J Exp Biol Advance Online Articles. First posted online on 24 January 2013 as doi:10.1242/jeb.080200
Access the most recent version at http://jeb.biologists.org/lookup/doi/10.1242/jeb.080200
Invasion and adaptation of a warm adapted species to montane
localities: effect of acclimation potential
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
Jyoti Chahal1*, Sudhir Kumar Kataria2 & Ravi Parkash1
*
Correspondence: Jyoti Chahal; Tel: +91 9050123129; Fax: +91 1262 243791; Email:
[email protected]
1
Present Address: Drosophila Research Lab, Department of Genetics, Maharshi Dayanand
University, Rohtak, India
2
Present Address: Department of Zoology, Maharshi Dayanand University, Rohtak, India
Running Title: Survival of a sensitive species in montane
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Copyright (C) 2013. Published by The Company of Biologists Ltd
Summary
Drosophila ananassae has successfully invaded the cold and dry montane localities of the
Western Himalayas in recent years. The ability of this desiccation and cold sensitive
tropical Drosophila species to evolve in response to seasonal changes in montane
localities is largely unknown. Here, we investigated how this sensitive species adapt to
seasonally varying environmental conditions that are lethal to its survival. We observed
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
change in the frequency of dark and light morphs of D. ananassae in five mid-altitude
localities during last decade (2000 to 2010). We document invasion of D. ananassae to
montane localities and increase in frequency of the dark morph. The stress tolerance of
morphs (dark and light) remained unaffected of developmental acclimation. However,
adult acclimation has shown significant effects on tolerance to various environmental
stresses in morphs and effect of this acclimation persist for long durations. Desiccation
and cold stress tolerance was increased after adult acclimation for respective stress in the
dark morph; while tolerance of the light morph was not affected. Further, heat tolerance
of the light morph was increased after adult heat acclimation with no influence on heat
tolerance of the dark morph. Our results suggest a possible role of adult acclimation in
successful invasion and adaptation of D. ananassae to montane localities. Future
experiments should be carried out to know if the survival in adverse conditions of low
versus high temperature and humidity during seasonal changes is assisted by different
acclimation abilities of the two morphs of D. ananassae.
Keywords: adult acclimation, developmental acclimation, persistence
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Introduction
Geographic distributions of species are constrained by several factors acting at
different scales, with climate assumed to be a major determinant at broad extents.
Environmental stresses like desiccation, cold and heat are main barriers which restrict
distribution and abundance of Drosophila species (Kellermann et al., 2009; 2012).
Recently, Drosophila ananassae has invaded successfully to the montane localities of the
Western Himalaya and climate change has been considered as the primary reason for
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
invasion of the cold sensitive D. ananassae (Rajpurohit et al., 2008a; Rajpurohit et al.,
2008b). Under the influence of changing climatic conditions, an organism can either
adapt or shift the range or undergo extinction. Drosophila species from the warm humid
tropics may be threatened if they can not adapt to drier and colder conditions of
montanes. Some studies have found evidence that desert insects are able to tolerate lower
water levels or store more water (Hadley, 1994; Gibbs and Matzkin, 2001; Gibbs et al.,
2003). Moreover, desiccation resistance in Drosophila can be increased by selection
causing accumulation of more bulk water in selected lines than controls (Gibbs et al.,
1997; Folk et al., 2001). Blows and Hoffmann (1993) observed that selection resulted in
reduced metabolic rates under desiccating conditions. They suggested that reduced
respiratory water loss was responsible for increased desiccation resistance. Further,
adaptations can be achieved by phenotypic plasticity (Bradshaw and Holzapfel, 2001).
Desiccation resistance can be increased with phenotypic plasticity either of cuticular
lipids (Parkash et al., 2008) or of melanisation (Parkash et al., 2009) under different
environmental conditions. Presence of melanism thickens the cuticle and acts as barrier to
evaporation through cuticle. In this way, melanism increases desiccation resistance by
reducing cuticular water loss (Parkash et al., 2009). Further, the thickness provided by
melanisation also insulates flies from cold (Parkash et al., 2010). However, in species like
D. ananassae, there is no reaction norm (thermal-induced plasticity) for melanisation
(Rajpurohit et al., 2008b), so alternative strategy of adaptation might be expected.
