果蝇 幼虫注射方案
Summary
果蝇黑色素体 成年蝇已被广泛用作模型生物，以研究宿主抗菌先天免疫反应和微生物感染策略的分子机制。为了促进 D. melanogaster 幼虫阶段作为附加或替代模型系统，描述了幼虫注射技术。
Abstract
使用非常规模型来研究先天免疫力和病原体毒力为哺乳动物模型提供了一种有价值的替代方案，哺乳动物模型可能代价高昂并引发伦理问题。众所周知，非常规模型非常便宜，易于处理和文化，并且不占用太多空间。它们在基因上是可适应的，并且拥有完整的基因组序列，并且它们的使用没有道德考虑。例如，果蝇 果蝇黑色素胃蝇为各种行为，发育，新陈代谢和免疫研究提供了很好的见解。更具体地说， D. melanogaster 成年苍蝇和幼虫具有与脊椎动物共享的几种先天防御反应。调节免疫反应的机制主要通过 D. melanogaster 模型中的遗传和分子研究揭示出来。本文提供了一种新型幼虫注射技术，将进一步促进 对黑腹 股蓝虫幼虫先天免疫过程的研究，并探索多种微生物感染的发病机制。
Introduction
几十年来，黑腹果蝇在生物学和生物医学研究中得到了极大的应用，因为一系列复杂的遗传和分子工具已经稳步发展，用于分析广泛的研究1，2，3，4。黑色素瘤蚜虫发育、稳态和先天免疫的进化保守方面使其成为研究各种人类和昆虫疾病的宝贵模式生物5，6。值得注意的是，D. melanogaster模型在研究免疫力方面的基本作用在成年苍蝇研究中得到了很大的体现。然而，D. melanogaster幼虫的研究也为目前的知识做出了贡献，并主要探索了细胞免疫反应，特别是通过昆虫角质层发生的黄蜂和线虫感染7，8，9，10。黑果蝇幼虫拥有三种不同类型的血细胞，统称为血细胞：浆细胞，晶体细胞和薄片细胞11，12，13。当黑色素体幼虫感染细菌，真菌，病毒和寄生虫等病原体时，这些细胞可以产生一系列免疫反应14，15，16。细胞免疫应答包括小分子或细菌的直接吞没（吞噬作用），黑色化，较大病原体（如寄生虫卵）的包封，以及活性氧（ROS）和一氧化氮合成酶（NOS）的产生17，18，19。
相比之下，关于使用 D. melanogaster 幼虫模型分析体液免疫反应的研究较少。这主要是由于对 D. melanogaster 幼虫的口腔感染的喂养测定的应用以及与微注射幼虫相关的一些挑战，包括幼虫的精确处理和微针的正确使用，特别是在渗透期间20，21。因此，对幼虫感染的有限知识和技术困难（即高死亡率）经常使 D. melanogaster 幼虫模型难以使用。幼虫模型将有可能确定新的分子机制，这些机制将为宿主 – 病原体相互作用以及诱导针对致病性感染的特定宿主先天免疫反应提供进一步的见解。
这里详细描述了一种简单有效的方案，可用于向 D.黑色素食虫 幼虫注射各种病原体，例如细菌。特别是， D. melanogaster 幼虫用于注射人类病原体 Photorhabdus asymbiotica 和非致病性细菌 大肠杆菌。该方法可用于操作和分析 D. melanogaster对 各种微生物感染的免疫反应。
Protocol
1. 苍蝇饲养 注意：D. melanogaster的生命周期分为四个阶段：胚胎，幼虫，蛹和成虫。在实验室中具有最佳饲养条件（~25°C，60%湿度和足够的食物）的生成时间约为从受精卵到关闭成虫的10天。雌性每天产下约100个胚胎，胚胎发生持续约24小时22。幼虫经历三个发育阶段（幼虫;L1-L3）在~4天内（L1和L2：24小时，L3：48小时）。第一龄幼虫立即开始?…
Representative Results
如果操作正确，注射 D. melanogaster 幼虫显示出细菌特异性作用。在感染 无菌疟原 虫（ATCC43943株）、 大肠杆菌 （菌株K12）和PBS（图4）后的几个时间点收集生存数据。虽然 D. melanogaster 幼虫对 P. asymbiotica敏感，这会迅速损害生存，但注射 大肠杆菌 或PBS对照的幼虫表现出更长的存活率24，25…
Discussion
黑腹果蝇 是最有价值的实验操纵模型之一，用于研究各种微生物感染的先天免疫和发病机制。这是由于其简单而快速的生命周期，实验室中的简单维护，完善的进化遗传学和多样化的遗传工具箱。以前的 D. melanogaster 幼虫注射方法，例如使用混合微流体装置或Narishige显微操纵器，需要高度专业化的设备，并且可能很昂贵21，27。在目前的方?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢乔治华盛顿大学（GWU）生物科学系的成员对手稿的批判性阅读。GT通过GWU的Harlan夏季奖学金获得支持。所有图形图形都是使用BioRender制作的。
Materials
| Fly Food B (Bloomington Recipe) | LabExpress | 7001-NV | Food B, in narrow vials, 100 vials/tray |
| 100 x 15, Mono Petri Dishes Fully Stackable | VWR | 25384-342 | Diameter 100 x 15 mm |
| 60 x 15, Mono Petri dishes Fully Stackable | VWR | 25384-092 | Diameter 60 x 15 mm |
| Glass capillaries | VWR | 53440-186 | |
| Grade 1 qualitative filter paper standard grade, circle | VWR | 28450-150 | Diameter 150 mm |
| Lab culture Class II Type A2 Biosafety Safety Cabinet | ESCO | LA2-4A2-E | |
| LB Agar | Fisher Scientific | BP1425-500 | LB agar miller powder 500 g |
| LB Broth | Fisher Scientific | BP1426-500 | LB broth miller powder 500 g |
| Mineral oil | Alfa Aesar, Thermo Fisher Scientific | 31911-A1 | |
| NanoDrop 2000/2000c Spectrophotometer | Thermo Fisher Scientific | ND-2000C | |
| Nanoject III Programmable Nanoliter Injector | Drummond | 3-000-207 | |
| Narrow Drosophila Vials, Polystyrene | Genesee Scientific | 32-109 | |
| Needles, hypodermic | VWR | 89219-316 | 22 G, 25 mm |
| Next Generation Micropipette Puller | World Precision Instruments | SU-P1000 | |
| PBS | VWR | 97062-732 | Buffer PBS tablets biotech grade 200tab |
| Prism | GraphPad | Version 8 | |
| Syringes – plastic, disposable | VWR | 76124-652 | 20 mL |
| Trypan Blue | Sigma-Aldrich | T8154 |
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Citar este artigo
Tafesh-Edwards, G., Kenney, E., Eleftherianos, I. Drosophila melanogaster Larva Injection Protocol. J. Vis. Exp. (176), e63144, doi:10.3791/63144 (2021).