Dynamics of seminal fluid production after mating.

Seminal fluid proteins (SFPs) play vital roles for optimizing reproductive success in diverse animals. Underlining their significance, SFP production and transfer are highly plastic, e.g., depending on the presence of rivals or mating status of partners. However, surprisingly little is known about replenishing SFPs after mating. This is especially relevant in species that mate multiple times, as they continuously produce and use SFPs throughout their reproductive life. Here we examined the expression pattern of SFP genes after mating in the great pond snail, Lymnaea stagnalis. Our results show that two out of the six SFP genes investigated here were upregulated 1 week after mating. Surprisingly, most SFP genes did not change their expression immediately after mating. Even after 1 week, when supposedly seminal fluid is fully replenished, the expression of SFP genes is rather high. In addition, the difference with previous studies hints at the possibility that SFP production after mating is plastic and depends on the mating history of female-acting snails. Our results shed light on unexplored aspects of SFP production, thereby expanding the understanding of reproductive strategies in animals.

Same same, but different: exploring the enigmatic role of the pituitary adenylate cyclase-activating polypeptide (PACAP) in invertebrate physiology.

Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.

Learning to anticipate mate presence shapes individual sex roles in the hermaphroditic pond snail, Lymnaea stagnalis.

Despite being simultaneously male and female, hermaphrodites may still need to assume the male or female sexual role in a mating encounter, with the option to swap roles afterwards. For the great pond snail, Lymnaea stagnalis, deciding which sexual role to perform has important consequences, since sperm transfer and male reproductive success can be decreased. We hypothesised that detecting cues that indicate a possible mating encounter could help them to adapt their mating behaviour. Therefore, we experimentally assessed whether signalling the presence of a conspecific with an odour can affect the sexual role of Lymnaea stagnalis. The results showed that learning resulted in either an increased ability to mate as a male or in faster mating compared to the control group. These findings reveal that learning shapes the mating dynamics of Lymnaea stagnalis, thus showing that cognitive processes not only affect mating in separate-sexed species but also in hermaphrodites.

Ion channel profiling of the Lymnaea stagnalis ganglia via transcriptome analysis.

BACKGROUND: The pond snail Lymnaea stagnalis (L. stagnalis) has been widely used as a model organism in neurobiology, ecotoxicology, and parasitology due to the relative simplicity of its central nervous system (CNS). However, its usefulness is restricted by a limited availability of transcriptome data. While sequence information for the L. stagnalis CNS transcripts has been obtained from EST libraries and a de novo RNA-seq assembly, the quality of these assemblies is limited by a combination of low coverage of EST libraries, the fragmented nature of de novo assemblies, and lack of reference genome. RESULTS: In this study, taking advantage of the recent availability of a preliminary L. stagnalis genome, we generated an RNA-seq library from the adult L. stagnalis CNS, using a combination of genome-guided and de novo assembly programs to identify 17,832 protein-coding L. stagnalis transcripts. We combined our library with existing resources to produce a transcript set with greater sequence length, completeness, and diversity than previously available ones. Using our assembly and functional domain analysis, we profiled L. stagnalis CNS transcripts encoding ion channels and ionotropic receptors, which are key proteins for CNS function, and compared their sequences to other vertebrate and invertebrate model organisms. Interestingly, L. stagnalis transcripts encoding numerous putative Ca2+ channels showed the most sequence similarity to those of Mus musculus, Danio rerio, Xenopus tropicalis, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that many calcium channel-related signaling pathways may be evolutionarily conserved. CONCLUSIONS: Our study provides the most thorough characterization to date of the L. stagnalis transcriptome and provides insights into differences between vertebrates and invertebrates in CNS transcript diversity, according to function and protein class. Furthermore, this study provides a complete characterization of the ion channels of Lymnaea stagnalis, opening new avenues for future research on fundamental neurobiological processes in this model system.

Divergence of seminal fluid gene expression and function among natural snail populations.

