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

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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 reventing 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.
 
Description Sea lice are small parasitic crustaceans feeding on the mucus, skin and blood of their fish hosts. Infections with sea lice constitute a major health challenge in the commercial farming of Atlantic salmon, with the salmon louse (Lepeophtheirus salmonis) being responsible for most infections in the North Atlantic. At farm sites, sea lice are controlled using integrated pest management strategies, which combine drug treatments and a variety of non-chemical control approaches. These latter include farm management approaches such as fallowing and a range of other preventative and responsive controls ranging from use of physical methods of delousing to the deployment of cleaner fish for biological control. While non-chemical approaches to sea louse control are now widely used, veterinary medicines remain essential for the control of infections, e.g. where alternatives are not available or are advised against from a fish health point of view. However, there is a limited range of available licensed salmon delousing agents and for many of these there has been a fall in treatment efficacy due to drug resistance development in the parasite. Despite the high relevance of drug resistance in sea lice, the molecular mechanisms that confer resistance are still poorly understood, complicating efficient resistance management. The aims of the present project were to obtain insights into the genetic basis of sea lice resistance against two major drugs, the pyrethroid deltamethrin (DM) used in the bath treatment AMX®, and the macrocyclic lactone emamectin benzoate (EMB), contained in the oral drug SLICE®. In the current project, experiments involving genetic crossing between different louse strains (resistant / susceptible) were combined with novel high-throughput gene sequencing technologies in order to help to identify the genetic basis for drug resistance in sea lice. Crosses between resistant and susceptible parasites generated a number of families spanning three generations, in which the individual parentage of animals was known, and all parasites were characterised regarding their resistance to treatment with DM or EMB. The results revealed that DM resistance was inherited maternally, meaning that the resistance status of the female parent in a cross was the main factor in deciding whether the next generation would be resistant or not, with only little effect of the male parent. This pattern of inheritance was unexpected and suggested that DM resistance in sea lice might be controlled by mitochondrial genes. Mitochondria are subcellular bodies involved in energy metabolism, which possess their own DNA and are transmitted to the next generation only through the female line. Analysis of the mitochondrial genome in resistant and susceptible sea lice from different origins confirmed this hypothesis and led to the identification of mitochondrial mutations associated to DM resistance. These exciting and novel results, which were published in the journal PLoS One in 2017, can now provide the basis for genetic tests that assist salmon farmers in predicting drug resistance and avoiding treatment failures. However, more research is needed to identify the exact mechanism underlying the observed resistance. The discovery of roles of mitochondrial genes in DM resistance in salmon lice has challenged the common assumption that the mechanisms of drug resistance in sea lice resemble those in insects. In insects, resistance to DM and other pyrethroids is usually caused by mutations in sodium channels of the nervous system, which represent the site of action for these drugs. Alternatively, pyrethroid resistance in insects can also be caused by the up-regulation of metabolic pathways that detoxify drugs. The potential roles of both these mechanisms in DM resistance in sea lice was also therefore studied. As reported in an article published in the journal Pest Management Science in 2018, salmon lice possess three sodium channel genes, one of which shows characteristic mutations in resistant strains, suggesting an additional contribution to DM resistance. In addition, the cytochrome P450s, a gene family of enzymes often involved in pesticide resistance, has been characterised in the salmon louse for the first time. The still unpublished study revealed that parasitic salmon lice have fewer P450 genes than free-living crustacean counterparts, and that P450 genes are not up-regulated in DM- resistant salmon lice, suggesting they are not involved in the resistance mechanism. In contrast to DM, the pattern by which EMB resistance was transmitted within families generated in the above crossing experiments agreed with the classical laws of genetics discovered by Gregor Mendel, with the degree of resistance in a parasite being determined by the combination of genetic factors received from both parents. Genome sequencing techniques and genetic association studies were used to analyse the nature of the genetic factors controlling EMB resistance. In our experiments, ~24,000 genetic markers spread along the full sea louse genome were discovered and employed to examine the genetic structure, as it relates to drug resistance, of selected families of the crossing experiments. To support further genetic analyses, the number of chromosomes in the salmon louse was assessed by cytogenetic studies for the first time, showing that the parasite possesses 15 pairs of chromosomes. After using ~5000 of the above genetic markers to generate a map of the relative positions of markers across the genome / chromosome complement, markers associated to EMB resistance were identified and located. The results of the study, which is currently under review for publication, revealed that EMB resistance is controlled by two regions of the same chromosome, suggesting that at least two genes are involved in the mechanism. Among more than 180 genetic markers associated with EMB resistance, it has been shown that use of a minimum set of three specific markers allows accurate prediction of resistance. In addition to identifying genetic determinants of DM and EMB resistance, this project has created a range of new genetic resources that will support ongoing and future research on the biology of sea lice and the development of strategies for their control. A multi-stage transcriptome generated by Illumina sequencing has been published in 2015 in PLoS One. Moreover, as detailed above, a large number of new genetic markers have been discovered. Mitochondrial markers have been published while nuclear markers will be made publicly available as soon as the corresponding research article has been accepted for publication. In addition, further transcriptome sequencing has been undertaken using different drug-resistant and -susceptible strains of sea lice, in order to produce an updated transcriptome assembly. Moreover, the salmon louse genome has been re-sequenced using long read single molecule real time sequencing (SMRT) technology. The obtained genome assembly is considerably improved compared to the best current existing genome assembly, which is highly fragmented (LSalAtl2s, Jan 2013, https://metazoa.ensembl.org). Both new assemblies are currently being annotated and will be published as soon as annotation is completed.
Exploitation Route Findings achieved so far will result in the development of new and refinement of existing diagnostic methodologies. Genetic resources created in this project will allow the early detection and management of potential resistance formation against non-medicinal control technologies. The generated genetic resources will further facilitate research into the biology of sea lice and the development of new treatment strategies.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Through publication of the findings of this research project in academic journals, as well as reporting research aims and outcomes in trade journals and at industry events, stakeholders are today much more aware of the risk of drug resistance developments, and actively follow the current state of knowledge about the underlying mechanisms. While initially sceptical, with the help of this project, salmon farming companies now routinely use genetic tests to detect resistance, and prefer this to bioassays, which were previously the gold standard for susceptibility assessment. At least with regards to the UK salmon farming industry, this research project has crucially contributed to improving the understanding of general resistance mechanisms and acceptance of genetic tests as means of monitoring resistance status. Within this project, the discovery of the maternal inheritance of deltamethrin resistance in sea lice, and the dissemination of the study results in fora beyond academia, such as industry events and publications in trade journals, has played a key role in increasing the acceptance of genetic tests in the industry. The current main provider of such tests is the diagnostic company Patogen, which was involved in an independent study that led to a patent for markers detecting deltamethrin resistance. While this precludes commercialisation of the markers identified in our study, which overlap with those marketed by Patogen, the full description of the maternal mechanism of inheritance was first provided by our study. Dissemination of these results helped salmon farmers understand why the spread of deltamethrin resistance was so fast in 2013, and has made an important contribution to increasing acceptance of genetic tests for deltamethrin resistance in the UK salmon farming industry. We are currently pursuing the commercialisation of further genetic markers that have arisen from this project, with the intent that they will soon become available to fish health professionals. Transcriptomic and genomic sequence resources and genetic marker panels created by this project are available for use by sea louse and drug resistance researchers worldwide.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Environment
Impact Types Societal,Economic

