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A fundamental requirement for all animals is to sense and respond to changes in environmental O 2 availability. Low O 2 (hypoxia) typically stimulates breathing, a universal and critical response termed the hypoxic ventilatory response (HVR). In this study, we test the hypothesis that taste-signaling pathways are used for O 2 sensing and activation of the HVR. We show that Merkel-like cells (MLCs), which are part of the taste-bud complex, function as O 2 chemoreceptor cells in larval zebrafish and that transduction of the O 2 signal uses taste-signaling pathways. Specifically, MLCs responded to hypoxia in vivo with an increase in Ca 2+ activity that can drive the HVR. In addition, MLCs transmit O 2 signals to afferent cranial nerves IX and X (nIX/X), which project into the area postrema within the hindbrain and synapse with interneurons that are in contact with vagal motor neurons. Hypoxia or chemo-activation of nIX/X caused Ca 2+ activity to increase within the area postrema and elicited hyperventilation. The results provide the first demonstration of an O 2 signaling pathway that commences with the activation of taste receptors (MLCs) to yield a critical physiological reflex, the HVR., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Zebrafish (Danio rerio) are unusual in having two paralogues of the serotonin re-uptake transporter (Sert), slc6a4a (serta) and slc6a4b (sertb), the transporter that serves in serotonin re-uptake from a synapse into the pre-synaptic cell or in serotonin uptake from the extracellular milieu into cells in the peripheral tissues. To address a knowledge gap concerning the specific roles of these paralogues, we used CRISPR/Cas9 technology to generate zebrafish knockout lines predicted to lack functional expression of Serta or Sertb. The consequences of loss-of-function of Serta or Sertb were assessed at the gene expression level, focusing on the serotonergic signalling pathway, and at the behaviour level, focusing on aggression. Whereas serta mRNA was expressed in all tissues examined, with high expression in the heart, gill and brain, only the brain displayed substantial sertb mRNA expression. In both serta -/- and sertb -/- fish, changes in transcript abundances of multiple components of the serotonin signalling pathway were detected, including proteins involved in serotonin synthesis (tph1a, tph1b, tph2, ddc), packaging (vmat2) and degradation (mao), and serotonin receptors (htr1aa, htr1ab). Using a mirror aggression test, serta -/- male but not female fish exhibited greater aggression than wildtype fish. However, both male and female sertb -/- fish displayed less aggression than their wildtype counterparts. These differences in behaviour between serta -/- and sertb -/- individuals hold promise for increasing our understanding of the neurophysiological basis of aggression in zebrafish., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Adrenaline and noradrenaline, released as hormones and/or neurotransmitters, exert diverse physiological functions in vertebrates, and teleost fishes are widely used as model organisms to study adrenergic regulation; however, such investigations often rely on receptor subtype-specific pharmacological agents (agonists and antagonists; see Glossary) developed and validated in mammals. Meanwhile, evolutionary (phylogenetic and comparative genomic) studies have begun to unravel the diversification of adrenergic receptors (ARs) and reveal that whole-genome duplications and pseudogenization events in fishes results in notable distinctions from mammals in their genomic repertoire of ARs, while lineage-specific gene losses within teleosts have generated significant interspecific variability. In this Review, we visit the evolutionary history of ARs (including α1-, α2- and β-ARs) to highlight the prominent interspecific differences in teleosts, as well as between teleosts and other vertebrates. We also show that structural modelling of teleost ARs predicts differences in ligand binding affinity compared with mammalian orthologs. To emphasize the difficulty of studying the roles of different AR subtypes in fish, we collate examples from the literature of fish ARs behaving atypically compared with standard mammalian pharmacology. Thereafter, we focus on specific case studies of the liver, heart and red blood cells, where our understanding of AR expression has benefited from combining pharmacological approaches with molecular genetics. Finally, we briefly discuss the ongoing advances in 'omics' technologies that, alongside classical pharmacology, will provide abundant opportunities to further explore adrenergic signalling in teleosts., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The study of breathing in fishes has featured prominently in Journal of Experimental Biology (JEB), particularly during the latter half of the past century. Indeed, many of the seminal discoveries in this important sub-field of comparative respiratory physiology were reported first in JEB. The period spanning 1960-1990 (the 'golden age of comparative respiratory physiology') witnessed intense innovation in the development of methods to study the control of breathing. Many of the guiding principles of piscine ventilatory control originated during this period, including our understanding of the dominance of O2 as the driver of ventilation in fish. However, a critical issue - the identity of the peripheral O2 chemoreceptors - remained unanswered until methods for cell isolation, culture and patch-clamp recording established that gill neuroepithelial cells (NECs) respond to hypoxia in vitro. Yet, the role of the NECs and other putative peripheral or central chemoreceptors in the control of ventilation in vivo remains poorly understood. Further progress will be driven by the implementation of genetic tools, most of which can be used in zebrafish (Danio rerio). These tools include CRISPR/Cas9 for selective gene knockout, and Tol2 systems for transgenesis, the latter of which enables optogenetic stimulation of cellular pathways, cellular ablation and in vivo cell-specific biosensing. Using these methods, the next period of discovery will see the identification of the peripheral sensory pathways that initiate ventilatory responses, and will elucidate the nature of their integration within the central nervous system and their link to the efferent motor neurons that control breathing., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Adjustments of ventilation in fishes to regulate the volume of water flowing over the gills are critically important responses to match branchial gas transfer with metabolic needs and to defend homeostasis during environmental fluctuations in O 2 and/or CO 2 levels. In this focused review, we discuss the control and consequences of ventilatory adjustments in fish, briefly summarizing ventilatory responses to hypoxia and hypercapnia before describing the current state of knowledge of the chemoreceptor cells and molecular mechanisms involved in sensing O 2 and CO 2 . We emphasize, where possible, insights gained from studies on early developmental stages. In particular, zebrafish ( Danio rerio ) larvae have emerged as an important model for investigating the molecular mechanisms of O 2 and CO 2 chemosensing as well as the central integration of chemosensory information. Their value stems, in part, from their amenability to genetic manipulation, which enables the creation of loss-of-function mutants, optogenetic manipulation, and the production of transgenic fish with specific genes linked to fluorescent reporters or biosensors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Perry, Pan and Gilmour.)

