- Email: [email protected]
The role of haemoglobin in the aquatic pulmonate, Planorbis corneus
Comp. Biochem. Physiol., 1964, Vol. 12, pp. 283 to 295. Pergamon Press Ltd. Printed in Great Britain
T H E ROLE OF HAEMOGLOBIN IN THE AQUATIC PULMONATE, P L A N O R B I S C O R N E U S j. D. JONES Department of Zoology, The University of Sheffield, England (Received 2 March 1964)
A b s t r a c t - - 1 . Limiting external pO2's have been determined for full and zero saturation of arterial and venous blood of Planorbis corneus and the pO 2 range for pigment function has been deduced. 2. For oxygen uptake from the lung the pigment is functional over the pulmonary pO2 range 20-60 mm; this is precisely the region over which Planorbis pulmonary oxygen utilization exceeds that in Lymnaea stagnalis lacking a respiratory pigment. 3. Cutaneous uptake is facilitated by haemoglobin down to a dissolved pO2 of about 30 mm. 4. The oxygen capacity of Planorbis blood was found to range from 0'94 to 2-49 vol per cent; the question of a possible storage function is discussed. INTRODUCTION IN AN earlier paper (Jones, 1961) some observations on the respiration of Planorbis corneus* and L y m n a e a stagnalis* were reported. Various aspects of gas exchange were examined in an attempt to form an overall picture from the various factors involved, in strict relation to the normal diving behaviour of these aquatic pulmonates. T h e principal conclusions based on that work were as follows: (1) total oxygen uptake is independent of the tension of dissolved oxygen because, as cutaneous uptake falls with declining dissolved pO2, pulmonary uptake increases in a complementary fashion; (2) in neither case is cutaneous uptake fully adequate even when dissolved pO2 is as high as 240 m m ; (3) pulmonary uptake is slightly more important in L y m n a e a than in Planorbis at all values of dissolved pO 2 up to this limit of observation; (4) the two species commence the dive with a similar slight deficiency (compared with the atmosphere) in pulmonary oxygen, but the final pulmonary oxygen content in Planorbis (2.8 per cent = 21 m m ) is very significantly lower than in L y m n a e a (8.8 per cent-= 65 ram); (5) the superior exploitation of the pulmonary oxygen store in Planorbis appears to facilitate longer diving periods and so fits this species to occupy a separate ecological niche, i.e. bottom-feeding as distinct from browsing on submerged vegetation. * Generic names only are used subsequently since no other species are referred to. The first species should be referred to the genus PIanorbarius (Baker, 1945) but in the present context it seems preferable to use the name by which this animal has been known to students of respiratory pigments for almost a century. 283
284
J.D. JONES
Finally, it was suggested that it is in the range of pulmonary pO~ from 21 to 65 mm which distinguishes the Planorbis dives that the high affinity haemoglobin of this species discharges its principal function. Although the shape of the oxygen dissociation curve of Planorbis haemoglobin is well established (Zaaijer & Wolvekamp, 1958), only one or two inadequately controlled observations of Leitch (1916) and of Fox (1945) existed to suggest that the diffusion gradient across the respiratory surface is such as to place the function of the pigment in the above range of external pO2. Ideally one would determine the pO2 and pCO 2 of venous and arterial blood separately, under a natural range of external conditions, and refer these to the appropriate dissociation curve. Such methods have been employed with success by some workers on larger decapod crustacea (Redmond, 1955, 1962; Spoek, 1962). Since direct blood gas tension determinations are impractical in the case of an animal as small as Planorbis, one may attempt a spectroscopic assessment of the degree of saturation of the pigment in vivo while varying the external pO 2. In the above-mentioned works Leitch and Fox attempted such observations on the blood in the foot in relation to changing dissolved oxygen tension only. No account was taken of the level of pulmonary oxygen, and the blood in the full thickness of the foot is of doubtful arterial/venous status. Further, the thickness of the blood viewed by the spectroscope in this way is very variable. The principal object of the present paper is to describe a method for and results of such observations in which external cutaneous and pulmonary pO2 are independently controlled and in which the states of arterial and venous blood are distinguished. The opportunity is also taken to present some new data on the oxygen capacity of Planorbis blood. Leitch (1916) briefly reported a single observation made by Barcroft's method which gave a value of 0.9 vol per cent. Borden (1931), using the Barcroft apparatus and the van Slyke apparatus, obtained mean values of 1.27 and 1-43 vol per cent respectively. These should represent respectively combined and total oxygen, leaving a difference (which was just significant) of 0.16 vol per cent for dissolved oxygen. Since the blood samples were equilibrated with atmospheric air beforehand, this difference is unexpectedly small. In the same paper Borden gave corresponding values for the blood of Arenicola marina of 8.7 and 9.7 vol per cent; here the difference due to dissolved oxygen is exceptionally large. The matter therefore seemed to merit re-examination. MATERIALS AND METHODS All the snails were originally obtained from an aquarist's supplier and subsequently kept for 6-12 months in balanced aquaria before use. For the oxygen capacity determinations, large- and medium-sized specimens of the normal dark-pigmented variety were used. Determinations of in vivo haemoglobin saturation were made on young specimens (about 0-4 g wt.) of the albino variety, which completely lacks the skin pigmentation, in order to permit spectroscopic examination of blood vessels inside the shell. This has the advantage over foot examination that a constant thickness of blood can be viewed and some attempt can
T H E R O L E OF H A E M O G L O B I N I N T H E A Q U A T I C P U L M O N A T E , P L A N O R B I S C O R N E U S
285
be made to select arterial and venous viewpoints. In larger albino specimens the shell becomes too opaque for this kind of observation. An observation cell made from Perspex (Fig. 1) enables the animal to be kept in a fixed position under the -~ in. objective of a high power binocular microscope, while being perfused with water of varying pO 2 as required. The shell is immobilized between the top and bottom plates of the cell by means of a small pellet of plasticine on each side and is orientated as shown. The wheel in the right half of
i
¢"
1 ,
I
,
'
I
'
•
I
L
:f
Ii
FIG. 1. Perspex observation/perfusion cell for in vivo haemoglobin saturation measurements. Above---plan view with snail in place; below--side view. The snail is secured between the upper and lower plates by a small lump of plasticine (not shown) on either side of the shell at the centre of the umbilicus, but is free to crawl along the outer edge of the treadmill. A triangular deflector block ensures that practically all the perfusion water flows over the snail. A thin strip of plasticine is applied to the midline of the outer whorl. the chamber functions as a sort of treadmill, giving the animal a substrate on which to crawl; this encourages a normal posture and activity. The lid of the cell (upper plate) is simply secured by pressing down on a thin smear of lanoline applied round the margin. This method of sealing facilitates speedy sampling of the pulmonary gas for analysis in the Krogh rnicrogas-analyser once the required state of pigment saturation has been observed. The analysis of the gas is carried out according to the slightly modified procedure previously described (Jones, 1959). The observation cell is manipulated by means of a normal mechanical stage and the required structure is centred in the field by observation through one of the eyepieces. The second eyepiece is replaced by a Zeiss spectroscopic ocular. Final disappearance of the separate oxyhaemoglobin absorption bands gives an adequate indication of complete deoxygenation, but for recognition of the initial departure from the fully oxygenated state a comparison spectrum is required. This is arranged by illumination of oxyhaemoglobin solution and distilled water
286
J . D . JONES