The acclimation ability or response to non lethal stress increases the resistance to
desiccation stress (Hoffmann 1990, 1991) and thermal stresses (Hoffmann and Watson,
1993; Kristensen et al., 2008). Different acclimation treatments (developmental, gradual
and rapid) had shown very beneficial effects on cold tolerance resulting in tolerant
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phenotypes of D. melanogaster (Colinet and Hoffmann, 2012). Many studies have
examined the effects of developmental acclimation in Drosophila (Kristensen et al.,
2008; Colinet and Hoffmann, 2012). Developmental acclimation has increased starvation
and heat knockdown time in D. buzzatii and thus affecting clinal variation of stress
resistance traits (Sarup and Loeschcke, 2010). The adult acclimation for cold resistance in
D. melanogaster increases the lifespan, reduces mortality and recovery time after
exposure to subzero temperatures (Rako and Hoffmann, 2006; Le Bourg, 2007). Further,
the dark morph of D. ananassae is more resistant to cold and desiccating conditions as
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
compared to the light morph; while the latter is more resistant to heat stress (Parkash et
al., 2012). Thus, we may expect evolutionary responses to natural selection on traits
related to desiccation and cold stress in subtropical populations of D. ananassae.
In the generalist Drosophila species, there is huge genetic variation for
adaptations to spatially and temporally varying climatic conditions (Powell 1997;
Hoffmann and Weeks, 2007). By contrast, two rainforest Drosophila species of montium
species subgroup - D. serrata (Hallas et al., 2002) and D. birchii (Hoffmann et al., 2003),
have low potential for climatic stress adaptations which match with their restricted
distribution patterns. It is possible that absence of acclimation ability in D. birchii
(Hoffmann, 1991) has resulted in its restricted distribution. However, desiccation and
cold sensitive D. ananassae has extended its boundaries to adverse climatic conditions of
the western Himalayas. But, it is not clear how it survive and proliferates under drier
environments as there are seasonal and diurnal variations in thermal as well as humidity
conditions in northern subtropics of the Indian subcontinent.
The objective of the present study is to explore the reason for successful
adaptation of a sensitive stenothermal species to montane localities of the Western
Himalayas. The questions addressed are1.
Does the frequency of D. ananassae body color morphs have changed in past few
years?
2.
Does the developmental and adult acclimation to stressful conditions increase
stress tolerance of the morphs?
3.
Do the morphs differ in their acclimation responses for various stresses?
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4.
Do the number of acclimation treatments and duration of stress make any
difference in stress tolerance?
The present study focuses on adaptation of a desiccation and cold sensitive
species, D. ananassae in the montane localities after successful invasion. We sampled D.
ananassae from 5 mid-altitude localities (Dharamshala, Nauni, Kandaghat, Solan and
Shoghi) of the Western Himalaya. We analysed that how D. ananassae copes with wet/
dry and high/ low temperature conditions inspite of its lack of plasticity for melanisation
and its sensitivity for dry environment. We investigated developmental and adult
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
acclimation ability of two body color morphs for environmental stresses and the
persistence of acclimation effect.
Material and methods
Collections and cultures
The D. ananassae flies were collected twice in every month from February to
May and August to November in year 2010 from five mid-altitude localities of the
Western Himalaya [Dharamshala (1219 m), Nauni (1300 m), Kandaghat (1432 m), Solan
(1440 m) and Shoghi (1833 m)] with bait trap and net sweeping methods. Wild-caught
flies showed dark vs. light total body color dimorphism for all the six abdominal
segments of both sexes. The wild-caught flies were examined for frequency of light and
dark phenotypes and the frequencies thus obtained were compared with frequencies in
2000 (unpublished data). Both collections (year 2000 and 2010) were made in a similar
manner.
Type of acclimation treatments
All acclimation experiments were carried out on ten homozygous (for phenotype)
strains for the dark as well as light morphs (used in Parkash et al., 2012). These strains
were originally derived from true breeding (for body color) iso-female lines from Solan.
These strains were grown at 25 °C for six generations on cornmeal-yeast-agar medium
under 12 hours light and 12 hours dark photoperiod prior to experiments. Two types of
acclimation treatments were conducted: Developmental and adult (partially followed
Hoffmann 1990). The experimental design is represented schematically in Fig. 1.
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A. Developmental acclimation treatment – involving manipulation of the
temperatures which pre-adult stages experienced but adult stages were kept at
similar conditions i.e., 25 °C.
B. Adult acclimation treatment – in which pre-adult stages developed at a common
temperature i.e., 25 °C, but adults were exposed to different temperature and
humidity conditions.
