Comparative Biochemistry and Physiology, Part A 178 (2014) 102–108

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High blood oxygen affinity in the air-breathing swamp eel Monopterus albus Christian Damsgaard a,⁎, Inge Findorf a, Signe Helbo a, Yigit Kocagoz a, Rasmus Buchanan a, Do Thi Thanh Huong b, Roy E. Weber a, Angela Fago a, Mark Bayley a, Tobias Wang a a b

Zoophysiology, Department of Bioscience, Aarhus University, 8000 Aarhus C, Denmark College of Aquaculture and Fisheries, Can Tho University, Can Tho City, Vietnam

a r t i c l e

i n f o

Article history: Received 25 May 2014 Received in revised form 4 August 2014 Accepted 12 August 2014 Available online 17 August 2014 Keywords: Air-breathing Blood Hemoglobin Myoglobin Respiration Swamp eel

a b s t r a c t 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 ± 0.4 mmHg at 27 °C and pH 7.7), 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 ± 0.19 mg ∙ 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. © 2014 Elsevier Inc. All rights reserved.

1. Introduction The Asian swamp eel (Monopterus albus) is an air-breathing member of the Synbranchidae that is widely distributed across South-east Asia (Rosen and Greenwood, 1976), where it inhabits slow flowing and often hypoxic waters. Air-breathing is believed to have evolved as an adaptation to aquatic hypoxia and/or seasonal water level fluctuations, and the vast majority of extant air-breathing fish are found in tropical hypoxic waters (Graham, 1997). Unlike most air-breathing fish, M. albus lacks a distinct air-breathing organ (ABO) and relies on extrabranchial gas exchange using a highly vascularised epithelium in the buccopharyngeal cavity as well as a vascularized esophagus and

Abbreviations: [CO2]pl, Total concentration of carbon dioxide in blood plasma; Hb, Hemoglobin; [Hb-O2], Concentration of oxygen bound to hemoglobin; Hct, Hematocrit; Mb, Myoglobin; n50, Hill’s cooperativity coefficient at half saturation; [O2]total, Total concentration of oxygen in blood; P50, Partial pressure of oxygen at half saturation; PCO2, Partial pressure of carbon dioxide; PO2, Partial pressure of oxygen; RBC, Red blood cell; Y, Fractional saturation; αCO2, Solubility coefficient of carbon dioxide in plasma; αO2,blood, Solubility coefficient of oxygen in blood; φ, Bohr factor. ⁎ Corresponding author at: Zoophysiology, Department of Bioscience, C.F. Møllers Alle 3, Aarhus University, DK-8000 Aarhus C, Denmark. Tel.: +45 87 15 43 27. E-mail address: [email protected] (C. Damsgaard).

http://dx.doi.org/10.1016/j.cbpa.2014.08.001 1095-6433/© 2014 Elsevier Inc. All rights reserved.

integument (Taylor, 1831; Liem, 1967; Lefevre et al., 2014). The buccal cavity of M. albus expands during air-breathing remaining initially inflated during submergence and exhalation occurs both under water and at the surface prior to the next inhalation (Wu and Kung, 1940). Given the reduced gills, M. albus was originally classified as an obligate air-breather, like its close relative M. cuchia (Carter, 1931; Wu and Lui, 1943; Lomholt and Johansen, 1976), but because M. albus maintains blood O2 concentrations during forced submersion in normoxic water, it was argued to be a facultative rather than obligate air-breather (Iversen et al., 2013). The pharyngeal air-breathing structures are characterised by respiratory islets divided by non-respiratory section (Iversen et al., 2013), where only the former is perfused by intraepithelial capillaries (Liem, 1967). This, in combination with a smaller respiratory surface area and presumably con-current gas exchange represent a considerably lower diffusion capacity and lower gas exchange efficiency than normal piscine gills (Hughes, 1972). Thus, to sustain O2 uptake with a low diffusion capacity it would seem beneficial to maintain a large PO2 (partial pressure of oxygen) gradient across this epithelium, which can be achieved by high O2 affinity and O2 carrying capacity of the blood (Hlastala and Berger, 2001). The correlation between blood O2 affinity across fish species and their natural environmental O2 availability was first noted by Krogh

