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The relation of oxygen consumption to ambient oxygen concentration during metamorphosis of the blowfly, Phormia regina
J. Ins. Physiol., 1960, Vol. 4, pp. 220 to 228. Pergamon Press Ltd., London. Printed in Great Britain
THE RELATION OF OXYGEN CONSUMPTION TO AMBIENT OXYGEN CONCENTRATION DURING METAMORPHOSIS OF THE BLOWFLY, PHORMIA REGINA HELEN
D. PARK and JOHN BUCK
National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda 14, Maryland (Received
18 Septembw
1959)
Abstract-At 1-5 days of pupal life in Phormia regk at 25”C, respiration becomes oxygen-limited at ambient oxygen concentrations between 15 and 10 per cent. In 1 per cent 0, it is reduced to about one-fifth of the corresponding control levels. One and 5 day pupae tend to be somewhat more sensitive to hypoxia than other ages. Pupae exposed to pure nitrogen for 4 hr show a subsequent respiratory overshoot in air as compared with controls. Considering the overshoot as repayment of oxygen debt, the theoretical extent of repayment in 7 hr is 14 or 26 + per cent, depending on whether development is considered to stop or to continue during anoxia. The corresponding repayments in pure oxygen are 26 and 33 + per cent, suggesting that the rate of postanoxic respiration in air is physically limited.
INTRODUCTION relation between environmental oxygen concentration and rate of oxygen consumption is often a useful indication of an organism’s ability to regulate its respiration, and of the relative importance of fermentative and aerobic processes in its overall metabolism. Although the relation has been investigated in a wide variety of invertebrates, there are very few studies on insects in which a full series of oxygen concentrations has been used, and only one, to our knowledge, dealing with metamorphosis. In that work, GAARDER (1918) concluded that during the period between 100 hr and 150 hr after pupation of the beetle, Tenebrio molitor, oxygen consumption is independent of oxygen concentration above 6 per cent. The fly Phormia regina was used in the present study because of its short pupal life and active respiration. THE
MATERIALS
AND METHODS
In the present investigation, ‘pupal life’ was taken as beginning at the ‘white pupa’ stage, reached after the pre-pupa has ceased feeding, emptied its gut, and become immobile and contracted (about 6 days after egg laying at 25°C). It is taken as ending when the adult fly emerges from the puparium, which, at 25°C occurs about 140 hr after white pupa formation. Actually, true pupal life (adult development) does not begin until the last larval moult, which in Phormiu 220
THE RELATION OF OXYGEN CONSUMPTION TO AMBIENT OXYGEN CONCENTRATION
221
takes place within the puparium about 20 hr after the formation of the white pupa (KEISTER, 1953). The time at which true pupal life ends (i.e. beginning of pharate adult development in the sense of HINTON, 1946) is unknown. White pupae of 58 mg mean weight were collected at 8 a.m. from horse meat cultures maintained at approximately 25”C, and washed thoroughly. Except for the ‘zero day’ pupae, which were used immediately, they were then stored at 25°C. Oxygen consumption of 0-, l-, 2-, 3-, 4-, and S-day-old individual pupae was measured at 25°C by WARBURG’S direct method using 15 ml flasks with 0.33 ml of 10 per cent KOH in the insets. During each experiment the oxygen consumption of the experimental pupae was measured in air from 9 a.m. to 11 a.m., then in a lower oxygen concentration from 11 a.m. to 1 p.m., and finally in air again from 1 p.m. to 3 p.m. Controls were on the same schedule except that only air flushes were used. With 1, 5, 10, and 15 per cent O,, a 5-min flush was adequate, but in order to attain complete anoxia it was necessary to use specially purified commercial nitrogen and to prolong the flush to 10 min. At the end of the flush the venting cocks were closed while nitrogen still flowed through the manifold, and a smaller flow of nitrogen was then maintained until the manometers were set at the end of the 15-min equilibration period. Manometers were read at 15-min intervals during each 2-hr period and oxygen-uptake rates were calculated from the last six readings. To eliminate the possibility of effects carried over from prior exposures, a given pupa was used at only one age. It was impracticable to make the necessary exposures to the six oxygen concentrations each day for six successive days, all on pupae from the same hatch of eggs. Therefore, three groups of at least five pupae each, all from the same hatch, were used each day, each group being exposed to a different oxygen concentration. The exposures were randomized so that all possible combinations of the six gas concentrations, taken three at a time, were used at least once with each pupal age. The total number of groups of pupae of each age exposed to each oxygen concentration varied from five to nine, i.e. there are never fewer than twenty-five pupae involved in any mean rate presented, and usually not fewer than thirty. Significance of intergroup differences in response was determined by Student’s t test, the 0.05 level of probability being used. In all figures, the cap lines on the individual points represent standard errors. RESULTS Oxygen consumption in air of control pupae from zero through 5 days of age is shown in Fig. 1, the three points for each age representing the mean values for the three 2-hr periods of measurement. The curve has the characteristic U shape of insect respiration during metamorphosis (e.g. NEEDHAM, 1950; AGRELL, 1953; KEISTER, 1953) and shows that aerobic metabolism in the Phormia pupa passes, at about 2 days of age, through a minimum which is only one-third of its initial and terminal values. Except for somewhat lower zero and 5-day values, and slightly shorter duration, the curve agrees well with that which KEISTER (1953) obtained in the same species by following individuals throughout development.
