Stress and metabolic responses to municipal wastewater effluent exposure in rainbow trout effluent

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

Physiological and growth responses to water deficit in the bioenergy crop Miscanthus x giganteus

High yielding perennial biomass crops of the species Miscanthus are widely recognized as one of the most promising lignocellulosic feedstocks for the production of bioenergy and bioproducts. Miscanthus is a C(4) grass and thus has relatively high water use efficiency. Cultivated Miscanthus comprises primarily of a single clone, Miscanthus x giganteus, a sterile hybrid between M. sacchariflorus and M. sinensis. M. x giganteus is high yielding and expresses desirable combinations of many traits present in the two parental species types; however, it responds poorly to low water availability. To identify the physiological basis of the response to water stress in M. x giganteus and to identify potential targets for breeding improvements we characterized the physiological responses to water-deficit stress in a pot experiment. The experiment has provided valuable insights into the temporal aspects of drought-induced responses of M. x giganteus. Withholding water resulted in marked changes in plant physiology with growth-associated traits among the first affected, the most rapid response being a decline in the rate of stem elongation. A reduction in photosynthetic performance was among the second set of changes observed; indicated by a decrease in stomatal conductance followed by decreases in chlorophyll fluorescence and chlorophyll content. Measures reflecting the plant water status were among the last affected by the drought treatment. Metabolite analysis indicated that proline was a drought stress marker in M. x giganteus, metabolites in the proline synthesis pathway were more abundant when stomatal conductance decreased and dry weight accumulation ceased. The outcomes of this study in terms of drought-induced physiological changes, accompanied by a proof-of-concept metabolomics investigation, provide a platform for identifying targets for improved drought-tolerance of the Miscanthus bioenergy crop

What evidence exists on the impacts of large herbivores on climate change? A systematic map protocol

Background- In recent years there has been an increased focus on the role of large herbivores in ecosystem restoration and climate change mitigation. There are multiple processes by which large herbivores could potentially influence climate feedback and forcing effects, but the evidence has not yet been synthesised in a systematic and accessible format. Grazing, browsing, trampling, defecation, and seed dispersal by large herbivores can influence vegetation and soils in ways that may directly or indirectly contribute to climate change or mitigation. For example, changes in vegetation could impact wildfire regimes, carbon storage, and albedo, with ultimate impacts on climate. These processes may be influenced by herbivore species composition, density, and functional traits. The main aim of this systematic map is to synthesise the range of research on climate feedback and forcing effects from large herbivores (≥ 10 kg) in terrestrial ecosystems. We also aim to identify knowledge clusters and gaps in the research base, as well as assessing the potential for quantitative analyses. Methods- A search of peer-reviewed and grey literature will be conducted using a range of bibliographic databases, search engines and websites. The search strategy will involve using a pre-defined search string with Boolean operators. All search results will be screened for relevance according to specific eligibility criteria. Screening will be conducted in two stages: all articles will initially be screened by title and abstract, then those that meet the eligibility criteria will be screened by full text. At both stages, articles will be excluded if they don’t meet all eligibility criteria or if they meet any exclusion criteria. All articles included as eligible after full text screening will be coded. At each stage (of screening and coding) a proportion of articles will be processed independently by two reviewers to assess inter-reviewer reliability and resolve differences. The evidence will be presented in a searchable database with accompanying visual outputs. A narrative synthesis will be provided outlining the range and distribution of evidence, knowledge gaps and clusters, potential bias, and areas for further research

In vivo microsampling to capture the elusive exposome

Bessonneau, V., Ings, J., McMaster, M., Smith, R., Bragg, L., Servos, M., & Pawliszyn, J. (2017). In vivo microsampling to capture the elusive exposome. Scientific Reports, 7, 44038. The final and definitive publication is available through Nature publishing Group via: http://dx.doi.org/10.1038/srep44038Loss and/or degradation of small molecules during sampling, sample transportation and storage can adversely impact biological interpretation of metabolomics data. In this study, we performed in vivo sampling using solid-phase microextraction (SPME) in combination with non-targeted liquid chromatography and high-resolution tandem mass spectrometry (LC-MS/MS) to capture the fish tissue exposome using molecular networking analysis, and the results were contrasted with molecular differences obtained with ex vivo SPME sampling. Based on 494 MS/MS spectra comparisons, we demonstrated that in vivo SPME sampling provided better extraction and stabilization of highly reactive molecules, such as 1-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoleoyl-glycero-3-phosphocholine, from fish tissue samples. This sampling approach, that minimizes sample handling and preparation, offers the opportunity to perform longitudinal monitoring of the exposome in biological systems and improve the reliability of exposure-measurement in exposome-wide association studies.Environment Canada, Environmental Damages Fund (Grant EC-129114) provided to Environment Canada through the Joint Oil Sands Monitoring Program