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and Leitch (1919). In addition, given the much greater O2 availability in air than in water, it has been postulated that the evolutionary transition from water to air-breathing would be associated with a decrease in blood O2 affinity (McCutcheon and Hall, 1937; Johansen et al., 1978b), as documented in at least two pairs of closely related species with different breathing mode (Powers et al., 1979). Although there are large variations in blood O2 binding affinities amongst air-breathing fish (Lenfant and Johansen, 1968; Lomholt and Johansen, 1976; Heisler, 1982) an analysis of the O2 affinity of whole blood from 40 genera of water and air-breathers from the Amazon basin (Powers et al., 1979) revealed no evidence of any systematic difference associated to breathing mode, while noting a trend that O2 affinity in species inhabiting slow-flowing hypoxic water was higher compared to species living in fast flowing waters. While the evolution of blood with a high O2 affinity would benefit branchial O2 uptake, the unloading of O2 in the tissues becomes more difficult (Brauner and Wang, 1997; Wang and Malte, 2011). This can be countered in part by the Bohr effect, where proton binding to Hb and stabilizes its low affinity T(ense)-state conformation, increases O2 unloading and causes blood PO2 to increase. Unloading of O2 in the tissues can further be enhanced by increasing the O2 flux from the blood to the tissues. Myoglobin (Mb) functions as an intracellular O2 carrier in the skeletal and heart muscles of most vertebrates (Wittenberg and Wittenberg, 2003) and when expressed at high concentrations it would increase the flux of O2 from blood to the mitochondria. Teleost fishes display the most extensive heterogeneity in adult Hb structure and function amongst vertebrates and can accordingly be categorized (Weber, 2000) as class I species like plaice and carp (Weber and de Wilde, 1976) that have multiple (electrophoretically-) anodal Hbs with almost identical O2 affinities and Bohr effects, and Class II species (anquillid eels, salmonids and catfish) (Weber et al., 1976; Weber and Lykkeboe, 1978) that additionally express electrophoretically-cathodal isoHbs that have high isoelectric points (pI N 8.2), commonly exhibit high O2 affinities, and show reverse or no Bohr effects in the absence of anionic effectors. The latter isoHbs have variously been postulated to function as a blood O 2 reserve that can be drawn upon during hypoxia or when blood pH decreases (e.g. due to increased physical activity in fast-flowing water) (Powers, 1972; Weber, 1990). No information appears to be available on the functional consequences of Hb multiplicity in Monopterus albus. We hypothesized that M. albus would exhibit high blood O 2 carrying capacity, a high blood O2 affinity, and high Mb concentrations in the O 2 consuming muscles. To examine these hypotheses, we measured hematocrit (Hct) and whole blood O 2 equilibria at two CO 2 levels to determine blood O 2 affinity and Bohr effect. Because the O 2 affinity of the Hb in blood depends on its intrinsic O 2 affinity and its interaction with protons and red blood cell (RBC) anionic effectors (Weber and Fago, 2004), we also measured O 2 equilibrium curves of the stripped (cofactor-free) Hb and the major isoHb components, variously in the absence and presence of chloride, and of physiological levels of RBC co-factors (unstripped Hb solutions). Finally, we measured the concentration of Mb in the heart and skeletal muscle and determined O2 equilibrium curves of purified Mb.

2. Materials and Methods 2.1. Fish Specimens of Monopterus albus (Zuiew, 1793) were obtained from a local aquaculture facility in Can Tho City (Vietnam) and transported to Aarhus University (Denmark). They were kept in large aquaria at 27 ± 0.5 °C, fed to satiation with mussels every third day and were acclimated for three months prior to the experiments.

