Effects of azadirachtin on insect cytochrome P-450 dependent ecdysone 20-monooxygenase activity

The effects of the insect growth and ecdysis inhibitor azadirachtin on ecdysone 20-monooxygenase 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 last instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations of azadirachtin and the ecdysone 20-monoxygenase activity quantified by radioassay. Azadirachtin was found to inhibit in a dose-response fashion the ecdysone 20-monooxygenase activity associated with all the insect preparations. The concentration of azadirachtin required to elicit approximately 50% inhibition of the ecdysone 20-monooxygenase activity ranged from a low of 1 x 10(-4) M for Drosophila to a high of 4 x 10(-4) M for Manduca midgut.     

Preface

The vector of Chagas' disease, Rhodnius prolixus, feeds exclusively on blood. The blood meals are slowly digested, and these insects wait some weeks before the next meal. During the life of an insect, energy‐requiring processes such as moulting, adult gonadal and reproductive growth, vitellogenesis, muscular activity, and fasting, lead to increased metabolism. Carbohydrates are a major source of energy and their mobilization is important. We determined the amounts of glycogen, trehalose, and glucose present in the fat body and/or hemolymph of adult males of R. prolixus and recorded the processes of accumulation and mobilization of these carbohydrates. We also tested our hypothesis that these processes are under endocrine control. The amount of glycogen in the fat body progressively increased until the fourth day after feeding (from 9.3±2.2 to 77. 3±7.5 µg/fat body), then declined to values around 36.3±4.9 µg/fat body on the fifteenth day after the blood meal. Glycogen synthesis was eliminated in decapitated insects and head‐transplanted insects synthesized glycogen. The amount of trehalose in the fat body increased until the sixth day after feeding (from 16. 6±1.7 to 40. 6±5.3 nmol/fat body), decreased abruptly, and stabilized between days 7 and 15 at values ranging around 15–19 nmol/fat body. Decapitated insects did not synthesize trehalose after feeding, and this effect was reversed in head‐transplanted insects. The concentration of trehalose in the hemolymph increased after the blood meal until the third day (from 0.07±0.01 to 0.75±0.05 mM) and at the fourth day it decreased until the ninth day (0.21±0.01 mM), when it increased again until the fourteenth day (0.79±0.06 mM) after the blood meal, and then declined again. In decapitated insects, trehalose concentrations did not increase soon after the blood meal and at the third day it was very low, but on the fourteenth day it was close to the control values. The concentration of glucose in the hemolymph of untreated insects remained low and constant (0.18±0.01 mM) during the 15 days after feeding, but in decapitated insects it progressively increased until the fifteenth day (2.00±0.10 mM). We recorded the highest trehalase activity in midgut, which was maximal at the eighth day after feeding (2,830±320 nmol of glucose/organ/h). We infer that in Rhodnius prolixus, the metabolism of glycogen, glucose, and trehalose are controlled by factors from the brain, according to physiological demands at different days after the blood meal. © 2009 Wiley Periodicals, Inc.     

Cellular immune response in Rhodnius prolixus: role of ecdysone in hemocyte phagocytosis

In this paper we investigate in vivo and in vitro effects of orally administered azadirachtin and ecdysone on the phagocytic responses of Rhodnius prolixus 5th-instar larval hemocytes to the yeast Saccharomyces cerevisiae. Groups of insects fed non-treated blood (control) and insects that received azadirachtin plus ecdysone in the blood meal were inoculated with yeast cells in the hemocele. The injected yeast cells disappeared rapidly from the hemolymph, being removed completely by 90min after inoculation. In the insects treated only with azadirachtin the clearance of free yeast circulating particles was significantly delayed compared to the two previously mentioned groups. It was demonstrated that the binding of yeast cells to hemocytes was reduced in the insects treated only with azadirachtin in comparison to both non-treated control and azadirachtin plus ecdysone-treated groups. Phagocytosis occurred when yeast cells were added to hemocyte monolayers prepared with hemolymph from blood fed insects, treated or not with azadirachtin plus ecdysone, so that yeast cells were rapidly bound to hemocytes and internalized in high numbers. By contrast, insects treated with azadirachtin exhibited a drastic reduction in the quantity of yeast cell-hemocyte binding and subsequent internalization. In all groups, the hemocytes attached to the glass slides were predominantly plasmatocytes. The magnitude and speed of the cellular response suggests that hemocyte phagocytosis is one of the main driving forces for the clearance of free circulating yeast cells from the hemolymph. We propose that ecdysone modulates phagocytosis in R. prolixus larvae, and that this effect is antagonized by azadirachtin.     

