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Tracheal volume in the pupa on the saturniid moth Hyalophora cecropia determined with inert gases
Respiration Physiology (1980) 40, 281 291 © Elsevier/North-Holland Biomedical Press
TRACHEAL V O L U M E IN THE PUPA OF THE SATURNIID M O T H HYALOPHORA CECROPIA DETERMINED WITH INERT GASES
C.R. BRIDGES, P. K E S T L E R ~ a n d P. SCHEID A bteilung Physiologie, Max-Planck-hlstitut j~r experimentelle Medizin. D-34 G6ttingen, F.R.G.
Abstract. Tracheal volume (Vlr) was measured in pupae of the Giant silkworm moth l~valophora
ceeropia (Saturniidac. l.cpidoptcra, lnsccta) using inert gas wash-out techniques. The animal was placed in a small vessel that was continuously ventilated (rate, V) by a gas mixture containing 20°/. 0 2 in N2; the inflowing (F[) and outflowing gas fractions (Fl=) of the vessel could be continuously measured by a respiratory mass spectrometer. At the onset of a spiracular constriction period, which was evidenced from the Fr:c.c): trace, the mixture was rapidly replaced by pure Ar. At the subsequent burst, the a m o u n t of N 2 cmcrging from the animal, M ,,~, was calculated from 'v' and the diffcrence (Fli- F z)N:. V Ir was calculated from MN, and the N 2 concentration in the tracheal system before constriction (assumed to equal that in the ventilating gas before replacement by Ar). Measurements were repeated with N 2 and Ar replacing each other. VTr avcragcd 48 lal. g - I (range 39 to 59) for animals of 5.8 g average body weight (range 3.4 to 9.9), when inert gas solubility in body fluids was accounted for. Both size and stage in pupal development appear to affect Vrr. These values show reasonable agreement with literature data, mostly obtained by emptying the tracheal gas space by mechanical compression. Insects Mass spectrometry
Tissue solubility
In terrestrial insects the gas-filled tracheal system ramifies throughout the tissues of the organism and so allows O z and CO 2 to exchange between the environment and the cells. The structure of the ramifications of the tracheal system has been studied in many species (of. Wigglesworth, 1965; Edwards, 1953; Miller, 1974), however, only few measurements exist of the volume of this system (see Discussion), an important parameter when analyzing respiratory gas exchange of insects. Respiratory mass spectrometers have recently been applied in the study of vertebrate respiratory physiology. Inert gas wash-out techniques have allowed rapid and reproducible determination of lung volume. This technique appears to be not Accepted for publication 22 December 1979 Present address: 1. Zoologisches Institut der Universitiit, D-34 G6ttingen, F.R.G. 281
c.R. BRIDGES et al.
282
directly applicable to insects with their discontinuous respiration, ventilatory movements apparently lacking entirely in pupae (Punt, 1944; Ito, 1954; Buck and Keister, 1955; Schneiderman and Williams, 1955; Schneiderman, 1960; Kestler, 1978). We have designed a method that allows determination of tracheal volume in insects by respiratory mass spectrometry. Application to the pupa of the Giant silkworm moth, Hyalophora cecropia, yielded reproducible results, comparable with literature data.
Principle of measurement
Consider an animal kept in a small vessel that can be ventilated with gas mixtures of known and variable composition (FI). Any gas component that evolves from the animal in a concentration that differs from FI will cause the concentration of the ventilating gas at the vessel outlet, FE, to rise above FI. Hence, evolving CO, permits one to follow the sequence of constriction, flutter and burst in the normal respiratory cycle (Kestler, 1971 ; fig. 1). Consider that air constitutes, for a number of cycles, the ventilating mixture, with FIN2 close to 0.8. Since N: is not metabolized by the animal, tracheal N: concentration, FTrs~, will be close to FI~,: during spiracular opening (burst) and the corresponding partial pressure, PTry~, will be given by PT"rx: = FIN~ (PR - P.20)
(1)
where PB constitutes barometric pressure; PH~O, water vapor pressure, obtained
4
. . . . . . . Constriction
I
Normal Flutter
Cycle .....
Burst
Co~Irictlor
InducedBurst
FC%0001I o"
i
o~_ oo~ FN 2 7-
O--
10[-
i Time Fig. 1. Principleof the method used to induce a respiratory burst, by ambient anoxia, during a normal respiratory cycle in a llyalophora pupa. 0"
TRACHEAL VOLUME IN INSECTS
283
by assuming full water saturation at the animal's temperature. The amount of N2 in the animal, M~,, comprises the amount in the tracheal gas phase, of volume VTr, and an amount dissolved in the body tissues, of volume VTis. If~N: is the solubility of N2 in the body tissues and ~if N2 has reached equilibration between tracheal gas and body tissues, the mass balance yields MN2 = PTrN, {/~g. VTr + ~ , . . VTis}
(2)
in which the solubility in the gas phase or capacitance coefficient, pg, is constant for all ideal gases and depends only on the (absolute) temperature, T, (cj~ Piiper et al., 1971) /~g = 1/RT
(3)
with the ideal gas constant, R = 0.0624 L - Torr • (K • mmol) - ~. (T, absolute temperature in Kelvin). To measure M N2, N2 in the ventilating gas mixture is rapidly rcplaced by argon (Ar) at the onset of a constriction period, when the spiracles are gas-tight. At the following spiracular opening, N2 evolves from the animal and may be measured in the outlet from the vessel. This measurement is facilitated by removing 02 from the ventilating gas creating an induced burst in which the spiracles remain wide open. FEN: will in this case display a distinct 'washout' curve, corresponding to the diffusional N2 loss from the tracheal system, which can be integrated to calculate, with known flow rate, M x~. Equation (2) contains, apart from V-n-, two unknown parameters, of which VTis may be approximated by the body mass, W, assuming a specific gravity of the tissues of unity. The other unknown parameter is ar~.- To eliminate c~y,, a second gas of low solubility, e.g. argon (Ar), may be used. If MAr, P'I'rAr, and ~,~r constitute amount, tracheal partial pressure, and tissue solubility of Ar, eq. (2), applied to both N2 and Ar, may be solved for the tracheal volume ( V'l'r =
M -)78 -PTr
)
[M/(/?g'PTr)]Ar--[M/(,Sg'PTr)]N, :w~ -
~-,,~,,,~Z-]
(4)
Solubility coefficients, 2, may be obtained from the literature for both N~ and Ar and Ibr a number of solvents (see below). Despitc considerable differences in values bctween various solvents and tissues, the ratio of ~,~,/~, is essentially identical. This value may thus be used for insect tissue. Using thc value of VTr, eq. (2) allows calculation of thc solubility coefficient,
and, with the assumed ratio ~.a,/~x:, the solubility :~.~ may be obtained.
