Infections by gastrointestinal parasitic nematodes are of major importance for both human and animal health. Control of these parasites mainly relies on the use of broad-spectrum anthelmintic molecules such as levamisole (LEV), benzimidazoles (BZs) and avermectins (AVMs). However, the intensive and repeated use of these anthelmintics has led to the emergence of resistant parasites (Kaplan and Vidyashankar, 2012, Sangster et al., 2018). This is currently a major concern for the small ruminant industry where highly pathogenic species such as Haemonchus contortus have developed resistance to the three major anthelmintic families (Peter and Chandrawathani, 2005, Kaplan and Vidyashankar, 2012). Importantly, LEV remains the anthelmintic with the lowest level of resistance compared with the other two main families of anthelmintics (Rose Vineer et al., 2020), thus stressing the need to identify and functionally validate robust resistance markers to optimize its use.

The pharmacological targets of LEV are nicotinic acetylcholine receptors (nAChRs) localized in worm body wall muscles. LEV binding results in permanent opening of the receptor, allowing entry of cations, causing continuous hyper-contraction of muscle and the irreversible paralysis of nematodes (Aceves et al., 1970, Aubry et al., 1970, Harrow and Gration, 1985, Martin et al., 2004). In the model nematode Caenorhabditis elegans, genetic screens for levamisole-resistant mutants (Lewis et al., 1980, 1987) allowed the identification of five nAChR subunits encoded by the unc-29, unc-38, unc-63, lev-1 and lev-8 genes, respectively (Fleming et al., 1997, Culetto et al., 2004, Towers et al., 2005). The nAChR subunits (approximately 500 amino acids) share conserved structural features including a signal peptide, four transmembrane domains and a large intra-cytoplasmic loop. Using the Xenopus laevis oocyte as a heterologous expression system, Boulin et al. (2008) demonstrated that the co-injection of these five subunits in combination with a set of ancillary proteins (RIC-3, UNC-74, UNC-50) resulted in the expression of functional heteropentameric levamisole-sensitive AChRs (L-AChRs) (Boulin et al., 2008), thus paving the way for the molecular characterization of their counterpart in parasitic nematodes.

In H. contortus and other parasitic trichostrongylid species such as Teladorsagia circumcincta and Trichostrongylus colubriformis, for which levamisole resistance is a major concern (Sangster et al., 1988, Martin and McKenzie, 1990, van Wyk et al., 1990, Sangster and Gill, 1999, Kaplan, 2004, Papadopoulos et al., 2012, Queiroz et al., 2020), the differential analysis of the expression of the unc-29, unc-63, unc-38 and lev-1 orthologues in LEV-susceptible versus LEV-resistant isolates revealed truncated isoforms of UNC-63 as potential molecular markers associated with LEV resistance (Neveu et al., 2010). The truncated Hco-unc-63 (Hco-unc-63b) transcript identified in the H. contortus LEV-resistant RHS6 isolate encodes a predicted protein of 343 amino acids including a signal peptide, the entire N-terminal extracellular domain, two transmembrane domains (TM1 and TM2) and 26 residues resulting from translation of the 3′ untranslated region (UTR; Neveu et al., 2010). Importantly, in the Xenopus oocyte, this truncated isoform of Hco-UNC-63 had a dominant negative effect on the expression of the recombinant L-AChR of H. contortus (made of UNC-63, UNC-38, UNC-29 and ACR-8), thus providing first, but nonetheless indirect, evidence of its potential involvement in the LEV resistance phenotype observed in H. contortus (Boulin et al., 2011).

In the present study, we took advantage of the recombinant Cel-L-AChR expressed in the Xenopus oocyte and the use of C. elegans as an expression system for AChR subunits to further investigate the involvement of truncated UNC-63 in LEV resistance. First, we tested the potential dominant negative effect of either Hco-UNC-63b, or an artificially truncated UNC-63 from C. elegans mimicking its parasitic nematode counterpart (Cel-UNC-63b), on C. elegans L-AChR expression. Second, we performed thrashing assays on transgenic C. elegans expressing either Hco-UNC-63b or Cel-UNC-63b to monitor a potential modulation of the worm’s LEV susceptibility. We report that in contrast with Hco-UNC-63b, Cel-UNC-63b has a drastic dominant negative effect on the expression of the Cel-L-AChR and confers LEV resistance to transgenic C. elegans. Our results represent the first known direct evidence of the role of a truncated AChR subunit in anthelmintic resistance, paving the way for the functional validation of a range of molecular markers associated with cholinergic agonist resistance.