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The pronephros (early-stage kidney) is an important osmoregulatory organ, and the onset of its function occurs relatively early in some teleost fishes. As such, any defects in kidney development and function are likely associated with a decreased ability to osmoregulate. Previous work has shown that early-life stage (ELS) zebrafish (Danio rerio) acutely exposed to Deepwater Horizon (DWH) crude oil exhibit transcriptional changes in key genes involved in pronephros development and function, as well as pronephric morphological defects and whole-animal osmoregulatory impairment. The objective of this study was to examine the acute effects of crude oil exposure during zebrafish ELS on pronephros function by assessing its fluid clearance capacity and glomerular filtration integrity. Following a 72-h exposure to control conditions, 20% or 40% dilutions of high-energy water-accommodated fractions (HEWAF) of DWH crude oil, zebrafish were injected into the common cardinal vein either with fluorescein-labeled (FITC) 70-kDa dextran to assess glomerular filtration integrity or with FITC-inulin to assess pronephric clearance capacity. Fluorescence was quantified after the injections at predetermined time intervals by fluorescence microscopy. The results demonstrated a diminished pronephric fluid clearance capacity and failed glomerular perfusion when larvae were exposed to 40% HEWAF dilutions, whereas only a reduced glomerular filtration selectivity was observed in zebrafish previously exposed to the 20% HEWAF dilution., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Crude oil is known to induce developmental defects in teleost fish exposed during early-life stages (ELSs). A recent study has demonstrated that zebrafish (Danio rerio) larvae acutely exposed to Deepwater Horizon (DHW) crude oil showed transcriptional changes in key genes involved in early kidney (pronephros) development and function, which were coupled with pronephric morphological defects. Given the osmoregulatory importance of the kidney, it is unknown whether ELS effects arising from short-term crude exposures result in long-term osmoregulatory defects, particularly within estuarine fishes likely exposed to DWH oil following the spill. To address this knowledge gap, an acute 72 h exposure to red drum (Sciaenops ocellatus) larvae was performed using high-energy water-accommodated fractions (HEWAFs) of DWH weathered oil to analyze transcriptional changes in genes involved in pronephros development and function by quantitative PCR. To test the latent effects of oil exposure on osmoregulation ability, red drum larvae were first exposed to HEWAF for 24 h. Larvae were then reared in clean seawater for two weeks and a 96 h acute osmotic challenge test was performed by exposing the fish to waters with varying salinities. Latent effects of ELS crude oil exposure on osmoregulation were assessed by quantifying survival during the acute osmotic challenge test and analyzing transcriptional changes at 14 dpf. Results demonstrated that ELS crude oil exposure reduced survival of red drum larvae when challenged in hypoosmotic waters and that latent transcriptional changes in some target pronephric genes were evident, indicating that an affected kidney likely contributed to the increased mortality., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Crude oil is known to induce developmental defects in teleost fish exposed during early life stages (ELSs). While most studies in recent years have focused on cardiac endpoints, evidence from whole-animal transcriptomic analyses and studies with individual polycyclic aromatic hydrocarbons (PAHs) indicate that the developing kidney (i.e., pronephros) is also at risk. Considering the role of the pronephros in osmoregulation, and the common observance of edema in oil-exposed ELS fish, surprisingly little is known regarding the effects of oil exposure on pronephros development and function. Using zebrafish (Danio rerio) ELSs, we assessed the transcriptional and morphological responses to two dilutions of high-energy water accommodated fractions (HEWAF) of oil from the Deepwater Horizon oil spill using a combination of qPCR and whole-mount in situ hybridization (WM-ISH) of candidate genes involved in pronephros development and function, and immunohistochemistry (WM-IHC). To assess potential functional impacts on the pronephros, three 24 h osmotic challenges (2 hypo-osmotic, 1 near iso-osmotic) were implemented at two developmental time points (48 and 96 h post fertilization; hpf) following exposure to HEWAF. Changes in transcript expression level and location specific to different regions of the pronephros were observed by qPCR and WM-ISH. Further, pronephros morphology was altered in crude oil exposed larvae, characterized by failed glomerulus and neck segment formation, and straightening of the pronephric tubules. The osmotic challenges at 96 hpf greatly exacerbated edema in both HEWAF-exposed groups regardless of osmolarity. By contrast, larvae at 48 hpf exhibited no edema prior to the osmotic challenge, but previous HEWAF exposure elicited a concentration-response increase in edema at hypo-osmotic conditions that appeared to have been largely alleviated under near iso-osmotic conditions. In summary, ELS HEWAF exposure impaired proper pronephros development in zebrafish, which coupled with cardiotoxic effects, most likely reduced or inhibited pronephros fluid clearance capacity and increased edema formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Crude oil has multiple toxic effects in fish, particularly during their early life stages. Recent transcriptomics studies have highlighted a potential effect on cholesterol homeostasis and biosynthesis, but have not investigated effects on steroid hormones, which are biosynthetically downstream metabolites of cholesterol. We exposed zebrafish (Danio rerio) embryos and larvae to 3 concentrations of a high energy water accommodated fraction (HEWAF) of crude oil and measured effects on cholesterol and steroid hormones at 48 and 96 h post fertilization (hpf). HEWAF exposure caused a small decrease in cholesterol at 96 hpf but not 48 hpf. HEWAF-exposed larvae had higher levels of androstenedione, testosterone, estradiol, cortisol, corticosterone, and progesterone at 96 hpf compared to controls, while effects at 48 hpf were more modest or not present. 2-Methoxyestradiol was lower following HEWAF exposure at both time points. Dihydrotestosterone was elevated in one HEWAF concentration at 48 hpf only. Our results suggest that hormone imbalance may be an important toxic effect of oil HEWAF exposure despite no major effect on their biosynthetic precursor cholesterol., (Copyright © 2021 Elsevier B.V. All rights reserved.)

There is evidence that the combination of polycyclic aromatic hydrocarbons (PAHs) released in the Deepwater Horizon oil spill impairs the glucocorticoid stress response of vertebrates in the Gulf of Mexico, but the mechanisms are unclear. We hypothesized that inhibition of cortisol release may be due to 1) overstimulation of the hypothalamic-pituitary-inter-renal (HPI) axis, or 2) an inhibition of cortisol biosynthesis through PAH activation of the aryl hydrocarbon receptor (AhR). Using a flow-through system, Gulf toadfish (Opsanus beta) were continuously exposed to control conditions or one of 3 environmentally relevant concentrations of PAHs from Deepwater Horizon oil (∑PAH 50  = 0-3 μg L -1 ) for up to 7 d. One group of toadfish was then exposed to a recovery period for up to 7 d. No changes in corticotrophin-releasing factor mRNA expression, adrenocorticotropic hormone (ACTH), or pituitary mass suggested that overstimulation of the HPI axis was not a factor. The AhR activation was measured by an elevation of cytochrome P4501A1 (CYP1A) mRNA expression within the HPI axis in fish exposed to high PAH concentrations; however, CYP1A was no longer induced after 3 d of recovery in any of the tissues. At 7 d of recovery, there was an impairment of cortisol release in response to an additional simulated predator chase that does not appear to be due to changes in the mRNA expression of the kidney steroidogenic pathway proteins steroidogenic acute regulatory protein, cytochrome P450 side chain cleavage, and 11β-hydroxylase. Future analyses are needed to determine whether the stress response impairment is due to cholesterol availability and/or down-regulation of the melanocortin 2 receptor. Environ Toxicol Chem 2021;40:1062-1074. © 2020 SETAC., (© 2020 SETAC.)

Increases in ambient salinity demand parallel increases in intestinal base secretion for maintenance of osmoregulatory status, which is likely the cause of a transient acidosis following transfer of euryhaline fish from freshwater to seawater. It was predicted that transfer of the marine Gulf toadfish (Opsanus beta) from seawater (35 ppt) to hypersaline (60 ppt) seawater (HSW) would lead to a transient acidosis that would be compensated by increases in branchial acid excretion to offset the acid-base disturbance. Toadfish exposed to HSW showed a significant decrease in blood pH and [HCO 3 - ] but no increase in pCO 2 , followed by a full recovery after 48-96 h. A similar metabolic acidosis and recovery was found when fish were exposed to 60-ppt HCO 3 - -free seawater (HEPES-buffered), which may suggest that compensation for intestinal base loss during hypersaline treatment is from gill H + excretion rather than gill HCO 3 - uptake. However, we cannot rule out that reduced branchial HCO 3 - excretion contributed to an increase in net acid excretion. Since colchicine prevents full compensation, translocation of H + and/or HCO 3 - transporters between cytosolic compartments and plasma membrane fractions might be involved in compensating for the hypersalinity-induced acidosis. Translocation of transporters rather than de novo synthesis may represent a faster and less energetically demanding response to rapidly fluctuating and high salinities encountered by toadfish in their natural environment.

