Soluble adenylyl cyclase is an acid-base sensor in rainbow trout red blood cells that regulates intracellular pH and haemoglobin-oxygen binding.

AIM: To identify the physiological role of the acid-base sensing enzyme, soluble adenylyl cyclase (sAC), in red blood cells (RBC) of the model teleost fish, rainbow trout. METHODS: We used: (i) super-resolution microscopy to determine the subcellular location of sAC protein; (ii) live-cell imaging of RBC intracellular pH (pHi) with specific sAC inhibition (KH7 or LRE1) to determine its role in cellular acid-base regulation; (iii) spectrophotometric measurements of haemoglobin-oxygen (Hb-O2) binding in steady-state conditions; and (iv) during simulated arterial-venous transit, to determine the role of sAC in systemic O2 transport. RESULTS: Distinct pools of sAC protein were detected in the RBC cytoplasm, at the plasma membrane and within the nucleus. Inhibition of sAC decreased the setpoint for RBC pHi regulation by ~0.25 pH units compared to controls, and slowed the rates of RBC pHi recovery after an acid-base disturbance. RBC pHi recovery was entirely through the anion exchanger (AE) that was in part regulated by HCO3 --dependent sAC signaling. Inhibition of sAC decreased Hb-O2 affinity during a respiratory acidosis compared to controls and reduced the cooperativity of O2 binding. During in vitro simulations of arterial-venous transit, sAC inhibition decreased the amount of O2 that is unloaded by ~11%. CONCLUSION: sAC represents a novel acid-base sensor in the RBCs of rainbow trout, where it participates in the modulation of RBC pHi and blood O2 transport though the regulation of AE activity. If substantiated in other species, these findings may have broad implications for our understanding of cardiovascular physiology in vertebrates.

The physiological significance of plasma-accessible carbonic anhydrase in the respiratory systems of fishes.

Carbonic anhydrase (CA) activity is ubiquitously found in all vertebrate species, tissues and cellular compartments. Most species have plasma-accessible CA (paCA) isoforms at the respiratory surfaces, where the enzyme catalyzes the conversion of plasma bicarbonate to carbon dioxide (CO2) that can be excreted by diffusion. A notable exception are the teleost fishes that appear to lack paCA at their gills. The present review: (i) recapitulates the significance of CA activity and distribution in vertebrates; (ii) summarizes the current evidence for the presence or absence of paCA at the gills of fishes, from the basal cyclostomes to the derived teleosts and extremophiles such as the Antarctic icefishes; (iii) explores the contribution of paCA to organismal CO2 excretion in fishes; and (iv) the functional significance of its absence at the gills, for the specialized system of O2 transport in most teleosts; (v) outlines the multiplicity and isoform distribution of membrane-associated CAs in fishes and methodologies to determine their plasma-accessible orientation; and (vi) sketches a tentative time line for the evolutionary dynamics of branchial paCA distribution in the major groups of fishes. Finally, this review highlights current gaps in the knowledge on branchial paCA function and provides recommendations for future work.

A novel perspective on the evolutionary loss of plasma-accessible carbonic anhydrase at the teleost gill.

The gills of most teleost fishes lack plasma-accessible carbonic anhydrase (paCA) that could participate in CO2 excretion. We tested the prevailing hypothesis that paCA would interfere with red blood cell (RBC) intracellular pH regulation by ß-adrenergic sodium-proton exchangers (ß-NHE) that protect pH-sensitive haemoglobin-oxygen (Hb-O2) binding during an acidosis. In an open system that mimics the gills, ß-NHE activity increased Hb-O2 saturation during a respiratory acidosis in the presence or absence of paCA, whereas the effect was abolished by NHE inhibition. However, in a closed system that mimics the tissue capillaries, paCA disrupted the protective effects of ß-NHE activity on Hb-O2 binding. The gills are an open system, where CO2 generated by paCA can diffuse out and is not available to acidifying the RBCs. Therefore, branchial paCA in teleosts may not interfere with RBC pH regulation by ß-NHEs, and other explanations for the evolutionary loss of the enzyme must be considered.

