Rapid antibody-based field test to distinguish between Helicoverpa armigera (Lepidoptera: Noctuidae) and Helicoverpa punctigera (Lepidoptera: Noctuidae)

Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) are the two most important insect pests of cotton production in Australia and require application of insecticides to control them. H. armigera has developed resistance to several insecticides but H. punctigera has not. Cost-effective management of insecticide resistance requires that growers be able to determine the proportion of H. armigera eggs or young larvae present on their crop before applying insecticides. This is impossible visually. We generated two monoclonal antibodies that reacted with the insect protein "lipophorin" and were capable of discriminating individuals of the two species at all life-stages. The antibodies were incorporated into a rapid test kit that was tested under field conditions over two growing seasons. Results obtained with the kit agreed closely with those obtained by rearing larvae through to second instar.     

Pyrethroid resistance and mechanisms of resistance in field strains of Helicoverpa armigera (Lepidoptera: Noctuidae)

Pyrethroid resistance was found in 54 field strains of Helicoverpa armigera collected between 1995 and 1999 from 23 districts in seven states of India. LD50 values of the field strains ranged from 0.06 to 72.2 microg/larva with slopes of 0.5-3.1. Resistance was highest in regions where pyrethroid use was frequent (four to eight applications per season). Resistance to deltamethrin was exceptionally high with resistance ratios of 13,570 and 27,160 in two strains collected during February 1998 in central India. Resistance to cypermethrin, fenvalerate and cyhalothrin also was high with resistance ratios of >1,000 in four strains collected from central and southern India. Resistance ratios were below 100 in >50% of the strains tested. Pyrethroid resistance was high in strains collected from the districts in Andhra Pradesh where a majority of the cotton farmer suicide cases in India were reported. Resistance to pyrethroids appeared to have increased over 1995-1998 in most of the areas surveyed. Studies carried out through estimation of detoxification enzyme activity and synergists indicated that enhanced cytochrome p450 and esterase activities were probably important mechanisms for pyrethroid resistance in field strains. Pyrethroid nerve insensitivity also was found to be a major mechanism in some parts of the country where the use of pyrethroids was high. The information presented illustrates the importance of proper insect management programs to avoid the consequences associated with improper insecticide use.     

In-season changes in resistance to insecticides in Helicoverpa armigera (Lepidoptera: Noctuidae) in India

Discriminating doses of fenvalerate, cypermethrin, quinalphos, and endosulfan were determined with an insecticide-susceptible Helicoverpa armigera (Hübner) strain. In-season changes in insecticide resistance were monitored with discriminating dose assays at weekly intervals throughout the cropping season for 6 yr from 1993 to 1999 in central India. Resistance to pyrethroids was high throughout all seasons. Resistance to 0.75 microg of quinalphos was consistent, with seasonal averages ranging from 23 to 27% survival over the 6 yr. Resistance to 10.0 microg of endosulfan was moderately high at an average of 40-47% survival during 1993-1994 and in 1997-1998. It was lower in 1996-1997 at 27%, and in 1998-1999 at 33%. The weekly monitoring data for all seasons were pooled and the consolidated 6-yr seasonal average profile indicated that resistance to quinalphos and endosulfan was low during September at 21 and 27% survival, respectively, but increased to 28 and 37% by the end of November. Resistance levels to organophosphates and endosulfan increased during the season, depending on the use of these compounds. At almost all monitoring sites, the within-season changes in quinalphos resistance for all seasons through the study period followed a trend similar to that of endosulfan. The results suggest the possibility of cross-resistance between these compounds. Based on this study and the existing information on cotton pest management, we have developed a "window strategy" for cotton pest management with specific emphasis on the management of insecticides for effective control of H. armigera. This strategy has contributed to improved control at reduced costs in extensive trials.     

A simple and reliable method for discriminating between Helicoverpa armigera and Helicoverpa assulta (Lepidoptera: Noctuidae)

Abstract The cotton bollworm Helicoverpa armigera and the oriental tobacco budworm H. assulta are sibling species, both being important agricultural pests. Morphologically, the two insects are almost indistinguishable at the egg, larval and pupal stages. One of the big challenges in the study of these insects, in particular in integrated pest management, is a timely and dependable identification of these insects at their early stages of development. Here, we report a H. armigera‐specific nuclear DNA marker, and demonstrate that it can be employed to reliably discriminate between H. armigera and H. assulta by simple polymerase chain reaction amplification experiment.     

