Micro-Computed Tomography Reveals a Remarkable Twin Intromittent Organ in Spiders – A Novelty for Arachnids With Direct Sperm Transfer

The modification of male pedipalps into secondary sexual intromittent organs is one of the hallmark characteristics of spiders, yet understanding the development and evolution of male genitalia across the order remains a challenging prospect. The embolus – the sclerite bearing the efferent spermatic duct or spermophor, and used to deliver sperm directly to the female genitalia during copulation – has always been considered the single unambiguously homologous palpal sclerite shared by all spider species, fundamental to the bauplan of the order and to the evolution and functional morphology of spider reproductive systems. Indeed, after two centuries of comparative research on spider reproduction, the presence of a single spermophor and embolus on each of a male spider’s two pedipalps remains a central tenet of evolutionary arachnology. Our findings challenge this premise, and reveal a remarkable twin intromittent organ sperm transfer system in a lineage of Australian palpimanoid spiders, characterized by a bifurcate spermophor and the presence of two efferent ducts leading to a pair of embolic sclerites on each pedipalp. This is the first time such a remarkable conformation has been observed in any group of arachnids with direct sperm transfer, complicating our understanding of palpal sclerite homologies, and challenging ideas about the evolution of spider genitalia.

Size, shape, and direction matters: Matching secondary genital structures in male and female mites using multiple microscopy techniques and 3D modeling

Studies of female genital structures have generally lagged behind comparable studies of male genitalia, in part because of an assumption of a lower level of variability, but also because internal genitalia are much more difficult to study. Using multiple microscopy techniques, including video stereomicroscopy, fluorescence microscopy, low-temperature scanning electron microscopy (LT-SEM), and confocal laser scanning microscopy (CLSM) we examined whether the complex sperm transfer structures in males of Megalolaelaps colossus (Acari: Mesostigmata) are matched by similarly complex internal structures in the female. While both LT-SEM and CLSM are well suited for obtaining high-quality surface images, CLSM also proved to be a valuable technique for observing internal anatomical structures. The long and coiled sperm transfer organ on the chelicera of the males (spermatodactyl) largely matches an equally complex, but internal, spiral structure in the females in shape, size, and direction. This result strongly suggests some form of genital coevolution. A hypothesis of sexual conflict appears to provide the best fit for all available data (morphology and life history).

Mating duration and prolonged post-copulatory associations in Arrenurus water mites (Actinotrichida: Parasitengonina: Hydrachnidiae)

Conflicts of interest between the sexes over control of mating can be reflected in various aspects of morphology and behaviour, including structure of genitalia and copulation duration. In Arrenurus water mites (Hydrachnidia: Arrenuridae) there are two main patterns of sperm transfer that differ in degree of potential male control of sperm uptake by females. In some species, males are equipped with an intromittent organ (the "petiole") that is used to forcefully insert sperm into the female reproductive tract. In others, males lack an intromittent organ and females appear to push sperm into their reproductive opening themselves. Theory suggests that the amount of time spent in courtship after sperm transfer should differ between males with and without an intromittent structure. We predicted that male Arrenurus able to push sperm into the female's reproductive tract (petiolate males) should spend less time courting females after transferring sperm than apetiolate males, which may have to "convince" females to take up their sperm. Here, we examined durations of mating for 10 species of Arrenurus with males that differ in genital morphology: six species with males equipped with a well developed petiole (= "petiolate" species) and four species with males that either completely lack a petiole or have a minute peg-like petiole that does not appear to function as an intromittent organ (= "apetiolate" species). We tested whether males of petiolate species spend less time in the stage of courtship that takes place after sperm transfer (= "post-transfer courtship") than apetiolate males. In contrast to our prediction, we found that species with well developed petioles spent significantly more time in post-transfer behaviours than species lacking petioles. The possible function of protracted post-transfer courtship in the genus Arrenurus is discussed.

Functional morphology of immature mating in a widow spider

Background Mating generally occurs after individuals reach adulthood. In many arthropods including spiders, the adult stage is marked by a final moult after which the genitalia are fully developed and functional. In several widow spider species (genus Latrodectus), however, immature females may mate a few days before they moult to adulthood, i.e. in their late-subadult stage. While the “adult” mating typically results in cannibalism, males survive the “immature” mating. During both “immature” and “adult” matings, males leave parts of their paired copulatory organs within female genitalia, which may act as mating plugs. To study potential costs and benefits of the two mating tactics, we investigated female genital morphology of the brown widow spider, L. geometricus. Light microscopy, histology and micro-computed tomography of early-subadult, late-subadult and adult females were conducted to determine the overall pattern of genital maturation. We compared genitalia of mated late-subadult and adult females to reveal potential differences in the genitalic details that might indicate differential success in sperm transfer and different environments for sperm storage and sperm competition. Results We found that the paired sperm storage organs (spermathecae) and copulatory ducts are developed already in late-subadult females and host sperm after immature mating. However, the thickness of the spermathecal cuticle and the staining of the secretions inside differ significantly between the late-subadult and adult females. In late-subadult females mating plugs were found with higher probability in both spermathecae compared to adult females. Conclusions Sperm transfer in matings with late-subadult females follows the same route as in matings with adult females. The observed differences in the secretions inside the spermathecae of adult and late-subadult females likely reflect different storage conditions for the transferred sperm which may lead to a disadvantage under sperm competition if the subadult female later re-mates with another male. However, since males mating with late-subadult females typically transfer sperm to both spermathecae they might benefit from numerical sperm competition as well as from monopolizing access to the female sperm storage organs. The assessment of re-mating probability and relative paternity will clarify the costs and benefits of the two mating tactics in light of these findings.