Adrenocorticotropic Hormone Suppresses Gonadotropin-Stimulated Estradiol Release from Zebrafish Ovarian Follicles

While stress is known to impact reproductive performance, the pathways involved are not entirely understood. Corticosteroid effects on the functioning of the hypothalamus-pituitary-gonadal axis are thought to be a key aspect of stress-mediated reproductive dysfunction. A vital component of the stress response is the pituitary secretion of adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. We recently reported MC2R mRNA abundance in fish gonads leading to the hypothesis that ACTH may be directly involved in gonadal steroid modulation. Using zebrafish (Danio rerio) ovarian follicles, we tested the hypothesis that acute ACTH stimulation modulates cortisol and estradiol (E2) secretion. ACTH neither affected cortisol nor unstimulated E2 release from ovarian follicles. However, ACTH suppressed human chorionic gonadotropin (hCG)-stimulated E2 secretion in a dose-related manner, with a maximum decrease of 62% observed at 1 I.U. ACTH mL−1. This effect of ACTH on E2 release was not observed in the presence of either 8-bromo-cAMP or forskolin, suggesting that the mechanism(s) involved in steroid attenuation was upstream of adenylyl cyclase activation. Overall, our results suggest that a stress-induced rise in plasma ACTH levels may initiate a rapid down-regulation of acute stimulated E2 biosynthesis in the zebrafish ovary, underscoring a novel physiological role for this pituitary peptide in modulating reproductive activity

Correctional Service of Canada Prison Libraries from 1980 to 2010

The last three decades have seen many developments in Canadian prison libraries. This article follows the history of the libraries in federal Correctional Service of Canada (CSC) from the 1980s to the present, concentrating on the libraries in the Pacific Region. A chronological overview of the major legislative changes, reports, and events of the last thirty years highlights the increased profile of prison libraries and their role in supporting Correctional Service of Canada's Mission and Goals. Some of these changes include the adoption in 1992 of the Corrections and Condition Release Act (CCRA) and Regulations, modifications to Commissioner's Directive 720 (2007a; under which libraries fall), and the adoption in the Pacific Region of Library Policy Guidelines. In addition to legislative and policy changes, Canadian society itself has also changed during this thirty-year period. As the face of Canada has become more diverse in age and ethnicity, as well as in social and technological expectations, so has the face of the prison population. These changes have, of course, also impacted on prison libraries. This article examines how prison libraries have met the challenges created by these societal and technological changes.published or submitted for publicatio

Tissue-specific melanocortin 2 receptor (MC2R) gene expression.

<p>Expression of melanocortin 2 receptor (MC2R) and β-actin in adult zebrafish tissues as determined with RT-PCR. Products were amplified (MC2R-36 cycles, β-actin- 32 cycles) from total RNA extracts. Images are representative of the results observed from three to five fish. Water replaced cDNA in the negative (Neg.) control treatments.</p

Temporal (3 or 8 h incubations) cortisol production by zebrafish ovarian follicles.

<p>Cortisol (grey bars) is not detected in the culture media of control or ACTH treated follicles, but is detected in the human chorionic gonadotropin (hCG, 10 I.U./mL) and hCG plus adrenocorticotropic hormone (ACTH, 1.5 I.U./mL; hatched bars) treatments. Control (open bar) and hCG-stimulated (black bar) estradiol (E₂) media concentrations after 3 h are provided as a reference. Values represent mean ± SEM (<i>N</i> = 3; each <i>N</i> is a pool of follicles from three fish). Treatments with different letters are significantly different, as determined by one-way ANOVA at each time point followed by Student-Newman-Keuls test for multiple comparisons (P<0.05). An asterisk (*) with the E₂ measurements indicates a significant difference determined by a Student's <i>t</i> test (P<0.05).</p