家蚕蜕皮与变态的内分泌调控

家蚕的蜕皮与变态是由前胸腺分泌的脱皮素（ｍｏｌｔｉｎｇ ｈｏｒｍｏｎｅ或 ｅｃｄｙｓｔｅｒｏｉｄ简称 ＭＨ）及由咽侧体分泌的保幼激素（ｊｕｖｅｎｉｌｅ ｈｏｒｍｏｎｅ）控制的,而促有前胸腺激素（ｐｒｏｔｈｏｒａｃｉｃｏｔｒｏｐｉｃ ｈｏｒｍｏｎｅ,以下简称ＰＴＴＨ）的功能为刺激前胸腺分泌蜕皮素。笔者近１０年来从家蚕内分泌体系的一系列研究中发现,蜕皮素浓度的变化可以通过控制咽侧体的保幼激素的生物合成来影响幼虫发育,而ＰＴＴＨ的信息传递可通过调控前胸腺的功能,进而影响血淋巴中蜕皮素浓度。     

Stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis by the prothoracic glands during the last larval instar of the silkworm, Bombyx mori

The stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori, were studied in the present study. When last instar larvae were starved beginning on day 1 of that instar, all larvae died between days 5 and 7 of the instar. Although the prothoracicotropic hormone (PTTH) release from the brain-corpus cardiacum-corpus allatum (BR-CC-CA) did not significantly change during starvation, a deficiency in PTTH signal transduction was maintained, which led to very low levels of hemolymph ecdysteroids after the beginning of starvation. However, when starvation began on day 3 of the last larval instar, the major hemolymph ecdysteroid peak, preceding larval-pupal transformation, occurred 1 day earlier than that in control larvae. Protein content of the prothoracic glands in day 3-starved larvae was maintained at a low level as compared to that of control larvae. The secretory activity of the prothoracic glands in day 3-starved larvae was maintained at a level similar to that of control larvae. However, the rate of ecdysteroidogenesis, expressed per microgram of glandular protein, was greatly enhanced in these starved larvae, indicating that upon starvation, larvae increased the ecdysteroid production rate to enhance the rate of survival.     

Effects of RH-5992 on ecdysteroidogenesis of the prothoracic glands during the fourth larval instar of the silkworm, Bombyx mori

Stage-dependent effects of RH-5992 on ecdysteroidogenesis of the prothoracic glands during the fourth larval instar of the silkworm, Bombyx mori, were studied in the present report. When larvae were treated with RH-5992 during the early stages of the fourth larval instar (between day 0 and day 1), initially ecdysteroid levels in the hemolymph were inhibited. However, 24 h after RH-5992 application, ecdysteroid levels were greatly increased as compared with those treated with acetone. The examination of the in vitro prothoracic gland activity upon RH-5992 application during the early stages of the fourth larval instar confirmed a short-term inhibitory effect. When RH-5992 was applied to the later stages of the fourth larval instar, no effects on both hemolymph ecdysteroid levels and prothoracic gland activity were observed. Addition of RH-5992 to incubation medium strongly inhibited ecdysteroid secretion by the prothoracic glands from the early fourth instar, indicating direct action of RH-5992 on ecdysteroidogenesis by prothoracic glands. Four hours after application with RH-5992 on day 1.5, prothoracic glands still showed an activated response to PTTH in both PTTH-cAMP signaling and the extracellular signal-regulated kinase (ERK) signaling. Moreover, addition of RH-5992 to incubation medium did not interfere with the stimulatory effect of the glands to PTTH in ecdysteroidogenesis. These results indicated that both PTTH-cAMP signaling and PTTH-ERK signaling may not be involved in short-term inhibitory regulation by RH-5992.     

