Bernard Mauchamp

3×P3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards

Transgenesis was recently achieved in Bombyx mori L., but it has proved difficult and time-consuming to screen the numerous progeny to identify the transgenic individuals. As the 3xP3-EGFP marker has been shown to be a suitable universal marker for transgenic insects (Nature 402 (1999) 370), we evaluated its use for embryonic-stage screening for B. mori L. germline transformation. Using the piggyBac-derived vector pBac[3xP3-EGFPaf], we were able to isolate four transgenic individuals from about 120,000 embryos (560 broods). The screening was straightforward due to EGFP production in the G1 embryonic stemmata, which was visible through the translucent egg chorion. EGFP was produced in the stemmata and central and peripheral nervous systems from the fifth day of embryonic development. It persisted at high levels in the stemmata throughout the larval stage, and was also present in the compound eyes and nervous tissues of the pupae and the compound eyes of the moths.

Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms

A gene construct was made by fusing the coding sequence of the red fluorescent protein (DsRed) to the exon 2 of the fibrohexamerin gene (fhx), that encodes a subunit of fibroin, the major silk protein of the silkworm Bombyx mori. The fusion gene was inserted into a piggyBac vector to establish a series of transgenic lines. The expression of the transgene was monitored during the course of larval life and was found restricted to the posterior silk gland cells as the endogenous fhx gene, in all the selected transgenic lines. The exogenous polypeptide was secreted into the lumen of the posterior silk gland together with fibroin, and further exported with the silk proteins as a foreign constituent of the cocoon fiber. The capacity of DsRed to emit fluorescence in the air-dried silk thread led to show that the recombinant protein was distributed over the whole length of the fiber. A remarkable property of the system lies in the localization of the globular protein at the periphery of the silk thread, allowing its rapid and easy recovery in aqueous solutions, without dissolving fibroin. The procedure represents a novel and promising strategy for the production of massive recombinant proteins of biomedical and pharmaceutical interest, with reduced cost.

Effects of the non-steroidal ecdysone mimic, RH-5849, on diapause and non-diapause larvae of the European corn borer, Ostrinia nubilalis Hbn.

Abstract Topical applications of the non-steroidal ecdysone mimic, RH-5849, as expected, initiated a premature moult in last larval instar of the European corn borer, Ostrinia nubilalis . However, RH-5849 induced a perfect supernumerary larval moult in diapause and non-diapause larvae when the application was performed very early in the instar. The compound induced larval-pupal intermediates and normal pupae if it was applied after 2 days in the last instar of non-diapause larvae and after 5–6 days in diapause larvae. The occurrence of larval-pupal moults and normal pupae demonstrated the onset of pupal commitment of the diapause larvae. Moulting to normal pupae was greatly improved by a double topical application of 1 μg RH-5849 onto diapause larvae. Although the rate of normal pupation was rather low, the ecdysone mimic could break the larval diapause of the European corn borer and produce normal pupae. From these results, the endocrine control of diapause induction is discussed.

Effects of ultralow doses of fenoxycarb on juvenile hormone-regulated physiological parameters in the silkworm, Bombyx mori L

Effects of fenoxycarb at ultralow doses were investigated on juvenile hormone (JH)–regulated parameters in the silkworm, B. mori. Like JH, this non-terpenoid carbamate is able to induce permanent larvae in the last larval instar. However, whereas micrograms of JH are needed to produce this effect, only a few picograms of fenoxycarb are necessary to induce the same effect. The effects of fenoxycarb observed in this study were only visible from day 4 of the last larval instar—that is, when the JH titer has dropped to undetectable levels and JH-repressed physiological parameters would naturally be expressed. We observed that the permanent larvae induced with low doses of fenoxycarb (100 pg/larva) had no 20-hydroxyecdysone (20E) peak. Their prothoracic glands (Pgs) were completely inactive and very weakly sensitive to prothoracicotropic hormone (PTTH). Fenoxycarb at doses of 1 ng/larva also significantly inhibited silk gland growth and coloration, whereas carotenoid content of the hemolymph was maintained at high levels, which could reflect an inhibition of its uptake by the silk glands. Total hemolymph protein levels in last instar larvae were also depressed at these doses. So, it seems that low doses of fenoxycarb are sufficient to maintain in a juvenilized status the physiological parameters that are normally expressed when JH titer has declined. Moreover, from an endocrinological viewpoint, we demonstrated that the corpora allata (CA) are not necessary for fenoxycarb to induce those effects and discussed its possible mode of action. Arch. Insect Biochem. Physiol. 37:178–189, 1998.

