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The effects of sea lice on the marine survival of wild salmonids are widely debated. In Norway this debate has reached a crescendo as the Norwegian government has recently ratified a management system where the growth in the salmonid aquaculture industry will be conditional on regional estimated impact of salmon lice on wild fish. Sea lice have thus become the most prominent obstacle to the stated political aim of quintupling aquaculture production in Norway by 2050. Scientific documentation that salmon lice impact the marine survival of salmon is robust. However, it is also evident that marine survival of salmon is strongly impacted by other factors, and that the effect of salmon lice is most likely an integral part of these other mortality factors. In this paper, our goal is to discuss and give advice on how managers and policy makers should handle this complexity, and to identify the greatest challenges in using scientific results to construct robust management rules. Inadequate extrapolation from the scale of known effects to the scale of management implementation may initially give a false impression of scientific certainty, but will eventually fuel upsetting disagreements among stakeholders as they gradually uncover the shaky foundation of the implemented policy. Thus, using a single model and parameter to determine management advice is not warranted, as no single data point reflects the natural complexity of nature. Furthermore, robust management rules should be based on unambiguous definitions of key concepts. Finally, despite the scientific consensus that salmon lice are a risk to salmon, studies on wild populations in situ that accurately quantify the impact of salmon lice are still urgently needed. We give advice on how this can be accomplished.

Wrasse (Labridae) species have been used as parasite cleaners in Atlantic salmon farming since the 1980s. However, their use has recently escalated, with millions now being introduced into salmon cages each year. Most fish are of wild origin, their exploitation potentially impacting native populations. Genetic information is urgently required to inform management decisions. We identified 174 microsatellite and 149 SNP markers from ddRAD sequence data. From these, 17 and 48 microsatellite and SNP markers, respectively, were validated by genotyping 150 goldsinny wrasse collected from five locations along the Norwegian and Swedish coasts. Two to 30 alleles were identified at the microsatellite loci, while gene diversity (H e ) ranged 0.101–0.907. All SNP loci were biallelic, with averaged H e per locus ranging between 0.063 and 0.495.

Wild goldsinny wrasse Ctenolabrus rupestris, corkwing wrasse Symphodus melops and ballan wrasse Labrus bergylta were collected at 8 sampling sites in Sweden and Norway during summer 2014. Brain tissue from 466 wrasses were analyzed for nervous necrosis virus (NNV) infections by real-time RT-PCR, and positive samples were subjected to sequencing and phylogenetic analysis of partial segments of the RNA2 and RNA1 genes. This study shows that NNV is present in wild ballan, corkwing and goldsinny wrasse along the coastline of Sweden and Norway. The overall prevalence in the sampled labrids was 6.7%. Prevalence was 6.4% in goldsinny, 6.3% in corkwing and 18% in ballan wrasse. The wrasse RNA2 NNV sequences revealed high genetic variability and were divided into 3 clusters within the cold water barfin flounder NNV (BFNNV) and warm water cluster red-spotted grouper NNV (RGNNV) genogroups. Within the BFNNV genogroup, wrasse NNVs clustered in 2 sub-genogroups, with grey mullet NNV (GMNNV) and with Atlantic halibut NNV (AHNNV). These groups were previously dominated by virus originating from Atlantic cod Gadus morhua and Atlantic halibut Hippoglossus hippoglossus from the northeast Atlantic. The presence of NNV in wild wrasse and the surprising high genetic variability observed in this study should be considered before moving wild-caught wrasse between geographically distant sites. The results show that use of wild-caught wrasse as brood fish in wrasse farming represents a risk of introducing NNV into aquaculture.

The salmon industry is heavily dependent on wrasse for delousing infected fish. The goldsinny wrasse is numerically the most important, and each year, millions are harvested from the wild and transported large distances into fish farms. Population genetic knowledge is required to sustainably exploit this species. Here, 1051 goldsinny wrasses from 16 locations across Scandinavia, the British Isles, and Spain were genotyped with 14 microsatellite and 36 SNP markers. Within-population genetic diversity decreased towards north, and a genetic break was observed across the North Sea. Samples from Northern Norway differed from rest of the Scandinavian samples, and samples from the British Isles differed from the Spanish ones. Within Scandinavia, isolation-by-distance was detected. Observed genetic patterns fitted well with expectations derived from oceanographic drift simulations. A sample from mid-Norway deviated from these patterns however, and was genetically very similar to southern Scandinavian samples. We conclude that the population structure of this species is primarily determined by the opposing evolutionary forces of passive drift, limited adult migration and spawning-site fidelity, whereas the deviation in isolation-by-distance observed in mid-Norway is potentially caused by inadvertent translocations of wrasse from southern Scandinavia via current aquaculture practise. Inclusion of outlier loci gave greater resolution, suggesting that diversifying selection may also affect population structuring among goldsinny wrasses. publishedVersion

