SEXUAL CONFLICT in ARACHNIDS Ph.D

MASARYK UNIVERSITY FACULTY OF SCIENCE DEPARTMENT OF BOTANY AND ZOOLOGY

SEXUAL CONFLICT IN ARACHNIDS Ph.D. Dissertation Lenka Sentenská

Supervisor: prof. Mgr. Stanislav Pekár, Ph.D. Brno 2017

Bibliographic Entry

Author Mgr. Lenka Sentenská Faculty of Science, Masaryk University Department of Botany and Zoology

Title of Thesis: Sexual conflict in arachnids

Degree programme: Biology

Field of Study: Ecology

Supervisor: prof. Mgr. Stanislav Pekár, Ph.D.

Academic Year: 2016/2017

Number of Pages: 199

Keywords: Sexual selection; Spiders; Scorpions; Sexual cannibalism; Mating plugs; Genital morphology; Courtship

Bibliografický záznam

Autor: Mgr. Lenka Sentenská Přírodovědecká fakulta, Masarykova univerzita Ústav botaniky a zoologie

Název práce: Konflikt mezi pohlavími u pavoukovců

Studijní program: Biologie

Studijní obor: Ekologie

Vedoucí práce: prof. Mgr. Stanislav Pekár, Ph.D.

Akademický rok: 2016/2017

Počet stran: 199

Klíčová slova: Pohlavní výběr; Pavouci; Štíři; Sexuální kanibalismus; Pohlavní zátky; Morfologie genitálií; Námluvy

ABSTRACT Sexual conflict is pervasive in all sexually reproducing taxa and is especially intense in carnivorous species, because the interaction between males and females encompasses the danger of getting killed instead of mated. Carnivorous arachnids, such as spiders and scorpions, are notoriously known for their cannibalistic tendencies. Studies of the conflict between arachnid males and females focus mainly on spiders because of the frequent occurrence of sexual cannibalism and unique genital morphology of both sexes. The morphology, in combination with common polyandry, further promotes the sexual conflict in form of an intense sperm competition and male tactics to reduce or avoid it. Scorpion females usually mate only once per litter, but the conflict between sexes is also intense, as females can be very aggressive, and so males engage in complicated mating dances including various components considered to reduce female aggression and elicit her cooperation. This thesis includes six studies which focus on three selected phenomena associated with sexual conflict. The first two publications investigate a rare case of reversed form of sexual cannibalism, recorded in the spider Micaria sociabilis. Mating experiments and field observation revealed that cannibalism is directed towards old females during years when the sex ratio is female biased. The reason why males attack females is likely the decrease in prey availability. Another three studies focus on mate plugging and genital morphology in two spider species, M. sociabilis and Philodromus cespitum. In both species, the female seems to be in control of mate plugging but the plug efficacy measured as plug removal success differed between the two species. The morphological study of male copulatory organ revealed the production site of the plug material and, more importantly, a sensory organ and a neural tissue located in the male copulatory device (i.e. bulbus genitalis), which has been always considered to be a numb, insensitive structure. The last study deals with the morphological aspect of sexual stinging performed during courtship in the scorpion Euscorpius alpha, a species where males possess bigger telsons (i.e. structures encapsulating the venom gland) than females. Our study revealed that both sexes possess similar types of secretory cells in their venom gland, but the proportional representation of the cells largely differs. It indicates that males produce venom of different properties than females, which is likely employed during sexual stinging.

ABSTRAKT Sexuální konflikt vychází z rozdílných zájmů samce a samice a můžeme se s ním setkat u všech pohlavně se množících organismů. U těch masožravých pak často bývá velmi intenzívní, neboť každá interakce mezi samcem a samicí přináší nebezpečí, že místo ke kopulaci dojde k zabití jednoho z jedinců. Karnivorní pavoukovci, mezi které patří například pavouci či štíři, jsou dobře známí svými kanibalistickými tendencemi. V rámci pavoukovců se studium konfliktu mezi pohlavími soustředilo nejvíce na pavouky, a to nejen kvůli častému výskytu sexuálního kanibalismu, ale také kvůli unikátní stavbě pohlavních orgánů obou pohlaví. Ta v kombinaci s častou polyandrií dále posiluje konflikt mezi samcem a samicí ve formě kompetice spermií, které se samci snaží vyhnout či ji redukovat pomocí nejrůznějších taktik. U štírů se samice obvykle páří pouze jednou před tím, než vyprodukují potomstvo, nicméně i u nich se s konfliktem mezi pohlavími setkáváme. Protože jsou samice často agresivní, samci předvádějí složité námluvy skládající se z mnoha různých komponentů, které slouží ke snížení agresivity samic a k vyvolání jejich spolupráce. Tato práce obsahuje šest studií zabývající se třemi vybranými jevy v rámci konfliktu mezi pohlavími. První dvě publikace se věnují vzácnému případu reversního sexuálního kanibalismu u pavouka mikarie pospolité (Micaria sociabilis). Pokusy v laboratoři a pozorování v terénu odhalily, že je kanibalismu namířen proti starým samicím v letech, kdy jsou výrazně početnější než samci. Důvodem, proč samci samice zabíjejí a konzumují, je pravděpodobně snížená potravní nabídka. Další tři studie se zaobírají pohlavními zátkami a morfologií kopulačních orgánů dvou pavoučích druhů, m. pospolité (M. sociabilis) a listovníka obecného (Philodromus cespitum). U obou druhů kontroluje zátkování samice, nicméně efektivita zátky měřená tím, zda-li je možné ji odstranit, se mezi zkoumanými druhy výrazně liší. Morfologická studie samčího kopulačního orgánu odhalila místo, kde je materiál tvořící zátku produkován. Rovněž bylo zjištěno, že orgán pro přenos spermií, který byl vždy považován za strukturu bez inervace, je vybaven smyslovým orgánem a nervovou tkání. Poslední studie se zabývá morfologickým aspektem samčího bodání samice během námluv u štíra západoalpského (Euscorpius alpha), u kterého samčí telzon dosahuje větších rozměrů než telzon samice. Naše studie ukázala, že jedové žlázy obou pohlaví jsou vybaveny podobnými buňkami, nicméně jejich proporcionální zastoupení se výrazně liší mezi samci a samicemi. Struktura buněk převládajících v jedové žláze samců napovídá, že samci produkují jed o jiných vlastnostech než samičí a je velmi pravděpodobné, že je tento jed použit právě při bodání, které provází námluvy.

ACKNOWLEDGEMENT

I would like to thank my supervisor Prof. Stano Pekár for introducing me to world of spiders and their intriguing behaviour and for his advice, ideas, scientific and financial support during the course of more than ten years of my study. He enabled me to attend conferences, internships and field trips and meet many interesting people for which I am very grateful. Many thanks go to people from our department, who made my study entertaining and adventurous due to numerous, excursions, conferences, parties and other occasions. Special thanks go to Radek, Eva, Veronika, and Michal for their support and friendship. I would also like to thank Honza who was willing to proofread the text of my thesis. Huge thanks go to Germany to Prof. Gabriele Uhl, who was of tremendous help during my doctoral study and who gave me the opportunity to learn morphological methods, and who hosted me so many times in her group where I always felt like home with her team, experiencing highly motivational atmosphere and having so much fun. Thanks go also to the people from the group: to Lizzi, who showed me how cool morphology can be and who patiently walked me through the first steps, to Peter for his advice with manuscripts and morphology but also for the tips for surviving in the scientific world, to Carsten who helped me greatly with understanding morphology, to Prof. Alberti who always found time for an advice, to Pierick, Philip, Anne, Anja, Heidi and the other members of the group for their help, scientific and non-scientific discussion, gaming nights, and the great fun I had with them. Another big thanks go to Switzerland to Prof. Wolfgang Nentwig and Prof. Christian Kropf, who kindly agreed to host me in their group for more than a year. This visit gave me great working experience but also a lot of fun and friends. I would like to thank them as well, above all to Thomas for his support and enthusiasm and million other things, to Tabea, Fränzi, Remo, Aline, Christopher, Benji, Fäbu, Deja, Michal, Katrin, and other members of the group for the nice times over fondue, table football and incredibly expensive Swiss beer. I would also like to thank the arachnological community, domestic and international, which is a bunch of amazing and funny people who are exceptionally interesting and a bit crazy and who kindly accepted me and my first nervous talks with enthusiasm and interest and who annually deliver a huge motivational kick. Special thanks go to Chri and Christian not only for the great companionship on the conferences but also on the travels around the world which I would have never done without them. At last but definitely not the least, I would like to thank to my family and friends, especially to my parents who have always supported me and have never doubted my decision to study spiders, but, on the contrary, were and are always proud of me.

TABLE OF CONTENTS

I. PREFACE ...... 17 II. INTRODUCTION ...... 17

1. SEXUAL CONFLICT ...... 17 1.1. Anisogamy and its consequences ...... 17 1.2. Mating and mate choice ...... 18 1.3. Polyandry and post-copulatory processes ...... 20 a. Cryptic female choice ...... 21 b. Sperm competition ...... 21 1.4. Male mate choice ...... 22

2. SEXUAL CONFLICT IN ARACHNIDS ...... 23 2.1. Spiders ...... 24 a. Mate search and courtship ...... 24 b. Sexual size dimorphism ...... 25 c. Sexual cannibalism ...... 26 d. Reversed sexual cannibalism ...... 28 e. Genital morphology ...... 30 f. Mating plugs ...... 34 2.2. Scorpions ...... 37 a. Courtship...... 38 b. Sexual stinging ...... 38 III. AIMS OF THE THESIS ...... 41 IV. CONCLUSION ...... 43 V. REFERENCES ...... 47 VI. SUPPLEMENT ...... 67

1. STUDY A ...... 69

2. STUDY B ...... 81

3. STUDY C ...... 91

4. STUDY D ...... 117

5. STUDY E ...... 133

6. STUDY F ...... 163

7. AUTHOR’S CONTRIBUTION ...... 195

8. LIST OF PUBLICATIONS ...... 197

9. CONFERENCE CONTRIBUTION ...... 198

I. PREFACE

This thesis deals with the sexual conflict in arachnids and encapsulates our findings on this topic, summarized in six research articles published in, or submitted to, scientific peer- reviewed journals. In all of them I am the first author. The data published in the first two articles were partly obtained during my bachelor’s and master’s studies. The remaining four studies were conducted and written up during my doctoral studies. The introductory part of this thesis encompasses the general theoretical background to the sexual conflict and describes the conflict between sexes and its examples in arachnids, predominantly in spiders. The main topics are sexual cannibalism in spiders, specifically in its reversed form, spider genital morphology with emphasis on mate plugging, and sexual stinging in scorpions. The introduction is followed by a short description of aims of the thesis and conclusions summarising the main findings of the six studies, which are enclosed at the end of the thesis along with a list of author’s contribution. The thesis is supplemented by a complete list of my publications and contributions to international and domestic conferences.

II. INTRODUCTION

1. SEXUAL CONFLICT Despite the common aim to produce viable offspring, interactions between males and females are characterized by conflicts over every aspect of breeding, from mate search to parental care (Trivers 1972, Kokko and Jenninson 2014). Sexual conflict, i.e. a conflict between the evolutionary interests of individuals of two sexes (Parker 1979), is a result of sexual reproduction and production of dissimilar gametes (i.e. anisogamy). This leads to an unequal investment into the offspring by males and females (Parker et al. 1972), setting a stage for a conflict between sexes (Arnqvist and Rowe 2005), which is pervasive in sexually reproducing taxa from cellular to behavioural levels (Gavrilets 2000).

1.1. Anisogamy and its consequences In anisogamous organisms, females produce a restricted number of large, energy-rich macrogametes (eggs), while males abound with small, highly motile microgametes (sperm). When these two gametes fuse, the large, food-rich egg provides all nutrition for the newly formed zygote, while all what the sperm brings is the male genetic information. That means

17 that the female initial investment into offspring is much higher than the male investment (Parker et al. 1972). In general, members of the sex that provides less to the offspring compete with each other for mating with members of the opposite sex (Trivers 1972). Therefore, females are typically the selective sex for which males compete. Males evolved a whole variety of traits maximizing their success in male-male competition (intrasexual selection) in order to drive away their rivals, to get access to a potential mate, and also to defend females or resources required by females for reproduction and survival (Miller 2013). Consequently, male body or its defence traits usually reach bigger sizes than those of females. Additionally, in order to excite or stimulate females, males are often equipped with various ornaments which are lacking in the opposite sex (intersexual selection). The competition between males persists also during interactions with the opposite sex as females choose among their suitors (mate choice) and grant access to their eggs only to the most agreeable partner(s) (Davies et al. 2012). Another consequence of anisogamy is that the sex with lower parental investment (typically males) has a higher potential rate of reproduction (Clutton-Brock and Parker 1992). Male fertilization rate is potentially higher than the rate at which female produces eggs (Bateman 1948). Males are therefore able to produce more offspring than females, being constrained mainly by accessability to females and their gametes (Trivers 1972). In males, each mating generally provides an opportunity to father offspring, and thus male fitness increases steadily with increasing number of matings. Mating that leads to inviable or unhealthy offspring represents a smaller proportion of the male reproductive output than it does for a female. Females, in contrast, maximize reproductive success by maximizing the amount of viable offspring produced through efficient transfer of resources, and so the mating optimum is often reached after a single or a few matings and any additional one might be detrimental for her (Arnqvist and Nilsson 2000). Therefore, males and females are expected to have different optimal mating rates over which a conflict arises.

1.2. Mating and mate choice Mating is usually non-random, containing an element of discrimination not only to distinguish individuals of the same species, but also to assess quality of a potential mating partner (Heisler et al. 1987; Andersson 1994). The choice can be either direct or indirect. A female chooses directly if she responds differently to males (accepting some of them and rejecting the others) according to a certain trait (e.g. size), and this choice leads to a differential male reproductive success. The choice is often indirect, though, being based on 18 certain female behaviour or attributes but not involving active choice or discrimination (Prenter et al. 2006). For example, a female resists all males, but the large ones are able to overcome her resistance. Alternatively, a female signals that she is ready to mate, and that stimulates a competition between the males, which favours those with higher competitive abilities (Wiley and Poston 1996). Consequently, although female approach to individual males does not differ, the male reproductive success does (Prenter et al. 2006). Traditionally, mate choice was perceived as a female bias or attraction to certain traits which, when expressed, lead to a higher mating success of a subset of males. The female mate preference coevolves with certain male trait, and this coevolution leads to a concurrent increase in trait display and attraction to the trait via positive feedback (Fisher 1930; Davies et al. 2012). The benefits gained by females due to choosing and mating a specific male may be direct, in the form of resources, such as egg-laying site, food provisioning, or parental care (Price et al. 1993). However, she often gets nothing more than a sperm, and the benefits are only indirect (genetic) in terms of conferring more attractive sons (“sexy sons” or Fisher’s hypothesis; Fisher 1930; Pomiankowski and Iwasa 1993) or offspring of a higher viability (good genes hypothesis; Zahavi 1975, 1977; Hamilton and Zuk 1982; Grafen 1990). Later studies suggest that female mate choice evolved from sexual conflict rather from acquisition of benefits (Rice 1996; Holland and Rice 1998; Rice and Holland 1999; Gavrilets et al. 2001; Chapman et al. 2003a, b). According to this theory, female choice is caused by general avoidance of costs associated with mating, such as loss of energy, increased exposure to predators, or transmission of diseases and/or parasites. Moreover, even if the costs are not direct, copulation with a low-quality partner is considered to be costly. Although the costs of mating are imposed on both sexes, their compensation by benefits of mating differs between males and females. In females, costs of additional matings or mating with a low-quality partner are usually not compensated by any benefits. Therefore, to avoid these costs, females evolved certain degree of resistance to male mating attempts. Hence, males are selected to overcome the female resistance and to manipulate her to mate. For example, males evolve display traits exploiting pre-existing sensory bias of females, and this bias exploitation may lead to cases when females mate suboptimally (e.g. too often; Holland and Rice 1998). Consequently, females are selected to evolve resistance to such traits, and males, in turn, are selected to overcome the resistance. The cycle of persistence and resistance is described as sexually antagonistic coevolution. It is characterised by complex and dynamic evolutionary chases between the sexes, in which

19 sexually antagonistic traits interact, and adaptations of one sex are harmful to the opposite sex (Holland and Rice 1998; Chapman et al. 2003a; Wigby and Chapman 2004). According to this interpretaion, the coevolution between the sexes is similar to that between a species and its parasites, or between predators and competitors (Van Valen 1973). The conflicting traits displace the sexes from their sex-specific optima, as the optimal mating rate are rarely identical for males and females. When this theory is applied to empirical data, it often gets unclear whether observed mating rate is a female decision or a result of male manipulation compromising female interests (Hosken et al. 2009). Various male morphological, physiological and behavioural traits that counteract the female resistance can impose physical damage to the female or manipulate her reproductive schedule, in both cases potentially lowering her life span (Bonduriansky et al. 2008). Consequently, females are counterselected to reduce the extent of the male-induced harm. Female resistance, however, can potentially serve as a tool to estimate male genetic quality – it allows a female to screen males and choose the most resilient one, and so, despite the costs caused by such mating, she can profit from production of highly successful sons (Eberhard 1996; Córdoba-Aguilar and Contreras-Garduño 2003). In general, it is not trivial to determine the adaptive significance of female choice, as it is difficult to disentangle sexual conflict from traditional models of sexual selection (Chapman et al. 2003a, b; Córdoba-Aguilar and Contreras-Garduño 2003; Cordero and Eberhard 2003). Nevertheless, no matter whether female rejects a male because of his low attractiveness or because of the high cost of mating, female mating resistance will always be in conflict with the reproductive interest of a rejected male (Parker 2006).

1.3. Polyandry and post-copulatory processes When both sexes mate multiple times, the sexual conflict is often very intense and costly (Rice 2000; Hosken et al. 2009). Despite low mating rates expected in females and the high costs associated with mating, females of most animal species mate with several partners (Arnqvist and Nilsson 2000). There are several hypotheses explaining this discrepancy between theory and empirical data. Female polygamy (i.e. polyandry) may occur for various reason: to gather sufficient amount of sperm (Walker 1980; Yasui 1998); to obtain direct benefits, such as food or egg laying sites (e.g. Lewis and South 2012; Reding 2015); to increase genetic quality of her offspring (e.g. Fedorka and Mousseau 2002; Fisher et al. 2006; Egan et al. 2016); or to simply avoid costs of resisting the mating (e.g. Parker 1970a; Lee and Hays 2004). Due to the risk of sperm limitation, virgin females are expected to be less choosy and increase their choosiness with the number of mating (Kokko and Mappes 2005). 20

Copulating with several males enables a female to correct her initial choice in case she encounters a higher-quality male. In animals with internal fertilization, copulation seldom leads directly to gamete fusion. Therefore, male paternity success is not ensured by genital coupling and not even by successful sperm transfer. If female mates with other males, his sperm do not have to be used for fertilization, or at least the likelihood of his paternity might be reduced (Eberhard 1985). The chances that copulation results in fertilization (and to what extent) are influenced by female choice inducing processes active after the onset of copulation (i.e. cryptic female choice) and by interaction between ejaculates (i.e. sperm competition; Eberhard 1996).

a. Cryptic female choice

Cryptic female choice occurs when a female mates with more than one male, and her biological or physiological responses to a given male bias his paternity success based on his particular behavioural or morphological trait (Thornhill 1983; Eberhard 1996). The choice is carried out through processes varying from prevention or early termination of sperm transfer to sperm dumping or zygote abortion (Eberhard 1996). It allows a female to choose not only whether to mate or not, but also how many resources (if any) to allocate to a given mate or mating. Additionally, physical contact with a male during genital coupling and sperm transfer may provide more accurate assessment of male quality than during precopulatory stage (Edward and Chapman 2011). Since the amount of sperm transferred increases with increased copulation duration, males typically try to prolong the copulation and often engage in courtship not only before the copulation, but also during its course (i.e. copulatory courtship) through e.g. genital stimulation, gustatory feeding, and tapping or stroking the female while copulating (Eberhard 1996; Kunz et al. 2012).

b. Sperm competition

If a female copulates with two or more males and their ejaculates spatially and temporally overlap, a competition between sperm for fertilization of a given set of eggs occurs (Parker 1970b, Birkhead and Moller 1998). Consequently, male-male competition persists even after a successful sperm transfer, but instead of males, their ejaculates compete. If a female mates with another male, the paternity success of the previous partner may be diluted, and so the male is selected to rule out the sperm of previous partners, and subsequently prevent this from happening to his sperm. This phenomenon shapes a whole range of behavioural,

21 physiological and morphological adaptations to reduce either mating success of previous males (offensive strategies) or further mating behaviour of the female (defensive strategies; Parker 1970b). Males evolved many different strategies of how to protect their paternity - they engage in mate guarding before and/or after copulation, transfer anti-aphrodisiacs with their ejaculate to reduce female attractiveness or receptivity, flush out, dilute, replace or displace ejaculates of previous males from female sperm storage organs, or apply mechanical barriers in or on her genitalia (Parker 1970b; Simmons 2001). In some cases, when a male attempts to induce female monandry, it may in turn lead to male monogamy (i.e. monogyny), when males give up additional mating in order to enhance and/or protect their paternity in the context of competition with other males (Fromhage et al. 2008).

