Water-breathing Fish Research Articles

A method for measuring meaningful physiological variables in fish blood without surgical cannulation

Gaining meaningful blood samples from water-breathing fish is a significant challenge. Two main methods typically used are grab ‘n’ stab and surgical cannulation. Both methods have benefits, but also significant limitations under various scenarios. Here we present a method of blood sampling laboratory fish involving gradual induction of anaesthesia within their home tank, avoiding physical struggling associated with capture, followed by rapid transfer to a gill irrigation system to maintain artificial ventilation via adequate gill water flow and then followed by sampling the caudal vasculature. This method negates many blood chemistry disturbances associated with grab ‘n’ stab (i.e., low pH and oxygen, elevated lactate, CO2 and stress hormones) and generates results that are directly comparable to cannulated fish under a wide range of experimentally-induced acid–base scenarios (acidosis and alkalosis). Crucially this method was successful in achieving accurate acid–base blood measurements from fish ten times smaller than are typically suitable for cannulation. This opens opportunities not previously possible for studies that relate to basic physiology, sustainable aquaculture, ecotoxicology, conservation, and climate change.

Comparative Transcriptomic Analysis of Regenerated Skins Provides Insights into Cutaneous Air-Breathing Formation in Fish.

Simple SummaryCutaneous air-breathing is one of the air-breathing patterns in bimodal respiration fishes, while little is known about its underlying formation mechanisms. The skin regeneration of loach (Misgurnus anguillicaudatus, a cutaneous air-breathing fish) and yellow catfish (Pelteobagrus fulvidraco, a water-breathing fish) were first investigated through morphological and histological observations. Then, the original skins (OS: MOS, POS) and regenerated skins (RS: MRS, PRS) when their capillaries were the most abundant during healing, of the two fish species were collected for high-throughput RNA-seq. A total of 56,054 unigenes and 53,731 unigenes were assembled in loach and yellow catfish, respectively. A total of 640 (460 up- and 180 down-regulated) and 4446 (2340 up- and 2106 down-regulated) differentially expressed genes (DEGs) were respectively observed in RS/OS of loach and yellow catfish. Subsequently, the two DEG datasets were clustered in GO, KOG, and KEGG databases, and further analyzed by comparison and screening. Consequently, tens of genes and thirteen key pathways were targeted, indicating that these genes and pathways had strong ties to cutaneous skin air-breathing in loach. This study provides new insights into the formation mechanism of cutaneous air-breathing and also offers a substantial contribution to the gene expression profiles of skin regeneration in fish.Cutaneous air-breathing is one of the air-breathing patterns in bimodal respiration fishes, while little is known about its underlying formation mechanisms. Here, we first investigated the skin regeneration of loach (Misgurnus anguillicaudatus, a cutaneous air-breathing fish) and yellow catfish (Pelteobagrus fulvidraco, a water-breathing fish) through morphological and histological observations. Then, the original skins (OS: MOS, POS) and regenerated skins (RS: MRS, PRS) when their capillaries were the most abundant (the structural foundation of air-breathing in fish) during healing, of the two fish species were collected for high-throughput RNA-seq. A total of 56,054 unigenes and 53,731 unigenes were assembled in loach and yellow catfish, respectively. A total of 640 (460 up- and 180 down-regulated) and 4446 (2340 up- and 2106 down-regulated) differentially expressed genes (DEGs) were respectively observed in RS/OS of loach and yellow catfish. Subsequently, the two DEG datasets were clustered in GO, KOG and KEGG databases, and further analyzed by comparison and screening. Consequently, tens of genes and thirteen key pathways were targeted, indicating that these genes and pathways had strong ties to cutaneous skin air-breathing in loach. This study provides new insights into the formation mechanism of cutaneous air-breathing and also offers a substantial contribution to the gene expression profiles of skin regeneration in fish.

