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2018 Distiphallus Morphology and its Role in Copulation Dynamics in Anastrepha Suspensa (Loew) Taylor J. Inboden

Recommended Citation Inboden, Taylor J., "Distiphallus Morphology and its Role in Copulation Dynamics in Anastrepha Suspensa (Loew)" (2018). Masters Theses. 4469. https://thekeep.eiu.edu/theses/4469

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Please submit in duplicate. DISTIPHALLUS MORPHOLOGY AND ITS ROLE IN COPULATION

DYNAMICS IN ANASTREPHA SUSPENSA (LOEW)

(TITLE)

BY

TAYLORJ. INBODEN

THESIS

SUBMITIED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE in BIOLOGICAL SCIENCES

IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS

2018

YEAR

I HEREBY RECOMMEND THAT THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE

'?-/T-/18 1!ftr-u DATE DEPARTM¢T/SCHOOL CHAIR DATE OR CHAIR'S DESIGNEE

THESIS COMMITIEE MEMBER DATE

THESIS COMMITIEE MEMBER DATE THESIS COMMITIEE MEMBER DATE DISTIPHALLUS MORPHOLOGY AND ITS ROLE IN COPULATION DYNAMICS IN

ANASTREPHA SUSPENSA (LOEW)

BY

TAYLOR INBODEN

Thesis ABSTRACT

Anastrepha suspensa (Diptera; Tephritidae) is an agricultural pest species causing severe economic damage and is controlled, in part, by applying knowledge of this species' reproduction (e.g., disrupting fertile copulations by SIT). During copulation, males transfersperm as well as protein rich fluids through an aedeagus and distiphallus, which females then store in three spermathecae and one ventral receptacle. Within the female reproductive tract, the ventral receptacle and the three spermathecae are strategically separated from each other. I hypothesized males' ability to direct sperm transferwithin the female reproductive tract is through the structures found in the distiphallus. Utilizing scanning electron microscopy, fluorescent light microscopy and light microscopy, coupled pairs were dissected and their genitalic positions relative to each other's structures, specifically the position of the distiphallus during copulation, was deduced. Bifurcation of the sperm ducts within the distiphallus were observed, suggesting males possess the ability to direct sperm toward the sperm storing structures. Changes in female inter­ genitalic distances was found to correspond to findings on female sperm storage previously reported. However, through analysis of the distiphallus position during copulation, the ability for females to control the length of their reproductive tract was discovered, which suggests that a thorough and detailed understanding of the morphologyof the male genitalia in relation to the morphologyof the female reproductive tract may illuminate the important role of co-evolution between the sexes.

ii DEDICATION

This thesis is dedicated to my parents and siblings. Thank you for your guidance, understanding and instilling an appreciation for nature.

iii ACKNOWLEDGMENTS

The completion of this thesis would not have been possible without the support and guidance of numerous people. I would like to show my appreciation to my committee members, Ors. Gary Fritz and Zhiwei Liu, as well as my advisor Dr.

Ann Fritz. Dr. Gary Fritz, I am grateful foryour insight and showing me how to think outside the box. Thank you, Dr. Liu for your help with microscopy and demonstrating the importance of morphological studies. I also have deep gratitude to my advisor, Dr. Ann Fritz, for teaching me to be ambitious, find my own answers and to look where no others have looked. Under her mentorship, I became a much better writer, scientist, and I am grateful for her unwavering patience. I would also like to thank Ors. Barbara Carlsward, Gary Bulla and Scott Meiners as well as Sandra

Baumgartner and Marschelle McCoy their support during my thesis research. The

Florida Department of Agriculture and Consumer Services, Division of Plant

Industry (FOACS-DPI) Biological Control Unit was vital to my project by providing

Anastrepha suspensa specimens. I am also thank the members of the Beckman

Institute Microscopy Unit at University of Illinois Urbana-Champaign for aiding in preparing my specimens for analysis, and for the opportunity for training and access to their scanning electron microscope. Lastly, I would like to thank my family for their continued support.

iv TABLE OF CONTENTS

Abstract...... ii

Dedication ...... iii

Acknowledgments ...... iv

List of Tables ...... vii

List of Figures...... viii

Introduction ...... 1

Sperm Storage and Preservation ...... 2

Sperm Competition ...... 6

Antagonistic Co-Evolution ...... 8

...... Species of Interest ...... 10

Genital Morphology...... 13

Study Objectives ...... 20

Methods ...... 20

Subject Acquisition ...... 20

Determining Distiphallus Length and Location During Copulation... 21

Scanning Electron Microscopy of Male Genitalia...... 23

v Fluorescent Light Microscopy of Male Genitalia ...... 23

Statistical Analysis ...... 24

Results ...... 24

Discussion ...... 25

Literature Cited ...... 30

Tables ...... 38

Figures ...... 39

vi LIST OF TABLES

Table 1. Data from the three time trials ...... 38

vii LIST OF FIGURES

Figure 1. Squash preparation of the female reproductive tract of A. suspensa, (two of the three spermathecae) under UV light (Fritz & Turner

2001) ...... 39

Figure 2. Male genitalia ofA. suspensa in insect saline (63x magnification) ...... 40

Figure 3. Inflation sac scales ( 400x magnification, UV light) ...... 40

Figure 4. Distiphallus in the bursa copulatrix in Anastrepha ludens (Briceno et al.

