Carbohydrate and fat as a fuel for insect flight. A comparative study

J. Insect Physiol., 1969, Vol. 15, pp. 353 to 361. Pergamon Press. Printed in Great Britain

CARBOHYDRATE AND FAT AS A FUEL FOR INSECT FLIGHT. A COMPARATIVE STUDY A. M. TH.

BEENAKKERS

Zoological Institute, Catholic University, Driehuixerweg 200, Nijmegen, The Netherlands (Received 14 October 1968) Abstract-From a metabolic point of view, three different types of insect flight muscles can be distinguished: muscles that degrade only carbohydrates, those depending on1.y on fatty acids, and a third type in which both substrates can be oxidized. In the flight muscles examined no quantitative differences in the activities of the citric acid cycle enzymes were observed, although between the various insects during flight differences exist in respiratory rate. On the other hand, strict relations are present between respiration and the glycolytic or t%oxidative pathway in Apis, Locusta, and Actias. The activities of the two enzymes of the glycerophosphate cycle are directly correlated with the glycolytic capacity of the flight muscles. Fatty acid oxidation is supposed to be advantageous for migratory animals and insects that do not feed in the adult stage. Before the onset of migratory flights carbohydrate-metabolic processes in the flight muscles might be responsible for reaching optimal thoracic temperatures. INTRODUCTION

THE IDEA that the energy for muscle contraction is not always derived from anaerobic glycolysis was corroborated by biochemical and physiological investigations on insects. The flight muscles of these animals are often extravagantly supplied with tracheoles (SMITH, 1961; BROSEMERet uZ., 1963 ; WEIS-FOGH, 1964) and densely packed with mitochondria (WILLIAMS and WILLIAMS, 1943 ; VOGELL et al., 1959); during flight a low respiratory quotient was measured in some insects (KROGH and WEIS-FOGH, 1951; ZEBE, 1954) ; sustained flight in locusts is accompanied by fat depletion in the body (WEIS-FOGH, 1952). Therefore, fat metabolic processes in insect muscles could be anticipated. The presence of fatty acid oxidizing enzymes in the flight muscles of Locusta migratoria (ZEBE, 1960; BEENAKKERS,1963), the oxidation of palmitic acid-lJ*C by flight muscle preparaAbbrewiutions: GAPDH = glyceraldehydephosphate dehydrogenase (E.C. 1.2.1.12); LDH = lactate dehydrogenase (E.C. 1 .1.1.27); GDH = glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8); GP-ox = glycerol-3-phosphate oxidase (E.C. 1.1.99.5); HOAD = 3-hydroxyacyl-CoA dehydrogenase (E.C. 1.1.1.35); CAT = camitine acetyltransferase (E.C. 2.3.1.7); CS = citrate synthase (E.C. 4.1.3.7); SDH = succinate dehydrogenase (EC. 1.3.99.1); AK = arginine kinase (E.C. 2.7.3.3.); EDTA = ethylenediaminetetracetic acid; TRA = triethanolamine; Tris = tris (hydroxymethyl) aminomethane. 23

353

A. M.

354

TH. BEENAKKERS

tions of Hyalophora cecropiu (DOMROESE and GILBERT, 1964), and the camitinedependent oxidation of externally supplied fatty acids by locust flight muscle homogenates and mitochondrial suspensions, as measured in Warburg experiments (BEENAKKERS, 1963; BODE and KLINGENBERG, 1965), finally proved the direct participation of fatty acids in the energy production in the flight muscles of at least some insects. In other insects flight performances only depend on carbohydrate utilization (JONGBLOEDand WIERSMA, 1935 ; SACKTOR, 1955). In the present comparative investigation the enzyme activities of various metabolic pathways are estimated in the flight muscles of species of four orders of insects. MATERIALS