A Novel Body Plan Alters Diversification of Body Shape and Genitalia in Live-Bearing Fish

Major evolutionary innovations can greatly influence subsequent evolution. While many major transitions occurred in the deep past, male live-bearing fishes (family Poeciliidae) more recently evolved a novel body plan. This group possesses a three-region axial skeleton, with one region—the ano-urogenital region—representing a unique body region accommodating male genitalic structures (gonopodial complex). Here we evaluate several hypotheses for the evolution of diversity in this region and examine its role in the evolution of male body shape. Examining Gambusia fishes, we tested a priori predictions for (1) joint influence of gonopodial-complex traits on mating performance, (2) correlated evolution of gonopodial-complex traits at macro- and microevolutionary scales, and (3) predator-driven evolution of gonopodial-complex traits in a post-Pleistocene radiation of Bahamas mosquitofish. We found the length of the sperm-transfer organ (gonopodium) and its placement along the body (gonopodial anterior transposition) jointly influenced mating success, with correlational selection favoring particular trait combinations. Despite these two traits functionally interacting during mating, we found no evidence for their correlated evolution at macro- or microevolutionary scales. In contrast, we did uncover correlated evolution of modified vertebral hemal spines (part of the novel body region) and gonopodial anterior transposition at both evolutionary scales, matching predictions of developmental connections between these components. Developmental linkages in the ano-urogenital region apparently play key roles in evolutionary trajectories, but multiple selective agents likely act on gonopodium length and cause less predictable evolution. Within Bahamas mosquitofish, evolution of hemal-spine morphology, and gonopodial anterior transposition across predation regimes was quite predictable, with populations evolving under high predation risk showing more modified hemal spines with greater modifications and a more anteriorly positioned gonopodium. These changes in the ano-urogenital vertebral region have facilitated adaptive divergence in swimming abilities and body shape between predation regimes. Gonopodium surface area, but not length, evolved as predicted in Bahamas mosquitofish, consistent with a previously suggested tradeoff between natural and sexual selection on gonopodium size. These results provide insight into how restructured body plans offer novel evolutionary solutions. Here, a novel body region—originally evolved to aid sperm transfer—was apparently co-opted to alter whole-organism performance, facilitating phenotypic diversification.

Mating behavior and spermatophore morphology of the whip scorpion Typopeltis dalyi Pocock, 1900 (Uropygi, Thelyphonida)

Whip scorpions are an enigmatic group of terrestrial raptorial arachnids that show remarkable mating and courtship behavior in which the male forms a complex spermatophore. While whip spiders (Amblypygi) are relatively well-studied, whip scorpions (Uropygi) are poorly known. The two orders form the Pedipalpi, whip scorpions (Uropygi include Thelyphonida and Schizomida) and whip spiders (Amblypygi). Two major groups have been described based on the mode of sperm transfer that differ in the duration and mode of the typical female–male tandem mating dance. Because comprehensive studies are lacking, in this study we add to our knowledge of the reproductive biology of whip scorpions by analyzing the mating behavior and spermatophore morphology of the previously unstudied species Typopeltis dalyi Pocock, 1900. Our observations show that this species belongs to the second group and supports the hypothesis of P. Weygoldt that their mode of sperm transfer appears more effective than that of the first group and that sufficient sperm can be supplied with one mating. The mating behavior and spermatophore morphology in T. dalyi are similar to those of closely related species and add additional characters applicable for species classification and phylogenetic inferences.

Baffling: A cheater strategy using self-made tools in tree crickets

Intense sexual selection in the form of mate choice can facilitate the evolution of different alternative reproductive strategies. These strategies can be condition-dependent, wherein genetically similar individuals express the strategy based on their condition. Our study shows that baffling, a mate attraction strategy using self-made acoustic amplifiers, employed by male tree crickets, is a classic example of a condition-dependent alternative strategy. We show that less preferred males, who are smaller or produce less loud calls, predominantly use this alternative strategy. Baffling allows these males to increase their call loudness and advertisement range, and attract a higher number of mates. Baffling also allows these males to deceive females into mating for longer durations with them. Our results suggest that the advantage of baffling in terms of sperm transfer is primarily limited to less preferred males, thus maintaining the polymorphism of calling strategies in the population.Impact statementThis study shows that less preferred tree cricket males use an alternative signaling strategy to call louder, thus attracting and mating with otherwise choosy females using deception.