ORIGINAL ARTICLE OA 000 EN

Functional
Ecology 1998
Induced thermotolerance and associated expression
12, 786–793 of the heat-shock protein Hsp70 in adult Drosophila
melanogaster
J. DAHLGAARD,* V. LOESCHCKE,† P. MICHALAK‡ and J. JUSTESEN§
*Centre for Tropical Biodiversity, Department of Population Biology, University of Copenhagen,
Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark, †Department of Ecology and Genetics, University
of Aarhus, Ny Munkegade, Bldg. 540, DK-8000 Aarhus C, Denmark, ‡Institute of Zoology, Jagiellonian
University, ul. Ingerdena 6, 30–060 Krakow, Poland, and §Department of Molecular Biology, University of
Aarhus, Universitetsparken Bldg. 135, DK-8000 Aarhus C, Denmark

Summary
1. Inducible heat-shock proteins are synthesized when temperatures are increased to
levels substantially above normal. The functional role of these proteins is well known
at the cellular level. Today increasing interest has been directed towards the impor-
tance of heat-shock proteins for resistance of whole organisms to high-temperature
stress and other environmental stressors.
2. Here the functional relationship between the heat-shock protein, Hsp70, and ther-
mal resistance in adult Drosophila melanogaster was examined by comparing thermal
resistance, i.e. survival at 39 °C for 85 min, and levels of Hsp70 at various times
elapsed (2, 4, 8, 16, 32 and 64 h) after thermotolerance was induced by short-term
acclimation/heat hardening at 37 °C for 55 min.
3. Levels of Hsp70 in both males and females were highest 2 h after heat hardening
and declined with longer times elapsed. The rate of decrease initially was very fast but
diminished with increasing time. After 32 h the level of Hsp70 approached the level in
flies that were not hardened. Levels of Hsp70 in males exceeded that of females during
the entire period.
4. Survival of both sexes increased with increasing time after heat hardening and reached
an optimum between 8 and 32 h. Thereafter resistance decreased with longer times
elapsed. Survival of females generally exceeded that of males except after 16 and 64 h.
5. Regression analysis applied to the data on Hsp70 levels revealed that the model
describing these data could not explain the data for survival. Also, higher levels of
Hsp70 in males compared with females were not associated with greater survival in
males. However, statistical analysis on paired measurements of Hsp70 and survival
revealed a positive association between Hsp70 level and survival at each time elapsed
after induction of thermotolerance.
Key-words: Acclimation, heat-shock proteins, high-temperature stress, insects, survival
Functional Ecology (1998) 12, 786–793

Introduction
Adult Drosophila that have been pre-exposed to high
temperatures benefit from an extended period of
increased thermotolerance (Loeschcke, Krebs &
Barker 1994). The build-up and subsequent decrease in
resistance that results after pre-exposure to high tem-
peratures (Krebs & Loeschcke 1994a) resembles the
regulation of heat-shock protein synthesis after expo-
sure to heat (Landry et al. 1982). We therefore exam-
ined whether heat hardening in adult Drosophila
© 1998 British melanogaster Meigen is correlated with the expression
Ecological Society of heat-shock proteins.
Heat-shock proteins (hsps) belong to highly con-
served protein families. Some of these proteins act as
molecular chaperones and participate in protein folding
and unfolding (Parsell & Lindquist 1994). At the cellu-
lar level heat-shock proteins are important for acquisi-
tion of thermal resistance (Lindquist 1986; Landry
et al. 1989; Parsell & Lindquist 1994), and for microor-
ganisms they are a major factor in coping with high
temperatures. Recently, the heat-shock protein Hsp70
has been found to improve thermotolerance in both
embryos (Welte et al. 1993) and larvae (Feder 1996;
Feder & Krebs 1997) of D. melanogaster, and natural

786
787 variation of Hsp70 expression in larvae has also been
Thermal found to be correlated with thermotolerance (Krebs &
resistance and Feder 1997). These studies constitute the first direct
Hsp70 in D. evidence revealing that heat-shock proteins improve
melanogaster thermal resistance of multicellular organisms.
