Ethanol and acetic-acid tolerances in Drosophila melanogaster: similar maternal effects in a cross between 2 geographic races

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Ethanol and acetic-acid tolerances in Drosophila

melanogaster: similar maternal effects in a cross

between 2 geographic races

M Chakir, P Capy, E Pla, J Vouidibio, Jr David

To cite this version:

Original

article

Ethanol and acetic-acid tolerances

in

_Drosophila

_{melanogaster:}

similar maternal

effects

in

a cross

between

2

_geographic

races

M

Chakir

_{P Capy}

E Pla

J Vouidibio

JR

David

1

CNRS,

Laboratoire de

_Biologie

et

_{Cenetique Evolutives,}

91198

_{Gif-sur-Yvette, France;}

2

Faculté

des

Sciences,

Laboratoire de

Biologie

des

Populations,

Brazzaville, Congo

(Received

3 March 1993;

accepted

14 October

₁₉₉₃₎

Summary - Ethanol and acetic-acid tolerances were studied in a cross between 2

geo-graphic

races of

Drosophila melanogaster,

ie a _very sensitive

_population

from

equatorial

Africa and a resistant French

_{population. Average}

values in the Fl and F2 were similar

and close to the

_mid-parent

value. A clear maternal genotype effect was,

however,

observed

for both traits between

reciprocal

Fis, and the difference

disappeared

in the F2. Further

investigations

demonstrated that for ethanol tolerance, the

_large

difference between the

parental

strains was not

_entirely

due to differences in their allelic

_frequencies

at the Adh locus. The

_possible

mechanisms of these

physiological

variations are discussed.

ethanol tolerance

_/

acetic-acid tolerance

_/

ADH _polymorphism

_/

maternal _genotype effect

_/

_geographic races

Résumé - Tolérances à l’éthanol et à l’acide _acétique chez _{Drosophila melanogaster :}

effets maternels similaires dans un croisement entre 2 races _{géographiques.} Les

tolérances à l’éthanol et à l’acide

acétique

ont été

étudiées,

à

partir

d’un croisement

entre 2 races

_{géographiques}

de D

_{melanogaster :}

une

_population

très sensible

_d’Afrique

équatoriale

et une

_population

française

résistante. Les valeurs _moyennes des

_génémtions

F

l et F2 sont semblables et

proches

de la valeur du parent moyen. Une

différence nette,

due au

_{génotype maternel,}

a été observée entre les Fi

réciproques.

Cette

_{différence disparaît}

en

F 2

. D’autres

expériences

ont montré que, pour la tolérance à

l’éthanol,

la

grande différence

observée entre les souches

parentales

n’est _pas entièrement

provoquée

par la

différence

des

fréquences alléliques

observées au locus Adh. Les mécanismes

_possibles

de ces variations

physiologiques

sont discutés.

tolérance à l’éthanol

_/

tolérance à l’acide _acétique

_/

_{polymorphisme} de l’ADH

_/

effet

INTRODUCTION

Of the 8

_species

of the

_{melanogaster subgroup,}

_only

_Drosophila

_melanogaster

presents

a

high

alcohol tolerance. In this

species, adaptation

to resources with a

high

ethanol content is considered as a

_major

ecological

and

evolutionary

event

(see

David, 1977;

Van

_{Delden, 1982;}

Lemeunier et

_{al 1986;}

David and

_Capy,

_1988;

Hoffman and

Parsons,

1991).

Ethanol tolerance is based on the _presence of a _very

abundant alcohol

_{dehydrogenase}

_(ADH),

and null mutants are _very sensitive

(David

et

_{al 1976).}

On the other

hand,

flies that are

heterozygous

for a null and a normal

allele exhibit a normal tolerance

(Kerver

and

_Rotman,

1987).

ADH is

_expressed

at various levels in most

_{developmental}

_stages

_{including embryos.}

It was

_recently

demonstrated

_(Kerver

and

_Rotman,

₁₉₈₇₎

that

_embryos

that are

_heterozygous

for a

null allele exhibit different ethanol sensitivities in relation with the

genotypes

of the

parents.

When the functional allele was inherited from the female

_parent,

embryonic

tolerance was much better than when it was inherited

_through

the _sperm.

_However,

this maternal effect

_disappeared

in later

stages.