AND iUETHODS

Chemicals Acetyl-CoA was synthesized according to the method of SIMON and SHEMIN (1953), and acetoacetyl-CoA according to DRU~CIMOND and STERN (1960). DLAcetylcarnitine was synthesized from DL-carnitine (Fluka A. G., Buchs A. G., Switzerland) following the procedure of BREMER (1962). Enzymes were obtained from Boehringer GmbH, Mannheim. All other chemicals used were commercial products of the highest attainable purity. Animals Some insects, e.g. Locusta migratoria (examined 12 days after the larval-adult ecdysis), Philosamia Cynthia, and Actias selene (both examined 4-5 days after ecdysis), were reared in the laboratory. Others, such as Apis mellij‘ica, Agrotis exclamationis, Amphimallus solstitialis, and Pieris brassicae, were caught in the field during flight. Preparation of muscle extracts In all experiments the enzyme activities were measured in the longitudinal indirect flight muscles. Freshly extirpated muscles were pooled and weighed (100-300 mg fresh wt.); using an Ultra-Turrax homogenizer (Janke-Kunkel) the flight muscles were disrupted in 3 ml O-1 M potassium phosphate buffer (pH 7*3), containing 2 mM sodium EDTA. This mixture was stirred for 30 min and then centrifuged at 14,000g for 12 min. Enzyme activities were estimated in both supematant and sediment, resuspended in 3 ml fresh buffer solution. GP-ox and SDH could only be demonstrated in the sedimental fraction, the other enzymes were mainly present in the supematant fraction. Their activities, given below, represent the summation of activities in both supematant and residue. Assay of enzyme activities Activities were estimated spectrophotometrically at 25°C using an Eppendorf photometer with automatic recording of absorption against time (method of the optical test: WARBURG and CHRISTIAN, 1936).

CARBOHYDRATE

AND FAT AS A FUEL FOR INSECT FLIGHT

355

The assay mixture for each of the measured enzymes was as follows: GAPDH: 0.05 M TRA-HCl buffer (pH 7.6), 5 mM EDTA, 3.3 mM MgSO,, 0.15 mM NADH, 2.4 mM reduced glutathione, l-5 mM ATP, 7 mM 3-phosphoglycerate, 3-phosphoglycero-kinase (200 units); changes in absorption were recorded at 366 mp. LDH: 0.05 M TRA-HCl buffer (pH 7*6), 5 mM EDTA, 0.15 mM NADH, 2.4 mM pyruvate; recording of absorption changes at 366 rnp. GDH: 0.05 M TRA-HCl buffer (pH 7*6), 5 mM EDTA, 0.15 mM NADH, O-4 mM dihydroxyaceton-phosphate; recording at 366 mp. GP-ox: 0.1 M potassium phosphate buffer (pH 7*4), 5 mM EDTA, 8 mM MgSO,, 1 mM KCN, 0.106 cytochrome-c, 20 mM cu-glycerophosphate; absorption changes at 546 rnp. HOhD: O-1 M TRA-HC! buffer (pH 7-O), 5 mM EDTA, 0.45 mM NADH, O-1 mM acetoacetyl-Coil; recording of absorption changes at 366 ml*. CAT: 0.2 M Tris-HCl buffer (pH 8*0), 5 mM EDTA, 5 mM NAD+, 50 mM L-malate, malate dehydrogenase (20 units), citrate synthase (10 units), O-3 mM CO-~-SH, 2 mM acetylcarnitine; time course-absorption curves recorded at 366 rnp. CS: 0.2 M Tris-HCl buffer (pH S-O), 5 mM EDTA, 5 mM NAD+, 50 mM L-malate, malate dehydrogenase (20 units), 0.15 mM acetyl-Co_4; absorption changes recorded at 366 mp. SDH: O-1 M potassium phosphate buffer (pH 7*4), 5 mM EDTA, 1 mM KCN, O-lo; cytochrome-c, 20 mM succinate ; recorded at 546 mp. AK: O-1 M TRA-HCl buffer (pH S-4), 5 mM EDTA, 8 mM MgSO,, 0.15 mM NADH, 3 mM ATP, 25 mM arginine, 0.8 mM phospho-enol pyruvate, pyruvate kinase (3 units), lactate dehydrogenase (5 units); recorded at 366 rnp RESULTS

Before reporting on the results of our activity measurements, it is important to evaluate the meaning of these enzyme activities. The latter were obtained under optimal conditions, which will be different from the actual conditions in viuo. Moreover, even within one cell we do not know exactly to what degree, for instance, the pH varies between the subcellular compartments. However, since our experiments were performed with the same kind of tissue, we may assume that the in oivo conditions in each muscle examined will differ comparably from the optimal conditions. As absolute enzyme activities are less important, a comparison of the activities of a particular enzyme in different muscles will give a better understanding of the role played by this enzyme in the metabolic system. The enzymes, measured in this experimental series, are chosen in such a way that the relative contribution of the main catabolic processes, dealing with energy generation, can be estimated in each muscle (cf. PETTE, 1965: constant proportion groups). Thus we measured the activities of GAPDH and LDH (glycolytic pathway), CAT and HOAD (fatty acid transport and oxidation), CS and SDH (citric acid cycle), GDH and GP-ox (glycerophosphate cycle), and in some muscles also AK.

il.