Linking environmental salinity to respiratory phenotypes and metabolic rate in fishes: a data mining and modelling approach.

The gill is the primary site of ionoregulation and gas exchange in adult teleost fishes. However, those characteristics that benefit diffusive gas exchange (large, thin gills) may also enhance the passive equilibration of ions and water that threaten osmotic homeostasis. Our literature review revealed that gill surface area and thickness were similar in freshwater (FW) and seawater (SW) species; however, the diffusive oxygen (O2) conductance (Gd) of the gill was lower in FW species. While a lower Gd may reduce ion losses, it also limits O2 uptake capacity and possibly aerobic performance in situations of high O2 demand (e.g. exercise) or low O2 availability (e.g. environmental hypoxia). We also found that FW fishes had significantly higher haemoglobin (Hb)-O2 binding affinities than SW species, which will increase the O2 diffusion gradient across the gills. Therefore, we hypothesized that the higher Hb-O2 affinity of FW fishes compensates, in part, for their lower Gd. Using a combined literature review and modelling approach, our results show that a higher Hb-O2 affinity in FW fishes increases the flux of O2 across their low-Gd gills. In addition, FW and SW teleosts can achieve similar maximal rates of O2 consumption (MO2,max) and hypoxia tolerance (Pcrit) through different combinations of Hb-O2 affinity and Gd. Our combined data identified novel patterns in gill and Hb characteristics between FW and SW fishes and our modelling approach provides mechanistic insight into the relationship between aerobic performance and species distribution ranges, generating novel hypotheses at the intersection of cardiorespiratory and ionoregulatory fish physiology.

Adrenergically induced translocation of red blood cell ß-adrenergic sodium-proton exchangers has ecological relevance for hypoxic and hypercapnic white seabass.

White seabass (Atractoscion nobilis) increasingly experience periods of low oxygen (O2; hypoxia) and high carbon dioxide (CO2, hypercapnia) due to climate change and eutrophication of the coastal waters of California. Hemoglobin (Hb) is the principal O2 carrier in the blood and in many teleost fishes Hb-O2 binding is compromised at low pH; however, the red blood cells (RBC) of some species regulate intracellular pH with adrenergically stimulated sodium-proton-exchangers (ß-NHEs). We hypothesized that RBC ß-NHEs in white seabass are an important mechanism that can protect the blood O2-carrying capacity during hypoxia and hypercapnia. We determined the O2-binding characteristics of white seabass blood, the cellular and subcellular response of RBCs to adrenergic stimulation, and quantified the protective effect of ß-NHE activity on Hb-O2 saturation. White seabass had typical teleost Hb characteristics, with a moderate O2 affinity (Po2 at half-saturation; P50 2.9 kPa) that was highly pH-sensitive (Bohr coefficient -0.92; Root effect 52%). Novel findings from super-resolution microscopy revealed ß-NHE protein in vesicle-like structures and its translocation into the membrane after adrenergic stimulation. Microscopy data were corroborated by molecular and phylogenetic results and a functional characterization of ß-NHE activity. The activation of RBC ß-NHEs increased Hb-O2 saturation by ∼8% in normoxic hypercapnia and by up to ∼20% in hypoxic normocapnia. Our results provide novel insight into the cellular mechanism of adrenergic RBC stimulation within an ecologically relevant context. ß-NHE activity in white seabass has great potential to protect arterial O2 transport during hypoxia and hypercapnia but is less effective during combinations of these stressors.

Molecular and biochemical characterization of the bicarbonate-sensing soluble adenylyl cyclase from a bony fish, the rainbow trout Oncorhynchus mykiss.

