Name: feeding and quantifying animal-derived blood and artificial meals in aedes aegypti mosquitoes
Uploaded: 2020-10-22T15:00:00.000+00:00
Duration: 9 min 42 s
Description: Watch this Scientific Journal Video about Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes at JoVE.com

我们感谢尼朋·巴斯鲁尔、阿德里亚娜·罗萨斯·维尔加斯、纳达夫·沙伊和特雷弗·索雷尔对手稿的评论，以及宫中燕和基罗洛斯·巴苏姆的技术援助。我们感谢亚历克斯·威尔德在 图1中使用的照片。K.V.得到了博林格·英格尔海姆基金会博士奖学金的支持。V.J.部分得到NIH T32-MH095246的支持。这项工作的部分支持来自洛克菲勒大学从霍华德休斯医学研究所通过詹姆斯H.吉利亚姆高级研究奖学金计划到V.J.该材料基于国家科学基金会研究生研究奖学金计划支持的工作，该奖学金项目由第1号赠款公司提供。NSF DGE-1325261 到 V.J.本材料中表达的任何意见、发现、结论或建议均为作者的意见，不一定反映国家科学基金会的观点。

血液喂养程序不使用活体动物或人类宿主进行，并符合洛克菲勒大学机构动物护理和使用委员会（IACUC）和机构审查委员会（IRB）制定的准则。
1. 膳食准备
<ol><li>法戈刺激剂，腺苷5+三磷酸酯的制备<ol>
<li>准备25米水性NaHCO3 溶液（分子量，兆瓦=84.006克/摩尔）。对于 100 mL 的 25 mM NaHCO3，将 210 毫克 NaHCO3 添加到体积烧瓶中，并填充双蒸馏水（ddH2O），总体积为 100 mL。使用磁搅拌棒，彻底混合溶液，直到所有 NaHCO3 溶解。</li>
		<li>在水性 25 mM NaHCO3 中重组 ATP 脱钠盐水合物 （MW = 551.14 g/mol），最终浓度为 200 mM ATP。对于总体积为 10 mL 的 200 mM ATP 在 25 mM NaHCO3 缓冲区，将 1.1 克 ATP 脱钠盐水合物添加到体积瓶中，并填充 25 mM NaHCO3 缓冲器，总体积为 10 mL。使用磁搅拌棒，彻底混合溶液，直到所有 ATP 溶解。 注:要尽量减少ATP的水解，必须通过纳赫科3等盐溶液进行缓冲。</li>
		<li>阿里引用ATP解决方案，并在-20°C存储。 注:ATP的这种库存解决方案通常每六个月新鲜一次，用于下面描述的所有膳食。为了防止退化，ATP 别名不应经历多个冷冻解冻周期，也不应与其他膳食组件一起加热。</li>
	</ol></li>
	<li>荧光示踪剂溶液、荧光素的制备<ol>
<li>准备2%（w/v）的液态荧光素库存溶液。对于总库存溶液量为 10 mL，在室温下用铝箔包裹的 15 mL 锥形管中混合 0.2 克荧光素脱钠盐和 10 mL 的 ddH2O。这种荧光素的库存溶液可用于以下所有膳食的稀释。 注:由于荧光素对光敏感，请用铝箔包装容器，避免暴露在光线下。</li>
	</ol></li>
	<li>准备动物源性血液餐<ol>
<li>计算喂养所有蚊子所需的膳食数量;每个格利图伯有一顿2mL的饭菜，喂养大约25只蚊子。准备一顿额外的膳食，以校准荧光读数的标准曲线。除非另有说明，否则本节中的所有步骤都描述了准备一顿饭所需的试剂量，最终体积为 2 mL。</li>
		<li>对于动物源性血液餐，将 1.98-2 mL 除颤羊血转移到 15 mL 锥形管中（参见步骤 3.3，以获得所需的血液量）。 注:商业去除脊椎动物血液来源，包括绵羊、豚鼠和人类，可使用13。使用前，确保所购血液未过期，并通过倒置瓶子将其混合好，尤其是在血液成分明显分离的情况下。</li>
		<li>为了获得最佳喂养，在水浴中将羊血加热到 45 °C 后，将 ATP 添加到 1-2 mM 的最终浓度中。对于 1 mM ATP 的最终浓度，在 200 mM ATP 库存解决方案中添加 10 μL，加入 1.99 mL 的预热血和混合液。对于 2 mM ATP 的最终浓度，将 200 mM ATP 库存中的 20 μL 添加到 1.98 mL 的预温血液和混合物中。如果不添加 ATP，加热 2 mL 的除颤羊血。</li>
		<li>如果随后要对膳食大小进行基于荧光的量化，则将荧光素溶液添加到最终浓度为 0.002% （2 μL 的 2% 荧光素库存在 2 mL 总膳食量中）。将血液量减少与添加的荧光素相同的量。保留含有 0.002% 荧光素的最终膳食配方的 1 mL 以生成参考标准曲线。将保留的体积与提供给蚊子的膳食相同;在整个实验过程中暴露在相同的光线和温度条件下，然后与送餐一起冷冻。</li>
	</ol></li>
	<li>制备人造血餐<ol>
<li>计算喂养所有蚊子所需的膳食数量;每个格利图伯有一顿2mL的饭菜，喂养大约25只蚊子。准备一顿额外的膳食，以校准荧光读数的标准曲线。除非另有说明，否则本节中的所有步骤都描述了准备一顿 2 mL 餐所需的试剂量。</li>
		<li>为了准备人造血液（改编自科根（1990年）22），如 表1，首先使库存溶液400 mM NaHCO3。 总容量为 10 mL 的 400 mM NaHCO3 （MW = 84.006 g/mol），将 336 毫克 NaHCO3 添加到体积烧瓶中，并填充双蒸馏水（ddH2O），总体积为 10 mL。使用磁搅拌棒，彻底混合溶液，直到所有 NaHCO3 溶解。</li>
