Venlafaxine exposure alters mitochondrial respiration and mitomiR abundance in zebrafish brains.

Wastewater treatment plant (WWTP) effluent often releases pharmaceuticals like venlafaxine (a serotonin-norephinephrine reuptake inhibitor antidepressant) to freshwater ecosystems at levels causing adverse metabolic effects on fish. Changes to fish metabolism can be regulated by epigenetic mechanisms like microRNA (small RNA molecules that regulate mRNA translation), including regulating mitochondrial mRNAs. Nuclear-encoded microRNAs regulate mitochondrial gene expression in mammals, and have predicted effects in fish. We aimed to identify whether venlafaxine exposure changed mitochondrial respiration and resulted in differentially abundant mitochondrial microRNA (mitomiRs) in zebrafish brains. In vitro exposure of brain homogenate to below environmentally relevant concentrations of venlafaxine (<1 µg/L) caused a decrease in mitochondrial respiration, although this was not driven by changes to mitochondrial Complex I or II function. To identify whether these effects occur in vivo, zebrafish were exposed to 1 µg/L venlafaxine for 0, 1, 6, 12, 24, and 96 h. In vivo, venlafaxine exposure had no significant effects on brain mitochondrial respiration; however, select mitomiRs (dre-miR-301a-5p, dre-miR-301b-3p, and dre-miR-301c-3p) were also measured, because they were bioinformatically predicted to regulate mitochondrial cytochrome c oxidase subunit I (COI) abundance. These mitomiRs were differentially regulated based on venlafaxine exposure (with miR-301c-3p abundance differing during the day and miR-301b-3p being lower in exposed fish at night), and with respect to sex and time sampled. Overall, the results demonstrated that in vitro venlafaxine exposure to zebrafish brain caused a decrease in mitochondrial respiration, but these effects were not seen after acute in vivo exposure. Results may have differed because in vivo exposure allows for fish to mitigate effects through mechanisms that could include mitomiR regulation, and because fish were only acutely exposed. Environ Toxicol Chem 2024;00:1-14. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Oxygen consumption rate during recovery from loss of equilibrium induced by warming, hypoxia, or exhaustive exercise in rainbow darter (Etheostoma caeruleum).

Animals routinely encounter environmental (e.g., high temperatures and hypoxia) as well as physiological perturbations (e.g., exercise and digestion) that may threaten homeostasis. However, comparing the relative threat or "disruptiveness" imposed by different stressors is difficult, as stressors vary in their mechanisms, effects, and timescales. We exploited the fact that several acute stressors can induce the loss of equilibrium (LOE) in fish to (i) compare the metabolic recovery profiles of three environmentally relevant stressors and (ii) test the concept that LOE could be used as a physiological calibration for the intensity of different stressors. We focused on Etheostoma caeruleum, a species that routinely copes with environmental fluctuations in temperature and oxygen and that relies on burst swimming to relocate and avoid predators, as our model. Using stop-flow (intermittent) respirometry, we tracked the oxygen consumption rate (MO2) as E. caeruleum recovered from LOE induced by hypoxia (PO2 at LOE), warming (critical thermal maximum, CTmax), or exhaustive exercise. Regardless of the stressor used, E. caeruleum recovered rapidly, returning to routine MO2 within ~3 h. Fish recovering from hypoxia and warming had similar maximum MO2, aerobic scopes, recovery time, and total excess post-hypoxia or post-warming oxygen consumption. Though exhaustive exercise induced a greater maximum MO2 and corresponding higher aerobic scope than warming or hypoxia, its recovery profile was otherwise similar to the other stressors, suggesting that "calibration" to a physiological state such as LOE may be a viable conceptual approach for investigators interested in questions related to multiple stressors, cross tolerance, and how animals cope with challenges to homeostasis.

Impact of turbidity on the gill morphology and hypoxia tolerance of eastern sand darter (Ammocrypta pellucida).