Evolution of vertebrate respiratory central rhythm generators

Tracing the evolution of the central rhythm generators associated with ventilation in vertebrates is hindered by a lack of information surrounding key transitions. To begin with, central rhythm generation has been studied in detail in only a few species from four vertebrate groups, lamprey, anuran amphibians, turtles, and mammals (primarily rodents). Secondly, there is a lack of information regarding the transition from water breathing fish to air breathing amniotes (reptiles, birds, and mammals). Specifically, the respiratory rhythm generators of fish appear to be single oscillators capable of generating both phases of the respiratory cycle (expansion and compression) and projecting to motoneurons in cranial nerves innervating bucco-pharyngeal muscles. In the amniotes we find oscillators capable of independently generating separate phases of the respiratory cycle (expiration and inspiration) and projecting to pre-motoneurons in the ventrolateral medulla that in turn project to spinal motoneurons innervating thoracic and abdominal muscles (reptiles, birds, and mammals). Studies of the one group of amphibians that lie at this transition (the anurans), raise intriguing possibilities but, for a variety of reasons that we explore, also raise unanswered questions. In this review we summarize what is known about the rhythm generating circuits associated with breathing that arise from the different rhombomeric segments in each of the different vertebrate classes. Assuming oscillating circuits form in every pair of rhombomeres in every vertebrate during development, we trace what appears to be the evolutionary fate of each and highlight the questions that remain to be answered to properly understand the evolutionary transitions in vertebrate central respiratory rhythm generation.

The weakly electric fish, Apteronotus albifrons, actively avoids experimentally induced hypoxia

Anthropogenic environmental degradation has led to an increase in the frequency and prevalence of aquatic hypoxia (low dissolved oxygen concentration, DO), which may affect habitat quality for water-breathing fishes. The weakly electric black ghost knifefish, Apteronotus albifrons, is typically found in well-oxygenated freshwater habitats in South America. Using a shuttle-box design, we exposed juvenile A. albifrons to a stepwise decline in DO from normoxia (> 95% air saturation) to extreme hypoxia (10% air saturation) in one compartment and chronic normoxia in the other. On average, A. albifrons actively avoided the hypoxic compartment below 22% air saturation. Hypoxia avoidance was correlated with upregulated swimming activity. Following avoidance, fish regularly ventured back briefly into deep hypoxia. Hypoxia did not affect the frequency of their electric organ discharges. Our results show that A. albifrons is able to sense hypoxia at non-lethal levels and uses active avoidance to mitigate its adverse effects.

Evolutionary and cardio-respiratory physiology of air-breathing and amphibious fishes.

Air-breathing and amphibious fishes are essential study organisms to shed insight into the required physiological shifts that supported the full transition from aquatic water-breathing fishes to terrestrial air-breathing tetrapods. While the origin of air-breathing in the evolutionary history of the tetrapods has received considerable focus, much less is known about the evolutionary physiology of air-breathing among fishes. This review summarizes recent advances within the field with specific emphasis on the cardiorespiratory regulation associated with air-breathing and terrestrial excursions, and how respiratory physiology of these living transitional forms are affected by development and personality. Finally, we provide a detailed and re-evaluated model of the evolution of air-breathing among fishes that serves as a framework for addressing new questions on the cardiorespiratory changes associated with it. This review highlights the importance of combining detailed studies on piscine air-breathing model species with comparative multi-species studies, to add an additional dimension to our understanding of the evolutionary physiology of air-breathing in vertebrates.

Evaluating the physiological significance of hypoxic hyperventilation in larval zebrafish (Danio rerio).

In water-breathing fishes, the hypoxic ventilatory response (HVR) represents an increase in water flow over the gills during exposure to lowered ambient O2 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 O2 tensions (Pcrit) remained constant (30-34 mmHg) over the same period of development. Micro-optrode O2 sensors were used to measure a significant decrease in buccal cavity water O2 tensions in 4 and 7 dpf larvae compared with the water they inspired, demonstrating significant extraction of O2 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 Pcrit was observed in larvae at 7 dpf but not 4 dpf, suggesting that buccal ventilation is important for O2 extraction by 7 dpf. Combined, these data indicate that branchial/buccal gas transfer plays a significant role in O2 uptake during hypoxia, and supports a physiological benefit of the HVR in early life stages of zebrafish.

The Air-Breathing Paradise Fish (Macropodus opercularis) Differs From Aquatic Breathers in Strategies to Maintain Energy Homeostasis Under Hypoxic and Thermal Stresses.

