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The respiratory function of gills in the larvae of Amblystoma punctatum
DEVELOPMENTAL
The
BIOLOGY,
Respiratory
7, 420431
( 1963 )
Function
of IGills
of Amblystoma E. J.
BOELL,
Department
PHYLLIS
Larvae
punctatoml
GREENFIELD,
of Biology, Yale University, Accepted
in the
AND
BERTIL
HILLE
New Haven, Connecticut
October 31, 1962
INTRODUCTION
It has now been well established that when larvae of certain amphibian species are reared in water saturated with a gas mixture containing less oxygen than the normal supply in air, the gills increase very considerably in size. This observation was first reported by Bab&k (1907) and was later confirmed by Drastich (1925). More recently, Bond (1960) made a careful analysis in three species of salamander of the response of the gills to reduced oxygen supply. Her work showed that the length, filament number, cell dimensions and number, mitotic index, and blood volume of gills were affected by low oxygen supply in such a way as to iead to approximately a threefold increase in gill surface.’ Such results have generally been interpreted as representing an adaptive response to environmental stress, in this instance an increase in total respiratory surface as a consequence of reduced oxygen supply in the environment (Krogh, 1941; Bishop, 1950). Although the gills of the salamander larva seem ideally suited by virtue of their structure, position, movements, and abundant circulation to perform a respiratory function, observations from a number of sources suggest indirectly that gills may be used, but they are not essential in respiration under ordinary and under some extraordinary circumstances. Harrison ( 1921) replaced the branchial ectoderm of Amblystoma punctatum embryos with flank ectoderm and noted that ‘This research was supported in part by grants from the National Science Foundation, G-8771 and G-16240. *In addition both Drastich (1925) and Bond (1960) reported that when larvae were reared in oxygen concentrations greater than normal, the gills became greatly reduced in size.
420
THE
RESPIRATORY
FUNCTION
OF
GILLS
421
the resulting larvae exhibited greatly impaired gill development. In one case, gills were almost completely lacking. Nevertheless, the development of this animal, apart from the absence of gills was normal. Apparently, the larva was able to adjust itself to the loss of its respiratory organs. In similar experiments, Severinghaus (1930) showed that larvae of A. punctatum and A. tigrinum developed essentially normally even though gill development was experimentally interfered with. Additional indirect evidence of the dispensability of gills (Boell, unpublished) has been obtained in a quantitative study of the swimming capacities of A. punctatum larvae by the techniques described by Detwiler ( 1946a,b). Gill-less animals were found to be as competent as normal ones in responding to repeated mechanical stimulation, and there was no evidence that the reduction of respiratory surface interfered in any way with sustained physical activity. The present study was undertaken in order to obtain direct information on the possible role of gills in respiration and involves a systematic investigation of the effect of gill removal on respiration and on development in A. punctatum larvae of different ages and under conditions of varying oxygen supply. MATERIAL
AND METHODS
The larvae used in these experiments were obtained from Amhlystoma punctatum eggs collected in the vicinity of New Haven. They were permitted to develop slowly at low temperature until approximately stage 35 when they were removed from their jellies and reared in fresh spring water at a temperature of approximately 23°C. During feeding stages larvae were fed on Daphnia or enchytraeid worms. Observations were confined to a period of development beginning at stage 38 and continuing for approximately 2 months after feeding had commenced. During this period, the gills are proportionately larger than at any other time during development, and might be expected, therefore, to have their greatest potential significance as respiratory structures. Respiration was measured in Warburg manometers at a temperature of 25°C. Flasks of 5 ml or 15 ml capacity, containing, respectively, I or 3 ml of spring water, were used depending upon the size of the larvae. The number of larvae per flask varied generally between five and one, but with some of the youngest stages eight larvae per flask were employed. In a series of preliminary experiments, it was
422
BOELL,
GREENFIELD,
AND
HILLE
determined that shaking at a rate of 70 cycles per minute over an amplitude of 3 cm was optimal for ensuring adequate equilibration of the liquid and gaseous phases in the manometer flasks without unduly stimulating the larvae. The procedure generally followed in the experiments reported below involved measurement of respiration in air-saturated water during a control period of from 1 to 3 hours and then again during an experimental period of approximately the same length. In these determinations, each animal or set of animals thus served as its own control for the experimental period which followed. As a consequence, the effect of biological variability, which may be considerable, even in larvae from the same clutch of eggs, was minimized. Gas mixtures were made up volumetrically in a calibrated spirometer having a capacity of approximately 10 liters from cylinder supplies of oxygen, nitrogen, and carbon monoxide. In the manometric experiments in which different oxygen concentrations were used, approximately 1.2 liters of gas were run through each manometer flask while it was being shaken to ensure equilibration with the new gas mixture. Gills were removed from larvae after light anesthetization in MS222, diluted one part in 3000 or in 6000 from a stock solution. Gill amputation could be accomplished with almost no loss of blood by the expedient of pinching the gill near its point of attachment with watchmaker’s forceps and then cutting along the edge of the forceps with a sharp scalpel. Animals whose gills were merely snipped off by scissors bled profusely. After completion of the operations, the larvae were washed in several changes of fresh spring water and allowed to recover before respiratory measurements were begun. In order to determine the effect of various concentrations of oxygen in the medium on viability and on gill development, larvae were reared in 500-ml Erlenmeyer flasks containing approximately 125 ml of spring water. Each flask was sealed by means of a two-holed rubber stopper through which passed two glass tubes for gas inlet and outflow. The inlet tube was of such length that it ended in the water near the bottom of the flask after the stopper was inserted. When it was desired to change the oxygen supply to the larvae, approximately 4 liters of a desired gas mixture were slowly bubbled through each flask. After this, the flask was tightly sealed by means of rubber tubing and screw clamps. Gas mixtures were renewed in each flask every 2 or 3 days, and, when necessary, the larvae were fed
THE
RESPIRATORY
FUNCTION
OF
423
GILLS
at appropriate intervals by dropping Daphnia or enchytraeid worms through the gas exit tube. Larvae in the various flasks were examined from time to time to take note of their viability, stage of development, behavior, and, particularly, the state of their gill development. RESULTS
Figure 1 depicts the results of a typical experiment on the respiration of larvae before and after gill removal in a medium saturated with air. In this experiment, two lots of three larvae at stage 46 were carefully selected for uniformity of size and development, and the respiratory rate for each was determined during a control period of
30
20
DEGILLED
IO
0 I______
-_I 0
I
2
3
HOURS FIG. 1. Graph of oxygen larvae at stage 46. At dotted vened, during which animals one set.
