The Role of Lipid in Adult Development and Flight-Muscle Metabolism in Hyalophora Cecropia^*

Lipid is known to be an important energy reserve for adult development in holo-metabolous insects (cf. Scoggin & Tauber, 1950) and the direct fuel for flight in some Orthoptera (Weis-Fogh, 1952) and a few Lepidoptera that feed as adults (Zebe, 1954). The biochemical aspects of lipid metabolism in insects have not been thoroughly studied and are obscure in most respects. Earlier work with flight-muscle preparations revealed that acetate was actively oxidized but butyrate and octanoate were not (Barron & Tahmisian, 1948; Rees, 1954). More recently, individual enzymes involved in fatty acid oxidation have been reported in insect tissues. Fatty acid activating enzymes are now known to exist in the fat body of the cockroach (Periplaneta americana) and several species of Lepidoptera, and condensing enzyme plus β-keto-acyl-thiolase in mitochondria from locust flight muscle (Nelson, 1958; Siakotos, 1959; Zebe, 1960). The only fatty acid oxidizing system described in insects is a particulate fraction from the thoracic muscle of the desert locust (Meyer, Preiss & Bauer, 1960).

The work of Kozhantshikov (1938), Zebe (1954) and Gilbert & Schneiderman (1961) has suggested the importance of lipid as a substrate for respiratory metabolism in various species of Lepidoptera. However, the oxidative pathway of lipid catabolism in this group of insects has not been described. The experiments reported herein describe the role of lipid in the pupal-adult transformation of the American silkworm, Hyalophora cecropia, and a fatty acid oxidation system in the flight muscle of the adult moth. Since many of the insect hormones appear to be lipid (cf. Gilbert, 1963), it is hoped that studies on the metabolism of lipids in insects may eventually lead to the elucidation of the pathways of hormone synthesis and breakdown.

The experimental animals were giant silkmoths, Hyalophora cecropia, obtained as pupae from dealers or reared from eggs on wild cherry trees covered with large nylon nets.

From a large number of pupae that had been chilled at 6° C. for 6 months, 27 males and 27 females within the weight range of 4 · 81 − 5 · 24 g. were placed at 25 ° C. to initiate adult development. The subsequent developmental stages were determined according to Schneiderman & Williams (1954). At predetermined developmental stages three animals of each sex were weighed and then frozen at − 20 ° C. for subsequent determinations of lipid, glycogen, and other carbohydrate.

Lipids were extracted with redistilled 95 % ethanol and ethyl ether. The ether was peroxide-free ‘ether for fat extraction’ (Fisher Chemicals) stored over metallic sodium and distilled prior to use. Alternatively, we used reagent grade, anhydrous, peroxidefree ether (Fisher Chemicals) within 24 hr. after opening a new container. The animals to be extracted were added to an equal volume of 95 % ethanol in an 80 ml. stainless steel container and homogenized at 8000 rev./min. for 3 min. in a Lourdes Multimixer. The homogenate was transferred quantitatively with several washings of 95 % alcohol into 40 ml. polypropylene tubes and centrifuged to obtain a clear supernatant. After centrifugation the supernatant was poured into a separatory funnel through fat-free filter-paper. The residue in the centrifuge tube was re-extracted several times with fresh ether until the supernatant was colourless after centrifugation. These ether extracts were added to the alcohol extract in the separatory funnel. The pooled extracts were then washed four times with an equal volume of 0 · 04% CaCl₂. The washed lipid extract was transferred with washings of ether from the separatory funnel to a round-bottomed flask and the solvent was evaporated under reduced pressure at 40 ° C. in a Flash Evaporator. Any water remaining in the lipid extract after evaporation of the solvent was removed by several washes with absolute ethanol and the whole was brought to dryness in a Flash Evaporator. The extracted lipid was then transferred to a tared vial with portions of anhydrous ether. This ether was removed under a stream of nitrogen and the vial was placed in a vacuum oven at 50° C. for 60 min. to remove all traces of solvent. After removal from the oven the vial was capped, allowed to cool to room temperature, and weighed.

