Do air-breathing fish suffer branchial oxygen loss in hypoxic water?

In hypoxia, air-breathing fish obtain O2 from the air but continue to excrete CO2 into the water. Consequently, it is believed that some O2 obtained by air-breathing is lost at the gills in hypoxic water. Pangasionodon hypophthalmus is an air-breathing catfish with very large gills from the Mekong River basin where it is cultured in hypoxic ponds. To understand how P. hypophthalmus can maintain high growth in hypoxia with the presumed O2 loss, we quantified respiratory gas exchange in air and water. In severe hypoxia (PO2: ≈ 1.5 mmHg), it lost a mere 4.9% of its aerial O2 uptake, while maintaining aquatic CO2 excretion at 91% of the total. Further, even small elevations in water PO2 rapidly reduced this minor loss. Charting the cardiovascular bauplan across the branchial basket showed four ventral aortas leaving the bulbus arteriosus, with the first and second gill arches draining into the dorsal aorta while the third and fourth gill arches drain into the coeliacomesenteric artery supplying the gut and the highly trabeculated respiratory swim-bladder. Substantial flow changes across these two arterial systems from normoxic to hypoxic water were not found. We conclude that the proposed branchial oxygen loss in air-breathing fish is likely only a minor inefficiency.

Psidium guajava L.- dichloromethane and ethyl acetate fractions ameliorate striped catfish (Pangasianodon hypophthalmus) status via immune response, inflammatory, and apoptosis pathways.

Psidium guajava L. is known to possess immune-modulatory properties in humans and other mammals. Although the positive effects of P. guajava-based diets on the immunological status have been shown for some fish species, the underlying molecular mechanisms of its protective effects remain to be investigated. The aims of this study were to evaluate the immune-modulatory effects of two guava fractions from dichloromethane (CC) and ethyl acetate (EA) on striped catfish with in vitro and in vivo experiments. Striped catfish head kidney leukocytes were stimulated with 40, 20, 10 and 0 µg/ml of each extract fraction, and the immune parameters (ROS, NOS, and lysozyme) were examined at 6 and 24 h post stimulation. A final concentration of each fraction at 40, 10 and 0 µg/fish was then intraperitoneally injected into the fish. After 6, 24, and 72 h of administration, immune parameters as well as the expression of some cytokines related to innate and adaptive immune responses, inflammation, and apoptosis were measured in the head kidney. Results indicated that the humoral (lysozyme) and cellular (ROS and NOS) immune endpoints were regulated differently by CC and EA fractions depending on dose and time in both, in vitro and in vivo experiments. With regards to the in vivo experiment, the CC fraction of the guava extract could significantly enhance the TLRs-MyD88-NF-κB signaling pathway by upregulating its cytokine genes (tlr1, tlr4, myd88, and traf6), following the upregulation of inflammatory (nfκb, tnf, il1ß, and il6) and apoptosis (tp53 and casp8) genes 6 h after injection. Moreover, fish treated with both CC and EA fractions significantly enhanced cytokine gene expression including lys and inos at the later time points - 24 h or 72 h. Our observations suggest that P. guajava fractions modulate the immune, inflammatory, and apoptotic pathways.

Salinity significantly affects intestinal microbiota and gene expression in striped catfish juveniles.

In the present study, juvenile striped catfish (Pangasianodon hypophthalmus), a freshwater fish species, have been chronically exposed to a salinity gradient from freshwater to 20 psu (practical salinity unit) and were sampled at the beginning (D20) and the end (D34) of exposure. The results revealed that the intestinal microbial profile of striped catfish reared in freshwater conditions were dominated by the phyla Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Alpha diversity measures (observed OTUs (operational taxonomic units), Shannon and Faith's PD (phylogenetic diversity)) showed a decreasing pattern as the salinities increased, except for the phylogenetic diversity at D34, which was showing an opposite trend. Furthermore, the beta diversity between groups was significantly different. Vibrio and Akkermansia genera were affected differentially with increasing salinity, the former being increased while the latter was decreased. The genus Sulfurospirillium was found predominantly in fish submitted to salinity treatments. Regarding the host response, the fish intestine likely contributed to osmoregulation by modifying the expression of osmoregulatory genes such as nka1a, nka1b, slc12a1, slc12a2, cftr, and aqp1, especially in fish exposed to 15 and 20 psu. The expression of heat shock proteins (hsp) hsp60, hsp70, and hsp90 was significantly increased in fish reared in 15 and 20 psu. On the other hand, the expression of pattern recognition receptors (PRRs) were inhibited in fish exposed to 20 psu at D20. In conclusion, the fish intestinal microbiota was significantly disrupted in salinities higher than 10 psu and these effects were proportional to the exposure time. In addition, the modifications of intestinal gene expression related to ion exchange and stressful responses may help the fish to adapt hyperosmotic environment. KEY POINTS: • It is the first study to provide detailed information on the gut microbiota of fish using the amplicon sequencing method. • Salinity environment significantly modified the intestinal microbiota of striped catfish. • Intestinal responses may help the fish adapt to hyperosmotic environment.

