The emergence of resistance to the benzimidazole anthlemintics in parasitic nematodes of livestock is characterised by multiple independent hard and soft selective sweeps

Abstract

Anthelmintic resistance is a major problem for the control of parasitic nematodes of livestock and of growing concern for human parasite control. However, there is little understanding of how resistance arises and spreads or of the "genetic signature" of selection for this group of important pathogens. We have investigated these questions in the system for which anthelmintic resistance is most advanced; benzimidazole resistance in the sheep parasites Haemonchus contortus and Teladorsagia circumcincta. Population genetic analysis with neutral microsatellite markers reveals that T. circumcincta has higher genetic diversity but lower genetic differentiation between farms than H. contortus in the UK. We propose that this is due to epidemiological differences between the two parasites resulting in greater seasonal bottlenecking of H. contortus. There is a remarkably high level of resistance haplotype diversity in both parasites compared with drug resistance studies in other eukaryotic systems. Our analysis suggests a minimum of four independent origins of resistance mutations on just seven farms for H. contortus, and even more for T. circumincta. Both hard and soft selective sweeps have occurred with striking differences between individual farms. The sweeps are generally softer for T. circumcincta than H. contortus, consistent with its higher level of genetic diversity and consequent greater availability of new mutations. We propose a model in which multiple independent resistance mutations recurrently arise and spread by migration to explain the widespread occurrence of resistance in these parasites. Finally, in spite of the complex haplotypic diversity, we show that selection can be detected at the target locus using simple measures of genetic diversity and departures from neutrality. This work has important implications for the application of genome-wide approaches to identify new anthelmintic resistance loci and the likelihood of anthelmintic resistance emerging as selection pressure is increased in human soil-transmitted nematodes by community wide treatment programs.

Fig 1. Prevalence of parasitic nematode species identified on the seven study farms and parasite population structure.

(A) The relative frequencies of the eight major ovine gastro-intestinal nematode species based on species-specific PCR assays on 90 individual L₁ larvae on the seven study farms. (B) Pairwise F_ST values based on genotyping with ten microsatellite loci for H. contortus and T. circumcincta respectively. F_ST values estimated before correction for null alleles below the diagonal and after correction above. Values underlined and in bold show significant differences based on bootstrapping (10, 000 replicates) using the software program FreeNA. (C) SplitsTrees4 Networks based on populations of thirty worms on each farm genotyped using ten microsatellite markers for each species.

Fig 2. The relative proportions of isotype-1 β-tubulin alleles encoding resistance-conferring polymorphisms at F167Y, E198L or F200Y.

Populations for (A) H. contortus and (B) T. circumcincta, consisting of 30 or 32 individual worms, were pyrosequence genotyped at the three benzimidzole resistance associated SNPs in separate assays to determine the proportion of each SNP in each population.

Fig 3. Relative frequency of individual resistant haplotypes in seven UK sheep parasite populations.

A total number of five resistant haplotypes for (A) H. contortus and twenty eight resistant haplotypes for (B) T. circumcincta were identified. Susceptible haplotypes are not included this figure but are show in S3 Fig.

Fig 4. Network analysis of sequences of isotype-1 β-tubulin sequences.

Split networks were generated with the neighbour-net method of SplitsTrees4 from (A) H. contortus sequences and (B) T. circumcincta sequences. The circles in Split networks represent the different haplotype and the size of the circles is proportional to the frequency in the population. The colours define the resistant haplotypes across positions P167, P198 and P200 (P167F:P198E:P200F, Susceptible = blue; P167F:P198E:P200Y, Resistant at P200 = Red; P167Y:P198E:P200F, Resistant at P167 = green; P167F:P198L:P200F, Resistant at P198 = orange).