Phospholipid Dependence of the Reversible,Energy-Linked,Mitochondrial Transhydrogenase in <Emphasis Type="Italic">Manduca sexta</Emphasis>

Midgut mitochondria from fifth larval instar Manduca sexta exhibit a membrane-associated transhydrogenase that catalyzes hydride ion transfer between NADP(H) and NAD(H). The NADPH-forming transhydrogenations occur as nonenergy- and energy-linked activities. The energy-linked activities couple with electron transport-dependent utilization of NADH/succinate, or with Mg²⁺-dependent ATPase. These energy-linked transhydrogenations have been shown to be physiologically and developmentally significant with respect to insect larval/pupal maturation. In the present study, isolated mitochondrial membranes were lyophilized and subjected to organic solvent or phospholipase treatments. Acetone extraction and addition of Phospholipase A₂ proved to be effective inhibitors of the insect transhydrogenase. Liberation of phospholipids was reflected by measured phosphorous release. Addition of phospholipids to organic solvent- and phospholipase-treated membranes was without effect. Employing a partially lipid-depleted preparation, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were reintroduced and transhydrogenase activity assessed. Of the phospholipids tested, only phosphatidylcholine significantly stimulated transhydrogenase activity. The results of this study suggest a phospholipid dependence of the M. sexta mitochondrial transhydrogenase.     

Midgut mitochondrial transhydrogenase in wandering stage larvae of the tobacco hornworm, Manduca sexta

Midgut mitochondria from fifth larval instar Manduca sexta exhibited a transhydrogenase that catalyzes the following reversible reaction: NADPH + NAD(+) <--> NADP(+) + NADH. The NADPH-forming transhydrogenation occurred as a nonenergy- and energy-linked activity. Energy for the latter was derived from the electron transport-dependent utilization of NADH or succinate, or from Mg++-dependent ATP hydrolysis by ATPase. The NADH-forming and all of the NADPH-forming reactions appeared optimal at pH 7.5, were stable to prolonged dialysis, and displayed thermal lability. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the NADPH --> NAD(+) and energy-linked NADH --> NADP(+) transhydrogenations, but not the nonenergy-linked NADH --> NADP(+) reaction. Oligomycin only inhibited the ATP-dependent energy-linked activity. The NADH-forming, nonenergy-linked NADPH-forming, and the energy-linked NADPH-forming activities were membrane-associated in M. sexta mitochondria. This is the first demonstration of the reversibility of the M. sexta mitochondrial transhydrogenase and, more importantly, the occurrence of nonenergy-linked and energy-linked NADH --> NADP(+) transhydrogenations. The potential relationship of the transhydrogenase to the mitochondrial, NADPH-utilizing ecdysone-20 monooxygenase of M. sexta is considered.     

Effects of plant flavonoids on Manduca sexta (tobacco hornworm) fifth larval instar midgut and fat body mitochondrial transhydrogenase

The reversible, membrane-associated transhydrogenase that catalyzes hydride-ion transfer between NADP(H) and NAD(H) was evaluated and compared to the corresponding NADH oxidase and succinate dehydrogenase activities in midgut and fat body mitochondria from fifth larval instar Manduca sexta. The developmentally significant NADPH-forming transhydrogenation occurs as a nonenergy- or energy-linked activity with energy for the latter derived from either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg++-dependent ATPase. In general, the plant flavonoids examined (chyrsin, juglone, morine, quercetin, and myricetin) affected all reactions in a dose-dependent fashion. Differences in the responses to the flavonoids were apparent, with the most notable being inhibition of midgut, but stimulation of fat body transhydrogenase by morin, and myricetin as also noted for NADH oxidase and succinate dehydrogenase. Although quercetin inhibited or stimulated transhydrogenase activity depending on the origin of mitochondria, it was without effect on either midgut or fat body NADH oxidase or succinate dehydrogenase. Observed sonication-dependent increases in flavonoid inhibition may well reflect an alteration in membrane configuration, resulting in increased exposure of the enzyme systems to the flavonoids. The effects of flavonoids on the transhydrogenation, NADH oxidase, and succinate dehydrogenase reactions suggest that compounds of this nature may prove valuable in the control of insect populations by affecting these mitochondrial enzyme components.     

