A partial cystatin cDNA from rainbow trout was generated by reverse transcription polymerase chain reaction with two degenerate primers. The partial cystatin PCR product was 168 bp and used to screen trout liver λgt 11 cDNA library. Four positive clones were isolated and designated as cstl, cst2, cst3 and cst4. Only cst2 contained the full-length cystatin cDNA which was 674 bp and included 5' untranslated region and the polyadenylation signal sequence AATAAA in the 3' region. Translation of the cDNA contains 132 amino acid residues. Comparison of the amino acid sequence with those of family II cystatin indicated that the 21 amino acids at N-terminal end is a signal peptide that leads to cystatin secretion, and the 111 amino acids are mature cystatin. Four cysteine residues in the cystatin may form two disulfide bonds for the secondary structure. Cst2 was subcloned into pGEM-3z for Northern and Southern blot experiments. Northern blot indicated that trout cystatin mRNA is about 750 bp. Cystatin is expressed in all tissues examined but at various levels. This difference may reflect the regulation of cysteine proteinase activities. Southern blot of trout genomic DNA showed that the copy number of the trout cystatin gene is probably one per haploid genome. / Graduation date: 1997

Dimethylbenz (a) anthracene (EMBA), a carcinogen that requires metabolic activation to produce active metabolites capable of binding to DNA, has been studied in the trout and other fish. Polycyclic aromatic hydrocarbons (PAH) are of importance as they are ubiquitous in the environment and their carcinogenic effects in fish from contaminated waters are an important indication of the pollution risks to man. Since such pollution risk assessment presents the involvement of multiple agents, the study of the modulation of PAH-DNA binding produced by other agents is important. In this study the effect, of dietary pretreatment at 500 ppm, 100 ppm and 2000 ppn, using BNF, Aroclor 1254, or indole-3-carbinol (I3C) respectively on DMBA-DNA binding was examined. To study the effect of age on sensitivity to DMBA-DNA binding, adult trout and fry were used in two separate studies. The fish were fed treatment diet for at least two weeks. Fry were then injected with [³H] DMBA, at 22.4 μCi/3.9 x 10⁻² μmole/fish and adult trout at 284 μCi/1.58 μmole/fish. Liver DNA was isolated, purified and binding of radioactivity to DNA was examined and computed as the covalent binding index (CBI). Mean CBI for control dietary group vising adult trout was 1000 fold lower than for fry. Statistical analysis of covalent binding index for the treatment groups revealed that a statistically significant (p < 0.05) inhibition in DNA-DMBA binding response in adult trout and fry was produced fcy the DNF dietary treatment only. Diethylnitrosamine (DENA), a potent hepatocarcinogen in several animal species belongs also to the class of compounds that require metabolic activation. Dietary treatment and continuous exposure of trout to the carcinogen in water, have produced hepatocellular carcinctnas. The water exposure also produced a dose related DNA ethylation of the O⁶ position of guanine, believed to be the promutagenic adduct produced after DENA exposure. This study examines two other routes of exposure to DENA, in vitro hepatocyte incubations and i.p. injection. Adult trout and fry were injected with [³H] DENA. Adult fish received 3.3, 16.5, and 33 mg/kg DENA, and fry received 10, 50 and 100 mg/kg. Hepatocyte incubation was performed with doses up to 220 μM [³H] DENA, or 1 mM unlabelled DENA. DNA from fish livers and from hepatocyte pellets was isolated, purified and examined for radioactive binding of the DENA metabolites or in the case of the unlabelled DENA, was analyzed for O⁶ and N7 adduct using an HPIC technique with fluorescence detection. O⁶-EtG adduct after DENA exposure, in DNA of hepatocytes obtained from trout pretreated with beta-naphthoflavone (BNF, a known inducer of cytodhrcme P-450 dependent enzyme activities involved in the activation of xenobiotics) was below the limits of detection of the HPDC-fluorescenoe detection procedure used. To examine further if the lack of DNA binding and absence of the O⁶-EtG adduct was due to rapid repair, the persistence of O⁶-EtG after exposure to 40 nM ethylnitrosourea (ENU, a direct ethylating agent) was studied in hepatocytes at 2, 4, and 5 hours after treatment. The activity of the alkyltransferase protein involved in the repair of alkylguanines also was determined using liver extracts from adult rainbow trout. The studies did not reveal a significantly high rate of repair. It is concluded that i.p. injection and in vitro hepatocyte incubations are not a good method for studying the kinetics of activation and DNA binding of DENA in the rainbow trout. The i.p. route may lead to substantial loss of the dose of the carcinogen administered and hepatocyte incubations are limited by the toxic effects of increasing carcinogen concentration. The reasons mentioned above, coupled with low levels of metabolism of nitrosamines in trout results in the inability to detect and study the appearance, persistence and repair of the O⁶-EtG adduct. / Graduation date: 1989

