Tor Einar Horsberg

Salmon lice – impact on wild salmonids and salmon aquaculture

Salmon lice, Lepeophtheirus salmonis, are naturally occurring parasites of salmon in sea water. Intensive salmon farming provides better conditions for parasite growth and transmission compared with natural conditions, creating problems for both the salmon farming industry and, under certain conditions, wild salmonids. Salmon lice originating from farms negatively impact wild stocks of salmonids, although the extent of the impact is a matter of debate. Estimates from Ireland and Norway indicate an odds ratio of 1.1:1-1.2:1 for sea lice treated Atlantic salmon smolt to survive sea migration compared to untreated smolts. This is considered to have a moderate population regulatory effect. The development of resistance against drugs most commonly used to treat salmon lice is a serious concern for both wild and farmed fish. Several large initiatives have been taken to encourage the development of new strategies, such as vaccines and novel drugs, for the treatment or removal of salmon lice from farmed fish. The newly sequenced salmon louse genome will be an important tool in this work. The use of cleaner fish has emerged as a robust method for controlling salmon lice, and aquaculture production of wrasse is important towards this aim. Salmon lice have large economic consequences for the salmon industry, both as direct costs for the prevention and treatment, but also indirectly through negative public opinion.

Determination of reduced sensitivity in sea lice (Lepeophtheirus salmonis Krøyer) against the pyrethroid deltamethrin using bioassays and probit modelling

Abstract Four Norwegian populations of the ectoparasitic copepod Lepeophtheirus salmonis K. were used to develop a bioassay for sensitivity determination to the pyrethroid deltamethrin (AlphaMax, Alpharma, Oslo, Norway, 10 mg deltamethrin ml −1 ). Bioassays using preadult II stage lice were performed with the sea lice exposed on salmon ( Salmo salar L.) and with the sea lice exposed in polystyrene boxes. The purpose was to investigate: (1) the difference in response in sea lice attached to salmon versus sea lice in polystyrene boxes, (2) the optimal time point for evaluation of the response after the end of the 30-min exposure period and (3) the difference between the immobilizing and the lethal concentrations of deltamethrin. The EC 50 (EC 50 : the concentration immobilizing 50% of the exposed sea lice) was calculated with probit modelling. The EC 50 value for sea lice in polystyrene boxes, evaluated at 24 h after termination of the exposure period, was considered a good indicator of sensitivity. The results were used to establish criteria for identification of strains with reduced sensitivity to deltamethrin. Bioassays with preadult II lice in polystyrene boxes demonstrated a significant ( P 50 =0.25 ppb). The other populations tested (Nord Trondelag, EC 50 =0.04 ppb; Sor Trondelag, EC 50 =0.06 ppb; Aust Agder, EC 50 =0.10 ppb) were not significantly ( P >0.05) different with respect to sensitivity towards deltamethrin.

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.

Single dose pharmacokinetic study of florfenicol in Atlantic salmon (Salmo salar) in seawater at 11°C

Abstract The pharmacokinetics of intravenously and orally administered florfenicol were determined in Atlantic salmon (Salmo salar) weighing 194±40 g (mean±s.d.). The study was performed at 10.8±1.5°C. A dose of 10 mg florfenicol/kg body weight was administered either intravenously or orally to groups of 85 fish each. At seven time points, from 3 h to 120 h after administration, blood was sampled from 10 individual fish in each group. The plasma was assayed for florfenicol using an HPLC method. The pharmacokinetic modelling of the results was performed using the computer program PCNONLIN. Following intravenous administration, the plasma concentration-time data of florfenicol were best described by a two-compartment open model. The volume of distribution at steady state, Vd(ss), and the total body clearance, ClT, were 1.122 l/kg and 0.086 l/h·kg, respectively. The elimination halflife, t 1 2 β , was estimated as 12.2 h. Following oral administration, the plasma concentration-time data of florfenicol were best described by a one-compartment open model with first order absorption and elimination. Peak plasma concentration, Cmax, was estimated at 4.0 μg/ml and was estimated to occur at 10.3 h (Tmax) following dosing. The bioavailability, F, was estimated at 96.5%. Based on the median minimum inhibitory concentrations (MICs) of 0.8 μg/ml reported for Aeromonas salmonicida, Vibrio anguillarum and Vibrio salmonicida, plasma concentrations should remain above the MIC for 36–40 h following a single oral dose of 10 mg florfenicol/kg.

