烟实夜蛾(Helicoverpa assulta Guenée)触角感器的超微结构观察

应用扫描电镜对烟实夜蛾成虫触角的外部形态结构进行了观察.结果表明,成虫触角上存在着7种感器,即毛形感器、刺形感器、腔锥形感器、锥形感器、耳形感器、柱形感器和腔乳头状感器.其中以毛形感器数目最多,并分为长毛形和短毛形2种.刺形感器分为长刺形和短刺形.并对烟实夜蛾雌、雄成虫触角感器的种类和分布差异及与其相近昆虫的差异进行了讨论.

烟夜蛾(Helicoverpa assulta Guenée)滞育激素基因时空表达的研究

利用RT PCR法,在转录水平上对烟夜蛾滞育激素(Diapausehormone,DH)基因的时空表达进行了研究.结果表明,该基因在滞育诱导条件下饲养的烟夜蛾3,4,5,6龄幼虫及滞育蛹中表达,在已解除滞育的蛹体内该基因同样表达.但在正常条件下饲养的烟夜蛾幼虫及非滞育蛹虫体中未见表达.对烟夜蛾滞育蛹脑部、胸部、腹部的RT PCR检测表明,脑部为DH基因的表达部位.

烟青虫(Helicoverpa assulta)性信息素的研究应用进展

对烟青虫(Helicoverpa assulta)性信息素的化学通讯和结构鉴定,以及其在预测预报、大量诱捕、干扰交配等方面的研究与应用进行了综述,并就其影响因子和今后的发展方向进行了扼要的介绍。

烟夜蛾(Helicoverpa assulta Guenée)滞育激素基因在大肠杆菌中的表达

采用RT PCR技术从烟夜蛾(HelicoverpaassultaGuen啨e)滞育蛹中扩增到滞育激素(diapausehormone,DH)基因cDNA,将其与pET 30a(+)质粒连接后转化至大肠杆菌BL21中,测序结果表明,重组质粒插入片段编码序列完整,读框正确;重组工程菌用IPTG诱导表达后,SDS聚丙烯酰胺凝胶电泳检测到一条约21kDa大小的外源蛋白,它与DH的大小相应.DH基因原核表达载体的成功构建为大量获取DH重组蛋白、研究DH的结构及功能奠定了基础.

Cloning and Expression Analysis of cDNA Encoding Or83b-Like Receptor from Helicoverpa assulta

An Or83b-like receptor gene was cloned from antennae of Helicoverpa assulta（Guenée） by using reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of cDNA ends（RACE）.Sequence analysis revealed that the transcript of the Or83b-like receptor gene from H.assulta consists of 1 946 nucleotides and the open reading frame（ORF） encodes a peptide of 473 amino acids with 7 putative transmembrane domains.Alignment analysis suggested that amino acid sequence of Or83b-like receptor from H.assulta shares high identity with other Or83b family receptors and this gene was hence named as HassOr83b.Tissues expression analysis showed that the HassOr83b transcript is clearly observed in the antennae,labial palps and proboscises,but not in bodies,wings and legs.The further development expression analysis suggested HassOr83b is also expressed in several preadult stages,including early-stage larvae,late-stage larvae and pupae,but not in embryos.Locked nucleic acid（LNA）-based in situ hybridization of antennal section indicated that HassOr83b is expressed in a very large number of antennal cells,which suggests that HassOr83b may play a special role in olfaction in H.assulta.

Gene Cloning and Expression Analysis of G Protein αq Subunit from Helicoverpa assulta (Guenée)

The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta （Guen6e） by reverse transcription polymerase chain reaction （RT-PCR） and named as HassGαq. Sequencing analysis showed that the fulllength of HassGαq open reading frame （ORF） is 1 062 bp, 353 amino acid residues are encoded. The predicted molecular weights （MW） and isoelectric point （PI） are 41.5 kD and 5.15, respectively. HassGαq gene was then constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside （IPTG）, the GST-HassGαq fusion protein is expressed in Escherichia coil BL21, and its MW was found to be about 66 kD nearly equal to the predicted. In addition, RT-PCR analysis showed that the expressions of HassGαq are not tissue specific.