Analysis of ecdysteroidogenic activity of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori

The cellular mechanism underlying ecdysteroidogenesis throughout the last larval instar of the silkworm, Bombyx mori, was analyzed by determining the in vitro ecdysteroid secretory activity of the prothoracic glands and cAMP accumulation of gland cells, as well as changes in responsiveness to stimulation by prothoracicotropic hormone (PTTH) and 1-methyl-3-isobutylxanthine (MIX). It was found that the prothoracic glands during the first 3 days of the last instar cannot produce detectable ecdysteroid and showed no response to stimulation by PTTH or 1-methyl-3-isobutylxanthine (MIX). However, artificial elevation of cellular cAMP levels by in vitro dibutyryl cAMP treatment stimulated the glands to secrete detectable ecdysteroid, implying the presence of a cAMP-dependent ecdysteroidogenic apparatus during this stage. From days 3 to 8, basal gland activities fluctuated, but the glands showed activation responses to PTTH and to the chemicals that increase cellular cAMP levels. After the occurrence of the peak in basal gland activity on day 9, glands on day 10 showed no response to PTTH, implying a refractory state of the glands to PTTH stimulation. For cAMP accumulation, it was found that glands on day 2 began to show increased cAMP accumulation to PTTH, implying that the acquisition of gland competency for elevation of cAMP levels after stimulation by PTTH precedes that of ecdysteroid production. Moreover, during most parts of the last larval instar (between days 3 and 8) and at the pupation stage, greatly increased cAMP accumulation upon stimulation by PTTH was observed only in the presence of MIX, indicating that cAMP phosphodiesterase levels may be high during these stages. From these results, we concluded that development-specific PTTH signal transduction during the last larval instar, which shows a different pattern from that of the penultimate larval instar, may play an important role in regulating changes in prothoracic gland activity and in leading to larval-pupal metamorphosis.     

In vitro and in vivo stimulation of extracellular signal-regulated kinase (ERK) by the prothoracicotropic hormone in prothoracic gland cells and its developmental regulation in the silkworm, Bombyx mori

In this study, we investigated activation of the extracellular signal-regulated kinase (ERK) by the prothoracicotropic hormone (PTTH) in prothoracic gland cells of the silkworm, Bombyx mori. The results showed that the PTTH stimulated ERK phosphorylation as this depends on time and dose and ecdysteroidogenic activity. The ERK phosphorylation inhibitors, PD 98059 and U0126, blocked both basal and PTTH-stimulated ERK phosphorylation and ecdysteroidogenesis. In addition, activation of glandular ERK phosphorylation by the PTTH appeared to be developmentally regulated with the refractoriness of gland cells to the PTTH occurring during the latter stages of both the fourth and last larval instars. Moreover, in vitro activation of ERK phosphorylation of prothoracic glands by the PTTH was also verified by in vivo experiments: injection of the PTTH into day 6 last instar larvae greatly increased the activity of glandular ERK phosphorylation and ecdysteroidogenesis. These results suggest that development-specific changes in ERK phosphorylation may play a role in PTTH stimulation of ecdysteroidogenesis.     

Temporal analysis of ecdysteroidogenic activity of the prothoracic glands during the fourth larval instar of the silkworm, Bombyx mori

The cellular mechanism underlying ecdysteroidogenesis during the fourth larval instar of the silkworm, Bombyx mori, was analyzed by determining the in vitro ecdysteroid biosynthetic activity of the prothoracic glands, cAMP accumulation of the gland cells, the in vitro release of prothoracicotropic hormone (PTTH), etc. According to the differential responsiveness of prothoracic glands to PTTH, dibutyryl cAMP (dbcAMP), and 1-methyl-3-isobutylxanthine (MIX), the following different stages were classified and changes in PTTH signal transduction were assumed. During the first stage (between days 0 and 1), the glands showed low basal and PTTH-stimulated activities in both cAMP accumulation and ecdysteroidogenesis, and PTTH release in vitro was maintained at low but detectable levels, implying that a low but sustained PTTH signal may be transduced to prothoracic gland cells. On day 1.5, when low basal ecdysteroid production of the prothoracic glands was being maintained, both the responsiveness of glands to the stimulation of PTTH and PTTH release in vitro dramatically increased, indicating greatly increased PTTH transduction. On day 3 (when the basal ecdysteroidogenesis became maximal) and afterwards, high PTTH release in vitro was maintained, but the gland showed no response to PTTH, implying that the refractoriness of gland cells to PTTH may occur at this stage. We assume that the development-specific changes in PTTH signal transduction during the penultimate larval instar may play a critical role in regulating changes in ecdysteroidogenesis of the prothoracic glands.     