The washings from the lipid extraction were collected in a volumetric flask and brought to 500 ml. by the addition of distilled water. An aliquot of this was analysed for anthrone-positive substances and this value was taken as the non-glycogen carbohydrate value.

The glycogen which was precipitated in the initial homogenization of the animals in 95 % ethanol was determined with anthrone reagent after isolation of the glycogen by the method of Good, Kramer & Somogyi (1933).

Thirteen male pupae were selected from a large number of animals that had been chilled at 6° C. for 6 months. The fresh weight of each animal selected fell within a range of 4 · 73 − 4 · 87 g. with an average of 4 · 78 g. These animals were incubated at 25 ° C. and allowed to complete adult development. After eclosion, nine animals were divided into three groups. Three animals were frozen at − 20 ° C. after wing expansion and excretion of the meconium. Three animals were placed in separate covered cardboard cartons (17 cm. high × 17 cm. diameter) and allowed to carry on normal activity at room temperature. A third group of three animals was treated similarly to the latter group except that they were prevented from flying by bringing the wings together over the dorsum of the animal and stapling them together. The animals of the second and third groups were sacificed at the end of five days and their lipid content was determined. The extraction and determination of total lipid was as described above except that homogenization was conducted in a mixture of 3:1 (v/v) distilled ethanol and peroxide-free ethyl ether.

The R.Q. of developing pupae was determined by a modification of the method in Umbreit, Burris & Staufer (1957) which utilizes the direct method for measuring CO₂ evolution. Gas exchange was measured at 25 ° C. in large Warburg flasks having a gas volume of approximately 60 ml. Readings were taken for an initial 3 hour period with alkali in the flask to determine Q_O2. This was followed immediately by another 3 hr. period of readings with acid in the flask to permit the calculation of the CO₂ evolved during a 3 hr. period. Valid R.Q. determinations cannot be made with this method prior to the 3rd day of adult development because of the discontinuous release of CO₂ by pupae (Schneiderman & Williams, 1955).

The endogenous R.Q. of muscle homogenates was determined by the direct method for measuring CO₂ described in Umbreit et al. (1957).

All labelled compounds were obtained from the California Corporation for Biochemical Research, Los Angeles, California. Soluble compounds were diluted to appropriate levels of radioactivity in distilled water or buffer. Insoluble palmitic acid was prepared as the albumin complex according to Masironi & Depocas (1961).

The oxidation of ¹⁴C-labelled substrates was measured in Warburg vessels using Hydroxide of Hyamine 10 − x (Reg. trademark of Rohm and Haas, Inc.) to absorb the CO₂. Hyamine was added to the Warburg flasks in small glass vials that could be dropped into the centre well of a Warburg vessel and supported there in a vertical position.

¹⁴C-labelled substrates were added to the main compartment and 0 · 5 ml. of 8 N H₂SO₄ to the side arm of Warburg vessels held in crushed ice. To initiate the experiment an aliquot of tissue homogenate was pipetted into the main compartment and a vial, previously filled with the necessary amount of Hyamine (usually 0 · 3 ml.), was placed in the centre well with forceps. The vessels were attached to manometers and placed in a 25 ° C. water bath with shaking (104 oscillations/min.). At the termination of the incubation period the acid from the side arm was tipped in to stop the reaction and shaking was continued for an additional period of 1 o min. for absorption of any CO₂ that was released from the reaction medium. After the collection of CO₂ was completed the vial was removed from the centre well and placed in the scintillator solution in a counting vial. It was previously determined that the presence of the glass vial in the counting vial had no effect on the efficiency of the counting system.

All counting procedures utilized a Packard Tri-Carb liquid scintillation spectrometer, with a toluene: 2,5-diphenyloxazolet,4-bis-2-(5-phenyloxazole)-benzene:(1000 ml. : 3 g. : 100 mg.) scintillator solution, in low-potassium glass counting vials. Counting was done for a sufficient time to give a count within a 2 % error at the 95 % level of confidence at the measured background rate of 12 − 22 counts/min. The recorded counts on the instrument were converted to actual counts/min. by correcting for background, efficiency of the instrument and for quenching when necessary.

