Downregulation of the cAMP signal transduction cascade in the prothoracic glands is responsible for the fenoxycarb-mediated induction of permanent 5th instar larvae in Bombyx mori

Fenoxycarb application at 48 h (day 2) of the 5th instar of Bombyx mori induced permanent larvae with prothoracic glands (PGs) exhibiting weak ecdysteroidogenic activity. Although glands from control and fenoxycarb-treated larvae exhibited similar responses to dibutyl cAMP and forskolin on day 2, forskolin could not stimulate ecdysteroid secretion from PGs of fenoxycarb-treated larvae on day 3. Glands from control larvae incubated with cholera toxin (CTX) on day 3 had increased cAMP content and enhanced ecdysteroid secretion. Cholera toxin did not stimulate ecdysteroid secretion and marginally increased cAMP content in day 3 PGs of fenoxycarb-treated larvae. After application of fenoxycarb on day 2, crude brain extracts (cBRAIN) could not increase the glandular cAMP content throughout the rest of the 5th instar of the treated larvae. Fenoxycarb did not affect the basal or cBRAIN-stimulated cAMP accumulation in control PGs on day 2 and day 3 in vitro. Application of fenoxycarb on day 2 did not affect the recombinant PTTH (rPTTH)-stimulated ecdysteroid secretion on day 3, but reduced the cBRAIN-stimulated ecdysteroid secretion on day 3 to levels similar to that of rPTTH. The combined results suggest that the cAMP signalling cascade in the PGs of B. mori becomes nonfunctional after fenoxycarb application on day 2 of the 5th instar.     

Differences between recombinant PTTH and crude brain extracts in cAMP-mediated ecdysteroid secretion from the prothoracic glands of the silkworm,Bombyx mori

The ability of recombinant prothoracicotropic hormone (rPTTH) or crude brain extract (cBRAIN) of Bombyx mori to stimulate ecdysteroid secretion from prothoracic glands (PGs) was investigated throughout the fifth instar and the first day of the pupal stage. Crude brain extracts could stimulate much higher ecdysteroid secretion than rPTTH during a 2h incubation. Recombinant PTTH did not increase the level of glandular cyclic AMP, except on days 4 and 5 of the fifth instar. Glandular cAMP levels were increased by cBRAIN from day 0 until day 5 of the fifth instar with the highest increase on day 3. On this day, rPTTH could not stimulate any increase of ecdysteroid secretion from the PGs during a 30min incubation. On the contrary, PGs incubated with cBRAIN for 30min showed increased secretory activity. Furthermore, on day 3 and in the absence of extracellular Ca(2+), rPTTH did not increase the glandular cAMP levels but cBRAIN did. Recombinant PTTH-stimulated ecdysteroid secretion from day 3 PGs was dependent on extracellular Ca(2+) in a dose-dependent manner. However, cBRAIN could stimulate ecdysteroid secretion even in the absence of extracellular Ca(2+). Taken together, the results of these experiments suggest the presence of a previously unknown cerebral prothoracicotropic factor that can stimulate glandular cAMP levels and ecdysteroid secretion from the PGs of Bombyx mori.     

Ecdysteroidogenic action of Bombyx prothoracicotropic hormone and bombyxin on the prothoracic glands of Rhodnius prolixus in vitro

An in-vitro assay for ecdysteroid synthesis by the prothoracic glands (PGs) of fifth instar Rhodnius prolixus has been employed to evaluate the actions of prothoracicotropic neuropeptides from the silkmoth, Bombyx mori. Crude prothoracicotropic hormone (PTTH) extracts of recently emerged adult brain complexes of Bombyx induced a dose-dependent stimulation of ecdysteroid synthesis by Rhodnius PGs, which was similar to that obtained using crude Rhodnius PTTH. In both cases, maximum stimulation was obtained with one brain equivalent. Rhodnius PGs were then challenged with incremental doses of recombinant Bombyx PTTH and synthetic bombyxin-II. Dose-response curves for the action of both peptides on Rhodnius PGs were very similar to those obtained for their action on the pupal PGs of Bombyx in vitro. Bombyx PTTH stimulated the PGs of Rhodnius at concentrations comparable to those effective on Bombyx. The curve for Bombyx PTTH showed a steep ascending region from 3 to 8ng/ml and a sharp peak. For bombyxin, concentrations 40-fold higher were required to elicit the same amount of stimulation as obtained using Bombyx PTTH. Therefore, Rhodnius PGs possess recognition sites for both Bombyx PTTH and bombyxin. This is the first study of the ecdysteroidogenic properties of the Bombyx peptides on a heterologous species. It is suggested that the function and conformation of PTTH may be conserved between distantly related insect groups.     

