Transcriptome and enzyme activity analyses of tolerance mechanisms in pearl oyster (Pinctada fucata) under high-temperature stress

Pearl oyster (Pinctada fucata) is an economically valuable shellfish for seawater pearl production. However, many pearl oysters are dying as the global climate warms and seawater temperatures rise. The breeding of high temperature-resistant strains is therefore urgent. We performed transcriptome analysis to reveal the response mechanism of P. fucata to long-term high-temperature stress. Nanopore sequencing identified 1787 loci and revealed high-quality full-length isoforms for 17,803 novel splice isoforms. The large-scale study suggested differential patterns of alternative splicing (AS) regulation and transcriptional regulation under heat stress. There were more AS events in the high-temperature group (HT) than the normal temperature (NT) control group. In total, 44 upregulated and 33 downregulated differentially expressed genes (DEGs) were evident between NT and HT. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that DEGs were mainly associated with defence mechanisms, phagosome signalling, cytoskeleton, posttranslational modification, protein turnover and chaperones under heat stress. Nine DEGs (c-fox, heat shock protein 60, heat shock protein 90, fucolectin-1, dermatopontin, caveolin-1, ETS homologous factor, glutathione S-transferase sigma 3 and fatty acyl-CoA hydrolase precursor) showed complex expression pattern changes from 1 h to 30 days of hightemperature induction. Furthermore, heat stress resulted in irregular shell microstructures and impacted enzyme activity, exemplified by continuous upregulation of lactate dehydrogenase (LDH), indicating that the energy needs of P. fucata needs were met in part by anaerobic metabolism. Integrated analyses demonstrated that longterm high-temperature stress can significantly impact internal bodily functions, and disrupt physiological and biochemical processes. High-temperature stress induces pearl oysters to activate gene expression and thereby alter immune responses, respiratory metabolism, antioxidant systems, biomineralisation, and secondary metabolism. These pathways likely coordinate to support P. fucata physiology under high-temperature stress. The findings deepen our understanding of the molecular mechanisms underlying the responses to long-term hightemperature stress, and provide valuable information for future breeding of thermotolerant P. fucata strains.