Phosphoprotein phosphatase activity during development of the insect Ceratitis capitata.

Phosphohistone phosphatase activity was determined in homogenates of the insect Ceratitis capitata at several stages of development. Enzyme activity varied differently during development; the highest values were found during larval instars and the lowest values coincided with apolysis. These results are discussed on the basis of the relationship between the cyclic nucleotide-protein kinase system and hormonal regulation previously described in this Diptera.     

Study of the RNA-degrading activities during the development of the insect Ceratitis capitata

1. Three different RNA-degrading activities have been characterized in the insect C. capitata. Two of them are non dependent on divalent cations and show acid and alkaline optimum pH values respectively. For the third one, the maximum activity is observed at pH 8.5, being this enzyme inhibited by EDTA. 2. Distribution of the enzyme levels during the development of the insect is reported. Results are interpreted in terms of the functional role of these enzymes.     

Polyadenylated-RNA during the development of Ceratitis capitata

Polyadenylated-RNA has been isolated at different stages of the life cycle of the insect Ceratitis capitata using chromatography on oligo-dT-cellulose. The molecular characterization of this RNA fraction has been carried out by melting, sedimentation, electrophoretic and CD studies. The distribution of this polynucleotide fraction during the development has been related to physiological changes of this holometabolous insect. The results obtained are discussed in terms of the presence of RNA-degrading activities previously reported for this insect.     

Lysozyme from the insect Ceratitis capitata eggs.

1. Lysozyme from eggs of the Dipterous Ceratitis capitata (Wiedeman) has been purified by ion-exchange chromatography and gel filtration and its physicochemical properties have been investigated. This is the first insect lysozyme characterized so far and it exhibits some properties different to those described for other animal lysozymes. 2. Lysozyme from the insect eggs has a molecular weight of about 23200 and a sedimentation coefficient of 2.4 S. Molecular weight determination by sodium dedecylsulphate gel electrophoresis indicates that the molecule consists of a single polypeptide chain. 3. This lysozyme preparation shows notable stability at acidic pH values and lability at alkline pH values. It shows a single optimum pH at about 6.5.4. Chitinase/muramidase specific activity ratio is around 350 times higher for the insect lysozyme than for the hen egg-white enzyme. 5. The amino-acid composition shows the presence of one tryptophan residue per molecule of enzyme. This fact differentiates the lysozyme from insect eggs from other animal and plant lysozymes. From the amino acid composition, the absorption coefficient and the partial specific volume are calculated. 6. Glycine is the N-terminal residue.     

Variation of the levels of cyclic AMP and cyclic GMP during development of the insect Ceratitis capitata.

Changes in the levels of adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) during development were studied in the Dipterous Ceratitis capitata. The developmental patterns were different to each other. Cyclic AMP showed a sharp maximum in the larval stage to decrease afterwards during adult development. Changes of cyclic GMP exhibited an opposite pattern, although its levels were always higher than those of cyclic AMP.     

Fatty acid synthetase complex from the insect Ceratitis capitata.

Fatty acid synthesis capacity of the insect Ceratitis capitata has been investigated in vitro from [1-14C]acetyl-CoA using homogenates at different stages of development. A maximum activity was observed after 5--6 days of larval development. But homogenates of the pharate adult insect did not show synthetic capacity of fatty acids. Fatty acid synthetase complex has been isolated from the particle-free supernatant fraction of homogenates from the 6-day C. capitata larvae. The enzyme complex was purified 182-fold with respect to the protein contained in the crude extract. The complex was homogeneous when analysed by gel filtration and by polyacrylamide-gel electrophoresis. The molecular weight was 5.2X10(5). The enzyme was dissociated into half-molecular subunits. Amino acid analysis, general properties, stability and kinetic constants (V and Km) for the substrates are reported. The fatty acid synthetase complex from the insect contains 42+/-1-SH residues and one phosphopatetheine moiety per 5.2X10(5). Activity was dependent on the presence of NADPH; FMN strongly inhibited the enzyme activity promoted by NADPH. The enzyme complex synthesized a range of fatty acid (10:0--18:0), palmitate being the predominant end product. The proportions of fatty acids synthesized varied with substrate concentrations. Fatty acids released from the complex were almost completely in the free form.     

Primary structure of cytochrome c from the insect Ceratitis capitata.

