Pharate Adult Homogenates Research Articles

The developmental profile of paramyosin and other myofibrillar proteins of larval and imaginal thoracic muscles of Lepidoptera

The synthesis of paramyosin and other myofibrillar proteins of the thoracic muscles of the tobacco hornworm Manduca sexta was studied by immunological and electrophoretical methods during the histolysis of the larval thoracic muscles and the differentiation of the indirect flight muscles. Antigens of the myofibrillar proteins in the thoracic muscles of the last-larval stage cross reacted with those in the flight muscles of the adults against polyspecific antibodies from actomyosin and monospecific antibodies from paramyosin. After the breakdown of the larval thoracic muscles (2 days from larval-pupal ecdysis) these antigens can no longer be detected in the thorax. The results indicate an almost complete removal of the larval thoracic muscles. Paramyosin could be identified again in a homogenate of the thoracic muscles of animals on the 13th day from larval-pupal ecdysis. Paramyosin is the first protein found during the differentiation of the flight muscles. The other myofibrillar proteins could be identified in thoracic homogenates of pharate adults of Manduca sexta on the 14th and 15th day from larval-pupal ecdysis. On the 14th day from larval-pupal ecdysis the dorso-longitudinal muscle and the tergosternal muscles show cross-striation, and the appearance of most of the electrophoretical results are in accordance with immunological and morphological findings. The myofibrillar proteins of the indirect flight muscles of Manduca sexta are synthesized de novo during metamorphosis.

Incorporation of fatty acids and the positional distribution analysis of fatty acids in acylglycerols of the insect Ceratitis capitata

1.1. In vitro time-course of the labelled palmitic acid incorporation into larval triacylglycerols of Ceratitis capitata exhibited a tendency to reach the analytical values of the sn-distribution.2.2. However, in the fatty acid incorporation by pharate adult homogenates, palmitic acid showed a tendency to get the mass analytical contents but oleic acid did not.3.3. It suggests the incorporation of unsaturated fatty acids into the position sn-2 by synthetic routes different from the de novo pathway.4.4. Stereospecific distribution of the fatty acids was analysed in diacylglycerols and triacylglycerols from the different stages of development of the insect.5.5. Position sn-1 was mainly esterified by saturated fatty acids; unsaturated fatty acids, palmitoleic acid and oleic acid, linked at the sn-2 whereas in the position sn-3 there was a mixture of saturated and unsaturated fatty acids.6.6. Triacyglycerols from larvae, pharate adults and adults were highly asymmetrical whereas triacylglycerols from eggs were more symmetrical because of the higher presence of unsaturated fatty acids at the position sn-1.7.7. Distribution of fatty acids in diacyl-sn-glycerols and triacyl-sn-glycerols where rather similar; some discrepancies are interpreted as due to transacylation reactions in the de novo triacyl-sn-glycerols to reach their characteristic final distribution.

The biosynthesis of phenethyl alcohol in the male bertha armyworm Mamestra configurata

The structure of the pheromone produced by adult male bertha armyworm, Mamestra configurata, is confirmed as phenethyl alcohol. Radioactive labelling experiments using male pharate adults have shown that the pheromone is biosynthesized from phenylalanine. The data indicate that this conversion is carried out via cinnamic acid rather than phenylpyruvic acid. This interpretation was supported by in vitro experiments with a crude enzyme preparation from homogenates of male pharate adults. A proposed pathway from phenylalanine to phenethyl alcohol is presented.

Biochemistry of development in insects. Incorporation of fatty acids into different lipid classes.

1. To study the different metabolic behaviour of various stages of development of the insect Ceratitis capitata, the incorporation of labelled decanoic, lauric, myristic, palmitic, stearic, oleic, and linoleic acids into triacylglycerols by insect homogenates was investigated. The time-course of incorporation of labelled fatty acids was firstly studied by using oleic acid; it showed that after 10 min of incubation the levels of radioactivity incorporated into triacylglycerols and those remaining in the free fatty acids were practically unchanged. 2. All labelled fatty acids were efficiently incorporated by larval homogenates; however, most of the radioactivity remained as free fatty acids in the presence of pharate adult homogenates, palmitic, and stearic acids being the most scarcely incorporated by this stage of development of the insect. 3. Plots of triacylglycerol and free fatty acid radioactivites versus the stage of development defined a crossing-zone in coincidence with the larval-pupal apolysis. This metabolic difference between larval and pharate adult homogenates could not be explained through differences in the acyl-CoA synthetase activity of the insect; this enzyme activity was notably higher in pharate adult homogenates than in the larval homogenates whatever would be the nature of the fatty acid. 4. [14C]Triolein was scarcely hydrolyzed by both larval and pharate adult homogenates. 5. Double-label experiments were carried out by incorporating either [3H]oleic acid or [3H]-palmitic acid and [14C]glycerol 3-phosphate by larval and pharate adult homogenates at different incubation intervals. Diacylglycerols, triacylglycerols, and phosphoglycerides were isolated and the 14C/3H molar ratio calculated. Results suggest the existence of a different acyltransferase activity in the different stages of development of the insect.

