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Effects of acute temperature changes on aerial and aquatic gas exchange, pulmonary ventilation and blood gas status in the South American lungfish, Lepidosiren paradoxa
Comparative Biochemistry and Physiology, Part A 138 (2004) 133 – 139 www.elsevier.com/locate/cbpa
Effects of acute temperature changes on aerial and aquatic gas exchange, pulmonary ventilation and blood gas status in the South American lungfish, Lepidosiren paradoxa J. Amin-Naves, H. Giusti, M.L. Glass * Department of Physiology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Avenida Bandeirantes 3900, Ribeira˜o Preto, SP 14049-900, Brazil Received 20 October 2003; received in revised form 18 February 2004; accepted 20 February 2004
Abstract Lungfish (Dipnoi) are probably sister group relative to all land vertebrates (Tetrapoda). The South American lungfish, Lepidosiren paradoxa, depends markedly on pulmonary gas exchange. In this context, we report on temperature effects on aquatic and pulmonary respiration, ventilation and blood gases at 15, 25 and 35 jC. Lung ventilation increased from 0.5 (15 jC) to 8.1 ml BTPS kg 1 min 1 (35 jC), while pulmonary O2-uptake increased from 0.06 (15 jC) to 0.73 ml STPD kg 1 min 1 (35 jC). Meanwhile aquatic O2-uptake remained about the same ( f 0.01 ml STPD kg 1 min 1) at all temperatures. Concomitantly, the pulmonary gas exchange ratio (RE) rose from 0.11 (15 jC) to 0.62 (35 jC), because a larger fraction of total CO2 output became eliminated by the lung. Accordingly, PaCO2 rose from 13 (15 jC) to 37 mm Hg (35 jC), leading to a significant decrease of pHa at higher temperature (pHa = 7.58 – 15 jC; 7.33 – 35 jC). The acid – base status of L. paradoxa was characterized by a generally low pH (7.4 – 7.5), high bicarbonate level (20 – 25 mM) and PaO2 ( f 80 mm Hg). The increased dependence on the lung at higher temperature parallels data for amphibians. Further, the effects of bimodal gas exchange on temperature-dependent acid – base regulation closely resemble those of anuran amphibians. D 2004 Elsevier Inc. All rights reserved. Keywords: Acid – base regulation; Bimodal respiration; Dipnoi; Gas exchange ratio; Lepidosiren paradoxa; Lungfish; Pulmonary ventilation; Temperature; Evolution; Sarcopterygii
1. Introduction Dipnoi may represent the last evolutionary ramification relative to tetrapods (Meyer and Dolven, 1992; Yokobori et al., 1994). Like early amphibians, lungfish possess a combination of reduced gills and a true lung (Carroll, 1988). The extant genera of lungfish are Neoceratodus (Australia), Protopterus (Africa) and Lepidosiren (South America). Neoceratodus possess a single lung combined with gills that suffice to sustain total metabolism in normoxic water (Fritsche et al., 1993). Conversely, Lepidosireniformes, including Lepidosiren and Protopterus, are obligatory airbreathers with well-developed bilateral lungs and reduced gills (Carroll, 1988; Johansen and Lenfant, 1967). Lenfant et al. (1970) suggested that Protopterus and, in particular, Lepidosiren essentially depend on pulmonary ventilation for ˙ O2, while their reduced gills play some role in O2-uptake V * Corresponding author. Tel.: +55-16-6023202; fax: +55-16-6330017. E-mail address: [email protected] (M.L. Glass). 1095-6433/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2004.02.016
˙ CO2). Consistently, Sawaya (1946) CO2-elimination (V reported that Lepidosiren paradoxa obtained a small frac˙ O2 by gill respiration. tion of total V However, it should be emphasized that temperature effects on bimodal gas exchange can be complex (Wang et al., 1998). In amphibians, the lung becomes increasingly important for gas exchange as temperature rises, because cutaneous respiration is mainly a diffusive process, which confines its upper level for gas exchange (Jackson, 1978). Consistently, air-breathing teleosts increase aerial respiration at high temperature (Lomholt and Johansen, 1974; Glass et al., 1986). In combined aquatic and aerial gas exchange, the tendency is that the aerial exchanger plays the ˙ O2 increases (Burggren et al., 1983). major role when total V A general tendency in ectothermic vertebrates is that pHa decreases with rising temperature (Reeves, 1972; Heisler, 1984). In some teleosts, this negative DpH/Dt is achieved by adjustments of plasma bicarbonate levels (Randall and Cameron, 1973), whereas PCO2-dependent adjustments dominate in amphibians and reptiles. Reptiles adjust pulmo-
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nary ventilation to CO2-output, whereas additional cutaneous CO2 elimination must be taken into account when studying temperature-dependent acid – base regulation in amphibian (Jackson, 1978; Wang et al., 1998). We hypothesize that temperature-dependent regulation of blood acid –base status in L. paradoxa is similar to that of amphibians and depends on bimodal gas exchange. In ectothermic air breathers, an evaluation of how acid – base status is regulated in relation to temperature requires a combination of measurements, including blood gases, pulmonary ventilation and gas exchange (Jackson, 1978, 1989). This assessment becomes more complicated in bimodal respiration, because the relative contribution of gas exchange surfaces must be considered (Wang et al., 1998). Therefore, we measured pulmonary ventilation, aerial and aquatic gas exchanges and blood gases of the dorsal aorta in L. paradoxa exposed to 15, 25 and 35 jC.
1983). In short, the animal was kept in a 10 l aquarium, equipped with a submerged screen that guided the animal to surface within a funnel-shaped chamber, open to the air. To monitor pulmonary ventilation, a pneumotachograph tube (Fleisch head) was placed onto the outlet of the chamber. A grid of tubes served to assure laminar gas flow, while a highly sensitive differential pressure transducer (Mod. DP45-14-2112, Valydine Instr., Northridge, CA, USA) monitored pressure gradients across this grid. Pressure gradients are proportional to inspired and expired flow (Principle of Poiseuille). The transducer was connected to a data acquisition system, based on hard- and software from National Instr. (Austin, TX, USA) and supplemented with specific software for pneumotachography, including tidal volume, respiratory frequency and pulmonary ventilation (FAC Instruments, Sa˜o Carlos, Sa˜o Paulo State, Brazil). Calibration was performed by injection of known air volumes into the chamber.
2. Materials and methods
2.4. Measurements of aerial gas exchange
2.1. Animals
A continuous flow to gas analyzers (Beckman LB-2 and OM-11, Beckman Cardiopulmonary Instr., Fullerton, CA, USA) was used to measure aerial gas exchange (Hill, 1972; Glass et al., 1983). To maintain a low and stable flow to the analyzers (5 ml min 1) a Harvard peristaltic pump (Harvard App., Millis, MA, USA) substituted the built-in flow control of the analyzers. Measurements were continued until steady O2 and CO2 gradients between room air and the animal chamber were reached. Gas exchanges were calculated as,
Specimens of L. paradoxa, Fitzinger (mean body mass 0.48 F 0.05 kg; mean F SEM, n = 8) were collected in the region of the city Cuiaba´, Mato Grosso, Brazil. The animals were transported to the central animal holding facility of University of Sa˜o Paulo in Ribeira˜o Preto, Sa˜o Paulo State, where they were kept in 1000 l tanks, containing shallow water kept at 25 jC. Mostly, the animals remained within PVC tubes placed at the bottom. A diet of chicken liver was provided except during the last 48 h before experimentation. All experiments were conducted within the more active season of the animals (October to May). 2.2. Surgical procedure The animal was immersed into a benzocaine solution (1 g l ) for about 10 min, after which reflexes disappeared. Then, the animal was placed into a support for surgery, while anesthesia was maintained at a level of 0.25 g l 1. An incision of about 3 cm was cut in the caudal region of the animal, starting from the left hind limb and extending in the frontal direction. Next, the dorsal artery was dissected free from tissue attached to the distal part of the lung and the vertebrate column. The dorsal aorta was catheterized, using a PE50 catheter, fixed into the artery and surrounding tissue. Finally, the catheter was exteriorized and incisions were closed with non-traumatic sutures. Placed into benzocainefree water at 25 jC, the animal recovered movements and normal breathing in less than 1 h.
