A. De Loof

Characterization of a cloned locust tyramine receptor cDNA by functional expression in permanently transformed Drosophila S2 cells

Abstract: The cDNA for Tyr‐Loc, a G protein‐coupled receptor that clearly shows homology to a number of mammalian and fruit fly receptors for biogenic amines, was cloned from the nervous system of Locusta migratoria. Functional expression of the cloned cDNA was obtained in cultured insect cells, i.e., in Spodoptera SF9 cells using a baculoviral expression system and in stably transformed Drosophila Schneider 2 (S2) cells. Multiple copies of the receptor expression construct are inserted into the genome of these permanently transformed cells. The expression of the receptor cDNA was driven by the upstream sequences of a Bombyx mori baculoviral immediate early gene. Tyramine shows a much higher binding affinity to this receptor than other possible endogenous ligands. It also reduces forskolin‐induced cyclic AMP production in the permanently transformed S2 cells. The pharmacological profile of the Tyr‐Loc receptor is distinct from that of any locust receptor‐type described so far, but it is similar to that of the Drosophila tyramine/octopamine receptor. In the locust CNS, the Tyr‐Loc mRNA is not present in the distal part of the optic lobes but has a widespread distribution in the brain and the ventral nerve cord.

Lom-AG-myotropin: A novel myotropic peptide from the male accessory glands of Locusta migratoria

A myotropic peptide, termed Lom-AG-myotropin, was isolated from extracts of 4400 accessory gland complexes of males of the locust, Locusta migratoria; the following sequence was derived: Gly-Phe-Lys-Asn-Val-Ala-Leu-Ser-Thr-Ala-Arg-Gly-Phe-NH2. This sequence is completely different from all presently known myotropic peptides from Locusta or other insects. The Lom-AG-myotropin is active on the oviduct and hindgut of Locusta migratoria and Leucophaea maderae. The stimulatory activity is, in both insects, 1000 times greater on the oviduct than on the hindgut, suggesting a specificity for the oviduct.

Hormonal control of synthesis of vitellogenic female protein in the Colorado beetle, Leptinotarsa decemlineata

Abstract The protein pattern of the haemolymph of short-day (SD) beetles is characterized by three specific ‘short-day proteins’ and by the absence of the vitellogenic female protein (VFP), which is found in ovipositing females. There are no indications that the SD proteins should be modified vitellogenic proteins. Allatectomy applied to long-day (LD) adult females at emergence or at pupal-adult ecdysis induces synthesis of SD proteins but does not influence the synthesis of VFP. After this operation vitellogenesis can persist for about 2 weeks. Allatocardiacectomy at pupal-adult ecdysis (but not at emergence) prevents synthesis of VFP. Injection of synthetic juvenile hormone (JH) in SD females results in a low synthesis of VFP and blocking of the synthesis of the SD proteins. Implantation of active corpora cardiaca in allatocardiacectomized SD females does not have any effect on the blood protein pattern. An injection of JH results in oviposition in allatectomized LD females and also in allatectomized SD females, which are subjected to LD after the operation. When injection is made in the lateral side of the abdominal cavity oogenesis is most pronounced in the ovary in contact with the injected JH-paraffin mixture. Two hormonal factors seem to be necessary for the synthesis of VFP: the neurosecretory hormone (s) from the brain and the JH. The latter hormone is also involved in the uptake of vitellogenic blood proteins by the follicle cells and in the regulation of the synthesis of the SD proteins.

Vacuum‐blotting: a new simple and efficient transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to nitrocellulose

The transfer of proteins from polyacrylamide gels to nitrocellulose paper has been of great interest since a wide variety of analytical procedures can be applied to the immobilized proteins. graphic naming of transfer techniques (Southern, Northern and Western) is not continued. Being Europeans, we find it hard to describe this method as Eastern blotting. Therefore we suggest to refer it as vacuum-blotting.

EFFECT OF ECDYSTERONE ON VITELLOGENIN CONCENTRATION IN HAEMOLYMPH OF MALE AND FEMALE SARCOPHAGA BULLATA

Using Polyacrylamide gel electrophoresis, cycloheximide, incorporation of 3H-labelled amino acids and immunological methods, we have demonstrated that injection of ecdy- sterone induces de novo synthesis and release of vitellogenin in both sexes of Sarcophaga bullata. Vitellogenin concentrations were measured by the Mancini-radial immunodiffusion technique. In males a dose as low as 1 ng always makes vitellogenin appear in the haemolymph but very reproducible results are only obtained when doses varying from 10 to 250 ng were injected. In this range, the dose-response curve was linear on a semi- logarithmic scale. In females, vitellogenin concentration remained low until a few hours after liver feeding and thereafter it rose sharply and reached its maximum about 24 h after the protein meal. 100 μg 6-hydroxydopamine HCl, injected before liver feeding in 4-day-old females, inhibited vitellogenin synthesis and yolk deposition, probably by interfering with the release of a brain hormone. This inhibitory effec...

