on
the intensity and risk of sperm competition. Sperm competition intensity relates to the
number of ejaculates from different males competing within a female’s genital tract,
while sperm competition risk relates to the probability that a female has mated before
or will mate again with a different male. On the one hand, males are expected to
transfer less ejaculate when sperm competition intensity is high because, after a certain
point, males will obtain diminished returns from their increased investment in
ejaculates. On the other hand, males are expected to transfer more ejaculate when
there is a high risk of sperm competition: if there is a high probability that sperm
competition will occur, the more sperm transferred to a female, the higher fertilization
success will be, assuming all else is equal (Parker et al. 1996, Parker et al. 1997,
reviewed by Wedell et al. 2002, Parker and Pizzari 2010). Because the models make
opposite predictions, it is important that experimental studies distinguish between
measurements of intensity and risk (Enqvist and Reinhold 2005). This is no easy task
because, for example, varying the density of rivals a male is exposed to can potentially
signal both risk and intensity to that male.
62
Males of several species have been shown to respond to sperm competition by changing their behavior. This includes changing courtship intensity and overall copulation duration (Lorch et al. 1993, Andrés and Cordero-Rivera 2000), as well as providing females with different quality nuptial gifts (Sakaluk 1985, Fox et al. 1995,
Oberhauser 1998, Vahed 1998, Wagner 2005). For example, in Drosophila melanogaster , males court females more vigorously when sperm competition intensity is lower (Tompkins and Hall 1981, Friberg 2006). In this case, they assess sperm competition intensity by detecting female mating status through the female cuticular hydrocarbon profile. In other species, males attempt to assess sperm competition risk by assessing the operational sex ratio before and during mating. Although there are a large number of examples showing male behavioral responses to sperm competition in the literature, only one study has investigated its effects on male fitness. (Bretman et al. 2009).
Males are also expected to selectively allocate resources (such as ejaculate size and nuptial gifts) when mating with females of different qualities, if doing so can increase their reproductive success. This process is known as cryptic male choice
(Bonduriansky 2001). Males have been shown to selectively allocate more resources to females with traits that relate to high fecundity in a number of species (Yasui 1996,
Gage and Barnard 1996, Wedell 1998, Gage 1998, Parker et al. 1999).
Males of the soldier fly Merosargus cingulatus respond to sperm competition by prolonging copulations when rival density at the oviposition site is high. They also increase copulation duration when mating with larger, more fecund females (Barbosa
63
2011). In this species, mating occurs at the oviposition sites, where males try to capture and mate with any female that approaches. Both males and females mate multiply and frequently. Males do not court females before mating, but they perform copulatory courtship: during the entire duration of copulation, they alternate bouts of tapping the female abdomen and waving their hind legs in the air. This copulatory courtship induces the female to lay eggs immediately following copulation (Barbosa 2009). Females oviposit multiple egg clutches in the same day. They often return to the same oviposition site and lay additional clutches after having already oviposited in that area
(F. Barbosa, personal observation).
Here, I tested the hypothesis that male behavioral response to sperm competition results in higher reproductive success for males. There are several, non- mutually exclusive ways this behavior could increase a male´s reproductive success. One possibility is that males stimulate females to lay a larger number of eggs through prolonged copulations or courtship (since copulatory courtship occurs during the entire copulation, copulation and courtship duration are equivalent in this species). Another possibility is that males that prolong copulations fertilize a larger proportion of a female’s eggs, either by transferring more sperm or accessory gland secretions, or through cryptic female choice for longer copulations, courtships, or more secretions. I tested both of these possibilities by submitting males to different treatments that simulated different amounts of sperm competition (high and low male density at the oviposition site). Although we do not know if and how males detect sperm competition in this species, it is possible that they use density at the oviposition site as a cue. This
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possible indicator of sperm competition will hereafter be referred to as “simulated sperm competition” or “sperm competition” for simplicity. In addition, female size plays a role in copulation duration in this species: males prolong copulations when mating with larger, more fecund females. Because of that, I also tested if male reproductive success under different amounts of sperm competition differed between males that were given either large or small females as mates. I found that sperm competition did not affect clutch size, but it affected fertilization success: males under higher simulated sperm competition fertilized a higher percentage of a female’s egg clutch. Female size did not affect the relationship between simulated sperm competition and clutch size, or between the former and fertilization success.
