Methoprene-tolerant is essential for embryonic development of the red flour beetle Tribolium castaneum.

Insect juvenile hormone (JH) is well known to regulate post-embryonic development and reproduction in concert with ecdysteroids in a variety of insect species. In contrast, our knowledge on the role of JH in embryonic development is limited and inconsistent. Preceding studies indicate that JH biosynthesis or JH signaling genes are dispensable in holometabolous Drosophila melanogaster and Bombyx mori, while essential in hemimetabolous Blattella germanica. In the red flour beetle Tribolium castaneum, we performed functional analyses of key factors in JH signaling, i.e. the JH receptor Methoprene-tolerant (Met) and the early JH-response gene Krüppel homolog 1 (Kr-h1) using parental RNA interference. Knockdown of Met resulted in a significant reduction in hatching rates and survival rates in the first and second larval instars. Meanwhile, knockdown of Kr-h1 caused no significant effect on hatching or survival. The unhatched embryos under Met knockdown developed up to the late embryonic stage, but their body shape was flat and tubby compared with the controls. Attempts to suppress JH biosynthesis by parental RNA interference of JH biosynthetic enzymes were unsuccessful due to insufficient knockdown efficiency. These results suggested that Met but not Kr-h1 is essential for the embryonic development of T. castaneum, although involvement of JH still remains to be examined. Taken together, the function of Met in embryonic development seems to be diverse among insect species.

Expressional and functional analysis of CYP15A1, a juvenile hormone epoxidase, in the red flour beetle Tribolium castaneum.

Juvenile hormone (JH) is synthesized and secreted by the corpora allata. In the final two steps of JH biosynthesis, farnesoic acid (FA) is converted to JH through methylation by JH acid O-methyltransferase (JHAMT) and epoxidation by the cytochrome P450 enzyme CYP15. In the present study, we identified a homolog of CYP15 from the red flour beetle Tribolium castaneum (TcCYP15A1), and analyzed its expression as well as its role in JH biosynthesis. Quantitative RT-PCR analysis showed that the level of TcCYP15A1 mRNA was high in the embryonic stage as well as in the middle of the final larval instar. In the embryonic stage, the transcript level of TcCYP15A1 started to increase 30h after egg laying (AEL), peaked 54-60h AEL, and was followed by an increase of TcJHAMT mRNA, suggesting that JH biosynthesis started at this time point. TcCYP15A1 mRNA was present, but not exclusively so in the larval corpora allata. The recombinant TcCYP15A1 protein epoxidized both FA and methyl farnesoate (MF) in highly stereo-specific manners. These results confirmed that TcCYP15A1 is involved in JH biosynthesis. The RNAi-mediated knockdown of TcCYP15A1 in the pre-final larval instar did not result in precocious metamorphosis to pupa, indicating that MF may exhibit JH-like activity in order to maintain the larval status. The double knockdown of TcJHAMT and TcCYP15A1 resulted in pupae and adults with shorter wings, suggesting that the precursors of JH, JH acid and MF, may be essential for wing expansion.