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Acclimation for desiccation resistance in Drosophila: Species and population comparisons
.I. InsectPhysiol.Vol. 37, No. 10, pp. 757-762, 1991
0022-1910/91 $3.00 + 0.00
Printed in Great Britain. ,411rights reserved
copyright 0 1991 Pergamon Press plc
ACCLIMATION FOR DESICCATION RESISTANCE I!~ROSOPHILA : SPECIES AND POPULATION COMPARISONS
IN
ARY A. HOFFMANN Department ol‘Genetics and Human Variation, La Trobe University, Bundoora, Victoria 3083, Australia (Received 23 April 1991; revised 3 June 1991)
Abstract-In previous work, it was found that Drosophila melanogaster females became more resistant to adesiccation after prior exposure to a non-lethal desiccation stress. Here, this acclimation response is shown to occur in three other widespread Drosophila species: D. simulans, D. immigrans and D. serrata. However, such acclimation was unsuccessful in D. birchii, a sib’ling species of D. serrata found in rainforest habitats. An acclimation response in all the widespread species was evident after a prior exposure period of a few hours, and acclimation was still evident after more than 24 h. Two D. serrata populations, one originating from a tropical location (Townsville) and one from a cooler coastal area (Forster) had a similar acclimation response. Although D. melanogaster and D. simulans populations originating from a tropical site (Cairns) were less resistant to desiccation than conspecific populations from a temperate site (Melbourne), the populations had similar acclimation responses. Hence, there was no evidence for geographic variation in acclimation ability. Key Word Index: Drosophila; acclimation;
desiccation; resistance; adaptation
INTRODUCTION
Insects may alter their levels of resistance to desiccation stress by becoming acclimated after prior exposure to a non-lethal stress period. Evidence for physiological changes likely to increase desiccation resistance has been found in a number of species (e.g. Miller, 1964; Krafsu.r, 1971; Toolson, 1982). More directly, the desiccation resistance of Drosophila melanogaster could be altered by prior exposure to a low humidity environment (Hoffmann, 1990). This response developed within 2 h of initial exposure to low humidity, and ;persisted more than 28 h after the prior stress period. There was also evidence for genetic variation in ,the acclimation response. Lines selected for resistance showed a markedly reduced acclimation response compared to unselected lines, presumably because the acclimation response and genetic variation for desiccation resistance were partly based on a similar mechanism. Dry periods of a few hours duration where humidity falls below 20% are regularly recorded in temperate localities where widespread Drosophila are collected, suggesting that acclimation responses of the type found in D. melanogaster may be of adaptive significance. If this is the case, then it is expected that acclimation ability will vary between Drosophila species depending cln the types of habitats they occupy. In particular, species from dry habitats might be expected to show a. greater acclimation ability than species restricted to rainforests where high relative
humidity is the normal expectation. Adaptation to dry habitats may also involve increased resistance although there are often costs involved with high levels of stress resistance (Hoffmann and Parsons, 1991). In a parallel way, differences in acclimation responses might be expected between populations of widespread species occupying a range of habitats. A number of Drosophila studies have demonstrated geographic variation in resistance to desiccation and other stresses, where population differences tend to match predictions based on climatic data (see Arlian and Eckstrand, 1975; Stanley and Parsons, 1981; Hoffmann and Parsons, 1991). However, there have been almost no attempts to examine geographic variation in acclimation ability in Drosophila with the exception of some work by Levins (1969) on heat resistance which showed interpopulation variation in some species. In this paper, I have tested these predictions by examining acclimation in four Drosophila species other than D. meIanogaster and by considering geographic variation in three of these species. The two cosmopolitan species D. immigrans and D. simulans were tested, as well as D. serrata which is more restricted rainforests
in its distribution.
D. serrata occurs
in
as well as in coastal areas of north-eastern Australia. I also examined acclimation in D. birchii, a sibling species of D. serrata largely restricted to tropical rainforests. It was predicted that D. birchii and to a lesser extent D. serrata would have low 757
758
ARY A. HOFFMANN
resistance and a reduced acclimation response. In the geographic comparisons, I examined populations of D. simulans and D. melanogaster originating from tropical and temperate locations, as well as D. serrata populations originating from a rainforest and a cooler coastal site. It was predicted that the tropical/ rainforest populations of these species would have relatively lower desiccation resistance and a reduced acclimation ability. MATERIALS
AND METHODS
Stocks and desiccation
The D. immigrans stock was established with the progeny of 25 females collected from a compost heap in Melbourne. The Melbourne D. simulans and D. melanogaster populations were started with the progeny of 25 (D. melanogaster) or 10 (D. simulans) females collected from an orchard. Cairns stocks of these species were established a month before the Melbourne stocks with progeny from 11 (D. simufans) or 25 (D. melanogaster) females collected from mangoes. It was important to initiate stocks at approximately the same time because acclimation responses may be altered during long-term laboratory culture (Toolson and Kuper-Simbron, 1989). The North Queensland D. serrata and D. birchii stocks were founded by the progeny of 16 and 20 females, respectively. These flies had been collected by fruit baiting in tropical rainforests. The 16 females which initiated the Forster D. serrata stock had been collected by fruit baiting in coastal vegetation between Forster and Seal Rocks in New South Wales, approx. 1600 km south of the tropical collection sites. This area marks the southern limit of the D. serrata distribution, while North Queensland is central in its distribution (Ayala, 1965). The two D. serrata populations were collected 2 months apart. All experiments were carried out within 15 months after stocks had been established in the laboratory. Stocks were maintained by mass transfer at 18-20°C. The culture medium for D. simulans, D. melanogaster and D. immigrans comprised of agar (3%), sucrose (9%) and dead yeast (6%) with propionic acid and methyl hydroxybenzoate as preservatives. Half the sucrose was replaced by instant potato mash for the D. serrata and D. birchii cultures. Adults to be used in the experiments were cultured at 24-25°C under continuous light. Humidity outside the culture bottles was 40-60%. Flies were collected from bottles when they were O-2 days old and aged (sexes mixed) in vials with laboratory medium at a density of 30-50 flies per vial prior to acclimation or desiccation. The ageing and sexing procedure differed for the species as detailed below. Flies were acclimated and tested for stress resistance in glass desiccators containing silica gel. Most experiments involved desiccation at 25°C except for the D. birchii experiment and the D. serrata population comparison (see below) which were carried out
