早熟素对棉铃虫滞育的终止作用(英文)

用外源激素和某些化合物处理棉铃虫的滞育蛹 ,结果发现早熟素与蜕皮素一样可终止棉铃虫蛹的滞育 ,使其进入到发育阶段。而保幼激素类似物ZR 5 1 5 ,环腺氨酸 (cAMP)和色氨类化合物 (5 HT)则完全不具有终止棉铃虫滞育的作用。结合早熟素还可终止蚜虫寄生蜂AphidiusmatricariaeHaliday和PraonvolucreHaliday的预蛹滞育的报道 ,建议早熟素应具有终止多种类昆虫滞育的功能     

滞育和非滞育棉铃虫血淋巴中蛋白质含量及图谱的比较

昆虫在滞育期间和非滞育期间,由于某些器官的生理功能产生差异,致使血淋巴中的化学成份发生了量或质的变化,特别是蛋白质的变化,如在浦育的马铃薯叶甲Leptinotarsadecemlineata[1,2]及西南玉米杆草螟Diatraeagrandiosella[3,4]的血淋巴中,均发现了特殊蛋白质,即滞育蛋白。为了弄清滞育棉铃虫Helicoverpaarmigera的血淋巴中是否也出现滞育蛋白或与滞有相关联的蛋白,及注定滞育和非滞育棉铃虫在不同发育阶段中蛋白质含量的变化和谱带特征,以进一步探索棉铃虫滞育的生理生化机制,我们通过电泳等手段对不同时期的滞育和非滞育棉铃虫…     

棉铃虫脑在控制滞育中的作用

脑摘除试验表明: 棉铃虫蛹的初期发育（化蛹后12 h 内）受脑的控制;化蛹12 h后的滞育蛹发育与脑无关。注射活性脑的匀浆液可阻止部分注定滞育的预蛹和化蛹后1～2天的蛹进入滞育状态,说明滞育的个体很有可能从预蛹期开始其脑的活性就已降低。     

不同氰戊菊酯抗性水平的棉铃虫滞育比较研究

本文利用人工诱导滞育的方法,对不同氰戊菊酯抗性水平的棉铃虫蛹滞育进行了比较研究。结果表明,氰戊菊酯抗性高的棉铃虫滞育率低,滞育蛹历期长,耐寒性差。不同氰戊菊酯抗性水平的棉铃虫蛹重无显著差异。     

葱蝇非滞育、夏滞育和冬滞育蛹体内抗氧化酶活性的比较分析（英文）

【目的】通过测定并比较分析抗氧化酶活性及谷胱甘肽氧化还原状态,探讨葱蝇Delia antiqua非滞育、夏滞育和冬滞育蛹体内抗氧化系统的差异。【方法】取葱蝇非滞育、夏滞育和冬滞育蛹,分别测定铜锌超氧化物歧化酶(Cu/Zn-SOD)、锰超氧化物歧化酶(Mn SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPx)和谷胱甘肽还原酶(GR)活性及还原型谷胱甘肽(GSH)和氧化性还原型谷胱甘肽(GSSG)含量及GSH/GSSG比随发育时间的变化;并对各抗氧化因子进行了典型判别分析。【结果】5种抗氧化酶的活性在整个蛹期是动态变化的。与非滞育蛹和夏滞育蛹相比,处于滞育前期的冬滞育蛹具有较高的Cu/Zn-SOD酶活性,但处于夏滞育及冬滞育的维持期和滞育后期的滞育蛹体内Cu/Zn-SOD酶活性则明显低于同一发育时期的非滞育蛹。在整个蛹期,非滞育蛹体内Mn SOD酶活性显著高于夏滞育和冬滞育蛹,而在夏滞育和冬滞育蛹之间则无明显差异。比较两种酶活性则发现同一发育时期的Mn SOD平均酶活性明显高于Cu/Zn-SOD酶活性。在头外翻之前,滞育蛹体内CAT酶活性高于非滞育蛹,但处于滞育维持期和后期的蛹体内CAT酶活性则低于非滞育蛹。无论在非滞育蛹还是滞育蛹中,GPx和GR酶活性变化基本上呈相反的趋势。典型判别分析进一步表明葱蝇蛹体内的抗氧化系统具有发育时期和滞育类型特异性。【结论】非滞育蛹与夏滞育和冬滞育蛹体内的氧化还原状态存在明显差异。滞育前期和滞育后期的蛹体内较高的抗氧化酶活性和较低的GSH/GSSG比提示氧化状态的变化与高的呼吸速率及发育过程密切相关。     