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2.2. Surgical procedure and experimental protocol Seven eels with a mean body mass of 145 ± 25 g (mean ± s.e.m) were anesthetized with benzocaine (0.3 g L-1, dissolved in a small volume of acetone) and transferred to an operating table when reflexes to pinching subsided. The dorsal aorta was accessed ventrally through a 5 cm incision anterior to the anus and cannulated with a PE50 catheter containing heparinized saline (50 IU mL-1). The fish were allowed to recover undisturbed for 24 h at 27 °C in normoxic water to allow blood gases to return to normal values before blood sampling. On the following day, the arterial catheter was extended and after having left the fish undisturbed for another 2 h, a 0.4 mL blood was drawn for in vivo measures of concentration of oxygen bound to Hb ([Hb-O2]), Hct, pH and total concentration of carbon dioxide in plasma ([CO2]pl) as described below (Sections 2.5 and 2.6). Thereafter, an additional 1.5 mL blood was sampled for measurement of in vitro blood O2 equilibrium curves. 2.3. Hemoglobin purification RBC were separated from plasma by centrifugation, washed 5 times in 0.9% NaCl and lysed in 4-fold volume of ice-cold distilled water. 1 mol L-1 Hepes (pH 7.4) was added to reach a final buffer concentration of 10 mM and the mixture was left on ice for 15 min. The hemolysate was centrifuged at 8,100 g for 10 min. The supernatant containing the Hb was collected and Hb heterogeneity was evaluated on isoelectrofocusing (IEF) polyacrylamide gels (GE-Healthcare, pH gradient 3-10). To strip Hb from organic allosteric effectors, the hemolysate was mixed with AG 501-X8 mixed bed resin, centrifuged for 10 min at 10,000 rpm and the supernatant dialyzed against 10 mmol L-1 Hepes pH 7.4 for 24 h. IsoHb composition was moreover investigated by preparative IEF on a 110 mL (Amersham Biosciences, type 8102) column as previously described (Larsen et al., 2003), using Amersham ampholytes in pH ranges of 3-10.5, 5-8 and 6.7 – 7.7 (10, 30 and 60%, respectively). 2.4. Myoglobin purification Heart and muscle tissue were dissected out of euthanized fish, washed in ice-cold saline, snap-frozen in liquid nitrogen and stored at -80 °C until further use. Mb concentrations were measured using a modified version of the method developed by Reynafarje (1963). Heart ventricle and muscle tissues were homogenized for 1 min in 40 mmol L-1 phosphate, pH 6.6 buffer (19.25 mL buffer/g wet tissue) on ice using an Ultra-Turrax T25 homogenizer (IKA, Staufen, Germany). Samples were centrifuged (50 min, 15,000 g) and the supernatant collected and equilibrated with CO gas for 2 min. A pinch of sodium dithionite was added and the sample was equilibrated with CO for 2 min. Finally, absorbance at 538 and 568 nm was measured in quartz cuvettes using a HP 8453 UV-visible spectrophotometer and the Mb concentrations (mg protein/g wet tissue) were calculated as: −1

C ðMbÞ ¼ ðA538nm −A568nm Þ  117:3mg g wet tissue

ð1Þ

To purify Mb heart ventricles and muscle tissue were homogenized on ice in buffer (~5 mL/g tissue, 50 mmol L-1 Tris, 0.5 mmol L-1 EDTA, 0.5 mg mL-1 DTT, pH 8.3) and centrifuged (Helbo and Fago, 2011). The supernatant was collected and submitted to two rounds of ammonium sulphate precipitation (40 and 80%) followed by desalting through a PD-10 column equilibrated with 50 mmol L-1 Tris, 0.5 mmol L-1 EDTA, 0.5 mg mL- 1 DTT, pH 8.3. Finally the sample was passed through a Tricorn Superdex 75 10/300 GL fast protein liquid chromatography (FPLC) gel filtration column (Amersham Biosciences) equilibrated with 50 mmol L-1 Tris, 0.5 mmol L- 1 EDTA, 0.5 mg mL- 1 DTT, 150 mmol L-1 NaCl, pH 8.3 at a flow rate of 0.7 mL min-1, to separate

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Mb from contaminants. Protein purity was assessed by SDS-PAGE (GE-Healthcare, Phast gel 20%) and IEF (GE-Healthcare, pH gradient 3-9). Prior to O2 equilibrium measurements the purified Mb was reduced to the ferrous form by addition of sodium dithionite crystals followed immediately by buffer exchange using Amicon Ultra centrifugal devices (3 × 25 min, 3000 g) in 50 mmol L-1 Tris, 0.5 mmol L-1 EDTA, 0.5 mg mL-1 DTT, pH 8.3.