Two major strategies are used by most fish to maintain energy homeostasis under hypoxia. One is to utilize alternative metabolic pathways to increase energy production, and the other is to limit energy expenditure by suppressing energy-consuming processes, especially ionoregulation. Some anabantoid fishes live in tropical rivers, where hypoxic environments occur frequently. We previously found that under ambient hypoxia, anabantoid fishes do not downregulate Na+/K+-ATPase (NKA) activity to conserve energy in gills but instead increase the frequency of air-breathing respiration (ABR). In addition to the hypoxic condition, another factor that may cause cellular hypoxia in fish is abnormally high environmental temperatures. The frequency of such extreme thermal events has increased due to global climate change. In the present study, we examined whether the anabantoid fish, Macropodus opercularis employs the two strategies mentioned above to resist both ambient hypoxic and elevated thermal (cellular hypoxic) conditions. Results indicate that neither glucose metabolism nor gill NKA activity were altered by hypoxia (DO = 1.5 ± 1 mg/L), but glucose metabolism was increased by thermal stress (34 ± 1°C). NH4+ excretion and ABR frequency were both increased under hypoxia, thermal or hypoxic-and-thermal treatments. In fish that were restricted from breathing air, increased mortality and glucose metabolism were observed under hypoxic or thermal treatments. These results suggest that for M. opercularis, increasing ABR is an important strategy for coping with unmet oxygen demand under hypoxic or thermal stress. This behavioral compensation allows anabantoid fish to physiologically withstand hypoxic and thermal stresses, and constitutes a mechanism of stress resistance that is unavailable to water-breathing fishes.

Insights into the evolution of polymodal chemoreceptors

Respiratory chemoreceptors in vertebrates are specialized cells that detect chemical changes in the environment or arterial blood supply and initiate autonomic responses, such as hyperventilation or changes in heart rate, to improve O2 uptake and delivery to tissues. These chemoreceptors are sensitive to changes in O2, CO2 and/or H+. In fish and mammals, respiratory chemoreceptors may be additionally sensitive to ammonia, hypoglycemia, and numerous other stimuli. Thus, chemoreceptors that affect respiration respond to different types of stimuli (or modalities) and are considered to be "polymodal". This review discusses the polymodal nature of respiratory chemoreceptors in vertebrates with a particular emphasis on chemoreceptors of the carotid body and pulmonary epithelium in mammals, and on neuroepithelial cells in water- and air-breathing fish. A major goal will be to examine the evidence for putative polymodal chemoreceptors in fish within the context of studies on mammalian models, for which polymodal chemoreceptors are well described, in order to improve our understanding of the evolution of polymodal chemoreceptors in vertebrates, and to aid in future studies that aim to identify putative receptors in air- and water-breathing fish.

Emersion behaviour underlies variation in gill morphology and aquatic respiratory function in the amphibious fish Kryptolebias marmoratus

Fishes acclimated to hypoxic environments often increase gill surface area to improve O2 uptake. In some species, surface area is increased via reduction of an interlamellar cell mass (ILCM) that fills water channels between gill lamellae. Amphibious fishes, however, may not increase gill surface area in hypoxic water because these species can, instead, leave water and breathe air. To differentiate between these possibilities, we compared wild amphibious mangrove rivulus Kryptolebias marmoratus from two habitats that varied in O2 availability - a hypoxic freshwater pool versus nearly anoxic crab burrows. Fish captured from crab burrows had less gill surface area (as ILCMs were enlarged by ∼32%), increased rates of normoxic O2 consumption and increased critical O2 tension compared with fish from the freshwater pool. Thus, wild mangrove rivulus do not respond to near-anoxic water by decreasing metabolism or increasing O2 extraction. Instead, fish from the crab burrow habitat spent three times longer out of water, which probably caused the observed changes in gill morphology and respiratory phenotype. We also tested whether critical O2 tension is influenced by genetic heterozygosity, as K. marmoratus is one of only two hermaphroditic vertebrate species that can produce both self-fertilized (inbred) or out-crossed (more heterozygous) offspring. We found no evidence for inbreeding depression, suggesting that self-fertilization does not impair respiratory function. Overall, our results demonstrate that amphibious fishes that inhabit hypoxic aquatic habitats can use a fundamentally different strategy from that used by fully aquatic water-breathing fishes, relying on escape behaviour rather than metabolic depression or increased O2 extraction ability.

