- Email: [email protected]
Aestivation in the South American lungfish, Lepidosiren paradoxa: Effects on cardiovascular function, blood gases, osmolality and leptin levels
Respiratory Physiology & Neurobiology 164 (2008) 380–385
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Aestivation in the South American lungfish, Lepidosiren paradoxa: Effects on cardiovascular function, blood gases, osmolality and leptin levels Glauber dos Santos Ferreira da Silva, Humberto Giusti, Adriana Paula Sanchez, Jussara Márcia do Carmo, Mogens Lesner Glass ∗ Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Avenida Bandeirantes 3.900, 14.049-900 SP, Brazil
a r t i c l e
i n f o
Article history: Accepted 29 August 2008 Keywords: Aestivation South American lungfish Osmolality Lepidosiren Blood gases Acid-base status
a b s t r a c t The African (Protopterus sp.) and South American lungfish (Lepidosiren paradoxa) inhabit shallow waters, that seasonally dry out, which induces aestivation and cocoon formation in Protopterus. Differently, L. paradoxa has no cocoon, and it aestivates in a simple burrow. In water PaCO2 is 21.8 ± 0.4 mmHg (mean values ± S.E.M.; n = 5), whereas aestivation for 20 days increased PaCO2 to as much as 37.6 ± 2.1 mmHg, which remained the same after 40 days (35.8 ± 3.3 mmHg). Concomitantly, the plasma [HCO3 − ]-values for animals in water were 22.5 ± 0.5 mM, which after 20 days increased to 40.2 ± 2.3 mM and after 40 days to 35.8 ± 3.3 mM. Initially in water, PaO2 was 87.7 ± 2.0 mmHg, but 20 days in aestivation reduced the value to 80.5 ± 2.2 and later (40 days) to 77.1 ± 3.0 mmHg. Meanwhile, aestivation had no effect on pHa and hematocrit. The blood pressures were equal for animals in the water or in the burrow (Pmean ∼ 30 mmHg), and cardiac frequency (fH ) fell from 31 beats min−1 to 22 beats min−1 during 40 days of aestivation. The osmolality (mOsm kg H2 O−1 ) was elevated after 20 and 40 days of aestivation but declined upon return to water. The transition from activity to aestivation involves new set-points for the variables that determine the acid–base status and PaO2 of the animals, along with a reduction of cardiac frequency. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In animals, aestivation is a temporary period of dormancy, which down-regulates O2 -uptake, pulmonary ventilation and cardiovascular activity, and this usually reflects adverse ambient conditions. The roman word “aestas” means summer, but aestivation is not necessarily linked to a specific part of the year. Aestivation occurs in lepidosirenid lungfish and some amphibians and reptiles, and this condition may occur without any change of ambient temperature (Abe, 1995; Abe and Steffensen, 1996a,b). In aestivating animals, the O2 -uptake usually is 20–55% of resting values for the awaken animal (Glass et al., 1997; DeLaney et al., 1974). The South American lungfish, Lepidosiren paradoxa, inhabits swamps that dry out on a seasonal basis within the Pantanal region (Mato Grosso, Brazil), which coincides with the winter in the middle of the year. Information on aestivation in L. paradoxa is highly limited, except for Harder et al. (1999), who reported on cardiac frequencies during formation of a burrow. When a lake dries out, L. paradoxa
∗ Corresponding author. Tel.: +55 16 36023202; fax: +55 16 36330017. E-mail address: mlglass@rfi.fmrp.usp.br (M.L. Glass). 1569-9048/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2008.08.009
digs a hole into the mud and clay, which allows movements and change of position. For the animals in water, these authors reported a cardiac frequency (fH ) of 31 beats min−1 which became downregulated by 46% during aestivation. As a major difference, Protopterus secretes a protective mucous from the skin to form a hardened cocoon (DeLaney et al., 1974). Much less is known about aestivation in L. paradoxa, in particular concerning blood gases and osmolality. Abe and Steffensen (1996a,b) reported that VO2 was reduced by no more than 30% after 4 months of aestivation at 25 ◦ C. Considerably more is known about aestivation in the African lungfish (P. aethiopicus). When the water dries out, this lungfish forms a burrow and secretes a gradually hardening mucous to form a cocoon, which is open at the top, where the animal respires. Aestivation in P. aethiopicus has spectacular consequences, including an increase from PaCO2 from 26 mmHg to ∼50 mmHg upon transition from water to a cocooned condition (DeLaney et al., 1977), and they reported that pHa remained within a rather narrow range, due to large increases of plasma [HCO3 − ] levels. Aestivation can persist for amazingly long periods such as 9 months in P. aethiopicus (DeLaney et al., 1974, 1977). A P. amphibius survived for 7 years in a cocoon, and a second animal with 6 years in the same condition reduced its VO2 from 0.840 (in water) to
G.d.S.F. da Silva et al. / Respiratory Physiology & Neurobiology 164 (2008) 380–385
0.039 mL kg−1 min−1 . In the latter animal, the end-expired PaCO2 was 40 mmHg (Lomholt, 1993). Recently, lungfish have received considerable interest, because Dipnoi represents the most likely sister group relative to the land vertebrates (Tetrapoda). The physiology of Protopterus sp. during activity and/or aestivation is, however, known in considerable detail (cf. DeLaney et al., 1974, 1977; Chew et al., 2003, 2004; Loong et al., 2005). When its lake dries out, the African lungfish secretes a mucous that gradually hardens to protect the animal (DeLaney et al., 1977). A straight forward hypothesis can explain blood gas values in L. paradoxa at 25 ◦ C. In this lungfish, 50% of total CO2 -output was eliminated to aerated water, while pulmonary ventilation eliminated the other half (Amin-Naves et al., 2004; Bassi et al., 2005). Aestivation in a burrow would favour CO2 -elimination by pulmonary ventilation. This would occur, because water was replaced by the limited space within a burrow. In this context Dejours (1981) stated that: “Compared to water breathers, air breathers are in a state of compensated hypercapnic acidosis”. A very high PaCO2 is expected for Protopterus sp., because the animal aestivates within a gastight cocoon. In this case, virtually the whole CO2 -output would be expired by pulmonary ventilation (Lomholt, 1993). Aestivation in lungfish is characterized by very high PaCO2 -values that can be maintained over long periods (DeLaney et al., 1974, 1977), which involves a proportional regulation of lung ventilation and CO2 -output. The effects of blood osmolality were also measured, and will be discussed in relation to activity or aestivation. In addition, plasmatic leptin levels were measured to evaluate possible metabolic effects of this hormone. The inclusion of leptin levels was motivated by the recent discovery, that low leptin levels can reduce pulmonary ventilation in mammals (O’Donnell et al., 1999, 2000). Furthermore, we also addressed down-regulation of cardiovascular function in relation to activity or aestivation (DeLaney et al., 1977; Glass et al., 1997). Our aim is to compare aestivation in Protopterus and L. paradoxa and evaluate the consequences of the cocooned state and the effects of a simple burrow. 2. Materials and methods Specimens of L. paradoxa (n = 20) were collected close to the city Cuiabá, Mato Grosso State, and then transported to the Central Animal Holding Facility of University of São Paulo, Ribeirão Preto, São Paulo State, Brazil, and were kept in 1000 L tanks at a water level of 1 m and at 25 ◦ C ± 1 ◦ C at all times (Fraéllio Electric Equipments, Ribeirão Preto, São Paulo State). All measurements were performed at that temperature. Food (snails and chicken liver) was provided outside the period of aestivation. Four groups were established with n = 5: (1) Control group of freely swimming animals, placed in normoxic and normocarbic water in April. W = 746 ± 23 g; (2) after 20 days of aestivation in July. W = 404 ± 15 g; (3) after 40 days of aestivation in July–August. W = 471 ± 19 g; (4) Control group of freely swimming animals during normoxic and normocarbic water in September. W = 509 ± 26 g; weight losses during aestivation ranged from 9 to 11% of pre-aestivation values. Collection of the animals was approved by IBAMA (Proc. IBAMA/MMA-No. 02027.002172/2005-68; IBAMA = Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis) and by the Ethics Committee of University of São Paulo, Faculty of Medicine of Ribeirão Preto (Proc. #076/2005).
