Non-lethal detection of Eubothrium crassum (Cestoda) in farmed Atlantic salmon, Salmo salar, using anal swabs and real-time PCR.

Detection of intestinal parasites in fish typically requires autopsy, resulting in the sacrifice of the fish. Here, we describe a non-lethal method for detecting the tapeworm Eubothrium crassum in fish using anal swabs and real-time PCR detection. Two assays were developed to detect cytochrome oxidase I (COI) mitochondrial DNA and 18S ribosomal DNA sequences of E. crassum, respectively. The assays were tested on swab samples from confirmed pathogen free Atlantic salmon (Salmo salar L.) and on samples from farmed Atlantic salmon, where the presence and intensity of parasites had been established through autopsy. The COI assay was shown to be specific to E. crassum, while the 18S assay also amplified the closely related E. salvelini, a species infecting Arctic charr (Salvelinus alpinus L.) in freshwater. The COI assay detected E. crassum in all field samples regardless of parasite load while the 18S assay failed to detect the parasite in two samples. The results thus demonstrates that this non-lethal approach can effectively detect E. crassum and can be a valuable tool in assessing the prevalence of infection in farmed salmon, aiding in treatment decisions and evaluating treatment effectiveness.

Trends in de-lousing of Norwegian farmed salmon from 2000-2019-Consumption of medicines, salmon louse resistance and non-medicinal control methods.

The salmon louse Lepeophtheirus salmonis has been a substantial obstacle in Norwegian farming of Atlantic salmon for decades. With a limited selection of available medicines and frequent delousing treatments, resistance has emerged among salmon lice. Surveillance of salmon louse sensitivity has been in place since 2013, and consumption of medicines has been recorded since the early 80's. The peak year for salmon lice treatments was 2015, when 5.7 times as many tonnes of salmonids were treated compared to harvested. In recent years, non-medicinal methods of delousing farmed fish have been introduced to the industry. By utilizing data on the annual consumption of medicines, annual frequency of medicinal and non-medicinal treatments, the aim of the current study was to describe the causative factors behind salmon lice sensitivity in the years 2000-2019, measured through toxicity tests-bioassays. The sensitivity data from 2000-2012 demonstrate the early emergence of resistance in salmon lice along the Norwegian coast. Reduced sensitivity towards azamethiphos, deltamethrin and emamectin benzoate was evident from 2009, 2009 and 2007, respectively. The annual variation in medicine consumption and frequency of medicinal treatments correlated well with the evolution in salmon louse sensitivity. The patterns are similar, with a relatively small response delay from the decline in the consumption of medicines in Norway (2016 and onward) to the decline in measured resistance among salmon louse (2017 and onward). 2017 was the first year in which non-medicinal treatments outnumbered medicinal delousing treatments as well as the peak year in numbers of cleanerfish deployed. This study highlights the significance of avoiding heavy reliance on a few substance groups to combat ectoparasites, this can be a potent catalyst for resistance evolution. Further, it demonstrates the importance of transparency in the global industry, which enables the industry to learn from poor choices in the past.

A selection study on a laboratory-designed population of salmon lic (Lepeophtheirus salmonis) using organophosphate and pyrethroid pesticides.

Resistance towards antiparasitic agents in the salmon louse (Lepeophtheirus salmonis) is a widespread problem along the Norwegian coast, reducing treatments efficacies and slowing down the envisioned expansion of Norwegian salmon production. The present study was conducted in order to assess the efficacies of two of the most widely used anti-parasitic substances-azamethiphos and deltamethrin-as well as assessing the benefit of having a resistant genotype compared to being fully sensitive when exposed to one of these substances. Atlantic salmon were exposed to a mix of salmon lice copepodids from a fully sensitive, a double resistant and a multi-resistant strain. Once the lice reached pre-adult stages, one group was exposed to 100 µg/L azamethiphos for 60 minutes, the other to 2 µg/L deltamethrin for 30 minutes, and the last was kept in a seawater control. Detached lice were collected at a series of time points following exposure, and all lice (immobilized and surviving) were analysed for both pyrethroid (sensitive "S" and resistant "R") and azamethiphos (fully sensitive "SS", heterozygous resistant "RS" and fully resistant "RR") resistance markers. We found that the efficacies of deltamethrin on parasites with genotype S and R were 70.3 and 13.2%, respectively. The overall efficacy of the deltamethrin treatment was 32.3%. The efficacies of azamethiphos on parasites with genotype SS, RS and RR were 100, 80 and 19.1%, respectively. The overall efficacy of the azamethiphos treatment was 80.4%. Survival analyses revealed that the median survival time in deltamethrin-sensitive and-resistant parasites were 16.8 and >172 hours, respectively. The differences were even more pronounced in the azamethiphos-treated group, where SS, RS and RR parasites survived for 0.26, 6.6 and >172 hours, respectively. The substantial differences in survival between sensitive and resistant lice following treatment demonstrate the ability of medicinal treatments to drive genetic selection towards a much more resistant salmon lice population within a very short time span if there is no influx of sensitive genotypes.