Salinity affects growth performance, physiology, immune responses and temperature resistance in striped catfish (Pangasianodon hypophthalmus) during its early life stages.

In this study, striped catfish larvae were gradually exposed to the increase of different salinities, and then they reached the levels of 0, 5, 10, 15, and 20 psu after 10 days, followed by heat shock at 39 °C to determine stress tolerance. After the 10-day experiment, the survival rate of fish exposed to the 20 psu treatment was only 28.6 ± 4%, significantly lower than that of the other treatments. The results showed that the osmolality of the whole-body (WB) homogenate was gradually and significantly increased with salinity elevation, except in fish exposed to freshwater and 5 psu treatments, while there were no significant changes in WB Na+/K+-ATPase activity. Digestive enzymatic activities, i.e., pepsin, α-amylase, alkaline phosphatase, and leucine alanine peptidase (leu-ala) generally increased with salinity, but not aminopeptidase and trypsin. Lysozyme and peroxidase activities increased in fish larvae exposed to 15 and 20 psu. These increases proportionally improved growth performance, with the lowest and the highest final weights observed in fish reared at 0 psu (0.08 ± 0.03 g/larvae) and 20 psu (0.11 ± 0.02 g/larvae), respectively, although the average growth recorded at 20 psu could be biased by the high mortality in this group. Occurrence of skeleton deformities, such as in caudal vertebrae and branchiostegal rays, was significantly higher in fish exposed to the higher osmotic conditions (15.0 ± 1.2% and 10.3 ± 2.1% respectively at 0 psu vs. 31.0 ± 2.9% and 49.0 ± 5.6%, respectively at 15 psu). After the 12.5-h heat shock, survival rates significantly differed between treatments with the highest survival observed in fish submitted to 5 psu (68.9%), followed by those exposed to 0 (27%) and 10 (20%) while all fish died at 15 psu. These findings suggest that the striped catfish larvae could be reared in salinity up to 5 to 10 psu with a higher survival and tolerance to thermal stress when compared to fish maintained in freshwater.

Effect of water pH and calcium on ion balance in five fish species of the Mekong Delta.

Acidic freshwater habitats disrupt ion-homeostasis in fishes, yet the often acidic waters of the Mekong host the second highest diversity of freshwater fish in the world. To investigate how five Mekong fish species tolerate water acidity, we measured: time to loss of equilibrium (LOE) at sustained (4 days) low pH (3.5) and net ion flux in acute low pH (3.5 and 3) in Chitala ornata, Pangasianodon hypophthalmus, Osphronemus goramy, Trichogaster pectoralis, and Monopterus albus. Our sustained low pH exposures revealed that C. ornata was least tolerant, P. hypophthalmus and M. albus were moderately tolerant, and O. goramy and T. pectoralis were highly tolerant to low pH. In general, net ion loss in acute low pH exposures was greatest in species with the shortest time to LOE in the sustained low pH exposure. We also explored how low water [Ca2+] (relative to current Mekong levels) affected ion flux at low water pH in the least tolerant C. ornata and highly tolerant T. pectoralis. In C. ornata, low water Ca2+ (56 ±â€¯1 µmol L-1) increased net ion loss relative to high Ca2+ (342 ±â€¯3 µmol L-1) water while no effect was observed in T. pectoralis. Finally, we find that T. pectoralis is among the most acid-tolerant fish species examined to date.

Ontogeny and morphometrics of the gills and swim bladder of air-breathing striped catfish Pangasianodon hypophthalmus.