respectively in a two-stage cup and plunger colorimeter and passage of the emergent beam via a 45 ° prism and condenser to the comparison prism of the spectroscope. Since light reaching the spectroscope from the snail passes not only through blood and tissues but also through the shell, two thicknesses of a similar shell are interposed between the condenser and comparison prism. This materially assists in matching the spectra by compensating for the diffuse absorption by the shell which is strongest at the blue end. This diffuse absorption somewhat reduces the sharpness of the oxyhaemoglobin absorption bands, so matching is somewhat less accurate than with pure haemoglobin solutions. However, discrimination tests with oxy- and deoxy-haemoglobin in the colorimeter cups indicated that departure from full and zero saturation could be recognized at not worse than 85 per cent and 15 per cent respectively. In making determinations of departure from full saturation it is sufficient to compare with full saturation at corresponding concentration. The first cup is therefore filled with oxygenated horse haemoglobin at -11oodilution and the second with water. Thus any thickness of fully saturated blood in the snail can be matched by simply racking the colorimeter up or down and adjusting the light intensity. Arrangements are made to perfuse the cell with any desired mixture of airsaturated and nitrogen-saturated water. The effluent from the cell passes into and overflows from a stoppered 1½ in. × ~ in. vial from which samples can be withdrawn for Winkler analysis in connexion with the cutaneous-limit observations. In determining the external pO 2 for cutaneous exchange it is essential to avoid stagnation of water close to the surface of the snail and to ensure that the uptake of oxygen does not significantly lower the pO 2 of the water. Only thus will the pOz of the effluent be virtually the same as that prevailing at the skin surface. Accordingly a rate of flow of about 1 1/hr is used as standard for both cutaneous and pulmonary-limit observations unless otherwise stated. It was calculated that, at this rate, oxygen depletion of the effluent would not exceed 2 per cent and in most cases would be substantially less. Determinations of limiting pulmonary pO 2 (i.e. corresponding to just observable departure from full or zero saturation of the pigment) commence by allowing the snail in the half-filled cell to fill its lung with air. The cell is then completely filled with water, placed on the microscope stage and, if departure from full saturation is sought, the comparison spectrum is matched as described above. Perfusion with N 2 water then commences and continues until the required change in absorption is observed. Then, with minimum delay, the animal is released from the cell and a small sample of pulmonary gas is collected under water into the cup of the microgas-analyser. When preparing to determine cutaneous limits the animal is again allowed to replenish the pulmonary gas if matching against the comparison standard is required. The cell is then perfused with Nz water until all trace of oxygenation has disappeared, indicating exhaustion of the pulmonary oxygen. Airsaturatedwater is then added to the perfusion fluid until a mixture is obtainedwhich sustains the pigment in a state bordering on complete oxygenation or deoxygenation as the case may be. This achieved, the effluent is sampled for micro-Winkler
THE ROLE OF HAEMOGLOBIN IN THE AQUATIC PULMONATE, P L A N O R B I S
CORNEUS
287
analysis using the procedure of van Dam (1935); the temperature within the cell is noted, to permit accurate conversion to partial pressure. The spectroscopic viewpoints were selected on the basis of personal observations on the gross anatomy of the living snail and upon the descriptions of the vascular system by Simroth & Hoffmann (1928) and by Baker (1945). These accounts admittedly lack detail in a number of respects but they will serve for the present purpose. All venous blood (except that in the renal vein) is collected into the circulus venosus which borders the free margin of the mantle. This blood is drawn from the lacunae (a) of the foot and (b) of other parts of the body. It appears that only the former portion is exposed to the possibility of oxygenation in the skin and accessory gill. With cutaneous uptake of oxygen eliminated, the circulus venosus contains mixed venous blood.* The most satisfactory point for viewing this blood was found to be close to the point where the right margin of the shell aperture meets the preceding whorl. From the circulus venosus the blood passes through the capillary system of the mantle roof (lung) so that arterial blood* collects in the pulmonary vein which runs back along the mid-dorsal line of the mantle roof, turning finally towards the right side to enter the atrium; close to this junction it receives the renal vein. The renal vein pursues a course parallel to the pulmonary vein somewhat to the left of the mid-dorsal line, and between these two vessels in the mantle roof lies the tubular part of the kidney. If the pulmonary vein is viewed laterally, as a strictly peripheral structure, from the right side of the shell, no light reaching the spectroscope will pass through the renal vein, though a little may pass through the tubular portion of the kidney. The latter does not appear to be well vascularized and so would not be expected to contribute significantly to the absorption pattern. In order to view the pulmonary vein from the side as a peripheral structure, it is necessary to outline the mid-dorsal line of the shell with a thin strip of plasticine as shown in Fig. 1, otherwise an excessive amount of light reaches the spectroscope which passes altogether outside the shell. The pulmonary vein provides an arterial viewpoint with respect to pulmonary and cutaneous uptake; the circulus venosus provides a venous viewpoint with respect to pulmonary uptake and arterial viewpoint with respect to cutaneous uptake. It would be very difficult to define a strictly venous point with respect to cutaneous uptake because only part of the total blood flow is exposed to the possibility of oxygenation in the skin or accessory gill. Minimally oxygenated blood draining from the viscera within the shell is not accessible to spectroscopic examination. Determinations of oxygen capacity were made with the Natelson microgasometer (Natelson & Menning, 1955). This represents a modification of the classical van Slyke manometric apparatus, ad~ipted for use with mammalian blood samples of 0.03 ml. Such small samples of Planorbis blood would yield very small pressure changes and accordingly the Natelson pipette was recalibrated for the introduction of samples of 0.1 ml, which could be obtained from single specimens of the larger snails or by pooling the blood of two of the medium-size specimens. * "Arterial" and "venous" are used throughout to signify respectively blood which has or has not been exposed to the possibility of reoxygenation after leaving the tissues.
288
J . D . JONES
After carefully drying the outside of the shell and mopping up any water inside the shell with wisps of soft paper tissue, the blood is obtained by foot-puncture. The freshly drawn blood is carefully equilibrated with several changes of air in a closed vial before the sample is measured along with the other reagents into the reaction chamber. By omission of the haemolysing reagent and reduction of the amount of anti-foam reagent it is possible to keep the total volume of liquid at the level specified in the original procedure. Thus it is only necessary to correct the original factors (for conversion of pressure differences to gas volumes per cent) with respect to the extraction of the measured gas from a larger sample. Reagent blanks were run on de-gassed reagents with de-gassed water in lieu of the blood sample. The pressure differences obtained on these blank runs did not exceed 0.5 mm and, being within the limits of error of the method, were disregarded. This method, involving vacuum extraction of both dissolved and combined gas, yields values of total gas content. Estimates of dissolved gas were made, so that capacity of the blood for combining oxygen could be calculated. This aspect of the procedure is described later alongside the statement of results. RESULTS In vivo saturation of haemoglobin Limiting oxygen tensions in the external medium were determined as follows: PFA--minimum pulmonary pO e for full saturation, arterial blood. PZA--maxlmum pulmonary pO e for zero saturation, arterial blood. PFV--minimum pulmonary pO e for full saturation, venous blood. PZV--maximum pulmonary pO e for zero saturation, venous blood. CFA--minimum cutaneous pO 2 for full saturation, arterial blood. CZA--maximum cutaneous pO 2 for zero saturation, arterial blood. Values for limiting tensions and pressures are given in Table 1 together with the corresponding values of pCO 2 in the case of pulmonary gas analyses. For reasons already mentioned, the observed saturation conditions probably more nearly represent 85 per cent and 15 per cent saturation respectively. Only six suitable animals were available for the pulmonary determinations and one determination was made on each; by the time the cutaneous observations were made the number of snails was reduced, so duplicate analyses were made on each animal. All the observations were made at room temperature which varied between 21 ° and 22°C. In pulmonary determinations, cutaneous uptake of oxygen was eliminated by perfusion of the cell with N e water. This proved not to be completely free of oxygen but had in fact a pOe of about 14 mm. However, this figure is sufficiently below the lowest value required for minimal oxygenation of the pigment at cutaneous sites (CZA) for the residual oxygen in the water to be without significance. It seemed possible that the perfusion flow of 1 1/hr might make for more effective loss of CO e from the skin than under natural conditions of near stagnation. Some pulmonary determinations were therefore made in static water. In all cases
THE ROLE OF HAEMOGLOBIN IN THE AQUATIC PULMONATE, PLA.NORBIS CORNEUS
289
this resulted in an appreciable rise in pulmonary pCO~ and in the case of P F A there was clearly a significant rise in limiting pO~ as well. T h i s is precisely what would be expected through the operation of the Bohr effect. In the case of PZA and P Z V the influence of pCO2 is less marked. Again this is to be expected since the Bohr effect (in absolute terms) is smaller at low percentage saturations, and at true zero saturation, of course, is nil. T h e values of p u l m o n a r y p C O 2 obtained T A B L E 1 - - L I M I T I N G EXTERNAL OXYGEN TENSIONS ( m m ) FOR FULL AND ZERO SATURATION OF P l a n o r b i $ HAEMOGLOBIN ill. ~i'l)O
Snail No.
pO2
pCO2
Snail No.
PFA 4
pCOz
Snail No.
