Conspecific Sperm Research Articles

The Effect of Synthetic Polyamine BPA-C8 on the Fertilization Process of Intact and Denuded Sea Urchin Eggs.

Sea urchin eggs are covered with layers of extracellular matrix, namely, the vitelline layer (VL) and jelly coat (JC). It has been shown that sea urchin eggs' JC components serve as chemoattractants or ligands for the receptor on the fertilizing sperm to promote the acrosome reaction. Moreover, the egg's VL provides receptors for conspecific sperm to bind, and, to date, at least two sperm receptors have been identified on the surface of sea urchin eggs. Interestingly, however, according to our previous work, denuded sea urchin eggs devoid of the JC and VL do not fail to become fertilized by sperm. Instead, they are bound and penetratedby multiple sperm, raising the possibility that an alternative pathway independent of the VL-residing sperm receptor may be at work. In this research, we studied the roles of the JC and VL using intact and denuded eggs and the synthetic polyamine BPA-C8. BPA-C8 is known to bind to the negatively charged macromolecular complexes in the cells, such as the JC, VL, and the plasma membrane of echinoderm eggs, as well as to the actin filaments in fibroblasts. Our results showed that, when added to seawater, BPA-C8 significantly repressed the Ca2+ wave in the intact P. lividus eggs at fertilization. In eggs deprived of the VL and JC, BPA-C8 binds to the plasma membrane and increases fibrous structures connecting microvilli, thereby allowing the denuded eggs to revert towards monospermy at fertilization. However, the reduced Ca2+ signal in denuded eggs was nullified compared to the intact eggs because removing the JC and VL already decreased the Ca2+ wave. BPA-C8 does not cross the VL and the cell membrane of unfertilized sea urchin eggs to diffuse into the cytoplasm at variance with the fibroblasts. Indeed, the jasplakinolide-induced polymerization of subplasmalemmal actin filaments was inhibited in the eggs microinjected with BPA-C8, but not in the ones bath-incubated with the same dose of BPA-C8.

Rapid sperm length divergence in a polygynandrous passerine: a mechanism of cryptic speciation?

When populations become geographically isolated, they begin to diverge in various traits and at variable rates. The dynamics of such trait divergences are relevant for understanding evolutionary processes such as local adaptation and speciation. Here we examine divergences in sperm and body structures in a polygynandrous songbird, the alpine accentor (Prunella collaris) between two allopatric high-altitude populations, in Morocco and Spain. The populations diverged around 82,000 years ago, as estimated with a coalescence-based phylogenetic analysis of genome-wide single-nucleotide polymorphisms. We found that birds in the two areas had nonoverlapping sperm lengths, which suggests adaptation to divergent female reproductive tract environments. Sperm length also showed an exceptionally low coefficient of among-male variation, a signal of strong stabilizing selection imposed by sperm competition. The evolutionary rate of sperm length was almost twice the rates for the most divergent morphological traits and more than three times higher than expected from literature data over a similar generational timescale. This rapid evolution of a key reproductive trait has implications for reproductive isolation and ultimately for speciation. Strong selection for different sperm length optima in allopatry predicts conspecific sperm precedence and disruptive selection in sympatry, hence a possible postcopulatory prezygotic barrier to gene flow.

Can cryptic female choice prevent invasive hybridization in external fertilizing fish?

Polyandrous mating systems result in females mating with multiple males, generating opportunities for strong pre-mating and post-mating sexual selection. Polyandry also creates the potential for unintended matings and subsequent sperm competition with hybridizing species. Cryptic female choice allows females to bias paternity towards preferred males under sperm competition and may include conspecific sperm preference when under hybridization risk. The potential for hybridization becomes particularly important in context of invasive species that can novelly hybridize with natives, and by definition, have evolved allopatrically. We provide the first examination of conspecific sperm preference in a system of three species with the potential to hybridize: North American native Atlantic salmon (Salmo salar) and brook char (Salvelinus fontinalis), and invasive brown trout (Salmo trutta) from Europe. Using naturalized populations on the island of Newfoundland, we measured changes in sperm swimming performance, a known predictor of paternity, to determine the degree of modification in sperm swimming to female cues related to conspecific sperm preference. Compared to water alone, female ovarian fluid in general had a pronounced effect and changed sperm motility (by a mean of 53%) and swimming velocity (mean 30%), but not linearity (mean 6%). However, patterns in the degree of modification suggest there is no conspecific sperm preference in the North American populations. Furthermore, female cues from both native species tended to boost the sperm of invasive males more than their own. We conclude that cryptic female choice via ovarian fluid mediated sperm swimming modification is too weak in this system to prevent invasive hybridization and is likely insufficient to promote or maintain reproductive isolation between the native North American species.

