Combined effects of salinity, temperature, hypoxia, and Deepwater Horizon oil on Fundulus grandis larvae.

Oil spills have polluted the marine environment for decades and continue to be a major source of polycyclic aromatic hydrocarbons (PAHs) to marine ecosystems around the globe, for example during the 2010 Deepwater Horizon spill. Although the toxicity of PAHs to fish has been well studied, their effects combined with abiotic stressors are poorly understood. The goal of this study was to describe the combined impacts of crude oil and environmental stressors on fish larvae, a sensitive life stage. Gulf killifish (Fundulus grandis) larvae (<24 h post-hatch) were exposed for 48 h to high energy water accommodated fractions (HEWAF; total PAHs 0-125 ppb) of Macondo oil from the Deepwater Horizon spill under different combinations of environmental conditions (dissolved oxygen 2, 6 ppm; temperature 20, 25, 30 °C; salinity 3, 10, 30 ppt). Even under optimal environmental conditions (25 °C, 10 ppt, 6 ppm) larval survival and development were negatively affected by PAHs, starting with the lowest concentration tested (∼15 ppb). Hypoxia and high temperature each increased the adverse effects of HEWAF on development and mortality. In contrast, salinity had little effect on any of the endpoints measured. Importantly, expression of the detoxifying gene cyp1a was highly induced in PAH-exposed larvae under normoxic conditions, but not under hypoxic conditions, potentially explaining the enhanced toxicity observed under hypoxia. This work highlights the importance of considering how suboptimal environmental conditions can exacerbate the effects of pollution on fish early life stages.

Behavioral and physiological responses of yellow perch (Perca flavescens) to moderate hypoxia.

While severe hypoxia can be lethal and is usually avoided by mobile aquatic organisms, moderate hypoxic conditions are likely more prevalent and may affect organisms, such as fishes, in a variety of systems. However, fishes have the potential to adjust physiologically and behaviorally and thus reduce the negative effects of hypoxia. Quantifying such physiological responses may shed light on the ability of fishes to tolerate reduced oxygen concentrations. This study assessed how two different hatchery populations of yellow perch Perca flavescens, a fish that is likely to encounter moderate hypoxic conditions in a variety of systems, responded to moderate hypoxic exposure through three experiments: 1) a behavioral foraging experiment, 2) an acute exposure experiment, and 3) a chronic exposure experiment. No marked behavioral or physiological adjustments were observed in response to hypoxia (e.g., hemoglobin, feeding rate, movement frequency, gene expression did not change to a significant degree), possibly indicating a high tolerance level in this species. This may allow yellow perch to utilize areas of moderate hypoxia to continue foraging while avoiding predators that may be more sensitive to moderately low oxygen.

Comparative in vitro toxicity assessment of perfluorinated carboxylic acids.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are synthetic fluorinated compounds that are highly bioaccumulative and persistent organic pollutants. Perfluorooctanoic acid (PFOA), an eight-carbon chain perfluorinated carboxylic acid, was used heavily for the production of fluoropolymers, but concerns have led to its replacement by shorter carbon chain homologues such as perfluorohexanoic acid (PFHxA) and perfluorobutanoic acid (PFBA). However, limited toxicity data exist for these substitutes. We evaluated the toxicity of PFOA, PFHxA and PFBA on a zebrafish liver cell line and investigated the effects of exposure on cell metabolism. Gross toxicity after 96 h of exposure was highest for PFOA and PFO- , while PFHxA and PFBA exhibited lower toxicity. Although the structural similarity of these compounds to fatty acids suggests the possibility of interference with the transport and metabolism of lipids, we could not detect any differential expression of peroxisome proliferator-activated receptor (ppar-α, -ß and -γ), fabp3 and crot genes after 96 h exposure to up to 10 ppm of the test compounds. However, we observed localized lipid droplet accumulation only in PFBA-exposed cells. To study the effects of these compounds on cell metabolism, we conducted fluorescence lifetime imaging microscopy using naturally fluorescent biomarkers, NADH and FAD. The fluorescence lifetimes of NADH and FAD and the bound/free ratio of each of these coenzymes decreased in a dose- and carbon length-dependent manner, suggesting disruption of cell metabolism. In sum, our study revealed that PFASs with shorter carbon chains are less toxic than PFOA, and that exposure to sublethal dosage of PFOA, PFHxA or PFBA affects cell metabolism. Copyright © 2016 John Wiley & Sons, Ltd.

Cloning and characterization of Na(+)/H(+) Exchanger isoforms NHE2 and NHE3 from the gill of Pacific dogfish Squalus suckleyi.

Na(+)/H(+) Exchanger (NHE) proteins mediate cellular and systemic homeostasis of sodium and acid and may be the major sodium uptake method for fishes. We cloned and sequenced NHE2 and NHE3 from the gill of the North Pacific Spiny Dogfish shark Squalus suckleyi and expressed them in functional form in NHE-deficient (AP-1) cell lines. Estimated IC50 for inhibition of NHE activity by amiloride and EIPA were 55 µmol l(-1) and 4.8 µmol l(-1), respectively, for NHE2 and 9 µmol l(-1) and 24 µmol l(-1), respectively, for NHE3. Phenamil at 100 µmol l(-1) caused less than 16% inhibition of activity for each isoform. Although the IC50 are similar for the two isoforms, dfNHE2 is less sensitive than human NHE2 to inhibition by amiloride and EIPA, while dfNHE3 is more sensitive than human NHE3. These IC50 estimates should be considered when selecting inhibitor doses for fishes and for reinterpretation of previous studies that use these pharmacological agents.

