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.
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