381
buccal cavity with benzocain (0.25 g L−1 ). The dorsal aorta was dissected free at a point 1 cm behind the caudal part of the lung. Then, a PE50 catheter was inserted into the vessel and fixed in place by sutures after which the incisions were closed, finally the catheter was flushed with heparinized Ringer solution (100 IU mL−1 ). The animals recovered movements after 10 min in aerated water. 2.2. Blood gas measurements Arterial blood samples (1 mL) were immediately analyzed after withdrawal, using an electrode (FAC 001 O2 ), connected to a 204 O2 analyzer (FAC Instr., São Carlos, SP, Brazil). This electrode was calibrated with N2 (zero) and with air-equilibrated water (PO2 = 145 mmHg—elevation 710 m; 25 ◦ C). A microelectrode for blood gas measurements (Mettler Toledo, Switzerland) was used after calibration with high precision buffer solutions (Queel, São Paulo, SP, Brazil). A BGM 200 Cameron Analyzer (Cameron, Port Aransas, TX, USA) was equipped with CO2 -electrodes and calibrated with CO2 mixtures provided by a Cameron GF3/MP gas flow meter. Total plasma CO2 was measured by means of a Capni-Con 5 Analyzer (Cameron), calibrated with standard [HCO3 − ] solutions. To obtain haematocrit, microtubes were filled with blood and centrifuged using a Hermle z.200 M/H at 7500 rpm during 1 min. To measure blood pressures and cardiac frequency (fH ), the implanted catheter (P50) was connected to an arterial pressure transducer (Statham PB23 GB, Palo Alto, USA). The signals were fed into a multipurpose amplifier at a sampling rate of 1000 Hz (Spectramed, Statham P10 Ez, Palo Alto, USA) and transmitted to a computer. Advanced CODAS (Dataq Instr., Akron, OH, USA) was applied to analyze the cardiovascular data, and the rate-pressure product (RPP) was calculated as fH ·Psyst (mmHg min−1 ) as a measure of myocardial activity. 2.3. Measurements of osmolality and leptin levels The plasmatic osmolality (mOsm kg H2 O−1 ) was measured using an osmometer (OS Mark 3, Fiske Associates, Needham Heights, MA, USA). Plasmatic leptin levels were measured by radioimmunoassay, using mice polyclonal leptin antibodies (Lincoˇıs Multi-Species Leptin Radioimmunoassay Kit) as described by Paolucci et al. (2001). About 0.4 mL blood was withdrawn from the animal and placed into a centrifuge (Hermle z200 M/H, Germany) during 10 min. Our Multi-Species Kit for leptin measurements was insufficient for covering the whole period of aestivation. 2.4. Experimental protocol The animals were divided in 4 separated groups, each with 5 animals: (1) Measurements of blood gases, cardiac frequency and blood pressures were obtained for active animals in water. (2) Aestivation was induced by a gradual reduction of water levels in a 50 cm deep mixture of mud and clay, and the digging out of the burrow would usually not take more than 45 min. The animal was then left in the burrow for 18 days, after which the arterial catheter was quickly implanted. After recovery, the animal spontaneously re-entered the burrow to complete 20 days of aestivation. Group (3) procedures were described above, but the aestivation was extended to 40 days. Group (4) second control group of active animals in water. 2.5. Statistics and calculations
2.1. Implantation of catheters Immersion into a solution of benzocain (1 g L−1 ) anaesthetized the animal within 10 min and was maintained by flushing the
The rate-pressure product (RPP) was calculated as: Psyst ·fH (mmHg min−1 ). ANOVA was followed by Bonferroni’s multiple comparison post-test to compare values for 4 independent groups:
382
G.d.S.F. da Silva et al. / Respiratory Physiology & Neurobiology 164 (2008) 380–385
Fig. 1. The numbers corresponds to the columns: (1) Animals were kept in aerated water for two weeks and PaCO2 was 21.8 ± 0.4 mmHg. (2) Aestivation for 20 days increased PaCO2 to 37.6 ± 2 mmHg. (3) Aestivation for 40 days resulted in a PaCO2 of 34.4 ± 3 mmHg. (4) Control measurements in well-aerated water resulted in a PaCO2 of 20.8 ± 1 mmHg. Mean ± S.E., N = 5; P < 0.05. (*) Indicates a difference between the animal in water and during aestivation.
(1) Control values for the animal in water; (2) 20 days; (3) 40 days of aestivation; (4) Control animals in water. Mean ± S.E.M.; n = 5 (P > 0.05). The total number of individuals was 20 since 4 groups were formed. 3. Results As shown in Fig. 1, PaCO2 was low 21.8 ± 0.4 mmHg when in water, whereas 20 days of aestivation increased PaCO2 to 37.6 ± 2.1 (73%), and after 40 days the value was slightly reduced to 34.4 ± 3.2. After aestivation a second control for the fish in water (=Group 4) was PaCO2 = 20.8 ± 1.2 mmHg. The animal in water had a plasma [HCO3 − ] of 22.5 ± 0.5 mM, but aestivation increased plasma [HCO3 − ] to 40.2 ± 2.3 mM (74%) in Group 2 (20 days of aestivation). Group 3 (40 days of aestivation) resulted in 35.8 ± 3.3 mM and, Group 4 (fish in water) reduced [HCO3 − ] to 23.0 ± 0.4 mM (Fig. 2). Group 1 (in water) had a PaO2 of 87.7 ± 2.0 mmHg, while 20 days of aestivation reduced PaO2 to 80.5 ± 2.2 mmHg which, after 40 days of aestivation, was cut further down to 77.1 ± 3.0. Group 4
Fig. 2. (1) In aerated water [HCO3 − ] was 22.5 ± 0.5 mM. (2) Aestivation during 20 days elevated [HCO3 − ] to 40.2 ± 2 mM. (3) 40 days of aestivation resulted in a plasma [HCO3 − ] of 35.8 ± 3 mM. (4) In water, plasma [HCO3 − ] was 23 ± 0.4 mM. Statistics and symbols as above.
Fig. 3. (1) In water, PaO2 was 87.7 ± 2 mmHg. (2) Aestivation reduced PaO2 to 80.5 ± 2 mmHg (20 days). (3) 40 days of aestivation further reduced PaO2 to 77 ± 3 mmHg. (4) Return to water increased PaO2 to 85.7 ± 2 mmHg. Statistics and symbols as above. Table 1 Blood pressures, pHa, leptin levels and hematocrit Before aestivation Psyst (mmHg) Pdiast (mmHg) Pmean (mmHg) pHa Leptin (ng/mL) Hct (%)
30.8 25.3 27.3 7.51 1.43 28
± ± ± ± ± ±
0.5 0.4 0.3 0.05 0.14 1
20 days of aestivation 30.4 26 27.8 7.49 1.53 25
± ± ± ± ± ±
0.8 0.5 0.7 0.02 0.08 2
40 days of aestivation
After aestivation
35.2 ± 27.1 ± 31.8 ± 7.53 ± – 26 ±
31.2 ± 25 ± 28 ± 7.49 ± – 27 ±
0.6 0.5 0.7 0.05 1
0.7 0.4 0.3 0.03 1
This table represents the variables that were not affected by aestivation. Values are expressed as mean values ± S.E.M.; n = 5. The values were obtained from separate groups.
(animal in water) had a PaO2 of 85.7 ± 1.8, which was identical to the values of Group 1 (Fig. 3). Blood pressures were the same during aestivation and in water (see Table 1). By contrast, the cardiac frequency (fH ) of the animals in water was fH = 30.8 ± 0.5 beats min−1 , which initially reduced fH slightly to 28.0 ± 0.5 (20 days of aestivation) and then to 22.0 ± 1.0 (40 days of aestivation; Fig. 4). This down-regulation can
Fig. 4. The effects of aestivation on osmolality (mOsm kg H2 O−1 ) were pronounced. (1) In water, the osmolality of the animal was 232 ± 3. (2) Aestivation for 20 days increased this to 261 ± 5. (3) Aestivation for 40 days increased osmolality to 267 ± 3. (4) The second control in water was 231 ± 3. Statistics and symbols as above.
G.d.S.F. da Silva et al. / Respiratory Physiology & Neurobiology 164 (2008) 380–385
Fig. 5. (1) When in water fH was 31 ± 1. (2) 20 days of aestivation slightly decreased fH to 28 ± 0.5 beats min−1 . (3) 40 days of aestivation reduced fH to 22 ± 1 beats min−1 . (4) Animals in water increased heart rate to 32 ± 1 beats min−1 . Symbols as above. In addition, the symbol (#) indicates a difference between 20 and 40 days aestivation.
