Histopathological Effects of the Protein Toxin from Xenorhabdus nematophila on the Midgut of Helicoverpa armigera

Xenorhabdus nematophila HB310, which is highly virulent for many insects, is symbiotic with Steinernema carpocapsae HB310. Toxin II was obtained using methods such as salting out and native-PAGE from the cells of X. nematophila HB310. The histopathology of toxin II on H. armigera larvae was studied by dissecting an olefin slice of the midgut. The symptoms showed that the histopathology of the H. armigera midgut was similar to that of other novel midgut-active toxins such as the δ-endotoxins from Bacillus thuringiensis, as well as Tca from Photorhabdus luminescens W14. The midgut tissues of H. armigera fourth-instar larvae began to transform after the oral intake of the toxin Ⅱ over 6 h. First, the anterior region of the peritrophic membrane （PM） began to degrade followed by the elongation of the columnar cells. The epithelium decomposed gradually, and the midgut tissues were either loose or disordered. The PM disappeared after 12 h but reappeared after 72 h following transient or sublethal exposure to the toxin Ⅱ. Toxin Ⅱ also directly destroyed in vitro PMs of H. armigera.

Cloning and Sequencing of a Gene Encoding Aminopeptidase N in the Midgut of Helicoverpa armigera(Hubner)

A cDNA encoding aminopeptidase N was cloned by degenerated PCR combined with RACE technique in this paper. The full-length of APN-Harm is 3 043 bp. Open reading frame is 2 856 bp in length, encoding 951 amino acid residues. Its predicted molecular weight and isoelectric point are 108. 3 kDa and 5.29, respectively. This deduced amino acid sequence shares some common structural features with aminopeptidase N from several moth species, including the consensus zinc-binding motif HEXXHX18E and the GAMEN motif common to gluzincin aminopeptidases. The first 20 amino acid residues at N-termini is hydrophobic transmembrane helix. The sequence of APN-Harm was deposited in GenBank and the accession number is AY181026.

Cryptochromes mRNA Expression Under Mating and Black light Treatment on Helicoverpa armigera

[ Objective ] The paper was to confirm the effect of mating and black light treatment on cryptochromes mRNA expression of Hclicoverpa armigera. [ Method ] Quantitative real-time PCR （ SYBR Green） technique was applied to detect the expression of cryptochromes gene （ cryl and cry2 ） of H. armigera under different conditions. Total RNA was extracted from the head of H. armigera, and carried out reverse transcription to synthesize cDNA after digested by DNase I. Specific primers were used to carry out quantitative real-time PCR on cryl, cry2 and EF-Ict gene, respectively. [ Result] The expression of cryl mRNA of H. ar- migera significantly decreased after exposure to black light for 2 h, the mRNA expression of cry2 was smaller than control, they had no significant difference with each other. Mating had significant effect on mRNA expression of cryl and cry2 of H. armigera, and the mRNA expression of cryl and cry2 of male and female adults showed decreasing trend with the prolongation of time after mating. [ Conclusion] The result had important significance for further studying on function of cry gene and the control of cotton bollwonn.

不同光波及光强度下棉铃虫(Helicoverpa armigera)成虫的行为反应

运用室内行为实验方法研究了不同波长单色光和白光刺激的棉铃虫[Helicoverpaarmigera(H櫣ｂｎｅｒ）］成虫趋光、避光反应及其与光强度之间的相互关系?峁允?:(1)一定时间暗适应的棉铃虫蛾对单色光和白光刺激均具有趋光行为反应 ,依其趋光反应率大小 ,其光谱反应曲线在340 -605nm波谱范围内 ,表现为大小较复杂的几个峰 ,其中两个峰分别在蓝光区483nm、紫外340nm ,另在近紫外的400nm、绿黄光区的538nm有峰。(2)一定光强度范围内 ,棉铃虫蛾的趋光反应率随单色光和白光光强度的增强而增大 ,至一定光强度时增加变缓 ,呈现近似S型曲线式样。(3)性别、日龄对棉铃虫蛾的光谱、光强度反应曲线均有一定影响 ,雌性较雄性的趋光反应率和可适应的光强度高 ,1、3、5日龄蛾中以3日龄的趋光反应率和可适应的光强度高。 (4)棉铃虫蛾的趋光、避光反应行为具有相似的波长及光强度选择机制。

