In this study, full coding sequences of Atlantic salmon (Salmo salar L.) muscle genes were cloned, including myogenic regulatory factors (MRFs) (myod1c, myog, mrf4, myf5), inhibitors of Myostatin (fst, decorin), markers of myogenic progenitor cell (MPC) proliferation (sox8) and fusion (calpastatin), a marker of slow muscle fibre differentiation (smlc1) and a novel eukaryotic gene involved in regulating growth (cee). Several of these genes were then characterised using a range of experimental and computational analyses with the aim to better understand their role in myogenesis and their evolution in teleosts. A series of experiments supported previous findings that teleosts have extra copies of many genes relative to tetrapods as a result of a whole genome duplication (WGD) event that occurred some 320-350 Mya. For example, it was shown that genes for myod and fst have duplicated in a common teleost ancestor, but were then specifically lost or retained in different lineages. Furthermore, several characterised Atlantic salmon genes were conserved as paralogues, likely from a later WGD event specific to the salmonid lineage. Phylogenetic reconstruction and comparative genomic approaches were used to characterise the evolution of teleost paralogues within a framework of vertebrate evolution. As a consequence of one experiment, a revised nomenclature for myod genes was proposed that is relevant to all diploid and polyploid vertebrates. The expression patterns of multiple myogenic genes were also established in Atlantic salmon embryos using specific complementary RNA probes and in situ hybridization. For example, co-ordinated embryonic expression patterns were revealed for six salmon MRFs (myod1a, myod1b, myod1c, myog, mrf4, myf5), as well as markers of distinct MPC populations (pax7, smlc1), providing insight into the regulatory networks governing myogenesis in a tetraploid teleost. Furthermore, it was shown that Atlantic salmon fst1 was expressed concurrently to pax7 in a recently characterised MPC population originating from the anterior domain of the epithelial somite, which is functionally analogous to the amniote dermomyotome. In another experiment, the individual expression domains of three Atlantic salmon myod1 paralogues were shown to together recapitulate the expression of the single myod1 gene in zebrafish, consistent with the partitioning of ancestral cis-acting regulatory elements among salmonid myod1 duplicates. Additionally, the in situ expression of cee a novel and highly conserved eukaryotic gene was revealed for the first time in a vertebrate and was consistent with an important role in development including myogenesis. Additionally, Atlantic salmon were reared at 2, 5, 8 or 10 ºC solely to a defined embryonic stage, which was just subsequent to the complete pigmentation of the eye. After this time, animals were provided an equal growth opportunity. Remarkably, changing temperature during this short developmental window programmed the growth trajectory throughout larval and adult stages. While 10 and 8 ºC fish were larger than those reared at 2 and 5 ºC at the point of smoltification, strong compensatory growth was subsequently observed. Consequently, after 18 months of on growing, size differences among 5, 8 and 10 ºC fish were not significant, although each group was heavier than 2 ºC fish. Furthermore, significant embryonic-temperature induced differences were observed in the final muscle fibre phenotype, including the number, size distribution and myonuclear density of muscle fibres. A clear optimum for the final muscle fibre number was observed in 5 ºC fish, which was up to 17% greater than other treatments. In a sub-sample of embryos, temperature induced heterochonies were recorded in the expression of some MRFs (myf5, mrf4) but not others (myod1a, myog). These results allowed the proposition of a potential mechanism explaining how temperature can program the muscle phenotype of adult teleosts through modification of the somitic external cell layer, a source of MPCs throughout teleost ontogeny.