Fig. 6. (1) For animals in water the rate-pressure product (RPP, mmHg min−1 ) was 968 ± 32. (2) 20 days of aestivation reduced RPP to 850 ± 21 mmHg min−1 . (3) Aestivation for 40 days reduced to RPP to 796 ± 31 mmHg min−1 . (4) Animals in water had a RPP of 998 ± 38 mmHg min−1 . Statistics and symbols as above.
383
fish and land vertebrates (Tetrapoda). These include central and peripheral acid–base CO2 /H+ -receptors that modify the ventilatory drive (Sanchez et al., 2001; Amin-Naves et al., 2007a,b). In addition, the specific O2 -stimulus in lungfish and tetrapods is partial pressure of O2 , while decreases of O2 content, [O2 ], and reduction of haemoglobin saturation (SO2 ) failed to stimulate pulmonary ventilation. By contrast, the primary ventilatory stimulus in holeost and teleost fish is O2 , and the receptors are located within the gill system along with CO2 /H+ -receptors of secondary importance (Milsom, 2002). Currently, only one study provided some evidence for central chemoreceptos in a teleost or holeost fish (Wilson et al., 2000). It turns out that Johansen et al. (1967) were correct, when he stated: “It is generally accepted that vertebrates acquired functional lungs before they possessed a locomotor apparatus for the terrestrial environment”. The lepidosirenid lungfish Protopterus and L. paradoxa aestivate, whereas the Australian lungfish (Neoceratodus forsteri) (Ceratodontiformes) inhabits river systems and aestivation is absent (Johansen et al., 1967; Kind et al., 2002). Smith (1935) was a pioneer of metabolic measurements in Protopterus, which stimulated an interest for aestivation. As a major effort, DeLaney et al. (1974, 1977) studied gas exchange, blood gases and cardiovascular function in P. aethiopicus initially in water and, later, during aestivation. Formation of a burrow was accompanied by secretion of mucous from the skin, which hardened to form a cocoon after one week or more. This transition to dormancy was accompanied by large decreases of O2 -uptake, which was 0.2–0.5 mL kg−1 min−1 when in water, but fell to 0.084 mL kg−1 min−1 during aestivation (DeLaney et al., 1974). Likewise, fH became reduced from 25 to 14–16 beats min−1 , and the mean blood pressure became reduced from 25 mmHg to15 mmHg. These down-regulated values were maintained stable for no less than 8 months. As a difference between the studies, L. paradoxa maintained the same mean blood pressure (27–32 mmHg), independent of being in the water or in the burrow. Aestivation for 40 days down-regulated fH from 32 to 22 beats min−1 .This is consistent with the pattern for the toad Chaunus schneideri (earlier Bufo paracnemis), in which blood pressures remained the same during dormancy and activity. On the other hand, VO2 , fH , and RPP were consistently down-regulated by 50% relative to uptakes from active toads (Glass et al., 1997). 4.2. Arterial acid–base status and O2 -levels
be expressed by the rate-pressure product (RPP—mmHg min−1 ), which became reduced during aestivation (Fig. 5). When in water the osmolality (mOsm kg H2 O−1 ) was 232 ± 2.6, which increased to 261 ± 4.5 (20 days) and 267 ± 2.2 (40 days) and then fell to 231 ± 2.5 (Fig. 6). Table 1 compiles the variables that remained constant in spite of changing ambient conditions. These include systolic, diastolic and mean blood pressures. Moreover, pHa, plasmatic leptin levels and hematocrit were constant in spite of large aestivation-induced changes of acid–base status and downregulation of cardiovascular function. 4. Discussion 4.1. Down-regulation of cardiovascular function Lungfish (Dipnoi) are currently considered as a highly probable sister group to the land vertebrates (cf. Toyama et al., 2000; Brinkmann et al., 2004), which is backed up by the discovery of a 417 million years old fish fossil (Styloichthys) with the expected features of a common ancestor to tetrapods and lungfish (Zhu and Yu, 2002). In addition, many features of respiratory control link lung-
When in water, L. paradoxa had a PaCO2 of about 25 mmHg (Amin-Naves et al., 2004), which rose to 38 mmHg during aestivation. In P. aethiopicus, the transition from life in a lake to a cocooned state increased PaCO2 from 26 to no less than 50 mmHg. An X-ray study (Lomholt, 1993) and a drawing by DeLaney et al. (1974) suggest a tight fit between the animal and the cocoon. Supposedly, this would seal better than a mixture of clay and mud, and would contribute to the highly elevated PaCO2 in P. aethiopicus. An important relationship is, V˙ Ef R·T = , PL CO2 V˙ CO2 in which V˙ Ef = effective ventilation of the lung; V˙ CO2 = CO2 output; R = the gas constant; T = absolute temperature (K); PL CO2 = the PCO2 of the lung gas. Only mammals have alveoli, and, therefore, effective ventilation substitutes “alveolar ventilation” and PL CO2 substitutes PA CO2 . The message of the equation above is that PL CO2 and PaCO2 can be kept constant, if V˙ Ef /V˙ CO2 are proportionally down-regulated, which seems to be the case. The equation can be expanded by
384
G.d.S.F. da Silva et al. / Respiratory Physiology & Neurobiology 164 (2008) 380–385
earlier data for P. aethiopicus (DeLaney et al., 1974). Leptin is a small protein hormone with a widespread distribution among vertebrates, and its functions are diverse. One of these is to interact with central chemoreceptor systems in mammals. In particular, ob/ob mice are unable to produce leptin, which results in highly reduced ventilatory responses to hypercarbia (O’Donnell et al., 1999, 2000; Polotsky et al., 2004). Our conclusion is that leptin is present in Lepidosiren, but an interaction with respiratory responses could not be established. In conclusion, the patterns of aestivation are very similar in Protopterus sp. and L. paradoxa. An important difference is that the former aestivates in a cocooned state, whereas the latter aestivates in a simple burrow of mud. Large aestivation-induced increases of PaCO2 result from a transition to an exclusive elimination by the lung. This is accompanied by large increases of plasma [HCO3 − ] and slightly reduced PaO2 -values. This study was supported by CNPq (Conselho Nacional de Desenvolvimento Cientíco e Tecnológico) and FAPESP (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo). References Fig. 7. This figure is a Davenport diagram, which illustrates the difference between animals in water and in aestivation. The values for the present study are represented by an open circle, whereas aestivation is marked by a full circle. Notice the practically stable pHa-values. An open triangle represents Protopterus aethiopicus in water, whereas the full triangle indicates aestivation. Notice the very high pHa values in P aetiopicus (DeLaney et al., 1977). The letter “i” stand for the initial value (before aestivation) and the letter “R” signifies return to the initial control conditions after aestivation. The pK 1 -value (6.09) value is from Sanchez et al. (2005) and the solubility was calculated from Reeves (1976).
