Hemolymph Ecdysteroid Titer Research Articles

Regulation of ecdysteroid secretion from the Y-organ by molt-inhibiting hormone in the American crayfish, Procambarus clarkii

In crustaceans, molt-inhibiting hormone (MIH) has been proposed to regulate molting by inhibiting the secretion of ecdysteroids from the Y-organ. Thus, MIH titer in the hemolymph should be inversely related to ecdysteroid titers during the molt cycle. However, it has not been demonstrated whether the MIH titer in the hemolymph changes during the molt cycle. The purpose of this study was to determine the changes in the MIH titers in the hemolymph during the molt cycle of the American crayfish, Procambarus clarkii, and to discuss the role of MIH in regulation of molting. As predicted by the hypothesis, the hemolymph MIH titer was high at the intermolt stage when the hemolymph ecdysteroid titer was low, and the MIH titer decreased to a basal level at the early premolt stage when the hemolymph ecdysteroid titer began to increase slightly. At the middle premolt stage when the hemolymph ecdysteroid titer increased, the MIH titer was restored to a level as high as that during the intermolt stage. This is in contradiction to the hypothesis. However, the Y-organs at this stage scarcely responded to MIH both in vitro and in vivo. The present findings suggest that ecdysteroid secretion from the Y-organ may be regulated not only by changes in the hemolymph MIH titer, but also by changes in the responsiveness of the Y-organ to MIH.

The hemolymph JH titer exhibits a large-amplitude, morph-dependent, diurnal cycle in the wing-polymorphic cricket, Gryllus firmus

The hemolymph juvenile hormone (JH) titer was measured in over 500 flight-capable and flightless, adult female Gryllus firmus at 3–6 h intervals during each of days 2–8 of adulthood. The flight-capable morph exhibited a large-amplitude daily cycle in the hemolymph JH titer, while the flightless morph exhibited a barely perceptible cycle. The JH titer cycle was observed on all days in the flight-capable morph, but the large amplitude cycle (>15–20 fold increase in mean titer; >100-fold increase in some individuals), began on day 5. For both the large and small amplitude cycles, the JH titer peaked near the end of the photophase–beginning of the scotophase. The hemolymph ecdysteroid titer did not exhibit a corresponding large amplitude daily cycle, although a low amplitude cycle (1–3-fold change) was seen in both morphs. The large magnitude rise in the JH titer in the flight-capable morph during the photophase was not due to decreased hemolymph volume or JH degradation. Daily cycles in the JH titer may be common, but may have gone unnoticed in other insect species due to restricted temporal sampling. Failure to identify these cycles can result in substantial errors in inferring biological roles for JH. Because JH regulates flight behaviors, morph-specific daily cycles in the JH titer may be especially common in dispersal-polymorphic insects, in which flight is restricted to one morph during a limited period of the day or night. However, because JH regulates numerous biological traits, analogous cycles may be common in insects exhibiting other types of complex (e.g. caste or phase) polymorphism, in which morphs differ in a biological characteristic that is restricted to a specific period of the photophase or scotophase.

Protein content and pattern during mucus gland maturation and its ecdysteroid control in honey bee drones

We analyzed mucus gland protein content and pattern for drones of Africanized honey bees. The effect of exogenous ecdysteroids on mucus gland maturation was judged against the endogenous ecdysteroid titer. During the first 5 days of adult life, the mucus protein content increases steeply, whereas the protein pattern becomes reduced in complexity. Subsequently, the protein content decreases, reaching a plateau level at day 8. The protein pattern of mature glands is characterized by three dominant polypeptides. Injection of 20-hydroxyecdysone into newly emerged drones abolished the normal increase in protein content and prolonged the persistence of the protein pattern typical for immature glands. Ecdysteroids thus appear to act as negative regulators in the maturation process of drone mucus glands. This hypothesis received support from analyses of the hemolymph ecdysteroid titer, which was found to rapidly decline soon after emergence. male accessory gland / ecdysone / radioimmunoassay / Apis mellifera / mucus protein

The relationship between ‘critical weight’ and 20-hydroxyecdysone in the female ixodid tick, Amblyomma hebraeum