Biochemistry of Development in Insects

1. Previous experiments showed that fatty acids were incorporated into triacylglycerols by homogenates of Ceratitis capitata larvae far more efficiently than by pharate adult homogenates. This metabolic behaviour of both stages of development of the insect has been interpreted throughout the existence of a different acyltransferase activity. To obtain new data on the acyltransferase mechanism, a time-course of the stereospecific incorporation of labelled myristic, palmitic, oleic and linoleic acids into the sn-positions of triacylglycerols has been followed. 2. Studies on the stereospecific incorporation of labelled fatty acids confirmed previous results. Palmitic acid was mainly incorporated into sn-1 and sn-3 positions whereas position 2 exhibited a low incorporation. Myristic acid acylated sn-3 position at a higher rate than it acylated the other sn-positions. Oleic acid was more specifically distributed than palmitic acid and linoleic acid was more efficiently incorporated than the monounsaturated acid. All these data reflect substrate differences in the acyltransferase activity of larval homogenates. Pharate adult homogenates incorporated fatty acids very scarcely and mainly into positions (1 + 3). 3. Kinetics of incorporation of labelled fatty acids into the sn-positions points to a non-random distribution with respect to the major saturated and unsaturated fatty acids in triacylglycerols of larvae of Ceratitis capitata.

On methylating activity of L-(methyl-14C)-methionine in metabolism of phospholipids by insect Ceratitis capitata.

The methylating activity of L-(methyl-14C)-methionine in different stages of developmenent of the insect Ceratitis capitata was studied in a series of in vitro and in vivo experiments. Larval and pharate adult homogenates of the insect were used in the in vitro conditions, and the utilization of the methyl group of methionine for the synthesis of different classes of phospholipids was evaluated. Incorporation of radioactivity in lipids by pharate adult homogenates was significantly higher than that by larval homogenates. In both cases, phosphatidyl ethanolamine showed the highest levels of radioactivity incorporation. Free bases from total lipid hydrolysates were resolved and identified by paper chromatography, and the labeling was investigated by radioactivity scanning of paper chromatograms. Significant differences were observed in the activity of both stages of development of the insect. Larval and pharate adult homogenates incorporated mainly the labeled methyl groups into ethanolamine. Monmethyl ethanolamine was the only methyl derivative that appeared in the hydrolysates of lipids synthesized by larval homogenates, whereas mono-,di-and trimethyl ethanolamine clearly were detected in those synthesized by pharate adult homogeneates. Administration of L-(methyl 14C)-methionine to larvae confirmed the existence of methylation reactions in the metabolic activity of the insect.

Lipogenesis from [ 14C]acetate during development of Ceratitis capitata

Lipogenesis from [ 14C]acetate has been precisely carried out during development of Ceratitis capitata. Acetyl-CoA carboxylase activity and fatty acid synthesis capacity were determined in vitro from eggs to 10-day adults; both activities exhibit a sharp peak in the larval stage and drop to a minimum value in the pharate adult stage. Fatty acids synthesized by the larvae were mainly incorporated into triglycerides, whereas fatty acids formed by the pharate adult homogenates were substantially recovered as phospholipids. Variations of the levels of labelled lipids reached by larvae fed on [ 14C]acetate at consecutive development ages (5, 6, and 7 days) parallel the changes that labelled lipids show in consequence of the metabolic behaviour of the 5-day-old larvae. Variations of the levels of the different lipid classes and patterns of specific radioactivity allow us to state some properties of the lipogenic system of the insect.

