Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mostly located in the post-synaptic membrane of cholinergic synapses. The natural neurotransmitter is acetylcholine, but they are also the direct targets for neonicotinoids, chemicals widely used against ectoparasites, arthropod vectors and agricultural pests. There are significant concerns regarding adverse effects of neonicotinoids on beneficial insects. In arthropods, functional nAChRs made of α subunits have been expressed from Drosophila genes, and hybrid receptors (sometimes also referred to as chimeric receptors) using species-specific α subunits and vertebrate β subunits have been expressed ex-vivo. Arthropod-specific nAChRs made of both α and β subunits from the target species have not been expressed ex-vivo. The aim of the current study was to express such receptors in Xenopus oocytes using only genes from Lepeophtheirus salmonis, to characterize them and study their modulation. Genes encoding α and β subunits of the nAChRs and three ancillary proteins, RIC-3, UNC-50 and UNC-74 were identified in the L. salmonis genome, subjected to RACE-PCR, cloned into an expression vector and the cRNA produced was then injected into Xenopus laevis oocytes. Co-expression of the ancillary proteins was essential for the successful expression of the L. salmonis nAChRs with both α and β subunits. Two functional nAChRs were identified: Lsa-nAChR1 consisting of α1, α2, β1 and β2 subunits, reconstituted to one distinct receptor, while Lsa-nAChR2, consisting of α3, β1 and β2 subunits reconstitutes receptors with two distinct characteristics. Out of seven neonicotinoids tested, six worked as partial agonist of Lsa-nAChR1 while only three did so for Lsa-nAChR2. Four non-neonicotinoid compounds tested had no effect on either of the nAChRs. The study demonstrated that fully functional, non-hybrid nAChRs containing both α and β subunits from an arthropod can be reconstituted ex-vivo by co-expression of essential ancillary proteins. Such models would be valuable for in-depth studies of effects by neonicotinoids and other compounds on target pests, as well as for studies of adverse effects on non-target arthropods. Nicotinic acetylcholine receptors, nAChRs, respond to the neurotransmitter acetylcholine or drugs like nicotine. These receptors are targets for neonicotinoids, the most commonly used compounds against ectoparasites and agricultural pests. In-depth studies of the function of these channels in arthropods are sparse, as no groups managed to reconstitute functional nAChRs made of both α and β subunits using genes only from the target arthropod in an ex-vivo system. We report the successful assembly of non-hybrid, fully functional nAChRs containing both α and β subunits from a marine arthropod, assembled and expressed in Xenopus laevis oocytes. We identified three possible combinations of α and β subunits producing functional receptors. We found ancillary proteins to be essential for successful expression and assembly of both α and β subunits into a functional receptor. The findings of the present study provide a basis for studying native nAChRs from arthropods, with a switch from hybrid nAChRs to species-specific native nAChRs.

Control of the sea louse Caligus rogercresseyi in the Chilean salmonid industry is reliant on chemical treatments. Azamethiphos was introduced in 2013, although other organophosphates were previously used. In 2014, reduced sensitivity to azamethiphos was detected in the Los Lagos Region using bioassays. The main target of organophosphates is the enzyme acetylcholinesterase (AChE). Mutations in the AChE gene are the main cause of organophosphate resistance in arthropods, including other sea lice. In the present study, we aimed to characterize C. rogercresseyi AChE(s) gene(s) and to study the association between AChE variants and azamethiphos resistance in this sea louse species. Samples of adult male and female C. rogercresseyi were collected in the Los Lagos Region in 2014. Twenty-four hour exposure bioassays with azamethiphos were performed to select sensitive and resistant lice. The full-length cDNA coding sequences encoding for two AChEs in C. rogercresseyi were molecularly characterized. One of the AChE genes was screened by direct sequencing in the azamethiphos-selected lice to search for variants. An additional louse sampling was performed before and after an azamethiphos treatment in the field in 2017 to validate the findings. The molecular analysis revealed two putative AChEs in C. rogercresseyi. In silico analysis and 3D modelling of the protein sequences identified both of them as invertebrate AChE type 1; they were named C. rogercresseyi AChE1a and 1b. AChE1a had the characteristics of the main synaptic AChE, while AChE1b lacked some of the important amino acids of a typical AChE. A missense change found in the main synaptic AChE (1a), F318F/V (F290 in Torpedo californica), was associated with survival of C. rogercresseyi at high azamethiphos concentrations (bioassays and field treatment). The amino acid change was located in the acyl pocket of the active-site gorge of the protein. The present study demonstrates the presence of two types of AChE1 genes in C. rogercresseyi. Although enzymatic assays are needed, AChE1a is most probably the main synaptic AChE. The function of AChE1b is unknown, but evidence points to a scavenger role. The AChE1a F/V318 variant is most probably involved in organophosphate resistance, and can be a good marker for resistance monitoring.