Parallel selection of ethanol and acetic-acid tolerance in Drosophila melanogaster populations from India

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Parallel selection of ethanol and acetic-acid tolerance in

Drosophila melanogaster populations from India

R Parkash, Shamina, Non Renseigné

To cite this version:

Original

article

Parallel

selection of ethanol

and

acetic-acid tolerance

in

_Drosophila

_melanogaster

populations

from

India

R Parkash

_Shamina,

Neena

Department of Biosciences,

Maharshi

_{Dayanand University, Rohtak, 124001,}

India

(Received

10

_{September 1993; accepted}

23 June

₁₉₉₄₎

Summary - Nine Indian

_{geographical populations}

of

_{Drosophila melanogaster,}

collected

along

the 20°N latitudinal _range, revealed a

significant

clinal variation at the alcohol

dehydrogenase

(Adh)

locus,

Adh

F

allelic

_{frequency increasing significantly}

with latitude

(0.036 !

0.004 for 1°

_{latitude; genetic divergence F}

_ST

=

0.25).

Patterns of ethanol and

acetic-acid tolerance in adult individuals revealed

_{significant genetic divergence.}

Parallel patterns of latitudinal ethanol tolerance

₍₁₀

to

_15%)

and acetic-acid tolerance

_(3.7

to

13.2%)

were observed in adult individuals from the 9

_{geographical populations. Thus,}

the northern and southern

_populations

revealed

_divergence

in the patterns of resource

utilisation. The

_parallel

latitudinal

_{genetic divergence}

at the Adh locus and for ethanol and acetic-acid tolerance in Indian

populations

of

_{Drosophila melanogaster}

could be

_explained

by balancing

natural selection

_{varying spatially along}

the north-south axis of the Indian subcontinent.

ADH _polymorphism

_/

ethanol tolerance

_/

acetic-acid tolerance

_/

latitudinal clines

_/

Indian _populations

_/

_{Drosophila melanogaster}

Résumé - Sélection _parallèle des tolérances à l’éthanol et à l’acide _acétique dans des _populations indiennes de _{Drosophila melanogaster.}

_{Neuf populations géographiques}

indiennes de

_{Drosophila melanogaster,}

échelonnées sur une latitude de

20°N,

révèlent

une variation clinale

_{significative}

au locus de l’alcool

_{déshydrogénase}

_(Adh),

avec un

accroissement

_significatif

de la

fréquence

de l’allèle

Adh

F

avec la latitude

_{(0, 036 ::L}

_0,004

par

degré

de

_latitude)

et un indice de

fixation F

_ST

= _{0, 25.} Des évolutions

_parallèles

_de

la tolérance à l’éthanol

₍₁₀

à

_15%)

et de la tolérance à l’acide

_acétique

_(3,7

à

_13,2%)

en

_fonction

de la latitude sont observées chez les adultes des 9

_{populations géographiques,}

révélant ainsi des

_divergences

dans le mode d’utilisation des ressources entre le nord et le

sud. La

_divergence

observée en

_fonction

de la latitude à la

_fois

au locus Adh _{et pour} les

*

Correspondence

and

_reprints:

_446/23

Near

_Park,

DLF

_{Colony, Rohtak, 124001, Haryana,}

tolérances à l’éthanol et à l’acide

acétique pourrait s’expliquer

par une sélection naturelle

équilibrante

variant selon l’axe nord-sud du sous-continent indien.

polymorphisme de l’Adh

_/

tolérance à l’éthanol et à l’acide acétique

/

clines de latitude

_/

_populations indiennes

_/

Drosophila melanogaster

INTRODUCTION

The

_evolutionary

potential

of a

_species

is a function of the amount of

_genetic

vari-ation it

_{undergoes. Colonising}

_species

such as

_{Drosophila melanogaster populations}

offer excellent material for

_{micro-evolutionary}

studies

_(Parsons,

_1983).