Soluble adenylyl cyclase (sAC) is a HC O 3 - -stimulated enzyme that produces the ubiquitous signalling molecule cAMP, and deemed an evolutionarily conserved acid-base sensor. However, its presence is not yet confirmed in bony fishes, the most abundant and diverse of vertebrates. Here, we identified sAC genes in various cartilaginous, ray-finned and lobe-finned fish species. Next, we focused on rainbow trout sAC (rtsAC) and identified 20 potential alternative spliced mRNAs coding for protein isoforms ranging in size from 28 to 186 kDa. Biochemical and kinetic analyses on purified recombinant rtsAC protein determined stimulation by HC O 3 - at physiologically relevant levels for fish internal fluids (EC50 ∼ 7 mM). rtsAC activity was sensitive to KH7, LRE1, and DIDS (established inhibitors of sAC from other organisms), and insensitive to forskolin and 2,5-dideoxyadenosine (modulators of transmembrane adenylyl cyclases). Western blot and immunocytochemistry revealed high rtsAC expression in gill ion-transporting cells, hepatocytes, red blood cells, myocytes and cardiomyocytes. Analyses in the cell line RTgill-W1 suggested that some of the longer rtsAC isoforms may be preferentially localized in the nucleus, the Golgi apparatus and podosomes. These results indicate that sAC is poised to mediate multiple acid-base homeostatic responses in bony fishes, and provide cues about potential novel functions in mammals.

Teleost red blood cells actively enhance the passive diffusion of oxygen that was discovered by August Krogh.

In the early 20th century, August and Marie Krogh settled one of the most controversial questions in physiology, showing through elegant experiments that oxygen (O2) uptake at the lung is driven by passive diffusion alone. Krogh's later work, on the regulation of local blood flow and capillary recruitment at the tissues, was awarded with the Nobel Prize in 1920. A century later it is still undisputed that O2 moves across tissues by diffusion, however, animals use active mechanisms to regulate and facilitate the passive process. Teleost fishes have evolved a mechanism by which adrenergic sodium-proton-exchangers (ß-NHEs) on the red blood cell (RBC) membrane actively create H+ gradients that are short-circuited in the presence of plasma-accessible carbonic anhydrase (CA) at the tissue capillaries. The rapid acidification of the RBC reduces the O2 affinity of pH-sensitive haemoglobin, which increases the O2 diffusion gradient to the tissues. When RBCs leave the site of plasma-accessible CA, ß-NHE activity recovers RBC pH during venous transit, to promote renewed O2 loading at the gills. This mechanism allows teleosts to unload more O2 at their tissues without compromising O2 diffusion gradients and therefore, to use the available O2 carrying capacity of the blood to a greater degree. In Atlantic salmon, ß-NHE short-circuiting reduces the requirements on the heart by up to 30% during moderate exercise and even at rest, with important ecological implications. Thus, in some teleosts, the RBCs participate in regulating the systemic O2 flux by actively altering the passive diffusion of O2 that Krogh discovered.

The ontogeny of Na+ balance during rapid smoltification in pink salmon (Oncorhynchus gorbuscha).

Pink salmon hatch in fresh water, but their highly anadromous life history requires them to migrate into the ocean immediately after gravel-emergence, at a very small size. During their down-river migration these larvae undergo rapid smoltification that completely remodels their osmoregulatory physiology. At this time, the larvae reportedly have high whole-body Na+ contents and we hypothesised that the active accumulation of internal Na+ occurs in preparation for ocean entry. Using a comparative approach, the present study characterised the ontogeny of Na+ regulation in larvae of the anadromous pink salmon and the fresh-water rainbow trout. Our results indicate that larvae from both species actively accumulated Na+; however, whole-body Na+ content was higher in rainbow trout larvae compared to pink salmon. The time-course of this response was similar in the two species, with highest Na+-uptake rates ([Formula: see text]) shortly after yolk sac absorption, but the mechanism of Na+ accumulation differed between the species. Rainbow trout larvae greatly increased [Formula: see text] to overcompensate for a large simultaneous increase in Na+-efflux rate ([Formula: see text]), whereas pink salmon mounted a smaller increase in [Formula: see text] while maintaining tight control over [Formula: see text], which is supported by a significantly lower paracellular permeability. Our results indicate that the transient accumulation of internal Na+ is not a unique feature of the highly anadromous life history in pink salmon and may be a common ontogenetic pattern during larval development in salmonids; and perhaps it is associated with the development of the cardiovascular system during the larvae's transition to a more active lifestyle.

