RNA Interference by Ingested dsRNA-Expressing Bacteria to Study Shell Biosynthesis and Pigmentation in Crassostrea gigas

RNA interference (RNAi) is an important molecular tool for analysis of gene function in vivo. Although the Pacific oyster Crassostrea gigas is an economically important species with fully sequenced genome, very few mechanistic studies have been carried out due to the lack of molecular techniques to alter gene expression without inducing stress. In this present study, we used unicellular alga Platymonas subcordiformis and Nitzschia closterium f. minutissima as a vector to feed oysters with Escherichia coli strain HT115 engineered to express double-stranded RNAs (dsRNAs) targeting specific genes involved in shell pigmentation. A C. gigas strain with black shell was used to target tyrosinase or peroxidase gene expression by RNAi using the above-mentioned approach. The results showed that feeding oyster with dsRNA of tyrosinase could knock down the expression of corresponding tyrosinase and hinder the developed shell growth. Feeding oyster with dsRNA of peroxidase could knock down the expression of the corresponding peroxidase and result in reduced black pigmentation in the newly developed shell. This non-invasive RNAi study demonstrated that tyrosinase played a vital role in the assembly and maturation of shell matrices and peroxidase was essential for black pigmentation in the shell. Moreover, the RNA interference by ingested dsRNA-expressing bacteria is a relatively simple and effective method for knockdown of a gene expression in adult oysters, thus further advances the use of C. gigas as model organism in functional genomic studies.