		<li>对于人造血液的蛋白质成分，在400 mM NaHCO3中制备50毫克/毫升γ球蛋白、在ddH2O中35毫克/mL的血红蛋白和在ddH2O中300毫克/毫升的白蛋白的库存溶液，蛋白质库存溶液可在4°C储存长达2个月。人造血液中人类蛋白质总量的最终浓度为125毫克/mL。这包括15毫克/mL γ球蛋白、8毫克/mL血红蛋白和102毫克/mL白蛋白的最终浓度。</li>
		<li>每2毫升餐，结合600μL的γ球蛋白，460μL的血红蛋白，680μL的白蛋白，和250μL的ddH2O从 表1中列出的股票解决方案。等待添加 10μL 的 200 mM ATP 库存解决方案，直到饭后加热到 45 °C，然后立即呈现餐食。</li>
		<li>如果随后要对膳食大小进行基于荧光的量化，则将荧光素溶液添加到最终浓度为 0.002% （2 μL 的 2% 荧光素库存在 2 mL 总膳食量中）。在步骤4.4中减少 ddH 2 O 的体积，其量与添加的荧光素相同。保留含有0.002%荧光素的最终膳食配方至少1 mL以生成参考标准曲线。将保留的体积与提供给蚊子的膳食相同;在整个实验过程中暴露在相同的光线和温度条件下，然后与送餐一起冷冻。</li>
	</ol></li>
	<li>准备无蛋白盐水餐（改编自杜瓦尔等人（2019）7） 注:无蛋白质盐水餐可以以多种方式准备7，27，32。这里介绍的盐水餐是上述人工血液配方的无蛋白质版本。<ol>
<li>计算喂养所有蚊子所需的膳食数量;每个 Glytube 都提供 2 mL 餐食，并喂养大约 25 只蚊子，准备一顿额外的膳食，以校准荧光测量的标准曲线。除非另有说明，否则本节中的所有步骤都描述了准备一顿 2 mL 餐所需的试剂量。</li>
		<li>要准备盐水餐，制作 400 mM NaHCO3的库存溶液。对于总体积为 10 mL 的 400 mM NaHCO3 （MW = 84.006 g/mol），将 336 毫克的 NaHCO3 添加到体积烧瓶中，并填充 ddH2O，总体积为 10 mL。使用磁搅拌棒，彻底混合溶液，直到所有 NaHCO3 溶解。</li>
		<li>对于每2毫升餐，结合在一个15毫升锥形管600μL的400 mM NaHCO3 与1.39毫升的ddH2O.等待添加10μL的200 mM ATP库存解决方案，直到饭后已加热到45°C在水浴。</li>
		<li>如果随后要对膳食大小进行基于荧光的量化，则将荧光素溶液添加到最终浓度为 0.002% （2 μL 的 2% 荧光素库存在总膳食量的 2 mL 中）。在步骤5.3中减少 ddH 2 O 的体积，其量与添加的荧光素相同。保留含有0.002%荧光素的最终膳食配方至少1 mL以生成参考标准曲线。将保留的体积与提供给蚊子的膳食相同;在整个实验过程中暴露在相同的光线和温度条件下，然后与送餐一起冷冻。</li>
	</ol></li>
</ol>2. 送餐给蚊子
<ol><li>设置供喂养的蚊子容器 注:只要符合以下标准，蚊子就可以在各种容器中喂养。确保容器足够大，蚊子可以飞来飞去，但不要太大，蚊子很难找到网状表面并开始进食。用于覆盖容器的网格在材料和孔大小上可能有所不同。孔必须足够大，雌性蚊子的风格穿透，但不是那么大，蚊子可以逃脱。牢牢地固定网格，使其绷紧，Glytube 在整个喂食期间可以稳定地放在其表面上。<ol>
<li>例如容器 （图 1）是一个经过改装的 946 mL （32 盎司） 高密度聚乙烯 （HDPE） 塑料桶。要复制此设置，请使用剃须刀刀片在桶盖中切割直径约 10 厘米的中央孔。要组装容器供蚊子占据，在桶顶上固定一块约 400 厘米2 平方的白色 0.8 毫米聚酯蚊帐，将穿孔盖牢牢地推倒在水桶上，以紧握。</li>
		<li>收集关闭后至少3天的雌性蚊子，以确保它们足够成熟，可以喂血。最佳喂养率在7天33后观察到。</li>
		<li>将雌性蚊子放入容器中，并盖上网状。如果容器中密布蚊子，则增加使用的胶合管数量。最佳喂养是通过+25蚊子/胶管实现的。这减少了获得喂食膜的竞争。</li>
		<li>留出一组未受控制的蚊子，这些蚊子不会得到一顿饭。在体重测量协议中，分别对未受约束的组进行称重，并使用此重量来估计以膳食为食的实验组的体重增加。在基于荧光的量化协议中，将未受约束的蚊子群添加到井中，用于标准曲线计算和负控制。为了匹配实验组的基线蚊子组织自荧光，确保标准曲线和负控制井包含未受蚊子。</li>
	</ol></li>
	<li>建造和建立格利图布 （改编自科斯塔 - 达西尔瓦等人 （2013）20）<ol>
<li>如 图 1所示，要生成热源，请将 50 mL 锥形管填充 40 mL 的 100% 甘油。用 5 厘米× 5 厘米的护膜密封打开的圆锥管，再加一块 5 厘米× 5 厘米的护膜，以最大限度地减少泄漏的机会。可选地，可以使用橡皮筋将准胶片放在原位。倒置管，以确保没有孔或缝隙。</li>
		<li>要创建送餐装置，只需使用锋利的剃须刀刀片或车床，在圆锥管螺钉盖上切割一个直径为 2.5 厘米的中心孔。均匀地伸展一块 5 厘米× 5 厘米的准胶片，使其大小大致翻倍。准胶片应该足够薄，蚊子很容易穿透它，但不应该有泄漏。密封螺钉盖的外表面，以完全覆盖孔，并设置盖子一边。 注意:为了增加对Glytube的吸引力，在拉伸护膜之前，用人类的气味来香水，轻轻揉在一块没有化妆品的人体皮肤上，注意不制造任何孔洞。如果实验的目的不是调查蚊子接近膳食所需的感官线索，建议这样做。</li>
		<li>在42-45°C的水浴中加热甘油和膳食的密封管（除ATP外的所有组件），至少15分钟。不要预热ATP:在开始实验前立即添加它。</li>
		<li>将 ATP 添加到加热的膳食中，并彻底转到漩涡中。移液器 2 mL 的加热餐进入螺丝帽的内室，轻轻地将倒置、加热、甘油填充的 50 mL 锥形管放入螺丝帽中。部分拧盖与膳食甘油填充管-只是足以防止泄漏的膳食或甘油。 注意:使用的膳食量在1 mL和2.5 mL之间。当餐食用于提供稀缺或昂贵的化合物时，较低的体积可能特别有用。在此步骤中快速工作非常重要，以便膳食不会冷却到环境温度，并降低最大进食的可能性。冷却速度将取决于执行这些步骤的房间的环境温度，但通常应在 5 分钟内在 25 °C 完成。</li>