2010) ...... 41

Figure 5. Full aedeagus penetration, claspers are engaged with female abdomen

(90x magnification)...... 42

Figure 6. Measurement of aedeagus length (in mm) denoted by yellow line (63x magnification) ...... 43

Figure 7. Distiphallus (650x magnification,

SEM) ...... 44

Figure 8. Terminus of the genital hook (650x magnification,

SEM) ...... 45

viii Figure 9. Terminus of the genital hook (3500x magnification,

SEM) ...... 46

Figure 10. Terminus of the trumpet (1500x magnification, SEM) ...... 47

Figure 11. Terminus of the trumpet (1000x magnification, SEM) ...... 48

Figure 12. Distiphallus under UV light ( 400x magnification) ...... 49

Figure 13. Distiphallus inserted into fe male reproductive tract; 5 minutes of

copulation (100x magnification) ...... 50

Figure 14. Distiphallus inserted into female reproductive tract; 10 minutes of

copulation (lOOx magnification) ...... 50

Figure 15. Distiphallus inserted into female reproductive tract; 10 minutes of

copulation (lOOx magnification) ...... 51

Figure 16: Distiphallus inserted into female reproductive ...... 51

ix INTRODUCTION:

Evolutionary processes have resulted in diverse insect genitalia and mating

systems across the taxa. Many species-specific identifying traits rely on the genitalia because of the great genital divergence among closely related species. Male genitalia are especially useful in taxonomic analysis (D'Hotman & Scholtz 1990). For example, genitalia are the primary mean of species identification in the beetle genus

Ptomaphaginus, where the 70 known species in the genus have little discernable difference in external morphology (Schilthuizen & Perreau 2003). Akin to the genus

Ptomaphaginus many insect species are identified by genital morphology

(Schilthuizen 2003). Although insect genitalia exhibit a large divergence between species, the common fundamental anatomy still exists with extreme modifications,

presumably driven by the forces of sexual selection, including those of female and

male interests in the reproductive dynamics. Male insect genitalia are thought to be under intense selection because effective coupling and sperm transfer increase male fi tness (e.g., males ability to sire offspring). Having species-specific genitalia also allows females to ensure incompatible inter-species mating does not occur. Intense selective pressures drive genitalic evolution at a rapid rate compared to other body structures, thus allowing for even closely related species to have largely divergent genitalic morphologies (Hosken & Stockley 2004).

1 SPERM STORAGE AND PRESERVATION:

A major adaptation found in the female arthropod reproductive tract is the specific structures capable of storing sperm. Sperm is stored in specialized organs called spermathecae, and in some circumstances also a fertilization chamber, both are apparently specific adaptations selected for by natural or sexual selection

(Brown 1985, Uhl 2000). Specialized sperm storage organs allow females to store sperm for extended periods of time before ova fertilization (Eberhard 1996,

Simmons 2001). By delaying the time between sperm ejaculation during copulation and ova fertilization, females may gain more control over paternity. In some insect species, females can reject a male's sperm after copulation has occurred rather than engaging in costly copulation resistance, as has been observed in Drosophila melanogaster (Manier et al. 2010). If a male mates with a female who ultimately voids his sperm rather than fertilizing her ova with the sperm, such copulations are thought not to cause undo harm to the female in the sense of a loss in resources. As a strategy, the female can accept the copulation and subsequently reject the sperm

(Manier et al. 2010). In species where females mate multiple times, sperm storage may ensure quality sperm is being used in fertilization of ova, and storing multiple males' sperm could increase the genetic diversity of her offspring. Sperm storage capacity and duration are also likely to be under strong selection because these characteristics impact reproductive success. A female may only need to copulate at one stage in her lifetime (e.g., a mating flight)with many males to garner the sperm she needs for her entire lifespan (Collins et al. 2006). Sperm storage capacity is the limiting factor in fire ant queen reproductive longevity. Since fire ant queens mate

2 with multiple males once, having larger sperm storage capacities would greatly benefit a female, increasing her reproductive longevity, and therefore reproductive success (Tschinkel 1987).

After the female structures for sperm storage had evolved, mechanisms for maintaining the viability of the sperm also developed (Pitnick et al. 1999). Sperm stored by females remain viable for several years in honeybees, and up to several decades in ants; sperm viability is ensured by the presence of proteinaceous enzymes and anti-oxidizing compounds (Collins et al. 2004, Pamilo 1991). Sperm must be protected not only upon storage in the sperm storage organs, but also during the initial movement to the sites of storage. In many cases, male insects ejaculate seminal fluids composed of a mixture of proteins with the spermatozoa.

The seminal fl uid proteins play varied roles, from protecting the sperm from damage before being stored in the female sperm storage sites, to manipulating female physiology and behavior (Chapman 2001, Wolfner 2002, Spencer et al. 1992,

Spencer et al. 1995). Females of some species also produce compounds to protect the sperm fr om degradation and allow for long-term sperm storage (Collins et al.

2004). In honeybees, and other social insects where sperm is stored for individual females' entire life, antioxidative enzymes are produced. Antioxidative enzymes produced by females increase the preservation of the stored sperm, protecting the sperm from reactive oxygen (Collins et al. 2004).

Both sexes' fitness is enhanced by sperm preservation, since sperm viability leads to successful ova fertilization. If the sperm within a sperm storage site becomes inviable, the cost of resources males and females expended into copulation,

3 sperm storage and egg production are wasted. Indeed, both sexes lose valuable energetic investments when sperm viability and integrity are lost, but the costs paid by fe males may be higher as females have a greater resource deficit (Daly 1978,

Rowe et al. 1994, Stockley 1997). Males possessing enhanced abilities to produce sperm-preserving compounds in their seminal fluid may have a selective advantage compared to males lacking the ability to produce such compounds. Natural selection may, then, act as a selective force fo r males whose sperm were viable longer.

However, it must be noted that male preservation compounds only remain functional fo r a limited period of time.

The sperm-protecting compounds fo und in male seminal fluid may also play a role in sperm preservation prior to ejaculation (Hunter & Birkhead 2002). Females continue the preservation process by producing endogenous compounds; the production of these compounds has been fo und to be costly for females. In the ant species Atta colombica, initial sperm storage has been fo und to reduce queen ants' immune response, making newly mated queens vulnerable to pathogens (Baer et al.

2006). As more males contribute to the stored sperm, the queen's immune response declines incrementally downward. The risk to the queen of compromising the immune system is warranted fo r Atta colombica, as females in this species do not re­ mate, making the female's reproductive success solely dependent on the sperm stored during the initial copulations (Baer et al. 2006).

Not all species store sperm for multiple years or until they die like honeybees and ants (Baer et al. 2006, Collins et al. 2004). When males of a mating system where sperm is only stored for one mating, or for the purpose of delaying the time

4 between sperm ejaculation and ova fertilization by the female, produce sperm

preserving compounds, they produce more of them. In this case, the metabolic

"burden" of producing more preserving compounds falls to males, because females have less interest in preserving sperm for long-term storage. The viability of sperm has been found to have an influence over paternity, suggesting sperm preservation by males is a trait potentially under selection (Garcia-Gonzalez & Simmons 2005).

