Screening And Identification Of The Candidate Genes Associated With Resistance To Ivermectin In Haemonchus Contortus

Helminthic parasites are an important group that has major impacts on public heath,veterinary,agriculture,and socio-economic sectors worldwide.The most important and broad-spectrum drug used to control the parasitic worms to date is ivermectin?IVM?.But resistance against IVM has been emerged in parasites,and preserving the efficacy of IVM is now becoming a serious issue.The parasitic nematode Haemonchus contortus is economically an important parasite of small ruminants across the globe.This parasite has a successful track record in IVM resistance.Resolving the genetic basis of IVM resistance is focus of increasing interest.There are growing evidences regarding the multigenic nature of IVM resistance.Although some genes have been proposed as candidates of IVM resistance using lower magnification of genome,the genetic basis of IVM resistance still remains poorly resolved.In this thesis we implemented the population genomics approach to identify the IVM resistance associated genes in H.contortus,and provide a new way to elucidate the molecular basis and mechanism of nematode drug resistance.In order to understand the geographical background variation we first analysed the various strains of H.contortus isolated from different geographical regions?Chapters 2 and 3?.Firstly,we applied genome-wide 2b-RAD pool sequencing on the three global populations of H.contours from: China?Tibet,TB;Hubei,HB;Inner Mongolia,IM;Sichuan,SC?;UK;and Australia?AS?,identified single nucleotide polymorphisms?SNPs?in all the strains,and analysed the genetic diversity among the different strains.The SNPs revealed a significant differentiation level?Average Fst = 0.34264?among them.Chinese populations TB and SC,along with the UK strain,were more divergent populations.Chinese populations IM and HB showed affinities to Australian strain.We then analysed signature of selection and detected 44?UK?,and 03?AS?private selective sweeps containing 49 and 05 genes,respectively.Finally,we did the functional annotation of selective sweeps and proposed biological significance to signature of selection.Although we detected signature of selection in UK and Australian strains,the background genetic differentiation was higher across the populations.This background genetic variation can lead to false positive conclusion,if not eliminated.So we next focused on the IVM-sensitive and resistant strains of H.contortus having very close genetic background.Therefore,we tested the six farm populations of H.contortus?IVM-sensitive and IVM-resistant?from northern China,and also compared them to H.contortus isolates from UK and Australia.We first prepared individual DNA samples from 240 adult worms,and generated genotyping data using eight microsatellite markers.Obtained data was then subjected to allelic frequency and population genetic analyses.The overall allelic richness?mean/locus/pop=7.375±0.844-10.125±1.109?,and expected heterozygosity?mean/locus/pop=0.646±0.040-0.735±0.025?indicated high degree of population genetic variation across the Chinese isolates.Low level of genetic differentiation?Fst=0.010-0.066?was observed across all the populations.AMOVA results showed high level of variation?93%?within the populations.PCA analysis revealed mixed clustering of all the populations with no visible geographical sub-structuring.Finally,the population admixture analysis resulted in extensive admixing of genotypes across all the populations.With these findings we conclude that there is no obvious population genetic structure with extensive gene flow across all the farm populations of H.contortus in northern China.We then subjected all these populations to whole genome resequencing in order to analyse their genome at the full magnification?Chapter 4?.The pooled samples of H.contortus strains both IVM-susceptible?Shan Xi-S,SXS;Australian-S,ASS?,and IVM-resistant?Wu Meng-R,WMR;Wu Shen-R1,WSR1;Shan Xi-R,SXR;UK-R,UKR;Wu Shen-R2.1,WSR2.1;Wu Shen-R2.2,WSR2.2?were sequenced?coverage=43X to 55.8X?,obtained reads data was mapped on the reference genome,and variant calling was performed across all the strains [SNPs?Indel?: 1135117?200119?in WMR;1269230?229384?in WSR;1306283?232273?in SXR;1338090?238944?in SXS;1573743?246766?in UKR;1303834?233877?in WSR2.1;1308649?234472?in WSR2.2;and 1079609?187012?in ASS].High levels of nucleotide diversity?0.030±0.008-0.041±0.011?were observed across all the autosomes as compared to the X chromosomal estimates?0.010±0.008-0.015±0.012?.The lowest genetic differentiation was observed between the SXS and SXR?Fst=0.014?,and the highest differentiation was shown by SXS vs UKR?Fst= 0.090?.Based on pairwise Fst data,candidate genes were identified in the elevated outlier windows?top 5 %?.The functional annotations of outliers revealed a total of 26 genes across the genome as candidates of IVM resistance.The candidate genes were falling under these areas: glutamate-gated chloride channels,oxidation-reduction process?cyt-p450?,GPCR,ABC transporter and pgp,nuclear hormone receptors,ligand-gated channels,acetylcholine-gated chloride channels,vesicle-mediated transport,and transmembrane transportation of ions.Finally,the analysis of Tajima's D further confirmed that all of these candidate genes are under the selection.In conclusion,we have zipped through the genetic architecture and nature of IVM selection in H.contortus,screening out a number of candidate genes associated with IVM resistance,which thus can further be subjected to functional validation assays.Finally,we selected two of the candidate genes?HCON₀0143950,a member of Cytochromes P450;and HCON₀0155510,a GPCR?for functional validation?Chapter 5?using RNAi based assays in H.contortus.The eggs of H.contortus were cultured into L2 larvae,which were then subjected to RNAi assays.After blocking the relevant genes the L2 larvae were subjected to different concentrations of IVM in serial dilutions.Lastly,a larval feeding inhibition assay?LFIA?revealed that blocking of HCON₀0143950 gene significantly reduced the feeding in larvae.With these findings we conclude that the gene HCON₀0143950 is involved in IVM resistance in H.contortus.Its possible mode of action is to take part in detoxification of xenobiotic in phase I of xenobiotic metabolism i.e.,biotransformation of drugs.In summary,we found that the H.contortus populations have high level of genetic diversity,and possess geographical background genetic variations.Using a genome-wide population genomics approach we have successfully identified IVM-resistant candidate genes.We have successfully validated functionally one of the candidate genes associated to IVM resistance.The Cyt-P450 gene?HCON₀0143950?significantly reduced the feeding in worms when it was silenced by RNAi,so it may be involved in IVM resistance.These results further enhance our understanding on the IVM resistance,and continue to provide further evidence in favour of multigenic nature of IVM resistance.