光因子对棉铃虫(Helicoverpa armigera)雌蛾性信息素产生及其求偶行为的影响

分为 3部分 :第 1部分为光因子对棉铃虫雌蛾求偶行为的影响 ;第 2部分为光因子对棉铃虫雌蛾性信息素 ( Z-1 1 -1 6:Ald)含量的影响 ;第 3部分为光因子对棉铃虫雌蛾脑因子活性的影响。研究结果表明 :用非正常光周期、间隔光照、不同光质处理棉铃虫雌蛾后 ,求偶峰值下降 ,求偶节律混乱 ,性信息素含量下降 ,脑因子活性受到抑制。主要研究目的是探讨一种不使用或少使用农药的新型生态防治方法。

The Sublethal Effects of Chlorantraniliprole on Helicoverpa armigera (Lepidoptera: Noctuidae)

Sublethal effects of chlorantraniliprole to Helicoverpa armigera （Hiibner） larvae were evaluated through exposure of third instar larvae to the insecticide incorporated into an artificial diet. When larvae were fed on the insecticide-containing diet for 7 d, the LC10, LC20, LC40, and LC50values were 3.790, 7.977, 21.577, and 33.121 μg active ingredient L^-1, respectively. Chlorantraniliprole at sublethal concentrations significantly reduced the larval body mass, emergence ratio, adult longevity and egg hatching rate in both the parental and offspring generations. The pupation and copulation rate in the parental generation and the pupal mass in the offspring also strongly decreased. Reproduction was seriously disturbed in both the parental and offspring groups even when only one of the partners was exposed to chlorantraniliprole at larval stages. However, at the lowest concentration of exposure （LC^0）, offspring fecundity was strongly reduced only when both partners were exposed. The net reproductive rate （R0）, intrinsic rate of increase （rm）, and gross reproduction rate （GRR） at LC20, LC40 concentrations were significantly lower than that of the control. Post-exposure effects also included an extended larval developmental time and increased male proportion in both generations. The doubling time （Dt） at LC20, LC40 concentrations as well as gross reproduction rate （GRR） at LC10 concentration were also significantly increased. Chlorantraniliprole might have significant effects on H. armigera population dynamics even at sublethal concentrations on both parental and offspring generations.

棉铃虫(Helicoverpa armigera)幼虫性特征和精子的发生

以棉铃虫精巢为材料,气干法制片,Giemsa染色,研究了棉铃虫精巢的形态变化和精子发生的动态过程。结果表明:棉铃虫的精巢自预蛹期开始会发生融合现象;雄性棉铃虫染色体数目为n=31。还发现雌性幼虫的腹部有4个腺体,可以作为幼虫的性别特征。

棉铃虫[Helicoverpa armigera(Hübner)]蛾复眼视网膜电位研究

运用视网膜电位（Ｅｌｅｃｔｒｏｒｅｔｉｎｏｇｒａｍ ， ＥＲＧ） 技术研究了棉铃虫［ Ｈｅｌｉｃｏｖｅｒｐａ ａｒｍｉｇｅｒａ（Ｈüｂｎｅｒ）］ 成虫在暗适应过程中对单色光和复合光（ 即白光） 刺激的光感受性变化， 并初步分析了ＥＲＧ 的波形成分组成及影响因素。结果显示：（１） 依ＥＲＧ 振幅大小（ 峰－ 峰值） ，棉铃虫蛾的光谱敏感曲线在３４０ － ６０５ｎｍ 波谱内有三个大小不等的峰，其中第一峰位于绿－ 黄光区的５６２ｎｍ ，第二峰位于蓝光区的４８３ｎｍ ，第三个峰在近紫外区的４００ｎｍ ，显示其复眼至少具有三种感受器类型。（２） 一定光强度范围内， 一定日龄的棉铃虫蛾的ＥＲＧ 值随单色光和白光刺激光强度的增强而增大， 至一定光强度时增加变缓，呈近似Ｓ 型曲线式样。表明该蛾复眼具有较强的光强度自调节和适应机制。（３） 棉铃虫蛾复眼的ＥＲＧ 波形成分由４ 个部分组成：开光反应、正相电位、持续负电位和闭光反应，波长、光强度及光刺激时程对其各成分有不同影响。

