Role of the cardiovascular system in ammonia excretion in early life stages of zebrafish (Danio rerio).

The purpose of this study was to investigate if the cardiovascular system is important for ammonia excretion in the early life stages of zebrafish. Morpholino knockdowns of cardiac troponin T (TNNT2) or vascular endothelial growth factor A (VEGFA) provided morphants with nonfunctional circulation. At the embryonic stage [30-36 h postfertilization (hpf)], ammonia excretion was not constrained by a lack of cardiovascular function. At 2 days postfertilization (dpf) and 4 dpf, morpholino knockdowns of TNNT2 or VEGFA significantly reduced ammonia excretion in all morphants. Expression of rhag, rhbg, and rhcgb showed no significant changes but the mRNA levels of the urea transporter (ut) were upregulated in the 4 dpf morphants. Taken together, rhag, rhbg, rhcgb, and ut gene expression and an unchanged tissue ammonia concentration but an increased tissue urea concentration, suggest that impaired ammonia excretion led to increased urea synthesis. However, in larvae anesthetized with tricaine or clove oil, ammonia excretion was not reduced in the 4 dpf morphants compared with controls. Furthermore, oxygen consumption was reduced in morphants regardless of anesthesia. These results suggest that cardiovascular function is not directly involved in ammonia excretion, but rather reduced activity and external convection may explain reduced ammonia excretion and compensatory urea accumulation in morphants with reduced cardiovascular function.

Tissue Accumulation and the Effects of Long-Term Dietary Copper Contamination on Osmoregulation in the Mudflat Fiddler Crab Minuca rapax (Crustacea, Ocypodidae).

We examined copper accumulation in the hemolymph, gills and hepatopancreas, and hemolymph osmolality, Na+ and Cl- concentrations, together with gill Na+/K+-ATPase and carbonic anhydrase activities, after dietary copper delivery (0, 100 or 500 Cu µg g-1) for 12 days in a fiddler crab, Minuca rapax. In contaminated crabs, copper concentration decreased in the hemolymph and hepatopancreas, but increased in the gills. Hemolymph osmolality and gill Na+/K+-ATPase activity increased while hemolymph [Na+] and [Cl-] and gill carbonic anhydrase activity decreased. Excretion likely accounts for the decreased hemolymph and hepatopancreas copper titers. Dietary copper clearly affected osmoregulatory ability and hemolymph Na+ and Cl- regulation in M. rapax. Gill copper accumulation decreased carbonic anhydrase activity, suggesting that dietary copper affects acid-base balance. Elevated gill Na+/K+-ATPase activity appears to compensate for the ion-regulatory disturbance. These effects of dietary copper illustrate likely impacts on semi-terrestrial species that feed on metal-contaminated sediments.

Ontogeny of urea and ammonia transporters in mahi-mahi (Coryphaena hippurus) early life stages.

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.

Behavioural salinity preference of juvenile yellow perch Perca flavescens.

The present study determined the behavioural salinity preference of a freshwater stock of juvenile yellow perch Perca flavescens acclimated to salinities of 0 and 10. The preferred salinities ranged between 7·3 and 13·0 (mean ± s.d. = 10·4 ± 1·7; n = 13) with no significant effect of acclimation salinity. The results showed that juvenile P. flavescens prefers near isoosmotic salinities, which could be due to a lowered energetic cost of osmoregulation.

Renal plasticity in response to feeding in the Burmese python, Python molurus bivittatus.