1.4. Male mate choice So far, males have been presented as the indiscriminative sex in the text, only competing for choosy females. This view, however, cannot be fully generalized, as systems with partial (competitive, but also choosy males) or complete (choosy males, competitive females) sex role reversal also exist. Male mate choice has been first attributed to species in which males provide a significant contribution to the offspring. Therefore, it has been considered that mate choice can be predicted based on male parental investment – when a male provides more care than a female, male mate choice and female-female competition is expected, and when both sexes contribute equally, mutual mate choice is likely to occur (Gwynne 1991; Jones et al. 2000). The level of parental investment is reflected in the number of sexually available males and females (i.e. operational sex ratio) and potential reproductive rate, as higher parental investment lowers future mating rate and prolongs time of sexually unreceptive stage (Emlen and Oring 1977). However, male mate choice has also been reported in systems where males do not provide any paternal care (Bonduriansky 2001; Edward and Chapman 2011). Therefore, an alternative explanation is required, such as choosiness due to the energetic costs of copulation (Dewsbury 1982; Petrie 1983), or limitation caused by sexually selected traits (Edward and Chapman 2011). These traits increase male attractiveness to females or provide an advantage in male-male competition (e.g. longer courtship, production of larger gametes or ejaculates) but reduce the number of matings a male can achieve (Wedell et al. 2002; Byrne and Rice 2006). A prerequisite for the evolution of male mate choice is variation in female quality, causing the benefit of mating to differ between females (Kokko and Johnstone 2002; Edward and Chapman 2011). The greater the variation, the more adaptive the male mate choice is. 22

Males tend to favour female phenotypes that indicate high fecundity (often related to female body size) or reduced sperm competition. There are many ways how a male can exert his choice – if courted, he can reject a given female (Berglund and Rosenqvist 2001). If courting, he may decide to court some females, but not others, or adjust courtship intensity or his involvement in male-male competition according to his preference (Tudor and Morris 2009; Lihoreau et al. 2008). Additionally, similar to females, males can allocate their investment based on female quality even after the onset of copulation, e.g. by different parental investment, ejaculate composition or simply the number of sperm transferred. This cryptic male mate choice is likely to be common because females often do not advertise their mating status and thus male can evaluate it only through a physical contact (Edward and Chapman 2011), often encompassing mechanisms which are difficult to be distinguished from copulatory behaviour (Bonduriansky 2001).

2. Sexual conflict in arachnids Arachnids are a group of over 100,000 named species (Cracraft and Donoghue 2004) exhibiting a wide range of mating tactics. The mechanisms of sperm transfer differ considerably within and among the arachnid orders. Males typically form a spermatophore which is transferred into female genitalia by various means, from its deposition without an encounter of the mating partners to a direct transfer during copulation (Polis and Sissom 1990; Proctor et al. 1995). The two exceptions are harvestmen, that transfer sperm directly into female genitalia with a penis (Pinto-da-Rocha et al. 2007) and spiders, that use secondary copulatory organs carried on pedipalps (Eberhard and Huber 2010). In many arachnid taxa, the oviposition does not take place right after copulation but is delayed, which in combination with polyandry facilities intense sperm competition. In studies of sexual selection and conflict, spiders received by far the most attention among arachnids. They are particularly interesting taxon to study conflict between sexes as they are predacious, sexually dimorphic, and often polygamous. This, in combination with their unique genital morphology, forms a potential for an intense sexual conflict, which may escalate into extreme forms not only before the copulation itself, but also afterwards. Spider mating behaviour involves various behavioural tactics and has become a popular subject for the study of sexual selection and conflict (Schneider and Lubin 1998, Eberhard 2004, Huber 2005; Schneider and Fromhage 2010). 23

2.1. Spiders

a. Mate search and courtship

Typically, spiders (and other arachnids) are solitary-living animals, engaging in intraspecific interactions only during mating (but see Choe and Crespi 1997 for exceptions). Before reaching adulthood, males and females have a similar lifestyle. However, after the maturation moult, the lifestyle of males changes. The change is especially dramatic in web- building spiders, as males stop to build webs to catch prey or even cease feeding and become vagrant to search for females (Foelix 2011, Foellmer and Fairbairn 2005). In actively hunting spiders, the change is less obvious, but adult males are typically the searching, more active sex (Foelix 2011). As spiders are usually dispersed over a large area, occur at low densities, and their females are rather sedentary, the chance of a random encounter with a mate may be low. Therefore, the search for a potential mate is largely dependent on female-produced chemicals (sex pheromones; reviewed in Gaskett 2007). Airborne pheromones play an essential part as they are effective over long distances. They indicate a general location of the female and elicit searching behaviour in males. Contact chemicals then provide specific information about the female and stimulate courtship. They are deposited either directly on female cuticle or on her silk. The latter option allows a male to gain various information about a particular female (e.g. age, mating history, quality, hunger level, mating status; Johnson 2001, Gaskett et al. 2004, Roberts and Uetz 2005, Aisenberg and Costa 2008, Pruitt and Riechert 2009; Johnson et al. 2011) without the necessity of a direct contact with her. Given the risk of sexual cannibalism, obtaining correct information about a female and her receptivity from a safe distance might be crucial for male survival (Gaskett 2007, Schneider and Andrade 2011). Because females are often voracious and aggressive, male spiders face a problem: in case of not being recognized by their potential mate, they might be mistaken for a prey and instead of mating they might end up as her next meal (Gould 1984; Schneider and Andrade 2011). Therefore, male spiders often engage in long-lasting and costly courtship. The courtship serves not only for identification of a suitable mate but also for induction of female receptivity and for sexual stimulation. The courtship behaviour is therefore critically important for male mating success. Spider males evolved elaborated epigamic signals through chemical, tactile, acoustic and/or visual channels (Uhl and Elias 2011). The complexity and duration of the signals varies among species, including those with complex

24 multimodal rituals, such as lycosids or salticids (e.g. Uetz et al. 2009; Elias et al. 2012), but also some rather rare cases of species without any obvious courtship behaviour (Sentenská and Pekár 2013). As the spider courtship signalling is often very complex, combining e.g. motion displays with complicated vibratory songs, female mate choice are usually affected by multiple cues (Schneider and Andrade 2011). Females have been found to prefer males based on a courtship quality (e.g. high drumming rate; Kotiaho et al. 1996; Parri et al. 1997; Sentenská et al., submitted b), physical attributes favouring larger males (Singer and Riechert 1995), or males with symmetrical secondary sexual traits (Uetz et al. 1996). Female choice is not invariable but changes with e.g. age, mating history and/or nutritional state (Watson 1991, 1998; Hebets 2003; Persons and Uetz 2005; Uetz and Norton 2007).

b. Sexual size dimorphism

The risk of being cannibalised is not fully averted after a correct identification, and persists for the whole time the sexes are in contact. Sexual cannibalism is facilitated by the fact that in the most of spider species, females reach bigger sizes, which makes an approaching male an easy prey. Evolutionary significance of size differences between the sexes is a subject of debate and remains controversial (Foellmer and Moya-Laraño 2007). In general, sexual size dimorphism is attributed to different selection pressures affecting males and females due to their different lifestyles. Despite the advantage of bigger size in male-male competition and during female choice, being small might be advantageous in spider males in order to avoid being cannibalized during courtship (Elgar et al. 1990, Elgar 1992, Elgar and Fahey 1996, but see Foellmer and Fairbairn 2004, Prenter et al. 2006) or being predated during the mate search (Elgar and Nash 1988, Gunnarsson 1998). A smaller body size provides locomotion advantage in terms of moving faster on vertical surfaces (Moya-Laraño et al. 2002, but see Foellmer and Fairbairn 2005) or walking upside-down using own silk bridges (Corcobado et al. 2010), which might be advantageous in competition with other males (reaching females sooner than bigger males) or in predator avoidance. The suppressed selection pressure on males to reach bigger size, which is normally favoured in male contests, has been explained by reduced male-male competition resulting from higher mortality during mate search (Vollrath and Parker 1992). Moreover, early maturation leading to small body size would enable a male to reach virgin females sooner than larger, later-matured males (advantageous due to sperm competition, see below). The degree of different body size ratios across species has been considered to be determined by different life-histories,

25 predatory strategies and mobility-dependent mortality, predicting that actively hunting predators should be less dimorphic than sit-and-wait predators (Vollrath and Parker 1992). The major component of a female reproductive success is the egg production. Since it is highly variable and dependent, among other features, on female size (Eberhard 1979, Head 1995), females are selected to increase body sizes in order to maximize egg production. The variation in sexual size dimorphism between species can be explained by varying ecological constrains and differences in foraging ability, which result in variation in the strength of fecundity selection (Head 1995). The original idea that size differences between sexes result from selection favouring male dwarfism (Elgar and Nash 1988; Elgar et al. 1990; Elgar 1992; Vollrath and Parker 1992; Gunnarsson 1998) has been supported by several studies (e.g. Piel 1996, Schneider and Lubin 1996, 1997; Legrand and Morse 2000; Maklakov et al. 2004; Danielson-François et al. 2012), including gravity hypothesis (Moya-Laraño et al. 2002; Corcobado et al. 2010), but is contradicted by comparative and cladistic analyses. These analyses suggest that sexual size dimorphism would be better explained by evolution of female gigantism (Coddington et al. 1997; Prenter et al. 1997, 1998, Hormiga et al., 2000). That means than females rather than males evolved from the original size due to the fecundity selection (Head 1995). Female-biased sexual size dimorphism, prevalent in spiders, has a significant impact on interactions of males and females. The size advantage enables females to dominate and control sexual interactions (Schneider and Fromhage 2010). Moreover, in predatory animals, both mating partners are selected to avoid and protect themselves against aggression of the opposite sex, but if one of the sexes is physically dominant, the selection pressure is asymmetrical and may lead to sexual cannibalism (Schneider 2014).

c. Sexual cannibalism

Sexual cannibalism is a situation when an individual (typically a female) attacks, kills, and consumes its mate before, during, or after copulation. The potential to avoid a cannibalistic attack is size-dependent (Elgar 1992, Wilder and Rypstra 2008), so in spiders, the roles of a potential cannibal and a potential victim are given. The frequencies of sexual cannibalism within and among the spider species are related to the degree of sexual size dimorphism (Wilder and Rypstra 2008) and, accordingly, rare cases of reversed sexual cannibalism (i.e. males kill and consume females before copulation) seem to be connected with male-biased

26 sexual size dimorphism, which is exceptional in spiders (Schutz and Taborsky 2005, 2011; Aisenberg et al. 2009, 2011; Sentenská and Pekár 2013, 2014). Sexual cannibalism is often interpreted as a pinnacle of sexual conflict, because the result of such interaction is extreme in terms of obvious limitation of victim’s reproductive future (Elgar and Schneider 2004). The degree of this limitation, however, depends on timing of the cannibalistic attack. Although there is no benefit for a male if cannibalism takes place before copulation, he may profit if it occurs during or after the sperm transfer (Buskirk et al. 1984; Johns and Maxwell 1997; Andrade 1996, 2003). Naturally, this could only be the case in the classical sexual cannibalism, where a female kills a male, since killing a female after copulation would be maladaptive. A widely-accepted explanation of female attacks towards males is because they are motivated by hunger (Wilder et al. 2009). According to the economic model (Newman and Elgar 1991), sexual cannibalism prior to insemination by virgin females results from foraging considerations based on assessment of male value as a prey compared to his value as a sexual partner. Females are expected to be more cannibalistic when food is scarce and mates abundant and accordingly, when mates are rare, they should restrict their cannibalistic tendencies due to the risk of remaining unmated (Newman and Elgar 1991). Based on observations of stereotypical female behaviour and the high rate of unmated cannibalistic females, the aggressive spillover hypothesis has been proposed (Arnqvist and Henriksson 1997). It suggests that sexual cannibalism is a result of genetic constrains given by voracious personality, which is advantageous in foraging but maladaptive in the mating context. This hypothesis has found some support (Morse 2004), but has been contradicted by recent studies (Johnson 2013; Pruitt and Keiser 2013; Kralj-Fišer et al. 2012, 2013, 2016). Rather than being maladaptive, sexual cannibalism can represent an effective mechanism of female mate choice (Elgar and Nash 1988, Eberhard 1996, Prenter et al. 2006) which utilizes rejected males as a meal. A female may switch between two behavioural modes: decreased aggression towards some males and increased aggression towards the others. Alternatively, in case of indirect mate choice, females attack all males but only some of them (typically the larger ones) are able to avoid the attack. In other words, the female attack behaviour does not differ but the attack success does (Prenter et al. 2006). Mate choice via cannibalistic attacks does not have to be not strictly binary, as a female may exert her choice by timing of the attack. The amount of sperm transferred usually increases with increasing copulation duration (e.g. Elgar et al. 2000; Schneider et al. 2000, 2006; but see Bukowski et al. 2001, Snow and Andrade 2004) and thus female can delay attack when

27 copulating with preferred males to allow them to transfer more sperm and fertilize more eggs. Therefore, sexual cannibalism can be an effective way of paternity control (Prenter et al. 2006). Interestingly, in some cases, males facilitate sexual cannibalism through copulatory posture (Andrade 1996) or even spontaneous death during copulation (Foellmer and Fairbairn 2003). It has been shown that by sacrificing themselves, males may gain paternity benefits by means of prolonging the copulation and decreasing female receptivity to further mating attempts (Andrade 1996). Since cannibalism during sperm transfer may lead to increased paternity, the male faces a trade-off between a current and a future reproductive investment. If his expected future reproductive value is low (e.g. he is unlikely to achieve additional copulations), being eaten can be an adaptive male strategy (i.e. paternal investment strategy; Parker 1979; Buskirk et al. 1984; Schneider 2012; Schwartz et al. 2016) leading to monogyny, in which males invest maximally into fertilization of a single female. In most cases, however, being eaten is not adaptive for males, and so they evolve traits effective against cannibalism (Elgar 1992). Males are selected to perceive signs of female receptivity in order to avoid females which are likely to be cannibalistic (e.g. mated or hungry females, Johnson 2001; Johnson et al. 2011; Rabaneda-Bueno et al. 2008). In some orb-web spider species, males perform courtship on a mating thread, from which they may drop in case of a female attack, and this strategy likely evolved due to the risk of sexual cannibalism (Elgar 1991). In other species, males mate with females when they are unable to attack, e.g. during moulting or feeding (Lubin 1986; Fromhage and Schneider 2005a; Biaggio et al. 2016). Although nuptial gifts are very rare among spiders, they most likely serve as a diversion of female voracity from the male to another prey item during copulation (Prokop and Maxwell 2009). Furthermore, some males emit chemicals that reduce female aggressiveness (Zhang et al. 2011) or induce catalepsy in females (Singer et al. 2000; Becker et al. 2005).

d. Reversed sexual cannibalism

The role of the cannibal and the victim can be switched in several taxa in which the restriction by female-biased sexual dimorphism is not that strong. This so-called reversed sexual cannibalism has been reported only in a handful of species (Jackson 1982; Jackson and Pollard 1990; Schutz and Taborsky 2005; Cross et al. 2007; Aisenberg et al. 2011; Sentenská and Pekár 2013, 2014) and appears to be associated with male-biased sexual size dimorphism, which is atypical with spiders (Schutz and Taborsky 2005, 2011; Aisenberg et

28 al. 2009, 2011; Sentenská and Pekár 2013, 2014). This behaviour has been investigated in detail only in two species and in both cases, the findings indicate that reversed sexual cannibalism follows similar rules as its classical counterpart, and can therefore be explained by similar hypotheses, especially as a mechanism of extreme mate choice. Male mate choice is common in systems in which the male investment into reproduction is high. This is the case of a sand-dwelling wolf spider species Allocosa brasiliensis in which the sex roles are reversed – adult males are larger than females and dig deep burrows in which they remain sedentary, while females are mobile, search for males and initiate the courtship. After mating, a male yields his burrow to the female; the burrow represents a valuable paternal investment (Aisenberg et al. 2007; Aisenberg and Costa 2008). Males of this species were observed to attack mated females of poor body condition, and so this case of cannibalism can be explained as an extreme male mate choice via cannibalism, a strategy used to avoid loss of the burrow to a low-quality female (Aisenberg et al. 2011). In an ant-mimicking gnaphosid spider Micaria sociabilis, males also occasionally attack, kill and consume females (Sentenská and Pekár 2013). In this species, cannibalism also seems to act as a mechanism of male mate choice. However, the cause of this extreme choosiness is not so trivial to determine, as males do not put any apparent investment into the offspring apart from sperm. Nevertheless, although male spiders generally invest nothing more than sperm into their progeny, they are often choosy. Male choice should occur especially in species in which males have limited number of matings, e.g. when they face the danger of sexual cannibalism or when they damage their genitalia in order to plug female copulatory ducts (see below) – this strategy potentially diminishes sperm competition but leaves the males functionally sterile (Huber 2005; Schneider and Andrade 2011). In web-builders, adult males often do not feed and they can maintain sufficient body condition to search, court, and mate only a limited number of females (Schneider and Andrade 2011). In addition, predation or sexual cannibalism may occasionally result into female-biased sex ratio and, consequently, male mate choice (Costa and Pérez-Miles 2002, Moya-Laraño et al. 2003). However, males can be choosy simply because of high variability in female quality. As female fecundity is strongly correlated with her size, males often favour bigger or heavier females (e.g. Beck and Connor 1992; Uhl 1998; Bridge et al. 2000; Danielson-François et al. 2002; Cross et al. 2007; Hoefler 2007). Furthermore, spider males commonly prefer to mate with virgin females in order to avoid sperm competition (e.g. Gaskett et al. 2004; Riechert and Singer 1995; Rypstra et al. 2003; Aisenberg et al. 2011).