Developmental acclimation treatment
The 20 true breeding strains (10 for the dark and 10 for light body color) were
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allowed to lay eggs at 25 °C in 3 replicates. One replicate (out of three) with eggs was
transferred to one of three different growth temperatures (i.e., one at 20 °C, one at 25 °C
and one at 30 °C. All flies developed at three different temperatures were placed at 25 °C
after eclosion. After 6 days, assays were performed to check the effect of developmental
acclimation on desiccation tolerance, cold recovery minutes (after 5 hours cold stress at 0
°C) and heat knockdown time.
Adult acclimation treatment
Two different experiments (acclimation and persistence effect) were carried out to
study adult acclimation benefits for stress related traits. Controls were not acclimatized.
Ten replicates of ten flies each from homozygous stains were used for every treatment for
both morphs.
Desiccation acclimation
Multiple acclimation – Both morphs were given a prior desiccation stress of 3 hours in
three groups in ten replicates each. Flies of first, second and third group were stressed for
once, twice and thrice respectively with an in-between interval of 12 hours before they
were left to recover for 12 hours prior to measurement of the desiccation resistance.
Persistence effect – Ten replicates each having ten flies from both (10 dark and 10 light)
strains were given desiccation treatment twice for three hours each with an interval of 12
hours and allowed to recover on food for 2, 4, 6, 8 and 10 days before they were tested
for resistance to desiccation stress.
Cold acclimation
Multiple acclimation – Ten replicates of five flies each for both morphs were given prior
chilling of 1 hour in three groups (once/ twice/ thrice) as described in desiccation
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acclimation section. Then flies were recorded for percent mortality and recovery time
after chill coma stress (at 0 °C) as a function of duration of stress (1 hour, 3 hour and 5
hour).
Persistence effect – Ten replicates having five flies each from the ten dark strains were
given cold treatment of one hour at 0 °C (similarly in three groups as in desiccation) and
then allowed to recover on cornmeal-yeast-agar food medium for 2, 4, 6, 8 and 10 days
respectively before checking percent mortality and recovery time after 5 hours cold stress
at 0 °C.
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
Heat hardening
Multiple acclimations – Flies were initially given 5 minutes heat stress at 39 °C in three
different groups per three acclimation treatments, with 12 hour interval between each
treatment and before measuring the final effect in form of knockdown time.
Persistence effect – Persistence of the acclimation ability for heat was checked by placing
the heat treated flies (ten replicates having five flies each from the ten light strains) on
food for 2, 4, 6, 8 and 10 days before finally scoring the heat knockdown period at 39 °C.
Desiccation Resistance
To measure desiccation resistance, 10 individuals from each of the 10 dark and
light strains from both acclimation treatments (developmental and adult) were isolated in
a dry plastic vial, which contained 2 g of silica gel at the bottom of each vial and were
covered with a disc of foam piece. Such vials with foam plugs were placed in a
desiccation chamber (Secador electronic desiccator cabinet) which maintains 1-2 %
relative humidity at 25 °C. The vials were inspected every hour and the number of dead
flies (completely immobile) was recorded.
Thermoresistance assays
For thermoresistance traits, seven days old flies from both acclimation treatments
(developmental and adult) were isolated in separate vials after mild anaesthesia with
vapors of diethyl ether for a maximum of 30 seconds followed by one day recovery
period at 25 °C. Heat knockdown was measured individually on ten homozygous strains
each of the dark as well as light morph (5 flies x 10 strains). For heat knockdown assay,
individual flies were placed in 5 ml glass vials submerged into a water bath at a constant
temperature of 39 ºC. Flies were scored for time (in minutes) taken to be knocked down.
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Further, for chill – coma recovery ten groups of ten flies either for the dark or
light morph from both acclimation treatments were transferred without anesthesia in
empty 5 ml glass vials. These vials were set in thermocol boxes (24 x 13 x 10 cm)
containing ice flakes (made with an ice flaking machine AICIL) which were kept at 0 ºC
in the refrigerator. The vials were removed after 6 hour and flies were transferred to petriplates (9 cm diameter) in a temperature controlled room at 25 °C and cold recovery
period (in minutes) for every individual fly was also recorded.
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
Statistical analyses
Data was checked for normality and homoscedasticity by using normal
probability plot and Kolmogorov–Smirnov test. All the variables have normal
distribution, so parametric statistics have been used. Two-way ANOVA was applied to
know the effect of morphs, developmental acclimation treatments and their interaction on
various stress related traits. One-way ANOVA was used to compare the effects of
multiple adult acclimation treatments on chill – coma resistance in dark and light morph.
Comparison of stress tolerance between multiple acclimation treatments for desiccation
and heat knockdown was done using one way ANOVA differently for the two morphs.
