Turquoise killifish: A natural model of age-dependent brain degeneration.

Turquoise killifish (Nothobranchius furzeri) are naturally short-lived vertebrates that display a wide range of spontaneous age-related changes, including onset of cancer, reduced mobility, and cognitive decline. Here, we focus on describing the phenotypic spectrum of the aging killifish brain. As turquoise killifish age, their dopaminergic and noradrenergic neurons undergo a significant decline in number. Furthermore, brain aging in turquoise killifish is associated with several glial-specific changes, such as an increase in the astrocyte-covered surface area and an increase in the numbers of microglial cells, i.e. the brain-specific macrophage population. Killifish brains undergo age-dependent reduced proteasome activity and increased protein aggregation, including the aggregation of the Parkinson's disease marker α-synuclein. Parallel to brain degeneration, turquoise killifish develop spontaneous age-related gut dysbiosis, which has been proposed to affect human neurodegenerative disease. Finally, aged turquoise killifish display declined learning capacity. We argue that, taken together, the molecular, cellular and functional changes that spontaneously take place during aging in killifish brains are consistent with a robust degenerative process that shares remarkable similarities with human neurodegenerative diseases. Hence, we propose that turquoise killifish represent a powerful model of spontaneous brain degeneration which can be effectively used to explore the causal mechanisms underlying neurodegenerative diseases.

The roles of calcium and salinity in protecting against physiological symptoms of waterborne zinc toxicity in the euryhaline killifish (Fundulus heteroclitus).

In fresh water, environmental Ca ameliorates Zn toxicity because Ca2+ and Zn2+ compete for uptake at the gills. Zn toxicity is also lower in sea water, but it is unclear whether this is due to increased Ca2+ concentration, and/or to the other ions present at higher salinity. Using the euryhaline killifish, we evaluated the relative roles of Ca2+ (as CaNO3) versus the other ions contributing to salinity in protecting against physiological symptoms of Zn2+ toxicity. Killifish were exposed to a sublethal level of Zn (500 µg/L, as ZnSO4) for 96 h in either fresh water (0 % salinity) at low (1 mmol/L) and High Ca (10 mmol/L) or 35 ppt sea water (100 % salinity) at low (1 mmol/L) and High Ca (10 mmol/L). At 0 % salinity, High Ca partly or completely protected against the following effects of Zn seen at Low Ca: elevated plasma Zn, hypocalcaemia, inhibited unidirectional Ca2+ influx, inhibited branchial Na+/K+ATPase and Ca2+ATPase activities, and oxidative stress in gills, liver, intestine, and muscle. At 100 % salinity, in the presence of 1 mmol/L (Low Ca), Zn caused no disturbances in most of these same parameters, showing that the "non-Ca" components of sea water alone provided complete protection. However, for a few endpoints (inhibited intestinal Ca2+ATPase activity, oxidative stress in gill and liver), High Ca (10 mmol/L) was needed to provide full protection against Zn in 100 % salinity. There was no instance where the combination of 100 % salinity and High Ca failed to provide complete protection against Zn-induced disturbances in sea water.

Focal adhesion kinase and osmotic responses in ionocytes of Fundulus heteroclitus, a euryhaline teleost fish.

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) anion channels are the regulated exit pathway in Cl- secretion by teleost salt secreting ionocytes of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase Focal Adhesion Kinase (FAK) phosphorylated at Y407 (FAKpY407) and FAKpY397 are colocalized at the apical membrane and in subjacent membrane vesicles of ionocytes. Hypotonic shock and the α-2 adrenergic agonist clonidine rapidly and reversibly inhibit Cl- secretion by isolated opercular epithelia, simultaneous with dephosphorylation of FAKpY407 and increased FAKpY397, located in the apical membrane of ionocytes in the opercular epithelium. FAKpY407 is re-phosphorylated at the apical membrane of ionocytes and Cl- secretion rapidly restored by hypertonic shock, detectable at 2 min., maximum at 5 min and still elevated at 30 min. In isolated opercular epithelia, the FAK phosphorylation inhibitor Y15 and p38MAP kinase inhibitor SB203580 significantly blunted the recovery of short-circuit current (Isc, equal to Cl- secretion rate) after hypertonic shock. The cSRC inhibitor saracatinib dephosphorylated FAKpY861 seen near tight junctions of pavement cells, and reduced the increase in epithelial resistance normally seen with clonidine inhibition of ion transport, while FAKpY397 was unaffected. The results show rapid osmosensitive responses in teleost fish ionocytes involve phosphorylation of CFTR by FAKpY407, an opposing role for FAKpY397 and a possible role for FAKpY861 in tight junction dynamics.

