Air-breathing Fish Species Research Articles

TOXICOLOGICAL ASSESSMENT OF COPPER SULPHATE ON CHANNA ORIENTALIS AND HETEROPNEUSTES FOSSILIS: LC50 VALUES AND MORTALITY ANALYSIS

The adverse effects of heavy metals, particularly copper sulphate, released from industrial effluents on aquatic life are well-documented. In this study, we determined the LC50 values of copper sulphate for two freshwater air-breathing fish species, Channa orientalis (Bloch) and Heteropneustes fossilis (Bloch). The LC50 values for Channa orientalis were determined to be 33 ppm at 24 hours, 32 ppm at 48 hours, 21 ppm at 72 hours, and 7.5 ppm at 96 hours. For Heteropneustes fossilis, the corresponding LC50 values were 15 ppm at 24 hours, 13 ppm at 48 hours, 9.5 ppm at 72 hours, and 5 ppm at 96 hours. We observed a significant increase in mucus secretion at higher concentrations of copper sulphate as compared to lower concentrations. The potential mechanisms of fish mortality under these conditions are also discussed in detail

Metabolism of β-naphthoflavone by cytochrome P450 isoforms in three air breathing fish, Channa punctatus, Heteropneustes fossilis and Clarias batrachus

The crucial phase I xenobiotic metabolising enzyme cytochrome P450 (CYP 450) has a variety of isoforms and a wide range of substrate specificities. While the roles of mammalian CYP 450 are well established, those of non-mammalian species are much less defined. These roles are frequently extrapolated from mammalian data and assumed to be similar across vertebrate species. In the present study, three air breathing fish species, Channa punctatus, Heteropneustes fossilis and Clarias batrachus were injected intraperitoneally (IP) with 50 mg/kg β-naphthoflavone (β-NF) and its effects on hepatic CYP 450 system were evaluated. β-NF treatment resulted in significant increase (p<0.01) in CYP 450 content and CYP1A mediated EROD (Ethoxyresorufin O-deethylase) activity in all three treated fish groups compared to control. CYP2B mediated N,N-dimethylaniline demethylase activity and CYP2E1 mediated aniline hydroxylase activity showed no response suggesting insensitivity to β-NF treatment. All three fish species showed an increase in CYP3A4 mediated erythromycin N-demethylase activity, but only H. fossilis displayed a significant difference (p<0.05). Of all the activities investigated, EROD activity mediated by CYP1A responded towards β-NF treatment the best and could serve as a sensitive biomarker to gauge the effects of chemical pollutants in the aquatic environment.

Choline Chloride Induces Growth Performance of Indian Major Carps and Air-Breathing Fish Species with an Outcome of Quality Food-Fish under a Semi-Intensive Culture System: A Biochemical Investigation.

The present study was intended to determine the possible influences of direct field application of choline chloride into pond water in addition to farm-made-aqua-feed under a semi-intensive culture system on the growth and biochemical parameters of two Indian major carps (IMCs), Catla catla (Catla) and Labeo rohita (Rahu), and two air-breathing species, Clarias batrachus (Magur) and Anabas testudineus (Koi), cultured in a ratio of 2:5:1:1 (Catla/Rahu/Magur/Koi) in three experimental ponds for a period of 90 days during the breeding season (June to August). Results were compared with control (C: fed only with farm-made-aqua-feed) and treatment (T: P1 and P2: farm-made-aqua-feed plus choline chloride into pond water directly at the rate of 350 g bigha–1 fortnightly or 350 g per 1600 square meter fortnightly). A significant increasing trend was observed in the growth parameters including total length-final (TLF), standard length-final (SLF), mean weight-final (MWF), % gain of mean total length (MTL), % gain of mean standard length (MSL), % weight gain (WG), specific growth rate (SGR) % per day, and survivability %. However, a reverse pattern was noticed in the food conversion ratio (FCR) both in IMCs and air-breathing fish species under choline supplementation. Serum biochemical responses, e.g., total protein (PRO), lactate dehydrogenase (LDH), glucose (GLU), and calcium (Ca) showed significant enhancement, and alkaline phosphatase (ALP), alanine amino transaminase (ALT), aspartate amino transaminase (AST), cholesterol (CHOLES), and triglycerides (Trig) showed gradual significant reduction during the breeding season under choline exposure. Treated fishes showed prevention from liver dysfunction and fatty liver formation, and increased body crude protein content. Results indicated favorable growth and yield, which may benefit fish farmers during their culture practices, and the output fish species under choline supplementation resulted in quality food-fish for human consumption.