The ontogeny of Na+ uptake in larval rainbow trout reared in waters of different Na+ content.

Teleost fish have a remarkable capacity to maintain ion homeostasis against diffusion gradients in hypo-ionic freshwater. In adult teleosts the gills are the primary site for ion uptake; however, in larvae, the gills are underdeveloped, and as ion-regulation is primarily cutaneous, branchial mechanisms of plasticity are not yet available. In larval rainbow trout, the gills become the primary site for Na+ uptake at ~ 15 days post hatch (dph). To address how Na+ uptake develops in response to differences in water [Na+], the present study characterised the ontogeny of Na+ uptake in rainbow trout larvae, at a time when ion regulation transitions from being a primarily cutaneous to a primarily branchial process. Results indicate that initially (0-15 dph), when ion-regulation is cutaneous, low-[Na+] reared larvae had a higher Na+ affinity (lower Km) compared to the high-[Na+] treatment. In addition, larvae reared in low-[Na+] water had a lower internal Na+ content, despite similar Na+-uptake rates ([Formula: see text]) across treatments. But, once the gills became the dominant site for ion-regulation (> 15 dph), larvae in all treatments maintained the same Na+ content, despite large differences in [Formula: see text], indicating plasticity in those mechanisms that control Na+ efflux ([Formula: see text]). The mechanisms of Na+ uptake in larval rainbow trout showed plasticity during all stages of development. However, in young larvae that relied on cutaneous Na+ uptake, the internal Na+ content was significantly affected by the [Na+] in the water, perhaps revealing challenges to ion homeostasis and a period of heightened vulnerability to external stressors during early larval development.

A novel acidification mechanism for greatly enhanced oxygen supply to the fish retina.

Previously, we showed that the evolution of high acuity vision in fishes was directly associated with their unique pH-sensitive hemoglobins that allow O2 to be delivered to the retina at PO2s more than ten-fold that of arterial blood (Damsgaard et al., 2019). Here, we show strong evidence that vacuolar-type H+-ATPase and plasma-accessible carbonic anhydrase in the vascular structure supplying the retina act together to acidify the red blood cell leading to O2 secretion. In vivo data indicate that this pathway primarily affects the oxygenation of the inner retina involved in signal processing and transduction, and that the evolution of this pathway was tightly associated with the morphological expansion of the inner retina. We conclude that this mechanism for retinal oxygenation played a vital role in the adaptive evolution of vision in teleost fishes.

Evolutionary links between intra- and extracellular acid-base regulation in fish and other aquatic animals.

The acid-base relevant molecules carbon dioxide (CO2 ), protons (H+ ), and bicarbonate (HCO3- ) are substrates and end products of some of the most essential physiological functions including aerobic and anaerobic respiration, ATP hydrolysis, photosynthesis, and calcification. The structure and function of many enzymes and other macromolecules are highly sensitive to changes in pH, and thus maintaining acid-base homeostasis in the face of metabolic and environmental disturbances is essential for proper cellular function. On the other hand, CO2 , H+ , and HCO3- have regulatory effects on various proteins and processes, both directly through allosteric modulation and indirectly through signal transduction pathways. Life in aquatic environments presents organisms with distinct acid-base challenges that are not found in terrestrial environments. These include a relatively high CO2 relative to O2 solubility that prevents internal CO2 /HCO3- accumulation to buffer pH, a lower O2 content that may favor anaerobic metabolism, and variable environmental CO2 , pH and O2 levels that require dynamic adjustments in acid-base homeostatic mechanisms. Additionally, some aquatic animals purposely create acidic or alkaline microenvironments that drive specialized physiological functions. For example, acidifying mechanisms can enhance O2 delivery by red blood cells, lead to ammonia trapping for excretion or buoyancy purposes, or lead to CO2 accumulation to promote photosynthesis by endosymbiotic algae. On the other hand, alkalinizing mechanisms can serve to promote calcium carbonate skeletal formation. This nonexhaustive review summarizes some of the distinct acid-base homeostatic mechanisms that have evolved in aquatic organisms to meet the particular challenges of this environment.