consumption of normal and degilled line on abscissa, two and three-quarters were narcotized in MS222 and gills
A. punctatutn hours interremoved from
424
BOELL,
GREENFIELD,
AND
HILLE
1 hour. The larvae were then removed from the manometer vessels, lightly anesthetized in 1: 3000 MS222 for 7 minutes, and the gills were then amputated from one lot. After a recovery period of approximately two and three quarters hours, respiration was measured again. As shown in Fig. 1, gill removal, at this stage of development, had absolutely no effect on the respiration of the larvae immediately after the operation. Nor did the absence of gills result in lowered respiratory activity later on. The same result, save for quantitative differences in the initial level of respiration, was obtained in all experiments of similar nature. It might be thought that older animals having a higher respiratory rate would find absence of gills disadvantageous, but the results in Table 1 do not support such a suggestion. No direct estimate of the age of the larvae included in the table can be made, for they represent individuals from different lots of eggs and with different temperature histories; chronological age and developmental age are, therefore, not synonymous. Although the respiration of the larvae in Table 1 covers a range of from 3.5 to 20 ~1 per hour, which during normal development would correspond to an age span of approximately 2 months, the respiratory rates before and after gill removal are not significantly different. TABLE RESPIRATION Before
gill removaln
3.5 5.0 5.4 20.0
n Oxygen
OF Amblystoma
LARVAE After
1 IN
AIR-SATURATED
gill removel~
3.5 4.6 5.i 20.8
WATER % 100
92 105 104
consumption given as microliters per larva per hour.
It appears from the data presented that gills are readily dispensable when larvae are reared in water saturated with air. It is possible, however, that gills might assume increased functional significance in media of lower oxygen concentrations. In order to obtain information on this question, the respiratory activity of normal and gill-less larvae was determined in gas mixtures containing 15, 10, and 5% oxygen. The results of these experiments are summarized in Fig. 2. The curve includes data for the respiration of normal and gill-less larvae
THF.
RESPIRATORY
FUNCTION
OF
425
GILLS
in water saturated with various oxygen concentrations and also for the respiration of normal larvae in 50% and 100% oxygen.3 The oxygen consumption of normal and gill-less larvae is essentially identical from 21 to 10% oxygen. However, in 5% oxygen, respiration of gill-less larvae, on the average, is depressed below that of controls. This concentration of oxygen is at or near the lower limit of tolerance for A. punctatum larvae. In experiments involving exposure to 5% oxygen 120 -
100 -
I
I
I
5
IO
15
PERCENTAGE
I
I
!
I
I
I
I
20
25
30
35
40
46
50
i&i
OXYGEN
FIG. 2. The relationship between respiration of A. punctatum larvae as percentage of the value in air (ordinate) and percentage oxygen concentration (abscissa) of gas mixture in equilibrium with water in manometer flasks. Filled circles, normal larvae; open circles, degilled larvae. The number of observations averaged as experimental points for normal (N) and degilled (D) larvae are tabulated below. In no case does the probable error of the average exceed the diameter of the point on the graph.
lOOL& 02
N D
8
-
50%
02 1
21%
15 27
c2
10%
il 33
O?
5%
02
16 16
for several days viability of the larvae was reduced, development was considerably retarded, and morphological abnormalities frequently a We have also found that the gills of larvae reared continuously in pure oxygen shrivel and become thickened and knobby in appearance. This is also true, but to a lesser extent, of the gills of larvae reared in 50% oxygen.
