棉铃虫不同寄主种群遗传分化的RAPD分析

应用RAPD技术对生活于辣椒,烟草和番茄3种不同寄主上的棉铃虫（Helicover pa armigera)种群,并以烟草烟青虫（H.assulta)为外群进行了种群遗传分化的研究,研究结果表明：4个处理内个体间最大的遗传距离均小于处理间最小的遗传距离,烟青虫的遗传距离为0．177－0．346,棉铃虫的辣椒种群为0．289－0．404,烟草种群为0．396－0．505,番茄种群为0．329－0．382,采用UPDGA法进行聚类,并构建系统发育树。各处理成聚的遗传距离为：烟青虫是0．334,棉铃虫的辣椒种群是0．372,烟草种群是0．463,番茄种群是0．360。各处理均首先成聚,棉铃虫与烟青虫成聚的遗传距离（0．703）,稍大于棉铃虫各种群的成聚值（0．639）。上述结果表明,棉铃虫不同寄主种群已经存在明显的遗传分化。     

烟草挥发物对2近缘种夜蛾产卵行为的影响及其成分分析

寡食性烟夜蛾Helicoverpa assulta (Guenée)和广食性棉铃虫H.armigera (Hübner)是铃夜蛾属2近缘种,烟草是其共同寄主.室内实验测定了1个普通烟草品种和4个黄花烟草品种叶片挥发物对二者电生理和行为反应的影响.结果表明,烟夜蛾处女雌蛾和交配雌蛾对4个黄花烟草品种叶片挥发物的EAG反应均显著高于普通烟草,而棉铃虫对普通烟草叶片挥发物的反应显著高于黄花烟草;二者的行为反应与EAG测试结果相似,黄花烟草叶片挥发物对烟夜蛾有较强的引诱作用,棉铃虫对普通烟草叶片挥发物有较强趋性;两种夜蛾雄蛾对这些挥发物的EAG和行为反应均没有雌蛾强烈,性别差异显著;GC-MS分析表明,与K326相比,马合烟叶片挥发物中尼古丁的相对含量高(76.91%),绿叶气味种类多但芳香族化合物种类少,挥发物种类和含量的不同是否与两种夜蛾产卵趋性差异相关,有待进一步研究.     

Comparative study of sex pheromone composition and biosynthesis in Helicoverpa armigera, H. assulta and their hybrid

Two Helicoverpa species, H. armigera and H. assulta use (Z)-11-hexadecenal and (Z)-9-hexadecenal as their sex attractant pheromone components but in opposite ratios. Since both female and male interspecific hybrids produced by female H. assulta and male H. armigera have been obtained in our laboratory, we can make a comparative study of sex pheromone composition and biosynthesis in the two species and their hybrid. With GC and GC-MS analyses using single gland extracts, the ratio of (Z)-9-hexadecenal to (Z)-11-hexadecenal was determined as 2.1:100 in H. armigera, and 1739:100 in H. assulta. The hybrid has a ratio of 4.0: 100, which is closer to that of H. armigera, but significantly different from H. armigera. We investigated pheromone biosynthesis with labeling experiments, using various fatty acid precursors in H. armigera, H. assulta and the hybrid. In H. armigera, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation; (Z)-9-hexadecenal results from delta11 desaturation of stearic acid, followed by one cycle of chain shortening, reduction and terminal oxidation. delta11 desaturase is the unique desaturase for the production of the two pheromone components. In our Chinese strain of H. assulta, palmitic acid is used as the substrate to form both the major pheromone component, (Z)-9-hexadecenal and the minor one, (Z)-11-hexadecenal. Our data suggest that delta9 desaturase is the major desaturase, and delta11 desaturase is responsible for the minor component in H. assulta, which is consistent with previous work. However, the weak chain shortening acting on (Z)-9 and (Z)-11-octadecenoic acid, which is present in the pheromone glands, does occur in this species to produce (Z)-7 and (Z)-9-hexadecenoic acid. In the hybrid, the major pheromone component, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation. The direct fatty acid precursor of the minor component, (Z)-9-hexadecenoic acid is mainly produced by delta9 desaturation of palmitic acid, but also by delta11 desaturation of stearic acid and one cycle of chain shortening. The greater relative amounts of (Z)-9-hexadecenal in the hybrid are due to the fact that both palmitic and stearic acids are used as substrates, whereas only stearic acid is used as substrate in H. armigera. The evolutionary relationships between the desaturases in several Helicoverpa species are also discussed in this paper.     

