Study On Physiological Response And Molecular Mechanism Of Bees To High Temperature And High Humidity Stress

Temperature and humidity are important factors that affect the physiological state of bees such as the individual reproduction and development and their pollination and collection behavior which greatly limits the development of the colony.Insects have evolved a series of physiological and biochemical mechanisms to resist the damage of high temperature and high humidity to the body during the long evolutionary process.The study of heat resistance mechanism of individual bees would provide a theoretical basis for revealing the resistance mechanism of insects during solitary or social evolution and the management of bee pollination in facilities.In the experiment,the survival time,tolerability and water loss of A.c.cerana and A.m.ligustica were measured under different temperature and humidity conditions.The activities of five antioxidant enzymes and five detoxifying enzymes in A.c.cerana and A.m.ligustica in conditions of high temperature or high humidity and both high temperature and high humidity treatments were measured.The experiment analyzed glucose,cholesterol,sorbitol,mannitol and trehalose,and changes in the content of free amino acids to explore the physiological response of bees under high temperature and humidity.The m RNA expression pattern of four endocrine-related genes(Jhe,Vg,CRH-BP,Aker)and three immune genes(Defensin,Abaecin,Hymenopt)in A.c.cerana and A.m.ligustica were measured,revealing the response of the endocrine system and immune system under high temperature and humidity.This study investigated the changes of heat resistance-related genes in honeybees by performing transcriptome sequencing on individual honeybee treated under different temperatures and humidity.Based on the results of transcriptome sequencing,the m RNA expression of six high temperature response genes(Hsp90,Hsp70 Ab,Zfp37,Stk-6,Pod,and Cyp450)in two species of bees were detected using q-RT PCR.Besides,m RNA expression patterns of two high humidity response genes(Pla2 and Afp)under different treatments of high humidity and high humidity treatment time were also determined.q-RT PCR and Western blot were used to determine the expression pattern of Hsp83 in the two species under different treatment temperature and time,and the correlation of the expression level with the concentration of each heat-resistant substances under the same treatment was analyzed.Hsp83 gene of A.c.cerana and A.m.ligustica was silenced by specific si RNA,and the changes of other heat-resistance related genes in the two honeybee species before and after silencing were detected.By recorded the survival time of A.c.cerana and A.m.ligustica before and after high temperature stress,the physiological function of honeybee Hsp83 was preliminarily estimated.The main results of this study were as follows:(1)The A.c.cerana and A.m.ligustica had different tolerance to high temperature and high humidity.In general,high temperature has a greater impact on bees,while high humidity has a relatively lesser effect on bees.Under high-temperature and high-humidity circumstances,the survival time of A.m.ligustica is slightly longer than that of A.c.cerana,and the amount of water loss is greater,but the A.c.cerana is more resistant to extreme high temperatures and the water loss rate is also higher.(2)The contents of antioxidant enzymes,detoxifying enzymes,glucose,cholesterol,sorbitol,mannitol and trehalose,and free amino acids in A.c.cerana and A.m.ligustica were significantly different between different temperature and humidity treatment groups.Under high temperature stress,the activities of antioxidant enzymes and detoxifying enzymes in the A.c.cerana and A.m.ligustica were increased.The high temperature treatment induced the content of glucose,cholesterol,sorbitol,mannitol and trehalose content in the A.c.cerana and A.m.ligustica to increased.The expression of multiple endocrine and immune genes in A.c.cerana and A.m.ligustica were affected by high temperature and treatment time.(3)After different temperature and humidity treatments,A.m.ligustica has found many differentially expressed genes.The GO analysis showed that the differentially expressed genes were involved in the biosynthesis and metabolism of lipids,proteins,amino acids,and polyols,ion transport,and signal transduction;in addition,there were some members of the heat-resistant protein family that were also different significantly between different temperature and humidity treatment groups.(4)The expression of multiple thermal response genes in A.c.cerana and A.m.ligustica were enhanced by high temperature and high temperature treatment time.Both high temperature and prolonged stress could significantly increase the expression of these genes,and the expression pattern of these genes were basically the same between the two bee species.The high-humidity response genes were also induced by high-humidity and treatment time,but the effects were mildly.The expression trends of these genes were similar between the two bee species.(5)Based on the bioinformatics analysis of Hsp83 in A.c.cerana and A.m.ligustica,it was found that the similarity between the sequences of Acer Hsp83 and Amel Hsp83 were more than 96%,and the physical and chemical properties and protein structure were high similarity.The expression of Hsp83 in A.c.cerana and A.m.ligustica was induced by high temperature and treatment time.Both high temperature and prolonged stress can significantly increase the expression of this gene,and its expression trend of was similar between the two bee species.The expression of Hsp83 in A.c.cerana and A.m.ligustica was significantly related to the concentration of trehalose and various amino acids in honeybees.After the Hsp83 of A.c.cerana and A.m.ligustica was silenced,the expression levels of most heat-resistant protein genes changed significantly.Preliminary studies speculated that Hsp83 contributed to the heat resistance of honeybees.This experiment analyzed the physiological response and resistance of A.c.cerana and A.m.ligustica to high temperature and high humidity.At the same time,the analysis of high temperature and humidity stress response gene expression patterns of A.c.cerana and A.m.ligustica,as well as the exploration of Hsp83 expression characteristics and function.It provides a theoretical basis for studying the stress resistance mechanism of bees in high temperature and high humidity environment,and also provides guidance for applying bees to pollinate facility crops and feeding and management of bees in facilities.