426
BOELL,
GREENFIELD,
AND
HILLE
appeared. Bond (1960) has also reported that 5% oxygen is approximately the minimum tolerated level for the larvae of A. opaca and A. jeffersonianurn. Normal animals were somewhat more resistant, but even short-term exposures to 5% oxygen often proved fatal to gill-less larvae. An illustrative example is shown in Fig. 3. On the day before the measurements recorded in the graph were made, several lots each consisting of three larvae of similar stage (hindlimb buds well developed) were selected. Gills were removed from one half the lots, and respiration
60 60 -
AIR
II ’
NORMAL
,
6Or
60 -
HOURS
FIG. 3. Typical experiment larvae in water saturated with of the experiment, see text.
of respiration of normal and degilled A. punctatum air, 10% oxygen, and 5% oxygen. For description
THE
RESPIRATORY
FUNCTION
OF
GILLS
427
of control and degilled larvae was then determined during consecutive periods in air, in 10% oxygen, and in 5% oxygen. The data for typical control and degilled animals (Fig. 3) show that the respiratory rates of normal and degilled animals were essentially identical in 10% oxygen (89% and 87%, respectively, of the values in air), but, at a level of 5% oxygen, the respiration of the gill-less larvae was reduced much more than that of the controls. The figure for gill-less larvae is 3% of the value in air; for normal larvae at the same oxygen concentration, it is 81%. At the end of the period of measurement all control and gill-less larvae were examined, and the following account, transcribed from the protocol for the experiment, describes the results: ControE Lartiae: appear normal in all respects; respond to tactile stimulation by vigorous swimming; circulation strong. Gill-less larvae: completely paralyzed; give no response to tactile stimulation; stomachs bloated and gas-filled; larvae float upside down; heart beat weak and circulation sluggish. The next morning several of the larvae were dead; the remainder were abnormal. Although in a medium saturated with 10% oxygen, respiration of normal and gill-less larvae is reduced somewhat (15% below the rate in air), such organisms appear normal in every respect even under conditions of prolonged exposure to the lowered oxygen supply. So far as rate of development, swimming, and food intake are concerned, the larvae cannot be distinguished from those developing in water saturated with air. The only observable effect of long-term exposure to lowered oxygen concentration is the development of gills which are much larger and more luxuriant than normal.’ DISCUSSION
From the results presented, it appears that the gills of Amblystoma larvae have little significance in respiration save under conditions of extremely low oxygen supply. Even here, however, the possession of gills does not give the larvae much advantage over their gill-less counterparts. Of course, it cannot be concluded from such experiments that gills do not participate in the respiratory exchange of the organ4 For Salamandru larvae, Drastich ( 1925) reported that exposure to 11% oxygen caused retarded development. The difference between his results and ours may be due to the fact that the larvae he used were older and were exposed to lowered oxygen for a much longer time.
428
BOELL,
GREENFIELD,
AND
HILLE
km, but it is apparent that they are not essential for the maintenance of the normal level of oxygen uptake. The curve in Fig. 2 shows that A. punctatum larvae, even when challenged by only a modest reduction in the environmental oxygen supply, can regulate respiration to only a very limited extent, and that except in 5% oxygen gill-less animals are not more handicapped than normal individuals. By contrast, many organisms and especially those with blood respiratory pigments (see Prosser and Brown, 1961, for examples) are able to regulate respiration at an essentially constant level over a wide range of oxygen concentrations. The results obtained with Amblystoma are perhaps somewhat surprising, for in larvae of the age used in these experiments, the circulation is well-developed; the blood appears red and presumably contains hemoglobin. We do not have data on the loading tension nor the oxygen capacity of larval Amblystoma blood. However, McCutcheon (1936) and Riggs (1951) have shown that hemoglobin from Rana catesbianu tadpoles, of somewhat older developmental age than the Amblystoma larvae used in the present investigation, has a very low loading tension for half saturation; Riggs gives a figure of 4.6 mm at pH 7.32. In this connection, it is of interest to recall that Helff and Stubblefield (1931) sh owed that respiration of Rana pipiens tadpoles was independent of oxygen supply down to an average PC+ of 35 mm, i.e., equivalent to saturation of water with 4.6% oxygen. It appears from the data presented that blood of Amblystoma has radically different properties from that of Rana, and one might question whether hemoglobin is significantly involved in oxygen transport at all. Unfortunately, it would be very difficult, owing to the small volumes of blood in larvae at this stage, to obtain direct information on this question. However, indirect evidence through the use of carbon monoxide can be obtained (Ewer, 1942; Johnson, 1942). A series of experiments was performed in which Amblystoma larvae of various ages were subjected to an atmosphere containing 29% carbon monoxide, 20% oxygen, and 66% nitrogen. The concentration of carbon monoxide in such a mixture is much lower than that required to inhibit cytochrome oxidase directly (Boell, 1945)) so that presumably any effect of the gas on respiration involves interference with oxygen transport by hemoglobin. The results are summarized in Fig. 4. From stage 38, the earliest tested, to approximately stage 43, respiration is not affected, or only slightly so, by carbon monoxide. Beginning with
THE
36 50
RESPIRATORY
FUNCTION
41 ’
8
’
IO
’
’
12 14
’
16
DAYS
’
46 t
18 20
OF
GILLS
’
(
429
0
’
22
’
24 26
--
___
28 - -
OF DEVELOPMENT
FIG. 4. Graph showing respiration of A. pun&turn larvae consisting of 20% CO, 20% O?, and 60% oxygen as percentage of (ordinate) at different ages (abscissa) after fertilization. Each a single determination. The last two points are for older larvae age. Numerals above baseline refer to Harrison’s stages.
