Recruitment of a threatened foundation oyster species varies with large and small spatial scales.

Understanding how habitat attributes (e.g., patch area and sizes, connectivity) control recruitment and how this is modified by processes operating at larger spatial scales is fundamental to understanding population sustainability and developing successful long-term restoration strategies for marine foundation species-including for globally threatened reef-forming oysters. In two experiments, we assessed the recruitment and energy reserves of oyster recruits onto remnant reefs of the oyster Saccostrea glomerata in estuaries spanning 550 km of coastline in southeastern Australia. In the first experiment, we determined whether recruitment of oysters to settlement plates in three estuaries was correlated with reef attributes within patches (distances to patch edges and surface elevation), whole-patch attributes (shape and size of patches), and landscape attributes (connectivity). We also determined whether environmental factors (e.g., sedimentation and water temperature) explained the differences among recruitment plates. We also tested whether differences in energy reserves of recruits could explain the differences between two of the estuaries (one high- and one low-sedimentation estuary). In the second experiment, across six estuaries (three with nominally high and three with nominally low sedimentation rates), we tested the hypothesis that, at the estuary scale, recruitment and survival were negatively correlated to sedimentation. Overall, total oyster recruitment varied mostly at the scale of estuaries rather than with reef attributes and was negatively correlated with sedimentation. Percentage recruit survival was, however, similar among estuaries, although energy reserves and condition of recruits were lower at a high- compared to a low-sediment estuary. Within each estuary, total oyster recruitment increased with patch area and decreased with increasing tidal height. Our results showed that differences among estuaries have the largest influence on oyster recruitment and recruit health and this may be explained by environmental processes operating at the same scale. While survival was high across all estuaries, growth and reproduction of oysters on remnant reefs may be affected by sublethal effects on the health of recruits in high-sediment estuaries. Thus, restoration programs should consider lethal and sublethal effects of whole-estuary environmental processes when selecting sites and include environmental mitigation actions to maximize recruitment success.

Transgenerational plasticity responses of oysters to ocean acidification differ with habitat.

Transgenerational plasticity (TGP) has been identified as a critical mechanism of acclimation that may buffer marine organisms against climate change, yet whether the TGP response of marine organisms is altered depending on their habitat is unknown. Many marine organisms are found in intertidal zones where they experience episodes of emersion (air exposure) daily as the tide rises and recedes. During episodes of emersion, the accumulation of metabolic carbon dioxide (CO2) leads to hypercapnia for many species. How this metabolic hypercapnia impacts the TGP response of marine organisms to climate change is unknown as all previous transgenerational studies have been done under subtidal conditions, where parents are constantly immersed. Here, we assess the capacity of the ecologically and economically important oyster, Saccostrea glomerata, to acclimate to elevated CO2 dependent on habitat, across its vertical distribution, from the subtidal to intertidal zone. Tidal habitat altered both the existing tolerance and transgenerational response of S. glomerata to elevated CO2. Overall, larvae from parents conditioned in an intertidal habitat had a greater existing tolerance to elevated CO2 than larvae from parents conditioned in a subtidal habitat, but had a lower capacity for beneficial TGP following parental exposure to elevated CO2. Our results suggest that the TGP responses of marine species will not be uniform across their distribution and highlights the need to consider the habitat of a species when assessing TGP responses to climate change stressors.

Ocean acidification but not warming alters sex determination in the Sydney rock oyster, Saccostrea glomerata.

Whether sex determination of marine organisms can be altered by ocean acidification and warming during this century remains a significant, unanswered question. Here, we show that exposure of the protandric hermaphrodite oyster, Saccostrea glomerata to ocean acidification, but not warming, alters sex determination resulting in changes in sex ratios. After just one reproductive cycle there were 16% more females than males. The rate of gametogenesis, gonad area, fecundity, shell length, extracellular pH and survival decreased in response to ocean acidification. Warming as a sole stressor slightly increased the rate of gametogenesis, gonad area and fecundity, but this increase was masked by the impact of ocean acidification at a level predicted for this century. Alterations to sex determination, sex ratios and reproductive capacity will have flow on effects to reduce larval supply and population size of oysters and potentially other marine organisms.

Intertidal oysters reach their physiological limit in a future high-CO2 world.