The number of matings of M. sociabilis males does not seem to be limited. At the same time, they are not in danger of female cannibalism as they are bigger than females. They also do not put much effort into the courtship as they directly mount the female and transfer their sperm (Sentenská and Pekár 2013). As mentioned above, however, male mate choice can arise from female-biased operational sex ratio (i.e. the number of females available for mating is higher than the number of males available for mating; Kvarnemo and Ahnesjö 1996) and/or high variability in female quality (Bonduriansky 2001). M. sociabilis seems to fit to both hypotheses. Four years of field and laboratory observations revealed that reversed sexual cannibalism was common in M. sociabilis only when the sex ratio was shifted towards females, while in years with sex ratio close to 1 (i.e. males and females reaching similar abundance), the cannibalism was very rare (Sentenská and Pekár 2014). Examination of female characteristics revealed that female size and mating history has no effect on the male cannibalistic behaviour. Female age, however, played an important role. M. sociabilis produces two generations a year, and the period of overlap of the two generations is also the time when most cases of cannibalism were observed. Specifically, old, spring-born females were cannibalized by young, summer-born males if the latter were large enough (Sentenská and Pekár 2013). Observation of potential prey and its availability during the season revealed that males of the summer generation develop in a period when prey occurrence is high, but when they moult to the adulthood, the prey availability decreases. Therefore, the male cannibalistic behaviour most likely represents a male foraging strategy of how to deal with the prey scarcity, but it is regulated by the abundance of their potential mates, and, if adopted, is directed to those of an inferior quality (Sentenská and Pekár 2014).

e. Genital morphology

Genitalia of spider males are unique among the animal kingdom in several aspects. Male copulatory organs are not associated with their primary genitalia, which are located in the abdomen, but are shifted to the cephalothorax, where they are located on a pair of modified legs, the pedipalps. Therefore, before the copulation itself, sperm has to be transferred from the site of its production into the copulatory organs. In order to do so, after the maturation moult, a spider male constructs a web in which he deposits the sperm. The sperm is consequently transported to a dead-end tube (spermophor), which is located within the genital bulbus, i.e. the part of the pedipalp specialized to receive and transfer sperm. Spider sperm leave the male reproductive tract inactive, coiled and encapsulated in a sheath, and

30 they remain in this condition during the transfer into the male copulatory organ and eventually into the female genitalia, where they are stored and activated by the female shortly before oviposition (Foelix 2011). The main copulatory device, the genital bulbus, consists of sclerites joint by folded membranes (hematodochae), which are expanded by a hydraulic pressure caused by haemolymph being pumped into the bulbus during copulation. Hematodochae cause changes in the bulbus conformation, as they rotate, extend, and tilt the sclerites, enabling highly complex movements of the whole organ (Huber 2004). Interestingly, the bulbus lacks muscles (Comstock 1910; Gering 1953; Levi 1961; Huber 2004) and setae (Kraus 1978; Eberhard and Huber 2010), and until recently it was considered to lack any sensory organs or neural tissue whatsoever (Osterloh 1922; Harm 1931; Lamoral 1973; Suhm et al. 1995; Eberhard and Huber 1998a; Eberhard and Huber 2010). The apparent lack of nerves and muscles brought up many questions about the function of male copulatory organ in spiders and its impact on male-female interactions. The absence of innervation is striking because a copulatory organ which is not innervated is incapable of providing any direct sensory feedback during copulation. The proximal parts of the palp (cymbium, tibia) are covered by setae and richly innervated, but as they do not participate directly in sperm transfer, they may provide only crude information about the bulbus position. That would suggest that a male is basically “sensorily blind” when it comes to activities crucial for a successful reproduction, such as sperm uptake, genital coupling, and sperm transfer (Eberhard and Huber 1998a, 2010). Moreover, the lack of innervation may prevent a male from an accurate assessment of female mating history. Such information is a highly relevant characteristic, considering female genital morphology (see below) and post-copulatory processes, and males may be favoured to adjust their mating investment accordingly (Lipke et al. 2015). The lack of any sensory feedback during genital coupling was considered to be compensated by the evolution of preliminary locking structures on genitalia of both sexes, providing anchoring points that facilitate alignment of genitalia and intromission. Nevertheless, high complexity and rapid divergent evolution of the spider genitalia (Eberhard 1985; Huber 2004) suggest that apart from their primary function to transfer sperm, they evolved to such diversity in the context of sexual selection. The male copulatory organ might be shaped by cryptic female mate choice and sperm competition, and may even function as internal courtship devices (Eberhard 1985).

Moreover, behavioural observations actually suggest sensitivity of the male copulatory organ. For example, males seem to sense whether the tip of their bulbus has entered a sperm droplet during the sperm induction (Eberhard and Huber 1998b). Furthermore, it has been observed that male spiders adjust their behaviour once they reach genitalia of a previously inseminated female, which suggests that this information can be perceived only through genital contact, and indicates sensitivity of the intromittent structure (Bukowski et al. 2001; Jones and Elgar 2008). Although genital coupling is often accompanied by various palpal movements interpreted as failed intromission attempts due to lack of sensory feedback (Watson 1991; Huber 1998), they may represent exploratory behaviour or even copulatory courtship behaviour used to female stimulation (Stratton et al. 1996; Eberhard 1994, 1996; Eberhard and Huber 2010). Two recent studies prove that despite the historically lacking evidence of innervation, male genital bulbus possesses not only nerves and neurons (Lipke et al. 2015), but also sensory organs (Sentenská et al. submitted a). Interestingly, although the two spider taxa used in these studies are phylogenetically distant, the similarity of the arrangement of the nerve and neurons is striking, suggesting that the presence and arrangement of the neural tissue represents a common pattern in all spiders. The function of the sensory organ inside the male bulbus has not been clarified yet. Nevertheless, based on its structure and association with the intromittent structure of the bulbus (i.e. embolus), it most likely enables the male to perceive mechanical and/or chemical cues from female genitalia (Sentenská et al, submitted a).

Female spiders typically possess paired sperm storage organs and up to three genital openings. Female genital system of haplogyne spiders represents a plesiomorphic state, as the primary genital opening (gonopore) is used both for copulation and for oviposition. During copulation, males insert both copulatory organs into the gonopore and deposit sperm into the spermathecae. The fertilization takes place in uterus externus which is connected to spermathecae with the same duct which males use for copulation and so the copulatory duct is at the same time used as a fertilization duct (cul-de-sac design). In entelegyne spiders (comprising about 80% of all known spider species), females possess so- called epigyne, which is a copulatory organ with two copulatory openings. Copulatory ducts lead from each opening to one of paired spermathecae, from which a fertilization duct leads to the uterus externus (conduit design). During mating, the tip of the male copulatory organ (i.e. embolus) is inserted into one of the copulatory openings, through the copulatory duct, and finally to the spermatheca, where sperm is deposited. To inseminate both 32 spermathecae, males have to achieve two insertions, one with each palp. Such morphology enables females to influence insemination and paternity likelihood by selectively allowing one or two insertions (Bukowski and Christenson 1997; Snow and Andrade 2005). Before oviposition, sperm in the spermatheca are activated, and migrate through the fertilization ducts to the uterus, where they fertilize egg cells that are laid through the gonopore (Foelix 2011). Sexual dimorphism and genital morphology of both spider sexes indicate that females can be hardly physically coerced into copulation by male genital structures (but see Řezáč 2009). As the external parts of entelegyne female genitalia, including the epigyne, the copulatory ducts, and parts of the spermathecae, are compact, rigid, sclerotized, and often complex structures, male spiders are not capable to push into or pull open the female genitalia in order to force entry or achieve deeper intromission, which are procedures often seen in insect (Eberhard and Huber 2010). The copulatory ducts into which males insert their emboli during copulation are typically much longer and more coiled than the fertilization ducts, which are out of the reach of male copulatory organ. Such a difference suggests that female genitalia have evolved to be selective filtering devices, facilitating successful sperm transfer by males whose genitalia have certain mechanical properties. Such design enables cryptic female choice by means of mechanical fit, favouring males who are able to adjust mechanically to complex female genitalia (Eberhard and Huber 2010). Unlike male genital structures in insects, which seize or clasp female genitalia to restrain movements of her abdomen, copulatory organs of spider males can hardly do so, as they lack muscles and powerful clasping structures. Therefore, successful coupling and sperm transfer can be easily affected by female behaviour. Female size advantage, in addition to the absence of clasping structures in male copulatory organs, therefore enables a female to control the male fertilization success simply by early termination of the copulation. Based on the female genital morphology corresponding with taxonomical classification of spiders (haplogynes and entelegynes), sperm precedence patterns have been proposed (Austad 1984). Assuming a female mates multiple times and several males deposit their sperm into the same spermatheca, the haplogyne cul-de-sac design favours sperm of the last male, because it is deposited closest to the fertilization site (last in - first out). Therefore, in order to avoid the sperm competition, males of haplogyne spiders were expected to guard females before egg-laying. In entelegynes, the first male sperm precedence rule was assumed as first sperm to enter should be the closest to the fertilization duct (conduit design; first in – first out). Entelegyne males should therefore tend to guard

33 subadult females in order to be the first ones to transfer their sperm into the female genitalia (Austad 1984). However, some entelegynes possess the cul-de-sac condition with one duct only, while some haplogynes evolved two ducts. Therefore, sperm precedence system cannot be determined based simply on spider taxonomical classification. Additionally, to estimate sperm precedence system in species with spermathecae equipped with two ducts, the shape of the spermatheca and the relative position of the ducts is crucial – first male sperm may also enter the fertilization duct first only if it is connected to an ovoid spermatheca on the opposite side of the copulatory duct. If both ducts open close to each other, which is a conformation often seen in entelegyne female (e.g. Sentenská et al. 2015, submitted b), first sperm in the spermatheca is likely to be pushed away from the fertilization duct entrance by following sperm, leading rather to last than the first male precedence predicted for entelegynes (Elgar 1998). Moreover, the priority patterns presume that sperm from different males are stratified within the female storage organs. That is possible due to the fact that spermatozoa are transferred and stored encapsulated, non-motile, and in distinct clusters. However, several studies described cases of sperm mixing within a female spermatheca, and so the sperm stratification cannot be fully generalized (Uhl 2000; Useta et al. 2007).

f. Mating plugs

Genital morphology of entelegyne females (rigid sclerotized nature of its external parts, separate openings for insemination and oviposition) in combination with female polyandry and ability to store sperm for long periods are most likely the reasons why mating plugs are so frequent among spiders (Elgar 1998; Uhl et al. 2010). Mating plugs, or copulatory plugs, are generally thought to be applied by males in order to protect their paternity by means of impeding access of rival males to female genitalia. However, they may also serve other functions, such as prevention from genital infection or sperm leakage, backflow, or desiccation (Hinton 1964; Huber 1995; Simmons 2001). Furthermore, mating plugs may reduce female attractiveness (Parker 1970b), alter her receptivity towards other males (Eberhard 2004; Avila et al. 2011), or affect female reproductive physiology and behaviour in favour of the male who applied the plug (Eberhard and Cordero 1995; Eberhard 1996). Most studies of the phenomenon of plugging indicate that the major factor driving the evolution of these devices is the sperm competition, as they often lower the female mating frequencies by means of applying a mechanical barrier into female genitalia, preventing or delaying her remating (Parker 1970b; Eberhard and Huber 2010; Uhl et al. 2010).

Females do not act idly when plugged but can facilitate, avoid, or otherwise affect the plugging efficacy by various means. A female may profit from a plug if deposited by a preferred male, as the high quality sperm she obtained can be protected, or the plug can suppress the harassment by other males (Fromhage 2012). Nevertheless, being plugged may compromise sequential cryptic mate choice, and so, especially in virgin females, a strong precopulatory mate choice according to a certain quality threshold is adaptive in order to avoid costs arising from mating with low quality males without possibility to remate. As the plug is placed usually in the end of copulation, a female can permit sperm transfer but prevent the plug deposition behaviourally simply by uncoupling male palp from her genitalia (Uhl et al. 2010). She can also prevent deposition of the plug by a cannibalistic attack (Snow et al. 2006). In species where female secretions need to be added in order to produce a functional plug, female simply does not add her part of the compound (Aisenberg and Eberhard 2009). Genital morphology of both sexes is an important feature affecting the possibilities of plugging and its potential control by females. As female copulatory openings in entelegynes are paired, a male has to insert his palps into each of them in order to inseminate both spermathecae. If the openings are widely separated, a female can selectively control number of insertions, as well as whether a male blocks one or both openings. This way a female can obtain enough sperm and still leave the other spermatheca for another male, which allows her to exhibit cryptic female choice (Schneider and Lesmono 2009; Welke and Schneider 2009). When the female epigynal plate forms a depression (i.e. atrium) in which both copulatory openings are located, a male can plug a female with a single insertion (Uhl et al. 2010; Sentenská et al. 2015; submitted b). Spider plugs consist either of an amorphous secretion, or parts of male body. Although both types are used as paternity control devices, their properties largely differ. A male can block female genitalia using parts of his body, typically his copulatory organs, their parts, or his body as a whole. Pieces of the intromittent, sperm transferring organ, i.e. the embolus, are often found in female copulatory openings, copulatory ducts, or even spermathecae. Although they do not always preclude subsequent insertions (but see e.g. Fromhage and Schneider 2005a), this so-called lesser genital damage (Kuntner et al. 2014) has been shown to limit the amount of sperm transferred by following males and thus may bring paternity advantage to the male who placed it (Fromhage and Schneider 2006; Snow et al. 2006; Nessler et al. 2007, 2009; Ghione and Costa 2011; Herberstein et al. 2012; but see Schneider et al. 2001). Findings of several embolic parts within a single duct or

35 spermatheca suggest that these plugs do not block female genitalia unless placed into an optimal location (e.g. a narrow opening of the spermatheca) in which they would prevent subsequent males from transferring their sperm (Berendonck and Greven 2002). In some species, however, the genital damage may be a side-effect of male attempts to prolong the copulation process rather than an attempt to block access to female genitalia (Schneider et al. 2001). Male genital mutilation is not restricted to embolic breakage; in fact, a male may break off his whole genital structure during the act of emasculation (Robinson and Robinson 1980). While the partial genital damage does not necessarily result in functional sterility of the mutilated palp (Snow et al. 2006, but see e.g. Fromhage and Schneider 2005b), a full emasculation causes the male to become a full or partial eunuch restricted to mating with one (monogyny) or two females (bigyny; Kuntner et al. 2014). The severed palp left in the female genitalia does not necessarily prevent further mating of the female, so the eunuchs often stay around the female after copulation and engage in mate guarding through male- male fights. As the emasculation often occurs in species in which males are considerably smaller than females, getting rid of the palps, which represent a significant proportion of their body mass, may enhance male stamina and/or agility in male-male competition, providing advantage over non-emasculated rivals (Ramos et al. 2004; Kuntner et al. 2009a, b; Kralj-Fišer et al. 2011; Lee at al. 2012). If males do not engage in post-copulatory mate guarding, emasculation is often associated with spontaneous male death during the copulation, typically combined with female cannibalism (Branch 1942; Knoflach 2002; Knoflach and van Harten 2000; Knoflach and van Harten 2001; but see Knoflach and Benjamin 2003). Interestingly, detached palps were observed to keep on transferring sperm (Knoflach and van Harten 2001). The evolution of emasculation has been shown to be associated with extreme sexual size dimorphism, sexual cannibalism, and intense competition for virgin females, and may be interpreted as a terminal investment strategy related to the evolution of spider monogyny (Robinson and Robinson 1980; Miller 2007; Kuntner et al. 2014). In some species males invariably die once they achieve the final insertion, and their whole body serves as a short-term mating plug (Foellmer and Fairbairn 2003; Knoflach and Benjamin 2003). While plugs consisting of male genitalia or their parts may leave the male functionally sterile, amorphous mating plugs do not seem to have any apparent costs for the male. Morphological studies investigating the origin of amorphous mating plugs have shown that the plug material is produced by glands in the genital bulbus (Suhm et al. 1996; Uhl et al. 2014; Sentenská et al. 2015; submitted). Additionally, observations of spider behaviour

36 suggest that the material might also originate from the male genital tract (Knoflach 1998, 2004), male mouth area (Braun 1956), and, as they can be also partly or entirely produced by a female, in an undetermined site inside the female body (Knoflach 1998, 2004; Aisenberg and Eberhard 2009). The properties of the amorphous plugs differ interspecifically as they can be flimsy, gelatinous, waxy, sticky, rubbery, or very hard (Uhl et al. 2010). Some plugs contain spermatozoa (Sentenská et al. submitted b) or even consist predominantly of sperm (Whitehouse and Jackson 1994; Huber 1995). The plug production is often a time-related process as the amount of secretion increases with the copulation duration (Uhl and Busch 2009; Sentenská et al. 2015; submitted b). The amount of plug material transferred determines the plug efficacy as incomplete plugs often do not hinder additional copulation (Masumoto 1993; Uhl and Busch 2009; Sentenská et al. 2015). Nevertheless, even complete plugs can sometimes be removed and may allow the female to exert sequential mate choice (Sentenská et al. submitted b). Fully effective plugs are expected to be evolutionary instable as they either cause plugged females to be avoided by males, which lowers the selection pressure on plugs, or select males to overcome whatever plug deposited by their predecessors (Parker 1984; Fromhage 2012). The cost of production of the plug material does not seem to be high, although it is possible that its supplies can be depleted (Uhl et al. 2014) and it also needs to be clarified whether its deposition may reduce the amount of sperm transferred (Moore et al. 2004; Uhl et al. 2010).

2.2. Scorpions The knowledge on reproductive biology of scorpions is poor and comes only from a handful of studies (Benton 2001). Scorpions are long-lived, large-bodied arthropods which, similarly to web-building spiders, hunt passively, using the sit-and-wait strategy. During the reproductive period, females reduce their surface activity, whereas males abandon their retreats and become vagrant, searching of females. Unlike spiders, scorpion females lack sperm storage organs, and thus sperm can be stored only inside a specialized glandular tissue (Kovoor 1987) or in the genital tract lumen (Benton 2001). Most sperm gets flushed out from the female genital tract during birth, and so, in most scorpion species, females have to remate before producing each litter. As females usually mate just once per each reproductive season, male scorpions often engage in pre-copulatory guarding of soon-to-be receptive females (e.g. those carrying offspring which is about to disperse; Benton 1992).

a. Courtship

The courtship (also termed as the mating dance or so-called promenade à deux) in scorpions is complex and consists of several components (Polis and Sissom 1990; Benton 2001). It is initiated by a male, which grips a female with his pedipalps, trying to lead her to a site suitable for spermatophore deposition. Once they reach the right spot, the male extrudes a spermatophore and pulls the female over it. She lowers her body and inserts it into her genital aperture. Once sperm is transferred, the male sometimes engages in a post-mating attack, occasionally stinging the female before losing his grip (Polis and Farley 1979). Although the sexual cannibalism in scorpions is not as common as previously thought (Peretti et al. 1999), females are often aggressive, bigger than males, and may attack at any stage of the copulation. The components of courtship are considered to minimize the probability of a cannibalistic attack and, moreover, to secure cooperation and coordination which is necessary for an indirect sperm transfer (Polis and Farley 1979; Polis and Sissom 1990). However, some behavioural experiments suggest that the courtship behaviour of scorpion males is not used to force the female to mate, but should be rather interpreted as luring and/or stimulation of the female, with female resistance functioning as a screening mechanism (Peretti and Carrera 2005). The courtship is initiated when a male grips a female with his pedipalps, which usually elicits a female response – the female attempts to push him away or even sting him with her tail. The male tries to protect himself with his tail and during this “tail wrestling” he may transfer secretions from the caudal gland onto the female, probably lowering her aggression (Polis and Sissom 1990). Sometimes the female resists as she walks back dragging the male with her. Then the male usually engages in juddering, i.e. rapidly rocking his body back and forth, which is considered to stimulate the female to cooperate. From time to time, the male stops during the promenade, retracts his palps, moving the female towards him, and performs a cheliceral massage, which is supposed to make the female more cooperative (Polis and Farley 1979). The male also hits the female with the stinger tucked away (i.e. clubbing) and in some species he even punctures the female body with his stinger and leaves it inside her body for several minutes (i.e. sexual stinging; Angermann 1955, 1957; Francke 1979; Tallarovic et al. 2000).

b. Sexual stinging

Surprisingly, the role of the male stinging behaviour during courtship is far from being fully understood. It is not known whether any venom is transferred during stinging or whether

38 the stinging is a purely mechanical stimulus. Nevertheless, it seems to decrease the female resistance (Polis and Sissom 1990). Sexual stinging has been observed in species showing sexual dimorphism in telson size – males, although smaller than females, possesses strikingly bigger telsons (Polis and Sissom 1990; Kraepelin 1908; Pavlovsky 1913; Fet et al. 2013; Sentenská et al. submitted c). However, the link between the telson dimorphism and sexual stinging has not been investigated. A single study comparing morphology of telson and the venom gland in males and females has shown that males possess considerably larger venom gland containing higher number of secretory cells (Sentenská et al. submitted c). Although both sexes possess the same types of secretory cells, their proportional representation differs markedly. While females are equipped mainly with cells containing granules, the male venom gland comprises predominantly secretory cells free of granules. Granules generally represent venom proteins (Kubota 1918, Keegan and Lockwood 1971, Farley 1999), but scorpions also possess so-called prevenom, a granule-free liquid which is more efficacious in prey immobilization but less potent as a venom (Inceoglu et al. 2003). Using such substance during sexual stinging may reduce the female resistance during the courtship without causing her a serious harm (Inceoglu et al. 2003). Therefore, sexual dimorphism in telson size and the secretory equipment of the venom gland may be a result of sexual selection associated with sexual stinging (Sentenská et al. submitted c).

III. AIMS OF THE THESIS

The aim of my thesis was to investigate sexual conflict on three different phenomena: 1) Reversed sexual cannibalism in the spider Micaria sociabilis (study A and B). 2) Mating plugs and genital morphology in the spiders Micaria sociabilis and Philodromus cespitum (study C, D and E). 3) Sexual stinging in the scorpion Euscorpius alpha (study F).