Natural

_populations

of D

_melanogaster

exhibit

_large

variations in their ethanol

tolerance,

often

_{arranged according}

to latitudinal clines

_(David

and

_Bocquet,

1975;

David et

al,

1988).

Among

all the

_{populations investigated}

so

far,

the

most sensitive are found in

_{equatorial Africa,}

which

probably

_correspond

to the ancestral

_populations

of the

_species

_(David

and

_Capy,

_1988),

with an

LC

50

of

about

6%

ethanol.

_Highly

tolerant

_populations

are found in

_{temperate countries,}

and

_especially

in

_Europe.

In

_France,

for

instance,

the average

LC

50

exceeds

17%.

These differences in ethanol tolerance are

_accompanied

_by

variations in allelic

frequencies

at the Adh locus

_(David

et

_al,

_1986).

More

_precisely,

the more active

Adh-F allele is more abundant in

_temperate

_populations,

while the less active Adh-S allele

_predominates

in

_tropical,

sensitive

_populations.

It has been

_{repeatedly argued}

(see

Van

_Delden,

_1982,

for a

review)

that a causal

_relationship

existed between the

2

_traits,

and that the

_high

ethanol tolerance was due to the

_{higher frequency}

of the Adh-F allele.

However,

this

_point

was never

_{correctly investigated}

in natural

populations.

It was

recently

shown

(Chakir

et

al,

1993)

that acetic-acid and ethanol tolerances were

always strongly

correlated both at intra- and

inter-specific

levels.

Morever,

the 2 tolerances involve the same metabolic

_{pathway, leading}

to the

_production

of an

increased amount of

Acetyl-CoA.

Acetic-acid

tolerance, however,

does not

_depend

on the _presence of active ADH.

These observations led us to

_investigate

the

_genetic

bases of ethanol and acetic-acid tolerances in crosses between African and

_{European populations}

of D

melanogaster.

The most

_interesting

observation is a maternal

_effect,

observed be-tween

_reciprocal

_F

_i

_s

and

_disappearing

in

_F

₂

_.

_Moreover,

strains that are

homozygous

for the Adh-F and Adh-S alleles were extracted from the 2

_types

of natural

popu-lations.

_Significant

differences

were found between flies

_according

to their

_geographic

MATERIAL AND METHODS

Two

_{geographic populations}

were

compared.

A French

population

was collected near Bordeaux. The other

_population

was collected in the

_Congo,

in a

locality

called

Loua in the suburbs of Brazzaville

_(see

Vouidibio et

al,

1989,

for a

_precise

location).

These 2

_populations

were

_polymorphic

at the Adh locus

_(left

arm of chromosome 2 at

_50.1,

see Grell et

al,

1965)

but the

_frequencies

of the 2

_widespread

alleles were

highly different, according

to the well-known latitudinal cline that occurs between

Europe

and

_tropical

Africa

_(David

et

al,

1986).

In

_Bordeaux,

the

_frequency

of Adh-F

was

94%

while it was

_only

4%

in Loua. Both

_populations

were

_kept

as mass cultures

in

_laboratory

bottles. More than 100 adults were transferred at each

_generation

in order to avoid

_inbreeding

and

_prevent

_laboratory

drift. These mass

_populations

were used in the

_toxicity

tests.

Simultaneously,

isofemale lines were established

_{by isolating}

wild collected

fe-males in

_single

vials. After larvae were

_observed,

each female was checked for its Adh

_genotype,

and lines

_harboring

the 2 alleles were conserved. From each initial

line,

several

F

I

pairs

were made and set in culture vials. After about 1

week,

when

offspring

larvae were

_observed,

the

_genotypes

of the 2

_parents

were established

_by

electrophoresis.

From these

F

1

pairs,

2 selections were

undertaken,

in order to

_get

homozygous

lines for the F or the S allele. For

_example,

for

_purifying

the F

_allele,

the

_F

_I

_pairs

with the

_highest

F

_frequencies

were

_chosen,

_F

₂

_pairs

were

_established,

allowed to

_oviposit,

checked for Adh

_genotypes,

and selected

_again

if _necessary. With this

procedure,

the

_{required homozygous}

lines were obtained after

_2,

3 or ₄

gen-erations. From the French

_population,

from 8 wild

_living

_females,

8

_homozygous

FF and 8 S’S lines were

available;

from the

_Congolian

_population,

7 lines of each

genotype

were obtained from 7 wild females.