Burmese pythons are sit-and-wait predators that are well adapted to go long periods without food, yet subsequently consume and digest single meals that can exceed their body weight. These large feeding events result in a dramatic alkaline tide that is compensated by a hypoventilatory response that normalizes plasma pH; however, little is known regarding how plasma HCO3(-) is lowered in the days post-feeding. The current study demonstrated that Burmese pythons contain the cellular machinery for renal acid-base compensation and actively remodel the kidney to limit HCO3(-) reabsorption in the post-feeding period. After being fed a 25% body weight meal plasma total CO2 was elevated by 1.5-fold after 1 day, but returned to control concentrations by 4 days post-feeding (d pf). Gene expression analysis was used to verify the presence of carbonic anhydrase (CA) II, IV and XIII, Na(+) H(+) exchanger 3 (NHE3), the Na(+) HCO3(-) co-transporter (NBC) and V-type ATPase. CA IV expression was significantly down-regulated at 3 dpf versus fasted controls. This was supported by activity analysis that showed a significant decrease in the amount of GPI-linked CA activity in isolated kidney membranes at 3 dpf versus fasted controls. In addition, V-type ATPase activity was significantly up-regulated at 3 dpf; no change in gene expression was observed. Both CA II and NHE3 expression was up-regulated at 3 dpf, which may be related to post-prandial ion balance. These results suggest that Burmese pythons actively remodel their kidney after feeding, which would in part benefit renal HCO3(-) clearance.

The effects of sustained aerobic swimming on osmoregulatory pathways in Atlantic salmon Salmo salar smolts.

Atlantic salmon Salmo salar smolts were exposed to one of the four different aerobic exercise regimens for 10 weeks followed by a 1 week final smoltification period in fresh water and a subsequent eight-day seawater transfer period. Samples of gill and intestinal tissue were taken at each time point and gene expression was used to assess the effects of exercise training on both branchial and intestinal osmoregulatory pathways. Real-time polymerase chain reaction (PCR) analysis revealed that exercise training up-regulated the expression of seawater relevant genes in the gills of S. salar smolts, including Na(+) , K(+) ATPase (nka) subunit α1b, the Na(+) , K(+) , 2 Cl(-) co-transporter (nkcc1) and cftr channel. These findings suggest that aerobic exercise stimulates expression of seawater ion transport pathways that may act to shift the seawater transfer window for S. salar smolts. Aerobic exercise also appeared to stimulate freshwater ion uptake mechanisms probably associated with an osmorespiratory compromise related to increased exercise. No differences were observed in plasma Na(+) and Cl(-) concentrations as a consequence of exercise treatment, but plasma Na(+) was lower during the final smoltification period in all treatments. No effects of exercise were observed for intestinal nkcc2, nor the Mg(2+) transporters slc41a2 and transient receptor protein M7 (trpm7); however, expression of both Mg(2+) transporters was affected by salinity transfer suggesting a dynamic role in Mg(2+) homeostasis in fishes.

Evaluation of pre- and post-zygotic mating barriers, hybrid fitness and phylogenetic relationship between Cyprinodon variegatus variegatus and Cyprinodon variegatus hubbsi (Cyprinodontiformes, Teleostei).

The euryhaline fish Cyprinodon variegatus variegatus (Cvv) is capable of tolerating ambient salinities ranging from 0.3 to 167 g l(-1) , but incapable of long-term survival in freshwater (< 2 mM Na(+) ). However, a population of this species, now designated as a subspecies (Cyprinodon variegatus hubbsi; Cvh), has been isolated in several freshwater (0.4-1 mM Na(+) ) lakes in central Florida for the past ~150 ky. We previously demonstrated that Cvh has a significantly higher affinity for Na(+) uptake suggesting that it has adapted to its dilute freshwater environment. We here evaluate whether Cvh should be considered a separate species by characterizing pre- and post-zygotic isolation, Na(+) transport characteristics of the two populations and their hybrids, and developing a molecular phylogeny of Cvv and Cvh populations in Florida using mtDNA sequence data. We found evidence of partial prezygotic isolation with Cvv females mating almost exclusively (89%) with con-specific males in choice mating experiments. Partial post-zygotic isolation was also observed with significant (59-89%) reductions in hatching success of hybrid embryos compared with con-specific embryos. Na(+) uptake kinetics in hybrids (both Cvv x Cvh and Cvh x Cvv) bred and raised under common garden conditions were intermediate to Cvh (high affinity) and Cvv (low affinity) indicating that observed differences are genetically based. Similar observations were made with respect to short-term (96 h) survival of juveniles acutely transferred from 7 mM Na(+) to a range of more dilute (0.1-2 mM Na(+) ) freshwater. Finally, although phylogenetic analysis of Cvv and Cvh populations using mtDNA sequence for ND2 were unable to fully resolve a polytomy between Cvh and Cvv populations from northeastern Florida, these data do not falsify the hypothesis that Cvh is of monophyletic origin. Overall, the available data suggest that Cvh should be considered a separate species or at a minimum an evolutionarily significant unit.