Control Effect and Cost Evaluation of Different Sex Pheromones and Trapping Lamps against Spodoptern litura,Helicoverpa assulta and Helicoverpa armigera in Tobacco

[ Objective] The aim was to study the control effect of different sex pheromones and trapping lamps against main pests in tobacco. [ Method] Control effect of sex pheromone of different matrix lures and Jiaduo trapping lamp against Spodoptern litura, Helicoverpa assulta and Helicoverpa armigera in tobacco in Teng- chong of Yunnan were determined and compared, and the control cast was evaluated. [Result] The use of insect sex pheromones and insecticidal light traps had certain effect against S. litura and H. armiger. The trapping effect of sex pheromone traps was better than that of Jiaduo trapping lamp, and the PVC matrix lure had better performance than rubber matrix lure, which had strongest capturing capacity, continuous control effect and significant effort against S. litura. The traps with different settled densities and hanging heights also had different trapping effect against S. litura and H. armiger, and the hanging height of 100 - 150 cm from ground was the best; as the cost was considered, the cast of the area with low density of traps was the lowest, which was reduced by over 44% than conventional chemical control area. [ Conclusion] It is safe and effective to use sex pheromone and insecticidal light traps to control S. litura and H. armigera, and it is a green environmental protection biological physical control technology, having extended application prospect in large area.

Behavioral and Electrophysiological Responses to Caffeine of Helicoverpa armigera Larvae and H. assulta Larvae

The dual-choices tests of behavioral test were used to study the gustatory behavioral responses to caffeine of Helicoverpa armigera larvae and H. assulta larvae. Electrophysiological responses were studied by electrophysiological tip recording. Behavioral experiments showed that caffeine was a feeding deterrent for both larvae, but it showed a stronger feeding deterring effect on the oligophagous H. assulta . The electrophysiological tip-recording results showed that both H. armigera larvae and H. assulta larvae had one caffeine-sensitive feeding deterrent neuron at the medial sensilla, and the electrophysiological pulse response of H. assulta larvae was significantly stronger than that of H. armigera larvae. Therefore, caffeine had a stronger feeding deterring effect on the oligophagous H. assulta than the polyphagous H. armigera . The difference in behavioral effect was closely related to the sensitivity of to caffeine the feeding deterrent neurons at the medial sensilla.

Screening of High Virulent Isolate of Beauveria spp. against Helicoverpa assulta and Changes in Protective Enzymes Activities of Infected Larvae

Eleven isolates of Beauveria spp., including B. bassiana(Bb062) newly isolated from Helicoverpa assulta, and other seven B. bassiana isolates and three B. brongniartii isolates that were originally isolated from different geographic origins and various hosts, were tested against the 3^(rd) instar larvae of H. assulta. The protective enzyme activity in the 3^(rd) larvae of H. assulta infected by highly virulent isolate was also assayed. The results showed that the isolate Bb062 had the highest virulence to the 3^(rd) instar larvae among eleven isolates of Beauveria spp., with a corrected mortality reaching 91.07% within 10 d post treatment, and the LT_(50) was 4.67 d. After inoculated with three concentrations(1.0 ×10~6, 1.0 ×10~7 and 1.0×10~8 conidia/mL) of Bb062 conidial suspension, the accumulative mortality of H. assulta larvae increased with the increase of concentration and observation time, and the LC_(50) was 1.82×10~7 conidia/mL. Activities of superoxide dismutase(SOD), peroxidase(POD) and catalase(CAT) in H. assulta larvae first increased rapidly then dropped sharply within 72 h. Bb062 showed high virulence to H. assulta and could inhibit activities of protective enzymes. Therefore, it will be a promising biocontrol agent against H. assulta.

