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The differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the South American lungfish, Lepidosiren paradoxa
Comparative Biochemistry and Physiology Part A 130 Ž2001. 677᎐687
The differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the South American lungfish, Lepidosiren paradoxa Adriana Sanchez a , Roseli Soncini a , Tobias Wang a,c,U , Pia Koldkjaer a,d, Edwin W. Taylor a,b, Mogens L. Glass a a
Department of Physiology, Faculty of Medicine of Ribeirao ˜ Preto, Uni¨ ersity of Sao ˜ Paulo, Sao ˜ Paulo, Brazil b School of Biosciences, The Uni¨ ersity of Birmingham, Birmingham, UK c Department of Zoophysiology, Aarhus Uni¨ ersity, DK 8000 Aarhus C, Denmark d School of Biological Science, The Uni¨ ersity of Li¨ erpool, Li¨ erpool, UK Received 19 September 2000; received in revised form 16 June 2001; accepted 26 June 2001
Abstract Lungfishes ŽDipnoi. occupy an evolutionary transition between water and air breathing and possess well-developed lungs and reduced gills. The South American species, Lepidosiren paradoxa, is an obligate air-breather and has the lowest aquatic respiration of the three extant genera. To study the relative importance, location and modality of reflexogenic sites sensitive to oxygen in the generation of cardio-respiratory responses, we measured ventilatory responses to changes in ambient oxygen and to reductions in blood oxygen content. Animals were exposed to aquatic and aerial hypoxia, both separately and in combination. While aerial hypoxia elicited brisk ventilatory responses, aquatic hypoxia had no effect, indicating a primary role for internal rather than branchial receptors. Reducing haematocrit and blood oxygen content by approximately 50% did not affect ventilation during normoxia, showing that the specific modality of the internal oxygen sensitive chemoreceptors is blood P O 2 per se and not oxygen concentration. In light of previous studies, it appears that the heart rate responses and the changes in pulmonary ventilation during oxygen shortage are similar in lungfish and tetrapods. Furthermore, the modality of the oxygen receptors controlling these responses is similar to tetrapods. Because the cardio-respiratory responses and the modality of the oxygen receptors differ from typical water-breathing teleosts, it appears that many of the changes in the mechanisms exerting reflex control over cardio-respiratory functions occurred at an early stage in vertebrate evolution. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Lungfish; Dipnoi; Lepidosiren; Hypoxia; Hyperoxia; Ventilation; Breathing pattern; Heart rate; Ventilatory response; Oxygen modality; Chemoreceptors
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Corresponding author. Tel.: q45-8942-2694; fax: q45-8619-4186. E-mail address: [email protected] ŽT. Wang..
1095-6433r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 1 . 0 0 3 9 5 - 6
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1. Introduction The lungfishes ŽDipnoi. belong to an ancient lineage that occupies the evolutionary transition between water and air breathing. In addition to their well-developed lungs, they have retained reduced gills and exhibit bimodal respiration, combined with a significant contribution of cutaneous gas exchange Žsee Abe and Steffensen, 1996, for data on Lepidosiren.. Among the three extant genera, the Australian lungfish, Neoceratodus, is a facultative air-breather that ventilates its lungs only when exposed to hypoxic water ŽJohansen et al., 1967; Fritsche et al., 1993., while the South American lungfish, Lepidosiren paradoxa, is an obligate air-breather with highly reduced gills ŽJohansen and Lenfant, 1967.. Potential stimuli for air-breathing in fish include hypoxia, and hypercapnia, both modulated by increased temperature and exercise, which increase oxygen demand and CO 2 production ŽJohansen, 1971; Smatresk, 1994; Graham, 1997.. Until recently it was not established whether the increases in air-breathing observed under these circumstances are stimulated solely by changes in oxygen availability, delivery, or demand, or whether lungfish also respond to changes in blood pH or P CO 2 . In the bowfin Amia cal¨ a, air breathing is only stimulated by changes in water or blood O 2 status ŽMcKenzie et al., 1991., and they do not appear to possess central chemosensitivity controlling gill ventilation or air breathing ŽHedrick et al., 1991.. In contrast, both Protopterus Žthe African lungfish. and Lepidosiren increase air breathing frequency during aquatic hypercapnia ŽJohansen and Lenfant, 1968; Sanchez and Glass, 2001.. Sanchez and Glass Ž2001. also showed that Lepidosiren displays a marked post-hypercapnic hyperpnea as described for ectothermic tetrapods ŽMilsom, 1995.. Collectively, these observations indicate resemblance between Dipnoi and the land vertebrates with respect to the ventilatory responses to hypercapnia. The relative importance and location of reflexogenic sites sensitive to oxygen in the generation of cardiorespiratory responses to changes in ambient or blood gas composition have not yet been clearly elucidated for Lepidosiren. Consequently, the present study set out to evaluate to what extent the hypoxic drive to pulmonary ventilation arises from stimulation of external or inter-
nal receptors. This was explored by exposing animals to aquatic and aerial hypoxia, both separately and in combination. Furthermore, the modality of the oxygen sensitive chemoreceptors has not been investigated in lungfish. Thus, it is not known whether they exhibit ventilatory responses to reduced blood oxygen content ŽwO 2 x. like some teleost fish ŽRandall, 1982; Smith and Jones, 1982; Soncini and Glass, 2000., or regulate ventilation in relation to oxygen partial pressure Ž P O 2 . like amphibians ŽWang et al., 1994.. To investigate this question, we compared the ventilatory responses during aerial hypoxia with the responses measured following similar reductions in blood oxygen content induced by reductions in haematocrit during normoxia. 2. Materials and methods 2.1. Animals Lungfish Ž Lepidosiren paradoxa, Fitz; 800᎐2000 . g of undetermined sex were collected in the Pantanal region near the city of Cuiaba ´ in the state of Mato Grosso do Sul, southwestern Brazil, and transported to The University of Sao Paulo, at Riberao Preto Žstate of Sao Paulo.. Here they were maintained for several weeks prior to experiments in 1000-l tanks, containing dechlorinated, aerated tapwater at a temperature of 25 " 3⬚C. The water was continuously renewed and a 12 h:12 h light:dark cycle was maintained. The animals were fed, mainly on chopped liver, several times a week, but food was withheld for more than 48 h prior to experimentation. 2.2. Surgical procedure The fish used for blood sampling and manipulation of blood oxygen carrying capacity were anaesthetised by immersion into a 1-g ly1 benzocaine solution until they ceased to exhibit responses to pinching of the skin. The fish were then transferred to a surgical table where the gills on one side were irrigated by a continuous flow of water containing 0.25 g ly1 benzocaine. The gill arches on the contralateral Žright. side were exposed by a 2-cm incision above the operculum and the afferent vessel on the fifth and last gill arch was dissected free and occlusively cannulated wsee Romer Ž1970. for gill structure and numberingx. The catheter ŽClay Adams PE50 con-
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taining heparinised Ringer. was forwarded into the ventral aorta and secured to the gill arch by sutures, after which the catheter was exteriorised and secured to the body wall. The incision was closed by sutures and tissue glue, and the animal was transferred to aerated water where it was allowed to recover for approximately 24 h before experimentation. There was no detectable effect of operation on the behaviour of the animals. The experimental chamber was covered to avoid visual disturbances to the animal 2.3. Analytical techniques Blood samples of 0.7 ml were withdrawn anaerobically from the ventral aortic cannula and analysed immediately Žfollowing analysis, 0.5 ml of the blood and 0.2 ml of Saline was re-injected into the animal.. Haematocrit was determined following 3-min centrifugation at 12 000 rpm in capillary tubes. Blood P O 2 and pH were determined using a FAC 204A O 2 analyser ŽFAC Instruments, Sao ˜ Carlos, Sao ˜ Paulo State, Brazil. and a Micronal B374 pH meter ŽSao ˜ Paulo, Brazil.. The oxygen electrodes were calibrated with pure nitrogen and humidified atmospheric air, taking the blood ᎐ gas sensitivity ratio into account ŽSiggaard-Andersen, 1976.. The pH electrode was adjusted using high precision buffers ŽS1500 and S1510; Siggaard-Andersen, 1976.. The electrode chamber was maintained at the temperature of the fish water Ž25⬚C.. Total content of O 2 was measured as described by Tucker Ž1967.. 2.4. Measurement of ¨ entilation and cardiac
¨ ariables
Ventilation at 25⬚C was measured directly, using a plethysmographic method ŽLomholt and Johansen, 1974.. In short, the animal was confined within a 10-l chamber, shaped like an inverted funnel and filled with water to slightly above the cylindrical neck. Due to the expansion of the lungs, inspiration increased the water level within the neck of the funnel, while the animal floated upwards. Oppositely, expiration decreased the water level. These vertical movements of the water column were recorded as pressure changes, using a Niho Kohoen Polygraph System ŽJapan., consisting of a venous pressure transducer, an AP6216 carrier amplifier and the recorder. Calibration was obtained by injection and withdrawal
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of known volumes of water, which provided a direct relationship between pressure change at the bottom of the water column and the amount of water added or withdrawn from the system. Between periods of blood sampling, the catheter was connected to an arterial pressure transducer that formed part of the Niho Kohoen system. Cardiac frequency and blood pressure were recorded, using the polygraph system. Calibration of the pressure transducer was performed daily using a static water column of known elevation. The experimental chamber was continuously flushed either with air or with gas mixtures obtained by feeding pure gases to a GF-3rMP gas mixer. The total flow was set at 1 l miny1 and gases were supplied to the air space in the funnel andror to the water space containing the fish. Using this system, water P O 2 could be changed within 10᎐15 min. 2.5. Experimental protocols Experiments were performed as three separate series, as described below. Series 1 addressed the effects of repeated exposure to hypoxia; series 2 described the effects of aerial andror aquatic hypoxia; and series 3 determined the effects of reduced blood oxygen content during normoxia and hypoxia. 2.5.1. Series 1 The effects of repeated exposure to aerial hypoxia was determined in nine lungfish. Each unrestrained and un-operated lungfish was placed into the experimental chamber overnight and ventilation during normoxia was recorded the following day. When a stable ventilatory pattern had been recorded for a minimum of 30 min, hypoxia Žaerial P O 2 s 69, then 35 mmHg. was applied as described below, after which the animals were returned to normoxia. Following 2᎐3 h in normoxia, the hypoxic exposures were repeated. 2.5.2. Series 2 The ventilatory responses to aerial andror aquatic hypoxia were measured in eight unrestrained and un-operated lungfish. Each animal was placed in the experimental chamber the day before experimentation. The animal never struggled and a regular breathing pattern soon developed. After a normoxic control period, established by 2 h of vigorous aeration of the cham-
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ber, the water P O 2 was, in sequence, changed to 69, 35 and then, 208 mmHg, corresponding to 10, 5 and 30% O 2 , respectively, delivered from the gas flow meter. Meanwhile, the aerial phase was maintained normoxic. Secondly, after a normoxic recovery period, the same sequence of gas mixtures was delivered to the aerial phase, while the water was kept normoxic. Thirdly, the hypoxic sequences were applied simultaneously to the water and to the aerial phase. Each experimental condition was maintained for 1 h. Meanwhile, tidal volume and frequency were continuously recorded.