284
C R . BRIDGES et al.
Materials and methods
Pupae of the Giant silkworm moth (Hyalophora cecropia) ranging in weight between 3.4 and 9.9 g were obtained from commercial suppliers in the USA and stored at 20°C and 100% humidity or, in the refrigerator, at 4°C and 80°/(, humidity. Animals kept at 20 °C normally remained in the diapause for 2-3 months during which time measurements could be made of the respiratory cycle length to ascertain the developmental stage of the pupae. Animals kept at 4 °C usually developed within 3 -6 weeks of being removed from the refi'igerator and placed at 20 °C.
TICHNIQUI.!S
Figure 2 shows the thermostatted flow-through respirometer used in this study (volume, 17 ml). Gas mixtures provided by gas mixing pumps (type M301/a-F, W6sthoff, Bochum, F.R.G.) were humidified at the desired temperature and passed through the respirometer at a rate of 45 m l . m i n -~. The selected temperature of the water jacket was maintained within +0.1 °C. A series of needle valves allowed adjustment of flow which was monitored continuously with a pneumotachograph (Model 17212, Godart-Statham, Bilthoven, the Netherlands). Concentrations of O2, CO,, N~ and Ar in gas at the inlet (F0 or outlet (FI!) of the respirometer were continuously and simultaneously analyzed by a respiratory mass spectrometer (Scheid et al., 1979), the output of which was electronically converted to measure fractional concentration (Slama and Scheid, 1975). Output signals from the mass spectrometer and pneumotachograph were recorded on an 8-channel recorder (Brush 481), and could be integrated electronically with respect to time. The mass spectrometer was calibrated with mixtures provided by separate gas mixing pumps (W6sthoff, see above).
Humidified Gases
C02 02 t
TemperatuControl re/ Joc~ei i
q
N2 Ar
Fig. 2. Schema of experimental set-up for continuous recording of gas exchange in a flow-through respirometer. Mass Spcc{. mass spectrometer.
TRACIIEAL VOLUME IN INSECI'S
285
EXPERIMENTAL PROTOCOL
A pupa was placed in the respirometer, which was initially kept at 20°C, and was ventilated with an O_,/N 2 mixture (Flo~ = 0.20, F!y~ = 0.80). FLc.o: was measured as a record of the respiratory cycle. If the constriction period was below 5 rain. the temperature of the respiromcter was lowered and the constriction period thereby prolonged. A period of 5 min was safely in excess of the time needed to change the respirometer atmosphere from one mixture to another. The pupa was left in the respirometer for about 12 hours to acclimate to the experimental conditions. A measurement was started by replacing, approximately half a minute alter the onset of a constriction period, the inflowing gas mixture by pure Ar. During the following burst, N2 evolving from the animal was recorded. At the end of the N2 wash-out, a k n o w n volume (approximately equal to Vvr) of pure N2 was injected into the respirometer, and the ensuing burst in FEN: was recorded for calibration purpose. Typically the time that elapsed between switching to pure Ar and completion of the measurement was 20 rain. Then, O~ was added to the ventilating Ar (FIo: = 0.20; FIA~= 0.80) and three cycles were allowed for the animal to equilibrate with thc Ar of this mixture, after which a measurement was performed, as described above, where Ar evolving from the animal was recorded with N2 as the background inert gas. A set of N2 and Ar measurements was completed within 4 hours. In some cases, multiple determinations were performed.
CALCULATIONS
Inert gas partial pressure in the tracheal system was calculated from eq. (1). The amount of inert gas, M x, evolving from the animal in the induced burst was obtained from the inert gas concentration in the respirometer outflow, Ft-, (inflow concentration. FI = 0) and from the ventilatory flow rate, 'q, which was constant U x = ~' .~(FE)~.dt
(6)
where x denotes N2 or Ar, and integration extends over the period in which FE Xis measurably different from zero (usually I-5 min). Tissue volume was obtained t¥om body mass assuming specific gravity of unity. Tracheal volume, VTr, was obtained from eq. (4). Literature values for the ratio ~Ar/~: needed in ec1. (4) range from 2.05 to 2.30 for water, saline, rabbit blood and brain (Altman and Dittmer, 1971; Battino and Clever, 1966; Ohta et al., 1979). We have adopted an intermediate value of 2.22 and have assumed the temperature dependence of ~.,xr to be identical with that listed by Altman and Dittmer (1971) for ~N2, thereby assuming ~N:/~Ar tO be independent of temperature in the experimental temperature range. Solubility for N2, ~x~, was calculated from eq. (5), and ~ , obtained from this value with the assumed solubility ratio.
286
C.R. BRIDGES et al. 0.001
-
Vco2 {
I ' ~ . _
c=., ,c,ioo
F,o.o
Bor., oos,r,c,ioo
F,u.er
IBors'lCoo"r'c"oo 1
l 1 hour Fig. 3. Recording of (:0 2 eMux from a Hyalophora pupa in diapause, at 20°C and 100'5o humidity.
Results Figure 3 displays a control record of the CO2 concentration in the respirometer outflow (FEco,) when its inflow, Flco: = 0. There is a clear distinction between the three phases in the respiratory cycle, constriction (no CO2 evolving from the animal), flutter (short spikes of CO2 corresponding to short periods of CO2 release), burst (initial phase of massive CO2 evolvement, fading off into greater spikes of CO2). It is apparent that the corresponding phases in successive cycles are of approximately equal length. In particular the time for constriction does not vary much between cycles, provided the experimental temperature is kept constant. Figure 4 constitutes an original record obtained for a determination of tracheal volume with N2 washout. It shows the concentrations in the respirometer outflow, FE, for both C02 and N2. At the onset of constriction, evidenced in the CO2 trace, N2 in the ventilating gas was replaced by Ar. Of interest is particularly the N2 record. Before switching to Ar, FEr% is 0.8 which is off-scale in the record. After switching to Ar, and at the end o f the measurement, the mass spectrometer was calibrated. During the induced burst, N2 washes out from the animal and produces a welldefined hump in FE. The wash-out of CO2 is prolonged and the CO2 amount, relative to the initial peak value, much larger than for the other gases. This phenomenon, which is due to the high tissue solubility of C02, will be analyzed in detail in a separate communication.