For adult Drosophila, evidence supports that selec-
tion for thermal resistance occurs in nature (Parsons
1980; Loeschcke et al. 1994), and recent published
data reveal that a selective response in adults does not
evolve as an indirect response from direct selection on
the larval life stage (Loeschcke & Krebs 1996).
However, multicellular organisms such as Drosophila
possess various mechanisms to cope with high tem-
peratures apart from the strictly biochemical ones.
Behaviour, morphology and physiology may all func-
tion to prevent potential damage from temperature
stress. Owing to the evolution of such features, the rel-
ative importance of heat-shock proteins in the most
complex organisms could have become subordinate.
Among the different life stages in Drosophila, the
adult stage is the most complex in terms of behaviour,
morphology and physiology. Nevertheless, despite the
possibility of adaptive changes in morphological and
physiological traits, McColl, Hoffmann &
McKechnie (1996) showed a response of two heat-
shock genes to selection for knockdown heat resis-
tance in adult D. melanogaster. Lessons from cell
cultures, embryos and larvae on the role of heat-shock
proteins for thermal resistance therefore also may
apply to adults.
In addition to the resemblance between the dynam-
ics of heat hardening and the induction of heat-shock
synthesis, other observations appear to link the two
phenomena. Fitness costs experienced by adults
shortly after exposure to high temperatures (Krebs &
Loeschcke 1994a,b) are consistent with two funda-
mental observations related to the heat-shock
response. First, costs may occur owing to repression
of normal cell activity after exposure to heat shock,
e.g. in cell lines of D. melanogaster (DiDomenico,
Bugaisky & Lindquist 1982a,b). Second, increased
consumption of energy for protein production follow-
ing a heat shock (Gething & Sambrook 1992) may
cause an energy debt (Krebs & Loeschcke 1994b) that
increases vulnerability against further stress.
Here we test for a functional relationship between
resistance at various times elapsed after induction of
thermotolerance, by exposing flies to a high non-
lethal stress (37 °C for 55 min), and expression of the
heat-shock protein Hsp70 in adult D. melanogaster.
Thermotolerance was induced in flies from a mass
population and groups of flies were randomly
assigned to either a stress resistance- or an enzyme-
linked immunosorbent assay (ELISA) at 2, 4, 8, 16, 32
and 64 h after induction. This procedure was repli-
© 1998 British cated in two blocks to obtain two replicates of paired
Ecological Society, measurements (survival vs Hsp70 level) for each time
Functional Ecology, elapsed after induction. Paired measurements of
12, 786–793 Hsp70 level and resistance from within each block
and sex, at each time elapsed after induction, were
then examined for potential associations between the
two traits.
Materials and methods
A mass population of D. melanogaster was founded
with adults from reciprocal crosses among three popu-
lations out of Mali in Africa, Italy (Bologna) and
Spain (Tenerife). Prior to the experiment, the popula-
tion was maintained for 21 generations at 25 °C on an
agar–sugar–yeast medium at a population size greater
than 600. For the experiment, 10 bottles with 20 pairs
each were set up and females were allowed to oviposit
for 24 h. During the next 3 days, flies were transferred
to fresh bottles to produce sufficient numbers of flies
for both the stress resistance assay and the ELISA.
INDUCTION OF THERMOTOLERANCE
After emergence, flies were collected and sexed under
light CO2 anaesthesia each day over a 4-day period.
