Regulation of gene expression is associated with tolerance of the Arctic copepod Calanus glacialis to CO2-acidified sea water

Abstract

Ocean acidification is the increase in seawater pCO ₂ due to the uptake of atmospheric anthropogenic CO ₂, with the largest changes predicted to occur in the Arctic seas. For some marine organisms, this change in pCO ₂, and associated decrease in pH, represents a climate change-related stressor. In this study, we investigated the gene expression patterns of nauplii of the Arctic copepod Calanus glacialis cultured at low pH levels. We have previously shown that organismal-level performance (development, growth, respiration) of C. glacialis nauplii is unaffected by low pH. Here, we investigated the molecular-level response to lowered pH in order to elucidate the physiological processes involved in this tolerance. Nauplii from wild-caught C. glacialis were cultured at four pH levels (8.05, 7.9, 7.7, 7.5). At stage N6, mRNA was extracted and sequenced using RNA-seq. The physiological functionality of the proteins identified was categorized using Gene Ontology and KEGG pathways. We found that the expression of 151 contigs varied significantly with pH on a continuous scale (93% downregulated with decreasing pH). Gene set enrichment analysis revealed that, of the processes downregulated, many were components of the universal cellular stress response, including DNA repair, redox regulation, protein folding, and proteolysis. Sodium:proton antiporters were among the processes significantly upregulated, indicating that these ion pumps were involved in maintaining cellular pH homeostasis. C. glacialis significantly alters its gene expression at low pH, although they maintain normal larval development. Understanding what confers tolerance to some species will support our ability to predict the effects of future ocean acidification on marine organisms.

Keywords: RNA‐seq; ocean acidification; pH; phenotypic buffering; stress response; transcriptomics.

Figure 1

“Volcano plot” illustrating the selection of genes that were significantly differently expressed with pH in C. glacialis. Plotted are ‐log‐transformed Ward test p‐values for the slope of expression with pH, corrected for multiple tests by the Benjamini‐Hochberg method, and presented as false discovery rate (FDR), and –(log2 fold change) of expression over one unit of pH. The log2 fold change (LFC) is negative to indicate that those with a negative value are downregulated as pH is reduced. Significantly differently expressed genes (DECs, in darkest gray) were defined as those with |LFC| > 1 and FDR < 0.1

Figure 2

Heatmap of rlog‐transformed gene expression in C. glacialis at different pH treatments, centered, and standardized by gene for each sample (in columns). The color of the cell indicates relative gene expression, with red being higher than the gene's average, and blue lower. Only the 151 significantly differentially expressed genes are shown. The pH treatment and biological replicate (tank) are indicated in shaded red‐yellow and gray, respectively, above the heatmap. On the left vertical axis, genes are arranged according to their position in the hierarchical clustering (similarity measure was Pearson correlation coefficient; clustering by complete linkage)

Figure 3

Cytoscape network of the 350 most significant downregulated biological process Gene Ontology (GO) terms in C. glacialis, linked by SimRel semantic similarity of the terms by ReViGO. The color of each GO term node (from white to dark red) indicates the adjusted p‐value, with more significant terms in darker red. Likewise, the larger the label name, the more significant the adjusted p‐value. The size of each node indicates the size of the GO term in the entire UniProt database

Figure 4

Cytoscape network of the most significant upregulated biological process Gene Ontology (GO) terms in C. glacialis, linked by SimRel semantic similarity of the terms by ReViGO. Plot components as in Figure 3