Resistance to heat and cold stress in Drosophila melanogaster: intra and inter population variation in relation to climate

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Resistance to heat and cold stress in Drosophila

melanogaster: intra and inter population variation in

relation to climate

D Guerra, S Cavicchi, Ra Krebs, V Loeschcke

To cite this version:

Original

article

Resistance

to

heat and cold

stress

in

_Drosophila

_{melanogaster:}

intra and inter

_population

variation

in relation

to

climate

D

Guerra

1

S Cavicchi

RA Krebs

2 V

Loeschcke

3

1

Di

P

artimento

di

_Biologia

evoluzionistica

s

P

erimentale,

Universita di

_Botogna,

via Selmi 3,

40126-Bologna, Italy;

2

Department of Organismal Biology

and

_Anatomy,

The

_{University of Chicago,}

1027 East 57th

_{Street, Chicago,}

IL 60637,

USA;

3

Department of Ecology

and

Genetics, University of Aarhus, Ny Munkegade,

Bldg

540 DK - 8000 Aarhus

C,

Denmark

(Received

13 June 1996;

accepted

9 June

₁₉₉₇₎

Summary - Genetic variation for resistance to

high

and low temperature stress and

wing

size was examined within and _among four

_{Drosophila melanogaster populations}

from

temperate

(Denmark

and

_Italy)

and

_subtropical

areas

_(Canary

Islands and

_Mali).

The

temperature of induction of the heat shock response was examined

_{by conditioning}

flies

to different

_high

temperatures in the range 34 to 40°C

_prior

to

_exposing

them to heat shock

_(41.5°C

for 0.5

_h).

Stress resistance

_appeared

to be related to climate:

_populations

from warm

_regions

were the most heat tolerant and those from cold

_regions

were the

most cold tolerant. This trend suggests that natural selection in the wild at non-extreme temperature can lead to a correlated response in tolerance to extreme temperature.

Wing

size varied

significantly,

and

generally

was

_larger

for flies from more

_northerly

populations. Populations

varied

_genetically

in all traits measured.

_{Among traits,}

a

_positive

correlation was _present between heat-shock resistance with

_conditioning

and resistance to

cold,

and the correlation was

_suggestive

between heat-shock resistance with and without a

conditioning

treatment, but no correlation was indicated between cold resistance and heat

resistance of non-conditioned individuals.

_Wing

size was not correlated with any stress type. The results _suggest that different groups of genes are involved in the resistance at extreme _temperature ranges.

acclimation

_/

heat shock resistance

_/

cold shock resistance

_/

_wing size

Résumé - Résistance au stress de chaleur et de froid chez Drosophila melanogaster : variation entre et intrapopulations en fonction du climat. La variation

_génétique

_pour

la résistance au stress à haute ou basse

_température

et la taille de l’aile ont été examinées

*

dans quatre

populations

de

Drosophila melanogaster

provenant des

_{régions tempérées}

(Danemark

et

_Italie)

et

_{subtropicales}

_(îles

Canaries et

_Mali).

La

_température

d’induction de la

_réponse

au choc

_thermique

a été examinée

après

conditionnement à

températures

différentes

(de

34

à

_!,0

°

_C)

avant le traitement _proprement dit

_{(41.5 ’}

° C

pendant

30

_min).

La résistance au stress est en relation avec le cLimat : les

_populations

des

régions

chaudes

montrent la

_{plus grande}

résistance à la chaleur et celles des

_{régions froides,}

la

_{plus grande}

résistance au

froid.

Ce résultat

_suggère

_que la sélection naturelle dans un milieu

_tempéré

peut amener à une

réponse

corrélée _pour la tolérance au stress

_thermique.

On a observé une

variation

_{significative}

de la taille de

_l’aile,

_{qui augmente} avec la latitude. Une variabilité

génétique

pour tous les caractères considérés a été aussi mise en évidence dans toutes

les

populations.

La résistance à la chaleur

après

conditionnement a été en corrélation positive avec la résistance au

froid

et une corrélation _presque

significative

a été trouvée

entre mouches conditionnées et non _pour la résistance au choc

_thermique.

D’un autre

côté,

on n’a _pas trouvé de corrélation entre la résistance à la chaleur et la résistance au

froid

chez les mouches non conditionnées. La taille de l’aile n’a été corrélée avec aucun

stress

_thermique.

