Andrew J. Esbaugh

Deepwater Horizon crude oil impacts the developing hearts of large predatory pelagic fish

Significance The 2010 Deepwater Horizon (MC252) disaster in the northern Gulf of Mexico released more than 4 million barrels of crude oil. Oil rose from the ocean floor to the surface where many large pelagic fish spawn. Here we describe the impacts of field-collected oil samples on the rapidly developing embryos of warm-water predators, including bluefin and yellowfin tunas and an amberjack. For each species, environmentally relevant MC252 oil exposures caused serious defects in heart development. Moreover, abnormalities in cardiac function were highly consistent, indicating a broadly conserved developmental crude oil cardiotoxicity. Losses of early life stages were therefore likely for Gulf populations of tunas, amberjack, swordfish, billfish, and other large predators that spawned in oiled surface habitats. The Deepwater Horizon disaster released more than 636 million L of crude oil into the northern Gulf of Mexico. The spill oiled upper surface water spawning habitats for many commercially and ecologically important pelagic fish species. Consequently, the developing spawn (embryos and larvae) of tunas, swordfish, and other large predators were potentially exposed to crude oil-derived polycyclic aromatic hydrocarbons (PAHs). Fish embryos are generally very sensitive to PAH-induced cardiotoxicity, and adverse changes in heart physiology and morphology can cause both acute and delayed mortality. Cardiac function is particularly important for fast-swimming pelagic predators with high aerobic demand. Offspring for these species develop rapidly at relatively high temperatures, and their vulnerability to crude oil toxicity is unknown. We assessed the impacts of field-collected Deepwater Horizon (MC252) oil samples on embryos of three pelagic fish: bluefin tuna, yellowfin tuna, and an amberjack. We show that environmentally realistic exposures (1–15 µg/L total PAH) cause specific dose-dependent defects in cardiac function in all three species, with circulatory disruption culminating in pericardial edema and other secondary malformations. Each species displayed an irregular atrial arrhythmia following oil exposure, indicating a highly conserved response to oil toxicity. A considerable portion of Gulf water samples collected during the spill had PAH concentrations exceeding toxicity thresholds observed here, indicating the potential for losses of pelagic fish larvae. Vulnerability assessments in other ocean habitats, including the Arctic, should focus on the developing heart of resident fish species as an exceptionally sensitive and consistent indicator of crude oil impacts.

The structure and function of carbonic anhydrase isozymes in the respiratory system of vertebrates

Carbonic anhydrase is a ubiquitous metalloenzyme that catalyzes the reversible hydration/dehydration of carbon dioxide. To date, 16 different CA isozymes have been identified in mammals, and several novel isozymes have also been identified in non-mammalian vertebrates. These isozymes are involved in many physiological processes; however, one of the most important roles is facilitating the transport and subsequent excretion of carbon dioxide. As such, CA isozymes are found at virtually every step of the process, including the metabolic site of CO(2) production (muscle), the circulating red blood cells, and the primary respiratory surface (gills/lungs). This review will examine the structural characteristics that are integral to CAs participation in respiration, as well as highlight the specific roles and tissues that the different CA isozymes are involved in.

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.

Cytoplasmic carbonic anhydrase isozymes in rainbow trout Oncorhynchus mykiss: comparative physiology and molecular evolution.

SUMMARY It is well established that the gills of teleost fish contain substantial levels of cytoplasmic carbonic anhydrase (CA), but it is unclear which CA isozyme(s) might be responsible for this activity. The objective of the current study was to determine if branchial CA activity in rainbow trout was the result of a general cytoplasmic CA isozyme, with kinetic properties, tissue distribution and physiological functions distinct from those of the red blood cell (rbc)-specific CA isozyme. Isolation and sequencing of a second trout cytoplasmic CA yielded a 780 bp coding region that was 76% identical with the trout rbc CA (TCAb), although the active sites differed by only 1 amino acid. Interestingly, phylogenetic analyses did not group these two isozymes closely together, suggesting that more fish species may have multiple cytoplasmic CA isozymes. In contrast to TCAb, the second cytoplasmic CA isozyme had a wide tissue distribution with high expression in the gills and brain, and lower expression in many tissues, including the red blood cells. Thus, unlike TCAb, the second isozyme lacks tissue specificity and may be expressed in the cytoplasm of all cells. For this reason, it is referred to hereafter as TCAc (trout cytoplasmic CA). The inhibitor properties of both cytoplasmic isozymes were similar (Ki acetazolamide 1.21±0.18 nmol l-1 and 1.34±0.10 nmol l-1 for TCAc and TCAb, respectively). However, the turnover of TCAb was over three times greater than that of TCAc (30.3±5.83 vs 8.90±1.95 e4 s-1, respectively), indicating that the rbc-specific CA isoform was significantly faster than the general cytoplasmic isoform. Induction of anaemia revealed differential expression of the two isozymes in the red blood cell; whereas TCAc mRNA expression was unaffected, TCAb mRNA expression was significantly increased by 30- to 60-fold in anaemic trout.

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

SUMMARY 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 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(J_{\mathrm{in}}^{\mathrm{Cl}^{-}}\) \end{document}) preceded the mRNA spike, occurring at 2–5 d.p.f. Raising zebrafish in water with a low [Cl–] caused marked increases in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(J_{\mathrm{in}}^{\mathrm{Cl}^{-}}\) \end{document} 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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(J_{\mathrm{in}}^{\mathrm{Cl}^{-}}\) \end{document} 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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(J_{\mathrm{in}}^{\mathrm{Cl}^{-}}\) \end{document} 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.

