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Respiratory gas exchange in the aquatic pulmonate, Biomphalaria sudanica
Comp. Biochem. Physiol., 1964, Vol. 12, pp. 297 to 310. PergamonPressLtd. Printedin Great Britain
RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE, B I O M P H A L A R I A S U D A N I C A j. D. J O N E S Department of Zoology, The University, Sheffield, England (Received 2 March 1964) A b s t r a c t - - 1 . Biomphalaria sudanica, a swamp-dwelling planorbid from East Africa, was examined with respect to a number of factors related to respiratory gas exchange: duration of the dive; effect of buoyancy on diving behaviour; pulmonary gas composition and volume; oxygen capacity and haemoglobinoxygen equilibrium of the blood. 2. The principal respiratory characteristics of the environment are reviewed. 3. The results are compared with those previously obtained for Planorbis corneus and Lymnaea stagnalis. 4. It is concluded that Biomphalaria is as well but not better fitted than Planorbis for life in water devoid of dissolved oxygen. However, the higher oxygen affinity of haemoglobin will permit maximal exploitation of pulmonary oxygen at the higher ambient pCO2 and temperature which characterize the swamp habitat of Biomphalaria. INTRODUCTION FOLLOWING some earlier work on respiratory gas exchange in Planorbis corneus* and Lymnaea stagnalis* (Jones, 1961) the opportunity arose, through the kind invitation of Professor L. C. Beadle, to pay a short visit to Uganda and there to repeat m a n y of the observations on Biomphalaria sudanica.* T h i s small planorbid snail is a characteristic part of the limited fauna of papyrus swamp waters in East Africa, although it is also frequently found in less anoxic environments including irrigation ditches, ponds and the margins of the great lakes. Over considerable areas of East Africa B. sudanica is an intermediate host of the h u m a n blood fluke Schistosoma mansoni. Since the earlier observations on Planorbis and Lymnaea had been made in relation to their life in waters of normal or exceptionally high oxygen content, it appeared particularly interesting to investigate conditions in a closely related species able to live in water completely devoid of dissolved oxygen. All the earlier kinds of observation have been repeated with the exception of simultaneous pulmonary/cutaneous oxygen uptake measurements. In addition the oxygen equilibrium of Biomphalaria blood has been studied and some determinations made of the oxygen capacity of the blood. T h r o u g h o u t the account which follows the parallel data for Planorbis and Lymnaea are included for comparison; * Since there is no occasion to refer to any other species of these three genera, generic names alone will be used subsequently. 297
J.D. JONES
298
all are taken from the earlier account (Jones, 1961) and all references are to this paper unless otherwise stated. MATERIALS AND METHODS Much of the work was carried out in Kampala using specimens collected from nearby valley swamps, but some of the measurements were made or completed in Sheffield using a culture which was very successfully established with specimens from Kampala. This culture was kept at 26°C inbalanced aquariaunder fluorescent tube illumination; the usual calcium alginate gell was added once a week to supplement the natural algal food supply.
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Fro. 1. Tonometer and accessories for equilibration of Biomphalaria blood. The 2 ml Ostwald pipette is mounted in a water bath by means of rubber tubing (cross hatched) passing through rubber grommets. At the right the pipette rotates inside the tubing which forms a connexion to the gas burette; on the left the tubing grips the pipette tip (but can rotate inside the grommet) and forms a mechanical link (v/a the T-shaped glass gas outlet) to the motor spindle at the extreme left. The pipette stem is provided with a shallow cylindrical plastics jacket (seen in plan and side view) filled with glycerin. The rectangular base of this jacket provides a means of exact registration of the capillary bore under the microspectroscope. All the methods were the same as those used in the earlier work with the following exceptions. Oxygen capacity of the blood was determined by the microgasometric method of Natelson (Natelson & Menning, 1955) with modifications described in a foregoing paper (Jones, 1964). Oxygen dissociation curves were determined by the use of a 2 ml Ostwald pipette as a tonometer; this with its viewing chamber is shown mounted in a temperature-controlled water bath in Fig. 1. T h e sample of freshly drawn blood (ca. 0-05 m l - - a s much as can be obtained from a single animal by foot puncture) is run into the capillary stem (1-5 m m bore) above the bulb and allowed to run down to a fixed point for observation with the Zeiss spectroscopic ocular. Observation in this cylindrical "cell" is much improved by enclosing it in a plastics chamber filled with glycerin. T h e rectangular base in association with a mechanical
RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE~ BIOMPHALARIA SUDAN1CA
299
stage facilitates precisely repeatable alignment under the microscope objective For equilibration with the various gas mixtures, which are dispensed from a 100 ml gas burette, the sample is run into the bulb and the pipette rotated at 30 rev/min by the arrangement shown. The gas mixture is fushed through intermittently to a ~
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total of about 80 ml over 45 min, the pipette tap being closed between flushings. To eliminate evaporation of water from the sample the gas burette includes 2 or 3 ml of water to saturate the mixture. The exact composition of the gas mixtures is determined in the Lloyd gas-analyser (an improved type of Haldane apparatus-Lloyd, 1958) or in the mierogas-analyser of Seholander (1947). Percentage saturation of the equilibrated samples is determined by visual matching of absorption
300
J.D. JONES
bands against a comparison spectrum of an optical mixture of haemoglobin and oxyhaemoglobin. For the initial determinations made in Kampala this was done by using the conventional wedge trough; later in Sheffield a two-stage cup-andplunger colorimeter was used (Jones, 1955). Equilibrations were carried out at 16° and 26°C and at the higher temperature with 0, 10 and 20 mm CO 2. Pulmonary volume determinations were made in essentially the same manner as in the previous work. A closed system in which the pulmonary gas constitutes the only gas phase is reduced in volume by a standard amount and the ensuing pressure increase (read on a manometer) provides a measure of the volume of the gas. However, certain modifications were made because the pulmonary volume in Biomphalaria (<0"1 ml) is less than one-fifth of that found in the other species. The original manometer was therefore replaced with one of half the bore (0"5 mm) and the standard volume change was measured as 1.5 cm rise in this narrower tube instead of the original 3.0 cm. Since the observed pressure changes were in the present instance converted to volumes exclusively by the use of a calibration curve, a more precise method of calibration was required. This is simply achieved by introducing calibration bubbles into the apparatus by the withdrawal of accurately measured volumes of water with an "Agla" micrometer syringe. A specimen calibration curve is reproduced in Fig. 2. The original apparatus was also used as a respirometer for determination of simultaneous cutaneous/pulmonary oxygen uptake. An account of the modifications which have been made in this direction will be given elsewhere. THE ENVIRONMENT OF BIOMPHALARIA As stated above, B. sudanica is found in a variety of habitats in East Africa. In the present context the conditions in the most extreme environment (from a respiratory point of view) are of most interest. An excellent review of the physical nature and biology of the various kinds of Uganda swamps has been given by Beadle & Lind (1960) while Carter (1955) has presented a very large collection of data on the temperature, oxygen and carbon dioxide content and chemical composition, etc. of the various kinds of swamp water. The observations of these authors have been fully summarized elsewhere (Jones, 1963) and the principal relevant conclusions only are given below. The swamps fringe the great lakes and frequently, in a modified form, fill the valleys of the slow-flowing rivers which drain the shallow saucer between the two great rift valleys. In the landward parts of the littoral swamps, and more generally in the valley swamps, dissolved oxygen seldom exceeds 5 per cent of air saturation and is frequently absent altogether.* Carbon dioxide also accumulates to unusually high levels in many of these habitats, and Carter recorded values for free CO 2 of 33-58 ppm in littoral swamps, 13-75 ppm in valley swamps. The method used, involving titration with sodium carbonate against phenolphthalein, may give * Confirmed by personal observation--several samples taken from the water between the papyrus stems in valley swamps were quite devoid of oxygen. These and the swamp tank oxygen determinations were kindly carried out for me by Mr. John Wilson.
RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE, B I O M P H A L A R I A SUDA:VICA
301
spurious results if samples contain acids other than carbonic. Since organic acids (including humic acids) are known to occur in swamp waters, Carter's results may be somewhat too high. Allowing for the temperatures which were also recorded, his values correspond to ranges of 16-26 mm and 6-35 mm pCO2 respectively. Temperatures were in all cases measured before midday but over the greater part of a year and showed a range from 18 ° to 29°C (18°-24 ° in the valley swamps and the drier parts of the littoral swamps; 23°-29 ° in the outer parts of the littoral swamps). It is quite impossible to observe the snails in these swamp waters because of the presence of a thick surface scum of ferric hydroxide. Accordingly, many of the measurements which should ideally have been made in the field had to be performed with animals housed in an aquarium in which it was attempted to simulate natural swamp conditions. The tank, which measured 4 ft x 2 ft x 2 ft, contained large amounts of decaying swamp vegetation and numerous living papyrus rhizomes, some of which produced active growing aerial shoots in the laboratory. The typical surface scum soon appeared in this artificial swamp, but observation could be made through the glass sides of the tank below water level. Several water samples taken within 2 or 3 cm of the surface gave zero for dissolved oxygen when subjected to the Winkler procedure modified for swamp waters by Beadle (1958). Two similar samples were analysed for CO 2 by equilibrating an air bubble of about 0.02 ml with the 20 ml sample in a syringe. Part of the bubble was then transferred for analysis in Krogh's microgas-analyser. The resulting values of 3"0 and 2.9 °/o CO 2 are equivalent (at 660 mm b.p. and 24 mm vapour pressure) to 19-2 and 18.4 mm pCO2. RESPIRATORY BEHAVIOUR In general, the diving behaviour of Biomphalaria resembles that of Planorbis and Lymnaea. On most occasions lung filling is followed by a deliberate move away from the surface; creeping on the surface film was not observed in the artificial swamp and only occasionally in aquaria filled with clean water. There is, however, in this species a tendency for snails to leave the water now and then and to move around for some time on the debris above the water level. This perhaps is as much related to opportunities for feeding as to respiratory needs. Duration of the dive was measured in the swamp tank, where dissolved oxygen concentration was nil, and in a small aquarium containing clean well-aerated water. Both tanks were at room temperature (25°-26°C). The results together with those for Planorbis and Lymnaea are given in Table 1. There is no basis for statistical comparison of the Biomphalaria figures with the others, though it is clear that there is no striking difference between them. The difference between the two sets of Biomphalaria figures is highly significant in spite of the fact that the animals in the well-aerated tank probably had easier access to the surface than those in the swamp tank. The influence of buoyancy on diving behaviour was tested in exactly the same way as previously, and a typical record is shown in Fig. 3. To the left of the double
J.D.
302 TABLE ]--DURATION
pO2
Planorbis
OF DIVES ( M I N ) AT DIFFERENT DISSOLVED OXYGEN T E N S I O N S *
285 m m
P
115 m m
P
66"5 + 8-9
0"05
39"8 + 6-1
0'02
(5)
Lymnaea
JoNEs
(25)
(5)
66"1 + 30"7 (4)
(5)
p02
Biomphalaria
20'1 _+ 1-2
(26)
(5)
35"1 _ 2"7
(28)
28 m m
<0"01
20"3 +_2"1
(41)
(5)
140 m m
P
0 mm
38"5 +_2"9
-< 0"01
24"3 + 2"5
(12)
(68)
(18)
(8)
(61)
(28)
* T h e figures in brackets indicate the n u m b e r of animals and the total n u m b e r of dives observed respectively. M e a n and s.E. based on the sum of the m e a n dive durations for each animal in the group. Significance of the differences between the means at different PO~ values indicated by values of P ; interspecific differences are not significant.
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FI~. 3. Activity record for two Biomphalaria in the pressure apparatus. Increase and decrease of pressure occurred at the times indicated by the arrows. T h i c k e n e d portions of the record show upward m o v e m e n t s under increased pressure. Open circles mark moments of lung-filling. T i m e intervals of 5 min are marked above the record. For further description see the text.
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RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE~ B I O M P H A L A R I A S U D A N 1 C A
303
dotted line two animals were observed together in the pressure flask, and the responses to a 760 mm increase in pressure were prompt and very closely synchronized. Snail Ba withdrew into its shell for about 20 min but on emergence the close synchronization of the responses reappeared. Compression periods 5 and 8 were deliberately terminated while the snail(s) were crawling rapidly towards the surface. In each case the upward movement was immediately reversed as the shell swung round to lie above the animal, giving every appearance of acting like a rudder. The right-hand portions of the record are for the same snails observed separately with smaller pressure changes of 250 mm unless otherwise stated. Some of the responses were then less prompt. For the last occasion with Bz the flask was decompressed (from an excess of 760 ram) after the closure of the lung and departure from the surface. This resulted in the loss of some gas from the lung followed by an uncompleted excursion to the surface, as though the reduction of buoyancy from an abnormally high to a normal level started a surface-excursion response which was countermanded. Biomphalaria evidently resembles Planorbis and Lymnaea in the effect of pressure on diving behaviour and in all cases it is probable that the return to the surface results at least in part from the stimulus of reduced buoyancy as pulmonary oxygen is consumed. RESPIRATORY GAS EXCHANGE
Pulmonary gas composition Pulmonary gas composition was determined at the beginning and end of dives in the artificial swamp tank, avoiding those in which snails made use of the direct route to the surface via the glass side of the tank. In this way it was hoped to avoid the possibility of getting spuriously high fina[ pO2 values due to snails getting back to the surface abnormally quickly following the onset of the stimulus to return. The results should therefore approximate closely to those that would be found under wholly natural conditions. Some analyses were also made for Biomphalaria in small well-aerated aquaria as for the other species. Table 2 gives the results alongside the earlier figures, as percentage composition and as partial pressure. In converting to partial pressure, account has been taken of the fact that in the laboratory at Makerere College (at an altitude of ca. 4000 ft) the normal barometric pressure is only 660 ram. The initial pO 2 in the Biomphalaria lung is significantly lower than in Lymnaea ( P < 0.01 for habitat figures), but the percentage of O3 at the beginning of natural dives shows no significant interspecific differences; lung opening leaves the pulmonary gas equally far from equilibrium with the atmosphere in each species. Final oxygen is the same in Biomphalaria and Planorbis natural dives, very significantly below that of Lymnaea on either basis of computation. In aquarium dives, Planorbis final oxygen is higher for reasons previously discussed but this does not happen in Biomphalaria or Lymnaea. The carbon dioxide values bring out two interesting points on either basis of comparison. First, in well-aerated water (ditch or aquarium) all three species
