Comparative overwintering physiology of Alaska and Indiana populations of the beetle Cucujus clavipes (Fabricius): roles of antifreeze proteins, polyols, dehydration and diapause
1 Department of Biological Sciences, University of Notre Dame, Notre Dame,
IN 46556, USA
2 Institute of Arctic Biology, University of Alaska, Fairbanks, AK 49775,
USA
3 Sycamore Community High School, 7400 Cornell Road, Cincinnati, OH 45242,
USA
4 Department of Chemistry and Biochemistry, University of Notre Dame, Notre
Dame, IN, 46556, USA
Author for correspondence (e-mail:
duman.1{at}nd.edu)
Accepted 20 September 2005
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Summary |
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Key words: beetle, insect, cold tolerance, antifreeze protein, subzero adaptation, vitrification, Cucujus clavipes
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Introduction |
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The beetle Cucujus clavipes (Cucujidae) has a broad latitudinal
range, from North Carolina (35°N) to northern interior Alaska above
the Arctic circle (
67°30' N). Consequently, this species
presents the opportunity to study overwintering physiology over a large
latitudinal expanse, including one of the coldest environments in North
America. Previous studies of overwintering adaptations of an Indiana
population of C. clavipes demonstrated the activity of AFPs and mean
lower lethal temperatures in winter ranging from 18 to 25°C
(Duman, 1979
,
1984
). Interestingly, C.
clavipes were freeze tolerant during the winter of 1979, but by 1983 they
had altered their overwintering mechanism to freeze avoidance
(Duman, 1984
). In contrast to
the Indiana population, Miller
(1982
) reported lower lethal
temperatures of C. clavipes from interior Alaska of 55°C
or colder. The supercooling points of the Alaskan insects were not reported,
and whether they were freeze tolerant or freeze avoiding was not known. Miller
did not screen the Alaskan insects for the presence of AFPs. The goal of our
present study was to compare the overwintering adaptations of populations of
C. clavipes from the northern limit of their range in arctic and
subarctic Alaska with those near the southern end of their range in northern
Indiana. Special attention was given to the role of antifreeze proteins
because AFPs had not previously been studied in Alaskan insects, even though
they are now known to be common in Alaskan terrestrial arthropods
(Duman et al., 2004
).
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Materials and methods |
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Cucujus clavipes (Coleoptera: Cucujidae) larvae were collected from each of these populations at various times in the year between 2001 and 2004 and studied for seasonal changes in supercooling point (SCP), hemolymph thermal hysteresis activity (THA), polyols, water content and respiration rate. Air and microhabitat temperatures were monitored using Hobo Pro Series data loggers along with BoxCar Par 4 software (Onset Computer Corporation, Bourne, MA, USA).
Outdoor enclosures and indoor acclimations
Although field collections of larvae were made during all seasons, to
ensure sufficient material for mid-winter experiments, Cucujus larvae
were collected around Fairbanks in September and placed in plastic food
storage containers (20x15x10 cm; N=2050 per box)
with moist bark from their native trees. To simulate field conditions, some
boxes were placed in an outside enclosure in a wooded area on the University
of Alaska, Fairbanks (UAF) campus either on small logs at ground level or
0.5 m above ground to reduce the insulating effect of snow cover and
expose them to colder winter air temperatures. Larvae collected in September
from Fairbanks were also placed in box enclosures outdoor in Indiana, and
September-collected Indiana larvae were placed outdoors in box enclosures in
South Bend and Fairbanks. Larvae collected near Wiseman were also placed into
containers in the field. Temperature data loggers (see below) were used to
monitor enclosures and air temperatures at these sites. Boxes were retrieved
in either mid-winter (January) or late winter/spring (March or April).
Cucujus survival, SCPs, thermal hysteresis activity, water content
and respiration rates were determined as described below. Additional boxes of
insects collected in September were cold-acclimated in a Tenney Series 942
environmental chamber at the University of Notre Dame according to the
following protocol. On days 13 the insects were held at 0°C, days
46 at 1°C, days 79 at 2°C, days
1014 at 3°C, days 1521 at 4°C and days
2230 at 4.5°C.
