棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解CrylAc毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigern（Hǔbner）与甜菜夜蛾Spodoptera exigm（Hǔbner）的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

棉铃虫围食膜蛋白的电泳分离技术

比较了棉铃虫Helicoverpa armigera(Hübner)围食膜不同处理方法,不同围食膜数量,在不同电泳下的分离效果,筛选最佳的围食膜蛋白分离技术。结果表明:冷冻干燥与非冷冻干燥处理围食膜后进行SDS-PAGE电泳分离效果基本相同,建议使用冷冻干燥后处理较好。取1、3、5条围食膜进行SDS-PAGE电泳,都能将围食膜蛋白分离,且分离效果基本一致,建议5条围食膜最合适,具有可靠性和代表性。浓缩胶为5%、分离胶分别为8%、10%和12%时棉铃虫围食膜的SDS-PAGE电泳分离效果差异不大,而分离胶为12%时效果较好。无水三氟利克酸处理围食膜后进行NuPAGE电泳,分离出的围食膜蛋白大约30多种,远远超过上述方法分离的16²0种,且需要的围食膜材料少,分离效果最佳,但费用高。     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解Cry1Ac毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigera(Hübner)与甜菜夜蛾Spodoptera exigua(Hübner)的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

Comparison of Metarhizium isolates for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) in chickpea

Metarhizium isolates from soil (53) and insect hosts (10) were evaluated for extracellular production of cuticle degrading enzyme (CDE) activities such as chitinase, chitin deacetylase (CDA), chitosanase, protease and lipase. Regression analysis demonstrated the relation of CDE activities with Helicoverpa armigera mortality. On basis of this relation, ten isolates were selected for further evaluation. Subsequently, based on LT₅₀ of the 10 isolates towards H. armigera, five isolates were selected. Out of these five isolates, three were selected on the basis of higher conidia production (60–75 g/kg rice), faster sedimentation time (ST₅₀) (2.3–2.65 h in 0.1% (w/v) Tween 80) and lower LC₅₀ (1.4–5.7×10³ conidia/mL) against H. armigera. Finally, three Metarhizium isolates were selected for the molecular fingerprinting using ITS sequencing and RAPD patterning. All three isolates, M34412, M34311 and M81123, showed comparable RAPD patterns with a 935G primer. These were further evaluated for their field performance against H. armigera in a chickpea crop. The percent efficacies with the three Metarhizium isolates were from 65 to 72%, which was comparable to the chemical insecticide, endosulfan (74%).     

An in vitro method for increasing UV-tolerance in a strain of Helicoverpa armigera (Lepidoptera: Noctuidae) nucleopolyhedrovirus

A method for increasing tolerance to ultraviolet (UV) radiation in a strain of nucleopolyhedrovirus of cotton bollworm, Helicoverpa armigera (Hübner) (HearNPV) using a solar simulator is described. The Coimbatore isolate (CBE I) of HearNPV was subjected to a five-step sequence of selection to increase its UV tolerance. Each step consisted of irradiation of wet deposits of the virus to near UV (at energy level of 300W/m²), bioassay against second instar H. armigera larvae and propagation in early fifth instar larvae. Selection steps carried out at 15, 30, 60 and 90 minutes of exposure revealed that the continuous exposure of HearNPV-CBE I at low doses of UV irradiation (270–540 KJ/m²) did not significantly affect the virus activity as measured by its biological activity against second instar larvae. Selection at higher doses (1620 KJ/m²) led to loss of viral activity in the first two exposure cycles; however, there was retention of virulence coupled with increased tolerance to UV doses from third cycle onwards. Further, studies on the persistence of UV tolerant strain of HearNPV-CBE I in comparison with original strain showed that the tolerant strain had more persistence even after 7 days of weathering both under exposed (18% original activity remaining) and shaded (26% original activity remaining) condition on potted cotton plant.     

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.     