In the aquatic environment, ubiquitous natural factors such as ultraviolet light (UV) and dissolved organic carbon (DOC) are likely to influence crude oil toxicity. The present study examined the interactive effects of DOC, UV, and DOC-UV co-exposure on the acute toxicity of Deepwater Horizon crude oil in larval red drum (Sciaenops ocellatus). Although DOC alone did not influence crude oil toxicity, it mildly reduced UV photo-enhanced toxicity. Environ Toxicol Chem 2020;39:2509-2515. © 2020 SETAC., (© 2020 SETAC.)

The failure of the swim bladder to inflate during fish development is a common and sensitive response to exposure to petrochemicals. Here, we review potential mechanisms by which petrochemicals or their toxic components (polycyclic aromatic hydrocarbons; PAHs) may affect swim bladder inflation, particularly during early life stages. Surface films formed by oil can cause a physical barrier to primary inflation by air gulping, and are likely important during oil spills. The act of swimming to the surface for primary inflation can be arduous for some species, and may prevent inflation if this behavior is limited by toxic effects on vision or musculature. Some studies have noted altered gene expression in the swim bladder in response to PAHs, and Cytochrome P450 1A (CYP1A) can be induced in swim bladder or rete mirabile tissue, suggesting that PAHs can have direct effects on swim bladder development. Swim bladder inflation failure can also occur secondarily to the failure of other systems; cardiovascular impairment is the best elucidated of these mechanisms, but other mechanisms might include non-inflation as a sequela of disruption to thyroid signaling or cholesterol metabolism. Failed swim bladder inflation has the potential to lead to chronic sublethal effects that are as yet unstudied., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Respiratory quotient (RQ) is commonly used to infer which substrates are oxidized, with glucose yielding RQ = 1 and fat normally thought to yield an average of RQ = 0.71. Because fat depot compositions differ among species, we examined how the various common fatty acids affect RQ. RQs ranged from less than 0.7 (e.g., stearic acid) to greater than 0.76 (e.g., docosahexaenoic acid). Furthermore, we conducted a survey of the fatty acid composition of fuel lipids of several vertebrate taxa to determine how the RQ for lipid oxidation during fasting should vary among species. Our survey indicates that most fasting vertebrates from terrestrial ecosystems oxidizing fat should have RQs equaling approximately 0.71, as normally expected. However, some fasting animals in aquatic or marine systems-particularly fish-should have RQs as high as 0.73 when oxidizing only fat. Selective mobilization of fatty acids increased the lipid RQ, but probably by a negligible amount. We conclude that researchers should take habitat and taxon into account when choosing a value for lipid RQ, and preferably should use fatty acid composition for their study species to determine an appropriate RQ for lipids. In the absence of species-specific fatty acid composition data, we suggest assuming a lipid RQ of 0.725 for cold-water fish., (© 2020 Wiley Periodicals LLC.)

Ectothermic animals are especially susceptible to temperature change, considering that their metabolism and core temperature are linked to the environmental temperature. As global water temperatures continue to increase, so does the need to understand the capacity of organisms to tolerate change. Sheepshead minnows (Cyprinodon variegatus) are the most eurythermic fish species known to date and can tolerate a wide range of environmental temperatures from - 1.9 to 43.0 °C. But little is known about the physiological adjustments that occur when these fish are subjected to acute thermal challenges and long-term thermal acclimation. Minnows were acclimated to 10, 21, or 32 °C for 4 weeks or acutely exposed to 10 and 32 °C and then assessed for swimming performance [maximum sustained swimming velocity (U crit ), optimum swimming velocity (U opt )] and metabolic endpoints (extrapolated standard and maximum metabolic rate [SMR, MMR), absolute aerobic scope (AS), and cost of transport (COT)]. Our findings show that the duration of thermal exposure (acute vs. acclimation) did not influence swimming performance. Rather, swimming performance was influenced by the exposure temperature. Swimming performance was statistically similar in fish exposed to 21 or 32 °C (approximately 7.0 BL s -1 ), but was drastically reduced in fish exposed to 10 °C (approximately 2.0 BL s -1 ), resulting in a left-skewed performance curve. There was no difference in metabolic end points between fish acutely exposed or acclimated to 10 °C. However, a different pattern was observed in fish exposed to 32 °C. MMR was similar between acutely exposed or acclimated fish, but acclimated fish had a 50% reduction in extrapolated SMR, which increased AS by 25%. However, this enhanced AS was not associated with changes in swimming performance, which opposes the oxygen-capacity limited thermal tolerance concept. Our findings suggest that sheepshead minnows may utilize two distinct acclimation strategies, resulting in different swimming performance and metabolic patterns observed between 10 and 32 °C exposures.

Aquaporins are the predominant water-transporting proteins in vertebrates, but only a handful of studies have investigated aquaporin function in fish, particularly in mediating water permeability during salinity challenges. Even less is known about aquaporin function in hypoxia (low oxygen), which can profoundly affect gill function. Fish deprived of oxygen typically enlarge gill surface area and shrink the water-to-blood diffusion distance, to facilitate oxygen uptake into the bloodstream. However, these alterations to gill morphology can result in unfavorable water and ion fluxes. Thus, there exists an osmorespiratory compromise, whereby fish must try to balance high branchial gas exchange with low ion and water permeability. Furthermore, the gills of seawater and freshwater teleosts have substantially different functions with respect to osmotic and ion fluxes; consequently, hypoxia can have very different effects according to the salinity of the environment. The purpose of this study was to determine what role aquaporins play in water permeability in the hypoxia-tolerant euryhaline common killifish (Fundulus heteroclitus), in two important osmoregulatory organs-the gills and intestine. Using immunofluorescence, we localized aquaporin-3 (AQP3) protein to the basolateral and apical membranes of ionocytes and enterocytes, respectively. Although hypoxia increased branchial AQP3 messenger-RNA expression in seawater and freshwater, protein abundance did not correlate. Indeed, hypoxia did not alter AQP3 protein abundance in seawater and reduced it in the cell membranes of freshwater gills. Together, these observations suggest killifish AQP3 contributes to reduced diffusive water flux during hypoxia and normoxic recovery in freshwater and facilitates intestinal permeability in seawater and freshwater., (© 2020 Wiley Periodicals LLC.)

Estuarine crocodiles, Crocodylus porosus , inhabit freshwater, estuarine and marine environments. Despite being known to undertake extensive movements throughout and between hypo-osmotic and hyperosmotic environments, little is known about the role of the cloaca in coping with changes in salinity. We report here that, in addition to the well-documented functional plasticity of the lingual salt glands, the middle of the three cloacal segments (i.e. the urodaeum) responds to increased ambient salinity to enhance solute-coupled water absorption. This post-renal modification of urine serves to conserve water when exposed to hyperosmotic environments and, in conjunction with lingual salt gland secretions, enables C. porosus to maintain salt and water balance and thereby thrive in hyperosmotic environments. Isolated epithelia from the urodaeum of 70% seawater-acclimated C. porosus had a strongly enhanced short-circuit current (an indicator of active ion transport) compared with freshwater-acclimated crocodiles. This enhanced active ion absorption was driven by increased Na + /K + -ATPase activity, and possibly enhanced proton pump activity, and was facilitated by the apical epithelial Na + channel (ENaC) and/or the apical Na + /H + exchanger (NHE2), both of which are expressed in the urodaeum. NHE3 was expressed at very low levels in the urodaeum and probably does not contribute to solute-coupled water absorption in this cloacal segment. As C. porosus does not appear to drink water of salinities above 18 ppt, observations of elevated short-circuit current in the rectum as well as a trend for increased NHE2 expression in the oesophagus, the anterior intestine and the rectum suggest that dietary salt intake may stimulate salt and possibly water absorption by the gastrointestinal tract of C. porosus living in hyperosmotic environments., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The understanding of the detection threshold and behavioral response of fishes in response to crude oil is critical to predicting the effects of oil spills on wild fish populations. The Deepwater Horizon oil spill released approximately 4.9 million barrels of crude oil into the northern Gulf of Mexico in 2010, overlapping spatially and temporally with the habitat of many pelagic fish species. Yet, it is unknown whether highly migratory species, such as mahi-mahi ( Coryphaena hippurus ), might detect and avoid oil contaminated waters. We tested the ability of control and oil-exposed juvenile mahi-mahi (15-45 mm) to avoid two dilutions of crude oil in a two-channel flume. Control fish avoided the higher concentration (27.1 μg/L Σ 50 PAH), while oil-exposed (24 h, 18.0 μg/L Σ 50 PAH) conspecifics did not. Electro-olfactogram (EOG) data demonstrated that both control and oil-exposed (24 h, 14.5 μg/L Σ 50 PAH) juvenile mahi-mahi (27-85 mm) could detect crude oil as an olfactory cue and that oil-exposure did not affect the EOG amplitude or duration in response to oil or other cues. These results show that a brief oil exposure impairs the ability of mahi-mahi to avoid oil and suggests that this alteration likely results from injury to higher order central nervous system processing rather than impaired olfactory physiology.