TRACHEAL VOLUME IN INSECTS Normal Burst
100% Argon
287
Induced Burst C~ib
oE
VI
J
....... Injectlor~
300/Jr m
m
m
~-
Colib
I
of N2 Calibration
F,~ 0.02f 0t_
........
U. ll,l:ill
IIIIIIIIIIIIIIII
il
!
I
........ [.
I
]
Time Ira,n) Fig. 4. Actual recording trace taken from a determination of tracheal volume in a 4.5 g pupa. After a p p a r e n t completion o f the N2 wash-out, a k n o w n a m o u n t o f pure N2 gas was injected into the respirometer which p r o d u c e d the discrete burst in the N2 line. At the end o f the record, pure A r was replaced by an Ar/02 mixture and the measurement, after some cycles for equilibration, was repeated with A r as the gas measured (see Methods). To calibrate the area underneath the induced burst o f N2, both the calibrated sensitivity o f the mass spectrometer, together with the measured flow rate, and the area o f the calibration burst were used. Both gave very similar results. Table 1 presents mean values for VTr determined for 12 specimens o f Hyalophora cecropia in the diapause. The variability in the values is mainly due to a variability between animals, reproducibility for a given specimen being a b o u t 1 ~tl .g-~ The tracheal volume accounts for approximately 5 ~ o f the b o d y volume. The solubility coefficients, calculated from eq. (5), averaged ~<~2= 0.0159 ml STPD. (ml .atm)-~ = 0.000934 m m o l • L- ' . T o r r - l , C
Weight (g) Temperature (°C) Tracheal volume (I.tl •g - 1)
Mean _+SD
Range
5.7 + 1.8 18.8 + 2.5 48.4 + 6.8
3.4- 9.9 15 22.5 39 -59
288
C.R. BRIDGES et ctl. 800 J-
L 600 " J
~ooi o#
•~"
•
~ o
f
i 0 !
0
1
I
2
1
...................
~.___.L
4 6 Weight (g)
.....
L
I
8
10
Fig. 5. Relationship between tracheal volume (VTr) and body weight in 11ya/ophora cecropia. Closed circles. pupae in diapat, se ; open circles, pupae before adult emergence, the number indicating the number of days before hatching. Regression line calculatcd for animals in diapausc [Vrr(lal) = 41 W(g) + 36].
Discussion (a) CRITIQUE OF METI-1ODS An important assumption o f the method is that the spiracles themselves arc gastight during constriction. This allows the exchange o f the external gas medium for another gas mixture o f different composition without dilution or loss o f the inert gas contained within the pupa. To test this assumption recordings were made with a pupa equilibrated in an 80~,,i N • 20~i O= mixture. At constriction the external gas mixture was changed to 100~°~i A r g o n and then returned, betbre the end o f constriction, to the initial mixture. Efflux o f argon from the pupa, monitored during the next flutter/burst period, was found to be zero, indicating that no Ar had leaked into the animal during the constriction. As further evidence lbr gas-tightness at constriction, it was found that there was no significant difference between the length o f the constriction phase when ~ o , , / N ,_" 20j0,~ O, mixture as the either pure inert gas (Ar o r N2) was used or an ,,,,~o ambient atmosphere during constriction. If there were any leakage, the constriction period would be shorter under pure N 2 or argon as 02 would leak out o f the tracheal system thus triggering the flutter/burst period earlier in the O~ free environment. F o r the calculation o f VTr according to eq. (4) it has been assumed that all b o d y tissues are equilibrated with inert gas in the tracheal system. This may be justified as the tracheal system extends into all b o d y tissues (q/~ Miller, 1974).
TRACHEAL VOLUME IN INSECTS
289
It was further assumed that this equilibration is fast enough to allow all inert gas to be removed, during the tracheal wash-out, from the tissues. Without exact knowledge of the geometry of the tracheolar system in relation to the body tissues, this assumption cannot be proved theoretically. However, the fact that in the induced burst the inert gas concentration, unlike the CO, concentration, returned to zero may be taken to indicate that the tissues effectively discharged their inert gases completely. Use of two inert gases of low, yet differing, solubility circumvents the need of assigning an exact value to the tissue solubility coefficient, as this value is dependent on the solvent and its constituents (cf Battino and Clever, 1966). Rather, the ratio of solubility coefficients is needed, which appears to be relatively constant at around ~Ar/~,~:=2.22 in water, oil, blood and other tissues (Altman and Dittmer, 1971 ; Battino and Clever, 1966; Ohta et al., 1979). The solubility coefficients for N: and Ar obtained from eq. (5) may be considered as the effective value for tissue solubility. Calculation of the tissue/water partition coefficient gave an average value of 1.01 (+0.2). This value should be considered a compromise between tissue constituents with solubility coefficients lower (e.g. electrolytes) or higher than those of pure water (e.g. lipids) (cf. Meyer et al., 1980). In fact, tissue water partition coefficients not far from unity have been measured for other tissues. Ohta et al. (1979) reported values of 1.12 and 1.08 for the brain/ blood partition coefficients of N2 and Ar, respectively. Campos Caries et al. (1975) also reported valucs for muscle/water partition coefficients for a number of inert gases which range, for most gases, between 1 and 2.
(b) COMPARISON WITH VALUES IN THE LITERATURE Table 2 gives details of literature data on tracheal volume in Hyalophora pupae that were obtained with different methods. In general the values in the literature are in agreement with those found in this study. Of the previous methods employed, vacuum extraction (Schneiderman and Williams, 1955) leads to an overestimation of the tracheal volume whilst the mechanical pressure used by Buck and Keister TABLE 2 Literature values for tracheal volume in IIyalophora Weight (g)
-
5.0 5.7
Methodof determination
Tracheal volume (p.l .g 1)
Reference
Vacuum extraction Mechanical compression Water compression Inert gas washout
I00 75 50 48
Schneiderman and Williams (1955) Buck and Keister (1958) Kanv,'isher (1966) This study
290
c.R. BRIDGES et aL
(1958) to s q u e e z e o u t the air f r o m the a n i m a l l e a d s to w a t e r - f i l l i n g o f t h e t r a c h e a o r to e v i s c e r a t i o n o f the p u p a e , p r e v e n t i n g m u l t i p l e d e t e r m i n a t i o n s o n the s a m e a n i m a l . K a n w i s h e r (1966) a p p l i e d a p l e t h y s m o g r a p h i c t e c h n i q u e by w h i c h t r a c h e a l v o l u m e is c a l c u l a t e d f r o m the v o l u m e i n c r e m e n t s n e c e s s a r y to c h a n g e the o v e r a l l p r e s s u r e in a c l o s e d w a t e r - f i l l e d s y s t e m c o n t a i n i n g a p u p a . B u c k a n d K e i s t e r (1958) also m a d e m e a s u r e m e n t s o n a s m a l l e r p u p a (0.7-1.1 g) of Agapema
g a l b i n a a n d f o u n d a m e a n t r a c h e a l v o l u m e o f 55 lal . g - ~ (_+ 12.6).
