茶尺蠖核型多角体病毒多角体蛋白单克隆抗体的制备

为了建立一种基于免疫反应检测茶尺蠖核型多角体病毒的方法,以纯化后的茶尺蠖核型多角体病毒作为抗原,免疫BALB/c小鼠,将小鼠脾脏细胞与小鼠骨髓瘤细胞Sp2/0融合,经间接ELISA筛选及克隆得到了一株稳定分泌单克隆抗体的杂交瘤细胞株,命名为7D3。同时克隆并在大肠杆菌中表达了EoNPV多角体蛋白基因,获得重组多角体蛋白。经Western blotting鉴定,该抗体可与EoNPV的多角体蛋白特异性结合。利用制备EoNPV多角体蛋白的单克隆抗体,建立了间接ELISA测定EoNPV的方法。     

茶核·苏云菌悬浮剂对灰茶尺蠖的田间防控应用

【目的】茶尺蠖核型多角体病毒EcobNPV是一种重要的昆虫病原微生物,可侵染茶尺蠖Ectropis obliqua和灰茶尺蠖Ectropisgrisescens2种宿主。为探明茶尺蠖核型多角体病毒制剂的田间应用技术,对影响防效的3个因子喷施方式、剂量和虫龄进行研究。【方法】采用植保无人机和人工喷施2种喷施方式,比较茶核·苏云菌悬浮剂（1×104 PIB·2 000 IU/μL）防治灰茶尺蠖的效果;设置75、150和375 mL/667 m²3种剂量,比较茶核·苏云菌悬浮剂对灰茶尺蠖的防治效果;并比较其对灰茶尺蠖1龄、2龄和3龄不同虫龄的防治效果。【结果】在相同剂量下,用植保无人机和人工喷施病毒制剂对灰茶尺蠖的总防效分别为69.0%和67.6%,两者无显著差异;在75、150和375 mL/667 m23种剂量,比较茶核·苏云菌悬浮剂对灰茶尺蠖的防治效果;并比较其对灰茶尺蠖1龄、2龄和3龄不同虫龄的防治效果。【结果】在相同剂量下,用植保无人机和人工喷施病毒制剂对灰茶尺蠖的总防效分别为69.0%和67.6%,两者无显著差异;在75、150和375 mL/667 m² 3种剂量下病毒制剂对灰茶尺蠖的防效差异显著,其中375 mL/667 m2 3种剂量下病毒制剂对灰茶尺蠖的防效差异显著,其中375 mL/667 m²剂量下的防效最高,150 mL/667 m2剂量下的防效最高,150 mL/667 m²其次,150 mL/667 m2其次,150 mL/667 m²和375 mL/667 m2和375 mL/667 m²剂量对灰茶尺蠖的总防效均在90%以上;病毒制剂对灰茶尺蠖1龄、2龄和3龄幼虫的防效分别为93.3%、78.7%和67.8%,对1龄幼虫的防效显著高于3龄。【结论】茶核·苏云菌悬浮剂防治灰茶尺蠖可选用植保无人机喷施,剂量可选用150-375 mL/667 m2剂量对灰茶尺蠖的总防效均在90%以上;病毒制剂对灰茶尺蠖1龄、2龄和3龄幼虫的防效分别为93.3%、78.7%和67.8%,对1龄幼虫的防效显著高于3龄。【结论】茶核·苏云菌悬浮剂防治灰茶尺蠖可选用植保无人机喷施,剂量可选用150-375 mL/667 m²,以1龄幼虫期喷施防效为最佳。     

Characterization of Adoxophyes honmai single-nucleocapsid nucleopolyhedrovirus: morphology,structure, and effects on larvae

A nucleopolyhedrovirus (NPV) was isolated from a diseased larva of the smaller tea tortrix, Adoxophyes honmai, collected from a tea field in Tsukuba, Ibaraki, Japan. Electron microscopic observations confirmed that A. honmai NPV (AdhoNPV) was a single-nucleocapsid type virus. The genome size of AdhoNPV was estimated to be 111.6 +/- 0.9kb (mean +/- SE) by restriction endonuclease analysis. AdhoNPV was also infectious to two other Adoxophyes species, the summer fruit tortrix Adoxophyes orana and Adoxophyes dubia. The LD50 values for neonatal, second, third, fourth, and fifth (final) instar larvae of A. honmai were determined as 61, 107, 688, 1,961, and 4,085 occlusion bodies/insect, respectively. Most of the infected larvae died 5-9 days after molting to the final instar, regardless of the timing of inoculation. However, when neonates were exposed to extremely high doses of AdhoNPV (greater than 100 x LD90), larval development was prevented and most of the larvae died in the first instar.     