Corresponding morphological and molecular indicators of crude oil toxicity to the developing hearts of mahi mahi.

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

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.

Modulation of hypothalamic–pituitary–interrenal axis function by social status in rainbow trout

Juvenile rainbow trout (Oncorhynchus mykiss) form stable dominance hierarchies when confined in pairs. These hierarchies are driven by aggressive competition over limited resources and result in one fish becoming dominant over the other. An important indicator of low social status is sustained elevation of circulating cortisol levels as a result of chronic activation of the hypothalamic-pituitary-interrenal (HPI) axis. In the present study it was hypothesized that social status modulates the expression of key proteins involved in the functioning of the HPI axis. Cortisol treatment and fasting were used to assess whether these characteristics seen in subordinate fish also affected HPI axis function. Social status modulated plasma adrenocorticotropic hormone (ACTH) levels, cortisol synthesis, and liver glucocorticoid receptor (GR) expression. Plasma ACTH levels were lower by approximately 2-fold in subordinate and cortisol-treated fish, consistent with a negative feedback role for cortisol in modulating HPI axis function. Although cortisol-treated fish exhibited differences in corticotropin-releasing factor (CRF) and CRF-binding protein (CRF-BP) mRNA relative abundances in the preoptic area and telencephalon, respectively, no effect of social status on CRF or CRF-BP was detected. Head kidney melanocortin 2 receptor (MC2R) mRNA relative levels were unaffected by social status, while mRNA relative abundances of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage (P450scc) enzyme were elevated in dominant fish. Liver GR2 mRNA and total GR protein levels in subordinate fish were lower than control values by approximately 2-fold. In conclusion, social status modulated the functioning of the HPI axis in rainbow trout. Our results suggest altered cortisol dynamics and reduced target tissue response to this steroid in subordinate fish, while the higher transcript levels for steroid biosynthesis in dominant fish leads us to propose an adaptive role for responding to subsequent stressors.

Comparative physiology and molecular analysis of carbonic anhydrase from the red blood cells of teleost fish

This study investigates the early evolution of vertebrate red blood cell (rbc) carbonic anhydrase (CA) by examining the physiological and molecular properties of rbc CA in teleost fish. When representatives of four different families of teleosts were compared, it was found that differences in overall rbc CA activity were due to different concentrations of CA, rather than differences in the enzyme’s kinetic properties. Additional molecular analysis of CA from the rbcs of rainbow trout provided further evidence that critical elements of the enzyme, such as the active site, have been highly conserved during vertebrate evolution. The active site of the trout CA differed from that of gar rbc CA at only two amino acid positions. The rainbow trout rbc CA sequence also showed high sequence homology with CA sequences from other fish tissues, and fits into an emerging group of fish CAs that are basal to mammalian CA I, II and III. Northern blot analysis of the tissue expression of the sequenced CA indicated that it is primarily found in the rbcs, but high amounts of cytosolic CA activity were also found in the gill, suggesting the presence of other cytosolic CA isozymes in this species.

The toxicity and physiological effects of copper on the freshwater pulmonate snail, Lymnaea stagnalis.

Several recent studies have demonstrated that the freshwater pulmonate snail Lymnaea stagnalis is extremely sensitive to metals (Co, Ni, Pb) in chronic exposures. The objective of the current study was to evaluate the acute and chronic sensitivity of L. stagnalis to Cu and investigate the underlying mechanism(s) of toxic action. A 96-h LC50 of 31μg L(-1) Cu was estimated indicating L. stagnalis was moderately acutely sensitive to Cu relative to other aquatic organisms. However, in a 30-day chronic exposure using juvenile snails an EC20 of 1.8μg L(-1) Cu was estimated for snail growth making L. stagnalis the most sensitive organism tested to date for Cu. Hardness-based and BLM-based water quality criteria for Cu at the water quality conditions used in this study were 7.8 and 1.5μg L(-1), respectively, indicating L. stagnalis is significantly under-protected by hardness-based WQC. Investigations into the mechanism(s) of toxic action for Cu were conducted on young adult snails necessitating higher Cu exposures. Exposure to Cu at 12μg L(-1) resulted in no detectable effects on hemolymph osmolality, net Ca(2+) uptake, titratable acid excretion, or ammonia excretion. Exposure to 48μg L(-1) Cu was shown to significantly reduce (91%) net Ca(2+) uptake which is strongly correlated with shell deposition and corresponding snail growth. Snails exposed to 48μg L(-1) Cu also exhibited reduced ammonia excretion, a marked hemolymph acidosis, and a compensatory increase in titratable acid excretion. The reduction in net Ca(2+) uptake was hypothesized to be a secondary effect of Cu-induced inhibition of carbonic anhydrase, but no reduction in carbonic anhydrase activity was detected. Overall, it remains unclear whether inhibition of Ca(2+) uptake is a direct result of Cu exposure or, along with the other observed physiological effects, is secondary to an unidentified primary mode of toxic action. Given the hypersensitivity of L. stagnalis to Cu, further study into the mechanisms of action and effects of varying water chemistry on Cu toxicity is clearly warranted.