Further, for testing differences in the acclimation treatments between two morphs
factorial ANOVA was applied to show interaction effect for various stress assays (Data
shown in Electronic Supplementary Material). Persistence of acclimation effect for the
traits was compared using Newman – Keuls post hoc test. Statistica software (Statsoft
Inc., Release 5.0, Tulsa, OK, USA and Statsoft Inc., Release 7.0, Tulsa, OK, USA) was
used for calculations as well as illustrations.
Results
Change in frequency
The 10 year collection record (2000 to 2010) has documented onset and increase
in frequency of the dark morph (from 0.0 to 0.18) and decrease in the light morph (1.0 to
0.61) in the five mid-altitude localities of the western Himalaya. In year 2000, only light
morph was prevalent; while in year 2010, the dark morph was also present at a
considerable frequency (See figure 1 of the Electronic Supplementary Material). An
intermediate morph (F1 of dark and light morph) was also abundant in collections of year
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2010 (See figure 1 of the Electronic Supplementary Material); but in the present study,
we are analyzing two contrasting pure body color morphs (the dark and light) only.
Developmental acclimation
The homozygous laboratory strains of dark and light morphs lack developmental
thermal acclimation (See figure 2 of the Electronic Supplementary Material), tested at
three temperatures (20, 25 and 30 °C). ANOVA results (Table 1) have shown significant
differences between morphs for all three stresses. However, no effect of developmental
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acclimation temperature was found on desiccation resistance, chill – coma recovery time
and heat knockdown time in D. ananassae (Table 1 ANOVA). Further, no significant
interaction effect between morphs and developmental acclimation treatments was
observed (Table 1 ANOVA).
Adult acclimation & persistence
Desiccation acclimation
One way ANOVA (Table 2) has shown significant effect of adult acclimation on
the dark morph, when controls were included (F = 168.06***, p < 0.001) but non
significant when controls were excluded (F = 1.62 ns, p = 0.121). There was no
difference between desiccation resistance of flies after once or twice or thrice acclimation
treatments (Table 2 ANOVA) that is why the data was pooled and showed as a single bar
(Fig 2A). The light morph has shown no acclimation potential for desiccation resistance
(Table 2 ANOVA). Further, there was a significant interaction effect between morphs
and multiple desiccation acclimation treatments (See table 1 of the Electronic
Supplementary Material). Acclimated dark flies has significantly high desiccation
resistance even after 10 days of prior acclimation as compared to control when tested
with Newman – Keuls post hoc test (p < 0.001***; df = 54 & MS error = 0.793; Fig 2B).
However, the desiccation resistance of flies did not differ significantly among 2nd day
versus 4th day (p = 0.47ns), 2nd day versus 6th day (p = 0.11ns) and 2nd day versus 8th day
(p = 0.12ns) after acclimation, showing high persistence of acclimation effect up to 8th
day according to Newman – Keuls post hoc test. But, the resistance differed significantly
among 8th and 10th day of acclimation (p = 0.001*** Newman – Keuls post hoc test; Fig.
2B) indicating that the effect of acclimation had declined after 8 days.
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Cold acclimation
Two way ANOVA for cold acclimation has shown significant effect of
acclimation treatment for the dark morph for percent cold mortality (Fig. 3A; Table 3)
and chill – coma recovery (Fig. 3B; Table 3); while non significant for the light morph
(Table 3). However, stress duration has shown significant effect for both morphs in
measures of cold resistance (Table 3). The interaction effects between morphs,
acclimation treatments and duration of stress were highly significant (See table 2 of the
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Electronic Supplementary Material). The differences between different acclimation
treatments were more distinct under 5 hour cold stress as compared to 1 hour or 3 hour
for both measures of cold resistance (Fig. 3A % mortality and Fig. 3B recovery time).
The flies of the dark morph differed significantly between control vs. 10 day after
acclimation for morality (Fig. 3C; p < 0.001***; df = 180 & MS error = 0.139) and for
recovery time (Fig. 3D; p < 0.001***; df = 180 & MS error = 0.139) in all three
acclimation treatments, when means were compared with Newman – Keuls post hoc test,
showing effect of cold acclimation. However, the % mortality of cold acclimated dark
flies did not differ among 2nd day, 4th day and 6th day after acclimation for three
treatments (1.00 < p > 0.05, Newman – Keuls post hoc test; Fig. 3C). Further, the % cold
mortality differed significantly among 6th and 8th day of relaxation on food for all three
acclimation treatments (p < 0.001***, Newman – Keuls post hoc test; Fig. 3C) indicating
decrease in effect of cold acclimation after 6 days. The cold recovery time did not differ
between 2nd and 4th day after thrice cold acclimation treatment (p = 0.75ns with Newman
– Keuls post hoc test); but differed significantly among 4th and 6th day (p < 0.001***)
indicating that acclimation effect persist up to 4 days and decline after that (Fig. 3D).