Sessile marine molluscs living in the intertidal zone experience periods of internal acidosis when exposed to air (emersion) during low tide. Relative to other marine organisms, molluscs have been identified as vulnerable to future ocean acidification; however, paradoxically it has also been shown that molluscs exposed to high CO2 environments are more resilient compared with those molluscs naive to CO2 exposure. Two competing hypotheses were tested using a novel experimental design incorporating tidal simulations to predict the future intertidal limit of oysters in a high-CO2 world; either high-shore oysters will be more tolerant of elevated PCO2 because of their regular acidosis, or elevated PCO2  will cause high-shore oysters to reach their limit. Sydney rock oysters, Saccostrea glomerata, were collected from the high-intertidal and subtidal areas of the shore and exposed in an orthogonal design to either an intertidal or a subtidal treatment at ambient or elevated PCO2 , and physiological variables were measured. The combined treatment of tidal emersion and elevated PCO2  interacted synergistically to reduce the haemolymph pH (pHe) of oysters, and increase the PCO2  in the haemolymph (Pe,CO2 ) and standard metabolic rate. Oysters in the intertidal treatment also had lower condition and growth. Oysters showed a high degree of plasticity, and little evidence was found that intertidal oysters were more resilient than subtidal oysters. It is concluded that in a high-CO2 world the upper vertical limit of oyster distribution on the shore may be reduced. These results suggest that previous studies on intertidal organisms that lacked tidal simulations may have underestimated the effects of elevated PCO2.

Fast-growing oysters show reduced capacity to provide a thermal refuge to intertidal biodiversity at high temperatures.

Ecosystem engineers that modify the thermal environment experienced by associated organisms might assist in the climate change adaptation of species. This depends on the ability of ecosystem engineers to persist and continue to ameliorate thermal stress under changing climatic conditions-traits that may display significant intraspecific variation. In the physically stressful intertidal, the complex three-dimensional structure of oysters provides shading and traps moisture during aerial exposure at low tide. We assessed variation in the capacity of a faster- and slower-growing population of the Sydney Rock Oyster, Saccostrea glomerata, to persist, form three-dimensional structure and provide a cool microhabitat to invertebrates under warmer conditions. The two populations of oysters were exposed to a temperature gradient in the field by attaching them to passively warmed white, grey and black stone pavers and their growth, survivorship and colonisation by invertebrates was monitored over a 12-month period. Oysters displayed a trade-off between fast growth and thermal tolerance. The growth advantage of the fast-growing population diminished with increasing substrate temperature, and at higher temperatures, the faster-growing oysters suffered greater mortality, formed less habitat, and were consequently less effective at ameliorating low-tide air temperature extremes than slower-growing oysters. The greater survivorship of slower-growing oysters, in turn, produced a cooler microclimate which fed back to further bolster oyster survivorship. Invertebrate recruitment increased with habitat cover and was greater among the slower than the faster-growing population. Our results show that the capacity of ecosystem engineers to serve as microhabitat refugia to associated organisms in a warming climate displays marked intraspecific variation. Our study also adds to growing evidence that fast growth may come at the expense of thermal tolerance.

Adult exposure to ocean acidification is maladaptive for larvae of the Sydney rock oyster Saccostrea glomerata in the presence of multiple stressors.

Parental effects passed from adults to their offspring have been identified as a source of rapid acclimation that may allow marine populations to persist as our surface oceans continue to decrease in pH. Little is known, however, whether parental effects are beneficial for offspring in the presence of multiple stressors. We exposed adults of the oyster Saccostrea glomerata to elevated CO2 and examined the impacts of elevated CO2 (control = 392; 856 µatm) combined with elevated temperature (control = 24; 28°C), reduced salinity (control = 35; 25) and reduced food concentration (control = full; half diet) on their larvae. Adult exposure to elevated CO2 had a positive impact on larvae reared at elevated CO2 as a sole stressor, which were 8% larger and developed faster at elevated CO2 compared with larvae from adults exposed to ambient CO2 These larvae, however, had significantly reduced survival in all multistressor treatments. This was particularly evident for larvae reared at elevated CO2 combined with elevated temperature or reduced food concentration, with no larvae surviving in some treatment combinations. Larvae from CO2-exposed adults had a higher standard metabolic rate. Our results provide evidence that parental exposure to ocean acidification may be maladaptive when larvae experience multiple stressors.