J . D . JONES
304
have an appreciable amount of CO2 in the lung and it is significantly lower in
Biomphalaria. Secondly, the initial/final differences show clearly that only in Biomphalaria swamp dives is there a statistically significant increase during the dive, indicating a limited elimination of CO 2 via the lung. T h e final p C O 2 in this TABLE 2 - - I N I T I A L AND FINAL PULMONARY GAS COMPOSITION*
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Habitat
Species %
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113 +_3"8 58.6 _+6"2
16'2+-0"99 (6) 2"8 -+0"52
120 +_6"7
F
15-2+_0"51 (18) 7-9 +_0"84
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19.0+_0.12
141 +_0"9
18.2-+0'48
135 +_3'5
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10'2+-0-53
75-7+_3"9
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16"8_+1.04 (8) 2.9+_0-84 (7)
121 _+7"5
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20"8 +_3'9
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20.9-+6.1
17-1+_0"68 (11 ) 2-9+_0'46 (12)
65"3+_6"0 109 +_4"3 18'5_+2'9
F
2'0+_0-45 (18) 1"9_+0'19
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4'3+_0'4
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14"8_+3"3
8"8+_0"81
14'1+_1"4
1'2+0"19 (6) 1"3+_0'19
8"9+_1"4 9'7+_1"4
CO2 Lymnaea
Biomphalaria F
4'3+__0"6
1"7-+0"17 (11 ) 2.5+_0.12 (11)
10'8-+1"1 15.9+0.8
* Means and S.E.--number of observations in brackets unless the same as the line above. A sixth final 02 per cent figure for Planorbis in the ditch has been omitted; comparison of this value (11'2 per cent) with the other five indicates clearly that it does not belong to this population, d/a being 7'3 ! This animal was probably brought to the surface prematurely by mechanical loss of part of the pulmonary gas. All aquaria values in clean well-aerated water; Biomphalaria habitat values from the artificial swamp tank. case is in close agreement with the value found in the swamp tank water. It may appear surprising that, however low the final pCO2, there is not a small drop after lung filling, if only due to dilution b y the intake of a certain volume of atmospheric air. T h e probable explanation of this anomaly is apparent when it is r e m e m b e r e d that the so-called initial samples are, in all cases, removed from the lung up to 1 min after closure of the pulmonary aperture. During this short but inevitable
RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE, B I O M P H . , 4 L A R I A S U D A N I C A
305
delay, the pulmonary gas may well come into equilibrium with the blood in respect of CO2, while during the dive a constant pressure gradient of a few millimetres suffices to cause the elimination of respiratory CO2, via the skin and the accessory gill, as fast as it is formed. Comparison of the initial and final pCO2 values in wellaerated aquaria suggests that the gradient necessary to maintain this steady state is significantly smaller in Biomphalaria than in the other two species. This situation may be due to nothing more than the superior surface/volume ratio in Biomphalaria (ca. 0.5 g) compared with Lymnaea (ca. 3.5 g) and Planorbis (ca. 3.0 g). For the swamp species in its natural habitat, however, the external pCO2 is high enough to raise the final pulmonary pCO2 to ca. 16 mm when some loss on lung-filling does become apparent.
Pulmonary gas volume Values for pulmonary gas volume at the beginning and end of the dive are given in Table 3. Where possible, percentage change has been calculated and for each snail the mean pulmonary volume per g (total weight) is given. For the first two animals the final volumes are probably reliable because the pattern of the dive was clear-cut. The third snail behaved rather as did Lymnaea in this apparatus. TABLE 3--INITIAL
Snail wt. (g)
Duration of dive (min)
Initial volume (ml)
Final volume (ml) --
25 23 27
0.076 0.081 0.082 0.082 0'085
60 70 29
0.081 0.071 0"079 0"076 0'073
48 61 60 40 (92)
0.305
0.292
0'268
0-271 0-204
AND FINAL VOLUMES OF PULMONARY GAS*
0.074 0.077 0.078
% change
Pulmonary volume (ml/g)
9.8 6.1 9.1
0-23
0-069 0.064 0'066
12'7 15-8 9"6
0'26
0"073 0"074 0-075 0.071
0.057 0.059 0-059 0.052
21 "9 20-3 21-3 26"2
0"082
0.059
27.7
0-053 0.050
---
--
0"27 0"26 0.25
* P u l m o n a r y v o l u m e in t h e final c o l u m n is b a s e d on the m e a n initial v o l u m e for each snail. T h e single dive for t h e f o u r t h snail was i n c o m p l e t e (see text).
306
J.D. JONES
On its second dive it rose to the top of the chamber after 35 min and final volume was determined. On presentation with air, however, it declined to fill the lung and returned to the bottom. The same thing happened after a further 12 min and only after a total of 61 min was the lung refilled. In two subsequent dives, also, several such abortive returns to the surface were made before lung opening. The fourth snail had difficulty in maintaining its grip on the glass and floated to the top of the chamber several times. Even after 92 min this animal was still buoyant and still not interested in filling the lung. It was subsequently found to have an abnormally light shell--26 per cent of total weight compared with a mean of 32 per cent for the other four snails. Final volumes could not be determined for the fifth snail as the initial volumes were already at the limit of the calibration curve. In view of the doubt about the interpretation of the dives for the third and fourth snails it is not possible to be certain whether the percentage change in volume is significantly different from that in Planorbis (mean 8.9 per cent) but it seems improbable. The overall mean value for initial pulmonary volume per g is 0-25 ml/g, slightly but significantly higher than in Planorbis (0-22 ml/g).
HAEMOGLOBIN OF BIOMPHALARIA
Oxygen capacity The small size of the available Biomphalaria made it necessary to pool the blood of a number of animals for each determination and no attempt was made to determine the solubility of oxygen in this blood. From the determined total oxygen a deduction of 0.44 vol per cent is made; this is the value found for dissolved oxygen in Planorbis blood (Jones, 1964). The resultant values for combined oxygen capacity are 2.08 (3), 1"74 (3), 1.91 (4), 0"98 (5) and 1-57 (6) vol per cent (numbers of animals from which blood was pooled in brackets). All these values lie within the range found in Planorbis (0-94-2-49 vol per cent); the larger spread of values in the latter case is probably due to the less extensive pooling of blood with the larger animals. Clearly the range of oxygen capacity is very similar in these two species.
The haemoglobin-oxygen equilibrium in Biomphalaria blood Oxygen-dissociation curves for freshly drawn undiluted blood are shown in Fig. 4. There is a considerable scattering of the points which probably reflects the fact that separate individual blood samples were used for the determination of only one or two points in order to avoid the possibility of deterioration of the pigment at the relatively high temperatures used for most of the equilibrations. If Biomphalaria blood exhibits the same diversity of buffering power found by Zaaijer & Wolvekamp (1958) in Planorbis blood, considerable variation in pH will result from the equilibration of different samples at the same pCO2. Nevertheless, the present results give a reasonable picture of the likely state of the functional pigment and provide the basis for a simple comparison with Planorbis (see Discussion).