356

M.

TH. BEZNAICXERS

Fig. 1 shows the activities of these enzymes in the flight muscles of a number of insects. The activities, expressed as pmoles substrate-conversion/g fresh weight per hr, are plotted in a logarithmic scale. The activities of CS and SDH in all muscles examined are of the same ratio (Table l), but even more important may be the fact that the absolute activities are about the same in all these muscles (CS rl: 10,000, SDH 2 600 pmoles/g fresh weight per hr). The activities of LDH are very low in comparison to LDH in vertebrate heart and skeletal muscles (DELBR~CK et al., 1959). Locusta

IOrthoptcral

Pieris

Agtotis

Philosamia

I Lepidoptenl

llcpidoptcral

I Lepidoptcra

Actias

I 1Lepidopter,

al

I

AK

B

CA1

GPO"

son

GPox

SDd

SDH

Sl

LGH

FIG.

1.

Enzyme

patterns in the indirect flight muscles of seven insect species. Activities are plotted in a logarithmic scale.

The ratio between the activities of GDH and GP-ox is almost the same in all flight muscles (in Amphimallus only GDH activity has been measured), demonstrating their close relationship (Table 1). The relationship between these enzymes of the glycerophosphate cycle and the activities of the glycolytic enzymes is of great interest. Table 1 indicates that, independent of the absolute activities of the enzymes concerned, the ratio GAPDH/GDH offers a striking resemblance in the muscles of these various insects (the flight muscles of the honey-bee show a somewhat higher figure, but still in the same order of magnitude).

357

CARBOHYDIL4TJZ AND FAT AS A FUEL FOR INSECT FLIGHT TABLE I-RELATION

OF ENZYME ACTIVITIES IN THE FLIGHT MUSCLES OF VARIOUS INSECTS*

Apis Citric acid cycle CS SDH Glycerophosphate cycle GDH GP-ox Glycolytic chain GAPDH R-Oxidative pathway HO_4D Ratio : CS/SDH GDH/GP-ox GAPDH/GDH GAPDH/HOAD

Amphimallus

Locusta

12,000 920

10,840 -

9600

16,000 1050

22,340 -

10,600 760

82,000

37,000

16,600

50

260

13.0 15.2 5.1 -

-

1.7

690

5400

13.9 14.0 1.6 3-l

Pieris

Agrotis

9300 590

11,000 570

8100 490

Philosamia

Actias

10,000 570

12,000 630

10,000 600

1600 100

2000 150

12,800

16,600

2500

3000

4300

23,000

9500

25,000

15.8 16.6 1.6 3.0

19.2 16.6 1.6 0.7

17.6 16.0 1.6 0.2

19.0 13.4 1.5 0.1

* Activities expressed as pmoles/g fresh weight per hr.

In muscles with measurable CAT activities a consistent ratio was demonstrated between these activities and the activities of HOAD, which again indicates the close co-operation of these two enzymes in muscular tissues (BEENAKKERS et al., 1967). A comparison of the various flight muscles with respect to glycolytic and fatty acid oxidative capacities clearly demonstrates the great variety in metabolic pattern of these muscles. In the flight muscles of both Apis and Amphimallus HOAD activities are very low (also in comparison to non-muscular tissues as rat liver and kidney; PETTE, 1965), GAPDH activities being high. The reverse situation exists in two species of the Lepidoptera: Philosamiu and Act&s. In their flight muscles the ratio GAPDH/HOAD is about 0.1 to O-2 (Table 1). In the muscles of other insects examined this ratio is higher: O-7 in Agrotis and 2 to 3 in Lvcusta and Pieris, indicating that the r81e of neither glycolysis nor p-oxidation is as absolute as in the muscles already mentioned. The activity of AK in the muscles of Locusta, Philosamia, and Pieris is high and comparable to creatine-kinase activity in various vertebrate muscles (JACOBS et al., 1964). DISCUSSION Although it has been made clear that the processes involved in energy production in insect tissues are qualitatively alike and comparable to analogous processes in vertebrate organs, studies about the quantitative participation of the various metabolic pathways in insect muscles are relatively scarce.

