我们承认, 对, 克里斯柏鼎言, 彼得约翰逊和克莱尔多伊格为我们的殖民地维护方法的发展作出了贡献, 三名匿名的评论家为他们的建议, 帮助改进了原稿。我们的研究得到了国家卫生与医学研究理事会的项目赠款和奖学金, 以及惠康信托基金的翻译资助。PAR 是澳大利亚政府研究培训项目奖学金获得者。

由墨尔本大学人类伦理委员会 (批准 #: 0723847) 批准对人体的蚊子进行血液喂养。所有志愿者都提供了书面同意。
1. 幼体饲养
注: 蚊子被关押在26±0.5 ° c 和50-70% 相对湿度, 与 12:12 h (光照: 暗) 光周期为这个殖民地维护协议。这些条件与澳大利亚凯恩斯的平均气候条件相似, 在Ae. 蚊生存和发展的最佳热范围内,44,45,46。高温可能导致来自蚊子菌落的沃尔巴克感染的丢失, 应避免47。我们每个人口至少保持500人, 以尽量减少近亲繁殖;维护更小的大小的殖民地可能有健身后果 [罗斯et al.未发布]。在这些条件下, 假设足够的营养, 平均生成时间为28天 (参见表 1)。
<ol><li>将卵浸入基质中的托盘 (图 1A) 中, 包含3升水 (反渗透水或老化自来水, 通过在使用前24小时将自来水放入托盘中而产生), 〜300毫克的鱼食品 (一粒压碎的药片, 请参见材料表) 和少量的活性干酵母颗粒可诱发孵化48。</li>
	<li>在孵化后的一天, 使用玻璃吸管将大约500幼虫转移到含有4升水的托盘 (图 1B), 使用一个遥控器计数器计数。在每个托盘上加两片碎鱼食品。如果需要, 使用不同大小的容器饲养幼虫 (图 1A), 但保持幼虫密度低于0.5 幼虫/毫升, 以避免过度拥挤。</li>
	<li>每天检查托盘, 确保幼虫有足够的食物;每两天将大约两片食物放到托盘上。提供食物ad 随意但确保在这段时间内有0.5 毫克/幼虫/日可供开发, 以确保同步和身体尺寸一致, 否则实验结果可能会被混淆 (见代表性结果)。</li>
	<li>注意避免幼虫的过度, 特别是在较小的饲养容器中, 水的表面积和体积较少。如果水看起来混浊或者有明显的幼虫死亡, 用淡水代替它;如果以最佳方式喂养幼虫, 死亡率应可忽略不计。</li>
</ol>2. 成人诞生
注意: 幼虫在孵化后五天内将开始蛹, 如果喂养良好, 多数应在孵化后七天蛹。成人将开始出现大约二天在蛹以后, 如果保持优选地在26° c (参见代表性结果)。幼虫发育通常不受沃尔巴克感染的影响, 当充足的食物提供23,39,49。
<ol><li>孵化七天后, 将托盘的全部内容通过细网格 (孔径 0.4 mm) 浇注。将过滤后的幼体水保留在 ovicups (参见 "血液喂养和产卵" 部分)。倒置的网格和蘸入一个塑料容器与200毫升的水转移蛹。如果有幼虫存在, 提供额外的食物。</li>
	<li>准备成人出现的笼子 (图 1C), 提供两杯10% 蔗糖溶液 (图 1F) 和两杯潮湿的棉绒以防止干燥 (图 1E)。</li>
	<li>如果蛹不需要按性别分类, 将蛹的盖容器放入笼中, 并将盖子略微开半掩, 使成虫进入笼中。或者, 在容器上放置一个倒漏斗, 以尽量减少溺水。确保所有的成年人在从笼子里取出容器之前就已经出现, 以防止对慢速开发人员的选择。</li>
</ol>3. 交的蛹性别鉴定
<ol><li>如果蛹确实需要按性别 (例如, 用于交), 将蛹从幼虫盘中移除, 并将性别 (图 2) 分离成塑料容器 (图 1A), 每24小时200毫升水, 直到每个性别的所需数量已经达到。把盖子放在容器上, 让它们闭上。</li>