The different stunning methods for Atlantic salmon can still be improved with regard to animal welfare. Salmon exposed to carbon monoxide expressed no aversive reactions towards CO as such. CO exposed fish showed an earlier onset of rigour mortis and a faster decrease in muscle pH due to depletion of oxygen during the treatment. Exposure to CO did increase the level of cortisol compared to undisturbed control fish, but the increase was less than in the water only control group. Neuroglobin, a CO binding globin, was found in salmon brain and Saccus vasculosus, a richly vascularized sac connected to the fish brain. Binding of CO to neuroglobin during sedation might possibly improve animal welfare.

Atlantic salmon were exposed to carbon monoxide (CO) before the fish were percussively killed and gill cut. The fish were compared against a control group treated identically, without CO. Salmon exposed to CO expressed no adverse reactions and were easily stunned by percussion. CO-treated salmon had an earlier onset of rigor mortis and a faster decrease in muscle pH than the control group. No significant difference in drip loss was found between salmon treated with CO and the control. A significantly deeper red colour of both gills and fillets of CO-treated salmon was observed 10 days post mortem. Significantly higher levels of plasma lactate and potassium were found in CO-treated salmon compared to control, as well as a lower level of pCO2. Exposure to CO did not increase plasma cortisol, sodium, haematocrit or glucose; however, lactate was high. Exposure of salmon or other fish to CO could improve quality and welfare when slaughtered.

The use of carbon monoxide (CO) to anesthetize fish is not common. However, CO is an efficient sedative that does not produce aversive reactions in fish. Combined with other slaughter methods (electrical stunning), CO is a promising candidate for future and humane fish slaughter. New data suggest that CO causes brain death due to oxygen displacement in the heme-groups of neuroglobin and Saccus vasculosis , two recently discovered brain components in fish. CO enhances flesh color by preventing discoloration caused by myoglobin and hemoglobin oxidation, and may improve quality in salmon and white fish. Using filtered smoke with CO, and CO packaging, favor quality. CO inhibits bacterial growth and reduces the contribution of heme-proteins to lipid oxidation. Chromatographic measurement of CO content in water enables continuous monitoring capabilities and excellent research possibilities. The effects of CO on fish quality can be assessed using Visible and Near Infrared Reflection spectrophotometry.

The salmon louse Lepeophtheirus salmonis (Kroyer, 1838) is a marine ectoparasitic copepod which feeds on skin, mucus and blood from its salmonid hosts [1,2]. The salmon louse is a major disease problem for the salmon farming industry in Norway, and also constitutes a considerable threat to wild salmonids. The present therapeutic treatment available to the aquaculture industry has several limitations, such as possible emergence of resistance [3], re-infection and environmental impacts. Alternatives are, therefore, sought, and an intimate knowledge of the molecular biology of the salmon louse is essential in order to understand the parasite–host interaction. The salmon louse feeds on a protein rich diet and adult female lice (taken from farmed salmon) frequently possess blood in the gut [2]. A strategy repressing protein digestion may be a way to arrest parasitic propagation. This approach will be especially attractive if blood is a pre-requisite for egg production. In species that feed on high protein food, for example, blood eating insects and parasites, proteolytic enzymes play a crucial role in digestion. This was highlighted in a recent publication [4] which reported an expanded serine protease superfamily in the Anopheles gambiae genome, compared to the carbohydrate feeding Drosophila melanogaster [5]. Trypsin-like proteases (EC 3.4.21.4) are secretory endopeptidases within the serine protease superfamily [6]. The trypsins are important digestive enzymes, constituting a ma