Since the male produced sperm preserving compounds keep the sperm viable for the length of time needed by females, fe males can minimize the production of sperm preserving compounds.

When copulation and sperm deposition are temporally separated from ova fertilization, a female that has stored sperm can easily mate with another male if the sperm previously stored is no longer viable, although resources such as time and energy may be wasted, and risk of predation rises, increasing mortality potential

(Cordero & Eberhard 2003, Gavrilets et al. 2001). However, mating with multiple males may have a direct increase on reproductive success in fe males (Arnqvist &

Nilsson 2000). In order to reduce wasted resources by attempting to fertilize eggs with inviable sperm, females would select males who had more prolonged sperm preservation, which may be denoted by pre-copulatory, copulatory, or post­ copulatory behavior, increasing the males' ability for sperm self-preservation

(Garcia-Gonzalez & Simmons 2005). Through natural selection, females may have been selected to choose males possessing behaviors that signal high sperm vitality.

Mating systems utilizing long-term sperm storage strategies rely on females to maintain the vitality of stored sperm, because the males' preservation

5 compounds do not have long-term viability. Thus, storing sperm long-term requires constant preservation, which can only be given through continuous production of sperm preserving compounds by the female. Such long-term sperm storage is costly for females, but females benefit by increasing their reproductive success; many species with long-term storage do not re-mate (Baer et al. 2003 & Baer et al. 2006).

In species with short-term storage, males are required to preserve sperm once ejaculated. The preserv ation compounds produced by the male can maintain short­ term storage, but not long-term storage. Being able to re-mate, females show less reliance on the sperm they have stored; therefore, females do not put the resources into ensuring relatively long-term storage. However, secretions produced by the female sperm storage organs have been found to play an important role in oviposition, suggesting females are reliant on producing endogenous reproductive secretions which may play dual roles of sperm preservation and oviposition thus allowing males to put less resources into preserving their own sperm after ejaculation (Schnakenberg et al. 2011).

SPERM COMPETITION:

Storing sperm from multiple males in the same site of sperm storage may drive competition between different males' spermatozoa or between male compounds (i.e., sperm competition). Males are thought to have been under selection to produce sperm that outcompetes other males' sperm in some manner.

For example, a competition between different males' spermatozoa may occur when sperm motility is needed to reach the ovum for fertilization; the spermatozoa

6 reaching the ovum, motility or "swimming speed" may be the character under selection by male-male competition (Parker 1982). In species where fertilization occurs directly after ejaculation the sperm compete to be first to fertilize the ovum.

The sperm traits under selection in this instance are length and speed; such sperm are short-Jived, as they do not have to be stored (Baer et al. 2003). Many researchers' findings suggest that greater sperm length and size increase fe rtilization success (Lamunyon & Ward 1998, Otronen et al. 1997, Radwan 1996).

Correlations between sperm morphology and female reproductive tract have been documented in many insect species, suggesting female mediated selection (Dybas &

Dybas 1981, Hihara & Kurukawa 1987, Pitnick & Markow 1994, Pitnick et al. 1999).

The female reproductive tract selects for longer sperm morphology, creating a bias in paternity outcome for males who have comparatively long spermatozoa (Miller &

Pitnick 2002).

Females are known to not only mediate sperm competition by providing the

"arena" under which sperm competition occurs, but also drive the selection for sperm removing structures found on the male genitalia of many insect species. The male sperm-removing structures allow males to remove the stored sperm of a previously mated male, eliminating any competition that may have been produced by the initial male's sperm being present within the female. An example of mechanical sperm removal by a male can be found in species of damselflies, which have structures on their genitalia that allow for the removal of the sperm from the previously mated male (Tsuchiya & Hayashi 2014). The existence of male conducted sperm removal explains why males who are second to copulate are more likely to

7 sire the offspring (Danielsson 1998), given a circumstance where females receive equal amounts of spermatozoa from each male. In this case, the order of male copulation and the temporal use of the sperm by the female may play an important role in paternity outcomes. In the medfly, sperm from the first male to copulate were found to be homogenously distributed in the distal portion of the fertilization chamber alveolus. The sperm of the second male to copulate was found to be concentrated in the central portion of the fertilization chamber alveolus. However, the separation of sperm was not maintained over time, thus the temporal use of the stored sperm may result in differing paternity outcome. Fertilization of the ova that occurs shortly after the second male's sperm is stored will increase the second male's chances of siring the offspring. Once the second male's sperm becomes depleted, the first male's sperm is readily available, however, sperm mixing eventually eliminates the possibility of second male precedence (Scolari et al. 2014).

ANTAGONISTIC CO-EVOLUTION:

In polygamous species, the control over paternity and the benefit for females to choose which male sires her offspring play a large role in the selection pressures on the females' reproductive tract. The battle over control for paternity leads to the evolution of traits resisting the control the opposite sex has in paternity outcome

(Gavrilets et al. 2001). Antagonistic coevolution is dependent on promiscuity. If sexual conflict between the sexes existed among a monogamous model, traits that lowered the reproductive success of one sex would equally lower the reproductive success of the opposite sex. In models where multiple mating exists, the overall

8 fecundity of an individual differs from its mates, leading to antagonistic coevolution.

Introducing promiscuity allows for the selection of traits that may reduce the reproductive success of the opposite sex (Holland & Rice 1999). As the fe male reproductive tract evolves to evade the male's mechanisms of ensuring the use and storage of his sperm, the males' genitalia should evolve to neutralize the female genitalia adaptation. The evolution of the male genitalia is thought to be mediated, at least partially, by cryptic female choice (Eberhard 1997, Eberhard 1996).

