Periodic Reporting for period 1 - Widow Spider Mating (Immature mating as a novel tactic of an invasive widow spider) Reporting period: 2020-02-10 to 2022-02-09 Summary of the context and overall objectives of the project Mating with immature females was previously considered impossible, as by definition only adult individuals were regarded sexually mature, possessing developed reproductive organs enabling them to copulate and reproduce. Nevertheless, in several widow species (genus Latrodectus) immature (subadult) females engage in copulation and, once reaching maturity, produce viable offspring. Although widow spider females are notoriously known for their cannibalistic tendencies, males are not attacked by subadult females. Apart from lack of cannibalism, immature mating is characterized by much-reduced courtship and, due to the lack of cannibalism, a higher number of insertions during copulation. However, other consequences of immature mating are not known. Within this project, we aim to evaluate the costs and benefits of immature mating in the brown widow spider L. geometricus. This project mainly focuses on basic, fundamental research of behavioural and morphological aspects of immature mating. Considering the invasive nature and medical importance of the widow spiders, my investigation of this flexible mating system may help to lay groundwork for developing a non-toxic pest control tool based on mate attraction in future. Previous studies indicate male preference for mating with adult females. Within this project, we examine the male mate choice into more depth. We also explore the underlying behavioural and morphological mechanisms and its impact on male and female reproductive success in comparison to suicidal mating with adult females to identify trade-offs of both mating tactics and to help us understand how they evolved. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The outgoing phase was realized in the lab of Prof. Maydianne Andrade at the University of Toronto. Prof. Andrade is a world expert on mating in widow spiders. Her expertise along with her lab and rearing facilities allowed me to conduct several behavioural experiments. First, we examined male mate choice in L. geometricus based on contact cues in female silk. Previous studies showed male preference for adults based on airborne pheromones. Male spiders typically locate females through airborne pheromones, but gain more specific information only upon contact with the female and her silk. Therefore, we asked whether the preference of adults is maintained after males reach female’s webs. To investigate male mate choice based on contact cues, we exposed males to silk of adults and subadults simultaneously. The experiment revealed that males prefer adults also based on silk-borne contact cues. Further, we examined courtship behaviours towards adults and subadults and cues triggering them. Previous studies report that males court adult females longer than subadults using more extensive repertoire of behaviours towards adults. To determine which types of cues trigger different courtship components, we swapped adults and subadults between webs. We showed that contact with webs and bodies of adults triggers majority of the behaviours, only vibrational signals occur regardless of the female developmental stage. Despite previous observations, in our experiments, subadults were less likely to mate than adults, potentially explaining why males prefer mating with adult females. This study is in press in one of the top-ranked journals in behavioural ecology (Sentenská et al. 2022).Another set of experiments explored paternity assurance in form of plugging in L. geometricus. Males of widow spider break off the tips of their paired copulatory organs, thereby plugging the female genitalia and hindering female re-mating. Although males mating with subadults typically achieve two insertion and so potentially block both of female paired sperm storage organ (spermathecae), the subadult genitalia are likely not yet hardened enough to hold the plugs. The plug placement has been investigated via examination of genitalia of mated males and females. Contrary to our expectations, the plugging success was higher in subadults as mating plugs were found in both of their spermathecae with higher probability compared to adults (Sentenská et al. 2021). A potential cost of immature mating for males can be higher re-mating rates of subadults, a behaviour that would decrease paternity of the first male to mate. Males typically engage in self-sacrificial behaviour when mating with adults as they somersault into their mouthparts during copulation, triggering female cannibalism. In the congener L. hasselti, this behaviour increases copulation duration and lowers re-mating probability of mated females. Since males do not sacrifice themselves to subadults, we expected their re-mating rate to be significantly higher than for adults experiencing self-sacrifice. The experiments exploring function of somersaulting in L. geometricus and the effect of the absence of this behaviour on subadult mating and re-mating are now finished and are currently being analysed. The final experiment focuses on paternity in double-mated females differing in the timing of their first mating (adult vs. subadult stage) using sterile male technique. We test the hypothesis that males mating with subadult females have lower paternity than expected for the first male. This may occur due to unfavourable sperm storage conditions in the immature spermathecae which may lead to lower competitive ability of the first male‘s sperm relative to the second male's sperm. We sterilized some males by irradiating them and paired these males with virgin and previously-mated females. Examination of egg sacs produced by double-mated females will reveal the paternity shares of both males since irradiated sperm fertilize eggs, but those eggs fail to develop. The experiments are now finished and we are currently assessing offspring production. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Our investigation so far revealed that brown widow males prefer to approach and court adult over subadult females based on contact cues. The shorter courtship and the lack of self-sacrifice during immature mating were previously considered to be benefits a male experiences when mating with subadults and therefore a preference of subadult females was expected. However, they rather indicate a lower investment into immature mating and further support the consistent male preference of adult females. This preference may be driven by the greater likelihood of mating success with adults and their readiness for oviposition, which likely outweigh the apparent energy and fitness costs of a long courtship ending with cannibalism. Our ongoing investigation will answer whether male preference for adult females arises from higher re-mating rates of subadults or lower paternity gained from mating with them. After finishing the outgoing phase in Prof. Andrade’s lab, the project will move into return phase realized at the University of Greifswald in the lab of Prof. Gabriele Uhl. Prof. Uhl studies aspects of mating systems using the combination of behavioural and morphological approaches. Her lab is equipped by state-of-the-art equipment for advanced morphological studies, which will be used in order to elucidate the mechanism of the immature mating in comparison to male suicidal tactic. This work will complement the behavioural observation conducted during the outgoing phase and will help us to better understand this unusual mating tactic in spider species notoriously known to public for its potent neurotoxic venom and dramatic mating ritual. Male of the brown widow spider mating with an immature female
