The mechanism of the dart's influence on paternity in the

snail, Cantareus aspersus

Katrina C. Blanchard

Department of Biology

McGill University

Montreal

October 2005

A thesis submitted to McGill University in the partial

fulfillment of the requirements of the degree of Master of Science

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Abstract...... i

Résumé ...... ü

Acknowledgements ...... iii

Chapter 1: Introduction ...... 1

The research problem ...... 2

Reproduction in Cantareus aspersus ...... 4

The function of the dart ...... 8

Sexual conflict in hermaphrodites ...... 13

Dart shooting and cryptic female choice ...... 16

Experimental design ...... 17

Figure 1...... 20

Figure 2 ...... 21

Chapter 2: Methods ...... 22

Snail care and maintenance ...... 23

Removal ofthe dart sac and digitiform mucus glands ...... 23

Digitiform gland mucus homogenate ...... 24

Mating trials ...... 25

Egg laying ...... 27

DNA extraction and genotyping ...... 27

Statistical analysis ...... 30 Chapter 3: Results ...... 32

Sample size ...... 33

Success of samples ...... 33

Spenn precedence ...... 34

The mucus effect ...... 35

Relationship between maternaI shell volume and the mucus injection ...... 36

Table 1 ...... 38

Figure 3 ...... 39

Figure 4 ...... 40

Figure 5 ...... 42

Figure 6 ...... 43

Chapter 4: Discussion ...... 44

Digitiform gland mucus carried on the dart increases paternity ...... 45

Increased detection of outside fathers ...... 45

Spenn precedence ...... 46

Mucus injection and maternaI shell volume ...... 46

Mating order and maternaI shell volume ...... 48

Future experimentation ...... 49

Dart shooting drives sexual selection ...... 51

References ...... " ...... 53 Abstract

The courtship behavior of the brown garden snail, Cantareus aspersus, inc1udes a bizarre component where one snail attempts to pierce its mating partner with a sharp, calcareous dart that is covered with mucus. In vitro, the mucus causes conformation al changes to the reproductive tract causing sperm to be stored rather than digested. In addition, successful dart shooters have an increased relative patemity compared to unsuccessful shooters. 1 have tested whether this increased patemity is caused by the mucus delivered on the dart or by the mechanical action of the dart. Mating trials were conducted using dartless and glandless snails, where a future mother was mated to two different potential fathers, receiving an injection of mucus with one mating, and an injection of saline with the second mating. The fathers accompanied by the mucus injection sired significantly more offspring than the fathers accompanied by the saline injection. 1 conc1ude that the mucus carried on the dart is responsible for increased patemity levels in Cantareus aspersus. Résumé

Les comportements de séduction de l'escargot de jardins brun, le Cantareus aspersus, inclus une composante assez bizarre où l'escargot essaie de percer son partenaire avec un dard à la fois pointu, aiguisé, calcaire et couvert de mucus. In vitro, ce mucus cause des changements de conformation au niveau des voies reproductives causant ainsi l'entreposage plutôt que la digestion du sperme. De plus, celui qui insère son dart avec succès voit son taux de paternité augmenter par rapport à celui qui a moins de succès. J'ai cherché à savoir si le taux élevé de paternité observé est causé par le mucus qui couvre le dard ou bien si il est tout simplement causé par l'action mécanique du dard lui-même. Des essais d'accouplement ont été faits en utilisant des escargots sans dard et sans glande productrice de mucus. La future mère était donc accouplé avec deux pères potentiels différents recevant une injection de mucus avec le premier et une injection saline avec le second. Les pères accompagnés d'une injection de mucus produisèrent significativement plus de progénitures que les pères accompagnés d'une injection saline.

Je conclue donc que le mucus recouvrant le dard de l'escargot est responsable pour les taux élevés de paternité chez les Cantareus aspersus.

11 Acknowledgements

Foremost, 1 would like to thank Dr. Chase for rus continuing support and dedication to this project. His enthusiasm was always a constant, which made work more stimulating and exciting. He would come to lab in the morning with additional insight or new ideas making his love of scholarship apparent. His critical evaluation was both challenging and essential to the leaming process. He was an excellent supervisor, 1 truly feel lucky to have been his student.

Donald Kramer and Laura Nilson, the other two members of my supervisory committee, provided invaluable guidance, suggestions, and critical insights. Angus

Davison suggested modifications for the DNA extraction protocol reported by Doyle and

Dickson (1987). Advice from Jim Ramsay regarding statistical procedures was also much appreciated. In addition, 1 want to thank Geoff Conrad and Julie Bernard, who provided the translation of the abstract to French.

1 would like to acknowledge the support 1 received from the other members of our labo This project would not have been possible without the help of Kristin Vaga, who perfonned surgery on 275 snails to remove their digitifonn mucus glands and dart sacs. 1 am grateful for Kris's presence for her helpful discussions and her friendship. 1 also thank

Robert Hutcheson for his discussions, revisions, and great companionship for the past two years.

1 would also like to thank Frédérick Robidoux and Amèlie Villeneuve, technicians at the Genome Quebec Innovation Center, for processing the microsatellites. They were extremely accommodating and patient when sharing their expertise with me.

iii Lastly, 1 would like to thank my family and friends for their unwavering support.

Will Sharp has been an incredible source of support and a best friend throughout this

Masters. My parents have supported me throughout all of my endeavors, and encouraged me to experience life to its fullest. 1 will always be grateful to them for their guidance and constancy.

iv Chapter 1:

Introduction The research problem

The terrestrial snail Cantareus aspersus (formerly known as Helix aspersa), engages in a long, complex courtship prior to mating. During courtship the snails attempt to stab their partners with a sharp, calcareous dart. This bizarre mating ritual often leads to instances where dart receipt is dramatic, such as the dart piercing right through the cerebral ganglion. Cantareus aspersus mates with several partners before oviposition, and can store sperm in the spermathecal sacs from a single mating for up to four years

(Duncan 1975). At the time of oviposition helicid snails may use sperm from an average of 3.2 different donors, with up to as many as five donors to fertilize a single clutch of eggs (Murray 1964). Recent studies suggest that the dart may have evolved as an aid to .. the male function in sperm competition. It has been demonstrated that dart shooting affects the amount of allosperm that will be stored, with successful shooters benefiting from increased sperm storage (Rogers and Chase 2001). Snails that shoot darts effectively (hit the recipient) therefore increase their chances of patemal reproductive success (Landolfa et al. 2001, Rogers and Chase 2002).

The primary objective of this thesis is to determine if the effects of dart shooting are caused by chemical or mechanical action. When expelled, the dart is covered with mucus from the digitiform glands that gets distributed within the hemolymph of the recipient after the dart pierces through its dermis. A study by Koene and Chase (1998b) suggested that the mucus carried on the dart initiates a physiological response within the recipient. Extracts of di gitiform gland mucus applied to the reproductive tract caused conformational changes that favored sperm storage rather than digestion when applied in vitro. The digitiform gland mucus may contain an allohormone, which is defined as a

2 honnone that is transferred from one individual to another that induces a physiological response (Koene and ter Maat 2001). When successfully shot, the dart delivers the mucus to the hemolymph of the recipient, enabling the allohonnone to distribute and cause the confonnational changes to the reproductive tract. These confonnational changes lead to increased spenn storage and ultimately, increased paternity.

Alternatively, it could be the mechanical action ofthe dart that causes increased spenn storage and greater paternal reproductive success. When the dart pierces the recipient it could excite mechanoreceptor cells that are located within the skin, which could then send a signal from the peripheral nervous system to the central nervous system

(CNS). The CNS could elicit a motor response that causes the confonnational change within the reproductive tract. Either one of these two mechanisms could be responsible for the association between successful dart shooting and increased reproductive paternal success.

The secondary objective of this thesis is to examine how maternai shell volume influences paternity. In 2001 and 2002 Rogers and Chase found a significant interaction between successful dart shooting and maternai shell volume. These results indicate that the effect of the dart is stronger in smaller animais. They suggest that the reason for these results is that the digitifonn gland mucus carried on the dart becomes diluted in the hemocoel of larger animais, lessening the effect of the dart. Furthennore, they examined whether the difference in size between the two potential fathers had an effect on paternity. They found that the difference in shell volume between the two potential fathers was significant. They explained this result with the reasoning that larger animaIs donate larger spennatophores, which contain more spenn, thus, increasing a snail' s

3 chances ofbecoming a father. The current study will control for this by using the difference in patemal shell volumes as a covariate in the statistical analysis. The effect between maternaI shell volume and the mucus injection will be investigated in this study to verify whether it plays an important role in the determination of paternity in Cantareus aspersus.