RESPIRATOR'/" GAS EXCHANGE I N THE AQUATIC PULMONATE) B I O M P t t A L A R I A S U D A N I C A
307
At 26°C all three curves are but slightly inflected, resembling the Planorbis curves at lower temperatures; only at 20°C do the Planorbis curves become obviously sigmoid. On a conventional linear transformation based on Hill's equation (log (y/100 - y ) v. log p) a slope indicating n = 1"4-1.5 is obtained for the Biomphalaria data at 26 ° and 10 mm pCO 2. The Bohr effect is well marked, Ps0 at 0, 10 and 20 mm pCO~ being 1.6, 3.4 and 6 mm respectively. Comparison with the two points determined at 16°C indicates that the temperature effect is normal in kind. I00]
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FIG. 4. Dissociation curves for undiluted blood of Biomphalaria. Small symbols-Kampala determinations; large symbols--Sheffield determinations. DISCUSSION In most of the respects for which a comparison is possible, the respiratory performances of Planorbis and Biomphalaria are strikingly similar. Pulmonary volume (and probably percentage change during the dive), initial and final pulmonary oxygen contents show no significant differences and the utilization of oxygen during the dive (Jones, 1964) is essentially the same. The oxygen capacity of the blood likewise appears to be the same in both species. There is nothing herc to indicate that Biomphalaria is in any way better fitted to exploit the possibilities of pulmonary gas exchange. In two respects only is there a significant difference between the species. The general pulmonary pCO2 in Biomphalaria is significantly higher than in Planorbis (or Lymnaea) and shows a significant increase during the dive. This increase (from 11 to 16 mm) is small compared with the drop in pO 2 (109-19 ram) so that CO 2 elimination via the lung is unimportant even in Biomphalaria, but it 21
J.D. JONES
308
does reflect the higher ambient pCO 2. With the ready loss of CO 2 via the skin and accessory gill, the vascular, pulmonary and ambient values of pCO 2 are unlikely to vary by more than a few millimetres, so that extremes of external CO e will provide a good guide to the conditions under which the respiratory pigment is called upon to work. The highest external pCO 2 recorded in the Planorbis studies was 15 mm at dawn in a ditch which was choked with aquatic plants. Planorbis will certainly spend much the greater part of its life in water with much lower pCO2. In the swamp waters, on the other hand, the diurnal fluctuation will be absent and pCO 2 will remain high, perhaps reaching 35 mm (Carter, 1955). 100 U
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FIG. 5. Comparison of dissociation curves for bloods of Biomphalaria ( ) and Planorbis ( . . . . ) at 26° and 20°C respectively. Data for Planorbis from Zaaijer & Wolvekamp (1958). In this context the second distinguishing feature is of particular interest. Fig. 5 shows the dissociation curves for Planorbis and Biomphalaria at 20 ° and 26°C respectively and at various pCOe levels. The data for Planorbis are taken from Zaaijer & Wolvekamp (1958) who did not study the Bohr effect above 20 °. For a strict comparison it might be necessary to imagine the Biornphalaria curves displaced somewhat to the left. On the other hand, the respective mean water temperatures in the temperate and tropical habitats are probably well represented by these two figures (see Jones, 1961, and the description of the swamp habitat above). The Pso values show a striking interspecific difference only in the absence of CO2, but in view of the different shapes of the upper parts of the curves it is clear that for any given value of pCO 2 the turnover of a substantial part of the combined
RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE~ B I O M P H . 4 L A R I A SUD.4NICA
309
oxygen can take place with markedly lower arterial pO 2 in the case of Biomphalaria. However, in view of the predominant difference in the ambient pCOz levels which characterize the respective habitats it is probably more meaningful to put this proposition the other way round: for a given arterial pO 2 Biomphalaria will be able to utilize the greater part of the dissociation range at considerably higher values of arterial pCO~ and of temperature than Planorbis. In conclusion it is suggested that there is no significant difference between Planorbis and Biomphalaria which can be related to the complete lack of dissolved oxygen in the swamp water habitat of the latter. It seems that Planorbis already possesses all the respiratory qualities necessary to make possible a reasonably aerobic life in oxygen-free water and Biomphalaria cannot do better than exploit these qualities to the full all the time. Only with respect to oxygen transport by the haemoglobin at generally higher levels of blood pCO 2 and temperature does Biomphalaria show any significant difference which can be related to the vicissitudes of the swamp environment. SUMMARY 1. Biomphalaria sudanica from the valley swamps of Uganda was examined in respect of a number of aspects of respiratory gas exchange and the results compared with those previously obtained for Planorbis corneus and Lymnaea stagnalis. 2. T h e haemoglobin-oxygen equilibrium and oxygen capacity of Biomphalaria blood were also investigated. 3. T h e respiratory characteristics of the swamp water environment are briefly summarized. 4. In most respects Biomphalaria closely resembles Planorbis and shows the same significant differences from Lymnaea. It is concluded that from the point of view of life in oxygen-free water Biomphalaria is as well, but not better, equipped as Planorbis. 5. T h e oxygen affinity of the haemoglobin is higher in Biomphalaria and this is seen as enabling utilization of the greater part of the dissociation range at higher levels of pCO~ and temperature than in Planorbis without raising the arterial pO 2. This will permit maximal use of pulmonary oxygen at the high ambient pCO~ which is a feature of the swamp habitat.
Acknowledgements--It is a great pleasure to be able to acknowledge my indebtedness to all those who contributed to the enjoyment and usefulness of my short stay at Makerere University College, Kampala. In particular I wish to thank Professor L. C. and Mrs. S. Beadle for their hospitality and help and encouragement in the work; Mr. T. R. Milburn (Department of Botany) for much practical help and encouragement; Mr. Semakula and his technical staff in the Department of Zoology for smoothing my path in many ways ; all staff and student members of Livingstone Hall for the delightful characteristics of my nonworking environment. I also wish to acknowledge my indebtedness to The Commonwealth Universities Interchange Scheme and to the Tropical Medicine Research Board and Department of Technical Co-operation for grants to cover travelling, living and research expenses respectively.
310
J . D . JONES
REFERENCES BEADLE L. C. (1958) The measurement of oxygen in swamp waters. Further modification of the Winkler method, ft. Exp. Biol. 35, 556-566. BEADLE L. C. & LIND E. M. (1960) Research on the swamps of Uganda. Uganda ft. 24, 84-98. CARTER G. S. (1955) The Papyrus Swamps of Uganda. Heifer, Cambridge. JONES J. D. (1955) Observations on the respiratory physiology and on the haemoglobin of the polychaete genus Nephthys, with special reference to N. hombergii (Aud. et M.-Edw.). ft. Exp. Biol. 32, 110-125. JONES J. D. (1961) Aspects of respiration in Planorbis corneus L. and Lymnaea stagnalis L. (Gastropoda: Pulmonata). Comp. Biochem. Physiol. 4, 1-29. JONES J. D. (1963) A study of the respiration of some aquatic pulmonates with special reference to the role of haemoglobin in Planorbis corneus. Thesis, University of Sheffield. JONES J. D. (1964) The role of haemoglobin in the aquatic pulmonate Planorbis corneus L. Comp. Biochem. Physiol. 12, 283-295. LLOYD B. B. (1958) A development of Haldane's gas-analysis apparatus. J. Physiol. 143, 5-6. NATELSON S. & MENNINC C. M. (1955) Improved methods of analysis for oxygen, carbon monoxide and iron on fingertip blood. Clin. Chem. 1, 165-179. SCROLANDE8 P. F. (1947) Analyser for accurate estimation of respiratory gases in one-half cubic centimeter samples, ft. Biol. Chem. 167, 235-250. ZAAIJER J. J. P. & WOLVEKAMP H. P. (1958) Some experiments on the haemoglobin-oxygen equilibrium in the blood of the ramshorn (Planorbis corneus L.). Acta Physiol. Pharmacol. N~erl. 7, 56-77.
RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE, B I O M P H A L A R I A S U D A N I C A j. D. J O N E S Department of Zoology, The University, Sheffield, England (Received 2 March 1964) A b s t r a c t - - 1 . Biomphalaria sudanica, a swamp-dwelling planorbid from East Africa, was examined with respect to a number of factors related to respiratory gas exchange: duration of the dive; effect of buoyancy on diving behaviour; pulmonary gas composition and volume; oxygen capacity and haemoglobinoxygen equilibrium of the blood. 2. The principal respiratory characteristics of the environment are reviewed. 3. The results are compared with those previously obtained for Planorbis corneus and Lymnaea stagnalis. 4. It is concluded that Biomphalaria is as well but not better fitted than Planorbis for life in water devoid of dissolved oxygen. However, the higher oxygen affinity of haemoglobin will permit maximal exploitation of pulmonary oxygen at the higher ambient pCO2 and temperature which characterize the swamp habitat of Biomphalaria. INTRODUCTION FOLLOWING some earlier work on respiratory gas exchange in Planorbis corneus* and Lymnaea stagnalis* (Jones, 1961) the opportunity arose, through the kind invitation of Professor L. C. Beadle, to pay a short visit to Uganda and there to repeat m a n y of the observations on Biomphalaria sudanica.* T h i s small planorbid snail is a characteristic part of the limited fauna of papyrus swamp waters in East Africa, although it is also frequently found in less anoxic environments including irrigation ditches, ponds and the margins of the great lakes. Over considerable areas of East Africa B. sudanica is an intermediate host of the h u m a n blood fluke Schistosoma mansoni. Since the earlier observations on Planorbis and Lymnaea had been made in relation to their life in waters of normal or exceptionally high oxygen content, it appeared particularly interesting to investigate conditions in a closely related species able to live in water completely devoid of dissolved oxygen. All the earlier kinds of observation have been repeated with the exception of simultaneous pulmonary/cutaneous oxygen uptake measurements. In addition the oxygen equilibrium of Biomphalaria blood has been studied and some determinations made of the oxygen capacity of the blood. T h r o u g h o u t the account which follows the parallel data for Planorbis and Lymnaea are included for comparison; * Since there is no occasion to refer to any other species of these three genera, generic names alone will be used subsequently. 297
J.D. JONES
298
all are taken from the earlier account (Jones, 1961) and all references are to this paper unless otherwise stated. MATERIALS AND METHODS Much of the work was carried out in Kampala using specimens collected from nearby valley swamps, but some of the measurements were made or completed in Sheffield using a culture which was very successfully established with specimens from Kampala. This culture was kept at 26°C inbalanced aquariaunder fluorescent tube illumination; the usual calcium alginate gell was added once a week to supplement the natural algal food supply.
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Fro. 1. Tonometer and accessories for equilibration of Biomphalaria blood. The 2 ml Ostwald pipette is mounted in a water bath by means of rubber tubing (cross hatched) passing through rubber grommets. At the right the pipette rotates inside the tubing which forms a connexion to the gas burette; on the left the tubing grips the pipette tip (but can rotate inside the grommet) and forms a mechanical link (v/a the T-shaped glass gas outlet) to the motor spindle at the extreme left. The pipette stem is provided with a shallow cylindrical plastics jacket (seen in plan and side view) filled with glycerin. The rectangular base of this jacket provides a means of exact registration of the capillary bore under the microspectroscope. All the methods were the same as those used in the earlier work with the following exceptions. Oxygen capacity of the blood was determined by the microgasometric method of Natelson (Natelson & Menning, 1955) with modifications described in a foregoing paper (Jones, 1964). Oxygen dissociation curves were determined by the use of a 2 ml Ostwald pipette as a tonometer; this with its viewing chamber is shown mounted in a temperature-controlled water bath in Fig. 1. T h e sample of freshly drawn blood (ca. 0-05 m l - - a s much as can be obtained from a single animal by foot puncture) is run into the capillary stem (1-5 m m bore) above the bulb and allowed to run down to a fixed point for observation with the Zeiss spectroscopic ocular. Observation in this cylindrical "cell" is much improved by enclosing it in a plastics chamber filled with glycerin. T h e rectangular base in association with a mechanical
RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE~ BIOMPHALARIA SUDAN1CA
299
stage facilitates precisely repeatable alignment under the microscope objective For equilibration with the various gas mixtures, which are dispensed from a 100 ml gas burette, the sample is run into the bulb and the pipette rotated at 30 rev/min by the arrangement shown. The gas mixture is fushed through intermittently to a ~
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total of about 80 ml over 45 min, the pipette tap being closed between flushings. To eliminate evaporation of water from the sample the gas burette includes 2 or 3 ml of water to saturate the mixture. The exact composition of the gas mixtures is determined in the Lloyd gas-analyser (an improved type of Haldane apparatus-Lloyd, 1958) or in the mierogas-analyser of Seholander (1947). Percentage saturation of the equilibrated samples is determined by visual matching of absorption
300
J.D. JONES
bands against a comparison spectrum of an optical mixture of haemoglobin and oxyhaemoglobin. For the initial determinations made in Kampala this was done by using the conventional wedge trough; later in Sheffield a two-stage cup-andplunger colorimeter was used (Jones, 1955). Equilibrations were carried out at 16° and 26°C and at the higher temperature with 0, 10 and 20 mm CO 2. Pulmonary volume determinations were made in essentially the same manner as in the previous work. A closed system in which the pulmonary gas constitutes the only gas phase is reduced in volume by a standard amount and the ensuing pressure increase (read on a manometer) provides a measure of the volume of the gas. However, certain modifications were made because the pulmonary volume in Biomphalaria (<0"1 ml) is less than one-fifth of that found in the other species. The original manometer was therefore replaced with one of half the bore (0"5 mm) and the standard volume change was measured as 1.5 cm rise in this narrower tube instead of the original 3.0 cm. Since the observed pressure changes were in the present instance converted to volumes exclusively by the use of a calibration curve, a more precise method of calibration was required. This is simply achieved by introducing calibration bubbles into the apparatus by the withdrawal of accurately measured volumes of water with an "Agla" micrometer syringe. A specimen calibration curve is reproduced in Fig. 2. The original apparatus was also used as a respirometer for determination of simultaneous cutaneous/pulmonary oxygen uptake. An account of the modifications which have been made in this direction will be given elsewhere. THE ENVIRONMENT OF BIOMPHALARIA As stated above, B. sudanica is found in a variety of habitats in East Africa. In the present context the conditions in the most extreme environment (from a respiratory point of view) are of most interest. An excellent review of the physical nature and biology of the various kinds of Uganda swamps has been given by Beadle & Lind (1960) while Carter (1955) has presented a very large collection of data on the temperature, oxygen and carbon dioxide content and chemical composition, etc. of the various kinds of swamp water. The observations of these authors have been fully summarized elsewhere (Jones, 1963) and the principal relevant conclusions only are given below. The swamps fringe the great lakes and frequently, in a modified form, fill the valleys of the slow-flowing rivers which drain the shallow saucer between the two great rift valleys. In the landward parts of the littoral swamps, and more generally in the valley swamps, dissolved oxygen seldom exceeds 5 per cent of air saturation and is frequently absent altogether.* Carbon dioxide also accumulates to unusually high levels in many of these habitats, and Carter recorded values for free CO 2 of 33-58 ppm in littoral swamps, 13-75 ppm in valley swamps. The method used, involving titration with sodium carbonate against phenolphthalein, may give * Confirmed by personal observation--several samples taken from the water between the papyrus stems in valley swamps were quite devoid of oxygen. These and the swamp tank oxygen determinations were kindly carried out for me by Mr. John Wilson.
RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE, B I O M P H A L A R I A SUDA:VICA
301
spurious results if samples contain acids other than carbonic. Since organic acids (including humic acids) are known to occur in swamp waters, Carter's results may be somewhat too high. Allowing for the temperatures which were also recorded, his values correspond to ranges of 16-26 mm and 6-35 mm pCO2 respectively. Temperatures were in all cases measured before midday but over the greater part of a year and showed a range from 18 ° to 29°C (18°-24 ° in the valley swamps and the drier parts of the littoral swamps; 23°-29 ° in the outer parts of the littoral swamps). It is quite impossible to observe the snails in these swamp waters because of the presence of a thick surface scum of ferric hydroxide. Accordingly, many of the measurements which should ideally have been made in the field had to be performed with animals housed in an aquarium in which it was attempted to simulate natural swamp conditions. The tank, which measured 4 ft x 2 ft x 2 ft, contained large amounts of decaying swamp vegetation and numerous living papyrus rhizomes, some of which produced active growing aerial shoots in the laboratory. The typical surface scum soon appeared in this artificial swamp, but observation could be made through the glass sides of the tank below water level. Several water samples taken within 2 or 3 cm of the surface gave zero for dissolved oxygen when subjected to the Winkler procedure modified for swamp waters by Beadle (1958). Two similar samples were analysed for CO 2 by equilibrating an air bubble of about 0.02 ml with the 20 ml sample in a syringe. Part of the bubble was then transferred for analysis in Krogh's microgas-analyser. The resulting values of 3"0 and 2.9 °/o CO 2 are equivalent (at 660 mm b.p. and 24 mm vapour pressure) to 19-2 and 18.4 mm pCO2. RESPIRATORY BEHAVIOUR In general, the diving behaviour of Biomphalaria resembles that of Planorbis and Lymnaea. On most occasions lung filling is followed by a deliberate move away from the surface; creeping on the surface film was not observed in the artificial swamp and only occasionally in aquaria filled with clean water. There is, however, in this species a tendency for snails to leave the water now and then and to move around for some time on the debris above the water level. This perhaps is as much related to opportunities for feeding as to respiratory needs. Duration of the dive was measured in the swamp tank, where dissolved oxygen concentration was nil, and in a small aquarium containing clean well-aerated water. Both tanks were at room temperature (25°-26°C). The results together with those for Planorbis and Lymnaea are given in Table 1. There is no basis for statistical comparison of the Biomphalaria figures with the others, though it is clear that there is no striking difference between them. The difference between the two sets of Biomphalaria figures is highly significant in spite of the fact that the animals in the well-aerated tank probably had easier access to the surface than those in the swamp tank. The influence of buoyancy on diving behaviour was tested in exactly the same way as previously, and a typical record is shown in Fig. 3. To the left of the double
J.D.
302 TABLE ]--DURATION
pO2
Planorbis
OF DIVES ( M I N ) AT DIFFERENT DISSOLVED OXYGEN T E N S I O N S *
285 m m
P
115 m m
P
66"5 + 8-9
0"05
39"8 + 6-1
0'02
(5)
Lymnaea
JoNEs
(25)
(5)
66"1 + 30"7 (4)
(5)
p02
Biomphalaria
20'1 _+ 1-2
(26)
(5)
35"1 _ 2"7
(28)
28 m m
<0"01
20"3 +_2"1
(41)
(5)
140 m m
P
0 mm
38"5 +_2"9
-< 0"01
24"3 + 2"5
(12)
(68)
(18)
(8)
(61)
(28)
* T h e figures in brackets indicate the n u m b e r of animals and the total n u m b e r of dives observed respectively. M e a n and s.E. based on the sum of the m e a n dive durations for each animal in the group. Significance of the differences between the means at different PO~ values indicated by values of P ; interspecific differences are not significant.
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FI~. 3. Activity record for two Biomphalaria in the pressure apparatus. Increase and decrease of pressure occurred at the times indicated by the arrows. T h i c k e n e d portions of the record show upward m o v e m e n t s under increased pressure. Open circles mark moments of lung-filling. T i m e intervals of 5 min are marked above the record. For further description see the text.
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RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE~ B I O M P H A L A R I A S U D A N 1 C A
303
dotted line two animals were observed together in the pressure flask, and the responses to a 760 mm increase in pressure were prompt and very closely synchronized. Snail Ba withdrew into its shell for about 20 min but on emergence the close synchronization of the responses reappeared. Compression periods 5 and 8 were deliberately terminated while the snail(s) were crawling rapidly towards the surface. In each case the upward movement was immediately reversed as the shell swung round to lie above the animal, giving every appearance of acting like a rudder. The right-hand portions of the record are for the same snails observed separately with smaller pressure changes of 250 mm unless otherwise stated. Some of the responses were then less prompt. For the last occasion with Bz the flask was decompressed (from an excess of 760 ram) after the closure of the lung and departure from the surface. This resulted in the loss of some gas from the lung followed by an uncompleted excursion to the surface, as though the reduction of buoyancy from an abnormally high to a normal level started a surface-excursion response which was countermanded. Biomphalaria evidently resembles Planorbis and Lymnaea in the effect of pressure on diving behaviour and in all cases it is probable that the return to the surface results at least in part from the stimulus of reduced buoyancy as pulmonary oxygen is consumed. RESPIRATORY GAS EXCHANGE
Pulmonary gas composition Pulmonary gas composition was determined at the beginning and end of dives in the artificial swamp tank, avoiding those in which snails made use of the direct route to the surface via the glass side of the tank. In this way it was hoped to avoid the possibility of getting spuriously high fina[ pO2 values due to snails getting back to the surface abnormally quickly following the onset of the stimulus to return. The results should therefore approximate closely to those that would be found under wholly natural conditions. Some analyses were also made for Biomphalaria in small well-aerated aquaria as for the other species. Table 2 gives the results alongside the earlier figures, as percentage composition and as partial pressure. In converting to partial pressure, account has been taken of the fact that in the laboratory at Makerere College (at an altitude of ca. 4000 ft) the normal barometric pressure is only 660 ram. The initial pO 2 in the Biomphalaria lung is significantly lower than in Lymnaea ( P < 0.01 for habitat figures), but the percentage of O3 at the beginning of natural dives shows no significant interspecific differences; lung opening leaves the pulmonary gas equally far from equilibrium with the atmosphere in each species. Final oxygen is the same in Biomphalaria and Planorbis natural dives, very significantly below that of Lymnaea on either basis of computation. In aquarium dives, Planorbis final oxygen is higher for reasons previously discussed but this does not happen in Biomphalaria or Lymnaea. The carbon dioxide values bring out two interesting points on either basis of comparison. First, in well-aerated water (ditch or aquarium) all three species