˙ 2 ðml STPD min1 Þ ¼ flowðml min1 Þ k VO ðFin O2 Fout O2 Þ
ð1Þ
1 1 ˙ VCO 2 ðml STPD min Þ ¼ flowðml min Þ k Fout CO2 ;
ð2Þ
1
2.3. Experimental set-up Lung ventilation was measured by pneumotachography, modified for free swimming aquatic animals (cf. Glass et al.,
˙ O2 = pulmonary oxygen uptake; flow = flow from where V the chamber to the analyzers; k = correction factor from BTPS to STPD; FinO2 FoutO2 = the steady state fractional concentration gradient between inlet and outlet O2 of the ˙ CO2 = pulmonary CO2 elimination; FoutCO2 = chamber; V the final concentration gradient for CO2. The pulmonary gas exchange ratio was calculated as, ˙ 1 ˙ RE ; pul ¼ VCO 2 VO2
ð3Þ
2.5. Aquatic gas exchange Oxygen uptake from water was measured in separate experiments, since the volume of animal chamber was too large (10 l) for accurate determination. Instead, the animal was placed into a PVC tube (1.5 l) with a stirrer at the bottom. The water was equilibrated overnight with room air. Then
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aeration was stopped, after which PO2 of the water was measured at 20-min intervals for 4 h. Samples were analyzed with FAC O2 electrodes (FAC Instruments) connected to a FAC 204 A O2 analyzer. The electrodes were calibrated using with water equilibrated with pure N2 (zero) and with room air. They were always maintained at the temperature of the animal. The (gas phase/water)-factor was evaluated on a daily basis (Siggaard-Andersen, 1974). Aquatic oxygen uptake was calculated as, ˙ 2 ðml STPD min1 Þ ¼ Vtot aO2 ðDPO2 =DtÞ; VO
ð4Þ
where Vtot = total water volume; aO2 = solubility of O2 in distilled water (Lomholt and Johansen, 1974; Dejours, 1981); DPO2/Dt = best fit regression for decrease of PO2 ˙ CO2 was derived as, with time. Aquatic V 1 ˙ ˙ VCO 2 ; aq:ðml STPD min Þ ¼ RQ VO2 ; total ˙ VCO 2 ; pul:
ð5Þ
135
using a thermostat (CR Fauvel, Sa˜ o Carlos, Brazil), connected to the animal chamber. For heat exchange, the water was circulated within spirals at the bottom of the chamber. Change of temperature took up to 2 h, and measurements were postponed to the last 5 h of each exposure. 2.8. Statistics Statistics were performed using one-way analysis of variance, followed by Bartlett’s test for equal variances. If variances were equal ( P < 0.05) we applied Bonferroni’s multiple comparison test for differences between individual means. In the case of unequal variance, we applied logarithmic transformations. If this failed, Friedman’s test was applied followed by Dunn’s multiple comparison test for differences between means. Values are expressed as mean F SEM. Significance level was P < 0.05. The n-value is 5 for all sets of data. The data sets are based on runs in which the same animal underwent all measurements and completed the three experimental temperatures.
where RQ represents the respiratory quotient for tissues. Based on the alimentation of the animals, an overall value of RQ = 0.75 was assumed from the food provided (liver). All ˙ O2 and V ˙ CO2 are expressed as mass-specific, values for V 1 i.e. ml STPD min kg 1. 2.6. Blood analysis Dorsal arterial PO2 was measured using the O2-electrode described above. Blood PCO2 and pH were measured using a Cameron Instr. CO2 electrode combined with a Mettler Toledo pH microelectrode (Mettler, Switzerland) and coupled to a BGM 200 Cameron Analyzer (Cameron Instr., Port Aransas, TX, USA). All data were transmitted to the data acquisition system. The CO2 electrode was calibrated with a GF3/MP Gas Flow Meter (Cameron Instr.), using 1% and 3% CO2, respectively, while the pH meter was calibrated using high precision buffer solutions (Queel, Sa˜o Paulo, Brazil). The electrodes were always kept and calibrated at the experimental temperature (15, 25 and 35 jC). Total plasma CO2 was obtained using a Capni-Con 5 Analyzer (Cameron Instr.), calibrated with standard bicarbonate solutions (see Nicol et al., 1983), while whole blood O 2 content was measured using an Oxycon (Cameron Instr.) calibrated with known volumes of atmospheric air. Hematocrit was measured by sampling into microtubes that were centrifuged using a HERMLE Z.200 M/H, Germany. 2.7. Experimental protocol After 24 h of recovery from surgery at 25 jC, the temperature was decreased to 15 jC, maintained until 48 h. Then the temperature was returned to and maintained at 25 jC until 72 h. Finally, the temperature was increased to and kept at 35 jC until 96 h. Temperature control was obtained
Fig. 1. In South American lungfish (L. paradoxa): (A) temperaturedependent changes in pulmonary ventilation (VI). (B) Tidal volume (VT) and respiratory frequency ( fR) in relation to temperature (15, 25 and 35 jC). Mean values F SEM, n = 5. Tidal volume (VT) was constant with temperature (ANOVA, Bonferroni), whereas respiratory frequency ( fR) and pulmonary ventilation (VI) increased significantly ( P < 0.05) with increases of temperature (Friedman, Dunn). Asterisk (*) denotes significant difference from the value at 25 jC, whereas (#) indicates that only the difference between 15 and 35 jC was significant.
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3. Results 3.1. Pulmonary ventilation and gas exchange Pulmonary ventilation increased considerably with temperature from 0.5 (15 jC) to 8.1 (35 jC) ml BTPS kg 1 min 1; BTPS = body temperature, ambient pressure, saturated with water vapor) (Fig. 1), which resulted from an increased frequency of surfacing ( fR = 0.03 min 1 at 15 jC and 0.24 min 1 at 35 jC). Meanwhile, tidal volume was rather constant with temperature (29 – 34 ml BTPS kg 1; ˙ O2 increased from 0.06 ml STPD kg 1 Fig. 1). Pulmonary V 1 min (15 jC) to 0.73 ml STPD kg 1 min 1 (35 jC); ˙ O2 Fig. 3. Pulmonary and aquatic gas exchanges as percentages of total V ˙ CO2 (% V ˙ O2: ANOVA, log-nat.-transform., Bonferroni; and total V ˙ CO2: Friedman, Dunn) in L. paradoxa. Notice that the relative %V ˙ CO2 decrease with rising temperature. ˙ O2 and V contributions of aquatic V ˙ CO2 was aquatic, while the contribution was 27% at 35 At 15 jC, 86% of V jC. Symbols as above. Mean F SEM, n = 5.
STPD = standard temperature, standard pressure, dry). Con˙ CO2 rose from 0.01 to 0.34 ml comitantly, pulmonary V ˙ O2 was low and constant STPD kg 1 min 1. Aquatic V with temperature (about 0.01 ml STPD kg 1 min 1), ˙ CO2 was much greater and increased whereas aquatic V with temperature from 0.04 (15 jC) to 0.21 ml STPD kg 1 min 1(35 jC) (Figs. 2A,B). Due to the scale in Fig. ˙ O2, it is difficult to 2A combined with a low aquatic V perceive the relative contribution of the gas exchange surfaces. For this reason, we have added Fig. 3, depicting ˙ O2 or cutaneous gas exchanges as percentage of total V ˙ CO2. With increase of temperature, the lung eliminated a V larger fraction of total CO2 output. Accordingly, the pulmonary gas exchange ratio (RE) increased from 0.11 (15 jC) to 0.62 (35 jC) (Fig. 4), which implies that intrapulmonary PCO2 increases with temperature.