The objective of the work described in the current thesis was to provide a better understanding of some of the key factors associated with sea louse, Lepeophtheirus salmonis, infection of farmed Atlantic salmon. In Chapter 2, initial work focused on establishing the vertical and horizontal distribution of sea lice copepodids and spatial patterns of on-farm infections. The louse distribution was investigated along the main current gradient across adjacent salmon production pens at three commercial sites. A depth profile for the distribution of larval lice was also established for the top 12 m of the water column at three different locations in close proximity to commercial salmon farms. Within all multi-pen sites there were clear patterns of distribution and infection along the main water current gradient with the abundance of lice in end pens at each site appearing to be different from the central pens. The vertical distribution pattern of free swimming L. salmonis larvae (nauplii and copepodids) showed that the surface 6 m harboured 85.5 ± 1.6 % of the lice present in the water body analysed (0 – 12 m depth), irrespective of sampling date and location. In Chapter 3, further environmental effects / influences on attachment success of the copepodids were analysed using controlled infection challenges. A flume with adjustable flow rates, and controlled light conditions was designed for this study. Flume current velocity was observed to be a significant factor in infection success, with higher infection levels observed at lower current velocities, while higher current velocities were demonstrated to reduce settlement success. At fixed velocity, higher copepodid exposure levels gave rise to higher infection levels, this having a linear relationship suggestive of a lack of competitive effects for space on the fish. Light was also shown to play an important role in host settlement. A positive correlation between increasing light intensity and higher louse attachment success was found for all tested light spectra / wavelengths (white - Halogen, blue 455 nm, green 530 nm and red 640 nm). Observation of an infecting cohort of copepodids showed maximal infection at four days post-moulting with a tail-off of infection by six days post-moulting. However, even under the optimal conditions represented by a flume challenge, including linear water flow, the constraint of copepodids to pass close to the salmon host and the very high exposure levels of copepodids per fish, louse attachment success was still relatively low. Chapter 4 examined implementation of a possible management approach based upon some of the environmental influences observed. This chapter described a study in which environmental manipulation of salmon swimming depth was employed on-site in an attempt to reduce farm infection of Atlantic salmon. The effects of submerged artificial lighting in combination with submerged feeding were tested with respect to salmon swimming depth and sea lice infection, following the hypothesis that L. salmonis infection in a commercial salmon population could be reduced through exposure to deep lighting and feeding. The results of the study suggest that swimming depth manipulation can indeed be used at a commercial scale to reduce salmon lice burdens on Atlantic salmon by physically minimising spatial interactions between the two animals. In the final research chapter (Chapter 5), this thesis examines the question of whether ploidy of the host impacts on sea louse infection levels and whether susceptibility of individual fish is consistent between replicate infections. Results showed that triploid salmon are not subject to higher sea louse infection levels under experimental challenge and farm infection conditions compared to diploid hosts. In addition, triploid fish subject to initial infection, did not become more or less resistant to infection compared to diploids when comparing repeated sea louse infections. In summary, this thesis describes work conducted to analyse key infection pathways and factors influencing infection of Atlantic salmon by sea lice and suggestions made as to how findings may be exploited to reduce louse burdens in Atlantic salmon farming. The practical solutions presented to exploit the results found in this work are currently under consideration by the Scottish salmon industry.

In 2005, Atlantic salmon (Salmo salar L.) migrating to the United Kingdom exhibited swollen, haemorrhagic vents, symptoms not previously recorded. The condition was latterly termed Red Vent Syndrome (RVS), and subsequently observed across the North Atlantic. RVS has been pathognomonically associated with one of the most abundant parasites within the marine environment, the ascaridoid nematode Anisakis simplex, which also causes Anisakiasis in humans. Although A. simplex is commonly found in Atlantic salmon, heavy infestation of the vent region is novel, and the expression of RVS has not been prevalent in other fish species. Red Vent Syndrome has been well studied, however, the causes of the condition, and the reasons driving the novel site of infestation exhibited by A. simplex, have not been clarified. The aim of this PhD therefore, is to provide new information regarding the underlying factors of the infestation of the vent region by A. simplex, and the emergence of RVS. This study therefore: i) assessed the relationship between nematode burdens within the viscera and musculature, in comparison to the vent in 117 adult Atlantic salmon; ii) compared the genetic structure of A. simplex present in the vent region and the viscera using the entire nuclear internal transcribed spacer (ITS) region; iii) investigated migratory route and feeding ground of Scottish salmon populations using stable isotope analysis of dorsal muscle tissue and parasite component communities and, iv) assessed the expression of the cytokine TNF-α1 within vent muscle tissue using (q)RT-PCR, in relation to RVS severity. Phylogenetic analyses have shown that it is A. simplex sensu stricto infesting the vent region. The results show that there is a significant positive relationship between the nematodue burden in the body (viscera and musculature) and in the vent region. Isotopic signatures of salmon populations showed no significant differences, however, A. simplex intensities between populations on the East and North coasts of Scotland suggest geographical differences in A. simplex transmission pathways. Finally, the expression of TNF-α1 is not significantly different between RVS severity, and nematode burden. Out of the four studied factors, increasing nematode intensities in Atlantic salmon populations, and the significant positive relationship of nematode intensities between the body (viscera and musculature) and the vent, are likely to explain the infestation of the vent by A. simplex. The underlying causes of RVS however remain uncertain and require further research. With incidences of RVS observed across a number of populations over a large spatial area, regional and global effectors such as warming sea surface temperatures, and the North Atlantic Oscillation are expected to play key roles in its aetiology.