Similarities in vitellogenin and control of vitellogenin synthesis within the genera Sarcophaga, calliphora, phormia and lucilia (diptera)

Abstract 1. 1. The major yolk polypeptides from the different species are identified by SDS-gradient gel electrophoresis. 2. 2. Vitellogenins of the 4 species have similarities in both minimum number and size of their polypeptides. They also react with antibodies raised against vitellin of Sarcophaga. 3. 3. In males of all species ecdysterone but not the JH analog methoprene induces the yolk polypeptides. 4. 4. In non-protein fed females only ecdysterone is able to replace the triggering effect of the protein meal on vitellogenin synthesis. 5. 5. The inability to stimulate vitellogenin synthesis in vivo by a JH analog is discussed.

Isolation, identification and synthesis of locustamyotropin (Lom-MT), a novel biologically active insect peptide

A peptide that stimulates the spontaneous contractions of the hindgut of Leucophaea maderae has been purified from extracts of brain-corpora cardiaca/corpora allata-subesophageal ganglion complexes of 9000 adult Locusta migratoria and was designated locustamyotropin or Lom-MT. The primary structure of this 12 residue peptide has been determined Gly-Ala-Val-Pro-Ala-Ala-Gln-Phe-Ser-Pro-Arg-Leu-NH2. The C-terminal sequence (Phe-Ser-Pro-Arg-Leu-NH2) is identical to the C-terminal pentapeptide of the pheromone biosynthesis activating neuropeptide, recently isolated from Heliothis zea, and is also similar to the C-terminal of leucopyrokinin of Leucophaea. Synthetic Lom-MT showed biological as well as chemical characteristics, indistinguishable from those of native Lom-MT. In locust preparations, Lom-MT provoked an increase in frequency, amplitude and tonus of contractions of the oviduct, but was inactive in the same conditions on the locust hindgut preparation.

Radioimmunological Determinations of Concentrations of 6 C-21, C-19 and C-18 Steroids During the Reproductive-cycle of Female Artemia Sp (crustacea, Anostraca)

1. 1. Progesterone, pregnenolone, 5α-dihydro testosterone, testosterone, estrone and estradiol were measured by radioimmunoassay in purified total body extracts of adult female Anemia during the vitellogenic cycle. 2. 2. Progestagens were present in high amounts (20–30 ng/g) between the vitellogenic cycles while estrogen concentrations had peak values during vitellogenesis (mean values of 269 pg/g and 2720 pg/g for estrone and estradiol, respectively). 3. 3. When testosterone concentrations were lowest (144 pg/g), concentrations of 5α-dihydrotestosterone increased up to 1190 pg/g.

The relation between haemolymph proteins and vitellogenesis in the Colorado beetle, Leptinotarsa decemlineata

Abstract In adult female Colorado beetles, reared under long-day conditions, three sex-specific proteins can be demonstrated in the haemolymph. One of these proteins, the vitellogenic female protein, is selectively absorbed by the follicle cells and accumulated in the oocytes during vitellogenesis. It is the main component (± 70 per cent) of the yolk proteins. Two weeks after castration, the protein concentration of the haemolymph of females is 12 per cent as against 3·5 per cent in normal ovipositing females. This is almost exclusively due to the accumulation of vitellogenic female protein. A specific antiserum against this protein was prepared. The female protein was purified and its amino-acid composition determined.

Neuroendocrinological and Molecular Aspects of Insect Reproduction

This review summarizes recent advances and novel concepts in the area of insect reproductive neuroendocrinology. The role of ‘classic’ hormones, such as ecdysteroids and juvenoids, to control reproduction is well documented in a large variety of insect species. In adult gonads, ecdysteroids appear to induce a cascade of transcription factors, many of which also occur during the larval molting response. Recent molecular and functional data have created opportunities to study an additional level of regulation, that of neuropeptides, growth factors and their respective receptors. As a result, many homologs of factors playing a role in vertebrate reproductive physiology have been discovered in insects. This review highlights several neuropeptides controlling the biosynthesis and release of the ‘classic’ insect hormones, as well as various peptides and biogenic amines that regulate behavioural aspects of the reproduction process. In addition, hormone metabolizing enzymes and second messenger pathways are discussed with respect to their role in reproductive tissues. Finally, we speculate on future prospects for insect neuroendocrinological research as a consequence of the recent ‘Genomics Revolution’.