Trends in de-lousing of Norwegian farmed salmon from 2000-2019-Consumption of medicines, salmon louse resistance and non-medicinal control methods.

The salmon louse Lepeophtheirus salmonis has been a substantial obstacle in Norwegian farming of Atlantic salmon for decades. With a limited selection of available medicines and frequent delousing treatments, resistance has emerged among salmon lice. Surveillance of salmon louse sensitivity has been in place since 2013, and consumption of medicines has been recorded since the early 80's. The peak year for salmon lice treatments was 2015, when 5.7 times as many tonnes of salmonids were treated compared to harvested. In recent years, non-medicinal methods of delousing farmed fish have been introduced to the industry. By utilizing data on the annual consumption of medicines, annual frequency of medicinal and non-medicinal treatments, the aim of the current study was to describe the causative factors behind salmon lice sensitivity in the years 2000-2019, measured through toxicity tests-bioassays. The sensitivity data from 2000-2012 demonstrate the early emergence of resistance in salmon lice along the Norwegian coast. Reduced sensitivity towards azamethiphos, deltamethrin and emamectin benzoate was evident from 2009, 2009 and 2007, respectively. The annual variation in medicine consumption and frequency of medicinal treatments correlated well with the evolution in salmon louse sensitivity. The patterns are similar, with a relatively small response delay from the decline in the consumption of medicines in Norway (2016 and onward) to the decline in measured resistance among salmon louse (2017 and onward). 2017 was the first year in which non-medicinal treatments outnumbered medicinal delousing treatments as well as the peak year in numbers of cleanerfish deployed. This study highlights the significance of avoiding heavy reliance on a few substance groups to combat ectoparasites, this can be a potent catalyst for resistance evolution. Further, it demonstrates the importance of transparency in the global industry, which enables the industry to learn from poor choices in the past.

Candidate genes for monitoring hydrogen peroxide resistance in the salmon louse, Lepeophtheirus salmonis.

BACKGROUND: Hydrogen peroxide (H2O2) is one of the delousing agents used to control sea lice infestations in salmonid aquaculture. However, some Lepeophtheirus salmonis populations have developed resistance towards H2O2. An increased gene expression and activity of catalase, an enzyme that breaks down H2O2, have been detected in resistant lice, being therefore introduced as a resistance marker in the salmon industry. In the present study the aim was to validate the use of catalase expression as a marker and to identify new candidate genes as additional markers to catalase, related to H2O2 resistance in L. salmonis. METHODS: A sensitive and an H2O2 resistant laboratory strain (P0 generation, not exposed to H2O2 for several years) were batch crossed to generate a cohort with a wide range of H2O2 sensitivities (F2 generation). F2 adult females were then exposed to H2O2 to separate sensitive and resistant individuals. Those F2 lice, the P0 lice and field-collected resistant lice (exposed to H2O2 in the field) were used in an RNA sequencing study. RESULTS: Catalase was upregulated in resistant lice exposed to H2O2 compared to sensitive lice. This was, however, not the case for unexposed resistant P0 lice. Several other genes were found differentially expressed between sensitive and resistant lice, but most of them seemed to be related to H2O2 exposure. However, five genes were consistently up- or downregulated in the resistant lice independent of exposure history. The upregulated genes were: one gene in the DNA polymerase family, one gene encoding a Nesprin-like protein and an unannotated gene encoding a small protein. The downregulated genes encoded endoplasmic reticulum resident protein 29 and an aquaporin (Glp1_v2). CONCLUSIONS: Catalase expression seems to be induced by H2O2 exposure, since it was not upregulated in unexposed resistant lice. This may pose a challenge for its use as a resistance marker. The five new genes associated with resistance are put forward as complementary candidate genes. The most promising was Glp1_v2, an aquaglyceroporin that may serve as a passing channel for H2O2. Lower channel number can reduce the influx or distribution of H2O2 in the salmon louse, being directly involved in the resistance mechanism.