Neuropeptides encoded by the genomes of the Akoya pearl oyster Pinctata fucata and Pacific oyster Crassostrea gigas: a bioinformatic and peptidomic survey.

BACKGROUND: Oysters impart significant socio-ecological benefits from primary production of food supply, to estuarine ecosystems via reduction of water column nutrients, plankton and seston biomass. Little though is known at the molecular level of what genes are responsible for how oysters reproduce, filter nutrients, survive stressful physiological events and form reef communities. Neuropeptides represent a diverse class of chemical messengers, instrumental in orchestrating these complex physiological events in other species. RESULTS: By a combination of in silico data mining and peptide analysis of ganglia, 74 putative neuropeptide genes were identified from genome and transcriptome databases of the Akoya pearl oyster, Pinctata fucata and the Pacific oyster, Crassostrea gigas, encoding precursors for over 300 predicted bioactive peptide products, including three newly identified neuropeptide precursors PFGx8amide, RxIamide and Wx3Yamide. Our findings also include a gene for the gonadotropin-releasing hormone (GnRH) and two egg-laying hormones (ELH) which were identified from both oysters. Multiple sequence alignments and phylogenetic analysis supports similar global organization of these mature peptides. Computer-based peptide modeling of the molecular tertiary structures of ELH highlights the structural homologies within ELH family, which may facilitate ELH activity leading to the release of gametes. CONCLUSION: Our analysis demonstrates that oysters possess conserved molluscan neuropeptide domains and overall precursor organization whilst highlighting many previously unrecognized bivalve idiosyncrasies. This genomic analysis provides a solid foundation from which further studies aimed at the functional characterization of these molluscan neuropeptides can be conducted to further stimulate advances in understanding the ecology and cultivation of oysters.

The interacting effects of diversity and propagule pressure on early colonization and population size.

We are now beginning to understand the role of intraspecific diversity on fundamental ecological phenomena. There exists a paucity of knowledge, however, regarding how intraspecific, or genetic diversity, may covary with other important factors such as propagule pressure. A combination of theoretical modelling and experimentation was used to explore the way propagule pressure and genetic richness may interact. We compare colonization rates of the Australian bivalve Saccostrea glomerata (Gould 1885). We cross propagule size and genetic richness in a factorial design in order to examine the generalities of our theoretical model. Modelling showed that diversity and propagule pressure should generally interact synergistically when positive feedbacks occur (e.g. aggregation). The strength of genotype effects depended on propagule size, or the numerical abundance of arriving individuals. When propagule size was very small (<4 individuals), however, greater genetic richness unexpectedly reduced colonization. The probability of S. glomerata colonization was 76% in genetically rich, larger propagules, almost 39 percentage points higher than in genetically poor propagules of similar size. This pattern was not observed in less dense, smaller propagules. We predict that density-dependent interactions between larvae in the water column may explain this pattern.

Resilience against the impacts of climate change in an ecologically and economically significant native oyster.

Climate change is acidifying and warming our oceans, at an unprecedented rate posing a challenge for marine invertebrates vital across the globe for ecological services and food security. Here we show it is possible for resilience to climate change in an ecologically and economically significant oyster without detrimental effects to the energy budget. We exposed 24 pair-mated genetically distinct families of the Sydney rock oyster, Saccostrea glomerata to ocean acidification and warming for 4w and measured their resilience. Resilience was identified as the capacity to defend their acid-base balance without a loss of energy available for Scope for Growth (SFG). Of the 24 families, 13 were better able to defend their acid-base balance while eight had no loss of energy availability with a positive SFG. This study has found oyster families with reslience against climate change without a loss of SFG, is an essential mitigation strategy, in a critical mollusc.

Intergenerational toxicity of 17α-ethinylestradiol (EE2): Effects of parental exposure on early larval development and transcriptomic profiles in the Sydney rock oyster, Saccostrea glomerata.

