The Genetics of Larval Fitness in the Pacific Oyster: Responses to Acidified Seawater and Temporally Dynamic Selection Processes

The Pacific Oyster (Crassostrea gigas) is one of the most economically and ecologically significant shellfish species worldwide. In the Pacific Northwest United States (PNW), the sustainability oyster stocks is increasingly threatened by ocean acidification (OA), which has had significant negative effects on the aquaculture industry in this region over the last decade. Currently, little is known with regards to stock-based differences in larval fitness for PNW populations of C. gigas in ambient or high pCO2 conditions. Furthermore, no studies have been performed that evaluate the genetic consequences of larval rearing in acidified seawater for Pacific oysters. Here I examined genetic components of larval fitness traits, both in ambient seawater as well as simulated OA conditions. In Chapter 2 my co-authors and I conducted two experiments to compare the relative fitness of larvae from selectively bred aquaculture stocks to those spawned from a naturalized source of broodstock in Willapa Bay, WA. We reared genetically diverse pools of larvae from each group in ambient (~400 μatm) and high (~1600 μatm) pCO2 seawater for 22-24 days from fertilization through settlement to juvenile stage. Overall, we found that the impacts of high pCO2 seawater on larval phenotypes were heterogeneous across larval developmental stages and variable between the two experiments. Nevertheless, larvae from selectively bred lines had consistently higher survival and greater developmental success through settlement than those from naturalized stocks across both conditions and experiments. In Chapter 3 we analyzed the overall changes in genetic composition of each larval pool created in the first experiment. We found an abundance of loci with significantly distorted allele frequencies across larval development, nearly all of which were specific to each broodstock type. There were additional genetic changes owing to high pCO2 culture that, also, were largely unique to each parental group. Overall, larvae from selectively bred stocks had significantly less genetic change than those from naturalized populations, both for general larval development as well as survival in acidified seawater. We performed functional enrichment analyses on genes associated with distorted loci and found that those with altered allele frequencies in acidified seawater were significantly over-represented by gene-ontology categories concerning membrane structure and function. This finding is consistent with previous studies which also highlighted this aspect of larval physiology as a critical component for survival in acidified conditions. The reduced genetic change in larvae from selected lines suggests that multiple generations of hatchery culture has had a domesticating effect on the genotypes of selectively bred oyster stocks. These results also support the phenotypic results from Chapter 2 which suggested that general improvements to larval fitness have crossover benefits for environmental stressors like OA. In Chapter 4 we further investigated the dynamic genetic changes taking place in larval populations during development, absent any overt environmental stress. By analyzing temporal patterns of changes in allele frequencies across development, we observed that genetic changes were highly dynamic, with more than a quarter that displayed significant shifts in allele frequencies changing in direction across developmental transitions. Importantly, this resulted in the detection of substantially more genetic changes taking place than were indicated from overall analyses from ‘start’ and ‘end’ points in larval culture examined in Chapter 3. These findings represent a novel and unparalleled analysis of the genetic impacts of genotype-dependent mortality in oysters. The patterns of viability selection that we describe likely have extensive consequences on the genetic diversity of this species, and its long term capacity for adaptation

Comparison of larval development in domesticated and naturalized stocks of the Pacific oyster Crassostrea gigas exposed to high pCO₂ conditions

Ocean acidification (OA) has had significant negative effects on oyster populations on the west coast of North America over the past decade. Many studies have focused on the physiological challenges experienced by young oyster larvae in high pCO₂/low pH seawater with reduced aragonite saturation state (Ωarag), which is characteristic of OA. Relatively few, by contrast, have evaluated these impacts upon fitness traits across multiple larval stages and between discrete oyster populations. In this study, we conducted 2 replicated experiments, in 2015 and 2016, using larvae from naturalized ‘wild’ and selectively bred stocks of the Pacific oyster Crassostrea gigas from the US Pacific Northwest and reared them in ambient (~400 µatm) or high (~1600 µatm) pCO₂ seawater from fertilization through final metamorphosis to juvenile ‘spat.’ In each year, high pCO₂ seawater inhibited early larval development and affected the timing, but not the magnitude, of mortality during this stage. The effects of acidified seawater on metamorphosis of pediveligers to spat were variable between years, with no effect of seawater pCO₂ in the first experiment but a ~42% reduction in spat in the second. Despite this variability, larvae from selectively bred oysters produced, on average, more (+ 55 and 37%) and larger (+ 5 and 23%) spat in ambient and high pCO₂ seawater, respectively. These findings highlight the variable and stage-specific sensitivity of larval oysters to acidified seawater and the influence that genetic factors have in determining the larval performance of C. gigas exposed to high pCO₂ seawater

