Small RNA Analysis Of Perna Viridis After Exposure To Prorocentrum Lima,a DSP Toxins-producing Dinoflagellate

Diarrheic shellfish poisoning toxins(DSP toxins)are one of the important phycotoxins produced by some dinoflagellates.Studies show that DSP toxins have a varity toxicities such as genotoxicity,cytotoxicity,and immunotoxicity and cause oxidative damages to bivalve molluscs.However,these toxicities appear decreasing with exposure time and concentration of DSP toxins.The underlying mechanism involved remains unclear.In this paper,small RNA sequencing was performed in digestive gland of the mussel Perna viridis after exposure to DSP toxins-producing microalge Prorocentrum lima for different time peroids.The potential roles of miRNA in response and detoxification to DSP toxins in mussel were analyzed.Small RNA sequencing of 12 individuals of P.viridis was conducted by BGISEQ-500.A total of 123 mature miRNAs were identified,including 90 conserved miRNAs,and 33 potential novel miRNAs.Among them,miR-1,miR-100,miR-1175,miR-184,miR-2,miR-375,and miR-71 are mollusk-specific miRNAs,which displayed high expression in P.viridis;miR-100,miR-153,and miR-34 that play key roles in many metabolic pathways deserved to be studied in the future.After exposure to P.lima,expression of miR-100＿2,miR-71＿5,miR-750＿1,novel＿mir14,and novel＿mir4 were changed at 6 h,while miR-71＿5,miR-750＿1 and novel＿mir4displayed some alterations at 96 h.q PCR demonstrated that the three miRNAs miR-71＿5,miR-750＿1 and novel＿mir4 were significantly up-regulated at 6 h after exposure to P.lima,while miR-100＿2 was significantly down-regulated after 96 h of exposure.KEGG analysis showed that the differentially expressed miRNA target genes enriched in focal adhesions,PI3K-Akt signaling pathway,mTOR signaling pathway,immune system,and ECM-receptor interaction effect.These outcomes suggested that miRNA might be related to the response of P.viridis to DSP toxins by regulating the expression of target genes.Further analyses on the differentially expressed miRNA and related target genes revealed some important genes involved in cytoskeleton,apoptosis,complement system,and immune stress.For example,mir-750＿1 targeted SIPA1L1 and NHLRC2 genes,novel＿mir4 targeted c1q-like genes,and mir-71＿5 targeted znf26-like genes.After 6 h of DSP toxin exposure,miR-750＿1 was significantly up-regulated,and NHLRC2 was significantly down-regulated,suggesting that miR-750＿1 might promote apoptosis by directly regulating NHLRC2.Meanwhile,the up-regulation of novel＿mir4 and down-regulation of C1q-like suggested that novel＿mir4 might directly target C1q-like for immune responses.After 96 h of DSP toxin exposure,miR-750＿1 level was not changed,but NHLRC2 was significantly up-regulated.Meanwhile,there was no significant difference in levels of novel＿mir4 and C1q-like.These outcomes suggested that the mussel recovered from the harm of DSP toxins after long-term exposure.In addition,down-regulation of ZnF26-like after 96 h of exposure to DSP toxins indicated that the mussel P.viridis might affect cAMP-PKA pathway,phagocytosis,and recombinant cytoskeleton pathway by regulating ZnF26-like.However further study for this presumption should be conducted in the future.In conclusion,the mussel P.viridis might respond to DSP toxins through mir-750＿1,NOVEL＿MIR4 and Mir-71＿5 regulating the expression of relevant target genes involved in apoptosis,cytoskeleton,and immune response,etc.This study might provide new clues to uncover the toxic response of bivalve to DSP toxins,and lay a foundation for revealing the role of miRNA in the environmental adaptation mechanism of shellfish.