INTEGRATING BARCODED MICROCRYSTAL TECHNOLOGY WITH ONGOING WEST NILE VIRUS SURVEILLANCE

One overarching goal of Aim 1 is to develop this multiplexing pipeline such that barcode DNA obtained at multiple sites can be tagged with unique identifiers and quantified in a single multiplexed NGS experiment. Barcodes adapted to a next-generation sequencing platform will permit multiplexed detection of mosquitoes traveling from multiple locations captured in the same surveillance traps. Barcode particle synthesis & mosquito marking. Prior to the project starting we anticipate completing the synthesis of a modular library of 256 NGS-optimized barcode sequences. During the project we will scale up our production of the host porous protein crystals using shake flask and bioreactor protein production. Distinct synthetic barcodes will be loaded into protein microcrystal batches to create at least 125 distinct barcode batches. DNA loading efficacy will be quantified by quantifying supernatant DNA before and after loading. Wild mosquito larvae will be naturally exposed to DNA-loaded crystals during their immature, aquatic developmental stages. Emerging adult mosquitoes will be captured in CDC light traps operated by VDCI, and barcodes will be detected in mosquito homogenates alongside WNV screening at CSU. With the assistance of VDCI collaborators, we have identified 25 larval habitat sites throughout Fort Collins that have been historically productive for Culex mosquitoes. In particular, the east side of town around the Running Deer and Riverbend Ponds Natural areas will be targeted. Each site is georeferenced and will be assigned a unique series of barcodes (SourceTags). Plastic basins will be placed at each site to serve as our standardized larval bait station (mimicking our successful 2020 field trial). Habitat water will be added to each basin, and treated with crystals containing the assigned SourceTag for that location during the first week of June 2021. Sites will be visited weekly by study personnel to replenish water, supply a batch of barcode crystals, and to take a water sample for NGS barcode quantification. Using a total of 125 distinct barcode sequences, we will mark the same 25 sites for 5 consecutive weeks. By altering the sequence at each site we will encode temporal data in addition to location data. Control experiments: barcode persistence inside the standardized plastic basins. To assess the importance of the host crystals, 5 habitats will be treated with a second free (naked) barcode DNA sequence in addition to the crystal-adsorbed DNA.We will quantify the relative recovery of protected versus free barcode. Lastly, a subsample of immature mosquitoes found in the larval bait station basins will be brought back to rear in the lab and confirm SourceTag barcode presence upon emergence.Barcode recovery. Existing light trap locations in the surveillance network that are in closest proximity to the marked larval sites will be prioritized for barcode testing. Once mosquito trap collections are sorted into pools by VDCI and sent to CSU for WNV testing, an aliquot of mosquito homogenate from Culex pipiens and Cx. tarsalis pools will be reserved for DNA extraction and barcode testing. DNA will be extracted from homogenized mosquitoes using the Mag-Bind® Viral DNA/RNA Kit (Omega Bio-tek) on a KingFisher Flex extraction robot (ThermoFisher Scientific). Mosquito pools will be initially screened for barcodes by quantitative PCR. The Illumina flow cell binding sequence plus DNA sequences unique to each of the surveillance traps (TrapTag) will be annealed to the end of each recovered barcode. More than one TrapTag will be assigned to each trap, in the event there are multiple pools processed from that location and time. Samples will be sent to the University of Colorado Anschutz Medical Campus next generation sequencing core facility for library preparation and sequencing. Upon bioinformatics analysis of these returned data, each recovered sequence from a mosquito pool will encode both the SourceTag and the TrapTag, so we can associate and map the location of the larval breeding site and the associated collection location of the adult mosquito. Additionally, we will link these data with the WNV infection status (and Vector Index), determined by collaborating CSU labs. By utilizing the NGS platform for barcode recovery, we will also have the power to identify multiple SourceTags from the same surveillance traps. Furthermore, the library of 2021 barcodes will be different from future barcode libraries, such that we could test for the remarkable ability to identify mosquitoes marked in 2021 that overwinter and are captured in 2022.Sugar bait stations. Upon emergence from their larval habitat and throughout their lifespan, mosquitoes imbibe plant nectar as an energy source. Therefore, in addition to the widespread deployment of larval bait stations, we will also pilot-test attractive sugar bait stations for marking adult mosquitoes. By spiking an artificial sugar meal with barcoded microcrystals, we hope to validate an additional marking strategy that may be more conducive tospecies with more expansive, temporary, or obscure larval habitats.Environmental persistence of barcodes. In Aim 2, we will further test the persistence of barcodes in different habitat types following a single application. This is to be distinguished from our surveillance activities with mosquitoes (Aim 1). To evaluate environmental persistence, we will mark multiple container types in triplicate with unique SourceTags: a plastic backyard container, a discarded tire, and a cement container to mimic a culvert pipe. Matched, identical containers will be treated with the naked barcode amplicon only, not protected in the crystallized protein shell. We hypothesize that the crystal encasement is necessary for barcode persistence and recovery in harsh, natural conditions. Every three days for the first month, and every 15 days thereafter for at least 3 months, 200ul water samples will be taken from the middle and bottom of the water column of each container (to account for crystal sedimentation) and frozen at -20C. Barcodes will be extracted from water samples as described above, screened by qPCR, and sequenced.Project Management.The Snow lab (Department of Chemical and Biological Engineering)has strategically designed the barcode library to be used, and will manage the synthesis of these barcodes and ligating them into the unique library of 256 SourceTags. The Snow lab will also mass-produce microcrystals and load barcodes into them in support of both project Aims. This will require considerable coordination and effort, to generate unique SourceTags for all larval sites and TrapTags for the supporting network of CDC light traps, as well as producing enough volume of each tag for sufficient marking . The Snow lab will oversee the bioinformatic decoding of the returned data, but this is an area where student cross-training could enable participation from both labs. The Kading lab will manage project approvals, field site access, and the weekly distribution of the crystals to the larval habitats, as well as coordination of these activities with industry and local government partners. Once mosquito pools have been tested for WNV through the established surveillance program, the Kading lab will receive an aliquot of the homogenate and perform DNA extractions and an initial qPCR testing for barcode presence. NGS library preparation and sequencing will be outsourced. The Kading lab will also subsample larvae/pupae from each site for rearing in the insectary and molecular confirmation of the barcode from emerging mosquitoes.