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Cyclic CO2 release in diapausing pupae—II
7. ins. Physiol.,
1958, Vol. 1, pp. 327 to 340.
CYCLIC
CO, RELEASE
TRACHEAL
ANATOMY,
VOLUME;
Pergamon Press Ltd., London
IN DIAPAUSING VOLUME
INTERBURST
AND $CO,;
CO, RELEASE
JOHN BUCK and MARGARET National
U.S.
Department
PUPAE-II BLOOD RATE
KEISTER
Laboratory of Physical Biology Institute of Arthritis and Metabolic Diseases National Institutes of Health Public Health Service of Health, Education, and Welfare, Bethesda, Maryland (Received
27 August
1957)
Abstract-An average one gram cyclically-respiring diapausing pupa of the saturniid moth Agapema gal&a has a tracheal voIume of about 60 ~1 and a tracheal pC0, of 45 mm Hg. This shows that nearly 90 per cent of the approximately 30 ~1 of gaseous CO, released during a “burst” must come out of solution and combination in tissue stores. Body water per g is distributed: blood, 330 ~1; tissues, 180 ~1; gut lumen, 100 ~1; and cuticle 110 ~1. Dimensions of the major parts of the tracheal path from environment to tissues are given, and it is shown by computation that the spiracular valve is the only site where significant resistance to difkion could occur. It is shown statistically that the rate of COs release increases gradually during the interburst period,. as it should if CO, is being impounded in body buffers. Arguments are advanced for cyclic CO, release being potentially widespread in insects under conditions where 0, supply is high relative to demand.
INTRODUCTION PREVIOUSwork on the remarkable “bursts” of CO, released periodically by certain insects has yielded a considerable body of facts (for review see SCHNEIDERMAN and WILLIAMS, 195.5; BUCK and KEISTER, 1955), but several data essential for a quantitative description and understanding of the physical mechanism of CO, retention are still lacking. Among these are the volume of the tracheal system, the dimensions of the environment-tissue diffusion pathway, pupal blood and tissue water content, and the composition of the tracheal gas during the cycle. The present paper deals with attempts to supply these and certain other informational deficiencies. Most of our data are derived from diapausing pupae of the saturniid moth Agapema galbina, but we wish particularly to thank Dr. HOWARD, SCHNEIDERMAN for sending us Cecropia pupae (~ryaZoph~~a) for comparison. 1. TRACHEAL
VOLUME
As an indication of the volume of endogenous gaseous CO, which is immediately available for release, in contrast to that which has first to diffuse out of tissue liquids, the volume of the pupal tracheal system is an important datum. SCHNEIDERMANand WILLIAMS have reported (of Hyalophora) that “Vacuum extraction of the gases in the tracheal system and dissolved in the tissue fluids. 327
328
JOHNBUCK
AND~WWGARETKEISTER
revealed the tracheal volume of Cecropia pupae to be considerably less than 10 per cent of the total volume of the insect”. In our opinion this, the usual method of estimating tracheal volume, is subject to two potentially serious sources of error. The first is that one has no way of knowing how much of the extracted gas is normally present as gas (i.e. in the tracheae) and how much is evacuated from solution and combination in blood and tissues. The second objection is that evacuation under water (as is usually done) does not remove by any means all of the gas in the pupal tracheal system. This is seen very clearly in the fact that the abdomen elongates greatly when the vacuum is applied, due to expansion of trapped tracheal gas, and then shortens when atmospheric pressure again prevails, and that this reversible telescoping continues with repeated applications of vacuum I;
/” I/s ml,
(--
CAPILLARY,
7cm
0.7 pl,/mm,
TE3 SYRINGE
lm
LONG
WATER
LEVEL
FUNNEL
PUPA
FIG. 1. Apparatus for measuring volume of expelled tracheal gas (see text for details).
regardless of whether or not gas bubbles actually issue from the spiracles during the first evacuation. Our method for measuring tracheal volume is based on the fact that in many insects a relatively small mechanical pressure applied to the exoskeleton and transmitted through the body liquids will completely collapse the tracheal system, driving the tracheal gas out through the spiracles where its volume can be measured. In practice this was accomplished as follows : The pupa was first picked up between the thumb and first two fingers of both hands; the fingers and pupa were then quickly dipped in 70 per cent alcohol (to prevent adhesion of small air bubbles), then immediately immersed in water directly under the liquid-filled capillaryfunnel arrangement shown in Fig. 1. Often within a few seconds the pupa contracted its abdomen spontaneously, driving gas bubbles out of the spiracles. After this occurred, or if it did not occur, the pupa was compressed firmly in a direction parallel to its longitudinal axis to drive out any remaining gas. The gas bubbles
CYCLIC CO, RELEASE IN DIAPAUSING
PUPAE
329
issuing from the spiracles were brushed off the pupa with the fingers if they did not detach spontaneously, and the total volume of gas accumulated at the funnelcapillary junction was then drawn into the horizontal part of the calibrated capillary by means of the syringe cemented to the end of the capillary. Finally the length of capillary liquid displaced was measured. The water with which the immersion basin, funnel and capillary were filled contained NaCl to saturation at 25°C (to reduce gas solubility), dilute HCl (to reduce solution of CO,), and a few drops of caprylic alcohol (to prevent foaming and to reduce possible resistance to gas escape by the capillarity of the air-water interface at the spiracles). It was found that after the tracheal system was emptied of gas expellable by “reasonable” pressure, an additional small amount could sometimes be obtained by pressure extreme enough to explode and completely eviscerate the animal. This procedure is not considered desirable, both because of the trapping of gas froth in the viscera floating up into the funnel, and because of the possibility that if gas were present elsewhere in the body than the tracheae-for example, in the gut or under the wings-it might be added to the tracheal gas. Another precaution taken was to avoid pupae with parasites, since in these very large extratracheal, gas spaces may occur if the parasite has started development (BUCK and KEISTER, 1956). If there were significant physical resistance to gas escape at the spiracles our method of measuring tracheal volume would be vulnerable to the objection that the pressure might have driven some of the tracheal gas into solution in the body liquids rather than to the exterior. As a check against this possibility we also squeezed pupae which had been in flowing pure N, for +2 hr just previously, a treatment that should have had the dual effects of forcing the spiracular valves to open and of replacing all the tracheal and tissue gases with a relatively insoluble single gas. Table 1 summarizes the results obtained with Agapema pupae. It will be seen that there was no consistent difference between pupae pre-treated with N, and those not treated, but that there was a great variation between individuals in volume of expressible gas. This variability is due, we believe, almost entirely to variation in degree of abdominal extension at the moment of submersion, low values presumably representing pupae which had contracted before being measured. This is suggested also by the complete lack of correlation between body weight and tracheal gas volume. Values marked with a dagger (t) represent pupae that were squeezed to complete evisceration. These indicate that no characteristically greater gas volume is obtained by this extreme procedure. The values marked with asterisks are regarded as particularly reliable in the sense that the gas was expelled with ease. In summary, it can be concluded that the tracheal volume of normal Agapema pupae varies, and probably is capable of changing in the individual, at least over the range lo-75 PI/g, and that a reasonable average figure for non-contracted individuals might be of the order of 62 $/g ( mean for the 7 “best” determinations). This would correspond to about 6 per cent of body volume, assuming an average tissue specific gravity of 1-O. Determinations on two Cecropia pupae gave a figure of 7.5 per cent. Determinations on three Bombyx pupae (one 16, one 9, and one 8
JOHN BUCKAND MARGARETKEISTER
330
days after spinning) gave the low value of O-8 per cent. Since in Agapenza about 30 ~1 of CO, are liberated in an average burst, the concentration of CO, in the tracheal gas would need to be almost 50 per cent if the burst represented tracheal TABLE ~-TRACHEAL VOLUMEIN 32 DIAPAUSING Agapema PUPAE Individual tracheal volumes above ( p.l/g)and weights below (mg)
--___
No Ns pre-treatment lo!;* 641 984 Ns pre-treatment
19 811
56” 1028
15 989
1st 1189
231 788
698
780
41” 790
8::
70;
8692
7;:
804 731
241 822
43t 8.56
9::
91:
7:;*
60* 707
45 802
19 978
_-
sz* 11::* 16 997
33t
922
w
25t 918
3@t
77.5
21-t
--
42t 984
381 706
49t 1024
--__
I I
* Best determinations on basis of spontaneous expulsion of bubbles, or slight pressure required. t Pupa pressed completely (to evisceration). CO, alone. In Cecropia the concentration would have to be nearly 80 per cent, judged from our rough estimate of 75 $/g for tracheal volume and SCHNEIDERMAN and WILLIAMS’ finding of 57 pi/g for average burst volume. 2. THE PUPAL TRACHEAE
AS A DIFFUSION
PATHWAY
Because of the empirical and theoretical importance of the tracheae and spiracles in CO, retention, an attempt was made to measure the main anatomical parameters of the respiratory system of the Agapema pupa for use in future computations. Dissection was confined to abdominal spiracles and adjacent tracheal “trees”, of which there are 6 functional pairs, and the assumption was made that these are anatomically and physiologically equivalent to the thoracic pair. The stigma or frame of the external opening into the tracheal system (Fig. 2A) consists of a rigid sclerotized “filter apparatus” (F) set in a shallow depression within the oval stigma1 border or peritreme (P) on the pupal surface. Actually, the two sides of the filter consist of transversely ribbed or fluted cylindrical lips ‘which curve steeply inward on either side of a narrow slit, thus much resembling the aperture of a cowrie shell. The main slit is about 535 x 27 p, and each of the 25 lateral spaces between the flutings on each lip is 3-5 x IS-20 k, giving a total area for the aperture of about 5qOO ~2 (5 x 10e5 -cm2). The length of the passage through the slit (perpendicular to the body surface) is 130 l_~(1.3 x 1O-2 cm). Just internal to the stigma1 slit is a spiracular chamber or atrium (AT, Fig. 2C), elliptical in cross-section (470 x 170 p) and 300 lo long. At the internal end of the atrium is the spiracular valve itself (Fig. 2B; V, Fig. 2C). Since this has been
CYCLIC COa RELEASE IN DIAPAUSING
exhaustively saturniid, no the valve lips valve passage
PUPAE
331
(Cecropia), another described by BECKEL (1955) in Hydophora details of its structure will be given except to state that the length of is about 400 p, that the thickness of the lips-that is, the length of the in the direction of diffusion-is about 50 EL.(5 x 10~~ cm), that the
FIG. 2. Diagrams of Agapema spiracular apparatus. A shows an external surface view of the stigma1 filter (F) within the peritreme (P). B is a view of the spiracular valve (V), in open position, as seen from the outside of the pupa looking through the atrium with peritreme and filter removed. C is a longitudinal section, taken at right angles to the long axes of filter and valve (see broken line) to indicate the restrictions in gas path at these points. AT = atrium, M = tracheal manifold.