PFV
6
7"4 16"8 18"0
3'1 1-3 1'9
5 3 2
38"9 45"3 35'2
9"9 7'8 7"4
1
pO2
1
PZA
48"2
pOz
CFA
6"7
6 6 2 2 4 4
PZV
39.2 52.1 61 "5 80-2 64"3 57"9
CZA
6 4 1 3
6.8 5"3 8"1 9'7
0 2.0 2-0 0
3 6 1
11.9 14"2 13"5
1 "8 1.3 1.9
2 5
15.9 18.4
3'8 5"1
4 5 2
23'1 20"3 11-8
6"4 4.3 3.1
4 4 2 2 5 5
20.5 33.4 35.0* 33.4* 36-3 24.0
* These values obtained from the viewpoint of the circulus venosus--all other arterial values from the pulmonary vein; venous values from the circulus venosus. Cutaneous values and those pulmonary values above the line with flowing water; pulmonary values below the line with static water. For key to abbreviations see the text. with the static conditions are closer to those found in Planorbis diving in the natural habitat (Jones, 1961) so the higher limiting pO2 values in T a b l e 1 below the line will give a better indication of the normal limits of pigment action with respect to p u l m o n a r y uptake. Since the method of determining limiting cutaneous pOz required a substantial rate of flow, it was not possible to determine these limits in static water. T h e pCO2 of the effluent from the cell was not determined, but would certainly be abnormally low compared with no normal values close to the skin. Accordingly, the observed values for limiting cutaneous p O 2 are certainly
J . D . JONES
290
too low, especially in the case of CFA, and should probably be scaled up by amounts similar to those indicated by the two sets of pulmonary values. Difficulties were experienced in measurements of P F V on account of variations in the degree of dilation of the circulus venosus, which made it very difficult to establish a fixed reference by which to judge the beginnings of deoxygenation. Only in one case was the initial absorption pattern judged sufficiently static to proceed with the determination; this was in a snail which remained fully extended from its shell, but inactive, for 30 rain. Since in these circumstances the demand TABLE 2--OXYGEN CAPACITY OF Planorbis HAEMOGLOBIN (VOL PER CENT)*
1.12 (1) 0.94 (1) 1"64 (1) 1"31 (1) 1.o3 (1)
2.02 1.85 1.94 2.49
(2) (2) (2) (3)
* Numbers of animals from which the blood was pooled in brackets. The values are for combined O2 and are obtained by deduction of 0'44 vol per cent from the determined values of total 02; the allowance for dissolved 02 being made on the basis of the following paired observations of total O2 : (a) Total Oo at 720 mm pO2 = 3.13 vol per cent ~ Total O,.~at 151 mm pO2 1.47 vol per cent L .'. Dissolved O2 at 56"-~mm pO2 1-66 vol per cent ~ at 21 C and dissolved 02 at 151 mm pO2 = 0.44 vol per cent J (b) Total 02 at 722 mm pO2 = 2.36 vol per cent ~ Total Oa at 151 mm pO2 = 0'76 vol per cent .'. Dissolved Ou at 57--i-mm pO~ 1.60 vol per cent at 2 2 C and dissolved O., at 151 mm pO2 0.43 vol per cent
J
for oxygen would be abnormally low, it would be easier than normal to satisfy these requirements solely from dissolved oxygen in the blood. U n d e r normal conditions of activity, therefore, the pigment would be called into action earlier in the course of pulmonary oxygen depletion, and the single determined value of P F V is probably too low. T h e four values of C Z A obtained from blood in the pulmonary vein are in good agreement with the two obtained from the circulus venosus, confirming the absence of pulmonary uptake in these cases. T h e oxygen capacity data which are given in T a b l e 2 represent combined oxygen only. Since the v a c u u m extraction used in the Natelson method gives a value for total oxygen content, a separate assessment of oxygen solubility was made. T h i s simply involves comparing the values obtained after equilibrating aliquots from a single sample (a) with atmospheric air and (b) with oxygen at atmospheric pressure, temperature of equilibration and total pressure being known. Since the pigment is fully saturated in both cases the difference represents the oxygen
THE ROLE OF HAEMOGLOBIN IN THE AQUATIC PULMONATE, P L A N O R B I S C O R N E U S
291
dissolved under a pressure equal to the difference in pO2 for the two equilibrations. Two such comparisons were made, with blood from a single animal in each case, and the results are shown in the lower part of Table 2. Combined oxygen values then range from 0.94 to 2-49 vol per cent. It is unfortunate that the highest value was obtained with the pooled blood of three smaller animals; evidently the maximum may be appreciably higher than this. There is also a suggestion, which has not yet been followed up, that the smaller animals (represented by the pooled samples) may have blood of higher oxygen capacity. This could conceivably be due to the necessity of supporting a higher metabolic rate. DISCUSSION The number of data from the in vivo saturation observations is too small to merit statistical treatment, and the limits can be regarded as established only approximately. Furthermore, for full and zero saturation one should probably read 85 per cent and 15 per cent saturation respectively. Nevertheless, these levels probably represent the most useful part of the saturation range in view of the sigmoid nature of the dissociation curve. With all the above limitations in mind, one may conclude that, with respect to pulmonary uptake, the haemoglobin will have a usefulness limited to a range of external pO 2 between about 15-20 mm (PZA) and 50-60 mm (PFV). Below PZA no appreciable oxygenation of the pigment in the lung will occur and above PFV all requirements will be met from oxygen in solution in the plasma and the pigment will not dissociate at all as it passes through the tissues. CZA values (21-36 ram) determined with flowing water may be up to 10 mm too low because of the abnormally small Bohr effect. Subject to such an adjustment they represent the lowest useful dissolved pO 2 for oxygenation of the haemoglobin via the skin and accessory gill. The PZA values should represent approximately the gradient across the pulmonary epithelium, for in this condition the arterial pO 2 would be close to zero. The gradient can also be deduced from the PFA values if arterial pO 2 is derived from the appropriate dissociation curve. The data of Zaaijer & Wolvekamp (1958) show 90 per cent saturation at 18 mm for 20°C and 9 mm pCO2; at 21-22 ° this figure could be raised 1 or 2 ram. Out of the 3 5 4 5 mm of the PFA values this would leave 15-25 mm for the gradient, in good agreement with the direct (PZA) estimate of 16-18 mm at slightly lower pCO a than has been found in natural diving. The normal pulmonary gradient can be fixed with confidence at about 20 mm. Owing to the mixing of blood in the circulus venosus it is not possible to make a deduction about the cutaneous gradient with equal confidence. The CZA values represent a mixture of uncertain proportions between the slightly oxygenated blood from the foot and accessory gill and the true venous blood from other parts of the body; the very considerable variation in these values may reflect variations in these proportions. As an estimate of the external tension needed to produce this minimal oxygenation of cutaneous-arterial blood, the observed CZA values are therefore too high. On the other hand, they were obtained with flowing 20
292
J . D . JONES
water which would largely eliminate the normal Bohr effect (see above), and for this reason the values would tend to be abnormally low. There is no means of deciding to what extent these two disturbing effects balance each other, and it can only be suggested tentatively that the cutaneous gradient lies somewhere within the range of observed values--21-36 ram. These data on in vivo saturation can now be related to the natural range of pulmonary pO 2 values given previously (Jones, 1961) and reproduced in Table 2 of a subsequent paper (Jones, 1964). It is at once apparent that the limiting external pO 2 values (lung gas) for significant transport by Planorbis haemoglobin from the lung coincide almost exactly with the range of pulmonary gas from 65 to 20 mm which distinguishes the natural dives of Planorbis from those of Lymnaea. It is more difficult to establish whether the pigment confers any advantage on Planorbis with respect to cutaneous uptake. The in vivo saturation measurements suggest a utility of the pigment down to about 30 mm whereas previous measurements (Jones, 1961) indicate an appreciable cutaneous uptake at least down to 15 mm. This discrepancy presumably represents use of oxygen by superficial tissues which can be reached directly by short diffusion pathways. In actual cutaneous uptake Planorbis and Lymnaea are equally matched at the lower end of the dissolved pO 2 scale (15 mm); at 240 mm Lymnaea uptake is a little higher but the difference is of doubtful significance. At all dissolved oxygen values the complementary pulmonary uptake is significantly higher in Lymnaea so that the total uptake and the percentage of the total which is pulmonary is higher in Lymnaea than in Planorbis (Jones, 1961). Some observations of Zaaijer & Wolvekamp (1958) also indicate that Planorbis and Lymnaea are equally proficient in lowering the dissolved oxygen concentration in a restricted volume of water. It therefore does not appear that the haemoglobin gives Planorbis any superiority over Lymnaea in cutaneous uptake. The pigment must make a contribution in the appropriate range (say 30-80 mm) but this simply matches more favourable diffusion conditions in Lymnaea. Although it is generally held that an accessory gill is lacking in the latter, Simroth & Hoffmann (1928) have suggested that the much expanded tentacles may function as such. In Table 3 some data are assembled which bear on the utilization of pulmonary oxygen during a single dive. Figures for Planorbis and Lymnaea are taken from the earlier account, those for Biomphalaria from a subsequent paper (Jones, 1964). The tissue weight figures are based on weighing seven or eight snails of each species and in the case of Planorbis and Lymnaea agree well with the values (73.7 and 86"3 per cent respectively) of F/isser & Krfiger (1951). The calculated utilization of 02 during the dive (ml/g) shows a marked inferiority in Lymnaea, in spite of which this species succeeds in maintaining a 30 per cent higher total oxygen consumption. This must in part depend on the maintenance of a higher mean pulmonary pO~ through more frequent filling of the lung. However, Hazelhoff (quoted by Jordan, 1930) found that over the range of 16-8 per cent (in which Planorbis haemoglobin is inactive) the rate of fall of pulmonary oxygen concentration in Lymnaea was 2.4 times that in Planorbis. Since the relative pulmonary
THE ROLE OF HAEMOGLOBIN I N THE AQUATIC PULMONATE, P L A N O R B I S C O R N E U S
293
volumes are 0-63-1 (Table 3), this gives further evidence of superior pulmonary uptake by Lymnaea but in this case not dependent on superior pO2. T h u s for similar external pO 2 there is a higher rate of oxygen exchange in Lymnaea, both through the lung and the skin, at least when the Planorbis pigment is inactive. This must rest on generally lower diffusion resistances and/or steeper diffusion gradients. A steeper diffusion gradient is likely to result from a lower venous pO 2 which in turn is likely to reflect a lower tissue tension. Offsetting this advantage, Lymnaea has a smaller pulmonary volume and (in the absence of a respiratory pigment) a restricted capacity for drawing on the pulmonary gas.