HELEN
222
D.
PARK AND JOHN BUCK
In Fig. 2 can be seen the rates of oxygen uptake at each of the six pupal ages in the six concentrations of oxygen. Although at all ages the rate of uptake in 15 per cent 0, tended to be lower than in the air controls, the reduction was statistically significant only in l-day-old pupae. At 10 per cent and lower concentrations the depression of respiration was significant at all ages.
0
0
I
2 PUPAL
3 AGE
FIG. 1. Pupal oxygen-uptake
4
5
IN DAYS
rate in air at six ages.
If the respiratory rates of Fig. 2 are plotted as percentages of air rate against oxygen concentration there does not appear to be any striking difference in the respiratory embarrassment caused at the different ages. Thus the rate drop in 15 per cent 0, ranges only from 1 to 7 per cent, in 10 per cent 0, from ‘10 to 25 per cent, in 5 per cent 0, from 28 to 46 per cent, and in 1 per cent 0, from 71 to 86 per cent. Statistically significant differences do, however, exist, both between the sensitivities of various pupal ages to a given reduction in oxygen concentration and between the trends of respiratory depression in pupae of a given age in progressively lower ambient oxygen concentrations. For example (Fig. Z), the uptake rates of zero and 5-day-old pupae in air were almost equally high, yet the uptake of 5-day-old pupae was significantly lower than that of zero-day pupae in 1 per cent, 5 per cent, and 10 per cent 0,. Other differences are brought out when the data are plotted against pupal age (Fig. 3). For example, the oxygen-uptake rates of Z-day-old pupae in 1 per cent and 5 per cent 0, are significantly less reduced than those of l-, 4-, and 5-day-old pupae, and the rates of 3- and 4-day-old pupae in 10 per cent 0, are less affected than those of l- and 5-day pupae. In concentrations lower than 15 per cent there seem to be two periods of relative sensitivity to oxygen lack, one on day 1 and the other beginning on day 3 or 4 and reaching its maximum on day 5. Periods of relative
THE
RELATION
OF
OXYGEN
CONSUMPTION
TO
AMBIENT
OXYGEN
CONCENTRATION
223
insensitivity occur at zero days and at 2-3 days, the latter period being close to the normal time of lowest uptake from air.
0
5
I
10
AMBIENT FIG.
2. Pupal
oxygen-uptake
15
O2 CONCENTRATION
rate in relation
21
(%)
to age and ambient
oxygen
concentration.
100
0 0
2
,
PUPAL
FIG. 3. Pupal
16
oxygen
3
4
5
AGE IN DAYS
uptake, as per cent of control rate, in relation ambient oxygen concentration.
to age and
HE~EN D.
224
PARK AND JOHN BUCK
As shown in Fig. 4, a period in reduced oxygen may cause either a depression or enhancement of subsequent oxygen-uptake rate in air as compared with that of controls. The decreases are significant in 3-, 4-, and ii-day-old pupae after sojourn in 5 per cent 0, and in S-day-old pupae after 10 per cent 0,. None of the apparent increases in oxygen-uptake rate is significant. However, the several instances of slightly elevated rates after 2 hr exposure to nitrogen do suggest the possibility that some oxygen debt accrues during complete anoxia. In order to test this possibility further, experiments were carried out with longer exposure to nitrogen. “0° L .so
80
t
0
I
2 PUPAL
FIG. 4. Pupal oxygen-uptake
5
AGE
IN
DAYS
rates in air following 2 hr exposure to a reduced oxygen concentration.