at 24°C. Humidity in the desiccators declines to 8-10% after 1 h and reduces to 3-5% in about 3 h. Flies were tested in groups of 20 which were acclimated and desiccated in empty glass vials covered with gauze. To determine stress resistance, vials were arranged around the perimeter of a desiccator so that mortality in the vials could be monitored without opening the desiccator. The time taken for 50% of the flies to die (LT,) was scored by counting the number of dead flies at 1 h intervals. The 50% mark was linearly interpolated when more than half the flies had died. Acclimation of dtflerent species D. immigrans. Flies were aged for 3 days before they were sexed under carbon dioxide anaesthesia, and held another 2 days before acclimation. Preliminary experiments indicated that D. immigrans females could be desiccated for 4 h before mortality started to occur, so females were acclimated for 3 or 4 h, and allowed to recover for 9 h. Eight replicate vials were set up for each treatment. The persistence of the acclimation effect was examined by desiccating females for 4 h and holding them for 5, 8, 29 or 53 h on laboratory medium before testing desiccation resistance. Flies which had not been acclimated served as controls. In addition, I tested females which had been desiccated for 4 h but were only given a 5 h recovery period. Flies for all treatments were aged for 2 days before sexing, so that they were 5-6 days old when they were tested for desiccation resistance. Ten replicates were set up per treatment. D. simulans. Previous experiments with D. melanogaster females indicated that the effect of acclimation increases with the length of the prior exposure period to low humidity (Hoffmann, 1990). This association was also examined in D. simulans females from the Melbourne stock. Flies were aged in vials for 3 days and females were separated by aspiration and aged in vials for another 1 day before exposure to the acclimation treatments. Females were acclimated for 3, 4, 5 and 6 h. They were left to recover for 9 h on laboratory medium prior to being tested for desiccation resistance. A control treatment where flies were not exposed to a prior stress was also included. Females were not acclimated for longer than 6 h to avoid mortality, reflecting the lower desiccation resistance of this species compared to D. melanogaster (Stanley et al., 1980). Five replicate vials were set up for each treatment. The persistence of the acclimation response was tested with females from the Melbourne stock. Flies were acclimated for 6 h, and held for 4, 9, 18, 24 and 48 h on laboratory medium before they were tested for desiccation resistence. Controls which had not been acclimated were also included. Five replicates per treatment were set up. D. serrata. The effects of different prior desiccation periods were examined using the Forster stock. Flies
Acclimation for desiccation resistance were aged (sexes mixed) for 5 days after eclosion. Because the sexes of D. serrata cannot be easily separated by aspiration, flies were sexed under carbon dioxide anaesthesia, and aged another day before acclimation. Flies were aged longer than D. melanogaster and D. simulans because D. serrata takes longer to reach sexual maturity (l-2 days after eclosion) and peak fecundity (7-12 days after eclosion) when they are ktept at 25°C (M. Blows, personal communication) than D. melanogaster and D. simuZans which mate less than 12 h after eclosion and reach peak fecundity after 34 days. Females were acclimated for 2, 3 and 4 h and an unacclimated control was also tested. The 4 h period corresponds to the maximal time that D. serrata can be desiccated before mortality starts to occur. Males were also tested for acclimation, using a 3 h prior desiccation period. Flies were allowed to recover for 9 h on laboratory medium before they were tested for stress resistance. Ten replicates were set up for each treatment. To test for persistence of the acclimation response, females from the Forster stock were acclimated for 4 h and then held on. laboratory medium for 18 or 24 h prior to testing for desiccation resistance. Controls which were not acclimated were also included. Twelve replicates were set up for the acclimation treatments, and 8 replicates for the controls. D. birchii. Preliminary experiments indicated that this species was more sensitive to desiccation than D. serrata because some mortality was already evident when females were desiccated for 4 h. The effect of a 3.5 h prior desiccation period on stress resistance was therefore compared to a non-acclimated control. Flies were treated prior to desiccation as described above for D. serrata. Ten replicate vials were set up for the acclimated and non-acclimated treatments. This experiment was set up at the same time as the D. serrata population comparison. Population comparisoiw D. serrata. Populations of this species from Cairns and Forster were compared for acclimation responses. In contrast to the above experiments with this species, flies were not sexed prior to acclimation to avoid the use of anaesthesia. This is unlikely to have much influence on the results because the earlier experiments had indicated that the sexes had similar resistance to desiccation and showed a similar acclimation response (see below). Nevertheless, the sex ratio of each vial was determined after desiccation to see if this variable influenced resistance. The resistance of flies acclimated for 3.5 h (with a 9 h recovery period) was compared with the resistance of non-acclimated flies. The experimental design (ignoring sex ratio as a covariate) is given by
Yijk= u + b, +
cl+
bc, + e,(i))t
(1)
where bi is the effect due to population, c, the effect due to acclimation, bc,, the acclimation by population
759
interaction and e,,,, the error term. Ten replicate vials with 20 flies were set up for each treatment. D. melanogaster and D. simulans. Acclimation responses of the Cairns and Melbourne populations of these species were compared by testing acclimated and non-acclimated females from each population. Flies were sexed by aspiration and treated as in the D. simuians experiments. D. simulans females were acclimated for 6 h and were allowed to recover for 6 h on laboratory medium. D. meianogaster females were acclimated for 8 h and allowed to recover for 6 h. This recovery period is shorter than the normal 9 h period, but previous experiments (Hoffman, 1990) indicated that this reduction would not adversely affect the acclimation response. The experimental design follows equation (1) and there were 8-10 replicates per treatment. RESULTS
Acclimation in different species D. immigrans. Mean LT,,s for the treatments were initially compared with a one-way analysis of variance (ANOVA) which indicated a significant effect of treatment (early desiccation exposure time) on desiccation resistance [F(,,, = 20.45, P < O.OOl]. The significance of differences between pairs of means was determined by computing the least significant difference as outlined in Sokal and Rohlf (1981, p. 244). The mean LT, for the control treatment (2 = 12.53, SD = 0.62 h) differed significantly from the means for the 3 h (2 = 13.69, SD = 1.04 h) and 4 h (2 = 15.51, SD = 1.09 h) treatment. Acclimation therefore increased the desiccation resistance of the females, the 4 h treatment increasing resistance (LT,,) by 24% of the control treatment. In the experiment testing the persistence of the acclimation response, treatment means differed significantly in an ANOVA [F& = 10.50, P < O.OOl] and acclimated treatments had a higher desiccation resistance than controls (Fig. 1). Comparisons of pairs of means after computing the least significant
20
Tim-
on
lab
mrdlum
(hourr)
Fig. 1. Persistence of the acclimation response of D. immigram females after holding them on laboratory medium following acclimation. Error bars represent upper 95% confidence limits of the samples.