	<li>成人将出现在容器中;在释放到笼子之前确认他们的性别 (图 2C)。在成年前24小时内性不正确的器, 以达到性成熟。一旦性别得到确认, 每24小时将成年人释放到笼子里。</li>
	<li>
要获得与自然种群相似的遗传背景的沃尔巴克-受感染的菌落, outcross 通过在实验室菌落中添加沃尔巴克-受感染的雌性从产卵收集的卵子中提取的未感染雄性的笼子字段39, 保持每个填充的500个人的规定密度。
	<ol>
<li>重复交至少三个世代, 以产生至少87.5% 个与田间人口的相似的菌落39。关键: 确保在这个阶段的性别是正确的 (见步骤 3.1)。</li>
	</ol>
</li>
	<li>雌性Ae. 蚊通常是在交配后的几个小时内进行人工授精的50。当交殖民地时, 允许雌性和雄性在各自的笼子里成熟两天, 然后将雌性吸进雄性笼子, 为所有男性提供均等的机会。</li>
</ol>4. 供血和产卵
<ol><li>
等待至少三天后, 最后一个女性出现之前, 血液喂养, 让充裕的时间成熟。血液喂养女性在两周内出现, 以防止过度死亡, 特别是对蚊子与沃尔巴克感染的负面影响长寿22,24,49。在喂养前的前一天去掉糖杯, 以提高喂养率。
	<ol>
<li>请志愿者把前臂插入笼子, 让雌性蚊子喂食。大多数雌性应在5分钟内喂养饱食, 但为了减少对慢喂食者的选择, 将前臂留在笼中15分钟, 或直到所有雌性都明显肿胀;保护手不受叮咬的乳胶手套是可选的, 但推荐使用。</li>
		<li>在血液喂养两天后, 放置两个装有幼体养育水的塑料杯, 并衬一条砂纸 (图 1G) (或滤纸 (图 1H)) 进入笼子, 雌性产卵。部分淹没在水中的砂纸条, 以保持其湿润。除去其他水源, 防止雌鸟在产卵杯外产卵。 注: 自来水可在杯中使用, 但幼体饲养的水可鼓励产卵51,52 , 雌鸟会更同步地产卵。</li>
	</ol>
</li>
</ol>5. 蛋的收集和调理
<ol><li>雌鸟会在水线上方的砂纸上产卵;每天收集和更换的砂纸条, 直到没有更多的鸡蛋奠定。注意产卵可能持续长达一周。</li>
	<li>部分干燥的砂纸条, 轻轻地涂抹在纸巾上, 三十年代, 注意不要把鸡蛋。然后, 用一张干纸巾把小条裹好, 放到一个密封的塑料袋里 (图 1I)。</li>
	<li>在解剖显微镜下检查卵的状况 (图 3)。如果砂纸条太湿, 鸡蛋可能孵化之前被淹没在水中 (图 3B), 但如果干燥太严厉, 鸡蛋可能会崩溃 (图 3C)。</li>
	<li>卵可以在三天 post-collection 的任何时间孵化;孵化所有的鸡蛋从每个殖民地, 收集了所有的日子, 在同一容器的水, 以确保下一代是抽样从一个大的, 随机的个人池。</li>
	<li>为了长期贮存, 在20° c 左右的高度 (> 80%) 湿度保持在密封容器中的鸡蛋。在这些条件下, 没有沃尔巴克的鸡蛋可以保存几个月, 同时保持高孵化率53,54。</li>
	<li>由于某些沃尔巴克感染会大大降低与年龄相关的卵子的生存能力4955, 在收集一周内将卵从沃尔巴克感染的行中孵化出来, 以防止有关株.一周后, 如果需要更多的卵子, 血液就会再次喂养雌性。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>天</td>
			<td colspan="3">步</td>
		</tr>
<tr>
<td>0</td>
			<td colspan="3">孵化蛋</td>
		</tr>
<tr>
<td>1</td>
			<td colspan="3">将幼虫计数成托盘</td>
		</tr>
<tr>
<td>7</td>
