开发一种用于评估植物化学物质对 Helicoverpa armigera 的杀虫作用的摄食试验系统
Summary
该方案描述了专性摄食测定法，以评估植物化学物质对鳞翅目昆虫幼虫的潜在毒性作用。这是一种高度可扩展的昆虫生物测定法，易于优化亚致死剂量和致死剂量、威慑活性和生理效应。这可用于筛选环保杀虫剂。
Abstract
Helicoverpa armigera是一种鳞翅目昆虫，是一种分布于全球的多食性害虫。这种草食性昆虫对植物和农业生产力构成威胁。作为回应，植物会产生几种植物化学物质，对昆虫的生长和生存产生负面影响。该方案展示了一种专性摄食测定方法，用于评估植物化学物质（槲皮素）对昆虫生长，发育和生存的影响。在受控条件下，新生儿在预定的人工饮食中维持到第二龄。这些二龄幼虫被允许以对照和含有槲皮素的人工饮食为食 10 天。隔天记录昆虫的体重、发育阶段、体重和死亡率。在整个测定过程中评估体重的变化、喂养模式的差异和发育表型。 所描述的强制性摄食测定模拟了自然的摄入模式，并且可以放大到大量昆虫。它允许人们分析植物化学物质对 H. armigera 的生长动态、发育过渡和整体适应性的影响。此外，该设置还可用于评估营养参数和消化生理过程的改变。本文提供了一种详细的喂养测定系统方法，该方法可能适用于毒理学研究、杀虫分子筛选以及了解植物-昆虫相互作用中的化学效应。
Introduction
影响作物生产力的生物因子主要是病原体和害虫。一些害虫造成 15% 至 35% 的农作物损失，并影响经济可持续性实践 1.属于鞘翅目、半翅目和鳞翅目昆虫的昆虫是毁灭性害虫的主要目。环境的高度适应性使鳞翅目动物进化出几种生存机制。在鳞翅目昆虫中，棉铃虫（Helicoverpa armigera）可以以大约 180 种不同的作物为食，并对其生殖组织造成重大损害2。在世界范围内，阿米格拉嗜血杆菌的侵扰已造成约 50 亿美元的损失3.棉花、鹰嘴豆、木豆、西红柿、向日葵和其他作物是 H. armigera 的宿主。它在寄主植物的不同部分完成其生命周期。雌性飞蛾产下的卵在叶子上孵化，然后在幼虫阶段以营养组织为食。幼虫阶段由于其贪婪和高度适应性而最具破坏性 4,5。由于其显著的属性，如多噬性、优异的迁徙能力、更高的繁殖力、强烈的滞育性以及对现有昆虫防治策略的抗性的出现，H. armigera显示出全球分布和对新领土的侵占6。
来自萜烯、类黄酮、生物碱、多酚、氰基葡糖苷等多种化学分子被广泛用于控制阿米氏嗜血杆菌侵染7。然而，化学分子的频繁应用会因其残留物的获得而对环境和人类健康产生不利影响。此外，它们对各种害虫捕食者产生不利影响，导致生态失衡 8,9。因此，有必要研究害虫防治化学分子的安全和环保选择。
植物产生的天然杀虫分子（植物化学物质）可用作化学杀虫剂的有前途的替代品。这些植物化学物质包括属于生物碱、萜类化合物和酚类的各种次生代谢物7,10。槲皮素是存在于各种谷物、蔬菜、水果和叶子中的最丰富的类黄酮（酚类化合物）之一。它显示出对昆虫的喂食威慑和杀虫活性;此外，它对害虫的天敌无害11,12.因此，该方案演示了使用槲皮素的喂养测定来评估其对 H. armigera 的毒性作用。
已经开发了各种生物测定方法来评估天然和合成分子对昆虫摄食、生长、发育和行为模式的影响13.常用的方法包括叶盘试验、选择喂养试验、液滴喂养试验、接触试验、日粮覆盖试验和专性喂养试验13,14。这些方法根据杀虫剂施用于昆虫的方式进行分类。专性喂养试验是测试可能的杀虫剂及其致死剂量的最常用、最灵敏、最简单和适应性最强的方法之一 14。在专性喂养测定中，将目标分子与人工饮食混合。这提供了对饮食成分的一致性和控制，产生了稳健且可重复的结果。影响摄食测定的重要变量是昆虫的发育阶段、杀虫剂的选择、环境因素和样本量。测定的持续时间、两次数据记录之间的间隔、日粮喂养的频率和数量、昆虫的健康状况以及操作人员的操作技能也会影响喂养测定的结果14,15。
本研究旨在证明专性摄食试验以评估槲皮素对 H. armigera 存活和健康的影响。对各种参数的评估，如昆虫体重、死亡率和发育缺陷，将提供对槲皮素杀虫作用的见解。同时，测量营养参数，包括摄入食物转化效率 （ECI）、消化食物转化效率 （ECD） 和近似消化率 （AD），将突出槲皮素的抗摄食属性。
Protocol
Representative Results
Discussion
实验室生物测定有助于预测结果，并在短时间内以合理的成本生成几种化合物的比较毒性数据。摄食生物测定有助于解释昆虫-杀虫剂和昆虫-植物-杀虫剂之间的相互作用。它是一种测量各种物质毒性的有效方法，大大简化了确定致死剂量50（LD50）、致死浓度50（LC50）或任何其他致死浓度或剂量24,25的过程。各种实验室生物测定用于确定?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
SM、YP 和 VN 承认新德里印度政府大学教育资助委员会颁发的奖学金。RJ 感谢印度科学与工业研究委员会 （CSIR） 和印度浦那的 CSIR-国家化学实验室根据项目代码 MLP036626、MLP101526 和 YSA000826 提供财政支持。
Materials
| Agar Agar | Himedia | RM666 | Solidifying agent |
| Ascorbic acid | Himedia | CMS1014 | Vitamin C source |
| Bengal Gram | NA | NA | Protein and carbohydrate source |
| Casein | Sigma | C-5890 | Protein source |
| Cholesterol | Sisco Research Laboratories | 34811 | Fatty acid source |
| Choline Chloride | Himedia | GRM6824 | Ammonium salt |
| DMSO | Sigma | 67-68-5 | Solvent |
| GraphPad Prism v8.0 | https://www.graphpad.com/guides/prism/latest/user-guide/using_choosing_an_analysis.htm | ||
| Methyl Paraben | Himedia | GRM1291 | Antifungal agent |