棉铃虫转录因子c-Myc基因在滞育和非滞育蛹脑中的表达分析及多克隆抗体制备

【目的】c-Myc是近年来研究较多的转录因子,也是受Wnt/β-catenin信号通路调节的重要靶标。本研究旨在克隆棉铃虫 Helicoverpa armigera c-Myc基因,从核酸水平初步调查 c-myc 在滞育和非滞育蛹脑中的表达情况,同时制备其蛋白的多克隆抗体。【方法】通过RACE方法克隆棉铃虫 c-myc 基因的cDNA,运用RT-PCR方法比较滞育和非滞育蛹脑中Har-c-myc基因的表达情况。根据获取的序列构建原核表达载体,在大肠杆菌 Escherichia coli 中进行表达,纯化后免疫新西兰兔,制备了多克隆抗体。【结果】克隆了棉铃虫 c-myc 基因,核酸水平的研究表明滞育蛹脑中 c-myc 表达水平明显低于非滞育蛹脑。成功地在大肠杆菌中表达了c-Myc部分肽段并通过镍柱纯化获得了较纯的重组蛋白。制备的c-Myc抗体效价达到了1:125 000。【结论】滞育蛹脑中 Har-c-myc 的表达下调。获得了抗棉铃虫c-Myc的多克隆抗体。本研究的成果为后续进一步深入研究棉铃虫Wnt/β-catenin信号通路在棉铃虫发育中的作用奠定了基础。     

葱蝇非滞育、 冬滞育和夏滞育蛹发育和形态特征比较

昆虫非滞育、 冬滞育和夏滞育蛹具有不同的生理和发育过程。本研究以葱蝇Delia antiqua作为模式种, 以黑腹果蝇Drosophila melanogaster蛹的发育形态特征和命名为参照, 用解剖、 拍照、 长度测量和图像处理等方法系统地比较研究了非滞育、 冬滞育和夏滞育蛹的发育历期和形态变化, 重点在头外翻和滞育相关发育和形态特征, 目的在于弄清非滞育、 冬滞育和夏滞育蛹发育和形态特征差异, 为滞育发育阶段的识别、 滞育分子机理研究奠定形态学基础。冬滞育蛹的滞育前期、 滞育期和滞育后期分别为4, 85和27 d, 夏滞育蛹的滞育前期、 滞育期和滞育后期分别为2, 8和22 d。从化蛹至头外翻完成为滞育前期, 头外翻完成约10 h内复眼中央游离脂肪体可见。头外翻的完成是滞育发生的前提, 非滞育、 夏滞育和冬滞育蛹头外翻发生在化蛹后的48, 36和83 h, 在头外翻过程中发育形态没有差异。头外翻的过程为： 首先, 头囊和胸部附肢从胸腔外翻, 头部形态出现; 然后, 腹部肌肉继续收缩, 将血淋巴和脂肪体推进头部及胸部附肢。葱蝇蛹在完成蛹期有效积温约15%时进入冬滞育或夏滞育。在滞育期, 蛹的形态一直停留在复眼中央游离脂肪体可见这一形态时期, 且冬滞育和夏滞育的蛹在形态上没有区别。在体长、 体宽和体重上, 冬滞育蛹最大, 夏滞育蛹次之, 非滞育蛹最小。在滞育后期, 在黄色体出现期间, 非滞育蛹的马氏管清楚可见, 呈绿色, 而滞育蛹的马氏管几乎不可见。本研究为认知昆虫滞育生理、 从发育历期和形态上推断滞育发育进程提供了依据。     

烟夜蛾滞育蛹和非滞育蛹的耐寒性

对烟夜蛾Helicoverpa assultaGuen?e不同日龄滞育蛹和非滞育蛹的过冷却点和结冰点测定结果表明,在预蛹至5日龄蛹期,过冷却点和结冰点均逐渐下降。在预蛹期和3日龄蛹期,滞育蛹的过冷却点与非滞育蛹差异不显著;5日龄蛹时,滞育蛹和非滞育蛹的过冷却点分别下降至(-15.7±1.8)℃和(-13.5±1.2)℃,结冰点分别降至(-12.7±2.7)℃和(-9.5±1.3)℃,两类蛹间的差异均达极显著水平。自然越冬后,滞育蛹的存活率显著高于非滞育蛹,在土壤不同深处越冬的蛹的存活率存在差异,距土表15cm深的蛹存活率最高。     

