Establishment of cryopreservation methods for sustainable maintenance of Culex spp. mosquito colonies

建立可持续维持库蚊属的低温保存方法。

• 批准号: 10183160
• 负责人: Claudia Rueckert
• 金额: $ 7.2万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-05 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10183160
• 关键词: Address; Adult; Aedes; Animals; Anopheles Genus; Arboviruses; Avian Malaria; Biology; Birds; California Encephalitis Virus; Communities; Conditioned Culture Media; Cryopreservation; Cryoprotective Agents; Culex (Genus); Culex pipiens; Culicidae; Development; Drosophila genus; Embryo; Equus caballus; Evaluation; Female; Fertility; Filaria bancrofti; Filarial Elephantiases; Freezing; Future; Generations; Great Plains; Hawaii; Hawaiian population; Health; Human; Infection; Injections; Insecta; La Crosse virus; Laboratories; Larva; Left; Liquid substance; Maintenance; Methodology; Methods; Microinjections; Midwestern United States; Nematoda; Nitrogen; Paper; Parasites; Partner in relationship; Phase; Plasmodium; Population; Protocols documentation; Public Health; Publications; Publishing; Research; Research Personnel; Resources; Risk; Scientist; St. Louis Encephalitis Virus; Standardization; Surface; Survival Rate; Technology; Temperature; Testing; Time; Transgenic Organisms; Virus; Water; West Nile virus; base; cold temperature; design; egg; embryo cryopreservation; enzootic; improved; neglect; novel; novel strategies; offspring; oocyte cryopreservation; pathogen; preservation; transmission process; vapor; vector; vector control; vector mosquito

Project Summary/Abstract The overall aim of the proposed project is to develop feasible strategies to preserve Culex mosquito colonies without continuous maintenance. Mosquito species of the genus Culex represent important vectors for arboviruses, filarial worms and protozoans threatening human and animal health worldwide. Despite their significance as pathogen vectors, few laboratories have focused their research efforts on Culex mosquitoes compared to Aedes or Anopheles. One likely reason for the lack of resources for Culex research is the need for continuous colony maintenance. Culex mosquitoes lay their eggs on the surface of water in egg-rafts consisting of hundreds of eggs. These rafts are sensitive to drying out or other physical damage and are generally left on the surface of water to hatch immediately into new larvae. Some labs keep colonies of 1-3 Culex species continuously for years, while other labs catch local Culex mosquitoes and keep colonies throughout the summer, for example. In contrast, due to the preservation of Aedes aegypti and Aedes albopictus eggs on filter paper for months, laboratories can feasibly maintain numerous lines even while these are not being actively used. For other insect species, such as Drosophila, cryopreservation protocols allow storage of a multitude of transgenic lines over long periods of time without loss of these colonies. The aim of this project is to develop new cryopreservation strategies for various stages of development, assess which methodology is the most efficient, safe and ultimately feasible way of reducing the effort associated with the maintenance of Culex mosquito colonies. Two different strategies will be applied: 1) the careful development, testing and optimization of a protocol for cryopreservation of Culex embryos at ultra-low temperature (liquid nitrogen), and 2) the evaluation of various medium-term cryopreservation methods for Culex mosquito larval and adult stages. Long-term cryopreservation of embryos would allow a way to generate banks of different Culex lines for future use, while medium-term cryopreservation could help reduce the immediate burden of maintaining multiple lines in parallel. Overall, the methodologies established in this project will be of high value to the mosquito research community. Less effort in the maintenance of Culex colonies may result in increased research focus on these somewhat neglected vectors and it will allow for the generation and feasible maintenance of future transgenic Culex lines. In addition, methods established here for Culex may help in the development of similar cryopreservation strategies for Anopheles or Aedes mosquitoes.

项目概要/摘要 拟议项目的总体目标是制定可行的策略来保护库蚊群落 无需持续维护。库蚊属蚊子是重要的传播媒介 虫媒病毒、丝虫和原生动物威胁着全世界人类和动物的健康。尽管他们的 库蚊作为病原体载体具有重要意义，但很少有实验室将研究重点放在库蚊身上 与伊蚊或按蚊相比。库蚊研究缺乏资源的一个可能原因是需要 用于持续的菌落维护。库蚊将卵产在水面的卵筏中 由数百个鸡蛋组成。这些筏对干燥或其他物理损坏很敏感，并且 通常留在水面上立即孵化成新的幼虫。一些实验室保留 1-3 个菌落 库蚊物种连续多年，而其他实验室则捕获当地库蚊并保留群落 例如，整个夏天。相比之下，由于保存了埃及伊蚊和伊蚊 白纹伊蚊卵在滤纸上放置数月，实验室可以维持大量的生产线，即使这些生产线 没有被积极使用。对于其他昆虫物种，例如果蝇，冷冻保存协议允许 长期储存大量转基因品系而不损失这些菌落。 该项目的目的是为不同的发展阶段制定新的冷冻保存策略，评估 哪种方法是减少与相关工作相关的工作量的最有效、最安全和最终可行的方法 库蚊群的维持。将应用两种不同的策略：1）谨慎 库蚊胚胎超低温冷冻保存方案的开发、测试和优化 温度（液氮），2）各种中期冷冻保存方法的评估 库蚊幼虫和成虫阶段。胚胎的长期冷冻保存将提供一种产生胚胎的方法 不同库蚊品系的库以供将来使用，而中期冷冻保存可能有助于减少 维持多条生产线并行的直接负担。 总体而言，该项目建立的方法对于蚊子研究界具有很高的价值。 减少库蚊群落维护工作可能会导致更多的研究关注这些问题 被忽视的载体，它将允许未来转基因库蚊品系的产生和可行的维持。 此外，这里为库蚊建立的方法可能有助于开发类似的冷冻保存 针对按蚊或伊蚊的策略。