Sperm motility of oysters from distinct populations differs in response to ocean acidification and freshening.

Species' responses to climate change will reflect variability in the effects of physiological selection that future conditions impose. Here, we considered the effects of ocean acidification (increases in pCO2; 606, 925, 1250 µatm) and freshening (reductions in salinity; 33, 23, 13 PSU) on sperm motility in oysters (Crassostrea gigas) from two populations (one recently invaded, one established for 60+ years). Freshening reduced sperm motility in the established population, but this was offset by a positive effect of acidification. Freshening also reduced sperm motility in the recently invaded population, but acidification had no effect. Response direction, strength, and variance differed among individuals within each population. For the established population, freshening increased variance in sperm motility, and exposure to both acidification and freshening modified the performance rank of males (i.e. rank motility of sperm). In contrast, for the recently invaded population, freshening caused a smaller change in variance, and male performance rank was broadly consistent across treatments. That inter-population differences in response may be related to environmental history (recently invaded, or established), indicates this could influence scope for selection and adaptation. These results highlight the need to consider variation within and among population responses to forecast effects of multiple environmental change drivers.

Oceanographic barriers to gene flow promote genetic subdivision of the tunicate Ciona intestinalis in a North Sea archipelago.

Pelagic larval development has the potential to connect populations over large geographic distances and prevent genetic structuring. The solitary tunicate Ciona intestinalis has pelagic eggs and a swimming larval stage lasting for maximum a few days, with the potential for a homogenizing gene flow over relatively large areas. In the eastern North Sea, it is found in a geomorphologically complex archipelago with a mix of fjords and open costal habitats. Here, the coastal waters are also stratified with a marked pycnocline driven by salinity and temperature differences between shallow and deep waters. We investigated the genetic structure of C. intestinalis in this area and compared it with oceanographic barriers to dispersal that would potentially reduce connectivity among local populations. Genetic data from 240 individuals, sampled in 2 shallow, and 4 deep-water sites, showed varying degrees of differentiation among samples (FST = 0.0-0.11). We found no evidence for genetic isolation by distance, but two distant deep-water sites from the open coast were genetically very similar indicating a potential for long-distance gene flow. However, samples from different depths from the same areas were clearly differentiated, and fjord samples were different from open-coast sites. A biophysical model estimating multi-generation, stepping-stone larval connectivity, and empirical data on fjord water mass retention time showed the presence of oceanographic barriers that explained the genetic structure observed. We conclude that the local pattern of oceanographic connectivity will impact on the genetic structure of C. intestinalis in this region.

Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity.

Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle B. improvisus, based on genome and transcriptome sequencing. Our analyses reveal that B. improvisus contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the B. improvisus aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU) compared to high salinity (33 PSU). Our study provides a base for future mechanistic studies on the role of aquaporins in osmoregulation.

Low sensitivity of reproductive life-stages in the Pacific oyster (Crassostrea gigas) to abamectin.

Hard surfaces submerged in the marine environment often become colonised by macro-organisms unless the surfaces have some form of biofouling protection. While protective paints that contain tributyltin or copper work well to prevent biofouling, release of these materials into the environment has been shown to have wider negative impacts. Consequently, new low-release antifouling paints are being developed with alternative active ingredients, such as avermectins, yet little is known about their potential effects on non-target organisms in marine environments. Here we investigated the toxicity of a key avermectin, specifically abamectin, on several aspects of reproduction (sperm motility, fertilisation success, early larval development) in the Pacific oyster, Crassostrea gigas. Oyster reproduction was generally insensitive to the low concentrations of abamectin, although greater concentrations of abamectin did negatively affect all three endpoints - LOECs were 1000 µg l-1, 500 µg l-1, and 100 µg l-1 abamectin for sperm motility, fertilisation success, and larval development, respectively. A similar pattern was found in the EC50s of the three endpoints (mean ± SE) 934 ± 59 µg l-1, 1076.26 ± 725.61 µg l-1, and 140 ± 78 µg l-1 abamectin (sperm motility, fertilisation success, and larval development, respectively). Together, these results clearly indicate that of the three endpoints considered, larval development was more sensitive to abamectin (lower LOEC, EC50) than fertilisation success and sperm motility. Although more data are needed from a wider range of marine species and environments to fully assess potential toxicity effects on non-target organisms, our results highlight the potential utility of abamectin in low-release antifouling paints.

