Chemokines are cytokines with chemoattractant ability, and comprise one of the major groups of molecules in immune system. These are small, secreted proteins cause the migration of leukocytes to the sites of injury. Over 40 mammalian chemokines have been identified to date, and they have been implicated in a number of immune mediated processes, including regulation of inflammation, antigen presentation, blood cell development, metastasis, viral infection and wound healing. In rainbow trout, there have been fewer chemokines reported and only one functional study has been published. Rainbow trout chemokine 2 (CK-2) is the only known CC chemokine with a mucin stalk, which has the potential for extensive <em>O</em>-glycosylation. However, no functional characterization has been performed on this molecule yet. CK-2 shares the presence of a mucin stalk with the mammalian chemokines, fractalkine (CX₃CL1), lymphotactin (XCL1), and CXCL16. Another related trout CC chemokine sequence, CK-2. 1, has been discovered recently, which has 98% nucleotide sequence identity with CK-2. CK-2. 1 was believed to be a separate gene due to its apparent differential regulation in challenged rainbow trout. The question remained, however, whether or not CK-2. 1 was a separate gene or an allele of CK-2. The goal of this project was to further characterize both CK-2 and CK-2. 1. <br /><br /> Through genomic PCR on several outbred individuals it was shown that CK-2. 1 is an allele of CK-2 but not a separate gene. Reverse transcriptase (RT) PCR analysis revealed an increased level of transcript both CK-2 and CK-2. 1 in response to phytohaemagglutinin (PHA) stimulation of head kidney leukocytes (HKL) and peripheral blood leukocytes (PBL) collected from fish with different allelic distributions. Similar results were also observed in the rainbow trout macrophage/monocyte cell line, RTS11. Moreover, an anti-CK-2 antiserum was developed in rabbits, which cross-reacted with CK-2. 1. This newly produced antibody was used to determine the protein expression levels in PHA stimulated rainbow trout tissues. RT-PCR was also performed on the same tissues in order to examine the transcript expression. Rainbow trout with both CK-2 and CK-2. 1 were used for this experiment. An overall decreasing pattern of transcript (both CK-2 and CK-2. 1) was observed in brain and HK over 24 hours, while protein was still detected at 24 hours post stimulation. However, in spleen the CK-2 transcript showed a slight upregulation at 4 hours post stimulation along with a very little or no CK-2. 1 expression, although no protein was detected in spleen. Liver showed a very low level of CK-2 and CK-2. 1 transcript at 8 hours post stimulation; while protein was again detected at 24 hours post stimulation. In addition, the sizes of the proteins found in different tissues were larger than expected (&le;30 kDa for CK-2 or &le;35 for CK-2. 1), perhaps due to the presence of extensive <em>O</em>-glycosylation at the mucin stalk of the protein. <br /><br /> A chemotaxis assay was carried out, which is the definitive assay for chemokine activity. This assay showed migration of peripheral blood leukocytes across a membrane with 5??m pores toward CK-2 at an optimal concentration of 500ng/ml (17nm). Moreover, by pre-treating the recombinant chemokine with the polyclonal antisera, it was shown that the chemokine was actually causing the chemotactic activity. Pre-treatment of the cells with pertussis toxin, an inhibitor of G-protein signalling inhibited the migration of PBLs, established the fact that CK-2 caused chemotaxis by binding to a 7 transmembrane, G-coupled receptor just like all other known chemokines. Interestingly, CK-2 was also shown to attract RTS-11 cells. <br /><br /> Overall, the above findings indicate that CK-2 is functionally a chemokine with two very different alleles in rainbow trout. It is probably heavily <em>O</em>-glycosylated and different tissues express different sizes of the protein. This is only the second functional study of a fish chemokine.

The aquaculture sector is currently thriving, and has expanded to meet the demand for fish and shellfish as an alternative protein source to meat. This is especially true for high value products such as Atlantic salmon, where in Scotland salmon farming is reported to be worth> £1 billion to the national economy. Currently around 40% of farmed fish and shellfish destined for human consumption are derived from aquaculture. Therefore, a great deal of attention is paid to problems that the industry faces, with fish diseases of paramount importance. A variety of species of bacteria, viruses and parasites are common in the aquatic environment, which can result in serious diseases amongst fish stocks. As a result, ways to improve disease resistance have been the focus of much attention, with the use of vaccines considered a desirable way forward. However, other approaches are also followed, such as the use of immunostimulants to improve fish health in a more limited, non-specific way, or the use of genetic markers to allow selective breeding of important disease resistance traits. For all of these approaches more information is needed on the pathways that give rise to disease resistance in fish in different situations, to allow their manipulation or monitoring, and the studies in this thesis are directed towards this goal. Fish has been used as a model to study the evolution of vertebrate immunolity for some deacades, especially work on humoral immune responses where knowledge on antibody production has dominated much of the literature on fish immunology. In contrast, little known about specific cell-mediated immunity in fish, even though it also likely plays an important role in the immune system and disease resistance. Therefore, this thesis has been focused on analysing such responses, taking advantage of the recently discovered cytokines of adaptive immune responses in fish, which allow transcriptomic studies in particular to look at the molecules turned on during infection and after vaccination. Thus the goal of this thesis was to take advantage of some successful vaccines that exist for rainbow trout, and examine the gene expression changes that occur in vaccinated trout post-challenge with the homologus pathogen, and to try to dissect pathways that may correlate with disease resistance in this species.

