Genes Genet. Syst. (2013) 88, p. 289–300

Cold tolerance and metabolic rate increased by cold
acclimation in Drosophila albomicans
from natural populations
Kotoha Isobe1, Aya Takahashi1,2 and Koichiro Tamura1,2*
1

Department of Biological Sciences, Tokyo Metropolitan University, 1-1 Minami-osawa,
Hachioji-shi, Tokyo 192-0397, Japan
2
Research Center for Genomics and Bioinformatics, Tokyo Metropolitan University,
1-1 Minami-osawa, Hachioji-shi, Tokyo 192-0397, Japan(Received 9 November 2013, accepted 16 January 2014)

Cold acclimation is one of the important factors in temperature adaptation for
insects needing to make rapid adjustment to the seasonal temperature changes intheir living environment. In a fruit fly species, Drosophila albomicans, which has
a tropical origin and currently has a wide geographic distribution extended into
Asian temperate regions, cold tolerance in terms of survival time at 1°C of adultflies reared at 25°C was substantially improved by a cold acclimation at 20°C for
several days. Examining 29 isofemale lines from widely distributed natural populations, we observed a substantial variation in their acclimation response.
However, the acclimation response was not necessarily stronger in the strainsfrom the recently colonized temperate regions. A significantly stronger acclimation response was detected in male flies of the temperate strains when comparedto those of the tropical strains. D. albomicans also showed stronger cold tolerancecompared to its closely related species belonging to the D. nasuta subgroup.
Among these strains, we detected a strong positive correlation between the cold
tolerance change and the metabolic rate change upon the cold acclimation, suggesting their strong physiological association regulated by common genetic factors,
which may have been the target of natural selection for the temperature
adaptation. The response to deacclimation and reacclimation suggested that asystematic change in gene expressions is the main molecular mechanism for thecold acclimation to have effects on the cold tolerance and metabolic rate changes.
Key words: cold acclimation, cold tolerance, Drosophila albomicans, metabolic
rate, temperature adaptation
INTRODUCTION
Cold tolerance is a critical trait in insects whose activity and survival are directly affected by changes in theirenvironmental temperature (Lee and Denlinger, 2010).
In Drosophila, species distributed in relatively colder
locations usually show higher cold tolerance compared tothose distributed in relatively warmer locations. Thistendency is consistent with the concept of environmental
adaptation and is often observed in geographic populations within a single species (Hoffmann et al., 2003 forreview). These facts clearly indicate that cold tolerance
is one of the key factors affecting fitness of insects,
enabling them to withstand conditions in their environ* Edited by Etsuko Matsuura* Corresponding author. E-mail: ktamura@tmu.ac.jp

ment.
To cushion the impact of seasonal temperature fluctuation, the capacity for adjusting physiological condition to
the temperature changes is thought to be an importantpart of cold tolerance. Many insects respond to an exposure to a cooler temperature than usual for several days
and enhance their cold tolerance in preparation for the
anticipated subsequent temperature changes. Such phenomenon is called acclimation and is observed as an
improved survival rate in a cold temperature (say ~0°C)
after experiencing a mildly lower temperature than theusual living temperature (Salt, 1961). Rako and Hoffmann(2006) classified this type of plastic response into three
categories: “rearing acclimation” as a continuous response
to a lower temperature than the optimal living temperature during the course of development (e.g., Ohtsu et al.,1999), “cold acclimation” as a response to a moderately