ARY A. HOFFMANN
760
_ Acclimation
T
T
5
6
I
_ Period
i 3 Afclimatlo”
(hours)
Period
(hours)
Fig. 2. Effect of acclimation period on the desiccation resistance of D. simulans females. Error bars are upper 95% confidence limits.
Fig. 4. Effect of acclimation period on the desiccation resistance of D. serrata females. Error bars are upper 95% confidence limits.
indicated that only the 53 h treatment did not differ significantly from the controls. The acclimation response therefore persisted for at least 29 h, but was reduced after 29 h compared to after 8 h. The 5 h recovery period was sufficient to produce an acclimation response. D.simulans. Mean LT,,s indicate that acclimation increased desiccation resistance, the magnitude of the effect increasing with the length of the prior exposure period (Fig. 2). Treatments differed significantly by an ANOVA [ Fc4,2,,j = 12.44, P < O.OOl].The 6 h treatment increased resistance by more than 4 h or 40% of the control resistance. All four acclimation treatments differed significantly from the controls by a least significant difference analysis, so that desiccation resistance was increased even when flies were exposed to low humidity for 3 h. A linear regression analysis was carried out using the means for each prior desiccation period to predict LT,,: the fitted equation (LT,, = 0.65 prior desiccation period + 10.76) accounted for a significant proportion of the variance (P < 0.05) in mean LT,, . The acclimation response declined with the length of time flies were held on laboratory medium (Fig. 3). Treatments differed significantly by an ANOVA and a least significant KS.24)= 9.67, P < O.OOl),
except the 48 h period differed significantly from the controls. Acclimation therefore persisted for more than 24h and a recovery period of 4 h or more was sufficient to produce an acclimation response. D. serrata. The treatments differed significantly by
difference
difference
analysis
indicated
that
all
treatments
an ANOVA [FC3,361 = 10.98, P < 0.001). Each of the acclimation treatments differed significantly from the controls by a least significant difference analysis. The 4 h acclimation period increased resistance by 2.5 h or 26% of the control resistance, while the 2 h treatment produced a smaller acclimation response (Fig. 4). The desiccation resistance of males was also increased by a 3 h acclimation period; the mean LT, for the controls was 7.86 h (SD = 0.3 1) compared to a mean of 9.52 h (SD = 6.33) for the 3 h treatment. These means differ significantly by a r-test (t = 7.46, d.f. = 18, P < 0.001). The resistance of both sexes was therefore increased after acclimation. Acclimation was detected even after females had been held on laboratory medium for 18 and 24 h after acclimation. These recovery periods differed significantly by an ANOVA [FC2,29j = 7.52, P < O.OOl].The mean LT,, for the control treatment (2 = 8.42, SD = 0.45 h) differed significantly from means for the 18 h (2 = 10.19, SD= 0.78 h) and 24h (2 = 9.31, SD = 1.14 h) periods as determined by a least significant difference analysis.
Table 1. Mean LT,,s for acclimated and non-acclimated flies from populations of D. serrata, D. melanogaster and D. simulans
Acclimated
Non-acclimated
zz
SD
2
SD
13.50 14.14
1.41 0.92
11.64 11.21
1.29 1.17
15.84 16.82
1.12 1.87
14.41 15.42
1.57 0.84
12.80 14.14
0.75 0.89
10.14 12.27
0.74 1.23
D. serrata
Townsville Forster D. melanogaster Time
on
Iaib
medium
(hours)
Fig. 3. Persistence of the acclimation response of D. simulans females after holding them on laboratory medium following acclimation. Error bars represent upper 95% confidence limits.
Cairns Melbourne D. simulans
Cairns Melbourne
Acclimation for desiccation resistance Table 2. Analyses af variance for comparisons of D. serrata, D. mdanagasier and D. simulans populations d.f. Mean square
F
D. serrata Population Acclimation Pop. x acclimatior. Error
32
0.12 58.12 2.60 1.37
D. melanogaster Population Acclimation Pop. x acclimation Error
1 1 1 35
9.57 19.38 0.00 1.97
4.85’ 9.83** 0.00
1 I I
27.60 47.10 1.39 0.82
33.64*** 57.41*** 1.70
D. simulans Population Acclimation Pop. x acclimation Error
1 1
1
33
0.09
42.46*** 1.90
761
being more resistant than the Cairns population. The magnitude of the acclimation response was similar in the two populations as evident from the non-significant interaction term. D. simuluns. Results were similar to those obtained with D. melanogaster, acclimation increasing desiccation resistance and having a similar effect in both populations. Cairns was less resistant to desiccation than Melbourne (Table 1) in agreement with the D. melanogaster results. The non-significant interaction term indicates that geographic variation in desiccation resistance was not associated with variation in acclimation ability. In summary, these experiments demonstrate geographic variation for desiccation resistance in two of the three species, but there is no evidence for variation in acclimation ability in any of the species.
*P 4 0.05; **P < 0.01; **P < 0.001. DISCUSSION
D. birchii. The mean LT,, for the control treatment
was 8.11 h (SD = 0.95) while the mean LTSo for the acclimated flies was 8 55 h (SD = 0.54). These means did not differ significantly by a r-test (t = 1.27, d.f. = 18), so acclimation did not influence the desiccation resistance of this species. The experiment was carried out at the same time as the D. serrata population comparison (see below) and D. serruta had higher desiccation resistance as expected (Table 1). In summary, the experiments indicate that a prior desiccation stress increased resistance in three Drosophila species, but not in a fourth species found only in the humid tropics. Population comparisons D. serrata. This experiment tested for geographic variation in desiccati’on resistance and acclimation responses. Sex ratio did not have a significant effect on desiccation resistance. The population term was not significant in the ANOVA (Table 2), indicating that populations had similar overall desiccation resistance. The effect of acclimation was highly significant in the ANOVA ,and the means (Table 1) show that the desiccation resistance of both populations was increased by acclimation. The LT,, for the controls was higher than in the previous experiments with this species, and this may have reflected the lower temperature (24°C) at which this experiment was carried out. The interaction between population and acclimation was not significant, so the acclimation response of the two populations was similar and there was no evidenc,e for geographic variation in acclimation ability. D. melanogaster. The acclimation term was significant in the ANOVA (Table 2) and the desiccation resistance of both populations was increased by acclimation (Table 1). The significant population term indicates that populations differed overall in desiccation resistance, the Melbourne population
Many Drosophila species apparently have the ability to become acclimated to desiccation stress. The acclimation responses of the species tested so far have several common features. First, in all species including D. melanogaster (Hoffmann, 1990) a prior stress exposure increased desiccation resistance by 2@40%. Second, the acclimation responses of the species persisted for at least 24 h after the initial stress period. Finally, the degree of resistance increased with the length of time the flies were initially exposed to low humidity. Despite these similarities, there were large differences between species in the conditions producing an acclimation response. For example, the largest response occurred in D. simulans after 6 h at low humidity, whereas this acclimation period had only a small influence on the resistance of D. melanogaster compared to 9 h of prior desiccation (Hoffmann, 1990). There may be an association between resistance to desiccation and the length of time required to produce an acclimation response because large responses occurred after only a few hours desiccation in the least resistant species (D. immigrans, D. serrata).
