CREB-binding protein regulates metamorphosis and compound eye development in the yellow fever mosquito,Aedes aegypti

Juvenile hormones (JH) and ecdysone coordinately regulate metamorphosis in Aedes aegypti. We studied the function of an epigenetic regulator and multifunctional transactivator, CREB binding protein (CBP) in A. aegypti. RNAi-mediated knockdown of CBP in Ae. aegypti larvae resulted in suppression of JH primary response gene, Krüppel-homolog 1 (Kr-h1), and induction of primary ecdysone response gene, E93, resulting in multiple effects including early metamorphosis, larval-pupal intermediate formation, mortality and inhibition of compound eye development. RNA sequencing identified hundreds of genes, including JH and ecdysone response genes regulated by CBP. In the presence of JH, CBP upregulates Kr-h1 by acetylating core histones at the Kr-h1 promoter and facilitating the recruitment of JH receptor and other proteins. CBP suppresses metamorphosis regulators, EcR-A, USP-A, BR-C, and E93 through the upregulation of Kr-h1 and E75A. CBP regulates the expression of core eye specification genes including those involved in TGF-β and EGFR signaling. These studies demonstrate that CBP is an essential player in JH and 20E action and regulates metamorphosis and compound eye development in Ae. aegypti.     

Sumoylation modulates 20-hydroxyecdysone signaling by maintaining USP protein levels in Drosophila

The nuclear receptor complex for the insect steroid hormone, 20-hydroxyecdysone (20E), is a heterodimer of EcR and USP. It has been shown that Drosophila EcR and USP can be sumoylated in mammalian cells, but it is unknown whether EcR-USP sumoylation naturally occurs in Drosophila. In Drosophila cells, USP, but not EcR, was sumoylated by Smt3, the only Drosophila SUMO protein. The presence of EcR enhanced USP sumoylation, which is further enhanced by 20E treatment. In addition to the Lys20 sumoylation site, five potential acceptor lysine residues in USP were predicted and verified. Mutation of the USP sumoylation sites or reduction of smt3 expression by RNAi attenuated 20E-induced reporter activity. Moreover, in the salivary glands, reducing smt3 expression by RNAi decreased 20E-induced reporter activity, gene expression, and autolysosome formation. Importantly, at least partially, the smt3 RNAi-mediated reduction in 20E signaling resulted from decreased protein levels of USP. In conclusion, sumoylation modulates 20E signaling by maintaining USP protein levels in Drosophila.     

Genome-wide comparison of genes involved in the biosynthesis,metabolism, and signaling of juvenile hormone between silkworm and other insects

Juvenile hormone (JH) contributes to the regulation of larval molting and metamorphosis in insects. Herein, we comprehensively identified 55 genes involved in JH biosynthesis, metabolism and signaling in the silkworm (Bombyx mori) as well as 35 in Drosophila melanogaster, 35 in Anopheles gambiae, 36 in Apis mellifera, 47 in Tribolium castaneum, and 44 in Danaus plexippus. Comparative analysis showed that each gene involved in the early steps of the mevalonate (MVA) pathway, in the neuropeptide regulation of JH biosynthesis, or in JH signaling is a single copy in B. mori and other surveyed insects, indicating that these JH-related pathways or steps are likely conserved in all surveyed insects. However, each gene participating in the isoprenoid branch of JH biosynthesis and JH metabolism, together with the FPPS genes for catalyzing the final step of the MVA pathway of JH biosynthesis, exhibited an obvious duplication in Lepidoptera, including B. mori and D. plexippus. Microarray and real-time RT-PCR analysis revealed that different copies of several JH-related genes presented expression changes that correlated with the dynamics of JH titer during larval growth and metamorphosis. Taken together, the findings suggest that duplication-derived copy variation of JH-related genes might be evolutionarily associated with the variation of JH types between Lepidoptera and other insect orders. In conclusion, our results provide useful clues for further functional analysis of JH-related genes in B. mori and other insects.     

Upregulation of the expression of prodeath serine/threonine protein kinase for programmed cell death by steroid hormone 20-hydroxyecdysone

Serine/threonine protein kinases phosphorylate protein substrates to initiate further cellular events. Different serine/threonine protein kinases have varied functions despite their highly conserved homology. We propose prodeath-S/TK, a prodeath serine/threonine protein kinase from the lepidopteran insect Helicoverpa armigera, promotes programmed cell death (PCD) during metamorphosis. Prodeath-S/TK is expressed in various tissues with a high expression level during molting and metamorphosis by 20-hydroxyecdysone (20E) induction. Prodeath-S/TK is localized in the larval midgut during metamorphosis. Prodeath-S/TK knockdown by injecting dsRNA into larval hemocoel suppresses the 20E-induced metamorphosis and PCD, as well as downregulates a set of genes involved in the PCD and 20E signaling pathway. 20E upregulates prodeath-S/TK expression through its nuclear receptor EcR-B1 and USP1. Prodeath-S/TK overexpression in the epidermal cell line leads to PCD with DNA fragmentation and the activation of caspases 3 and 7. Prodeath-S/TK plays role in the cytoplasm. The N-terminal and C-terminal sequences of prodeath-S/TK determine its subcellular location. These data indicate that prodeath-S/TK participates in PCD by regulating gene expression in the 20E signaling pathway.     

Hormonal regulation and developmental role of Krüppel homolog 1, a repressor of metamorphosis,in the silkworm Bombyx mori

Juvenile hormone (JH) has an ability to repress the precocious metamorphosis of insects during their larval development. Krüppel homolog 1 (Kr-h1) is an early JH-inducible gene that mediates this action of JH; however, the fine hormonal regulation of Kr-h1 and the molecular mechanism underlying its antimetamorphic effect are little understood. In this study, we attempted to elucidate the hormonal regulation and developmental role of Kr-h1. We found that the expression of Kr-h1 in the epidermis of penultimate-instar larvae of the silkworm Bombyx mori was induced by JH secreted by the corpora allata (CA), whereas the CA were not involved in the transient induction of Kr-h1 at the prepupal stage. Tissue culture experiments suggested that the transient peak of Kr-h1 at the prepupal stage is likely to be induced cooperatively by JH derived from gland(s) other than the CA and the prepupal surge of ecdysteroid, although involvement of unknown factor(s) could not be ruled out. To elucidate the developmental role of Kr-h1, we generated transgenic silkworms overexpressing Kr-h1. The transgenic silkworms grew normally until the spinning stage, but their development was arrested at the prepupal stage. The transgenic silkworms from which the CA were removed in the penultimate instar did not undergo precocious pupation or larval–larval molt but fell into prepupal arrest. This result demonstrated that Kr-h1 is indeed involved in the repression of metamorphosis but that Kr-h1 alone is incapable of implementing normal larval molt. Moreover, the expression profiles and hormonal responses of early ecdysone-inducible genes (E74, E75, and Broad) in transgenic silkworms suggested that Kr-h1 is not involved in the JH-dependent modulation of these genes, which is associated with the control of metamorphosis.