Effect of environmental salinity on sea lice Lepeophtheirus salmonis settlement success

The sea louse Lepeophtheirus salmonis (Kr?yer, 1837) (Copepoda: Caligidae) is an ectoparasite of salmonid fish. It has earlier been proposed that the free-swimming infectious copepodid stage of L. salmonis gather at river mouths to infect wild Atlantic salmon Salmo salar L. and sea trout S. trutta L. smolts during their seaward migration. This study used aquarium-based methods to investigate the survival, infective ability and behaviour of L. salmonis copepodids exposed to short periods of low salinity levels, such as those encountered at river mouths. Survival of free-swimming copepodids was found to be severely compromised at salinity levels below 29 parts per thousand (ppt). Attachment to an S. salar host did not aid copepodid survival during post-infection exposure to low salinity environment, and a reduction in salinity appears to reduce the ability of copepodids to remain attached to S. salar smolts. Pre-infection exposure of copepodids to reduced salinity levels reduced infection of S. salar. Infection levels at reduced salinity were lower than predicted from the free-swimming survival experiment, suggesting that low salinity compromises the copepodids' ability to sense or respond to the presence of a host. In salinity gradients, copepodids demonstrated avoidance of salinities below 27 ppt, by both altering their swimming behaviour and changing the orientation of passive sinking. Avoidance of low salinity levels may be due to their adverse effects on copepodid physiology, as suggested by the reduction in survival. Sinking rates were also faster in reduced salinity, suggesting that remaining in the water column would be more energetically demanding for the copepodids at reduced salinity. These results show that both survival and host infectivity of L. salmonis are severely compromised by short-term exposure to reduced salinity levels.     

Seasonal occurrence of sea lice Lepeophtheirus salmonis on sea trout in two north Norwegian fjords

Seasonal occurrence of the parasitic copepod Lepeophtheirus salmonis (sea lice) was studied from March to December 2001 in two large north Norwegian sill fjords without fish farming activity, the Ranafjord and the Balsfjord. Anadromous brown trout Salmo trutta (sea trout) in both fjords had a low infestation rate during all sampling periods, but followed a seasonal pattern. During early and late winter (November to December and March to April) and spring (May to June), the prevalence varied from 0 to 25% and the abundance was <0·5 sea lice. Adults dominated (92%) during this period, particularly gravid females. In both fjords, the highest prevalence was during September (80–81%, all stages represented). In Ranafjord, the abundance and mean intensity during this month was 6·8 and 8·6 sea lice, respectively, while in Balsfjord it was 3·6 and 4·5 sea lice, respectively. Fish were captured at temperatures down to 1° C and at full strength sea water which is supposed to cause osmoregulatory problems for the fish. This observation has implications for the understanding of high‐latitude sea trout behaviour and can also make the fish more vulnerable to heavy sea lice infestation during this period. It is suggested that winter running sea trout help to maintain a self replicating local population of sea lice within such fjord systems where other possible hosts ( e.g . farmed Atlantic salmon Salmo salar ) are not present during a whole year cycle.     

Changes in physiological parameters and feeding behaviour of Atlantic salmon Salmo salar infected with sea lice Lepeophtheirus salmonis

Atlantic salmon Salmo salar L. artificially infected with salmon lice Lepeophtheirus salmonis (Kr?yer 1837) recovered from detrimental physiological changes and skin damage induced by preadult lice as the parasites matured. Growth rates of Atlantic salmon remained unaffected by lice infection, but food consumption decreased with increasing feeding and movement of the lice prior to and post-mating, correlating with the appearance of head erosions and detrimental changes in physiological integrity. Food consumption of the fish increased as the lice moulted to the adult stage and gravid female lice settled in a posterior location on the fish, subsequently reducing the impact of infection and allowing recovery of the skin damage. However, the impact of preadults was limited, as the decrease in food consumption of fish at 21 d post-infection had no effect on either the specific growth rate or condition factor of the fish. Furthermore, the intensity of lice infections at each of the sample days was not correlated with food consumption, specific growth rate or any of the haematological or physiological parameters measured, either before or after infection, indicating that lice intensity was independent of social dominance/subordinance. This work has provided the first evidence that infected fish can recover from the detrimental changes caused by lice infection, even when they are still infected with lice. If fish can survive the preadult stage of lice, then the mortal impact of lice infections is greatly reduced.     

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.     

