高温条件下棉铃虫化蛹率、夏滞育率和蛹重的变化

在预蛹期,高温处理能诱导棉铃虫蛹进入夏滞育。本实验着重就33～39℃的变温下滞育蛹和未滞育蛹的失重动态进行了对比研究,同时以常温（27℃）下蛹作为参照。研究发现： 在33～39℃的变温条件下,棉铃虫化蛹率显著低于其在常温下的化蛹率,且所化蛹中有63.2%的雄性和10.9%的雌性进入高温夏滞育,其中高温滞育蛹和未滞育蛹分别都轻于正常发育蛹。化蛹后第2日至第5日期间,高温滞育蛹失重量显著低于高温未滞育蛹和正常发育蛹的失重量,分别为3.62、13.30和5.49 mg;蛹期总失重量结果与化蛹后第2～5日间蛹失重量趋势一致,高温滞育蛹、未滞育蛹和正常发育蛹失重量分别为15.60、49.35和26.30 mg。蛹失重动态研究发现高温滞育蛹在夏滞育期间其失重曲线平缓,显著低于高温未滞育蛹和正常发育蛹;高温滞育蛹滞育解除后,其失重曲线与正常发育蛹的失重趋势基本一致。结果表明,棉铃虫夏滞育蛹能通过维持低的代谢水平来度过不利环境,具有一定的生态适应意义。     

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。     

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。     

光周期和温度对大草蛉滞育解除及滞育后发育和繁殖的影响

【目的】大草蛉Chrysopa pallens(Rambur)是自然界重要的天敌昆虫,以预蛹兼性滞育越冬。本研究旨在明确光周期和温度对大草蛉滞育解除及滞育后发育和繁殖的影响。【方法】在室内观测了不同温度(22℃和25℃)和光周期(15L∶9D及9L∶15D)条件下大草蛉预蛹的滞育解除及滞育解除后的蛹期、蛹存活率、成虫鲜重、成虫寿命、产卵前期和单雌产卵量等生物学特性,以及5℃低温处理对预蛹滞育解除的作用,并分析了滞育持续时间对大草蛉滞育解除后发育和繁殖的影响。【结果】在光周期15L∶9D和9L∶15D下,25℃下大草蛉预蛹期(分别为50.09和49.47 d)显著短于22℃下(分别为80.80和82.20 d)。5℃低温处理极显著延长了大草蛉预蛹期(P0.01),且缩小了预蛹期的个体差异。22℃下,与非滞育预蛹相比,滞育后预蛹的存活率显著降低,蛹期和产卵前期显著延长,雌成虫寿命显著缩短,成虫鲜重和单雌产卵量显著下降。22℃,光周期15L∶9D下大草蛉的滞育持续时间为50~170 d,且能影响滞育后发育:随着滞育持续时间的延长,蛹期逐渐延长,雌、雄成虫的鲜重逐渐降低,雄成虫寿命呈先延长后缩短的趋势,蛹存活率、雌成虫寿命、产卵前期和单雌产卵量没有显著性差异。【结论】试验条件下,两种光周期对大草蛉滞育解除、滞育后发育和繁殖没有明显的影响,而温度是调节大草蛉滞育发育和繁殖的重要因子。较高温度能促进滞育的解除,低温处理能够同步种群的滞育发育。大草蛉的滞育存在生殖代价,滞育持续时间影响滞育解除后的部分生物学特性。     

南京地区棉铃虫越冬蛹滞育的解除与发育

南京地区棉铃虫Heliclverpa armigera (Hubner)越冬蛹滞育的解除时间及解除后发育与温度的关系等问题。结果表明,该地区越冬蛹于12月中旬前后解除滞育,12月下旬至3月上旬处于休眠状态,3月下旬至4月上旬温度上升至约10℃～12℃后眼点开始移动。发现该虫在滞育后的发育中,眼点移动前期的发育速率、发育起始温度及血淋巴总蛋白含量动态明显不同于眼点移动后期或非滞育蛹。     

