Maurice Charlet

Insect immunity: Isolation of three novel inducible antibacterial defensins from the vector mosquito, Aedes aegypti

The injection of Escherichia coli and Micrococcus luteus into the hemocoel of Aedes aegypti induces a potent antibacterial activity in the hemolymph. We have purified and fully characterized three 40-residue antibacterial peptides from the hemolymph of bacteria-challenged mosquitoes that are absent in naive mosquitoes. The peptides are potently active against Gram-positive bacteria and against one of the Gram-negative bacteria that were tested. The amino acid sequences clearly show that the three peptides are novel isoforms of the insect defensin family of antibacterial peptides. They differ from each other by one or two amino acid residues. We present here the complete amino acid sequences of the three isoforms and the activity spectrum of the predominant Aedes defensin.

Experimental evidence for a neuroendocrine control of ecdysone biosynthesis in adult females of Locusta migratoria

Abstract The considerable increase in ecdysteroid concentration which occurs in normal Locusta ovaries at the end of each cycle of oocyte maturation is prevented if the median neurosecretory cells of the pars intercerebralis are cauterized, or if the corpora cardiaca are excised 24 hr before the onset of ecdysone synthesis in normal females. Implantation of additional brain-corpora cardiaca complexes into young vitellogenic females advances the time of ecdysone synthesis by some 12 hr. Oocyte growth itself is not affected in these different types of experiments. It is inferred from the data of the present study that ecdysone synthesis in the follicle cell epithelium of maturing Locusta ovaries is stimulated by a neurohormone produced in the median neurosecretory cells of the pars intercerebralis and secreted into the blood via the corpora cardiaca.

Immunocytochemical localization of Bombyx-PTTH-like molecules in neurosecretory cells of the brain of the migratory locust, Locusta migratoria

SummaryUsing a monoclonal antibody directed against a synthetic pentadecapeptide corresponding to the N-terminus of the prothoracicotropic hormone (PTTH) of Bombyx mori, we report the presence of immunoreactive molecules in a large number of median neurosecretory cells of the pars intercerebralis of the migratory locust, Locusta migratoria. These cells correspond to the A1 cell type which we show to contain also neuroparsins, a family of predominant neurohormones of the migratory locust. In contrast, PTTH-like molecules are absent from A2 cells of the pars intercerebralis which contain Locusta insulin-related peptide (LIRP). Developmental studies show the presence of PTTH-related substances in neurosecretory cells of Locusta migratoria from late embryogenesis to adult development, including ageing vitellogenic female adults.

Antimicrobial Peptides in Insect Immunity

With around one million named species and probably 8–10 times that number unnamed, insects account for the great majority of animal species on earth. Insects can be found in almost all terrestrial and freshwater habitats, from the driest deserts to freshwater ponds, from the canopy of a tropical rainforest (where their diversity is unbelievably great) to the arctic wastes. They are a tremendously successful group. They are a fundamental part of our ecosystem. They are responsible for the pollination of many plants; the decomposition of organic materials, facilitating the recycling of carbon, nitrogen, and other essential nutrients; the control of populations of harmful invertebrate species (including other insects); the direct production of certain foods (honey, for example); and the manufacture of useful products such as silk. However, they are often considered as negative, mainly because they are important vectors of animal and human diseases.

Developmental and morphogenetic alterations in larvae of Locusta migratoria reared on plant diet with a selectively modified sterol profile

Abstract Wheat seedlings germinating in the presence of the systemic fungicide fenpropimorph accumulate 9β, 19-cyclopropylsterols (95% of total sterols) in place of Δ 5 -sterols which are normally produced in these plants. Larvae of the phytophagous insect Locusta migratoria show reduced titres of ecdysteroids and considerably delayed development when reared on fenpropimorph-treated wheat. In addition, a significant number of such larvae exhibit anticipated wing reversal and reach the adult stage after four larval instars in place of the normal number of five. These anticipated morphogenetic events and the reduction of the number of larval instars had been induced in earlier experiments by implantation of supernumerary prothoracic glands into juvenile larvae, but not by injections of ecdysone. The prothoracic glands of experimental larvae reared on fenpropimorph-treated wheat show ultrastructural signs indicative of an intense protein synthesis in the rough-surfaced endoplasmic reticulum, and we hypothesize that proteins secreted by the prothoracic glands are responsible for the morphogenetic changes observed in the experimental larvae.