The DNA repair capabilities of rainbow trout (Salmo gairdneri) were studied vising the method of autoradiography. Trout were fed a semi-purified control diet containing 0 ppm, 50 ppm, or 300 ppm cyclopropenoid fatty acids (CPFA) for 6-9 weeks. Liver slices were prepared and exposed in vitro to a control treatment, ultraviolet irradiation (UV), ethidium bromide (EB), UV/EB in succession, or aflatoxin B₁. The degree of DNA repair was analyzed in terms of net grains per cell. Except following the EB treatment, fish on the control diet revealed an absence of ongoing DNA repair. Trout fed 50 ppm CPFA exhibited a consistently low level of repair over time following the in vitro control treatment. Fish fed 300 ppm CPFA revealed a relatively higher degree of ³H-Me-thymidine incorporation indicative of induced DNA repair following the in vitro control treatment, and the degree of repair increased with time on the diet. UV-irradiation caused a marked increase in the degree of induced DNA repair in 300 ppm CPFA fish at 6 and 7.5 weeks, and in 50 ppm CPFA fish at 7.5 weeks. Follcwing UV-irradiation, liver slices were exposed to EB, a DNA intercalating agent used to inhibit normal DNA replication. However, in contrast to the desired effect, EB caused a marked decrease in the degree of repair synthesis observed in 300 ppm CPFA fish at 6 and 7.5 weeks. Indicative of intercalation, the in vitro EB treatment caused a moderate degree of ³H-Me-thymidine incorporation in fish fed the control diet. Repair was also induced in 300 ppm CPFA fish following exposure to EB at 6 and 7.5 weeks. Aflatoxin B₁ induced DNA repair to various degrees in fish on all diets at 7.5 and 9 weeks. In comparison to the in vitro control treatment, it was observed that the degree of induced DNA repair was decreased significantly - "completely" following the UV, UV/EB, and EB treatments - in fish fed the 300 ppm CPFA diet for 9 weeks. In view of the low level of DNA repair observed in rainbow trout using autoradiography, the repair capabilities were studied using a more sensitive assay, bromodeoxyuridine (BrdU) photolysis. Isolated hepatocytes were prepared from fish fed the various diets and exposed in vitro to a control treatment, UV-irradiation, or 4-nitroquinoline-N-oxide. The obtained results were nonconclusive indicating technical improvements on the assay need to be made. / Graduation date: 1988

The introduction of trout has been implicated in the declines in native fish fauna in New Zealand and worldwide. Since the introduction of brown (Salmo trutta) and rainbow (Oncorhynchus mykiss) trout to New Zealand in 1867, their distribution has spread and they have been implicated in the fragmentation of native fish distributions, particularly native non-migratory galaxiids. However, in the Upper Waimakariri basin the co-occurrence of trout and galaxiid populations is relatively common, even in streams where trout reach sizes known to be piscivorous. To investigate mechanisms that may regulate trout and galaxiid co-occurrence, I investigated differences in antipredator behaviour and growth rate between stream types with varying levels of trout presence. Using quantitative survey data collected between 1997 and 2006, I found that trout abundance was low and varied annually in frequently disturbed sites compared their high abundance in stable streams. This finding was used to classify streams into three population types, barrier (trout absent), disturbed (trout presence intermittent) and sympatric (constant trout presence). Using this classification, I tested the effects of trout chemical cues on galaxiid activity and refuge use in artificial channels. There were no differences in activity or refuge use between trout odour and there were no effects of population type or galaxiid size during both the day and the night. Using otolith weight-fish length relationships in galaxiids collected from each population type, I found that galaxiid growth rate was higher in disturbed streams than in stable streams either with or without trout. An experiment manipulating trout size and presence, over two months in a natural stream, found galaxiids from treatments without trout grew slower than those with trout. Slow growth rates in galaxiids above trout-migration barriers and in sympatry, combined with low growth rates in treatments without trout suggest that the mechanisms that regulate galaxiid growth are more complex than previously thought.