The complete amino acid sequence of cytochrome c from the Dipterous Ceratitis capitata (serie Acalypterae) has been determined by combining automatic and manual methods of sequence analysis. No overlaps between positions 79 and 80, 86 and 87, 91 and 92 as well as between 99 and 100 were obtained. The alignment of these peptides was done by homology with other sequences of cytochromes c from insects already described. Comparison with the sequences of cytochromes c of other Diptera studied so far shows three changes (positions 50, 60 and 61, according to vertebrate cytochrome c numeration) from the Acalypteran Drosophila melanogaster and five changes (positions 9, 36, 50, 60 and 61) from that of the Calypteran Haematobia irritans.     

Ceratitis capitata brain adenylate cyclase and its membrane environment

Adenylate cyclase activation by GTP and octopamine as well as basal activity (in the presence of Mg2+) have been studied as a function of membrane structure in plasma membranes from brain of the dipterous Ceratitis capitata. Benzyl alcohol and lidocaine, but not phenobarbital, inhibited the three activities to the same extent. Triton X-100-solubilized adenylate cyclase was also inhibited by benzyl alcohol and lidocaine, but not by phenobarbital. Results could be explained by an effect on the catalytic unit lipid environment, which would be maintained after solubilization, counteracting the effect of these drugs to facilitate lateral diffusion and coupling of adenylate cyclase components in the lipid bilayer. The observation that the insect adenylate cyclase is relatively insensitive to changes in bulk bilayer fluidity is strengthened by the absence of effect of phenobarbital on enzyme activities. Indeed, this compound was as active as lidocaine or benzyl alcohol in increasing bulk membrane fluidity. The response of C. capitata adenylate cyclase to changes in membrane fluidity is different from that recorded in mammalian systems. This may be functionally important and result from the fact that insects are not warm-blooded.     

Variation of superoxide dismutases during the development of the fruit fly Ceratitis capitata L.

Superoxide dismutase levels were estimated in eggs, larvae and pupae of the fruit fly $\text{Ceratitis capitata}$, as well as in adult flies. No changes occur in the first three stages, but development of the adult fly is accompanied by a large increase in mitochondrial superoxide dismutase per gm of material, and a much smaller relative increase in the cytoplasmic enzyme.     

Fatty acid synthetase content during development of the fly,Ceratitis capitata

Immunochemical titrations of different enzyme preparations from larval, pharate adult, and emerged adult Ceratitis capitata indicated that the changes in fatty acid synthetase activity during development of the insect are not related entirely to changes in the content of the enzyme. Changes in catalytic efficiency during larval and pharate adult development were clearly paralleled by the amounts of immunoprecipitate; however, the changes of enzyme activity with adult age were not correlated to the changes of enzyme content. Dietary manipulations of the larval stage of the insect Ceratitis capitata show the adaptive nature of the fatty acid synthetase. Fasting produced a clear decrease of activity and level of the enzyme from the larvae and refeeding restored practically normal values.     

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.     

Turnover of the glycerol moiety of different lipid classes during development of Ceratitis capitata

• 1.1. Labelling of triacylglycerols, diacylglycerols, phosphatidylelthanolamine, phosphatidylcholine and their lysoderivatives was followed during the development of Ceratitis capitata after feeding larvae with [³H]glycerol. Both, specific activity and dpm/individual were plotted versus the time of development.
• 2.2. Decay curve for diacylglycerols had two exponential components accounting for two metabolic pools of this lipid class. Decay curves for phosphoglycerides and triacylglycerols exhibited a monophasic behaviour.
• 3.3. Triacylglycerols had large values of the turnover time parameters consistent with their reserve nature; phosphoglycerides exhibited a turnover time lower than that of triacylglycerols and similar to that of diacylglycerols in agreement with their metabolic relationships.
• 4.4. Half-life values of lysoderivatives were shorter than that of diacylglycerols suggesting a rapid deacylation-acylation mechanism for the phosphoglycerides.

