Project Summary Leishmaniasis is the second biggest parasitic killer after malaria. It is estimated to kill an estimated 40,000 people every year, and accounts for an estimated 2.4 million disability-adjusted life-years (DALYs). The Leishmania parasite is transmitted to humans when female sand flies bite humans to obtain blood meals that they require for reproduction. A better understanding of sand fly biology and populations is important for the control and monitoring of this disease vector. This proposal will generate a high-quality genome references to support future sand fly and Leishmania control efforts. New and ongoing methods of integrated vector control are improved by genomic analysis. Descriptions of species complexes, estimation of effective population sizes, monitoring of pesticide resistance allele emergence, and assessment of changes in population structure can all be accomplished using genetic analyses. New vector control methods based on CRISPR-Cas9 manipulation to create gene drives to prevent transmitting malaria and microbiome manipulation with Wolbachia to prevent transmission of dengue are being trialed in mosquitoes. Unfortunately, our early attempts to create high-quality genome reference resources for sand flies were not successful. This study will generate new, very high-quality reference genome sequences for two critical sand fly species: Lutzomyia longipalpis, found in the New World, is the primary vector of American visceral leishmaniasis and Phlebotomus papatasi, found in the Old World, including northern Africa, the Middle East and India, is a vector of cutaneous leishmaniasis. New DNA sequencing and genome assembly techniques have improved the quality and reduced the cost to generate new reference genomes. This study will generate multiple high-quality DNA sequence information datasets for new high-quality genome assemblies. The primary sequence information will be long read (10-15 thousand bases) high accuracy (> 99.8%) DNA sequences using new DNA sequencing platforms from a single individual of each species. HiC chromatin sequence data will be generated to understand the relative chromosome position of the sequences. Optical mapping data will enable better scaffolding and validation of the final assembly. A genome assembly software pipeline based on the vertebrate genome assembly pipeline currently generating the highest quality reference genome assemblies will be used to assemble the data as much as possible separating haplotypes. Long read high accuracy transcriptome data will be generated to support a high-quality genome annotation, and the sand flies' microbiomes to fully characterize the environment of Leishmania in these vectors. The final product will be a high-quality genome reference foundation for future efforts to monitor and control the vector of this devastating disease.