Metabolic cost of osmoregulation by the gastro-intestinal tract in marine teleost fish.

Introduction: Although dozens of studies have attempted to determine the metabolic cost of osmoregulation, mainly by comparing standard metabolic rates (SMR) in fish acclimated to different salinities, consensus is still lacking. Methods: In the present study, using the Gulf toadfish, Opsanus beta, we aimed to determine the metabolic cost of esophageal and intestinal osmoregulatory processes by estimating ATP consumption from known ion transport rates and pathways and comparing these estimates with measurements on isolated tissues. Further, we performed whole animal respirometry on fish acclimated to 9, 34 and 60 ppt. Results and Discussion: Our theoretical estimates of esophageal and intestinal osmoregulatory costs were in close agreement with direct measurements on isolated tissues and suggest that osmoregulation by these tissues amounts to ∼2.5% of SMR. This value agrees well with an earlier attempt to estimate osmoregulation cost from ion transport rates and combined with published measurements of gill osmoregulatory costs suggests that whole animal costs of osmoregulation in marine teleosts is ∼7.5% of SMR. As in many previous studies, our whole animal measurements were variable between fish and did not seem suited to determine osmoregulatory costs. While the esophagus showed constant metabolic rate regardless of acclimation salinity, the intestine of fish acclimated to higher salinities showed elevated metabolic rates. The esophagus and the intestine had 2.1 and 3.2-fold higher metabolic rates than corresponding whole animal mass specific rates, respectively. The intestinal tissue displays at least four different Cl- uptake pathways of which the Na+:Cl-:2 K+ (NKCC) pathway accounts for 95% of the Cl- uptake and is the most energy efficient. The remaining pathways are via apical anion exchange and seem to primarily serve luminal alkalinization and the formation of intestinal CaCO3 which is essential for water absorption.

Regulation of apical H⁺-ATPase activity and intestinal HCO3⁻ secretion in marine fish osmoregulation.

The absorption of Cl(-) and water from ingested seawater in the marine fish intestine is accomplished partly through Cl(-)/HCO(3)(-) exchange. Recently, a H(+) pump (vacuolar-type H(+)-ATPase) was found to secrete acid into the intestinal lumen, and it may serve to titrate luminal HCO(3)(-) and facilitate further Cl(-)/HCO(3)(-) exchange, especially in the posterior intestine, where adverse concentration gradients could limit Cl(-)/HCO(3)(-) exchange. The H(+) pump is expressed in all intestinal segments and in gill tissue of gulf toadfish (Opsanus beta) maintained in natural seawater. After acute transfer of toadfish to 60 ppt salinity, H(+) pump expression increased 20-fold in the posterior intestine. In agreement with these observations was a fourfold-increased H(+)-ATPase activity in the posterior intestine of animals acclimated to 60 ppt salinity. Interestingly, Na(+)-K(+)-ATPase activity was elevated in the anterior intestine and gill, but not in the posterior intestine. Apical acid secretion by isolated intestinal tissue mounted in Ussing chambers fitted with pH-stat titration systems increased after acclimation to hypersalinity in the anterior and posterior intestine, titrating >20% of secreted bicarbonate. In addition, net base secretion increased in hypersalinity-acclimated fish and was ∼70% dependent on serosal HCO(3)(-). Protein localization by immunohistochemistry confirmed the presence of the vacuolar-type H(+)-ATPase in the apical region of intestinal enterocytes. These results show that the H(+) pump, especially in the posterior intestine, plays an important role in hypersaline osmoregulation and that it likely has significant effects on HCO(3)(-) accumulation in the intestinal lumen and, therefore, the continued absorption of Cl(-) and water.

