Table_1_Ocean Acidification Induces Subtle Shifts in Gene Expression and DNA Methylation in Mantle Tissue of the Eastern Oyster (Crassostrea virginica).XLSX

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 adult eastern oyster (Crassostrea virginica) exposed to experimental OA over 80 days. Oysters were reared under three experimental pCO2 treatments (“control,” 580 μatm; “moderate OA,” 1,000 μatm; “high OA,” 2,800 μatm) and sampled at 6 time points (24 h−80 days). We found that high OA initially induced an increase in the pH of the extrapallial fluid (pHEPF) relative to the external seawater that peaked at day 9, but then 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 days 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 under OA, the pHEPF was significantly 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 pHEPF-correlated gene clusters. These results suggest that OA induces a subtle response in a large number of genes in C. virginica, but also indicate that plasticity at the molecular level may be limited. Our study highlights the need to reassess our understanding 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.

早期研究证据表明，DNA甲基化（DNA methylation）可介导海洋钙化物种对海洋酸化（ocean acidification, OA）的表型响应。然而，鲜有研究通过时间序列明确探究钙化组织中的DNA甲基化变化。本研究针对暴露于实验性海洋酸化环境长达80天的成年美洲牡蛎（Crassostrea virginica），对其钙化位点处的外套膜（mantle）组织与外套腔液（extrapallial fluid）的表型与分子响应展开分析。实验设置3种二氧化碳分压（pCO2）处理组：对照组（580 μatm）、中度酸化组（1000 μatm）、高度酸化组（2800 μatm），并在6个时间节点（24小时至80天）进行取样。研究发现，高度酸化处理组最初会使外套腔液pH（pHEPF）相较于外界海水升高，该效应在第9天达到峰值，随后随时间推移逐渐减弱。高度酸化组的钙化速率显著低于其余两组。为探究牡蛎如何调控外套腔液，本研究在第9天与第80天，分别对对照组与高度酸化组牡蛎的外套膜边缘组织进行基因表达与DNA甲基化分析。外套膜组织对海洋酸化产生了显著的全局性分子响应（涵盖转录组（transcriptome）与甲基化组（methylome）），且该响应随时间发生动态变化。尽管未发现单一基因在海洋酸化处理下存在显著差异表达，但外套腔液pH与多个共表达基因簇（co-expressed gene clusters）的特征基因表达量（eigengene expression）显著相关。本研究仅发现少量由海洋酸化诱导的差异甲基化位点（differentially methylated loci），其对应关系为：全基因组基因体DNA甲基化（gene body DNA methylation）的酸化诱导变化与基因表达变化存在弱关联。然而，基因体DNA甲基化与外套腔液pH相关基因簇的特征基因表达量并无显著相关性。上述结果表明，海洋酸化会诱导美洲牡蛎大量基因产生细微响应，但同时也提示其分子层面的可塑性可能较为有限。本研究强调，有必要重新审视我们对海洋钙化物种组织特异性分子响应的认知，以及DNA甲基化与基因表达在介导生理响应与生物矿化（biomineralization）响应以应对海洋酸化过程中所发挥的作用。