The hypoxic ventilatory response (HVR) in fish is an important reflex that aids O2 uptake when low environmental O2 levels constrain diffusion. In developing zebrafish (Danio rerio), the acute HVR is multiphasic, consisting of a rapid increase in ventilation frequency (fV) during hypoxia onset, followed by a decline to a stable plateau phase above fV under normoxic conditions. In this study, we examined the potential role of catecholamines in contributing to each of these phases of the dynamic HVR in zebrafish larvae. We showed that adrenaline elicits a dose-dependent β-adrenoreceptor (AR)-mediated increase in fV that does not require expression of β1-ARs, as the hyperventilatory response to β-AR stimulation was unaltered in adrb1-/- mutants, generated by CRISPR/Cas9 knockout. In response to hypoxia and propranolol co-treatment, the magnitude of the rapidly occurring peak increase in fV during hypoxia onset was attenuated (112±14 breaths min-1 without propranolol to 68±17 breaths min-1 with propranolol), whereas the increased fV during the stable phase of the HVR was prevented in both wild type and adrb1-/- mutants. Thus, β1-AR is not required for the HVR and other β-ARs, although not required for initiation of the HVR, are involved in setting the maximal increase in fV and in maintaining hyperventilation during continued hypoxia. This adrenergic modulation of the HVR may arise from centrally released catecholamines because adrenaline exposure failed to activate (based on intracellular Ca2+ levels) cranial nerves IX and X, which transmit O2 signals from the pharyngeal arch to the central nervous system., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Across species, including humans, elevated levels of brain estrogen receptor (ER) α are associated with enhanced cognitive aging, even in the absence of circulating estrogens. In rodents, short-term estrogen treatment, such as that commonly used in the menopausal transition, results in long-term increases in ERα levels in the hippocampus, leading to enhanced memory long after termination of estrogen treatment. However, mechanisms by which increased levels of brain ERα enhances cognitive aging remain unclear. Here we demonstrate in aging female rats that insulin-like growth factor-1 (IGF-1), which can activate ER via ligand-independent mechanisms, requires concomitant synthesis of brain-derived neuroestrogens to phosphorylate ERα via MAPK signaling, ultimately resulting in enhanced memory. In a rat model of menopause involving long-term ovarian hormone deprivation, hippocampal neuroestrogen activity decreases, altering IGF-1 activity and resulting in impaired memory. However, this process is reversed by short-term estradiol treatment. Forty days of estradiol exposure following ovariectomy results in maintenance of neuroestrogen levels that persist beyond the period of hormone treatment, allowing for continued interactions between IGF-1 and neuroestrogen signaling, elevated levels of hippocampal ERα, and ultimately enhanced memory. Collectively, results demonstrate that short-term estradiol use following loss of ovarian function has long-lasting effects on hippocampal function and memory by dynamically regulating cellular mechanisms that promote activity of ERα in the absence of circulating estrogens. Translational impacts of these findings suggest lasting cognitive benefits of short-term estrogen use near menopause and highlight the importance of hippocampal ERα, independent from the role of circulating estrogens, in regulating memory in aging females. SIGNIFICANCE STATEMENT Declines in ovarian hormones following menopause coincide with increased risk of cognitive decline. Because of potential health risks, current recommendations are that menopausal estrogen therapy be limited to a few years. Long-term consequences for the brain and memory of this short-term midlife estrogen therapy are unclear. Here, in a rodent model of menopause, we determined mechanisms by which short-term midlife estrogen exposure can enhance hippocampal function and memory with cognitive benefits and molecular changes enduring long after termination of estrogen exposure. Our model indicates long-lasting benefits of maintaining hippocampal estrogen receptor function in the absence of ongoing estrogen exposure and suggests potential strategies for combating age-related cognitive decline., (Copyright © 2022 Baumgartner et al.)