equilibration with 1% and 3% CO2. pH was measured on whole blood using a Radiometer BMS MK2 system with a microcapillary electrode (G299A) (Radiometer, Copenhagen, Denmark). [CO2]pl was determined as described by Cameron (1971). Hct was measured as volume percentage of RBC after centrifugation of blood (12,000 rpm for 3 min.). All parameters were measured in duplicate. Plasma bicarbonate concentrations [HCO-3] were calculated as:

2.5. O2 equilibrium curves

½HCO− 3  ¼ ½CO2 pl −αCO2  PCO2

Blood O2 equilibrium curves were determined using an Eschweiler tonometer at 27 °C coupled to two serial-linked Wösthoff gas pumps (Bochum, Germany) for mixing pure N2 and O2 to generate watersaturated gas mixtures with known PO2 values. Initially, blood was equilibrated with 30% O2 (PO2 ~ 217 mmHg) for 40 min to determine the its O2 carrying capacity ([O2]total), while PCO2 (partial pressure of carbon dioxide) was kept constant at 1% (7.3 mmHg) or 3% (23.0 mmHg). Subsequently, [Hb-O2] was measured in the 20-80% saturation range by lowering the O2 fraction in the equilibration gas mixtures to 1.68%, 1.05%, 0.42% and 0.21% (PO2 tensions of 1.5, 3.1, 7.1 and 12.3 mmHg). Blood was equilibrated to each O2 level for a minimum of 20 min. Blood O2 concentration was measured as described by Tucker (1967) and blood O2 saturation (Y) was calculated as the fractional [Hb-O2] relative to the [Hb-O2] value at 30% O2, which was calculated as:

where αCO2 is the temperature-compensated solubility of CO2 in trout plasma (Boutilier et al., 1985) and PCO2 is the partial pressure of CO2 in the gas mixture prepared by the Wösthoff pump. pK´ values were calculated from the Henderson-Hasselbach equation as:

½Hb−O2  ¼ ½O2 total −αO2 ; blood  PO2

ð2Þ

0

pK ¼ pH− log

½HCO− 3  αCO2  PCO2

ð5Þ

! ð6Þ

In vivo PCO2 was calculated using the pH-compensated pK´ values: PCO2 ¼

½CO2 pl  0 αCO2  1 þ 10pH−pK

ð7Þ

3. Results 3.1. Blood oxygen binding

where αO2,blood is the blood O2 solubility (Christoforides and HedleyWhyte, 1969). O2 equilibrium curves of Mb and purified Hb were determined using a modified diffusion chamber (Weber, 1981, 1992), where a thin smear (4 μl) of Mb/Hb solution was placed in a diffusion chamber equipped with a photomultiplier (model RCA 931-A) and an Eppendorf model 1100 M photometer coupled to a potentiometric linear recorder. Water-saturated mixtures of O2 or air and ultrapure (N 99.998%) nitrogen gas (N2) prepared by two serial-coupled Wösthoff (Bochum, Germany) gas mixing pumps were allowed to equilibrate the sample. PO2 was increased in steps while light absorption was recorded at 436 nm. Zero and 100% O2 saturation levels were obtained by equilibrating with pure N2 and O2, respectively. Mb O2 equilibrium curves were measured in 50 mmol L-1 Tris buffer, 0.5 mmol L-1 EDTA, 0.5 mg mL-1 DTT, pH 8.3 at 25 °C. Hb O2 equilibrium curves were determined at pH values in the range 6.9-7.8 in 100 mmol L-1 Hepes buffer at 25 °C. For O2 equilibrium curves P50 and n50 (partial pressure of oxygen at half saturation and cooperativity coefficient, respectively) were calculated from the zero-intercept and slope, respectively, of Hill plots (log(Y)/(1-Y) vs. logPO2, where Y is the fractional saturation, based on ≥4 saturation steps. O2 equilibrium curves were derived from the Hill equation using the estimated P50 and n50 values: n

