用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究
Summary
为了获得轴性昆虫，其卵表面被灭菌，然后使用轴性叶子饲养孵化的幼虫。这种方法为抗焦虑昆虫的制备提供了一种有效的方法，而无需施用抗生素或开发人造饮食，这也可以应用于其他食叶昆虫。
Abstract
昆虫的肠道被各种细菌定植，这些细菌可以深刻地影响宿主的生理特征。将特定的细菌菌株引入轴性昆虫中是验证肠道微生物功能并阐明肠道微生物 – 宿主相互作用机制的强大方法。给予抗生素或对卵子表面进行消毒是两种常用的方法，用于清除昆虫的肠道细菌。然而，除了抗生素对昆虫的潜在不利影响外，以前的研究表明，喂食抗生素并不能消除肠道细菌。因此，无菌人工饲料通常用于维持轴性昆虫，这是一个繁琐且劳动密集型的过程，不能完全类似于天然食品中的营养成分。这里描述的是一种有效且简单的方案，用于制备和维持叶甲虫（Plagiodera versicolora）的轴性幼虫。具体来说，甲虫卵的表面被消毒，然后使用无菌的杨树叶来饲养轴系幼虫。通过培养依赖性和培养无关性测定进一步证实了昆虫的轴性状态。通过结合鸡蛋消毒和无菌栽培，共同开发了一种有效且方便的方法来获得轴性 花叶假单胞菌，为其他食叶昆虫提供了易于转移的工具。
Introduction
与哺乳动物类似，昆虫消化道是食物消化和吸收的空腔。大多数昆虫含有多种共生细菌，这些细菌在肠道中茁壮成长，并以宿主1提供的营养为生。肠道共生群落对昆虫的多种生理过程具有深远的影响，包括食物消化和解毒2，3，4，营养和发育5，6，7，对病原体和寄生虫的防御8，9，10，11，化学通讯12，13 和行为14，15.有趣的是，一些肠道微生物群可能兼性致病性或被入侵病原体操纵以加重感染，这表明肠道细菌在某些情况下可能是有害的16，17，18。肠道细菌还可以作为生物技术应用和害虫管理的微生物资源。例如，来自植物噬菌和木食虫的木质纤维素消化细菌用于消化植物细胞以开发生物燃料19。表达生物活性分子的工程肠道共生体的分散是一种新颖而有前途的策略，用于管理传播传染病的农林害虫和蚊子19，20，21，其也可用于改善益虫的适应性22。因此，说明肠道细菌 在体内 的行为被认为是充分利用其功能并将其进一步用于各种应用的优先事项。
动物可以在肠道中携带1至>1000种共生微生物物种1。因此，很难准确地验证个体细菌分类群或其组装在动物体内的表现，以及宿主或其微生物伙伴是否驱动特定功能。因此，制备轴性幼虫以通过单物种或多物种定植获得无菌昆虫对于研究细菌功能和与昆虫的相互作用是必要的23。目前，给予抗生素鸡尾酒和对昆虫卵表面进行灭菌是去除肠道细菌的常用方法14，24，25，26。然而，抗生素饮食不能完全消除肠道细菌，并对宿主昆虫的生理学产生负面影响27，28。因此，使用抗生素治疗的昆虫可能会掩盖某些肠道细菌的真正能力。幸运的是，卵的表面灭菌可以消除这个问题23，29，这对实验昆虫没有或可以忽略不计的影响。此外，人工饮食不能完全类似于天然昆虫食品，开发人造饮食是一个昂贵且耗费人力的过程30，31。
柳叶甲虫，Plagiodera versicolora（Laicharting）（鞘翅目：Chrysomelidae），是一种广泛的食叶害虫，主要以水杨树为食，如柳树（柳树）和杨树（杨树L）。32，33.在这里，柳叶甲虫被用作代表性的食叶昆虫，以制定一种方案来准备和饲养无菌昆虫。我们利用植物组织培养物获得无菌的杨树叶，以从灭菌卵中培育出杂色P.通过培养依赖性和培养无关性测定验证了花斑蚴幼虫的轴性状态。该协议可以维持轴系昆虫，这些昆虫比人工饮食的昆虫饲养更能模仿野生条件。更重要的是，这种方法以非常低的成本方便，这增加了获得轴性昆虫用于未来昆虫 – 肠道微生物群相互作用研究的可行性，特别是对于没有发达人工饲料的非模型昆虫。
Protocol
1. 昆虫饲养 在生长室中维持花 斑芽孢杆菌 种群，条件为27°C和70±5%相对湿度，光周期为16小时光照/ 8小时黑暗。将它们放在带有瓷砖湿吸纸的穿孔塑料盒中，并喂它们新鲜的杨树枝。在吸收纸上喷洒清水以保持水分，并每两天更换一次树枝。 分离成虫在化蛹后产卵。给它们喂食嫩叶以获得更多的卵。 收集新产卵（24小时内）。将卵放在潮湿的吸收纸上60?…
Representative Results
P. versicolora的寿命阶段如图1所示。成年男性比成年女性小（图1A）。在田野里，甲虫将其卵聚集在叶子上;在这里，四个卵从叶子上分离出来（图1B）。用于斧头昆虫饲养的杨树茎段和幼苗如图2所示。3龄幼虫的肠道如图3所示，肠道段用白色括号标记。 <p class="jove_content…
Discussion
制备无菌幼虫和通过重新引入特定细菌菌株获得无菌幼虫是阐明宿主 – 微生物相互作用机制的有力方法。新孵化的幼虫以两种主要方式获得肠道微生物群：从母亲到后代的垂直传播或从兄弟姐妹和环境的水平获取34。前者可以通过亲本通过污染卵表面35将后代转移给后代来实现。因此，通过对昆虫卵表面27、28、29</…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由中国国家自然科学基金（31971663）和CAST青年精英科学家赞助计划（2020QNRC001）资助。
Materials