Flight-muscle sarcosomes (mitochondria) were prepared according to Sacktor (1953).

Dialysis was conducted at 4 ° C. in seamless cellulose tubing with an inflated diameter of 0 · 75 in. The dialysis sac was placed in a 400 ml. beaker containing distilled water which was agitated by a Teflon-covered bar driven by a magnetic stirrer. The distilled water was replaced every 20 min. during dialysis.

Glycogen and other carbohydrates were determined by the anthrone method of Seifter, Dayton, Novic & Muntwyler (1950).

Activated fatty acids were trapped as their hydroxamic acid derivatives according to the method of Kornberg & Pricer (1953). The incubation medium was the same except that the reaction mixture did not include cysteine, since addition did not influence the result. The enzyme source was an aqueous extract of flight-muscle homogenate dialysed against distilled water for 3 hr. Incubation was at 25 ° C. for 60 min.

Acid-insoluble dry-weight was determined by the method of Werkheiser & Bartley (1957).

The total lipid and carbohydrate content of males and females of H. cecropia during adult development and adult life is seen in Table 1. The most meaningful interpretation of the data is obtained by comparison of the quantity of extracted materials in terms of absolute amounts. The animals used in this determination were originally selected within a narrow weight range to permit this type of analysis. Analysis of the data on a fresh-weight basis is complicated by the constant water loss during development and the severe weight loss at ecdysis. Thus in the male the amount of lipid on a fresh-weight basis would show an increase from the 20th day of development to the 2nd day of adult life whereas in absolute amounts it can be seen that the amount of lipid remains essentially constant.

The data in Table 1 reveal that the pattern of lipid utilization during development is related to the sex of the animal. In the female both total lipid and carbohydrate fall progressively during adult development. During adult life the lipid and non-glycogen carbohydrate continue to fall whereas the glycogen content increases over the amount present on day 20 of adult development. Since the extraction of adult, females included the eggs, it is possible that this increase in glycogen represents the synthesis of egg glycogen during this period.

In the male the lipid content remains essentially constant throughout adult development while total carbohydrate progressively falls. In the adult, total lipid declines precipitously between day 2 and day 5 and continues to decrease thereafter. Total carbohydrate in the adult male falls to insignificant values and it is probable that very little energy for metabolism is available from this source during adult life.

In both males and females there is an apparent net increase in total lipid between days 2 and 8 of adult development, suggesting a conversion of other material to lipid during this period.

The sexual dimorphism in ether-extractable lipid in H. cecropia previously reported by Gilbert & Schneiderman (1961) is also apparent from the data in Table 1. At day 2 of adult development the males contain 36 % more lipid on an absolute basis than the females and this dimorphism continues to express itself throughout the course of development.

To explore lipid utilization further, R.Q.’S were determined during the pupal-adult transformation. The results of those determinations show a difference in pattern between males and females. In the female the R.Q. decreases from higher values early in development to values between 0 · 75 and 0 · 80 shortly after the 1st week of develop-ment (Fig. 1). The R.Q. remains at this characteristic level throughout the rest of development and in adult life. In terms of the role of lipid as an energy source for development the R.Q. pattern suggests that after the 1st week of development lipid provides a greater portion of the energy expended in the female. The early and continuous decline in total lipid previously cited during development agrees with this interpretation.

In the male, the R.Q. is maintained for the most part at a level well above 0 · 80 until the 17th day of development, at which time it falls to levels indicative of greater lipid utilization (Fig. 2). The R.Q. persists at this lower value during the remainder of adult development and in adult life. This drop in R.Q. characteristically occurred simultaneously with the appearance of the large dark pigmented wing spot on the 17th day of development. This R.Q. pattern as well as the data previously cited on changes in total lipid content indicates that the male uses more of the non-lipid substrates for energy during adult development with a conservation of the lipid stores for use during adult life.