The feeding cycle of adult female ticks (Acari: Ixodidae) is divided into preparatory, slow and rapid feeding phases. At the transition from slow to rapid feeding, Amblyomma hebraeum (Koch) females reach a ‘critical weight’ (CW; approx. 10× the unfed weight) that is characterized by several behavioural and physiological changes. Five of these changes were used as criteria to establish a more precise estimate of CW than we have to date. The CW as defined by re-attachment to the host was 9× the unfed weight, while for haemolymph ecdysteroid titre, salivary gland degeneration, ovary weight, oocyte length and oocyte vitellin content the CW was 10×, 10×, 12×, 12× and 13×, respectively. CW thus varies depending on the parameter measured. Although previous studies have established the influence of ecdysteroids on salivary gland degeneration and vitellogenesis, here we demonstrate a further effect of ecdysteroids: inhibiting re-attachment to the host.

Developmental profile of the changes in the prothoracicotropic hormone titer in hemolymph of the silkworm Bombyx mori: correlation with ecdysteroid secretion

A very sensitive time-resolved fluoroimmunoassay for the prothoracicotropic hormone (PTTH) of the silkworm Bombyx mori has been established. The lower limit of detection in this assay was 0.1 pg. With this assay method, the amounts of PTTH in the central nervous system and hemolymph were quantified. PTTH was detected only in the brain within the central nervous system, and, in the fifth instar, its content in the brain increased gradually with larval growth and decreased rapidly after the beginning of wandering. A substantial amount of PTTH was also found in the retrocerebral complex of day-3 fifth instar larvae, accounting for 28% of total PTTH. The PTTH titer in hemolymph changed dramatically during Bombyx development, with a small peak in the middle of the fourth instar, medium-sized peaks at the wandering and prepupal stages in the fifth instar, and a large prolonged peak during early pupal–adult development. The changes were overall closely correlated with those in hemolymph ecdysteroid titer. However, some unexpected aspects of PTTH dynamics in hemolymph have also been disclosed. Based on these observations, the significance of PTTH secretion in the control of insect development is discussed.

Ecdysteroid titer, ovary status, and dominance in adult worker and queen bumble bees ( Bombus terrestris)

The role of ecdysteroids in the regulation of dominance and reproduction in social Hymenoptera is little explored. In the current study we compared ecdysteroid titers in hemolymph of individual queen and worker bumble bees ( Bombus terrestris) that differ in their behavior, reproductive status and social environment. Egg-laying queens that head colonies and have ovaries exhibiting all stages of follicle development, had a higher ecdysteroid titer than virgin queens whose ovaries contained only follicles at initial stages. In workers, the relationship between ecdysteroid titers and follicle development appears to be more complex and to be influenced by the bee's social status and social environment. Shortly after emergence, young workers had only follicles at the initial stages of oogenesis and they exhibited a low ecdysteroid titer. No significant correlation was detected between ovary status and ecdysteroid titer in workers, with some workers showing activated ovaries but low ecdysteroid titers. However, at six days of age, a trend towards higher ecdysteroid titer was observed for workers in queenless groups, a condition characterized by rapid follicle development relative to queenright conditions. In these queenless groups, high social status was associated with high ecdysteroid titers. By contrast, in queenright workers ecdysteroid titers were low, even for bees with presumably high social status that had activated ovaries and were observed performing oviposition behavior. This study suggests that ecdysteroids are involved in regulation of reproduction in B. terrestris.

Ecdysteroid levels/profiles of the parasitoid wasp, Diapetimorpha introita, reared on its host, Spodoptera frugiperda and on an artificial diet

