0000000000146923
AUTHOR
Jean-paul Delbecque
Diflubenzuron-Induced alterations during in vitro development ofTenebrio molitor pupal integument
The effects of diflubenzuron (DFB) in Tenebrio molitor pupae were first investigated on cuticle secretion induced by 20-hydroxyecdysone in vitro. The sternal integuments were treated by DFB either 3 days before culture or during culture. DFB, when applied before culture, did not prevent the molting hormone from inducing a new cuticle deposition by integument explants in vitro. However, this cuticle showed several architectural alterations and a thickness reduction. When applied during the culture in the presence of 20-hydroxyecdysone, DFB at high dose (≥ 20 μg/ml) was able to inhibit cuticle secretion, but lower doses (⩽ 10 μg/ml) resulted in epicuticle deposition. These observations confir…
Is tubulin the sole antigen recognized by a putative anti-bursicon antibody?
Abstract A 56-kDa polypeptide suspected to be the tanning hormone `bursicon' was analyzed using the monoclonal antibody (mAb) 01C10 of Song and Ma. We studied the beetle Tenebrio molitor, for which data on bursicon have been recently published. After purification by two-dimensional gel electrophoresis of brain proteins, the immunoreactive 56-kDa polypeptide was trypsinated and microsequenced. The obtained sequences revealed a high homology with α- and β-tubulins. In a complementary study, immunoreactive clones were isolated, using the 01C10 mAb, from a library in expression vector obtained from Drosophila melanogaster head cDNAs. Again, the isolated clones were found, after cDNA sequencing,…
Sterol profiles of Serratula tinctoria, a plant producing ecdysteroids: plant and cell cultures
National audience
2-Deoxyecdysone is a circulating ecdysteroid in the beetle Zophobas atratus
A qualitative analysis of ecdysteroids has been performed during the post-embryonic development of the tenebrionid beetle, Zophobas atratus, by high performance liquid chromatography (HPLC) combined with enzyme immunoassay (EIA) using two different antibodies. Three HPLC peaks were found to be immunoreactive, in hemolymph extracts of both sexes. Moreover, these peaks had ecdysteroid-like UV spectra, determined using a photodiode array detector. The use of two different HPLC systems (reverse and normal phases), in combination with two different EIA antibodies, allowed us to identify 20-hydroxyecdysone (20E) and ecdysone (E), as the two main ecdysteroids, but also suggested the presence of 2-…
Postembryonic development of Zophobas atratus Fab. (Coleoptera: Tenebrionidae) under crowded or isolated conditions and effects of juvenile hormone analogue applications
Abstract Pupation in Zophobas atratus is dependent on isolation and this provides a model for study of the onset of metamorphosis in beetles. We have analyzed the effects of isolation vs grouping on certain developmental parameters; the number of larval instars; their duration and weight. Sex differences were detected, such as the occurrence of regressive moults in females. Moreover, the development of Z. atratus is discussed in relation to Tenebrio molitor , the physiological timing of which appeared in part extendable to Z. atratus . Experiments show that 4–6 days of isolation are required for commencement of metamorphosis, as indicated by an immobile crooked posture observed at the end o…
Biological activity of flucycloxuron, a novel benzoylphenylurea derivative, onTenebrio molitor: comparison with diflubenzuron and triflumuron
Flucycloxuron, a novel benzoylphenylurea (BPU) derivative, exhibited insecticidal activity when injected into newly ecdysed pupae ofTenebrio molitor. Mortality occurs because of defective adult ecdysis. Treatment caused a reduction in both cuticle thickness and incorporation of14C-labelled precursor into chitin, although it had no significant effect on the protein synthesis. The potencies of other BPU compounds as inhibitors of chitin biosynthesis have been examined and results showed that diflubenzuron was less effective than either flucycloxuron or triflumuron.
Calcium inhibits ovarian steroidogenesis in the blowfly Phormia regina.