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-1kg-1, respectively, for a 100 g fish and the ADF of the swim bladder was found to be 308 cm2 µm-1kg-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.

Analysis of the nicotinamide phosphoribosyltransferase family provides insight into vertebrate adaptation to different oxygen levels during the water-to-land transition.

One of the most important events in vertebrate evolutionary history is the water-to-land transition, during which some morphological and physiological changes occurred in concert with the loss of specific genes in tetrapods. However, the molecular mechanisms underlying this transition have not been well explored. To explore vertebrate adaptation to different oxygen levels during the water-to-land transition, we performed comprehensive bioinformatics and experimental analysis aiming to investigate the NAMPT family in vertebrates. NAMPT, a rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, is critical for cell survival in a hypoxic environment, and a high level of NAMPT significantly augments oxidative stress in normoxic environments. Phylogenetic analysis showed that NAMPT duplicates arose from a second round whole-genome duplication event. NAMPTA existed in all classes of vertebrates, whereas NAMPTB was only found in fishes and not tetrapods. Asymmetric evolutionary rates and purifying selection were the main evolutionary forces involved. Although functional analysis identified several functionally divergent sites during NAMPT family evolution, in vitro experimental data demonstrated that NAMPTA and NAMPTB were functionally conserved for NAMPT enzymatic function in the NAD+ salvage pathway. In situ hybridization revealed broad NAMPTA and NAMPTB expression patterns, implying regulatory functions over a wide range of developmental processes. The morpholino-mediated knockdown data demonstrated that NAMPTA was more essential than NAMPTB for vertebrate embryo development. We propose that the retention of NAMPTB in water-breathing fishes and its loss in air-breathing tetrapods resulted from vertebrate adaptation to different oxygen levels during the water-to-land transition.

Air‐breathing fishes

Air‐breathing fishes

Metabolism, gas exchange, and acid‐base balance of giant salamanders

The giant salamanders are aquatic and paedomorphic urodeles including the genera Andrias and Cryptobranchus (Cryptobranchidae), Amphiuma (Amphiumidae), Siren (Sirenidae), and Necturus (Proteidae, of which only N. maculosus is considered 'a giant'). Species in the genera Cryptobranchus and Necturus are considered aquatic salamanders well adapted for breathing water, poorly adapted for breathing air, and with limited abilities to compensate acid-base disturbances. As such, they are water-breathing animals with a somewhat fish-like respiratory and acid-base physiology, whose habitat selection is limited to waters that do not typically become hypoxic or hypercarbic (although this assertion has been questioned for N. maculosus). Siren and Amphiuma species, by contrast, are dependent upon air-breathing, have excellent lungs, inefficient (Siren) or no (Amphiuma) gills, and are obligate air-breathers with an acid-base status more similar to that of terrestrial tetrapods. As such, they can be considered to be air-breathing animals that live in water. Their response to the aquatic hypercarbia that they often encounter is to maintain intracellular pH (pH(i) ) and abandon extracellular pH regulation, a process that has been referred to as preferential pH(i) regulation. The acid-base status of some present-day tropical air-breathing fishes, and of Siren and Amphiuma, suggests that the acid-base transition from a low PCO(2) -low [] system typical of water-breathing fishes to the high PCO(2) -high [] systems of terrestrial tetrapods may have been completed before emergence onto land, and likely occurred in habitats that were typically both hypoxic and hypercarbic.