In addition, this thesis will investigate sperm precedence in Cantareus aspersus.

In organisms that store sperm there is the possibility for both first and last (most recent) male sperm precedence. With first male sperm precedence it is advantageous to mate first because the sperm that is received will be used preferentially relative to sperm that is received from any secondary matings. Contrarily, in organisms where last male sperm precedence predominates, sperm from the last donor will most likely be used for fertilizations, and consequently, it is beneficial to be the last sperm donor before oviposition. Sperm precedence in Cantareus aspersus has been investigated several times. Rogers and Chase (2002) reported a trend for first male sperm precedence, and more recently, Evanno et al. (2005) used twice-mated virgin snails in their experiment and also reported significant results towards first male sperm precedence.

Reproduction in Cantareus aspersus

Courtship behavior

Cantareus aspersus (Gastropoda: Pulmonata: Stylommatophora: Helicidae) is a simultaneous hermaphrodite that does not self-fertilize. Although these animaIs are hermaphrodites, they will sometimes be referred to as males and females in this text, but it is important to understand that it is only the male and female functions that are being referred to. Cantareus aspersus is a simultaneous hermaphrodite that mates reciprocally,

4 meaning that during each mating both snails donate and receive sperm. Snails that are

sexually aroused are identified by genital eversion, which is the protrusion of the genitals

from the right side of the neck. When two sexually aroused snails come into contact with

one another courtship behavior begins. There are three main stages to courtship behavior:

introductory behavior, dart shooting, and copulation.

During introductory behavior the snails orient themselves face-to-face, and begin

tactile stimulation of each other using their tentacles. This is followed by mouth-to-mouth

contact, and subsequently by mouth-to-genital contact. Adamo and Chase (1988) defined

six stages of genital eversion during courtship, ranging from the initial swelling of the

genital pore (stage 1) to full penile eversion (stage 6). During introductory behavior

genital eversion levels increase until both penile and vaginal lobes are visible (stage 4 of

eversion) (Adamo and Chase 1988). During mouth-to-genital contact many snails scrape

each other with their odontophores, a tooth bearing structure that is normally used for

feeding (Chung 1987, Chase 2002). Courting snails often break mutual contact for brief

periods oftime by circling one another, or by simply withdrawing from one another and

remaining still. Eversion levels usually decrease during break periods, but increase again

when contact is reestablished (Adamo and Chase, 1988). The function ofthese breaks

remains unclear. The snails may lose contact with each other as a result ofbeing blind, or

the breaks may allow the snails to realign themselves, or test the commitment of their

mating partner.

The second stage of courtship, dart shooting, begins when snails reach stage five

of genital eversion, which is marked by the swelling of the vaginal lobe. In preparation

for dart shooting the courting snails align themselves to facilitate genital-to-genital

5 contact. Dart shooting requires tactile stimulation of the vaginal lobe, so it is in this position that dart shooting most often occurs. In order to shoot a dart, a snail ceases movement and presses against its mating partner. The body anterior to the genital pore

contracts causing the posterior body section to bulge with ex cess blood, and by using this hydrostatic pressure the dart is pushed into the recipient (Adamo and Chase 1988).

Before expulsion the dart is housed in the dart sac with the base of the dart attached to the

tubercule (Tompa 1982). To shoot the dart a snail everts its dart sac (figure 1), and upon

contact with the partner' s dermis the dart becomes disconnected from the tubercule.

Consequently, darts that miss the recipient are retracted by the shooter and digested

within the bursa copulatrix (Adamo and Chase 1988). Just before expulsion, the dart is

coated with mucus that is derived from the digitiform glands which are closely associated

with the dart sac (figure 1). The dart is a hollow spear with four blades (Hunt 1979), that

most likely facilitate the delivery ofthe mucus (figure 2).

Many times darts are shot poorly, and the shooter will completely miss the

recipient, or the dart may not stay lodged within the recipient' s dermis. Although dart

shooting is not al ways successful, it seems to be obligatory to courtship (Chung 1987).

Snails always display dart shooting behavior even when they do not expel a dart.

Dissections of the dart sac from snails that did not shoot a dart during courtship revealed

that they did not posses one (Chase and Vaga in press). Once a snail expels a dart,

regeneration of a new dart begins within six hours and takes 6-8 days to complete

(Tompa 1982). It is possible that snails that did not have a dart are in the process of

regenerating one, or are virgins. Virgin snails do not possess darts, and only generate

them after they have everted their dart sacs for the first time (Chung 1986).

6 The third and final stage of courtship begins after dart expulsion. At this point the snails reach the sixth stage of genital eversion, which is marked by penile eversion. Once both snails have reached this stage they try to intromit. Intromission is achieved only when both snails simultaneously insert their penes into their partner's genital atrium.

Snails remain in intromission for 5-8 hours and ex change spermatophores during this time.

The exchange ofspermatophores

The spermatophore is formed in the epiphallus and flagellum within two minutes of achieving intromission (figure 1). However, it takes anywhere from 2-6 hours to fill the spermatophore with sperm and for it to move into the penis (Adamo and Chase 1988).

When the spermatophore is complete it is transferred from the penis into the partner' s bursa tract diverticulum (Lind 1973, Tompa 1984). The spermatophore may be slightly longer than the bursa tract diverticulum, and as a result the tail of the spermatophore may extend beyond the base of the bursa tract diverticulum and into the genital atrium (Tompa

1984; figure 1). The sperm escape from the tail of the spermatophore and have two possible destinations. Firstly, they can be drawn into the bursa copulatrix by strong peristaltic contractions where they will be digested. The bursa copulatrix is filled with a reddish mass that serves to break down and absorb items such as allosperm, autosperm that is expelled at times other than copulation, spermatophore shells, and darts (Lind

1973). Altematively, the sperm can be drawn into the spermathecal sacs, where they are stored for later fertilizations (figure 1). The spermathecal sacs are a series ofblind tubes that are located within the fertilization pouch-spermathecal complex (FPSC) which is embedded in the albumen gland. Typically, only 0.025% of allosperm make it to the

7 spermathecal sacs while the rest are digested (Rogers and Chase 2001). The spenn that reach the spennathecal sacs may be used to fertilize eggs at the time of oviposition.

The function of the dart

Dart shooting has been under investigation for hundreds of years, and many different hypotheses have been developed to explain its function. Piercing your mating partner with a 9 mm sharp, calcareous dart may not seem conducive to mating at first glance, however, dart shooting prior to copulation is an important part of the courtship behavior in Cantareus aspersus. Over the last few centuries, people have come up with many theories to explain why dart shooting evolved. Sorne believed that dart shooting evolved as an aphrodisiac, invoking passion in the recipient, while others believed that the dart is a nuptial gift. It is even possible that the story of Cupid originated from watching the mating behavior of dart shooting snails. The most recent and most viable hypotheses regarding the function of the dart are outlined below.

Reproductive isolation

One theory behind dart shooting lies in the morphology of the dart. Darts from different species of snails vary markedly in their morphology, ranging from simple conical shapes to complex bladed structures (Koene and Schulenburg 2005). Both Diver

(1940) and Webb (1952) hypothesized that dart shooting is a mechanism to prevent interspecific mating between different species of snails. However, there are several reasons why this hypothesis is not valid.

If dart shooting were used for species recognition the snail would have to detennine the shape of the dart upon penetration. Snails do not posses a dart receiving organ, and although dart receipt is often near the genital pore (Chase and Vaga in press),

8 darts can be received aIl over the body, and vary with their depth of penetration (Chung

1987). It is highly unlikely that a snail would be able to perceive the exact shape of a dart by a simple puncture wound. It is also possible to argue that it is the mucus on the dart that aids snails in species recognition. If the digitiform gland mucus were responsible for species recognition, then it should only indu ce genital eversion within conspecifics.

However, Chung (1986) reported that mucus from Cantareus aspersus that was injected into Cepaea nemoralis induced genital eversion. This clearly shows that neither the dart nor the mucus carried on the dart is responsible for species recognition.

Furthermore, if dart shooting were the critical step in courtship behavior that aIlowed snails to identify the species of potential mates, then the dart would always have to penetrate the dermis of its courting partner. However, many times the dart misses its recipient completely, or hits and faIls out immediately. In addition, if a snail was not hit by a dart, it should refuse to mate because it wouldn't know with what species it was mating; yet snails readily mate with their partner even in the absence ofbeing hit with a dart. It is apparent that dart shooting did not evolve as a method to recognize conspecifics during courtship.

