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.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv. / Härtill 4 uppsatser. I publ. anges Department of Aquaculture som utgivande institution. Med sammanfattning på svenska och franska.

Atlantic salmon (Salmo salar L.) has been domesticated since the 1960s and has undergone over 10 generations of artificial selection for economically important traits. As a result, domesticated salmon have diverged with respect to a number of phenotypic, genotypic and behavioural traits from their wild counterparts. Since the selection pressures that are present in the wild differ greatly from the ones that shape salmon under culture conditions, domesticated salmon stocks are considered to be maladapted to natural conditions. Despite strict regulations, insoluble issues pertaining to large-scale cage rearing of farmed fish mean that there is a continuous presence of farm escapees in the wild. Gene flow from escapees has been perceived as a factor in the decline of wild populations, suggested to occur through disruption of local adaptation. This study aims to improve understanding of the genetic differences between wild and domesticated stocks by comparing the transcriptomes of Figgjo (wild) and Mowi (domesticated) strains. A series of common garden experiments have been performed, utilizing pure and reciprocal hybrid crosses of the wild and domesticated stocks, reared under two different conditions and sampled at four time points and three distinct life stages (embryo, sac-fry and feeding fry). Microarray interrogations were performed employing a 44K custom microarray design to identify genes and gene pathways that are differentially expressed between the stocks. KEGG-based functional analyses have been implemented using different gene set enrichment packages, and dominance and additive parameters were calculated from normalized expression values to predict the mode of heritability of the genes identified as differentially expressed between stocks. Most biological functions represented in wild and domesticated crosses were consistent across life stages and environments. The transcriptomic differences detected between stocks in multiple developmental stages likely reflected adaptations to selection pressures differing between natural and aquaculture environments. Down-regulated environmental information processing and immune and nervous system functions in domesticated vs. wild fish may be due to local adaptation to captivity. These included reduced information acquisition and processing systems, altered stress responsiveness and changes in feeding behaviour. In line with the resource allocation theory of production trait animals, reduced immune function was coupled with increased expression of growth and development related pathways in domesticated salmon, compared to wild counterparts. Although there is support for this trade-off in all life-stages, resource allocation showed a shift over time; possibly reflecting variation in the utilization of energy sources during the transition from endogenous to exogenous feeding. Differences in cell communication and signalling pathways between wild and domesticated stocks, associated with organogenesis during the embryo stage, reflect sampling time and are indicative of altered organ development in response to domestication. Stress responses common across stocks included the down-regulation of cellular processes, including cell cycle and meiosis, and genetic information processing, such as replication and repair, transcription and translation pathways, probably reflecting the reallocation of energy resources away from growth and towards the restoration of homeostasis. Moreover, the mobilization of energy to cover the increased demands of maintaining homeostasis was indicated by the up-regulation of some metabolic pathways, mostly involved in energy, lipid and carbohydrate metabolism in response to stress. The analysis also revealed cross-specific stress responses, including indicators of a non-additive stress response in hybrid crosses. Most differentially expressed transcripts exhibited additive (31-59%) or maternal dominant (19-33%) inheritance patterns, although maternal over-dominance (23-26%) was also significant in the embryo stage. The mode of heritability of some immune transcripts was suggestive of maternal environmental influence having been affected by aquaculture. This study has demonstrated that biological functions affected by domestication include those associated with allocation of resources, involve reduction of information acquisition and processing systems and may lead to loss of local adaptation to wild conditions. Since such changes may affect key systems, such as immunity and responsiveness to stress, they can potentially have serious negative consequences under natural conditions. Transcriptomic differences observed between wild and domesticated stocks primarily exhibited additive and maternal dominant inheritance modes. Since gene-flow from farmed fish can be frequent and primarily concerns farmed females, this suggests that introgression due to repeated large scale escape events has the capacity to significantly erode local adaptation.