Basolateral NBCe1 plays a rate-limiting role in transepithelial intestinal HCO3- secretion, contributing to marine fish osmoregulation.

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.

Cytosolic carbonic anhydrase in the Gulf toadfish is important for tolerance to hypersalinity.

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.

Salinity selection and preference of the grey snapper Lutjanus griseus: field and laboratory observations.

Field observations were supplemented with laboratory experiments to reveal patterns of salinity selection and preference for grey snapper Lutjanus griseus (c. 21 cm total length, L(T)), an ecologically and economically important species in the south-eastern U.S.A. Fish abundance data were examined from a long-term field survey conducted in the mangrove habitats of Biscayne Bay, Florida, where salinities ranged from <1 to 40. First, regression analyses indicated significant, positive linear relationships with salinity for both L. griseus frequency of occurrence and concentration (density when present). These patterns are inconsistent with physiological expectations of minimizing energetic osmoregulatory costs. Next, the salinity preference and swimming activity of 11 L. griseus (ranging from 18 to 23 cm L(T)) were investigated using a newly developed electronic shuttlebox system. In the laboratory, fish preferred intermediate salinities in the range of 9-23. Swimming activity (measured in terms of spontaneous swimming speed) followed a parabolic relationship with salinity, with reduced activity at salinity extremes perhaps reflecting compensation for higher osmoregulatory costs. It is suspected that the basis of the discrepancy between laboratory and field observations for size classes at or near maturity ultimately relates to the reproductive imperative to move towards offshore (high-salinity) coral-reef habitats, a necessity that probably overrides the strategy of minimizing osmoregulatory energetic costs.

Evidence that SLC26 anion transporters mediate branchial chloride uptake in adult zebrafish (Danio rerio).

Experiments were performed to test the hypothesis that three members of the SLC26 anion transporter gene family (SLC26a3, A4, and A6; hereafter termed za3, za4, and za6) mediate branchial Cl(-)/HCO(3)(-) exchange in adult zebrafish (Danio rerio). Real-time RT-PCR demonstrated that the gill expressed relatively high levels of za6 mRNA; za3 and za4 mRNA, while present, were less abundant. Also, za4 and za6 were expressed at relatively high levels in the kidney. The results of in situ hybridization or immunocytochemistry (za3 only) experiments performed on gill sections revealed that the SLC26 transporters were predominantly expressed on the filament epithelium (especially within the interlamellar regions) and to a lesser extent on the lamellar epithelium at the base of lamellae. This distribution pattern suggests that the SLC26 anion transporters are localized to mitochondrion-rich cells (ionocytes). Transferring fish to water containing low [Cl(-)] (0.02 mmol/l) resulted in significant increases in branchial SLC26 mRNA expression after 5-10 days of exposure relative to fish raised in normal water [Cl(-)] (0.4 mmol/l); transferring fish to Cl(-)-enriched water (2.0 mmol/l) was without effect on mRNA levels. Transferring fish to water containing elevated levels of NaHCO(3) (10-12.5 mmol/l) caused marked increases in branchial SLC26 mRNA expression between 3 and 10 days of transfer that was associated with a significant 40% increase in Cl(-) uptake (as measured upon return to normal water after 7 days). A decrease in whole body net acid excretion (equivalent to an increase in net base excretion) in fish previously maintained in high [NaHCO(3)] water, concurrent with increases in Cl(-) uptake and SLC26 mRNA levels, suggests a role for these anion transporters in Cl(-) uptake and acid-base regulation owing to their Cl(-)/HCO(3)(-) exchange activities.

The intestinal response to feeding in seawater gulf toadfish, Opsanus beta, includes elevated base secretion and increased epithelial oxygen consumption.