Artificial fertilisation method for the production of pen shell Atrina pectinata juveniles in hatcheries

In juvenile production of pen shell Atrina pectinata, broodstocks are usually induced to spawn by a combination of a slight increase in water temperature and addition of conspecific sperm to rearing water, but the stimulus is not always effective in eliciting a positive response from ripe adults. To solve the problem of unstable spawning induction, we developed an artificial fertilisation method suitable for the hatchery production of pen shell juveniles. The method was based on our recent finding that all-trans retinoic acid (at-RA) strongly induced the maturation of pen shell oocytes. Experiments were conducted to assess the following: 1) suitable at-RA concentration for oocyte treatment, 2) necessity of at-RA treatment of sperm, 3) optimum egg to sperm ratio, 4) allowable time lag between the at-RA treatment of oocytes and insemination, and 5) possible changes in the performance of the method during the spawning season. In addition, we confirmed the effectiveness of the method by observing the early growth of hatched larvae and the actual production of pen shell juveniles in two separate hatcheries. The recommended conditions were clarified as follows: 1) 1.0 or 0.1 μM at-RA was suitable to prepare mature oocytes, 2) at-RA treatment of sperm was not necessary, 3) optimum egg to sperm ratio was 1:1000, 4) mature oocytes were competent for insemination until 2 h after the at-RA treatment, and 5) fertilisation and hatching rates changed in accordance with alterations in the maturity of broodstocks. In the actual production of juveniles, benthic juveniles of pen shells were successfully obtained at two different hatcheries, although the number of produced juveniles was still small. Increasing familiarity with this new method through repetitive practice, together with the improvement of rearing techniques, would improve the performance of the method. The artificial fertilisation method presented in this article offers a novel alternative for stable production of juvenile pen shells.

Testing potential mechanisms of conspecific sperm precedence in Drosophila pseudoobscura.

Drosophila pseudoobscura females that co-occur with sister species D. persimilis show elevated fertilization by conspecific sperm when they mate with both a heterospecific and a conspecific male. This phenomenon, known as conspecific sperm precedence (CSP), has evolved as a mechanism to avoid maladaptive hybridization with D. persimilis. In this study, we assessed pericopulatory (during mating) and postcopulatory (after mating) traits in crosses with sympatric or allopatric D. pseudoobscura females and conspecific or heterospecific males to evaluate potential mechanisms of CSP in this system. We observed shorter copulation duration in crosses with sympatric females, but found no difference in quantity of sperm transferred or female reproductive tract toxicity between sympatry and allopatry. Our data show some support for the hypothesis that parasperm, a short, sterile sperm morph, can protect fertile eusperm from the D. pseudoobscura female reproductive tract, though it is unclear how this might affect patterns of sperm use in sympatry vs. allopatry. Overall, these results suggest that copulation duration could potentially contribute to the elevated CSP observed in sympatry.

Reproductive interference hampers species coexistence despite conspecific sperm precedence.