cardiorespiratory responses to hypoxia ŽSeries 3. were evaluated by two-way ANOVAs for repeated measures. The effect of aquatic and aerial hypoxia on ventilation in the uninstrumented animals ŽSeries 2. was evaluated by a one-way ANOVA for repeated measures. In all cases, means that were significantly different from the normoxic level were subsequently identified by a Student᎐Newman᎐Keuls post-hoc test. We applied a fiducial limit for significance of P- 0.05 and all data are presented as mean " 1 S.E.M.
3. Results 2.5.3. Series 3 Cannulated fish were placed in the experimental chamber in order to record heart rate and ventilation. After obtaining a blood sample during normoxia, hypoxia was applied as described above ŽSeries 1. and blood samples were obtained at the end of each exposure. The animals were returned to normoxia, and a volume of blood Ž2% of body weight. was removed to render the animal anaemic, down to a haematocrit of approximately 50% of normal. Blood volume was maintained by replacing the removed blood with an equal volume of lungfish Ringer. Ventilation and heart rate were measured under normoxic conditions, 2᎐3 h after the reduction in blood oxygen carrying capacity.
3.1. The effects of ambient hypoxia on un-operated and unrestrained animals The responses of un-operated and unrestrained animals to repeated exposure to aerial hypoxia ŽSeries 1. are illustrated in Table 1. The frequency of air breathes Ž f R . increased significantly during both sets of exposures. Coupled with an insignificant trend towards an increased tidal volume Ž VT ., this resulted in a highly significant, three-fold increase in ventilation volume Ž VI . at a PI O 2 of 38 mmHg, compared to the normoxic control value. There was no significant difference between the responses measured during the first and the second exposures to hypoxia. In a separate series of single exposures ŽSeries . 2 , illustrated in Fig. 1, aerial hypoxia, with or without aquatic hypoxia, caused a progressive increase in f R reaching a four-fold increase at a P O 2 of 35 mmHg, compared to the control, normoxic rate. As the amplitude of air breaths was
2.6. Statistics and data analysis The effects of repeated exposure to hypoxia ŽSeries 1., and the effects of reducing blood oxygen carrying capacity on blood gases and the
Table 1 Effects of repeated exposure to aerial hypoxia in unrestrained and un-operated lungfish First hypoxic exposure ŽPO 2 in mmHg.
VT Žml kgy1 . fR Žhy1 . VI Žml kgy1 hy1 .
Second hypoxic exposure run ŽPO 2 in mmHg.
150 Ž9.
72 Ž9.
38 Ž9.
150 Ž9.
72 Ž9.
38 Ž7.
26.5" 1.8 10.4" 1 272 " 32.5
28.3" 2.8 17.2" 3.5 483 " 88.3
34.7" 4.1 23.3" 5.6 791 " 194
31.2" 2.3 9.1" 1.8 259 " 61
29.3" 2.5 18.3" 4.3 566 " 133
33.5" 4.6 26.5" 5.7 834 " 174
Values are presented as mean q 1 S.E.M., and the number of animals is listed in brackets; there was no significant difference between the first and second set of exposures Žtwo-way ANOVA for repeated measures.. Abbre¨ iations: VT s tidal volume; f R s respiratory frequency; VI s inspired volume.
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in fish exposed to aerial hyperoxia in hyperoxic water that, when combined with a small decrease in VT , resulted in a significant reduction in mean VI to 60% of the control, normoxic value ŽFig. 1.. In this second series of experiments, the breathing frequencies were generally lower than those obtained in series 1 and 3. Because the animals studied in series 1 were un-operated, as in series 2, we are not able to identify a simple cause for this difference. Since series 2 was performed on a separate group of fish during the winter season, it is possible that seasonal effects may account for the lower breathing frequency. 3.2. Changes in blood gas ¨ alues during hypoxia and following induced anaemia The changes in blood gas values during a twostep reduction in ambient P O 2 are shown in Fig. 2. The oxygen partial pressure changed in direct proportion to ambient hypoxia and the other data are related to these values. Hypoxic exposure caused a progressive reduction in blood oxygen content ŽCaO 2 ., while haematocrit and pH were unaffected. The substitution of approximately half of the blood volume by Ringer resulted in a 50% reduction in normoxic haematocrit, a proportional decrease in blood oxygen content, but did not affect pH. 3.3. Ventilation and heart rate in relation to ¨ entral aortic PO2 during hypoxia and following induced anaemia Fig. 1. Changes in ventilation rate Ž f R ., tidal volume Ž VT . and ventilation volume Ž VI . of lungfish Ž Lepidosiren. exposed to two levels of hypoxia and to hyperoxia. The changes in P O 2 were imposed in the water alone Žopen circles., in the air space alone Žgrey circles., or in both media simultaneously Žsolid circles.. While aquatic hypoxia alone was without effect on ventilation, hypoxia imposed in the air space from which the fish breathed caused significant increases in f R and VT , with or without aquatic hypoxia. Mean values that are significantly different from the normoxic value are marked with an asterisk.
unaffected, this resulted in a proportional increase in VI . Aquatic hypoxia, with access to normoxic air, had no significant effect on the mean rate or amplitude of the air-breaths, so that ventilation volume was unaffected. Neither aquatic nor aerial hyperoxia alone, had any affect on the frequency or amplitude of air breaths. However, there was a 30% reduction in mean f R
The changes in ventilation and heart rate in relation to changes in ventral aortic P O 2 during progressive hypoxia and in response to anaemia, induced by a 50% reduction in haematocrit, are illustrated in Fig. 3. Hypoxia had no effect on heart rate or VT , but caused a significant increase in f R , with the result that VI was markedly increased to more than double its normoxic value. Anaemia induced a 10% increase in normoxic heart rate, but had no effect on the respiratory variables. 4. Discussion 4.1. Critique of blood sampling The blood samples were obtained through a cannula inserted into the fifth gill arch, from
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separation in the bulbus. Therefore, the blood gases measured in our study are likely to reflect a mixture of systemic venous blood and blood returning from the lungs. Thus, the ventral aortic blood gases reported here are unlikely to represent arterial systemic blood. Arterial blood is normally sampled for descriptions of cardiorespiratory response curves. However, the location of oxygen-sensitive chemoreceptors within the cardiovascular system are unknown in lungfish and the primary purposes of cannulation was to monitor the manipulation of blood oxygen carrying capacity and to establish whether vascular P O 2 levels were significantly affected. In this context, the collected blood samples answered the questions addressed. 4.2. Ventilatory pattern and responses to hypoxia in water and air
Fig. 2. Blood gas values in lungfish exposed to hypoxic gas mixtures in the air space of the experimental chamber and subsequently, to induced anaemia. Blood oxygen content was markedly reduced both by hypoxia and by anaemia. Hypoxia was without effect on haematocrit, but it was reduced by approximately 50% by anaemia. Neither imposed variable affected pH. Mean values that are significantly different from the normoxic value are marked with an asterisk.
where it was advanced into the bulbus. This cannulation is relatively non-invasive and the animals recovered quickly from the surgery. The blood perfusing the functional gill arches predominantly stems from the systemic venous circulation, and is preferentially distributed towards the pulmonary circulation Že.g. Johansen et al., 1968a; Lenfant and Johansen, 1968; Fishman et al., 1985, 1989.. In addition, it is uncertain whether the insertion of the cannulae interfered with the blood flow
During normoxia, and when undisturbed, Lepidosiren remained quietly submerged and surfaced at regular intervals to breathe. Each breath consisted of a single prolonged and continuous expiration that was followed immediately by a bout of consecutive small inspirations Žbetween 5 and 14, with a mean of approx. 9 inspirations.. The same breathing pattern was recently observed in caecilian amphibians ŽGardner et al., 2000. and resembles the inflation bouts of anuran amphibians ŽKruhøffer et al., 1987.. From a functional point of view the repeated inspirations reduce the functional dead space. In the African lungfish, Protopterus, the inspirations are caused by a buccal force pump mechanism ŽMcMahon, 1969; Delaney and Fishman, 1977. and visual inspection during the present study suggests that this is also the case for Lepidosiren. During hypoxia, the ventilatory pattern continued to consist of single breaths and the increased overall lung ventilation was accomplished by a reduction of the breathhold periods, whereas tidal volume only increased very slightly. This points to an important role of pulmonary stretch receptor feedback exerting the Hering᎐Breuer reflex, where inflation reflexly terminates inspiration. Protopterus possesses pulmonary stretch receptors ŽDelaney et al., 1983., and manipulation of lung volume alters the duration of the non-ventilatory periods ŽPack et al., 1992.. Stretch receptors in the air-breathing organs are also important in determining tidal volume and breathing pattern in other air breath-
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Fig. 3. The effect of hypoxia and anaemia on ventilation and heart rate in lungfish, plotted against mean levels of oxygen partial pressure in the ventral aorta Ž P VA O 2 .. While tidal volume Ž VT . was unaffected, ventilation rate Ž f R . increased during hypoxia, causing a proportional increase in ventilation volume Ž VI .. Heart rate was unaffected by hypoxia, but increased by 10% in anaemic fish.