Ammonia excretion in fish excretory epithelia is a complex interplay of multiple membrane transport proteins and mechanisms. Using the model system of zebrafish (Danio rerio) larvae, here we identified three paralogues of a novel ammonia transporter, hippocampus-abundant transcript 1 (DrHiat1), also found in most vertebrates. When functionally expressed in Xenopus laevis oocytes, DrHiat1a and DrHiat1b promoted methylamine uptake in a competitive manner with ammonia. In situ hybridization experiments showed that both transporters were expressed as early as the 4-cell stage in zebrafish embryos and could be identified in most tissues 4 days post-fertilization. Larvae experiencing morpholino-mediated knockdown of DrHiat1b exhibited significantly lower whole-body ammonia excretion rates compared with control larvae. Markedly decreased site-specific total ammonia excretion of up to 85% was observed in both the pharyngeal region (site of developing gills) and the yolk sac (region shown to have the highest NH4+ flux). This study is the first to identify DrHiat1b/DrHIAT1 in particular as an important contributor to ammonia excretion in larval zebrafish. Being evolutionarily conserved, these proteins are likely involved in multiple other general ammonia-handling mechanisms, making them worthy candidates for future studies on nitrogen regulation in fishes and across the animal kingdom., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The sensing of environmental fluctuations and initiation of appropriate physiological responses is crucial to homeostasis. Neuroepithelial cells (NECs) in fishes are putative chemoreceptors, resembling mammalian Type I (glomus) cells, that respond in vitro to changes in O 2 , CO 2 , NH 3 , and pH. Cytosolic carbonic anhydrase (Ca17a) is thought to be involved in CO 2 sensing owing to its presence in NECs. Zebrafish ( Danio rerio ) lacking functional Ca17a were generated via CRISPR/Cas9 technology and used to assess the role of Ca17a in initiating the cardiorespiratory responses to elevated CO 2 (hypercapnia). Unfortunately, the homozygous knockout mutants ( ca17a -/- ) did not survive more than ∼12-14 days postfertilization (dpf), restricting experiments to early developmental stages (4-8 dpf). Changes in ventilation ( f V ) and cardiac ( f H ) frequency in response to hypercapnia (1% CO 2 ) in wild-type ( ca17a +/+ ), heterozygous ( ca17a +/- ) and ca17a -/- fish were used to investigate Ca17a-dependent CO 2 sensing and downstream signaling. Wild-type fish exhibited hyperventilation during hypercapnia as indicated by an increase in f V . In the ca17a -/- fish, the hyperventilatory response was attenuated markedly but only at 8 dpf. Hypercapnic tachycardia was observed for all genotypes and did not appear to be influenced by the absence of Ca17a. Interestingly, ca17a -/- fish exhibited a significantly lower resting f H that became more pronounced as the fish aged. The decrease in resting f H was prevented ("rescued") when ca17a -/- embryos were injected with ca17a mRNA. Collectively, the results of this study support a role for Ca17a in promoting hyperventilation during hypercapnia in larval zebrafish and suggest a previously unrecognized role for Ca17a in determining resting heart rate.

Aim: Although zebrafish are gaining popularity as biomedical models of cardiovascular disease, our understanding of their cardiac control mechanisms is fragmentary. Our goal was to clarify the controversial role of the ß1-adrenergic receptor (AR) in the regulation of heart rate in zebrafish., Methods: CRISPR-Cas9 was used to delete the adrb1 gene in zebrafish allowing us to generate a stable adrb1 -/- line. Larval heart rates were measured during pharmacological protocols and with exposure to hypercapnia. Expression of the five zebrafish adrb genes were measured in larval zebrafish hearts using qPCR., Results: Compared with genetically matched wild-types (adrb1 +/+ ), adrb1 -/- larvae exhibited ~20 beats min -1 lower heart rate, measured from 2 to 21 days post-fertilization (dpf). Nevertheless, adrb1 -/- larvae exhibited preserved positive chronotropic responses to pharmacological treatment with AR agonists (adrenaline, noradrenaline, isoproterenol), which were blocked by propranolol (general ß-AR antagonist). Regardless of genotype, larvae exhibited similar increases in heart rate in response to hypercapnia (1% CO 2 ) at 5 dpf, but tachycardia was blunted in adrb1 -/- larvae at 6 dpf. adrb1 gene expression was abolished in the hearts of adrb1 -/- larvae, confirming successful knockout. While gene expression of adrb2a and adrb3a was unchanged, adrb2b and adrb3b mRNA levels increased in adrb1 -/- larval hearts., Conclusion: Despite adrb1 contributing to the setting of resting heart rate in larvae, it is not strictly essential for zebrafish, as we generated a viable and breeding adrb1 -/- line. The chronotropic effects of adrenergic stimulation persist in adrb1 -/- zebrafish, likely due to the upregulation of other ß-AR subtypes., (© 2022 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Hypoxia-inducible factor 1-α (Hif-1α), an important transcription factor regulating cellular responses to reductions in O 2 , previously was shown to improve hypoxia tolerance in zebrafish ( Danio rerio ). Here, we examined the contribution of Hif-1α to hypoxic survival, focusing on the benefit of aquatic surface respiration (ASR). Wild-type and Hif-1α knockout lines of adult zebrafish were exposed to two levels (moderate or severe) of intermittent hypoxia. Survival was significantly compromised in Hif-1α knockout zebrafish prevented from accessing the surface during severe (16 mmHg) but not moderate (23 mmHg) hypoxia. When allowed access to the surface in severe hypoxia, survival times did not differ between wild-type and Hif-1α knockouts. Performing ASR mitigated the negative effects of the loss of Hif-1α with the knockouts initiating ASR at a higher P O 2 threshold and performing ASR for longer than wild-types. The loss of Hif-1α had little impact on survival in fish between 1 and 5 days post-fertilization, but as the larvae aged, their reliance on Hif-1α increased. Similar to adult fish, ASR compensated for the loss of Hif-1α on survival. Together, these results demonstrate that age, hypoxia severity and, in particular, the ability to perform ASR significantly modulate the impact of Hif-1α on survival in hypoxic zebrafish.