Mechanisms of premating isolation between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae)

Helicoverpa armigera and Helicoverpa assulta are sympatric sibling species, and in the laboratory they can interbreed and produce viable offspring. To assess the contributions of temporal barriers and sexual barriers to premating isolation, we investigated both the temporal rhythms of calling behavior and pheromone titers of H. armigera and H. assulta females and the behavioral responses of males to conspecific and heterospecific calling females in a wind tunnel. Both H. armigera and H. assulta females called throughout the scotophase, and there was more calling during the second half of the scotophase than during the first half. Maximal pheromone titer and maximal calling activity in H. armigera synchronously occurred at the sixth hour into the scotophase, whereas, in H. assulta, the maximal pheromone titer occurred 2 h before the peak of calling. Pheromone blend ratios of the two species were opposite and, within each species, changes in the ratio within the scotophase and at different ages were relatively small. Males of both H. armigera and H. assulta responded strongly to their conspecific calling females in the wind tunnel and completed the whole courtship sequence. In contrast, they did not land and had no copulation attempts in response to heterospecific calling females. These results show that the two species do not have obvious temporal differences in calling behavior and pheromone production, and the specificity of sex pheromone blend emitted by females plays a key role in their premating isolation. In addition, we summarized the potential isolation mechanisms of H. armigera and H. assulta.     

Molecular markers to discriminate among four pest species of Helicoverpa (Lepidoptera: Noctuidae)

The four significant pest species in the Helicoverpa genus (H. armigera, H. assulta, H. punctigera and H. zea) are morphologically similar and can only be reliably distinguished through dissection of adult genitalia. Two partial regions of the mitochondrial DNA (mtDNA), the cytochrome oxidase subunit I (COI) and the cytochrome b (Cyt b) genes were amplified by PCR and digested with restriction endonucleases. The restriction patterns, generated by the endonucleases BstZ17I and HphI, demonstrated reliable differentiation of the four Helicoverpa pest species. This technique is fast, reliable and effective at distinguishing specimens irrespective of their life stages and offers support to conventional taxonomic differentiation based on morphological characters.     

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Helicoverpa assulta和棉铃虫H. armigera 5龄幼虫48 h,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。     

烟夜蛾性肽受体基因的克隆与表达模式分析

【目的】克隆烟夜蛾Helicoverpa assulta (Guenée)性肽受体基因并分析其表达模式, 为深入研究性肽与交配后反应的关系奠定基础。【方法】采用RT-PCR方法, 从烟夜蛾雌蛾性信息素腺体中得到性肽受体基因cDNA全序列。利用荧光定量PCR方法, 分析该基因的表达模式。【结果】序列分析结果显示, 烟夜蛾性肽受体基因cDNA全长2 048 bp, 命名为HassSPR（GenBank登录号: AFH53182.1）。该基因的开放阅读框长1 275 bp, 编码424个氨基酸残基, 序列中含有7个跨膜域结构, 预测分子量和等电点分别为48.6 kDa和9.25。序列比对分析表明, HassSPR与近缘种棉铃虫H. armigera和其他蛾类性肽受体的氨基酸序列一致性分别达98.35%和超过84%, 与已经报道的其他昆虫的性肽受体的氨基酸序列一致性也在64%以上。不同组织表达分析表明, HassSPR在测定的1日龄雌蛾不同组织中均有表达, 以在脑中的表达量最高。时序表达分析表明, 在羽化前1 天至羽化后6日龄雌蛾的信息素腺体中均有表达, 以3日龄表达量最高。雌蛾交配后, HassSPR在性信息素腺体和脑中的表达量显著上调, 而在交配囊和卵巢中的表达量显著下调。【结论】从烟夜蛾雌蛾性信息素腺体中克隆得到性肽受体基因HassSPR, 其表达模式提示该基因的表达水平与雌蛾的生殖生理和生殖行为有关。     