Stocking, transfer and introductions of hatchery-reared salmonids are commonly used to enhance recreational or commercial fisheries and to preserve or re-establish threatened populations (Cowx, 1994). Whilst a lot of effort has been directed toward understanding the production and stocking methods of hatchery-reared salmonids, relatively little is known about the post-stocking survival and behaviour of these fish (Hickley, 1994). In particular there is little available information concerning the post-stocking dietary habits, dispersion and behaviour of hatchery-reared brown trout; particularly following release into standing waters. Consequently, a study was instigated to examine the relative post-stocking dispersion and temporal changes in the diets of hatchery-reared brown trout released to support a commercial recreational fishery, Carron Valley Reservoir. Further laboratory studies examined if resident brown trout display a prior-resident competitive advantage over stocked conspecifics, if hatchery-reared brown trout display non-cost effective aggressive behaviour and the short-term changes in the feeding efficiency of naive hatchery-reared brown trout when experiencing novel prey. 1000 commercially produced hatchery-reared brown trout were marked with a subcutaneous alcian blue tattoo and released into Carron Valley Reservoir at the start of the 1999 fishing season. Stomach samples were taken from angler recaptured hatchery-reared fish along with consecutively captured resident brown trout. The diets of the two groups were compared to assess the relative post-stocking temporal changes in the diets of the hatchery-reared brown trout. This study found hatchery-reared brown trout to consume lower weights and numbers of prey, and they appeared to exhibit a preconditioned 'look up' dietary response to surface prey immediately following release than resident conspecifics. It was further observed that although hatchery-reared brown trout did not immediately adapt to natural diets, their relative foraging efficiency increased over the sample period. In laboratory experiments naive hatchery-reared brown trout further demonstrated the improved feeding efficiency with experience. 1000 commercially produced hatchery-reared brown trout were tagged with a combination of Visible Implant (VI) and 'Floy' style tags prior to release during the 1998,1999 and 2000 fishing seasons. An angler survey programme was instigated to record the reported recapture positions of these tagged fish in order to assess the post-stocking gross dispersion patterns of hatchery-reared brown trout in Carron Valley Reservoir. A further 3 trout were radiotagged to elucidate the fine scale post-stocking dispersion of these fish. This study found hatchery-reared brown trout to disperse quickly from their respective release sites, although over a relatively restricted area. Individual hatchery-reared brown trout were observed to exhibit high levels of activity immediately following release, during which period they covered relatively large total distances within a relatively restricted area. In an artificial stream environment, established wild brown trout displayed a prior-resident competitive advantage over later introductions of both hatchery-reared and wild conspecifics. Established wild fish initiated more aggressive acts and maintained home stations closer to a point source of feed than introduced trout. Introduced hatchery-reared brown trout were more aggressive and exhibited a lower mean specific growth rate than simultaneously stocked wild conspecifics, suggesting that excessive expenditure of energy for unnecessary aggression may contribute to the poor post-stocking survival in hatchery-reared brown trout.

No description available.

This paper is about analyzing water quality of a stream that flows in southwest of Swedencalled Hasslebäcken. The stream is a tributary of Suseån and has its springs in the naturereserve Biskopstorp and flows through a mixed landscape of mostly spruce forests andagriculture land. Little is known about Hasslebäcken and its water and therefore a study by request of Suseåns vattenråd was performed. The study consisted of analyzing the water ofphosphorous, nitrogen, pH, suspended particles and conductivity. To broaden the examination of water quality sampling of benthic macroinvertebrates and two index (ASPTindex and Shannon’s diversity index) of water quality were used. Another part of the study were to search for fish barrier in the stream and to evaluate the possibilities for troutspawning and habitats. The whole stretch of the stream were examined and eleven differentsampling sites were used. The results show that Hasslebäckens upper parts have low levelsof nutrients while the lower parts show intermediate to extremely high levels. The indexresults points at low ecological values for Shannon and high ecological values for ASPT. Thisis normal for a stream that flows through an agricultural landscape. The results concerning pH show a lower pH in the upper parts than in the lower parts. This is also normal for a stream in a production forest of spruce in these parts of Sweden. Hasslebäcken also have several fish barrier that prevent various fish species to migrate up along the stream from thesea. The evaluation of trout habitats and spawning area show decent possibilities for successful reproduction and growth.