in a gas mixture the value in air point represents of undetermined
stage 43, however, respiration is increasingly depressed throughout the period of development studied. Even at the latest stages tested, however, the effect of carbon monoxide appears to be relatively low. If the data summarized in Fig. 4 can be interpreted in the usual way, they suggest that the respiratory pigment in Amblystoma blood begins to take on the function of oxygen transport at about stage 43. Before this, and perhaps also to a considerable extent afterward, oxygen is transported by blood predominantly in solution rather than in combination with hemoglobin. In this connection, it is of interest to note that adult vertebrates (Xenopus and the hagfish) are occasionally found with hemoglobin-free blood and that the so-called ice fish, of the family Chaenichthyidae, normally has no hemoglobin or erythrocytes in its blood. Although the oxygen capacity of the blood of these forms is the same as that of the plasma of other vertebrates, the animals appear to have no difficulty maintaining the activity necessary for survival (Lehmann and Huntsman, 1961). It should not be surprising, therefore, to find that in larval Amblystoma, hemoglobin has limited significance in oxygen transport.
430
BOELL,
GREENFIELD,
AND
HILLE
Although the foregoing results suggest that the respiratory function of gills in Amblystoma larvae may not be particularly impressive, it seems unlikely that these elaborately developed structures have no function. As Krogh (1941) has pointed out, “Both lungs and gills serve other purposes besides respiration and it is sometimes difficult to decide whether cavities or appendages have any definite respiratory function.” In a number of forms, gills are concerned with the uptake of ions and the excretion of inorganic materials or the products of metabolism. It remains for future work to discover whether these or other functions are carried out by the gills of Amblystoma. SUMMARY
A study has been made of the effect of gill removal on the oxygen consumption of developing Amblystoma punctatum larvae. In water saturated with 21%, l!%, and 10% oxygen, the rate of oxygen consumption of gill-less animals is indistinguishable from that of normal individuals. At saturation with 5% oxygen, respiration of gill-less animals is less than that of controls with gills. Although gills may function in respiratory exchange, they are not essential for normal oxygen uptake Indirect evidence indicates that the under ordinary circumstances. hemoglobin of Amblystoma blood, at the stages studied, has limited significance in oxygen transport. REFERENCES BAB~~K, E. ( 1907). ifber die funktionelle Anpassung die susseren Kiemen bei Sauerstoffmangels. Zen&. Physiol. 21, 97-99. BISHOP, D. W. ( 1950). Respiration and metabolism. In “Comparative Animal Physiology” (C. L. Prosser, ed.), Chap. VIII. W. B. Saunders, Philadelphia. BOELL, E. J. (1945). Functional differentiation in embryonic development. II. Respiration and cytochrome oxidase activity in Amblystoma punctatum. J. Exptl. Zool. 100, 331352. BOND, A. N. ( 1960). An analysis of the response of salamander gills to changes in the oxygen concentration of the medium. huelop. Biol. 2, l-20. DETWILER, S. R. (1946a). Experiments upon the midbrain of Amblystoma embryos. Am. J. Anat. 78, 115-138. DETWILER, S. R. (1946b). A quantitative study of locomotion in larval Amhlystoma following either midbrain or forebrain excision. J. ExptZ. ZooZ. 102, 321-332. DRASTICH, E. (1925). Wber das Leben der Salamandru-Larven bei hohem und niedrigem Sauerstoffpartialdruck. 2. uergleich. PhysioZ. 2, 632-657. EWER, R. F. ( 1942). On the function of hemoglobin in Chironomus. J. ExptZ. BioZ. 18, 197-205.
THE
RESPIRATORY
FUNCTION
OF
GILLS
431
HARRISON, R. G. (1921). Experiments on the development of the gills in the amphibian embryo. Bio2. Bull. 41, 156-170. HELFF, 0. M., and STUBBLEFIELD, K. I. ( 1931). The influence of oxygen tension on the oxygen consumption of Rana pipiens larvae. Physiol. Zod. 4, 271-286. JOHNSON, M. L. (1942). The respiratory function of the hemoglobin of the earthworm. J. Exptl. Biol. 18, 266-277. KROGH, A. ( 1941) . “The Comparative Physiology of Respiratory Mechanisms.” Univ. of Pennsylvania Press, Philadelphia. LEHMANN, H., and HUNTSMAN, R. G. ( 1961). Why are red cells the shape they are? In “Functions of the Blood” (R. G. Macfarlane and A. H. T. Robb-Smith, eds.), Chap. II. Academic Press, New York. MCCUTCHEON, F. H. (1936). Hemoglobin function during the life history of the bullfrog. J. Cellular Comp. Physiol. 8, 63-81. PROSSER, C. L., and BROWN, F. A., JR. ( 1961). “Comparative Animal Physiology,” Chap. VII. W. B. Saunders, Philadelphia. RIGGS, A. (1951). The metamorphosis of hemoglobin in the bullfrog. J. Gen. Physiol. 35, 2340. SEVEEUNGHAUS, A. E. (1930). Gill development in Amblystoma punctatum. J. Exptl. Zool. 56, l-29.