addition of a CO2 -output by the skin (Glass and Soncini, 1995), PaCO2 =
V˙ CO2 tot (Dcut + VEf · ˇg)
,
where V˙ CO2 tot = total CO2 output; Dcut = cutaneous CO2 -output to the water and ˇg = the capacitance coefficient for gases (ˇg = 1/RT). The above equation predicts that pulmonary CO2 -output will increase, if cutaneous CO2 -elimination is restricted or absent, which would be the case during aestivation. Our simple model seems to agree with the data. Protopterus and L. paradoxa possess highly reduced gills with a minute contribution to the CO2 -output which, in the latter, has been estimated to only 0.0013% of the respiratory surface area (Moraes et al., 2005). Recently, Perry et al. (2008) studied P. dolloi, which, however, does not aestivate within its region (Greenwood, 1986). Nevertheless, it secreted a cocoon that hardened, but a downregulation was missing, since its O2 -uptake increased from 0.35 to 0.45 mL kg−1 min−1 , reducing PaCO2 from 18 to14 mmHg (Glass, 2008). DeLaney et al. (1977) discussed their very high plasma [HCO3 − ]levels and stated that a slowly rising plasma [HCO3 − ] and suggested a role of dehydration as an alternative. Consistently, the concentration of red blood cells increased nearly two-fold during the first month of aestivation. A Davenport diagram presents the data for our study and from on P. aethiopicus (DeLaney et al., 1977), in which amazingly high PaCO2 -values were recorded (PaCO2 ∼ 50 mmHg), while L. paradoxa reached 40 mmHg. The emergency strategies of the two genera are clearly different. Aestivation without a cocoon permits to escape from an unfavourable site. On the other hand, the cocoon may protect the animal for years, but as reported by Lomholt (1993) this may lead to death (Fig. 7). The osmolality was the same before and after aestivation (∼232 ± 2.6 Osmol), but increased significantly to the range 261 (20 days) to 267 Osmol (40 days). These values are in agreement with
Abe, A.S., 1995. Estivation in South American amphibians and reptiles. Brazilian Journal of Medical and Biological Research 28, 1241–1247. Abe, A.S., Steffensen, J.F., 1996a. Perda cutânea de oxigênio na pirambóia, Lepidosiren paradoxa, exposta à água hipóxica (Osteichthyes, Dipnoi). Revista. Brasileira da Biologia 56, 211–216. Abe, A.S., Steffensen, J.F., 1996b. Respirac¸ão pulmonar e cutânea na pirambóia, Lepidosiren paradoxa, durante a atividade e a estivac¸ão (Osteichthyes, Dipnoi). Revista. Brasileira da Biologia 56, 485–489. Amin-Naves, J., Giusti, H., Hoffman, A., Glass, M.L., 2007a. Components to the acid base related ventilatory drives in the South American lungfish Lepidosiren paradoxa. Respiratory Physiology & Neurobiology 155, 35–40. Amin-Naves, J., Giusti, H., Hoffman, A., Glass, M.L., 2007b. Central ventilatory control in the South American lungfish, Lepidosiren paradoxa: contributions of pH and CO2 . Journal of Comparative Physiology B 177, 529–534. Amin-Naves, J., Giusti, H., Glass, M.L., 2004. Effects of the acute temperature changes on aerial and aquatic gas exchange, pulmonary ventilation and blood gases status in the South American lungfish, Lepidosiren paradoxa. Comparative and Biochemistry Physiology A 138, 133–139. Bassi, M., Klein, W., Fernandes, M.N., Perry, S.F., Glass, M.L., 2005. Pulmonary oxygen diffusing capacity of the South American lungfish Lepidosiren paradoxa: physiological values by the Bohr integration method. Physiological and Biochemical Zoology 78, 560–569. Brinkmann, H., Denk, A., Zitzle, J., Joss, J.M.P., Meyer, A., 2004. Complete mitochondrial genome sequence of the South American and the Australian Lungfish: testing of the phylogenetic performance of mitochondrial data sets for phylogenetic problems in tetrapod relationships. Journal of Molecular Evolution 59, 834–848. Chew, S.F., Chan, N.K., Loong, A.M., Hiong, K.C., Tam, W.L., Ip, Y.K., 2004. Nitrogen metabolism in the African lungfish (Protopterus dolloi) aestivating in a mucus cocoon on land. Journal of Experimental Biology 207, 777–786. Chew, S.F., Ong, T.F., Ho, L., Tam, W.L., Loong, A.M., Hiong, K.C., Wong, W.P., Ip, Y.K., 2003. Urea synthesis in the African lungfish Protopterus dolloi—hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure. Journal of Experimental Biology 206, 3615–3624. Dejours, P., 1981. Principles of Comparative Respiratory Physiology, 2nd Ed. Elsevier, North Holland Publishing Company. DeLaney, R.G., Lahiri, S., Fishman, A.P., 1974. Aestivation of the African lungfish Protopterus aethiopicus: cardiovascular and pulmonary function. Journal of Experimental Physiology 6, 111–128. DeLaney, R.G., Lahiri, S., Hamilton, R., Fishman, A.P., 1977. Acid-base balance and plasma composition in the aestivating lungfish (Protopterus). American Journal of Physiology 232, R10–R17. Glass, M.L., 2008. The enigma of aestivation in the African lungfish Protopterus dolloi—commentary on the paper by Perry et al. Respiratory Physiology & Neurobiology 160, 18–20. Glass, M.L., Fernandes, M.S., Soncini, R., Glass, H., Wasser, J., 1997. Effects of dry season dormancy on oxygen uptake, heart rate, and blood pressures in the toad Bufo paracnemis. Journal of Experimental Zoology 279, 330–336. Glass, M.L., Soncini, R., 1995. Regulation of acid-base status in ectothermic vertebrates: the consequences for oxygen pressures in lung gas and arterial blood. Brazilian Journal of Medical and Biological Research 28, 1161–1166. Greenwood, P.H., 1986. The natural history of African lungfishes. Journal Morphological Supplement 1, 163–179. 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. 99–110.
G.d.S.F. da Silva et al. / Respiratory Physiology & Neurobiology 164 (2008) 380–385 Johansen, K., Lenfant, C., Grigg, G.C., 1967. Respiratory control in the lungfish Neoceratodus forsteri (Krefft). Comparative Biochemistry 20, 835–854. Kind, P.K., Grigg, G.C., Booth, D.T., 2002. Physiological responses to prolonged aquatic hypoxia in the Quensland lungfish, Neoceratodus forsteri. Respiratory Physiology & Neurology 132, 179–190. Lomholt, J.P., 1993. Breathing in the Aestivating African Lungfish, Protopterus amphibious. Advances in Fish Research 1, 17–34. Loong, A.M., Hiong, K.C., Lee, S.M., Wong, W.P., Chew, S.F., Ip, Y.K., 2005. Ornithineurea cycle and urea synthesis in African lungfishes, Protopterus aethiopicus and Protopterus annectens, exposed to terrestrial conditions for six days. Journal Experimental Zoology A Comparative Experimental Biology 303, 354–365. Milsom, W.K., 2002. Phylogeny of CO2 /H+ chemoreception in vertebrates. Respiratory Physiology & Neurobiology 131, 29–41. Moraes, M.F.P.G., Fernandes, M.N., Höller, S., Costa, O.P.F., Glass, M.L., Perry, S.F., 2005. Morphometric comparison of the respiratory organs of the South American lungfish Lepidosiren paradoxa (Dipnoi). Physiological and Biochemical Zoology 78, 546–559. O’Donnell, C.P., Schaub, C.D., Haines, A.S., Berkowitz, D.E., Tankerley, C., Schwartz, A.R., Smiths, P.L., 1999. Leptin prevents respiratory depression in obesity. American of Jouranl of Respiratory Critical Care Medicine 159, 1417–1484. O’Donnell, C.P., Tankerley, C.G., Polotky, V.P., Schwartz, A.R., Smith, P.L., 2000. Leptin, obesity and respiratory function. Respiration Physiology 119, 173–180. Paolucci, M., Rocco, M., Varicchio, E., 2001. Leptin presence in plasma, liver and fat bodies in the lizard Podarcis sicula: fluctuations throughout the reproductive cycle. Life Science 69, 2399–23408. Perry, S.F., Euverman, R., Wang, T., Loong, A.M., Chew, S.F., Ip, Y.K., Gilmour, K.M., 2008. Control of breathing in African lungfish (Protopterus dolloi): a compari-
385
son of aquatic and cocooned (terrestrialized) animals. Respiratory Physiology & Neurobiology 160, 8–17. Polotsky, V.Y., Smaldone, M.C., Schardf, M.T., Li, J., Tankersley, C.G., Smith, P.L., Schwartz, A.R., O’Donnel, C.P., 2004. Impact of interrupted leptin pathways on ventilatory control. Journal of Applied Physiology 96, 991–998. Reeves, R.B., 1976. Temperature-induced changes in blood acid-base status: pH and PCO2 in a binary buffer. Journal of Applied Physiology 40, 752–761. Sanchez, A.P., Giusti, H., Bassi, M., Glass, M.L., 2005. Acid-base regulation in the South American lungfish Lepidosiren paradoxa: effects of prolonged hypercarbia on blood gases and pulmonary ventilation. Physiological and Biochemical Zoology 78, 908–915. Sanchez, A.P., Hoffman, A., Rantin, F.T., Glass, M.L., 2001. The relationship between pH of the cerebro-spinal fluid and pulmonary ventilation of the South American lungfish, Lepidosiren paradoxa. Journal of Experimental Zoology 290, 421– 425. Smith, H.M., 1935. The metabolism of a lungfish I. General considerations of the fasting metabolism in an active fish. Journal of Cellular Comparative Physiology 6, 43–67. Toyama, Y., Ichimiya, T., Kasama-Yoshida, H., Cao, Y., Hasegava, M., Kojima, H., Tamai, Y., Kurihari, T., 2000. Phylogenetic relation of lungfish indicated by the amino acid sequence of myelin DM20. Molecular Brain Research 8, 256–259. Wilson, R.J., Harris, M.B., Remmers, J.E., Perry, S.F., 2000. Evolution of air-breathing and central CO2 /H+ respiratory chemosensitivity: new insights from an old fish? Journal of Experimental Biology 203, 3505–3512. Zhu, M., Yu, X., 2002. A primitive fish close to the common ancestor of tetrapods and lungfish. Nature 418, 767–770.
