P.S. Chen

Paragonial substance (sex peptide) and other free ninhydrin-positive components in male and female adults of Drosophila melanogaster.

Abstract By using preparative ion-exchange chromatography the paragonial substance (sex peptide) has been isolated from male adults of Drosophila melanogaster . On the amino acid analyser this substance has an effluent volume of about 50 ml at pH 3·28 and 50°C, and appears as a prominent peak in the acidic region between phosphoserine and glycerophosphoethanolamine. Analyses of extracts from the paragonia alone and studies of the chromatographic behaviour and amino acid composition showed that the present peak contains the sex peptide. The fact that a low but distinct peak appears in the corresponding position in mated females, but is absent in virgin females, suggests that this substance is introduced into the female by the male fly at mating. Injection of the isolated and purified peptide into virgin females resulted in a two to three times increase of oviposition. The stimulating effect is correlated with the concentration of the sample used for injection. Quantitative evaluation of other chromatographic peaks on the analyser demonstrated that 8-day-old female flies contain twice as much free methionine as males of the same age. A similar situation has been found for a number of other amino acids. Conversely, the content of β-alanine amounts to about 50 per cent higher in male than in female adults. The metabolic significance of these sex-specific differences is discussed.

A sex-specific ninhydrin-positive substance found in the paragonia of adult males of Drosophila melanogaster☆

Abstract The paragonia (accessory glands) of male adults of Drosophila melanogaster were found to contain a ninhydrin-positive substance which, judging from its position on the two-dimensional chromatogram, obviously corresponds to the sex peptide reported by others. The content of the paragonia substance increases as the paragonia grow in the course of adult development. Two lines of evidence indicate that this substance occurs only in the paragonia of males: (a) extraction of free ninhydrin-reacting components from different organs; (b) after transplantation of male genital discs into larvae of the same sex, the concentration of this substance in the adult was found to increase. The fact that this substance was also found in female flies which had received male genital discs by transplantation indicates that its formation is autonomous. No distinct evidence of the occurrence of the paragonia substance could be found in adult males of Culex pipiens and C. fatigans . The chemical nature and the possible function of this substance are discussed.

Studies on the protein metabolism of Culex pipiens L.—I: Metabolic changes of free amino acids during larval and pupal development☆

Abstract Using paper partition chromatography, the free amino acids and related substances in the methanol extracts and in the blood from various larval and pupal stages of Culex pipiens (autogenous form) were studied. The ninhydrin-positive pattern is the same as that described earlier ( Laven and Chen , 1956), and no qualitative differences were found between the developmental periods. In the course of larval development the quantities of proline, tyrosine, and glutamine per individual increase rapidly. According to previous workers, these compounds are particularly involved in the synthesis of cuticular proteins in the fully grown larvae. However, the concentration as well as the amount of total free ninhydrin-positive components per unit body weight or total N remain essentially unchanged during the larval life. The free amino acid content in the developing pupae is low. At about 24 hr after pupation tyrosine, proline, glutamic acid, glutamine, and valine exhibit a slight increase. This fact suggests that the histolytic process is at its maximum at this period.

Studies on the protein metabolism of Culex pipiens L.—II: Quantitative differences in free amino acids between male and female adult mosquitoes☆

Abstract The free ninhydrin-positive substances in the adults of Culex pipiens (autogenous form) have been studied by partition chromatography. The methanol extract from individuals shortly after emergence contains α-alanine, β-alanine, arginine, aspartic acid, cystine, glutamic acid, glutamine, glycine, histidine, leucine (and/or iso -leucine), lysine, methionine sulphoxide, proline, serine, threonine, tyrosine, valine, and 2 peptides. Adults maintained for 4–6 days on a sucrose diet become poor in free amino acids. This is especially true for valine, leucine, threonine, tyrosine, and lysine, all of which have been shown to be essential for higher organisms. Male and female adult mosquitoes 4–6 days old differ distinctly in their chromatographic patterns. There is an accumulation of free β-alanine in males and methionine sulphoxide in females. Evidence has been brought forward to show that this difference is not directly due to materials accumulated in the gonads or other sexual organs. The metabolic significance of these amino acids in connexion with the reproductive processes is discussed.

Secretory proteins and sex peptides of the male accessory gland in Drosophila sechellia

Abstract The electrophoretic protein pattern of the male accessory gland in Drosophila sechellia differs from those in the sibling species D. melanogaster, D. simulans and D. mauritiana. The male-specific sex peptides isolated from D. melanogaster and D. sechellia are qually efficient in repressing female sexual receptivity in these four sibling species, but have no effect on females of a distantly related species D. funebris. Sequencing of the sechellia-peptide shows that it consists of 36 amino acids and differs from the melanogaster-peptide reported previously only in three amino acids at positions 8, 11 and 12.