Anthropogenic stressors such as agriculture and urbanization can increase river turbidity, which can negatively impact fish gill morphology and growth due to reduced oxygen in the benthic environment. We assessed the gill morphology, field metabolic rate (FMR), and two hypoxia tolerance metrics (oxygen partial pressure at loss of equilibrium, PO2 at LOE, and critical oxygen tension, Pcrit ) of eastern sand darter (Ammocrypta pellucida), a small benthic fish listed as threatened under the Species at Risk Act in Canada, from rivers in southern Ontario. Field trials were conducted streamside in the Grand River (August 2019; mean NTU 8) and in the comparatively more turbid Thames River (August 2020; mean NTU 94) to test the effect of turbidity on each physiological endpoint. Gills were collected from incidental mortalities and museum specimens, and were assessed using hematoxylin and eosin and immunofluorescent staining. The between-river comparison indicated that turbidity significantly increased interlamellar space and filament width but had no significant influence on other gill morphometrics or FMR. Turbidity significantly increased PO2 at LOE (i.e., fish had a lower hypoxia tolerance) but did not significantly impact Pcrit . Therefore, although turbidity influences hypoxia tolerance through LOE, turbidity levels were not sufficiently high in the study rivers to contribute to measurable changes in gill morphology or metabolism in the wild. Determining whether changes in gill morphology or metabolism occur under higherturbidity levels would help resolve the ecological importance of turbidity on species physiology in urban and agricultural ecosystems.

Impacts of temperature and turbidity on the gill physiology of darter species.

Fish gills are complex organs that have direct contact with the environment and perform numerous functions including gas exchange and ion regulation. Determining if gill morphometry can change under different environmental conditions to maintain and/or improve gas exchange and ion regulation is important for understanding if gill plasticity can improve survival with increasing environmental change. We assessed gill morphology (gas exchange and ion regulation metrics), hematocrit and gill Na+/K+ ATPase activity of wild-captured blackside darter (Percina maculata), greenside darter (Etheostoma blennioides), and johnny darter (Etheostoma nigrum) at two temperatures (10 and 25 °C) and turbidity levels (8 and 94 NTU). Samples were collected August and October 2020 in the Grand River to assess temperature differences, and August 2020 in the Thames River to assess turbidity differences. Significant effects of temperature and/or turbidity only impacted ionocyte number, lamellae width, and hematocrit. An increase in temperature decreased ionocyte number while an increase in turbidity increased lamellae width. Hematocrit had a species-specific response for both temperature and turbidity. Findings suggest that the three darter species have limited plasticity in gill morphology, with no observed compensatory changes in hematocrit or Na+/K+ ATPase activity to maintain homeostasis under the different environmental conditions.

Physiological differences between wild and captive animals: a century-old dilemma.

Laboratory-based research dominates the fields of comparative physiology and biomechanics. The power of lab work has long been recognized by experimental biologists. For example, in 1932, Georgy Gause published an influential paper in Journal of Experimental Biology describing a series of clever lab experiments that provided the first empirical test of competitive exclusion theory, laying the foundation for a field that remains active today. At the time, Gause wrestled with the dilemma of conducting experiments in the lab or the field, ultimately deciding that progress could be best achieved by taking advantage of the high level of control offered by lab experiments. However, physiological experiments often yield different, and even contradictory, results when conducted in lab versus field settings. This is especially concerning in the Anthropocene, as standard laboratory techniques are increasingly relied upon to predict how wild animals will respond to environmental disturbances to inform decisions in conservation and management. In this Commentary, we discuss several hypothesized mechanisms that could explain disparities between experimental biology in the lab and in the field. We propose strategies for understanding why these differences occur and how we can use these results to improve our understanding of the physiology of wild animals. Nearly a century beyond Gause's work, we still know remarkably little about what makes captive animals different from wild ones. Discovering these mechanisms should be an important goal for experimental biologists in the future.

Diploid and triploid Chinook salmon (Oncorhynchus tshawytscha) have altered microRNA responses in immune tissues after infection with Vibrio anguillarum.