柑橘大实蝇滞育和非滞育蛹体内海藻糖含量的调控

【目的】通过比较柑橘大实蝇Bactrocera minax蛹滞育期与滞育前和滞育后以及滞育蛹与非滞育蛹体内海藻糖和葡萄糖含量的变化、海藻糖合成代谢途径中关键酶的活力变化以及关键酶基因的表达量变化,明确蛹滞育期间海藻糖合成代谢途径中关键酶对海藻糖含量的调控。【方法】利用分光光度法检测柑橘大实蝇滞育前(1日龄蛹)、滞育期(30,60和90日龄蛹)以及滞育后(120和150日龄蛹)蛹体内海藻糖与葡萄糖含量的变化,以及海藻糖合成代谢途径中的海藻糖-6-磷酸合成酶(TPS)、海藻糖-6-磷酸磷酸酯酶(TPP)和海藻糖酶(Tre)活力的变化;利用实时定量荧光PCR(qPCR)检测TPS,TPPB,TPPC-1,TPPC-2和Tre-1基因表达量的变化。向1日龄蛹体内注射20-羟基蜕皮酮(20E)作为处理(以注射10%乙醇为对照),分别于注射后1和30 d比较处理组与对照组蛹体内海藻糖与葡萄糖含量、关键酶活力以及上述基因表达量的差异。【结果】柑橘大实蝇蛹进入滞育后,海藻糖含量显著升高,葡萄糖含量无显著变化; TPS和TPP的酶活力以及TPS,TPPC-1和TPPC-2表达量在化蛹后逐渐升高,于滞育期达到最高水平,维持至羽化前显著下降;TPPB表达量在整个蛹期无显著差异; Tre酶活力以及Tre-1表达量在化蛹后逐渐升高,于滞育早期达到最高水平,随后显著下降,羽化前再次显著上升。注射20E后1 d,与对照组相比,处理组蛹体内海藻糖与葡萄糖含量、关键酶(TPS,TPP和Tre)活力以及TPS,TPPC-2和Tre-1表达量无显著变化,TPPB表达量显著下降,TPPC-1表达量显著上升;注射后30 d,与对照组滞育蛹相比,处理组非滞育蛹海藻糖含量显著上升,葡萄糖含量、TPS和Tre酶活力、TPS和Tre-1表达量显著下降,TPP酶活力以及TPPB和TPPC-2表达量无显著差异。【结论】柑橘大实蝇蛹体内海藻糖的含量在合成代谢途径中关键酶的调控下,随着滞育状态发生变化,表明海藻糖与滞育之间存在密切的关系,但其作用机理仍待进一步研究。     

棉铃虫Wnt-1基因克隆、多克隆抗体制备及在滞育和非滞育蛹脑中的表达检测

【目的】Wnt1蛋白是Wnt/β-catenin信号通路的重要成员。本研究旨在克隆棉铃虫Helicoverpa armigera Wnt1基因,制备多克隆抗体,进而从蛋白水平初步研究该蛋白在滞育和非滞育蛹脑中的表达情况。【方法】通过RACE的方法克隆棉铃虫Wnt1基因。根据获取的序列构建Wnt1的真核表达载体并调查其亚细胞定位,同时构建原核表达载体,在大肠杆菌Escherichia coli BL21(DE3)中进行表达、纯化后免疫兔子,制备多克隆抗体。最后用Western blot的方法检测Wnt1蛋白在两种发育状态蛹脑中的表达情况。【结果】成功克隆了棉铃虫Wnt1基因(Gen Bank登录号为KJ206240)。Wnt1定位在细胞质,通过镍柱纯化获得了较纯的重组蛋白。制备的Wnt1抗体效价高达1∶625 000。Western blot结果表明滞育蛹脑中Wnt1蛋白水平明显低于非滞育蛹脑。【结论】获得了高效价的棉铃虫Wnt1多克隆抗体。我们的结果表明滞育蛹脑中Wnt/β-catenin通路很有可能受到抑制。本研究结果为进一步深入研究棉铃虫Wnt/β-catenin信号通路在棉铃虫发育中的作用奠定了基础。     