Ecdysone 20-monooxygenase in mitochondria and microsomes of Manduca sexta (L.) Midgut: Is the dual localization real?

The dual localization of ecdysone 20-monooxygenase in mitochondria and microsomes of Manduca sexta larval midgut was investigated. Cosubstrate requirements and response to osmolarity of the microsomal ecdysone 20-monooxygenase system were found to be different from those previously reported for the mitochondrial enzyme system. The microsomal monooxygenase utilized NADPH and, less efficiently, NADH as cosubstrates. NADPH and NADH effects were neither additive nor synergistic. NADPH yielded identical activities in isotonic and hypotonic incubations. Mitochondria and microsomes showed no synergistic interaction for ecdysone 20-hydroxylation. After washing of the mitochondria, a large proportion of their ecdysone 20-monooxygenase activity was lost. The extent of the loss was inversely correlated to the concentration of mitochondria in the incubation mixture. The addition of bovine serum albumin to the incubations (2 mg/ml) largely restored the original activities. The microsomal contamination in mitochondrial pellets after each of three successive washings was determined by measuring the activity of a microsomal marker enzyme, NADPH-cytochrome c reductase. At each step of the purification, the ecdysone 20-monooxgenase activity of the mitochondrial preparations far exceeded the activity attributable to the microsomal contamination. These results confirm the existence of two independent ecdysone 20-monooxygenase systems in the midgut of M. sexta larvae.     

Ecdysone 20-hydroxylation in Manduca sexta midgut: Kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases

The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20-monooxygenase (E20MO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The K_m⁵ (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 × 10⁻⁵ and 3.67 × 10⁻⁷ M, respectively. The V_max was 82.7 pmol/min/mg protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondrial E20MO has the higher V_max, at physiological ecdysone concentrations (10⁻⁷ − 10⁻⁸ M) it is only one-eighth to one-tenth as active as the microsomal enzyme. It is concluded that the microsomal E20MO is the primary, if not the only, enzyme involved in ecdysone 20-hydroxylation in M. sexta midgut. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Ecdysone oxidase and 3-oxoecdysteroid reductases in Manduca sexta: Developmental changes and tissue distribution

The activities of ecdysone oxidase (EO), 3-oxoecdysteroid 3α-reductase (3α-R), and 3-oxoecdysteroid 3β-reductase (3β-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 3α-R, and both increased up to the seventh day after ecdysis. Hemolymph and fat body had only moderate to high 3β-R and low EO activites, and the epidermis did not contain significant activity of any of the enzymes. On the ninth day after the last larval ecdysis the larval midgut epithelium was replaced by a new pupal midgut epithelium. After this event only 3β-R was restored to high activities, whereas EO and 3α-R showed only low to marginal activities. It is concluded that only the larval midgut has a role in the inactivation of ecdysteroids by 3-epimerization. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

ECDYSONE 20-MONOOXYGENASE ACTIVITY IN THE PROTHORACIC GLANDS OF LAST INSTAR LARVAE OF BOMBYX MORI*

Abstract Using cell free radioassay, activities of ecdysone 20-monooxygenase (E-20-M) were determined in homogenates of prothoracic glands (PGs) and fat bodies at 24 h intervals during last instar larval development of Bombyx mori. It was found that the profile of E-20-M activity in PG homogenates was characterized by a basal line from day 0 to day 3 which begins to rise on 4th day and reaches a peak on 5th day. Nevertheless, in comparison with PGs, E-20-M activity in fat body was much higher.     