Production of sterile fishes through artificial retention of a third set of chromosomes (triploidy) is a sustainable alternative for aquaculture since it reduces escapee pressure on wild populations. However, these fishes have reduced survival in stressful conditions and in response to infection. In this study, the impact of Vibrio anguillarum infection on diploid and triploid Chinook salmon (Oncorhynchus tshawytscha) was investigated to identify if there was any significant immune regulation by microRNAs (miRNA). Small RNAs from hindgut, head kidney, and spleen were sequenced to determine if miRNA transcript abundance was altered due to ploidy and infection in nine-month old full-sibling diploids and triploids. All three tissues had differentially expressed miRNA prior to infection, indicating subtle changes in epigenetic regulation due to increased ploidy. Additionally, miRNA were altered by infection, but there was only a difference in spleen miRNA expression between diploids and triploids at three days of infection. Furthermore, one miRNA (ssa-miR-2188-3p) was confirmed as having an altered response to infection in triploids compared to diploids, implicating potential immune dysregulation due to increased ploidy. The miRNAs identified in this study are predicted to target immune pathways, providing evidence for their importance in regulating responses to pathogens. This study is the first to investigate how increased ploidy alters miRNA expression in response to infection. Additionally, it provides evidence for epigenetic dysregulation in triploid fishes, which may contribute to their poor performance in response to stress.

Sex matters: Gamete-specific contribution of microRNA following parental exposure to hypoxia in zebrafish.

Oxygen availability varies among aquatic environments, and oxygen concentration has been demonstrated to drive behavioral, metabolic, and genetic adaptations in numerous aquatic species. MicroRNAs (miRNAs) are epigenetic modulators that act at the interface of the environment and the transcriptome and are known to drive plastic responses following environmental stressors. An area of miRNA that has remained underexplored is the sex specific action of miRNAs following hypoxia exposure and its effects as gene expression regulator in fishes. This study aimed to identify differences in mRNA and miRNA expression in the F1 generation of zebrafish (Danio rerio) at 1 hpf after either F0 parental male or female were exposed to 2 weeks of continuous (45 %) hypoxia. In general, F1 embryos at 1 hpf demonstrated differences in mRNA and miRNAs expression related to the stressor and to the specific sex of the F0 that was exposed to hypoxia. Bioinformatic pathway analysis of predicted miRNA:mRNA relationships indicated responses in known hypoxia signaling and mitochondrial bioenergetic pathways. This research demonstrates the importance of examining the specific male and female contributions to phenotypic variation in subsequent generations and provides evidence that there is both maternal and paternal contribution of miRNA through eggs and sperm.

A comparative analysis of the partitioning behaviour of SARS-CoV-2 RNA in liquid and solid fractions of wastewater.

As fragments of SARS-CoV-2 RNA can be quantified and measured temporally in wastewater, surveillance of concentrations of SARS-CoV-2 in wastewater has become a vital resource for tracking the spread of COVID-19 in and among communities. However, the absence of standardized methods has affected the interpretation of data for public health efforts. In particular, analyzing either the liquid or solid fraction has implications for the interpretation of how viral RNA is quantified. Characterizing how SARS-CoV-2 or its RNA fragments partition in wastewater is a central part of understanding fate and behaviour in wastewater. In this study, partitioning of SARS-CoV-2 was investigated by use of centrifugation with varied durations of spin and centrifugal force, polyethylene glycol (PEG) precipitation followed by centrifugation, and ultrafiltration of wastewater. Partitioning of the endogenous pepper mild mottled virus (PMMoV), used to normalize the SARS-CoV-2 signal for fecal load in trend analysis, was also examined. Additionally, two surrogates for coronavirus, human coronavirus 229E and murine hepatitis virus, were analyzed as process controls. Even though SARS-CoV-2 has an affinity for solids, the total RNA copies of SARS-CoV-2 per wastewater sample, after centrifugation (12,000 g, 1.5 h, no brake), were partitioned evenly between the liquid and solid fractions. Centrifugation at greater speeds for longer durations resulted in a shift in partitioning for all viruses toward the solid fraction except for PMMoV, which remained mostly in the liquid fraction. The surrogates more closely reflected the partitioning of SARS-CoV-2 under high centrifugation speed and duration while PMMoV did not. Interestingly, ultrafiltration devices were inconsistent in estimating RNA copies in wastewater, which can influence the interpretation of partitioning. Developing a better understanding of the fate of SARS-CoV-2 in wastewater and creating a foundation of best practices is the key to supporting the current pandemic response and preparing for future potential infectious diseases.

