ABSTRACT Drosophila melanogaster is one of the leading animal models for biomedical research, with researchers generating new lines of Drosophila every year for studies in numerous cancers, genetic disorders, and other maladies. Currently there are greater than 160,000 Drosophila stocks held at different stock centers around the world. As reliable and cost-effective approaches for long-term preservation of Drosophila stocks are lacking, individual labs and stock centers must maintain their lines as living populations, which is resource-intensive and puts the stocks at risk of loss. In 2021, our group developed an easily implemented and robust cryopreservation protocol for Drosophila melanogaster embryos which can be applied in any lab without the need for specialized instruments. This protocol is broadly applicable, and it has been successfully used to preserve 25 distinct strains from different sources. For most strains, >50% of the embryos hatch and >25% of the resulting larvae develop into fully functioning adults after cryopreservation and rewarming (normalized survival to control embryos), providing sufficient numbers of adults to revive the strain. To accelerate the cryopreservation and archival of critical Drosophila stocks, we will develop, standardize, and disseminate resources to individual labs and stock centers. Significant efforts will be devoted to developing reliable methods for cryogenic storage and shipping of Drosophila embryos as well as to study if any significant mutagenic changes occur due to cryopreservation. We will be working with multiple groups within the cryobiology and fly community to gather feedback on and refine our approach. With the collaboration of the Bloomington Drosophila Stock Center (BDSC), we will identify and prioritize the stocks that need cryopreservation to prevent their total loss in the event of a disaster or genetic drift. We will also demonstrate the efficacy of our cryopreservation protocol on a large scale with a selected group of stocks identified by BDSC. Finally, we will also estimate the costs for cryopreservation in comparison to the traditional method of Drosophila maintenance to inform future efforts to protect these precious genetic resources. This is a multi-PI project led by investigators with significant experience in Drosophila genetics and cryobiology.

PROJECT NARRATIVE The proposed project aims to further develop innovative, easy-to-follow protocols for cryopreserving Drosophila (fruit fly) embryos in labs and stock centers around the world. These protocols, which likely prevent the genetic drift common to live stock continuation, will be a highly useful tool for reliable and cost-effective long-term preservation of thousands of Drosophila stocks.