The absence of an acciimation response in D. interesting, in light of the marked effect of acclimation in its sibling species D. serrata. Both species occur in the wet tropical rainforests of North Queensland and New Guinea (Ayala, 1965). However, D. birchii seems to be largely confined to the tropics whereas D. serrata is more widespread and can be collected from habitats well away from rainforests (Ayala, 1965; Parsons, 1982; M. Blows, personal communication). The ability of D. serrata to become acclimated may therefore represent an adaptation to drier conditions, enabling this species to colonize areas outside rainforests. This is consistent with the higher desiccation resistance of D. serrata compared to D. birchii. Interspecific variation birchii is particularly
762
ARY A. HOFFMANN
in desiccation resistance has previously been associated with habitat type in many Drosophila species (Parsons, 1983), but an association between acclimation responses and habitat type has not been considered. Additional comparisons of sibling species groups from different habitats would be of interest. While most of the species I tested are widespread, D. melanogaster and D. simulans have African relatives restricted to the tropics which are stress-sensitive (Stanley et al., 1980) and could be useful for such comparisons. The difference in desiccation resistance between the Cairns and Melbourne D. melanogaster populations is consistent with the results of Stanley and Parsons (1981) who found that D. melanogaster from Melbourne were more resistant than those from Townsville, a tropical location approx. 300 km south of Cairns. The D. simulans findings indicate that a similar difference in desiccation resistance exists in a sibling species. This geographic variation is probably adaptive because minimum and mean monthly humidities are much lower in Melbourne than in the tropics (Stanley and Parsons, 1981). The population comparisons for D. serrata, D. melanogaster and D. simulans provide no evidence for genetic differences in acclimation ability at the geographic level. It might be expected that populations of these species exposed to drier conditions would have a greater acclimation ability than populations from the tropics. One possible explanation for the lack of geographic differentiation in D. melanogaster and D. simulans is that the effects of selection for acclimation ability were not detected because genetic variation in desiccation resistance and acclimation ability are not independent. In previous experiments (Hoffmann, 1990) I found that D. melanogaster females from Melbourne lines successfully selected for increased desiccation resistance had a reduced acclimation response compared to females from unselected lines. This suggested that the response to selection and the acclimation response might involve a similar mechanism so that females from the selected lines always expressed a phenotype similar to that triggered by acclimation. On the basis of these results, the Cairns populations of D. melanogaster and D. simulans are expected to show a larger acclimation response than the Melbourne populations of these species because of the lower desiccation resistance of the Cairns populations. The similar acclimation responses of these populations might therefore reflect selection for increase acclimation ability in the Melbourne population. However, this argument assumes that findings based on variation in desiccation resistance within the Melbourne population can be extrapolated to variation in desiccation resistance between populations. Another explanation is that selection may only have favoured genes influencing desiccation resistance directly rather than genes influencing acclimation ability. Acclimation represents an example of phenotypic plasticity where the expression of a geno-
type is altered by the environment, and there are a number of reasons why such plastic changes might not be altered by selection whereas non-plastic changes in stress resistance are altered (Hoffmann and Parsons, 1991). These include the absence of much genetic variation for acclimation ability or costs associated with increased plasticity. Laboratory selection experiments on the Cairns stocks of D. simulans and D. melanogaster under conditions likely to favour plastic responses could provide a test of such possibilities, as well as providing data on the interaction between plastic and non-plastic changes in stress resistance. The mechanisms underlying acclimation to desiccation stress are not known. As discussed in Hoffmann (1990), possibilities include the induction of protective stress proteins, a reduction in metabolic rate, and changes in the composition of epicuticular lipids. It should be possible to measure physiological changes in acclimated flies to elucidate the importance of these factors. Acknowledgements-I am grateful to Zena Cacoyianni and Cameron Black for expert technical assistance. Steve McKechnie and Peter Parsons provided many useful comments on an earlier draft of this paper. REFERENCES
Arlian L. G. and Eekstrand I. A. (1975) Water balance in Drosophilapseudoobscura,and its ecological implications. Ann. en?. Sot. Am. 68, 827-832.
Ayala (1965)Sibling speciesof the Drosophila serrata group. Evolution 19, 5388545.
Hoffmann A. A. (1990) Acclimation for desiccation resistance in Drosophila melanogaster and the association between acclimation responses and genetic variation. J. Insect Physiol. 36, 885-891.
Hoffmann A. A. and Parsons P. A. (1991) Evolutionary Genetics and Environmental Stress. OUP, Oxford. Krafsur E. S. (1971) Behavior of thoracic spiracles of Aedes mosquitoes in controlled relative humidities. Ann ent. Sot. Am. 64, 93-102.
Levins R. (1969)Thermal acclimation and heat resistance in Drosophila species. Am. Nat. 103, 483-499. Miller P. L. (1964) Factors altering spiracle control in adult dragonflies: water balance. J. exp. Biol. 41, 331-343. Parsons P. A. (1982) Evolutionary ecology of Australian Drosophila. Evol. Biol. 14, 297-350.
Parsons P. A. (1983) The Evolutionary Biology of Colonizing Species. Cambridge University Press, New York. Sokal R. R. and Rohlf F. J. (1981) Biometrv. Freeman. New York. Stanley S. M. and Parsons P. A. (1981) The response of the cosmopolitan species, Drosophila melanogaster, to ecological gradients. Proc. Ecol. Sot. Aust. 11, 121-130. Stanley S. M., Parsons P. A., Spence G. E. and Weber L. (1980) Resistance of species of the Drosophila melanogaster subgroup to environmental extremes. Aust. J. Zool. 28, 413-421. Toolson E. C. (1982) Effects of rearing temperature on cuticle permeability and epicuticular lipid composition in Drosophila pseudoobscura. J. exp. Zool. 222. 249-253. Toolson- E. C. and Kuper-Simbron R. (1989) Laboratory evolution of epicuticular hydrocarbon composition and cuticular permeability in Drosophila pseudoobscura: effects on sexual dimorphism and- thermal acclimation ability. Evolution 43, 468472.