Negative interspecific mating interactions, known as reproductive interference, can hamper species coexistence in a local patch and promote niche partitioning or geographical segregation of closely related species. Conspecific sperm precedence (CSP), which occurs when females that have mated with both conspecific and heterospecific males preferentially use conspecific sperm for fertilization, might contribute to species coexistence by mitigating the costs of interspecific mating and hybridization. We discussed whether two species exhibiting CSP can coexist in a local environment in the presence of reproductive interference. First, using a behaviorally explicit mathematical model, we demonstrated that two species characterized by negative mating interactions are unlikely to coexist because the costs of reproductive interference, such as loss of mating opportunity with conspecific partners, are inevitably incurred when individuals of both species are present. Second, we experimentally examined differences in mating activity and preference in two Harmonia ladybird species known to exhibit CSP. These behavioral differences may lead to local extinction of H. yedoensis because of reproductive interference by H. axyridis. This prediction is consistent with field observations that H. axyridis uses various food sources and habitats whereas H. yedoensis is confined to a less preferred prey item and a pine tree habitat. Finally, by a comparative approach, we observed that niche partitioning or parapatric distribution, but not sympatric coexistence in the same habitat, is maintained between species with CSP belonging to a wide range of taxa, including vertebrates and invertebrates living in aquatic or terrestrial environments. Taken together, it is possible that reproductive interference may destabilize local coexistence even in closely related species that exhibit CSP.

Within-population sperm competition intensity does not predict asymmetry in conpopulation sperm precedence.

Postcopulatory sexual selection can generate evolutionary arms races between the sexes resulting in the rapid coevolution of reproductive phenotypes. As traits affecting fertilization success diverge between populations, postmating prezygotic (PMPZ) barriers to gene flow may evolve. Conspecific sperm precedence is a form of PMPZ isolation thought to evolve early during speciation yet has mostly been studied between species. Here, we show conpopulation sperm precedence (CpSP) between Drosophila montana populations. Using Pool-seq genomic data we estimate divergence times and ask whether PMPZ isolation evolved in the face of gene flow. We find models incorporating gene flow fit the data best indicating populations experienced considerable gene flow during divergence. We find CpSP is asymmetric and mirrors asymmetry in non-competitive PMPZ isolation, suggesting these phenomena have a shared mechanism. However, we show asymmetry is unrelated to the strength of postcopulatory sexual selection acting within populations. We tested whether overlapping foreign and coevolved ejaculates within the female reproductive tract altered fertilization success but found no effect. Our results show that neither time since divergence nor sperm competitiveness predicts the strength of PMPZ isolation. We suggest that instead cryptic female choice or mutation-order divergence may drive divergence of postcopulatory phenotypes resulting in PMPZ isolation.This article is part of the theme issue ‘Fifty years of sperm competition’.

Spice up your life!

Cryptic female choice is the ability of a polyandrous mating female to favour - or hinder - the sperm of a particular male from fertilizing her ova, under sperm competition. It operates under varied mechanisms and occurs in both internal and external fertilizers. In fishes, a key process is driven by the effect of ovarian fluid - the fluid released with the ova at spawning - on the swimming characteristics of sperm. Due to the limited functioning life-span of fish gametes, researchers have previously had to use fresh ovarian fluid and sperm, thus limiting the geographical and temporal range of their studies, or freeze one, or the other, or both. Much work has been done on the cryo-preservation of fish sperm; however, it was unclear - until now - whether the freezing of ovarian fluid affects its sperm-influencing properties. This is where Craig Purchase and Anna Rooke of Memorial University of Newfoundland, Canada come in (Purchase & Rooke, 2020). They took ovarian fluid and semen from three species of salmonids - lake tout, brown trout and Atlantic salmon - and tested both frozen and fresh ovarian fluid on the swimming characteristics of sperm. They found quite clearly that freezing did not reduce the effect of ovarian fluid on the motility, swimming speed or linearity of conspecific sperm. This is good news for researchers; it allows them to build up collections of ovarian fluids from different females - the “spice-rack” of the title - in the knowledge that freezing them will not alter the very properties that they are investigating. Now, studies can include different populations, even different years, to investigate how ovarian fluids influence the sperm of different males, and thus the extent and importance of this effect in reproductive isolation, for example. It will also help in the ultimate identification of the proximate factor(s) involved, but this is still some way ahead.