Freshwater- and seawater-acclimated Fundulus heteroclitus were exposed to acute hypoxia (10% air saturation, 3 h), followed by normoxic recovery (3 h). In both salinities, ventilation increased and heart rate fell in the classic manner, while Ṁ O 2  initially declined by ∼50%, with partial restoration by 3 h of hypoxia, and no O 2 debt repayment during recovery. Gill paracellular permeability (measured with [ 14 C] PEG-4000) was 1.4-fold higher in seawater, and declined by 50% during hypoxia with post-exposure overshoot to 188%. A similar pattern with smaller changes occurred in freshwater. Drinking rate (also measured with [ 14 C] PEG-4000) was 8-fold higher in seawater fish, but declined by ∼90% during hypoxia in both groups, with post-exposure overshoots to ∼270%. Gill diffusive water flux (measured with 3 H 2 O) was 1.9-fold higher in freshwater fish, and exhibited a ∼35% decrease during hypoxia, which persisted throughout recovery, but was unchanged during hypoxia in seawater fish. Nevertheless, freshwater killifish gained mass while seawater fish lost mass during hypoxia, and these changes were not corrected during normoxic recovery. We conclude that this hypoxia-tolerant teleost beneficially reduces gill water permeability in a salinity-dependent fashion during hypoxia, despite attempting to simultaneously improve Ṁ O 2 , but nevertheless incurs a net water balance penalty in both freshwater and seawater., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

In fishes, olfactory cues evoke behavioral responses that are crucial to survival; however, the receptors, olfactory sensory neurons, are directly exposed to the environment and are susceptible to damage from aquatic contaminants. In 2010, 4.9 million barrels of crude oil were released into the northern Gulf of Mexico from the Deepwater Horizon disaster, exposing marine organisms to this environmental contaminant. We examined the ability of bicolor damselfish ( Stegastes partitus ), exposed to the water accommodated fraction (WAF) of crude oil, to respond to chemical alarm cue (CAC) using a two-channel flume. Control bicolor damselfish avoided CAC in the flume choice test, whereas WAF-exposed conspecifics did not. This lack of avoidance persisted following 8 days of control water conditions. We then examined the physiological response to CAC, brine shrimp rinse, bile salt, and amino acid cues using the electro-olfactogram (EOG) technique and found that WAF-exposed bicolor damselfish were less likely to detect CAC as an olfactory cue but showed no difference in EOG amplitude or duration compared to controls. These data indicate that a sublethal WAF exposure directly modifies detection and avoidance of CAC beyond the exposure period and may suggest reduced predator avoidance behavior in oil-exposed fish in the wild.

Deepwater Horizon crude oil is comprised of polycyclic aromatic hydrocarbons that cause a number of cardiotoxic effects in marine fishes across all levels of biological organization and at different life stages. Although cardiotoxic impacts have been widely reported, the mechanisms underlying these impairments in adult fish remain understudied. In this study, we examined the impacts of crude oil on cardiomyocyte contractility and electrophysiological parameters in freshly isolated ventricular cardiomyocytes from adult mahi-mahi ( Coryphaena hippurus ). Cardiomyocytes directly exposed to oil exhibited reduced contractility over a range of environmentally relevant concentrations (2.8-12.9 μg l -1 ∑PAH). This reduction in contractility was most pronounced at higher stimulation frequencies, corresponding to the upper limits of previously measured in situ mahi heart rates. To better understand the mechanisms underlying impaired contractile function, electrophysiological studies were performed, which revealed oil exposure prolonged cardiomyocyte action potentials and disrupted potassium cycling (9.9-30.4 μg l -1 ∑PAH). This study is the first to measure cellular contractility in oil-exposed cardiomyocytes from a pelagic fish. Results from this study contribute to previously observed impairments to heart function and whole-animal exercise performance in mahi, underscoring the advantages of using an integrative approach in examining mechanisms of oil-induced cardiotoxicity in marine fish.

The simplified nervous system of Aplysia californica (Aplysia) allows for detailed studies of physiological and molecular changes in small sets of neurons. Sensory neurons of the biting and tail withdrawal reflexes are glutamatergic and show reduced L-Glutamate current density in aged animals, making them a good candidate to study age-related changes in glutamatergic responses. To examine if changes in ionotropic L-Glu receptor (iGluR) transcription underlie reduced physiology, mRNA expression of iGluR was quantified in two sensory neuron clusters of two cohorts of Aplysia at both sexual maturity (~8 months) and advanced age (~12 months). Sensory neuron aging resulted in a significant overall decrease in expression of iGluR subunits in both sensory neuron clusters and cohorts. Although the individual subunits differentially expressed varied between sensory neuron clusters and different cohorts of animals, all differentially expressed subunits were downregulated, with no subunits showing significantly increased expression with age. Overall declines in transcript expression suggest that age-related declines in L-Glu responsiveness in Aplysia sensory neurons could be linked to overall declines in iGluR expression, rather than dysregulation of specific subunits. In both sensory neuron clusters tested the N-methyl-D-aspartate receptor subtype was expressed at significantly greater levels than other iGluR subtypes, suggesting an in vivo role for NMDAR-like receptors in Aplysia sensory neurons., Competing Interests: The authors have declared no competing interests exist.

Background: Mahi-mahi (Coryphaena hippurus) is a commercially and ecologically important fish species that is widely distributed in tropical and subtropical waters. Biological attributes and reproductive capacities of mahi-mahi make it a tractable model for experimental studies. In this study, life development of cultured mahi-mahi from the zygote stage to adult has been described., Results: A comprehensive developmental table has been created reporting development as primarily detailed observations of morphology. Additionally, physiological, behavioral, and molecular landmarks have been described to significantly contribute in the understanding of mahi life development., Conclusion: Remarkably, despite the vast difference in adult size, many developmental landmarks of mahi map quite closely onto the development and growth of Zebrafish and other warm-water, active Teleost fishes., (© 2019 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.)