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Aestivation in the South American lungfish, Lepidosiren paradoxa: Effects on cardiovascular function, blood gases, osmolality and leptin levels Glauber dos Santos Ferreira da Silva, Humberto Giusti, Adriana Paula Sanchez, Jussara Márcia do Carmo, Mogens Lesner Glass ∗ Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Avenida Bandeirantes 3.900, 14.049-900 SP, Brazil
a r t i c l e
i n f o
Article history: Accepted 29 August 2008 Keywords: Aestivation South American lungfish Osmolality Lepidosiren Blood gases Acid-base status
a b s t r a c t The African (Protopterus sp.) and South American lungfish (Lepidosiren paradoxa) inhabit shallow waters, that seasonally dry out, which induces aestivation and cocoon formation in Protopterus. Differently, L. paradoxa has no cocoon, and it aestivates in a simple burrow. In water PaCO2 is 21.8 ± 0.4 mmHg (mean values ± S.E.M.; n = 5), whereas aestivation for 20 days increased PaCO2 to as much as 37.6 ± 2.1 mmHg, which remained the same after 40 days (35.8 ± 3.3 mmHg). Concomitantly, the plasma [HCO3 − ]-values for animals in water were 22.5 ± 0.5 mM, which after 20 days increased to 40.2 ± 2.3 mM and after 40 days to 35.8 ± 3.3 mM. Initially in water, PaO2 was 87.7 ± 2.0 mmHg, but 20 days in aestivation reduced the value to 80.5 ± 2.2 and later (40 days) to 77.1 ± 3.0 mmHg. Meanwhile, aestivation had no effect on pHa and hematocrit. The blood pressures were equal for animals in the water or in the burrow (Pmean ∼ 30 mmHg), and cardiac frequency (fH ) fell from 31 beats min−1 to 22 beats min−1 during 40 days of aestivation. The osmolality (mOsm kg H2 O−1 ) was elevated after 20 and 40 days of aestivation but declined upon return to water. The transition from activity to aestivation involves new set-points for the variables that determine the acid–base status and PaO2 of the animals, along with a reduction of cardiac frequency. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In animals, aestivation is a temporary period of dormancy, which down-regulates O2 -uptake, pulmonary ventilation and cardiovascular activity, and this usually reflects adverse ambient conditions. The roman word “aestas” means summer, but aestivation is not necessarily linked to a specific part of the year. Aestivation occurs in lepidosirenid lungfish and some amphibians and reptiles, and this condition may occur without any change of ambient temperature (Abe, 1995; Abe and Steffensen, 1996a,b). In aestivating animals, the O2 -uptake usually is 20–55% of resting values for the awaken animal (Glass et al., 1997; DeLaney et al., 1974). The South American lungfish, Lepidosiren paradoxa, inhabits swamps that dry out on a seasonal basis within the Pantanal region (Mato Grosso, Brazil), which coincides with the winter in the middle of the year. Information on aestivation in L. paradoxa is highly limited, except for Harder et al. (1999), who reported on cardiac frequencies during formation of a burrow. When a lake dries out, L. paradoxa
∗ Corresponding author. Tel.: +55 16 36023202; fax: +55 16 36330017. E-mail address: mlglass@rfi.fmrp.usp.br (M.L. Glass). 1569-9048/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2008.08.009
digs a hole into the mud and clay, which allows movements and change of position. For the animals in water, these authors reported a cardiac frequency (fH ) of 31 beats min−1 which became downregulated by 46% during aestivation. As a major difference, Protopterus secretes a protective mucous from the skin to form a hardened cocoon (DeLaney et al., 1974). Much less is known about aestivation in L. paradoxa, in particular concerning blood gases and osmolality. Abe and Steffensen (1996a,b) reported that VO2 was reduced by no more than 30% after 4 months of aestivation at 25 ◦ C. Considerably more is known about aestivation in the African lungfish (P. aethiopicus). When the water dries out, this lungfish forms a burrow and secretes a gradually hardening mucous to form a cocoon, which is open at the top, where the animal respires. Aestivation in P. aethiopicus has spectacular consequences, including an increase from PaCO2 from 26 mmHg to ∼50 mmHg upon transition from water to a cocooned condition (DeLaney et al., 1977), and they reported that pHa remained within a rather narrow range, due to large increases of plasma [HCO3 − ] levels. Aestivation can persist for amazingly long periods such as 9 months in P. aethiopicus (DeLaney et al., 1974, 1977). A P. amphibius survived for 7 years in a cocoon, and a second animal with 6 years in the same condition reduced its VO2 from 0.840 (in water) to
G.d.S.F. da Silva et al. / Respiratory Physiology & Neurobiology 164 (2008) 380–385
0.039 mL kg−1 min−1 . In the latter animal, the end-expired PaCO2 was 40 mmHg (Lomholt, 1993). Recently, lungfish have received considerable interest, because Dipnoi represents the most likely sister group relative to the land vertebrates (Tetrapoda). The physiology of Protopterus sp. during activity and/or aestivation is, however, known in considerable detail (cf. DeLaney et al., 1974, 1977; Chew et al., 2003, 2004; Loong et al., 2005). When its lake dries out, the African lungfish secretes a mucous that gradually hardens to protect the animal (DeLaney et al., 1977). A straight forward hypothesis can explain blood gas values in L. paradoxa at 25 ◦ C. In this lungfish, 50% of total CO2 -output was eliminated to aerated water, while pulmonary ventilation eliminated the other half (Amin-Naves et al., 2004; Bassi et al., 2005). Aestivation in a burrow would favour CO2 -elimination by pulmonary ventilation. This would occur, because water was replaced by the limited space within a burrow. In this context Dejours (1981) stated that: “Compared to water breathers, air breathers are in a state of compensated hypercapnic acidosis”. A very high PaCO2 is expected for Protopterus sp., because the animal aestivates within a gastight cocoon. In this case, virtually the whole CO2 -output would be expired by pulmonary ventilation (Lomholt, 1993). Aestivation in lungfish is characterized by very high PaCO2 -values that can be maintained over long periods (DeLaney et al., 1974, 1977), which involves a proportional regulation of lung ventilation and CO2 -output. The effects of blood osmolality were also measured, and will be discussed in relation to activity or aestivation. In addition, plasmatic leptin levels were measured to evaluate possible metabolic effects of this hormone. The inclusion of leptin levels was motivated by the recent discovery, that low leptin levels can reduce pulmonary ventilation in mammals (O’Donnell et al., 1999, 2000). Furthermore, we also addressed down-regulation of cardiovascular function in relation to activity or aestivation (DeLaney et al., 1977; Glass et al., 1997). Our aim is to compare aestivation in Protopterus and L. paradoxa and evaluate the consequences of the cocooned state and the effects of a simple burrow. 2. Materials and methods Specimens of L. paradoxa (n = 20) were collected close to the city Cuiabá, Mato Grosso State, and then transported to the Central Animal Holding Facility of University of São Paulo, Ribeirão Preto, São Paulo State, Brazil, and were kept in 1000 L tanks at a water level of 1 m and at 25 ◦ C ± 1 ◦ C at all times (Fraéllio Electric Equipments, Ribeirão Preto, São Paulo State). All measurements were performed at that temperature. Food (snails and chicken liver) was provided outside the period of aestivation. Four groups were established with n = 5: (1) Control group of freely swimming animals, placed in normoxic and normocarbic water in April. W = 746 ± 23 g; (2) after 20 days of aestivation in July. W = 404 ± 15 g; (3) after 40 days of aestivation in July–August. W = 471 ± 19 g; (4) Control group of freely swimming animals during normoxic and normocarbic water in September. W = 509 ± 26 g; weight losses during aestivation ranged from 9 to 11% of pre-aestivation values. Collection of the animals was approved by IBAMA (Proc. IBAMA/MMA-No. 02027.002172/2005-68; IBAMA = Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis) and by the Ethics Committee of University of São Paulo, Faculty of Medicine of Ribeirão Preto (Proc. #076/2005).
