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Fish aquaculture facilities strive to maintain abiotic conditions that optimise somatic growth. Two physiological systems that are critically linked to environmental abiotic factors are ion and acid‐base regulation. Given the detrimental effects of ionic and acid‐base imbalances coupled with the potential energetic costs of ion and acid‐base regulation, it is important to understand how changes in abiotic variables such as salinity, temperature and pH alter ionoregulatory and acid‐base physiology. The gill is the predominant site of ion and acid‐base regulation, two linked processes that are vital to the health of fishes. Most species maintain ion levels in the blood plasma within a narrow range, independent of external ion concentrations. In freshwater (FW), fishes must actively take up ions from their dilute environment to counteract passive ion losses, while in seawater (SW), ion excretion serves to counteract passive salt gain. Ion uptake (FW) and excretion (SW) are coordinated by different subtypes of ion‐transporting cells (ionocytes) expressed in the gill epithelium. In this review, we discuss the function of FW and SW ionocytes in the movement of ions across the gill epithelium and address other features of the gill that are integral to ionoregulation. We also discuss the mechanisms of acid‐base regulation by the gill, which is intimately related to ion regulation because it is coordinated by Na+‐ and Cl−‐linked fluxes of acid (H+) and base (HCO3‐) equivalents. These fundamental physiological principles are integral in understanding how fishes in aquaculture respond to relevant disturbances that may occur under culture conditions. [ABSTRACT FROM AUTHOR]

Adrenergic regulation, acting via the sympathetic nervous system, provides a major mechanism to control cardiac function. It has recently been shown that hypoxia inducible factor‐1α (Hif‐1α) is necessary for normal development of sympathetic innervation and control of cardiac function in the mouse. To investigate whether this may represent a fundamental trait shared across vertebrates, we assessed adrenergic regulation of the heart in wild‐type and Hif‐1α knockout (hif‐1α−/−) zebrafish (Danio rerio). Wild‐type and hif‐1α−/− zebrafish larvae (aged 4 and 7 days postfertilisation) exhibited similar routine heart rates within a given age group, and β‐adrenergic receptor blockade with propranolol universally reduced heart rate to comparable levels, indicating similar adrenergic tone in both genotypes. In adult fish, in vivo heart rate measured during anaesthesia was identical between genotypes. Treatment of spontaneously beating hearts in vitro with adrenaline revealed a similar positive chronotropic effect and similar maximum heart rates in both genotypes. Tyrosine hydroxylase immunohistochemistry with confocal microscopy demonstrated that the bulbus arteriosus (outflow tract of the teleost heart) of adult fish was particularly well innervated by sympathetic nerves, and nerve density (as a percentage of bulbus arteriosus area) was similar between wild‐types and hif‐1α−/− mutants. In summary, we did not find any evidence that adrenergic cardiac control was perturbed in larval or adult zebrafish lacking Hif‐1α. We conclude that Hif‐1α is not essential for the normal development of cardiovascular control or adult sympathetic cardiac innervation in zebrafish, although it is possible that it plays a redundant or auxiliary role. Research Highlights: Hypoxia inducible factor‐1α (Hif‐1α) is involved in the development of cardiac innervation in mammals.By contrast, cardiac adrenergic control in Hif‐1α knockout zebrafish is similar to wild‐type, indicating that Hif‐1α is nonessential. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that hypoxia tolerance is improved in zebrafish (Danio rerio) larvae after prior exposure to lowered ambient O2 levels. Such improved hypoxia performance was attributed in part, to increased levels of hypoxia-inducible factor 1α (Hif-1α) exerting downstream effects on various physiological processes including promotion of trunk skin angiogenesis. Since O2 uptake ( M ˙ O 2 ) in larvae is facilitated largely by O2 diffusion across the skin, enhanced cutaneous vascularization is expected to enhance M ˙ O 2 during hypoxia and thus contribute to improved hypoxia tolerance. In this study, we used the scanning micro-optrode technique together with quantification of cutaneous vascularity in wild types (WT) and Hif-1α knockouts (hif1aa−/−ab−/−) to test the hypothesis that improved hypoxia tolerance after hypoxia acclimation in larvae at 4 or 7 days post-fertilization (dpf) was associated with Hif-1α-dependent increases in skin vascularity and regional cutaneous O2 fluxes (JO2). Hypoxia tolerance, as determined by measurements of critical PO2 (Pcrit), was unaltered by hypoxia pre-exposure in larvae at 4 dpf and there were no significant differences in Pcrit between WT and hif1aa−/−ab−/− larvae at this developmental stage. However, at 7 dpf there was a significant effect of genotype with WT larvae showing a lower Pcrit than hif1aa−/−ab−/− larvae, an effect that was being driven by a reduced Pcrit in the WT larvae after hypoxia pre-exposure (19.2 ± 1.9 mmHg) compared to hif1aa−/−ab−/− fish (35.5 ± 3.5 mmHg). Regardless of genotype, pre-exposure status or developmental age, JO2 decreased along the body in the anterior-to-posterior direction. Neither hypoxia pre-exposure nor genotype affected JO2 at any region along the body. The lack of any effect of hypoxia pre-exposure or genotype on JO2 was consistent with the lack of any effect on skin vascularity as measured in Tg(fli1:EGFP)yl transgenic larvae. Thus, the decreased hypoxia performance (increased Pcrit) at 7 dpf in the hif1aa−/−ab−/− larvae did not appear to be reliant on changes in trunk vascularity or cutaneous O2 diffusion. [ABSTRACT FROM AUTHOR]

The structural features of the fish gill necessary for oxygen uptake also favor undesirable, passive movements of ions and water. Reversible gill remodeling is one solution to this conflict. Cell masses that limit functional surface area are lost when oxygen availability decreases in hypoxia or oxygen demand increases with exercise or high temperature. However, much remains to be learned about how widespread reversible gill remodeling is among fish species, and how and why it occurs. [ABSTRACT FROM AUTHOR]