Blood and Gill Carbonic Anhydrase in the Context of a Chondrichthyan Model of CO2 Excretion.

Pacific spiny dogfish (Squalus suckleyi) have been widely used as a representative species for chondrichthyan CO2 excretion. Pacific spiny dogfish have a slower red blood cell (RBC) carbonic anhydrase (CA) isoform than teleost fishes, extracellular CA activity, no endogenous plasma CA inhibitor, and plasma-accessible CA IV at the gills. Thus, both the RBC and plasma compartments contribute to bicarbonate ion (HCO3-) dehydration at the gills for CO2 excretion in contrast to teleost fishes, in which HCO3- dehydration is restricted to RBCs. We compared CA activity levels, subcellular localization, and presence of plasma CA inhibitors in the blood and gills of 13 chondrichthyans to examine the hypothesis that the dogfish model of CO2 excretion applies broadly to chondrichthyans. In general, blood samples from the 12 other chondrichthyans examined had lower RBC CA activity than teleosts, some extracellular CA activity, and no endogenous plasma CA inhibitor. While type IV-like membrane-associated CA was found in the gills in all four of the chondrichthyans examined, S. suckleyi had three times more CA activity (183±13.2 µmol CO2 min-1 mg protein-1) in the microsomal (membrane) fraction of gills than the other three. In addition, unexpected variation in CA characteristics was observed between chondrichthyan species. Thus, in general, it appears that the pattern of CA distribution in fishes can be generally categorized as either chondrichthyan or teleost models. However, further studies should examine the functional significance of the within-chondrichthyan differences we observed and investigate whether CO2 excretion patterns exist along a continuum or in discrete groups.

Metabolic fuel use after feeding in the zebrafish (Danio rerio): a respirometric analysis.

We used respirometric theory and a new respirometry apparatus to assess, for the first time, the sequential oxidation of the major metabolic fuels during the post-prandial period (10 h) in adult zebrafish fed with commercial pellets (51% protein, 2.12% ration). Compared with a fasted group, fed fish presented peak increases of oxygen consumption (78%), and carbon dioxide (80%) and nitrogen excretion rates (338%) at 7-8 h, and rates remained elevated at 10 h. The respiratory quotient increased slightly (0.89 to 0.97) whereas the nitrogen quotient increased greatly (0.072 to 0.140), representing peak amino acid/protein usage (52%) at this time. After 48-h fasting, endogenous carbohydrate and lipid were the major fuels, but in the first few hours after feeding, carbohydrate oxidation increased greatly, fueling the first part of the post-prandial specific dynamic action, whereas increased protein/amino acid usage predominated from 6 h onwards. Excess dietary protein/amino acids were preferentially metabolized for energy production.

A solution to Nature's haemoglobin knockout: a plasma-accessible carbonic anhydrase catalyses CO2 excretion in Antarctic icefish gills.

