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 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.
 
Description Sea lice are small parasitic crustaceans feeding on the mucus, skin and blood of their fish hosts. Infestations with sea lice constitute a major health challenge in the commercial farming of Atlantic salmon. To protect the health and welfare of farmed fish, and prevent effects of farm-origin parasites on wild fish populations, control of sea lice infections at salmon mariculture sites is mandatory. Sea lice control follows integrated pest management strategies, which combine drug treatments and a range of non-chemical control approaches. Salmon delousing has for long relied on a limited range of licensed veterinary medicines; however the continual use of the same or similarly acting treatments in the absence of rotation has led to the development of resistance against most of the available drug treatments.
Despite the high relevance of drug resistance in sea lice, the molecular mechanisms involved are still poorly understood, complicating efficient management of infestations. 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) and the macrocyclic lactone emamectin benzoate (EMB), and develop methods to detect resistance in the field, aiding salmon farmers to select the most effective treatments.
In order to shed light on the question of how sea lice can become resistant to drugs, we focused on the salmon louse (Lepeophtheirus salmonis), the most common sea lice species causing infestations of farmed salmon in Scotland. Genetic crosses were made between resistant and susceptible parasites, producing a number of hybrid families. DNA was obtained from the families and subjected to novel high-throughput gene sequencing, allowing to identify genetic variants that are associated with the resistance. We further elucidated the number of chromosomes present in the salmon louse, and re-sequenced the genome of the species, allowing to map genetic variants to their genomic location. Finally, homologues to genes that are known to have roles in drug resistance in other invertebrate pests were identified in the salmon louse and analysed for potential roles in causing resistance in the salmon louse.
The results obtained revealed that DM resistance was inherited mostly maternally, meaning that genes transferred by females to their offspring were key in causing resistance, while the genetic contribution of males to resistance was rather small. This observation suggested that DM resistance in salmon lice might be mainly controlled by mitochondria, which are small bodies within cells that have roles in energy metabolism and possess their own DNA. Further analyses confirmed a major contribution of mitochondrial mutations to DM resistance. In contrast, the pattern by which EMB resistance was transmitted within families agreed with the classical laws of genetics discovered by Gregor Mendel, with genes received from both parents contributing to resistance. After mapping of genetic variants affecting EMB resistance to the genome, two regions of the same chromosome were highlighted, suggesting that at least two genes are involved. These exciting and novel results, some of which were published in the journal PLoS One in 2017, are currently used to develop genetic tests that will assist salmon farmers in predicting drug resistance and selecting the most efficient treatments.
In addition, genetic analyses in salmon lice included genes with known roles in drug resistance in other pests. Drug resistance is often caused by mutations causing changes in the site of action of a drug, or genes involved the metabolism of drug inactivation. In DM resistant salmon lice, sodium channels that represent the drug's site of action showed characteristic mutations, which may contribute to DM resistance in addition to the mitochondrial mutations mentioned above. In contrast, the study of genes with roles in drug inactivation did not provide evidence for their roles in salmon lice resistance against DM or EMB.

Over the course of this project, sea lice control strategies at farm sites have shifted from employing mainly drug treatments to the current wider use of non-medicinal control approaches, which include thermal and mechanical delousing, and biological control through cleaner fish. These innovations expand the arsenal of weapons available to combat sea lice, and reduce the selection pressure driving drug resistance. At the same time, if overused, the novel treatments could in the future potentially themselves be affected by resistance development. Genomic resources for the salmon louse generated in the present project allow the detection of genetic changes in the parasites indicating early resistance development, allowing to adjust control strategies to prevent resistance formation
Exploitation Route Findings achieved are currently used for 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