Four novel trypsin-like S1A peptidase transcripts (LsTryp2–5) from the marine parasitic copepod Lepeophtheirus salmonis were characterised based on analyses of 1918 expressed sequence tags from two adult female libraries. In addition, one previously described salmon louse trypsin, LsTryp1, has been further characterised. The five peptidases possessed all residues typically found in trypsins in correct sequence contexts. Interestingly, two cysteine residues, possibly involved in a disulphide bridge not previously reported in trypsins are conserved in all louse trypsin sequences. Phylogenetic analyses showed that the five louse peptidases form a monophyletic group with other crustacean trypsins (Brachyurin Ts). Quantitative PCR analyses demonstrated increased transcript levels from planktonic to early host-attached stages and from preadult to sexually mature adult stages. Furthermore, sex-specific differences in transcription regulation were found. In situ hybridisation demonstrated that all five trypsin-like peptidases are transcribed throughout the undifferentiated midgut, indicating a digestive function. The sequence characteristics, histological localisation and transcript regulation suggest that LsTryp1–4 encode typical digestive trypsins. LsTryp5, however, showed some sequence and regulatory peculiarities that rendered its function less clear. Our findings support earlier suggestions for the function of the midgut cells and suggest the existence of an additional undifferentiated cell-type.

The Atlantic salmon, Salmo salar L, is an important species both for traditional fishery and fish farming. Many Atlantic salmon stocks have been declining and a suspected main contributor to this decline is the salmon louse (Lepeophtheirus salmonis); a parasitic copepod living off the salmonid hosts epidermal tissues and blood. Contributing to the growing body of knowledge on the molecular biology of the salmon louse we have utilized a microarray containing 11,100 salmon louse genes to study the gene expression patterns in selected tissues. This approach has yielded information about potential functions of the transcripts and tissues. Microarray analyses were preformed on subcuticular and frontal (neuronal and gland enriched tissue) tissues, as well as gut, ovary and testes of adult lice. Tissue specific transcriptomes were evident, allowing us to address main traits of functional partitioning between tissues and providing valuable insight into the biology of the louse. The results furthermore represent an important tool and resource for further experiments.

The effect of carbon monoxide (CO) as stunning method in Atlantic salmon (Salmo salar L.) on stress indicators (adrenaline, A; noradrenaline, NAD) and on fillets quality during the shelf life has been investigated. The CO was dissolved into tanks with salmon for 8 and 20 min to obtain fish groups CO8 and CO20, respectively. These groups were compared to a non-stressed control group (C). All the fish were hauled out from the tank and killed by percussion. Adrenaline content of CO20 group was 1.8 and 1.7-fold higher than CO8 and C groups respectively (P

Norwegian aquaculture has grown from its pioneering days in the 1970s to be a major industry. It is primarily based on culturing Atlantic salmon and rainbow trout and has the potential to influence the surrounding environment and wild populations. To evaluate these potential hazards, the Institute of Marine Research initiated a risk assessment of Norwegian salmon farming in 2011. This assessment has been repeated annually since. Here, we describe the background, methods and limitations of the risk assessment for the following hazards: genetic introgression of farmed salmon in wild populations, regulatory effects of salmon lice and viral diseases on wild salmonid populations, local and regional impact of nutrients and organic load. The main findings are as follows: (i) 21 of the 34 wild salmon populations investigated indicated moderate-to-high risk for genetic introgression from farmed escaped salmon. (ii) of 109 stations investigated along the Norwegian coast for salmon lice infection, 27 indicated moderate-to-high likelihood of mortality for salmon smolts while 67 stations indicated moderate-to-high mortality of wild sea trout. (iii) Viral disease outbreaks (pancreas disease, infectious pancreatic necrosis, heart and skeletal muscle inflammation, and cardiomyopathy syndrome) in Norwegian salmon farming suggest extensive release of viruses in many areas. However, screening of wild salmonids revealed low to very low prevalence of the causal viruses. (iv) From ∼500 yearly investigations of local organic loading under fish farms, only 2% of them displayed unacceptable conditions in 2013. The risk of eutrophication and organic load beyond the production area of the farm is considered low. Despite several limitations, especially limited monitoring data, this work represents one of the world’s first risk assessment of aquaculture. This has provided the Norwegian government with the basis upon which to take decisions for further development of the Norwegian aquaculture industry.

