Triploid Pacific oysters exhibit stress response dysregulation and elevated mortality following heatwaves.

Polyploidy has been suggested to negatively impact environmental stress tolerance, resulting in increased susceptibility to extreme climate events. In this study, we compared the genomic and physiological response of diploid (2n) and triploid (3n) Pacific oysters (Crassostrea gigas) to conditions present during an atmospheric heatwave that impacted the Pacific Northwestern region of the United States in the summer of 2021. Climate stressors were applied either singly (single stressor; elevated seawater temperature, 30°C) or in succession (multiple stressor; elevated seawater temperature followed by aerial emersion at 44°C), replicating conditions present within the intertidal over a tidal cycle during the event. Oyster mortality rate was elevated within stress treatments with respect to the control and was significantly higher in triploids than diploids following multiple stress exposure (36.4% vs. 14.8%). Triploids within the multiple stressor treatment exhibited signs of energetic limitation, including metabolic depression, a significant reduction in ctenidium Na+ /K+ ATPase activity, and the dysregulated expression of genes associated with stress response, innate immunity, glucose metabolism, and mitochondrial function. Functional enrichment analysis of ploidy-specific gene sets identified that biological processes associated with metabolism, stress tolerance, and immune function were overrepresented within triploids across stress treatments. Our results suggest that triploidy impacts the transcriptional regulation of key processes that underly the stress response of Pacific oysters, resulting in downstream shifts in physiological tolerance limits that may increase susceptibility to extreme climate events that present multiple environmental stressors. The impact of chromosome set manipulation on the climate resilience of marine organisms has important implications for domestic food security within future climate scenarios, especially as triploidy induction becomes an increasingly popular tool to elicit reproductive control across a wide range of species used within marine aquaculture.

Proteomic response of early juvenile Pacific oysters (Crassostrea gigas) to temperature.

Pacific oysters (Crassostrea gigas) are a valuable aquaculture product that provides important ecosystem benefits. Among other threats, climate-driven changes in ocean temperature can impact oyster metabolism, survivorship, and immune function. We investigated how elevated temperature impacts larval oysters during settlement (19-33 days post-fertilization), using shotgun proteomics with data-independent acquisition to identify proteins present in the oysters after 2 weeks of exposure to 23 °C or 29 °C. Oysters maintained at elevated temperatures were larger and had a higher settlement rate, with 86% surviving to the end of the experiment; these oysters also had higher abundance trends of proteins related to metabolism and growth. Oysters held at 23 °C were smaller, had a decreased settlement rate, displayed 100% mortality, and had elevated abundance trends of proteins related to immune response. This novel use of proteomics was able to capture characteristic shifts in protein abundance that hint at important differences in the phenotypic response of Pacific oysters to temperature regimes. Additionally, this work has produced a robust proteomic product that will be the basis for future research on bivalve developmental processes.

Acclimatory gene expression of primed clams enhances robustness to elevated pCO2.

Sublethal exposure to environmental challenges may enhance ability to cope with chronic or repeated change, a process known as priming. In a previous study, pre-exposure to seawater enriched with pCO2 improved growth and reduced antioxidant capacity of juvenile Pacific geoduck Panopea generosa clams, suggesting that transcriptional shifts may drive phenotypic modifications post-priming. To this end, juvenile clams were sampled and TagSeq gene expression data were analysed after (i) a 110-day acclimation under ambient (921 µatm, naïve) and moderately elevated pCO2 (2870 µatm, pre-exposed); then following (ii) a second 7-day exposure to three pCO2 treatments (ambient: 754 µatm; moderately elevated: 2750 µatm; severely elevated: 4940 µatm), a 7-day return to ambient pCO2 and a third 7-day exposure to two pCO2 treatments (ambient: 967 µatm; moderately elevated: 3030 µatm). Pre-exposed geoducks frontloaded genes for stress and apoptosis/innate immune response, homeostatic processes, protein degradation and transcriptional modifiers. Pre-exposed geoducks were also responsive to subsequent encounters, with gene sets enriched for mitochondrial recycling and immune defence under elevated pCO2 and energy metabolism and biosynthesis under ambient recovery. In contrast, gene sets with higher expression in naïve clams were enriched for fatty-acid degradation and glutathione components, suggesting naïve clams could be depleting endogenous fuels, with unsustainable energetic requirements if changes in carbonate chemistry persist. Collectively, our transcriptomic data indicate that pCO2 priming during post-larval periods could, via gene expression regulation, enhance robustness in bivalves to environmental change. Such priming approaches may be beneficial for aquaculture, as seafood demand intensifies concurrent with increasing climate change in marine systems.