This study exposed adult Sydney rock oysters, of either sex or both, to the synthetic estrogen 17α-ethinylestradiol (EE2) at 50 ng/L for 21 days, followed by an examination of developmental endpoints and transcriptomic responses in unexposed larvae. Reduced survival was observed at 1 day post-fertilisation (dpf) in larvae from bi-parental exposure (FTMT). Motile larvae at 2 dpf were fewer from maternal (FTMC), paternal (FCMT), and FTMT exposures. Additionally, shell length at 7 dpf decreased in larvae from FTMC and FTMT parents. RNA sequencing (RNA-seq) revealed 1064 differentially expressed genes (DEGs) in 1-dpf larvae from FTMT parents, while fewer DEGs were detected in larvae from FTMC and FCMT parents, with 258 and 7, respectively. GO and KEGG analyses showed significant enrichment of DEGs in diverse terms and pathways, with limited overlap among treatment groups. IPA results indicated potential inhibition of pathways regulating energy production, larval development, transcription, and detoxification of reactive oxygen species in FTMT larvae. qRT-PCR validation confirmed significant downregulation of selected DEGs involved in these pathways and relevant biological processes, as identified in the RNA-seq dataset. Overall, our results suggest that the intergenerational toxicity of EE2 is primarily maternally transmitted, with bi-parental exposure amplifying these effects.

Organic enrichment reduces sediment bacterial and archaeal diversity, composition, and functional profile independent of bioturbator activity.

Eutrophication is a worldwide issue that can disrupt ecosystem processes in sediments. Studies have shown that macrofauna influences sediment processes by engineering environments that constrain microbial communities. Here, we explored the effect of different sizes of the Sydney cockle (Anadara trapezia), on bacterial and archaeal communities in natural and experimentally enriched sediments. A mesocosm experiment was conducted with two enrichment conditions (natural or enriched) and 5 cockle treatments (small, medium, large, mixed sizes and a control). This study was unable to detect A. trapezia effects on microbial communities irrespective of body size. However, a substantial decrease of bacterial richness, diversity, and structural and functional shifts, were seen with organic enrichment of sediments. Archaea were similarly changed although the magnitude of effect was less than for bacteria. Overall, we found evidence to suggest that A. trapezia had limited capacity to affect sediment microbial communities and mitigate the effects of organic enrichment.

Estrogenic mixtures induce alterations in lipidomic profiles in the gonads of female oysters.

This study aimed to reveal possible alterations to lipidomic profiles in Sydney rock oysters, Saccostrea glomerata, exposed to estrogenic mixtures (i.e., estrone, E1; 17ß-estradiol, E2; estriol, E3; 17α-ethinylestradiol, EE2; bisphenol A, BPA; 4-t-octylphenol, 4-t-OP; and 4-nonylphenol, 4-NP) at "low" and "high" concentrations, typical of those detected in Australian and global receiving waters. A seven-day acute exposure window exhibited significantly lower abundances of many non-polar metabolites in digestive gland, gills, and gonads. Overall, there was a strong effect of the carrier solvent ethanol (despite a low exposure of 0.0002%), with all solvent containing treatments exhibiting lower abundances of lipidic metabolites, especially in the gill and digestive gland. No significant changes of the lipidome were exhibited in the male gonad by estrogenic exposure. However, in the female gonad, significant reductions of phospholipids and phosphatidylcholine were associated with exposure to high estrogenic mixtures. We hypothesise that the decreases in these phospholipids in the female gonad may be attributable to 1) lower algal consumption and thus lower uptake of lipidic building blocks; 2) a reduction of available substrates for phospholipid and phosphatidylcholine synthesis; and/or 3) induction of reactive oxygen species via estrogen metabolism, which may cause lipid peroxidation and lower abundance of phospholipids.

Microbiomes of an oyster are shaped by metabolism and environment.

Microbiomes can both influence and be influenced by metabolism, but this relationship remains unexplored for invertebrates. We examined the relationship between microbiome and metabolism in response to climate change using oysters as a model marine invertebrate. Oysters form economies and ecosystems across the globe, yet are vulnerable to climate change. Nine genetic lineages of the oyster Saccostrea glomerata were exposed to ambient and elevated temperature and PCO2 treatments. The metabolic rate (MR) and metabolic by-products of extracellular pH and CO2 were measured. The oyster-associated bacterial community in haemolymph was characterised using 16 s rRNA gene sequencing. We found a significant negative relationship between MR and bacterial richness. Bacterial community composition was also significantly influenced by MR, extracellular CO2 and extracellular pH. The effects of extracellular CO2 depended on genotype, and the effects of extracellular pH depended on CO2 and temperature treatments. Changes in MR aligned with a shift in the relative abundance of 152 Amplicon Sequencing Variants (ASVs), with 113 negatively correlated with MR. Some spirochaete ASVs showed positive relationships with MR. We have identified a clear relationship between host metabolism and the microbiome in oysters. Altering this relationship will likely have consequences for the 12 billion USD oyster economy.