Seasonal, environmental and individual effects on hypoxia tolerance of eastern sand darter (Ammocrypta pellucida).

Metabolic rate and hypoxia tolerance are highly variable among individual fish in a stable environment. Understanding the variability of these measures in wild fish populations is critical for assessing adaptive potential and determining local extinction risks as a result of climate-induced fluctuations in temperature and hypoxic conditions. We assessed the field metabolic rate (FMR) and two hypoxia tolerance metrics, oxygen pressure at loss of equilibrium (PO2 at LOE) and critical oxygen tolerance (Pcrit) of wild-captured eastern sand darter (Ammocrypta pellucida), a threatened species in Canada, using field trials (June to October) that encompassed ambient water temperatures and oxygen conditions typically experienced by the species. Temperature was significantly and positively related to hypoxia tolerance but not FMR. Temperature alone explained 1%, 31% and 7% of the variability observed in FMR, LOE, and Pcrit, respectively. Environmental and fish-specific factors such as reproductive season and condition explained much of the residual variation. Reproductive season significantly affected FMR by increasing it by 159-176% over the tested temperature range. Further understanding the impact of reproductive season on metabolic rate over a temperature range is crucial for understanding how climate change could impact species fitness. Among-individual variation in FMR significantly increased with temperature while among-individual variation in both hypoxia tolerance metrics did not. A large degree of variation in FMR in the summer might allow for evolutionary rescue with increasing mean and variance of global temperatures. Findings suggest that temperature may be a weak predictor in a field setting where biotic and abiotic factors can act concurrently on variables that affect physiological tolerance.

Nuclear microRNAs may regulate mitochondrial gene expression following effluent exposure in darter (Etheostoma) species.

Wastewater effluent is a metabolic stressor to aquatic organisms, though the mechanisms regulating metabolic rate in fish are not fully understood. Changes in metabolism may be regulated by microRNA (miRNA), small RNA molecules that post-transcriptionally regulate target mRNA translation in fish. Nuclear encoded miRNA are present in mammalian mitochondria where they regulate translation of mitochondrial genes, namely subunits for oxidative phosphorylation complexes; though this mechanism has not been identified in fish. This study aimed to identify if miRNA are present in darter (Etheostoma spp.) mitochondria, and if the metabolic stress occurring in darters in the Grand River, Waterloo, is partly regulated by miRNAs supressing translation of target mitochondrial genes. Three species of darters (E. caeruleum; E. nigrum; E. flabellare) were collected from upstream and downstream of the Waterloo wastewater treatment plant, and qPCR analysis confirmed the presence of four miRNA bioinformatically predicted to target mitochondrial mRNAs within the mitochondria, namely let-7a, miR-1, miR-122 and miR-20. E. caeruleum collected from downstream had lower cytochrome c oxidase activity, with a respective higher miR-1 abundance in the mitochondria, while E. nigrum had both a higher miR-20 abundance and cytochrome c oxidase activity downstream. E. flabellare was the only species that exhibited a lower miR-122 abundance downstream, despite no difference in cytochrome c oxidase activity between sites. Overall, this study confirmed the presence of miRNA within the mitochondria of daters, predicted a relationship between miR-1, and miR-20 abundance and cytochrome c oxidase activity, and identified one sex-specific miRNA, miR-20.