Diapetimorpha introita is an ichneumonid ectoparasitoid of the fall armyworm, Spodoptera frugiperda. Since it has been reported that D. introita wasps reared on an artificial diet exhibit a significantly lower percentage of adult eclosion and fecundity than host-reared wasps, this study was undertaken to elucidate the factors responsible for the reduced viability observed in diet-reared wasps. A system of markers has been devised to track the development (from the initiation of cocooning through adult eclosion) of D. introita. Although wasps reared on artificial diet developed more slowly than did those reared on host pupae, both diet- and host-reared wasps passed through the same stages of development — the eyes enlarged and moved backward, the gut was purged and upon ecdysis the exarate pupa emerged. The thorax was the first to darken, followed by the head and then the abdomen. Pharate pupal formation occurred before gut purge. Two peaks of hemolymph ecdysteroids were observed, one in wasps in which gut purge was almost complete and the second in day-2 exarate pupae. Ecdysone and 20-hydroxyecdysone were the major ecdysteroids present in hemolymph sampled at these times. Small quantities of 20,26-dihydroxyecdysone, polar ecdysteroids and/or possibly 26-hydroxyecdysone were also present. In six stages of development, hemolymph ecdysteroid titers were significantly higher in host-reared than in diet-reared wasps (Eye 1, Eye 2, Gut Purge 2, Pharate Pupa, Head/Thorax Dark, and Abdomen Dark). Relatively high percentages of mortality were observed in diet-reared wasps in four of these stages and in two others which occurred in close proximity to one of the stages, the Abdomen Dark stage. Thus, insufficient ecdysteroid in the hemolymph may be responsible, in part, for the relatively high percentage of mortality that occurred in wasps reared on an artificial diet.

Synthesis and Precocious Metamorphosis-Inducing Activity of 3-(1-Alkenyl) pyridines

A series of 3-pyridine derivatives with an 1-alkenyl group was synthesized and evaluated against 4th instar (penultimate) larvae of the silkworm, Bombyx mori, for their activity to induce precocious metamorphosis. Of the compounds tested, 3-(2-methyl-1-phenyl-1-propenyl)pyridine (18) was the most active, indicating that both a phenyl substituent at the 1 position and a short alkyl chain at the 2 position in the ethenyl moiety were significant for activity. Precocious pupation induced by 18 was accompanied by the prolongation of the larval period and the increase of body weights. Both 20-hydroxyecdysone and methoprene, a juvenile hormone agonist, could fully counteract precocious metamorphosis induced by 18. When applied to newly molted 4th instar larvae, 18 suppressed the increase of hemolymph ecdysteroid titers leading to larval ecdysis to 5th instar. These results indicated that 18 acted as an anti-ecdysteroid agent and temporarily depressed the ecdysteroid titer in the hemolymph to induce precocious metamorphosis.

Larval Growth and Diapause in a Tropical Moth, Omphisa fuscidentalis Hampson

The bamboo borer, Omphisa fuscidentalis, is a moth found in northern Thailand, Lao and Myanmar and its larvae feed on the inner pulp of bamboo shoots. In a tropical highland (about 500 m sea level) forest at 19°N near Chiang Mai, Thailand, the larvae feed on at least 5 bamboo species. Nucleotide sequence analysis of the region of mitochondrial cytochrome C oxidase subunit 1 gene amplified by the polymerase chain reaction (PCR) verified that larvae collected from different bamboos belong to the same species. Adults appeared in early August and laid clusters of eggs on the newly grown bamboo shoot. The newly hatched larvae bore a hole in the shoot, enter an internode of the shoot and feed on the inner pulp. After maturation in September, the larvae remain in an internodal cavity of bamboo for up to 9 months, from September to the following June. Number of larval instars was estimated by measuring the width of head capsules remained in internodes of bamboo shoots. The growth curve of the width fitted to Dyar's law and the mature larvae were estimated to be 5th instar. Mature larvae were collected in the field each month and their body weight, head capsule width, protein and fat contents and hemolymph ecdysteroid titer were measured. Body weight continuously decreased during the 9 months whereas head capsule width remained constant. Fat content fluctuated during this period while protein level remained at a similar level until March, after which it significantly increased. During this period, hemolymph ecdysteroid concentrations remained low. Current results show that the bamboo borer larvae enter diapause at the end of feeding period of the fifth (last) larval instar and the larval diapause lasts until June.