Les résultats

_suggèrent

que des groupes

différents

de

_gènes

contrôlent la résistance à

différentes températures

extrêmes.

climatisation

_/

résistance à la chaleur

_/

résistance au froid

_/

taille de l’aile

INTRODUCTION

Variation in resistance to environmental stress has been observed among related

species

and

_populations

of

_Drosophila

from

_climatically

different

_regions,

particu-larly

for heat

_(Hosgood

and

_Parsons,

_1968;

_{Parsons, 1979;}

_Coyne

et

_al,

_1983),

and cold shock resistance

_(Jefferson

et

_{al, 1974; Tucic, 1979;}

Marinkovic et

_{al, 1980;}

Kimura,

1982;

Fukatami, 1984;

Heino and

_{Lumme, 1989;}

Hoffmann and

_Watson,

1993),

and this variation _appears to be an

_evolutionary

_response to the

environ-ment

_(Hoffmann

and

_Parsons,

_1991;

Loeschcke et

_al,

_1994).

Success in

_selecting

for stress resistance indicates that a

_significant

additive

_genetic

_component

also

is

_present

within

_populations

_(Morrison

and

Milkman, 1978;

Kilias and

Alahio-tis, 1985;

Quintana

and

_Prevosti,

1990

_b;

Jenkins and

_Hoffmann,

_1994;

Krebs and

Loeschcke,

1996).

Maintenance of

_Drosophila

_populations

at different

_temperatures

in the

labora-tory

indicates that

adaptation

to non- extreme

_temperatures

_may

_yield

correlated

responses to tolerance to extreme

_high

_temperatures

_(Stephanou

and

_Alahiotis,

1983; Huey

et

_al,

_1991,

Cavicchi et

_al,

_1995),

and that these correlated effects

in-clude

_changes

in the induction of the heat shock response

(Cavicchi

et

al,

1995).

Conditioning

individuals with a short _exposure to

_high

_temperatures

before heat shock increases resistance relative to that without a

_conditioning

_treatment,

and

multiple

treatments _may increase survival more than a

_single

treatment

_(Loeschcke

et

_{al, 1994;}

Krebs and

_Loeschcke,

_1995).

The molecular basis of the

_regulation

of the heat shock response

(Maresca

and

Lindquist, 1991;

Morimoto et

_{al, 1990,}

_1994),

which occurs across all

_kingdoms

of life

(Landry

et

_al,

_1982;

_{Vierling, 1991;}

Parsell and

_Lindquist,

_1994),

_provides

a link between

_conditioning

treatments that induce heat shock

protein

production

and those

_increasing

survival under thermal stress

or other stress

_types

_(Landry

et

_{al, 1982;}

_Lindquist,

_1986;

_Brown,

_1991).

Our aim was to

_identify

if this resistance relates to the climate of the localities of

origin.

If so, natural selection in the wild at non-extreme

_temperature

has led to

a

_genetically

correlated _{response in} tolerance to extreme

_temperature.

The ques-tion of

_general

interest is: does selection for increased fitness at a

_given

_range of

temperature

lead to a

genetically

correlated _{response in} the resistance to extreme

temperature

close to the

_optimum?

If _so, are the same or different _groups of _genes

involved in the

_adaptation

to the

_optimum

_and/or

to either hot or cold

tempera-ture extremes

_(Huey

and

_Kingsolver,

_1993)?

Our

_previous

works on chromosomal

analysis

of

_{laboratory populations}

of

_{Drosophila adapted}

to different

_temperatures

(Cavicchi

et

al, 1989,

1995)

showed that the genes

responsible

for

adaptation

to

intermediate

_temperature

are located on chromosomes different from those

control-ling

survivorship

at extreme

_heat,

_although

heat resistance evolved as a correlated

response to natural selection at non-extreme

temperature.

Does the same

relation-ship

occur in natural

_populations

from different climatic areas? Here we

_analysed

the

_survivorship

of

_genotypes

from different

_populations

at

high

and low

temper-ature extremes. The correlation between

_performances

of different isofemale lines

could be a useful tool to assess whether the same or different

_evolutionary

mecha-nisms are at work in the

_laboratory

and in the wild.

Because

_phenotypic

differences in

_body

size _may have

_impact

on resistance to

temperature

extremes

_(Quintana

and

_Prevosti,

_1990a;

Loeschcke et

_al,

_1994),

_wing

size variation _among

_populations

was

_compared

and the correlation with stress

resistance

_analysed.