0022-1910/91 $3.00 + 0.00
Printed in Great Britain. ,411rights reserved
copyright 0 1991 Pergamon Press plc
ACCLIMATION FOR DESICCATION RESISTANCE I!~ROSOPHILA : SPECIES AND POPULATION COMPARISONS
IN
ARY A. HOFFMANN Department ol‘Genetics and Human Variation, La Trobe University, Bundoora, Victoria 3083, Australia (Received 23 April 1991; revised 3 June 1991)
Abstract-In previous work, it was found that Drosophila melanogaster females became more resistant to adesiccation after prior exposure to a non-lethal desiccation stress. Here, this acclimation response is shown to occur in three other widespread Drosophila species: D. simulans, D. immigrans and D. serrata. However, such acclimation was unsuccessful in D. birchii, a sib’ling species of D. serrata found in rainforest habitats. An acclimation response in all the widespread species was evident after a prior exposure period of a few hours, and acclimation was still evident after more than 24 h. Two D. serrata populations, one originating from a tropical location (Townsville) and one from a cooler coastal area (Forster) had a similar acclimation response. Although D. melanogaster and D. simulans populations originating from a tropical site (Cairns) were less resistant to desiccation than conspecific populations from a temperate site (Melbourne), the populations had similar acclimation responses. Hence, there was no evidence for geographic variation in acclimation ability. Key Word Index: Drosophila; acclimation;
desiccation; resistance; adaptation
INTRODUCTION
Insects may alter their levels of resistance to desiccation stress by becoming acclimated after prior exposure to a non-lethal stress period. Evidence for physiological changes likely to increase desiccation resistance has been found in a number of species (e.g. Miller, 1964; Krafsu.r, 1971; Toolson, 1982). More directly, the desiccation resistance of Drosophila melanogaster could be altered by prior exposure to a low humidity environment (Hoffmann, 1990). This response developed within 2 h of initial exposure to low humidity, and ;persisted more than 28 h after the prior stress period. There was also evidence for genetic variation in ,the acclimation response. Lines selected for resistance showed a markedly reduced acclimation response compared to unselected lines, presumably because the acclimation response and genetic variation for desiccation resistance were partly based on a similar mechanism. Dry periods of a few hours duration where humidity falls below 20% are regularly recorded in temperate localities where widespread Drosophila are collected, suggesting that acclimation responses of the type found in D. melanogaster may be of adaptive significance. If this is the case, then it is expected that acclimation ability will vary between Drosophila species depending cln the types of habitats they occupy. In particular, species from dry habitats might be expected to show a. greater acclimation ability than species restricted to rainforests where high relative
humidity is the normal expectation. Adaptation to dry habitats may also involve increased resistance although there are often costs involved with high levels of stress resistance (Hoffmann and Parsons, 1991). In a parallel way, differences in acclimation responses might be expected between populations of widespread species occupying a range of habitats. A number of Drosophila studies have demonstrated geographic variation in resistance to desiccation and other stresses, where population differences tend to match predictions based on climatic data (see Arlian and Eckstrand, 1975; Stanley and Parsons, 1981; Hoffmann and Parsons, 1991). However, there have been almost no attempts to examine geographic variation in acclimation ability in Drosophila with the exception of some work by Levins (1969) on heat resistance which showed interpopulation variation in some species. In this paper, I have tested these predictions by examining acclimation in four Drosophila species other than D. meIanogaster and by considering geographic variation in three of these species. The two cosmopolitan species D. immigrans and D. simulans were tested, as well as D. serrata which is more restricted rainforests
in its distribution.
D. serrata occurs
in
as well as in coastal areas of north-eastern Australia. I also examined acclimation in D. birchii, a sibling species of D. serrata largely restricted to tropical rainforests. It was predicted that D. birchii and to a lesser extent D. serrata would have low 757
758
ARY A. HOFFMANN
resistance and a reduced acclimation response. In the geographic comparisons, I examined populations of D. simulans and D. melanogaster originating from tropical and temperate locations, as well as D. serrata populations originating from a rainforest and a cooler coastal site. It was predicted that the tropical/ rainforest populations of these species would have relatively lower desiccation resistance and a reduced acclimation ability. MATERIALS
AND METHODS
Stocks and desiccation
The D. immigrans stock was established with the progeny of 25 females collected from a compost heap in Melbourne. The Melbourne D. simulans and D. melanogaster populations were started with the progeny of 25 (D. melanogaster) or 10 (D. simulans) females collected from an orchard. Cairns stocks of these species were established a month before the Melbourne stocks with progeny from 11 (D. simufans) or 25 (D. melanogaster) females collected from mangoes. It was important to initiate stocks at approximately the same time because acclimation responses may be altered during long-term laboratory culture (Toolson and Kuper-Simbron, 1989). The North Queensland D. serrata and D. birchii stocks were founded by the progeny of 16 and 20 females, respectively. These flies had been collected by fruit baiting in tropical rainforests. The 16 females which initiated the Forster D. serrata stock had been collected by fruit baiting in coastal vegetation between Forster and Seal Rocks in New South Wales, approx. 1600 km south of the tropical collection sites. This area marks the southern limit of the D. serrata distribution, while North Queensland is central in its distribution (Ayala, 1965). The two D. serrata populations were collected 2 months apart. All experiments were carried out within 15 months after stocks had been established in the laboratory. Stocks were maintained by mass transfer at 18-20°C. The culture medium for D. simulans, D. melanogaster and D. immigrans comprised of agar (3%), sucrose (9%) and dead yeast (6%) with propionic acid and methyl hydroxybenzoate as preservatives. Half the sucrose was replaced by instant potato mash for the D. serrata and D. birchii cultures. Adults to be used in the experiments were cultured at 24-25°C under continuous light. Humidity outside the culture bottles was 40-60%. Flies were collected from bottles when they were O-2 days old and aged (sexes mixed) in vials with laboratory medium at a density of 30-50 flies per vial prior to acclimation or desiccation. The ageing and sexing procedure differed for the species as detailed below. Flies were acclimated and tested for stress resistance in glass desiccators containing silica gel. Most experiments involved desiccation at 25°C except for the D. birchii experiment and the D. serrata population comparison (see below) which were carried out
