Over the past few years one of the major concerns in the Atlantic salmon (Salmo salar) farming industry has been the increasing incidence and severity of inflammatory viral diseases. Heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS) are currently two of the most prevalent viral diseases in commercial Atlantic salmon farms in Norway. Mortality levels in both diseases are generally low but morbidity can be very high with the associated chronic inflammatory response lasting for several months. The consequent reduced growth performance is causing considerable financial impact as HSMI has become increasingly widespread in recent years. The impact of CMS is further exacerbated as it generally affects large fish close to harvest. HSMI lesions occur in the atrium and ventricle in the heart including inflammation and necrosis in epi- endo- and myocardium along with myositis of red skeletal muscle. CMS lesions are commonly observed in the spongy myocardium in the atrium and ventricle of the heart with severe mononuclear inflammation and necrosis. Furthermore, circulatory disturbances associated with reduced cardiac function cause multifocal liver steatosis and necrosis in both diseases. Currently there are no vaccines or any other effective treatments for these diseases and so alternative therapies that could potentially modulate the intensity of the inflammatory response could be crucial to improve the clinical manifestation of the diseases. Therefore, the overall aim of the present study was to evaluate the concept of “clinical nutrition” to improve the clinical symptoms of both viral diseases, HSMI and CMS, through the use of functional feeds formulated with reduced lipid content and increased proportions of anti-inflammatory fatty acids to moderate the apparently uncontrolled inflammatory response in the heart tissue associated with both diseases and also alleviate the secondary hepatic lesions. The experimental work consisted of three major dietary trials in Atlantic salmon in seawater. Two large trials investigated the effects of functional feeds in Atlantic salmon challenged with Atlantic salmon piscine reovirus (ASRV) and piscine myocarditis virus (PMCV), the causal agents of HSMI and CMS, respectively. In both trials, heart transcriptome, heart and liver histopathology and tissue lipid and fatty acid compositions and metabolism were determined post-infection in fish fed with the functional feeds in comparison with fish fed with a standard commercial feed formulation considered as a reference diet. All the functional feeds were formulated to have reduced digestible energy through lower dietary lipid and higher protein contents, and increased levels and proportions of anti-inflammatory long-chain polyunsaturated fatty acids (LC-PUFA), particularly eicosapentaenoic acid (EPA) compared with the reference diets. Histopathology, fatty acid composition and gene expression of heart were assessed over a long time-period of 16 weeks and 14 weeks post-challenge with ASRV and PMCV, respectively. Viral load in heart tissue, hepatic histopathology and fatty acid composition of liver and head kidney along with expression of the genes involved in the eicosanoid and LC-PUFA and eicosanoid biosynthesis pathways were also determined in the HSMI trial. The third trial was a nutritional trial evaluating the effects of dietary digestible energy content on lipid and fatty acid metabolism in salmon fed diets containing graded amounts of lipid. Fatty acid composition of liver and heart were assessed over 12 weeks, along with the hepatic expression of genes of lipid and fatty acid metabolism. The results of this research are presented in four chapters (Chapters 2-5) as four paper manuscripts. The manuscripts/Papers are either published (Chapter 2), in review (Chapter 3 and 4) or drafted for submission (Chapter 5) in appropriate peer-reviewed international journals. Chapter 2 and 3 correspond to the HSMI trial, Chapter 4 to the nutritional trial, and Chapter 5 to the CMS trial. Chapter 2 showed that viral load and histopathology scores were lower in fish fed the functional feeds, especially diet FF1, which displayed better performance. Diet strongly influenced the expression of genes related with the immune and inflammatory responses, with delayed expression in fish fed the functional feeds. Up-regulation of pro-inflammatory genes was correlated with the higher viral load observed at early-mid stages of the disease in fish fed the reference diet (ST). Expression of genes related with the immune response at 16-weeks post challenge reflected the differences in immunomodulation between the functional feeds, with fish fed diet FF1 showing lower expression. Therefore, severity of the heart lesions was correlated with the intensity of the immune response and could be associated with tissue anti-inflammatory LC-PUFA levels. Chapter 3 was focused on liver histopathology, fatty acid composition and LC-PUFA biosynthesis, along with phospholipid fatty acid composition and eicosanoid production in head kidney and heart tissue at early and late stages of ASRV infection. Liver was severely affected by the virus at the beginning of the infection in fish fed the reference ST diet, but the level of lesions were similar in all dietary groups at the end of the trial. Hepatic expression of fatty acyl desaturases was significantly depressed in fish fed the ST diet compare with fish fed the functional feeds despite the lower levels of dietary LC-PUFA in that feed. Thus endogenous production and bioavailability of anti-inflammatory LC-PUFA was potentially enhanced in fish fed the functional feeds. Changes in tissue lipid content, mobilization of fatty acids involved in inflammatory responses and changes in expression of transcription factors and genes involved in eicosanoid biosynthesis were more prominent in head kidney, confirming the important role of this organ in dietary immunomodulation after viral infection. To a lesser extent similar changes were observed in heart tissue, suggesting in situ production of eicosanoids could also be important. The unexpected effects of diet on expression of genes of LC-PUFA biosynthesis were specifically investigated in