Size variations _may not be

easily separated

from variation

in

_resistance,

as

_geographical

clines for

_body

size follow

_temperature

_gradients

in several

_{Drosophila species}

_(Stalker

and

_Carson,

_{1947; Prevosti,}

_1955;

Misra and

_{Reeve, 1964;}

David et

_al,

_1977;

David and

_Capy,

_1988;

_Capy

et

_{al, 1993;}

Imasheva et

_al,

_1994).

A

_genetic

and

_{phenotypic relationship}

between

_body

size and

_temperature

also has been shown in the

_laboratory

_(Anderson,

_1973;

Cavicchi

et

_al,

_1985,

_1989),

where adult

_body

size

_negatively

correlated with

_temperature

(Starmer

and

_{Wolf, 1989; Thomas,}

_1993),

except at

_temperatures

_approaching

the limit for

_development

_(David

et

_al,

_1994).

MATERIALS AND METHODS

Origin of populations

The founder

_populations

derived from 50-100 females of D

_{rnelanogaster}

collected in

nature from

_Hov,

Denmark in late

_{October, 1992;}

from

_{Bologna, Italy}

in

_October,

1993;

from southwestern

_{Teneriffe, Canary Islands;}

and from

Bamako,

southern Mali in

_December,

1993. Table I describes differences in thermal extremes for each

region.

Single

females were

_put

in vials. From those identified as

_{melanogaster,}

ten

isofemale lines for each

_population

were established. The lines were reared in bottles with discrete

_{generations, avoiding}

_{overcrowding.}

Mass

_populations

were obtained

by pooling

lines of each

_population

_{in cages} with

_{overlapping generations.}

Flies were

maintained on a medium of

_yeast,

sugar, cornmeal and agar at 25°C.

Experiments

Heat resistance and induced thermotolerance

Flies were heat shocked

_using

the

_{procedures adopted}

in

_{previous experiments}

(Cavicchi

et

_al,

_1995).

Males and females were collected

_using

ether anaesthesia

and

partitioned

into about 50 flies per vial. Females and males were considered

together because,

under our

_{experimental conditions,}

_they

survived

_similarly

in

replicated experiments

at different shock

_{temperatures.}

Flies were restrained at the bottom of

_weighted

_plastic

vials

_(without

_food)

_by

_sponge

_plugs

and were shocked in a water bath at 41.5°C for 30 min. Care was taken to treat

_only

_4-7-day-old

flies as

resistance declines in older individuals

_(Quintana

and

_{Prevosti, 1990b;}

_Dahlgaard

et

_al,

_1995).

_During

_treatment,

_humidity

was not controlled within

vials,

but the

water bath was a saturated

_humidity

environment that minimised _any desiccation effects

_(Maynard

Smith,

1956;

Hoffmann and

_Parsons,

_1989).

_Following

heat

_shock,

flies were transferred to new vials

_{containing food,}

and

_survivorship

was scored 24 h later. As almost all individuals were

_{knocked-down,}

_survivorship

was taken as

the

_proportion

of individuals that reacted when touched with

_forceps.

Heat shock

was

_applied

both on mass

_populations

and on the individual isofemale lines. For

comparing populations,

three

_replicate

measurements were obtained in each of two

independent

blocks. For isofemale

_lines,

two

replicates

were

subjected

to the heat

treatment,

but, owing

to the bath

_size,

at different times for various

_populations.

Data were arcsin transformed before statistical

_analysis.

To determine differences _among the four mass

_populations

for the threshold

tem-perature

that induces

_{thermotolerance,}

two

replicates

of 50 flies each in one or two

independent

blocks were conditioned for 5 min at one of a

_graded

series of

temper-atures

_ranging

from 34 to

_40°C,

returned to 25°C for 0.5 h and then heat shocked

as described

(Cavicchi

et

_al,

_1995).

As

_only

two

_conditioning

_temperatures

could be tested _{at any one}

_time,

non-conditioned control flies from each mass

_population

were also

_{simultaneously}

heat shocked.