Y¼

PO250

n PO250

n

þ P 5050

ð3Þ

The in vivo arterial blood parameters for M. albus listed in Table 1 reveal a high arterial PCO2 and low arterial Hb-O2 saturation. Blood has a high Hct (42.4 ± 4.5%) and binds O2 with low cooperativity (n50 = 1.3 ± 0.05) and high affinity (P50 = 2.8 ± 0.4 mmHg and 4.1 ± 0.5 mmHg at pH 7.7 ± 0.1 (1% CO2) and 7.5 ± 0.1 (3% CO2), respectively) (1 mmHg = 133 Pa) (Fig. 1). The blood Bohr factor calculated from the O2 equilibrium curves measured at 3% CO2 and 1% CO2 equals -0.79 (Fig. 1) compared to the Bohr factors of -0.27, -0.20 and -0.35 observed in the unstripped Hb and the stripped Hb in the absence and presence of 100 mmol L-1 KCl, respectively (Figs. 2, 3). An arterial O2 equilibrium curve derived from Eq. (3) and P50 and n50-values at the measured arterial pH (Table 1, Fig. 1) reveals a mean arterial blood O2 saturation of 69.3 ± 5.4% corresponding to a calculated mean arterial PO2 of 6.9 mmHg (Fig. 1). The calculated in vivo PCO2 was 24.1 mmHg. 3.2. Hemoglobin heterogeneity and oxygen binding Remarkably the measurements revealed the same O2 affinity in the stripped (organic effector-free) Hb in the absence and in the presence of 100 mmol L-1 KCl, and in the unstripped Hb (hemolysate that does contain organic phosphates) at high pH, but a higher O2 affinity in the stripped Hb in the absence of Cl- at low pH (Fig. 2). Cooperativity of O2 binding is high in stripped Hb (n50 = 2.5-3.0) compared to whole blood (n50 = 1.0-1.5).

The pH-sensitivity of O2 affinity was quantified as the Bohr factor (φ): ΔlogP 50 ϕ¼ ΔpH

ð4Þ

2.6. Measurements and calculations of blood parameters Arterial O2 saturation was calculated as the arterial [Hb-O2] relative to the O2 carrying capacity for the individual fish. Plasma pH and [CO2]pl as well as Hct were measured at 100% and 20-30% O2 saturation during

Table 1 Arterial blood parameters in M. albus. Hct pH⁎ [Hb-O2] (mM) O2 saturation (%) [CO2] (mM)⁎ [HCO-3] (mM)⁎ PCO2 (mmHg) Data are mean ± s.e.m (n = 6). ⁎ Indicate plasma values.

42.4 ± 4.5 7.5 ± 0.03 7.5 ± 1.2 69.3 ± 5.4 18.8 ± 1.6 18.2 ± 1.6 24.1

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Fig. 1. O2 equilibrium curves for M. albus blood equilibrated with 1% CO2 (black circles) and 3% CO2 (white circles) at 27 °C showing a high blood O2 affinity (P50 = 2.75 ± 0.4 and 4.1 ± 0.5 mmHg for 1% CO2 and 3% CO2, respectively). The curves were generated by applying average P50 and n50 values (n = 5) to Eq. (3). The red curve represents the arterial O2 equilibrium calculated using P50 and n50-values at arterial blood pH (7.5; Table 1, Eq. (3)). Horizontal and vertical red dashed drop lines indicate arterial O2 saturation and PO2, respectively. Horizontal black dotted line indicates half saturation. Data points and error bars represent mean ± s.e.m (n = 5).

Native IEF gels (not shown) indicated the presence of 5 distinct Hb isoforms in M. albus. The marked heterogeneity is confirmed by preparative IEF that revealed 6 isoHb components (Hbs I – VI in Fig. 3A) with isoelectric points (pI) between 7.15 and 7.36 (Fig. 3A), indicating that M. albus lacks cathodic components. In accordance with this heterogeneity pattern of multiple anodic components, the two major isoHbs, Hb I and Hb V, show virtually identical O2 affinities (P50 = 4.8 – 5.2 mmHg at 25 °C and pH 7.7) and similar, normal Bohr effects (φ = − 0.24 and − 0.21, respectively). The isolated Hbs show the same cooperativity (n50 ~ 2.7) as in the composite Hb (stripped hemolysate). Strikingly, increasing CO2 tensions to 14.7 and 44.1 mmHg (2% and 6% CO2, respectively) disclosed a distinct specific, pH-independent CO2 effect (Fig. 3B). The effect was greater at pH 7.7 Fig. 3. A. Preparative isoelectric focusing of M. albus Hb showing optical density (O.D.) at 540 nm (circles) and pH at 15 °C (triangles) of the collected fractions, and (horizontal bars) the fractions of isoHbs (I to VI) that were pooled. B. pH dependence of P50 and n50 values of Hb I (solid symbols) and Hb V (open symbols), measured at 25 °C in 0.1 M HEPES buffer, in the absence of CO2 (squares), and in the presence of 14.7 mm Hg CO2 (inverted triangles) and 44.1 mmHg (pyramidal triangles) (2 and 6% CO2, respectively).