the trial described in Chapter 4. One aim of this study was to clarify whether dietary lipid content or viral infection was the cause of altered expression of desaturase genes between the different diets. Hepatic expression of other genes of lipid and fatty acid metabolism were also determined to evaluate metabolic changes associated with dietary lipid/energy level. In general, reduction of dietary energy and lipid contents while maintaining similar proportions of dietary fatty acids, led to a general up-regulation of genes involved in lipid biosynthetic pathways. Thus salmon fed lower energy diet showed increased liver expression of fatty acyl desaturases in comparison with fish fed higher energy levels. Heart transcriptomic data in Chapter 5 showed a similar delay in the inflammatory response in fish fed the functional feeds after PCMV infection as observed in the HSMI study. Modulation of inflammatory responses, similar to that previously described after ASRV infection, was also observed in fish fed the functional feeds. However, the differences in the expression of immune related genes and the level of heart lesions were not as prominent at mid-late stages of the disease as in fish fed FF1 in the HSMI trial. The present study demonstrated the beneficial effects of a clinical nutrition approach via functional feeds in two viral inflammatory diseases, HSMI and CMS, currently affecting farmed Atlantic salmon.

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Considering the response of organisms to their environment is difficult; it is made more so if population numbers cannot be closely monitored. In such cases different methods of population assessment are required. This thesis uses lipids as a measure of Atlantic salmon (Salmo salar L.) quality and investigates its usefulness in indicating fish condition. The first study examines the relationship between fish total lipid content and W[sub]R condition factor; this study clearly demonstrates that there is a significant positive relationship between the condition factor of a fish and its total lipid content. In the following study the lipid storage between the different tissues of the Atlantic salmon is considered. This indicates that the red muscle and the adipose tissues hold higher concentrations of lipid than the white muscle. However, the white muscle makes up the majority of lipid tissue mass in the Atlantic salmon so contains the bulk of stored lipid in a fish, at low concentration. The next study investigates the effect of spawning on Atlantic salmon condition. Salmon can be seen preferentially conserving lipid in their musculature and drawing down the lipid stored in their adipose tissues. The following study looked at one key lipid group, triacylglycerides, in salmon. Triacylglycerides are energetically important in fish and this study found that the spawning process depleted triacylglyceride reserves, but that the red muscle conserves triacylglycerides even after spawning. The final study considers the relationship between maternal quality and egg quality, identifying that longer Atlantic salmon produce eggs with more lipid after spawning migration. Egg lipid concentrations were comparably maintained between fish. Monitoring quality in this way is a useful tool to determine population wellbeing and help indicate where populations are compromised.

Flesh colour and fillet fat percentage are the two most important attributes to salmon fillet quality. A medium genetic component to body lipid percentage within commercial lines has previously been shown (h2 = 0.17-0.24). A low level of heritability (h2 = 0.16) has also been reported for flesh colour in Atlantic salmon. To investigate whether this genetic component includes loci of major effect, a genome-wide QTL scan was performed with commercially bred Atlantic salmon (Landcatch Natural Selection). Five large full-sib families (10 parents with 153 offspring) were genotyped using microsatellite markers. To utilize the large difference between sire and dam recombination rate, a two-stage genotyping was employed. Initially, the parents and offspring were genotyped for two microsatellite markers per linkage group, and sire based QTL analysis was used to detect linkage groups with significant effects on those flesh quality traits. A linear-regression based interval as analytical method was applied for QTL detection. The results revealed evidence of QTLs affecting percentage fat percentage and flesh colour on linkage groups LNS16 and LNS1 respectively. To confirm the QTL and to provide an improved estimate of position, a dam-based analysis was then employed. One major QTL was located on the genome-wide significance level for percentage fat percentage. Microsatellite marker Ssa0016NVH (at position of 1.3 cM) was found to be tightly linked to QTL affecting percentage fat percentage. In addition, a QTL affecting flesh colour was found to be flanked by microsatellite markers Ssa9.44NUIG at position of 68.7 cM and Ssa0021NVH at position of 50.6 on linkage group LNS16. The evidence for suggestive QTL affecting flesh colour on linkage group LNS1 was also revealed. In order to increase marker density within these and other linkage groups, AFLP markers were employed, 24 primer combinations resulted in a total of 489 polymorphic fragments. Among 11 fragments that were found to be linked to the microsatellite markers on linkage group LNS16, four fragments (AAG-CAC328, AGG-CAG447, AGG-CTA237 and AGG-CTC237) were tightly linked to microsatellite marker Ssa9.44NUIG, but none were found to be linked to microsatellite Ssa0021NVH. Moreover, none of the AFLP markers were found to be linked to microsatellites residing on linkage group LNS1. Using a constructed map of microsatellite and AFLP markers for linkage group LNS16, the dam based analysis revealed a significant QTL for flesh colour at the location of 189 cM, while the sire based analysis detected a significant QTL for fat percentage at the location of 80 cM. Considering the dominant nature and clustering character of AFLP markers, it was concluded that a certain primer combination in AFLP markers could be of limited use for fine mapping and QTL detection in Atlantic salmon.