Four blocks with flies of similar age (± 1 day) were
obtained. For the stress-resistance assay, two blocks
contained 56 vials with 20 flies each (two sexes, seven
treatments and four replicates). For the ELISA assay,
four blocks contained 28 vials with 30 flies each (two
sexes, seven treatments and two replicates). Thirty
flies per vial were used in order to reduce random
variation in body size among vials. Three days after
emerging, flies were transferred to fresh food vials. To
induce thermotolerance, flies were exposed to 37 °C
for 55 min in fresh vials containing agar. This temper-
ature is known to be very effective in inducing Hsp70
synthesis in Drosophila cell lines although a tempera-
ture as low as 33 °C is also effective (Lindquist 1980;
DiDomenico et al. 1982a). Preliminary experiments
revealed that 33 °C also results in elevated levels of
Hsp70 in adult D. melanogaster. After induction of
thermotolerance, all flies were transferred back to the
food vials. Flies were allowed to recover for various
lengths of time (2, 4, 8, 16, 32 or 64 h), following
induction, before the ELISA- and the stress-resistance
assays on day six. In the control group tolerance was
not induced.
THERMAL STRESS RESISTANCE
On day six postemergence, adults for the stress-resis-
tance assay, of the seven different acclimation/harden-
ing treatments, were transferred to fresh vials to be
exposed to a potentially lethal stress (39 °C for
85 min). Induction of thermotolerance and heat shock
both took place in inverted food vials containing a 2%
agar solution. Stoppers were moistened and inserted
fully to obtain nearly saturated humidity (treatment
details are described in Krebs & Loeschcke 1994b).
The proportion of surviving flies was scored 22 h
after exposure to heat stress to allow time to recover
788 mobility. Flies were considered alive if they were
J. Dahlgaard able to walk following a slight touch with a brush.
et al.
HSP70 LEVEL
On day six postemergence, flies for the ELISA assay
of the seven different acclimation/induction treat-
ments were transferred to empty glass vials and
stored at – 70 °C. To make an Hsp70 standard, flies
of an Australian D. melanogaster population were
exposed to 38 °C for 45 min and allowed to recover
for 5 h before being stored at – 70 °C. Protein sam-
ples for measurement of Hsp70 were obtained by
homogenizing frozen flies in an ice-cold phosphate
buffered saline (PBS) containing 2 mM phenyl
methyl sulphonyl fluoride (PMSF) and 1% (per vol-
ume) antiprotease cocktail (100 µg ml–1 pepstatin A,
50 µg ml–1 leupeptin, 10 mM benzamidine, 10 mM
sodium metabisulphite). Homogenates were cen-
trifuged at 14 000 g for 30 min at 4–6 °C, and the
supernatants were stored at – 70 °C. Storing adults at
– 70 °C did not affect the level of Hsp70 compared
with adults frozen using liquid nitrogen. Likewise,
storing homogenates at – 70 °C did not affect the
level of Hsp70 compared with the method where
homogenates were used immediately after homoge-
nization, without freezing. Before ELISA, the pro-
tein concentrations of samples and standards were
measured (BCA Assay, Pierce Biochemicals,
Rockford, IL, USA). Samples and standards were
diluted in coating buffer to 30 µg protein ml–1 and
incubated in microplate wells at 37 °C for 1 h to
allow proteins to adsorb to the plate. To make sure
that all protein binding sites were blocked, 150 µl of
2% BSA in PBS was placed in each well at 37 °C for
1 h. After extensive washing, bound Hsp70 was
detected with an Hsp70-specific monoclonal pri-
mary antibody (7.FB) (Velazquez, DiDomenico &
Lindquist 1980; Velazquez et al. 1983) and an HRP-
conjugated secondary antibody (antirat IgG, DAKO
A/S, Glostrup, Denmark). The primary antibody is
specific for the 70-kDa inducible member of the
Hsp70 family in Drosophila, and does not recognize
either Hsp68 or the various Hsc70s. Plates were
incubated with a solution of orthophenylenediamine
(OPD) tablets (DAKO A/S) and the coloured reac-
tion product was measured at 490 nm in a microplate
reader. Each plate contained seven samples (accli-
mation/induction treatments) and two standards in
four replicates. Two blanks (without primary anti-
body) of each sample and standard were included to
allow correction for non-specific signal. Prior to the
experiment, different concentrations of both the pri-
mary and secondary antibodies as well as of the sam-
ples were tested to ensure that the proper range was
© 1998 British worked within (i.e. that the corrected signal was lin-
Ecological Society, early dependent on the Hsp70 concentration) by
Functional Ecology, mixing samples of flies, of which thermotolerance
12, 786–793 was either induced or not, in different proportions.