Sperm Accumulated Against Surface: A novel alternative bioassay for environmental monitoring.

Forecasting the impacts of changes in water quality on broadcast spawning aquatic organisms is a key aspect of environmental monitoring. Rapid assays of reproductive potential are central to this monitoring, and there is a need to develop a variety of methods to identify responses. Here, we report a proof-of-concept study that assesses whether quantification of "Sperm Accumulated Against Surface" (SAAS) of tissue culture well-plates could be a rapid and simple proxy measure of fertilisation success. Our results confirm that motile sperm (but not immotile sperm) actively accumulate at surfaces and that the pattern of accumulation reflects fertilisation success in the model oyster species Crassostrea gigas. Furthermore, we confirm these patterns of SAAS for another marine species, the polychaete Galeolaria caespitosa, as well as for a freshwater species, the fish Gasterosteus aculeatus. For all species considered, SAAS reflected changes in sperm performance caused by experimentally manipulated differences in water quality (here, salinity). These findings indicate that SAAS could be applied easily to a range of species when examining the effects of water quality. Measurement of SAAS could, therefore, form the basis of a rapid and reliable assay for bioassessments of broadcast spawning aquatic organisms.

Climate envelope modeling and dispersal simulations show little risk of range extension of the Shipworm, Teredo navalis (L.), in the Baltic sea.

The shipworm, Teredo navalis, is absent from most of the Baltic Sea. In the last 20 years, increased frequency of T. navalis has been reported along the southern Baltic Sea coasts of Denmark, Germany, and Sweden, indicating possible range-extensions into previously unoccupied areas. We evaluated the effects of historical and projected near-future changes in salinity, temperature, and oxygen on the risk of spread of T. navalis in the Baltic. Specifically, we developed a simple, GIS-based, mechanistic climate envelope model to predict the spatial distribution of favourable conditions for adult reproduction and larval metamorphosis of T. navalis, based on published environmental tolerances to these factors. In addition, we used a high-resolution three-dimensional hydrographic model to simulate the probability of spread of T. navalis larvae within the study area. Climate envelope modeling showed that projected near-future climate change is not likely to change the overall distribution of T. navalis in the region, but will prolong the breeding season and increase the risk of shipworm establishment at the margins of the current range. Dispersal simulations indicated that the majority of larvae were philopatric, but those that spread over a wider area typically spread to areas unfavourable for their survival. Overall, therefore, we found no substantive evidence for climate-change related shifts in the distribution of T. navalis in the Baltic Sea, and no evidence for increased risk of spread in the near-future.

Interactive effects of ocean acidification, elevated temperature, and reduced salinity on early-life stages of the pacific oyster.

Ocean acidification (OA) effects on larvae are partially attributed for the rapidly declining oyster production in the Pacific Northwest region of the United States. This OA effect is a serious concern in SE Asia, which produces >80% of the world's oysters. Because climate-related stressors rarely act alone, we need to consider OA effects on oysters in combination with warming and reduced salinity. Here, the interactive effects of these three climate-related stressors on the larval growth of the Pacific oyster, Crassostrea gigas, were examined. Larvae were cultured in combinations of temperature (24 and 30 °C), pH (8.1 and 7.4), and salinity (15 psu and 25 psu) for 58 days to the early juvenile stage. Decreased pH (pH 7.4), elevated temperature (30 °C), and reduced salinity (15 psu) significantly delayed pre- and post-settlement growth. Elevated temperature lowered the larval lipid index, a proxy for physiological quality, and negated the negative effects of decreased pH on attachment and metamorphosis only in a salinity of 25 psu. The negative effects of multiple stressors on larval metamorphosis were not due to reduced size or depleted lipid reserves at the time of metamorphosis. Our results supported the hypothesis that the C. gigas larvae are vulnerable to the interactions of OA with reduced salinity and warming in Yellow Sea coastal waters now and in the future.