Reproductive strategies in the intercrossing corals Acropora donei and A. tenuis to prevent hybridization

Although many sympatric species of the coral Acropora spawns on the same day, hybridization is partly prevented by their species-specific fertilization manner. On the other hand, several species show bidirectional intercrossing, but their hybridization is prevented by slight time differences in their spawning and fertilization preference with conspecific sperm. However, it is scarcely described how the intercrossing species avoid hybridization. Here we demonstrate that hybridization of the intercrossing species Acropora tenuis and A. donei is likely prevented by differences in the timing of spawning, gametogenesis (differences in production numbers of matured sperm and eggs), population density, and fertilization preferences. We investigated three locations in Okinawa, Japan: Aka Island, Sesoko Island, and Oku. The timing of gamete release in the two species overlapped at Aka Island and Oku, but A. donei did not spawn vigorously at Sesoko Island. Histological observations indicated a decrease in the number of mature sperm and eggs in the weakly spawning colonies of A. donei at Sesoko Island. From the population density, as assessed by field surveys, we predicted that the encounter rate with heterospecific gametes for A. donei around Sesoko Island was higher than at the other locations. Thus, one predictable reason for the suppression of their gonadal development may be the avoidance of hybridization. Although synchronous spawning of A. tenuis and A. donei was observed at Aka Island and Oku, hybridization is likely infrequent, because sperm choice experiments demonstrated that the eggs preferred fertilization by conspecific sperm. Moreover, microsatellite analyses suggested that hybridization rarely occurred even at synchronous spawning sites, as the population structures of the two species at both Aka Island and Oku were not mixed. Collectively, the findings of this study highlight several strategies, tactics, and mechanisms of avoiding hybridization that are likely to be active in other species that hybridize in vitro.

The effects of heterospecific mating frequency on the strength of cryptic reproductive barriers.

Heterospecific mating frequency is critical to hybrid zone dynamics and can directly impact the strength of reproductive barriers and patterns of introgression. The effectiveness of post-mating prezygotic (PMPZ) reproductive barriers, which include reduced fecundity via heterospecific matings and conspecific sperm precedence, may depend on the number, identity and order of mates. Studies of PMPZ barriers suggest that they may be important in many systems, but whether these barriers are effective at realistic heterospecific mating frequencies has not been tested. Here, we evaluate the strength of cryptic reproductive isolation in two leaf beetles (Chrysochus auratus and C.cobaltinus) in the context of a range of heterospecific mating frequencies observed in natural populations. We found both species benefited from multiple matings, but the benefits were greater in C.cobaltinus and extended to heterospecific matings. We found that PMPZ barriers greatly limited hybrid production by C.auratus females with moderate heterospecific mating frequencies, but that their effectiveness diminished at higher heterospecific mating frequencies. In contrast, there was no evidence for PMPZ barriers in C.cobaltinus females at any heterospecific mating frequency. We show that integrating realistic estimates of cryptic isolation with information on relative abundance and heterospecific mating frequency in the field substantially improves our understanding of the strong directional bias in F1 production previously documented in the Chrysochus hybrid zone. Our results demonstrate that heterospecific mating frequency is critical to understanding the impact of cryptic post-copulatory barriers on hybrid zone structure and dynamics, and that future studies of such barriers should incorporate field-relevant heterospecific mating frequencies.

Conspecific sperm precedence is reinforced, but postcopulatory sexual selection weakened, in sympatric populations of Drosophila.

Sexual selection can accelerate speciation by driving the evolution of reproductive isolation, but forces driving speciation could also reciprocally feedback on sexual selection. This might be particularly important in the context of 'reinforcement', where selection acts directly to increase prezygotic barriers to reduce the cost of heterospecific matings. Using assays of sperm competition within and between two sister species, we show a signature of reinforcement where these species interact: populations of Drosophila pseudoobscura that co-occur with sister species D. persimilis have an elevated ability to outcompete heterospecific sperm, consistent with selection for increased postcopulatory isolation. We also find these D. pseudoobscura populations have decreased sperm competitive ability against conspecifics, reducing the opportunity for sexual selection within these populations. Our findings demonstrate that direct selection to increase reproductive isolation against other species can compromise the efficacy of sexual selection within species, a collateral effect of reproductive traits responding to heterospecific interactions.