Body size affects lethal and sublethal responses to organic enrichment: Evidence of associational susceptibility for an infaunal bivalve.

Eutrophication is an increasing problem worldwide and can disrupt ecosystem processes in which macrobenthic bioturbators play an essential role. This study explores how intraspecific variation in body size affects the survival, mobility and impact on sediment organic matter breakdown in enriched sediments of an infaunal bivalve. A mesocosm experiment was conducted in which monocultures and all size combinations of three body sizes (small, medium and large) of the Sydney cockle, Anadara trapezia, were exposed to natural or organically enriched sediments. Results demonstrate that larger body sizes have higher tolerance to enriched conditions and can reduce survival of smaller cockles when grown together. Also, large A. trapezia influenced sediment organic matter breakdown although a direct link to bioturbation activity was not clear. Overall, this study found that intraspecific variation in body size influences survival and performance of bioturbators in eutrophic scenarios.

Energetic lipid responses of larval oysters to ocean acidification.

Climate change will increase energetic demands on marine invertebrate larvae and make planktonic food more unpredictable. This study determined the impact of ocean acidification on larval energetics of the oysters Saccostrea glomerata and Crassostrea gigas. Larvae of both oysters were reared until the 9-day-old, umbonate stage under orthogonal combinations of ambient and elevated p CO 2 (340 and 856 µatm) and food was limited. Elevated p CO 2 reduced the survival, size and larval energetics, larvae of C. gigas being more resilient than S. glomerata. When larvae were fed, elevated p CO 2 reduced lipid levels across all lipid classes. When larvae were unfed elevated p CO 2 resulted in increased lipid levels and mortality. Ocean acidification and food will interact to limit larval energetics. Larvae of S. glomerata will be more impacted than C. gigas and this is of concern given their aquacultural status and ecological function.

Climate change alters the haemolymph microbiome of oysters.

The wellbeing of marine organisms is connected to their microbiome. Oysters are a vital food source and provide ecological services, yet little is known about how climate change such as ocean acidification and warming will affect their microbiome. We exposed the Sydney rock oyster, Saccostrea glomerata, to orthogonal combinations of temperature (24, 28 °C) and pCO2 (400 and 1000 µatm) for eight weeks and used amplicon sequencing of the 16S rRNA (V3-V4) gene to characterise the bacterial community in haemolymph. Overall, elevated pCO2 and temperature interacted to alter the microbiome of oysters, with a clear partitioning of treatments in CAP ordinations. Elevated pCO2 was the strongest driver of species diversity and richness and elevated temperature also increased species richness. Climate change, both ocean acidification and warming, will alter the microbiome of S. glomerata which may increase the susceptibility of oysters to disease.

Exposure to estrogenic mixtures results in tissue-specific alterations to the metabolome of oysters.

The current study investigated the effect of environmentally relevant mixtures of estrogens at levels representative of receiving waters on the metabolome of the Sydney rock oyster, Saccostrea glomerata. Oysters were exposed to a "low" and a "high" mixture of (xeno) estrogens (representative of Australian and global receiving waters respectively) for 7 days and digestive gland, gill, and gonad tissue were sampled for quantification of polar metabolites by 1H NMR spectroscopy. Exposure to both mixtures lowered body mass and altered the metabolite profile in the digestive glands. Comparatively, gills, and ovaries demonstrated lesser sensitivity to the mixtures, with significant metabolomic alterations observed only for the high mixture. The male gonad did not respond to either estrogenic exposure. In the responsive tissues, major metabolites including amino acids, carbohydrates, intermediates of the tricarboxylic acid cycle and ATP were all down-regulated and exhibited tissue-specific patterns of down-regulation with the greatest proportion of metabolites down-regulated due to estrogenic exposure in the digestive gland. Exposure to (xeno) estrogen mixtures representative of concentrations reported in receiving waters in Australia and globally can impact the metabolome and associated energy metabolism, especially in the digestive gland, translating to lower pools of available ATP energy for potential cellular homeostasis, somatic maintenance and growth, reproduction and fitness.

