Name: oral bacterial infection and shedding in drosophila melanogaster
Uploaded: 2018-05-31T14:45:00.000+00:00
Duration: 9 min 32 s
Description: Watch this Scientific Journal Video about Oral Bacterial Infection and Shedding in Drosophila melanogaster at JoVE.com

这项工作得到了来自免疫、感染和进化中心的康威信托基金 (http://ciie.bio.ed.ac.uk) 的一项战略奖的支持。PFV 得到了 Branco 维斯奖学金 (https://brancoweissfellowship.org/) 和总理奖学金 (爱丁堡大学生物科学学院) 的支持;JASJ 得到了 NERC E3 DTP 博士奖学金的支持。

1. 保养苍蝇
<ol><li>保持苍蝇在23毫升塑料瓶含有7毫升新鲜的刘易斯培养基 (修改从参考31; 1 升三蒸馏 H2O, 6.1 克琼脂, 93.6 克红糖, 68 克玉米, 18.7 克速溶酵母, 15 毫升 Tegosept 抗真菌剂) 在孵化器中在 25 1 °c, 在 12 h:12 h 光: 黑暗的周期以 ~ 60% 湿气。用不吸棉的羊毛塞住瓶子。</li>
	<li>每14天后, 将20–30成人转移到一个新的食品瓶, 用即时, 干酵母添加到表面, 2–3天, 允许产卵发生。在这个时间段之后, 确保鸡蛋在食物表面可见。移除成年苍蝇。 注意: 这会使小瓶中的苍蝇成为单代、年龄匹配的种群。</li>
	<li>让鸡蛋发育。 注: 在25摄氏度, 成年苍蝇开始 eclose 从蛹在11天, 并继续几天12–14。</li>
</ol>2. 准备实验苍蝇
<ol><li>在一个75毫升的苹果琼脂板 (1 升三重蒸馏 H2O, 收集在人口/胚胎收集笼中的母体后代的卵; 30 克琼脂, 33 克蔗糖, 330 毫升苹果汁, 7 毫升 Tegosept 抗真菌剂) 与酵母糊传播 (混合干酵母与水的花生黄油一样的一致性)。在笼中加入水浸棉绒, 以提供水分。 注意: 为了避免幼虫饲养密度差异引起的混杂效应, 在不同的瓶子中, 实验苍蝇在相似的密度下饲养是很重要的。执行上述步骤以避免混淆效果。</li>
	<li>孵化24小时在25°c 在 12 h:12 h 光: 黑暗的循环, 直到产卵已经发生。如果24小时后卵子过少, 则提供较长的适应期。更换苹果琼脂板, 并允许产卵再发生24小时。</li>
	<li>从人口笼中拿出富含鸡蛋的苹果琼脂盘子。从琼脂表面除去剩余的酵母膏和任何死苍蝇。</li>
	<li>将琼脂浸泡在20毫升的1x 磷酸盐缓冲盐水 (PBS) 中, 用细画笔轻轻地将鸡蛋从苹果琼脂中取出。当悬浮在 PBS, 转移鸡蛋到一个50毫升离心管和离开5分钟, 所以鸡蛋下沉到底部。 注: 大多数鸡蛋是在琼脂的外边缘发现的。</li>
	<li>通过切割 p1000 过滤的吸管尖端的底部4毫米, 并使用吸管尖端绘制1毫升的溶液, 从50毫升离心管底部取出。把这个转移到1.5 毫升离心管, 让它安定下来。 注意: 当吹打蛋时, 快速释放柱塞比温和的释放更有效。</li>
	<li>通过切割 p20 过滤的吸管尖端的底部4毫米删除。将吸管设置为所需的体积并从离心管底部抽出。 注: 在实践中, 5 µL 的体积包含大约100个卵。</li>
	<li>把收集到的鸡蛋放到食物上, 让它们在所需的时间内发展。</li>
</ol>3. 细菌培养
<ol><li>要种植entomophila和绿脓杆菌区域性, 请分别接种10毫升的 Luria-Bertani (磅) 汤, 其100µL 冷冻细菌储存在30摄氏度 (p entomophila)和37°c (p. 铜绿假单胞菌)。一夜之间摇晃150转。确保细菌培养达到饱和阶段。</li>
	<li>为了确保用于接种苍蝇的细菌处于指数阶段, 迅速复制, 将隔夜文化接种到一个新的亚文化中, 在第二天早上就能达到预期的体积。确保前接种是亚文化的总容量的10%。 注意: 口腔感染需要高。因此, 有必要增加大量的细菌培养, 以便能够为所需剂量和实验尺寸生产足够的接种培养。计算使用等式 msV = M iVi来生成所需传染性剂量需要多少亚文化, 其中 m 表示以 600 nm (OD600) 值测量的区域性光学密度, V 代表其体积。下标字母指的是作为亚文化或传染性剂量 (i) 的区域性。</li>
	<li>将这个亚文化生长在一个体积为2升的锥形烧瓶中, 这样, 亚文化的表面就会下降 (最多) 就在烧瓶的斜坡的起始处。不要在这个标记上面填充, 因为它会阻碍细菌的生长。</li>
	<li>通过测量每30分钟的 OD, 确保该亚文化中的细菌处于指数生长阶段。 注意: 这发生在 3–5 h 之后, 其中亚文化在0.6–0.8 之间达到 OD600 。</li>
	<li>在50毫升离心管上倒入同样数量的亚文化, 在 2500 x g 处旋转亚文化为15分钟, 在4摄氏度时将细菌颗粒化。一旦颗粒化, 取出后再旋转上清, 在上述条件下确认清除绝大多数细菌。 注: 可忽略大小的颗粒 (高度小于1毫米) 证实了这一点。</li>