Phenol oxidases from Rhodnius prolixus: Temporal and tissue expression pattern and regulation by ecdysone

Rhodnius prolixus 5th instar nymphs have significant PO enzymatic activity in the anterior midgut, fat body and hemolymph. The tissue with the major amount of PO activity is the anterior midgut while those with higher specific activities are the fat body and hemolymph. In this work the temporal pattern of PO enzymatic activity in different tissues was investigated. In fat body, PO peaks occur at 7, 12 and 16 days after a blood meal. In hemolymph, PO diminishes until day 7, and then recovers by day 14. In the anterior midgut tissue, PO peaks on day 9 and just before ecdysis; a similar pattern was observed in the anterior midgut contents. Some of these activities are dependent on the release of ecdysone, as feeding blood meal containing azadirachtin suppresses them and ecdysone treatment counteracts this effect. These results suggest that during the development of the 5th instar, the insect has natural regulating cycles of basal PO expression and activation, which could be related to the occurrence of natural infections. The differences in temporal patterns of activity and the effects of azadirachtin and ecdysone in each organ suggest that, at least in R. prolixus, different tissues are expressing different PO genes.     

Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta

The titer of ecdysone in whole animal extracts of Manduca sexta was determined by radioimmunoassay during the fifth (last) larval instar, pharate pupal development and pupation. A subtle peak in ecdysone concentration was noted at day 4 (just prior to the onset of the wandering stage) and a second and greater peak at day 8.5 (coincident with pharate pupal development). The titer fluctuations during development were a result of changes in tissue ecdysone and not of alterations in the ecdysone content of the gut. When prothoracic gland secretory activity was analyzed in vitro at the same stages, the most rapid rate of α-ecdysone secretion was shown to occur on day 7 (one day prior to the peak in whole-animal ecdysone concentration). An earlier peak in prothoracic gland activity may occur at day 4–5. Thin layer and gas-liquid chromatographic analyses revealed developmental changes in the ratio of β:α-ecdysone in hemolymph and whole-animal extracts. It is suggested that the steroid-hydroxylating capacity of the insect increases during the instar.     

Prothoracic gland activity and blood titres of ecdysone and ecdysterone during the last larval instar of Locusta migratoria L.

The kinetics of secretion of ecdysone by the prothoracic glands of Locusta migratoria were studied during the last larval instar. Three stages of intense production of ecdysone (α-ecdysone) were monitored during this developmental period: they correspond to three peaks of moulting hormone concentration in the blood, which indicates that the main regulation of the moulting hormone titre is achieved through variations in prothoracic gland activity. In the haemolymph the ratio of ecdysone to ecdysterone (20-hydroxy-ecdysone) is in favour of ecdysone during the two first moulting hormone peaks ecdysterone being by far predominant over ecdysone at the time of the third (major) peak; these results support previous studies on the metabolic fate of injected labelled ecdysone during the same developmental period in Locusta migratoria.     

Effects of the compounds 2-methoxynaphthoquinone, 2-propoxynaphthoquinone, and 2-isopropoxynaphthoquinone on ecdysone 20-monooxygenase activity

The effects of the natural compound 2-methoxy-1,4-naphthoquinone, isolated from the leaves of Impatiens glandulifera and the synthetic compounds 2-propoxy-1,4-naphthoquinone and 2-isopropoxy-1,4-naphthoquinone on ecdysone 20-monooxygenase (E-20-M) activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the three compounds. All three compounds were found to inhibit in a dose-dependent fashion the E-20-M activity in the three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 3 x 10(-5) to 7 x 10(-4) M.     

家蚕(Bombyx mori)末龄幼虫前胸腺内蜕皮素20-单氧酶活性(英文)

应用无细胞放射实验技术,逐日测定了家蚕末龄幼虫发育期间前胸腺和脂肪体匀浆液内蜕皮素20单氧酶(E-20-M)的活性。结果发现前胸腺内E-20-M活性特点是:从蜕皮到第3天幼虫前胸腺缺乏或具较低水平E-20-M活性;第4天前胸腺E-20-M活性开始升高;到第5天前胸腺E-20-M活性达到高峰。然而,与前胸腺相比,脂肪体E-20-M活性要高的多。     

A larval serum protein of the silkworm,Bombyx mori: cDNA sequence and developmental specificity of the transcript