Baseline toxicity of emamectin and spinosad to Helicoverpa armigera (Lepidoptera: Noctuidae) for resistance monitoring

Acute toxicity studies of emamectin and spinosad against Helicoverpa armigera revealed that the pest is highly susceptible to both the insecticides. The median lethal dose (LD₅₀) of emamectin is 3.86 × 10⁻³ µg per larva. The median lethal concentrations (LC₅₀) of emamectin and spinosad were found to be 0.09 and 2.94 ppm, respectively. The discriminating doses were fixed based on the LC₉₅ of the susceptible population of H. armigera as 0.80 ppm for emamectin and 10 ppm for spinosad. Resistance was not observed when the discriminating doses of emamectin and spinosad were applied on field-collected populations of H. armigera from two intensive cotton growing areas, Coimbatore and Madurai, India.     

飞行过程中棉铃虫对温度的主动选择

为了探讨温度对迁飞性昆虫空中群体聚集成层等行为的作用机制,在室内利用自行设计的连续温度梯度发生装置对棉铃虫Helicoverpa armigera在飞行过程中的温度选择行为进行了研究.结果表明：在存在明显温差的连续温度梯度中,所有棉铃虫飞行个体均对温度具有显著的选择行为.棉铃虫试虫群体表现出对空间最优飞行温度共同的主动选择,选择的温度范围是20~22℃之间;在这一温度范围内棉铃虫的振翅频率最高,持续振翅时间最长.对不同温度梯度条件下各温度区间内试虫的飞行时间进行定量比较发现,在16~22℃温度梯度场中的棉铃虫群体对最适温度的选择比在19~30℃的温度梯度场中的群体更显著,表明在温度较低的迁飞季节中温度对迁飞棉铃虫空中虫群聚集成层的影响要比在高温季节更明显.持续飞行时间对棉铃虫振翅频率的影响明显,表明昆虫迁飞高度与昆虫自身能量的消耗存在联系.     

Hybridization between Helicoverpa armigera and Helicoverpa assulta (Lepidoptera: Noctuidae): development and morphological characterization of F1 hybrids

Reciprocal hybridizations between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) were studied. The cross between females of H. armigera and males of H. assulta yielded only fertile males and sterile individuals lacking an aedeagus, valva or ostium bursae. A total of 492 larvae of the F1 generation were obtained and 374 of these completed larval development and pupated. Only 203 pupae were morphologically normal males, the remaining 171 pupae were malformed. Larvae and pupae that gave rise to morphologically abnormal adults exhibited longer development times. Sterility was not only associated with malformed external sex organs, but also a range of abnormalities of the internal reproductive system: (i) loss of internal reproductive organs, (ii) with one to three copies of an undeveloped bursa copulatrix; or (iii) with one or two undeveloped testes. Normal male hybrid adults showed higher flight activity in comparison with males of both species. In contrast, the cross between females of H. assulta and males of H. armigera yielded morphologically normal offspring (80 males and 83 females). The interaction of the Z-chromosome from H. assulta with autosomes from H. armigera might result in morphological abnormalities found in hybrids and backcrosses, and maternal-zygotic incompatibilities might contribute to sex bias attributed to hybrid inviability.     

Exon-primed intron-crossing (EPIC) PCR markers of Helicoverpa armigera (Lepidoptera: Noctuidae)

Applying microsatellite DNA markers in population genetic studies of the pest moth Helicoverpa armigera is subject to numerous technical problems, such as the high frequency of null alleles, occurrence of size homoplasy, presence of multiple copies of flanking sequence in the genome and the lack of PCR amplification robustness between populations. To overcome these difficulties, we developed exon-primed intron-crossing (EPIC) nuclear DNA markers for H. armigera based on ribosomal protein (Rp) and the Dopa Decarboxylase (DDC) genes and sequenced alleles showing length polymorphisms. Allele length polymorphisms were usually from random indels (insertions or deletions) within introns, although variation of short dinucleotide DNA repeat units was also detected. Mapping crosses demonstrated Mendelian inheritance patterns for these EPIC markers and the absence of both null alleles and allele 'dropouts'. Three examples of allele size homoplasies due to indels were detected in EPIC markers RpL3, RpS6 and DDC, while sequencing of multiple individuals across 11 randomly selected alleles did not detect indel size homoplasies. The robustness of the EPIC-PCR markers was demonstrated by PCR amplification in the related species, H. zea, H. assulta and H. punctigera.     