_Therefore,

induction of thermotolerance was

measured for each

_population

as the difference between the

_proportion

of flies that

survived heat shock with

_conditioning

in each

_replicate

and the mean for each

pop-ulation that survived without

_{conditioning.}

A total of 44 vials were non-conditioned

(12

for Mali and

_Denmark,

10 for

_Canary

Islands and

_Bologna)

while 78 vials were

the

_Canary

Island and Danish

_populations

therefore were first

_exposed

to a

_slightly

lower

_temperature

_(36°C)

than those from Mali or

Bologna

(38°C).

In this case

also,

the

_{preconditioning}

and heat shock treatments were

performed separately

for

each

_population.

Cold resistance

Flies both from mass

populations

and isofemale lines were

subjected

to cold

treatment of 0°C for 48 h in a thermostatic chamber with saturated

humidity.

The

initial

_temperature

was

_20-22°C,

and the

_temperature

declined to 0°C in about 15 min. As for heat

shock,

about 50 flies were

_placed

in

_empty

_plastic

vials. Two

replicates

in three blocks were treated for

_{comparing populations.}

For

_comparing

lines,

two

_replicates

for each isofemale line were cold shocked at the same

_time,

while,

as for heat

shock,

various

_populations

were treated at different times.

_Again,

resistance was scored as the

_proportion

of flies

_reacting

when touched with

forceps

and the data were arcsin transformed before statistical

analysis.

Wing

size

After

_rearing

individuals of each isofemale line in uncrowded

conditions,

the

_right

wing

of five females per line was removed and mounted on

slides,

from which

_wing

area was measured

_(MTV3

_program of Data Crunch

_Corporation,

South

_Clemente,

CA).

The overall mean was taken as the

population

mean size.

Statistical

_analysis

In the first

_experiment,

_significance

of

_population

differences for heat

_resistance,

cold resistance

_(after

arcsin

_{transformation)}

and

_wing

size was tested

_by

Anova

and a

_{posteriori hypotheses}

of

_pair-wise

differences were examined

_{using Tukey’s}

multiple

comparisons

test.

_Significance

_{of differences among}

_populations

and

differ-ent acclimatization treatments in the second

_experiment

was tested in a

_two-way

fixed effects Anova

_{(SAS, 1989).}

Intraclass correlations

_(t)

were derived from Anova

only

for

_wing

size. For

survivorship,

which is a threshold

_trait,

we followed the method

_proposed

_by

Robertson and Lerner

₍₁₉₄₉₎

in which:

where x

2

is the

_{heterogeneity}

in the 2 x N

_table,

as flies can be classified

only

as

dead or

_alive,

N is the number of isofemale lines and

where n is the number of flies heat or cold shocked for each isofemale line. In

our

_experiment,

N = 10 isofemale lines and _{n >} 50 flies for each

line, averaging

_two

The observed variance in binomial data is correlated with the mean. Hence

correction for

_comparing

intraclass correlations from different treatments and

populations

can be made

_{by transforming}

t on the

_probit

scale

_by

_multiplying

t by

where

_p

is the fraction which survives

_{(or dies)}

and z is the ordinate of the normal

curve at the

_point

where the tail area is

_equal

to

_p.

Standard errors of intraclass correlations were

computed

_following

Falconer

(1989)

for

_wing

size and Fisher

₍₁₉₄₁₎

for

_{survivorships.}

Overall t values were

reported

on the basis of a

pooling

procedure

both for stress

resistances and

_wing

size.

Standard

_parametric

correlation coefficients _among the four traits were obtained

for each

_{population using}

the mean stress resistance

_(after

arcsin

_{transformation)}

or size of each line. Overall correlations also were

_reported

on the basis of the

_pooled

variance-covariance matrix.

RESULTS

Interpopulation analysis

Mean

_survivorship

_(%)

for each

population

following

either a heat or cold

_treatment,

and mean

_wing

size of females are

_presented

in table II

_(rows

identified

_by

No

_1).

For cold

_resistance,

_{variation among} blocks was

significant

(P

<

_0.01).

For neither heat

nor cold shock was the

_{population by}

block interaction

_significant.

Variation due to

the

_origin

of

_populations

was

_{highly significant}

for all three traits

_(P

<

_0.001),

and

two

_by

two

_comparisons

_(Tukey’s

_{multiple comparisons}

_test)

revealed

_significant

differences between

_geographic

areas

(table II).