Positive feedback regulation of prothoracicotropic hormone secretion by ecdysteroid – A mechanism that determines the timing of metamorphosis

When insect larvae have fully grown, prothoracicotropic hormone (PTTH) is released from the brain, triggering the initiation of metamorphic development through stimulation of ecdysteroid secretion by the prothoracic glands. The present study analyzes the mechanism that regulates the occurrence of this PTTH surge. In the silkworm Bombyx mori, the PTTH surge occurs on day 6 of the fifth instar and is preceded by a small rise in hemolymph ecdysteroid titer, which occurs late on day 5. We therefore hypothesized that this rise of ecdysteroid titer is involved in the induction of the PTTH surge. To test this hypothesis, two experiments were conducted. First, a small amount of 20-hydroxyecdysone was injected on day 4, two days before the expected day of the PTTH surge, to simulate the small rise in hemolymph ecdysteroid titer on day 5. This injection led to a precocious surge of PTTH the next day. Next, the hemolymph ecdysteroid titer on day 5 was artificially lowered by injecting ecdysteroid-22-oxidase, which inactivates 20-hydroxyecdysone. After this treatment, the PTTH surge did not occur on day 6 in 80% of the animals. These results indicate that a small rise of the hemolymph ecdysteroid titer plays a critical role in the induction of the PTTH surge. Since basal ecdysteroidogenic activity of the prothoracic glands increases with larval growth, a circulating level of ecdysteroids may convey information about larval maturity to the brain, to coordinate larval growth and metamorphosis. This is the first report in invertebrates to demonstrate positive feedback regulation of the surge of a tropic hormone by a downstream steroid hormone.     

Analysis of precocious metamorphosis induced by application of an imidazole derivative to early third instar larvae of the silkworm,Bombyx mori

When an imidazole derivative (KK-42) was applied to day 1 third instar larvae of the silkworm, Bombyx mori, 100% underwent precocious metamorphosis at the end of the fourth instar. Thus, the fourth instar becomes the last instar in these KK-42–treated larvae. The endocrine systems underlying the precocious metamorphosis were analyzed in the present study. Hydroprene application during the prolonged third instar after KK-42 treatment can prevent precocious metamorphosis, and the results showed dose-dependent and stage-specific effects. From analysis of the developmental changes in ecdysteroid levels in both KK-42–treated larvae and KK-42– and hydroprene-treated larvae, we conclude that changes in JH levels during the third larval instar can modify the secretion pattern of prothoracic glands and that during the next larval instar, very low ecdysteroid levels during the early stages of the presumptive last (fourth) larval instar are directly related to precocious metamorphosis. Arch. Insect Biochem. Physiol. 36:349–361, 1997. © 1997 Wiley-Liss, Inc.     

Bombyx mori prothoracicostatic peptide inhibits ecdysteroidogenesis in vivo

Bombyx prothoracicostatic peptide (Bom-PTSP) is a brain neuropeptide that has recently been reported to have in vitro inhibitory activity to prothoracicotropic hormone (PTTH)-stimulated ecdysteroid biosynthesis in the prothoracic gland of the silkworm, Bombyx mori. In the present report, Bom-PTSP has been shown to significantly decrease hemolymph ecdysteroid titer in the fifth instar larvae when Bom-PTSP was injected into the fifth instar day 8 silkworm larvae, resulting in significant delay in spinning behavior. This is the first evidence that Bom-PTSP inhibits in vivo ecdysteroidogenesis in the silkworm.     

Insulin stimulates ecdysteroidogenesis by prothoracic glands in the silkworm,Bombyx mori

