Calcifying marine organisms, including the eastern oyster (Crassostrea virginica), are vulnerable to ocean acidification (OA) because it is more difficult to precipitate calcium carbonate (CaCO3) under acidic conditions. We previously investigated the molecular mechanisms associated with resilience to OA in C. virginica. There were significant differences in SNP and gene expression profiles among oysters reared under normal and OA conditions. Converged evidence generated by both of these approaches highlighted the role of genes related to biomineralization, including perlucin, in resilience to OA. Perlucin is important for the nucleation of CaCO3 crystals during bivalve shell formation, which has been shown to be negatively impacted by OA in C. virginica.
In this study, we used RNAi or gene silencing to validate findings and confirm the protective role of perlucin associated with resilience to OA. Larvae were exposed to short dicer-substrate small interfering RNA (DsiRNA) to silence perlucin or to one of two control treatments (control DsiRNA or seawater), before cultivation under OA conditions (pH ~7.3) or control (pH ~8) conditions. Two different transfection methods were performed in parallel, one during fertilization and one during early larval development (6 hours post fertilization). Both transfection methods were successful at significantly reducing the expression of perlucin; however, the transfection method did influence results. Larval viability, size, development, and biomineralization were monitored daily. Silenced oysters under acidification stress were the smallest, had shell abnormalities, and had significantly reduced shell mineralization, thereby indicating that perlucin does help larvae mitigate the effect of OA.