Preservation Time on Ice Impacts Global Gene Expression in Oyster Spat

The pearling industry is one of Australia's most valuable and iconic industries, creating significant economic and employment opportunities across Northern Australia. To improve our understanding of the relationship between the silver-lipped pearl oyster and its biotic and abiotic environment using transcriptomics, oyster spat are collected weekly from several offshore sites in Northwestern Australia. However, due to the remoteness of the farming locations, collected spat must be transported on ice for intervals before snap-freezing becomes possible. In this study, we investigate the impact of incubation on ice for intervals of up to 90 min on the global gene expression of collected spat. Preserving spat on ice for up to 90 min did not significantly impact the RNA concentration or quality. On average, more than 100 ng/µL of RNA (RIN 7.0–8.5) was extracted per sample to generate sequencing libraries, and no significant impact on sequencing and mapping reads to the genome was observed between treatments. However, prolonged incubation on ice did alter gene expression, with a significant increase in differential gene expression observed after 60 min. Furthermore, changes in the number of predicted genes, potentially in response to stress and cold, including stress-related heat shock protein (HSP) and Cytochrome P450 (CYP) genes, immune-related perlucin-like genes, and solute carrier (SLC) transporters, were observed with prolonged incubation. The observed stress-induced changes can alter the global gene expression status of the spat at the time of collection and may lead to misinterpretations upon analysing the impact of abiotic and biotic factors on the health of silver-lipped pearl oyster spat. Prolonged incubation intervals of oyster spat on ice destined for RNASeq analysis are therefore not recommended. Remote sites for longitudinal and comparative studies were therefore selected with this guideline in mind. This study also demonstrates the importance of conducting pilot trials in the development of research study protocols.