Oviposition deterrents from eggs of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae): chemical identification and analysis by electroantennogram

Our previous results showed that an extract of the abdomens of mated females of the cotton bollworm, Helicoverpa armigera, deterred oviposition of conspecifics. In this paper, we found that the extract, in addition to reducing conspecifics’ oviposition, could elicit strong electroantennogram responses from mated females. The EAG elicitors mainly came from mature chorionated eggs in the ovarioles. Laid eggs or their surface extract evoked similar EAG responses. The main chemical components of the EAG elicitors from mature chorionated and laid eggs were myristic, palmitic, stearic, and oleic acids. A blend of authentic fatty acids at the ratio found in the laid eggs and in an amount equivalent to 100 laid eggs mimicked the EAG response and deterring effect. Moreover, these four oviposition-deterring fatty acids and their corresponding methyl esters evoked significantly higher EAG responses from both mated females and males than hexane blank and background. The EAG values differed among the test chemicals and between sexes. These results demonstrate that the four fatty acids from eggs are at least partially responsible for the oviposition-deterring effect of the extract from the abdomens of mated female H. armigera and that the moths may detect these chemicals olfactorily by antennae.     

栗精碱对棉铃虫中肠组织可溶型海藻糖酶的抑制效应

【目的】研究一种植物源化合物栗精碱对棉铃虫Helicoverpa armigera(Hübner)5龄幼虫中肠组织可溶型海藻糖酶的活体和离体抑制效果,初步探索其抑制作用。【方法】经过离子交换层析和疏水层析从棉铃虫5龄幼虫中肠组织分离纯化得到可溶型海藻糖酶,通过凝胶过滤层析确定其分子量。分别用不同浓度的抑制剂进行离体和活体抑制研究,得出栗精碱对可溶型海藻糖酶的抑制率和抑制中浓度(IC50),并采用Lineweaver-Burk和Dixon作图法分析抑制类型。【结果】棉铃虫中肠可溶型海藻糖酶比活力为2.022 U/mg,回收率为67.57%,纯化倍数为23.84,分子量约为67 k Da。离体抑制研究表明,3.75,7.5,15和30μmol/L的栗精碱对可溶型海藻糖酶的抑制率分别为42.00%,50.30%,58.32%和67.33%;抑制中浓度(IC50)为7.32μmol/L。通过LineweaverBurk和Dixon作图法,确定栗精碱是棉铃虫中肠组织可溶型海藻糖酶的一种有效的竞争性抑制剂,Ki值为4.9μmol/L。活体抑制研究表明,分别注射浓度为30,15和7.5μmol/L栗精碱10和20 h后,棉铃虫中肠可溶型海藻糖酶活性被显著抑制(P0.05)。相应地,注射后随着时间的延长,血淋巴海藻糖含量连续增加。而注射栗精碱20 h后,血淋巴葡萄糖浓度显著下降,使棉铃虫的能量供给出现障碍。【结论】结果表明,栗精碱是棉铃虫中肠可溶型海藻糖酶的一种有效的抑制剂,可为下一步开发有效的棉铃虫杀虫剂提供理论支持。     

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.     

Effects of age and mated status on flight potential of Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract The effects of ageing and female mated status on the flight potential of Helicoverpa armigera (Hübner), collected as larvae from a pigeonpea crop in southern India, were investigated using a tethered-flight technique. In non-mated moths fed sugar solution, from the first night after adult eclosion, the durations of both total and longest continuous flight per night increased up to night 4 and remained at this level until at least night 6. Ovarian maturation was rapid with 77% of unmated moths having commenced oviposition by the third night. On the basis of field evidence it is likely that most females would be mated by the third or fourth night, provided plants with nectar or sugary exudates were locally available. In successfully-mated females a 15-fold decrease in total flight duration and a 28-fold decrease in longest continuous flight duration was observed in contrast to non-mated females of similar age. As host plants suitable for adult feeding and oviposition were locally available during the time of feral adult emergence, synchronous pre-reproductive migration was unlikely to occur in the population studied.     