The air-breathing fish Pangasianodon hypophthalmus has been shown to have highly plastic branchial surfaces whose area (SA) increases with temperature and aquatic hypoxia. This modulation occurs through development of inter-lamellar cell mass (ILCM). Paradoxically, in conditions where this fish has been shown capable of covering its entire aerobic scope from the water phase, it has been shown to have a very small branchial SA. To address this paradox, we measured the SA, harmonic mean diffusion distance (τh) and calculated the anatomic diffusion factor (ADF) of the branchial and swim bladder surfaces in fish ranging from 3 to 1900 g at 27°C in normoxia. Since the lamellae were distinguishable from the ILCM, we measured the actual SA as well as the potential SA if ILCM were lost. As a result of low τh, P. hypophthalmus has a high capacity for branchial oxygen uptake with or without ILCM. Actual and potential gill ADF were 361 and 1002 cm2 µm-1 kg-1, respectively, for a 100 g fish and the ADF of the swim bladder was found to be 308 cm2 µm-1 kg-1 By swimming fish to exhaustion at different temperatures, we show that modulation of this SA is rapid, indicating that the apparent paradox between previous studies is eliminated. Regression analysis of log-log plots of respiratory SA in relation to body mass shows that the gill scales with mass similarly to the SA in active water-breathing fish, whereas the swim bladder scales with mass more like the mammalian lung does. This fish presents a combination of respiratory surfaces not previously seen in air-breathing fish.

Acid-base regulation in the air-breathing swamp eel (Monopterus albus) at different temperatures.

Vertebrates reduce arterial blood pH (pHa) when body temperature increases. In water breathers, this response occurs primarily by reducing plasma HCO3- levels with small changes in the partial pressure of CO2 (PCO2 ). In contrast, air breathers mediate the decrease in pHa by increasing arterial PCO2  (PaCO2 ) at constant plasma HCO3- by reducing lung ventilation relative to metabolic CO2 production. Much less is known about bimodal breathers, which utilize both water and air. Here, we characterized the influence of temperature on arterial acid-base balance and intracellular pH (pHi) in the bimodal-breathing swamp eel, Monopterus albus This teleost uses the buccopharyngeal cavity for gas exchange and has very reduced gills. When exposed to ecologically relevant temperatures (20, 25, 30 and 35°C) for 24 and 48 h, pHa decreased by -0.025 pH units (U) °C-1 in association with an increase in PaCO2 , but without changes in plasma [HCO3-]. pHi was also reduced with increased temperature. The slope of pHi of liver and muscle was -0.014 and -0.019 U °C-1, while the heart muscle showed a smaller reduction (-0.008 U °C-1). When exposed to hypercapnia (7 or 14 mmHg) at either 25 or 35°C, M. albus elevated plasma [HCO3-] and therefore seemed to defend the new pHa set-point, demonstrating an adjusted control of acid-base balance with temperature. Overall, the effects of temperature on acid-base balance in M. albus resemble those in air-breathing amniotes, and we discuss the possibility that this pattern of acid-base balance results from a progressive transition in CO2 excretion from water to air as temperature rises.

Clown knifefish (Chitala ornata) oxygen uptake and its partitioning in present and future temperature environments.

It has been argued that tropical ectotherms are more vulnerable to the projected temperature increases than their temperate relatives, because they already live closer to their upper temperature limit. Here we examine the effects of a temperature increase in environmental temperature to 6°C above the present day median temperature (27°C) in the freshwater air-breathing fish Chitala ornata, on aspects of its respiratory physiology in both normoxia and in hypoxia. We found no evidence of respiratory impairment with elevated temperature. The standard metabolic rate (SMR) and routine metabolic rate (RMR) in the two temperatures in normoxia and hypoxia increased with Q10 values between 2.3 and 2.9, while the specific dynamic action (SDA) and its coefficient increased from 7.8 to 14.7% in 27°C and 33°C, respectively. In addition, Chitala ornata exhibited significantly improved growth at the elevated temperature in both hypoxic and normoxic water. While projected temperature increases may negatively impact other essential aspects in this animal's environment, we see no evidence of a negative impact on this species itself.

Bioconcentration and half-life of quinalphos pesticide in rice-fish integration system in the Mekong Delta, Vietnam.

