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Time-course for attainment and reversal of acclimation to constant
temperature in two Ceratitis species
Christopher W. Weldon a,n, John S. Terblanche b, Steven L. Chown a
a Centre for Invasion Biology, Department of Botany and Zoology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa
b Department of Conservation Ecology and Entomology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa
a r t i c l e i n f o
Article history:
Received 8 June 2011
Accepted 19 August 2011
Available online 26 August 2011
Keywords:
Chill-coma recovery time
Heat knock-down time
Thermal tolerance
Ceratitis capitata
Ceratitis rosa
Tephritidae
a b s t r a c t
Acclimation in the thermal tolerance range of insects occurs when they are exposed to novel temperatures
in the laboratory. In contrast to the large number of studies that have tested for the ability of insects to
acclimate, relatively few have sought to determine the time-course for attainment and reversal of thermal
acclimation. In this study the time required for the Mediterranean fruit fly, Ceratitis capitata Wiedemann,
and the Natal fruit fly, Ceratitis rosa Karsch, to acclimate to a range of constant temperatures was tested by
determining the chill-coma recovery time and heat knock-down time of flies that had been exposed to
novel benign temperatures for different durations. The time required for reversal of acclimation for both
Ceratitis species was also determined after flies had been returned to the control temperature. Acclimation
to 31 1C for only one day significantly improved the heat knock-down time of C. capitata, but also led to
slower recovery from chill-coma. Heat knock-down time indicated that acclimation was achieved after only
one day in C. rosa, but it took three days for C. rosa to exhibit a significant acclimation response to a novel
temperature of 33 1C when measured using chill-coma recovery time. Reversal of acclimation after return
to initial temperature conditions was achieved after only one day in both C. capitata and C. rosa. Adult
C. capitata held at 31.5 1C initially exhibited improved heat knock-down times but after 9 days the heat
knock-down time of these flies had declined to levels not significantly different from that of control flies
held at the baseline temperature of 24 1C. In both Ceratitis species, heat knock-down time declined with age
whereas chill-coma recovery time increased with age, indicating an increased susceptibility to high and low
temperatures, respectively.
& 2011 Elsevier Ltd. All rights reserved.
1. Introduction
It has been demonstrated widely that the thermal tolerance of
insects is affected by prior thermal experience. Developmental
temperature can have enduring morphological and physiological
consequences that influence fitness (Gibert et al., 2001; Kingsolver
and Huey, 1998). Furthermore, plastic changes in the thermal
tolerance range of insects occur when they are exposed to novel
temperatures (e.g. Overgaard and Sørensen, 2008; Rako and
Hoffmann, 2006; Marais and Chown, 2008). Acclimation is a rapid
and reversible change in phenotype (be it physiological, biochem-
ical, or anatomical) in response to chronic exposure to a new
environmental condition (Woods and Harrison, 2002), and these
plastic changes are often beneficial for the survival of insects in a
changing environment (e.g. Fay and Meats, 1987; Terblanche et al.,
2006). Across insect taxa, acclimation to temperature is most
pronounced at the lower temperature threshold, while there is little
flexibility in the upper thermal limit (e.g. Hoffmann and Watson,
1993; Chown, 2001; Chown and Terblanche, 2006).
In contrast to the large number of studies that have tested for the
ability of insects to acclimate to changes in temperature, relatively
few have sought to determine the time-course for attainment of
thermal acclimation. This is surprising given the wide range of
temperatures and durations that have been used to induce acclima-
tion, as well as hardening. In the Drosophila literature alone, Sinclair
and Roberts (2005) identified 27 different published methods to
induce changes in thermal tolerance. Hardening, a form of acclima-
tion, has been induced by subjecting animals to rapid changes in
temperature in the laboratory (Hoffmann et al., 2003). However, the
magnitude of temperature change used in hardening treatments is
considerably larger than that of acclimation treatments, and their
duration is typically for only 1–3 h followed by a test for change in
thermal tolerance (Hoffmann et al., 2003). The physiological changes
associated with acclimation and hardening at upper and lower
temperatures can be shared, which has led to suggestions that
acclimation and hardening represent the same phenomenon, but
expressed over different time scales due to the relative levels
of stress involved (Bowler, 2005; Loeschcke and Sørensen, 2005).