than 7.0 and correspondingly decreased the Bohr effect (φ = − 0.18 and − 0.16 in Hbs I and V, respectively, at PCO2 of 44.1 mmHg) – as characterizes carbamino formation (Dahms et al., 1972; Weber et al., 2013). 3.3. Myoglobin oxygen binding The concentrations of Mb in the heart and skeletal muscle were 5.16 ± 0.99 and 1.08 0.19 mg g wet tissue- 1, respectively (n = 4). Mb was successfully purified as judged from SDS gels (not shown), with a molecular mass estimated to ~ 16 kDa. The P50 of M. albus Mb was 1.11 ± 0.08 mmHg and n50 0.96 ± 0.01 consistent with lacking cooperativity of the monomeric Mb (Fig. 4). 3.4. Whole blood buffering Fig. 2. pH dependence of cooperativity coefficient (n50) (upper panel) and O2 affinity (logP50) (lower panel) for M. albus unstripped hemolysate (closed circles) and stripped Hb measured at 25 °C, and in the presence of 100 mmol L-1 HEPES buffer and of 100 mmol L-1 KCl (open triangles).

The calculated pK’-values of CO2 hydration (CO2 + H2O⇌H++HCO-3) were 6.1-6.3. [CO2]pl and were not affected by blood deoxygenation when equilibrated with 1% CO2, but increased during deoxygenation

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Fig. 4. Hyperbolic fit of O2 equilibrium curve measured at 25 °C of myoglobin from M. albus that binds O2 non-cooperatively (n50 = 0.96 ± 0.01) and with a high affinity (P50 = 1.11 mmHg). Data points and error bars represent mean ± s.e.m (n = 2).

when equilibrated with 3% CO2, indicating the presence of a Haldane effect (Table 2). Correspondingly, plasma [HCO-3] increased 1.3 fold compared to the arterial value during 70-80% deoxygenation when equilibrated with 3% CO2, but only showed a moderate increase during deoxygenation when equilibrated with 1% CO2 (Table 2). Based on in vitro measurements of pH and calculations of [HCO-3], the nonbicarbonate buffering capacity was -22.3 slykes (Δ[HCO-3] ΔpH-1). 4. Discussion 4.1. Whole blood oxygen affinity Our study documents that M. albus has a very high blood O2-affinity (P50 = 2.8 mmHg at pH 7.7 and 27 °C) compared to other species of Synbranchidae (P50 = 7.0 mmHg at pH 7.8 and 30 °C in Synbranchus marmoratus (Heisler, 1982), P50 = 7.9 mmHg at pH 7.6 and 30 °C in Monopterus cuchia (Lomholt and Johansen, 1976)), other air-breathing fish with more complex and conceivably more efficient ABO’s (P 50 = 10 mmHg at PCO2 = 6 mmHg and 25 °C in Protopterus aethiopicus (Lenfant and Johansen, 1968)), and even compared to hypoxia-acclimated carp (Cyprinus carpio) (P50 = 3.0 mmHg at pH = 7.9 and 20 °C (Weber and Lykkeboe, 1978)). As blood passes through the thick respiratory epithelium in the buccopharyngeal cavity of M. albus, it is likely that the low PO2 of the blood serves to maintain a large trans-epithelial PO 2-difference facilitating O 2 uptake. Thus, the high blood O2 affinity will to some extent counteract the low diffusion capacity for O2 air-breathing structures (Liem, 1967). We propose a relationship between the complexity of the extrabranchial gas-exchange surfaces and blood O2 affinity, where O2 uptake across simple respiratory surface with high trans-epithelial resistance benefits Table 2 In vitro respiratory parameters in blood and plasma equilibrated to O2 tensions of 217 and 1.52 mmHg and 1% and 3% CO2. PO2 = 217 mmHg