Results of the ELISA are presented as a percentage
of the signal of the standard. In addition, Western
blotting was performed on a subsample of male
homogenates. Proteins were electrophoretically sep-
arated on 10% SDS-PAGE (sodium dodecyl sulphate
polyacrylamide gel electrophoresis) gels by the
method of Laemmli (1970) and following were
transferred to an Immobilon membrane (Millipore).
The membrane was blocked with PBS containing 5%
dry skim milk. The primary and secondary antibod-
ies described above were used to identify Hsp70 and
the antibodies were visualized with enhanced
chemoluminescence (ECL, Amersham) according to
the manufacturer’s instructions.
THE ASSOCIATION BETWEEN HSP70 LEVEL AND
THERMAL RESISTANCE
One paired measurement (xtbs, ytbs) of survival (x) and
Hsp70 level (y) was obtained for each time (t) elapsed
after heat hardening from each of the two blocks (b)
and for each sex (s). As Hsp70 could not be detected
after 64 h, this treatment was not included in the anal-
ysis. To test whether increases in the level of Hsp70
are accompanied by increases in survival, the between
block differences in Hsp70 level (xt1s – xt2s) and in
survival (yt1s – yt2s) were calculated for each time (t)
and sex (s). For a parametric test of this hypothesis,
the two sets of data were transformed before differ-
ences were calculated in order to obtain homogeneity
of variances within each time group, or at least to
reduce the heterogeneity of these variances. All
survival data were arcsine-square-root transformed
and homogeneity of variances was confirmed
(Bartlett’s test) allowing the estimation of a variance
(vbs) common to all time groups for each block and
sex. The differences, xt1s – xt2s , were adjusted accord-
ing to the formula:
xt1s – xt2s
√
v1s v2s eqn 1
––– + –––
n n
where (n) is the number of samples within each time
group per block per sex.
For the ELISA data, a log10-transformation was
applied in order to remove a dependence of the vari-
ance on the mean. The differences, yt1s – yt2s, were
then calculated. Associations between the differences,
xt1s – xt2s and yt1s – yt2s, were examined by calculating
the Pearson correlation coefficient for each sex sepa-
rately and for both sexes together.
For a non-parametric test, the difference in block
means between block 1 and block 2 of each sex was
added to block 2 in order to obtain the same grand
mean in the two blocks. Associations between the dif-
ferences, xt1s – xt2s and yt1s – yt2s, were then examined
by comparing their signs. If these differences are not
associated the probability of two identical signs in one
comparison is 50% and the probability of two differ-
789 ent signs likewise is 50%. The binomial probability
Thermal for any outcome can then easily be computed.
resistance and
Hsp70 in D.
Results
melanogaster
THERMAL STRESS RESISTANCE
The time for which flies were at 25 °C after induction
of thermotolerance prior to heat shock explained a
significant proportion of the variation in survival
(F5,82 = 2·31, P = 0·05). Survival without prior induc-
tion was low (males: 0·027 ± 0·014; females:
0·070 ± 0·025). Male survival increased with increas-
ing time after thermal induction with highest survival
after 16 h and lower survival after 32 h and 64 h
(Fig. 1). Female survival also increased with time
after induction, except for the 16-h group, with high-
est survival after 32 h and lower survival after 64 h.
