昆虫激素和抗激素类在蚕业上的应用研究进展

控制家蚕幼虫蜕变和变态的内分泌系统是脑一咽侧体一前胸腺。咽侧体分泌的保幼激素（JH）和前胸腺分泌的蜕皮激素（MH）调节着家蚕的幼虫蜕皮、变态等生命现象,而脑分泌的促咽侧体激素和促前胸腺激素又控制着这两种腺体的分泌活动。家蚕的MH和JH的化学结构早在60年代中期被先后阐明,70年代后对这两种主要激素在血淋巴中的浓度已能精确定量,从而阐明了发生幼虫蜕皮和变态的激素环境。与此同时,发现了一些天然化合物能影响家蚕正常的内分泌活动,导致早熟变态和其他生理变化（如体色变化、生有障碍等）,称之为抗激素类物质。家蚕内分…     

成虫滞育的主要特点及神经内分泌调控

成虫滞育的主要特点是生殖受到了抑制,其调控涉及到咽侧体、脑和前胸腺的作用,但主要以咽侧体的作用为主.滞育期间,咽侧体的活性很低,分泌的保幼激素量极微,而咽侧体的活性高低直接受脑所分泌的神经激素所调控.     

昆虫变态的激素与基因调控

昆虫变态是一个有趣而复杂的生物学现象,有不完全变态和完全变态。昆虫通过变态获得飞翔与生殖能力,扩大了生活范围,增强了适应能力。昆虫变态主要由咽侧体分泌的保幼激素和前胸腺分泌的蜕皮激素协同调控,众多基因参与。本文综合近几年的研究进展,重点介绍昆虫变态过程中保幼激素与蜕皮激素通过级联反应基因和microRNA调控的机制,以及保幼激素和蜕皮激素的合成调节机制。     

昆虫蛹滞育的神经内分泌调控

从神经内分泌的角度,分别介绍了脑、前胸腺和咽侧体在昆虫蛹滞育中所起的作用。脑主要是通过对促前胸腺激素的合成和释放的控制来参与滞育调节的;而前胸腺的活性过低,分泌的蜕皮激素远远低于启动成虫发育所需要的量,是导致蛹滞育的最根本原因;咽侧体在某些虫种中对蛹滞育可能起到间接的调控作用。     

植物中的昆虫变态激素

昆虫在一生中要进行多次蜕皮才能长成成虫,如蚕进行4次蜕皮,然后变态成蛹,再变态成蛾。昆虫的蜕皮受其体内的激素所支配。昆虫头部的咽侧体分泌保幼激素,由胸部的前胸腺分泌蜕皮激素,这两种激素保持平衡即能完成正常发育。     

桑蚕促前胸腺激素的作用与前胸腺分泌活动的某些特点

本工作以前胸腺的体外器官培养技术和蜕皮激素的放射免疫分析法(MH-RIA)相结合,研究了桑蚕(Bombyx mori)促前胸腺激素(PTTH)的作用与前胸腺分泌的某些特点。结果表明,被PTTH激活后的前胸腺,在一定的时相过程内合成并分泌脱皮甾类激素;前胸腺本体不积累蜕皮甾类激素;PTTH对前胸腺的作用是积累性的;五龄不同天数的前胸腺合成分泌脱皮甾类激素的能力不同,并有不同的剂量反应。     

三眠蚕诱导剂咪唑类物质KK-42对桑蚕内分泌系统的作用

为了研究作为三眠现诱导剂的一种唑化合物RK-42的作用机制,本文应用侧休(CA)短期体化学测定Bombyx motx则的活性,通过记蟆皮(MH)的垫射免疫分析(RIA)法测定了血苏巴内根据的放射免疫法成功珍KK-42作用后的肢导泌侃前胸蚊腺面的试验,提出了KK-42作用的新解说,吧调体活性的湖汇KK-42与侧体外支明KK-42应用下桑蚕四龄前期时,把器官首先见侧休,咽侧体看成性反使用分泌PTTH产生相应变化,从而引起蜕皮激素高峰的推迟.推迟了的蜕皮素峰与扣对馈管的保幼激素JH共同作用的结果引起了化蛹蜕皮,产生了三眠蚕.     

昆虫促前胸腺激素研究进展

昆虫促前胸腺激素研究进展李毅平龚和（中国科学院动物研究所,北京１０００８０）关键词促前胸腺激素受体信号系统促前胸腺激素（ｐｒｏｔｈｏｒａｃｉｃｏｔｒｏｐｉｃｈｏｒ－ｍｏｎｅＰＴＴＨ）因其促进前胸腺（ＰＧ）合成和分泌蜕皮激素而得名,以前也称为脑激素,因...     

