Assessment of early life stage mahi-mahi windows of sensitivity during acute exposures to Deepwater Horizon crude oil.

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.

A novel system for embryo-larval toxicity testing of pelagic fish: Applications for impact assessment of Deepwater Horizon crude oil.

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.

Guanylin peptides regulate electrolyte and fluid transport in the Gulf toadfish (Opsanus beta) posterior intestine.

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.

Acute embryonic or juvenile exposure to Deepwater Horizon crude oil impairs the swimming performance of mahi-mahi (Coryphaena hippurus).

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.

Effects of potassium ion supplementation on survival and ion regulation in Gulf killifish Fundulus grandis larvae reared in ion deficient saline waters.

Teleost fish often live in an environment in which osmoregulatory mechanisms are critical for survival and largely unknown in larval fish. The effects of a single important marine ion (K(+)) on survival and ion regulation of larval Gulf killifish, an estuarine, euryhaline teleost, were determined. A four-week study was completed in four separate recirculating systems with newly hatched larvae. Salinity in all four systems was maintained between 9.5 and 10‰. Two systems were maintained using crystal salt (99.6% NaCl) with K(+) supplementation (1.31±0.04mmol/L and 2.06±0.04mmol/L K(+); mean±SEM), one was maintained with crystal salt and no K(+) supplementation (0.33±0.05mmol/L K(+)), the fourth system was maintained using a standard marine mix salt (2.96±0.04mmol/L K(+)), the salt mix also included standard ranges of other ions such as calcium and magnesium. Larvae were sampled throughout the experiment for dry mass, Na(+)/K(+)-ATPase (NKA) activity, whole body ion composition, relative gene expression (NKA, Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR)), and immunocytochemistry staining for NKA, NKCC, and CFTR. Larvae stocked into water with no K(+) supplementation resulted in 100% mortality within 24h. Mortality and dry mass were significantly influenced by K(+) concentration (P≤0.05). No differences were observed among treatment groups for NKA activity. At 1dph NKA mRNA expression was higher in the 0.3mmol [K(+)] group than in other treatment groups and at 7dph differences in intestinal NKA and CFTR staining were observed. These data indicate that the rearing of larval Gulf killifish may be possible in ion deficient water utilizing specific ion supplementation.

Osmoregulatory response to low salinities in the European sea bass embryos: a multi-site approach.

Embryonic osmoregulation effected by embryonic ionocytes in the European sea bass Dicentrarchus labrax has been studied at several sites, including the yolk sac membrane, the first gill slits and the gut ionocytes. D. labrax embryos, spawned in seawater (SW) (39 ‰), were exposed to dilute seawater (DSW) (5 ‰) during 48 h, from stage 10 pairs of somites (10S) to hatching time (HT). Control embryos originating from the same spawn were maintained in SW. Both SW and DSW embryos were examined after 24- and 48-h exposure. Nanoosmometric measurements of the embryonic fluids osmolality suggest that late embryos are confronted with the variations in external salinity and that they were able to slightly regulate their osmolality. Immunolocalization of Na⁺/K⁺ ATPase, NKCC and CFTR has shown that DSW-exposed embryos can limit ion losses due to compensatory physiological mechanisms. CFTR and NKCC were not observed in DSW embryos in the yolk sac ionocytes and in the tegumentary ionocytes of the gill slits. The quantification of mRNA indicated that NKA, NKCC1 and CFTR transcript levels increased from stage 10S to stage HT. At stage HT, following 48 h of DSW- or SW-exposure, different responses were observed according to salinity. These results, when compared to those obtained in D. labrax juveniles and adults long-term exposed to fresh water (FW), show that in embryos the physiological response following a short-term DSW exposure is different. The mechanisms of hyper-osmoregulation observed in D. labrax embryos, although not fully efficient, allow their survival for several days in DSW.

Effects of low salinity media on growth, condition, and gill ion transporter expression in juvenile Gulf killifish, Fundulus grandis.