However, once and twice acclimated flies of the dark morph differed among all days after
acclimation in persistence (p < 0.001*** with Newman – Keuls post hoc test), showing
continuous decline in acclimation effect (Fig. 3D).
Heat hardening
One way ANOVA has shown significant effects of acclimation/ hardening
treatments on the light morph in the three groups, whether controls were included (F =
2701.3***) or excluded (F =1579.9***; Table 4; Fig. 4A); but effects were non
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significant for the dark morph (Table 4; Fig. 4A). Further, both morphs differ
significantly in their interaction with subsequent acclimation treatments (See table 3 of
the Electronic Supplementary Material). The heat knockdown minutes between 2nd to 6th
day after heat acclimation treatment did not differ significantly (1.00 < p > 0.05, df = 180
& MS error = 0.302 with Newman – Keuls post hoc test; Fig. 4B) for three heat
acclimation treatments showing strong persistence of heat acclimation up to 6 days after
treatment. However, after 6th day the effect of acclimation decreased (as p < 0.001
between 6th and 8th day of relaxation with Newman – Keuls post hoc test; Fig. 4B). The
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
difference in heat resistance between control versus acclimated flies has completely
vanished on 10th day of relaxation (p = 0.32ns with Newman – Keuls post hoc test) in
case of once acclimated flies; while for twice and thrice acclimated flies differences were
still there (p < 0.001*** with Newman – Keuls post hoc test).
Discussion
Insects, particularly drosophilids, are known as the good biological indicators for
climate change as they respond to minor fluctuations in the temperature either by
changing their adaptive behavior or by shifting their boundaries. Climatic suitability
depends upon three factors- first, deviations from optimum conditions; second, extent of
variation among years and third, extent of variation within a year (Danks 1999; 2007).
Further, spatial complexity of habitats is one template for evolution of seasonal
adaptations. Most organisms experience high thermal variations in the environment and
this poses substantial challenges for key elements of fitness such as survival and
reproduction (Dahlhoff and Rank, 2007), therefore temperature is considered as an
important selective agent (Hoffmann et al., 2003). In the present study, an onset and
increase in the frequency of dark morph of D. ananassae has been observed at midaltitude localities in past ten years. The adaptation of individual species in a particular
habitat depends on two main factors: first is constraint i.e. adversity, which in the present
case may be seasonal cycle with changes in temperature and humidity. Second is need i.e.
suitability; which may be the increased temperature as a result of global warming
creating optimum conditions for the species in montanes. The first factor possibly has led
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to the evolution and increase in the frequency of dark morph and the second factor may
have helped the species to invade montanes.
The flies developed at fluctuating temperature had higher resistance than flies at a
constant temperature, thus affecting clinal variation and proving role of developmental
acclimation in D. buzzatii (Sarup and Loeschcke, 2010). Further, field releases of D.
melanogaster on two continents across a range of temperatures had shown enormous
benefits of developmental as well as adult acclimation at low temperature in field
(Kristensen et al., 2008). However, in the present study, no benefit of developmental
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temperature acclimation was found on stress resistance of body color morphs in D.
ananassae. This is in accordance with the fact that D. ananassae also lack developmental
plasticity for body color and melanisation at range of temperatures (Rajpurohit et al.,
2008b; Parkash et al., 2012). One reason may be that the flies were kept at a common
temperature after eclosion which did not provide adult acclimation. While D.
melanogaster flies were kept at the same temperature as for development, in the study by
Kristensen et al. (2008), which possibly had resulted in adult acclimation. Further, a
study on the tsetse fly Glossina pallidipes demonstrated that the stage at which
acclimation occurs has significant effects on adult physiological traits (Terblanche and
Chown, 2006). The study on Glossina pallidipes has showed that adult acclimation had a
larger effect on critical thermal minima while developmental acclimation has a little
effect on that. Furthermore, Colinet and Hoffmann (2012) reported that gradual thermal
acclimation of few days was beneficial for cold recovery time while no such result was
found for developmental acclimation in D. melanogaster. Similarly, in the present study
there was no increase in stress resistance as a result of developmental acclimation in D.
ananassae. Thus, we can say that developmental temperature does not show any
significant contribution towards adaptation of D. ananassae, when adults face a different
temperature than pre - adult stages.