Evaluation of Helicoverpa armigera Nucleopolyhedrovirus (HearNPV) for Control of Helicoverpa armigera (Lepidoptera: Noctuidae) on Citrus in South Africa

A concentration of 1.15×10⁷ OBs/mL of HearNPV, sprayed using a knapsack, resulted in a 100% reduction in Helicoverpa armigera larval infestation within 7 days on tomato plants in a hot house environment. A 10-fold lower concentration, 1.15×10⁶ OBs/mL, resulted in a 100% reduction within 16 days. These two concentrations reduced damage to tomatoes by 81 and 69%, respectively. In two field trials on navel oranges, the lower concentration and an additional even lower concentration of 7.26×10⁵ OBs/mL, both resulted in a 100% reduction in H. armigera infestation within 14 days or longer. H. armigera damage to fruit was reduced by up to 84 and 75%, respectively, in the two trials. Rejection for export was reduced by 96 and 62%. This could imply a saving of US$339 per hectare compared to the untreated control. These results were better than those achieved with Bacillus thuringiensis and various standard chemical insecticides used in the same trials. Reasons for these results are discussed.     

Antifeedant and cytotoxic activity of gibberellic acid against Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

The present study investigates the effect of gibberellic acid (GA3), a terpenoid and phytohormone, on the digestive physiology and intermediary metabolism of the cotton bollworm Helicoverpa armigera Hübner (Lepidoptera, Noctuidae). Incorporation of GA3 (800 μg g^?1 diet) in an artificial diet results in significant reductions in the rates of diet consumption and the efficiency of conversion of food consumption by by H. armigera larvae. The relative growth rate decreases as the concentration increases. The relative α‐amylase activity in sixth‐instar larvae of H. armigera decreases significantly after ingestion of four concentrations of GA3. Histological studies of the midgut in GA3‐treated larvae (800 μg g^?1 diet) show degeneration of the epithelial cells. The alanine and aspartate aminotransferase activity decreases at the highest concentration. However, acid phosphatase, alkaline phosphatase, γ‐glutamyl transferase and lactate dehydrogenase activity increase significantly compared with the control. The results clearly demonstrate the adverse effects of GA3 on H. armigera via interruption of nutritional physiology and metabolism.     

Gene-flow between populations of cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) is highly variable between years

Both large and small scale migrations of Helicoverpa armigera Hübner in Australia were investigated using AMOVA analysis and genetic assignment tests. Five microsatellite loci were screened across 3142 individuals from 16 localities in eight major cotton and grain growing regions within Australia, over a 38-month period (November 1999 to January 2003). From November 1999 to March 2001 relatively low levels of migration were characterized between growing regions. Substantially higher than average gene-flow rates and limited differentiation between cropping regions characterized the period from April 2001 to March 2002. A reduced migration rate in the year from April 2002 to March 2003 resulted in significant genetic structuring between cropping regions. This differentiation was established within two or three generations. Genetic drift alone is unlikely to drive genetic differentiation over such a small number of generations, unless it is accompanied by extreme bottlenecks and/or selection. Helicoverpa armigera in Australia demonstrated isolation by distance, so immigration into cropping regions is more likely to come from nearby regions than from afar. This effect was most pronounced in years with limited migration. However, there is evidence of long distance dispersal events in periods of high migration (April 2001-March 2002). The implications of highly variable migration patterns for resistance management are considered.     

