Photoperiod is an important signal involved in the timing and co-ordination of many processes such as growth and reproduction in salmonids. Both growth and reproduction appear to be controlled by endogenous rhythms, which under natural conditions, is entrained by the seasonal changes in daylength, that is accurately reflected by the diel pattern of melatonin. This thesis investigates the influence of photoperiod on growth and reproduction in rainbow trout (Oncorhynchus mykiss), and examines the effect on plasma insulin-like growth factor-I (IGF-I). These studies aim to further our scientific understanding of the endocrine mechanisms controlling growth and reproduction and transfer this knowledge to commercial trout farming practices. Studies at fry (<5g) and fingerling (>25g) stages investigated the effect of exposure to constant long-days (LD18:6) and constant light (LL) on growth performance. In all experiments, there was a significant growth enhancing effect following exposure to extended light regimes relative to those under ambient or constant short-day photoperiods. Growth enhancement appeared to be caused through direct photo-stimulation or a phaseshift in an underlying endogenous rhythm dependent on the timing of photoperiod application. Measurement of plasma IGF-I accurately reflected growth rate during juvenile development. Furthermore, a clear autumnal increase in IGF-I was observed that was apparently up-regulated by long-day photoperiods. Additionally, it was shown that melatonin implants significantly reduced growth rate below that of unimplanted controls irrespective of photoperiod, but did not reduce circulating IGF-I levels suggesting that elevated melatonin levels masked the perception of daylength but did not act directly on the somatotropic axis to control growth. As size or one of its correlates (growth rate, energy balance or nutritional status) is regarded as an important determinant of the ability to undergo puberty, the interaction of natural (SNP) and advancing (ADV: long-short day) photoperiods with growth, IGF-I and reproduction was investigated in virgin female broodstock. Under SNP 63% of the population attained maturity while only 29% spawned in the ADV regime. Under SNP both size and growth rate in late spring/early summer appeared to determine whether an individual may initiate maturation. Conversely, under ADV, condition factor appeared to be a better predictor. A complete seasonal relationship between plasma IGF-I, daylength and temperature was demonstrated for the first time in rainbow trout under natural conditions, and provides direct evidence for the relationship between maturation and IGF-I. IGF-I levels showed a negative correlation with testosterone as fish initiated maturation. Furthermore, IGF-I levels accurately reflected growth rate prior to the initiation of vitellogenesis, suggesting that IGF-I may provide an endocrine signal between the somatotropic and reproductive axes that nutritional status, growth rate and/or size is sufficient to initiate maturation. In addition, maturing individuals under both photoperiods typically expressed higher circulating IGF-I levels than those that remained immature and may reflect a greater opportunity for IGF-I to act on the pituitary to stimulate gonadotropin production. In this sense, the observation of elevated levels for 3 months under SNP compared to only 1 month under ADV may reflect a reduction in the window of opportunity to initiate maturation under advancing photoperiods and hence explain the reduction in fish spawning. Finally, using a commercially available ELISA kit provided evidence of a possible detection method for measurement of a leptin-like peptide as absolute levels differed significantly between mature and immature fish. This provides preliminary evidence for a possible involvement of a leptin-like peptide in rainbow trout reproduction and energy homeostasis. Given the above evidence of a growth enhancing effect of photoperiod in covered systems, the transfer of constant light (LL) regimes to uncovered commercial trout farming practices was also examined. The application of LL regimes during either fry grow-out in tanks using floating lights or ongrowing to harvest size during winter in cages using submersible lights was investigated. In all trials conducted, the exposure to LL in autumn significantly enhanced winter growth rate and feeding efficiency by up to 30% and 25% respectively relative to those maintained under ambient light. Furthermore, the effect of light intensity was clearly shown to be an important requirement in successfully applying photoperiod regimes onto the ambient photoperiod in order to enhance commercial production in "uncovered systems". These studies clearly indicate the importance of photoperiod in influencing growth and reproduction in rainbow trout. Furthermore, the use of plasma IGF-I as an indicator of growth provides a practical tool for studying growth-photoperiod-reproduction interactions in this species. However, further studies are necessary to further our understanding of the endocrine pathways governing physiological mechanisms, especially growth and reproduction. Overall, this work has provided important information to improve both scientific understanding and commercial development although it is clear that substantial research is still required.