The advancement of male traits that only improve a male's attractiveness to a female can be explained through the "sexy sons" hypothesis. "Sexy sons" selection is self-perpetuated, in that it selects for females that choose males with attractive traits, not necessarily traits improving offspring viability (Cameron et al. 2003,

Hosken & Stockley 2004, Simmons 2001). Females selecting for attractive male traits will produce offspring that possess the same traits, gaining fitness through the inherited male traits of attractiveness. The male offspring will have improved reproductive success through possession of traits attractive to females and the female offspring will benefit by choosing males that will produce offspring with the same attractive traits (Andersson & Simmons 2006). The selection of males with such traits often coincides with females selecting for antagonistic adaptations in males. Although the antagonistic characteristics of males reduce the direct reproductive success of a female, the female benefits by producing sons with the same traits that control or manipulate offspring paternity (Iwasa & Pomiankowski

1995, Cordero & Eberhard 2003).

9 SPECIES OF INTEREST:

Tephritid flies are economically important agricultural pests of over 300 fruit and vegetable plants (Morse 1995). The new world endemic genus, Anastrepha, is composed of 192 species, which are all restricted to tropical and subtropical environments (Aluja 2000, Aluja 1994). Originating in South America, Anastrepha have relatively rapidly and recently speciated (Hernandez-Ortiz & Ortiz 1992,

Morgante et al. 1980). In fact, species of the genus Anastrepha have been foundto be too recent for coevolution with host plants, which may contribute to the generalist feeding of some of the pest Anastrepha species as well as the severe and sometimes detrimental effect on host plants (Norrbom 1985). Ranging from the southern parts of the United States and most of Mexico, Central America, South America and the

Caribbean Islands, Anastrepha species have become the bane of many agriculturists due to their mass destruction of crops via larval infestations (Aluja et al. 1993).

Anastrepha suspensa (Leow) is a new world fruit fly pest belonging to the dipteran familyTephritidae and is endemic to the Greater Antilles, including the islands of Cuba, Jamaica, Hispaniola, Puerto Rico, and also Florida (Norrbom & Foote

1989). The migration into Florida was thought to have occurred in three separate and isolated incidences, but recent genetic analyses have found that there is constant gene flow from the Caribbean into Florida, most likely due to the movement of infested fruits through human traffic (Boykin et al. 2010, Kendra et al.

2007).

10 The female A. suspensa deposit their eggs in the host species' ripening fruit, where the larvae feed on the pulp of the fruit. The fruit characteristics (i.e., pH, temperature, level of rotting) help determine the development of the larvae, but after three instars of larval development, the third instar leaves the fruit and burrows into the ground to pupate, where the depth of pupation is subject to the soil chemistry and composition (Aluja 1994, Bressan & Coast-Teles 1990). The length of time required for development into an adult is greatly influenced by environmental factorsand sex of the individual (Aluja 1994, Baker 1945, Celedonio­

Hurtado el al. 1988, Leyva et al. 1991, Shaw 1946, Sivinski 1990). After the pupal stage, the adult fly emerges from the ground and goes through a maturation period before becoming sexually active (Aluja 1994, Christenson & Foote 1960).

Since A. suspensa specifically is a pest of at least 80 different plant hosts found in Florida, such as economically important citrus (e.g., oranges and grapefruits) control measures to prevent or slow the migration of A. suspensa into other parts of the United States have been instituted (Swanson & Baranowski 1972).

Large-scale economic damage has instigated various control strategies, including the use of pesticides and biological control methods.

Pesticide application has been an important historical component in containing the growth ofestablished A. suspensa populations, but pesticides have been found to lose effectiveness over time due to resistance evolving in the targeted species (Vontas et al. 2011). Pesticides have also been found to cause environmental damage and allow for an increase in secondary pests (Ehler & Endicott 1984, Soto­

Manitiu et al. 1987). Along with killing the pest species, pesticides also kill many

11 other benign or even beneficial insect species, including those species that prey on the pest species. The release of predatory species such as hymenopteran parasitoids may become a useful biological control method, though currently rarely used

(Ovruski et al. 2000). Another biological control method used against A. suspensa is the sterile insect technique (SIT). SIT relies on the release of massive numbers of sterile males (male flies whose sperm are rendered sterile due to treatment by ionizing radiation) into feral fly populations. The sterile males mate with fe ral females causing a reduction in fe rtilized eggs. However, since female A. suspensa have the ability to store multiple males' sperm, sterile males' sperm may or may not be used in ova fertilization, or it may be used in addition to viable sperm the female has stored fro m feral males, both affects reduce the overall effectiveness of SIT

(Klassen & Curtis 2005). The males used in SIT may also lack desired traits found in the males of feral populations. SIT uses males raised in mass rearing facilities,where

"negative" artificial selection may be facilitated. The couples with shorter copulation times may be selected over couples with long copulation times due to time management of the facility. The couples that have finished copulation may be taken for egg production, where the couples still copulating are not used and no offspring are reared. Selecting for males that may have shorter copulation times may contribute to lowering the success of SIT in controlling A. suspensa. Having shorter copulation times, males may have reduced chances of their sperm being stored. Since females can reject males' sperm after ejaculation, maintaining copulation post-ejaculation may increase a male's chance of having sperm stored rather than rejected (Manier et al. 2010).

12 GENITAL MORPHOLOGY:

Anastrepha suspensa possesses complex genitalic structures in both males and females; these structures are likely under the previously discussed selective pressures. The parts of the female reproductive tract that are of interest are the three spermathecae (each having a separate duct, duct musculature and valves, which converge to a single opening tubercle in the bursa copulatrix) and the ventral receptacle (Fig. 1, courtesy of Fritz & Turner 2001). The role of the spermathecae and the ventral receptacle is sperm storage, with the duration of sperm storage varying between the spermathecae and the ventral receptacle. The spermathecae are found at the base ofthe lateral oviducts and lie across from the ventral receptacle, putting the spermathecae and ventral receptacle on opposite sides ofthe reproductive tract (Fig. 1). However, when the female reproductive tract is represented in its natural form the tubercle and base of the ventral receptacle are in close proximity. Producing glandular secretions, the spermathecae may be used in long-term sperm storage, compared to the ventral receptacle, which lacks glandular cells. Lying across from the ventral receptacle, the spermathecae may transfer sperm to the ventral receptacle via osmoregulatory actions. The ventral receptacle also stores sperm but no evidence of long-term storage has been observed (Dhakal

2008, Fritz 2004, Fritz & Turner 2001, Twig & Yuval 2005).