Seminal fluid proteins (SFPs) can trigger drastic changes in mating partners, mediating post-mating sexual selection and associated sexual conflict. Also, cross-species comparisons have demonstrated that SFPs evolve rapidly and hint that post-mating sexual selection drives their rapid evolution. In principle, this pattern should be detectable within species as rapid among-population divergence in SFP expression and function. However, given the multiple other factors that could vary among populations, isolating divergence in SFP-mediated effects is not straightforward. Here, we attempted to address this gap by combining the power of a common garden design with functional assays involving artificial injection of SFPs in the simultaneously hermaphroditic freshwater snail, Lymnaea stagnalis. We detected among-population divergence in SFP gene expression, suggesting that seminal fluid composition differs among four populations collected in Western Europe. Furthermore, by artificially injecting seminal fluid extracted from these field-derived snails into standardized mating partners, we also detected among-population divergence in the strength of post-mating effects induced by seminal fluid. Both egg production and subsequent sperm transfer of partners differed depending on the population origin of seminal fluid, with the response in egg production seemingly closely corresponding to among-population divergence in SFP gene expression. Our results thus lend strong intraspecific support to the notion that SFP expression and function evolve rapidly, and confirm L. stagnalis as an amenable system for studying processes driving SFP evolution.

Identification, presence, and possible multifunctional regulatory role of invertebrate gonadotropin-releasing hormone/corazonin molecule in the great pond snail (Lymnaea stagnalis).

In the last years, our interpretation of the origin and function of the gonadotropin-releasing hormone (GnRH) neuropeptide superfamily has changed substantially. A main driver for these conceptual changes came from increased investigations into functions and evolutionary lineage of previously identified molluscan GnRH molecules. Emerging evidence suggests not only reproductive, but also diverse biological effects of these molecules and proposes they should most likely be called corazonin (CRZ). Clearly, a more global understanding requires further exploration of species-specific functions and structure of invGnRH/CRZ peptides. Towards this goal, we have identified the full-length cDNA of invGnRH/CRZ peptide in an invertebrate model species, the great pond snail Lymnaea stagnalis, termed ly-GnRH/CRZ, and characterized the transcript and peptide distribution in the central nervous system (CNS) and peripheral organs. Our results are consistent with previous data that molluscan GnRHs are more related to CRZs and serve diverse functions. Hence, our findings support the notion that peptides originally termed molluscan GnRH are multifunctional modulators and that nomenclature change should be taken into consideration.

A "Love" Dart Allohormone Identified in the Mucous Glands of Hermaphroditic Land Snails.

Animals have evolved many ways to enhance their own reproductive success. One bizarre sexual ritual is the "love" dart shooting of helicid snails, which has courted many theories regarding its precise function. Acting as a hypodermic needle, the dart transfers an allohormone that increases paternity success. Its precise physiological mechanism of action within the recipient snail is to close off the entrance to the sperm digestion organ via a contraction of the copulatory canal, thereby delaying the digestion of most donated sperm. In this study, we used the common garden snailCornu aspersumto identify the allohormone that is responsible for this physiological change in the female system of this simultaneous hermaphrodite. The love dart allohormone (LDA) was isolated from extracts derived from mucous glands that coat the dart before it is stabbed through the partner's body wall. We isolated LDA from extracts using bioassay-guided contractility measurement of the copulatory canal. LDA is encoded within a 235-amino acid precursor protein containing multiple cleavage sites that, when cleaved, releases multiple bioactive peptides. Synthetic LDA also stimulated copulatory canal contractility. Combined with our finding that the protein amino acid sequence resembles previously described molluscan buccalin precursors, this indicates that LDA is partially conserved in helicid snails and less in other molluscan species. In summary, our study provides the full identification of an allohormone that is hypodermically injected via a love dart. More importantly, our findings have important consequences for understanding reproductive biology and the evolution of alternative reproductive strategies.

Sex determination and gender expression: Reproductive investment in snails.

Sex determination is generally seen as an issue of importance for separate-sexed organisms; however, when considering other sexual systems, such as hermaphroditism, sex allocation is a less-binary form of sex determination. As illustrated here, with examples from molluscs, this different vantage point can offer important evolutionary insights. After all, males and females produce only one type of gamete, whereas hermaphrodites produce both. In addition, sperm and accessory gland products are donated bidirectionally. For reciprocal mating, this is obvious since sperm are exchanged within one mating interaction; but even unilaterally mating species end up mating in both sexual roles, albeit not simultaneously. With this in mind, I highlight two factors that play an important role in how reproductive investment is divided in snails: First, the individual's motivation to preferentially donate rather than receive sperm (or vice versa) leads to flexible behavioral performance, and thereby investment, of either sex. Second, due to the presence of both sexual roles within the same individual, partners are potentially able to influence investment in both sexual functions of their partner to their own benefit. The latter has already led to novel insights into how accessory gland products may evolve. Moreover, the current evidence points towards different ways in which allocation to reproduction can be changed in simultaneous hermaphrodites. These often differ from the separate-sexed situation, highlighting that comparison across different sexual systems may help identify commonalities and differences in physiological, and molecular mechanisms as well as evolutionary patterns. Mol. Reprod. Dev. 84: 132-143, 2017. © 2016 Wiley Periodicals, Inc.