Triggers of spawning and oocyte maturation in the commercial sea cucumber Cucumaria frondosa

The present study investigated methods to trigger spawning and artificially induce oocyte maturation in the commercial cold-water suspension-feeding sea cucumber Cucumaria frondosa. Spawning occurred after exposure to live phytoplankton, commercial phytoplankton paste, and conspecific sperm. Live phytoplankton at 1 × 105 cells ml−1 induced the highest proportion of females to spawn (mean of 56%); promoted the greatest oocyte release (mean of 19,200 oocytes female−1), best quality of eggs and highest survival of embryos (65%; 20 d post fertilization). More concentrated live phytoplankton (1 × 106 cells ml−1) triggered 43% of the females to release ~16,100 oocytes female−1 and survival rates 20 d post fertilization were lower (50%). Both phytoplankton paste treatments (1 × 105 and 1 × 106 cells ml−1) yielded intermediate results in terms of percent spawning females (26%), abundance of oocytes released (11,000 and 7300 oocytes female−1, respectively), quality of eggs, and survival of embryos (44 and 50%, respectively). Sperm from conspecific (2 and 10 × 106 spermatozoa ml−1) induced the lowest proportion of females to spawn (16%) with release of 3000 and 1700 oocytes female−1, respectively; furthermore, all embryos died within 10 d post fertilization. Results for males were slightly different: spawning was more frequent (63%) in the sperm treatment at 2 × 106 spermatozoa ml−1 followed by live phytoplankton at 1 × 106 and 1 × 105 cells ml−1 (43%), and phytoplankton paste at 1 × 105 cells ml−1 (46%); the lowest spawning success was observed for phytoplankton paste at 1 × 106 cells ml−1 and conspecific sperm at 10 × 106 spermatozoa ml−1 (26%). Thermal shock, desiccation, potassium chloride (injection or bath), and serotonin (injection) did not induce spawning in C. frondosa. When oocytes isolated from mature ovaries were exposed to three different concentrations of 1-Methyladenine (1-MA), Dithiothreitol (DTT), 2,3-Dimercapto-1-propanol (BAL), and L-cysteine (L-cyst), DTT at 10−1 M induced the highest proportion of oocytes (48%) to shed follicle cells (i.e. ovulate); whereas other treatments induced only 2–6% of ovulation, similar to the control (seawater). All oocytes that shed follicle cells remained unfertilizable. Overall, live phytoplankton emerged as the most suitable spawning trigger for C. frondosa, a Dendrochirotida that produces maternally-provisioned oocytes. In the context of expanding sea cucumber aquaculture, this finding provides a tool to maximize the availability of gametes in this cold-water species, with possible applicability to similar commercial dendrochitotids.

In vitro oocyte maturation by radial nerve extract and early development of the black sea cucumber (Holothuria leucospilota)

The popularity of sea cucumbers has led to overexploitation of wild populations and increasing need to develop their aquaculture. In vitro induction of oocyte maturation followed by fertilization represents an innovative approach that could help increase their production. The inducers of oocyte maturation are naturally synthesised within neural tissue, which in sea cucumbers includes longitudinal radial nerves and a circumoral nerve ring. In this study, we showed that reproductive maturation of the black sea cucumber (Holothuria leucospilota Brandt, 1835) in South-East Queensland, Australia, peaked in December, based on biometric analysis of gonads. Radial nerve extract (RNE; 233 μg/ml) prepared from reproductive mature females induced in vitro oocyte maturation at 98.9% efficiency, based on germinal vesicle breakdown. RNE-matured oocytes were fertilized (99%) with conspecific sperm and larvae developed to late auricularia stage (25 days post-fertilization). The NGIWYamide neuropeptide (cubifrin), which induces oocyte maturation in the sea cucumber Apostichopus japonicus, could not induce oocyte maturation in H. leucospilota. These results provide information that could be used for large-scale artificial breeding of H. leucospilota, and a platform towards the identification of the bioactive RNE factor(s).

A Sperm Spawn-Inducing Pheromone in the Silver Lip Pearl Oyster (Pinctada maxima).