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low temperature for a few days to several weeks (e.g.,
Goto, 2000; Bubliy et al., 2002) and “rapid cold hardening,” as a rapid response to a sub-lethal temperature fora very short time from a few minutes to hours (e.g.,
Czajka and Lee, 1990). Since the seasonal temperaturefluctuation is relatively mild in the temperate regions, thecold acclimation is likely to be an effective strategy for
insects like Drosophila to adapt to temperature changes
in their habitats.
Changes in lipid composition of cell membrane havebeen identified as a molecular mechanism underlying therearing acclimation and the rapid cold hardening in
insects (Ohtsu et al., 1998; Lee et al., 2006; Overgaard etal., 2008), whereas changes in metabolic rate are thought
to be a mechanism for the cold acclimation. For instance,
Berrigan (1997) showed that D. melanogaster flies rearedat 18°C had a higher respiration rate (metabolic rate) at
15°C compared to the flies reared at 25°C or 29°C.
Terblanche et al. (2005) showed that metabolic rate of atsetse fly species, Glossina morsitans morsitans, reared at
24°C was increased after experiencing 19°C for 10 days.
Using its closely related species, G. pallidipes, Terblanche
and Chown (2006) measured the critical thermal minima(CTmin) defined as the temperature, at which loss of coordinated muscle function is observed in a gradually
decreasing temperature condition (–0.25°C/min), andreported that the flies acclimatized at 21°C had a lower
CTmin with a higher metabolic rate compared to the control flies reared at 25°C. These studies suggested that
there is a connection between cold acclimation and metabolic rate.
In general, metabolic rate decreases as the ambient
temperature decreases in ectothermic animals including
Drosophila (Fleming and Miquel, 1983; Berrigan andPartridget, 1997; Oikawa et al., 2006), and this tendency
is regarded as a response to temperature changes (e.g.,
Gillooly et al., 2001). However, in the studies with
Drosophila and the tsetse fly species mentioned above(Berrigan, 1997; Terblanche et al., 2005; Terblanche andChown, 2006), significant effects of the rearing temperature on the response to temperature changes were alsosuggested. The connection between cold tolerance andmetabolic rate was also suggested in studies of D.
melanogaster mutants. A mutant of dystroglycan gene,atsugari, which causes an increase in mitochondrial oxidative metabolic rate, showed a higher cold tolerance(Takeuchi et al., 2009). Similarly, a dopamine transporter-defective mutant, fumin, showed a higher metabolic rate and a higher cold tolerance (Ueno et al., 2012).
These observations suggest that the cold tolerance of flies
is improved by the increased metabolic rate.
Another approach to examine if the elevation of metabolic rate is a major response to the cold acclimation
would be to analyze intraspecific variations of cold tolerance and of the metabolic rate and see if they have acorrelation. It is also intriguing to see if they parallel
each other in response to the cold acclimation. For thispurpose, D. albomicans belonging to the D. nasuta subgroup is particularly suitable, because this species had awide geographic distribution in Southeast Asia until late1980’s (Kitagawa et al., 1982) and has expanded the distribution to the Japanese archipelago in the temperateregions recently (Mikasa, 1991; Chen et al., 1994; Fujino
et al., 2006; Hoshina et al., 2007), whereas other closely
related species of the D. nasuta subgroup have stayed in
the tropics or subtropics. This suggests the possibility
that D. albomicans has a wider variation in cold tolerance
to be able to distribute from the tropics to the temperateregions. Therefore, it is an intriguing question to ask
whether the distribution expansion of D. albomicans has
been accompanied by an increase in cold tolerance withan increase in metabolic rate upon cold acclimation.
In this study, we first investigate the basic thermal
physiology, i.e. cold tolerance and acclimation response, of
D. albomicans in comparison to that of its closely related
species in the D. nasuta subgroup and also to that of D.
melanogaster, a well-studied canonical model species.
Then, to explore the possibility that the metabolic ratechange caused by the cold acclimation is responsible forthe higher cold tolerance in Drosophila, we analyze theeffects of cold acclimation on the cold tolerance and on themetabolic rate using geographic populations of D.
albomicans. We examined whether there is a strong
association between the cold tolerance and the metabolic
rate changes in response to the cold acclimation. This
study could be a step forward in understanding molecularmechanisms of evolutionary adaptation to thermal environment by organisms.
MATERIALS AND METHODS
Fly strains Twenty-nine strains of D. albomicans wereoriginally collected from 23 localities in 1979–1992 by
Kitagawa et al. (1982), each of which was establishedfrom a single inseminated female as an isofemale line andhas been maintained by sib-mating in a small mass culture with the standard cornmeal medium (containing 90 g
cornmeal, 100 g glucose, 40 g dead beer-yeast and 8 g agarin 1 litter water) at 20°C under constant light condition
before use. The abbreviation of the strain names andtheir collection sites and years are described in Supplementary Table S1. Its closely related species belonging
to the D. nasuta subgroup, i.e., 13 strains of D. nasuta, 4strains each of D. kepulauana, D. kohkoa, D. sulfurigasterbilimbata and D. pallidifrons, and 3 strains each of D. s.
albostrigata and D. pulaua, were also used (Supplementary Table S2). In addition, a wild type strain (CantonS) of D. melanogaster was maintained at 25°C in otherwise the same condition as the D. albomicans strains.

Cold tolerance of Drosophila
Categorization of geographic populations To investigate the cold tolerance of D. albomicans with regard to
the temperature condition of their habitat, we categorized
23 collection sites of the D. albomicans strains into 3 temperature zones according to the lowest monthly mean
temperature and the lowest monthly mean of daily minimum temperature obtained from World MeteorologicalOrganization, Central Weather Bureau, or Japan Meteorological Agency dependent on the location of each collection site. The detailed information on the meteorological
data is described in Supplementary Tables S1 and S2.
The lowest monthly mean temperature and the lowest
monthly mean of daily minimum temperature from all
the collection sites were plotted on a scatter diagram (Fig.
1). The two measurements as indicators of winter severity showed a high correlation with each other (Spearman’sr = 0.98, P < 10–15). However, since the latter on the xaxis showed a discontinuous distribution, the collection
sites were categorized into 3 temperature zones separated
by the intervals indicated by the dotted lines. As theresult, the criteria for the 3 temperature zones became asfollows. Temperature Zone 1: the lowest monthly meanof daily minimum temperature –5–5°C, Temperature
Zone 2: the lowest monthly mean of daily minimum temperature 5–20°C, and Temperature Zone 3: the lowest
monthly mean of daily minimum temperature 20–25°C.
Temperature Zone 1 consists of the newly colonized populations in Japan and China since 1980s (Supplementary
Table S1). It should be noted that the populations in
Temperature Zone 3 are unlikely to regularly experiencetemperature below 20°C even in the coldest month of ayear.
Cold acclimation and deacclimation To control for

Lowest monthly mean
temperature (ºC)

30
25
20
15
10
5
0
-5
-5

0

5

10

15

20

25

Lowest monthly mean of daily minimum
temperature (ºC)
Fig. 1. Correlation between the lowest monthly mean of daily
minimum temperature and the lowest monthly mean temperature in 23 collection sites of the D. albomicans strains used inthis study (Spearman’s r = 0.98, P < 10–15). Dotted lines indicate where three temperature zones were divided.