The effects of acute exposure to acidic water on Na+ and Cl- homeostasis, and the mechanisms underlying their compensatory regulation, were investigated in the larval zebrafish Danio rerio. Exposure to acidic water (pH 4.0; control pH 7.6) for 2 h significantly reduced Na+ uptake and whole body Na+ content. Nevertheless, the capacity for Na+ uptake was substantially increased in fish preexposed to acidic water but measured in control water. Based on the accumulation of the Na+-selective dye, Sodium Green, two ionocyte subtypes exhibited intracellular Na+ enrichment after preexposure to acidic water: H+-ATPase rich (HR) cells, which coexpress the Na+/H+ exchanger isoform 3b (NHE3b), and a non-HR cell population. In fish experiencing Na+-Cl- cotransporter (NCC) knockdown, we observed no Sodium Green accumulation in the latter cell type, suggesting the non-HR cells were NCC cells. Elimination of NHE3b-expressing HR cells did not prevent the increased Na+ uptake following acid exposure. On the other hand, the increased Na+ uptake was abolished when the acidic water was enriched with Na+ and Cl-, but not with Na+ only, indicating that the elevated Na+ uptake after acid exposure was associated with the compensatory regulation of Cl-. Further examinations demonstrated that acute acid exposure also reduced whole body Cl- levels and increased the capacity for Cl- uptake. Moreover, knockdown of NCC prevented the increased uptake of both Na+ and Cl- after exposure to acidic water. Together, the results of the present study revealed a novel role of NCC in the compensatory regulation of Na+ and Cl- uptake following acute acidosis. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO) is a gaseous signaling molecule and is produced in vivo from the intracellular breakdown of heme via the heme oxygenase (HO) family of enzymes. In this study we investigated the role of the HO-1/CO system in the control of ventilation in zebrafish, Danio rerio. Immunohistochemistry revealed the presence of HO-1 in the chemoreceptive neuroepithelial cells (NECs) of larvae (4 days postfertilization) and adults, indicating the potential for endogenous CO production in the NECs. Hypoxia (20 min, water Po2 of 30 mmHg) caused a significant increase in HO-1 activity in whole larvae and in the gills of adult fish. Zebrafish with reduced HO-1 activity (via HO-1 knockdown in larvae or zinc protoporphyrin IX treatment in adults) exhibited increased ventilation frequency (Vf) under normoxic but not hypoxic conditions. The addition of exogenous CO restored resting Vf in fish with diminished CO production, and in some cases (e.g., hypoxic sham larvae) CO modestly reduced Vf below resting levels. Larval fish were treated with phenylhydrazine (PHZ) to eliminate the potential confounding effects of CO-hemoglobin interactions that might influence ventilation. PHZ treatment did not cause changes in Vf of normoxic larvae, and the addition of CO to PHZ-exposed larvae resulted in a significant decrease in sham and HO-1-deficient fish under normoxic conditions. This study demonstrates for the first time that CO plays an inhibitory role in the control of breathing in larval and adult zebrafish. [ABSTRACT FROM AUTHOR]

Clifford et al shrug off these complexities and by using classical approaches, provide direct evidence for a novel Na SP + sp uptake mechanism that serves to sustain Na SP + sp balance in acidic water. Reassessing the contribution of the Na + /H + exchanger Nhe3b to Na + uptake in zebrafish (Danio rerio) using CRISPR/Cas9 gene editing. Over the past century, studies of fish gills have provided much insight into the fundamental mechanisms of ion movements across epithelia. [Extracted from the article]

Carbon monoxide (CO) is a gaseous neurotransmitter produced from the breakdown of heme via heme oxygenase-1 (HO-1; hypoxia-inducible isoform) and heme oxygenase-2 (HO-2; constitutively expressed isoform). In mammals, CO is involved in modulating cardiac function. The role of the HO-1/CO system in the control of heart function in fish, however, is unknown and investigating its physiological function in lower vertebrates will provide a better understanding of the evolution of this regulatory mechanism. We explored the role of the HO-1/CO system in larval zebrafish (Danio rerio) in vivo by investigating the impact of translational gene knockdown of HO-1 on cardiac function. Immunohistochemistry revealed the presence of HO-1 in the pacemaker cells of the heart at 4 days post-fertilization and thus the potential for CO production at these sites. Sham-treated zebrafish larvae (experiencing normal levels of HO-1) significantly increased heart rate ( fH) when exposed to hypoxia (PwO2=30 mmHg). Zebrafish larvae lacking HO-1 expression after morpholino knockdown (morphants) exhibited significantly higher fH under normoxic (but not hypoxic) conditions when compared with sham-treaded fish. The increased fH in HO-1 morphants was rescued ( fH was restored to control levels) after treatment of larvae with a CO-releasing molecule (40 μmol l-1 CORM). The HO-1-deficient larvae developed significantly larger ventricles and when exposed to hypoxia they displayed higher cardiac output (Q) and stroke volume (SV). These results suggest that under hypoxic conditions, HO-1 regulates Q and SV presumably via the production of CO. Overall, this study provides a better understanding of the role of the HO-1/CO system in controlling heart function in lower vertebrates. We demonstrate for the first time the ability for CO to be produced in presumptive pacemaker cells of the heart where it plays an inhibitory role in setting the resting cardiac frequency. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) can act as a signalling molecule for various ion channels and/or transporters, however, little is known about its potential involvement in Ca2+ balance. Using developing zebrafish (Danio rerio) as an in vivo model system, the present study demonstrated that acute exposure to H2S donors increased Ca2+ influx at 4 days post fertilization (dpf) while chronic (3-day) exposure caused a rise in whole-body Ca2+ levels. The mRNA expression of Ca2+-transport related genes was unaffected by H2S exposure, suggesting that post-transcriptional modifications were responsible for the altered rates of Ca2+ uptake. Indeed, treatment of fish with the protein kinase A (PKA) inhibitor H-89 abolished the H2S-mediated stimulation of Ca2+ influx, suggesting that H2S increased Ca2+ influx by activating cAMP-PKA pathways. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are two key enzymes in the endogenous synthesis of H2S. Using an antisense morpholino knockdown approach, we demonstrated that Ca2+ influx was reduced in CBSb- but not in CSE-deficient fish. Interestingly, the reduction in Ca2+ influx in CBSb-deficient fish was observed only in fish that were acclimated to low Ca2+ water (i.e., 25 μM Ca2+; control: 250 μM Ca2+). Similarly, mRNA expression of cbsb but not cse was increased in fish acclimated to low Ca2+ water. Results from whole-mount immunohistochemistry further revealed that CBSb was expressed in Na+/K+-ATPase-rich cells (NaR cells), which are implicated in Ca2+ uptake in zebrafish larvae. Collectively, the present study suggests a novel role for H2S in promoting Ca2+ influx, particularly in a low Ca2+ environment. [ABSTRACT FROM AUTHOR]

Highlights: [•] Heme oxygenase-1 (HO-1) was present in the neuroepithelial cells (NECs; putative branchial O2 chemoreceptors) of 7°C goldfish only. [•] Hypoxia exposure increased gill HO-1 activity in hypoxic fish. [•] Inhibition of HO-1 activity with zinc protoporphyrin IX (ZnPPIX) increased the ventilation frequency response to acute hypoxia (30mmHg) in 7°C goldfish. [•] 7°C fish injected with ZnPPIX did not significantly decrease breathing frequency during acute hyperoxia (∼500mmHg). [•] Inhibition of HO-1 activity did not affect the ventilatory response of 25°C goldfish exposed to acute hypoxia or hyperoxia. [ABSTRACT FROM AUTHOR]