			<td colspan="3">将幼虫和蛹转移到菌落笼</td>
		</tr>
<tr>
<td>17</td>
			<td colspan="3">血液喂养女性成人</td>
		</tr>
<tr>
<td>21</td>
			<td colspan="3">开始收集鸡蛋</td>
		</tr>
<tr>
<td>25</td>
			<td colspan="3">完成收集鸡蛋</td>
		</tr>
<tr>
<td>28</td>
			<td colspan="3">孵化蛋</td>
		</tr>
</tbody></table>表 1: Ae. 蚊殖民地维护计划的概述在26° c.雌性供血和卵孵化的时机是灵活的, 但应避免这些阶段的长时间, 特别是对感染了沃尔巴克的蚊子, 以减少死亡率。按照这一计划, 在所有生命阶段, 只要幼虫得到最佳喂养, 就可以最大限度地减少对蚊子的选择。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56124/56124fig1.jpg"> 图 1: 实验室中用于饲养Ae. 蚊的设备.(A) 用于孵化卵或饲养幼虫的塑料容器, 体积为500、750和5000毫升 (从左至右)。(B) 用于在受控密度下饲养幼虫的托盘, 通常为4升水中的500只幼虫。(C) 19.7 l 和 (D) 3 l 笼用于住房成人。应保持25成人或少于每公升的密度, 以提供足够的空间。(E) 35 毫升杯, 用湿棉绒作为水的来源, 供成年人使用。(F) 35 毫升杯, 蔗糖溶液通过脐带或牙科灯芯作为糖的来源提供。(G-H)盛满幼虫的水杯, 用砂纸或滤纸 (分别为G和H) 的产卵衬底。(I) 用于存储砂纸条或滤纸的拉链锁袋。砂纸上的黑点是蚊子的卵。<a href="//cloudflare.jove.com/files/ftp_upload/56124/56124fig1large.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/56124/56124fig2.jpg"> 图 2: 外侧 (a) 和背部 (B) 的蛹和成虫的意见Ae. 蚊(C) 展示其性异形.男性被安置在左边和女性在右边在每个小组。当最佳喂养时, 雄性和雌性蛹按大小区分;雌性比雄性大 (A), 相对于有平坦两侧 (B) 的雄性, 雌虫的头比较高。在所有的饲养条件下, 雄性成虫很容易与雌性区别开来, 主要由其羽触角和长触须。<a href="//cloudflare.jove.com/files/ftp_upload/56124/56124fig2large.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/56124/56124fig3.jpg"> 图3。四天旧Ae. 蚊鸡蛋在不同的条件下。(一个) 在砂纸条上保持完好的卵在高 (> 80%) 湿度, 但没有任何可见的湿气。如果正确维护了野生类型的Ae. 蚊, 则孵化率应高于90%。(B) 在被水淹没之前孵化的卵 (早熟孵化) 由分离的蛋帽和可见的幼虫区分。这表明砂纸条被保持太潮湿。(C) 干燥得太厉害的鸡蛋可能会倒塌, 而且它们的凹形外观明显可见。如果砂纸变得僵硬, 这也表明, 鸡蛋可能太干燥。<a href="//cloudflare.jove.com/files/ftp_upload/56124/56124fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