METHODS
This work was conducted at the Smithsonian Tropical Research Institute field station in Gamboa, Panama, between July and September, 2009. All experiments were conducted in a 1.8 x 1.8 x 1.8 meter mesh enclosure containing an area of 80x40 cm covered with fruit peels to create suitable oviposition sites. This enclosure was located in an open area adjacent to small patches of forest in a residential area. All individuals used were field caught. To attract individuals, piles of fruit peels were set up in the field.
Females were caught as they arrived at the sites, before they had a chance to oviposit.
When individuals were not introduced to the experimental enclosure as soon as they were captured, they were temporarily housed individually in 50 ml plastic jars. They
65
were not housed in the plastic jars for longer than 30 minutes. All individuals used were weighed.
Sperm competition and clutch size
Males were randomly assigned to a treatment that simulated either low or high sperm competition, and were mated to either small or large females, resulting in a 2x2 design. To create the groups with different simulated amounts of sperm competition, males were assigned to different density treatments. In the low density treatment they were introduced into the enclosure alone; in the high density treatment they were introduced with four other males. The amount of oviposition substrate was the same for both treatments. Natural male density at oviposition sites in the field varies considerably, and the two treatments used are within the natural range (Barbosa 2011).
For both treatments, males had an acclimation period of one hour, during which they established territories on the oviposition substrate. After acclimation, the four extra males in the high density treatment were removed. Following the density treatments, I introduced a female from one of two female size categories, small (<42 mg) or large
(>42 mg). These size categories correspond to the top and bottom halves of a female size distribution that I obtained from previous measurements of field-caught individuals
(unpublished data). Females were introduced into the enclosure with an entomological net. I allowed the pair to mate and the female to oviposit. Females were allowed to terminate oviposition naturally, and therefore were not captured until they left the oviposition substrate. I video recorded all copulations. This experimental design resulted
66
in four groups: males that mated under conditions mimicking high sperm competition with large females (n=12) or small females (n=8), and males that mated under conditions mimicking low sperm competition with large females (n=10) or small females
(n=10).
I measured two aspects of female oviposition behavior that may be affected by copulation duration: clutch size and the percentage of available mature eggs laid by the female. I focused on the percentage of mature eggs laid because larger M. cingulatus females are more fecund than small ones (Barbosa 2011), so female size likely affects clutch size. To determine clutch size, I counted the number of eggs laid by the female after mating. To determine the percentage of eggs laid, I dissected the females and counted the mature eggs that were left in their ovaries after they oviposited. I performed 2x2 ANOVAs with density (low, high) and female size (large, small) as between-subjects factors to compare clutch size, percentage of eggs laid and copulation duration between treatments. Data were transformed if necessary to achieve normal distributions (arcsine for percentages, log and square root).
Sperm competition and paternity
I used AFLP profiles to determine what percentage of the eggs laid by the female after mating in the previous experiment was fertilized by the experimental male. After oviposition, I collected the mating pair and the eggs laid. I allowed the eggs to develop to larvae and randomly selected 25 larvae from each family for analysis. For pairs with less than 25 offspring, all larvae were analyzed. DNA extraction, AFLPs and analysis were
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done as in Barbosa (2009). For four of the 40 pairs, it was not possible to obtain genotype data for one of the parents, because the PCR failed. Since data from both parents are necessary to determine paternity, those families were not analyzed. Some of the offspring of the other families were excluded from the analyses because of PCR failure.
Barbosa (2011) showed that copulations are longer when sperm competition is high, so I tested if there was an effect of simulated sperm competition on copulation duration and on copulatory courtship. I quantified different components of copulatory courtship (rate of taps, rate of waves, duration of tap bouts and duration of wave bouts) by measuring them from the video recordings and comparing them between the treatment groups.
I performed 2x2 ANOVAs with density (low, high) and female size (large, small) as between-subjects factors to compare clutch size, percentage of eggs laid and copulation duration between treatments. I also performed linear regression analyses to test the effects of male and female size, sperm competition, and the interactions between these variables on copulation duration. I initially tested a model with all the interactions, then removed the statistically non-significant interactions to arrive at a reduced model with higher power. Data were transformed if necessary (arcsine for percentages, log and square root). I used dummy coding to code for sperm competition
(0=high sperm competition, 1=low sperm competition).
