Shona K. Whyte

A fixed-dose approach to conducting emamectin benzoate tolerance assessments on field-collected sea lice, Lepeophtheirus salmonis.

In New Brunswick, Canada, the sea louse, Lepeophtheirus salmonis, poses an on-going management challenge to the health and productivity of commercially cultured Atlantic salmon, Salmo salar. While the in-feed medication, emamectin benzoate (SLICE® ; Merck), has been highly effective for many years, evidence of increased tolerance has been observed in the field since late 2008. Although bioassays on motile stages are a common tool to monitor sea lice sensitivity to emamectin benzoate in field-collected sea lice, they require the collection of large numbers of sea lice due to inherent natural variability in the gender and stage response to chemotherapeutants. In addition, sensitive instruments such as EC(50) analysis may be unnecessarily complex to characterize susceptibility subsequent to a significant observed decline in efficacy. This study proposes an adaptation of the traditional, dose-response format bioassay to a fixed-dose method. Analysis of 657 bioassays on preadult and adult stages of sea lice over the period 2008-2011 indicated a population of sea lice in New Brunswick with varying degrees of susceptibility to emamectin benzoate. A seasonal and spatial effect was observed in the robustness of genders and stages of sea lice, which suggest that mixing different genders and stages of lice within a single bioassay may result in pertinent information being overlooked. Poor survival of adult female lice in bioassays, particularly during May/June, indicates it may be prudent to consider excluding this stage from bioassays conducted at certain times of the year. This work demonstrates that fixed-dose bioassays can be a valuable technique in detecting reduced sensitivity in sea lice populations with varying degrees of susceptibility to emamectin benzoate treatments.

Sea lice monitoring on Atlantic salmon farms in New Brunswick, Canada: comparing audit and farm staff counts

Sea lice audits were performed by the Atlantic Veterinary College on commercial aquaculture sites in New Brunswick, Canada, in 2011. Although the primary objective was to verify that farms were reporting similar lice counts to third-party counts, more detailed comparisons were made to identify when lice counts were more likely to differ between the audit team and farm employees. A total of 28 sea lice audits were conducted on 16 sites between June and December 2011. During each audit, 10 cages were evaluated per site where possible, with ten fish per cage being evaluated by an audit technician and a further ten by a farm employee. Data analysis included descriptive statistics of lice counts by stage and limits of agreement plots. A random effects negative binomial model that accounted for clustering of cages within sites was applied to assess the effect of counter type and season on lice counts by stage. The results indicate that farms counts were generally in agreement with audit counts. However, when the average counts for chalimus and preadult (male and female) and adult male lice stages were high, farm counters were more likely to report a lower value. Higher lice counts were observed during autumn compared to summer especially for the adult female stage. Finally, there was a significant clustering effect for site and cage, with most of the variation attributable to site.

Cypermethrin exposure induces metabolic and stress-related gene expression in copepodid salmon lice (Lepeophtheirus salmonis)

Cypermethrin has been administered for decades to control salmon lice (Lepeophtheirus salmonis) infestations in Atlantic salmon farming regions globally. However, resistance to cypermethrin and other available therapeutants has threatened the sustainability of this growing industry. To better understand the effects of cypermethrin on L. salmonis, a 38K oligonucleotide microarray and RT-qPCR analyses were applied to pools of copepodid larvae exposed to 1.0ppb cypermethrin or seawater controls for 24h. Phenotypic assessments and global gene expression profiles showed a significant disruption of homeostasis in copepodid L. salmonis exposed to cypermethrin. Multiple degradative enzymes were overexpressed in cypermethrin-treated lice including five trypsin-like serine proteases and three cytochrome p450s CYP3a24 (p=0.03, fold change (FC)=3.8; GenBank accession no. JP326960.1), CYP6w1 (p=0.008, FC=5.3; GenBank accession no. JP317875.1), and CYP6d4 (p=0.01; FC=7.9; GenBank accession no. JP334550.1). These enzymes represent preliminary markers for understanding the physiological response of L. salmonis to cypermethrin exposure. A general stress response was also observed in cypermethrin-treated lice which included differential expression of cell signaling genes involved in the induction of cell growth, solute transport, and metabolism. Lastly, a consensus-based analysis was completed with two previously published L. salmonis transcriptome studies revealing genes that respond to cypermethrin, emamectin benzoate (another delousing agent) and hyposalinity. This included concordant differential expression of heat shock beta-1, ammonium transporter Rh types B, and 72kDa type IV collagenase across different L. salmonis studies. This is currently the most comprehensive transcriptome assessment of chemical exposure on the first infectious stage of L. salmonis, providing novel markers for studying drug resistance and general stress in this important parasite.