Epidermal Mucus Extracts of three Freshwater Air-Breathing Fish Species against Gram Positive and Gram Negative Human Pathogenic Bacteria

Mucus layer from epidermal secretions of the fish act as a first line of defense between fish and pathogens in their environment. Fish skin mucus has been reported to prevent colonisation of pathogenic bacteria. The objective of the present study was to explore the antibacterial activity of epidermal mucus extracts from three freshwater air-breathing fish species (Clarias gariepinus, Heteropneustis fossilis and Channa punctatus) against human bacterial pathogens. The crude, acidic and organic extracts of skin mucus were prepared and tested for antibacterial activity by disc diffusion method against three Gram positive (Enterococcus faecalis, Staphylococcus aureus and Streptococcus pneumoniae) and 5 Gram negative (Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Vibrio inaba and Pseudomonas aeruginosa) bacteria. Out of 72 tests performed (nine types of mucus extracts against eight different bacterial strains), 59 tests showed antibacterial activity. The order of the level of antibacterial activity of the mucus of the three fish species observed in the present study was, C. gariepinus > C. punctatus > H. fossilis and the acidic extract of C. gariepinus showed the maximum antibacterial activity against Gram negative bacteria.

Differential expression of epidermal growth factor receptor (EGFR) in stomach and diverticulum of Otocinclus affinis (Steindachner, 1877) as a potential element of the epithelium remodeling mechanism

It is suggested that due to the several stomach modifications, Otocinculus affinis (dwarf sucking catfish) possess the ability to breathe air during hypoxia, however, the exact mechanism remains unknown. The aim of this study was detailed analysis of the expression of EGFR in the stomach and diverticulum of the O. affinis at the mRNA and protein levels together with the immunohistochemical localization of EGFR in these organs. The intensity of band fluorescence corresponding to the EGFR gene expression level is significantly higher in the stomach than in the diverticulum. Further, quantitative analysis of EGFR protein abundance also revealed its higher synthesis in the stomach than in the diverticulum and the immunohistochemistry method confirmed these results. As regional localization of respiratory function in gut air-breathing fishes seems to be connected with “morphological remodeling” of the epithelium of their gut, the present research demonstrated the potential efficiency of the O. affinis stomach as a respiratory organ. Having the potential possibility to create an air-blood barrier in the gastrointestinal tract allowing gas diffusion and respiration in hypoxic states seems to be very beneficial for these fish. It seems that facultative gut air breathing fish species are a relevant vertebrate model for high throughput screening, vascular biology and evolution.

The effect of seasonal temperature on endogenous gut enzyme activity in four air-breathing fish species

Abstract The digestion process in fish is a complex phenomenon and is accomplished with the help of both endogenous and exogenous enzymes. A wide range of enzymes have been reported in fish and their secretion and concentrations highly depend on feeding habits, gut structure and environmental conditions. In this present investigation, we have demonstrated the activity of two endogenous enzymes (protease and amylase) in four economically important air-breathing fish species (Clarias batrachus, Channa punctatus, Anabas testudineus and Heteropneustes fossilis) at different seasonal temperatures. Our results demonstrated that protease activity was higher in all fish during the month of May, followed by September and December. It was also detected that enzyme activity was higher in the hindgut region (HG) compared to the foregut (FG), which suggests that HG is more active in digestion compared to FG. The optimization of feeding efficiency in aquaculture industries is very important, as the cost of feed is high and its availability is also limited. The information presented may be of importance when developing optimized diets and feeding regimes for the species considered.

The nitric oxide: A molecule of possible therapeutic use against aquatic stress in fishes.