valve appears capable of closing completely, and that by pulling upon appropriate structures in a dissection the lips can be made to separate to give an apertural area up to the order of 40,000 Al. 2. In living pupae the valve can be seen to flutter open to areas estimated to be nearly 6000 p2, but this figure has little value because of possible foreshortening. In any case no’ conclusion can be drawn about normal dimensions during either burst or interburst because the valve cannot even be seen inthe living pupa without an operation involving removal of most of the stigma1 plate. Immediately internal to the spiracular valve is a 720 x 535 x 355 EL.chamber, which we call the “tracheal manifold” (M, Fig. 2C) because of the 17 or so large tracheal trunks given off radially to the surrounding tissues, where they arboresce profusely. Careful measurement of all the trunks issuing from the manifold gives a total cross-sectional area of the order of 38,000 p2 (3.8 x lOA cm2). The maximum diffusion distance in the pupa, i.e. the distance from either member of a pair of spiracles of one segment to the point in that segment most remote from both spiracles, is about 5 mm (5 x 10-r cm). In spite of the evidence that the spiracular valves control the CO, burst cycle, it is necessary, before gradients or valve areas can be computed, to make certain that there is no other point in the gas transfer path at which diffusion limitation
332
JOHN BUCK AND
MARGARET EEISTER
could occur. Since the rate of total diffusive transfer of a particular gas must be the same at all levels of the path, and since the rate of oxygen uptake is known, the relative resistances of various regions of the respiratory unit consisting of a single spiracle and its associated tracheae can be obtained readily by computing the concentration gradient of 0, necessary to maintain the known transfer rate through each anatomical step in the overall diffusion path. For these computations we use C, = RLJ -, where C, - C, is DA the concentration difference (cms/cms) over the diffusion path, R. (cm3/sec) the rate of gas transfer, L (cm) the length of the path, D (cmz/sec) the diffusivity of the gas in question, and A (cm2) the cross-sectional area of the diffusion pathway.* In addition to parameters already given we need values for rate of 0, uptake (16 kl/g/hr = 3 x 10 -’ cm3/sec/spiracle) and for the diffusivity of 0, at 25” (2 x 10 -l cm2/sec). Also we make the assumption that the total cross-sectional area of all the tracheae and branches supplying a single manifold is constant at all distances from the manifold. This assumption is in accord with measurements of KROGH (1920) and of THORPE and CRISP (1947).t Now, for transfer through the stigma1 slit, a rearrangement
of the familiar Fick equation, C, -
C _ C, = @ 0 z DA
= (3 x IO-‘) x (1.3 x IO-? (2 x 10-l) x (5 x 10-b)
_ 4 x 1O-g = 4 x 10-4 cm3/cms, - 10 x 10-s
which is about 0.3 mm Hg in partial pressure units (negligible). A similar computation for the atrium, using 6.2 x 10d4 cm2 for the cross-section computed as an ellipse, gives also a negligible concentration drop (7 x 10e5 cms/cm3 = 0.05 mm Hg), and for one spiracular manifold and its entire associated tracheal tree the pressure drop is less than 0.1 mm Hg, even assuming that all the oxygen travels the maximum 5 mm path to the tissues.2 Since then, neither the stigma1 plate, spiracular atrium, manifold nor tracheae offers any significant resistance to gas diffusion it follows that the spiracular valve must be the site and cause of any gas concentration fall that occurs between environment and tissue, or in the opposite direction. The actual dimensions of the limiting valve aperture will vary under different conditions and cannot be computed without knowing the gas concentration gradients involved. These will be considered in future .papers. 3. BLOOD VOLUME; BODY AND TISSUE WATER CONTENT For use in estimating COs storage capacity, the partitioning of pupal water was investigated in Agapema. Blood was collected by cracking off the head capsule, * We are much indebted to Dr. J. VERDUIN for instruction in the computational method. t NUNOME (1944) reported a constantly decreasing total area beyond the main tracheae, but since he did not report the number of branches at each level, an essential though elusive datum, it is possible that sufficient account had not been taken of the enormous distal increase in numbers of collaterals. $ Use of the “pore-diffusion” correction factor for L (BROWN and ESCOMBE, 1900; VERDUIN, 1954) would increase the computed gradient values from 7 to 50 per cent, depending on the dimensions involved, but since the gradients would still be negligible we have omitted the corrections in the interest of simplicity.
CYCLIC CO, RELEASE IN DIAPAUSING
PUPAE
333
making slits into the wings, and putting the animal head-down into a sedimentation tube graduated to 4 ~1. The tube was then centrifuged briefly at low speed and the resulting weight and volume of clear, tissue-free blood were recorded. This was repeated several times until the volume of extracted blood was constant. Then the centrifugal force was increased until the gut ruptured and added its black liquid content to the blood. Centrifugation was continued until no more black liquid appeared and the tissues began to be displaced out of the pupal shell. The difference between true blood (which averaged about 33 per cent of pupal fresh weight) and total free liquid extractable by centrifugation was recorded as “gut liquid” (which averaged about 10 per cent of fresh body weight). Fresh exoskeleton, scraped free of tissues, averaged from 15 per cent to 24 per cent of total body weight depending on whether the pupa was large (1.0-1.2 g) or small (0.5-0.6 g), and contained about 60 per cent water. (Since these pupae were 7-8 months old at the time of analysis it is possible that cuticle dry weight was somewhat higher than average, as compared with total dry weight, because of loss of body water.) Fresh tissue alone (“fat”) accounted for about 34 per cent of total body weight and contained about 53 per cent water. Whole pupae dried to constant weight at 110°C averaged about 28 per cent of their live weights, indicating that an average 1 g pupa would contain 720 ~1 of water. Judging from the analyses given above this 720 pl should include roughly 330 ~1 free in the blood, 100 ~1 free in the gut, 180 ~1 in the tissues and 110 ~1 in the cuticle. Estimates of body CO, capacity are contained in Part III of this paper (BUCK and FREIDMAN, 1958). 4. INTERBURST TRACHEAL pC0, For two reasons the concentration of CO, in the tracheal system is a key datum for the understanding of CO, release cycles. First, as long as tracheal COs concentration is not definitely known to be too high for the observed O,/CO, transfer ratio during interburst to be explained on the basis of pure diffusion, the periodic intervention of a qualitatively different type of metabolism cannot be ruled out. In other words, in spite of the apparent improbabilities of a metabolic shift occurring in the presence of unchanged 0, uptake rate and of its being synchronized with spiracular valve changes, the possibility still exists that the rate of CO, release is low during interburst simply because CO, is being fixed during this period, and high during the burst because at that time a sudden decarboxylation occurs which raises tracheal pC0,. Second, regardless of how CO, is retained during the interburst period, the actual escape of that gas seemingly can involve only diffusion. Hence any quantitative description or. explanation of cyclic CO, release will require values for both spiracular valve area and trans-spiracular CO, concentration difference. Either of these parameters can be computed via the Fick equation, if valve length and CO, release rate are known, but the other must be actually measured. PUNT (1944) guessed that CO, might rise to 10 per cent in the tracheae of the adult bug TTiatoma before a burst occurred, but his value was based on the unsupported assumptions that the spiracles were tightly closed during interburst
334
JOHN BUCK AND MARGARET KEISTER
(20 min) and that the tracheae occupied 60 per cent of body volume (DEMOLL’S 1928 estimate for the adult beetle Melolontha). In the absence of techniques ,for direct analysis of tracheal gas in situ, we have estimated tracheal CO, concentration in Agapema by observing the direction of gas movement when the pupa equilibrates with various CO,-Ns-0, mixtures in Warburg respirometers in the absence of alkali. The idea behind this method is that when the spiracles open wide, as they do at burst time, CO, should leave the pupa (“burst”) or enter (“negative burst”) depending on whether the outside pC0, is lower or higher, respectively, than tracheal pCO,.* In the procedure, TABLE ~-EFFECT DIRECTION
OF DIFFERENT'ENVIRONMENTAL 0F ~.4s EXCHANGEAT TIME
I
I
I
;::, ;:; 9.0 12 25 49 I
1
I
No.