Adult acclimation is the other form of plasticity used to encounter environmental
stresses (Hoffmann 1990, 1991; Hoffmann and Watson, 1993). Acclimation to a
particular environment enhances performance which proves beneficial for adaptation
(Leroi et al., 1994). Tropical and rainforest species inhabiting high humidity conditions
has been found more desiccation sensitive than the temperate or arid species (Parsons,
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1983) suggesting adaptive significance of the acclimation responses. The variations
between species and geography have been examined for acclimation potential for heat
resistance (Levins, 1969) and for desiccation tolerance (Hoffmann, 1990; 1991).
However, to the best of our knowledge acclimation potential of morphs of any
Drosophila species had not been analysed so far.
Acclimation responses were indicated after different types of thermal treatments
in populations of D. melanogaster and D. simulans (Hoffmann and Watson, 1993;
Watson and Hoffmann, 1996) and of D. buzzatii (Krebs and Loeschcke, 1996). Long
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term cold survival of D. melanogaster was increased significantly after low temperature
acclimation (Overgaard et al., 2008). Apart from temperature acclimation, geographical
variation in acclimation responses for desiccation stress in D. melanogaster and D.
simulans were also found (Hoffmann, 1990). Acclimation to low humidity can
significantly increase the desiccation tolerance. Some studies have shown strong
responses of insect desiccation rate towards changes in relative humidity (Gibbs et al.,
2003; Hoffmann, 1990, 1991). Further, in the present study the two morphs differ in their
acclimation response towards desiccation stress. The dark morph has increased the
desiccation tolerance after acclimation but there was no effect in tolerance of the light
morph.
Majority of research work on cold tolerance of insects has been focused on
seasonal acclimation, which is result of cold hardiness acquired during overwintering.
Cold hardening is described as a process which is induced by exposure to lower
temperature for a short time (Lee et al., 1987); with in some time protection is gained by
the insect to otherwise lethal temperature. Further, combined cold acclimation treatment
has evidenced strong impact on field stress resistance in D. melanogaster (Colinet and
Hoffmann 2012). In present study, the dark morph of D. ananassae has gained some
protection against low temperature and low humidity by acclimation, while the light
morph is still sensitive. The dark morph of D. ananassae is more desiccation and cold
tolerant as compared to the light morph while the latter is more tolerant for heat (Parkash
et al., 2012). Inspite of the above fact, there is high acclimation ability of the dark morph
for desiccation and cold while of light morph for heat resistance. From this observation
we may postulate that these morphs are still under divergent selection for stress resistant
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traits in nature and the selection will be complete when there will be no response to
further acclimation or prior stress; as desiccation selected D. melanogaster did not
respond to prior desiccation treatment or acclimation (Hoffmann, 1990).
Interspecific studies on heat acclimation have shown significant differences
between D. melanogaster and D. simulans (Levins, 1969). Hardening treatments at high
temperature has shown increase in thermo-tolerance for 8 Drosophila species (Kellett et
al., 2005) and in codling moths, Cydia pomonella (Chidawankiya and Terblanche, 2011).
However, acclimation potential for heat has been a potential gap for morphs of a species.
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
In present study, the light morph of D. ananassae has shown the higher acclimation
potential for heat stress than the dark morph, with increasing heat tolerance after
subsequent heat treatments. The heat knockdown time of the dark morph was unaffected
of heat hardening treatments.
The frequency and duration of stress experienced are major factors which strongly
influence and define acclimation ability of a species. However, number of times of
exposure (acclimation) to desiccation stress has not affected the level of increase in
desiccation resistance in the dark morph in present study. But, with increasing frequency
of cold exposures the cold acclimation potential of the dark morph has increased; thus
percent mortality and recovery time during cold stress decreased. Further, duration of
cold stress has also affected the mortality and recovery of morphs (Fig. 3A & B).
However, the dark morph was not acclimated to heat but the heat tolerance (in terms of
knockdown time) of light morph was increased with every acclimation treatment.
The acquired tolerance to stress persists for several days after initial stress
exposure. The persistence of desiccation acclimation effect is known up-to 24 hours in D.
melanogaster (Hoffmann, 1990). Here, in the present study, the acquired resistance
persists up to longer durations (10 days; Fig. 2B). Further, effect of cold acclimation also
persist up to ten days; however, with every advancing day the acclimation effect becomes
less effective (Fig. 3C & D). In the light morph also, the hardening effect persist in form
of high heat knockdown time in hardened flies in comparison to control flies (Fig. 4B).