Potentialities of methoxyfenozide for the integrated management of Helicoverpa armigera (Lepidoptera: Noctuidae) on cotton in Benin,West Africa

Intensive use of chemical insecticides against Helicoverpa armigera has led to the development of resistance to the major chemical families of insecticides. Consequently, management of H. armigera using conventional chemical insecticides is increasingly difficult. Methoxyfenozide is an agonist of the insect moulting hormone 20-hydroxyecdysone that acts faster than chitin synthesis inhibitors. The present work is aimed to assess the effectiveness of methoxyfenozide for use against H. armigera on cotton in Benin, West Africa. Laboratory tests and field experiments have been carried out. For laboratory studies, topical application of methoxyfenozide was done using L2 and L5 instar larva of H. armigera. Tested methoxyfenozide concentrations varied from 24 μl/ml to 144 μl/ml and the control is water only. For field experiments, a complete randomised block design was used with methoxyfenozide (72 μl/ml) and a control (no spraying). Berthoud Ultra Low Volume sprayer was used to spray methoxyfenozide suspension at 60-l per hectare. Two sprays were applied, 7 days apart. Laboratory tests indicated that 24 h after application of methoxyfenozide, about 100% of treated L2 and L5 larva were morbid or dead compared with 0% for the control larva. There are no significant differences between tested concentrations. Morbid larvae died within 2 days. In field experiments, cotton yield harvested on methoxyfenozide treated plants was double of that obtained on untreated plants (9375 kg and 4875 kg per hectare respectively). Thus methoxyfenozide may be used as component of Integrated Pest Control Programme on cotton in Benin, West Africa.     

Effect of repeated in vitro sub-culturing on the virulence of Metarhizium anisopliae against Helicoverpa armigera (Lepidoptera: Noctuidae)

The effect of repeated conidial sub-culturing of Metarhizium anisopliae on its virulence against Helicoverpa armigera (Hübner) was studied. The LT₅₀ observed against third instar larvae of H. armigera for the first sub-culture was 3.4 days; it increased to 4.5 and 5.6 days for the 20th and the 40th sub-cultures, respectively. The LT₅₀ values after passage of the 40th sub-culture on H. armigera decreased to 4.4 and 3.7 days for the 40th (first in vivo) and the 40th (fifth in vivo) passages, respectively. Similarly, the LC₅₀ of M. anisopliae towards third instar larvae of H. armigera increased from the first sub-culture (0.17×10⁴) to (3.0×10⁴) for the 40th conidial transfers on potato dextrose agar and again decreased to 0.74×10⁴ and 0.23×10⁴ in the 40th (first in vivo) and the 40th (fifth in vivo) passage, respectively. Similar trends for LC₅₀ and LT₅₀ values were seen when sugarcane woolly aphid, Ceratovacuna lanigera Zehntner was used as a host. Significant variation in appressorium formation and cuticle-degrading enzyme production such as chitinase, chitin deacetylase, chitosanase and protease during subsequent sub-culturing and passage through H. armigera was observed. Though there was no effect on internal transcribed spacer (ITS) sequence pattern, interestingly, in randomly amplified polymorphic DNA (RAPD), significant differences in the band intensities and in the banding pattern for different sub-cultures of M. anisopliae were observed. As stable virulence towards the insect pest is desirable for commercialisation of a mycoinsecticide, such changes in virulence due to repeated in vitro transfer need to be monitored and minimised.     

Laboratory investigations on the potential of entomopathogenic fungi for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) larvae and pupae

The susceptibility of third instar Helicoverpa armigera to seven strains of three entomopathogenic fungal species, i.e. Metarhizium anisopliae, Beauveria bassiana and Paecilomyces fumosoroseus, was tested under laboratory conditions using the larval immersion method. High efficacies ranging from 68 to 100% corrected mortality were recorded with more profound effects in treatments with B. bassiana and P. fumosoroseus strains. The median lethal concentration (LC₅₀) for L3 was 6.0×10⁵ in M. anisopliae 79, 1.5×10⁵ in B. bassiana 124 and 4.2×10⁴ in P. fumosoroseus 14. These three strains were further used to characterize the age-dependent mortality of different larval stages (L2-L5) and the effect against pupae of H. armigera. Larval stages did not differ in their mortality but differed i in median lethal time, with shorter values recorded in the second instar. Tested fungi also caused a high reduction between 74.4 and 100% in the emergence of pupae using the soil inoculation method and the pupal immersion technique. All three fungal species, especially P. fumosoroseus, have a high potential for biocontrol of H. armigera larvae and also as a soil treatment targeting the pupae.     