Seven experiments were done, each involving four groups of six to eight pupae each. After an hour’s preliminary run in air the pupae were treated as follows : group lin air 4 hr, then in fresh air for an additional 7 hr; group 2-in air 4 hr, then in oxygen 7 hr; group 3-in nitrogen 4 hr, then in air 7 hr ; group &in nitrogen 4 hr, then in oxygen 7 hr. One-day-old pupae were used because they have undergone the last larval moult and hence are operating on truly pupal metabolism, yet are respiring in the steepest part of the oxygen uptake curve, where their post-anoxic respiration should be, in the absence of debt repayment, significantly lower than their pre-anoxic respiration. Respiration was found to be markedly elevated for some hours after removal of the pupae from nitrogen, but there is some question as to how to express this finding quantitatively. A priori it seems possible either that some pupal development proceeds during anoxia, or that progress halts. Some preliminary results on
THE
RELATION
OF
OXYGEN
CONSUMPTION
TO
AMBIENT
OXYGEN
CONCENTRATION
22.5
Galleria (obtained by Dr. MARGARET KEISTER in our laboratory) suggest that anoxia interrupts development of the pupae and that upon return to air they simply prolong their pupal period by a corresponding amount. If the same situation holds in Phormia, post-anoxic oxygen uptake rate should be compared with that of controls that are 4 hr younger, rather than with that of controls of the same chronological age (but which are presumably developmentally older). The broken lines in Fig. 5 show the oxygen uptake rates of the experimental pupae on this basis. If, on the contrary, it be considered that development proceeds during anoxia and that post-anoxic respiration corresponds to that of contemporaneous controls rather than chronologically younger controls, the comparison would be between the control rates from hours 4-11 and corresponding experimental rates, as indicated by the solid lines in Fig. 5.
25
26
27
28
29
PUPAL
30
31
32
33
34
35
36
AGE IN HOURS
FIG. 5. Oxygen-uptake rates of 1-day,oldpupae in air andoxygenfollowing a 4-hr exposure to nitrogen. n Experimentals in mygen; L7 Experimentals in air; 0 Controls in oxygen; 0 Controls in air.
Analysis of the data of Fig. 5 shows first, that the oxygen-uptake rates of the air and oxygen controls are not significantly different. Second, if the pupae are considered to have halted development during anoxia, the respiratory overshoot has disappeared by the end of the seventh hour after return to air. Third, if the pupae are considered to have developed contemporaneously with the controls, post-anoxic respiration is still significantly above the control level at the end of the seventh hour. Fourth, during the first 4 hr after removal from nitrogen the increase in oxygen-uptake rates over the control level is significantly higher in oxygen than in air. The average debt contracted, taken as the mean cumulative oxygen uptake of the seven sets of controls over the 4 hr during which the experimentals were in nitrogen, was 1.65 L 0.02 ,ul./mg. If the pupae exposed to nitrogen are considered
226
HELEN D. PARK ANDJOHN BUCK
to have stopped development at the beginning of anoxia, the cumulative respiratory overshoot at the end of 7 hr, computed as the excess oxygen uptake in air after nitrogen over the oxygen uptake of 4-hr younger controls, was 14 rt 3 per cent in air, 22+ 2 per cent in oxygen. These quantities are significantly different from both their control rates and from each other. If the experimental pupae are considered to have developed along with the controls the debt repayment at the end of 7 hr was 26 + 2 per cent in air and 33 + 2 per cent in oxygen, again significantly above the rates of contemporaneous controls and significantly different from each other. Furthermore, the trend of the curves indicates that some additional repayment would occur after the 7-hr period studied. DISCUSSION From the shapes of the curves showing oxygen-uptake rates with increasing concentrations of ambient oxygen (Fig. 2) we can conclude that during metamorphosis Phormia regina has little capacity for maintaining its respiratory rate in the face of lowered ambient oxygen concentration. One-day-old pupae show a significantly decreased oxygen-uptake rate in 15 per cent &, and all ages are limited by 10 per cent and lower oxygen concentrations. This dipterous pupa, therefore, stands in striking contrast to the larva of Cdliphora (FRAENKEL and HERFORD, 1938) and to certain lepidopterous pupae in which oxygen uptake remains normal even in 1 per cent oxygen (BUCK and KEISTER, 1955). That the differing sensitivities of the various pupal ages to oxygen deprivation reflect qualitative differences in underlying metabolic systems seems quite possible, particularly such a difference as that between the respiratory rates of zero day ‘pupae’ (essentially larvae) and 5-day pupae (essentially adults) in 1, 5, and 10 per cent 0, (Fig. 2). However, in spite of, or perhaps because of, the availability of numerous chemical’ and enzymatic fluctuations during insect development (cf. HAUB and HITCHCOCK, 1941; AGRELL, 1953), it would be premature to attempt a specific association of any such changes with overall oxygen uptake. One-day-old Phormia pupae join the small number of insects in which oxygen debt has been demonstrated directly, though the repayment is incomplete and spread out over a relatively 1ongYnterval. Whether repayment would be greater with longer anoxia or at other ages, and the nature of the materials being oxidized during repayment, are questions of great interest, but, as emphasized by VONBRAND (1953), the theoretical degree of repayment is not likely to give any useful clue to biochemical events. One possible explanation of the fact that post-anoxic respiration is greater in pure oxygen than in air is that in the l-day-old pupa, at least, the oxygen concentration of air is insufficient to support all the potential respiratory metabolism. Inability to operate at optimum capacity can reasonably be attributed to the pupal spiracular apertures, which, on the one hand, are presumably set at an area which minimizes transpiratory water loss, and, on the other, lack any mechanical means of dilatation.
