Abstract:The activities of ecdysone oxidase (EO), 3-oxoecdysteroid 3a-reductase (3a-R), and 3-oxoecdysteroid 3p-reductase (3P-R) were determined for epidermis, hemolymph, and fat body of wandering fifth instar Manduca sexta larvae and for midguts of various developmental stages between 3 days after the last larval and 14 days after the pupal ecdysis. The larval midgut was the only organ showing substantial specific activities of EO and 3a-R, and both increased up to the seventh day after ecdysis. Hernolymph and fat bod… Show more
“…The fact that high expression of ecdysone oxidase occurs in the late stage of the last larval instar, when the active molting hormone titer is highest (6), suggests that the enzyme may play a role in deactivation of endogenous ecdysteroids. In Manduca sexta, ecdysone oxidase activity also increases at a similar developmental stage as observed in S. littoralis (36). Also, the developmental profile showed that ecdysone oxidase is highly expressed in the early pupal stage.…”
Section: Figsupporting
confidence: 63%
“…Ecdysone oxidase activity has been demonstrated in midgut, fat body, hemolymph, and integument of larvae in various insect species, although the predominant activity appears to be in midgut during the larval stage (11,35,36). Our Northern blot analysis shows that the mRNA for ecdysone oxidase was highly expressed in the midgut but undetectable in other tissues examined at the prepupal stage in S. littoralis under our conditions (Fig.…”
One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalyzed conversion into 3-dehydroecdysteroid followed by irreversible reduction by 3-dehydroecdysone 3␣-reductase to 3-epiecdysone. We have purified from Spodoptera littoralis the first ecdysone oxidase and subjected it to limited amino acid sequencing. A reverse-transcriptase polymerase chain reaction-based approach has been used to clone the cDNA (2.8 kilobases) encoding this 65-kDa protein.Northern blotting showed that the mRNA transcript was expressed in midgut during the prepupal stage of the last larval instar at a time corresponding to an ecdysteroid titer peak. Conceptual translation of the ecdysone oxidase cDNA and data base searching revealed that the enzyme is an FAD flavoprotein that belongs to the glucose-methanol-choline oxidoreductase superfamily. Ecdysone oxidase represents the only oxidase in eukaryotic animals known to catalyze oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via NAD(P) ؉ -linked dehydrogenases. The injection of RH-5992, an ecdysteroid agonist, induced the transcription of ecdysone oxidase, suggesting that ecdysone oxidase is an ecdysteroid-responsive gene. The gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and FTZ-F1 have been found in the 5-flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a singlecopy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.
“…The fact that high expression of ecdysone oxidase occurs in the late stage of the last larval instar, when the active molting hormone titer is highest (6), suggests that the enzyme may play a role in deactivation of endogenous ecdysteroids. In Manduca sexta, ecdysone oxidase activity also increases at a similar developmental stage as observed in S. littoralis (36). Also, the developmental profile showed that ecdysone oxidase is highly expressed in the early pupal stage.…”
Section: Figsupporting
confidence: 63%
“…Ecdysone oxidase activity has been demonstrated in midgut, fat body, hemolymph, and integument of larvae in various insect species, although the predominant activity appears to be in midgut during the larval stage (11,35,36). Our Northern blot analysis shows that the mRNA for ecdysone oxidase was highly expressed in the midgut but undetectable in other tissues examined at the prepupal stage in S. littoralis under our conditions (Fig.…”
One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalyzed conversion into 3-dehydroecdysteroid followed by irreversible reduction by 3-dehydroecdysone 3␣-reductase to 3-epiecdysone. We have purified from Spodoptera littoralis the first ecdysone oxidase and subjected it to limited amino acid sequencing. A reverse-transcriptase polymerase chain reaction-based approach has been used to clone the cDNA (2.8 kilobases) encoding this 65-kDa protein.Northern blotting showed that the mRNA transcript was expressed in midgut during the prepupal stage of the last larval instar at a time corresponding to an ecdysteroid titer peak. Conceptual translation of the ecdysone oxidase cDNA and data base searching revealed that the enzyme is an FAD flavoprotein that belongs to the glucose-methanol-choline oxidoreductase superfamily. Ecdysone oxidase represents the only oxidase in eukaryotic animals known to catalyze oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via NAD(P) ؉ -linked dehydrogenases. The injection of RH-5992, an ecdysteroid agonist, induced the transcription of ecdysone oxidase, suggesting that ecdysone oxidase is an ecdysteroid-responsive gene. The gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and FTZ-F1 have been found in the 5-flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a singlecopy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.