CO,
I
1
1
VOLUME
AND
29.9 i: 31.6 f 2.3 1.0
43*
19 ;
11* 4 17x
31.2 14 & 4.5 39.4 * 3.1
z:;9 9
16 (1) 20 (4) 18
28 33 29
35.2 & 2.3 5.0 31.8 27.8 + 2.2
:: 18 I
I
I
33;*
I
ON
1
No.
:zIi] I
CONCENTRATIONS
0s SPIRACULAROPENING (Agapema)
I
+ 26.3 + 17.4 5 pos., 35.5 - 79.0 92.2 -215 -368
.
I
f 1.8 + 1.7 6 neg. + 2.7 jz 8.0 + 6-7 &- 8.8 f 7.5
83 58
ll&
200 262 677 1320 I
Numbers in parentheses after number of pupae indicate number used twice (i.e. on a second day). All individuals listed as being tested in CO, concentrations of 8 per cent or higher gave at least one negative burst. The values for negative burst volume in 9 per cent, 12 per cent and 25 per cent CO, include the extra CO, which leaked in during the flush-burst interval. This correction was too small to be estimated accurately with 8 per cent CO,, and with 49 per cent CO, the bursts occurred too soon. * Include at least 2 measurements on almost every pupa. 7 The 5 positive bursts averaged 144 PI/g and the 6 negative bursts 17 pi/g. Eight pupae gave no detectable burst in either direction in at least 5 hr. 1 Assuming that a larger sampling would have shown an air burst volume close to the usual 30 PI/g. CO, release rate was measured in air until (usually) two normal bursts had occurred, then the flasks were flushed with a known mixture for 5 min at a flowrate of 500 ~1 per min. The gas combinations were made by mixing CO, and air in the desired for spiracular proportions via calibrated flowmeters. Because the triggering $0, opening in certain adult insects has been reported to vary with ambient ~0, (WIGGLESWORTH, 1953; CASE, 1956), we took care to bleed into each mixture the amount of 0, necessary to keep the total 0, concentration at 21 per cent as monitored with a Pauling meter.
* We are indebted to Dr. H. SPECHT for originally interpreting the volumetric changes occurring with high ambient pC0,; and to Dr. RUBERTANDZFLSON for showing us how such measurements could be used to estimate tracheal pC0,.
CYCLIC
COz RELEASE
IN
DIAPAUSING
PUPAE
335
Table 2 summarizes the results of tests with several mixtures, and shows that the predicted transfer of CO, in both directions actually occurred. Another confirmation of the soundness of the experiment is the fact that the average interval between flushing and spiracular dilatation varied inversely with the CO, concentration of the ambient gas (2$ hr with 9 per cent; 2+ hr with 12 per cent; 1 hr with 24 per cent; 35 min with 49 per cent) as would be expected if the CO, were leaking into the tracheal system and accelerating the rise to triggering concentration. The actual volumes of CO, exchanged may be slightly in error because of simultaneous exchange of other gases, but both the direct tests with 5.9 per cent CO, and interpolation from other concentrations (Fig. 3) indicate that tracheal CO, concentration at the time of spiracular opening is about 6 per cent @CO2 = 45 mm Hg). This value is the same as in the honey-bee (BISHOP, 1923), an insect not known to exhibit cyclic CO, release.
I
I
I
I
I
5
-10
15
20
25
AMBIENT
CO2 CONCENTRATION
(%I
FIG. 3. Relation between ambient CO, concentration
and volume of CO2 moving out of (+ values on ordinates) or into (- values on ordinates) Agrqpema pupae at the time of presumed spiracular opening. V&es expressed both in terms of absolute volume (left ordinate) and percentage of normal burst volume in air (right ordinate). Dashed portions of lines in upper right corner indicate course to values for 49 per cent ambient CO,. Vertical broken line indicates normal tracheal CO, concentration (6 per cent) with zero gas exchange.
It should be pointed out that although the decrease in burst volume in 2.5 per cent and 5-O per cent CO, probably resulted from changed triggering level of CO,, in ambient concentrations above 6 per cent the valves do not constrict again, once they have dilated, hence the volume of CO, that enters is the amount required to fully equilibrate the blood and tissues. We were unable to use the above technique in estimating the tracheal pCOz of the Cecropia pupa because the negative bursts always occurred before the end of the period of temperature equilibration after flushing.
336
JOHNBucx ANDMARGARET KEISTER
The 6 per cent value for estimated tracheal CO, concentration, when considered in relation to the average tracheal volume of 60 ~1, shows that not more than 3.6 ~1 of the average burst volume of about 30 ~1 could come from CO, initially in the gas phase. This finding thus supports the opinion of SCHNEIDERMANand WILLIAMS in regard to the Cecropia burst. Besides indicating the need for information on the CO, capacity of the blood and tissue liquids-a matter considered in Part III of this paper-the fact that the CO, in the burst must come from two sources may have some bearing on the characteristically asymmetrical time course of CO, release during the burst. Thus the rate of CO, release typically rises almost instantaneously to a peak from which it declines much more slowly. This is shown even in Warburg records of bursts (e.g. SCHNEIDERMANand WILLIAMS, Figs. 2,4) but is particularly striking in PUNT’S fast recordings (e.g. 1950, Figs. 3, 9, 12). Part of this skewing may be related to the sort of spiracular valve flutter observed by SCHNEIDERMAN (1956) or, conceivably, to asynchronous opening or closing among the different individual or pairs of valves. However, it would also be expected that the burst would involve an initial very rapid diffusive escape of the CO, already in the tracheal gas phase, followed by a slower escape of CO, from blood and tissues. Such a differentiation between the escape rates of gas phase and dissolved phase CO,, apparently before the spiracles constrict, is perhaps shown in Fig. 1 of PUNT, PARSER and KUCHLEIN (1957). Evidence that release of CO, from body liquids is by no means instantaneous is also seen in our observation that even after equilibration with CO, concentrations as low as 12 per cent, the giving back of the extra CO,, upon return of the pupa to air, was detectable for 30 min. Similarly, the “purging” of body CO, which occurs when the valves are forced to dilate widely by putting the pupa in N, (BUCK and KEISTER, 1955) lasted an average of 32 min (17 pupae) as compared with 12 min for normal bursts in air. 5. COz RELEASE RATE DURING THE INTERBURST PERIOD If a near equilibrium between gas in tissue liquid and tracheal gas exists throughout the interburst period, the concentration of CO, in the gas phase should increase as the metabolic CO, is impounded and the concentration of free gas and its combination products with water increase in the liquid phase. Quite aside from changes due to possible spiracular flutter, therefore, the rate of CO, leakage should rise throughout the interburst period. Curiously enough no such rise was observed by SCHNEIDERMANand WILLIAMS (1955) nor is any perceptible in our individual Warburg records (e.g. BUCK and KEISTER, 1955, Fig. l).” This could be due to a progressive diminution in spiracular valve area during interburst. Since, however, it is difficult to imagine either a reason why the pupa should keep the rate of CO, release constant, or a control mechanism for accomplishing this, we investigated the alternative possibility that the rate of increase is too slight to show in individual records. By computing the net rate of gas exchange during the * It is difficult to be certain about the baseline in PUNT'Sdiaferometer records because of possible galvanometer drift and lack of absolute concentration calibration, but some of his records seem, disturbingly, to show no sign of an increased rate of CO2 leakage between bursts.
CYCLIC COP RELEASE IN DIAPAUSING
337
PUPA.6
three hours immediately preceding the burst in 95 cycles of 3-6 hr duration (mean = 4Q hr) it was shown clearly (Fig. 4) that the rate of change in volume declines as the time of the burst approaches. (It will be remembered that in the “direct” Warburg method, CO, release is not measured directly but is obtained as the difference between 0, uptake measured in the presence of alkali and the net gas volume change (0, minus CO,) when there is no absorption of CO,.) The apparently accelerated rate of change just before the burst may be an artifact due to valve flutter (cf. SCHNEIDERMAN, 1956), but considered as either a straight or 6.2
I
I
I
r
I
I
6.0
i .