| Multivitamin capsule | GalaxoSmithKline | NA | Vitamin source |
| Quercetin | Sigma | Q4951-10G | Phytochemical |
| Sorbic Acid | Himedia | M1880 | Antimicrobail agent |
| Streptomycin | Himedia | CMS220 | Antibiotic |
| Vitamin E capsule | Nukind Healthcare | NA | Vitamin E source |
| Yeast Extract | Himedia | RM027 | Amino acid source |
Riferimenti
- Popp, J., Pető, K., Nagy, J. Pesticide productivity and food security. A review. Agronomy for Sustainable Development. 33 (1), 243-255 (2013).
- da Silva, F. R., et al. Comparative toxicity of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) to selected insecticides. Insects. 11 (7), 431 (2020).
- Usman, A., Ali, M. I., Shah, M., e Amin, F., Sarwar, J. Comparative efficacy of indigenous plant extracts and a synthetic insecticide for the management of tomato fruit worm (Helicoverpa armigera Hub.) and their effect on natural enemies in tomato crop. Pure and Applied Biology. 7 (3), 1014-1020 (2018).
- Honnakerappa, S. B., Udikeri, S. S. Abundance of Helicoverpa armigera (Hubner) on different host crops. Journal of Farm Science. 31, 436-439 (2018).
- Edosa, T. T. Review on bio-intensive management of African bollworm, Helicoverpa armigera (Hub.): Botanicals and semiochemicals perspectives. African Journal of Agricultural Research. 14 (1), 1-9 (2019).
- Zhou, Y., et al. Migratory Helicoverpa armigera (Lepidoptera: Noctuidae) exhibits marked seasonal variation in morphology and fitness. Environmental Entomology. 48 (3), 755-763 (2019).
- Souto, A. L., et al. Plant-derived pesticides as an alternative to pest management and sustainable agricultural production: Prospects, applications and challenges. Molecules. 26 (16), 4835 (2021).
- Özkara, A., Akyıl, D., Konuk, M. Pesticides, environmental pollution, and health. Environmental Health Risk-Hazardous Factors to Living Species. , (2016).
- Alengebawy, A., Abdelkhalek, S. T., Qureshi, S. R., Wang, M. -. Q. Heavy metals and pesticides toxicity in agricultural soil and plants: Ecological risks and human health implications. Toxics. 9 (3), 42 (2021).
- Tlak Gajger, I., Dar, S. A. Plant allelochemicals as sources of insecticides. Insects. 12 (3), 189 (2021).
- Riddick, E. W. Potential of quercetin to reduce herbivory without disrupting natural enemies and pollinators. Agriculture. 11 (6), 476 (2021).
- Gao, Y. -. L., et al. The effect of quercetin on the growth, development, nutrition utilization, and detoxification enzymes in Hyphantria cunea Drury (Lepidoptera: Arctiidae). Forests. 13 (11), 1945 (2022).
- Durmuşoğlu, E., Hatipoğlu, A., Gürkan, M. O., Moores, G. Comparison of different bioassay methods for determining insecticide resistance in European Grapevine Moth, Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae). Turkish Journal of Entomology. 39 (3), 271-276 (2015).
