Fish gill chemosensing: knowledge gaps and inconsistencies.

In this review, we explore the inconsistencies in the data and gaps in our knowledge that exist in what is currently known regarding gill chemosensors which drive the cardiorespiratory reflexes in fish. Although putative serotonergic neuroepithelial cells (NEC) dominate the literature, it is clear that other neurotransmitters are involved (adrenaline, noradrenaline, acetylcholine, purines, and dopamine). And although we assume that these agents act on neurons synapsing with the NECs or in the afferent or efferent limbs of the paths between chemosensors and central integration sites, this process remains elusive and may explain current discrepancies or species differences in the literature. To date it has been impossible to link the distribution of NECs to species sensitivity to different stimuli or fish lifestyles and while the gills have been shown to be the primary sensing site for respiratory gases, the location (gills, oro-branchial cavity or elsewhere) and orientation (external/water or internal/blood sensing) of the NECs are highly variable between species of water and air breathing fish. Much of what has been described so far comes from studies of hypoxic responses in fish, however, changes in CO2, ammonia and lactate have all been shown to elicit cardio-respiratory responses and all have been suggested to arise from stimulation of gill NECs. Our view of the role of NECs is broadening as we begin to understand the polymodal nature of these cells. We begin by presenting the fundamental picture of gill chemosensing that has developed, followed by some key unanswered questions about gill chemosensing in general.

Ocean acidification alters foraging behaviour in Dungeness crab through impairment of the olfactory pathway.

Crustacean olfaction is fundamental to most aspects of living and communicating in aquatic environments and more broadly, for individual- and population-level success. Accelerated ocean acidification from elevated CO2 threatens the ability of crabs to detect and respond to important olfactory-related cues. Here, we demonstrate that the ecologically and economically important Dungeness crab (Metacarcinus magister) exhibits reduced olfactory-related antennular flicking responses to a food cue when exposed to near-future CO2 levels, adding to the growing body of evidence of impaired crab behaviour. Underlying this altered behaviour, we find that crabs have lower olfactory nerve sensitivities (twofold reduction in antennular nerve activity) in response to a food cue when exposed to elevated CO2 . This suggests that near-future CO2 levels will impact the threshold of detection of food by crabs. We also show that lower olfactory nerve sensitivity in elevated CO2 is accompanied by a decrease in the olfactory sensory neuron (OSN) expression of a principal chemosensory receptor protein, ionotropic receptor 25a (IR25a) which is fundamental for odorant coding and olfactory signalling cascades. The OSNs also exhibit morphological changes in the form of decreased surface areas of their somata. This study provides the first evidence of the effects of high CO2 levels at multiple levels of biological organization in marine crabs, linking physiological and cellular changes with whole animal behavioural responses.

Invited review - the effects of anthropogenic abiotic stressors on the sensory systems of fishes.

Climate change is a growing global issue with many countries and institutions declaring a climate state of emergency. Excess CO2 from anthropogenic sources and changes in land use practices are contributing to many detrimental changes, including increased global temperatures, ocean acidification and hypoxic zones along coastal habitats. All senses are important for aquatic animals, as it is how they can perceive and respond to their environment. Some of these environmental challenges have been shown to impair their sensory systems, including the olfactory, visual, and auditory systems. While most of the research is focused on how ocean acidification affects olfaction, there is also evidence that it negatively affects vision and hearing. The effects that temperature and hypoxia have on the senses have also been investigated, but to a much lesser extent in comparison to ocean acidification. This review assembles the known information on how these anthropogenic challenges affect the sensory systems of fishes, but also highlights what gaps in knowledge remain with suggestions for immediate action. Olfaction, vision, otolith, pH, freshwater, seawater, marine, central nervous system, electrophysiology, mechanism.

Acidification can directly affect olfaction in marine organisms.