The effects of cortisol on epithelial permeability and sodium (Na+) handling during acid exposure were investigated in larval zebrafish (Danio rerio). The results demonstrated that the whole-body absorption of the paracellular permeability marker polyethylene glycol- 4000 (PEG-4000) decreased with increasing levels of exogenous cortisol. Western blot analysis revealed that the abundance of the epithelial tight junction proteins occludin-a and claudin-b was increased after cortisol treatment. Furthermore, translational gene knockdown of claudin-b using an antisense morpholino oligonucleotide caused an increase in the permeability to PEG-4000, which was mitigated by cortisol treatment, further suggesting a role for cortisol in reducing paracellular permeability. Exposure to acidic water (pH 4.0 vs 7.6) caused an expected increase in the diffusive loss of Na+ and a decrease in whole-body Na+ levels. These disruptive effects of acute acid exposure on Na+ balance were reduced by treatment of larvae with exogenous cortisol. Translational knockdown of the glucocorticoid receptor (GR) abolished the effects of cortisol on epithelial PEG permeability, suggesting that activation of GR was probably the major signaling pathway for reducing epithelial permeability. During acid exposure, the epithelial PEG permeability in the GR morphants was significantly higher than in the control fish. Additionally, GR morphants exhibited a more pronounced diffusive loss of Na+ than the control fish during acid exposure. These findings suggest that cortisolmay help to minimize the negative consequences of acid exposure onNa+ homoeostasis via GR-mediated reductions in epithelial permeability and paracellular Na+ loss. [ABSTRACT FROM AUTHOR]

The functional role of the tight junction protein claudin-b in larval zebrafish (Danio rerio) was investigated. We showed that claudin-b protein is expressed at epithelial cell-cell contacts on the skin. Translational gene knockdown of claudin-b protein expression caused developmental defects, including edema in the pericardial cavity and yolk sac. Claudin-b morphants exhibited an increase in epithelial permeability to the paracellular marker polyethylene glycol (PEG-4000) and fluorescein isothiocyanate- dextran (FD-4). Accumulation of FD-4 was confined mainly to the yolk sac and pericardial cavity in the claudin-b morphants, suggesting these regions became particularly leaky in the absence of claudin-b expression. Additionally, Na+ efflux was substantially increased in the claudin-b morphants, which contributed to a significant reduction in whole-body Na+ levels. These results indicate that claudin-b normally acts as a paracellular barrier to Na+. Nevertheless, the elevated loss of Na+ in the morphants was compensated by an increase in Na+ uptake. Notably, we observed that the increased Na+ uptake in the morphants was attenuated in the presence of the selective Na+/Cl--cotransporter (NCC) inhibitor metolazone, or during exposure to Cl--free water. These results suggested that the increased Na+ uptake in the morphants was, at least in part, mediated by NCC. Furthermore, treatment with an H+-ATPase inhibitor bafilomycin A1 was found to reduce Na+ uptake in the morphants, suggesting that H+-ATPase activity was essential to provide a driving force for Na+ uptake. Overall, the results suggest that claudin-b plays an important role in regulating epithelial permeability and Na+ handling in zebrafish. [ABSTRACT FROM AUTHOR]

Abstract: Fish possess chemoreceptors able to sense increasing levels of ambient CO2 and initiate various cardiorespiratory reflexes including hyperventilation and bradycardia. These chemoreceptors are localized predominantly to the gills, are oriented to sense the external environment and typically are stimulated by changes in environmental molecular CO2 rather than H+ (although increasing H+ may be the proximate intracellular stimulus). In zebrafish, a subset of branchial neuroepithelial cells (NECs) act as bimodal sensors of CO2 and O2, similar to the Type I (glomus) cells of the mammalian carotid body. Like O2 sensing, the mechanisms underlying CO2 detection involve the inhibition of a background K+ current leading to membrane depolarization and subsequent elevation of intracellular Ca2+ levels. Carbonic anhydrase, by catalysing the hydration of CO2 to H+ and HCO3 −, appears to play a critical role in reducing NEC response times and increasing the magnitude of membrane depolarization accompanying hypercapnia. In larval zebrafish, CA activity is essential for the rapid development of hypercapnic bradycardia. [Copyright &y& Elsevier]

Abstract: Mechanisms of ion uptake by freshwater (FW) fish have received considerable attention over the past 80 years. Through an assortment of techniques incorporating whole animal physiology, electrophysiology and molecular biological approaches, three models have been proposed to account for Na+ uptake. (1) Direct exchange of Na+ and H+ via one or more types of Na+/H+ exchanger (slc9), (2) uptake of Na+ through epithelial Na+ channels energized by an electrical gradient created by H+-ATPase and (3) Na+/Cl− co-transport (slc12). While each mechanism is supported at least in part by theoretical or experimental data, there are several outstanding questions that have not yet been fully resolved. Furthermore, there are few details concerning how these Na+ uptake mechanisms are fine tuned in response to the fluctuating FW environments. In this review, we summarize the current understanding of these three Na+ uptake mechanisms and discuss their regulation by endocrine (cortisol and prolactin) and neurohumoral (catecholamines) factors. [Copyright &y& Elsevier]

Abstract: If severe enough, periods of acute stress in animals may be associated with the release of catecholamine hormones (noradrenaline and adrenaline) into the circulation; a response termed the acute humoral adrenergic stress response. The release of catecholamines from the sites of storage, the chromaffin cells, is under neuroendocrine control, the complexity of which appears to increase through phylogeny. In the agnathans, the earliest branching vertebrates, the chromaffin cells which are localized predominantly within the heart, lack neuronal innervation and thus catecholamine secretion in these animals is initiated solely by humoral mechanisms. In the more advanced teleost fish, the chromaffin cells are largely confined to the walls of the posterior cardinal vein at the level of the head kidney where they are intermingled with the steroidogenic interrenal cells. Catecholamine secretion from teleost chromaffin cells is regulated by a host of cholinergic and non-cholinergic pathways that ensure sufficient redundancy and flexibility in the secretion process to permit synchronized responses to a myriad of stressors. The complexity of catecholamine secretion control mechanisms continues through the amphibians, reptiles and birds although neural (cholinergic) regulation may become increasingly important in birds. Discrete adrenal glands are present in the non-mammalian tetrapods but unlike in mammals, there is no clear division of a steroidogenic cortex and a chromaffin cell enriched medulla. However, in all groups, there is an obvious intermingling of chromaffin and steroiodogenic cells. The association of the two cell types may be particularly important in the amphibians and birds because like in mammals, the enzyme catalysing the methylation of noradrenaline to adrenaline, PNMT, is under the control of the steroid cortisol. [Copyright &y& Elsevier]