Larval development in the Pacific oyster and the impacts of ocean acidification : differential genetic effects in wild and domesticated stocks

The adaptive capacity of marine calcifiers to ocean acidification (OA) is a topic of great interest to evolutionary biologists and ecologists. Previous studies have provided evidence to suggest that larval resilience to high pCO2 seawater for these species is a trait with a genetic basis and variability in natural populations. To date, however, it remains unclear how the selective effects of OA occur within the context of complex genetic interactions underpinning larval development in many of the most vulnerable taxa. Here we evaluated phenotypic and genetic changes during larval development of Pacific oysters (Crassostrea gigas) reared in ambient (~400 µatm) and high (~1600 µatm) pCO2 conditions, both in domesticated and naturalized “wild” oysters from the Pacific Northwest, USA. Using pooled DNA samples, we determined changes in allele frequencies across larval development, from early “D-stage” larvae to metamorphosed juveniles (spat), in both groups and environments. Domesticated larvae had ~26% fewer loci with changing allele frequencies across developmental stages and <50% as many loci affected by acidified culture conditions, compared to larvae from wild broodstock. Functional enrichment analyses of genetic markers with significant changes in allele frequency revealed that the structure and function of cellular membranes were disproportionately affected by high pCO2 conditions in both groups. These results indicate the potential for a rapid adaptive response of oyster populations to OA conditions; however, underlying genetic changes associated with larval development differ between these wild and domesticated oyster stocks and influence their adaptive responses to OA conditions

Gene expression correlated with delay in shell formation in larval Pacific oysters (Crassostrea gigas) exposed to experimental ocean acidification provides insights into shell formation mechanisms

Background: Despite recent work to characterize gene expression changes associated with larval development in oysters, the mechanism by which the larval shell is first formed is still largely unknown. In Crassostrea gigas, this shell forms within the first 24 h post fertilization, and it has been demonstrated that changes in water chemistry can cause delays in shell formation, shell deformations and higher mortality rates. In this study, we use the delay in shell formation associated with exposure to CO2-acidified seawater to identify genes correlated with initial shell deposition. Results: By fitting linear models to gene expression data in ambient and low aragonite saturation treatments, we are able to isolate 37 annotated genes correlated with initial larval shell formation, which can be categorized into 1) ion transporters, 2) shell matrix proteins and 3) protease inhibitors. Clustering of the gene expression data into co-expression networks further supports the result of the linear models, and also implies an important role of dynein motor proteins as transporters of cellular components during the initial shell formation process. Conclusions: Using an RNA-Seq approach with high temporal resolution allows us to identify a conceptual model for how oyster larval calcification is initiated. This work provides a foundation for further studies on how genetic variation in these identified genes could affect fitness of oyster populations subjected to future environmental changes, such as ocean acidification

Gene expression correlated with delay in shell formation in larval Pacific oysters (Crassostrea gigas) exposed to experimental ocean acidification provides insights into shell formation mechanisms

Background: Despite recent work to characterize gene expression changes associated with larval development in oysters, the mechanism by which the larval shell is first formed is still largely unknown. In Crassostrea gigas, this shell forms within the first 24 h post fertilization, and it has been demonstrated that changes in water chemistry can cause delays in shell formation, shell deformations and higher mortality rates. In this study, we use the delay in shell formation associated with exposure to CO2-acidified seawater to identify genes correlated with initial shell deposition. Results: By fitting linear models to gene expression data in ambient and low aragonite saturation treatments, we are able to isolate 37 annotated genes correlated with initial larval shell formation, which can be categorized into 1) ion transporters, 2) shell matrix proteins and 3) protease inhibitors. Clustering of the gene expression data into co-expression networks further supports the result of the linear models, and also implies an important role of dynein motor proteins as transporters of cellular components during the initial shell formation process. Conclusions: Using an RNA-Seq approach with high temporal resolution allows us to identify a conceptual model for how oyster larval calcification is initiated. This work provides a foundation for further studies on how genetic variation in these identified genes could affect fitness of oyster populations subjected to future environmental changes, such as ocean acidification.Keywords: Crassostrea gigas, Gene expression, Larvae, Ocean acidification, Aragonite, Calcificatio

Applications of hyperspectral imaging for documenting smoltification status and welfare in Atlantic salmon

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Seawater carbonate chemistry and larval development in domesticated and naturalized stocks of the Pacific oyster Crassostrea gigas

