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Thirteen-lined ground squirrels (TLGSs) are obligate hibernators that cycle between torpor (low metabolic rate and body temperature) and interbout euthermia (IBE; typical euthermic body temperature and metabolism) from late autumn to spring. Many physiological changes occur throughout hibernation, including a reduction in liver mitochondrial metabolism during torpor, which is reversed during arousal to interbout euthermia. Nuclear-encoded microRNA (miRNA, small posttranscriptional regulator molecules) differ in abundance throughout TLGS hibernation and have been shown to regulate mitochondrial gene expression in mammalian cell culture (where they are referred to as mitomiRs). This study characterized differences in mitomiR profiles from TLGS liver mitochondria isolated during summer, torpor, and IBE, and predicted their mitochondrial targets. Using small RNA sequencing, differentially abundant mitomiRs were identified between hibernation states, and using quantitative PCR analysis, we quantified the expression of predicted mitochondrial mRNA targets. Most differences in mitomiR abundances were seasonal (i.e., between summer and winter) with only one mitomiR differentially abundant between IBE and torpor. Multiple factor analysis (MFA) revealed three clusters divided by hibernation states, where clustering was predominantly driven by mitomiR abundances. Nine of these differentially abundant mitomiRs had predicted mitochondrial RNA targets, including subunits of electron transfer system complexes I and IV, 12S rRNA, and two tRNAs. Overall, mitomiRs were predicted to suppress the expression of their mitochondrial targets and may have some involvement in regulating protein translation in mitochondria. This study found differences in mitomiR abundances between seasons and hibernation states of TLGS and suggests potential mechanisms for regulating the mitochondrial electron transfer system. NEW & NOTEWORTHY: During the hibernation season, thirteen-lined ground squirrels periodically increase metabolism remarkably between torpor and interbout euthermia (IBE). This process involves rapid reactivation of mitochondrial respiration. We predicted that mitochondrial microRNA (mitomiRs) might be altered during this response. We found that the abundance of 38 liver mitomiRs differs based on hibernation state (summer, IBE, and torpor). Small RNA sequencing identified mitomiR profiles, including some mitomiRs that are predicted to bind to mitochondrial RNAs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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;43:1569–1582. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Resuspension and redistribution of sediments induced by propeller wash may significantly influence aquatic ecosystems at contaminated sediment sites. This study describes a numerical modeling method developed to predict the sediment resuspension and subsequent transport processes resulting from ship traffic, with a fully coupled simulation of hydrodynamics, sediment transport, and propeller wash. By including propeller momentum effects in the flow field computation, the advection and dispersion of resuspended sediments are better represented than in previously available methods. To achieve this improvement, a computational algorithm was first developed to calculate the propeller wash effects from one or more ships (e.g., erosion rate and momentum flux); these results were then dynamically linked to a hydrodynamic and sediment transport computation using Environmental Fluid Dynamics Code Plus (EFDC+). This modeling framework was evaluated using a field experiment conducted by the US Navy. The model was calibrated with flow velocities and sediment erosion depths, and then validated with resuspended sediment concentrations in the water column. The model results reproduced the horizontal and vertical distributions of resuspended sediments better when the propeller-induced momentum was incorporated into the flow field computation. The sensitivity test indicated that the increased flow energy from propeller momentum resulted in significant dispersion of resuspended sediments in both longitudinal and lateral directions. The maximum scour was dependent on the propeller revolution speed, the ship engine power, and the distance between the propellers and the sediment bed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Environmental Fluid Dynamics Code, a numerical estuarine and coastal ocean circulation hydrodynamic and eutrophication model, was used to simulate the distributions of dissolved oxygen (DO), salinity, water temperature, and nutrients in the Caloosahatchee River Estuary. Modeled DO, salinity, and water temperature were in good agreement with field observational data from the Florida Department of Environmental Protection and South Florida Water Management District. Sensitivity analyses identified the effects of fiver discharge, atmospheric winds, and tidal forcing on the spatial and temporal distributions of DO. Simulation results indicated that vertical mixing due to wind forcing increased the bottom DO concentration. River discharge enhanced stratification in deep locations but propagated vertical mixing in the shallow upper estuary. Finally, tidal forcing heavily influenced bottom layer DO concentrations throughout the whole fiver estuary. DOI: 10.1061/(ASCE)EE.1943-7870.0000239 CE Database subject headings: Coastal processing; Oceans; Dissolved oxygen; Estuaries; Florida. Author keywords: Caloosahatchee; EFDC; Coastal ocean; Dissolved oxygen.