Induction of dauer pupae by fenoxycarb in the silkworm,Bombyx mori

Topical application of fenoxycarb (1 μg per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb at 132 h of the fifth instar elicited a high ecdysteroid titre in the pharate pupal stage and a steadily high ecdysteroid titre in the pupal stage. The fenoxycarb-induced permanent pupae had non-degenerating prothoracic glands that secreted low amounts of ecdysteroid and did not respond to recombinant prothoracicotropic hormone (rPTTH) late in the pupal stage. The Bombyx PTTH titre in the haemolymph, determined by a time-resolved fluoroimmunoassay, was lower than that of controls at the time of pupal ecdysis, but higher than controls later in the pupal stage in fenoxycarb-treated animals. After application of fenoxycarb, its haemolymph level, measured by ELISA, reached a peak at pupal ecdysis, then remained low. These results suggest that the fenoxycarb-mediated induction of permanent pupae is only partially a brain-centred phenomenon. It also involves alterations in the hormonal interplay that govern both the initiation of pupal-adult differentiation and changes in the steroidogenic pathway of the prothoracic glands of B. mori.     

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.     

An in vitro study on regulation of prothoracic gland activity in the early last-larval instar of the silkworm Bombyx mori

The endocrine mechanisms that regulate prothoracic gland (PG) activity in early stages of final larval instar of the silkworm Bombyx mori were investigated using a newly developed long-term cultivation system of the gland. The PGs dissected from day-0 fifth instar larvae did not secrete detectable amounts of ecdysone for the first 24 h in culture but started secretion within the next 2 days. The amount of secreted ecdysone increased day by day. When day-0 PGs were co-cultivated with corpora allata, however, they remained inactive for at least 8 days. PGs dissected from 1-day younger larvae (day-3 fourth instar larvae) secreted ecdysone for the first 24 h but stopped secretion for the next 24 h, followed by recovery of ecdysone secretory activity. By contrast, PGs from day-1 fourth instar larvae remained active throughout a cultivation period without any sign of inactivation. However, when the same glands were exposed to a high titer of 20-hydroxyecdysone for the second 24h in culture, they gradually lost their activity. These results indicate that PGs of fourth instar larvae are inactivated by ecdysteroid through a negative feedback mechanism and that thus inactivated PGs spontaneously recover ecdysone secretory activity in the early fifth instar unless inhibited by juvenile hormone.     

Tissue-specific regulation of juvenile hormone esterase gene expression by 20-hydroxyecdysone and juvenile hormone in Bombyx mori

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.     

Involvement of calcium, inositol-1,4,5 trisphosphate and diacylglycerol in the prothoracicotropic hormone-stimulated ecdysteroid synthesis and secretion in the prothoracic glands of Bombyx mori