Studies on the transamination reactions in the larval fat body of Drosophila melanogaster

Abstract The in vitro experiments demonstrated the presence of active transaminases in the fat body of Drosophila larvae. The alanine/glutamate reaction proceeded most rapidly. The aspartate/α-ketoglutarate reaction was also active, but the reciprocal combination glutamate/oxalacetate started initially at a high rate and then dropped down very soon to a low level. Combinations of α-ketoglutarate with other amino acids ( dl -leucine, glycine, dl -valine, dl -threonine, and l -arginine) always proceeded at a low speed. One transamination reaction which did not involve either aspartate or glutamate was the formation of glycine from dl -alanine and glyoxylate. Synthesis of glutamine from glutamate and ammonia in the presence of Mg 2+ and ATP was observed. No activity of arginase could be detected. Malpighian tubules and midgut were shown to be also active in transamination. On a wet weight basis, the Malpighian tubules are more active and the midgut less active than the fat body.

Studies on the protein metabolism of Culex pipiens L.—IV. Separation of free amino acids and peptides in adult mosquitoes by column chromatography☆

Separation of free amino acids and peptides in methanol extracts of adult mosquitoes (Culex pipiens and C. fatigans) was accomplished with Dowex-50 column chromatography in a gradient elution system. In addition to the amino acids previously reported, tryptophan and phenylalanine were detected and leucine and isoleucine could be separated distinctly. The relative concentrations of the various amino acids are in good agreement with those previously estimated by paper chromatography. Furthermore, the detailed analyses revealed the presence of at least thirty different peptides and amino acid derivatives, which amount to about 14–19 per cent of the total ninhydrin-positive components. Some preliminary data on the amino acid compositions of these compounds are given. The present results do not suggest any unusual features, at least not at the level of free amino acids and peptides, which could be responsible for the initiation of ovarian development in autogenous mosquitoes.

Ultrastructure and nature of secretory proteins in the male accessory gland of Drosophila funebris

The ultrastructural differentiation of the secretory cells and the nature of secretory proteins in the male accessory gland of Drosophila funebris have been studied by electron-microscopic and immunological methods. (1) In the pupae at 112 days before eclosion, secretory products can be detected in the lumen, even though most glandular cells are at the initial phase of differentiation. At the time of eclosion both main and secondary cells are fully differentiated, but the whole set of five immunologically active proteins are detectable only on the second to third day of adult life. (2) The secondary cells contain giant protein granules, the so-called filamentous bodies, which become partially fused and the filaments assume a twisted form. Randomly dispersed filaments and closely packed filament bundles are also visible in the gland lumen. Antigenic labelling of ultrathin sections and immunoreplica electrophoresis yielded no evidence for the microtubular nature of these filaments. The secretion stored in the lumen contains in addition a large quantity of flocculent proteins which have their origin in the main cells. (3) During the period of high secretory activity in the 7-day-old male flies no vacuolization and disintegration of either the main or secondary cells have been observed. We conclude that both types of cells have the merocrine secretory mechanism. (4) Ultrastructural alterations in the glandular cells confirmed our previous observation that copulation stimulates RNA and protein synthesis.

Accumulation of glutamic acid in the paragonial gland of Drosophila nigromelanica

Abstract The paragonial gland of Drosophila nigromelanica is characterized by accumulating a large quantity of glutamic acid. Its concentration in the gland of flies 10 to 11 days after ecdysis amounts to 60 to 70% of the total free ninhydrin-positive components. The accumulation of glutamic acid is paralleled by a high activity of the enzyme l -alanine aminotransferase in the secretory tissue. The possible role of glutamic acid in the reproductive process is discussed.

Studies on the protein metabolism of Culex pipiens L.—III: A comparative analysis of the protein contents in the larval haemolymph of autogenous and anautogenous forms☆

The protein contents in the larval haemolymph of Culex pipiens (autogenous form) and Culex fatigans (anautogenous form) have been determined by paper electrophoresis. In both mosquito strains the electropherograms showed only one protein fraction which increased rapidly in its concentration during the later period of larval development. The fully grown larva of the anautogenous form was found to contain more proteins per unit volume blood than that of the autogenous form. On the other hand, the total blood concentration of the free ninhydrin-reacting components, as estimated by the method of paper chromatography, is higher in the autogenous form than in the anautogenous form. On the basis of data presented in this study, the relation of nutritional reserves to the phenomenon of autogenous egg production in mosquitoes is discussed.