MitosRNAs and extreme anoxia tolerance in embryos of the annual killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrate. Annual killifish inhabit ephemeral ponds, producing drought and anoxia-tolerant embryos, which allows the species to persist generation after generation. Anoxia tolerance and physiology vary by developmental stage, creating a unique opportunity for comparative study within the species. A recent study of small ncRNA expression in A. limnaeus embryos in response to anoxia and aerobic recovery revealed small ncRNAs with expression patterns that suggest a role in supporting anoxia tolerance. MitosRNAs, small ncRNAs derived from the mitochondrial genome, emerged as an interesting group of these sequences. MitosRNAs derived from mitochondrial tRNAs were differentially expressed in developing embryos and isolated cells exhibiting extreme anoxia tolerance. In this study we focus on expression of mitosRNAs derived from tRNA-cysteine, and their subcellular and organismal localization in order to consider possible function. These tRNA-cys mitosRNAs appear enriched in the mitochondria, particularly near the nucleus, and also appear to be present in the cytoplasm. We provide evidence that mitosRNAs are generated in the mitochondria in response to anoxia, though the precise mechanism of biosynthesis remains unclear. MitosRNAs derived from tRNA-cys localize to numerous tissues, and increase in the anterior brain during anoxia. We hypothesize that these RNAs may play a role in regulating gene expression that supports extreme anoxia tolerance.

Cell migration driven by substrate deformation gradients.

Identifying the cues followed by cells is key to understand processes as embryonic development, tissue homeostasis, or several pathological conditions. Based on a durotaxis model, it is shown that cells moving on predeformed thin elastic membrane follow the direction of increasing strain of the substrate. This mechanism, straintaxis, does not distinguish the origin of the strain, but the active stresses produce large strains on cells or tissues being used as substrates. Hence, straintaxis is the natural realization of duratoaxis in vivo. Considering a circular geometry for the substrate cells, it is shown that if the annular component of the active stress component increases with the radial distance, cells migrate toward the substrate cell borders. With appropriate estimation for the different parameters, the migration speeds are similar to those obtained in recent experiments (Reig et al 2017 Nat. Commun. 8 15431). In these, during the annual killifish epiboly, deep cells that move in contact with the epithelial enveloping cell layer (EVL), migrate toward the EVL cell borders with speeds of microns per minute.

Functionalized silver nanoparticles depress aerobic metabolism in the absence of overt toxicity in brackish water killifish, Fundulus heteroclitus.

Engineered nanomaterials (ENMs) tend to precipitate in saline waters so the majority of aquatic toxicity studies have focused on freshwaters, where bioavailability is presumed to be higher. Recent studies have illustrated that some ENM formulations are bioavailable and bioactive in salt water and that their effects are more pronounced at the physiological than biochemical level. These findings raise concerns regarding the effects of ENMs on marine organisms. Therefore, our goal was to characterize the effects of polyvinylpyrolidone-functionalized silver ENMs (nAg) on aerobic performance in the killifish (Fundulus heteroclitus), a common euryhaline teleost. Fish were exposed to 80 µg L-1 of 5 nm nAg for 48 h in brackish water (12 ppt) and routine (MO2min) and maximum (MO2max) rates of oxygen consumption were quantified. Silver dissolution was minimal and nAg remained well dispersed in brackish water, with a hydrodynamic diameter of 21.0 nm, compared to 19.3 in freshwater. Both MO2min and MO2max were significantly lower (by 53 and 30%, respectively) in killifish exposed to nAg and a reduction in MO2 variability suggested spontaneous activity was suppressed. Neither gill Na+/K+-ATPase activity, nor various other biochemical markers were affected by nAg exposure. The results illustrate that a common ENM formulation is bioactive in salt water and, as in previous studies on functionalized copper ENMs, that effects are more pronounced at the whole animal than the biochemical level.