Lake trout populations were extirpated from the lower four Great Lakes by 1960 and from Lake Champlain by 1900. The decline of lake trout populations fueled a wave of restoration-based research that spanned the Great Lakes and filled in many of the gaps in our knowledge of lake trout behavior and ecology. However, remarkably little is known about lake trout spawning behavior, even less about sex-specific differences in spawning site use. Lake trout use specific spawning sites, and may return to the same site year after year. More males are caught on spawning sites than females and are present at spawning sites earlier in the spawning season. The focus of this project is to describe the spawning movements of male and female lake trout within and among spawning seasons and spawning sites. I used acoustic telemetry in Lake Champlain to look at specific questions of spawning site fidelity and whether or not there were differences in male and female movements. I hypothesized that males show site fidelity and remain at a preferred site during the spawning season, whereas females 'sample' multiple spawning reefs to maximize their reproductive success. I established an acoustic telemetry array of ten acoustic receivers placed over eight spawning sites and implanted acoustic transmitters (tags) in 44 male and 48 female lake trout over two years. During two spawning seasons, males spent more time on spawning sites than females. Both male and female lake trout that were active on monitored sites during the spawning season selected a single preferred site. There was no difference in the number of sites visited by males and females. Of the lake trout detected during both spawning seasons, most returned to their capture site in the subsequent spawning season, showing evidence of site fidelity. I also developed a binomial rolling residence test (BRR test) to improve the current method of assessing the duration of a fish's residence at a single receiver. I measured daily detection probabilities (DP) at a given distance from a receiver site. The BRR test evaluates a tag's residence every minute by moving a one-hour time window centered on time t across the duration of the data. The daily DPs are incorporated into a binomial test of the null hypothesis that a fish is not within x meters of the receiver at time t. I performed a 48-hour stationary residence test using two onsite tags and two offsite tags and compared the performance of the BRR test to three residence assessment methods found in the literature. The results showed that the BRR test performs better than all of the time-threshold residency evaluations in our 48-hour stationary residence test. We suggest that this method has the potential to advance the field of telemetry by improving the interpretation of telemetry data.

Rainbow trout macrophages were stimulated with PMA to produce 02- and H2O2 as detected by the reduction of nitroblue tetrazolium (NBT) and the oxidation of phenol red respectively. Addition of DDC or nitroprusside, inhibitors of superoxide dismutase (SOD) increased O2-levels and decreased H2O2 levels, whereas addition of exogenous SOD had the reverse effect. Such data are indicative of a respiratory burst pathway in teleost macrophages comparable with that of mammals. Respiratory burst activity, acid phosphatase activity and RNA synthesis in rainbow trout macrophages which have been stimulated in vitro with the mitogen Concanavalin A (Con A) or in vivo by injection of formalin-fixed Aeromonas salmonicida in Freund's incomplete adjuvant (FIA) was analysed. With Con A, in vitro stimulated head kidney (HK) or elicited macrophages had increased O2-production and RNA synthesis but no significant increases in H2O2 or acid phosphatase activity after 72h post-stimulation with Con A. In contrast, all functions were increased in in vivo stimulated macrophages compared with FIA-elicited peritoneal macrophages. In a bactericidal assay, Con A stimulated macrophages did not show an increase in killing of an avirulent strain of A. salmonicida (004) above control levels whereas in vivo stimulated macrophages not only displayed increased killing of the avirulent strain of bacteria but also acquired the ability to kill a virulent strain (048). Thus, Con A stimulated macrophages only possessed some of the features of activation whereas in vivo stimulated macrophages were activated as defined by the increased bactericidal activity. Peritoneal washes obtained in the collection of activated macrophages were able to increase NBT reduction in normal HK macrophages suggesting the presence of a soluble activating factor. Lymphokine (LK)-containing supernatants produced using either HK or blood derived leucocytes, by pulsing with 10ug/ml Con and 5ng/ml PMA, were able to increase O2- and H2O2 production, to enhance the killing of an avirulent strain of A. Salmonicida and conferred the ability to kill a virulent strain of A. salmonicida. The LK present in these supernatants was therefore designated a macrophage activating factor (MAF). The use of potential second signals to enhance the killing of bacteria by LK-treated macrophages, met with limited success. Only A. salmonicida (strain 004) LPS was able to produce a small increase in killing above LK-treatment. The MAF produced in this study was tested for antiviral/interferon (IFN) activity. The results showed that the supernatants did contain IFN activity. Attempts to semi-purify the MAF from antiviral activity showed the two activities to co-purify, indicating that both activities may be due to the same molecular species. The retention time of the MAF/IFN, coupled with the results of SDS-PAGE analysis showed the molecular weight of the moiety to be approximately 19K daltons. Both activities were sensitive to low pH (pH 2), high temperature (60oC) and trypsin, providing further evidence that the MAF and IFN activity produced in these studies may be due to the same molecular species, possibly akin to IFN- of higher vertebrates.