Hypoxia (low oxygen) exposure generally leads to decreased reproductive capacity, exhibited by reductions in testicular mass, reproductive hormones, and sperm swimming speed. However, in many fish species, reproduction occurs either periodically or exclusively under hypoxic conditions. In this study we assessed how hypoxia influences sperm performance in the plainfin midshipman (Porichthys notatus Girard, 1854), a species that lives in intertidal nests that become hypoxic during low tides. We exposed sperm from the same male to normoxic or hypoxic conditions and compared sperm characteristics and oxygen consumption between treatments. Sperm exposed to hypoxic water swam faster and consumed more oxygen than sperm swimming in normoxic conditions. Sperm swimming speed was positively related with oxygen consumption. For each male, the percentage of motile spermatozoa did not differ between treatments, suggesting that the same number of sperm were active but their performance was dependent on the dissolved oxygen content in the water. We discuss the implications of our results in the context of sperm competition and fertilization success under hypoxic conditions. Une exposition a l'hypoxie (faibles concentrations d'oxygene) mene generalement a une diminution de la capacite reproductive, ce qui se manifeste par une reduction de la masse testiculaire, des hormones reproductives et de la vitesse de nage des spermatozoides. Cependant, chez plusieurs especes de poissons, la reproduction a lieu periodiquement on meme exclusivement dans des conditions d'hypoxie. Dans notre etude, nous evaluons les influences de l'hypoxie sur la performance des spermatozoides chez le pilotin tachete (Porichthys notatus Girard, 1854), une espece qui vit dans des nids intertidaux qui deviennent hypoxiques a maree basse. Nous avons expose les spermatozoides d'un meme male a des conditions normoxiques on hypoxiques et avons compare les caracteristiques des spermatozoides et leur consommation d'oxygene dans les deux conditions experimentales. Les spermatozoides exposes a l'eau hypoxique nagent plus rapidement et consomment plus d'oxygene que les spermatozoides qui nagent en conditions normoxiques. Il y a une relation positive entre la vitesse de nage des spermatozoides et leur consommation d'oxygene. Chez un meme male, le pourcentage de spermatozoides mobiles ne varie pas d'un traitement a 1'autre, ce qui laisse croire qu'il y a le meme nombre de spermatozoides en activite, mais que leur performance depend de la concentration d'oxygene dissous dans l'eau. Nous discutons des implications de nos resultats dans le cadre de la competition spermatique et du succes de la fecondation dans des conditions hypoxiques. [Traduit par la Redaction], Introduction Environmental exposure to hypoxia (low oxygen) typically impairs reproductive functions in males. For example, in humans, rodents, and primates, males exposed to hypobaric hypoxia have disrupted testicular development, produce [...]

Metals like iron (Fe) and copper (Cu) function as integral components in many biological reactions, and, in excess, these essential metals are toxic, and organisms must control metal acquisition and excretion. We examined the effects of chronic waterborne Cu exposure and the interactive effects of elevated dietary Fe on gene expression and tissue metal accumulation in zebrafish. Softwater acclimated zebrafish exposed to 8 [micro]g/l Cu, with and without supplementation of a diet high in Fe (560 vs. 140 mg Fe/kg food) for 21 days demonstrated a significant reduction in liver and gut Cu load relative to waterborne Cu exposure alone. Gene expression levels for divalent metal transport (DMT)-I, copper transporter (CTR)-I, and the basolateral metal transporter ATP7A in the gills and gut increased when compared with controls, but the various combinations of Cu and high-Fe diet revealed altered levels of expression. Further examination of the basolateral Fe transporter, ferroportin, showed responses to waterborne Cu exposure in the gut and a significant increase with Fe treatment alone in the liver. Additionally, we examined metallothionein 1 and 2 (MT1 and MT2), which indicated that MT2 is more responsive to Cu. To explore the relationship between transcription and protein function, we examined both CTR-1 protein levels and gill apical uptake of radiolabeled [Cu.sup.64], which demonstrated decreased Cu uptake and protein abundance in the elevated Cu treatments. This study shows that high dietary Fe can significantly alter the genetic expression pattern of Cu transporters at the level of the gill, liver, and gastrointestinal tract. divalent metal transport; copper transporter; metallothionein