Reversed sexual cannibalism was investigated by means of mating experiments in the laboratory to clarify how often males of M. sociabilis cannibalize on females, and which females are targeted. Furthermore, an observation in the field was conducted to estimate phenology of the species and potential prey availability in order to reveal factors affecting male cannibalistic behaviour. Mate plugging was studied from behavioural and morphological perspective. The occurrence and factors affecting the plug deposition and removal were examined through mating experiments, while the morphological study added up information on genital morphology of both sexes and casted light on mechanism of plug extrusion and its potential for female control. The last study dealt with the morphological aspect of sexual stinging performed during courtship by males of the scorpion E. alpha, which shows sexual dimorphism in telson size. We compared internal morphology of the venom glands of males and females to clarify whether males possess different secretory epithelium than females.

IV. CONCLUSION

Carnivorous arachnid taxa, such as spiders and scorpions, are particularly intriguing organisms, well-suited for study of the conflict between sexes. Both arachnid sexes are carnivorous, sexually dimorphic and, especially in spiders, a unique morphology of their genitalia facilitates an intense sperm competition and evolution of various male tactics in order to control the fertilization process. In my thesis, I focused on three phenomena encompassing apparent conflict between males and females. They were investigated by a combination of methods and this approach enables better understanding of occurrence and significance of the studied phenomena. Although female spiders are notoriously known for their cannibalistic behaviour, the role of the cannibal and the victim is not fully fixed, as shown by observations of rare cases of reversed sexual cannibalism. It may seem detrimental at the first sight, but our observations of mating in M. sociabilis throughout the whole season for several years suggest that it represents an extreme form of male mate choice exerted by large young males and directed against old females. These females are likely to be of low quality, which might be represented by an inability to produce offspring or simply by a shorter life-span in comparison to young females. The low rates of cannibalism observed in years when the sex ratio was more or less equal suggest that males can suppress their cannibalistic behaviour when females are less abundant. Thus it is possible that they may mate with old females, which might still be able to produce offspring. However, it remains unclear what is the underlying mechanism of different male approach towards young and old females and whether the choice is direct (i.e. males discriminate between young and old females and behaves differently towards them) or indirect (i.e. males attack all females, but the young ones are able to resist). Nevertheless, the reversed sexual cannibalism is most likely far from being an erroneous behaviour, as it seems to help males to cope with a decrease in prey availability by feeding on low quality females. The combination of our observations in the laboratory along with the field survey helped us to understand that this rare behaviour is not driven only by characteristics of males and females but also by ecological conditions, and that it follows similar rules as the classical form of sexual cannibalism. Genitalia of spider males and females are used as tools in the conflict between the sexes. Investigation of internal morphology of male copulatory organ of M. sociabilis and P. cespitum revealed that males possess glands that produce material to plug female genitalia. Moreover, a sensory organ and a neural tissue have been found in the bulbus of P. cespitum, although the bulbus was considered to be a numb structure for a long time. These findings 43 change our perception of the possibilities and constraints of mating in spiders and also cast light on the mechanisms of the sperm release and the plug production, as both processes are probably under neural control. Nevertheless, the function of the sensory organ needs to be clarified by futher morphological investigations to determine what kind of information (mechanical or chemical) it provides to the male (Sentenská et al, submitted a).

Mating plugs are very common among spiders, but the factors affecting their deposition as well as their impact on female mating possibilities is far from being fully understood. We investigated the process of amorphous plug deposition and removal in two spider species and found out that the plug occurrence, properties and consequences of plugging differ significantly between the two species. Both species share similar morphology of female genitalia, as both copulatory openings are situated in a single depression (i.e. atrium) which allows the male to monopolize the female with a single insertion. The plugging, however, has a different effect in both species. While in M. sociabilis, complete plugs indeed prevent the female from further copulations, in P. cespitum the plugs can be removed by subsequent males even if they fully cover the atrium, and so the female can modify the effect of plugging simply by deciding to remate. In M. sociabilis, females seem to control the plug deposition process as they may terminate the copulation before a complete plug is deposited. The combination of morphological and behavioural studies helped us to understand the mechanism of plug extrusion and its implications for plugging rate and its potential for female control. However, as we were able to only estimate the extent of plug removal, genetic methods revealing paternity and its proportion should be employed in future studies to elucidate whether sperm could be transferred by the second male and to what extent they could be used for egg fertilization. Components of scorpion courtship, such as sexual stinging, are often interpreted as tools to reduce aggression of a female and to elicit her cooperation. We investigated the morphology of the stinger in the sexually dimorphic scorpion E. alpha. Our results suggest that the dimorphism in telson size has evolved under sexual selection as it is most likely associated with the male stinging behaviour during courtship. Our conclusion is based on the fact that although both sexes possess similar glandular equipment, its composition greatly differs between males and females. Males are equipped mainly with secretory cells without granules, which likely produce prevenom, a substance which is less lethal but more paralytic than metabolically expensive venom. Such substance may modify female muscular contraction during courtship without exposing her to venom components which might kill her. Of course, the necessary step to test this hypothesis is to verify whether any substances 44 are transferred during sexual stinging and whether it indeed modifies female behaviour. It could be achieved by observation of female behaviour during courtship by exposing her to males prevented from transferring any substances during sexual stinging.

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VI. SUPPLEMENT

Study A Sentenská, L., & Pekár, S. (2013). Mate with the young, kill the old: reversed sexual cannibalism and male mate choice in the spider Micaria sociabilis (Araneae: Gnaphosidae). Behavioral Ecology and Sociobiology, 67(7), 1131-1139.

Study B Sentenská, L., & Pekár, S. (2014). Eat or not to eat: reversed sexual cannibalism as a male foraging strategy in the spider Micaria sociabilis (Araneae: Gnaphosidae). Ethology, 120(5), 511-518.

Study C Sentenská, L., Müller, H. G. C., Pekár, S., & Uhl, G. Male copulatory organ of spiders is not a numb structure as evidenced by presence of neurons and a sensory organ. Scientific Reports (submitted).

Study D Sentenská, L., Pekár, S., Lipke, E., Michalik, P., & Uhl, G. (2015). Female control of mate plugging in a female-cannibalistic spider (Micaria sociabilis). BMC Evolutionary Biology, 15(1), 18.

Study E Sentenská, L., Pekár, S., & Uhl, G. How spider females control deposition and removal of mating plugs. Animal Behaviour (submitted).

Study F Sentenská, L., Graber, F., Richard, M., & Kropf, C. Sexual dimorphism in venom gland morphology in a sexually stinging scorpion. Biological Journal of the Linnean Society (submitted).

1. STUDY A

MATE WITH THE YOUNG, KILL THE OLD: REVERSED SEXUAL CANNIBALISM

AND MALE MATE CHOICE IN THE SPIDER MICARIA SOCIABILIS

(ARANEAE: GNAPHOSIDAE) Sentenská, L., & Pekár, S. (2013). Behavioral Ecology and Sociobiology, 67(7), 1131-1139.

2. STUDY B

EAT OR NOT TO EAT: REVERSED SEXUAL CANNIBALISM AS A MALE FORAGING

STRATEGY IN THE SPIDER MICARIA SOCIABILIS (ARANEAE: GNAPHOSIDAE) Sentenská, L., & Pekár, S. (2014). Ethology, 120(5), 511-518.

3. STUDY C

MALE COPULATORY ORGAN OF SPIDERS IS NOT A NUMB STRUCTURE AS EVIDENCED

BY PRESENCE OF NEURONS AND A SENSORY ORGAN Sentenská, L., Müller, H. G. C., Pekár, S., & Uhl, G. Scientific Reports (submitted).

Male copulatory organ of spiders is not a numb structure as evidenced by presence of neurons and a sensory organ

Lenka Sentenská1*, Carsten H.G. Müller2, Stano Pekár1 & Gabriele Uhl2*

1 Department of Botany and Zoology, Masaryk University, Brno, Czech Republic.

2 Zoological Institute and Museum, Department of General and Systematic Zoology, Ernst-Moritz-Arndt-

University Greifswald, Germany.

* [email protected], [email protected]

The primary function of male copulatory organs is depositing spermatozoa directly into the female reproductive tract but their complexity can only be understood in the context of sexual selection. Accordingly, typical male genitalia are sensorily active. This is in contrast to the genital bulb of male spiders, which has been considered to lack nerves and muscles. Recently, however, neurons have been found within the bulb of the spider Hickmania troglodytes, a taxon basal to all Neocribellata. We provide the first record for neurons and an internalized multi-sensillar sensory organ in the bulb of the entelegyne spider Philodromus cespitum. The sensory organ likely provides mechanical or chemical feedback from the intromitting structure, the embolus.

We found further neurons associated with two glands within the bulb, one of which is likely responsible for sperm extrusion during mating. Our study provides a new framework for studies on reproductive behaviour and sexual selection in spiders.

KEY WORDS: sensilla, epidermal exocrine glands, sperm transfer, X-ray microscopy, electron microscopy, functional morphology

INTRODUCTION

The primary function of male copulatory organs is depositing spermatozoa directly into the female reproductive tract. However, the complexity of most animal genitalia and their rapid and divergent evolution strongly suggest that copulatory organs are not functionally restricted to sperm transfer but evolved to such diversity in the context of sexual selection. The evolution of such complicated machinery is considered favourable for cryptic female mate choice and sperm competition, and may even function as internal courtship devices1. Accordingly, typical male genitalia are equipped with a wealth of muscles and nerves and are able to expand and move and are sensorily active. This is in contrast to the “secondary” male genitalia of spiders, that are unique in that they lack nerves. In male spiders, transfer sperm to females is accomplished with the pedipalps, a pair of modified legs that is also present in females. The male pedipalp has become highly specialised: sperm are taken up into, stored in and released from the distal part of the pedipalp - the so called bulbus genitalis. The bulbus is considered to be derived from hypodermal cells that in females and immatures secrete a claw2-4. Accordingly, the bulbus was found free of muscles5-9, setae or sensilla10-11, and neural tissue 4,11-14. Despite this unique lack, spider genitalia share the evolutionary trend of a rapid divergent evolution1,9. The bulbus evolved into a wealth of structures consisting of a sperm reservoir, several sclerites and membranes in most spiders. Driven by internal pressure changes applied by more proximal muscles, bulbus conformation can change considerably in the process of mating. The sclerites are engaged in grasping, bracing, pushing, intromitting, and even damaging the female counterparts11,15-17. The numb and sensorily blind copulatory organs of spiders that cannot receive feedback in the mating process have attracted much attention in sexual selection research18 and have been used a test case for various hypothesis that attempted to explain genitalic diversity in general11.

Conflicting with the notion that spider genital bulbs are sensorily blind, behavioural observations suggest that male spiders sense whether the tip of their bulbus has entered a sperm droplet during sperm induction or not19. Although the various palpal movements accompanying genital coupling are often interpreted as failed intromission attempts that are a consequence of the numb structures having difficulties in achieving the proper alignment with the female structures20,21, they may represent exploratory behaviour or even copulatory courtship serving to stimulate the female11,22-24. Furthermore, it has been recorded that males adjust their behaviour once they reach genitalia of previously mated female suggesting that this information is perceived through genital contact and indicates a sensitivity of the intromitting structure25,26.

The discrepancy between morphological and behavioural studies was resolved by a recent study in which it was shown that the simply structured bulbus of the cave spider Hickmania troglodytes is innervated27. Several neurons were found attached to the cuticle, suggesting a proprioreceptive function that may enable the male to gain sensory input during sperm induction and copulation. Furthermore, the epidermal exocrine glands within the bulbus appear connected to the nerve suggesting that the secretory activity might be under neural control27. H. troglodytes belongs to the species poor family Austrochilidae which is considered sister group to the two major spider clades Haplogynes and Entelegynes, the so called Neocribellates28,29, which together comprise about 90% of all known araneomorph spiders. It remains to be assessed if the neural equipment was lost at the base of the neocribellates or if all spider genitalia possess nerves. Here, we provide the starting point for revisiting the supposedly sensorily blind male spider bulbs by scrutinizing common running crab spider Philodromus cespitum (Walckenaer, 1802)

(Philodromidae). For this entelegyne spider, we provide evidence for not only the presence of neural tissue but also for a multisensillar sensory organ. This was achieved by means of histological, ultrastructural and micro-computer tomographic analyses.

RESULTS

The male copulatory organ of P. cespitum consists of conical cymbium and an egg-shaped bulbus genitalis (Fig. 1A). The bulbus contains the spermophor, a coiled blind tube that functions as an interims storage device for the seminal fluid (Fig. 1B-D). There are two sclerites connected to the bulbus: the crest-like conductor and the embolus. The embolus has a wide base that narrows distally and bears an opening close to its tip. The embolus is the actual intromitting organ; it releases the sperm into the female genital tract.

At the base of the cymbium, a nerve forks into two distinct nerve bundles. They continue and branch inside the cymbium which is typically densely equipped with sensilla. One of the branches enters the bulbus at the contact point between both structure (Figs. 1C, 2A-B), and is called bulbus nerve in the following. The bulbus nerve consists of neurite bundles surrounded by glial cells. The glial cells form radial processes that divide the bulbus nerve into three large and one small compartment (fascicles according to Foelix30) each carrying a distinct neurite bundle (Fig.

2B’). The bulbus nerve projects inside the bulbus and connects to a cluster of somata adjacent to a gland at the blind end of the spermophor (Fig. 2A, C). Since the blind end is defined as the fundus, we term the gland fundus gland. The cluster of somata very likely belong to sensillar receptor cells (Fig. 2C´). The somata contain circular nuclei with little heterochromatin. The cytoplasm around the nuclei is weakly osmiophilic and equipped with numerous, often elongated or even branched mitochondria of the cristae type, dispersed cisternae of the rough ER, some microtubules, and polymorphic vesicles. The cluster of somata is surrounded and traversed by radiating processes of glial cells (Fig. 2C’) typically associated with neurons. The somata turn into thin distal cytoplasmic processes that project along the ventral margin of the spermophor. These cytoplasmic processes connect to a deeply internalized sense organ situated at the base of the embolus nestled inside the embolus gland (Fig 1D, 2D, 3A).

The sensory organ consists of multiple, tightly adjoined sensilla (Fig. 2D, 3A). Each sensillum is innervated by two or three receptor cells, and is surrounded by several sheath cells (Figs. 2D’,

3A,B). Based on the terminology that was established for insect sensilla (e.g. 31,32), the distal process of a sensillar receptor cell soma has to be termed the dendritic inner segment. The dendritic inner segments are assembled in pairs or triads and contain numerous microtubules, mitochondria and small, electron-lucent vesicles (Figs. 2D’, 3A, E). Most distally, each dendritic inner segment exhibits a single (sensory) cilium comprising the basal body and axonemal part

(ciliary segment, Fig. 3E) as well as the outer dendritic segment - a cilium with microtubules lacking axonemal formation (Fig. 3B-D). Two or three dendritic outer segments pass through the sensillum lymph space which is lined by the thick, highly osmiophilic dendritic sheath (Fig. 3C-E).

The rough appearance of the inner surface of the dendritic sheath is caused by fibrillous or knob

-like protrusions extending into the sensillum lymph space (Fig. 3D). The dendritic sheath is surrounded and built up by the second sheath cell, whereas the first, basal sheath cell enwraps the dendritic inner segments and builds up and supplies the sensillum lymphs. Along its apex, the first sheath cell emits numerous elongated microvilli protruding into the sensillum lymph space

(Fig. 3C, E). Both sheath cell types are characterized by a moderately electron-dense cytoplasm including high abundance of microtubules, mitochondria of the cristae type as well as nuclei with high amounts of heterochromatin, dispersed cisternae of rough and smooth ER, polysomes, Golgi stacks, and polymorphic vesicles with highly osmiophilic, heterogeneous contents resembling lysosomes (Fig. 3C-E). Possibly, there are more than two sheath cells in each sensillum as indicated by the high number of cell layers (6-8) between sensillum lymph spaces (compare Fig.

3C, E). The dendritic outer segments pass through the cuticle of the embolus (Fig. 3A). Here, the sensillum lymph space is lined by remains of the dendritic sheath (Fig. 3B). The course of the dendritic outer segments could not be followed further due to difficulties in sectioning the highly sclerotized embolus. 97

A further neurite bundle (Fig. 2D’) follows the sensillar dendrites, then bends further ventrally where it connects to the epithelium of the embolus gland situated at the base of the embolus

(Fig. 2D). The embolus gland consists of numerous secretory cells arranged in a single but highly prismatic epithelium. The epithelium lines a huge reservoir that narrows anteriorly into a thin canal that runs ventrally of the sensillar apparatus and ends in the embolus canal through which sperm have to be released.

Another neurite bundle is found in the epithelium of the fundus gland close to the cluster of somata (Figs. 2C’, 4A). The fundus gland consists of a thin, single-layered vacuolated epithelium that surrounds a voluminous reservoir filled by an amorphous substance of low electron density

(Figs. 2C’, 4A). The reservoir connects to the porous wall of the spermophor fundus region. The substance in the spermophor shows similar staining affinities towards toluidine blue (Fig. 2C) suggesting that it originated from the fundus gland. The existence of neurites within fundus gland is indicated by neurotubules extending through a weakly osmiophilic cytoplasm (Fig. 4B). Single neurites were occasionally observed in between secretory cells (Fig. 4C).

A third gland, the spermophor gland runs along the spermophor wall between fundus gland and embolus gland for most of its length. It connects to the wall of the spermophor that is porous in the region of contact (Fig. 2 C,D). The spermophor gland does not show any indication of innervation. It comprises many secretory cells that form a thick glandular epithelium rich in rough endoplasmic reticulum and microtubules (not illustrated). The lumen of the spermophor that is surrounded by this gland is filled with sperm and spherical vesicles of high electron density (Fig.

2C,D).

Comparison of male palps before and after copulation revealed differences in the distribution of sperm and glandular secretions: in virgin males, that charged their palp with sperm but did not mate yet, a mixture of sperm and circular vesicles fills three quarters of the spermophor lumen,

98 whereas the substance of the fundus gland fills the distal portion of the spermophor, the fundus and the voluminous reservoir. In palps fixed right after copulation, the substance of the fundus gland fills two thirds of the spermophor lumen, and the apically remaining lumen contains sperm and vesicles. The other two glands do not show any obvious differences before and after copulation.

DISCUSSION

Our study demonstrates that the genital bulb of an entelegyne spider possesses neural tissue.

Moreover, we provide first evidence of a multisensillar sensory organ. For several decades it has been accepted that male spider genitalia are sensorily blind during the mating process due to the lack neurons in the part of copulatory organ that is involved in sperm transfer. This notion built on numerous histological studies2,9,12-14. The apparent lack of sensory feedback during genital coupling has been considered to be compensated by the evolution of a number of sclerites that interlock with structures of the female genitalia as is typical for most spider species11. Recently, however, neural tissue was found in the bulb of the austrochilid cave spider Hickmania troglodytes27 which gave the starting point for revisiting the neurobiological properties of the bulbus in a representative of the majority of spiders, the Neocribellates. Interestingly, the arrangement of the nerve and neurons in both species is nearly identical. Similar to H. troglodytes27, the neural tissue in the bulbus of P. cespitum consists of a nerve originating in the cymbium and a somata cluster inside the bulbus. In both species, the neural tissue reaches the embolus base and seems to be tightly associated with glands as the neurites penetrate their tissue. The striking similarity in neural anatomy of two widely separated taxa may suggest that the presence and arrangement of the neural tissue represents a ground pattern of all spiders.

The nerve that runs through the bulbus of P. cespitum is clearly distinguishable from other tissues when using the combination of methods applied in our study. This may explain why the studies mentioned above failed to find neural tissue in bulbi of other neocribellate spider species.

Depending on the quality of the chemical fixation process and magnification, the presence of neurons may have been obscured. Especially in paraffin and semi-thin sectioning, which were applied in most of these studies, the neural tissue does not conspicuously stain with the common staining protocols. Also, in specimens only fixed in ethanol the neural tissue does not seem to be preserved well enough to be distinguishable, which was the case for Philodromus dispar9.

Therefore, we predict to find nerves in the genital bulbs of all spider families. Whether the nerves have a sensory or motor function or both needs to be assessed in follow up studies. Our results suggest that both directions are conceivable, since the bulbus nerve contains several neurite bundles (Fig. 2B´). The fact that we found a senory organ requires that the input perceived by this organ is transfered and processed to an integrating unit. Consequently, some of the neurite bundles must be sensory. Since the neurites connect to at least two of the three glands found inside the bulbus, a further motor function of some of the bundles is plausible. These nerves may be involved in release of secretion and consequently in the expulsion of sperm during mating, a process that likewise is not understood at present. In the following, we first discuss our data on the sensory and motor aspect of the neural equipment of the P. cespitum bulbus.