作者声明他们没有竞争的金融利益。

按照此处提供的用于维护沃尔巴克感染的Ae. 蚊的协议, 应确保为实验和公开的现场发布提供一致质量的健康蚊子。与其他优先生产蚊子数量的协议相比 (参见参考57), 这些方法的重点是通过实施宽松的饲养条件, 在几代人内最大限度地提高他们的身体素质, 并跨越世代通过减少近亲繁殖, 选择和实验室适应。此协议也专门为ae. 蚊和沃尔巴克感染而设计, 但...

伊蚊蚊蚊子负责传播世界上一些最重要的媒, 包括登革热、Zika 和基孔肯亚1。这些病毒正日益成为全球健康的威胁, 因为在热带地区的Ae. 蚊的广泛分布继续扩展2、34。雌性Ae. 蚊优先于人血的5 , 因此倾向于与人类紧密地生活在一起, 特别是在人口最稠密的城市地区。通过与人类的密切联系, 他们也适应在人工生境中繁殖, 包括轮胎、花盆、水槽和水箱6,7。Ae. 蚊还可以很容易地适应实验室环境, 在从字段直接收集后, 它们无需任何特殊要求即可进行维护, 这与伊蚊属属8中的其他物种不同, 9,10。他们的易于维护已经看到他们广泛的研究在实验室中的广泛领域, 最终旨在控制蚊子可能传播的疾病。
传统上, arboviral 控制在很大程度上依赖于杀虫剂的使用来减少蚊子的数量。然而, 越来越多的兴趣的方法, 在实验室饲养的改良蚊子, 然后释放到自然的人口。释放蚊子可以修改基因11,12,13, 生物14,15, 通过辐照16, 化学处理17,18,或者使用组合技术19来抑制蚊子的数量, 或者用不耐 arboviral 传输的蚊子来替代它们, 如20。
沃尔巴克是当前用作媒的生物控制代理的细菌,.最近在Ae. 蚊中引入了几株沃尔巴克, 实验使用胚胎微注射21,22,23,24。这些菌株降低了媒在蚊子中传播和复制的能力, 减少了它们的传输电位23,25,26,27,28.沃尔巴克感染由母亲传给后代 , 但某些菌株在感染的男性与未感染的女配时会导致不孕 ,22。因此,沃尔巴克-受感染的雄性可以大量释放, 以抑制自然蚊子的数量, 最近在其他的伊蚊属物种1529中演示了这一点。但是, 由于沃尔巴克还会在Ae. 蚊中抑制 arboviral 传输, 因此蚊子也可以被释放, 以更差的向量替换本地种群。使用沃尔巴克进行实验感染的Ae. 蚊现在正被释放到多个国家的字段中, 这些应用后一种方法是14,3031。
基于沃尔巴克的 arboviral 控制方法依赖于对沃尔巴克、蚊子和环境之间的交互的正确理解。沃尔巴克在广泛的昆虫中自然发生, 而引入蚊子的菌株在它们的效果中是多种多样的32。由于新的沃尔巴克感染类型被引入到Ae. 蚊24中, 因此有必要在各种情况下对其对蚊子的适应性、繁殖和 arboviral 干扰的影响进行描述。因此, 需要在实验室进行严格的试验, 以评估沃尔巴克菌株在该领域取得成功的可能性。
使用沃尔巴克感染的Ae. 蚊的开放字段版本通常需要每发布区域数千到数万蚊子, 每个星期都要饲养14,3031。初始释放的成功可以通过释放蚊子的大尺寸, 以最大限度地提高其繁殖力33和交配成功3435。蚊子也应该适应他们将在野外经历的条件, 但是长期的实验室饲养可能会导致行为和生理学的变化, 这可能会影响到现场性能36,37, 38。
我们描述了一个简单的协议, 在实验室中使用基本设备来饲养Ae. 蚊。该协议适用于野生型和沃尔巴克感染的蚊子, 后者可能需要特别注意, 因为有些沃尔巴克菌株对蚊子的生命有实质性影响-历史特征39,40. 饲养条件避免过度拥挤和争夺食物以生产出一致大小的蚊子, 这对媒介能力和体能实验都是至关重要的, 并确保蚊子在野外释放时是健康的41.我们还采取预防措施, 以减少选择压力和确保下一代从一个大的, 随机池取样, 以减少实验室适应和近亲繁殖。然而, 实验室环境与野外条件明显不同, 在放松条件下的长期维护可能会使蚊子在释放时的适应度降低到37,42,43.因此, 我们周期性地将雌性从实验室线交叉到野外收集的雄性, 从而在基因上类似于实验比较的菌落, 并适应目标领域的人口39。这些方法不需要任何专门的设备, 可以扩展到每星期为现场发布的数万人的后方。该议定书还优先考虑蚊子在几代人之内和不同的适宜性, 这是在自然种群中建立的昆虫的重要考量。该协议适用于大多数需要维护Ae. 蚊的实验室, 特别是在实验比较中, 蚊子和 relatability 的质量是很重要的。