Gift ofcalcium

Darts are composed of calcium, which is essential to growth and reproduction in snails (Tompa 1980). The developing embryo of Cantareus aspersus needs calcium to produce its embryonic sheIl (CroweIlI973). Chamoy (1979) and Leonard (1992) both proposed that the dart could be a nuptial gift of calcium for the female. Nuptial gifts are intended to increase female fecundity or survival, thus enhancing the male's own mating success (Andersson 1994). In many organisms males have developed strategies to secure

9 their spenn for fertilizations, these include parental care, mate guarding, sperm plugs, or nuptial gifts (Gwynne 1984). Snails do not guard their mates, use spenn plugs, or exhibit parental care; therefore, the dart was hypothesized to be a gift to the female that would enhance the survival ofher offspring, and therefore the male's fitness.

However, only 6.3% of received darts are intemalized (Koene and Chase 1998a), and those that are intemalized take months to dissolve (Rogers 2001), making it unlikely for the dart to be able to contribute calcium to the shooter's young. The dart contains roughly the same amount of calcium as a single egg; therefore, it can not significantly contribute to the development of an average clutch size of 59 eggs (Koene and Chase

1998a). Moreover, an experiment by Tompa (1975) revealed that the amount of calcium that is required to produce a clutch of eggs is taken directly from the mother' s shell. This evidence demonstrates that the dart has not evolved as a nuptial gift of calcium.

Sexual stimulation

One of the oldest hypotheses behind the function of the dart is that it has a stimulatory effect on the recipient. These ideas originated with de Maupertuis (1753), when he suggested that the dart's function is to invoke passion in mating snails. A stimulatory effect could either come from the physical trauma of dart receipt, or from the mucus that is carried on the dart. Goddard (1962) tested this hypothesis; he found that pinching the skin near the genital pore, a common area for dart receipt, caused increased tonus in the penis sheath muscles. He also reported that injection of digitifonn gland mucus did not have an effect on the recipient, and concluded that it was the piercing action of the dart that caused increased stimulation, rather than the mucus on the dart.

10 Contrary to this, both Dorello (1925) and Chung (1986) found that when they injected digitiform gland mucus into Cantareus aspersus it caused increased stiffening of the penis and genital eversion. Adamo and Chase (1988) found that the time between dart shooting and copulation was reduced by an average of26 minutes (a reduction of5.5%) when the first dart shooter was successful, compared to pairs where the first dart shooter was unsuccessful. This indicates that dart shooting may have a stimulatory effect that results in shortened courtship duration. Adamo and Chase (1990) conducted a follow up study in which they completed courtship trials with dartless and glandless snails. They found that both glandless and dartless pairs required more time to reach copulation than the controls that had been sham-operated. This provided evidence that the mucus on the dart causes stimulatory effects, not the dart alone, and that the mucus could contain a substance that reduces courtship duration.

Despite this evidence, there are several studies which report that dart receipt does not stimulate courtship. Chase and Vaga (in press) found that when the first dart shot was successful, it did not affect the time to the next dart shot, or shorten courtship duration, contradicting Adamo and Chase's (1988) findings. Jeppesen (1976), and Giusti and

Lepri (1980) both observed that dart receipt did not cause stimulation of courtship or help facilitate copulation.

Giusti and Lepri (1980) suggested that dart shooting was in place to deter unmotivated snails during courtship. They reasoned that because dart receipt could be painful, only the snails that were serious about mating would continue courtship after being hit with a dart. Complementary to this, Lind (1976) observed that snails that were hit with a dart were more likely to break off courtship than those that were missed.

11 However, these ideas seem improbable because dart shooting is at the end of courtship, and by this time the snails have spent valuable time courting and have reached stage 5 of eversion. Circ1ing and pauses in courtship behavior prior to dart shooting provide uncommitted snails with the chance to break off courtship; if they were going to lose interest in mating it would probably be at this critical step in courting behavior. Snails that make it to dart shooting are dedicated to mating, and the need for the dart to further stimulate courtship would be extraneous at this point.

Post-copulatory sperm competition

Post-copulatory sperm competition occurs when sperm from more than one male compete for fertilization of the ova. Males have developed many physiological, anatomical, and behavioral strategies that facilitate the use oftheir sperm over others.

Examples inc1ude, the use of accessory gland fluids that cause the female to seem undesirable to others, sperm plugs and mate guarding (Parker 1998). Cantareus aspersus provides an environment where post-copulatory sperm competition is likely due to the storage of sperm from multiple males. The dart may have evolved as a method for males to increase the chances of their sperm being used over that of others. Recent studies have shown that successful dart shooters benefit from higher levels of patemity than unsuccessful shooters (Landolfa et al. 2001, Rogers and Chase 2002). Chung (1987) and

Adamo and Chase (1996) both proposed that the digitiform gland mucus on the dart causes changes in the reproductive tract of the recipient that may benefit the shooter.

Subsequently, Koene and Chase (1998b) demonstrated with in vitro experiments that when extracts of digitiform gland mucus were applied directly to the reproductive tract, conformational changes were observed. The entrance to the bursa copulatrix contracted,

12 the opening to the copulatory canal became wider, and the bursa tract diverticulum underwent peristaltic contractions that caused sperm to be pushed into the genital atrium,

from where the sperm could then travel on to the spermathecal sacs. This provides

evidence that mucus carried on the dart causes a reconfiguration of the reproductive tract

that hinders sperm digestion and facilitates sperm storage. In 2001, Rogers and Chase

tested the effect of dart receipt on the number of sperm stored in Cantareus aspersus.

They found that snails that were hit by a dart stored 116% more sperm than those that

were missed.

If successful dart shooting results in increased storage of the shooter' s sperm, then

it implies that snails that are successful dart shooters should benefit from increased

patemity levels as well. Indeed, Landolfa et al. (2001) and Rogers and Chase (2002) both

found that snails that shot successful darts also benefited from increased patemity levels.

These experiments provide evidence for the hypothesis that dart shooting is a trait that

has evolved to benefit the male function. The dart serves as an aid in sperm competition;

sperm from those that shoot successfully escape digestion, thus outcompeting sperm from

snails that do not shoot successfully. In addition, dart receivers may also benefit because

most of the sperm that they store would be from successful dart shooters, therefore, their

offspring may be better dart shooters, and thus, more fit.

Sexual contlict in hermaphrodites

Parker (1979) defined sexual conflict to be when one sex of a species reduces the

fitness of the other sex while maximizing its own reproductive success. The difference in

male and female mating strategies stems from the cost of their respective gametes; sperm

are relatively cheap, while eggs are more costly to produce (Bateman 1948). Locher and

13 Baur (2000) reported that resource allocation to gametes in Arianta arbustorum, a pumonate land snail, was highly female biased, reinforcing Bateman's principle in snails.

It may seem that simultaneous hermaphrodites are doubtful candidates for sexual conflict, but recent studies have supported the idea that conflict occurs between the sexes in

terrestrial snails (Michiels 1998, Koene and Schulenburg 2005). Evidence of sexual

conflict is demonstrated with examples of evolutionary arms races occurring between the

male and female functions, which result in harmful traits and counter adaptations becoming more extreme over time (Koene and Schulenburg 2005).

Due to the different reproductive strategies of the male and female functions in

Cantareus aspersus, several evolutionary arms races may have evolved. Cantareus

aspersus receives more sperm than is required for fertilizations because oflarge ejaculate

sizes and multiple matings. As a result, most of the received sperm (99.98%) is digested

in the bursa copulatrix (Rogers and Chase 2001). Males have established a few pro cesses

that counteract the effect of the bursa copulatrix, and maximize their own reproductive

success. As detailed below, these inc1ude large ejaculate sizes, lengthening oftheir

flagellum, and ultimately- dart shooting.

Greeff and Michiels (1999) provided the tirst argument of an evolutionary arms

race existing between the male and female functions in helicid snails. They hypothesized

that males increase their ejaculate sizes trying to increase the amount of their sperm that

will be stored, and females respond by digesting yet even more sperm. The amount of

sperm contained in the spermatophore has been documented in two helicid land snails,

Arianta arbustorum and Cantareus aspersus. The average amount of sperm they deliver

per c1utch is 2.21XI06 and 5.56X106 respectively (Baur et al. 1998, Rogers and Chase

14 2001). Both of these animaIs have large ejaculate sizes relative to the amount of sperm that reach the spermathecal sacs.