T A B L E 3 - - U T I L I Z A T I O N OF PULMONARY OXYGEN DURING THE DIVE
Planorbis Biomphalaria Lymnaea Tissue as o/ /o of total wt.
68'3
Initial pulmonary gas vol (ml/g)
"~ f
Total wt. Tissue wt.
Change during dive
; J
% 02 mm pO2
Utilized O~ vol (ml/g)*
; 3
Total wt. Tissue wt.
0"22 0"32 13"4 99"2 0-030 0'044
69'0 0'25 0-36 14-2 90'5 0"032 0'045
85"9 0-17 0"20 9"4 70-0 0'018 0-021
* The utilized 02 is expressed as ml at 760 mm pressure in all cases but in the case of
Biomphalaria the gas parameters were actually measured at 660 mm barometric pressure (Jones, 1964). Since there is some uncertainty about the per cent change in volume during the dive, the calculation of 02 utilization is based on an assumed common volume change of 10 per cent. For each species the volume change probably lies within the range of 10-15 per cent so the error introduced by this approximation will be very small. These restrictions limit the duration of the dive compared with Planorbis. T h e r e can be no doubt that the possession of haemoglobin in the latter plays an important part in the more extensive exploitation of the lung oxygen store. This, together with the higher pulmonary volume and lower metabolic rate, allows Planorbis a much longer diving period, in consequence of which it can occupy a different ecological niche as previously described (Jones, 1961). Leitch (1916) and Borden (1931), on the basis of their oxygen capacity and other measurements, calculated the potential oxygen storage value of the pigment. In terms of the length of time that normal activity could be sustained by the maximal volume of bound oxygen, their results were 3 and 18 min respectively. These periods were regarded as insignificant. Total blood volume has not been redetermined but the present oxygen capacity figures (maximum 2.4 vol per cent) indicate that the earlier higher estimate (based on 1.27 vol per cent) may be up to 50 per cent too low. It is not reasonable to dismiss this storage potential, for
294
J . D . JONES
in an animal largely dependent on periodic excursions to the surface, a storage period of 20 or more minutes could substantially extend the diving range. However, storage needs to be viewed in a different light. Consider any animal endowed with a respiratory pigment and faced with a declining ambient pO2, whether it be in the true external medium or in an infrequently ventilated lung. Once arterial pO2 drops below the level for full saturation, the total combined oxygen in the arterial blood falls short of the maximum. The pigment may be regarded as having given up part of its "store" but this is entirely incidental to its role as transporter. When external and arterial pOz fall so low as to make continued significant transport impossible, the pigment will be virtually deoxygenated and its storage function nil. It is therefore meaningful to speak of a circulating* pigment as having a storage function per se only when the animal is cut off suddenly from external oxygen supplies with its store in a well-charged condition. Such a situation will not be found in the normal conditions of life of aquatic pulmonates. In any other declining ambient oxygen situation all respiratory pigments are incidentally potential oxygen stores. The role of haemoglobin in Planorbis may be summarized as follows. So long as pulmonary pO2 exceeds about 60 mm, venous pO2 does not fall low enough to cause dissociation of the oxygenated pigment, all requirements being met from solution. As pulmonary pO2 falls below this level the pigment is active in transport of oxygen from the lung. In the region below about 40 mm, arterial blood is no longer fully saturated, so that total combined oxygen is diminishing and the oxygen capacity will become a factor in determining how long the tissues can continue to receive significant amounts of oxygen. By the time pulmonary pO2 reaches 20 mm both transport and storage functions are virtually exhausted. At the same time, so long as dissolved pO 2 exceeds something of the order of 30 mm, part of the pigment may pick up oxygen via the skin and so spare pulmonary oxygen. The dissolved pO2 which will spare pulmonary oxygen entirely has not been determined, but it exceeds 240 mm. SUMMARY 1. A method is described for the in vivo spectroscopic determination of percentage saturation of haemoglobin in albino specimens of Planorbis corneus. 2. The method has been'used to determine the limiting external pO2's for full and zero saturation of haemoglobin in arterial and venous blood with respect to both pulmonary and cutaneous oxygen uptake. 3. For pulmonary uptake the external range of pO~ for pigment function is between 20 and 50-60 mm; for cutaneous uptake the lower limit only has been fixed at about 30 mm. 4. The pulmonary range corresponds well with that which has previously been found to distinguish the dives of Planorbis from those of Lymnaea stagnalis lacking a respiratory pigment. * It has been argued elsewhere that non-circulating coelomic pigments can only act as oxygen stores (Jones, 1963).
THE ROLE OF HAEMOGLOBIN IN THE AQUATIC PULMONATE, P L A N O R B 1 S C O R N E U S
295
5. T h e new data are discussed in relation to earlier observations on these two species, and it is concluded that in Lymnaea conditions for diffusion of oxygen both through the skin and the lung epithelium are easier than in Planorbis. 6. T h e oxygen capacity of the blood of Planorbis has been found to range from 0-94 to 2.49 vol per cent and the question of a possible storage function is discussed. REFERENCES BAKERF. C. (1945) The Molluscan Family Planorbidae. Univ. Illinois Press, Urbana. BORDEN M. A. (1931) A study of the respiration and of the function of haemoglobin in Planorbis corneus and Arenicola marina. J. Mar. biol. Ass. U.K. 17, 709-738. DAM L. VAN (1935) A method for determining the amount of oxygen dissolved in 1 cc of water. J. Exp. Biol. 12, 80--85. Fox H. M. (1945) The oxygen affinities of certain invertebrate haemoglobins. J. Exp. Biol. 21, 161-165. F~2SSER H. & KRt~GER F. (1951) Vergleichende Versuche zur Atmungsphysiologie von Planorbis corneus und Limnaea stagnalis (Gastropoda Pulmonata). Z. vergl. Physiol. 33, 14-52. JONES J. D. (1959) A new tonometric method for the determination of dissolved oxygen and carbon dioxide in small samples. J. Exp. Biol. 36, 177-190. 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) The functions of the respiratory pigments of invertebrates. In Problems in Biology, Vol. 1, (Edited by KERKUTG.). Pergamon Press, Oxford. JONES J. D. (1964) Respiratory gas exchange in the aquatic pulmonate BiomphalaHa sudanica. Comp. Biochem. Physiol. 12, 297-310. JORDAN H. J. (1930) Le r~glage de la consommation de l'oxyg~ne chez les animaux "tension gazeuse alv~olaire inconstante". Arch. nderl. Physiol. 15, 198-212. LEITCH I. (1916) The function of haemoglobin in invertebrates with special reference to Planorbis and Chironomus larvae. J. Physiol. 50, 370-379. NATELSONS. & MENNINGC. M. (1955) Improved methods of analysis for oxygen, carbon monoxide and iron on fingertip blood. Clin. Chem. 1, 165-179. REOMOND J. R. (1955) The respiratory function of haemocyanin in crustacea. J. Cell. Comp. Physiol. 46, 209-247. REDMOND J. R. (1962) Oxygen-hemocyanin relationships in the land crab, Cardisoma guanhumi. Biol. Bull., Woods Hole 122, 252-262. SIMROTH H. & HOFFMANN H. (1928) In Bronn's Klassen und Ordnungen des Tierreichs. Bd. 3, Abt. II, Buch 2. Pulmonata. Akad. Verlagsges, Leipzig. SPOEK G. L. (1962) Oxygen dissociation curves and arterial and venous oxygen content ot bloods of Homarus gammarus and Maia squinado. Versl. gewone Vergad. Akad. ~.Jmst. 71, 29-34. ZAAUER J. J. P. & WOLVEKAMPPI. P. (1958) Some experiments on the haemoglobinoxygen equilibrium in the blood of the ramshorn (Planorbis corneus L.). Acta Physiol. Pharmacol. Nderl. 7, 56-77.