at 24°C. Humidity in the desiccators declines to 8-10% after 1 h and reduces to 3-5% in about 3 h. Flies were tested in groups of 20 which were acclimated and desiccated in empty glass vials covered with gauze. To determine stress resistance, vials were arranged around the perimeter of a desiccator so that mortality in the vials could be monitored without opening the desiccator. The time taken for 50% of the flies to die (LT,) was scored by counting the number of dead flies at 1 h intervals. The 50% mark was linearly interpolated when more than half the flies had died. Acclimation of dtflerent species D. immigrans. Flies were aged for 3 days before they were sexed under carbon dioxide anaesthesia, and held another 2 days before acclimation. Preliminary experiments indicated that D. immigrans females could be desiccated for 4 h before mortality started to occur, so females were acclimated for 3 or 4 h, and allowed to recover for 9 h. Eight replicate vials were set up for each treatment. The persistence of the acclimation effect was examined by desiccating females for 4 h and holding them for 5, 8, 29 or 53 h on laboratory medium before testing desiccation resistance. Flies which had not been acclimated served as controls. In addition, I tested females which had been desiccated for 4 h but were only given a 5 h recovery period. Flies for all treatments were aged for 2 days before sexing, so that they were 5-6 days old when they were tested for desiccation resistance. Ten replicates were set up per treatment. D. simulans. Previous experiments with D. melanogaster females indicated that the effect of acclimation increases with the length of the prior exposure period to low humidity (Hoffmann, 1990). This association was also examined in D. simulans females from the Melbourne stock. Flies were aged in vials for 3 days and females were separated by aspiration and aged in vials for another 1 day before exposure to the acclimation treatments. Females were acclimated for 3, 4, 5 and 6 h. They were left to recover for 9 h on laboratory medium prior to being tested for desiccation resistance. A control treatment where flies were not exposed to a prior stress was also included. Females were not acclimated for longer than 6 h to avoid mortality, reflecting the lower desiccation resistance of this species compared to D. melanogaster (Stanley et al., 1980). Five replicate vials were set up for each treatment. The persistence of the acclimation response was tested with females from the Melbourne stock. Flies were acclimated for 6 h, and held for 4, 9, 18, 24 and 48 h on laboratory medium before they were tested for desiccation resistence. Controls which had not been acclimated were also included. Five replicates per treatment were set up. D. serrata. The effects of different prior desiccation periods were examined using the Forster stock. Flies
Acclimation for desiccation resistance were aged (sexes mixed) for 5 days after eclosion. Because the sexes of D. serrata cannot be easily separated by aspiration, flies were sexed under carbon dioxide anaesthesia, and aged another day before acclimation. Flies were aged longer than D. melanogaster and D. simulans because D. serrata takes longer to reach sexual maturity (l-2 days after eclosion) and peak fecundity (7-12 days after eclosion) when they are ktept at 25°C (M. Blows, personal communication) than D. melanogaster and D. simuZans which mate less than 12 h after eclosion and reach peak fecundity after 34 days. Females were acclimated for 2, 3 and 4 h and an unacclimated control was also tested. The 4 h period corresponds to the maximal time that D. serrata can be desiccated before mortality starts to occur. Males were also tested for acclimation, using a 3 h prior desiccation period. Flies were allowed to recover for 9 h on laboratory medium before they were tested for stress resistance. Ten replicates were set up for each treatment. To test for persistence of the acclimation response, females from the Forster stock were acclimated for 4 h and then held on. laboratory medium for 18 or 24 h prior to testing for desiccation resistance. Controls which were not acclimated were also included. Twelve replicates were set up for the acclimation treatments, and 8 replicates for the controls. D. birchii. Preliminary experiments indicated that this species was more sensitive to desiccation than D. serrata because some mortality was already evident when females were desiccated for 4 h. The effect of a 3.5 h prior desiccation period on stress resistance was therefore compared to a non-acclimated control. Flies were treated prior to desiccation as described above for D. serrata. Ten replicate vials were set up for the acclimated and non-acclimated treatments. This experiment was set up at the same time as the D. serrata population comparison. Population comparisoiw D. serrata. Populations of this species from Cairns and Forster were compared for acclimation responses. In contrast to the above experiments with this species, flies were not sexed prior to acclimation to avoid the use of anaesthesia. This is unlikely to have much influence on the results because the earlier experiments had indicated that the sexes had similar resistance to desiccation and showed a similar acclimation response (see below). Nevertheless, the sex ratio of each vial was determined after desiccation to see if this variable influenced resistance. The resistance of flies acclimated for 3.5 h (with a 9 h recovery period) was compared with the resistance of non-acclimated flies. The experimental design (ignoring sex ratio as a covariate) is given by
Yijk= u + b, +
cl+
bc, + e,(i))t
(1)
where bi is the effect due to population, c, the effect due to acclimation, bc,, the acclimation by population
759
interaction and e,,,, the error term. Ten replicate vials with 20 flies were set up for each treatment. D. melanogaster and D. simulans. Acclimation responses of the Cairns and Melbourne populations of these species were compared by testing acclimated and non-acclimated females from each population. Flies were sexed by aspiration and treated as in the D. simuians experiments. D. simulans females were acclimated for 6 h and were allowed to recover for 6 h on laboratory medium. D. meianogaster females were acclimated for 8 h and allowed to recover for 6 h. This recovery period is shorter than the normal 9 h period, but previous experiments (Hoffman, 1990) indicated that this reduction would not adversely affect the acclimation response. The experimental design follows equation (1) and there were 8-10 replicates per treatment. RESULTS
Acclimation in different species D. immigrans. Mean LT,,s for the treatments were initially compared with a one-way analysis of variance (ANOVA) which indicated a significant effect of treatment (early desiccation exposure time) on desiccation resistance [F(,,, = 20.45, P < O.OOl]. The significance of differences between pairs of means was determined by computing the least significant difference as outlined in Sokal and Rohlf (1981, p. 244). The mean LT, for the control treatment (2 = 12.53, SD = 0.62 h) differed significantly from the means for the 3 h (2 = 13.69, SD = 1.04 h) and 4 h (2 = 15.51, SD = 1.09 h) treatment. Acclimation therefore increased the desiccation resistance of the females, the 4 h treatment increasing resistance (LT,,) by 24% of the control treatment. In the experiment testing the persistence of the acclimation response, treatment means differed significantly in an ANOVA [F& = 10.50, P < O.OOl] and acclimated treatments had a higher desiccation resistance than controls (Fig. 1). Comparisons of pairs of means after computing the least significant
20
Tim-
on
lab
mrdlum
(hourr)
Fig. 1. Persistence of the acclimation response of D. immigram females after holding them on laboratory medium following acclimation. Error bars represent upper 95% confidence limits of the samples.
