Characterization Of Energy Metabolism Regulation In Clam Meretrix Petechialis In Response To Vibrio Infection And And Its Relationship With Death

Meretrix petechialis is an important marine economic shellfish.In recent years,due to the deterioration of the breeding environment and the invasion of pathogenic bacteria,massive death of M.petechialis occurs frequently,which has become an important problem restricting the development of M.petechialis aquaculture industry.Therefore,analysing the disease resistance mechanism of M.petechialis infected Vibrio parahaemolyticus and selecting the Vibrio-resistant varieties is an effective way to solve this problem.With the further study of Vibrio resistance traits,it has been found that the survival of M.petechialis infected V.parahaemolyticus is not only related to the defense of host immune system,but also closely related to the regulation of energy and material metabolism during V.parahaemolyticus infection.Therefore,it is significant to understand the relationship between the mechanism of energy and material metabolism changes and fate of survival or death during V.parahaemolyticus infection.This study focuses on the energy metabolism regulation center Mp AMPK of M.petechialis and the key energy metabolism pathway-lipid metabolism,and investigates the energy metabolism response of M.petechialis to V.parahaemolyticus infection revealing the key role of energy metabolism in response to V.parahaemolyticus infection.Then,candidate genes related to energy metabolism were screened for resistant biomarker,based on resistant and sensitive clam families,and the basic expression level of these genes is closely related to the survival rate after infection.The above research provides new molecular markers for the Vibrio-resistant clam selection.The main results are as follows:1.The Mp AMPK-α2 sequence of M.petechialis was obtained.It is found that the sequence of Mp AMPK-α2 has a degree of conservation by homologous sequence analysis.The response of AMP/ATP and ADP/AMP in muscle tissue of M.petechialis were clarified to V.parahaemolyticus infection by artificial V.parahaemolyticus infection experiment.Correspondingly,it was detected that Mp AMPK gene expression and P-Mp AMPK activity had a positive response on AMP/ATP and ADP/ATP level.We further explored metabolic regulation of glucose,lipid and protein downstream of Mp AMPK and the change of NAD~+/NADH value in the process of V.parahaemolyticus infection.It is clear that glycolysis process enhanced and the metabolism of glucose,protein and lipid showed a trend of enhanced anabolism,which was also aligned to the increase of NAD~+/NADH ratio.These results suggested that coping with V.parahaemolyticus infection for M.petechialis is an energy-consuming process,and the host may enhance glycolysis process,and glucose,protein and lipid synthesis to meet the energy and material supply in the establishment of immune defense and damage repair during V.parahaemolyticus infection.2.It was suggested that V.parahaemolyticus infection has a significant effect on energy metabolism-related gene expression in M.petechialis based on transcriptome data.So this part of the study focused on the changes of key enzyme gene expression and key content indicators of products in triglyceride anabolism and catabolism,fatty acid de novo synthesis,and fatty acidβoxidation of lipid metabolism pathway in the hepatopancreas of M.petechialis during V.parahaemolyticus infection.The results show that levels of triglycerides(TG)decreased and free fatty acids(FFA)increased in the infected clams.Correspondingly,the synthase activities of TG were significantly reduced after V.parahaemolyticus infection.Additionally,expression of key enzymes related to FFA de novo synthesis was strengthened,but key genes of the fatty acidβ-oxidation pathway showed no significant changes after V.parahaemolyticus infection.The results supported the relationship existed between lipid metabolism changes and the emergence of massive mortality induced by bacterial infection in clams,which will help to better understand the role of lipid metabolism in energy regulation when the host responds to V.parahaemolyticus infection.3.The resistant and sensitive clam populations were identified based on the mortality difference by V.parahaemolyticus infection experiment.Based on the significant response of glucose,lipid and protein metabolism to V.parahaemolyticus infection,we examined the m RNA expression of the 14 key genes of these three main energy metabolism pathways in the resistant and susceptible groups kept in both uninfected and infected conditions.The basic expression levels of 10 genes(GS,m TOR,e EF2K,ATGL,MDY,SREBP,FAS,CPT-1,ACS and ECH)in the uninfected state and 9 genes(GS,m TOR,e EF2K,SREBP,FAS,ACC,CPT-1,ACAD and ECH)under infection condition were significantly different,and the expression in the resistant group was higher.It revealed that the higher metabolic level may be the metabolic characteristic of the resistant group.Among them,the gene expression of10 genes(GS,m TOR,e EF2K,ATGL,MDY,SREBP,FAS,CPT-1,ACS and ECH)in the uninfected state can be used to determine different Vibrio resistance of M.petechialis without V.parahaemolyticus challenge as potential markers,which provide theoretical and technical support for the breeding of Vibrio-resistant clams.