Analysis Of Transcriptomics And Proteomics And Functional Reseach Of IVM-Resistant Candidate Genes In Haemonchus Contortus

Haemonchus contortus(H.contortus)is representative gastrointestinal nematode parasitizing the abomasum of ruminants,causing incalculable economic losses to most countries and regions,including China.Control of this parasitic disease currently relies on the use of anthelmintics such as ivermectin.However,the repeated use and abuse of ivermectin has led to the widespread drug resistance problems.The mechanisms by which the parasites develop resistance to ivermectin-based drugs are complex and are not yet clear.The formation of drug resistance has been reported to require the presence of drug resistance genes and is polygenic in nature.Therefore,exploring the mechanisms of ivermectin resistance and uncovering possible resistance genes are key to parasite resistance research.In this thesis,the drug resistance of H.contortus isolated strains WS-R and YC-S were tested by larval development inhibition test,larval migration inhibition test,and locomotion behavior testing.The results showed that the LD50 for ivermectin resistance of YC-S was 2.17 ng/mL in the larval developmental inhibition test,which was less than the reported ivermectin LD50 sensitivity threshold of 9 ng/mL,indicating that YC-S was ivermectin-susceptible strain,while the LD50 for ivermectin resistance of WS-R was 15.28 ng/mL,which was higher than the LD50 sensitivity of ivermectin.The resistance ratio(RR)of WS-R to YC-S was greater than 7,which was significantly higher than that of the reported resistant strain(1.7),indicating that WS-R was an ivermectin-resistant strain.The results of larval migration inhibition test and locomotor behavior assay further confirmed that WS-R was ivermectin-resistant strain and YC-S was ivermectin-susceptible strain.Subsequently,the transcriptome changes of H.contortus susceptible and resistant strains before and after ivermnectin treatment were investigated,and the results showed that 39 genes were significantly regulated in all four comparison groups,and the GO annotations of these genes were mostly related to the functions of metabolic processes,catalytic activity and heat stress.KEGG pathway analysis revealed that these genes were significantly enriched in the pathways of substance and energy metabolism,TCA cycle,drug metabolism-cytochrome P450 and metabolism of xenobiotics by cytochrome P450,suggesting that after ivermectin treatment,the body turns on the process of transport,metabolism,or excretion of xenobiotics.In addition,reported drug resistance genes such as MRPs,P-gps,and cytochrome P450(CYPs),as well as UDP-glycosyltransferase(UGT)genes,were found to be significantly regulated.On the basis of transcriptomic studies,the proteomics of the susceptible and resistant strains of H.contortus before and after ivermectin treatment was further investigated,and the analysis showed that 354 differentially expressed proteins in the susceptible strain and 89 differentially expressed proteins in the resistant strain after the ivermectin treatment,which were annotated into redox,catabolism,and material transport functions,as well as into various pathways of material metabolism and biosynthesis,ABC transport proteins,and the TCA cycle.The proteomic and transcriptomic association analysis revealed that numerous drug resistance genes such as CYPs,P-gps,unc,and glc were significantly regulated on H.contortus after ivermectin treatment.In addition,3 genes and their corresponding proteins showed changes in expression,1 up-regulated and 2 down-regulated,in the susceptible strain before and after ivermectin treatment,and 1 gene and its corresponding protein expression was down-regulated in the resistant strain before and after ivermectin treatment.10 genes and their corresponding proteins showed the same expression trend,1 up-regulated and 9 down-regulated,in the resistant strain before ivermectin treatment compared to the susceptible strain before ivermectin treatment.3 genes and their corresponding protein expressions were down-regulated in the drug-resistant strain after ivermectin treatment compared to the susceptible strain after ivermectin treatment.KEGG Pathway analysis of these genes and proteins revealed significant enrichment into various pathways related to drug metabolism-cytochrome P450,metabolism of xenobiotics by cytochrome P450 and glutathione metabolism,suggesting that these genes play a role in the interaction between ivermectin and the worm.Among these differentially expressed genes and proteins,GST,UGT,Hsp70,pgp-9,and the hypothetical proteins TRINITY_DN30265_c0_g1₁5₁ were screened as ivermectin-resistant candidate genes in H.contortus.Finally,the candidate genes obtained from two omics sequencing screens were functionally investigated.Firstly,the feasibility of RNAi by soaking was verified,followed by gene silencing of the five genes,and then the locomotor behavior and feeding of the larvae were examined after gene silencing,showing a significant increase in sensitivity to ivermectin in larvae after Pgp-9,UGT,and GST gene silencing,suggesting that these three genes are associated with resistance to ivermectin in H.contortus.In summary,the present study examined the drug resistance of two isolated strains of H.contortus by several resistance assays.Using high-throughput sequencing techniques(transcriptomics and proteomics),we investigated the changes at the gene and protein levels of H.contortus under ivermectin treatment.Finally,we determined that GST,UGT and pgp-9 were associated with ivermectin resistance in H.contortus by two omics correlation analyses and RNAi.This study not only provides useful biological information for better understanding the interaction mechanism between ivermectin and H.contortus and the research of genes related to drug resistance,but also provides some valuable data for the prediction of drug targets and the identification and control of drug resistance of H.contortus.