Spilled heavy fuel oil (HFO) sinks within the water column and accumulates in sediments, affecting aquatic organisms that are not typically exposed to oils that float. Previously, the 3-4 ring alkyl substituted polycyclic aromatic hydrocarbons (PAHs) have been identified as the major toxic components in crude oil. Since HFO is comprised of higher concentrations of 3-4 ringed alkyl PAH and an abundance of 5-6 ringed PAH relative to crude oil, it is predicted to be more toxic to the early life stages of fish. An effects-driven chemical fractionation (EDCF) of HFO 7102 was undertaken to establish the toxicity relative to crude oil, and to identify the compounds that are bioavailable and chronically toxic to the early life stages of fish. In this EDCF, the complex HFO 7102 mixture was separated by low temperature vacuum distillation into three distinct fractions, 2, 3 and 4. Each fraction was assessed using a chronic bioassay to determine whether it contained components that caused toxicity to rainbow trout embryos similar to that of the whole oil. Acute bioassays with juvenile trout demonstrated the presence of compounds that induce cytochrome P450 enzymes, an indicator of exposure to PAH. Fraction 3, the fraction more toxic than the parent mixture, was further separated by cold acetone extraction into fraction 3-1 (PAH-rich extract) and fraction 3-2 (wax residue), and assessed with the same bioassays. Simultaneous chemical analysis with bioassays guided the fractionation, and identified compounds abundant and consistently present in toxic fractions. Due to resistance to dispersion of HFO, a chemical dispersant was used with vigorous mixing to drive the maximum amount of oil into solution to minimize the potential for false negatives and the volume of test material used. The potency of HFO 7102 and its fractions were also measured using water accommodated fractions (WAFs) produced by a continuous flow system of water flowing through oil coated gravel. Both exposure methods traced the toxicity from whole oil into fractions containing higher concentrations of 3-4 ring alkyl PAH, similar to crude oil. This research is the first toxicological assessment of HFO 7102, which is essential for determining the risk of spills of HFO to fish, and whether the risk of oils can be predicted from their alkyl PAH composition. / Thesis (Master, Biology) -- Queen's University, 2013-01-24 14:14:16.278