Fig. 2. In South American lungfish (L. paradoxa): (A) the effects of ˙ O2 was ˙ O2. Aquatic V temperature on pulmonary, aquatic and total V constant with temperature ( P>0.05; one-way ANOVA, Bonferroni’s test), ˙ O2 changed significantly with all whereas pulmonary and total V temperature transitions ( P < 0.05; ANOVA, log-nat.-transform., Bonfer˙ CO2 increased significantly with temperature ( P < 0.05; roni). (B) Total V ˙ CO2 ANOVA, log-nat.-transform., Bonferroni), whereas pulmonary V increased significantly between 15 and 35 jC (Friedman, Dunn). Aquatic CO2 elimination accounted for a substantial fraction of total CO2 output and increased with temperature ( P < 0.05; ANOVA, log-nat.-transform., Bonferroni). Symbols as for Fig. 1. Mean F SEM, n = 5.
Fig. 4. The pulmonary gas exchange ratio (RE) in L. paradoxa increased with temperature. Symbols as above (ANOVA, Bonferroni). Symbols as above. Mean F SEM, n = 5.
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4.2. Consequences of bimodal respiration for temperaturedependent acid – base regulation
Fig. 5. Increases of PaCO2 with rising temperature and the corresponding decreases of pHa (ANOVA, Bonferroni) in South American lungfish (L. paradoxa). Mean F SEM, n = 5. Plasma [CO2] was constant with temperature (see Table 1). Symbols as above.
3.2. Blood gases and pH As a consequence of the temperature-induced increases of RE, PaCO2 rose significantly from 13 (15 jC) to 37 mm Hg (35 jC). Concomitantly, pHa was constant between 15 and 25 jC, but decreased significantly between 25 and 35 jC (DpHa/Dt = 0.014 units jC 1; Fig. 5). Arterial PO2 was constant with temperature (range 75 – 105 mm Hg) (Table 1). This differs from data on amphibians and reptiles, where PaO2 increases with temperature due to large central vascular shunts (Wood and Hicks, 1985). As in most ectothermic lung breathers, [CO2]tot, [O2]a and hematocrit were constant with temperature (cf. Ultsch and Jackson, 1996).
4. Discussion 4.1. Gas exchange patterns In its Pantanal region habitat, L. paradoxa is exposed to temperatures ranging from 18 to 34 jC (Harder et al., 1999). The lung must play a predominant role in respiratory adjustments to temperature, since the gills are rudimentary. ˙ O2. Consistently, we confirmed a low level for aquatic V However, aquatic uptake became more important at the ˙ O2 and lowest temperature (15 jC), when pulmonary V ˙ CO2 was always ventilation were highly reduced. Aquatic V ˙ O2 and, moreover, increased with temperagreater than V ture. This could indicate recruitment of more skin capillaries at higher temperature (Burggren and Moalli, 1984) or an influence of gill respiration. ˙ O2 in Johansen et al. (1976) reported that aquatic V Protopterus amphibius was reduced with increasing size of the animal. At 30 jC, a 0.5 kg P. amphibius obtained 10 – ˙ O2 from the water. Our values for L. para15% of total V doxa are much lower, indicating a greater dependence on the lung.
As temperature increased, L. paradoxa eliminated a larger fraction of total CO2 production by pulmonary ventilation (Fig. 5). Thereby, the pulmonary gas exchange ratio (R E ) rose with temperature, which predictably increases intrapulmonary PCO2 for a given PO2 (cf. Fenn et al., 1946). The resulting increase of PaCO2 at higher temperature caused reductions of pH with increasing temperature, while [CO2]a was constant. In turn, this lead to the predicted decrease of blood pH with temperature. Studying the toad Bufo paracnemis, Wang et al. (1998) expressed similar relationships in terms of conductance equations for pulmonary and cutaneous CO2-elimination. In principle, L. paradoxa could eliminate CO2 to the water by the skin and/or by the gills, but their relative role is unknown. A conductance model for gas exchange cannot be applied without this information on the respiratory role of the gills (Glass and Soncini, 1985). 4.3. Comparison to amphibians and air-breathing teleosts Amphibians increase lung ventilation at high temperature, while the gas conductance of the skin remains virtually constant (Gottlieb and Jackson, 1976; Jackson, 1978; Mackenzie and Jackson, 1978; Wang et al., 1998). As temperature increases, amphibians redirect a larger fraction of total CO2 output to the lung (Wang et al., 1998). Consistent data have been obtained for urodele salamanders (for a review see ˙ CO2 Ultsch and Jackson, 1996). At 5 jC, 90 –100% of V was aquatic in Amphiuma means, while at 25 jC only 40– ˙ CO2 was cutaneous (Guimond and Hutchin80% of the V son, 1974). There are further similarities between amphibians and Lepidosireniformes. These concern composition of pulmonary surfactant (Orgeig and Daniels, 1995) and the types of pulmonary stretch receptors. Lepidosireniformes slow adapting pulmonary stretch receptors that decrease firing rate in response to elevated pulmonary CO2 and cause characteristic ventilatory ‘‘off’’ responses upon transition from hypercarbia to air (DeLaney et al., 1983; Sanchez and Glass, 2001). This CO2-inhibition has also been documented in the bullfrog (Rana catesbeiana) and in
Table 1 Blood gas variables, including PaO2, O2 content-[O2]a, total blood plasma CO2-[CO2]pl, and hematocrit, in L. paradoxa in relation to temperature PaO2 (mm Hg) [O2]a (vol.%) [CO2]pl (mM) Hematocrit (%)
15 jC
25 jC
35 jC
86.0 F 1.2 5.9 F 0.9 23.2 F 3.4 18 F 3
79.4 F 0.9 6.9 F 1.1 24.8 F 2.0 20 F 3
101.1 F 1.7 4.2 F 0.6 27.2 F 2.6 19 F 3
Mean F SEM, n = 5. There were no temperature effects on these variables using one-way ANOVA combined with Bonferroni’s multiple comparison test.
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other non-mammalian tetrapods (Kinkead and Milsom, 1997). This specific ventilatory response also has been reported for L. paradoxa (Sanchez and Glass, 2001). Moreover, central pH receptors are also present like in tetrapods (Sanchez et al., 2001). Finally, Protopterus shows a response strikingly similar to the Hering – Breuer reflex in tetrapods (Pack et al., 1990). Bimodal (aquatic and aerial) respiration also characterizes a number of teleosts. Studying the air-breathing Amphipnous cuchia, Lomholt and Johansen (1974) reported that RE for the air-breathing organ increased with temperature. Data for the snakehead fish, Channa argus are consistent (Glass et al., 1986). Data for some of these bimodal breathers are compiled in Table 2, which also includes values for aerial ventilation, O2-extraction from the lung, pulmonary gas exchange, and aquatic exchange. The table shows that gas exchange levels are similar, in particular between lungfish and air-breathing teleosts.
levels of PaCO2 reflect an increased dependence on aerial gas exchange (Rahn, 1966; Dejours, 1981). L. paradoxa maintained a constant PaO2 within its relevant temperature range. This differs from amphibians and reptiles that increase PaO2 with rising temperature due to large central cardiovascular shunts (Wood, 1982). This may indicate that central vascular shunts in L. paradoxa are negligible which could result from selective regulation of gill and lung perfusion (Fishman et al., 1988). A very limited shunt in L. paradoxa is also supported by a substantial increase of PaO2 with application of hyperoxia (Johansen and Lenfant, 1967). In the pioneering study, Johansen and Lenfant (1967) pointed out that Lepidosiren presents an intriguing combination of piscine and amphibian characters. The present study points out that L. paradoxa resembles typical amphibians since bimodal respiration provides a mechanism for temperature-dependent adjustments to PaCO2 which, in turn, causes a negative DpH/Dt. The interaction of aquatic and aerial gas exchange accounted for temperature-dependent adjustments of blood acid – base status. This is distinct from temperature-dependent acid – base regulation of exclusively gill-breathing teleosts, that mainly achieve the required decline by modulation of plasma bicarbonate levels. It is also different from that of typical reptiles, that proportion DpHa/Dt by modulations of the ˙ I/V ˙ O2). The bimodal (aerial and aquatic) gas exratio (V change is undoubtedly an ancient condition that accounts for many similarities of basic pulmonary function in amphibians and lungfish.