Intestinal HCO3- secretion is essential to marine teleost fish osmoregulation and comprises a considerable source of base efflux attributable to both serosal HCO3- and endogenous CO2 hydration. The role of intestinal HCO3- secretion in dynamic acid-base balance regulation appears negligible in studies of unfed fish, but evidence of high intestinal fluid [HCO3-] in fed marine teleosts led us to investigate the source of this HCO3- and its potential role in offsetting the postprandial 'alkaline tide' commonly associated with digestion. Specifically, we hypothesized that elevated metabolic rate and thus endogenous CO2 production by intestinal tissue as well as increased transepithelial intestinal HCO3- secretion occur post-feeding and offset a postprandial alkaline tide. To test these hypotheses changes in HCO3- secretion and O2 consumption by gulf toadfish (Opsanus beta) isolated intestine were quantified 0, 3, 6, 12, 24 and 48 h post-feeding. Intestinal tissue of unfed fish in general showed high rates of HCO3- secretion (15.5 mumol g(-1) h(-1)) and O2 consumption (8.9 mumol g(-1) h(-1)). Furthermore, postprandial increases in both intestinal HCO3- secretion and O2 consumption (1.6- and 1.9-fold peak increases, respectively) were observed. Elevated intestinal HCO3- secretion rates preceded and outlasted those of O2 consumption, and occurred at a magnitude and duration sufficient to account for the lack of alkaline tide. The dependence of these high rates of postprandial intestinal base secretion on serosal HCO3- indicates transepithelial HCO3- transport increases disproportionately more than endogenous CO2 production. The magnitude of postprandial intestinal HCO(3)(-) secretion indicates the intestine certainly is capable of postprandial acid-base balance regulation.

The involvement of SLC26 anion transporters in chloride uptake in zebrafish (Danio rerio) larvae.

After demonstrating phylogenetic relatedness to orthologous mammalian genes, tools were developed to investigate the roles of three members (A3, A4 and A6c) of the SLC26 anion exchange gene family in Cl- uptake and HCO3 excretion in embryos and larvae of zebrafish (Danio rerio). Whole-mount in situ hybridization revealed the presence of SLC26 mRNA in gill primordia, mesonephros and heart (slc26a3 and a4 only) at 5-9 days postfertilization (d.p.f.). SLC26A3 protein was highly expressed in lateral line neuromasts and within the gill, was localized to a sub-population of epithelial cells, which often (but not always) coexpressed Na+/K+-ATPase. SLC26 mRNA levels increased with developmental age, peaking at 5-10 d.p.f.; the largest increases in rates of Cl- uptake (JinCl-) preceded the mRNA spike, occurring at 2-5 d.p.f. Raising zebrafish in water with a low [Cl-] caused marked increases in JinCl- at 3-10 d.p.f. and was associated with increased levels of SLC26 mRNA. Raising fish in water of high [Cl-] was without effect on JinCl- or SLC26 transcript abundance. Selective gene knockdown using morpholino antisense oligonucleotides demonstrated a significant role for SLC26A3 in Cl- uptake in larval fish raised in control water and roles for A3, A4 and A6c in fish raised in water with low [Cl-]. Prolonged (7 days) or acute (24 h) exposure of fish to elevated (2 or 5 mmol l(-1)) ambient [HCO3-] caused marked increases in Cl- uptake when determined in water of normal [HCO3-] that were accompanied by elevated levels of SLC26 mRNA. The increases in JinCl- associated with high ambient [HCO3-] were not observed in the SLC26 morphants (significant only at 5 mmol l(-1) HCO3- for A4 and 2 mmol l(-1) HCO3- for A6c). Net base excretion was markedly inhibited in the slc26a3 and a6c morphants thereby implicating these genes in Cl-/HCO3- exchange. The results suggest that under normal conditions, Cl- uptake in zebrafish larvae is mediated by SLC26A3 Cl-/HCO3- exchangers but under conditions necessitating higher rates of high affinity Cl- uptake, SlC26A4 and SLC26A6c may assume a greater role.

The effects of Deepwater Horizon crude oil on ammonia and urea handling in mahi-mahi (Coryphaena hippurus) early life stages.