1479-6805 0022-0795; Calcium is frequently involved in the stimulation of steroidogenesis in gonads and endocrine glands, generally in association with cAMP. However, our present observations show that it has the opposite effect in the ovary of the blowfly Phormia regina. Our in vitro experiments first showed that extracellular calcium does not play a role during the stimulation of steroidogenesis in fly ovaries; indeed steroidogenesis was activated in vitro as efficiently in a medium with or without calcium, either by pharmacological compounds mimicking cAMP signaling or by active brain extracts. When calcium was experimentally introduced into biosynthetic cells by ionophores or liberated …
Genetic Transformation of Serratula tinctoria (Dyer’s Savory) for Ecdysteroid Production
Serratula tinctoria is a perennial plant of the Compositae family with medium-sized, serrated leaves and purple flowers (Loste 1937). This plant, also known as dyer’s savory, is widespread in Europe but with an irregular distribution. Inflorescences (capitula) are purple and are usually unisexual, staminate, or pistillate. In Europe, the flowering period extends from July to September. More than 40 species have been described in Europe, North Africa, and Asia. The plants produce large amounts of secondary metabolites, in particular ecdysteroids at very high concentration in roots (up to 2% dry wt.), in flowers, and in leaves (Bathori et al. 1986; Rudel et al. 1992; Corio-Costet et al. 1993b…
Ecdysteroid titres in a tenebrionid beetle, Zophobas atratus: effects of grouping and isolation.
Metamorphosis in Zophobas atratus is dependent on isolation: when kept in grouped conditions, larvae undergo numerous supernumerary moults, growing in size, without pupating. This beetle thus represents an interesting model for the analysis of possible differences in the endocrine regulation of normal vs. supernumerary larval moults. In this study, the ecdysteroid titres have been analysed in this species, using enzyme immunoassay. The hormonal variations of larvae undergoing normal or supernumerary larval cycles were particularly examined, in either grouped or isolated conditions. Normal larval cycles presented very similar ecdysteroid variations in grouped as well as isolated conditions, …
Developmental changes and acetylcholinesterase activity in the metamorphosing brain ofTenebrio molitor: Correlation to ecdysteroid titers
The brain of Tenebrio molitor exhibited marked fluctuations in acetylcholinesterase (AChE) activity throughout metamorphosis. This was true AChE activity, since it was inhibited by high substrate concentrations and by 10 μM of the specific AChE inhibitor BW284C51 [(1,5-bis'4-allyldimethylammoniumphenyl)-pentan-3-one dibromide] but not by iso-OMPA (tetraisopropylpyrophosphoramide), a cholinesterase (but not AChE) inhibitor. The histochemical AChE activity was localized in the neuropile and the nuclear envelope of neurons and glial cells. The enzyme extracted from brains with 1% Triton X-100 and 1 M NaCl sedimented as a single peak in a sucrose density gradient, with a sedimentation coefficie…
Chenopodium album L. (Fat Hen): In Vitro Cell Culture, and Production of Secondary Metabolites (Phytosterols and Ecdysteroids)
The name Chenopodium is derived from the Greek words chenos (goose) and podos (foot), because the leaves often resemble goose feet. This genus consists of ca. 120 species, widely distributed over the world, 45 of which have been described in India.
Cyclic AMP-dependent and independent stimulations of ovarian steroidogenesis by brain factors in the blowfly, Phormia regina.
0303-7207 doi: DOI: 10.1016/S0303-7207(00)00312-9; The involvement of cyclic-AMP (cAMP) as a potential second messenger in the neurohormonal control of ovarian steroidogenesis was investigated in the adult female blowfly Phormia regina. Individual measurements of ovarian cAMP concentrations and of ovarian biosynthesis of ecdysteroids, stimulated after a protein meal, demonstrated that steroidogenesis is preceded by a peak of cAMP in the ovaries. In vitro, ovarian steroidogenesis was stimulated by cell-permeable analogues of cAMP and by forskolin. Crude brain extracts were also able to elicit a rise of cAMP in the ovaries in vitro and the secretion of ecdysteroids into the medium: such extra…
Serratula tinctoria (L.) (Dyer's savory): In vitro culture and the production of ecdysteroids and other secondary metabolites
The name Serratula is derived from the Latin word serra, which means saw. It designates perennial plants of the Compositae family with medium-sized, serrated leaves and purple flowers (Loste 1937). More than 40 species have been described in Europe, North Africa, and Asia. The most common is Serratula tinctoria, also known as dyer’s savory. This species, 30 to 80 cm high, grows on all kind of soils, and is widely distributed in central and northern Europe (synonym: Carduus tinctoria, Scop; French name: serratule des teinturiers; German name: Faberscharte). In France, for example, Serratula tinctoria is widespread but with an irregular distribution, rare in the north and near the Mediterrane…
Inhibition of ovarian steroidogenesis by cyclic-GMP in a fly
