Physiological Entomology (1991) 16, 447-456
Physiological aspects of diapause and cold tolerance
during overwintering in Pieris brassicue
A . S . PULLTN, J . S . B A L E * a n d X . L . R . F O N T A I N E t
Department of Biological Sciences, University of Keele, *Department of Pure and
Applied Biology, University of Leeds, and 'School of Chemistry, University of Leeds
Abstract. The relationship between diapause-associated metabolic suppression and carbohydrate metabolism linked with cold tolerance was
investigated in pupae of Pieris brassicae L. Cold tolerance was assessed
by measuring the crystallization temperature ( T,) and by estimates
of pre-freeze mortality. Metabolic suppression was measured using
31P nmr and carbohydrates by GLC.
Sorbitol (a possible cryoprotectant) accumulated from the onset of
diapause in October until December reaching a concentration of c. 40
mMolal in both years of the study, but then declined from January until
adult eclosion in May. The T, remained between -23 and -25°C
throughout the winter except for a slight rise before eclosion in May.
The absence of a significant T, suppression is as predicted from the
low concentration of sorbitol accumulated. The pre-freeze mortality
experiments indicate that pupae are most cold tolerant in the period
December-February when sorbitol concentration is high, suggesting
an alternative cryoprotective role for sorbitol. Glycogen declined at the
beginning of diapause until February after which there was some
recovery suggesting that it may be the source of carbon for sorbitol
synthesis.
Diapause-associated metabolic suppression is evident in the low 31P
nmr resonances of ATP during November-February compared with
non-diapause pupae and diapause pupae soon after pupation. The
suppression of metabolism at this time may have a direct role in
cryoprotection and by itself (rather than sorbitol) account for the
increased pre-freeze cold tolerance. ATP appears to increase slowly
from February until a sharp increase occurs shortly before eclosion.
Arginine phosphate remains high during diapause until late FebruaryMarch when it begins a decline which continues until eclosion. A period
of change in energy and carbohydrate metabolism is apparent at the
same time which may indicate diapause termination and related changes
in cold tolerance mechanisms.
It is argued that in P.brassicae sorbitol accumulates as a result of
Correspondence: Dr A. S. Pullin, Department of Biological Scienccs, University of Keele, Keele, Staffs
STS 5BG.
447
448
A . S. Pullin, J . S . Bale and X . L. R . Fontaine
metabolic suppression and may have no cryoprotective role. However,
for species living in, or colonizing, low temperature environments it is a
short evolutionary step to exploit this pathway and accumulate high
concentrations of polyols as a specialized cold tolerance strategy.
Key words. Diapause, cold tolerance, metabolic suppression, Pieris,
cryoprotectant, overwintering.
Introduction
Insects employ two major strategies to increase
their chances of surviving the winter. The first
of these, termed diapause, is a hormonally
mediated state, induced in advance of adverse
conditions, often in response to specific environmental stimuli, enabling the insect to accumulate energy reserves in preparation for a
long period without food. The diapause state is
usually characterized by some degree of metabolic suppression and the insect may go through
stages of responsiveness to environmental stimuli which eventually terminate the diapause
(Tauber & Tauber, 1976; Danks, 1987). The
second strategy, increased cold tolerance, is
characterized by a suite of adaptations which
enhance survival at low temperatures. A few
insects are tolerant of extracellular freezing,
whilst the majority are intolerant. Cold tolerance is thought to be achieved by an array of
physiological mechanisms including accumulation of cryoprotectants and production of
antifreeze proteins (Zachariassen, 1985).
Since the earliest investigations of both diapause and cold tolerance it has been apparent
that there is great diversity within these strategies and any synthesis of understanding of
their evolutionary relationship remains elusive.
In many insects, increasing cold tolerance during
autumn coincides with the onset of diapause,
but the relationship between the two strategies
is unclear. Salt (1961), reviewing evidence up to
1960, concluded that cold tolerance mechanisms
were separate from diapause and their apparent
relationship was due only to their coincident
timing. Danks (1987) considered that diapause
was only rarely interrelated with general cold
hardiness. However, Chino (1957) reported that
the accumulation of the cryoprotectant sorbito1 was associated with the diapause state in
eggs of Bombyx mori and considerable evidence
has emerged since that time to suggest that in
many species diapause has a controlling influence over some cold tolerance mechanisms
(Somme, 1982), particularly cryoprotectant
synthesis (Yamashita et al., 1975; Yaginuma &
Yamashita, 1978; Tsumuki & Kanehisa, 1981),
if not over general cold hardiness, often
measured only in terms of crystallization temperatures (supercooling points). In some insects, such as Ips acuminatus, diapause has some
influence over cold tolermce and cryoprotectant synthesis, but low temperature also plays a
key role (Gehrken, 1985). In a few insects,
particularly from arctic and antarctic regions,
such as some collembolans (Cannon & Block,
1988) and Coleoptera such as Scolytus ratzeburgi (Ring, 1977), cold tolerance is increased
without diapause.
