Cell lines derived from the western corn rootworm larvae, Diabrotica virgifera virgifera (Chrysomelidae: Coleoptera)
Cell lines derived from the western corn rootworm larvae, Diabrotica virgifera virgifera (Chrysomelidae: Coleoptera)
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DOI：
10.1007/s11626-020-00460-7
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发表时间：
2020-04
期刊：
影响因子：
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通讯作者：
Kaile Zhou;C. Goodman;J. Ringbauer;D. Stanley
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文献类型：
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作者：
Kaile Zhou;C. Goodman;J. Ringbauer;D. Stanley
昆虫细胞系是筛选潜在杀虫剂、生产重组蛋白和了解昆虫生物学的宝贵工具（Smagghe 等人，2009 年；Smagghe 和 Swevers，2012 年；Zitzmann 等人，2017 年）。我们的实验室致力于建立由特定组织或代表性不足的物种产生的“下一代”昆虫细胞系（Reall 等人，2019 年；Zhou 等人，2019 年）。迄今为止，尚未建立来自西部玉米根虫 (WCR)、Diabrotica virgifera virgifera 的细胞系。这种昆虫是玉米的主要害虫，广泛发生，包括北美和欧洲（Gray et al. 2009，Wesseler and Fall 2010，Marchioro and Krechemer 2018，https://www.cabi.org/isc/datasheet/18637# toDistributionMaps）。幼虫吃玉米根，成虫吃玉米丝、花粉、叶子和玉米粒。全球范围内 WCR 造成的农作物损失以及相关控制成本超过 20 亿美元（Wechsler 和 Smith，2018）。此外，WCR 已经对控制技术产生了抵抗力，包括杀虫剂、轮作和产生杀虫毒素的玉米（Paolino 和 Gassmann 2017）。唯一的叶甲属。目前可用的细胞系来自南方玉米根虫 Diabrotica undecimpunctata（Lynn 和 Stoppleworth 1984），这是一种植物寄主范围截然不同的害虫（Marchioro 和 Krechemer 2018）。 WCR 细胞系的可用性将使生物制品的物种特异性测试以及对基因组和生理特征的深入研究成为可能。我们在此报告从 D. virgifera virgifera 幼虫建立的三种细胞系。昆虫取自密苏里州哥伦比亚市美国农业部农业研究局植物遗传学研究实验室 (Man Huynh, B. Hibbard)，并在 96 孔板中、完全黑暗、25°C 下以人工饲料饲养（Huynh 等人，2017）。使用 95% 乙醇（麻醉，3 分钟）、0.525% 次氯酸钠（5 分钟）和 70% 乙醇（5 分钟）对第三龄幼虫进行表面消毒，用培养基冲洗，并在 0.5 mL 培养基中用塑料杵轻轻压碎，然后用微型剪刀切碎。将组织悬浮液离心（800×g，5分钟，4℃），并将所得沉淀在CMF-PBS（不含钙镁的磷酸盐缓冲盐水）中洗涤3次。最终沉淀在含有 50 U/mL 青霉素和 0.05 mg/mL 链霉素（Sigma-Aldrich，圣路易斯，密苏里州）的 EX-CELL 420+ 9% FBS 中培养，并转移至三个 T25 烧瓶（Falcon，Fisher Scientific，Waltham，MA）中。培养物维持在28°C，大约每两周更换1/2培养基，然后每周更换一次，然后完全更换培养基。培养于2017年6月17日开始，DvWL1（通过移液）于2018年2月12日、DvWL2（使用0.5%胰蛋白酶-EDTA，Sigma-Aldrich）于2017年10月23日、DvWL3（使用胰蛋白酶）于2018年10月2日产生第一代。目前使用胰蛋白酶对生长活跃的培养物进行传代（3 分钟，DvWL1；4-5 分钟，DvWL2 和 DvWL3）。所有这三个系均已被传代超过 40 次。
Insect cell lines serve as valuable tools in screening potential insecticides, the production of recombinant proteins, and the understanding of insect biology (Smagghe et al. 2009; Smagghe and Swevers 2012; Zitzmann et al. 2017). Our lab is focusing on the establishment of “next generation” insect cell lines that are generated from specific tissues or from under-represented species (Reall et al. 2019; Zhou et al. 2019). To date, no cell lines have been established from the western corn rootworm (WCR), Diabrotica virgifera virgifera. This insect is a major pest of maize and occurs very broadly, including North America and Europe (Gray et al. 2009, Wesseler and Fall 2010, Marchioro and Krechemer 2018, https://www. cabi. org/isc/datasheet/18637# toDistributionMaps). Larvae consume maize roots and adults feed on silks, pollen, leaves, and kernels. Crop losses due to WCR, and associated control costs, exceeded $2 billion worldwide (Wechsler and Smith 2018). Additionally, the WCR has become resistant to control technologies, including insecticides, crop rotation, and insecticidal toxin-producing maize (Paolino and Gassmann 2017). The only Diabrotica spp. cell line currently available is from the southern corn rootworm, Diabrotica undecimpunctata (Lynn and Stoppleworth 1984), a pest with a much different plant host range (Marchioro and Krechemer 2018). Availability of WCR cell lines will enable speciesspecific testing of biologicals as well as in-depth investigations into genomic and physiological characteristics.We report here on three cell lines established from D. virgifera virgifera larvae. Insects were obtained from the Plant Genetics Research Lab, USDA-ARS laboratories (Man Huynh, B. Hibbard) in Columbia, MO, and reared on artificial diet in 96-well-plates in complete darkness at 25 C (Huynh et al. 2017). Third instar larvae were surfaced sterilized using 95% ethanol (to anesthetize, 3 min), 0.525% sodium hypochlorite (5 min), and 70% ethanol (5 min), rinsed with media, and gently crushed with a plastic pestle in 0.5-mL medium, then minced with micro scissors. The tissue suspension was centrifuged (800× g, 5 min, 4 C) and the resulting pellet was washed three times in CMF-PBS (calcium magnesium-free phosphate buffered saline). The final pellet was brought up in EX-CELL 420+ 9% FBS containing 50 U/mL penicillin and 0.05 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO) and transferred into three T25 flasks (Falcon, Fisher Scientific, Waltham, MA). Cultures were maintained at 28 C, and 1/2 media was replaced approximately biweekly, then weekly, then full media replacement. Cultures were initiated on June 17, 2017, and first passages were generated on February 12, 2018, for DvWL1 (via pipetting), October 23, 2017, for DvWL2 (using 0.5% trypsin-EDTA, Sigma-Aldrich), and October 2, 2018, for DvWL3 (using trypsin). Actively growing cultures are currently passaged using trypsin (3 min, DvWL1; 4–5 min, DvWL2 and DvWL3). All three lines have each been passaged over 40 times.