Relationship between Temperature and Occurrence of Adult Helicoverpa armigera(Hübner)

[Objective] We aimed to investigate the relationship between temperature and occurrence of Helicoverpa armigera （H0bner） in Shihezi Zong Chang. [Method] The data of occurrence of adult Helicoverpa armigera （Hubner） and temperature in 2007-2010 was analyzed for building the prediction model. [Result] The amount of overwintering generation of Helicoverpa armigera （Hubner） increased with the in- crease of temperature and its population dynamics showed a positive correlation with temperature. The amount of first and second generation of Helicoverpa armigera （Hubner） decreased with the increase of temperature and its population dynamics showed a negative correlation with temperature. [Conclusion] This research would provide a basis for the forecast and prevention of Helicoverpa armigera （Hubner） in North Xinjiang.

Structure,Binding Characteristics,and 3D Model Prediction of a Newly Identified Odorant-Binding Protein from the Cotton Bollworm,Helicoverpa armigera (Hübner)

The full-length sequence of the odorant binding protein 5 gene,HarmOBP5,was obtained from an antennae cDNA library of cotton bollworm,Helicoverpa armigera （Hübner）.The cDNA contains a 444 bp open reading frame,encoding a protein with 147 amino acids,namely HarmOBP5.HarmOBP5 was expressed in Escherichia coli and the recombinant protein was purified by affinity chromatography.SDS-PAGE and Western blot analysis demonstrated that the purified protein can be used for further investigation of its binding characteristics.Competitive binding assays with 113 odorant chemicals indicated that HarmOBP5 has strong affinity to some special plant volatiles,including （E）-β-farnesene,ethyl butyrate,ethyl heptanoate,and acetic acid 2-methylbutyl ester.Based on three-dimensional （3D） model of AaegOBP1 from Aedes aegypti,a 3D model of HarmOBP5 was predicted.The model revealed that some key binding residues in HarmOBP5 may play important roles in odorant perception of H.armigera.This study provides clues for better understanding physiological functions of OBPs in H.armigera and other insects.

Response of successive three generations of cotton,bollworm,Helicoverpa armigera (Hübner),fed on cotton bolls under elevated CO_2

The growth, development and consumption of successive three generations of cotton bollworm, Helicoverpa armigera （Htibner）, fed on cotton bolls grown under elevated CO2 （double-ambient vs. ambient） in open-top chambers were examined. Significant decreases in protein, total amino acid, water and nitrogen content and increases in free fatty acid were observed in cotton bolls. Changes in quality of cotton bolls affected the growth, development and food utilization of H. armigera. Significantly longer larval development duration in three successive generations and lower pupal weight of the second and third generations were observed in cotton bollworm fed on cotton bolls grown under elevated CO2. Significantly lower fecundity was also found in successive three generations of H. armigera fed on cotton bolls grown under elevated CO2. The consumption per larva occurred significant increase in successive three generations and frass per larva were also significantly increased during the second and third generations under elevated CO2. Significantly lower relative growth rate, efficiency of conversion of ingested food and significant higher relative consumption rate in successive three generations were observed in cotton bollworm fed on cotton bolls grown under elevated CO2. Significantly lower potential female fecundity, larval numbers and population consumption were found in the second and third generations of cotton bollworm fed on cotton bolls grown under elevated CO2. The integrative effect of higher larval mortality rate and lower adult fecundity resulted in significant decreases in potential population consumption in the latter two generations. The results show that elevated CO2 adversely affects cotton bolls quality, which indicates the potential population dynamics and potential population consumption of cotton bollworm will alleviate the harm to the plants in the future rising CO2 atmosphere.