		<li>将组装好的 Glytube 放在蚊子容器的顶部，让蚊子至少食用 15 分钟，以达到最大喂养率。</li>
		<li>为了获得最佳的喂养，请将蚊子容器放置在装有 CO2 垫的室内，并在送餐前在 25-28 °C 和 70-80% 的湿度下进行至少 15 分钟的适应。这里使用的检测室是一个简单和低成本的修改，以前公布的设置16。它使用半透明聚丙烯存储盒大小 36 厘米 L × 31 厘米 W × 32 厘米 H 与可拆卸盖。室壁上直径为 1.5 厘米的孔允许通过硅胶管输送 CO2。 CO2 扩散垫贴在盖子的内侧中心，用于输送净化空气和 CO2， 以在试验期间调节室内气氛。 注意:确保宿主提示（热和二氧化碳，可选宿主气味16）存在，以便蚊子被吸引到膜喂食器。如果蚊子没有挤在 Glytube 下面，请检查 CO2 是否正确传递，膳食和 Glytube 是否足够温暖。如果外部 CO2 源不可用，CO2 可以通过呼出的人类呼吸气泡传递。</li>
		<li>喂养后，Glytube 帽可以作为生物危害废物丢弃，或浸泡在低百分比漂白液中并彻底冲洗在水中后再使用。</li>
	</ol></li>
</ol>3. 消费膳食的量化
<ol><li>称量蚊子用于进一步实验 注:称量蚊子以量化膳食大小允许它们用于进一步的活体实验，但这种方法需要从一组5只蚊子中测量体重。由于使用大多数实验室平衡很难精确测量单个蚊子的重量，因此无法通过测量重量轻松量化单个膳食大小的变异性。仅建议女性明显在用餐时进行称重。<ol>
<li>冷麻醉蚊子通过移动他们的容器到4°C的冷室或把它放在冰上。</li>
		<li>称量组5女性从未提供一组（即蚊子从来没有提供一顿饭），并计算他们的平均体重作为估计的"喂养前"重量。未受蚊子的平均重量取决于基因型、性别和饲养条件。未受雌性 Ae. aegypti 蚊子饲养， 与 广告利比图姆 接触蔗糖通常每个重约 2 毫克。</li>
		<li>从实验队列（即提供膳食的蚊子），根据眼睛7可观察到的腹部分量，将雌性分成"喂"和"不喂"的桩。将每个"喂"和"不喂"的堆分别分成5组蚊子进行称量。每组5人中的蚊子应来自同一实验组，用于进行组重量测量。计算实验组每个"喂养"和"未喂养"堆中每个女性的平均体重。</li>
	</ol></li>
	<li>荧光测量，用于终点分析7,27,34 注意:要从不再需要进一步活体实验的单个蚊子那里获得精确的膳食大小测量，请在进食后立即将含有0.002%荧光素的1mL食物储存在-20°C。实验可以在这里暂停。此方法概述在 图2.<ol><li>要生成参考标准曲线，则准备对提供给实验组蚊子的含有 0.002% 荧光素的同一餐进行连续稀释。总共将有 8 个标准曲线解决方案。在每种解决方案中，含有 0.002% 荧光素的最终膳食量为 5、2.5、1.25、0.625、0.3125、 0.15625， 0.078125， 或 0 μL， 每个将在 1x 磷酸盐缓冲盐水 （PBS） 总体积 100 μL （例如， 5 μL 的膳食含有 0.002% 荧光素在 95 μL 的 1x PBS）.</li>
		<li>要使标准曲线的第一个解决方案，添加 50 μL 的膳食含有 0.002% 的荧光素到 950 μL 的 1x PBS 和漩涡彻底（最终体积:5 μL 的膳食含有 0.002% 的荧光素在 95μL 的 1x PBS）。要制作其余标准曲线解决方案，请从上一个管子中取出 500μL 并将其添加到包含 500μL 1 倍 PBS 的新管中，为每一步执行 2 倍稀释。在准备下一次 2 倍稀释之前， 漩涡很好。</li>
		<li>要准备用于生成参考标准曲线的油井，在 96 井 PCR 板的第一列中，每个标准曲线解决方案的移液器 100 μL 进入每个 8 口油井。在板的第一列相同的 8 口井中添加 1 个未受控制的蚊子。在板的第二列中重复以进行复制测量。 注:如果实验组提供不同的膳食类型，则必须为每种膳食类型准备一个单独的参考标准曲线。</li>
		<li>在未对照组和实验组的每个剩余井中添加 100 μL 的 1x PBS。如果组织要在随后的步骤中使用珠磨同质化剂或旋涡中断，则在每口井中添加一个 3 mm 的玻利西酸固体玻璃珠。</li>
		<li>作为负控制，在板的后 2 列中，在每口井中添加 1 只未清除的蚊子。该组测量的荧光设置基线截止，以解释组织自荧光，并将用于确定实验组中的蚊子是否在膳食中喂养。</li>
		<li>在提供膳食的实验组的剩余油井中，每口井增加1只蚊子。</li>
		<li>小心密封板，通过手动研磨破坏组织。腹部应彻底同质化以释放膳食。破坏组织的方法包括使用珠磨机同质化剂，具有3毫米的波罗西酸固体玻璃珠（30赫兹30秒），涡流搅拌机与3毫米波罗西酸盐固体玻璃珠，或无珠子的刺磨机。</li>
		<li>以 2000 rpm 的速度将盘子离心 1-2 分钟，以收集碎液。</li>
		<li>准备一个黑色的 96 井板，每口井有 180μL 的 1x PBS。</li>
		<li>将 20 μL 的利酸盐转移到每口井，并混合 180μL 的 1x PBS 并混合。如果可用，请在此步骤中使用多通道移液器，以提高速度和更好的一致性。</li>
		<li>使用 485/520 激发/排放通道上的板式读取器测量每口井的荧光强度。通过根据相应的荧光强度测量绘制已知的膳食量，生成参考标准曲线。</li>
		<li>使用生成的参考标准曲线，推断每个实验组蚊子摄入的膳食量。从每个实验组个体的荧光强度读数中减去未受蚊子负对照组的平均荧光强度读数，以校正基线组织自荧光。</li>
	</ol></li>
</ol>