Supercooling points
To determine SCPs, thermocouples were fixed to the dorsal surface of
individual larvae using a small amount of beeswax, and larvae were suspended
in 1.5 ml plastic tubes that were placed inside a larger glass container that
was submerged in a cooling bath. Once equilibrated to 0°C, the container
temperature was reduced at a rate of 0.2 deg. min1. The
lowest larval temperature recorded before the release of the latent heat of
fusion of body water, as evidenced by an exotherm, was recorded as the SCP
(Lee and Denlinger, 1991). To
determine their susceptibility to inoculative freezing during different
seasons, SCPs were also determined on larvae in contact with ice. In these
cases, larvae were equilibrated at 2 to 5°C to ensure that
surrounding water was frozen before further cooling.
Thermal hysteresis activity
THA is an indication of the presence and activity of antifreeze proteins
(AFPs) and was determined according to the method of DeVries
(1986). Hemolymph samples
(
26 µl) were drawn from punctured individual larvae when
possible or pooled from larvae as necessary and sealed in glass capillary
tubes. The sample was partially frozen by freezing the outside of the
capillary tube with a spray freeze (Fisher Brand Super Friendly Spray Freeze;
Fisher Scientific, Pittsburgh, PA, USA) and the temperature slowly raised to
melt the ice until the ice crystal was just visible or disappeared under the
microscope (= melting point). Beginning again with a seed crystal
0.25 mm
in diameter, the temperature was lowered very slowly until it was observed to
grow (= freezing point). In the absence of AFPs, if the temperature is lowered
0.010.02°C below the melting point the crystal will immediately
grow (i.e. melting point = freezing point). However, if AFPs are present, the
crystal will not grow until the temperature has been lowered to the hysteretic
freezing point, whereupon the crystal grows rapidly (i.e. melting point and
freezing point are not equal). The difference between melting point and
freezing point is taken as the THA.
Water content
Total body water content was determined according to Rojas et al.
(1986). Individual larval
fresh mass was determined to the nearest 0.1 mg. Larvae were then dried at
60°C to constant dry mass (
48 h). Body water content was calculated
as the percentage of initial fresh mass lost during drying. The absolute body
water content of larvae was also calculated (g water g1 dry
mass; Hadley, 1994
).
Polyol determinations
13C NMR was used to determine the presence of polyols and other
potentially important solutes in the hemolymph of cold acclimated C.
clavipes larvae. The 13C{1H} NMR spectrum was
obtained on a Varian Unity Plus 600-MHz NMR spectrometer equipped
with dual 1H/13C 3-mm microprobes (Nalorac, Martinez,
CA, USA), operating at 150.86 MHz for 13C. The hemolymph sample
(250 µl) was diluted with 30 µl of 2H2O and
transferred to the NMR tube prior to data collection. Data acquisition
conditions were as follows: 31 000 transients; 2.5 s recycle time; 303 K;
1-230 p.p.m. spectral window. The resulting free induction decay (FID) was
zero-filled (yielding a final digital resolution of 0.14 Hz per point), and a
1-Hz line-broadening function was applied prior to Fourier transformation.
Chemical shifts were referenced to the most intense C1/C3 signal of glycerol
(64.2 p.p.m.) observed in the spectrum
(Kukal et al., 1988).
Glycerol concentrations in the hemolymph were determined using a
colorimetric assay (Boehringer Mannheim/R-Biopharm, Marshall, MI, USA)
(Kreutz, 1962).
Respirometry
Insect resting rates of CO2 production were measured using a
flow-through respirometry system (Sable Systems International, Las Vegas, NV,
USA) with a LiCor model LI-6252 CO2 analyzer (Lincoln, NB, USA).
The incoming air stream (baseline) was scrubbed of water vapor and
CO2 using Molecular SieveTM and AscariteTM and magnesium
perchlorate, respectively. Air flow rate was 25 ml min1.
Carbon dioxide production by individual larvae was recorded for 20 min with 10
min of baseline recording between larvae. Summer larvae were removed from food
for 24 h before recordings to clear the digestive tract. This was not
necessary for winter larvae since they had ceased feeding. Each larva was
recorded at ambient temperatures of 0, 10 and 20°C and allowed to
equilibrate in each condition for one hour before recording. To prevent
desiccation between recordings, insects were flushed with air humidified to
83% relative humidity by bubbling the air through a 20% KOH solution at a rate
of 25 ml min1 (Solomon,
1951). Insects were weighed to the nearest 0.1 mg both before and
after respirometry and typically showed less than 10% change in fresh mass.