The Bombyx mori larval serum protein (BmLSP) is a major component of larval hemolymph proteins until early in the last instar. The cDNA for BmLSP was cloned from a library constructed from fat body RNA of penultimate instar larvae, and the complete nucleotide sequence of the 909 base pair cDNA insert was determined. The deduced 262 amino acid polypeptide included a 16 amino acid residue signal peptide and a 15 amino acid sequence prosegment. A homology search showed that BmLSP has significant similarity with microvitellogenin of Manduca sexta and the 30K proteins of B. mori. Tissue distribution and developmental profile of BmLSP mRNA were analyzed by northern hybridization. BmLSP mRNA was abundant in fat body but not detected in midgut and silk gland. BmLSP mRNA was present during the feeding periods of the fourth and fifth instar larvae, but absent during the larval molt and after the onset of cocoon spinning.     

Allelochemical stimulation of ecdysone 20-monooxygenase in fall armyworm larvae

The influence of dietary allelochemical on ecdysone 20-monooxygenase activity was studied in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Feeding the indoles (indole-3-carbinol, indole-3-acetonitrile), flavonoids (flavone, β-naphthoflavone), monoterpenes (menthol, menthone, peppermint oil), and a coumarin (xanthotoxin) to the larvae stimulated midgut microsomal ecdysone 20-monooxygenase activity from 28 to 200% as compared with the controls. β-Naphthoflavone was the most potent inducer among those tested. Phenobarbital, a well-known cytochrome P450 inducer, also caused a 2-fold increase in the microsomal ecdysone 20-monooxygenase activity. Ecdysone 20-monooxygenase activity was 2.7-fold higher in the microsomal fraction than in the mitochondrial fraction isolated from larval midguts. Microsomal ecdysone 20-monooxygenase activity was highest in the fat body, followed by the midgut and Malpighian tubules. Tissue localization and enzyme inducibility were different between ecdysone 20-monooxygenase and xenobiotic-metabolizing cytochrome P450 monooxygenases, including aldrin epoxidase, biphenyl hydroxylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, p-chloro-N-methylaniline N-demethylase, and phorate sulfoxidase in fall armyworm larvae. © 1995 Wiley-Liss, Inc.     

Ecdysones and imaginal disc development during the last larval instar of Pieris brassicae

Ecdysone haemolymph levels and in vivo development of imaginal wing discs have been studied during the last larval instar of Pieris brassicae.During this period, β-ecdysone variations show two successive peaks, the first one related to the induction of wandering stage, and the second (main) one to pupal cuticle synthesis. The observed situation is very similar to that of Manduca sexta. Imaginal wing disc growth is composed of several genetically programmed steps that need the presence of ecdysone, but do not appear very closely linked to circulating hormone levels. It seems that ecdysone haemolymph peaks should be considered as periods where ecdysone levels are above a threshold value.     

Severe developmental timing defects in the prothoracicotropic hormone (PTTH)-deficient silkworm,Bombyx mori

The insect neuropeptide prothoracicotropic hormone (PTTH) triggers the biosynthesis and release of the molting hormone ecdysone in the prothoracic gland (PG), thereby controlling the timing of molting and metamorphosis. Despite the well-documented physiological role of PTTH and its signaling pathway in the PG, it is not clear whether PTTH is an essential hormone for ecdysone biosynthesis and development. To address this question, we established and characterized a PTTH knockout line in the silkworm, Bombyx mori. We found that PTTH knockouts showed a severe developmental delay in both the larval and pupal stages. Larval phenotypes of PTTH knockouts can be classified into three major classes: (i) developmental arrest during the second larval instar, (ii) precocious metamorphosis after the fourth larval instar (one instar earlier in comparison to the control strain), and (iii) metamorphosis to normal-sized pupae after completing the five larval instar stages. In PTTH knockout larvae, peak levels of ecdysone titers in the hemolymph were dramatically reduced and the timing of peaks was delayed, suggesting that protracted larval development is a result of the reduced and delayed synthesis of ecdysone in the PG. Despite these defects, low basal levels of ecdysone were maintained in PTTH knockout larvae, suggesting that the primary role of PTTH is to upregulate ecdysone biosynthesis in the PG during molting stages, and low basal levels of ecdysone can be maintained in the absence of PTTH. We also found that mRNA levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in PTTH knockouts. Our results provide genetic evidence that PTTH is not essential for development, but is required to coordinate growth and developmental timing.     