In fish, environmental pollution is one factor that induces oxidative stress, and this can disturb the natural antioxidant defense system. Oxidative stress has been well characterized in vitro, yet the in vivo effects of metal-induced oxidative stress have not been extensively studied. In two experiments we examined the impacts of copper (Cu) on gene expression, oxidative damage, and cell oxidative capacity in liver and gill of zebrafish. In the first experiment, soft water-acclimated zebrafish were exposed to 8 and 15 [micro]g/1 Cu for 48 h. This exposure resulted in significant increases in gene expression of cytochrome c oxidase subunit 17 (COX-17) and catalase, associated with both increased Cu load and protein carbonyl concentrations in the gill and liver after 48 h. In addition, we examined the potential protective effects of increased waterborne [Ca.sup.2+] (3.3 mM) and [Na.sup.+] (10 mM) on acute Cu toxicity. While both treatments were effective at reducing liver and/or gill Cu loads and attenuating oxidative damage at 48 h, 10 mM [Na.sup.+] was more protective than 3.3 mM [Ca.sup.2+]. There were variable changes in the maximal activities of COX and citrate synthase (CS), indicating possible alterations in cell oxidative capacity. Moreover, Cu affected COX-to-CS ratios in both gill and liver, suggesting that Cu alters normal mitochondrial biogenic processes, possibly though metallochaperones like COX-17. Overall, this study provides important steps in determining the transcriptional and physiological endpoints of acute Cu toxicity in a model tropical species. gene expression; oxidative capacity; catalase; COX-17; protein-carbonyls

Hypoxia stress suppresses appetite in a variety of fish species, but the mechanisms mediating this response are not known. Therefore, given their anorexigenic and hypophysiotropic properties, we investigated the contribution of forebrain corticotropin-releasing factor (CRF) and urotensin I (UI) to the regulation of food intake and the hypothalamic-pituitary-interrenal (HPI) stress axis in hypoxic rainbow trout. Exposure to 50 and 35% 02 saturation for 24 h decreased food intake by 28 and 48%, respectively. The 35% [O.sub.2] treatment also increased forebrain CRF and UI mRNA levels, plasma cortisol, and lactate. Exposure for 72 h to the same conditions resulted in similar reductions in food intake, increases in plasma cortisol proportional to the hypoxia severity, and increases in forebrain CRF and UI mRNA levels in the 50% [O.sub.2] treatment. Relative to saline-infused fish, chronic intracranial infusion of the CRF receptor antagonist [alpha]-helical CR[F.sub.(9-41)] reduced the appetite-suppressing effects of 24-h exposure to 35% [O.sub.2] and blocked the hypoxia-induced increase in plasma cortisol. Finally, forebrain microdissection revealed that 50 and 35% [O.sub.2] exposure for 24 h specifically increases preoptic area CRF and UI mRNA levels in proportion to the severity of the hypoxic challenge and either has no effect or elicits small decreases in other forebrain regions. These results show that CRF-related peptides play a physiological role in regulating the HPI axis and in mediating at least a portion of the reduction in food intake under hypoxic conditions in rainbow trout and demonstrate that the response of forebrain CRF and UI neurons to this stressor is region specific. feeding behavior; anorexia; hypothalamic-pituitary-interrenal axis; cortisol; Oncorhynchus mykiss

