Identification of critical enzymes in the salmon louse chitin synthesis pathway as revealed by RNA interference-mediated abrogation of infectivity.

Treatment of infestation by the ectoparasite Lepeophtheirus salmonis relies on a small number of chemotherapeutant treatments that currently meet with limited success. Drugs targeting chitin synthesis have been largely successful against terrestrial parasites where the pathway is well characterised. However, a comparable approach against salmon lice has been, until recently, less successful, likely due to a poor understanding of the chitin synthesis pathway. Post-transcriptional silencing of genes by RNA interference (RNAi) is a powerful method for evaluation of protein function in non-model organisms and has been successfully applied to the salmon louse. In the present study, putative genes coding for enzymes involved in L. salmonis chitin synthesis were characterised after knockdown by RNAi. Nauplii I stage L. salmonis were exposed to double-stranded (ds) RNA specific for several putative non-redundant points in the pathway: glutamine: fructose-6-phosphate aminotransferase (LsGFAT), UDP-N-acetylglucosamine pyrophosphorylase (LsUAP), N-acetylglucosamine phosphate mutase (LsAGM), chitin synthase 1 (LsCHS1), and chitin synthase 2 (LsCHS2). Additionally, we targeted three putative chitin deacetylases (LsCDA4557, 5169 and 5956) by knockdown. Successful knockdown was determined after moulting to the copepodite stage by real-time quantitative PCR (RT-qPCR), while infectivity potential (the number of attached chalimus II compared with the initial number of larvae in the system) was measured after exposure to Atlantic salmon and subsequent development on their host. Compared with controls, infectivity potential was not compromised in dsAGM, dsCHS2, dsCDA4557, or dsCDA5169 groups. In contrast, there was a significant effect in the dsUAP-treated group. However, of most interest was the treatment with dsGFAT, dsCHS1, dsCHS1+2, and dsCDA5956, which resulted in complete abrogation of infectivity, despite apparent compensatory mechanisms in the chitin synthesis pathway as detected by qPCR. There appeared to be a common phenotypic effect in these groups, characterised by significant aberrations in appendage morphology and an inability to swim. Ultrastructurally, dsGFAT showed a significantly distorted procuticle without distinct exo/endocuticle and intermittent electron dense (i.e. chitin) inclusions, and together with dsUAP and dsCHS1, indicated delayed entry to the pre-moult phase.

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.

Transcriptomic responses to emamectin benzoate in Pacific and Atlantic Canada salmon lice Lepeophtheirus salmonis with differing levels of drug resistance.

Salmon lice Lepeophtheirus salmonis are an ecologically and economically important parasite of wild and farmed salmon. In Scotland, Norway, and Eastern Canada, L. salmonis have developed resistance to emamectin benzoate (EMB), one of the few parasiticides available for salmon lice. Drug resistance mechanisms can be complex, potentially differing among populations and involving multiple genes with additive effects (i.e., polygenic resistance). Indicators of resistance development may enable early detection and countermeasures to avoid the spread of resistance. Here, we collect sensitive Pacific L. salmonis and sensitive and resistant Atlantic L. salmonis from salmon farms, propagate in laboratory (F1), expose to EMB in bioassays, and evaluate either baseline (Atlantic only) or induced transcriptomic differences between populations. In all populations, induced responses were minor and a cellular stress response was not identified. Pacific lice did not upregulate any genes in response to EMB, but downregulated degradative enzymes and transport proteins at 50 ppb EMB. Baseline differences between sensitive and now resistant Atlantic lice were much greater than responses to exposures. All resistant lice overexpressed degradative enzymes, and resistant males, the most resistant group, overexpressed collagenases to the greatest extent. These results indicate an accumulation of baseline expression differences related to resistance.

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.

Sub-chronic toxicity of Senilia senilis hepatopancreas in albino Wistar mice.

This study investigates the sub-chronic effects of shellfish possibly contaminated by algal toxins in albino Wistar mice. Following in-feed treatment with hepatopancreas, there were significant (p < 0.05) differences in packed cell volume, haemoglobin concentration, red blood cell count, lymphocyte count and alanine aminotransferase between the different treatment groups and control. Post mortem revealed inflammation and congestion in the gastrointestinal tract of the middle and highest dose groups. The stomach of the mice fed on the highest dose of the hepatopancreas showed severe ulcerations, leaving behind deep cavities and necrotic tissues on the glandular epithelium of the mucosal wall.

'Diarrhetic' type shellfish poisoning in Nigeria.

The safety of shellfish found and consumed in Nigeria is doubtful because no investigations have been carried out on their toxicity. The occurrence and toxicity of toxins in the commonly consumed Nigerian shellfish from Lagos, Warri, Oron, and Port Harcourt (PH) were investigated. Albino Wistar mice treated with chloroform extract of hepatopancreas (HP) from PH shellfish died 7-8 min post treatment. They convulsed for 30-60 s prior to death. The aqueous phase obtained from the diethyl ether extraction of the same HP resulted in the death of one out of two mice injected with it, 39 min post treatment.