Midgut and fatbody mitochondrial transhydrogenase activities during larval-pupal development of the tobacco hornworm, Manduca sexta

Midgut and fatbody mitochondria from fifth larval instar Manduca sexta display a membrane-associated transhydrogenase that catalyzes a reversible hydride ion transfer between NADP(H) and NAD(H). The NADPH-forming activity 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. During the ten-day developmental period preceding the larval-pupal molt (fifth larval instar), significant peaks in the mitochondrial transhydrogenase activities of midgut and fatbody tissues were noted and these peaks were coincident with the onset of wandering behavior and with the fifty-fold increase in ecdysone 20-monooxygenase (E20-M) activity previously reported for M. sexta midgut. Since E20-M preferentially uses NADPH in catalyzing ecdysone conversion to the physiologically active molting hormone, 20-hydroxyecdysone, the physiological and developmental significance of the mitochondrial, NADPH-forming energy-linked transhydrogenations were made apparent. Moreover, that the increases in all transhydrogenase activities resulted from de novo enzyme synthesis were indicated by the cycloheximide-dependent reductions in these activities.     

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-hydroxylation and 3-epimerization in larvae of the gypsy moth,Lymantria dispar L.: tissue distribution and developmental changes

The potential for ecdysone metabolism was determined for various larval tissues of the gypsy moth, Lymantria dispar. Homogenates of fat body, midguts, and Malpighian tubules, taken on different days during the second half of the fifth instar, were incubated with [(3)H]ecdysone, and the products were analyzed by reversed-phase and normal-phase HPLC. All tissues showed conversion to 20-hydroxyecdysone, and midguts also produced 3-epiecdysone. Ecdysone 20-monooxygenase (E20MO) activity in the fat body increased from a low level on day 5 to a peak on day 11, coinciding with the peak in the hemolymph ecdysteroid titer on the penultimate day of the instar. Midguts and Malpighian tubules showed E20MO activity only during the last 3 or 4days of the instar, with the highest activity also occurring on the penultimate day. For the midguts, the appearance of the E20MO coincided with the transition from larval to pupal tissue. No activity was detected in larval midguts. 3-Epiecdysone formation, however, was mainly found in larval midguts, with only marginal activity detectable in pupal midguts.     

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.     

Molecular cloning,expression analysis and functional confirmation of two ecdysone receptor isoforms from the rice stem borer Chilo suppressalis

PCR techniques were used to clone and identify cDNAs for ecdysone receptor A and B1 (EcR-A and EcR-B1) isoforms from the rice stem borer, Chilo suppressalis. They differ only in the N-terminal A/B regions and show high sequence identities to other insects' EcRs. At the wandering stage, EcR-B1 mRNA was expressed more abundantly in the midgut than in the epidermis and fat body, whereas expression levels of EcR-A mRNA were similar in the three tissues. In the epidermis of the last instar larvae, the maximal mRNA expression of both EcR-A and EcR-B1 was observed from the wandering to prepupal stages prior to the peak of ecdysteroid titer in the hemolymph. In gel mobility shift assays, in vitro translated C. suppressalis EcR-B1 (CsEcR-B1) and Bombyx mori ultraspiracle (BmUSP) proteins bound to the Pal 1 and Drosophila melanogaster hsp27 ecdysone response element as a heterodimer. These results indicate that the cDNAs isolated here encode functional ecdysone receptors.     

Ecdysone,juvenile hormone and oögenesis in Rhodnius prolixus

Oviposition and oögenesis can be inhibited in female Rhodnius prolixus by ecdysone given by the digestive tract. The inhibition is dose-dependent, and doses higher than 4.0 ng ecdysone/mg body weight drastically reduce the size and shape of the whole ovaries. In ecdysone-treated insects, normal oviposition and oögenesis can be re-established by a subsequent blood meal without ecdysone, or by the application of a juvenile hormone analogue.These results suggest that ecdysone inhibits juvenile hormone production.     

