Ocean acidification induces subtle shifts in gene expression and DNA methylation in mantle tissue of the Eastern oyster (Crassostrea virginica)

Abstract

Word Count: 350 Manuscript Word Count: 15658 1 Abstract Early evidence suggests that DNA methylation can mediate phenotypic responses of marine calcifying species to ocean acidification (OA). Few studies, however, have explicitly studied DNA methylation in calcifying tissues through time. Here, we examined the phenotypic and molecular responses in the extrapallial fluid and mantle (fluid and tissue at the calcification site) in the Eastern oyster ( Crassostrea virginica ) exposed to experimental OA over 80 days. Oysters were reared under three experimental pCO 2 treatments ('control', 580 μatm; 'moderate OA', 1000 uatm; 'high OA', 2800 μatm) and sampled at 6 time points (24 hours -80 days). We found that high OA initially induced changes in the pH of the extrapallial fluid (pH EPF ) relative to the external seawater, but the magnitude of this difference was highest at 9 days and diminished over time. Calcification rates were significantly lower in the high OA treatment compared to the other treatments. To explore how oysters regulate their extrapallial fluid, gene expression and DNA methylation were examined in the mantle-edge tissue of oysters from day 9 and 80 in the control and high OA treatments. Mantle tissue mounted a significant global molecular response (both in the transcriptome and methylome) to OA that shifted through time. Although we did not find individual genes that were significantly differentially expressed to OA, the pH EPF was correlated with the eigengene expression of several co-expressed gene clusters. A small number of OA-induced differentially methylated loci were discovered, which corresponded with a weak association between OA-induced changes in genome-wide gene body DNA methylation and gene expression. Gene body methylation, however, was not significantly correlated with the eigengene expression of pH EPF correlated gene clusters. These results suggest that in C. virginica , OA induces a subtle response in a large number of genes, but also indicates that plasticity at the molecular level may be limited. Our study highlights the need to re-assess the plasticity of tissue-specific molecular responses in marine calcifiers , as well as the role of DNA methylation and gene expression in mediating physiological and biomineralization responses to OA.

Juvenile Eastern Oysters More Resilient to Extreme Ocean Acidification than Their Mud Crab Predators

“…Juvenile eastern oysters investigated in the present study exhibit a nonlinear threshold response to decreasing Ω C (7.3‐0.6), consistent with work by Downey et al. (2020) on adult eastern oysters, but contrasting the linear declines in calcification rate of adult eastern oysters observed by Gazeau et al. (2007) and Ries et al.…”

“…Previous studies investigating oysters' calcification response to ocean acidification have either found no change in net calcification rates (Dickinson et al., 2012) or decreasing net calcification rates with increasing p CO 2 across a wide range of life stages (Beniash et al., 2010; Downey‐Wall et al., 2020; Gazeau et al., 2007; Miller et al., 2009; Ries et al., 2009). Oyster larvae utilize the aragonite polymorph of CaCO 3 in shell development and are therefore considered more vulnerable to ocean acidification than juveniles or adults that utilize the less soluble calcite polymorph.…”

“…Short‐term exposure of Pacific oyster spat to decreasing Ω C (5.7‐2.0) yielded similar linear decreases in net calcification rates (Gazeau et al., 2007). Likewise, the net calcification rate of adult eastern oysters declined linearly when exposed for 60 days to Ω C ranging from 4.0 to 1.1 (Ries et al., 2009) and in a nonlinear, threshold manner when exposed for 80 days to Ω C ranging from 3.0 to 0.9 (Downey‐Wall et al., 2020). Juvenile eastern oysters investigated in the present study exhibit a nonlinear threshold response to decreasing Ω C (7.3‐0.6), consistent with work by Downey et al.…”

“…Recent studies have shown that the pH of the calcifying extrapallial fluid of eastern oysters is less than seawater pH under control (i.e., ambient) pCO 2 conditions (Crenshaw, 1972; Sutton et al., 2018), but increases relative to seawater pH with increasing p CO 2 (Downey‐Wall et al., 2020; Liu et al., 2020)—suggesting that oysters regulate extrapallial fluid pH to mitigate the impacts of ocean acidification on shell formation. However, the onset of differences in microstructure of the interior portions of the oyster shells (i.e., the actively growing portion of shell adjacent to the extrapallial fluid and mantle), combined with significantly reduced net calcification rates in the highest p CO 2 treatment, suggest that the oysters' regulation of extrapallial fluid pH is insufficient to completely mitigate the impacts of ocean acidification on juvenile oyster calcification.…”

“…This vulnerability may be exacerbated by the oyster's patchily distributed periostracum, which may confer less protection to their shell than the periostraca of other bivalves that possess more hydrophobic and continuous periostraca, such as clams and mussels (Ries et al., 2009). Eastern oysters also maintain their extrapallial fluid—their putative calcifying fluid—at lower pH than the surrounding seawater (Crenshaw, 1972; Crenshaw & Neff, 1969; Downey‐Wall et al., 2020; Liu et al., 2020; Sutton et al., 2018), which may also increase their vulnerability to CO 2 ‐induced ocean acidification (Ries, 2011).…”