Importance of plasticity and local adaptation for coping with changing salinity in coastal areas: a test case with barnacles in the Baltic Sea.

BACKGROUND: Salinity plays an important role in shaping coastal marine communities. Near-future climate predictions indicate that salinity will decrease in many shallow coastal areas due to increased precipitation; however, few studies have addressed this issue. The ability of ecosystems to cope with future changes will depend on species' capacities to acclimatise or adapt to new environmental conditions. Here, we investigated the effects of a strong salinity gradient (the Baltic Sea system--Baltic, Kattegat, Skagerrak) on plasticity and adaptations in the euryhaline barnacle Balanus improvisus. We used a common-garden approach, where multiple batches of newly settled barnacles from each of three different geographical areas along the Skagerrak-Baltic salinity gradient were exposed to corresponding native salinities (6, 15 and 30 PSU), and phenotypic traits including mortality, growth, shell strength, condition index and reproductive maturity were recorded. RESULTS: We found that B. improvisus was highly euryhaline, but had highest growth and reproductive maturity at intermediate salinities. We also found that low salinity had negative effects on other fitness-related traits including initial growth and shell strength, although mortality was also lowest in low salinity. Overall, differences between populations in most measured traits were weak, indicating little local adaptation to salinity. Nonetheless, we observed some population-specific responses--notably that populations from high salinity grew stronger shells in their native salinity compared to the other populations, possibly indicating adaptation to differences in local predation pressure. CONCLUSIONS: Our study shows that B. improvisus is an example of a true brackish-water species, and that plastic responses are more likely than evolutionary tracking in coping with future changes in coastal salinity.

Sperm swimming in the polychaete Galeolaria caespitosa shows substantial inter-individual variability in response to future ocean acidification.

The rapidity of ocean acidification intensifies selection pressure for resilient phenotypes, particularly during sensitive early life stages. The scope for selection is greater in species with greater within-species variation in responses to changing environments, thus enhancing the potential for adaptation. We investigated among-male variation in sperm swimming responses (percent motility and swimming speeds) of the serpulid polychaete Galeolaria caespitosa to near- (ΔpH -0.3) and far-future ocean acidification (ΔpH -0.5). Responses of sperm swimming to acidification varied significantly among males and were overall negative. Robust sperm swimming behavior under near-future ocean acidification in some males may ameliorate climate change impacts, if traits associated with robustness are heritable, and thereby enhance the potential for adaptation to far-future conditions. Reduced sperm swimming in the majority of male G. caespitosa may decrease their fertilization success in a high CO2 future ocean. Resultant changes in offspring production could affect recruitment success and population fitness downstream.

Investigating a possible role for the bacterial signal molecules N-acylhomoserine lactones in Balanus improvisus cyprid settlement.