The external genitalia of the male, which interacts directly with the female reproductive tract, is comprised of an aedeagus and a distiphallus (Fig. 2). The

13 aedeagus is a thin, flexible, chitinous tube connected to the testes via genital ducts and used to deliver sperm and ejaculate fluids. The aedeagus of many insect species have been found to have little allometric growth, with the length of the aedeagus complementing the female reproductive tract (Ohno et al. 2003). The coevolution between the sexes drives the aedeagus length to match the reproductive tract length of the female. A male with an aedeagus length falling short of that needed to deposit sperm close to the sperm storage sites is disadvantaged. An aedeagus, which is too long relative to the female's structures, may reduce a male's ability to continue copulation after the fe male makes attempts to terminate copulation. Aedeagus that are too Jong will create space between the male's and fe male's external, abdominal structures during coupling due to the aedeagus "bottoming out" and not being able to be inserted any farther in the female reproductive tract. The increased space between the copulating couple will generate difficulty for the male, in the sense of continuing copulation, when the female attempts to terminate copulation. (Jaspers

(Fig. 2), which are found at the base of the aedeagus and aid the male during copulation by holding on to the female's abdomen, will have reduced function when the aedeagus length is too long compared to female reproductive tract length.

Practically speaking, the void between the copulating couple, created by the male not being able to fully penetrate the female, will not allow the claspers to engage the female's abdomen. Removing one or both claspers from aiding males during copulation have been found to have detrimental effects on successful copulations.

By removing the claspers in the heteropteran species Stenomacra marginella, intromission success decreased from 62%-0%. The claspers of S. marginel/a have

14 been found to play a direct role in sperm transfer, however this does not reduce the importance of claspers in Tephritids (Moreno-Garcfa & Cordero 2008). The removal of claspers in tephritids may not show the persuasive results found in S. marginella but claspers do play a role in controlling females during copulation and increasing copulation time (Aluja et al. 1993, Tychsen 1978).

Males having an aedeagus that complement the dimensions of the female reproductive tract will be able to both deposit sperm near the sperm storage organs and counter a female's attempt to terminate copulation due to the reasons previously discussed, which puts the female at a disadvantage in controlling the paternity of offspring at this point in copulation. A male's ability to copulate as long as the he is not dislodged is a trait in A. suspensa. Fritz (2009) showed that normal males mating with decapitated fe males (e.g., females unable to mount a "dislodging" behavior) mated for prolonged periods of time (e.g., up to 160 min).

As discussed earlier, sexual selection in promiscuous species will lead to a reduction in reproductive success in one sex or the other, which in the current example the fe male reproductive success is reduced. Being able to prevent sexual selection from shaping male genitalia to reduce the control females have over paternity, would prove advantageous to females. One possible mechanism to prevent males from increasing paternity control via aedeagus length is for females to possess a dynamic reproductive tract, or a reproductive tract that can be shortened or lengthened during copulation via musculature. A dynamic reproductive tract may prevent uni-directional sexual selection from allowing males to gain an advantage through aedeagus length, for females could cryptically apply

15 selective pressures that would produce an aedeagus length that is beneficial to females. Having a dynamic reproductive tract may allow females to control the distance between the site of sperm deposition and the sperm storage organs. By altering the distnnce to the sperm storage organs, females can increase control of paternity by dictating where sperm is deposited within the reproductive tract. If a female encounters a male with an adeagus that is too long compared to the contracted reproductive tract of the female (which would bias control toward the male allowing access to deposit sperm at the sperm storage sites) the female could retract the sperm storage organs where the male cannot reach the sperm storage sites, reducing the male's reproductive success while increasing the female's.

Another defense females may have against a "long" aedeagus is to maintain the contracted reproductive tract, which could create difficulty for the male's claspers to engage the female because the male is not granted full penetration. Since having an aedeagus that is too long, compared to the aedeagus length females are selecting for according to the dynamic reproductive tract model, males with "shorter" aedeagus rather than "longer" would be selected. However, males who have too short of aedeagus may not be able to reach the sperm storage sites, for the female may be able to only constrict the reproductive tract to a certain extent. Studies of Tephritid copulation have fo und the distiphallus falls short of the sperm storage sites

(Eberhard & Pereira 1995, Eberhard 2005). The distiphallus falling just short of the sperm storage sites allows females to utilize the dynamic reproductive tract and easily grant or deny access to the sperm storage sites. Non-allometric growth of the aedeagus might be understood as the result of selection against aedeagus that are

16 too long and too short, in the constraining presence of the dynamic female reproductive tract.

The distiphallus, like the aedeagus, functions in sperm delivery. However, unlike the aedeagus, the distiphallus has evolved into a much more complex structure used in the deposition of sperm. Within the distiphallus there are multiple substructures, including the trumpet, genital hook and inflation sac, each playing an important role in improving the male's chances of reproduction and influencing cryptic femalechoice. In Anastrepha ludens the trumpet has been found to be the only channel for sperm delivery, and is directed toward the spermathecae (Briceno et al. 2010). In A. ludens, the genital hook and the inflationsac is used to securely lodge the distiphallus in the female reproductive tract. The genital hook has been observed to firmly affix into the opening of the ventral receptacle, blocking the opening of the ventral receptacle to sperm deposition via the trumpet and directing all sperm deposition to the spermathecae. The inflationsac, once inserted into the female, inflatescreating difficulty for the female in terminating copulation by dislodging the male. Scales present on the outside of the inflationsac (Fig. 3) are oriented to resist the removal of the male genitalia and aid in securing the male genitalia in the female reproductive tract (Briceno et al. 2010). The layout of the distiphallus in A. ludens coincides with the arrangement of the female reproductive tract, consistent with a co-evolutionary history (Fig. 4). The genital hook interacts with the ventral receptacle and the trumpet delivers sperm to the spermathecae.

The separation of sperm storage sites to possibly reduce the control males have in sperm storage is mitigated by the distiphallus. The process of sperm storage and

17 transfer from spermathecae to ventral receptacle adds limitations when increasing distance between the two sperm storage sites. Using the spermathecae to replenish the ventral receptacle for egg fertilization means that the proximity of long term to short term storage sites is a constrained distance. However, the females' dynamic reproductive tract allows for the increase in distance from sperm deposition by males and the sperm storage sites. The ability to control the distance of the sperm storage sites from the distiphallus may be what allows the spermathecae and ventral receptacle to remain in close proximity, offsetting the influence males may have toward sperm storage.