291

epigenetic effects, fly strains were cultured at 25°C on thestandard corn-meal medium for at least 3 consecutive
generations before all the experiments. Female flies
were separated from males within 48 hours after eclosion
without using anesthesia. For the cold acclimation, 20–
60 flies were put in a vial with fresh medium and kept at
15°C or 20°C for 7 days (designated as “25/15” or “25/20”,
respectively). The non-acclimatized control flies were
kept in 25°C for the same period (designated as “25/25”).
Flies of WL1 and Canton S strains were acclimatized atboth 15°C and 20°C, and flies of other 28 D. albomicans
strains were acclimatized at 20°C.
Experiments for deacclimation were performed under 3different temperature schemes. (1) Flies reared at 20°C
for at least 3 consecutive generations were kept at 25°C
for 7 days (designated as “20/25”), whereas the control
flies were kept at 20°C for the same period (designated as“20/20”). For this condition, we used 8 D. albomicans
strains: X108, WL1, IOM2, NG3, YAK1, TAK2, KKU202
and KKU204. (2) Flies reared at 25°C were exposed to
20°C for 3 days and then placed back into 25°C for 4 days
(designated as “25/20/25”). The control flies reared at
25°C were exposed to 20°C for 3 days and subsequently
another 4 days. WL1 and X108 strains were used forthis condition. (3) Flies reared at 20°C were exposed to
25°C for 3 days and then placed back into 20°C for 4 days
(designated as “20/25/20”). The control flies reared at
20°C were exposed to 25°C for 3 days and subsequently
another 4 days. WL1 and X108 strains were used forthis condition.
Measurement of cold tolerance To examine the cold
tolerance of flies, we measured the survival rate of flies
after exposed to 1°C for various time lengths and drew thesurvival curve against the exposure time. We used 29
strains of D. albomicans and Canton S strain of D.
melanogaster for this purpose. For each strain, 20 individuals were transferred into a glass vial (15 mm diameter and 105 mm long) containing 2 ml of a sugar agar
medium [10% sucrose; 1% agar; 1% butyl parahydroxybenzoate] and kept in an incubator, in which air temperature was regulated to be 1 ± 0.5°C for the period of 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 36 or 48 hours. To measure
the actual air temperature in the incubator during theexperiment, we used temperature data loggers(Thermochron G type) and collected temperature data.
After each period of 1°C exposure, the air temperature
was programed to become 20°C in approximately 25 minutes. Then, we counted the number of survivors after 48
hours, following Rako and Hoffmann (2006). If a fly
stood on its legs and walk, we identified the fly as asurvivor. For each time length and sex, the survival ratemeasurement was independently replicated 5 times. The
number of time points varied among experiments as thesurvival rate varied among strains. The time point at

K. ISOBE et al.

RESULTS
Cold tolerance and cold acclimation response of
Drosophila The survival rate curves obtained from maleand female flies with the 7 day cold acclimation at 15°C or
20°C are shown in Fig. 2, A and B for D. albomicans (WL1)and Fig. 2, C and D for D. melanogaster (Canton
S). They reveal substantial variations in the cold tolerance and the response to the cold acclimation between

Survival rate (%)

100%

50%

0%
5

15

25

35

45

35

45

35

45

35

45

(B) D. albomicans female
100%

Survival rate (%)

Measurement of metabolic rate Following Hulbert
et al. (2004), we measured metabolic rate in terms of theconsumption of O2 of flies at 7 days after the eclosion for
10 strains: X108, SWB101, KM3, HNZ12, WL1, NG3,
KMT21, KKU202, TAK502 and SMT503. We assembled
a respiration chamber connecting a plastic syringe of 1 ml
size containing a small amount of CO2 absorber (Sodalime, Wako) to a 50 μl micropipette via a plastic adaptor
and a short rubber tube. For this experiment, only maleflies were used, because they have smaller body size variation irrespective of their reproductive state. We placed
20 flies in the syringe body and inserted the plunger toclose the respiration chamber. We then placed the respiration chamber on a flat plate in a water bath filledwith ice-cold water for at least 1 hour to equilibrate theflies’ respiration rate and put 4 μl of ink into the micropipette at the tip. Finally, we measured the movement of
the ink corresponding to the oxygen consumption at every
1 hour over the next 3 hours. The measurements for theacclimatized flies and for the control flies were conductedsimultaneously in the same water bath with an empty
chamber to control unknown effects from ambient temperature, pressure, etc. Each set of the measurement
were replicated 30 times independently to obtain the
average and the variance.
The metabolic rate at 20°C was also measured for theacclimatized flies and the control flies of WL1 and X108
strains. In this case, the oxygen consumption was measured at every 30 minutes over 3 to 4 hours after theequilibration for 1 hour. The measurement was replicated 30 times. An empty chamber was also preparedfor each replicate as the control.