<ol>
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作者没有什么可透露的。

对于许多实验室应用，人工膜喂食器通过让研究人员能够直接操纵餐食内容，与活体宿主相比具有明显的好处。虽然有多种方法可用于人工膜喂养，但此处描述的方法在灵活性、成本和吞吐量方面具有优势。与其他商业膜喂食器相比，Glytube检测需要少量的膳食量，通过最小化所需的总体积7，35，使其成为昂贵的试剂（包括药物或病原体）的有效输送机制。由于无蛋白盐水和人工血餐都促进活力，化合物或病原体可以添加到任何一餐作为高通量和非侵入性替代注射。此外，Glytube 的每个组件都很容易被洗涤、更换或放大，以提供和量化多种膳食类型，而不会交叉污染喂养设备。
为了量化蚊子的膳食量，荧光法比在喂食前后称量蚊子更精确的膳食大小量化。应当指出，这种方法是一种终点检测。相比之下，称量使蚊子能够存活下来进行进一步的实验。通过使用板式读取器，基于荧光的方法可以轻松扩展，以高通量量化数百名女性食用的膳食。
为了达到高喂养率，必须存在足够的宿主提示组合，以激活女性寻宿主行为，并吸引女性到喂食器。如果蚊子没有挤在 Glytube 下面，膳食可能无法适当加热，或者二氧化碳 的输送可能不够。在膜表面添加人类气味可可靠地增加人造膜的吸引力。如果在 Glytube 下观察到蚊子，但无法进食，则膳食成分可能有错。如果膳食本身不温暖，血液太旧，或者如果膳食中的添加剂本质上是反常的，或引起不良的化学反应，女性可能无法进食。额外的ATP还可以可靠地提高喂养率，并且可以在所提供的每个食谱中扩展到2mM的最终浓度。如果准胶片没有拉紧格莱图帽，女性可能无法进食:护身膜应均匀透明，不应扣扣，因为这样可以防止女性能够有效地用她的风格刺穿准胶片。如果餐食通过 Glytube 泄漏到网状物上，则准胶片可能在拉伸过程中撕裂，应更换。
改变膳食成分还可以使研究人员能够操纵从中餐中清除膳食所需的时间长度以及随后的寻宿行为。这里提供的膳食需要24-36小时消化7 类似于动物源性血液。吃完这些饭菜后，雌性会在消化时间窗口内抑制寻找宿主。由于盐水餐缺乏蛋白质，女性在餐后返回寻求宿主。如果更快的回报是可取的，研究人员可以选择替代"快速清除"盐水餐，排泄在大约6小时27。虽然这里呈现的盐粉成分与人工血餐直接比较结果，但"快速清除"膳食更符合脊椎动物血液中的生理盐含量。
此处描述的方法有局限性，在选择最适合研究人员实验目标的测定之前应考虑这些限制。所述的荧光素测量不允许蚊子再次用于额外的实验。但是，可以使用荧光素检测在膳食大小量化之前进行体重测量。如果给定膳食的多个试验中体重和膳食大小是一致的，则重量可以用作未来实验的代理。此外，该议定书没有区分寻求宿主和喂血行为的缺陷:发现膜喂食器有损伤的蚊子会降低进食率和/或膳食大小。通过添加摄像头来记录整个测定过程中的行为，研究人员可以确定雌性是否找不到Glytube，或者他们是否找到了Glytube，但不会进食。
此处描述的检测可以适应探索与蚊子喂养行为相关的许多未决问题。例如，可以通过改变成分蛋白的比例或人工血餐中蛋白质总浓度来探讨特定血液蛋白的贡献。为了评估多个喂养事件的膳食大小，可以添加具有明显荧光光谱的染料，以区分膳食与独特来源37。该协议也可以修改，以单独刺激内口部分检测血液和用于摄入（即风格），和化疗附属物接触皮肤（即阴唇，腿）作为蚊子土地开始血液喂养36。例如，如果将配体直接添加到膳食中，它们不会接触阴唇和腿部，因为膜仅通过样式穿孔。如果配体被添加到副膜的外表面，他们仍然与膳食分离，并可能接触的实验室和腿36。最后，对血液喂养行为的详细动能没有很好的理解，这里提出的方法可以修改为将高分辨率跟踪与机器学习工具相结合，以提取运动、姿势和喂食动力学的行为读数38。
该协议旨在用户友好且具有成本效益，能够为使用药理学和基因操作来研究蚊子血液喂养和血液喂养后行为的研究人员提供服务。