东方粘虫末龄幼虫血淋巴蜕皮甾类滴度的变化及促前胸腺激素对前胸腺的活化作用

东方粘虫六龄幼虫血淋巴蜕皮甾类滴度,在幼虫的取食生长期一直处在很低的水平(<6pg/μl血淋巴),其后于徘徊期的前一天开始升高,至预蛹期形成唯一的1个高峰(～450pg/μl血淋巴)。前胸腺离体培养的结果表明,前胸腺分泌活力与血淋巴蜕皮甾类滴度的动态呈基本平行的趋势,只是较后者超前了约24小时。促前胸腺激素粗提物能直接活化离体前胸分泌蜕皮甾类。粘虫六龄2日龄(LVI_2)幼虫的前胸腺已能被促前胸腺激素活化,说明此时的前胸腺对促前胸腺激素已具感受性。     

家蚕幼虫-蛹变态期前胸腺和脂肪体细胞解离和程序性细胞死亡的比较分析

【目的】检测家蚕Bombyx mori变态期前胸腺细胞的解离、自噬与凋亡,并与脂肪体的进行对比,从而解析昆虫幼虫-蛹变态期过程中不同组织重塑的异同。【方法】以家蚕5龄期、游走期、预蛹期和蛹期前胸腺和脂肪体组织为材料,在光学显微镜下观察前胸腺和脂肪体细胞解离情况;分别利用Lyso-Tracker和TUNEL染色,在荧光共聚焦显微镜下观察细胞自噬和细胞凋亡的发生情况;利用qRT-PCR检测家蚕前胸腺中自噬发生标志基因Atg8的表达水平;利用透射电镜观察前胸腺和脂肪体细胞自噬小体和前胸腺线粒体;利用Caspase3酶活性检测试剂盒测定Caspase3酶活性;利用qRT-PCR检测前胸腺中蜕皮酮(ecdysone)合成相关基因Spo,Phm,Dib和Sad的表达水平;利用酶免疫试验(enzyme-immunoassay, EIA)测定前胸腺中蜕皮酮的含量,进而检测合成蜕皮酮的活力。【结果】在家蚕幼虫到蛹的变态发育过程中,在化蛹第1天家蚕前胸腺和脂肪体细胞中同时开始出现细胞解离;脂肪体细胞自噬和凋亡分别在游走期和预蛹第1天开始出现并逐渐增强;而前胸腺一直到化蛹第2天都没有发生明显的细胞自噬和凋亡;此外,前胸腺中线粒体的形态变化和蜕皮酮合成相关基因的转录水平均与对应时期前胸腺合成蜕皮酮的活力一致。【结论】在变态发育时家蚕不同组织消亡发生的时间不同,虽然前胸腺和脂肪体在化蛹第1天同时出现细胞解离,但是前胸腺直到化蛹第2天都不发生细胞自噬和凋亡,可能与其持续合成蜕皮酮的功能有关。本研究为昆虫幼虫-蛹变态发育时期组织消亡的深入研究提供了理论依据与工作基础。     

Decreased JH biosynthesis is related to precocious metamorphosis in recessive trimolter (rt) mutants of the silkworm, Bombyx mori

In recessive trimolter (rt) mutants of the silkworm, Bombyx mori, that have four larval instars rather than five larval instars of normal B. mori, a decrease after a small increase in the hemolymph ecdysteroid titer during the early stages of the last (fourth) larval instar appeared to be a prerequisite for larvae to undergo precocious metamorphosis. The present study was carried out to investigate the possible mechanism underlying this decrease in the ecdysteroid titer. It was found that juvenile hormone (JH) biosynthetic activity of the corpora allata (CA) increased during the first day of the last larval instar, but its absolute JH biosynthesis activity was relatively lower compared to that of normal fourth-instar larvae in tetramolters. This lowered JH biosynthetic activity appeared to be related to a decrease in prothoracic gland ecdysteroidogenesis during the second day of the last instar, because hydroprene application prevented this decrease in prothoracic gland ecdysteroidogenesis, leading to the induction of a supernumerary larval molt. The in vitro incubation of prothoracic glands with hydroprene showed that hydroprene did not directly exert its action on prothoracicotropic hormone (PTTH) release. Further study showed that the application of hydroprene enhanced the competency of the glands to respond to PTTH. From these results, it was supposed that the lowered JH biosynthesis of the CA during the first day of last instar in rt mutants was related to decreased ecdysteroidogenesis in the prothoracic glands during the second day, thus playing a role in leading to precocious metamorphosis.     