Ecdysiotropic activity in brains and corpora cardiaca of larvae and adults of Locusta migratoria: in vitro assays

Summary Under in vitro conditions the excised left and right prothoracic glands of a larva of Locusta migratoria produce similar amounts of ecdysone, as monitored by RIA. When brain-corpora cardiaca extracts from last instar larvae of the same species are added to one of the two glands, its ecdysone production is significantly increased over that of the homologous gland. The stimulation is dose dependent. This in vitro system has been used as an assay to probe the presence of ecdysiotropic factors in brain-corpora cardiaca complexes of larvae and adult females of Locusta migratoria. The results show that the apparent titres of ecdysiotropic factors in these extracts fluctuate during larval development and in adult females. These fluctuations are discussed in relation to our present information on the role of brain and corpora cardiaca in the control of ecdysone synthesis in prothoracic glands and follicle cell epithelia in this species.

De novo sequencing by nano-electrospray multiple-stage tandem mass spectrometry of an immune-induced peptide of Drosophila melanogaster

To combat infection, the fruit fly Drosophila melanogaster responds by rapid synthesis of a series of immune-induced molecules reported as Drosophila immune-induced molecules (DIMs). Characterization of the primary structure of the DIMs is required to establish their exact function. In order to get such information, previous studies on the elucidation of primary structures of the DIMs were developed using a methodology combining matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), high-performance liquid chromatography (HPLC), enzymatic digestion and Edman degradation. Nevertheless, some of the DIMs were resistant to classical Edman sequencing. Therefore, mass spectrometry was used to characterize the primary structure of one of the DIMs, namely the N-blocked DIM13 peptide. The complete sequence of DIM13 was established by means of a strategy of nano-electrospray ionisation (ESI)combined with multiple-stage tandem mass spectrometry (MS n )and then was partially confirmed with a combination of enzymatic digestions and MALDI-MS analyses. Interestingly, most of the amino acid sequences have been determined using three-stage (MS3)and four-stage (MS4)tandem experiments, whereas classical tandem mass spectrometry (MS2)yielded only incomplete sequence information. Finally, DIM13 is a 23 amino acid peptide with a pyroglutamic modification at the N terminal position. This work illustrates the remarkable advantages of MS3 and MS4 compared with the MS2 experiment for de novo peptide sequencing. The use of nano-ESI also makes these experiments compatible with the low amount (picomolar level)of DIM13 peptide available for sequencing by ESI-MS n .

Recherches sur les ecdysteroïdes présents dans les cocons de la sangsue Hirudo medicinalis au cours de l'embryogenèse / Investigations on the ecdysteroids in the cocoons of the leech Hirudo medicinalis during embryogenesis

Summary In earlier studies, we demonstrated that leeches contain ecdysone and 20-hy- droxyecdysone. The titre of these molecules was found to fluctuate during the moult/intermoult cycle which is suggestive of a role of ecdysteroids in the control of cuticulogenesis in Hirudinea similar to that observed in Arthropods. We have now extended our investigations to embryonic development in the leech Hirudo medicinalis. During this period of development, which lasts some 30 days, 5–25 embryos grow inside a large cocoon at the expense of a proteolipidic gel (‘albumen’) synthesized by clitellian glandular cells of the parent leech. We have found that the albumen already contains ecdysteroids before the onset of embryogenesis (‘parental ecdysteroids’). At this time, and during the early stages of embryogenesis, several as yet unidentified low polar ecdysteroids predominate in the albumen; the titre of these molecules shows a dramatic decrease in the albumen at mid-embryogenesis which is concomitant with a marked ri...

Presence of bombyxin-like molecules in the brain of Eisenia fetida (Oligochaeta, Lumbricidae)

Summary A monoclonal antibody directed against a synthetic decapeptide corresponding to the N-terminal part of the neurohormone bombyxin reacts with a limited number of neurosecretory cells of the brain of the Oligochete Eisenia fetida. Bombyxin is a member of the superfamily of insulins initially isolated from brains of the Lepidopteran insect Bombyx mori. Our results therefore suggest that Oligochetes use insulin-related molecules as neurohormonal messengers.