Resistance towards chemotherapeutants in Caligus species

Resistance in sea lice towards available chemical treatments is an increasing problem in salmonid aquaculture. Sea lice species belonging to the genus Caligus represent a potential danger for salmonids farmed on the Northern hemisphere (Caligus elongatus and C. clemensi) and a serious threat for the Southern hemisphere salmonid aquaculture (C. rogercresseyi). This project focus on the identification of resistance development and characterization of the resistance mechanisms in C. elongatus and C. rogercresseyi. Sensitivity patterns in Caligus elongatus (Norway) towards chemical treatments using bioassays: Two isolates of C. elongatus were collected and tested: I) C. elongatus sampled from a salmon farm in Aukra commune. Lice were kept in a continuous culture and bioassays were run on the first generation. II) C. elongatus from wild saithe obtained close to Florø. These lice are kept in a continuous culture using Atlantic salmon as host at the University of Bergen (Sea Lice Research Center). Bioassays were run on the fourth and fifth generation. Bioassays on C. elongatus were performed using the four main anti-louse chemicals used to control salmon lice (Lepeophtheirus salmonis): deltamethrin, azamethiphos, emamectin benzoate and hydrogen peroxide. Bioassay results from C. elongatus collected on wild saithe showed the natural sensitivity of the species. Sensitivity levels were in the range of the values reported for L. salmonis never exposed to the chemicals. In the case of C. elongatus collected from the salmon farm, only deltamethrin and azamethiphos were tested since these were the main anti-louse chemicals used at the studied farm. Sensitivity levels were similar to C. elongatus from wild fish, thus, reduced sensitivity was not detected in the C. elongatus specimens from this farm. Sensitivity levels in Caligus rogercresseyi (Chile) towards chemical treatments using bioassays and study of the resistance mechanisms: Samples of C. rogercresseyi were collected from farms rearing Atlantic salmon and rainbow trout located in Region X (Los Lagos, Chile). Bioassays were performed for assessing the sensitivity of C. rogercresseyi towards four anti-louse drugs (deltamethrin, emamectin benzoate, azamethiphos and hydrogen peroxide). Drug concentration (five and two dose) used in the bioassays were adjusted for C. rogercresseyi based on previous knowledge. The bioassays performed in the present study confirmed the loss of sensitivity of C. rogercresseyi towards pyrethroids and emamectin benzoate, as previously reported in Chile. Regarding azamethiphos and hydrogen peroxide, results from the bioassays indicated that C. rogercresseyi is sensitive to hydrogen peroxide (it has been used very seldom in Chile) and that the parasite could be in the process of developing resistance to azamethiphos (it was introduced in Chile in 2013, but other organophosphates were used for 20 years in the past). Two additional studies were performed in collaboration with the company Cermaq Chile and the University Austral of Chile: I) The effect of anti-louse drugs was tested on the hatching viability of C. rogercresseyi egg strings. The results showed that the development of the eggs, the hatching rate and the larva survival were negatively affected by the drugs. II) The correlation between the sensitivity level of C. rogercresseyi to anti-louse chemicals and the treatment efficacy in several farms was explored. Results showed an updated picture of the sea lice resistance problems in Chile, and provided better tools for resistance monitoring and identification of problematic areas. Bioassays were also used for selecting resistant and sensitive C. rogercresseyi for every drug in order to study the mechanisms that cause the resistance at the molecular level. The molecular mechanism responsible for the azamethiphos resistance in L. salmonis has been already elucidated: A mutation in the acetylcholinesterase gene. The sequence analysis of that gene in C. rogercresseyi has shown that the mutation found in L. salmonis is not present in C. rogercresseyi individuals displaying reduced azamethiphos sensitivity. Mutations in other regions of the gene might also be responsible of organophosphate resistance in other arthropods, therefore, a search for mutations in the whole C. rogercresseyi acetylcholinesterase gene is currently being performed. C. rogercresseyi gene sequences were obtained with the assistance of Dr. Ben Koop (University of Victoria, Canada). Additional studies to understand the molecular mechanisms responsible for the resistance towards deltamethrin in L. salmonis and C. rogercresseyi are currently in progress.

Resistance in sea lice towards available treatments (pyrethroids, organophosphates and avermectins) is an increasing problem in Norwegian and international salmonid aquaculture. A considerable effort and good progress has been made on the salmon louse, Le peophtheirus salmonis, but little has been done regarding reduced sensitivity towards treatments in the parasitic copepods Caligus elongatus and C. rogercresseyi. The project will start with a bioassay surveillance of the sensitivity of C. elongatus towar ds the commonly used treatments against sea lice in Norwegian strains. If insensitivity is detected against one or more treatments, molecular methods will be used to amplify and sequence the genes coding for the target proteins in the search for mutations . Quantitative methods will be used to explore the expression levels of important detoxifying enzymes and other known resistance mechanisms in the parasites. If the mechanism(s) can be identified, rapid molecular assays can be developed, as is already the case for pyrethroid- and azamethiphos resistance in L. salmonis. In C. rogercresseyi, there is already ample evidence for resistance and this part of the project will focus on collecting representative samples for laboratory assays of mutations and expre ssion levels of known resistance mechanisms. The project will be integrated in the Sea Lice Research Centre, and performed at the Norwegian School of Veterinary Science. It will be a 2-year post doc project for Celia Agusti Ridaura.