Identifying epidemiological factors affecting sea lice Lepeophtheirus salmonis abundance on Scottish salmon farms using general linear models

The variation in Lepeophtheirus salmonis sea lice numbers across 40 Scottish salmon farm sites during 1996 to 2000 was analysed using mean mobile abundance for 3 important 6 mo periods within the production cycle. Using statistical regression techniques, over 20 management and environmental variables suspected to have an effect on controlling lice populations were investigated as potential risk factors. The findings and models developed provide a picture of mobile L. salmonis infestation patterns on Scottish farm sites collectively. The results identified level of treatment, type of treatment, cage volume, current speed, loch flushing time and sea lice levels in the preceding 6 mo period to be key explanatory factors. Factors such as stocking density, site biomass, water temperature and the presence of neighbours, previously cited to be important correlates of sea lice risk from analysis of individual sites over time, were not found to be important. Variation in mobile abundance in the first half of the second year of production could be adequately explained (adjusted R2 between 55 and 72%) by the recorded data, suggesting that there is scope for management to control L. salmonis abundance, though much of the variation remains unexplained.     

Commercial trials using emamectin benzoate to control sea lice Lepeophtheirus salmonis infestations in Atlantic salmon Salmo salar

Two trials were conducted at commercial salmon farms to evaluate the efficacy of emamectin benzoate (Slice, 0.2% aquaculture pre-mix, Schering-Plough Animal Health) as a treatment for sea lice Lepeophtheirus salmonis (Kr?yer) and Caligus elongatus Nordmann infestations in Atlantic salmon Salmo salar L. Trials were carried out in 15 m2 commercial sea pens, at temperatures of 5.5 to 7.5 degrees C and 10.8 to 13.8 degrees C. Each pen was stocked with 14,000 to 17,500 fish with mean weights of 0.44 to 0.74 and 1.33 to 1.83 kg. Fish were naturally infested with sea lice at the start of each trial. At Day -1, samples of 10 or 15 fish were taken from each pen to determine pre-treatment numbers of lice. Emamectin benzoate was administered in feed, to 4 replicate pens, at a dose of 50 micrograms kg-1 biomass d-1 for 7 consecutive days (Days 0 to 6). Sea lice were counted again, between Days 7 and 77, and comparisons made with untreated control fish. Despite adverse weather conditions, wide variations in fish weights and exposure to new infestations, treatment was effective against chalimus and motile stages of L. salmonis. In the autumn trial, efficacy at Day 27 was 89%, and lice numbers remained lower on treated fish than on control fish 64 d from the start of treatment. In the winter trial, reductions in lice numbers at low temperatures were slower but good efficacy was achieved by Day 35. Although control fish had to be treated with hydrogen peroxide at Day 21, fish treated only with emamectin benzoate on Days 0 to 6 still had 89% fewer lice than control fish at Day 35. There were very few C. elongatus present, but at the end of both trials numbers were lower on treated fish. No adverse effects were associated with treatment of fish with emamectin benzoate.     

Novel point mutation in the sodium channel gene of pyrethroid-resistant sea lice Lepeophtheirus salmonis (Crustacea: Copepoda)

Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4-S6) region of the channel protein, where several different mutation sites have been identified across a range of insect species. To investigate the possibility that a kdr-type mechanism is responsible for pyrethroid resistance in sea lice, a domain II region of the Lepeophtheirus salmonis sodium channel gene was PCR amplified and sequenced. To our knowledge, this is the first published sodium channel sequence from a crustacean. Comparison of sequences from a range of samples, including several individuals from areas in which control failures had been reported, failed to identify any of the mutations within this region that have previously been linked with resistance. Instead, a novel glutamine to arginine mutation, Q945R, in transmembrane segment IIS5 was consistently found in the samples from areas of control failure and may therefore be associated with resistance to pyrethroids in this species.     

Large-scale use of fish traps for monitoring sea trout (Salmo trutta) smolts and sea lice (Lepeophtheirus salmonis) infestations: efficiency and reliability

Lice-infected sea trout populations were monitored using fish traps in the Romsdalsfjord (Norway). The reliability and efficiency of this capture technique, which allows estimation of lice infestation rates without killing the fish, was evaluated through a mark–recapture study. A total of 2447 sea trout smolts were captured, tagged and released over a three-year period. There was a considerable variation in capture rates (range: 0.4–17.7 fish per day) and sea lice numbers (number of lice per fish: 2.8–30.3; number of lice per gram body weight: 0.02–0.69) among localities, sampling times and years. Recapture rates of tagged fish with traps, which were low (2% or 0.11 fish per day), showed that the risk for pseudoreplication was minor, in terms of counting lice on the same fish several times. Most of the tagged sea trout (90%) were recaptured within the first two months after release, and no significant variations in lice numbers were found between tagging and recapture. The lack of differences in lice levels between tagging and recapture during the first week after tagging indicated that the method most likely would not significantly underestimate the lice infestations due to loss of lice during handling. Therefore, our results confirm that the use of fish traps is a suitable method for estimation of lice numbers on wild salmonids.     