In the current study, adult zebrafish (Danio rerio) were exposed to 72 h hypoxia (90 mmHg) to assess the time domains of the hypoxia ventilatory response (HVR) and the consequence on a subsequent more severe (40 mmHg) bout of acute hypoxia. Experiments were performed on wild-type fish and mutants in which one or both paralogs of hypoxia inducible factor-1α (hif-1α) were knocked out. Although there were subtle differences among the wild-type and knockout fish, resting f V was reestablished after 2-8 h of continuous hypoxia in both groups, a striking example of hypoxic ventilatory decline (HVD). When fish were subsequently exposed to more severe hypoxia, a rapid increase in f V was observed, the magnitude of which was independent of genotype or prior exposure history. During recovery, fish that had been exposed to 72 h of 90 mmHg hypoxia exhibited a pronounced undershoot in f V , which was absent in the hif-1α double knockouts. Overall, the results revealed distinct time domains of the HVR in zebrafish that were largely Hif-1α-independent., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The hypoxia-inducible factor (HIF) pathway is a key regulator of cellular O2 homeostasis and an important orchestrator of the physiological responses to hypoxia (low O2) in vertebrates. Fish can be exposed to significant and frequent changes in environmental O2, and increases in Hif-α (the hypoxia-sensitive subunit of the transcription factor Hif) have been documented in a number of species as a result of a decrease in O2. Here, we discuss the impact of the Hif pathway on the hypoxic response and the contribution to hypoxia tolerance, particularly in fishes of the cyprinid lineage, which includes the zebrafish (Danio rerio). The cyprinids are of specific interest because, unlike in most other fishes, duplicated paralogs of the Hif-α isoforms arising from a teleost-specific genome duplication event have been retained. Positive selection has acted on the duplicated paralogs of the Hif-α isoforms in some cyprinid sub-families, pointing to adaptive evolutionary change in the paralogs. Thus, cyprinids are valuable models for exploring the evolutionary significance and physiological impact of the Hif pathway on the hypoxic response. Knockout in zebrafish of either paralog of Hif-1α greatly reduces hypoxia tolerance, indicating the importance of both paralogs to the hypoxic response. Here, with an emphasis on the cardiorespiratory system, we focus on the role of Hif-1α in the hypoxic ventilatory response and the regulation of cardiac function. We explore the effects of the duration of the hypoxic exposure (acute, sustained or intermittent) on the impact of Hif-1α on cardiorespiratory function and compare relevant data with those from mammalian systems., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© 2021 Wiley Periodicals LLC.)

Previous studies have demonstrated that hypoxia tolerance is improved in zebrafish (Danio rerio) larvae after prior exposure to lowered ambient O 2 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 O 2 uptake ([Formula: see text]) in larvae is facilitated largely by O 2 diffusion across the skin, enhanced cutaneous vascularization is expected to enhance [Formula: see text] 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 O 2 fluxes (JO 2 ). Hypoxia tolerance, as determined by measurements of critical PO 2 (P crit ), was unaltered by hypoxia pre-exposure in larvae at 4 dpf and there were no significant differences in P crit 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 P crit than hif1aa -/- ab -/- larvae, an effect that was being driven by a reduced P crit 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, JO 2 decreased along the body in the anterior-to-posterior direction. Neither hypoxia pre-exposure nor genotype affected JO 2 at any region along the body. The lack of any effect of hypoxia pre-exposure or genotype on JO 2 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 P crit ) at 7 dpf in the hif1aa -/- ab -/- larvae did not appear to be reliant on changes in trunk vascularity or cutaneous O 2 diffusion.

Oxygen uptake (ṀO 2 ) in larval zebrafish prior to maturation of the gill relies on cutaneous O 2 transfer. Under normoxic conditions, rates of cutaneous O 2 transfer are unaffected by haemoglobin availability but are diminished in fish lacking a functional circulatory system, suggesting that internal convection is critically involved in setting the resting ṀO 2 in zebrafish larvae, even when relying on cutaneous O 2 transfer. The reliance of ṀO 2 on blood circulation led to the first objective of the current study, to determine whether loss of internal convection would reduce acute hypoxia performance (as determined by measuring critical PO 2 ; P crit ) in larval zebrafish under conditions of moderate hypoxia (PO 2  = 55 mmHg) at 28.5 and 34 °C. Internal convection was eliminated by preventing development of blood vessels using morpholino knockdown of vascular endothelial growth factor (VEGF); these fish are termed VEGF morphants. Breathing frequency (f V ) and heart rate (f H ) also were measured (at 28.5 °C) to determine whether any detriment in performance might be linked to cardiorespiratory dysfunction. Although ṀO 2 was reduced in the VEGF morphants, there was no significant effect on P crit at 28.5 °C. Raising temperature to 34 °C resulted in the VEGF morphants exhibiting a higher P crit than the shams, suggesting an impairment of hypoxia tolerance in the morphants at the higher temperature. The usual robust increase in f V during hypoxia was absent or attenuated in VEGF morphants at 4 and 5 days post fertilization (dpf), respectively. Resting f H was reduced in the VEGF morphants and unlike the sham fish, the morphants did not exhibit hypoxic tachycardia at 4 or 5 dpf. The number of cutaneous neuroepithelial cells (presumptive O 2 chemoreceptors) was significantly higher in the VEGF morphants and thus the cardiorespiratory impairment in the morphants during hypoxia was unlikely related to inadequate peripheral O 2 sensing.