Nicotinic acetylcholine receptors: Ex-vivo expression of functional, non-hybrid, heteropentameric receptors from a marine arthropod, Lepeophtheirus salmonis.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mostly located in the post-synaptic membrane of cholinergic synapses. The natural neurotransmitter is acetylcholine, but they are also the direct targets for neonicotinoids, chemicals widely used against ectoparasites, arthropod vectors and agricultural pests. There are significant concerns regarding adverse effects of neonicotinoids on beneficial insects. In arthropods, functional nAChRs made of α subunits have been expressed from Drosophila genes, and hybrid receptors (sometimes also referred to as chimeric receptors) using species-specific α subunits and vertebrate ß subunits have been expressed ex-vivo. Arthropod-specific nAChRs made of both α and ß subunits from the target species have not been expressed ex-vivo. The aim of the current study was to express such receptors in Xenopus oocytes using only genes from Lepeophtheirus salmonis, to characterize them and study their modulation. Genes encoding α and ß subunits of the nAChRs and three ancillary proteins, RIC-3, UNC-50 and UNC-74 were identified in the L. salmonis genome, subjected to RACE-PCR, cloned into an expression vector and the cRNA produced was then injected into Xenopus laevis oocytes. Co-expression of the ancillary proteins was essential for the successful expression of the L. salmonis nAChRs with both α and ß subunits. Two functional nAChRs were identified: Lsa-nAChR1 consisting of α1, α2, ß1 and ß2 subunits, reconstituted to one distinct receptor, while Lsa-nAChR2, consisting of α3, ß1 and ß2 subunits reconstitutes receptors with two distinct characteristics. Out of seven neonicotinoids tested, six worked as partial agonist of Lsa-nAChR1 while only three did so for Lsa-nAChR2. Four non-neonicotinoid compounds tested had no effect on either of the nAChRs. The study demonstrated that fully functional, non-hybrid nAChRs containing both α and ß subunits from an arthropod can be reconstituted ex-vivo by co-expression of essential ancillary proteins. Such models would be valuable for in-depth studies of effects by neonicotinoids and other compounds on target pests, as well as for studies of adverse effects on non-target arthropods.

Deltamethrin resistance in the salmon louse, Lepeophtheirus salmonis (Krøyer): Maternal inheritance and reduced apoptosis.

Resistance towards deltamethrin (DMT) in the crustacean ectoparasite Lepeophtheirus salmonis (Caligidae) is a problem on fish farms lining the North Atlantic Ocean. Two Norwegian strains with different susceptibility towards DMT were crossed in the parental generation (P0), females from a sensitive strain were crossed with males from a resistant strain and vice versa. Individual susceptibility towards DMT was assessed in the second filial generation (F2). DMT resistance was only found in F2 descendants when the P0 females were from the resistant strain, pointing to maternal inheritance. Since maternal inheritance might be linked to the mitochondrial (mt) genome, the nucleotide sequences and the gene expressions of mt-genes were analysed. Twenty non-synonymous single nucleotide polymorphisms (SNPs) were identified in mt-transcripts from resistant F2 parasites, including SNPs in two cytochrome C oxidase subunits (COX1 and COX3) and two subunits of the NADH dehydrogenase complex (ND1 and ND5) previously linked to DMT resistance in the salmon louse. Differential expression analysis between the sensitive and resistant strain revealed strain effect in seven out of twelve mt-genes. The current study also show that DNA fragmentation (indicating apoptosis) was affected by DMT exposure in skeletal muscle tissue and that resistant parasites undergo less apoptosis than sensitive parasites.

Identification and characterisation of the ecdysone biosynthetic genes neverland, disembodied and shade in the salmon louse Lepeophtheirus salmonis (Copepoda, Caligidae).

The salmon louse is a marine ectoparasitic copepod on salmonid fishes. Its lifecycle consists of eight developmental stages, each separated by a molt. In crustaceans and insects, molting and reproduction is controlled by circulating steroid hormones such as 20-hydroxyecdysone. Steroid hormones are synthesized from cholesterol through catalytic reactions involving a 7,8-dehydrogenase Neverland and several cytochrome P450 genes collectively called the Halloween genes. In this study, we have isolated and identified orthologs of neverland, disembodied and shade in the salmon louse (Lepeophtheirus salmonis) genome. Tissue-specific expression analysis show that the genes are expressed in intestine and reproductive tissue. In addition, levels of the steroid hormones ecdysone, 20-hydroxyecdysone and ponasterone A were measured during the reproductive stage of adult females and in early life stages.