The sensory organ of P. cespitum is composed of aggregated and internalized sensilla. It is situated close to the base of the embolus, the intromitting structure of the bulb. The cuticle of the embolus is penetrated by the outer dendritic segments of the sensilla, however, we could not trace their course in the cuticle all along the way. We tentatively suggest that this sensory organ is chemo and mechanoreceptive. We base our hypothesis on ultrastructural details (see below), the connection with the various bulbus glands, and the position of the sense organ at the

100 embolus. In Hickmania trogylodytes, neurites were likewise found attached to the cuticle close to the embolus base. The assumed proprioreceptive function was derived from stress and strain finite-element modelling analysis27. However, our ultrastructural data reveal that the sensilla of the bulbus of P. cespitum resemble those of the tarsal organ, a chemoreceptor found on the tarsus of the walking legs as well as on the pedipalp of various spiders (e.g. 33-35). Since the function of the tarsal organs is not resolved yet, the internalized sensilla in the bulbus may function either as pheromone receptors36 or as combined thermo- and hygroreceptors37 as was previously suggested for the tarsal organ. Proprioreception in addition to one of these potential functions is likewise conceivable.

Our study also documents two neurite bundles that make contact and branch within the glandular epithelia associated with the embolus and fundus. However, we yet failed to depict synapses, probably beacuase the fixative does not infiltrate easily into the bulbus. However, there is reason to assume that these two neurite bundles (see Figs. 2D‘ and 4A) include axons that innervate and thus regulate the activity of the embolus and fundus gland. Our arguments for assuming that these neurite bundles carry efferents and would therefore have to be interpreted as branches of the bulbus nerve are the following. First, the bulbus nerve is made up of four distinct compartments of unequal size, so called fascicles30. In insects, such nerve compartments carry axons of strictly either sensory or motor neurons when distant from the ganglia38. Accordingly, we suggest that the fascicles that are small in diameter contain efferents coming from interneurons located in the brain, each invervating a particular gland, whereas the largest fascicles of the bulbus nerve contains afferents that come from the sensory organ.

The neural equipment of the male spider bulb does not only impact on our perception of the possibilities and constraint that occur during the mating process in spiders. The finding that neurites enter the fundus and embolus glands may considerably improve our knowlegde on the

101 mechanism by which sperm is released during mating from the interim sperm storage site, the spermophor. It has been assumed that sperm expulsion is achieved through increasing internal hemolymph pressure applied on the bulbus by more proximal muscles or by means of a secretion from a gland attached to the spermophor12,13,39. Particularly, the spermophor gland was suspected to discharge its product through the porous wall into the lumen of the spermophor during mating2,13,40. Our histological examinations on P. cespitum reveal that the reservoir of the fundus gland is filled by a material identical to the one present in the lumen of the spermophor.

In virgin males, large amounts of the material are deposited in the glandular reservoir and a small portion can be found in the fundus region of the spermophor. After copulation, however, the gland appears shrunken and the material fills more than a half of the spermophor lumen. Our data strongly suggest that the fundus gland of P. cespitum plays a crucial role in sperm extrusion and that the process is triggered by a neural stimulus. Since the mating process in spiders generally entails changes in pedipalp and bulbus conformation by internal pressure changes, the pressure pump and the neural stimulus may be coordinated for successful sperm transfer.

Lamoral13 already wondered why sperm is only released during mating after embolus insertion despite the fact that the pressure pump is active much earlier in the mating sequence, e.g. for external alignment with the female genitalia. He suspected that the release of secretion for sperm transfer from the gland is triggered by neurohormones produced in more proximal parts of the male pedipalp, when the palp achieved the right position. However, since sperm transfer often occurs very rapidly, a neurohormonal activation seemed unlikely14. Our study provides a tentative answer to this question since the fundus gland seems to be directly innervated by neurites projecting through the bulbus nerve. The sperm expulsion may be triggered when the sensory organ sends information about the correct positioning of the palp during mating. This afferent transmission may cause a signal to the fundus gland that triggers release of the

102 substance from its reservoir into the spermophor lumen and to flush out the seminal fluid stored therein.

Since the male bulbus of P. cespitum contains several glands the function of the embolus gland and the spermophor gland remain to be investigated. The spermophor gland seems to release secretion into the spermophor even before mating in many spider species (Uhl, unpublished).

Consequently, it may be involved in sperm uptake which may explain why there were no obvious differences between the spermophor gland of virgin and mated males in our study. The function of the embolus gland remains enigmatic. However, since P. cespitum males produce a mating plug by which males can hinder females from remating with rival males (Sentenská et al., unpublished) as was shown for other spiders41-43, one of these glands will be responsible for producing the mating plug material.

In conclusion, our finding of a nerve and sensory organ in the palp of an entelegyne spider together with the previous finding on nerves in the palp of a basally branching Austrolichilid spider requires not only revising the common notion that genitalia of spider male are numb structures. The finding also calls for a reanalysis of the origin of the spider bulb as a derivative of the tarsal claw2,44. This explanation has already been challenged by the observation that traces of a claw as well as of a bulbus occur during early development of male pedipalps11,44. Evo-devo studies on precursor cells might solve this ridle.

METHODS

Subadult males and females of Philodromus cespitum (Walckenaer, 1802) were collected with a beating-net in the canopy and understorey of an abandoned fruit orchard within the city boundary of Brno, Czech Republic in April 2015. Spiders were housed individually in a Petri dish

(diameter 5 cm, height 1 cm) with a piece of filter paper, which was moistened with few drops of 103 water at 2-day intervals to maintain the required humidity. Spiders were kept at room temperature (approx. 22 °C), at 40% RH, and under a natural LD regime and fed with fruit flies

(Drosophila melanogaster Meigen) to satiation at 2-day intervals. A day after the final moult the spiders were sent to Greifswald University, Germany and their pedipalps were fixed in Karnovsky fixation (see below) except of two which were fixed in 70% alcohol. Three of the virgin males were mated with virgin females (approximately five days after the final moult) and their pedipalps were fixed right after the copulation in Karnovsky solution.

A high definition stacking photograph of the external anatomy of the male pedipalp fixed in 70% alcohol of P. cespitum was produced with a Visionary Digital BK Plus Lab System (duninc.com/bk- plus-lab-system.html) equipped with a Canon EOS 6D and a customized Canon EF 100mm f2.8 IS

USM professional macro lens at the University of Greifswald. The images were processed with

Adobe Photoshop CS6.

For histological and ultrastructural analysis, pedipalps of 10 males were fixed overnight in a fresh, cold fixative solution modified after Karnovsky45 containing 2.5% glutaraldehyde, 2.5% paraformaldehyde, 1.5% NaOH and 5% D-glucose, buffered with 0.1 M sodium phosphate buffer adjusted at pH 7.4. After rinsing the palps three times in the buffer solution for 5 min, postfixation in 1% OsO4 solution (same buffer) was conducted at room temperature for 4 h followed by dehydration in graded series of ethanols and embedding in Spurr media (SIGMA-ALDRICH).

Semi-thin (500 nm) and ultrathin (50–65 nm) cross sections were made using a Leica ultramicrotome UC6. The semi-thin sections were stained with 1% toluidine blue in a solution of

1% sodium tetraborate (borax; modified after Richardson46), and examined and photographed under an Olympus BX60 connected to a Zeiss MCr digital camera. Ultrathin sections were mounted on Formvar-coated slotgrids (PLANO: model G2500C), stained with uranyl acetate and lead citrate for 4 min each, and then examined under a JEOL JEM-1011 transmission electron

104 microscope operated at 80 kV. Images were obtained with an Olympus Mega View III digital camera using iTEM software.

For non-invasive X-ray microscopy (XRM) three pedipalps were prepared after incubation in

Karnovsky’s prefixative overnight (see protocol above), in the following way: after dehydration, samples were stained overnight using 1% iodine solution (in pure ethanol). After washing in pure ethanol, samples were critical point dried (Leica) and subsequently mounted on insect pins using super glue. Scans were performed in an Xradia XCT-200 (Carl Zeiss Microscopy GmbH) using the

20x and 40x objective lens unit with the following scan parameters: 40 kV, 8 W, 200 µA, exposure time 30 sec/frame. Reconstructed image stacks were created using XMReconstructor software

(Carl Zeiss Microscopy GmbH). Subsequent segmentation (delineation) of the structures of interest in the male was performed with Amira 5.4.5 (Visualization Science Group, FEI). The reconstruction of neural tissue was done by comparing MicroCT images with semi-thin sections.

The terminology for the description of the neural tissue is according to the neuroglossary established by Richter et al.47 (2010).

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ACKNOWLEDGMENTS

Helpful comments were given by Elisabeth Lipke, Peter Michalik and Gerd Alberti.

We dedicate this manuscript to the memory of the late Gerd Alberti, an expert in ultrastructural analysis.

University of Greifswald provided funding for activities between partner universities granted for research visits of LS to Greifswald. The 3D X-ray microscope was co-funded by the state of

Mecklenburg-Vorpommern and the German Science Foundation (INST 292/119-1 FUGG and INST

292/120-1 FUGG), which is gratefully acknowledged. LS was supported by grant no.

MUNI/A/1484/2014 provided by Masaryk University.

AUTHORS’ CONTRIBUTION

LS participated in microscopy and wrote the manuscript. CHGM participated in the microscopy and writing the manuscript. GU and SP conceived the study, participated in its design and coordination, and contributed to the writing of the manuscript. All authors read and approved the final manuscript.

COMPETING FINANCIAL INTERESTS

The authors declare that they have no financial competing interests.

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FIGURE LEGENDS

Fig. 1A-D. Male copulatory organ of Philodromus cespitum. A: Ventral aspect (Visionary Digital

BK Plus Lab System). B-C: Volume and surface reconstruction of copulatory organ based on 3D X- ray microscopy, virtual longitudinal sections showing ventral (B) and prolateral (C) view. D.

Delineated focal parts including neural tissue, spermophore, three glandular tissues and embolus.

BG – bulbus genitalis, Con – conductor, Cy – cymbium, Em – embolus, RTA – retrotibial apophysis,

S – spermophor, Ti – tibia

Fig. 2A-D. Histological organization and ultrastructure of the bulbus of Philodromus cespitum. A.

Virtual medio-longitudinal section of bulbus based on 3D X-ray microscopy. Neural and sensory tissues are highlighted in yellow: bulbus nerve passing through the cymbium and entering the bulbus. Section planes illustrated in B, C and D are indicated by black lines. Note that further branches of the pedipalp nerve projecting through the cymbium are not highlighted. B-D: Light microscopy; B‘-D‘: TEM. B-D: Sequence of semithin cross-sections of the bulbus showing the location and extension of aggregated internalized sensilla and associated nerve: B exhibits cross- profile of the bulbus nerve, C shows the deeply sunk cluster of somata of sensillar receptor cells, while D highlights distal (dendritic) components of the sensilla. B‘-D‘: Ultrastructural insights from cross-cut bulbus nerve (B‘) showing neurite bundles in four distinctive compartments as separated by glial cell sheaths. C‘: Cell cluster adjoining fundus gland, it contains somata of sensillar receptor cells enwrapped and invaded by glial cells. D‘: Proximal aspect of dendritic system of sensilla, pairs or (occasionally) clusters of more than ten dendritic inner segments are visible surrounded by sheath cell somata.

BH – basal haematodocha, BN – bulbus nerve, BV – blood vessel, Cy – cymbium, DIS – dendritic inner segment, Em – embolus, EGC – embolus gland cells, FG – fundus gland, FGL – fundus gland

111 lumen, GC – glia cell, HeS – hemolymph space, InS – internalized sensilla, S – spermophor, SC – somata cluster, SF – seminal fluid, SG – spermophor gland, ShC – sheath cell, NeB – neurite bundle, So(RC) – soma of receptive cell

Fig. 3A-E. Ultrastructure of aggregated internalized sensilla in the bulbus of Philodromus cespitum. A provides overview of distal sensilla components comprising dendrites and sensillum lymph spaces, note that part of embolus gland tissue surrounds the sensilla. B. Most distal, endocuticular part of sensillum penetrating embolus cuticle, electron-lucent sensillum lymph space bears two dendritic outer segments. The aggregation of fibrillous material are potential remains of the dendritic sheath (arrowhead). C-D. In the proximal region the dendritic outer segments project through sensillum lymph space which is lined by highly electron-dense dendritic sheaths (C), D close-up of sector boxed in C, sensillum lymph space contains three outer dendritic segments in oblique view. Arrowheads point at knob-like protrusions of the dendritic sheath. E. Transition zone between outer and inner dendritic segments (basal body region), one basal body is visible. The three dendritic inner segments are encompassed by innermost (type-1) sheath cell projecting elongated microvilli into sensillum lymph space.

BB – basal body, Cu – cuticle, DIS – dendritic inner segment, DOS – dendritic outer segment, DS

– dendritic sheath, Em – embolus, EGC – embolus gland cells, EGL – embolus gland lumen EpC – epidermal cells, InS – internalized sensilla, Ly – lysozomes, Mi – mitochondrium, Mt – microtubuli,

Mv – microvilli, SLS – sensillum lymph space, ShC – sheath cell, SN – sheath cell nuclei

Fig. 4A-C. Neural tissue associated with the fundus gland in the bulbus of Philodromus cespitum.

A. Numerous electron-lucent neuritic inclusions found within the glandular epithelium. B. Neural origin is indicated by presence of neurotubules dispersed in a weakly osmiophilic cytoplasm also

112 containing numerous mitochondria. C. A solitary neuritic process projecting through lateral interspace of two fundus gland cells.

DOS – dendritic outer segment, ExS – extracellular space, FGC – fundus gland cells, FGL – fundus gland lumen, HeS – hemolymph space, Mi – mitochondrium, Mt – microtubuli, Ne – neurite, NeB

- neurite bundle, Nt – neurotubuli, Nu – nucleus, RER – rough endoplasmatic reticulum

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4. STUDY D

FEMALE CONTROL OF MATE PLUGGING IN A FEMALE-CANNIBALISTIC SPIDER

(MICARIA SOCIABILIS) Sentenská, L., Pekár, S., Lipke, E., Michalik, P., & Uhl, G. (2015). BMC Evolutionary Biology, 15(1), 18.

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5. STUDY E

HOW SPIDER FEMALES CONTROL DEPOSITION AND REMOVAL OF MATING PLUGS Sentenská, L., Pekár, S., & Uhl, G. Animal Behaviour (submitted).

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How spider females control deposition and removal of mating plugs

Lenka Sentenská1*, Stano Pekár1* & Gabriele Uhl2*

1 Department of Botany and Zoology, Masaryk University, Brno, Czech Republic

2 Zoological Institute and Museum, Department of General and Systematic Zoology, Ernst Moritz

Arndt University of Greifswald, Germany

*corresponding authors: [email protected], [email protected], [email protected]

ABSTRACT

In order to avoid sperm competition males of many taxa apply physical barriers into the female genital tract, so called mating plugs. Due to potential costs imposed on females, female control may focus on strong pre-copulatory mate choice or on influencing plug efficacy. We investigated behavioural and morphological aspects of plug deposition and removal in a promiscuous spider,

Philodromus cespitum (Philodromidae). We performed laboratory mating trials with virgin individuals to investigate factors affecting plug deposition. The copulation in this species is brief and terminated by the female. The plugging material is produced in the male copulatory organ and transferred to the female together with sperm. After mating, plugging material was found in the female genital atrium in all cases, however to varying amounts. The extent of coverage of the atrium was affected by the number of taps male delivered with his legs on the body of the female during courtship. The tapping movements are correlated with male body size measured as foreleg length. Subsequent mating trials with females with a plug that covered the atrium revealed that plugs can partly or entirely be removed. The extent of plug removal was affected

135 by the foreleg length ratio between the male who removed the plug and the one who deposited it. Our results suggest that females control deposition and removal of plugs based on male size.

KEY WORDS

Araneae, female mate choice, courtship, polyandry, X-ray microscopy, Philodromus

INTRODUCTION

Males of many species apply barriers, so called mating plugs, into the female copulatory tract to hinder the access of other males to ova of the female they copulated with (Parker, 1970; Wigby

& Chapman, 2004). Since plugs may oppose female reproductive interests as females often benefit from mating multiply (Eberhard, 1996; Arnqvist & Rowe, 2005; Uhl, Schmitt, & Schäfer,

2005) and incur costs from obtaining sperm of low quality males with no possibility to remate

(Uhl, Nessler, & Schneider, 2010), strong precopulatory female choice is expected to occur in plug producing species. Alternatively, females may have evolved means to affect and control plug deposition, e.g. by selectively adding substances influencing plug efficacy (Eberhard & Huber,

1998; Knoflach, 1998, 2004; Aisenberg & Eberhard, 2009) or dislodging the male before he applies the plug material (Uhl & Busch, 2009; Gutiérrez & Cordero, 2015; Sentenská et al., 2015).

The amount of plugging material seems to affect plug efficacy strongly, as incomplete plugs (i.e. plugs covering only a portion of female genital opening) often do not constrain female remating

(Hartung & Dewsbury, 1978; Wallach & Hart, 1983; Jackson, 1980; Matsumoto & Suzuki, 1992;

Masumoto, 1993; Uhl & Busch, 2009; Simmons, 2001; Kunz, Witthuhn, & Uhl, 2014; Sentenská et al., 2015). Even if a complete mating plug is deposited, males may be able to remove plugs transferred by their rivals (Parker, 1984) and thus the fate of the plug can be affected by the female´s decision to remate or not. Consequently, female choice likely operates not only before and during plug deposition, but also during its removal. A male´s ability to remove the plug can 136 itself be an indicator of his quality (Eberhard, 1996). If a female accepts the first male according to a quality threshold and the plug helps her to protect high quality sperm (Fromhage, 2012), she may allow plug removal only to males of higher quality than her previous partner.

Mating plugs can be found in a variety of animals, vertebrates (e.g., Shine, Olsson, &

Mason, 2000; Dixson & Anderson, 2002) and invertebrates (e.g., Barker, 1994; Baer, Morgan, &

Schmid-Hempel, 2001; Uhl, Nessler, & Schneider 2010). They are usually effective only for a short time because otherwise they would interfere with oviposition. An exception represents ditrysian

Lepidoptera, entelegyne spiders and some mites, where long-lasting plugs occur due to the unique morphology of female genitalia (Uhl, Nessler, & Schneider 2010). Females of entelegyne spiders possess three separate genital openings: two of them are used for copulation and one for oviposition (Foelix, 1996). Hence, sealing of one or both copulatory openings does not interfere with oviposition. Such morphology may explain the widespread occurrence of mating plugs in spider reproductive biology (Uhl, Nessler, & Schneider, 2010; Uhl et al., 2014).

Spider males can plug female genitalia by parts of their copulatory organs located on pedipalps

(i.e. genital plug) or by amorphous substances of glandular origin (i.e. amorphous or secretory plug). Amorphous plugs are produced in the bulbus genitalis of the copulatory organ (Suhm,

Thaler, & Alberti, 1996; Uhl et al., 2014; Sentenská et al., 2015), in the genital tract (Knoflach,

1998, 2004) or in the mouth area (Braun, 1956). As spider females possess two copulatory openings, a male has to achieve two insertions to monopolize the female if the openings are widely separated. Such morphology facilitates female control through selectively allowing males a second insertion. In species where female copulatory openings are close to each other or situated in a depression (i.e. atrium), a male can plug and monopolize the female with a single insertion (Uhl, Nessler, & Schneider, 2010).

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The goal of our study was to investigate plug deposition and removal in a promiscuous spider, Philodromus cespitum (Walckenaer) (Philodromidae) for which mating plugs had been reported (Michalko, personal communication). We aimed to explore whether and how a female can affect the deposition of the mating plug and its efficacy. First, we observed the courtship and mating behaviour of virgin females and males to elucidate how and when the plug is deposited and whether the deposition is under female control. Further, we estimated the extent to which the plug covered the female atrium as a proxy for plug quality and related it to male courtship behaviour and morphological traits of both partners. We hypothesized that plug coverage is a result of female choice based on courtship quality and/or physical attributes of the male, with favoured males being allowed to deposit more plug material. Then we performed mating trials with plugged females to investigate the ability of males to remove the plug and female ability to control the removal. We expected females to allow plug removal only to males of superior quality to her first partner. Further, we conducted a morphological study of the male and female genitalia of P. cespitum to elucidate the origin and storage of plug material, the mechanism of its extrusion and how far it extends into the female copulatory ducts.