The mechanism(s) of ammonia and urea excretion in freshwater fish have received considerable attention; however, parallel investigations of seawater fish, specifically in the early life stages are scarce. The first objective of this study was to evaluate the patterns of ammonia and urea excretion in mahi-mahi (Coryphaena hippurus) up to 102  hours post fertilization (hpf). Similar to other teleosts, mahi embryos are ureotelic before hatch and gradually switch to being ammoniotelic around the time of hatch. The second objective was to characterize mRNA levels of ammonia transporters (Rhag, Rhbg, Rhcg1 and Rhcg2), as well as urea transporter (UT) and sodium hydrogen exchangers (NHE3 and NHE2) during mahi development. As predicted, the mRNA levels of the Rhesus glycoprotein (Rh) genes, especially Rhag, Rhbg and the UT gene were highly consistent with the ontogeny of ammonia and urea excretion rates. Further, the localization of each transporter was examined in larvae collected at 60 and 102 hpf using in situ hybridization. Rhag was expressed in the gills, yolk sac, and operculum. Rhbg was expressed in the gills and upper mouth. Rhcg1 and NHE3 were co-localized in the sub-operculum, and Rhcg2 was expressed in the skin. Together, these results indicate that urea excretion is critical for ammonia detoxification during embryonic development and that Rh proteins are involved in ammonia excretion via gills and yolk sac, possibly facilitated by NHE3., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The 2010 Deepwater Horizon oil spill coincided with the spawning season of many pelagic fish species in the Gulf of Mexico. Yet, few studies have investigated physiological responses of larval fish to interactions between anthropogenic crude oil exposure and natural factors (e.g. temperature, oxygen levels). Consequently, mahi mahi (Coryphaena hippurus) embryos were exposed for 24 hours to combinations of two temperatures (26 and 30°C) and six concentrations of oiled fractions of weathered oil (from 0 to 44.1 μg ∑50PAHs·L-1). In 56 hours post-fertilization larvae, heart rate, stroke volume and cardiac output were measured as indicators of functional cardiac phenotypes. Fluid accumulation and incidence of edema and hematomas were quantified as indicators of morphological impairments. At both 26 and 30°C, oil-exposed larvae suffered dose-dependent morphological impairments and functional heart failure. Elevation of temperature to 30°C appeared to induce greater physiological responses (bradycardia) at PAH concentrations in the range of 3.0-14.9 μg·L-1. Conversely, elevated temperature in oil-exposed larvae reduced edema severity and hematoma incidence. However, the apparent protective role of warmer temperature does not appear to protect against enhanced mortality. Collectively, our findings show that elevated temperature may slightly decrease larval resilience to concurrent oil exposure., Competing Interests: The authors have declared that no competing interests exist.

This study examined potential interactive effects of co-exposure to Deepwater Horizon (DWH) crude oil (∼30 μg L -1 ΣPAHs) for 24 h and either hypoxia (2.5 mg O 2 L -1 ; 40% O 2 saturation) or elevated temperature (30 °C) on the swimming performance of juvenile mahi-mahi (Coryphaena hippurus). Additionally, effects of shorter duration exposures to equal or higher doses of oil alone either prior to swimming or during the actual swim trial itself were examined. Only exposure to hypoxia alone or combined with crude oil elicited significant decreases in critical swimming speed (U crit ) and to a similar extent (∼20%). In contrast, results indicate that elevated temperature might ameliorate some effects of oil exposure on swimming performance and that effects of shorter duration exposures are either reduced or delayed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The objective of the present study was to determine whether the polycyclic aromatic hydrocarbons (PAHs) associated with the Deepwater Horizon (DWH) oil spill impacted the stress response of teleost fish. The hypothesis was that intraperitoneal (IP) treatment with PAHs associated with the DWH oil spill or waterborne exposure to DWH oil high energy water-accommodated fraction (HEWAF) would result in the downregulation of the stress response of Gulf toadfish, Opsanus beta, a benthic marine teleost fish that resides in the Gulf of Mexico. In vivo plasma cortisol levels and adrenocorticotropic hormone (ACTH)-mediated cortisol secretion by in vitro isolated kidney tissue were measured. Toadfish at rest IP-treated with naphthalene had higher plasma cortisol compared to fluorene-treated and control fish; phenanthrene-treated fish tended to have higher plasma cortisol levels that fluorene-treated and controls. When subjected to an additional crowding stress, naphthalene and phenanthrene-treated fish were no longer able to mount a stress response compared to fluorene-treated and control fish, suggesting exhaustion of the stress response. Supporting this in vivo data, there tended to be less cortisol released by the kidney in vitro from naphthalene and phenanthrene-treated fish in response to ACTH compared to controls. In contrast, toadfish at rest exposed to 3% Slick A HEWAF did not have significantly different plasma cortisol levels compared to controls. But, exposed fish did have significantly less cortisol released by the kidney in vitro in response to ACTH. When toadfish were subjected to an additional stress, there were no significant differences in plasma cortisol or ACTH, suggesting the action of a secondary secretagogue to maintain plasma cortisol in vivo. Combined, these data suggest that in response to acute PAH exposure, there may be internalization or downregulation of the melanocortin 2 receptor (MC2R) that mediates the action of ACTH., (Copyright © 2017. Published by Elsevier B.V.)

Mahi-mahi (Coryphaena hippurus) is a commercially and ecologically important species of fish occurring in tropical and temperate waters worldwide. Understanding early life events is crucial for predicting effects of environmental stress, which is largely restricted by a lack of genetic resources regarding expression of early developmental genes and regulation of pathways. The need for anchoring developmental stages to transcriptional activities is highlighted by increasing evidence on the impacts of recurrent worldwide oil spills in this sensitive species during early development. By means of high throughput sequencing, we characterized the developmental transcriptome of mahi-mahi at three critical developmental stages, from pharyngula embryonic stage (24 hpf) to 48 hpf yolk-sac larva (transition 1), and to 96 hpf free-swimming larva (transition 2). With comparative analysis by multiple bioinformatic tools, a larger number of significantly altered genes and more diverse gene ontology terms were observed during transition 2 than transition 1. Cellular and tissue development terms were more significantly enriched in transition 1, while metabolism related terms were more enriched in transition 2, indicating a switch progressing from general embryonic development to metabolism during the two transitions. Special focus was given on the most significant common canonical pathways (e.g. calcium signaling, glutamate receptor signaling, cAMP response element-binding protein signaling, cardiac β-adrenergic signaling, etc.) and expression of developmental genes (e.g. collagens, myosin, notch, glutamate metabotropic receptor etc.), which were associated with morphological changes of nervous, muscular, and cardiovascular system. These data will provide an important basis for understanding embryonic development and identifying molecular mechanisms of abnormal development in fish species.

Windows of exposure to a weathered Deepwater Horizon oil sample (slick A) were examined for early life stage mahi-mahi (Coryphaena hippurus) to determine whether there are developmental periods of enhanced sensitivity during the course of a standard 96-h bioassay. Survival was assessed at 96 h following oil exposures ranging from 2 h to 96 h and targeting 3 general periods of development, namely the prehatch phase, the period surrounding hatch, and the posthatch phase. In addition, 3 different oil preparations were used: high- and low-energy water accommodated fractions of oil and very thin surface slicks of oil (∼1 μm). The latter 2 were used to distinguish between effects due to direct contact with the slick itself and the water underlying the slick. Considering the data from all 3 exposure regimes, it was determined that the period near or including hatch was likely the most sensitive. Furthermore, toxicity was not enhanced by direct contact with slick oil. These findings are environmentally relevant given that the concentrations of polycyclic aromatic hydrocarbons eliciting mortality from exposures during the sensitive periods of development were below or near concentrations measured during the active spill phase. Environ Toxicol Chem 2017;36:1887-1895. © 2016 SETAC., (© 2016 SETAC.)

Cardiovascular performance is altered by temperature in larval fishes, but how acute versus chronic temperature exposures independently affect cardiac morphology and physiology in the growing larva is poorly understood. Consequently, we investigated the influence of water temperature on cardiac plasticity in developing mahi-mahi. Morphological (e.g. standard length, heart angle) and physiological cardiac variables (e.g. heart rate f H , stroke volume, cardiac output) were recorded under two conditions by imaging: (i) under acute temperature exposure where embryos were reared at 25°C up to 128 h post-fertilization (hpf) and then acutely exposed to 25 (rearing temperature), 27 and 30°C; and (ii) at two rearing (chronic) temperatures of 26 and 30°C and performed at 32 and 56 hpf. Chronic elevated temperature improved developmental time in mahi-mahi. Heart rates were 1.2-1.4-fold higher under exposure of elevated acute temperatures across development ( Q 10 ≥2.0). Q 10 for heart rate in acute exposure was 1.8-fold higher compared to chronic exposure at 56 hpf. At same stage, stroke volume was temperature independent ( Q 10 ∼1.0). However, larvae displayed higher stroke volume later in stage. Cardiac output in developing mahi-mahi is mainly dictated by chronotropic rather than inotropic modulation, is differentially affected by temperature during development and is not linked to metabolic changes., Competing Interests: Competing interestsM. Grosell is a Maytag chair of Ichtyology., (© 2017. Published by The Company of Biologists Ltd.)

