J. Insect Phyriol., 1970, Vol. 16, pp. 979 to 990. Pergamon Press.

Printed in Great Britain

VARIATIONS IN GLYCEROL CONTENT AND ITS

INFLUENCE ON COLD HARDINESS IN THE
ALASKAN CARABID BEETLE,
PTEROSTICHUS BREVICORNIS”

JOHN G. BAUST and L. KEITH MILLER

Institute of Arctic Biology, University of Alaska, College, Alaska 99701 t

(Received 5 June 1969; revised 12 September 1969)

Abstract-Quantitative paper chromatographic determinations of glycerol

content in the haemolymph of adult P. brevicornis have revealed seasonal
variations in the concentration of this substance. Mean glycerol concentrations
in excess of 22 gq/, have been measured during winter with the values decreasing
to less than 1 g% during spring and summer. Acclimation studies gave similar
results. In the artificially warmed winter beetles glycerol content decreased to
less than 1 9% within 36 hr at 20°C. Glycerol concentrations were closely
correlated with the changing supercooling points and haemolymph freezing
points in both the naturally acclimatized and laboratory acclimated specimens.
Temperature preferences showed a positive correlation with changes in cold
tolerance.

INTRODUCTION

THE MECHANISMS allowing for winter survival of northern insects have been studied
by relatively few investigators. Most notable was the work of SALT (1956) in which
the effects of both glycerol and water content were considered. Water content,
expressed as the ability to supercool, was found to be of little protective significance
to the insect unless severe desiccation occurred. In a later study SALT (1959)
showed that glycerol played a direct r81e in cold hardening larvae of Bracon cephi
by depressing the melting point of haemolymph as much as 17*5”C and lowering
the supercooling point to - 47.2”C.
SDMRIE(1964) found that in several species, cold hardening was facilitated by
increased glycerol levels resulting in depression of the supercooling point. Other
authors (CHINO, 1957; ASAHINA,1966; BAUST,1968) have considered this question
with similar results.
However, the question of glycerol involvement is not resolved. A number of
workers feel that high concentrations of glycerol are only coincidental with cold-
hardening. Based upon the detection of significant glycerol concentrations in the
haemolymph of non-hibernating insects and its absence in certain overwintering
species, SBMME(1964) h as supported this view. This paper describes the adaptative
* Supported in part by NIH Grant GM-10402.
t Publication No. 93, Institute of Arctic Biology.

979
980 JOHN G. BAUSTANDL. KEITH MILLER

advantages possessed by an insect with the ability to regulate its glycerol content
seasonally. It should be noted that previous studies were conducted almost
exclusively on larvae and eggs of various insects. This study is concerned with the
adult overwintering form.

METHODS AND MATERIALS

Pterostichus a carabid beetle (average weight, 8 mg) which over-
brevicornis,
winters in both the adult and larval stages was the principal subject of study.
The adults were collected in wooded regions near Fairbanks and were abundant
in decaying stumps in which the beetles aggregate during autumn and winter.
During the latter period exposure to temperatures as low as - 60°C is possible and
continued exposure to -40°C for several weeks can occur. Extreme daily tem-
perature fluctuations within the microhabitat may reach 40°C with occasional
winter thawing evident. In early summer (May-June) the adults leave the stumps
and may be found on the forest floor. Acclimatization studies utilized outdoor
specimens while acclimation experiments were conducted in temperature regulated
environmental chambers.
Glycerol content in the haemolymph was determined utilizing a chromato-
graphic technique described by PERKINS and ARONOFF(1959) and modified by
S~MME (1964). Ascending chromatograms were run on Whatman No. 1 paper
(20 x 40 cm) with a single phase solvent, n-butanol-acetic acid-water (12 : 3 : 5).
Haemolymph was collected with a 1~1 syringe supported in a micromanipulator.
The samples as well as standards were then applied to the paper. Chromatograms
were run for 18 hr at 20°C. After air drying, chromatograms were sprayed with
a solution of 0.01 M aqueous potassium periodate, dried, and sprayed with a
solution of 35% saturated sodium tetraborate (v/v), 0.8% potassium iodide (w/v),
0.9% sodium tetraborate (w/v), and 3% soluble starch (w/v). Glycerol and other
polyhydroxy alcohols yield white spots on a blue-brown background (Table 1)
following this treatment.