<table><tbody><tr><td>Wild type &lt;em&gt;Aedes aegypti&lt;/em&gt;</td><td></td><td></td><td>Collected from field locations in Queensland, Australia, see Yeap and others&lt;sup class=&quot;xref&quot;&gt;39&lt;/sup&gt; for details</td></tr><tr><td>&lt;em&gt;w&lt;/em&gt; Mel-infected &lt;em&gt;Aedes aegypti&lt;/em&gt;</td><td></td><td></td><td>Provided by Monash University. Refer to Walker and others&lt;sup class=&quot;xref&quot;&gt;23&lt;/sup&gt; for information on the strain</td></tr><tr><td>&lt;em&gt;w&lt;/em&gt; AlbB-infected &lt;em&gt;Aedes aegypti&lt;/em&gt;</td><td></td><td></td><td>Provided by Monash University. Refer to Xi and others&lt;sup class=&quot;xref&quot;&gt;21&lt;/sup&gt; for information on the strain</td></tr><tr><td>&lt;em&gt;w&lt;/em&gt; MelPop-infected &lt;em&gt;Aedes aegypti&lt;/em&gt;</td><td></td><td></td><td>Provided by Monash University. Refer to McMeniman and others&lt;sup class=&quot;xref&quot;&gt;22&lt;/sup&gt; for information on the strain</td></tr><tr><td>Instant dried yeast</td><td>Lowan</td><td></td><td>Stimulates egg hatching. Found in general grocery stores. Other brands may be used</td></tr><tr><td>5 L plastic tub</td><td>Quadrant</td><td>Q110950</td><td>Used for hatching and rearing larvae. Other products may be used</td></tr><tr><td>Fish Food (Tetramin Tropical Tablets)</td><td>Tetra</td><td>16152</td><td>Provided to larvae as a source of food. Web address: https://www.amazon.com/Tetra-16152-TetraMin-Tropical-10-93-Ounce/dp/B00025Z6SE</td></tr><tr><td>Plastic containers</td><td></td><td></td><td>Used for rearing larvae. Any plastic container above 500 mL should be suitable</td></tr><tr><td>Glass pipette</td><td></td><td></td><td>Used for transferring larvae and pupae between containers. Web address: https://www.aliexpress.com/item/10Pcs-Durable-Long-Glass-Experiment-Medical-Pipette-Dropper-Transfer-Pipette-Lab-Supplies-With-Red-Rubber-Cap/32704471109.html?spm=2114.40010308.4.2.py4Kez</td></tr><tr><td>Clicker counter</td><td>RS Pro</td><td>710-5212</td><td>Used to assist in the counting of larvae, pupae and eggs. Web address: http://au.rs-online.com/web/p/products/7105212/?grossPrice=Y</td></tr><tr><td>Rearing trays</td><td>Gratnells</td><td></td><td>Used for rearing larvae. Web address: http://www.gratnells.com</td></tr><tr><td>Nylon mesh</td><td></td><td></td><td>Used to transfer larvae and pupae to containers of fresh water. Other brands may be used. Web address: https://www.spotlightstores.com/fabrics-yarn/specialty-apparel-fabrics/nettings-tulles/nylon-netting/p/BP80046941001-white</td></tr><tr><td>Cages</td><td>BugDorm</td><td>DP1000</td><td>Houses adult mosquitoes. Alternative products may be used. Web address: http://bugdorm.megaview.com.tw/bugdorm-1-insect-rearing-cage-30x30x30-cm-pack-of-one-p-29.html</td></tr><tr><td>35 mL plastic cup</td><td>Huhtamaki</td><td>AA272225</td><td>Used to provide water or sucrose to adult mosquitoes. Other brands may be used</td></tr><tr><td>35 mL plastic cup lid</td><td>Huhtamaki</td><td>GB030005</td><td>Used to provide sucrose to adult mosquitoes. Other brands may be used</td></tr><tr><td>Cotton wool</td><td>Cutisoft</td><td>71841-13</td><td>Moist cotton wool is provided as a source of water to adults. Other brands may be used</td></tr><tr><td>White Sugar</td><td></td><td></td><td>Provided as a source of sugar to adult mosquitoes. Found in general grocery stores</td></tr><tr><td>Rope</td><td>M Recht Accessories</td><td>C323C/W</td><td>Used to provide sucrose solution to adults. Other brands may be used. Web address: https://mrecht.com.au/haberdashery/braids-cords-and-tapes/cords/plaited-cord/cotton/</td></tr><tr><td>Plastic cup (large)</td><td></td><td></td><td>Used as an oviposition container. Any plastic cup that holds 100 mL of water should be suitable</td></tr><tr><td>Sandpaper</td><td>Norton Master Painters</td><td>CE015962</td><td>Provided as an oviposition substrate. Alternative products may be used, but we use this brand because it is relatively odorless. Lighter colors are used for contrast with eggs. Web address: https://www.bolt.com.au/115mm-36m-master-painters-bulk-roll-p80-medium-p-9396.html</td></tr><tr><td>Filter paper</td><td>Whatman</td><td>1001-150</td><td>Used as an alternative oviposition substrate. Other brands may be used</td></tr><tr><td>Latex gloves</td><td>SemperGuard</td><td>Z560979</td><td>Prevents mosquito bites on hands when blood feeding. Other brands may be used. Web address: http://www.sempermed.com/en/products/detail/semperguardR_latex_puderfrei_innercoated/</td></tr></tbody></table>