It is becoming clear that major adjustments
in metabolism are often made to increase cold
tolerance, such as the commitment of glycogen
reserves to glycerol and sorbitol synthesis in
Eurosta solidaginis and to glycerol in Epiblema
scudderiana (Storey, 1990). Since many species
exhibit a diapause which also involves major
metabolic adjustments, usually in terms of a
suppression of respiratory or energy metabolism, the probability of some interrelationship
would seem high.
Diapausing pupae of the cabbage white
butterfly, Pieris brassicae, accumulate a common cryoprotectant, sorbitol, in the absence of
low temperature exposure, whilst non-diapause
pupae show no accumulation, even at low temperature (Pullin & Bale, 1989a). Additionally,
artificial termination of diapause by injection of
ecdysone causes rapid depletion of sorbitol to
non-diapause levels (Pullin & Bale, 1989b).
Thus in this species there is good circumstantial
evidence for a link between diapause and
carbohydrate metabolism associated with cold
tolerance. Guillet (1976) has shown that dia-
Diapause and cold tolerance
pausing P. brassicae have greatly reduced oxygen consumption after pupation indicating a
suppressed metabolism. The study presented
here investigates the relationship between
metabolic suppression measured by 31P nmr
and carbohydrate metabolism, cryoprotectant
synthesis and cold hardiness in field ovenvintering populations of P. brassicae.
Methods
Rearing and overwintering regimes. Diapausing and non-diapausing pupae were reared by
exposing larvae taken from a laboratory culture
to short (LD 12:12 h) and long (LD 18:6 h) daylengths respectively at a temperature of 20°C.
Non-diapausing pupae were kept at 20°C
throughout development, whilst diapausing
pupae were exposed to field overwintering conditions by placing them inside open-topped
plastic cuvettes (exposed to air but sheltered
from rain and direct sunlight) on the roof of a
building in Leeds, West Yorkshire, U.K.,
during the winter of 1988/89 and in Keele,
Staffordshire, U.K., during the winter 1989/90.
Maximum and minimum temperatures were
recorded daily. Groups of pupae were sampled
periodically for cold tolerance and biochemical
analysis. Only crystallization temperatures ( T,)
and sorbitol concentration were measured
during 1988/89.
Crystallization temperature. The Tc or supercooling point of individual pupae was measured
at a cooling rate of 1°C min-’ using a thermoelectric cooling system. Insects were in contact
with thermocouples recording the temperature
via a thermocouple converter linked to a computer based recording system (Bale et al., 1984).
Groups of overwintering pupae were measured
at 2-week intervals during the winter 1988/89.
Measuring pre-freeze mortality. Cold tolerance at temperatures above the T, was assessed
by exposing pupae to - 10°C for periods from 1
to 28 days. Assessment of survival was based on
the successful emergence of the adult. On four
occasions in winter 1989/90 groups of 100 diapausing pupae were taken from their overwintering site for the required exposure period
to -10°C and immediately returned to the
field. Non-diapause pupae were given the same
regime 5 days after pupation but were kept at
20°C before and after exposure. The period of
449
exposure which would be expected to kill 50%
of the group (LTso) was then calculated as an
indicator of the level of pre-freeze cold tolerance using probit analysis after correcting for
mortality in controls using the formula of
Abbott (1925).
Biochemical analyses. Glycogen content
of pupae was measured using the enzymatic
method of Keppler & Decker (1974). Carbohydrates were measured using gas-liquid
chromatography after Pullin & Bale (1989~).
Freeze-dried pupae were crushed and suspended in ice-cold 60% ethanol, centrifuged at
12,000 g, the supernatant removed and the process repeated. Pooled supernatants were dried
under nitrogen and derivatized using sigma silA. Quantification was achieved using a SE-30
WCOT capillary column and flame ionization
detector. The column temperature was held
at 110°C for 5 min then warmed to 250°C at
3”C/min and held there for 25 min. Helium
was used as the carrier gas and arabitol as
an internal standard. Each treatment group
consists of six pupae which were analysed
individually.
