Graduation date: 1994

The neuropeptides arginine-vasotocin (AVT) and corticotropin-releasing hormone (CRH) are involved in the hypothalamic-pituitary-interrenal (HPI) axis. During stress, the HPI axis is activated and cortisol is released into the blood. In addition to their role in the HPI axis, AVT and CRH also have behavioural effects. The roles of AVT and CRH in stress responses and agonistic behaviour were studied in this thesis, using two different models. In the first model, two strains of rainbow trout (Onchorhynchus mykiss) divergent in stress-induced release of cortisol were investigated. This was done by observing behaviour and stress responses under different conditions. These strains were found to have divergent stress coping strategies based on the observed behaviour and levels of plasma cortisol. This divergence in behaviour could be associated with the CRH system, since the mRNA levels of CRH differed between the strains during stress. However, no differences between strains were observed in AVT or its receptor expressions. In the second model, non-selected rainbow trout were paired and the effect of intracerebroventricular (icv) injections of an active substance (AVT, CRH or the CRH related peptide Urotensin-I (UI)) on fights for dominance was investigated. One fish of the pair received the active substance icv and the other received saline icv. Fish receiving AVT became subordinate in accordance with the suggestion that AVT attenuates aggression in territorial vertebrates. Fish receiving CRH became subordinate whereas UI showed no effect on fights for dominance. Further, both CRH and UI induced an anxiety-related behaviour similar to non-ambulatory motor activity in rats. In addition, CRH appeared to affect the dopaminergic and serotonergic systems. In this thesis, it is suggested that CRH is involved in the behavioural modulation of the stress coping strategies in teleost fish. Further, AVT and CRH seem to act inhibitory on aggressive behaviour.

Aquatic organisms in response to toxic insults from environmental pollutants activate defence systems including the aryl hydrocarbon receptor (AhR) in an attempt to metabolize and excrete these toxicants and their metabolites. These detoxification mechanisms however may come with certain energetic costs. I hypothesize that the activation of the AhR by β-Naphthoflavone (β-NF), a model AhR agonist, results in increased energetic costs requiring metabolic reorganization in rainbow trout hepatocytes. While the results obtained suggest that there are no significant energetic costs of AhR activation, analysis of enzyme activities suggests possible metabolic reorganization. This study also showed significant changes in cellular processes in hepatocytes over the incubation periods which previously were not reported. Furthermore, for the first time in fish hepatocytes, metabolic flux analysis (MFA) was used to examine intra-cellular metabolism, the applicability of which is discussed.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

Pharmaceuticals and personal care products are a growing concern in the aquatic environment. Compounds from the class of non-steroidal anti-inflammatory drugs are commonly detected in surface waters and have the potential to negatively affect aquatic organisms. The purpose of this experiment was to determine the acute and chronic effects of ibuprofen on rainbow trout (Oncorhynchus mykiss). Cyclooxygenase (COX) activity, vitellogenin (VTG) concentration and ethoxyresorufin-O-deethylase (EROD) activity were evaluated following waterborne ibuprofen exposure of trout to 1 and 10 mg/L in the acute exposure and 1, 32 and 1000 μg/L in the chronic exposure, along with an experimental control, E2 control of 1000 μg/L and an E2-ibuprofen mixed treatment. Ibuprofen did not inhibit COX enzyme activity in either gill or kidney tissue. To evaluate the estrogenic effects of ibuprofen, VTG concentrations were measured; by the end of the 56 day chronic exposure VTG concentrations significantly increased in all of the ibuprofen treatments relative to the controls. EROD activity may have been inhibited by ibuprofen but definitive conclusions could not be made. These findings indicate that more research needs to be done studying ibuprofen in aquatic systems. / UOIT