4.4. Blood gas levels in L. paradoxa Johansen and Lenfant (1967) obtained some blood gas samples from L. paradoxa with PaO2 f 35 mm Hg (20 jC), which is considerably lower than measured in the present study (PaO2 f 90 mm Hg). Their animals had both the pulmonary artery and the dorsal aorta catheterized, which might have influenced blood gas levels. However, it should be pointed out that their animals were small (104 – 212 g) with non-functional traces of external gills. The acid – base status of L. paradoxa is characterized by a high [CO2]pl level combined with a low pHa (7.4 – 7.5) relative to values for both teleosts (normal range: 7.7 < pH < 8.1, 25 jC; cf. Heisler, 1984; Cameron, 1984) and amphibians (normal range: 7.6 < pH < 7.9, 25 jC; cf. Boutilier et al., 1986; Kruhøffer et al., 1987). Arterial PCO2 of L. paradoxa ( f 25 mm Hg at 25 jC) was considerably higher than in teleosts (1– 3 mm Hg; cf. Rahn, 1966) and in amphibians (7– 12 mm Hg; Kruhøffer et al., 1987). Higher
Acknowledgements This study was supported by FAPESP (Fundacß a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo). Proc. #98/ 06731-5, CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico). Proc. #520769/93-7 and FAEPA
Table 2 Oxygen uptake and aerial ventilation in selected vertebrates of bimodal respiration ˙ O2 (air) Species Group Temperature Weight V (jC) (kg) ml STPD/ (kg min) Lepidosiren paradoxaa Lepidosiren paradoxab Protopterus sp.c Bufo paracnemisd Channa arguse Amphipnous cuchiaf a
lungfish lungfish lungfish anuran amphibian teleost teleost
VT ml BTPS/ kg
fR (l/min)
25 20 30 27
0.48 f 0.15 0.3 – 0.8 0.2 – 0.8
0.26 0.89 0.43 0.61
0.01
29
0.07 0.21
35
0.09 0.18 0.14
25 30
1–2 f 0.35
0.28 0.37
0.19
25.6 35.4
0.11 0.31
Present study. Johansen and Lenfant (1967). c Johansen et al. (1976); Lomholt (1993). d Wang et al. (1998). e Glass et al. (1986). f Lomholt and Johansen (1974,1976). b
˙ O2 (water) V ml STPD/ (kg min)
˙ AIR ml BTPS/ V (kg min) 2.7 5.0 24.0 2.9 5.3
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(Fundacß a˜o de Apoio ao Ensino, Pesquisa e Assisteˆncia do Hospital das Clı´nicas da FMRPUSP). We acknowledge fellowship support from CNPq.
References Boutilier, R.G., Glass, M.L., Heisler, N., 1986. Blood gases, and extracellular/intracellular acid – base status as a function of temperature in the anuran amphibians Xenopus laevis and Bufo marinus. J. Exp. Biol. 130, 13 – 25. Burggren, W.W., Moalli, R., 1984. Active regulation of cutaneous gas exchange in amphibians experimental evidence and a revised model for skin respiration. Respir. Physiol. 55, 379 – 392. Burggren, W.W., Feder, M.E., Pinder, A.W., 1983. Temperature and the balance between aerial and aquatic respiration in larvae of Rana berlandieri and Rana catesbeiana. Physiol. Zool. 56, 263 – 273. Cameron, J.N., 1984. Acid – base status of fish at different temperatures. Am. J. Physiol. 246, R452 – R459. Carroll, R.L., 1988. Vertebrate Paleontology and Evolution Freeman, New York, NY. Dejours, P., 1981. Principles of Comparative Respiratory Physiology, 2nd ed. Elsevier/North-Holland, Amsterdam. DeLaney, R.G., Laurent, P., Galante, R., Pack, A.I., Fishman, A.P., 1983. Pulmonary mechanoreceptors in the dipnoi lungfish Protopterus and Lepidosiren. Am. J. Physiol. 244, R418 – R428. Fenn, W.O., Rahn, H., Otis, A.B., 1946. A theoretical study of the composition of alveolar air at altitude. Am. J. Physiol. 146, 637 – 653. Fishman, A.P., Galante, R.J., Pack, A.I., 1988. Diving physiology. Lungfish. In: Johansen, K., Burggren, W.W. (Eds.), Cardiovascular Shunts, Phylogenetic, Ontogenetic and Clinicals Aspects. Munksgaard, Copenhagen, pp. 645 – 676. Fritsche, R., Axelsson, M., Franklin, C.E., Grigg, G.G., Holmgren, S., Nilsson, S., 1993. Respiratory and cardiovascular responses to hypoxia in the Australian lungfish. Respir. Physiol. 94, 173 – 187. Glass, M.L., Soncini, R., 1985. Regulation of acid – base status in ectothermic vertebrates: the consequences for oxygen pressures in lung gas and arterial blood. Braz. J. Med. Res. 28, 11 – 12. Glass, M.L., Boutilier, R.G., Heisler, N., 1983. Ventilatory control of arterial PO2 in the turtle, Chrysemys picta bellii: effects of temperature and hypoxia. J. Comp. Physiol., B 151, 145 – 153. Glass, M.L., Ishimatsu, A., Johansen, K., 1986. Responses of aerial ventilation to hypoxia and hypercapnia in Channa argus, an air-breathing fish. J. Comp. Physiol., B 156, 425 – 430. Gottlieb, G., Jackson, D.C., 1976. Importance of pulmonary ventilation in respiratory control in the bullfrog. Am. J. Physiol. 230, 608 – 613. Guimond, R.W., Hutchinson, V.H., 1974. Aerial and aquatic respiration in the congo eel, Amphiuma means means (Garden). Respir. Physiol. 20, 147 – 159. Harder, V., Souza, R.H.S., Severi, W., Rantin, F.T., Bridges, C.R., 1999. The South American lungfish—adaptations to an extreme habitat. In: Val, A.L., Almeida-Val, V.M.F. (Eds.), Biology of Tropical Fishes. INPA, Manaus, pp. 87 – 98. Heisler, N., 1984. Acid – base regulation in fishes. Fish Physiol. 10A, 315 – 401. Hill, R.W., 1972. Determination of oxygen consumption by use of the paramagnetic oxygen analyzer. J. Appl. Physiol. 33, 261 – 263. Jackson, D.C., 1978. Respiratory control and CO2 conductance: temperature effects in a turtle and a frog. Respir. Physiol. 33, 103 – 114. Jackson, D.C., 1989. Control of breathing: effects of temperature. In: Wood, S.C. (Ed.), Comparative Pulmonary Physiology: Current Concepts. Marcel Dekker, New York, pp. 621 – 637. Johansen, K., Lenfant, C., 1967. Respiratory function in the South American lungfish, Lepidosiren paradoxa. J. Exp. Biol. 46, 205 – 218.