ing fish such as Amia and Lepisosteus ŽJohansen et al., 1970; Smatresk and Cameron, 1982; Hedrick and Jones, 1999.. As pointed out by Hedrick and Jones Ž1993, 1999., the control of tidal volume is particularly important for aquatic animals where the degree of lung inflation significantly affects buoyancy. In most species of teleost fish, gill ventilation increases markedly during aquatic hypoxia, due to stimulation of receptors that are located principally on the first gill arch ŽDaxboeck and Holeton, 1978; Milsom and Brill, 1986.. Some of these receptors are located externally and screen the inspired water, while others are internal and monitor blood O 2 levels ŽMilsom and Brill, 1986; Smatresk et al., 1986; Burleson and Smatresk, 1990; Burleson et al., 1992.. As a consequence, ventilatory responses to hypoxic water may occur without any changes in blood O 2 levels ŽGlass et al., 1990; Soncini and Glass, 2000.. Pulmonary ventilation in Lepidosiren did not increase in response to aquatic hypoxia and it does not appear,
therefore, that branchial O 2 sensitive receptors are involved in the control of pulmonary ventilation in this animal. The existence of external receptors cannot, however, be excluded, since addition of nicotine or cyanide into the opercular cavity stimulates both branchial and pulmonary ventilation in Protopterus ŽJohansen et al., 1968a; Lahiri et al., 1970.. The inability of aquatic hypoxia to stimulate lung ventilation is consistent with responses of Neoceratodus and Protopterus, the air-breathing teleosts Channa argus and Electrophorus electricus, and the aquatic caecilian Typhlonectus natans ŽJohansen et al., 1967, 1968b; Johansen and Lenfant, 1968; Glass et al., 1986; Gardner et al., 2000.. However, in Amia, Ancistris, Hypostomus and Lepisosteus, stimulation of external oxygen sensitive receptors stimulate breathing ŽJohansen et al., 1970; Graham and Baird, 1982; Smatresk et al., 1986; Hedrick and Jones, 1993.. The ventilatory response to aerial hypoxia concords with the effects observed in the other species
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of lungfish Že.g. Jesse et al., 1967; Johansen et al., 1967; Johansen and Lenfant, 1968; Fritsche et al., 1993.. These responses must be attributed to internal oxygen sensitive receptors, most likely sensing blood P O 2 , but the specific location of these remain unknown ŽFishman et al., 1989.. It is not surprising that Lepidosiren resembles the other genera of lungfishes in this context, as the members of the Lepidosirenidae are obligate air breathers, while the other genera are more or less facultative air-breathers. According to Johansen et al. Ž1976., adult specimens of Protopterus obtain 70% of total O 2 uptake by pulmonary ventilation. Because the gills of Lepidosiren are considerably reduced so that the filaments are nearly absent on the first two gill arches ŽJohansen and Lenfant, 1967; Lenfant et al., 1970., it would be expected that branchial gas exchange is lower than in any other species of lungfish. However, Abe and Steffensen Ž1996. showed that almost 40% of the oxygen uptake of quietly resting Lepidosiren occur over the skin and gills. Pulmonary ventilation decreased during exposure to hyperoxia in Lepidosiren ŽFig. 1., showing that oxygen provides a tonic drive to ventilation in normoxia. Similar effects have been observed in Protopterus ŽJesse et al., 1967., other airbreathing fish, amphibians and reptiles Že.g. Lomholt and Johansen, 1974; Heisler, 1982; Hughes and Singh, 1971; Smatresk et al., 1986; McKenzie et al., 1992.. Gill ventilation in teleosts is also reduced during hyperoxia. 4.3. Heart rate during hypoxia In Lepidosiren and Protopterus, earlier studies have shown an increased heart rate during the intermittent breathing episodes ŽAxelsson et al., 1989; Burggren and Johansen, 1986. and Fritsche et al. Ž1993. also recorded increased pulmonary blood flow associated with lung ventilation in Neoceratodus. In our study there were no apparent changes in heart rate during ventilation. The heart rates recorded in the present study are in general agreement with previous studies on Lepidosiren ŽAxelsson et al., 1989., but as pointed out by these authors, these heart rates appear relatively high. In the study by Axelsson et al. Ž1989., the respiratory-related tachycardia did occur in animals with low heart rate, possibly due to a
larger vagal tone on the heart that can be released during ventilation. There was no significant change in heart rate during hypoxia in Lepidosiren ŽFig. 3.. This in agreement with earlier observations on Neoceratodus, although this species does increase pulmonary blood flow, possibly due to redistribution of blood, during progressive aquatic hypoxia ŽFritsche et al., 1993.. A reflex tachycardia in response to a reduced oxygen supply is characteristic of committed lung breathers such as mammals that show the so-called ‘secondary response’ to hypoxia following stimulation of lung stretch receptors during hypoxic hyperventilation ŽDaly, 1997.. In this respect, Lepidosiren shows physiological responses that are typical for lung breathers possessing pulmonary mechanoreceptors ŽDelaney et al., 1983.. However, because heart rate remained elevated during the breath hold periods in the anaemic animals, stimulation of pulmonary stretch receptors is not obligatory for the hypoxaemic tachycardia. Lepidosiren did show a significant tachycardia in normoxic hypoxaemia. The cardiac responses of lungfish are diametrically opposed to that of typical water-breathing fish, where environmental hypoxia normally induces a reflex bradycardia ŽTaylor, 1992.. This response is predominantly in response to stimulation of externally located branchial oxygen receptors sensitive to water P O 2 ŽBurleson et al., 1992.. Thus, the absence of a hypoxic bradycardia is in agreement with the lack of evidence for external receptors on the gills of Lepidosiren. 4.4. Modality of the oxygen response and the effects of reducing haematocrit The specific oxygen stimulus that elicits ventilatory responses in vertebrates is a matter of contention Že.g. Boggs, 1995., but most studies on air-breathing vertebrates point to P O 2 being the driving stimulus Že.g. Lahiri et al., 1981; Wang et al., 1994, 1997; Boggs, 1995; McKenzie et al., 1991.. The responses of Lepidosiren are consistent with this view, as the large reduction in haematocrit and ventral aortic oxygen content during normoxia did not augment ventilation above the normoxic values previously attained ŽFigs. 2 and 3.. The fact that the lungfish studied in series 1 maintained a brisk hypoxic ventilatory response during the second hypoxic exposure
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shows that they are still able to respond to reduction in partial pressure. Thus, from our data it appears that the chemoreceptors responsible for the hypoxic ventilatory response are sensitive to P O 2 and that the lungfish resembles other airbreathing vertebrates, in this respect. Again, the hypoxic responses of Lepidosiren are opposed to typical, water-breathing fish, where systemic hypoxaemia, even in the absence of an associated hypoxia Že.g. anaemia. appears to stimulate ventilation ŽRandall, 1982; Smith and Jones, 1982.. Heart rate increased following the reduction in haematocrit in Lepidosiren ŽFig. 3.. A tachycardia in response to lowered blood oxygen content, in the absence or presence of ventilatory changes, appears to be a general response of water and air-breathing vertebrates Že.g. Wood and Shelton, 1980; Boggs, 1995; Wang et al., 1994, 1997.. In mammals, this response may arise from stimulation of chemoreceptors in the aortic arch ŽDaly, 1997. and it seems that other vertebrates also possess oxygen sensitive chemoreceptors that primarily exert a cardiovascular control. However, in studies that involve withdrawal of blood, it is difficult to dismiss the possibility that the heart rate changes are due to regulation of blood pressure rather than stimulation of oxygen-sensitive chemoreceptors per se. 4.5. Perspecti¨ es and conclusions These and previous studies demonstrate that the location with respect to the oxygen cascade and the functional characteristics of oxygen sensitive receptors controlling the heart and ventilation, plus the stimulus modality of these receptors, are similar in lungfish and tetrapods. Thus, many of the changes in the mechanisms exerting reflex control over cardio-respiratory functions, accompanying the transition between water and air breathing, may have occurred at an early stage in vertebrate evolution.
Acknowledgements This study was supported by FAPESP ŽProc. no. 1998r06731-5 . and CNPq wProc. no. 300603r91-6ŽRN.x and The Danish Research Council ŽSNF.. We gratefully acknowledge technical help from Humberto Giusti.