	<li>将分离管中的细菌小球重新悬浮在5毫升亚文化上清, 并在单个50毫升管中重组这些溶液。以 2500 x g 为15分钟, 在4摄氏度旋转这种浓缩的培养细菌。</li>
	<li>取出上清液, 并在5% 蔗糖水溶液中重新悬浮最后的细菌颗粒。检查 od 并调整到所需的传染性剂量 (od600 = 100 为p. entomophila8和 OD600 = 25 P. 铜绿假单胞菌16,28), 通过重新悬浮小球在5%蔗糖水溶液达到所需体积。 注: 可使用步骤3.2.1 中的等式 (msVs = MiVi) 计算要添加的5% 蔗糖水溶液的数量。</li>
</ol>4. 口腔感染蝇类
<ol><li>为了确保口腔感染, 在接触细菌之前将苍蝇饿死 2–4 h, 将苍蝇转移到标准琼脂瓶 (1 升三重蒸馏 h2O, 20 克琼脂, 84 克红糖, 7 毫升 Tegosept 抗真菌剂)。</li>
	<li>在苍蝇饿死的时候准备感染瓶。通过将标准糖琼脂的500µL 吹打到7毫升样品管的盖子上, 使铜绿假单胞菌感染小瓶并使其干燥。将一碟滤纸放在盖子上, 将100µL 细菌培养液直接放到过滤盘上。为了控制感染, 在滤纸上用相同体积的5% 蔗糖水溶液取代细菌培养。</li>
	<li>添加单飞到样品管和离开 18–24 h。</li>
	<li>为了确认口腔感染, 首先在细菌暴露后立即对苍蝇进行表面杀菌, 将其放置在100µL 70% 乙醇中 20–30. 在除去水之前, 将乙醇取出并添加100µL 的三重蒸馏水。添加100µL 1x PBS 和融汇的苍蝇。</li>
	<li>将匀浆输送到96井板的顶排, 并将90µL 的 1x PBS 添加到下面的每一个井中。</li>
	<li>连续稀释此样本以区分 CFU 值的范围。将匀浆的10µL 在顶井中, 并将其添加到下面的井中。重复此步骤与第二个井, 转移10µL 到第三井, 等等, 为尽可能多的串行稀释的要求。 注意: 重要的是, 新的吸管提示用于每组稀释。</li>
	<li>在5µL 液滴上, 将系列稀释在 LB 营养琼脂板上, 以确保所有水滴保持离散。</li>
	<li>分别在30°c 和37°c 上孵化出 LB 琼脂盘, 用于p. entomophila和铜绿假单胞菌, 并计数可见 CFUs。 注意: 虽然果蝇肠道微生物需要不同的厌氧生长条件, 但选择性培养基 (如铜绿假单胞菌隔离培养基 (PIM)) 可用于确保只计算假单胞菌CFUs。</li>
	<li>计算每只苍蝇的 CFUs 数, 计数在 10–60 CFUs 明显可见的序列稀释中存在的菌落数。然后乘以稀释因子的存在来计算每只苍蝇的细菌数量。</li>
	<li>进行统计分析。如有必要, 将 CFUs 每飞转换为正常分布。通过日志转换来完成此过程。一旦转换, 使用广义线性模型 (GLMs)30,31,32测试处理组每飞 CFUs 的差异 (使用常用的统计软件包 (如 R33))。 注: 剩余的苍蝇匀浆可用于定量反向转录 PCR (qPCR) 分析测定基因表达。在50µL 的 rna 隔离试剂中, 提取 rna, 并通过 RT-qPCR (参见例如, 古普塔和淡水河谷16来确定详细的协议) 来固定匀浆。具体免疫基因转录的表达应规范化到管家基因的转录水平 (即, rp49), 并表示为一个折叠变化相对于控制苍蝇使用 2−ΔΔCt方法31, 32,33,34。</li>
</ol>5. 感染后的生存记录
<ol><li>如步骤4.2 所述, 口服苍蝇。</li>
	<li>转移感染或控制苍蝇从他们各自的感染小瓶成标准的刘易斯瓶, 并保持在一个孵化器在25°c 在 12 h:12 h 光黑暗周期 (或期望的条件)。保持苍蝇, 直到他们死。</li>
	<li>每天计算每瓶中的活蝇或死苍蝇的数量, 或按需要经常计数。</li>
	<li>每5天将苍蝇转到新瓶子, 以避免苍蝇卡在食物中。</li>
	<li>将这些数据呈现为卡普兰-梅尔 (公里) 生存曲线或平均值的生存地块。要分析几个因素和/或它们各自相互作用的影响, 请使用一个统计包 (例如, R33中的包 "生存") 来运行生存分析, 如 Cox 比例危险模型35。</li>
</ol>6. 测量细菌负荷
<ol><li>在所需的时间点, 将一只受感染的苍蝇转移到不育的1.5 毫升离心管。</li>
	<li>表面消毒苍蝇如步骤4.4 所述。</li>
	<li>使用步骤4.5–4.10 中描述的协议, 融汇苍蝇并量化细菌负荷。</li>
</ol>7. 测量细菌脱落
<ol><li>测量与内部负载一起脱落。</li>
	<li>感染后, 转移单飞到1.5 毫升的离心管含有50µL 的刘易斯培养基24小时。</li>
	<li>卸下用于内部负载测量的苍蝇 (见步骤 6), 用100µL 1x PBS 冲洗管子, 涡流3秒。</li>
	<li>使用步骤4.6–4.8 中描述的相同协议, 测量 CFUs 在 LB 营养琼脂上的电镀。</li>
	<li>感染后, 转移单飞到1.5 毫升的离心管含有50µL 的刘易斯培养基24小时。</li>
	<li>将苍蝇转移到新的离心管, 其中含有50µL 的刘易斯培养基, 进一步 24 h. 用100µL 1x PBS 清洗受污染的管子, 涡流严重的3秒。</li>
	<li>使用步骤4.6–4.8 中描述的相同协议, 在 LB 营养琼脂上进行电镀, 测量 CFUs。</li>
	<li>重复步骤7.2 和7.3 并记录每次转移时的飞行死亡率。</li>
</ol>