Fluctuating water temperatures affect development, physiological responses and cause sex reversal in fathead minnows.

Natural and human activities can result in both high temporal and spatial variability in water temperature. Rapid temperature changes have the potential to dramatically affect physiological processes in aquatic organisms and, due to their limited mobility, fish early life stages are particularly vulnerable to ambient temperature fluctuations. In this study, we examined how the magnitude and frequency of temperature fluctuations affect survival, growth, development, expression of thermoresponsive genes, and gonadal differentiation in fathead minnows, Pimephales promelas. We exposed individuals (0 to 4 days post fertilization) of known genotypic sex to fluctuations of Δ4 °C over 12-h, Δ8 °C over 12- and 24-h, and three stable temperatures (21, 25, and 29 °C) for up to 45 d. Expression of hsp70 in fish exposed to the highest-magnitude, highest-frequency fluctuating treatment cycled in concert with temperature and was upregulated initially during exposure, and may have contributed to temperature fluctuations having little effect on time to and size at hatching (whole-organism responses). This treatment also caused fish to undergo nondirectional sex reversal. These results indicate that hsp70 may be involved in mediating thermal stress from subdaily temperature fluctuations and that sex determination in fathead minnows can be influenced by cycling temperatures.

Phosphate absorption across multiple epithelia in the Pacific hagfish (Eptatretus stoutii).

Inorganic phosphate (Pi) is an essential nutrient for all organisms, but in seawater, Pi is a limiting nutrient. This study investigated the primary mechanisms of Pi uptake in Pacific hagfish (Eptatretus stoutii) using ex vivo physiological and molecular techniques. Hagfish were observed to have the capacity to absorb Pi from the environment into at least three epithelial surfaces: the intestine, skin, and gill. Pi uptake in all tissues was concentration dependent, and saturable Pi transport was observed in the skin and gill at <2.0 mmol/l Pi. Gill and intestinal Pi uptake was sodium dependent, but Pi uptake into the skin increased under low sodium conditions. Gill Pi transport exhibited an apparent affinity constant ~0.23-0.6 mmol/l Pi. A complete sequence of a type II sodium phosphate cotransporter (Slc34a) was obtained from the hagfish gill. Phylogenetic analysis of the hagfish Slc34a transporter indicates that it is earlier diverging than, and/or ancestral to, the other identified vertebrate Slc34a transporters (Slc34a1, Slc34a2, and Slc34a3). With the use of RT-PCR, the hagfish Slc34a transcript was detected in the intestine, skin, gill, and kidney, suggesting that this may be the transporter involved in Pi uptake into multiple epithelia in the hagfish. This is the first measurement of Pi uptake across the gill or skin of any vertebrate animal and first sodium phosphate cotransporter identified in hagfish.

Time course of the acute response of the North Pacific spiny dogfish shark (Squalus suckleyi) to low salinity.

Dogfish are considered stenohaline sharks but are known to briefly enter estuaries. The acute response of North Pacific spiny dogfish (Squalus suckleyi) to lowered salinity was tested by exposing sharks to 21‰ salinity for 48 h. Temporal trends in blood pH, plasma osmolality, CO2, HCO3(-), Na(+), Cl(-), K(+), and urea concentrations, and in the rates of urea efflux and O2 consumption, were quantified. The rate of O2 consumption exhibited cyclic variation and was significantly depressed by lowered salinity. After 9 h, plasma [Cl(-)] stabilized at 9% below initial levels, while plasma [Na(+)] decreased by more than 20% within the first 12 h. Plasma [urea] dropped by 15% between 4 and 6 h, and continued to decrease. The rate of urea efflux increased over time, peaking after 36 h at 72% above the initial rate. Free-swimming sharks subjected to the same salinity challenge survived over 96 h and differed from cannulated sharks with respect to patterns of Na(+) and urea homeostasis. This high-resolution study reveals that dogfish exposed to 21‰ salinity can maintain homeostasis of Cl(-) and pH, but Na(+) and urea continue to be lost, likely accounting for the inability of the dogfish to fully acclimate to reduced salinity.

Extrabranchial mechanisms of systemic pH recovery in hagfish (Eptatretus stoutii).

This study investigates the role of branchial and extrabranchial processes in acid-base regulation in the Pacific Hagfish (Eptatretus stoutii). Hagfish were injected with one of the following solutions: acid saline (250mM HCl [pH=0.60], 250mM NaCl), alkaline saline (250mM NaHCO3, 250mM NaCl, [pH≈8.43]) or control saline (500mM NaCl) in order to achieve an acid/alkaline/saline load of 6000µmol·kg(-1). Using a custom designed hagfish compartmentalizing flux chamber, we partitioned flux of net acid or base equivalents and ammonia into the anterior (gill+skin) and posterior (skin+intestinal/renal/cloacal) components. We found that Pacific hagfish excrete H(+) primarily via branchial mechanisms but base excretion occurs through extrabranchial mechanisms located in the posterior region. In addition, we demonstrate that hagfish are able to excrete ammonia via the skin although this flux was not involved in compensation from an acid-base disturbance.