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Johansen, K., Lomholt, J.P., Maloiy, G.M., 1976. Importance of air and water breathing in relation to size of the African lungfish Protopterus amphibius Peters. J. Exp. Biol. 65, 395 – 399. Kinkead, R., Milsom, W.K., 1997. Role of pulmonary stretch receptor feedback in control of breathing in the bullfrog. Am. J. Physiol. 272, R497 – R508. Kruhøffer, M., Glass, M.L., Abe, A.S., Johansen, K., 1987. Control of breathing in an amphibian Bufo paracnemis: effects of temperature and hypoxia. Respir. Physiol. 69, 267 – 275. Lenfant, C., Johansen, K., Hanson, D., 1970. Bimodal gas exchange and ventilation – perfusion relationship in lower vertebrates. Fed. Proc. 29, 1124 – 1129. Lomholt, J.P., 1993. Breathing in the aestivating African lungfish, Protopterus amphibius. Adv. Fish Res. 1, 17 – 34. Lomholt, J.P., Johansen, K., 1974. Control of breathing in Amphipnous cuchia, an amphibious fish. Respir. Physiol. 21, 325 – 340. Lomholt, J.P., Johansen, K., 1976. Gas exchange in the amphibious fish, Amphipnous cuchia. J. Comp. Physiol. 107, 141 – 157. Mackenzie, J.A., Jackson, D.C., 1978. The effect of temperature on cutaneous CO2, loss and conductance in the bullfrog. Respir. Physiol. 32, 313 – 323. Meyer, A., Dolven, S.I., 1992. Molecules, fossils, and the origin of tetrapods. J. Mol. Evol. 35, 102 – 113. Nicol, S.C., Glass, M.L., Heisler, N., 1983. Comparison of directly determined and calculated plasma bicarbonate concentration in the turtle Chrysemys picta bellii at different temperatures. J. Exp. Biol. 107, 521 – 525. Orgeig, S., Daniels, C.B., 1995. The evolutionary significance of pulmonary surfactant in lungfish (Dipnoi). Am. J. Respir. Cell Mol. Biol. 13, 161 – 166. Pack, A.I., Galante, R.J., Fishman, A.P., 1990. Control of interbreath interval in the African lungfish. Am. J. Physiol. 259, R139 – R146. Rahn, H., 1966. Aquatic gas exchange: theory. Respir. Physiol. 1, 1 – 12. Randall, D.J., Cameron, J.N., 1973. Respiratory control of arterial pH as temperature changes in rainbow trout Salmo gairdneri. Am. J. Physiol. 225, 997 – 1002. Reeves, R.B., 1972. An imidazole alphastat hypothesis for vertebrate acid – base regulation: tissue carbon dioxide content and body temperature in bullfrogs. Respir. Physiol. 14, 219 – 236. Sanchez, A.P., Glass, M.L., 2001. Effects of environmental hypercapnia on pulmonary ventilation of the South American lungfish. J. Fish Biol. 58, 1181 – 1189. Sanchez, A.P., Hoffmann, A., Rantin, F.T., Glass, M.L., 2001. Relationship between cerebro-spinal fluid pH and pulmonary ventilation in the South American lungfish, Lepidosiren paradoxa (Fitz.). J. Exp. Zool. 290, 421 – 425. Sawaya, P., 1946. Sobre a biologia de alguns peixes de respiracßa˜o ae´rea (Lepidosiren paradoxa Fitzinger e Arapaima gigas Cuvier). Bol. Fac. Filos., Cienc. Let. Univ. Sa˜o Paulo, Bot. 11, 255 – 286. Siggaard-Andersen, O., 1974. The Acid – Base Status of the Blood, 4th ed. Munksgaard, Copenhagen. Ultsch, G.R, Jackson, D.C., 1996. pH and temperature in ectothermic vertebrates. Bull. Ala. Mus. Nat. Hist. 18, 1 – 41. Wang, T., Abe, A.S., Glass, M.L., 1998. Temperature effects on lung and blood gases in the toad Bufo paracnemis: consequences of bimodal gas exchange. Respir. Physiol. 113, 231 – 238. Wood, S.C., 1982. Effect of O2 affinity on arterial PO2 in animals with central vascular shunts. J. Appl. Physiol. 53, 1360 – 1364. Wood, S.C., Hicks, J.W., 1985. Oxygen homeostasis in vertebrates with cardiovascular shunts. In: Johansen, K., Burggren, W.W. (Eds.), Cardiovascular Shunts, Phylogenetic, Ontogenetic and Clinicals Aspects. Munksgaard, Copenhagen, pp. 354 – 372. Yokobori, S., Hasegawa, M., Ueda, T., Okada, N., Nishikawa, K., Watanabe, K., 1994. Relationship among coelacanths, lungfishes, and tetrapods: a phylogenetic analysis based on mitochondrial cytochrome oxidase I gene sequences. J. Mol. Evol. 38, 602 – 609.
Effects of acute temperature changes on aerial and aquatic gas exchange, pulmonary ventilation and blood gas status in the South American lungfish, Lepidosiren paradoxa J. Amin-Naves, H. Giusti, M.L. Glass * Department of Physiology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Avenida Bandeirantes 3900, Ribeira˜o Preto, SP 14049-900, Brazil Received 20 October 2003; received in revised form 18 February 2004; accepted 20 February 2004
Abstract Lungfish (Dipnoi) are probably sister group relative to all land vertebrates (Tetrapoda). The South American lungfish, Lepidosiren paradoxa, depends markedly on pulmonary gas exchange. In this context, we report on temperature effects on aquatic and pulmonary respiration, ventilation and blood gases at 15, 25 and 35 jC. Lung ventilation increased from 0.5 (15 jC) to 8.1 ml BTPS kg 1 min 1 (35 jC), while pulmonary O2-uptake increased from 0.06 (15 jC) to 0.73 ml STPD kg 1 min 1 (35 jC). Meanwhile aquatic O2-uptake remained about the same ( f 0.01 ml STPD kg 1 min 1) at all temperatures. Concomitantly, the pulmonary gas exchange ratio (RE) rose from 0.11 (15 jC) to 0.62 (35 jC), because a larger fraction of total CO2 output became eliminated by the lung. Accordingly, PaCO2 rose from 13 (15 jC) to 37 mm Hg (35 jC), leading to a significant decrease of pHa at higher temperature (pHa = 7.58 – 15 jC; 7.33 – 35 jC). The acid – base status of L. paradoxa was characterized by a generally low pH (7.4 – 7.5), high bicarbonate level (20 – 25 mM) and PaO2 ( f 80 mm Hg). The increased dependence on the lung at higher temperature parallels data for amphibians. Further, the effects of bimodal gas exchange on temperature-dependent acid – base regulation closely resemble those of anuran amphibians. D 2004 Elsevier Inc. All rights reserved. Keywords: Acid – base regulation; Bimodal respiration; Dipnoi; Gas exchange ratio; Lepidosiren paradoxa; Lungfish; Pulmonary ventilation; Temperature; Evolution; Sarcopterygii
1. Introduction Dipnoi may represent the last evolutionary ramification relative to tetrapods (Meyer and Dolven, 1992; Yokobori et al., 1994). Like early amphibians, lungfish possess a combination of reduced gills and a true lung (Carroll, 1988). The extant genera of lungfish are Neoceratodus (Australia), Protopterus (Africa) and Lepidosiren (South America). Neoceratodus possess a single lung combined with gills that suffice to sustain total metabolism in normoxic water (Fritsche et al., 1993). Conversely, Lepidosireniformes, including Lepidosiren and Protopterus, are obligatory airbreathers with well-developed bilateral lungs and reduced gills (Carroll, 1988; Johansen and Lenfant, 1967). Lenfant et al. (1970) suggested that Protopterus and, in particular, Lepidosiren essentially depend on pulmonary ventilation for ˙ O2, while their reduced gills play some role in O2-uptake V * Corresponding author. Tel.: +55-16-6023202; fax: +55-16-6330017. E-mail address: [email protected] (M.L. Glass). 1095-6433/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2004.02.016
˙ CO2). Consistently, Sawaya (1946) CO2-elimination (V reported that Lepidosiren paradoxa obtained a small frac˙ O2 by gill respiration. tion of total V However, it should be emphasized that temperature effects on bimodal gas exchange can be complex (Wang et al., 1998). In amphibians, the lung becomes increasingly important for gas exchange as temperature rises, because cutaneous respiration is mainly a diffusive process, which confines its upper level for gas exchange (Jackson, 1978). Consistently, air-breathing teleosts increase aerial respiration at high temperature (Lomholt and Johansen, 1974; Glass et al., 1986). In combined aquatic and aerial gas exchange, the tendency is that the aerial exchanger plays the ˙ O2 increases (Burggren et al., 1983). major role when total V A general tendency in ectothermic vertebrates is that pHa decreases with rising temperature (Reeves, 1972; Heisler, 1984). In some teleosts, this negative DpH/Dt is achieved by adjustments of plasma bicarbonate levels (Randall and Cameron, 1973), whereas PCO2-dependent adjustments dominate in amphibians and reptiles. Reptiles adjust pulmo-
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nary ventilation to CO2-output, whereas additional cutaneous CO2 elimination must be taken into account when studying temperature-dependent acid – base regulation in amphibian (Jackson, 1978; Wang et al., 1998). We hypothesize that temperature-dependent regulation of blood acid –base status in L. paradoxa is similar to that of amphibians and depends on bimodal gas exchange. In ectothermic air breathers, an evaluation of how acid – base status is regulated in relation to temperature requires a combination of measurements, including blood gases, pulmonary ventilation and gas exchange (Jackson, 1978, 1989). This assessment becomes more complicated in bimodal respiration, because the relative contribution of gas exchange surfaces must be considered (Wang et al., 1998). Therefore, we measured pulmonary ventilation, aerial and aquatic gas exchanges and blood gases of the dorsal aorta in L. paradoxa exposed to 15, 25 and 35 jC.