Pharmacological evidence that DAPI inhibits NHE2 in Fundulus heteroclitus acclimated to freshwater.

Ionoregulation in the euryhaline killifish Fundulus heteroclitus has been intensively studied over the last two decades using a variety of techniques. However, there has been limited use of pharmacological inhibitors to identify proteins involved in ion transport for this species. In this study, we used a range of pharmacological inhibitors (EIPA, DAPI, ethoxzolamide, bumetanide, bafilomycin, phenamil, hydrochlorothiazide) to investigate the proteins involved in Na+ transport in freshwater (1 mM Na+) acclimated F. heteroclitus. Our results indicate that Na+ uptake under these conditions is sensitive to both EIPA (NHE-specific inhibitor) and DAPI (putative ASIC-specific inhibitor), but not to any of the other inhibitors. Results for EIPA are consistent with previous studies indicating F. heteroclitus relies solely on NHE2 for Na+ transport across the apical membrane of ionocytes. In contrast, results for DAPI are surprising given previous studies that have indicated the H+-ATPase is basolaterally located in F. heteroclitus and so cannot contribute to Na+ uptake via ASIC. The lack of bafilomycin sensitivity in the current study is consistent with a basolaterally located H+-ATPase. This suggests that DAPI is not an ASIC-specific inhibitor as has been previously assumed, and that it may also inhibit NHE2. Finally, we did not observe Na+ uptake to be sensitive to ethoxzolamide, suggesting that carbonic anhydrase may not be involved in generating the H+ needed to maintain NHE activity in freshwater as has been previously proposed.

Nitric oxide inhibition of NaCl secretion in the opercular epithelium of seawater-acclimated killifish, Fundulus heteroclitus.

Nitric oxide (NO) modulates epithelial ion transport pathways in mammals, but this remains largely unexamined in fish. We explored the involvement of NO in controlling NaCl secretion by the opercular epithelium of seawater killifish using an Ussing chamber approach. Pharmacological agents were used to explore the mechanism(s) triggering NO action. A modified Biotin-switch technique was used to investigate S-nitrosation of proteins. Stimulation of endogenous NO production via the nitric oxide synthase (NOS) substrate l-arginine (2.0 mmol l-1), and addition of exogenous NO via the NO donor SNAP (10-6 to 10-4 mol l-1), decreased the epithelial short-circuit current (Isc). Inhibition of endogenous NO production by the NOS inhibitor l-NAME (10-4 mol l-1) increased Isc and revealed a tonic control of ion transport by NO in unstimulated opercular epithelia. The NO scavenger PTIO (10-5 mol l-1) supressed the NO-mediated decrease in Isc, and confirmed that the effect observed was elicited by release of NO. The effect of SNAP on Isc was abolished by inhibitors of the soluble guanylyl cyclase (sGC), ODQ (10-6 mol l-1) and Methylene Blue (10-4 mol l-1), revealing NO signalling via the sGC/cGMP pathway. Incubation of opercular epithelium and gill tissues with SNAP (10-4 mol l-1) led to S-nitrosation of proteins, including Na+/K+-ATPase. Blocking of NOS with l-NAME (10-6 mol l-1) or scavenging of NO with PTIO during hypotonic shock suggested an involvement of NO in the hypotonic-mediated decrease in Isc Yohimbine (10-4 mol l-1), an inhibitor of α2-adrenoceptors, did not block NO effects, suggesting that NO is not involved in the α-adrenergic control of NaCl secretion.

AHR-related activities in a creosote-adapted population of adult atlantic killifish, Fundulus heteroclitus, two decades post-EPA superfund status at the Atlantic Wood Site, Portsmouth, VA USA.

