Sea lice population and sex differences in P-glycoprotein expression and emamectin benzoate resistance on salmon farms in the Bay of Fundy, New Brunswick, Canada.

BACKGROUND: Parasitic sea lice are a major challenge for salmon aquaculture. This is especially due to the recent development of resistance to emamectin benzoate (EMB) in the parasite. We investigated: (1) whether EMB treatment success in Grand Manan, Bay of Fundy, NB, Canada can be explained through EMB bioassay and P-glycoprotein (P-gp) mRNA expression studies; (2) if other populations of sea lice not under EMB selective pressure possess similar EMB sensitivity as Grand Manan sea lice populations; and (3) the heritability of EMB resistance in Lepeophtheirus salmonis. RESULTS: EMB bioassay results indicated population, species, sex and temporal differences in EMB EC50 values. RT-qPCR analyses revealed population and sex differences in P-gp mRNA levels, correlating with the bioassay results. Laboratory-reared sea lice maintained their EMB sensitivity status up to the F3 generation. Caligus elongatus, collected from Grand Manan showed more than twofold lower EMB EC50 values compared with L. salmonis collected from the same site. Concurrent exposure to EMB and verapamil yielded no increase in C. elongatus sensitivity to the parasiticide. CONCLUSION: Sea lice bioassay and P-gp mRNA studies can be used to track EMB resistance and sex differences in EMB sensitivity and P-gp mRNA levels exist in the parasite.

Optimization and field use of a bioassay to monitor sea lice Lepeophtheirus salmonis sensitivity to emamectin benzoate.

A bioassay for sea lice Lepeophtheirus salmonis sensitivity towards emamectin benzoate (EMB) was validated for field use. A probit regression model with natural responsiveness was used for the number of affected (moribund or dead) sea lice in bioassays involving different concentrations of EMB. Bioassay optimization included an evaluation of the inter-rater reliability of sea lice responsiveness to EMB and an evaluation of gender-related differences in susceptibility. Adoption of a set of bioassay response criteria improved the concordance (evaluated using the concordance correlation coefficient) between raters' assessments and the model estimation of EC50 values (the 'effective concentration' leading to a response of 50% of the lice not prone to natural response). An evaluation of gender-related differences in EMB susceptibility indicated that preadult stage female sea lice exhibited a significantly larger sensitivity towards EMB in 12 of 19 bioassays compared to preadult males. In order to evaluate sea lice sensitivity to EMB in eastern Canada, the intensive salmon farming area in the Bay of Fundy in southwestern New Brunswick was divided into 4 distinct regions based on industry health management practices and hydrographics. A total of 38 bioassays were completed from 2002 to 2005 using populations of preadult stage sea lice collected from Atlantic salmon Salmo salar farms within the 4 described regions. There was no significant overall effect of region or year on EC50 values; however, analysis of variance indicated a significant effect of time of year on EC50 values in 2002 and a potential effect in 2004 to 2005. Although the range of EC50 values obtained in this 3 yr study did not appear sufficient to affect current clinical success in the control of sea lice, the results suggest a seasonal- or temperature-associated variation in sensitivity to EMB. This will need to be considered if changes in EMB efficacy occur in the future.

Evidence for changes in the transcription levels of two putative P-glycoprotein genes in sea lice (Lepeophtheirus salmonis) in response to emamectin benzoate exposure.

Overexpression of P-glycoproteins (Pgps) is assumed to be a principal mechanism of resistance of nematodes and arthropods to macrocyclic lactones. Quantitative RT-PCR (Q-RT-PCR) was used to demonstrate changes in transcription levels of two putative P-glycoprotein genes, designated here as SL0525 and SL-Pgp1, in sea lice (Lepeophtheirus salmonis) following exposure to emamectin benzoate (EMB). Pre-adult L. salmonis were challenged in an EMB bioassay for 24h and gene expression was studied from lice surviving EMB concentrations of 0, 10, and 30ppb. Gene expression was measured using Q-RT-PCR with elongation factor 1 (eEF1alpha) as an internal reference gene. The results show that both target genes, SL0525 and SL-Pgp1, had significantly increased levels of expression with exposure to 10ppb EMB (p=0.11 and p=0.17, respectively) whereas the group exposed to 30ppb was on the verge of being significant (p=0.053) only in the expression of SL-Pgp1. Gene expression for SL0525 and SL-Pgp1 were increased over five-fold at 10ppb EMB. Therefore, the upregulation of these target genes may offer protection by increasing Pgp expression when lice are exposed to EMB. Our optimized Q-RT-PCR can be used to determine if over-expression of these genes could be the basis for development of resistance in sea lice and thus allow suitable alternative chemotherapeutic options to be assessed.

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.

Susceptibility of rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and coho salmon Oncorhynchus kisutch to experimental infection with sea lice Lepeophtheirus salmonis.

Physiological, immunological and biochemical parameters of blood and mucus, as well as skin histology, were compared in 3 salmonid species (rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and coho salmon O. kisutch) following experimental infection with sea lice Lepeophtheirus salmonis. The 3 salmonid species were cohabited in order to standardize initial infection conditions. Lice density was significantly reduced on coho salmon within 7 to 14 d, while lice persisted in higher numbers on rainbow trout and Atlantic salmon. Lice matured more slowly on coho salmon than on the other 2 species, and maturation was slightly slower on rainbow trout than on Atlantic salmon. Head kidney macrophages from infected Atlantic salmon had diminished respiratory burst and phagocytic capacity at 14 and 21 d post-infection (dpi), while infected rainbow trout macrophages had reduced respiratory burst and phagocytic capacities at 21 dpi, compared to controls. The slower development of lice, coupled with delayed suppression of immune parameters, suggests that rainbow trout are slightly more resistant to lice than Atlantic salmon. Infected rainbow trout and Atlantic salmon showed increases in mucus lysozyme activities at 1 dpi, which decreased over the rest of the study. Mucus lysozyme activities of infected rainbow trout, however, remained higher than controls over the entire period. Coho salmon lysozyme activities did not increase in infected fish until 21 dpi. Mucus alkaline phosphatase levels were also higher in infected Atlantic salmon compared to controls at 3 and 21 dpi. Low molecular weight (LMW) proteases increased in infected rainbow trout and Atlantic salmon between 14 and 21 dpi. Histological analysis of the outer epithelium revealed mucus cell hypertrophy in rainbow trout and Atlantic salmon following infection. Plasma cortisol, glucose, electrolyte and protein concentrations and hematocrit all remained within physiological limits for each species, with no differences occurring between infected and control fish. Our results demonstrate that significant differences in mucus biochemistry and numbers of L. salmonis occur between these species.