High level efficacy of lufenuron against sea lice ( Lepeophtheirus salmonis ) linked to rapid impact on moulting processes

Drug resistance in the salmon louse Lepeophtheirus salmonis is a global issue for Atlantic salmon aquaculture. Multiple resistance has been described across most available compound classes with the exception of the benzoylureas. To target this gap in effective management of L. salmonis and other species of sea lice (e.g. Caligus spp.), Elanco Animal Health is developing an in-feed treatment containing lufenuron (a benzoylurea) to be administered prior to seawater transfer of salmon smolts and to provide long-term protection of salmon against sea lice infestations. Benzoylureas disrupt chitin synthesis, formation, and deposition during all moulting events. However, the mechanism(s) of action are not yet fully understood and most research completed to date has focused on insects. We exposed the first parasitic stage of L. salmonis to 700 ppb lufenuron for three hours and observed over 90% reduction in survival to the chalimus II life stage on the host, as compared to vehicle controls. This agrees with a follow up in vivo administration study on the host, which showed >95% reduction by the chalimus I stage. Transcriptomic responses of salmon lice exposed to lufenuron included genes related to moulting, epithelial differentiation, solute transport, and general developmental processes. Global metabolite profiles also suggest that membrane stability and fluidity is impacted in treated lice. These molecular signals are likely the underpinnings of an abnormal moulting process and cuticle formation observed ultrastructurally using transmission electron microscopy. Treated nauplii-staged lice exhibited multiple abnormalities in the integument, suggesting that the coordinated assembly of the epi- and procuticle is impaired. In all cases, treatment with lufenuron had rapid impacts on L. salmonis development. We describe multiple experiments to characterize the efficacy of lufenuron on eggs, larvae, and parasitic stages of L. salmonis, and provide the most comprehensive assessment of the physiological responses of a marine arthropod to a benzoylurea chemical.

Acquired Protective Immunity in Atlantic Salmon Salmo salar against the Myxozoan Kudoa thyrsites Involves Induction of MHIIβ+ CD83+ Antigen-Presenting Cells

ABSTRACT The histozoic myxozoan parasite Kudoa thyrsites causes postmortem myoliquefaction and is responsible for economic losses to salmon aquaculture in the Pacific Northwest. Despite its importance, little is known about the host-parasite relationship, including the host response to infection. The present work sought to characterize the immune response in Atlantic salmon during infection, recovery, and reexposure to K. thyrsites. After exposure to infective seawater, infected and uninfected smolts were sampled three times over 4,275 degree-days. Histological analysis revealed infection severity decreased over time in exposed fish, while in controls there was no evidence of infection. Following a secondary exposure of all fish, severity of infection in the controls was similar to that measured in exposed fish at the first sampling time but was significantly reduced in reexposed fish, suggesting the acquisition of protective immunity. Using immunohistochemistry, we detected a population of MHIIβ+ cells in infected muscle that followed a pattern of abundance concordant with parasite prevalence. Infiltration of these cells into infected myocytes preceded destruction of the plasmodium and dissemination of myxospores. Dual labeling indicated a majority of these cells were CD83+/MHIIβ+. Using reverse transcription-quantitative PCR, we detected significant induction of cellular effectors, including macrophage/dendritic cells (mhii/cd83/mcsf), B cells (igm/igt), and cytotoxic T cells (cd8/nkl), in the musculature of infected fish. These data support a role for cellular effectors such as antigen-presenting cells (monocyte/macrophage and dendritic cells) along with B and T cells in the acquired protective immune response of Atlantic salmon against K. thyrsites.