In several toxicological research studies involving fish as animal model, it has been seen that fish die primarily because of suffocation caused due to dismantling of the vascular components followed by wear and tear and sloughing of epithelium layer of the respiratory organs such as gills and accessory respiratory organs (ARO) especially the air-breathing organs (ABO) found in some air-breathing fish species like Clarias batrachus, Clarias gariepinus, Anabas sp. etc. The gills are main respiratory organs and are fully aquatic in respiratory function while the ABO, on the other hand, are said to be aerial in nature i.e., extract the oxygen from atmospheric air. The ABO is hence more important for air-breathing fishes as they enable them to survive even in a condition of stress produced by ambient water contaminants/pollutants.

Using an acoustic telemetry array to assess fish volumetric space use: a case study on impoundments, hypoxia and an air-breathing species (Neoceratodus forsteri)

Facultative air-breathing fish can persist in hypoxic waters due to their capacity to acquire atmospheric oxygen. Most studies examining responses of air-breathing fish to aquatic hypoxia have occurred under experimental conditions. How air-breathing fish respond to hypoxic conditions in the field has received less attention. Using depth sensor transmitters and an array of acoustic receivers to monitor the facultative air-breathing Australian lungfish (Neoceratodus forsteri), we investigated habitat preferences and behavioural responses to seasonal hypoxic zones in a riverine impoundment. Three-dimensional (3-D) kernel utilisation distribution (KUD) models revealed that during stratified conditions, lungfish remained above the oxycline, rarely venturing into hypoxic waters, whereas during holomixis lungfish used a wider range of depths. Total volumetric space utilisation did not change significantly during stratified periods, but the distribution of space used changed, constrained by the oxycline. Despite N. forsteri having lungs to supplement oxygen requirements, the presence of a hypoxic zone constrained the core (50% 3-D-KUD) volumetric space used by lungfish to <1.6% of the total available space of the study area. With increasing demand for new impoundments in many tropical and subtropical regions, the present study provides insights to how air-breathing fish species may respond to altered riverine conditions from impoundments.

Bacterial Diversity in the Digestive Tracts of Four Indian Air-Breathing Fish Species Investigated by PCR Based Denaturing Gradient Gel Electrophoresis

ABSTRACT An investigation was conducted to identify the allochthonous microbiota (entire intestine) and the autochthonous microbiota in proximal intestine (PI) and distal intestine (DI) of four species of Indian air-breathing fish (climbing perch; Anabas testudineus, murrel; Channa punctatus, walking catfish; Clarias batrachus and stinging catfish; Heteropneustes fossilis) by PCR based denaturing gradient gel electrophoresis (DGGE). High similarities of the allochthonous microbiota were observed between climbing perch and murrel, walking catfish and stinging catfish, indicating similar food behavior. The autochthonous microbiota of PI and DI from climbing perch and murrel revealed more similarity, than the result obtained from walking catfish and stinging catfish. The autochthonous microbiota of climbing perch and murrel were similar with regard to the allochthonous microbiota, but no such similarity was observed in case of walking catfish and stinging catfish. The fish genotype and intestinal bacteria are well matched and show co-evolutionary relationship. Three fish species has its unique bacteria; autochthonous Enterobacter cloacae, Edwardsiella tarda and Sphingobium sp. in DI of climbing perch, Pseudomonas sp.; allochthonous and autochthonous in PI of walking catfish and uncultured bacterium (EU697160.1), uncultured bacterium (JF018065.1) and uncultured bacterium (EU697160.1) for stinging catfish. In murrel, no unique bacteria were detected.

Transcriptomic Analysis of Compromise Between Air-Breathing and Nutrient Uptake of Posterior Intestine in Loach (Misgurnus anguillicaudatus), an Air-Breathing Fish.