The impacts of Deepwater Horizon (DWH) oil on morphology and function during embryonic development have been documented for a number of fish species, including the economically and ecologically important pelagic species, mahi-mahi (Coryphaena hippurus). However, further investigations on molecular events and pathways responsible for developmental toxicity have been largely restricted due to the limited molecular data available for this species. We sought to establish the de novo transcriptomic database from the embryos and larvae of mahi-mahi exposed to water accommodated fractions (HEWAFs) of two DWH oil types (weathered and source oil), in an effort to advance our understanding of the molecular aspects involved during specific toxicity responses. By high throughput sequencing (HTS), we obtained the first de novo transcriptome of mahi-mahi, with 60,842 assembled transcripts and 30,518 BLAST hits. Among them, 2,345 genes were significantly regulated in 96hpf larvae after exposure to weathered oil. With comparative analysis to a reference-transcriptome-guided approach on gene ontology and tox-pathways, we confirmed the novel approach effective for exploring tox-pathways in non-model species, and also identified a list of co-expressed genes as potential biomarkers which will provide information for the construction of an Adverse Outcome Pathway which could be useful in Ecological Risk Assessments.

The timing and location of the 2010 Deepwater Horizon (DWH) incident within the Gulf of Mexico resulted in crude oil exposure of many commercially and ecologically important fish species, such as mahi-mahi (Coryphaena hippurus), during the sensitive early life stages. Previous research has shown that oil exposure during the embryonic stage of predatory pelagic fish reduces cardiac function - a particularly important trait for fast-swimming predators with high aerobic demands. However, it is unclear whether reductions in cardiac function translate to impacts on oxygen consumption in these developing embryos and larvae. A 24-channel optical-fluorescence oxygen-sensing system for high-throughput respiration measurements was used to investigate the effects of oil exposure, temperature and developmental stage on oxygen consumption rates in embryonic and larval mahi-mahi. Oil-exposed developing mahi-mahi displayed increased oxygen consumption, despite clear cardiac deformities and bradycardia, confirming oxygen uptake and delivery from a source other than the circulatory system. In addition to metabolic rate measurements, nitrogenous waste excretion was measured to test the hypothesis that increased energy demand was fueled by protein catabolism. This is the first study to our knowledge that demonstrates increased energy demand and energy depletion in oil-exposed developing mahi-mahi., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Exposure to polycyclic aromatic hydrocarbons (PAH) negatively impacts exercise performance in fish species but the physiological modifications that result in this phenotype are poorly understood. Prior studies have shown that embryonic and juvenile mahi-mahi (Coryphaeus hippurus) exposed to PAH exhibit morphological abnormalities, altered cardiac development and reduced swimming performance. It has been suggested that cardiovascular function inhibited by PAH exposure accounts for the compromised exercise performance in fish species. In this study we used in-situ techniques to measure hemodynamic responses of young adult mahi-mahi exposed to PAH for 24h. The data indicate that stroke volume was reduced 44% in mahi-mahi exposed to 9.6±2.7μgl -1 geometric mean PAH (∑PAH) and resulted in a 39% reduction in cardiac output and a 52% reduction in stroke work. Maximal change in pressure over change in time was 28% lower in mahi-mahi exposed to this level of ∑PAH. Mean intraventricular pressures and heart rate were not significantly changed. This study suggests exposure to environmentally relevant PAH concentrations impairs aspects of cardiovascular function in mahi-mahi., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Key differences in the developmental process of pelagic fish embryos, in comparison to embryos of standard test fish species, present challenges to obtaining sufficient control survival needed to successfully perform traditional toxicity testing bioassays. Many of these challenges relate to the change in buoyancy, from positive to negative, of pelagic fish embryos that occurs just prior to hatch. A novel exposure system, the pelagic embryo-larval exposure chamber (PELEC), has been developed to conduct successful bioassays on the early life stages (ELSs; embryos/larvae) of pelagic fish. Using this unique recirculating upwelling system, it was possible to significantly improve control survival in pelagic fish ELS bioassays compared to commonly used static exposure methods. Results demonstrate that control performance of mahi-mahi (Coryphaena hippurus) embryos in the PELEC system, measured as percent survival after 96-hrs, significantly outperformed agitated static exposure and static exposure systems. Similar significant improvements in 72-hr control survival were obtained with yellowfin tuna (Thunnus albacares). The PELEC system was subsequently used to test the effects of photo-induced toxicity of crude oil to mahi-mahi ELSs over the course of 96-hrs. Results indicate a greater than 9-fold increase in toxicity of Deepwater Horizon (DWH) crude oil during co-exposure to ambient sunlight compared to filtered ambient sunlight, revealing the importance of including natural sunlight in 96-hr DWH crude oil bioassays as well as the PELEC system's potential application in ecotoxicological assessments., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The temporal and geographic attributes of the Deepwater Horizon incident in 2010 likely exposed pelagic game fish species, such as mahi-mahi, to crude oil. Although much of the research assessing the effects of the spill has focused on early life stages of fish, studies examining whole-animal physiological responses of adult marine fish species are lacking. Using swim chamber respirometry, the present study demonstrates that acute exposure to a sublethal concentration of the water accommodated fraction of Deepwater Horizon crude oil results in significant swim performance impacts on young adult mahi-mahi, representing the first report of acute sublethal toxicity on adult pelagic fish in the Gulf of Mexico following the spill. At an exposure concentration of 8.4 ± 0.6 µg L -1 sum of 50 selected polycyclic aromatic hydrocarbons (PAHs; mean of geometric means ± standard error of the mean), significant decreases in the critical and optimal swimming speeds of 14% and 10%, respectively (p < 0.05), were observed. In addition, a 20% reduction in the maximum metabolic rate and a 29% reduction in aerobic scope resulted from exposure to this level of ΣPAHs. Using environmentally relevant crude oil exposure concentrations and a commercially and ecologically valuable Gulf of Mexico fish species, the present results provide insight into the effects of the Deepwater Horizon oil spill on adult pelagic fish. Environ Toxicol Chem 2016;35:2613-2622. © 2016 SETAC., (© 2016 SETAC.)

The Deepwater Horizon (DWH) oil spill contaminated the spawning habitats for numerous commercially and ecologically important fishes. Exposure to the water accommodated fraction (WAF) of oil from the spill has been shown to cause cardiac toxicity during early developmental stages across fishes. To better understand the molecular events and explore new pathways responsible for toxicity, RNA sequencing was performed in conjunction with physiological and morphological assessments to analyze the time-course (24, 48, and 96 h post fertilization (hpf)) of transcriptional and developmental responses in embryos/larvae of mahi-mahi exposed to WAF of weathered (slick) and source DWH oils. Slick oil exposure induced more pronounced changes in gene expression over time than source oil exposure. Predominant transcriptomic responses included alteration of EIF2 signaling, steroid biosynthesis, ribosome biogenesis and activation of the cytochrome P450 pathway. At 96 hpf, slick oil exposure resulted in significant perturbations in eye development and peripheral nervous system, suggesting novel targets in addition to the heart may be involved in the developmental toxicity of DHW oil. Comparisons of changes of cardiac genes with phenotypic responses were consistent with reduced heart rate and increased pericardial edema in larvae exposed to slick oil but not source oil.

To better understand the impact of the Deepwater Horizon (DWH) incident on commercially and ecologically important pelagic fish species, a mahi-mahi spawning program was developed to assess the effect of embryonic exposure to DWH crude oil with particular emphasis on the effects of weathering and dispersant on the magnitude of toxicity. Acute lethality (96 h LC50) ranged from 45.8 (28.4-63.1) μg l(-1) ΣPAH for wellhead (source) oil to 8.8 (7.4-10.3) μg l(-1) ΣPAH for samples collected from the surface slick, reinforcing previous work that weathered oil is more toxic on a ΣPAH basis. Differences in toxicity appear related to the amount of dissolved 3 ringed PAHs. The dispersant Corexit 9500 did not influence acute lethality of oil preparations. Embryonic oil exposure resulted in cardiotoxicity after 48 h, as evident from pericardial edema and reduced atrial contractility. Whereas pericardial edema appeared to correlate well with acute lethality at 96 h, atrial contractility did not. However, sub-lethal cardiotoxicity may impact long-term performance and survival. Dispersant did not affect the occurrence of pericardial edema; however, there was an apparent reduction in atrial contractility at 48 h of exposure. Pericardial edema at 48 h and lethality at 96 h were equally sensitive endpoints in mahi-mahi., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Crude oils from distinct geological sources worldwide are toxic to developing fish hearts. When oil spills occur in fish spawning habitats, natural resource injury assessments often rely on conventional morphometric analyses of heart form and function. The extent to which visible indicators correspond to molecular markers for cardiovascular stress is unknown for pelagic predators from the Gulf of Mexico. Here we exposed mahi (Coryphaena hippurus) embryos to field-collected crude oil samples from the 2010 Deepwater Horizon disaster. We compared visible heart defects (edema, abnormal looping, reduced contractility) to changes in expression of cardiac-specific genes that are diagnostic of heart failure in humans or associated with loss-of-function zebrafish cardiac mutants. Mahi exposed to crude oil during embryogenesis displayed typical symptoms of cardiogenic syndrome as larvae. Contractility, looping, and circulatory defects were evident, but larval mahi did not exhibit downstream craniofacial and body axis abnormalities. A gradation of oil exposures yielded concentration-responsive changes in morphometric and molecular responses, with relative sensitivity being influenced by age. Our findings suggest that 1) morphometric analyses of cardiac function are more sensitive to proximal effects of crude oil-derived chemicals on the developing heart, and 2) molecular indicators reveal a longer-term adverse shift in cardiogenesis trajectory.