Many ecologically important fishes, including mahi-mahi (Coryphaena hippurus), and their offspring were directly exposed to crude oil following the Deepwater Horizon (DWH) oil spill. Early life stage fish are especially vulnerable to the toxicity of crude oil-derived polycyclic aromatic hydrocarbons (PAHs). In teleosts, yolk sac proteins are the main energy source during development and are usually catabolized into ammonia or urea among other byproducts. Although excretion of these waste products is sensitive to oil exposure, we know little about the underlying mechanisms of this process. In this study, we examined the effects of crude oil on ammonia and urea handling in the early life stages of mahi. Mahi embryos exposed to 30-32 µg L-1 ∑PAH exhibited increased urea excretion rates and greater accumulation of urea in the tissues before hatch suggesting that ammonia, which is highly toxic, was converted into less-toxic urea. Oil-exposed embryos (6.3-32 µg L-1 ∑PAH) displayed significantly increased tissue ammonia levels at 42 hpf and upregulated mRNA levels of ammonia transporters (Rhag, Rhbg and Rhcg1) from 30 to 54 hpf. However, despite increased accumulation and higher expression of ammonia transporters, the larvae exposed to higher ∑PAH (30 µg L-1 ∑PAH) showed reduced ammonia excretion rates after hatch. Together, the increased production of nitrogenous waste reinforces previous work that increased energy demand in oil-exposed embryos is fueled, at least in part, by protein metabolism and that urea synthesis plays a role in ammonia detoxification in oil-exposed mahi embryos. To our knowledge, this study is the first to combine physiological and molecular approaches to assess the impact of crude-oil on both nitrogenous waste excretion and accumulation in the early life stages of any teleosts.

Is Cl- protection against silver toxicity due to chemical speciation?

In freshwater teleosts, the primary mechanism of acute silver toxicity is inhibition of Na(+)/K(+) ATPase and carbonic anhydrase at the gill, leading to net Na(+) and Cl(-) loss due to the continued diffusion of these ions into the hypoosmotic external environment. External Cl(-) has been shown to protect rainbow trout (Oncorhychus mykiss) against silver toxicity presumably by complexation to form AgCl. However, Cl(-) does not appear to greatly influence silver toxicity to at least two other species, the European eel (Anguilla Anguilla) and the fathead minnow (Pimephales promelas). We hypothesized that differences in protective effects of Cl(-) at the gill were due to differing requirements or mechanisms for Cl(-) uptake among fish species. To test this hypothesis, we exposed Fundulus heteroclitus, which does not take up Cl(-) across the gills, and Danio rerio and P. promelas, which do rely on Cl(-) uptake across the gills, to Ag(+) in waters of varying Cl(-) concentration. The 96-h LC50s of F. heteroclitus exposed to Ag(+) in soft water with 10 microM Cl(-), 1mM KCl, and 0.5mM MgCl(2) were 3.88, 1.20, and 3.20 microg/L, respectively, and not significantly different. The 96-h LC50s for D. rerio exposed to Ag(+) in soft water with 10 microM Cl(-) and 1mM KCl were 10.3 and 11.3 microg/L, respectively and P. promelas exposed under the same conditions were 2.32 and 2.67 microg/L, respectively. Based on these results, increasing external Cl(-) concentration by as much as 1mM (35.5mg/L) did not offer protection against Ag(+) toxicity to any fish species tested. Although previous results in our laboratory have demonstrated that P. promelas do take up Cl(-) at the gill, a mechanism of uptake has not been identified. Additional experiments, investigating the mechanisms of Na(+) and Cl(-) influx at the gill of P. promelas and the influence of silver, demonstrated that Cl(-) uptake in P. promelas acclimated to soft water occurs through both a Na(+):K(+):2Cl(-) co-transporter and a Cl(-)/HCO(3)(-) exchanger, but is not dependent on carbonic anhydrase. Further, acclimation water chemistry was found to greatly influence subsequent branchial silver accumulation, but Cl(-) uptake was not sensitive to 10 microg/L Ag(+).

Physiology is pivotal for interactions between salinity and acute copper toxicity to fish and invertebrates.