In the determination of the lipid content of males during development and adult life it was observed that the male shows a sudden increase in the utilization of total lipid between day 2 and day 5 of adult life, whereas prior to this time the lipid content remains essentially constant (Table 1). The value of the R.Q. of resting metabolism during this same period of adult life also indicated that lipid stores were being expended for energy (Fig. 2). This sudden drop in lipid was thought to be due to the rather abrupt assumption of flight activity in young adults.

The effect of limiting flight activity on the lipid content of young male moths is seen in Table 2. The initial selection of the experimental animals within a narrow weight range permits analysis of the data in terms of the absolute weight of the extracted lipid. The data reveal that the prevention of flight results in a conservation of lipid. The decrease of total lipid in the flightless animals represents a conservation of 57% of the amount used in the flying animals when compared to the control. These results support the suggestion that the onset of flight activity is the major causative factor in the sudden decrease in lipid of male moths during the early days of adult life, and indicate the importance of lipid in supporting the energy requirement of flight-muscle metabolism.

The adult moths do not possess functional mouth parts and thus cannot feed. Of necessity the metabolic substrates of the adult are limited to those available after the requirements of metamorphosis have been satisfied. As indicated in Table 1 the carbohydrate store of the adult male is exhausted during the pupal-adult transformation and the R.Q. studies indicate that the animals utilize lipid during this stage. To determine whether the flight muscles still retain the ability to oxidize carbohydrates, exogenous carbohydrate was added to flight-muscle homogenate. The effects of adding exogenous glucose are seen in Table 3. The addition of glucose had no observable effect on either the O₂ uptake or the R.Q. and indicated the inability of muscle tissue to oxidize directly glucose supplied exogenously. The stability of the R.Q. value also indicates there is no significant conversion of added glucose to lipid. If such a conversion was proceeding the R.Q. would be expected to show considerable increase.

Additional information concerning substrate preference for flight-muscle metabolism was obtained by using various ¹⁴C-labelled compounds and measuring the production of ¹⁴CO₂. The results of this determination are seen in Table 4. The 2-, 4-, 10- and 16-carbon fatty acids are all actively oxidized. It appears that the longer chain fatty acids are preferred as seen by the complete oxidation of the palmitic acid and about 50 % oxidation of the shorter chain fatty acids. However, it is possible that the necessity of adding the water-insoluble palmitic acid as the albumin complex may make it more readily available to the fatty acid oxidizing enzymes. Thus the apparent speed of oxidation of this long-chain fatty acid may only reflect its greater availability when compared to the shorter chained fatty acids. The citric acid cycle intermediate, succinate, was also oxidized to a degree comparable to that of the shorter chain fatty acids. Glucose, pyruvate, and glycerol are oxidized very weakly. The amino acid alanine, while not as completely oxidized as the fatty acids, is oxidized to a greater extent than the carbohydrate derivatives.

The results cited above demonstrate the almost exclusive lipid nature of flightmuscle metabolism. Since lipid is the available substrate and the apparent substrate of choice, the endogenous respiration of flight muscle is essentially a reflexion of fatty acid oxidation. We can thus assume that procedures designed specifically to modify the rate of endogenous respiration are essentially doing so by their effect on fatty acid oxidation. This was used to advantage in subsequent studies designed to investigate the nature of fatty acid oxidation in flight muscle. Metab olic inhibitors were added to homogenates to investigate the pathway of fatty acid oxidation.

The effects of the addition of various metabolic inhibitors on the endogenous respiration of H. cecropia flight-muscle homogenates are revealed in Table 5. All the substances tested were potent inhibitors of flight-muscle respiration. The inhibition brought about by 2,4-dinitrophenol, which exerts its effect by uncoupling oxidative phosphorylation, was not reversed by the addition of ATP to a concentration of 6 · 10 ⁻³ M.