According to the present study, adult acclimation ability has increased the stress
tolerance which possibly has led the species to adaptation. Increased adult acclimation
ability of morphs of D. ananassae can be a result of absence of developmental plasticity
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in these genotypes as some cost have been postulated as a way of accounting for the
absence of genotypes with a very high degree of plasticity (Heslop-Harrison, 1964;
Bradshaw, 1965). D. ananassae have responded towards stress in different way;
evidencing inter-morph variation in acclimation potential suggesting that two morphs of
same species may use different adaptive strategy according to their need. For instance,
the dark morph have better acclimation potential for desiccating conditions as compared
to light morph, while the light morph has more acclimation potential for heat resistance
and thus may help the species to survive during hot summer days. The high acclimation
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potential of the dark morph for cold and desiccating conditions has helped this sensitive
species to survive during adverse dry climatic conditions during seasonal change and thus
supports invasion. The respective acclimation potential of the two morphs can also justify
the changing frequency of morphs during two different seasons in the Western Himalaya.
According to Colinet and Hoffmann (2012), stress acclimation experienced by adults just
before the chronic stress is more influential than acclimation experienced during
development. The present study supports Colinet and Hoffmann (2012), as
developmental acclimation has not shown any increase in stress resistance while adult
acclimation had shown enormous benefits.
In conclusion, increased frequency of the dark morph in past few years may have
enabled D. ananassae to invade and adapt successfully in seasonally varying montane
localities. This suggests that natural selection and evolution are more firmly associated
with gradual changes in environment of any organism; giving rise to a graded response.
Further, it seems like acclimation to one extreme decrease the capacity to acclimate under
opposite extreme; as the dark morph can acclimate to colder and desiccation conditions
but not to high temperatures while the reverse is true for light morph. Also, number of
acclimation treatments increases the tolerance for thermal stresses and this increase in
stress tolerance persists for longer durations in D. ananassae. Thus, different acclimation
abilities of two morphs have enabled this sensitive species to increase some tolerance to
stress. Further, field testing should be done to know if high acclimation potential of D.
ananassae enables this species to adapt under adverse conditions of montane localities.
15
Acknowledgements
We are indebted to the two anonymous reviewers for constructive comments towards
improvement of the manuscript.
Funding
Financial assistance from the Council of Scientific and Industrial Research, New Delhi in
the form of Senior Research Fellowship [09/382(0152)2012-EMR-1] to J.C. is gratefully
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
acknowledged.
16
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20
Legend for figures:
Fig. 1 Schematic representation of the experimental design used to investigate the
contribution of developmental and adult acclimation in physiological performance of
morphs of D. ananassae.
Fig. 2 Desiccation resistance of the dark and light morph (A) as a function of times of
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
prior acclimation (B) persistence of acclimation effect after relaxation period.
Fig. 3 (A) Percent mortality & (B) recovery period during chill coma stress as a function
of duration of stress and number of times the flies of the dark morph acclimated to the
stress; Persistence of acclimation response for (C) percent mortality & (D) recovery time
as a function of relaxation days on food, after chill coma stress of four hours. Controls
were not acclimated.
Fig. 4 Comparison in the dark and light morph for (A) heat knockdown time as a function
of times of acclimation prior to stress & (B) persistence of acclimation effect for the heat
stress after relaxation period on food.
21
Developmental
Acclimation
Eggs
25 °C, 9 days
30 °C, 7 days
25 °C, 9 days
20 °C, 21 days
Flies (at eclosion)
Flies (at eclosion)
25 °C, 6 days
Adults
25 °C, 6 days
Adults
Stress resistance measurement
Desiccation
Cold
Heat
Adult
Acclimation
Common conditions
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Eggs
Common conditions
Acclimation Treatments
Desiccation
(3 hours at
25 °C)
Cold
(1 hour at
0 °C)
Heat
(5 min.
at 39 °C)
Acclimations were done once, twice and thrice with
an interval of 12 hours between every treatment
Stress resistance measurement
Desiccation
Cold
Heat
Fig. 1 Schematic representation of the experimental design used to investigate the contribution of developmental and
adult acclimation in physiological performance of morphs of D. ananassae.
22
Desiccation Hours
Dark morph
25
Light morph
20
15
10
5
0
Control
Acclimated
(Once/ twice/ thrice)
(B)
Desiccation survival hours
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(A) 30
30
25
20
Dark morph
Light morph
15
10
5
Control
2
4
6
8
10
Time on food after stress (days)
Fig. 2 Desiccation resistance of the dark and light
morph (A) as a function of times of prior acclimation
(B) persistence of acclimation effect after relaxation
period.
