Three new NILT genes were successfully cloned and characterized from rainbow trout, with one NILT alternatively spliced into a long isoform containing two Ig domains and a short isoform containing one Ig domain.  The expression of NILTs was studied in six different tissues and two different cell lines, with expression apparent in immunologically important tissues.  Furthermore, phylogenetic analysis showed that NILTs are more closely related to triggering receptor expressed on myeloid (TREM) cells and Nkp44 from humans than to NITRs from rainbow trout. The genomic organisation and structure of the multigene family of NILTs in Atlantic salmon was investigated using a BAC sequencing approach.  This revealed the presence of six novel NILT genes, which either contained one or two Ig domains and several immunoreceptors tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region. By homology search two NILT-like genes in zebrafish (<i>Danio rerio</i>) located on chromosome 1 have been obtained. Chromosome 1 in zebrafish also contains the <i>Dare</i>-ZE genes, which are equivalent to the human MHC class I genes located on chromosome 6.  The distance between the later and the TREM genes on chromosome 6 is similar to the distance between the NILT-like genes and <i>Dare</i>-ZE genes on zebrafish chromosome 1.  In addition, two NILT-like Ig domains were obtained from the green spotted pufferfish (<i>Tetraodon nigroviridis</i>), putatively part of the same receptor. The results will contribute to our knowledge of the immune system in fish and provide useful information for the control of inflammatory processes in salmonids.

Sterile triploid Atlantic salmon represent a solution to the issues of pre-harvest sexual maturation and mature escapees from open aquaculture systems. Although the initial problems of reduced performance and increased deformities in triploids have been thoroughly researched, there is a continued lack of information on their susceptibility and response to disease and routine on-farm treatments compared to diploids. Thus, the main aim of this thesis was to enhance the current understanding of triploid health and immunity through experimental disease challenges and treatments, and aid in determining their robustness and, therefore, suitability for aquaculture. A commercial furunculosis vaccine equally protected diploids and triploids against challenge with Aeromonas salmonicida, and adhesion scores were similar between ploidy (Chapter 2). Interestingly, triploids had lower white blood cell counts but increased cellular activity, e.g. respiratory burst, compared to diploids. Following experimental cohabitation infection with Neoparamoeba perurans, causative agent of Amoebic Gill Disease (AGD), ploidy did not affect the manifestation or severity of AGD-associated gill pathology, or the serum innate immune response (Chapter 3). Hydrogen peroxide, used to treat against parasitic diseases, elicited similar primary and secondary stress responses in both ploidy, but led to differences in the expression of stress (cat, gpx1, gr, hsp70, sod1, sod2) and immune (saa5, crp/sap1a, crp/sap1b, il1β) genes (Chapter 4). Finally, vaccination with different vaccine treatments (4 commercial vaccines, 6 different vaccine combinations and a sham-vaccinated control) showed no ploidy differences in adhesion score or antibody response, although vertebral deformities remained higher in triploids (Chapter 5). Increasing severity of vaccine treatments negatively affected weight, length and thermal growth coefficient in both ploidy. Triploids were heavier than diploids at smolt (+ 14 %) and post smolt (+ 32 %). Overall, this research shows that triploid Atlantic salmon respond as well as diploids to disease and treatment challenges, and supports their application into full-scale commercial aquaculture.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 198-219).