Koene and Schulenburg (2005) reported that an evolutionary arms race also exists between the length of the flagellum and the length of the bursa tract diverticulum. The spermatophore is formed in the flagellum and transferred to the bursa tract diverticulum within the female reproductive tract. To avoid digestion, sperm actively swim out of the tail of the spermatophore, enter the genital atrium, and travel on to the spermathecal sacs where they will be stored. The longer the tail of the spermatophore, the less distance the sperm have to travel to get to the genital atrium, which results in less sperm being drawn into the bursa copulatrix where they will be digested (figure 1). Over evolutionary time, the female has increased the length of the bursa tract diverticulum causing the spermatophore tail to be farther away from the genital atrium, and in response, the male has increased the length ofhis flagellum to produce a longer spermatophore.

Lastly, males may have evolved dart shooting as another mechanism to overcome the digestive actions of the bursa copulatrix. When a snail shoots a dart successfully it benefits from increased sperm storage and increased patemity levels (Landolfa et al.

2001, Rogers and Chase 2001, 2002). It appears that males use dart shooting as a method to control the fate of their sperm, by causing it to bypass the bursa copulatrix and travel on to the spermathecal sacs.

Despite the above evidence that is provided for antagonistic coevolution, it is possible that the traits that are evolving in the male and female functions are actually cooperatively coevolving, with the male and female functions working together towards successful fertilization (Rice and Holland 2005).

15 Dart shooting and cryptic female choice

Cryptic female choice occurs after copulation has been achieved, and is based on the idea that a female chooses which sperm to use when fertilizing her eggs. This phenomenon can result from a female-controlled structure that selectively favors patemity by males with a particular physiological trait over that of others who lack the trait. Or, females could preferentially select sperm from their storage organ from males that displayed a certain trait at the time of mating (Eberhard 1996).

In helicid snails there are three opportunities for females to display cryptic female choice. First, dart shooting may be a male sexual signal that females use as an indicator of mate viability. Successful dart shooting could be an indicator of good genes, and females should then choose sperm from males that shot successfully when fertilizing their eggs (Landolfa 2002). Second, the lengthening ofthe bursa tract diverticulum in females causes them to select for sperm from males that have a longer flagellum. It is possible that males that produce longer flagella are considered more fit to females than males that have shorter flagella, therefore, a female would benefit because her offspring would be more fit. Lastly, sperm digestion may allow the female to dispose of excess sperm that is not needed for fertilization; or it may allow the female to filter out weaker, slower sperm.

Sperm digestion organs may be in place as a postcopulatory mechanism for selecting the best sperm (Birkhead et al. 1993).

If females used dart shooting as an indicator of fitness, they would have to select sperm from their spermathecal sacs from individuals based on dart traits. Although an interesting idea, post-copulatory selection of sperm from the spermathecal sacs is highly unlikely because it would require an individual to be able to select sperm from males that

16 had shot a dart successfully during courtship. To facilitate this, spenn from different donors would most likely have to be stored in different tubules of the spennathecal sacs.

Furthennore, females would have to be able to differentiate between the tubules to use a certain individual' s spenn, and release the correct spenn at the time of oviposition.

Several studies have been conducted to test if this is plausible. The spermathecal sacs are all accessed via the spennoviduct, consequently, sperm that is moving towards the spennathecal sacs has to pass through the spennoviduct (figure 1). Hasse and Baur

(1995) suggest that the cilia located within the spennoviduct are capable of sequestering spenn from different don ors into different spennathecal sacs, and that at the time of oviposition specific spennathecal sacs could be chosen to fertilize the eggs. Following up on this idea Bojat et al. (2001a, 2001b) tested whether spenn can be manipulated mechanically once it reaches the spennoviduct, through muscles and ciliary action. These studies gave insight into the structure of the spennoviduct and spennathecal sacs, but did not demonstrate that sperm from different donors is stored separately, or that a female could control which spennathecal sacs to use when fertilizing her eggs. Moreover,

Rogers (2001) found that when a snail receives spenn into the spennathecal sacs it is not sequestered into one tubule, but it is instead spread throughout all the sacs, thus discounting the idea that females can sequester different individuals' spenn into separate tubules of the spermathecal sacs. Therefore, this would make it virtually impossible for females to select spenn from a particular individual for fertilization.

Experimental Design

The primary objective of this thesis is to detennine whether the dart functions chemically or mechanically. If the confonnational changes that occur in the reproductive

17 tract are chemically induced then an allohormone contained within the mucus that is carried on the dart may be responsible for these conformational changes. Alternatively, the dart piercing the dermis of its partner could excite the mechanoreceptor cells in the dermis, and cause a signal to be transported to the CNS, which then causes a motor response changing the conformation of the reproductive tract. In order to determine which mechanism is responsible an experimental study was designed.

The experiment consisted of mating triads, where one "mother" snail was mated to two different "fathers". Prior to the mating trials all snails had their dart sacs surgi calI y removed to eliminate dart shooting from courtship behavior. Mating trials were conducted so that when a mother snail mated with one father it received an injection of digitiform gland mucus, mimicking a dart shot in nature (to test whether the dart functions chemicalIy). When the same mother snail mated with the second father it received an injection of saline, mimicking a dart shot without any mucus carried on it (to test whether the dart functions mechanicalIy). The experiment was balanced so that half of the mothers received a mucus injection with their first mating, and half of the mothers received a saline injection with their first mating. This measure was taken to ensure that the injection type is not dependent on the order of mating. Because Cantareus aspersus is a hermaphrodite, an individual in each mating can serve as both a mother and a father at the same time. This factor adds complexity to the design, but also reduces the number of total matings to be completed. Once a snail had mated with two other snails (receiving a different injection with each snail) it was placed on soil to promote egg laying. The eggs were allowed to hatch and grow to a specifie size for DNA extraction. The DNA of the mother, the two potential fathers and thirty of their offspring was extracted for paternity

18 analysis. The DNA was genotyped using microsatellites, which provided information that allowed the paternity of the babies to be determined.

If it is found that significantly more babies are fathered by a mucus-donating father than by a saline-donating father, it can be concluded that it is the mucus that is carried on the dart that is responsible for the reproductive advantage. Conversely, ifthere is no difference between the number ofbabies fathered by a mucus-donating father and a saline-donating father then the mucus can not be responsible for the effects caused by dart shooting, and the dart must function mechanically rather than chemically.

The secondary objectives of this thesis include examining the interaction between maternaI shell volume and the mucus injection and investigating sperm precedence. In order to determine whether the maternai shell volume interacts with the mucus injection, the sizes of aIl the mother' s shells were measured. If a significant interaction is found between the size of the mother and mucus injection, then it could indicate that the effects ofthe mucus are becoming diluted in larger animaIs. To control for sperm precedence half of the mothers received an injection of mucus with the first mating, and half of them received an injection of saline with the first mating. By implementing this strategy we can be confident that the effect of sperm precedence is not dependent on that of the injection type.

19 spermathecal sacs

albumen gland

genital pore

Figure 1. Reproductive anatomy of Cantareus aspersus. The bursa tract diverticulum contains a representation of a spermatophore (black) that would have been received from the mating partner during copulation. The dart sac contains a dart that is normally shot during courtship behavior. When the dart sac and penis become everted they extend through the genital pore which is located on the dermis of the body wall. c.c, copulatory canal; g.a., genital atrium.

20 Figure 2. Photograph of a dart from Cantareus aspersus. This dart was shot during courtship behavior, but missed its recipient. There are 4 vanes on the dart that help facilitate the transfer of digitiform gland mucus. This dart was collected and cleaned ovemight in a solution of sodium hypochlorite to remove the digitiform gland mucus.

21 Chapter 2:

Methods

22 Snail care and maintenance

Snails were supplied from the towns ofPacific Grove, Glendale, Santa Cruz and

Fresno, alllocated within Califomia. In order to distinguish the separate populations of snails, each population was marked with a different color nail polish. AdditionallY' each snail was labeled with a number to keep track of individuals. Ninety-eight snails from each population were housed in Lucite boxes, with 49 individual chambers (5x5x8 cm) in each box. The chambers kept the snails isolated from one another to prevent them from mating. Lucite boxes were kept in a humidified refrigerator at a temperature of 15-18° C under a reversed 16 h light: 8 h dark photoperiod. Snails were showered and fed a diet of nutrients and chicken feed three times a week, every other week.