Metabolic detoxification of capsaicin by UDP-glycosyltransferase in three Helicoverpa species

Capsaicin β-glucoside was isolated from the feces of Helicoverpa armigera, Helicoverpa assulta, and Helicoverpa zea that fed on capsaicin-supplemented artificial diet. The chemical structure was identified by NMR spectroscopic analysis as well as by enzymatic hydrolysis. The excretion rates of the glucoside were different among the three species; those in the two generalists, H. armigera and H. zea, were higher than in a specialist, H. assulta. UDP-glycosyltransferases (UGT) enzyme activities measured from the whole larval homogenate of the three species with capsaicin and UDP-glucose as substrates were also higher in the two generalists. Compared among five different larval tissues (labial glands, testes from male larvae, midgut, the Malpighian tubules (MT), and fat body) from the three species, the formation of the capsaicin glucoside by one or more UGT is high in the fat body of all the three species as expected, as well as in H. assulta MT. Optimization of the enzyme assay method is also described in detail. Although the lower excretion rate of the unaltered capsaicin in H. assulta indicates higher metabolic capacity toward capsacin than in the other two generalists, the glucosylation per se seems to be insufficient to explain the decrease in capsaicin in the specialist, suggesting that H. assulta might have another important mechanism to deal with capsaicin more specifically.     

棉铃虫和烟青虫性信息素腺体脂肪醇和乙酸酯转化的比较研究

棉铃虫Helicoverpa armigera和烟青虫H. assulta属于可同域发生的近缘种昆虫,通过产生比例相反的两种性信息素化合物——顺9-十六碳烯醛和顺11-十六碳烯醛维持种间生殖隔离。本研究应用外源不饱和脂肪醇及乙酸酯在棉铃虫和烟青虫性信息素腺体进行在体转化,利用气相色谱法分析转化产物,从酶学角度探讨了上述两近缘种昆虫性信息素腺体组分差异的形成原因。实验结果表明,两种昆虫信息素腺体表皮伯醇氧化酶对外源顺9-十六碳烯醇、顺11-十六碳烯醇和反10-十六碳烯醇无催化专一性,说明末端氧化过程对于醛类性信息素组分特定比例的形成不起作用。棉铃虫性信息素腺体组织具有较高的乙酸酯酶活性,可水解外源乙酸酯,但烟青虫性信息素腺体乙酸酯酶活性很低。这些发现对于进一步了解两种昆虫的生殖隔离机制有重要参考价值。     

棉铃虫对萎蔫枫杨气味的行为反应

利用嗅觉仪测定了棉铃虫Helicoverpaarmigera (H櫣ｂｎｅｒ)成虫对萎蔫的枫杨Pterocaryastenoptera气味的行为反应。结果证实 ,萎蔫的枫杨气味仅能引诱棉铃虫处女雌蛾 ,但对已交配的雌蛾和雄蛾无显著的引诱作用 ;萎蔫的枫杨气味能显著地加强棉铃虫处女雌蛾对雄蛾的引诱作用。并讨论了枫杨枝把引诱棉铃虫的生物学意义     

Using COI gene sequence to barcode two morphologically alike species: the cotton bollworm and the oriental tobacco budworm (Lepidoptera: Noctuidae)