The close proximity of net-pen salmon farms and wild Pacific salmon stocks in British Columbia (BC) is an incentive for precautionary management of the environmentally and economically damaging parasites known as sea lice. Bivalves cultured as part of an integrated multi-trophic aquaculture (IMTA) system may contribute natural, preventative louse control through the ingestion of planktonic sea lice larvae. A field trial was conducted to test sea lice mitigation by bivalves at a commercial Atlantic salmon (Salmo salar) farm in BC using Pacific oysters (Crassostrea gigas). Oysters were cultured in trays around one end of the farm and at a reference site approximately 150 m away from August 2013 until August 2014. Parasitic and planktonic sea lice (Lepeophtheirus salmonis and Caligus clemensi) were monitored before and during oyster deployment, beginning in December 2012. Parasite abundance peaked in January 2013 (6.5 lice/fish, >85% C. clemensi), and the following year in February 2014 (3.3 lice/fish, >80% L. salmonis). Larval density within cages peaked in January, both in 2013 (1.28 larvae/m3) and 2014 (0.96 larvae/m3). Parasite abundance was significantly correlated with both surface salinity (r2= 0.28, p=0.04) and sea lice larval density (r2= 0.65, p=0.01). Observed densities were significantly lower (t=3.41, p=0.009) than those calculated for the site based on water temperature and salinity, the number of adult female lice present, and the approximate number of fish. Sea lice mitigation by oysters was assessed by comparing monthly sea lice larval densities inside bivalve and non-bivalve fish cages, and by analyzing preserved oyster digestive tracts from January 2014 (when larval densities were highest) for presence of L. salmonis DNA using PCR. Using these methods, no significant evidence of sea lice mitigation was detected. Oyster growth was monitored by measuring whole wet weight, soft tissue wet, dry, and ash-free dry weight, and shell length, width, and height approximately every four months. Oysters were sampled equally across different sides of the farm and at the reference site (~150 m away from the farm) at three depths: 1, 3, and 6 m. All seven measurements increased significantly over time. Effects of side and depth varied by growth parameter; in general, oysters at 1 and 3 m were significantly larger than those at 6 m, and oysters cultured at the reference site were either significantly smaller or the same size as those cultured around the farm. Oysters from select sides were consistently, significantly larger than those from other sides and from the reference site. Overall, the findings suggest that sea lice larvae quickly dispersed away from the farm after hatching and were not significantly impacted by bivalve presence around the fish cages. Bivalves grew significantly larger over time and size was significantly impacted by both depth and side of the fish cage. While no evidence of larval sea lice reduction/ingestion by cultured bivalves was detected, this study provides information on all sea lice stages present throughout an Atlantic salmon production cycle, as well as the first detailed growth analysis of Pacific oysters cultured alongside farmed Atlantic salmon in BC. / Graduate / 0792 / byrneaa@gmail.com

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The efficacy of a range of anti-parasitic chemotherapeutic agents against the salmon louse Lepeophtheirus salmonis following topical application was studied in vitro and in vivo. In general, adult and preadult lice were susceptible to a wide range of compounds with dose rates, following 1 hour exposures, ranging several orders of magnitude (10.0 - 0.001 mg/L). Overall the pyrethroid compounds which were tested were found to have the widest therapeutic ratios, indicating the potential of this group of chemotherapeutants for sea lice control. Resistance to the organophosphorus (OP) compounds dichlorvos and azamethiphos was detected in isolated populations of lice. Field trials with azamethiphos indicated that the compound was highly efficacious against sensitive lice (@ 0.1 mg/L; however, where resistance was present, efficacy (@ 0.2 mg/L) was highly variable. When used at the above dose rates, azamethiphos was found to be well tolerated by fish as indicated by a lack of significant brain acetylcholinesterase inhibition. Results on cross resistance (between pyrethroids and OPs) were inconclusive which was believed to be, primarily, due to the overall high toxicity of the group; but also to the variable responses from exposed lice. In a series of preliminary trials, one of the pyrethroid compounds, PHRDL-D, was found to effectively remove lice when administered orally to infected salmon, indicating the potential of pyrethroids as oral chemotherapeutants. A comparison of the relative toxicity of azamethiphos (OP), resmethrin (pyrethroid), ivermectin (avermectin) and the structurally similar compound SKB7 (milbemycin), indicated that chalimus stages were only susceptible to ivermectin and SKB7 following topical and intra-peritoneal injection to lice infected fish. In contrast, azamethiphos and resmethrin were found to be non toxic to chalimus larvae at dose rates which were highly toxic to both adult lice and treated fish. Preliminary studies on the uptake of r4C]azamethiphos in adult lice indicated that uptake was both concentration and time dependant, reaching a plateau at the onset of toxicity. Uptake appeared to be primarily associated with frontal plates, 1st antennae and anus. The fmdings indicated that several compounds/compound classes are highly active against lice and, given the limited number of compounds available for sea lice control and the development of resistance to OPs, might be considered as alternatives. In light of these findings, the potential of chemotherapy for the future control of sea lice is discussed.