Repeat exposure to hypercapnic seawater modifies growth and oxidative status in a tolerant burrowing clam.

Although low levels of thermal stress, irradiance and dietary restriction can have beneficial effects for many taxa, stress acclimation remains little studied in marine invertebrates, even though they are threatened by climate change stressors such as ocean acidification. To test the role of life-stage and stress-intensity dependence in eliciting enhanced tolerance under subsequent stress encounters, we initially conditioned pediveliger Pacific geoduck (Panopea generosa) larvae to ambient and moderately elevated PCO2 (920 µatm and 2800 µatm, respectively) for 110 days. Then, clams were exposed to ambient, moderate or severely elevated PCO2 (750, 2800 or 4900 µatm, respectively) for 7 days and, following 7 days in ambient conditions, a 7-day third exposure to ambient (970 µatm) or moderate PCO2 (3000 µatm). Initial conditioning to moderate PCO2 stress followed by second and third exposure to severe and moderate PCO2 stress increased respiration rate, organic biomass and shell size, suggesting a stress-intensity-dependent effect on energetics. Additionally, stress-acclimated clams had lower antioxidant capacity compared with clams under ambient conditions, supporting the hypothesis that stress over postlarval-to-juvenile development affects oxidative status later in life. Time series and stress intensity-specific approaches can reveal life-stages and magnitudes of exposure, respectively, that may elicit beneficial phenotypic variation.

Coupled microbiome analyses highlights relative functional roles of bacteria in a bivalve hatchery.

BACKGROUND: Microbial communities are ubiquitous throughout ecosystems and are commensal with hosts across taxonomic boundaries. Environmental and species-specific microbiomes are instrumental in maintaining ecosystem and host health, respectively. The introduction of pathogenic microbes that shift microbiome community structure can lead to illness and death. Understanding the dynamics of microbiomes across a diversity of environments and hosts will help us to better understand which taxa forecast survival and which forecast mortality events. RESULTS: We characterized the bacterial community microbiome in the water of a commercial shellfish hatchery in Washington state, USA, where the hatchery has been plagued by recurring and unexplained larval mortality events. By applying the complementary methods of metagenomics and metaproteomics we were able to more fully characterize the bacterial taxa in the hatchery at high (pH 8.2) and low (pH 7.1) pH that were metabolically active versus present but not contributing metabolically. There were shifts in the taxonomy and functional profile of the microbiome between pH and over time. Based on detected metagenomic reads and metaproteomic peptide spectral matches, some taxa were more metabolically active than expected based on presence alone (Deltaproteobacteria, Alphaproteobacteria) and some were less metabolically active than expected (e.g., Betaproteobacteria, Cytophagia). There was little correlation between potential and realized metabolic function based on Gene Ontology analysis of detected genes and peptides. CONCLUSION: The complementary methods of metagenomics and metaproteomics contribute to a more full characterization of bacterial taxa that are potentially active versus truly metabolically active and thus impact water quality and inter-trophic relationships.

Temporal proteomic profiling reveals insight into critical developmental processes and temperature-influenced physiological response differences in a bivalve mollusc.