     

Regulation by forskolin of octopamine-stimulated adenylate cyclase from brain of the dipterous Ceratitis capitata

Forskolin, a diterpene that exerts several pharmacological effects, activates adenylate cyclase in brain and in some other mammalian tissues. Properties of forskolin activation of adenylate cyclase from central nervous system of the dipterous Ceratitis capitata are described. The interaction of forskolin with the insect adenylate cyclase system was studied by evaluating its effect on metal-ATP kinetics, protection against thermal inactivation, membrane fluidity and enzyme modulation by fluoride, guanine nucleotides, octopamine, and ADP-ribosylation by cholera toxin. The diterpene stimulated basal enzyme activity both in membranes and Triton X-100-solubilized preparations, apparently devoid of functional regulatory unit, this effect being rapidly reversed by washing the membranes. An increase of Vmax accounts for the activation of soluble and membrane adenylate cyclase preparations by forskolin, whereas the affinity of the enzyme for the substrate was not affected. Forskolin apparently protects the membrane enzyme from thermal inactivation, and at concentrations that promote the enzyme activity the diterpene does not alter membrane microviscosity. Forskolin does not appear to alter the sensitivity of insect adenylate cyclase to sodium fluoride, guanine nucleotide, or regulatory subunit ADP ribosylated by cholera toxin, the combined effect of these factors with the diterpene resulting in a nearly additive enzymatic activation. However, forskolin blocks the octopamine stimulatory input. Results obtained with the insect adenylate cyclase system are discussed and compared to what is known about mammalian systems to propose a mechanism of enzyme activation by forskolin.     

Inter-population variation among Ceratitis capitata flies in host acceptance pattern

Significant inter-populational differences in propensity to attempt boring into (accept) various types of fruit for oviposition were found among Ceratitis capitata females from two wild sources and one laboratory source. Evidence suggests that (a) fruit size had a strong influence whereas fruit taxonomic status had little influence on the acceptance pattern of each population, and (b) at least a portion of the inter-populational variation had a genetic basis.

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Zusammenfassung Bei zwei Wildherkünften und einem Laboratoriumsstamm von Ceratitis capitata wurden Weibchen auf ihre Neigung zur Probebohrung in Früchte vor der Eiablage untersucht; dabei ergaben sich gesicherte Unterschiede zwischen den Populationen. Die Resultate deuten drauf hin, (a) dass die Fruchtgrösse einen grossen, und die taxonomische Stellung der Früchte einen kleinen Einfluss auf das Annahmemuster der Populationen ausübt, und (b) dass mindestens ein Teil der Unterschiede zwischen den Populationen genetisch bedingt ist.

     

Solubilization of adenylate cyclase from Ceratitis capitata neural membranes and incorporation into liposomes

Solubilization of the adenylate cyclase from neural membranes of the dipterous Ceratitis capitata, by using several detergents, and regulatory characteristics of the solubilized enzyme were examined. Triton X-100 is the most effective detergent in solubilizing this enzyme activity. The adenylate cyclase in Triton X-100-solubilized preparations (105,000 g supernatant) does not respond to either guanine nucleotides or fluoride and it apparently seems to be devoid of a functional regulatory component. When this preparation is centrifuged again at 300,000 g for 30 min no enzyme activity is detectable in the supernatant, however only 8% of total activity is recovered in the pellet. The activation pattern for the enzyme in the 300,000 g pellet is similar to that observed for the enzyme in the 105,000 g supernatant. Incorporation of solubilized enzyme into dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) or cholesterol-enriched DOPC liposomes increases the 300,000 g pellet adenylate cyclase activity in a similar extension; thus, this increase in enzyme activity appears to be independent not only on the phospholipid composition but also on the liposome fluidity.     

Modes of programmed cell death during Ceratitis capitata oogenesis

In the present study, we demonstrate the existence of two distinct apoptotic patterns in nurse cells during Ceratitis capitata oogenesis. One is developmentally regulated and normally occurs during stages 12 and 13, and the other is stage specific and is sporadically observed during stages 7 and 8. The pre-apoptotic manifestation of the first pattern begins at stage 11 and is characterized by the formation of actin bundles. Subsequently, at stages 12 and 13, the nurse cell nuclei exhibit condensed chromatin and contain fragmented DNA, as revealed by TUNEL assay. The apoptotic nurse cell remnants are phagocytosed by the neighboring follicle cells at the end of oogenesis during stages 13 and 14. In the second apoptotic pattern, which occurs sporadically during stages 7 and 8, the nurse cells degenerate and are phagocytosed by the follicular epithelium that contains apoptotic cell bodies. The data presented herein, compared to previous reported results in Drosophila melanogaster and Dacus oleae (Nezis et al., 2000, 2001), strongly suggest that nurse cell apoptosis is a developmentally regulated and phylogenetically conserved mechanism in higher Dipteran. They also suggest that, the sporadic apoptotic pattern consists of a possible protective mechanism throughout oogenesis when damaged or abnormal egg chambers, are eliminated before they reach maturity.