THE RELATION
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TO AMBIENT
OXYGEN
CONCENTRATION
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Our experiments give no clear bases for deciding whether development stops or is slowed during oxygen deprivation. The normal time of eclosion is sufficiently variable that the delay conceivably caused by the 4-hr exposure to nitrogen would be extremely difficult to detect. It is only possible to say that the apparent debt repayment is larger if the experimental and control pupae are compared on the basis of chronological age than on the assumption that development ceases during anoxia. Two final mysteries of pupal respiration deserve mention even though no The first is the consistent dip in control explanation is presently apparent. respiration at the fifth hour after setup (age 30 hr in Fig. 5). Since the dip occurs in the first hour after the flush, the two events could conceivably be related. However, in a trial experiment using ten pupae, a decrease followed by a rise in respiration was also shown by 30-hr pupae in flasks which were not flushed. The consistent dip has no bearing on results and conclusions reported but does raise the possibility of some diurnal influence on respiratory rate with a minimum at 4 p.m. The second puzzle is the significant depression in oxygen-uptake rate in air following 2-hr exposure to 5 per cent 0, at ages 3, 4, and 5 days (Fig. 4). In these pupae, in other words, release from a mild respiratory stress produces the opposite effect to what release from severe stress (2-4 hr in pure nitrogen) produces in pupa of 0, 1, and 2 days of age (Figs. 4, 5). This is particularly curious in that control respiration is rising steeply during the third to fifth pupal day (Fig. 1) so that the post-hypoxic rates in air might be expected to be, if anything, higher than before exposure to 5 per cent 0,. SUMMARY
At l-5 days of pupal life in Phormia regina at 25”C, respiration becomes oxygenlimited at ambient oxygen concentrations between 15 per cent and 10 per cent, and, in 1 per cent O,, is reduced to about one-fifth of the corresponding control levels. One- and 5-day pupae tend to be somewhat more sensitive to hypoxia than other ages. Pupae exposed to pure nitrogen for 4 hr show a subsequent respiratory overshoot in air as compared with controls. Considering the overshoot as repayment of oxygen debt, the theoretical extent of repayment in 7 hr is 14 per cent or 26+ per cent, depending on whether development is considered to stop or to continue during anoxia. The corresponding repayments in pure oxygen are 26 per cent and 33 + per cent, suggesting that the rate of post-anoxic respiration in air is physically limited. REFERENCES AGRELL I. (1953) The aerobic and anaerobic utilization of metabolic energy during insect metamorphosis. Acta physiol. stand. 28, 306-335. VON BRAND T. and MEHLMAN B. (1953) Relations between pre- and post-anaerobic oxygen consumption and oxygen tension in some fresh water snails. Biol. Bull., Woods Hole
104, 301-312. BUCK J. and KEISTER
Bull.,
M.
(1955) Cyclic CO,
Woods Hole 109, 144-163.
release in diapausing Agapema
pupae.
Biol.
HELEN D. PARK AND JOHN BUCK
228
FRAENKEL G. and HERFORD G. V. B. (1938) Th e respiration of insects through the skin. -7. exp. Biol. 15, 266-280. GAARDER T. (1918) Uber den Einfluss des Sauerstoffdruckes auf den StofIwechsel-I. Nach Versuchen an Mehlwurmpuppen. Biochem. 2. 89, 48-93. HAUB J. G. and HITCHCOCK F. A. (1941) The interconversion of foodstuffs in the blowfly (Phormiu regina) during metamorphosis-III. Chemical composition of larvae, pupae and adults. Ann. ent. SOL. Amer. 34, 32-37. HINTON H. E. (1946) Concealed phases in the metamorphosis of insects. Nature, Lond.
157, 552-553. HITCHCOCK F. A. and HAUB J. G. (1941) The interconversion of foodstuffs in the blowfly Respiratory metabolism and nitrogen (Phormiu regina) during metamorphosis-I. excretion. Ann. ent. Sot. Amer. 34, 17-25. KEISTER M. L. (1953) Some observations on pupal respiration in Phormia regina. J. Morph.
