CHARACTERIZATION OF INTRAEMBRYONIC FREEZING IN ANOPHELES-GAMBIAE EMBRYOS

Intraembryonic freezing (IEF) in Anopheles mosquito embryos has been e valuated by differential scanning calorimetry with respect to embryo a ge, temperature, rate and duration of cooling, and absence or presence of extraembryonic ice. The initial temperatures for intraembryonic ic e nucleation were -30.1 +/- 0.3, -28.4 +/- 0.4, and -29.1 +/- 0.2 degr ees C for embryos incubated for 15 h at 17 degrees C, 15 h at 26 degre es C, and 24 h at 26 degrees C, respectively, after oviposition. The f irst value is slightly but significantly lower than the latter two. Th ese values were obtained on embryos in which the surface water was rem oved by brief drying; however, the values were nearly identical when e xternal water and ice were present. Not only were the embryos of all t hree ages able to supercool at least transiently to -26 degrees C, but they could remain supercooled for up to 4 h at -20 degrees C after be ing cooled to -20 degrees C at 10 degrees C/min or (in the case of emb ryos incubated for 15 h at 26 degrees C) at 100 degrees C/min. The amo unt 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 mu g for embryos incubated for 15 h at 17 degrees C, 15 h at 26 degrees C, and 24 h at 26 degrees C, respecti vely. The differences are not significant. The corresponding values fo r the total water contents for embryos of the three ages were 4.04 +/- 0.20, 3.72 +/- 0.16, and 3.98 +/- 0.10 mu g, values that also did not differ significantly. Water thus makes up similar to 74% of the total weight of the embryo (similar to 5.3 mu g) and about 91% of that wate r is freezable. Total water contents were determined gravimetrically a fter extensive air and vacuum drying. The kinetics of dehydration were determined during the air drying. They differed substantially among t he three ages. The embryos incubated for 15 h at 17 degrees C lost wat er at about four times the rate of those incubated for 15 h at 26 degr ees C and 10 times the rate of the embryos incubated for 24 h at 26 de grees C. (C) 1996 Academic Press, Inc.