Thesis / Master of Science (MS)

This thesis examined the effects of exhaustive exercise and post-exercise recovery on metabolism and respiratory gas exchange in juvenile rainbow trout, in relation to the classical oxygen (O₂) debt hypothesis (Hill & Lupton, 1923). The initial study was a detailed quantitative analysis of the metabolic cost of post-exercise recovery in terms of O₂ and ATP equivalents. A 5 min bout of exhaustive exercise resulted in about a 2.0-2.5 fold increase in O₂ consumption (MO₂), a 6-8 fold increase in whole-body lactate (LAC) levels and a near depletion in whole-body glycogen (GLY), adenosine triphosphate (ATP) and creatine phosphate (CP) stores. Recovery of MO₂, LAC and GLY was usually complete by 6 h, though GLY did not always return to resting levels. Recovery of resting whole-body ATP required 1.0-1.5 h, whereas restoration of CP required only 5 min. Quantitative budgets of the cost of recovery were prepared, based on two opposite assumptions. The first scenario (A) assumed that all the GLY resynthesized came from LAC, and that the remaining 25% of the total LAC cleared was oxidized. This scenario accounted for 48% of the excess post-exercise O₂ consumption (EPOC) and 94% of the ATP budget. The alternative scenario (B), attributed 100% of the EPOC to LAC oxidation (64% of the total LAC cleared), while the remaining LAC (36%) was resynthesized into GLY. As scenario B accounted for only 35% of the ATP budget, scenario (A) appeared more probable. Subsequent experiments attempted to experimentally dissociate LAC disappearance from EPOC, via repetitive exercise bouts and prior GLY depletion. A 2nd bout of exhaustive exercise, given 6 h after the 1st, significantly reduced EPOC by 40%, whereas metabolite status (LAC disappearance and GLY, ATP, and CP restoration) remained essentially unchanged. In contrast, prior GLY depletion by 5 days starvation significantly lowered the LAC burden while EPOC remained unchanged. Neither the two scenarios of the theoretical analysis could adequately explain the relationships between EPOC and the various metabolites. It is likely that some blend of the two approaches may be more realistic. Nevertheless, these approaches all point to the conclusion that the EPOC is not directly related to the metabolism of LAC after exercise in the rainbow trout. Thus, in contrast to the classical O₂ debt hypothesis, LAC disappearance does not determine the magnitude EPOC after exhaustive exercise in rainbow trout. / Thesis / Master of Science (MS)

Juvenile rainbow trout were chronically exposed to cadmium in hard water, soft water, and in calcium-supplemented soft water in order to understand the effects of long term cadmium exposure in freshwater fish. A particular goal was to characterize changes in gill cadmium burden and the cadmium-binding properties of the gills during chronic sublethal exposures, so as to examine the applicability of the acute gill surface metal binding model or Biotic Ligand Model to trout chronically exposed to cadmium. Trout were exposed for 30 days to sublethal concentrations of cadmium in: a) moderately hard, Hamilton tap water (Ca = 1000 μM), b) synthetic soft water (Ca = 130 μM), or c) calcium-supplemented soft water (Ca = 260, 470, 770, and 1200 μM Ca). For both the hard and soft water cadmium exposures, no effects were observed on growth, swimming performance, and whole body ions. Growth and whole body and plasma Ca²⁺ concentrations were similar for all treatments in the calcium-supplemented soft water experiment; however, swimming performance was significantly reduced for the 470 μM Ca + Cd exposed fish. Acclimation to cadmium occurred in the hard water and lower concentrations of calcium-supplemented exposures but not in the soft water exposure. Cadmium accumulation was greatest in kidneys and gills and was directly related to cadmium exposure concentration. Tissue metal burdens were reduced with increased water calcium concentrations. Affinity of the gill for cadmium and the number of binding sites for cadmium decreased at higher water calcium concentrations. Affinity of the gill for Cd decreased with chronic cadmium exposure but binding site numbers increased with chronic Cd exposure. The acute gill binding model or Biotic Ligand Model, originally developed in soft water, was successfully applied to fish in both hard and soft water; however, complications arose when extending the model to fish chronically exposed to cadmium at various water calcium concentrations. / Thesis / Master of Science (MS)

Thesis / Master of Science (MS)

Trout genetically engineered to possess extra functioning growth homone genes were simulated, using injections of fish and bovine growth hormone. This was done to test potential and ecological impact of such genetic engineering on trout. Through analysis of growth and dry-mass budgets, it was determined that elevated levels of growth hormone resulted in increased growth and consumption. Potential constraints pertaining to respiration, density and evolutionary history were considered. Surprisingly, bovine growth hormone appeared to act as a super-normal stimuli, being more potent than the natural trout hormone. In fact the natural hormone inhibited growth at high dosages. Juvenile growth rates were less sensitive to elevated levels of growth hormone than those in more mature fish. Thus, improved growth was achieved by altering its normal ontogeny. This suggests that duplicating copies of growth hormone gene in an organism is not equivalent to a speciation event. Evidently, other manipulations would be required to increase the intrinsic metabolic power. Growth rate apparently is used as a cue for determining a particular ontogenetic trajectory (Stearns and Crandall, 1984). The fact that various species conform to one of at least four plastic developmental trajectories that are shaped by natural constraints and mortality patterns, means that growth hormone may elicit different responses in different species. The findings and implications of this study underscore the importance of using the holistic scope of ecology to achieve effective and efficient genetic engineering of target species. / Thesis / Master of Science (MS)

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No description available.

