CYROBIOLOGICAL PRESERVATION OF ANOPHELES EMBRYOS

疟疾每年感染2亿多人， 200万，大部分是孩子。 在许多方面， 按蚊和疟原虫的基因 控制，由于这些原因，昆虫的分子遗传学是 深入研究的课题。分子遗传学方法 需要创建和分析数百条候选线路， 获得具有所需遗传特征的基因。 步伐 即使是大型实验室也无法 同时保持多个繁殖系。这个障碍 如果胚胎或幼虫的冷冻保存被 就像果蝇胚胎一样。 在任何形式的 冷冻时，细胞必须对水和冷冻保护剂都是可渗透的 溶质。 大多数符合该要求的细胞可以通过以下方法冷冻保存： 缓慢冷却，使其渗透脱水， 细胞内冷冻 幼按蚊卵的渗透性很差， 水并且对于冷冻保护剂如乙二醇是不可渗透的。 他们 可以像果蝇一样，通过暴露于庚烷而渗透，但即使 那么它们就不能在缓慢冷却中存活，因为它们表现出极端的 激冷敏感性 事实上，年轻的卵对寒冷非常敏感 他们的冷冻保存似乎排除即使使用非常高的 冷却和升温速度，导致成功的冷冻保存 果蝇的 较老的按蚊卵基本上不那么冷 敏感，但它们对庚烷的透化反应是难处理的。 庚烷可以去除卵黄膜上的蜡层。 耐火 在较老的卵子中，由于酪氨酸的交联， 这些反应由酚氧化酶或过氧化物酶驱动。 我们有 发现没有办法显著降低年轻人对寒冷的敏感性， 鸡蛋，因此，我们已经并建议应用组合 化学、物理和遗传方法来破坏或规避 老鸡蛋的不渗透性。 建议的化学方法包括 间苯三酚和低氧张力阻碍酚氧化酶， 过氧化物酶反应物理方法是电穿孔。 的 与疾病控制中心合作的遗传学方法 将是创造和分离A.冈比亚缺乏 在这些交联途径中，导致黑色素沉着的途径 鸡蛋。 这样的突变体已经在一个相关的 四斑按蚊Anopheles quadrimaculatus 我们还建议与 美国国立卫生研究院寄生虫病实验室 冈比亚的幼虫。

Malaria infects over 200,000,000 people annually and kills nearly 2,000,000, mostly children. Many aspects of the interaction between the Anopheles mosquito and the Plasmodium parasite are under genetic control, and for these reasons the molecular genetics of the insect are the subject of intensive research. The molecular genetic approach requires the creation and analysis of hundreds of candidate lines to obtain ones possessing the desired genetic characteristics. The pace of such research is impeded by the inability of even large labs to maintain more than a few breeding lines concurrently. That impediment would largely disappear if cryopreservation of embryos or larvae were possible as it is with Drosophila embryos. To survive any form of freezing, a cell must be permeable to both water and cryoprotective solutes. Most cells that meet that requirement can be cryopreserved by cooling them slowly so that osmotic dehydration prevents lethal intracellular freezing. Young Anopheles eggs are poorly permeable to water and are impermeable to cryoprotectants like ethylene glycol. They can be permeabilized, like Drosophila, by exposure to heptane, but even then they can not survive slow cooling because they exhibit extreme chilling sensitivity. Indeed, the young eggs are so chill sensitive that their cryopreservation appears precluded even using the very high cooling and warming rates that led to the successful cryopreservation of Drosophila. Older Anopheles eggs are substantially less chill sensitive, but they are refractory to permeabilization by heptane. Heptane removes a wax layer in the vitelline membrane. The refractory barrier in older eggs appears due to the cross linking of tyrosine in that membrane, reactions driven by phenoloxidase or peroxidase. We have found no way to significantly reduce the chill sensitivity of young eggs, and, therefore, we have been and propose to apply combinations of chemical, physical, and genetic approaches to breach or circumvent the impermeability of the older eggs. The proposed chemical methods include phloroglucinol and low oxygen tensions to impede the phenoloxidase and peroxidase reactions. The physical method is electroporation. The genetic approach in collaboration with the Centers for Disease Control will be to create and isolate mutants of A. gambiae that are deficient in these cross-linking pathways, pathways that lead to melanization of the eggs. Such a mutant has been isolated in a related species, Anopheles quadrimaculatus. We also propose to collaborate with the Laboratory of Parasitic Diseases at NIH in the cryopreservation of A gambiae larvae.