Importance of sperm density in assessing the toxicity of metals to the fertilization of broadcast spawners.

Many marine invertebrates reproduce through broadcast spawning, where sperm and eggs are released into the water column and are vulnerable to toxicants present in the environment. The potential impacts of toxicants on spawning success are often assessed through laboratory-based fertilization tests. In most cases, these tests assess toxicant impacts at a single, pre-defined sperm density, based on a sperm:egg ratio that ensures high fertilization success (≥70-80%) in a filtered seawater control. Here we show that use of a single sperm density can considerably underestimate toxicity and that assessments over a range of sperm densities can provide more ecologically relevant, conservative and informative toxicity data. Fertilization assays were run for the polychaete Galeolaria caespitosa exposed to four heavy metals (Cu, Zn, Pb, Cd) across multiple sperm densities. There was a three-fold difference in the toxicity of copper and zinc when calculated at sperm densities of 104 and 106 sperm mL-1, both of which result in over 80% fertilization in FSW controls. By testing across multiple sperm densities, we identified that metals impact the sperm of G. caespitosa during the fertilization process. Assessing toxicity across multiple sperm densities is not always practical. This is due to the increased effort required to complete full fertilization curves, across enough concentrations of a toxicant, to establish a concentration-response relationship. In such cases, we provide recommendations for adopting aspects of fertilization assays that will improve on standard spermiotoxicity tests but which are still manageable for routine toxicity testing.

Nonequilibrium Conditions Explain Spatial Variability in Genetic Structuring of Little Penguin (Eudyptula minor).

Factors responsible for spatial structuring of population genetic variation are varied, and in many instances there may be no obvious explanations for genetic structuring observed, or those invoked may reflect spurious correlations. A study of little penguins (Eudyptula minor) in southeast Australia documented low spatial structuring of genetic variation with the exception of colonies at the western limit of sampling, and this distinction was attributed to an intervening oceanographic feature (Bonney Upwelling), differences in breeding phenology, or sea level change. Here, we conducted sampling across the entire Australian range, employing additional markers (12 microsatellites and mitochondrial DNA, 697 individuals, 17 colonies). The zone of elevated genetic structuring previously observed actually represents the eastern half of a genetic cline, within which structuring exists over much shorter spatial scales than elsewhere. Colonies separated by as little as 27 km in the zone are genetically distinguishable, while outside the zone, homogeneity cannot be rejected at scales of up to 1400 km. Given a lack of additional physical or environmental barriers to gene flow, the zone of elevated genetic structuring may reflect secondary contact of lineages (with or without selection against interbreeding), or recent colonization and expansion from this region. This study highlights the importance of sampling scale to reveal the cause of genetic structuring.

"Nested" cryptic diversity in a widespread marine ecosystem engineer: a challenge for detecting biological invasions.

BACKGROUND: Ecosystem engineers facilitate habitat formation and enhance biodiversity, but when they become invasive, they present a critical threat to native communities because they can drastically alter the receiving habitat. Management of such species thus needs to be a priority, but the poorly resolved taxonomy of many ecosystem engineers represents a major obstacle to correctly identifying them as being either native or introduced. We address this dilemma by studying the sea squirt Pyura stolonifera, an important ecosystem engineer that dominates coastal communities particularly in the southern hemisphere. Using DNA sequence data from four independently evolving loci, we aimed to determine levels of cryptic diversity, the invasive or native status of each regional population, and the most appropriate sampling design for identifying the geographic ranges of each evolutionary unit. RESULTS: Extensive sampling in Africa, Australasia and South America revealed the existence of "nested" levels of cryptic diversity, in which at least five distinct species can be further subdivided into smaller-scale genetic lineages. The ranges of several evolutionary units are limited by well-documented biogeographic disjunctions. Evidence for both cryptic native diversity and the existence of invasive populations allows us to considerably refine our view of the native versus introduced status of the evolutionary units within Pyura stolonifera in the different coastal communities they dominate. CONCLUSIONS: This study illustrates the degree of taxonomic complexity that can exist within widespread species for which there is little taxonomic expertise, and it highlights the challenges involved in distinguishing between indigenous and introduced populations. The fact that multiple genetic lineages can be native to a single geographic region indicates that it is imperative to obtain samples from as many different habitat types and biotic zones as possible when attempting to identify the source region of a putative invader. "Nested" cryptic diversity, and the difficulties in correctly identifying invasive species that arise from it, represent a major challenge for managing biodiversity.