Impacts on antioxidative enzymes and transcripts in darter (Etheostoma spp.) brains in the Grand River exposed to wastewater effluent.

The Grand River watershed is the largest in southern Ontario and assimilates thirty wastewater treatment plants (WWTP) with varied degrees of treatment. Many WWTPs are unable to effectively eliminate several contaminants of emerging concern (CECs) from final effluent, leading to measurable concentrations in surface waters. Exposures to CECs have reported impacts on oxidative stress measured through antioxidative enzymes (SOD, CAT, GPX). This study focuses on the effects of WWTP effluent on four Etheostoma (Darter) species endemic to the Grand River, by investigating if increased antioxidative response markers are present in darter brains downstream from the effluent outfall compared to an upstream reference site relative to the Waterloo, ON WWTP across two separate years (Oct 2020 and Oct 2021). This was assessed using transcriptional and enzyme analysis of antioxidant enzymes and an enzyme involved in serotonin synthesis, tryptophan hydroxylase (tph). In fall 2020, significant differences in transcript markers were found between sites and sexes in GSD with SOD and CAT showing increased expression downstream, in JD with both sexes showing increased SOD downstream, and an interactive effect for tph in RBD. Changes in transcripts aligned with enzyme activity where interactive effects with sex-related differences were observed in fish collected fall 2020. In contrast, transcripts measured in fall 2021 were increased upstream compared to downstream species in RBD and GSD. This study additionally displayed yearly, species and sex differences in antioxidant responses. Continued investigation on the impacts of CECs in effluent in non-target species is required to better understand WWTP effluent impacts.

Responses of microRNA and predicted mRNA and enzymatic targets in liver of two salmonids (Oncorhynchus mykiss and Salvelinus fontinalis) following air exposure.

The acute stress response is well-characterized, with rainbow trout as a teleost model for physiological and molecular responses. Air exposure, which stimulates an acute stress response, modulates liver microRNAs in rainbow trout; however, these highly conserved non-coding RNAs that bind to mRNA and repress translation, have never been measured in brook trout and it is unknown how miRNA expression responds following air exposure in this less studied salmonid. Our objective was to characterize the effects of air exposure on rainbow and brook trout liver miRNA expression, as well as the mRNA expression and enzyme activity that the miRNAs are predicted to target. Brook and rainbow trout were sampled pre- and 1-, 3-, and 24-h post- a three-minute air exposure. Plasma cortisol, glucose, and lactate were measured. Relative expression of miR-21a-5p, miR-143-3p, let-7a-5p and relative expression and enzyme activities of five predicted targets (pyruvate kinase, glucokinase, citrate synthase, cytochrome c oxidase, and catalase) were measured in liver. Rainbow and brook trout both had increases in plasma cortisol and lactate, while only rainbow trout had significant post-stress increases in plasma glucose. Furthermore, both trout species had increased miR-143-3p and miR-21a-5p relative expression 24-h post-stress. Four of the five enzymes measured had altered activity following stress. Brook trout miRNAs had inverse relative expression with relative catalase mRNA expression and cytochrome c oxidase enzyme activity, but no relationship was found in rainbow trout. Therefore, we have further characterized the transcriptional and enzymatic response to air exposure in two salmonids.

Chronic exposure to anthropogenic and climate related stressors alters transcriptional responses in the liver of zebrafish (Danio rerio) across multiple generations.