381
buccal cavity with benzocain (0.25 g L−1 ). The dorsal aorta was dissected free at a point 1 cm behind the caudal part of the lung. Then, a PE50 catheter was inserted into the vessel and fixed in place by sutures after which the incisions were closed, finally the catheter was flushed with heparinized Ringer solution (100 IU mL−1 ). The animals recovered movements after 10 min in aerated water. 2.2. Blood gas measurements Arterial blood samples (1 mL) were immediately analyzed after withdrawal, using an electrode (FAC 001 O2 ), connected to a 204 O2 analyzer (FAC Instr., São Carlos, SP, Brazil). This electrode was calibrated with N2 (zero) and with air-equilibrated water (PO2 = 145 mmHg—elevation 710 m; 25 ◦ C). A microelectrode for blood gas measurements (Mettler Toledo, Switzerland) was used after calibration with high precision buffer solutions (Queel, São Paulo, SP, Brazil). A BGM 200 Cameron Analyzer (Cameron, Port Aransas, TX, USA) was equipped with CO2 -electrodes and calibrated with CO2 mixtures provided by a Cameron GF3/MP gas flow meter. Total plasma CO2 was measured by means of a Capni-Con 5 Analyzer (Cameron), calibrated with standard [HCO3 − ] solutions. To obtain haematocrit, microtubes were filled with blood and centrifuged using a Hermle z.200 M/H at 7500 rpm during 1 min. To measure blood pressures and cardiac frequency (fH ), the implanted catheter (P50) was connected to an arterial pressure transducer (Statham PB23 GB, Palo Alto, USA). The signals were fed into a multipurpose amplifier at a sampling rate of 1000 Hz (Spectramed, Statham P10 Ez, Palo Alto, USA) and transmitted to a computer. Advanced CODAS (Dataq Instr., Akron, OH, USA) was applied to analyze the cardiovascular data, and the rate-pressure product (RPP) was calculated as fH ·Psyst (mmHg min−1 ) as a measure of myocardial activity. 2.3. Measurements of osmolality and leptin levels The plasmatic osmolality (mOsm kg H2 O−1 ) was measured using an osmometer (OS Mark 3, Fiske Associates, Needham Heights, MA, USA). Plasmatic leptin levels were measured by radioimmunoassay, using mice polyclonal leptin antibodies (Lincoˇıs Multi-Species Leptin Radioimmunoassay Kit) as described by Paolucci et al. (2001). About 0.4 mL blood was withdrawn from the animal and placed into a centrifuge (Hermle z200 M/H, Germany) during 10 min. Our Multi-Species Kit for leptin measurements was insufficient for covering the whole period of aestivation. 2.4. Experimental protocol The animals were divided in 4 separated groups, each with 5 animals: (1) Measurements of blood gases, cardiac frequency and blood pressures were obtained for active animals in water. (2) Aestivation was induced by a gradual reduction of water levels in a 50 cm deep mixture of mud and clay, and the digging out of the burrow would usually not take more than 45 min. The animal was then left in the burrow for 18 days, after which the arterial catheter was quickly implanted. After recovery, the animal spontaneously re-entered the burrow to complete 20 days of aestivation. Group (3) procedures were described above, but the aestivation was extended to 40 days. Group (4) second control group of active animals in water. 2.5. Statistics and calculations
2.1. Implantation of catheters Immersion into a solution of benzocain (1 g L−1 ) anaesthetized the animal within 10 min and was maintained by flushing the
The rate-pressure product (RPP) was calculated as: Psyst ·fH (mmHg min−1 ). ANOVA was followed by Bonferroni’s multiple comparison post-test to compare values for 4 independent groups:
382
G.d.S.F. da Silva et al. / Respiratory Physiology & Neurobiology 164 (2008) 380–385
Fig. 1. The numbers corresponds to the columns: (1) Animals were kept in aerated water for two weeks and PaCO2 was 21.8 ± 0.4 mmHg. (2) Aestivation for 20 days increased PaCO2 to 37.6 ± 2 mmHg. (3) Aestivation for 40 days resulted in a PaCO2 of 34.4 ± 3 mmHg. (4) Control measurements in well-aerated water resulted in a PaCO2 of 20.8 ± 1 mmHg. Mean ± S.E., N = 5; P < 0.05. (*) Indicates a difference between the animal in water and during aestivation.
(1) Control values for the animal in water; (2) 20 days; (3) 40 days of aestivation; (4) Control animals in water. Mean ± S.E.M.; n = 5 (P > 0.05). The total number of individuals was 20 since 4 groups were formed. 3. Results As shown in Fig. 1, PaCO2 was low 21.8 ± 0.4 mmHg when in water, whereas 20 days of aestivation increased PaCO2 to 37.6 ± 2.1 (73%), and after 40 days the value was slightly reduced to 34.4 ± 3.2. After aestivation a second control for the fish in water (=Group 4) was PaCO2 = 20.8 ± 1.2 mmHg. The animal in water had a plasma [HCO3 − ] of 22.5 ± 0.5 mM, but aestivation increased plasma [HCO3 − ] to 40.2 ± 2.3 mM (74%) in Group 2 (20 days of aestivation). Group 3 (40 days of aestivation) resulted in 35.8 ± 3.3 mM and, Group 4 (fish in water) reduced [HCO3 − ] to 23.0 ± 0.4 mM (Fig. 2). Group 1 (in water) had a PaO2 of 87.7 ± 2.0 mmHg, while 20 days of aestivation reduced PaO2 to 80.5 ± 2.2 mmHg which, after 40 days of aestivation, was cut further down to 77.1 ± 3.0. Group 4
Fig. 2. (1) In aerated water [HCO3 − ] was 22.5 ± 0.5 mM. (2) Aestivation during 20 days elevated [HCO3 − ] to 40.2 ± 2 mM. (3) 40 days of aestivation resulted in a plasma [HCO3 − ] of 35.8 ± 3 mM. (4) In water, plasma [HCO3 − ] was 23 ± 0.4 mM. Statistics and symbols as above.
Fig. 3. (1) In water, PaO2 was 87.7 ± 2 mmHg. (2) Aestivation reduced PaO2 to 80.5 ± 2 mmHg (20 days). (3) 40 days of aestivation further reduced PaO2 to 77 ± 3 mmHg. (4) Return to water increased PaO2 to 85.7 ± 2 mmHg. Statistics and symbols as above. Table 1 Blood pressures, pHa, leptin levels and hematocrit Before aestivation Psyst (mmHg) Pdiast (mmHg) Pmean (mmHg) pHa Leptin (ng/mL) Hct (%)
30.8 25.3 27.3 7.51 1.43 28
± ± ± ± ± ±
0.5 0.4 0.3 0.05 0.14 1
20 days of aestivation 30.4 26 27.8 7.49 1.53 25
± ± ± ± ± ±
0.8 0.5 0.7 0.02 0.08 2
40 days of aestivation
After aestivation
35.2 ± 27.1 ± 31.8 ± 7.53 ± – 26 ±
31.2 ± 25 ± 28 ± 7.49 ± – 27 ±
0.6 0.5 0.7 0.05 1
0.7 0.4 0.3 0.03 1
This table represents the variables that were not affected by aestivation. Values are expressed as mean values ± S.E.M.; n = 5. The values were obtained from separate groups.
(animal in water) had a PaO2 of 85.7 ± 1.8, which was identical to the values of Group 1 (Fig. 3). Blood pressures were the same during aestivation and in water (see Table 1). By contrast, the cardiac frequency (fH ) of the animals in water was fH = 30.8 ± 0.5 beats min−1 , which initially reduced fH slightly to 28.0 ± 0.5 (20 days of aestivation) and then to 22.0 ± 1.0 (40 days of aestivation; Fig. 4). This down-regulation can
Fig. 4. The effects of aestivation on osmolality (mOsm kg H2 O−1 ) were pronounced. (1) In water, the osmolality of the animal was 232 ± 3. (2) Aestivation for 20 days increased this to 261 ± 5. (3) Aestivation for 40 days increased osmolality to 267 ± 3. (4) The second control in water was 231 ± 3. Statistics and symbols as above.
G.d.S.F. da Silva et al. / Respiratory Physiology & Neurobiology 164 (2008) 380–385
Fig. 5. (1) When in water fH was 31 ± 1. (2) 20 days of aestivation slightly decreased fH to 28 ± 0.5 beats min−1 . (3) 40 days of aestivation reduced fH to 22 ± 1 beats min−1 . (4) Animals in water increased heart rate to 32 ± 1 beats min−1 . Symbols as above. In addition, the symbol (#) indicates a difference between 20 and 40 days aestivation.
Fig. 6. (1) For animals in water the rate-pressure product (RPP, mmHg min−1 ) was 968 ± 32. (2) 20 days of aestivation reduced RPP to 850 ± 21 mmHg min−1 . (3) Aestivation for 40 days reduced to RPP to 796 ± 31 mmHg min−1 . (4) Animals in water had a RPP of 998 ± 38 mmHg min−1 . Statistics and symbols as above.