The involvement of a Na+/H+ exchanger (NHE) in mediating Na+ uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na+ uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na+ uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na+ uptake in fish reared in acidic water (pH ~ 4.0). A role for NHE in Na+ uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na+/H+ exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na+ uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na+ uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na+ uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H+-ATPase) significantly reduced Na+ uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na+ uptake in larval zebrafish in acidic water: one in which Na+ uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H+-ATPase. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that chronic dietary salt loading causes hypertension and a decreased sensitivity of the systemic vasculature to a-adrenergic stimulation and other hypertensive stimuli (e.g. hypercapnia) in rainbow trout (Oncorhynchus mykiss). This reduced sensitivity to hypertensive stimuli is consistent with a possible blunting of homeostatic responses normally aimed at raising blood pressure. To test this idea, we examined the consequences of long-term salt feeding and the associated hypertension on the interactive capacities of the renin angiotensin system (RAS) and adrenergic systems to elevate blood pressure in trout. Secretion of catecholamines in response to a range of doses of homologous ANG II in vivo and in situ (using a perfused posterior cardinal vein preparation) was reduced in the salt-fed fish. The reduced sensitivity to ANG II could not be explained by alterations in stored catecholamine (adrenaline or noradrenaline) levels or the general responsiveness of the chromaffin cells to depolarizing stimuli (60 mmol/l KCl). Despite the decreased responsiveness of the chromaffin cells to ANG II, plasma catecholamines were increased to a greater extent in the salt-fed fish during acute hypoxia (a condition that activates the RAS). Interestingly, the pressor effects of ANG II in vivo were actually heightened in the salt-fed fish. The increased pressor response to exogenous ANG II was likely attributable to its direct interaction with vascular ANG II receptors because the effect persisted even after blockade of a-adrenergic receptors. Treating fish with the vascular smooth muscle relaxant papaverine caused similar reductions in blood pressure and increases in plasma ANG II levels regardless of diet. Similarly, inhibition of angiotensin converting enzyme with lisinopril reduced blood pressure equally in control and salt-fed fish. These results indicate that, while long-term dietary salt loading blunts the response of trout chromaffin cells to ANG II, the RAS itself appears to be unaffected. Indeed, the capacity of ANG II to elevate blood pressure is not compromised nor do fish exhibit a reduced capacity to mount an acute humoral adrenergic stress response during acute hypoxia. [ABSTRACT FROM AUTHOR]

Abstract: The nature of ammonia and urea excretion was examined in the Pacific hagfish (Eptatretus stoutii), which, under resting conditions, excreted similar quantities of nitrogen as either ammonia or urea. In the presence of high external ammonia (HEA) concentrations, ammonia was taken up at high rates and then excreted at similarly high rates upon return to normal water. However, although elevated by HEA, plasma ammonia levels were maintained at approximately 1–4μmolNg−1, reflecting time-dependent decreases in the rates of ammonia uptake, the possible conversion of ammonia to urea, and the potential active excretion of ammonia against a gradient. Internal injections of NH4Cl caused marked increases in the rate of ammonia excretion and a delayed increase in urea excretion that may have resulted from increasing urea levels in the plasma. Conversely, when the rate of urea excretion was reduced in the presence of 0.1mM phloretin, ammonia excretion was significantly elevated. Rates of urea excretion were initially increased by approximately 1000-fold following internal urea injections while the presence of high external urea levels (5–100mM final concentration) resulted in associated linear increases in plasma urea levels. Using hagfish skin mounted in Ussing chambers, the rate of diffusion of ammonia across the skin exceeded that of urea by approximately four times when equivalent gradients were imposed. Based on western blotting and immunocytochemistry, hagfish gill appears to possess Rh proteins (Rhag, Rhbg and Rhcg1) and urea transporter proteins. Despite the tolerance of hagfish to high levels of ammonia and urea, it is suggested that the presence of ammonia and urea transporter proteins may be required during the period of time hagfish spend in burrows or while feeding, when conditions of high ammonia and/or urea might be encountered. [ABSTRACT FROM AUTHOR]

At temperatures below 15°C the gill lamellae of goldfish (Carassius auratus) are largely covered by an interlamellar cell mass (ILCM) which decreases the functional surface area of the gill. The presence of the ILCM in goldfish acclimated to cold water conceivably could lead to a covering of the neuroepithelial cells (NECs), which are believed to be important for sensing ambient O2 and CO2 levels. In this study we tested the hypothesis that goldfish with covered lamellae (and presumably fewer NECs exposed to the water) exhibit a decreased capacity to hyperventilate in response to hypoxic stimuli. Measurements of ventilation amplitude and frequency were performed during exposure to acute hypoxia (PwO2=30mmHg) or following injections of the O2 chemoreceptor stimulant NaCN into the buccal cavity or caudal vein of fish acclimated to 25°C (uncovered lamellae) or 7°C (covered lamellae) to stimulate predominantly the externally or internally oriented NECs, respectively. The results demonstrated no significant differences in the response to hypoxia, with each group exhibiting similar percentage increases in ventilation amplitude (90-91%) and frequency (34-43%). Similarly, with the exception of a rightward shift of the ventilation frequency dose-response in the fish acclimated to 7°C, there were no significant differences between the two groups of fish in the ED50 values. These findings suggest that goldfish with covered lamellae retain the capacity to sense external hypoxic stimuli. Using immunohistochemistry to identify serotonin-enriched NECs, it was demonstrated that the presence of the ILCM results in the NECs being redistributed towards the distal regions of the lamellae. In 25°C-acclimated fish, the NECs were distributed evenly along the length of the lamellae with 53±3% of them in the distal half, whereas in fish acclimated to 7°C, 83±5% of the NECs were confined to the distal half. Using the neuronal marker antibody ZN-12, it was demonstrated that the NECs at the distal edges of the lamellae are innervated by nerve fibres. Thus, it is hypothesised that the capacity to sense external hypoxic stimuli in goldfish acclimated to cold water is maintained despite the increasing coverage of the gill epithelial surfaces because of a redistribution of innervated NECs to the exposed distal regions of the Iamellae. [ABSTRACT FROM AUTHOR]

