Jean-Paul Delbecque

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 glands possibly mitosis, meiosis, reprogramming, regeneration or early steps of embryogenesis).

Prothoracic gland involution related to moulting hormone levels during the metamorphosis of Tenebrio molitor L.

Abstract Prothoracic gland (PG) of Tenebrio shows ultrastructural changes which can be correlated with ecdysteroid levels (measured by radioimmunoassay) during larval-pupal development. However, the gland cells begin to degenerate before pupal-adult ecdysis: the PG involution is completed before the moulting hormone peak which triggers pupal-adult development. These facts strongly suggest that another endocrine organ produces moulting hormone needed for adult development.

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 of this period, characterizing the beginning of prepupal development. During this period, return to grouped conditions, as well as applications of a juvenile hormone analogue (ZR 515) prevented metamorphosis and precipitated another larval moult. This suggests that grouped conditions may delay the onset of metamorphosis through the modulation of juvenile hormone titres.

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.

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-pupal) development by delaying or preventing larval apolysis. Our observations confirm that the onset of metamorphosis is linked to a loss of sensitivity to starvation and to an increased requirement of isolation. In isolated conditions, larval-pupal apolysis was found to be preceeded by a characteristic behaviour (transitory period), homologous to the wandering stage of Lepidoptera. Ecdysteroid titres, measured with a radioimmunoassay, showed a very different pattern in a larval-larval cycle (a single high peak beginning at apolysis) compared to a larval-pupal one (several smaller peaks, the first one during the transitory period). The involvement of ecdysteroids and other hormones in the control of developmental variability is discussed.

Effects of the ingestion of Serratula tinctoria extracts, a plant containing phytoecdysteroids, on the development of the vineyard pest Lobesia 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 (particularly with Sep-Pak fractions SP60, SP80, and SP100). Pathophysiological effects were also observed: Larval development was significantly faster on experimental diets and a weight loss, 14% for males and from 12% to 22% for females, was noted (particularly when reared on hairy roots and SP20, SP60, and SP80 extracts). Moreover, sterol and ecdysteroid contents were disturbed after rearing on experimental diets. Arch. Insect Biochem. Physiol. 35:227–235, 1997.

Sterol and ecdysteroid 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 (0.1–1.2% dry wt) than in cell cultures (0.01–0.03%). Analysis confirmed the presence of 20-hydroxyecdysone, 20-hydroxyecdysone-3-acetate and polypodine B as the main compounds. Roots were not only the richest organ in ecdysteroids, but also in cholesterol, lathosterol and 24-methylene-cholesterol. The results obtained suggest interesting relationships between free or esterified sterols and ecdysteroid contents.

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-deoxyecdysone (2dE) as the third hemolymph component. Secretion of putative 2dE, together with E (but not 20E) was also demonstrated in vitro from incubations of prothoracic glands and of tegumental explants. In these experiments, either in vivo or in vitro, 3-dehydroecdysone was never observed. Our observations thus strongly suggest that 2dE is a circulating ecdysteroid in Z. atratus and may function as a prohormone during the development of some insects.

Efficacy of hexaflumuron against the fungus-growing termite Pseudacanthotermes spiniger (Sjostedt) (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 fungus combs. This chitin synthesis inhibitor did not act as a fungicide, since growth of the mycelium of this fungus was not inhibited in vitro.

Polar and apolar metabolites of ecdysteroids during the metamorphosis of Tenebrio molitor

Abstract Radioactive ecdysone was injected into last-instar larvae and pupae of Tenebrio molitor ; then the metabolites, after various incubation times, were analysed using reverse-phase HPLC. In addition to a rapid transformation of ecdysone into 20-hydroxyecdysone, several other compounds were evidenced in small amounts, comigrating with 26-hydroxyecdysone, 20,26-dihydroxyecdysone and ecdysonic acids. However, these compounds did not accumulate and, within a few days, radioactivity was recovered only as two classes of metabolites, a class of polar products and a class of apolar products. Though their identification has not yet been possible, they appeared different from polar and apolar ecdysteroid metabolites described so far. Polar products were hardly studiable using reverse-phase HPLC because they were not retained at all, but anion-exchange HPLC appeared more suitable for their separation. Since in Tenebrio pupae, a peak of ecdysteroids occurs after the degeneration of the prothoracic glands, the hypothesis of a possible recycling of such metabolites into active ecdysteroids has been investigated. However, after injection of tritiated ecdysone into larvac, no radioactivity was recovered as ecdysone or 20-hydroxyecdysone at the pupal peak time. Morcover, hydrolysis experiments using esterases or Helix pomatia enzymes did not transform polar products into active hormones, suggesting that these compounds were not conjugates. Apolar products, on the contrary, were hydrolysable, but liberated almost exclusively polar products. In conclusion, it appears unlikely that the pupal ecdysteroid peak in Tenebrio could be formed from metabolites of ecdysone stored in larvae.