The
BIOLOGY,
Respiratory
7, 420431
( 1963 )
Function
of IGills
of Amblystoma E. J.
BOELL,
Department
PHYLLIS
Larvae
punctatoml
GREENFIELD,
of Biology, Yale University, Accepted
in the
AND
BERTIL
HILLE
New Haven, Connecticut
October 31, 1962
INTRODUCTION
It has now been well established that when larvae of certain amphibian species are reared in water saturated with a gas mixture containing less oxygen than the normal supply in air, the gills increase very considerably in size. This observation was first reported by Bab&k (1907) and was later confirmed by Drastich (1925). More recently, Bond (1960) made a careful analysis in three species of salamander of the response of the gills to reduced oxygen supply. Her work showed that the length, filament number, cell dimensions and number, mitotic index, and blood volume of gills were affected by low oxygen supply in such a way as to iead to approximately a threefold increase in gill surface.’ Such results have generally been interpreted as representing an adaptive response to environmental stress, in this instance an increase in total respiratory surface as a consequence of reduced oxygen supply in the environment (Krogh, 1941; Bishop, 1950). Although the gills of the salamander larva seem ideally suited by virtue of their structure, position, movements, and abundant circulation to perform a respiratory function, observations from a number of sources suggest indirectly that gills may be used, but they are not essential in respiration under ordinary and under some extraordinary circumstances. Harrison ( 1921) replaced the branchial ectoderm of Amblystoma punctatum embryos with flank ectoderm and noted that ‘This research was supported in part by grants from the National Science Foundation, G-8771 and G-16240. *In addition both Drastich (1925) and Bond (1960) reported that when larvae were reared in oxygen concentrations greater than normal, the gills became greatly reduced in size.
420
THE
RESPIRATORY
FUNCTION
OF
GILLS
421
the resulting larvae exhibited greatly impaired gill development. In one case, gills were almost completely lacking. Nevertheless, the development of this animal, apart from the absence of gills was normal. Apparently, the larva was able to adjust itself to the loss of its respiratory organs. In similar experiments, Severinghaus (1930) showed that larvae of A. punctatum and A. tigrinum developed essentially normally even though gill development was experimentally interfered with. Additional indirect evidence of the dispensability of gills (Boell, unpublished) has been obtained in a quantitative study of the swimming capacities of A. punctatum larvae by the techniques described by Detwiler ( 1946a,b). Gill-less animals were found to be as competent as normal ones in responding to repeated mechanical stimulation, and there was no evidence that the reduction of respiratory surface interfered in any way with sustained physical activity. The present study was undertaken in order to obtain direct information on the possible role of gills in respiration and involves a systematic investigation of the effect of gill removal on respiration and on development in A. punctatum larvae of different ages and under conditions of varying oxygen supply. MATERIAL
AND METHODS
The larvae used in these experiments were obtained from Amhlystoma punctatum eggs collected in the vicinity of New Haven. They were permitted to develop slowly at low temperature until approximately stage 35 when they were removed from their jellies and reared in fresh spring water at a temperature of approximately 23°C. During feeding stages larvae were fed on Daphnia or enchytraeid worms. Observations were confined to a period of development beginning at stage 38 and continuing for approximately 2 months after feeding had commenced. During this period, the gills are proportionately larger than at any other time during development, and might be expected, therefore, to have their greatest potential significance as respiratory structures. Respiration was measured in Warburg manometers at a temperature of 25°C. Flasks of 5 ml or 15 ml capacity, containing, respectively, I or 3 ml of spring water, were used depending upon the size of the larvae. The number of larvae per flask varied generally between five and one, but with some of the youngest stages eight larvae per flask were employed. In a series of preliminary experiments, it was
422
BOELL,
GREENFIELD,
AND
HILLE
determined that shaking at a rate of 70 cycles per minute over an amplitude of 3 cm was optimal for ensuring adequate equilibration of the liquid and gaseous phases in the manometer flasks without unduly stimulating the larvae. The procedure generally followed in the experiments reported below involved measurement of respiration in air-saturated water during a control period of from 1 to 3 hours and then again during an experimental period of approximately the same length. In these determinations, each animal or set of animals thus served as its own control for the experimental period which followed. As a consequence, the effect of biological variability, which may be considerable, even in larvae from the same clutch of eggs, was minimized. Gas mixtures were made up volumetrically in a calibrated spirometer having a capacity of approximately 10 liters from cylinder supplies of oxygen, nitrogen, and carbon monoxide. In the manometric experiments in which different oxygen concentrations were used, approximately 1.2 liters of gas were run through each manometer flask while it was being shaken to ensure equilibration with the new gas mixture. Gills were removed from larvae after light anesthetization in MS222, diluted one part in 3000 or in 6000 from a stock solution. Gill amputation could be accomplished with almost no loss of blood by