Ecdysteroid titers in mated and unmated Drosophila melanogaster females

Radioimmunoassay was used to determine ecdysteroid titers in mated or unmated Drosophila melanogaster females. Whole-body ecdysteroid titers increase after mating and this response is more pronounced after 12–24 hours than it is immediately after mating. In one experiment, females were mated to transgenic males deficient in accessory gland proteins to test whether these peptides mediate the observed increase in female whole-body ecdysteroid titers. Females mated to such transgenic males do not show a pronounced increase in whole-body ecdysteroid titers. The effect of mating on female hemolymph ecdysteroid titers was also investigated. Hemolymph ecdysteroid titers decrease after mating. The ecdysteroid titer change in the hemolymph may result from yolk protein uptake of ecdysteroids into developing vitellogenic oocytes as a consequence of male accessory gland protein stimulation of female oocyte maturation and yolk protein synthesis following mating.

Profile of the ecdysteroid hormone and its receptor in the salivary gland of the adult female tick, Amblyomma hebraeum

We examined the hemolymph ecdysteroid titer (by radioimmunoassay) and profile of the ecdysteroid receptor (EcR/USP; by [ 3H]ponasterone A binding, gel mobility shift assay, Western blot) in the salivary gland of the ixodid tick, Amblyomma hebraeum Koch (Acari: Ixodidae) throughout the tick feeding period and first 6 days post-engorgement. Throughout the slow phase of feeding, the hemolymph ecdysteroid titer was ∼18 pg/μl. The titer peaked at ∼52 pg/μl during the rapid phase of feeding, falling back to ∼22 pg/μl on the day of engorgement. Ecdysteroid titer rose again to ∼750 pg/μl by day 6 post-engorgement. EcR was undetectable by any of the three assays in unfed ticks. Following the onset of feeding, there appeared both specific ponasterone A binding and two major EcR bands detected by Western blot analysis. Both measurements were sustained throughout the feeding period, but declined after detachment when the salivary glands were degenerating. After ticks reached about 100 mg (by which time most females are mated), a discrete DNA-binding band was shown by gel mobility shift assay using Drosophila hsp27 EcRE as a probe. Moreover, the band intensified when hemolymph ecdysteroid titer reached its peak during the rapid phase of feeding; it declined along with decreasing EcR/USP levels, and with specific ligand binding activity following engorgement. This study suggests a role for the small hemolymph ecdysteroid peak during the rapid phase of feeding in initiating salivary gland degeneration.

Synthesis/release of ecdysteroids by Cotesia congregata, a parasitoid wasp of the tobacco hornworm, Manduca sexta

Parasitized Manduca sexta undergo developmental arrest in the instar (typically the fifth) from which the parasitoid wasp Cotesia congregata will emerge. Host hemolymph ecdysteroid titers as well as levels of other regulatory molecules are manipulated by the parasitoid to create an environmental milieu which is most suitable for the wasp to complete its life cycle. Based on the results of in vivo studies (reported earlier), the parasitoid appears to require a host hemolymph ecdysteroid titer of 300–400 pg/μl, which it effects in its host, in part, by synthesizing and releasing ecdysteroid. Here we used in vitro incubation to characterize C. congregata's synthesis and release of this group of steroid hormones. In addition, we identified physical characteristics as markers for tracking parasitoid ecdysteroid content as well as hormone synthesis and release. Ecdysteroid content of whole body homogenates increased with parasitoid age. Body length, anal vesicle size and time of ecdysis provided more precise indicators of times when ecdysteroid content would be high or low. Ecdysteroid levels peaked at the time of the parasitoid's molt from the 1st to the 2nd instar and again in the largest parasitoids that were preparing for their molt to the 3rd instar. Ecdysteroid synthesis and release also increased with parasitoid age. When body length was the independent variable, synthesis and release peaked in larvae that were 3.0 mm (often undergoing a molt), dipped in larvae that were 3.6 mm (post-molt), and rose again as larvae increased in length. E, 20HE and polar ecdysteroids were the predominant ecdysteroids released on days 1 through 4. Thus, an ecdysteroid known to be physiologically active is released by the parasitoid. This is the first time that a precise, detailed system of markers has been identified for tracking C. congregata development. Our results support the view that parasitoid synthesis and release of ecdysteroids are associated with specific events in the parasitoid's life cycle and can be predicted by selected physical markers including parasitoid size and instar, size/degree of ballooning of the anal vesicle, and shedding of the 1st instar cuticle. Arch. Insect Biochem. Physiol. 40:17–29, 1999. Published 1999 Wiley-Liss, Inc.