The disposition of 14C-florfenicol in Atlantic salmon (Salmo salar)

Abstract The absorption, distribution, metabolism and excretion of 14C-labelled florfenicol in Atlantic salmon held in sea water at 8.5–11.5°C were studied by means of whole body autoradiography (WBA), liquid scintillation counting (LSC) and high performance liquid chromatography (HPLC). Uniformly ring-labelled 14C-florfenicol was mixed in capelin oil, and coated on ordinary fish feed. Each of 28 fish (139.6±18.4 g) was gavaged with a single dose of 10 mg/kg bodyweight of florfenicol, corresponding to 119.05 μCi/kg bodyweight. Whole fish were sampled from 3 h to 56 days after administration, and were analyzed by WBA and LSC. Muscle samples from fish sampled 6 h to 3 days after administration were analysed by HPLC, and LSC of fractions of the eluate. Peak radioactivity levels were detected in most tissues at 12 h after administration (3 days in the kidney), and declined thereafter. The radioactivity in the brain was low compared with that in muscle and blood, suggesting that the passage of the blood-brain barrier by florfenicol or its metabolites is limited. Radioactivity levels in blood and muscle were similar at all times. Accumulation of radioactivity could be seen in the kidney and the choroidea of the eye, indicating a possible affinity to melanin. The radioactivity was excreted both via urine and bile. There were trace amounts of radioactivity in skin, liver and kidney tissues of fish sampled 56 days post administration, while no radioactivity was detected in any blood or muscle samples taken later than 28 days after administration. The fraction of parent florfenicol in muscle decreased from approximately 90% (6 h) to about 20% (3 days), while the fraction of florfenicol amine increased from about 7% to about 70% during the same period. Other metabolites were detected, but were quantitatively of minor importance. The results strongly suggest that florfenicol is rapidly metabolized in the body of Atlantic salmon, and that the dominant metabolite is florfenicol amine.

Single-dose field bioassay for sensitivity testing in sea lice, Lepeophtheirus salmonis: development of a rapid diagnostic tool.

Sea lice on farmed salmonids are often treated with chemicals. Sensitivity testing of sea lice can reduce the number of treatments by identifying substances the sea lice are susceptible to. This study describes a simpler protocol for field sensitivity testing than todays six-dose bioassay. The protocol, which uses a single dose of the delousing agents deltamethrin, azamethiphos and emamectin benzoate, was developed on four different strains of sea lice and their subsequent generations. A sensitive strain and a strain showing reduced sensitivity were identified for each chemical after performing traditional bioassays and small-scale treatments. The single doses for each chemical were established by modelling dose-response curves from 24-h bioassays on strains with differences in sensitivity. The largest difference between the lower 80% prediction interval for the sensitive strain and the upper 80% prediction interval for the strain showing reduced sensitivity was identified for each delousing agent. The concentration of the chemical and the % mortality corresponding to each of the 80% prediction intervals were subsequently established. To validate the protocol for field use, further studies on both sensitive and resistant strains of sea lice under field conditions are required.

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.

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.

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.

Single dose kinetic study of sarafloxacin after intravenous and oral administration of different formulations to Atlantic salmon (Salmo salar) held in sea water at 8.5°C

Abstract The pharmacokinetics of sarafloxacin hydrochloride (Sarafin™) following intravenous and oral administration were determined in Atlantic salmon (Salmo salar) weighing 163±36 g (mean±s.d.). The study was performed at 8.5±0.7°C. A dose of 10 mg sarafloxacin/kg body weight was administered intravenously to one group and orally to four groups of 28 fish each. Two of the oral groups were given drug formulations prepared as medicated feeds, the other two oral groups being given formulations prepared using corn oil or capelin oil as the drug vehicle. At four time points, from 6 h to 48 h after administration, blood was sampled from each of five fish per group. The plasma was assayed for sarafloxacin using an HPLC method. Following intravenous administration, the plasma concentration-time data of sarafloxacin were best described by a one-compartment open model. The volume of distribution Vd(area), and the total body clearance ClT, were 4.1 l/kg and 0.18 l/h·kg, respectively. The elimination half-life, t 1 2 , was estimated to be 15.9 h. Following oral administration, the peak plasma concentration, Cmax, ranged from 0.08 to 0.70 μg/ml, and the time to reach Cmax (Tmax) from 6 to 24 h, depending on the formulation used. The bioavailability, F, ranged from 3.6 to 23.9%. There were no significant differences in observed plasma concentrations between fish in the two medicated feed groups, but highly significant differences between fish in the two oil groups. After oral administration, the group using corn oil as the drug vehicle showed the highest plasma concentrations.