T r a c h e a l v o l u m e has also b e e n d e t e r m i n e d in larval a n d a d u l t stages. K r o g h (1920a) r e p o r t e d a v a l u e o f 14 Ixl • g-~ in the C o s s u s l a r v a w h i c h c o m p a r e s w i t h 6 6 - 9 9 lal • g in D y t i s c u s l a r v a e ( K r o g h , 1920b). N u n o m e
(1944) c a l c u l a t e d f r o m m e a s u r e m e n t s
o f the i n t e r n a l d i m e n s i o n s o f t h e t r a c h e a l system a t r a c h e a l v o l u m e o f 33 lal • g in the s i l k w o r m larva. In the a d u l t c o c k c h a f e r M e l o l o n t h a , D e m o l l (1927) r e p o r t s a t r a c h e a l v o l u m e o f 585 l a l - g ~ .
A v a l u e o f 265 lal . g - ~ c a n be c a l c u l a t e d f r o m
the d a t a o f C l a r k e (1958) for t h e t r a c h e a l v o l u m e o f L o c u s t a m i g r a t o r i a at the fifth instar.
References Altman, P. L. and D. S. Dittmer (1971). Biological Handbooks: Respiration and Circulation. Federation of American Societies for Experimental Biology. Bethesda, MD, U.S.A. Battino, R. and H.L. Clever (1966). Solubility of gases in liquids. Chem. Rev. 66: 395-463. Buck, J. and M. Keister (1955). Cyclic CO..2 release in diapausing Agapema pupae. Biol. Bull. Mar. Biol. Lab., Woods tlolc 109: 144-163. Buck, J. and M. Keister (1958). Cyclic CO 2 release in diapausing pupae. It. Tracheal anatomy, volume and Pco2; blood volume; interburst CO 2 release rate. J. Insect. Physiol. 1 : 327-340. Campos Caries, A., T. Kawashiro and J. Piiper (1975). Solubility of various inert gases in rat skeletal muscle. Pfliigers Arch. 359: 209-218. Clarke, K. U. (1958). Studies on the relationship between changes in the volume of the tracheal system and growth in Locusta migratoria L. Proe. lOth Int. Congr. Entomol. 2:205-211. Demoll, R. (1927). Untersuchungen fiber die Atmung der Insekten. Z. Biol. 87: 8-22. Edwards, G. A. (1953). Respiratory Mechanisms. In: Insect Physiology, edited by K. Roeder. New York, Wiley, pp. 55-96. lto, T. (1954). Discontinuous output of carbon dioxide by undifferentiated Bombyx pupae. Jap. J. Appl. Zool. 19: 98. Kanwisher, J.W. (1966). Tracheal gas dynamics in pupae of the Cecropia silkworm. Biol. Bull. Mar. Biol. Lab., Woods Hole 130: 96-105. Kestler, P. (1971). Die diskontinuierliche Ventilation bei Periplaneta americana L. und anderen Insekten. Ph.D. Thesis, Wi.irzburg, F.R.G. Kestler, P. (1978). Atembewegungen und Gasaustausch bei der Rubeatmung adulter terrestrischer lnsekten. Verh. Dtsch. Zool. Ges. 1978: 269. Krogh, A. (1920a). Studien fiber Trachcenrespiration. II. ~ber Gasdiffusion in den Tracheen. Pfli~g. Arch. Ges. Physiol. 179:95 112. Krogh, A. (1920b). Studien tiber Trachcenrespiration. II1. Die Kombination von mechanischer Ventilation mit Gasdiffusion nach Versuchen an Dytiscus Larven. Pfliig. Arch. Ges. Physiol. 179: 113-120. Miller, P.L. (1974). Respiration-Aerial Gas Transport. In: The Physiology of Insecta, edited by M. Rockstein, Vol. VI, Chapt. 5. London, Academic Press, pp. 346 397.
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Meyer, M., U. Tebbe and J. Piiper (1980). Solubility of inert gases in dog blood and skeletal muscle. Pfliigers Arch. (submitted). Nunome, J. (1944). Studies on the respiration of the silkworm. II. Several measurements in the respiratory organ. Bull. Seric. Exp. Sta. Jap. 12: 41-90. Ohta, Y., A. Ar and L.E. Farhi (1979), Solubility and partition coefficients for gases in rabbit brain and blood. J. Appl. Physiol. 46:1169-1170. Piiper, J., P. Dejours, P. Haab and H. Rahn (1971). Concepts and basic quantities in gas exchange physiology. Respir. Physiol. 13 : 292 304. Punt, A. (1944). De gaswisseling van enkelc bloedzuigende parasieten van warmbloedige dieren ( Cirnex, Rhodinius, Triatorna), Onderz. Physiol. Lab. Rijks-Univ. Utrecht, 8th Ser., 3: 122-141. Scheid, P., M. Meyer and H. Slama (1979). Use of a mass spectrometer to measure lung diffusing capacity for 0 2 and CO by rebreathing stable isotopes at tracer levels. Bull. Eur. Physiopathol. Resp. 15:11P.-14P. Schneiderman, H.A. and C.M. Williams (1955). An experimental analysis of the discontinuous respiration of the Cecropia silkworm. Biol. Bull. Mar. Biol. Lab., Woods Hole 112:106-119. Schneiderman, H. A. (1960). Discontinuous respiration in insects. Role of the spiracles. Biol. Bull. Mar. Biol. lab., Woods Hole 119 : 494-528. Slama, H. and P. Scheid (1975). Electronic feedback circuit for increasing the signal to noise ratio in a mass spectrometer. Pneumonologie 151:247-249. Wigglesworth, V.B. (1965). The Principles of Insect Physiology. 6th ed., London, Methuen.