358

A. Vi. TH. BEENAKKER~

It has been stated repeatedly that regarding substrate utilization three types of insects can be distinguished : insects deriving their energy from carbohydrates, those depending on lipids, and a third type which can use both substrates (ZEBE and M&HAN, 1957; cf. SACKTOR, 1965). As far as flight muscles are concerned, a direct proof of fatty acid oxidation was obtained in Locusta m@ztoriu (BEENAKKERS,1963 ; BODE and KLINGENBERG, 1965) and in Hyalophora cecropia (DOMROESE and GILBERT, 1964). However, so far no studies have been dealing with a comparison of the main metabolic pathways in the flight muscles of members of different insect orders. The results of our experiments clearly indicate the great variety in metabolic Energy generation during flight in Apis and pattern in insect flight muscles. Amphimallus depends exclusively on the breakdown of carbohydrates, in Philosamia and Actias only on fatty acid oxidation. GAPDH activity in the muscles of the lastmentioned insects is very low. -4ctually it is the lowest ever measured in both vertebrate and insect tissues. Moreover, it may be assumed that this glycolytic capacity is only meant to supply the mitochondria with essential citric acid cycle intermediates. Between these two extremes a third type of flight muscle can be distinguished, in which both metabolic pathways exist in a ratio determined by the species. It is surprising to see that the activity of CS (and consequently also the activity of SDH, as these two enzymes are members of the same constant proportion group) has practically the same value in all muscles examined. In these highly aerobic tissues we would expect some consistent relation between the activity of the citric acid cycle and the amount of oxygen used during flight. In the honey-bee oxygen uptake during flight is about 100,000 $/g animal per hr (JONGBLOEDand WIE&MA, 1935). Since the flight muscles account for about 12 per cent of the total fresh weight, and since during flight most of the oxygen will be consumed by these muscles, the respiratory rate can be calculated to be 13,800 pi/g flight muscle per min. In the locust Schistocerca, KROGH and WEIS-FOGH (1951) measured an oxygen uptake of maximum 30 l/kg animal per hr in flying animals. Assuming a similar rate in Locusta, the respiration in this insect (the flight muscle weight being 18 per cent of the total body weight) is 2770 ,LLI0,/g flight muscle per min. Even without taking into consideration a difference in the respiratory quotient between these animals, the respiratory rate differs by a factor of 5, although the citric acid cycle enzymes have the same activity. In Philosamia and Actias the flight muscle activity mainly depends on fatty acid oxidation. Nevertheless, in Actias HOAD activity is more than twice as high as CS activity, but in Philosamia the ratio HOADICS is only about O-95. Ahhough it might be possible that certain other biochemical pathways, not taken into consideration in our enzyme patterns, are responsible for these discrepancies, it might also indicate that the total mitochondrial content of the distinct flight muscles is more or less independent of the ultimate oxygen need of the flight muscle. CS, SDH, and the other citric acid cycle enzymes may be considered as constitutive enzymes, synthesized to the same extent with the other mitochondrial structures. If we can consider the enzymes of

CARBOHYDRATEAND FAT AS A FURL FOR INSECT FLIGHT

359

the glycolytic and /3-oxidative pathway as adaptive enzymes, the activities of GAPDH and HOAD should be related in some way to the respiratory rate. The locust is capable of metabolizing both carbohydrates and fatty acids during flight. WEIS-FOGH (1952) demonstrated that in the first period of prolonged flight the energy is derived from carbohydrates, whereas later lipids act as substrate. As in both periods the oxygen uptake can reach values as indicated above, both GAPDH and HOAD activities must be capable of meeting this respiratory rate. Table 2 shows the ratio between G_4PDH activities of the flight muscles of the TABLE 2-GAF’DH AND HOAD ACTIVITIES IN THE FLIGHT MUSCLES OF THREE INSECTS COMPAREDWITH THE RESPIRATORYRATS OF THE ANIMALS DURING FLIGHT

GAPDH

HOAD

(pmoles/g muscle

*moles/g muscle per hr)

per hr) Locust Honey-bee Moth Ratio : Honey-bee/locust Moth/locust For further explanation: * Expected.