| 0.22 µm syringe filters | Millipore | SLGP033RB | |
| 1 mg/mL NAA stock solution | a. Prepare 0.1 M NaOH solution (dissolve 0.8 g NaOH in 200 mL of distilled water). b. Add 0.2 g NAA in a 250 mL beaker, add little 0.1 M NaOH solution until NAA dissolved, and adjust the final volume to 200 mL with distilled water. c. Filter the solution to remove bacteria with a 0.22 µm syringe filter and a 50 mL sterile syringe, subpackage the solution in 1.5 mL centrifuge tubes and restore at -20 °C. |
||
| 1.5 mL microcentrifuge tubes | Sangon Biotech | F600620 | |
| 10x PBS stock solution | Biosharp Life Sciences | BL302A | |
| 2 M KOH solution | Dissolve 22.44 g KOH (molecular weight: 56.1) in 200 mL of distilled water and autoclave it for 20 min at 121 °C. | ||
| 250 mL and 2,000 mL beakers | Shubo | sb16455 | |
| 50 mL sterile syringes | Jinta | JT0125789 | |
| 500 mL measuring cylinder | Shubo | sb1601 | |
| 50x TAE stock solution | a. Dissolve 242 g Tris and 18.612 g EDTA in 700 mL of distilled water. b. Adjust pH to 7.8 with about 57.1 mL of acetic acid. c. Adjust the final volume to 1,000 mL. d. The stock solution was diluted to 1x TAE buffer when used. |
||
| 75% ethanol | Xingheda trade | ||
| α-naphthalene acetic acid (NAA) | Solarbio Life Sciences | 86-87-3 | |
| Absorbing paper | 22.3 cm x 15.3 cm x 9 cm | ||
| Acetic acid | Sinopharm Chemical Reagent Co. Ltd | ||
| Agar | Coolaber | 9002-18-0 | |
| Agarose | Biowest | 111860 | |
| Autoclave | Panasonic | MLS-3781L-PC | |
| Bead-beating homogenizer | Jing Xin | XM-GTL64 | |
| DNA extraction kit | MP Biomedicals | 116560200 | |
| EDTA | Saiguo Biotech | 1340 | |
| Filter paper | Jiaojie | 70 mm diameter | |
| Gel electrophoresis unit | Bio-rad | 164-5052 | |
| Gel Signal Green nucleic acid dye | TsingKe | TSJ003 | |
| Germ-free poplar seedlings | Shan Xin poplar from Ludong University in Shandong Province | ||
| Golden Star Super PCR Master Mix (1.1×) | TsingKe | TSE101 | |
| Growth chamber | Ruihua | HP400GS-C | |
| LB agar medium | a. Dissolve 5 g tryptone, 5 g NaCl, 2.5 g yeast extract in 300 mL of distilled water. b. Adjust the final volume to 500 mL, transfer the solution to a 1,000 mL conical flask, and add 7.5 g agar. c. Autoclave the medium for 20 min at 121 °C. |