Allatotropin Modulates Myostimulatory and Cardioacceleratory Activities in Rhodnius prolixus (Stal).

Haematophagous insects can ingest large quantities of blood in a single meal and eliminate high volumes of urine in the next few hours. This rise in diuresis is possible because the excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of intensification of aorta contractions combined with an increase of the anterior midgut peristaltic waves. It has been previously described that haemolymph circulation during post-prandial diuresis is stimulated by the synergistic activity of allatotropin (AT) and serotonin in the kissing bug Triatoma infestans; resulting in an increase in aorta contractions. In the same species, AT stimulates anterior midgut and rectum muscle contractions to mix urine and feces and facilitate the voiding of the rectum. Furthermore, levels of AT in midgut and Malpighian tubules increased in the afternoon when insects are getting ready for nocturnal feeding. In the present study we describe the synergistic effect of AT and serotonin increasing the frequency of contractions of the aorta in Rhodnius prolixus. The basal frequency of contractions of the aorta in the afternoon is higher that the observed during the morning, suggesting the existence of a daily rhythmic activity. The AT receptor is expressed in the rectum, midgut and dorsal vessel, three critical organs involved in post-prandial diuresis. All together these findings provide evidence that AT plays a role as a myoregulatory and cardioacceleratory peptide in R. prolixus.     

TMOF-like factor controls the biosynthesis of serine proteases in the larval gut of Heliothis virescens.

Proteolytic enzyme biosynthesis in the midgut of the 4th instar larva of Heliothis virescens is cyclical. Protease activity increases immediately after the molt from the 3rd to the 4th instar larvae and declines just before the molt into the 5th instar. Characterization of the midgut proteases using soybean tryspin inhibitor (SBTI) Bowman Birk Inhibitor (BBI) 4-(2-aminoethyl)benzensulfonylfluoride (AEBSF) and N-tosyl-L-phenylalanine chloromethylketone (TPCK) indicate that protease activity is mostly trypsinlike (80%) with a small amount of chymotrypsinlike activity (20%). Injections of late 3rd and 4th instar larval hemolymph into H. virescens larvae inhibited tryspin biosynthesis in the larval midgut. Similar results were obtained when highly purified 4th instar larval hemolymph that crossreacted with Aea-TMOF antisurm using ELISA was injected into 2nd instar larvae. Injections of Aea-TMOF and its analogues into 2nd instar, and Aea-TMOF alone into 4th instar larvae stopped trypsin biosynthesis 24 and 48 h after the injections, respectively. Injections of 4th instar H. virescens larval hemolymph into female Aedes aegypti that took a blood meal stopped trypsin biosynthesis and egg development. These results show that the biosynthesis of trypsin-like enzymes in the midgut of a lepidoptera is modulated with a hemolymph circulating TMOF-like factor that is closely related to Aea-TMOF. Arch.     

Rhipicephalus sanguinius: localization of vitellogenin synthesis by immunological methods and electron microscopy

In ovipositing Rhipicephalus sanguinius (Latrelle), complete immunological identity existed between vitellogenin from the midgut, fat body, and hemolymph and vitellin from eggs. This supported the hypothesis that the same vitellogenin was synthesized by both the midgut and fat body, then released into the hemolymph and transported to the ovary. Vitellogenin was taken up unaltered by the oocytes during vitellogenesis to become vitellin. Antivitellogenin did not react with host (dog) hemoglobin. Transmission electron microscopy showed specialized cells with large amounts of rough endoplasmic reticulum, Golgi complexes, and secretory granules in the midgut and fat body of ovipositing females that were absent in the midgut and fat body of fed males. It is suggested that these cells synthesize vitellogenin.     

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm,Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.     