Atlantic killifish, Fundulus heteroclitus, are adapted to creosote-based PAHs at the US EPA Superfund site known as Atlantic Wood (AW) on the southern branch of the Elizabeth River, VA USA. Subsequent to the discovery of the AW population in the early 1990s, these fish were shown to be recalcitrant to CYP1A induction by PAHs under experimental conditions, and even to the time of this study, killifish embryos collected from the AW site are resistant to developmental deformities typically associated with exposure to PAHs in reference fish. Historically, however, 90 +% of the adult killifish at this site have proliferative hepatic lesions including cancer of varying severity. Several PAHs at this site are known to be ligands for the aryl hydrocarbon receptor (AHR). In this study, AHR-related activities in AW fish collected between 2011 and 2013 were re-examined nearly 2 decades after first discovery. This study shows that CYP1A mRNA expression is three-fold higher in intestines of AW killifish compared to a reference population. Using immunohistochemistry, CYP1A staining in intestines was uniformly positive compared to negative staining in reference fish. Livers of AW killifish were examined by IHC to show that CYP1A and AHR2 protein expression reflect lesions-specific patterns, probably representing differences in intrinsic cellular physiology of the spectrum of proliferative lesions comprising the hepatocarcinogenic process. We also found that COX2 mRNA expression levels were higher in AW fish livers compared to those in the reference population, suggesting a state of chronic inflammation. Overall, these findings suggest that adult AW fish are responsive to AHR signaling, and do express CYP1A and AHR2 proteins in intestines at a level above what was observed in the reference population.

Mummichog (Fundulus heteroclitus) continue to successfully produce eggs after exposure to high levels of 17α-ethinylestradiol.

17α-Ethinylestradiol (EE2) is a potent estrogen used in birth-control pills. Previous laboratory and field studies have shown negative impacts in a variety of fish species after exposure to low levels of EE2, most notably a nearly complete shutdown of egg production. The present study demonstrates that mummichog (Fundulus heteroclitus), a small-bodied estuarine species, is able to continue to produce eggs after exposure for 28 d to 100 ng of EE2/L. No effect of EE2 on egg production was observed, whereas a >35-fold increase in vitellogenin (vtg 1) gene expression in males was found. The lack of response in egg production in fish exposed to high levels of EE2 warrants further investigations on species-specific responses to estrogens and endocrine disruptors in general.

The evolution of an annual life cycle in killifish: adaptation to ephemeral aquatic environments through embryonic diapause.

An annual life cycle is characterized by growth, maturity, and reproduction condensed into a single, short season favourable to development, with production of embryos (seeds, cysts, or eggs) capable of surviving harsh conditions which juveniles or adults cannot tolerate. More typically associated with plants in desert environments, or temperate-zone insects exposed to freezing winters, the evolution of an annual life cycle in vertebrates is fairly novel. Killifish, small sexually dimorphic fishes in the Order Cyprinodontiformes, have adapted to seasonally ephemeral water bodies across much of Africa and South America through the independent evolution of an annual life history. These annual killifish produce hardy desiccation-resistant eggs that undergo diapause (developmental arrest) and remain buried in the soil for long periods when fish have perished due to the drying of their habitat. Killifish are found in aquatic habitats that span a continuum from permanent and stable to seasonal and variable, thus providing a useful system in which to piece together the evolutionary history of this life cycle using natural comparative variation. I first review adaptations for life in ephemeral aquatic environments in killifish, with particular emphasis on the evolution of embryonic diapause. I then bring together available evidence from a variety of approaches and provide a scenario for how this annual life cycle evolved. There are a number of features within Aplocheiloidei killifish including their inhabitation of marginal or edge aquatic habitat, their small size and rapid attainment of maturity, and egg properties that make them particularly well suited to the colonization of ephemeral waters.

Alternative intrapopulation life-history strategies and their trade-offs in an African annual fish.

In ephemeral habitats, the same genotypes cope with unpredictable environmental conditions, favouring the evolution of developmental plasticity and alternative life-history strategies (ALHS). We tested the existence of intrapopulation ALHS in an annual killifish, Nothobranchius furzeri, inhabiting temporary pools. The pools are either primary (persisting throughout the whole rainy season) or secondary (refilled after desiccation of the initial pool), representing alternative niches. The unpredictable conditions led to the evolution of reproductive bet-hedging with asynchronous embryonic development. We used a common garden experiment to test whether the duration of embryonic period is associated with post-embryonic life-history traits. Fish with rapid embryonic development (secondary pool strategy, high risk of desiccation) produced phenotypes with more rapid life-history traits than fish with slow embryonic development (primary pool strategy). The fast fish were smaller at hatching but had larger yolk sac reserves. Their post-hatching growth was more rapid, and they matured earlier. Further, fast fish grew to a smaller body size and died earlier than slow fish. No differences in fecundity, propensity to mate or physiological ageing were found, demonstrating a combination of plastic responses and constraints. Such developmentally related within-population plasticity in life history is exceptional among vertebrates.