灰茶尺蠖核型多角体病毒对灰茶尺蠖的致病性研究

采取室内毒力测定和田间防效调查相结合的方法,对灰茶尺蠖核型多角体病毒(Ectropis grisescensnucleopolyhedrovir-us)的致病力进行了研究。毒力测定结果表明,该病毒对2龄灰茶尺蠖幼虫的剂量对数-死亡机率值回归方程为y=-0.8774 0.9498x,LC50=1.5417×106PIB.mL-1;田间用浓度为1×107、1.5×107和2×107PIB.mL-1的EgNPV防治灰茶尺蠖,防效分别达58.81%、89.51%和94.81%。     

Horizontal and vertical transmission of wild-type and recombinant Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus

Transmission plays a central role in the ecology of baculoviruses and the population dynamics of their hosts. Here, we report on the horizontal and vertical transmission dynamics of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV-WT) and a genetically modified variant (HaSNPV-AaIT) with enhanced speed of action through the expression of an insect-selective scorpion toxin (AaIT). In caged field plots, horizontal transmission of both HaSNPV variants was greatest when inoculated 3rd instar larvae were used as infectors, transmission was intermediate with 2nd instar infectors and lowest with 1st instar infectors. Transmission was greater at a higher density of infectors (1 per plant) than at a lower density (1 per 4 plants); however, the transmission coefficient (number of new infections per initial infector) was lower at the higher density of infectors than at the lower density. HaSNPV-AaIT exhibited a significantly lower rate of transmission than HaSNPV-WT in the field cages. This was also the case in open field experiments. In the laboratory, the vertical transmission of HaSNPV-AaIT from infected females to offspring of 16.7+/-2.1% was significantly lower than that of HaSNPV-WT (30.9+/-2.9%). Likewise, in the field, vertical transmission of HaSNPV-AaIT (8.4+/-1.1%) was significantly lower than that of HaSNPV-WT (12.6+/-2.0%). The results indicate that the recombinant virus will be transmitted at lower rates in H. armigera populations than the wild-type virus. This may potentially affect negatively its long-term efficacy as compared to wild-type virus, but contributing positively to its biosafety.     

烟碱对假眼小绿叶蝉和茶尺蠖的忌避(拒食)活性

【目的】尼古丁是茄科植物的重要生物碱,本文首次确定了硫酸烟碱对假眼小绿叶蝉Empoasca vitis G?the的驱避作用和田间防效,以及不同浓度硫酸烟碱对茶尺蠖Ectropis obliqua Prout的拒食作用。【方法】使用Y形嗅觉仪和T形观测室研究了假眼小绿叶蝉对硫酸烟碱的行为反应,使用叶碟法测试了硫酸烟碱对茶尺蠖的拒食作用。【结果】硫酸烟碱600倍稀释液和800倍稀释液处理茶梢分别对叶蝉若虫的寄主选择行为和雌成虫的产卵具有显著的驱避作用;田间喷施硫酸烟碱对假眼小绿叶蝉的防治效果较好。此外,硫酸烟碱对茶尺蠖幼虫具有拒食作用,并且拒食作用随着取食时间的延长而增强,取食8 d以后,死亡率显著高于对照。【结论】本研究结果为开发茶树害虫的植物源忌避(拒食)剂提供一定的科学参考。     

Genetic engineering of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus as an improved pesticide

The Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) has been registered and is commercially produced in China as a biopesticide to control the bollworm in cotton. However, the virus has a relatively slow speed of action. To improve its efficacy, recombinant HearNPVs were generated by deleting the ecdysteroid UDP-glucosyltransferase (egt) gene (HaCXW1 and HaLM2) or by inserting the insect-specific toxin gene AaIT in the egt locus (HaCXW2) of HearNPV using conventional recombination strategies in insect cell culture. The various recombinants remained genetically stable when cultured in HzAM1 insect cells. Bioassay data showed a significant reduction in the time required for all HearNPV recombinants to kill second instar H. armigera larvae. The LT(50) of the egt deletion recombinants HaCXW1 and HaLM2 was about 27% faster than that of wild-type HearNPV. The largest reduction in LT(50) was achieved by inserting the gene for the insect-specific neurotoxin, AaIT, in the egt locus, giving a reduction in LT(50) of 32% compared to wild-type HearNPV. The ability to genetically improve the properties of HearNPV as a biopesticide provides a further opportunity to develop this virus into a commercially viable product to control the bollworm in China.     