(A) D. albomicans male

50%

0%
5

15

25

(C) D. melanogaster male
100%

Survival rate (%)

which 50% of flies died (50% lethal time: LT50) or 25%
flies died (25% lethal time: LT25) was estimated by thelogistic regression analysis implemented in R version
2.13.0 software (R Core Team, 2008).
The cold tolerance was compared between D. albomicans
and its closely related species within the D. nasuta subgroup in terms of the survival rate after 16 hour exposure
to 1°C. The survival rate was examined for each sex
reared at 25°C with or without the cold acclimation for 36
strains from 7 species in total (Supplementary Table
S2). We used 20 individuals for each experiment which
was replicated 5 times.

50%

0%
5

15

25

(D) D. melanogaster female
100%

Survival rate (%)

292

50%

0%
5

15

25

Time exposed to 1 ºC (hours)
Fig. 2. Survival rate at 1°C for male (A) and female (B) flies of
the D. albomicans WL1 strain, and for male (C) and female (D)flies of the D. melanogaster Canton S strain after the cold acclimations at 15°C (dotted line) or 20°C (broken line). Solid line
represents the curve for the non-acclimatized control flies. The
error bars indicate the standard errors.

Cold tolerance of Drosophilamales and females within the same species and between
the species. The overall cold tolerance of D. melanogaster was substantially higher than that of D. albomicans regardless of the fact that the flies were acclimatizedor not. Another distinct between-species difference wasintheacclimation
responsetodifferent
temperatures. For D. albomicans, the acclimation at
15°C made almost the same (males) or only slightly better(females) effect compared to the acclimation at 20°C (Fig.
2, A and B), whereas the flies acclimatized at 15°C had ahigher survival rate than those acclimatized at 20°C inboth sexes in D. melanogaster (Fig. 2, C and D). This
result suggests that the effective temperature for the cold
acclimation is different between D. albomicans and D.
melanogaster: 20°C is sufficient to induce a full acclimation response in D. albomicans whereas lower temperature is more effective in D. melanogaster.
The effective time length of the cold acclimation for D.
albomicans was measured by the survival rate at 1°C for
14 hours (X108 and YAK) or 16 hours (WL1 and KKU204)
after various time lengths of cold acclimation at 20°C.
Although the response to the different time lengths of cold(A) Non-acclimatized male293

acclimation varied among the strains, its maximum wasreached within 4–5 days in all the strains (Supplementary Fig. S1). Based on this result, we determined theacclimation condition to be at 20°C for 7 days throughout
the experiments in order to induce the full acclimation
response. ANOVA showed that the survival time at 1°C
for acclimatized flies was significantly longer than thatfor non-acclimatized flies and that the survival time ofmales was significantly longer than that of females
regardless of whether the flies were acclimatized or not(Supplementary Table S3).
Geographic variation of cold tolerance in D.
albomicans and its closely related species To examine whether adaptation to the cooler climate in the temperate regions was involved in the distribution expansionof D. albomicans, we investigated the variation of cold tolerance among the geographic populations. We compared
the 50% lethal time (LT50) at 1°C among D. albomicans
strains from different temperature zones (Fig. 3, A and
B). The expected adaptation scenario that the cold tolerance is higher in the regions with severe winter was(B) Non-acclimatized female

20

20

LT50 at 1 ºC (hours)

LT50 at 1 ºC (hours)

*
15

10

5

0

15

10

5

0
1

2

3

1

Temperature Zones(C) Acclimatized male20

LT25 at 1 ºC (hours)

LT25 at 1 ºC (hours)

3(D) Acclimatized female

*

20

2

Temperature Zones

15

10

5

15

10

5

0

0
1

2

3

Temperature Zones

1

2

3

Temperature Zones

Fig. 3. The 50% lethal time at 1°C (LT50) for non-acclimatized male (A) and female (B) flies and the 25% lethal timeat 1°C (LT25) for acclimatized male (C) and female (D) flies of D. albomicans. The asterisks indicate a statistical significance of the difference between temperature zones 1 and 3 (Tukey’s HSD P < 0.05). The error bars indicate thestandard errors.

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K. ISOBE et al.

observed but the magnitude was rather small. A marginally significant difference among the zones was
detected in non-acclimatized male flies by one-way
ANOVA (F2,26 = 4.03, P = 0.03), whereas the difference
was not significant in female flies (F2,26 = 1.50, P = 0.24)(Fig. 3, A and B). The significant difference in the maleflies was attributed to the difference between Temperature Zones 1 and 3 by Tukey’s HSD test (P < 0.05) (Fig.
3A).
The responses to the cold acclimation by D. albomicans
strains from different temperature zones were also

compared. We used the 25% lethal time (LT25) rather
than LT50 to compare the cold tolerance after cold acclimation at 20°C, because 4 out of 28 strains showed survival rates higher than 50% at the 24-hour point when theexperiment was terminated (Supplementary Table S4).
Strains from colder locations tend to have a higher tolerance to cold temperature upon the cold acclimation,although the tendency was not strong. Again, the difference was detected only in male flies by one-way ANOVA(Fig. 3C, F2,26 = 3.31, P = 0.05) and the significant difference was detected between Temperature Zones 1 and 3(A) Non-acclimatized

Survival rate (%) at 1 ºC

100%
N = 12

N = 13

N=4

N=4

Zone11
Zone

Zone 2Zone
2

Zone 3
Zone 3

N = 13

N=4

N=3

N=3

N=3

N=4

40%

20%

0%
D. kepulauanaD. nasuta D. kohkoa
D.s. albostrigata
D.s. bilimbataD. pulaua
D. albomicans