Environmental pollution by pharmaceuticals is an issue of concern that is currently attracting attention around the world. Although environmental effects of pharmaceutical contaminants are not yet well documented, studies have shown that these substances may have the potential to disrupt the biotic component of an ecosystem. Particularly worrisome contaminants are the neuroactive pharmaceuticals which have the potentiality to induce behavioral modifications in non-target species. In the present study, I examined the effects of a benzodiazepine anxiolytic pharmaceutical (oxazepam) on the behavior of Atlantic salmon (Salmo salar) juveniles (fry). The hypothesis of this study was that oxazepam reduces the anxiety-like behavior of the Atlantic salmon juveniles. To test the hypothesis and assess the impact of oxazepam exposure on Atlantic salmons fry behavior, two different concentrations of this drug; a low-level (1.9 ug L-1) and a high-level concentration (1000 ug L-1) were used. Exposures lasted for 48 hours and afterwards, the fish were recorded to evaluate their behavioral responses. The results of this study reveal that oxazepam in a high concentration (1000 ug L-1 ) alters specific behavioral endpoints related to the fitness (feeding/predator avoidance) of Atlantic salmons fry. Individuals exposed to the high oxazepam concentration exhibited significant lower average speed and acceleration as well as they traveled a shorter mean distance compared to the unexposed (control) individuals. These findings confirm the hypothesis and show that psychotropic pharmaceutical contaminants modify animal behaviors, which can ultimately lead to ecological consequences. However, the concentration that generated behavioral effects in this study was three magnitudes higher than concentrations measured in the environment and thus, should not be viewed representative for oxazepam contaminated ecosystems.

Photoperiod manipulations are widely used throughout the Atlantic salmon (Salmo salar) farming industry as a means of producing a product of uniform quality all-year round. However, farmers still remain sceptical over their effectiveness to regulate growth and maturation during the on-growing stage. Furthermore, reports of a characteristic growth-dip following light exposure suggest that light may negatively affect the physiological performance of fish in the short-term. Thus, this thesis investigates the effects of light characteristics (spectral quality, intensity and photoperiod) on growth and maturation of salmonid fish and addresses some of the uncertainties surrounding photoperiod use currently reported within the industry. Rainbow trout (Oncorhynchus mykiss) are seemingly an ideal model species for examining photoperiod effects on growth. Consequently, the application of constant light exposure (LL) at two different intensities (28W and 16W) during two different thermal conditions (summer and winter) was examined on individually tagged fish. Feed intake and growth appeared to be related to the ambient water temperature and did not appear to be affected by intensity or photoperiod, although the onset of constant light did appear to initially affect growth rate. This may indicate that LL has a limiting effect on the growth of trout or that the prevailing water temperature at which light is applied may override the photoperiodic effect. Furthermore, the lack of enhanced growth in trout exposed to LL, unlike that demonstrated for other salmonids, suggest that there may be a species-specific response to environmental variables. Thus, questions regarding photoperiod effects should be limited to the species in question. The main source of variation in results observed under photoperiod manipulations stems from the salmon industry. Atlantic salmon post-smolts were reared in seawater tanks and either maintained under a natural photoperiod (NP) or exposed to a simulated natural photoperiod (SNP), constant light superimposed on the natural light (NPLL) or constant light only (LL). Artificial light onset, irrespective of photoperiod, resulted in an apparent trend for a reduced appetite lasting up to 60 days. Furthermore, the onset of constant light resulted in a significant chronic elevation of plasma cortisol levels and changes to growth and thyroid hormone levels, providing direct evidence that constant light exposure induces stress. In addition, fish exposed to SNP failed to exhibit a stress response despite a low feed intake. However, differences in the plasma melatonin levels during twilight times, as compared to NP, suggest that gradual changes in the natural light intensity throughout the day, particularly around dawn and dusk, may be important for synchronizing daily events. No differences in growth were observed between the NP and NPLL regimes, although fish reared in an enclosed regime (SNP and LL) exhibited a significantly