棉铃虫蜕皮调节转录因子在大肠杆菌中的重组表达及其包涵体纯化

蜕皮调节转录因子(hormone receptor 3,HR3)在昆虫蜕皮过程中启动蜕皮相关早期基因簇表达,并抑制蜕皮相关晚期基因簇表达,对昆虫蜕皮级联反应起着关键的调控作用。利用合成的特异性引物通过RT-PCR扩增了棉铃虫 Helicoverpa armigera 蜕皮调节转录因子(HHR3),并与pGEX-4T-1载体连接,在大肠杆菌Escherichia coli DH5α内进行扩增,经过PCR筛选获得了HHR3-pGEX-4T-1重组质粒。 用该质粒转化大肠杆菌表达菌株BL21并进行诱导表达,获得了与谷胱甘肽-S转移酶（GST）融合表达的HHR3包涵体,分子量在94 kD左右,通过无离子去垢剂CAPS（3-［cyclohexylamino］-1-propanesulfonic acid）变性、复性后获得了可溶性GST-HHR3融合蛋白,经凝血酶裂解和SDS-PAGE分离得到纯化的HHR3,经蛋白质N-端测序确认表达正确。用重组表达的HHR3免疫家兔,制备了兔抗HHR3多克隆抗体,免疫印迹检测显示该抗体对HHR3有特异性识别能力, 可以用于HHR3功能与调控等下游研究。免疫印迹检测结果还表明,HHR3在5龄向6龄蜕皮的幼虫脂肪体中高表达,在进入6龄24 h 的幼虫脂肪体中含量明显下降,在6 龄72 h 的幼虫中肠中没有检测到HHR3表达;成虫卵巢中有HHR3表达。     

两株棉铃虫胚胎新细胞系的建立及其对杆状病毒侵染的反应

昆虫细胞系的建立在病毒学和昆虫学等领域的研究和应用中发挥着重要的作用。本研究由棉铃虫Helicoverpa armigera胚胎组织建立了两株细胞系, 分别命名为QB-Ha-E-1和QB-Ha-E-5, 在含10％胎牛血清的TNM-FH培养基中已传代60余代。两株细胞系均以圆形和短梭形细胞为主。DAF鉴定结果表明, 两株细胞系均来源于棉铃虫胚胎, 其扩增谱带与其他几种昆虫细胞系明显不同; QB-Ha-E-1和QB-Ha-E-5的第30代细胞群体倍增时间分别为63.7 h和66.9 h。两株细胞系均能被棉铃虫核型多角体病毒（HaSNPV）感染, 4 d的感染率分别为86.6％和56.5％, 对甘蓝夜蛾Mamestra brassicae核型多角体病毒（MbNPV）7 d的感染率均为15％左右, 但对苜蓿银纹夜蛾Autographa californica核型多角体病毒（AcMNPV）侵染的反应不同。DAPI染色和基因组DNA电泳结果表明, AcMNPV可诱导QB-Ha-E-5细胞发生凋亡, 极少数细胞内可形成多角体, 但不能诱导QB-Ha-E-1细胞发生凋亡, 其感染率为55.3％; 两株细胞系均可被1.25 μg/mL的放线菌素D诱导发生凋亡。两株细胞系具有相同的遗传背景, 但对AcMNPV侵染的反应不同, 可作为昆虫病毒和细胞之间相互关系以及细胞凋亡机制研究的理想材料。     

Polymorphic microsatellite loci for the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) and some remarks on their isolation

Five polymorphic tri‐ and tetranucleotide microsatellite loci suitable for population genetic analysis were identified in the cotton bollworm Helicoverpa armigera from two partial phagemid genomic libraries enriched for microsatellite inserts. The overall microsatellite‐cloning efficiency in H. armigera is 2.5%, which is approximately eightfold lower than that for the gadoid fishes (20%) employing the same enrichment protocol, supporting the notion of a relative low frequency of microsatellite sequences in lepidopteran genomes. In addition, a large proportion of cloned microsatellite sequences turned out to be repetitive DNA, thus further increasing the difficulty of developing such markers in butterflies and moths.     

