用半发酵无菌日粮饲养 Axenic Delia antiqua
Summary
描述了用半发酵无菌日粮饲养 axenic Delia antiqua 的简单程序。使用PCR方法在每龄的Axenic D. antiqua 中仅检测到一种沃尔巴克氏菌株。
Abstract
Axenic昆虫是从使用无菌培养基的无菌人工饲养系统中获得的。这些昆虫的特点是体型小、生长周期短、饲料要求低，是研究微生物与宿主之间关系的理想选择。肠道菌群显著影响昆虫宿主的生理特性，将特定菌株引入昆虫中提供了一种验证肠道微生物功能的方法。 Delia antiqua 是双翅目、Anthomyiidae 科和 Delia 属的一种威胁性害虫，主要以洋葱、大蒜、韭菜和百合科的其他蔬菜为食。它的幼虫以鳞茎为食，导致整株植物腐烂、枯萎甚至死亡。通过饲养蛤蜊幼虫，可以进行后续研究，观察肠道菌群对 古蛾生长发育的影响。与涉及抗生素消除相关微生物的方法不同，本文提出了一种低成本和高效的方法来培养 axenen D. antiqua。对 古蛾 卵进行表面灭菌后，采用半发酵无菌日粮饲养幼虫，通过培养依赖性和培养非依赖性试验验证 了古蛾 的轴质状态。总之，昆虫卵灭菌和幼虫培养无菌日粮的制备相结合，能够开发出一种有效和简单的方法来获得 Axenic D. antiqua。该方法为研究昆虫-微生物群落的相互作用提供了一种强有力的方法。
Introduction
Axenic 动物，定义为无法检测到活微生物或寄生虫的动物，是研究宿主-微生物相互作用的有价值的实验模型 1,2。昆虫是最大的无脊椎动物类群，可以与微生物形成共生关系3.Axenic 昆虫可用于研究共生系统中的宿主-共生体相互作用4.例如，Nishide等人[5]为恶臭蠕虫Plautia stali建立了一种实用的无菌饲养程序，能够可靠而严格地分析模型共生系统中的宿主-共生体相互作用。Axenic昆虫可以通过对卵阶段进行消毒并向幼虫和成虫提供无菌食物来产生6,7。Axenic昆虫具有重要意义，在生物学研究中被广泛使用。例如，Somerville等人进行的一项研究[8]表明，接种阴沟肠杆菌的菱形蛾提高了转基因雄性的适应性。
Delia antiqua Meigen 是全球洋葱和其他百合科作物的一种具有重要经济意义的害虫，其幼虫会破坏洋葱和其他百合科作物的鳞茎9.D. antiqua 主要分布在温带气候中，广泛分布于美洲、欧洲和亚洲的洋葱种植区。如果控制不当，可导致洋葱（洋葱）、大蒜（Allium sativum L.）、青葱（Allium fistulosum L.）和韭菜（Alliumchoenoprasum L.）的作物损失，从50%到100%不等10,11。幼虫以植物的地下部分为食，这种进食导致幼苗枯萎并最终死亡。此外，受损的植物可以使病原体进入，导致鳞茎腐烂12。即使这些植物没有被幼虫完全吃掉，它们造成的损害也会使洋葱植物无法销售并导致经济损失。
昆虫与肠道微生物群密切相关，大多数昆虫肠道含有多种共生细菌，这些细菌在宿主提供的营养物质中茁壮成长13,14。Jing等[15]表明，肠道共生群落的主要功能是提供必需的营养物质，其次是消化和解毒相关的功能。在某些情况下，肠道细菌可以作为害虫管理目的的微生物资源。因此，研究单个肠道细菌在 D. antiqua 体内的表现和特定功能是可取的。因此，制备轴幼虫对于研究特定细菌菌株与昆虫之间的相互作用尤为重要16。目前，消除昆虫肠道细菌的常用方法是使用抗生素组合来根除相关微生物17,18,19。与单独使用抗生素只能减少微生物数量不同，昆虫的轴性饲养可以控制微生物的组成和数量，从而更准确地验证肠道微生物群的功能。
因此，本文介绍了一种制备和饲养 axenic D. antiqua 的方案。Axenic幼虫食品是通过利用天然饮食的高温杀菌与半发酵食品相结合而获得的。按照实验方案对卵进行灭菌以获得斧头虫卵，最后，从斧头卵中培养斧头幼虫。在实验中，轴性饲养系统仅进行了一代。这将为研究昆虫与肠道微生物群之间的相互作用提供便利。
Protocol
D. antiqua 产自泰安市樊镇田间。 1.无菌饮食的制备 剥掉葱的外层，丢弃绿叶。保留葱的白色部分（图1A）并用无菌水清洗，重复漂洗过程三次。用剪刀将葱的白色部分切成1-2厘米的丁块（见 材料表），用75%EtOH溶液（在步骤2.5中提到）灭菌，以备后用。 称取 50 克葱丁并将它们放入食品加工机中（见 材…
Representative Results
D. antiqua 的生命阶段如图 4 所示。完整的生命周期包括卵、幼虫、蛹（图4C）和成虫（图4D）。它们在无菌离心管中培养，其外观和存活率与在非轴性条件下饲养的 D. antiqua 没有区别。D. antiqua每个阶段的生长和发育时间如图6所示。非轴培的古蛾的卵期为2.83±0.29 d，轴向饲养的 D. …
Discussion
昆虫具有高度复杂的肠道微生物群20,21，因此需要使用接种特定肠道微生物菌株的昆虫来研究昆虫与微生物的相互作用。昆虫的制备对于此类研究工作至关重要。抗生素治疗是一种用于消除肠道微生物群的方法。例如，Jung 和Kim 22 用青霉素喂养草地贪夜蛾，而Raymond 23 用含有利福平的人工饮食喂养木叶假单</e…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
