miRNA-seq provides novel insight into the response to hyper- and hypo- salinity acclimation in Crassostrea hongkongensis.

The Hong Kong oyster, Crassostrea hongkongensis, is a significant bivalve species with economic importance. It primarily inhabits the estuarine intertidal zones in southern China, making it susceptible to salinity fluctuations. Consequently, investigating the molecular mechanisms governing salinity regulation in C. hongkongensis is essential. In this study, we conducted miRNA-seq on C. hongkongensis to compare miRNA expression differences under varying salinities (5‰, 25‰, and 35‰). The miRNA sequencing revealed 51 known miRNAs and 95 novel miRNAs across nine small RNA libraries (S5, S25, and S35). Among these miRNAs, we identified 6 down-regulated differentially expressed (DE) miRNAs in response to hypo-salinity stress (5‰), while 1 up-regulated DE miRNA and 5 down-regulated DE miRNAs were associated with hyper-salinity stress (35‰). Additionally, we predicted 931 and 768 potential target genes for hypo- and hyper-salinity stress, respectively. Functional gene annotation indicated that the target genes under hypo-salinity stress were linked to vesicle-mediated transport and metal ion binding. Conversely, those under hyper-salinity stress were primarily involved in signal transduction and metabolic processes. These findings have provided insights into the regulatory role of miRNAs, their potential target genes and associated pathways in oyster hypo- and hyper-salinity stress, which establish a foundation for future studies on the roles of miRNAs in salinity acclimation mechanisms in C. hongkongensis.

Transcriptional responses of Crassostrea hongkongensis under high and low salinity stress.

Salinity, a key limiting factor, affects the distribution and survival of marine species. The Hong Kong oyster (Crassostrea hongkongensis), a euryhaline species found along the coast of the South China Sea, has become a major aquaculture bivalve species. To determine the molecular mechanism by which oysters respond to coastal waters with varying salinity levels, we used RNA-seq to sequence the gill samples of oysters exposed to normal (25 ‰, S25), low (5 ‰, S5) and high (35 ‰, S35) salinity conditions for one month. The results revealed different expression transcriptome levels among oysters living under low and high salinity conditions. Using high-throughput sequencing, we identified 811 up-regulated genes and 769 down-regulated genes. As determined by KEGG pathway mapping, the differentially expressed genes (DEGs) were significantly enriched in the prion diseases, histidine metabolism, arginine and proline metabolism, and beta-alanine metabolism pathways in both the S5 vs. S25 and S35 vs. S25 group comparison. Several DEGs including heat shock 70 kDa protein 12B-like, poly (ADP-ribose) polymerase (PARP), and tripartite motif-containing protein 2 (TRIM2), and low-density lipoprotein receptor-like, as well as KEGG pathways, including arginine and proline metabolism, apoptosis, PPAR signaling pathway, the thyroid hormone signaling pathway, were concerning response to salinity stress. Additionally, eight DEGs involved in salinity adaptation were selected for RT-qPCR validation, and the results confirmed the credibility of the transcriptome sequencing data. Overall, we designed a one-month, medium-term experiment to examine the responses of C. hongkongensis exposed to different levels of salinity stress and performed transcriptome analysis using high-throughput sequencing. Our results enhance current understanding of the molecular mechanisms of salinity stress responses in C. hongkongensis and provided insights into the osmotic biology of oysters.

Toll-like receptor may be involved in acquired immune response in pearl oyster Pinctada fucata.

The increasing experimental evidence suggests that there are some forms of specific acquired immunity in invertebrates, in which Toll-like receptors (TLRs) play vital roles in activating innate and adaptive immunity and have been comprehensively investigated in mammalian species. Yet, the immune mechanisms underlying TLR mediation in mollusks remain obscure. In this study, we identified a TLR13 gene in the pearl oyster Pinctada fucata for the first time and named it PfTLR13 which consists of a 5'-untranslated terminal region (5'-UTR) of 543 bp, an open reading frame (ORF) of 2667 bp, and a 3'-UTR of 729 bp. We found that PfTLR13 mRNA was expressed in all tissues examined, with the highest level in the gills. The expression of PfTLR13 in the gills of oysters exposed to Vibrio alginolyticus or pathogen-associated molecular patterns (PAMPs) (including LPS, PGN, and poly(I:C)) was significantly higher than in the control group. Interestingly, the immune response to the first stimulation was weaker than the response to the second stimulation, suggesting that the primary stimulation may lead to immune priming of TLR in pearl oysters, similar to acquired immunity in vertebrates. Furthermore, we found that PfTLR13 expression was differentially associated with allograft and xenograft in the pearl oyster P. fucata, with the highest expression levels observed at 12 h post-allograft and 24 h post-xenograft. Overall, our findings provide new insights into the immune mechanisms underlying TLR mediation in mollusks and suggest that PfTLR13 may play a crucial role in the specific acquired immunity of pearl oysters.