16,000 82,000 -

4.9 -

5400 25,000

4.6

Oxygen uptake during flight @l/g animal per mm) 2770 13,800 12,740*

5.0 4.6*

see text.

honey-bee and locust. This ratio is almost equal to the ratio of oxygen uptake in both animals during intensive flight. Comparing HOAD activities in the flight muscles of Actias and Locusta, a ratio of 4.6 is obtained. If the proportion in respiratory rate is the same, we would anticipate the oxygen consumption in Actias during flight to be 4.6 x 2770 = 12,740 pi/g flight muscle per min. As the flight muscles account for 8.5 per cent of the total body weight, the oxygen uptake will correspond to 1500 pi/g animal per min. This value is very well within the range found by &BE (1954) for several moths (700-1660 pi/g animal per min). Thus the results of these calculations are in correspondence with the view that a strict relation exists between the enzyme activities of the glycolytic respectively fl-oxidative pathway and the respiratory rate in these insect muscles. There seems to be no doubt that the activities of the enzymes GDH and GP-ox are directly correlated with the glycolytic possibilities of the aerobic insect muscles (Table 1). No relation between GDH and enzymes of the fatty acid oxidation cycle can be established. These results are consistent with their importance in the glycerophosphate cycle, in the sense of reoxidation of NADH during carbohydrate degradation (ZEBE et al., 1959; SACKTOR and DICK, 1962). The physiological meaning of the demonstrated differences in substrate

360

A. M. TH. BEENAKKERS

utilization in insects has partly been evaluated by BEEN~KKERS (1965), GILBERT (1967), WEIS-FOGH (1967), and others. In migratory insects, as Locusta, fat has the advantage over carbohydrates, because an isocaloric quantity of fat is less voluminous than the same quantity of carbohydrate. Also, during metabolism lipids produce twice as much water as carbohydrates, which is particularly important in these terrestrial animals. For the same reasons lipids are also more advantageous than other fuel stores in some moths (for instance, Philosamia and Actias), as these insects do not feed in the adult stage. Other species of the Lepidoptera feed in the adult stage, e.g: Pieris and Agrotis. Their relatively high muscular GAPDH activities-in comparison with the other Lepidoptera-may be related to this physiological characteristic. But as Pieris is also capable of prolonged flight (WILLI~IS, 1958; NIELSEN, 1967) and as another species of Agrotis, i.e. Agrotis ipsilon, is well known for migratory flights (WILLIAMS, 1958), it seems quite likely that their muscular enzyme pattern, which is comparable to that of the locust (cf. Table l), is related to their flight abilities. Many migratory insects start their flight only if their thoracic temperatures are sufficiently high (PRINGLE, 1957), and it may be that carbohydrate degradation is important to reach that temperature, probably by wing-vibration. AcknomZedgements-I am indebted to bliss L. M. STOFFELS,Miss M. A. H. G. MEISEN, and Mr. F. N. C. M. KRIJZER for their assistance in the estimation of enzyme activities. REFERENCES BEENAKKEFUA. M. T. (1963) Fatty acid oxidation in insect muscles. Acta physiol. pharmacol. nierl. 12, 332-335. BEENAKKER~A. M. T. (1965) Transport of fatty acids in Locusta migratoria during sustamed flight. J. Insect Ph_vsiol. 11, 879-888. BEEN~UUCERS A. M. T., DEWAIDEJ. H., HENDERSONP. T., and LUTGERHORSTA. (1967) Fatty acid oxidation and some participating enzymes in animal organs. Camp. Biochem. Physiol. 22, 675-682. BODE C. and KLINCEXBERGM. (1965) Die Veratmung von Fettsluren in isolierten Mitochondrien. Biochem. 2. 341, 271-299. BREMERJ. (1962) Carnitine in intermediary metabolism: Reversible acetylation of carnitine by mitochondria. J. biol. Chem. 237, 2228-2231. BROSEMERR. W., VOGELLW., und B~CHER T. (1963) Morphologische und enzymatische Muster bei der Entwicklung indirekter Flugmuskeln von Locusta migratoria. Biochem. 2. 338, 854-910. DELBRUCK A., SCHIMASSEKH., BARTSCH K., and BOCHER T. (1959) Enzym-Verteilungsmuster in einigen Organen und in experimentellen Tumoren der Ratte und der Maus. Biochem. Z. 331, 297-311. DOMROESEK. A. and GILBERT L. I. (1964) Th e role of lipid in adult development and flight muscle metabolism in Hyalophora cecropia. J. exp. Biol. 41, 573-590. DRUMMONDG. I. and STERNJ. R. (1960) Enzymes of ketone body metabolism-II. J. biol. Chem. 235, 318-325. GILBERT L. I. (1967) Lipid metabolism and function in insects. Adz. Insect Physiol. 4, 69-211. JACOBSH., HELDT H. W., and KLINGENBERGM. (1964) High activity of creatine kinase in mitochondria from muscle and brain and evidence for a separate mitochondrial isoenzyme of creatine kinase. Biochem. biophys. Res. Commun. 16, 516-521.

