Love bites: male frogs (Plectrohyla, Hylidae) use teeth scratching to deliver sodefrin precursor-like factors to females during amplexus.

BACKGROUND: Efficient transfer of chemical signals is important for successful mating in many animal species. Multiple evolutionary lineages of animals evolved direct sex pheromone transmission during traumatic mating-the wounding of the partner with specialized devices-which helps to avoid signal loss to the environment. Although such direct transmission modes of so-called allohormone pheromones are well-documented in invertebrates, they are considered rare in vertebrates. Males of several species of the frog genus Plectrohyla (Hylidae, Anura) have elongated teeth and develop swollen lips during the breeding season. Here we investigated the possibility that these structures are used to scratch the females' skin and apply allohormone pheromones during traumatic mating in several Plectrohyla species. RESULTS: Our behavioural observations revealed that males press their upper jaw onto the females' dorsum during amplexus, leaving small skin scratches with their teeth. Histological examinations of the males' lips identified specialized mucus glands, resembling known amphibian pheromone glands. Whole-transcriptome sequencing of these breeding glands showed high expression of sodefrin precursor-like factor (SPF) proteins, which are known to have a pheromone function in multiple amphibian species. CONCLUSIONS: Our study suggests SPF delivery via traumatic mating in several anuran species: the males have specialized breeding glands in the lips for production and secretion and use their elongated teeth as wounding devices for application. We hypothesize that these SPF proteins end up in the females' circulatory system, where understanding their exact function will require further molecular, physiological and behavioural testing.

A targeted in situ hybridization screen identifies putative seminal fluid proteins in a simultaneously hermaphroditic flatworm.

BACKGROUND: Along with sperm, in many taxa ejaculates also contain large numbers of seminal fluid proteins (SFPs). SFPs and sperm are transferred to the mating partner, where they are thought to play key roles in mediating post-mating sexual selection. They modulate the partner's behavior and physiology in ways that influence the reproductive success of both partners, thus potentially leading to sexual conflict. Despite the presumed general functional and evolutionary significance of SFPs, their identification and characterization has to date focused on just a few animal groups, predominantly insects and mammals. Moreover, until now seminal fluid profiling has mainly focused on species with separate sexes. Here we report a comprehensive screen for putative SFPs in the simultaneously hermaphroditic flatworm Macrostomum lignano. RESULTS: Based on existing transcriptomic data, we selected 150 transcripts known to be (a) predominantly expressed in the tail region of the worms, where the seminal fluid-producing prostate gland cells are located, and (b) differentially expressed in social environments differing in sperm competition level, strongly implying that they represent a phenotypically plastic aspect of male reproductive allocation in this species. For these SFP candidates, we then performed whole-mount in situ hybridization (ISH) experiments to characterize tissue-specific expression. In total, we identified 98 transcripts that exhibited prostate-specific expression, 76 of which we found to be expressed exclusively in the prostate gland cells; additional sites of expression for the remaining 22 included the testis or other gland cells. Bioinformatics analyses of the prostate-limited candidates revealed that at least 64 are predicted to be secretory proteins, making these especially strong candidates to be SFPs that are transferred during copulation. CONCLUSIONS: Our study represents a first comprehensive analysis using a combination of transcriptomic and ISH screen data to identify SFPs based on transcript expression in seminal fluid-producing tissues. We thereby extend the range of taxa for which seminal fluid has been characterized to a flatworm species with a sequenced genome and for which several methods such as antibody staining, transgenesis and RNA interference have been established. Our data provide a basis for testing the functional and evolutionary significance of SFPs.

Hidden female physiological resistance to male accessory gland substances in a simultaneous hermaphrodite.

To increase fertilization chances compared with rivals, males are favoured to transfer accessory gland proteins to females during mating. These substances, by influencing female physiology, cause alteration of her sperm usage and remating rate. Simultaneously hermaphroditic land snails with love-darts are a case in point. During courtship, a love-dart is pierced through the partner's body wall, thereby introducing accessory mucous gland products. This mucus physiologically increases paternity by inhibiting the digestion of donated sperm. The sperm, which are packaged in a spermatophore, are exchanged and received in an organ called the diverticulum. Because of its length, this organ was previously proposed to be a female anatomical adaptation that may limit the dart interference with the recipient's sperm usage. For reproductive success of the donor, an anatomically long spermatophore, relative to the partner's diverticulum, is beneficial as sperm can avoid digestion by exiting through the spermatophore's tail safely. However, the snail Eobania vermiculata possesses a diverticulum that is three times longer than the spermatophore it receives. Here, we report that the love-dart mucus of this species contains a contraction-inducing substance that shortens the diverticulum, an effect that is only properly revealed when the mucus is applied to another helicid species, Cornu aspersum This finding suggests that E. vermiculata may have evolved a physiological resistance to the manipulative substance received via the love-dart by becoming insensitive to it. This provides useful insight into the evolution of female resistance to male manipulations, indicating that it can remain hidden if tested on a single species.

