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Rewitz KF, Rybczynski R, Warren JT, Gilbert LI. Identification, characterization and developmental expression of Halloween genes encoding P450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology

Department of Life Sciences and Chemistry, Roskilde University, P.O. Box 260, 4000 Roskilde, Denmark.
Insect Biochemistry and Molecular Biology (Impact Factor: 3.45). 04/2006; 36(3):188-99. DOI: 10.1016/j.ibmb.2005.12.002
Source: PubMed

ABSTRACT

The insect molting hormone 20-hydroxyecdysone (20E) plays a central role in regulating gene expression during development and metamorphosis. In many Lepidoptera, the pro-hormone 3-dehydroecdysone (3DE), synthesized from cholesterol in the prothoracic gland, is rapidly converted to ecdysone (E) by a hemolymph reductase, and E is subsequently converted to 20E in various peripheral target tissues. Recently, four Drosophila melanogaster P450 enzymes, encoded by specific Halloween genes, were cloned and functionally characterized as mediating the last hydroxylation steps leading to 20E. We extended this work to the tobacco hornworm Manduca sexta, an established model for endocrinological and developmental studies. cDNA clones were obtained for three Manduca orthologs of CYP306A1 (phantom; phm, the 25-hydroxylase), CYP302A1 (disembodied; dib, the 22-hydroxylase) and CYP315A1 (shadow; sad, the 2-hydroxylase), expressed predominantly in the prothoracic gland during the fifth (final) larval instar and during pupal-adult development, with fifth instar mRNA levels closely paralleling the hemolymph ecdysteroid titer. The data indicate that transcriptional regulation of phm, dib and sad plays a role in the developmentally varying steroidogenic capacities of the prothoracic glands during the fifth instar. The consistent expression of the Halloween genes confirms the importance of the prothoracic glands in pupal-adult development. These studies establish Manduca as an excellent model for examining the regulation of the Halloween genes.