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JONGBLOED J. and WIERSMAC. A. G. (1935) Der StoffwechseI der Honigbiene w&rend des Fluges. 2. verg2. Physiol. 21, 519-533. KROCH A. and WEIS-FOCH T. (1951) The respiratory exchange of the desert locust (Schistocerca gregaria) before, during and after flight. J. exp. Biol. 28, 344-357. NIELSENE. T. (1967) Insekten auf Reisen. Springer, Berlin. PFXTE D. (1965) Plan und Muster im zellularen Stofhvechsel. Naturwissenschaften 22, 597-616. PRINCLEJ. W. S. (1957) Insect FZight. Cambridge University Press, London. SACKTORB. (1955) Cell structure and the metabolism of insect flight muscle. g. biophys. biochem. Cytol. I, 29-46. SACKTORB. (1965) Energetics and respiratory metabolism of muscular contraction. In The Physiology qf Insecta (Ed. by ROCKSTEINM. ) 2, 483-580. Academic Press, New York. SACKTORB. and DICK A. (1962) Pathways of hydrogen transport in the oxidation of extramitochondrial reduced diphosphopyridine nucleotide in flight muscle. J. biol. Chem. 237, 3259-3263. SIPHONE. J. and SHEMIN D. (1953) The preparation of S-succinyl coenzyme A. J. Am. them. Sot. 75, 2520. SMITH D. S. (1961) The structure of insect fibrillar flight muscle. r. biophys. biochem. Cytol. 10,123-158. VOGELLW., BISHAIF. R., B&HER T., KLINGENBERG M., PETTED., and ZEBE E. C. (1959) Ober strukturelle und enzymatische Muster in Muskeln von Locusta migratoria. Biochem. Z. 332, 81-117. WARBURG0. and CHRISTIANW. (1936) Pyridin, der wasserstoffiibertragende Bestandteil von G&-ungsfermenten (Pyridin-Nucleotide). Biochem. 2. 287, 291-328. WEIS-FOGH T. (1952) Fat combustion and metabolic rate of flying locusts (Schistocerca gregaria Forsk.). Phil. Trans. R. Sot. (B) 237, l-36. WEIS-FOGH T. (1964) Diffusion in insect wing muscle, the most active tissue known. J. e@. Biol. 41, 229-256. WEIS-FOGH T. (1967) Metabolism and weight economy in migrating animals, particularly birds and insects. In Insects and Physiology (Ed. by BEAMENTJ. W. I,. and TREHERNE J. E.), pp. 143-159. Oliver & Boyd, Edinburgh. WILLIAMS C. B. (1958) Insect Migration. Collins, London. WILLIAMS C. M. and WILLIAMS M. V. (1943) The flight muscles of Drosophila repleta. J. Morph. 72, 589-599. ZEBE E. C. (1954) uber den Stoffwechsel der Lepidopteren. 2. oergl. Physiol. 36, 290-317. ZEBE E. C. (1960) Condensing enzyme und /3-keto-acylthiolase in verschiedenen Muskeln. Biochem. 2. 332, 328-332. ZEBE E. C., DELBR~CKA., und BUCHERTH. (1959) Uber den Glycerin-l-Phosphat-Cyklus in den Flugmuskeln von Locusta migratoria. Biochem. Z. 331, 254-272. ZEBE E. C. and MCSHAN W. H. (1957) Lactic and n-glycerophosphate dehydrogenases in insects. J. gen. Physiol. 40, 779-790.