Although the functions of the distiphallus of A. ludens have been elucidated, the findings of the genital hook blocking the opening to the ventral receptacle contradict previous studies of sperm storage patterns in A. suspensa. The ventral receptacle has been found to be the most likely of the sperm storage sites to store sperm and store the most sperm (Fritz 2004). If the genital hook blocks the opening to the ventral receptacle and the deposition of sperm there, the ventral receptacle would not be expected to show higher frequencies of sperm storage compared to the three spermathecae. One would predict that no sperm would be found in the ventral receptacle directly after copulation if the opening were blocked. The sperm found in the ventral receptacle is also used in egg fertilization, making the ventral receptacle the optimal sperm storage site for a male to have sperm stored in terms of reproductive fitness (Solinas & Nuzzaci 1984). Having sperm in the ventral receptacle greatly increases a male's chance of reproduction, and blocking access to the ventral receptacle with the genital hook seems counter to male fitness interests.

18 Having sperm ducts within the genital hook would support the sperm storage patterns seen in A. suspensa and the reproductive benefit granted to males by having sperm stored in the ventral receptacle.

If the trumpet delivers sperm as well as the genital hook, a bifurcation of the sperm ducts within the distiphallus would be evident. Distiphallus bifurcation has been observed in many insect taxa, but not in the genus Anastrepha. Given what is understood about the function of the sperm storage organs in A. suspensa, with regards to how the sperm storage organs utilize the stored sperm, the existence of distiphallus bifurcation can be seen as benefiting male interests. By possessing the ability to direct sperm deposition toward both the ventral receptacle and the confluenceof the spermathecal ducts (e.g., the female tubercle located in the bursa copulatrix), males can ensure their sperm is positioned to be stored for egg fertilization (ventral receptacle) and long-term storage of their sperm for future offspring (spermathecae), iftaken up by the female. If males where to only deposit sperm at the spermathecae, then the use of their sperm for egg fertilization is not guaranteed, for the female may not transfer the sperm into the ventral receptacle.

Depositing sperm only at the ventral receptacle may increase a male's chance of fertilizing an egg, but the sperm in the ventral receptacle is for short-term use, reducing a male's fecundity over time. Having sperm in both storage sites grants a male increased chance of egg fertilization over an extended period of time, increasing the overall amount of eggs fertilized by the male. Although males may increase the chances of reproduction through the distiphallus bifurcation, females

19 can still choose to store sperm after copulation or reject the sperm (Manier et al.

2010).

STUD Y OBJECTIVES:

The purpose of this study was to test the hypothesis of distiphallus bifurcation and sperm transfer control in male Anastrepha suspensa. Understanding the morphology of the male genitalia may elucidate relationships among male and female reproductive tract structures, which could potentially be a subject of future work on antagonistic co-evolution between the sexes. Males' ability to direct sperm in the female reproductive tract would increase sperm storage and use in egg fertilization. Ifmales do have the ability to assist probable sperm storage, the mechanisms and morphology of the adeagus and specifically the distiphallus will complement the female reproductive tract (Thornhill & Alcock 1983). As the female reproductive tract changes to combat male control tactics, the male reproductive tract will be selected to combat the female control tactics, creating an evolutionary arms race between the sexes. The inter-sexual competition will create more complex and elaborate reproductive tracts and structures (Thornhill & Alcock

1983).

METHODS:

SUBJECT ACQUISITION:

To test the hypothesis, A. suspensa pupae were obtained from the Florida

Department of Agriculture and Consumer Services, Division of Plant Industry

20 (FDACS-DPI) Biological Control Unit and mass-rearing facility and housed in the

Department of Biological Sciences USDA Quarantine Facility at Eastern Illinois

University. Pupae were placed in plastic brood chambers consisting of 1.5L plastic jars with a stockinet sleeve for access. The brood chambers were held under a photoperiod of 14h light : lOh dark, temperature of 25±22C, and a relative humidity level of :::::80% RH. Twenty-four hours after pupal emergence, teneral adult flies were segregated by sex, using an insect aspirator, to ensure unmated status in the individuals. The adult flieswere supplied water, via wicked plastic containers, and fed 1:3 ratio of hydrolyzed yeast and sugar. Each adult chamber housed 100 individuals of a single sex.

DETERMINING DIST/PHALLUS LENGTH AND LOCATION DURING COPULATION:

Copulating couples were studied to further elucidate the precise location of the male genitalia, specifically that of the distiphallus, within the female reproductive tract. Thirty males and thirty females were combined in a plastic container during the last 5 hours of photophase. Individuals used in the copulation trials were two weeks old to ensure full sexual maturity (Kendra et al. 2006). After copulation began (i.e. male genital insertion into the female gonopore) the couples were frozen at 5 minutes in a standard chest freezer, terminating copulation. The same protocol was repeated with the copulations being terminated at 10 minutes into copulation, and again at 15 minutes into copulation. After being frozen, the females' reproductive tract including the male genitalia, were dissected in situ under a stereoscope and photographed under a compound-scope at lOOx magnification to

21 reveal the placement of the male genitalia in referenceto the female's reproductive tract. Only couples that had full aedeagus penetration, which was determined by the claspers engaging the female abdomen, were used (Figure 5). If the adeagus did not have full penetration the measurements would not represent the inter-genital distance of couples during sperm transfer. The distance between the genital trumpet and the entrance of the ventral receptacle (inter-genital distance) were measured using lmagej version 1.440, with a scale of 100X magnification.

Aedeagus length of the copulated males was measured to determine the variation among the individuals, which was used to compare the placement within the female tract. Afterthe distance between the genital trumpet and the ventral receptacle was measured, each aedeagus was removed from the already copulated males and isolated from each other in individual baths of 60% acetic acid and 40% insect saline to break down the chitin in the aedeagus. By reducing the chitinous outer layer, the coiled aedeagus was straightened to allow for an easier measurement of length to be taken. Each aedeagus was soaked in the 60% acetic acid bath for 72 hours, washed fourtimes with DI water and soaked in cellosolve for

72 hours. The cellosolve removed acetic acid to allow for proper slide mounting.