The objective of this study was to determine which intracellular second messenger systems are activated by prothoracicotropic hormone in the prothoracic glands (PGs) of Bombyx mori. Recombinant prothoracicotropic hormone (rPTTH) could stimulate ecdysteroid synthesis and secretion from day 6 PGs of the 5th instar of Bombyx mori within 30 min of in vitro incubation. However, rPTTH did not stimulate any increases in the glandular content of inositol 1,4,5-trisphosphate and cAMP during this short incubation period. Extracellular Ca2+ influenced the basal and rPTTH-stimulated ecdysteroid synthesis and release in a dose-dependent manner. The L-type Ca2+ channel antagonist, nitrendipine, inhibited the rPTTH-stimulated ecdysteroid synthesis and secretion (IC50-28 microM). The phospholipase C inhibitor, 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate, inhibited the rPTTH-stimulated ecdysteroid synthesis (IC50-19 microM). The protein kinase C inhibitor, chelerythrine chloride, inhibited the rPTTH-stimulated ecdysteroid synthesis (IC50-14 microM). The protein kinase C activator, phorbol-12-myristate 13-acetate (PMA), could stimulate basal ecdysteroid synthesis and secretion (EC50-1 microM) and its inactive alpha-isomer (4 alpha-PMA) was ineffective. The combined results suggest that the PTTH-stimulated ecdysteroid synthesis and release in the PGs of Bombyx is dependent on extracellular Ca2+ and the bifurcating second messenger signalling cascade of inositol 1,4,5-triphosphate and diacylglycerol.     

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.     

Involvement of reactive oxygen species in PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm,Bombyx mori

In the present study, the possible involvement of reactive oxygen species (ROS) in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis of Bombyx mori prothoracic glands (PGs) was investigated. Results showed that PTTH treatment resulted in a rapidly transient increase in the intracellular ROS concentration, as measured using 2′,7′-dichlorofluorescin diacetate (DCFDA), an oxidation-sensitive fluorescent probe. The antioxidant, N-acetylcysteine (NAC), abolished PTTH-induced increase in fluorescence. Furthermore, PTTH-induced ROS production was partially inhibited by the NAD(P)H oxidase inhibitor, apocynin, indicating that NAD(P)H oxidase is one of the sources for PTTH-stimulated ROS production. Four mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and diphenylene iodonium (DPI)) significantly attenuated ROS production induced by PTTH. These data suggest that the activity of complexes I and III in the electron transport chain and the mitochondrial inner membrane potential (ΔΨ) contribute to PTTH-stimulated ROS production. In addition, PTTH-stimulated ecdysteroidogenesis was greatly inhibited by treatment with either NAC or mitochondrial inhibitors (rotenone, antimycin A, FCCP, and DPI), but not with apocynin. These results indicate that mitochondria-derived, but not membrane NAD(P)H oxidase-mediated ROS signaling, is involved in PTTH-stimulated ecdysteroidogenesis of PGs in B. mori.     

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.     

Regulation of capacitative Ca2+ entry by prothoracicotropic hormone in the prothoracic glands of the silkworm, Bombyx mori

Measurements of Ca2+ influx in Fura-2/AM loaded steroidogenic cells (prothoracic glands; PGs) of the silkworm, Bombyx mori showed that application of the neuropeptide prothoracicotropic hormone (PTTH) can increase the intracellular [Ca2+]i. This PTTH-mediated Ca2+ influx in PG cells had kinetic patterns and pharmacological characteristics similar to those induced by thapsigargin. Namely, it produced increases in intracellular Ca2+ levels only in the presence of extracellular Ca2+, it was blocked by Gd3+ and 2-Aminoethoxydiphenylborate (2-APB), and it was unaffected by several toxins or compounds that block voltage-activated Ca2+ channels. Moreover, the PTTH-stimulated increase of Ca2+ levels was eliminated in the presence of heparin (an IP3 receptor blocker), and by TMB-8 which also blocked any PTTH-dependent increase of ecdysteroid secretion. The PTTH-mediated increase of Ca2+ levels was not affected by the non-hydrolysable GDP analogue, GDPbetaS, an indication that a G protein is not downstream of the PTTH receptor. These results argue strongly in favor of gating by the PTTH receptor of capacitative Ca2+ entry (CCE) channels (or store-operated Ca2+ channels (SOCs)) by a mechanism that does not involve any G proteins but requires the presence of functional IP3 receptors. Because the ability of PTTH to stimulate the [Ca2+]i levels of PG cells was completely mimicked by thapsigargin and exhibited a pharmacological profile similar to CCE mechanisms, we believe that PTTH directly regulates a CCE pathway in PG cells thereby activating a plethora of downstream regulators responsible for ecdysteroid secretion by the PGs of Bombyx mori.     