In order to determine the distribution and enable the elimination of quinalphos, a popular active pesticide compound used in the Mekong Delta, an experiment was set up in a rice-fish integration system in Can Tho City, Vietnam. Fish was stocked into the field when the rice was two-months old. Quinalphos was applied twice in doses of 42.5 g per 1000 m2. Water, fish and sediment samples were collected at time intervals and analyzed by a Gas Chromatography Electron Capture Detector system. The results show that quinalphos residues in fish muscles were much higher than those of the water and the bioconcentration factor (logBCF) was above 2 for the fish. The half-life of first and second quinalphos applications were 12.2 and 11.1 days for sediment, 2.5 and 1.1 days for silver barb, 1.9 and 1.3 days for common carp, and 1.1 and 1.0 days for water, respectively.

Gill remodelling and growth rate of striped catfish Pangasianodon hypophthalmus under impacts of hypoxia and temperature.

Gill morphometric and gill plasticity of the air-breathing striped catfish (Pangasianodon hypophthalmus) exposed to different temperatures (present day 27°C and future 33°C) and different air saturation levels (92% and 35%) during 6weeks were investigated using vertical sections to estimate the respiratory lamellae surface areas, harmonic mean barrier thicknesses, and gill component volumes. Gill respiratory surface area (SA) and harmonic mean water - blood barrier thicknesses (HM) of the fish were strongly affected by both environmental temperature and oxygen level. Thus initial values for 27°C normoxic fish (12.4±0.8g) were 211.8±21.6mm2g-1 and 1.67±0.12µm for SA and HM respectively. After 5weeks in same conditions or in the combinations of 33°C and/or PO2 of 55mmHg, this initial surface area scaled allometrically with size for the 33°C hypoxic group, whereas branchial SA was almost eliminated in the 27°C normoxic group, with other groups intermediate. In addition, elevated temperature had an astounding effect on growth with the 33°C group growing nearly 8-fold faster than the 27°C fish.

Ambient CO2, fish behaviour and altered GABAergic neurotransmission: exploring the mechanism of CO2-altered behaviour by taking a hypercapnia dweller down to low CO2 levels.

Recent studies suggest that projected rises of aquatic CO2 levels cause acid-base regulatory responses in fishes that lead to altered GABAergic neurotransmission and disrupted behaviour, threatening fitness and population survival. It is thought that changes in Cl(-) and HCO3 (-) gradients across neural membranes interfere with the function of GABA-gated anion channels (GABAA receptors). So far, such alterations have been revealed experimentally by exposing species living in low-CO2 environments, like many oceanic habitats, to high levels of CO2 (hypercapnia). To examine the generality of this phenomenon, we set out to study the opposite situation, hypothesizing that fishes living in typically hypercapnic environments also display behavioural alterations if exposed to low CO2 levels. This would indicate that ion regulation in the fish brain is fine-tuned to the prevailing CO2 conditions. We quantified pH regulatory variables and behavioural responses of Pangasianodon hypophthalmus, a fish native to the hypercapnic Mekong River, acclimated to high-CO2 (3.1 kPa) or low-CO2 (0.04 kPa) water. We found that brain and blood pH was actively regulated and that the low-CO2 fish displayed significantly higher activity levels, which were reduced after treatment with gabazine, a GABAA receptor blocker. This indicates an involvement of the GABAA receptor and altered Cl(-) and HCO3 (-) ion gradients. Indeed, Goldman calculations suggest that low levels of environmental CO2 may cause significant changes in neural ion gradients in P. hypophthalmus. Taken together, the results suggest that brain ion regulation in fishes is fine-tuned to the prevailing ambient CO2 conditions and is prone to disruption if these conditions change.

High affinity and temperature sensitivity of blood oxygen binding in Pangasianodon hypophthalmus due to lack of chloride-hemoglobin allosteric interaction.