383
fish and land vertebrates (Tetrapoda). These include central and peripheral acid–base CO2 /H+ -receptors that modify the ventilatory drive (Sanchez et al., 2001; Amin-Naves et al., 2007a,b). In addition, the specific O2 -stimulus in lungfish and tetrapods is partial pressure of O2 , while decreases of O2 content, [O2 ], and reduction of haemoglobin saturation (SO2 ) failed to stimulate pulmonary ventilation. By contrast, the primary ventilatory stimulus in holeost and teleost fish is O2 , and the receptors are located within the gill system along with CO2 /H+ -receptors of secondary importance (Milsom, 2002). Currently, only one study provided some evidence for central chemoreceptos in a teleost or holeost fish (Wilson et al., 2000). It turns out that Johansen et al. (1967) were correct, when he stated: “It is generally accepted that vertebrates acquired functional lungs before they possessed a locomotor apparatus for the terrestrial environment”. The lepidosirenid lungfish Protopterus and L. paradoxa aestivate, whereas the Australian lungfish (Neoceratodus forsteri) (Ceratodontiformes) inhabits river systems and aestivation is absent (Johansen et al., 1967; Kind et al., 2002). Smith (1935) was a pioneer of metabolic measurements in Protopterus, which stimulated an interest for aestivation. As a major effort, DeLaney et al. (1974, 1977) studied gas exchange, blood gases and cardiovascular function in P. aethiopicus initially in water and, later, during aestivation. Formation of a burrow was accompanied by secretion of mucous from the skin, which hardened to form a cocoon after one week or more. This transition to dormancy was accompanied by large decreases of O2 -uptake, which was 0.2–0.5 mL kg−1 min−1 when in water, but fell to 0.084 mL kg−1 min−1 during aestivation (DeLaney et al., 1974). Likewise, fH became reduced from 25 to 14–16 beats min−1 , and the mean blood pressure became reduced from 25 mmHg to15 mmHg. These down-regulated values were maintained stable for no less than 8 months. As a difference between the studies, L. paradoxa maintained the same mean blood pressure (27–32 mmHg), independent of being in the water or in the burrow. Aestivation for 40 days down-regulated fH from 32 to 22 beats min−1 .This is consistent with the pattern for the toad Chaunus schneideri (earlier Bufo paracnemis), in which blood pressures remained the same during dormancy and activity. On the other hand, VO2 , fH , and RPP were consistently down-regulated by 50% relative to uptakes from active toads (Glass et al., 1997). 4.2. Arterial acid–base status and O2 -levels
be expressed by the rate-pressure product (RPP—mmHg min−1 ), which became reduced during aestivation (Fig. 5). When in water the osmolality (mOsm kg H2 O−1 ) was 232 ± 2.6, which increased to 261 ± 4.5 (20 days) and 267 ± 2.2 (40 days) and then fell to 231 ± 2.5 (Fig. 6). Table 1 compiles the variables that remained constant in spite of changing ambient conditions. These include systolic, diastolic and mean blood pressures. Moreover, pHa, plasmatic leptin levels and hematocrit were constant in spite of large aestivation-induced changes of acid–base status and downregulation of cardiovascular function. 4. Discussion 4.1. Down-regulation of cardiovascular function Lungfish (Dipnoi) are currently considered as a highly probable sister group to the land vertebrates (cf. Toyama et al., 2000; Brinkmann et al., 2004), which is backed up by the discovery of a 417 million years old fish fossil (Styloichthys) with the expected features of a common ancestor to tetrapods and lungfish (Zhu and Yu, 2002). In addition, many features of respiratory control link lung-
When in water, L. paradoxa had a PaCO2 of about 25 mmHg (Amin-Naves et al., 2004), which rose to 38 mmHg during aestivation. In P. aethiopicus, the transition from life in a lake to a cocooned state increased PaCO2 from 26 to no less than 50 mmHg. An X-ray study (Lomholt, 1993) and a drawing by DeLaney et al. (1974) suggest a tight fit between the animal and the cocoon. Supposedly, this would seal better than a mixture of clay and mud, and would contribute to the highly elevated PaCO2 in P. aethiopicus. An important relationship is, V˙ Ef R·T = , PL CO2 V˙ CO2 in which V˙ Ef = effective ventilation of the lung; V˙ CO2 = CO2 output; R = the gas constant; T = absolute temperature (K); PL CO2 = the PCO2 of the lung gas. Only mammals have alveoli, and, therefore, effective ventilation substitutes “alveolar ventilation” and PL CO2 substitutes PA CO2 . The message of the equation above is that PL CO2 and PaCO2 can be kept constant, if V˙ Ef /V˙ CO2 are proportionally down-regulated, which seems to be the case. The equation can be expanded by
384
G.d.S.F. da Silva et al. / Respiratory Physiology & Neurobiology 164 (2008) 380–385
earlier data for P. aethiopicus (DeLaney et al., 1974). Leptin is a small protein hormone with a widespread distribution among vertebrates, and its functions are diverse. One of these is to interact with central chemoreceptor systems in mammals. In particular, ob/ob mice are unable to produce leptin, which results in highly reduced ventilatory responses to hypercarbia (O’Donnell et al., 1999, 2000; Polotsky et al., 2004). Our conclusion is that leptin is present in Lepidosiren, but an interaction with respiratory responses could not be established. In conclusion, the patterns of aestivation are very similar in Protopterus sp. and L. paradoxa. An important difference is that the former aestivates in a cocooned state, whereas the latter aestivates in a simple burrow of mud. Large aestivation-induced increases of PaCO2 result from a transition to an exclusive elimination by the lung. This is accompanied by large increases of plasma [HCO3 − ] and slightly reduced PaO2 -values. This study was supported by CNPq (Conselho Nacional de Desenvolvimento Cientíco e Tecnológico) and FAPESP (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo). References Fig. 7. This figure is a Davenport diagram, which illustrates the difference between animals in water and in aestivation. The values for the present study are represented by an open circle, whereas aestivation is marked by a full circle. Notice the practically stable pHa-values. An open triangle represents Protopterus aethiopicus in water, whereas the full triangle indicates aestivation. Notice the very high pHa values in P aetiopicus (DeLaney et al., 1977). The letter “i” stand for the initial value (before aestivation) and the letter “R” signifies return to the initial control conditions after aestivation. The pK 1 -value (6.09) value is from Sanchez et al. (2005) and the solubility was calculated from Reeves (1976).