The mechanism (Phe362Tyr mutation) behind resistance in Lepeophtheirus salmonis pre-dates organophosphate use in salmon farming.

The salmon louse is an ectoparasitic copepod of salmonids in the marine environment, and represents a global challenge to salmon aquaculture. A major issue is the reliance of the industry on a limited number of chemicals to delouse salmonids on farms, and the high levels of resistance that lice have developed to all of these agents. However, for most of these chemicals, resistance and dispersal mechanisms are unknown. We recently demonstrated that the Phe362Tyr mutation is the primary cause of organophosphate resistance in lice collected on Norwegian farms. In the present study, we genotyped >2000 lice collected throughout the entire North Atlantic in the period 1998-2016, using Phe362Tyr and nine tightly linked SNPs. Our results showed that the Phe362Tyr mutation is strongly linked to lice survival following chemical treatment on farms located throughout the North Atlantic, demonstrating for the first time, that this mutation represents the primary mechanism for organophosphate resistance in salmon lice across the North Atlantic. Additionally, we observed multiple and diverse high frequency haplotypes linked with the allele conveying resistance to organophosphate. We, therefore, conclude that Phe362Tyr is not a de novo mutation, but probably existed in salmon lice before the introduction of organophosphates in commercial aquaculture.

A selection study on a laboratory-designed population of salmon lic (Lepeophtheirus salmonis) using organophosphate and pyrethroid pesticides.

Resistance towards antiparasitic agents in the salmon louse (Lepeophtheirus salmonis) is a widespread problem along the Norwegian coast, reducing treatments efficacies and slowing down the envisioned expansion of Norwegian salmon production. The present study was conducted in order to assess the efficacies of two of the most widely used anti-parasitic substances-azamethiphos and deltamethrin-as well as assessing the benefit of having a resistant genotype compared to being fully sensitive when exposed to one of these substances. Atlantic salmon were exposed to a mix of salmon lice copepodids from a fully sensitive, a double resistant and a multi-resistant strain. Once the lice reached pre-adult stages, one group was exposed to 100 µg/L azamethiphos for 60 minutes, the other to 2 µg/L deltamethrin for 30 minutes, and the last was kept in a seawater control. Detached lice were collected at a series of time points following exposure, and all lice (immobilized and surviving) were analysed for both pyrethroid (sensitive "S" and resistant "R") and azamethiphos (fully sensitive "SS", heterozygous resistant "RS" and fully resistant "RR") resistance markers. We found that the efficacies of deltamethrin on parasites with genotype S and R were 70.3 and 13.2%, respectively. The overall efficacy of the deltamethrin treatment was 32.3%. The efficacies of azamethiphos on parasites with genotype SS, RS and RR were 100, 80 and 19.1%, respectively. The overall efficacy of the azamethiphos treatment was 80.4%. Survival analyses revealed that the median survival time in deltamethrin-sensitive and-resistant parasites were 16.8 and >172 hours, respectively. The differences were even more pronounced in the azamethiphos-treated group, where SS, RS and RR parasites survived for 0.26, 6.6 and >172 hours, respectively. The substantial differences in survival between sensitive and resistant lice following treatment demonstrate the ability of medicinal treatments to drive genetic selection towards a much more resistant salmon lice population within a very short time span if there is no influx of sensitive genotypes.

Surveillance of the Sensitivity towards Antiparasitic Bath-Treatments in the Salmon Louse (Lepeophtheirus salmonis).