Those that lost more than 10% mass or that defecated while in recording
chambers were excluded from analysis. Recordings were analyzed using DataCan
(Sable Systems International) or LabGraph (developed by Oivind Toien,
University of Alaska, Fairbanks). For each larva, mean CO2
production (µl h1 g1 dry mass),
corrected for standard temperature and pressure, assuming a respiratory
quotient of 0.85, was calculated from the most stable 25 min of the 20
min recording to exclude fluctuations due to animal movement. (Note that even
the use of respiratory quotient values very different from 0.85 would not
affect the results of this study.)
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Results |
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Air and microhabitat temperatures were monitored over the course of three years in the three primary collecting sites: near South Bend (Indiana) and Fairbanks and Wiseman (Alaska). Fig. 1 illustrates the diversity of temperatures among these climates and permits the physiological adaptations to be placed into context. Fig. 1A demonstrates that larvae in a fallen log (near Fairbanks) that was suspended off the ground, and therefore had little insulation, experienced low temperatures similar to air, a minimum of 37°C during winter 20012002. Fig. 1B shows winter air and microhabitat temperatures at Wiseman, Alaska, near the northern limit of the range of C. clavipes. Air temperatures here are somewhat lower than at Fairbanks, and the winter is longer. However, snow depths are generally greater and in these circumstances provide additional, prolonged insulation for those larvae under the snow. Consequently, larvae in this well-insulated site experienced a minimum temperature of only 13°C in October prior to heavy snow cover. Microhabitat temperatures in other sites varied according to the amount of insulation provided by snow (data not shown). Fig. 1C illustrates the shorter and less severe winters experienced by C. clavipes near South Bend, Indiana. During the winter of 20022003, the minimum temperature experienced by larvae in this `above ground' log was 13°C, the same minimum temperature in the insulated site at Wiseman (Fig. 1B). (It should be mentioned that recent winters at all three sites were warmer than average.) These three examples illustrate the variation in microhabitat temperatures at the collecting sites. Although air temperatures are lower in Alaska, the temperatures experienced by C. clavipes depend on their microhabitat temperatures, and these can vary drastically within a given region. Obviously, the duration of the winter is much longer in Alaska.
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Populations of C. clavipes must be adapted to survive a range of minimum temperatures, depending on the air temperatures occurring during a given winter and the extent of insulation provided by a given microhabitat. This ability is illustrated by an experiment where larvae collected from habitats near Fairbanks in October 2002 were placed into two enclosures, one placed at ground level and the other above the eventual snowline. Temperatures experienced by the two groups are shown in Fig. 2. In early January, larvae in the ground level box insulated by snow experienced a minimum temperature of 15°C while the group above the snow had a minimum temperature of 35°C. In spite of these large differences, both groups had >90% survivorship when the boxes were retrieved in mid-January (95.7% in the ground box and 93.4% in the high, uninsulated box).
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Of a group of March 2003 larvae collected from Wiseman and placed there in
a box enclosure the previous September, only 1 of 16 showed an exotherm
indicative of an SCP (at 48.9°C), while the other 15 failed to
freeze when cooled to 64°C (the lowest temperature our equipment at
that time was capable of reaching; Fig.
3). These larvae were then held at 64°C for 10 h.
After then being held at 4°C for several days, seven of 15 were alive,
indicating that these had SCPs below 64°C. In early January 2004,
none of the 32 Wiseman larvae tested exhibited exotherms when cooled to
80°C, the lowest temperature our equipment at that time was capable
of recording (Fig. 3). By
contrast, Fairbanks larvae in early January had a mean SCP of
26.5°C, considerably higher than in January of the previous year or
in the Wiseman larvae of January 2004. However, by 25 January 2004, the
Fairbanks larvae did not freeze when cooled to 60°C (32 of 32).
Fairbanks larvae collected on 10 March 2004 had a mean SCP of
37.2°C, not including five individuals (of 16) that did not exhibit
exotherms down to 80°C. The absence of freezing at such a low
temperature as 80°C hints that the water in these larvae may have
been vitrified rather than liquid.
Some individual larvae on most of the winter dates in Fig. 3 have SCPs that are suspiciously higher than might be expected (i.e. 9 January 2004), sometimes even higher than the ambient temperatures experienced by the larvae. At least some of these SCPs are probably real and therefore illustrate considerable variation in the population. However, they may also represent an artifact resulting from the difficulty encountered in the process of removing larvae from under the bark for collection. Considerable amounts of ice are present in this habitat, especially in the sites at the base of standing trees, and it is common for larvae to be partially, or completely, encased by ice. Consequently, the cuticle may be damaged when the larvae are collected, thereby affecting the SCP. Larvae with missing legs or antennae were not used, but more subtle damage, such as broken bristles or abrasions to the cuticle, may have been overlooked. As a consequence, the mean winter SCPs may well be lower than shown.