Synthesis/release of ecdysteroids by Cotesia congregata , a parasitoid wasp of the tobacco hornworm, Manduca sexta

Parasitized Manduca sexta undergo developmental arrest in the instar (typically the fifth) from which the parasitoid wasp Cotesia congregata will emerge. Host hemolymph ecdysteroid titers as well as levels of other regulatory molecules are manipulated by the parasitoid to create an environmental milieu which is most suitable for the wasp to complete its life cycle. Based on the results of in vivo studies (reported earlier), the parasitoid appears to require a host hemolymph ecdysteroid titer of 300–400 pg/μl, which it effects in its host, in part, by synthesizing and releasing ecdysteroid. Here we used in vitro incubation to characterize C. congregata's synthesis and release of this group of steroid hormones. In addition, we identified physical characteristics as markers for tracking parasitoid ecdysteroid content as well as hormone synthesis and release. Ecdysteroid content of whole body homogenates increased with parasitoid age. Body length, anal vesicle size and time of ecdysis provided more precise indicators of times when ecdysteroid content would be high or low. Ecdysteroid levels peaked at the time of the parasitoid's molt from the 1st to the 2nd instar and again in the largest parasitoids that were preparing for their molt to the 3rd instar. Ecdysteroid synthesis and release also increased with parasitoid age. When body length was the independent variable, synthesis and release peaked in larvae that were 3.0 mm (often undergoing a molt), dipped in larvae that were 3.6 mm (post-molt), and rose again as larvae increased in length. E, 20HE and polar ecdysteroids were the predominant ecdysteroids released on days 1 through 4. Thus, an ecdysteroid known to be physiologically active is released by the parasitoid. This is the first time that a precise, detailed system of markers has been identified for tracking C. congregata development. Our results support the view that parasitoid synthesis and release of ecdysteroids are associated with specific events in the parasitoid's life cycle and can be predicted by selected physical markers including parasitoid size and instar, size/degree of ballooning of the anal vesicle, and shedding of the 1st instar cuticle. Arch. Insect Biochem. Physiol. 40:17–29, 1999. Published 1999 Wiley-Liss, Inc.

Ecdysteroid titres in haemolymph and other tissues during moulting and reproduction in the terrestrial isopod, Oniscus asellus (L.)

Summary Haemolymph ecdysteroid titres were determined throughout the intermoult cycle of Oniscus asellus and related to ecdysteroid-related physiological events. In non-breeding females, ecdysteroid titres rise during premoult to a peak two days prior to ecdysis in synchrony with formation of a new cuticle in the posterior half of the animal. However, the anterior half does not form cuticle during this time, but instead accumulates calcium. Thus, anterior and posterior halves of the epidermis respond differently to the same levels of ecdysteroids. The anterior half secretes a new cuticle at a later time and at a very low ecdysteroid titre. During the first breeding moult of a season, differentiation of a brood pouch commences in early premoult and the rise in ecdysteroid titre is delayed by two weeks. Both vitellogenin synthesis by the fat body and oocyte growth closely parallel the ecdysteroid titre. Although both of these processes are thought to be influenced by ecdysteroids, the titres of breeding and non-breeding females did not differ, indicating that other factors determine whether or not vitellogenesis will occur in the presence of ecdysteroids. Together, these findings show that Oniscus displays ecdysteroid-related events in strikingly unusual ways which raise questions concerning the regulation of cellular responses to ecdysteroids. The hepatopancreas accumulates ecdysteroids to levels well above those in the haemolymph during late premoult and appears to be the main route for their elimination from the haemolymph. Ecdysteroids in the hepatopancreas are translocated rapidly into the hindgut and discarded with the hindgut cuticle at the posterior ecdysis.