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. [ABSTRACT FROM AUTHOR]

The effluent from municipal wastewater treatment plants is a major point source of contamination in Canadian waterways. The improvement of effluent quality to reduce contaminants, such as pharmaceuticals and personal care products, before being released into the environment is necessary to reduce the impacts on organisms that live in the river downstream. Here, we aimed to characterize the metabolic and gill physiological responses of rainbow (Etheostoma caeruleum), fantail (Etheostoma flabellare), and greenside (Etheostoma blennioides) darters to the effluent in the Grand River from the recently upgraded Waterloo municipal wastewater treatment plant. The routine metabolism of darters was not affected by effluent exposure, but some species had increased maximum metabolic rates, leading to an increased aerobic scope. The rainbow darter aerobic scope increased by 2.2 times and the fantail darter aerobic scope increased by 2.7 times compared to the reference site. Gill samples from effluent-exposed rainbow darters and greenside darters showed evidence of more pathologies and variations in morphology. These results suggest that darters can metabolically adjust to effluent-contaminated water and may also be adapting to the urban and agricultural inputs. The modification and damage to the gills provide a useful water quality indicator but does not necessarily reflect how well acclimated the species is to the environment due to a lack of evidence of poor fish health. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Warm acclimation of rainbow trout can cause a decrease in the collagen content of the heart. This ability to remove cardiac collagen is particularly interesting considering that collagen deposition in the mammalian heart, following an injury, is permanent. We hypothesized that collagen removal can be facilitated by microRNA-29b (miR-29b), a highly conserved, small, non-coding RNA, as a reduction in this microRNA has been reported during the development of fibrosis in the mammalian heart. We also used a bioinformatics approach to investigate the binding potential of miR-29b to the seed sequences of vertebrate collagen isoforms. Cultured trout cardiac fibroblasts were transfected with zebrafish mature miR-29b mimic for 7 days with re-transfection occurring after 3 days. Transfection induced a 17.8-fold increase in miR-29b transcript abundance (P<0.05) as well as a 54% decrease in the transcript levels of the col1a3 collagen isoform, compared with non-transfected controls (P<0.05). Western blotting demonstrated that the level of collagen type I protein was 85% lower in cells transfected with miR-29b than in control cells (P<0.05). Finally, bioinformatic analysis suggested that the predicted 3'-UTR of rainbow trout col1a3 has a comparatively higher binding affinity for miR-29b than the 3'-UTR of col1a1. Together, these results suggest that miR-29b is a highly conserved regulator of collagen type I protein in vertebrates and that this microRNA decreases collagen in the trout heart by targeting col1a3. [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase is an enzyme that mediates communication between cellular energy status and diverse effector proteins, particularly those that play roles in determining the metabolic phenotype. By phosphorylating metabolic enzymes, transcriptional regulators and proteins involved in cellular structure, it can modify energy metabolism in both the short term and long term. Its basic features are highly conserved, with homologues in all eukaryotes. Gene and/or genome duplications endowed early vertebrates with paralogs of AMPK subunits, though the nature of their subfunctionalization remains uncertain, even in mammals. While most research focuses on the role of the enzyme in human health, a great deal can be learned from comparative studies targeting non-traditional model animals. Fish, in particular, are interesting models because of the diversity in the metabolic properties and complex relationships between metabolism and environmental challenges. In this review, we examine what is known about AMPK structure and function though the lens of comparative physiology, looking for opportunities to better understand how this vital energy sensor has evolved in animals. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • MitomiRs let-7a, miR-1, miR-122 and miR-20 are present within fish mitochondria. • MitomiR miR-20 is sex-specific within rainbow and fantail darters. • MitomiRs may suppress translation of mitochondrial mRNA in darters. • Cytochrome c oxidase activity may be regulated by mitomiRs in darter livers. [ABSTRACT FROM AUTHOR]

