A mosquito feeding assay to examine Plasmodium transmission to mosquitoes using small blood volumes in 3D printed nano-feeders

BACKGROUND: To understand the dynamics of malaria transmission, membrane feeding assays with glass feeders are used to assess the transmission potential of malaria infected individuals to mosquitoes. However, in some circumstances, use of these assays is hindered by both the blood volume requirement and the availability of fragile, specially crafted glass feeders. 3D printed plastic feeders that require very small volumes of blood would thus expand the utility of membrane feeding assays. METHODS: Using two 3D printing production methods, MultiJet (MJ) and Digital Light Processing (DLP), we developed a plastic version of the most commonly used standard glass feeder (the mini-feeder) with an improved design, and also a smaller feeder requiring only 60 µl of blood (the nano-feeder). Performance of the 3D printed feeders was compared to standard glass mini-feeders by assessing infectivity of gametocytes to mosquitoes in standard membrane feeding assays with laboratory reared Anopheles stephensi mosquitoes and cultured Plasmodium falciparum gametocytes. In addition, the optimum number of mosquitoes that can feed on the nano-feeder was determined by evaluating fully fed mosquitoes visually and by assessing blood- meal volume with a colorimetric haemoglobin assay. RESULTS: The 3D printing methods allowed quick and inexpensive production of durable feeders. Infectivity of gametocytes to mosquitoes was comparable for MJ and DLP 3D printed feeders and glass feeders, and the performance of the 3D printed feeders was not influenced by repeated washing with bleach. There was no loss in transmission efficiency when the feeder size was reduced from mini-feeder to nano-feeder, and blood-meal volume assessment indicated ~10 An. stephensi mosquitoes can take a full blood-meal (median volume 3.44 µl) on a nano-feeder. CONCLUSIONS: Here we present 3D printed mini- and nano-feeders with comparable performance to the currently used glass mini-feeders. These feeders do not require specialized glass craftsmanship, making them easily accessible. Moreover, the smaller nano-feeders will enable evaluation of smaller blood volumes that can be collected from finger prick, thus expanding the utility of membrane feeding assays and facilitating a more thorough evaluation of the human infectious reservoir for malaria. [Image: see text]