| Although Insect-resistant cotton of widespread planting reduced the application of a large amount of pesticides,the damage of Spodoptera litura as the secondary target pest became increasingly serious.Spodoptera litura is a polyphagous pest that has caused substantial losses in many economically important crops.However,the long-term use of chemical pesticides as the main measure of control for S.litura has led to the increasingly prominent problem of pesticide resistance.Many studies have been conducted on the resistance of S.litura,but the mechanisms remain unclear.In this study,we first analyzed the changes in biological characteristics of S.litura,such as life history,pupation rate,and eclosion rate,after feeding on insect-resistant cotton and herbicide-resistant cotton under different treatment conditions.Then,16 s rRNA high-throughput sequencing was performed to analyze the effects of insect-and herbicide-resistant cotton and their various tissues on the gut microbial diversity of S.litura.Subsequently,tissue-level dynamic transcriptome was used to analyze the changes in the pesticide metabolism pathways of S.litura in its pesticide response process,as well as key pesticide-responsive pathways and related genes.In addition,we conducted a genome-wide identification of key Toll-like receptor(TLR)genes that affect gut microbes and are involved in pesticide-responsive metabolic pathways and the validation of these genes that mediate pesticide resistance.This study is of great significance in the clarification of the effects of insect-and herbicide-resistant cotton on the gut microbial diversity and pesticide resistance of S.litura,which will improve the management of S.litura resistance while also providing new methods and a theoretical basis for the study and management of pest resistance.The following results were obtained:1.Using insect-resistant cotton as the control,S.litura fed with glyphosate-resistant cotton showed no significant differences in age-related weight,pupation rate,eclosion rate,or life history,thus indicating that transgenic glyphosate-resistant cotton could reach the safety levels of insect-resistant cotton against the non-target organism S.litura.However,after treating the glyphosate-resistant cotton with glyphosate herbicide,the pupation rate,eclosion rate,life history,and female pupal weight of the treatment group were significantly lower than those of the control group,which suggests that glyphosate could affect the growth and development of the non-target organism S.litura.In field experiments,S.litura larvae were fed with glyphosateresistant cotton with or without glyphosate.The gut microflora abundance of Corynebacterium was significantly different in fourth instar larvae and was negatively correlated with the treatment;in fifth instar larvae,abundance of Ralstonia was significantly different and was positively correlated with the chemical treatment.Further analysis of the functions of the differential bacteria revealed the enhancement of four functions,namely,amino acid transport and metabolism,carbohydrate transport and metabolism,ribosomal structure and biogenesis,and translation,which suggests that the microorganisms involved in these functions play crucial roles in the response to the herbicide glyphosate.2.After feeding the third instar larvae of S.litura artificial feed containing common pesticides(avermectin and matrine)for 72 h,midgut and integument samples were taken to analyze the distribution and abundance of gut microflora using 16 S rRNA high-throughput sequencing.The results showed that Enterococcus was the dominant bacterium shared between the pesticide-treated and the control groups;there were significant differences in gut flora(P <0.05),and Verrucomicrobia were positively correlated with pesticide treatment;there were eight other bacteria observed at a significantly different abundance at the genus level.The analysis of differences in the abundance of bacterial taxa beyond the larval gut revealed that one significantly different bacteria at the phylum level between the treatment and the control groups,Saccharibacteria,was positively correlated with the pesticide treatment.The significantly different abundance of bacteria at the genus level were all positively correlated with the treatment groups,involving up to 26 genera.These bacteria were mostly associated with pesticide metabolism and resistance formation.3.Comparative transcriptome analysis was performed on fifth instar larvae of S.litura at6 h,12 h,24 h,48 h,and 72 h after being fed artificial feed alone and artificial feed containing insecticide,and the dynamic transcriptome profile of pesticide metabolism in S.litura was constructed.The results showed that P450 was the main mechanism of insecticide metabolism in the larvae at 12 h,after which the number of genes involved in this mechanism gradually increased.After 48 h,the main mechanism of response to insecticide shifted to Toll and IMD metabolic mechanisms,and RT-q PCR verified that the expression trends of these genes were consistent with those of the transcriptome.A weighted correlation network analysis was performed to further identify co-expression modules that were significantly correlated with the insecticide response at 12 h and 24 h(r = 0.403,P = 0.0371;r = 0.436,P = 0.023),from which48 candidate genes associated with insecticide response were further identified.4.The genome-wide identification of the TLR genes was carried out using the published genome of S.litura.Seven TLR genes were identified,and bioinformatics tools were further used to analyze the evolutionary relationships among the TLR gene family members of S.litura.It was found that the TLR genes of S.litura were highly homologous to those of Helicoverpa armigera.Tissue-specific expression analysis of these seven genes showed that LOC111354111 was highly expressed in the gut,and five genes were found to respond positively to pesticide treatment.These results were combined with transcriptome data to identify the gene expression network of pesticide-responsive TLRs,which revealed coexpression relationships between LOC111357546 and 30 other genes.These genes were mainly involved in cell surface receptor signaling pathways,cellular localization,signal transduction,cellular regulatory processes,and biological regulatory processes.The discovery of these genes has laid the foundation for further research on the pesticide resistance mechanisms of S.litura. |
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