addition of a CO2 -output by the skin (Glass and Soncini, 1995), PaCO2 =
V˙ CO2 tot (Dcut + VEf · ˇg)
,
where V˙ CO2 tot = total CO2 output; Dcut = cutaneous CO2 -output to the water and ˇg = the capacitance coefficient for gases (ˇg = 1/RT). The above equation predicts that pulmonary CO2 -output will increase, if cutaneous CO2 -elimination is restricted or absent, which would be the case during aestivation. Our simple model seems to agree with the data. Protopterus and L. paradoxa possess highly reduced gills with a minute contribution to the CO2 -output which, in the latter, has been estimated to only 0.0013% of the respiratory surface area (Moraes et al., 2005). Recently, Perry et al. (2008) studied P. dolloi, which, however, does not aestivate within its region (Greenwood, 1986). Nevertheless, it secreted a cocoon that hardened, but a downregulation was missing, since its O2 -uptake increased from 0.35 to 0.45 mL kg−1 min−1 , reducing PaCO2 from 18 to14 mmHg (Glass, 2008). DeLaney et al. (1977) discussed their very high plasma [HCO3 − ]levels and stated that a slowly rising plasma [HCO3 − ] and suggested a role of dehydration as an alternative. Consistently, the concentration of red blood cells increased nearly two-fold during the first month of aestivation. A Davenport diagram presents the data for our study and from on P. aethiopicus (DeLaney et al., 1977), in which amazingly high PaCO2 -values were recorded (PaCO2 ∼ 50 mmHg), while L. paradoxa reached 40 mmHg. The emergency strategies of the two genera are clearly different. Aestivation without a cocoon permits to escape from an unfavourable site. On the other hand, the cocoon may protect the animal for years, but as reported by Lomholt (1993) this may lead to death (Fig. 7). The osmolality was the same before and after aestivation (∼232 ± 2.6 Osmol), but increased significantly to the range 261 (20 days) to 267 Osmol (40 days). These values are in agreement with
Abe, A.S., 1995. Estivation in South American amphibians and reptiles. Brazilian Journal of Medical and Biological Research 28, 1241–1247. Abe, A.S., Steffensen, J.F., 1996a. Perda cutânea de oxigênio na pirambóia, Lepidosiren paradoxa, exposta à água hipóxica (Osteichthyes, Dipnoi). Revista. Brasileira da Biologia 56, 211–216. Abe, A.S., Steffensen, J.F., 1996b. Respirac¸ão pulmonar e cutânea na pirambóia, Lepidosiren paradoxa, durante a atividade e a estivac¸ão (Osteichthyes, Dipnoi). Revista. Brasileira da Biologia 56, 485–489. Amin-Naves, J., Giusti, H., Hoffman, A., Glass, M.L., 2007a. Components to the acid base related ventilatory drives in the South American lungfish Lepidosiren paradoxa. Respiratory Physiology & Neurobiology 155, 35–40. Amin-Naves, J., Giusti, H., Hoffman, A., Glass, M.L., 2007b. Central ventilatory control in the South American lungfish, Lepidosiren paradoxa: contributions of pH and CO2 . Journal of Comparative Physiology B 177, 529–534. Amin-Naves, J., Giusti, H., Glass, M.L., 2004. Effects of the acute temperature changes on aerial and aquatic gas exchange, pulmonary ventilation and blood gases status in the South American lungfish, Lepidosiren paradoxa. Comparative and Biochemistry Physiology A 138, 133–139. Bassi, M., Klein, W., Fernandes, M.N., Perry, S.F., Glass, M.L., 2005. Pulmonary oxygen diffusing capacity of the South American lungfish Lepidosiren paradoxa: physiological values by the Bohr integration method. Physiological and Biochemical Zoology 78, 560–569. Brinkmann, H., Denk, A., Zitzle, J., Joss, J.M.P., Meyer, A., 2004. Complete mitochondrial genome sequence of the South American and the Australian Lungfish: testing of the phylogenetic performance of mitochondrial data sets for phylogenetic problems in tetrapod relationships. Journal of Molecular Evolution 59, 834–848. Chew, S.F., Chan, N.K., Loong, A.M., Hiong, K.C., Tam, W.L., Ip, Y.K., 2004. Nitrogen metabolism in the African lungfish (Protopterus dolloi) aestivating in a mucus cocoon on land. Journal of Experimental Biology 207, 777–786. Chew, S.F., Ong, T.F., Ho, L., Tam, W.L., Loong, A.M., Hiong, K.C., Wong, W.P., Ip, Y.K., 2003. Urea synthesis in the African lungfish Protopterus dolloi—hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure. Journal of Experimental Biology 206, 3615–3624. Dejours, P., 1981. Principles of Comparative Respiratory Physiology, 2nd Ed. Elsevier, North Holland Publishing Company. DeLaney, R.G., Lahiri, S., Fishman, A.P., 1974. Aestivation of the African lungfish Protopterus aethiopicus: cardiovascular and pulmonary function. Journal of Experimental Physiology 6, 111–128. DeLaney, R.G., Lahiri, S., Hamilton, R., Fishman, A.P., 1977. Acid-base balance and plasma composition in the aestivating lungfish (Protopterus). American Journal of Physiology 232, R10–R17. Glass, M.L., 2008. The enigma of aestivation in the African lungfish Protopterus dolloi—commentary on the paper by Perry et al. Respiratory Physiology & Neurobiology 160, 18–20. Glass, M.L., Fernandes, M.S., Soncini, R., Glass, H., Wasser, J., 1997. Effects of dry season dormancy on oxygen uptake, heart rate, and blood pressures in the toad Bufo paracnemis. Journal of Experimental Zoology 279, 330–336. Glass, M.L., Soncini, R., 1995. Regulation of acid-base status in ectothermic vertebrates: the consequences for oxygen pressures in lung gas and arterial blood. Brazilian Journal of Medical and Biological Research 28, 1161–1166. Greenwood, P.H., 1986. The natural history of African lungfishes. Journal Morphological Supplement 1, 163–179. 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. 99–110.
G.d.S.F. da Silva et al. / Respiratory Physiology & Neurobiology 164 (2008) 380–385 Johansen, K., Lenfant, C., Grigg, G.C., 1967. Respiratory control in the lungfish Neoceratodus forsteri (Krefft). Comparative Biochemistry 20, 835–854. Kind, P.K., Grigg, G.C., Booth, D.T., 2002. Physiological responses to prolonged aquatic hypoxia in the Quensland lungfish, Neoceratodus forsteri. Respiratory Physiology & Neurology 132, 179–190. Lomholt, J.P., 1993. Breathing in the Aestivating African Lungfish, Protopterus amphibious. Advances in Fish Research 1, 17–34. Loong, A.M., Hiong, K.C., Lee, S.M., Wong, W.P., Chew, S.F., Ip, Y.K., 2005. Ornithineurea cycle and urea synthesis in African lungfishes, Protopterus aethiopicus and Protopterus annectens, exposed to terrestrial conditions for six days. Journal Experimental Zoology A Comparative Experimental Biology 303, 354–365. Milsom, W.K., 2002. Phylogeny of CO2 /H+ chemoreception in vertebrates. Respiratory Physiology & Neurobiology 131, 29–41. Moraes, M.F.P.G., Fernandes, M.N., Höller, S., Costa, O.P.F., Glass, M.L., Perry, S.F., 2005. Morphometric comparison of the respiratory organs of the South American lungfish Lepidosiren paradoxa (Dipnoi). Physiological and Biochemical Zoology 78, 546–559. O’Donnell, C.P., Schaub, C.D., Haines, A.S., Berkowitz, D.E., Tankerley, C., Schwartz, A.R., Smiths, P.L., 1999. Leptin prevents respiratory depression in obesity. American of Jouranl of Respiratory Critical Care Medicine 159, 1417–1484. O’Donnell, C.P., Tankerley, C.G., Polotky, V.P., Schwartz, A.R., Smith, P.L., 2000. Leptin, obesity and respiratory function. Respiration Physiology 119, 173–180. Paolucci, M., Rocco, M., Varicchio, E., 2001. Leptin presence in plasma, liver and fat bodies in the lizard Podarcis sicula: fluctuations throughout the reproductive cycle. Life Science 69, 2399–23408. Perry, S.F., Euverman, R., Wang, T., Loong, A.M., Chew, S.F., Ip, Y.K., Gilmour, K.M., 2008. Control of breathing in African lungfish (Protopterus dolloi): a compari-
385
son of aquatic and cocooned (terrestrialized) animals. Respiratory Physiology & Neurobiology 160, 8–17. Polotsky, V.Y., Smaldone, M.C., Schardf, M.T., Li, J., Tankersley, C.G., Smith, P.L., Schwartz, A.R., O’Donnel, C.P., 2004. Impact of interrupted leptin pathways on ventilatory control. Journal of Applied Physiology 96, 991–998. Reeves, R.B., 1976. Temperature-induced changes in blood acid-base status: pH and PCO2 in a binary buffer. Journal of Applied Physiology 40, 752–761. Sanchez, A.P., Giusti, H., Bassi, M., Glass, M.L., 2005. Acid-base regulation in the South American lungfish Lepidosiren paradoxa: effects of prolonged hypercarbia on blood gases and pulmonary ventilation. Physiological and Biochemical Zoology 78, 908–915. Sanchez, A.P., Hoffman, A., Rantin, F.T., Glass, M.L., 2001. The relationship between pH of the cerebro-spinal fluid and pulmonary ventilation of the South American lungfish, Lepidosiren paradoxa. Journal of Experimental Zoology 290, 421– 425. Smith, H.M., 1935. The metabolism of a lungfish I. General considerations of the fasting metabolism in an active fish. Journal of Cellular Comparative Physiology 6, 43–67. Toyama, Y., Ichimiya, T., Kasama-Yoshida, H., Cao, Y., Hasegava, M., Kojima, H., Tamai, Y., Kurihari, T., 2000. Phylogenetic relation of lungfish indicated by the amino acid sequence of myelin DM20. Molecular Brain Research 8, 256–259. Wilson, R.J., Harris, M.B., Remmers, J.E., Perry, S.F., 2000. Evolution of air-breathing and central CO2 /H+ respiratory chemosensitivity: new insights from an old fish? Journal of Experimental Biology 203, 3505–3512. Zhu, M., Yu, X., 2002. A primitive fish close to the common ancestor of tetrapods and lungfish. Nature 418, 767–770.