For males, survival after 64 h was still significantly
higher compared with flies that were not hardened
(P < 0·05, Duncan’s multiple comparisons test). For
females, survival after 32 h exceeded that of flies
without heat hardening (P < 0·05, Duncan’s multiple
comparisons test). Survival of females exceeded that
of males (F1,96 = 4·01, P < 0·05) except after 16 h and
64 h. The sex × treatment interaction was significant
(F6,96 = 2·74, P < 0·05). Survival in the two blocks dif-
fered (F1,96 = 26·62, P < 0·001) which is normal for
this stress resistance assay but the rank order of the
acclimation/hardening treatments also differed
between the two blocks. The treatment × block inter-
action therefore was significant (F6,83 = 4·9,
Fig. 1. Percentage survival (mean ± SE) of Drosophila melanogaster males and
females after 55 min at 37 °C. Survival was estimated at different times elapsed (h)
between acclimation/heat hardening and exposure to heat shock. Survival without
prior induction was low (males: 0·027 ± 0·014; females: 0·070 ± 0·025).
P < 0·001). The treatment × block × sex interaction
also was significant (F6,83 = 3·54, P < 0·01). These
interaction terms with block, however, were pooled
within the error mean square and thus served only to
make the F-test more conservative.
HSP70 LEVEL
Induction of thermotolerance increased the level of
Hsp70, with the highest concentration 2 h after expo-
sure to 37 °C (Figs 2 and 3). The time at 25 °C after
induction explained a significant proportion of the
variation in the level of Hsp70 (F5,81 = 51·1,
P < 0·001). The level of Hsp70 in males was signifi-
cantly greater than in females (F1,81 = 39·2, P < 0·001).
The difference in Hsp70 level between the sexes
decreased and finally disappeared with increasing time
after induction and the time × sex interaction therefore
was significant (F5,81 = 7·59, P < 0·001). The block
effect was significant (F3,81 = 9·16, P < 0·001). Rank
order of the different acclimation/hardening treatments
generally was consistent between blocks except after
32 h and 64 h where the level of Hsp70 approached
that of flies without heat hardening. The block × time
interaction therefore was significant (F15,63 = 2·6,
P < 0·01). This term, however, was pooled within the
error mean square and thus served only to make the F-
test more conservative. Regression analysis applied to
male and female data suggested that the level of Hsp70
is an inverse function of time elapsed after induction:
(1) Cmale = 26·1 + (212·6/T); (R2 = 0·70;
F1,46 = 108·30, P < 0·001) and (2)
Cfemale = 23·8 + (96·1/T); (R2 = 0·75; F1,46 = 138·49,
P < 0·001). The subsequent decrease in the level of
Hsp70, although initially fast, diminished with time.
The level of Hsp70 thus remained elevated for several
hours, approaching the level of the male (0·19 ± 0·03)
and female (0·22 ± 0·04) control group only after 32 h.
Results from Western blotting were consistent with
those of the ELISA assay (Figs 2 and 3). With increas-
ing exposure time, the ECL detection system revealed
the presence of a faint band at 32 h after induction (not
shown). Furthermore, the background seen in the
ELISA (Fig. 2) is not present in the Western blot
(Fig. 3).
THE ASSOCIATION BETWEEN HSP70 LEVEL AND
THERMAL RESISTANCE
To test for the possibility that an excess in the level of
Hsp70 is accompanied by an excess in survival, the
between block differences in Hsp70 level and in sur-
vival were calculated (see materials and methods) for
each time (t) and sex (s). For a parametric test of this
hypothesis, associations among differences were
examined by calculating the Pearson correlation coef-
ficient for each sex separately (rmale = 0·1, NS;
rfemale = 0·92, P < 0·05) and for both sexes together
(r = 0·44, NS; Table 1).
790
J. Dahlgaard
et al.
Fig. 2. Levels of Hsp70 (in percentage of standard) in Drosophila melanogaster males (a) and females (b) at different times
elapsed after acclimation/heat hardening (the grand means across blocks are indicated by diamonds). The curves connect the
predicted values of Hsp70 estimated by the regression analysis (indicated by dotted lines). Horizontal lines show the level of
Hsp70 in the male and female control group. Males and females were exposed to 37 °C for 55 min. Aliquots of homogenates of
adult flies, that were exposed to 38 °C for 45 min and allowed to recover for 5 h, were used as a standard reference.