In all vertebrates studied to date, CO2 excretion depends on the enzyme carbonic anhydrase (CA) that catalyses the rapid conversion of HCO3- to CO2 at the gas-exchange organs. The largest pool of CA is present within red blood cells (RBCs) and, in some vertebrates, plasma-accessible CA (paCA) isoforms participate in CO2 excretion. However, teleost fishes typically do not have paCA at the gills and CO2 excretion is reliant entirely on RBC CA - a strategy that is not possible in icefishes. As the result of a natural knockout, Antarctic icefishes (Channichthyidae) are the only known vertebrates that do not express haemoglobin (Hb) as adults, and largely lack RBCs in the circulation (haematocrit <1%). Previous work has indicated the presence of high levels of membrane-bound CA activity in the gills of icefishes, but without determining its cellular orientation. Thus, we hypothesised that icefishes express a membrane-bound CA isoform at the gill that is accessible to the blood plasma. The CA distribution was compared in the gills of two closely related notothenioid species, one with Hb and RBCs (Notothenia rossii) and one without (Champsocephalus gunnari). Molecular, biochemical and immunohistochemical markers indicate high levels of a Ca4 isoform in the gills of the icefish (but not the red-blooded N. rossii), in a plasma-accessible location that is consistent with a role in CO2 excretion. Thus, in the absence of RBC CA, the icefish gill could exclusively provide the catalytic activity necessary for CO2 excretion - a pathway that is unlike that of any other vertebrate.

Time course of red blood cell intracellular pH recovery following short-circuiting in relation to venous transit times in rainbow trout, Oncorhynchus mykiss.

Accumulating evidence is highlighting the importance of a system of enhanced hemoglobin-oxygen (Hb-O2) unloading for cardiovascular O2 transport in teleosts. Adrenergically stimulated sodium-proton exchangers (ß-NHE) create H+ gradients across the red blood cell (RBC) membrane that are short-circuited in the presence of plasma-accessible carbonic anhydrase (paCA) at the tissues; the result is a large arterial-venous pH shift that greatly enhances O2 unloading from pH-sensitive Hb. However, RBC intracellular pH (pHi) must recover during venous transit (31-90 s) to enable O2 loading at the gills. The halftimes ( t1/2) and magnitudes of RBC ß-adrenergic stimulation, short-circuiting with paCA and recovery of RBC pHi, were assessed in vitro, on rainbow trout whole blood, and using changes in closed-system partial pressure of O2 as a sensitive indicator for changes in RBC pHi. In addition, the recovery rate of RBC pHi was assessed in a continuous-flow apparatus that more closely mimics RBC transit through the circulation. Results indicate that: 1) the t1/2 of ß-NHE short-circuiting is likely within the residence time of blood in the capillaries, 2) the t1/2 of RBC pHi recovery is 17 s and within the time of RBC venous transit, and 3) after short-circuiting, RBCs reestablish the initial H+ gradient across the membrane and can potentially undergo repeated cycles of short-circuiting and recovery. Thus, teleosts have evolved a system that greatly enhances O2 unloading from pH-sensitive Hb at the tissues, while protecting O2 loading at the gills; the resulting increase in O2 transport per unit of blood flow may enable the tremendous athletic ability of salmonids.

Enhanced hemoglobin-oxygen unloading in migratory salmonids.

Recent findings indicate that some teleost fishes may be able to greatly enhance hemoglobin-oxygen (Hb-O2) unloading at the tissues under conditions that result in catecholamine release. The putative mechanism relies on the high pH sensitivity of teleost hemoglobin (Hb), intracellular red blood cell (RBC) pH regulation via ß-adrenergic Na+/H+ exchanger (ß-NHE) activity, and plasma-accessible carbonic anhydrase at the tissues that short-circuits RBC pH regulation. Previous studies have shown that in rainbow trout, this system may double Hb-O2 unloading to red muscle compared to a situation without short-circuiting. The present study determined that: (1) in rainbow trout this system may be functional even at low concentrations of circulating catecholamines, as shown by conducting a dose-response analysis; (2) Atlantic and coho salmon also possess ß-NHE activity, as shown by changes in hematocrit in adrenergically stimulated cells; and (3) with ß-NHE short-circuiting, Atlantic and coho salmon may be able to increase Hb-O2 unloading by up to 74 and 159%, respectively, as determined by modeling based on O2 equilibrium curves. Together, these results indicate that a system to enhance Hb-O2 unloading may be common among salmonids and may be operational even under routine conditions. In view of the life histories of Atlantic and coho salmon, a system to enhance Hb-O2 unloading during exercise may help determine a successful spawning migration and thus reproductive success.