Studies on

biogeography,

ecology

and

_{adaptive physiological}

traits in

_global

_populations

of D

_melanogaster

have revealed that

_{Afrotropical populations}

constitute ancestral

populations,

which later colonised Eurasia and more

_recently

America and

Aus-tralia

_(David

and

_{Capy, 1988).}

Most studies on

_{allozymic polymorphism}

have been

made on American and Australian

populations

of D

_melanogaster

while Asian

pop-ulations remain

unexplored

(David,

1982; Oakeshott

et

_{al, 1982;}

Anderson et

_al,

1987).

Gel

_{electrophoretic analysis}

has

_helped

in

_elucidating

the

_genetic

structure

of

_geographical

populations

of diverse

_taxa,

and it was therefore considered worth-while

_{characterising}

the extent of

_{genic divergence}

at the alcohol

dehydrogenase

(Adh)

locus in

_{latitudinally}

_varying

Indian natural

_populations

of D

_{melanogaster.}

D

_{melanogaster populations exploit}

a wide _array of

fermenting

and

decaying

fruit

and

_vegetables,

_organic

materials and man-made alcoholic environments. Ethanol is the end

_product

of fermentation and ethanol vapours

provide

a normal _energy

source in D

_melanogaster

_{(Parson, 1983).}

Ethanol is converted into acetic acid via

acetaldehyde

and thus concentrations of these 2 metabolites are

_generally

found

in natural habitats of the

_{Drosophila species.}

The alcohol

_{dehydrogenase}

_(ADH)

of D

_melanogaster

converts a wide range of alcohols into

aldehydes

and more than

90%

of the external alcohols are metabolised in a

pathway

initiated

by

this _enzyme

(Geer

et

_al,

_1989).

Most studies on ethanol tolerance have been made on D

_melanogaster

populations

from

Europe

and Africa

_(David

et

_al,

₁₉₈₆₎

and Australia

_(McKenzie

and

_{Parsons, 1972; Parsons, 1979,}

_1980a),

but information on D

melanogaster

from

India as well as other

_tropical

_parts

of the world is still

_{lacking. Recently,}

acetic

acid has been found to be a

parallel

resource to ethanol in D

_melanogaster

_(Chakir

et

_{al, 1993,}

_1994).

The

_objective

of this

_study

is to

_analyse

acetic-acid and ethanol utilisation

by

D

_melanogaster

_populations

from the Indian subcontinent.

MATERIALS

AND METHODS

Isofemale lines of D

_melanogaster

from 9 Indian

_geographical

sites

_(Madras

to

Dalhousie,

13°04’N

to

_33°N;

_fig

_1,

table

_I)

were established for 2-3

_generations

and used for measurements of ethanol and acetic-acid utilisation as well as ADH

polymorphism.

Homogenates

of

_single

individuals from each isofemale line were

subjected

to

_{electrophoresis}

at 250 V and 25 mA at 4°C for 4 h and

_gel

slices were

patterns

was

_interpreted

from the

_segregation

patterns

of enzyme

electromorphs

of

parents, F

l

and

F

2

progeny of several

single-pair

matings.

The

_genetic

indices were

Ethanol and acetic-acid tolerance

_patterns

of mass cultures of each of 9

popu-lations of D

_melanogaster

were assessed

following

the

procedure

of Starmer et

al,

(1977).

Groups

of 10 males or 10

_females,

_grown on a killed

_yeast

medium

(without

any

ethanol),

were

_aged

for 3 d on fresh

Drosophila

food medium and then

trans-ferred with the

_help

of an

_aspirator

to

_air-tight

_plastic

vials

₍₄₀

_ml;

4 x

_{1 inches).}

The flies were admitted to the upper

vial,

which was

_{separated by}

fine

_terylene

cloth

from the lower vial

_containing

10 ml of ethanol or concentrated acetic acid absorbed on 1 _g cellulose wool. Such

_paired

vials were sealed with

_cellophane

_tape

and all

experiments

were conducted at 23°C. The alcoholic solutions were not

changed

during

the

_experiment.

The flies were not etherised

_during

different

_experiments.