Goldfish acclimated to cold water (e.g. 7°C) experience a marked reduction in functional lamellar surface area owing to the proliferation of an interlamellar cell mass (ILCM), a phenomenon termed gill remodelling. The goal of the present study was to assess the consequences of the reduced functional surface area on the capacity of goldfish to excrete ammonia. Despite the expected impact of ambient temperature on functional surface area, fish acclimated to 7°C and 25°C exhibited similar rates of ammonia excretion (Jnet,amm) the Q10 values for fed and starved fish were 1.07 and 1.20, respectively. To control for possible temperature-related differences in rates of endogenous ammonia production, Jnet,amm was determined at the two acclimation temperatures after loading fish with 1.12μmolg-1 of NH4CI. In the 3h post-injection period, Jnet,amm was elevated to a greater extent in the 25°C fish. To estimate the potential contribution of increased ventilation and cardiac output to ammonia clearance in the warmer fish, the ammonia loading experiment was repeated on the 7°C fish immediately after they were exercised to exhaustion. The rate of excretion of ammonia was significantly increased in the exercised 7°C fish (presumably experiencing increased ventilation and cardiac output for at least some of the measurement period) suggesting that differences in external and internal convection may at least partially explain the enhanced capacity of the 25°C fish to clear the ammonia load. To more specifically assess the contribution of the different functional surface areas on the differing rates of ammonia clearance at the two acclimation temperatures, the 7°C fish were exposed for 7days to hypoxia (PO2=10mmHg=1.33kPa), a treatment known to cause the disappearance of the ILCM. The results demonstrated that the hypoxia-associated loss of the ILCM was accompanied by a significant increase in the rate of ammonia clearance in the 7°C fish when returned to normoxic conditions. To determine whether compensatory changes in the ammonia transporting proteins might be contributing to sustaining Jnet,amm under conditions of reduced functional lamellar surface area, the relative expression and branchial distribution of four Rh proteins were assessed by western blotting and immunocytochemistry. Although the relative expression of the Rh proteins was unaffected by acclimation temperature, there did appear to be a change in the spatial distribution of Rhag, Rhbg and Rhcg1. Specifically, these three Rh proteins (and to a lesser extent Rhcg2) appeared to localize in cells on the outer edge of the ILCM that were enriched with Na+/K+- ATPase. Thus, we suggest that despite the impediment to ammonia excretion imposed by the ILCM, goldfish acclimated to 7°C are able to sustain normal rates of excretion owing to the redistribution of ammonia transporting cells. [ABSTRACT FROM AUTHOR]

The plainfin midshipman ( Porichthys notatus) possesses an aglomerular kidney and like other marine teleosts, secretes base into the intestine to aid water absorption. Each of these features could potentially influence acid-base regulation during respiratory acidosis either by facilitating or constraining HCO accumulation, respectively. Thus, in the present study, we evaluated the capacity of P. notatus to regulate blood acid-base status during exposure to increasing levels of hypercapnia (nominally 1-5% CO). Fish exhibited a well-developed ability to increase plasma HCO levels with values of 39.8 ± 2.8 mmol l being achieved at the most severe stage of hypercapnic exposure (arterial blood PCO = 21.9 ± 1.7 mmHg). Consequently, blood pH, while lowered by 0.15 units (pH = 7.63 ± 0.06) during the final step of hypercapnia, was regulated far above values predicted by chemical buffering (predicted pH = 7.0). The accumulation of plasma HCO during hypercapnia was associated with marked increases in branchial net acid excretion ( JH) owing exclusively to increases in the titratable alkalinity component; total ammonia excretion was actually reduced during hypercapnia. The increase in JH was accompanied by increases in branchial carbonic anhydrase (CA) enzymatic activity (2.8×) and CA protein levels (1.6×); branchial Na/K-ATPase activity was unaffected. Rectal fluids sampled from control fish contained on average HCO concentrations of 92.2 ± 4.8 mmol l. At the highest level of hypercapnia, rectal fluid HCO levels were increased significantly to 141.8 ± 7.4 mmol l but returned to control levels during post-hypercapnia recovery (96.0 ± 13.2 mmol l). Thus, the impressive accumulation of plasma HCO to compensate for hypercapnic acidosis occurred against a backdrop of increasing intestinal HCO excretion. Based on in vitro measurements of intestinal base secretion in Ussing chambers, it would appear that P. notatus did not respond by minimizing base loss during hypercapnia; the increases in base flux across the intestinal epithelium in response to alterations in serosal HCO concentration were similar in preparations obtained from control or hypercapnic fish. Fish returned to normocapnia developed profound metabolic alkalosis owing to unusually slow clearance of the accumulated plasma HCO. The apparent inability of P. notatus to effectively excrete HCO following hypercapnia may reflect its aglomerular (i.e., non-filtering) kidney coupled with the normally low rates of urine production in marine teleosts. [ABSTRACT FROM AUTHOR]

We tested the hypothesis that endogenously produced hydrogen sulfide (H2S) can potentially contribute to the adrenergic stress response in rainbow trout by initiating catecholamine secretion from chromaffin cells. During acute hypoxia (water P02 = 35 mmHg), plasma H2S levels were significantly elevated concurrently with a rise in circulating catecholamine concentrations. Tissues enriched with chromaffin cells (posterior cardinal vein and anterior kidney) produced H2S in vitro when incubated with L-cysteine. In both tissues, the production of H2S was eliminated by adding the cystathionine β-synthase inhibitor, aminooxyacetate. Cystathionine β-synthase and cystathionine γ-lyase were cloned and sequenced and the results of real-time PCR demonstrated that with the exception of white muscle, mRNA for both enzymes was broadly distributed within the tissues that were examined. Electrical field stimulation of an in situ saline-perfused posterior cardinal vein preparation caused the appearance of H2S and catecholamines in the outfiowing perfusate. Perfusion with the cholinergic receptor agonist carbachol (1 X 10-6 M) or depolarizing levels of KCI (1 X 10-2 M) caused secretion of catecholamines without altering H2S output, suggesting that neuronal excitation is required for H2S release. Addition of H2S (at concentrations exceeding 5 X 10-7 M) to the perfusion fluid resulted in a marked stimulation of catecholamine secretion that was not observed when Ca2+-free perfusate was used. These data, together with the finding that H2S-induced catecholamine secretion was unaltered by the nicotinic receptor blocker hexamethonium, suggest that H2S is able to directly elicit catecholamine secretion via membrane depolarization followed by Ca2+-mediated exocytosis. [ABSTRACT FROM AUTHOR]

Presently, three α1-adrenoceptor (AR) types are recognized in vertebrates: α1A-, α1B-, and α1D-ARs. These α1-subtypes have distinct pharmacology and molecular profiles, play crucial roles in metabolic and vascular control, and are the targets for numerous pharmaceuticals, especially those affecting blood pressure and vascular resistance. To better understand the functional divergence within the α1-AR gene family, we sequenced these α1-AR paralogs in the rainbow trout and performed an extensive phylogenetic analysis. We show that these AR genes evolved by duplication events just before the origin of the jawed vertebrates. Our computational analyses suggest that the differences between the three α1-AR subtypes may affect their tissue specificity, ligand specificity, and possibly signal transduction processes and desensitization. We also show that, within each subtype, differences exist between fish and mammalian receptors, both at the transcriptional and at the physiological level. These differences, however, suggest that the role of α1-ARs in fish is more complex than previously thought. Our integrated analysis of the α1-AR gene family suggests that these receptors evolved these distinct features very early within vertebrates. [ABSTRACT FROM AUTHOR]