BACKGROUND: Protein expression patterns underlie physiological processes and phenotypic differences including those occurring during early development. The Pacific oyster (Crassostrea gigas) undergoes a major phenotypic change in early development from free-swimming larval form to sessile benthic dweller while proliferating in environments with broad temperature ranges. Despite the economic and ecological importance of the species, physiological processes occurring throughout metamorphosis and the impact of temperature on these processes have not yet been mapped out. RESULTS: Towards this, we comprehensively characterized protein abundance patterns for 7978 proteins throughout metamorphosis in the Pacific oyster at different temperature regimes. We used a multi-statistical approach including principal component analysis, ANOVA-simultaneous component analysis, and hierarchical clustering coupled with functional enrichment analysis to characterize these data. We identified distinct sets of proteins with time-dependent abundances generally not affected by temperature. Over 12 days, adhesion and calcification related proteins acutely decreased, organogenesis and extracellular matrix related proteins gradually decreased, proteins related to signaling showed sinusoidal abundance patterns, and proteins related to metabolic and growth processes gradually increased. Contrastingly, different sets of proteins showed temperature-dependent abundance patterns with proteins related to immune response showing lower abundance and catabolic pro-growth processes showing higher abundance in animals reared at 29 °C relative to 23 °C. CONCLUSION: Although time was a stronger driver than temperature of metamorphic proteome changes, temperature-induced proteome differences led to pro-growth physiology corresponding to larger oyster size at 29 °C, and to altered specific metamorphic processes and possible pathogen presence at 23 °C. These findings offer high resolution insight into why oysters may experience high mortality rates during this life transition in both field and culture settings. The proteome resource generated by this study provides data-driven guidance for future work on developmental changes in molluscs. Furthermore, the analytical approach taken here provides a foundation for effective shotgun proteomic analyses across a variety of taxa.

Larval Geoduck (Panopea generosa) Proteomic Response to Ciliates.

The innate immune response is active in invertebrate larvae from early development. Induction of immune response pathways may occur as part of the natural progression of larval development, but an up-regulation of pathways can also occur in response to a pathogen. Here, we took advantage of a protozoan ciliate infestation of a larval geoduck clam culture in a commercial hatchery to investigate the molecular underpinnings of the innate immune response of the larvae to the pathogen. Larval proteomes were analyzed on days 4-10 post-fertilization; ciliates were present on days 8 and 10 post-fertilization. Through comparisons with larval cultures that did not encounter ciliates, proteins implicated in the response to ciliate presence were identified using mass spectrometry-based proteomics. Ciliate response proteins included many associated with ribosomal synthesis and protein translation, suggesting the importance of protein synthesis during the larval immune response. There was also an increased abundance of proteins typically associated with the stress and immune responses during ciliate exposure, such as heat shock proteins, glutathione metabolism, and the reactive oxygen species response. These findings provide a basic understanding of the bivalve molecular response to a mortality-inducing ciliate and improved characterization of the ontogenetic development of the innate immune response.

Metabolic recovery and compensatory shell growth of juvenile Pacific geoduck Panopea generosa following short-term exposure to acidified seawater.

While acute stressors can be detrimental, environmental stress conditioning can improve performance. To test the hypothesis that physiological status is altered by stress conditioning, we subjected juvenile Pacific geoduck, Panopea generosa, to repeated exposures of elevated pCO2 in a commercial hatchery setting followed by a period in ambient common garden. Respiration rate and shell length were measured for juvenile geoduck periodically throughout short-term repeated reciprocal exposure periods in ambient (~550 µatm) or elevated (~2400 µatm) pCO2 treatments and in common, ambient conditions, 5 months after exposure. Short-term exposure periods comprised an initial 10-day exposure followed by 14 days in ambient before a secondary 6-day reciprocal exposure. The initial exposure to elevated pCO2 significantly reduced respiration rate by 25% relative to ambient conditions, but no effect on shell growth was detected. Following 14 days in common garden, ambient conditions, reciprocal exposure to elevated or ambient pCO2 did not alter juvenile respiration rates, indicating ability for metabolic recovery under subsequent conditions. Shell growth was negatively affected during the reciprocal treatment in both exposure histories; however, clams exposed to the initial elevated pCO2 showed compensatory growth with 5.8% greater shell length (on average between the two secondary exposures) after 5 months in ambient conditions. Additionally, clams exposed to the secondary elevated pCO2 showed 52.4% increase in respiration rate after 5 months in ambient conditions. Early exposure to low pH appears to trigger carryover effects suggesting bioenergetic re-allocation facilitates growth compensation. Life stage-specific exposures to stress can determine when it may be especially detrimental, or advantageous, to apply stress conditioning for commercial production of this long-lived burrowing clam.