23
(A)
(C)
30
30
Percent mortality
Percent mortality
20
10
0
Once acclimatized
T wice acclimatized
T hrice acclimatized
20
10
0
1 hour
3 hour
5 hour
(B)
Control 2
4
6
8
10
Control 2
4
6
8
10
(D)
50
50
Dark morph
Recovery time (minutes)
Recovery time (minutes)
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
Dark morph
Control
Once acclimatized
T wice acclimatized
T hrice acclimatized
40
30
20
10
0
1 hour
3 hour
5 hour
Duration of cold stress
40
30
20
10
0
Time on food after stress (days)
Fig. 3 (A) Percent mortality & (B) recovery period during chill coma stress as a function of
duration of stress and number of times the flies of the dark morph acclimated to the stress;
Persistence of acclimation response for (C) percent mortality & (D) recovery time as a function of
relaxation days on food, after chill coma stress of four hours. Controls were not acclimated.
24
Heat knockdown (min)
20
Dark m orph
Light m orph
15
10
5
0
control
once acclimated
twice acclimated thrice acclimated
Conditions
(B)
Heat Knockdown (min)
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(A)
20
Light
15
Once Acclim atized
T wice Acclim atized
T hrice Acclim atized
10
5
Dark
0
CONTROL 2
4
6
8
10
Time on Food after stress (days)
Fig. 4 Comparison in the dark and light morph for (A)
heat knockdown time as a function of times of
acclimation prior to stress & (B) persistence of
acclimation effect for the heat stress after relaxation
period on food.
25
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
Table 1 Two-way ANOVA for stress related traits as a function of morph and developmental acclimation treatments
(DAT) in D. ananassae. *** = p < 0.001; ns = non-significant.
Stress related Traits
df
Morph (M)
1
DAT (T)
2
M X DAT
2
Error
294
Desiccation
MS
F
9303.5
3903.5 ***
3.61
1.51 ns
0.34
0.14 ns
2.38
-
Cold Recovery
MS
F
87245.2
13151.5 ***
15.1
2.28 ns
3.3
0.50 ns
6.6
-
Heat Knockdown
MS
F
9462.8
283.9 ***
70.39
2.12 ns
32.72
0.98 ns
3.22
-
26
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
Table 2 Analysis of variance for two morphs of D. ananassae as a function of adult acclimation
conditions for desiccation stress (*** = p < 0.001).
Term
With Control
Acclimation
Error
Without Control
Acclimation
Error
df
3
36
MS
89.72
0.53
2
27
0.86
0.53
Desiccation Acclimation
Dark Morph
F
% Var.
MS
168.06***
93.3
0.72
6.66
0.47
1.62 ns
-
10.74
89.25
0.62
0.44
Light Morph
F
% Var.
1.52 ns
11.3
88.8
1.14 ns
-
9.48
90.51
27
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
Table 3 Analysis of variance between two morphs of D. ananassae as a function of adult acclimation conditions and duration of
chill coma stress (*** = p < 0.001).
Traits
Cold Mortality
Acclimation (1)
Stress duration (2)
1X2
Error
df
3
2
6
108
Chill-coma
recovery
Acclimation (1)
Stress duration (2)
1X2
Error
3
2
6
108
Dark Morph
MS
F
109.31
85.92***
1195.45
939.69***
96.64
75.96***
1.27
-
% Var.
9.54
69.6
16.8
3.99
809.59
2298.45
160.94
0.88
30.03
56.84
11.94
1.18
916.76***
2602.72***
182.25***
-
MS
2.37
29185.9
4.03
6.35
Light Morph
F
0.37
4598.89***
0.64
-
% Var.
0.012
98.78
0.041
1.16
0.8
13016.9
0.1
1.7
0.4
7735.9***
0.1
-
0.01
99.29
0.002
0.69
28
The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT
Table 4 Analysis of variance between two morphs of D. ananassae as a function of adult acclimation
conditions for heat stress; with or without controls (*** = p < 0.001).
Term
With Control
Acclimation
Error
Without Control
Acclimation
Error
Heat Hardening
df
3
196
MS
0.036
0.166
Dark Morph
F
0.21 ns
-
% Var.
0.33
99.69
MS
347.50
0.13
Light Morph
F
2701.3***
-
% Var.
97.63
2.36
2
147
0.053
0.169
0.31 ns
-
0.43
99.56
213.84
0.14
1579.9***
-
95.55
4.44
29