Increased settlement on bacterial biofilms has been demonstrated for a number of marine invertebrate larvae, but the nature of the cue(s) responsible is not well understood. We tested the hypothesis that the bay barnacle Balanus improvisus utilizes the bacterial signal molecules N-acylhomoserine lactones (AHLs) as a cue for the selection of sites for permanent attachment. Single species biofilms of the AHL-producing bacteria Vibrio anguillarum, Aeromonas hydrophila and Sulfitobacter sp. BR1 were attractive to settling cypris larvae of B. improvisus. However, when AHL production was inactivated, either by mutation of the AHL synthetic genes or by expression of an AHL-degrading gene (aiiA), the ability of the bacteria to attract cyprids was abolished. In addition, cyprids actively explored biofilms of E. coli expressing the recombinant AHL synthase genes luxI from Vibrio fischeri (3-oxo-C6-HSL), rhlI from Pseudomonas aeruginosa (C4-HSL/C6-HSL), vanI from V. anguillarum (3-oxo-C10-HSL) and sulI from Sulfitobacter sp. BR1 (C4-HSL, 3-hydroxy-C6-HSL, C8-HSL and 3-hydroxy-C10-HSL), but not E. coli that did not produce AHLs. Finally, synthetic AHLs (C8-HSL, 3-oxo-C10-HSL and C12-HSL) at concentrations similar to those found within natural biofilms (5 µm) resulted in increased cyprid settlement. Thus, B. improvisus cypris exploration of and settlement on biofilms appears to be mediated by AHL-signalling bacteria in the laboratory. This adds to our understanding of how quorum sensing inhibition may be used as for biofouling control. Nonetheless, the significance of our results for larvae settling naturally in the field, and the mechanisms that underlay the observed responses to AHLs, is as yet unknown.

Individual variability in reproductive success determines winners and losers under ocean acidification: a case study with sea urchins.

BACKGROUND: Climate change will lead to intense selection on many organisms, particularly during susceptible early life stages. To date, most studies on the likely biotic effects of climate change have focused on the mean responses of pooled groups of animals. Consequently, the extent to which inter-individual variation mediates different selection responses has not been tested. Investigating this variation is important, since some individuals may be preadapted to future climate scenarios. METHODOLOGY/PRINCIPAL FINDINGS: We examined the effect of CO(2)-induced pH changes ("ocean acidification") in sperm swimming behaviour on the fertilization success of the Australasian sea urchin Heliocidaris erythrogramma, focusing on the responses of separate individuals and pairs. Acidification significantly decreased the proportion of motile sperm but had no effect on sperm swimming speed. Subsequent fertilization experiments showed strong inter-individual variation in responses to ocean acidification, ranging from a 44% decrease to a 14% increase in fertilization success. This was partly explained by the significant relationship between decreases in percent sperm motility and fertilization success at ΔpH = 0.3, but not at ΔpH = 0.5. CONCLUSIONS AND SIGNIFICANCE: The effects of ocean acidification on reproductive success varied markedly between individuals. Our results suggest that some individuals will exhibit enhanced fertilization success in acidified oceans, supporting the concept of 'winners' and 'losers' of climate change at an individual level. If these differences are heritable it is likely that ocean acidification will lead to selection against susceptible phenotypes as well as to rapid fixation of alleles that allow reproduction under more acidic conditions. This selection may ameliorate the biotic effects of climate change if taxa have sufficient extant genetic variation upon which selection can act.

The effectiveness of yoga for the improvement of well-being and resilience to stress in the workplace.

OBJECTIVES: Recent research has indicated concern for the degree of stress and emotional well-being among university staff. This study examined the effectiveness of yoga in enhancing emotional well-being and resilience to stress among university employees. METHODS: In a randomized controlled trial at a British university, we recruited 48 employees and randomized them into either a yoga or a wait-list control group. The yoga group was offered six weeks of Dru Yoga, comprising one 60-minute class per week. These classes were offered by a certified Dru Yoga instructor at lunchtime from January-March 2008. The wait-list control group received no intervention during this six-week study. Baseline and end-program measurements of self-reported mood and well-being were self-assessed with the Profile of Mood States - Bipolar (POMS-Bi) and the Inventory of Positive Psychological Attitudes (IPPA). RESULTS: This six-week yoga intervention resulted in significantly improved POMS-Bi and IPPA scores for the yoga compared to the wait-list control group for seven of eight measures of mood and well-being. In comparison to the wait-list control group at baseline and the end of the program, the yoga group reported marked improvements in feelings of clear-mindedness, composure, elation, energy, and confidence. In addition, the yoga group reported increased life purpose and satisfaction, and feelings of greater self-confidence during stressful situations. CONCLUSION: These results show that even a short program of yoga is effective for enhancing emotional well-being and resilience to stress in the workplace. We suggest that employers should consider offering yoga classes to their employees.