Micro ribonucleic acids (miRNAs) are recently discovered small regulatory molecules that control messenger RNA (mRNA) translation in plants and animals and have been implicated in a variety of hormone-related physiological pathways. Estrogens, thyroid hormones, and gonadotropins are all known to act on miRNA abundance to cause major shifts in cellular activity, physiology, and homeostatic control mechanisms. Research on cancer biology has also recently considered miRNA as therapeutic targets, because the deregulation of specific miRNAs in various tissues has been correlated with tumorigenesis and other carcinogenic responses. Because many pharmaceuticals are considered to be endocrine-disrupting chemicals (EDCs), their effects on miRNAs may be important to our understanding of basic physiological control and phenotypic outcomes of wildlife exposed to EDCs. Presented is a brief overview of the synthesis, control, and action of miRNAs, focusing on endocrine systems. The antidepressant fluoxetine will be used as an example for miRNA studies in aquatic species, one of the few examples in ecotoxicology. Given the mounting evidence that miRNAs are regulated by hormones, a clear need exists to investigate the potential for environmental EDCs to deregulate miRNA expression and action. Environ Toxicol Chem 2016;35:788-793. © 2015 SETAC [ABSTRACT FROM AUTHOR]

This study examined the similarities in microRNA profiles between fasted and fluoxetine (FLX) exposed zebrafish and downstream target transcripts and biological pathways. Using a custom designed microarray targeting 270 zebrafish miRNAs, we identified 9 differentially expressed miRNAs targeting transcripts in biological pathways associated with anabolic metabolism, such as adipogenesis, cholesterol biosynthesis, triacylglycerol synthesis, and insulin signaling. Exposure of female zebrafish to 540 ng/L FLX, an environmentally relevant concentration and a known metabolic repressor, increased specific miRNAs indicating greater inhibition of these pathways in spite of continued feeding. Further examination revealed two specific miRNAs, dre-let-7d and dre-miR-140-5p, were predicted in silico to bind to a primary regulator of metabolism, adenosine monophosphate-activated protein kinase (AMPK), and more specifically the two isoforms of the catalytic subunit, AMPKα1 and α2, respectively. Real-time analysis of the relative transcript abundance of the α1 and α2 mRNAs indicated a significant inverse relationship between specific miRNA and target transcript. This suggests that AMPK-related pathways may be compromised during FLX exposure as a result of increased miRNA abundance. The mechanism by which FLX regulates miRNA abundance is unknown but may be direct at the liver. The serotonin transporter, slc6a4, is the target of FLX and other selective serotonin reuptake inhibitors (SSRI) and it was found to be expressed in the liver, although treatment did not alter expression of this transporter. Exposure to FLX disrupts key hepatic metabolic pathways, which may be indicative of reduced overall fitness and these effects may be linked to specific miRNA abundance. This has important implications for the heath of fish because concentrations of SSRIs in aquatic ecosystems are continually increasing. [ABSTRACT FROM AUTHOR]

Abstract: The role of methionine supplementation in fish metabolism remains largely unexplored. This study investigates the effects of methionine deprivation (MD) on glucose uptake in rainbow trout (Oncorhynchus mykiss) hepatocytes. To this end, primary hepatocytes were incubated in the presence (+M) or the absence (−M) of methionine for 48h and glucose uptake was assessed using a novel non-radioactive, fluorescent-linked enzymatic assay. Evidence indicated that glucose uptake increased under methionine deprivation, primarily due to the increased abundance of membrane bound sodium-glucose transporter 2 (SGLT2), which was likely facilitated by the cellular reduction in [ATP] resulting from increased mitochondrial uncoupling, as supported by elevated transcript levels of uncoupling protein 2a (UCP2a). This study is the first to suggest that the mechanisms responsible for the rapid glucose uptake associated with MR are facilitated by the greater abundance of SGLT2 glucose transporter and mitochondrial uncoupling. [Copyright &y& Elsevier]