Serotonergic neuroepithelial cells (NECs) in larval zebrafish are believed to be O 2 chemoreceptors. Serotonin (5-HT) within these NECs has been implicated as a neurotransmitter mediating the hypoxic ventilatory response (HVR). Here, we use knockout approaches to discern the role of 5-HT in regulating the HVR by targeting the rate limiting enzyme for 5-HT synthesis, tryptophan hydroxylase (Tph). Using transgenic lines, we determined that Tph1a is expressed in skin and pharyngeal arch NECs, as well as in pharyngeal arch Merkel-like cells (MLCs), whereas Tph1b is expressed predominately in MLCs. Knocking out the two tph1 paralogs resulted in similar changes in detectable serotonergic cell density between the two mutants, yet their responses to hypoxia (35 mmHg) were different. Larvae lacking Tph1a (tph1a -/- mutants) displayed a higher ventilation rate when exposed to hypoxia compared to wild-types, whereas tph1b -/- mutants exhibited a lower ventilation rate suggesting that 5-HT located in locations other than NECs, may play a dominant role in regulating the HVR., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The effects of high external ammonia (HEA) exposure on breathing and the potential involvement of ammonia transporting Rh proteins in ammonia sensing were assessed in larval and adult zebrafish. Acute exposure of adults to either 250 or 500 μM (NH 4 ) 2 SO 4 caused increases in ventilation amplitude (A VENT ) without affecting frequency (f VENT ), resembling the ventilatory response to hypercapnia rather than hypoxia, during which f VENT was increased exclusively. The hyperventilatory response to HEA was prevented by hyperoxia, indicating that control of breathing through ammonia sensing is likely secondary to O 2 chemoreception. Neuroepithelial cells (NECs) isolated from gill filaments exhibited a significant increase of intracellular [Ca 2+ ] in response to 1 mM NH 4 Cl but this response was small (roughly 30%) compared to the response to hypercapnia (37.5 mmHg; ~800% increase). Immunohistochemistry (IHC) failed to reveal the presence of Rh proteins (Rhcgb, Rhbg or Rhag) in gill filament NECs. Knockout of rhcgb did not affect the ventilatory response of adults to HEA. Larvae at 4 days post fertilization (dpf) responded to HEA with increases in f VENT (A VENT was not measured). The hyperventilatory response of larvae to HEA was attenuated (60% reduction) after treatment from 0 to 4 dpf with the sympathetic neurotoxin 6-hydroxydopamine. In larvae, Rhcgb, Rhbg and Rhag were undetectable by IHC in cutaneous NECs yet the f VENT to HEA following Rhbg knockdown was slightly (22%) attenuated. Thus, the hyperventilatory response to external ammonia in adult zebrafish, while apparently initiated by activation of NECs, does not require Rhcgb, nor is the entry of ammonia into NECs reliant on other Rh proteins. The lack of colocalization of Rh proteins with NECs suggests that the entry of ammonia into NECs in larvae, also is not facilitated by this family of ammonia channels., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish ( Danio rerio ) is expressed abundantly in Na + -absorbing/H + -secreting H + -ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a -/- mutants exhibiting a significant decrease in survival beginning at ∼12 days postfertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a -/- mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na + uptake rates were significantly increased by up to 300% in homozygous mutants compared with wild-type individuals at 4 and 9 dpf; however, whole body Na + content remained constant. While Cl - uptake was significantly reduced in ca17a -/- mutants, Cl - content was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl - uptake, implicating Ca17a in the mechanism of Cl - uptake by larval zebrafish. H + secretion, O 2 consumption, CO 2 excretion, and ammonia excretion were generally unaltered in ca17a -/- mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl - uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.

In zebrafish ( Danio rerio ), a specific ionocyte subtype, the H + -ATPase-rich (HR) cell, is presumed to be a significant site of transepithelial Na + uptake/acid secretion. During acclimation to environments differing in ionic composition or pH, ionic and acid-base regulations are achieved by adjustments to the activity level of HR cell ion transport proteins. In previous studies, the quantitative assessment of mRNA levels for genes involved in ionic and acid-base regulations relied on measurements using homogenates derived from the whole body (larvae) or the gill (adult). Such studies cannot distinguish whether any differences in gene expression arise from adjustments of ionocyte subtype numbers or transcriptional regulation specifically within individual ionocytes. The goal of the present study was to use fluorescence-activated cell sorting to separate the HR cells from other cellular subpopulations to facilitate the measurement of gene expression of HR cell-specific transporters and enzymes from larvae exposed to low pH (pH 4.0) or low Na + (5 μM) conditions. The data demonstrate that treatment of larvae with acidic water for 4 days postfertilization caused cell-specific increases in H + -ATPase ( atp6v1aa ), ca17a, ca15a , nhe3b , and rhcgb mRNA in addition to increases in mRNA linked to cell proliferation. In fish exposed to low Na + , expression of nhe3b and rhcgb was increased owing to HR cell-specific regulation and elevated numbers of HR cells. Thus, the results of this study demonstrate that acclimation to low pH or low Na + environmental conditions is facilitated by HR cell-specific transcriptional control and by HR cell proliferation.