BmSUC1 is essential for glycometabolism modulation in the silkworm, Bombyx mori

Sucrose is the most commonly transported sugar in plants and is easily assimilated by insects to fulfill the requirement of physiological metabolism. BmSuc1 is a novel animal β-fructofuranosidase (β-FFase, EC 3.2.1.26)-encoding gene that was firstly cloned and identified in silkworm, Bombyx mori. BmSUC1 was presumed to play an important role in the silkworm-mulberry enzymatic adaptation system by effectively hydrolyzing sucrose absorbed from mulberry leaves. However, this has not been proved with direct evidence thus far. In this study, we investigated sucrose hydrolysis activity in the larval midgut of B. mori by inhibition tests and found that sucrase activity mainly stemmed from β-FFase, not α-glucosidase. Next, we performed shRNA-mediated transgenic RNAi to analyze the growth characteristics of silkworm larvae and variations in glycometabolism in vivo in transgenic silkworms. The results showed that in the RNAi-BmSuc1 transgenic line, larval development was delayed, and their body size was markedly reduced. Finally, the activity of several disaccharidases alone in the midgut and the sugar distribution, total sugar and glycogen in the midgut, hemolymph and fat body were then determined and compared. Our results demonstrated that silencing BmSuc1 significantly reduced glucose and apparently activated maltase and trehalase in the midgut. Together with a clear decrease in both glycogen and trehalose in the fat body, we conclude that BmSUC1 acts as an essential sucrase by directly modulating the degree of sucrose hydrolysis in the silkworm larval midgut, and insufficient sugar storage in the fat body may be responsible for larval malnutrition and abnormal petite phenotypes.     

Haemolymph ecdysteroid titres during larval-adult development in Rhodnius prolixus: Correlations with moulting hormone action and brain neurosecretory cell activity

A haemolymph ecdysteroid titre of the fifth (last)-larval instar of the hemipteran, Rhodnius prolixus has been determined by radioimmunoassay. During the last-larval stadium the ecdysteroid titre increases from a negligible level in the unfed insect to a detectable level within minutes following a blood meal. The titre reaches a plateau of ～50–70 ng/ml at 3–4 hr and this level is maintained until day 5–6, the time of the head-critical period in Rhodnius. At the head-critical period the titre begins to increase again, this time dramatically, reaching a peak of ～ 3500 ng/ml at day 13. From day 14 to ecdysis (day 21) the titre declines to a low level, ～ 30 ng/ml. Basal levels of ecdysteroids, ～ 15 ng/ml, were detectable in young adult males and females. A survey of haemolymph volumes during the last-larval instar indicates that the changes in the ecdysteroid titre reflect changes in the rates of ecdysteroid synthesis, and not changes in haemolymph volume. Excretion of ecdysteroids varies systematically during the instar, suggesting that control of ecdysteroid excretion may be important in regulation of the haemolymph titre. Qualitative analysis of the haemolymph ecdysteroid RIA activity revealed the presence of only ecdysone and 20-hydroxy-ecdysone. For the large peak preceding larval-adult ecdysis, 20-hydroxy-ecdysone was the predominant hormone. These results indicate that there may be two periods of release of prothoracicotropic hormone (PTTH) from the brain in Rhodnius, one immediately following the blood meal and the second on day 5 or 6. The significance of these times of PTTH release is discussed in relation to classical evidence of the timing of moulting hormone action, the response of target tissues, and with more recent findings on the timing of release of neurosecretory material from the brain of Rhodnius during moulting.     

Sn-protoporphyrin inhibits both heme degradation and hemozoin formation in Rhodnius prolixus midgut

Hematophagy is a feeding habit that involves the ingestion of huge amounts of heme. The hematophagous hemipteran Rhodnius prolixus evolved many genetic resources to protect cells against heme toxicity. The primary barrier against the deleterious effects of heme is the aggregation of heme into hemozoin in the midgut lumen. Hemozoin formation is followed by the enzymatic degradation of heme by means of a unique pathway whose end product is dicysteinyl-biliverdin IX-γ (Rhodnius prolixus biliverdin, RpBv). These mechanisms are complemented by a heme-binding protein (RHBP) in the hemolymph that attenuates the pro-oxidant effects of heme. In this work, we show that when insects are fed with blood enriched with a heme analog, Sn-protoporphyrin (SnPP-IX), both hemozoin synthesis and RpBv production are inhibited in a dose-dependent manner. These effects are accompanied by increased oxidative damage to the midgut epithelium and inhibition of oviposition, indicating that hemozoin formation and heme degradation are protective mechanisms that work together and contributed to the adaptation of this insect to successfully feed on vertebrate blood.