Hemolymph ecdysteroid titer and ecdysteroid-dependent developmental events in the last-larval stadium of the silkworm, Bombyx mori: role of low ecdysteroid titer in larval–pupal metamorphosis and a reappraisal of the head critical period

The endocrine regulation of larval–pupal metamorphosis was studied in the silkworm, Bombyx mori, by measuring the following changes: hemolymph ecdysteroid titer, the secretory activity of prothoracic glands and the responsiveness of larvae to ecdysteroids and prothoracicotropic hormone (PTTH), with regard to developmental events such as the occurrence of spinneret pigmentation, initiation of cocoon spinning and onset of wandering stage as indicated by gut purge. These measurements were concentrated especially on the time before and after the head critical period (HCP) which falls 3–4 days before the gut purge ( Sakurai, 1984). A small increase in the hemolymph ecdysteroid titer was first found during the HCP, and then the titer increased with daily fluctuations. Small but significant titer peaks were found prior to the occurrence of both spinneret pigmentation and gut purge, indicating that an individual titer peak could possess a specific role in development. Responsiveness of larvae to exogenous 20-hydroxyecdysone (20E) after the HCP was markedly higher than that before the HCP. The sensitivity of the prothoracic gland to PTTH also changed during the HCP. The results thus showed that the HCP is not the period after which an additional PTTH release is not required for the developmental events occurring on schedule, but rather it is the period during which complex events occur not only in the endocrine glands but also in the peripheral tissues. In addition, various developmental phenomena before gut purge are brought about by the hemolymph ecdysteroid whose concentration gradually increased with daily fluctuations, and these precise changes in the titer appeared to be important for the sequential occurrence of developmental events in the larval–pupal metamorphosis.

Manipulation of fifth-instar host ( Manduca sexta) ecdysteroid levels by the parasitoid wasp Cotesia congregata

Although 5th (last) instar parasitized Manduca sexta larvae undergo developmental arrest and do not wander, they exhibit a small hemolymph ecdysteroid peak (300–400 pg/ μl) which begins one day prior to the parasitoid's molt to the 3rd (last) instar and concomitant emergence from the host. Ecdysteroids present in this peak were 20-hydroxyecdysone, 20,26-dihydroxyecdysone and one or more very polar ecdysteroids, as well as small amounts of 26-hydroxyecdysone and ecdysone. In parasitized day-1 and -2 5th instars ligated just behind the 1st abdominal proleg, hemolymph ecdysteroid levels increased in both anterior and posterior portions (100–500 pg/ μl), while in unparasitized larvae, hormone levels only increased in the anterior portion (100–350 pg/ μl). Thus, the ecdysteroid peak observed in host 5th instars was probably produced, at least in part, by the parasitoids. It may serve to promote Cotesia congregata's molt from the second to the third instar and/or to facilitate parasitoid emergence from the host. In parasitized day-1 and -2 5th instars ligated between the last thoracic and 1st abdominal segments, hemolymph ecdysteroid titers reached much higher levels (500–3500 pg/ μl) in the anterior portion (no parasitoids present) than in the posterior portion (150–450 pg/ μl). Therefore, it appears that the parasitoid's regulation of hemolymph ecdysteroid titers occurs at two levels. First, parasitization neutralizes the host's ability to maintain its normal hemolymph ecdysteroid levels. Second, in a separate action, the parasitoid manipulates the ecdysteroid-producing machinery so that hemolymph levels are maintained at the 200–400 pg/ μl characteristic of day 3–4 hosts. This is the first report of a parasitoid's ability to interfere with the normal inhibitory mechanisms which prevent prothoracic gland production of ecdysteroid at inappropriate periods of insect growth and development.