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    • "The findings reported in the present study are the first to establish the gut as a major organ of spook expression in any arthropod. Typically the Halloween genes (except shade) are expressed in prothoracic glands of immature insects (Rewitz et al., 2006; Iga & Smagghe, 2010) or in ovaries of adults (Petryk et al., 2003; Rewitz et al., 2007), where the enzymes contribute to the production of ecdysteroids during moulting or reproduction, respectively. In crustaceans , the enzymatic activities for ecdysteroidogenesis are found in the Y organ or ovaries (Mykles, 2011; Tom et al., 2013). "
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    ABSTRACT: The ecdysteroid biosynthetic pathway involves sequential enzymatic hydroxylations by a group of enzymes collectively known as Halloween gene proteins. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), were identified in varroa mites and sequenced. Phylogenetic analyses of predicted amino acid sequences for Halloween orthologues showed that the acarine orthologues were distantly associated with insect and crustacean clades indicating that acarine genes had more ancestral characters. The lack of orthologues or pseudogenes for remaining genes suggests these pathway elements had not evolved in ancestral arthropods. Vdspo transcript levels were highest in gut tissues, while Vddib transcript levels were highest in ovary-lyrate organs. In contrast, Vdshd transcript levels were lower overall but present in both gut and ovary-lyrate organs. All three transcripts were present in eggs removed from gravid female mites. A brood cell invasion assay was developed for acquiring synchronously staged mites. Mites within 4 h of entering a brood cell had transcript levels of all three that were not significantly different from mites on adult bees. These analyses suggest that varroa mites may be capable of modifying 7-dehydro-cholesterol precursor and hydroxylations of other steroid precursors, but whether the mites directly produce ecdysteroid precursors and products remains undetermined.
    Full-text · Article · Jun 2015 · Insect Molecular Biology
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    • "Genes with specific functions in development are noted in orange (Hwn Halloween: Gilbert 2004, Namiki et al. 2005, Rewitz et al. 2006a, nompH1: Willingham and Keil 2004; bb: Rewitz and O'Connor 2011; cutl: Sztal et al. 2012 cutHC: Qiu et al. 2012) those associated with insecticide resistance are in purple (DDT-R; Daborn et al. 2002, Rst(DDT): Amichot et al. 2004, and Rst(luf): Bogwitz et al. 2005) and others that have been the focus of publications are represented by blue lettering (Hrd: Hardstone et al. 2006, lau: Helvig et al. 2004, temp: Kang et al. 2011, agg: Dierick and Greenspan 2006, and SXE1: Fujii et al. 2008). RNAi-L refers to genes shown to be lethal in an RNAi screen of Chung et al. (2009) and RNAi-S are sublethal in that screen. "
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    ABSTRACT: We map 114 gene gains and 74 gene losses in the P450 gene family across the phylogeny of twelve Drosophila species by examining the congruence of gene trees and species trees. While the number of P450 genes varies from 74-94 in the species examined, we infer that there were at least 77 P450 genes in the ancestral Drosophila genome. One of the most striking observations in the data set is the elevated loss of P450 genes in the Drosophila sechellia lineage. The gain and loss events are not evenly distributed among the P450 genes - with 30 genes showing no gene gains or losses whereas others show as many as 20 copy number changes among the species examined. The P450 gene clades showing the fewest number of gene gain and loss events tend to be those evolving with the most purifying selection acting on the protein sequences, although there are exceptions, such as the rapid rate of amino acid replacement observed in the single copy phantom (Cyp306a1) gene. Within D. melanogaster we observe gene copy number polymorphism in ten P450 genes including multiple cases of inter-paralog chimeras. Non-allelic homologous recombination (NAHR) has been associated with deleterious mutations in humans, but here we provide a second possible example of an NAHR event in insect P450s being adaptive. Specifically we find that a polymorphic Cyp12a4/Cyp12a5 chimera correlates with resistance to an insecticide. While we observe such inter-paralog exchange in our within-species datasets, we have little evidence of it between species, raising the possibility that such events may occur more frequently than appreciated but are masked by subsequent sequence change.
    Full-text · Article · Apr 2014 · Genome Biology and Evolution
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    • "Thus, it can be speculated that the temporal expression pattern of Sfdib and Lsdib coincides with E and 20E titers in the haemolymph. Similarly, the variations of expression levels of Bmdib and Msdib were correlated with the changes in E and 20E titers in the haemolymph of B. mori [20] and M. sexta [21]. Moreover, we found that Sfdib and Lsdib were expressed at higher levels in the thoraces where prothoracic glands were located, compared to the mRNA levels in corresponding heads or abdomens. "
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    ABSTRACT: Sogatella furcifera and Laodelphax striatellus are economically important rice pests in China by acting as vectors of several rice viruses, sucking the phloem sap and blocking the phloem vessels. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. A cytochrome P450 monooxygenase CYP302A1 (22-hydroxylase), encoded by the Halloween gene disembodied (dib), plays a critical role in ecdysteroidogenesis. The objective of this study is to test whether dib genes are potential targets for RNA interference-based management of S. furcifera and L. striatellus. We cloned and characterized Sfdib and Lsdib. The open reading frame regions of dib genes were generated and used for designing and constructing dsRNA fragments. Experiments were conducted using oral delivery of dsdib to investigate the effectiveness of RNAi in S. furcifera and L. striatellus nymphs. Real-time quantitative reverse transcriptase-PCR analysis demonstrated that continuous ingestion of dsdib at the concentration of 0.01, 0.05 and 0.50 mg/ml diminished Sfdib expression levels by 35.9%, 45.1% and 66.2%, and ecdysone receptor (SfEcR) gene mRNA levels by 34.0%, 36.2% and 58.5% respectively in S. furcifera, and decreased Lsdib expression level by 18.8%, 35.8% and 56.7%, and LsEcR mRNA levels by 25.2%, 46.8% and 68.8% respectively in L. striatellus. The reduction in dib and EcR transcript abundance resulted in observable phenotypes. The development of nymphs was impaired and the survival was negatively affected. Our data will enable the development of new insect control strategies and functional analysis of vital genes in S. furcifera and L. striatellus nymphs.
    Full-text · Article · Jan 2014 · PLoS ONE
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