Eastern oysters in the northern Gulf of Mexico are routinely infected with the protistan parasite Perkinsus marinus, the cause of the disease commonly known as dermo. Recent experimental challenges among Atlantic coast populations have identified both resistant and susceptible genotypes using comparative transcriptomics. While controlled experimental challenges are essential first assessments, expanding this analysis to field reared individuals provides an opportunity to identify key genomic signatures of infection that appear both in the laboratory and in the field. In this study we combined reduced representation bisulfite sequencing with 3 RNA sequencing (Tag-seq) to describe two molecular phenotypes associated with infection in oysters outplanted at a common garden field site. These combined approaches allowed us to examine changes in DNA methylation and gene expression for a large number of individuals (n = 40) that developed infections during the course of a common garden outplant experiment. Our epigenetic analysis of DNA methylation identified significant changes in gene body methylation associated with increasing infection intensity, across genes associated with immune responses. There was a smaller transcriptomic response to increasing infection intensities with 32 genes showing differential expression; however, only 40% of these genes were found to also be differentially methylated. While there was no clear pattern between direction of differential methylation and gene expression, there was a significant effect of percent methylation on gene-by-gene expression levels and the coefficient of variation in gene body methylation between treatments. These results show that in C. virginica, heavily methylated genes have high levels of gene expression with low levels of variation. Comparing our differential expression results with previously published experimental P. marinus challenges identified overlapping expression patterns for genes associated with C1q-domain-containing and V-type proton ATPase proteins. Through our comparative transcriptomic approach using field reared individuals and co-expression network analysis we have also been able to identify a network of genes

It has been hypothesized that environmentally induced changes to gene body methylation could facilitate adaptive transgenerational responses to changing environments. We compared patterns of global gene expression (Tag‐seq) and gene body methylation (reduced representation bisulfite sequencing) in 80 eastern oysters Crassostrea virginica from six full‐sib families, common gardened for 14 months at two sites in the northern Gulf of Mexico that differed in mean salinity. At the time of sampling, oysters from the two sites differed in mass by 60% and in parasite loads by nearly two orders of magnitude. They also differentially expressed 35% of measured transcripts. However, we observed differential methylation at only 1.4% of potentially methylated loci in comparisons between individuals from these different environments, and little correspondence between differential methylation and differential gene expression. Instead, methylation patterns were largely driven by genetic differences among families, with a PERMANOVA analysis indicating nearly a two orders of magnitude greater number of genes differentially methylated between families than between environments. An analysis of CpG observed/expected values (CpG O/E) across the C. virginica genome showed a distinct bimodal distribution, with genes from the first cluster showing the lower CpG O/E values, greater methylation and higher and more stable gene expression, while genes from the second cluster showed lower methylation, and lower and more variable gene expression. Taken together, the differential methylation results suggest that only a small portion of the C. virginica genome is affected by environmentally induced changes in methylation. At this point, there is little evidence to suggest that environmentally induced methylation states would play a leading role in regulating gene expression responses to new environments.

Eastern oysters in the northern Gulf of Mexico are facing rapid environmental changes and can respond to this change via plasticity or evolution. Plasticity can act as an immediate buffer against environmental change, but this buffering could impact the organism's ability to evolve in subsequent generations. While plasticity and evolution are not mutually exclusive, the relative contribution and interaction between them remains unclear. In this study, we investigate the roles of plastic and evolved responses to environmental variation and Perkinsus marinus infection in Crassostrea virginica by using a common garden experiment with 80 oysters from six families outplanted at two field sites naturally differing in salinity. We use growth data, P. marinus infection intensities, 3′ RNA sequencing (TagSeq) and low‐coverage whole‐genome sequencing to identify the effect of genotype, environment and genotype‐by‐environment interaction on the oyster's response to site. As one of first studies to characterize the joint effects of genotype and environment on transcriptomic and morphological profiles in a natural setting, we demonstrate that C. virginica has a highly plastic response to environment and that this response is parallel among genotypes. We also find that genes responding to genotype have distinct and opposing profiles compared to genes responding to environment with regard to expression levels, Ka/Ks ratios and nucleotide diversity. Our findings suggest that C. virginica may be able to buffer the immediate impacts of future environmental changes by altering gene expression and physiology, but the lack of genetic variation in plasticity suggests limited capacity for evolved responses.

Salinity conditions in oyster breeding grounds in the Gulf of Mexico are expected to drastically change due to increased precipitation from climate change and anthropogenic changes to local hydrology. We determined the capacity of the eastern oyster, Crassostrea virginica , to adapt via standing genetic variation or acclimate through transgenerational plasticity (TGP). We outplanted oysters to either a low- or medium-salinity site in Louisiana for 2 years. We then crossed adult parents using a North Carolina II breeding design, and measured body size and survival of larvae 5 dpf raised under low or ambient salinity. We found that TGP is unlikely to significantly contribute to low-salinity tolerance since we did not observe increased growth or survival in offspring reared in low salinity when their parents were also acclimated at a low-salinity site. However, we detected genetic variation for body size, with an estimated heritability of 0.68 ± 0.25 (95% CI). This suggests there is ample genetic variation for this trait to evolve, and that evolutionary adaptation is a possible mechanism through which oysters will persist with future declines in salinity. The results of this experiment provide valuable insights into successfully breeding low-salinity tolerance in this commercially important species.

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