Salmon Pancreas Disease Virus (SPDV) is the only viral species of the genus Alphavirus, family Togaviridae, affecting fish. SPDV induces two conditions historically recognised independently as Pancreas disease (PD) and Sleeping disease (SD), affecting Atlantic salmon (Salmo salar L) and rainbow trout (Oncorhynchus mykiss), respectively. Infection by SPDV can lead to clinical disease with characteristic acinar pancreatic necrosis and a range of myopathies of the skeletal and heart muscle. Mortality is not a necessary outcome of the disease and usually is not significant. However, affected fish stop eating and therefore present a reduced growth rate and the disease can also leave visible lesions at the fillet level that lead to downgrading at slaughter. SPDV can affect in the fresh and sea water environments, but a higher and most relevant impact reported in the latter. Historically, PD has posed a significant challenge to the Atlantic salmon farming industry in the UK, as well as in other salmon producing countries. This thesis was developed and conducted at Marine Scotland Science (MSS), the Scottish National Reference Laboratory, with the aim to contribute to knowledge gaps identified by the industry and research communities. The focus was on development and improvement of in vivo and in vitro infection models to assist with host pathogen interaction studies. In vivo work was to establish an experimental challenge model to induce SPDV infection in a more natural way than by intra-peritoneal (IP) injection. The first step involved selection of an infective SPDV isolate through a comparative IP challenge study. An infective isolate was then used to establish a co-habitation challenge model in "post smolts", the sea-water stage predominantly affected by PD. Additionally, during this experiment assessment of viral tissue tropism along time and potential intra-subtype differences in infectivity was undertaken. In vitro work accounted for the more innovative part of this thesis with the development, optimization and application of an ex vivo cardiac primary culture originated from Atlantic salmon embryos. While fish origin aggregates of self-contracting cardiomyocytes had been previously isolated and suggested as a robust tool on human biomedical research and pharmacological and toxicology testing, paradoxically very little has been done to explore the approach of ex vivo primary cultures as a disease model with the specific goal for health issues affecting fish. The work involved an adaptation and refinement to produce salmon cardiac primary cultures (SCPCs). Once this was achieved, SCPCs could be kept under laboratory conditions with minimal maintenance for periods up to 6 months. Following this work, SCPCs were successfully challenged with different SPDV isolates as well as another cardiotropic viral agent (Infectious Salmon Anaemia, ISA). The kinetics of SPDV and ISA viral infection and one element of the immune response (i.e. expression of mx gene) were studied. As part of this study, the comparative response of SCPCs of diverse genetic backgrounds (i.e. IPN resistant vs. IPN sensitive) was also assessed. Differences were observed, which highlights potential usefulness of SCPCs to examine genotype-based differences in response to viral disease. Finally, SCPCs were used to examine the SPDV infection cycle ultrastructure by transmission electron microscopy (TEM). This work resulted in novel insights on the replication cycle of SPDV, drawing from the extensive literature in mammalian alphavirus work. With SPDV and other virus associated myocarditis severely affecting Atlantic salmon aquaculture at present, I believe that the SCPCs model represents the most relevant contribution of this PhD.