Interendocrine regulation of the corpora allata and prothoracic glands of Manduca sexta

Regulation of the haemolymph titres of ecdysteroids and the juvenile hormones (JH) during larval-pupal development of the tobacco hornworm, Manduca sexta, involves the interendocrine control of the synthesis of each hormone by the other. Temporal relationships between the ecdysteroid titre peaks in the fourth and early fifth larval instar and the increases in corpora allata (CA) activity at these times suggests that ecdysteroids are evoking the increases. Incubation of brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes and isolated CA from these stages with 20-hydroxyecdysone (20-HE) revealed that 20-HE stimulates CA activity and that it does this indirectly via the Br-CC. The resulting increase in the JH titre after the commitment (first) peak in the fifth instar stimulates the fat body to secrete a factor which appears to be the same as a haemolymph stimulatory factor for the prothoracic glands. This moiety acts as a secondary effector that modulates the activity of the prothoracic glands and thus the ecdysteroid titre. These findings together have begun to elucidate the mechanisms by which the principal developmental hormones in the insect interact to regulate postembryonic development.     

Correlations between juvenile hormone esterase activity,ecdysone titre and cellular reprogramming in Galleria mellonella

Juvenile hormone esterase (JHE) activity, ecdysone titre, and developmental competence of the epidermis were determined in last instar larvae and pupae of Galleria mellonella. Haemolymph JHE activity reaches a peak before increases are observed in ecdysone titre both during larval-pupal and pupal-adult metamorphosis. JHE activity is low during the penultimate larval instar although general esterase activity is relatively high. In last instar larvae two ecdysone peaks are noted after the increase in JHE activity. Furthermore, epidermal cell reprogramming occurs just after the increase in haemolymph JHE activity and possibly before the first increase in ecdysone titre. This was tested by injection of high doses of β-ecdysone into last instar larvae of different ages resulting in rapid cuticle deposition. Reprogramming occurred if the resulting cuticle was of the pupal type. These correlative observations may increase our understanding of the relative importance of an ecdysone surge in the absence of JH in reprogramming of the insect epidermis.     

大豆胰蛋白酶抑制剂和防御信号物质对斜纹夜蛾解毒酶的影响

在昆虫与植物漫长的相互作用中,植物合成多种抗虫物质并采用防御信号转导系统抵御昆虫,昆虫也具有多种解毒酶系统保护其免受植物毒素的毒害.本文研究了人工添加大豆胰蛋白酶抑制剂和植物防御信号物质对斜纹夜蛾幼虫羧酸酯酶和谷胱甘肽-S-转移酶活性的影响.结果表明:持续6代自幼虫2龄或3龄开始喂养含有大豆胰蛋白酶抑制剂的人工饲料,其5龄幼虫中肠和脂肪体内羧酸酯酶、谷胱甘肽-S-转移酶活性显著升高,2、3龄处理的继代幼虫中肠和脂肪体内羧酸酯酶活性均在第二代达到最大值,分别为对照的2.06、2.40倍和1.96、2.70倍;其谷胱甘肽-S-转移酶活性则分别于第4、2代达到最大值,分别为对照的7.03、11.58倍和5.71、3.60倍,并呈现先升高再降低的趋势.预先接触外源信号物质茉莉酸甲酯、水杨酸甲酯48 h和添加大豆胰蛋白酶抑制剂均可使斜纹夜蛾幼虫中肠、脂肪体内羧酸酯酶和谷胱甘肽-S-转移酶的活性显著升高,且预先接触茉莉酸甲酯和水杨酸甲酯48 h可减缓大豆胰蛋白酶抑制剂对幼虫中肠和脂肪体内羧酸酯酶、谷胱甘肽-S-转移酶活性的作用效果.