Physiology and immunology of Lepeophtheirus salmonis infections of salmonids

'Sea lice' is a common name for a large number of species of marine ectoparasitic copepods, many of which are widespread and important disease-causing agents that infect both cultured and wild fish. Of these copepods, the salmon louse Lepeophtheirus salmonis is the most extensively studied because of its economic impact on the salmonid aquaculture industry and its possible impacts on wild salmonid populations. Different levels of infection by this parasite can affect the long-term survival and viability of its hosts. In this article, we review the nature of the interactions between L. salmonis and it hosts to identify crucial areas that warrant further research to aid understanding of the impact of infection with L. salmonis.     

The chemical cues of male sea lice Lepeophtheirus salmonis encourage others to move between host Atlantic salmon Salmo salar

Adult male sea lice Lepeophtheirus salmonis were more likely to leave host fish Atlantic salmon Salmo salar if they detected the chemical cues of other adult male lice than if they detect cues of female lice. The detection of both male and female chemical cues yielded an intermediate response. These results suggest that males use chemical cues to balance competition for resources and mate acquisition, and they highlight the need for further studies of the chemical ecology of this important parasite.     

Frontal filament morphogenesis in the salmon louse Lepeophtheirus salmonis.

The objective of the present study was to understand how and when the frontal filament (FF) in the salmon louse Lepeophtheirus salmonis is produced by examining the sequence of morphological changes leading to FF production in the copepodid and early chalimus stages. Atlantic salmon (Salmo salar) were heavily infested with newly molted copepodids. Sea lice were sampled prior to infestation and at 1, 2, 3, 4, 5, 6, 7, 8, and 9 days postinfestation. FF morphogenesis from newly molted copepodid to chalimus II, i.e., through 2 molts, was studied using high-resolution light microscopy of serial transverse and sagittal resin sections. Three groups of cells, identified as A, B, and C, are thought to be involved in the production of the secretions (S1 and S2) that form the filament material. The amount and shape of S1 and S2 and their association with B- and C-group cells, respectively, changed with the molt cycle. The following scenario for FF development is proposed: the first secretion to form after the molt for both copepodid and chalimus stages is S1, and it is formed by B-group cells and becomes the basal plate of the external FE C-group cells produce S2 during mid-intermolt to premolt stage. The S2 becomes the stem of the external FE In premolt larvae, S1 and S2 were contained within a cuticle-lined invagination that had a form similar to that of the extruded filament. The axial duct present in both copepodid and chalimus originates from the A-group cells and probably carries a secretion used to attach the filament to the host. This study provides strong evidence that L. salmonis produces a new filament with each molt, creating the possibility of using a sea lice control method based on interference with filament production more feasible.     

Identification and localization of a putative ATP-binding cassette transporter in sea lice (Lepeophtheirus salmonis) and host Atlantic salmon (Salmo salar)

Some members of the ABC-transporter superfamily, such as P-glycoprotein and the multidrug resistance associated protein, may confer resistance to the avermectin subclass of macrocyclic lactones. The aim of this study was to examine the presence of ABC transporters in both sea lice (Lepeophtheirus salmonis) and its Atlantic salmon host (Salmo salar) using monoclonal antibodies (C219 and JSB-1, with high selectivity for P-gp) and a new polyclonal antibody (SL0525) generated against a putative sea louse ABC transporter. The antibody raised to SL0525 did not react with rat P-gp, suggesting that an ABC transporter, not necessarily P-gp, was isolated. C219 was the only antibody to localize P-gp in all 3 salmon tissues (intestine, kidney and liver). American lobster (Homarus americanus) was used as a reference crustacean for L. salmonis immunostaining reactions and showed positive staining in the hepatopancreatic and intestinal tissues with all 3 antibodies. The L. salmonis showed positive staining in the intestinal epithelial lining with all antibodies. This report represents the first documented evidence for the expression of ABC transporters in L. salmonis, its Atlantic salmon host, and the American lobster.     

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.     

Sequence variation in four mitochondrial genes of the salmon louse Lepeophtheirus salmonis

A total of 210 Lepeophtheirus salmonis collected from 7 locations (Scotland, Russia, Canada, Japan and 3 locations in Norway), were screened for sequence variation in 4 mitochondrial genes; ATPase subunit 6 (A6), Cytochrome b oxidase subunit I (COI), Cytochrome b (Cyt b) and 16S rRNA. A high level of intraspecific variation was observed within all genes. The majority of polymorphisms were present in single individuals only, which resulted in a high number of private haplotypes within each gene. Little evidence of genetic differentiation was observed among the 3 Norwegian locations or between L. salmonis samples from Norway, Scotland and Russia. Pairwise FST values indicated that a weak degree of sub-division between L. salmonis sampled in Canada and the Northeast Atlantic might, however, exist. All samples collected in the Atlantic were highly different from the Japanese sample. It is suggested that the lack of genetic differentiation among lice samples from the North Atlantic is a result of extensive gene flow mediated by passive transport of L. salmonis larvae, and the migratory pattern of its salmonid hosts.     