Recent years have seen considerable advances in the study of biological rhythms and the underlying molecular mechanisms that drive the daily and seasonal physiology of vertebrates. Amongst teleosts the majority of work in this field has focused on the model species the zebrafish to characterise clock genes and the molecular feedback loop that underpins circadian rhythms and physiology. Daily profiles of clock gene expression in a wide variety of tissues and cell types are now relatively well described. However the zebrafish is a tropical species that does not display distinct seasonality and therefore may not be the species of choice to investigate the entrainment of circannual physiology. In contrast, Atlantic salmon is a highly seasonal teleost that displays considerable temporal organisation of most physiological processes. In salmonids photoperiod is widely known to synchronise physiology to the environmental conditions and as such photoperiod manipulation is routinely used by the salmon industry throughout the production cycle to control and manipulate spawning, smoltification and puberty. Previous studies in salmonid species have already identified a set of clock genes that are linked to these seasonal physiological processes. However, to date, the molecular mechanisms regulating daily and seasonal physiology are largely unknown despite the strong commercial relevance in the Atlantic salmon. In the Atlantic salmon, Davie et al (2009) was the first to report the photoperiod dependent circadian expression of clock genes (Clock, Bmal and Per2 and Cry2) in the brain of the Atlantic salmon. In the same investigation the expression of clock genes was reported in a wide variety of peripheral tissues, however 24h profiles of expression in peripheral tissues were not characterised. In order to examine further the role of seasonal photoperiod on the circadian expression of clock genes, the present work first aimed to characterise diel profiles of Clock, Per1 and Per 2 expression in the brain together with plasma melatonin levels in II Atlantic salmon acclimated to either long day (LD), short day (SD), 12L:12D (referred to as experiment 1 throughout) and SNP (referred to as experiment 2 throughout). Photoperiod dependent clocks were also investigated in peripheral tissues, namely in the fin and liver. Results showed circadian profiles of melatonin under all photoperiods. In experiment 1 both Clock and Per2 displayed significant circadian expression in fish exposed to LD. This is in contrast to previous results where rhythmic clock gene expression was observed under SD. In addition, clock gene expression differed in response to experimental photoperiod in the liver, and diel rhythm differed to that of the brain. No rhythmic expression was observed in the fin. Levels of plasma melatonin exhibited a circadian rhythm peaking during the nocturnal phase as expected. However the amplitude of nocturnal melatonin was significantly elevated under LD (experiment 1) and the SNP long day photoperiod and 2010 autumnal equinox samples (experiment 2). Overall results from these experiments suggested that the control of clock gene expression would be photoperiod dependent in the brain and the liver however photoperiod history is also likely to influence clock gene expression. Interestingly, the gradual seasonal changes in photoperiod under SNP did not elicit similar profiles of clock gene expression as compared to experimental seasonal photoperiods and clock gene expression differed between experimental photoperiod and SNP treatments. In experiment 2 significant seasonal differences were also observed in the amplitude of individual clock gene expression. The mechanisms underlying this and potential impact on seasonal physiology are unknown. Developmental changes such as the smoltification process or abiotic factors such as temperature or salinity should be further investigated. In mammals previous work has focused on the molecular switch for photoperiod response and regulation of thyroid hormone bioactivity via deiodinase mediated conversion of T4 to the biologically active form T3. In mammals and birds expression of key seasonal molecular markers i.e. Tsh, Eya3 and Dio2, are up-regulated hours after exposure to the first LD and III persist under chronic LD conditions. In order to confirm the involvement of these genes in the seasonal photoperiodic response in salmon, a microarray study was first carried out. Results displayed transcriptome level differences in the seasonal expression of a wide variety of target genes including Eya3 and Dio1-3 in relation to LD and SD photoperiod suggesting that these genes may have a conserved role in salmon. qPCR validations of selected genes of interest were then performed (Dio1, Dio2 and Dio3, Eya3 and Tshover diel cycles in fish exposed to LD and SD photoperiod (autumn acclimated fish). In addition an unrelated qPCR study was undertaken in salmon parr acclimated to LD, 12L12D and SD photoperiod (spring acclimated fish)(Dio2, Eya3 and Tsh. Consistent with findings obtained in other vertebrate species, circadian expression of Dio2 was observed under LD. However expression of Eya3 and Tsh appeared to be dependent on photoperiod history prior to acclimation to the experimental photoperiods as already suggested for clock gene expression in this thesis. This is potentially a consequence of direct regulation by clock genes. To our knowledge, this is the first report on the expression of key molecular components that drive vertebrate seasonal rhythms in a salmonid species. The thesis then focused on another key component of the photoneuroendocrine axis in fish, the pineal organ. In the Atlantic salmon, as in other teleosts the photoreceptive pineal organ is considered by many to be essential to the generation, synchronisation and maintenance of circadian and seasonal rhythms. This would be primarily achieved via the action of melatonin although direct evidence is still lacking in fish. In salmonids the production of pineal melatonin is regulated directly by light and levels are continually elevated under constant darkness. In non salmonid teleosts the rhythmic high at night/ low during day melatonin levels persists endogenously under constant conditions and is hypothesised to be governed by light and intra- pineal clocks. The aims of the present in vitro and in vivo trials were to determine if circadian clocks and Aanat2 expression, the rate limiting enzyme for melatonin IV production, are present in salmon, test the ability of the pineal to independently re-entrain itself to a different photoperiod and establish whether the candidate clock genes and Aanat2 expression can be sustained under un-entrained conditions. Expression of clock genes was first studied in vitro with pineal organs exposed to either 12L:12D photoperiod, reversed 12D:12L photoperiod and 24D. Clock gene expression was also determined in vivo, in fish exposed to 12L:12D. Results were then contrasted with an in vitro (12L:12D) investigation in the European seabass, a species displaying endogenous melatonin synthesis. Results revealed no rhythmic clock gene (Clock, per1 and per2) expression in isolated salmon pineals in culture under any of the culture conditions. In the seabass, Clock and Per1 did not also display circadian expression in vitro. However rhythmic expression of Cry2 and Per1 was observed in vivo in the salmon pineal. This suggested some degree of extra-pineal regulation of clocks in the Atlantic salmon. In terms of Aanat2 no rhythmic expression was observed in the Atlantic salmon under any experimental conditions while rhythmic expression of Aanat2 mRNA was observed in seabass pineals. This is consistent with the hypothesis that in salmonids AANAT2 is regulated directly at the protein level by light while in other teleosts, such as seabass, AANAT2 is also regulated by clocks at a transcriptional level.