In those species where the time required for complete acclimation
Contents lists available at SciVerse ScienceDirect
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Journal of Thermal Biology
0306-4565/$ - see front matter & 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jtherbio.2011.08.005
n Corresponding author. Tel.: þ27 21 808 2835; fax: þ27 21 808 2995.
E-mail address: cwweldon@gmail.com (C.W. Weldon).
Journal of Thermal Biology 36 (2011) 479–485

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has been determined, acclimation is relatively rapid and asymptotic
(Table 1). In the Queensland fruit fly, Bactrocera tryoni (Froggatt)
acclimation to low temperatures is very rapid for the cold torpor
threshold (Meats, 1973). In B. tryoni, the time required to acclimate to
a new lower temperature is shorter if the change in temperature is
small; for a full acclimation response in cold torpor threshold, only
1 min is required for a drop in temperature of 1 1C (if the new lower
temperature is not below torpor temperature; Meats, 1973).
Conversely, acclimation to a new higher temperature is fastest at
higher temperatures, regardless of the magnitude of change (Meats,
1973). The findings of Meats (1973) suggest that the magnitude of
temperature change has important implications for the rate of change
in insect tolerance to high and low temperatures. However, data for
other species summarised in Table 1 provide inconsistent support for
the role of temperature change magnitude in contributing to the
rate of acclimation attainment and reversal. Thus, a broader set of
information concerning the time course of acclimation would be
useful for further understanding of thermal tolerance in this group.
Here we provide such information for two species of fruit flies.
The Mediterranean fruit fly, Ceratitis capitata Wiedemann,
is one of the world’s most destructive horticultural pests and
represents an enormous economic burden due to lost productiv-
ity, control costs and trade barriers. Molecular genetic evidence
suggests that the endemic range of C. capitata was central eastern
Africa (Baliraine et al., 2004), but in just over 100 years it
has spread through human activities and unassisted dispersal
throughout Africa, the Mediterranean Basin and Middle East,
Central and South America, and parts of North America and
Australia (for a review, see Malacrida et al., 2007). In contrast,
the Natal fruit fly, Ceratitis rosa Karsch, which also originates in
central eastern Africa and utilises a similar range of hosts, has
a naturalised range restricted to south-eastern Africa and the
Mauritius and Re´union islands (Baliraine et al., 2004). The
discrepancy between the invaded distribution of C. capitata and
C. rosa poses the question of why the former has so successfully
established in regions where it has been introduced? Work to
date suggests that differences in the bioclimatic potential of
C. capitata and C. rosa could provide one explanation for their
current distribution and changes therein that might follow as a
result of climate change. The distribution and abundance of
C. capitata and C. rosa on La Re´union, where they are both
introduced and invasive, suggests that each species has a well-
defined bioclimatic niche: C. capitata is more prevalent at lowland
sites where it is warmer and drier, whereas C. rosa is found at
higher altitudes that experience cooler and wetter conditions
(Duyck et al., 2006). Work on the critical thermal limits (the lower
or upper temperature at which muscular function is lost) of the
two species indicates that the thermal tolerance of C. capitata is
broader than that of C. rosa; the critical thermal minimum (CTmin)
of both species does not differ (5.4–6.6 1C) but the critical thermal
maximum (CTmax) of C. capitata (42.4–43.0 1C) is significantly
higher than that of C. rosa (41.8–42.4 1C) (Nyamukondiwa and
Terblanche, 2009).
In both C. capitata and C. rosa it has also been demonstrated that
thermal experience during the adult phase can lead to small but
significant reversible and irreversible changes in their tolerance of
extreme temperatures (Nyamukondiwa and Terblanche, 2010).
Acclimation for 7 days to 30 or 20 1C, after being initially held at
25 1C, led to improved heat tolerance and cold resistance, respec-
tively (Nyamukondiwa and Terblanche, 2009). However, the dura-
tion of acclimation prior to testing used by Nyamukondiwa and
Terblanche (2009) was based only on the few studies that have
reported the time required for thermal acclimation to be attained.