The simultaneous presence of neuroepithelial cells and neuroepithelial bodies in the respiratory gas bladder of the longnose gar, Lepisosteus osseus, and the spotted gar, L. oculatus

Anatomical and functional studies on the autonomic innervation as well as the location of airway receptors in the air-bladder of lepisosteids are very fragmentary. These water-breathing fishes share in common with the bichirs the presence of a glottis (not a ductus pneumaticus) opening into the esophagus. In contrast to a high concentration of neuroepithelial cells (NECs) contained in the furrowed epithelium in the lung of Polypterus, these cells are scattered as solitary cells in the glottal epithelium, and grouped to form neuroepithelial bodies (NEBs) in the mucociliated epithelium investing the main trabeculae in the air-bladder of Lepisosteus osseus and L. oculatus. The present immunohistochemical studies also demonstrated the presence of nerve fibers in the trabecular striated musculature and a possible relation to NEBs in these species, and identified immunoreactive elements of this innervation. Tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), 5-HT and neuropeptide immunoreactivities were detected in the intramural nerve fibers. 5-HT and VIP immunopositive nerve fibers are apparently associated with NEBs. TH, VIP and SP immunoreactivities are also present in nerve fibers coursing in the radially arranged striated muscle surrounding the glottis and its submucosa. 5-HT positive neurons are also found in submucosal and the muscle layers of the glottis. The physiological function of the adrenergic and inhibitory innervation of the striated muscle as well as the neurochemical coding and morphology of the innervation of the NEBs are not known. Future studies are needed to provide evidence for these receptors with the capacity of chemoreceptors and/or mechanoreceptors.

Gross and fine anatomy of the respiratory vasculature of the mudskipper, Periophthalmodon schlosseri (Gobiidae: Oxudercinae)

To illustrate vascular modification accompanying transition from aquatic to amphibious life in gobies, we investigated the respiratory vasculatures of the gills and the bucco-opercular cavities in one of the most terrestrially-adapted mudskippers, Periophthalmodon schlosseri, using the corrosion casting technique. The vascular system of Pn. schlosseri retains the typical fish configuration with a serial connection of the gills and the systemic circuits, suggesting a lack of separation of O(2)-poor systemic venous blood and O(2)-rich effluent blood from the air-breathing surfaces. The gills appear to play a limited role in gas exchange, as evidenced from the sparsely-spaced short filaments and the modification of secondary lamellar vasculature into five to eight parallel channels that are larger than red blood cell size, unlike the extensive sinusoidal system seen in purely water-breathing fishes. In contrast, the epithelia of the bucco-opercular chamber, branchial arches, and leading edge of the filaments are extensively laden with capillaries having a short (<10 μm) diffusion distance, which strongly demonstrate the principal respiratory function of these surfaces. These capillaries form spiral coils of three to five turns as they approach the epithelial surface. The respiratory capillaries of the bucco-opercular chamber are supplied by efferent blood from the gills and drained by the systemic venous pathway. We also compared the degree of capillarization in the bucco-opercular epithelia of Pn. schlosseri with that of the three related intertidal-burrowing gobies (aquatic, non-air-breathing Acanthogobius hasta; aquatic, facultative air-breathing Odontamblyopus lacepedii; amphibious air-breathing Periophthalmus modestus) through histological analysis. The comparison revealed a clear trend of wider distribution of denser capillary networks in these epithelia with increasing reliance on air breathing, consistent with the highest aerial respiratory capacity of Pn. schlosseri among the four species.

Response of mucous cells of the gills of traíra (Hoplias malabaricus) and jeju (Hoplerythrinus unitaeniatus) (Teleostei: Erythrinidae) to hypo- and hyper-osmotic ion stress

The mucous cells (MC) of traíra, Hoplias malabaricus, and jeju, Hoplerythrinus unitaeniatus, two ecologically distinct erythrinid species, were analyzed in fish exposed to deionized (DW) and high ion concentration (NaCl and Ca2+) water (HIW) during 15 days to evaluate the MC responses to ion challenge. MCs are localized in the leading and trailing edge and, interlamellar region of the gill filament epithelium but, in H. unitaeniatus, they are also found in the breathing or lamellar epithelium. MC density is lower in H. malabaricus, the exclusively water-breathing fish, than in H. unitaeniatus, a facultative air-breathing fish. The transference to DW or HIW did not change the MC density and surface area, excepting in H. malabaricus, in the first day of exposure to DW. A single MC containing three types of glycoproteins (neutral, acidic and sulphated) was identified in the gill epithelium of both, H. malabaricus and H. unitaeniatus. The amount (based on the intensity of histochemistry reaction) of these glycoproteins differed between the species and were altered after exposure to DW and HIW showing little adjustments in the amount of mucosubstances in the MC of H. malabaricus and reduction of acidic and sulphated glycoproteins in H. unitaeniatus. The decreasing of these glycoproteins in H. unitaeniatus reduced the mucus protection against desiccation of gill tissue when change the ion concentration in water.