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我们提出了一个可靠的口服感染的细菌病原体的D.腹腹病毒的协议。我们专注于铜绿假单胞菌和entomophila, 但该协议可以很容易地被修改, 以使其他细菌种类的感染,例如,沙雷质7.本议定书的关键方面将因细菌种类而异。因此, 最有效的感染剂量, 相应的毒力, 和宿主基因型易感性, 应考虑和理想的试验研究。将苍蝇暴露在各种光学密度的细菌培养中, 并测量其传染性剂量和存活率, 是与新的细菌物种或飞行线合作的适当起点。
在5% 蔗糖溶液喂养和重新悬浮细菌颗粒之前, 诸如苍蝇饥饿等协议步骤在口腔感染中司空见惯, 并提高了暴露期间细菌感染的可靠性7,8,9,10. 然而, 重要的是要注意, 在暴露期间, 苍蝇基本上生活在细菌培养的表面上。在这种文化的行走过程中, 细菌将会被放在苍蝇的表面上, 特别是在角质层或刚毛的24附近。这些表皮细菌, 不反映成功的肠道感染, 但仍将检测到的飞行均匀化和电镀。为了减少误报的可能性, 必须通过浸泡在70% 乙醇中对苍蝇进行表面杀菌, 以达到1分钟。
在考虑细菌脱落率时, 口腔感染是必不可少的。主机发布到环境中的病原体数量通常很难测量, 内部负载通常被视为感染严重性的代理, 因此传输2627。在细菌脱落的同时测量细菌负荷, 可以检查疾病严重性这两个重要组成部分之间的关系, 并进行38的传播。所提出的方法的一个局限性是, 测定苍蝇的内部细菌负荷需要破坏性抽样。这使得在同一个人中调查病原体生长和清除的纵向趋势变得困难。然而, 可以通过破坏性地抽样在不同感染阶段的个体群来克服这一限制, 假定每个队列中的平均微生物负荷反映了任何给定的纵向病原体动力学。个人。细菌脱落并没有受到同样的限制, 我们提供了如何在横断面样本中量化脱落的例子, 或者是纵向研究在一段时间内脱落的变化。
许多宿主和病原体特征共同决定了个人传播疾病的倾向25,26,39。虽然这些性状的意义可能不同于寄主病原体系统, 但脱落可能是粪便-口服传播的主要决定因素。测量细菌脱落的能力打开了测试这个假设的机会。实验者在所需的飞行线组中具有寄主病原体动力学特征, 可以对个体进行口服感染, 并在感染期间将其与未感染的敏感寄主一起放置。然后, 这些 ' 受体 ' 苍蝇可以检测的内部细菌负荷在不同的时间点作为直接测量传播的方式。