This current study determined the time required for C. capitata and
C. rosa to acclimate to a range of constant temperatures. Acclima-
tion of each species was tested by determining the chill-coma
recovery time and heat knock-down time of flies that had been
exposed to novel benign temperatures for different durations. The
time required for reversal of acclimation for both Ceratitis species
was also determined by measuring chill-coma recovery and
heat knock-down time after flies had been returned to the control
temperature.
Table 1
Time required for attainment and reversal of thermal acclimation in insects. All studies report time of response by adults to novel temperature treatments unless otherwise
indicated. Arrows indicate the direction of the temperature change.
Taxon Attainment Reversal Reference
Treatment Time Treatment Time
Blattaria
Blatta orientalis Linnaeus 15 1C230 1C r20 h 15-30 1C r20 h Mellanby (1939)
30-15 1C 2–3 days
Hemiptera
Cimex lectularius Linnaeus 15 1C230 1C r20 h 15230 1C r20 h Mellanby (1939)
Rhodnius prolixus Sta˚l 15 1C230 1C r20 h 15230 1C r20 h Mellanby (1939)
Diptera
Bactrocera oleae (Rossi) 25 1C-20 & 30 1C 3–5 days N/A N/A Fletcher and Zervas (1977)
25 1C-15 1C 5 days
25 1C-5 & 10 1C 411 days
Bactrocera tryoni (Froggatt)
Third instar larvae 25 1C-35 1C 8 h 35 1C-25 1C 6–8 h Beckett and Evans (1997)
Adults 25 1C-8 1C 3 h 8 1C-25 1C 3 min Meats (1973)
12 1C-8 1C 2 min 8 1C-12 1C 3 h
Drosophila melanogaster Meigen 25 1C-29 1C 1 day 29 1C-25 1C 2 days Levins (1969)
25 1C-18 1C 7 days N/A N/A Gibert et al. (2001)
Calliphora vicina Robineau-Desvoidy 15 1C230 1C r 20 h 15 1C230 1C r20 h Mellanby (1939)
Lucilia sericata (Meigen) 15 1C230 1C r 20 h 15 1C230 1C r20 h Mellanby (1939)
Glossina pallidipes Austen 24 1C-19 1C 5 days 19 1C-24 1C r9 days Terblanche et al. (2006)
24 1C-16 1C 5 days 16 1C-24 1C
Lepidoptera
Bicyclus anynana (Butler) 20 1C-27 1C 2 days 27 1C-20 1C 43 days Fischer et al. (2010)
Danaus plexippus Kluk 4–5 1C-23–24 1C 4–6 days 23–24 1C-4–5 1C 44 days Kammer (1971)
Lycaena tityrus Poda 27 1C-20 1C 3 days N/A N/A Zeilstra and Fischer (2005)
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2. Material and methods
2.1. Source and maintenance of insects
Pupae of C. capitata and C. rosa were obtained from large,
outbred cultures maintained at Citrus Research International,
Nelspruit, South Africa. The cultures were held indoors under
variable though buffered temperatures (annual temperature
range: 15–30 1C). Adult females oviposited through a nylon mesh
screen (1 mm mesh size). The eggs were collected in water, held
for 24 h, and then transferred to a bran-based larval rearing
medium. Third instar ‘hopping’ larvae burrowed to the surface
of the larval rearing medium and migrated to a layer of fine
vermiculite where they commenced the pupal phase. Pupae were
then shifted from the vermiculite before being sent by express
courier to Stellenbosch University, Matieland, South Africa. Upon
arrival at Stellenbosch University, the pupae of each species were
distributed into nine ventilated 5-l plastic cages, with approxi-
mately 250 pupae per cage. Cages were furnished with cotton
wool soaked in water, granulated sucrose, and yeast extract
powder (Atiss YE, Borregaard Ingredients, Sarpsborg, Norway).
Each cage was placed in an incubator set to 25 1C with a 12:12 LD
photo cycle until adult emergence.