In vitro and in vivo fate of saturated and unsaturated fatty acids during development of insects

1. 1. In vitro elongation, saturation and desaturation reactions of fatty acids using labelled 10:0, 12:0, 14:0, 18:0, 18:1 and 18:2 have been investigated with homogenates of larvae and pharate adults of the Dipterous Ceratitis capitata. 2. 2. Larval homogenates desaturate and elongate the labelled substrates according to their chain length. Direct unsaturation and elongation of fatty acids by pharate adult homogenates are insignificant. Unsaturated fatty acids are scarcely hydrogenated to stearic acid by larval homogenates; pharate adult homogenates does not exhibit any saturating activity. 3. 3. Larval and pharate adult homogenates show a different pattern of incorporation of labelled fatty acids into the main classes of lipids. As a general rule, labelled fatty acids are preferentially incorporated into triacylglycerols by larval homogenates; C 18 fatty acids exhibit a different incorporating pattern according to the degree of unsaturation. Labelled fatty acids remain mainly as free fatty acids in the presence of pharate adult homogenates. This different behaviour of larval and pharate adult homogenates can not be explained through differences in the fatty acid activating capacity of both stages of development. 4. 4. In vivo experiments of feeding larvae with 16:0, 18:0, 18:1 and 18:2 have been also carried out. Transformations of these fatty acids are followed through the different stages of development of the insect. The observed elongation activity is in general agreement with the in vitro results. Palmitic and stearic acids are increasingly desaturated during the larval stage of development, that is also in agreement with the in vitro results. A de novo synthesis from labelled acetate, obtained from oxidative degradation of labelled fatty acids, may explain the presence in the lipid fraction of shorter-chain labelled fatty acids than the substrates. 5. 5. Incorporation of 18:0 and 18:2 into the main classes of lipids was also determined in the in vivo experiments. An incorporation of free fatty acids into triacylglycerols during larval and pharate adult stages of development is noticed. Incorporation of 18:2 into phospholipids was higher than that of 18:0. In the main, the in vivo results of incorporation do not reproduce the in vitro experiments, as far as the triacylglycerols-free fatty acids relationship is concerned.

Glycogen phosphorylase activity in pharate adults of the stable fly and the effects of a juvenile hormone analogue

Glycogen phosphorylase was assayed in homogenates of pharate adults of the stable fly, Stomoxys calcitrans, by the release of inorganic phosphorous (Pi) from glucose-1-phosphate (G-1-P) in the presence of glycogen. Activity was determined as active and total phosphorylase present by the absence or presence of adenosine-5-monophosphate (AMP) in homogenates. Homogenates of the pharate adults (that were treated topically immediately after larval-pupal apolysis with a synthetic insect juvenile hormone analogue, ( E)-4-[(6,7-epoxy-3-ethyl-7-methyl-2-nonenyl)oxy]-1,2-(methylenedioxy)benzene) displayed no inhibition or enhancement of phosphorylase activity when compared to untreated pharate adults.

In vitro and in vivo [ 14C]acetate incorporation during development of insects

A comparative study on the in vitro and in vivo incorporation of [ 14C]acetate into different classes of lipids by the insect Ceratitis capitata has been carried out. The pattern of labelled lipids obtained in the in vitro experiments depends on both the time of incubation and the stage of development of the insect. Larval homogenates and pharate adult homogenates incorporate rapidly [1- 14C]acetate during the first 60 minutes, mainly into phospholipids. In the in vivo experiments triglycerides account for the highest percentages of incorporation in both stages of development, whereas free fatty acids and phospholipids exhibit lower levels of incorporation. Radioactivity of individual fatty acids present in the lipid classes was determined by gas-liquid radio-chromatography. The specific activity of fatty acids present as free fatty acids in triglycerides increased during the larval-pharate adult transition, whereas that of those present in phospholipids decreased. Relative abundance of monoenoic fatty acids synthesized de novo generally decreased from larvae to pharate adults. Triglycerides from both larvae and pharate adults showed the highest levels of unsaturation.

