An investigation into potential emamectin benzoate resistance mechanisms in sea lice Lepeophtheirus salmonis

Due to the limited chemotherapeutic options available, there is a continued reliance on emamectin benzoate (EMB) for the control of sea lice ( Lepeophtheirus salmonis) infestation on farmed Atlantic salmon ( Salmo salar), making resistance development a major concern. Previous studies indicate target site mutations and/or over-expression of P-glycoprotein (P-gp) to be the predominant mechanisms involved in the development of an avermectin resistance phenotype. This study primarily focused on the expression of P-gp in sea lice and its potential role in EMB resistance development. Two candidate genes, SL0525 and SL-Pgp1, were identified in sea lice from a sea lice expressed sequence tag (EST) database and RT-PCR on total RNA, respectively. Western blot analysis using mammalian P-gp-specific monoclonal antibodies, C219 and JSB-1, and the polyclonal antibody against SL0525 generated a signal at 160kDa which resembled P-gp with respect to the expected molecular mass. Immunohistochemistry using these antibodies localized P primarily in sea lice intestinal epithelial layer. The optimized Western blot analysis and immunohistochemistry methods offer novel tools for detection of P-gp in sea lice in future studies. Real time RT-PCR was used to examine both SL0525 and SL-P mRNA levels in response to EMB exposure. The results of this study demonstrated significantly increased levels of both SL0525 and SL-Pgp1 in sea lice exposed to 10ppb EMB for 24 hours. These results represent the first evidence of increased levels of putative P-gps in L. salmonis in response to a xenobiotic, in this case EMB. This study is also the first to examine expression levels of P-gp in a parasitic organism in response to drug exposure using Q-RT-PCR. The information gained from this project-like immunoreactivity is the first evidence of P-gp expression in sea lice and the methods developed in this study allow for the detection of its over-expression, should resistance to EMB develop. In this study we also identified the genes for avermectin target sites, GluCl and GABA-gated chloride channel alpha subunits. These genes showed a high level of homology to their respective counterparts in other organisms and phylogenetic analysis also grouped these receptors within their respective channels. These receptor subunits are the primary binding regions for avermectins. The isolation of a GluCl-gated chloride channel gene in sea lice is the first to be identified in a crustacean species. Further identification of receptor polymorphisms in sea lice populations will assist in detecting resistance to avermectin resistance due to altered target sites. Additionally, receptor characterization will facilitate improved therapeutic efficacy. The knowledge gained from this study can be transferred to aquaculture to offer both surveillance methods for the early detection of resistance, and possibly advice for alternative treatments. Decreasing the selection pressure for such resistance, thus maintaining EMB use in an already limited available drug pool, is therefore important for the Atlantic salmon industry.