Pheromones are considered to play an important role in broadcast spawning in aquatic animals, facilitating synchronous release of gametes. In oysters, the sperm has been implicated as a carrier for the spawn-inducing pheromone (SIP). In hatchery conditions, male pearl oysters (Pinctata maxima) can be stimulated to spawn through a variety of approaches (e.g. rapid temperature change), while females can only be induced to spawn through exposure to conspecific sperm, thus limiting development of targeted pairing, required for genetic research and management. The capacity for commercial production and improvement of genetic lines of pearl oysters could be greatly improved with access to a SIP. In this study, we prepared and sequenced crude and semi-purified P. maxima sperm extracts that were used in bioassays to localise the female SIP. We report that the P. maxima SIP is proteinaceous and extrinsically associated with the sperm membrane. Bioactivity from pooled RP-HPLC fractions, but not individual fractions, suggests that the SIP is multi-component. We conclude that crude sperm preparations, as described in this study, can be used as a sperm-free inducer of female P. maxima spawning, which enables for a more efficient approach to genetic breeding.

Do Sperm Really Compete and Do Eggs Ever Have a Choice? Adult Distribution and Gamete Mixing Influence Sexual Selection, Sexual Conflict, and the Evolution of Gamete Recognition Proteins in the Sea.

The evolution of gametic compatibility and the effectiveness of compatibility, within and across species, depend on whether sperm from different males directly compete for an egg and whether eggs ever have a choice. Direct sperm competition and egg choice depend on whether sperm from different males arrive at an egg in the brief interval between first sperm contact and fertilization. Although this process may be relevant for all sexually reproducing organisms, it is most easily examined in aquatic external fertilizers. When sperm are released into the sea, packets of seawater at the spatial scale relevant to single eggs might contain sperm from only one male, eliminating the potential for direct sperm competition and egg choice. Field experiments and a simple heuristic model examining the degree of sperm mixing for the sea urchin Strongylocentrotus franciscanus indicate that degree of competitive fertilization depends on density and distribution of competing males and that the nature of this competition influences whether males with high- or low-affinity gamete recognition protein genotypes have higher reproductive success. These results provide a potential explanation for the generation and maintenance of variation in gamete recognition proteins and why effectiveness of conspecific sperm precedence can be density dependent.

Individual female differences in chemoattractant production change the scale of sea urchin gamete interactions

Sperm selection by females is an important process influencing fertilization and, particularly in broadcast-spawning organisms, often occurs before sperm reach the egg. Waterborne sperm chemoattractants are one mechanism by which eggs selectively influence conspecific sperm behavior, but it remains an open question whether the eggs from different females produce different amounts of sperm chemoattractant, and how that might influence sperm behavior. Here, we quantify the differences in attractant production between females of the sea urchin species Lytechinus pictus and use computational models and microfluidic sperm chemotaxis assays to determine how differences in chemoattractant production between females affects their ability to attract sperm. Our study demonstrates that there is significant individual female variation in egg chemoattractant production, and that this variation changes the scope and strength of sperm attraction. These results provide evidence for the importance of individual female variability in differential sperm attraction and fertilization success.

Sorted gene genealogies and species-specific nonsynonymous substitutions point to putative postmating prezygotic isolation genes in Allonemobius crickets.

In the Allonemobius socius complex of crickets, reproductive isolation is primarily accomplished via postmating prezygotic barriers. We tested seven protein-coding genes expressed in the male ejaculate for patterns of evolution consistent with a putative role as postmating prezygotic isolation genes. Our recently diverged species generally lacked sequence variation. As a result, ω-based tests were only mildly successful. Some of our genes showed evidence of elevated ω values on the internal branches of gene trees. In a couple of genes, these internal branches coincided with both species branching events of the species tree, between A. fasciatus and the other two species, and between A. socius and A. sp. nov. Tex. In comparison, more successful approaches were those that took advantage of the varying degrees of lineage sorting and allele sharing among our young species. These approaches were particularly powerful within the contact zone. Among the genes we tested we found genes with genealogies that indicated relatively advanced degrees of lineage sorting across both allopatric and contact zone alleles. Within a contact zone between two members of the species complex, only a subset of genes maintained allelic segregation despite evidence of ongoing gene flow in other genes. The overlap in these analyses was arginine kinase (AK) and apolipoprotein A-1 binding protein (APBP). These genes represent two of the first examples of sperm maturation, capacitation, and motility proteins with fixed non-synonymous substitutions between species-specific alleles that may lead to postmating prezygotic isolation. Both genes express ejaculate proteins transferred to females during copulation and were previously identified through comparative proteomics. We discuss the potential function of these genes in the context of the specific postmating prezygotic isolation phenotype among our species, namely conspecific sperm precedence and the superior ability of conspecific males to induce oviposition in females.

