实验海洋生物学重点实验室知识库

The clam Meretrix petechialis is an important commercial aquaculture species in China. During the clam culture period, mass mortality events often occur due to the Vibrio infection. Understanding the mechanism of disease occurrence and host immune response can provide a scientific basis for disease control and genetic breeding of resistance varieties. In this study, M. petechialis was challenged with Vibrio parahaemolyticus immersion to simulate a natural infection, and the dynamic immune and metabolism response of clam hepatopancreas to Vibrio infection was analyzed by transcriptome analysis. We investigated the variation of hepatopancreas Vibrio load and immune-related signaling pathways during early bacterial infection, and identified the key signaling pathways related to gluconeogenesis at death outbreak phase. In addition, we analyzed the correlation between the infection doses and the Vibrio load. The main results were as follows:

After M. petechialis were challenged with V. parahaemolyticus immersion, the infection process was divided into four phases including latency, prodrome, onset and recovery phases based on the clam mortality data. The dynamic response of clams to Vibrio infection at different infection phases were investigated by transcriptome analysis. A total of 38067 differentially expressed genes (DEGs) were identified at different infection phases. DEG annotations showed that immune-related and metabolism-related signaling pathways were enriched, indicating that immune defense and metabolism process play key roles during bacterial infection. Three kinds of expression pattern were classified by cluster analysis, including U-shape, L-shape and inverted V-shape, and the KEGG analysis were performed, respectively. Anabolism and cellular growth proliferation related signaling pathways were repressed (long-lasting or transient) during bacterial infection. However, the immune related signaling pathways with different immune functions showed induction expression or repression expression against bacterial infection, which indicated that immune system take different strategies against bacterial infection. Furthermore, some signaling pathways such as PI3K-Akt signaling pathway both involved in immune defense and cell metabolism. This study provides a sight that the dynamic immunity and metabolic responses may be integrated to improve the host survival and shift more energy for immune defense.

In this study, the Vibrio load variation in hepatopancreas were analyzed during early phase of Vibrio infection (i.e., latency, prodrome) and the results showed that Vibrio load was high at 1 dpi (days post-infection), and then dramatically decreased at 2 dpi and kept low level at following time points. The results of transcriptome analysis and quantitative real time PCR indicated the Toll and Imd pathway and ROS (reactive oxygen) system play important immune defense roles during early bacterial infection. The Toll pathway and ROS system were activated to release antibacterial peptide (AMP) and H₂O₂, respectively, while the Imd pathway might be repressed to prevent from the bacteria proliferation. The RNAi experiment further supported that Toll pathway and ROS system could directly control the Vibrio abundance via producing AMP and H₂O₂. In addition, we identified the Vibrio-load related genes in the above pathways, which provide the candidate molecular markers for genetic breeding of Vibrio-resistant clam strain.

The transcriptome analysis of clam hepatopancreas during the death outbreak phase showed the significantly enriched of multiple metabolism-related signaling pathways containing PPAR, AMPK, PI3K-Akt signaling pathways and glycolysis/ gluconeogenesis, which revealed the involvement of metabolism-related signaling pathways in host immune defense. The weighted correlation network analysis (WGCNA) exhibited that several key enzymes involved in gluconeogenesis were enriched at this phase, indicating that the gluconeogenesis was related to death outbreak of clams. The detection of content change of metabolic markers (i.e., glucose and phosphoenolpyruvate carboxykinase (PEPCK)) and expression variation of key genes (i.e., glucose-6-phosphatase (G6Pase), PEPCK and etc.) in gluconeogenesis showed that the gluconeogenesis was repressed during Vibrio infection. Further analysis of the differences of metabolic markers and key genes in PI3K-Akt signaling pathway between moribund and survival individuals implied that gluconeogenesis was related with clam mass mortality. The results could help us to understand the molecular mechanism of death outbreak event and provide gene marker for healthy status detection of this clam.

Two clam groups with different survival rates (group A and group B) were challenged with four Vibrio doses and the result confirmed that group A had higher survival rates than group B in all the four doses. The survival rate decreased with the increase of the infection dose while the begin time and peak of death were similar under different doses. The change tendency of Vibrio load in hepatopancreas were similar during infection period among different infection doses, and Vibrio load increased with the infection dose increment at 3 dpi. The clam resistance (bactericidal ability) was evaluated based on the Vibrio load, group A is high resistant family and group B is low resistant family, respectively, which is consistent with the survival rate. In addition, we detected the expression level of immune-related genes containing dual oxidase (Duox), myeloid differential protein-88 (Myd88), Tube and catalase (CAT) among the individuals in group A and group B and its’ correlation with Vibrio load variation and screened several molecular indicators for clam resistance.