<p>During the smoltification the Atlantic salmon (Salmo salar) develop different adaptations to survive in oceanic environment. One of the most important adaptations is the ability to excrete the surplus of salt through the gills. The excretion is controlled by an enzyme called Na+/K+-ATPase which is produced in an α-subunit by two gene isoforms called α1a and α1b. Enzyme activity is increasing during the smoltification process and is a strong indicator for that the process is taking place. The aim of this study was to investigate a landlocked strain of Atlantic salmon and see how the enzyme activity is developing in comparison with the gene expression of the mRNA that is coded for the enzyme. The study was carried out between March and May in the hatchery in Brattfors, Värmland. Fish were sampled at four occasions. The enzyme activity was compared between two groups of salmon where one group had full ration of food, 100% and the other group had a 15% food ration. The enzyme activity for the 100% group was then compared with the gene expression from the same group. The hypothesis was that food availability should effect smoltification and that the 15% group would have a faster increase in activity compared with the 100% group. There should also be some correlation between enzyme activity and gene expression. Na+/K+-ATPase enzyme activity showed no major differences between the groups except for a significant difference at the last sampling. Both groups had a large increase in activity from the second to the third sampling with a peak on 3.16 µmol ADP/mg/h at most. This was followed by a drop in activity at the last sampling date. The gene expression showed a fast increase of the α1b gene over the study with drop in the last sampling and the α1a gene had a constant increase from the first to the last sampling. The comparison with enzyme activity and gene expression showed a weak correlation. Compared with studies done on anadromous salmon and the land locked salmon in this study had a different development in gene expression. This could be explained that the different life strategies play an important role how the genes are expressed.</p> / <p>Under smoltifieringen utvecklar atlantlaxen (Salmo salar) olika anpassningar för att överleva i havsmiljö. En av de vikigaste anpassningarna är att utsöndra överskott av salt via gällarna. Exkretionen är kontrollerad av ett enzym som heter Na+/K+-ATPas som produceras i en α-subenhet av två isoformer av gener som heter α1a och α1b. Enzym aktiviteten ökar under smoltifieringen och är en stark indikator på att processen sker. Målet med denna studie var att undersöka en sjövandrande stam av atlantlax och se hur enzymaktiviteten utvecklas i jämförelse med gen expressionen av mRNA som kodar för produktionen av enzymet. Studien genomfördes vid fiskodlingen i Brattfors, Värmland där prover togs vid fyra tillfällen. Enzymaktiviteten jämfördes mellan två grupper av lax där en grupp fick full matranson 100 % och en grupp fick 15 % matranson. Senare jämfördes enzymaktiviteten för 100 % gruppen med gen expressionen inom samma grupp. Hypotesen var att tillgängligheten på mat skulle påverka smoltifieringen och att 15 % gruppen skulle ha en snabbare ökning i jämförelse med 100 % gruppen. Det skulle också vara en viss korrelation mellan enzymaktivitet och gen expression. Na+/K+-ATPas enzym aktiviteten visade inga större skillnader mellan grupperna förutom vid sista provtagningen. Båda grupperna hade en stor ökning från den andra till den tredje provtagningen med den högsta aktiviteten på 3.16 µmol ADP/mg/h. Detta följdes av ett fall i aktivitet vid sista provtagningen. Gen expressionen visade en snabb ökning av α1b genen över studien med en nedgång vid sista provtagningen och α1a hade en konstant men mindre ökning från första till sista provtagningen. Jämförelsen mellan enzymaktivitet och gen expression visade på en svag korrelation. Det fanns en skillnad i gen expression mellan studier gjorda på anadrom lax och sjövandrande lax i denna studie. Detta kan förklaras av att de olika livsstrategierna spelar en betydande roll i hur generna uttrycks.</p>

<p> </p><p>ABSTRACT</p><p> </p><p>Migration is common among populations of brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). However, not all individuals in the same population migrate, a phenomenon referred to as partial migration. The aim of this thesis was to investigate if an individual’s behaviour and metabolic rate influences its decision to migrate and how such knowledge may be used when trying to produce hatchery-raised smolts with as high a proportion of migrating individuals as possible. In paper I the influence of reduced food ration on the proportion and swimming speed of migrating brown trout and Atlantic salmon smolts was investigated. Furthermore, the standard metabolic rate (SMR) of migrating and non-migrating individuals was compared. In paper II, a laboratory experiment, SMR was correlated to the behaviour of individual brown trout and Atlantic salmon. Dominant fish of both species had a higher SMR than subordinates (paper II). In addition, migrant brown trout had a higher SMR than non-migrant trout