A new family of glutamate-gated chloride channels in parasitic sea louse Caligus rogercresseyi: A subunit refractory to activation by ivermectin is dominant in heteromeric assemblies.

Sea louse ectoparasitosis is a major threat to fish aquaculture. Avermectins such as ivermectin and emamectin have been effectively used against sea louse infestation, but the emergence of resistance has limited their use. A better understanding of the molecular targets of avermectins is essential to the development of novel treatment strategies or new, more effective drugs. Avermectins are known to act by inhibiting neurotransmission through allosteric activation of glutamate-gated chloride channels (GluCls). We have investigated the GluCl subunit present in Caligus rogercresseyi, a sea louse affecting aquaculture in the Southern hemisphere. We identify four new subunits, CrGluCl-B to CrGluCl-E, and characterise them functionally. CrGluCl-A (previously reported as CrGluClα), CrGluCl-B and CrGluCl-C all function as glutamate channel receptors with different sensitivities to the agonist, but in contrast to subunit -A and -C, CrGluCl-B is not activated by ivermectin but is rather antagonised by the drug. CrGluCl-D channel appears active in the absence of any stimulation by glutamate or ivermectin and CrGluCl-E does not exhibit any activity. Notably, the expression of CrGluCl-B with either -A or -C subunits gives rise to receptors unresponsive to ivermectin and showing altered response to glutamate, suggesting that coexpression has led to the preferential formation of heteromers to which the presence of CrGluCl-B confers the property of ivermectin-activation refractoriness. Furthermore, there was evidence for heteromer formation with novel properties only when coexpressing pairs E/C and D/B CrGluCl subtypes. Site-directed mutagenesis shows that three transmembrane domain residues contribute to the lack of activation by ivermectin, most crucially Gln 15' in M2, with mutation Q15'T (the residue present in ivermectin-activated subunits A and C) conferring ivermectin activation to CrGluCl-B. The differential response to avermectin of these Caligus rogercresseyi GluClsubunits, which are highly conserved in the Northern hemisphere sea louse Lepeophtheirus salmonis, could have an influence on the response of these parasites to treatment with macrocyclic lactones. They could serve as molecular markers to assess susceptibility to existing treatments and might be useful molecular targets in the search for novel antiparasitic drugs. Author summary: Fish is used the world over as a source of protein for a growing population. Fish farm productivity is severely menaced by outbreaks of sea lice, small crustaceans which feed on the skin of fish damaging their muscle and fat, making them unmarketable. Macrocyclic lactone compounds known as avermectins have been used to combat sea louse infestation. Avermectins act by irreversibly activating sea louse glutamate-gated chloride channels, GluCl, disrupting nerve signals leading to paralysis and death. Unfortunately, resistance to avermectins has developed markedly, decreasing their usefulness. To better understand the interaction between avermectins and its target in sea louse we have studied the GluCl subunits in Caligus rogercresseyi, the main farmed-fish pest in the Southern Hemisphere. We found that C. rogercresseyi GluCls form a family of five, named A to E, subunits encoded by separate genes. Of note, subunit B is not activated by avermectins and is able to confer this property to other GluCls when expressed together. We find that C. rogercresseyi GluCl subunits are highly conserved in the main Northern Hemisphere parasitic sea louse Lepeophtheirus salmonis. Together they might serve as molecular markers to assess susceptibility to existing treatments and could become useful molecular targets in the search for novel antiparasitic drugs. [ABSTRACT FROM AUTHOR]