It is generally accepted that the prothoracicotropic hormone (PTTH) is the stimulator of ecdysteroidogenesis by prothoracic glands in larval insects. In the present study, we investigated activation of ecdysteroidogenesis by bovine insulin in prothoracic glands of the silkworm, Bombyx mori. The results showed that the insulin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. In addition, insulin also stimulated both DNA synthesis and viability of prothoracic glands. Insulin-stimulated ecdysteroidogenesis was blocked by either LY294002 or wortmannin, indicating involvement of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Activation of ecdysteroidogenesis by insulin appeared to be developmentally regulated. Moreover, in vitro activation of ecdysteroidogenesis of prothoracic glands by insulin was also verified by in vivo experiments: injection of insulin into day 6 last instar larvae greatly increased both hemolymph ecdysteroid levels and ecdysteroidogenesis 24 h after the injection, indicating its possible in vivo function. Phosphorylation of Akt and the insulin receptor was stimulated by insulin, and stimulation of Akt phosphorylation appeared to be PI3K-dependent and developmentally regulated. Insulin did not stimulate extracellular signal-regulated kinase (ERK) signaling of the prothoracic glands. These results suggest that in silkworm prothoracic glands, in addition to the PTTH and an autocrine factor, ecdysteroidogenesis is also stimulated by insulin during development.     

Adenylate cyclase in prothoracic glands during the last larval instar of the silkworm, Bombyx mori

We have previously reported that the absence of prothoracicotropic hormone (PTTH) signal transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of the corpora allata, as well as larval-pupal transformation. In the present study, adenylate cyclase was characterized in crude preparations of prothoracic gland cell membranes in an effort to localize the cause of refractoriness to PTTH. It was found that cyclase activity of the prothoracic glands from the day 6 last instar showed activation responses to fluoride, a guanine nucleotide analogue, as well as calmodulin (CaM) in dose-dependent fashions. The additive effects of day 5 prothoracic gland adenylate cyclase stimulation by fluoride and CaM imply that there may exist Gs protein-dependent and CaM-dependent forms of adenylate cyclase. For day 1 last instar prothoracic glands, which showed no response to stimulation by PTTH in either cAMP generation or ecdysteroidogenesis, adenylate cyclase activity exhibited far less responsiveness to Ca(2+)/CaM than did that from day 5 glands. These findings suggest that day 1 prothoracic glands may possess some lesions in the receptor-Ca(2+) influx-adenylate cyclase signal transduction pathway and these impairments in PTTH signal transduction may be, at least in part, responsible for decreased ecdysteroidogenesis.     

Regulation and significance of ecdysteroid titre fluctuations in lepidopterous larvae and pupae

The last larval moult of Galleria mellonella is induced by an elevation of ecdysteroid titre to more than 200 ng/g. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr, is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 ng/g) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500–2500 ng/g) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 ng/g) in the middle of pupal instar.Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 30–40 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing the prothoracicotropic neurohormone (PTTH). In either case, the pupation-inducing increase of ecdysteroids is 3 times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed.Neither in G. mellonella larvae, nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid production or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5–15 times lower than usual ecdysteroid concentration when the last-instar larvae are exposed to JH.     

Juvenile hormone coordinates the regulation of the hemolymph ecdysteroid titer during pupal commitment in Manduca sexta

The timing and magnitude of the pupal commitment peak in the hemolymph ecdysteroid titer of fifth instar Manduca sexta larvae are controlled by the combined effects of prothoracicotropic hormone (PTTH), a prothoracic gland-stimulating factor present in the hemolymph, and the biosynthetic competence of the prothoracic glands themselves. The present data indicate those individual effects are coordinated by juvenile hormone (JH): (1) Treatment of larvae with the JH analog (7S)-hydroprene prevents the normal precommitment drop in the titer of the stimulatory factor; (2) treatment of larvae with (7S)-hydroprene suppresses in a dose- and time-dependent manner the biosynthetic competence of the prothoracic glands; and (3) (7S)-hydroprene acts directly on the brain to inhibit the release of PTTH in vitro. Thus, during Manduca development, a drop in the JH titer early in the fifth instar results in a rapid drop in the titer of the stimulatory factor, the gradual acquisition by prothoracic glands of biosynthetic competence, and lastly, the gated release of PTTH into the hemolymph. The resulting increase in ecdysone synthesis by the prothoracic glands gives rise to the small peak in the ecdysteroid titer that drives pupal commitment.     