In the past decade, many studies have investigated the effects of low pH/high CO2 as a proxy for ocean acidification on olfactory-mediated behaviours of marine organisms. The effects of ocean acidification on the behaviour of fish vary from very large to none at all, and most of the maladaptive behaviours observed have been attributed to changes in acid-base regulation, leading to changes in ion distribution over neural membranes, and consequently affecting the functioning of gamma-aminobutyric acid-mediated (GABAergic) neurotransmission. Here, we highlight a possible additional mechanism by which ocean acidification might directly affect olfaction in marine fish and invertebrates. We propose that a decrease in pH can directly affect the protonation, and thereby, 3D conformation and charge distribution of odorants and/or their receptors in the olfactory organs of aquatic animals. This can sometimes enhance signalling, but most of the time the affinity of odorants for their receptors is reduced in high CO2/low pH; therefore, the activity of olfactory receptor neurons decreases as measured using electrophysiology. The reduced signal reception would translate into reduced activation of the olfactory bulb neurons, which are responsible for processing olfactory information in the brain. Over longer exposures of days to weeks, changes in gene expression in the olfactory receptors and olfactory bulb neurons cause these neurons to become less active, exacerbating the problem. A change in olfactory system functioning leads to inappropriate behavioural responses to odorants. We discuss gaps in the literature and suggest some changes to experimental design in order to improve our understanding of the underlying mechanisms and their effects on the associated behaviours to resolve some current controversy in the field regarding the extent of the effects of ocean acidification on marine fish.

New insights into organ-specific oxidative stress mechanisms using a novel biosensor zebrafish.

BACKGROUND: Reactive oxygen species (ROS) arise as a result from, and are essential in, numerous cellular processes. ROS, however, are highly reactive and if left unneutralised by endogenous antioxidant systems, can result in extensive cellular damage and/or pathogenesis. In addition, exposure to a wide range of environmental stressors can also result in surplus ROS production leading to oxidative stress (OS) and downstream tissue toxicity. OBJECTIVES: Our aim was to produce a stable transgenic zebrafish line, unrestricted by tissue-specific gene regulation, which was capable of providing a whole organismal, real-time read-out of tissue-specific OS following exposure to a wide range of OS-inducing environmental contaminants and conditions. This model could, therefore, serve as a sensitive and specific mechanistic in vivo biomarker for all environmental conditions that result in OS. METHODS: To achieve this aim, we exploited the pivotal role of the electrophile response element (EpRE) as a globally-acting master regulator of the cellular response to OS. To test tissue specificity and quantitative capacity, we selected a range of chemical contaminants known to induce OS in specific organs or tissues, and assessed dose-responsiveness in each using microscopic measures of mCherry fluorescence intensity. RESULTS: We produced the first stable transgenic zebrafish line Tg (3EpRE:hsp70:mCherry) with high sensitivity for the detection of cellular RedOx imbalances, in vivo in near-real time. We applied this new model to quantify OS after exposure to a range of environmental conditions with high resolution and provided quantification both of compound- and tissue-specific ROS-induced toxicity. DISCUSSION: Our model has an extremely diverse range of potential applications not only for biomonitoring of toxicants in aqueous environments, but also in biomedicine for identifying ROS-mediated mechanisms involved in the progression of a number of important human diseases, including cancer.

A role for nitric oxide in the control of breathing in zebrafish (Danio rerio).

Nitric oxide (NO) is a gaseous neurotransmitter, which, in adult mammals, modulates the acute hypoxic ventilatory response; its role in the control of breathing in fish during development is unknown. We addressed the interactive effects of developmental age and NO in the control of piscine breathing by measuring the ventilatory response of zebrafish (Danio rerio) adults and larvae to NO donors and by inhibiting endogenous production of NO. In adults, sodium nitroprusside (SNP), a NO donor, inhibited ventilation; the extent of the ventilatory inhibition was related to the pre-existing ventilatory drive, with the greatest inhibition exhibited during exposure to hypoxia (PO2=5.6 kPa). Inhibition of endogenous NO production using L-NAME suppressed the hypoventilatory response to hyperoxia, supporting an inhibitory role of NO in adult zebrafish. Neuroepithelial cells (NECs), the putative oxygen chemoreceptors of fish, contain neuronal nitric oxide synthase (nNOS). In zebrafish larvae at 4 days post-fertilization, SNP increased ventilation in a concentration-dependent manner. Inhibition of NOS activity with L-NAME or knockdown of nNOS inhibited the hypoxic (PO2=3.5 kPa) ventilatory response. Immunohistochemistry revealed the presence of nNOS in the NECs of larvae. Taken together, these data suggest that NO plays an inhibitory role in the control of ventilation in adult zebrafish, but an excitatory role in larvae.