Role of chemical cues from cotton in mediating host selection and oviposition behaviour in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

We investigated the role of chemoreception in the host selection and oviposition behaviour of Helicoverpa armigera in the laboratory using five cotton genotypes and synthetic volatile terpenes. Female moths oviposited on substrates treated with methanol, ethanol, acetone and pentane extracts of leaves, squares and flowers of the cotton genotypes. Phytochemicals soluble in pentane were the most efficient in eliciting oviposition behaviour. In a two-way bioassay, pentane extracts of leaves or squares of a Multiple Host-plant Resistance genotype (MHR11), Deltapine commercial (DP90), and Smith Red Leaf (SRL) received significantly more eggs than solvent-treated controls. Extracts of squares of the native genotype Gossypium nelsonii did not receive more eggs. Females preferred DP90 and MHR11 to SRL and G. nelsonii. Female moths also laid more eggs on pentane extracts of MHR11 flowers than MHR11 leaves from preflowering, early flowering and peak-flowering plants. In a flight chamber, female moths used olfactory cues at short range to mediate oviposition and discrimination between host plants. Egg-laying, mated females were attracted at a distance (1.5 m) to volatile compounds released by whole plants and odours emanating from filter papers treated with synthetic volatile terpenes. Individually, the terpenes did not stimulate any significant oviposition response. However, there was a significant oviposition response to a mixture of equal volumes of the terpenes ( trans -β-caryophyllene, α-pinene, β-pinene, myrcene, β-bisabolol, and α-humulene). Conversely, antennectomised (moths with transected antennae), egg-laying, mated females did not stimulate any significant oviposition response. The significance of these findings in relation to H. armigera host-plant selection are discussed.     

Impact of Cheiracanthium pelasgicum (Araneae: Miturgidae) and Chrysoperla carnea (Neuroptera: Chrysopidae) intraguild predation on the potential control of cotton pest Helicoverpa armigera (Lepidoptera: Noctuidae)

Antagonist interactions such as intraguild predation (IGP) or cannibalism among predatory arthropods can reduce the impact of these invertebrates on pest limitation in agroecosystems. Here, the effects of IGP between two major natural enemies of cotton pests, the cursorial spider Cheiracanthium pelasgicum (C.L. Koch) and the common green lacewing Chrysoperla carnea (Stephens), were studied under laboratory conditions. First, a feeding preference test was carried out to determine the degree of C. pelasgicum preference for lacewing larvae, using second-instar Helicoverpa armigera larvae as alternative prey. In a second bioassay, the effects of predator interactions on potential predation of H. armigera larvae were analysed using three treatment combinations (plus a control with no predator): (1) spider alone, (2) lacewing larvae alone, (3) spider + lacewing larvae. Potential predation by C. pelasgicum on lacewing eggs was also studied. C. pelasgicum showed no significant preference for either of the two species, indicating that this spider may impact negatively on the green lacewing population. Findings revealed no additive effects and an antagonist interaction between C. pelasgicum and green lacewing larvae, which adversely affected H. armigera suppression; both predators displayed lower predation rates when kept together than either predator alone. However, presence of lacewing larvae and subsequent unidirectional IGP did not affect the predation capacity of C. pelasgicum. Finally, predation rates of C. pelasgicum on lacewing eggs were very low (mean 2.35 ± 0.71 eggs, 24 h after offering) indicating that the impact of C. pelasgicum on lacewing populations may be limited.