The antidepressant, venlafaxine (VFX), and climate change stressors, such as increased water temperature and decreased dissolved oxygen, are current threats to aquatic environments. This study aimed to determine how microRNAs (miRNAs) and predicted targeted transcripts were altered in livers of zebrafish exposed to these stressors, and livers of their un-exposed F1 and F2 offspring. Following a 21 day exposure to multiple stressors (1 µg/L VFX, +5 °C ambient, 50% O2), then a subsequent 21 day recovery, relative abundances of cyp3a65, hsp70, hsp90, and ppargc1a and miRNAs predicted to target them (miR-142a, miR-16c, miR-181c, and miR-129, respectively) were measured in the liver via quantitative PCR (RT-qPCR). There were significant decreases in miR-142a in the exposed F0 generation and the exposed F1 generation. While there were no changes detected in cyp3a65 relative abundance, there was a significant inverse relationship between cyp3a65 and miR-142a. Hsp70 expression significantly increased in the F1 generation, which persisted to the F2 generation and the relative abundance of hsp90 significantly increased in all generations. There was a significant reduction in miR-181c in the F1 generation, but there was no significant relationship between miR-181c and hsp90. Finally, there was a significant decrease in ppargc1a relative abundance in the F1 generation which was associated with an increase in miR-129. Combined, these results suggest that parental exposure to multiple, environmentally relevant stressors can confer transcriptional and epigenetic responses in the F1 and F2 generations, although identifying which stressor is a driving force becomes unclear.

Increased metabolic rate associated with immune stimulation of heat-killed Vibrio anguillarum at different temperatures in zebrafish (Danio rerio).

The action of the immune response in zebrafish (Danio rerio) has been a target of many studies. However, the energetic demands involved in the immune response are poorly understood in ectothermic poikilotherms, such as fish. This research aims to characterize the energetic response of zebrafish to an immune challenge of heat-killed Vibrio anguillarum at 22 °C and 27.5 °C. Zebrafish were either not injected, injected intraperitoneally with 10 µl of saline and Freund's incomplete adjuvant (sham), or heat-killed Vibrio anguillarum & Freund's incomplete adjuvant (1.21 × 1010 cfu/ml). Respirometry was then performed on these zebrafish for a period of 27 h. Following this, spleen was collected for quantitative PCR analysis of the catalytic subunit of AMPK (ampka1 & ampka2), the nuclear factor kappa-light-chain-enhancer of activated B cells (nf-kb), and several cytokines (tnfa, il-1b, il-8, il-10). While there was no increase in oxygen consumption with any treatment at 22 °C, there was a marked 30% increase in oxygen consumption in zebrafish injected with heat-killed Vibrio at 27.5 °C. Furthermore, temperature had a strong effect on the timing of the immune response. At 22 °C, there was a 2-3-fold increase in the cytokines measured associated with heat-killed Vibrio injection, whereas there were no differences found at 27.5 °C. Furthermore, while there was an increase in ampka2 at 22 °C, there was a sharp decrease in ampka2 at 27.5 °C, although the changes in ampka2 transcript abundance could not be solely attributed to heat-killed Vibrio, as there were similar changes associated with the sham group. The results of this study demonstrate some of the first evidence that zebrafish increase routine metabolic rate associated with immune stimulation.

Time Series Resolution of the Fish Necrobiome Reveals a Decomposer Succession Involving Toxigenic Bacterial Pathogens.