1479-6805 0022-0795; Previous investigations in the female blowfly Phormia regina have shown that 3-isobutyl-1-methylxanthine (IBMX), a broad spectrum inhibitor of phosphodiesterases (PDEs), fails to mimic the steroidogenic effects of cAMP on ovaries, although it efficiently increases the concentrations of this second messenger. In this study, experiments carried out to clear up this contradiction demonstrated that IBMX, besides its effect on cAMP, also increased cGMP concentrations in blowfly ovary and that these two cyclic nucleotides controlled ovarian steroidogenesis antagonistically. In particular, a selective inhibitor of cGMP-specific PDEs, unlike IBMX, had a very strong negative eff…
Efficacy of hexaflumuron against the fungus-growing termitePseudacanthotermesspiniger(Sjöstedt) (isoptera, macrotermitinae)
The efficacy of hexaflumuron, a benzophenylurea insecticide, has been studied for the first time on a fungus-growing termite (Pseudacanthotermes spiniger Sjostedt, Macrotermitinae). Results show that hexaflumuron could be useful in treating infestations of such pest species, which are of great economic importance in many tropical and equatorial countries. Foraging workers harvested and introduced treated food into the nest and subsequently contaminated the brood by trophallaxis. Hexaflumuron showed potent larvicidal activity. The compound did not appear to be rapidly degraded by the digestive enzymes of termite workers, nor by the symbiotic fungus Termitomyces eurhizus Heim growing on fungu…
Sterol and ecdysteroids profiles of Serratula tinctoria (L.) : plant and cell cultures producing steroids
Abstract Cell suspension cultures have been obtained from Serratula tinctoria, a plant producing ecdysteroids. Sterol profiles and ecdysteroid contents have been analysed and compared in plants and cell cultures. In particular, the composition of free and esterified sterols was investigated using appropriate analytical techniques. In plants, esterified sterols were more abundant (50–70% of the total sterol) than in cell cultures (13–36%). A selectivity for sterol esterification was noted: in plants, the triterpenes (as amyrins) were esterified, whereas it was the 4-desmethylsterols (sitosterol and cholesterol) in cell cultures. Ecdysteroids were present in higher quantities in plant (0.1–1.…
The onset of metamorphosis in Tenebrio molitor larvae (Insecta, Coleoptera) under grouped, isolated and starved conditions
Abstract The post-embryonic development of the beetle Tenebrio molitor presents a variable number of larval instars. Several parameters (instar length, time of apolysis and cuticle deposition) were compared during the larval-larval and larval-pupal cycles of mealworms over 50 mg, reared in grouped or isolated conditions. In grouped conditions comparable to mass breeding, larval-larval and larval-pupal apolyses were found to occur at the same time, but instar duration was longer in the case of prepupae. However, isolation was found to accelerate larval-pupal (but not larval-larval) apolyses and to reduce the number of larval instars, whereas starvation inhibited larval-larval (but not larval…
Evolution of ecdysteroids and of their apolar conjugates during the post-embryonic development of the tickOrnithodoros moubata
The ecdysteroid (ES) content of the soft tick Ornithodoros moubata was investigated during the five successive nymphal molting cycles by means of an enzyme immunoassay (EIA). Samples were submitted to esterase hydrolysis in order to release free ecdysteroids from the acyl-ester conjugates (AP = apolar products). Crude and hydrolysed extracts were then analyzed by EIA using two different antibodies, a monoclonal raised against 20-hydroxyecdysone (20E) and a polychlonal raised against ecdysone (E). With the crude extracts, each molting cycle was associated with an ES peak, occurring in the middle of the instar. 20E was preponderant during the first 2 nymph cycles, but the proportion of E and …
Alternative sites for ecdysteroid production in insects
Summary Several evidences have been obtained in various insect species demonstrating that, besides prothoracic glands and ovaries, other tissues could be alternative sites of moulting hormone production. After a detailed review on the various methods of investigation and criteria required to validate such observations, the nature of these sites, namely oenocytes, epidermis and testes, is discussed. Their possible involvement in moulting and/or reproduction is analyzed, giving the opportunity to put forward several new hypotheses. In particular, autocrine and paracrine secretions of ecdysteroids could play a role in localized developmental events, more difficult to control from endocrine gla…
Quantification of ecdysteroids by immunoassay: comparison of enzyme immunoassay and radioimmunoassay.
Abstract The performance of enzyme immunoassay (EIA) and radioimmunoassay (RIA) in the quantitative analysis of ecdysteroids was compared. The EIA was found to be at least equivalent to the RIA with respect to analytical range and sensitivity and to be more comfortable with respect to safety and time saving. When biological samples were analyzed by both assays a good correlation (r = 0.83) was found. Since the EIA has certain advantages over the RIA, we now recommend the use of the former assay for the quantification of ecdysteroids.