我们提出了一个协议，使用人工膜喂食器向 伊 蚊提供改性血液膳食，并精确测量每只蚊子的膳食量。此协议可以灵活地调整，以改变膳食内容或比较不同实验组蚊子消耗的膳食量。
艾吉普提蚊子传播病原体，导致黄热病、登革热、基孔肯雅病和寨卡1、2、3、4、5等疾病，威胁全球健康。艾吉普蒂女性是义务献血者:他们必须消耗脊椎动物的血液，以获得卵子发育所需的蛋白质，每个离合器的鸡蛋需要从至少一个主机6，7，8全血餐。雌性蚊子首先咬她的主人，用她的风格刺穿皮肤和注射唾液，其中含有化合物，触发宿主的免疫反应9。然后，她通过抽血通过她的风格到她的中口水喂养。在食用受感染宿主的血液餐时，她可能会摄入血液传播的病原体6、8，然后从蚊子的中游转移到唾液腺10。以这种方式感染的雌性蚊子在叮咬随后的宿主11、12时，会通过注射病原体和唾液来传播疾病。了解和量化供血行为的机制是控制蚊媒疾病传播的关键步骤。
许多用于蚊子饲养和实验的实验室协议使用活体动物，包括老鼠、豚鼠或人类作为血源13、14、15、16。活体动物的使用对人员培训、动物住房和护理以及遵守机构动物护理和使用委员会（IACUC）的政策提出了道德问题和复杂的要求。它还限制了可以输送到蚊子的化合物的类型，这限制了可以进行的研究17。
人工供血设备通常使用膜系统来模拟宿主皮肤，是研究血液喂养行为的有用工具，可以规避维持活体宿主的需要。全血可以从一些供应商购买，并喂蚊子使用加热，水衣人造膜喂食器或类似的设备18，19。在此协议中，我们演示了使用称为"Glytubes"的小型一次性膜馈线器。这种膜喂食器以前由科斯塔-达-西尔瓦等人（2013年）20日出版，可以很容易地从标准的实验室设备组装，使其非常适合向适量的蚊子提供血液膳食，并直接扩大规模，以测试更大的群体或多种膳食配方。Glytube 是一种廉价和高效的替代其他商业人工喂食器，它可能需要更大的膳食量，更适合批量喂养大群蚊子的单餐配方21。
该协议包括两个部分:准备/提供人工餐食和量化消费。在第一节中，Glytubes 被用作提供操纵饮食的有效手段。全血可以用完全人造的膳食代替，以比较血液替代品代替血液餐的效果。一个食谱改编自科根（1990）22在这里提出，虽然多个人造膳食配方已经开发23，24。此外，与注射相比，喂养是一种侵入性更小、更不费力的方法来引入药理化合物。由于每餐所需的总容量较低（1-2 mL），此协议提供了一个有吸引力的递送方法，以减少昂贵的试剂量。Ae. aegypti女性容易食用无蛋白质的盐水溶液与腺苷 5 +三磷酸酯 （ATP）25，26，它提供了一个基线，用于测量单膳食成分的影响.例如，Ae.aegypti的神经肽 Y 样受体 7 （NPYLR7） 已知在富含蛋白质的血液餐后会调解宿主求抑制，当 NPYLR7 激动剂添加到无蛋白质盐水餐中时，雌性蚊子表现出类似于那些消耗了全血的雌性蚊子寻求宿主的抑制。
在第二节中，介绍了量化雌性蚊子每餐消费量的步骤。这种测定基于荧光，捕获喂养状态的分辨率高于女性根据腹部分化的视觉评估被归类为"喂养"或"未喂养"的方法。通过在进食前将荧光素添加到膳食中，个人摄入的膳食量可以通过将每只蚊子同质化在 96 口井盘中并测量荧光强度作为读取来量化。这种检测可以测量喂养活力的差异，以响应诸如膳食成分或蚊子的遗传背景等变量。精确量化对于中餐规模至关重要，例如，当女性获得含有喂养威慑力的次优膳食时，或当她们食用可变大小为27的蔗糖餐时。如果餐量大小量化后需要喂养蚊子进行后续行为检测，则可以通过分组称量麻醉女性并估计每个人平均增加的质量来计算膳食大小。虽然重量不如荧光素标记精确，但仍提供膳食体积的汇总估计值，并允许检查膳食对下游过程的影响，如粪便或随后的宿主吸引力。虽然血餐的大小是可变的，并可能受到无数因素的影响11，28，29，摄入膳食大小测量与这里描述的方法是一致的以前的量化7，30，31。

<table><tbody><tr><td>15 mL conical tubes</td><td>Fisher Scientific</td><td>14-959-70C</td><td></td></tr><tr><td>3 mm diameter borosilicate solid-glass bead</td><td>MilliporeSigma</td><td>Z143928</td><td>For use for bead mill homogenizer; not required if using pellet pestle grinder</td></tr><tr><td>32 oz. high-density polyethylene (HDPE) plastic cup</td><td>VWR</td><td>89009-668</td><td>Example mosquito container used for feeding assays shown; alternate options can be used</td></tr><tr><td>50 mL conical tubes</td><td>Fisher Scientific</td><td>14-959-49A</td><td></td></tr><tr><td>96-well black polystyrene plate</td><td>ThermoFisher</td><td>12-566-09</td><td></td></tr><tr><td>96-well PCR plate sealing film</td><td>Bio-Rad</td><td>MSB1001</td><td>Alternate options can be used</td></tr><tr><td>96-well PCR plates</td><td>Bio-Rad</td><td>HSP9621</td><td>Alternate options can be used</td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate (ATP) disodium salt hydrate</td><td>MilliporeSigma</td><td>A6419</td><td></td></tr><tr><td>Albumin (human serum)</td><td>MilliporeSigma</td><td>A9511</td><td></td></tr><tr><td>Aluminum foil</td><td>Fisher Scientific</td><td>01-213</td><td>Alternate options can be used to block light entering fluorescein container</td></tr><tr><td>Balance</td><td>Fisher Scientific</td><td>01-911</td><td>Alternate options can be used</td></tr><tr><td>Bead mill homogenizer</td><td>Qiagen</td><td>85300</td><td>Not required if using pellet pestle grinder</td></tr><tr><td>Cotton ball</td><td>Fisher Scientific</td><td>22456880</td><td>For nectar-feeding; alternate options can be used</td></tr><tr><td>Defibrinated sheep blood</td><td>Hemostat Laboratories</td><td>DSB100</td><td>Alternate options can be used</td></tr><tr><td>Drosophila CO&lt;sub&gt;2&lt;/sub&gt; fly pad</td><td>Tritech Research</td><td>MINJ-DROS-FP</td><td>Alternate options can be used</td></tr><tr><td>Fluorescein</td><td>MilliporeSigma</td><td>F6377</td><td></td></tr><tr><td>Fluorescence plate-reader</td><td>ThermoFisher</td><td>VL0000D0</td><td>Alternate options can be used</td></tr><tr><td>Gamma-globulin (human blood)</td><td>MilliporeSigma</td><td>H7379</td><td></td></tr><tr><td>Glycerol</td><td>MilliporeSigma</td><td>G7893</td><td></td></tr><tr><td>Hemoglobin (human)</td><td>MilliporeSigma</td><td>G4386</td><td></td></tr><tr><td>Laboratory wrapping film - parafilm</td><td>Fisher Scientific</td><td>13-374</td><td></td></tr><tr><td>Magnetic stirrer</td><td>Fisher Scientific</td><td>90-691</td><td>Alternate magnetic stirrers can be used</td></tr><tr><td>Microcentrifuge for 96-well plate</td><td>VWR</td><td>80094-180</td><td>Alternate options can be used</td></tr><tr><td>Microcentrifuge Tubes</td><td>MilliporeSigma</td><td>2236412</td><td>Alternate options can be used</td></tr><tr><td>Pellet pestle grinder</td><td>VWR</td><td>KT749521-1500</td><td>Not required if using bead mill homogenizer</td></tr><tr><td>Phosphate buffered solution (PBS)</td><td>Fisher Scientific</td><td>BW17-516F</td><td>Optional</td></tr><tr><td>Razor blades</td><td>Fisher Scientific</td><td>12-640</td><td>Alternate options can be used, such as a lathe for better consistency of cutting</td></tr><tr><td>Rubber bands</td><td></td><td></td><td></td></tr><tr><td>Silicone tubing</td><td>McMaster Carr</td><td></td><td>Needed if using a fly pad for CO&lt;sub&gt;2&lt;/sub&gt; delivery</td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>S233</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>MilliporeSigma</td><td>S9888</td><td></td></tr><tr><td>Stir bars</td><td>Fisher Scientific</td><td>14-512</td><td>Alternate magnetic stir bars can be used</td></tr><tr><td>Translucent polypropylene storage box with removable lid</td><td></td><td></td><td>Example box used for feeding assays shown</td></tr><tr><td>Vortex mixer</td><td></td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td>Alternate heating device may be used</td></tr><tr><td>White 0.8 mm polyester mosquito netting</td><td>American Home &amp;amp; Habit Inc.</td><td>F03A-PONO-MOSQ-M008-WT</td><td>Alternate options can be used</td></tr></tbody></table>