Hydrogen sulfide inhibits Na+ uptake in larval zebrafish, Danio rerio.

The present study investigated the role of hydrogen sulfide (H2S) in regulating Na(+) uptake in larval zebrafish, Danio rerio. Waterborne treatment of larvae at 4 days post-fertilization (dpf) with Na2S or GYY-4137 (chemicals known to generate H2S) significantly reduced Na(+) uptake. Exposure of larvae to water enriched with NaCl (1 mM NaCl) caused a pronounced reduction in Na(+) uptake which was prevented by pharmacological inhibition of cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE), two key enzymes involved in the endogenous synthesis of H2S. Furthermore, translational gene knockdown of CSE and CBSb significantly increased the basal rate of Na(+) uptake. Waterborne treatment with Na2S significantly decreased whole-body acid excretion and reduced Na(+) uptake in larval zebrafish preexposed to acidic (pH 4.0) water (a condition shown to promote Na(+) uptake via Na(+)-H(+)-exchanger 3b, NHE3b). However, Na2S did not affect Na(+) uptake in larvae depleted of NHE3b-containing ionocytes (HR cells) after knockdown of transcription factor glial cell missing 2 (gcm2) in which Na(+) uptake occurs predominantly via Na(+)-Cl(-) co-transporter (NCC)-containing cells. These observations suggest that Na(+) uptake via NHE3b, but not NCC, is regulated by H2S. Whole-mount immunohistochemistry demonstrated that ionocytes expressing NHE3b also express CSE. These data suggests a physiologically relevant role of H2S as a mechanism to lower Na(+) uptake in zebrafish larvae, probably through its inhibitory action on NHE3b.

Characterisation of putative oxygen chemoreceptors in bowfin (Amia calva).

Serotonin containing neuroepithelial cells (NECs) are putative oxygen sensing cells found in different locations within the gills of fish. In this study we wished to determine the effect of sustained internal (blood) hypoxaemia versus external (aquatic) hypoxia on the size and density of NECs in the first gill arch of bowfin (Amia calva), a facultative air breather. We identified five different populations of serotonergic NECs in this species (Types I-V) based on location, presence of synaptic vesicles (SV) that stain for the antibody SV2, innervation and labelling with the neural crest marker HNK-1. Cell Types I-III were innervated, and these cells, which participate in central O2 chemoreflexes, were studied further. Although there was no change in the density of any cell type in bowfin after exposure to sustained hypoxia (6.0 kPa for 7 days) without access to air, all three of these cell types increased in size. In contrast, only Type II and III cells increased in size in bowfin exposed to sustained hypoxia with access to air. These data support the suggestion that NECs are putative oxygen-sensing cells, that they occur in several locations, and that Type I cells monitor only hypoxaemia, whereas both other cell types monitor hypoxia and hypoxaemia.

The role of hydrogen sulphide in the control of breathing in hypoxic zebrafish (Danio rerio).