68
RESULTS
Sperm competition and clutch size
Simulated sperm competition did not affect clutch size (figure 1). A 2x2 ANOVA on square-root transformed clutch size showed no effect of density (F 1, 36 = 0.121, p =
0.730) or female size (F 1, 36 = 2.991, p = 0.092), and there was no interaction between
density and female size (F 1, 36 = 0.001, p = 0.972). Simulated sperm competition also did
not affect the percentage of eggs laid (figure 2). A 2x2 ANOVA on arc-sine transformed
percentage of eggs showed no effect of density (F 1, 36 = 0.548, p = 0.464). There was an effect of female size on percentage of eggs laid with smaller females tending to lay a larger percentage of their available eggs (F 1, 36 = 5.287, p = 0.027). There was no interaction between density and female size (F 1, 36 = 0.474, p = 0.469). Female size
averaged 29.31±8.98 mg for the small female group and 61.09±14.84 mg (xˉ ±SD) for the
large female group. Male size averaged 44.66±20.09 mg (xˉ ±SD).
Sperm competition and paternity
The fertilization success of the experimental male was influenced by simulated
sperm competition: when sperm competition was higher, males fertilized a higher
percentage of the clutch than when sperm competition was low (for small females, low
sperm competition: 79.21±13.86%, high sperm competition: 91.78±10.41%; for large
females, low sperm competition: 77.21±17.81%, high sperm competition:
87.57±14.04%, (mean±SD), figure 3). A 2x2 ANOVA on arc-sine transformed fertilization
success revealed a main effect of sperm competition (F 1, 31 = 5.758, p = 0.023). There
69
was no effect of female size (F 1, 31 = 0.195, p=0.662), and there was no interaction between sperm competition and female size (F 1, 31 = 0.067, p =0.797).
As expected, copulation duration was longer when simulated sperm competition was higher (table 1). Female and male size also affected copulation duration: copulations were longer for smaller males and for larger females. A linear regression model containing male size, female size, simulated sperm competition, and the interaction between sperm competition and male size on copulation duration was statistically significant (Table 1).
A linear regression model with fertilization success as the dependent variable showed a statistically significant interaction between copulation duration, male size and female size (Table 2). Males of a similar size needed to mate longer with a large female than with a small one to obtain the same fertilization success. In addition, for a given female body size, smaller males would need to mate for longer than larger males to obtain the same fertilization success.
Since copulatory courtship occurs for the entire duration of copulation, the duration of copulatory courtship was different in the groups of low and high sperm competition. However, sperm competition did not affect the composition of the courtship display. There were no statistically significant differences among the treatments in the rate and the average duration of bouts of taps, or of leg waves (Mann-
Whitney tests, p=0.81, 0.54, 0.54 and 0.72, respectively).
70
DISCUSSION
This study shows that male behavioral response to variation in sperm competition results in higher fertilization success. Male soldier flies prolong copulations when mating under high simulated sperm competition, and under those conditions, males fertilized on average 14.4% more of a female’s clutch than males under low simulated sperm competition. This result is important, because it demonstrates not only that males are detecting and responding to variation in sperm competition, but also that this behavioral response affects male fitness. To the best of my knowledge, this is the only species in which increased fitness has been demonstrated other than D. melanogaster (Bretman et al. 2009); this is also and the first time this phenomenon has been demonstrated under field conditions. The fact that males prolong copulations under high simulated sperm competition is consistent with previous work in this species
(Barbosa 2011).
The relationship between copulation duration and fertilization success is complex, since the effect of copulation duration depended on both male and female size. There was a negative relationship between copulation duration and male size, but a positive relationship between copulation duration and female size. Female body size is known to play a role in copulation duration in this species (Barbosa 2011). Body size, copulation duration, and interactions between these factors, all play a role in determining fertilization success in this species (see table 2). For a given female body size, a small male would need to mate for a longer time than would a large male to obtain the same
71
fertilization success. Likewise, males of similar size need to mate for a longer time with a large female than with a small one to obtain the same fertilization success.
The sperm competition treatments used in this study, where males were exposed to different densities of rivals, can potentially signal both risk and intensity to that male. Therefore, it is unclear whether soldier fly males were responding to risk or intensity of sperm competition. The results found here are comparable to those found by Bretman et al. (2009) in D. melanogaster . In that study, males prolonged copulation duration under high risk of sperm competition but, as expected, copulations were shorter under high sperm competition intensity. Soldier flies are highly polygamous; both males and females mate multiply (Barbosa 2009), so sperm competition is likely to play an important role in this species. The levels of both risk and intensity of sperm competition are likely to be high for this species, but it is unknown whether males assess and respond to these components differently.