METHODS

Juvenile and subadult individuals of P. cespitum were collected with a beating-net in the canopy and understorey of an abandoned fruit orchard within the city boundary of Brno, Czech Republic.

Spiders were housed individually in a Petri dish (diameter 5 cm, height 1 cm) with a piece of filter paper, which was moistened with few drops of water every second day. The dishes with spiders were kept at room temperature (approx. 22 °C), at 40% RH, and under a natural LD regime. The spiders were fed with fruit flies (Drosophila melanogaster Meigen) to satiation at 2-day intervals.

A minimum of five days and maximum of eight days after the final moult the spiders were used in the behavioural experiments and for the morphological study.

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Mating behaviour

All mating trials were performed in the laboratory between April and June 2012. The prosoma and the femur length of all individuals was measured before the trial using an ocular micrometer on a stereomicroscope (Olympus SZ61).

At the beginning of a mating trial a male was introduced into the dish occupied by a female and the mating behaviour was recorded using an analogous camera (CV-735) connected to a video recorder (SONY GV-HD700 HDV). The following behaviours were recorded: the number of taps delivered by a male on female body with his forelegs and abdomen twitches (jerky lifts of abdomen while touching the female with his legs) during courtship; the number of lateral palpal movements during genital coupling; the number and total duration of partial haematodochal expansions after the palp fixation; the number and total duration of full haematodochal expansions after the palp fixation and the number of pedipalp insertions (i.e. the palp was coupled to the female genitalia and haematodochal expansions were observed). The trials were terminated when no contact between male and female occurred within 10 minutes. For detailed description see Data analysis and Results.

Mating trials with virgin females

To investigate plug production, 24 virgin males were paired with 24 virgin females and behaviours described above were video recorded. The relative body size of the pair was calculated by dividing the male prosoma size by female prosoma size and male femur length by female femur length. After each trial, the female was anesthetised with CO2 and her genital area was checked for the presence of a plug under a stereomicroscope (Olympus SZX9) at ×20 magnification and photographed using Leica digital camera attached to the stereomicroscope. When a plug material was present, the extent to which it covered the atrium was visually estimated in ten

139 percent intervals (from 0 - no plug material visible in the atrium to 100% - atrium fully covered by plug material).

Mating trials with plugged females

To study whether and under which conditions plug removal occurred, females with plugs in the atrium covering both copulatory openings (i.e. atrium coverage ranging from 80 to 100%) from the previous mating experiments (N = 19) were paired with a second male each. Rematings were staged after three days from first matings with males from the previous mating experiment (N =

19), but females were paired with a different male than before. After the trials, females were anesthetised with CO2 and their genital area was checked again, photographed and the extent of the removal of the original plug was estimated. The proportion of removed plug was acquired by subtracting the removed plug material after the second mating from the original plug coverage recorded after the first mating (e.g. if half of the plug material was removed from the previously deposited plug with 100% coverage, the removal was 50%). In addition, the relative size of subsequent males was calculated by dividing the second male prosoma and femur length by the prosoma and femur length of the first male. As there was no significant difference in amount of

plug removal between 80 to 100% plug coverages (GLM-qb, F1,17 = 1.48, P =0.24), we pooled the data and analysed these trials together.

Data analysis

To evaluate effects of the measured variables (see below) on plug production and removal, the proportion of the plug material covering the female atrium was analysed using a generalized linear model (GLM) with a quasibinomial setting as appropriate for overdispersed data (Pekár &

Brabec 2016). The effect of male femur size on the number of taps was analysed using a generalized linear model (GLM) with a gamma error structure. 140

Due to the high number of variables compared to the number of trials and their inter- correlation we grouped similar variables by means of Principal Component Analysis and extracted scores along the first axis, which were then used as auxiliary explanatory variables. Specifically, the following explanatory variables were formed: male size (length of male prosoma and length of femur), female size (length of female prosoma and length of femur), pair size ratio (length ratio of prosoma and length ratio of femur in the pair), courtship (number of taps and number of twitches), coupling (number of lateral palpal movements and number of pedipalp insertions), partial expansions (number and duration of partial haematodochal expansions), and full expansions (number and duration of full haematodochal expansions). All auxiliary variables were included into an initial model, which was then simplified using a stepwise selection procedure based on the F test. Once the initial model included only significant auxiliary predictors a new model was fitted in which auxiliary variables were replaced by the original ones. The new model was also simplified using stepwise selection.

In the analysis of data on plug removal, two initial models were fitted due to a high number of original as well as auxiliary variables compared to the data. The first initial model consisted of auxiliary variables representing sizes (male size, female size, 2nd/1st male size ratio,

2nd male/female size ratio and 1st male/female size ratio), the second one included auxiliary variables representing behaviours (courtship, coupling, partial expansions and full expansions).

The initial models were simplified using stepwise selection and a new model was fitted with the original variables.

All statistical analyses were performed in R environment (R Development Core Team

2010). Descriptive statistics are given as arithmetic mean ± standard error.

141

Morphology of male and female genitalia

For histological and ultrastructural analysis, female opisthosomata of virgin (N=1) and mated female with a plug in the genital area (N = 5) and male pedipalps (N = 4) were fixed in a fresh fixative solution modified after Karnovsky (1965) containing 2.5% glutaraldehyde, 2.5% paraformaldehyde, 1.5% NaOH and 5% D-glucose, buffered with 0.1 M sodium phosphate buffer adjusted at pH 7.4. After rinsing the palps three times for each 5 min in the same buffer solution, postfixation in 1% OsO4 solution (same buffer) was conducted at room temperature for 4 h followed by dehydration in graded ethanols and propylene oxid and embedding in Agar Low

Viscosity Resin. Semithin (500 nm) and ultrathin (50–65 nm) cross sections were made using a

Leica ultramicrotome UC6. After staining with 1% toluidine blue in a solution of 1% sodium tetraborate (borax; modified after Richardson, Jarett & Finke, 1960), the semithin sections were examined and photographed under an Olympus BX60 connected to a Zeiss MCr digital camera.

Ultrathin sections were mounted on Formvar-coated slotgrids (PLANO: model G2500C), stained with uranyl acetate and lead citrate for 4 min each, and then examined under a JEOL JEM-1011 transmission electron microscopes operated at 80 kV. Images were obtained with an Olympus

Mega View III digital camera using iTEM software.

3D X-ray microscopy (XRM) and 3D reconstruction

After fixation (see protocol above), three female opisthosomata (two with and one without a plug in the genital area) were prepared for an X-ray microscopy scan (Xradia XCT-200, Carl Zeiss

Microscopy GmbH) in the following way: after dehydration, samples were stained overnight using 1% iodine solution (in pure ethanol). After washing in pure ethanol, samples were critical point dried (BalTec 30) and subsequently mounted on insect pins using super glue. Scans were obtained using the 40x objective lens unit with the following scan parameters: 40 kV, 8 W, 200

µA, exposure time 30 sec/frame. Reconstructed image stacks were created using 142

XMReconstructor software (Carl Zeiss Microscopy GmbH) and the subsequent segmentation

(delineation) of sclerotized structures of female genitalia was performed with Amira 5.6

(Visualization Science Group, FEI).

RESULTS

Mating behaviour

After the first contact, the male always started to court the female – he tapped her body with his forelegs and twitched his abdomen while touching the female. Initially, the female was escaping the courting males, often pushing male legs away by her own forelegs. After several chases she stopped moving and allowed the male to climb over her body while he continued tapping and twitching. The female usually did not remain motionless and shook off the male several times before he managed to adopt mating position and insert one of his pedipalps (Video S1).

Spiders mated in the type III posture (von Helversen 1976), meaning that the male positioned himself above the female with his front facing toward the tip of her abdomen. The male reached around her opisthosoma to insert one of his pedipalps. His right palp was inserted into her left copulatory duct and vice versa. Attempts to couple the pedipalp to the female genital plate (i.e. genital coupling) were accompanied by lateral palpal movements. Once the pedipalp was coupled, several rhythmic partial expansions of the membranous part (haematodocha) of the sperm transfer organ followed and they were terminated by a single long expansion during which the haematodocha inflated entirely. The duration of one partial expansion was less than a second, while the full haematodochal expansions lasted on average (mean ± SE) 3.5 ± 0.28 s

(N = 24). Males typically performed one full expansion per insertion (except three males who performed two full expansions) and after that the female jerked her body and shook off the male thereby ending the copulation abruptly. After the separation, the male resumed courtship and tried to mount the female again. Male normally achieved two to three insertions but in many 143 cases (N=14) one of the insertions did not result into the full haematodochal expansion as female shook off the male before he perfomed it.

Mating trials with virgin females

The components of courtship and copulation with virgin females are listed and quantified in Table 1. The atrium of virgin females was devoid of any plug material before copulation (N =

24). After mating, a complete plug (i.e. covering 100% of the atrium) was found in 45.9% of cases

(N = 24). In 33.3% of cases the plug did not cover the atrium completely (80-90% coverage) but it covered both copulatory openings. In 12.5% of the cases an incomplete plug was found, i.e. the plug material was found in the atrium in varying amounts (30-70% coverage) but it covered only one copulatory opening. In two cases (8.3%) only a small amount of plug material was found in the atrium (10% coverage) covering neither of the copulatory openings.

GLM revealed that the extent of plug coverage was significantly affected by two variables: it increased with increasing number of taps a male delivered on the female body during courtship

(GLM-qb, F1,22 = 5.96, P = 0.024) and with the total duration of the long haematodochal expansion

(GLM-qb, F1,22 = 12.31, P = 0.002, Fig. 1). The number of taps was positively related to the femur length ratio of the pair (GLM-g, F1, 22 = 8.97, P = 0.006, Fig. 2), meaning that males with femora proportionally longer than female femora performed more taps.

Mating trials with plugged females

The components of courtship and copulation are listed and quantified in Table 1. All females initially were aggressive towards the second male (N = 19). Despite numerous bites directed to male legs, the males readily courted the females and all remated. Courtship took longer than with virgin females as males performed more taps and abdomen twitches (Table 1). Once allowing the male to reach the mating position, the females usually remained motionless for a longer time than the virgin females. They did not shake off the male after each insertion, which 144 allowed him to change and insert his palps several times without dismounting the female. The copulation ended when the female shook off the male and started to be aggressive towards him.

Males continued courting the females but the females usually vigorous bit the males and did not allow them to remount. Females with plugs showed higher values in all recorded behaviours compared to virgin females except for full haematodochal expansions that where both in number as well as duration lower compared to virgin females (Table 1).

After remating (N=19), a substantial part of the plug (70 to 100%) was removed in 47.4% of cases. Only a small part of the plug (10 to 30%) was removed in 42.1% and the plug remained intact in 10% of cases. Statistical analysis revealed that only the femur length ratio between the

nd st 2 and 1 male (GLM-qb, F1,17 = 7.87, P = 0.012) and the number of palpal movements during coupling (GLM-qb, F1,17 = 10.69, P = 0.005) significantly affected the probability of plug removal

(Fig. 3). A new plug was produced in five cases (26%, N = 19) – in three cases the new plug covered half of the atrium, in two cases the whole atrium was covered. In all cases full haematodochal expansions were observed.

Morphology of genitalia

In the male, the bulbus contains three glands, spermophor, embolus and fundus, associated with the spermophor (Fig. 4A). The spermophor gland runs along its wall for most of its length. This gland comprises many secretory cells forming a thick glandular epithelium (Fig. 4B) rich in rough endoplasmic reticulum and microtubules. The part of spermophor surrounded by this gland is filled with sperm and round vesicles of a high electron density (Fig. 4C). Vesicles similar to those stored within the spermophor are present also in the spermophor gland lumen, specifically, in the portion directly adjacent to the spermophor cuticle and in the pores within the cuticle (Fig.

4D). Lumina of the two remaining glands (fundus and embolus gland) contain an amorphous substance of low electron density devoid of vesicles.

145

In females of P. cespitum, the epigynal plate forms an oval cavity, an atrium, with two laterally-situated copulatory openings (Fig. 5A, B). The openings lead to copulatory ducts, each distally connecting to the bottom of a globular spermatheca (Fig. 5A). Proximally, a short fertilization duct leaves from the spermatheca in the direction of the uterus (Fig. 5A). The plug is a mixture of compressed circular or oval vesicles and spermatozoa (Fig. 5B, C). Histology revealed that the plug extends into both copulatory ducts approximately up to a half of their length (Fig.

5A, D), while the remaining part of the ducts and the spermathecae are filled mainly by spermatozoa (Fig. 5D).

DISCUSSION

We investigated mating plug production and removal in the spider Philodromus cespitum.

Behavioural experiments revealed that the deposition of plug material onto female genitalia is an obligatory part of copulation with virgin females of P. cespitum since plug material was found in the female atrium after the copulation in all cases. Experiments with plugged females showed that even complete plugs do not hinder access to female genitalia irreversibly as half of the males managed to remove substantial parts of the plug and mate. The morphological study confirmed that the plug material originates from the male as the material filling the female atrium and copulatory ducts after copulation correspond to the material found in the male copulatory organ.

Mating plugs may represent a powerful tool to monopolize females. Because they likely collide with female interests, virgin females are expected to be particularly choosy (Uhl, Nessler,

& Schneider, 2010). In P. cespitum, however, virgin females readily mated with the first male they encountered and consequently bore plug material in their atrium after copulation. The extent of the coverage differed resulting in none, one or both copulatory openings being blocked. Most of the males produced mating plugs covering both copulatory openings. The deposition of plug material could not have been visually distinguished through differences in behaviour from sperm

146 transfer as found in other species (Knoflach, 1998, 2004; Sentenská et al., 2015). It seems that after an intromission is achieved, the female is unable to prevent plug deposition completely since the copulation is very short and, as demonstrated by our morphological investigation, the sperm and the plug material are stored together in male copulatory organ and transferred together via the same structures during several partial and typically one full haematodochal expansion per insertion. However, as the amount of material on female genitalia increased with the total duration of full expansions the transfer of considerable amount of plug material likely takes place during the full haematodochal expansions. Since there is typically only one full haematodochal expansion per insertion, the female can selectively allow or reject additional insertion.

Nevertheless, full plug was observed to be deposited even after a single insertion. In general, a female can affect the degree of monopolization of her genitalia even after the onset of the transfer of plug material. By dislodging a male during mating the female can reduce plug efficacy - incomplete plugs were shown to be easily overcome by subsequent males (Jackson,

1980; Matsumoto & Suzuki, 1992; Masumoto, 1993; Uhl & Busch, 2009; Kunz, Witthuhn, & Uhl,

2014). In P. cespitum, the copulation was terminated by females and the amount of plug material in their atrium differed, indicating that females might be capable of controlling how much plug material is deposited. As the amount of plug material in female atrium was related to the total duration of full haematodochal expansions, the female may control plug deposition by interrupting the copulation before the full expansion starts. Indeed, most males performed less full expansions than they achieved insertions, indicating that they were prevented to perform full haematodochal expansion during some insertions due to premature termination of the copulation by the female.

The ability of females to control plug efficacy may represent a mechanism of cryptic

147 female choice exercised on a certain male trait (Eberhard, 1996). For example, in Leucauge mariana the female cooperates in plug formation with those males who perform more copulatory courtship movements during mating (Aisenberg & Eberhard, 2009). In Micaria sociabilis, no observable courtship occurs before or during copulation, however, females copulated longer with males that were quick in genital coupling (Sentenská et al., 2015). In the current study, the amount of plug material in the female atrium was affected by male courtship activity, specifically by the number of taps delivered by male forelegs onto the female body. Even though none of the measured size characteristics had a significant direct effect on the extent of plug coverage, the number of taps increased significantly with increasing relative foreleg size of the males. Specifically, the longer the femur of the male foreleg than the one of the female, the higher the number of taps the male delivered. This can be explained by female behaviour during courtship when she pushes the male forelegs away with her own during his attempts to tap her.

If the male has longer forelegs than the female, he is able to reach her more easily and thus perform more taps. Therefore, taps may be considered as a proxy for male quality represented by the relative length of his forelegs. Although our results indicate that males with higher courtship rate transfer more plug material and are possibly favoured by females, it is impossible to disentangle whether these males are indeed permitted to deposit larger plugs or rather to pass more sperm (or both). Considering the joint storage and extrusion of sperm and plug material, females may allow preferred males to transfer more sperm, which at the same time leads to the deposition of a more effective plug. The majority of males managed to transfer enough material to plug both copulatory openings. No matter whether the material covered 80% of the atrium or filled it completely, these plugs were equally likely removed by subsequent males. Males readily courted females with plugs covering both copulatory openings even after contact with their sealed genitalia and despite their initial aggression and the females allowed

148 the males to mount them. In half of cases, males removed more than a half of the plug. In comparison to matings with virgin females, males mating with plugged females performed more taps and twitches during the courtship and made more insertions and lateral palpal movements during the copulation. More vigorous courtship is likely linked to higher aggressiveness of mated females and their reluctance to remate. The higher number of palpal movements can be explained by failed attempts to fix the palp on the sealed genitalia. These movements also seem to be related to plug removal since the amount of plug material removed was positively correlated with the number of palpal movements.

No matter whether a female exerted mate choice during her first mating or not, she can benefit from acquisition of another male´s sperm if he is of better quality than her previous mating partner (Watson, 1998; Jennions & Petrie, 2000; Simmons, 2001; Pitcher et al., 2003).

In P. cespitum, mated females typically accepted second males, and after mating with them, the amount of plug material removed from their atrium differed. As the copulation is terminated by the female, she may behaviourally either facilitate or prevent the plug removal. In P. cespitum even complete plugs can be overcome by the next male which is in contrast to e.g. M. sociabilis where only 7% of males manage to remove a complete plug (Sentenská et al., 2015). This together with a high incidence of plugging indicates that females are not under strong selection to prevent males from plug deposition. It is possible that female control of plug transfer could only evolve at the expense of sperm transfer and may thus not be strongly selected for.

In general, females must accept and reject mates without precise knowledge of future mate encounters (Kokko & Mappes, 2013). Since errors can be made during initial assessment, a female can correct her previous choice if she mates multiply (Watson, 1998) and she can gain more information about male quality after the onset of copulation (Eberhard, 1996). In P. cespitum, the first copulation is very brief and provides a female with a small time window to

149 acquire information on male quality. However, once mated, a plug in her genitalia may secure enough time to assess the subsequent male without receiving his sperm. The female can reject the second male while he tries to remove the plug, or, if he is of better quality than the first one, correct her initial choice by letting him remove the plug and transfer his sperm.

The male trait(s) assessed by females are expected to remain the same across all instances of mate assessment, even if the mechanisms of the assessment changes over the female’s life history (Watson, 1990, 1991, 1998). In our study, the amount of material removed was not affected by the number of taps performed by second males during courtship, which was a trait affecting the amount of plug deposited in the first mating. However, the extent of plug removal was affected by relative length of forelegs of the second male - a trait affecting male tapping behaviour while mating with virgin females. In the second mating trials, the plugging material was removed to a higher extent by males whose foreleg femora were longer than those of the male who applied the plug. Virgin females seem to assess the leg length of the first partner indirectly during the courtship and once mated they estimate it directly during the second mating. The mechanisms of mate assessment and comparison, however, remain unknown.

The amorphous mating plugs in spiders can be a product of male, female or both sexes

(Eberhard & Huber, 1998; Knoflach, 1998, 2004; Uhl, Nessler, & Schneider, 2010). In P. cespitum the material found in the genitalia of mated females consisted of products found in male copulatory organs only. The plug consists of a matrix containing numerous circular vesicles and several spermatozoa, both found within the male spermophor before mating. In other species, plugs are devoid of sperm, a feature likely related to a storage and/or release of the plugging material separate from sperm (Uhl et al., 2014; Sentenská et al., 2015). The presence of lumps of spermatozoa in the plug of P. cespitum likely results from the joint storage of sperm and the plugging material within the spermophor and its release via the same exit. Histology revealed

150 that the plugging material fills the atrium and extends into the copulatory ducts, while the spermathecae are filled predominantly by spermatozoa. It remains unclear how these two materials, can be deposited separately in and on the female genitalia. It is possible that one of the other glands within the male bulbus, the embolus gland, adds a product to ejaculate altering coherence or viscosity of its various components.