Despite progress understanding microbial communities involved in terrestrial vertebrate decomposition, little is known about the microbial decomposition of aquatic vertebrates from a functional and environmental context. Here, we analyzed temporal changes in the "necrobiome" of rainbow darters, which are common North American fish that are sensitive indicators of water quality. By combining 16S rRNA gene and shotgun metagenomic sequence data from four time points, we studied the progression of decomposers from both taxonomic and functional perspectives. The 16S rRNA gene profiles revealed strong community succession, with early decomposition stages associated with Aeromonas and Clostridium taxa and later stages dominated by members of the Rikenellaceae (i.e., Alistipes/Acetobacteroides genera). These results were reproducible and independent of environmental perturbation, given that exposure to wastewater treatment plant effluent did not substantially influence the necrobiome composition of fish or the associated water sample microbiota. Metagenomic analysis revealed significant changes throughout decomposition in degradation pathways for amino acids, carbohydrates/glycans, and other compounds, in addition to putrefaction pathways for production of putrescine, cadaverine, and indole. Binning of contigs confirmed a predominance of Aeromonas genome assemblies, including those from novel strains related to the pathogen Aeromonas veronii These bins of Aeromonas genes also encoded known hemolysin toxins (e.g., aerolysin) that were particularly abundant early in the process, potentially contributing to host cell lysis during decomposition. Overall, our results demonstrate that wild-caught fish have a reproducible decomposer succession and that the fish necrobiome serves as a potential source of putative pathogens and toxigenic bacteria.IMPORTANCE The microbial decomposition of animal tissues is an important ecological process that impacts nutrient cycling in natural environments. We studied the microbial decomposition of a common North American fish (rainbow darters) over four time points, combining 16S rRNA gene and shotgun metagenomic sequence data to obtain both taxonomic and functional perspectives. Our data revealed a strong community succession that was reproduced across different fish and environments. Decomposition time point was the main driver of community composition and functional potential; fish environmental origin (upstream or downstream of a wastewater treatment plant) had a secondary effect. We also identified strains related to the putative pathogen Aeromonas veronii as dominant members of the decomposition community. These bacteria peaked early in decomposition and coincided with the metagenomic abundance of hemolytic toxin genes. Our work reveals a strong decomposer succession in wild-caught fish, providing functional and taxonomic insights into the vertebrate necrobiome.

Acute air exposure modulates the microRNA abundance in stress responsive tissues and circulating extracellular vesicles in rainbow trout (Oncorhynchus mykiss).

The hypothalamic-pituitary-interrenal axis is an important regulator of stress and metabolism in teleosts. Cortisol is secreted by the head kidney where it increases gluconeogenesis in the liver to increase circulating glucose levels. MicroRNAs (miRNAs) are small, non-coding RNA molecules that bind to the 3' untranslated region of specific mRNA to regulate their expression. MicroRNAs can also be secreted into circulation by association with extracellular vesicles (EVs) where they can influence the phenotype of other tissues. In this study, adult rainbow trout were exposed to a 3-minute acute air stress and allowed to recover for 1-, 3-, or 24-h to determine how miRNAs were altered. MicroRNAs measured in this study were chosen based on their high relative abundance in tissues that drive the stress response (miR-21a-3p, let-7a-5p, miR-143-3p) or their role in regulating DNA methylation (miR-29a-3p). In general, miRNAs increased in circulating EVs during the recovery period while decreasing in head kidney and liver at the same timepoints. Predicted targets for these miRNAs were analyzed using KEGG and DAVID functional enrichment analysis. Pathways involved in metabolism and cell signaling were predicted to be upregulated. Future studies can use these results to investigate how pathways are regulated after stress. Overall, our results indicate that miRNAs are regulated during teleost stress responses and could be supporting the cortisol-mediated changes that occur.

A novel sigma coordinate system to simulate abrupt changes of underwater terrain in a hydrodynamic model: application to Lake Mead, USA.

The sigma (SIG) coordinate system in ocean circulation simulation models results inevitably in horizontal pressure gradient error. This problem also emerges in models of deep lakes or reservoirs with the same characteristics of underwater terrain mutation. SIG coordinates reflect vertical relative stratification but cannot be used to calculate horizontal pressure gradient force in places with drastic topographic changes; this results in vertical water temperature and circulation errors. In deep lakes or reservoirs, differences in water density caused by the temperature difference between upper and lower water bodies is the primary cause of thermal stratification phenomena. Lake Mead was used as a case study on steep topography based on Environmental Fluid Dynamics Code (EFDC) model in this study. SIG coordinates result in close agreement between the calibrated temperature time series at the top and middle water layers, but disparity in the bottom water layer. The error emerges in the horizontal pressure gradient error due to the SIG coordinate transformation. Neither increasing the vertical resolution nor adjusting the horizontal viscosity coefficient resolve this error. We test the sigma-zed (SGZ) coordinate which combines Z coordinate and SIG coordinate as a replacement for the SIG coordinate to find that they effectively reduce the model's runtime and simulation efficiency. The vertical temperature distribution in SGZ coordinate mode is more accurate than the distribution in SIG coordinate mode. The Navier-Stokes horizontal gradient and advection diffusion equation results under SIG coordinates are very sensitive to the pressure gradient. The replacement also enhances resolution near the thermocline, facilitates reclosing of the water bottom and the equal sigma surface, lends significant advantages in terms of vertical temperature in the simulation for local deep water with steep terrain, and shortens runtime for 0.14 h. SGZ mixed coordinates are recommended in the simulation of deep lakes or reservoirs wherein the underwater topography is large (with abundant continuous deep trenches or reefs).