Indiscriminate males: mating behaviour of a marine snail compromised by a sexual conflict?

BACKGROUND: In promiscuous species, male fitness is expected to increase with repeated matings in an open-ended fashion (thereby increasing number of partners or probability of paternity) whereas female fitness should level out at some optimal number of copulations when direct and indirect benefits still outweigh the costs of courtship and copulation. After this fitness peak, additional copulations would incur female fitness costs and be under opposing selection. Hence, a sexual conflict over mating frequency may evolve in species where females are forced to engage in costly matings. Under such circumstance, if females could avoid male detection, significant fitness benefits from such avoidance strategies would be predicted. METHODOLOGY/PRINCIPAL FINDINGS: Among four Littorina species, one lives at very much higher densities and has a longer mating season than the other three species. Using video records of snail behaviour in a laboratory arena we show that males of the low-density species discriminate among male and female mucous trails, trailing females for copulations. In the high-density species, however, males fail to discriminate between male and female trails, not because males are unable to identify female trails (which we show using heterospecific females), but because females do not, as the other species, add a gender-specific cue to their trail. CONCLUSIONS/SIGNIFICANCE: We conclude that there is likely a sexual conflict over mating frequency in the high-density species (L. saxatilis) owing to females most likely being less sperm-limited in this species. This has favoured the evolution of females that permanently or optionally do not release a cue in the mucus to decrease excessive and costly matings resulting in unusually high frequencies of male-male copulating attempts in the wild. This is one of few examples of masking gender identity to obtain fewer matings.

Barriers to cross-fertilization between populations of a widely dispersed polychaete species are unlikely to have arisen through gametic compatibility arms-races.

Although there are theoretical reasons to suspect that gametic incompatibility may develop readily among populations of broadcast spawning marine invertebrates, there have been very few studies documenting geographic patterns of interpopulation incompatibility for any species. To address this we determined how successfully individuals of the intertidal serpulid polychaete, Galeolaria caespitosa, can cross-fertilize within and among populations from across temperate Australia. Fertilization assays revealed asymmetrical differences between very distantly located populations from different coasts, with near-complete incompatibility between eggs from Sydney with sperm from Adelaide, but the reverse cross (Adelaide eggs, Sydney sperm) was reasonably compatible. Although that pattern was congruent with a clear difference in Cytochrome B sequences between worms on the south and east coasts of Australia, we also detected some indication of interpopulation incompatibility within the genetic grouping on east coast, between two populations separated by only 220 km. We then assessed whether commonly proposed gametic compatibility arms-races could account for these patterns. Our results suggest reduced gametic compatibility may reduce a female's maximum fertilization potential, resulting in a cost to this potential mechanism for reducing polyspermy. Consequently, the apparently rapid development of reproductive barriers here seems unlikely to have been driven by arms-races involving sexual conflict over fertilization rate.

Male discrimination of female mucous trails permits assortative mating in a marine snail species.

Recent research has shown the potential for nonallopatric speciation, but we lack an adequate understanding of the mechanisms of prezygotic barriers and how these evolve in the presence of gene flow. The marine snail Littorina saxatilis has distinct ecotypes in different shore microhabitats. Ecotypes hybridize in contact zones, but gene flow is impeded by assortative mating. Earlier studies have shown that males and females of the same ecotype copulate for longer than mates of different ecotype. Here we report a new mechanism that further contributes to reproductive isolation between ecotypes in the presence of gene flow. This mechanism is linked to the ability of males to track potential partners by following their mucous trail. We show that cliff ecotype males follow the trails of females of the same ecotype for longer than females of the alternate (boulder) ecotype. In addition, cliff males are more likely to follow the mucous trail in the correct direction if the trail is laid by a cliff-female. The capacity to discriminate the ecotype of female mucous trails combined with differential copulation times creates a strong prezygotic reproductive barrier between ecotypes of L. saxatilis that reduces gene flow from cliff to boulder ecotypes by >/=80%.