Removal of the dart sac and digitiform mucus glands

A total of275 snails underwent surgery to remove their dart sacs and digitiform mucus glands, thus, preventing them from shooting a dart during courtship behavior. The surgeries followed a slightly modified procedure from that outlined by Adamo and Chase

(1990). Prior to surgery snails were injected in the foot with an anesthetic composed of

2% MgCl, 0.02% succinylcholine chloride in snail saline (Kerkut and Meech 1966). A small (3-5 mm) incision was made dorsal and posterior to the genital pore, and parallel with the body axis. The dart sac and digitiform mucus glands were drawn out through the incision, cut at their base, and removed. The incision was closed with two sutures.

Following surgery, snails were given an injection of antibiotic (50 J.ll penicillin and streptomycin; Sigma P3539) in 500 J.ll sterile snail saline. Snails were isolated for a two week recovery period. The mortality rate from surgeries was less than 5.6 %. Both Chung

(1987) and Adamo and Chase (1990) demonstrated that dartless snails exhibit aIl the

23 same behaviors as intact snails, including the typical dart-shooting posture, a behavior first labeled by Lind (I976) in Helix pomatia. Dart-shooting posture is marked by shortened tentacles, significant genital eversion, and swelling around the genital pore.

From this position the snail will evert its dart sac and dislodge a dart. Moreover, dartless snails mated as readily as normal snails.

Digitiform gland mucus homogenate

The digitiform mucus glands that were removed during surgeries were collected and frozen at -200 C. The frozen glands were homogenized in snail saline to create a homogenate for the mucus injections during mating trials. The homogenate contained both digitiform gland mucus and digitiform gland tissue. It needed to be at least equivalent in strength to the pure mucus that is delivered on the dart in normal dart shooting. Since digitiform gland mucus can become toxic at high levels, the homogenate could not be so strong that it caused deleterious effects in the recipient. After completing dosage trials, the strongest homogenate that did not cause adverse reactions in the snails was determined to be 1 t digitiform mucus glands in 500 ~l of saline per dose.

In order to determine the amount of digitiform gland mucus contained in one injection of mucus homogenate, 20 digitiform glands were removed from 10 animaIs.

The mucus was extruded from the digitiform glands by placing them on a glass slide and covering aIl but the severed end of the stalk with a plastic coverslip. The mucus was squeezed out of the glands by applying gentle pressure to the slide. Each gland contained an average of 4.4 mg of mucus (data provided by R. Chase). Since each 500 ~l injection of mucus contains 1 t glands it means that one mucus injection contained an average of

24 5.85 mg ofmucus. Thus, one injection ofmucus is equal to 2.9 times the amount of digitifonn gland mucus that is nonnally carried on the dart (Chung 1986).

For preparation of the homogenate 16 glands were homogenized in 1 ml snail saline with a 1.5 ml hand homogenizer. The homogenate was transferred to a 1.5 ml microtube and centrifuged for two minutes at 5,000 g to remove solid debris. These steps were repeated until aIl available glands were used. It was advantageous to collect glands and use as many as possible in the preparation of the homogenate to equalize the dosage given with each injection. The supernatant from each microtube was decanted into a beaker, where it was brought to the appropriate concentration with snail saline. The homogenate was then divided into 500 J.lI aliquots and frozen at -200 C. Hereafter, the mucus homogenate will be referred to as the mucus injection.

Mating trials

Mating trials were conducted in order to fonn mating triads comprising a future mother snail and two potential fathers. AlI three snails originated from a different population. Snails lack the ability to travel far distances; consequently, snails live in spatially segregated groups that typically do not intennate. This keeps populations isolated from one another, which causes them to become more and more differentiated genetically (Arnaud et al. 2003). By obtaining snails from different areas in California we increased the genetic diversity between individuals. This presumably reduced the number of snails that carry the same genotype, making microsatellite analysis more effective.

Prior to mating trials the snails were isolated for 61.5 ± 17.6 (mean ± SD) days to reduce the amount of stored spenn they had from previous donors. Spenn that has been stored for over 70 days (received during the prior breeding season) is less likely to be

25 used for fertilizations than spenn that was received in the CUITent breeding season (Baur

1994). For mating trials snails were showered and placed in a large Lucite box (36x36x8 cm) where they were free to initiate courtship behavior. To increase the likelihood of mating, snails that were selected to participate in mating trials were used immediate1y after their first feeding following a week of starvation (Adamo and Chase 1991). Once two snails originating from different populations displayed courtship behavior they were removed and placed on a glass plate (lOx13 cm). The courting snails were carefully observed until dart shooting posture was assumed. Once this posture was observed in one snail, its courtship partner received an injection in the foot of either mucus or saline with a 26 gage hypodennic needle. When the second snail in the courting pair displayed the dart shooting posture, the recipient snail also received an injection of either mucus or saline. The injection of mucus simulated a dart shot in nature, while the injection of saline simulated a dart shot without any mucus carried on it. After receiving their injections, the two snails were allowed to finish courting and intromit. Following copulation, snails were isolated for a minimum of 21 days to allow them to have sufficient time to replenish their spenn stores (Locher and Baur 1999). After this time had elapsed snails were allowed to re-mate with a second partner (an average of27.1 ±

9.44 days later, mean ± SD). During the second mating snails received the injection that they did not receive during their first mating. For example, if a snail received an injection of mucus during its first mating, then during its second mating it received an injection of saline. A triad was complete when a snail (future mother) mated with two spenn donors

(potential fathers), receiving an injection of mucus with one spenn donor and an injection of saline with the other spenn donor. Mating trials were designed so that in one half of

26 the triads the future mother received an injection of mucus with the tirst donor, and in the other half of the triads the future mother received an injection of saline with the tirst donor. This strategy controlled for a sperm precedence effect.

To investigate whether the size of the triad participants affects patemity, shell

3 measurements (in millimeters) of each snail were taken. Shell volume (cm ) was calculated with the following formula, (length x width x height) (4.0 x 10-4) - 0.3129

(Chase and Vaga in press).

Egg laying

After a mating triad was complete the future mother was isolated for 7 days to ensure that aIl of the sperm from the second donor had time to reach the spermathecal sacs or be digested (Lind 1973). After the week had passed the snail was placed in an individual plastic chamber (8x14 cm) with 2-5 cm of a 3: 1 mixture oftopsoil and sand.

When building a nest for the eggs, the snail digs a hole in the soil and lays the eggs at the bottom, making them visible through the clear plastic. The chambers were maintained under a reversed 16 h light: 8 h dark photoperiod with high humidity. The snails were fed every other day, altemating between carrots and snail feed mixture. Containers were checked daily for new eggs, which were allowed to hatch. Offspring were fed daily and kept active until they reached an average size of70 mg at which point they were frozen at

-800 C.

DNA extraction and genotyping

Following egg laying each parental snail had a 5-7 mm section of tail removed from the foot using a scalpel. Thirty of the frozen offspring from each clutch were haphazardly selected for DNA extraction. DNA was extracted from the parental tissue

27 sampI es and the offspring folIowing a procedure that was modified from Doyle and

Dickson (1987). Microtubes containing a solution of350 III 2% Hexadecyltrimethyl­ ammonium (CTAB; Sigma H6269) isolation buffer and 20 III of 10 mg/ml proteinase K

(Invitrogen 25530-015) were pre-heated in a 600 C water bath. Parental tissue sampI es and whole frozen offspring were each homogenized separately in the pre-heated 1.5 ml microtubes with an electric tissue grinder. Samples were incubated in a 600 C water bath for 30-60 minutes, and then homogenized a second time with the tissue grinder. AlI samples were extracted by adding 370 III ofphenol:chloroform:isoamyl a1cohol (Sigma

P3803) to each microtube. The microtubes were centrifuged at 8,000 g for 8-10 minutes, producing two phases in each microtube. The aqueous phase was removed and added to a clean 1.5 ml microtube. Another 370 III ofphenol:chloroform:isoamyl a1cohol was added to the once extracted aqueous phase, and it was centrifuged a second time. The aqueous phase was transferred to a clean 1.5 ml microtube, and approximately 600 III of 70% ethanol was added to each microtube. The microtubes were hand shaken to precipitate the

DNA into a white and viscous globule within the ethanol. The DNA was removed by adhering it to the side of a micropipette tip, and then transferred to another microtube containinglOO III Ultra Pure water (Invitrogen 10977-015). The microtubes were incubated in a 600 C water bath ovemight to dissolve the DNA in the water. The DNA samples were stored at 40 C.