茶尺蠖和灰茶尺蠖内共生菌Wolbachia的分子检测及序列分析

【目的】对茶尺蠖Ectropis obliqua及其近缘种灰茶尺蠖E.grisescens体内共生菌Wolbachia进行分子鉴定,确定两者体内Wolbachia的感染率及其进化地位,为进一步探讨其对茶尺蠖和灰茶尺蠖的潜在影响提供科学依据。【方法】采用Wolbachia的16S r RNA、fts Z和wsp基因特异性引物,通过PCR扩增法检测了我国3个茶尺蠖地理种群(浙江杭州、余杭和江苏无锡)和3个灰茶尺蠖地理种群(浙江新昌、湖北浠水和江西南昌)中Wolbachia的感染情况,并进行测序和序列分析。【结果】茶尺蠖和灰茶尺蠖都感染了Wolbachia,灰茶尺蠖的Wolbachia感染率为100%,但茶尺蠖的Wolbachia感染率在22%~95%,且PCR产物电泳得到的条带微弱。wsp序列在茶尺蠖和灰茶尺蠖种间、种内无差异;但16S r RNA序列在茶尺蠖和灰茶尺蠖种间、种内差异为0.362%~0.727%之间;茶尺蠖样本未成功扩增出fts Z序列,灰茶尺蠖样本获得2条fts Z基因序列差异为1.647%。基于Wolbachia的16S r RNA和wsp基因构建的系统发育树表明,本研究中茶尺蠖和灰茶尺蠖种群所感染的Wolbachia全部属于B组的Pip亚组。【结论】茶尺蠖和灰茶尺蠖均被B组Pip亚组的Wolbachia感染,但感染率相差很大,这为研究Wolbachia对茶尺蠖和灰茶尺蠖生物学及生态学的影响奠定了基础。     

草间钻头蛛、大草蛉和中华通草蛉对茶尺蠖、小绿叶蝉的选择效应

室内研究了草间钻头蛛Hylyphantes graminicola雌蛛对茶尺蠖Ectropis oblique1龄幼虫和小绿叶蝉Em-poesca flavescens成虫、大草蛉Chrysopa pallens和中华通草蛉Chrysoperla sinica的2龄幼虫对茶尺蠖卵、1龄幼虫的选择捕食作用。对试验数据应用Ivelev指数、Jacobs指数和Manly指数及Murdoch作图法进行了比较分析。结果显示,草间钻头蛛对茶尺蠖1龄幼虫和小绿叶蝉成虫的选择性不明显,选择性不因猎物数量的增加而转移。而大草蛉和中华通草蛉对茶尺蠖卵均有明显的正选择效应。     

游猎蛛在茶尺蠖高峰日的种间竞争作用

为了合理保护和充分利用天敌,明确茶园游猎蛛天敌捕食茶尺蠖Ectropis obliqua hypulina的种间竞争力及其差异。本文运用通经分析法、灰色关联度分析法、竞争系数分析法及竞争强度指数分析法对龙井43、黄山大叶种、乌牛早、安吉白茶、平阳特早和农抗早6类茶园中茶尺蠖高峰日时的7种游猎蛛捕食茶尺蠖的种间竞争作用进行分析。结果表明,按相关系数比较,乌牛早茶园茶尺蠖与条纹蝇虎Plexippus setipet的相关系数为-0.888,平阳特早茶园茶尺蠖与三突花蟹蛛Misumenops tricuspidatus和斜纹猫蛛Oxyopes Sertatws的相关系数分别为0.981和-0.902,均达相关水平,天敌间鞍型花蟹蛛Xysticus ephippiafus、三突花蟹蛛、斑管巢蛛Clubiona reichlini和斜纹猫蛛均与黑色跳蛛Plexippus paykulli相关,鞍型花蟹蛛、粽管巢蛛Clubiona japonicola和黑色跳蛛均与三突花蟹蛛相关。用灰色关联度法分析,与茶尺蠖数量上关系密切的前4种天敌依次是三突花蟹蛛、粽管巢蛛、黑色跳蛛和鞍型花蟹蛛。按竞争系数大小...     