Numerous fish species, including rainbow trout (Oncorhynchus mykiss), are able to inhabit both freshwater and seawater and routinely migrate between the two environments. One of the most critical adjustments allowing such successful migrations is a remodelling of the gill in which a suite of morphological and molecular changes ensure optimal function in the face of reversing requirements for salt and water balance. The remodelling leads to specific freshwater and seawater gill phenotypes that are readily identified by the orientation and/or quantities of specific ion transporters and the presence or absence of specific cell types. The proximate cues promoting gill phenotypic plasticity are unknown. Here, by assessing the consequences of a salt-enriched diet (in the absence of any changes in external salinity) in the freshwater rainbow trout, we demonstrate that internal salt loading alone, is able to induce various elements of the seawater gill phenotype. Specifically, we show upregulation of three ion transport genes, cystic fibrosis transmembrane conductance regulator (CFTR), Na+/K+/2Cl- co-transporter (NKCC1) and Na+/K+-ATPase, which are essential for ionic regulation in seawater, and the appearance of chloride cell-accessory cell complexes, which are normally restricted to fish inhabiting seawater. These data provide compelling evidence that gill remodelling during migration from freshwater to seawater may involve sensing of elevated levels of internal salt. [ABSTRACT FROM AUTHOR]

We indirectly tested the idea that the epithelial Ca2+ channel (ECaC) of the trout gill is regulated in an appropriate manner to adjust rates of Ca2+ uptake. This was accomplished by assessing the levels of gill ECaC rnRNA and protein in fish exposed to treatments known to increase or decrease Ca2+ uptake capacity. Exposure of trout to soft water ([Ca2+] = 20-30 nmol/l) for 5 days (a treatment known to increase Ca2+ uptake capacity) caused a significant increase in ECaC mRNA levels and an increase in ECaC protein expression. The inducement of hypercalcemia by infusing fish with CaCl2 (a treatment known to reduce Ca2+ uptake) was associated with a significant decrease in ECaC mRNA levels, yet protein levels were unaltered. ECaC mRNA and protein expression were increased in fish treated with the hypercalcemic hormone cortisol. Finally, exposure of trout to 48 h of hypercapnia (~7.5 mmHg, a treatment known to increase Ca2+ uptake capacity) elicited an ~100-fold increase in the levels of ECaC mRNA and a significant increase in protein expression. immunocytochemical analysis of the gills from hypercapnic fish suggested a marked increase in the apical expression of ECaC on pavement cells and a subpopulation of mitochondria-rich cells. The results of this study provide evidence that Ca2+ uptake rates are, in part, regulated by the numbers of apical membrane Ca2+ channels that, in turn, modulate the inward flux of Ca2+ into gill epithelial cells. [ABSTRACT FROM AUTHOR]

Experiments were performed to test the hypothesis that exposure of rainbow trout to repetitive hypoxia would result in a decreased capacity of chromaffin cells to secrete catecholamines owing to increased production of nitric oxide (NO), a potent inhibitor of catecholamine secretion. A partial sequence of trout neuronal nitric oxide synthase (nNOS) was cloned and its mRNA was found to be present in the posterior cardinal vein (PCV), the predominant site of chromaffin cells in trout. Using heterologous antibodies, nNOS and endothelial NOS (eNOS) were localized in close proximity to the chromaffin cells of the PCV. Exposure of trout to acute hypoxia (5.33 kPa for 30 mm) in vivo resulted in significant increases in plasma catecholamine and NO levels. However, after 4 days of twice-daily exposures to hypoxia, the elevation of plasma catecholamine levels during hypoxia was markedly reduced. Associated with the reduction in plasma catecholamine levels during acute hypoxia was a marked increase in basal and hypoxia-evoked circulating levels of NO that became apparent after 2-4 days of repetitive hypoxia. The capacity of the chromaffin cells of the hypoxia-exposed fish to secrete catecholamine was assessed by electrical stimulation of an in situ saline-perfused PCV preparation. Compared with control (normoxic) fish, the PCV preparations derived from fish exposed to repeated hypoxia displayed a significant reduction in electrically evoked catecholamine secretion that was concomitant with a marked increased in NO production. This additional rise in NO secretion in preparations derived from hypoxic fish was prevented after adding NOS inhibitors to the perfusate; concomitantly, the reduction in catecholamine secretion was prevented. The increased production of NO during hypoxia in vivo and during electrical stimulation in situ was consistent with significant elevations of nNOS mRNA and protein; eNOS protein was unaffected. These results suggest that the reduced capacity of trout chromaffin cells to secrete catecholamines after repeated hypoxia reflects an increase in the expression of nNOS and a subsequent increase in NO production during chromaffin-cell activation. [ABSTRACT FROM AUTHOR]

Abstract: This study examined the effect of acute hypoxic and hypercapnic cardiorespiratory stimuli, superimposed on existing cardiorespiratory disturbances in tambaqui. In their natural habitat, these fish often encounter periods of hypoxic hypercapnia that can be acutely exacerbated by water turnover. Tambaqui were exposed to periods of normoxia, hypoxia, hyperoxia and hypercapnia during which, externally oriented O2 and CO2 chemoreceptors were further stimulated, by administration into the inspired water of sodium cyanide and CO2-equilibrated water, respectively. Hyperoxic water increased the sensitivity of the NaCN-evoked increase in breathing frequency (fR) and decrease in heart rate. Hypoxia and hypercapnia attenuated the increase in fR but, aside from blood pressure, did not influence the magnitude of NaCN-evoked cardiovascular changes. Water PO2 influenced the magnitude of the CO2-evoked cardiorespiratory changes and the sensitivity of CO2-evoked changes in heart rate and blood flow. The results indicate that existing respiratory disturbances modulate cardiorespiratory responses to further respiratory challenges reflecting both changes in chemosensitivity and the capacity for further change. [Copyright &y& Elsevier]

This study addressed the hypothesis that the secretion of catecholamines from trout (Oncorhynchus mykiss) chromaffin cells, during hypoxia, is triggered by stimulation of O[sub 2] chemoreceptors located within the gills. Sodium cyanide was administered into the inspired water (external cyanide) or injected into the gill circulation (internal cyanide) to pharmacologically stimulate external (water sensing) or internal (blood sensing) O[sub 2] chemoreceptors, respectively. Both of these treatments caused an elevation of circulating catecholamine levels. The response to external, but not internal, cyanide was abolished by removal of the first gill arch. Hypoxia produced an increase in circulating catecholamine levels that was unaffected by removal of the first gill arch or by denervation of the pseudobranch. Cyanide and hypoxia both caused the well-documented cardiorespiratory reflexes normally observed in this species. This study demonstrates, for the first time, that gill O[sub 2] chemoreceptors can initiate the reflex that leads to catecholamine release from the chromaffin cells and that stimulation of internally oriented O[sub 2] receptors on all gill arches appears to be the physiologically important mechanism for initiating release. [ABSTRACT FROM AUTHOR]

