Malaria infects over 200,000,000 people annually and kills nearly 2,000,000, mostly children. Many aspects of the transmission of the Plasmodium parasite are under genetic influence of the Anopheles mosquito. For this reason, the molecular genetics of the insect is a subject of intensive research. One specific research area is to develop mutant lines of Anopheles with genes that block the development or transmission of the parasite, genes that would spread throughout the indigenous mosquito population when introduced into that population. Whether for this specific purpose or more generally, the molecular genetic approach usually requires the creation and analysis of hundreds of candidate lines to obtain one possessing the required genetic characteristics. The pace of such research is severely impeded by the inability of even large labs to maintain more than a few breeding lines concurrently. That impediment would largely disappear if cryopreservation were possible. This proposal is concerned with achieving that goal. The likelihood of success has been enhanced by recent success in cryopreserving Drosophila embryos. However, although Anopheles and Drosophila embryos exhibit cryobiologically relevant similarities, our recent research indicates there are also important differences. To survive cryogenic temperatures, cells must be permeable to both water and cryoprotective solutes. Neither Drosophila nor Anopheles embryos meet this requirement. Drosophila and Anopheles can both be rendered permeable by exposure to alcohol and alkanes. Although innocuous to the former, the treatment is unfortunately lethal to the latter because, paradoxically, their water permeability is too high. We now, however, have achieved survival of permeabilized young Anopheles embryos by substituting air-drying for alcohol. A second serious problem with both Drosophila and Anopheles is that they exhibit high sensitivity to chilling even in the absence of freezing. High sensitivity to chilling forces one to cryopreservation by the more demanding vitrification approach. Older Anopheles embryos are much less chill sensitive but they are refractory to the permeabilization procedures effective in younger embryos. We propose approaches to circumventing the chill sensitivity of younger embryos and we propose a number of sequential physical and chemical approaches to permeabilizing older embryos. The physical approaches include bombardment with alpha particles and electroporation. The chemical approaches include extraction with supercritical CO2.
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