93, 573-587. NEEDHAM J. (1950) The biochemistry of insect metamorphosis. genesis, Part 2, Sect. 285. Cambridge University Press.
Biochemistry
and Morpho-
THE RELATION OF OXYGEN CONSUMPTION TO AMBIENT OXYGEN CONCENTRATION DURING METAMORPHOSIS OF THE BLOWFLY, PHORMIA REGINA HELEN
D. PARK and JOHN BUCK
National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda 14, Maryland (Received
18 Septembw
1959)
Abstract-At 1-5 days of pupal life in Phormia regk at 25”C, respiration becomes oxygen-limited at ambient oxygen concentrations between 15 and 10 per cent. In 1 per cent 0, it is reduced to about one-fifth of the corresponding control levels. One and 5 day pupae tend to be somewhat more sensitive to hypoxia than other ages. Pupae exposed to pure nitrogen for 4 hr show a subsequent respiratory overshoot in air as compared with controls. Considering the overshoot as repayment of oxygen debt, the theoretical extent of repayment in 7 hr is 14 or 26 + per cent, depending on whether development is considered to stop or to continue during anoxia. The corresponding repayments in pure oxygen are 26 and 33 + per cent, suggesting that the rate of postanoxic respiration in air is physically limited.
INTRODUCTION relation between environmental oxygen concentration and rate of oxygen consumption is often a useful indication of an organism’s ability to regulate its respiration, and of the relative importance of fermentative and aerobic processes in its overall metabolism. Although the relation has been investigated in a wide variety of invertebrates, there are very few studies on insects in which a full series of oxygen concentrations has been used, and only one, to our knowledge, dealing with metamorphosis. In that work, GAARDER (1918) concluded that during the period between 100 hr and 150 hr after pupation of the beetle, Tenebrio molitor, oxygen consumption is independent of oxygen concentration above 6 per cent. The fly Phormia regina was used in the present study because of its short pupal life and active respiration. THE
MATERIALS
AND METHODS
In the present investigation, ‘pupal life’ was taken as beginning at the ‘white pupa’ stage, reached after the pre-pupa has ceased feeding, emptied its gut, and become immobile and contracted (about 6 days after egg laying at 25°C). It is taken as ending when the adult fly emerges from the puparium, which, at 25°C occurs about 140 hr after white pupa formation. Actually, true pupal life (adult development) does not begin until the last larval moult, which in Phormiu 220
THE RELATION OF OXYGEN CONSUMPTION TO AMBIENT OXYGEN CONCENTRATION
221
takes place within the puparium about 20 hr after the formation of the white pupa (KEISTER, 1953). The time at which true pupal life ends (i.e. beginning of pharate adult development in the sense of HINTON, 1946) is unknown. White pupae of 58 mg mean weight were collected at 8 a.m. from horse meat cultures maintained at approximately 25”C, and washed thoroughly. Except for the ‘zero day’ pupae, which were used immediately, they were then stored at 25°C. Oxygen consumption of 0-, l-, 2-, 3-, 4-, and S-day-old individual pupae was measured at 25°C by WARBURG’S direct method using 15 ml flasks with 0.33 ml of 10 per cent KOH in the insets. During each experiment the oxygen consumption of the experimental pupae was measured in air from 9 a.m. to 11 a.m., then in a lower oxygen concentration from 11 a.m. to 1 p.m., and finally in air again from 1 p.m. to 3 p.m. Controls were on the same schedule except that only air flushes were used. With 1, 5, 10, and 15 per cent O,, a 5-min flush was adequate, but in order to attain complete anoxia it was necessary to use specially purified commercial nitrogen and to prolong the flush to 10 min. At the end of the flush the venting cocks were closed while nitrogen still flowed through the manifold, and a smaller flow of nitrogen was then maintained until the manometers were set at the end of the 15-min equilibration period. Manometers were read at 15-min intervals during each 2-hr period and oxygen-uptake rates were calculated from the last six readings. To eliminate the possibility of effects carried over from prior exposures, a given pupa was used at only one age. It was impracticable to make the necessary exposures to the six oxygen concentrations each day for six successive days, all on pupae from the same hatch of eggs. Therefore, three groups of at least five pupae each, all from the same hatch, were used each day, each group being exposed to a different oxygen concentration. The exposures were randomized so that all possible combinations of the six gas concentrations, taken three at a time, were used at least once with each pupal age. The total number of groups of pupae of each age exposed to each oxygen concentration varied from five to nine, i.e. there are never fewer than twenty-five pupae involved in any mean rate presented, and usually not fewer than thirty. Significance of intergroup differences in response was determined by Student’s t test, the 0.05 level of probability being used. In all figures, the cap lines on the individual points represent standard errors. RESULTS Oxygen consumption in air of control pupae from zero through 5 days of age is shown in Fig. 1, the three points for each age representing the mean values for the three 2-hr periods of measurement. The curve has the characteristic U shape of insect respiration during metamorphosis (e.g. NEEDHAM, 1950; AGRELL, 1953; KEISTER, 1953) and shows that aerobic metabolism in the Phormia pupa passes, at about 2 days of age, through a minimum which is only one-third of its initial and terminal values. Except for somewhat lower zero and 5-day values, and slightly shorter duration, the curve agrees well with that which KEISTER (1953) obtained in the same species by following individuals throughout development.