when given a normal food ration, whereas no difference in SMR between migrating and non-migrating salmon could be seen (paper I). When administered low food rations, smolts of both species migrated faster than smolts given a normal food ration, and the proportion of migrating smolts was higher for salmon given less food when the size difference for smolts from the two feeding regimes was large (paper I). Other factors that influenced migration speed were the degree of smolt development and water temperature (paper I). SMR was not correlated with aggressiveness, or with different measurements of boldness. Moreover, aggression and boldness were not correlated with each other (paper II). Trout showed a higher level of aggressiveness and acclimated more rapidly to laboratory conditions than salmon (paper II). In summary, there was no support for the existence of coping styles in migratory Atlantic salmon and brown trout. Instead, metabolic rates were related to both migratory behaviour and social status. Furthermore, an individual’s decision to migrate was influenced by ration size.</p>

Anthropogenic influences and climate change are warming rivers in New Brunswick and threatening the cold water habitats of native salmonids. When ambient river temperatures in summer exceed the tolerance level of Atlantic salmon and brook trout, individuals behaviourally thermoregulate by seeking out cold water refugia. These critical thermal habitats are often created by tributaries and concentrated groundwater discharge. Thermal infrared imagery was used to map cold water anomalies along a 53 km reach of the Cains River on 23 July 2008. Although efficient and useful for mapping surface temperature of a continuous stream reach, the fish did not use all identified thermal anomalies as refugia. Overall, 100 % of observed large brook trout >35 cm in length were found in 30 % of the TIR-mapped cold water anomalies. Ninety eight percent of observed small brook trout 8 – 30 cm in length were found in 80 % of the mapped cold water anomalies and their densities within anomalies were significantly higher than densities outside of anomalies. Fifty nine percent of observed salmon parr were found in 65 % of the mapped anomalies; however, they were dispersed within study sites and their densities were not significantly different within anomalies compared to outside of the anomalies. No brook trout were observed at the seven noncold water study sites that were investigated. Preference curves for various habitat variables including velocity, temperature, depth, substrate, and deep water availability near cold water anomalies were developed based on field investigations during high temperature events (ambient river temperature >21 oC). Combined with thermal imagery, managers can use the physical descriptions of thermal refugia developed here as a tool to help conserve and restore critical thermal refugia for Atlantic salmon and brook trout on the Cains River, and potentially similar river systems.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 62-69.

ABSTRACT   Migration is common among populations of brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). However, not all individuals in the same population migrate, a phenomenon referred to as partial migration. The aim of this thesis was to investigate if an individual’s behaviour and metabolic rate influences its decision to migrate and how such knowledge may be used when trying to produce hatchery-raised smolts with as high a proportion of migrating individuals as possible. In paper I the influence of reduced food ration on the proportion and swimming speed of migrating brown trout and Atlantic salmon smolts was investigated. Furthermore, the standard metabolic rate (SMR) of migrating and non-migrating individuals was compared. In paper II, a laboratory experiment, SMR was correlated to the behaviour of individual brown trout and Atlantic salmon. Dominant fish of both species had a higher SMR than subordinates (paper II). In addition, migrant brown trout had a higher SMR than non-migrant trout when given a normal food ration, whereas no difference in SMR between migrating and non-migrating salmon could be seen (paper I). When administered low food rations, smolts of both species migrated faster than smolts given a normal food ration, and the proportion of migrating smolts was higher for salmon given less food when the size difference for smolts from the two feeding regimes was large (paper I). Other factors that influenced migration speed were the degree of smolt development and water temperature (paper I). SMR was not correlated with aggressiveness, or with different measurements of boldness. Moreover, aggression and boldness were not correlated with each other (paper II). Trout showed a higher level of aggressiveness and acclimated more rapidly to laboratory conditions than salmon (paper II). In summary, there was no support for the existence of coping styles in migratory Atlantic salmon and brown trout. Instead, metabolic rates were related to both migratory behaviour and social status. Furthermore, an individual’s decision to migrate was influenced by ration size.

