Fig 7. Melanization of embryos over time.

Preferred melanization stages for injection are indicated with dotted brackets. (A) Aedes aegypti embryos are best injected between 45 min and 1 h 30 min, when they are getting darker, so their chorion is hardened enough to protect the embryo from bursting but not too hard that they would break the needle. Scale bar = 500 μm. Aedes aegypti (Liverpool strain, LMVR, NIH) gravid female mosquitoes were transferred to a 50-ml tube (Falcon, Fisher Scientific) with a humid filter paper on the bottom to promote egg laying. (B) Lutzomyia longipalpis takes longer to start melanizing. The time frame for optimal Lu. longipalpis embryo microinjections is between 1 h 30 min and 3 h after oviposition. Scale bar = 250 μm. For visualization of the melanization process, both Ae. aegypti and Lu. longipalpis were allowed to lay eggs for five min.