After the two chemical baths, each aedeagus was mounted on a standard microscope slide using Euparol as the mounting medium. Each slide was cured in an oven at 260C for 5 days. Once the slides had cured, each slide was photographed under a dissecting scope at 63X magnification. The photograph of each aedeagus was uploaded to ImageJ, set to the scale for 63X magnification and the aedeagus was

22 measured from the proximal point of the aedeagus to the end of the distiphallus, measuring the outside edge (Figure 6).

SCANNING ELECTRON MICROSCOPY OF MALE GENITALIA:

Scanning electron microscopy (SEM) was also used to examine the morphology of the aedeagus and distiphallus. Genitalia were removed from male flies and placed in 2.0% paraformaldehyde and 2.5% gluteraldehyde solution in a

0.1M Na-cacodylate buffer, which had a pH of 7.4 (both para formaldehyde and gluteraldehyde were E.M. grade). Once in the solution, the specimens were placed on ice for 4 hours. After being on ice, the specimens were rinsed in O.lM Na­ cacodylate buffer for 10min on a shaker table. The specimens were dehydrated by being placed for 10min each in ethanol solution of 37% Etoh, 67% Etoh, 95% Etoh and 100% Etoh, respectively, three times. Dehydrated specimens were placed and kept in a critical point dryer until being sputter coated with gold-palladium and analyzed by SEM.

FLUORESCENT LIGHT MICRSCOPY OF MA LE GENITALIA:

In addition to SEM, fl uorescent light microscopy was employed to understand the morphology of the male genitalia. Aedeagus specimens were removed from males and slides were made using squash-prep technique in an insect saline solution. The specimens were examined under ultra-violet light

(wavelength=450nm. Due to the auto-fluorescing properties of the chitin within the aedeagus and distiphallus, additional staining was unnecessary to illuminate the

23 morphological structures. The auto-fluorescence also allowed for observations of what structures within the aedeagus and distiphallus were non-chitinous. Images were taken of the structures under ultra-violet light for analysis of the genital structures.

STATISTICA L ANAL YSIS:

Two tailed, two sample equal variance t-tests were used to determine the correlation between the three copulation trials (5 minutes, 10 minutes and 15 minutes) and the aedeagus length ofthe individuals in each trial.

RESULTS:

The three trials had varying sample sizes, ranging from 11-21 subjects (Table

1). When compared, the inter-genital distances of the five minute and ten minute trials yielded a t-stat of <0.01, the ten minute and fifteen minute trials had a t-stat of

<0.01, and the five minute and fifteen minute trials t-stat equaled 0.96 (Table 1). The mean inter-genital distance was 0.93mm for five minutes, 0.63mm for ten minutes and 0.93mm forfifteen minutes. The aedeagus length of males within each trial showed t-stats of 0.97 for five minutes and ten minutes, 0.95 forten minutes and fifteen minutes and 0.93 for five minutes and fifteen minutes (Table 1). The mean aedeagus length was 1.49mm for all three trials (Table 1).

The heretofore un-described distiphallus of A. suspensa, composed of a genital hook, trumpet and inflation sac (Fig. 7). The terminus of the genital hook showing the opening of the sperm duct (Fig. 8-9). The terminus of the trumpet and

24 trumpet sperm duct opening (Fig. 10-11). The distiphallus under ultra-violet light, showing the heretofore-undiscovered bifurcation of the sperm ducts in A. suspensa

(Fig. 12). The inter-genital distance at five minutes of copulation (Fig. 13). The inter­ genital distance at ten minutes of copulation (Fig. 14-15), and the inter-genital distance at fifteen minutes of copulation (Fig. 16).

DISCUSSION:

Sperm ducts in the genital hook, along with bifurcation, have been seen in other Tephritid species, but neither bifurcation nor sperm ducts in the genital hook have been observed in A. suspensa. Since the morphology of the male genitalia has been demonstrated to be the most species-specific organ, morphological studies of other species' aedeagus and distiphallus cannot be substituted for studies of A. suspensa genitalia (Briceno et al 2010, Gornostaev et al. 1998, Marchini et al. 2001).

The images obtained from the SEM did provide evidence showing the presence of bifurcation in the distiphallus. The opening of the genital hook can be seen from the dorsal side of the distiphallus (Fig. 7), and from the proximal side (Fig. 8-9). The opening in the trumpet can be seen, along with a channel between the genital hook opening and the trumpet opening (Fig. 10-11). The channel along the proximal end of the distiphallus does not appear to have openings for sperm transfer (Fig. 12).

The chitinous regions of the distiphallus can be seen where the chitin auto­ fluoresces under UV light (Fig. 12). Both the genital hook and trumpet are heavily chitinous, with the structures forming apparent bifurcating ducts. The channel that is seen in the SEM images does not appear to have chitinous ducts, suggesting there

25 are no sperm ducts within the channel proper. When the distiphallus is compared to

' A. suspensa s female reproductive tract (Fig. 1), the bifurcation directly compliments the female's sperm storage sites. The genital hook directs sperm toward the ventral receptacle and the trumpet directs sperm toward the spermathecae. It is intriguing to speculate that having the ability to direct sperm to the sperm storage sites greatly increase a male's chance of having his sperm stored by the fe male. Ejaculating sperm shallow in the reproductive tract, fa r from the sperm storage sites, may allow a female to reject the sperm easier (Manier et al. 2010). The advantage a male gains over other males by having bifurcation is what may drive the selection for the ability to direct sperm, but the expansion of the female tract is what appears to set the conditions for competition among males, and drives the morphology of the distiphallus. As the fe male tracts' sperm storage sites separate within the genitalia to reduce the control males have when depositing sperm, selective pressures are put on the male genitalia to maintain control in sperm deposition and ensure sperm deposition occurs at a prime location within the female (i.e. at the sperm storage sites).