Dojo loach (Misgurnus anguillicaudatus) is an air-breathing fish species by using its posterior intestine to breathe on water surface. So far, the molecular mechanism about accessory air-breathing in fish is seldom addressed. Five cDNA libraries were constructed here for loach posterior intestines form T01 (the initial stage group), T02 (mid-stage of normal group), T03 (end stage of normal group), T04 (mid-stage of air-breathing inhibited group), and T05 (the end stage of air-breathing inhibited group) and subjected to perform RNA-seq to compare their transcriptomic profilings. A total of 92,962 unigenes were assembled, while 37,905 (40.77%) unigenes were successfully annotated. 2298, 1091, and 3275 differentially expressed genes (fn1, ACE, EGFR, Pxdn, SDF, HIF, VEGF, SLC2A1, SLC5A8 etc.) were observed in T04/T02, T05/T03, and T05/T04, respectively. Expression levels of many genes associated with air-breathing and nutrient uptake varied significantly between normal and intestinal air-breathing inhibited group. Intraepithelial capillaries in posterior intestines of loaches from T05 were broken, while red blood cells were enriched at the surface of intestinal epithelial lining with 241 ± 39 cells per millimeter. There were periodic acid-schiff (PAS)-positive epithelial mucous cells in posterior intestines from both normal and air-breathing inhibited groups. Results obtained here suggested an overlap of air-breathing and nutrient uptake function of posterior intestine in loach. Intestinal air-breathing inhibition in loach would influence the posterior intestine's nutrient uptake ability and endothelial capillary structure stability. This study will contribute to our understanding on the molecular regulatory mechanisms of intestinal air-breathing in loach.

Mode of Association, Enzyme Producing Ability and Identification of Autochthonous Bacteria in the Gastrointestinal Tract of Two Indian Air-Breathing Fish, Murrel (Channa punctatus) and Stinging Catfish (Heteropneustes fossilis)

The mode of association of epithelium-associated bacteria in the gastrointestinal (GI) tract of two Indian air-breathing fish species, the murrel, Channa punctatus and the stinging catfish, Heteropneustes fossilis was demonstrated through scanning and transmission electron microscopy (SEM and TEM). The SEM examination revealed substantial numbers of rod shaped bacterial cells associated with the microvillus brush borders of enterocytes in proximal (PI) and distal regions (DI) of the GI tract of both the fish species. The TEM investigation indicated endocytosis and translocation of bacteria in the microvilli. The isolated bacterial strains (two each from the PI and DI of murrel and stinging catfish) were quantitatively evaluated for their extracellular amylase, cellulase and protease production. All the bacterial strains exhibited high cellulolytic activity than that of amylolytic and proteolytic enzymes. Only two strains, CPF1 and CPF2, isolated from the PI of murrel exhibited high proteolytic activity. Maximum amylase activity was exhibited by the strain, HFH5, isolated from the DI of stinging catfish. Totally six most promising enzyme-producing autochthonous bacterial strains were identified based on partial 16S rRNA gene sequence analytical results. All the strains showed close (92–99 %) similarity to Bacillus licheniformis.

Development of gas exchange and ion regulation in two species of air-breathing fish, Betta splendens and Macropodus opercularis

Aquatic air-breathing anabantoids, a group of fish species characterized by the presence of a labyrinth organ and some gills, exhibit morphological variations. This study aimed to examine whether unequal gill growth begins during the early stages and described the sequence of the early gill developmental events in Betta splendens and Macropodus opercularis. To determine when the ion regulatory and gas exchange abilities first appear in the gills, mitochondria-rich cells (MRCs) and neuroepithelial cells (NECs) were examined in young B. splendens. To evaluate the relative importance of the gills and the labyrinth organ under different levels of oxygen uptake stress, the levels of carbonic anhydrase II (CAII) and Na+/K+–ATPase (NKA) protein expressions in 2 gills and the labyrinth organ were examined in M. opercularis. We found that the first 3 gills developed earlier than the 4th gill in both species, an indication that the morphological variation begins early in life. In B. splendens, the MRCs and NECs clearly appeared in the first 3 gills at 4dph and were first found in the 4th gill until 11dph. The oxygen-sensing ability of the gills was concordant with the ionoregulatory function. In M. opercularis, the hypoxic group had a significantly higher air-breathing frequency. CAII protein expression was higher in the labyrinth organ in the hypoxic group. The gills exhibited increased NKA protein expression in the hypoxic and restricted groups, respectively. Functional plasticity in CAII and NKA protein expressions was found between the gills and the labyrinth organ in adult M. opercularis.