No description available.

Understanding that disease is a limiting factor to the aquaculture industry together with the knowledge that drugs and chemotherapeutics can cause newly resistant bacterial strains, has driven attention to finding new prophylactic measures to control diseases that include vaccination and the use of “functional feeds” to modulate the fish immune system. The supplementation of immunostimulants into fish diets is already widely used in aquaculture. However, searching for new and effective substances is one of the targets of many fish feed suppliers, including EWOS Ltd. who have co-funded the work presented in this study. There are a number of immunostimulant molecules used at present. Some bacterial components such as LPS are used to enrich fish diets and have been described to improve the natural immune defences. In contrast, peptidoglycan (PG), another ubiquitous component of the bacterial cell wall, has so far received less attention and is therefore investigated in this present study. Its ability to stimulate innate immunity is assessed using antimicrobial peptides (AMPs) as molecular markers, which are known to be involved in the early response against a broad range of pathogens. To date, AMPs in fish are not well characterised and in most cases the mechanisms of pathogen killing as well as the pathways inducing their expression still remain to be elucidated. Initially the cloning and characterisation of three novel trout β-defensin genes (omDB-2, omDB-3, omBD-4) was performed, and the molecules compared to the previously reported omDB-1. Each β-defensin gene was fully cloned and preliminary expression work in vivo and in vitro revealed the ability of these genes to be induced by bacteria and viruses. Analysis of the gene organization found that all three new genes contained three exons divided by two introns. Constitutive expression of these genes was detected by real time PCR ofmucosal and systemic tissues from healthy fish, with omDB-3 and omDB-4 showing the highest expression levels. Following bacterial challenge in vivo, the defensin genes were induced at the three mucosal sites examined (skin, gill, gut), with levels of omDB-2 and omDB-3 increased some 16-fold in gut and gill respectively. Using polyinosinic polycytosinic RNA (polyI:C) as a viral mimic, all of the four trout -defensin genes were induced in head kidney primary leucocyte cultures at 4h post-stimulation, with omDB-1 and omDB-3 showing particularly high expression. To determine the -defensin spectrum of activity against 10 strains of Gram negative and Gram positive bacteria, transfected RTG-2 cell lines over expressing GFP and the target genes omDB-1, omDB-3 and omDB-4 -defensins were produced and their supernatants used. Results showed highest bioactivity against Gram negative bacteria, in particular the supernatant from omDB-1 transfected cells showed the widest range of activity towards the majority of selected bacteria. In addition immune relevant genes (Toll-like receptors, genes involved in the anti-inflammatory response and in the apoptosis process) were screened in normal cell lines stimulated with the supernatant of omDB-1, as well as in the RTG-2 cells transfected with the three different defensins. Results showed for all the cell lines, a clear link with the viral recognition receptors TLR 3 and TLR 9, which supported the poly I:C data reported in Chapter 2 and by the induction in omDB-1 and omDB-3 transfected cell lines of the IFN- gene known to be involved in the antiviral response. Trout β-defensins also up-regulated MHC II and the CCR6 receptor. To determine the effects of fish diets enriched with different concentrations of PG, three in vivo feeding trial experiments in rainbow trout were carried out. Effectiveness of the diets was assessed using gene expression of selected AMPs, including β-defensins, cathelicidins and liver expressed antimicrobial peptide molecules. Fish fed with diets containing either 10 mg/Kg or 50 mg/Kg of PG respectively, showed the highest up-regulation of AMPs at 14 days of feeding. Data showed omDB-2 in the gut as the most inducible gene in agreement with the results obtained in the first experiment and omDB-3 was the fastest to respond in skin and gill. In addition, after ceasation of feeding the enriched diet, modulation of AMP expression was still detectable 28 days later, although a lower degree of induction was found in such fish relative to those maintained on the enriched diet. A final PG feeding trial was combined with a Yersinia ruckeri bacterial challenge which used two PG supplemented diets containing 10 mg/Kg and 50 mg/Kg of immunostimulant, and a commercial β-glucan supplemented diet (as a positive control), and fed to trout for 7 and 14 days before intraperitoneal injection challenge of the fish. Only a delay in the mortality rate was found in fish fed for 14 days with the 10 mg/Kg diet, with no clear protection from any of the functional feeds assessed. Finally, at least 500 bp of the regulatory 5’ end flanking region of two defensin (omDB-1 and omDB-2) and two liver expressed (hepcidin and LEAP-2A) genes were cloned and sequenced. In addition, the promoter sequence already known for the cathelicidin-1 gene was used in this study. Bioinformatic tools were used to search for putative transcription factor binding sites, and revealed the presence in all promoters of regulatory elements which could enhance or inhibit the expression of these genes, in response to different stimuli.

Approximately 100 brook trout were transplanted into each of four Virginia streams in September 2008. Garth Run and Kinsey Run were re-introductions, and Wildcat Hollow and Sweet Run were new introductions. Single pass electrofishing (EF) surveys were conducted with a backpack electrofisher five times during the study. Selected trout > 70 mm but < 100 mm and all trout > 100 mm received a Biomark® 134.2 kHz passive integrated transponder (PIT) tag to identify individual fish upon recapture. Trout were present in all streams almost three years post introductions. Catches decreased across the first four surveys. Young of year catch rates severely decreased from June 2009 (n = 77) to June 2010 (n = 7). Adult refuge under low flow conditions and environmental factors such as elevated stream temperatures and drought were determined to be potential limiting factors of brook trout success. Future monitoring is needed to further assess the populations.