Flight-muscle homogenates were dialysed against distilled water to determine if removal of any dialysable materials depressed the endogenous respiratory rate. If such was the case the restoration of the respiratory rate by the addition of known cofactors would indicate the requirements of the enzyme systems for oxidation of fatty acids. Preliminary studies indicated that muscle homogenates lost none of their endogenous lipid during a 3 hr. dialysis period. Dialysis of muscle homogenates inhibited respiration and this inhibition could be reversed by the addition of a mixture of known co-factors to the dialysed homogenate (Table 6). The addition of the cofactor mixture increased the respiration of the dialysed homogenate to 95 % of that of the undialysed control. This increase in respiration cannot be accounted for by the presence of citrate as a respiratory substrate in the co-factor mixture but represents an actual stimulation of oxidation of the endogenous substrate. If the theoretical O₂ uptake (100 · 8 μ.) for the complete oxidation of the added citrate is subtracted from the total, the stimulation of respiration by the added co-factor mixture is still 65 % that of the undialysed control.

Which of the added co-factors, if not all, were required to restore endogenous respiration of dialysed homogenates was determined by the deletion of single co-factors from the complete medium. These results (Table 7) show that ATP, magnesium, and citrate are required to restore the endogenous respiration of dialysed homogenates to the level of the non-dialysed control.

As previously noted, we equated the endogenous respiration of flight muscle with fatty acid oxidation. The finding that ATP and magnesium are necessary for the restoration of endogenous respiration in dialysed muscle homogenates indicates that these co-factors may be essential for fatty acid oxidation. The stimulation of respiration by citrate may be due to some extent to its oxidation as an added substrate or perhaps to its stimulation of oxidation of endogenous lipid by priming the citric acid cycle. To test these two alternatives the oxidation of palmitic acid-1-¹⁴C by dialysed flight-muscle homogenate was measured in the presence of added co-factors. The results (Table 8) clearly indicate that added citrate stimulates fatty acid oxidation and verify the need for ATP and magnesium.

The stimulatory effect of citrate is not specific and other citric acid cycle intermediates can stimulate fatty acid oxidation (Table 9). Glutamic acid, which can readily enter the citric acid cycle by transamination, is also an effective stimulator of fatty acid oxidation. The fact that glucose-1-phosphate is ineffective is in keeping with the previously cited observation that glycolytic products are not readily metabolized by the muscle enzyme systems. Butyric acid was likewise unable effectively to stimulate fatty acid oxidation.

The pH optimum for the oxidation of palmitic acid-1-¹⁴C by dialysed flight-muscle homogenate with added co-factors is 6 · 4 (Fig. 3). This pH optimum coincides with that of endogenous respiration of undialysed flight-muscle homogenate (Fig. 3).

Table 5 shows that malonate is an effective inhibitor of endogenous flight-muscle respiration. This observation, together with the demonstration that citric acid cycle intermediates stimulate fatty acid oxidation, indicates that the pathway of fatty acid oxidation is via the citric acid cycle. Additional evidence for the participation of this pathway was obtained by measuring the production of ¹⁴CO₂ from a fatty acid labelled in different numbered carbons. Citrate is a symmetrical compound but biologically it is handled as if it were asymmetrical (White, Handler, Smith & Stetten, 1959). Because of the non-randomization of citrate the carboxyl carbon of added acetate will appear in the CO₂ after two turns of the citric acid cycle whereas the second carbon will require three turns. If fatty acids enter the citric acid cycle as two-carbon fragments a fatty acid labelled in an even carbon will show a time lag in ¹⁴CO₂ production compared to a fatty acid labelled in an odd carbon. With this in mind flight-muscle homogenates were incubated with either butyrate-1-¹⁴C or butyr-ate-2-¹⁴C and the quantity of ¹⁴CO₂ was determined after 30 min. (Table 10). In 30 min. the butyrate labelled in the odd carbon had produced about 50% more ¹⁴CO₂ than the even-labelled butyrate, indicating that butyrate is oxidized via the citric acid cycle.