Phosphorus nrnr. A group of pupae was kept
in the same field overwintering conditions as
the above group at Keele during 1989/90 and
one male and one female were removed for
measurement of their 31P nmr spectra at approximately monthly intervals. The male pupa
survived throughout the overwintering period
but female pupae had to be replaced twice
because of mortality (possibly due to bacterial
or fungal infection). Therefore one series of
spectra is from one male individual but the
second is a composite of three female pupae.
Additionally one male, non-diapausing pupa
was measured three times over its 10 day
developmental period at 20°C.
The 31P nmr measurements were carried out
at 9.35T on a Bruker AM-400 spectrometer
equipped with a 10 mm tuneable multinuclear
(high frequency range) probehead and operating at 162 MHz for phosphorus. Each experiment was performed on a single pupa held in a
10 mm outside diameter borosilicate glass tube.
The experiments were run at ambient magnet
temperatures (c. 293 K or 20°C) without sample
spinning and without lock stabilization (field
sweep off); the magnet drift ( c . 0.01 ppm/h) did
not significantly affect the experiments.
Typically 20-50 thousand transients of 1 K
450
A . S. Pullin, J. S . Bale and X . L . R . Fontaine
points were collected for each measurement. A
90" pulse width (17.3 ps in our instrument configuration) was used with a pre-acquisition delay
of 1 s. The free induction decays were multiplied by a decreasing exponential function
(equivalent to 20 Hz line broadening) prior to
Fourier transformation to improve the signal to
noise ratios (typical 31P resonance linewidths
were larger than 200 Hz without the use of any
broadening functions). Chemical shifts are given
to high frequency (low field) of 85% H3P04
taken as -40 480 730 Hz and are accurate to
1 PPm.
-16
1
-30!. ,
SEP OCT
. , . , . , . ,
NOV
DEC
JAN
FER
, . , .
.
MAR
APR
I
MAY
overwintering time
Fig. 1. Mean (SE) crystallization temperatures of
Pieris brussicae pupae during overwintering in the
field at Leeds, U.K., during 1988/89. n = 9.
Results
Cold tolerance
The T, of diapause pupae show little change
during overwintering, ranging between -23
and -25°C with a small rise just before adult
eclosion to a mean of -22.5"C (Fig. 1). This
approaches the mean figure for non-diapause
pupae of -21.4"C reported by Pullin & Bale
(1989a).
The pre-freeze mortality tests indicate that
diapause pupae are most cold tolerant in midwinter (December-February). During this time
over 50% of the population can survive 28 days
(670 h) at -10°C (Table 1). Early and late in
winter cold tolerance is significantly reduced
resulting in SO% of the population dying after
exposure to -10°C for 108 h and 166 h respectively. Non-diapause pupae appear to have a
similar cold tolerance 5 days after pupation,
with an LTso value of 172 h. The effect of
exposure time also differs between groups.
In mid-diapause, increasing exposure time has
little effect on mortality as indicated in the 142day diapausing group in which the mortality
was 22% after 1 day and 30% after 28 days at
-10°C. In contrast, in non-diapause pupae
exposure time had a marked effect on mortality
there being 100% survival after 1 and 3 days at
- 10°C but 100% mortality after 14 and 28 days.
Both of the winters over which the experiments were undertaken were unusually mild.
In 1988/89 subzero temperatures were experienced only during four short spells of only a few
days with a minimum temperature of -2°C. In
1989/90 subzero temperatures were more frequent, most notably during November and
April, but the minimum temperature was only
-4°C. This indicates that pupae would not be in
danger of freezing and that pre-freeze mortality
would also be low. This was supported by the
level of mortality recorded which was below
10% in both years and could not be attributed
to low temperature exposure.
Overwintering physiology
At the start of overwintering diapausing
pupae contain a mean concentration of
55.6 mg/g dry weight glycogen, but this then
declines rapidly to a minimum concentration of
6.1 mglg 3 months later (Fig. 2). Thereafter
Table 1. Tolerance of Pieris brussicue pupae to a pre-freeze temperature (- 10°C) at different stages of diapause
shown as time in hours taken to kill 50% of the experimental group (LTs0). n = 20 for each treatment.
