Regulation Of Glucose Homeostasis And The Effect Of Starvation Stress On The Immunity Of Macrobrachium Rosenbergii

The giant freshwater prawn,Macrobrachium rosenbergii,is a significant aquaculture species in China,originating from the Indian and Pacific Oceans.Due to its glucose intolerance,the proportion of carbohydrates in diet is limited.Understanding the glucose tolerance and metabolic regulation pathways of M.rosenbergii would be beneficial in comprehending glucose intolerance in this species.Besides,glucose is the primary energy source in organisms.When confronted with the stress of starvation,organisms typically redistribute their energy resources to ensure their survival.To investigate the glucose tolerance and regulation of glucose homeostasis in M.rosenbergii,as well as its effects on immunity,we conducted glucose tolerance tests,starvation experiments and artificial infection challenge.Our findings are as follows:1.A glucose tolerance test was performed to assess the metabolic response of M.rosenbergii to a glucose load.The prawns were fasted for 24 hours,then given an injection of 1 mg of glucose per gram of body weight.Plasma glucose and trehalose levels peaked at 0.5 and 1 hour after injection,respectively,and returned to baseline levels between 2-4 hours,indicating a rapid clearance of exogenous glucose.The content of trehalose,glycogen,and triglyceride in both the hepatopancreas and muscle significantly increased after the glucose load,and variations in the transcriptional levels of metabolism-related genes were observed.The transcriptional levels of the gluconeogenesis rate-limiting enzyme,phosphoenolpyruvate carboxykinase,and catabolism-related enzymes,including trehalase,glycogen debranching enzyme,and adipose triglyceride lipase,were inhibited by the glucose load.In contrast,glycolysis and anabolism were stimulated,as indicated by increased mRNA levels of glucokinase,trehalose-6-phosphate synthase,glycogen branching enzyme,and diacylglycerol acyltransferase.Furthermore,the glucose injection decreased the expression of crustacean hyperglycemic hormone(CHH)and increased the expression of insulin-like peptide 1(ILP 1),while insulin-like peptide 2(ILP 2)expression remained unchanged.These findings suggest that a glucose load may induce the expression of ILP 1,stimulate glycolysis and the conversion of glucose to trehalose,glycogen,and triglyceride,while decreasing the expression of CHH and inhibiting gluconeogenesis to aid in the clearance of exogenous glucose in M.rosenbergii.2.To investigate the effects of starvation and refeeding on glucose metabolism in M.rosenbergii,prawns(8±0.7 g)were starved for 14 days and then refed for 7 days.The results showed that glucose and trehalose content in hemolymph,hepatopancreas,and muscle tissues decreased significantly during the first 2 days of starvation.hepatopancreas glycogen content decreased significantly from the fourth day of starvation,while muscle glycogen content decreased significantly from the first day of starvation.Triglyceride content in hepatopancreas and muscle decreased significantly from the 10th and 7th day of starvation,respectively,while protein content in hepatopancreas and muscle decreased significantly from the 14th and 10th day of starvation,respectively.Results indicate that during starvation,glucose and trehalose are the first energy sources to be consumed,followed by glycogen,triglycerides,and proteins in order.Compared to the hepatopancreas,muscle tissue responds more quickly to starvation stress.The mRNA levels of glycolysis rate-limiting enzyme and anabolism-related enzymes decreased during starvation,while transcriptional levels of gluconeogenic enzymes and catabolism-related enzymes(glycogen debranching enzyme,GDE,adipose triglyceride lipase,ATGL,and cathepsin B,CB)were increased.Starvation also stimulated the expression of CHH and ILP 1 expression at the beginning of starvation.During the subsequent food deprivation,no significant difference was observed between the two groups.Refeeding for 7 days led to a recovery of physiological and biochemical indices and transcriptional levels of metabolism-related genes.3.Between January 2021 and September 2022,pathogen detection was carried out in M.rosenbergii farming ponds,and 11 strains of pathogens were identified.Three strains of pathogens,including non-O1/O139 Vibrio cholerae,Citrobacter freundii,and Aeromonas hydrophila were selected for artificial infection experiments.The 24-hour median lethal concentrations(LD50)values of non-O1/O139 V.cholerae,C.freundii,and A.hydrophila in M.rosenbergii were found to be 1.6×107,2.1 ×106,and 1.2×107 CFU/mL respectively.To investigate the effect of starvation on the immunity,the prawns were starved for 2,4,7,10,and 14 days,and then artificially infected with the three pathogens.The results showed that the mortality rate was significantly reduced in 4,7,10,and 14 days starved groups.To further explore the immune response from the molecular level,14-day starved prawns were infected with A.hydrophila,and non-specific immune factors were measured at different time points after infection.The results showed that the activity of SOD,ACP,and AKP in the starved group was significantly higher than that in the feeding group,and the MDA content in the starvation group was significantly lower than that in the feeding group.These findings indicate that short-term starvation can stimulate the activity of non-specific immune factors,enhancing the resistance of M.rosenbergii to bacterial infections.