- Paramasivam, M., Selvi, C. Laboratory bioassay methods to assess the insecticide toxicity against insect pests-A review. Journal of Entomology and Zoology Studies. 5 (3), 1441-1445 (2017).
- Clark, E. L., Isitt, R., Plettner, E., Fields, P. G., Huber, D. P. W. An inexpensive feeding bioassay technique for stored-product insects. Journal of Economic Entomology. 107 (1), 455-461 (2014).
- Waldbauer, G. P., Cohen, R. W., Friedman, S. An improved procedure for laboratory rearing of the corn earworm, Heliothis zea (Lepidoptera: Noctuidae). The Great Lakes Entomologist. 17 (2), 10 (2017).
- Friesen, K., Berkebile, D. R., Zhu, J. J., Taylor, D. B. Laboratory rearing of stable flies and other muscoid Diptera. JoVE. (138), e57341 (2018).
- Zheng, M. -. L., Zhang, D. -. J., Damiens, D. D., Lees, R. S., Gilles, J. R. L. Standard operating procedures for standardized mass rearing of the dengue and chikungunya vectors Aedes aegypti and Aedes albopictus (Diptera: Culicidae)-II-Egg storage and hatching. Parasites & Vectors. 8, 1-7 (2015).
- Nagarkatti, S., Prakash, S. Rearing Heliothis armigera (Hubn.) on an artificial diet. Technical Bulletin Commonwealth Institute of Biological Control. , (1974).
- Adhav, A. S., Kokane, S. R., Joshi, R. S. Functional characterization of Helicoverpa armigera trehalase and investigation of physiological effects caused due to its inhibition by Validamycin A formulation. International Journal of Biological Macromolecules. 112, 638-647 (2018).
- Abbasi, B. H., et al. Rearing the cotton bollworm, Helicoverpa armigera, on a tapioca-based artificial diet. Journal of Insect Science. 7 (1), 35 (2007).
- Armes, N. J., Jadhav, D. R., Bond, G. S., King, A. B. S. Insecticide resistance in Helicoverpa armigera in South India. Pesticide Science. 34 (4), 355-364 (1992).
- Waldbauer, G. P. The consumption and utilization of food by insects. Advances in Insect Physiology. 5, 229-288 (1968).
- Carpinella, M. C., Defago, M. T., Valladares, G., Palacios, S. M. Antifeedant and insecticide properties of a limonoid from Melia azedarach (Meliaceae) with potential use for pest management. Journal of Agricultural and Food Chemistry. 51 (2), 369-374 (2003).
- Diaz Napal, G. N., Palacios, S. M. Bioinsecticidal effect of the flavonoids pinocembrin and quercetin against Spodoptera frugiperda. Journal of Pest Science. 88, 629-635 (2015).
- ffrench-Constant, R. H., Roush, R. T. Resistance detection and documentation: the relative roles of pesticidal and biochemical assays. Pesticide Resistance in Arthropods. , 4-38 (1990).
- Gikonyo, N. K., Mwangi, R. W., Midiwo, J. O. Toxicity and growth-inhibitory activity of Polygonum senegalense (Meissn.) surface exudate against Aedes aegypti larvae. International Journal of Tropical Insect Science. 18 (3), 229-234 (1998).
- Sharma, R., Sohal, S. K. Bioefficacy of quercetin against melon fruit fly. Bulletin of Insectology. 66 (1), 79-83 (2013).
- Després, L., David, J. -. P., Gallet, C. The evolutionary ecology of insect resistance to plant chemicals. Trends in Ecology & Evolution. 22 (6), 298-307 (2007).
- Shi, G., Kang, Z., Ren, F., Zhou, Y., Guo, P. Effects of quercetin on the growth and expression of immune-pathway-related genes in silkworm (Lepidoptera: Bombycidae). Journal of Insect Science. 20 (6), 23 (2020).
- Selin-Rani, S., et al. Toxicity and physiological effect of quercetin on generalist herbivore, Spodoptera litura Fab. and a non-target earthworm Eisenia fetida Savigny. Chemosphere. 165, 257-267 (2016).
- Ateyyat, M., Abu-Romman, S., Abu-Darwish, M., Ghabeish, I. Impact of flavonoids against woolly apple aphid, Eriosoma lanigerum (Hausmann) and its sole parasitoid, Aphelinus mali (Hald). Journal of Agricultural Science. 4 (2), 227 (2012).
- Brito-Sierra, C. A., Kaur, J., Hill, C. A. Protocols for testing the toxicity of novel insecticidal chemistries to mosquitoes. JoVE. (144), e57768 (2019).
- Mitchell, C., Brennan, R. M., Graham, J., Karley, A. J. Plant defense against herbivorous pests: exploiting resistance and tolerance traits for sustainable crop protection. Frontiers in Plant Science. 7, 1132 (2016).