果蝇 (也称为醋蝇), D.黑腹, 已被广泛用作感染和免疫的模型有机体的各种病原体1,2。这项工作提供了对感染的生理后果的基本洞察力, 也开创了解开宿主免疫应答的分子路径寄生蜂, 细菌, 真菌和病毒感染。这种知识不仅有助于了解昆虫和其他无脊椎动物的先天免疫反应, 而且由于许多免疫机制在昆虫和哺乳动物之间进化保存, 因此,果蝇也促使发现哺乳动物的主要免疫机制, 包括人类的3。
大多数关于果蝇感染和免疫的工作都集中在全身感染上, 使用接种方法, 通过刺或注射4,5,6,将病原体直接送到昆虫体内。这些方法在允许分娩受控的传染性剂量方面的优势是明确的, 并得到了大量的全身性感染工作的支持。然而, 许多自然发生的细菌病原体的D.腹腹是通过喂养分解有机物, 在那里肠道活性发挥重要作用的主机防御7,8,9,10,11,12,13,14,15. 使用全身感染的实验绕过这些防御, 因此, 提供了一个完全不同的图片, 如何对昆虫安装防御的自然病原体。如果这项工作的目的是测试对感染的生态学和进化的预测, 那么使用自然病原体和感染途径是重要的16、17, 这一点尤其相关。最近的工作突出了病原体的路线如何对疾病结果产生重大影响18,19, 引出不同的免疫通路20,21, 可以确定的保护效果继承的 endosymbionts16, 甚至可能在主机防御的演化过程中扮演重要角色17。
使用口服感染途径的另一个原因是, 它允许通过测量口腔感染后粪便排泄过程中的细菌脱落来调查病原体传播的变化22,23, 24. 了解疾病传播中宿主异质性的来源在自然种群中具有挑战性25,26, 但在受控实验室中测量传播的成分, 如病原体脱落。条件提供了一种有用的替代方法27。通过在受控实验条件下, 通过喂养苍蝇细菌和测量细菌脱落的各种遗传和环境背景, 可以确定寄主之间传播的变异来源。
在这里, 我们描述了一个口头感染D.腹腹病毒与细菌病原体的协议, 并对细菌的生长和脱落进行量化 (图 1)。我们描述了两个铜绿假单胞菌的这个协议: 一个致命的菌株的机会病原体p. 绿脓杆菌(PA14), 和一个较小的毒株自然蝇病原体P. entomophila。Pseudomonads 是常见的革兰阴性菌, 具有广泛的寄主范围, 感染昆虫, 线虫, 植物和脊椎动物, 并在大多数环境中发现4,6。果蝇的肠道感染由铜绿假单胞菌和P. entomophila导致病理到肠道上皮细胞12,13,14,15, 28。虽然我们关注这两种细菌病原体, 这里所描述的方法原则上可以适用于任何细菌病原体的轻微修改。在口腔暴露后, 我们测量感染后的生存, 并测量的微生物负荷在个别苍蝇和可行的微生物流入环境中, 表示在菌落形成单位 (CFUs)。最后, 由于肠道活性的结果是上皮屏障和体液反应的结合, 我们也测量的生存, 这些防御中断的飞行线。具体地说, Drosocrystallin (Dcy)变种人以前被证明更容易受到口腔细菌感染, 原因是肠道中的 peritrophic 基质已耗尽29。我们还测量在一个津津有味 (相对) 突变体的生存, 这是阻碍了生产抗菌肽抗革兰阴性菌通过 IMD 路径30。