The current study investigated the role of hydrogen sulphide (H2S) in oxygen sensing, intracellular signalling and promotion of ventilatory responses to hypoxia in adult and larval zebrafish (Danio rerio). Both larval and adult zebrafish exhibited a dose-dependent increase in ventilation to sodium sulphide (Na2S), an H2S donor. In vertebrates, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are enzymes that catalyse the endogenous production of H2S. In adult zebrafish, inhibition of both CBS and CSE with aminooxyacetate (AOA) and propargyl glycine (PPG) blunted or abolished the hypoxic hyperventilation, and the addition of Na2S to the water partially rescued the effects of inhibiting endogenous H2S production. In zebrafish larvae (4 days post-fertilization), gene knockdown of either CBS or CSE using morpholinos attenuated the hypoxic ventilatory response. Furthermore, the intracellular calcium concentration of isolated neuroepithelial cells (NECs), which are putative oxygen chemoreceptors, increased significantly when these cells were exposed to 50 µm Na2S, supporting a role for H2S in Ca(2+)-evoked neurotransmitter release in these cells. Finally, immunohistochemical labelling showed that NECs dissociated from adult gill contained CBS and CSE, whereas cutaneous NECs in larval zebrafish expressed only CSE. Taken together, these data show that H2S can be produced in the putative oxygen-sensing cells of zebrafish, the NECs, in which it appears to play a pivotal role in promoting the hypoxic ventilatory response.

Time domains of the hypoxic cardio-respiratory response in bowfin (Amia calva).

The aim of this study was to determine whether time domains exist in the hypoxic ventilatory (HVR) and cardiac responses (HCR) of bowfin (Amia calva), a facultative air breather, exposed to sustained hypoxia (SH) (26mmHg at 8°C or 45mmHg at 22°C). It was hypothesized that time domains would be evident in the HVR and HCR of bowfin when denied access to air during SH, as have been reported in mammals. It was also hypothesized that they would not be present in bowfin with access to air during SH because their oxygen supply should not be limited due to air breathing. Bowfin without access to air during SH exhibited time domains of the HVR and some time domains of the HCR. As hypothesized, bowfin with access to air did not exhibit time dependent changes in the gill breathing, air breathing, or cardiac responses to SH. The extent to which these reflect homologous processes to those underlying time domains in mammals remains to be determined.

Serotonin directly stimulates cortisol secretion from the interrenals in goldfish.

While serotonin (5-HT) can stimulate the hypothalamic-pituitary-interrenal stress axis in fish, the specific site(s) of 5-HT action are poorly understood. In this study, goldfish (Carassius auratus) were injected intraperitoneally with either saline or the 5-HT1A/7 receptor agonist 8-OH-DPAT at a dose of 100 or 400 µg/kg body weight and sampled 1.5 and 8 h post-injection. Relative to unhandled controls, the saline and 100 µg/kg 8-OH-DPAT treatments elicited similar transient 5- to 7-fold increases in plasma cortisol and the 400 µg/kg 8-OH-DPAT dosage resulted in a sustained 16-fold increase in cortisol levels. Although the 5-HT1A receptor is expressed in the brain preoptic area (POA), the pituitary and the head kidney, neither the saline nor the 8-OH-DPAT treatments affected the mRNA abundance of POA corticotropin-releasing factor and pituitary pro-opiomelanocortin or plasma adrenocorticotropic hormone (ACTH) levels. To assess the direct actions of 5-HT on cortisol secretion relative to those of ACTH, head kidney tissue were superfused with 10(-7)M 5-HT, ACTH or a combined 5-HT/ACTH treatment. Overall, the ACTH and 5-HT/ACTH treatments resulted in higher peak cortisol and total cortisol release than in the 5-HT treatment but the response time to peak cortisol release was shorter in the combined treatment than in either the 5-HT or ACTH alone treatments. Both 8-OH-DPAT and cisapride, a 5-HT4 receptor agonist, also stimulated cortisol release in vitro and their actions were reversed by selective 5-HT1A and 5-HT4 receptor antagonists, respectively. Finally, double-labeling with anti-tyrosine hydroxylase and anti-5-HT revealed that the chromaffin cells of the head kidney contain 5-HT. Thus, in goldfish, 5-HT can directly stimulate cortisol secretion from the interrenals via multiple 5-HT receptor subtypes and the chromaffin cells may be involved in the paracrine regulation of cortisol secretion via 5-HT.

Distribution of acetylcholine and catecholamines in fish gills and their potential roles in the hypoxic ventilatory response.

