BACKGROUND AND PURPOSE The cholinergic agonist levamisole is widely used to treat parasitic nematode infestations. This anthelmintic drug paralyses worms by activating a class of levamisole‐sensitive acetylcholine receptors (L‐AChRs) expressed in nematode muscle cells. However, levamisole efficacy has been compromised by the emergence of drug‐resistant parasites, especially in gastrointestinal nematodes such as Haemonchus contortus. We report here the first functional reconstitution and pharmacological characterization of H. contortus L‐AChRs in a heterologous expression system. EXPERIMENTAL APPROACH In the free‐living nematode Caenorhabditis elegans, five AChR subunit and three ancillary protein genes are necessary in vivo and in vitro to synthesize L‐AChRs. We have cloned the H. contortus orthologues of these genes and expressed them in Xenopus oocytes. We reconstituted two types of H. contortus L‐AChRs with distinct pharmacologies by combining different receptor subunits. KEY RESULTS The Hco‐ACR‐8 subunit plays a pivotal role in selective sensitivity to levamisole. As observed with C. elegans L‐AChRs, expression of H. contortus receptors requires the ancillary proteins Hco‐RIC‐3, Hco‐UNC‐50 and Hco‐UNC‐74. Using this experimental system, we demonstrated that a truncated Hco‐UNC‐63 L‐AChR subunit, which was specifically detected in a levamisole‐resistant H. contortus isolate, but not in levamisole‐sensitive strains, hampers the normal function of L‐AChRs, when co‐expressed with its full‐length counterpart. CONCLUSIONS AND IMPLICATIONS We provide the first functional evidence for a putative molecular mechanism involved in levamisole resistance in any parasitic nematode. This expression system will provide a means to analyse molecular polymorphisms associated with drug resistance at the electrophysiological level.

Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important “classical” anthelmintics like levamisole and pyrantel, as well as “novel” anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.

Haemonchus contortus is a haematophagous parasitic nematode of veterinary interest. We have performed a survey of its genome-wide diversity using single-worm whole genome sequencing of 223 individuals sampled from 19 isolates spanning five continents. We find an African origin for the species, together with evidence for parasites spreading during the transatlantic slave trade and colonisation of Australia. Strong selective sweeps surrounding the β-tubulin locus, a target of benzimidazole anthelmintic drug, are identified in independent populations. These sweeps are further supported by signals of diversifying selection enriched in genes involved in response to drugs and other anthelmintic-associated biological functions. We also identify some candidate genes that may play a role in ivermectin resistance. Finally, genetic signatures of climate-driven adaptation are described, revealing a gene acting as an epigenetic regulator and components of the dauer pathway. These results begin to define genetic adaptation to climate in a parasitic nematode.

The candidate gene strategy developed in this study revealed an unexpectedly high diversity of L-AChR subunits specific to the trichostrongylid parasites that are a principal target for the drug LEV. Abbreviated variants, predicted to produce nonfunctional unc-63, were associated with LEV resistance. This study contributes significantly to a better understanding of LEV receptor constitution in parasitic nematodes and highlights the putative role of aberrant mRNA encoding L-AChR subunits in LEV resistance.