Air-breathing fishes represent interesting organisms in terms of understanding the physiological changes associated with the terrestrialization of vertebrates, and, further, are of great socio-economic importance for aquaculture in Southeast Asia. To understand how environmental factors, such as high temperature, affect O2 transport in air-breathing fishes, this study assessed the effects of temperature on O2 binding of blood and Hb in the economically important air-breathing fish Pangasianodon hypophthalmus. To determine blood O2 binding properties, blood was drawn from resting cannulated fishes and O2 binding curves made at 25°C and 35°C. To determine the allosteric regulation and thermodynamics of Hb O2 binding, Hb was purified, and O2 equilibria were recorded at five temperatures in the absence and presence of ATP and Cl(-). Whole blood had a high O2 affinity (O2 tension at half saturation P50 = 4.6 mmHg at extracellular pH 7.6 and 25°C), a high temperature sensitivity of O2 binding (apparent heat of oxygenation ΔH(app) = -28.3 kcal/mol), and lacked a Root effect. Further, the data on Hb revealed weak ATP binding and a complete lack of Cl(-) binding to Hb, which, in part, explains the high O2 affinity and high temperature sensitivity of blood O2 binding. This study demonstrates how a potent mechanism for increasing O2 affinity is linked to increased temperature sensitivity of O2 transport and provides a basic framework for a better understanding of how hypoxia-adapted species will react to increasing temperatures.

Oxygen delivery does not limit thermal tolerance in a tropical eurythermal crustacean.

In aquatic environments, rising water temperatures reduce water oxygen content while increasing oxygen demand, leading several authors to propose cardiorespiratory oxygen transport capacity as the main determinant of aquatic animal fitness. It has also been argued that tropical species, compared with temperate species, live very close to their upper thermal limit and hence are vulnerable to even small elevations in temperature. Little, however, is known about physiological responses to high temperatures in tropical species. Here we report that the tropical giant freshwater shrimp (Macrobrachium rosenbergii) maintains normal growth when challenged by a temperature rise of 6°C above the present day average (from 27°C to 33°C). Further, by measuring heart rate, gill ventilation rate, resting and maximum oxygen uptake, and hemolymph lactate, we show that oxygen transport capacity is maintained up to the critical maximum temperature around 41°C. In M. rosenbergii heart rate and gill ventilation rate increases exponentially until immediately below critical temperatures and at 38°C animals still retained more than 76% of aerobic scope measured at 30°C, and there was no indication of anaerobic metabolism at the high temperatures. Our study shows that the oxygen transport capacity is maintained at high temperatures, and that other mechanisms, such as protein dysfunction, are responsible for the loss of ecological performance at elevated temperatures.

High blood oxygen affinity in the air-breathing swamp eel Monopterus albus.

The Asian swamp eel (Monopterus albus, Zuiew 1793) is a facultative air-breathing fish with reduced gills. Previous studies have shown that gas exchange seems to occur across the epithelium of the buccopharyngeal cavity, the esophagus and the integument, resulting in substantial diffusion limitations that must be compensated by adaptations in others steps of the O2 transport system to secure adequate O2 delivery to the respiring tissues. We therefore investigated O2 binding properties of whole blood, stripped hemoglobin (Hb), two major isoHb components and the myoglobin (Mb) from M. albus. Whole blood was sampled using indwelling catheters for blood gas analysis and determination of O2 equilibrium curves. Hb was purified to assess the effects of endogenous allosteric effectors, and Mb was isolated from heart and skeletal muscle to determine its O2 binding properties. The blood of M. albus has a high O2 carrying capacity [hematocrit (Hct) of 42.4±4.5%] and binds O2 with an unusually high affinity (P50=2.8±0.4mmHg at 27°C and pH7.7), correlating with insensitivity of the Hb to the anionic allosteric effectors that normally decrease Hb-O2 affinity. In addition, Mb is present at high concentrations in both heart and muscle (5.16±0.99 and 1.08±0.19mg ∙ g wet tissue⁻¹, respectively). We suggest that the high Hct and high blood O2 affinity serve to overcome the low diffusion capacity in the relatively inefficient respiratory surfaces, while high Hct and Mb concentration aid in increasing the O2 flux from the blood to the muscles.

A High-Quality Genome Assembly of Striped Catfish (Pangasianodon hypophthalmus) Based on Highly Accurate Long-Read HiFi Sequencing Data.

