Epigenetics in teleost fish: from molecular mechanisms to physiological phenotypes

The final publication is available at Elsevier via https://doi.org/10.1016/j.cbpb.2018.01.006. © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/While the field of epigenetics is increasingly recognized to contribute to the emergence of phenotypes in mammalian research models across different developmental and generational timescales, the comparative biology of epigenetics in the large and physiologically diverse vertebrate infraclass of teleost fish remains comparatively understudied. The cypriniform zebrafish and the salmoniform rainbow trout and Atlantic salmon represent two especially important teleost orders, because they offer the unique possibility to comparatively investigate the role of epigenetic regulation in 3R and 4R duplicated genomes. In addition to their sequenced genomes, these teleost species are well-characterized model species for development and physiology, and therefore allow for an investigation of the role of epigenetic modifications in the emergence of physiological phenotypes during an organism's lifespan and in subsequent generations. This review aims firstly to describe the evolution of the repertoire of genes involved in key molecular epigenetic pathways including histone modifications, DNA methylation and microRNAs in zebrafish, rainbow trout, and Atlantic salmon, and secondly, to discuss recent advances in research highlighting a role for molecular epigenetics in shaping physiological phenotypes in these and other teleost models. Finally, by discussing themes and current limitations of the emerging field of teleost epigenetics from both theoretical and technical points of view, we will highlight future research needs and discuss how epigenetics will not only help address basic research questions in comparative teleost physiology, but also inform translational research including aquaculture, aquatic toxicology, and human disease

High Throughput Sequencing of MicroRNA in Rainbow Trout Plasma, Mucus, and Surrounding Water Following Acute Stress

Circulating plasma microRNAs (miRNAs) are well established as biomarkers of several diseases in humans and have recently been used as indicators of environmental exposures in fish. However, the role of plasma miRNAs in regulating acute stress responses in fish is largely unknown. Tissue and plasma miRNAs have recently been associated with excreted miRNAs; however, external miRNAs have never been measured in fish. The objective of this study was to identify the altered plasma miRNAs in response to acute stress in rainbow trout (Oncorhynchus mykiss), as well as altered miRNAs in fish epidermal mucus and the surrounding ambient water. Small RNA was extracted and sequenced from plasma, mucus, and water collected from rainbow trout pre- and 1 h-post a 3-min air stressor. Following small RNA-Seq and pathway analysis, we identified differentially expressed plasma miRNAs that targeted biosynthetic, degradation, and metabolic pathways. We successfully isolated miRNA from trout mucus and the surrounding water and detected differences in miRNA expression 1-h post air stress. The expressed miRNA profiles in mucus and water were different from the altered plasma miRNA profile, which indicated that the plasma miRNA response was not associated with or immediately reflected in external samples, which was further validated through qPCR. This research expands understanding of the role of plasma miRNA in the acute stress response of fish and is the first report of successful isolation and profiling of miRNA from fish mucus or samples of ambient water. Measurements of miRNA from plasma, mucus, or water can be further studied and have potential to be applied as non-lethal indicators of acute stress in fish.This research was funded through the Global Water Futures Grant #419205. HI is supported by an NSERC PGS-D