The ability of sarcosomes isolated from flight muscle of H. cecropia to oxidize labelled fatty acids was investigated. This work is preliminary to further studies with these subcellular particles. We found that sarcosomes isolated by differential centrifugation required the addition of exogenous co-factors for fatty acid oxidation. Table 11 indicates that the co-factor requirements are similar to those necessary for the restoration of endogenous respiration in dialysed muscle homogenates, e.g. ATP, magnesium, and citric acid cycle intermediate. Sarcosomes from thoracic muscle of male S. cynthia moths had the same co-factor requirements as found in H. cecropia.

It has been demonstrated in tissues from organisms other than insects that the oxidation of fatty acids requires prior activation resulting in the formation of fatty-acyl-CoA. Considering the high rate of fatty acid oxidation in flight muscle, we assayed this tissue for fatty acid activating enzymes. Hydroxamic acid derivatives of fatty acid were accumulated in the reaction mixture using octanoate as substrate and a dialysed aqueous extract of flight-muscle homogenate as the enzyme source. In a typical determination the experimental tube had an accumulation of 0 · 59 μ moles of hydroxamic acid while the control showed no hydroxamic acid formation. Preliminary determinations had shown that the formation of hydroxamic acid derivatives under these conditions was dependent on the addition of CoA, ATP, and fatty acid. Magnesium was not essential but was stimulatory.

The results reported in Table 1 on the changes in total lipid content of male H. cecropia confirm those reported by Gilbert & Schneiderman (1961) on the lipid content of abdomens of the same species during various stages of adult development. During the pupal-adult transformation there is a definite conservation of lipid. This finding is supported by the continuous drop in total carbohydrate and the relatively high R.Q. during most of this period. The same authors report a drop in total lipid in the female during the pupal-adult transformation on the basis of determining the lipid content of pupae and adults. The actual extraction of female lipid at various stages of development (Table 1) confirms this observation. The continuous drop in total female lipid indicates that lipid is utilized throughout the period of pupal-adult transformation. This finding is supported by the low R.Q. characteristic of female metabolism during most of this period. The above suggests that females catabolize appreciable lipid to underwrite adult development whereas males utilize other substrates and conserve their lipid store for adult life.

The data in Table 1 and Figs. 1 and 2 indicate that lipid is the most important energy-supplying substrate for both sexes during adult life. This is apparent from the almost undetectable carbohydrate content, the continual decrease in total lipid content, and the low R.Q. recorded during this period. In the female the lipid content, which drops continuously during development, decreases even further during adult life. The low R.Q. values recorded for adult females are also indicative of lipid oxidation. In male moths the lipid content that has remained essentially constant during pupal-adult development drops precipitously between days 2 and 5 and continues to drop thereafter. The low R.Q. values recorded for adult males also show an intense lipid utilization.

In summary, it appears that the female utilizes her lipid stores continuously throughout pupal-adult development and during adult life. In the male, on the other hand, the pupal-adult transformation is characterized by a lipid-sparing metabolism. In the adult, however, lipid serves as the major energy source. This sexual difference in the pattern of lipid utilization has been observed in other species of Lepidoptera. Demyanovsky & Zubova (1957) showed that the lipid content of Antheraea pernyi falls throughout the development of the female. In the male there is a drop in lipid until the 16th day of development, followed by an increase until ecdysis, so that the adult male has more lipid than the pupa. They suggest that lipid stores are provided for the adult male by an active synthesis of lipid from other substrates during the latter period of development. In Bombyx, both sexes have about the same quantity of lipid in the prepupal stage. During pupal-adult development the female uses about 50% and the male 30% of the initial lipid so that the male moth contains more lipid (Yamafuji, 1937; Niemierko, Wlodauer & Wojtczak, 1956). It thus appears that development in the Lepidoptera shows a metabolic specialization such that the male of the species is provided with adequate lipid stores for use in the adult stage.

The significance of this difference in pattern of lipid utilization in some Lepidoptera is believed to be correlated with difference in flight activity (Gilbert & Schneiderman, 1961). The male must fly long distances seeking out the virgin female and the lipid conserved during development provides the necessary flight fuel. The female, on the other hand, uses the energy of her lipid stores to underwrite the production of eggs during adult development and is not an active flier as an adult.