Non-diapause
LTso (-10°C)
95% CI
172
143-204
Days in diapause
22
92
142
205
108
39-220
>670
>670
-
.-
166
83-344
Diapause and cold tolerance
451
60
E
$50
s
2 40
U
0
SEP
OCT
NOV
DEC
JAN
FEB
MAR
APR
MAY
SEP
OCT
NOV
time in diapause
DEC
JAN
FEB
MAR
APR
MAY
time in diapause
Fig. 2. Mean (SE) concentration of glycogen (M) and
sorbitol (V)in Pieris brassicae pupae overwintering
in thc field at Keele, U.K., during 1989190. n = 6.
Fig. 4. Mean (SE) concentration of sorbitol in Pieris
brassicae pupae overwintering in the field at Leeds,
U.K., during 1988/89 (M) and Kcele, U.K., during
1989/90 (V).n = 6.
changes are not so marked but there is an
increase during February -March followed by a
decrease in the month before eclosion.
Glucose concentration remains stable during
most of the winter but shows a decline at the
end of February and a sharp increase before
emergence in May (Fig. 3). After an initial
increase during October -November fructose
reaches a similar concentration to glucose but
declines to significantly lower concentrations by
March.
Trehalose appears to be the most dynanfk of
the carbohydrates measured (Fig. 3). It shows
two major peaks of concentration, one at the
beginning of overwintering in October and
another at the end in early April. Two more
minor peaks occur between these times. The
lowest concentration of trehalose corresponds
M
with that of glucose at the end of February.
However, it is difficult to attribute any significance to this pattern without further work.
Sorbitol shows the same pattern of accumulation in both winters, reaching similar peaks of
concentration but slightly earlier in 1988189
(Fig. 4). In this year the subsequent decline
begins very early in December but is only
gradual. In 1989190 the decline does not begin
until late January but is more rapid in February.
Sorbitol concentration returns to pre-diapause
levels immediately prior to adult eclosion. If
sorbitol levels are expressed in mglg dry weight
of pupae to enable comparison with glycogen,
as in Fig. 2, it is evident that the increase in
sorbitol can be accounted for by the breakdown
of glycogen.
During overwintering the water content of
pupae gradually increases from 73% to 18%
wet weight, and the fresh weight loss during
both winters was a little over 10% by the
beginning of May.
Phosphorus nmr and energy metabolism
30
3
10
0
SEP
OCT
NOV
.
.
.
.
.
.
DEC
JAN
FEB
MAR
APR
MAY
time in diapause
Fig. 3. Mean (SE) concentration of glucose (M),
fructose (V) and trehalose (0)in Pieris brussicue
pupae overwintering in the field at Keele, U.K.,
during 1989/W. n = 6.
The overwintering 3 1 nmr
~
profile of male
and female P. brassicae is shown in Fig. 5. Seven
peaks are clearly resolved at the beginning of
diapause representing (from left to right) sugar
phosphates, inorganic phosphate, arginine
phosphate, yATP, aATP, uridine diphosphoglucose (UDPG) and BATP. Subsequent
spectra show an initial decline in ATP from
October to December indicating metabolic suppression, after which low levels are maintained
452
A . S. Pullin, J. S. Bale and X . L . R. Fontaine
n
n,
h1
YATP
> ' . . . , . . ' , * . . . . I . ' .
15
10
5
aATP
BATP
. 1 . " ' 1 ' " ' 1 ' ~ " 1 . " ' 1
0
-5
PPM
-10
15
-20
-25
15
10
5
0
-5
-10
15
-20
-25
PPM
Fig. 5. 31P nmr spectra of a male (a) and female (b) Pieris brassicae pupa overwintering in the field at Keele,
U.K., during 1989/90. Chemical shift in ppm from 85% H3P04. Date of analysis is shown on the left of each
spectrum. SP = sugar phosphates, IP
diphosphoglucose.
=
inorganic phosphate, AP
until February when ATP begins to increase
again. A more marked increase occurs in the
last spectra taken in May just before adult
emergence. The male emerged on 14 May, 4
days after the last measurement, but the female
emerged on 13 May only 2 days after the last
measurement. This may therefore account for
the higher ATP resonances observed in the last
female spectrum. Arginine phosphate remains
high throughout the period October-March
after which levels decline, most markedly in the
male pupa. The inorganic phosphate resonance,
which is distinguishable at the beginning of
overwintering, is progressively lost in the broad
resonance of sugar phosphates. This large sugar
phosphate resonance is a combination of unresolved resonances representing a range of
sugar phosphates (Asakura et al., 1988). In
some spectra different sugar phosphate reson-
=
arginine phosphate, UDPG
=
uridine
ances are partially resolved such as that of the
female pupa on 4 October. The UDPG resonance is resolved only in the October spectra of
each pupa.