TRACHEAL V O L U M E IN THE PUPA OF THE SATURNIID M O T H HYALOPHORA CECROPIA DETERMINED WITH INERT GASES
C.R. BRIDGES, P. K E S T L E R ~ a n d P. SCHEID A bteilung Physiologie, Max-Planck-hlstitut j~r experimentelle Medizin. D-34 G6ttingen, F.R.G.
Abstract. Tracheal volume (Vlr) was measured in pupae of the Giant silkworm moth l~valophora
ceeropia (Saturniidac. l.cpidoptcra, lnsccta) using inert gas wash-out techniques. The animal was placed in a small vessel that was continuously ventilated (rate, V) by a gas mixture containing 20°/. 0 2 in N2; the inflowing (F[) and outflowing gas fractions (Fl=) of the vessel could be continuously measured by a respiratory mass spectrometer. At the onset of a spiracular constriction period, which was evidenced from the Fr:c.c): trace, the mixture was rapidly replaced by pure Ar. At the subsequent burst, the a m o u n t of N 2 cmcrging from the animal, M ,,~, was calculated from 'v' and the diffcrence (Fli- F z)N:. V Ir was calculated from MN, and the N 2 concentration in the tracheal system before constriction (assumed to equal that in the ventilating gas before replacement by Ar). Measurements were repeated with N 2 and Ar replacing each other. VTr avcragcd 48 lal. g - I (range 39 to 59) for animals of 5.8 g average body weight (range 3.4 to 9.9), when inert gas solubility in body fluids was accounted for. Both size and stage in pupal development appear to affect Vrr. These values show reasonable agreement with literature data, mostly obtained by emptying the tracheal gas space by mechanical compression. Insects Mass spectrometry
Tissue solubility
In terrestrial insects the gas-filled tracheal system ramifies throughout the tissues of the organism and so allows O z and CO 2 to exchange between the environment and the cells. The structure of the ramifications of the tracheal system has been studied in many species (of. Wigglesworth, 1965; Edwards, 1953; Miller, 1974), however, only few measurements exist of the volume of this system (see Discussion), an important parameter when analyzing respiratory gas exchange of insects. Respiratory mass spectrometers have recently been applied in the study of vertebrate respiratory physiology. Inert gas wash-out techniques have allowed rapid and reproducible determination of lung volume. This technique appears to be not Accepted for publication 22 December 1979 Present address: 1. Zoologisches Institut der Universitiit, D-34 G6ttingen, F.R.G. 281
c.R. BRIDGES et al.
282
directly applicable to insects with their discontinuous respiration, ventilatory movements apparently lacking entirely in pupae (Punt, 1944; Ito, 1954; Buck and Keister, 1955; Schneiderman and Williams, 1955; Schneiderman, 1960; Kestler, 1978). We have designed a method that allows determination of tracheal volume in insects by respiratory mass spectrometry. Application to the pupa of the Giant silkworm moth, Hyalophora cecropia, yielded reproducible results, comparable with literature data.
Principle of measurement
Consider an animal kept in a small vessel that can be ventilated with gas mixtures of known and variable composition (FI). Any gas component that evolves from the animal in a concentration that differs from FI will cause the concentration of the ventilating gas at the vessel outlet, FE, to rise above FI. Hence, evolving CO, permits one to follow the sequence of constriction, flutter and burst in the normal respiratory cycle (Kestler, 1971 ; fig. 1). Consider that air constitutes, for a number of cycles, the ventilating mixture, with FIN2 close to 0.8. Since N: is not metabolized by the animal, tracheal N: concentration, FTrs~, will be close to FI~,: during spiracular opening (burst) and the corresponding partial pressure, PTry~, will be given by PT"rx: = FIN~ (PR - P.20)
(1)
where PB constitutes barometric pressure; PH~O, water vapor pressure, obtained
4
. . . . . . . Constriction
I
Normal Flutter
Cycle .....
Burst
Co~Irictlor
InducedBurst
FC%0001I o"
i
o~_ oo~ FN 2 7-
O--
10[-
i Time Fig. 1. Principleof the method used to induce a respiratory burst, by ambient anoxia, during a normal respiratory cycle in a llyalophora pupa. 0"
TRACHEAL VOLUME IN INSECTS
283
by assuming full water saturation at the animal's temperature. The amount of N2 in the animal, M~,, comprises the amount in the tracheal gas phase, of volume VTr, and an amount dissolved in the body tissues, of volume VTis. If~N: is the solubility of N2 in the body tissues and ~if N2 has reached equilibration between tracheal gas and body tissues, the mass balance yields MN2 = PTrN, {/~g. VTr + ~ , . . VTis}
(2)
in which the solubility in the gas phase or capacitance coefficient, pg, is constant for all ideal gases and depends only on the (absolute) temperature, T, (cj~ Piiper et al., 1971) /~g = 1/RT
(3)
with the ideal gas constant, R = 0.0624 L - Torr • (K • mmol) - ~. (T, absolute temperature in Kelvin). To measure M N2, N2 in the ventilating gas mixture is rapidly rcplaced by argon (Ar) at the onset of a constriction period, when the spiracles are gas-tight. At the following spiracular opening, N2 evolves from the animal and may be measured in the outlet from the vessel. This measurement is facilitated by removing 02 from the ventilating gas creating an induced burst in which the spiracles remain wide open. FEN: will in this case display a distinct 'washout' curve, corresponding to the diffusional N2 loss from the tracheal system, which can be integrated to calculate, with known flow rate, M x~. Equation (2) contains, apart from V-n-, two unknown parameters, of which VTis may be approximated by the body mass, W, assuming a specific gravity of the tissues of unity. The other unknown parameter is ar~.- To eliminate c~y,, a second gas of low solubility, e.g. argon (Ar), may be used. If MAr, P'I'rAr, and ~,~r constitute amount, tracheal partial pressure, and tissue solubility of Ar, eq. (2), applied to both N2 and Ar, may be solved for the tracheal volume ( V'l'r =
M -)78 -PTr
)
[M/(/?g'PTr)]Ar--[M/(,Sg'PTr)]N, :w~ -
~-,,~,,,~Z-]
(4)
Solubility coefficients, 2, may be obtained from the literature for both N~ and Ar and Ibr a number of solvents (see below). Despitc considerable differences in values bctween various solvents and tissues, the ratio of ~,~,/~, is essentially identical. This value may thus be used for insect tissue. Using thc value of VTr, eq. (2) allows calculation of thc solubility coefficient,
and, with the assumed ratio ~.a,/~x:, the solubility :~.~ may be obtained.