亚洲玉米螟越冬幼虫存活和滞育解除与水分摄入的关系

在22～28℃和16～22℃变温条件下研究了水分对亚洲玉米螟Ostrinia furnacalis (Guenée)越冬幼虫存活、滞育解除及成虫生殖潜力的影响。结果表明：在越冬幼虫复苏后至化蛹阶段,水分是影响越冬幼虫完成滞育发育的关键因子,只有满足其饮水需求,才能解除滞育而化蛹。幼虫的存活和化蛹率随幼虫饮水后体重的增加而增加。幼虫化蛹的早晚受温度和水分的综合影响,在22～28℃下越冬幼虫化蛹进度较快,在复苏后35天时为最佳饮水时间,比在16～22℃下早5～10天。此阶段如能满足幼虫的需水量,幼虫死亡率低,化蛹率高,而只在滞育发育早期或后期饮水时,死亡率高,化蛹率低。同一变温条件下,其滞育发育因饮水时间的推迟而延长。因此可以推测春季温度偏高、干旱的气候条件不利于亚洲玉米螟越冬代的大发生,反之亦然。     

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。     

滞育和非滞育棉铃虫血淋巴类固醇蜕皮素含量变化的比较

采用放射免疫分析法对不同时期的注定滞育和非滞育棉铃虫的血淋巴中的类固醇蜕皮素的含量进行了测定,发现在预蛹期间,注定滞育的棉铃虫的类固醇蜕皮素含量高于非滞育的棉铃虫,化蛹后,注定滞育的棉铃虫的类固醇蜕皮素含量则迅速降到极低的水平,明显低于非滞育棉铃虫。用20-羟基蜕皮素处理不同时期的滞育蛹,均能打破滞育。由此可见,类固醇蜕皮素含量的降低或缺乏乃是导致棉铃虫滞育的关键因子之一。     

光周期对大草蛉(Chrysopa pallens)滞育及发育的影响

在22℃条件下,研究大草蛉对光周期的敏感性,结果表明:大草蛉属短日照滞育型,在短光照条件下饲养获得的预蛹进入滞育状态,诱导预蛹滞育的临界光周期为10.5L-13.5D到11L-13D.2龄幼虫期是诱导预蛹滞育的敏感虫期,只有当2龄幼虫期处于短光照条件下时才能进入滞育状态.1龄和3龄幼虫期也在滞育诱导条件下进行滞育诱导,能够提高滞育率.光周期对大草蛉幼虫历期及预蛹重的影响研究结果表明:光周期对幼虫历期有一定的影响,特别对2龄幼虫期的影响比较明显.幼虫期的光周期条件影响预蛹的重量,滞育预蛹的重量显著高于非滞育预蛹.     

鞭角华扁叶蜂蜕皮甾类激素滴度的变化

用放射免疫分析法测定了鞭角华扁叶蜂Chinolyda flagellicornis末龄幼虫及滞育预蛹血淋巴中蜕皮甾类激素滴度。结果表明,末龄幼虫血淋巴中蜕皮甾类激素滴度在第2和4天各有一高峰;滞育预蛹血淋巴中保持一定滴度的蜕皮甾类激素,并随发育时期的不同有所波动;预蛹化蛹前一周血淋巴中蜕皮甾类激素滴度存在两个与变态相对应的峰值。表明鞭角华扁叶蜂的滞育与蜕皮甾类激素相关。     

The diapause hormone-pheromone biosynthesis activating neuropeptide gene of Helicoverpa armigera encodes multiple peptides that break, rather than induce, diapause

FXPRLamide peptides encoded by the DH-PBAN (diapause hormone-pheromone biosynthesis activating neuropeptide) gene induce embryonic diapause in Bombyx mori, but terminate pupal diapause in Helicoverpa armigera (Har). Here, we explore the mechanisms of terminating pupal diapause by the FXPRLamide peptides. Using quantitative RT-PCR, we observed that expression of Har-DH-PBAN mRNA in the SG of nondiapause-type pupae was significantly higher than in diapause-type pupae. Immunocytochemical results indicated that the level of FXPRLamide peptides and axonal release are related to the diapause decision. Ecdysteroidogenesis in prothoracic glands (PGs) was stimulated by synthetic Har-DH in vivo and in vitro, and labeled Har-DH bound to the membrane of the PG, thus suggesting that DH breaks diapause by activating the PG to synthesize ecdysone. Furthermore, the response of DH in terminating diapause was temperature dependent. Decerebration experiments showed that the brain can control pupal development through the regulation of DH, and DH can terminate diapause and promote development without the brain. This result suggests a possible mechanism of response for the signals of DH and other FXPRLamide peptides in H. armigera.     