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.

Alternative delivery of male accessory gland products.

To increase fertilization success, males transfer accessory gland products (Acps). Several species have evolved unconventional Acps transfer modes, meaning that Acps are transferred separately from the sperm. By surveying the sperm-free Acps transfer cases, we show that these animals have evolved a common strategy to deliver Acps: they all inject Acps directly through the partner's body wall into the hemolymph. Our review of this mode of Acps transfer reveals another striking similarity: they all transfer sperm in packages or via the skin, which may leave little room for Acps transfer via the conventional route in seminal fluid. We synthesise the knowledge about the function, and the effects in the recipients, of the Acps found in the widely diverse taxa (including earthworms, sea slugs, terrestrial snails, scorpions and salamanders) that inject these substances. Despite the clearly independent evolution of the injection devices, these animals have evolved a common alternative strategy to get their partners to accept and/or use their sperm. Most importantly, the evolution of the injection devices for the delivery of Acps highlights how the latter are pivotal for male reproductive success and, hence, strongly influence sexual selection.

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.

Emerging issues in the evolution of animal nuptial gifts.

Uniquely positioned at the intersection of sexual selection, nutritional ecology and life-history theory, nuptial gifts are widespread and diverse. Despite extensive empirical study, we still have only a rudimentary understanding of gift evolution because we lack a unified conceptual framework for considering these traits. In this opinion piece, we tackle several issues that we believe have substantively hindered progress in this area. Here, we: (i) present a comprehensive definition and classification scheme for nuptial gifts (including those transferred by simultaneous hermaphrodites), (ii) outline evolutionary predictions for different gift types, and (iii) highlight some research directions to help facilitate progress in this field.

Sublethal heat reduces overall reproductive investment and male allocation in a simultaneously hermaphroditic snail species.

The exposure to sublethally high temperature reduces reproductive performance in diverse organisms. Although this effect has been particularly emphasized for males or male reproductive functioning, it remains largely unknown whether the effect of heat on fertility is sex-specific. Here we examined the impact of sublethally high temperature on male and female functions in a simultaneously hermaphroditic snail species, Lymnaea stagnalis. Examining hermaphrodites is useful to evaluate the sex-specific impacts of heat exposure, since they possess male and female functions within a single individual, sharing genetic and environmental factors. Moreover, previously developed sex allocation theory allows us to compare the differential performance of sex functions. In this study, we exposed snails to 20°C (control), 24°C and 28°C for 14 days and assessed their egg and sperm production, sperm transfer, mating behaviour and growth. Both types of gamete production were significantly reduced by higher temperature, leading to an overall reduction of reproductive investment. By quantifying sex allocation, we furthermore revealed that the heat-stressed snails reduced the relative investment in their male function. This study illustrates that examining simultaneous hermaphrodites can provide significant insights for the impact of heat, and the proximate mechanism, on reproduction in diverse organisms.

Sensitivity of isolated eggs of pond snails: a new method for toxicity assays and risk assessment.

The concentration of heavy metals in the environment is normally low. We here address whether using the development of isolated pond snail Radix auricularia eggs would provide a more sensitive endpoint and whether the gelatinous matrix of the egg mass surrounding the eggs indeed protects the snail embryos. In the present study, artificial removal of the gelatinous matrix of egg masses greatly increased the sensitivity of developing eggs to a heavy metal (cadmium). The sensitivity of isolated eggs to cadmium was determined using several convenient endpoints, including mortality, hatching rate, and heart rate, with an acute toxicity test and a subchronic test. In the acute toxicity test, a 96-h LC(50) value of 58.26 µg/L cadmium was determined. In the subchronic toxicity test, sublethal effects in terms of a significant reduction in hatching rate could be found in the 25-µg/L treatment, and a significant decrease of heart rate was observed in both treatments (5 and 25 µg/L). The high sensitivity of isolated eggs indicates that such tests can be efficient for toxicity assays and risk assessment, although one needs to keep in mind that the ecologically relevant measure of toxicity will be how eggs are affected when they are still inside the egg mass.

Dominant gingers - Discovery and inheritance of a new shell polymorphism in the great pond snail Lymnaea stagnalis.