PO2 = 1.52 mmHg

3% CO2 1% CO2 3% CO2 1% CO2 (7.3 mmHg) (22.0 mmHg) (7.3 mmHg) (22.0 mmHg) pH⁎ [Hb-O2] (mM) [CO2] (mM)⁎ [HCO-3] (mM)⁎ Hb-O2 Saturation (%)

7.72 ± 0.05 11.2 ± 2.0 11.1 ± 1.5 8.5 ± 0.3 100

Data are mean ± s.e.m. ⁎ Indicate plasma values.

7.51 ± 0.04 9.7 ± 1.5 18.9 ± 0.9 17.9 ± 1.3 100

7.74 3.9 11.2 9.6 31.5

± ± ± ± ±

0.03 1.1 0.8 0.5 3.5

7.54 2.1 24.7 23.8 22.3

± ± ± ± ±

0.05 0.3 2.5 3.6 4.9

from high blood O2 affinities, whereas a lower blood O2 affinity is beneficial for species with a well-developed ABO, allowing for the full exploitation of the high and stable PO2 in air. Species differences in the diffusion resistances in the ABO may accordingly explain some of the variation in the blood O2 affinity between air-breathing fish and the high blood O2 affinity in M. albus. Based on our measurements of arterial Hb-O2 saturation, we predict that the very high blood O2 affinity results from the low arterial PO2 of around 7 mmHg in normoxic water (see the modelled arterial O2 equilibrium curve in Fig. 1). Thus, even with high blood O2 affinity, diffusion rates may be insufficient to attain full saturation of arterial blood due to the high epithelial resistance and the high Hct. However, blood in the dorsal aorta of M. albus is admixed with venous blood and it is thus difficult to discern the extent to which the lack of full arterial blood O2 saturation is due to diffusion limitations of the ABO or venous arterial blood mixing. A previous study on fish reporting high O2 carrying capacities in the air-breathing fish Synbranchus and Pterygoplichthys (Johansen et al., 1978a) similarly suggests that the reduced saturation results from mixing of afferent blood from the ABO with deoxygenated venous blood before it enters the circulation. Furthermore, it is suggested that this low arterial O2 saturation necessitates high perfusion requirements to maintain an adequate O2 supply to the tissues. Arterial blood that is not fully saturated allows O2 transport to occur on the steepest part of the O2 equilibrium curve. Combined with a high O2 carrying capacity, this results in a very high O2 capacitance of the blood (illustrated as the functional slope of the blood O2 equilibrium curve) and thereby a proportionally lowered convective requirement of the heart. This study documents higher Hct and [CO2]pl as well as lower pH compared to a previous study on this species (Iversen et al., 2013). Both studies identify large inter-individual variation in Hct, which with the small sample size in this study could result in the variant Hct values. The Hct value reported in this study, however, agrees with the high Hct found in S. marmoratus and M. cuchia (Graham, 1997). pH was lower in this study compared to the value reported by Iversen et al. (2013), which partly explains the high [CO2]pl resulting from non-bicarbonate buffering.

4.2. Effect of allosteric effectors This study documents comparable O2 affinities for stripped Hb and hemolysate in the physiological pH range, but shows a higher affinity of stripped Hb in the absence of KCl at low pH (Fig. 2). In fish RBC, allosteric effectors such as ATP and GTP typically bind to Hb and stabilize its low affinity tense-state conformation (Perutz and Brunori, 1982; Weber et al., 1987; Weber and Jensen, 1988), thus decreasing Hb-O2 affinity. Normally in vertebrates, the P50 for stripped Hb is therefore markedly lower than that in the hemolysate that contains red cell anionic co-factors. Thus, it seems that the insensitivity of M. albus Hb to these anionic effectors explains the unusually high O2 affinity of the blood. The current data does not allow us to document if Hb is insensitive towards ATP/GTP, as Cl- that competes for the same binding sites could mask possible ATP/GTP effects (Damsgaard et al., 2013). The O2 affinity of the stripped Hb and the isolated major isoHbs was lower than that for whole blood, even when taking into account that intraerythrocytic pH is lower than plasma pH. While this finding is unexpected it is consistent with the lack of a significant effect of allosteric effectors that normally reduce affinity. It may moreover reflect the fact that the O2 affinities of whole blood and purified Hb were determined by different methods and that the very high affinity renders both methods very sensitive to small experimental errors. Values for cooperativity of O2 binding (n50) show consistent differences between stripped Hb and whole blood. This is also observed for blood and Hb in other fish, where n50 values were higher for hemolysate compared to whole blood (Jensen, 1991; Mandic et al., 2009).