Naphthenic acids (NA) are used in a variety of commercial and industrial applications, and are primary toxic components of oil sands wastewater. We investigated developmental and metabolic responses of tadpoles exposed to sub-lethal concentrations of a commercial NA blend throughout development. We exposed Lithobates pipiens tadpoles to 1 and 2 mg/L NA for 75 days and monitored growth and development, condition factor, gonad and liver sizes, and levels of liver glucose, glycogen, lipids and cholesterol following exposure. NA decreased growth and development, significantly reduced glycogen stores and increased triglycerides, indicating disruption to processes associated with energy metabolism and hepatic glycolysis. Effects on liver function may explain reduced growth and delayed development observed in this and previous studies. Our data highlight the need for greater understanding of the mechanisms leading to hepatotoxicity in NA-exposed organisms, and indicate that strict guidelines may be needed for the release of NA into aquatic environments. [Copyright &y& Elsevier]

Mammalian studies report that methionine restriction (MR) as a dietary regimen extends life span, delays the onset of age-related diseases and enhances fat oxidation in obese subjects with metabolic syndromes. However, the underlying cellular signalling pathways are poorly understood. Rainbow trout (Oncorhynchus mykiss) is a glucose-intolerant species, providing an excellent model for the study of carbohydrate metabolism. MR diets in combination with 12 % (+/ − ) and 22 % (+/ − ) carbohydrate-rich meals were fed to rainbow trout for a period of 8 weeks and phenotypic and transcript expression changes in the liver and white muscle were assessed. Fish fed MR diets, irrespective of carbohydrate load, were shown to abolish the glucose-intolerant phenotype 6 h post-feeding. There was a distinct switch in glucose and glycogen content in the liver of fish fed MR diets, with a significantly higher concentration of glycogen, suggesting reduced glycolytic capacity. Transcriptional responses to MR demonstrated decreased expression of hepatic fatty acid synthase, sterol regulatory binding protein 1, PPARγ coactivator 1-α and PPARα, indicative of a reduction in the de novo synthesis of fatty acids and cholesterol, and a potential decrease in hepatic fat oxidative capacity. Muscle adenylate charge was depressed under MR, and increased expression of AMP-activated protein kinase α1 was detected, indicative of reduced energy availability. Total DNA methylation showed that carbohydrate load, rather than MR, dictated hypomethylation of genomic DNA. This is the first study which demonstrates that MR can abolish a glucose-intolerant phenotype in trout, and identifies trout as a suitable model for studying metabolic syndromes. [ABSTRACT FROM PUBLISHER]

AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, is proposed to function as a ''fuel gauge'' to monitor cellular energy status in response to nutritional environmental variations. However, in fish, few studies have addressed the metabolic consequences related to the activation of this kinase. This study demonstrates that the rainbow trout (Oncorhynchus mykiss) possesses paralogs of the three known AMPK subunits that co-diversified, that the AMPK protein is present in the liver and in isolated hepatocytes, and it does change in response to physiological (fasting-refeeding cycle) and pharmacological (AICAR and metformin administration and incubations) manipulations. Moreover, the phosphorylation of AMPK results in the phosphorylation of acetyl-CoA carboxylase, a main downstream target of AMPK in mammals. Other findings include changes in hepatic glycogen levels and several molecular actors involved in hepatic glucose and lipid metabolism, including mRNA transcript levels for glucokinase, glucose-6-phosphatase and fatty acid synthase both in vivo and in vitro. The fact that most results presented in this study are consistent with the recognized role of AMPK as a master regulator of energy homeostasis in living organisms supports the idea that these functions are conserved in this piscine model. [ABSTRACT FROM AUTHOR]

Environmental fluid dynamic code (EFDC), a numerical estuarine and coastal ocean circulation hydrodynamic model, was used to simulate the distribution of the salinity, temperature, nutrients, and dissolved oxygen (DO) in Perdido Bay and adjacent Gulf of Mexico. External forcing factors included the coupled effects of the astronomical tides, river discharge, and atmospheric winds on the spatial and temporal distributions of salinity and DO. Modeled time series were in good agreement with field observations of water level, nutrients, temperature, salinity, and DO. Perdido Bay and adjacent northern Gulf of Mexico coasts can be divided into two areas according to salinity, water level, and DO concentrations. The first area was lower Perdido Bay and the associated Gulf of Mexico coasts, acting primarily under the influence of tidal forcing, which increases the vertical stratification. The second division was upper Perdido Bay, which was influenced by both tidal forcing and freshwater inflow. Simulations also indicated winds influenced the salinity and DO distributions, with an enhanced surface pressure gradient. Tidal effects were also important for conducting salinity and water quality simulations in Perdido Bay. Low amplitude tides induced relatively weak vertical mixing and favored the establishment of stratification at the bay, especially along deeper bathymetry. Flood tides influenced the distribution of salinity and DO more than ebb tides, specifically along shallow bathymetry. [ABSTRACT FROM AUTHOR]

