Surveillance of mosquito and arbovirus dispersal using smart microcrystals

使用智能微晶监测蚊子和虫媒病毒的传播

• 批准号: 9807932
• 负责人: Rebekah Crockett Kading
• 金额: $ 22.03万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-16 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9807932
• 关键词: Adult; Arboviruses; Arthropods; Back; Binding; Biological Markers; Biosensing Techniques; Biosensor; Capsid Proteins; Color; Confocal Microscopy; Crystallization; Culex (Genus); Culicidae; DNA; DNA sequencing; Data; Detection; Disease Vectors; Dust; Engineering; Entomology; Environment; Fluorescein; Fluorescence; Future; Geography; Habitats; In Vitro; Individual; Infection; Ingestion; Label; Larva; Length; Life; Location; Logistics; Mass Spectrum Analysis; Methodology; Methods; Molecular; Monitor; Movement; Nature; Oligonucleotides; Optics; Oral Ingestion; Outcome; Pathogen detection; Pattern; Performance; Population Density; Population Sizes; Protein Engineering; Proteins; RNA; Recovery; Reporting; Reproduction spores; Research Personnel; Signal Transduction; Standardization; Techniques; Technology; Testing; Time; Tissues; Trace Elements; Translational Research; Validation; West Nile virus; analytical method; cost; crosslink; crystallinity; design; disease transmission; epidemiologic data; field study; fitness; in vivo imaging system; millimeter; nanobiotechnology; novel; pathogen; protein crosslink; stable isotope; survivorship; synthetic construct; tool; vector; viral RNA; viral detection

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.

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， 决定着节肢动物疾病媒介的媒介能力。虽然这些研究是一种有价值的手段， 估计蚊子种群密度、生殖营养周期长度、种群大小、存活率和模式 尽管蚊子在整个环境中传播，但现有的技术和后勤挑战仍然存在， 方法论。最广泛使用的局部施用荧光粉的技术受到以下限制： 释放大量蚊子的必要性，标记技术对蚊子的潜在影响 健身，以及随着时间的推移缺乏持久性的痕迹。最近，微量元素和稳定同位素 已经提供了标记蚊子幼虫栖息地的自然方法，但是使用多个独特的标记物， 涉及质谱法的有限的分析方法通常成本过高。在本提案中，我们 将开发一种新的应用，用工程蛋白质微晶标记蚊子， DNA条形码无论是幼虫还是成虫，蚊子都会在野外吞食这些晶体， 一种独特的、信息丰富的DNA特征，可以通过PCR从监测池中扩增。我们 假设成年蚊子和幼虫蚊子都可以持续标记有独特的DNA条形码 通过摄入智能微晶。独特的条形码与地理参考诱饵站相关联， 从而通知标记获取的位置和时间， 监视陷阱我们还将把离体病原体生物传感能力整合到晶体中， 加强监控应用。我们假设，DNA信标安装在高浓度内 宿主晶体可以提供放大的光信号， 收集蚊子。预计的成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信标分子工程进入现有晶体， 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，我们将 优化微晶向成蚊和幼蚊的递送方法，并确定组织定位 和坚持。在第二年，我们将部署装有独特DNA条形码的智能微晶， 他们的现场能力，以报告季节性运动模式的Cx。跗骨蚊沿着栖息地走廊。 到目前为止，我们已经证明，DNA条形码可以存储，回收，并从晶体扩增， qPCR表明，条形码设计中使用的空聚体序列不扩增Cx的DNA。tarsalis或Ae. aegypti 蚊子（图2b），成年蚊子可以摄取载有荧光素的智能微晶， 随后，可以使用体内成像系统检测荧光（图5）。拟议的研究 为这项技术未来的大规模现场应用奠定基础。