1983). In short, the animal was kept in a 10 l aquarium, equipped with a submerged screen that guided the animal to surface within a funnel-shaped chamber, open to the air. To monitor pulmonary ventilation, a pneumotachograph tube (Fleisch head) was placed onto the outlet of the chamber. A grid of tubes served to assure laminar gas flow, while a highly sensitive differential pressure transducer (Mod. DP45-14-2112, Valydine Instr., Northridge, CA, USA) monitored pressure gradients across this grid. Pressure gradients are proportional to inspired and expired flow (Principle of Poiseuille). The transducer was connected to a data acquisition system, based on hard- and software from National Instr. (Austin, TX, USA) and supplemented with specific software for pneumotachography, including tidal volume, respiratory frequency and pulmonary ventilation (FAC Instruments, Sa˜o Carlos, Sa˜o Paulo State, Brazil). Calibration was performed by injection of known air volumes into the chamber.
2. Materials and methods
2.4. Measurements of aerial gas exchange
2.1. Animals
A continuous flow to gas analyzers (Beckman LB-2 and OM-11, Beckman Cardiopulmonary Instr., Fullerton, CA, USA) was used to measure aerial gas exchange (Hill, 1972; Glass et al., 1983). To maintain a low and stable flow to the analyzers (5 ml min 1) a Harvard peristaltic pump (Harvard App., Millis, MA, USA) substituted the built-in flow control of the analyzers. Measurements were continued until steady O2 and CO2 gradients between room air and the animal chamber were reached. Gas exchanges were calculated as,
Specimens of L. paradoxa, Fitzinger (mean body mass 0.48 F 0.05 kg; mean F SEM, n = 8) were collected in the region of the city Cuiaba´, Mato Grosso, Brazil. The animals were transported to the central animal holding facility of University of Sa˜o Paulo in Ribeira˜o Preto, Sa˜o Paulo State, where they were kept in 1000 l tanks, containing shallow water kept at 25 jC. Mostly, the animals remained within PVC tubes placed at the bottom. A diet of chicken liver was provided except during the last 48 h before experimentation. All experiments were conducted within the more active season of the animals (October to May). 2.2. Surgical procedure The animal was immersed into a benzocaine solution (1 g l ) for about 10 min, after which reflexes disappeared. Then, the animal was placed into a support for surgery, while anesthesia was maintained at a level of 0.25 g l 1. An incision of about 3 cm was cut in the caudal region of the animal, starting from the left hind limb and extending in the frontal direction. Next, the dorsal artery was dissected free from tissue attached to the distal part of the lung and the vertebrate column. The dorsal aorta was catheterized, using a PE50 catheter, fixed into the artery and surrounding tissue. Finally, the catheter was exteriorized and incisions were closed with non-traumatic sutures. Placed into benzocainefree water at 25 jC, the animal recovered movements and normal breathing in less than 1 h.
˙ 2 ðml STPD min1 Þ ¼ flowðml min1 Þ k VO ðFin O2 Fout O2 Þ
ð1Þ
1 1 ˙ VCO 2 ðml STPD min Þ ¼ flowðml min Þ k Fout CO2 ;
ð2Þ
1
2.3. Experimental set-up Lung ventilation was measured by pneumotachography, modified for free swimming aquatic animals (cf. Glass et al.,
˙ O2 = pulmonary oxygen uptake; flow = flow from where V the chamber to the analyzers; k = correction factor from BTPS to STPD; FinO2 FoutO2 = the steady state fractional concentration gradient between inlet and outlet O2 of the ˙ CO2 = pulmonary CO2 elimination; FoutCO2 = chamber; V the final concentration gradient for CO2. The pulmonary gas exchange ratio was calculated as, ˙ 1 ˙ RE ; pul ¼ VCO 2 VO2
ð3Þ
2.5. Aquatic gas exchange Oxygen uptake from water was measured in separate experiments, since the volume of animal chamber was too large (10 l) for accurate determination. Instead, the animal was placed into a PVC tube (1.5 l) with a stirrer at the bottom. The water was equilibrated overnight with room air. Then
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aeration was stopped, after which PO2 of the water was measured at 20-min intervals for 4 h. Samples were analyzed with FAC O2 electrodes (FAC Instruments) connected to a FAC 204 A O2 analyzer. The electrodes were calibrated using with water equilibrated with pure N2 (zero) and with room air. They were always maintained at the temperature of the animal. The (gas phase/water)-factor was evaluated on a daily basis (Siggaard-Andersen, 1974). Aquatic oxygen uptake was calculated as, ˙ 2 ðml STPD min1 Þ ¼ Vtot aO2 ðDPO2 =DtÞ; VO
ð4Þ
where Vtot = total water volume; aO2 = solubility of O2 in distilled water (Lomholt and Johansen, 1974; Dejours, 1981); DPO2/Dt = best fit regression for decrease of PO2 ˙ CO2 was derived as, with time. Aquatic V 1 ˙ ˙ VCO 2 ; aq:ðml STPD min Þ ¼ RQ VO2 ; total ˙ VCO 2 ; pul:
ð5Þ
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using a thermostat (CR Fauvel, Sa˜ o Carlos, Brazil), connected to the animal chamber. For heat exchange, the water was circulated within spirals at the bottom of the chamber. Change of temperature took up to 2 h, and measurements were postponed to the last 5 h of each exposure. 2.8. Statistics Statistics were performed using one-way analysis of variance, followed by Bartlett’s test for equal variances. If variances were equal ( P < 0.05) we applied Bonferroni’s multiple comparison test for differences between individual means. In the case of unequal variance, we applied logarithmic transformations. If this failed, Friedman’s test was applied followed by Dunn’s multiple comparison test for differences between means. Values are expressed as mean F SEM. Significance level was P < 0.05. The n-value is 5 for all sets of data. The data sets are based on runs in which the same animal underwent all measurements and completed the three experimental temperatures.