N = 12

N = 13N=4

N=4N = 13

N=4

N=3

N=4

N=3

N=4

N = 13

N=4

N=3

N=4

N=3

N=4

(B) Acclimatized

Survival rate (%) at 1 ºC

100%
N = 12

N = 13

N=4

Zone 11
Zone

Zone 22
Zone

Zone 3
Zone 3

N=4

50%

0%
D. kepulauanaD. nasuta D. kohkoa
D.s. albostrigata
D.s. bilimbataD. pulaua
D. albomicans

N = 12

N = 13N=4

N=4N = 13

N=4

N=3

N=4

N=3

N=4

Fig. 4. The survival rates (± 1SE) of non-acclimatized (A) and acclimatized (B) male (filled bar) and female (open bar)flies at 1°C for 16 hours for D. albomicans populations from 3 temperature zones and its closely related species in theD. nasuta subgroup. N denotes the number of strains involved in the populations or species.

Cold tolerance of Drosophila
Table 1.

295

The 50% lethal time (LT50) and metabolic rate measurements for 10 D. albomicans
strains without or with cold acclimation
Metabolic rate (μl/mg.hour) ± S.E.

LT50 (hours)
Strainmale

female

male

25/25

25/20

25/25

25/20

25/25

25/20

X108

10.66

12.29

9.08

10.56

0.171 (± 0.03)

0.145 (± 0.02)

SWB101

9.81

17.57

6.22

13.73

0.170 (± 0.02)

0.185 (± 0.03)

KM3

11.67

20.21

8.36

20.49

0.124 (± 0.01)

0.156 (± 0.01)

HNZ12

13.52

20.18

10.73

16.72

0.146 (± 0.02)

0.141 (± 0.03)

WL1

7.86

23.34

5.33

17.98

0.162 (± 0.02)

0.221 (± 0.02)

NG3

15.63

24.55

13.38

22.20

0.129 (± 0.02)

0.148 (± 0.02)

KMT21

9.68

18.70

10.41

18.48

0.159 (± 0.01)

0.191 (± 0.02)

KKU202

11.41

20.74

10.60

20.66

0.135 (± 0.01)

0.183 (± 0.01)

TAK502

8.52

19.17

6.58

15.01

0.166 (± 0.01)

0.201 (± 0.01)

SMT503

15.52

19.27

10.16

19.83

0.180 (± 0.02)

0.159 (± 0.01)(A)

Effect of cold acclimation on metabolic rate in D.
albomicans To investigate the effect of the cold acclimation on metabolism of flies, the metabolic rate wasmeasured in terms of oxygen consumption in ice-coldwater (ca. 0°C) for 10 D. albomicans strains (Table 1). A
marginally significant increase in the metabolic rate upon
the cold acclimation was observed in 8 of the 10 strains(P < 0.05 in the sign test), suggesting that the metabolic
rate was affected by the cold acclimation to some extent.
When the metabolic rate was measured at 20°C for WL1
and X108 strains as the representatives showing respectively the maximum and minimum acclimation responses
in the cold tolerance at 0°C, they also showed a large andsmall acclimation response, respectively (Supplementary
Fig. S2). In this case, however, since the flies were notimmobilized in such warm temperature, the resting state
metabolic rate was unable to be obtained separately fromthe active state metabolic rate. Nevertheless, the metabolic rate of the acclimatized flies was higher than that of
the control flies with a statistical significance in WL1(B)Metabolic rate (μl /mg.hour)(P < 0.05 by Tukey’s HSD test). The difference was not
significant in female flies (Fig. 3D, F2,26 = 2.67, P = 0.09).
The cold tolerance was also compared between D.
albomicans and 7 its closely related species mostly distributed in the tropics and subtropics (Supplementary
Table S2) in terms of the survival rate after incubation at
1°C for 16 hours. The D. albomicans strains from Zones
1 and 2 showed the highest cold tolerance both in theacclimatized (“25/20”) and the non-acclimatized (“25/25”)
flies of both sexes (Fig. 4). The cold tolerance of the D.
albomicans strains from Zone 3 was comparable to thatof the closely related species in both acclimatized andnon-acclimatized flies.

0.25
0.20
0.15
0.10
0.05
0.00
0

5

10

15

20

25

Δ Metabolic rate (μl /mg.hour)

LT50 (hours)

0.06
0.04
0.02
0.00
-0.02
-0.04
0

2

4

6

8

10

12

14

16

18

Δ LT50 (hours)
Fig. 5. Relationship between metabolic rate and the 50% lethal
time (LT50) at 1°C of the non-acclimatized flies (open circle) andthe acclimatized flies (filled circle) for 10 D. albomicans strains(A) and relationship between the metabolic rate change andLT50 change upon the cold acclimation, where black, grey andwhite marks indicate the strains from Temperature Zones 1, 2
and 3, respectively (B).

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K. ISOBE et al.