Mitochondrial COI and nuclear RAG1 DNA sequences and analyses of specimens of the three morphologically established species in the genus Trichopsis (Perciformes: Osphronemidae) reveal new/cryptic species

Air-breathing fish species of the genus Trichopsis have been reported in Cambodia, Lao PDR, Indonesia, Malaysia, Singapore, Thailand and Vietnam. It is only in Thailand that all three recognized species (Trichopsis vittata, Trichopsis schalleri and Trichopsis pumila), as judged by distinct external features, are found. Cambodia and Lao PDR harbor two species each. The present work involves first-time DNA sequencing and analysis based on mitochondrial (COI) and nuclear (RAG1) DNA of numerous specimens of these species and specimens of a controversial Phetchaburi (Thailand) fish population with a mixed outward appearance. In addition to confirming the morphologically clear-cut taxonomic division of the three fish species, our DNA results show that whereas the T. pumila populations form one single species, there are cryptic species in the T. vittata and T. schalleri populations and possibly a new one in the latter. Members of the putative Phetchaburi fish population have been proven to be hybrids between T. pumila and T. vittata. In addition, a new the phylogenetic tree indicating ancestral relationships is also presented. This study should generate further research to find new/cryptic species of the genus Trichopsis in all countries harboring the fish.

Air‐breathing fishes

Air‐breathing fishes

Air‐breathing fishes in aquaculture. What can we learn from physiology?

During the past decade, the culture of air-breathing fish species has increased dramatically and is now a significant global source of protein for human consumption. This development has generated a need for specific information on how to maximize growth and minimize the environmental effect of culture systems. Here, the existing data on metabolism in air-breathing fishes are reviewed, with the aim of shedding new light on the oxygen requirements of air-breathing fishes in aquaculture, reaching the conclusion that aquatic oxygenation is much more important than previously assumed. In addition, the possible effects on growth of the recurrent exposure to deep hypoxia and associated elevated concentrations of carbon dioxide, ammonia and nitrite, that occurs in the culture ponds used for air-breathing fishes, are discussed. Where data on air-breathing fishes are simply lacking, data for a few water-breathing species will be reviewed, to put the physiological effects into a growth perspective. It is argued that an understanding of air-breathing fishes' respiratory physiology, including metabolic rate, partitioning of oxygen uptake from air and water in facultative air breathers, the critical oxygen tension, can provide important input for the optimization of culture practices. Given the growing importance of air breathers in aquaculture production, there is an urgent need for further data on these issues.

Water quality and productivity of the traditional aquaculture system (whedos) in Oueme delta (Benin)

In regard to improve the fish yield of the whedos (traditional fish ponds) by stocking and rearing during the draw-down period, we assessed their water quality comparatively to those of the Oueme River on which they depend. Variables such as pH, K25, NO2- and cladocerans density were similar in both systems while temperature, dissolved oxygen, transparency and microalgal biomass were significantly lower (P<0.05) in the whedos than the river channel. Using multivariate analyses, it was demonstrated that the variability of water quality in the whedos depends not only on their size but also on their width and water height. Moreover, many fish species were observed in the whedos (Clarias, Ctenopoma, Parachanna, Protopterus, Polypterus, Malapterurus, Xenomystus and Brienomyrus) and all display adaptations for survival in low dissolved oxygen conditions. The macrophytes were also abundant in the whedos and very diversified (24 species). In summary, the observations carried out in the whedos during this study show that the quality of their water limits consequently the possibilities to stock many fish species. However, potentiality of improving the fish yield of the whedos by stocking and rearing air breathing fish species during the draw-down period is currently debated.Keywords : Whedo, fisheries management, water quality, plankton, fish stocking.