Heat resistance was

_higher

for flies

from the

_Canary

Islands and from Mali than for flies from Denmark and

_Italy;

while cold resistance was

_highest

for flies from

_Italy,

followed

_by

those from

_Denmark,

Mali and the

_Canary

_{Islands, respectively,}

_{although significance}

levels

_overlapped

between some

populations.

Wing

size was

significantly

_larger

for flies from

_Italy

and Denmark than for those from the

_Canary

Islands

_population,

and

_wing

size of flies from Mali was

_{significantly}

smaller than that of all other

populations.

Mean

_survivorship

differences between flies heat shocked with and without

conditioning

at

_temperatures

from 34 to 40°C are

presented

in table IIIA. The two

populations subjected

to

_higher

summer

_temperatures

in nature

_(Mali

and

_Italy)

showed a

_larger

induction of thermotolerance at

_higher

_temperatures

than the other

two

₍₃₈

versus

_36°C).

The increase in survival was not

_{significantly}

different among

the four

_populations

conditioned with _any of the

_{temperatures.}

Similar results for all

conditioning

treatments enabled us to

_pool

across

_temperatures

and test differences

in survival among

populations

either with or without

_conditioning

(table IIIB).

Conditioning significantly

increased

_survival,

and as

before,

the

populations

varied in survival after thermal

_stress,

while the

_{population by}

treatment interaction

was not

_significant.

Survival of flies from the

_Canary

Islands

_population

and from Mali was

_{significantly}

_higher

than that for flies from

Denmark,

and flies from the

Comparisons

are

_{given only}

between

comparable

groups.

Equal

letters denote groups not

statistically

different based on

_{Tukey’s multiple comparisons}

test. For mass

_populations,

three

_replicates

of about 50 flies in two

_independent

blocks were considered for heat and

two

_replicates

in three blocks were considered for cold shock

_{(experiment 1);}

_in

_experiment

2,

a total of 44 vials were not conditioned

₍₁₂

for Mali and

_Denmark,

10 for

_Canary

Islands

and

_Bologna)

while 78 vials were conditioned

(18

for Denmark and

_Italy,

20 for

_Canary

Islands and 22 for

_Mali).

For lines

_{(experiment 3),}

two

_replicates

of about 50 flies for 10

isofemale lines were

exposed

to thermal stress.

_Wing

size refers to five female

_{right wings}

from ten isofemale lines.

values are lower than those of the

_previous

_experiment

(No 1)

_owing

to a

_slight

increase

_(less

than 0.5 of a

_degree)

of the water bath

_temperature.

Intrapopulation

analyses

From

_analyses

on individual isofemale

_lines,

mean values

(table

_II,

rows identified

by

No

₃₎

and intraclass correlations

_{(table IV)}

were obtained in each

_population

for heat shock resistance with and without

_{conditioning,}

for cold

_resistance,

and for

_wing

size.

Comparisons

among

populations

were not

_given

as each

_population

also

repre-sents a different

_experimental

block. In

_spite

of

_that,

with the

exception

of the

Canary

Islands

_{population subjected}

to heat shock without

_{conditioning,}

the

inter-population

differences were

_comparable

to those of the

_{previous experiments.}

Intraclass correlations were not different _{among stress}

_types,

but those for

Correlations among stress

types

and

_wing

size

Table V

_gives

correlation coefficients between each

_pair

of traits

_separately

for each

population

and the overall correlations. At the

_{population level,}

a

_significant

correlation is observed between cold and heat shock resistance without

_conditioning

resistance with

_conditioning

in the Danish

_population.

The

_analysis

of covariance

showed

_homogeneity

_among

_populations

for the correlations between _any

_pair

of traits. The overall

correlations,

based on the

pooled

variances-covariances

within

_populations,

revealed that

_body

size is not correlated with _any stress

_type.

Heat shock resistance with

_conditioning

and cold shock resistance were correlated

significantly

and

_positively.