At 2 days after adult emergence, a plastic dish filled with
saturated NaCl solution and covered by insect screen mesh was
inserted into each of the 5-l plastic cages containing flies to regulate
humidity. Each cage of flies was then placed inside a clear plastic
bag that was then wrapped around the cage to seal it so that the
humidity in the cage could be increased by the saturated salt
solution. In a sealed container, saturated NaCl solution maintains a
constant relative humidity of 76%, 75.5% and 75.5% at 20, 25 and
30 1C, respectively (Winston and Bates, 1960). Three cages each of
C. capitata and C. rosa were then introduced to incubators set to 20
or 30 1C while the remaining three cages of each species remained in
the incubator set to 25 1C. After acclimation for 10 days, each group
of flies was then returned to the incubator set to 25 1C to test for the
rate of loss of acclimation.
The actual temperature and relative humidity experienced by
flies in each incubator was recorded at hourly intervals using data
loggers attached to the inside wall of each cage (DS1923, iButtons,
Maxim Integrated Products, Sunnyvale CA, USA). Actual acclimation
temperatures (mean71 standard error) experienced by C. capitata in
incubators set to 20, 25 and 30 1C were 20.970.0 1C, 23.970.0 1C
and 31.670.4 1C, respectively. The relative humidity maintained
within cages using saturated NaCl solutions was 8370% at 20.9 1C,
8070% at 23.9 1C, and 7770% at 31.6 1C. Actual acclimation
temperatures experienced by C. rosa in incubators set to 20, 25 and
30 1Cwere 20.871.3 1C, 24.470.3 1C, and 33.071.0 1C, respectively.
At each respective actual acclimation temperature, the relative
humidity maintained by saturated NaCl solutions in the cages that
housed C. rosa was 8570%, 8172%, and 7770%. The results are
reported and interpreted using the actual temperatures (to the
nearest 0.5 1C) experienced by flies.
2.2. Chill-coma recovery time
Chill-coma recovery times (CCRT) for adult C. capitata and C. rosa
were assessed for each acclimation temperature after different
durations of exposure and recovery. Flies (n¼25–30) of each species
were tested after 1, 3, 5, 7 and 9 days of exposure to acclimation
temperatures, and after 1 and 3 days of being returned to the
incubator set at 25 1C. Flies were transferred to and weighed in
individual 7 ml screw-cap plastic vials of known weight with two
1 mm diameter holes pierced through the cap for ventilation. The
vials were then placed into a large zip-lock bag that was plunged into
a water bath (GP200-R4, Grant Instruments Inc., Cambridge, UK) held
at 0 1C for one hour. The plastic vials were then placed on their side
on a table in a roommaintained at 2571 1C and the time required for
each fly to regain the ability to stand was recorded. A small number
of C. capitata that did not recover from the cold shock within two
hours (n¼27 drawn from all acclimation temperatures) were
excluded from analysis. Flies were weighed immediately before the
assay and their sex was recorded after the assay was completed.
2.3. Heat knock-down time
In this assay, heat knock-down time (HKDT) was determined
for both C. capitata and C. rosa in relation to duration of exposure
to a novel thermal environment. Flies were weighed in ventilated
7 ml plastic vials of known weight before being exposed to a test
temperature of 43.970.3 1C on a thermal stage. A temperature of
43 1C is known to result in loss of motor control in C. capitata and
C. rosa regardless of acclimation temperature, age or heating rate
(Nyamukondiwa and Terblanche, 2009, 2010). The thermal
stage was a sealed Perspex box with aluminium top through
which water was circulated from a programmable water bath
(GP200-R4, Grant Instruments Inc., Cambridge, UK) set at 54 1C.
Walls and a removable cover of Perspex enclosed the aluminium
stage top to stabilise the temperature of the apparatus. The
conditions experienced by the flies led to increased locomotor
activity followed by uncoordinated flight, spasms, and then loss of
activity. The time (in minutes) at which activity was lost by flies
after placing vials on the thermal stage was recorded. Flies
(n¼25–30) of each species were tested after 1, 3, 5, 7 and 9 days
of exposure to acclimation temperatures, and after 1 and 3 days of
being returned to the incubator set at 25 1C. The sex of each fly
was recorded after the assay was completed.