Evidence for biased use of sperm sources in wild female giant cuttlefish (Sepia apama).

In species where females store sperm from their mates prior to fertilization, sperm competition is particularly probable. Female Sepia apama are polyandrous and have access to sperm from packages (spermatangia) deposited by males onto their buccal area during mating and to sperm stored in internal sperm-storage organs (receptacles) located below the beak. Here, we describe the structure of the sperm stores in the female's buccal area, use microsatellite DNA analyses to determine the genetic diversity of stored sperm and combine these data with offspring genotypes to determine the storage location of paternal sperm. The number of male genotypes represented in the sperm receptacles was significantly lower than that found among the spermatangia. Estimation of the volumes of sperm contained in the receptacles and the spermatangia were statistically comparable; however, paternal sperm were more likely to have come from spermatangia than from the sperm receptacles. These results confirm a genetic polyandrous mating system in this species and suggest that fertilization pattern with respect to the sperm stores used is not random.

Behavioural ecology: transient sexual mimicry leads to fertilization.

Sexual mimicry among animals is widespread, but does it impart a fertilization advantage in the widely accepted 'sneak-guard' model of sperm competition? Here we describe field results in which a dramatic facultative switch in sexual phenotype by sneaker-male cuttlefish leads to immediate fertilization success, even in the presence of the consort male. These results are surprising, given the high rate at which females reject copulation attempts by males, the strong mate-guarding behaviour of consort males, and the high level of sperm competition in this complex mating system.

Reproductive behavior in the squid Sepioteuthis australis from South Australia: ethogram of reproductive body patterns.

Squids use a diverse range of body patterns for communication. Each pattern consists of a series of chromatic, postural, and locomotor components that are under neural control and can change within fractions of a second. Here we describe an ethogram of 48 body pattern components from in situ observations of reproductively active Sepioteuthis australis. In addition, we identify the total time and average duration that each component is shown, to a resolution of 1 s. Our results suggest that only a few components (e.g., "Golden epaulettes," "Stitchwork fins," and "Rigid arms") are temporally common, that is, shown for more than 80% of the time. In contrast to the component classification reported for other species of squid, for this species we suggest a classification system consisting of "short acute" (lasting for < 10 s); some of these same components were also classified as "medium acute" (11-60 s) or "chronic" (> 60 s). Several body patterning components were previously unreported, as were some of the combinations observed. The significance of these patterning components is discussed within the context of the associated behaviors of the squid on the spawning grounds.

Reproductive behavior in the squid Sepioteuthis australis from South Australia: interactions on the spawning grounds.

Squid behavior is synonymous with distinctive body patterns, postures, and movements that constitute a complex visual communication system. These communications are particularly obvious during reproduction. They are important for sexual selection and have been identified as a potential means of species differentiation. Here we present a detailed account of copulation, mating, and egg deposition behaviors from in situ observations of the squid Sepioteuthis australis from South Australia. We identified four mating types from 85 separate mating attempts: "Male-upturned mating" (64% of mating attempts); "Sneaker mating" (33%); "Male-parallel" (2%); and "Head-to-head" (1%). Intervals between successive egg deposition behaviors were clearly bimodal, with modes at 2.5 s and 70.0 s. Ninety-three percent of egg capsules contained 3 or 4 eggs (mean = 3.54), and each egg cluster contained between 218 and 1922 egg capsules (mean = 893.9). The reproductive behavior of S. australis from South Australia was different from that described for other cephalopod species. More importantly, comparison between these results and those for other populations of S. australis suggests that behavior may differ from one population to another.