Purification of Aminopeptidase N Protein and Differences in cDNAs Encoding APN1 Between Susceptible and Resistant Helicoverpa armigera Strains to Bacillus thuringiensis Toxins

The brush border membrane vesicles (BBMVs) in midgut of Helicoverpa armigera weresuccessfully separated, and most of the Aminopeptidase N (APN) activities in BBMV werepreserved. The 3-[(3-chlor-amidopropyl) dimethylammonio]-1-propane-sulphonate (CHAPS)can enhance the dissolution of BBMV, and phosphatidylinositol-specific phosopholipase C(PI-PLC) can cleave the APN from midgut membrane. The APN was primarily purified usinga Mono-Q column. The results of immunoblotting showed that the 120 and 170kDa proteinsin the BBMV could bind Cry1Ac, and 120kDa APN was a glycosylphosphalidylinositol(GPI)-anchored protein. Two Bt-resistant strains (Bt-P, Bt-M) were obtained after beingselected for more than five years in laboratory using Bt insecticides and Bt transgeniccotton incorporated into diet separately. The resistance of Bt-P and Bt-M were 1083.3and 48.7 times that of susceptible strain. The genes encoding APN1 in midgut ofsusceptible and resistant H.armigera were cloned by PCR and RACE techniques. Theinferred amino acid sequences of APN1 possessed the common character of APN family ininsects. In comparison with APN1 in susceptible strain, three nucleotide mutations wereobserved in the APN1 of Bt-M strain and resulted in two amino acid replace in theputative protein sequences, and eight nucleotide mutations were observed in Bt-P strainand resulted in five amino acid replace.

Functional Characteristics of a Novel Chemosensory Protein in the Cotton Bollworm Helicoverpa armigera (Hübner)

A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera （Hvbner） was obtained from antennal eDNA libraries and expressed in Escherichia coll. The real time quantitative PCR （RT-qPCR） results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings. Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles. The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde, whereas methyl phenylacetate, 2-decanone, 1-pentanol, carvenol, isobomeol, nerolidol, 2- nonanone and ethyl heptanoate have relatively weak binding affinity. Moreover, the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33-38 （αl）, 40-48 （α2）, 62-72 （α3）, 80-96 （α4）, 98-108 （α5）, and 116-119 （α6）, two pairs of disulfide bridges Cys49-Cys55, Cys75-Cys78. The two amino acid residues, Ile94 and Trpl01, may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.

棉铃虫Helicoverpa armigera的环境友好型诱杀技术研究

2003年和2004年，在山东惠民采用杀虫灯、性诱剂、杨树枝把等措施开展了诱杀棉铃虫Helicoverpa armigera成虫的防治试验。杀虫灯＋10支性诱剂，杀虫灯＋5支性诱剂＋5支杨树枝把，杀虫灯＋10支杨树枝把，杀虫灯4种措施的日均诱蛾量分别为11．4、10．4、8．6和7．0头，性诱剂和杨树枝把均未显著提高对棉铃虫的诱杀效果。不同诱捕方案对棉铃虫累积诱蛾量随日期的变化符合Gompertz方程。普通灯和时控灯对棉铃虫的日均诱蛾量分别为19．9头和17．4头，二者差异不显著；时控灯对异色瓢虫和龟纹瓢虫的日均诱捕量均少于普通灯；减少开灯时间可在不显著降低棉铃虫诱杀量的同时，有效保护捕食性瓢虫。与普通灯相比，设置时间合理的时控灯（夜间19：00—23：00和3：00—5：00开灯）未显著降低棉铃虫的日均诱蛾量，而对异色瓢虫和龟纹瓢虫的日均诱捕量分别为2．0头和4．9头，均显著低于普通灯的3．7头和9．2头，合理设置开关灯控制模式可在不显著降低对棉铃虫诱杀效果的同时，降低近50％的天敌杀伤率，节约用电40％。