The evolution of drug resistant parasitic sea lice is of major concern to the salmon farming industry worldwide and challenges sustainable growth of this enterprise. To assess current status and development of L. salmonis sensitivity towards different pesticides used for parasite control in Norwegian salmon farming, a national surveillance programme was implemented in 2013. The programme aims to summarize data on the use of different pesticides applied to control L. salmonis and to test L. salmonis sensitivity to different pesticides in farms along the Norwegian coast. Here we analyse two years of test-data from biological assays designed to detect sensitivity-levels towards the pesticides azamethiphos and deltamethrin, both among the most common pesticides used in bath-treatments of farmed salmon in Norway in later years. The focus of the analysis is on how different variables predict the binomial outcome of the bioassay tests, being whether L. salmonis are immobilized/die or survive pesticide exposure. We found that local kernel densities of bath treatments, along with a spatial geographic index of test-farm locations, were significant predictors of the binomial outcome of the tests. Furthermore, the probability of L. salmonis being immobilized/dead after test-exposure was reduced by odds-ratios of 0.60 (95% CI: 0.42-0.86) for 2014 compared to 2013 and 0.39 (95% CI: 0.36-0.42) for low concentration compared to high concentration exposure. There were also significant but more marginal effects of parasite gender and developmental stage, and a relatively large random effect of test-farm. We conclude that the present data support an association between local intensities of bath treatments along the coast and the outcome of bioassay tests where salmon lice are exposed to azamethiphos or deltamethrin. Furthermore, there is a predictable structure of L. salmonis phenotypes along the coast in the data, characterized by high susceptibility to pesticides in the far north and far south, but low susceptibility in mid Norway. The study emphasizes the need to address local susceptibility to pesticides and the need for restrictive use of pesticides to preserve treatment efficacy.

Phe362Tyr in AChE: A Major Factor Responsible for Azamethiphos Resistance in Lepeophtheirus salmonis in Norway.

Organophosphates (OP) are one of the major treatments used against the salmon louse (Lepeophtherius salmonis) in Norwegian salmonid aquaculture. The use of OP since the late 1970s has resulted in widespread resistant parasites. Recently, we reported a single mutation (Phe362Tyr) in acetylcholinesterase (AChE) as the major mechanism behind resistance in salmon louse towards OP. The present study was carried out to validate this mechanism at the field level. A total of 6658 salmon louse samples were enrolled from 56 different fish farms across the Norwegian coast, from Vest Agder in the south to Finnmark in the north. All the samples were genotyped using a TaqMan probe assay for the Phe362Tyr mutation. A strong association was observed between areas with frequent use of the OP (azamethiphos) and the Phe362Tyr mutation. This was confirmed at 15 sites where results from independently conducted bioassays and genotyping of parasites correlated well. Furthermore, genotyping of surviving and moribund parasites from six bioassay experiments demonstrated a highly significant negative correlation between the frequency of resistance alleles and the probability of dying when exposed to azamethiphos in a bioassay. Based on these observations, we could strongly conclude that the Phe362Tyr mutation is a major factor responsible for OP resistance in salmon louse on Norwegian fish farms.

Identification and Molecular Characterization of Two Acetylcholinesterases from the Salmon Louse, Lepeophtheirus salmonis.

Acetylcholinesterase (AChE) is an important enzyme in cholinergic synapses. Most arthropods have two genes (ace1 and ace2), but only one encodes the predominant synaptic AChE, the main target for organophosphates. Resistance towards organophosphates is widespread in the marine arthropod Lepeophtheirus salmonis. To understand this trait, it is essential to characterize the gene(s) coding for AChE(s). The full length cDNA sequences encoding two AChEs in L. salmonis were molecularly characterized in this study. The two ace genes were highly similar (83.5% similarity at protein level). Alignment to the L. salmonis genome revealed that both genes were located close to each other (separated by just 26.4 kbp on the L. salmonis genome), resulting from a recent gene duplication. Both proteins had all the typical features of functional AChE and clustered together with AChE-type 1 proteins in other species, an observation that has not been described in other arthropods. We therefore concluded the presence of two versions of ace1 gene in L. salmonis, named ace1a and ace1b. Ace1a was predominantly expressed in different developmental stages compared to ace1b and was possibly active in the cephalothorax, indicating that ace1a is more likely to play the major role in cholinergic synaptic transmission. The study is essential to understand the role of AChEs in resistance against organophosphates in L. salmonis.

Mechanism behind Resistance against the Organophosphate Azamethiphos in Salmon Lice (Lepeophtheirus salmonis).