棉铃虫P450基因HarmCYP9A33的克隆、 序列分析及原核表达

夜间活动昆虫如夜蛾类主要通过嗅觉来寻找配偶、 寄主植物和产卵场所, 是研究昆虫嗅觉分子机制的理想材料。P450为多功能单加氧酶, 在昆虫对各种内源与外源物质的代谢中起重要作用。为研究P450在昆虫嗅觉中的作用, 本研究采用RT-PCR和RACE技术, 从夜蛾科昆虫棉铃虫Helicoverpa armigera (Hübner)雄蛾触角中扩增得到一条全长1 772 bp的P450基因, 命名为HarmCYP9A33 (GenBank登录号为JX486677)。序列分析表明, HarmCYP9A33开放阅读框全长1 590 bp, 编码529个氨基酸残基, 预测蛋白质分子量和等电点分别为61. 62 kD和7. 97; HarmCYP9A33与甘蓝夜蛾Mamestra brassicae触角毛形感器中高表达的MbraCYP9A13蛋白的氨基酸序列一致性最高, 达75%, 蛋白二级结构相似, 6个底物识别位点(substrate recognition sites, SRSs)序列一致性达61%, 其中底物与酶结合通道开关Ⅰ螺旋中SRS4序列完全相同, 与棉铃虫CYP9A亚家族蛋白有一定的结构相似性。Real-time PCR检测表明, HarmCYP9A33在雌、 雄蛾各组织中均有表达, 以腹部表达量最高, 其次为头部; 在卵至成虫各个时期也均表达, 以蛹中表达量最高; 在触角中的表达量随羽化时间而变化, 且多高于卵和幼虫中的表达量。SDS-PAGE检测和Western blot鉴定表明HarmCYP9A33体外融合表达成功。本研究为深入探讨该基因在棉铃虫触角感器细胞中的定位及其生物学功能奠定了基础。     

棉铃虫围食膜的蛋白质组鉴定

【目的】围食膜(peritrophic membrane, PM)是昆虫抵御随食物摄入的病原微生物入侵的第一道天然屏障。本研究旨在鉴定出农业重大害虫棉铃虫Helicoverpa armigera围食膜的总蛋白成分,为进一步揭示昆虫围食膜的形成机制及研发新颖的害虫控制策略奠定基础。【方法】剥离棉铃虫5龄幼虫PM,用三氟甲磺酸(trifluoromethane sulfonic acid, TFMS)处理,采用液质联用技术(LC-MS/MS)鉴定围食膜蛋白质组,然后对鉴定结果进行生物信息学分析。【结果】本研究共鉴定出棉铃虫幼虫围食膜蛋白质169个,是目前鉴定最多的棉铃虫围食膜蛋白。通过GO分析,可以将这些鉴定的蛋白分为细胞组分、分子功能和生物学过程三大类;KEGG富集结果显示,鉴定蛋白可以富集在12条代谢通路中;蛋白互作分析(protein protein interaction, PPI)结果表明,以ACC和CG3011等蛋白为核心可以形成蛋白互作网络。【结论】本研究鉴定了169个棉铃虫幼虫围食膜蛋白质,并对其进行了GO, KEGG和PPI分析,结果有助于人们全面理解昆虫围食膜的分子结构和功能。     

微粒体多功能氧化酶系与棉铃虫对氰戊菊酯抗药性的关系

测定了棉铃虫Helicoverpa armigera抗氰戊菊酯种群及相对敏感种群不同组织微粒体的甲氧试卤灵-O-脱甲基酶、乙氧试卤灵-O-脱乙基酶、乙氧香豆素-O-脱乙基酶、芳烷基羟基化酶和艾氏剂环氧化酶的活性。结果表明：抗性种群棉铃虫中肠组织的这5种酶活性分别比敏感种群的活性提高了11.29、4.10、2.66、6.30和2.34倍,其脂肪体及体壁的相应酶活性则分别为敏感种群的1.46、6.80、1.36、4.05、1.48倍和12.32、2.2、1.33、0.80和0.51倍。两种群中,棉铃虫不同组织部位的各单加氧酶活性均不同,活性高低顺序在两种群间也不同。总体而言,均是中肠或脂肪体微粒体对不同底物的氧化代谢能力最强。     

应用毛细管气相色谱法检测棉铃虫细胞色素P450 O-脱甲基活性

依据毛细管气相色谱法灵敏度高 ,样品用量少 ,分辨率高的特点 ,建立了一个用于检测棉铃虫Helicoverpaarmigera幼虫中肠和脂肪体细胞色素P45 0O -脱甲基活性产物的小型反应体系 ( 875 μL) ,其中含 0 1mol L ,pH 7 8Tris HCl缓冲液 ,0 5 μmol,NADPH ,7 5 μmolMgCl2 ,0 0 85 %BSA ,0 1 5 7mmol对硝基苯甲醚及酶液。利用毛细管气相色谱直接检测该反应体系中棉铃虫细胞色素P45 0的O -脱甲基产物对硝基苯酚。     

Isolations of Cry2Ab resistance in Australian populations of Helicoverpa armigera (Lepidoptera: Noctuidae) are allelic