EAG and behavioral responses of Helicoverpa armigera males to volatiles from poplar leaves and their combinations with sex pheromone

Electroantennogram (EAG) evaluation of selected compounds from wilted leaves of black poplar, Populus nigra, showed that phenyl acetaldehyde, methyl salicylate, (E)-2-hexenal elicited strong responses from male antennae of Helicoverpa armigera. When mixed with sex pheromone (Ph), some volatiles, e.g. phenyl acetaldehyde, benzyl alcohol, phenylethanol, methylsalicylate, linalool, benzaldehyde, (Z)-3-hexenol, (Z)-3-hexenylacetate, (Z)-6-nonenol, cineole, (E)-2-hexenal, and geraniol elicited stronger responses from male antennae than Ph alone. Wind tunnel bioassay demonstrated that various volatiles could either enhance or inhibit the effect of synthetic sex pheromone. (E)-2-hexenal, (Z)-3-hexenol and linalool in combination with Ph could not induce any male to land on source at all, whereas phenyl acetaldehyde, benzaldehyde, (Z)-6-nonenol and salicylaldehyde combined with Ph enhanced male response rates by 58.63%, 50.33%, 51.85% and 127.78%, respectively, compared to Ph alone. These results suggested that some volatiles shouldmodify sex pheromone caused behavior and that some of them could possibly be used as a tool for disrupting mating or for enhancing the effect of synthetic sex pheromone in the field.

Analysis of phototactic responses in Spodoptera frugiperda using Helicoverpa armigera as control

Light traps are widely utilized to monitor and manage insect pest populations.In late 2018,the fall armyworm(FAW),Spodoptera frugiperda,invaded China through Yunnan Province representing a huge threat to grain production.To estimate the efficiency of light traps on FAW moths,we first identified the opsin genes from FAW by using the transcriptome.Phylogenetic analysis indicated that the four opsins of FAW were clustered with those of other Noctuidae species.The expressed levels of opsins in S.frugiperda were lower than in Helicoverpa armigera,suggesting a different phototactic response between the two species.Then,we determined the phototactic behavior of FAW using H.armigera as a control,which is widely monitored and managed using light traps in China.Our results indicated that the two moths species showed significantly different phototactic behavior and both female and male FAW displayed faster flight-to-light speed than H.armigera.This may be due to a faster flight capacity in FAW compared to H.armigera.However,the capture rate of both female and male of S.frugiperda was significantly lower than that of H.armigera,which was consistent with the expression levels of opsins.These results support the positive phototaxis of S.frugiperda moths and suggest light traps could be used for monitoring and managing the pests,but with a lower efficiency than H.armigera.

棉铃虫(Helicoverpa armigera)FK506结合蛋白基因的克隆、表达与纯化

为了深入了解FK506结合蛋白基因的作用和探索调控棉铃虫CYP6B6的表达机制,基于单酵母杂杂交结果采用RT-PCR的方法从棉铃虫的中肠cDNA中扩增得到了FK506结合蛋白基因,将测序正确的目的片段克隆至原核表达载体pET32a中,在大肠杆菌BL21中用异丙基-β-D-硫代半乳糖(IPTG)诱导表达,通过镍柱离子亲和层析纯化目的蛋白。用SDS-PAGE检测目的蛋白的表达和纯化结果,并用Western blotting进行验证。结果表明,克隆得到的目的基因大小为327 bp,编码108个氨基酸残基,预测蛋白质分子量和等电点分别是11.78 kD和7.87。氨基酸序列分析表明该序列具有完整的开放阅读框,且没有信号肽。重组质粒pET32a-FKBP在大肠杆菌BL21中获得表达,主要以可溶性形式存在,经亲和层析柱纯化获得目的蛋白,Western blotting检测发现纯化的目的蛋白大小正确且纯度高。

Response dynamics of three defense related enzymes in cotton leaves to the interactive stress of Helicoverpa armigera (Hübner) herbivory and omethoate application