2.4. Data analysis
All analyses were performed in R 2.10.1 (R Development Core
Team, 2010). CCRT and HKDT did not meet assumptions of
constant variance and normal errors that are implicit in linear
models. Due to this and because CCRT and HKDT are both
measured as the time until an event, the main effects and two-
way interactions of acclimation temperature, time and sex on
CCRT and HKDT for each Ceratitis species were determined using
Cox’s proportional hazards survival regression (function ‘‘coxph’’
in the ‘‘survival’’ library) and log-likelihood tests. Acclimation
time and temperature were treated as categorical variables. The
reference category for acclimation temperature was set to ‘‘25 1C’’
(function ‘‘relevel’’). Weight of flies was included in the model as
a covariate to account for the influence of size on CCRT and HKDT.
CCRT and HKDT were known for all individuals in the analysis so a
censoring indicator vector was created and included in the
analysis to indicate that no data were censored. The proportion-
ality of hazards assumption for a Cox regression fit (i.e. that the
effects of model variables were constant over time) was met in all
analyses (function ‘‘cox.zph’’ in the ‘‘survival’’ library). Post-hoc
pairwise survival analyses were performed to examine significant
main effects and interaction terms.
3. Results
3.1. Ceratitis capitata
Heat knock-down time of C. capitata was significantly affected by
temperature, time, and their interaction (Table 2). After acclimation to
31.5 1C for one day, HKDT of adult C. capitata was significantly longer
than acclimation to 21 or 24 1C (Fig. 1A). HKDT of 31.5 1C-acclimated
C. capitata steadily declined but continued to be higher than the
C.W. Weldon et al. / Journal of Thermal Biology 36 (2011) 479–485 481

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HKDT of flies acclimated to 21 or 24 1C until 7 days after acclima-
tion commenced. However, by 9 days after the commencement
of acclimation there was no significant difference in HKDT between
C. capitata acclimated to 21, 24 or 31.5 1C, and this continued to be
the case after all acclimation treatments were returned to 24 1C after
10 days of acclimation (Fig. 1A). The interaction of acclimation
temperature and sex also had a significant effect on HKDT of C.
capitata (Table 2). HKDT of C. capitata that were acclimated at 21 1C
was marginally shorter in females (median, lower - upper 95%
confidence interval¼4 min, 3–4 min) than males (4 min, 4–4 min),
but there was no difference between the sexes when acclimated to
24 1C (female: 4 min, 3–4 min; male: 4 min, 4–4 min) or 31.5˚C
(female: 4 min, 4–4 min; male: 5 min, 4–5 min).
CCRT of adult C. capitata was significantly affected by acclima-
tion temperature (Table 2). At all ages, including after only 1 day,
adult C. capitata acclimated to 31.5 1C had a CCRT that was
significantly longer than those acclimated to 21 or 24 1C, while
CCRT of 21 and 24 1C-acclimated C. capitata did not differ (Fig. 1B).
CCRT of C. capitata also varied significantly with time. After 9 days
of acclimation, CCRT was significantly longer than after only one
day of acclimation, but following return of flies to 24 1C after 10
days of acclimation, CCRT at 11 and 13 days did not differ
significantly from when it was assessed at 1 day of acclimation.
Sex of adult C. capitata significantly affected CCRT (Table 2), with
male CCRT (median¼10 min) being slightly higher than that of
females (median¼9 min). There was also a significant effect of
weight on adult C. capitata CCRT (Table 2); heavier flies were
more likely to have a shorter CCRT.
3.2. Ceratitis rosa
Acclimation temperature had a significant effect on HKDT of
C. rosa (Table 3). Overall, adult C. rosa acclimated to 24.5 1C had a
HKDT that was marginally but significantly longer than that of
C. rosa acclimated to 21 or 33 1C (Fig. 2A). HKDT of C. rosa was also
significantly affected by time (Table 3); HKDT of C. rosa after
acclimation for 3, 5, 7 and 9 days was significantly longer than after
only 1 day of acclimation, and after all acclimation treatments were
returned to 24.5 1C at 10 days (i.e. 11 and 13 days) (Fig. 2A).