Diapause and development in the tobacco budworm,Heliothis virescens: A comparison of haemolymph ecdysteroid titres

The signal to induce diapause in H. virescens comes early in development (prior to the third instar in most insects), but the signal to break diapause can come shortly after entrance into diapause at pupation. Haemolymph ecdysteroid titres in both diapause-bound and non-diapause-bound Heliothis virescens larvae were similar in the first two thirds of the last-larval instar, when similar changes in morphology and behaviour occurred. However, the number of stepwise increases in titre and the timing of the steps was different in the two groups of larvae. Haemolymph ecdysteroid titres in the last third of the instar were approx, five times higher in non-diapause than in diapause-bound larvae. In diapausing pupae, haemolymph ecdysteroid titres dropped to levels found in larvae which had completed two thirds of the last instar. When diapausing pupae were warmed to break diapause, haemolymph ecdysteroid titres rose again. However, 2 of the 4 high ecdysteroid levels detected in pupae developing after diapause break were considerably lower than those detected for non-diapause pupae.     

棉铃虫蛹期血淋巴的蜕皮甾类

目前为止仅在少数几种昆虫中研究过蛹期的蜕皮激素。关于蜕皮甾类的性质分析,结果也颇不一致。本文采用放射免疫分析、薄层层析、高压液相色谱及质谱对棉铃虫Heliothis armigera蛹血淋巴内的蜕皮激素进行了研究。结果如下:1.物理-化学方法证明蛹血淋巴内存在二种蜕皮甾类:蜕皮酮和20-羟基蜕皮酮。2.蛹期蜕皮甾类滴度呈一宽峰,高峰出现在化蛹后的第5天(3435ng/ml)。3.在高峰时,蜕皮酮与20-羟基蜕皮酮的比例为1:3.57,说明20-羟基蜕皮酮是主要的蜕皮甾类。4.比较雌雄两性蛹的蜕皮甾类滴度,未见明显差异。研究表明在棉铃虫中影响成虫发育的主要激素是20-羟基蜕皮酮而不是蜕皮酮。     

A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.     

Levels of ecdysteroids in diapause and non-diapause eggs of the Australian plague locust,Chortoicetes terminifera (Walker)

The levels of ecdysteroids during embryogenesis in Chortoicetes terminifera were measured by radioimmunoassay. Soon after laying, eggs laid by females reared under non-diapause conditions contained three times as much as those laid by females reared under diapause inducing conditions. In eggs incubated at 32°C which did not enter diapause there was a sharp rise in ecdysteroid levels which coincided with the formation of the first-instar cuticle, but this did not occur in eggs incubated at 20°C even though they developed and hatched normally. Comparisons are made with previous results in other locust species and the possible role of ecdysteroids in embryonic diapause is discussed.     

Ecdysteroid synthesis and molting by the tobacco hornworm, Manduca sexta, in the absence of prothoracic glands

When a pair of prothoracic glands (PGs) were removed from Manduca sexta pupae on the day of pupation, the hemolymph ecdysteroid titer remained at a low level. When a portion of the gland pair was extirpated from pupae after the critical period for prothoracicotropic hormone release, the maximum hemolymph ecdysteroid titer was reduced in proportion to the mass of the PGs removed. These findings clearly showed that the PGs in intact pupae are responsible for the elevated ecdysteroid titer required to elicit adult development on schedule. When brains were removed on the day of pupation, the initiation of adult development was delayed for weeks or months. In contrast, pupae whose PGs were removed on the day of pupation initiated development only 7 days late, indicating the existence of an additional source of pupal ecdysteroids. Further, abdomens of male M. sexta that were isolated on the day of pupation initiated adult development spontaneously within 70 days. The implantation of day 0 pupal brains into these isolated abdomens accelerated the initiation of adult development and elicited synchronous adult development. The hemolymph ecdysteroid titer of those isolated abdomens receiving implants of brains increased within 5 days and reached a maximum level of 1.5 micrograms/ml. The analysis of hemolymph ecdysteroids by reverse-phase HPLC revealed that ecdysone was the major moiety and that the ecdysteroid composition was similar to that of normal, intact pupae that had just initiated adult development. These results demonstrate that the PGs are not requisite for adult development. An increased hemolymph ecdysteroid titer was also observed in isolated abdomens from which the testes were removed and in abdomens devoid of their digestive tract. Indeed, in the latter case, the ecdysteroid titer attained much higher levels than those observed for abdomens with intact guts. Despite numerous attempts to identify the tissue(s) in the isolated abdomens responsible for the increase in ecdysteroid titer, its identity remains unknown.     