Effects of sublethal concentrations of diazinon on surfacing and hanging behaviors of snakeheadChanna striata

The air-breathing fish Channa striata is widely distributed in South-East Asia, and is an important source of protein for local inhabitants. The fish can be found in various water bodies including lakes, reservoirs, ponds, canals, drains and rice fields where agro-chemicals are applied. This species has been reported to die if surface breathing is prevented. The distinctive surfacing (where the fish swallow air from the surface) and hanging (where the fish remain motionless just below the surface) behaviors in this species depend on the dissolved oxygen level. Surfacing frequency was negatively correlated with the partial pressure of oxygen (PO2) but hanging behavior was positively correlated with PO2. Thus, although this fish is an obligate air breather, it can alter its respiratory strategy depending on the dissolved oxygen concentration (DO) in the water. Increased air-breathing activity and reduced oxygen uptake over the gill have been found when C. striata was exposed to metasystox at the concentration causing 50% mortality during 48 hours of exposure (LC50-48 h). This effect of toxins on surfacing behavior has also been shown in another species of air-breathing fish,Macropodus cupanus, which increases surfacing frequency with increasing sublethal concentrations of carbaryl. This shift can be viewed in terms of chemical avoidance since the gills are often the most important route of chemical uptake in fish. Swimming down concentration gradients is the only possibility for toxin avoidance in fish species without the ability to breathe air, but air-breathing fishmay be able to avoid the toxin by shifting the source of oxygen from contaminated water to air. Diazinon is one of the most commonly used insecticides in rice fields of the Mekong River Delta (MRD). Although diazinon is not persistent in the environment, it is highly toxic to vertebrates including fish. Diazinon is a neurotoxin through its ability to inhibit acetylcholinesterase, causing abnormal swimming behavior and altered respiration and social interactions in aquatic organisms. Water contamination with insecticides occurs mostly at concentrations that do not cause massmortality, and sublethal effects are bothmore widespread and more important for population dynamics. This study aimed to determine whether low sublethal concentrations of the organophosphate diazinon, which the fish may encounter in its natural habitat (0.008 and 0.079 mg/L), caused rapid alterations in behaviors related to respiratory strategy, particularly surfacing and hanging. Adult C. striata were obtained from a laboratory stock population (Can Tho University, Vietnam) at a size of 40.3 3.5 g (mean standard deviation). The fish had been raised from larvae in chlorinefree tap water (pH 7.60 0.03, DO 7.9 0.1 mg/L, water temperature 26.7 0.4°C) for one year. The commercial insecticide Basudin 50EC, containing 50% wt/wt [6-methyl-2-(1-methylethyl)-4pyrimidinyl] ester, common name diazinon, was purchased (Angiang Plant Protection and Services Co., Long Xuyen City, Vietnam). The insecticide *Corresponding author: Tel: 84-71-83-5701. Fax: 84-71-83-0323. Email: ntphuong@ctu.edu.vn Received 13 March 2006. Accepted 17 August 2006. FISHERIES SCIENCE 2008; 74: 1330–1332

Brain cholinesterase response in the snakehead fish ( Channa striata) after field exposure to diazinon

The snakehead Channa striata is an economically important air-breathing fish species in the Mekong delta of Vietnam. Rice paddies, which are disturbed by the frequent application of agro-chemicals, are among the preferred habitats for this species during the rainy season. Diazinon is one of most commonly used chemicals in rice paddies. In the present study, exposure of adult snakehead fish to a single diazinon application in cages within a rice field resulted in long-term brain cholinesterase inhibition, while the water concentration of this insecticide fell below the detection limit within 3 days. In addition, incubation of brain homogenates with 2-PAM caused reactivation of the cholinesterase diazinon complex to within 80% of the control level. These experiments also showed that chemical ageing of the diazinon cholinesterase binding occurred, which may explain the long-term effects of this pesticide.

