Surveillance of mosquito and arbovirus dispersal using smart microcrystals

项目摘要 标记-释放-再捕获(MRR)研究是估计昆虫学变量的标准方法， 控制节肢动物病媒的传播能力。虽然这些研究是一种有价值的手段 估计蚊子种群密度、性腺营养周期长度、种群大小、存活率和模式 关于蚊子在整个环境中传播的问题，现有的技术和后勤挑战仍然存在 方法论。最广泛使用的局部应用荧光粉尘的技术受到 大量释放蚊子的必要性，标记技术对蚊子的潜在影响 体能，以及随着时间的推移缺乏毅力的印记。最近，微量元素和稳定同位素 提供了一种标记蚊子幼虫栖息地的自然方法，但使用多个独特的标记是 涉及质谱学的有限和分析方法通常成本过高。在这项提案中，我们 将开发利用携带独特基因的工程蛋白微晶体来标记蚊子的新应用 DNA条形码。蚊子在野外摄取这些晶体，无论是幼虫还是成虫，都会被标记为 一种独特的、信息丰富的DNA签名，可以通过从监测池中进行聚合酶链式反应进行扩增。我们 假设成虫和幼虫都可以被持久地标记上独特的DNA条形码 通过摄取智能微晶体。唯一的条形码与地理参考诱饵站相关联， 从而在蚊子被收集和分类时通知标记获取的位置和时间 监视陷阱。我们还将把体外病原体生物传感能力集成到晶体中，以进一步 增强监控应用。我们假设DNA信标在体内浓度很高 寄主晶体可以提供放大的光学信号，报告田间病原体的感染状态- 收集蚊子。预计结果包括从标记的条形码恢复的优化 蚊子，针对西尼罗河病毒(WNV)的DNA信标进入现有晶体的分子工程， 并在体外以及在含有西尼罗河病毒的蚊子匀浆中验证这些信标。此外，我们还将 优化微晶对成蚊和幼蚊的给药方法，并确定组织定位 和坚持不懈。在第二年，我们将部署装有独特DNA条形码的智能微晶进行测试 他们的实地能力，以报告Cx的季节性移动模式。栖息地走廊上的塔尔萨里蚊子。 到目前为止，我们已经证明了DNA条形码可以通过以下方式从晶体中存储、恢复和放大 QPCR，条形码设计中使用零体序列不会扩增Cx的DNA。Tarsalis或Ae.埃及伊蚊 蚊子(图2b)，成年蚊子可以摄取装有荧光素的智能微晶体，以及 随后，可以使用体内成像系统来检测荧光(图5)。建议进行的研究 为该技术未来的大规模现场应用奠定了基础。

Project Summary Mark-release-recapture (MRR) studies are a standard method for estimating the entomological variables that govern the vectorial capacity of arthropod disease vectors. While these studies are a valuable means of estimating mosquito population density, length of gonotrophic cycle, population size, survivorship, and patterns of mosquito dispersal throughout the environment, technical and logistical challenges remain with existing methodologies. The most widely-used technique of topically-applying a fluorescent dust is limited by the necessity to release large numbers of mosquitoes, the potential impact of the marking technique on mosquito fitness, and the lack of persistence of the marks over time. More recently, trace elements and stable isotopes have provided a natural means of labeling mosquito larval habitat, but the use of multiple unique markers is limited and analytical methods involving mass spectrometry are typically cost-prohibitive. In this proposal, we will develop the novel application of marking mosquitoes with engineered protein microcrystals carrying unique DNA barcodes. Mosquitoes ingesting these crystals in the field as either larvae or adults are thus marked with a unique and information-rich DNA signature that can be amplified by PCR from surveillance pools. We hypothesize that both adult and larval mosquitoes can be persistently marked with unique DNA barcodes through ingestion of smart microcrystals. Unique barcodes are associated with georeferenced bait stations, thereby informing the location and time of mark acquisition, when mosquitoes are collected and sorted from surveillance traps. We will also integrate ex vivo pathogen biosensing capability into the crystals to further enhance surveillance applications. We hypothesize that DNA beacons installed at high concentration within host crystals can provide an amplified optical signal that reports on the pathogen infection status of field- collected mosquitoes. Projected outcomes include the optimization of barcode recovery from marked mosquitoes, the molecular engineering of DNA beacons targeting West Nile virus (WNV) into existing crystals, and validation of these beacons in vitro as well as in mosquito homogenates containing WNV. Further, we will optimize the delivery method of microcrystals to adult and larval mosquitoes, and determine tissue localization and persistence. During year 2, we will deploy smart microcrystals loaded with unique DNA barcodes to test their field capacity to report the seasonal movement patterns of Cx. tarsalis mosquitoes along habitat corridors. To date, we have demonstrated that DNA barcodes can be stored, recovered, and amplified from crystals by qPCR, that nullomer sequences used in barcode design do not amplify the DNA of Cx. tarsalis or Ae. aegypti mosquitoes (Fig 2b), and that adult mosquitoes can ingest smart microcrystals loaded with fluorescein, and subsequently the fluorescence can be detected using an in vivo imaging system (Fig 5). The proposed studies lay the groundwork for future large-scale field applications of this technology.