Vaccination and the use of immune stimulants are two important ways to mitigate the costs of disease in fish aquaculture. A vaccine to Enteric redmouth disease (ERM) was the first licensed fish vaccine in the world. Although effective in protecting fish from the motile bacterial (Yersinia ruckeri) infection, ERM can occur in ERM vaccinated fish due to the rise of non-motile Y. ruckeri that does not express flagellin. This highlighted the need for continual improvement of vaccine efficacy and the importance of flagellin in fish immune responses. In this thesis the immune response to the ERM vaccine was studied first to give insights for vaccine development. A recombinant flagellin from Y. ruckeri (YRF) was then produced and its bioactivities were investigated in vitro and in vivo. The immune response to ERM vaccination was studied in rainbow trout in two major and relevant immune organs, the spleen and gills. Intraperitoneal injection of the ERM vaccine induces an early balanced expression of pro- and anti-inflammatory cytokines and adaptive cytokines in the spleen, with a heightened expression of acute phase proteins (APPs) and anti-microbial peptides (AMPs) in both spleen and gills. The analysis suggests that ERM vaccination activates host innate immunity and the expression of specific IL-12 and IL-23 isoforms leading to a Th1 and Th17 biased immune responses. This study has increased our understanding of the host immune response to ERM vaccination and the adaptive pathways involved. The early responses of a set of genes established in this study may prove useful as biomarkers in future vaccine development in aquaculture. YRF was next produced in a bacterial system, and purified. Its bioactivity was investigated first in the trout macrophage cell line RTS-11 and head kidney primary cell cultures. YRF is a potent activator of pro-inflammatory cytokines, APPs, AMPs and subunits of the IL-12 cytokine family in vitro. This property was further confirm in vivo in multiple tissues after intraperitoneal injection of YRF. These results suggest that flagellins are important pathogen-associated molecular patterns (PAMPs) that can activate an inflammatory response in fish not only in vitro but also in vivo. Furthermore, YRF was shown to be the most potent PAMP in vitro, in terms of activation of an inflammatory response, compared to pure LPS and peptidoglycan. In addition, YRF mixed with complete Freund's adjuvant can induce YRF-specific IgM antibodies in rainbow trout. These antibodies are able to neutralize YRF bioactivity, and react against the middle domain of YRF, as assessed in Western blot analysis. When YRF was fused with a protein antigen, it increased the antigen-specific IgM antibody response. This analysis reveals that YRF is a potent activator of host immune responses and can be used as an immune stimulant and adjuvant to improve vaccine efficacy.

Graduation date: 2001

Resident bull trout (Salvelinus confluentus) may be particularly vulnerable to human related disturbance, however very few studies have focused on resident bull trout populations. The abundance of bull trout is one measure of the strength and potential for persistence of a population. Habitat characteristics may influence resident bull trout abundance to differing degrees and by varying means at multiple spatial scales. We used day and night snorkel counts to assess relative bull trout abundance. A modification of the Forest Service R1/R4 Fish and Fish Habitat Inventory was used to assess habitat characteristics associated with resident bull trout. Logistic and multiple linear regression were used to assess the relationships between resident bull trout abundance and habitat characteristics at the patch (1 to 5 km), reach (0.5 to 1 km) and habitat unit (1 to 100 m) scales. Site categorical variables were used along with quantitative habitat variables to explain among-site and across-site variation in the data. The significance of both quantitative habitat variables and categorical site variables at various spatial scales suggest that relationships between bull trout abundance and habitat characteristics are complex and in part dependent on scale. The characteristics of individual habitat units explained little of the variation in bull trout presence/absence (logistic regression; Somers' D=0.44) and density (multiple linear regression; adjusted R��=0.08) in habitat units, however there were habitat characteristics that were significantly (P���0.05) correlated to bull trout presence/absence and density in habitat units. The relationships between habitat characteristics and bull trout presence/absence and density varied between habitat unit types. There was a strong quadratic relationship between bull trout abundance and mean summer water temperature at the reach (P=0.004) and patch scales (P=0.001). The mean temperature of patches appears to explain some of the variation in bull trout density at smaller spatial scales, such as reaches and habitat units. An appreciation of the complex nature of scale dependent interactions between bull trout abundance and habitat characteristics may help resource managers make wiser decisions regarding conservation of resident bull trout populations. / Graduation date: 2000