TABLE1-Rf VALUEFROMCHROMATOGRAMS OFVARIOUS

~RYOPRO~XC-~IVE
COM~~~JNDS(SUOARSAND POLYHYDRIC
ALCOHOLS)

Compound Rf Value
-
Glycerol 053
Sorbital 0.10
Glucose 0.19
Galactose 0.08
Fructose 0.16
Mannose 0.14
Sucrose 0.03
Trehalose 0.01

Solvent: n-Butanol-acetic acid-water (12 : 3 : 5).

 GLYCEROL CONTENT AND ITS INFLUBNCE ON THE CARABID BEETLE 981

Detection limits of this method are better than 1.0 per cent. A standard
solution containing 10 pg glycerol per 0.1~1 water is resolvable. Sample concen-
trations were determined by reference to a standard curve constructed by plotting
either weight or area of developed spots against the standard concentrations.
This curve was linear for standards ranging from 2 to 60% glycerol (w/v).
Supercooling points were measured with a 32 gauge copper-constantan
thermocouple on the abdomen while cooling at 1 or 3°C per min. Temperatures
were indicated on a recording potentiometer. Initiation of spontaneous freezing
results in termination of supercooling and is indicated by a rise in the temperature
curve due to released heat of fusion. The temperature at which spontaneous
freezing occurred was termed the supercooling point. Haemolymph freezing points
in the non-supercooled insect were measured using a modified Scholander freezing
point apparatus. Haemolymph samples were extracted rapidly, placed in a capillary
tube, covered with mineral oil and ‘flash’ frozen. The freezing point was defined
as the temperature at which ice crystals in haemolymph remain constant in size,
with neither growth nor melting being observed.
Glucose concentrations of the haemolymph were determined utilizing thin-
layer chromatography. Standard and unknown samples were run on commercially
prepared silica gel plates (Eastman Chromagram 6061). Plates were pretreated
in a 2% solution of sodium bisulphite in 60% ethanol, dried, and activated at
100°C for 15 min. The migrating solvent was ethyl acetate-methanol-acetic acid-
water (12 : 3 : 3 : 2). Visualization was accomplished by spraying the dried sheets
with a solution of 5% aniline hydrogen phthalate in glacial acetic acid and heating
at 85°C. Zone detection was made under long wave U.V. light. Glucose R values
were O-33.
Temperature preferences were determined in a gradient chamber with very
dim light and near absolute humidity. The temperature gradient was nearly
linear having a total span of approximately 25°C with the lowest temperature
being between - 5” and - 11°C.

RESULTS
Acclimatization experiments
Glycerol concentrations in haemolymph varied seasonally as did supercooling
points, haemolymph freezing points, and temperature preferences (Table 2).
Fig. 1 illustrates the temporal relationships between supercooling and freezing
points and glycerol concentration.
Glycerol concentration was observed to increase during autumn and winter
following the first frost. This initial increase appears to be temperature related
with the continued fluctuations during mid-winter (December to February) also
apparently being temperature dependent. As glycerol concentrations increased
(August to December), supercooling and freezing points decreased. Glycerol
content and freezing points correlated well over this period. Supercooling points
and glycerol content were closely related during autumn and early winter, but the
relationship was somewhat askew during spring and summer (Fig. 2).
982 JOHN G. BAUST AND L. KEITH MILLER

As glycerol concentrations decreased at winter’s end, supercooling points

increased. However, in early spring supercooling points were observed to plateau
while glycerol content continued to decrease. It was not until glycerol levels were

TABLE 2-SEAsoNAL CHANGES IN HAEMOLYMPH GLYCEROL CONTENT AND FREEZING POINTS

AND IN SUPERCOOLING POINTS OF Pterostichus brevicornis

Glycerol Supercooling Freezing

concentration points points
Date k%> CC) CC)