feeding and quantifying animal-derived blood and artificial meals in aedes aegypti mosquitoes

某些蚊子物种的女性可以传播疾病，同时咬脊椎动物宿主，以获得卵子发育所需的富含蛋白质的血液膳食。在实验室里，研究人员可以通过膜喂食器向蚊子提供动物源性血液和人造血液餐，从而能够操纵膳食成分。在这里，我们 介绍的方法， 喂血和人工血液餐伊蚊和量化个别女性的消费量。
有针对性的喂养和定量人工/血液膳食具有广泛的用途，包括测试膳食成分对蚊子行为和生理的影响，不注射就提供药理化合物，以及用特定病原体感染蚊子。在进食前将荧光素染料添加到膳食中，可进行后续膳食尺寸的量化。蚊子消耗的膳食量可以测量体重，如果女性以后要用于行为实验，或者通过在96口井板中使个体雌性同质化，并使用板读器作为终点检测测量荧光水平。膳食大小量化可用于确定更改膳食成分是否会改变摄入的膳食量，或者蚊子菌株之间的膳食消耗是否不同。精确的膳食大小量化对于下游检测也至关重要，例如测量对宿主吸引力或丰盛度的影响。这里介绍的方法可以进一步适应跟踪膳食消化在几天内，或包括多个可区分的标记添加到不同的膳食（如花蜜和血液），以量化每餐由一只蚊子的消费。
这些方法使研究人员能够单枪匹马地进行高通量测量，以比较数百只蚊子的膳食量。因此，这些工具对于蚊子研究人员群体回答各种生物问题将大有用处。