DNA samples were delivered to the Genome Quebec Innovation Centre located on the McGill University Campus. AlI individuals were genotyped at six microsatelIite loci: Ha2, Ha5, Ha6, Ha8, Ha9, and HalO, developed by Guiller et al. (2000). Other markers (Ha7, Hall, Hal2 and Ha13) were used in production but the chromatographs

28 were not clearly defined, which lowered the confidence in the calls obtained resulting in poor success rates for these markers. Success rate indicates the percentage of time that a call (defined allele length) could be made for each marker.

Samples were amplified using PCR, with the forward primer labeled with fluorescent dye (either HEX or F AM). All PCR reactions were carried out using 4.0 !lI of

(5ng/!ll) template DNA, 0.8 !lllOx PCR buffer, 0.8 !lI MgCb, 0.08 !lI dNTP, 0.2 !lI primers, 0.04 Amplitaq Gold polymerase and 2.08 !lI dH20. The standard PCR conditions were 96° C for 10 min, followed by 35 cycles of95° C for 30 s,50° C for 30 s,72° C for 1 min. Labeled products were run on a 3730xl ABI DNA Analyzer with an internaI size standard and analyzed using Genemapper (version 3.7). The software output gave two allele lengths for each sample at every marker. The data were organized into families, with the mother, two sperm donors and thirty offspring, each in a different Excel spreadsheet. All parental identities were coded so paternity assignments could not be biased. The technicians at Genome Quebec assigned one ofthe offspring's alleles to the mother, and the other allele to one of the putative fathers at each locus. They reported these results using a color code to denote the paternity ofthe o ffspring , salleles.

1 evaluated the paternity assignments for each offspring across all mark ers in order to decide whether the offspring was sired by the first sperm donor, the second sperm donor, or an unidentified sperm donor. My paternity judgments were based on the following predetermined guidelines. When a marker failed to specify an allele length for one or more parents, the marker was disregarded and was not used for the determination of paternity. If an offspring was assigned conflicting paternities across any two of the six markers, the paternity was taken to be from an unidentified father. If neither one of the

29 offspring's two aneles from a marker could be assigned to the mother, the marker was disregarded for that offspring. There are several reasons why the latter situation could arise. The sample may have been contaminated, the intensity of the marker may have been too weak to specify aneles, or there may have been too much background noise.

Statistical analysis

Paternity analysis

The number of offspring sired by each sperm donor in a triad was expressed as the proportion of offspring sired by the first donor (P 1) and the proportion of offspring sired by the second donor (P2). Offspring that were not sired by either of the two fathers in a triad were included in the proportion calculations. These unidentified fathers had previously donated sperm in the wild before the snails were brought to the lab, they will be referred to as outside sperm donors or outside fathers (PO). To normalize the distribution ofP-values, an proportions were transformed using the arcsine transformation (Sokal and Rohlf 1995). Statistical analyses were performed using SPSS version Il.0. An means are reported as the mean ± standard deviation.

Statistical analysis was performed using a generallinear model. The dependent variable was the arcsine transformed proportion of offspring fathered by either the first or the second donor in the triad. The type of injection given and the mating order were included in the design as categorical factors. MaternaI shen volume and the difference in paternal shen volumes were used as covariates. The difference in patemal shen volumes was calculated as the shen volume of the first donor niinus the shen volume of the second donor. It was included as a control for larger animaIs donating larger spermatophores. In addition to these four variables, the interaction between the type of injection given and

30 the mating order was incIuded in sorne of the models. The generallinear mode1 was run multiple times using all possible combinations of predictor variables to obtain the most parsimonious mode!. This method required performing a generallinear model for each possible subset of independent variables. The subset of independent variables that produced the model with the greatest statistical significance and lowest p-value was selected. To test wh ether an interaction existed between the mucus injection and the maternaI shell volume, the P- values ofthe snails that received the mucus injection were regressed against maternaI shell volume; this was done separately for Pl and P2.

Sperm precedence analysis

To examine whether sperm precedence affected paternity, an independent samples

{-test was performed using the arcsine transformed Pl and P2 values. The null hypothesis stated that the mean Pl and P2 values are equal, indicating no precedence effect. The Pl and P2 values were ca1culated after removing the offspring sired by outside fathers from each cIutch. Outside fathers were excIuded from the proportion calculations in this test because the mating history of each snail prior to collection was unknown. The reported p­ value for this test is two tailed.

31 Chapter 3:

Results

32 Sample size

Over 260 matings were observed resulting in a total of 56 mothers that had each mated with two potential fathers. Of the 56 mothers, eight did not lay eggs (14%), and four mothers laid eggs that were either nonviable or too few in number (7%). This left 44 mothers, 88 potential fathers, and 1,320 offspring, or 44 complete triads. The 44 triads were sent to the Genome Quebec Innovation Centre for analysis. After analysis 3 triads were discarded because they were comprised of a mating that was unsatisfactory, and after genotyping, two triads produced inconclusive results, leaving 39 final triads. Of the

39 triads, 19 contained mothers that had received the mucus injection with the first mating, and 20 contained mothers that had received the saline injection with the first mating. One of the triads in which the mother received the saline injection first was randomly discarded to provide a sample size of 19 triads where the mucus injection was received first, and 19 triads where the saline injection was received first.

Success of samples

Ofthe 1,467 parental and offspring DNA sampI es delivered to the Genome

Quebec Innovation Centre only 17 (1 %) failed to contain material that could be used for microsateUite analysis. An average success rate of 85% was obtained for the six loci

(Ha2, Ha5, Ha6, Ha8, Ha9, and HalO) that were used for analysis. AUeles for sorne of the markeTS were found outside the expected size range reported by Guiller et al. (2000)

(table 1). This discrepancy may have arisen because the expected ranges for these markers were based on specifie snail populations in France, while the snails used in this experiment were from California. The parental exclusion probability (Jamieson and

33 Taylor 1997) was calculated from the allele frequencies of 112 adult snails. The exclusion probability from all six loci is 0.9985, indicating a powerful genetic assay (data provided by R. Chase).

Sperm precedence

Outside fathers were detected in 34 (89%) of the 38 clutches. Overall, 494 (44%) of 1117 offspring were from outside fathers. Figure 3 shows the amount of offspring that were sired by each sperm donor. The proportion of offspring sired by outside fathers (PO) ranged from 0.00 tol.00 with a mean of 0.442 ± 0.339. The proportion of offspring assigned to the tirst donor (P 1) ranged from 0.00 to 1.00 with a mean of 0.331 ± 0.320.

The proportion of offspring sired by the second donor (P2) ranged from 0.00 to 0.97 with a mean of 0.227 ± 0.257. The outside sperm donors sired the highest overall proportion of offspring, followed by the tirst sperm donors and the second sperm donors respectively.

The frequency distributions of the proportions of offspring from each sperm donor also reflect this result (tigure 4). The proportions of offspring from outside donors are fairly evenly distributed, while the proportions of offspring from the tirst sperm donor are slightly skewed to the right, and the proportions of offspring from the second sperm donor are highly skewed to the right.

To investigate whether the tirst sperm donor benetited from tirst sperm precedence, an independent sampI es (-test was conducted where the means of the arcsine transformed Pl and P2 values were compared. Outside fathers were excluded from the Pl and P2 proportion calculations for this test because the number of outside sperm donors for each snail is unknown. The mean Pl value was 0.546 ± 0.382 (arsine transformed mean = 0.840 ± 0.565), and the mean P2 value was 0.454 ± 0.382 (arcsine transformed

34 mean = 0.731 ± 0.565). These means are not significantly different from each other

(1 = 0.829, df= 72,p = 0.410). However, the sample size of the offspring was reduced by

44% after the exclusion of outside fathers, causing the Pl and P2 values to become

unreliable estimates due to the smaller number of offspring included in their calculations.

The mucus effect

A generallinear model with the inclusion of covariates was used to investigate the

effects of injection type, mating order, maternaI shell volume, and the difference in size

of the paternal shell volumes on paternity. Outside fathers were included in the

proportion ca1culations for this test. The generallinear model was run multiple times to

obtain the most parsimonious model (see Methods). The resulting model included only

one variable, the type of injection received. The type of injection administered to the

mother snail greatly affected the paternity of the two fathers in the triad (MS = 1.913,

F I,74 = 13.756,p < 0.001). The mean proportion of offspring that were sired by the father

that accompanied the mucus injection was 0.389 ± 0.321, and the mean proportion of

offspring sired by the father that accompanied the saline injection was 0.169 ± 0.213.

Neither the order in which the snails mated, nor the interaction between the injection type

and mating order significantly affected paternity.