Fishes living in fresh water counter the passive loss of salts by actively absorbing ions through specialized cells termed ionocytes. Ionocytes contain ATP-dependent transporters and are enriched with mitochondria; therefore ionic regulation is an energy-consuming process. The purpose of this study was to assess the aerobic costs of ion transport in larval zebrafish ( Danio rerio ). We hypothesized that changes in rates of Na + uptake evoked by acidic or low Na + rearing conditions would result in corresponding changes in whole-body oxygen consumption ( Ṁ O 2 ) and/or cutaneous oxygen flux ( J O 2 ), measured at the ionocyte-expressing yolk sac epithelium using the scanning micro-optrode technique (SMOT). Larvae at 4 days post-fertilization (dpf) that were reared under low pH (pH 4) conditions exhibited a higher rate of Na + uptake compared with fish reared under control conditions (pH 7.6), yet they displayed a lower Ṁ O 2  and no difference in cutaneous J O 2 Despite a higher Na + uptake capacity in larvae reared under low Na + conditions, there were no differences in Ṁ O 2  and J O 2  at 4 dpf. Furthermore, although Na + uptake was nearly abolished in 2 dpf larvae lacking ionocytes after morpholino knockdown of the ionocyte proliferation regulating transcription factor foxi3a , Ṁ O 2   and J O 2  were unaffected. Finally, laser ablation of ionocytes did not affect cutaneous J O 2 Thus, we conclude that the aerobic costs of ion uptake by ionocytes in larval zebrafish, at least in the case of Na + , are below detection using whole-body respirometry or cutaneous SMOT scans, providing evidence that ion regulation in zebrafish larvae incurs a low aerobic cost., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Peripheral chemosensitivity in fishes is thought to be mediated by serotonin-enriched neuroepithelial cells (NECs) that are localized to the gills of adults and the integument of larvae. In adult zebrafish ( Danio rerio ), branchial NECs are presumed to mediate the cardiorespiratory reflexes associated with hypoxia or hypercapnia, whereas in larvae, there is indirect evidence linking cutaneous NECs to hypoxic hyperventilation and hypercapnic tachycardia. No study yet has examined the ventilatory response of larval zebrafish to hypercapnia, and regardless of developmental stage, the signaling pathways involved in CO 2 sensing remain unclear. In the mouse, a background potassium channel (TASK-2) contributes to the sensitivity of chemoreceptor cells to CO 2 . Zebrafish possess two TASK-2 channel paralogs, TASK-2 and TASK-2b, encoded by kcnk5a and kcnk5b , respectively. The present study aimed to determine whether TASK-2 channels are expressed in NECs of larval zebrafish and whether they are involved in CO 2 sensing. Using immunohistochemical approaches, TASK-2 protein was observed on the surface of NECs in larvae. Exposure of larvae to hypercapnia caused cardiac and breathing frequencies to increase, and these responses were blunted in fish experiencing TASK-2 and/or TASK-2b knockdown. The results of these experiments suggest that TASK-2 channels are involved in CO 2 sensing by NECs and contribute to the initiation of reflex cardiorespiratory responses during exposure of larvae to hypercapnia.

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

The rapid increase in critical thermal maximum (CT max ) in fish (or other animals) previously exposed to critically high temperature is termed 'heat hardening', which likely represents a key strategy to cope with increasingly extreme environments. The physiological mechanisms that determine acute thermal tolerance, and the underlying pathways facilitating heat hardening, remain debated. It has been posited, however, that exposure to high temperature is associated with tissue hypoxia and may be associated with the increased expression of hypoxia-inducible factor-1 (Hif-1). We studied acute thermal tolerance in zebrafish ( Danio rerio ) lacking functional Hif-1 α paralogs (Hif-1aa and Hif-1ab double knockout; Hif-1 α -/- ), which are known to exhibit markedly reduced hypoxia tolerance. We hypothesized that Hif-1 α -/- zebrafish would suffer reduced acute thermal tolerance relative to wild type and that the heat hardening ability would be lost. However, on the contrary, we observed that Hif-1 α -/- and wild-type fish did not differ in CT max , and both genotypes exhibited heat hardening of a similar degree when CT max was re-tested 48 h later. Despite exhibiting impaired hypoxia tolerance, Hif-1 α -/- zebrafish display unaltered thermal tolerance, suggesting that these traits are not necessarily functionally associated. Hif-1 α is accordingly not required for short-term acclimation in the form of heat hardening.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The coordination of the hypoxic response is attributed, in part, to hypoxia-inducible 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 O 2 tension ( P crit ; the partial pressure of oxygen (PO 2 ) at which O 2 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 P crit (decreased hypoxia tolerance) in larval fish. Prior exposure of larvae to hypoxia decreased P crit 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 P crit . 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.