3.1.68 17.8 C!Z

0.8 -9.0 -
22.1.68 16.5 + 2.5 -9.7 -
27.2.68 23.0 - 9.4 f 0.3 -
19.3.68 21.0 - -
25.3.68 18.0 f 0.0 -8.3 -
30.4.68 1.5 -8.1 -3.0
14.5.68 0.0 + 0.0 - 6.0 f 0.4 - 3.0 * 0.0
28.5.68 0.0 * 0.0 -6.8f0.3 - 1.4 + 0.0
11.6.68 0.0 IL0.0 - 6.0 f 0.0 -1.1 f0.1
5.7.68 0.0 f 0.0 - 4.6 f 0.1 -
23.7.68 0.0 f 0.0 - 4.2 +I0.6 -
29.7.68 0.0 f 0.0 - 4.7 f 0.5 -
5.8.68 Trace - 5.6 f 0.4 -0.6kO.l
23.8.68 0.5 * 0.0 -7.6fO.l -
6.9.68 0.5 f 0.0 - 7.4 f 0.2 - 0.8 + 0.0
8.10.68 4.5 f 0.5 - 8.9 zk0.3 - 1.8 f 0.0
22.10.68 9.5 f 0.0 - 10.3 f 0.3 - 2.8 + O-0
5.11.68 - - 3.5 f 0.0
11.11.68 - -11.2f0.2 -
19.11.68 16.8 k 1.6 - 10.7 + 0.4 -
5.12.68 22.4 f 0.1 -11*5f0*4 -
10.1.69 14.3 f 2.3 - 10.6 f 0.3 - 4.2 f 0.0
22.1.69 22.5 f 6.5 - 10.2 L-0.3 -
14.2.69 10.5 f 0.5 - 10.1 f 0.2 -4.3 f 0.1
4.3.69 12.1 * 1.5 -11.1 f0.2 -5*OfO.l
17.3.69 19.8 + 0.6 - 10.9 f 0.2 - 4.9 * 0.1
2.4.69 8.1 f 0.7 - 10.7 + 0.1 -3.5 f 0.1
21.4.69 3.0 * 0.0 -7.7 f 0.1 - 2.5 LIZ
0.1

Values are mean + standard error.

low ( N 6 per cent) that supercooling points commenced to further increase. Glycerol
levels were below detectable levels by mid-May while supercooling points con-
tinued to rise.
A similar but inverse response was observed after mid-summer (July). Super-
cooling points commenced falling prior to glycerol appearance. This decrease
continued while glycerol was first observed during late August. During the initial
gradual and later rapid glycerol increase, supercooling points plateaued but soon
fell again as glycerol levels increased.
 GLYCEROL CONTENT AND ITS INFLUENCE ON THE CARABID BEETLE 983

The plot of mean freezing points was a near mirror image of the glycerol curve
(Fig. 1). The relationship between glycerol concentration and freezing point was
linear over most of the year, with mid-late winter excepted. (Fig. 2). During
spring and autumn, the haemolymph freezing was depressed 1°C per 5% glycerol

Jon. Feb. Mar Apr. May JuneJuly Aug Sepi.Oct. Nov. Dec. Jan. Feb Mar Apr.

Month

FIG. 1. Seasonal variations in haemolymph glycerol content, supercooling points

and haemolymph freezing points. Supercooling points are whole body deter-
minations. All measurements are on adult Pterostichus brevicornis. Values are
mean L-S.E.

.“‘.. . .._.,.__, ,F Freezing points

.
‘.’ w ..._.._,,
. “.....,,
.
““‘..... . ..... ..._..._..

Super cooling points

0
-Ye - 0

-12
0 3 6 9 12 15 21 24
Glycerol concentration, g%

SZZZr Fall Winter

and
Spring

FIG. 2. Mean freezing and supercooling points vs. mean glycerol concentration
in adult Pterostichus brmicornis during different seasons.