亚洲玉米螟末龄幼虫促前胸腺激素与蜕皮甾类激素滴度的变化

用放射免疫分析法(Radioimmunoassay,RIA)以12小时间隔测定了亚洲玉米螟Ostrinia tfurnacalis末龄非滞育幼虫血淋巴中蜕皮甾类激素滴度.通过前胸腺体外培养,以12小时间隔测定了前胸腺体外分泌活性的变化.发现二者的变化在相同发育阶段是一致的.在亚洲玉米螟上建立了促前胸腺激素(PTTH)体外测定法,并用此法以24小时间隔测定了末龄幼虫脑和血淋巴中PTTH滴度.发现血淋巴中PTTH滴度在末龄第5和7天各有一高峰,脑中只在第5天有一高峰.     

Effects of juvenile hormone on the secretion of prothoracicotropic hormone in the last- and penultimate-instar larvae of the silkworm Bombyx mori

The effect of juvenile hormone (JH) on the secretion of the prothoracicotropic hormone (PTTH) was investigated, by examining the changes in hemolymph PTTH titer after the topical application of JH-I on the larvae of the silkworm, Bombyx mori. The titer of PTTH was determined by the time-resolved fluoroimmunoassay. JH-I application at very early stages of development in the fifth (last) instar resulted in a significant increase in the PTTH titer, but this effect became less evident thereafter. After the onset of wandering (day 6 of the fifth instar), JH-I did not affect the hemolymph PTTH titer. JH-I application on day 5 resulted in the delay of spinneret pigmentation on day 6, which is induced by an increase in the ecdysteroid titer on day 5 and is the first visible indication of larval-pupal transformation. However, the JH-I application did not suppress the increase in either PTTH or ecdysteroid titer on day 5, suggesting that JH-I acts on the spinneret to inhibit the response of the tissue to ecdysteroids. JH-I also exhibited a PTTH titer-elevating effect in the fourth instar. These results suggest that JH has a role as a potent stimulator of PTTH secretion in both the penultimate and last instar of the silkworm.     

cDNA cloning and expression of a hormone-regulated heat shock protein (hsc 70) from the prothoracic gland of Manduca sexta

The brain neuropeptide prothoracicotropic hormone (PTTH) stimulates a rapid increase in ecdysteroid hormone synthesis that is accompanied by general and specific increases in protein synthesis, including that of a 70 kDa cognate heat shock protein (hsc 70). To further understand the possible roles of hsc 70, hsc 70 cDNA clones were isolated from a tobacco hornworm (Manduca sexta) prothoracic gland cDNA library. All sequenced clones were highly homologous to the Drosophila hsc 70-4 isoform. Manduca hsc 70 mRNA levels during the last larval instar exhibited a peak at the onset of wandering and a peak that coincided with the major pre-metamorphic peak of ecdysteroid synthesis. Manipulations of the glands' hormonal milieu showed that hsc 70 mRNA levels respond to 20-hydroxyecdysone, dibutyryl cAMP, PTTH and the JH analogue hydroprene. The protein and mRNA data suggest that hsc 70 could be involved in a negative feedback loop regulating assembly of the ecdysone receptor complex.     

Effects of ecdysteroid agonist RH-2485 reveal interactions between ecdysteroids and juvenile hormones in the development of Sesamia nonagrioides

Larvae of Sesamia nonagrioides developing under long day (LD) conditions pupate in the 5th or 6th instar, whereas under the short day (SD) conditions, they undergo several supernumerary larval molts and are regarded as diapausing. The development in early larval instars occurs in the LD larvae at a moderate and in the SD larvae at a high juvenile hormone (JH) titer; ecdysteroid titer cycles similarly under both conditions. The transformation to pupa is initiated by a burst of ecdysteroids at undetectable JH levels, whereas extra larval molts in the diapausing larvae are associated with moderate JH titer and irregular rises of ecdysteroids. Application of 0.2 ppm RH-2485 to the diet of the 6th instar larvae promotes hormonal changes supporting metamorphosis in the LD larvae and slightly accelerates larval molts in the diapausing SD larvae. The 0.5- and 1-ppm doses revert these patterns of endocrine regulations to a mode typical for early larval instars. Particularly dramatic is a JH titer increase provoked within 24 h in the LD larvae. After the treatment, both the LD and SD larvae undergo a series of larval molts, suggesting that hormonal programming of the larval development has been stabilized. A few insects receiving 1 ppm RH-2485, and a high proportion of those fed with 5 ppm RH-2485, deposit two cuticles within a single apolysis and die.