图 1提供了组装 Glytube 的示意图，而图 2显示了使用此处描述的基于荧光的检测来测量膳食大小的实验设计概述。图 3提供了血液喂养实验中具有代表性的荧光素膳食尺寸测量结果。图 4、图 5和图 6显示了可使用本协议解决的生物问题的样本。该协议的应用范围很广，包括改变血粉成分、喂养药理化合物、精确量化次优血餐或小型花蜜餐，以及比较蚊子基因型的喂养行为。
为了生成膳食量计算的标准曲线，荧光读数从指定的参考井中绘制，每个油井都含有未受蚊虫和已知卷，含 0.002% 荧光素（图 3A）。剩余水井的荧光读数与此标准曲线进行比较，以量化每只蚊子消耗的膳食量 （μL） （图 3B）。要验证此检测中的基线读数，应确认未受负对照组的蚊子未分配消耗的 μL 正值（图 3B，左图）。虽然实验组的所有女性都得到了血餐，但有些蚊子喂食（图3B，中间），有些没有（图3B，右图）。这一结果表明，可以从本协议中获取两种类型的数据:1） 以给定膳食为食的女性总数的百分比，以及 2） 以给定膳食为食的女性摄入的体积。
此协议可用于提供和量化含有各种蛋白质成分的膳食。 图 4A，B 显示使用添加荧光素的膳食收集的数据。喂养的蚊子比例和它们摄入的膳食量分别根据荧光读数计算。这些读数非常敏感，允许精确量化μL，但有蚊子不能用于未来活体实验的局限性。 图4C，D 显示了从对蚊子的独立实验中收集的数据，这些蚊子在没有荧光素的膳食后被评分为喂养或没有眼睛。膳食大小计算为5组蚊子的平均体重/雌性。虽然这些重量测量比荧光测量更不敏感，但它们允许女性被恢复并用于进一步的活体实验。喂养的蚊子比例可能因实验日而异，如 图4A 和 图4C所反映的那样。
图 5 显示了含有调节蚊子宿主寻找行为的药物的膳食消耗量。在这些实验中，女性获得血液、盐水+ATP或盐水+ATP餐食，其中100μM为人类NPY Y2受体激动剂TM30338。这种药物通过激活 类似 Ae. aegypti NPY 的受体 7 来改变寻宿主行为。测量膳食大小对于解释实验以评估这种药物对血液喂养后行为的影响至关重要，因为它允许研究人员计算每个女性消耗的剂量。
在以前的例子中，女性要么喂血，要么用血代替，所有这些都导致3-5μL餐（图3，图4，图5）。这种基于荧光的检测方法还可用于测量从平均组重量测量中无法准确辨别的更小和/或更可变的膳食大小。在图6中，同样的荧光量化方案被用来测量花蜜喂养行为，用Glytube交换一个棉球饱和10%的蔗糖含有0.002%的荧光素。花蜜糖不能在Glytube检测中呈现，因为女性无法检测花蜜糖的存在与风格，不启动喂养27。这些数据使研究人员能够确定糖膳食总是小于血餐，这与以前的工作34（图6）一致。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61835/61835fig1v2.png"> 图1:设置用于向蚊子喂食的Glytube方法。（A） 用于向蚊子喂血和其他膳食的解构胶管的示意图。（B） 格利图布的示意图呈现在一个带网状盖的蚊子容器上。雌性蚊子可以穿透网盖喂食。（C） 格利图布（上图）和雌性伊蚊在喂食前、喂食期间和喂食后（从下到右）的照片。蚊子通过覆盖容器的网眼穿透，进入膜馈线。（D） 照片显示雌性Ae. aegypti蚊子的外观，这些蚊子没有（左），并且已经注入了人工血餐（右，顶部）或盐水 + ATP 餐（右，下）。格利图布方法之前发表在科斯塔-达-西尔瓦等人（2013）20。照片在 （C） 和（D） 由亚历克斯 · 威尔德提供。<a href="https://www.jove.com/files/ftp_upload/61835/61835fig1v2large.png" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61835/61835fig2v4.png"> 图2:胶管供血协议后如何量化膳食大小的示意图。（A） 蚊子提供荧光素（顶部，实验组）或无餐（底部，无负对照组）的膳食。（B） 终止喂食实验后，个别蚊子会加入96井盘内。（C） 标准曲线是使用已知含有0.002%荧光素的膳食量生成的。（D） 蚊子是同质化的，以释放任何消耗的荧光素，每个井的荧光水平使用板读取器进行量化。这种荧光量化方法从利施等人（2013）34中修改而成。<a href="https://www.jove.com/files/ftp_upload/61835/61835fig2v4large.png" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61835/61835fig3v2.png"> 图3:用基于荧光素的量化进行胶管供血实验。（A） 从未受控制组的蚊子被添加到已知数量的膳食中，含有0.002%荧光素（y轴尺度=任意单位）的井中获得的标准曲线测量结果。（B） 餐量使用未对照组（左、黑、n =40）、以血液为食的实验组（中、红色、n = 37）和不以血液为食的实验组（右、红、n=23）的女性计算。每个点代表来自女性个体的测量结果。数据显示为带范围的中位数。字母表示统计学上不同的组，克鲁斯卡尔-瓦利斯测试与邓恩的多重比较，p<0.01。这些数据发表在Jové等人（2020）27。<a href="https://www.jove.com/files/ftp_upload/61835/61835fig3v5large.png" target="_blank">请点击这里查看此数字的较大版本。</a>

<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61835/61835fig5v2.png"> 图5:用药理化合物量化膳食。女性食用相同大小的羊血（红色）、盐水+ATP（水）和盐水+ATP+100 μM剂量的人类NPY Y2受体激动剂TM30338（深蓝色）。使用荧光读数计算的膳食量。每个点代表来自女性个体的测量结果。数据显示为带范围的中位数，n = 12。字母表示统计学上不同的组，克鲁斯卡尔-瓦利斯测试与邓恩的多重比较，p<0.05。 <a href="https://www.jove.com/files/ftp_upload/61835/61835fig5v5large.png" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61835/61835fig6v2.png"> 图6:小花蜜餐的量化。（A） 花蜜喂养检测的示意图。（B） 在花蜜喂养检测中，使用野生型雌性荧光读数计算的膳食量提供水（蓝色、n = 36）或10%蔗糖（绿色，n = 53），每餐均带有0.002%的荧光素。每个点代表来自女性个体的测量结果。数据显示为带范围的中位数。字母表示统计学上不同的组，曼恩-惠特尼测试，p<0.05。这些数据发表在Jové等人（2020）27。<a href="https://www.jove.com/files/ftp_upload/61835/61835fig6v5large.png" target="_blank">请点击这里查看此数字的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="4">人造血餐</td>
		</tr>
<tr>
<td></td>
			<td>库存解决方案的集中（毫克/mL）</td>
			<td>膳食中库存解决方案的体积 （μL/mL）</td>
			<td>最终膳食浓度（毫克/mL）</td>
		</tr>
<tr>
<td colspan="4">蛋白质成分*</td>
		</tr>
<tr>
<td>γ-球蛋白</td>
			<td>50</td>
			<td>300</td>
			<td>15</td>
		</tr>
<tr>
<td>血红蛋白</td>
			<td>35</td>
			<td>230</td>
			<td>8</td>
		</tr>
<tr>
<td>白 蛋白</td>
			<td>300</td>
			<td>340</td>
			<td>102</td>
		</tr>
<tr>
<td>蛋白质总量</td>
			<td>-</td>
			<td>-</td>
			<td>125</td>
		</tr>
<tr>
<td colspan="4">非蛋白质组件</td>
		</tr>
<tr>
<td></td>
			<td>库存解决方案的集中 （mM）</td>
			<td>膳食中库存解决方案的体积 （μL/mL）</td>
			<td>最终膳食浓度 （mM）</td>
		</tr>
<tr>
<td>Nacl</td>
			<td>以γ球蛋白股票</td>
			<td>-</td>
			<td>5-10</td>
		</tr>
<tr>
<td>纳科3
</td>
			<td>以γ球蛋白股票</td>
			<td>-</td>
			<td>120</td>
		</tr>
<tr>
<td>Atp</td>
			<td>200</td>
			<td>5</td>
			<td>1</td>
		</tr>
<tr>
<td>水</td>
			<td>-</td>
			<td>125</td>
			<td>-</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td colspan="4">*蛋白质成分在双蒸馏水的库存溶液中制备，γ球蛋白除外，这些蛋白溶解在 400 mM NaHCO3 中，包括可变量的 NaCl （2-4%）在产品中。</td>
		</tr>
</tbody></table>表1:制备人工血餐的食谱（改编自科根（1990）22）。人造血液由人类血液中经常发现的蛋白质和非蛋白质成分组成，并提供改变这些成分比例的选择。蚊子可以产生鸡蛋后，喂养人工血液7，22。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="4">盐粉</td>
		</tr>
<tr>
<td>组件</td>
			<td>库存解决方案的集中 （mM）</td>
			<td>膳食中库存解决方案的体积 （μL/mL）</td>
			<td>最终膳食浓度 （mM）</td>
		</tr>
<tr>
<td>Nacl</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
<tr>
<td>纳科3
</td>
			<td>400</td>
			<td>300</td>
			<td>120</td>
		</tr>
<tr>
<td>Atp</td>
			<td>200</td>
			<td>5</td>
			<td>1</td>
		</tr>
<tr>
<td>水</td>
			<td>-</td>
			<td>695</td>
			<td>-</td>
		</tr>
</tbody></table>表2:与ATP的盐水餐食谱（改编自杜瓦尔等人（2019）7）。无蛋白盐水餐可用于向蚊子提供感兴趣的化合物，同时仍模仿血液喂养后发生的腹部分化，但不会触发蛋白质摄入时发生的卵子发育。