Metabolic and embryonic responses to terrestrial incubation of Fundulus grandis embryos across a temperature gradient.

This study simulated terrestrial incubation and measured rates of embryogenesis, nitrogen elimination, heart rate, lactate production, maximum length of time a hatch could be delayed and developmental responses of terrestrially incubated Gulf killifish Fundulus grandis embryos at temperatures ranging from 20 to 30° C. Temperature had a positive relationship with rate of embryogenesis, but a negative relationship with extent of extended incubation. The 30° C treatment reached embryonic maturity 6 days before the 20° C treatment. Embryos hatched between intervals of 240 and 336, 144 and 288, 96 and 240 and 96 and 192 h after reaching developmental maturity for the 20, 23, 26 and 30° C treatments. Significantly higher concentrations of total nitrogen, in the form of ammonia and urea, were recorded in the 20 and 30° C treatments. While temperature significantly influenced lactate and ATP concentrations, no significant influence of time of incubation was detected. Terrestrial embryos displayed an ability to develop quickly during embryogenesis and prolong incubation for an extended period of time after reaching embryonic maturity. This adaptation may be a life-history trait used to minimize asynchronous hatching, cannibalism and cohort size heterogeneity.

Functional and population genomic divergence within and between two species of killifish adapted to different osmotic niches.

Adaptation to salinity affects species distributions, promotes speciation, and guides many evolutionary patterns in fishes. To uncover the basis of a complex trait like osmoregulation, genome-level analyses are sensible. We combine population genomic scans with genome expression profiling to discover candidate genes and pathways associated with divergence between osmotic environments. We compared transcriptome sequence divergence between multiple freshwater and saltwater populations of the rainwater killifish, Lucania parva. We also compared sequence divergence between L. parva and its sister species, Lucania goodei, a freshwater specialist. We found highly differentiated single nucleotide polymorphisms (SNPs) between freshwater and saltwater L. parva populations in cell junction and ion transport genes, including V-type H(+) ATPase. Between species, we found divergence in reproduction and osmotic stress genes. Genes that were differentially expressed between species during osmotic acclimation included genes involved in ion transport and cell volume regulation. Gene sets that were divergent in coding sequence and divergent in expression did not overlap, although they did converge in function. Like many studies using genomic scans, our approach may miss some loci that contribute to adaptation but have complicated patterns of allelic variation. Our study suggests that gene expression and coding sequence may evolve independently as populations adapt to a complex physiological challenge.

The influence of salinity on acute nickel toxicity to the two euryhaline fish species, Fundulus heteroclitus and Kryptolebias marmoratus.

Nickel (Ni) is a common pollutant found in aquatic environments and may be harmful at elevated concentrations. Increasing salinity has been shown to decrease the bioavailability and toxicity of other metals to aquatic organisms. In the present study, acute Ni toxicity experiments (96-h) were conducted at various salinities (0-36 ppt) to determine the effects of salinity on Ni toxicity to 2 euryhaline fish species, Kryptolebias marmoratus and Fundulus heteroclitus. Nickel concentrations causing lethality to 50% of the fish ranged from 2 mg/L in moderately hard freshwater to 66.6 mg/L in 36 ppt saltwater. Nickel toxicity to F. heteroclitus decreased linearly with increasing salinity; however, Ni toxicity to K. marmoratus was only lowered by salinities above 6 ppt, demonstrating potential physiological differences between the 2 species when they are functioning as freshwater fish. Furthermore, the authors investigated the influence of Mg(2+) , Ca(2+) , Na(+) , and Cl(-) on Ni toxicity to F. heteroclitus. Freshwater with up to 120 mg/L Ca(2+) as CaSO4 , 250 mg/L Mg(2+) as MgSO4 , or 250 mg/L Na(+) as NaHCO3 did not provide protection against Ni toxicity. Alternatively, 250 mg/L Na(+) , as NaCl, was protective against Ni toxicity; and the extent of protection was similar to that demonstrated from salt water with the same Cl(-) concentration. These results suggest that Cl(-) is the predominant ion responsible for reducing Ni toxicity to K. marmoratus and F. heteroclitus in higher salinity waters.