Ocean acidification (OA) has had significant negative effects on oyster populations on the west coast of North America over the past decade. Many studies have focused on the physiological challenges experienced by young oyster larvae in high pCO2/low pH seawater with reduced aragonite saturation state (Omega arag), which is characteristic of OA. Relatively few, by contrast, have evaluated these impacts upon fitness traits across multiple larval stages and between discrete oyster populations. In this study, we conducted 2 replicated experiments, in 2015 and 2016, using larvae from naturalized 'wild' and selectively bred stocks of the Pacific oyster Crassostrea gigas from the US Pacific Northwest and reared them in ambient (~400 µatm) or high (1600 µatm) pCO2 seawater from fertilization through final metamorphosis to juvenile 'spat.' In each year, high pCO2 seawater inhibited early larval development and affected the timing, but not the magnitude, of mortality during this stage. The effects of acidified seawater on metamorphosis of pediveligers to spat were variable between years, with no effect of seawater pCO2 in the first experiment but a 42% reduction in spat in the second. Despite this variability, larvae from selectively bred oysters produced, on average, more (+ 55 and 37%) and larger (+ 5 and 23%) spat in ambient and high pCO2 seawater, respectively. These findings highlight the variable and stage-specific sensitivity of larval oysters to acidified seawater and the influence that genetic factors have in determining the larval performance of C. gigas exposed to high pCO2 seawater

Temporally balanced selection during development of larval Pacific oysters (Crassostrea gigas) inherently preserves genetic diversity within offspring

Balancing selection is one of the mechanisms which has been proposed to explain the maintenance of genetic diversity in species across generations. For species with large populations and complex life histories, however, heterogeneous selection pressures may create a scenario in which the net effects of selection are balanced across developmental stages. With replicated cultures and a pooled sequencing approach, we show that genotype-dependent mortality in larvae of the Pacific oyster (Crassostrea gigas) is largely temporally dynamic and inconsistently in favour of a single genotype or allelic variant at each locus. Overall, the patterns of genetic change we observe to be taking place are more complex than what would be expected under classical examples of additive or dominant genetic interactions. They are also not easily explained by our current understanding of the effects of genetic load. Collectively, temporally heterogeneous selection pressures across different larval developmental stages may act to maintain genetic diversity, while also inherently sheltering genetic load within oyster populations

Seawater carbonate chemistry and bioinformatic quality statistics

The adaptive capacity of marine calcifiers to ocean acidification (OA) is a topic of great interest to evolutionary biologists and ecologists. Previous studies have provided evidence to suggest that larval resilience to high pCO2 seawater for these species is a trait with a genetic basis and variability in natural populations. To date, however, it remains unclear how the selective effects of OA occur within the context of complex genetic interactions underpinning larval development in many of the most vulnerable taxa. Here we evaluated phenotypic and genetic changes during larval development of Pacific oysters (Crassostrea gigas) reared in ambient (400 µatm) and high (1600 µatm) pCO2 conditions, both in domesticated and naturalized 'wild' oysters from the Pacific Northwest, USA. Using pooled DNA samples, we determined changes in allele frequencies across larval development, from early “D-stage” larvae to metamorphosed juveniles (spat), in both groups and environments. Domesticated larvae had 26% fewer loci with changing allele frequencies across developmental stages and < 50% as many loci affected by acidified culture conditions, compared to larvae from wild brood stock. Functional enrichment analyses of genetic markers with significant changes in allele frequency revealed that the structure and function of cellular membranes were disproportionately affected by high pCO2 conditions in both groups. These results indicate the potential for a rapid adaptive response of oyster populations to OA conditions; however, underlying genetic changes associated with larval development differ between these wild and domesticated oyster stocks and influence their adaptive responses to OA conditions

Seawater carbonate chemistry and gene expression, shell formation in larval Pacific oysters (Crassostrea gigas)

Background: Despite recent work to characterize gene expression changes associated with larval development in oysters, the mechanism by which the larval shell is first formed is still largely unknown. In Crassostrea gigas, this shell forms within the first 24 h post fertilization, and it has been demonstrated that changes in water chemistry can cause delays in shell formation, shell deformations and higher mortality rates. In this study, we use the delay in shell formation associated with exposure to CO2-acidified seawater to identify genes correlated with initial shell deposition. Results: By fitting linear models to gene expression data in ambient and low aragonite saturation treatments, we are able to isolate 37 annotated genes correlated with initial larval shell formation, which can be categorized into 1) ion transporters, 2) shell matrix proteins and 3) protease inhibitors. Clustering of the gene expression data into co-expression networks further supports the result of the linear models, and also implies an important role of dynein motor proteins as transporters of cellular components during the initial shell formation process. Conclusions: Using an RNA-Seq approach with high temporal resolution allows us to identify a conceptual model for how oyster larval calcification is initiated. This work provides a foundation for further studies on how genetic variation in these identified genes could affect fitness of oyster populations subjected to future environmental changes, such as ocean acidification