Spectra from a non-diapausing pupa shown in
Fig. 6 indicate that high levels of ATP are
maintained throughout the pupal and pharate
adult stage and arginine phosphate declines
only slightly. Inorganic phosphate appears to
decline becoming indistinguishable from the
broader sugar phosphate resonance and similarly UDPG becomes indistinguishable from
the ATP resonance.
Discussion
The supercooling capacity of diapausing and
non-diapausing P.brassicae pupae is sufficient
Diupuuse and cold tolerunce
Fig. 6. 3'P nmr spectra of a non-diapausing male pupa
of Pieris brussicue during its 10-day development at
20°C. Chemical shift in ppm from 85% H3P04. Time
of analysis in days after pupation is shown on the left
of each spectrum. SP = sugar phosphates, IP =
inorganic phosphate, AP = arginine phosphate,
UDPG = uridine diphosphoglucose.
to avoid freezing during normal British winters.
This cannot be accounted for by accumulation
of cryoprotectant since sorbitol is at very low
concentration in non-diapause pupae and immediately after pupation in diapause pupae and
only accumulates to a maximum concentration
of around 40 mMolal which is sufficient to depress the T, by only a fraction of 1 degree. This
is confirmed by the lack of T, depression observed during overwintering, and suggests that
the supercooling capacity is achieved by other
means. Zachariassen (1985) observes that many
insects can supercool to below -20°C without
accumulation of cryoprotectants and this may
be achieved by voiding of ice nucleators in the
gut and masking of proteins that may act as
intracellular ice nucleators. In P. brussicue this
process may be related to pupation since nondiapause pupae have a mean T, of -21°C. The
mean T, of diapause pupae may represent the
maximum achievable by this mechanism since
S0mme (1967) and Hansen & Merivee (1971)
reported similar T, values for this species from
the colder climates of Norway and Estonia respectively where temperatures below the T, are
frequently experienced. However, it seems
453
likely that the diapause state is responsible for
the observed depression of the T, in diapause
compared with non-diapause pupae.
The results of the pre-freeze mortality
measurements suggest that this may be an important source of mortality in colder climates
but there are no data to confirm this. British
winters are unlikely to cause significant prefreeze mortality based on these results; however, only one exposure temperature was used
and data using higher temperatures could be
revealing. The increase in pre-freeze cold tolerance in mid-diapause compared with early and
late diapause pupae coincides with high concentrations of sorbitol at this time, suggesting a
possible cryoprotective role for this polyol other
than T, depression. It has been suggested that
polyols may inhibit changes in protein structure
caused by low temperature. This was prompted
by the observation of Gekko & Timasheff
(1981a, b) that glycerol appears to inhibit protein denaturation caused by desiccation.
Alternatively, Williams (1990) has suggested
that polyols may stabilize membrane structures
at low temperatures. The need for such protection may account for the accumulation of low
concentrations of polyols observed in many
temperate insects. The relatively high level of
cold tolerance shown by the T, and LTso of
non-diapause pupae may be indicative of the
level of pre-adaptation to overwintering. However, when comparing the LTs0 values it should
be remembered that the diapause groups had to
survive a longer period between cold exposure
and eclosion than the non-diapause group. This
also holds for comparison between early and
late diapausers, perhaps explaining the relatively low LTso exposure time in the former
group.
The decrease in glycogen at the beginning of
diapause is probably due to breakdown to glucose resulting from the activation of glycogen
phosphorylase (Storey & Storey, 1988). In some
insects the stimulus for this action could be low
temperature exposure as reported by Hayakawa
(1985). Sorbitol may then be produced by diversion of carbon from the glycolytic pathway
by blocking of the pathway by PFK inhibition.
For example, Storey (1982) found that sorbitol
synthesis in Eurostu soliduginis was triggered by
low temperature inhibition of PFK diverting
carbon from the synthesis of glycerol to sorbitol.