Microbiome response differs among selected lines of Sydney rock oysters to ocean warming and acidification.

Oyster microbiomes are integral to healthy function and can be altered by climate change conditions. Genetic variation among oysters is known to influence the response of oysters to climate change and may ameliorate any adverse effects on oyster microbiome; however, this remains unstudied. Nine full-sibling selected breeding lines of the Sydney rock oyster (Saccostrea glomerata) were exposed to predicted warming (ambient = 24°C, elevated = 28°C) and ocean acidification (ambient pCO2 = 400, elevated pCO2 = 1000 µatm) for 4 weeks. The haemolymph bacterial microbiome was characterized using 16S rRNA (V3-V4) gene sequencing and varied among oyster lines in the control (ambient pCO2, 24°C) treatment. Microbiomes were also altered by climate change dependent on oyster lines. Bacterial α-diversity increased in response to elevated pCO2 in two selected lines, while bacterial ß-diversity was significantly altered by combinations of elevated pCO2 and temperature in four selected lines. Climate change treatments caused shifts in the abundance of multiple amplicon sequence variants driving change in the microbiome of some selected lines. We show that oyster genetic background may influence the Sydney rock oyster haemolymph microbiome under climate change and that future assisted evolution breeding programs to enhance resilience should consider the oyster microbiome.

Heatwaves alter survival of the Sydney rock oyster, Saccostrea glomerata.

Heatwaves are an increasing threat to organisms across the globe. Marine and atmospheric heatwaves are predicted to impact sessile intertidal marine organisms, especially when exposed at low tide and unable to seek refuge. The study aimed to determine whether a simulated atmospheric heatwave will alter the survival of selectively bred families of Sydney rock oysters (Saccostrea glomerata), and whether survival is dependent on morphological and physiological traits. The survival of S. glomerata families to a simulated atmospheric heatwave varied from 25 to 60% and was not correlated with morphology or physiology. Survival may depend on the presence of genotypes that translate into molecular defenses such as heat-shock proteins and inhibitor of apoptosis proteins that provide oysters with resilience. Understanding the responses among families of oysters to heatwaves is critical if we are to restore the ecological services of oyster reefs and sustain oyster aquaculture.

Parental exposure to the synthetic estrogen 17α-ethinylestradiol (EE2) affects offspring development in the Sydney rock oyster, Saccostrea glomerata.

Very little is currently known regarding the effects of estrogenic endocrine disrupting chemicals on embryonic and larval development in molluscs, nor the potential effects of parental (F0) exposure on resultant F1 offspring. In this study, we assessed the embryotoxic impacts of exposure to environmentally relevant concentrations of the synthetic estrogen, 17α-ethinylestradiol (EE2), to male and female parents (50 ng/L) and their offspring (5 and 50 ng/L) in the native Australian Sydney rock oyster, Saccostrea glomerata. There were no detectable effects of parental exposure on fertilisation success, proportions of early larval (F1) morphs and unfertilised eggs. Offspring impacts were evidenced in terms of developmental delays, with decreased percentages of D-veligers retained by 45 µm mesh, along with a reduction of swimming capabilities of larvae at 2 days post-fertilisation (dpf) when both parents had been exposed to 50 ng/L EE2. Although no significant parental effects were found on the survival of F1 larvae at 9 dpf, retardation of shell growth was observed on F1 larvae in treatments where both parents had been exposed to 50 ng/L EE2. Subsequent larval exposure from 2 to 9 dpf caused declines in survival and reduction of shell length in F1 larvae at both 5 and 50 ng/L EE2 across all parental exposure treatments. Collectively, parental EE2 imparts effects on offspring in terms of retardation of larval development, and subsequent offspring exposure to EE2 further exacerbates impacts to development. Future research should aim to understand the potential mechanisms of EE2 induced toxicity and its transmission resulting in altered phenotypes of the F1 generation.