ARY A. HOFFMANN
760
_ Acclimation
T
T
5
6
I
_ Period
i 3 Afclimatlo”
(hours)
Period
(hours)
Fig. 2. Effect of acclimation period on the desiccation resistance of D. simulans females. Error bars are upper 95% confidence limits.
Fig. 4. Effect of acclimation period on the desiccation resistance of D. serrata females. Error bars are upper 95% confidence limits.
indicated that only the 53 h treatment did not differ significantly from the controls. The acclimation response therefore persisted for at least 29 h, but was reduced after 29 h compared to after 8 h. The 5 h recovery period was sufficient to produce an acclimation response. D.simulans. Mean LT,,s indicate that acclimation increased desiccation resistance, the magnitude of the effect increasing with the length of the prior exposure period (Fig. 2). Treatments differed significantly by an ANOVA [ Fc4,2,,j = 12.44, P < O.OOl].The 6 h treatment increased resistance by more than 4 h or 40% of the control resistance. All four acclimation treatments differed significantly from the controls by a least significant difference analysis, so that desiccation resistance was increased even when flies were exposed to low humidity for 3 h. A linear regression analysis was carried out using the means for each prior desiccation period to predict LT,,: the fitted equation (LT,, = 0.65 prior desiccation period + 10.76) accounted for a significant proportion of the variance (P < 0.05) in mean LT,, . The acclimation response declined with the length of time flies were held on laboratory medium (Fig. 3). Treatments differed significantly by an ANOVA and a least significant KS.24)= 9.67, P < O.OOl),
except the 48 h period differed significantly from the controls. Acclimation therefore persisted for more than 24h and a recovery period of 4 h or more was sufficient to produce an acclimation response. D. serrata. The treatments differed significantly by
difference
difference
analysis
indicated
that
all
treatments
an ANOVA [FC3,361 = 10.98, P < 0.001). Each of the acclimation treatments differed significantly from the controls by a least significant difference analysis. The 4 h acclimation period increased resistance by 2.5 h or 26% of the control resistance, while the 2 h treatment produced a smaller acclimation response (Fig. 4). The desiccation resistance of males was also increased by a 3 h acclimation period; the mean LT, for the controls was 7.86 h (SD = 0.3 1) compared to a mean of 9.52 h (SD = 6.33) for the 3 h treatment. These means differ significantly by a r-test (t = 7.46, d.f. = 18, P < 0.001). The resistance of both sexes was therefore increased after acclimation. Acclimation was detected even after females had been held on laboratory medium for 18 and 24 h after acclimation. These recovery periods differed significantly by an ANOVA [FC2,29j = 7.52, P < O.OOl].The mean LT,, for the control treatment (2 = 8.42, SD = 0.45 h) differed significantly from means for the 18 h (2 = 10.19, SD= 0.78 h) and 24h (2 = 9.31, SD = 1.14 h) periods as determined by a least significant difference analysis.
Table 1. Mean LT,,s for acclimated and non-acclimated flies from populations of D. serrata, D. melanogaster and D. simulans
Acclimated
Non-acclimated
zz
SD
2
SD
13.50 14.14
1.41 0.92
11.64 11.21
1.29 1.17
15.84 16.82
1.12 1.87
14.41 15.42
1.57 0.84
12.80 14.14
0.75 0.89
10.14 12.27
0.74 1.23
D. serrata
Townsville Forster D. melanogaster Time
on
Iaib
medium
(hours)
Fig. 3. Persistence of the acclimation response of D. simulans females after holding them on laboratory medium following acclimation. Error bars represent upper 95% confidence limits.
Cairns Melbourne D. simulans
Cairns Melbourne
Acclimation for desiccation resistance Table 2. Analyses af variance for comparisons of D. serrata, D. mdanagasier and D. simulans populations d.f. Mean square
F
D. serrata Population Acclimation Pop. x acclimatior. Error
32
0.12 58.12 2.60 1.37
D. melanogaster Population Acclimation Pop. x acclimation Error
1 1 1 35
9.57 19.38 0.00 1.97
4.85’ 9.83** 0.00
1 I I
27.60 47.10 1.39 0.82
33.64*** 57.41*** 1.70
D. simulans Population Acclimation Pop. x acclimation Error
1 1
1
33
0.09
42.46*** 1.90
761
being more resistant than the Cairns population. The magnitude of the acclimation response was similar in the two populations as evident from the non-significant interaction term. D. simuluns. Results were similar to those obtained with D. melanogaster, acclimation increasing desiccation resistance and having a similar effect in both populations. Cairns was less resistant to desiccation than Melbourne (Table 1) in agreement with the D. melanogaster results. The non-significant interaction term indicates that geographic variation in desiccation resistance was not associated with variation in acclimation ability. In summary, these experiments demonstrate geographic variation for desiccation resistance in two of the three species, but there is no evidence for variation in acclimation ability in any of the species.