The glucose produced by glycogen breakdown
454
A . S . Pullin, J . S. Bale and X . L . R. Fontaine
in P.brassicae is more than sufficient to account
for the sorbitol produced and approximately
60% of the carbon must travel along other
pathways. However, low temperature exposure
cannot be the trigger for this process, because
previous experiments have shown that sorbitol
is synthesized in preference to glycerol in the
absence of low temperature exposure (Pullin &
Bale, 1989a). Storey (1990) suggests that glycogen breakdown by activation of glycogen
phosphorylase may result from hormonal induction via CAMP,thus providing an alternative
pathway of induction (if low temperature is not
responsible for hormone release) and this is
certainly worthy of further investigation.
Although the relationship between the prefreeze mortality levels and the pattern of sorbito1 accumulation suggest a cryoprotective role
for sorbitol it is also possible that the metabolic
suppression itself provides the cryoprotection
since this a h e l a t e s equally well with the prefreeze data. The suppression of some metabolic
pathways in advance of low temperature exposure may prevent the damaging imbalance which
may occur when enzyme activities change relative to each other as temperature decreases.
The suppression of energy yielding pathways
during diapause is presumably under hormonal
control and the effect of decreasing the concentration of free adenylates is apparent in the nmr
spectra. Whilst ATP concentration is low in
mid-diapause, inorganic phosphate is high, indicating a reduced number of high energy phosphate bonds. However, in the early stages of
overwintering metabolism P. brassicae does not
appear to rely on arginine phosphate as an
energy store. This molecule acts as a buffer to
changes in energy demand and might be expected to decrease along with ATP (Newsholme
& Start, 1973). Although some fluctuations do
occur, the generally high levels of arginine
phosphate maintained until February-March
are suggestive of a well-balanced energy state
during diapause. Arginine phosphate levels
peak during late February at the beginning of
the period of ATP increase. The subsequent
decline, presumably to provide ATP, is suggestive of a switch in metabolism after this time. In
this context the decrease in sorbitol during mid
winter is interesting because it occurs over the
same period in which glycogen increases,
suggesting a reversal of carbon flow, and at a
time when metabolic activity appears to be in-
creasing as shown by a progressive increase
in ATP. This is accompanied by a decline in
trehalose, glucose and fructose. Such a dynamic
period may indicate a change in the diapause
state and a relaxation of the suppression of
metabolism. This unblocking of metabolic pathways may explain the decrease in sorbitol and
supports the hypothesis that the initial sorbitol
accumulation is a consequence of a suppressed
metabolism. The increase in metabolic activity
in late February may be the result of a period of
high endogenous ecdysone titre associated with
termination of pupal diapause (Chippendale,
1983). Termination of diapause by injection
of synthetic ecdysone has been shown to decrease sorbitol concentration in diapausing
P .brassicae, suggesting that sorbitol synthesis may be under hormonal control or be
dependent on the hormonal suppression of
metabolism (Pullin & Bale, 1989b).
Whether the accumulation of low concentrations of sorbitol (and other polyols) is an
incidental result of this metabolic suppression
in P. brassicae (and other temperate insects) or
is specifically an adaptive mechanism for cold
tolerance is still a matter of debate. Merivee
(1978) suggested that species showing an intensive diapause such as P . brassicae possessed no
special cold tolerance adaptations, but survived
because of their high level of diapause induced
metabolic suppression. From an evolutionary
point of view the accumulation of modest
amounts of polyols in temperate insects may
not be an adaptation to cold tolerance but may
simply reflect a feature of diapause (or low
temperature) metabolism. If this is the case,
then the accumulation of high concentrations of
polyols in some species can be seen as a short
evolutionary step involving selection for individuals exhibiting extreme characteristics of a
mechanism or synthetic pathway which already
exists. Alternatively, as mentioned above, such
low cryoprotectant concentrations may play a
vital role in protection against pre-freeze mortality and may represent one end of the spectrum
of a highly evolved cold tolerance strategy. A
consideration of the diversity of both diapause
and cold tolerance strategies shown within insects suggests the evolutionary pathways are
likely to be complex but diapause related metabolic suppression deserves consideration as a
basis for cold tolerance strategy in some insect
groups.
Diapause and cold tolerance
Acknowledgments
Our thanks t o David Blakeley, Ann Cornes and
Steve Coulson. The Leeds experiments were
funded by SERC grant GR/D 75441 to J.S.B.
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Accepted 14 January 1991