Color polymorphism is a classic study system for evolutionary genetics. One of the most color-polymorphic animal taxa is mollusks, but the investigation of the genetic basis of color determination is often hindered by their life history and the limited availability of genetic resources. Here, we report on the discovery of shell color polymorphism in a much-used model species, the great pond snail Lymnaea stagnalis. While their shell is usually beige, some individuals from a Greek population show a distinct red shell color, which we nicknamed Ginger. Moreover, we found that the inheritance fits simple, single-locus Mendelian inheritance with dominance of the Ginger allele. We also compared crucial life-history traits between Ginger and wild-type individuals, and found no differences between morphs. We conclude that the relative simplicity of this polymorphism will provide new opportunities for a deeper understanding of the genetic basis of shell color polymorphism and its evolutionary origin.

Socially transferred materials: why and how to study them.

When biological material is transferred from one individual's body to another, as in ejaculate, eggs, and milk, secondary donor-produced molecules are often transferred along with the main cargo, and influence the physiology and fitness of the receiver. Both social and solitary animals exhibit such social transfers at certain life stages. The secondary, bioactive, and transfer-supporting components in socially transferred materials have evolved convergently to the point where they are used in applications across taxa and type of transfer. The composition of these materials is typically highly dynamic and context dependent, and their components drive the physiological and behavioral evolution of many taxa. Our establishment of the concept of socially transferred materials unifies this multidisciplinary topic and will benefit both theory and applications.

Cognition and Its Shaping Effect on Sexual Conflict: Integrating Biology and Psychology.

While genetic variation is of crucial importance for organisms to be able to adapt to their ever-changing environments over generations, cognitive processes can serve the same purpose by acting at shorter time scales. Cognition, and its resulting behaviour, allows animals to display flexible, fast and reversible responses that, without implying a genetic change, are crucial for adaptation and survival. In the research field on sexual conflict, where studies focus on male and female mating strategies that increase the individual's reproductive fitness while forcing a cost on the partner, the role that cognition may play in how such strategies can be optimised has been widely overlooked. However, a careful analysis of behavioural studies shows that animals can develop and change their responses depending on what they perceive as well as on what they can predict from their experience, which can be of prime importance for optimising their reproductive fitness. As will be reviewed here, largely psychological processes, such as perception, memory, learning and decision-making, can not only modulate sexual conflict, but can also have a big impact on the reproductive success of a given individual. This review highlights the need for a more integrative view of sexual conflict where cognitive processes are also considered as a fundamental part of an animal's adaptive mating response.

Consequences of artificial light at night on behavior, reproduction, and development of Lymnaea stagnalis.

Light is an important zeitgeber that regulates many behavioral and physiological processes in animals. These processes may become disturbed due to the changes in natural patterns of light and dark via the introduction of artificial light at night (ALAN). The present study was designed to determine the effect of possible consequences of ALAN on reproduction, hatching success, developmental success, growth rate, feeding rate, mortality rate, and locomotor activity of the simultaneous hermaphrodite pond snail Lymnaea stagnalis. Snails were exposed to different light intensities at night that simulate actual ALAN measurements from the snail's night environment. The data revealed that exposure to ALAN at a low level significantly affected the cumulative number of laid eggs. At the same time, snails exposed to ALAN laid smaller eggs than those laid under normal light-dark cycles. Additionally, high light-intensity of ALAN delayed development and hatching of eggs of L. stagnalis while it showed no effect on hatching percentage. Furthermore, ALAN increased both the feeding and growth rates but did not lead to mortality. The results also show that snails exposed to dark conditions at night travel longer distances and do so faster than those exposed to ALAN. In light of these findings, it is clear that ALAN may have an influence on snails and their abundance in an environment, possibly disturbing ecological stability.

Slowly seeing the light: an integrative review on ecological light pollution as a potential threat for mollusks.

Seasonal changes in the natural light condition play a pivotal role in the regulation of many biological processes in organisms. Disruption of this natural condition via the growing loss of darkness as a result of anthropogenic light pollution has been linked to species-wide shifts in behavioral and physiological traits. This review starts with a brief overview of the definition of light pollution and the most recent insights into the perception of light. We then go on to review the evidence for some adverse effects of ecological light pollution on different groups of animals and will focus on mollusks. Taken together, the available evidence suggests a critical role for light pollution as a recent, growing threat to the regulation of various biological processes in these animals, with the potential to disrupt ecosystem stability. The latter indicates that ecological light pollution is an environmental threat that needs to be taken seriously and requires further research attention.