外源激素处理后家蚕吡哆醛激酶和磷酸吡哆醇氧化酶转录水平的变化

【目的】研究家蚕 Bombyx mori 经蜕皮激素(20-hydroxyecdysone, 20-E)和保幼激素类似物(juvenile hormone analogue, JHA)处理后引起吡哆醛激酶(pyridoxal kinase, PLK)和磷酸吡哆醇氧化酶(pyridoxine-5′-phosphate oxidase, PNPO)的转录水平变化,为进一步研究激素对蚕体营养代谢等工作奠定基础。【方法】以20-E和JHA分别喂食不同发育时期(5龄第1, 3和5天)的家蚕幼虫,以喂食蒸馏水的家蚕为对照,采用实时荧光定量PCR(real-time quantitative PCR)方法在处理后24 和48 h对各组幼虫后部丝腺中PLP合成酶PLK和PNPO的转录水平进行分析。【结果】5龄第1天幼虫经20-E处理24和48 h后,PLK和PNPO的转录水平出现上调且与对照的差异达到极显著 (P＜0.01);5龄第3天幼虫经20-E处理,PLK的转录水平在48 h出现下调且与对照的差异达到显著(P＜0.05),PNPO的转录水平在24 和48 h均出现上调且与对照的差异达到极显著 (P＜0.01);5龄第5天幼虫经20-E处理后PLK和PNPO的转录水平无变化。5龄第1天幼虫经JHA处理后PLK和PNPO的转录水平未受到影响;5龄第3天幼虫经JHA处理后,PLK的转录水平在48 h出现显著下调且与对照的差异达到显著(P＜0.05),PNPO的转录水平在24和48 h后均出现显著下调且与对照的差异达到极显著(P＜0.05);5龄第5天幼虫经JHA处理24和48 h后,PLK和PNPO的转录水平出现下调且与对照的差异达到极显著 (P＜0.01)。【结论】20-E和JHA显著影响家蚕5龄幼虫PLK和PNPO的转录水平,20-E提高5龄前期家蚕PLK和PNPO的转录水平,JHA降低5龄后期它们的转录水平,为深入研究激素对VB₆的调控奠定基础。     

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.     

The effect of the juvenile hormone analog, fenoxycarb, on ecdysone receptor B1 expression in the midgut of Bombyx mori during larval-pupal metamorphosis

The Bombyx mori (Lepidoptera: Bombycidae) midgut undergoes remodeling during the larval-pupal metamorphosis. All metamorphic events in insects are controlled by mainly two hormones: 20-hydroxyecdysone (20E) and juvenile hormone (JH). Fenoxycarb, O-ethyl N-(2-(4-phenoxyphenoxy)-ethyl) carbamate, has been shown to be one of the most potent juvenile hormone analogs against a variety of insect species. In this study, the effect of fenoxycarb on EcR-B1 protein expression in the midgut of Bombyx mori during the remodeling processwas investigated. Fenoxycarb was topically treated to the beginning of the fifth instar Bombyx larvae. Its application prolonged the last instar and prevented metamorphic events. Analyses were performed from day 6 of the fifth instar to 24 hr after pupation in controls and to day 14 of the fifth instar in the fenoxycarb treated group. According to our results, the presence of EcR-B1 in the midguts of the fenoxycarb treated group during the feeding period suggested that EcR-B1 was involved in the functioning of larval cells and during this period fenoxycarb did not affect EcR-B1 status. Immediately after termination of the feeding stage, the amount of EcR-B1 protein increased, which indicated that it may strengthen the ecdysone signal for commitment of remodeling process. In the fenoxycarb treated group, its upregulation was delayed, which may be related to the inhibition of ecdysone secretion from the prothoracic gland.     

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.