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References Abe, A.S., Steffensen, J.F., 1996. Lung and cutaneous respiration in awake and estivating South American lungfish, Lepidosiren paradoxa. Rev. Bras. Biol. 56, 485᎐489. Axelsson, M., Abe, A.S., Bicudo, J.E.P.W., Nilsson, S., 1989. On the cardiac control in the South American lungfish, Lepidosiren paradoxa. Comp. Biochem. Physiol. 93A, 561᎐565. Boggs, D.F., 1995. Hypoxic ventilatory control and hemoglobin oxygen affinity. In: Sutton, J.R., Houston, C.S., Coates, G. ŽEds.., Hypoxia and the Brain. Queen City Printers, Burlington, Vermont, pp. 69᎐86. Burggren, W.W., Johansen, K., 1986. Circulation and respiration in lungfishes ŽDipnoi.. J. Morph. 1, 217᎐236. Burleson, M.L., Smatresk, N.J., 1990. Evidence for two oxygen-sensitive chemoreceptor loci in channel catfish, Ictalurus punctatus. Physiol. Zool. 63, 208᎐221. Burleson, M.L., Smatresk, N.J., Milsom, W.K., 1992. Afferent inputs associated with cardioventilatory control in fish. In: Hoar, W.S., Randall, D.J., Farrell, A.P. ŽEds.., Fish Physiology, vol. 12B, The Cardiovascular System. Academic Press, New York, NY, pp. 390᎐426. Daly, M.B., 1997. Peripheral Arterial Chemoreceptors and Respiratory᎐Cardiovascular Regulation. Oxford Medical Publications, Oxford 739 pp. Daxboeck, C., Holeton, G.F., 1978. Oxygen receptors in the rainbow trout, Salmo gairdneri. Can. J. Zool. 56, 1254᎐1259. Delaney, R.G., Fishman, A.P., 1977. Analysis of lung ventilation in the aestivating lungfish Protopterus aethiopicus. Am. J. Physiol. 233, R181᎐R187. 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. 13, R418᎐R428. Fishman, A.P., Delaney, R.G., Laurent, P., 1985. Circulatory adaptation to bimodal respiration in the dipnoan lungfish. J. Appl. Physiol. 59, 285᎐294. Fishman, A.P., Galante, R.J., Pack, A.I., 1989. Diving Physiology. Lungfish. In: Wood, S.C. ŽEd.., Comparative Pulmonary Physiology: Current Concepts, vol. 39, Lung Biology in Health and Disease. Marcel Dekker, New York. 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. Gardner, M.N., Smits, A.W., Smatresk, N.J., 2000. The ventilatory responses of the caecilian Typlonectus natans to hypoxia and hypercapnia. Physiol. Biochem. Zool. 73, 23᎐29.
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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. 156, 425᎐430. Glass, M.L., Andersen, N.A., Kruhøffer, M., Williams, E.M., Heisler, N., 1990. Combined effects of environmental P O 2 and temperature on ventilation and blood gases in the carp, Cyprinus carpio L. J. Exp. Biol. 148, 1᎐17. Graham, J.B., 1997. Air-Breathing Fishes: Evolution Diversity and Adaptation. Academic Press, San Diego. Graham, J.B., Baird, T., 1982. The transition to air breathing in fishes. I. Environmental effects on the facultative air breathing of Ancistrus chagresi and Hypostomus plecostomus ŽLoricariidae.. J. Exp. Biol. 96, 53᎐67. Hedrick, M.S., Jones, D.R., 1993. The effects of altered aquatic and aerial respiratory gas concentrations on air-breathing patterns in a primitive fish Ž Amia cal¨ a.. J. Exp. Biol. 181, 81᎐94. Hedrick, M.S., Jones, D.R., 1999. Control of gill ventilation and air-breathing in the bowfin Amia cal¨ a. J. Exp. Biol. 202, 87᎐94. Hedrick, M.S., Burleson, M.L., Jones, D.R., Milsom, W.K., 1991. An examination of central chemosensitivity in an air-breathing fish Ž Amia cal¨ a.. J. Exp. Biol. 155, 165᎐174. Heisler, N., 1982. Intracellular and extracellular acid᎐base regulation in the tropical freshwater teleost fish Synbranchus marmoratus in response to the transition from water breathing to air breathing. J. Exp. Biol. 99, 9᎐28. Hughes, G.M., Singh, B.N., 1971. Gas exchange with air and water in an air-breathing catfish, Saccobranchus (Heteropneustes) fossilis. J. Exp. Biol. 55, 667᎐682. Jesse, M.J., Shub, C., Fishman, A.P., 1967. Lung and gill ventilation of the African lungfish. Respir. Physiol. 3, 267᎐287. Johansen, J., Lenfant, C., 1967. Respiratory function in the South American lungfish, Lepidosiren paradoxa ŽFitz.. J. Exp. Biol. 46, 205᎐218. Johansen, K., 1971. Comparative physiology: gas exchange and circulation in fishes. Ann. Rev. Physiol. 33, 569᎐612. Johansen, K., Lenfant, C., Grigg, G.C., 1967. Respiratory control in the lungfish, Neoceratodus forsteri ŽKrefft.. Comp. Biochem. Physiol. 20, 835᎐854. Johansen, K., Lenfant, C., 1968. Respiration in the African lungfish, Protopterus aethiopicus. II. Control of breathing. J. Exp. Biol. 49, 453᎐468. Johansen, K., Lenfant, C., Hanson, D., 1968a. Cardiovascular dynamics in the lungfishes. Z. Vergl. Physiol. 59, 157᎐186. Johansen, K., Lenfant, C., Schmidt-Nielsen, K., Petersen, J., 1968b. Gas exchange and control of
breathing in the electric eel, Electrophorus electricus. Z. Vergl. Physiol. 61, 137᎐163. Johansen, K., Hanson, D., Lenfant, C., 1970. Respiration in a primitive air breather, Amia cal¨ a. Respir. Physiol. 9, 162᎐174. Johansen, K., Lomholt, J.P., Maloiy, G.M.O., 1976. Importance of air and water breathing in relation to size of the African lungfish Protopterus amphibius Peters. J. Exp. Biol. 65, 395᎐399. 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. Lahiri, S., Szidon, J.P., Fishman, A.P., 1970. Potential respiratory and circulatory adjustments to hypoxia in the African lungfish. Ann. Rev. Physiol. 29, 1141᎐1148. Lahiri, S., Mulligan, E., Nishino, T., Mokashi, A., Davies, R.O., 1981. Relative responses of aortic body and carotid body chemoreceptors to carboxyhemoglobinemia. J. Appl. Physiol. 50, 580᎐586. Lenfant, C., Johansen, K., 1968. Respiration in the African lungfish Protopterus aethiopicus. I. Respiratory properties of blood and normal patterns of breathing and gas exchange. J. Exp. Biol. 49, 437᎐452. 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., Johansen, K., 1974. Control of breathing in Amphipnous cuchia, an amphibious fish. Respir. Physiol. 21, 325᎐340. McKenzie, D.J., Aota, S., Randall, D.J., 1991. Ventilatory and cardiovascular responses to blood pH, plasma P CO 2 , blood O 2 content, and catecholamines in an air-breathing fish, the bowfin Ž Amia cal¨ a.. Physiol. Zool. 64, 432᎐450. McMahon, B.R., 1969. A functional analysis of the aquatic and aerial respiratory movements of an African lungfish, Protopterus aethiopicus, with reference to the evolution of the lung-ventilation mechanism in vertebrates. J. Exp. Biol. 51, 407᎐430. Milsom, W.K., 1995. The role of CO 2rpH chemoreceptors in ventilatory control. Braz. J. Med. Biol. Res. 28, 1147᎐1160. Milsom, W.K., Brill, R.W., 1986. Oxygen sensitive afferent information arising from the first gill arch of yellowfin tuna. Respir. Physiol. 66, 193᎐203. Pack, A.I., Galante, R.J., Fishman, A.P., 1992. Role of lung inflation in control of air breath duration in African lungfish Ž Protopterus annectens.. Am. J. Physiol. 31, R879᎐R884. Randall, D.J., 1982. The control of respiration and circulation in fish during exercise and hypoxia. J. Exp. Biol. 100, 275᎐288. Romer, A.S., 1970. The Vertebrate Body, 4th ed. Saunders, Philadelphia.
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Sanchez, A., Glass, M.L., 2001. Effects of environmental hypercapnia on pulmonary ventilation of the Southern American lungfish, Lepidosiren paradoxa ŽFitz... J. Fish Biol. 58, 1181᎐1189. Siggaard-Andersen, O., 1976. The Acid᎐Base Status of the Blood, 4th ed. Munksgaard, Copenhagen. Smatresk, N.J., 1994. Respiratory control in the transition from water to air breathing in vertebrates. Am. Zool. 34, 264᎐279. Smatresk, N.J., Cameron, J.N., 1982. Respiration and acid᎐base physiology of the spotted gar, a bimodal breather. III. Response to a transfer from freshwater to 50% sea water, and control of ventilation. J. Exp. Biol. 96, 295᎐306. Smatresk, N.J., Burleson, M.L., Azizi, S.Q., 1986. Chemoreflexive responses to hypoxia and NaCN in longnose gar, evidence for two chemoreceptor loci. Am. J. Physiol. 20, R116᎐R125. Smith, F.M., Jones, D.R., 1982. The effect of changes in blood oxygen-carrying capacity on ventilation volume in the rainbow trout Ž Salmo gairdneri.. J. Exp. Biol. 97, 325᎐334.
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Soncini, R., Glass, M.L., 2000. Oxygen and acid᎐base status-related drives to gill ventilation in carp. J. Fish Biol. 56, 528᎐541. Taylor, E.W., 1992. Nervous control of the heart and cardiorespiratory interactions. In: Hoar, W.S., Randall, D.J., Farrell, A.P. ŽEds.., Fish Physiology, vol. 12B, The Cardiovascular System. Academic Press, New York, NY, pp. 343᎐387. Tucker, V.A., 1967. Method for oxygen content and dissociation curves on microliter blood samples. J. Appl. Physiol. 23, 410᎐414. Wang, T., Branco, L.G.S., Glass, M.L., 1994. Ventilatory responses to hypoxia in the toad Bufo paracnemis before and after a decrease in haemoglobin oxygen-carrying capacity. J. Exp. Biol. 186, 1᎐8. Wang, T., Krosniunas, E., Hicks, J.W., 1997. The role of cardiac shunts in the regulation of arterial blood gases. Am. Zool. 37, 12᎐22. Wood, C.M., Shelton, G., 1980. The reflex control of heart rate and cardiac output in the rainbow trout: interactive influences of hypoxia, haemorrhage, and systemic vasomotor tone. J. Exp. Biol. 87, 271᎐284.
The differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the South American lungfish, Lepidosiren paradoxa Adriana Sanchez a , Roseli Soncini a , Tobias Wang a,c,U , Pia Koldkjaer a,d, Edwin W. Taylor a,b, Mogens L. Glass a a
Department of Physiology, Faculty of Medicine of Ribeirao ˜ Preto, Uni¨ ersity of Sao ˜ Paulo, Sao ˜ Paulo, Brazil b School of Biosciences, The Uni¨ ersity of Birmingham, Birmingham, UK c Department of Zoophysiology, Aarhus Uni¨ ersity, DK 8000 Aarhus C, Denmark d School of Biological Science, The Uni¨ ersity of Li¨ erpool, Li¨ erpool, UK Received 19 September 2000; received in revised form 16 June 2001; accepted 26 June 2001
Abstract Lungfishes ŽDipnoi. occupy an evolutionary transition between water and air breathing and possess well-developed lungs and reduced gills. The South American species, Lepidosiren paradoxa, is an obligate air-breather and has the lowest aquatic respiration of the three extant genera. To study the relative importance, location and modality of reflexogenic sites sensitive to oxygen in the generation of cardio-respiratory responses, we measured ventilatory responses to changes in ambient oxygen and to reductions in blood oxygen content. Animals were exposed to aquatic and aerial hypoxia, both separately and in combination. While aerial hypoxia elicited brisk ventilatory responses, aquatic hypoxia had no effect, indicating a primary role for internal rather than branchial receptors. Reducing haematocrit and blood oxygen content by approximately 50% did not affect ventilation during normoxia, showing that the specific modality of the internal oxygen sensitive chemoreceptors is blood P O 2 per se and not oxygen concentration. In light of previous studies, it appears that the heart rate responses and the changes in pulmonary ventilation during oxygen shortage are similar in lungfish and tetrapods. Furthermore, the modality of the oxygen receptors controlling these responses is similar to tetrapods. Because the cardio-respiratory responses and the modality of the oxygen receptors differ from typical water-breathing teleosts, it appears that many of the changes in the mechanisms exerting reflex control over cardio-respiratory functions occurred at an early stage in vertebrate evolution. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Lungfish; Dipnoi; Lepidosiren; Hypoxia; Hyperoxia; Ventilation; Breathing pattern; Heart rate; Ventilatory response; Oxygen modality; Chemoreceptors
U
Corresponding author. Tel.: q45-8942-2694; fax: q45-8619-4186. E-mail address: [email protected] ŽT. Wang..
1095-6433r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 1 . 0 0 3 9 5 - 6
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1. Introduction The lungfishes ŽDipnoi. belong to an ancient lineage that occupies the evolutionary transition between water and air breathing. In addition to their well-developed lungs, they have retained reduced gills and exhibit bimodal respiration, combined with a significant contribution of cutaneous gas exchange Žsee Abe and Steffensen, 1996, for data on Lepidosiren.. Among the three extant genera, the Australian lungfish, Neoceratodus, is a facultative air-breather that ventilates its lungs only when exposed to hypoxic water ŽJohansen et al., 1967; Fritsche et al., 1993., while the South American lungfish, Lepidosiren paradoxa, is an obligate air-breather with highly reduced gills ŽJohansen and Lenfant, 1967.. Potential stimuli for air-breathing in fish include hypoxia, and hypercapnia, both modulated by increased temperature and exercise, which increase oxygen demand and CO 2 production ŽJohansen, 1971; Smatresk, 1994; Graham, 1997.. Until recently it was not established whether the increases in air-breathing observed under these circumstances are stimulated solely by changes in oxygen availability, delivery, or demand, or whether lungfish also respond to changes in blood pH or P CO 2 . In the bowfin Amia cal¨ a, air breathing is only stimulated by changes in water or blood O 2 status ŽMcKenzie et al., 1991., and they do not appear to possess central chemosensitivity controlling gill ventilation or air breathing ŽHedrick et al., 1991.. In contrast, both Protopterus Žthe African lungfish. and Lepidosiren increase air breathing frequency during aquatic hypercapnia ŽJohansen and Lenfant, 1968; Sanchez and Glass, 2001.. Sanchez and Glass Ž2001. also showed that Lepidosiren displays a marked post-hypercapnic hyperpnea as described for ectothermic tetrapods ŽMilsom, 1995.. Collectively, these observations indicate resemblance between Dipnoi and the land vertebrates with respect to the ventilatory responses to hypercapnia. The relative importance and location of reflexogenic sites sensitive to oxygen in the generation of cardiorespiratory responses to changes in ambient or blood gas composition have not yet been clearly elucidated for Lepidosiren. Consequently, the present study set out to evaluate to what extent the hypoxic drive to pulmonary ventilation arises from stimulation of external or inter-
nal receptors. This was explored by exposing animals to aquatic and aerial hypoxia, both separately and in combination. Furthermore, the modality of the oxygen sensitive chemoreceptors has not been investigated in lungfish. Thus, it is not known whether they exhibit ventilatory responses to reduced blood oxygen content ŽwO 2 x. like some teleost fish ŽRandall, 1982; Smith and Jones, 1982; Soncini and Glass, 2000., or regulate ventilation in relation to oxygen partial pressure Ž P O 2 . like amphibians ŽWang et al., 1994.. To investigate this question, we compared the ventilatory responses during aerial hypoxia with the responses measured following similar reductions in blood oxygen content induced by reductions in haematocrit during normoxia. 2. Materials and methods 2.1. Animals Lungfish Ž Lepidosiren paradoxa, Fitz; 800᎐2000 . g of undetermined sex were collected in the Pantanal region near the city of Cuiaba ´ in the state of Mato Grosso do Sul, southwestern Brazil, and transported to The University of Sao Paulo, at Riberao Preto Žstate of Sao Paulo.. Here they were maintained for several weeks prior to experiments in 1000-l tanks, containing dechlorinated, aerated tapwater at a temperature of 25 " 3⬚C. The water was continuously renewed and a 12 h:12 h light:dark cycle was maintained. The animals were fed, mainly on chopped liver, several times a week, but food was withheld for more than 48 h prior to experimentation. 2.2. Surgical procedure The fish used for blood sampling and manipulation of blood oxygen carrying capacity were anaesthetised by immersion into a 1-g ly1 benzocaine solution until they ceased to exhibit responses to pinching of the skin. The fish were then transferred to a surgical table where the gills on one side were irrigated by a continuous flow of water containing 0.25 g ly1 benzocaine. The gill arches on the contralateral Žright. side were exposed by a 2-cm incision above the operculum and the afferent vessel on the fifth and last gill arch was dissected free and occlusively cannulated wsee Romer Ž1970. for gill structure and numberingx. The catheter ŽClay Adams PE50 con-
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taining heparinised Ringer. was forwarded into the ventral aorta and secured to the gill arch by sutures, after which the catheter was exteriorised and secured to the body wall. The incision was closed by sutures and tissue glue, and the animal was transferred to aerated water where it was allowed to recover for approximately 24 h before experimentation. There was no detectable effect of operation on the behaviour of the animals. The experimental chamber was covered to avoid visual disturbances to the animal 2.3. Analytical techniques Blood samples of 0.7 ml were withdrawn anaerobically from the ventral aortic cannula and analysed immediately Žfollowing analysis, 0.5 ml of the blood and 0.2 ml of Saline was re-injected into the animal.. Haematocrit was determined following 3-min centrifugation at 12 000 rpm in capillary tubes. Blood P O 2 and pH were determined using a FAC 204A O 2 analyser ŽFAC Instruments, Sao ˜ Carlos, Sao ˜ Paulo State, Brazil. and a Micronal B374 pH meter ŽSao ˜ Paulo, Brazil.. The oxygen electrodes were calibrated with pure nitrogen and humidified atmospheric air, taking the blood ᎐ gas sensitivity ratio into account ŽSiggaard-Andersen, 1976.. The pH electrode was adjusted using high precision buffers ŽS1500 and S1510; Siggaard-Andersen, 1976.. The electrode chamber was maintained at the temperature of the fish water Ž25⬚C.. Total content of O 2 was measured as described by Tucker Ž1967.. 2.4. Measurement of ¨ entilation and cardiac
¨ ariables
Ventilation at 25⬚C was measured directly, using a plethysmographic method ŽLomholt and Johansen, 1974.. In short, the animal was confined within a 10-l chamber, shaped like an inverted funnel and filled with water to slightly above the cylindrical neck. Due to the expansion of the lungs, inspiration increased the water level within the neck of the funnel, while the animal floated upwards. Oppositely, expiration decreased the water level. These vertical movements of the water column were recorded as pressure changes, using a Niho Kohoen Polygraph System ŽJapan., consisting of a venous pressure transducer, an AP6216 carrier amplifier and the recorder. Calibration was obtained by injection and withdrawal
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of known volumes of water, which provided a direct relationship between pressure change at the bottom of the water column and the amount of water added or withdrawn from the system. Between periods of blood sampling, the catheter was connected to an arterial pressure transducer that formed part of the Niho Kohoen system. Cardiac frequency and blood pressure were recorded, using the polygraph system. Calibration of the pressure transducer was performed daily using a static water column of known elevation. The experimental chamber was continuously flushed either with air or with gas mixtures obtained by feeding pure gases to a GF-3rMP gas mixer. The total flow was set at 1 l miny1 and gases were supplied to the air space in the funnel andror to the water space containing the fish. Using this system, water P O 2 could be changed within 10᎐15 min. 2.5. Experimental protocols Experiments were performed as three separate series, as described below. Series 1 addressed the effects of repeated exposure to hypoxia; series 2 described the effects of aerial andror aquatic hypoxia; and series 3 determined the effects of reduced blood oxygen content during normoxia and hypoxia. 2.5.1. Series 1 The effects of repeated exposure to aerial hypoxia was determined in nine lungfish. Each unrestrained and un-operated lungfish was placed into the experimental chamber overnight and ventilation during normoxia was recorded the following day. When a stable ventilatory pattern had been recorded for a minimum of 30 min, hypoxia Žaerial P O 2 s 69, then 35 mmHg. was applied as described below, after which the animals were returned to normoxia. Following 2᎐3 h in normoxia, the hypoxic exposures were repeated. 2.5.2. Series 2 The ventilatory responses to aerial andror aquatic hypoxia were measured in eight unrestrained and un-operated lungfish. Each animal was placed in the experimental chamber the day before experimentation. The animal never struggled and a regular breathing pattern soon developed. After a normoxic control period, established by 2 h of vigorous aeration of the cham-
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ber, the water P O 2 was, in sequence, changed to 69, 35 and then, 208 mmHg, corresponding to 10, 5 and 30% O 2 , respectively, delivered from the gas flow meter. Meanwhile, the aerial phase was maintained normoxic. Secondly, after a normoxic recovery period, the same sequence of gas mixtures was delivered to the aerial phase, while the water was kept normoxic. Thirdly, the hypoxic sequences were applied simultaneously to the water and to the aerial phase. Each experimental condition was maintained for 1 h. Meanwhile, tidal volume and frequency were continuously recorded.