Prothoracicotropic Hormone Acts as a Neuroendocrine Switch between Pupal Diapause and Adult Development

Diapause is a programmed developmental arrest that has evolved in a wide variety of organisms and allows them survive unfavorable seasons. This developmental state is particularly common in insects. Based on circumstantial evidence, pupal diapause has been hypothesized to result from a cessation of prothoracicotropic hormone (PTTH) secretion from the brain. Here, we provide direct evidence for this classical hypothesis by determining both the PTTH titer in the hemolymph and the PTTH content in the brain of diapause pupae in the cabbage army moth Mamestra brassicae. For this purpose, we cloned the PTTH gene, produced PTTH-specific antibodies, and developed a highly sensitive immunoassay for PTTH. While the hemolymph PTTH titer in non-diapause pupae was maintained at high levels after pupation, the titer in diapause pupae dropped to an undetectable level. In contrast, the PTTH content of the post-pupation brain was higher in diapause animals than in non-diapause animals. These results clearly demonstrate that diapause pupae have sufficient PTTH in their brain, but they do not release it into the hemolymph. Injecting PTTH into diapause pupae immediately after pupation induced adult development, showing that a lack of PTTH is a necessary and sufficient condition for inducing pupal diapause. Most interestingly, in diapause-destined larvae, lower hemolymph titers of PTTH and reduced PTTH gene expression were observed for 4 and 2 days, respectively, prior to pupation. This discovery demonstrates that the diapause program is already manifested in the PTTH neurons as early as the mid final instar stage.     

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly,Delia antiqua

The influence of pupal diapause on adult eclosion rhythm of Delia antiqua was investigated. When non-diapause and diapause pupae were exposed to various photoperiods at 15, 20 and 25 °C, both of them emerged as adults close to the light-on time, but the phase of eclosion varied with photoperiod and temperature. Moreover, there was a significant difference in the eclosion time between non-diapause and diapause pupae; the eclosion peak of diapause pupae was earlier than that of non-diapause pupae. When non-diapause and diapause pupae were transferred to constant darkness (DD) after having experienced LD 12:12 at 15, 20 and 25 °C, both showed circadian rhythmicity in eclosion. Although the free-running period (τ) decreased slightly as temperature increased in both non-diapause and diapause pupae, the latter tended to show shorter τ than the former. This observation suggests that the observed difference in eclosion time in LD cycles between non-diapause and diapause pupae is due to differences in τ.     

Possible involvement of ecdysteroids in embryonic diapause of Locusta migratoria

In the Ibaraki population (Japan) of Locusta migratoria, adult locusts produce diapause eggs under short-day (SD) conditions and non-diapause eggs under long-day (LD) conditions. The identity and titre of ecdysteroids in the ovaries and eggs from LD and SD adult females were investigated by RIA/HPLC. Maternal ecdysteroids accumulated in the developing ovaries represented about 90% polar conjugates, 5% free ecdysteroids and 5% non-hydrolyzable metabolites. Before oviposition the quantity of ecdysteroids reached 29.8±1.85 ng 20-hydroxyecdysone equiv. per mg tissue ovaries from LD females and 13.1±3.55 ng 20E equiv./mg in ovaries from SD females. The sum of RIA-positive materials in newly laid eggs was more than three times higher in non-diapause eggs than in diapause eggs. Ecdysteroids present in egg extracts comprised about 85% polar conjugates, 5% free ecdysteroids and 10% non-hydrolyzable metabolites. On the other hand, after diapause termination the amount of ecdysteroids increased drastically. Also, the composition of ecdysteroids differed from that observed during diapause and became comparable to that of non-diapause eggs. The significant differences in the ecdysteroids between non-diapause and diapause eggs may suggest the possible involvement of these compounds in the control of embryonic diapause of this locust.