A number of insects, for example, survive the winter by freeze
avoidance [71, 72, 184, 185], achieved by suppressing the presence
of ice-nucleating substances, synthesizing high concentrations of
cryoprotectants such as glycerol, and producing AFPs that bind to
ice and prevent it from growing (see also Subheadings 1.2 and 2.6
for more discussion of AFPs and ice blockers). In one case, that of
the larval Alaskan red flat bark beetle (Cucujus clavipes puniceus)
[185], more than half of the individuals tested supercooled to
below À60 to À70
C and none showed exotherms indicative of
freezing when cooled to À150
C in a DSC. All showed large whole
body glass transitions between À58 and À76
C (DSC curve inflection points; mean T G , À71
C). Two large larvae had a second small
T G at À96 or À98
C. When unselected larvae were cooled to
À71.5 Æ 1.5
C or to À100
C, the survival rates were about 50%
and 7%, respectively, although the latter rate was probably reduced
by mechanical damage sustained due to the methods used. In any
case, at least some larvae appear able to survive cooling to below
even the lowest observed T G s. Further, the coldest temperatures
recorded in nature (À79.8
C in Alaska [186], À89.2
C in air at
the Russian Vostok station in Antarctica [187], and most recently,
at another site in eastern Antarctica, ~À98
C on the ice surface and
À94 Æ 4
C in the overlying air [188]) are all below this insect’s
main T G and well below the highest measured T G of À58
C. This
suggests that some organisms have actually survived low temperatures in a vitreous state under natural conditions using endogenous
cryoprotectants similar in both molecular weight and concentration (in C. c. puniceus, up to 6.5 M glycerol was measured in the
cited study, and up to 10 M glycerol has been reported elsewhere
[189]) to those being used for artificial vitrification in cryobiological laboratories.
C. c. puniceus, in addition to elaborating protective substances,
concentrates them to vitrifiable levels in part by water loss to the
environment. Species whose water content varies with the ambient
humidity are said to be “poikilohydric” and can lose sufficient water
to induce cytoplasmic vitrification [144, 187, 190, 191]. These
partially or fully desiccation-tolerant organisms may make vitrification under natural conditions more common than survival by
freezing tolerance [187, 192]. As one example, soil nematodes
dried to below 0.3 g of water per g dry weight survived cooling
in liquid nitrogen and showed no evidence of freezing [193]. Vitrification by dehydration in anhydrobiotic organisms enables survival
at high temperatures as well as at low temperatures [13, 14],
although high-temperature survival in the vitreous state requires
specific adaptations in addition to the presence of vitrification per se
[194]. The lowest common terrestrial temperatures are typically
between about À30 and À60
C [185, 187], but the cells of many
species have been shown to have glass transition temperatures
above À50
C [71, 190, 195]. Twigs of Populus balsamifera were
42
Gregory M. Fahy and Brian Wowk
avoidance [71, 72, 184, 185], achieved by suppressing the presence
of ice-nucleating substances, synthesizing high concentrations of
cryoprotectants such as glycerol, and producing AFPs that bind to
ice and prevent it from growing (see also Subheadings 1.2 and 2.6
for more discussion of AFPs and ice blockers). In one case, that of
the larval Alaskan red flat bark beetle (Cucujus clavipes puniceus)
[185], more than half of the individuals tested supercooled to
below À60 to À70
C and none showed exotherms indicative of
freezing when cooled to À150
C in a DSC. All showed large whole
body glass transitions between À58 and À76
C (DSC curve inflection points; mean T G , À71
C). Two large larvae had a second small
T G at À96 or À98
C. When unselected larvae were cooled to
À71.5 Æ 1.5
C or to À100
C, the survival rates were about 50%
and 7%, respectively, although the latter rate was probably reduced
by mechanical damage sustained due to the methods used. In any
case, at least some larvae appear able to survive cooling to below
even the lowest observed T G s. Further, the coldest temperatures
recorded in nature (À79.8
C in Alaska [186], À89.2
C in air at
the Russian Vostok station in Antarctica [187], and most recently,
at another site in eastern Antarctica, ~À98
C on the ice surface and
À94 Æ 4
C in the overlying air [188]) are all below this insect’s
main T G and well below the highest measured T G of À58
C. This
suggests that some organisms have actually survived low temperatures in a vitreous state under natural conditions using endogenous
cryoprotectants similar in both molecular weight and concentration (in C. c. puniceus, up to 6.5 M glycerol was measured in the
cited study, and up to 10 M glycerol has been reported elsewhere
[189]) to those being used for artificial vitrification in cryobiological laboratories.
C. c. puniceus, in addition to elaborating protective substances,
concentrates them to vitrifiable levels in part by water loss to the
environment. Species whose water content varies with the ambient
humidity are said to be “poikilohydric” and can lose sufficient water
to induce cytoplasmic vitrification [144, 187, 190, 191]. These
partially or fully desiccation-tolerant organisms may make vitrification under natural conditions more common than survival by
freezing tolerance [187, 192]. As one example, soil nematodes
dried to below 0.3 g of water per g dry weight survived cooling
in liquid nitrogen and showed no evidence of freezing [193]. Vitrification by dehydration in anhydrobiotic organisms enables survival
at high temperatures as well as at low temperatures [13, 14],
although high-temperature survival in the vitreous state requires
specific adaptations in addition to the presence of vitrification per se
[194]. The lowest common terrestrial temperatures are typically
between about À30 and À60
C [185, 187], but the cells of many
species have been shown to have glass transition temperatures
above À50
C [71, 190, 195]. Twigs of Populus balsamifera were
42
Gregory M. Fahy and Brian Wowk