Jové Veronica, Venkataraman Krithika, Gabel Thomas M, Duvall Laura B
Laboratory of Neurogenetics and Behavior, The Rockefeller University.
Department of Biological Sciences, Columbia University.
J Vis Exp. 2020 Oct 22(164). doi: 10.3791/61835.
Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females. Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito. These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.
某些蚊种的雌性蚊子在叮咬脊椎动物宿主以获取卵子发育所需的富含蛋白质的血餐时，会传播疾病。在实验室中，研究人员可以通过膜饲器向蚊子提供动物源性和人工血餐，从而能够控制餐食成分。在此，我们介绍了向埃及伊蚊喂食血液和人工血餐并量化单个雌性蚊子摄入量的方法。对人工/血餐进行有针对性的喂食和量化具有广泛的用途，包括测试餐食成分对蚊子行为和生理的影响、无需注射即可递送药理化合物以及用特定病原体感染蚊子。在喂食前向餐食中添加荧光素染料，以便后续对餐食大小进行量化。如果雌性蚊子随后要用于行为实验，可以通过称重来测量蚊子摄入的餐食体积，或者将单个雌性蚊子在96孔板中匀浆，并使用酶标仪测量荧光水平作为终点测定法来测量。餐食大小量化可用于确定改变餐食成分是否会改变摄入的餐食体积，或者不同蚊子品系之间的餐食摄入量是否存在差异。精确的餐食大小量化对于下游测定也至关重要，例如那些测量对宿主吸引力或繁殖力影响的测定。这里介绍的方法可以进一步调整，以跟踪数天内餐食的消化情况，或者包括添加到不同餐食（如花蜜和血液）中的多个可区分标记，以量化单个蚊子对每餐的摄入量。这些方法使研究人员能够独立进行高通量测量，以比较数百只单个蚊子摄入的餐食体积。因此，这些工具对于蚊子研究群体回答各种生物学问题将具有广泛的用途。