HELEN
222
D.
PARK AND JOHN BUCK
In Fig. 2 can be seen the rates of oxygen uptake at each of the six pupal ages in the six concentrations of oxygen. Although at all ages the rate of uptake in 15 per cent 0, tended to be lower than in the air controls, the reduction was statistically significant only in l-day-old pupae. At 10 per cent and lower concentrations the depression of respiration was significant at all ages.
0
0
I
2 PUPAL
3 AGE
FIG. 1. Pupal oxygen-uptake
4
5
IN DAYS
rate in air at six ages.
If the respiratory rates of Fig. 2 are plotted as percentages of air rate against oxygen concentration there does not appear to be any striking difference in the respiratory embarrassment caused at the different ages. Thus the rate drop in 15 per cent 0, ranges only from 1 to 7 per cent, in 10 per cent 0, from ‘10 to 25 per cent, in 5 per cent 0, from 28 to 46 per cent, and in 1 per cent 0, from 71 to 86 per cent. Statistically significant differences do, however, exist, both between the sensitivities of various pupal ages to a given reduction in oxygen concentration and between the trends of respiratory depression in pupae of a given age in progressively lower ambient oxygen concentrations. For example (Fig. Z), the uptake rates of zero and 5-day-old pupae in air were almost equally high, yet the uptake of 5-day-old pupae was significantly lower than that of zero-day pupae in 1 per cent, 5 per cent, and 10 per cent 0,. Other differences are brought out when the data are plotted against pupal age (Fig. 3). For example, the oxygen-uptake rates of Z-day-old pupae in 1 per cent and 5 per cent 0, are significantly less reduced than those of l-, 4-, and 5-day-old pupae, and the rates of 3- and 4-day-old pupae in 10 per cent 0, are less affected than those of l- and 5-day pupae. In concentrations lower than 15 per cent there seem to be two periods of relative sensitivity to oxygen lack, one on day 1 and the other beginning on day 3 or 4 and reaching its maximum on day 5. Periods of relative
THE
RELATION
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OXYGEN
CONSUMPTION
TO
AMBIENT
OXYGEN
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223
insensitivity occur at zero days and at 2-3 days, the latter period being close to the normal time of lowest uptake from air.
0
5
I
10
AMBIENT FIG.
2. Pupal
oxygen-uptake
15
O2 CONCENTRATION
rate in relation
21
(%)
to age and ambient
oxygen
concentration.
100
0 0
2
,
PUPAL
FIG. 3. Pupal
16
oxygen
3
4
5
AGE IN DAYS
uptake, as per cent of control rate, in relation ambient oxygen concentration.
to age and
HE~EN D.
224
PARK AND JOHN BUCK
As shown in Fig. 4, a period in reduced oxygen may cause either a depression or enhancement of subsequent oxygen-uptake rate in air as compared with that of controls. The decreases are significant in 3-, 4-, and ii-day-old pupae after sojourn in 5 per cent 0, and in S-day-old pupae after 10 per cent 0,. None of the apparent increases in oxygen-uptake rate is significant. However, the several instances of slightly elevated rates after 2 hr exposure to nitrogen do suggest the possibility that some oxygen debt accrues during complete anoxia. In order to test this possibility further, experiments were carried out with longer exposure to nitrogen. “0° L .so
80
t
0
I
2 PUPAL
FIG. 4. Pupal oxygen-uptake
5
AGE
IN
DAYS
rates in air following 2 hr exposure to a reduced oxygen concentration.