Bifurcation in the distiphallus does not automatically increase a male's control over paternity due to a wide range of processes that are known to affect paternity even after sperm ejaculation and copulation has occurred (Eberhard

1996). One of the processes possibly affecting paternity is the female's ability to control the length of her genital tract (Table 1). The inter-genital distances between the five-minute trials and the fifteen-minute trials are statistically similar, not rejecting the null hypothesis of the inter-genital distance being different among

26 trials. However, the inter-genital distances recorded from the ten-minute trials are significantly different when compared to both the five-minute trials and fifteen­ minute trials. The mean inter-genital distance for the ten-minute trial is less than the means from the five-minute and fifteen-minute trials, suggesting that the female contracts her genital tract between five and fifteen minutes of copulation duration, then returning it to the initial length. The genital tract contraction seen in the ten­ minute trials corresponds with previous studies in sperm storage patterns in A. suspensa, which found sperm storage to increase near the ten minute mark of copulation duration (Dhaka! et al. 2017, Fritz 2004). The findings of Dhaka! et al.

(2017) and Fritz (2004), combined with the results of the copulation trials, further suggest females are contracting the genital tract, decreasing the inter-genital distance and beginning to store sperm near the ten minute mark of copulation.

With the data of the aedeagus lengths, which are convergent with the findings of Dodson 1978, showing much less variation than the variation found in the inter-genital distances across the three trials, the discrepancies among the inter­ genital distances lie within the female (Table 1). All the trials were significantly similar among aedeagus lengths, and ensuring all couples recorded had full aedeagus penetration removed any bias (Fig. 5). Having full aedeagus penetration will allow for the comparison of aedeagus length variation and inter-genital distance variation to be accurate.

The ventral receptacle has been found in previous studies to receive sperm before the spermathecae, which may be explained through this study (De Carlo et al.

1991, Fritz 200'1·,Scolari et al. 2014). The distiphallus fallsshort of the ventral

27 receptacle in many cases (Fig. 13-14,16), if sperm were deposited by males short of the sperm storage sites and stored by the female it would obviously reach the ventral receptacle before the spermathecae due to the anatomy of the female reproductive tract

Although the female may contract her genitalia to allow a male access to the sperm storage sites (Fig. 15), the distiphallus being short of the sperm storage sites shows that females can control where a male's genitalia lie with reference to her sperm storage sites. Having the ability to control the length of their tract, females may have the ability to ensure paternity control is reduced in males. The selective pressures for genital control in females may arise, in part, from the battle for paternity control, having control of the genital tract length reduces the effect of bifurcation in the distiphallus, for a female can control where the distiphallus lies within her genital tract. If a male does copulate with an unconsenting female, that female has the ability to prevent the male from depositing sperm deep within her genital tract. Having a shallower ejaculation, the female may eject the sperm more easily than if the ejaculation were deep within her tract (Manier et al. 2010). The bifurcation within males plays a role when the female's genital tract is contracted to where the distiphallus lies at the sperm storage sites. Once a male has access to the ventral receptacle and spermathecae, distiphallus bifurcation allows for sperm deposition at both sperm storage sites within the female. Ejaculation may most likely occur close to ten minutes into copulation, which is why the results show female contraction of the reproductive tract being greatest in the ten-minute trials

(compared to the five and fifteen minute trials). However, the ten-minute trials

28 showed variation in inter-genital distance as well as the other trials, which suggest factors other than time of copulation, may play a role in the dynamics of the female reproductive tract.

Through understanding the mechanisms of intromission and the role each sex plays, control over an invasive species such as A. suspensa may become easier through non-pesticide methods. The currently employed SIT will only work if the released males' have their sperm stored and used in egg fertilization. Understanding which male traits fe males' preferand which male traits are selected for in male­ male gametic competition gives SIT mass-rearing programs a means to craft breeding programs. Currently, artificial selection exerted in breeding facilitiesmay enhance the frequency of traits that are less beneficial. Copulation duration in laboratory-raised A. suspensa has been foundto be much Jess than feral flies

(Masomenos el al. 1977). By reducing the copulation duration, the probability of a sterile male's sperm being stored by a wild fe male is reduced (Manier et al. 2010).

The reduction in copulation duration may be due to morphological changes in the

male flies' genitalia, which may lead to a reduction in SIT effectiveness. With altered genitalia, a mass-reared male may not be able to out compete the feral males.

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37 Tables:

Table 1: Data from the three time trails

Trial n Mean Standard Mean Standard Inter- Aedeagus (minutes) Inter- Deviation adeagus Deviation genital Length trail genital Inter- length Aedeagus Distance comparison Distance genital (mm) Length trial (t-stat) (mm) Distance (mm) comparison mm t-stat 5 13 0.929 0.332 1.490 0.024 5-10min 5-10min

<0.01 0.97

10 21 0.626 0.135 1.490 0.027 10-15min 10-15min

<0.01 0.95

15 11 0.931 0.105 1.490 0.017 5-15min 5-15min

0.96 0.93

38 Figures:

Figure 1: Squash preparation of the female reproductive tract of A. suspensa, (two of the three spermathecae) under UV light (Fritz & Turner 2001)

39 Figure 2: Male genitalia of A. suspensa in insect saline (63x magnification)

Figure 3: Inflationsac scales ( 400x magnification, UV light)

40 Figure 4: Distiphallus in the bursa copulatrix in Anastrepha ludens (Briceno et al.

2010)

41 Figure 5: Full aedeagus penetration, claspers are engaged with female abdomen

(90x magnification)

42 Figure 6: Measurement of aedeagus length (in mm) denoted by yellow line (63x magnification)

43 Figure 7: Distiphallus (650x magnification, SEM)

44 Figure 8: Terminus of the genital hook (650x magnification, SEM)

45 Figure 9: Terminus of the genital hook (3500x magnification, SEM)

46 Figure 10: Terminus of the trumpet (1500x magnification,SEM)

47 Figure 11: Terminus of the trumpet (1000x magnification,SEM)

48 Figure 12: Distiphallus under UV light (400x magnification)

49 Figure 13: Distiphallus inserted into female reproductive tract; 5 minutes of copulation (lOOx magnification)

Figure 14: Distiphallus inserted into female reproductive tract; 10 minutes of copulation (lOOx magnification)

50 Figure 15: Distiphallus inserted into female reproductive tract; 10 minutes of copulation (100x magnification)

Figure 16: Distiphallus inserted into female reproductive

51