Watch this Scientific Journal Video about Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes at JoVE.com

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

该议定书的目标是通过人工膜喂食器向 伊 蚊提供动物源和人工血液膳食，并精确量化摄入的膳食量。

在 伊 蚊中喂养和量化动物衍生的血液和人工膳食

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

feeding-quantifying-animal-derived-blood-artificial-meals-aedes

Research

JoVE Journal

Immunology and Infection

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

7.7K 次观看.  The Rockefeller University. 该议定书的目标是通过人工膜喂食器向 伊 蚊提供动物源和人工血液膳食，并精确量化摄入的膳食量。

视频: Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

甲病毒转导系统：可视化，以免蚊虫感染的工具

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

科研

免疫与感染

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

生物学

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

环境科学

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

使用寨卡病毒对 埃及伊 蚊的载体能力分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

经口喂养和显微注射对成年蚊子进行实验性病毒感染

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

强迫流涎法分析蚊虫的载体能力

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

A Blood-Free Diet to Rear Anopheline Mosquitoes

Malaria research requires large-scale breeding and production conditions for mosquitoes (Anopheles spp.) in captivity. The sustainable and reliable production of mosquitoes is currently inhibited by the supply of fresh vertebrate blood. Alternatives to blood are required to promote efficient control strategies for malaria and other vector borne diseases that are transmitted by blood feeding insects. With this in mind, artificial liquid diets were formulated as substitutes for fresh vertebrate blood. Herein we report a blood-free artificial liquid diet that delivers feeding rates similar to blood and mimics the physiological effects of a fresh vertebrate blood meal. The diet induces ovarian and egg maturation of Anopheles mosquitoes and also produces good larval survival and development of functional adults. The formulated blood-free liquid diet is an important advance towards sustainable mosquito breeding in captivity and will reduce the maintenance costs of mosquito colonies and eliminate the need for fresh vertebrate blood.

A protocol is presented for formulation of a blood-free artificial diet to feed Anopheles mosquitoes in captivity. This diet has a similar performance to vertebrate blood and triggers oogenesis and egg maturation and produces viable adult progeny.

后尾性异草蚊子的无血饮食

Malaria research requires large-scale breeding and production conditions for mosquitoes (Anopheles spp.) in captivity. The sustainable and reliable ...

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness parameters, in particular egg production and hatch rates at the individual level, conventional methods have put a substantial burden on investigators due to high labor intensity and laboratory space requirements. Described is a simple method using 24 well tissue culture plate with agarose in each well and digital imaging of each well to determine egg numbers and hatch rates at an individual level with substantially reduced time and space requirements.

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates

Described is a time and space-saving method to count eggs and determine hatch rates of individual mosquitoes using 24 well tissue culture plates, which can substantially increase the scale and speed of fecundity and fertility assays.

使用 24 个井组织培养板 （EAgaL 板） 改进 了伊蚊 的丰度和生育率测定

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness ...

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism.
This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species.

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

抗生素处理蚊子的细菌口服喂养测定

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life parameter data from genetically modified mosquitoes and comparing them to mosquitoes lacking transgenes from the same genetic background. This manuscript illustrates how to measure common life history traits in the mosquito Aedes aegypti, including fecundity, wing size and shape, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature. The representative results quantify fitness costs associated with either a gene knock-out or a single insertion of a gene drive element. Standardizing how life parameter data are collected is important because such data may be used to compare the health of transgenic mosquitoes generated across studies or to model the transgene fixation rate in a simulated wild-type mosquito population. Although this protocol is specific for transgenic Aedes aegypti, the protocol may also be used for other mosquito species or other experimental treatment conditions, with the caveat that certain biological contexts may require special adaptations.

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes.

量化转基因 埃及伊蚊 的适应性成本

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life ...

在伊蚊中喂养和量化动物衍生的血液和人工膳食

摘要

探索更多视频

此视频中的章节

甲病毒转导系统：可视化，以免蚊虫感染的工具

蚊子养育的议定书“（冈比亚按蚊）

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

使用寨卡病毒对埃及伊蚊的载体能力分析

经口喂养和显微注射对成年蚊子进行实验性病毒感染

强迫流涎法分析蚊虫的载体能力

后尾性异草蚊子的无血饮食

使用 24 个井组织培养板（EAgaL 板）改进了伊蚊的丰度和生育率测定

抗生素处理蚊子的细菌口服喂养测定

量化转基因埃及伊蚊的适应性成本

在 伊 蚊中喂养和量化动物衍生的血液和人工膳食

摘要

探索更多视频

此视频中的章节

甲病毒转导系统：可视化，以免蚊虫感染的工具

蚊子养育的议定书“（冈比亚按蚊）

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

使用寨卡病毒对 埃及伊 蚊的载体能力分析

经口喂养和显微注射对成年蚊子进行实验性病毒感染

强迫流涎法分析蚊虫的载体能力

后尾性异草蚊子的无血饮食

使用 24 个井组织培养板 （EAgaL 板） 改进 了伊蚊 的丰度和生育率测定

抗生素处理蚊子的细菌口服喂养测定

量化转基因 埃及伊蚊 的适应性成本

在伊蚊中喂养和量化动物衍生的血液和人工膳食

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