Interspecific hybridization of Helicoverpa armigera and H.assulta (Lepidoptera:Noctuidae)

Reciprocal hybridization between Helicoverpa armigera (Hubner) and H. assulta Guenee followed by back-crossing of the hybrids (F1) with H. armigera produced backcross (BC) lines consisting of fertile females and males. The F1 of H. armigera female and H. assulta male had only male, no female sex. In this case Haldane’s rule applies, and therefore it is proved that the sex chromosomes of Helicoverpa species are of ZW type, and the female is the heterozygous sex. This hybrid also showed significant het-erosis with the heaviest pupal weight, and when it was back-crossed with H. armigera female, the sex ratio of the BC offspring was distorted as 1 : 4. The potential utilization of this hybrid in genetic controlling H. armigera was finally discussed.

Comparative study on the responses of maxillary sensilla styloconica of cotton bollworm Helicoverpa armigera and Oriental tobacco budworm H. assulta larvae to phytochemicals

Using the electro-physiological technique, the sensory mechanisms of maxillary sensilla styloconica to stimulants and deterrents were explored on two closely related species, the generalist Helicoverpa armigera and the specialist H. assulta. The results showed that: (i) in both species, cells sensitive to sucrose and azadirachtin were mainly in the lateral sensillum styloconicum, and those to inositol were in the medial sensillum styloconicum; (ii) sensitivity of medial sensillum styloconicum in H. assulta to inositol was higher than that in H. armigera; (iii) among 6 tested deterrents, only azadirachtin evoked high impulse discharge from the lateral sensillum styloconicum in both insects; (iv) the deterrents could disturb stimulants evoking impulse discharge from maxillary sensilla styloconica of both species in different degrees: To sucrose evoking impulses on lateral sensillum styloconicum, for H. armigera capsaicin had a strong inhibition and gossypol had a weak inhibition, while for H. assulta

Genetic differentiation of Helicoverpa armigera （Hübner） and H. assulta （Guenée） （Lepidoptera： Noctuidae） based on AFLP markers

Here we use amplified fragment length polymorphism （AFLP） to assess genetic differentiation of Helicoverpa armigera and H. assulta. The results indicated that both species-specific fingerprints and cluster analysis showed the ability of AFLP technique to discriminate the two sibling species; among a total 1963 AFLP markers amplified from nine primer combinations： 777 （39.6%） were H. armigera-specific, 602 （30.7%） were H. assulta- specific, and 584 （29.7%） were common bands. The mean number ofH. armigera-specific bands was significantly more than that of H. assulta-specific bands for nine primer combinations （P 〈 0.05）; the intraspecific distance of H. armigera and H. assulta was 0.123 0 and 0.110 7 respectively, and the interspecific distance was 0.178 3. In addition, the percentage of polymorphic loci and estimated average heterozygosity were used to estimate genetic diversity of the two species. This study therefore demonstrates that AFLP analysis is a sensitive and reliable technique to study genetic differentiation and genetic relationships between species and provides sufficient molecular markers for future linkage map conslruction, location and eventual cloning of genes involved in traits differentiation.

Biology and life table studies of the oriental tobacco budworm, Helicoverpa assulta （Lepidoptera： Noctuidae）, influenced by different larval diets

Development, survivorship, pupal weight, oviposition, and life table parameters of the oriental tobacco budworm, Helicoverpa assulta Guenée, were evaluated in the laboratory on an artificial diet, pepper （Capsicumfrutescens L.）, and tobacco （Nicotiana tobacum L.）. We found that the average developmental time of immature stages was longest on tobacco （36.2 d）, intermediate on pepper （34.4 d）, and shortest on artificial diet （33.5 d）. Immature survival from egg to pupa varied from 31% on tobacco, 43% on pepper, and 74% on artificial diet. Pupal weight ranged from 197.4 mg/pupa on tobacco, 233.1 mg/pupa on pepper and 253.4 mg/pupa on artificial diet. The average numbers of eggs laid by adults reared as larvae on the artificial diet, pepper, or tobacco were 614, 421 and 334 eggs/female, respectively. Numbers of remaining eggs in ovaries of the adult females reared as larvae on the artificial diet, pepper, or tobacco were 16, 26, and 42 eggs/female, respectively. The longevity of adult females developed from larvae reared on the three diets was not significantly different, whereas the longevity of male adults from the larvae reared on artificial diet was longer （16.8 d） than that for males reared on tobacco （13.8 d） and pepper （13.3 d）. The intrinsic, finite, gross, and net rates of increase were highest for females reared as larvae on artificial diet, lowest for females emerging from larvae reared on tobacco, and intermediate for females emerging from larvae reared on pepper. Generation times and doubling time of H. assulta were shortest for larvae fed artificial diet, intermediate from larvae reared on pepper, and longest from larvae reared on tobacco. We concluded that the artificial diet was the most suitable larval diet ofH. assulta followed by pepper, and tobacco.