Control of ovarian steroidogenesis in insects: A locust neurohormone is active in vitro on blowfly ovaries
0016-6480 doi: DOI: 10.1016/j.ygcen.2009.04.034; Ovarian steroidogenesis controlling insect reproduction is mainly regulated by brain gonadotropins liberated from corpora cardiaca (CC). Till now, different neurohormones have been identified in two insect groups only, locusts and mosquitoes, and it is unknown whether they could be active in other insects. In order to complete previous observations on the control of ovarian steroidogenesis in the blowfly, Phormia regina, we examined whether neuropeptides isolated from locust CC have an effect in vitro on ovarian steroidogenesis in our dipteran model. Our experiments showed that crude extracts from locust CC efficiently stimulated steroidogene…
Biosynthèse des stérols chez Chenopodium album et Serratula tinctoria: deux plantes produisant des ecdystéroïdes
National audience
Analysis of Hemolymph Ecdysteroids in the Female Earwig: Labidura riparia
International audience; The nature of hemolymph ecdysteroids was analyzed by high performance liquid chromatography-radioimmunoassay, during the two main phases of the reproductive cycles of Labidura riparia. 20-hydroxyecdysone and ecdysone were present together with polar and apoiar products.Changes in the ecdysone/20-hydroxyecdysone ratio were evidenced according to the ovarian development. During natural (feeding and sexual phase of the cycle) or induced ovarian development, an increase of the ratio occurred whereas, during ovarian rest (fasting and/or parental phase), a significant decrease was observed.Up to 40 days after ovariectomy, immunoreactive compounds were observed in the hemol…
Effects of the ingestion ofSerratula tinctoria extracts, a plant containing phytoecdysteroids, on the development of the vineyard pestLobesia botrana (Lepidoptera: Tortricidae)
We describe here the effects of extracts from Serratula tinctoria, a plant producing phytoecdysteroids, on the growth and development of Lobesia botrana, an economically important pest in vineyards. Leaves, hairy roots, or semi-purified (by Sep-Pak procedure) methanolic extracts from this plant were incorporated into an artificial diet given to insects. Larval growth, mortality, weight, and sex-ratio were investigated, as well as sterol and ecdysteroid contents. Experimental diets induced an important mortality in the first, second, and third larval instars, but also in pupae. As males appeared more sensitive to extracts, sex ratio was significantly modified on experimental diets (particula…
Control of ovarian steroidogenesis by insulin-like peptides in the blowfly (Phormia regina).
0022–0795/04/0181–147; This study investigated the ability of insulin and of insect insulin-like peptides (ILPs) to stimulate ovarian steroidogenesis in the blowfly Phormia regina. Bovine insulin was active on ovaries isolated in vitro, which showed an age-dependent sensitivity; this peptide progressively stimulated steroidogenesis in ovaries isolated from the third day after adult molt, but not in younger ones, and had maximal activity after the fifth day. This stimulatory effect was observed equally from females reared in the presence or in the absence of males, excluding a regulatory effect of mating. The mode of action of insulin in blowflies did not involve cAMP, but triggered a specif…
Cloning of Two Putative Ecdysteroid Receptor Isoforms from Tenebrio Molitor and their Developmental Expression in the Epidermis during Metamorphosis
Using the Drosophila EcR-B1 cDNA as a probe, we have cloned the putative ecdysteroid receptor from the mealworm Tenebrio molitor. We have isolated two cDNAs with different 5' termini that contain a complete open reading frame. These two cDNAs encode two proteins with distinct N-terminal regions corresponding to two isoforms. The coleopteran receptor is obviously related to the ecdysteroid receptor of other insects, but shares only 89% and 61% amino acid identities with the DNA-binding and ligand-binding domains of the Drosophila receptor, respectively. Its expression pattern has been examined in the epidermis during the last larval instar and pupal stage of T. molitor, in correlation with t…
Ecdysone and ecdysterone in physogastric termite queens and eggs of Macrotermes bellicosus and Macrotermes subhyalinus
Abstract Physogastric queens and freshly laid eggs of two species of termites ( Macrotermes bellicosus and Macrotermes subhyalinus ) are found to contain high levels of ecdysteroids (molting hormones) as indicated by radioimmunoassay and Musca bioassay. Ecdysteroids are accumulated in the ovaries of the queen and then stored in the eggs since newly laid eggs contain ecdysteroid concentrations similar to those found in the ovaries. Gas chromatography-mass spectrometry demonstrates that ecdysone (α-ecdysone) as well as ecdysterone (β-ecdysone) are present in queen ovaries and in eggs and that ecdysone is quantitatively the more important ecdysteroid in both ovary and eggs.