Dynamic response in the larval geoduck (Panopea generosa) proteome to elevated pCO2.

Pacific geoducks (Panopea generosa) are clams found along the northeast Pacific coast where they are important components of coastal and estuarine ecosystems and a major aquaculture product. The Pacific coastline, however, is also experiencing rapidly changing ocean habitat, including significant reductions in pH. To better understand the physiological impact of ocean acidification on geoduck clams, we characterized for the first time the proteomic profile of this bivalve during larval development and compared it to that of larvae exposed to low pH conditions. Geoduck larvae were reared at pH 7.5 (ambient) or pH 7.1 in a commercial shellfish hatchery from day 6 to day 19 postfertilization and sampled at six time points for an in-depth proteomics analysis using high-resolution data-dependent analysis. Larvae reared at low pH were smaller than those reared at ambient pH, especially in the prodissoconch II phase of development, and displayed a delay in their competency for settlement. Proteomic profiles revealed that metabolic, cell cycle, and protein turnover pathways differed between the two pH and suggested that differing phenotypic outcomes between pH 7.5 and 7.1 are likely due to environmental disruptions to the timing of physiological events. In summary, ocean acidification results in elevated energetic demand on geoduck larvae, resulting in delayed development and disruptions to normal molecular developmental pathways, such as carbohydrate metabolism, cell growth, and protein synthesis.

Transcriptomic and metabolic response to chronic and acute thermal exposure of juvenile geoduck clams Panopea globosa.

The Cortes geoduck Panopea globosa is a large bivalve with a high commercial value distributed from the southern Pacific coast of the Baja California Peninsula to the northern Gulf of California, inhabiting a wide range of subtropical temperatures. A new record of this species in shallower waters suggests that it can tolerate a warmer environment than previously thought. To better understand the whole-body and molecular response mechanisms to different temperatures, we assessed the metabolic rate of juvenile individuals exposed to chronic and acute thermal conditions and analyzed the transcriptomic response in ctenidial tissues. Whole-body metabolic rate measurements showed that juveniles were able to acclimate at least partially within three weeks from 20 °C (C20) to 29 °C (C29), while organisms acutely exposed to 29 °C (A29) significantly increased their metabolic rate. This was coincident with transcriptomic results, as similar gene expression patterns were found in clams chronically exposed to C29 and C20, but different from those acutely exposed to 29 °C (A29) and 31 °C (A31). High degree of expression of genes involved in DNA repair and transcription regulation were found in C29 juveniles, whereas protective genes against oxidative stress were highly expressed in A29 organisms. A high expression of genes involved in protein re-folding was also observed in A31 juveniles. In conclusion, the combined results of whole-body metabolism and transcriptomic expression patterns suggest that P. globosa juveniles have a high physiological plasticity and are well adapted to inhabit an environment with broad temperature fluctuations.

Differential response to stress in Ostrea lurida as measured by gene expression.