<table><tbody><tr><td>Vials</td><td>Sarstedt Ltd.</td><td>58.490</td><td>Polystyrene flat base tube 75mm x 23.5mm, 23ml</td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A7002</td><td>Agar, ash 2.0-4.5%</td></tr><tr><td>Brown sugar</td><td>Bidvest</td><td>66032</td><td>Light brown soft sugar</td></tr><tr><td>Maize</td><td>Dove&#039;s Farm</td><td></td><td>Organic maize flour</td></tr><tr><td>Fermipan yeast</td><td>Bidvest</td><td>96360</td><td>Dry, instant yeast</td></tr><tr><td>Methyl-4-hydroxybenzoate</td><td>Sigma-Aldrich</td><td>H5501</td><td>&gt;=99.0%, crystalline</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84097</td><td>Sucrose BioUltra, for molecular biology, &gt;=99.5% (HPLC)</td></tr><tr><td>Petri dishes</td><td>Fisher Scientific UK Ltd.</td><td>15788517</td><td>X600 Petri Dish 90 X 16.2MM Sterile Triple Vent</td></tr><tr><td>Falcon tubes (50ml)</td><td>Greiner Bio-one Inc</td><td>210261</td><td>Centrifuge tube, 50ml, skirted, bagged, sterile</td></tr><tr><td>Eppendorf tube (0.5ml)</td><td>Sarstedt Ltd.</td><td>72.699</td><td>Sarstedt Micro Tube Conical Base Push Cap 0.5ml Standard Neutral</td></tr><tr><td>Eppendorf tube (1.5ml)</td><td>Sarstedt Ltd.</td><td>72.690.001</td><td>Sarstedt Micro Tube Conical Base Push Cap 1.5ml Standard Neutral</td></tr><tr><td>Ethanol</td><td>VWR International Ltd.</td><td>20821.33</td><td>ETHANOL ABSOLUTE ANALAR NP ACS/R.PE - Analytical Grade</td></tr><tr><td>2 L Conical Flask</td><td>VWR International Ltd.</td><td>214-0038</td><td>Narrow neck, 2 L Erlenmeyer flask</td></tr><tr><td>Sterile filter paper</td><td>Fisher Scientific UK Ltd.</td><td>1001-020</td><td>Plain circle and sheets; Particle Retention: greater than11um; Filtration speed: 150 herzberg; Air flow: 10.5s/100mL/in2; Medium porosity; Smooth surface; Grade 1; Type: circle; Dia: 20mm</td></tr><tr><td>Bijou sample container</td><td>Fisher Scientific UK Ltd.</td><td>129A</td><td>7ml polystyrene sample container</td></tr><tr><td>Pseudomonas isolation agar</td><td>Sigma-Aldrich</td><td>17208</td><td>Contains agar 13.6 g/L, magnesium chloride 1.4 g/L, peptic digest of animal tissue 20g/L, potassium sulfate 10g/L, triclosan 0.025g/L</td></tr><tr><td>LB broth, Miller</td><td>Fisher Scientific UK Ltd.</td><td>BP1426</td><td>Contains 10g tryptone, 5g yeast extract, 10g sodium chloride per litre</td></tr><tr><td>Large Embryo Collection Cages</td><td>Scientific Laboratory Supplies Ltd.</td><td>59-101</td><td>Flystuff- fits 100mm petri dish</td></tr><tr><td>TRI reagent solution</td><td>Life Technologies</td><td>AM9738</td><td></td></tr><tr><td>96-well microplate</td><td>Scientific Laboratory Supplies Ltd.</td><td>353072</td><td>Falcon 96 Well Clear Flat Bottom TC-Treated Polystyrene Cell Culture Microplate with Lid Sterile</td></tr><tr><td>Pestle</td><td>Fisher Scientific UK Ltd</td><td>12649595</td><td>Pestle, Presterilized; Axygen; Tissue grinder; Blue; Inert polypropylene construction; Fits 1.5 and 2.0mL centrifuge tubes; Individually wrapped; 100/Pk</td></tr><tr><td>Glycerol</td><td>Scientific Laboratory Supplies Ltd.</td><td>CHE2068</td><td>Glycerol A.R. 99.5% 2.5 L</td></tr><tr><td>Cotton wool- non absorbent</td><td>Cowens Ltd</td><td>ABL</td><td>Non Absorbent Large quantity 20 bags x 500</td></tr><tr><td>Cotton wool- absorbent</td><td>Cowens Ltd</td><td>ABS</td><td>BP small quantity 20 bags x 500</td></tr><tr><td>Vortex</td><td>Fisherbrand</td><td></td><td>Whirlimixer 75W 50-6-Hz 220-240V</td></tr><tr><td>Orbital incubator</td><td>Gallenkamp</td><td>INR-200-010V</td><td>220 V, 50 Hz, 5 A. Nominal temperature: 70&amp;ordm;C. Shaking frequency: 0...400 rpm.</td></tr><tr><td>Absorbance Microplate Reader</td><td>Biotek Instruments Ltd</td><td></td><td>ELx808&amp;trade; Absorbance Microplate Reader</td></tr><tr><td>Gen 5 Microplate Reader and Imager Software</td><td>Biotek Instruments Ltd</td><td></td><td>For Absorbance Microplate Reader</td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>392304</td><td>Allegra X-12R Benchtop Centrifuge, refridgerated 50Hz 230V</td></tr><tr><td>Step One Plus Real Time qPCR System</td><td>Thermofisher Scientific</td><td>4376600</td><td>Applied biosystems Step One Plus real time qPCR system</td></tr><tr><td>Step One Software 2.3</td><td>Thermofisher Scientific</td><td></td><td>For Stepone and SteponePlus real time qPCR systems</td></tr><tr><td>R Statistical Software</td><td>https://cran.r-project.org/</td><td></td><td></td></tr><tr><td>10 &amp;micro;L pipette tips</td><td>Greiner Bio-one Inc</td><td>741015</td><td>Easlyload gilson-style. Graduated, clear, refill, 960 pcs</td></tr><tr><td>200 &amp;micro;L pipette tips</td><td>Greiner Bio-one Inc</td><td>741065</td><td>Easlyload gilson-style. Graduated, clear, refill, 960 pcs</td></tr><tr><td>1000 &amp;micro;L pipette tips</td><td>Greiner Bio-one Inc</td><td>741045</td><td>Easlyload gilson-style. Graduated, clear, refill, 960 pcs</td></tr><tr><td>20 &amp;micro;L filtered pipette tips</td><td>Greiner Bio-one Inc</td><td>774288</td><td>Filter tip gilson-style. Clear, rack blue, single packed, R/Dnase free, 10 racks of 96pcs</td></tr><tr><td>200 &amp;micro;L filtered pipette tips</td><td>Greiner Bio-one Inc</td><td>739288</td><td>Filter tip gilson-style. Clear, rack blue, single packed, R/Dnase free, 10 racks of 96pcs</td></tr><tr><td>1000 &amp;micro;L filtered pipette tips</td><td>Greiner Bio-one Inc</td><td>740288</td><td>Filter tip gilson-style. Clear, rack blue, single packed, R/Dnase free, 10 racks of 60pcs</td></tr></tbody></table>

oral bacterial infection and shedding in drosophila melanogaster

果蝇是一种最发达的感染和先天免疫模型系统. 虽然大多数工作集中在全身感染, 最近有兴趣的机制肠道活性的病原体, 这需要方法, 口服感染苍蝇。在这里, 我们提出一个口头揭露个人苍蝇的机会细菌病原体 (铜绿假单胞菌) 和一个自然细菌病原体D... 本议定书的目的是提供一种可靠的方法, 以揭露男性和女性苍蝇对这些病原体。我们提供了具有代表性的结果, 显示生存表型, 微生物负荷和细菌脱落, 这是有关研究的异质性的病原体传播。最后, 我们确认Dcy突变体 (缺乏肠道上皮中的保护性 peritrophic 基质) 和津津乐道突变体 (缺乏功能性免疫缺陷 (IMD) 通路), 显示了细菌口腔感染的敏感性增加。因此, 本议定书描述了一种通过口服感染途径感染苍蝇的健壮方法, 可扩展到对肠道感染结局和细菌传播的各种遗传和环境来源的研究。