Impacts of acute and anthropogenic stress on fish microRNA

Fishes play crucial roles in the ecology of aquatic environments and contribute to the multi-billion-dollar fisheries industry. The integrity of their populations and health needs to be maintained for future generations and research on the biology and effects of stress on fish can contribute to this cause. While much is understood about the adrenergic response to stress which results in the secretion of catecholamines and cortisol, there is much to be understood about molecular mechanisms of stress, such as the role of microRNAs. MicroRNAs (miRNAs) regulate post-transcriptional molecular responses by binding to mRNA and labelling them for degradation or blocking translation, effectively decreasing target protein translation levels. The response of miRNA transcript levels to environmental stressors, such as increased water temperatures, have been measured in fish since 2009. However, there is still much that is poorly understood about the effects of fish stress on miRNA levels, such as how time sensitive the response is, whether the response is tissue specific, and whether it is possible to measure miRNAs in non-lethal or non-invasive samples, such as mucus or the water surrounding fish. Furthermore, there are many gaps in understanding of how miRNA levels are altered in non-model species, such as salmonids. Most studies of fish stressors often focus on single stressor studies to elucidate the molecular mechanisms however, fish are not exposed to stressors individually in the aquatic environment. Therefore, it is important to study the simultaneous effects of multiple, emerging anthropogenic stressors of concern (e.g., increased water temperature, decreased dissolved oxygen, and pharmaceutical contaminants), on fish and their miRNA levels. The overall goal of my thesis is to determine how transcript levels of miRNAs are regulated when fishes are exposed to stress. More specifically, I wanted to further characterize the miRNA response in different tissues and at different timepoints, in both model and non-model fish species, to determine the conservation or specificity of the miRNA response. I also aimed to determine if it was possible to sample miRNAs in non-lethally collected samples as a novel method of measuring stress in fish. Furthermore, I measured predicted downstream responses (mRNA transcript levels, protein abundances, and enzyme activities) to understand the functional implications of changes to miRNA transcript levels and to describe the molecular response to acute and anthropogenic stressors. In studying the effects of chronic exposure to anthropogenic stressors on zebrafish gonads, I found that the miRNA response was reversible and associated with adverse reproductive impacts (Chapter 2). In studying the effects of different lengths of exposure to anthropogenic stressors on zebrafish liver and muscle tissues, I determined that the miRNA response was specific to length of exposure, tissue type, as well as the sex of the fish, and that fish were activating cell stress, decontamination, metabolic, and reproductive responses (Chapter 3). In studying the effects of acute stress on rainbow and brook trout liver tissues, I found that miRNA transcript levels, mRNA transcript levels, and metabolic enzyme activities were altered in a time-dependent manner post-stress and that there was much intra-species and inter-species variability (Chapter 4). In studying the effects of acute stress on rainbow trout blood plasma, mucus, and the surrounding water, I found that miRNAs were able to be measured and transcript levels were altered following stress in all three non-lethal sampling locations (Chapter 5). Altogether, I have contributed further to identifying specific transcript levels of miRNA that respond to acute and anthropogenic stressors in multiple fish species. I have also characterized how the miRNA response is associated with the presence of the stressor, the length of exposure to the stressor, and the length of time following exposure to the stressor. These data are helpful in understanding the molecular regulation and response to stress and broadly contribute to understanding how miRNAs play a role in how organisms can adapt to transient or ongoing stressors. In addition, the downstream molecular responses associated with changes in miRNA transcript levels were also measured in response to these stressors and highlight the metabolic, reproductive, and cellular stress responses that the fish were activating when exposed to anthropogenic stressors, as well as filling in gaps of metabolic enzymes that are part of the acute stress response. My research also highlights the complex role that miRNAs play in finetuning the molecular response to stress, as there are still many gaps in understanding what the altered miRNA transcript levels are targeting and post transcriptionally regulating. In the future, instead of focusing on identifying miRNAs that are regulating a particular transcript or pathway of interest, priority can be given to identifying miRNAs that are crucial in driving the stress response or in allowing a particular individual or species to adapt to stress

Omicron COVID-19 Case Estimates Based on Previous SARS-CoV-2 Wastewater Load, Regional Municipality of Peel, Ontario, Canada

We determined correlations between SARS-CoV-2 load in untreated water and COVID-19 cases and patient hospitalizations before the Omicron variant (September 2020–November 2021) at 2 wastewater treatment plants in the Regional Municipality of Peel, Ontario, Canada. Using pre-Omicron correlations, we estimated incident COVID-19 cases during Omicron outbreaks (November 2021–June 2022). The strongest correlation between wastewater SARS-CoV-2 load and COVID-19 cases occurred 1 day after sampling (r = 0.911). The strongest correlation between wastewater load and COVID-19 patient hospitalizations occurred 4 days after sampling (r = 0.819). At the peak of the Omicron BA.2 outbreak in April 2022, reported COVID-19 cases were underestimated 19-fold because of changes in clinical testing. Wastewater data provided information for local decision-making and are a useful component of COVID-19 surveillance systems