\ \ \ 0
-
Y = 5.12
+0.26x
--
Y = 4.63
+0.98x-0~208x2
I
1
I
I
3
2.5
2
I.5
HOURS
PRIOR
TO
I I
_
i
0.5
BURST
decrease in rate of net pupal gas uptake with time, presumably reflecting an increased rate of CO, leakage. Cap-lines indicate standard errors.
FIG. 4. Pre-burst
curved line the regression is significant by F test at the 5 per cent level, and corresponds roughly to a 20 per cent decrease in net rate of gas uptake during the interburst portion of a mean cycle length of 4s hr. If oxygen uptake were constant throughout, an increase in rate of CO, release of about 20 per cent per cycle would be indicated. Though 0, uptake appears to be constant with time in individual records, it cannot be safely concluded that no mean change would have been detectable in the 95 records if it had been possible to measure 0, and CO, exchange simultaneously. An approach to this problem was made by averaging 23 other 0, uptake records in which it was possible to identify the time of occurrence of bursts by small but significant artifacts previously observed (BUCK and KEISTER, 1955). In these records there was an apparent decrease in mean 0, uptake rate with
JOHNBUCKANDMARGARET KIZSTER
338
approach to burst time, but the linear regression on time was not statistically significant. It can be concluded tentatively, therefore, that the rate of CO, release during the interburst period does increase progressively. If we consider 6 per cent to be the triggering CO, concentration for the burst, the maximum range of change during the interburst period would be from 50 to 6-O per cent or from 38 to 45 mm. This conclusion resolves what had previously appeared to be a contradiction to the idea that most of the metabolic CO, produced between bursts is impounded in the pupal buffer systems, and, together with the information discussed in section 2, reinforces the evidence that cyclic CO, retention in insects involves constriction of the spiracular valves. 6. RELATION
OF METABOLIC
RATE TO CYCLIC RESPIRATION
The prevalence and striking nature of cyclic CO, release in diapausing lepidopterous pupae has tended to create the impression that the phenomenon is somehow characteristically or causally connected with a low metabolic rate. This impression has been strengthened by such findings as those of ITO(1954) in which the normally non-diapausing and non-cyclic Bombyx pupa was converted into a burst producing type by paralysis caused by ligaturing behind the brain, and of SCHNEIDERMANand WILLIAMS (1955) in which Cecropia pupae that had ceased to give CO, bursts because they had broken diapause and begun adult development were induced to resume “bursting” when the temperature (metabolic rate) was lowered. We report below (Table 3) a similar result in that Bombyx pupae exhibiting continuous CO, release at 25°C developed unmistakable CO, cycles at 15” and lo”, in agreement with a prediction of SCHNEIDERMANand WILLIAMS (1955). TABLE3-CO2 BURST PRODUCTION IN Bombyx PUPAE.ALL PUPAEl-4 DAYSAFTER PUPATION (CHANGES crvs~ IN ARBITRARY MANOMJ~TER UNITS,IN THE.ABSENCE OFFLASK ALKALI)
Temperature
25” 15p 15” 10”
Duration of measurement (hr)
No. pupae
4
10
ir
:: 14
4$
Range
and sign of volume
changes
-4
to -14 ; no bursts 2 pupae gave bursts (f 1 to + 5) 10 pupae gave bursts (+2 to +ll) 7 pupae gave bursts C+ 2 to + 8)
Since PUNT discovered cyclic CO, release originally in adults of Triatoma at rest and has since found it in adults of other species (Locusta, Melee, Rhodnius, Carabus, HadrocaTabus), it cannot be argued that the phenomenon is associated with some metabolic or structural peculiarity of immature insects. Furthermore, such meagre evidence as exists gives no grounds for believing that insects in which cyclic CO, release has been demonstrated differ systematically in relative tracheal volume, blood volume or tracheal pC0, from those in which it has not been
CYCLIC CO, RELEASEIN DIAPAUSING PUPAE
339
demonstrated; and Part III of this paper shows that the same is true for CO, capacity. However, the adult insects mentioned above, being either lethargic (when fed) or having been studied at low temperatures (e.g. lS”C), do have inactivity in common with the lepidopterous pupae. It is accordingly possible that it is not low metabolic rateper se that is necessary for cyclic CO, release, but low metabolism in relation to 0, supply. There could, in other words, be no CO, retention without valve constriction, and no sufficient valve constriction unless respiratory requirements’ were low. This would be in accord with our previous (1955) suggestion that interburst valve area is held to “ the minimum compatible with adequate respiratory 0, supply” in the interests of water conservation. If this hypothesis of minimal interburst valve area is valid, it might be anticipated that discontinuous respiration could be induced in species not normally showing it, by increasing the This experiment has not actually been done, but ambient 0, concentration. confirmation in principle is offered by a Cecropia pupa reported to exhibit only continuous CO, release in air, but a typical cycle in 40 per cent 0, (SCHNEIDERMAN and WILLIAMS, 1955). In
sum,
widespread, individual and
(c)
it seems
reasonable
previous
failure
in the respirometer, testing
the
insects
valve setting could
ture).
this connexion,
In
insects
shown
not known
(b)
cyclic
CO,
due to (a)
conditions
not be sustained
the triggering
to have much
to exhibit
that
it being
use of respirometers
under
spiracular
cycle has been
to guess
to detect
of
release having
with
in which spiracular with
than
one
poor time resolution,
a sufficiently
(for example,
in common
is potentially more
constricted
at too high a tempera-
valve normal
activity spiracular
during control
the in
cyclic CO, release (BUCK, 1957).
REFERENCES BECKEL W. E. (1955) The ,morphology and histology of the spiracular regulatory apparatus of the giant silkworm Hyalophora cecropiu (L.). Thesis, Cornell Univ. Also in press in Proc. X Int. Congr. Entomol. BISHOP G. H. (1923) Body fluid of the honeybee larva. I. Osmotic pressure, specific gravity, pH, O2 capacity, CO, capacity and buffer value, and their changes with larval activity and metamorphosis. J. biol. Chem. 58, 567-582. ’ BROWN H. T. and ESCOMBE F. (1900) Static diffusion of gases and liquids in relation to the assimilation of carbon dioxide and translocation in plants. Philos. Trans. (B) 193,223-291. BUCK J. (1957) Triggering of insect spiracular valves. Physiological Triggers and Discontinuous Rate Processes (Edited by T. H. BULLOCK) pp. 72-79. American Physiological Society. BUCK J. and FRIEDMAN S. (1958). To be published. BUCK J. and KEISTER M. (1955) Cyclic CO2 release in diapausing Agapema pupae. Biol. Bull.,
Woods Hate 109, 144-163. BUCK J. and KEISTER M. (1956) Host-parasite relations in Agapema pupae (Lepidoptera, Saturniidae). Ann. ent. Sot. Amer. 49, 94-97. CASE J. F. (1956) Carbon dioxide and oxygen effects on the spiracles of flies. Physiol. Zool.
29, 163-171. DEMOLL R. (1928) Untersuchungen 45, 513-534.
iiber die Atmung
der Insekten. III.
Zool. Jb. (Allg.)
340
JOHN BUCKAND MARGARETKEISTER
IT0 T. (1954) Discontinuous output of carbon dioxide by undifferentiated Bombyx pupae. Jap.J_ appl. Zool. 19, 98. KROGHA. (1920) Studien iiber Tracheenrespiration. II. ijber Gasdiffusion in den Tracheen. P$iig. Arch. ges. Physiol. 179, 95-112. NUNOME2. (1944) Studies on the respiration of the silkworm. I. Diffusion of oxygen in the respiratory system of the silkworm. Bull. seric. Exp. Sta. Japan 12, 17-39. PUNT A. (1944) De gaswisseling van enkele bloedzuigende parasieten van warmbloedige dieren (Cinzex, Rhodnius, Triatoma). Onderz. physiol. Lab. Utrecht. Hoogesch. Ser. 8, Pt. 3, pp. 122-141. PUNT A. (1950) The respiration of insects. Physiol. Comp. 2, 59-74. PUNT A., PARSERW. J., and KUCHLEINJ. (1957) Oxygen uptake in insects with cyclic CO, release. Biol. Bull., Woods Hole 112, 108-119. SCHNEIDERMAN H. A. (1956) Spiracular control of discontinuous respiration in insects. Nature, Lond. 177, 1169-1171. SCHNEIDERMAN H. A. and WILLIAMS C. M. (1955) An experimental analysis of the discontinuous respiration of the Cecropia silkworm. Biol. Bull., Woods Hole 109, 123-143. THORPEW. H. and CRISPD. J. (1947) Studies on plastron respiration. II. The respiratory efficiency of the plastron in Aphelocheks. J.exp. Biol. 24, 270-303. VERDUIN J. (1954) Estimation of the gaseous CO, concentration in intercellular spaces during photosynthesis. OhioJ. Sci. 54, 353-359. WIGGLESWORTH V. B. (1953) Surface forces in the tracheal system of insects. Quart. J. micr. Sci. 94, 507-522.