284
C R . BRIDGES et al.
Materials and methods
Pupae of the Giant silkworm moth (Hyalophora cecropia) ranging in weight between 3.4 and 9.9 g were obtained from commercial suppliers in the USA and stored at 20°C and 100% humidity or, in the refrigerator, at 4°C and 80°/(, humidity. Animals kept at 20 °C normally remained in the diapause for 2-3 months during which time measurements could be made of the respiratory cycle length to ascertain the developmental stage of the pupae. Animals kept at 4 °C usually developed within 3 -6 weeks of being removed from the refi'igerator and placed at 20 °C.
TICHNIQUI.!S
Figure 2 shows the thermostatted flow-through respirometer used in this study (volume, 17 ml). Gas mixtures provided by gas mixing pumps (type M301/a-F, W6sthoff, Bochum, F.R.G.) were humidified at the desired temperature and passed through the respirometer at a rate of 45 m l . m i n -~. The selected temperature of the water jacket was maintained within +0.1 °C. A series of needle valves allowed adjustment of flow which was monitored continuously with a pneumotachograph (Model 17212, Godart-Statham, Bilthoven, the Netherlands). Concentrations of O2, CO,, N~ and Ar in gas at the inlet (F0 or outlet (FI!) of the respirometer were continuously and simultaneously analyzed by a respiratory mass spectrometer (Scheid et al., 1979), the output of which was electronically converted to measure fractional concentration (Slama and Scheid, 1975). Output signals from the mass spectrometer and pneumotachograph were recorded on an 8-channel recorder (Brush 481), and could be integrated electronically with respect to time. The mass spectrometer was calibrated with mixtures provided by separate gas mixing pumps (W6sthoff, see above).
Humidified Gases
C02 02 t
TemperatuControl re/ Joc~ei i
q
N2 Ar
Fig. 2. Schema of experimental set-up for continuous recording of gas exchange in a flow-through respirometer. Mass Spcc{. mass spectrometer.
TRACIIEAL VOLUME IN INSECI'S
285
EXPERIMENTAL PROTOCOL
A pupa was placed in the respirometer, which was initially kept at 20°C, and was ventilated with an O_,/N 2 mixture (Flo~ = 0.20, F!y~ = 0.80). FLc.o: was measured as a record of the respiratory cycle. If the constriction period was below 5 rain. the temperature of the respiromcter was lowered and the constriction period thereby prolonged. A period of 5 min was safely in excess of the time needed to change the respirometer atmosphere from one mixture to another. The pupa was left in the respirometer for about 12 hours to acclimate to the experimental conditions. A measurement was started by replacing, approximately half a minute alter the onset of a constriction period, the inflowing gas mixture by pure Ar. During the following burst, N2 evolving from the animal was recorded. At the end of the N2 wash-out, a k n o w n volume (approximately equal to Vvr) of pure N2 was injected into the respirometer, and the ensuing burst in FEN: was recorded for calibration purpose. Typically the time that elapsed between switching to pure Ar and completion of the measurement was 20 rain. Then, O~ was added to the ventilating Ar (FIo: = 0.20; FIA~= 0.80) and three cycles were allowed for the animal to equilibrate with thc Ar of this mixture, after which a measurement was performed, as described above, where Ar evolving from the animal was recorded with N2 as the background inert gas. A set of N2 and Ar measurements was completed within 4 hours. In some cases, multiple determinations were performed.
CALCULATIONS
Inert gas partial pressure in the tracheal system was calculated from eq. (1). The amount of inert gas, M x, evolving from the animal in the induced burst was obtained from the inert gas concentration in the respirometer outflow, Ft-, (inflow concentration. FI = 0) and from the ventilatory flow rate, 'q, which was constant U x = ~' .~(FE)~.dt
(6)
where x denotes N2 or Ar, and integration extends over the period in which FE Xis measurably different from zero (usually I-5 min). Tissue volume was obtained t¥om body mass assuming specific gravity of unity. Tracheal volume, VTr, was obtained from eq. (4). Literature values for the ratio ~Ar/~: needed in ec1. (4) range from 2.05 to 2.30 for water, saline, rabbit blood and brain (Altman and Dittmer, 1971; Battino and Clever, 1966; Ohta et al., 1979). We have adopted an intermediate value of 2.22 and have assumed the temperature dependence of ~.,xr to be identical with that listed by Altman and Dittmer (1971) for ~N2, thereby assuming ~N:/~Ar tO be independent of temperature in the experimental temperature range. Solubility for N2, ~x~, was calculated from eq. (5), and ~ , obtained from this value with the assumed solubility ratio.
286
C.R. BRIDGES et al. 0.001
-
Vco2 {
I ' ~ . _
c=., ,c,ioo
F,o.o
Bor., oos,r,c,ioo
F,u.er
IBors'lCoo"r'c"oo 1
l 1 hour Fig. 3. Recording of (:0 2 eMux from a Hyalophora pupa in diapause, at 20°C and 100'5o humidity.
Results Figure 3 displays a control record of the CO2 concentration in the respirometer outflow (FEco,) when its inflow, Flco: = 0. There is a clear distinction between the three phases in the respiratory cycle, constriction (no CO2 evolving from the animal), flutter (short spikes of CO2 corresponding to short periods of CO2 release), burst (initial phase of massive CO2 evolvement, fading off into greater spikes of CO2). It is apparent that the corresponding phases in successive cycles are of approximately equal length. In particular the time for constriction does not vary much between cycles, provided the experimental temperature is kept constant. Figure 4 constitutes an original record obtained for a determination of tracheal volume with N2 washout. It shows the concentrations in the respirometer outflow, FE, for both C02 and N2. At the onset of constriction, evidenced in the CO2 trace, N2 in the ventilating gas was replaced by Ar. Of interest is particularly the N2 record. Before switching to Ar, FEr% is 0.8 which is off-scale in the record. After switching to Ar, and at the end o f the measurement, the mass spectrometer was calibrated. During the induced burst, N2 washes out from the animal and produces a welldefined hump in FE. The wash-out of CO2 is prolonged and the CO2 amount, relative to the initial peak value, much larger than for the other gases. This phenomenon, which is due to the high tissue solubility of C02, will be analyzed in detail in a separate communication.