本研究得到了国家自然科学基金（32272530）、济南大学新二十政策项目（2021GXRC040）、山东省重大科技创新项目（2021TZXD002）和齐鲁工业大学科教融合项目（2022PYI009、2022PY016、2022PT105）的支持。
Materials
| 0.22 μM filter bottle | Thermo Scientific | 450-0045 | |
| 0.22 μM Syringe Filter | Biosharp | BS-QT-011 | |
| 100-mesh sieve | Zhejiang Shangyu Jinding Standard Sieve Factory | No Catalog numbers | |
| 1x PBS solution | Solarbio | P1020 | |
| 2x Taq PCR Master Mix | GENVIEW | GR1113-1ML | |
| 5.2% NaClO solution | Sinopharm Chemical Reagent Co., Ltd. | 80010428 | |
| 500 mL Conical flask | Thermo Scientific | 4103-0500 | |
| 50 mL vented centrifuge tube | JET BIOFIL | BRT-011-050 | |
| 50x TAE buffer | GENVIEW | GT1307 | |
| Agar powder | Ding Guo | DH010-1.1 | |
| Biochemical incubator | STIK | 21040121500010 | |
| Cell sieve | SAINING | 5022200 | |
| Choline chloride | Sangon Biotech | A600299-0100 | |
| ddH2O | Ding Guo | PER018-2 | |
| Disposable grinding pestle | JET BIOFIL | CSP-003-002 | |
| DNA extraction kit | Sangon Biotech | B518221-0050 | |
| DNA Marker | Sangon Biotech | B600335-0250 | |
| Ethanol absolute | Sinopharm Chemical Reagent Co., Ltd. | 10009218 | |
| Filter paper | NEWSTAR | 1087309025 | |
| Food processor | Guangdong Midea Life Electric Appliance Manufacturing Co., Ltd. | WBL25B26 | |
| Illuminated incubator | Shanghai ESTABLISH Instrumentation Co., Ltd. | A16110768 | |
| L-Ascorbic acid | Sangon Biotech | A610021-0100 | |
| L-shaped spreader | SAINING | 6040000 | |
| Nutrient agar medium | Hope Bio | HB0109 | |
| Scissors | Bing Yu | BY-103 | Purchase on Jingdong |
| Shock incubator | Shanghai Zhichu Instrument Co., Ltd. | 2020000014 | |
| Sucrose | GENVIEW | CS326-500G | |
| Super Green nucleic acid dye | Biosharp | BS355A | |
| Super-clean table | Heal Force | AC130052 | |
| TSB | Hope Bio | HB4114 | |
| Vacuum pump | Zhejiang Taizhou Seeking Precision Vacuum Pump Co., Ltd. | 22051031 | |
| Yeast extract | Thermo Scientific | LP0021B |
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Diesen Artikel zitieren
Cao, X., Liang, Q., Li, M., Wu, X., Fan, S., Zhang, X., Zhou, F., Zhao, Z. Rearing Axenic Delia antiqua with Half-Fermented Sterile Diets. J. Vis. Exp. (202), e66259, doi:10.3791/66259 (2023).