Long-Term survival of anoxia despite rapid ATP decline in embryos of the annual killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus can survive for months in the complete absence of oxygen. Survival of anoxia is associated with entry into a state of metabolic dormancy known as diapause. However, extreme tolerance of anoxia is retained for several days of post-diapause development. Rates of heat dissipation in diapause II and 4 days post-diapause II embryos were measured under aerobic conditions and during the transition into anoxia. Phosphorylated adenylate compounds were quantified in embryos during entry into anoxia and after 12 hr of aerobic recovery. Rates of heat dissipation were not affected by exposure to anoxia in diapause II embryos, while post-diapause II embryos experienced a profound decrease in heat dissipation. ATP decreased substantially in both developmental stages upon exposure to anoxia, and all indicators of cellular energetic status indicated energetic stress, at least based on the mammalian paradigm. The rate of decline in ATP is the most acute reported for any vertebrate. The mechanisms responsible for cellular survival despite a clear dysregulation between energy production and energy consumption remain to be identified. Necrotic and apoptotic cell death in response to hypoxia contribute to poor survival during many diseases and pathological conditions in mammals. Understanding the mechanisms that are in place to prevent maladaptive cell death in embryos of A. limnaeus may greatly improve treatment strategies in diseases that involve hypoxia and reperfusion injuries.

Mitogen activated protein kinase 14-1 regulates serum glucocorticoid kinase 1 during seawater acclimation in Atlantic killifish, Fundulus heteroclitus.

The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies of environmental toxicants and osmoregulation. Previous research in our laboratory has shown that acute acclimation to seawater is mediated by an increase in SGK1. SGK1 promotes the trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane of the gill within the first hour in seawater resulting in increased chloride secretion. Although we have shown that the increase in gill SGK1 does not require activation of the glucocorticoid receptor, the mechanisms that mediate the rise SGK1 during acute acclimation is unknown. To test the hypothesis that mitogen activated protein kinase (MAPK14) is responsible for the rise in SGK1 we identified the coding sequence of killifish MAPK14-1 and designed a translational blocking vivo-morpholino targeting MAPK14-1. Injection of the MAPK14-1 vivo-morpholino resulted in a 30% reduction of MAPK14-1 and a 45% reduction in phosphorylated-MAPK14-1 protein in the gill of killifish transitioned from freshwater to seawater. Knock down of phosphorlyated-MAPK14-1 completely blocked the rise in SGK1 mRNA and protein in the killifish gill, providing the first direct and in vivo evidence that MAPK14-1 is necessary for acute seawater acclimation.

Oxidative stress parameters and antioxidant response to sublethal waterborne zinc in a euryhaline teleost Fundulus heteroclitus: protective effects of salinity.

Zinc is an essential trace metal, but many aspects of its toxicity remain unclear. In this study, we investigated zinc effects on oxidative stress parameters and antioxidant profile in four tissues (gill, liver, kidney, and white muscle) of Fundulus heteroclitus. Possible interactive effects of salinity were also studied. Killifish were exposed to sublethal level (500 µgL(-1)) of waterborne zinc for 96 h in 0% (fresh water), 10% (3.5 ppt), 30% (10.5 ppt) and 100% sea water (35 ppt). Salinity per se had no effect on any parameter in the control groups. Zinc exposure clearly induced oxidative stress, and responses were qualitatively similar amongst different tissues. Salinity acted as a strong protective factor, with the highest levels of reactive oxygen species (ROS) and greatest damage (protein carbonyls, lipid peroxidation as indicated by thiobarbituric acid reactive substances (TBARS) in 0 ppt, the least in 100% sea water (35 ppt), and gradations in between in many of the observed responses. Increases in total oxidative scavenging capacity (TOSC) occurred at higher salinities, correlated with increases in the activities of superoxide dismutase (SOD) and glutathione-S-tranferase (GST), as well as in tissue glutathione (GSH) concentrations. However, TOSC was depleted in zinc-exposed fish at 0 ppt, accompanied by decreases in SOD, GST, GSH, and also catalase (CAT) activity. Our results confirm that sublethal waterborne zinc is an oxidative stressor in fish, and highlight the important protective role of higher salinities in ameliorating the oxidative stress associated with zinc toxicity in this model estuarine teleost.