This is the first study to implement a genomic approach to ascertain both transcriptional and functional end points of chronic Cu toxicity in fish associated with experimentally manipulated water chemistries. Over 21 d, zebrafish acclimated to softwater (Na+ = 0.06 mM, Ca2+ = 0.08 mM, Mg2+ = 0.03 mM) were exposed to the following: soft-water (Ctrl); 12 μg L-1 Cu (Cu); 3.3 mM Na+ (Na); 3.3 mM Na+ + 12 μg L-1 Cu (Na + Cu); 3.3 mM Ca2+ (Ca); or 3.3 mM Ca2+ + 12 μg L-1 Cu (Ca + Cu). Although effective at reducing Cu load in all tissues, Na+ in the presence of Cu did not decrease the degree of oxidative damage, particularly in the gill and gut. In contrast, Ca + Cu treatment decreased Cu accumulation in gill, but not liver or gut, with no reduction in oxidative damage. Transcriptional analysis of candidate genes (atp7a, ctr1, ECaC, esr1) showed principally a down regulation of transcripts with the Cu only treatment, while Ca + Cu treatment restored some of the genes to control levels. Conversely, the Na + Cu treatment had a strong, opposing affect when compared to that of Cu alone. Zebrafish Affymetrix GeneChips revealed significantly clustered patterns of expression. Changes in expression induced by Cu appeared to be opposite to the majority of the other treatments. Our data on the preventative or enhancing effects of Na+ and Ca2+ both alone and in the presence of Cu, may, in the future, facilitate the incorporation of gene expression end points into a biotic ligand model predicting chronic Cu toxicity in this tropical model species of genomic importance. [ABSTRACT FROM AUTHOR]

Little is known regarding the ionoregulatory abilities of zebrafish exposed to soft water despite the popularity of this model organism for physiology and aquatic toxicology. We examined genomic and nongenomic changes to gills of zebrafish as they were progressively acclimated from moderately hard freshwater to typical soft water over 7 days and held in soft water for another 7 days. Gills were sampled daily and mRNA expression levels of gill Na+-K+-ATPase (NKA) a1a subunit, epithelium calcium channel (ECaC), carbonic anhydrase-1 and 2 (CA-1, CA-2), Na+/H+ exchanger (NHE-2), V-type proton (H+)-ATPase, and copper transport protein (CTR-1) were quantified by real-time PCR. Changes in enzyme activities of gill NKA were determined and protein levels of NKA and ECaC were quantified by Western blotting. Levels of mRNA for ECaC increased fourfold after day 6, with an associated increase in ECaC protein levels after 1 wk in soft water. CA-1 and CA-2 exhibited a 1.5- and 6-fold increase in gene expression on days 6 and J, respectively. Likewise, there was a fivefold increase in NHE-2 expression after day 6. Surprisingly, CTR-1 mRNA showed a large transient increase (over threefold) on day 6, while H+-ATPase mRNA did not change. These data demonstrate a high degree of phenotypic plasticity in zebrafish gills exposed to an ion-poor environment. This not only enhances our understanding of ionoregulatory processes in fish but also highlights the need for proper experimental design for studies involving preacclimation to soft water (e.g., metal toxicity). [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • There was a 30% increase in routine metabolic rate associated with heat-killed Vibrio anguillarum in zebrafish at 27.5oC • Temperature appeared to play a significant role in the timing of transcriptional expression differences between 22oC and 27.5oC • While heat-killed Vibrio stimulated a response, likely a live pathogen exposure would have been more robust. • This is some of the first evidence that indicates there is a metabolic cost associated with infection in teleost fish. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • Rainbow trout were exposed to a 3 min air stressor and recovered for 1, 3 or 24 h. • Specific microRNAs were increased in extracellular vesicles. • Specific microRNAs were decreased in head kidney and liver. • These microRNAs are predicted to target pathways associated with stress. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • Fish stress due to pharmaceutical and climate change exposure needs to be measured. • MicroRNA are altered in response to increased water temperature and venlafaxine. • Altered microRNA are associated with ovarian pathologies and the stress response. • Specific microRNA can be used as biomarkers of environmental stress. [ABSTRACT FROM AUTHOR]