The plug material extends partly to the copulatory ducts, but we were not able to determine whether the plug stays there or is removed by a subsequent male together with the material in the atrium. However, as half of males performed full haematodochal expansions after they removed a considerable amount of plug material deposited by previous male, it is likely that they achieved insertion and transfer their sperm to the spermatheca. The absence of full expansions and/or new plug material in some cases is likely caused by female behaviour as she dislodged the male before he managed to fully expand the haematodocha and deposit a plug.

Our study demonstrates that the plugging material in P. cespitum is produced by the male but that the plug deposition and fate is at least partly under female control. Plug deposition is alleviated for males that performed high courtship activity but the female control of deposition is complicated by the joint storage and extrusion of the plugging material and sperm. Female choice seems most pronounced in this species during remating. Since the amount of the plug material removed was higher in males who had legs longer than the previous partner of the female, we conclude that female choice uses size as an indicator of male quality.

ACKNOWLEDGMENTS

We thank the University of Greifswald for providing funding for activities between partner universities granted to GU for research visits of LS to Greifswald. The 3D X-ray microscope was funded by the German Science Foundation (INST 292/119-1 FUGG und INST 292/120-1 FUGG).

We would like to thank Radek Michalko for help with spider collecting in the field and helpful 151 comments. Helpful comments were also given by Pierick Mouginot, Carsten H.G. Müller and

Peter Michalik. LS was supported by grant no. MUNI/A/1484/2014 provided by Masaryk

University.

COMPETING INTERESTS

The authors declare that they have no competing interests.

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Fig. 1. Plug coverage (proportion of plug material in female atrium) in relation to number of taps during courtship and duration of long haematodochal inflations. Estimated logit model is shown.

Fig. 2. Number of taps in relation to femur length ratio in a pair (male to female). Estimated gamma model is shown.

Fig. 3. Plug removal (proportion of plug material removed from female atrium) in relation to femur length ratio of second to first male and number of lateral palpal movements. Estimated logit model is shown.

Fig. 4. Male copulatory organ of Philodromus cespitum. A: Volume and surface reconstruction of male copulatory organ based on 3D X-ray microscopy, proventral view. Delineated focal parts include spermophor, three glandular tissues and embolus. Section plane illustrated in B is indicated by black solid line. B. Semi-thin cross section of male pedipalp showing genital bulb containing spermophor and spermophor gland. C. Ultrathin section of the spermophor lumen containing vesicles and spermatozoa. D. Ultrathin section of the porous cuticle of the spermophor. Note the circular vesicles within the pores and within spermophor lumen.

BG – bulbus genitalis, bH – basal haematodocha, Cu – cuticle, Cy – cymbium, S – spermophor, SF

– seminal fluid, SG – spermophor gland, SL – spermophor lumen, Spz – spermatozoa, V – vesicle

Fig. 5. Female genitalia of Philodromus cespitum. Volume and surface reconstruction of female genitalia based on 3D X-ray microscopy. Ventral view without plug (A) and ventral (B), prodorsal

(C) and dorsal (D) view of female genitalia with plug. E. Semi-thin cross section of the female atrium and one of the copulatory ducts filled by plug material and spermatozoa. F. Ultrathin section of the plug in the atrium containing compressed circular vesicles and spermatozoa. G.

Semi-thin cross section of female copulatory ducts and spermatheca filled by plug material and spermatozoa. CD – copulatory duct, P – plug, Spth – spermathecal, arrows indicate copulatory openings

156

Fig. 1

157

Fig. 2

158

Fig. 3

159

Fig. 4

160

Fig. 5 161

Table 1: Descriptive data on mating trials with Philodromus cespitum females without plugs (virgin) and plugged females (previously mated) and

statistical comparison of the mating behaviours between the two groups

Mating trials with females Statistical comparison

without plug with plug Response variable Test statistics P-value Model (N=24) (N=19)

No. of taps 16.9 ± 5.1 97.7 ± 23.8 F1, 43=7.63 P=0.01 GEE-gamma

No. of abdomen twitches 10.3 ± 2.4 85.8 ± 23.6 F1, 43= 14.80 P< 0.001 GEE-gamma

No. of palpal movement 22.5 ± 7.9 103 ± 27 F1, 43=4.89 P=0.03 GEE-gamma

No. of partial haematodochal expansions 26.4 ± 3.7 149 ± 36 F1, 43=31.70 P< 0.001 GEE-gamma

Total duration of partial haematodochal expansions [s] 16.5 ± 2.6 41.6 ± 10.6 F1, 31=10.40 P= 0.001 GEE-gamma

2 No. of full haematodochal expansions 2 ± 0.3 0.4 ± 0.2 χ 1= 9.22 P= 0.002 GEE-poisson

2 Total duration of full haematodochal expansions [s] 6.7 ± 0.8 1.1 ± 0.6 χ 1= 13.20 P< 0.001 GEE-poisson

No. of insertions 2.4 ± 0.1 5.8 ± 1.1 F1, 31=20.50 P< 0.001 GEE-poisson

162

6. STUDY F

SEXUAL DIMORPHISM IN VENOM GLAND MORPHOLOGY IN A SEXUALLY STINGING

SCORPION Sentenská, L., Graber, F., Richard, M., & Kropf, C. Biological Journal of the Linnean Society (submitted).

163

164

Sexual dimorphism in venom gland morphology in a sexually stinging scorpion

Lenka Sentenská1*, Franziska Graber2, Miguel Richard3,4 and Christian Kropf3,4

1 Department of Botany and Zoology, Faculty of Sciences, Masaryk University, Kotlářská 2, 611 37

Brno, Czech Republic

2 Institute of Anatomy, Department of Topographic and Clinical Anatomy, University of Bern,

CH-3012 Bern, Switzerland

3 Natural History Museum Bern, Department of Invertebrates, CH-3005 Bern, Switzerland

4 Institute of Ecology and Evolution, Division of Community Ecology, University of Bern, CH-3012

Bern, Switzerland

*Corresponding author: [email protected]

RUNNING TITLE: Gland morphology in a sexually stinging scorpion

165

ABSTRACT

Males of several scorpion species possess bigger telsons than females. In at least some of these species males repeatedly sting females during mating. This behaviour (“sexual stinging”) is likely correlated with a sexual dimorphism in telson and venom gland size. As natural selection theory predicts bigger female venom glands (females need more nutrients for their offspring), we hypothesise that this sexual venom gland dimorphism evolved under sexual selection in relation to the male’s sexual stinging behaviour. We investigated morphometrics and morphology of male and female telsons and venom glands by means of light and transmission electron microscopy in the sexually stinging scorpion Euscorpius alpha Caporiacco, 1950 (Euscorpiidae). Male telsons are significantly bigger and more voluminous than those of females. Four different secretory cells can be distinguished with their representation varying considerably between sexes. While the female secretory epithelium consists mainly of cells filled by granules, males carry mainly cells containing dissolvable vesicles. This cell type likely produces transparent venom that had been identified in other scorpions as so-called “prevenom”. The function of this secretion is discussed in relation to sexual stinging behaviour.

KEY WORDS: courtship – Euscorpius alpha – prevenom – sexual selection – telson

166

INTRODUCTION

Sexual dimorphism in scorpions occurs in several forms. Males and females may differ from each other in body size, shape of various body structures and/or presence or development of certain features. As in many invertebrates, female scorpions are generally larger or more robust than males (Polis & Sissom, 1990; Fox, Cooper & Hayes, 2015; but see Alexander, 1959). They feed proportionately more often than mature males as they have to catch more prey to suffice enough food for themselves and the offspring they are gestating (Polis & Farley, 1979a, b; Polis & Sissom,

1990; Carlson, McGinley & Rowe, 2014; Fox et al., 2015). However, despite the bigger body sizes of females, in the genus Euscorpius males bear bigger and more bulbous telsons than females even though they are slightly smaller (Polis & Sissom, 1990; Kraepelin, 1908; Pavlovsky, 1913; Fet et al., 2013) and there are several more species showing sexual dimorphism in telson size and/or shape with male telson being bigger, longer or equipped with additional structures which are absent in females (Polis & Sissom, 1990; Soleglad & Fet, 2004; Booncham et al., 2007; Lourenco

& Duhem 2010).

Scorpions show consistent sexual dimorphism in pectines, i.e. structures which are widely employed by males during courtship and mating. As they typically reach disproportionally bigger sizes in males than in females, they are considered to represent secondary sexual traits in males evolved under sexual selection (Polis & Farley, 1979a; Fox et al., 2015). Therefore, to explain dimorphism in telson size one should consider a role of sexual selection as a force shaping the size or form of this structure in males. Indeed, the male telson is often employed during courtship in several ways (Baerg, 1954; Angermann, 1955, 1957; Polis & Sissom, 1990; Benton, 1993). In general, scorpion mating is a complex process involving several characteristic behaviours (Polis

& Sissom, 1990). Typically, the male leads the female in a classical promenade à deux - he grasps the female’s pedipalps in his own and pulls and pushes the female to a suitable location for spermatophore deposition. During the promenade, the male engages in various activities such 167 as sand scraping, juddering, cheliceral massage, clubbing or sexual stinging (Angermann, 1955,

1957; Alexander, 1959; Benton ,1993; Polis & Sissom, 1990), the latter two involving use of the male metasoma and telson. During clubbing, the male strikes the partner with the metasoma with sting tucked away, while during sexual stinging the male sinks his sting into membranous articulations of the female up to several minutes.

Sexual stinging was recorded in representatives of families Bothriuridae, Euscorpiidae (incl. genus

Euscorpius), Chactidae, Iuridae, Scorpionidae and Vaejovidae (Francke, 1979; Polis & Sissom,

1990; Tallarovic, Melville & Brownell, 2000; Peretti, Acosta & Martínez, 2000; Booncham et al.,

2007; Jiao & Zhu, 2010; Toscano-Gadea, 2010). Despite its uniqueness and peculiarity, there was little attention paid to this behaviour in the literature, and it was never investigated in detail.

Although sexual stinging is considered to be a predation suppressant or tool to surmount female reluctance or passivity (Polis & Sissom, 1990), it may also represent a stimulating impulse

(Inceoglu et al., 2003). Moreover, it is not even known whether venom is transferred during these stings or whether it is just a mechanical stimulus.

There is no study linking sexual dimorphism in telson size and occurrence of sexual stinging in scorpions. Nevertheless, the coincidence in the genus Euscorpius lead us to the hypothesis that males are sexually selected to have larger telsons to produce a substance of a higher amount or a different composition than it is produced in females and this substance is transferred during sexual stinging to the female body. To test this hypothesis morphologically, we inspected the content of the telson of Euscorpius alpha Caporiacco, 1950, a scorpion species in which males sting fenales during courtship (Kropf, personal observation) to find out whether the glandular equipment differs between sexes (i.e. males possess unique secretory cells that are not present in females) or whether the difference is simply in size and/or in the number of secretory cells.

This study presents the first comparison of male and female venom gland morphology in a sexually dimorphic scorpion and discusses its possible relation to the sexual stinging. 168

MATERIAL AND METHODS

Adult males and females of the scorpion Euscorpius alpha were collected in Mendrisiotto district in canton of Ticino, Switzerland in June 2014. They were placed in transparent containers and fed by crickets at the first day in captivity. After one week the individuals were anesthetized with ether and processed accordingly (see below).

Light microscopy

For the histological study 6 telsons (3 from each sex) were inspected. The telson was removed by fine surgical scissors. The tip of the telson (i.e. aculeus or stinger) was removed to enable penetration of the fixative to the telson. The samples were fixed in 2.5% glutaraldehyde in Hepes buffer (0.15 M, pH 7.34) and then subjected to postfixation in buffered 1% osmium tetroxide.

The samples were afterwards washed in maleate buffer, dehydrated in graded ethanols, and embedded in Epon.

Due to the incomplete penetration of the fixative with the previous procedure 4 additional telsons (2 from each sex) were processed. They were removed by fine surgical scissors. To enable better penetration of the fixative to the glands, a tip of the telson was removed and the rest of the telson was cut longitudinally in half with a razor blade. Both halves were fixed in 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4, 1.8% sucrose) and then subjected to postfixation in buffered 1% osmium tetroxide. The samples were afterwards washed in sodium cacodylate buffer, dehydrated in graded ethanols, and embedded in Agar Low Viscosity Resin.

Serial semi-thin sections (1 μm, cross sections) of male and female telsons were obtained using a Diatome Histo Jumbo diamond knife on a Reichert Jung Ultracut microtome. The sections were placed on glass slides, heated and stained with 1% toluidine blue. Examination of the sections was performed with a Zeiss Axioplan 2 microscope connected to a Zeiss Axiocam MCR digital camera. A number of different gland cell types was recorded for one of the paired gland on every

20th section. The relative amount of each cell type (i.e. a number of a given gland cell type divided 169 by total number of gland cells on the section) was calculated and transformed using angular transformation to homogenize variance. The relative amount of each cell type and total cell number on the inspected sections were compared between sexes using linear mixed-effects models (LME) with individual and section number as random effects.

Transmission electron microscopy

Ultrathin sections (60 nm, cross sections) were obtained from the same blocks described above using a Diatome Ultra 45° diamond knife on a Reichert Jung Ultracut microtome and placed on copper slot grids. Post-processing included staining with saturated uranyl acetate and lead citrate. The sections were examined with a transmission electron microscope (TEM) Philips EM

400. Images were obtained with a Morada 12 Megapixel camera using AnalySIS ITEM software.

Measurements

Length, width and height of telsons of males (N=11) and females (N=15) from the same population were measured using a Keyence VHX-2000 digital microscope. The measurements were compared using a Welch two sample t-test with Bonferroni correction. In addition, width of fifth metasomal segment was also recorded to control for the possibility that telson size varies with overall body size (i.e. male telsons are bigger simply because males are bigger).

RESULTS

Overall appearance of the venom gland.

The venom apparatus of E. alpha is composed of a pair of venom glands situated in the telson

(Fig. 1). Each gland is partly covered by cuticle and partly by muscles. The gland itself shows no folding, the glandular cells are of equal length and more or less alike in height throughout the gland (Fig. 1) which are features characteristic for simple type glands (Type 1) according to

Pavlovsky (1913, 1924). The glandular tissue is composed of venom-producing secretory cells and non-secretory supportive cells. The secretory cells are filled with different vesicles which are 170 released into a narrow, centrally located reservoir. The glands run bilaterally through the whole telson length. Distally, they narrow into two separated venom ducts which open to the outside just beneath the tip of the aculeus.

Muscles and connective tissues.

Each gland is fixed to 1/3 (close to the tip) or 1/2 (more proximally in the telson) at the cuticle; the rest is covered by striated ring-musculature (Fig. 1). The muscles occur in several bundles (5-

9) which can be separated by thin unicellular layers of connective tissue containing numerous vesicles (Fig. 2A). The M and H zones are undistinguishable and the Z lines show an irregular structure. Between muscle fibrils mitochondria and rows of glycogen granules can be found. The muscle fibrils attach to the cuticle in numerous branches by complicated tendon cells (Fig. 2B).

These tendon cells are attached to outgrowths of the cuticle (Fig. 2B, C). The muscles and the cuticle are separated from the glandular epithelium by a thick membrane formed by supportive cells (see below, Fig. 2D). Between the membrane and the gland cells a row of voluminous cells is found (Fig. 2D). The cuticle and muscles adjoin to the membrane mostly directly; however, there may also be cells containing vesicles that are located in between (Fig. 2D, E). On both sides of the membrane lie nerves (Fig. 2F). Sometimes proliferations of the membranes towards both sides can be seen, leading to a tighter connection between the membrane and the adjacent tissues (muscles, connective tissue cells, epidermal cells, 2E). The connective tissue cells have complex folded cell membranes, pointing to a high transport activity.

Secretory cells.

The secretory cells are columnar and show cell membranes with septate desmosomes (Fig. 3A), forming ladder- or grid-like structures (Fig. 3A). In the basal (peripheral) part of the cell the nucleus and densely packed ER with interspersed mitochondria are situated (Fig. 3B), likely forming a “factory” part of the cell where the vesicles are produced. Some cells contain predominantly the ER with only few or no vesicles, they likely represent emptied cells. Normally, 171 most of the cell is filled by vesicles of different size, shape and electron density. Four distinct secretory cell types can be distinguished with both light and transmission electron microscope according to the vesicles they contain:

Type A cells are filled with large more or less circular vesicles and/or a fine-grained

substance (Fig. 3A, 4A). The diameter of the vesicles is usually bigger than 3 μm and

reaches up to 10 μm. They are covered by a membrane. The vesicles occurs in two forms,

both have the same size range but some vesicles shows high electron density and appear

homogenous, while others are less electron dense and finely granulated. In some cells

both vesicle types occur, and transitions between the two can be seen (Fig. 3B, 4A). The

vesicles may unite into a big mass of substance and then gradually dissolve. During this

process they get more and more granulated, forming a coarse to fine-grained substance

(Fig. 3B, 4A). Sometimes the cell filled by this substance also contains one or a few large

vesicles described above.

Type B cells contain small (max. 1 μm long), electron-dense oval, barbell or kidney-shaped

vesicles (Fig. 4B). Within the cell, some of the vesicles are unified in a cluster surrounded

by a membrane, but most of them lie freely in the plasma. The vesicles are covered by a

membrane.

Type C cells are filled with electron-dense vesicles of median size (1-3 μm) and rather

irregular shape (Fig. 4C). The vesicles are smaller and never so round as in type A, and

bigger and never kidney-shaped as in type B. The vesicles are surrounded by a membrane.

Type D cells are typically formed by a row of compartments with electron-dense droplets

in the centre, the compartments with clusters of droplets are separated by thin thread-

like “bridges” (Fig. 4D). The droplets show no membranous cover.

The fine-grained substance of the A cells and the vesicles of type B and C cells were found in the reservoir (Fig. 4E); rarely also the homogeneous, electron-dense vesicles of cell type A are found 172 in the reservoir. However, this could be an artefact related to squeezing during opening the telson for fixative penetration.

Supportive cells.

Between the secretory cells, there is an extensive net of long and slim supportive cells of high electron-density (Fig. 5A). Muscle fibrils are found within the supportive net (Fig. 5A). The cells form the thick membrane surrounding the glandular epithelium. Proliferations of this membrane may be leading in all directions. In this basal part lies also the nucleus. The supportive cells expand towards the reservoir and cover the gland cells like a grid or net. These cell expansions are packed with lots of granules of different size and electron density, with numerous tubes and double or multiple membranes (Fig. 5A). The cells branch into numerous slim processes that again split into microvilli that contain microfilaments and penetrate into the reservoir (Fig. 5B). The cell expansions with their slim processes surround large parts of the gland cells where these open into the reservoir. The microvilli of the supportive cells have their highest density at the periphery of the reservoir, the lowest density in the centre. Interestingly, the tubes within the cell expansions look identical to the microvilli and also contain microfilaments.

Comparison of male and female venom apparatus

Male telsons in E. alpha are considerably bigger and more voluminous in comparison to the telsons of the females (Fig. 6; ♂ N=11, width = 1.45–1.90 mm, height = 1.25–1.64 mm, length =

2.43–3.22 mm; ♀ N=15, width = 1.03–1.26 mm, height = 0.85–1.09 mm, length = 1.76–2.24 mm).

All differences were highly significant for the telson measurements (Welch two sample t-test, p<0.001) while the width of the 5th metasomal segment does not differ between the sexes (Welch two sample t-test, p=0.983).

There are several differences between the sexes in the cell equipment (Table 1). The male gland contains nearly three times more secretory cells than the female gland. The relative amount of three of the four cell types differs between the sexes. Type A cells are significantly more abundant 173 in males than in females. In females these cells are situated mainly close to the tip of the telson and they are nearly absent more proximally in the gland. In males this cell type is the most abundant one throughout the whole gland and represents the most of all secretory cells. Type B cells occur throughout the glands of both sexes but they are significantly more abundant in females. The same holds true for type C cells, which are the most abundant cell type in females comprising nearly half of all secretory cells present in the gland. Type D cells occur with similarly low abundance in both sexes and represent the rarest cell type. The cells were counted throughout the whole gland length only in glands which were fixed in Agar Low Viscosity Resin

(see above) due to the complete penetration of the fixative. However, we inspected also glands in EPON which were not fixed throughout and despite an incomplete fixation the cell equipment of males and females showed the same pattern as described above.