increase. The relationship between mean glycerol concentrations and supercooling

points as illustrated in Fig. 2 was close to linear during autumn and early winter.
Total body supercooling points were depressed 0.7”C per 5% glycerol increase.
That is, the freezing point depression was 1.3 times that of the supercooling point
984 JOHN G. BAUSTAND L.KEITH MILLER

depression with the same incremental change in glycerol. However, since super-
cooling is a statistically random phenomenon dependent on variable factors (time
at a given low temperature and rate of temperature decrease), there is no reason to
expect a perfect 1 : 1 relationship.
During December and January glycerol concentrations were observed to
decrease followed by an increase during February and March. Supercooling
points changed as predicted: in general they increased as glycerol concentrations
decreased and vice versa. Freezing points, however, did not change as expected.
A continued gradual lowering was observed while glycerol levels diminished.
However, following a sharp increase in glycerol concentration, freezing points
decreased sharply. The haemolymph glucose concentration was measured during
this later period. No substantial amount was found. Therefore, it seems unlikely
that conversion of glycerol to glucose can explain the apparently anomalous
relationship between glycerol levels and freezing points. It should be noted that
seasonal variations in haemolymph trehalose levels have been detected but as yet
not quantitated.

Acclimation experiments
The change in glycerol content following acute warming was striking (Fig. 3).
Winter beetles collected at an ambient temperature of - 10°C and rapidly (within

4-

I I
0 6 12 I6 24 30 36 42 46 60 72

Hoursat 20%
FIG. 3. Changes in mean haemolymph glycerol concentrations resulting from
acute exposure of naturally cold-hardened, winter adults of Pterostichus brevicornis
to 20°C.

minutes) warmed to 20°C demonstrated a decrease in mean glycerol level of 18 g%

after 36 hr. The initial glycerol concentration of 18 g% decreased to near 0%.
This loss proved irreversible. That is, returning the specimens to low temperatures
either in stepwise increments or by acute freezing did not result in the re-synthesis
 GLYCEROL CONTENT AND ITS INFLUENCE ON THE CARABID BEETLE 985

of glycerol. If a critical temperature or temperature-time stimulus is involved in

this change, it has not been determined.
Specimens collected at an ambient temperature of - 10°C and warmed step-
wise over 5-day intervals to 0,5, and 7°C were also observed to lose glycerol rapidly
(Fig. 4). Mean glycerol levels dropped from 19 g% to 6 g% after 5 days at 0°C

0,
-IO -5 0 5

Temperature, OC

FIG. 4. Changes in mean haemolymph glycerol concentrations of naturally cold-

hardened, winter adults of Pterostichus brewicornis chronically exposed to various
temperatures for 5 days. Values are mean + S.E.

to 3.5 g% after 5 days at + 5°C and to 2.5 g% after 5 days at 7°C. Such a change
in temperature is consistent with that encountered in the wild, hence reflecting the
possible mode of glycerol fluctuation in a natural situation. Freezing points
increased from - 5*O”C to - 2.0%’ over the latter 5 day period.

Temperature preferences
Fig. 5 represents the mean and individual seasonal temperature preferences
demonstrated by P. brevicornis. Thermal preferences ranged from a mean high
of 13.3”C during July to a mean winter low of -5.5”C (January), the latter tem-
perature being well within the lower limit of mobility. Thermal responses indicated
as mean temperatures have a less than clear-cut meaning. It may be useful to the
investigator to quantitate in such a manner but of questionable importance to the
31
986 JOHN G. BAUST AND L. KEITH MILLER

insect. This is especially evident in Fig. 5 where temperature preferences as low

as - 11°C are indicated.

-14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 IO I2 14 16 Is 20 22

Temperature, OC

FIG. 5. Seasonal variations in individual and mean temperature preferences

demonstrated by adult Pterostichus brevicornis. Mean values are f SD.