1958, Vol. 1, pp. 327 to 340.
CYCLIC
CO, RELEASE
TRACHEAL
ANATOMY,
VOLUME;
Pergamon Press Ltd., London
IN DIAPAUSING VOLUME
INTERBURST
AND $CO,;
CO, RELEASE
JOHN BUCK and MARGARET National
U.S.
Department
PUPAE-II BLOOD RATE
KEISTER
Laboratory of Physical Biology Institute of Arthritis and Metabolic Diseases National Institutes of Health Public Health Service of Health, Education, and Welfare, Bethesda, Maryland (Received
27 August
1957)
Abstract-An average one gram cyclically-respiring diapausing pupa of the saturniid moth Agapema gal&a has a tracheal voIume of about 60 ~1 and a tracheal pC0, of 45 mm Hg. This shows that nearly 90 per cent of the approximately 30 ~1 of gaseous CO, released during a “burst” must come out of solution and combination in tissue stores. Body water per g is distributed: blood, 330 ~1; tissues, 180 ~1; gut lumen, 100 ~1; and cuticle 110 ~1. Dimensions of the major parts of the tracheal path from environment to tissues are given, and it is shown by computation that the spiracular valve is the only site where significant resistance to difkion could occur. It is shown statistically that the rate of COs release increases gradually during the interburst period,. as it should if CO, is being impounded in body buffers. Arguments are advanced for cyclic CO, release being potentially widespread in insects under conditions where 0, supply is high relative to demand.
INTRODUCTION PREVIOUSwork on the remarkable “bursts” of CO, released periodically by certain insects has yielded a considerable body of facts (for review see SCHNEIDERMAN and WILLIAMS, 195.5; BUCK and KEISTER, 1955), but several data essential for a quantitative description and understanding of the physical mechanism of CO, retention are still lacking. Among these are the volume of the tracheal system, the dimensions of the environment-tissue diffusion pathway, pupal blood and tissue water content, and the composition of the tracheal gas during the cycle. The present paper deals with attempts to supply these and certain other informational deficiencies. Most of our data are derived from diapausing pupae of the saturniid moth Agapema galbina, but we wish particularly to thank Dr. HOWARD, SCHNEIDERMAN for sending us Cecropia pupae (~ryaZoph~~a) for comparison. 1. TRACHEAL
VOLUME
As an indication of the volume of endogenous gaseous CO, which is immediately available for release, in contrast to that which has first to diffuse out of tissue liquids, the volume of the pupal tracheal system is an important datum. SCHNEIDERMANand WILLIAMS have reported (of Hyalophora) that “Vacuum extraction of the gases in the tracheal system and dissolved in the tissue fluids. 327
328
JOHNBUCK
AND~WWGARETKEISTER
revealed the tracheal volume of Cecropia pupae to be considerably less than 10 per cent of the total volume of the insect”. In our opinion this, the usual method of estimating tracheal volume, is subject to two potentially serious sources of error. The first is that one has no way of knowing how much of the extracted gas is normally present as gas (i.e. in the tracheae) and how much is evacuated from solution and combination in blood and tissues. The second objection is that evacuation under water (as is usually done) does not remove by any means all of the gas in the pupal tracheal system. This is seen very clearly in the fact that the abdomen elongates greatly when the vacuum is applied, due to expansion of trapped tracheal gas, and then shortens when atmospheric pressure again prevails, and that this reversible telescoping continues with repeated applications of vacuum I;
/” I/s ml,
(--
CAPILLARY,
7cm
0.7 pl,/mm,
TE3 SYRINGE
lm
LONG
WATER
LEVEL
FUNNEL
PUPA
FIG. 1. Apparatus for measuring volume of expelled tracheal gas (see text for details).
regardless of whether or not gas bubbles actually issue from the spiracles during the first evacuation. Our method for measuring tracheal volume is based on the fact that in many insects a relatively small mechanical pressure applied to the exoskeleton and transmitted through the body liquids will completely collapse the tracheal system, driving the tracheal gas out through the spiracles where its volume can be measured. In practice this was accomplished as follows : The pupa was first picked up between the thumb and first two fingers of both hands; the fingers and pupa were then quickly dipped in 70 per cent alcohol (to prevent adhesion of small air bubbles), then immediately immersed in water directly under the liquid-filled capillaryfunnel arrangement shown in Fig. 1. Often within a few seconds the pupa contracted its abdomen spontaneously, driving gas bubbles out of the spiracles. After this occurred, or if it did not occur, the pupa was compressed firmly in a direction parallel to its longitudinal axis to drive out any remaining gas. The gas bubbles
CYCLIC CO, RELEASE IN DIAPAUSING
PUPAE
329
issuing from the spiracles were brushed off the pupa with the fingers if they did not detach spontaneously, and the total volume of gas accumulated at the funnelcapillary junction was then drawn into the horizontal part of the calibrated capillary by means of the syringe cemented to the end of the capillary. Finally the length of capillary liquid displaced was measured. The water with which the immersion basin, funnel and capillary were filled contained NaCl to saturation at 25°C (to reduce gas solubility), dilute HCl (to reduce solution of CO,), and a few drops of caprylic alcohol (to prevent foaming and to reduce possible resistance to gas escape by the capillarity of the air-water interface at the spiracles). It was found that after the tracheal system was emptied of gas expellable by “reasonable” pressure, an additional small amount could sometimes be obtained by pressure extreme enough to explode and completely eviscerate the animal. This procedure is not considered desirable, both because of the trapping of gas froth in the viscera floating up into the funnel, and because of the possibility that if gas were present elsewhere in the body than the tracheae-for example, in the gut or under the wings-it might be added to the tracheal gas. Another precaution taken was to avoid pupae with parasites, since in these very large extratracheal, gas spaces may occur if the parasite has started development (BUCK and KEISTER, 1956). If there were significant physical resistance to gas escape at the spiracles our method of measuring tracheal volume would be vulnerable to the objection that the pressure might have driven some of the tracheal gas into solution in the body liquids rather than to the exterior. As a check against this possibility we also squeezed pupae which had been in flowing pure N, for +2 hr just previously, a treatment that should have had the dual effects of forcing the spiracular valves to open and of replacing all the tracheal and tissue gases with a relatively insoluble single gas. Table 1 summarizes the results obtained with Agapema pupae. It will be seen that there was no consistent difference between pupae pre-treated with N, and those not treated, but that there was a great variation between individuals in volume of expressible gas. This variability is due, we believe, almost entirely to variation in degree of abdominal extension at the moment of submersion, low values presumably representing pupae which had contracted before being measured. This is suggested also by the complete lack of correlation between body weight and tracheal gas volume. Values marked with a dagger (t) represent pupae that were squeezed to complete evisceration. These indicate that no characteristically greater gas volume is obtained by this extreme procedure. The values marked with asterisks are regarded as particularly reliable in the sense that the gas was expelled with ease. In summary, it can be concluded that the tracheal volume of normal Agapema pupae varies, and probably is capable of changing in the individual, at least over the range lo-75 PI/g, and that a reasonable average figure for non-contracted individuals might be of the order of 62 $/g ( mean for the 7 “best” determinations). This would correspond to about 6 per cent of body volume, assuming an average tissue specific gravity of 1-O. Determinations on two Cecropia pupae gave a figure of 7.5 per cent. Determinations on three Bombyx pupae (one 16, one 9, and one 8
JOHN BUCKAND MARGARETKEISTER
330
days after spinning) gave the low value of O-8 per cent. Since in Agapenza about 30 ~1 of CO, are liberated in an average burst, the concentration of CO, in the tracheal gas would need to be almost 50 per cent if the burst represented tracheal TABLE ~-TRACHEAL VOLUMEIN 32 DIAPAUSING Agapema PUPAE Individual tracheal volumes above ( p.l/g)and weights below (mg)
--___
No Ns pre-treatment lo!;* 641 984 Ns pre-treatment
19 811
56” 1028
15 989
1st 1189
231 788
698
780
41” 790
8::
70;
8692
7;:
804 731
241 822
43t 8.56
9::
91:
7:;*
60* 707
45 802
19 978
_-
sz* 11::* 16 997
33t
922
w
25t 918
3@t
77.5
21-t
--
42t 984
381 706
49t 1024
--__
I I
* Best determinations on basis of spontaneous expulsion of bubbles, or slight pressure required. t Pupa pressed completely (to evisceration). CO, alone. In Cecropia the concentration would have to be nearly 80 per cent, judged from our rough estimate of 75 $/g for tracheal volume and SCHNEIDERMAN and WILLIAMS’ finding of 57 pi/g for average burst volume. 2. THE PUPAL TRACHEAE
AS A DIFFUSION
PATHWAY
Because of the empirical and theoretical importance of the tracheae and spiracles in CO, retention, an attempt was made to measure the main anatomical parameters of the respiratory system of the Agapema pupa for use in future computations. Dissection was confined to abdominal spiracles and adjacent tracheal “trees”, of which there are 6 functional pairs, and the assumption was made that these are anatomically and physiologically equivalent to the thoracic pair. The stigma or frame of the external opening into the tracheal system (Fig. 2A) consists of a rigid sclerotized “filter apparatus” (F) set in a shallow depression within the oval stigma1 border or peritreme (P) on the pupal surface. Actually, the two sides of the filter consist of transversely ribbed or fluted cylindrical lips ‘which curve steeply inward on either side of a narrow slit, thus much resembling the aperture of a cowrie shell. The main slit is about 535 x 27 p, and each of the 25 lateral spaces between the flutings on each lip is 3-5 x IS-20 k, giving a total area for the aperture of about 5qOO ~2 (5 x 10e5 -cm2). The length of the passage through the slit (perpendicular to the body surface) is 130 l_~(1.3 x 1O-2 cm). Just internal to the stigma1 slit is a spiracular chamber or atrium (AT, Fig. 2C), elliptical in cross-section (470 x 170 p) and 300 lo long. At the internal end of the atrium is the spiracular valve itself (Fig. 2B; V, Fig. 2C). Since this has been
CYCLIC COa RELEASE IN DIAPAUSING
exhaustively saturniid, no the valve lips valve passage
PUPAE
331
(Cecropia), another described by BECKEL (1955) in Hydophora details of its structure will be given except to state that the length of is about 400 p, that the thickness of the lips-that is, the length of the in the direction of diffusion-is about 50 EL.(5 x 10~~ cm), that the
FIG. 2. Diagrams of Agapema spiracular apparatus. A shows an external surface view of the stigma1 filter (F) within the peritreme (P). B is a view of the spiracular valve (V), in open position, as seen from the outside of the pupa looking through the atrium with peritreme and filter removed. C is a longitudinal section, taken at right angles to the long axes of filter and valve (see broken line) to indicate the restrictions in gas path at these points. AT = atrium, M = tracheal manifold.