Accurate measurements of energy content and body composition are essential to effectively assess the well-being of fish. Bomb calorimetry and proximate analysis are currently the most dependable and accurate methods to estimate energy content and body composition. However, bioenergetic studies that employ the traditional methodology necessitate the killing of fish to determine physiological composition and energy content in a target tissue. The killing of the individual negates the ability for repeated measures on the same individual, and also suppresses compositional studies involving endangered or threatened species. Recent research has shown Bioelectrical Impedance Analysis (BIA), a quick, easy-to-use, non-invasive, and most importantly, non lethal technique to be an effective method for estimating the proximate composition and energy content of fish. The focus of this research is to evaluate the capability of BIA to accurately assess the bioenergetics of juvenile rainbow trout (Oncorhynchus mykiss), and to develop species-specific indices to predict energy content, total body water and dry mass. To do this, juvenile rainbow trout were subjected to one of three ration regimes: maintenance (0.4 % bw/day), optimum (1.9 % bw/day) and satiation (3.4 % bw/day) for 90 days. Subsamples from each treatment were taken every 30 days to be subjected to BIA testing. Tissue samples were collected from the subsampled trout for future caloric and compositional analysis via bomb calorimetry and proximate analysis. It was found that BIA demonstrated a strong predictive relationship with regard to energy content (r2 = 0.90), total body water (r2 = 0.89) and dry mass (r2 = 0.80). BIA was also able to successfully reflect a notable statistical difference between treatments with regard to total energy content, energy density, total body water, dry mass. These results, along with much of the existing literature, indicate that BIA may be an accurate and reliable tool to estimate the bioenergetics and proximate composition of fish. / UOIT

Graduation date: 1993

Aquatic organisms in response to toxic insults from environmental pollutants activate defence systems including the aryl hydrocarbon receptor (AhR) in an attempt to metabolize and excrete these toxicants and their metabolites. These detoxification mechanisms however may come with certain energetic costs. I hypothesize that the activation of the AhR by β-Naphthoflavone (β-NF), a model AhR agonist, results in increased energetic costs requiring metabolic reorganization in rainbow trout hepatocytes. While the results obtained suggest that there are no significant energetic costs of AhR activation, analysis of enzyme activities suggests possible metabolic reorganization. This study also showed significant changes in cellular processes in hepatocytes over the incubation periods which previously were not reported. Furthermore, for the first time in fish hepatocytes, metabolic flux analysis (MFA) was used to examine intra-cellular metabolism, the applicability of which is discussed.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

The physiological responses to stressor exposure can be broadly grouped into the organismal and the cellular stress responses. The organismal stress response involves the release of hormones into general circulation, while the cellular stress response involves the synthesis of proteins, the most important being the heat shock proteins (HSPs), which play a role in maintaining protein homeostasis. Elevated HSP70 expression in response to stressors has been demonstrated in trout (Oncorhynchus mykiss) red blood cells (RBCs). The ease of repeated sampling of blood suggests the possibility of using this tissue as a non-lethal marker of cellular stress in fish. This study tested the hypothesis that stressor exposure will elevate HSP70 expression in trout RBCs and the role of stress hormones in mediating this response. Acute heat shock exposure (+12oC) significantly elevated plasma cortisol, glucose and lactate levels in heat shocked fish over 24 h. A tissue-specific response was seen in HSP70 expression in liver, brain, gill and RBCs. To enable measurement of RBC HSP70 concentrations, an enzyme-linked immunosorbent assay (ELISA) was developed using a commercially available rabbit anti-salmon HSP70 and a recombinant chinook salmon (Oncorhynchus tshawytscha) HSP70. To determine effects of chronic exposure, two studies were conducted exposing trout to either cadmium (0, 0.75 or 2.0 µg/L over 28 d) or municipal wastewater effluent (0, 20 or 90% over 14 d). However, neither exposure elicited a significant HSP70 response. Effects of stress hormones on RBC HSP70 levels were tested by exposing cells in vitro to either cortisol (10 and 100 ng/mL) or epinephrine (10 nM) with or without heat shock. Heat shock elevated HSP70 content in trout RBCs but no modulation by stress hormones was seen. It was shown for the first time that RBCs release HSP70 content into the medium in response to an acute heat shock and this release is attenuated by stress hormones. Overall, HSP70 levels in RBCs have the potential to be a reliable non-lethal marker of acute cellular stress effects in fish. The release of HSP70 from RBCs leads to the hypothesis that HSP70 may also have an extracellular role in fish, and warrants further study.