The mechanism that leads to higher fertilization success after longer copulations in this species is unknown. One possibility is cryptic female choice: females may prefer longer copulations (or longer courtships), and bias fertilization towards these preferred males. For example, females might control sperm transfer and storage, biasing fertilization towards males that perform more intense copulatory courtship as in the cucumber beetle (Tallamy et al. 2002, Tallamy et al. 2003) and flour beetle (Fedina and Lewis 2004, Edvardsson and Arnqvist 2000, Edvardsson and Arnqvist 2005). Cryptic female choice occurs in this species by female control of whether oviposition occurs
72
immediately following copulation (Barbosa 2009), but it is unknown if females can also control sperm transfer, sperm storage and fertilization.
Another possibility is that longer copulations result in increased transfer of sperm or of other seminal products, which in turn leads to higher fertilization success.
Previous studies in a variety of species have shown that males adjust ejaculate size in response to sperm competition (Gage 1991, Wedell and Cook 1999, Engqvist 2007,
Ramm and Stockley 2007). Other studies have shown that males had higher fertilization success when they increased ejaculate transfer (Simmons 1987, Simmons et al. 1996,
Arnqvist and Danielsson 1999b). The relationship between copulation duration and sperm transfer is unknown for soldier flies, so this hypothesis also remains to be explored.
Finally, there is no evidence that the changes in male behavior caused by increased simulated sperm competition affected female egg production in the soldier fly. This contrasts with the results of Bretman et al. in D. melanogaster (2006), where increased sperm competition risk resulted in larger clutch sizes. In that study, it was hypothesized that larger clutch sizes were caused by an increase in seminal fluid transfer, which can affect female egg production behavior. In other species, females have been shown to increase clutch size, as well as the amount of resources allocated to eggs, as a result of cryptic female choice (Thornhill 1983, Wedell 1996, Arnqvist and
Danielsson 1999a, Arnqvist and Danielsson 1999b, Bretman et al. 2004, Aquiloni and
Gherardi 2008). Changes in clutch size do not seem to occur in this context in M. cingulatus .
73
In summary, these results demonstrate that sperm competition affect male copulatory behavior in soldier flies, and this in turn results in increased reproductive success. Under high simulated sperm competition, males prolong copulations and obtain higher fertilization success, which could be due to higher sperm transfer, or cryptic female choice.
FUNDING
This work was supported by the American Philosophical Society Lewis and Clark
Fund and Sigma Xi.
ACKNOWLEDGEMENTS
I am grateful to William Eberhard and Rex Cocroft for sharing invaluable ideas on the experiments and data analysis, and for comments on the manuscript. I also thank
Michael Reichert, Carl Gerhardt and Lori Eggert for comments on the manuscript, the staff at Smithsonian Tropical Research Institute for their assistance, and the Autoridad
Nacional del Ambiente (ANAM) at the Republic of Panama for issuing research permits.
74
TABLES
Table 1 – The effect of female size, male size, and sperm competition risk on copulation duration. Regression analysis with copulation duration as the dependent variable, showing the unstandardized coefficients (B), standard errors of the unstandardized coefficients (SE) and significance values (p) of the individual predictors present in the final model.
Predictor B SE p
Constant 12.465 1.976
Sperm competition risk -7.720 2.777 0.009
Female size 0.063 0.029 0.035
Male size -0.114 0.047 0.020
Male size*Sperm competition risk 0.104 0.058 0.084
Full model: R 2=0.401, p=0.003
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Table 2 – The effect of copulation duration, female size and male size on fertilization success. Regression analysis with fertilization success as the dependent variable. Table shows the unstandardized coefficients (B), standard errors of the unstandardized coefficients (SE) and significance values (p) of the individual predictors that were entered into the model.
Predictor B SE P
Constant 0.213 0.336 0.532
Copulation duration 0.058 0.033 0.092
Male size 0.013 0.008 0.144
Female size 0.020 0.008 0.014
Female size*Male size -4.925E-4 0.000 0.012
Female size*Copulation duration -0.002 0.001 0.009
Male size*Copulation duration 0.000 0.001 0.386
Male size*Female size*Copulation duration 4.578E-5 0.000 0.020
Full model: R 2=0.342, p=0.092
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FIGURES
Figure 1- Clutch size was not affected when females (small or large) mated with males
under high or low simulated sperm competition. Box plots show median (horizontal
bars), upper and lower quartiles (borders of the box). Whiskers extend from the 10 th to the 90 th percentiles.