The autonomic control and functional significance of the changes in heart rate associated with air breathing in the jeju,Hoplerythrinus unitaeniatus

The jeju is a teleost fish with bimodal respiration that utilizes a modified swim bladder as an air-breathing organ (ABO). Like all air-breathing fish studied to date, jeju exhibit pronounced changes in heart rate (fH) during air-breathing events, and it is believed that these may facilitate oxygen uptake (MO2) from the ABO. The current study employed power spectral analysis (PSA) of fH patterns, coupled with instantaneous respirometry, to investigate the autonomic control of these phenomena and their functional significance for the efficacy of air breathing. The jeju obtained less than 5% of total MO2 (MtO2) from air breathing in normoxia at 26 degrees C, and PSA of beat-to-beat variability in fH revealed a pattern similar to that of unimodal water-breathing fish. In deep aquatic hypoxia (water PO2)=1 kPa) the jeju increased the frequency of air breathing (fAB) tenfold and maintained MtO2 unchanged from normoxia. This was associated with a significant increase in heart rate variability (HRV), each air breath (AB) being preceded by a brief bradycardia and then followed by a brief tachycardia. These fH changes are qualitatively similar to those associated with breathing in unimodal air-breathing vertebrates. Within 20 heartbeats after the AB, however, a beat-to-beat variability in fH typical of water-breathing fish was re-established. Pharmacological blockade revealed that both adrenergic and cholinergic tone increased simultaneously prior to each AB, and then decreased after it. However, modulation of inhibitory cholinergic tone was responsible for the major proportion of HRV, including the precise beat-to-beat modulation of fH around each AB. Pharmacological blockade of all variations in fH associated with air breathing in deep hypoxia did not, however, have a significant effect upon fAB or the regulation of MtO2. Thus, the functional significance of the profound HRV during air breathing remains a mystery.

LUNGFISHES' BALANCING ACT

Our bodies are constantly working to keep everything in balance; when we exercise, not only do we breathe harder to get more oxygen to our muscles, but also to get rid of the painful lactic acid `burn'. All air breathing land dwellers deal with acid – an excess of protons – by reacting protons with bicarbonate ions to create water and CO2, which is breathed out. To deal with too much base, breathing slows down, keeping CO2 and therefore protons in the body. Water breathing fish take a different approach, relying on metabolic processes at their gills and kidneys to restore a normal blood pH. But how will animals that can breathe in both air and water deal with pH changes? `Lungfish are poised between air breathing and water breathing, and are a beautiful model to study this,' says Katie Gilmour of the University of Ottawa, who investigated with her colleagues if lungfish cope with pH changes like air breathers or water breathers(p. 1944).Lungfish bridge the divide between land and water, dependent on their watery environment for food but dying if they can't breathe air with their lungs. They also have gills, which they use to water breathe and get rid of some CO2. Once the team's fish had arrived in the lab from their African home, they trained them to sit in water-filled tube respirometers and pop their heads above the water's surface when they needed a gulp of air.To find out how the fish would deal with an excess of acid or base, they delicately operated on them to insert a cannula into the dorsal aorta, so that they could change blood pH by injecting acid or base into the blood stream. To find out how the fish dealt with an acid injection, they counted the number of air and water breaths, finding that they took twice as many of both and breathed out more CO2, much like a land dweller. To see if the gills or kidneys were also getting rid of excess acid, the team measured acid excretion into the water in the respirometer. They didn't see a rise in excretion from normal levels, suggesting that lungfish don't rely on their gills and kidneys much to get rid of excess protons.Injecting base and raising blood pH caused the fish to halve their water breathing rate; they also breathed out much less CO2 into the water. Their air breathing was unaffected, however, probably because they wouldn't get enough oxygen otherwise. The team measured that the fish were excreting much higher levels of base into the water, so to separate out how much the gills and kidneys were each contributing, they collected the fishes'urine via a catheter. They found that the kidneys excreted around 20% of the total base into the urine, and the gills around 80% into the water, much like fish, showing that the lungfish rely more on the water breathers' strategy to get rid of excess base and rebalance pH.`The lungfish have the best of both worlds,' says Gilmour. Like land dwellers, they rely more on air breathing to redress a more acidic blood pH,but their gills and kidneys deal with an excess of base to return blood pH back to normal, just like water breathing fish.

