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.

中文翻译：

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海洋酸化导致东部牡蛎（Crassostrea virginica）地幔组织中基因表达和DNA甲基化的微妙变化

早期证据表明 DNA 甲基化可以介导海洋钙化物种对海洋酸化 (OA) 的表型反应。然而，很少有研究明确研究钙化组织随时间推移的 DNA 甲基化。在这里，我们检查了暴露于实验性 OA 80 天以上的成年东部牡蛎（Crassostrea virginica）的外层液体和地幔（钙化部位的液体和组织）的表型和分子反应。牡蛎在三种实验 pCO2 处理下饲养（“对照”，580 μatm；“中等 OA”，1,000 μatm；“高 OA”，2,800 μatm），并在 6 个时间点（24 h-80 天）取样。我们发现，高 OA 最初会导致外层液体 (pHEPF) 相对于外部海水的 pH 值升高，在第 9 天达到峰值，但随着时间的推移逐渐减弱。与其他治疗相比，高 OA 治疗的钙化率显着降低。为了探索牡蛎如何调节其外层液，在对照和高 OA 处理的第 9 天和第 80 天的牡蛎的地幔边缘组织中检查了基因表达和 DNA 甲基化。地幔组织对 OA 产生了显着的全球分子反应（在转录组和甲基化组中），随着时间的推移而变化。虽然我们没有发现在 OA 下显着差异表达的单个基因，但 pHEPF 与几个共表达基因簇的特征基因表达显着相关。发现了少量 OA 诱导的差异甲基化位点，这与 OA 诱导的全基因组基因体 DNA 甲基化变化与基因表达之间的弱关联相对应。然而，基因体甲基化与 pHEPF 相关基因簇的特征基因表达没有显着相关性。这些结果表明 OA 在 C. virginica 中的大量基因中诱导了微妙的反应，但也表明分子水平的可塑性可能是有限的。我们的研究强调需要重新评估我们对海洋钙化物中组织特异性分子反应的理解，以及 DNA 甲基化和基因表达在介导对 OA 的生理和生物矿化反应中的作用。这些结果表明 OA 在 C. virginica 中的大量基因中诱导了微妙的反应，但也表明分子水平的可塑性可能是有限的。我们的研究强调需要重新评估我们对海洋钙化物中组织特异性分子反应的理解，以及 DNA 甲基化和基因表达在介导对 OA 的生理和生物矿化反应中的作用。这些结果表明 OA 在 C. virginica 中的大量基因中诱导了微妙的反应，但也表明分子水平的可塑性可能是有限的。我们的研究强调需要重新评估我们对海洋钙化物中组织特异性分子反应的理解，以及 DNA 甲基化和基因表达在介导对 OA 的生理和生物矿化反应中的作用。