Acetylcholinesterase (AChE) is the primary target for organophosphates (OP). Several mutations have been reported in AChE to be associated with the reduced sensitivity against OP in various arthropods. However, to the best of our knowledge, no such reports are available for Lepeophtheirus salmonis. Hence, in the present study, we aimed to determine the association of AChE(s) gene(s) with resistance against OP. We screened the AChE genes (L. salmonis ace1a and ace1b) in two salmon lice populations: one sensitive (n=5) and the other resistant (n=5) for azamethiphos, a commonly used OP in salmon farming. The screening led to the identification of a missense mutation Phe362Tyr in L. salmonis ace1a, (corresponding to Phe331 in Torpedo californica AChE) in all the samples of the resistant population. We confirmed the potential role of the mutation, with reduced sensitivity against azamethiphos in L. salmonis, by screening for Phe362Tyr in 2 sensitive and 5 resistant strains. The significantly higher frequency of the mutant allele (362Tyr) in the resistant strains clearly indicated the possible association of Phe362Tyr mutation in L. salmonis ace1a with resistance towards azamethiphos. The 3D modelling, short term survival experiments and enzymatic assays further supported the imperative role of Phe362Tyr in reduced sensitivity of L. salmonis for azamethiphos. Based on all these observations, the present study, for the first time, presents the mechanism of resistance in L. salmonis against azamethiphos. In addition, we developed a rapid diagnostic tool for the high throughput screening of Phe362Tyr mutation using High Resolution Melt analysis.

Drug resistance in sea lice: a threat to salmonid aquaculture.

Sea lice are copepod ectoparasites with vast reproductive potential and affect a wide variety of fish species. The number of parasites causing morbidity is proportional to fish size. Natural low host density restricts massive parasite dispersal. However, expanded salmon farming has shifted the conditions in favor of the parasite. Salmon farms are often situated near wild salmonid migrating routes, with smolts being particularly vulnerable to sea lice infestation. In order to protect both farmed and wild salmonids passing or residing in the proximity of the farms, several measures are taken. Medicinal treatment of farmed fish has been the most predictable and efficacious, leading to extensive use of the available compounds. This has resulted in drug-resistant parasites occurring on farmed and possibly wild salmonids.

Behavioural effects of the commonly used fish anaesthetic tricaine methanesulfonate (MS-222) on zebrafish (Danio rerio) and its relevance for the acetic acid pain test.

The pros and cons of using anaesthesia when handling fish in connection with experiments are debated. A widely adopted practice is to wait thirty minutes after anaesthesia before behavioural observations are initiated, but information about immediate effects of a treatment is then lost. This is pertinent for responses to acute stressors, such as acid injection in the acetic acid pain test. However, omission of anaesthetics in order to obtain data on immediate responses will compromise the welfare of fish and contribute to experimental noise due to stress. We therefore tested the effect of tricaine methanesulfonate on the behaviour of zebrafish. We predicted that tricaine (MS 222) would decrease swimming velocity and that the control fish would show an increased level of anxiety- and stress-related behaviours compared to the tricaine group. Following acclimatization to the test tank, baseline behaviour was recorded before immersion in either tricaine (168 mg l(-1), treatment group, N = 8) or tank water (control group, N = 7). Latencies to lose equilibrium and to lose response to touch were registered. The fish was then returned to the test tank, and the latency to regain equilibrium was registered in anaesthetized fish. When equilibrium was regained, and at five, thirty and sixty minutes after the fish had been returned to the test tank, behaviour was recorded. The tricaine fish showed the following responses (mean ± sd): latency to lose equilibrium 22.6 s±3.9; latency to lose response to touch 101.9 s±26.8; latency to regain equilibrium 92.0 s±54.4. Contrary to our predictions, neither treatment caused a change in any of the behaviours registered. This indicates that tricaine has no effect on several commonly used behavioural parameters, and that it may be unnecessary to postpone behavioural observations to 30 min after anaesthesia.

Kinetic properties of saxitoxin in Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua).

The disposition of STX in Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua) was studied after intraperitoneal (IP) injection (5 microg STX/kg bm and 3.43 microg (3)H-STXeq/kg bw respectively), intravenous (IV) injection (5 microg STX/kg bm, only salmon) and waterborne exposure (50 microg STXeq/L, only salmon). Plasma concentrations in salmon were quantified using a receptor binding assay and cod tissues were analyzed using scintillation counting of tissue extracts and autoradiography of whole fish slices. The estimated elimination half-life (T(1/2)) after IV administration of STX in salmon was 102.6 min. The volume of distribution (Vz) was observed to be 467.2 mL/kg and the total body clearance (Cl(T)) was 3.2 mL/min/kg. Waterborne exposure clearly showed that salmon absorbed PSP toxins directly from the water. In cod, (3)H-STX was observed in gills, muscle, brain, liver and posterior kidney from 30 to 480 min. The lowest concentrations of (3)H-STX were found in brain and muscle, whereas posterior kidney contained the majority of the toxin. Autoradiograms confirmed the high levels of (3)H-STX in the kidneys, indicating that renal excretion was the main elimination route. Buildup of harmful levels in edible tissue is not very likely due to the low concentrations accumulated in muscle tissue and rapid excretion.

