CYROBIOLOGICAL PRESERVATION OF ANOPHELES EMBRYOS

疟疾每年感染超过2亿人，并导致近 200万人，其中大部分是儿童。两国之间相互作用的许多方面 按蚊和疟原虫处于遗传状态 由于这些原因，昆虫的分子遗传学是 深入研究的主题。分子遗传学方法 需要创建和分析数百条候选线以 获得具有所需基因特征的基因。步调 这类研究的进展受阻于即使大型实验室也无法 同时保持几个以上的繁育系。那个障碍 如果胚胎或幼虫的超低温保存 就像果蝇的胚胎一样。为了在任何形式的 冷冻时，细胞必须对水和低温都具有渗透性。 溶质。大多数符合这一要求的细胞可以通过以下方式进行超低温保存 慢慢冷却它们，以便渗透脱水防止致命 细胞内冻结。幼年按蚊卵对 水，对乙二醇类低温保护剂不透水。他们 可以像果蝇一样，通过接触庚烷而被渗透，但即使 那么它们就不能在缓慢的冷却中存活下来，因为它们表现出极端的 令人不寒而栗的敏感性。事实上，幼卵对寒冷非常敏感 他们的冷冻保存似乎是不可能的，即使使用非常高的 导致成功冷冻保存的冷却和升温速率 果蝇。较老的按蚊卵的温度要低得多。 它们很敏感，但对庚烷的渗透作用很难。 正庚烷去除卵黄膜上的蜡层。耐火材料 在较老的鸡蛋中出现屏障是由于酪氨酸的交联性 由酚氧化酶或过氧化物酶驱动的反应。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，我们一直并建议应用以下组合 化学、物理和遗传方法来破坏或绕过 较老的卵子不透水。建议的化学方法包括 间苯三酚和低氧分压阻碍酚氧化酶和 过氧化物酶反应。物理方法是电穿孔。这个 与疾病控制中心合作的遗传方法 将是创造和分离冈比亚按蚊的缺乏的突变体 在这些交叉连接的通路中，导致黑化的通路 鸡蛋。这种突变体已经在一种相关的 种，四斑按蚊。我们还建议与 美国国立卫生研究院寄生虫病实验室超低温保存 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.