An Efficient Rearing and Microinjection Protocol for Genetic Modification of the Stable Fly, Stomoxys calcitrans

Stable flies are blood-feeding pests of livestock, companion animals, and humans, and are found on all continents except Antarctica. The painful bites of both male and female adults induce stress-related behaviors in livestock, resulting in weight loss and substantial economic losses for producers. Both male and female stable flies are obligate blood-feeders, which poses a challenge to sterile insect technique and other pest management strategies that require the mass release of males. Current stable fly pest management strategies often fail to adequately control fly populations. Although numerous genetic manipulation methods for introducing exogenous genetic material or inducing gene knockouts have been developed for mosquitoes, Drosophila, and other model insect groups, methods for the genetic transformation of stable flies remain limited. This protocol describes a reliable and improved approach for introducing foreign genetic material into stable flies using a hyperactive variant of the piggyBac transposase and a novel solid larval rearing medium that supports larval recovery, hatching, feeding, and fluorescent screening after embryonic microinjection.