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.

Chronic exposure to venlafaxine and increased water temperature reversibly alters microRNA in zebrafish gonads (Danio rerio).

MicroRNA (miRNA) are short, non-coding RNA that act by downregulating targeted mRNA transcripts. Only recently have they been used as endpoints in studies of aquatic toxicology. The purpose of this study was to determine the effect of an antidepressant contaminant, venlafaxine (VFX), and increased temperature on specific microRNA levels in zebrafish (Danio rerio) reproductive tissue. Adult zebrafish were exposed to one of four conditions; control, 1 µg/L VFX (VFX), 32 °C (Temp), or 1 µg/L VFX + 32 °C (VFX & Temp) for 21 days. Half of the fish were returned to control conditions for a 21-day recovery period. RT-qPCR was performed to measure relative abundances of several miRNAs known to respond to antidepressant exposure: dre-miR-22b-3p, dre-miR-301a, dre-miR-140-5p, dre-let-7d-5p, dre-miR-210-5p, and dre-miR-457b-5p. After the exposure period, dre-miR-22b-3p and dre-miR-301a showed a significant downregulation in response to all treatments. In contrast, after the recovery period, there were no significant differences in microRNA abundance. These altered microRNA are predicted to target several genes, including phosphofructokinase, and are associated with ovarian pathologies. Combined, we have shown that VFX and increased water temperature alter miRNA abundances in zebrafish reproductive tissue, an effect correlated with a functional stress response and cell cycle dysregulation.

Multiple Stressors in the Environment: The Effects of Exposure to an Antidepressant (Venlafaxine) and Increased Temperature on Zebrafish Metabolism.

Aquatic organisms are continuously exposed to multiple environmental stressors working cumulatively to alter ecosystems. Wastewater-dominated environments are often riddled by a myriad of stressors, such as chemical and thermal stressors. The objective of this study was to examine the effects of an environmentally relevant concentration of a commonly prescribed antidepressant, venlafaxine (VFX) [1.0 µg/L], in addition to a 5°C increase in water temperature on zebrafish metabolism. Fish were chronically exposed (21 days) to one of four conditions: (i) 0 µg/L VFX at 27°C; (ii) 1.0 µg/L VFX at 27°C; (iii) 0 µg/L VFX at 32°C; (iv) 1.0 µg/L VFX at 32°C. Following exposure, whole-body metabolism was assessed by routine metabolic rate (RMR) measurements, whereas tissue-specific metabolism was assessed by measuring the activities of major metabolic enzymes in addition to glucose levels in muscle. RMR was significantly higher in the multi-stressed group relative to Control. The combination of both stressors resulted in elevated pyruvate kinase activity and glucose levels, while lipid metabolism was depressed, as measured by 3-hydroxyacyl CoA dehydrogenase activity. Citrate synthase activity increased with the onset of temperature, but only in the group treatment without VFX. Catalase activity was also elevated with the onset of the temperature stressor, however, that was not the case for the multi-stressed group, potentially indicating a deleterious effect of VFX on the anti-oxidant defense mechanism. The results of this study highlight the importance of multiple-stressor research, as it able to further bridge the gap between field and laboratory studies, as well as have the potential of yielding surprising results that may have not been predicted using a conventional single-stressor approach.