Activity of kk-42 in combinated treatment with RH-0345 or 20-hydroxyecdysone on morphometric measurements and free ecdysteroid in eggs of mealworms.

RH-0345 (halofenozide), a bisacylhydrazine derivative, is a nonsteroidal ecdysteroid agonist that mimics the action of the moulting hormones, while KK-42, an imidazole compound, is a potent inhibitor of ecdysteroid biosynthesis. Recent experiments with Tenebrio molitor suggested that the reduction of ecdysteroid titer, leading to a reduction of reproductive capacity, is due to a direct and rapid action of KK-42 on ecdysterold biosynthesis. Moreover, RH-0345 could partly restore the effects on reproductive events induced by KK-42. On the other hand, RH-0345 was found to affect growth and development of ovaries in a manner similar to that 20-hydroxyecdysone (20-E) and increased the ecdysteroid production. Therefore, the present study evaluates KK-42 applied topically (10 microg/insect) in combination with RH-0345 or 20-E in order to obtain more information on the mode of action of this compound on reproduction in T. molitor. In a first series of experiments, the compounds were assayed on morphometric measurements of freshly laid eggs. Results showed that both the weight and the volume of eggs increase significantly in series treated by KK-42 followed by 20-hydroxyecdysone as compared to controls and treated series by KK-42 followed by RH-0345. Data from enzyme immunoassay measurements revealed that KK-42 applied before 20-E was found to reduce significantly the amounts of free ecdysteroids in eggs comparatively to the others series.

In vitro effects of RH-0345 and KK-42 on ecdysteroid levels and cuticle synthesis by pupal integument of mealworms.

In this study, the effects of two insect growth regulators (KK-42 and RH-0345), applied either alone or in combination, were evaluated on the ecdysteroid production in vitro. Integument explants from the abdominal sternites of newly ecdysed pupae of mealworms, Tenebrio molitor, were cultured and the amounts of ecdysteroids, released into the culture medium, were determined by an enzyme immunoassay (EIA) after various intervals of incubation. In combined treatments, explants were cultured for the first two days in a medium added with the first compound and then transferred in new medium containing the second compound. EIA measurements showed that RH-0345, either alone or followed by KK-42, resulted in higher amounts of ecdysteroids as compared to controls. In contrast, KK-42 alone caused a significant reduction. But, when KK-42 was followed by RH-0345, the ecdysteroid amounts were equal to controls. In another series of experiments, the effects on cuticle secretion were examined. Only in the case that explants were treated with RH-0345, either alone or followed by KK-42, new cuticle synthesis was observed.

RH-0345 restored partly the effects induced by KK-42 on reproductive events in mealworms.

In the mealworm, Tenebrio molitor L., KK-42, an imidazole derivative, was found to reduce ovarian ecdysteroid production, while RH-0345, a benzoylhydrazine analogue, has been reported to affect growth and development of mealworm ovaries in a manner similar to 20-hydroxyecdysone. In order to determine whether the reduction of reproduction events is dependent on inhibition of ecdysteroid production by ovaries, RH-0345 (10 micrograms/insect) was applied before or after topical application of KK-42 (10 micrograms/insect) on newly emerged adult females. KK-42 applied alone increased significantly the pre-oviposition period compared to controls. It also significantly delayed egg-laying compared to combined treatments using KK-42 before or after RH-0345. KK-42 applied alone or with and before RH-0345, was found to reduce significantly the duration of oviposition period. Moreover, there was a significant difference in the duration of oviposition period between the two combined treatments. The fecundity was significantly reduced in all treated series compared to controls. In addition, the fecundity recorded in treated series by RH-0345 alone or before KK-42 was significantly higher than in KK-42 treated series. Lastly, the viability of eggs laid was significantly reduced in all treated series compared to controls. Moreover, the combined treatment pronounced the reduction in egg viability. Free ecdysteroids (ecdysone and 20-hydroxyecdysone) in eggs of control and RH-0345 treated females were analyzed by an enzymo-immunoassay using two specific antibodies.