The HiFi sequencing technology yields highly accurate long-read data with accuracies greater than 99.9% that can be used to improve results for complex applications such as genome assembly. Our study presents a high-quality chromosome-scale genome assembly of striped catfish (Pangasianodon hypophthalmus), a commercially important species cultured mainly in Vietnam, integrating HiFi reads and Hi-C data. A 788.4 Mb genome containing 381 scaffolds with an N50 length of 21.8 Mb has been obtained from HiFi reads. These scaffolds have been further ordered and clustered into 30 chromosome groups, ranging from 1.4 to 57.6 Mb, based on Hi-C data. The present updated assembly has a contig N50 of 14.7 Mb, representing a 245-fold and 4.2-fold improvement over the previous Illumina and Illumina-Nanopore-Hi-C based version, respectively. In addition, the proportion of repeat elements and BUSCO genes identified in our genome is remarkably higher than in the two previously released striped catfish genomes. These results highlight the power of using HiFi reads to assemble the highly repetitive regions and to improve the quality of genome assembly. The updated, high-quality genome assembled in this work will provide a valuable genomic resource for future population genetics, conservation biology and selective breeding studies of striped catfish.

Autonomic control of the heart in the Asian swamp eel (Monopterus albus).

The Asian swamp eel (Monopterus albus) is an air-breathing teleost with very reduced gills that uses the buccal cavity for air-breathing. Here we characterise the cardiovascular changes associated with the intermittent breathing pattern in M. albus and we study the autonomic control of the heart during water- and air-breathing. The shift from water- to air-breathing was associated with a rise in heart rate from 27.7 ± 1.6 to 41.4 ± 2.6 min(-1) and an increase in cardiac output from 23.1 ± 3.0 to 58.7 ± 6.5 mLmin(-1)kg(-1), while mean systemic blood pressure did not change (39.0 ± 3.5 and 46.4 ± 1.3 cmH(2)O). The autonomic control of the heart during water- and air-breathing was revealed by infusion of the ß-adrenergic antagonist propranolol and muscarinic antagonist atropine (3 mgkg(-1)) in eels instrumented with an arterial catheter. Inhibition of the sympathetic and parasympathetic innervations of the heart revealed a strong vagal tone on the heart of water-breathing eels and that the tachycardia during air-breathing is primarily mediated by withdrawal of cholinergic tone.

Hypoxia tolerance and partitioning of bimodal respiration in the striped catfish (Pangasianodon hypophthalmus).

Air-breathing fish are common in the tropics, and their importance in Asian aquaculture is increasing, but the respiratory physiology of some of the key species such as the striped catfish, Pangasianodon hypophthalmus Sauvage 1878 is unstudied. P. hypophthalmus is an interesting species as it appears to possess both well-developed gills and a modified swim bladder that functions as an air-breathing organ indicating a high capacity for both aquatic and aerial respiration. Using newly developed bimodal intermittent-closed respirometry, the partitioning of oxygen consumption in normoxia and hypoxia was investigated in P. hypophthalmus. In addition the capacity for aquatic breathing was studied through measurements of oxygen consumption when access to air was denied, both in normoxia and hypoxia, and the critical oxygen tension, Pcrit, was also determined during these experiments. Finally, gill ventilation and air-breathing frequency were measured in a separate experiment with pressure measurements from the buccal cavity. The data showed that P. hypophthalmus is able to maintain standard metabolic rate (SMR) through aquatic breathing alone in normoxia, but that air-breathing is important during hypoxia. Gill ventilation was reduced during air-breathing, which occurred at oxygen levels below 8 kPa, coinciding with the measured Pcrit of 7.7 kPa. The findings in this study indicate that the introduction of aeration into the aquaculture of P. hypophthalmus could potentially reduce the need to air-breathe. The possibility of reducing air-breathing frequency may be energetically beneficial for the fish, leaving more of the aerobic scope for growth and other activities, due to the proposed energetic costs of surfacing behavior.

Striped catfish (Pangasianodon hypophthalmus) use air-breathing and aquatic surface respiration when exposed to severe aquatic hypercarbia.