maintaining aedes aegypti mosquitoes infected with wolbachia

伊蚊蚊实验感染了沃尔巴克的蚊子被用于控制媒的传播, 如登革热、基孔肯亚和 Zika。沃尔巴克-受感染的蚊子可以被释放到田间, 通过不相容的交配减少人口的大小, 或者通过不耐病毒传播的蚊子来改变种群。为了使这些策略成功, 从实验室释放到野外的蚊子必须与本土蚊子竞争。然而, 在实验室中维持蚊子会导致近亲繁殖、遗传漂移和实验室适应, 从而减少他们在野外的体能, 并可能混淆实验结果。为了测试不同的沃尔巴克感染在该领域中的适用性, 有必要在多个世代的受控实验室环境中维护蚊子。我们描述了在实验室中维护Ae. 蚊蚊子的简单协议, 它适用于沃尔巴克感染和野生类型的蚊子。这些方法最大限度地减少实验室适应和实施交, 以提高实验对野外蚊子的相关性。此外, 殖民地是保持在最佳条件下, 以最大限度地提高他们的适用性开放的野外发布。

图 4演示了次优营养对Ae. 蚊幼虫发育的影响。当容器提供每只幼虫每天或少于0.25 毫克的食物时, 雄性和雌性的发育时间就会增加, 而且与0.5 毫克食物的容器相比, 它的同步性更低。如果在幼体发育的整个过程中没有提供足够的食物, 这可能会对维护计划产生不利影响。缓慢发展的个体有被选择的危险, 血液喂养可能被延迟, 并且有更高的风险成?...