Enzymes released from Lepeophtheirus salmonis in response to mucus from different salmonids

Adult and mobile preadult sea lice Lepophtheirus salmonis were incubated with mucus samples from rainbow trout (Oncorhynchus mykiss), coho salmon (O. kisutch), Atlantic salmon (Salmo salar), and winter flounder (Pseudopleuronectes americanus) to determine the response of L. salmonis to fish skin mucus as assessed by the release of proteases and alkaline phosphatase. There was variation in the release of respective enzymes by sea lice in response to different fish. As well, sealice collected from British Columbia responded differently than New Brunswick sea lice to coho salmon mucus. Fish mucus and seawater samples were also analyzed using protease gel zymography to observe changes in the presence of low molecular weight (LMW) proteases after L. salmonis incubation. Significantly higher proportions of sea lice secreted multiple bands of L. salmonis-derived LMW proteases after incubation with rainbow trout or Atlantic salmon mucus in comparison with seawater, coho salmon, or winter flounder mucus. Susceptibility to L. salmonis infections may be related to the stimulation of LMW proteases from L. salmonis by fish mucus. The resistance of coho salmon to L. salmonis infection may be due to agents in their mucus that block the secretion of these LMW proteases or factors may exist in the mucus of susceptible species that stimulate their release.     

Life history and virulence are linked in the ectoparasitic salmon louse Lepeophtheirus salmonis

Models of virulence evolution for horizontally transmitted parasites often assume that transmission rate (the probability that an infected host infects a susceptible host) and virulence (the increase in host mortality due to infection) are positively correlated, because higher rates of production of propagules may cause more damages to the host. However, empirical support for this assumption is scant and limited to microparasites. To fill this gap, we explored the relationships between parasite life history and virulence in the salmon louse, Lepeophtheirus salmonis, a horizontally transmitted copepod ectoparasite on Atlantic salmon Salmo salar. In the laboratory, we infected juvenile salmon hosts with equal doses of infective L. salmonis larvae and monitored parasite age at first reproduction, parasite fecundity, area of damage caused on the skin of the host, and host weight and length gain. We found that earlier onset of parasite reproduction was associated with higher parasite fecundity. Moreover, higher parasite fecundity (a proxy for transmission rate, as infection probability increases with higher numbers of parasite larvae released to the water) was associated with lower host weight gain (correlated with lower survival in juvenile salmon), supporting the presence of a virulence–transmission trade‐off. Our results are relevant in the context of increasing intensive farming, where frequent anti‐parasite drug use and increased host density may have selected for faster production of parasite transmission stages, via earlier reproduction and increased early fecundity. Our study highlights that salmon lice, therefore, are a good model for studying how human activity may affect the evolution of parasite virulence.     

Molecular cloning and characterisation of a novel P-glycoprotein in the salmon louse Lepeophtheirus salmonis

The salmon louse, Lepeophtheirus salmonis, is a crustacean ectoparasite of salmonid fish. At present, sea louse control on salmon farms relies heavily upon chemical treatments. Drug efflux transport, mediated by ABC transporters such as P-glycoprotein (Pgp), represents a major mechanism for drug resistance in parasites. We report here the molecular cloning of a new Pgp from the salmon louse, called SL-PGY1. A partial Pgp sequence was obtained by searching sea louse ESTs, and extended by rapid amplification of cDNA ends (RACE). The open reading frame of SL-PGY1 encodes a protein of 1438 amino acids that possesses typical structural traits of P-glycoproteins, and shows a high degree of sequence homology to invertebrate and vertebrate P-glycoproteins. In the absence of drug exposure, SL-PGY1 mRNA expression levels did not differ between a drug-susceptible strain of L. salmonis and a strain showing a ~7-fold decrease in sensitivity against emamectin benzoate, the active component of the in-feed sea louse treatment SLICE (Merck Animal Health). Aqueous exposure of the hyposensitive salmon louse strain to emamectin benzoate (24h, 410 μg/L) provoked a 2.9-fold upregulation of SL-PGY1. Adult male lice of both strains showed a greater abundance of SL-PGY1 mRNA than adult females.