The control vials contained 10 ml of distilled water absorbed on cellulose wool. Four

replicates

were

_performed

for all the

_experiments.

For each

concentration,

40 males

and 40 females were treated with a _range of 6-8 different concentrations of ethanol

or acetic acid. The male and female individuals did not reveal _any

_significant

dif-ference in ethanol or acetic-acid tolerance and thus the data for the 2 sexes were

averaged

for all

_experiments.

The effects of metabolic alcoholic _vapours were

as-sessed from the number of flies alive after various time intervals and

LT

50

values

were

_expressed

as the number of hours after which

50%

of the flies had died and were

estimated

_by

linear

_{interpolation.}

The ethanol and acetic-acid threshold values were

used as

_indices,

ie if _vapours were utilised as the source then

LT

50

ethanol/LT

50

control was found to be more than

1;

if this ratio was less than

1,

then it acted as

stress. The threshold values were determined when

LT

50

ethanol/LT

SO

control = 1

(Parsons, 1983).

RESULTS

Populational genetic

structure at the Adh locus

Data on the number of isofemale lines and

Adh

F

frequency

in D

melanogaster

populations

are

_given

in table I. The clinal variation at the Adh locus was found

to be

_significant

_(3.6%

with 1°

latitude;

r =

_0.96;

b = 0.036 f

0.004).

The data on

Wright’s

fixation index

_(F

_ST

=

0.25)

revealed

significant

genic

divergence

at Adh

locus in Indian

_{populations. Contingency}

chi-squared

analysis

revealed

_significant

interpopulation genotypic heterogeneity

(75.8)

and allelic

_{heterogeneity (738.4)}

at

the Adh locus in Indian

_populations

of D

_{melanogaster.}

Adult ethanol tolerance

The adult individuals were

analysed

for their

potential

to utilise the ethanol _vapours

in a closed

_system

and the data on the ethanol and acetic-acid tolerance of 9

geographical

populations

are

_given

in table I. Data on 5

_geographical

_populations

of D

_melanogaster

are shown in

figures

2 and 3. The

intraspecific

variation for

ethanol tolerance was found to be

_{significantly}

different

_along

the north-south axis of the Indian subcontinent. The data on

LT

50

ethanol/LT

50

control

(which

are the measures of source versus

_stress)

show a latitudinal variation

(table

_I,

fig

2a).

The adult ethanol threshold values were found to _vary

_clinally

in the

localities

_{(table I).}

The ethanol concentrations _{up to}

15%

served as a source for

north Indian

populations

while a maximum of

10%

ethanol concentration could be utilised

_by

south Indian

_populations.

The

_LC

₅₀

ethanol concentrations were

calculated from

_mortality

data of adults after 6 d of ethanol treatment and

LC

50

values revealed clinal variation in the range of 9.0 to

_12.0%,

ie southern

_populations

of D

_melanogaster

showed

_{significantly}

lower ethanol tolerance than north Indian

populations

(table

I,

fig

3a).

The

_longevity

data on

6%

ethanol revealed that

northern

_populations

under

_experimental

conditions survived for 3 weeks

_(18-22

_d)

as

_compared

with 2 weeks duration in southern

populations

(fig 3c).

Adult acetic-acid tolerance

The south Indian

_population

of Madras revealed the minimum value of

LT

50

acetic

acid/LT

SO

control

_{(1.0) compared}

with

_higher

_LT

₅₀

acetic

_acid/LT

₅₀

control

_(2.38)

in the Dalhousie

_population

when adult individuals were

_exposed

to

3%

acetic acid

_(fig

_2b).

The adult acetic-acid threshold values also revealed latitudinal clines

in the _range of 3.7 to 13.2

_{(table I).}

The

_LC

₅₀

acetic-acid concentrations were

calculated from the

mortality

data at 3 d and the

LC

50

values revealed a clinal

variation of

5.6-11.7%

_(table

_I).

The acetic-acid tolerance indices of 5

_populations

of D

_melanogaster

are shown in

figures

2b and

3b,d.