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。     

Effect of the brain and suboesophageal ganglion on pupal development in Helicoverpa armigera through regulation of FXPRLamide neuropeptides

Recent studies in Helicoverpa armigera report a novel role for diapause hormone (DH), pheromone biosynthesis activating neuropeptide (PBAN) and three other FXPRLamide neuropeptides secreted from suboesophageal ganglion (SG) in terminating pupal diapause. In the present paper, we investigated the role of these five FXPRLamide family neuropeptides on pupal development. Although removal of SG could not make nondiapause-destined pupae enter diapause-like status, it did make them eclose approximately 0.6-1.2 days later when compared with the controls. The results of competitive ELISAs showed a high level of FXPRLamide titer in the hemolymph of the SG-removed pupae and this may be due to the expression of the DH-PBAN gene in tissues other than SG. DH-PBAN mRNA and peptides were also detected in the thoracic ganglia (TGs) by RT-PCR and immunocytochemistry. The expression of DH-PBAN gene in the TGs of the SG-removed pupae is significantly higher than that in normal pupae by quantitative PCR and immunocytochemistry. Decerebration experiments proved that the decerebrated pupae could enter diapause-like status through down-regulation of FXPRLamide titer in hemolymph. Our studies confirm that the brain plays an important role in the determination of pupal development by regulating the synthesis and release of FXPRLamide neuropeptides in H. armigera. Thus, the function of FXPRLamide peptides in H. armigera is closely correlated with pupal development.     

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。     

Pupal diapause of Helicoverpa armigera (Lepidoptera: Noctuidae): sensitive stage for thermal induction in the Okayama (western Japan) population

Helicoverpa armigera (Hübner) exhibits a facultative pupal diapause, which depends on temperature and photoperiod. Pupal diapause is induced at 20 degrees C by short photoperiods and inhibited by long photoperiods during the larval stage. However, in some pupae (35% of males and 57% of females) of a non-selected field population from Okayama Prefecture (34.6 degrees N), diapause is not induced by short photoperiods. In the present experiment, the importance of temperature for diapause induction was studied in the non-diapausing strain, which was selected from such individuals reared at 20 degrees C under a short photoperiod of 10L:14D. Furthermore, the sensitive stage for thermal determination of pupal diapause was determined by transferring larvae of various instars and pupae between 20 degrees C and 15 degrees C. Diapause was induced by 15 degrees C without respect to photoperiod. When larvae or pupae reared from eggs at 20 degrees C under a short or a long photoperiod were transferred to 15 degrees C in the periods of the middle fifth instar to the first three days after pupation, the diapause induction rate was significantly reduced in both males and females, especially in females. In contrast, when larvae or pupae reared at 15 degrees C were transferred to 20 degrees C in the same periods, diapause was induced in males, but not in females. However, the diapause induction rate of pupae transferred to 20 degrees C on the fourth day after pupation was significantly increased in females. The results show that temperature is the major diapause cue in the photoperiod-insensitive strain and the periods of middle fifth larval instar to early pupal stage are the thermal sensitive stages for pupal diapause induction with some different responses to temperatures between males and females in H. armigera.     