In order to explore the response dynamics of the activities of defense related enzymes in cotton leaves towards the interactive stress of Helicoverpa armigera herbivory and omethoate application, the activities of phenylalanine ammonia-lyase（PAL）, lipoxygenase（LOX）, and polyphenol oxidase（PPO） were examined from 6 to 126 h after cotton leaves were treated 12 h of H. armigera herbivory, and then sprayed with 800 mg L–1 omethoate. The results showed that the changes in the activities of PAL, LOX and PPO that occured under the interactive stress of H. armigera herbivory and omethoate application reflected the interactive effects of the two stresses on cotton defense. The similarity between the response dynamics of PAL, LOX, and PPO activities in cotton leaves under the interactive stress and that under H. armigera herbivory treatment alone showed that the induction of H. armigera herbivory on the activities of PAL, LOX and PPO in cotton leaves played a leading role in the interactive effects, and the effect of omethoate application played only a minor role. A joint factor analysis was performed according to a method which has been used to analyze the joint toxicity of pesticides; this analysis sought to clarify if there was a synergistic, antagonistic, or additive effect on PAL, LOX, and PPO activity in cotton leaves resulting from the interactive H. armigera herbivory and omethoate treatment. In the interactive effect on the response of PAL activity in cotton leaves, antagonistic effects of the omethoate application towards H. armigera herbivory were observed at 6 and 12 h. Synergistic effects were then observed at 18 and 30 h. Antagonistic effects were observed from 54 to 78 h and synergistic effects were finally observed at 126 h. The correlation between H. armigera herbivory and omethoate application in the interactive effect on cotton defense responses of LOX activity also fluctuated from synergism to antagonism during the time course. In the interactive effect on PPO activity, only antagonism was observed between H. armigera herbivory and omethoate application. In the interactive stress of H. armigera herbivory and omethoate application on cotton defense responses, omethoate affected the defense responses of cotton to H. armigera herbivory by producing antagonistic and synergistic effects. These results will be useful to understand the relationship between host plant and herbivorous pest.

土壤特性对棉铃虫(Helicoverpa armigera Hübner)发生的影响及发生程度分区

统计分析表明 ,土壤的多种理化性质与棉铃虫的发生程度间存在着密切的关系 ,其中起主要作用的是土壤容重、砂粒含量和粘粒含量 3个因子 ,其直接通径系数依次为 -0 .652 1、0 .673 5和 0 .3 4 56。这些因子使土壤表现出不同的粘性 ,决定了土壤的透气性、持水量和板结程度等 ,从而影响到棉铃虫幼虫入土后蛹的存活率。用上述 3个因子进行聚类分析 ,可将通州市划分为 3个棉铃虫发生区 ,各区域在测报和防治上应进行区别对待。

Efficiency of Different Methods for dsRNA Delivery in Cotton Bollworm(Helicoverpa armigera)

RNAi trigged by dsRNA not only facilitates the development of molecular biology, but also initiates a new way for pest control by silence of fatal genes. However, one of the key limitations in pest control is lack of the convenient and efficient method for dsRNA delivery. In this study, different dsRNA delivery methods at their own optimum conditions were evaluated comparatively for their efficiency with Helicoverpa armigera as test animal. It was found that the popular one- time injection of larvae with dsRNA could reduce the pupation rate by 43.0% and enhance larva mortality by 11.7%. One- time ingestion of dsRNA did not result in any significant effect on phenotype. Continuous ingestion of in vitro synthesized dsRNA by refreshing the bait diet every day caused 40.4% decrease in successful pupation and 10.0% increase in larval mortality, which was similar as one-time injection. The most efficient method was found to be the continuous ingestion of the bacteria containing dsRNA expressed, which reduced the rate of pupation by 68.7% and enhanced the larval mortality by 34.1%. Further analysis found that dsRNA was degraded faster in midgut juice than in hemolymph. However, the cell of bacteria could protect dsRNA and delay the degradation in the midgut juice of H. armigera. These results throw light on the application of dsRNA in pest management with proper ways.