A helium burst biolistic device adapted to penetrate fragile insect tissues

Abstract To compensate for the extremely low penetration efficiency of the original PDS/1000-He Bio Rad biolistic® device and the deleterious blast effect, design modifications have been made to the launching module. These modifications were evaluated on Bombyx mori embryos and fragile tissues, such as oocytes and imaginal wing disks. The original floppy macrocarrier was replaced by a rigid macrocarrier to avoid the effects of the helium blast. The efficiency of the gene gun bombardment was reinforced by the addition of a focusing nozzle. The reduced blast effect allowed us to carry out high-pressure shootings to small organs with improved penetration. This system allowed potentially all the internal embryonic tissues to be transfected with optimal survival rates. The new module was effective on tissues that are difficult to transfect, such as the epithelial wing disk that is covered by a peripodial membrane, and the ovarian follicle cells that lie under the ovariole cell membrane. The new macrocarrier allowed both an aqueous delivery of particles and an ethanolic dry delivery. No significant differences were noted between these two modes of delivery. The major improvement is the possibility of high pressure shooting correlated with appreciable penetration and a weak blast effect.

Gas chromatography-mass spectroscopy analysis of juvenile hormone released by insect corpora allata

Corpora allata incubated in appropriate medium release several compounds including juvenile hormones. Juvenile hormones (14C labeled or unlabeled) were extracted with hexane and directly analyzed by gas chromatography-mass spectroscopy. This method allowed the qualitative and quantitative analysis of total released juvenile hormone. It could also be used as a routine assay for evaluation of corpus allatum activity. Data obtained by this method were compared to those obtained by radiochemical assay.

Biological, radiochemical and physicochemical evidence for the low activity of disconnected corpora allata in locust

Abstract The rate of juvenile hormone biosynthesis by locust corpora allata after transection of the nervi corporis allati 1, was measured in vitro using both radiochemical assay and gas chromatography—mass spectroscopy analyses. Incubations in different culture media or in pure haemolymph result in a low rate of juvenile hormone biosynthesis by disconnected glands. In vivo studies using juvenile, chromatotropic and gonadotropic effects of the corpora allata confirm the low activity of the disconnected glands. Furthermore, animals with disconnected corpora allata appear to be more sensitive to corpora allata implantation than control hosts.

Influence of the packing material and the column filters on the reliability of a high-performance liquid chromatograph—mass spectrometer interface based on the direct liquid inlet principle

Abstract The direct liquid introduction interface for coupled high-performance liquid chromatography(HPLC)-mass spectrometry consists of a 1–3-μm pinhole in a nickel diaphragm. This interface produces an axial jet of HPLC eluent into the ionization source. Variations in the jet affect the mass fragmentation pattern. Physical modifications of the pinhole also induce substantial changes in the jet. The influence of the packing material and the column filters has been examined and solution are given for preventing plugging of the pinhole.

4′-OH-JH-III: an additional hydroxylated juvenile hormone produced by locust corpora allata in vitro

Abstract In the present paper, we report the identification of 4′OH JH-III, an additional hydroxy-JH-III, produced in vitro by the corpora allata of the African locust Locusta migratoria. This shows that the three methyl group of JH-III linked to carbon 4, 8 and 12 can be hydroxylated generating three new putative juvenile hormones namely 4′OH-JH III, 8′-OH-JH-III and 12′-OH-JH-III. Both the role and the biosynthetic pathway of hydroxy-JH is discussed, opening new perspectives for the studies on juvenile hormones and isoprenoid compounds.

MS/MS analyses of ecdysteroids in developing eggs of Dysdercus fasciatus

Abstract Several techniques are now available for analysing ecdysteroids. In this paper we describe a new rapid technique to obtain chemical characterization of the ecdysteroids. MS/MS is a convenient technique that can be used for mixtures or partly purified compounds. Conditions of analyses are described to allow the detection of small amounts (50 ng) of ecdysteroids in partly purified biological samples. First, using standard solutions, we optimized the conditions to obtain the pseudo-molecular ion (MH + ) with the highest relative intensity (100%). Such conditions were obtained using DCI/NH 3 with a pressure of 1.2 10 −4 torr. This ion was selected through the first analyser and introduced into the collision cell. After collision with neural atoms of argon in the collision cell, it was dissociated to give daughter ions. The residual parent ion and the daughter ions were then analysed by the second analyser. Collision activated dissociation of the parent ion generates the daughter ions or MS/MS spectrum of the molecule. This technique gives a good characterization of the molecules to be analysed. Using the conditions determined with standards, we developed the analysis from partly purified biological extracts. Sample from pooled HPLC fractions was introduced into the ionization chamber where all the molecules were ionized. From these ions, we selected an ion corresponding to a known ecdysteroid (RT of HPLC fractions). This ion was introduced into the collision cell to produce daughter ions. By comparison with the daughter ion spectrum of a standard recorded under the same conditions we can confirm the nature of the ecdysteroid detected. We improved this technique for analysing ecdysteroids contained in Dysdercus fasciatus eggs. Both makisterone A and makisterone C were detected and characterized through diagnostic daughter ions in the eggs.