维护伊蚊蚊蚊子感染沃尔巴克
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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<…
Representative Results
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?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Wild type Aedes aegypti | Collected from field locations in Queensland, Australia, see Yeap and others39 for details | ||
| w Mel-infected Aedes aegypti | Provided by Monash University. Refer to Walker and others23 for information on the strain | ||
| w AlbB-infected Aedes aegypti | Provided by Monash University. Refer to Xi and others21 for information on the strain | ||
| w MelPop-infected Aedes aegypti | Provided by Monash University. Refer to McMeniman and others22 for information on the strain | ||
| Instant dried yeast | Lowan | Stimulates egg hatching. Found in general grocery stores. Other brands may be used | |
| 5 L plastic tub | Quadrant | Q110950 | Used for hatching and rearing larvae. Other products may be used |
| Fish Food (Tetramin Tropical Tablets) | Tetra | 16152 | Provided to larvae as a source of food. Web address: https://www.amazon.com/Tetra-16152-TetraMin-Tropical-10-93-Ounce/dp/B00025Z6SE |
| Plastic containers | Used for rearing larvae. Any plastic container above 500 mL should be suitable | ||
| Glass pipette | 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 | ||
| Clicker counter | RS Pro | 710-5212 | Used to assist in the counting of larvae, pupae and eggs. Web address: http://au.rs-online.com/web/p/products/7105212/?grossPrice=Y |
| Rearing trays | Gratnells | Used for rearing larvae. Web address: http://www.gratnells.com | |
| Nylon mesh | 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 | ||
| Cages | BugDorm | DP1000 | 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 |
| 35 mL plastic cup | Huhtamaki | AA272225 | Used to provide water or sucrose to adult mosquitoes. Other brands may be used |
| 35 mL plastic cup lid | Huhtamaki | GB030005 | Used to provide sucrose to adult mosquitoes. Other brands may be used |
| Cotton wool | Cutisoft | 71841-13 | Moist cotton wool is provided as a source of water to adults. Other brands may be used |
| White Sugar | Provided as a source of sugar to adult mosquitoes. Found in general grocery stores | ||
| Rope | M Recht Accessories | C323C/W | 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/ |
| Plastic cup (large) | Used as an oviposition container. Any plastic cup that holds 100 mL of water should be suitable | ||
| Sandpaper | Norton Master Painters | CE015962 | 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 |
| Filter paper | Whatman | 1001-150 | Used as an alternative oviposition substrate. Other brands may be used |
| Latex gloves | SemperGuard | Z560979 | 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/ |
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Cite This Article
Ross, P. A., Axford, J. K., Richardson, K. M., Endersby-Harshman, N. M., Hoffmann, A. A. Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia. J. Vis. Exp. (126), e56124, doi:10.3791/56124 (2017).