On the effect specificity of accessory gland products transferred by the love-dart of land snails.

BACKGROUND: Sexual selection favours the evolution of male bioactive substances transferred during mating to enhance male reproductive success by affecting female physiology. These effects are mainly well documented for separate-sexed species. In simultaneous hermaphrodites, one of the most peculiar examples of transfer of such substances is via stabbing a so-called love-dart in land snails. This calcareous stylet delivers mucous products produced by accessory glands into the mate's haemolymph. In Cornu aspersum, this mucus temporarily causes two changes in the recipient. First, the spermatophore uptake into the spermatophore-receiving organ, called diverticulum, is probably favoured by contractions of this organ. Second, the amount of stored sperm increases by contractions of the copulatory canal, which close off the tract leading to the sperm digesting organ. However, it has yet to be determined whether these effects are similar across species, which would imply a common strategy of the dart in increasing male reproductive success. RESULTS: We performed a cross-reactivity test to compare the in vitro response of the diverticulum and copulatory canal of C. aspersum (Helicidae) to its own and other species' mucus (seven helicids and one bradybaenid). We found that the contractions in the diverticulum were only induced by dart mucus of certain species, while the copulatory canal responded equally to all but one species' mucus tested. In addition, we report a newly-discovered effect causing the shortening of the diverticulum, which is also only caused by dart mucus of certain species. The advantage seems to be a distance reduction to the sperm storage organ. CONCLUSIONS: All these findings are the first to shed light on the evolution of the different functions of accessory gland products in dart-bearing species. These functions may be achieved via common physiological changes caused by the substances contained in the dart mucus, since the responses evoked were similar across species' mucus. Moreover, while these substances can act similarly in separate-sexed species as in simultaneous hermaphrodites, differences may occur in their evolution between the two sexual systems.

Common effect of the mucus transferred during mating in two dart-shooting snail species from different families.

Several taxa of pulmonate land snails exhibit a conspicuous mating behaviour, the shooting of so-called love darts. During mating, such land snail species stab a mating partner with a mucus-coated dart. It has previously been shown that the sperm donor physiologically influences the sperm recipient via the mucus covering the dart and thereby decreases the number of sperm digested by the recipient. However, the generality of this effect of the dart's mucus is unclear, because almost all the previous studies on the effect of the mucus used the brown garden snail Cornu aspersum from the family Helicidae. Therefore, the relationship between the acquisition of the mucus effect on the recipient and the evolution of the dart itself, and its mucus, is still open to debate. To test the commonality of the physiological effect of the dart mucus, we examined this in Euhadra peliomphala, a species from the Bradybaenidae family, and compared our findings with the results of previous work using C. aspersum. Our experiments showed that in E. peliomphala, the dart mucus had a physiological effect and lowered the accessibility of the gametolytic organ, as found in C. aspersum. This indicates that in various dart-bearing species the mucus from the dart glands targets the same organ and that the inhibition of sperm digestion has played a crucial role in the evolution of the dart and its mucus.

High level of sperm competition may increase transfer of accessory gland products carried by the love dart of land snails.

Postcopulatory adaptations that increase reproductive success compared to rivals, like the transfer of accessory gland products that promote paternity, are common when sperm competition occurs among males. In land snails, the dart shooting behavior and its adaptive significance, in promoting individual fitness through enhanced paternity of the successful dart shooter, have been considered such an adaptation. The fitness result gained is mediated by the transfer of mucus components on the love dart capable of altering the physiology of the receiver's reproductive tract. In this context, dart shooting and mucus transfer could be considered as processes targeted by sexual selection. While the effect of dart mucus is beneficial for the dart user, so far it has remained unknown whether its transport is greater when snails experience a higher level of sperm competition. Here, we report results of a study on inter- and intraspecific variations of dart and mucus gland morphometry, considered to be traits reflecting the ability of snails to adjust the production and transfer of mucus under varying sperm competition scenarios. We investigated four populations with different densities from four dart-bearing species, Arianta arbustorum, Cepaea nemoralis, Cornu aspersum, and Helix lucorum. The results indicate that different adaptations of these traits occur among the studied species that all seem to achieve the same goal of transferring more mucus when sperm competition is higher. For example, the presence of longer and more branched mucous glands or an increase in dart surface most likely reflect increased mucus production and enhanced ability of mucus transport, respectively. Interestingly, the species for which the use of the dart is reported to be facultative, A. arbustorum, did not show any variation among the examined traits. To conclude, sexual selection in the form of sperm competition intensity seems to be an important selective force for these simultaneously hermaphroditic dart-bearing snails, driving differences in sexual traits.