Identification, characterization and developmental expression of Halloween genes encoding P450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm, Manduca sexta.
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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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.Insect Molecular Biology 01/2015; DOI:10.1111/imb.12155 · 2.98 Impact Factor
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ABSTRACT: Cytochrome P450 family (CYP) is a group of proteins virtually found in all living organisms. The main role of most CYPs is to metabolize endo and xenobiotics. Most of the studies on CYP have been carried out in mammals and other vertebrates, however recently a growing interest has been devoted to the identification of CYP isoforms in invertebrates. A gene belonging to the CYP sub-family, CYP356A1, was identified in sanitary sewage-exposed Pacific oysters, Crassostrea gigas. Through heterologous expression, we produced CYP356A1 purified protein and raised a mouse polyclonal antibody. Dot blot tests showed that oysters exposed in situ for 14 days to untreated urban effluent discharges had significantly higher levels of CYP356A1 in digestive gland. Using immunohistochemical techniques we observed that the lining epithelial cells of mantle, stomach and intestine showed a strong CYP356A1 staining, but the mucus and secretory cells were negative. Digestive diverticulum parenchyma and gills lining cells showed strong CYP356A1 reaction, while the filamentary rod (connective tissue) was negative. Free cells, as hemocytes and brown cells also showed CYP356A1 immunoreactions indicating the presence of biotransformation activity in these cells. Male germ cells at early stages expressed CYP356A1 but not sperm mature cells, suggesting that this protein could be involved in the male gonadal development. This study shows the use of a specific antibody to a mollusk CYP isoform and that this protein is inducible in oysters environmentally exposed to urban sewage effluents.Aquatic Toxicology 12/2014; 159:267-275. DOI:10.1016/j.aquatox.2014.12.021 · 3.51 Impact Factor
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ABSTRACT: A cytochrome P450 gene belonging to family9 was isolated from the midgut transcriptome of the termite Coptotermes formosanus Shiraki, for screening enzymes related to biomass degeneration. Some studies show that insect P450 enzymes have ligninase activities for catalyzing lignin degrada-tion. We employed the RACE method to clone this cytochrome P450 gene, named CYP9AX1 (GenBank accession No.JN969113). To the best of our knowledge, CYP9AX1 is the first member of the CYP9 family cloned from this termite. The full-length CYP9AX1 cDNA was 2242 bp long and in-cluded a 1599bp open-reading-frame (ORF), a 61-bp 5'-untranslated region (UTR) and a 592-bp 3'-UTR (excluding the poly-A tail). The CYP9AX1 protein deduced from the ORF contains 532 amino acids with a predicted signal peptide composed of 20 amino acid at its N-terminal and the classic heme-binding domain FXXGXXXCXG (residues 468-477). At position 473, residue Arg (R) changes to Gln (Q), this suggests that CYP9AX1 is a new type of CYP subfamily 9A. The phylogenetic tree showed that C. formo-sanus has high genetic relationship with Blattella germanica and Diploptera punctata. Quantitative RT-PCR assays demonstrated that CYP9AX1 was expressed most abundantly in malpighian tubules, and slightly lower in the head, foregut, midgut and hindgut. The results suggested that CYP9AX1 may be involved in enzymatic detoxification systems of the delignification process in C. formosanus.Sociobiology 12/2012; · 0.36 Impact Factor