The crustacean ectoparasite L. salmonis is the biggest problem in Atlantic salmon commercial aquaculture. In addition to financial losses, it severely affects fish welfare and public perception of the industry. The available antiparasitic measures are costly, stressful and traumatic for the fish and do not provide a satisfactory solution to the problem. A large-scale international project funded by the Norwegian Seafood Research Fund (FHF) is working to elucidate mechanisms underlying host resistance to the parasite. This work is expected to contribute to the development of effective antiparasite measures. Susceptibility to the parasite is markedly different between species in the Salmo vs. Oncorhynchus genera. The project is using state-of-the-art methods to study differences in chemical signaling, molecular and cellular responses in differentially susceptible salmon species. The use of CRISPR Cas gene silencing provides a powerful experimental approach to the search for genes responsible for lice resistance.
The success of lice infestation is determined by the ability of free- swimming copepodid stages to find and contact salmon, attach themselves and resist immune and cellular responses directed against the parasite. The project investigates both phases of parasite-host interactions: before and after attachment. Here we present chemical signaling research. Chemical cues (semiochemicals or kairomones ) play a key role in host detection. Water conditioned with Atlantic salmon but not with non-salmonid sea fish species attracts copepods. Given that lice attracting kairomones are not produced by the majority of fish species, blocking their biosynthesis is unlikely to cause any negative effect. The i dentification of genes involved in this pathway may open a practical way to improve resistance to the parasite. Semiochemical studies include three parts: water chemistry, lice behavior assays, and genomics. Water conditioned with Atlantic salmon, Pacific salmon and other marine species was collected from several farms and research stations in Norway and Canada, processed with solid phase extraction (SPE) , and analy zed by GC-MS and LC-MS. Putative semiochemical compounds were selected for testing on the free-swimming life stages of the parasite. Lice behavior studies use two complementary approaches. The immediate response is assessed by observing and recording directional movement towards the stimulus in an arena chamber. Another method evaluates response to a light flash following exposure to water containing test material . Lice copepods consistently show strong reactions to Atlantic salmon conditioned water but not to water conditioned with non-salmonid fish species. Comparison of lice behavioral responses to water conditioned with Atlantic and three Pacific salmon species is underway. Filtration through SPE columns removes semiochemicals and the response of lice to various sorbents has been evaluated. Recovery of the chemical stimuli in eluates from the columns was confirmed. A suite of putative semiochemicals was selected and tests of the lice behavioral response to them are underway.
The most ambitious goal of the project is termination of semiochemical production by gene silencing. It is important to find out in which tissues semiochemicals are produced and how they are released into water. The directional assay suggests that skin is a stronger attractant than other tissues and biological materials (blood plasma, mucus, and feces). Another assay indicates a dose-dependent effect of skin extract and the possible presence of semiochemicals in other tissues and mucus. In search for candidate genes, priority was given to cytochromes P450 (CYP). These enzymes play the key part in various pathways of secondary metabolism. In addition to the conserved CYP present in all vertebrates, some genes are specific to certain taxonomic groups. Their substrate specificity and physiological roles are unknown. Comparison of the genomes of salmons and other marine fish species has found several salmon-specific cyp with high expression in the skin. CRISPR Cas silencing of cyp2m1 was performed in Atlantic salmon.
Studies conducted by scientists from five countries combined parasitology and fish health, marine biology and invertebrate behavior, chemistry, genomics, and molecular biology. Research is ongoing and has reached a level where synergy is expected from pooled competency.