DISCUSSION

Although venom glands of several scorpion species were examined ultrastructurally (Kubota,

1918; Rosin 1965; Keegan & Lockwood, 1971; Mazurkiewicz & Bertke, 1972; Kovoor, 1973; Yahel-

Niv & Zlotkin, 1979; Halse et al., 1980; Kanwar, Sharma & Nagpal, 1981; Kanwar & Nagpal, 1983;

Taib & Jarrar, 1993; Jarrar & Al-Rowaily, 2008; Yigit & Benli, 2007, 2008, 2009, 2010), none of the studies provides a comparative description of male and female gland or states whether intersexual differences occur.

Sexual dimorphism in the telson of Euscorpius alpha was found in telson and venom gland size, the number of the secretory cells, and their proportional representation. Measurements confirmed that males possess significantly bigger, bulkier and more voluminous telsons which harbour venom glands bigger than are those of females. However, natural selection theory predicts exactly the opposite: As females need to subdue more and/or bigger prey in order to provide nutrients for their offspring, they should show bigger venom glands. This is known from 174 other scorpions, e.g. from bothriurids (e.g. Ojanguren-Affilastro, 2007). Considering the strange behaviour of “sexual stinging” in this species, we assume that the sexual dimorphism in venom gland size in Euscorpius alpha is related to sexual selection.

The male gland is equipped mainly with type A cells. They comprise nearly 90% of male secretory equipment, while they cover less than 25% of secretory cells in females. Based on the numerous transitional stages (Fig. 3B, 4A; homogenous dark vesicles - finely granulated lighter vesicles - finely granulated light substance filling whole cell), we consider the homogenous granules of high electron density as the early stages within type A cells, which gradually dissolve and unite in a big mass of finely granulated substance. They fill not only some of the cells but also extensively the reservoir, while its early stage, the vesicles, was only rarely found in the reservoir. Cells filled by the finely granulated substance are often described in literature as mucus cells (Kubota, 1918;

Keegan & Lockwood, 1971; Kanwar & Nagpal, 1983; Farley, 1999), but already Keegan &

Lockwood (1971) pointed out that the content of these cells is not entirely typical of mucus cells found elsewhere.

The female secretory epithelium is mainly composed of cells containing granules. In general, granules are considered to represent venom proteins (Kubota, 1918; Keegan & Lockwood, 1971;

Farley, 1999). Iceoglu et al. (2003) distinguished two types of venoms – prevenom with a low concentration of proteins & high concentration of K+ salts and a protein-rich venom. Therefore, we conclude that females of Euscorpius alpha possess mainly venom-producing cells, while males show lots of prevenom cells (probably erroneously termed “mucus cells”, see above) and much less venom-producing ones.

Prevenom is a transparent liquid that differs chemically and functionally from the venom – it is more efficacious but less potent than the venom and it is metabolically less expensive to produce

(Iceoglu et al., 2003). Venom is more lethal, but the regeneration of proteins is slower (Nisani,

Dunbar & Hayes, 2007) and it may take up to 5 days until most of its essential components (i.e. 175 major venom peptides) is restored (Nisani & Hayes, 2011). Given the effects of the prevenom, its use during mating is well thinkable (however, data on this are entirely missing) as it may modify muscular contractions of the female during the promenade without exposing her to venom components which are potentially toxic to her (Iceoglu et al., 2003). Our observation of the gland morphology support this hypothesis as the male gland contains mainly type A cells (“mucous” substance cells or its early stages) which are in their late stage devoid of granules (that likely would represent venom proteins) while the female gland possesses mainly cells filled by granules

(Type B and C). In addition, cell type A in females is mostly found in the distal parts of the gland while in the proximal parts this cells are lacking. This again supports the idea that it is cell type A that produces the prevenom.

Despite the lack of histological investigation, it has been shown that venom-producing animals may be sexually dimorphic in venom composition. In Scorpio maurus palmatus, the female venom contains more components (Abdel-Rahman et al., 2009) while in the spider Tegenaria agrestis both sexes possess same components but the venom composition differs between sexes in their relative abundance (Binford, 2001). However, the venom composition of male and female of E. alpha may still differ even though the morphology shows the same cell types. For example, two scorpion species of the genus Centruroides which differ greatly in their toxicity show the same gland morphology (Keegan & Lockwood, 1971).

Sexual dimorphism in telson size and venom gland morphology may be indeed a result of sexual selection related to the sexual stinging. However, it may also arise from different lifestyles of male and female and so different selective pressures may act on the sexes. While females dwell rather hidden, males become vagrant during mating period and so they are more exposed to predators during this time (Polis & Farley, 1979b; Polis & Sissom, 1990; Benton, 1992). Bigger telsons might be favoured in males because they are exposed to a higher predation pressure and therefore needs more venom for a defence. Nevertheless, such explanation seems rather unlikely 176 as male vagrancy is widespread among scorpions but there are only few species where males possess bigger telsons. Similar ambiguity holds for sexual dimorphism in pedipals and chelicerae in scorpions (Carrera, Mattoni & Peretti, 2009). Intersexual differences in these structures might be explained by their employment during mating as well as due to the differences in life style of male and female (e.g. burrowing behaviour, Polis, 1990; Prendini, 2001). There is, however, a strong correlation between sexual dimorphism in chelicerae and the occurrence of the cheliceral grip during mating (Carrera et al., 2009). So far, sexual stinging has been observed in few scorpion species (Table 2) but reports about sexual dimorphism in telson size are mostly lacking. Therefore a morphological and behavioural survey across scorpion taxa would be necessary to shed a light on sexual dimorphism in venom gland and its relation to sexual stinging.

In both sexes an elaborated system of supportive cells and their processes and microvilli was found. These microvilli were also reported by Keegan & Lockwood (1971) and Yigit & Benli (2007).

The observed muscle fibers within the processes of the supportive cells suggest that they are mobile. Our observation that masses of “intracellular” tubes look exactly like the microvilli could support an extreme mobility of these cells; depending on the filling state of the reservoir, the microvilli could be expelled or retracted. The microvilli also point to a high degree of material exchange between the reservoir and the supportive cells. Another possible function of the supportive cells could be the control of extrusion of the products of the gland cells.

To sum up, we found considerable intersexual differences in venom gland morphology. Although we cannot generalise that sexual dimorphism in venom gland is exclusively related to sexual stinging behaviour, we hypothesise that males possess bigger venom glands in order to produce a substance employed during sexual stinging. The most abundant type of secretory cells in males do not contain granules (that are considered to represent proteins). Such a substance was identified as a prevenom by Iceoglu et al. (2003) who predicted its use during sexual stinging and our study supports this prediction. Therefore, we hypothesize that the higher amount of cell 177 types B and C in females (that presumably contain the venom proteins) was shaped under natural selection, while the much higher amount of type A cells in males (that presumable produce the prevenom) is related to sexual selection, in particular to the behaviour of “sexual stinging”.

ACKNOWLEDGMENTS

We would like to thank Hannes Baur for his help with statistical analyses, Werner Graber and

Peter Michalik for technical support and their comments. This study was funded by Natural

History Museum Bern.

COMPETING INTERESTS

The authors declare that they have no competing interests.

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FIGURE LEGENDS

Fig. 1. External and internal (semi-thin cross section) morphology of male and female telson of

Euscorpius alpha showing size differences between the sexes. A simple (Type 1) paired venom gland runs longitudinally through the telson of both sexes. cu – cuticle, gt – glandular tissue, m

– muscles. Scale bar = 500 μm

Fig. 2. Muscles and connective tissue of the venom gland of E. alpha. A: Muscle fibrils organized in muscle bundles separated by connective tissue with vesicles. B: Attachment of the muscle fibrils to the tendon cells. C: Attachment of the muscle fibrils to the membrane of supportive

183 cells. D: Thick membrane of supportive cells adjoining directly to the muscle and to a row of voluminous connective tissue cells. E: Same membrane separated from the cuticle by vesicle- containing cells. F: Nerve associated with the thick membrane of supportive cells. cu – cuticle, ctc – connective tissue cell, m – muscle, mb – muscle bundle, me –membrane formed by supportive cells, mf – muscle fibrils, nctc – nucleus of a connective tissue cell, ne - nerve, nm – muscle nucleus, , tc – tendon cells, v - vesicle. Scale bar = 2 μm

Fig. 3. Glandular tissue of the venom gland of E. alpha. A: Glandular cells filled by different vesicles separated by cell membranes with septate desmosomes. The inset shows detail of a septate desmosome forming a grid-like structure. B: Basally located factory part of glandular cells with ER and few vesicles (different stages of Type A cells). ag – grained substance of Type A cells, av – vesicles of Type A cells, b – Type B cells, cm – cell membrane, er – endoplasmatic reticulum, v – vesicles. Scale bar = 5 μm, inset = 500 nm

Fig. 4. Four types of glandular cells of the venom gland of E. alpha. A: Type A cell filled by circular vesicles and grained substance. B: Type B cells containing small oval vesicles. C: Type C cells filled by irregular vesicles of medium size. D: Type D cells formed by a row of compartments with electron-dense droplets in the centre. E: Venom gland reservoir filled by vesicles of Type A (grained substance), B and C. Scale bar = 3 μm

Fig. 5. Supportive cells within the venom gland of E. alpha. A. Basal part of a supportive cell branching between factory parts of glandular cells. The supportive cell is packed with granules and membranes; inset shows muscle fibrils within the cell. B: Microvilli penetrating glandular reservoir. b – Type B cell, er- endoplasmatic reticulum, mf – muscle fibrils, mi – mitochondria, mv – microvilli, ngc – nucleus of a gland cell, res – reservoir, sc – supportive cell. Scale bar = 1

μm

Fig. 6. Comparison of male and female telsons in E. alpha.

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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Table 1. Differences in the cell equipment of the venom gland in males and females of E. alpha.

male female LME

cell type count % count % F test p

A 3534 88.3 339 23.6 37.3 0.025

B 172 4.3 384 26.7 81.2 0.012

C 235 5.9 701 48.7 33.1 0.03

D 62 1.5 14 1.0 8.7 0.1

total 4003 1438

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Table 2. Compilation of information available on the occurrence of sexual dimorphism and sexual stinging in scorpions.

Species Sexual stinging Telson dimorphism Reference Bothriuridae Bothriurus buecherli yes ? Toscano-Gadea (2010)

Bothriurus cordubensis yes ? Peretti et al. (2000)

Bothriurus noa yes ? Peretti et al. (2000)

Bothriurus prospicuus yes ? Peretti et al. (2000)

Urophonius brachycentrus yes ? Polis (1990) Buthidae Centruroides vittatus ? yes (different shape) Polis (1990)

Chactidae Uroctonus spp. ? yes (male telson swollen, vesicle-aculeus juncture very distinct) Soleglad & Fet (2004)

Anuroctonus spp. ? yes (highly swollen vesicle and aculeus base Soleglad & Fet (2004)

Chaerilidae Chaerilus pictus ? yes (male telson prominently elongated) Lourenco & Duhem (2010)

Euscorpiidae Euscorpius alpha yes yes this study

Euscorpius candiota ? yes Fet V et al. (2013)

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Euscorpius carpathicus yes ? Polis (1990) Euscorpius flavicaudis yes ? Polis (1990)

Euscorpius italicus yes ? Polis (1990)

Euscorpius ossae ? yes Fet V et al. (2013) Euscorpius scaber ? yes Fet V et al. (2013) Megacormus gertschi yes ? Francke (1979)

Scorpiops luridus yes (during initial ? Jiao & Zhu (2010) phase) Hemiscorpiidae Hemiscorpius sp. ? yes (male teslon elongated and bilobed distally) Polis (1990)

Iuridae Anuroctonus phaidocatylus ? yes (swelling at the base of aculeus in males) Polis (1990)

Hadrurus arizonensis yes ? Tallarovic et al. (2000)

Scorpionidae Pandinus imperator yes ? Garnier & Stockman (1972)

Heteromerus laoticus yes yes (males wider and longer telson) Booncham et al. (2007)

Vaejovidae Paruroctonus mesaensis yes (during escape ? Polis & Farley (1979b) behaviour) Vaejovis carolinianus yes ? Polis (1990)

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7. AUTHOR’S CONTRIBUTION

SP and LS designed the study, LS collected spiders in the field and performed laboratory experiments. LS and SP analysed data and wrote the manuscript. Contribution of LS was 75%.

SP and LS designed the study, LS collected spiders in the field, performed laboratory and field observations and analysed the data. The manuscript was written by LS with comments of SP. Contribution of LS was 80%.

Study C Sentenská, L., Müller, H. G. C., Pekár, S., & Uhl, G. Male copulatory organ of spiders is not a numb structure as evidenced by presence of neurons and a sensory organ. Proceedings of the Royal Society of London B: Biological Sciences (submitted).

LS and GU designed the study, LS collected spiders in the field, performed mating trials and analysed the data. The morphological study was performed by LS and CHGM. The manuscript was written by LS, CHGM and GU with comments of SP. Contribution of LS was 70%.

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LS, SP and GU designed the study, LS collected spiders in the field, performed mating trials and analysed the data. The morphological study was performed by LS under guidance by EL and PM. The manuscript was written by LS with comments of GU and SP. Contribution of LS was 80%.

Study E Sentenská, L., Pekár, S., & Uhl, G. How spider females control deposition and removal of mating plugs. Animal Behaviour (submitted).

LS designed the study, collected spiders in the field, performed mating trials, morphological study and analysed the data. The manuscript was written by LS with comments of GU and SP. Contribution of LS was 80%.

Study F Sentenská, L., Graber, F., Richard, M., & Kropf, C. Sexual dimorphism in venom gland morphology in a sexually stinging scorpion. Biological Journal of the Linnean Society (submitted).

The study was designed by CK, LS performed the morphological study under initial guidance of FG and CK. LS and CK wrote the manuscript. Contribution of LS was 65%.

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8. LIST OF PUBLICATIONS Publications with IF:

Petráková, L., Michalko, R., Loverre, P., Sentenská, L., Korenko, S., & Pekár, S. (2016): Intraguild predation among spiders and their effect on the pear psylla during winter. Agriculture, Ecosystems & Environment, 233, 67-74.

Petráková, L., Líznarová, E., Pekár, S., Haddad, C. R., Sentenská, L., & Symondson, W. O. (2015): Discovery of a monophagous true predator, a specialist termite-eating spider (Araneae: Ammoxenidae). Scientific Reports, 5, 14013.

Sentenská, L., Pekár, S., Lipke, E., Michalik, P., & Uhl, G. (2015): Female control of mate plugging in a female-cannibalistic spider (Micaria sociabilis). BMC Evolutionary Biology, 15(1), 18.

Sentenská, L., & Pekár, S. (2014): Eat or not to eat: reversed sexual cannibalism as a male foraging strategy in spider Micaria sociabilis (Araneae: Gnaphosidae). Ethology, 120(5), 511-518.

Sentenská, L., & Pekár, S. (2013): Mate with the young, kill the old: reversed sexual cannibalism and male mate choice in the spider Micaria sociabilis (Araneae: Gnaphosidae). Behavioral Ecology and Sociobiology, 67(7), 1131-1139.

Pekár, S., Michalko, R., Korenko, S., Šedo, O., Líznarová, E., Sentenská, L., & Zdráhal, Z. (2013): Phenotypic integration in a series of trophic traits: tracing the evolution of myrmecophagy in spiders (Araneae). Zoology, 166(1), 27-35.

Líznarová, E., Sentenská, L., García, L. F., Pekár, S., & Viera, C. (2013). Local trophic specialisation in a cosmopolitan spider (Araneae). Zoology, 116(1), 20-26.

Pekár, S., Šmerda, J., Hrušková, M., Šedo, O., Muster, C., Cardoso, P., Zdráhal, Z., Korenko, S., Bureš, P., Líznarová, E., & Sentenská, L. (2012): Prey‐race drives differentiation of biotypes in ant‐eating spiders. Journal of Animal Ecology, 81(4), 838-848.

Nedvěd, O., Pekár, S., Bezděčka, P., Líznarová, E., Řezáč, M., Schmitt, M., & Sentenská, L. (2011): Ecology of Arachnida alien to Europe. BioControl, 56(4), 539-550.

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Submitted manuscripts:

Sentenská, L., Müller, H. G. C., Pekár, S., & Uhl, G. Male copulatory organ of spiders is not a numb structure as evidenced by presence of neurons and a sensory organ. Scientific Reports. Sentenská, L., Pekár, S., & Uhl, G. How spider females control deposition and removal of mating plugs. Animal Behaviour. Sentenská, L., Graber, F., Richard, M., & Kropf, C. Sexual dimorphism in venom gland morphology in a sexually stinging scorpion. Biological Journal of the Linnean Society.

Publications aimed at a wider public:

Sentenská, L., & Líznarová, E. (2015): Samci vracejí úder: sexuální kanibalismus naruby/Males strike back: Sexual cannibalism inside out. Živa 5/2015, 259-260.

Sentenská, L., & Líznarová, E. (2010): Nový řád pavoukovců pro faunu České republiky/New arachnid order found in Czech Republic. Živa 3/2010, 126-127.

9. CONFERENCE CONTRIBUTION International:

Sentenská, L., Pekár, S., & Uhl, G. (2016). Are males really so insensitive? Palp morphology of the spider Philodromus cespitum reveals innervation. 20th International Congress of Arachnology, Golden, Colorado (oral presentation).

Sentenská, L., Graber, F., Richards, M, & Kropf, C. (2016). Sexual dimorphism in venom gland anatomy in a sexually stinging scorpion. 20th International Congress of Arachnology, Golden, Colorado (poster).

Sentenská, L., Pekár, S., & Uhl, G. (2015). Efficacy of mate plugging is a result of an interplay between male and female behaviour in Philodromus cespitum (Philodromidae). 29th European Congress of Arachnology, Brno, Czech Republic (oral presentation).

Sentenská, L., Lasut, L., Nentwig, W., Pekár, S., & Kropf, C. (2014). Indication of postmating isolation between two sibling species of spiders. 28th the European Congress of Arachnology, Torino, Italy (oral presentation).

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Sentenská, L., Graber, F., & Kropf, C. (2014). Male and female venom glands in a sexually stinging scorpion (Arachnida, Scorpiones). 28th the European Congress of Arachnology, Torino, Italy (poster).

Sentenská L., Uhl G., Lipke E., Michalik P., & Pekár S. (2013). Mating plugs in the spider Micaria sociabilis (Gnaphosidae): origin, efficacy and female control. 19th International Congress of Arachnology, Kenting National Park, Taiwan (oral presentation).

Sentenská, L., Cardénas, M., & Pekár, S. (2013). Kairomone use for microhabitat search in an ant-mimicking spider. 19th International Congress of Arachnology, Kenting National Park, Taiwan (poster).

Sentenská, L., Cardénas, M., & Pekár, S. (2012). Kairomone use for microhabitat search in an ant-mimicking spider. ASAB Winter Meeting, London (poster).

Sentenská, L., & Pekár, S. (2012). Mate young, kill old: reversed sexual cannibalism and male mate choice in spiders. 27th European Congress of Arachnology, Ljubljana, Slovenia (oral presentation).

Sentenská, L., & Pekár, S. (2011). Factors affecting frequency of reversed sexual cannibalism in Micaria sociabilis. 26th European Congress of Arachnology, Sede Boqer, Israel (oral presentation).

Domestic:

Sentenská, L., Müller, C. H. G., Pekár, S., & Uhl, G. (2017). Jsou samci pavouků opravdu tak necitliví? Smyslový organ nalezen v kopulačním orgánu pavouka. Zoologické Dny 2017, Brno (oral presentation).

Sentenská, L., Cardénas, M., & Pekár, S. (2013). Kairomone use for microhabitat search in an ant-mimicking spider. Zoologické Dny 2017, Brno (poster).

Sentenská, L., & Pekár, S. (2011). Reversní sexuální kanibalismus u druhu Micaria sociabilis. Zoologické dny 2011, Brno (oral presentation).

Sentenská, L., Líznarová, E., & Pekár, S. (2011). Lokální specializace a kondiční strategie v lovu kořisti u pavouka Oecobius navus. Konference České společnosti pro ekologii, Kostelec nad Černými lesy (poster).

Sentenská, L., & Pekár, S. (2010). Reversní sexuální kanibalismus u druhu Micaria sociabilis. Ethological Congress, Smolenice (oral presentation). 199