Biochemical And Intestinal Microecological Response Mechanisms Of Lymantria Dispar Asiatica Larvae Against Stresses

Lymantria dispar asiatica larvae were employed to undergo more acid/alkali diets administration,acute environmental temperature treatment,30%lethal MnCl2 contaminated diets administration,and 30%lethal avermectin stress in the present study.Here,more acid/alkali(compared with of ordinary pH)diets included 30%lethal DPB6(pH=5.00)and 10%lethal DPB8(pH=6.50).Diets DPB7(pH=6.05)was employed as control for DPB6 and DPB8.Environmental temperature included none lethal low temperature(15 and 20?),none lethal high temperature(30 and 35?),and 30%lethal high temperature(40?).The rearing temperature 25? was employed as control for stress temperature.After administration,the survive larvae were employed for analyses of digestive enzymic activities,antioxidant enzymic activities,total antioxidant capacity(T-AOC),and the structure and function of gut microbial community(V3-V4 region of 16S rRNA gene).Then,superoxide dismutase(SOD)and catalase(CAT)full sequences were cloned with rapid amplification of cDNA ends(RACE)technology.Finally,we detected the relative expression levels of Cu/ZnSOD(LdCZS),MnSOD(LdMS),and CAT(LdCAT)gene in different instar Lymantria dispar asiatica larvae,as well as the tissue specific and temporal profile of gene expression of these genes.Main results were showed as follows:1.Dietary pH significantly affected the activities of protease,amylase(AMS),and lipase(LPS)(p<0.05).The highest digestive activities were observed at different temperature,i.e.,protease at 30?,AMS and THL at 25?,and LPS at 20?.Digestive enzyme activities were inhibited when larvae were at lower or higher temperature condition,especially at 40?.LC30 MnCl2 contaminated diets significantly improved larval protease,AMS,and LPS activities(p<0.05).LC30 avermectin spraying significantly increased larval protease,THL,and LPS activities,and significantly decreased AMS activities(p<0.05).It suggests that larval digestive enzyme activities were affected by dietary pH,ambient temperature,manganese ion,and avermectin.2.Diets DPB8 significantly inhibited larval SOD activities(p<0.05).Diets DPB6 significantly increased larval polyphenol oxidase(PPO)activities(p<0.05).Larval T-AOC under DPB6 and DPB8 administrations were significantly higher than DPB7(p<0.05).LC30 MnCl2 contaminated diets significantly improved larval T-AOC through increasing the glutathione S-transferase(GST),carboxylesterase(CarE),peroxidase(POD),SOD,and CAT activities(p<0.05).LC30 avermectin significantly decreased larval SOD,POD,CAT,and PPO activities(p<0.05),but significantly improved larval CarE activities and T-AOC(p<0.05).These results suggest that dietary pH,manganese ion,and avermectin can affect larval antioxidant enzyme activities,and improve the larval T-AOC.In addition,results showed that larval T-AOC were inhibited by environmental temperature stress.But CAT could help Lymantria dispar asiatica larvae defensing none lethal high temperature stress(30 and 35?),and PPO could help larvae defensing none lethal low temperature(15 and 20?),none lethal high temperature(30 and 35?),and 30%lethal high temperature(40?).3.Diets DPB6 significantly affected larval gut microbial relative abundance,?-diversity,and five function strategies(p<0.05).Treating at 15?,the microbial relative abundance,dominant phylum and genus,and function of posttranslational modification,protein turnover,and chaperones(p<0.05)were affected.LC30 MnCl2 contaminated diets significantly affected the gut microbial relative abundance,?-diversity,and function of coenzyme transport and metabolism(p<0.05).Additionally,LC30 avermectin stress affected larval gut microbial relative abundance,dominant genus,?-diversity,and five function strategies(p<0.05).These results suggested that larval gut microbiota could be affected by dietary pH,ambient temperature,manganese ion,and avermectin.4.LdCZS,LdMS,and LdCAT expressed in all instar larvae and all detected tissues,which indicate their important roles in larval growth and development.The relative expression level of LdCZS,LdMS,and LdCAT were increased by sublethal avermetin stress.Cuticula was the tissue maintained highest expression levels rather than head,fat body,Malpighian tubule,and midgut after spraying sublethal avermectin(p<0.05).In addition,under LC90 avermetin stress,LdCZS showed higher expression than LdMS;LdCZS showed highest expression on the second hour,and LdMS showed the highest expression on the sixth hour.This suggested LdCZS respond more rapider and stronger than LdMS under avermetin stress.Furthermore,LdCAT showed the highest expression on the second hour,and then the relative expression of LdCAT was significantly reduced three orders of magnitude(p<0.05),and the relative expression of LdCAT from 4 to 12 h had no significant difference(p>0.05).It indicates the defense of LdCAT in the early stages under avermectin stress.In conclusion,stresses can differently affect the digestive enzyme activities,antioxidant enzyme activities,T-AOC,as well as the structure and function of gut microbial community of Lymantria dispar asiatica larvae.It also revealed the tissue specific and temporal profile of LdCZS,LdMS,and LdCAT under avermectin stress after full length cloning and bioinformatics analysis.The present study furthered our understanding about biochemical and intestinal microecological response mechanisms of Lymantria dispar asiatica larvae against stresses.