the expedient of pinching the gill near its point of attachment with watchmaker’s forceps and then cutting along the edge of the forceps with a sharp scalpel. Animals whose gills were merely snipped off by scissors bled profusely. After completion of the operations, the larvae were washed in several changes of fresh spring water and allowed to recover before respiratory measurements were begun. In order to determine the effect of various concentrations of oxygen in the medium on viability and on gill development, larvae were reared in 500-ml Erlenmeyer flasks containing approximately 125 ml of spring water. Each flask was sealed by means of a two-holed rubber stopper through which passed two glass tubes for gas inlet and outflow. The inlet tube was of such length that it ended in the water near the bottom of the flask after the stopper was inserted. When it was desired to change the oxygen supply to the larvae, approximately 4 liters of a desired gas mixture were slowly bubbled through each flask. After this, the flask was tightly sealed by means of rubber tubing and screw clamps. Gas mixtures were renewed in each flask every 2 or 3 days, and, when necessary, the larvae were fed
THE
RESPIRATORY
FUNCTION
OF
423
GILLS
at appropriate intervals by dropping Daphnia or enchytraeid worms through the gas exit tube. Larvae in the various flasks were examined from time to time to take note of their viability, stage of development, behavior, and, particularly, the state of their gill development. RESULTS
Figure 1 depicts the results of a typical experiment on the respiration of larvae before and after gill removal in a medium saturated with air. In this experiment, two lots of three larvae at stage 46 were carefully selected for uniformity of size and development, and the respiratory rate for each was determined during a control period of
30
20
DEGILLED
IO
0 I______
-_I 0
I
2
3
HOURS FIG. 1. Graph of oxygen larvae at stage 46. At dotted vened, during which animals one set.
consumption of normal and degilled line on abscissa, two and three-quarters were narcotized in MS222 and gills
A. punctatutn hours interremoved from
424
BOELL,
GREENFIELD,
AND
HILLE
1 hour. The larvae were then removed from the manometer vessels, lightly anesthetized in 1: 3000 MS222 for 7 minutes, and the gills were then amputated from one lot. After a recovery period of approximately two and three quarters hours, respiration was measured again. As shown in Fig. 1, gill removal, at this stage of development, had absolutely no effect on the respiration of the larvae immediately after the operation. Nor did the absence of gills result in lowered respiratory activity later on. The same result, save for quantitative differences in the initial level of respiration, was obtained in all experiments of similar nature. It might be thought that older animals having a higher respiratory rate would find absence of gills disadvantageous, but the results in Table 1 do not support such a suggestion. No direct estimate of the age of the larvae included in the table can be made, for they represent individuals from different lots of eggs and with different temperature histories; chronological age and developmental age are, therefore, not synonymous. Although the respiration of the larvae in Table 1 covers a range of from 3.5 to 20 ~1 per hour, which during normal development would correspond to an age span of approximately 2 months, the respiratory rates before and after gill removal are not significantly different. TABLE RESPIRATION Before
gill removaln
3.5 5.0 5.4 20.0
n Oxygen
OF Amblystoma
LARVAE After
1 IN
AIR-SATURATED
gill removel~
3.5 4.6 5.i 20.8
WATER % 100
92 105 104
consumption given as microliters per larva per hour.
It appears from the data presented that gills are readily dispensable when larvae are reared in water saturated with air. It is possible, however, that gills might assume increased functional significance in media of lower oxygen concentrations. In order to obtain information on this question, the respiratory activity of normal and gill-less larvae was determined in gas mixtures containing 15, 10, and 5% oxygen. The results of these experiments are summarized in Fig. 2. The curve includes data for the respiration of normal and gill-less larvae
THF.
RESPIRATORY
FUNCTION
OF
425
GILLS
in water saturated with various oxygen concentrations and also for the respiration of normal larvae in 50% and 100% oxygen.3 The oxygen consumption of normal and gill-less larvae is essentially identical from 21 to 10% oxygen. However, in 5% oxygen, respiration of gill-less larvae, on the average, is depressed below that of controls. This concentration of oxygen is at or near the lower limit of tolerance for A. punctatum larvae. In experiments involving exposure to 5% oxygen 120 -
100 -
I
I
I
5
IO
15
PERCENTAGE
I
I
!
I
I
I
I
20
25
30
35
40
46
50
i&i
OXYGEN
FIG. 2. The relationship between respiration of A. punctatum larvae as percentage of the value in air (ordinate) and percentage oxygen concentration (abscissa) of gas mixture in equilibrium with water in manometer flasks. Filled circles, normal larvae; open circles, degilled larvae. The number of observations averaged as experimental points for normal (N) and degilled (D) larvae are tabulated below. In no case does the probable error of the average exceed the diameter of the point on the graph.
lOOL& 02
N D
8
-
50%
02 1
21%
15 27
c2
10%
il 33
O?
5%
02
16 16
for several days viability of the larvae was reduced, development was considerably retarded, and morphological abnormalities frequently a We have also found that the gills of larvae reared continuously in pure oxygen shrivel and become thickened and knobby in appearance. This is also true, but to a lesser extent, of the gills of larvae reared in 50% oxygen.