Comparative pathogenesis of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus in noctuid hosts of different susceptibility

Neonate larvae of the noctuid moth Spodoptera exigua were susceptible to an infection by Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). Biological activity (LD(50),ST(50)) of the virus was considerably reduced as compared to its activity in the homologous host, H. armigera. Pathogenesis was studied using a recombinant HaSNPV carrying a green fluorescent protein gene, which induces fluorescence in infected cells to mark infection. In larvae of H. armigera, fluorescence was pronounced in the fat body after 2.9 days post infection and could also be detected in several other tissues. In contrast, fluorescence was not observed in tissues of S. exigua until 9 days post infection and was restricted almost exclusively to cells of the ganglia. Examination of serial sections of wildtype HaSNPV-infected S. exigua-larvae revealed a similar pattern of tissue tropism. Apparently, HaSNPV does not undergo the usual steps in host invasion and infection in this insect species, but targets specifically to nervous tissue.     

棉铃虫单核衣壳核多角体病毒几丁质酶基因的克隆与表达

根据棉铃虫单核衣壳核多角体病毒(HaSNPV)几丁质酶基因的序列,设计引物,引入适当的酶切位点,利用PCR扩增出不含N末端信号肽以及C末端内质网定位肽的几丁质酶基因片段.将该基因片断克隆至原核表达载体pProEXHTb,经IPTG诱导,在大肠杆菌DH5α中获得了高效表达,表达产物的大小为60kD,含量占菌体总蛋白量的40%.利用来源于AcMNPV 几丁质酶的抗体对表达蛋白进行检测,获得特异性的显色信号,证实所获原核表达产物与杆状病毒的几丁质酶具有同源性.     

棉铃虫单核衣壳核多角体病毒几丁质酶基因的克隆与表达

根据棉铃虫单核衣壳核多角体病毒 (HaSNPV)几丁质酶基因的序列 ,设计引物 ,引入适当的酶切位点 ,利用PCR扩增出不含N末端信号肽以及C末端内质网定位肽的几丁质酶基因片段。将该基因片断克隆至原核表达载体 pProEXHTb ,经IPTG诱导 ,在大肠杆菌DH5α中获得了高效表达 ,表达产物的大小为 6 0kD ,含量占菌体总蛋白量的 4 0 %。利用来源于AcMNPV几丁质酶的抗体对表达蛋白进行检测 ,获得特异性的显色信号 ,证实所获原核表达产物与杆状病毒的几丁质酶具有同源性     

茶尺蠖幼虫脑的解剖结构

【目的】明确茶尺蠖Ectropis obliqua Prout 5龄幼虫脑解剖结构,并分析和构建幼虫脑以及脑内部各神经髓结构的三维结构模型。【方法】采用免疫组织化学方法,利用突触蛋白抗体,染色标记脑内神经突触,定位突触联系密集分布的区域,获得脑内部神经髓的结构。利用激光共聚焦显微镜获取脑扫描图像,然后利用三维图像分析软件AMIRA进行图像分析,构建脑的三维结构模型,并计算脑以及脑内各神经髓结构的体积。【结果】突触蛋白抗体染色显示,茶尺蠖5龄幼虫脑内具有很多神经突触联系密集分布的区域,这些不同区域即为脑的不同神经髓结构。茶尺蠖幼虫脑主要包括前脑、中脑和后脑3个组成部分。其中前脑最大,包括成对的视叶、蕈形体、前脑桥和侧副叶以及不成对的中央体。视叶位于前脑的两侧后端。蕈形体位于脑半球正中间位置。侧副叶在中央体的下前方两侧。中央体在脑的正中心。前脑桥在中央体的上方后侧。除这些形态结构明显的神经髓区域外,前脑还包括大量内部边界不明显的神经髓区域,位于前脑左右两侧以及背侧和腹侧,这些区域被总称为中间脑,占整个脑神经髓的66%。触角叶为中脑的主要组成部分,在脑的下部最前端,为一对球状结构。后脑在脑的腹侧和触角叶下方,即围咽神经索进入脑的入口处。【结论】构建了茶尺蠖5龄幼虫脑以及各神经髓结构三维模型,分析了脑内各个神经髓之间的空间位置关系,明确了各神经髓的体积。茶尺蠖幼虫脑体积小而且结构简单的特征与其幼期视觉、嗅觉等感觉器官不发达、活动能力弱、行为简单的生物学习性相对应。     

