Transgenic mosquitoes are widely used in mosquito research. To distinguish transgenic individuals from wild types, genes for fluorescent proteins are the most commonly used genetic markers in transgenic constructs, offering all the advantages of visual selection. Although manual selection under a fluorescence binocular microscope is perfect for the selection of first-generation transgenics, managing established fluorescent lines can be facilitated by complex object parametric analyzer and sorter (COPAS) sorting, which we describe in this protocol. COPAS sorting allows researchers to purify large mosquito larval populations containing only homozygous transgenic individuals, only heterozygotes, or a mix of homozygous, wild types, and heterozygotes in desired proportions. Sorting large populations of a single sex is also possible. Finally, especially when several transgenes of different fluorescence colors are inserted in the same docking site (a recombination site previously inserted in the mosquito genome, which can be used to insert new transgenes into the same locus), they can be maintained together in a single mosquito population to save insectarium space and labor. COPAS sorting can then be used to extract the desired genotype when needed and to readjust transgene frequencies every few generations in case drift is observed.
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