Olympia oysters are the only oyster native to the west coast of North America. The population within Puget Sound, WA has been decreasing significantly since the early 1900's. Current restoration efforts are focused on supplementing local populations with hatchery bred oysters. A recent study by Heare et al. (2017) has shown differences in stress response in oysters from different locations in Puget Sound however, nothing is known about the underlying mechanisms associated with these observed differences. In this study, expression of genes associated with growth, immune function, and gene regulatory activity in oysters from Oyster Bay, Dabob Bay, and Fidalgo Bay were characterized following temperature and mechanical stress. We found that heat stress and mechanical stress significantly changed expression in molecular regulatory activity and immune response, respectively. We also found that oysters from Oyster Bay had the most dramatic response to stress at the gene expression level. These data provide important baseline information on the physiological response of Ostrea lurida to stress and provide clues to underlying performance differences in the three populations examined.

Genotoype-by-sequencing of three geographically distinct populations of Olympia oysters, Ostrea lurida.

Olympia oysters are found along the west coast of North America and as the only native oyster species in the region, receive considerable attention with regard to restoration and conservation. Knowledge of genetic structure of this species is essential for resource managers. Here we provide genetic data for three distinct populations of Olympia oysters in Puget Sound, Washington, USA in the form of genotype-by-sequencing data (GBS). Specifically, this includes description of sequence data and a derived table that provides single nucleotide polymorphism (SNP) information for 10,363 loci. These data are valuable not only for resource managers responsible for restoration aquaculture practices, but can provide insight into ecological drivers of selection and diversity.

Integrating Discovery-driven Proteomics and Selected Reaction Monitoring To Develop a Noninvasive Assay for Geoduck Reproductive Maturation.

Geoduck clams (Panopea generosa) are an increasingly important fishery and aquaculture product along the eastern Pacific coast from Baja California, Mexico, to Alaska. These long-lived clams are highly fecund, although sustainable hatchery production of genetically diverse larvae is hindered by the lack of sexual dimorphism, resulting in asynchronous spawning of broodstock, unequal sex ratios, and low numbers of breeders. The development of assays of gonad physiology could indicate sex and maturation stage as well as be used to assess the status of natural populations. Proteomic profiles were determined for three reproductive maturation stages in both male and female clams using data-dependent acquisition (DDA) of gonad proteins. Gonad proteomes became increasingly divergent between males and females as maturation progressed. The DDA data were used to develop targets analyzed with selected reaction monitoring (SRM) in gonad tissue as well as hemolymph. The SRM assay yielded a suite of indicator peptides that can be used as an efficient assay to determine geoduck gonad maturation status. Application of SRM in hemolymph samples demonstrates that this procedure could effectively be used to assess reproductive status in marine mollusks in a nonlethal manner.

Development and validation of a quantitative PCR assay for Ichthyophonus spp.

Members of the genus Ichthyophonus are trophically transmitted, cosmopolitan parasites that affect numerous fish species worldwide. A quantitative PCR (qPCR) assay specific for genus Ichthyophonus 18S ribosomal DNA was developed for parasite detection and surveillance. The new assay was tested for precision, repeatability, reproducibility, and both analytical sensitivity and specificity. Diagnostic sensitivity and specificity were estimated using tissue samples from a wild population of walleye pollock Theragra chalcogramma. Ichthyophonus sp. presence in tissue samples was determined by qPCR, conventional PCR (cPCR), and histology. Parasite prevalence estimates varied depending upon the detection method employed and tissue type tested. qPCR identified the greatest number of Ichthyophonus sp.-positive cases when applied to walleye pollock skeletal muscle. The qPCR assay proved sensitive and specific for Ichthyophonus spp. DNA, but like cPCR, is only a proxy for infection. When compared to cPCR, qPCR possesses added benefits of parasite DNA quantification and a 100-fold increase in analytical sensitivity. Because this novel assay is specific for known members of the genus, it is likely appropriate for detecting Ichthyophonus spp. DNA in various hosts from multiple regions. However, species-level identification and isotype variability would require DNA sequencing. In addition to distribution and prevalence applications, this assay could be modified and adapted for use with zooplankton or environmental samples. Such applications could aid in investigating alternate routes of transmission and life history strategies typical to members of the genus Ichthyophonus.