Isolation and characterisation of four partial cDNA sequences encoding multidrug resistance-associated proteins (MRPs) in the salmon louse Lepeophtheirus salmonis (Krøyer, 1837)

Abstract

The salmon louse Lepeophtheirus salmonis (Krøyer, 1837) is an ectoparasite of salmonid fish severely affecting cultured salmon production in the North Atlantic. Salmon louse control on farms currently relies in part upon the use of veterinary drugs; however, as only a limited number of salmon delousing agents are available, there are concerns about development of drug resistance in salmon lice. The common anti-salmon louse drug SLICE® (Merck Animal Health) contains the avermectin emamectin benzoate (EMB). Members of the large gene superfamily of ABC (ATP-binding cassette) transporters have been identified as potential avermectin resistance factors in parasitic nematodes. In salmon lice, only three ABC transporters have been cloned and studied to date. We report here upon the isolation of four novel L. salmonis ABC transporters, and employ an inhibitor-based approach to assess roles of L. salmonis ABC transporters in the toxicology of EMB. To isolate salmon louse ABC transporters, publicly available L. salmonis expressed sequence tags (ESTs) were subjected to homology searches, and the retrieved ESTs assembled into contiguous sequences and annotated. Potential ABC drug transporters isolated by this approach included four multidrug resistance-associated proteins (MRPs). In addition, five ABC proteins likely having roles unrelated to drug resistance were obtained. Quantitative real time PCR (RT-qPCR) was used to analyse mRNA levels of the MRPs in L. salmonis strains differing in EMB susceptibility. In the absence of EMB, all studied MRPs showed similar transcript levels in the drug-susceptible strain S and the moderately EMB-resistant strain R. Moreover, mRNA expression of the four studied MRPs remained unaffected by exposure to EMB. Taken together, the results of RT-qPCR analyses did not provide evidence for roles of the studied MRPs as factors affecting EMB susceptibility. Further experiments used an inhibitor-based approach to investigate the roles of ABC transporters in EMB toxicity. In immotility bioassays, salmon lice of the two strains were exposed to EMB, provided alone or in combination with cyclosporin A or verapamil, which are known inhibitors of P-glycoprotein and MRPs. Cyclosporin A increased EMB toxicity to a similar degree with both strains, suggesting the biochemical factors affected by this inhibitor show similar levels in both strains. In contrast, verapamil increased EMB effects only in the R strain. This result could be indicative of an enhanced expression of hitherto unknown ABC transporters in the moderately EMB-resistant R strain. More research is required to identify the target of verapamil in salmon lice.