Ethanol and acetic-acid tolerances were measured

_according

to

_previously

de-scribed

_techniques

_(Chakir

et

_al,

_1993).

Adults were _grown at 25°C in bottles on

a killed

_yeast

food.

_Upon

_emergence

_they

were

_lightly

etherized and distributed in groups of 20 males or 20 females. After a _recovery of 3-4 d on the same

food, they

were transferred to

_air-tight

_plastic

vials

_containing

_experimental

concentrations of ethanol or acetic acid. Dead flies were counted after 2 d and survival curves drawn

separately

for

_males,

females and both sexes. The

_LC

₅₀

values

_(ie

the concentrations

killing

50%

of the

_flies)

were estimated

_graphically

from each

_experiment.

ADH

_activity

was measured on adult males

aged

4

d, according

to

_Merqot

and

Higuet

(1987).

Activities are

expressed

as variation of

optical density

_{per mg} of

flies.

RESULTS

Ethanol and acetic-acid tolerances were measured on mass cultures from Bordeaux

and Loua.

_Reciprocal

_F

_I

and

_F

₂

cultures were also

investigated.

When

LC

50

s

are

measured on the same strain over successive

_experiments,

_significant

_differences,

due to unknown and uncontrolled

_variations,

may be observed

(Chakir

et

_al,

_1993).

It is thus _necessary to

_repeat

the measurements several times on the same strain in order to calculate an _average value for the

parental

strains and _{progeny. Mean}

LC

50

The ethanol tolerance

_(mean

of both

_sexes)

was on the _average

17%

in

Bor-deaux and

5.7%

in Loua. The difference

_(11.3%)

is

_{highly significant.}

A

_slight,

non-significant

difference exists between sexes, and the males are a little more

tol-erant than the females. This sex difference became

_significant

when all the data

(parents,

F

I

and

_F

₂

₎

were considered

simultaneously.

F

I

flies exhibit an

intermedi-ate tolerance

_(10.1%)

which is not

_{statistically}

different from the

_mid-parent

value

(11.33

t

_0.27%).

The

_reciprocal

F

i

s

are,

however, significantly

different

(see

ta-ble

_I),

and the flies from a Bordeaux mother

(F

lB

)

are more tolerant than those

from an African mother

(F

lA

)-

In such a case, male and female progeny must be

analysed separately,

since females are

_genetically

identical while males inherit their

X chromosome from their mother. Table I shows that males of the

_reciprocal

_F

_i

_s

are

clearly

different

(d

= 2.05 t

0.41%

alcohol),

and the direction of the

differ-ence _agrees

with

the mother’s characteristics. A similar observation is also valid for females

_(d

= 1.5 !

0.60%).

The difference is _a little smaller than in

males,

but

not

_{significantly}

so. The

_reciprocal

_F

₂

_s

are almost identical and the mean value

(both

sexes

pooled)

of 10.32 t

0.48%

is similar to the average

F

i

s

and not different

from the

_mid-parent

value. All these observations demonstrate that the difference

between the

_reciprocal

_F

_i

_s

is

_mostly

due to a maternal

_genotype

influence which

persists

until the adult

stage

but

_{totally disappears}

in the

F

2

s.

For acetic-acid tolerance the results are

_quite

similar

_{(table I).}

The difference

between the

_parental

strains

_(6.1%)

is less

pronounced

than for

_ethanol,

but still

highly significant

with no

overlap

of the distributions. A

general tendency

also

exists for the males to be a little more tolerant than the females. The

_reciprocal

F

i

s

are

_{statistically}

different and the difference is the same in females and males.

This difference

disappears

in the

reciprocal

F

2

s.

The mean values of the

F

i

s

(8.38%)

and

F

2

s

(8.23%)

are also _very close to the

_mid-parent

_(8.31%).

The occurrence of a maternal

_genotype

effect,

_disappearing

in the

_F

₂

_s,

is also a valid

_{interpretation.}

The

_experiments

described above were

_performed

with mass

_cultures,

polymor-phic

for the 2 alleles at the Adh locus. ADH is a

_key

_enzyme for ethanol

detoxific-ation,

and the most active allele

_(Adh-F)

was

_present

with a

_{high frequency}

(94%)

in the Bordeaux

population

but with a _very low

_frequency

_(4%)

in the Loua popu-lation. The

_big

difference in ethanol tolerance between these 2

_populations

could be due to this difference in allelic

_frequencies.