Due to limited morphological difference, the two closely related sister species, the cotton bollworm, Helicoverpa armigera (Hübner) and the oriental tobacco budworm, H. assulta (Guenée) (Lepidoptera: Noctuidae), are very difficult to distinguish, especially at the larvae stage. Recently, DNA sequence has been widely used as a bio-barcode for species identification. In this study, we attempted to distinguish H. armigera and H. assulta using the mitochondrial cytochrome C oxidase subunit I gene (COI) gene sequence as the barcode. We determined a 658 bp segment of the COI gene for 28 individuals of H. armigera, 8 individuals of H. assulta, and 10 individuals of Mamestra brassicae (as the outgroup) in Yunnan Province, southwest of P. R. China, together with one H. assulta and two H. armigera reported sequences from GenBank. Twenty-three haplotypes were identified in all 49 samples. As expected, network analysis of the haplotypes of the three species presented a clustering pattern consistent with the respective species status. Haplotypes of the same species differed from each other by no more than three nucleotide substitutions. However, each haplotype of H. armigera differed from that of H. assulta by at least 22 nucleotide substitutions. Both species differed from M. brassicae by more than 50 nucleotide substitutions. 17 unique diagnostic nucleotides were also used to discriminate the two species. The finding of large COI sequence differences between H. armigera and H. assulta suggested that it could be used to distinguish the two morphologically alike species and be employed for quick species identification during pest control.     

表皮碳氢化合物分析用于棉铃虫与烟青虫幼虫分类鉴别

本文利用ＧＣ－ＭＳ技术分析了棉铃虫与烟青虫幼虫的表皮碳氢化合物,并与亚洲玉米螟幼虫表皮碳氢化合物进行了比较。结果表明,棉铃虫与烟青虫幼虫表皮碳氢化合物的组分与含量均有显著差异,与玉米螟相比,这二者之间的差异明显小于它们与玉米螟的差异,其表现与三者之间自然的系统发育关系相一致。     

Expression in antennae and reproductive organs suggests a dual role of an odorant-binding protein in two sibling Helicoverpa species

Odorant-binding proteins (OBPs) mediate both perception and release of semiochemicals in insects. These proteins are the ideal targets for understanding the olfactory code of insects as well as for interfering with their communication system in order to control pest species. The two sibling Lepidopteran species Helicoverpa armigera and H. assulta are two major agricultural pests. As part of our aim to characterize the OBP repertoire of these two species, here we focus our attention on a member of this family, OBP10, particularly interesting for its expression pattern. The protein is specifically expressed in the antennae of both sexes, being absent from other sensory organs. However, it is highly abundant in seminal fluid, is transferred to females during mating and is eventually found on the surface of fertilised eggs. Among the several different volatile compounds present in reproductive organs, OBP10 binds 1-dodecene, a compound reported as an insect repellent. These results have been verified in both H. armigera and H. assulta with no apparent differences between the two species. The recombinant OBP10 binds, besides 1-dodecene, some linear alcohols and several aromatic compounds. The structural similarity of OBP10 with OBP1 of the mosquito Culex quinquefasciatus, a protein reported to bind an oviposition pheromone, and its affinity with 1-dodecene suggest that OBP10 could be a carrier for oviposition deterrents, favouring spreading of the eggs in these species where cannibalism is active among larvae.     

Mating Effect on Sex Pheromone Production of the Oriental tobacco budworm,Helicoverpa assulta

This study was undertaken to clarify the suppression phenomenon of sex pheromone production after mating and its relationship to the physiological mechanism in adult females of Helicoverpa assulta, and determine the mating factor from males causing depletion of sex pheromonc production. Sex pheromone production of H. assulta females was mostly terminated in 3 hours after mating. Mated females maintained with a low titer of sex pheromone until 3 days when it started to increase again, which showed a characteristic of species mating more than once. The mated female again produced pheromone upon injection of pheromone biosynthesis activating neuropeptide (PBAN) or extracts of brain-suboesophageal ganglion complexes (Br-Sg) of mated female, which were shown similar pheromonotropic activities as compared with virgin females. These results indicated that the mating did not inhibit the receptivity of pheromone gland itself and PBAN biosynthesis in suboesophageal ganglion of the mated females. And it seems to support that the depletion of sex pheromone production is responsible for blocking of PBAN release from head. To investigate the mating factor from adult males, when extracts of reproductive organs of male were injected into hemocoel of virgin females evoking depletion of sex pheromone production as shown in mated female. The results suggest that a chemical substance(s) from the male reproductive organs could be responsible for the loss of sex pheromone biosynthesis in H. assulta.     