TRACHEAL VOLUME IN INSECTS Normal Burst
100% Argon
287
Induced Burst C~ib
oE
VI
J
....... Injectlor~
300/Jr m
m
m
~-
Colib
I
of N2 Calibration
F,~ 0.02f 0t_
........
U. ll,l:ill
IIIIIIIIIIIIIIII
il
!
I
........ [.
I
]
Time Ira,n) Fig. 4. Actual recording trace taken from a determination of tracheal volume in a 4.5 g pupa. After a p p a r e n t completion o f the N2 wash-out, a k n o w n a m o u n t o f pure N2 gas was injected into the respirometer which p r o d u c e d the discrete burst in the N2 line. At the end o f the record, pure A r was replaced by an Ar/02 mixture and the measurement, after some cycles for equilibration, was repeated with A r as the gas measured (see Methods). To calibrate the area underneath the induced burst o f N2, both the calibrated sensitivity o f the mass spectrometer, together with the measured flow rate, and the area o f the calibration burst were used. Both gave very similar results. Table 1 presents mean values for VTr determined for 12 specimens o f Hyalophora cecropia in the diapause. The variability in the values is mainly due to a variability between animals, reproducibility for a given specimen being a b o u t 1 ~tl .g-~ The tracheal volume accounts for approximately 5 ~ o f the b o d y volume. The solubility coefficients, calculated from eq. (5), averaged ~<~2= 0.0159 ml STPD. (ml .atm)-~ = 0.000934 m m o l • L- ' . T o r r - l , C
Weight (g) Temperature (°C) Tracheal volume (I.tl •g - 1)
Mean _+SD
Range
5.7 + 1.8 18.8 + 2.5 48.4 + 6.8
3.4- 9.9 15 22.5 39 -59
288
C.R. BRIDGES et ctl. 800 J-
L 600 " J
~ooi o#
•~"
•
~ o
f
i 0 !
0
1
I
2
1
...................
~.___.L
4 6 Weight (g)
.....
L
I
8
10
Fig. 5. Relationship between tracheal volume (VTr) and body weight in 11ya/ophora cecropia. Closed circles. pupae in diapat, se ; open circles, pupae before adult emergence, the number indicating the number of days before hatching. Regression line calculatcd for animals in diapausc [Vrr(lal) = 41 W(g) + 36].
Discussion (a) CRITIQUE OF METI-1ODS An important assumption o f the method is that the spiracles themselves arc gastight during constriction. This allows the exchange o f the external gas medium for another gas mixture o f different composition without dilution or loss o f the inert gas contained within the pupa. To test this assumption recordings were made with a pupa equilibrated in an 80~,,i N • 20~i O= mixture. At constriction the external gas mixture was changed to 100~°~i A r g o n and then returned, betbre the end o f constriction, to the initial mixture. Efflux o f argon from the pupa, monitored during the next flutter/burst period, was found to be zero, indicating that no Ar had leaked into the animal during the constriction. As further evidence lbr gas-tightness at constriction, it was found that there was no significant difference between the length o f the constriction phase when ~ o , , / N ,_" 20j0,~ O, mixture as the either pure inert gas (Ar o r N2) was used or an ,,,,~o ambient atmosphere during constriction. If there were any leakage, the constriction period would be shorter under pure N 2 or argon as 02 would leak out o f the tracheal system thus triggering the flutter/burst period earlier in the O~ free environment. F o r the calculation o f VTr according to eq. (4) it has been assumed that all b o d y tissues are equilibrated with inert gas in the tracheal system. This may be justified as the tracheal system extends into all b o d y tissues (q/~ Miller, 1974).
TRACHEAL VOLUME IN INSECTS
289
It was further assumed that this equilibration is fast enough to allow all inert gas to be removed, during the tracheal wash-out, from the tissues. Without exact knowledge of the geometry of the tracheolar system in relation to the body tissues, this assumption cannot be proved theoretically. However, the fact that in the induced burst the inert gas concentration, unlike the CO, concentration, returned to zero may be taken to indicate that the tissues effectively discharged their inert gases completely. Use of two inert gases of low, yet differing, solubility circumvents the need of assigning an exact value to the tissue solubility coefficient, as this value is dependent on the solvent and its constituents (cf Battino and Clever, 1966). Rather, the ratio of solubility coefficients is needed, which appears to be relatively constant at around ~Ar/~,~:=2.22 in water, oil, blood and other tissues (Altman and Dittmer, 1971 ; Battino and Clever, 1966; Ohta et al., 1979). The solubility coefficients for N: and Ar obtained from eq. (5) may be considered as the effective value for tissue solubility. Calculation of the tissue/water partition coefficient gave an average value of 1.01 (+0.2). This value should be considered a compromise between tissue constituents with solubility coefficients lower (e.g. electrolytes) or higher than those of pure water (e.g. lipids) (cf. Meyer et al., 1980). In fact, tissue water partition coefficients not far from unity have been measured for other tissues. Ohta et al. (1979) reported values of 1.12 and 1.08 for the brain/ blood partition coefficients of N2 and Ar, respectively. Campos Caries et al. (1975) also reported valucs for muscle/water partition coefficients for a number of inert gases which range, for most gases, between 1 and 2.
(b) COMPARISON WITH VALUES IN THE LITERATURE Table 2 gives details of literature data on tracheal volume in Hyalophora pupae that were obtained with different methods. In general the values in the literature are in agreement with those found in this study. Of the previous methods employed, vacuum extraction (Schneiderman and Williams, 1955) leads to an overestimation of the tracheal volume whilst the mechanical pressure used by Buck and Keister TABLE 2 Literature values for tracheal volume in IIyalophora Weight (g)
-
5.0 5.7
Methodof determination
Tracheal volume (p.l .g 1)
Reference
Vacuum extraction Mechanical compression Water compression Inert gas washout
I00 75 50 48
Schneiderman and Williams (1955) Buck and Keister (1958) Kanv,'isher (1966) This study
290
c.R. BRIDGES et aL
(1958) to s q u e e z e o u t the air f r o m the a n i m a l l e a d s to w a t e r - f i l l i n g o f t h e t r a c h e a o r to e v i s c e r a t i o n o f the p u p a e , p r e v e n t i n g m u l t i p l e d e t e r m i n a t i o n s o n the s a m e a n i m a l . K a n w i s h e r (1966) a p p l i e d a p l e t h y s m o g r a p h i c t e c h n i q u e by w h i c h t r a c h e a l v o l u m e is c a l c u l a t e d f r o m the v o l u m e i n c r e m e n t s n e c e s s a r y to c h a n g e the o v e r a l l p r e s s u r e in a c l o s e d w a t e r - f i l l e d s y s t e m c o n t a i n i n g a p u p a . B u c k a n d K e i s t e r (1958) also m a d e m e a s u r e m e n t s o n a s m a l l e r p u p a (0.7-1.1 g) of Agapema
g a l b i n a a n d f o u n d a m e a n t r a c h e a l v o l u m e o f 55 lal . g - ~ (_+ 12.6).
