Abstract
Haemonchus contortus is arguably one of the most economically important and ubiquitous parasites of livestock globally and commonly involved in cases of anthelmintic resistance. Here, we performed reciprocal genetic crosses using susceptible (MHco3(ISE)) and multiple anthelmintic resistant (MHco18(UGA2004)) H. contortus isolates. Resultant admixed populations were designated MHco3/18 or MHco18/3, where the lead isolate reflects the origin of the females. Three independent filial generations were generated for each cross, which were subjected to bioassays, molecular approaches and population genetic analyses to investigate the phenotypic and genotypic inheritance of benzimidazole (BZ) resistance at each stage. A panel of microsatellite markers confirmed the success of the genetic cross as markers from both parents were seen in the F1 crosses. Egg hatch tests revealed a stark difference between the two F1 crosses with ED50 estimates for MHco18/3 being 9 times greater than those for MHco3/18. Resistance factors based on ED50 estimates ranged from 6 to 57 fold in the filial progeny compared to MHco3(ISE) parents. Molecular analysis of the F167Y and F200Y SNP markers associated with BZ resistance were analysed by pyrosequencing and MiSeq deep amplicon sequencing, which showed that MHco3/18.F1 and MHco18/3.F1 both had similar frequencies of the F200Y resistant allele (45.3% and 44.3%, respectively), whereas for F167Y, MHco18/3.F1 had a two-fold greater frequency of the resistant-allele compared to MHco3/18.F1 (18.2% and 8.8%, respectively). Comparison between pyrosequencing and MiSeq amplicon sequencing revealed that the allele frequencies derived from both methods were concordant at codon 200 (rc = 0.97), but were less comparable for codon 167 (rc = 0.55). The use of controlled reciprocal genetic crosses have revealed a potential difference in BZ resistance phenotype dependent on whether the resistant allele is paternally or maternally inherited. These findings provide new insight and prompt further investigation into the inheritance of BZ resistance in H. contortus.
Highlights
Resistance to each of the major broad-spectrum classes of anthel mintics has emerged rapidly in parasitic nematodes of small ruminants and is widespread in several species, including Teladorsagia cir cumcincta and Haemonchus contortus (Kaplan, 2004)
A number of non-synonymous single nucleotide polymorphisms (SNPs) on the β-tubulin isotype 1 gene have been asso ciated with BZ resistance in several helminth species, and include a phenylalanine to tyrosine substitution at codon 200 (F200Y) (Kwa et al, 1994), phenylalanine to tyrosine (F167Y) (Ghisi et al, 2007) or
A change at codon 198 was reported in H. contortus and T. circumincta where glutamic acid switched to leucine (E198L) and was found to be independent of F167Y and F200Y (Mohammedsalih et al 2020; Martínez-Valladares et al, 2020)
Summary
Resistance to each of the major broad-spectrum classes of anthel mintics has emerged rapidly in parasitic nematodes of small ruminants and is widespread in several species, including Teladorsagia cir cumcincta and Haemonchus contortus (Kaplan, 2004). Understanding the evolution and inheritance of anthelmintic resis tance has been a global research focus for many years. Developments in the field of BZ resistance are the most advanced for any of the anthel mintic classes, questions relating to the inheritance of resis tance genes still exist. A change at codon 198 was reported in H. contortus and T. circumincta where glutamic acid switched to leucine (E198L) and was found to be independent of F167Y and F200Y (Mohammedsalih et al 2020; Martínez-Valladares et al, 2020). In H. contortus, the β-tubulin isotype 1 gene (HCON_00005260) is auto somal and is located on chromosome 1 at position 7027492-7031447; in globally distributed populations, this genomic locus remains highly differentiated as a result of longterm and widespread use of BZ drugs that have selected for resistance (Doyle et al, 2020). The presence of the β-tubulin SNPs appears to be well correlated with phenotypic expression of BZ resistance (von Samson-Himmelstjerna et al, 2007), the relative impact of the different SNPs towards the resistance phenotype and interactions between them remain unclear (Kotze et al, 2014)
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