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.

Integration of environmental aspects in modelling and optimisation of water supply chains.

Climate change becomes increasingly more relevant in the context of water systems planning. Tools are necessary to provide the most economic investment option considering the reliability of the infrastructure from technical and environmental perspectives. Accordingly, in this work, an optimisation approach, formulated as a spatially-explicit multi-period Mixed Integer Linear Programming (MILP) model, is proposed for the design of water supply chains at regional and national scales. The optimisation framework encompasses decisions such as installation of new purification plants, capacity expansion, and raw water trading schemes. The objective is to minimise the total cost incurring from capital and operating expenditures. Assessment of available resources for withdrawal is performed based on hydrological balances, governmental rules and sustainable limits. In the light of the increasing importance of reliability of water supply, a second objective, seeking to maximise the reliability of the supply chains, is introduced. The epsilon-constraint method is used as a solution procedure for the multi-objective formulation. Nash bargaining approach is applied to investigate the fair trade-offs between the two objectives and find the Pareto optimality. The models' capability is addressed through a case study based on Australia. The impact of variability in key input parameters is tackled through the implementation of a rigorous global sensitivity analysis (GSA). The findings suggest that variations in water demand can be more disruptive for the water supply chain than scenarios in which rainfalls are reduced. The frameworks can facilitate governmental multi-aspect decision making processes for the adequate and strategic investments of regional water supply infrastructure.

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.

Is monitoring for mass spawning events in coral assemblages in north Western Australia likely to detect spawning?

In north Western Australia coral reefs occur near ports being developed to support rapidly expanding resources industries. Dredging for port construction is required to stop during significant mass coral spawning events due to the sensitivity of gametes and larvae to increases in turbidity and sedimentation, but the timing of this event can vary between seasons and years so monitoring is used to predict when spawning is imminent. Here we used simulations to mimick sampling strategies currently used in some coral spawning monitoring programmes in Western Australia, to assess the ability of these programmes to be able to predict multi-specific mass spawning events. We found that current practices may sometimes miss spawning events that are likely to be considered large enough to warrant stopping dredging. Generally, sampling fewer individuals in a large number of species is a better way of monitoring for upcoming spawning than sampling a large number of individuals in a small number of species, but overall, greater sampling efforts than are currently undertaken are needed if moderately sized events are to be detected reliably. Determining exactly how many samples are needed, however, depends on having a clearer definition of what actually constitutes a "significant mass spawning" event in the first place.

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.

Elucidating the ecology of bucephalid parasites using a mutation scanning approach.

Nucleotide variation in a portion of the mitochondrial cytochrome c oxidase subunit1 (cox1) gene from asexual stages of bucephalids of southern Australian scallops (Chlamys asperrima, Chlamys bifrons and Pecten fumatus) was investigated using a mutation scanning-sequencing approach. Single-strand conformation polymorphism (SSCP) analysis revealed three main profile types (A, B and C) for parasites isolated from scallops. Sequence analysis revealed that samples represented by profiles B and C had a high degree (97.3%) of sequence similarity, whereas they were approximately 21% different in sequence from those represented by profile A. These findings suggested that at least two types or species (represented by profile A, or profile B or C) of bucephalid infect scallops, of which both were detected in South Australia, while only one was found in Victoria. The prevalence of bucephalids (and their SSCP haplotypes) appeared to differ among the three species of scallop in South Australia as well as between the two scallop species in Victoria, indicating a degree of host specificity. Adult bucephalids were collected from Eastern Australian Salmon (Arripis trutta), in an attempt to match them with the asexual stages from the scallop hosts. Neither of the two taxa of adult bucephalid (Telorhynchus arripidis and an un-named Telorhynchus species) shared SSCP profiles with the bucephalids from scallops, but were genetically similar, suggesting that the asexual stages from scallops may represent the genus Telorhynchus. This study, which assessed nucleotide sequence variation in a portion of the mitochondrial cox1 gene for bucephalids found in scallops and arripid fish, illustrates the usefulness of the mutation scanning approach to elucidate complex life-cycles of marine parasites.