valve appears capable of closing completely, and that by pulling upon appropriate structures in a dissection the lips can be made to separate to give an apertural area up to the order of 40,000 Al. 2. In living pupae the valve can be seen to flutter open to areas estimated to be nearly 6000 p2, but this figure has little value because of possible foreshortening. In any case no’ conclusion can be drawn about normal dimensions during either burst or interburst because the valve cannot even be seen inthe living pupa without an operation involving removal of most of the stigma1 plate. Immediately internal to the spiracular valve is a 720 x 535 x 355 EL.chamber, which we call the “tracheal manifold” (M, Fig. 2C) because of the 17 or so large tracheal trunks given off radially to the surrounding tissues, where they arboresce profusely. Careful measurement of all the trunks issuing from the manifold gives a total cross-sectional area of the order of 38,000 p2 (3.8 x lOA cm2). The maximum diffusion distance in the pupa, i.e. the distance from either member of a pair of spiracles of one segment to the point in that segment most remote from both spiracles, is about 5 mm (5 x 10-r cm). In spite of the evidence that the spiracular valves control the CO, burst cycle, it is necessary, before gradients or valve areas can be computed, to make certain that there is no other point in the gas transfer path at which diffusion limitation
332
JOHN BUCK AND
MARGARET EEISTER
could occur. Since the rate of total diffusive transfer of a particular gas must be the same at all levels of the path, and since the rate of oxygen uptake is known, the relative resistances of various regions of the respiratory unit consisting of a single spiracle and its associated tracheae can be obtained readily by computing the concentration gradient of 0, necessary to maintain the known transfer rate through each anatomical step in the overall diffusion path. For these computations we use C, = RLJ -, where C, - C, is DA the concentration difference (cms/cms) over the diffusion path, R. (cm3/sec) the rate of gas transfer, L (cm) the length of the path, D (cmz/sec) the diffusivity of the gas in question, and A (cm2) the cross-sectional area of the diffusion pathway.* In addition to parameters already given we need values for rate of 0, uptake (16 kl/g/hr = 3 x 10 -’ cm3/sec/spiracle) and for the diffusivity of 0, at 25” (2 x 10 -l cm2/sec). Also we make the assumption that the total cross-sectional area of all the tracheae and branches supplying a single manifold is constant at all distances from the manifold. This assumption is in accord with measurements of KROGH (1920) and of THORPE and CRISP (1947).t Now, for transfer through the stigma1 slit, a rearrangement
of the familiar Fick equation, C, -
C _ C, = @ 0 z DA
= (3 x IO-‘) x (1.3 x IO-? (2 x 10-l) x (5 x 10-b)
_ 4 x 1O-g = 4 x 10-4 cm3/cms, - 10 x 10-s
which is about 0.3 mm Hg in partial pressure units (negligible). A similar computation for the atrium, using 6.2 x 10d4 cm2 for the cross-section computed as an ellipse, gives also a negligible concentration drop (7 x 10e5 cms/cm3 = 0.05 mm Hg), and for one spiracular manifold and its entire associated tracheal tree the pressure drop is less than 0.1 mm Hg, even assuming that all the oxygen travels the maximum 5 mm path to the tissues.2 Since then, neither the stigma1 plate, spiracular atrium, manifold nor tracheae offers any significant resistance to gas diffusion it follows that the spiracular valve must be the site and cause of any gas concentration fall that occurs between environment and tissue, or in the opposite direction. The actual dimensions of the limiting valve aperture will vary under different conditions and cannot be computed without knowing the gas concentration gradients involved. These will be considered in future .papers. 3. BLOOD VOLUME; BODY AND TISSUE WATER CONTENT For use in estimating COs storage capacity, the partitioning of pupal water was investigated in Agapema. Blood was collected by cracking off the head capsule, * We are much indebted to Dr. J. VERDUIN for instruction in the computational method. t NUNOME (1944) reported a constantly decreasing total area beyond the main tracheae, but since he did not report the number of branches at each level, an essential though elusive datum, it is possible that sufficient account had not been taken of the enormous distal increase in numbers of collaterals. $ Use of the “pore-diffusion” correction factor for L (BROWN and ESCOMBE, 1900; VERDUIN, 1954) would increase the computed gradient values from 7 to 50 per cent, depending on the dimensions involved, but since the gradients would still be negligible we have omitted the corrections in the interest of simplicity.
CYCLIC CO, RELEASE IN DIAPAUSING
PUPAE
333
making slits into the wings, and putting the animal head-down into a sedimentation tube graduated to 4 ~1. The tube was then centrifuged briefly at low speed and the resulting weight and volume of clear, tissue-free blood were recorded. This was repeated several times until the volume of extracted blood was constant. Then the centrifugal force was increased until the gut ruptured and added its black liquid content to the blood. Centrifugation was continued until no more black liquid appeared and the tissues began to be displaced out of the pupal shell. The difference between true blood (which averaged about 33 per cent of pupal fresh weight) and total free liquid extractable by centrifugation was recorded as “gut liquid” (which averaged about 10 per cent of fresh body weight). Fresh exoskeleton, scraped free of tissues, averaged from 15 per cent to 24 per cent of total body weight depending on whether the pupa was large (1.0-1.2 g) or small (0.5-0.6 g), and contained about 60 per cent water. (Since these pupae were 7-8 months old at the time of analysis it is possible that cuticle dry weight was somewhat higher than average, as compared with total dry weight, because of loss of body water.) Fresh tissue alone (“fat”) accounted for about 34 per cent of total body weight and contained about 53 per cent water. Whole pupae dried to constant weight at 110°C averaged about 28 per cent of their live weights, indicating that an average 1 g pupa would contain 720 ~1 of water. Judging from the analyses given above this 720 pl should include roughly 330 ~1 free in the blood, 100 ~1 free in the gut, 180 ~1 in the tissues and 110 ~1 in the cuticle. Estimates of body CO, capacity are contained in Part III of this paper (BUCK and FREIDMAN, 1958). 4. INTERBURST TRACHEAL pC0, For two reasons the concentration of CO, in the tracheal system is a key datum for the understanding of CO, release cycles. First, as long as tracheal COs concentration is not definitely known to be too high for the observed O,/CO, transfer ratio during interburst to be explained on the basis of pure diffusion, the periodic intervention of a qualitatively different type of metabolism cannot be ruled out. In other words, in spite of the apparent improbabilities of a metabolic shift occurring in the presence of unchanged 0, uptake rate and of its being synchronized with spiracular valve changes, the possibility still exists that the rate of CO, release is low during interburst simply because CO, is being fixed during this period, and high during the burst because at that time a sudden decarboxylation occurs which raises tracheal pC0,. Second, regardless of how CO, is retained during the interburst period, the actual escape of that gas seemingly can involve only diffusion. Hence any quantitative description or. explanation of cyclic CO, release will require values for both spiracular valve area and trans-spiracular CO, concentration difference. Either of these parameters can be computed via the Fick equation, if valve length and CO, release rate are known, but the other must be actually measured. PUNT (1944) guessed that CO, might rise to 10 per cent in the tracheae of the adult bug TTiatoma before a burst occurred, but his value was based on the unsupported assumptions that the spiracles were tightly closed during interburst
334
JOHN BUCK AND MARGARET KEISTER
(20 min) and that the tracheae occupied 60 per cent of body volume (DEMOLL’S 1928 estimate for the adult beetle Melolontha). In the absence of techniques ,for direct analysis of tracheal gas in situ, we have estimated tracheal CO, concentration in Agapema by observing the direction of gas movement when the pupa equilibrates with various CO,-Ns-0, mixtures in Warburg respirometers in the absence of alkali. The idea behind this method is that when the spiracles open wide, as they do at burst time, CO, should leave the pupa (“burst”) or enter (“negative burst”) depending on whether the outside pC0, is lower or higher, respectively, than tracheal pCO,.* In the procedure, TABLE ~-EFFECT DIRECTION
OF DIFFERENT'ENVIRONMENTAL 0F ~.4s EXCHANGEAT TIME
I
I
I
;::, ;:; 9.0 12 25 49 I
1
I
No.