(Supplementary Fig. S2, t = 2.38, df = 58, P < 0.05). By
contrast, the difference was negligible in X108 (Supplementary Fig. S2, t = 1.57, df = 58, P > 0.10).
Association between cold tolerance and metabolic
rate If the metabolic rate is closely associated with thecold tolerance, it is expected that LT50 becomes greater as
the metabolic rate increases. However, as shown in Fig.
5A, there was no clear correlation between LT50 and themetabolic rate either for the acclimatized flies (Spearman’sr = 0.24, P > 0.1) or for the non-acclimatized control flies
Table 2.

The 50% lethal time (LT50) for 8 D. albomicans
strains without or with deacclimation
LT50 - Half Lethal Time (hours)
malea

Strainfemaleb

20/20

20/25

20/20

20/25

X108

17.49

13.55

15.24

9.49

WL1

> 24.00c

10.47

22.13

9.45

IOM2

> 24.00

c

15.52

20.69

12.64

NG3

> 24.00c

20.13> 24.00c

15.14

YAK1

23.60

22.73

22.04

15.36

TAK2

20.14

16.50

15.56

10.87

KKU202> 24.00c

19.64> 24.00c

17.65

KKU204

19.73

18.62

17.70

13.13

a

Pairwise t test shows a significant difference between 20/20
and 20/25 (t = 7.65, df = 7, P < 0.01).
b
Pairwise t test shows a significant difference between 20/20
and 20/25 (t = 3.32, df = 7, P < 0.05).
c
Survival rate larger than 50% at the 24 hour time point.

Table 3.

The 50% lethal time (LT50) for two D. albomicans
strains with deacclimation or reacclimation
Deacclimation

Strain

WL1

X108

a

Reacclimation

25/20/20

25/20/25

20/25/25b

20/25/20

male

23.34

11.28

10.47

> 24.00c

female

17.98

5.93

9.45

20.08

male

12.29

8.58

11.21

12.22

female

10.56

6.54

8.91

9.70

a

Data from 25/20 in Table 1.
Data from 20/25 in Table 2.
cSurvival rate larger than 50% at the 24 hour time point.
b

Table 4.

bMetabolic rate (mean ± S.E.) of two D. albomicans strains experiencing various temperature conditions
25/25/25a

25/20/20b

25/20/25

20/20/20

20/25/25

20/25/20

WL1

0.162 (± 0.02)

0.221 (± 0.02)

0.158 (± 0.03)

0.194 (± 0.03)

0.131 (± 0.01)

0.185 (± 0.01)

X108

0.171 (± 0.03)

0.145 (± 0.02)

0.165 (± 0.03)

0.143 (± 0.01)

0.119 (± 0.01)

0.123 (± 0.01)

Straina(Spearman’s r = 0.28, P > 0.1). However, Fig. 5B showeda significant positive correlation between the change inthe cold tolerance and the change in the metabolic rate
upon the cold acclimation (Spearman’s r = 0.93, P <0.001). These results suggest that the metabolic rate
may not be a major factor of determining the cold tolerance per se, although they have a shared physiologicalbackground affected by the cold acclimation.
To examine the plasticity of the acclimatized physiological state, we performed the deacclimation experiments.
To examine the effect of deacclimation on the cold tolerance, we first used the flies reared at 20°C and thenexposed to 25°C for 7 days. The pairwise t-test showedthat the LT50 values from the deacclimatized flies weresignificantly lower than those from the control acclimatized flies in both males (t = 7.65, df = 7, P < 0.01) andfemales (t = 3.32, df = 7, P < 0.05) (see Table 2). Thissuggests that the increase in the temperature from 20 to25°C causes a relaxation of the effect of the cold
acclimation. The largest reduction in LT50 by the deacclimation was observed in WL1, one of the strains thatshowed the highest response to the cold acclimation(Table 1; Supplementary Table S4). The effect of thedeacclimation is consistent when flies were reared at
25°C and then acclimatized at 20°C (Table 3). Table 4shows that the reduction in the metabolic rate by thedeacclimation was highly significant in WL1 (0.194 vs.
0.131, t = 3.65, df = 58, P < 0.001) and in X108 (0.143 vs.
0.119, t = 2.85, df = 58, P < 0.01). When flies were rearedat 25°C and acclimatized at 20°C, the reduction by thesubsequent deacclimation was also highly significant in
WL1 (0.221 vs. 0.158, t = 4.74, df = 58, P < 0.001) but not
in X108 (0.145 vs. 0.165, t = 1.25, df = 58, P > 0.20).
We further used a reacclimation scheme, 20/25/20(reared at 20°C, deacclimatized at 25°C and reacclimatized at 20°C). The reacclimatized flies showed longer
LT50 values compared to the control flies in both WL1and X108 (Table 3). The difference in LT50 is larger in
WL1 than in X108. The metabolic rate of the reacclimatized flies was also increased significantly in WL1 (Table
4; 0.131 vs. 0.185, t = 3.14, df = 58, P < 0.01) but not in
X108 (Table 4; 0.119 vs. 0.123, t = 0.44, df = 58, P > 0.60).
In all the schemes for temperature changes, the resulted
changes in the metabolic rate paralleled the changes in
the cold tolerance.

Data from 25/25 in Table 1.
Data from 25/20 in Table 1.

