Time-to-response toxicity analysis as a method for drug susceptibility assessment in salmon lice.
The salmon louseLepeophtheirus salmonis(Krøyer, 1837) is an ectoparasite causing infections of wild and farmed Atlantic salmon (Salmo salarL.) in the Northern hemisphere. WhileL. salmoniscontrol at commercial mariculture sites increasingly employs non-medicinal approaches, such as cage designs reducing infection rates and biological control through cleaner fish, anti-parasitic drugs are still a requirement for effective fish health care. With only a limited range of salmon delousing agents available, all of which have been in use for more than a decade, drug resistance formation has been reported for different products. Successful resistance management requires reliable susceptibility assessment, which is usually achieved throughL. salmonisbioassays. These tests involve the exposure of parasites to different drug concentrations and require significant numbers of suitableL. salmonisstages. The present study reports an alternative bioassay that is based on time-to-response toxicity analyses and can be carried out with limited parasite numbers. The assay determines the median effective time (ET50), i.e., the time required until impaired swimming and/or attachment behaviour becomes apparent in 50% of parasites, by conducting repeated examinations of test animals starting at the time point where exposure to a set drug concentration commences. This experimental approach further allows the estimation of the apparent drug susceptibility of individualL. salmonisby determining their time to response, which may prove useful in experiments designed to elucidate associations between genetic factors and the drug susceptibility phenotype of parasites. Three laboratory strains ofL. salmonisdiffering in susceptibility to emamectin benzoate were characterised using standard 24 h bioassays and time-to-response toxicity assays. While both the median effective concentration (EC50) and the ET50showed variability between experimental repeats, both types of bioassay consistently discriminated susceptible and drug-resistantL. salmonislaboratory strains.



Infections by sea lice cause significant costs to the global salmon farming industry, which have been estimated to exceed €300 million per year worldwide. Control of sea lice still relies to a significant extent on chemical delousing; however, chemical control is threatened by resistance formation. Resistance can be combated by rotation between different drugs and strategic implementation of non-medicinal strategies. However, resistance management requires reliable and feasible methods of susceptibility assessment. The present study is a technical note introducing a novel approach to susceptibility assessments in sea lice. The method can be applied in susceptibility assessments on farms, where it offers the advantage of a reduced requirement of parasites for testing. In addition, the novel method allows deriving the times of parasite require to show a response after drug treatment has started, thus providing a variable characterizing the drug susceptibility phenotype of individual parasites. Accordingly, the bioassay approach presented here will be useful for studies aiming at unravelling the genetic determinants of drug resistance.
Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, Scotland, UK.
Greta
G
Carmona-Antoñanzas
Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, Scotland, UK.
Joseph L
JL
Humble
Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, Scotland, UK.
Stephen N
SN
Carmichael
Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, Scotland, UK.
Jan
J
Heumann
Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, Scotland, UK.
Hayden R L
HR
Christie
Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, Scotland, UK.
Darren M
DM
Green
Marine Environmental Research Laboratory, University of Stirling, Machrihanish, Argyll, PA28 6PZ, Scotland, UK.
David I
DI
Bassett
Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, Scotland, UK.
James E
JE
Bron
Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, Scotland, UK.
Armin
A
Sturm
Drug susceptibility; EC50, median effective concentration; EMB, emamectin benzoate; ET50, median effective time; Emamectin benzoate; LC50, median lethal concentration; LT50, median lethal time; PEG 300, polyethylene glycol 300; Parasite; Salmon delousing agent; Sea lice