Abstract:With the expansion of aquaculture and deterioration of farming environments, diseases are a major issue in Portunus trituberculatus farming, among which Vibrio parahaemolyticus is the primary pathogen causing mortality. Rab GTPases, which belong to the Rab protein family within the small GTPase Ras superfamily, play critical roles in regulating intracellular membrane trafficking in eukaryotic cells. Studies have shown that Rab enhances immunity in invertebrates against pathogenic infection by regulating phagocytosis in hemolymph tissues. DNA methylation, a common epigenetic regulatory mechanism, is involved in controlling the expression of key genes. However, the molecular mechanism by which DNA methylation regulates gene expression and thus influences the immune response of P. trituberculatus upon pathogen infection remains unclear. To explore the epigenetic regulatory mechanism involving DNA methylation in response to V. parahaemolyticus infections in P. trituberculatus, the gene structure, sequence similarity, and phylogenetic analysis of PtRab2A were characterized. In addition, the PtRab2A expression and its DNA methylation patterns in response to V. parahaemolyticus infection were investigated. Preliminary experiments were conducted with 90 randomly selected P. trituberculatus specimens, weighing approximately (30±5) g. Python was used to calculate the median lethal concentration (LC50) of V. parahaemolyticus infection at 72 h post-infection, which was determined to be 5×106 CFU/mL. This concentration was then applied in the formal experiment. A total of 40 temporarily cultured crabs were selected for the formal infection test, and hemolymph tissues were collected at 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h after infection for subsequent DNA methylation and gene expression analysis. The RNAi experiment was divided into two groups, each consisting of 80 individuals. At 12 and 24 h post-injection, hemolymph tissues were collected from five crabs each in both the blank control and experimental groups. Following 24 h of RNAi treatment, surviving crabs were infected with V. parahaemolyticus (5×106 CFU/mL). Mortality rates were recorded at 1-h intervals until complete mortality occurred. The results showed that the full length of PtRab2A was 1,824 bp, including a 15 bp 5′ UTR, an 822 bp ORF region, and a 987 bp 3′ UTR. Two conserved cysteine residues were identified at the C-terminus of PtRab2A. The predicted molecular formula was C1322H2084N382O399S12, with a molecular weight of 30.10 kDa and a theoretical isoelectric point (pI) of 7.05. The gene encodes a total of 274 amino acids. Homology analysis revealed that the conserved amino acid sequence of PtRab2A showed the highest similarity with that of Scylla paramamosain. SMART analysis identified a RAB domain within PtRab2A. Phylogenetic analysis using MEGA11 indicated that P. trituberculatus PtRab2A clustered most closely with S. paramamosain, and then grouped with Eriocheir sinensis. These crabs subsequently clustered with Penaeus monodon, Penaeus chinensis, and Drosophila melanogaster. Meanwhile, Homo sapiens, Mus musculus, Danio rerio, Oncorhynchus mykiss, Larimichthys crocea, Ruditapes philippinarum, and Crassostrea gigas formed another large cluster. Notably, D. melanogaster and crustaceans were grouped together, while Rab2A genes of invertebrates (excluding mollusks) such as P. trituberculatus were distinctly separated from those of vertebrates. The methylation profile of the PtRab2A gene following V. parahaemolyticus infection was analyzed using IGV visualization software. The monoclonal sequencing results were further analyzed using the QUMA online platform. After 72 h of V. parahaemolyticus challenge, the average methylation rate of PtRab2A was 40%, compared to 69% at 0 h, indicating a significant reduction in PtRab2A methylation levels. This trend was consistent with the IGV visualization results. Pearson correlation analysis revealed a correlation between PtRab2A methylation levels and gene expression levels in hemolymph tissue after V. parahaemolyticus infection, with a correlation coefficient of −1.000. This indicates a significant negative correlation between PtRab2A methylation and its gene expression levels. The RNAi results showed that the expression level of PtRab2A was significantly reduced at 12 h after injection of the interfering RNA (P＜0.05), and knockdown efficiency reached 80% at 24 h. After 24 h of interference, V. parahaemolyticus was injected into both the experimental and control groups. Mortality statistics indicated that in the experimental group, the mortality rate began to increase 2–3 h after infection, rising from 34.3% to 78.1%, representing a 2.27-fold increase, with all individuals dying by 4 h post-infection. In contrast, the mortality rate in the control group did not begin to rise until 4–5 h after infection, increasing from 15.0% to 62.5%, a 4.17-fold increase, with complete mortality occurring at 6 h. These results indicate that the mortality rate in the experimental group was significantly higher than that in the control group at the same time points, and peak mortality occurred earlier in the experimental group. Therefore, under V. parahaemolyticus infection, P. trituberculatus may enhance the expression of immune-related genes by reducing DNA methylation levels, thereby improving its immune capacity. These findings enrich the understanding of the molecular regulatory mechanisms involved in the defense against pathogen invasion in P. trituberculatus.