SCPs presented in Fig. 3 were gathered using `dry' larvae, i.e. those not in contact with ice. However, condensation can occur as air in the container holding the insects is cooled during the SCP measurement. This water can freeze on the surface of the insects. To more closely approximate microhabitat conditions in which larvae are often in contact with ice, SCPs were recorded with Alaskan larvae (Fairbanks and Wiseman) both in contact with ice and dry (Fig. 4). SCPs of the two groups were not significantly different. Larvae either in contact with ice or dry were included among those cooled to 60 or 80°C without showing exotherms.
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In September of both 2002 and 2003, larvae collected in Indiana were taken to Fairbanks and placed in outdoor box enclosures on the ground, and Fairbanks larvae were placed in enclosures in Indiana. The following spring, mortality of Indiana larvae in Alaska was 100% in both years, while mortality of Fairbanks larvae in Indiana was under 10% in both years. Indiana larvae in box enclosures in Indiana had mortalities of <10%, while mortality of Fairbanks larvae in box enclosures in Fairbanks was 14.3% in 2003. In February 2003, SCPs (mean, 24.0°C) of C. clavipes larvae collected near Fairbanks in September 2002 and placed in box enclosures in Indiana were comparable to those of Indiana larvae placed in enclosures in Indiana but were significantly higher than those of Alaska larvae from January 2003 (compare with Fig. 3).
Water content and dehydration
When winter C. clavipes were first collected in Alaska (in January
2002), most of the larvae appeared dead, perhaps resulting from the near
absence of insulating snow and a period of temperatures near 40°C
in the previous month. The larvae were so desiccated that hemolymph samples
could not be obtained. When warmed on moist paper towels to 4°C, or
higher, most did not become mobile, even after several days. However, in
March, mortality of larvae in the field was just 17.4%. Obviously, most of the
larvae were not dead in January. Two factors may be responsible for the
immobility of the larvae in midwinter. These are metabolic diapause (see
below) and dehydration.
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Antifreezes: glycerol and antifreeze proteins
Antifreezes are expected to be major factors in achieving the very low SCPs
seen in winter C. clavipes larvae, especially in Alaska. In fact, the
larvae produce both polyols and AFPs. Glycerol is the primary colligative
antifreeze in both Alaska and Indiana larvae. A typical winter hemolymph
glycerol concentration of Indiana larvae is 0.5 mol l1,
while that of Alaska larvae is considerably greater. Fairbanks larvae
collected in late September and cold acclimated for 1 month to a final
temperature of 4.5°C had a hemolymph glycerol concentration of 2.2
mol l1. However, the water content of these acclimated
larvae was 63.1% (1.701 g H2O g1 dry mass),
similar to that of summer larvae. Recall that we were unable to collect
hemolymph from Alaska larvae in January because they were highly desiccated.
The water content of Fairbanks larvae in January 2003 was 35.2% (0.532 g
H2O g1 dry mass), a
3.2-fold reduction in
water content relative to summer. If the hemolymph of the cold-acclimated
larvae with normal summer body water content and hemolymph glycerol
concentrations of 2.2 mol l1 was concentrated 3.2-fold, the
glycerol concentration would be 7.0 mol l1. This value
probably closely represents the true hemolymph glycerol concentration of
midwinter Alaska larvae following dehydration. 13C NMR of hemolymph
from cold-acclimated Fairbanks larvae (Fig.
8) demonstrated that glycerol is the only solute present in
unusually high concentrations, and consequently glycerol is the only polyol
antifreeze produced by C. clavipes larvae. Trehalose is the next most
abundant substrate. Proline is also present.
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As illustrated in Table 1, the level of thermal hysteresis indicative of antifreeze protein activity in winter is generally 34°C in both Alaska larvae prior to dehydration and in Indiana larvae. However, Alaska larvae produce antifreeze proteins much earlier in the autumn and lose them much later in the spring than do Indiana larvae. Alaska larvae collected in early September already had THA nearly equal to that of mid-winter Indiana larvae, while Indiana larvae do not begin increasing THA until late September or October. THA in Alaska insects collected in winter cannot be directly assessed since hemolymph samples cannot be obtained from dehydrated midwinter larvae. Using the hemolymph of the cold acclimated (but not dehydrated) larvae mentioned above in relation to glycerol, we determined a reasonable thermal hysteresis activity of January larvae in the field. THA in a hemolymph pool from the cold-acclimated larvae was 3.28°C. After this hemolymph was concentrated 3.2-fold to reflect the dehydration of the January larvae in the field, the measured THA was 12.85°C (Table 1). This value is by far the highest THA ever measured in any organism. It is interesting that this concentrated THA value is more than 3.2-fold greater than the THA prior to concentration. The high AFP activity in combination with the high concentrations of glycerol and other solutes results in a freezing point depression of the concentrated hemolymph of approximately 24°C.