cardiorespiratory responses to hypoxia ŽSeries 3. were evaluated by two-way ANOVAs for repeated measures. The effect of aquatic and aerial hypoxia on ventilation in the uninstrumented animals ŽSeries 2. was evaluated by a one-way ANOVA for repeated measures. In all cases, means that were significantly different from the normoxic level were subsequently identified by a Student᎐Newman᎐Keuls post-hoc test. We applied a fiducial limit for significance of P- 0.05 and all data are presented as mean " 1 S.E.M.
3. Results 2.5.3. Series 3 Cannulated fish were placed in the experimental chamber in order to record heart rate and ventilation. After obtaining a blood sample during normoxia, hypoxia was applied as described above ŽSeries 1. and blood samples were obtained at the end of each exposure. The animals were returned to normoxia, and a volume of blood Ž2% of body weight. was removed to render the animal anaemic, down to a haematocrit of approximately 50% of normal. Blood volume was maintained by replacing the removed blood with an equal volume of lungfish Ringer. Ventilation and heart rate were measured under normoxic conditions, 2᎐3 h after the reduction in blood oxygen carrying capacity.
3.1. The effects of ambient hypoxia on un-operated and unrestrained animals The responses of un-operated and unrestrained animals to repeated exposure to aerial hypoxia ŽSeries 1. are illustrated in Table 1. The frequency of air breathes Ž f R . increased significantly during both sets of exposures. Coupled with an insignificant trend towards an increased tidal volume Ž VT ., this resulted in a highly significant, three-fold increase in ventilation volume Ž VI . at a PI O 2 of 38 mmHg, compared to the normoxic control value. There was no significant difference between the responses measured during the first and the second exposures to hypoxia. In a separate series of single exposures ŽSeries . 2 , illustrated in Fig. 1, aerial hypoxia, with or without aquatic hypoxia, caused a progressive increase in f R reaching a four-fold increase at a P O 2 of 35 mmHg, compared to the control, normoxic rate. As the amplitude of air breaths was
2.6. Statistics and data analysis The effects of repeated exposure to hypoxia ŽSeries 1., and the effects of reducing blood oxygen carrying capacity on blood gases and the
Table 1 Effects of repeated exposure to aerial hypoxia in unrestrained and un-operated lungfish First hypoxic exposure ŽPO 2 in mmHg.
VT Žml kgy1 . fR Žhy1 . VI Žml kgy1 hy1 .
Second hypoxic exposure run ŽPO 2 in mmHg.
150 Ž9.
72 Ž9.
38 Ž9.
150 Ž9.
72 Ž9.
38 Ž7.
26.5" 1.8 10.4" 1 272 " 32.5
28.3" 2.8 17.2" 3.5 483 " 88.3
34.7" 4.1 23.3" 5.6 791 " 194
31.2" 2.3 9.1" 1.8 259 " 61
29.3" 2.5 18.3" 4.3 566 " 133
33.5" 4.6 26.5" 5.7 834 " 174
Values are presented as mean q 1 S.E.M., and the number of animals is listed in brackets; there was no significant difference between the first and second set of exposures Žtwo-way ANOVA for repeated measures.. Abbre¨ iations: VT s tidal volume; f R s respiratory frequency; VI s inspired volume.
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in fish exposed to aerial hyperoxia in hyperoxic water that, when combined with a small decrease in VT , resulted in a significant reduction in mean VI to 60% of the control, normoxic value ŽFig. 1.. In this second series of experiments, the breathing frequencies were generally lower than those obtained in series 1 and 3. Because the animals studied in series 1 were un-operated, as in series 2, we are not able to identify a simple cause for this difference. Since series 2 was performed on a separate group of fish during the winter season, it is possible that seasonal effects may account for the lower breathing frequency. 3.2. Changes in blood gas ¨ alues during hypoxia and following induced anaemia The changes in blood gas values during a twostep reduction in ambient P O 2 are shown in Fig. 2. The oxygen partial pressure changed in direct proportion to ambient hypoxia and the other data are related to these values. Hypoxic exposure caused a progressive reduction in blood oxygen content ŽCaO 2 ., while haematocrit and pH were unaffected. The substitution of approximately half of the blood volume by Ringer resulted in a 50% reduction in normoxic haematocrit, a proportional decrease in blood oxygen content, but did not affect pH. 3.3. Ventilation and heart rate in relation to ¨ entral aortic PO2 during hypoxia and following induced anaemia Fig. 1. Changes in ventilation rate Ž f R ., tidal volume Ž VT . and ventilation volume Ž VI . of lungfish Ž Lepidosiren. exposed to two levels of hypoxia and to hyperoxia. The changes in P O 2 were imposed in the water alone Žopen circles., in the air space alone Žgrey circles., or in both media simultaneously Žsolid circles.. While aquatic hypoxia alone was without effect on ventilation, hypoxia imposed in the air space from which the fish breathed caused significant increases in f R and VT , with or without aquatic hypoxia. Mean values that are significantly different from the normoxic value are marked with an asterisk.
unaffected, this resulted in a proportional increase in VI . Aquatic hypoxia, with access to normoxic air, had no significant effect on the mean rate or amplitude of the air-breaths, so that ventilation volume was unaffected. Neither aquatic nor aerial hyperoxia alone, had any affect on the frequency or amplitude of air breaths. However, there was a 30% reduction in mean f R
The changes in ventilation and heart rate in relation to changes in ventral aortic P O 2 during progressive hypoxia and in response to anaemia, induced by a 50% reduction in haematocrit, are illustrated in Fig. 3. Hypoxia had no effect on heart rate or VT , but caused a significant increase in f R , with the result that VI was markedly increased to more than double its normoxic value. Anaemia induced a 10% increase in normoxic heart rate, but had no effect on the respiratory variables. 4. Discussion 4.1. Critique of blood sampling The blood samples were obtained through a cannula inserted into the fifth gill arch, from
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separation in the bulbus. Therefore, the blood gases measured in our study are likely to reflect a mixture of systemic venous blood and blood returning from the lungs. Thus, the ventral aortic blood gases reported here are unlikely to represent arterial systemic blood. Arterial blood is normally sampled for descriptions of cardiorespiratory response curves. However, the location of oxygen-sensitive chemoreceptors within the cardiovascular system are unknown in lungfish and the primary purposes of cannulation was to monitor the manipulation of blood oxygen carrying capacity and to establish whether vascular P O 2 levels were significantly affected. In this context, the collected blood samples answered the questions addressed. 4.2. Ventilatory pattern and responses to hypoxia in water and air
Fig. 2. Blood gas values in lungfish exposed to hypoxic gas mixtures in the air space of the experimental chamber and subsequently, to induced anaemia. Blood oxygen content was markedly reduced both by hypoxia and by anaemia. Hypoxia was without effect on haematocrit, but it was reduced by approximately 50% by anaemia. Neither imposed variable affected pH. Mean values that are significantly different from the normoxic value are marked with an asterisk.
where it was advanced into the bulbus. This cannulation is relatively non-invasive and the animals recovered quickly from the surgery. The blood perfusing the functional gill arches predominantly stems from the systemic venous circulation, and is preferentially distributed towards the pulmonary circulation Že.g. Johansen et al., 1968a; Lenfant and Johansen, 1968; Fishman et al., 1985, 1989.. In addition, it is uncertain whether the insertion of the cannulae interfered with the blood flow
During normoxia, and when undisturbed, Lepidosiren remained quietly submerged and surfaced at regular intervals to breathe. Each breath consisted of a single prolonged and continuous expiration that was followed immediately by a bout of consecutive small inspirations Žbetween 5 and 14, with a mean of approx. 9 inspirations.. The same breathing pattern was recently observed in caecilian amphibians ŽGardner et al., 2000. and resembles the inflation bouts of anuran amphibians ŽKruhøffer et al., 1987.. From a functional point of view the repeated inspirations reduce the functional dead space. In the African lungfish, Protopterus, the inspirations are caused by a buccal force pump mechanism ŽMcMahon, 1969; Delaney and Fishman, 1977. and visual inspection during the present study suggests that this is also the case for Lepidosiren. During hypoxia, the ventilatory pattern continued to consist of single breaths and the increased overall lung ventilation was accomplished by a reduction of the breathhold periods, whereas tidal volume only increased very slightly. This points to an important role of pulmonary stretch receptor feedback exerting the Hering᎐Breuer reflex, where inflation reflexly terminates inspiration. Protopterus possesses pulmonary stretch receptors ŽDelaney et al., 1983., and manipulation of lung volume alters the duration of the non-ventilatory periods ŽPack et al., 1992.. Stretch receptors in the air-breathing organs are also important in determining tidal volume and breathing pattern in other air breath-
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Fig. 3. The effect of hypoxia and anaemia on ventilation and heart rate in lungfish, plotted against mean levels of oxygen partial pressure in the ventral aorta Ž P VA O 2 .. While tidal volume Ž VT . was unaffected, ventilation rate Ž f R . increased during hypoxia, causing a proportional increase in ventilation volume Ž VI .. Heart rate was unaffected by hypoxia, but increased by 10% in anaemic fish.