To see whether the significant difference in injection type is limited to the first or

second donor, two separate generallinear models were conducted. The group means are

presented in figure 5. The first generallinear model used the arcsine transformed Pl

values as the dependent variable, the type of injection given with the first mater as the

categorical independent variable, and maternaI shell volume and the difference in

paternal shell volume as covariates. The most parsimonious model included the variables

35 ofinjection type and maternaI shell volume. Both the injection type (MS = 0.814, FI,35 =

5.086,p < 0.05) and the maternaI shell volume (MS = 0.695, FI, 35 = 4.343,p < 0.05)

were signiticant. The most parsimonious generallinear model for the second donor used

the arcsine transformed P2 values as the dependent variable, and the type of injection

given with the second mater as a categorical independent variable. The type of injection

given was signiticant (MS = 1.086, FI, 36 = 10.652,p < 0.01). This model did not incIude

maternaI shen volume as a covariate because when maternaI shen volume was analyzed it

was not signiticant (MS = 0.002, FI, 35 = 0.015, p =0.904). The resuIts of these two

analyses demonstrate that injection treatments make a signiticant difference for both the

tirst and second donors (tirst donor: MS = 0.814, FI,35 = 5.086,p < 0.05, second donor:

MS = 1.086, FI,36= 10.652,p < 0.01). The mean proportion ofoffspring sired by the tirst

donor when accompanied with the mucus injection was 0.441 ± 0.355, while the mean

proportion of offspring sired by the tirst donor when accompanied by the saline injection

was 0.222 ± 0.242. The mean proportion of offspring sired by the second donor with the

mucus injection was 0.337 ± 0.284, and the mean proportion of offspring sired by the

second donor with the saline injection was 0.116 ± 0.170.

Relationship between maternaI shell volume and the mucus injection

Two linear regressions were used to look at the relationship between maternaI

shen volume and the mucus injection (tigure 6). In the tirst linear regression the

proportion of offspring that was sired by the tirst donor accompanied by the mucus

injection was regressed against maternaI shen volume (R2 = 0.181, FI, 18= 3.769, p = 0.069). In the second linear regression the proportion of offspring sired by the second

donor accompanied by the mucus injection was regressed against maternaI shell volume

36 2 (R = 0.013, FI, 18 = 0.225,p = 0.641). Since paternity did not decrease for either the first or second mucus donor as maternaI shell volume increased, there is no evidence that mucus was significantly diluted in larger animaIs.

37 Table 1. Charaeteristies of polymorphie mierosatellites in Cantareus aspersus. The observed eharaeteristies are based on 1,467 individuals.

Locus Success rate Previously Observed Previously Observed Previously Observed 2 (%) reported size range (bp) reported heterozygosity reported NA l range (bp)l heterozygosityl NA

Ha2 91 302-310 295-319 0.68 0.78 5 13

Ha5 84 183-195 181-198 0.60 0.83 6 11

Ha6 79 162-232 166-224 0.79 0.84 21 16

Ha8 91 152-220 155-190 0.91 0.89 18 16

Ha9 79 138-173 138-171 0.52 0.75 11 14

HalO 86 225-241 225-244 0.82 0.69 8 9

1 The previously reported base pair ranges, heterozygosities and number of alleles are from Guiller et al. (2000).

2 Number of alleles. Based on the results of mierosatellite analysis of parental snails (mother and two potential fathers) from 44 triads (N = 112). Parental snails may have been used in more than one triad; for example, a snail may have been a mother in one triad and a father in a different triad, resulting in less parental snails in total. Data provided by R. Chase.

38 1.0

0.9

0.8

0> c 0.7 ·C c.. :a::1/) 0.6 0 0 0.5 -c :e0 0 0.4 c.. 0 a.L- 0.3 0.2

0.1

0.0 outside donors tirst donor second donor

Figure 3. Mean P-values for outside sperm donors, the first sperm donor, and the second sperm donor. The error bars represent one standard deviation from the mean.

39 [figure on next page]

Figure 4. Observed frequency distributions of the proportion of offspring sired by outside sperm donors (PO), the tirst sperm donor (P1), and the second sperm donor (P2). The graphs are fit with a sigmoidal curve containing 4 parameters.

40 10

8

rh a> oC .B :::l 6 "0 0 -(jj .0 4 E :::l Z

2

0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 PO

10

8 rh a> ..c: ::;0 6 "0 '0 Qi .0 4 E :::l Z

2

0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 P1

10

8 rh a> ..c: .B 6 :::l "0 '0 .... a> .0 4 E :::l Z

2

0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 P2

41 1.0 ,..------,

0.9 c:::J outside don ors _ mucus injection 0.8 _ saline injection

g> 0.7 ï:::::a. ~ 0.6 * o 0.5 c o ~ 0.4 a. a..e 0.3

0.2

0.1

0.0 -'------~--.---'----- Outside donors First donor Second donor

Figure 5. The graph depicts paternity results fram 38 triads. In half of the triads

(N = 19) the offspring were sired by outside donors, the first donor accompanied by the mucus injection, and the second donor accompanied by the saline injection. In the other half of the triads (N = 19) the offspring were sired by the outside donors, the tirst donor accompanied by the saline injection, and the second donor accompanied by the mucus injection. Error bars represent one standard deviation fram the mean. * Effect of injection type on paternity gained by tirst donor, p < 0.05. ** Effect of injection type on paternity gained by second donor, p < 0.01.

42 1.2

1.0 0 • 0 ....CI) 00. 0 0 c .8 0 0 '0 CI) • :::l .6 (,) 0 :::l ce E 0• • 0 .4 -CI) • ·0 Q) Ce :::l 0 • al .2 • 0 • >1 0... .0 o• • ·0 • 0.0 0 0 0 • •

-.2 2 3 4 5 6 7 8

Maternai Shell Volume

Figure 6. The Y- axis is the proportion of offspring sired by fathers that were accompanied by the mucus injection. The open circles represent the proportion of offspring sired by the first donor and the mucus injection (fil = 0.181, F1. 18 =

3.769, P =0.069) and the closed circles represent the proportion of offspring sired by the second donor and the mucus injection (fil = 0.013, F1. 18 = 0.225, p = 0.641).

43 Chapter 4:

Discussion

44 Digitiform gland mucus carried on the dart increases paternity

1 have detennined that the digitifonn gland mucus that is carried on the dart causes increased patemity in Cantareus aspersus. The spenn donors that mated with the mother when she received the mucus injection sired significantly more offspring than the spenn donors that mated with the mother when she received the saline injection. This result demonstrates that the digitifonn gland mucus that is carried on the dart is responsible for increased patemity levels in Cantareus aspersus. This is consistent with

Koene and Chase's (1 998b) findings that showed that extracts of digitifonn gland mucus caused confonnational changes in the reproductive tract. It is also consistent with

Landolfa et al. (2001) and Rogers and Chase (2002) who both demonstrated that successful dart shooting gamers a reproductive advantage.

Increased detection of outside fathers

The use of microsatellites in this study greatly increased the detection of offspring that were sired by outside fathers. This is compared to previous studies that used allozymes for patemity analysis. 1 found outside fathers in 89% of clutches examined.

This measure was severely underestimated in the past; Landolfa et al. (2001) found outside fathers in only 27% oftheir clutches, while Rogers and Chase (2002) found outside fathers in 49% oftheir clutches. Similarly, 1 attributed 44% of offspring to outside fathers, while Landolfa et al. (2001) attributed 10% of offspring to outside fathers, and Rogers and Chase (2002) attributed Il % of offspring to outside fathers. The use of micro satellites in this study produced more accurate results, providing more insight into the extent of clutches with mixed patemity in Cantareus aspersus.

45 In addition, the increased detection of outside fathers demonstrates that spenn that has been stored for an average minimum of 61 days is still viable and contributes considerably to patemity. Contrary to Baur's (1994) conclusion, this suggests that spenn received during a previous breeding season may still compete effectively with spenn received within the same breeding season. The discrepancy between our results and

Baur's results may be explained by smaller sample sizes and a less powerful genetic assay (shell color) in Baur's study. This discovery implies that male first spenn precedence carries over to different breeding seasons.

Sperm precedence

The greatest number of offspring was sired by outside fathers, followed by the first donor, and subsequently the second donor (figure 3), demonstrating a first spenn precedence effect. This is consistent with Rogers and Chase (2002) and Evanno et al.

