Identification And Functional Analysis Of Factors Involved In Sterol Metabolism In Helicoverpa Armigera Gut

Sterols play important roles in many physiological processes in insects.However,unlike mammals,insects cannot de novo synthesize cholesterol.Herbivorous insects must acquire phytosterols from plants and convert them into cholesterol.Previous studies suggested sterols with different structures incorporated into insect tissues with different efficiency,but NPC1 b and SCP-2 proteins which mediate the uptake of dietary sterols did not show selectivity for different sterols.Our previous work indicated that ABC transporters may be involved in sterol excretion in insects and the studies in mammals proved that transportation mediated by ABC transporters can be selective.Except for the sterols being excreted out,some sterols can be converted into sterol esters for storage.Furthermore,the gut-bacteria may be actively involved in sterol metabolism in the caterpillar.However,the underlying molecular mechanisms are still unclear.In this study,we used the agricultural pest Helicoverpa armigera as the experimental model to screen and investigate the factors that may be involved in metabolism and regulation of sterol nutrition in the gut.We had 4 major findings:1.We screened a pool of the ABC transporters that may selectively excrete cholestanol from the intestinal tract of H.armigera by the transcriptome sequencing analysis.The functions of these candidate genes were verified by CRISPR/Cas9 technology;The Ha OG200342 was selected as the target gene and it was futher verified in vivo by strain purification and ABC transporter inhibitor tests;Tissue soaking experiments and protein docking experiments verified that the gene could specifically excrete cholesterol in vitro.The gene involved in the selective excretion of cholestanol from the H.armigera gut was finally identified as the ABC transporter Ha OG200342.2.Taking the advantage of the homozygous strain of H.armigera Ha OG200342 mutants that we obtained in the last study.We analyzed the transcriptional responses of the H.armigera caterpillar feeding on cholesterol and cholestanol diets.Seven genes that may be involved in the intestinal sterol metabolism were screened out,and the mammal homologs of these genes suggested they had the functions related to sterol metabolism.In the future,the functions of these genes in sterol metabolism and utilization warrant further examination.3.Based on the phylogenetic analysis,we screened out two MBOAT genes Harm.010114.1 and Harm.001362.1 that may be involved in the storage of sterols.The functions of these candidate genes were investigated by CRISPR/Cas9,GC-MS and protein docking analysis.The results indicated that Harm.010114.1 was involved in the storage of sterols in the intestine of H.armigera.Interestingly,the enzyme coded by Harm.010114.1showed preference to cholesterol over cholestanol,indicating that it may have selectivity for different sterols.Moreover,the expression of Harm.010114.1 was likely regulated by the expression of the ABC transporter Ha OG200342.Harm.001362.1 was not the target gene in the storage of sterols H.armigera,but it was involved in the synthesis of triglycerides in the intestinal tract of H.armigera.Mutations of this gene retard larval growth and affect ovary development in female adults.4.Many insects including lepidopterans have a large number of bacteria dwelling inside the gut.Many bacteria have cholesterol oxidase gene in their genomes and can use sterol as their nutritional supply.So gut-bacteria can potentially compete with their hosts for sterols.In this study,we identified the gut-microbiota in the intestine of five H.armigera populations by high throughput sequencing analysis.Species and abundance of bacteria were consistent with previous studies,and the differernce of populations were mainly caused by the host;Neutral model analysis indicated that some bacterial taxa may colonize inside the caterpillar gut.Among these bacteria,seven taxa can use sterol as their nutritional supply,and are good candidates for the further studies.In conclusion,our work demonstrated that the ABC transporter Ha OG200342 and the MBOAT protein Harm.010114.1 are involved in the selective excretion and storage of different sterols in the H.armigera gut.In addition,we screened out the target genes and the gut microbial candidates that may be involved in sterol metabolism in the H.armigera gut.These work substantially improves our understanding of sterol utilization and metabolism in insects.