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| Mini centrifuge | DRAGONLAB | D1008 | |
| MS basic medium | Coolaber | PM1121-50L | M0245 |
| MS solid medium for germ-free poplar seedling culture | a. Dissolve 4.43 g MS basic medium powder and 30 g sucrose in 800 mL of distilled water. b. Adjust the pH to about 5.8 with 2 M KOH by a pH meter. c. Adjust the final volume to 1,000 mL, separate into two parts, transfer into two 1,000 mL conical flasks, and add 2.6 g agar per 500 mL. d. Autoclave for 20 min at 121 °C. |
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| NanoDrop 1000 spectrophotometer | Thermo Fisher Scientific | ||
| Paintbrush | 1 cm width, used to collect the eggs | ||
| Parafilm | Bemis | PM-996 | |
| PCR Thermal Cyclers | Eppendorf | 6331000076 | |
| Petri dishes | Supin | 90 mm diameter | |
| pH meter | METTLER TOLEDO | FE20 | |
| Pipettes 0.2-2 µL | Gilson | ECS000699 | |
| Pipettes 100-1,000 µL | Eppendorf | 3120000267 | |
| Pipettes 20-200 µL | Eppendorf | 3120000259 | |
| Pipettes 2-20 µL | Eppendorf | 3120000232 | |
| Plant tissue culture container | Chembase | ZP21 | 240 mL |
| Plastic box | 2.35 L | ||
| Potassium hydroxide (KOH) | Sinopharm Chemical Reagent Co. Ltd | ||
| Primers for amplifying the bacterial 16S rRNA gene | Sangon Biotech | 27-F: 5’-ACGGATACCTTGTTACGAC-3’, 1492R: 5’-ACGGATACCTTGTTACGAC-3’ | |
| Sodium chloride (NaCl) | Sinopharm Chemical Reagent Co. Ltd | ||
| Sodium hydroxide (NaOH) | Sinopharm Chemical Reagent Co. Ltd | ||
| Steel balls | 0.25 mm | used to grind tissues | |
| Stereomicroscope | OLYMPUS | SZ61 | |
| Sucrose | Sinopharm Chemical Reagent Co. Ltd | ||
| Trans2K plus II DNA marker | Transgene Biotech | BM121-01 | |
| Tris base | Biosharp Life Sciences | 1115 | |
| Tryptone | Thermo Fisher Scientific | LP0037 | |
| UV transilluminator | Monad Biotech | QuickGel 6100 | |
| Vortexer | Scilogex | MX-S | |
| Willow branches | Sha Lake Park, Wuhan, China | ||
| Willow leaf beetle | Huazhong Agricultural University, Wuhan, China | ||
| Yeast extract | Thermo Fisher Scientific | LP0021 |
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Cite This Article
Ma, M., Liu, P., Yu, J., Han, R., Xu, L. Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle. J. Vis. Exp. (176), e63195, doi:10.3791/63195 (2021).