在这里, 我们提出了实验的说明结果, 其中D.腹腹被口服感染了铜绿假单胞菌或p. entomophila。 图 2演示了在12小时或24小时暴露时间后, 在p. 铜绿假单胞 (图 2A) 和p. entomophila (图 2B ) 的细菌培养中, 苍蝇的口腔感染是否成功。、C) 分别。图 2B说明了使用更集中的P. entomophila的区域性的重要性, 如在苍蝇接触到更大的光学密度的细菌培养物时细菌负荷的增加。男性和女性俄勒冈州 R (不再回应买主) 以相同的速率(图 3)清除铜绿假单胞菌感染, 并流出相同数量的 P . 铜绿假单胞 CFUs(图 4A)。但是, 当感染entomophila时, 雄性和雌性不再回应买主苍蝇在细菌脱落的数量上不同, 这种方式随时间而变化(图 4B)。男性和女性死于铜绿假单胞菌(图 5A) 和P. entomophila (图 5B) 以不同的速率死亡。我们还看到, Dcy突变体 (缺乏肠道上皮中的保护性 peritrophic 基质) 和津津乐道突变体 (缺乏功能的 IMD 免疫通路), 显示在p. entomophila和铜绿假单胞菌后存活率下降口腔感染 (图 5C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57676/57676fig1.jpg"> 图 1: 在果蝇的口腔感染后测量生存、脱落和内部细菌负荷的协议示意图.一例3的潜在实验, 在口腔感染后, D.通过将单只苍蝇转移到瓶子并记录感染的寿命来测量 "生存"。测量 ' 脱落 ' 通过转移单飞到1.5 毫升离心管与50µL 的刘易斯介质在 cap。在管24小时后, 用100µL 1x PBS 去除苍蝇和涡流管。去掉并板上这一溶液的 LB 营养琼脂, 以计算细菌脱落。在相同的苍蝇纵向测量脱落, 通过转移苍蝇到新鲜管与刘易斯介质在帽24小时后, 并清洗和电镀现在被污染的管。一只苍蝇的 "内部负荷" 可以通过服用受感染的苍蝇, 表面杀菌, 并将其均匀化, 最后在 LB 营养琼脂上电镀匀浆。这可以执行后, 脱落已经测量, 以计算如何 ' 内部负荷 ' 和脱落的关联。此图中使用的苍蝇插图最初是由 b Nuhanen36绘制的。作者修改了它伴随的例子卡普兰-Meier 曲线, 这是取自维基共享37。所有其他插图都是原创的。<a href="/files/ftp_upload/57676/57676fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57676/57676fig2.jpg"> 图 2: 口腔感染后感染的细菌剂量。(A)在接触到铜绿假单胞菌养殖 (OD600 = 25) 的情况下, 在 12 h 的情况下, 对俄勒冈州-R 蝇的传染性剂量。平均和 SE 由3个男性和3位女性计算。(B)在接触到四个entomophila区域性 (OD600 = 100、75、50和 25) 或控制 5% h 的蔗糖溶液后, outcrossed 野生型雌性的传染性剂量。暴露治疗之间的感染剂量 (F376 = 18.567、 p < 0.001) 的统计差异由不同的字母在条形上表示。从5只苍蝇中计算出的方法为 OD600 = 0 剂量, 18-20 用于所有其他剂量。(C)在暴露于P. entomophila区域性 (OD600 = 100) 之后, 男性和女性的俄勒冈州 R 蝇的传染性剂量为 24 h。平均和 SE 由20个男性和20位女性计算。<a href="/files/ftp_upload/57676/57676fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57676/57676fig3.jpg"> 图 3: 口腔感染后苍蝇的内部铜绿假单胞菌负荷。在口腔感染后, 男性和女性的俄勒冈-R 的细菌负荷的平均值为铜绿假单胞菌(OD600 = 25), 感染后168小时。每个时间点的平均值和 SE 由3个人计算。一只苍蝇的内部细菌负荷随着时间的推移显著变化 (p < 0.001)。<a href="/files/ftp_upload/57676/57676fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57676/57676fig4.jpg"> 图 4: 口腔感染后的细菌脱落。(A) P. 铜绿假单胞菌由相同的苍蝇所使用的图 3, 高达 120 h 后感染。平均和 SE 由3个男性和3位女性计算。(B) p. entomophila由男性和女性在entomophila (OD600 = 100) 的口腔感染后所流出的, 由 120 h 后感染引起。平均和 SE 由34个男性和38位女性计算。对于铜绿假单胞菌和entomophila, CFUs 的数量随着时间的推移而显著变化 (P < 0.001)。<a href="/files/ftp_upload/57676/57676fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57676/57676fig5.jpg"> 图 5: 细菌性口腔感染后苍蝇的存活.卡普兰-梅尔 (公里) (A)的生存曲线在口服感染与铜绿假单胞杆菌(OD600 = 25) 或控制5% 蔗糖溶液后, 俄勒冈州 R 的雄性和雌性苍蝇。公里生存曲线是从每治疗组4瓶20只苍蝇计算出来的。(B)不再回应买主在口腔感染后与 P . entomophila (OD 600 = 100) 后的男性和女性苍蝇。从4只单控制蝇和34只受感染的苍蝇中, 对雄性和雌性进行了公里生存曲线的计算。(C)免疫突变体: Dcy (Drosocrystallin-peritrophic 矩阵突变体) 和相对 (津津有味-IMD), 它暴露于p. entomophila (Pe), p. 铜绿假 (Pa14), 或控制5% 蔗糖溶液. 所有感染组的死亡速度明显高于控制苍蝇 (p < 0.001)。<a href="/files/ftp_upload/57676/57676fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Oral Bacterial Infection and Shedding in Drosophila melanogaster at JoVE.com