77
Figure 2- The percentage of mature eggs laid was not affected when females (small or large) mated with males under high or low simulated sperm competition. Box plots show median (horizontal bars), upper and lower quartiles (borders of the box). Whiskers extend from the 10 th to the 90 th percentiles.
78
Figure 3 – Male fertilization success increased significantly under higher simulated
sperm competition. Box plots show median (horizontal bars), upper and lower quartiles
(borders of the box), and whiskers extend from the 10 th to the 90 th percentiles, of the percentage of eggs fertilized by the experimental male under low and high simulated sperm competition when mating with small and large females.
79
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Bretman A, Fricke C, Chapman T. 2009. Plastic responses of male Drosophila
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5. Discussion
SUMMARY OF RESULTS
The experiments described in this dissertation amount to an unusually complete study of postcopulatory sexual selection, since I have examined cryptic female choice mechanisms, selected traits, and their interactions with cryptic male choice in a single species. The soldier flies are an excellent model system for studies of postcopulatory sexual selection, and particularly for field studies. In chapter 1, I demonstrated that cryptic female choice occurs in M. cingulatus through a novel mechanism: female control of oviposition timing. I showed that females are less likely to oviposit immediately after mating when a male does not perform copulatory courtship than when he does (Barbosa 2009). Through parentage analysis, I determined that last male sperm precedence occurs in this species. These results show that failure to oviposit before remating by the female decreases the previous male’s reproductive success. This study was the first demonstration of cryptic female choice in the field in any species, as well as the first demonstration of cryptic female choice by female control of oviposition timing. Because last male sperm precedence is a common pattern (Thornhill and Alcock
1983), it is possible that this mechanism of cryptic female choice is widespread among other species. In chapter 2, I demonstrated that males prolong copulation duration in situations when the risk of sperm competition is high. In addition, I demonstrated that cryptic male choice occurs in this species as well: males selectively allocate resources, investing more in copulations with larger females (Barbosa 2011). In chapter 3, I demonstrated that longer copulations result in higher reproductive success for males
85
(Barbosa, in review). Although I found no effect of copulation duration on the number of eggs laid by females, I found that fertilization success is influenced by copulation duration: males that mated for longer fertilized a higher percentage of the clutch than those with shorter copulations.
One important finding of this dissertation is the evidence that males of this species can control copulation duration. In Chapter 2, an increase in copulation duration was observed as a result of an experimental manipulation that affected males, but not females. This suggested that the difference in copulation duration observed was caused by the males (Barbosa 2011). Although this experiment does not rule out the possibility that females can also influence copulation duration, it demonstrates that males play a role. Determining the roles each sex plays in copulation duration can be challenging, since internal as well as external events may be involved, and it may be difficult to find an experimental manipulation that will affect one sex without being perceived by the other (Carazo et al. 2007; Wilder and Rypstra 2007; Holwell 2008;
Sakaluk and Muller 2008; Bretman et al. 2009; Carlos and Seiji 2010; Herberstein et al.
2010; Morse 2010). The methodology used here was successful in affecting males only.
In addition, the methods used in Chapters 2 and 3 also provide an important experimental tool to manipulate copulation duration. Manipulating male density is a much less disruptive technique than interrupting copulations, making it less likely to confound results or change the individuals’ behaviors.
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FUTURE DIRECTIONS
To the best of my knowledge, this is the first field study of postcopulatory sexual selection. Although field studies have some limitations and lack the control of laboratory experiments, they are extremely important, since they are relevant to natural populations. Sexual selection can play out very differently in the field than it does in the laboratory (Jennions and Petrie 1997). Several factors that are present in the field, such as predation risk, or the cost of mate sampling, are often not replicated in the laboratory. As a result of that, we may see levels of choosiness in laboratory experiments that would not be observed in the field. In spite of that, the vast majority of studies of postcopulatory sexual selection have been conducted in the laboratory, and as a consequence, little is known about the effects of postcopulatory sexual selection on natural populations. Therefore, more field studies of postcopulatory sexual selection with different species are necessary. Field studies are fundamental to assess the importance of cryptic mate choice and sperm competition as agents of sexual selection in natural populations.