The present paper presents original data and a review of the copper (Cu) toxicity literature for estuarine and marine environments. For the first time, acute Cu toxicity across the full salinity range was determined. Killifish, Fundulus heteroclitus, eggs were hatched in freshwater (FW), 2.5, 5, 10, 15, 22 and 35 ppt (seawater, SW) and juveniles were allowed to acclimate for 7 days prior to acute toxicity testing. Sensitivity was highest in FW (96 h LC50: 18 microg/l), followed by SW (96 h LC50: 294 microg/l) with fish at intermediate salinities being the most tolerant (96 h LC50 > 963 microg/l at 10 ppt). This approximately 50-fold, non-linear variation in sensitivity could not be accounted for by Cu speciation or competition among cations but can be explained by physiology. The relative Na(+) gradient from the blood plasma to the water is greatest in FW followed by SW and is smallest at 10 ppt. Regression of Cu toxicity versus the equilibrium potential for Na(+), which reflects the relative Na(+) gradient, revealed that 93% of the variation can be attributed to Na(+) gradients and thus osmoregulatory physiology. Examination of the existing literature on acute Cu toxicity in SW (defined as >25 ppt) confirmed that early life stages generally are most sensitive but this pattern may be attributable to size rather than developmental stage. Regardless of developmental stage and phylogeny, size clearly matters for Cu sensitivity. The existing literature on the influence of salinity on acute Cu toxicity as well as studies of mechanisms of Cu toxicity in fish and invertebrates are reviewed.

Altered brain ion gradients following compensation for elevated CO2 are linked to behavioural alterations in a coral reef fish.

Neurosensory and behavioural disruptions are some of the most consistently reported responses upon exposure to ocean acidification-relevant CO2 levels, especially in coral reef fishes. The underlying cause of these disruptions is thought to be altered current across the GABAA receptor in neuronal cells due to changes in ion gradients (HCO3(-) and/or Cl(-)) that occur in the body following compensation for elevated ambient CO2. Despite these widely-documented behavioural disruptions, the present study is the first to pair a behavioural assay with measurements of relevant intracellular and extracellular acid-base parameters in a coral reef fish exposed to elevated CO2. Spiny damselfish (Acanthochromis polyacanthus) exposed to 1900 µatm CO2 for 4 days exhibited significantly increased intracellular and extracellular HCO3(-) concentrations and elevated brain pHi compared to control fish, providing evidence of CO2 compensation. As expected, high CO2 exposed damselfish spent significantly more time in a chemical alarm cue (CAC) than control fish, supporting a potential link between behavioural disruption and CO2 compensation. Using HCO3(-) measurements from the damselfish, the reversal potential for GABAA (EGABA) was calculated, illustrating that biophysical properties of the brain during CO2 compensation could change GABAA receptor function and account for the behavioural disturbances noted during exposure to elevated CO2.

Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio).

While the zebrafish is commonly used for studies of developmental biology and toxicology, very little is known about their osmoregulatory physiology. The present investigation of Na(+) and Cl(-) transport revealed that the zebrafish is able to tolerate extremely low ambient ion concentrations and that this is achieved at least in part by a greatly enhanced apparent uptake capacity and affinity for both ions. Zebrafish maintain plasma and whole body electrolyte concentrations similar to most other freshwater teleosts even in deionized water containing only 35 microM NaCl, i.e soft water. We recorded an extremely low transport affinity constant (K(m)) of 8+/-1 microM for the active uptake of Cl(-) in soft water acclimated fish, while other transport kinetic parameters were in agreement with reports for other freshwater organisms. While both Na(+) and Cl(-) uptake in soft water clearly depends on apical proton pump activity, changes in abundance and possibly localization of this protein did not appear to contribute to soft water acclimation. Active Cl(-) uptake was strongly dependent on branchial carbonic anhydrase (CA) activity regardless of water type, while the response of Na(+) transport to a CA inhibitor was more variable. Differential response of Na(+) uptake to amiloride depending on acclimation medium suggests that different Na(+) transport mechanisms are employed by zebrafish acclimated to soft and hard water.