Cold tolerance of Drosophila
DISCUSSION
Cold adaptation of D. albomicans Drosophila albomicans is a widely distributed species originated from thetropics in Southeast Asia (Kitagawa et al., 1982). In
Drosophila species, the ability to overwinter is a criticalfactor to limit their geographical distribution (Kimura,
1988, 2004). Therefore, an expected trend is that strains
collected from colder areas have a higher cold tolerance.
This trend was observed in D. albomicans, although thedifference in cold tolerance among the strains from thethree temperature zones was not very large (Fig. 3) asreported for other Drosophila species as well (Kimura,2004). This may be partly due to frequent migrations
throughout their distribution range. In this regard, astrain from Taihsan, China, was noteworthy since thelowest monthly mean of daily minimum temperature of
the collection site is –4.70°C (Supplementary Table S4).
Since this species unlikely overwinters in such temperature condition, wind likely carried those flies from a distant location. Therefore, this strain was possibly
established from a migrant from a warmer location, suggesting that adaptation to local thermal environment
could be obscured by seasonal migration in comparative
studies among geographic populations.
Alternatively, our results suggest that the acclimation
response was not a critical factor for this species during
the recent colonization in Japan and China (Temperature
Zone 1). Nevertheless, the comparison among closely
related species revealed that the cold tolerance of the D.
albomicans strains from the subtropics to temperateregions (Temperature Zones 1 and 2) was higher thanthat of its closely related species belonging to the D.
nasuta subgroup distributed in the tropics and subtropics
irrespective of their experience of the cold acclimation (Fig.
4). This implies that the distribution expansion of D.
albomicans is likely attributed to the intraspecific variation including such strains that have a high cold tolerance
and a strong acclimation response. It is noteworthy that
D. pallidifrons, which is the most ancestral species in theD. nasuta subgroup (Wakahama et al., 1983; Suzuki etal., 1990), showed a relatively high cold tolerance after
the cold acclimation (Fig. 4). This implies that D.
albomicans may have retained the ability to respond tothe cold acclimation from the ancestral species, whereas
many of the closely related species have lost it. Such
preadapted trait was likely a critical factor for this species to have the potential of expanding its distribution tocolder regions. In either case, our results suggest that theability to adapt to temperature fluctuations varies among
species and among strains and that D. albomicans has
the best ability in the D. nasuta subgroup.
It might be worth discussing the effects of in-lab artificial selection during the long-term maintenance of thestrains used in this study. Every wild strain used was

297

originated from a single inseminated female fly so that at
each locus there might be genetic variations among four(or more by multiple inseminations) alleles carried by theancestral female in her eggs and the sperms that fertilized the eggs. Since all the strains have been propagatedby sib-mating, there has been no other source of genetic
variation except mutations newly occurred during the last
thirty years, which is considered to be too short for the
mutations to generate de novo genetic variation. Becauseof this small extent of genetic variation due to the small
number of original alleles within a strain, the effects of inlab selection should be largely restricted as selection does
not operate without genetic variation. Therefore, it isunlikely that the observed differences in the cold tolerance and acclimation response were largely influenced by
the effects of in-lab artificial selection during the longterm maintenance of the strains.
In contrast to the small difference among geographic
populations, there was a drastic difference in cold tolerance between D. albomicans and a cosmopolitan species,
D. melanogaster. The latter showed a much higher cold
tolerance than the former in all the tested temperature
conditions (Fig. 2). Cosmopolitan species are generally
considered to have higher tolerances to various environmental stresses such as heat, cold, and desiccation(Levins, 1969; Parsons and McDonald, 1978; Stanley etal., 1980; Jenkins and Hoffmann, 1999). For example,
Jenkins and Hoffmann (1999) showed that three cosmopolitan species, D. melanogaster, D. simulans and D.
immigrans, were more tolerant of cold than D. serrata
that is distributed in the subtropics and tropics of eastern
Australia. This is consistent with our observation of differences between D. albomicans and D. melanogaster.
Furthermore, the level of acclimation response to different temperature conditions differed between these species.
When flies were acclimatized at 15°C or 20°C, the effectwas very similar for D. albomicans (Fig. 2, A and B),
whereas the acclimation at 15°C was more effective thanthat at 20°C for D. melanogaster (Fig. 2, C and D).
In the previous studies on the effects of acclimation on
cold tolerance using D. melanogaster, the acclimation
temperature was set between 11–15°C. For example,
Goto (2000) showed that the half lethal temperature (temperature that kills half of the individuals for 24 hours)
was 1°C for the adult flies with an acclimation at 15°C for
1 day, whereas it was 3°C for those without acclimation.
Bubliy et al. (2002) showed that when flies reared at 25°C
were acclimatized at 11°C for 5 days before an exposure
to 0°C for 24 hours, the survival rate of the acclimatizedflies was about 5–7 times higher than that of the control
flies. Rako and Hoffmann (2006) showed that cold acclimatized flies at 12°C after reared at 19°C had lower mortality after a cold shock at −5°C or −2°C. Compared tothe acclimation responses of D. melanogaster described inthese studies, our results showed that a full acclimation

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K. ISOBE et al.