Alleles conferring resistance to Cry2Ab toxin occur at a frequency of 0.0033 in Australian populations of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), and it is evident that detectable levels of resistance predated the introduction of transgenic cotton expressing this toxin. From 2002 until 2006, 10 such resistant alleles were scored. Here, we examine colonies established from five of the 10 isolates by using complementation tests to determine their genetic relationships. The results demonstrate that the resistance in each colony is due to alleles at the same locus and that for each allele the resistance is recessive. This latter finding is in conflict with the frequency of apparently resistant individuals occurring in the initial F2 tests that were used to identify alleles that confer resistance. These frequencies were variable (range 6.7-35.6%, mean 16.2%), but they generally indicated a measure of dominance (i.e., were >6.25% expected for recessive resistance). We hypothesize that this conflict is the result of differences in the genetic background of the laboratory adapted resistant colonies and the initial field isolations.     

Elevated CO2 may alter pheromonal communication in Helicoverpa armigera (Lepidoptera: Noctuidae)

Carbon dioxide (CO₂) as a greenhouse gas has been increasing in recent decades. Because an elevated atmospheric CO₂ influences insect physiology and behaviour, we hypothesize that pheromone–mediated communication in the moth is affected by an increased CO₂ level. We test the behavioural responses of male Helicoverpa armigera to sex pheromone in a wind tunnel, demonstrating a significant reduction of approaching behaviour to the odour source at a high CO₂ level (1000 ppm). Electroantennogram (EAG) responses of male to the pheromone component are also significantly suppressed in high CO₂ environments (600 and 1000 ppm), indicating that a high CO₂ level inhibits both behavioural and electrophysiological responses of male to the sex pheromone. Interestingly, the EAG response of the whole head preparation of males is influenced more by the elevated CO₂ level than that of the antenna‐cut preparation. A sequential increase of CO₂ levels from an ambient CO₂ level also decreases the EAG response of the whole head but not of the labial palp‐removed head, implying a potential mediation of labial palp in the head where the CO₂ receptor is located. By contrast, sex pheromone production in females reared under or shifted to an elevated CO₂ condition is increased, and the putative underlying mechanism for this is discussed. The present study provides an insight into the adaptive strategy of moth pheromone communication in a changing environment.     

Return migration of Helicoverpa armigera (Lepidoptera: Noctuidae) during autumn in northern China

The autumn migration of Helicoverpa armigera (Hübner) was observed with radar and two types of light-trap at Langfang, Hebei province, China in 2001 and 2002. The sudden increase in the proportion of H. armigera moths in the searchlight trap indicated migration into the area and catches increased 10-fold during the second half of the night due to the landing of migrants before dawn. The moths' migratory flights took place at up to 2000 m above the ground, and moths flew differentially at times, and heights, when favourable (i.e. northerly) winds occurred. This facilitated the maximum displacement of moths towards the south during these 'return' migrations. The moths flew over the radar site at consistently high densities through the night, and the resulting flight durations of c. 10 h, at displacement speeds of 30-33 km h-1, would allow moths emerging in the far northeast of China (i.e. Liaoning and Jilin provinces and the Inner Mongolia autonomous region) to migrate into northern China (Hebei, Shandong and Henan provinces). The association of the seasonal migratory movements of H. armigera with crops in northern China is briefly discussed.     

Overwintering survival and spring emergence of Helicoverpa armigera (Lepidoptera: Noctuidae) in northern Greece

Overwintering survival of Helicoverpa armigera (Hübner) was studied under field conditions during the winter of 2004-2005 and 2005-2006 to clarify whether a local population of this insect is established in northern Greece. Additionally, the postdiapause eclosion times of the overwintering generation was compared with adult male dynamics using pheromone traps. Our study supplies strong evidence that part, if not all, of the late-season generation of H. armigera overwinters as diapausing pupa in northern Greece, suggesting that a local population exists in this area. Pupae enter diapause during September and October in response to declining temperatures and photoperiod. A limited number of degree-days in autumn prevented part of the population from developing to the diapausing pupal stage. Larvae of H. armigera that were placed in field conditions late in October were not able to pupate and died. The termination of diapause and the eclosion of adults in the following spring were determined by the local soil temperatures. Diapause terminated between mid-April and early May, and adult emergence occurred within a 4- to 6-wk period from late April to early June. Females emerged slightly earlier than the males. The emergence of 10, 25, 50, 75, and 90% of adults required 153, 199, 252, 303, and 347 DD, respectively. Differences in timing and degree of overlap between adult emergence from the experimental population and pheromone trap catches suggests that the spring population of this species also includes immigrants.