Atlantic salmon post-smolts were exposed to either chronic hypoxic (Hy) or normal oxygen (No) conditions in seawater tanks for 58 days, mimicking conditions typical of sea cages for farmed salmon at some periods of the year. By day 29 head kidney macrophages were isolated and subjected to in vitro poly I:C stimulation to simulate viral infection, and samples were collected over 48 h. By day 58 fish were subjected to in vivo stimulation using poly I:C or a Vibrio water-based vaccine to simulate viral or bacterial infection, respectively. The fish were monitored for stress responses and expression of several pro-inflammatory genes in head kidney and intestinal tissue up to five days post-injection. Stress load was monitored by plasma cortisol estimation at days 29 and 58, and on days 1, 2, 3 and 5 post-injection in the in vivo trial. Hy exposure resulted in elevated plasma cortisol levels on day 29 compared to No, while on day 58 cortisol levels were higher in the control group. Additionally, both poly I:C and the Vibrio vaccine gave significantly increased cortisol levels one day post-injection compared to PBS treated controls, irrespective of previous oxygen exposure. In vitro stimulation of macrophages with poly I:C revealed higher IFNα mRNA levels at 6, 12 and 24 h and for Mx at 12 and 24 h post-stimulation, for both No and Hy individuals. Moreover, IFNα levels were higher in No than in Hy individuals at all time points, and a similar difference was seen in Mx at 48 h. In vivo stimulation with poly I:C elicited strong elevation of the IL-1β, IFNγ, Mx and IP10 mRNA transcripts in head kidney, while TNFα1 and IFNα were found unaffected. The Vibrio vaccine elicited a strong up regulation of IL-1β, IFNγ and IP10 mRNA, whereas Mx, TNFα1 and IFNα appeared unchanged. Significant differences in expression between different oxygen exposure groups were found for all genes and both stimuli. The overall trend suggests that long-term hypoxia either reduces or delays the expression of these genes in head kidney. Expression of IFNγ and Mx in intestinal tissues also showed a strong up regulation of the genes following poly I:C stimulation, and also here the overall trend suggests that chronic hypoxia results in a lower or delayed expression of the measured genes. In summary, our results indicate that chronic hypoxia modulates the expression of important immune related genes putatively altering the immune response. As the effect is present in isolated macrophages as well as head kidney and intestinal tissue the modulation appears to be affecting local as well as systemic responses.

Neuroglobin (Ngb) exists only in small amounts in salmon brain. In order to study the protein in more detail salmon neuroglobin (sNgb) was cloned, hetereologously expressed in E.coli and purified. The protein had red color and showed the characteristic peaks at 411 nm (metNgb), 415 nm (carboxyNgb) and 424 nm (deoxyNgb). Western analysis showed that sNgb reacted weakly against a rabbit anti human neuroglobin (hNgb) and strongly to a sNgb specific antibody. Our 3Dhomology model of the sNgb indicated modifications adjacent to and in the O2/CO binding site. This may correlate to differences in substrate affinities for the sNgb compared to the hNgb. Also sNgb contained shorter helixes and longer interhelical loops typical for psycrophilic proteins. submittedVersion

Journal homepage: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1439-0426 Vertebral body compression is a common problem in commercial farming of Atlantic salmon. Although risk factors, such as vaccination and alnutrition, have been identified, the etiology is largely unknown. Histological studies of Atlantic salmon have shown that in a compressed deformity (platyspondyly) the length of the compact bone is reduced while the notochord start to form atypical chrondrogenic structures. In mammals, similar remodeling activities have been linked to inflammatory processes in the tissue. Hence, we wanted to investigate whether the compressed vertebrae in Atlantic salmon showed presence of local (IL-1β, TNF-α1), systemic (IgM) and chronic (MMP-13, MMP-9) immune responses (measured with quantitative PCR). Unvaccinated groups of Atlantic salmon that would later develop high or low prevalence of vertebral compression during on-growth in seawater were sampled at seawater transfer, and 3 and 6 weeks after seawater transfer. In addition, compressed and normal vertebrae from the high deformity prevalence group were sampled 44 weeks after transfer to seawater. MMP-13 was significantly up-regulated in the group that developed a high prevalence of deformity, and also significantly up-regulated in compressed vertebrae, 44 weeks after seawater transfer. In compressed vertebrae, MMP-13 was equally up-regulated in the notochord, compact bone and trabecular bone. The results of the present study suggest that MMP-13 may serve as an early indicator for bone remodeling which may lead to vertebral compression, and that there is a relationship between the development of vertebral compression and increased remodeling activities in farmed Atlantic salmon.