The

longevity

periods

at

3%

acetic-acid were found to be more than 2 weeks in northern

populations compared

with about 10 d in southern

populations

(fig 3d).

DISCUSSION

In order to test whether the Adh allelic

_{frequency changes}

and ethanol tolerance

potential

are correlated with

latitude,

a statistical

analysis

of correlation was carried

out for all 9

_geographical

populations

of D

_{melanogaster.}

The statistical correlations

were found to be

_{significantly}

_higher

_(0.96)

for latitudinal variation of adult ethanol tolerance versus

Adh

F

allelic

_frequency.

_Thus,

the

_present

data on clinal variation

at the Adh locus in Indian

_populations

of D

_melanogaster

further

_support

and validate the

hypothesis

that the occurrence of

parallel

or

complementary

latitudinal

clines across different continental

populations provides

_strong

evidence of natural

selection

_maintaining

such clinal

allozymic

variation

_(David,

_1982;

Oakeshott et al

1982;

Anderson et

_al,

_1987).

Latitudinal clines have been

reported

in American

_(Vigue

and

_Johnson,

_1973),

Australian

_(Oakeshott

et

_al,

_1982),

_Afrotropical

_(David

et

_al,

_1986,

_1989),

_Japanese

(Watada

et

_al,

₁₉₈₆₎

and Chinese

populations

(Jiang

et

_al,

_1989).

The occurrence

of clinal variation across diverse

biogeographical

_regions

cannot be

_explained

on

the basis of stochastic processes such as

_genetic

drift

and/or

_gene flow since the

continental

_populations

differ

_{significantly}

in their

_{evolutionary history}

as well as

ecogeographical

conditions. The existence of

_parallel

clinal allelic

_{frequency changes}

at the Adh locus

_provides

strong

evidence for the action of

_{latitudinally}

related

environmental

_gradients.

The Indian

_{geographical populations}

of D

_{!nelanogaster}

revealed

_significant

genetic divergence

in their

_potential

to use acetic acid. The adult

_longevity

1-13%

for north Indian

_populations.

The acetic-acid threshold values were found

to vary

clinally

in the range of 3.7 to

13.2%

for adults in

_{geographical populations}

from south to north localities. The

_LC

₅₀

values revealed clinal variation in the

range of 5.6 to

11.7%

acetic

_acid,

ie the southern

_populations

had lower acetic-acid tolerance than the northern

_populations.

Indian

_populations

of D

_melanogaster

revealed

_significant

_{parallel genetic divergence}

in their

potential

to utilise ethanol and acetic acid. The

parallel

utilisation of ethanol and acetic acid in Indian

_tropical

and

_subtropical

_populations

of D

_melanogaster

concur with such data on

_temperate

populations

of D

_{melanogaster (Chakir}

et

al,

1994).

High

ethanol and acetic-acid-rich environments seem to be

_exploited

_by

D

_{melanogaster.}

D

_melanogaster

utilises lower alcoholic concentrations but

_mainly

detoxifies the

_higher

ethanol concentrations

_occurring

in its natural and man-made habitats. The observed

_genetic

differentiation of ethanol tolerance in

_geographical

Indian

populations

of D

_melanogaster

concurs with other continental

_populations

from Africa and Australia

_(Parsons,

_1980b;

David et

_{al, 1986; David,}

_1988).

The ethanol and acetic-acid tolerance threshold values in adult individuals were found

to vary

latitudinally

in different Indian

_populations.

The

_present

observations are

in

_agreement

with other

_reports

on the evidence of action of natural selection at the

Adh locus as well as for ethanol tolerance in some

_{allopatric populations}

(Hickey

and

_{McLean, 1980;}

Van

_Herrewege

and

David,

1980).

Thus,

these traits have

adap-tive

_significance

and are

being

maintained

by

natural selection mechanisms.

ACKNOWLEDGMENTS

The financial assistance from

CSIR,

New Delhi is

_{gratefully acknowledged.}

We are

_grateful

to the reviewers for their

_helpful

comments and M Weber for

_drawing

the

_figures.

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