The guanylin family of peptides are effective regulators of intestinal physiology in marine teleosts. In the distal intestinal segments, they inhibit or reverse fluid absorption by inhibiting the absorptive short-circuit current (Isc). The present findings demonstrate that mRNA from guanylin and uroguanylin, as well as at least one isoform of the guanylin peptide receptor, apical guanylyl cyclase-C (GC-C), was highly expressed in the intestine and rectum of the Gulf toadfish (Opsanus beta). In the posterior intestine, GC-C, as well as the cystic fibrosis transmembrane conductance regulator and basolateral Na(+)/K(+)/2Cl(-) cotransporter, which comprise a Cl(-)-secretory pathway, were transcriptionally upregulated in 60 parts per thousand (ppt). The present study also shows that, in intestinal tissues from Gulf toadfish held in 35 ppt, renoguanylin (RGN) expectedly causes net Cl(-) secretion, inhibits both the absorptive Isc and fluid absorption, and decreases HCO3(-) secretion. Likewise, in intestinal tissues from Gulf toadfish acclimated to 60 ppt, RGN also inhibits the absorptive Isc and fluid absorption but to an even greater extent, corresponding with the mRNA expression data. In contrast, RGN does not alter Cl(-) flux and, instead, elevates HCO3(-) secretion in the 60-ppt group, suggesting increased apical Cl(-)/HCO3(-) exchange activity by SLC26a6. Overall, these findings reinforce the hypotheses that the guanylin peptide system is important for salinity acclimatization and that the secretory response could facilitate the removal of solids, such as CaCO3 precipitates, from the intestine., (Copyright © 2015 the American Physiological Society.)

The euryhaline pupfish, Cyprinodon variegatus variegatus (Cvv), can successfully osmoregulate in ≥2 mM Na(+) and a freshwater population (Cyprinodon variegatus hubbsi; Cvh) osmoregulates at ≥0.1mM Na(+). We previously demonstrated that Cvv relies on an apical NKCC and NHE in the gill for Na(+) uptake in high (7mM) and intermediate (2 mM) Na(+) concentrations, while Cvh relies only on NHE for Na(+) uptake. This study investigated whether differential NHE isoform use explains differences in Na(+) uptake kinetics between these two populations. We further studied whether Cvh uses a NHE-Rh metabolon or carbonic anhydrase (CA) to overcome thermodynamic challenges of NHE function in dilute freshwater. Transfer to more dilute freshwater resulted in upregulation of nhe-2 (Cvv only) and nhe-3 (Cvv and Cvh). Relative expression of nhe-3 compared to nhe-2 was 2-fold higher in Cvv, but 200-fold higher in Cvh suggesting that nhe-3 expression is an important freshwater adaptation for Cvh. Simultaneous measurement of Na(+) and Tamm flux under various conditions provided no support for a NHE-Rh metabolon in either population. Carbonic anhydrase activity in Cvv was comparable in 7 and 2 mM Na(+) acclimated fish. In Cvh, CA activity increased by 75% in 0.1 mM Na(+) acclimated fish compared to 7 mM Na(+) fish. Ethoxzolamide had variable effects, stimulating and reducing Na(+) uptake in Cvv acclimated to 7 and 2 mM Na(+), while reducing Na(+) uptake in 7 and 0.1mM Na(+) acclimated Cvh. This suggests that CA plays important, but different roles in regulating Na(+) uptake in Cvv and Cvh., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The physiological effects of guanylin (GN) and uroguanylin (UGN) on fluid and electrolyte transport in the teleost fish intestine have yet to be thoroughly investigated. In the present study, the effects of GN, UGN, and renoguanylin (RGN; a GN and UGN homolog) on short-circuit current (Isc) and the transport of Cl-, Na+, bicarbonate (HCO3-), and fluid in the Gulf toadfish (Opsanus beta) intestine were determined using Ussing chambers, pH-stat titration, and intestinal sac experiments. GN, UGN, and RGN reversed the Isc of the posterior intestine (absorptive-to-secretory), but not of the anterior intestine. RGN decreased baseline HCO3- secretion, but increased Cl- and fluid secretion in the posterior intestine. The secretory response of the posterior intestine coincides with the presence of basolateral NKCC1 and apical cystic fibrosis transmembrane conductance regulator (CFTR), the latter of which is lacking in the anterior intestine and is not permeable to HCO3- in the posterior intestine. However, the response to RGN by the posterior intestine is counterintuitive given the known role of the marine teleost intestine as a salt- and water-absorbing organ. These data demonstrate that marine teleosts possess a tissue-specific secretory response, apparently associated with seawater adaptation, the exact role of which remains to be determined., (Copyright © 2014 the American Physiological Society.)

The Deepwater Horizon incident likely resulted in exposure of commercially and ecologically important fish species to crude oil during the sensitive early life stages. We show that brief exposure of a water-accommodated fraction of oil from the spill to mahi-mahi as juveniles, or as embryos/larvae that were then raised for ∼25 days to juveniles, reduces their swimming performance. These physiological deficits, likely attributable to polycyclic aromatic hydrocarbons (PAHs), occurred at environmentally realistic exposure concentrations. Specifically, a 48 h exposure of 1.2 ± 0.6 μg L(-1) ΣPAHs (geometric mean ± SEM) to embryos/larvae that were then raised to juvenile stage or a 24 h exposure of 30 ± 7 μg L(-1) ΣPAHs (geometric mean ± SEM) directly to juveniles resulted in 37% and 22% decreases in critical swimming velocities (Ucrit), respectively. Oil-exposed larvae from the 48 h exposure showed a 4.5-fold increase in the incidence of pericardial and yolk sac edema relative to controls. However, this larval cardiotoxicity did not manifest in a reduced aerobic scope in the surviving juveniles. Instead, respirometric analyses point to a reduction in swimming efficiency as a potential alternative or contributing mechanism for the observed decreases in Ucrit.

The Deepwater Horizon disaster released more than 636 million L of crude oil into the northern Gulf of Mexico. The spill oiled upper surface water spawning habitats for many commercially and ecologically important pelagic fish species. Consequently, the developing spawn (embryos and larvae) of tunas, swordfish, and other large predators were potentially exposed to crude oil-derived polycyclic aromatic hydrocarbons (PAHs). Fish embryos are generally very sensitive to PAH-induced cardiotoxicity, and adverse changes in heart physiology and morphology can cause both acute and delayed mortality. Cardiac function is particularly important for fast-swimming pelagic predators with high aerobic demand. Offspring for these species develop rapidly at relatively high temperatures, and their vulnerability to crude oil toxicity is unknown. We assessed the impacts of field-collected Deepwater Horizon (MC252) oil samples on embryos of three pelagic fish: bluefin tuna, yellowfin tuna, and an amberjack. We show that environmentally realistic exposures (1-15 µg/L total PAH) cause specific dose-dependent defects in cardiac function in all three species, with circulatory disruption culminating in pericardial edema and other secondary malformations. Each species displayed an irregular atrial arrhythmia following oil exposure, indicating a highly conserved response to oil toxicity. A considerable portion of Gulf water samples collected during the spill had PAH concentrations exceeding toxicity thresholds observed here, indicating the potential for losses of pelagic fish larvae. Vulnerability assessments in other ocean habitats, including the Arctic, should focus on the developing heart of resident fish species as an exceptionally sensitive and consistent indicator of crude oil impacts.