Oral Bacterial Infection and Shedding in Drosophila melanogaster

本议定书描述了用细菌病原体口服和感染果蝇果蝇 melanogaster 的方法, 并测定肠道感染后感染细菌的数量。进一步描述了免疫突变体对口腔细菌感染后蝇存活的影响。

果蝇的口腔细菌感染和脱落

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

oral-bacterial-infection-and-shedding-in-drosophila-melanogaster

Research

JoVE Journal

Biology

Oral Bacterial Infection and Shedding in Drosophila melanogaster

11.5K 次观看.  University of Edinburgh. 本议定书描述了用细菌病原体口服和感染果蝇果蝇 melanogaster 的方法, 并测定肠道感染后感染细菌的数量。进一步描述了免疫突变体对口腔细菌感染后蝇存活的影响。

视频: Oral Bacterial Infection and Shedding in Drosophila melanogaster

Wolbachia Bacterial Infection in Drosophila

My name is Voia Friedman. I am associate researcher at Princeton University. And today I'm going to tell you about a few protocols that I developed to trans infect, to I initially to isolate, to go back from the heli of infected flies, and later on help to inject the heli feed to unaffected flights.So basically, this is a very simple protocol to isolate him oly for basics. And you will use this device from, from mepo that contains a filter that goes on top of an E.So you basically needs to put you, we start from around a hundred flies. You anize the flies, you put them into the filter device, you can just use a funnel and that will add our other fit very easily on this device and wash it with 70%ethanol.You just take the, take the future away, you can throw away the ethanol, and then after three times your last wash you do with molecule water because you don't want have the bacteria from the, with traces of ethanol. Well, and then you just go to a new weapon door and, and then you spin down on a micro, real micro for two minutes And later on how you can inject that. He only feed do unaffected flies.So basically take a double stick tape and usually these double stick tapes have two side, one side is a sticker than the other. So leave the sticker side up because that's the side that you're gonna glue that flies on top of it. And basically you'll, the gas apparatus, if you have this light on top of it, you'll be no gas flow and the flies you wake up if they're on top of the slide.So you can just direct the flow, making a little hood, a little chamber using tape. Then you direct the gas over the flies. So then you put your, this light here with the tape and you, you have your flies.Stick the flies on the top of the tape, all of them the same orientation, and put their head towards the direction that you'll be bringing the needle from. Because then you can just over the, the wing, you make sure since they fly, you don't want to fly. If they woke up during the procedure, don't want them to go away.So make sure that you take a little tip and pressure against the, the, the wings of the fly on the tail. And it's okay if they wings get damaged. And when you get rid, when you take the flies up the injection, most likely they'll lose their wings anyway, so they'll survive.That can, that can be useful because you're not gonna lose the flies that, so he puts a mineral oil on the micrometer for the back of the needle, then the needle injection polar, and then the, you can just adjust the pressure to bring the material up to the tip of the needle. I, I did to inject Somewhere between 10 to 15 nanoliters, but as, as long, as long as I'm consistents. Okay, so one important detail there is a cushion of this experiment is that the, the fly, once the apply is being injected when you saw the needle needs to come as far as you can through the upper part of the abdomen.Because here, basically when the needle penetrates, you have a, a, a, a, a bigger space to go up and down. And basically if it's close, closer to the cuticle, it's less likely they're gonna penetrate vital organs in the flight. You, it is very likely unlikely that you're gonna punch a hole in your gut, for instance, if that is gonna kill the flight that you are performing the surgery.My name is Voia Friedman. I am associate researcher at Princeton University in the lab of Eric Hels. And today I would like to tell you about this protocol that you just saw, the demonstration.So this protocol was done because we wanted to prove that UBA is capable of crossing tissues. So UBA is one of the most successful intracellular paras on the face of the earth. Infects from 20 to 70%of insects and many particles.And also is, is very important in terms of human diseases because also infect nematodes that transmit diseases like river blindness or s and ssis. And so, and CCA is our obligatory endo in, in these worms. And there's a great, a very big interest in using CCA also as means to control parasitic diseases transmitter by mosquito and Danny and this and by mosquito such as malaria and dengue.So this, this, this protocol basically was developed to show that once CCA inside the abdominal cavity of a fly, they can cross tissues and reach the germ. So basically what you can see here is the oph of the drosophila ovary and go back once present in the he leaf it, it can cross a pink peronial. She that is around the ovary.And then they cross a muscle layer. And actually one of the first places that they, they go inside the ovary is the somatic stem cell image. And then they can reach the germline.And this bacteria is transmitted the same way that mitochondria is transmitted is through, is through the mother transmitted, through the mother cyop plants present in the egg. So of course they needs to be present into the germline. So that's why this protocol is developed.And this protocol is, can be very useful to basically traditionally the way that people would try to trans infect wbaa basically take wbaa and introduce a new insect species. It would be through doing micro injections into the, into the embryo. But those are much more complicated.And basically here we are just taking advantage of the o biology since they are capable to cross tissues and moving into the stem cell tissues from, they can pass to the, through to the germline. So that's make easier taking UBA from certain, from, from certain species of insects that are infected and they can be transmitted to uninfected species. Prob I think it's the injection itself.Basically you need to be careful to mobilize the fly as well as you can. We are using double sided stick tape and you need to penetrate the needle basically as close to the, as parallel to the fly as possible so you don't go to deep. And it's less likely that we're gonna kill the fly after they're performing the surgery.

