Permeabilization of eggs of the malaria mosquito Anopheles gambiae

Although Anopheles gambiae embryos that have developed for 15 h at 17 degrees C are slightly permeable to water, they are impermeable to ethylene glycol, the cryoprotectant used in the cryopreservation of Drosophila melanogaster embryos. Success in cryopreservation requires that they be made permeable to protective solutes. Permeabilization of D. melanogaster was achieved by 1) dechorionation with 50% Clorox (household bleach) followed by 2) a water flush; 3) immersion in isopropyl alcohol to remove most extraembryonic water; 4) 2 min air drying to remove most isopropyl alcohol; 5) 90-s exposure to n-heptane containing 0.3% 1-butanol; and 6) 15-s exposure to pure n-heptane. The permeability of A. gambiae embryos was assessed by determining the times required for the initial dehydration of embryos in 1 M ethylene glycol in 260 mOsm Drosophila culture medium (permeability to water) and the times required for their return to normal volume (permeability to ethylene glycol). Based on these criteria, the above D. melanogaster procedure effectively permeabilizes 15 h/17 degrees C to 19 h/17 degrees C A. gambiae embryos. Nearly all collapsed in <5 min, and most returned to normal volume in approximately 40 min. Although permeable, all were killed by the permeabilization procedure. In analyzing the effect of each step on viability, 50% Clorox caused some lethality, and Clorox followed by isopropyl alcohol was 100% lethal. Decreasing the Clorox concentration to 10% still dechorionated eggs, but with reduced toxicity; substitution of a 10% solution of reagent-grade sodium hypochlorite for Clorox further decreased toxicity. The isopropyl alcohol step was also toxic. Consequently, the removal of residual surface water was achieved by substituting air drying for isopropyl alcohol. The drying took place under direct microscope observation and was continued until the embryos began to shrink. The combination of this modified method for dechorionation and controlled air drying before exposure to heptane resulted in permeabilization of all embryos in most runs and in approximately 30% survival of the permeabilized embryos.