A Transcriptomic Analysis of Phenotypic Plasticity in Crassostrea virginica Larvae under Experimental Acidification

Abstract

Ocean acidification (OA) is a major threat to marine calcifiers, and little is known regarding acclimation to OA in bivalves. This study combined physiological assays with next-generation sequencing to assess the potential for recovery from and acclimation to OA in the eastern oyster (Crassostrea virginica) and identify molecular mechanisms associated with resilience. In a reciprocal transplant experiment, larvae transplanted from elevated pCO₂ (~1400 ppm) to ambient pCO₂ (~350 ppm) demonstrated significantly lower mortality and larger size post-transplant than oysters remaining under elevated pCO₂ and had similar mortality compared to those remaining in ambient conditions. The recovery after transplantation to ambient conditions demonstrates the ability for larvae to rebound and suggests phenotypic plasticity and acclimation. Transcriptomic analysis supported this hypothesis as genes were differentially regulated under OA stress. Transcriptomic profiles of transplanted and non-transplanted larvae terminating in the same final pCO₂ converged, further supporting the idea that acclimation underlies resilience. The functions of differentially expressed genes included cell differentiation, development, biomineralization, ion exchange, and immunity. Results suggest acclimation as a mode of resilience to OA. In addition, the identification of genes associated with resilience can serve as a valuable resource for the aquaculture industry, as these could enable marker-assisted selection of OA-resilient stocks.

Keywords: RNASeq; acclimation; ocean acidification; oyster.

Figure 1

(A) Percent mortality for 7 day old oyster larvae before transplantation into the alternative pCO₂ treatment (* denotes significant difference for (A), G-test of independence, p < 0.001; n = 4, with a minimum of 100 larvae per replicate). Error bars are the 95% confidence intervals. (B) Length in micrometers for 7 day old oyster larvae before transplantation into the alternative pCO₂ treatment in the reciprocal transplant experiment (** denotes significant difference for (B), Mann–Whitney U test, p < 0.001; n = 4, with a minimum of 65 larvae per replicate). Error bars denote standard error of the mean.

Figure 2

Percent mortality 1 week post-transplant (different letters a, b above error bars represent significant difference, G-test of independence, p < 0.001; n = 4 per treatment, with a minimum of 340 larvae per replicate). Error bars are the 95% confidence intervals. Sample code names: first letter indicates original pCO₂ treatment (A: ambient, E: elevated); second letter indicates final pCO₂ treatment (first and second letters are different for transplanted treatments).

Figure 3

Length in micrometers for 15 day old oyster larvae transplanted (diagonal lines) and kept as controls (solid) (different letters a, b, c above error bars denote significant difference, Kruskal–Wallis rank sum test, p = 0.013; n = 4, with a minimum of 180 individuals per replicate). Error bars denote standard error of the mean. Sample code names: first letter indicates original pCO₂ treatment (A: ambient, E: elevated); second letter indicates final pCO₂ treatment (first and second letters are different for transplanted treatments).

Figure 4

(A) Hierarchical cluster analysis of DEGs (n = 4 replicates per treatment coalesced into 1 column). Over- and under-expressed genes are represented in red and blue, respectively. Data are normalized by average expression in all samples. (B) Principal component analysis of the normalized RNASeq data. Sample code names: first letter signifies original pCO₂ treatment (A: ambient, E: elevated); second letter signifies final pCO₂ treatment (first and second letters are different for transplanted treatments).

Figure 5

(A): Venn diagram showing the number of genes uniquely expressed within each group, with the overlapping regions showing the number of genes that are expressed in two or more groups. (B) DEGs in common/shared between comparisons; * indicates the group with higher expression for the gene. Purple: AA vs. EE; Blue: AA vs. AE; Green EE vs. EA; Red: AE vs. EA.