CO,
I
1
1
VOLUME
AND
29.9 i: 31.6 f 2.3 1.0
43*
19 ;
11* 4 17x
31.2 14 & 4.5 39.4 * 3.1
z:;9 9
16 (1) 20 (4) 18
28 33 29
35.2 & 2.3 5.0 31.8 27.8 + 2.2
:: 18 I
I
I
33;*
I
ON
1
No.
:zIi] I
CONCENTRATIONS
0s SPIRACULAROPENING (Agapema)
I
+ 26.3 + 17.4 5 pos., 35.5 - 79.0 92.2 -215 -368
.
I
f 1.8 + 1.7 6 neg. + 2.7 jz 8.0 + 6-7 &- 8.8 f 7.5
83 58
ll&
200 262 677 1320 I
Numbers in parentheses after number of pupae indicate number used twice (i.e. on a second day). All individuals listed as being tested in CO, concentrations of 8 per cent or higher gave at least one negative burst. The values for negative burst volume in 9 per cent, 12 per cent and 25 per cent CO, include the extra CO, which leaked in during the flush-burst interval. This correction was too small to be estimated accurately with 8 per cent CO,, and with 49 per cent CO, the bursts occurred too soon. * Include at least 2 measurements on almost every pupa. 7 The 5 positive bursts averaged 144 PI/g and the 6 negative bursts 17 pi/g. Eight pupae gave no detectable burst in either direction in at least 5 hr. 1 Assuming that a larger sampling would have shown an air burst volume close to the usual 30 PI/g. CO, release rate was measured in air until (usually) two normal bursts had occurred, then the flasks were flushed with a known mixture for 5 min at a flowrate of 500 ~1 per min. The gas combinations were made by mixing CO, and air in the desired for spiracular proportions via calibrated flowmeters. Because the triggering $0, opening in certain adult insects has been reported to vary with ambient ~0, (WIGGLESWORTH, 1953; CASE, 1956), we took care to bleed into each mixture the amount of 0, necessary to keep the total 0, concentration at 21 per cent as monitored with a Pauling meter.
* We are indebted to Dr. H. SPECHT for originally interpreting the volumetric changes occurring with high ambient pC0,; and to Dr. RUBERTANDZFLSON for showing us how such measurements could be used to estimate tracheal pC0,.
CYCLIC
COz RELEASE
IN
DIAPAUSING
PUPAE
335
Table 2 summarizes the results of tests with several mixtures, and shows that the predicted transfer of CO, in both directions actually occurred. Another confirmation of the soundness of the experiment is the fact that the average interval between flushing and spiracular dilatation varied inversely with the CO, concentration of the ambient gas (2$ hr with 9 per cent; 2+ hr with 12 per cent; 1 hr with 24 per cent; 35 min with 49 per cent) as would be expected if the CO, were leaking into the tracheal system and accelerating the rise to triggering concentration. The actual volumes of CO, exchanged may be slightly in error because of simultaneous exchange of other gases, but both the direct tests with 5.9 per cent CO, and interpolation from other concentrations (Fig. 3) indicate that tracheal CO, concentration at the time of spiracular opening is about 6 per cent @CO2 = 45 mm Hg). This value is the same as in the honey-bee (BISHOP, 1923), an insect not known to exhibit cyclic CO, release.
I
I
I
I
I
5
-10
15
20
25
AMBIENT
CO2 CONCENTRATION
(%I
FIG. 3. Relation between ambient CO, concentration
and volume of CO2 moving out of (+ values on ordinates) or into (- values on ordinates) Agrqpema pupae at the time of presumed spiracular opening. V&es expressed both in terms of absolute volume (left ordinate) and percentage of normal burst volume in air (right ordinate). Dashed portions of lines in upper right corner indicate course to values for 49 per cent ambient CO,. Vertical broken line indicates normal tracheal CO, concentration (6 per cent) with zero gas exchange.
It should be pointed out that although the decrease in burst volume in 2.5 per cent and 5-O per cent CO, probably resulted from changed triggering level of CO,, in ambient concentrations above 6 per cent the valves do not constrict again, once they have dilated, hence the volume of CO, that enters is the amount required to fully equilibrate the blood and tissues. We were unable to use the above technique in estimating the tracheal pCOz of the Cecropia pupa because the negative bursts always occurred before the end of the period of temperature equilibration after flushing.
336
JOHNBucx ANDMARGARET KEISTER
The 6 per cent value for estimated tracheal CO, concentration, when considered in relation to the average tracheal volume of 60 ~1, shows that not more than 3.6 ~1 of the average burst volume of about 30 ~1 could come from CO, initially in the gas phase. This finding thus supports the opinion of SCHNEIDERMANand WILLIAMS in regard to the Cecropia burst. Besides indicating the need for information on the CO, capacity of the blood and tissue liquids-a matter considered in Part III of this paper-the fact that the CO, in the burst must come from two sources may have some bearing on the characteristically asymmetrical time course of CO, release during the burst. Thus the rate of CO, release typically rises almost instantaneously to a peak from which it declines much more slowly. This is shown even in Warburg records of bursts (e.g. SCHNEIDERMANand WILLIAMS, Figs. 2,4) but is particularly striking in PUNT’S fast recordings (e.g. 1950, Figs. 3, 9, 12). Part of this skewing may be related to the sort of spiracular valve flutter observed by SCHNEIDERMAN (1956) or, conceivably, to asynchronous opening or closing among the different individual or pairs of valves. However, it would also be expected that the burst would involve an initial very rapid diffusive escape of the CO, already in the tracheal gas phase, followed by a slower escape of CO, from blood and tissues. Such a differentiation between the escape rates of gas phase and dissolved phase CO,, apparently before the spiracles constrict, is perhaps shown in Fig. 1 of PUNT, PARSER and KUCHLEIN (1957). Evidence that release of CO, from body liquids is by no means instantaneous is also seen in our observation that even after equilibration with CO, concentrations as low as 12 per cent, the giving back of the extra CO,, upon return of the pupa to air, was detectable for 30 min. Similarly, the “purging” of body CO, which occurs when the valves are forced to dilate widely by putting the pupa in N, (BUCK and KEISTER, 1955) lasted an average of 32 min (17 pupae) as compared with 12 min for normal bursts in air. 5. COz RELEASE RATE DURING THE INTERBURST PERIOD If a near equilibrium between gas in tissue liquid and tracheal gas exists throughout the interburst period, the concentration of CO, in the gas phase should increase as the metabolic CO, is impounded and the concentration of free gas and its combination products with water increase in the liquid phase. Quite aside from changes due to possible spiracular flutter, therefore, the rate of CO, leakage should rise throughout the interburst period. Curiously enough no such rise was observed by SCHNEIDERMANand WILLIAMS (1955) nor is any perceptible in our individual Warburg records (e.g. BUCK and KEISTER, 1955, Fig. l).” This could be due to a progressive diminution in spiracular valve area during interburst. Since, however, it is difficult to imagine either a reason why the pupa should keep the rate of CO, release constant, or a control mechanism for accomplishing this, we investigated the alternative possibility that the rate of increase is too slight to show in individual records. By computing the net rate of gas exchange during the * It is difficult to be certain about the baseline in PUNT'Sdiaferometer records because of possible galvanometer drift and lack of absolute concentration calibration, but some of his records seem, disturbingly, to show no sign of an increased rate of CO2 leakage between bursts.
CYCLIC COP RELEASE IN DIAPAUSING
337
PUPA.6
three hours immediately preceding the burst in 95 cycles of 3-6 hr duration (mean = 4Q hr) it was shown clearly (Fig. 4) that the rate of change in volume declines as the time of the burst approaches. (It will be remembered that in the “direct” Warburg method, CO, release is not measured directly but is obtained as the difference between 0, uptake measured in the presence of alkali and the net gas volume change (0, minus CO,) when there is no absorption of CO,.) The apparently accelerated rate of change just before the burst may be an artifact due to valve flutter (cf. SCHNEIDERMAN, 1956), but considered as either a straight or 6.2
I
I
I
r
I
I
6.0
i .