在果蝇的Wolbachia细菌感染

科研

生物学

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

饲养果蝇<em&gt;果蝇</em&gt;在无菌和悉生条件

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

发育生物学

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

全身细菌感染与免疫防御的表型<em&gt;果蝇</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

免疫与感染

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

从果蝇幼虫中提取包囊微生物感染及分析

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

果蝇、果蝇细胞免疫应答的评估

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Drosophila melanogaster Larva Injection Protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly and raise ethical issues. Unconventional models are notoriously cheap, easy to handle and culture, and do not take much space. They are genetically amenable and possess complete genome sequences, and their use presents no ethical considerations. The fruit fly Drosophila melanogaster, for instance, has provided great insights into a variety of behavior, development, metabolism, and immunity research. More specifically, D. melanogaster adult flies and larvae possess several innate defense reactions that are shared with vertebrate animals. The mechanisms regulating immune responses have been mostly revealed through genetic and molecular studies in the D. melanogaster model. Here a novel larval injection technique is provided, which will further promote investigations of innate immune processes in D. melanogaster larvae and explore the pathogenesis of a wide range of microbial infections.

Drosophila melanogaster Larva Injection Protocol

Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

果蝇 幼虫注射方案

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly ...

Infecting Flies with Bacterial Suspension Using a Nanoinjector

Source: Khalil, S. et al., <a href="https://app.jove.com/v/52613">Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster.</a>&#160;J. Vis. Exp.&#160; (2015)
This video demonstrates the nano-injection of bacterial suspension into the fly body cavity, resulting in bacterial multiplication and infection. The fly&#39;s immune response clears some bacteria, while others escape immune clearance and survive within the host causing an infection.

Source: Khalil, S. et al., Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster.&#160;J. Vis. Exp.&#160; (2015)
This ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host-Pathogen Interaction

A Septic Pinprick Technique to Introduce Systemic Bacterial Infection in Flies

Source: Khalil, S., et al. <a href="https://app.jove.com/v/52613">Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster.</a> J. Vis. Exp. (2015)
This video demonstrates a technique to infect Drosophila by introducing a pathogenic bacteria directly into their body cavity. This method bypasses epithelial barriers and other passive defense mechanisms, allowing for the induction of systemic infection. Upon infection, the resulting immune response is assessed by monitoring fly mortality and performing downstream assays.

一种在果蝇中引入全身性细菌感染的脓毒性针刺技术

Source: Khalil, S., et al. Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster. J. Vis. Exp. (2015)
This video ...

Measuring E. coli Bacterial Load in Drosophila melanogaster following E. coli Infection

Source: Khalil, S., et al. <a href="https://app.jove.com/v/52613">Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster.</a> J. Vis. Exp. &#160;(2015)
In this video, we demonstrate a technique for measuring the E. coli bacterial load after injecting E. coli into the thorax of Drosophila melanogaster.

测量大肠杆菌感染后黑腹果蝇中的大肠杆菌细菌载量

Source: Khalil, S., et al. Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster. J. Vis. Exp. &#160;(2015)
In this video, ...

果蝇的口腔细菌感染和脱落

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在果蝇的Wolbachia细菌感染

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摘要

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此视频中的章节

在果蝇的Wolbachia细菌感染

饲养果蝇

全身细菌感染与免疫防御的表型

从果蝇幼虫中提取包囊微生物感染及分析

果蝇病毒感染的建立及宿主病毒相互作用分析

果蝇、果蝇细胞免疫应答的评估

果蝇 幼虫注射方案

Infecting Flies with Bacterial Suspension Using a Nanoinjector

一种在果蝇中引入全身性细菌感染的脓毒性针刺技术

测量大肠杆菌感染后黑腹果蝇中的大肠杆菌细菌载量

果蝇幼虫注射方案