where RQ represents the respiratory quotient for tissues. Based on the alimentation of the animals, an overall value of RQ = 0.75 was assumed from the food provided (liver). All ˙ O2 and V ˙ CO2 are expressed as mass-specific, values for V 1 i.e. ml STPD min kg 1. 2.6. Blood analysis Dorsal arterial PO2 was measured using the O2-electrode described above. Blood PCO2 and pH were measured using a Cameron Instr. CO2 electrode combined with a Mettler Toledo pH microelectrode (Mettler, Switzerland) and coupled to a BGM 200 Cameron Analyzer (Cameron Instr., Port Aransas, TX, USA). All data were transmitted to the data acquisition system. The CO2 electrode was calibrated with a GF3/MP Gas Flow Meter (Cameron Instr.), using 1% and 3% CO2, respectively, while the pH meter was calibrated using high precision buffer solutions (Queel, Sa˜o Paulo, Brazil). The electrodes were always kept and calibrated at the experimental temperature (15, 25 and 35 jC). Total plasma CO2 was obtained using a Capni-Con 5 Analyzer (Cameron Instr.), calibrated with standard bicarbonate solutions (see Nicol et al., 1983), while whole blood O 2 content was measured using an Oxycon (Cameron Instr.) calibrated with known volumes of atmospheric air. Hematocrit was measured by sampling into microtubes that were centrifuged using a HERMLE Z.200 M/H, Germany. 2.7. Experimental protocol After 24 h of recovery from surgery at 25 jC, the temperature was decreased to 15 jC, maintained until 48 h. Then the temperature was returned to and maintained at 25 jC until 72 h. Finally, the temperature was increased to and kept at 35 jC until 96 h. Temperature control was obtained
Fig. 1. In South American lungfish (L. paradoxa): (A) temperaturedependent changes in pulmonary ventilation (VI). (B) Tidal volume (VT) and respiratory frequency ( fR) in relation to temperature (15, 25 and 35 jC). Mean values F SEM, n = 5. Tidal volume (VT) was constant with temperature (ANOVA, Bonferroni), whereas respiratory frequency ( fR) and pulmonary ventilation (VI) increased significantly ( P < 0.05) with increases of temperature (Friedman, Dunn). Asterisk (*) denotes significant difference from the value at 25 jC, whereas (#) indicates that only the difference between 15 and 35 jC was significant.
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3. Results 3.1. Pulmonary ventilation and gas exchange Pulmonary ventilation increased considerably with temperature from 0.5 (15 jC) to 8.1 (35 jC) ml BTPS kg 1 min 1; BTPS = body temperature, ambient pressure, saturated with water vapor) (Fig. 1), which resulted from an increased frequency of surfacing ( fR = 0.03 min 1 at 15 jC and 0.24 min 1 at 35 jC). Meanwhile, tidal volume was rather constant with temperature (29 – 34 ml BTPS kg 1; ˙ O2 increased from 0.06 ml STPD kg 1 Fig. 1). Pulmonary V 1 min (15 jC) to 0.73 ml STPD kg 1 min 1 (35 jC); ˙ O2 Fig. 3. Pulmonary and aquatic gas exchanges as percentages of total V ˙ CO2 (% V ˙ O2: ANOVA, log-nat.-transform., Bonferroni; and total V ˙ CO2: Friedman, Dunn) in L. paradoxa. Notice that the relative %V ˙ CO2 decrease with rising temperature. ˙ O2 and V contributions of aquatic V ˙ CO2 was aquatic, while the contribution was 27% at 35 At 15 jC, 86% of V jC. Symbols as above. Mean F SEM, n = 5.
STPD = standard temperature, standard pressure, dry). Con˙ CO2 rose from 0.01 to 0.34 ml comitantly, pulmonary V ˙ O2 was low and constant STPD kg 1 min 1. Aquatic V with temperature (about 0.01 ml STPD kg 1 min 1), ˙ CO2 was much greater and increased whereas aquatic V with temperature from 0.04 (15 jC) to 0.21 ml STPD kg 1 min 1(35 jC) (Figs. 2A,B). Due to the scale in Fig. ˙ O2, it is difficult to 2A combined with a low aquatic V perceive the relative contribution of the gas exchange surfaces. For this reason, we have added Fig. 3, depicting ˙ O2 or cutaneous gas exchanges as percentage of total V ˙ CO2. With increase of temperature, the lung eliminated a V larger fraction of total CO2 output. Accordingly, the pulmonary gas exchange ratio (RE) increased from 0.11 (15 jC) to 0.62 (35 jC) (Fig. 4), which implies that intrapulmonary PCO2 increases with temperature.
Fig. 2. In South American lungfish (L. paradoxa): (A) the effects of ˙ O2 was ˙ O2. Aquatic V temperature on pulmonary, aquatic and total V constant with temperature ( P>0.05; one-way ANOVA, Bonferroni’s test), ˙ O2 changed significantly with all whereas pulmonary and total V temperature transitions ( P < 0.05; ANOVA, log-nat.-transform., Bonfer˙ CO2 increased significantly with temperature ( P < 0.05; roni). (B) Total V ˙ CO2 ANOVA, log-nat.-transform., Bonferroni), whereas pulmonary V increased significantly between 15 and 35 jC (Friedman, Dunn). Aquatic CO2 elimination accounted for a substantial fraction of total CO2 output and increased with temperature ( P < 0.05; ANOVA, log-nat.-transform., Bonferroni). Symbols as for Fig. 1. Mean F SEM, n = 5.
Fig. 4. The pulmonary gas exchange ratio (RE) in L. paradoxa increased with temperature. Symbols as above (ANOVA, Bonferroni). Symbols as above. Mean F SEM, n = 5.
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4.2. Consequences of bimodal respiration for temperaturedependent acid – base regulation
Fig. 5. Increases of PaCO2 with rising temperature and the corresponding decreases of pHa (ANOVA, Bonferroni) in South American lungfish (L. paradoxa). Mean F SEM, n = 5. Plasma [CO2] was constant with temperature (see Table 1). Symbols as above.
3.2. Blood gases and pH As a consequence of the temperature-induced increases of RE, PaCO2 rose significantly from 13 (15 jC) to 37 mm Hg (35 jC). Concomitantly, pHa was constant between 15 and 25 jC, but decreased significantly between 25 and 35 jC (DpHa/Dt = 0.014 units jC 1; Fig. 5). Arterial PO2 was constant with temperature (range 75 – 105 mm Hg) (Table 1). This differs from data on amphibians and reptiles, where PaO2 increases with temperature due to large central vascular shunts (Wood and Hicks, 1985). As in most ectothermic lung breathers, [CO2]tot, [O2]a and hematocrit were constant with temperature (cf. Ultsch and Jackson, 1996).
4. Discussion 4.1. Gas exchange patterns In its Pantanal region habitat, L. paradoxa is exposed to temperatures ranging from 18 to 34 jC (Harder et al., 1999). The lung must play a predominant role in respiratory adjustments to temperature, since the gills are rudimentary. ˙ O2. Consistently, we confirmed a low level for aquatic V However, aquatic uptake became more important at the ˙ O2 and lowest temperature (15 jC), when pulmonary V ˙ CO2 was always ventilation were highly reduced. Aquatic V ˙ O2 and, moreover, increased with temperagreater than V ture. This could indicate recruitment of more skin capillaries at higher temperature (Burggren and Moalli, 1984) or an influence of gill respiration. ˙ O2 in Johansen et al. (1976) reported that aquatic V Protopterus amphibius was reduced with increasing size of the animal. At 30 jC, a 0.5 kg P. amphibius obtained 10 – ˙ O2 from the water. Our values for L. para15% of total V doxa are much lower, indicating a greater dependence on the lung.