*P 4 0.05; **P < 0.01; **P < 0.001. DISCUSSION
D. birchii. The mean LT,, for the control treatment
was 8.11 h (SD = 0.95) while the mean LTSo for the acclimated flies was 8 55 h (SD = 0.54). These means did not differ significantly by a r-test (t = 1.27, d.f. = 18), so acclimation did not influence the desiccation resistance of this species. The experiment was carried out at the same time as the D. serrata population comparison (see below) and D. serruta had higher desiccation resistance as expected (Table 1). In summary, the experiments indicate that a prior desiccation stress increased resistance in three Drosophila species, but not in a fourth species found only in the humid tropics. Population comparisons D. serrata. This experiment tested for geographic variation in desiccati’on resistance and acclimation responses. Sex ratio did not have a significant effect on desiccation resistance. The population term was not significant in the ANOVA (Table 2), indicating that populations had similar overall desiccation resistance. The effect of acclimation was highly significant in the ANOVA ,and the means (Table 1) show that the desiccation resistance of both populations was increased by acclimation. The LT,, for the controls was higher than in the previous experiments with this species, and this may have reflected the lower temperature (24°C) at which this experiment was carried out. The interaction between population and acclimation was not significant, so the acclimation response of the two populations was similar and there was no evidenc,e for geographic variation in acclimation ability. D. melanogaster. The acclimation term was significant in the ANOVA (Table 2) and the desiccation resistance of both populations was increased by acclimation (Table 1). The significant population term indicates that populations differed overall in desiccation resistance, the Melbourne population
Many Drosophila species apparently have the ability to become acclimated to desiccation stress. The acclimation responses of the species tested so far have several common features. First, in all species including D. melanogaster (Hoffmann, 1990) a prior stress exposure increased desiccation resistance by 2@40%. Second, the acclimation responses of the species persisted for at least 24 h after the initial stress period. Finally, the degree of resistance increased with the length of time the flies were initially exposed to low humidity. Despite these similarities, there were large differences between species in the conditions producing an acclimation response. For example, the largest response occurred in D. simulans after 6 h at low humidity, whereas this acclimation period had only a small influence on the resistance of D. melanogaster compared to 9 h of prior desiccation (Hoffmann, 1990). There may be an association between resistance to desiccation and the length of time required to produce an acclimation response because large responses occurred after only a few hours desiccation in the least resistant species (D. immigrans, D. serrata).
The absence of an acciimation response in D. interesting, in light of the marked effect of acclimation in its sibling species D. serrata. Both species occur in the wet tropical rainforests of North Queensland and New Guinea (Ayala, 1965). However, D. birchii seems to be largely confined to the tropics whereas D. serrata is more widespread and can be collected from habitats well away from rainforests (Ayala, 1965; Parsons, 1982; M. Blows, personal communication). The ability of D. serrata to become acclimated may therefore represent an adaptation to drier conditions, enabling this species to colonize areas outside rainforests. This is consistent with the higher desiccation resistance of D. serrata compared to D. birchii. Interspecific variation birchii is particularly
762
ARY A. HOFFMANN
in desiccation resistance has previously been associated with habitat type in many Drosophila species (Parsons, 1983), but an association between acclimation responses and habitat type has not been considered. Additional comparisons of sibling species groups from different habitats would be of interest. While most of the species I tested are widespread, D. melanogaster and D. simulans have African relatives restricted to the tropics which are stress-sensitive (Stanley et al., 1980) and could be useful for such comparisons. The difference in desiccation resistance between the Cairns and Melbourne D. melanogaster populations is consistent with the results of Stanley and Parsons (1981) who found that D. melanogaster from Melbourne were more resistant than those from Townsville, a tropical location approx. 300 km south of Cairns. The D. simulans findings indicate that a similar difference in desiccation resistance exists in a sibling species. This geographic variation is probably adaptive because minimum and mean monthly humidities are much lower in Melbourne than in the tropics (Stanley and Parsons, 1981). The population comparisons for D. serrata, D. melanogaster and D. simulans provide no evidence for genetic differences in acclimation ability at the geographic level. It might be expected that populations of these species exposed to drier conditions would have a greater acclimation ability than populations from the tropics. One possible explanation for the lack of geographic differentiation in D. melanogaster and D. simulans is that the effects of selection for acclimation ability were not detected because genetic variation in desiccation resistance and acclimation ability are not independent. In previous experiments (Hoffmann, 1990) I found that D. melanogaster females from Melbourne lines successfully selected for increased desiccation resistance had a reduced acclimation response compared to females from unselected lines. This suggested that the response to selection and the acclimation response might involve a similar mechanism so that females from the selected lines always expressed a phenotype similar to that triggered by acclimation. On the basis of these results, the Cairns populations of D. melanogaster and D. simulans are expected to show a larger acclimation response than the Melbourne populations of these species because of the lower desiccation resistance of the Cairns populations. The similar acclimation responses of these populations might therefore reflect selection for increase acclimation ability in the Melbourne population. However, this argument assumes that findings based on variation in desiccation resistance within the Melbourne population can be extrapolated to variation in desiccation resistance between populations. Another explanation is that selection may only have favoured genes influencing desiccation resistance directly rather than genes influencing acclimation ability. Acclimation represents an example of phenotypic plasticity where the expression of a geno-
type is altered by the environment, and there are a number of reasons why such plastic changes might not be altered by selection whereas non-plastic changes in stress resistance are altered (Hoffmann and Parsons, 1991). These include the absence of much genetic variation for acclimation ability or costs associated with increased plasticity. Laboratory selection experiments on the Cairns stocks of D. simulans and D. melanogaster under conditions likely to favour plastic responses could provide a test of such possibilities, as well as providing data on the interaction between plastic and non-plastic changes in stress resistance. The mechanisms underlying acclimation to desiccation stress are not known. As discussed in Hoffmann (1990), possibilities include the induction of protective stress proteins, a reduction in metabolic rate, and changes in the composition of epicuticular lipids. It should be possible to measure physiological changes in acclimated flies to elucidate the importance of these factors. Acknowledgements-I am grateful to Zena Cacoyianni and Cameron Black for expert technical assistance. Steve McKechnie and Peter Parsons provided many useful comments on an earlier draft of this paper. REFERENCES
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Hoffmann A. A. and Parsons P. A. (1991) Evolutionary Genetics and Environmental Stress. OUP, Oxford. Krafsur E. S. (1971) Behavior of thoracic spiracles of Aedes mosquitoes in controlled relative humidities. Ann ent. Sot. Am. 64, 93-102.
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Parsons P. A. (1983) The Evolutionary Biology of Colonizing Species. Cambridge University Press, New York. Sokal R. R. and Rohlf F. J. (1981) Biometrv. Freeman. New York. Stanley S. M. and Parsons P. A. (1981) The response of the cosmopolitan species, Drosophila melanogaster, to ecological gradients. Proc. Ecol. Sot. Aust. 11, 121-130. Stanley S. M., Parsons P. A., Spence G. E. and Weber L. (1980) Resistance of species of the Drosophila melanogaster subgroup to environmental extremes. Aust. J. Zool. 28, 413-421. Toolson E. C. (1982) Effects of rearing temperature on cuticle permeability and epicuticular lipid composition in Drosophila pseudoobscura. J. exp. Zool. 222. 249-253. Toolson- E. C. and Kuper-Simbron R. (1989) Laboratory evolution of epicuticular hydrocarbon composition and cuticular permeability in Drosophila pseudoobscura: effects on sexual dimorphism and- thermal acclimation ability. Evolution 43, 468472.