LOSP: A putative marker of parasperm lineage in male reproductive system of the prosobranch mollusk Littorina obtusata.

Reproductive isolation is the key attribute of biological species and establishment of the reproductive barriers is an essential event for speciation. Among the mechanisms of reproductive isolation, gamete incompatibility due to the variability of gamete interaction proteins may drive fast divergence even in sympatry. However, the number of available models to study this phenomenon is limited. In case of internally fertilized invertebrates, models to study gamete incompatibility and sperm competition mechanisms are restricted to a single taxon: insects. Here, we propose a group of closely related Littorina species as a new model for such studies. Particularly since periwinkles are already thoroughly studied in terms of morphology, physiology, ecology, phylogeny, and ecological speciation. Earlier, we have identified the first species-specific Littorina sperm protein (LOSP) with no known conservative domains or homologies. LOSP is relatively abundant component of sperm extracts and might be involved in gamete incompatibility. Here, we characterize its definitive localization and mRNA expression pattern in the male reproductive system by immunocytochemistry and RNA in situ hybridization. We demonstrate that LOSP distribution is limited to the parasperm cells. Losp gene expression occurs only at the early stages of parasperm development. The protein is stored within granules of mature parasperm and, most likely, is released after ejaculation inside female reproductive system. Thus, LOSP is the only described molluscan paraspermal protein to date, and there is a possibility for LOSP to be involved in gamete incompatibility since heterospermy is a common phenomenon among Littorina.

Species-Specific Proteins in the Oviducts of Snail Sibling Species: Proteotranscriptomic Study of Littorina fabalis and L. obtusata.

Genus Littorina subgenus Neritrema (Mollusca, Caenogastropoda) includes the "obtusata" group of closely related species (Littorina obtusata and L. fabalis). The anatomy of the adult reproductive system (pallial oviduct) is the only reliable feature used for species identification in females of these species. Reproductive system anatomy and reproduction-associated proteins often diverge between sibling species. Despite being of high evolutionary interest, the molecular basis of this divergence remains poorly understood. We performed proteotranscriptomic comparison of oviducts of L. obtusata and L. fabalis by RNA-seq on Illumina HiSeq 2500 and two-dimensional protein electrophoresis (2D DIGE) with MS/MS identification of the species-specific proteins. The interspecies differences in the oviduct were associated with (1) metabolic proteins reflecting overall physiological differences between L. obtusata and L. fabalis, (2) receptor proteins, and (3) transcripts related to transposable elements (TEs). Various receptors identified may recognize a wide variety of ligands from pathogen-associated molecular patterns to specific carbohydrates on the sperm surface. Therefore, these may participate in immune defense as well as in sperm storage and regulation. Species-specificity of multiple TE sequences (coding for reverse transcriptase and ribonuclease H) may indicate the important role of these genomic elements in the Littorina species divergence, which has not been reported previously.

Premating barriers in young sympatric snail species.

Sympatric coexistence of recently diverged species raises the question of barriers restricting the gene flow between them. Reproductive isolation may be implemented at several levels, and the weakening of some, e.g. premating, barriers may require the strengthening of the others, e.g. postcopulatory ones. We analysed mating patterns and shell size of mates in recently diverged closely related species of the subgenus Littorina Neritrema (Littorinidae, Caenogastropoda) in order to assess the role of premating reproductive barriers between them. We compared mating frequencies observed in the wild with those expected based on relative densities using partial canonical correspondence analysis. We introduced the fidelity index (FI) to estimate the relative accuracy of mating with conspecific females and precopulatory isolation index (IPC) to characterize the strength of premating barriers. The species under study, with the exception of L. arcana, clearly demonstrated preferential mating with conspecifics. According to FI and IPC, L. fabalis and L. compressa appeared reliably isolated from their closest relatives within Neritrema. Individuals of these two species tend to be smaller than those of the others, highlighting the importance of shell size changes in gastropod species divergence. L. arcana males were often found in pairs with L. saxatilis females, and no interspecific size differences were revealed in this sibling species pair. We discuss the lack of discriminative mate choice in the sympatric populations of L. arcana and L. saxatilis, and possible additional mechanisms restricting gene flow between them.