ing fish such as Amia and Lepisosteus ŽJohansen et al., 1970; Smatresk and Cameron, 1982; Hedrick and Jones, 1999.. As pointed out by Hedrick and Jones Ž1993, 1999., the control of tidal volume is particularly important for aquatic animals where the degree of lung inflation significantly affects buoyancy. In most species of teleost fish, gill ventilation increases markedly during aquatic hypoxia, due to stimulation of receptors that are located principally on the first gill arch ŽDaxboeck and Holeton, 1978; Milsom and Brill, 1986.. Some of these receptors are located externally and screen the inspired water, while others are internal and monitor blood O 2 levels ŽMilsom and Brill, 1986; Smatresk et al., 1986; Burleson and Smatresk, 1990; Burleson et al., 1992.. As a consequence, ventilatory responses to hypoxic water may occur without any changes in blood O 2 levels ŽGlass et al., 1990; Soncini and Glass, 2000.. Pulmonary ventilation in Lepidosiren did not increase in response to aquatic hypoxia and it does not appear,
therefore, that branchial O 2 sensitive receptors are involved in the control of pulmonary ventilation in this animal. The existence of external receptors cannot, however, be excluded, since addition of nicotine or cyanide into the opercular cavity stimulates both branchial and pulmonary ventilation in Protopterus ŽJohansen et al., 1968a; Lahiri et al., 1970.. The inability of aquatic hypoxia to stimulate lung ventilation is consistent with responses of Neoceratodus and Protopterus, the air-breathing teleosts Channa argus and Electrophorus electricus, and the aquatic caecilian Typhlonectus natans ŽJohansen et al., 1967, 1968b; Johansen and Lenfant, 1968; Glass et al., 1986; Gardner et al., 2000.. However, in Amia, Ancistris, Hypostomus and Lepisosteus, stimulation of external oxygen sensitive receptors stimulate breathing ŽJohansen et al., 1970; Graham and Baird, 1982; Smatresk et al., 1986; Hedrick and Jones, 1993.. The ventilatory response to aerial hypoxia concords with the effects observed in the other species
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of lungfish Že.g. Jesse et al., 1967; Johansen et al., 1967; Johansen and Lenfant, 1968; Fritsche et al., 1993.. These responses must be attributed to internal oxygen sensitive receptors, most likely sensing blood P O 2 , but the specific location of these remain unknown ŽFishman et al., 1989.. It is not surprising that Lepidosiren resembles the other genera of lungfishes in this context, as the members of the Lepidosirenidae are obligate air breathers, while the other genera are more or less facultative air-breathers. According to Johansen et al. Ž1976., adult specimens of Protopterus obtain 70% of total O 2 uptake by pulmonary ventilation. Because the gills of Lepidosiren are considerably reduced so that the filaments are nearly absent on the first two gill arches ŽJohansen and Lenfant, 1967; Lenfant et al., 1970., it would be expected that branchial gas exchange is lower than in any other species of lungfish. However, Abe and Steffensen Ž1996. showed that almost 40% of the oxygen uptake of quietly resting Lepidosiren occur over the skin and gills. Pulmonary ventilation decreased during exposure to hyperoxia in Lepidosiren ŽFig. 1., showing that oxygen provides a tonic drive to ventilation in normoxia. Similar effects have been observed in Protopterus ŽJesse et al., 1967., other airbreathing fish, amphibians and reptiles Že.g. Lomholt and Johansen, 1974; Heisler, 1982; Hughes and Singh, 1971; Smatresk et al., 1986; McKenzie et al., 1992.. Gill ventilation in teleosts is also reduced during hyperoxia. 4.3. Heart rate during hypoxia In Lepidosiren and Protopterus, earlier studies have shown an increased heart rate during the intermittent breathing episodes ŽAxelsson et al., 1989; Burggren and Johansen, 1986. and Fritsche et al. Ž1993. also recorded increased pulmonary blood flow associated with lung ventilation in Neoceratodus. In our study there were no apparent changes in heart rate during ventilation. The heart rates recorded in the present study are in general agreement with previous studies on Lepidosiren ŽAxelsson et al., 1989., but as pointed out by these authors, these heart rates appear relatively high. In the study by Axelsson et al. Ž1989., the respiratory-related tachycardia did occur in animals with low heart rate, possibly due to a
larger vagal tone on the heart that can be released during ventilation. There was no significant change in heart rate during hypoxia in Lepidosiren ŽFig. 3.. This in agreement with earlier observations on Neoceratodus, although this species does increase pulmonary blood flow, possibly due to redistribution of blood, during progressive aquatic hypoxia ŽFritsche et al., 1993.. A reflex tachycardia in response to a reduced oxygen supply is characteristic of committed lung breathers such as mammals that show the so-called ‘secondary response’ to hypoxia following stimulation of lung stretch receptors during hypoxic hyperventilation ŽDaly, 1997.. In this respect, Lepidosiren shows physiological responses that are typical for lung breathers possessing pulmonary mechanoreceptors ŽDelaney et al., 1983.. However, because heart rate remained elevated during the breath hold periods in the anaemic animals, stimulation of pulmonary stretch receptors is not obligatory for the hypoxaemic tachycardia. Lepidosiren did show a significant tachycardia in normoxic hypoxaemia. The cardiac responses of lungfish are diametrically opposed to that of typical water-breathing fish, where environmental hypoxia normally induces a reflex bradycardia ŽTaylor, 1992.. This response is predominantly in response to stimulation of externally located branchial oxygen receptors sensitive to water P O 2 ŽBurleson et al., 1992.. Thus, the absence of a hypoxic bradycardia is in agreement with the lack of evidence for external receptors on the gills of Lepidosiren. 4.4. Modality of the oxygen response and the effects of reducing haematocrit The specific oxygen stimulus that elicits ventilatory responses in vertebrates is a matter of contention Že.g. Boggs, 1995., but most studies on air-breathing vertebrates point to P O 2 being the driving stimulus Že.g. Lahiri et al., 1981; Wang et al., 1994, 1997; Boggs, 1995; McKenzie et al., 1991.. The responses of Lepidosiren are consistent with this view, as the large reduction in haematocrit and ventral aortic oxygen content during normoxia did not augment ventilation above the normoxic values previously attained ŽFigs. 2 and 3.. The fact that the lungfish studied in series 1 maintained a brisk hypoxic ventilatory response during the second hypoxic exposure
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shows that they are still able to respond to reduction in partial pressure. Thus, from our data it appears that the chemoreceptors responsible for the hypoxic ventilatory response are sensitive to P O 2 and that the lungfish resembles other airbreathing vertebrates, in this respect. Again, the hypoxic responses of Lepidosiren are opposed to typical, water-breathing fish, where systemic hypoxaemia, even in the absence of an associated hypoxia Že.g. anaemia. appears to stimulate ventilation ŽRandall, 1982; Smith and Jones, 1982.. Heart rate increased following the reduction in haematocrit in Lepidosiren ŽFig. 3.. A tachycardia in response to lowered blood oxygen content, in the absence or presence of ventilatory changes, appears to be a general response of water and air-breathing vertebrates Že.g. Wood and Shelton, 1980; Boggs, 1995; Wang et al., 1994, 1997.. In mammals, this response may arise from stimulation of chemoreceptors in the aortic arch ŽDaly, 1997. and it seems that other vertebrates also possess oxygen sensitive chemoreceptors that primarily exert a cardiovascular control. However, in studies that involve withdrawal of blood, it is difficult to dismiss the possibility that the heart rate changes are due to regulation of blood pressure rather than stimulation of oxygen-sensitive chemoreceptors per se. 4.5. Perspecti¨ es and conclusions These and previous studies demonstrate that the location with respect to the oxygen cascade and the functional characteristics of oxygen sensitive receptors controlling the heart and ventilation, plus the stimulus modality of these receptors, are similar in lungfish and tetrapods. Thus, many of the changes in the mechanisms exerting reflex control over cardio-respiratory functions, accompanying the transition between water and air breathing, may have occurred at an early stage in vertebrate evolution.
Acknowledgements This study was supported by FAPESP ŽProc. no. 1998r06731-5 . and CNPq wProc. no. 300603r91-6ŽRN.x and The Danish Research Council ŽSNF.. We gratefully acknowledge technical help from Humberto Giusti.