\ \ \ 0
-
Y = 5.12
+0.26x
--
Y = 4.63
+0.98x-0~208x2
I
1
I
I
3
2.5
2
I.5
HOURS
PRIOR
TO
I I
_
i
0.5
BURST
decrease in rate of net pupal gas uptake with time, presumably reflecting an increased rate of CO, leakage. Cap-lines indicate standard errors.
FIG. 4. Pre-burst
curved line the regression is significant by F test at the 5 per cent level, and corresponds roughly to a 20 per cent decrease in net rate of gas uptake during the interburst portion of a mean cycle length of 4s hr. If oxygen uptake were constant throughout, an increase in rate of CO, release of about 20 per cent per cycle would be indicated. Though 0, uptake appears to be constant with time in individual records, it cannot be safely concluded that no mean change would have been detectable in the 95 records if it had been possible to measure 0, and CO, exchange simultaneously. An approach to this problem was made by averaging 23 other 0, uptake records in which it was possible to identify the time of occurrence of bursts by small but significant artifacts previously observed (BUCK and KEISTER, 1955). In these records there was an apparent decrease in mean 0, uptake rate with
JOHNBUCKANDMARGARET KIZSTER
338
approach to burst time, but the linear regression on time was not statistically significant. It can be concluded tentatively, therefore, that the rate of CO, release during the interburst period does increase progressively. If we consider 6 per cent to be the triggering CO, concentration for the burst, the maximum range of change during the interburst period would be from 50 to 6-O per cent or from 38 to 45 mm. This conclusion resolves what had previously appeared to be a contradiction to the idea that most of the metabolic CO, produced between bursts is impounded in the pupal buffer systems, and, together with the information discussed in section 2, reinforces the evidence that cyclic CO, retention in insects involves constriction of the spiracular valves. 6. RELATION
OF METABOLIC
RATE TO CYCLIC RESPIRATION
The prevalence and striking nature of cyclic CO, release in diapausing lepidopterous pupae has tended to create the impression that the phenomenon is somehow characteristically or causally connected with a low metabolic rate. This impression has been strengthened by such findings as those of ITO(1954) in which the normally non-diapausing and non-cyclic Bombyx pupa was converted into a burst producing type by paralysis caused by ligaturing behind the brain, and of SCHNEIDERMANand WILLIAMS (1955) in which Cecropia pupae that had ceased to give CO, bursts because they had broken diapause and begun adult development were induced to resume “bursting” when the temperature (metabolic rate) was lowered. We report below (Table 3) a similar result in that Bombyx pupae exhibiting continuous CO, release at 25°C developed unmistakable CO, cycles at 15” and lo”, in agreement with a prediction of SCHNEIDERMANand WILLIAMS (1955). TABLE3-CO2 BURST PRODUCTION IN Bombyx PUPAE.ALL PUPAEl-4 DAYSAFTER PUPATION (CHANGES crvs~ IN ARBITRARY MANOMJ~TER UNITS,IN THE.ABSENCE OFFLASK ALKALI)
Temperature
25” 15p 15” 10”
Duration of measurement (hr)
No. pupae
4
10
ir
:: 14
4$
Range
and sign of volume
changes
-4
to -14 ; no bursts 2 pupae gave bursts (f 1 to + 5) 10 pupae gave bursts (+2 to +ll) 7 pupae gave bursts C+ 2 to + 8)
Since PUNT discovered cyclic CO, release originally in adults of Triatoma at rest and has since found it in adults of other species (Locusta, Melee, Rhodnius, Carabus, HadrocaTabus), it cannot be argued that the phenomenon is associated with some metabolic or structural peculiarity of immature insects. Furthermore, such meagre evidence as exists gives no grounds for believing that insects in which cyclic CO, release has been demonstrated differ systematically in relative tracheal volume, blood volume or tracheal pC0, from those in which it has not been
CYCLIC CO, RELEASEIN DIAPAUSING PUPAE
339
demonstrated; and Part III of this paper shows that the same is true for CO, capacity. However, the adult insects mentioned above, being either lethargic (when fed) or having been studied at low temperatures (e.g. lS”C), do have inactivity in common with the lepidopterous pupae. It is accordingly possible that it is not low metabolic rateper se that is necessary for cyclic CO, release, but low metabolism in relation to 0, supply. There could, in other words, be no CO, retention without valve constriction, and no sufficient valve constriction unless respiratory requirements’ were low. This would be in accord with our previous (1955) suggestion that interburst valve area is held to “ the minimum compatible with adequate respiratory 0, supply” in the interests of water conservation. If this hypothesis of minimal interburst valve area is valid, it might be anticipated that discontinuous respiration could be induced in species not normally showing it, by increasing the This experiment has not actually been done, but ambient 0, concentration. confirmation in principle is offered by a Cecropia pupa reported to exhibit only continuous CO, release in air, but a typical cycle in 40 per cent 0, (SCHNEIDERMAN and WILLIAMS, 1955). In
sum,
widespread, individual and
(c)
it seems
reasonable
previous
failure
in the respirometer, testing
the
insects
valve setting could
ture).
this connexion,
In
insects
shown
not known
(b)
cyclic
CO,
due to (a)
conditions
not be sustained
the triggering
to have much
to exhibit
that
it being
use of respirometers
under
spiracular
cycle has been
to guess
to detect
of
release having
with
in which spiracular with
than
one
poor time resolution,
a sufficiently
(for example,
in common
is potentially more
constricted
at too high a tempera-
valve normal
activity spiracular
during control
the in
cyclic CO, release (BUCK, 1957).
REFERENCES BECKEL W. E. (1955) The ,morphology and histology of the spiracular regulatory apparatus of the giant silkworm Hyalophora cecropiu (L.). Thesis, Cornell Univ. Also in press in Proc. X Int. Congr. Entomol. BISHOP G. H. (1923) Body fluid of the honeybee larva. I. Osmotic pressure, specific gravity, pH, O2 capacity, CO, capacity and buffer value, and their changes with larval activity and metamorphosis. J. biol. Chem. 58, 567-582. ’ BROWN H. T. and ESCOMBE F. (1900) Static diffusion of gases and liquids in relation to the assimilation of carbon dioxide and translocation in plants. Philos. Trans. (B) 193,223-291. BUCK J. (1957) Triggering of insect spiracular valves. Physiological Triggers and Discontinuous Rate Processes (Edited by T. H. BULLOCK) pp. 72-79. American Physiological Society. BUCK J. and FRIEDMAN S. (1958). To be published. BUCK J. and KEISTER M. (1955) Cyclic CO2 release in diapausing Agapema pupae. Biol. Bull.,
Woods Hate 109, 144-163. BUCK J. and KEISTER M. (1956) Host-parasite relations in Agapema pupae (Lepidoptera, Saturniidae). Ann. ent. Sot. Amer. 49, 94-97. CASE J. F. (1956) Carbon dioxide and oxygen effects on the spiracles of flies. Physiol. Zool.
29, 163-171. DEMOLL R. (1928) Untersuchungen 45, 513-534.
iiber die Atmung
der Insekten. III.
Zool. Jb. (Allg.)
340
JOHN BUCKAND MARGARETKEISTER
IT0 T. (1954) Discontinuous output of carbon dioxide by undifferentiated Bombyx pupae. Jap.J_ appl. Zool. 19, 98. KROGHA. (1920) Studien iiber Tracheenrespiration. II. ijber Gasdiffusion in den Tracheen. P$iig. Arch. ges. Physiol. 179, 95-112. NUNOME2. (1944) Studies on the respiration of the silkworm. I. Diffusion of oxygen in the respiratory system of the silkworm. Bull. seric. Exp. Sta. Japan 12, 17-39. PUNT A. (1944) De gaswisseling van enkele bloedzuigende parasieten van warmbloedige dieren (Cinzex, Rhodnius, Triatoma). Onderz. physiol. Lab. Utrecht. Hoogesch. Ser. 8, Pt. 3, pp. 122-141. PUNT A. (1950) The respiration of insects. Physiol. Comp. 2, 59-74. PUNT A., PARSERW. J., and KUCHLEINJ. (1957) Oxygen uptake in insects with cyclic CO, release. Biol. Bull., Woods Hole 112, 108-119. SCHNEIDERMAN H. A. (1956) Spiracular control of discontinuous respiration in insects. Nature, Lond. 177, 1169-1171. SCHNEIDERMAN H. A. and WILLIAMS C. M. (1955) An experimental analysis of the discontinuous respiration of the Cecropia silkworm. Biol. Bull., Woods Hole 109, 123-143. THORPEW. H. and CRISPD. J. (1947) Studies on plastron respiration. II. The respiratory efficiency of the plastron in Aphelocheks. J.exp. Biol. 24, 270-303. VERDUIN J. (1954) Estimation of the gaseous CO, concentration in intercellular spaces during photosynthesis. OhioJ. Sci. 54, 353-359. WIGGLESWORTH V. B. (1953) Surface forces in the tracheal system of insects. Quart. J. micr. Sci. 94, 507-522.