Seven experiments were done, each involving four groups of six to eight pupae each. After an hour’s preliminary run in air the pupae were treated as follows : group lin air 4 hr, then in fresh air for an additional 7 hr; group 2-in air 4 hr, then in oxygen 7 hr; group 3-in nitrogen 4 hr, then in air 7 hr ; group &in nitrogen 4 hr, then in oxygen 7 hr. One-day-old pupae were used because they have undergone the last larval moult and hence are operating on truly pupal metabolism, yet are respiring in the steepest part of the oxygen uptake curve, where their post-anoxic respiration should be, in the absence of debt repayment, significantly lower than their pre-anoxic respiration. Respiration was found to be markedly elevated for some hours after removal of the pupae from nitrogen, but there is some question as to how to express this finding quantitatively. A priori it seems possible either that some pupal development proceeds during anoxia, or that progress halts. Some preliminary results on
THE
RELATION
OF
OXYGEN
CONSUMPTION
TO
AMBIENT
OXYGEN
CONCENTRATION
22.5
Galleria (obtained by Dr. MARGARET KEISTER in our laboratory) suggest that anoxia interrupts development of the pupae and that upon return to air they simply prolong their pupal period by a corresponding amount. If the same situation holds in Phormia, post-anoxic oxygen uptake rate should be compared with that of controls that are 4 hr younger, rather than with that of controls of the same chronological age (but which are presumably developmentally older). The broken lines in Fig. 5 show the oxygen uptake rates of the experimental pupae on this basis. If, on the contrary, it be considered that development proceeds during anoxia and that post-anoxic respiration corresponds to that of contemporaneous controls rather than chronologically younger controls, the comparison would be between the control rates from hours 4-11 and corresponding experimental rates, as indicated by the solid lines in Fig. 5.
25
26
27
28
29
PUPAL
30
31
32
33
34
35
36
AGE IN HOURS
FIG. 5. Oxygen-uptake rates of 1-day,oldpupae in air andoxygenfollowing a 4-hr exposure to nitrogen. n Experimentals in mygen; L7 Experimentals in air; 0 Controls in oxygen; 0 Controls in air.
Analysis of the data of Fig. 5 shows first, that the oxygen-uptake rates of the air and oxygen controls are not significantly different. Second, if the pupae are considered to have halted development during anoxia, the respiratory overshoot has disappeared by the end of the seventh hour after return to air. Third, if the pupae are considered to have developed contemporaneously with the controls, post-anoxic respiration is still significantly above the control level at the end of the seventh hour. Fourth, during the first 4 hr after removal from nitrogen the increase in oxygen-uptake rates over the control level is significantly higher in oxygen than in air. The average debt contracted, taken as the mean cumulative oxygen uptake of the seven sets of controls over the 4 hr during which the experimentals were in nitrogen, was 1.65 L 0.02 ,ul./mg. If the pupae exposed to nitrogen are considered
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HELEN D. PARK ANDJOHN BUCK
to have stopped development at the beginning of anoxia, the cumulative respiratory overshoot at the end of 7 hr, computed as the excess oxygen uptake in air after nitrogen over the oxygen uptake of 4-hr younger controls, was 14 rt 3 per cent in air, 22+ 2 per cent in oxygen. These quantities are significantly different from both their control rates and from each other. If the experimental pupae are considered to have developed along with the controls the debt repayment at the end of 7 hr was 26 + 2 per cent in air and 33 + 2 per cent in oxygen, again significantly above the rates of contemporaneous controls and significantly different from each other. Furthermore, the trend of the curves indicates that some additional repayment would occur after the 7-hr period studied. DISCUSSION From the shapes of the curves showing oxygen-uptake rates with increasing concentrations of ambient oxygen (Fig. 2) we can conclude that during metamorphosis Phormia regina has little capacity for maintaining its respiratory rate in the face of lowered ambient oxygen concentration. One-day-old pupae show a significantly decreased oxygen-uptake rate in 15 per cent &, and all ages are limited by 10 per cent and lower oxygen concentrations. This dipterous pupa, therefore, stands in striking contrast to the larva of Cdliphora (FRAENKEL and HERFORD, 1938) and to certain lepidopterous pupae in which oxygen uptake remains normal even in 1 per cent oxygen (BUCK and KEISTER, 1955). That the differing sensitivities of the various pupal ages to oxygen deprivation reflect qualitative differences in underlying metabolic systems seems quite possible, particularly such a difference as that between the respiratory rates of zero day ‘pupae’ (essentially larvae) and 5-day pupae (essentially adults) in 1, 5, and 10 per cent 0, (Fig. 2). However, in spite of, or perhaps because of, the availability of numerous chemical’ and enzymatic fluctuations during insect development (cf. HAUB and HITCHCOCK, 1941; AGRELL, 1953), it would be premature to attempt a specific association of any such changes with overall oxygen uptake. One-day-old Phormia pupae join the small number of insects in which oxygen debt has been demonstrated directly, though the repayment is incomplete and spread out over a relatively 1ongYnterval. Whether repayment would be greater with longer anoxia or at other ages, and the nature of the materials being oxidized during repayment, are questions of great interest, but, as emphasized by VONBRAND (1953), the theoretical degree of repayment is not likely to give any useful clue to biochemical events. One possible explanation of the fact that post-anoxic respiration is greater in pure oxygen than in air is that in the l-day-old pupa, at least, the oxygen concentration of air is insufficient to support all the potential respiratory metabolism. Inability to operate at optimum capacity can reasonably be attributed to the pupal spiracular apertures, which, on the one hand, are presumably set at an area which minimizes transpiratory water loss, and, on the other, lack any mechanical means of dilatation.