A true summer diapause induced by high temperatures in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Summer diapause in the cotton bollworm, Helicoverpa armigera (Hübner), which prolongs the pupal stage, particularly in males, is induced by high temperatures. In the laboratory, summer-diapausing pupae of H. armigera were induced at high temperatures (33-39 degrees C) with a photoperiod of LD8:16; winter-diapausing and non-diapausing pupae, cultured at 20 degrees C with a photoperiod of LD8:16 and at 27 degrees C, LD16:8, respectively, acted as a control. Retention time of eye spots, weight, and lipid and glycogen levels were compared. At high temperatures, both body weight and energy storage capacity were much higher in summer-diapausing pupae than in non-diapausing pupae reared at 33-39 degrees C. At temperatures (>33 degrees C) high enough to maintain summer diapause, the eye spots of summer-diapausing pupae did not move during the 30-day experiment. However, eye spots of summer-diapausing pupae placed at 30 degrees C began to move about 10 days after they were transferred, significantly later than in non-diapausing pupae reared at 33-39 degrees C or non-diapausing pupae reared at 27 degrees C, which initiated eye spot movement 2 days after pupation. The differences in retention time of eye spots between summer- and winter-diapausing pupae shows that winter diapause is more intense than summer diapause in this insect. The weight loss, and lipid and glycogen metabolism curves indicate that the summer-diapausing pupae's metabolism is very low. We conclude that summer diapause in the cotton bollworm is a true diapause and that the summer diapause enables the cotton bollworm to withstand the high temperatures of summer.     

Cloning and expression of the cDNA encoding the FXPRL family of peptides and a functional analysis of their effect on breaking pupal diapause in Helicoverpa armigera

Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are encoded by a single mRNA in the suboesophegeal ganglion (SG) and are responsible for induction of embryonic diapause in Bombyx mori and sex pheromone biosynthesis in lepidopteran insects. PBAN cDNA analyses revealed that the DH-like peptide is present in several species that have a pupal diapause. However, the function of the DH-like peptide remains unknown. In the present study, we cloned the cDNA encoding DH-PBAN in Helicoverpa armigera utilizing the rapid amplification of the cDNA ends method. The nucleotide se quence analysis revealed that the longest open reading frame of this cDNA encodes a 194-amino acid precursor protein that con tains a 33-aa PBAN, a 24-aa DH-like peptide, and three other neuropeptides, all of which have a common C-terminal pentapeptide motif FXPR/KL ( X=G, T, S). A homology search showed that H. armigera DH-like and PBAN are highly homologous to those from other insects. Northern blot analysis demonstrated a single message RNA corresponding to the size of Har-DH-PBAN cDNA from pupal SG with significantly higher expression in the SG of nondiapause pupae than diapausing pupae. Western blot analysis showed DH-like peptide expression from SG of both males and females. When DH-like peptide was injected into nondiapause larvae and pupae, it did not induce diapause, but rather efficiently broke pupal diapause in H. armigera. The ED(50) of DH to terminate pupal diapause is 20 pmol/pupae. The other four FXPRLamide neuropeptides from the DH-PBAN polyprotein precursor have cross activity for diapause termination. These observations therefore suggest a potential role for these FXPRL family peptides in promoting continuous development in several noctuid species. The high expression of this gene in pharate adults and adults indicates that the FXPRL family peptides may have multiple physiological functions.     

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。     

Effects of constant and changing temperature conditions on diapause induction in Helicoverpa armigera (Lepidoptera: Noctuidae)

The effects of photoperiod and temperature on the induction and termination of facultative pupal diapause in Helicoverpa armigera (Lepidoptera: Noctuidae) were investigated under laboratory conditions. Exposing H. armigera larvae to both constant and fluctuating temperature regimes with a mean of 25°C and 20°C resulted in a type-III photoperiodic response curve of a short-long day insect. The long-day critical daylengths for diapause induction were ten hours and 12 hours at the constant temperatures of 25°C and 20°C, respectively. Higher incidences of diapause and higher values both for the longer and the shorter critical photoperiods for diapause induction were observed at fluctuating regimes compared with the corresponding constant ones. At alternating temperatures, the incidence of diapause ranged from 4.2% to 33.3% and was determined by the temperature amplitude of the thermoperiod and by the interaction of cryophase or thermophase with the photoperiod. Helicoverpa armigera larvae seem to respond to photoperiodic stimuli at temperatures >15°C and <30°C; all insects entered diapause at a constant temperature of 15°C, whereas none did so at a constant temperature of 30°C under all the photoperiodic regimes examined. Although chilling was not a prerequisite for diapause termination, exposure of diapausing pupae to chilling conditions significantly accelerated diapause development and the time of adult emergence. Therefore, temperature may be the primary factor controlling the termination of diapause in H. armigera.