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

Abstract 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 of H. 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 construction, location and eventual cloning of genes involved in traits differentiation.     

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.     

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。     

复配杀虫剂BtA对棉铃虫的杀虫效果及对天敌中红侧沟茧蜂蛹和成虫的触杀毒性(英文)

为了科学施药, 合理保护和利用天敌对棉铃虫Helicoverpa armigera进行综合防治, 本研究在室内测定和比较了复配杀虫剂BtA和7种常用杀虫剂（阿维菌素、 Bt、 β-氯氰菊酯、辛硫磷、定虫隆、氰戊菊酯和丁硫克百威）对棉铃虫的杀虫效果及对其天敌中红侧沟茧蜂Microplitis mediator蛹和雌成虫的触杀毒性。采用浸叶法测定杀虫剂在室内72 h内对棉铃虫3龄幼虫的杀虫效果, 结果表明： 与其他杀虫剂相比, BtA对棉铃虫幼虫具有更高的毒性和致死效应 (LC₅₀=0.7364 mg/mL)。将棉铃虫3龄幼虫接到用浓度4 mg/mL上述杀虫剂分别处理过的大白菜上24, 48和72 h, 发现其死亡率之间存在显著差异（P≤0.05）。但在72 h后, BtA和其他杀虫剂对棉铃虫幼虫的杀虫效果之间并无显著差异(P>0.05)。BtA施药后, 随着时间的延长, 棉铃虫幼虫的死亡率也在增加。另外, 通过杀虫剂对寄生蜂中红侧沟茧蜂蛹和雌成虫的触杀毒性的生物测定发现： 与β-氯氰菊酯、 氰戊菊酯和丁硫克百威相比, BtA对中红侧沟茧蜂蛹和雌成虫的毒性较低, 对其蛹和雌成虫致死率分别仅为13.82%和7.33%。本研究证明BtA对鳞翅目害虫具有中等毒性, 而对寄生蜂中红侧沟茧蜂则具有较低毒性。     

The ecology of Helicoverpa spp. (Lepidoptera: Noctuidae) in the Riverina region of south-eastern Australia and the implications for tactical and strategic management

Decisions on the choice of appropriate tactical and strategic control techniques require an understanding of the seasonal distribution and ecology of the target species. To address this need, data were collected from 1997 to 2000 using crop surveys, field sampling and pheromone trapping to monitor the population trends of Helicoverpa armigera (Hübner) and H. punctigera (Wallengren) in the Riverina region of south-eastern Australia. The data gathered are compared to predictions from the HElicoverpa Armigera and Punctigera Simulation (HEAPS) model, that simulates Helicoverpa population dynamics to assist in identifying the drivers of regional populations and provide a framework to make informed decisions. The results highlight the contrasting seasonal dynamics of H. punctigera and H. armigera within the region. Helicoverpa punctigera populations peak in the spring, driven by large scale spring migration into the region and subsequently decline. Helicoverpa armigera populations, conversely, are characterized by a period of spring recruitment of adults from overwintering pupae within the region, followed by a within-season population increase leading to severe late season pressure. The within-season increase of H. armigera is a result of the succession of crop and non-crop hosts with the main driver being sequentially sown, unsprayed maize. Tactical and strategic management options are discussed in light of these findings.     

Genetic basis of sex pheromone blend difference between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae)

The two closely related moth species, Helicoverpa armigera and H. assulta, are sympatric in China. Both species use a mixture of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald) as their sex pheromones but in widely different ratios. Hybridization and backcrossing experiments between H. armigera and H. assulta were conducted and sex pheromone compositions of the parent species, their F(1) hybrids and backcrosses were compared to study the genetic basis of the production of their sex pheromone blend composition. Results show that the difference in sex pheromone blend ratios of these Helicoverpa species is mainly controlled by an autosomal locus with two alleles, with the allele from H. armigera being almost completely dominant over that derived from H. assulta.