DISCUSSION
A question arises concerning the significance of the two plateaus in the super-
cooling curve (Fig. 1). The following theory, which integrates a number of factors,
is proposed. Supercooling points would be expected to rise due to decreasing
glycerol content. However, other cryoprotective compounds such as glucose,
trehalose, or low molecular weight lipids might account for the stabilization of the
supercooling points. This is supported by the discovery of WYATT (1967) that
glycerol is first converted to glucose before synthesis to glycogen. This increased
glucose concentration would tend to stabilize supercooling. As glucose is converted
to glycogen at the expense of glycerol along with the increased presence of nucleators
due to feeding (SALT, 1961), supercooling points would increase. During late
summer glycerol would again be synthesized from mobilized glucose or trehalose.
The presence of both these compounds would be expected to decrease super-
cooling points but a plateau is evident. This plateau may represent the inhibition
of the protective action of glycerol and glucose acting to decrease supercooling
points. This inhibition may take the form of the continued build-up of nucleators
during prehibernation feeding.
A second group of compounds may play a role equivalent to that suspected of
sugars. Kaufmann (personal communication) has found that total body fat varies
seasonally in an inverse manner to that of glycerol. The interconversion of glycerol
to an appropriate low molecular weight lipid cannot be overlooked as possibly
playing a key rdle in the understanding of overwintering.
 GLYCEROL CONTENT AND ITS INFLUENCE ON THE CARABID BEETLE 987

In Fig. 2 the skewed relationship of freezing and supercooling points to glycerol

content may be due to feeding resulting in the presence of increased nucleators
which, while not directly affecting the freezing, will certainly increase the probability
of freezing and raise the supercooling points. The most efficient nucleator, inde-
pendent of other less efficient nucleating compounds, will determine the lowest
temperature to which any system may be supercooled. The linear relationship
between supercooling points and glycerol concentrations during autumn and early
winter lends support to this statement. This interval reflects a period of gut
evacuation which diminishes the concentration of highly efficient atmospheric
nucleators (SALT, 1968) within the gut.
The decrease in glycerol during mid-winter is an apparent anachronism
(Fig. 1). Since glycerol is considered to be the most efficient cryoprotective
compound occurring naturally (SMITH, 1961; DOEBBLER,1966), the search for
other protective compounds would not appear fruitful (i.e. Table 1). Those other
compounds listed in Table 1 have not been detected in concentrations high enough
to play an independent r6le in cold hardening. However, concentration of a number
of substances changed concurrent with the change in glycerol. Kaufmann (personal
communication) has found that during the period of decreasing glycerol concen-
trations, total body fat increases. She has also noted that egg development is
initiated over this period and even indications of feeding activity have been
detected.
It is evident from Fig. 6 that the fluctuations in winter glycerol content are
nearly directly related to variations in temperature. This figure represents a plot

22 -60 -

24 t -70 ’ ’ ’ ’ ’ ’ ’ ’ ’
Mar. Apr. hloy June July Aug. Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr.

FIG.6. Seasonal variations in high-low ambient temperatures (“F) vs. mean

haemolymph glycerol concentrations. Latter scale is inverted. Arrows indicate
major snow accumulations.
988 JOHNG. BAUSTANDL. KEITH MILLER

of seasonal ambient temperatures (high-low) versus mean glycerol content (inverted