Experiments were performed to test the hypothesis that the marked ventilatory and cardiovascular responses to hypercarbia in rainbow trout Oncorhynchus mykiss arise from specific stimulation of chemoreceptors localised to the first gill arch. This was accomplished by measuring cardiorespiratory variables during acute hypercarbia (20 min at P[sub CO[sub 2]]=8mmHg; 1mmHg=0.133kPa) in fish subjected to selective bilateral extirpation of the first gill arch. The cardiovascular responses to hypercarbia in the intact fish included a significant bradycardia (from 75.0±1.6 to 69.0±2.0 beats min[sup -1]; means ± S.E.M.; N=16), an increase in dorsal aortic blood pressure (from 30.8±1.5 to 41.9±2.5 mmHg; N=16) and a rise in systemic vascular resistance (from 1.1±0.1 to 1.4±0.1 mmHg ml[sup -1] kg[sup -1] min[sup -1]; N=16). Removal of the first gill arch or pre-treatment with the muscarinic receptor antagonist atropine prevented the hypercarbic bradycardia without affecting the pressure or resistance responses. Correlation analysis, however, revealed shallow but significant inverse relationships between water P[sub CO[sub 2]] and cardiac frequency in both atropinised (r²=0.75) and gill-extirpated (r²=0.90) fish, suggesting a direct mild effect of CO[sub 2] on cardiac function. The ventilatory response to hypercarbia in the intact fish consisted of an increase in ventilation amplitude (from 0.62±0.06 to 1.0±0.13cm; N=16) with no change in breathing frequency. Removal of the first gill arch lowered resting breathing frequency and prevented the statistically significant elevation of breathing amplitude. Gill extirpation, however, did not totally abolish the positive correlation between water P[sub CO[sub 2]] and ventilation amplitude (r²=0.84), suggesting the presence of additional (although less important) chemoreceptive sites that are not confined to the first gill arch. Plasma catecholamine... [ABSTRACT FROM AUTHOR]

Assesses the role of renin-angiotensin system in catecholamine release during hypoxia periods in rainbow trout. Increase of plasma catecholamine levels upon nicotinic receptor desensitization; Activation of the renin-angiotensin system during hypoxia; Role of angiotensin II in facilitating catecholamine release.

Focuses on a study which assessed the potential direct and modulating effects of acute hypoxia on catecholamine secretion in rainbow trout, Oncorhynchus mykiss using a perfused cardinal vein preparation and chronically cannulated fish. Materials and methods; Effects of hypoxic perfusate on in situ basal catecholamine secretion rates; Influence of acute moderate hypoxia on nicotine-evoked catecholamine release in vivo.

Deals with a study which cloned and characterized the B subunit of the trout H...V-ATPase to examine by northern analysis the expression and tissue distribution of the B subunit mRNA during external hypercapnia. Materials and methods; Results and discussion.

The tambaqui (Colossoma macropomum), migrates annually between whitewater and blackwater rivers of the Amazon. Unlike the whitewater, blackwater is characterized by higher levels of dissolved organic carbon (DOC), including humic acids (HA). Because humic substances impair sensory processes, the current study tested the hypothesis that O 2 and/or CO 2 chemoreception is impeded in blackwater owing to the presence of HA. Thus, the ventilatory responses of tambaqui to hypoxia or hypercapnia were assessed in well water transported from Manaus, local blackwater, and in well water containing HA either extracted from Rio Negro water or obtained commercially (Sigma Aldrich; SA). In well water, tambaqui exhibited typical hyperventilatory responses to hypoxia or hypercapnia. These responses were prevented by simultaneously exposing fish to SA HA (20 mg l−1). The negative effects of SA HA on ventilation were prevented when natural DOC (30 mg l−1; extracted from Rio Negro water after first removing the endogenous HA fraction) was added concurrently, indicating a protective effect of this non-humic acid DOC fraction. The hyperventilatory responses were unaffected during acute exposure or after acclimation of fish to Rio Negro water. HA extracted from Rio Negro water did not impair the hyperventilatory responses to hypoxia or hypercapnia. This study, while demonstrating a negative effect of SA HA derived from peat (coal) on the control of breathing in tambaqui, failed to reveal any detrimental consequences of HA (derived from the decomposition of a variety of lignin-rich plants) naturally occurring in the blackwaters of the Rio Negro. • This study tested the hypothesis that reflex ventilatory responses to would be impaired in Tambaqui in natural blackwater. • Commercial humic acid impaired the ventilatory response to hypoxia or hypercapnia. • Natural blackwater from the Rio Negro or humic acid derived from blackwater was without effect. • The negative effects of commercial humic acid were absent in the presence of DOC (minus the humic acid fraction) [ABSTRACT FROM AUTHOR]

Discusses a study which assessed the relative involvement of nicotinic and muscarinic cholinergic receptors in the neuronal control of catecholamine secretion from the chromaffin tissue of rainbow trout. Background information on the mechanism of catecholamine secretion in teleosts; Materials and methods; Results; Discussion.

Determines the contribution of angiotensin II (Ang-II)-elicited increases in humoral catecholamines levels to rainbow trout cardiovascular control. Effect of homologous rainbow trout Ang-II in plasma catecholamine levels; Cardiovascular effects of Ang-II with and without alpha-adrenoreceptor blockade; Relationship between plasma concentrations and their cardiovascular effects.

Focuses on the structure and function of the branchial chloride cell in freshwater fishes. Role of the chloride cell in ionic regulation and in acid-base regulation; Effects of chloride cell proliferation on blood-to-water diffusion distance; Effects of chloride cell proliferation on respiratory gas transfer.

Investigates the relationships between angiotensins and catecholamine release from the chromaffin tissue of rainbow trout. Evidence that angiotensins can elicit catecholamine release; Method in which angiotensins effects this release.

Investigates the relationships between angiotensins and catecholamine release from the chromaffin tissue of rainbow trout, using immunohistochemical and pharmacological techniques. Methodology and materials used for the study; Results of the study.

Assesses the control of catecholamine and serotonin release from the chromaffin tissue of the Atlantic hagfish. Saline-perfused systemic heart/posterior cardinal vein preparation; Potential modulatory effects of NECA, an adenosine receptor antagonist, on catecholamine release; Response to adrenocorticotropic hormone.

Evaluates the involvement of catecholamines in the ventilatory control and blood oxygen transport during hypoxia and hypercapnia in dogfish and rainbow trout. Plasma catecholamine levels; Hyperventilatory responses during hypoxia and hypercapnia; Effect of catecholamine administration on blood oxygen transport.

Reports that repeated physical stress significantly enhances the adrenergic responsiveness of the rainbow tout red blood cell. Experiments; Results; Discussion; Interrelationships between chronic and acute stress responses in fish.

Characterizes the acid-base disequilibrium in the arterial blood of rainbow trout Oncorhynchus mykiss during environmental hypoxia, hyperoxia or hypercapnia. Extracorporeal circulation; Measurement of pH, partial pressure of oxygen and carbon dioxide; Ventilation.