While it is increasingly being recognized that biotic ligand models (BLMs) are valuable in the risk assessment of metals in aquatic systems, the development of chronic BLMs has been less advanced for lead than for other metals. The authors investigated the univariate effects of Ca and pH on the chronic reproductive toxicity of Pb to Ceriodaphnia dubia at 4 levels. Calcium influenced chronic Pb toxicity to C. dubia only to a relatively small extent, whereas a high pH (8.2) provided strong protection against Pb toxicity (compared with lower pH levels). Based on this data set, a chronic Pb BLM for C. dubia was developed. The effect of pH was modeled as a single biotic ligand site competition by H(+) with a log stability constant for binding of H(+) to the biotic ligand (K(HBL)) of 7.6, while no other competitive constants were needed. The developed BLM was shown, in an independent validation with 3 other data sets, to be capable of predicting chronic Pb toxicity to different clones of C. dubia by an error of less than a factor of 2 in most synthetic and natural waters considered. The results add to the growing evidence that BLM-based risk assessment or water-quality criteria for Pb are likely to be more appropriate relative to hardness-based assessments or criteria., (© 2013 SETAC.)

The toxicity of many metals is impacted by environmental pH, through both competition and complexation by hydroxide and carbonate ions. To establish safe environmental regulation it is important to properly define the relationship between pH and metal toxicity, a process that involves manipulating the pH of test water in the lab. The current study compares the effects of the three most common pH manipulation methods (carbon dioxide, acid-base addition, and chemical buffers) on acute Pb toxicity of a model fish species, Pimephales promelas. Acidification of test water revealed that the Pb and Pb(2+) LC50 values were impacted by the pH manipulation method, with the following order of effects: HCl

Based on early pharmacological work, the serotonin 2A (5-HT(2A)) receptor subtype is believed to be involved in the regulation of toadfish pulsatile urea excretion. The goal of the following study was to characterize the toadfish 5-HT(2A) receptor at a molecular level, to determine the tissues in which this receptor is predominantly expressed and to further investigate the pharmacological specificity of toadfish pulsatile urea excretion by examining the effect of ketanserin, a 5-HT(2A) receptor antagonist, on resting rates of pulsatile urea excretion. The full-length toadfish 5-HT(2A) receptor encodes a 496 amino acid sequence and shares 57-80% sequence identity to 5-HT(2A) receptors of other organisms, with 100% conservation among important ligand-binding residues. Toadfish 5-HT(2A) receptor mRNA expression was highest in the swim bladder and gonad, followed by the whole brain. All other tissues tested (esophagus, stomach, anterior intestine, posterior intestine, rectum, liver, kidney, heart, muscle and gill) had mRNA expression levels that were significantly less than whole brain. Toadfish 5-HT(2A) receptor mRNA expression within the brain was highest in the hindbrain, telencephalon and midbrain/diencephalon regions. Treatment with the 5-HT(2A) receptor antagonist, ketanserin, resulted in a significant decrease in the pulsatile component of spontaneous urea excretion due to a reduction in urea pulse size with no significant change in pulse frequency. These results lend further support for the 5-HT(2A) receptor in the regulation of pulsatile urea excretion in toadfish., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The current study examined the chronic toxicity of lead (Pb) to three invertebrate species: the cladoceran Ceriodaphnia dubia, the snail Lymnaea stagnalis and the rotifer Philodina rapida. The test media consisted of natural waters from across North America, varying in pertinent water chemistry parameters including dissolved organic carbon (DOC), calcium, pH and total CO(2). Chronic toxicity was assessed using reproductive endpoints for C. dubia and P. rapida while growth was assessed for L. stagnalis, with chronic toxicity varying markedly according to water chemistry. A multi-linear regression (MLR) approach was used to identify the relative importance of individual water chemistry components in predicting chronic Pb toxicity for each species. DOC was an integral component of MLR models for C. dubia and L. stagnalis, but surprisingly had no predictive impact on chronic Pb toxicity for P. rapida. Furthermore, sodium and total CO(2) were also identified as important factors affecting C. dubia toxicity; no other factors were predictive for L. stagnalis. The Pb toxicity of P. rapida was predicted by calcium and pH. The predictive power of the C. dubia and L. stagnalis MLR models was generally similar to that of the current C. dubia BLM, with R(2) values of 0.55 and 0.82 for the respective MLR models, compared to 0.45 and 0.79 for the respective BLMs. In contrast the BLM poorly predicted P. rapida toxicity (R(2)=0.19), as compared to the MLR (R(2)=0.92). The cross species variability in the effects of water chemistry, especially with respect to rotifers, suggests that cross species modeling of invertebrate chronic Pb toxicity using a C. dubia model may not always be appropriate., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The current study examined the acute toxicity of lead (Pb) to Ceriodaphnia dubia and Pimephales promelas in a variety of natural waters. The natural waters were selected to range in pertinent water chemistry parameters such as calcium, pH, total CO(2) and dissolved organic carbon (DOC). Acute toxicity was determined for C. dubia and P. promelas using standard 48h and 96h protocols, respectively. For both organisms acute toxicity varied markedly according to water chemistry, with C. dubia LC50s ranging from 29 to 180μg/L and P. promelas LC50s ranging from 41 to 3598μg/L. Additionally, no Pb toxicity was observed for P. promelas in three alkaline natural waters. With respect to water chemistry parameters, DOC had the strongest protective impact for both organisms. A multi-linear regression (MLR) approach combining previous lab data and the current data was used to identify the relative importance of individual water chemistry components in predicting acute Pb toxicity for both species. As anticipated, the P. promelas best-fit MLR model combined DOC, calcium and pH. Unexpectedly, in the C. dubiaMLR model the importance of pH, TCO(2) and calcium was minimal while DOC and ionic strength were the controlling water quality variables. Adjusted R(2) values of 0.82 and 0.64 for the P. promelas and C. dubia models, respectively, are comparable to previously developed biotic ligand models for other metals., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Fathead minnows were subjected to an incremental velocity test using swim tunnel respirometry for the analysis of aerobic scope and swimming performance, as critical aerobic swim speed (U(crit)), following chronic exposures (33-57 ) to 0.9±0.4, 157±18 or 689±66 nmol L⁻¹ Pb and an acute exposure (24 h) to 672±35 nmol L⁻¹ Pb (mean±SEM). Assessment of Pb-induced anemia and neurological impairment were evaluated by blood hemoglobin (Hb) concentrations and a cost of transport (COT) analysis, respectively. Fish from the acute 672±35 nmol L⁻¹ Pb (24.4±1.2 BL s⁻¹) and chronic 689±66 nmol L⁻¹ Pb (24.6±0.9 BL s⁻¹) treatments exhibited reduced U(crits) compared to control fish (27.6±0.8 BL s⁻¹). Aerobic scope was reduced by acute Pb exposure (8.6±2.6 μmol O₂ g⁻¹ h⁻¹ vs. 22.6±3.8 μmol O₂ g⁻¹ h⁻¹ from controls) owing to a decrease in maximum oxygen consumption rate (38.8±0.8 μmol O₂ g⁻¹ h⁻¹ vs. 54.0±4.2 μmol O₂ g⁻¹ h⁻¹ from controls). However, no effect on aerobic scope was observed with fish chronically exposed to Pb. Significant differences were not observed for Hb concentrations or COT. These findings suggest that the impaired swimming performances arising from acute and chronic Pb exposures reflect different mechanisms of toxicity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