As temperature increased, L. paradoxa eliminated a larger fraction of total CO2 production by pulmonary ventilation (Fig. 5). Thereby, the pulmonary gas exchange ratio (R E ) rose with temperature, which predictably increases intrapulmonary PCO2 for a given PO2 (cf. Fenn et al., 1946). The resulting increase of PaCO2 at higher temperature caused reductions of pH with increasing temperature, while [CO2]a was constant. In turn, this lead to the predicted decrease of blood pH with temperature. Studying the toad Bufo paracnemis, Wang et al. (1998) expressed similar relationships in terms of conductance equations for pulmonary and cutaneous CO2-elimination. In principle, L. paradoxa could eliminate CO2 to the water by the skin and/or by the gills, but their relative role is unknown. A conductance model for gas exchange cannot be applied without this information on the respiratory role of the gills (Glass and Soncini, 1985). 4.3. Comparison to amphibians and air-breathing teleosts Amphibians increase lung ventilation at high temperature, while the gas conductance of the skin remains virtually constant (Gottlieb and Jackson, 1976; Jackson, 1978; Mackenzie and Jackson, 1978; Wang et al., 1998). As temperature increases, amphibians redirect a larger fraction of total CO2 output to the lung (Wang et al., 1998). Consistent data have been obtained for urodele salamanders (for a review see ˙ CO2 Ultsch and Jackson, 1996). At 5 jC, 90 –100% of V was aquatic in Amphiuma means, while at 25 jC only 40– ˙ CO2 was cutaneous (Guimond and Hutchin80% of the V son, 1974). There are further similarities between amphibians and Lepidosireniformes. These concern composition of pulmonary surfactant (Orgeig and Daniels, 1995) and the types of pulmonary stretch receptors. Lepidosireniformes slow adapting pulmonary stretch receptors that decrease firing rate in response to elevated pulmonary CO2 and cause characteristic ventilatory ‘‘off’’ responses upon transition from hypercarbia to air (DeLaney et al., 1983; Sanchez and Glass, 2001). This CO2-inhibition has also been documented in the bullfrog (Rana catesbeiana) and in
Table 1 Blood gas variables, including PaO2, O2 content-[O2]a, total blood plasma CO2-[CO2]pl, and hematocrit, in L. paradoxa in relation to temperature PaO2 (mm Hg) [O2]a (vol.%) [CO2]pl (mM) Hematocrit (%)
15 jC
25 jC
35 jC
86.0 F 1.2 5.9 F 0.9 23.2 F 3.4 18 F 3
79.4 F 0.9 6.9 F 1.1 24.8 F 2.0 20 F 3
101.1 F 1.7 4.2 F 0.6 27.2 F 2.6 19 F 3
Mean F SEM, n = 5. There were no temperature effects on these variables using one-way ANOVA combined with Bonferroni’s multiple comparison test.
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other non-mammalian tetrapods (Kinkead and Milsom, 1997). This specific ventilatory response also has been reported for L. paradoxa (Sanchez and Glass, 2001). Moreover, central pH receptors are also present like in tetrapods (Sanchez et al., 2001). Finally, Protopterus shows a response strikingly similar to the Hering – Breuer reflex in tetrapods (Pack et al., 1990). Bimodal (aquatic and aerial) respiration also characterizes a number of teleosts. Studying the air-breathing Amphipnous cuchia, Lomholt and Johansen (1974) reported that RE for the air-breathing organ increased with temperature. Data for the snakehead fish, Channa argus are consistent (Glass et al., 1986). Data for some of these bimodal breathers are compiled in Table 2, which also includes values for aerial ventilation, O2-extraction from the lung, pulmonary gas exchange, and aquatic exchange. The table shows that gas exchange levels are similar, in particular between lungfish and air-breathing teleosts.
levels of PaCO2 reflect an increased dependence on aerial gas exchange (Rahn, 1966; Dejours, 1981). L. paradoxa maintained a constant PaO2 within its relevant temperature range. This differs from amphibians and reptiles that increase PaO2 with rising temperature due to large central cardiovascular shunts (Wood, 1982). This may indicate that central vascular shunts in L. paradoxa are negligible which could result from selective regulation of gill and lung perfusion (Fishman et al., 1988). A very limited shunt in L. paradoxa is also supported by a substantial increase of PaO2 with application of hyperoxia (Johansen and Lenfant, 1967). In the pioneering study, Johansen and Lenfant (1967) pointed out that Lepidosiren presents an intriguing combination of piscine and amphibian characters. The present study points out that L. paradoxa resembles typical amphibians since bimodal respiration provides a mechanism for temperature-dependent adjustments to PaCO2 which, in turn, causes a negative DpH/Dt. The interaction of aquatic and aerial gas exchange accounted for temperature-dependent adjustments of blood acid – base status. This is distinct from temperature-dependent acid – base regulation of exclusively gill-breathing teleosts, that mainly achieve the required decline by modulation of plasma bicarbonate levels. It is also different from that of typical reptiles, that proportion DpHa/Dt by modulations of the ˙ I/V ˙ O2). The bimodal (aerial and aquatic) gas exratio (V change is undoubtedly an ancient condition that accounts for many similarities of basic pulmonary function in amphibians and lungfish.
4.4. Blood gas levels in L. paradoxa Johansen and Lenfant (1967) obtained some blood gas samples from L. paradoxa with PaO2 f 35 mm Hg (20 jC), which is considerably lower than measured in the present study (PaO2 f 90 mm Hg). Their animals had both the pulmonary artery and the dorsal aorta catheterized, which might have influenced blood gas levels. However, it should be pointed out that their animals were small (104 – 212 g) with non-functional traces of external gills. The acid – base status of L. paradoxa is characterized by a high [CO2]pl level combined with a low pHa (7.4 – 7.5) relative to values for both teleosts (normal range: 7.7 < pH < 8.1, 25 jC; cf. Heisler, 1984; Cameron, 1984) and amphibians (normal range: 7.6 < pH < 7.9, 25 jC; cf. Boutilier et al., 1986; Kruhøffer et al., 1987). Arterial PCO2 of L. paradoxa ( f 25 mm Hg at 25 jC) was considerably higher than in teleosts (1– 3 mm Hg; cf. Rahn, 1966) and in amphibians (7– 12 mm Hg; Kruhøffer et al., 1987). Higher
Acknowledgements This study was supported by FAPESP (Fundacß a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo). Proc. #98/ 06731-5, CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico). Proc. #520769/93-7 and FAEPA
Table 2 Oxygen uptake and aerial ventilation in selected vertebrates of bimodal respiration ˙ O2 (air) Species Group Temperature Weight V (jC) (kg) ml STPD/ (kg min) Lepidosiren paradoxaa Lepidosiren paradoxab Protopterus sp.c Bufo paracnemisd Channa arguse Amphipnous cuchiaf a
lungfish lungfish lungfish anuran amphibian teleost teleost
VT ml BTPS/ kg
fR (l/min)
25 20 30 27
0.48 f 0.15 0.3 – 0.8 0.2 – 0.8
0.26 0.89 0.43 0.61
0.01
29
0.07 0.21
35
0.09 0.18 0.14
25 30
1–2 f 0.35
0.28 0.37
0.19
25.6 35.4
0.11 0.31
Present study. Johansen and Lenfant (1967). c Johansen et al. (1976); Lomholt (1993). d Wang et al. (1998). e Glass et al. (1986). f Lomholt and Johansen (1974,1976). b
˙ O2 (water) V ml STPD/ (kg min)
˙ AIR ml BTPS/ V (kg min) 2.7 5.0 24.0 2.9 5.3
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(Fundacß a˜o de Apoio ao Ensino, Pesquisa e Assisteˆncia do Hospital das Clı´nicas da FMRPUSP). We acknowledge fellowship support from CNPq.
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