J . D . JONES
304
have an appreciable amount of CO2 in the lung and it is significantly lower in
Biomphalaria. Secondly, the initial/final differences show clearly that only in Biomphalaria swamp dives is there a statistically significant increase during the dive, indicating a limited elimination of CO 2 via the lung. T h e final p C O 2 in this TABLE 2 - - I N I T I A L AND FINAL PULMONARY GAS COMPOSITION*
Aquaria
Habitat
Species %
p.p. (ram)
o, /o
p.p. (mm)
113 +_3"8 58.6 _+6"2
16'2+-0"99 (6) 2"8 -+0"52
120 +_6"7
F
15-2+_0"51 (18) 7-9 +_0"84
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19.0+_0.12
141 +_0"9
18.2-+0'48
135 +_3'5
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10'2+-0-53
75-7+_3"9
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16"8_+1.04 (8) 2.9+_0-84 (7)
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20"8 +_3'9
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20.9-+6.1
17-1+_0"68 (11 ) 2-9+_0'46 (12)
65"3+_6"0 109 +_4"3 18'5_+2'9
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1"1+_0'12
8'2+0"9
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4'3+_0'4
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14"8_+3"3
8"8+_0"81
14'1+_1"4
1'2+0"19 (6) 1"3+_0'19
8"9+_1"4 9'7+_1"4
CO2 Lymnaea
Biomphalaria F
4'3+__0"6
1"7-+0"17 (11 ) 2.5+_0.12 (11)
10'8-+1"1 15.9+0.8
* Means and S.E.--number of observations in brackets unless the same as the line above. A sixth final 02 per cent figure for Planorbis in the ditch has been omitted; comparison of this value (11'2 per cent) with the other five indicates clearly that it does not belong to this population, d/a being 7'3 ! This animal was probably brought to the surface prematurely by mechanical loss of part of the pulmonary gas. All aquaria values in clean well-aerated water; Biomphalaria habitat values from the artificial swamp tank. case is in close agreement with the value found in the swamp tank water. It may appear surprising that, however low the final pCO2, there is not a small drop after lung filling, if only due to dilution b y the intake of a certain volume of atmospheric air. T h e probable explanation of this anomaly is apparent when it is r e m e m b e r e d that the so-called initial samples are, in all cases, removed from the lung up to 1 min after closure of the pulmonary aperture. During this short but inevitable
RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE, B I O M P H . , 4 L A R I A S U D A N I C A
305
delay, the pulmonary gas may well come into equilibrium with the blood in respect of CO2, while during the dive a constant pressure gradient of a few millimetres suffices to cause the elimination of respiratory CO2, via the skin and the accessory gill, as fast as it is formed. Comparison of the initial and final pCO2 values in wellaerated aquaria suggests that the gradient necessary to maintain this steady state is significantly smaller in Biomphalaria than in the other two species. This situation may be due to nothing more than the superior surface/volume ratio in Biomphalaria (ca. 0.5 g) compared with Lymnaea (ca. 3.5 g) and Planorbis (ca. 3.0 g). For the swamp species in its natural habitat, however, the external pCO2 is high enough to raise the final pulmonary pCO2 to ca. 16 mm when some loss on lung-filling does become apparent.
Pulmonary gas volume Values for pulmonary gas volume at the beginning and end of the dive are given in Table 3. Where possible, percentage change has been calculated and for each snail the mean pulmonary volume per g (total weight) is given. For the first two animals the final volumes are probably reliable because the pattern of the dive was clear-cut. The third snail behaved rather as did Lymnaea in this apparatus. TABLE 3--INITIAL
Snail wt. (g)
Duration of dive (min)
Initial volume (ml)
Final volume (ml) --
25 23 27
0.076 0.081 0.082 0.082 0'085
60 70 29
0.081 0.071 0"079 0"076 0'073
48 61 60 40 (92)
0.305
0.292
0'268
0-271 0-204
AND FINAL VOLUMES OF PULMONARY GAS*
0.074 0.077 0.078
% change
Pulmonary volume (ml/g)
9.8 6.1 9.1
0-23
0-069 0.064 0'066
12'7 15-8 9"6
0'26
0"073 0"074 0-075 0.071
0.057 0.059 0-059 0.052
21 "9 20-3 21-3 26"2
0"082
0.059
27.7
0-053 0.050
---
--
0"27 0"26 0.25
* P u l m o n a r y v o l u m e in t h e final c o l u m n is b a s e d on the m e a n initial v o l u m e for each snail. T h e single dive for t h e f o u r t h snail was i n c o m p l e t e (see text).
306
J.D. JONES
On its second dive it rose to the top of the chamber after 35 min and final volume was determined. On presentation with air, however, it declined to fill the lung and returned to the bottom. The same thing happened after a further 12 min and only after a total of 61 min was the lung refilled. In two subsequent dives, also, several such abortive returns to the surface were made before lung opening. The fourth snail had difficulty in maintaining its grip on the glass and floated to the top of the chamber several times. Even after 92 min this animal was still buoyant and still not interested in filling the lung. It was subsequently found to have an abnormally light shell--26 per cent of total weight compared with a mean of 32 per cent for the other four snails. Final volumes could not be determined for the fifth snail as the initial volumes were already at the limit of the calibration curve. In view of the doubt about the interpretation of the dives for the third and fourth snails it is not possible to be certain whether the percentage change in volume is significantly different from that in Planorbis (mean 8.9 per cent) but it seems improbable. The overall mean value for initial pulmonary volume per g is 0-25 ml/g, slightly but significantly higher than in Planorbis (0-22 ml/g).
HAEMOGLOBIN OF BIOMPHALARIA
Oxygen capacity The small size of the available Biomphalaria made it necessary to pool the blood of a number of animals for each determination and no attempt was made to determine the solubility of oxygen in this blood. From the determined total oxygen a deduction of 0.44 vol per cent is made; this is the value found for dissolved oxygen in Planorbis blood (Jones, 1964). The resultant values for combined oxygen capacity are 2.08 (3), 1"74 (3), 1.91 (4), 0"98 (5) and 1-57 (6) vol per cent (numbers of animals from which blood was pooled in brackets). All these values lie within the range found in Planorbis (0-94-2-49 vol per cent); the larger spread of values in the latter case is probably due to the less extensive pooling of blood with the larger animals. Clearly the range of oxygen capacity is very similar in these two species.
The haemoglobin-oxygen equilibrium in Biomphalaria blood Oxygen-dissociation curves for freshly drawn undiluted blood are shown in Fig. 4. There is a considerable scattering of the points which probably reflects the fact that separate individual blood samples were used for the determination of only one or two points in order to avoid the possibility of deterioration of the pigment at the relatively high temperatures used for most of the equilibrations. If Biomphalaria blood exhibits the same diversity of buffering power found by Zaaijer & Wolvekamp (1958) in Planorbis blood, considerable variation in pH will result from the equilibration of different samples at the same pCO2. Nevertheless, the present results give a reasonable picture of the likely state of the functional pigment and provide the basis for a simple comparison with Planorbis (see Discussion).