T H E ROLE OF HAEMOGLOBIN IN THE AQUATIC PULMONATE, P L A N O R B I S C O R N E U S j. D. JONES Department of Zoology, The University of Sheffield, England (Received 2 March 1964)
A b s t r a c t - - 1 . Limiting external pO2's have been determined for full and zero saturation of arterial and venous blood of Planorbis corneus and the pO 2 range for pigment function has been deduced. 2. For oxygen uptake from the lung the pigment is functional over the pulmonary pO2 range 20-60 mm; this is precisely the region over which Planorbis pulmonary oxygen utilization exceeds that in Lymnaea stagnalis lacking a respiratory pigment. 3. Cutaneous uptake is facilitated by haemoglobin down to a dissolved pO2 of about 30 mm. 4. The oxygen capacity of Planorbis blood was found to range from 0'94 to 2-49 vol per cent; the question of a possible storage function is discussed. INTRODUCTION IN AN earlier paper (Jones, 1961) some observations on the respiration of Planorbis corneus* and L y m n a e a stagnalis* were reported. Various aspects of gas exchange were examined in an attempt to form an overall picture from the various factors involved, in strict relation to the normal diving behaviour of these aquatic pulmonates. T h e principal conclusions based on that work were as follows: (1) total oxygen uptake is independent of the tension of dissolved oxygen because, as cutaneous uptake falls with declining dissolved pO2, pulmonary uptake increases in a complementary fashion; (2) in neither case is cutaneous uptake fully adequate even when dissolved pO2 is as high as 240 m m ; (3) pulmonary uptake is slightly more important in L y m n a e a than in Planorbis at all values of dissolved pO 2 up to this limit of observation; (4) the two species commence the dive with a similar slight deficiency (compared with the atmosphere) in pulmonary oxygen, but the final pulmonary oxygen content in Planorbis (2.8 per cent = 21 m m ) is very significantly lower than in L y m n a e a (8.8 per cent-= 65 ram); (5) the superior exploitation of the pulmonary oxygen store in Planorbis appears to facilitate longer diving periods and so fits this species to occupy a separate ecological niche, i.e. bottom-feeding as distinct from browsing on submerged vegetation. * Generic names only are used subsequently since no other species are referred to. The first species should be referred to the genus PIanorbarius (Baker, 1945) but in the present context it seems preferable to use the name by which this animal has been known to students of respiratory pigments for almost a century. 283
284
J.D. JONES
Finally, it was suggested that it is in the range of pulmonary pO~ from 21 to 65 mm which distinguishes the Planorbis dives that the high affinity haemoglobin of this species discharges its principal function. Although the shape of the oxygen dissociation curve of Planorbis haemoglobin is well established (Zaaijer & Wolvekamp, 1958), only one or two inadequately controlled observations of Leitch (1916) and of Fox (1945) existed to suggest that the diffusion gradient across the respiratory surface is such as to place the function of the pigment in the above range of external pO2. Ideally one would determine the pO2 and pCO 2 of venous and arterial blood separately, under a natural range of external conditions, and refer these to the appropriate dissociation curve. Such methods have been employed with success by some workers on larger decapod crustacea (Redmond, 1955, 1962; Spoek, 1962). Since direct blood gas tension determinations are impractical in the case of an animal as small as Planorbis, one may attempt a spectroscopic assessment of the degree of saturation of the pigment in vivo while varying the external pO 2. In the above-mentioned works Leitch and Fox attempted such observations on the blood in the foot in relation to changing dissolved oxygen tension only. No account was taken of the level of pulmonary oxygen, and the blood in the full thickness of the foot is of doubtful arterial/venous status. Further, the thickness of the blood viewed by the spectroscope in this way is very variable. The principal object of the present paper is to describe a method for and results of such observations in which external cutaneous and pulmonary pO2 are independently controlled and in which the states of arterial and venous blood are distinguished. The opportunity is also taken to present some new data on the oxygen capacity of Planorbis blood. Leitch (1916) briefly reported a single observation made by Barcroft's method which gave a value of 0.9 vol per cent. Borden (1931), using the Barcroft apparatus and the van Slyke apparatus, obtained mean values of 1.27 and 1-43 vol per cent respectively. These should represent respectively combined and total oxygen, leaving a difference (which was just significant) of 0.16 vol per cent for dissolved oxygen. Since the blood samples were equilibrated with atmospheric air beforehand, this difference is unexpectedly small. In the same paper Borden gave corresponding values for the blood of Arenicola marina of 8.7 and 9.7 vol per cent; here the difference due to dissolved oxygen is exceptionally large. The matter therefore seemed to merit re-examination. MATERIALS AND METHODS All the snails were originally obtained from an aquarist's supplier and subsequently kept for 6-12 months in balanced aquaria before use. For the oxygen capacity determinations, large- and medium-sized specimens of the normal dark-pigmented variety were used. Determinations of in vivo haemoglobin saturation were made on young specimens (about 0-4 g wt.) of the albino variety, which completely lacks the skin pigmentation, in order to permit spectroscopic examination of blood vessels inside the shell. This has the advantage over foot examination that a constant thickness of blood can be viewed and some attempt can
T H E R O L E OF H A E M O G L O B I N I N T H E A Q U A T I C P U L M O N A T E , P L A N O R B I S C O R N E U S
285
be made to select arterial and venous viewpoints. In larger albino specimens the shell becomes too opaque for this kind of observation. An observation cell made from Perspex (Fig. 1) enables the animal to be kept in a fixed position under the -~ in. objective of a high power binocular microscope, while being perfused with water of varying pO 2 as required. The shell is immobilized between the top and bottom plates of the cell by means of a small pellet of plasticine on each side and is orientated as shown. The wheel in the right half of
i
¢"
1 ,
I
,
'
I
'
•
I
L
:f
Ii
FIG. 1. Perspex observation/perfusion cell for in vivo haemoglobin saturation measurements. Above---plan view with snail in place; below--side view. The snail is secured between the upper and lower plates by a small lump of plasticine (not shown) on either side of the shell at the centre of the umbilicus, but is free to crawl along the outer edge of the treadmill. A triangular deflector block ensures that practically all the perfusion water flows over the snail. A thin strip of plasticine is applied to the midline of the outer whorl. the chamber functions as a sort of treadmill, giving the animal a substrate on which to crawl; this encourages a normal posture and activity. The lid of the cell (upper plate) is simply secured by pressing down on a thin smear of lanoline applied round the margin. This method of sealing facilitates speedy sampling of the pulmonary gas for analysis in the Krogh rnicrogas-analyser once the required state of pigment saturation has been observed. The analysis of the gas is carried out according to the slightly modified procedure previously described (Jones, 1959). The observation cell is manipulated by means of a normal mechanical stage and the required structure is centred in the field by observation through one of the eyepieces. The second eyepiece is replaced by a Zeiss spectroscopic ocular. Final disappearance of the separate oxyhaemoglobin absorption bands gives an adequate indication of complete deoxygenation, but for recognition of the initial departure from the fully oxygenated state a comparison spectrum is required. This is arranged by illumination of oxyhaemoglobin solution and distilled water
286
J . D . JONES
