Research On Adaptation Mechanism Of Phosphine Resistant Liposcelis Entomophila To Hypoxia Environment

Liposcelis entomophila,as a tiny stored-grain pest species,has developed resistance to a variety of insecticides.In recent years,they have been widespread outbreaks in southern China,which seriously threaten the security of grain storage in China.At present,L.entomophila has widely developed phosphine（PH₃）resistance.Meanwhile,with the popularization and promotion of enriched Nitrogen controlled atmosphere technology,L.entomophila may have developed the adaptability to hypoxia（2%-5%O₂ level）,which may threaten the use of this technology.The hypoxia adaptability of L.entomophila is a critical research gap,especially for PH₃ resistant populations.This study mainly includes the following sections:1.The analysis of PH3 resistance and hypoxia adaptability of L.entomophilaThe PH₃ resistance and hypoxia adaptability of L.entomophila from the areas with serious occurrence in south and central China were investigated in this study.It was found that 8strains of L.entomophila in China had developed strong PH₃ resistance,and no susceptible strains were surveyed by using FAO recommended discriminate doses.The average mortality of the 8 strains was below 30%under a strong resistance discriminate dose（0.25 mg/L for 20h）,and the strongest resistance was Le ZJ strain,with an average mortality of 8.67%.By analyzing the polymorphism of the DLD gene,it is found that the PH₃ resistance of L.entomophila is caused by the mutation of dihydrolipoamide dehydrogenase encoded by the DLD gene.N505H is the most prevalent mutation site in the resistant strains of L.entomophila.By evaluating the hypoxia adaptability of different geographical strains of L.entomophila,PH₃ resistant strains Le ZJ and Le ZS had lower mortality,with an average mortality of only9.04%and 12.12%,which means PH₃ resistant strains have initially shown hypoxia adaptability.The population fitness and fecundity parameters,including survival rate at each stage,egg production,sex ratio and population trend index,were evaluated after two kinds of sublethal stress of PH₃ fumigation and hypoxia treatment.The results showed that hypoxia treatment had a significant inhibitory effect on egg production and the hatching rate of eggs,and there was no significant change in sex ratio and nymph survival rate.After sublethal doses of PH₃ fumigation and hypoxia treatment,although they can not completely kill PH₃ resistant L.entomophila,it shows fitness cost to these stress.PH₃and hypoxia treatment had inhibitory effects on the population of PH₃ resistant L.entomophila,but the inhibitory effect weakened after the second generation,which indicates the PH₃ resistant L.entomophila has the potential to develop into hypoxic adaptation.2.Comparative transcriptomic and metabolomic analysis of hypoxia adaptation mechanism of phosphine resistant L.entomophilaTo reveal the hypoxia adaptation mechanism of PH₃ resistant L.entomophila,comparative transcriptomic analysis was used to study the differentially expressed genes（DEGs）of PH₃resistant booklice after 48 h of PH₃ fumigation and hypoxia stress.A total of 11929 DEGs were found after PH₃ fumigation,including 5520 up-regulated genes and 6409 down-regulated genes.After hypoxia treatment,a total of 5160 DEGs were found,of which 3479 were up-regulated and 1681 were down-regulated.The GO enrichment analysis of these DEGs showed that the genes regulated by PH₃ resistant L.entomophila in response to hypoxia stress were mainly enriched in GO functional clusters such as nutrient metabolism,ribosome and mitochondrial function.KEGG enrichment analysis showed that hypoxia treatment enhanced some important pathways of PH₃ resistant L.entomophila,such as PI3K Akt,HIF and MAPK signaling pathways,and then regulated nutrient metabolism.Antioxidant enzymes,heat shock proteins and some signal pathways related to insect immunity were also involved in the regulation of L.entomophila on hypoxia stress.Based on the results of transcriptome analysis,PH₃ resistant L.entomophila may develop hypoxic adaptability through two mechanisms:The first is metabolic remodeling,which can enhance pentose phosphate pathway and pyruvate metabolism by changing the metabolic mode of carbohydrates,and compensatory enhance amino acid metabolism and phospholipid metabolism,so as to ensure the level of energy metabolism in hypoxic environment and maintain free amino acids,fatty acids and glucose to adapt to the hypoxic environment.The second is oxidative damage repair,which can enhance the damage repair and improve the adaptability under hypoxia by enhancing the antioxidant enzyme system and a series of signal transduction and immune mechanisms.To verify the metabolic regulation mechanism revealed on the transcriptional level,this study analyzed the changes in metabolome level of PH₃ resistant L.entomophila under hypoxia stress by using metabolomics technology.The analysis of differential