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Respirometry
Based on CO2 production rates
(Fig. 9), it appears that
Alaska, but not Indiana, Cucujus enter a winter diapause indicated by
seasonal depression of metabolic rates. In an analysis of variance using a
general linear, full-factorial model, significant between-subjects effects
were detected for both population (F1,45=4.86,
P<0.05) and season (F3,45=34.01,
P<0.0001), indicating that both location and time of year when
insects were collected influenced CO2 production rates. There was
also a significant interaction between population and season
(F2,45=23.23, P<0.0001), indicating that the
seasonal effects differ between the Indiana and Alaska populations. Within
subjects, a univariate repeated measures ANOVA (Greenhouse-Geisser corrected
P-values) showed that temperature had a significant effect on
CO2 production rate as expected
(F1.3,58.0=208.87, P<0.0001). Significant
interaction terms were detected between temperature and season
(F3.9,58.0=16.23, P<0.0001) and for the
three-way interaction between temperature, season and population
(F2.6,58.0=22.71, P<0.0001), indicating that
season altered the effect of temperature on CO2 production rate and
that this alteration also varied between populations. However, the
temperaturepopulation interaction was not significant
(F1.3,58.0=1.62, P=0.211), suggesting that
temperature had a similar effect on CO2 production rates in both
Indiana and Alaska populations of Cucujus.
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Discussion |
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The low SCPs of Alaska C. clavipes are noteworthy. The mean SCP in
January 2003 was 42°C, with individual SCPs reaching as low as
58°C. However, even more interesting is the lack of freezing
exotherms in Wiseman larvae cooled to 64°C in late March 2003 and
the subsequent survival of half of these larvae. Likewise, 32 Wiseman larvae
in early January 2004 and 32 Fairbanks larvae collected in late January 2004
failed to exhibit freezing exotherms when cooled to 80 and
60°C, respectively, even with half of the individuals in contact
with ice. It is interesting that during the entire three years of the study no
exotherms were recorded below 58°C. A possible explanation is that
these larvae were vitrified, although there is no experimental evidence to
prove this. Miller (1982)
reported that two adult C. clavipes survived temperatures of
55°C. He speculated that they were freeze tolerant, although SCPs
were not measured.
Other insects with extremely low SCPs have been identified. Larvae of the
beetle Pytho deplanatus from high altitude in the Canadian Rocky
Mountains had a mean SCP of 54°C, yet the larvae survived freezing
(Ring, 1982). Populations of
three species of willow gall insects from interior Alaska exhibited mean SCPs
of 56 to 58°C (Miller,
1982
). These were two species of Diptera (Cecidomyiidae:
Rhabdophaga strobiloides and Mayetiola rigidae) and a
hymenopteran (Euura sp.). These freeze-avoiding, gall-forming (and
therefore uninsulated) species require extremely low SCPs as they are found
primarily in low-lying areas where cold dense air pools in winter, and they
are therefore exposed to very cold air temperatures. However, failure to
exhibit freezing exotherms was not reported in these species.