For a non-parametric test, associations among
differences were examined by comparing their signs.
For females, five out of five comparisons had identi-
cal signs whereas for males only three of five com-
parisons were identical in sign. For both sexes
therefore 8 of 10 comparisons were of similar sign.
Table 1 shows the different outcomes as well as the
Fig. 3. Western blot showing Hsp70 expression in
Drosophila melanogaster males at different times elapsed
after acclimation/heat hardening. Flies were exposed to
37 °C for 55 min and were allowed to recover for various
lengths of time: 2 h, 4 h, 8 h, 16 h, 32 h and 64 h. For a con-
trol group, tolerance was not induced. A group of adult flies
that were exposed to 38 °C for 45 min and allowed to
recover for 5 h also was included (the standard from the
ELISA assay). Marker: Sigma-prestained ‘blue’ marker.
There were no signs of proteolytic degradation of Hsp70 in
any of the lanes on the blot.
Table 1. Associations between the level of Hsp70 and survival after heat shock of
Drosophila melanogaster estimated on the basis of differences between sets of pair-
wise measurements at five different times elapsed after induction of thermotolerance.
All probabilities given refer to one tailed tests
Binomial ‘sign test’ Pearson correlation coefficient
Males 3:2 (P = 0·50) 0·1 (P = 0·44)
Females 5:0 (P = 0·03) 0·92 (P = 0·01)
Both sexes 8:2 (P = 0·05) 0·44 (P = 0·10)
binomial probability for obtaining an outcome being
at least as extreme as the one obtained (one tailed
probabilities).
Discussion
Inducing thermotolerance by short-term acclima-
tion/heat hardening at 37 °C for 55 min substantially
increased the concentration of Hsp70 in adult D.
melanogaster. For up to 32 h after induction of ther-
motolerance, the level of Hsp70 remained elevated
compared with that of flies without hardening despite
an initially rapid decay rate. The level of Hsp70 in
males greatly exceeded that in females. As synthesis
of Hsp70 is proportional to the severity of the stress
in Drosophila cell lines (DiDomenico et al. 1982a) as
well as in adults (J. Dahlgaard, V. Loeschcke, P.
Michalak & J. Justesen, unpublished observations),
heat hardening may be experienced as more stressful
by males than by females. However, although males
are smaller than females, having a surface to volume
ratio that enhances a faster internal temperature
increase, the main reason for the observed sex differ-
ence in large remains unknown. With increasing
times elapsed after induction, differences in Hsp70
level between the sexes became smaller and finally
disappeared.
Female survival generally exceeded that of males,
as previously reported for this species (Krebs &
Loeschcke 1994b). Survival after heat shock at differ-
ent times after induction resembled that reported by
Krebs & Loeschcke (1994a). For both sexes, resis-
tance initially increased with time after induction,
reached a maximum between 8 and 32 h, and then
791 decreased. The present results, however, suggest a
Thermal somewhat later maximum than do the results of Krebs
resistance and & Loeschcke (1994a). The location of the maximum
Hsp70 in D. may vary depending on the intensity and duration of
melanogaster the acclimation/hardening treatment as recovery from
heat shock in Drosophila cell lines (DiDomenico
et al. 1982b) is proportional to the severity of the
stress. Although it is unclear whether results from cell
lines apply to adults, a process of recovery after heat
hardening may explain why protection is not maximal
2 h after induction, when levels of Hsp70 were high-
est. As thermotolerance in the present study was
induced using a higher temperature than did Krebs &
Loeschcke (1994a), it is likely that higher acclima-
tion/induction temperatures may cause maximal resis-
tance to be delayed and prolonged.
Resistance was not proportional to the level of
Hsp70. In fact the initial increase in resistance was
almost balanced by a decrease in the level of Hsp70.