metabolites in carbohydrate,lipid,and amino acid metabolism showed that the content of metabolites maintaining blood glucose stability increased.The contents of free fatty acids and free amino acids remained relatively stable or increased.Hypoxia partially inhibited the process of tricarboxylic acid cycle and enhanced pyruvate metabolism in glycolysis and pentose phosphate pathway.The PH₃ resistant L.entomophila can adapt to the hypoxic environment by enhancing amino acid metabolism and phospholipid metabolism,ensuring normal energy metabolism,and maintaining the normal levels of free amino acids,fatty acids and blood glucose.3.Function analysis of hypoxia inducible factor（HIF）in hypoxic adaptation of L. entomophilaFurthermore,to further clarify the role of hypoxia inducible factor（HIF）in the hypoxic adaptation of L.entomophila,three key genes in the HIF pathway,HIF1α,HIF1βand PHD,were cloned,and their expression patterns under hypoxia stress were studied.HIF1αhas a significant hypoxic response,while HIF1βand PHD did not change significantly under hypoxia.Meanwhile,the expression stability of 12 reference genes of L.entomophila under grain storage environmental stress was evaluated,and the most stable reference genes expressed in the process of PH₃ fumigation and hypoxia treatment were ACTB and EF1a,which laid a foundation for the further study of gene expression and functional analysis.For in-depth study the function of HIF1αin hypoxia adaptation of L.entomophila,RNAi method based on feeding ds RNA was used to knock down Le HIF1α.Results showed that Le HIF1αcould be successfully knocked down after continues feeding ds Le HIF1αfor 2 d.After knocking down Le HIF1α,the mortality of L.entomophila increased significantly（3.1-fold）under hypoxia stress,which confirmed HIF1αin the regulation of hypoxic adaptation of insecticidal L.entomophila.After knocking down Le HIF1α,the expression of key regulatory enzyme genes,HK,PFKL,GLUT1,ALDO,ENO1 and LDHA,related to glucose metabolism in the downstream of HIF pathway decreased significantly.These results confirmed the regulation role of HIF1αto these genes and supported that HIF1αcan positively regulate the glucose metabolism under hypoxia.Knocking down HIF1αdisturbed the metabolic regulation of L.entomophila under hypoxia,resulting in metabolism disorder and increased mortality.4.Role of antioxidant enzymes in hypoxic adaptation of L.entomophilaThe antioxidant enzyme system plays an important role in insect resistance and adaptation to environmental stress.Transcriptome analysis showed that hypoxia and PH₃ could enhance the antioxidant enzyme system of L.entomophila.To clarify the role of antioxidant enzymes in hypoxic adaptation of L.entomophila,the changes of ROS level and antioxidant enzyme activity in PH₃ resistant L.entomophila after PH₃ fumigation and hypoxia stress were detected.Results showed that both PH₃ fumigation and hypoxia treatment induced lipid peroxidation in L.entomophila,and PH₃ fumigation caused greater oxidative damage and H₂O₂ accumulation,but hypoxia stress had no such effect.The activities of Catalase（CAT）,Superoxide dismutase（SOD）and Peroxidase（POD）increased significantly after hypoxia treatment.These results provide a basis for exploring the resistance of PH₃ resistant L.entomophila to oxidative damage caused by PH₃ fumigation and hypoxia.This study also cloned and identified 10 genes in the antioxidant enzyme system of L.entomophila and clarified their expression profiles under PH₃ fumigation and hypoxia stress.SOD2,PRDX1,PRDX3 and PRDX6 responded significantly after hypoxia stress,indicating that they play an important role in eliminating oxidative damage caused by hypoxia.These results laid a foundation for understanding the role of antioxidant enzyme system in the adaptation of L.entomophila to adverse environmental stress.In conclusion,PH₃ resistant L.entomophila has become a dominant species in Chinese grain storage ecology,and its hypoxia adaptability will threaten the safety of grain storage in China.From the ecological test results,PH₃ resistant L.entomophila has the potential to develop into hypoxia adaptability.From the perspective of molecular mechanism,L.entomophila can regulate metabolic activities,enhance amino acid metabolism,stabilize the normal levels of free amino acids,fatty acids,and glucose in the Haemolymph,to ensure normal energy supply in hypoxic environment.Also,it can rely on signal transduction and other regulatory mechanisms to enhance HIF pathway and antioxidant enzyme system,regulate energy metabolism,and eliminate oxidative damage,to adapt to hypoxic environment.The results of this study provided a theoretical basis for next step studying the molecular mechanism of hypoxia adaptability of PH₃ resistant L.entomophila population,formulating scientific and effective fumigation and controlled atmosphere control schemes,and for revealing the molecular mechanism of stored grain pests in response to environmental stress.