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appeared. Bond (1960) has also reported that 5% oxygen is approximately the minimum tolerated level for the larvae of A. opaca and A. jeffersonianurn. Normal animals were somewhat more resistant, but even short-term exposures to 5% oxygen often proved fatal to gill-less larvae. An illustrative example is shown in Fig. 3. On the day before the measurements recorded in the graph were made, several lots each consisting of three larvae of similar stage (hindlimb buds well developed) were selected. Gills were removed from one half the lots, and respiration
60 60 -
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FIG. 3. Typical experiment larvae in water saturated with of the experiment, see text.
of respiration of normal and degilled A. punctatum air, 10% oxygen, and 5% oxygen. For description
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of control and degilled larvae was then determined during consecutive periods in air, in 10% oxygen, and in 5% oxygen. The data for typical control and degilled animals (Fig. 3) show that the respiratory rates of normal and degilled animals were essentially identical in 10% oxygen (89% and 87%, respectively, of the values in air), but, at a level of 5% oxygen, the respiration of the gill-less larvae was reduced much more than that of the controls. The figure for gill-less larvae is 3% of the value in air; for normal larvae at the same oxygen concentration, it is 81%. At the end of the period of measurement all control and gill-less larvae were examined, and the following account, transcribed from the protocol for the experiment, describes the results: ControE Lartiae: appear normal in all respects; respond to tactile stimulation by vigorous swimming; circulation strong. Gill-less larvae: completely paralyzed; give no response to tactile stimulation; stomachs bloated and gas-filled; larvae float upside down; heart beat weak and circulation sluggish. The next morning several of the larvae were dead; the remainder were abnormal. Although in a medium saturated with 10% oxygen, respiration of normal and gill-less larvae is reduced somewhat (15% below the rate in air), such organisms appear normal in every respect even under conditions of prolonged exposure to the lowered oxygen supply. So far as rate of development, swimming, and food intake are concerned, the larvae cannot be distinguished from those developing in water saturated with air. The only observable effect of long-term exposure to lowered oxygen concentration is the development of gills which are much larger and more luxuriant than normal.’ DISCUSSION
From the results presented, it appears that the gills of Amblystoma larvae have little significance in respiration save under conditions of extremely low oxygen supply. Even here, however, the possession of gills does not give the larvae much advantage over their gill-less counterparts. Of course, it cannot be concluded from such experiments that gills do not participate in the respiratory exchange of the organ4 For Salamandru larvae, Drastich ( 1925) reported that exposure to 11% oxygen caused retarded development. The difference between his results and ours may be due to the fact that the larvae he used were older and were exposed to lowered oxygen for a much longer time.
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km, but it is apparent that they are not essential for the maintenance of the normal level of oxygen uptake. The curve in Fig. 2 shows that A. punctatum larvae, even when challenged by only a modest reduction in the environmental oxygen supply, can regulate respiration to only a very limited extent, and that except in 5% oxygen gill-less animals are not more handicapped than normal individuals. By contrast, many organisms and especially those with blood respiratory pigments (see Prosser and Brown, 1961, for examples) are able to regulate respiration at an essentially constant level over a wide range of oxygen concentrations. The results obtained with Amblystoma are perhaps somewhat surprising, for in larvae of the age used in these experiments, the circulation is well-developed; the blood appears red and presumably contains hemoglobin. We do not have data on the loading tension nor the oxygen capacity of larval Amblystoma blood. However, McCutcheon (1936) and Riggs (1951) have shown that hemoglobin from Rana catesbianu tadpoles, of somewhat older developmental age than the Amblystoma larvae used in the present investigation, has a very low loading tension for half saturation; Riggs gives a figure of 4.6 mm at pH 7.32. In this connection, it is of interest to recall that Helff and Stubblefield (1931) sh owed that respiration of Rana pipiens tadpoles was independent of oxygen supply down to an average PC+ of 35 mm, i.e., equivalent to saturation of water with 4.6% oxygen. It appears from the data presented that blood of Amblystoma has radically different properties from that of Rana, and one might question whether hemoglobin is significantly involved in oxygen transport at all. Unfortunately, it would be very difficult, owing to the small volumes of blood in larvae at this stage, to obtain direct information on this question. However, indirect evidence through the use of carbon monoxide can be obtained (Ewer, 1942; Johnson, 1942). A series of experiments was performed in which Amblystoma larvae of various ages were subjected to an atmosphere containing 29% carbon monoxide, 20% oxygen, and 66% nitrogen. The concentration of carbon monoxide in such a mixture is much lower than that required to inhibit cytochrome oxidase directly (Boell, 1945)) so that presumably any effect of the gas on respiration involves interference with oxygen transport by hemoglobin. The results are summarized in Fig. 4. From stage 38, the earliest tested, to approximately stage 43, respiration is not affected, or only slightly so, by carbon monoxide. Beginning with
THE
36 50
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FIG. 4. Graph showing respiration of A. pun&turn larvae consisting of 20% CO, 20% O?, and 60% oxygen as percentage of (ordinate) at different ages (abscissa) after fertilization. Each a single determination. The last two points are for older larvae age. Numerals above baseline refer to Harrison’s stages.