A

_positive

correlation between heat shock resistance with and without

_{conditioning approached significance.}

DISCUSSION

We

_investigated

heat and cold resistance and the induction of thermotolerance in

four

_populations

of D

_{melanogaster,}

two from

_temperate

and two from

_subtropical

areas. Our aim was to evaluate

_i)

the amount of

_genetic

_variability

for different resis-tance traits and

_ii)

their

_{correlations;}

to

_identify

whether

_iii)

this resistance relates

to the climate of the localities of

_origin

and to determine whether

_iv)

body size,

which varies

_{latitudinally,}

correlates at an

_{intrapopulational}

level with resistance

to

_temperature

extremes.

shown in the same and other

Drosophila species by

the authors

_quoted

in the introduction to this work

_(Morrison

and

_{Milkman, 1978;}

_Stephanou

and

_Alahiotis,

1983; Quintana

and

Prevosti, 1990b;

Jenkins and

_Hoffmann,

1994;. Tucic,

1979;

Heino and

_Lumme,

1989 for

_{temperature stresses;}

David and

_Capy,

_1988;

_Capy

et

_{al, 1993,}

1994 for

_size).

Intraclass correlations for isofemale lines estimate the

genetic

component

of

variance in a broad _sense,

_including

the

_additive,

dominance,

interaction and

maternal

_components.

When the additive variance is the

_prevailing

_component,

the intraclass correlation includes half the

_heritability

_{(Parsons, 1983).}

Direct estimates of

_heritability

for

_survivorship

under

_temperature

stress in D

_melanogaster

are

reported

for cold shock

_by

Tucic

₍₁₉₇₉₎

after

_long-term

artificial selection on a

population captured

near

_Belgrade.

He

_reported

an estimate of

14%

on adult

flies,

slightly

lower than the value we obtain

_{by averaging}

our four

_populations

(25% ),

but

similar to the _average of the two

_populations

from

_temperate

climates

_(15%).

For heat resistance we found

heritability

estimates of

25-28%.

Other direct estimates of

heritability

in this

_species

are available

_only

for knockdown

_temperature

(28%;

_Huey

et

_al,

_1992).

_Experiments

of indirect selection for heat

_survivorship

_(Stephanou

and

Alahiotis,

1983)

confirmed that D

_melanogaster

possesses

genetic

resources

to survive heat shock. For

_{wing size,}

our estimates are similar to those

_reported

by Capy

et al

_(1994),

with the

exception

of the

_Canary

Islands

population

whose

heritability

exceeded 1

_(t

=

0.789).

Wings

of one isofemale line were

_consistently

20%

shorter than the

population

mean. A

single

mutational event rather than

quantitative

variation _may have caused this result. In the absence of this

_line,

the intraclass correlation reduces to

_0.49,

which is in line with other estimates.

For heat resistance in the Mali

_population

and cold resistance in the Italian

popu-lation,

the lowest level of

_genetic

variation and the maximum

_performance

for these

traits were observed.

_Also,

the

_highest

levels of

_genetic

variation were found for

the reverse

_comparison,

cold resistance in the Mali

_population

and heat resistance in the Italian

_population,

where minimal

performance

was observed.

Populations

subjected

to novel stress conditions often exhibit

_genetic

variance at the

_highest

levels

_(Hoffmann

and

_Parsons,

_1991).

In

_general,

the low level of variation in the Mali

_population,

_may reflect a

_relatively

homozygous population following

continu-ous directional selection for

_adaptation

to heat extremes in nature

_{(Parsons, 1983),}

though,

for

_{morphological}

traits, tropical populations

show

_{phenotypic variability}

larger

than that exhibited

_by

_temperate

_populations

and

_genetic

_variability

that is

almost the same

_(Capy

et

_al,

_1993).

Relative

performance

under heat and cold stress seemed related to the mean summer maximum

_temperature

and the mean winter minimum

_temperature,

re-spectively,

for the four areas from which the flies were collected

_(comparing

tables I

and

_II).

Clear differences were

_present

_only

between _very

_separate

_geographic

re-gions.

For most

_traits,

differences between Mali and the

_Canary

Islands or between

Italy

and Denmark were

_small,

_although

_wing

size of Mali flies was smaller than

that of flies from the

_Canary

Islands.

Perhaps

behavioural traits that enable escape

from unfavourable climatic conditions

_(Jones

et

_al,

₁₉₈₇₎

are

_possible

within a

_given

temperature

range and these reduce

physiological

differences between

populations.

Migration

by

fruit

_trading

also could be relevant and

_give

a reason for the

_relatively

locality

would have

_given

more information for a

_comparison

of relative thermal re-sistance in relation to the climatic conditions at the

_sample

sites.

_However,

we chose

to

_keep

_up the number of

_geographic

populations

and traits instead of

increasing

sample

number _per

_locality.