Carotid body glomus cells in mammals contain a plethora of different neurochemicals. Several hypotheses exist to explain their roles in oxygen-chemosensing. In the present study we assessed the distribution of serotonin, acetylcholine and catecholamines in the gills of trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) using immunohistochemistry, and an activity-dependent dye, Texas Red hydrazide (TXR). In fish the putative oxygen sensing cells are neuroepithelial cells (NECs) and the focus in recent studies has been on the role of serotonin in oxygen chemoreception. The NECs of trout and goldfish contain serotonin, but, in contrast to the glomus cells of mammals, not acetylcholine or catecholamines. Acetylcholine was expressed in chain and proximal neurons and in extrinsic nerve bundles in the filaments. The serotonergic NECs did not label with the HNK-1 antibody suggesting that if they are derived from the neural crest, they are no longer proliferative or migrating. Furthermore, we predicted that if serotonergic NECs were chemosensory, they would increase their activity during hypoxia (endocytose TXR), but following 30 min of hypoxic exposure (45 Torr), serotonergic NECs did not take up TXR. Based on these and previous findings we propose several possible models outlining the ways in which serotonin and acetylcholine could participate in oxygen chemoreception in completing the afferent sensory pathway.

Acid water interferes with salamander-green algae symbiosis during early embryonic development.

The inner egg capsule of embryos of the yellow-spotted salamander (Ambystoma maculatum) are routinely colonized by green algae, such as Oophila amblystomatis, that supply O(2) in the presence of light and may consume nitrogenous wastes, forming what has been proposed to be a mutualistic relationship. Given that A. maculatum have been reported to breed in acidic (pH <5.0) and neutral lakes, we hypothesized that low water pH would negatively affect these symbiotic organisms and alter the gradients within the jelly mass. Oxygen gradients were detected within jelly masses measured directly in a natural breeding pond (pH 4.5-4.8) at midday in full sunlight. In the lab, embryo jelly masses reared continuously at pH 4.5 had lower P(O)2and higher ammonia levels relative to jelly masses held at pH 8.0 (control). Ammonia and lactate concentrations in embryonic tissues were approximately 37%-93% higher, respectively, in embryos reared at water pH 4.5 compared with pH 8.0. Mass was also reduced in embryos reared at pH 4.5 versus pH 8.0. In addition, light conditions (24 h light, 12L : 12D, or 24 h dark) and embryonic position (periphery vs. center) in the jelly mass affected P(O)2but not ammonia gradients, suggesting that algal symbionts generate O(2) but do not significantly impact local ammonia concentrations, regardless of the pH of the water. We conclude that chronic exposure to acidic breeding ponds had a profound effect on the microenvironment of developing A. maculatum embryos, which in turn resulted in an elevation of potentially harmful metabolic end products and inhibited growth. Under acidic conditions, the expected benefit provided by the algae to the salamander embryo (i.e., high O(2) and low ammonia microenvironment) is compromised, suggesting that the A. maculatum-algal mutualism is beneficial to salamanders only at higher water pH values.

Neurotransmitter profiles in fish gills: putative gill oxygen chemoreceptors.

In fish, cells containing serotonin, ACh, catecholamines, NO, H(2)S, leu-5-enkephalin, met-5-enkephalin and neuropeptide Y are found in the gill filaments and lamellae. Serotonin containing neuroepithelial cells (NECs) located along the filament are most abundant and are the only group found in all fish studied to date. The presence of NECs in other locations or containing other transmitters is species specific and it is rare that any one NEC contains more than one neurochemical. The gills are innervated by both extrinsic and intrinsic nerves and they can be cholinergic, serotonergic or contain both transmitters. Some NECs are presumed to be involved in paracrine regulation of gill blood flow, while others part of the reflex pathways involved in cardiorespiratory control. There is both direct and indirect evidence to indicate that the chemosensing cells involved in these latter reflexes sit in locations where some monitor O(2) levels in water, blood or both, yet the anatomical data do not show such clear distinctions.