This study also leads to several questions that must be answered so that we can
gain a better understanding of the mechanisms of postcopulatory selection that are
operating in M. cingulatus . One of these questions is what mechanism males use to
obtain a higher fertilization success after longer copulations. The experiments described
here show evidence that males are in control of copulation duration and that they are
responding to sperm competition, but they do not allow a distinction between different
sperm competition mechanisms. One possibility is that longer copulations result in
87
increased ejaculate transfer. This could lead to higher reproductive success simply due to a larger number of sperm being present, or due to an increase in transferring of seminal fluids that could potentially increase the male’s reproductive success (Parker et al. 1996, 1997). In other species, there are several examples of males that adjust ejaculate size as a response to sperm competition (Gage 1991, Cook and Gage 1995,
Gage and Barnard 1996, Marconato and Shapiro 1996, Simmons and Kvarnemo 1997,
Fuller 1998, Parker et al. 1999) and theory predicts that all else being equal, larger ejaculates will fare better in sperm competition (Parker et al. 1996, 1997). Therefore, it will be important to know what is the relationship between sperm transfer and copulation duration for M. cingulatus . This question remains to be explored.
Another possibility is that longer copulations lead to higher reproductive success for males due to cryptic female choice. Females may prefer longer copulations
(or longer courtships), and bias fertilization towards these preferred males. It is also possible that longer copulations result in larger ejaculates, and females prefer larger ejaculates. Females of several other species control sperm transfer, sperm storage and fertilization (reviewed by Eberhard 1996), favoring males with preferred traits. It is possible that female M. cingulatus can also exert choice using one or more of these mechanisms, but this question also remains for future studies.
Finally, these studies have been conducted under field conditions and with field-caught individuals. Although, as discussed above, field studies are important, they have certain limitations. Soldier flies mate multiply and frequently. The results of the paternity analysis studies showed that multiple paternity occurred in the vast majority
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of cases, implying that most females used in the study had mated with at least two males (Barbosa 2009, in review). I am unable to tell exactly how many times each female used in these studies had mated previously, and the number of ejaculates competing inside a female can affect patterns of sperm precedence, sperm competition and cryptic female choice (Wedell 2002, Zeh and Zeh 1994). Therefore, studies that control for how many times a female has mated can also shed more light on the issues described here.
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92
VITA
Flavia Araújo Barbosa was born in Belo Horizonte, Minas Gerais, Brazil on
February 5 th , 1983 to José Araújo de Oliveira and Myriam Barbosa Motta. She had her
first research experience in high school, as an intern at the Laboratory of Molecular
Parasitology at the Universidade Federal de Minas Gerais (UFMG), under the
mentorship of Dr. Élida Rabelo. She graduated from Colégio Técnico da UFMG in 2000
and enrolled in the Biological Sciences undergraduate program at Universidade Federal
de Minas Gerais in 2001. As an undergraduate, she continued working in Dr. Rabelo’s
laboratory, where she learned several molecular biology techniques. In 2003, she did a
semester-long study abroad program at the University of Texas – Austin. At UT, she took
an animal behavior class with Dr. Andrea Aspbury, where she discovered a love for the
subject. Through Dr. Aspbury, she met Dr. Mike Ryan, and worked as a research
assistant for one of his graduate students, Dr. Ray Engeszer. She then returned to UFMG
and started working as a research assistant in the Laboratory of Insect Behavior. Dr.
Julio Fontenelle, who was a graduate student in the laboratory, and Dr. Élida Rabelo
served as co-mentors for her undergraduate thesis, where she studied last male sperm 93
precedence in two species of soldier flies. In 2005, Flavia earned her Bachelor of Science degree in Biological Sciences, with a specialization in Vertebrate Zoology. She started
Graduate School at the University of Texas soon afterwards, under the supervision of Dr.
Mike Ryan. She worked as a research assistant at the Smithsonian Tropical Research
Institute (STRI) in Panama for Dr. Ryan the summer before starting Graduate School, where she studied female choice in Túngara frogs. She became interested in studying postcopulatory selection in soldier flies for her dissertation work. At STRI, she met Dr.
William Eberhard, who became a collaborator. In 2007, she transferred to the University of Missouri under the co-mentorship of Dr. Reginald Cocroft and Dr. Carl Gerhardt. She earned her doctoral degree in December 2011. She accepted a postdoctoral position with Dr. Rafael Rodriguez at the University of Wisconsin – Milwaukee, where she plans to study genotype-by-environment interactions in postcopulatory sexual selection.
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