Project Summary/Abstract The overall aim of the proposed project is to develop feasible strategies to preserve Culex mosquito colonies without continuous maintenance. Mosquito species of the genus Culex represent important vectors for arboviruses, filarial worms and protozoans threatening human and animal health worldwide. Despite their significance as pathogen vectors, few laboratories have focused their research efforts on Culex mosquitoes compared to Aedes or Anopheles. One likely reason for the lack of resources for Culex research is the need for continuous colony maintenance. Culex mosquitoes lay their eggs on the surface of water in egg-rafts consisting of hundreds of eggs. These rafts are sensitive to drying out or other physical damage and are generally left on the surface of water to hatch immediately into new larvae. Some labs keep colonies of 1-3 Culex species continuously for years, while other labs catch local Culex mosquitoes and keep colonies throughout the summer, for example. In contrast, due to the preservation of Aedes aegypti and Aedes albopictus eggs on filter paper for months, laboratories can feasibly maintain numerous lines even while these are not being actively used. For other insect species, such as Drosophila, cryopreservation protocols allow storage of a multitude of transgenic lines over long periods of time without loss of these colonies. The aim of this project is to develop new cryopreservation strategies for various stages of development, assess which methodology is the most efficient, safe and ultimately feasible way of reducing the effort associated with the maintenance of Culex mosquito colonies. Two different strategies will be applied: 1) the careful development, testing and optimization of a protocol for cryopreservation of Culex embryos at ultra-low temperature (liquid nitrogen), and 2) the evaluation of various medium-term cryopreservation methods for Culex mosquito larval and adult stages. Long-term cryopreservation of embryos would allow a way to generate banks of different Culex lines for future use, while medium-term cryopreservation could help reduce the immediate burden of maintaining multiple lines in parallel. Overall, the methodologies established in this project will be of high value to the mosquito research community. Less effort in the maintenance of Culex colonies may result in increased research focus on these somewhat neglected vectors and it will allow for the generation and feasible maintenance of future transgenic Culex lines. In addition, methods established here for Culex may help in the development of similar cryopreservation strategies for Anopheles or Aedes mosquitoes.