respectively in a two-stage cup and plunger colorimeter and passage of the emergent beam via a 45 ° prism and condenser to the comparison prism of the spectroscope. Since light reaching the spectroscope from the snail passes not only through blood and tissues but also through the shell, two thicknesses of a similar shell are interposed between the condenser and comparison prism. This materially assists in matching the spectra by compensating for the diffuse absorption by the shell which is strongest at the blue end. This diffuse absorption somewhat reduces the sharpness of the oxyhaemoglobin absorption bands, so matching is somewhat less accurate than with pure haemoglobin solutions. However, discrimination tests with oxy- and deoxy-haemoglobin in the colorimeter cups indicated that departure from full and zero saturation could be recognized at not worse than 85 per cent and 15 per cent respectively. In making determinations of departure from full saturation it is sufficient to compare with full saturation at corresponding concentration. The first cup is therefore filled with oxygenated horse haemoglobin at -11oodilution and the second with water. Thus any thickness of fully saturated blood in the snail can be matched by simply racking the colorimeter up or down and adjusting the light intensity. Arrangements are made to perfuse the cell with any desired mixture of airsaturated and nitrogen-saturated water. The effluent from the cell passes into and overflows from a stoppered 1½ in. × ~ in. vial from which samples can be withdrawn for Winkler analysis in connexion with the cutaneous-limit observations. In determining the external pO 2 for cutaneous exchange it is essential to avoid stagnation of water close to the surface of the snail and to ensure that the uptake of oxygen does not significantly lower the pO 2 of the water. Only thus will the pOz of the effluent be virtually the same as that prevailing at the skin surface. Accordingly a rate of flow of about 1 1/hr is used as standard for both cutaneous and pulmonary-limit observations unless otherwise stated. It was calculated that, at this rate, oxygen depletion of the effluent would not exceed 2 per cent and in most cases would be substantially less. Determinations of limiting pulmonary pO 2 (i.e. corresponding to just observable departure from full or zero saturation of the pigment) commence by allowing the snail in the half-filled cell to fill its lung with air. The cell is then completely filled with water, placed on the microscope stage and, if departure from full saturation is sought, the comparison spectrum is matched as described above. Perfusion with N 2 water then commences and continues until the required change in absorption is observed. Then, with minimum delay, the animal is released from the cell and a small sample of pulmonary gas is collected under water into the cup of the microgas-analyser. When preparing to determine cutaneous limits the animal is again allowed to replenish the pulmonary gas if matching against the comparison standard is required. The cell is then perfused with Nz water until all trace of oxygenation has disappeared, indicating exhaustion of the pulmonary oxygen. Airsaturatedwater is then added to the perfusion fluid until a mixture is obtainedwhich sustains the pigment in a state bordering on complete oxygenation or deoxygenation as the case may be. This achieved, the effluent is sampled for micro-Winkler
THE ROLE OF HAEMOGLOBIN IN THE AQUATIC PULMONATE, P L A N O R B I S
CORNEUS
287
analysis using the procedure of van Dam (1935); the temperature within the cell is noted, to permit accurate conversion to partial pressure. The spectroscopic viewpoints were selected on the basis of personal observations on the gross anatomy of the living snail and upon the descriptions of the vascular system by Simroth & Hoffmann (1928) and by Baker (1945). These accounts admittedly lack detail in a number of respects but they will serve for the present purpose. All venous blood (except that in the renal vein) is collected into the circulus venosus which borders the free margin of the mantle. This blood is drawn from the lacunae (a) of the foot and (b) of other parts of the body. It appears that only the former portion is exposed to the possibility of oxygenation in the skin and accessory gill. With cutaneous uptake of oxygen eliminated, the circulus venosus contains mixed venous blood.* The most satisfactory point for viewing this blood was found to be close to the point where the right margin of the shell aperture meets the preceding whorl. From the circulus venosus the blood passes through the capillary system of the mantle roof (lung) so that arterial blood* collects in the pulmonary vein which runs back along the mid-dorsal line of the mantle roof, turning finally towards the right side to enter the atrium; close to this junction it receives the renal vein. The renal vein pursues a course parallel to the pulmonary vein somewhat to the left of the mid-dorsal line, and between these two vessels in the mantle roof lies the tubular part of the kidney. If the pulmonary vein is viewed laterally, as a strictly peripheral structure, from the right side of the shell, no light reaching the spectroscope will pass through the renal vein, though a little may pass through the tubular portion of the kidney. The latter does not appear to be well vascularized and so would not be expected to contribute significantly to the absorption pattern. In order to view the pulmonary vein from the side as a peripheral structure, it is necessary to outline the mid-dorsal line of the shell with a thin strip of plasticine as shown in Fig. 1, otherwise an excessive amount of light reaches the spectroscope which passes altogether outside the shell. The pulmonary vein provides an arterial viewpoint with respect to pulmonary and cutaneous uptake; the circulus venosus provides a venous viewpoint with respect to pulmonary uptake and arterial viewpoint with respect to cutaneous uptake. It would be very difficult to define a strictly venous point with respect to cutaneous uptake because only part of the total blood flow is exposed to the possibility of oxygenation in the skin or accessory gill. Minimally oxygenated blood draining from the viscera within the shell is not accessible to spectroscopic examination. Determinations of oxygen capacity were made with the Natelson microgasometer (Natelson & Menning, 1955). This represents a modification of the classical van Slyke manometric apparatus, ad~ipted for use with mammalian blood samples of 0.03 ml. Such small samples of Planorbis blood would yield very small pressure changes and accordingly the Natelson pipette was recalibrated for the introduction of samples of 0.1 ml, which could be obtained from single specimens of the larger snails or by pooling the blood of two of the medium-size specimens. * "Arterial" and "venous" are used throughout to signify respectively blood which has or has not been exposed to the possibility of reoxygenation after leaving the tissues.
288
J . D . JONES
After carefully drying the outside of the shell and mopping up any water inside the shell with wisps of soft paper tissue, the blood is obtained by foot-puncture. The freshly drawn blood is carefully equilibrated with several changes of air in a closed vial before the sample is measured along with the other reagents into the reaction chamber. By omission of the haemolysing reagent and reduction of the amount of anti-foam reagent it is possible to keep the total volume of liquid at the level specified in the original procedure. Thus it is only necessary to correct the original factors (for conversion of pressure differences to gas volumes per cent) with respect to the extraction of the measured gas from a larger sample. Reagent blanks were run on de-gassed reagents with de-gassed water in lieu of the blood sample. The pressure differences obtained on these blank runs did not exceed 0.5 mm and, being within the limits of error of the method, were disregarded. This method, involving vacuum extraction of both dissolved and combined gas, yields values of total gas content. Estimates of dissolved gas were made, so that capacity of the blood for combining oxygen could be calculated. This aspect of the procedure is described later alongside the statement of results. RESULTS In vivo saturation of haemoglobin Limiting oxygen tensions in the external medium were determined as follows: PFA--minimum pulmonary pO e for full saturation, arterial blood. PZA--maxlmum pulmonary pO e for zero saturation, arterial blood. PFV--minimum pulmonary pO e for full saturation, venous blood. PZV--maximum pulmonary pO e for zero saturation, venous blood. CFA--minimum cutaneous pO 2 for full saturation, arterial blood. CZA--maximum cutaneous pO 2 for zero saturation, arterial blood. Values for limiting tensions and pressures are given in Table 1 together with the corresponding values of pCO 2 in the case of pulmonary gas analyses. For reasons already mentioned, the observed saturation conditions probably more nearly represent 85 per cent and 15 per cent saturation respectively. Only six suitable animals were available for the pulmonary determinations and one determination was made on each; by the time the cutaneous observations were made the number of snails was reduced, so duplicate analyses were made on each animal. All the observations were made at room temperature which varied between 21 ° and 22°C. In pulmonary determinations, cutaneous uptake of oxygen was eliminated by perfusion of the cell with N e water. This proved not to be completely free of oxygen but had in fact a pOe of about 14 mm. However, this figure is sufficiently below the lowest value required for minimal oxygenation of the pigment at cutaneous sites (CZA) for the residual oxygen in the water to be without significance. It seemed possible that the perfusion flow of 1 1/hr might make for more effective loss of CO e from the skin than under natural conditions of near stagnation. Some pulmonary determinations were therefore made in static water. In all cases
THE ROLE OF HAEMOGLOBIN IN THE AQUATIC PULMONATE, PLA.NORBIS CORNEUS
289
this resulted in an appreciable rise in pulmonary pCO~ and in the case of P F A there was clearly a significant rise in limiting pO~ as well. T h i s is precisely what would be expected through the operation of the Bohr effect. In the case of PZA and P Z V the influence of pCO2 is less marked. Again this is to be expected since the Bohr effect (in absolute terms) is smaller at low percentage saturations, and at true zero saturation, of course, is nil. T h e values of p u l m o n a r y p C O 2 obtained T A B L E 1 - - L I M I T I N G EXTERNAL OXYGEN TENSIONS ( m m ) FOR FULL AND ZERO SATURATION OF P l a n o r b i $ HAEMOGLOBIN ill. ~i'l)O
Snail No.
pO2
pCO2
Snail No.
PFA 4
pCOz
Snail No.