response was induced in D. albomicans at much higher
temperature. This suggests that the response to aslightly cooler temperature (20°C) is important for D.
albomicans in its habitat in the temperate regions.
There was another notable difference observed between
D. melanogaster and D. albomicans. In D. albomicans,male flies showed a higher cold tolerance than female
flies with a few exceptions (Fig. 2, A and B; Supplementary Table S4). This tendency was observed irrespectiveof their experience of cold acclimation. Such betweensex difference was not very clear in D. melanogaster (Fig.
2, C and D) or in other species belonging to the D. nasuta
subgroup. However, the previous studies on D.
melanogster and other species (D. takahashii, D. lutescens,
D. triauraria, D. trapezifrons, D. watanabei, D. simulans,
D. auraria and D. immigrans) showed that female flies
tend to have a higher cold tolerance than male flies(Kimura, 1982; Goto and Kimura, 1998; Goto et al., 2000;
Bubliy et al., 2002; Kojima and Kimura, 2003), although
Kimura (1982) reported that the sex showing higher cold
tolerance was dependent on the acclimation temperature
in D. lutescens. Therefore, the observed between-sex difference in the cold tolerance of D. albomicans was rather
exceptional among Drosophila species and may be reflecting some physiological traits specific to this species.
Metabolic rate and cold acclimation In this study,
there was no correlation found between cold tolerance andmetabolic rate among different strains (Fig. 5A). This isconsistent with Oikawa et al. (2006) showing no significant correlation between mass-adjusted oxygen consumption and thermal tolerance among 28 species belonging to
Drosophila and its related genera. This may be because
cold tolerance is a complex trait influenced by various factors such as presence of cryoprotective agents, antifreeze
proteins, ice-nucleating proteins and heat-shock proteinsand modified condition of cell membrane (Chown andNicolson, 2004, for review; Lee and Denlinger, 2010;
Michaud and Denlinger, 2010; Koštál, 2010; Chown and
Sinclair, 2010; Huey, 2010; Overgaard et al., 2010; Storey
and Storey, 2012). Differences in genetic background forthese factors likely cause diversities in cold tolerance
among strains and dispel the correlation with metabolic
rate, which is also a complex physiological trait influenced by various factors.
By contrast, we found a significant positive correlation
between the increase in cold tolerance and the increase inmetabolic rate upon the cold acclimation among different
strains of D. albomicans (Fig. 5B). In this case, the effectof cold acclimation was successfully extracted, because
the influence of the differences in genetic background wascontrolled. This result supports the hypothesis that thechanges in metabolic rate and the changes in cold tolerance are involved in common physiological processes. Similar metabolic rate changes upon cold acclimation havebeen shown by previous studies using D. melanogasterflies. For instance, Berrigan (1997) showed that the flies
developed at 29°C had a lower mass-specific metabolic
rate than the flies with cold-acclimation at 18°C. Overgaard et al. (2008) reported that the flies reared at 25°C
had a lower cold tolerance compared to the control flies
acclimatized at 20°C. Although these studies were
designed to examine the effect of the rearing temperature
rather than the acclimation, we clearly showed in thisstudy that the effect of rearing temperature is mostly
overridden by the temperature experienced at the adultstage using the deacclimation and reacclimation
experiments. Therefore, their results can be interpreted
as the response to the lower temperature experienced at
the adult stage rather than the rearing temperature. In
addition, using a tsetse fly species, G. pallidipes,
Terblanche et al. (2005) showed that the flies developed
at 19°C had a higher metabolic rate than those at 24°C
and 29°C when measured at either 20°C, 24°C, 28°C or
32°C. Furthermore, Terblanche and Chown (2006)
directly showed that both cold tolerance and metabolic
rate became higher in response to cold acclimation at theadult stage as shown in this study for Drosophila species.
Molecular mechanism of cold acclimation
responses Recent studies have suggested that changes
in gene expression are not the major driver of a shortterm response to a more abrupt temperature change inthe case of rapid cold hardening (Sinclair et al., 2007;
Teets et al., 2012, 2013). However, for the cold acclimation response to a longer term milder temperature change
observed in this study, there are several lines of circumstantial evidence suggesting that the changes in gene
expression play a major role. First of all, there havebeen cases reporting that the alteration of transcription
level of some genes can induce similar physiologicalresponses to those observed in our study. For example,
both metabolic rate and cold tolerance were enhanced in
atsugari and fumin mutants due to the altered transcription level of the corresponding and/or downstream genes(Takeuchi et al., 2009; Ueno et al., 2012). Secondly,
Smp30 (Dca), known as a candidate gene for cold tolerance, was up-regulated during the cold acclimation at
15°C for a few days in D. melanogaster originally rearedat 25°C, a condition similar to that of our study (Goto,
2000). Finally in this study, the deacclimation and reacclimation experiments showed that the switches between
acclimatized and non-acclimatized physiological states inflies required at least several days (Tables 3 and 4), suggesting that a rapidly triggered calcium signaling during
rapid cold hardening for example was unlikely to be theunderlying molecular mechanism. Therefore, so far,those circumstantial evidences imply that a systematicchange in gene expression is responsible for the cold acclimation responses observed in this study. Genome-wideCold tolerance of Drosophilatranscriptome analyses and intensive transgenetic experiments should shed light on the molecular mechanisms ofcold tolerance and help our understanding of the evolutionary adaptation to thermal environment by organisms.
We would like to thank M Toda and MT Kimura for valuable
discussions regarding the ecology and the distribution of D.
albomicans and its related species. This study is supported by
JSPS grants-in-aid for scientific research 23370096 and a grantin-aid from Tokyo Metropolitan University to KT.

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