Characterization of intraembryonic freezing in Anopheles gambiae embryos

Intraembryonic freezing (IEF) in Anopheles mosquito embryos has been evaluated by differential scanning calorimetry with respect to embryo age, temperature, rate and duration of cooling, and absence or presence of extraembryonic ice. The initial temperatures for intraembryonic ice nucleation were -30.1 ± 0.3, -28.4 ± 0.4, and -29.1 ± 0.2°C for embryos incubated for 15 h at 17°C, 15 h at 26°C, and 24 h at 26°C, respectively, after oviposition. The first value is slightly but significantly lower than the latter two. These values were obtained on embryos in which the surface water was removed by brief drying; however, the values were nearly identical when external water and ice were present. Not only were the embryos of all three ages able to supercool at least transiently to -26°C, but they could remain supercooled for up to 4 h at -20°C after being cooled to -20°C at 10°C/min or (in the case of embryos incubated for 15 h at 26°C) at 100°C/min. The amount of freezable water in single embryos has been calculated from the differential scanning calorimetry measurements to be 3.45 ± 0.08, 3.46 ± 0.08, and 3.53 ± 0.06 μg for embryos incubated for 15 h at 17°C, 15 h at 26°C, and 24 h at 26°C, respectively. The differences are not significant. The corresponding values for the total water contents for embryos of the three ages were 4.04 ± 0.20, 3.72 ± 0.16, and 3.98 ± 0.10 μg, values that also did not differ significantly. Water thus makes up ∼74% of the total weight of the embryo (∼5.3 μg) and about 91% of that water is freezable. Total water contents were determined gravimetrically after extensive air and vacuum drying. The kinetics of dehydration were determined during the air drying. They differed substantially among the three ages. The embryos incubated for 15 h at 17°C lost water at about four times the rate of those incubated for 15 h at 26°C and 10 times the rate of the embryos incubated for 24 h at 26°C.