The Gulf killifish, Fundulus grandis, is a euryhaline teleost which has important ecological roles in the brackish-water marshes of its native range as well as commercial value as live bait for saltwater anglers. Effects of osmoregulation on growth, survival, and body condition at 0.5, 5.0, 8.0 and 12.0‰ salinity were studied in F. grandis juveniles during a 12-week trial. Relative expression of genes encoding the ion transport proteins Na(+)/K(+)-ATPase (NKA), Na(+)/K(+)/2Cl(-) cotransporter(NKCC1), and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel was analyzed. At 0.5‰, F. grandis showed depressed growth, body condition, and survival relative to higher salinities. NKA relative expression was elevated at 7 days post-transfer but decreased at later time points in fish held at 0.5‰ while other salinities produced no such increase. NKCC1, the isoform associated with expulsion of ions in saltwater, was downregulated from week 1 to week 3 at 0.5‰ while CFTR relative expression produced no significant results across time or salinity. Our results suggest that Gulf killifish have physiological difficulties with osmoregulation at a salinity of 0.5‰ and that this leads to reduced growth performance and survival while salinities in the 5.0-12.0‰ are adequate for normal function.

Genomic and physiological footprint of the Deepwater Horizon oil spill on resident marsh fishes.

The biological consequences of the Deepwater Horizon oil spill are unknown, especially for resident organisms. Here, we report results from a field study tracking the effects of contaminating oil across space and time in resident killifish during the first 4 mo of the spill event. Remote sensing and analytical chemistry identified exposures, which were linked to effects in fish characterized by genome expression and associated gill immunohistochemistry, despite very low concentrations of hydrocarbons remaining in water and tissues. Divergence in genome expression coincides with contaminating oil and is consistent with genome responses that are predictive of exposure to hydrocarbon-like chemicals and indicative of physiological and reproductive impairment. Oil-contaminated waters are also associated with aberrant protein expression in gill tissues of larval and adult fish. These data suggest that heavily weathered crude oil from the spill imparts significant biological impacts in sensitive Louisiana marshes, some of which remain for over 2 mo following initial exposures.

Ontogeny of osmoregulation and salinity tolerance in the gilthead sea bream Sparus aurata.

The gilthead sea bream, Sparus aurata, is a euryhaline teleost that hatches in the open sea. The larvae drift to the coast and juveniles migrate into estuaries and lagoons where the salinity of the water may vary from brackish to hyper-saline. The ontogeny of osmoregulation in Sparus aurata was studied at successive stages, from day 1 (D1) post-hatch to the late juvenile stage (D300) after exposure to different salinities ranging from fresh water to 45.1 per thousand, at 18 degrees C. Survival ranged from between 5.1 and 39.1 per thousand at D3, and from 1.0 to 45.1 per thousand from D75. The fish were hyper-hypo-osmotic regulators at all studied stages. The acquisition of the full ability to hypo- and hyper-regulate occurred in four steps. The osmoregulatory capacity appeared age-dependent and reached its maximum level after D96, and the localization of ionocytes in the integument and gills occurred concurrently during development of the sea bream. However, the main site of osmoregulation shifted from the integument to the gills from D30 to D70, with a corresponding sharp increase in the osmoregulatory ability. Our results suggest that the early development of osmoregulatory ability, and thus of salinity tolerance in the sea bream may provide an advantageous flexibility for the timing of the migration between sea and estuaries and lagoons.

Influence of salinity on the localization and expression of the CFTR chloride channel in the ionocytes of juvenile Dicentrarchus labrax exposed to seawater and freshwater.

The European sea-bass, Dicentrarchus labrax is a euryhaline teleost whose high osmoregulatory abilities allow sea-lagoon migrations. In order to investigate the mechanism underlying the acclimation of juvenile fish to salinity, CFTR was studied in long-term (6 months) freshwater (FW)- and seawater (SW)-exposed fish, and in short-term (from day 0 to day 30) FW-exposed fish. Cellular and molecular approaches were combined to determine the functions of CFTR in the gills, posterior intestine and kidney. In the kidney, the expression of CFTR transcripts and protein is low. After a direct transfer from SW to FW, the CFTR mRNA is down-regulated in the gills within 1 day, followed by a protein decrease over 7 days. In the posterior intestine, first there is a protein decrease within one day and secondly at the mRNA level in 14 days. While in the gills the regulation is transcriptional, in the posterior intestine, there is first a post-transcriptional regulation followed by a transcriptional regulation after 14 days in FW. Over a long-term exposure, there is a transcriptional regulation in both organs. Coupled to other ion transports, CFTR contributes to ion regulation and thus to the adaptation of the European sea-bass to sea-lagoon transitions.