In vitro elongation and desaturation of fatty acids during development of insects

1. 1. In vitro elongation and desaturation reactions of fatty acids using labelled 10:0, 12:0, 14:0 and 16:0 have been investigated with homogenates of larvae and pharate adults of Ceratitis capitata. The obtained results show clear differences in the behaviour of larval and pharate adult stages of development. 2. 2. Larval homogenates desaturate and elongate the labelled substrates according to their chain length. The longer the length of the fatty acids used as substrates the more pronounced the abundance of the corresponding monounsaturated fatty acids, as well as the smaller activity of the elongation pathway. Direct unsaturation and elongation of fatty acids by pharate adult homogenates are insignificant. 3. 3. Labelled acetate incorporation into fatty acids by the larval homogenates is notably stimulated in the presence of malonate, whereas the fatty acid synthesis by pharate adult homogenates is practically unaffected. The fatty acids 10: 0 and 12: 0 are elongated by larval homogenates more effectively in the presence of acetate than that of malonate. 4. 4. The larval homogenate incorporates considerably more labelled fatty acids into triglycerides than into phospholipids, whereas the radioactivity remains mainly as free fatty acids in the presence of pharate adult homogenates. 5. 5. Pharate adult mitochondria preparations incorporate more acetate than larval itochondria preparations. Incorporation by larval mitochondria was not influenced by the presence of malonate and the resulting fatty acids are present mainly as phospholipids, in a clear contrast with the incorporation achieved by the whole larval homogenates.

In vitro regulation by nadph of fatty acid biosynthesis with larval and pharate adult homogenates of the fly, Ceratitis capitata

Acetyl-CoA carboxylase and fatty acid synthetase activities have been studied in larval and pharate adult homogenates of Ceratitis capitata under the influence of various concentrations of NADPH. NADPH, 0.5–1.0 μmole per ml., activates acetyl-CoA carboxylase, whereas 1.0–5.0 μmoles per ml. produces a marked decrease, using either labelled acetate or acetyl-CoA as substrates. This NADPH influence cannot be interpreted through a concomitant activation of the synthesis of fatty acids from malonyl-CoA, since the incorporation of labelled acetate and acetyl-CoA into fatty acids becomes negatively regulated by NADPH in the same interval, 1.0–5.0 μmoles per ml. The NADPH effect is discussed through its competitive influence with respect to malonyl-CoA on the fatty acid synthetase.

Time course of [ 32P] orthophosphate incorporation into different classes of phospholipids by larval and pharate adult homogenates of Ceratitis capitata

[ 32P]phosphate incorporation into the major phospholipids of larval and pharate adult homogenates of the fly Ceratitis capitata was investigated in a series of in vitro experiments. Phospholipid classes, levels, and specific radio-activity were evaluated. De novo biosynthesis of phosphatidylethanolamine and phosphatidylcholine and acylation of the lyso-derivatives take place during the first few hours of the experiment. Ethanolamine phosphoglycerides exhibit the highest metabolic activity. A certain degree of specificity in the metabolic activity of the different classes of phospholipids, according to the development stage of the insect, has been shown.

In vitro fatty acid and lipid biosynthesis during development of insects

1. 1. In vitro biosynthesis of fatty acids from [1- 14C]acetate, desaturation of [1- 14C] palmitic and [1- 14C]stearic acids and their incorporation into different classes of lipids have been investigated with homogenates of larvae and pharate adults of Ceratitis capitata. 2. 2. The time-course of [1- 14C]acetate incorporation shows clear differences in the behaviour of larval and pharate adult homogenates. Specific radioactivities of fatty acids synthesised by larval homogenates exhibit a net distinction between mediumchain and long-chain fatty acids. This distinction is not exhibited in the [ 14C]acetate incorporation by pharate adult homogenates. 3. 3. Both developmental stages also show differences in the incorporation of radioactivity into different classes of lipids. Triglycerides or phospholipids become rapidly labelled by either larval or pharate adult homogenates, respectively. This different behaviour of both developmental stages also applies to the specific radioactivity of the individual fatty acids present in the lipid classes. Decanoic acid has the highest specific radioactivity of the fatty acids incorporated into triglycerides by larval homogenates. The fatty acids of triglycerides from pharate adults, on the other hand, do not exhibit any incorporation of radioactivity. 4. 4. Desaturation of [1- 14C]palmitic and [1- 14C]stearic acids has been investigated in larval and pharate adult homogenates. Pharate adult homogenates do not desaturate these fatty acids, whereas larval homogenates exhibit a certain degree of desaturation. [ 14C]Palmitic and [ 14C]stearic acids are effectively incorporated by larval homogenates while they remain in a free form during incubation with pharate adult homogenate. However, stearic acid shows a lower metabolic efficiency than palmitic acid. Desaturation of palmitic acid is favoured by aerobic conditions and palmitoleic acid is preferentially incorporated into phospholipids.