Jean Delachambre

Effects of diflubenzuron on the pupal-adult development of Tenebrio molitor L. (Coleoptera, Tenebrionidae): Growth and development, cuticle secretion, epidermal cell density, and DNA synthesis

Diflubenzuron (DFB), applied by dipping on newly emerged pupae of Tenebrio molitor L., disturbs the pupal-adult development. Four main types of treated insects were obtained according to external morphology: blocked pupae, adults unable to ecdyse, adults partially ecdysed, and adults completely ecdysed. The proportion of these four types varied with the time of treatment during the pupal life. When DFB was administered by dipping at a 10 g/liter concentration to newly emerged pupae, HPLC measurements showed that the different responses were due to a difference in the levels actually incorporated. Histological and ultrastructural observations of sternal cuticles revealed that DFB disturbs the cuticle deposition: reduction of its thickness and modifications of its architecture. Tritiated thymidine incorporation and cell density measurements in sternal epidermis show that DFB affects both mitoses and DNA synthesis in blocked pupae. These findings suggest that DFB interferes with other biochemical processes besides the chitin biosynthesis; whether these effects are primary or secondary remain to be determined.

Abdominal production of ecdysterone and pupal-adult development in Tenebrio molitor (Insecta, Coleoptera).

Abstract Isolated abdomens of Tenebrio molitor of both sexes, ligated at pupal ecdysis, can undergo the adult molt because of their own production of the molting hormone (MH): A great peak in MH is detected by radioimmunoassay 6 to 10 days after ligation. Mass spectrometry indicates that MH consists principally of ecdysterone; small amounts of ecdysone are also detected. Our results indicate that hormonal concentrations in ligated abdomens are close to normal values in intact pupae and that the hormone is released into the hemolymph. The hormonal peak is clearly correlated with the initiation of adult development (apolysis and deposition of adult cuticle). Our data lead to the conclusion that this abdominal production of ecdysterone also occurs in nonligated animals and triggers pupal-adult development.

The in vitro development of the pupal integument and the effects of ecdysteroids in Tenebrio molitor (Insecta, Coleoptera)

SummaryIn order to study the pupal-adult metamorphosis of Tenebrio in vitro, pupal sternites of different ages were cultured in Landureaus medium and their development systematically observed by electron microscopy. In hormone-free medium, explants taken from young pupae do not secrete pupal postecdysial cuticle in vitro, and the epidermis spontaneously detaches from the pupal cuticle. On the contrary, explants taken from pharate adults continue to secrete adult preecdysial cuticle in vitro, and the epidermis never detaches from the cuticle. Ecdysterone in physiological concentrations (0.2 to 4 μg/ml) induces the secretion of a new cuticle in explants from young pupae but the epidermis remains undifferentiated. Ecdysone is necessary for the induction of some adult differentiation. Moreover, the quality of the cuticle secreted in vitro is increased by the addition of 2% foetal calf serum; the best results have thus far been obtained in a medium containing 0.2 μg/ml ecdysone, 1 μg/ml ecdysterone, and 2% foetal calf serum.

cDNA cloning of an adult male putative lipocalin specific to tergal gland aphrodisiac secretion in an insect (Leucophaea maderae)

Lma‐P22 is a cuticular surface protein specific to the tergal gland secretion of Leucophaea maderae adult males which is ingested by females just before copulation. The complete Lma‐P22 cDNA sequence was determined by RT‐PCR using primers based on Edman degradation fragments. The recombinant protein expressed in Escherichia coli was recognized by an anti‐Lma‐P22 antibody. Northern blot analysis indicates that the corresponding mRNA is transcribed only in the epidermis of male tergites. Sequence analysis indicated that Lma‐P22 deduced protein belongs to the lipocalin family. Lipocalins are extracellular proteins which carry hydrophobic compounds and some of them can bind sexual pheromone in vertebrates. Lma‐P22 is the first example of a lipocalin‐like protein involved in insect sexual behavior.

A water-soluble protein specific to the adult cuticle in Tenebrio

Abstract The water-soluble proteins of abdominal cuticle of larvae, pupae and adults subjected to electrophoresis, revealed a specific pattern for each stage. An antibody against an adult specific 18 kdaltons protein was produced. Electrophoresis analysis of SDS slab gel and Western-blots revealed that the 18 kdaltons adult protein is not present in the water-soluble fraction from haemolymph or fat body of pupae which secreted pre-ecdysial adult cuticle. Immuno-histochemical methods showed that the cross-reaction of the immune serum was limited to the apical part of the adult epidermis and to the cuticle lamellae which are not stabilized. Therefore, the 18 kdaltons protein is specific for the adult cuticle and originates in the epidermis.

Total and epidermal cyclic AMP levels related to the variations of ecdysteroids and bursicon during the metamorphosis of the mealworm Tenebrio molitor L.

Abstract Cyclic AMP levels, determined by a sensitive radioimmunoassay in whole Tenebrio molitor and in its epidermis, during metamorphosis, show marked variations which are related to the moulting hormone (MH) levels and to the bursicon activity in the haemolymph. The results show: (1) an inverse relation between MH levels and cyclic AMP concentrations in both the whole body and the epidermis; (2) a good correlation between the maximum value of bursicon activity in the haemolymph and the maximum value of epidermal cyclic AMP. This good temporal relationship suggests that cyclic AMP could be a mediator of bursicon in the epidermis.

Inhibition of ecdysteroid increase by diflubenzuron in Tenebrio molitor pupae and compensation of diflubenzuron effect on cuticle secretion by 20-hydroxyecdysone

Summary Dipping of newly ecdysed Tenebrio molitor pupae in an acetone solution of diflubenzuron prevents most of them (73%) from carrying on further development. Such blocked animals cannot secrete adult cuticle and remain apolysed. Their ecdysteroid levels analysed by radioimmunoassay do not present any increase. However, injection of 20-hydroxyecdysone (2–10 μg) several days after diflubenzuron application allows the secretion of a new cuticle with an abnormal architecture but with a high content in N-acetyl-aminosugars as revealed by fluorescent wheat germ agglutinin.

Epidermal polyamine levels related to cell cycle events during the metamorphosis of Tenebrio molitor L. (insecta, coleoptera): Effect of juvenoid application

Abstract 1. 1. Three polyamines were separated by reverse-phase HPLC and detected by fluorescence in Tenebrio epidermis: putrescine, spermidine and spermine. 2. 2. The levels of these compounds varied during metamorphosis: one peak was observed during the G 2 -arrest preceding the pupal-adult mitotic crisis and a second occurred when cells were again G 2 -arrested after the mitotic period. 3. 3. A juvenile hormone analogue, which inhibits the G 2 -M transition preparing cells to the adult mitoses, was unable to prevent the first polyamine increase suggesting that juvenile hormone does not act on further development via inhibition of polyamine synthesis.

Induction of epidermal cyclic AMP by bursicon in mealworm,Tenebrio molitor

Bursicon active hemolymph ofTenebrio, injected into host pupae deprived in bursicon, induces a 2-fold increase of cyclic AMP in the epidermis of the hosts 5 min after the injection. No increase is observed by injecting bursicon inactive hemolymph or insect Ringer. From these experiments it can be concluded that cyclic AMP is a second messenger of bursicon.