Studies On The Macronutrient Regulation And Its Molecular Mechanism In Mythimna Separata

Protein and carbohydrate are two essential nutrients for insects.Protein is the source of amino acids for insect growth,while carbohydrate is the source of energy for insect.These two nutrients play important roles in their growth and reproduction.In nature,insect herbivores experience heterogeneous nutritional landscapes,i.e.the contents of protein and carbohydrate in the plants generally vary upon different species,different tissues and different growth stages.Thus,herbivores can face with great challenge to find the foods that contain the proper nutritional composition in their living environment.To achieve the optimal nutrients,they must regulate food intake to meet their physiological demands.Geometric framework model has been demonstrated to be a powerful tool to investigate nutritional regulation in insects.This model has been extensively used in investigating protein,carbohydrate and other nutrients regulation in insects.Although the fitness response of many insects on the macronutrient has been widely determined using this model,the molecular mechanism involved in nutritional regulation is largely unknown in insects and the functions of related genes are unclear.This study used the phytophagous Mythimna separata as the model insect to investigate the macronutrient regulation.Furthermore,the fitness response of M.separata to the different protein and carbohydrate contents(both ratios and concentrations)in food was determined.Finally,the differential gene expression on transcription level involved in protein/carbohydrate regulation was studied through transcriptomic analysis,and the functions of related genes were investigated.The main research results are showed as follows:1.Analyses of protein and carbohydrate regulation and gene expression on transcription level in M.separata using geometric framework model.Under the choice and no-choice experiments,we confirmed that M.separata could reach their optimal intake target through self-selection and had a tight regulation to the protein.Moreover,M.separata performed well on the food containing the closest nutritional composition to the intake target,and performed bad on the food deviating from the intake target.The results of body nutrient growth showed that M.separata maintain the more lipid content in female.Transcriptome analysis showed that M.separata regulated a small number of genes when consuming the protein-biased food,and regulated a great number of genes when consuming the carbohydratebiased food,and the PEPCK gene was differently expressed under two extreme protein/carbohydrate ratio diets.2.The function of M.separata PEPCK gene in protein and carbohydrate regulation.The results of q PCR showed that the PEPCK gene expression was upregulated by the highprotein diet and downregulated by high-carbohydrate diet.M.separata PEPCK gene is cytosolic isoform and highly expressed in the fat body.The food intake of M.separata was significantly increased under p35:c7 and p21:c21 treatments when the PEPCK gene were knocked out by CRISPR/Cas9.However,the nitrogen accumulation in the pupa were significantly decreased,while the lipid accumulation in the pupa were significantly increased.In addition,a substantial number of amino acid transporter genes was highly expressed in the fat body,indicating that these amino acid transporters may facilitate the glycogenic amino acids into the fat body and participate in gluconeogenesis.3.The fitness response of M.separata under different nutrients ratios and concentrations.The intake targets of M.separata was distinguishable under different macronutrient concentration,but the final protein/carbohydrate ratio is closed to 1:1.The food intake was significantly decreased when the insects fed on the foods having high nutritional concentration,and significantly increased on the low concentration ones.However,the final nutrient intake did not reach the same level.Moreover,the lifetime fecundity was higher and body lipid content in the pupa was lower on the high protein ratio diets when the insects fed on high macronutrient content diets.In contrast,the results were inverse under high carbohydrate ratio when the insects fed on high macronutrient content diets.4.Protein and carbohydrate regulation and functional analysis of NPY gene function under solitary(1 neonate)and crowded(10 neonates)rearing density.The nutritional regulation strategies of M.separata were different under two rearing density.The larvae may sense the poor nutritional status of food and had less food intake in the p7:c35 diet under the crowed treatment.Moreover,the oriental armyworm had lighter body color,larger wing load and bigger ovary under solitary treatment than those under crowed treatment.The food intake of M.separata was significantly decreased under all treatments,and the mortality of larvae was significantly increased under p35:c7 and p7:c35 diets when the NPY gene were knocked out by CRISPR/Cas9.In summary,the present study investigated the nutritional regulation strategies to protein and carbohydrate and gene responses on transcription level for M.separata.The results indicated that M.separata have different nutritional regulation strategies under different protein/carbohydrate ratio diets.We then found that PEPCK gene played important role in the macronutrient regulation in M.separata,and NPY gene can induced different larval physiological responses under different protein/carbohydrate ratio diets.The study of macronutrient regulation of M.separata under two rearing density revealed that there was different nutritional regulation between two insect biotypes.The findings substantially improved our understandings of the physiological roles of nutrients in these insects,and provided fundamental information on how to manage and utilize these insects.