Also, while the level of Hsp70 in males greatly
exceeded that of females, male resistance generally was
lower than that of females. From these results we may
come to either of two conclusions: (1) Hsp70 is not
important for resistance of adult D. melanogaster or (2)
Hsp70 is involved in resistance in some way but sur-
vival from heat shock is complex and depends on the
cumulative stress level and the physiological state of
cells and organism as well as on sex. DiDomenico et al.
(1982b) showed that restoration of normal protein syn-
thesis is co-ordinate with repression of Hsp70. The co-
ordinate increase in resistance with decreased levels of
Hsp70 that we recorded resembles the pattern found by
DiDomenico et al. (1982b), and may be due to the fact
that normal cell activity has not recovered until at least
2 h after induction (DiDomenico et al. 1982a,b). Krebs
& Loeschcke (1994a) suggested that an energy debt
may increase the susceptibility of flies immediately
after induction. In addition, heat hardening may consti-
tute a stress in itself, as mentioned above, and it is pos-
sible that stress from the acclimation/hardening
treatment and the subsequent heat shock are cumula-
tive. Therefore, it is likely that flies experience an over-
all greater stress level when heat shocked shortly after
induction and, since normal cell activity is not yet
attained, benefits from highly elevated levels of Hsp70
may be outweighed. Attempts to associate resistance
directly with Hsp70 levels thus will confound different
cumulative stress levels and different levels of normal
cell activity with different times elapsed after induc-
tion. Sex differences, whether physiologically or genet-
ically determined, will also confound the analysis.
The experimental design, however, allows another
way to test for associations between Hsp70 level and
resistance. If higher concentrations of Hsp70 enhance
survival after heat shock, paired measurements of
© 1998 British Hsp70 level and resistance from within each block and
Ecological Society, sex, at each time elapsed after induction, are expected
Functional Ecology, to be associated. This approach avoids the confounding
12, 786–793 effects mentioned above in that associations are based
on comparisons within each time group for each sex
separately. A positive association was found between
Hsp70 level and resistance at each time elapsed after
induction, although this was significant only for
females. The trend, however, was similar for the male
data and the combined results therefore suggest a func-
tional relationship between the level of Hsp70 and heat-
shock tolerance in adult D. melanogaster.
Almost no attention has been paid to the ecological
significance of heat-shock protein expression in adults
for two reasons. First, adult Drosophila are highly
mobile and therefore should be able to avoid high
temperatures behaviourally, perhaps limiting the
attention to the molecular stress protection system
offered by the heat-shock proteins. Second, detection
of heat-shock protein expression in nature is techni-
cally difficult and not until recently (Feder 1996) was
the first report published on natural expression of
Hsp70 in larvae. That variation in heat-shock genes is
present and potentially could play an important eco-
logical role was demonstrated by McColl et al. (1996)
who detected a correlated response in the allele fre-
quencies of hsp68 and hsr-omega after selection for
increased knockdown heat resistance in D.
melanogaster. As the geographical distribution of D.
melanogaster extends into regions of high tempera-
tures, it is possible that Hsp70 expression in adults
play an important ecological role. Activity of adult D.
melanogaster has been observed in central Chaco,
Argentina, at temperatures of at least 33 °C in the
shade (J. Dahlgaard, E. Hasson & J. S. F. Barker,
unpublished data from March 1997). Temperatures
inside the collecting buckets, when placed in direct
sunlight, typically were 2–5 °C higher in the centre of
the buckets. Adults were nevertheless still attracted to
these buckets, allowing for the possibility that the
heat-shock response at least temporarily may be acti-
vated in adults in the field. Experiments with free-
ranging adult flies also revealed that the heat-shock
response, at least temporarily, may be activated in
nature (M. Feder, personal communication). Krebs &
Loeschcke (1994a) measured the cost of activating the
heat-shock response, and showed that when food con-
ditions were improved, the negative consequences of
expressing the heat-shock response may be reduced.
