Physiology, pharmacology and immunology of ion-channels in Atlantic salmon and the salmon louse, Lepeophtheirus salmonis

Ion channels are important cellular mechanisms in all living organisms. They regulate the ion flow across cell membranes in cells such as neurons and muscle cells. Ion channels are the target proteins for a wide range of drugs. In this four-year project, an attempt will be made to identify the various ion channels present in both Atlantic salmon and salmon lice. The genomes from salmon and salmon lice have been fully sequenced, and the tools for a thorough, comparative bioinformatics analysis in both species are now available. The aim of the project is to identify the ion channels found in salmon and salmon lice, and to evaluate whether any of these are possible targets for chemical and/or immunological control. This will be done by 1) Bioinformatic identification and comparison of ion channels in salmon and salmon lice 2) Evaluation of selected ion channels as target proteins for control, based on toxicity tests and RNA interference studies, and after expression in Xenopus eggs and tests with model substances 3) Evaluation of selected ion channels as possible immunological targets, based on bioinformatics evaluation of immunological properties and immunization experiments The project is organized in four work packages. WP 1 - Bioinformatics In the first period, bioinformatics tools were used to search the genomes and transcriptomes of salmon and salmon lice to identify genes encoding ion channels. Several open data sources have been used: Salmobase (https://salmobase.org/) as well as NCBI (https://www.ncbi.nlm.nih.gov/Traces/wgs/?val=GIYK01) for salmon, and Licebase (https://licebase.org/) as well as NCBI (https://www.ncbi.nlm.nih.gov/genome/?term=Lepeophtheirus+salmonis) for salmon lice. Genes encoding voltage-gated sodium channels have been investigated specifically since they are the first focus in WP 2 and WP 3. WP 2 - Expression of selected ion channels in Xenopus eggs Before the project started, the salmon lice's nicotinic acetylcholine receptors were cloned. This is the target protein for the newly approved anti-sealice agent imidacloprid (Ectosan). At the beginning of this project, genes encoding the same receptor in salmon were identified, and cRNA to be injected into the Xenopus eggs was synthesized. Cloning and testing with compounds will be done in early 2022. In addition, focus has been on voltage-gated sodium channels in salmon lice, presumed target proteins for pyrethroids. It was difficult to express a plasmid that could be propagated in E. coli with this gene. Instead, parts of the gene as well as the rest of the plasmid are synthesized by PCR, and the pieces are spliced together. The approach seems to work. Some tests in the laboratory remain before cRNA can be isolated and injected into Xenopus eggs in early 2022. WP 3 - Validation of the Xenopus model against in vivo results Expression of genes encoding ion channels in Xenopus eggs and testing of these with electrophysiological techniques provide valuable information. However, it is not a given that they have the same effect in a complete individual. In WP 3, effective protocols have been established to study the effect of model substances and drugs on copepodids. Groups of parasites are exposed to 2-fold dilutions of the agent. Previously, each group was exposed in 1-liter bottles of seawater, but we have demonstrated that this can easily be done in wells of 350 microliters in 96-well microtiter plates without affecting the results. Ten copepodides are added to each well. Tests with 15 different substances believed to act on voltage-gated sodium channels have been completed. The results are compared with the results from WP 2. In the project, we expect to identify ion channels where the differences between salmon and salmon lice are large. These can be target proteins for drugs or vaccines. To test whether they are essential for survival, we have set up the method RNA interference. The first larval stage is exposed to a variant of the gene to be repressed. The parasite mobilizes a defense mechanism that cuts it into small pieces. At the same time, the parasite's self-produced mRNA for the protein is cut up, and the ion channel is not produced. The method is established in microtiter plates. It works, but there have been challenges in getting the larvae to develop into copepodids, also among controls. Some method development remains. WP 4 - Immunological potential This work package is not planned to start before the end of 2022.

Ion-channels are ancient cellular mechanisms present in all living organisms. A major function is regulation of ion flow across cell membranes in excitable cells like neurons and muscle cells. Ion-channels are targets for numerous drugs used against a wide variety of conditions such as elevated blood pressure, anxiety and parasites. The anti-sealice agents azamethiphos, deltamethrin and emamectin benzoate all act – by different mechanisms - on ion-channels. In this four-year project, the various ion-channels present in both Atlantic salmon and the salmon louse will be sought identified. The physiological function for the most relevant will be described using molecular, toxicological, electrophysiological and bioinformatics tools. Ion-channels in salmon lice that either are not present in the salmon, or show large differences between the species, can be potential targets for chemical or immunological control. The project has four interacting work packages. In WP 1, the salmon and salmon louse genomes will be screened for genes coding for various ion-channels using bioinformatic tools. These will be compared to elucidate significant differences between the host (salmon) and the parasite (salmon louse). In WP 2, a selection of identified ion-channels from the host and the parasite will be attempted cloned into Xenopus laevis oocytes and tested with two-electrode voltage clamp electrophysiological techniques. The influence of model substances on the gating properties will be tested. WP 3 will be a validation of ex vivo results from WP 2 against in vivo toxic effects on different developmental stages of the parasite using model substances or RNAi. In WP 4, selected recombinant proteins will be tested as vaccine antigens in small-scale proof-of-concept challenge trials.