PFV
6
7"4 16"8 18"0
3'1 1-3 1'9
5 3 2
38"9 45"3 35'2
9"9 7'8 7"4
1
pO2
1
PZA
48"2
pOz
CFA
6"7
6 6 2 2 4 4
PZV
39.2 52.1 61 "5 80-2 64"3 57"9
CZA
6 4 1 3
6.8 5"3 8"1 9'7
0 2.0 2-0 0
3 6 1
11.9 14"2 13"5
1 "8 1.3 1.9
2 5
15.9 18.4
3'8 5"1
4 5 2
23'1 20"3 11-8
6"4 4.3 3.1
4 4 2 2 5 5
20.5 33.4 35.0* 33.4* 36-3 24.0
* These values obtained from the viewpoint of the circulus venosus--all other arterial values from the pulmonary vein; venous values from the circulus venosus. Cutaneous values and those pulmonary values above the line with flowing water; pulmonary values below the line with static water. For key to abbreviations see the text. with the static conditions are closer to those found in Planorbis diving in the natural habitat (Jones, 1961) so the higher limiting pO2 values in T a b l e 1 below the line will give a better indication of the normal limits of pigment action with respect to p u l m o n a r y uptake. Since the method of determining limiting cutaneous pOz required a substantial rate of flow, it was not possible to determine these limits in static water. T h e pCO2 of the effluent from the cell was not determined, but would certainly be abnormally low compared with no normal values close to the skin. Accordingly, the observed values for limiting cutaneous p O 2 are certainly
J . D . JONES
290
too low, especially in the case of CFA, and should probably be scaled up by amounts similar to those indicated by the two sets of pulmonary values. Difficulties were experienced in measurements of P F V on account of variations in the degree of dilation of the circulus venosus, which made it very difficult to establish a fixed reference by which to judge the beginnings of deoxygenation. Only in one case was the initial absorption pattern judged sufficiently static to proceed with the determination; this was in a snail which remained fully extended from its shell, but inactive, for 30 rain. Since in these circumstances the demand TABLE 2--OXYGEN CAPACITY OF Planorbis HAEMOGLOBIN (VOL PER CENT)*
1.12 (1) 0.94 (1) 1"64 (1) 1"31 (1) 1.o3 (1)
2.02 1.85 1.94 2.49
(2) (2) (2) (3)
* Numbers of animals from which the blood was pooled in brackets. The values are for combined O2 and are obtained by deduction of 0'44 vol per cent from the determined values of total 02; the allowance for dissolved 02 being made on the basis of the following paired observations of total O2 : (a) Total Oo at 720 mm pO2 = 3.13 vol per cent ~ Total O,.~at 151 mm pO2 1.47 vol per cent L .'. Dissolved O2 at 56"-~mm pO2 1-66 vol per cent ~ at 21 C and dissolved 02 at 151 mm pO2 = 0.44 vol per cent J (b) Total 02 at 722 mm pO2 = 2.36 vol per cent ~ Total Oa at 151 mm pO2 = 0'76 vol per cent .'. Dissolved Ou at 57--i-mm pO~ 1.60 vol per cent at 2 2 C and dissolved O., at 151 mm pO2 0.43 vol per cent
J
for oxygen would be abnormally low, it would be easier than normal to satisfy these requirements solely from dissolved oxygen in the blood. U n d e r normal conditions of activity, therefore, the pigment would be called into action earlier in the course of pulmonary oxygen depletion, and the single determined value of P F V is probably too low. T h e four values of C Z A obtained from blood in the pulmonary vein are in good agreement with the two obtained from the circulus venosus, confirming the absence of pulmonary uptake in these cases. T h e oxygen capacity data which are given in T a b l e 2 represent combined oxygen only. Since the v a c u u m extraction used in the Natelson method gives a value for total oxygen content, a separate assessment of oxygen solubility was made. T h i s simply involves comparing the values obtained after equilibrating aliquots from a single sample (a) with atmospheric air and (b) with oxygen at atmospheric pressure, temperature of equilibration and total pressure being known. Since the pigment is fully saturated in both cases the difference represents the oxygen
THE ROLE OF HAEMOGLOBIN IN THE AQUATIC PULMONATE, P L A N O R B I S C O R N E U S
291
dissolved under a pressure equal to the difference in pO2 for the two equilibrations. Two such comparisons were made, with blood from a single animal in each case, and the results are shown in the lower part of Table 2. Combined oxygen values then range from 0.94 to 2-49 vol per cent. It is unfortunate that the highest value was obtained with the pooled blood of three smaller animals; evidently the maximum may be appreciably higher than this. There is also a suggestion, which has not yet been followed up, that the smaller animals (represented by the pooled samples) may have blood of higher oxygen capacity. This could conceivably be due to the necessity of supporting a higher metabolic rate. DISCUSSION The number of data from the in vivo saturation observations is too small to merit statistical treatment, and the limits can be regarded as established only approximately. Furthermore, for full and zero saturation one should probably read 85 per cent and 15 per cent saturation respectively. Nevertheless, these levels probably represent the most useful part of the saturation range in view of the sigmoid nature of the dissociation curve. With all the above limitations in mind, one may conclude that, with respect to pulmonary uptake, the haemoglobin will have a usefulness limited to a range of external pO 2 between about 15-20 mm (PZA) and 50-60 mm (PFV). Below PZA no appreciable oxygenation of the pigment in the lung will occur and above PFV all requirements will be met from oxygen in solution in the plasma and the pigment will not dissociate at all as it passes through the tissues. CZA values (21-36 ram) determined with flowing water may be up to 10 mm too low because of the abnormally small Bohr effect. Subject to such an adjustment they represent the lowest useful dissolved pO 2 for oxygenation of the haemoglobin via the skin and accessory gill. The PZA values should represent approximately the gradient across the pulmonary epithelium, for in this condition the arterial pO 2 would be close to zero. The gradient can also be deduced from the PFA values if arterial pO 2 is derived from the appropriate dissociation curve. The data of Zaaijer & Wolvekamp (1958) show 90 per cent saturation at 18 mm for 20°C and 9 mm pCO2; at 21-22 ° this figure could be raised 1 or 2 ram. Out of the 3 5 4 5 mm of the PFA values this would leave 15-25 mm for the gradient, in good agreement with the direct (PZA) estimate of 16-18 mm at slightly lower pCO a than has been found in natural diving. The normal pulmonary gradient can be fixed with confidence at about 20 mm. Owing to the mixing of blood in the circulus venosus it is not possible to make a deduction about the cutaneous gradient with equal confidence. The CZA values represent a mixture of uncertain proportions between the slightly oxygenated blood from the foot and accessory gill and the true venous blood from other parts of the body; the very considerable variation in these values may reflect variations in these proportions. As an estimate of the external tension needed to produce this minimal oxygenation of cutaneous-arterial blood, the observed CZA values are therefore too high. On the other hand, they were obtained with flowing 20
292
J . D . JONES
water which would largely eliminate the normal Bohr effect (see above), and for this reason the values would tend to be abnormally low. There is no means of deciding to what extent these two disturbing effects balance each other, and it can only be suggested tentatively that the cutaneous gradient lies somewhere within the range of observed values--21-36 ram. These data on in vivo saturation can now be related to the natural range of pulmonary pO 2 values given previously (Jones, 1961) and reproduced in Table 2 of a subsequent paper (Jones, 1964). It is at once apparent that the limiting external pO 2 values (lung gas) for significant transport by Planorbis haemoglobin from the lung coincide almost exactly with the range of pulmonary gas from 65 to 20 mm which distinguishes the natural dives of Planorbis from those of Lymnaea. It is more difficult to establish whether the pigment confers any advantage on Planorbis with respect to cutaneous uptake. The in vivo saturation measurements suggest a utility of the pigment down to about 30 mm whereas previous measurements (Jones, 1961) indicate an appreciable cutaneous uptake at least down to 15 mm. This discrepancy presumably represents use of oxygen by superficial tissues which can be reached directly by short diffusion pathways. In actual cutaneous uptake Planorbis and Lymnaea are equally matched at the lower end of the dissolved pO 2 scale (15 mm); at 240 mm Lymnaea uptake is a little higher but the difference is of doubtful significance. At all dissolved oxygen values the complementary pulmonary uptake is significantly higher in Lymnaea so that the total uptake and the percentage of the total which is pulmonary is higher in Lymnaea than in Planorbis (Jones, 1961). Some observations of Zaaijer & Wolvekamp (1958) also indicate that Planorbis and Lymnaea are equally proficient in lowering the dissolved oxygen concentration in a restricted volume of water. It therefore does not appear that the haemoglobin gives Planorbis any superiority over Lymnaea in cutaneous uptake. The pigment must make a contribution in the appropriate range (say 30-80 mm) but this simply matches more favourable diffusion conditions in Lymnaea. Although it is generally held that an accessory gill is lacking in the latter, Simroth & Hoffmann (1928) have suggested that the much expanded tentacles may function as such. In Table 3 some data are assembled which bear on the utilization of pulmonary oxygen during a single dive. Figures for Planorbis and Lymnaea are taken from the earlier account, those for Biomphalaria from a subsequent paper (Jones, 1964). The tissue weight figures are based on weighing seven or eight snails of each species and in the case of Planorbis and Lymnaea agree well with the values (73.7 and 86"3 per cent respectively) of F/isser & Krfiger (1951). The calculated utilization of 02 during the dive (ml/g) shows a marked inferiority in Lymnaea, in spite of which this species succeeds in maintaining a 30 per cent higher total oxygen consumption. This must in part depend on the maintenance of a higher mean pulmonary pO~ through more frequent filling of the lung. However, Hazelhoff (quoted by Jordan, 1930) found that over the range of 16-8 per cent (in which Planorbis haemoglobin is inactive) the rate of fall of pulmonary oxygen concentration in Lymnaea was 2.4 times that in Planorbis. Since the relative pulmonary
THE ROLE OF HAEMOGLOBIN I N THE AQUATIC PULMONATE, P L A N O R B I S C O R N E U S
293
volumes are 0-63-1 (Table 3), this gives further evidence of superior pulmonary uptake by Lymnaea but in this case not dependent on superior pO2. T h u s for similar external pO 2 there is a higher rate of oxygen exchange in Lymnaea, both through the lung and the skin, at least when the Planorbis pigment is inactive. This must rest on generally lower diffusion resistances and/or steeper diffusion gradients. A steeper diffusion gradient is likely to result from a lower venous pO 2 which in turn is likely to reflect a lower tissue tension. Offsetting this advantage, Lymnaea has a smaller pulmonary volume and (in the absence of a respiratory pigment) a restricted capacity for drawing on the pulmonary gas.