The main parasite affecting the Scottish Atlantic salmon farming industry is Lepeophtheirus salmonis costing approximately £29 million annually through lost production and treatments. As such, the primary aim of this study was to investigate the infection pressure, fecundity and infection dynamics of L. salmonis in a west coast sea loch containing salmonid aquaculture. Atlantic salmon held in small sentinel cages were used to monitor sea lice infection pressure in Loch Shieldaig. Investigation of the factors affecting infection pressure within the loch found no evidence of a direct link between monthly gravid lice counts on local farmed salmon and lice burdens on sentinel cage fish. Copepodid L. salmonis were present throughout the year despite there being low numbers of wild salmonids in the system during much of the year and as such the source of this infection was unclear. In subsequent laboratory trials, L. salmonis from wild salmon were found to be more fecund than those from farmed salmon and similarly, fecundity and survival were also higher in summer compared to winter populations of L. salmonis. These findings raise the possibility that L. salmonis infecting wild salmonids may “seed” previously fallowed systems. Alternatively, trials with atypical hosts found that L. salmonis of farmed origin did re-infect Atlantic cod and produce viable egg strings which moulted through to the infective copepodid stage. Infection challenges demonstrated that L. salmonis copepodids were observed to settle on atypical hosts such as saithe and Atlantic cod in low numbers although failed to develop to the chalimus stages and complete its lifecycle. Challenges utilising the mobile pre-adult stage were similarily unsuccesful in allowing L. salmonis to moult to the adult stage on these atypical hosts. Previous work conducted on Pacific three-spined sticklebacks demonstrated the species could support large burdens. In infection trials with three-spined sticklebacks from Scotland’s west coast, settlement was found to be low, which suggests they play no role in the Scottish inshore infection dynamics of L. salmonis. These series of studies provide novel and timely data on the biology and infection dynamics of L. salmonis on typical and atypical hosts in Scottish marine aquaculture areas. The data will form part of the knowledge used to make informed pest management and policy decisions assisting the future development of the aquaculture industry in Scotland.

The present work reports on extensive plankton sampling surveys in a Scottish sea loch used by wild and farmed salmonids and describes the spatial and temporal distribution of planktonic larval sea lice and identifies factors which affect the larval distribution. The planktonic louse larvae recovered were predominantly <i>Lepeophtheirus salmonis</i> (Krøyer) and so the findings of this thesis refer primarily to this species of sea louse. Copepodids occurred in significantly greater densities at 0 m than at a depth of 5 m, whereas nauplii occurred in significantly greater densities at 5 m than at 0 m depth. Densities of caligid nauplii and <i>L. salmonis</i> copepodids recovered from the open-waters of Loch Shieldaig correlated significantly with the counts of gravid <i>L. salmonis</i> on farmed Atlantic salmon at the farm in Loch Shieldaig. Furthermore, the densities of <i>L. salmonis</i> copepodids were significantly correlated  with the estimated total numbers of gravid <i>L. salmonis</i> on all farmed Atlantic salmon in the Loch Torridon management area. Densities of <i>L. salmonis</i> copepodids did not correlate with estimated numbers of gravid <i>L. salmonis</i> on wild salmonids. Atlantic salmon farms were identified as important sources of <i>L. salmonis</i> larvae and these larvae can be transported several kilometres from the point of release. Husbandry louse control practices were indicated to be able to significantly reduce the densities of <i>L. salmonis</i> larvae in the water column. These findings support the principle of synchronised sea louse management at a hydrographic/management area level.