Fish increase ventilation during hypoxia, a reflex termed the hypoxic ventilatory response (HVR). The HVR is an effective mechanism to increase O 2 uptake, but at a high metabolic cost. Therefore, when hypoxia becomes severe enough, ventilation declines, as its benefit is diminished. The water oxygen partial pressure ( P w O 2 ) at which this decline occurs is expected to be near the critical P w O 2  ( P crit ), the P w O 2  at which O 2 consumption begins to decline. Our results indicate that in zebrafish ( Danio rerio ), the relationship between peak HVR and P crit is dependent on developmental stage. Peak ventilation occurred at P w O 2  values higher than P crit in larvae, but at a P w O 2  significantly lower than P crit in adults. Larval zebrafish use cutaneous respiration to a greater extent than branchial respiration and the cost of sustaining the HVR may outweigh the benefit, whereas adult zebrafish, which rely on branchial respiration, may benefit from using HVR at P w O 2  below P crit ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The H + -ATPase-rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na + uptake and H + extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. Here, a ratiometric imaging technique was developed and validated to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo Gene knockdown or CRISPR/Cas9 knockout approaches were used to evaluate the roles of the two principal apical membrane acid excretory pathways, the Na + /H + exchanger (NHE3b; slc9a3.2 ) and the H + -ATPase ( atpv1aa ). Additionally, the role of HR cell cytosolic carbonic anhydrase (CAc) was investigated because of its presumed role in providing H + for Na + /H + exchange and H + -ATPase. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO 2 and the extent of post-CO 2 alkalisation were altered markedly in fish experiencing knockdown/knockout of CAc, NHE3b or H + -ATPase. Although there were slight differences among the three knockdown/knockout experiments, the typical response was a greater degree of intracellular acidification during CO 2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. The metabolic alkalosis and subsequent acidification associated with 20 mmol l -1 NH 4 Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Freshwater fishes absorb Na + from their dilute environment using ion-transporting cells. In larval zebrafish ( Danio rerio ), Na + uptake is coordinated by (1) Na + /H + exchanger 3b (Nhe3b) and (2) H + -ATPase-powered electrogenic uptake in H + -ATPase-rich (HR) cells and by (3) Na + -Cl - -cotransporter (Ncc) expressed in NCC cells. The present study aimed to better understand the roles of these three proteins in Na + uptake by larval zebrafish under 'normal' (800 µmol l -1 ) and 'low' (10 µmol l -1 ) Na + conditions. We hypothesized that Na + uptake would be reduced by CRISPR/Cas9 knockout (KO) of slc9a3.2 (encoding Nhe3b), particularly in low Na + where Nhe3b is believed to play a dominant role. Contrary to this hypothesis, Na + uptake was sustained in nhe3b KO larvae under both Na + conditions, which led to the exploration of whether compensatory regulation of H + -ATPase or Ncc was responsible for maintaining Na + uptake in nhe3b KO larvae. mRNA expression of the genes encoding H + -ATPase and Ncc was not altered in nhe3b KO larvae. Moreover, morpholino knockdown of H + -ATPase, which significantly reduced H + flux by HR cells, did not reduce Na + uptake in nhe3b KO larvae, nor did rearing larvae in chloride-free conditions, thereby eliminating any driving force for Na + -Cl - -cotransport via Ncc. Finally, simultaneously treating nhe3b KO larvae with H + -ATPase morpholino and chloride-free conditions did not reduce Na + uptake under normal or low Na + These findings highlight the flexibility of the Na + uptake system and demonstrate that Nhe3b is expendable to Na + uptake in zebrafish and that our understanding of Na + uptake mechanisms in this species is incomplete., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Convective water flow across respiratory epithelia in water-breathing organisms maintains transcutaneous oxygen (O 2 ) partial pressure (Po 2 ) gradients that drive O 2 uptake. Following hatch, larval fishes lack a developed gill and the skin is the dominant site of gas transfer, yet few studies have addressed the contribution of convective water flow to cutaneous O 2 uptake in larvae. We hypothesized that the pectoral fins, which can generate water flow across the skin in larvae, promote transcutaneous O 2 transfer and thus aid in O 2 uptake. In zebrafish ( Danio rerio ), the frequency of pectoral fin movements increased in response to hypoxia at 4 days postfertilization (dpf), but the response was blunted by 15 dpf, when the gills become the dominant site of O 2 uptake, and was absent by 21 dpf. In rainbow trout ( Oncorhynchus mykiss ), Po 2 measured at the skin surface of ventilating larvae was lower when the pectoral fins had been surgically removed, directly demonstrating that fins contribute to convective flow that dissipates cutaneous Po 2 boundary layers. Lack of pectoral fins compromised whole animal O 2 consumption in trout during hypoxia, but this effect was absent in zebrafish. Overall, our findings support a respiratory role of the pectoral fins in rainbow trout, but their involvement in zebrafish remains equivocal.

Freshwater (FW) fishes inhabit dilute environments and must actively absorb ions in order to counteract diffusive salt loss. Neuroendocrine control of ion uptake in FW fishes is an important feature of ion homeostasis and several important neuroendocrine factors have been identified. The role of serotonin (5-HT), however, has received less attention despite several studies pointing to a role for 5-HT in the control of ion balance. Here, we used a gene knockout approach to elucidate the role of 5-HT in regulating Na + and Ca 2+ uptake rates in larval zebrafish. Tryptophan hydroxylase (TPH) is the rate-limiting step in 5-HT synthesis and we therefore hypothesized that ion uptake rates would be altered in zebrafish larvae carrying knockout mutations in tph genes. We first examined the effect of tph1b knockout (KO) and found that tph1bKO larvae, obtained from Harvard University, had reduced rates of Na + and Ca 2+ uptake compared to wild-type (WT) larvae from our institution (uOttawa WT), lending support to our hypothesis. However, further experiments controlling for differences in genetic background demonstrated that WT larvae from Harvard University (Harvard WT) had lower ion uptake rates than those of uOttawa WT, and that ion uptake rate between Harvard WT and tph1bKO larvae were not significantly different. Therefore, our initial observation that tph1bKO larvae (Harvard source) had reduced ion uptake rates relative to uOttawa WT was a function of genetic background and not of knockout itself. These data provide a cautionary tale of the importance of controlling for genetic background in gene knockout experiments., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In water-breathing fishes, the hypoxic ventilatory response (HVR) represents an increase in water flow over the gills during exposure to lowered ambient O 2 levels. The HVR is a critical defense mechanism that serves to delay the negative consequences of hypoxia on aerobic respiration. However, the physiological significance of the HVR in larval fishes is unclear as they do not have a fully developed gill and rely primarily on cutaneous gas transfer. Using larval zebrafish (4, 7, 10 and 15 days post-fertilization; dpf), we examined HVR under three levels of hypoxia (25, 45 and 60 mmHg). The larvae exhibited widely different HVRs as a function of developmental age and level of the hypoxia. Yet, critical O 2 tensions ( P crit ) remained constant (30-34 mmHg) over the same period of development. Micro-optrode O 2 sensors were used to measure a significant decrease in buccal cavity water O 2 tensions in 4 and 7 dpf larvae compared with the water they inspired, demonstrating significant extraction of O 2 from the buccal cavity. To assess the physiological significance of the HVR, ventilatory water flow was prevented in larvae at 4 and 7 dpf by embedding their heads in agar. An increase in P crit was observed in larvae at 7 dpf but not 4 dpf, suggesting that buccal ventilation is important for O 2 extraction by 7 dpf. Combined, these data indicate that branchial/buccal gas transfer plays a significant role in O 2 uptake during hypoxia, and supports a physiological benefit of the HVR in early life stages of zebrafish., (© 2019. Published by The Company of Biologists Ltd.)