Attractiveness of tobacco volatiles induced by Helicoverpa armigera and Helicoverpa assulta to Campoletis chlorideae

The attraction of Helicoverpa armigera- and Helicoverpa assulta-induced and mechanical damage-in- duced tobacco volatiles to Campoletis chlorideae was investi-gated, and the induced volatiles were analyzed. In wind- tunnel, C. chlorideae was strongly attracted by herbivore- induced tobacco volatiles. Mechanically damaged tobacco leaves, whether treated with caterpillar regurgitant or water, were more attractive to the parasitoid than undamaged to-bacco leaves. GC-MS analysis revealed that only 4 com-pounds were released from undamaged tobacco leaves, whereas 13 compounds were commonly emitted from herbi-vore-infested and mechanically damaged tobacco leaves. Compound β-pinene was specifically induced by the infesta- tion of H. armigera, and (Z)-3-hexenal was only induced by the infestation of H. armigera and H. assulta, whereas hexyl acetate was only induced by mechanical damage. Tobacco leaves infested by H. armigera and H. assulta released larger amounts of volatiles than undamaged tobacco leaves did. Tobacco leaves treated with artificial damage plus caterpil- lars regurgitant or water emitted the same levels of volatiles, which were higher than that emitted by undamaged tobacco leaves. The emission amounts of single compounds were also different between differently treated plants. The differences were large between herbivore-induced and mechanical dam-age-induced compounds, and small between H. armigera- and H. assulta-induced compounds, and among compounds emitted from mechanically damaged plants treated with wa-ter or caterpillar regurgitant.

烟青虫气味结合蛋白基因的克隆与序列分析

根据已经报道的棉铃虫普通气味结合蛋白I(GOBP1)和性信息素结合蛋白(PBP)基因的cDNA序列,设计了2对引物,以烟青虫Helicoverpaassulta触角cDNA为模板,分别进行PCR扩增,获得2条特异性条带,约400bp。将以上两条片段分别连接到T-easy载体上,获得重组子T-GOBP1-Hass和T-PBP-Hass。序列测定和结构分析表明,Hass-GOBP1开放阅读框全长441bp,编码147个氨基酸残基,预测分子量和等电点分别为17.2kD和4.71。Hass-PBP开放阅读框全长405bp,编码135个氨基酸残基,预测分子量和等电点分别为15.1kD和5.2。Hass-GOBP1和Hass-PBP具有昆虫气味结合蛋白的典型特征,即氨基酸序列中具有6个保守的半胱氨酸残基,呈酸性。这2个基因已在GenBank中登记,序列号分别是AY864774和AY864775。

几种虫生真菌对烟青虫的致病性研究

用 4种真菌共 9个菌株的固体培养物以及真菌与 Bt的混合物 ,分别对烟青虫 2龄幼虫进行了毒力测定 ,结果表明 :( 1 )球孢白僵菌和莱氏野村菌两个菌种对烟青虫幼虫有明显的防治效果 ,且前者优于后者 ;在白僵菌中 5 S0 3菌株毒力最高 ,对烟青虫 2龄幼虫的致死中时 ( LT50 )为 6.1 0 d,校正死亡率为 86.96% ,僵虫率为 33.33% ;另外两个菌株 Bb3和 Bb98-1的毒力也较高。 ( 2 )真菌 ( 5 S0 3)与 Bt混用 ,对防治烟青虫具有相互增效作用。

