Digital image analysis to estimate numbers of Aedes eggs oviposited in containers

Monitoring reproductive rates in experiments involving aedine mosquitoes is tedious and time demanding. Here, we demonstrate a protocol for rapid estimation of aedine mosquito egg number. The protocol uses ImageJ, a publicly available image analysis program developed at the U.S. National Institutes of Health. The method relies upon the oviposition behavior of Aedes (i.e., ovipositing on a moist substrate instead of water surface) and upon the contrast between dark-colored aedine eggs and a light, uniformly colored paper that is used as an oviposition substrate. The results for 3 Aedes species show that, following the generation of separate calibration curves for each species, the protocol allows for the accurate and repeatable estimation of samples containing hundreds of aedine eggs. We discuss the use of the protocol for monitoring immature aedine populations in both laboratory and field applications.