CCRT of C. rosa was significantly affected by temperature, time,
and their interaction (Table 3). There was no significant difference
in CCRT of C. rosa between acclimation treatments after one day
(Fig. 2B). After three days of acclimation, CCRT of 33 1C-accli-
mated C. rosa was significantly longer than 21 and 24.5 1C-
acclimated flies, and this difference continued to be evident at
5, 7 and 9 days. There was no significant difference between CCRT
of C. rosa acclimated at 21 and 24.5 1C at any tested time.
However, the CCRT of 21 and 24.5 1C-acclimated C. rosa increased
with time: CCRT at 3 days was significantly longer than when
Table 2
Analysis of deviance table for Cox’s proportional hazards survival regression of
heat knock-down time (HKDT) and chill-coma recovery time (CCRT) of Ceratitis
capitata with respect to acclimation temperature, acclimation time (days), sex and
weight.
Thermal tolerance assay Effect w2 d.f. p
HKDT Temperature 27.159 2 o0.0001
Time 91.545 6 o0.0001
Sex 2.227 1 0.1356
Weight 0.254 1 0.6146
Temperature time 28.002 12 0.0055
Temperature sex 7.176 2 0.0277
Time sex 9.081 6 0.1691
CCRT Temperature 59.799 2 o0.0001
Time 30.684 6 o0.0001
Sex 16.014 1 o0.0001
Weight 17.573 1 o0.0001
Temperature time 17.568 12 0.1295
Temperature sex 2.0285 2 0.3627
Time sex 6.3024 6 0.3902
Fig. 1. Change in median heat knock-down time (A) and chill-coma recovery time
(B) of adult Ceratitis capitata transferred from 24 1C to 31.5, 24 or 21 1C. Error bars
indicate the upper and lower 95% confidence interval. The vertical dashed line
indicates the return of flies to 24.5 1C after 10 days.
Table 3
Analysis of deviance table for Cox’s proportional hazards survival regression of
heat knock-down time (HKDT) and chill-coma recovery time (CCRT) of Ceratitis
rosa with respect to acclimation temperature, acclimation time, sex and weight.
Thermal tolerance assay Effect w2 d.f. p
HKDT Temperature 6.388 2 0.0410
Time 18.573 6 0.0050
Sex 0.530 1 0.4667
Weight 0.242 1 0.6228
Temperature time 18.025 12 0.1149
Temperature sex 2.881 2 0.2368
Time sex 3.576 6 0.7339
CCRT Temperature 42.665 2 o0.0001
Time 174.869 6 o0.0001
Sex 3.003 1 0.0831
Weight 0.514 1 0.4735
Temperature time 27.078 12 0.0075
Temperature sex 3.173 2 0.2047
Time sex 1.826 6 0.9350
C.W. Weldon et al. / Journal of Thermal Biology 36 (2011) 479–485482

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initially tested, but was significantly shorter than at 5, 7, 9, 11 and
13 days. Following return to 24.5 1C after 10 days of acclimation,
within one day CCRT of 33 1C-acclimated C. rosa did not differ
significantly from that of 21 and 24.5 1C-acclimated C. rosa of the
same age (Fig. 2B).
4. Discussion
It appears that acclimation is a relatively rapid process for pest
tephritid flies. Only one day was required for C. capitata to acclimate
to a novel constant temperature. Acclimation to 31 1C for one day
significantly improved the heat shock resistance of C. capitata, but
also led to slower recovery from a cold shock. Rapid acclimation has
also been reported in Bactrocera tryoni (Meats, 1973). Meats (1973)
demonstrated that the mean torpor threshold of adult B. tryoni
declined to a level commensurate with a novel temperature (from
25 to 8 1C) after only three hours. Results for acclimation by C. rosa
are mixed. Only 1 day was required for a significant difference in
heat knock-down time of 21 1C- and 33 1C-acclimated C. rosa from
those kept at 24.5 1C whereas a significant difference in chill-coma
recovery time was not detected until 3 days. However, the difference
between chill-coma recovery time of C. rosa acclimated to 33 1C and
flies acclimated to other temperatures was greatest after having
been exposed for 5 days. Fletcher and Zervas (1977) reported that it
took 3–5 days for the cold torpor threshold of adult Bactrocera oleae
to shift in response to a change in constant temperature of 5 1C. It is
possible that delayed acclimation in C. rosa relative to C. capitata
with regard to cool temperature tolerance may have resulted from
the 2 1C difference in temperature experienced in incubators set to
30 1C. However, the difference between C. capitata and C. rosa in the
time required for acclimation to novel temperatures is in agreement
with results by Nyamukondiwa et al. (2010) that C. capitata develops
a rapid cold hardening response significantly faster than C. rosa.
