Developmental expression of Manduca shade, the P450 mediating the final step in molting hormone synthesis
ABSTRACT The ecdysone 20-monooxygenase (E20MO; 20-hydroxylase) is the enzyme that mediates the conversion of ecdysone (E) to the active insect molting hormone, 20-hydroxyecdysone (20E), which coordinates developmental progression. We report the identification and developmental expression of the Halloween gene shade (shd; CYP314A1) that encodes the E20MO in the tobacco hornworm, Manduca sexta. Manduca Shd (MsShd) mediates the conversion of E to 20E when expressed in Drosophila S2 cells. In accord with the central dogma, the data show that Msshd is expressed mainly in the midgut, Malpighian tubules, fat body and epidermis with very low expression in the prothoracic gland and nervous system. Developmental variations in E20MO enzymatic activity are almost perfectly correlated with comparable changes in the gene expression of Msshd in the fat body and midgut during the fifth instar and the beginning of pupal-adult development. The results indicate three successive and overlapping peaks of expression in the fat body, midgut and Malpighian tubules, respectively, during the fifth larval instar. The data suggest that precise tissue-specific transcriptional regulation controls the levels, and thereby the activity, of the Manduca E20MO.
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- "E20M) is the insect cytochrome P450-dependent steroid hydroxylase responsible for the conversion of the arthropod molting hormone ecdysone (E) to its more active metabolite, namely, 20-hydroxyecdysone (20E)  . The nature, regulation, and molecular biology of E20M were elucidated predominantly employing the tobacco hornworm, Manduca sexta, as the model      . Indeed, the developmental impact of E20M was made evident in midgut tissue of the M. sexta fifth larval instar where E20M activity increases 50-fold between days four and five of the stadium and this increase is inextricably tied to the onset of wandering stage behavior   . "
ABSTRACT: The plant allelochemical, quinizarin (1,4-dihydroxy-9,10-anthraquinone), and five anthraquinones that were synthesized from quinizarin, viz., 1,4-anthraquinone; 2-hydroxy-1,4-anthraquinone; 2-methoxy-1,4-anthraquinone; 9-hydroxy-1,4-anthraquinone; and 9-methoxy-1,4-anthraquinone, were assessed as to their effects on the essential, P450-dependent ecdysone 20-monooxygenase system of the insect model Manduca sexta (tobacco hornworm). This steroid hydroxylase converts the arthropod molting hormone, ecdysone, to the physiologically required 20-hydroxyecdysone form. M. sexta fifth larval instar midgut homogenates were incubated with increasing concentrations (10-8 to 10-3 M) of each of the six anthraquinones followed by ecdysone 20-monooxygenase assessments using a radioenzymological assay. Four of the five anthraquinones exhibited I50’s of about 4 x 10-6 to 6 x 10-2 M. The most effective inhibitors were 2-methoxy-1,4-anthraquinone and 1,4-anthraquinone followed by 9-hydroxy-1,4 anthraquinone and 9-methoxy-1,4 –anthraquinone. At lower concentrations the latter anthraquinone stimulated E20M activity. Quinizarin was less inhibitory and 2-hydroxy-1,4-anthraquinone was essentially without effect. Significantly, these studies make evident for the first time that anthraquinones can affect insect E20M activity, and thus insect endocrine regulation and development, and that a relationship between anthraquinone structure and effectiveness is apparent. These studies represent the first demonstrations of anthraquinones affecting any steroid hydroxylase system.International Journal of Zoology 10/2014; 2014:8. DOI:10.1155/2014/261512
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- "This suggests that other tissues may have roles in ecdysteroid biosynthesis. According to previous studies, Malphigian tubules may release 20E into the hemolymph, whereas the midgut accumulates polar ecdysteroid metabolites prior to their excretion (Feyereisen et al. 1978, 1980; Rewitz et al. 2006b), which indicates that the Malpighian tubules may function not only in the excretion of 20E but also in maintaining the hemolymph 20E titer that is elicited during molting to the pupa. Moreover, recent work showed that 20E was involved in the differentiation of stem cells from the midgut of the caterpillar S. littoralis (Smagghe et al. 2005). "
ABSTRACT: Abstract 20-Hydroxyecdyone, an active form of ecdysteroid, is the key hormone in insect growth and development. Halloween genes encode ecdysteroidogenic enzymes, including cytochrome P450 monooxygenase. CYP307A1 (spook) is accepted as an enzyme acting in the so-called 'black box' that includes a series of hypothetical and unproven reactions that finally result in the oxidation of 7-dehydrocholesterol to diketol. In this study, the Holcocerus hippophaecolus Hua (Lepidoptera: Cossidae) CYP307A1 (HhSpo) gene was identified and characterized. The obtained cDNA sequence was 2084 base pairs with an open reading frame of 537 animo acids, in which existed conserved motifs of CYP450 enzymes. The transcript profiles of HhSpo were analyzed in various tissues of final instar larvae. The highest expression was observed in the prothoracic gland, while expression level was low but significant in other tissues. These results suggest that the sequence character and expression profile of HhSpo were well conserved and provided the basic information for its functional analysis.Journal of Insect Science 06/2013; 13(56):56. DOI:10.1673/031.013.5601 · 1.03 Impact Factor
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- "Despite the large numbers of P450s found in insects, the number of orthologous P450s conserved between insects is small. In general, it has been observed that conserved P450s have developmental roles, such as the P450s involved in 20E biosynthesis in D. melanogaster and Manduca sexta, –. The conservation of Cyp301a1 in insects indicates that Cyp301a1 is likely to play an important role. "
ABSTRACT: The sequencing of numerous insect genomes has revealed dynamic changes in the number and identity of cytochrome P450 genes in different insects. In the evolutionary sense, the rapid birth and death of many P450 genes is observed, with only a small number of P450 genes showing orthology between insects with sequenced genomes. It is likely that these conserved P450s function in conserved pathways. In this study, we demonstrate the P450 gene, Cyp301a1, present in all insect genomes sequenced to date, affects the formation of the adult cuticle in Drosophila melanogaster. A Cyp301a1 piggyBac insertion mutant and RNAi of Cyp301a1 both show a similar cuticle malformation phenotype, which can be reduced by 20-hydroxyecdysone, suggesting that Cyp301a1 is an important gene involved in the formation of the adult cuticle and may be involved in ecdysone regulation in this tissue.PLoS ONE 05/2012; 7(5):e36544. DOI:10.1371/journal.pone.0036544 · 3.23 Impact Factor