During the smoltification the Atlantic salmon (Salmo salar) develop different adaptations to survive in oceanic environment. One of the most important adaptations is the ability to excrete the surplus of salt through the gills. The excretion is controlled by an enzyme called Na+/K+-ATPase which is produced in an α-subunit by two gene isoforms called α1a and α1b. Enzyme activity is increasing during the smoltification process and is a strong indicator for that the process is taking place. The aim of this study was to investigate a landlocked strain of Atlantic salmon and see how the enzyme activity is developing in comparison with the gene expression of the mRNA that is coded for the enzyme. The study was carried out between March and May in the hatchery in Brattfors, Värmland. Fish were sampled at four occasions. The enzyme activity was compared between two groups of salmon where one group had full ration of food, 100% and the other group had a 15% food ration. The enzyme activity for the 100% group was then compared with the gene expression from the same group. The hypothesis was that food availability should effect smoltification and that the 15% group would have a faster increase in activity compared with the 100% group. There should also be some correlation between enzyme activity and gene expression. Na+/K+-ATPase enzyme activity showed no major differences between the groups except for a significant difference at the last sampling. Both groups had a large increase in activity from the second to the third sampling with a peak on 3.16 µmol ADP/mg/h at most. This was followed by a drop in activity at the last sampling date. The gene expression showed a fast increase of the α1b gene over the study with drop in the last sampling and the α1a gene had a constant increase from the first to the last sampling. The comparison with enzyme activity and gene expression showed a weak correlation. Compared with studies done on anadromous salmon and the land locked salmon in this study had a different development in gene expression. This could be explained that the different life strategies play an important role how the genes are expressed. / Under smoltifieringen utvecklar atlantlaxen (Salmo salar) olika anpassningar för att överleva i havsmiljö. En av de vikigaste anpassningarna är att utsöndra överskott av salt via gällarna. Exkretionen är kontrollerad av ett enzym som heter Na+/K+-ATPas som produceras i en α-subenhet av två isoformer av gener som heter α1a och α1b. Enzym aktiviteten ökar under smoltifieringen och är en stark indikator på att processen sker. Målet med denna studie var att undersöka en sjövandrande stam av atlantlax och se hur enzymaktiviteten utvecklas i jämförelse med gen expressionen av mRNA som kodar för produktionen av enzymet. Studien genomfördes vid fiskodlingen i Brattfors, Värmland där prover togs vid fyra tillfällen. Enzymaktiviteten jämfördes mellan två grupper av lax där en grupp fick full matranson 100 % och en grupp fick 15 % matranson. Senare jämfördes enzymaktiviteten för 100 % gruppen med gen expressionen inom samma grupp. Hypotesen var att tillgängligheten på mat skulle påverka smoltifieringen och att 15 % gruppen skulle ha en snabbare ökning i jämförelse med 100 % gruppen. Det skulle också vara en viss korrelation mellan enzymaktivitet och gen expression. Na+/K+-ATPas enzym aktiviteten visade inga större skillnader mellan grupperna förutom vid sista provtagningen. Båda grupperna hade en stor ökning från den andra till den tredje provtagningen med den högsta aktiviteten på 3.16 µmol ADP/mg/h. Detta följdes av ett fall i aktivitet vid sista provtagningen. Gen expressionen visade en snabb ökning av α1b genen över studien med en nedgång vid sista provtagningen och α1a hade en konstant men mindre ökning från första till sista provtagningen. Jämförelsen mellan enzymaktivitet och gen expression visade på en svag korrelation. Det fanns en skillnad i gen expression mellan studier gjorda på anadrom lax och sjövandrande lax i denna studie. Detta kan förklaras av att de olika livsstrategierna spelar en betydande roll i hur generna uttrycks.