We investigated the extent to which the facultative air-breathing fish, the striped catfish (Pangasianodon hypophthalmus), uses air-breathing to cope with aquatic hypercarbia, and how air-breathing is influenced by the experimental exposure protocol and level of hypercarbia. We exposed individuals to severe aquatic hypercarbia (up to Pw CO2 = 81 mmHg) using step-wise and progressive exposure protocols while measuring gill ventilation rate, heart rate, mean arterial blood pressure, and air-breathing frequency, as well as arterial blood pH and PCO2 . We confirm that P. hypophthalmus is tolerant of hypercarbia. Under both protocols gill ventilation rate, heart rate, and mean arterial blood pressure were maintained near control levels even at very high CO2 levels. We observed a marked amount of individual variation in the PwCO2 at which air-breathing was elicited, with some individuals not responding at all. The experimental protocol also influenced the onset of air-breathing. Air-breathing began at lower Pw CO2 in the step-wise protocol (23 ± 4.1 mmHg) compared with the progressive protocol (46 ± 7.8 mmHg). Air-breathing was often followed by aquatic surface respiration, at higher PCO2 (71 ± 5.2 mmHg) levels. On average, the blood PCO2 was approximately 43% lower (46 ± 2.5 mmHg) than water Pw CO2 (~81 mmHg) at our highest tested CO2 level. While this suggests that aerial CO2 elimination is an effective, and perhaps critical, respiratory strategy used by P. hypophthalmus to cope with severe hypercarbia, this observation may also be explained by a long lag time required for equilibration.

Ventilatory responses of the clown knifefish, Chitala ornata, to arterial hypercapnia remain after gill denervation.

The aim of this study was to corroborate the presence of CO2/H+-sensitive arterial chemoreceptors involved in producing air-breathing responses to aquatic hypercarbia in the facultative air-breathing clown knifefish (Chitala ornata) and to explore their possible location. Progressively increasing levels of CO2 mixed with air were injected into the air-breathing organ (ABO) of one group of intact fish to elevate internal PCO2 and decrease blood pH. Another group of fish in which the gills were totally denervated was exposed to aquatic hypercarbia (pH ~ 6) or arterial hypercapnia in aquatic normocarbia (by injection of acetazolamide to increase arterial PCO2 and decrease blood pH). Air-breathing frequency, gill ventilation frequency, heart rate and arterial PCO2 and pH were recorded during all treatments. The CO2 injections into the ABO induced progressive increases in air-breathing frequency, but did not alter gill ventilation or heart rate. Exposure to both hypercarbia and acetazolamide post-denervation of the gills also produced significant air-breathing responses, but no changes in gill ventilation. While all treatments produced increases in arterial PCO2 and decreases in blood pH, the modest changes in arterial PCO2/pH in the acetazolamide treatment produced the greatest increases in air-breathing frequency. These results strengthen the evidence that internal CO2/H+ sensing is involved in the stimulation of air breathing in clown knifefish and suggest that it involves extra-branchial chemoreceptors possibly situated either centrally or in the air-breathing organ.

Impact and tissue metabolism of nitrite at two acclimation temperatures in striped catfish (Pangasianodon hypophthalmus).

Elevated concentrations of nitrite develop occasionally in various aquatic habitats and aquaculture facilities, providing a potential danger for freshwater fish that take up nitrite via the gill chloride uptake mechanism. We studied the uptake, effects and metabolism of nitrite in blood, heart and skeletal muscle at two temperatures in striped catfish Pangasianodon hypophthalmus, a facultative air-breathing fish that is heavily cultivated in Southeast Asia. Exposure to 0.8 mM ambient nitrite increased blood [nitrite] and [methaemoglobin] (metHb) to high values at day 1, but values subsequently decreased towards controls at day 7. Blood [nitrite] and metHb content were unexpectedly higher at 27 °C (∼1.2 mM; 69% at day 1) than at 33 °C (∼0.9 mM; 55%), reflecting a lower nitrite uptake at the highest temperature, possibly via an increased reliance on air-breathing relative to water-breathing with temperature increase. A large fraction of the nitrite taken up was effectively eliminated by being detoxified to nitrate. Further, erythrocyte metHb reductase activity was increased during nitrite exposure, efficiently reducing metHb to functional haemoglobin. The uptake of nitrite into white skeletal musculature (main part of the fish) was much lower than into heart tissue. While heart [nitrite] was close to blood plasma levels, muscle [nitrite] peaked at ∼0.2 mM at day 1 and subsequently declined to ∼0.05 mM at day 7, which is below levels reported in various commercial cured meat products. Nitrite was partly metabolized to iron-nitrosyl, S-nitroso and N-nitroso compounds. The increase in nitros(yl)ated compounds was marginal in skeletal muscle and more pronounced in heart tissue.