Beyond just hemoglobin: Red blood cell potentiation of hemoglobin-oxygen unloading in fish.

Teleosts comprise 95% of fish species, almost one-half of all vertebrate species, and represent one of the most successful adaptive radiation events among vertebrates. This is thought to be in part because of their unique oxygen (O2) transport system. In salmonids, recent in vitro and in vivo studies indicate that hemoglobin-oxygen (Hb-O2) unloading to tissues may be doubled or even tripled under some conditions without changes in perfusion. This is accomplished through the short circuiting of red blood cell (RBC) pH regulation, resulting in a large arterial-venous pH difference within the RBC and induced reduction in Hb-O2 affinity. This system has three prerequisites: 1) highly pH-sensitive hemoglobin, 2) rapid RBC pH regulation, and 3) a heterogeneous distribution of plasma-accessible CA in the cardiovascular system (presence in the tissues and absence at the gills). Although data are limited, these attributes may be general characteristics of teleosts. Although this system is not likely operational to the same degree in other vertebrates, some of these prerequisites do exist, and the generation and elimination of pH disequilibrium states at the RBC will likely enhance Hb-O2 unloading to some degree. In human disease states, there are conditions that may partly satisfy those for enhanced Hb-O2 unloading, tentatively an avenue for future work that may improve treatment efficacy.

A novel technique for the precise measurement of CO2 production rate in small aquatic organisms as validated on aeshnid dragonfly nymphs.

The present study describes and validates a novel yet simple system for simultaneous in vivo measurements of rates of aquatic CO2 production (MCO2 ) and oxygen consumption (MO2 ), thus allowing the calculation of respiratory exchange ratios (RER). Diffusion of CO2 from the aquatic phase into a gas phase, across a hollow fibre membrane, enabled aquatic MCO2  measurements with a high-precision infrared gas CO2 analyser. MO2  was measured with a PO2  optode using a stop-flow approach. Injections of known amounts of CO2 into the apparatus yielded accurate and highly reproducible measurements of CO2 content (R2=0.997, P<0.001). The viability of in vivo measurements was demonstrated on aquatic dragonfly nymphs (Aeshnidae; wet mass 2.17 mg-1.46 g, n=15) and the apparatus produced precise MCO2  (R2=0.967, P<0.001) and MO2  (R2=0.957, P<0.001) measurements; average RER was 0.73±0.06. The described system is scalable, offering great potential for the study of a wide range of aquatic species, including fish.

Evidence for a plasma-accessible carbonic anhydrase in the lumen of salmon heart that may enhance oxygen delivery to the myocardium.

Oxygen supply to the heart of most teleosts, including salmonids, relies in part or in whole on oxygen-depleted venous blood. Given that plasma-accessible carbonic anhydrase (CA) in red muscle of rainbow trout has recently been shown to facilitate oxygen unloading from arterial blood under certain physiological conditions, we tested the hypothesis that plasma-accessible CA is present in the lumen of coho salmon (Oncorhynchus kisutch) hearts, and may therefore assist in the luminal oxygen supply to the spongy myocardium, which has no coronary circulation. We demonstrate a widespread distribution of CA throughout the heart chambers, including lumen-facing cells in the atrium, and confirm that the membrane-bound isoform ca4 is expressed in the atrium and ventricle of the heart. Further, we confirm that CA catalytic activity is available to blood in the atrial lumen using a modified electrometric ΔpH assay in intact atria in combination with either a membrane-impermeable CA inhibitor or specific cleavage of the Ca4 membrane anchor. Combined, these results support our hypothesis of the presence of an enhanced oxygen delivery system in the lumen of a salmonid heart, which could help support oxygen delivery when the oxygen content of venous blood becomes greatly reduced, such as after burst exercise and during environmental hypoxia.