Atlantic salmon (Salmo salar) parr-smolts were exposed to sublethal concentrations of cypermethrin, chlorothalonil, quintozene or atrazine to determine if they affected osmoregulation. After 96 h of exposure to a pesticide, Na+K+-ATPase, hematocrit, liver somatic index (LSI), plasma sodium, chloride, and cortisol concentrations were determined. There were no mortalities observed following a 24-h seawater challenge. No effects were seen with cypermethrin exposure. Chlorothalonil exposure resulted in increases in plasma Na+ concentrations following the seawater challenge in the 0.18 and 3.6 μg/L groups. For quintozene, decreases in LSI was seen at each concentration, and decreases in Na+K+-ATPase activity was seen at 0.55 μg/L as well as a decrease in Na+ concentrations at the highest exposure concentration. Atrazine exposure increased Na+K+-ATPase activity in the 1 and 100 μg/L groups, and plasma cortisol concentrations at100 μg/L. Overall, the pesticides examined had minimal effects on fish osmoregulation and stress at the concentrations tested.

The use of wrasse (Pisces: Labridae) as cleaner fish to combat infections with the parasitic copepods Lepeophtheirus salmonis (Kroyer) and Caligus elongatus (Nordmann) (sea-lice) in the culture of Salmo salar L. (Atlantic salmon) is now common. Infections with these parasites has caused considerable losses in the industry since its formative years. The use of the wrasse species Ctenolabrus rupestris (L. ) (goldsinny), Centrolabrus exoletus (L. ) (rockcook), Symphodus melops (= Crenilabrus melops) (L. ) (corkwing) and Labrus mixtus L. (cuckoo) as cleaner fish was first suggested in 1988. The use of these species in the industry is now widespread in Scotland, Ireland and Norway. The fish used are normally caught from the wild before being stocked with S. salar smolts during their first year at sea. The fish are routinely collected from waters close to the farm sites to be stocked. As most of the S. salar sea production sites in Scotland are located on the west coast of the country, the wrasse to be used in these sites are normally collected from these waters. The movement of wild fish into farm pens presents a risk of disease transfer from wrasse to S. salar and vice versa. Prior to their use as cleaner fish, these four species of wrasse had received little attention as subjects of scientific study. As a result, there was very little information available in the literature regarding their diseases. The present study was undertaken to investigate the potential pathogens present in wild populations in Scottish coastal waters, and, in particular, which of these pathogens, if any, could be transmitted to the S. salar. The study also investigated the susceptibility of wrasse to the two major viral diseases of S. salar to which they would be exposed in pens. In order to fully assess the pathogenicity of the potential disease agents under farm conditions, it was first necessary to establish the normal morphology of the wrasse species. Hence, a study of the morphological features of wrasse, with particular emphasis on those features important in the health of the fish was undertaken. Wrasse were shown to differ in many aspects from salmonids but shared many morphological features with other perciforme fish. Major differences from salmonids were evident in the skin, fins, pancreas, intestine, gonads and heart. There were also aspects of their morphology which differed from other perciforme fish, notably the structure of the heart. These features were regarded to be adaptations to the specific demands of their feeding strategies and habitats. This study was the first of its kind undertaken for wrasse and showed some early contraindications for the use of wrasse in culture; most notable was the marked lipid accumulation in, and resultant degeneration of, the liver resulting from the consumption of high energy S. salar feeds. Once the normal morphological features were established, it was possible to examine the disease status of wrasse. Wild fish were sampled from three different locations on the west coast of Scotland. These sites were all geographically distinct and were all used as sources of wrasse for the S. salar farming industry. Samples of wrasse were also obtained from farm sites supplied with wrasse from these wild sites, and an additional number of other geographically distinct farm locations. As a comparison wrasse were also obtained from a wrasse captive breeding facility and another captive location unrelated to the S. salar industry, a public aquarium. The fish from all of these sampling sites were examined fully for the presence of parasites, bacteria and, in some cases, viruses. Histological examination was also