The

_populations

differed much more for heat than for cold

_resistance,

a result

that could

_depend

either on the kind of treatment

_performed

or _upon different

reaction norms to hot or cold

_temperature

extremes.

The

_dependence

of heat tolerance on the

_temperature

at which a

_given

population

evolves has been well documented for

_{populations adapted}

to different

_temperatures

in the

_laboratory

_(Stephanou

and

_{Alahiotis, 1983; Huey}

et

_{al, 1991;}

Cavicchi

et

_al,

_1995).

_Populations

held at warmer

_temperatures

_may also show

_genetic

differences for induction of

_{thermotolerance, expressing}

the heat shock response

at a

_higher

_temperature

than those

_adapted

to cold

_(Cavicchi

et

_al,

_1995).

This trend

suggests

that natural selection in the wild at non-extreme

_temperature

has led to a

genetically

correlated _{response in} tolerance to extreme

_temperature,

but that

_adaptation

to one

_part

of the thermal

_performance

curve reduces

_adaptation

at

_temperature

extremes farther _away. Previous work on relative chromosomal

contributions to fitness

_{components suggests}

that different _{groups of genes} are

involved for

_adaptation

at intermediate

temperature

(Cavicchi

et

_al,

₁₉₈₉₎

and

resistance to extreme heat

_(Cavicchi

et

_al,

_1995).

The

_present

_{results, though}

not

concerning

intermediate

temperatures, suggest

that different groups of genes are

important

at the two extremes.

However,

the correlation between heat shock resistance with

_conditioning

and cold shock resistance in lines derived from natural

_populations

was

_significant,

suggesting

that similar _groups of _{genes may} affect resistance at the two

_temperature

extremes.

_Perhaps

this

relationship

is due to a

general

hardiness or weakness of some lines that is

_independent

of the shock _response.

_Inbreeding

is

_expected

within isofemale lines and uncontrolled

_genetic

drift or

_inbreeding

_may lead to

_positive

associations among fitness traits

_(Dahlgaard

et

_al,

_1995).

The

_performances

of different isofemale lines with or without

_conditioning

show a

low

_{correlation, suggesting}

that the role of heat shock _genes is

_unimportant

for heat tolerance when a

population

is

_{rapidly subjected}

to a

_potentially

lethal heat stress

(41.5°C

for 0.5 h without

_{conditioning).}

Molecular data

support

this

observation,

in that the maximal

_{transcription}

level of a more inducible heat shock _gene

(hsp-70)

is reached after about half an hour after a severe heat

_treatment,

while for

others

_{(hsp-82, -27)}

the maximum is observed after a

_longer

time

_(DiDomenico

et

_al,

1982

_a,b).

Suggestions

on the mechanistic basis

_underlying

how evolution in a

_population

at intermediate

_temperature

_may affect tolerance to extreme

_temperature

stress

are,

however, speculative. Nevertheless,

studies of enzymes

suggest

that natural selection at different

_temperatures

can be associated with variation in their kinetic

parameters

(Alahiotis,

1982;

Hoffmann and

_Parsons,

_1991;

_Somero,

₁₉₉₅₎

in such

a _way that _enzymes show a

_greater

_efficiency

under the conditions an

_organism

normally

encounters.

For the minimum

_temperature

for induction of

_{thermotolerance,}

the four D

_{melanogaster populations,}

which come from _very different

_regions,

were

populations adapted

to different

_temperature

_optima

_(Cavicchi

et

_al,

_1995).

These

response

types

may be a

general

rule across

_species,

and relate to the activation of heat shock genes

(Lindquist

and

Craig,

1988; Huey

and

_Bennett,

_1990).

The heat shock _{response is} a

_{physiologically}

_plastic

_response to deal with stress, and

in natural variable

environments,

induction

temperatures

may

change

little.

There-fore,

above some threshold

_temperature,

the level of acclimation that occurs _may

be similar.

ACKNOWLEDGEMENTS

The research was

_{supported by}

a _grant from MURST

_(Italy)

to S Cavicchi. The _stay of R Krebs in Aarhus was funded

_through

a _grant from the Danish Natural Science Research

Council

_{(No 11-9639-2).}

The authors are

_grateful

to R

Huey

for

_reading

the manuscript

and for valuable

_suggestions

and to Vittoria La Torre for

help

in the

_experimental

work.

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