Behavioural evasion therefore may not always be nec-
essary and perhaps benefits in terms of access to
resources for oviposition and feeding outweighs the
cost of activating the heat-shock response. Activity of
other Drosophila species, e.g. Drosophila simulans
Sturtevant and Drosophila buzzatii Patterson and
Wheeler, also has been reported at high temperatures,
of at least up to 35 °C in the shade (Vilela, Sene &
Pereira 1980).
The present results suggest a functional role of
Hsp70 that for two reasons could be of considerable
ecological importance. First, in nature temperatures
can increase rapidly (Dahlgaard & Loeschcke 1997),
and a molecular stress resistance system able to
792 respond at least as fast conceivably is beneficial. In
J. Dahlgaard the present study, the response to short-term acclima-
et al. tion/heat hardening was very fast, causing high levels
of Hsp70 to accumulate within a short time. Second,
as temperature in nature fluctuates diurnally, current
temperature extremes may predict future extremes.
Thus, because acquired thermotolerance does not
decay rapidly, it may protect against both an immedi-
ate thermal stress and those stresses experienced on
subsequent days. The extended period in which Hsp70
is detectable in adults after induction also is interest-
ing from the perspective that it contrasts with the situ-
ation in embryos. At the early embryo stages, Hsp70 is
sequestered into granules within minutes after return
to normal temperatures (Parsell & Lindquist 1994).
The detrimental effects of Hsp70 on growth and cell
division (Feder et al. 1992) therefore are most pro-
nounced in embryos or at least have the greatest con-
sequences at this life stage.
From our results it is reasonable to conclude that
the initial discovery of Hsp70 as a critical factor for
heat-shock resistance in early life stages of a multi-
cellular organism, i.e. of embryos (Welte et al. 1993)
and larvae (Feder 1996) of D. melanogaster, now
possibly can be extended to the adult life stage.
However, the average level of Hsp70 in whole ani-
mals cannot be the only critical factor for survival.
Higher levels of Hsp70 in males than in females, for
instance, do not correspond to males being more
resistant than females. Tissue-specific expression of
Hsp70 may imply that the level of Hsp70 in
homogenates of whole-bodied animals perhaps is not
the major determinant for survival. Instead, levels of
Hsp70 in tissue with low levels of Hsp70 expression
may be critical. Also, Krebs & Loeschcke (1996)
found that selected autosomal genes can have differ-
ent quantitative effects in males and females. Thus,
sex differences in resistance are not straightforward
to explain and may eventually be physiologically
determined. That Hsp70 is not the only factor respon-
sible for thermal resistance (Bader et al. 1992; Fisher
et al. 1992; Parsell & Lindquist 1994) was also sup-
ported by the fact that after the level of Hsp70 had
fallen to base level, resistance remained elevated. In
consequence, Hsp70 may be an important part in a
molecular cascade possibly involving other heat-
shock proteins with the entire cascade being respon-
sible for the regulation of thermal stress resistance in
adult D. melanogaster.
Acknowledgements
Flies from Mali and Bologna were kindly provided by
Sandro Cavicchi. The monoclonal antibody, 7.FB, was
generously provided by Susan Lindquist. We thank
Jørgen Granfeldt from the Department of Theoretical
© 1998 British
Ecological Society, Statistics, University of Aarhus, for advice on statisti-
Functional Ecology, cal analyses and Martin Feder for many helpful sug-
12, 786–793 gestions to the manuscript. Martin Feder provided a
protocol for the ELISA assay that was subsequently
modified. We are grateful to Doth Andersen for excel-
lent technical help, to Margit Christensen for homoge-
nizing flies and to Inger Bjørndal, Jure Piskur and
Lisbeth Hoenberg for helping with the protein assay.
We thank Jesper G. Sørensen for assessing different
techniques of protein/sample storage. The study was
supported by a grant from the Carlsberg Foundation
(No. 96–0359–40) and a Tempus grant to support the
stay of PM in Aarhus.
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Received 19 December 1996; revised 8 January 1998;
accepted 9 January 1998