Examines the response of cannulated rainbow trout to acute hypoxia in fish acclimated to two temperatures (5 and 15 degrees Celsius). Effects of acclimation on catecholamine release; Effects of acclimation temperature on hemoglobin oxygen-affinity; Other potential causes/correlates of catecholamine release.

Presents results of in vivo and in situ experiments on the Atlantic hagfish Myxine glutinosa. Characterization of the levels of circulating catecholamines during acute stresses; Potential mechanisms of catecholamine release from the systemic heart and posterior cardinal vein; Possible involvement of pituitary hormones.

Evaluates several potential factors limiting carbon dioxide excretion in the rainbow trout. Hematocrit level; Lysis of red blood cells (RBC); Carbonic anhydrase; RBC intracellular acidity.

In zebrafish (Danio rerio), the ammonia-transporting Rhesus glycoprotein Rhcgb is implicated in mechanisms of ammonia excretion and Na+ uptake. In particular, Rhcgb is thought to play an important role in maintaining ammonia excretion in response to alkaline conditions and high external ammonia (HEA) exposure, in addition to facilitating Na+ uptake via a functional metabolon with the Na+/H+-exchanger Nhe3b, specifically under low Na+ conditions. In the present study, we hypothesized that CRISPR/Cas9 knockout of rhcgb would reduce ammonia excretion and Na+ uptake capacity, particularly under the conditions listed above that have elicited increases in Rhcgb-mediated ammonia excretion and/or Na+ uptake. Contrary to this hypothesis, however, larval and juvenile rhcgb knockout (KO) mutants showed no reductions in ammonia excretion or Na+ uptake under any of the conditions tested in our study. In fact, under control conditions, rhcgb KO mutants generally displayed an increase in ammonia excretion, potentially due to increased transcript abundance of another rh gene, rhbg. Under alkaline conditions, rhcgb KO mutants were also able to maintain ammonia excretion, similar to wild-type fish, and stimulation of ammonia excretion after HEA exposure also was not affected by rhcgb KO. Surprisingly, ammonia excretion and Na+ uptake were unaffected by rhcgb or nhe3b KO in juvenile zebrafish acclimated to normal (800 μmol/L) or low (10 μmol/L) Na+ conditions. These results demonstrate that Rhcgb is expendable for ammonia excretion and Na+ uptake in zebrafish, highlighting the plasticity and flexibility of these physiological systems in this species. Unlabelled Image • The ammonia-transporting protein Rhcgb was knocked out by CRISPR/Cas9 gene editing. • Ammonia excretion rates were elevated in rhcgb knockout (KO) mutant zebrafish. • Transcript abundance of another rh gene, rhbg , was increased in rhcgb KO mutants. • rhcgb KO had no effect on ammonia excretion in response to high pH or ammonia •.Na+ uptake and Na+ balance were unaffected by rhcgb or nhe3b KO in mature zebrafish. [ABSTRACT FROM AUTHOR]

Beginning with the discovery more than 35 years ago that oxygen chemoreceptors of the fish gill are enriched with serotonin, numerous studies have examined the importance of this, and other neuroendocrine factors in piscine chemoreceptor function, and in particular on the chemoreceptor-mediated reflex control of breathing. However, despite these studies, there is continued debate as to the role of neuroendocrine factors in the initiation or modulation of breathing during environmental disturbances or physical activity. In this review, we summarize the state-of-knowledge surrounding the neuroendocrine control of oxygen chemoreception in fish and the associated reflex adjustments to ventilation. We focus on neurohumoral substances that either are present in chemosensory cells or those that are localised elsewhere but have also been implicated in the direct control of breathing. These substances include serotonin, catecholamines (adrenaline and noradrenaline), acetylcholine, purines and gaseous neurotransmitters. Despite the growing indirect evidence for an involvement of these neuroendocrine factors in chemoreception and ventilatory control, direct evidence awaits the incorporation of novel methods currently under development. [ABSTRACT FROM AUTHOR]

The coordination of the hypoxic response is attributed, in part, to hypoxiainducible factor 1α (Hif-1α), a regulator of hypoxia-induced transcription. After the teleost-specific genome duplication, most teleost fishes lost the duplicate copy of Hif-1α, except species in the cyprinid lineage that retained both paralogues of Hif-1α (Hif1aa and Hif1ab). Little is known about the contribution of Hif-1α, and specifically of each paralogue, to hypoxia tolerance. Here, we examined hypoxia tolerance in wild-type (Hif1aa+/+ab+/+) and Hif-1α knockout lines (Hif1aa−/−; Hif1ab−/−; Hif1aa−/−ab−/−) of zebrafish (Danio rerio). Critical O2 tension (Pcrit; the partial pressure of oxygen (PO2) at which O2 consumption can no longer be maintained) and time to loss of equilibrium (LOE), two indices of hypoxia tolerance, were assessed in larvae and adults. Knockout of both paralogues significantly increased Pcrit (decreased hypoxia tolerance) in larval fish. Prior exposure of larvae to hypoxia decreased Pcrit in wild-type fish, an effect mediated by the Hif1aa paralogue. In adults, individuals with a knockout of either paralogue exhibited significantly decreased time to LOE but no difference in Pcrit. Together, these results demonstrate that in zebrafish, tolerance to hypoxia and improved hypoxia tolerance after pre-exposure to hypoxia (pre-conditioning) are mediated, at least in part, by Hif-1α. [ABSTRACT FROM AUTHOR]

Purely diffusive O2 transport typically is insufficient to sustain aerobic metabolism in most multicellular organisms. In animals that are small enough, however, a high surface-to-volume ratio may allow passive diffusion alone to supply sufficient O2 transfer. The purpose of this study was to explore the impacts of internal convection on respiratory gas transfer in a small complex organism, the larval zebrafish (Danio rerio). Specifically, we tested the hypothesis that internal convection is required for the normal transfer of the respiratory gases O2 and CO2 and maintenance of resting aerobic metabolic rate in larvae at 4 days postfertilization (dpf). Morpholino knockdown of the vascular endothelial growth factor (VEGF) or cardiac troponin T (TNNT2) proteins allowed an examination of gas transfer in two independent models lacking internal convection. With the use of a scanning micro-optrode technique to measure regional epithelial O2 fluxes (JO2), it was demonstrated that larvae lacking convection exhibited reduced JO2 in regions spanning the head to the trunk. Moreover, the acute loss of internal convection caused by heart stoppage resulted in reduced rates of cutaneous JO2, an effect that was reversed upon the restoration of internal convection. With the use of whole body respirometry, it was shown that loss of internal convection was associated with reduced resting rates of O2 consumption and CO2 excretion in larvae at 4 dpf. The results of these experiments clearly demonstrate that internal convection is required to maintain resting rates of respiratory gas transfer in larval zebrafish. [ABSTRACT FROM AUTHOR]