Marit Jørgensen Bakke

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.

Attenuation of stress-induced anorexia in brown trout (Salmo trutta) by pre-treatment with dietary l-tryptophan.

The general consensus is that brain serotonin (5-HT) inhibits feed intake in teleost fishes and other vertebrates. Dietary manipulations with the 5-HT precursor tryptophan (TRP) have, however, yielded contradictory effects on feed intake, while studies of the endocrine response to stress indicate that the effects of TRP-enriched feed are context dependent. A characteristic behavioural response to stress is a reduction in feed intake, and in the present study we investigated whether pre-treatment with TRP-enriched feed affected stress-induced changes in feeding behaviour in brown trout (Salmo trutta). After acclimatisation in observation aquaria, isolated fish were fed control or TRP-supplemented feed for 7 d, whereupon they were transferred to a novel environment, in which all fish were fed control feed. Transfer to a new environment resulted in decreased feeding in both the TRP pre-treated and the control-treated group. However, this decrease was more pronounced in the control-treated group. Previous experiments have concluded that stimulation of brain 5-HT systems by TRP enhancement does not affect feed intake in salmonid fishes, but in these studies food intake was observed in unstressed animals only. The present study suggests that pre-treatment with dietary TRP attenuates stress-induced anorexia. Hence, it appears that the effect of dietary manipulations of TRP on feeding behaviour is dependent on the stress levels experienced by experimental animals. These behavioural data are discussed in the context of the involvement of 5-HT in appetite regulation.

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.

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.

The effect of dietary lipid composition on the intestinal uptake and tissue distribution of benzo[a]pyrene and phenanthrene in Atlantic salmon (Salmo salar).

Uptake of polycyclic aromatic hydrocarbons (PAHs) across the intestine is suggested to occur in association with dietary lipids. Partial replacement of fish ingredients by vegetable ingredients in aquafeeds has led to increased levels of PAHs in marine farmed fish. We therefore investigated, intestinal uptake, tissue distribution and PAH metabolism after a single dose of (14)C-benzo[a]pyrene (BaP) or (14)C-phenanthrene (PHE) given to Atlantic salmon (Salmo salar) acclimatized to a fish oil or vegetable oil based diet. Both BaP and PHE were absorbed along the intestine. Fish oil based feed increased BaP concentration in the pyloric caeca and that of PHE in the proximal intestine. In contrast, vegetable oil increased BaP concentrations in the distal intestine. Extraction of whole body autoradiograms removed PHE-associated radiolabeling almost completely from the intestinal mucosa, but not BaP-associated radiolabeling, indicating the presence of BaP metabolites bound to cellular macromolecules. This observation correlates with the increased cyp1a expression in the proximal intestine, distal intestine and liver in the BaP exposed group. Furthermore, BaP-induced cyp1a expression was higher in the distal intestine of salmon fed fish oil compared to the vegetable oil fed group. PHE had no significant effect on cyp1a expression in any of these tissues. We conclude that dietary lipid composition affects intestinal PAH uptake. Fish oil based feed increased intestinal PAH concentrations probably due to an enhanced solubility in micelles composed of fish oil fatty acids. Increased BaP accumulation in the distal intestine of vegetable oil fed fish seems to be associated with a reduced Cyp1a-mediated BaP metabolism.

Mutations in voltage-gated sodium channels from pyrethroid resistant salmon lice (Lepeophtheirus salmonis): Kdr mutations in L. salmonis

BACKGROUND Parasitic salmon lice (Lepeophtheirus salmonis) cause high economic losses in Atlantic salmon farming. Pyrethroids, which block arthropod voltage-gated sodium channels (Nav 1), are used for salmon delousing. However, pyrethroid resistance is common in L. salmonis. The present study characterized Nav 1 homologues in L. salmonis in order to identify channel mutations associated to resistance, called kdr (knockdown) mutations. RESULTS Genome scans identified three L. salmonis Nav 1 homologues, LsNav 1.1, LsNav 1.2 and LsNav 1.3. Arthropod kdr mutations map to specific Nav 1 regions within domains DI-III, namely segments S5 and S6 and the linker helix connecting S4 and S5. The above channel regions were amplified by RT-PCR and sequenced in deltamethrin-susceptible and deltamethrin-resistant L. salmonis. While LsNav 1.1 and LsNav 1.2 lacked nucleotide polymorphisms showing association to resistance, LsNav 1.3 showed a non-synonymous mutation in S5 of DII occurring in deltamethrin-resistant parasites. The mutation is homologous to a previously described kdr mutation (I936V, numbering according to Musca domestica Vssc1) and was present in two pyrethroid-resistant L. salmonis strains (allele frequencies of 0.800 and 0.357), but absent in two pyrethroid-susceptible strains. CONCLUSIONS The present study indicates that a kdr-mutation in LsNaV 1.3 may contribute to deltamethrin resistance in L. salmonis.