Research on the autophagy regulatory gene CgSVIP of Crassostrea gigas as a biomarker of pathogen stress

To explore the expression characteristics of CgSVIP in response to pathogen-associated molecular patterns (PAMPs) and pathogen stimulation in Crassostrea gigas, to discover the application of CgSVIP as a biomarker for disease early warning, and to analyze the mechanism of CgSVIP in regulating immune responses. Bioinformatics, RT-qPCR and WB techniques were used in this experiment. It was found that after stimulation with lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid Poly(I:C) for 12 h, the mRNA expression level of CgSVIP in haemocytes of C. gigas significantly increased under all four PAMPs stimulations, and the average expression difference level was the most significant among the differentially expressed genes in the transcriptome, with an average FPKM value of 106.938. Further, when C. gigas was stimulated with LPS and Vibrio splendidus, the absolute expression value of CgSVIP in haemocytes significantly increased at 12, 24 and 48 h after LPS stimulation, reaching 3.71, 3.52 and 3.55 lg copies/mL respectively; and significantly increased at 3, 6, 12, 48 and 72 h after V. splendidus stimulation, reaching 3.31, 3.12, 3.11, 3.42 and 3.26 lg copies/mL respectively. After stimulation with V. splendidus for 12 h from March to October 2023, the expression level of CgSVIP in haemocytes significantly increased except in March, reaching 3.52, 3.57, 3.45, 4.42, 3.77, 3.75 and 4.06 lg copies/mL respectively, with the highest expression in July samples. Therefore, the absolute expression value of CgSVIP was within the range of 2.93-3.35 lg copies/mL without PAMPs and pathogen stimulation, and within the range of 3.18-3.79 lg copies/mL with stimulation. It was preliminarily determined that the threshold for CgSVIP to respond to PAMPs and pathogen stress was 3.35 lg copies/mL. To analyze the mechanism of CgSVIP in regulating immune responses, the expression level of CgSVIP protein in haemocytes of C. gigas significantly increased after stimulation with carbamazepine (CBZ), reaching 1.67 fold that of the DMSO group. After inhibiting the expression of CgSVIP in C. gigas by RNAi and stimulating with LPS for 12 h, the expression levels of CgSVIP and autophagy-related genes CgLC3, CgBeclin1, CgAtg5 and CgP62 in haemocytes significantly decreased, reaching 0.53, 0.16, 0.38, 0.19 and 0.40 fold that of the NC-RNAi+LPS group respectively. The ratio of CgLC3II/CgLC3I was significantly decreased in the CgSVIP-RNAi+LPS group compared with the NC-RNAi+LPS group, reaching 0.73 fold that of the NC-RNAi+LPS group. CgSVIP can be used as a potential biomarker for disease early warning in C. gigas, and it participates in the regulation of immune responses by affecting autophagy in haemocytes and mediating antibacterial immune responses. Through changes in gene expression as a biomarker, early warning of C. gigas aquaculture can be achieved to reduce mortality and production losses caused by diseases.