Subclinical effects of saxitoxin and domoic acid on aggressive behaviour and monoaminergic turnover in rainbow trout (Oncorhynchus mykiss).

The algal produced neurotoxins saxitoxin and domoic acid may have serious effects on marine life and can be responsible for the intoxication of for instance sea mammals, sea birds and fish. Given that farmed fish cannot escape algal blooms, they may be more susceptible to intoxication than wild stocks. In the present study, subclinical effects of saxitoxin and domoic on aggressive behaviour and monoaminergic systems in the brain of the rainbow trout (Oncorhynchus mykiss) were investigated. The resident-intruder test was used to measure aggression where only the resident fish were subjected to the toxins and analysed for monoamines and their metabolites. The resident-intruder test was carried out on two consecutive days. On day one basal aggression was measured in the four groups. On day two three of the groups were injected with subclinical doses of one of the following: saxitoxin (1.752 microg/kg bw), domoic (0.75 mg/kg bw) or 0.9% saline solution. This was performed 30 min prior to the aggression test. Handling stress and injection affected aggressive behaviour, cortisol and the serotonergic system in telencephalic brain regions. Cortisol levels were elevated in all of the injected groups when compared to the control group. An increase in serotonergic turnover was evident when all injected groups were pooled and compared to the control group. All together this suggests that the handling stress in connection with the injection was similar in all of the three injected groups. In contrast to both the undisturbed control group and the toxin-injected groups, the saline-injected group displayed a reduction in aggressive behaviour which was evident in increased attack latency. Furthermore the domoic injected group displayed more aggressive attacks towards their conspecifics than the saline-injected group. Consequently the two toxins appear to mask the stress induced alteration in aggressive behaviour. Monoamine levels and monoaminergic turnover could not be demonstrated to be directly affected by the two toxins at the given doses in the investigated brain regions (dorsal and ventral parts of telencephalon, optic tectum, locus coeruleus, raphe nucleus, molecular and granular layer of cerebellum). This could indicate that the toxins mediate aggressive behaviour either through other systems than the monoaminergic systems, such as neuroactive amino acids, or that the mediation occurs in other brain regions.

Trace classical conditioning in rainbow trout (Oncorhynchus mykiss): what do they learn?

There are two main memory systems: declarative and procedural memory. Knowledge of these two systems in fish is scarce, and controlled laboratory studies are needed. Trace classical conditioning is an experimentally tractable model of declarative memory. We tested whether rainbow trout (Oncorhynchus mykiss) can learn by trace conditioning and form stimulus-stimulus, as opposed to stimulus-response, associations. We predicted that rainbow trout trained by trace conditioning would show appetitive behaviour (conditioned response; CR) towards the conditioned stimulus (CS; light), and that the CR would be sensitive to devaluation of the unconditioned stimulus (US; food). The learning group (L, N = 14) was trained on a CS + US contingency schedule with a trace interval of 3.4 s. The control group (CtrL, N = 4) was kept on a completely random schedule. The fish that learnt were further trained as either an experimental (L, N = 6) or a memory control (CtrM, N = 3) group. The L group had the US devalued. The CtrM group received only food. No fish in the CtrL group, but nine fish from the L group conditioned to the light. When tested, five L fish changed their CRs after US devaluation, indicating learning by stimulus-stimulus association of the light with the food. CtrM fish retained their original CRs. To the best of our knowledge, this experiment is the first to show that rainbow trout can learn by trace classical conditioning. The results indicate that the fish learnt by 'facts-learning' rather than by reflex acquisition in this study.

Pharmacokinetics of morphine after intramuscular injection in common goldfish Carassius auratus and Atlantic salmon Salmo salar.