T A B L E 3 - - U T I L I Z A T I O N OF PULMONARY OXYGEN DURING THE DIVE
Planorbis Biomphalaria Lymnaea Tissue as o/ /o of total wt.
68'3
Initial pulmonary gas vol (ml/g)
"~ f
Total wt. Tissue wt.
Change during dive
; J
% 02 mm pO2
Utilized O~ vol (ml/g)*
; 3
Total wt. Tissue wt.
0"22 0"32 13"4 99"2 0-030 0'044
69'0 0'25 0-36 14-2 90'5 0"032 0'045
85"9 0-17 0"20 9"4 70-0 0'018 0-021
* The utilized 02 is expressed as ml at 760 mm pressure in all cases but in the case of
Biomphalaria the gas parameters were actually measured at 660 mm barometric pressure (Jones, 1964). Since there is some uncertainty about the per cent change in volume during the dive, the calculation of 02 utilization is based on an assumed common volume change of 10 per cent. For each species the volume change probably lies within the range of 10-15 per cent so the error introduced by this approximation will be very small. These restrictions limit the duration of the dive compared with Planorbis. T h e r e can be no doubt that the possession of haemoglobin in the latter plays an important part in the more extensive exploitation of the lung oxygen store. This, together with the higher pulmonary volume and lower metabolic rate, allows Planorbis a much longer diving period, in consequence of which it can occupy a different ecological niche as previously described (Jones, 1961). Leitch (1916) and Borden (1931), on the basis of their oxygen capacity and other measurements, calculated the potential oxygen storage value of the pigment. In terms of the length of time that normal activity could be sustained by the maximal volume of bound oxygen, their results were 3 and 18 min respectively. These periods were regarded as insignificant. Total blood volume has not been redetermined but the present oxygen capacity figures (maximum 2.4 vol per cent) indicate that the earlier higher estimate (based on 1.27 vol per cent) may be up to 50 per cent too low. It is not reasonable to dismiss this storage potential, for
294
J . D . JONES
in an animal largely dependent on periodic excursions to the surface, a storage period of 20 or more minutes could substantially extend the diving range. However, storage needs to be viewed in a different light. Consider any animal endowed with a respiratory pigment and faced with a declining ambient pO2, whether it be in the true external medium or in an infrequently ventilated lung. Once arterial pO2 drops below the level for full saturation, the total combined oxygen in the arterial blood falls short of the maximum. The pigment may be regarded as having given up part of its "store" but this is entirely incidental to its role as transporter. When external and arterial pOz fall so low as to make continued significant transport impossible, the pigment will be virtually deoxygenated and its storage function nil. It is therefore meaningful to speak of a circulating* pigment as having a storage function per se only when the animal is cut off suddenly from external oxygen supplies with its store in a well-charged condition. Such a situation will not be found in the normal conditions of life of aquatic pulmonates. In any other declining ambient oxygen situation all respiratory pigments are incidentally potential oxygen stores. The role of haemoglobin in Planorbis may be summarized as follows. So long as pulmonary pO2 exceeds about 60 mm, venous pO2 does not fall low enough to cause dissociation of the oxygenated pigment, all requirements being met from solution. As pulmonary pO2 falls below this level the pigment is active in transport of oxygen from the lung. In the region below about 40 mm, arterial blood is no longer fully saturated, so that total combined oxygen is diminishing and the oxygen capacity will become a factor in determining how long the tissues can continue to receive significant amounts of oxygen. By the time pulmonary pO2 reaches 20 mm both transport and storage functions are virtually exhausted. At the same time, so long as dissolved pO 2 exceeds something of the order of 30 mm, part of the pigment may pick up oxygen via the skin and so spare pulmonary oxygen. The dissolved pO2 which will spare pulmonary oxygen entirely has not been determined, but it exceeds 240 mm. SUMMARY 1. A method is described for the in vivo spectroscopic determination of percentage saturation of haemoglobin in albino specimens of Planorbis corneus. 2. The method has been'used to determine the limiting external pO2's for full and zero saturation of haemoglobin in arterial and venous blood with respect to both pulmonary and cutaneous oxygen uptake. 3. For pulmonary uptake the external range of pO~ for pigment function is between 20 and 50-60 mm; for cutaneous uptake the lower limit only has been fixed at about 30 mm. 4. The pulmonary range corresponds well with that which has previously been found to distinguish the dives of Planorbis from those of Lymnaea stagnalis lacking a respiratory pigment. * It has been argued elsewhere that non-circulating coelomic pigments can only act as oxygen stores (Jones, 1963).
THE ROLE OF HAEMOGLOBIN IN THE AQUATIC PULMONATE, P L A N O R B 1 S C O R N E U S
295
5. T h e new data are discussed in relation to earlier observations on these two species, and it is concluded that in Lymnaea conditions for diffusion of oxygen both through the skin and the lung epithelium are easier than in Planorbis. 6. T h e oxygen capacity of the blood of Planorbis has been found to range from 0-94 to 2.49 vol per cent and the question of a possible storage function is discussed. REFERENCES BAKERF. C. (1945) The Molluscan Family Planorbidae. Univ. Illinois Press, Urbana. BORDEN M. A. (1931) A study of the respiration and of the function of haemoglobin in Planorbis corneus and Arenicola marina. J. Mar. biol. Ass. U.K. 17, 709-738. DAM L. VAN (1935) A method for determining the amount of oxygen dissolved in 1 cc of water. J. Exp. Biol. 12, 80--85. Fox H. M. (1945) The oxygen affinities of certain invertebrate haemoglobins. J. Exp. Biol. 21, 161-165. F~2SSER H. & KRt~GER F. (1951) Vergleichende Versuche zur Atmungsphysiologie von Planorbis corneus und Limnaea stagnalis (Gastropoda Pulmonata). Z. vergl. Physiol. 33, 14-52. JONES J. D. (1959) A new tonometric method for the determination of dissolved oxygen and carbon dioxide in small samples. J. Exp. Biol. 36, 177-190. 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) The functions of the respiratory pigments of invertebrates. In Problems in Biology, Vol. 1, (Edited by KERKUTG.). Pergamon Press, Oxford. JONES J. D. (1964) Respiratory gas exchange in the aquatic pulmonate BiomphalaHa sudanica. Comp. Biochem. Physiol. 12, 297-310. JORDAN H. J. (1930) Le r~glage de la consommation de l'oxyg~ne chez les animaux "tension gazeuse alv~olaire inconstante". Arch. nderl. Physiol. 15, 198-212. LEITCH I. (1916) The function of haemoglobin in invertebrates with special reference to Planorbis and Chironomus larvae. J. Physiol. 50, 370-379. NATELSONS. & MENNINGC. M. (1955) Improved methods of analysis for oxygen, carbon monoxide and iron on fingertip blood. Clin. Chem. 1, 165-179. REOMOND J. R. (1955) The respiratory function of haemocyanin in crustacea. J. Cell. Comp. Physiol. 46, 209-247. REDMOND J. R. (1962) Oxygen-hemocyanin relationships in the land crab, Cardisoma guanhumi. Biol. Bull., Woods Hole 122, 252-262. SIMROTH H. & HOFFMANN H. (1928) In Bronn's Klassen und Ordnungen des Tierreichs. Bd. 3, Abt. II, Buch 2. Pulmonata. Akad. Verlagsges, Leipzig. SPOEK G. L. (1962) Oxygen dissociation curves and arterial and venous oxygen content ot bloods of Homarus gammarus and Maia squinado. Versl. gewone Vergad. Akad. ~.Jmst. 71, 29-34. ZAAUER J. J. P. & WOLVEKAMPPI. P. (1958) Some experiments on the haemoglobinoxygen equilibrium in the blood of the ramshorn (Planorbis corneus L.). Acta Physiol. Pharmacol. Nderl. 7, 56-77.