As the first step toward parameterization of a chronic lead (Pb) biotic ligand model (BLM) for Ceriodaphnia dubia, 7-d toxicity tests were performed in waters modified to evaluate the influences of hardness, DOM (as Suwannee River NOM and Aldrich humic acid (HA)), pH (buffered with 4 mM MOPS) and alkalinity on the chronic toxicity of Pb. Calculated EC(20)s for the control base water test and each of the most extreme modified test waters were as follows in μg L(-1) Pb (95% confidence interval): base water control=45 (14-53), 5 mM CaSO(4)=22 (12-30), 32 mg L(-1) DOM=523 (388-573), 2.5 mM NaHCO(3)=73 (21-120) and pH 6.4 buffered with MOPS=3.9 μg L(-1) Pb (1-5). Results indicate that hardness does not protect against chronic toxicity of Pb to C. dubia, whereas HA does protect at the highest concentration tested (597 μM). Additionally, our findings suggest that low pH increases the chronic toxicity of Pb whereas increased alkalinity is protective. The findings reported herein support the need for a chronic Pb BLM as an alternative approach to hardness-based regulations., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The acute toxicity of lead (Pb) was examined for fathead minnows (Pimephales promelas; 96-h) and daphnids (Ceriodaphnia dubia; 48-h) in waters modified for hardness (as CaSO₄), dissolved organic carbon (DOC; as Aldrich humic acid) and alkalinity (as NaHCO₃) for parameterization of an acute freshwater biotic ligand model (BLM). Additionally, acute (96-h) and chronic (30-d) bioassays were performed for P. promelas to more clearly define the influence of pH (5.5-8.3) on Pb toxicity as modified by addition of HCl or NaOH using an automated titration system. Results indicate that Ca(2+) is protective against acute Pb toxicity to P. promelas but not C. dubia. Strong protection was afforded by DOC and NaHCO(3) against acute Pb toxicity to P. promelas, whereas milder protection was observed for C. dubia with both parameters. Dissolved Pb LC50s from the P. promelas pH bioassays revealed a complex effect of pH on Pb toxicity, likely explained in part by Pb speciation and the competitive interaction of H(+) with ionic Pb(2+). Chronic pH bioassays also demonstrated that 30-d growth is not impaired in fathead minnows at relevant Pb concentrations. The findings reported herein suggest that development of separate BLMs for P. promelas and C. dubia should be considered., (Copyright © 2010 Elsevier Inc. All rights reserved.)

It is well established that serotonin (5-HT; 5-hydroxytryptamine) plays a role in mammalian regulation of the hypothalamic-pituitary-adrenal (HPA) axis via the 5-HT receptor subtype 1A (5-HT(1A)). To date, there has not been a comprehensive investigation of the molecular, pharmacological and physiological aspects of the 5-HT(1A) receptor and its role in the activation of the hypothalamic-pituitary-interrenal (HPI) axis in teleost fish. The 5-HT(1A) receptor of the Gulf toadfish (Opsanus beta) was cloned and sequenced, showing 67.5% amino acid similarity to the human homologue. The 5-HT(1A) receptor was distributed throughout the brain, with the whole brain containing significantly higher levels of 5-HT(1A) mRNA compared to all other tissues and the midbrain/diencephalon region containing significantly higher levels of transcript than any other brain region. Substantial levels of transcript were also found in the pituitary, while very low levels were in the kidney that contains the interrenal cells. Xenopus oocytes injected with toadfish 5-HT(1A) receptor cRNA displayed significantly higher binding of [(3)H]5-HT that was abolished by the mammalian 5-HT(1A) receptor agonist, 8-OH-DPAT, indicating a conserved binding site of the toadfish 5-HT(1A) receptor and a high specificity for the agonist. Supporting this, binding of [(3)H]5-HT was not affected by the mammalian 5-HT(1B) receptor agonist, 5-nonyloxytryptamine, the 5-HT(7) receptor antagonist, SB269970, or the 5-HT(2) receptor agonist, alpha-methylserotonin. Confirming these molecular and pharmacological findings, intravenous injection of 8-OH-DPAT stimulated the HPI axis to cause a 2-fold increase in circulating levels of cortisol. The present study of the 5-HT(1A) receptor in a single teleost species illustrates the high conservation of this 5-HT receptor amongst vertebrates., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Carbonic anhydrase (CA) is a ubiquitous enzyme involved in acid-base regulation and osmoregulation. Many studies have demonstrated a role for this enzyme in fish osmoregulation in seawater as well as freshwater. However, to date CA responses of marine fish exposed to salinities exceeding seawater (approximately 35 ppt) have not been examined. Consequently, the aim of the present study was to examine CA expression and activity in osmoregulatory tissues of the Gulf Toadfish, Opsanus beta, following transfer to 60 ppt. A gene coding, for CAc of 1827 bp with an open reading frame of 260 amino acids was cloned and showed high expression in all intestinal segments and gills. CAc showed higher expression in posterior intestine and rectum than in anterior and mid intestine and in gills of fish exposed to 60 ppt for up to 4 days. The enzymatic activity, in contrast, was higher in all examined tissues two weeks following transfer to 60 ppt. Comparing early expression and later activity levels of acclimated fish reveals a very different response to hypersalinity among tissues. Results highlight a key role of CAc in osmoregulation especially in distal regions of the intestine; moreover, CAc play a role in the gill in hypersaline environments possibly supporting elevated branchial acid extrusion seen under such conditions., (Copyright 2010 Elsevier Inc. All rights reserved.)

Although endogenous CO2 hydration and serosal HCO3- are both known to contribute to the high rates of intestinal HCO3- secretion important to marine fish osmoregulation, the basolateral step by which transepithelial HCO3- secretion is accomplished has received little attention. Isolated intestine HCO3- secretion rates, transepithelial potential (TEP) and conductance were found to be dependent on serosal HCO3- concentration and sensitive to serosal DIDS. Elevated mucosal Cl- concentration had the unexpected effect of reducing HCO3- secretion rates, but did not affect electrophysiology. These characteristics indicate basolateral limitation of intestinal HCO3- secretion in seawater gulf toadfish, Opsanus beta. The isolated intestine has a high affinity for serosal HCO3- in the physiological range (Km=10.2 mmol l(-1)), indicating a potential to efficiently fine-tune systemic acid-base balance. We have confirmed high levels of intestinal tract expression of a basolateral Na+/HCO3- cotransporter of the electrogenic NBCe1 isoform in toadfish (tfNBCe1), which shows elevated expression following salinity challenge, indicating its importance in marine fish osmoregulation. When expressed in Xenopus oocytes, isolated tfNBCe1 has transport characteristics similar to those in the isolated tissue, including a similar affinity for HCO3- (Km=8.5 mmol l(-1)). Reported affinity constants of NBC1 for Na+ are generally much lower than physiological Na+ concentrations, suggesting that cotransporter activity is more likely to be modulated by HCO3- rather than Na+ availability in vivo. These similar functional characteristics of isolated tfNBCe1 and the intact tissue suggest a role of this cotransporter in the high HCO3- secretion rates of the marine fish intestine.

Historically, the USEPA has only considered water hardness when establishing acute and chronic water quality criteria (WQC) for lead (Pb) in freshwater. Yet, recent evidence suggests that hardness may not be protective during chronic Pb exposure and that other factors (e.g., dissolved organic carbon (DOC) and alkalinity) influence toxicity. In fact, we have recently shown that Ca(2+) (as CaSO(4)) does not protect against Pb accumulation in fathead minnows (Pimephales promelas) during chronic exposures whereas DOC as humic acid (HA) clearly does. To more clearly define the water chemistry parameters mediating chronic Pb toxicity we carried out 300 d exposures to study the influence of DOC and alkalinity on Pb accumulation and toxicity to fathead minnows at 2 different Pb concentrations (170 and 580 nM (35 and 120 microg/L)). Alkalinity was adjusted by addition of 500 microM NaHCO(3) and DOC by addition of 4 mg/L HA. Fish were collected at 4, 30, 150 and 300 d of exposure to measure growth and Pb accumulation. Breeding assays (21 d) were performed at the end of these exposures to assess reproductive and larval behavioral endpoints. To determine whether effects were acute or chronic, switched breeding exposures were performed in which control breeders were transferred to either high or low Pb conditions and Pb-exposed breeders transferred to tap water without Pb. Mortality and growth effects were observed primarily in the high Pb treatments and within the first 10 d of exposure. Strong protection against Pb accumulation was afforded by increased DOC at both Pb concentrations. Increased alkalinity also appeared to moderately reduce Pb accumulation although not to the level of statistical significance. Tissue distribution of Pb was analyzed at 300 d and was found to accumulate mostly in bone, gill, intestine and kidney. Unexpectedly, high Pb reduced total reproductive output and increased average egg mass in the HCO(3)(-) and DOC treatments but not in the control water (+Pb) treatments. No statistically significant differences in egg hatchability or egg Pb accumulation were observed. Results from switched exposures suggest that embryo Pb accumulation arose from acute exposure to embryos rather than parental transfer. Finally, prey capture assays revealed potential Pb-induced motor/behavioral impairment in 10-d-old F1 larvae exposed to high Pb in all water treatments., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)