Integrating barcoded microcrystal technology with ongoing West Nile virus surveillance

A number of viruses significant to animal health are transmitted by biting arthropods. Encephalitic arboviruses such as West Nile virus, Eastern, Western, and Venezuelan equine encephalitis viruses are transmitted by mosquitoes and lethal to horses. Rift Valley fever virus, which has not yet invaded the United States, is a mosquito-borne virus at risk of introduction that causes significant morbidity and mortality in cattle and other small ruminants. Biting midges transmit blue-tongue virus to sheep and cattle, and epizootic hemorrhagic disease to deer. In addition to pathogen transmission, biting flies such as stable flies, horn flies, deer flies, and horse flies cause significant irritation to livestock, could mechanically vector infectious agents, and reduce feeding and grazing behaviors,all of which which negatively impacts their health. Understanding how vectors andpathogens move around the landscape, including identification of primary breeding sources of vectors and where virus amplification is taking place,is important to disease surveillance and mitigation efforts, but difficult to accomplish.Currently, one of the most standard and comprehensive tools entomologists use to measure arthropod vector movement in nature is mark-release-recapture (MRR). By marking a subsample of vectors in the environment and monitoring their recapture rates and distances from release site, MRR offers a standard approach to gathering this epidemiologically-significant information on vector behavior and ecology directly from field populations. Despite their utility,MRR studies represent a research area that has posed significant challenges to entomologists for decades. For mosquito dispersal studies, topical fluorescent powders and paints, ingestible dyes, or larval habitat marking with rubidium or stable isotopes are typically used. Despite being the most popular and cheapest option, fluorescent powders are difficult to use for large numbers of mosquitoes, they have limited surface stability on the mosquito, and they can introduce biases by negatively affecting mosquito behavior and survivorship. Mosquitoes reared from larval habitats enriched in stable isotopes or rubidium can be detected via mass spectrometry or x-ray fluorescence spectrophotometry, respectively. Nevertheless, these methods only provide a handful of distinguishable markers, and detection via mass spectrometry is expensive and training intensive.To overcome these challenges, we have developed a new class of information-dense MRR markers based on synthetic DNA barcodes. Our method utilizes durable microscopic particles that are composed of crosslinked proteins, similar to bacterial spores which protect DNA under hostile conditions. These particles have an array of nanopores suitable to sequester and protect DNA, release DNA under the right settings, and retain stability under a wide range of environmental conditions. Host crystals will be loaded with synthetic DNA barcodes that can be tailored to encode vital information needed for MRR studies (i.e. a time point, a geographic location, etc.). We hypothesize that integration of DNA-loaded microcrystals into an existing arbovirus surveillance programs will reveal critical mosquito production and movement patterns that can be exploited to suppress the mosquito populations responsible for arbovirus transmission. Knowledge of mosquito movement data in the context of arbovirus surveillance will identify the hot spots of local mosquito production, directional movement of these mosquitoes following emergence, and relative contribution to the infectious mosquito population. Once this approach has been validated in an existing mosquito surveillance program, this will open the door toexpansion into additional vector-borne diseasesystems. This information will allow for the prioritized implementation of limited vector control resources to prevent the spread of viruses important to human and animal health.Aim 1. Utilize information rich microcrystals to identify local mosquito breeding hot spots. Historically productive Culex mosquito breeding sites in the City of Fort Collins, CO will each be marked with microcrystals containing a unique DNA barcode and georeferenced. Mosquitoes emerging from these sites will be identified by molecular detection of barcode signatures from surveillance trap collections concurrent to WNV infection status determination. Mosquito marking and capture sites will be mapped to visualize a spatial network of mosquito movement and WNV detections within the City over time. We will also pilot the deployment of attractive sugar bait stations targeting adult mosquitoes.Aim 2. Evaluate environmental persistence of crystals and enclosed barcode DNA in natural habitats The environmental stability of microcrystals placed in larval breeding habitats (i.e. backyard containers, tires, culvert pipe) will determine the frequency of crystal application to maintain efficacious mosquito marking in future practice. Sentinel containers will be marked once and re-sampled weekly throughout the summer to quantify barcode DNA persistence despite harsh environmental conditions.