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

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

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.     

Prothoracicotropic hormone regulates developmental timing and body size in Drosophila

In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. In this report, we examine the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion of larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the overgrowth and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth-rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval.     

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.     

Juvenile hormone acid and ecdysteroid together induce competence for metamorphosis of the Verson's gland in Manduca sexta

In the last larval instar of Lepidoptera, ecdysteroid in the absence of juvenile hormone (JH) is believed to cause the shift from larval to pupal development. In Manduca sexta, tissues such as the Verson's gland and crochet epidermis become pupally committed before the earliest pulse of ecdysteroid that occurs on day 2. What causes the change in commitment in these tissues? First it was necessary to determine at what stage these tissues become competent to express the pupal program. Last instar larvae of different ages were induced to molt prematurely by feeding the ecdysteroid analog RH5992 and Verson's gland proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Glands became competent to make pupal proteins between 24 and 32 h after the last larval ecdysis. Next, hormonal regulation of competence was examined in ligated abdomens of 12h last instar larvae. Treatment with JH II acid or methoprene acid plus a low dose (1/50th of the molt inducing dose) of RH5992 induced competence, whereas RH5992 alone, methoprene acid alone or methoprene plus RH5992 did not. Verson's glands maintained in vitro produced pupal proteins in response to methoprene acid together with RH5992 but not with RH5992 alone. Likewise, crochet epidermis lost the ability to make crochets (metamorphic change) only in isolated abdomens treated with JH II acid or methoprene acid and low doses of RH5992. In conclusion, JH acid in the presence of basal levels of ecdysteroid induces tissue competence for metamorphosis. Metamorphic competence is followed by commitment, induced by a small pulse of ecdysteroid in the absence of JH, and finally by expression caused by a high titer of ecdysteroid. It is proposed that JH acid is an essential metamorphic hormone.     

昆虫蜕皮的分子机理及应用

昆虫蜕皮是一个由PTTH启始的、激素介导的基因序列表达和相互作用的级联反应过程。阐明昆虫蜕皮的分子机理,不仅可以解释发育生物学的科学问题,为害虫控制提供新的思路,还可以从中发现新的可资生产应用的分子。作者通过蛋白质组学方法从棉铃虫Helicoverpa armigera Hubner蜕皮幼虫鉴定到30个差异表达的蛋白质。通过抑制性消减杂交技术,从棉铃虫蜕皮幼虫、变态决定幼虫和5龄取食幼虫鉴定到100个表达序列标签(EST)。证明其中的11个EST在蜕皮或变态时差异表达。通过RT-PCR方法克隆棉铃虫激素接受子3基因,研究该基因在发育中的表达模式。用该基因构建具有绿色荧光蛋白标记和多角体蛋白的基因重组病毒(AcMNPV-GFP-HHR3-Polh)。实验结果表明,AcMNPV-GFPHHR3-Polh病毒可以通过注射或口服感染棉铃虫,导致棉铃虫幼虫非正常蜕皮、生长延缓、半数存活时间下降。该研究显示昆虫蜕皮功能基因在害虫控制中有很好的应用前景。蜕皮功能基因的表达与调控、蜕皮激素介导的信号转导通路、变态过程中组织解体和重建的分子机理、激素调控基因顺序表达的分子机理、变态起始因子、JH受体等是本领域今后的主要研究方向。     

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.     

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.     

Early events in adult eye development of the moth, Manduca sexta

The eye imaginal disc of Manduca sexta is created early in the final larval instar from the adult eye primordium, which is composed of fully differentiated cells of the larval head capsule epidermis. Concomitant with the down-regulation of the larval epidermal program, expression of broad, a marker of pupal commitment, is activated in the primordium. The cells then detach from the cuticle, fold inward, and begin to proliferate at high levels to produce the inverted, eye imaginal disc. These and other events that begin on the first day of the final larval instar appear to mark the initiation of metamorphosis. Little is known about the endocrine control of the initiation of metamorphosis in any insect. The hemolymph titer of juvenile hormone (JH) declines to low levels during this period and the presence of JH is sufficient to repress development in cultured eye primordia. However, maintenance of JH at high levels in vivo by treatment with long-lasting JH mimics has no apparent effect on early steps in eye imaginal disc development. We discuss our findings in the context of the endocrine control of metamorphosis. The initiation of metamorphosis in Manduca, and perhaps a wide range of insect species, appears to involve the overcoming of JH repression by an unidentified, nutrient-dependent, hormonal factor.