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

项目摘要/摘要 拟议项目的总体目标是制定可行的策略来保存库蚊群落。 不需要持续的维护。库蚊属的蚊子物种是重要的媒介 虫媒病毒、丝虫和原生动物在世界范围内威胁着人类和动物的健康。尽管他们 作为病原体媒介的重要性，很少有实验室将研究重点放在库蚊上 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。库蚊在卵筏中的水面上产卵 由数百个蛋组成的。这些木筏对干燥或其他物理损坏很敏感，并且 一般留在水面上立即孵化成新的幼虫。一些实验室保留了1-3个菌落 库蚊物种持续多年，而其他实验室捕获当地的库蚊并保持群体 例如，整个夏天。相比之下，由于埃及伊蚊和伊蚊的保存 白纹伊蚊卵子放在滤纸上几个月，实验室可以可行地维持无数条线路，即使这些 没有被积极使用。对于其他昆虫物种，如果蝇，冷冻保存协议允许 在不损失这些菌落的情况下，长期储存大量转基因品系。 该项目的目的是为发育的不同阶段开发新的冷冻保存策略，评估 哪种方法是最有效、最安全且最终可行的方法，可以减少与 库蚊蚊子群落的维护。将采用两种不同的策略：1)细心 库蚊胚胎超低温冷冻保存方案的建立、测试与优化 温度(液氮)，以及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允许一种方法来产生 不同库雷克斯品系的银行供未来使用，而中期超低温保存可以帮助减少 维护多条线路并行的直接负担。 总体而言，该项目建立的方法学将对蚊子研究界具有很高的价值。 在维护库蚊群体方面的较少努力可能会导致在一定程度上增加对这些群体的研究 被忽视的载体，它将允许产生和可行的未来转基因库蚊品系的维护。 此外，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。

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.