昆虫<em>大蜡螟</em作为一个强大的的感染模型探讨细菌致病
Summary
大蜡蛾幼虫的口腔内和haemocolic的感染<em>大蜡螟</em>进行说明。这种昆虫可以用于研究昆虫病原以及哺乳动物机会主义细菌的毒力因子。饲养的昆虫，感染的方法和实施例的<em在体内</em>分析说明。
Abstract
细菌毒力的研究，往往需要一个合适的动物模型。哺乳动物感染模型是昂贵的，并且可能引发的伦理问题。利用害虫感染模型提供了一个有价值的替代方案。相比其他无脊椎动物型号的主机，如线虫，昆虫有比较先进的抗菌防御系统，因而更容易产生哺乳动物感染过程中的相关信息。像哺乳动物，昆虫具有一个复杂的先天免疫系统1。血淋巴中的细胞能够吞噬或微生物入侵封装，和体液免疫应答的诱导生产的溶菌酶和小抗菌肽2,3。此外，昆虫的幼虫中肠哺乳动物的消化系统和肠道细胞的上皮细胞被发现之间的类比。最后，如细胞粘附，抗细菌感染的过程中必需的几个基本元件抗菌肽，组织退化和适应氧化应激是在昆虫和哺乳动物1可能是重要的。因此，昆虫是多价的工具，参与哺乳动物感染的微生物毒力因子的识别和表征。
已经示出的大蜡蛾蜡螟幼虫提供一个有用的洞察发病机制中的范围广泛的包括哺乳动物真菌（ 尖孢镰孢 ， 烟曲霉 ， 白色念珠菌 ）和细菌病原体的微生物感染，如金黄色葡萄球菌 ， 变形杆菌粘质沙雷氏菌绿脓杆菌 ， 单核细胞增生李斯特氏菌或粪肠球菌 4-7的 。无论是细菌种类，得到的结果与广场通过直接注射感染的幼虫通过角质层不断与那些相似LAR哺乳动物研究：在哺乳动物模型是减毒的细菌菌株，表现出较低的广场 ，并造成了严重的人类感染菌株的毒力也非常致命的广场模型8-11。口腔感染的广场是不常用的和额外的化合物，如特定的毒素，需要达到的死亡率。
蜡螟幼虫本几个技术的优点：它们是比较大的（末龄幼虫化蛹前约2厘米长，重250毫克），使定义的注射剂量的细菌，它们可以被饲养在不同温度下（20° C至30℃）和感染的研究可以进行15℃之间，以上述37℃，12,13，允许模仿的哺乳动物的环境的实验。此外，养虫很容易和相对便宜的。感染的幼虫可以通过以下几种方式，其中包括监测细菌的毒力LD 50 14克计算，测量的细菌存活15,16和检查感染过程17。在这里，我们描述了饲养的昆虫，覆盖所有生命阶段的G.大蜡螟 。通过两种途径接种：口腔内haemocoelic我们提供了一个详细的协议的感染。在该协议中使用的细菌模型是蜡状芽孢杆菌（Bacillus cereus），革兰氏阳性病原体在胃肠道，以及在其他严重的局部或全身的机会性感染18,19牵连。
Protocol
1。养虫末龄幼虫从卵到整个周期持续大约5个星期，在25°C。需要一个或2个星期获得成年蝴蝶。 将至少100蛹或新合并的成人G.中大蜡螟的蝴蝶在一个5升的金属丝网笼。雄性蝴蝶测量10到15毫米。成年的雄蛾是米色微弱的灯光和深色斑纹。约20毫米的女蝴蝶措施。女性比男性与棕色/灰色暗。 在产蛋笼换暂停四层两包纸。术后第2天，成年女性会下蛋的边缘之?…
Representative Results
细菌的内部haemocoelic注射到G.蜡螟已被证明是非常有用的许多毒力因子的识别处理与组织损伤和抗几种人类病原体的先天免疫因素。作为一个例子， 图2A表示B 的各种剂量注射后的昆虫死亡率蜡状芽孢杆菌菌（野生型和突变株）22。 图2B表示细菌感染后存活的G.蜡螟由铜绿假单胞菌 4。 使用口腔感染模型…
Discussion
的昆虫，特别是幼虫阶段，对多种致病菌的感染模型，使用频繁。一个模式的选择是果蝇的某些方面（飞行模式），用于成人和幼虫期1,2。鳞翅目昆虫G.蜡螟也已主要通过注射用于测定细菌的毒力。承受较高的温度（高于37℃）比果蝇 （最多25℃）的优点是很重要的，当哺乳动物病原体以进行研究。此外，大小广场更方便的感染动力学研究和组织损伤。最后，我?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们想，克里斯托夫·比松感谢伊丽莎白Guillemet的优秀的技术援助和Ludovic Bridoux。我们非常感激西尔维Salamitou和Sinda Fedhila的初始系统设置。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Wax and pollen | La Ruche Roanaise | 303000 | Any honey producer |
| Automated syringe pump | KD Scientific | KDS 100 | |
| Syringe 1 ml | Terumo | BS 01T | |
| Needle 0.45 x 12 mm | Terumo | NN 2613R | |
| Petri dish 5 cm | VWR | 89000-300 | |
| Needle 30G, 25 mm hypodermic | Burkard Mfg. Co. Ltd. | PDE0005 |
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Tags
Insect Galleria MellonellaBacterial PathogenesisInfection ModelAnimal ModelInsects As Infection ModelsNon-vertebrate Model HostsAntimicrobial DefensesInnate Immune SystemPhagocytosisEncapsulationHumoral ResponsesLysozymeAntibacterial PeptidesEpithelial CellsLarval MidgutsMammalian Digestive SystemsMicrobial Virulence FactorsGreater Wax Moth Galleria Mellonella
Cite This Article
Ramarao, N., Nielsen-Leroux, C., Lereclus, D. The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis. J. Vis. Exp. (70), e4392, doi:10.3791/4392 (2012).