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Our experiments give no clear bases for deciding whether development stops or is slowed during oxygen deprivation. The normal time of eclosion is sufficiently variable that the delay conceivably caused by the 4-hr exposure to nitrogen would be extremely difficult to detect. It is only possible to say that the apparent debt repayment is larger if the experimental and control pupae are compared on the basis of chronological age than on the assumption that development ceases during anoxia. Two final mysteries of pupal respiration deserve mention even though no The first is the consistent dip in control explanation is presently apparent. respiration at the fifth hour after setup (age 30 hr in Fig. 5). Since the dip occurs in the first hour after the flush, the two events could conceivably be related. However, in a trial experiment using ten pupae, a decrease followed by a rise in respiration was also shown by 30-hr pupae in flasks which were not flushed. The consistent dip has no bearing on results and conclusions reported but does raise the possibility of some diurnal influence on respiratory rate with a minimum at 4 p.m. The second puzzle is the significant depression in oxygen-uptake rate in air following 2-hr exposure to 5 per cent 0, at ages 3, 4, and 5 days (Fig. 4). In these pupae, in other words, release from a mild respiratory stress produces the opposite effect to what release from severe stress (2-4 hr in pure nitrogen) produces in pupa of 0, 1, and 2 days of age (Figs. 4, 5). This is particularly curious in that control respiration is rising steeply during the third to fifth pupal day (Fig. 1) so that the post-hypoxic rates in air might be expected to be, if anything, higher than before exposure to 5 per cent 0,. SUMMARY
At l-5 days of pupal life in Phormia regina at 25”C, respiration becomes oxygenlimited at ambient oxygen concentrations between 15 per cent and 10 per cent, and, in 1 per cent O,, is reduced to about one-fifth of the corresponding control levels. One- and 5-day pupae tend to be somewhat more sensitive to hypoxia than other ages. Pupae exposed to pure nitrogen for 4 hr show a subsequent respiratory overshoot in air as compared with controls. Considering the overshoot as repayment of oxygen debt, the theoretical extent of repayment in 7 hr is 14 per cent or 26+ per cent, depending on whether development is considered to stop or to continue during anoxia. The corresponding repayments in pure oxygen are 26 per cent and 33 + per cent, suggesting that the rate of post-anoxic respiration in air is physically limited. REFERENCES AGRELL I. (1953) The aerobic and anaerobic utilization of metabolic energy during insect metamorphosis. Acta physiol. stand. 28, 306-335. VON BRAND T. and MEHLMAN B. (1953) Relations between pre- and post-anaerobic oxygen consumption and oxygen tension in some fresh water snails. Biol. Bull., Woods Hole
104, 301-312. BUCK J. and KEISTER
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M.
(1955) Cyclic CO,
Woods Hole 109, 144-163.
release in diapausing Agapema
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Biol.
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FRAENKEL G. and HERFORD G. V. B. (1938) Th e respiration of insects through the skin. -7. exp. Biol. 15, 266-280. GAARDER T. (1918) Uber den Einfluss des Sauerstoffdruckes auf den StofIwechsel-I. Nach Versuchen an Mehlwurmpuppen. Biochem. 2. 89, 48-93. HAUB J. G. and HITCHCOCK F. A. (1941) The interconversion of foodstuffs in the blowfly (Phormiu regina) during metamorphosis-III. Chemical composition of larvae, pupae and adults. Ann. ent. SOL. Amer. 34, 32-37. HINTON H. E. (1946) Concealed phases in the metamorphosis of insects. Nature, Lond.
157, 552-553. HITCHCOCK F. A. and HAUB J. G. (1941) The interconversion of foodstuffs in the blowfly Respiratory metabolism and nitrogen (Phormiu regina) during metamorphosis-I. excretion. Ann. ent. Sot. Amer. 34, 17-25. KEISTER M. L. (1953) Some observations on pupal respiration in Phormia regina. J. Morph.
93, 573-587. NEEDHAM J. (1950) The biochemistry of insect metamorphosis. genesis, Part 2, Sect. 285. Cambridge University Press.
Biochemistry
and Morpho-