The importance of lipid in the oxidative metabolism of Lepidoptera was indicated by Kozhantshikov (1938) and Zebe (1954). From their observations it appears that Lepidoptera are unable to use carbohydrates directly as a fuel for flight, but require fat. The observations made in this study also indicate a similar relationship between lipid and flight-muscle respiration in H. cecropia males. The 40% drop in the total lipid of adult males between days 2 and 5 coincides with the time that newly emerged adults would be expected to engage in active flight. Prevention of flight activity during this period significantly reduces the amount of lipid catabolized (Table 2), indicating that flight activity is a causative factor in the sudden drop of adult male lipid, and emphasizes the importance of lipid in flight metabolism. The 13 % decrease in total lipid in the non-flying group emphasizes the importance of lipid in the resting metabolism of the moth.

The use of ¹⁴C-labelled substrates revealed that fatty acids are actively oxidized by flight-muscle homogenates whereas glucose, pyruvate, and glycerol were oxidized only to a limited extent. The underlying basis for this inactivity presents an intriguing problem in the comparative physiology of insect muscle. Zebe (1959) has already suggested that insect flight-muscle is of three physiological types: the exclusive carbohydrate users, the flies; the exclusive fat users, the moths and butterflies; and those that can use both fat and carbohydrate, the locusts. Studies on the biochemistry of fly and locust muscle have shown no qualitative differences correlated with the type of fuel used (Zebe, 1959; Boettiger, 1960). Whether differences are exhibited by flight muscle of Lepidoptera is not known at present but future investigations along this line are planned. That some differences do in fact exist between these different groups of insects is suggested by the observation that after flight there is a continued elevation of respiration for an hour or more in locusts and moths whereas this does not occur in the Diptera (Chadwick & Gilmour, 1940; Krogh & Weis-Fogh, 1951 ; Zebe, 1954). Our finding that flight muscle of H. cecropia cannot appreciably oxidize glucose and glycolytic end-products indicates that further experiments relating to the biochemical pathways of this tissue may reveal significant quantitative differences when compared to flight muscle from other orders.

The results on the nature of flight-muscle respiration show that fatty acid oxidation in flight muscle of H. cecropia is stimulated by the addition of ATP, magnesium, and a citric acid cycle intermediate. Glutamic acid was as effective as citric acid cycle intermediates in stimulating fatty acid oxidation (Table 9). This effect is probably due to the conversion of glutamic acid to a-ketoglutarate, which effectively stimulates fattyacid oxidation. A glutamate-aspartate transaminase is present in flight muscle of H. cecropia (McAllen & Chefurka, 1961) but it is unlikely that this enzyme is involved in this conversion since it does not result in a net increase in citric acid cycle intermediates. This suggests that another pathway of glutamic acid conversion is present, possibly another transaminase or glutamic dehydrogenase.

In respect to the requisites for fatty acid oxidation the system we studied shows similarities to the requirements for fatty acid oxidation in rat-liver mitochondria (Kennedy & Lehninger, 1949). The insect system differs from rat mitochondrial preparations by the lack of cytochrome-c dependence. This may be due to the naturally high concentration of cytochrome-c in moth flight-muscle sarcosomes. Margoliash (personal communication) found that moth flight muscle contains the highest concentration of cytochrome-c yet described. Because of the naturally high concentration of cytochrome-c in moth flight muscle it may persist in sufficient quantities during tissue preparation, eliminating the necessity of an exogenous supply.

The requirements of the flight-muscle fatty acid oxidizing system of H. cecropia are similar to those known in other invertebrates. At present the only well characterized invertebrate systems have been described in the hepatopancreas of Carcinus (Munday & Munn, 1962) and the thoracic muscles of the locust (Meyer et al. 1960). The system in Carcinus requires ATP, magnesium, inorganic phosphate with trace amounts of succinate being stimulatory. This system was able to oxidize butyric, lauric, myristic, octanoic, and palmitic acids.