RESPIRATOR'/" GAS EXCHANGE I N THE AQUATIC PULMONATE) B I O M P t t A L A R I A S U D A N I C A
307
At 26°C all three curves are but slightly inflected, resembling the Planorbis curves at lower temperatures; only at 20°C do the Planorbis curves become obviously sigmoid. On a conventional linear transformation based on Hill's equation (log (y/100 - y ) v. log p) a slope indicating n = 1"4-1.5 is obtained for the Biomphalaria data at 26 ° and 10 mm pCO 2. The Bohr effect is well marked, Ps0 at 0, 10 and 20 mm pCO~ being 1.6, 3.4 and 6 mm respectively. Comparison with the two points determined at 16°C indicates that the temperature effect is normal in kind. I00]
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FIG. 4. Dissociation curves for undiluted blood of Biomphalaria. Small symbols-Kampala determinations; large symbols--Sheffield determinations. DISCUSSION In most of the respects for which a comparison is possible, the respiratory performances of Planorbis and Biomphalaria are strikingly similar. Pulmonary volume (and probably percentage change during the dive), initial and final pulmonary oxygen contents show no significant differences and the utilization of oxygen during the dive (Jones, 1964) is essentially the same. The oxygen capacity of the blood likewise appears to be the same in both species. There is nothing herc to indicate that Biomphalaria is in any way better fitted to exploit the possibilities of pulmonary gas exchange. In two respects only is there a significant difference between the species. The general pulmonary pCO2 in Biomphalaria is significantly higher than in Planorbis (or Lymnaea) and shows a significant increase during the dive. This increase (from 11 to 16 mm) is small compared with the drop in pO 2 (109-19 ram) so that CO 2 elimination via the lung is unimportant even in Biomphalaria, but it 21
J.D. JONES
308
does reflect the higher ambient pCO 2. With the ready loss of CO 2 via the skin and accessory gill, the vascular, pulmonary and ambient values of pCO 2 are unlikely to vary by more than a few millimetres, so that extremes of external CO e will provide a good guide to the conditions under which the respiratory pigment is called upon to work. The highest external pCO 2 recorded in the Planorbis studies was 15 mm at dawn in a ditch which was choked with aquatic plants. Planorbis will certainly spend much the greater part of its life in water with much lower pCO2. In the swamp waters, on the other hand, the diurnal fluctuation will be absent and pCO 2 will remain high, perhaps reaching 35 mm (Carter, 1955). 100 U
=
/
,'/ "
/
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20 m m CO 2
.~ 60
.........
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20
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0
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4
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FIG. 5. Comparison of dissociation curves for bloods of Biomphalaria ( ) and Planorbis ( . . . . ) at 26° and 20°C respectively. Data for Planorbis from Zaaijer & Wolvekamp (1958). In this context the second distinguishing feature is of particular interest. Fig. 5 shows the dissociation curves for Planorbis and Biomphalaria at 20 ° and 26°C respectively and at various pCOe levels. The data for Planorbis are taken from Zaaijer & Wolvekamp (1958) who did not study the Bohr effect above 20 °. For a strict comparison it might be necessary to imagine the Biornphalaria curves displaced somewhat to the left. On the other hand, the respective mean water temperatures in the temperate and tropical habitats are probably well represented by these two figures (see Jones, 1961, and the description of the swamp habitat above). The Pso values show a striking interspecific difference only in the absence of CO2, but in view of the different shapes of the upper parts of the curves it is clear that for any given value of pCO 2 the turnover of a substantial part of the combined
RESPIRATORY GAS EXCHANGE IN THE AQUATIC PULMONATE~ B I O M P H . 4 L A R I A SUD.4NICA
309
oxygen can take place with markedly lower arterial pO 2 in the case of Biomphalaria. However, in view of the predominant difference in the ambient pCOz levels which characterize the respective habitats it is probably more meaningful to put this proposition the other way round: for a given arterial pO 2 Biomphalaria will be able to utilize the greater part of the dissociation range at considerably higher values of arterial pCO~ and of temperature than Planorbis. In conclusion it is suggested that there is no significant difference between Planorbis and Biomphalaria which can be related to the complete lack of dissolved oxygen in the swamp water habitat of the latter. It seems that Planorbis already possesses all the respiratory qualities necessary to make possible a reasonably aerobic life in oxygen-free water and Biomphalaria cannot do better than exploit these qualities to the full all the time. Only with respect to oxygen transport by the haemoglobin at generally higher levels of blood pCO 2 and temperature does Biomphalaria show any significant difference which can be related to the vicissitudes of the swamp environment. SUMMARY 1. Biomphalaria sudanica from the valley swamps of Uganda was examined in respect of a number of aspects of respiratory gas exchange and the results compared with those previously obtained for Planorbis corneus and Lymnaea stagnalis. 2. T h e haemoglobin-oxygen equilibrium and oxygen capacity of Biomphalaria blood were also investigated. 3. T h e respiratory characteristics of the swamp water environment are briefly summarized. 4. In most respects Biomphalaria closely resembles Planorbis and shows the same significant differences from Lymnaea. It is concluded that from the point of view of life in oxygen-free water Biomphalaria is as well, but not better, equipped as Planorbis. 5. T h e oxygen affinity of the haemoglobin is higher in Biomphalaria and this is seen as enabling utilization of the greater part of the dissociation range at higher levels of pCO~ and temperature than in Planorbis without raising the arterial pO 2. This will permit maximal use of pulmonary oxygen at the high ambient pCO~ which is a feature of the swamp habitat.
Acknowledgements--It is a great pleasure to be able to acknowledge my indebtedness to all those who contributed to the enjoyment and usefulness of my short stay at Makerere University College, Kampala. In particular I wish to thank Professor L. C. and Mrs. S. Beadle for their hospitality and help and encouragement in the work; Mr. T. R. Milburn (Department of Botany) for much practical help and encouragement; Mr. Semakula and his technical staff in the Department of Zoology for smoothing my path in many ways ; all staff and student members of Livingstone Hall for the delightful characteristics of my nonworking environment. I also wish to acknowledge my indebtedness to The Commonwealth Universities Interchange Scheme and to the Tropical Medicine Research Board and Department of Technical Co-operation for grants to cover travelling, living and research expenses respectively.
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J . D . JONES
REFERENCES BEADLE L. C. (1958) The measurement of oxygen in swamp waters. Further modification of the Winkler method, ft. Exp. Biol. 35, 556-566. BEADLE L. C. & LIND E. M. (1960) Research on the swamps of Uganda. Uganda ft. 24, 84-98. CARTER G. S. (1955) The Papyrus Swamps of Uganda. Heifer, Cambridge. JONES J. D. (1955) Observations on the respiratory physiology and on the haemoglobin of the polychaete genus Nephthys, with special reference to N. hombergii (Aud. et M.-Edw.). ft. Exp. Biol. 32, 110-125. JONES J. D. (1961) Aspects of respiration in Planorbis corneus L. and Lymnaea stagnalis L. (Gastropoda: Pulmonata). Comp. Biochem. Physiol. 4, 1-29. JONES J. D. (1963) A study of the respiration of some aquatic pulmonates with special reference to the role of haemoglobin in Planorbis corneus. Thesis, University of Sheffield. JONES J. D. (1964) The role of haemoglobin in the aquatic pulmonate Planorbis corneus L. Comp. Biochem. Physiol. 12, 283-295. LLOYD B. B. (1958) A development of Haldane's gas-analysis apparatus. J. Physiol. 143, 5-6. NATELSON S. & MENNINC C. M. (1955) Improved methods of analysis for oxygen, carbon monoxide and iron on fingertip blood. Clin. Chem. 1, 165-179. SCROLANDE8 P. F. (1947) Analyser for accurate estimation of respiratory gases in one-half cubic centimeter samples, ft. Biol. Chem. 167, 235-250. ZAAIJER J. J. P. & WOLVEKAMP H. P. (1958) Some experiments on the haemoglobin-oxygen equilibrium in the blood of the ramshorn (Planorbis corneus L.). Acta Physiol. Pharmacol. N~erl. 7, 56-77.