Teleost fish have a nociceptive system and likely perceive pain. This warrants the development of analgesic protocols both for experimental surgery and for various husbandry procedures. Morphine is the standard analgesic against which the efficacy of other analgesics is assessed, and is the analgesic that has been most used in fish. The aims of this study were to describe the pharmacokinetics of morphine after an intramuscular (i.m.) injection in common goldfish and Atlantic salmon, and to illustrate the whole-body distribution of morphine in salmon following i.m. injection of tritiated morphine. In the kinetic experiment, goldfish and salmon were respectively i.m. injected with 40 and 100 mg morphine kg(-1) in the right dorsal epaxial musculature. Blood was drawn at predetermined time points. Plasma was analysed for morphine and metabolites using liquid chromatography-mass spectrometry (LC-MS/MS). Morphine had a Tmax (time at which the maximum plasma concentration was measured) of 0.5 h in both species. The Cmax (maximum plasma concentration) showed substantial inter-individual variation, with a mean (90% CI) of 187 (167 to 199) mg l(-1) in salmon and 37 (29 to 43) mg l(-1) in goldfish, as determined by bootstrap analysis. The mean elimination half-lives were 12.5 and 13.5 h in goldfish and in salmon, respectively. The degree of metabolism to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was low, with levels of M3G exceeding those of M6G. The distribution study demonstrated that the levels of tritiated morphine in the anterior kidney surpassed those in the other organs. A substantial amount seemed to be excreted through the gastrointestinal tract, while little tritium activity could be detected in the central nervous system.

Somatosensory evoked potentials in the telencephalon of Atlantic salmon (Salmo salar) following galvanic stimulation of the tail.

Electric activity in the brain which is time-locked to a given stimulation of the somatosensory system can be recorded as a somatosensory evoked potential (SEP). We investigated whether a galvanic stimulation of the tail base in Atlantic salmon (Salmo salar) would elicit a SEP in the telencephalon. The telencephalon is central in learning and memory, and activity here may be a prerequisite for processing of external stimuli on a cognitive or emotional level. Anaesthetized salmon (n = 11) were subjected to craniotomy and a recording electrode was inserted into the telencephalon. The fish were given stimulations of four intensities, i.e., 2, 5, 10 and 20 mA. A SEP was elicited in the contralateral dorsal telencephalon for all intensities. This result agrees with findings in other fish species. Furthermore, there was a significant difference between the maximum peak amplitude and mean amplitude of the SEP elicited by putative non-noxious (2 mA) and putative noxious (20 mA) stimulation intensities (P < 0.01). The stronger stimulation intensities also tend to introduce longer-latencies components in the SEP. The results added to the body of literature indicates that the exteroceptive senses are represented by processing within the telencephalon of the fish.

Effects of algal-produced neurotoxins on metabolic activity in telencephalon, optic tectum and cerebellum of Atlantic salmon (Salmo salar).

Neurotoxins from algal blooms have been reported to cause mortality in a variety of species, including sea birds, sea mammals and fish. Farmed fish cannot escape harmful algal blooms and their potential toxins, thus they are more vulnerable for exposure than wild stocks. Sublethal doses of the toxins are likely to affect fish behaviour and may impair cognitive abilities. In the present study, changes in the metabolic activity in different parts of the Atlantic salmon (Salmo salar) brain involved in central integration and cognition were investigated after exposure to sublethal doses of three algal-produced neurotoxins; saxitoxin (STX), brevetoxin (BTX) and domoic acid (DA). Fish were randomly selected to four groups for i.p. injection of saline (control) or one of the neurotoxins STX (10 microg STX/kg bw), BTX (68 microg BTX/kg bw) or DA (6 mg DA/kg bw). In addition, 14C-2-deoxyglucose was i.m. injected to measure brain metabolic activity by autoradiography. The three regions investigated were telencephalon (Tel), optic tectum (OT) and cerebellum (Ce). There were no differences in the metabolic activity after STX and BTX exposure compared to the control in these regions. However, a clear increase was observed after DA exposure. When the subregions with the highest metabolic rate were pseudocoloured in the three brain regions, the three toxins caused distinct differences in the respective patterns of metabolic activation. Fish exposed to STX displayed similar patterns as the control fish, whereas fish exposed to BTX and DA showed highest metabolic activity in subregions different from the control group. All three neurotoxins affected subregions that are believed to be involved in cognitive abilities in fish.

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.