Watch this Scientific Journal Video about Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia at JoVE.com

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

维护伊蚊蚊蚊子感染沃尔巴克

伊蚊蚊感染了沃尔巴克的蚊子被释放到自然种群中, 以抑制媒的传播。我们描述了将Ae. 蚊与沃尔巴克感染在实验室中进行实验和现场发布的方法, 并采取预防措施以尽量减少实验室的适应和选择。

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

maintaining-aedes-aegypti-mosquitoes-infected-with-wolbachia

Research

JoVE Journal

Biology

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

14.1K 次观看.  Bio21 Institute and University of Melbourne. 伊蚊蚊感染了沃尔巴克的蚊子被释放到自然种群中, 以抑制媒的传播。我们描述了将Ae. 蚊与沃尔巴克感染在实验室中进行实验和现场发布的方法, 并采取预防措施以尽量减少实验室的适应和选择。

视频: Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3. A control method known as RIDL&#160;(Release of Insects carrying a Dominant Lethal)4, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8. A RIDL&#160;strain of Ae. aegypti was successfully tested in the field in Grand Cayman9,10; further field use is planned or in progress in other countries around the world.
Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL&#160;program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program&#160;there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL&#160;control program,&#160;released as either pupae or adults11,12.
To suppress a mosquito population using RIDL&#160;a large number of high quality male adults need to be reared13,14. The following describes the methods for the mass rearing of OX513A, a RIDL&#160;strain of Ae. aegypti 8, for release and covers the techniques required for the production of eggs and mass rearing RIDL&#160;males for a control program.

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

To achieve population suppression of Aedes aegypti using the RIDL&#174; (Release of Insects carrying a Dominant Lethal) system, large numbers of male mosquitoes need to be released. This requires the use of mass rearing techniques and technology to provide reliable systems to obtain the maximum number of high quality male mosquitoes.

巴西田间放生转基因 伊 蚊的大规模生产

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the ...

科研

环境科学

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

维持细胞培养基的Wolbachia

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

生物学

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...

遗传学

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.

The protocols reported here illustrate three alternative ways to assess the performance of genetically-engineered mosquitoes destined for vector control in laboratory-contained small cage trials. Each protocol is tailored to the specific modification the mosquito strain bears (gene drive or non-gene drive) and the types of parameters measured.

基因工程按蚊的小笼实验室试验

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control ...

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation of RNA virus transmission using Wolbachia-transfected mosquitoes. Key strategies to control viruses include the manipulation of host reproduction via cytoplasmic incompatibility and the inhibition of viral transcripts via immune priming and competition for host-derived resources. However, the underlying mechanisms of the responses of Wolbachia-transfected mosquitoes to viral infection are poorly understood. This paper presents a protocol for the in vitro identification of Wolbachia infection at the nucleic acid and protein levels in Aedes albopictus (Diptera: Culicidae) Aa23 cells to enhance the understanding of the interactions between Wolbachia and its insect vectors. Through the combined use of polymerase chain reaction (PCR), quantitative PCR, western blot, and immunological analytical methods, a standard morphologic protocol has been described for the detection of Wolbachia-infected cells that is more accurate than the use of a single method. This approach may also be applied to the detection of Wolbachia infection in other insect taxa.

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

Four methods were used to detect intracellular Wolbachia, which complemented each other and improved the detection accuracy of Wolbachia infection of Aedes albopictus-derived Aa23 and Aa23-T&#160;cured of native Wolbachia infection using antibiotics.

检测白纹伊蚊细胞系中的沃尔巴克氏体菌株 wAlbB

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation ...

免疫与感染

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，<em&gt;白纹伊蚊</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

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 ...