carried out on all of the fish studied. A total of 24 new parasite host records, and two tentative ones, were recorded from the four wrasse species studied. These new parasite records included protozoa, digeneans, nematodes and crustacea. Parasite infections were found to vary in prevalence, abundance and intensity in respect to the geographical characteristics of sampling sites and also the length of time spent in S. salar pens. It was concluded that the separation of wrasse from their natural diet and habitat influenced the degree of parasitism. None of the parasites found to infect wrasse were observed to cause any significant pathology in their hosts other than localised tissue responses. The possibility of transfer of wrasse parasites to S. salar was also investigated experimentally in a series of infections in which parasites dissected from wrasse were introduced to S. salar smolts by means of a novel gavage method. None of the parasites used established in the S. salar, indicating that there is little risk of transfaunation of parasites between wrasse and S. salar. However, this aspect requires further work due to the low number of parasites available and the subsequent low numbers of S. salar infected. Bacterial isolates were obtained from wrasse held in S. salar pens but were not found in any of the fish collected from the wild. Most of the bacterial strains isolated would normally be considered as opportunistic pathogens of fish. It was concluded that the relatively high levels of stress, both environmental and physical, that wrasse are subjected to under farm conditions were instrumental in the number of bacterial infections seen in wrasse. Only one pathogenic bacterial infection was seen in any of the fish sampled. This was an isolate of Aeromonas salmonicida, the agent known to cause the disease furunculosis, isolated from a wrasse obtained from one of the farm samples. Other authors have reported that this bacterium has already caused substantial losses of wrasse under farm conditions. It was concluded that Aeromonas salmonicida will prove to be a major pathogen of wrasse held in S. salar pens. No viruses wereI isolated from any of the wrasse studied. The susceptibility of wrasse to the most significant pathogens of S. salar under farm conditions was also subjected to investigation. In addition to sea-lice infection, the industry lists Infectious Pancreatic Necrosis (IPN) and Pancreas Disease (PD) as of primary importance for further research. Both of these diseases cause substantial losses in the industry. The susceptibility of wrasse to both of these disease conditions was investigated by means of experimental infections. In the case of IPN wrasse were infected by bathing with two different infective doses, a low dose which would be expected to induce the disease in S. salar parr and a second dose substantially higher than the first. The C. rupestris used were found to be susceptible to IPN. The wrasse developed some of the pathological characteristics typical of the disease in S. salar, however, other pathological signs were peculiar to wrasse. The recovery rate from the disease seen in wrasse was far more rapid than that recorded from S. salar. Shedding of the virus in the faeces of infected C. rupestris was also demonstrated. This study has illustrated for the first time the susceptibility of wrasse to IPN and that they can shed the virus in their faeces. This suggests that infected wrasse could be a source of continual reinfection in an affected sea site. Experimental infections of C. rupestris with PD followed a standard protocol for the reproduction of the disease in S. salar. Infection was by means of intraperitoneal injection with putatively infective material obtained from S. salar affected with PD. Two infection doses were used, the lowest dose used had been proven to be effective in inducing the disease in S. salar parr while the second dose, ten times higher than the first, had been shown to be effective in reproducing PD in S. salar smolts. The C. rupestris infected did not develop any of the typical signs of the disease seen in S. salar. It was, therefore, concluded that wrasse were not susceptible to PD.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv. / Härtill 3 uppsatser.

Thesis (M.Sc.)--Acadia University, 2000. / Includes bibliographical references (leaves 38-51). Also available on the Internet via the World Wide Web.

Thesis (M. Sc.)--Acadia University, 1999. / Includes bibliographical references (leaves 143-150). Also available on the Internet via the World Wide Web.

Thesis (M. Sc.)--Acadia University, 1999. / Includes bibliographical references (leaves 143-150). Also available on the Internet via the World Wide Web.