Influence of salinity on the localization and expression of the CFTR chloride channel in the ionocytes of Dicentrarchus labrax during ontogeny.

The expression and localization of the cystic fibrosis transmembrane conductance regulator (CFTR) were determined in four osmoregulatory tissues during the ontogeny of the sea-bass Dicentrarchus labrax acclimated to fresh water and sea water. At hatch in sea water, immunolocalization showed an apical CFTR in the digestive tract and integumental ionocytes. During the ontogeny, although CFTR was consistently detected in the digestive tract, it shifted from the integument to the gills. In fresh water, CFTR was not present in the integument and the gills, suggesting the absence of chloride secretion. In the kidney, the CFTR expression was brief from D4 to D35, prior to the larva-juvenile transition. CFTR was apical in the renal tubules, suggesting a chloride secretion at both salinities, and it was basolateral only in sea water in the collecting ducts, suggesting chloride absorption. In the posterior intestine, CFTR was located differently from D4 depending on salinity. In sea water, the basolateral CFTR may facilitate ionic absorption, perhaps in relation to water uptake. In fresh water, CFTR was apical in the gut, suggesting chloride secretion. Increased osmoregulatory ability was acquired just before metamorphosis, which is followed by the sea-lagoon migration.

The Na+/K+/2Cl- cotransporter in the sea bass Dicentrarchus labrax during ontogeny: involvement in osmoregulation.

This study combines a cellular and molecular analysis of the Na(+)/K(+)/2Cl(-) cotransporter (NKCC) to determine the osmoregulatory role of this protein in different tissues during the ontogeny of the sea bass. We have characterized the complete sequence of the NKCC1 isoform isolated from the sea bass gills and have identified, by immunofluorescence, NKCC1, and other isoforms, within the epithelium of the major osmoregulatory organs. Different (absorptive and secretory) functions have been attributed to this protein according to the tissue and salinity. The effects of short- (1-4 days), medium- (7-21 days) and long (6 months)-term freshwater (FW) adaptations were investigated, in comparison with seawater (SW)-maintained sea bass. In adult sea bass after long-term adaptation to FW and SW, the gills had the highest expression of NKCC mRNA compared with the median/posterior kidney and to the posterior intestine. Expression of NKCC mRNA in the kidney was 95% (SW) and 63% (FW) lower, and in the intestine 98% (SW) and 77% (FW) lower. Compared to SW-maintained sea bass, long-term FW adaptation induced a significant 5.6-fold decrease in the branchial NKCC gene expression whereas the intestinal and renal expressions did not vary significantly. The cells of the intestine and collecting ducts as well as a part of the epithelium lining the urinary bladder expressed NKCC apically. Within the gill chloride cells, NKCC was found basolaterally in SW-acclimated fish; some apically stained cells were detected after 7 days of FW exposure and their relative number increased progressively following FW acclimation. The appearance of FW-type chloride cells induces a functional shift of the gills from a secretory to an absorptive epithelium, which was only completed after long-term exposure to FW. Short- and medium-term exposure to FW induced a progressive decrease in total NKCC content and an increase in functionally different branchial chloride cells. During development, the cotransporter was already expressed in tegumentary ionocytes and along the digestive tract of late embryos. NKCC was recorded in the branchial chamber and along the renal collecting ducts in prelarvae and also in the dorsal part of the urinary bladder in larvae. The expression of NKCC along the osmoregulatory epithelial cells and the presence of Na(+)/K(+)-ATPase within these cells contribute to the increase of the osmoregulatory capacity during sea bass ontogeny.