A number of studies of insects and other invertebrates have demonstrated a
correlation between dehydration and ability to prevent freezing
(Zachariassen, 1985;
Lundheim and Zachariassen,
1993
; Gehrken,
1989
; Rickards et al.,
1987
; Worland,
1996
; Block, 2003
;
Worland and Block, 2003
;
Danks, 2000
; Holmstroup, 1995;
Worland et al., 1998
). Over
the winter, it is not uncommon for dormant insects to experience water stress,
since they typically do not eat or drink. This is especially true of
freeze-avoiding species since at low temperatures in the presence of ice in
the hibernaculum the partial pressure of water in the air is lower than that
of the insect hemolymph. Thus, the insect loses water by evaporation
(Lundheim and Zachariassen,
1993
). This may be especially problematic for Alaska
Cucujus because of the length and severity of the winters; in fact,
Alaska C. clavipes exhibit severe dehydration in winter. Larvae
desiccate from mean values of
6365% body water (1.701.85 g
H2O g1 dry mass) in summer to 2840% body
water (0.400.68 g H2O g1 dry mass) in
mid-winter. Although this several-fold reduction in water volume may cause
water stress in the larvae, it should also promote supercooling by
concentrating antifreezes and reducing the volume of freezable water. The
hemolymph glycerol concentration in cold-acclimated autumn Fairbanks larvae
was 2.2 mol l1 in these non-dehydrated animals. Following a
3.2-fold reduction in water volume, the glycerol concentration would be at
least 7 mol l1, if there were no downward adjustments in
glycerol concentration during the desiccation process. (Actually, mean fold
reductions as large as 4.7 were measured. This would result in a glycerol
concentration of
10 mol l1.) Likewise, the 3.2-fold
dehydration of the cold-acclimated larval hemolymph produced THAs of nearly
13°C, much higher than has ever been reported. Therefore, although C.
clavipes larvae appear to complete AFP synthesis during a short period in
late summer, desiccation later in the season effectively concentrates the AFPs
several fold. Consequently, Alaska C. clavipes may not need to
synthesize more AFP than do Indiana larvae. Both cold-acclimated and
field-collected Alaskan larvae in late autumn prior to desiccation have
approximately the same levels of hemolymph thermal hysteresis as do Indiana
larvae in mid-winter. We were unable to extract hemolymph samples from Alaska
larvae after desiccation in mid-winter, but presumably the antifreeze
concentrations reflect the 34-fold increases consistent with the levels
of dehydration.
To achieve the extreme levels of supercooling characteristic of Alaska
C. clavipes larvae requires (1) the inhibition of inoculative
freezing initiated by external ice across the cuticle and (2) the removal
and/or inactivation of potential ice nucleators. AFPs are known to assist
supercooling by both of these mechanisms in larvae of the beetle
Dendroides canadensis (Olsen et
al., 1998; Olsen and Duman
1997a
,b
;
Duman, 2002
). It is important
to note that the level of protection afforded to the insect by AFPs greatly
exceeds the magnitude of thermal hysteresis activity measured in the insect
hemolymph, both with respect to inhibition of inoculative freezing and masking
of ice nucleators. The absence of freezing in some Alaska C. clavipes
suggests that their AFPs are able to inhibit ice nucleators to very low
temperatures and that they may even inhibit homogeneous nucleation, thereby
promoting vitrification. The absence of endotherms between 58°C
(the lowest SCP measured) and 80°C (and perhaps lower) indicates
that there may be a threshold effect operating such that, beyond a certain
level of dehydration (which concentrates the AFPs, glycerol and perhaps other
factors leading to high viscosity), vitrification, rather than freezing, may
occur.
Another overwintering adaptation present in Alaska, but not Indiana, larvae
is diapause. While Indiana larvae may continue to feed well into November and
resume feeding in March, the winter season is much more extended in Alaska.
This may necessitate the reduced metabolic state in the Alaska larvae. In
addition, the downregulated metabolism characteristic of larval diapause can
also contribute directly to supercooling capacity. For example, the stag
beetle Ceruchus piceus (Lucanidae) removes hemolymph lipoproteins
with ice nucleating activity in winter, permitting them to supercool
significantly without the production of antifreezes
(Neven et al., 1986). It is
unlikely that the normal lipid shuttle function of the hemolymph lipoproteins
could be spared in winter without a concomitant reduction in metabolic rate in
diapausing larvae.
In keeping with the more extreme temperatures experienced by Alaska populations of C. clavipes, Alaska larvae exhibited a considerably greater capacity to supercool than did Indiana larvae. As noted earlier, at certain times Alaska larvae failed to freeze even when cooled to 80°C, perhaps suggesting the involvement of vitrification. This level of supercooling may appear to be greater than necessary based on ambient temperatures measured at the Alaska collecting sites over the past three years, when minimum winter air temperatures did not exceed 42°C. However, these winters were abnormally mild. Air temperatures in the interior of Alaska commonly reach 50°C, sometimes for extended periods. In addition, such temperatures can occur at times (i.e. spring) when insulating snow cover is minimal. It appears that the combination of AFPs, glycerol, dehydration and diapause combine to produce extreme levels of supercooling, and perhaps vitrification, in Alaska C. clavipes.
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Acknowledgments |
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Footnotes |
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References |
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