scale). The peak December and March glycerol concentrations are directly related
to peak low temperatures of the same period. The January ‘peaks’ of glycerol and
low ambient temperature are offset. Doubtless this is due to the accumulation of
an insulative snow cover during December. That is, the stumps were no longer
exposed as in early December, but covered with 1 to 2 ft of snow. This insulating
layer would tend to dampen the velocity of thermal fluctuation within the stump.
The rapid temperature drop in late December, about 55-60”F (31-34”(Z) in 5 days,
was not effective in cooling the well-insulated microhabitat until mid-January.
The rate of advance of the ‘frost front’ was quite slow. Both the snow cover and
the decayed stump have low thermal conductivities resulting in a slow heat loss.
Warmer temperatures during February resulted in a decrease in insulative
cover and greater exposure of the beetles to variations in ambient temperature.
This is evident during mid-March. A sudden ambient temperature decrease
resulted in a concomitant increase in glycerol concentration.
In the light of these additional observations a number of questions arise.
Since the glycerol concentration within the haemolymph varies directly with
ambient temperature (November to March), is this fluctuation a consequence of
changing metabolic activity evident within the ‘frozen’ insect ? That is, do cold-
activated enzymes commence functioning at a given low ambient temperature ?
With fat build-up and increased ovarian development, is glycerol being trans-
formed to lipid material only to be replaced during January to March as a result
of early feeding on ‘warm’ days ? This theory warrants further consideration.
A second explanation of the fluctuating glycerol levels may be mentioned.
Glycerol is known to build up during autumn in the unfrozen and even super-
cooled insect. During this period glycerol might be entering individual cells but
a decrease within the haemolymph would go undetected. Upon ‘freezing’, the
glycerol would still be expected to diffuse intracellularly. The evaluation of this
theory might serve to explain the protective role played by glycerol within the
cold hardened insect. It should be noted that an ‘accumulation’ of glycerol may be
occurring in specific organ systems, i.e. reproductive organs or fat body, rather
than all cells.
In the light of the information presented in Fig. 5 it is apparent that a survival
advantage is indicated by such a cryotaxic response. Warm spells of relatively
short duration (i.e. a few days) are a common but irregular winter occurrence.
During these periods temperatures may rise as much as 30 to 40°C over a 24 hr
period. Beetles exposed to such a change would be expected to lose glycerol at a
moderate rate. However, this loss might be of sufficient magnitude to decrease
cold-hardiness to a point at which a sudden cooling would prove lethal (e.g.
SPIMME, 1964).
Also, on clear, cold days, exposed stumps are warmed superficially by infra-red
radiation. The author has measured a temperature of 0°C beneath the bark of a
frozen stump while the noonday air temperature was -40°C. This is certainly
an extreme case but indicative of the great thermal fluctuations experienced by
 GLYCEROL CONTENT
ANDITS INFLUENCE
ONTHECABABID
BEETLE 989

P. brevicornis. A positive cryotaxic response would allow the insect to seek a colder
and therefore more thermostable ecoclimate.

CONCLUSION
It is now apparent that cold-hardiness (freezing tolerance) in at least one insect
entails more than just an autumn increase and spring decrease in glycerol content.
Seasonal fluctuations in glycerol have been observed by SALT (1959), S~MME
(1964, 1965), and ASAHINA (1966). However, in P. breoicornis, haemolymph
glycerol content is observed to vary during winter without any apparent loss in
ability to survive low temperatures. MILLER (1968) has found that lower lethal
temperatures do not vary substantially over this period. Kaufmann (personal
communication) has shown that over this same period the total body fat content
is inversely related to glycerol variations. These changes along with initial egg
development occur at a time when stump temperatures range from - 15°C to
- 40°C.
This situation is difficult to resolve. With a decrease in glycerol, an attenuation
of freezing resistance is expected. This is not the case in P. brevicornis during
mid-winter. Haemolymph glycerol decreased without a loss of cold-hardiness.
Other cryoprotective compounds have been checked for and not found to be
present. Since the protective action of a compound is attributed to its affinity to
bind water, that is, the number of hydroxyl groups (SALT, 1961), and no other
polyols have been detected, it appears unlikely that other typical cryoprotective
compounds play a role.
Earlier, it was suggested that glycerol might not be lost during the mid-winter
period but accumulated either intracellularly throughout the organism or within
a particular organ system. Investigation along these lines warrants consideration
in the light of the observations of ovarian development, fat fluctuations, constant
lethal temperatures, and measurements of total body glycerol. An explanation for
the maintenance of a low haemolymph freezing point while glycerol levels decrease
is not available.
Pterostichus brmicornis presents a unique picture. On the basis of the data
presented, it is apparent that a review of experimental procedures utilized by
earlier investigators (SP)MME, 1964, 1965 ; ASAHINA, 1966) be given consideration.
Initial collection and storage temperatures are crucial to a meaningful study.
Maintenance of a cold-hardened condition is tenuous in an insect exposed to or
stored at ‘warm’ temperatures even though below their freezing points. Glycerol
may be lost rapidly but appears at a ‘high’ or ‘normal’ winter level to the investi-
gator. These ‘high’ levels may be below the critical level required for functional
cryoprotection resulting in the erroneous conclusion that a given species is not
freezing-tolerant.

Acknowledgements-The authors wish to thank Dr. L. IRVING for his continued interest
and critical advice related to this study. The technical assistance provided by Mrs. KAREN
COADY and Mr. DON DRAPER has proved invaluable.
990 JOHN G. BAUST AND L. KEITH MILLER

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