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no prevention. Control of mosquito vectors can rely on chemicals applied to the immature and adult stages, which can contribute to the mortality of non-targets and more importantly, lead to insecticide resistance in the vector. The sterile insect technique (SIT) is a method of controlling populations of pests through the release of sterilized adult males that mate with wild females to produce non-viable offspring. This paper describes the process of producing sterile males for use in an operational SIT program for the control of Aedes aegypti mosquitoes. Outlined here are the steps used in the program including rearing and maintaining a colony, separating male and female pupae, irradiating and marking adult males, and shipping Aedes aegypti males to the release site. Also discussed are procedural caveats, program limitations, and future objectives.

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The sterile insect technique (SIT) is used to control specific, medically important mosquito populations that may be resistant to chemical controls. Here, we describe a method of mass rearing and preparation of sterile male mosquitoes for release in an operational SIT program targeting the Aedes aegypti mosquito.

准备辐照和标记的雄性 埃及伊蚊 ，以便在操作性昆虫技术计划中释放

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no ...

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for wild-type females and induce sterility in the target population. Hence, laboratory assessment of male mating competitiveness is essential for evaluating the release strain&#8217;s fitness before field release. Conventionally, such an assay is performed by determining the proportion of viable eggs produced by the females after being simultaneously exposed to two sets of males (wild-type and release strains) for copulation. However, this process is time-consuming and laborious due to the need to first blood-feed the females for egg production and then hatch and enumerate the hatched eggs to determine egg viability.&#160;
Moreover, this method cannot discern the degree of competitiveness between two sterile or Wolbachia-infected mosquito lines as wild-type female mosquitoes will only produce non-viable eggs upon mating with both. To circumvent these limitations, this paper describes a more direct method of measuring male mosquito mating competitiveness in laboratory settings using the fluorescent dye, rhodamine B (RhB), which can be used to mark males by feeding them in sucrose solution containing RhB. After the mating assay, the presence of fluorescing sperms in the spermathecae of a female can be used to determine her mating partner. This method is cost-effective, reduces the experimental time by 90% and allows comparison of mating fitness between two sterile or Wolbachia-infected lines.&#160;

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

Here, we present a protocol for studying mating competitiveness of male Aedes aegypti using fluorescent dye as a marker. Female mosquitoes are exposed to both marked and unmarked males for copulation. Post mating, their spermathecae are examined under a fluorescence microscope to determine their mating partner.

使用荧光染料，罗达明B，研究雄性 伊蚊 的交配竞争力

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for ...

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 ...

维护伊蚊蚊蚊子感染沃尔巴克

摘要

探索更多视频

此视频中的章节

巴西田间放生转基因伊蚊的大规模生产

维持细胞培养基的Wolbachia

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

基因工程按蚊的小笼实验室试验

检测白纹伊蚊细胞系中的沃尔巴克氏体菌株 wAlbB

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，

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

准备辐照和标记的雄性埃及伊蚊，以便在操作性昆虫技术计划中释放

使用荧光染料，罗达明B，研究雄性伊蚊的交配竞争力

量化转基因埃及伊蚊的适应性成本

维护伊蚊蚊蚊子感染沃尔巴克

摘要

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巴西田间放生转基因 伊 蚊的大规模生产

维持细胞培养基的Wolbachia

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

基因工程按蚊的小笼实验室试验

检测白纹伊蚊细胞系中的沃尔巴克氏体菌株 wAlbB

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，

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

准备辐照和标记的雄性 埃及伊蚊 ，以便在操作性昆虫技术计划中释放

使用荧光染料，罗达明B，研究雄性 伊蚊 的交配竞争力

量化转基因 埃及伊蚊 的适应性成本

巴西田间放生转基因伊蚊的大规模生产

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

准备辐照和标记的雄性埃及伊蚊，以便在操作性昆虫技术计划中释放

使用荧光染料，罗达明B，研究雄性伊蚊的交配竞争力

量化转基因埃及伊蚊的适应性成本