Reversal of acclimation after return to initial temperature condi-
tions was achieved after only one day in both C. capitata and C. rosa.
When returned to the initial control temperature of 24–24.5 1C, chill-
coma recovery time of warm temperature-acclimated flies improved
to levels not significantly different from that of flies held constantly at
the control temperature. This rapid loss of acclimation reflects the
rapid loss of a cold hardening response in C. capitata and C. rosa
within 16 and 0.5 h, respectively (Nyamukondiwa et al., 2010).
For insects, much variation exists in the time reported for
reversal of acclimation (Table 1). In general, the magnitude of
change seems to have no bearing on the time required for reversal
of acclimation. After having initially been acclimated to a higher
temperature, reversal of acclimation has been reported to occur
in 2 days (adult D. melanogaster) and over 3 days (adult Bicyclus
anyana) where the magnitude of temperature change was similar to
that used in this study (Table 1). Similarly, increasing and decreasing
temperature changes in excess of 10 1C have led to reversal of
acclimation in under 20 h but up to 4 days in a range of taxa
(Table 1). Overgaard and Sørensen (2008) demonstrated that
thermal adaptation of Drosophila melanogaster in response to field
temperature variation was a continuous process. In D. melanogaster,
thermal acclimatisation tracked field temperature so that heat shock
survival improved with increasing temperature while cold shock
survival declined, with commensurate changes as field temperature
declined (Overgaard and Sørensen, 2008). Their results are in accord
with previous studies on the same species (Kelty and Lee, 2001;
Kelty, 2007).
There are multiple combinations of temperatures that can be
used to induce acclimation and test thermal stress. This is evident
from the range of temperatures that have been used to test for the
time-course for attainment and loss of acclimation (Table 1). While
the magnitude of temperature change does not seem to bear on the
time required for attainment of acclimation by a population, have
these temperature changes been ecologically relevant? In the case of
C. capitata and C. rosa, published data indicate that the temperatures
used to induce acclimation in this study are experienced in the
geographic range of both species (Duyck and Quilici, 2002; Duyck
et al., 2006), exceed the minimum temperature required for their
reproductive development (Duyck and Quilici, 2002), and are well
below the critical thermal maximum of adults (Nyamukondiwa and
Terblanche, 2009). The wide range in the times required for
acclimation across species may represent evolutionary responses
to different levels of temperature variability and predictability in
their environment. Theoretical modelling (Gabriel et al., 2005) and
empirical evidence (Kingsolver and Huey, 1998; Deere et al., 2006)
suggest that organisms living in environments with predictable
temperature fluctuations are more likely to evolve plastic pheno-
typic traits, while selection does not favour plasticity in those that
experience stable or unpredictable temperatures due to an inability
to match optimal phenotype with actual conditions. Diurnal tem-
perature fluctuations in part of the geographic range of C. capitata
and C. rosa can exceed 20 1C but are generally predictable from
day-to-day (Nyamukondiwa and Terblanche, 2010), so this may
contribute to the rapid attainment and loss of acclimation in these
species.
Fig. 2. Change in median heat knock-down time (A) and chill-coma recovery time
(B) of adult Ceratitis rosa transferred from 24.5 1C to 33, 24.5 or 21 1C. Error bars
indicate the upper and lower 95% confidence interval. The vertical dashed line
indicates the return of flies to 24.5 1C after 10 days.