Abstract: The antibacterial properties of silver nanoparticles (AgNPs) are widely exploited in a variety of medical and consumer products. AgNPs in these products can be released into the aquatic environment, however, the potential toxicity of AgNPs to organisms, including fish, is yet to be fully understood. The present study aimed to investigate the effects of the early life exposure to AgNPs on the hypothalamic–pituitary–interrenal (HPI) axis-mediated stress response in zebrafish (Danio rerio) larvae and adults. Zebrafish embryos were treated with AgNPs (0.5μg/mL) or Ag+ (0.05μg/mL) starting at 2h post fertilization (hpf). At 96hpf the larvae were either subjected to a swirling stress and euthanized, or raised to adulthood (10months) in silver-free water and then net-stressed, euthanized, and sampled. Whole-body basal or stress-induced cortisol levels in larvae were not affected by either AgNPs or Ag+; however, the transcript levels of corticotropin releasing factor (CRF), CRF-binding protein (CRF-BP), CRF-receptor 2 (CRF-R2), and pro-opiomelanocortin (POMCb) were significantly decreased by Ag+. The ability of the adult fish to release cortisol in response to a stressor was also not affected, although the transcript levels of CRF, CRF-BP, and CRF-R1 in the telencephalon were differentially affected in fish exposed to Ag+ as embryos. This is the first study that investigated the potential endocrine-disrupting effects of AgNPs during the early life stages and although AgNPs or Ag+ did not affect the ability of zebrafish to elevate cortisol levels in response to a stressor, the effects on transcript levels by Ag+ should be investigated further since CRF does not solely regulate the HPI axis but is also implicated in other physiological processes. [Copyright &y& Elsevier]

The objective of this study was to assess the effects of municipal wastewater treatment plant effluent on the energetics and stress response of rainbow darter ( Etheostoma caeruleum ). Male and female rainbow darter were collected upstream and downstream of the Waterloo WWTP in the Grand River watershed, ON, Canada. To assess the effects of wastewater treatment plant effluent on whole-body and tissue specific metabolic capacity, closed-chamber respirometry and muscle-enzyme activity analyses were performed. Plasma cortisol was also collected from fish before and after an acute air-exposure stressor to evaluate the cortisol stress response in fish exposed to additional stressors. Male and female rainbow darter collected downstream of the effluent had higher oxygen consumption rates, while differences in enzyme activities were primarily associated with sex rather than collection site. No impairment in the cortisol stress response between downstream and upstream fish was observed, however baseline cortisol levels in female fish from the downstream site were significantly higher compared to other baseline groups. Stress-induced cortisol levels were also higher in female fish from both sites when compared to their male counterparts. Overall, this study demonstrates that chronic exposure to WWTP effluent impacts whole-body metabolic performance. This study was also able to demonstrate that sex-differences are a key determinant of various metabolic changes in response to physiological stress, thereby, providing a novel avenue to be considered and further explored. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Characterized rainbow and brook trout response to 3-min air exposure. • Plasma metabolites increased post-air exposure with some variability. • Inverse relationships between miRNAs and predicted targets in brook trout. • Altered liver miRNAs and enzyme activities are linked to oxidative stress. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

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., (Copyright © 2020 Lobb et al.)