Molt-Inhibiting Hormone mRNA Levels and Ecdysteroid Titer during a Molt Cycle of the Blue Crab,Callinectes sapidus

Synthesis of ecdysteroid molting hormones by crustacean Y-organs is believed to be regulated by a neuropeptide, molt-inhibiting hormone (MIH), produced in eyestalk neural ganglia. In the present study, steady-state MIH mRNA and hemolymph ecdysteroid levels were determined by Northern blot and radioimmunoassay, respectively, during the molt cycle of the blue crab,Callinectes sapidus.The level of MIH mRNA dropped steadily during premolt (D1–D4), reaching a minimum in D3/D4, then increased by 10-fold in postmolt (A/B) and remained elevated during intermolt (C4). These stage-specific changes in MIH mRNA levels were accompanied by significant fluctuations in the hemolymph ecdysteroid titer. The ecdysteroid titer increased steadily to a peak of 377.0 ng/ml in D3 of premolt, then dropped to 120.0 ng/ml in D4 (just prior to molting), and was low during postmolt (A/B, 4.4 ng/ml) and intermolt (C4, 3.3 ng/ml). The results represent the first report of developmental changes in MIH gene expression and are generally consistent with the hypothesis that MIH negatively regulates ecdysteroid synthesis in crustaceans.

Transition from larva to pupa: morphogenesis, cell proliferation and protein synthesis in Bombyx wing disc

Summary In the present study, an attempt was made to clarify the timing of the transition from proliferation stage to pupal preparation stage of Bombyx wing discs and the correlation of its timing and ecdysteroid release. An histological study was carried out during the period of ecdysteroid increase in hemolymph in the last larval instar. The number of mitoses in wing discs from the feeding stage to the spinning stage was counted. During the feeding stage, the number of mitoses was about 200/disc, with the number increasing up to about 850/disc on the day of the beginning of spinning (S0); it then decreased to about 250/disc at the S1 stage, and finally mitotic figures were absent at S2. Comparing the mitotic number with hemolymph ecdysteroid titer, ecdysteroid during the high mitotic period is close to 100ng/ml, while ecdysteroid during the low mitotic period is about 10ng/ml at day 5 and 500ng/ml at S1, respectively. Two-dimensional gel electrophoresis was carried out to identify the ecdysone-induced polypeptides or the polypeptides synthesized during the spinning stage. Five ecdysteroid-inducible polypeptides were observed during the spinning stage or after 20-hydroxyecdysone (20E) treatment. From these findings, low concentrations of ecdysteroid induce cell proliferation, whereas high concentrations inhibit cell proliferation and induce new protein synthesis.

An Immunophilin is a Component of the Insect Ecdysone Receptor (EcR) Complex

The ecdysone receptor (EcR) complex has been identified in the prothoracic gland of Manduca sexta by specific immunoprecipitation and Western blot analyses, and includes EcR, ultraspiracle (USP) and FKBP46. The EcR complex binds ponasterone A in a dose-dependent manner with a Kd of 7.04×10−9M. Immunocytochemistry revealed that EcR, USP and FKBP46 were localized within the nucleus of the prothoracic gland cells, and suggested that the developmental expression patterns of EcR and USP changed in concert with the hemolymph ecdysteroid titer whereas that of FKBP46 did not. The composite results suggest that the hemolymph ecdysteroid titer, of which 20 hydroxyecdysone is the major component, modulates the expression of both EcR and USP in the prothoracic gland to achieve feedback regulation.

Cloning of two putative ecdysteroid receptor isoforms from Tenebrio molitor and their developmental expression in the epidermis during metamorphosis.

Using the Drosophila EcR-B1 cDNA as a probe, we have cloned the putative ecdysteroid receptor from the mealworm Tenebrio molitor. We have isolated two cDNAs with different 5' termini that contain a complete open reading frame. These two cDNAs encode two proteins with distinct N-terminal regions corresponding to two isoforms. The coleopteran receptor is obviously related to the ecdysteroid receptor of other insects, but shares only 89% and 61% amino acid identities with the DNA-binding and ligand-binding domains of the Drosophila receptor, respectively. Its expression pattern has been examined in the epidermis during the last larval instar and pupal stage of T. molitor, in correlation with the hemolymph ecdysteroid titer. Hybridizations revealed two transcripts of 7 kb and 6.5 kb detected in most stages during metamorphosis and corresponding to the A and B1 isoforms. These two mRNAs are highly evident just before the rise of each ecdysteroid peak both in prepupae and in pupae. They show almost the same expression pattern in epidermis except for the second part of the pupal stage, during which only the A isoform is detected.