Ammonia as a respiratory gas in water and air-breathing fishes

Ammonia is produced in the liver and excreted as NH(3) by diffusion across the gills. Elevated ammonia results in an increase in gill ventilation, perhaps via stimulation of gill oxygen chemo-receptors. Acidification of the water around the fish by carbon dioxide and acid excretion enhances ammonia excretion and constitutes "environmental ammonia detoxification". Fish have difficulties in excreting ammonia in alkaline water or high concentrations of environmental ammonia, or when out of water. The mudskipper, Periphthalmodon schlosseri, is capable of active NH(4)(+) transport, maintaining low internal levels of ammonia. To prevent a back flux of NH(3), these air-breathing fish can increase gill acid excretion and reduce the membrane NH(3) permeability by modifying the phospholipid and cholesterol compositions of their skin. Several air-breathing fish species can excrete ammonia into air through NH(3) volatilization. Some fish detoxify ammonia to glutamine or urea. The brains of some fish can tolerate much higher levels of ammonia than other animals. Studies of these fish may offer insights into the nature of ammonia toxicity in general.

Phylogeny of central CO2/pH chemoreception in vertebrates

The traditional view has been that respiratory chemoreceptors responsive to changes in PCO2/pH first evolved in air-breathing vertebrates at both peripheral and central sites. Levels of arterial PCO2 in water breathing fish are typically less than 2–3 torr and it has been assumed that the ventilatory responses of fish to changes in aquatic PCO2/pH were due to the effects of acidosis on haemoglobin oxygen transport. There is growing evidence, however, to suggest that fish also possess peripheral chemoreceptors responsive to changes in PCO2/pH per se that reside primarily in the gills, are innervated by the glassopharyngeal and vagus nerves, and respond primarily to changes in aquatic rather than arterial PCO2. Their distribution overlaps extensively with that of the gill O2 chemoreceptors in fish and it is not yet clear whether both responses arise from the same sensory cells. To date, there is no convincing evidence that strictly water breathing fish possess central chemoreceptors. There is, however, growing evidence to suggest that some species of air-breathing fish possess central CO2 chemoreceptors. Central chemosensitivity has been reported in in vitro brainstem-spinal cord preparations from both a primitive (holostean) and a modern (teleost) actinopterygian (ray finned) fish. Stimulation of these putative receptors had no effect on fictive gill ventilation but stimulated fictive air breathing. Unfortunately, the fictive breathing rates of these preparations were more than 25 times the resting rates reported for intact animals raising questions about the physiological significance of changes in the fictive motor output identified as air breathing under these conditions. In the South American lung-fish (a sarcopterygian fish belonging to the lineage giving rise to higher vertebrates), on the other hand, central perfusion with mock csf of differing pH stimulated air breathing at rates similar to those seen in control animals. While these data suggest that central chemoreceptors have arisen several times in evolutionary history, hand in hand with the evolution of air breathing, this issue is not yet totally resolved. An intriguing and related finding is that central CO2 chemosensitivity appears to develop slowly in amphibian tadpoles. It is not present in young tadpoles but develops over time. The receptors initially stimulate gill ventilation but transfer their influence to lung ventilation just prior to metamorphosis from the aquatic larval stage into the air breathing adult form. In association with this the primary location of the receptors in the brainstem shifts from a diffuse distribution to a rostral concentration. While central chemoreceptors have been demonstrated in reptiles and birds, not much is known of their properties or distribution. They have been well studied in mammals where there is growing evidence for multiple sites of central CO2/pH chemoreception and evidence to suggest that the mechanism of sensory transduction may vary both within and between receptor populations. While there has been much recent interest in the membrane channels, receptors and electrical coupling of several chemosensitive sites, this work has largely been on cells with downstream respiratory rhythmicity in preparations whose responses are very different (in terms of changes in frequency and amplitude of phrenic output) from that which is seen in intact animals. The phylogenetic trends that are emerging indicate that the use of CO2 chemoreception for cardio-respiratory processes may have arisen much earlier than previously believed, that CO2/pH chemoreception arose in the periphery before the evolution of central CO2/pH chemoreceptors, that the sites of CO2/pH chemoreception (both peripheral and central) have increased throughout the course of vertebrate evolution and that the mechanism of CO2/pH chemotransduction may vary. The sum of the data suggests that CO2/pH chemoreceptors have arisen multiple times, at multiple sites during vertebrate evolution.