Our system is comparable to the locust particulate system in that both require ATP and magnesium for optimum fatty acid oxidation. It differs, however, in that addition of a citric acid cycle intermediate enhances oxidative activity. The action of the citric acid cycle intermediate in H. cecropia is similar to that of the citric acid cycle intermediate necessary as a primer in liver mitochondria, whereas the primer in the oxidation of higher fatty acids by the locust thoracic particle is butyrate. Butyrate is unable to replace the citric acid cycle intermediate in our system.

The pH optimum for oxidation of fatty acids in flight muscle of H. cecropia was identical with that in the locust (6 · 4). This value is somewhat lower than that found in mammalian systems but is in keeping with the fact that the pH of the blood of various Lepidoptera and locusts has been reported to fall on the acid side of neutrality and has a range that frequently includes this particular level (Buck, 1953).

The inhibition of endogenous respiration in flight-muscle homogenates by malonate and cyanide was as expected for a system functioning via the citric acid cycle and the cytochrome chain. The production of more ¹⁴CO₂ from Na-butyrate labelled in the first carbon than from Na-butyrate labelled in the second also indicates utilization of the citric acid cycle for fatty acid oxidation.

Inhibition of oxidation in flight muscle of H. cecropia by iodoacetic acid (IAA) indicates the importance of sulphydryl groups in the oxidation of fatty acids. Although IAA is generally considered a specific glycolytic inhibitor, its action is generally extended to sulphydryl-containing enzymes (Hallaway, 1959). Since it was found that glucose is not actively oxidized by flight muscle of H. cecropia it is possible that the site of action of IAA is at a point other than the triosephosphate dehydrogenase. A possible site is the fatty acid activating enzymes that were shown to be present in this muscle. Although the effect of IAA on these enzymes of H. cecropia is not as yet determined, IAA at concentrations of 3 × 10 ^{− 3}M has been shown to inhibit irreversibly the action of fatty acid activating enzymes isolated from hog liver (Jencks, 1962). Other sites of action of IAA have also been suggested in other insects. The addition of IAA to vertebrate muscle causes contracture that can be reversed by the addition of pyruvate but not glucose. In Periplaneta americana contracture of the trochanter extensor after addition of IAA is not reversed by the addition of pyruvate (Bettini & Boccacci, 1959). It is suggested that the difference may be due to a permeability factor or a different site of inhibition.

The β-oxidation scheme of fatty acid oxidation in mammalian tissue has not been worked out in detail in any insect. However, recent work has identified individual enzymes in insect tissue that are similar to those found in mammalian tissue, suggesting the presence of the β-oxidative pathway. The acetate-activating enzyme was detected in the flight muscle of the honeybee (Hoskins, Cheldelin & Newburgh, 1956). Nelson (1958) showed the presence of fatty acid activating enzymes in the fat body of the cockroach and of several species of Lepidoptera including H. cecropia. In 1960 Zebe identified the condensing enzyme that leads activated acetate into the citric acid cycle and β-keto-acyl-thiolase that leads β-keto-acyl into the fatty acid cycle in locust flight muscle. In this study fatty acid activating enzymes have been identified in flight-muscle homogenates of H. cecropia, indicating that similar pathways of fatty acid oxidation are also present in this tissue. The exact nature of these fatty acid activating enzymes awaits further work in a more defined system than the crude systems used in the present study.

In summary, it appears from our observations that lipid metabolism in flight muscle of H. cecropia proceeds via pathways that are commonly found in fatty acid oxidation in other animals. It does not appear that the specialization toward lipid as the fuel for flight has involved any special modifications in lipid oxidative pathways. Carbohydrate pathways, however, appear to be unimportant. Only further work with sarcosomal preparations and individual enzymes will reveal any specific modifications of the basic pattern of flight-muscle oxidative metabolism in H. cecropia.

Supported by grant AM-02818 from The National Institutes of Health. K. A. Domroese was an NSF pre-doctoral fellow during the course of this work.