lower weight gain than fish in an open environment (NP and NPLL). This further highlights the impact that the rearing environment has on the growth performances of fish and the need for commercially run trials. Advances in lighting technologies and a greater understanding of how light is transformed through the water column have focussed research on the spectral sensitivity of fish. Therefore the lighting efficiency of novel blue narrow bandwidth LED lighting units through the water column and their effects on growth and maturation performances of salmon reared in commercial production cages were compared against the standard metal halide units currently utilized throughout the industry. LL application, irrespective of intensity or spectrum, reduced the numbers of fish maturing as compared to fish reared under a natural photoperiod. However, this was greatest under the standard metal halide units reflecting a greater light penetration and perception as determined by plasma melatonin levels. The metal halide groups exhibited the greatest relative weight gain over the trial period as compared to control fish. No evidence was observed for a growth-dip under metal halide light, although blue lit treatments exhibited an initial significant reduction in food consumption, suggesting a possible welfare issue. Nevertheless, the prototype blue LED units showed possible potential for commercial application by penetrating the water depth at half the distance of the metal halide units for only one eighth the power and one fifth the brightness. However, further tests of these prototype spectral units are required to examine the potential welfare and physiological growth and reproductive effects. These studies have shown that the efficacy of artificial light regimes is largely dependent upon the effectiveness of the light source through the underwater environment and its perception by fish, providing a sufficient intensity is emitted exceeding the physiological threshold level for the species cultured. Moreover, whilst the onset of artificial light may elicit a stress response and demonstrate a trend for a suppression of appetite for salmon reared in experimental tanks, no compelling evidence for a suppression of appetite or growth was found under normal commercial cage conditions. This suggests that the growth-dip observed within the industry may in part be a combination of a physiological response to the onset of light further exaggerated by the farmer’s perception and altered judgement in feeding. In addition, the results obtained from this study have helped to standardize the use of light regimes within the industry. Nevertheless, further studies are necessary to fully elucidate the underlying mechanisms which may govern growth and maturation in fish following the onset of light exposure.

Efficient control of sea lice is a major challenge for the sustainable production of farmed Atlantic salmon (Salmo salar (Linnaeus, 1758)). These marine ectoparasites feed on mucus, skin and blood of their hosts, thereby reducing the salmon’s growth rate and overall health. In the northern hemisphere, the most prevalent species is Lepeophtheirus salmonis (Krøyer, 1837). In 2006, global costs of sea lice infections are estimated to have exceeded €300 million, with the majority spent on a limited number of chemical delousing agents. Emamectin benzoate (EMB; SLICE®), an avermectin, has been widely used since its introduction in 2000, due to its convenient administration as an in-feed medication and its high efficacy against all parasitic stages of L. salmonis. However, over-reliance on a single or limited range of medicines favours the emergence of drug resistance and, as a result, the efficacy of this compound in treating L. salmonis has decreased in recent years, as reported from e.g. Chile, Norway, Scotland and Canada. Declining efficacy underlines the need for an improved understanding of the molecular mechanisms underlying EMB drug resistance in L. salmonis. Elucidation of these mechanisms would allow for improved monitoring tools, earlier detection of developing resistance, extended usability of current delousing agents and development of new parasiticides. The work described in this thesis sets out to examine the molecular mechanisms underlying EMB resistance in L. salmonis. In earlier studies, research in nematodes and arthropods has linked drug efflux transporters belonging to the family of ATP-binding cassette (ABC) transporters to ivermectin (IVM) resistance, a parasiticide with high chemical similarity to EMB. ABC transporters such as permeability glycoprotein (P-gp), transport a wide range of substrates, including drugs, and have been suggested to provide a potential molecular mechanism through which EMB resistance might be mediated in sea lice. As an example of such mechanisms, increased expression of P-gp is one of the