Aquaculture is growing rapidly worldwide and is projected to become the major source of fish used for human consumption. A major factor that limits aquaculture reaching its full potential is an adequate supply of the raw materials necessary for formulated fish feeds. The dependence of modern aquaculture on fishmeal obtained from wild fisheries is not environmentally sustainable and replacements for fishmeal must be found. Some animal by-products are viable replacements for fishmeal, and can provide sufficent nutrition for high growth rates, but little is known about the potential of animal by-products to adversely affect fish health. The objectives of these experiments were to determine if animal by-products used in fish feeds impair immune response or alter bone physiology in cultured juvenile rainbow trout. Four animal by-product containing diets (poultry meat meal (PMM)/ PMM plus feathermeal / PMM plus bloodmeal) and two reference diets (fishmeal or soya) were evaluated to determine their effect on innate immune response, the ability of fish to cope with normal husbandry stressors, and bone physiology. PMM was then selected due to its favourable amino acid profile and high digestibility and assessed to determine if the high levels of fishmeal replacement that may be required in the future, impact the health of rainbow trout. Due to the lack of reliable indicators of bone quality and quantity in salmonids the effects of exercise and phosphorus deficiency in rainbow trout were also examined. Relative to the fishmeal control diet, fish fed diets with PMM [(PMM) 50% crude protein, by substitution], PMM plus two percent blood meal, or PMM plus five percent feather meal, did not have an impaired innate immunity (lysozyme, alternative complement, phagocytosis, intracellular respiratory burst, differential counts of peripheral blood leukocytes) or changes in bone physiology as assessed by dynamic bone histomorphometry. Higher levels of PMM (0-70% digestible protein, by substitution) caused a reduction in apparent net mineral retention of phosphorus and calcium (P<0.001), a lower vertebral bone mineral content (P<0.001) and reduced vertebral mechanical properties (compressive extension (P=0.04), Young’s Modulus (P=0.03)), but fish growth was not affected. Exercise influenced bone modelling, with exercised animals having a reduced bone area and trabecular thickness (P=0.01), increased autocentrum width (P=0.04), and higher bone mineral content (P= 0.02); however, bone mechanical properties were unaffected. Induction of genes (receptor activator nuclear factor kappa beta and osteoprotogenerin), involved in the resorption of mineralised tissue, was not observed in fish fed phosphorus deficient diets although scales were evidenced to be an important source of labile minerals. Overall our results indicate that low level replacement of fish meal by poultry meat meal, and blends of poultry meat meal with blood or feathermeal do not affect fish innate immune response, bone physiology, or growth however the greatly elevated levels of poultry meat meal that may be required in future salmonid aquafeeds could increase the risk of spinal malformations. Thus the category 3 animal by products tested are valuable fishmeal replacements for aquaculture based on the endpoints measured in this study.

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.

An Investigation Into the ecology of benthic macro-invertebrates in earthen ponds subjected to intensive trout (Salmo trutta L.) culture practices at Howletoun, Central Scotland, was conducted between May 1984 and January 1986. Soil and water quality, seasonal changes in benthos, its role in the trout diet and the Interaction between fish and benthos were studied. Pond benthos mainly comprised 6 major groups including Oligochaeta. (10 species), Chironomldae (18 species), Molluscs and Hirudinea (2 species each) and an asellid and a sialid species. Oligochaeta formed 78 to 90% of benthic fauna, dominated by Tubifex tubifex. Limnodrilus hoffmeisteri, L. udekemianus and Psammoryctides barbatus, with an average population density of 68,400 - 191,200 worms mˉ², and exhibited peaks in summer and late autumn corresponding to two major breeding periods. The principal species of Chironomidae were Chironomus spp., Procladius spp. and Prodiamesa olivacea, with a population density of 5,400 to 14,900 ind. mˉ² and forming 7 to 13% of the total benthos with peaks in spring and autumn. Dry biomass of total benthos varied from 24-59 g mˉ² in the cultured ponds with oligochaetes accounting for 14-49 g mˉ² and chironomids 4-7 g mˉ². The mean annual dry weight production of total benthos varied from 130-215 g mˉ² in the cultured ponds, with oligochaete production of 94-160 g mˉ² and chironomid production of 20.6-33.5 g mˉ². An investigation into the ecology of benthic macro-invertebrates in earthen ponds subjected to intensive trout (Salmo trutta L.) culture practices at Howietoun, Central Scotland, was conducted between May 1984 and January 1986. Soil and water quality, seasonal changes in benthos, its role in the trout diet and the interaction between fish and benthos were studied. Pond benthos mainly comprised 6 major groups including Oligochaeta (10 species), Chironomidae (I8 species), Mollusca and Hirudinea (2 species each) and an asellid and a sialid species. Oligochaeta formed 78 to 90% of benthic fauna, dominated by Tubifex tubifex. Limnodrilus hoffmeisteri, L. udekemianus and Psammoryctides barbatus, with an average population density of 68,400 - 191,200 worms mˉ², and exhibited peaks in summer and late autumn corresponding to two major breeding periods. The principal species of Chironomidae were Chironomus spp., Procladius spp. and Prodiamesa olivacea, with a population density of 5,400 to 14,900 ind. mˉ² and forming 7 to 13% of the total benthos with peaks in spring and autumn. Dry biomass of total benthos varied from 24-59 g mˉ² in the cultured ponds with oligochaetes accounting for 14-49 g mˉ² and chironomids 4-7 g mˉ². The mean annual dry weight production of total benthos varied from 130-215 g mˉ² in the cultured ponds, with oligochaete production of 94-160 g mˉ² and chironomid production of 20.6-33.5 g mˉ². In an unstocked control pond the total production was 55 g mˉ², 35 g mˉ² of which was accounted for by oligochaetes and 8.06 g mˉ² by chironomids. Analyses of gut contents of the farmed trout showed that 12% of the diet by volume consisted of natural food, mainly benthos. Fish selectively fed on chironomid larvae, Mollusca, Asellus aquaticus and Sialis lutaria. Fish took maximum natural food in the morning and evening. Experimental enclosures to exclude fish from selected areas of the ponds demonstrated that number of species increased outside the enclosures but, except for chironomids, population density, biomass and production generally increased inside the enclosure. The possibility of explaining this result in terms of differential predation is discussed.

