Transgenic mosquitoes are used in many aspects of mosquito research, and convenient selection markers are crucial to identifying transgenic individuals even among large numbers of wild types. Visual markers, in the form of fluorescent proteins expressed in larval and adult mosquito tissues, are the most commonly used. This requires observing large numbers of mosquitoes under the fluorescence microscope and recognizing positive individuals expressing the fluorescent genetic marker. As research models, mosquito larvae possess the following two advantages over many other insects, greatly facilitating fluorescence screening: (1) Being aquatic, they can be isolated in a drop of clear water, an ideal medium for live observations under the binocular microscope; and (2) synchronous hatching from many eggs is easily obtained, so that large populations of larvae can be screened in batches of several tens of thousands at a time. Screening at the neonate stage, when larvae are ∼1-mm-long, allows the concentration of hundreds of larvae in a drop of water that fits in the observation field of the microscope at medium magnification. Thus, many individuals can be screened rapidly. We strongly recommend screening larvae at the neonate stage and before any feeding for two reasons, as follows: (1) Food particles can be strongly autofluorescent, thereby dramatically increasing the fluorescence background noise around larvae, and (2) tissue autofluorescence increases during development, notably in the digestive tract, significantly decreasing the specific signal-to-noise ratio. In this protocol, we guide the experimenter step-by-step for a fast and efficient medium-throughput manual screening for fluorescent larvae.
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