C.W. Weldon et al. / Journal of Thermal Biology 36 (2011) 479–485 483

Author's personal copy
In both Ceratitis species, age had a significant effect on heat
knock-down and chill-coma recovery time. Heat knock-down time
declined with age whereas chill-coma recovery time increased with
age, indicating an increased susceptibility to heat and cold-shock,
respectively. A similar pattern has been found in D. melanogaster
(reviewed in Bowler and Terblanche, 2008) for both heat knock-
down (Pappas et al., 2007) and chill-coma recovery time (David et al.,
1998). The results of Pappas et al. (2007) indicated that declines in
heat knock-down time in the early adult are likely a developmental
rather than an aging phenomenon, and are not strongly associated
with levels of inducible heat shock protein expression. For both
Ceratitis species, the duration of the current study represented a
relatively short period of their reported mean life expectancy. At a
constant temperature of 25 1C, the life expectancy of C. capitata is
65.8 days for females and 75.6 days for males (Carey et al., 2008). The
life expectancy of C. rosa at a constant temperature of 25 1C exceeds
100 days (Duyck et al., 2010). The results of the current study need to
be reconciled with those of Nyamukondiwa and Terblanche (2009)
who, measuring the critical thermal maximum and minimum (CTmax
and CTmin, respectively) of both species, found that there was a
general improvement in thermal tolerance with age up to 14 days
after adult emergence. This apparent conflict between age-related
patterns likely arises due to the processes that the two types of assay
measure. Both CTmax and CTmin represent the temperatures at which
neuromuscular function is lost, and evidence suggests that this
results from disruption of membrane composition, permeability,
and ion channel and ATPase activity (e.g. Folk et al., 2007;
Macmillan and Sinclair, 2011). On the other hand, heat knock-down
and chill-coma recovery times measure the time required for
enzyme activity and/or membrane permeability to be lost or return
to levels sufficient for neuromuscular function, respectively. Inter-
preted in this way, it seems that there is progressive increase with
age after adult eclosion in the ability for ion regulation to be
maintained at warmer and cooler temperatures in C. capitata and
C. rosa. However, as individuals age, neuromuscular function is lost
more rapidly when exposed to high temperatures, and recovery of
neuromuscular function after exposure to low temperatures is
impaired.
Heavier C. capitata were more likely to have a shorter chill-
coma recovery time. Correlation of clinal variation in size and
stress resistance traits of D. melanogaster and D. serrata also
indicates a negative relationship between adult size and chill-
coma recovery time (James et al., 1995; Hallas et al., 2002;
Hoffmann et al., 2002). Populations of D. melanogaster and
D. simulans with high resistance to cold stress have been shown
to possess poor desiccation resistance and low levels of extrac-
table lipids as a proportion of body weight (Hoffmann et al., 2005;
Kenny et al., 2008), which suggests an evolutionary trade-off
between cold-resistance and starvation resistance that may be
controlled by lipid metabolism (Hoffmann et al., 2005). However,
the mechanisms that link chill-coma recovery time and body size
are yet to be identified with certainty.
Acclimation to benign temperatures by the two Ceratitis
species investigated in this study occurred rapidly. Earlier studies
on thermal acclimation in C. capitata and C. rosa (Nyamukondiwa
et al., 2010) safely assumed that exposure to novel temperatures
for 7 days would be sufficient for a full acclimation response;
acclimation by C. capitata can take as little as one day, while
1–5 days were required for C. rosa to acclimate to the novel
temperatures experienced in this study. Acclimation and its
reversal occurs in many insect taxa, and recent efforts are
establishing the processes involved at the organismal, cellular
and molecular level of organisation. These advances, as well as
data from a wider range of taxa, provide an opportunity to explain
the diversity of rates of attainment and loss of acclimation in
insects.
Acknowledgements
Ceratitis pupae were kindly provided by Dr. Aruna Manrakhan,
Citrus Research International. We wish to thank Ross van Eetveld for
assistance with conduct of experiments and Dr. Peter le Roux for
statistical and R programming advice. Dr. Susana Clusella-Trullas and
two anonymous referees provided insightful comments on earlier
versions of the manuscript. This work was supported by funding
provided to JST by Sub-Committee B, Stellenbosch University, and a
NRF THRIP award to Dr. Pia Addison.
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