River flow regime is fundamental in determining lotic fish communities and populations, and especially of salmonid fishes. Quantifying the effects of human induced flow alteration on salmonids is a key question for conservation and water resources management. While qualitative responses to flow alteration are well characterised, a more intractable problem is quantifying responses in a way that is practical for environmental management. Using data drawn from the Environment Agency national database, I fitted generalised linear mixed models (GLMMs) using Bayesian inference to quantify the response of salmonid populations to the effects of impounding rivers, flow loss from rivers due to water abstraction, and the mitigating effects of flow restoration. I showed that in upland rivers downstream of impounded lakes, the magnitude of antecedent summer low flows had an important effect on the late summer abundance of 0+ salmonids Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). In contrast, the abundance of 1+ salmon and brown trout appeared to be largely unresponsive to the same flows. I demonstrated that short-term flow cessation had a negative impact on the abundance of 1+ brown trout in the following spring, but that recovery was rapid with negligible longer-term consequences. I further established that flow restoration in upland streams impacted by water abstraction provided limited short-term benefits to salmonid abundance when compared with changes at control locations. However, while benefits to salmonid abundance were limited, I detected important benefits to the mean growth rates of 0+ and 1+ brown trout from flow restoration. I discuss the implications of my findings for salmonid management and conservation and propose a more evidence-based approach to fishery management based on robust quantitative evidence derived using appropriate statistical models. The current approach to flow management for salmonids requires revision and I recommend an alternative approach based on quantitative evidence.

Triploid (sterile) trout potentially offer a more risk-averse option for stocking popular non-native sport fish; however the relative performance (e.g., survival and growth) of triploid versus diploid fish in natural settings is not well understood. I evaluated the relative performance of triploid versus diploid brook trout (Salvelinus fontinalis) stocked in high mountain Uinta lakes in response to food availability and lake morphology. I chose a set of 9 lakes that included a range of elevation and lake morphology. I observed no difference in CPUE or relative weight (Wr) of both types of trout in all lakes. Food availability (e.g., zooplankton and macroinvertebrates) varied substantially among lakes; however I observed no discernible difference between diploid and triploid diets, diet preference, or isotopic trophic signatures. Physical lake characteristics (e.g., dissolved oxygen [DO, mg/L] and temperature [°C]) were within or near optimal brook trout conditions (metabolically beneficial range) during the summer, but over-winter DO reached near lethal levels under the ice. In sum, between the two strains, I did not observe any significant differences in relative performance measured across a number of indices; however the size distribution of diploid fish was marginally (20 mm) skewed towards larger fish (a difference likely not great enough to be detectable by the average angler). In contrast to the similarity in performance between strains, I did observe considerable variability in performance of brook trout across lakes as a function of lake productivity, food availability, and most importantly fish density. Fish performance was greater in lakes with a lower density of stocked fish. Overall, the results from this study indicate triploid brook trout offer a viable and risk-averse alternative to stocking diploid fish in Uinta mountain lakes. Stocking triploid fish should decrease the threat of uncontrolled expansion into adjacent water bodies, while still allowing managers to maintain a popular non-native sport fishery.