Monooxygenase mediated pyrethroid detoxification in sea lice (Lepeophtheirus salmonis).

The role of monooxygenases in detoxification of the pyrethroids cypermethrin and deltamethrin was examined. Four strains of sea lice (Lepeophtheirus salmonis Krøyer) with normal or moderately reduced sensitivity towards the pyrethroids were tested in bioassays by exposure to the pyrethroid alone and in combination with an oxygenase inhibitor, piperonyl butoxide (PBO). The normal (baseline) sensitivity was considered as the sensitivity range for the two most sensitive strains. Pre-treatment with PBO elevated the sensitivity (P < 0.01) compared with groups exposed to the pyrethroid only. A positive, but not statistically significant, correlation between the activity of haem peroxidases and the pyrethroid concentration immobilizing 50% of the parasites was demonstrated (rho = 0.500 for deltamethrin and rho = 0.310 for cypermethrin). The results indicate that cytochrome P450 monooxygenases are involved in detoxification of pyrethroids in sea lice. 14C-Deltamethrin was absorbed in a lesser amount in a group of sea lice exposed to a mixture of the compound and PBO than in a group exposed to 14C-deltamethrin alone. A significant difference could be demonstrated both immediately after exposure (P < 0.01) and 24 h after exposure (P < 0.05). No significant differences were found between groups pre-treated with PBO and groups exposed to 14C-deltamethrin only. 14C-Deltamethrin was taken up mainly through the cuticle, especially the cuticle on the extremities of the ventral surface, and subsequently distributed throughout the body of the parasite.

Evidence for occurrence of an organophosphate-resistant type of acetylcholinesterase in strains of sea lice (Lepeophtheirus salmonis Krøyer).

Acetylcholinesterase (AChE) is the target of a major pesticide family, the organophosphates, which were extensively used as control agents of sea lice on farmed salmonids in the early 1990s. From the mid-1990s the organophosphates dichlorvos and azamethiphos were seriously compromised by the development of resistance. AChE insensitive to organophosphate chemotherapeutants has been identified as a major resistance mechanism in numerous arthropod species, and in this study, target-site resistance was confirmed in the crustacean Lepeophtheirus salmonis Krøyer isolated from several fish-farming areas in Norway and Canada. A bimolecular rate assay demonstrated the presence of two AChE enzymes with different sensitivities towards azamethiphos, one that was rapidly inactivated and one that was very slowly inactivated. To our knowledge this is the first report of target-site resistance towards organophosphates in a third class of arthropods, the Crustacea.

Analysis and management of resistance to chemotherapeutants in salmon lice, Lepeophtheirus salmonis (Copepoda: Caligidae).

In Northern Europe and Canada, the salmon louse, Lepeophtheirus salmonis (Krøyer), seriously affects the marine phase of salmon production. Although the problem is long-standing, the development of sustainable methods of pest management has been unable to keep pace with the intensification of production, leading to large-scale reliance on very few chemotherapeutants. This runs the risk of selecting for genetically determined resistance in target organisms. There are many examples of similar evolutionary adaptations in arthropod pests of arable crops, livestock and human health. Several hundred pest species are now documented as being resistant to one or more chemical classes of insecticides and acaricides. Many of these compounds are identical or closely related to ones currently employed against salmon lice. It is, therefore, opportune to consider what lessons have been learnt from contending with resistance in terrestrial organisms, the implications for sustainable use of chemotherapeutants in aquaculture, and the potential for developing effective resistance management strategies. An EU-funded project named SEARCH (QLK2-CT-2000-00809) has been initiated to explore in more detail the diagnosis, incidence, dynamics and management of resistance to chemotherapeutants in L salmonis.