 
Title Lepeophtheirus salmonis mitochondrial genome, complete sequence 
Description The complete sequence of the salmon louse (Lepeophtheirus salmonis) mitochondrial genome is provided, including annotation. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact The mitochondrial genome sequences avaiable in this data set complement and update earlier mitochondrial genome sequences in the species. The data can be expected to be of value with regard to the diagnostics of resistance against pyrethroids, which has been found to be maternally inherited. 
URL http://www.ebi.ac.uk/ena/data/view/PRJEB15628
 
Title Reference transcriptome for the salmon louse, Lepeophtheirus salmonis 
Description A multi-life stage reference transcriptome of Lepeophtheirus salmonis (Salmon louse) was generated using Illumina sequencing (RNA-seq). In the reference transcriptome, which comprised 33,933 transcripts corresponding to 30,159 putative genes, a total of 27,086 putative genes were represented as unique transcriptswhile 3,073 exhibited alternatively spliced transcripts. The raw sequence data from this study were submitted to the EBI Sequence Read Archive (SRA) study PRJEB1804. Annotated transcript sequences were deposited at the EBI European Nucleotide Archive (ENA) reference ERS237607 (contigs accession range HACA01000001-HACA01033933). 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact The publicly available transcriptome significantly contributes to genomic resources in L.salmonis, thereby enhancing research into the biology and control of this economically and ecologically important parasite 
URL http://www.ebi.ac.uk/ena/data/view/PRJEB1804
 
Description Aquaculture Europe Meeting, Edinburgh, 21-23 September, 2016. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Great Carmin-Antonanzas presented a study conducted within the project (QTL analysis of emamectin benzoate susceptibility in the salmon louse, Lepeophtheirus salmons) to an international audience. The talk sparked interested from science and industry representatives. Both the genome genomic resources developed in the project and the specific marker for drug resistance identified in the study are of high interest to fish health professionals working on the control of the salmon louse, an economically important parasite of farmed salmon.
Year(s) Of Engagement Activity 2016
 
Description Sealice Conference 2016, 26-28 September 2016, Westport, Ireland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Greta Carmona Antonanzas held a platform presentation entitled "QTL mapping of emamectin benzoate resistance in the salmon louse, Lepeophteirus salmonis" in the afternoon session on Wednesday 28t of September, 2016. The presentation showcased our research activities and specifically the genomic approaches talent to obtain genetic markers to detect drug resistance in sea lice to academics, regulators, industry representatives and fish health practitioners. A number of international contacts, specifically in Ireland, the Faroe islands and Chile were made.
Year(s) Of Engagement Activity 2016