T r a c h e a l v o l u m e has also b e e n d e t e r m i n e d in larval a n d a d u l t stages. K r o g h (1920a) r e p o r t e d a v a l u e o f 14 Ixl • g-~ in the C o s s u s l a r v a w h i c h c o m p a r e s w i t h 6 6 - 9 9 lal • g in D y t i s c u s l a r v a e ( K r o g h , 1920b). N u n o m e
(1944) c a l c u l a t e d f r o m m e a s u r e m e n t s
o f the i n t e r n a l d i m e n s i o n s o f t h e t r a c h e a l system a t r a c h e a l v o l u m e o f 33 lal • g in the s i l k w o r m larva. In the a d u l t c o c k c h a f e r M e l o l o n t h a , D e m o l l (1927) r e p o r t s a t r a c h e a l v o l u m e o f 585 l a l - g ~ .
A v a l u e o f 265 lal . g - ~ c a n be c a l c u l a t e d f r o m
the d a t a o f C l a r k e (1958) for t h e t r a c h e a l v o l u m e o f L o c u s t a m i g r a t o r i a at the fifth instar.
References Altman, P. L. and D. S. Dittmer (1971). Biological Handbooks: Respiration and Circulation. Federation of American Societies for Experimental Biology. Bethesda, MD, U.S.A. Battino, R. and H.L. Clever (1966). Solubility of gases in liquids. Chem. Rev. 66: 395-463. Buck, J. and M. Keister (1955). Cyclic CO..2 release in diapausing Agapema pupae. Biol. Bull. Mar. Biol. Lab., Woods tlolc 109: 144-163. Buck, J. and M. Keister (1958). Cyclic CO 2 release in diapausing pupae. It. Tracheal anatomy, volume and Pco2; blood volume; interburst CO 2 release rate. J. Insect. Physiol. 1 : 327-340. Campos Caries, A., T. Kawashiro and J. Piiper (1975). Solubility of various inert gases in rat skeletal muscle. Pfliigers Arch. 359: 209-218. Clarke, K. U. (1958). Studies on the relationship between changes in the volume of the tracheal system and growth in Locusta migratoria L. Proe. lOth Int. Congr. Entomol. 2:205-211. Demoll, R. (1927). Untersuchungen fiber die Atmung der Insekten. Z. Biol. 87: 8-22. Edwards, G. A. (1953). Respiratory Mechanisms. In: Insect Physiology, edited by K. Roeder. New York, Wiley, pp. 55-96. lto, T. (1954). Discontinuous output of carbon dioxide by undifferentiated Bombyx pupae. Jap. J. Appl. Zool. 19: 98. Kanwisher, J.W. (1966). Tracheal gas dynamics in pupae of the Cecropia silkworm. Biol. Bull. Mar. Biol. Lab., Woods Hole 130: 96-105. Kestler, P. (1971). Die diskontinuierliche Ventilation bei Periplaneta americana L. und anderen Insekten. Ph.D. Thesis, Wi.irzburg, F.R.G. Kestler, P. (1978). Atembewegungen und Gasaustausch bei der Rubeatmung adulter terrestrischer lnsekten. Verh. Dtsch. Zool. Ges. 1978: 269. Krogh, A. (1920a). Studien fiber Trachcenrespiration. II. ~ber Gasdiffusion in den Tracheen. Pfli~g. Arch. Ges. Physiol. 179:95 112. Krogh, A. (1920b). Studien tiber Trachcenrespiration. II1. Die Kombination von mechanischer Ventilation mit Gasdiffusion nach Versuchen an Dytiscus Larven. Pfliig. Arch. Ges. Physiol. 179: 113-120. Miller, P.L. (1974). Respiration-Aerial Gas Transport. In: The Physiology of Insecta, edited by M. Rockstein, Vol. VI, Chapt. 5. London, Academic Press, pp. 346 397.
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Meyer, M., U. Tebbe and J. Piiper (1980). Solubility of inert gases in dog blood and skeletal muscle. Pfliigers Arch. (submitted). Nunome, J. (1944). Studies on the respiration of the silkworm. II. Several measurements in the respiratory organ. Bull. Seric. Exp. Sta. Jap. 12: 41-90. Ohta, Y., A. Ar and L.E. Farhi (1979), Solubility and partition coefficients for gases in rabbit brain and blood. J. Appl. Physiol. 46:1169-1170. Piiper, J., P. Dejours, P. Haab and H. Rahn (1971). Concepts and basic quantities in gas exchange physiology. Respir. Physiol. 13 : 292 304. Punt, A. (1944). De gaswisseling van enkelc bloedzuigende parasieten van warmbloedige dieren ( Cirnex, Rhodinius, Triatorna), Onderz. Physiol. Lab. Rijks-Univ. Utrecht, 8th Ser., 3: 122-141. Scheid, P., M. Meyer and H. Slama (1979). Use of a mass spectrometer to measure lung diffusing capacity for 0 2 and CO by rebreathing stable isotopes at tracer levels. Bull. Eur. Physiopathol. Resp. 15:11P.-14P. Schneiderman, H.A. and C.M. Williams (1955). An experimental analysis of the discontinuous respiration of the Cecropia silkworm. Biol. Bull. Mar. Biol. Lab., Woods Hole 112:106-119. Schneiderman, H. A. (1960). Discontinuous respiration in insects. Role of the spiracles. Biol. Bull. Mar. Biol. lab., Woods Hole 119 : 494-528. Slama, H. and P. Scheid (1975). Electronic feedback circuit for increasing the signal to noise ratio in a mass spectrometer. Pneumonologie 151:247-249. Wigglesworth, V.B. (1965). The Principles of Insect Physiology. 6th ed., London, Methuen.