causative factors for drug resistance in human cancer cells and avermectin resistance in nematode parasites such as Caenorhabditis elegans or Haemonchus contortus. Initial research involved screening for novel salmon lice P-gps that might contribute to EMB resistance. A novel P-gp, SL-PGY1, was discovered using a combined bioinformatic and molecular biological approach. The expression was compared in two well-characterised L. salmonis strains differing in their susceptibility to EMB (S = susceptible, R = resistant). Prior to EMB exposure, mRNA levels did not differ from each other, while, after 24 h exposure, a 2.9-fold increase in SL-PGY1 mRNA expression was observed in the R strain. SL-PGY1 appears not to be a major factor contributing to reduced EMB susceptibility, although it could play a role, as expression levels increased upon exposure to EMB. A further four additional drug transporters (ABC C subfamily) were also discovered showing high homology to multidrug-resistance proteins (MRP). The relative expression levels of each MRP was compared in the strains S and R, before and after exposure to EMB. No significant changes were found in their expression patterns. If ABC drug transporters mediate the efflux of EMB and thereby reduce the intracellular concentrations of the drug in exposed animals, the inhibition of those ABC drug transporters was expected to lead to higher intracellular levels of EMB. This could result in an enhanced toxic effect when EMB is co-administered with an inhibitor. Two known inhibitors of human P-gps and MRPs, cyclosporin A (CSA) and verapamil (VER), were co-administered with EMB. CSA increased the toxic effect of EMB in both tested strains, implying that the targets of CSA are expressed at comparable levels and that they may be part of the mechanism conferring EMB resistance. VER increased the toxic effect of EMB in the R strain, but had no significant effects on the S strain. This implies that the expression of factors inhibited by VER differs between the two L. salmonis strains. It is hypothesised that a number of ABC transporters with distinct, yet overlapping patterns of inhibitor specificity are affected by those inhibitors. The search for drug-resistance conferring genes was complemented with a systematic, genome-wide survey of ABC transporters in L. salmonis to find additional members of this important gene family. Next-generation high-throughput RNA sequencing (RNA-seq) was employed to assemble a reference transcriptome from pooled total RNA of salmon lice at different development stages. The transcriptome was assembled against the L. salmonis genome and annotated. Thirty-nine putative ABC transporters were found. Of further interest were transcripts of the subfamily B, C and G, as they contain drug-transporting ABC proteins. For the ABC B subfamily, one full (SL-PGY1) and three half transporter transcripts were found. Only full transporters are known to transport drugs and SL-PGY1 is apparently not a major factor contributing to EMB resistance. Fourteen ABCC sequences were found – 11 MRPs and 3 homologues to sulfonylurea receptors. Of interest are MRPs, as they contribute to drug detoxification in humans and invertebrates. Four MRPs had been identified previously and their expression ratios did not differ between S and R strain parasites. Seven sequences belonging to ABCG subfamily were found. However, none of the L. salmonis ABCG transcripts identified showed sufficient homology to known drug transporters in other species. With the currently limited understanding of the mechanisms conferring EMB resistance, monitoring the susceptibility of L. salmonis subpopulations is essential. Dose-response bioassays are currently widely used. Tests with pre-adult II or adult parasites requires relatively large numbers of parasites (~150) to conduct this type of bioassay, which may not always be available. Addressing this issue, we tested the feasibility of a single-dose bioassay (requiring fewer test animals than dose-response bioassays) to discriminate between L. salmonis strains with differing EMB susceptibility. This alternative approach uses time-course toxicity analysis, where the toxic effect of EMB is monitored over time. After clearly defining the effect criteria, we found that it is possible to discriminate between those L. salmonis strains. However, while requiring fewer test animals, time course toxicity analysis is more labour-intensive, but the alternative design can be suitable under certain circumstances. The work reported here has provided new knowledge concerning the mechanisms of EMB resistance in sea lice. Several novel putative drug transporters have been identified, an important first step toward unravelling the complex interactions of genes involved in EMB resistance in this commercially important parasite.

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.