Identifying molecular determinants of drug susceptibility in salmon lice (Lepeophtheirus salmonis)

The salmon louse, Lepeophtheirus salmonis, is a crustacean ectoparasite infecting wild and farmed salmonid fishes, which causes significant problems in marine Atlantic salmon aquaculture. Salmon louse infections on farms require control to maintain good fish health and welfare, and to minimise potential impacts of farm-origin parasites on wild fish populations. Currently, salmon louse control on fish farms depends heavily upon the use of drugs, supplemented by farm management measures. A number of non-chemical control strategies are currently under development, and these include the biological control of lice through cleaner fish and attempts to render salmon resistant to lice through vaccination and selective breeding programmes. However, these and other alternative control strategies are presently not sufficiently developed to allow full implementation at an industrial scale.

While anti-parasitic drugs offer efficient salmon louse control, relatively few types of delousing agents are available. It is known from other pests and parasites that the repeated use of the same or similar chemicals increases the risk that parasites may develop drug resistances, driven by the mechanisms of natural selection discovered by Charles Darwin. Certain genetic features, called "resistance alleles", are usually rare in organisms as they provide no benefits to their carriers under normal conditions. Parasites possessing resistance alleles, however, are able to survive drug treatments better than those without, hence if the same drug is over-used, resistance alleles can increase their frequency in treated parasite populations as susceptible individuals are killed by treatment. This may ultimately result in drug resistance of the entire population.

Very little is known about the mechanisms responsible for drug resistance in salmon lice. In the proposed project, drug-resistant strains of salmon lice generated in the laboratory will be used to investigate potential resistance mechanisms. Two strategies will be followed to find out how these lice have managed to become drug resistant. Firstly, breeding crosses will be made between resistant and drug-susceptible salmon lice, and genetic sequences that are associated with resistance will be identified using the latest DNA sequencing methodologies. Secondly, differences in gene expression between resistant and drug-susceptible salmon lice will be determined, which will provide new insights into the involvement of particular molecular pathways in development of resistance to particular drugs. Such knowledge will be extremely useful in the identification of drugs which can break the resistance mechanism.

Choices among available treatment options on salmon farms are currently based on the results of "bioassays", which are small-scale laboratory treatments of salmon lice used to establish susceptibility to treatment. However, salmon louse bioassays are error-prone and regularly fail to correctly predict the success of subsequent farm treatments. The present project will develop fast tests to detect genetic susceptibility to treatment that are expected to be more specific and accurate than bioassays in identifying the best treatment choice for a given farm situation. Using the best available treatment has obvious economic and environmental benefits, and can help to prevent resistance development. New, unexploited drug classes represent a limited natural resource of high value for future food security. By helping to combat the development of drug resistance, this project will help to extend the life of current and novel delousing agents, and thus improve the sustainability of intensive salmon farming.

The overarching goal of this proposal is to provide a fundamental understanding of the molecular determinants of drug susceptibility in the salmon louse, a key pathogen of farmed Atlantic salmon. The study combines genetic mapping and gene expression studies, using drug susceptible and resistant L. salmonis laboratory strains as models. The study will generate a high-density genetic map based on an extensive library of single nucleotide polymorphism (SNP) markers, identify SNPs showing association to drug susceptibility status, and elucidate molecular pathways potentially involved in drug resistance using whole transcriptome sequencing. The utility of molecular markers in predicting treatment success will be validated by experiments on field isolates.

Methodology. A number of susceptible and resistant strains of L. salmonis are in culture at the Institute of Aquaculture and have displayed stable phenotypes over multiple generations (>10). Using two drug-resistant and one drug-susceptible sea louse strains, eight families will be made in F2 mapping crosses, beginning with dual strain parental mate pairs. Per family, one pair of F1 siblings will be crossed to produce many F2 animals, which will be assessed for their drug susceptibility. DNA from all family members will be used in restriction-associated DNA sequencing (RAD-seq) for SNP identification and parallel genotyping. A genetic map will be made and SNPs associated with drug susceptibility phenotype identified by QTL analyses. PCR-based genotyping assays will confirm this association for selected SNP in additional families. Transcriptomic profiles of resistant and drug-susceptible sea louse strains will be determined by Illumina RNA-seq technology to identify candidate genes and molecular pathways involved in resistance. To validate candidate markers, adult L. salmonis will be sampled at salmon farms, characterised regarding their drug susceptibility, and subjected to molecular analyses in the laboratory.

Outputs from this research will impact at many levels, including academia, the salmon production industry, independent agencies and regulatory bodies involved in fish health management, veterinary drug companies, government policy makers and consumers.

The main non-academic beneficiaries of this project are the UK salmon industry. With an annual production retail value of more than £1 billion, the UK is one of the world's leading salmon producers. Economic losses due to L. salmonis infections have been estimated at 33.6 million euros annually in the UK. The genome-wide genetic marker discovery and mapping approach of this project aims at identifying markers capable of predicting the drug-susceptiblity profile of L. salmonis populations. If successful, this approach will tremendously improve the accuracy with which veterinarians can identify effective, and avoid ineffective delousing treatments. This will have positive economic effects on the competitiveness of the UK salmon production. We have many years of successful engagement with the salmon industry as evidenced by our successful previous collaborations and the Letters of Support from this sector. By improving control of sea lice, this study will provide a major contribution to the sustainability of the UK salmon industry and thereby, UK food security in this sector.

Further beneficiaries of this study include various societal groups that will benefit from improved salmon louse control. Knowledge arising from this proposal is expected to help avoid inefficient drug treatments of farmed salmon, which, in addition to having economic implications, also carries environmental costs. Accordingly, this project will contribute to the reduction of potential adverse environmental impacts of salmon farming related to salmon louse treatments, and by doing so the study will indirectly contribute to maintaining a positive image of the UK salmon farming industry. Efficient L. salmonis control on farms is also important in preventing potential negative impacts of the parasite on wild salmonids. The proposed study will therefore have significant benefits for the conservation of UK biodiversity, and in the long term could have positive effects for communities in which fisheries are an important asset for tourism.

Another group of beneficiaries of this research project are pharmaceutical companies producing fish medicines. The proposed research attempts to elucidate, for the first time, the molecular mechanisms of drug resistance in a member of the large arthropod group of crustacea. The comparative analysis of transcriptomic profiles among L. salmonis strains differing in drug susceptibility, achieved by whole transcriptome sequencing, will identify molecular pathways potentially involved in drug resistance. This knowledge will be essential for finding strategies to overcome resistance, and thus potentially useful for pharmaceutical companies. Close collaborative links exists to different drug companies, some of which have an interest in developing novel products against L. salmonis. Three international producers of veterinary drugs have provided letters of support.

Finally, the proposed research will have long-term benefits for society by contributing to the safeguarding of the sustainability of aquaculture practices. Drug classes as yet unexploited as veterinary medicines represent a limited natural resource of paramount importance for future food security. By elucidaing the mechanism of drug resistance in salmon lice, this proposal will contribute to developing strategies to combat drug resistance and thus extend the lifetime of extant salmon delousing agents. Apart from immediate benefits related to current drug resistance, this study will provide a research framework for the dissection of the molecular mechanisms associated with other resistance problems in the future.