Municipal wastewater effluent (MWWE) is an important source of pollution in the aquatic environment impacting fish. MWWE is a complex mixture of chemicals including pharmaceuticals, personal care products, industrial chemicals and pesticides. A link between reproductive endocrine disruption and MWWE exposure has been established in fish, but less is known about the effects of MWWE on non-reproductive endocrine disruption. The overall objective of this thesis was to examine the impacts of MWWE exposure on the stress response and intermediary metabolism in rainbow trout (Oncorhynchus mykiss). In fish, the primary adaptive organismal stress response involves the activation of hypothalamic-sympathetic-chromaffin axis to produce catecholamines, predominantly epinephrine, and the hypothalamic-pituitary-interrenal (HPI) axis to produce cortisol. Both of these hormones play a key role in elevating plasma glucose levels that is essential to fuel the increased energy demand associated with stress. Along with the organismal stress response, the cellular stress response, involving the synthesis of a suite of heat shock proteins (hsps), also plays an important role in protecting cellular protein homeostasis in response to stressors, including toxicants. The impact of MWWE on stress-related pathways were identified using a low-density trout cDNA microarray enriched with genes encoding for proteins involved in endocrine-, stress- and metabolism-related processes. This was further confirmed by assessing plasma hormone and metabolite levels and stress-related targeted genes and proteins expression and enzyme activities in select tissues in rainbow trout. Studies were carried out in controlled field (caging) and laboratory experiments to examine the impacts of MWWE on stress and tissue-specific metabolic responses in rainbow trout. Further in vitro studies using rainbow trout hepatocytes in primary cultures were carried out to investigate the mechanism of action of two pharmaceuticals, atenolol and venlafaxine, found in relatively high concentrations in MWWE in impacting the stress-mediated glucose response. In caged fish, MWWE exposure significantly elevated plasma cortisol and glucose concentrations, and altered the mRNA abundance of a number of stress-related genes, hormone receptors, glucose transporter 2 and genes related to immune function. When fish were exposed to an acute handling stress following a 14 d exposure to MWWE, the cortisol response was abolished and the glucose response was attenuated. The effects on cortisol did not correlate with changes in the expression of genes involved in cortisol biosynthesis, but were associated with an increase in hepatic glucocorticoid receptor (GR) protein expression. Upon further investigation in controlled laboratory studies, MWWE exposure elevated constitutive hsp 70 and hsp90 expression after 8 d exposure, which correlated with a decrease in glycogen levels in the liver in fish exposed to a high concentration of MWWE compared to control fish, pointing to a MWWE-induced increase in liver energy demand. By 14 d, glycogen stores were replenished, and this was commensurate with increases in liver gluconeogenic capacity, including increases in the activities of phosphoenolpyruvate carboxykinase (PEPCK) and alanine aminotransferase (AlaAT), along with a decrease in liver GR expression. In the heart, GR protein expression increased in treated fish, and the activity of pyruvate kinase increased, indicating an increase in glycolytic capacity. Subjecting the MWWE exposed fish to a secondary handling disturbance (acute stress) led to an attenuated plasma cortisol and glucose response compared to the control group. This corresponded with a reduced liver gluconeogenic capacity and a lower liver and heart glycolytic capacities, reflecting a disturbance in the energy substrate repartitioning that is essential to cope with stress. While it is difficult to establish causative agents from a complex mixture such as MWWE, the two pharmaceutical that were tested impacted glucose production. Specifically, atenolol and venlafaxine disrupted the epinephrine-induced glucose production, but did not modify cortisol-mediated glucose production in trout hepatocytes. The suppression of epinephrine-mediated glucose production by atenolol and venlafaxine was abolished by cAMP analogue (8-bromo cAMP) or glucagon (a metabolic hormone that increases glucose production). This suggests that both drugs disrupt β-adrenoceptor signaling, while it remains to be determined if the response is receptor isoform-specific. Altogether MWWE exposure disrupts the organismal and cellular stress responses in trout. Key targets for MWWE impact leading to the impaired cortisol and metabolic responses to stress include liver and heart GR expression, liver gluconeogenic capacity, and liver, heart and gill glycolytic capacities. Most significantly, MWWE impairs the ability to metabolically adjust to a secondary acute stressor, which is an important adaptive process that is integral to successful stress performance. From an environmental stand-point, long-term exposure to MWWE will lead to reduced fitness and will compromise the capacity of fish to cope with additional stressor, including escape from predators.

Proteinase P-II purified from parasitized Pacific whiting muscle was previously identified to be one form of cathepsin L. It appeared to be present in three isozymatic forms on non-denaturing PAGE gel stained for activity. Its autolytic degradation was observed on SDS-PAGE gel under its optimum condition, 55°C and pH 5.5, in the absence of substrate. Amino acid composition analysis revealed that this enzyme had a considerably greater proportion of hydrophobic amino acids than cathepsin L from other fish species, and monosaccharide analysis showed it was not glycosylated. The N-terminal amino acid sequence of the enzyme was 60-65% identical with cathepsin L from chicken and mammalian species, but only 39% identical with mammalian cathepsin B. The moderate identity of the N-terminal amino acid sequence of P-II with other cathepsin L revealed that this cysteine proteinase from Pacific whiting might be encoded by a cathepsin L-related gene. Two degenerate primers were designed to clone cathepsins cDNA from rainbow trout. The 500-bp PCR product from rainbow trout liver cDNA contained at least three different cysteine proteinase sequences, referred to as SFL2, SFL5, and SFL17. SFL5 was the partial cDNA of trout cathepsin L, which was over 80% identical with chicken cathepsin L amino acid sequence. SFL5 was labeled with Dig-11-dUTP and used to screen a trout liver cDNA library. One positive clone referred to as LC was identified and contained a 700-bp insertion overlapping with SFL5. By combining the two overlapping sequences, a 895-bp cDNA sequence was identified, which included 88% of the mature enzyme and a 307-bp 3' end untranslated part. Its deduced amino acid sequences had 83% identity, 91% similarity with chicken cathepsin L and 73% identity, 86% similarity with human cathepsin L. SFL2 might be the partial cDNA of a novel cathepsin L-related cysteine proteinase. SFL17 may be the partial cDNA of trout cathepsin K. It had 70% identity and 89% similarity with rabbit and human cathepsin K at the amino acid level. / Graduation date: 1996

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 59-61). Also available via the Internet.

Thesis (PhD)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Thomas E. McMahon. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains viii, 89 p. : ill. Includes abstract. Includes bibliographical references (p. 67-73).

Thesis (Ph. D. biochemistry)--Washington State University, May 2008. / Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [164]-176).

Thesis (M.S.)--Montana State University--Bozeman, 2004. / Typescript. Chairperson, Graduate Committee: Alexander V. Zale. Includes bibliographical references (leaves 54-60).

Thesis (Ph. D.)--West Virginia University, 2004 / Title from document title page. Document formatted into pages; contains xiii, 119 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.