植物次生物质对烟青虫和棉铃虫食物利用及中肠解毒酶活性的影响

用分别添加 0 5 % (干重比 )棉酚、烟碱、番茄苷和辣椒素 4种植物次生物质的人工饲料饲养烟青虫Helicoverpaassul ta和棉铃虫H .armigera 5龄幼虫 48h ,测定这些次生物质对烟青虫和棉铃虫的营养效应和中肠谷胱甘肽S 转移酶 (GST)及羧酸酯酶 (CarE)活性的影响。结果表明 :在实验浓度下 ,棉酚可显著降低烟青虫的相对消化率 ,但对棉铃虫却有助食作用 ;番茄苷抑制烟青虫的取食和生长 ,对其近似消化率和食物利用率也有显著的抑制作用 ,但食物转化率有明显升高 ,对棉铃虫的各营养指标无显著影响 ;烟碱对烟青虫和棉铃虫的相对生长率均无影响 ;辣椒素使烟青虫的取食量有大幅度的提高 ,对棉铃虫的取食量无影响 ,但引起其相对消化率的提高。由此可见 ,棉铃虫对 4种次生物质有普遍的适应性 ,而烟青虫只对寄主植物所含的烟碱和辣椒素有较好的适应性。烟青虫和棉铃虫幼虫中肠CarE活性不受 4种次生物质的影响 ,烟碱和辣椒素对烟青虫GST有显著的诱导作用 ,番茄苷对烟青虫GST活性则有抑制作用 ,4种次生物质对棉铃虫GST均无显著影响。

三种夜蛾成虫口器感器的超微形态

为确定不同种类夜蛾口器及其感器在超微结构上的差异,采用扫描电子显微镜对棉铃虫Helicoverpa armigera(Hübner)、烟夜蛾H.assulta(Guenée)和银纹夜蛾Argyrogramma agnata(Staudinger)3种鳞翅目(Lepidoptera)夜蛾科(Noctuidae)重要农业害虫雌、雄成虫口器感器的超微形态进行了观察和比较。结果表明:3种夜蛾雌、雄成虫口器感器类型均无明显差异。棉铃虫和烟夜蛾口器感器在类型和形状上十分类似,均具有毛形、锥形和栓锥形感器;喙管末端的栓锥感器粗、密,棱纹明显。银纹夜蛾口器感器与两种铃夜蛾区别明显,除上述3类感器外,还具有腔锥形感器;其喙管末端的栓锥感器细、疏,棱纹不明显。结果显示口器感器可用于夜蛾的分类及亲缘关系研究。

烟实夜蛾信息素结合蛋白3 cDNA的克隆、序列分析与原核表达

利用RT-PCR技术从烟实夜蛾Helicoverpa assulta( Hass)雄虫触角中扩增得到了信息素结合蛋白3( HassPBP3)。克隆和测序结果表明,该基因核苷酸序列全长495 bp,编码164个氨基酸残基,预测分子量18.5 kD。并预测N-末端疏水区包含由22个氨基酸组成的信号肽。因此,成熟蛋白应包括142个氨基酸,预测分子量为16.1 kD,等电点为5.44。经氨基酸序列同源性分析发现,此序列与已知昆虫PBP3有较高的同源性,而且具有气味结合蛋白的典型特征。将该基因重组到表达载体pGEX-4T-2中进行原核表达。经IPTG诱导、SDS-PAGE分析和Western印迹检测,结果表明烟实夜蛾PBP3基因能在大肠杆菌BL21中表达,电泳检测到一条大约42 kD的外源蛋白,与预测的融合蛋白分子量相符。

烟夜蛾滞育激素的cDNA克隆和序列测定

运用RT -PCR法克隆了烟夜蛾 (HelicoverpaassultaGuen啨ｅ)滞育激素的cDNA ,并对其进行了序列分析 .结果表明 ,烟夜蛾滞育激素由 2 4个氨基酸组成 ,属于FXPRL神经肽家族 ,与家蚕 (BombyxmoriLinnaeus)、棉铃虫 (HelicoverpaarmigeraH櫣ｂｎｅｒ)滞育激素氨基酸序列相比较 ,其同源性分别为 6 2 %和 99% .