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Blood of M. albus exhibits a normal Bohr effect (φ = −0.79) determined from the right-shift of the O2 equilibrium curve (Fig. 1) with increasing PCO2. The Bohr effect is high compared to other air-breathing fish and lies in the range expected for water breathing fish (Shartau and Brauner, 2014). Given the very high O2 affinity, the Bohr effect may play a role in unloading of oxygen in the tissues. The marked specific (pH-independent) CO2 effect is noteworthy, given the fact that physiologically-significant carbamino formation has not been observed in water-breathing fish that have low blood CO2 tensions and the fact that the N-terminal residue of the α-chains (a major CO2 binding site in vertebrate Hbs) commonly is blocked by acetylation in teleost fish Hbs (Kleinschmidt and Sgouros, 1987; Weber, 2000). Supporting this inference, stripped carp Hb exhibits a modest specific CO2 effect, that is reduced by ATP, which competes for binding at the N-terminal residue of the β-chains, and is obliterated by GTP that binds more strongly at the same site (Weber and Lykkeboe, 1978). Taken together these findings predict that the Nterminal residues of the α-chains of M. albus Hb are free, and that CO2 binding at the β-chain N-terminal is not materially inhibited by phosphate binding, which is consistent with the lacking ATP sensitivity of M. albus Hb. The data call for deeper analysis of the molecular mechanism of the CO2 effect in swamp eels, and its occurrence and biological significance in air-breathing fish generally. 4.3. Myoglobin concentration and oxygen binding To maintain a sufficient O2 flux in the face of a low off-loading PO2, M. albus should have a high Mb-O2 affinity and/or high Mb concentration compared to other fish. The P50 of M. albus Mb lies within the range expected for vertebrate Mbs (Nichols and Weber, 1989; Helbo et al., 2013). However, Mb is found in very high concentration in both the heart and the skeletal muscle (5.2 ± 1.0 and 1.1 ± 0.2 mg g wet tissue - 1 , respectively) compared to other, non-tuna fish species (Giovane et al., 1980). Mb facilitates intra-cellular O 2 transport and a high Mb concentration will thus decrease the diffusion resistance between blood and mitochondria. This illustrates that all levels in the O2 transport cascade respond to changes in blood O2 affinity. As a high resistance step is introduced into the O2 transport system (the buccopharangeal epithelial O2 exchange), resistance-countering adaptations must be expected, exemplified here by the possession of high blood O2 affinity and Mb being present in high concentrations. Mb also serves as a cellular O2 store that, in combination with high blood Hct, may contribute to M. albus‘s significant anoxia tolerance of more than 30 min at 30 °C (Iversen et al., 2013). 4.4. Whole blood buffer effect Air-breathing is typically associated with a rise in blood PCO2 because the ventilation requirements are reduced due to the high O2 capacitance in air compared to water (Dejours, 1975). This is illustrated by the high blood PCO2 of 24.1 mmHg in vivo, which is similar to that of other air-breathing fish but higher than most water breathing fish (Shartau and Brauner, 2014). A high buffering capacity of the blood stabilizes blood pH and the high non-bicarbonate buffering capacity of -22.3 slykes in M. albus is consistent with the high Hct (Brauner and Berenbrink, 2007). 5. Conclusions The high blood and Hb-O2 affinities in M. albus appear to compensate for the low diffusion capacity of the extra-branchial gas exchange structures, while the high Mb-concentration will serve to maintain steep a PO2 gradient in the second diffusion step of the O2 transport system to secure O2 transport in the face of high ABO diffusion resistance. This study identifies novel respiratory adaptations associated with the evolution of air-breathing in fish and shows how adaptations

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