in a gas mixture the value in air point represents of undetermined
stage 43, however, respiration is increasingly depressed throughout the period of development studied. Even at the latest stages tested, however, the effect of carbon monoxide appears to be relatively low. If the data summarized in Fig. 4 can be interpreted in the usual way, they suggest that the respiratory pigment in Amblystoma blood begins to take on the function of oxygen transport at about stage 43. Before this, and perhaps also to a considerable extent afterward, oxygen is transported by blood predominantly in solution rather than in combination with hemoglobin. In this connection, it is of interest to note that adult vertebrates (Xenopus and the hagfish) are occasionally found with hemoglobin-free blood and that the so-called ice fish, of the family Chaenichthyidae, normally has no hemoglobin or erythrocytes in its blood. Although the oxygen capacity of the blood of these forms is the same as that of the plasma of other vertebrates, the animals appear to have no difficulty maintaining the activity necessary for survival (Lehmann and Huntsman, 1961). It should not be surprising, therefore, to find that in larval Amblystoma, hemoglobin has limited significance in oxygen transport.
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Although the foregoing results suggest that the respiratory function of gills in Amblystoma larvae may not be particularly impressive, it seems unlikely that these elaborately developed structures have no function. As Krogh (1941) has pointed out, “Both lungs and gills serve other purposes besides respiration and it is sometimes difficult to decide whether cavities or appendages have any definite respiratory function.” In a number of forms, gills are concerned with the uptake of ions and the excretion of inorganic materials or the products of metabolism. It remains for future work to discover whether these or other functions are carried out by the gills of Amblystoma. SUMMARY
A study has been made of the effect of gill removal on the oxygen consumption of developing Amblystoma punctatum larvae. In water saturated with 21%, l!%, and 10% oxygen, the rate of oxygen consumption of gill-less animals is indistinguishable from that of normal individuals. At saturation with 5% oxygen, respiration of gill-less animals is less than that of controls with gills. Although gills may function in respiratory exchange, they are not essential for normal oxygen uptake Indirect evidence indicates that the under ordinary circumstances. hemoglobin of Amblystoma blood, at the stages studied, has limited significance in oxygen transport. REFERENCES BAB~~K, E. ( 1907). ifber die funktionelle Anpassung die susseren Kiemen bei Sauerstoffmangels. Zen&. Physiol. 21, 97-99. BISHOP, D. W. ( 1950). Respiration and metabolism. In “Comparative Animal Physiology” (C. L. Prosser, ed.), Chap. VIII. W. B. Saunders, Philadelphia. BOELL, E. J. (1945). Functional differentiation in embryonic development. II. Respiration and cytochrome oxidase activity in Amblystoma punctatum. J. Exptl. Zool. 100, 331352. BOND, A. N. ( 1960). An analysis of the response of salamander gills to changes in the oxygen concentration of the medium. huelop. Biol. 2, l-20. DETWILER, S. R. (1946a). Experiments upon the midbrain of Amblystoma embryos. Am. J. Anat. 78, 115-138. DETWILER, S. R. (1946b). A quantitative study of locomotion in larval Amhlystoma following either midbrain or forebrain excision. J. ExptZ. ZooZ. 102, 321-332. DRASTICH, E. (1925). Wber das Leben der Salamandru-Larven bei hohem und niedrigem Sauerstoffpartialdruck. 2. uergleich. PhysioZ. 2, 632-657. EWER, R. F. ( 1942). On the function of hemoglobin in Chironomus. J. ExptZ. BioZ. 18, 197-205.
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HARRISON, R. G. (1921). Experiments on the development of the gills in the amphibian embryo. Bio2. Bull. 41, 156-170. HELFF, 0. M., and STUBBLEFIELD, K. I. ( 1931). The influence of oxygen tension on the oxygen consumption of Rana pipiens larvae. Physiol. Zod. 4, 271-286. JOHNSON, M. L. (1942). The respiratory function of the hemoglobin of the earthworm. J. Exptl. Biol. 18, 266-277. KROGH, A. ( 1941) . “The Comparative Physiology of Respiratory Mechanisms.” Univ. of Pennsylvania Press, Philadelphia. LEHMANN, H., and HUNTSMAN, R. G. ( 1961). Why are red cells the shape they are? In “Functions of the Blood” (R. G. Macfarlane and A. H. T. Robb-Smith, eds.), Chap. II. Academic Press, New York. MCCUTCHEON, F. H. (1936). Hemoglobin function during the life history of the bullfrog. J. Cellular Comp. Physiol. 8, 63-81. PROSSER, C. L., and BROWN, F. A., JR. ( 1961). “Comparative Animal Physiology,” Chap. VII. W. B. Saunders, Philadelphia. RIGGS, A. (1951). The metamorphosis of hemoglobin in the bullfrog. J. Gen. Physiol. 35, 2340. SEVEEUNGHAUS, A. E. (1930). Gill development in Amblystoma punctatum. J. Exptl. Zool. 56, l-29.