冷驯化对茶尺蠖抗寒性生理指标的影响

【目的】冷驯化作为一种有效提高耐寒能力的途径,在昆虫对环境温度变化的适应中发挥重要作用,是当前昆虫耐寒性研究的热点。为了解茶尺蠖Ectropis oblique(Prout)对冷驯化的生理响应机制,本文研究了不同冷驯化对茶尺蠖抗寒性生理指标的影响。【方法】分别对茶尺蠖快速冷驯化0℃2 h、5℃2 h,长时间冷驯化5℃24 h、5℃48 h,利用热电偶方法测定了茶尺蠖的过冷却点,采用生理生化测定法研究了其体内的抗寒性生理指标的变化。【结果】长时间冷驯化处理使茶尺蠖过冷却点显著降低;虫体内含水量显著降低;脂质和糖原含量显著增加;蛋白质含量略有增加但无显著差异;过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)3种保护酶活性显著升高及ATP能量代谢酶活显著降低。短时间冷驯化使茶尺蠖过冷却点显著降低;虫体内含水量降低但差异不显著;脂质、糖原和蛋白质含量显著升高;POD、CAT、SOD 3种保护酶活性及ATP能量代谢酶活分别显著升高和降低。【结论】长时间冷驯化和快速冷驯化均能提高茶尺蠖耐寒性,这是虫体内的水分、脂肪、糖原、蛋白质等含量以及抗逆酶活性变化的综合反映。本研究结果对深入研究茶尺蠖越冬策略以及结合气候条件确定茶尺蠖的分布,为评价田间死亡率及预测翌年发生程度提供重要依据,对制定防治措施具有重要现实意义。     

报喜斑粉蝶华南亚种的形态特征和生物学特性研究

通过自然种群观察及实验种群饲养,系统研究了报喜斑粉蝶华南亚种Delias pasithoe porsenna(Cramer,1776)的形态特征及生物学特性.自然种群在广州1年发生6代,无越冬现象.在温度28℃、相对湿度70%、光照L:D=12 h:12 h条件下,实验种群卵期7.95±0.78 d,幼虫期27.69±0.82 d,蛹期11.33±0.98 d,成虫寿命9.68±1.69 d.     

Comparison of male antennal morphology and sensilla physiology for sex pheromone olfactory sensing between sibling moth species: Ectropis grisescens and Ectropis obliqua (Geometridae)

Ectropis grisescens and Ectropis obliqua (Lepidoptera: Geometridae) are sibling pest species that co‐occur on tea plants. The sex pheromone components of both species contain (Z,Z,Z)‐3,6,9‐octadecatriene and (Z,Z)‐3,9‐cis‐6,7‐epoxy‐octadecadiene. E. obliqua has (Z,Z)‐3,9‐cis‐6,7‐epoxy‐nonadecadiene as an additional sex pheromone component, which ensures reproductive segregation between the two species. To ascertain the detection mechanism of olfactory organs for sex pheromone components of E. grisescens and E. obliqua, we applied scanning electron microscopy and single sensillum recording to compare antennal morphology and sensillum physiology in the two species. There was no apparent morphological difference between the antennae of the two species. Both species responded similarly to all three sex pheromone components, including, E. obliqua specific component. The distribution patterns of antennal sensilla trichodea differed between the two species. Sex pheromone olfactory sensing in these sibling species appears to be determined by the density of different types of olfactory sensing neurons. Dose‐dependent responses of sensilla trichodea type 1 to (Z,Z)‐3,9‐cis‐6,7‐epoxy‐octadecadiene, the most abundant component, showed an “all or none” pattern and the other two components showed sigmoidal dose‐response curves with a half threshold of 10^?4 (dilution equal to the concentration of 10 μg/μl). These results suggest that the major sex pheromone component functions as an on–off controller while secondary components function as modulators during olfactory transmission to the primary olfactory center.     

有效积温法预测茶尺蠖防治适期的研究

利用22年历史气象资料和有效积温法,由茶尺蠖幼虫期或成虫发生高峰期预测下一代防治适期,并进行验证。1993年在室内外经5代39次预测验证,总符合率达96．6％。由幼虫期预测可使预报提前20～35天。