To check this

_hypothesis,

_homozygous

FF and SS lines were extracted from each natural

population

(see

Materials and

Methods)

and ethanol tolerances measured. These results are shown in

figure

1 and

the statistics are

_given

in table II.

A broad

_variability

is evidenced between

_lines,

and this _may be due to the

_high

inbreeding

which occurred

_during

the process of

purifying

the

homozygous

lines.

The

_phenomenon

is

_relatively

more

_pronounced

_among the

Congolian

_lines,

with

coefficients of variation around

35%.

No

_significant

differences exist

_according

to the Adh

_genotype.

On the other

hand,

there is no

overlap

of the values between

French and

_Congolian

lines and the differences

_according

to

_geographic

_origins

are

highly significant.

A final observation is that the mean values in table II are

somewhat

_superior

to those of the

_parental

lines in table

_I,

but the differences are

not

_significant.

Acetic-acid tolerance was not measured in these lines since this trait

does not

_depend

on the _presence of an active ADH

(Chakir

et

al,

1993).

It remained

(see

Kreitman,

1983),

African strains could have a lower ADH

_activity

due to different

_genetic

_regulations.

To check this

_hypothesis,

the

_homozygous

strains from the same

_geographic

area were

_pooled

in a

single

mass

_culture,

and ADH

_activity

measured on males. The results in table II

_clearly

show that this was not the case:

the well-known difference between F and S alleles was confirmed. No difference was

found, however,

between French and

_Congolian

flies with the same allele.

DISCUSSION AND CONCLUSION

Two main conclusions _may be drawn from this work.

_First,

the

_large

difference that exists between the ethanol tolerance of the French and

_equatorial

African

popula-tions of D

_melanogaster

is correlated with a difference in Adh allelic

_frequencies

(David

et

_al,

₁₉₈₆₎

but is not

_entirely

due to this

_genetic

_divergence.

_Moreover,

the

compare

geographically

distant

populations,

ethanol tolerance is

mainly

mediated

by

a

different,

but still

unknown,

physiological

_process. This was

_{already suggested}

by

Middleton and Kacser

₍₁₉₈₃₎

and David et al

_(1989).

One mechanism could

be that the

_sensitivity

of the

_target

_organs, ie the nervous

_system,

is _very

differ-ent between

_European

and

_Congolian

flies. But another mechanism is that ethanol

tolerance could

depend

not

_only

on ADH

_activity,

but also on ACS

(Acetyl-CoA-synthetase)

activity.

This

_possibility,

which was excluded for larvae

_by

Freriksen et al

_(1991),

seems worth consideration for adults

_according

to the work of Chakir et al

_(1993).

_Obviously,

this

problem

deserves further

_{investigation.}

A

second,

and

_novel,

conclusion is the

_magnitude

of a maternal effect which

discriminates the

_reciprocal

_F

_i

_s,

and also the fact that this effect concerns both acetic-acid and ethanol tolerances.

Various processes may

explain

maternal

influences,

including

the transmission of

cytoplasmic

organelles

(Sager, 1977)

or

symbionts

(Nardon, 1993),

a

perturbation

of

the mother’s

_{physiology by}

environmental effects

_{(David, 1962)}

or the

_asymmetric

contribution of

_paternal

and maternal

_genotypes

to the formation of the

_embryo

(see

Lawrence,

1992).

Our observations are

probably explained

_by

this last

_type,

ie maternal

_genotype

effect. The most

_striking

observation is that

_{physiological}

differences between

reciprocal F

i

s,

which are initiated in the

embryo, persist

until the adult

stage.

By

contrast, maternal effects on ADH

_activity,

described

by

Kerver and Rotman

(1987),

disappeared

much earlier in the larval

_stages.

We do not know which

metabolic

_pathway

is involved in the maternal influence observed in crosses between

European

and

_Afrotropical

flies. The

_similarity

between ethanol and acetic-acid

tolerances

_suggests

that the ACS

_activity

could be involved. But the tolerance of the

_target

_organs,

_especially

of the nervous

_system,

could also be modified

_(David

et

al,

1989).

In

conclusion,

the

_large

difference in ethanol tolerance between

_European

polymorphism.

Natural

selection, however,

may also use other as

_yet

unidentified

processes, for

increasing

the

_difference,

as evidenced in this _paper.

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