Lectin staining and flow cytometry reveals female-induced sperm acrosome reaction and surface carbohydrate reorganization.

All cells are covered by glycans, an individually unique layer of oligo- and polysaccharides that are critical moderators of self-recognition and other cellular-level interactions (e.g. fertilization). The functional similarity between these processes suggests that gamete surface glycans may also have an important, but currently overlooked, role in sexual selection. Here we develop a user-friendly methodological approach designed to facilitate future tests of this possibility. Our proposed method is based on flow cytometric quantification of female-induced sperm acrosome reaction and sperm surface glycan modifications in the Mediterranean mussel Mytilus galloprovincialis. In this species, as with many other taxa, eggs release water-soluble factors that attract conspecific sperm (chemoattraction) and promote potentially measurable changes in sperm behavior and physiology. We demonstrate that flow cytometry is able to identify sperm from other seawater particles as well as accurately measure both acrosome reaction and structural modifications in sperm glycans. This methodological approach can increase our understanding of chemically-moderated gamete-level interactions and individual-specific gamete recognition in Mytilus sp. and other taxa with similar, easily identifiable acrosome structure. Our approach is also likely to be applicable to several other species, since carbohydrate-mediated cellular-level interactions between gametes are universal among externally and internally fertilizing species.

Intraspecific sperm competition genes enforce post-mating species barriers in Drosophila.

Sexual selection and sexual conflict are considered important drivers of speciation, based on both theoretical models and empirical correlations between sexually selected traits and diversification. However, whether reproductive isolation between species evolves directly as a consequence of intrapopulation sexual dynamics remains empirically unresolved, in part because knowledge of the genetic mechanisms (if any) connecting these processes is limited. Here, we provide evidence of a direct mechanistic link between intraspecies sexual selection and reproductive isolation. We examined genes with known roles in intraspecific sperm competition (ISC) in D. melanogaster and assayed their impact on conspecific sperm precedence (CSP). We found that two such genes (Acp36DE and CG9997) contribute to both offensive sperm competition and CSP; null/knockdown lines both had lower competitive ability against D. melanogaster conspecifics and were no longer able to displace heterospecific D. simulans sperm in competitive matings. In comparison, Sex Peptide (Acp70A)-another locus essential for ISC-does not contribute to CSP. These data indicate that two loci important for sperm competitive interactions have an additional role in similar interactions that enforce post-mating reproductive isolation between species, and show that sexual selection and sexual isolation can act on the same molecular targets in a gene-specific manner.

Positive selection at a seminal fluid gene within a QTL for conspecific sperm precedence.

The role of sexual selection in driving the rapid evolution of male reproductive proteins has been tested in a wide variety of organisms. Sperm competition is a form of postmating sexual selection that can contribute to reproductive isolation between species by biasing the proportion of progeny fathered by conspecific over heterospecific males. This phenomenon is known as conspecific sperm precedence (CSP). A previous quantitative trait loci study between Drosophila simulans and D. sechellia identified a locus associated with CSP within the second chromosome centered at the 53 cytogenetic map position. Male accessory gland proteins (ACPs) are associated with triggering postmating physiological responses in D. melanogaster females that can contribute to differential male reproductive success. Moreover, a large number of ACPs evolve rapidly and under positive selection among closely-related species of Drosophila. Here we have sequenced five candidate Acp genes (Acp53C14a, Acp53C14b, Acp53C14c, Acp53Ea and Acp54A1) within the previously mapped D. simulans-D. sechellia CSP locus from different D. simulans and D. sechellia strains. Polymorphism data analysis shows evidence of a selective sweep at Acp53Ea within D. simulans. In the context of CSP, the combined use of polymorphism and interspecies sequence divergence shows that Acp53C14c gene tree topology separates D. simulans and D. sechellia. Moreover, Acp53C14c is the only gene showing evidence of positive selection with five fixed amino acid substitutions between species. Our results highlight Acp53C14c as a candidate gene for future gene targeting studies to elucidate its role in CSP between D. simulans and D. sechellia.