Loss-of-function technologies, such as morpholino- and RNAi-mediated gene knockdown, and TALEN- and CRISPR/Cas9-mediated gene knockout, are widely used to investigate gene function and its physiological significance. Here, we provide a general overview of the various knockdown and knockout technologies commonly used in comparative physiology and discuss the merits and drawbacks of these technologies with a particular focus on research conducted in zebrafish. Despite their widespread use, there is an ongoing debate surrounding the use of knockdown versus knockout approaches and their potential off-target effects. This debate is primarily fueled by the observations that, in some studies, knockout mutants exhibit phenotypes different from those observed in response to knockdown using morpholinos or RNAi. We discuss the current debate and focus on the discrepancies between knockdown and knockout phenotypes, providing literature and primary data to show that the different phenotypes are not necessarily a direct result of the off-target effects of the knockdown agents used. Nevertheless, given the recent evidence of some knockdown phenotypes being recapitulated in knockout mutants lacking the morpholino or RNAi target, we stress that results of knockdown experiments need to be interpreted with caution. We ultimately argue that knockdown experiments should not be discontinued if proper control experiments are performed, and that with careful interpretation, knockdown approaches remain useful to complement the limitations of knockout studies (e.g. lethality of knockout and compensatory responses)., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Purely diffusive O 2 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 O 2 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 O 2 and CO 2 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 O 2 fluxes ( Jo 2 ), it was demonstrated that larvae lacking convection exhibited reduced Jo 2 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 Jo 2 , 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 O 2 consumption and CO 2 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.

Hypoxia-inducible factor (Hif) 1α, an extensively studied transcription factor, is involved in the regulation of many biological processes in hypoxia including the hypoxic ventilatory response. In zebrafish, there are two paralogs of Hif-1α (Hif-1A and Hif-1B), but little is known about the specific roles or potential sub-functionalization of the paralogs in response to hypoxia. Using knockout lines of Hif-1α paralogs, we examined their involvement in the hypoxic ventilatory response, measured as ventilation frequency ( f V ) in larval and adult zebrafish ( Danio rerio ). In wild-type zebrafish, f V increased across developmental time (4, 7, 10 and 15 days post--fertilization, dpf) in response to hypoxia (55 mmHg). In contrast, the Hif-1B knockout fish did not exhibit an increase in hypoxic f V at 4 dpf. Similar to wild-type, as larvae of all knockout lines developed, the magnitude of f V increased but to a lesser degree than in the wild-type larvae, until 15 dpf at which point there was no difference among the genotypes. In adult zebrafish, only in Hif-1B knockout fish was there an attenuation in f V during sustained exposure to 30 mmHg for 1 h but there was no effect when fish were exposed for a shorter duration to progressive hypoxia. The mechanism of action of Hif-1α, in part, may be through its downstream target, nitric oxide synthase, and its product, nitric oxide. Overall, the effect of each Hif-1α paralog on the hypoxic ventilatory response of zebrafish varies over development and is dependent on the type of hypoxic stress., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Na + uptake in larval zebrafish (Danio rerio) is coordinated by three mechanisms: Na + /H + -exchanger 3b (NHE3b) expressed in H + -ATPase-rich (HR) cells, an unidentified Na + channel coupled to electrogenic H + -ATPase expressed in HR cells, and Na + -Cl - -cotransporter (NCC) expressed in NCC cells. Recently, acid-sensing ion channels (ASICs) were proposed to be the putative Na + channel involved in H + -ATPase-mediated Na + uptake in adult zebrafish and rainbow trout. In the present study, we hypothesized that ASICs also play this role in Na + uptake in larval zebrafish. In support of this hypothesis, immunohistochemical analyses revealed that ASIC4b was expressed in HR cells on the yolk sac skin at 4 days post-fertilization (dpf). However, neither treatment with the ASIC-specific blocker 4,6-diamidino-2-phenylindole (DAPI) nor morpholino knockdown of ASIC4b reduced Na + uptake in circumneutral conditions at 4 dpf. However, because ASIC4b knockdown led to significant increases in the mRNA expression of nhe3b and ncc and a significant increase in HR cell density, it is possible that Na + influx was sustained by increased participation of non-ASIC4b pathways. Moreover, when fish were reared in acidic water (pH = 4), ASIC4b knockdown led to a stimulation of Na + uptake at 3 and 4 dpf, results which also were inconsistent with an essential role for ASIC-mediated Na + uptake, even under conditions known to constrain Na + uptake via NHE3b. Thus, while ASIC4b clearly is expressed in HR cells, the current functional experiments cannot confirm its involvement in Na + uptake in larval zebrafish., (Copyright © 2018 Elsevier Inc. All rights reserved.)