Mechanisms of acid–base regulation in the African lungfishProtopterus annectens

African lungfish Protopterus annectens utilized both respiratory and metabolic compensation to restore arterial pH to control levels following the imposition of a metabolic acidosis or alkalosis. Acid infusion (3 mmol kg(-1) NH(4)Cl) to lower arterial pH by 0.24 units increased both pulmonary (by 1.8-fold) and branchial (by 1.7-fold) ventilation frequencies significantly, contributing to 4.8-fold and 1.9-fold increases in, respectively, aerial and aquatic CO(2) excretion. This respiratory compensation appeared to be the main mechanism behind the restoration of arterial pH, because even though net acid excretion (J(net)H(+)) increased following acid infusion in 7 of 11 fish, the mean increase in net acid excretion, 184.5+/-118.5 micromol H(+) kg(-1) h(-1) (mean +/- s.e.m., N=11), was not significantly different from zero. Base infusion (3 mmol kg(-1) NaHCO(3)) to increase arterial pH by 0.29 units halved branchial ventilation frequency, although pulmonary ventilation frequency was unaffected. Correspondingly, aquatic CO(2) excretion also fell significantly (by 3.7-fold) while aerial CO(2) excretion was unaffected. Metabolic compensation consisting of negative net acid excretion (net base excretion) accompanied this respiratory compensation, with J(net)H(+) decreasing from 88.5+/-75.6 to -337.9+/-199.4 micromol H(+) kg(-1) h(-1) (N=8). Partitioning of net acid excretion into renal and extra-renal (assumed to be branchial and/or cutaneous) components revealed that under control conditions, net acid excretion occurred primarily by extra-renal routes. Finally, several genes that are involved in the exchange of acid-base equivalents between the animal and its environment (carbonic anhydrase, V-type H(+)-ATPase and Na(+)/HCO (-)(3) cotransporter) were cloned, and their branchial and renal mRNA expressions were examined prior to and following acid or base infusion. In no case was mRNA expression significantly altered by metabolic acid-base disturbance. These findings suggest that lungfish, like tetrapods, alter ventilation to compensate for metabolic acid-base disturbances, a mechanism that is not employed by water-breathing fish. Like fish and amphibians, however, extra-renal routes play a key role in metabolic compensation.

Beta-adrenergic control of plasma glucose and free fatty acid levels in the air-breathing African catfishClarias gariepinusBurchell 1822

In several water-breathing fish species, beta-adrenergic receptor stimulation by noradrenaline leads to a decrease in plasma free fatty acid (FFA) levels, as opposed to an increase in air-breathing mammals. We hypothesised that this change in adrenergic control is related to the mode of breathing. Therefore, cannulated air-breathing African catfish were infused for 90 min with noradrenaline or with the nonselective beta-agonist, isoprenaline. To identify the receptor type involved, a bolus of either a selective beta1-antagonist (atenolol) or a selective beta2-antagonist (ICI 118,551) was injected 15 min prior to the isoprenaline infusion. Both noradrenaline and isoprenaline led to an expected rise in glucose concentration. Isoprenaline combined with both the beta1- and beta2-antagonist led to higher glucose concentrations than isoprenaline alone. This could indicate the presence of a stimulatory beta-adrenoceptor different from beta1 and beta2-adrenoceptors; these two receptors thus seemed to mediate a reduction in plasma glucose concentration. Both noradrenaline and isoprenaline led to a significant decrease in FFA concentration. Whereas the beta1-antagonist had no effect, the beta2-antagonist reduced the decrease in FFA concentration, indicating the involvement of beta2-adrenoceptors. It is concluded that the air-breathing African catfish reflects water-breathing fish in the adrenergic control of plasma FFA and glucose levels.