“…Ecdysone oxidase, 3-DE 3α-reductase, and 3-DE 3β-reductase catalyze the metabolism of ecdysone [35] (Fig. 4a) and ecdysone is converted into 20E by 20-hydroxylase in many tissues [36]. It is 20E, which initiates molting and metamorphosis in insects [34, 37].…”
Section: Resultsmentioning
confidence: 99%
“…It is 20E, which initiates molting and metamorphosis in insects [34, 37]. The midgut also contains both ecdysone oxidase and 3-DE 3α-reductase simultaneously, thus facilitating 20E metabolism to 3-epiecdysone in this tissue [36]. Fig.…”
BackgroundInsects can be models for understanding human intestinal infection and pathology. Molting, a special period during which the old insect cuticle is shed and a new one is produced, is crucial for insect development. Holometabolous insects may experience several larva-to-larva moltings to become larger, a pupal molt and adult eclosion to become adults. During the larval molts, they stop feeding and become quiescent. Although the molting larvae become quiescent, it is not known if changes in microbiome, physiology, development and immunity of midguts occur.ResultsTranscriptome analysis indicated that functions such as metabolism, digestion, and transport may become reduced due to the downregulated expression of many associated genes. During the molting stage, midguts harbor less microflora and DNA synthesis decreases. Both ecdysone and juvenile hormone in the larval midgut likely degrade after entering the larva-to-larva molting stage. However, at 12 h after ecdysis, the feeding larvae of 5th instars that were injected with 20-hydroxyecdysone entered a molting-like stage, during which changes in midgut morphology, DNA synthesis, gene expression, and microflora exhibited the same patterns as observed in the actual molting state.ConclusionThis study is important for understanding insect midgut physiology, development and immunity during a special development stage when no food is ingested. Although the molting larva becomes immobile and quiescent, we demonstrate that numerous changes occur in midgut morphology, physiology, metabolism and microbiome during this period.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3162-8) contains supplementary material, which is available to authorized users.
Molting in insects is regulated by molting hormones (ecdysteroids). The major active hormone, 20-hydroxyecdysone, is formed by ecdysone 20-monooxygenase-catalyzed hydroxylation of ecdysone. During times of decreasing hormone titers, inactivation occurs by several routes including (i) 26-hydroxylation and further oxidation to the 26-oic acid, (ii) formation of various conjugates (e.g. phosphates), and (iii) in Lepidoptera in particular, ecdysone oxidase-catalyzed formation of 3-dehydroecdysteroid, which is reduced to 3-epiecdysteroid, followed by phosphotransferase-catalyzed formation of phosphate conjugates. Administration of the nonsteroidal ecdysteroid agonist RH-5849 (1,2-dibenzoyl-1-tert-butylhydrazine), but not 20-hydroxyecdysone, to tobacco hornworm (Manduca sexta) resulted in induction of midgut cytosolic ecdysone oxidase and ecdysteroid phosphotransferase activities. In addition, both 20-hydroxyecdysone and RH-5849 caused induction of ecdysteroid 26-hydroxylase activity in midgut mitochondria and microsomes, whereas 20-hydroxylase was induced to a lesser extent by 20-hydroxyecdysone in mitochondria and by either RH-5849 or 20-hydroxyecdysone in microsomes. Commensurate with induction of the enzymes by ecdysteroid and RH-5849 is a requirement for RNA and protein synthesis, without precluding indirect mechanisms. These results indicate that molting hormone stimulates at least one universal route of its own inactivation by inducing ecdysteroid 26-hydroxylase activity and are discussed in relation to an analogous phenomenon observed for vitamin D inactivation in vertebrates.Molting hormones (ecdysteroids) regulate molting in immature stages of insects (1). Conversion of ecdysone into the major active hormone 20-hydroxyecdysone occurs in certain peripheral tissues and is catalyzed by the cytochrome P450-dependent ecdysone 20-monooxygenase (2, 3). The activity of this enzyme undergoes developmental change, exhibiting a distinct peak during the final larval instar in several species (2, 4).Inactivation of ecdysteroids occurs by various reactions including conversion of ecdysteroid via the 26-hydroxy derivative into the 26-oic acid, formation of various conjugates (e.g. phosphates), and conversion into 3-epi(3␣-hydroxy)-ecdysteroids (5, 6). 3-Epiecdysteroid formation is prominent in lepidopteran midgut cytosol and involves ecdysone oxidase-catalyzed formation of 3-dehydroecdysteroid followed by NAD(P)H-dependent irreversible reduction to 3-epiecdysteroid, which may also be phosphorylated. The 3-dehydroecdysteroid may also undergo NAD(P)H-dependent reduction back to 3-hydroxy ecdysteroid (for reviews, see Refs. 3 and 7). These enzymatic activities in the midgut cytosol ( Fig. 1) also exhibit developmental changes (8, 9).In the tobacco hornworm (Manduca sexta), the subject of this investigation, midgut ecdysone 20-monooxygenase is localized in both mitochondrial and microsomal fractions (10). During development of the fifth instar, the midgut 20-monooxygenase undergoes a 50-fold increase in activity, temporally c...
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