(2005), who both reported first spenn precedence in Cantareus aspersus. However, the results of an independent sampi es t-test comparing the mean number of offspring sired by the first donor (Pl) and the second donor (P2) revealed that the means are not significantly different from each other. The Pl and P2 values were calculated after removing the offspring sired by outside fathers from each clutch. It is important to note that the sample size of the offspring was reduced by 44% when the offspring sired by outside fathers were excluded. This reduction of the net sample size changed the calculated Pl and P2 values, and caused them to be unreliable, and thus, may be the reason that the t-test produced non-significant results.

46 Mucus injection and maternaI shell volume

In 2001 and 2002 Rogers and Chase found a significant interaction between successful dart shooting and maternaI shell volume. Their results indicated that the effect of the dart was stronger in smaller animaIs than in larger animaIs. They suggested that the reason for this result was that the digitiform gland mucus carried on the dart became diluted in the hemocoel of larger animaIs, lessening the effect of the dart. Contrary to their results, the regression analysis in this experiment demonstrated that there was not a significant interaction between maternaI shell volume and the mucus injection (figure 6).

The difference in the dosage of mucus received by the mothers between these two experiments may provide a plausible explanation for this discrepancy. The experiments carried out by Rogers and Chase used mating trials that involved natural dart shooting events to investigate the effects of dart shooting on paternity. This differs from the current experimental design, which used the mucus injection to simulate dart shooting.

During natural dart shooting 2 mg of digitiform gland mucus are carried on the dart, whereas the mucus injection used in this experiment contained 5.85 mg of digitiform gland mucus per dose, or 2.9 times the amount of mucus that is carried on the dart. It is possible that the dosage of the mucus injection used in this experiment was saturating, meaning that it is so much stronger than the digitiform gland mucus that is naturally carried on the dart that it masks the dilution effect found by Rogers and Chase.

Furthermore, in natural dart shooting it is possible that not all of the mucus carried on the dart is ingested by the recipient. Sorne of the mucus may be lost by adhering to the skin of the recipient; or the dart may not fully penetrate the dermis of the

47 recipient, preventing the mucus that is on the exposed portion of the dart from entering the hemocoel.

Mating order and maternai shell volume

Maternai shen volume was used as a covariate in the paternity anaIysis; it was significant for the first donor, but was not significant for the second donor. One plausible explanation for the significant result of the maternai shen volume for the first donor may be due to the variable morphology of the spennathecaI sacs between individuaIs. The

spennathecaI sacs are composed of one main tubule and severaI smaIler branching tubules. The number of spennathecaI tubules that an individuaI has can vary. For ex ample, Arianta arbustorum has between 2 and 9 tubules (B~nger and Hasse 1999).

Most of the allospenn that is received is primarily stored in the main tubule, with the branching tubules containing less allospenn (Bojat and Hasse 2(02). Baminger and Hasse

(1999) examined six populations of snails and found significant correlations in two of the populations between shen height and the length of the main tubule. They aIso found a significant correlation between both shen height and shen breadth with the cumulative lepgth of the ail the tubules. These significant correlations suggest that as a sn ail increases in size it aIso increases its spenn storage capacity. IndividuaIs with a higher spenn storage capacity can store more spenn, and possibly store spenn from more mating partners (Baminger and Hasse 1999). This explains why maternai shen volume plays a significant role in paternity, as a snail increases in size it may aIso increase its spenn storage capacity. Snails that mate with larger animais may benefit from increased paternity because more of their spenn may be stored in the recipient due to increased storage capacity.

48 However, maternaI shell volume was only significant for the first donor. Two factors (individuaI sperm storage capacity and time intervaI between matings) provide a speculative explanation for why maternaI shell volume was significant for the first donor but not the second donor. The snails used in this experiment had a variable number of mating partners before they came to the lab; hence, they each had varying amounts of stored aIlosperm in their spermathecaI sacs. It is possible that the sperm from the frrst donor in the triad took up most of the remaining available space in the spermathecal tubules of the mother snail, resulting in less available storage room for the second donor in the triad. Moreover, more time elapsed between the snail's last mating in the wild and il' s first mating in the triad (average of 61 days), than the amount of time between the first mating and the second mating in the lab (average of 27 days). Because more time elapsed between a snail' s last mating in the wild and its first mating in the triad, than between the first and second matings of the triad, more stored sperm may have been have discarded during this time providing room for sperm from the first donor. Since only half the amount of time passed between the frrst and second matings in the triad it was not sufficient time for the snail to discard any more sperm to create room for sperm from the second mating of the triad. Thus, an interaction between individu al storage capacity and intermating interval provide a reasonable explanation as to why maternai shell volume was significant for the first donor only. The size of the mother's shell may still have had an impact for the second donor, but it was not significant.

Future experimentation

The use of microsatellites in this experiment revealed how common it was for offspring to be sired by outside fathers. Due to the influence prior sperm donors had on

49 patemity, future experiments involving mating trials and patemity determination should use virgin snails that were raised in the lab, rather than snails collected from the wild. The use of virgin snails in this experiment would have provided clearer results regarding sperm precedence because the net sample size of the offspring would not be reduced due to large proportion of offspring sired by outside fathers. It would also be much more economical, as 44% of the babies processed were not used in sorne ofthe statistical tests.

Previously, it has been difficult to raise virgins due to a low survival rate and time constraints, however, raising virgins has currently had a higher success rate than in the past.

Now that 1 have determined that the digitiform gland mucus is responsible for the increased patemity that is associated with dart shooting, it would be interesting to determine what the active ingredient is within the digitiform gland mucus that causes the conformational change to the reproductive tract. A study conducted by Bômchen (1967) reported that the digitiform gland cells contain secretory droplets that are manufactured in the golgi apparatus and are composed of mucopoly-saccharides and proteins. The secretory droplets produce secretions containing both proteins and small amounts of polysaccharides, either ofwhich could be the active ingredient in the mucus. Based on results from a bioassay, Chung (1986) suggested that the active ingredient in the mucus was a polypeptide. In addition, a preliminary study by Nagle and Chase (unpublished) discovered that the mucus contained a novel peptide that may have been responsible for the conformational change in the reproductive tract. Although these studies are insightful, they do not successfully determine the exact composition of the mucus, or define the ingredient responsible for the conformational changes in the reproductive tract. It is

50 important to reinvestigate this topic with new methods. This would be possible by removing digitiform gland mucus from the digitiform glands, and isolating the proteins and peptides. The isolated proteins and peptides could be individually applied to the reproductive tract in vitro to confirm which peptide is the active ingredient within the digitiform gland mucus. Once the protein or peptide responsible for the conformational change in the reproductive tract is isolated it can then be identified.

Dart shooting drives sexual selection

A trait or behavior is said to be sexually selected when it is associated with differences in reproductive success (Darwin 1871). Landolfa et al. (2001) and Rogers and

Chase (2002) hypothesized that the dart serves as an aid in sperm competition, thus, benefiting the male function because sperm from snails that shot darts successfully escaped digestion outcompeting sperm from those that shot poorly. The results from this thesis also provide supporting evidence that dart shooting benefits the male function, therefore, dart shooting is a sexually selected behavior in the mating system of Cantareus aspersus.

A recent study by Koene and Schulenburg (2005) provides further support that dart shooting evolved for the purpose of mucus transfer. By using phylogenetic analyses they discovered that aIl dart shooting land snails also have the associated digitiform mucus glands. In addition, the different shapes (for example, blades and curvatures) of darts facilitate mucus transport. This suggests that the dart evolved in order to transport mucus from one individual to another.

AlI of the aforementioned studies hypothesize that dart shooting benefits the male function, and is therefore sexuaIly selected. However, these organisms are

51 hennaphrodites, begging the question of wh eth er dart shooting also benefits the female function. This would be possible if individuals shot successful darts consistently, indicating that dart shooting ability could be a heritable trait that is passed on to offspring. F emales would benefit from mating with males that are good dart shooters because their offspring would in tum be better dart shooters, and therefore, increase their direct fitness. It has not yet been demonstrated that dart shooting is heritable, thus, it remains unknown whether dart shooting could benefit the female function in this manner.

Dart shooting is one of the most complex and remarkable mating behaviors seen in molluscs. Researchers have been investigating dart shooting in terrestrial snails for over 250 years in order to determine its function, yet it was only discovered within the last five years that successful dart shooting increased the patemity of the shooter. The results from this thesis demonstrate that it is the digitifonn gland mucus carried on the dart during dart shooting which causes increased patemity. This result contributes to the knowledge and understanding ofthe act of dart shooting in Cantareus aspersus.

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