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References Abe, A.S., Steffensen, J.F., 1996. Lung and cutaneous respiration in awake and estivating South American lungfish, Lepidosiren paradoxa. Rev. Bras. Biol. 56, 485᎐489. Axelsson, M., Abe, A.S., Bicudo, J.E.P.W., Nilsson, S., 1989. On the cardiac control in the South American lungfish, Lepidosiren paradoxa. Comp. Biochem. Physiol. 93A, 561᎐565. Boggs, D.F., 1995. Hypoxic ventilatory control and hemoglobin oxygen affinity. In: Sutton, J.R., Houston, C.S., Coates, G. ŽEds.., Hypoxia and the Brain. Queen City Printers, Burlington, Vermont, pp. 69᎐86. Burggren, W.W., Johansen, K., 1986. Circulation and respiration in lungfishes ŽDipnoi.. J. Morph. 1, 217᎐236. Burleson, M.L., Smatresk, N.J., 1990. Evidence for two oxygen-sensitive chemoreceptor loci in channel catfish, Ictalurus punctatus. Physiol. Zool. 63, 208᎐221. Burleson, M.L., Smatresk, N.J., Milsom, W.K., 1992. Afferent inputs associated with cardioventilatory control in fish. In: Hoar, W.S., Randall, D.J., Farrell, A.P. ŽEds.., Fish Physiology, vol. 12B, The Cardiovascular System. Academic Press, New York, NY, pp. 390᎐426. Daly, M.B., 1997. Peripheral Arterial Chemoreceptors and Respiratory᎐Cardiovascular Regulation. Oxford Medical Publications, Oxford 739 pp. Daxboeck, C., Holeton, G.F., 1978. Oxygen receptors in the rainbow trout, Salmo gairdneri. Can. J. Zool. 56, 1254᎐1259. Delaney, R.G., Fishman, A.P., 1977. Analysis of lung ventilation in the aestivating lungfish Protopterus aethiopicus. Am. J. Physiol. 233, R181᎐R187. 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. 13, R418᎐R428. Fishman, A.P., Delaney, R.G., Laurent, P., 1985. Circulatory adaptation to bimodal respiration in the dipnoan lungfish. J. Appl. Physiol. 59, 285᎐294. Fishman, A.P., Galante, R.J., Pack, A.I., 1989. Diving Physiology. Lungfish. In: Wood, S.C. ŽEd.., Comparative Pulmonary Physiology: Current Concepts, vol. 39, Lung Biology in Health and Disease. Marcel Dekker, New York. 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. Gardner, M.N., Smits, A.W., Smatresk, N.J., 2000. The ventilatory responses of the caecilian Typlonectus natans to hypoxia and hypercapnia. Physiol. Biochem. Zool. 73, 23᎐29.
686
A. Sanchez et al. r Comparati¨ e Biochemistry and Physiology Part A 130 (2001) 677᎐687
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. 156, 425᎐430. Glass, M.L., Andersen, N.A., Kruhøffer, M., Williams, E.M., Heisler, N., 1990. Combined effects of environmental P O 2 and temperature on ventilation and blood gases in the carp, Cyprinus carpio L. J. Exp. Biol. 148, 1᎐17. Graham, J.B., 1997. Air-Breathing Fishes: Evolution Diversity and Adaptation. Academic Press, San Diego. Graham, J.B., Baird, T., 1982. The transition to air breathing in fishes. I. Environmental effects on the facultative air breathing of Ancistrus chagresi and Hypostomus plecostomus ŽLoricariidae.. J. Exp. Biol. 96, 53᎐67. Hedrick, M.S., Jones, D.R., 1993. The effects of altered aquatic and aerial respiratory gas concentrations on air-breathing patterns in a primitive fish Ž Amia cal¨ a.. J. Exp. Biol. 181, 81᎐94. Hedrick, M.S., Jones, D.R., 1999. Control of gill ventilation and air-breathing in the bowfin Amia cal¨ a. J. Exp. Biol. 202, 87᎐94. Hedrick, M.S., Burleson, M.L., Jones, D.R., Milsom, W.K., 1991. An examination of central chemosensitivity in an air-breathing fish Ž Amia cal¨ a.. J. Exp. Biol. 155, 165᎐174. Heisler, N., 1982. Intracellular and extracellular acid᎐base regulation in the tropical freshwater teleost fish Synbranchus marmoratus in response to the transition from water breathing to air breathing. J. Exp. Biol. 99, 9᎐28. Hughes, G.M., Singh, B.N., 1971. Gas exchange with air and water in an air-breathing catfish, Saccobranchus (Heteropneustes) fossilis. J. Exp. Biol. 55, 667᎐682. Jesse, M.J., Shub, C., Fishman, A.P., 1967. Lung and gill ventilation of the African lungfish. Respir. Physiol. 3, 267᎐287. Johansen, J., Lenfant, C., 1967. Respiratory function in the South American lungfish, Lepidosiren paradoxa ŽFitz.. J. Exp. Biol. 46, 205᎐218. Johansen, K., 1971. Comparative physiology: gas exchange and circulation in fishes. Ann. Rev. Physiol. 33, 569᎐612. Johansen, K., Lenfant, C., Grigg, G.C., 1967. Respiratory control in the lungfish, Neoceratodus forsteri ŽKrefft.. Comp. Biochem. Physiol. 20, 835᎐854. Johansen, K., Lenfant, C., 1968. Respiration in the African lungfish, Protopterus aethiopicus. II. Control of breathing. J. Exp. Biol. 49, 453᎐468. Johansen, K., Lenfant, C., Hanson, D., 1968a. Cardiovascular dynamics in the lungfishes. Z. Vergl. Physiol. 59, 157᎐186. Johansen, K., Lenfant, C., Schmidt-Nielsen, K., Petersen, J., 1968b. Gas exchange and control of
breathing in the electric eel, Electrophorus electricus. Z. Vergl. Physiol. 61, 137᎐163. Johansen, K., Hanson, D., Lenfant, C., 1970. Respiration in a primitive air breather, Amia cal¨ a. Respir. Physiol. 9, 162᎐174. Johansen, K., Lomholt, J.P., Maloiy, G.M.O., 1976. Importance of air and water breathing in relation to size of the African lungfish Protopterus amphibius Peters. J. Exp. Biol. 65, 395᎐399. 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. Lahiri, S., Szidon, J.P., Fishman, A.P., 1970. Potential respiratory and circulatory adjustments to hypoxia in the African lungfish. Ann. Rev. Physiol. 29, 1141᎐1148. Lahiri, S., Mulligan, E., Nishino, T., Mokashi, A., Davies, R.O., 1981. Relative responses of aortic body and carotid body chemoreceptors to carboxyhemoglobinemia. J. Appl. Physiol. 50, 580᎐586. Lenfant, C., Johansen, K., 1968. Respiration in the African lungfish Protopterus aethiopicus. I. Respiratory properties of blood and normal patterns of breathing and gas exchange. J. Exp. Biol. 49, 437᎐452. 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., Johansen, K., 1974. Control of breathing in Amphipnous cuchia, an amphibious fish. Respir. Physiol. 21, 325᎐340. McKenzie, D.J., Aota, S., Randall, D.J., 1991. Ventilatory and cardiovascular responses to blood pH, plasma P CO 2 , blood O 2 content, and catecholamines in an air-breathing fish, the bowfin Ž Amia cal¨ a.. Physiol. Zool. 64, 432᎐450. McMahon, B.R., 1969. A functional analysis of the aquatic and aerial respiratory movements of an African lungfish, Protopterus aethiopicus, with reference to the evolution of the lung-ventilation mechanism in vertebrates. J. Exp. Biol. 51, 407᎐430. Milsom, W.K., 1995. The role of CO 2rpH chemoreceptors in ventilatory control. Braz. J. Med. Biol. Res. 28, 1147᎐1160. Milsom, W.K., Brill, R.W., 1986. Oxygen sensitive afferent information arising from the first gill arch of yellowfin tuna. Respir. Physiol. 66, 193᎐203. Pack, A.I., Galante, R.J., Fishman, A.P., 1992. Role of lung inflation in control of air breath duration in African lungfish Ž Protopterus annectens.. Am. J. Physiol. 31, R879᎐R884. Randall, D.J., 1982. The control of respiration and circulation in fish during exercise and hypoxia. J. Exp. Biol. 100, 275᎐288. Romer, A.S., 1970. The Vertebrate Body, 4th ed. Saunders, Philadelphia.
A. Sanchez et al. r Comparati¨ e Biochemistry and Physiology Part A 130 (2001) 677᎐687
Sanchez, A., Glass, M.L., 2001. Effects of environmental hypercapnia on pulmonary ventilation of the Southern American lungfish, Lepidosiren paradoxa ŽFitz... J. Fish Biol. 58, 1181᎐1189. Siggaard-Andersen, O., 1976. The Acid᎐Base Status of the Blood, 4th ed. Munksgaard, Copenhagen. Smatresk, N.J., 1994. Respiratory control in the transition from water to air breathing in vertebrates. Am. Zool. 34, 264᎐279. Smatresk, N.J., Cameron, J.N., 1982. Respiration and acid᎐base physiology of the spotted gar, a bimodal breather. III. Response to a transfer from freshwater to 50% sea water, and control of ventilation. J. Exp. Biol. 96, 295᎐306. Smatresk, N.J., Burleson, M.L., Azizi, S.Q., 1986. Chemoreflexive responses to hypoxia and NaCN in longnose gar, evidence for two chemoreceptor loci. Am. J. Physiol. 20, R116᎐R125. Smith, F.M., Jones, D.R., 1982. The effect of changes in blood oxygen-carrying capacity on ventilation volume in the rainbow trout Ž Salmo gairdneri.. J. Exp. Biol. 97, 325᎐334.
687
Soncini, R., Glass, M.L., 2000. Oxygen and acid᎐base status-related drives to gill ventilation in carp. J. Fish Biol. 56, 528᎐541. Taylor, E.W., 1992. Nervous control of the heart and cardiorespiratory interactions. In: Hoar, W.S., Randall, D.J., Farrell, A.P. ŽEds.., Fish Physiology, vol. 12B, The Cardiovascular System. Academic Press, New York, NY, pp. 343᎐387. Tucker, V.A., 1967. Method for oxygen content and dissociation curves on microliter blood samples. J. Appl. Physiol. 23, 410᎐414. Wang, T., Branco, L.G.S., Glass, M.L., 1994. Ventilatory responses to hypoxia in the toad Bufo paracnemis before and after a decrease in haemoglobin oxygen-carrying capacity. J. Exp. Biol. 186, 1᎐8. Wang, T., Krosniunas, E., Hicks, J.W., 1997. The role of cardiac shunts in the regulation of arterial blood gases. Am. Zool. 37, 12᎐22. Wood, C.M., Shelton, G., 1980. The reflex control of heart rate and cardiac output in the rainbow trout: interactive influences of hypoxia, haemorrhage, and systemic vasomotor tone. J. Exp. Biol. 87, 271᎐284.