Trypsins constitute a subclass of the S1A family of serine peptidases found in all groups of animal and some bacteria. At present, no information about the genomic organisation of trypsins is available for copepods. The only data of copepod trypsins indicate several different trypsins in the marine parasitic copepod Lepeophtheirus salmonis. In the present study, 31.7 kbp of genomic DNA surrounding the previously described LsTryp1-5 sequences was sequenced. The sequenced regions contain nine full-length and three partial trypsin genes. A conservative estimate based on PCR analysis and genomic sequence indicated at least 22 different trypsin genes in L. salmonis, of which 18 are most similar to the previously described LsTryp1 and -2 cDNA sequences. Four of these genes are putative pseudogenes. In addition, a putative mariner like transposase gene was identified. The genomic sequences suggest that the L. salmonis trypsin genes reside within one or more gene clusters. Three different LsTryp intron exon structures were identified, and all three are different from the intron exon organisation previously reported for other S1A peptidases. This implies several intron loss and gain events in the evolution of the L. salmonis trypsin genes.

Background: Ten generations of domestication selection has caused farmed Atlantic salmon Salmo salar L. to deviate from wild salmon in a range of traits. Each year hundreds of thousands of farmed salmon escape into the wild. Thus, interbreeding between farmed escapees and wild conspecifics represents a significant threat to the genetic integrity of wild salmon populations. In a previous study we demonstrated how domestication has inadvertently selected for reduced responsiveness to stress in farmed salmon. To complement that study, we have evaluated the expression of seven stress-related genes in head kidney of salmon of farmed, hybrid and wild origin exposed to environmentally induced stress. Results: In general, the crowding stressor used to induce environmental stress did not have a strong impact on mRNA expression levels of the seven genes, except for insulin-like growth factor-1 (IGF-1) that was downregulated in the stress treatment relative to the control treatment. mRNA expression levels of glutathione reductase (GR), Cu/Zn superoxide dismutase (Cu/Zn SOD), Mn superoxide dismutase (Mn SOD), glutathione peroxidase (GP) and IGF-1 were affected by genetic origin, thus expressed significantly different between the salmon of farmed, hybrid or wild origin. A positive relationship was detected between body size of wild salmon and mRNA expression level of the IGF-1 gene, in both environments. No such relationship was observed for the hybrid or farmed salmon. Conclusion: Farmed salmon in this study displayed significantly elevated mRNA levels of the IGF-1 gene relative to the wild salmon, in both treatments, while hybrids displayed a non additive pattern of inheritance. As IGF-1 mRNA levels are positively correlated to growth rate, the observed positive relationship between body size and IGF-1 mRNA levels detected in the wild but neither in the farmed nor the hybrid salmon, could indicate that growth selection has increased IGF-1 levels in farmed salmon to the extent that they may not be limiting growth rate. publishedVersion

Background Trypsin-like serine proteases are involved in a large number of processes including digestive degradation, regulation of developmental processes, yolk degradation and yolk degradome activation. Trypsin like peptidases considered to be involved in digestion have been characterized in Lepeophtheirus salmonis. During these studies a trypsin-like peptidase which differed in a number of traits were identified. Results An intronless trypsin-like serine peptidase (LsTryp10) from L., salmonis was identified and characterized. LsTryp10 mRNA is evenly distributed in the ovaries and oocytes, but is located along the ova periphery. LsTryp10 protein is deposited in the oocytes and all embryonic cells. LsTryp10 mRNA translation and concurrent degradation after fertilization was found in the embryos demonstrating that LsTryp10 protein is produced both by the embryo and maternally. The results furthermore indicate that LsTryp10 protein of maternal origin has a distribution pattern different to that of embryonic origin. Conclusion Based on present data and previous studies of peptidases in oocytes and embryos, we hypothesize that maternally deposited LsTryp10 protein is involved in regulation of the yolk degradome. The function of LsTryp10 produced by the embryonic cells remains unknown. To our knowledge a similar expression pattern has not previously been reported for any protease.