Ethanol and acetic-acid tolerance in Indian geographical populations of Drosophila immigrans

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Ethanol and acetic-acid tolerance in Indian geographical

populations of Drosophila immigrans

R Parkash, Neena, Non Renseigné

To cite this version:

Original

article

Ethanol

and

acetic-acid tolerance

in

Indian

_{geographical populations}

of

_Drosophila

_immigrans

R

Parkash

_Neena,

Shamina

Department of Biosciences,

Maharshi

_{Dayanand University, Rohtak, 124001,}

India

(Received

10

_September

1993;

accepted

25 March

₁₉₉₄₎

Summary - Indian

_{geographical populations}

of

_{Drosophila immigrans}

revealed a lack of

allozymic

variation at the alcohol

_{dehydrogenase}

locus. Latitudinal clines of ethanol

toler-ance

(1.5-4.2%)

and acetic-acid tolerance

(2.9-4.9%)

were observed in adult individuals of

4

_{geographical populations}

of

_{Drosophila immigrans. Thus,}

both ethanol and acetic-acid tolerance decreased towards the _equator. The

_parallel

_patterns of utilisation of ethanol and

acetic acid seem to be correlated with the concentrations of these 2 metabolites in natural food resources.

_{Thus, intra-specific}

differences for ethanol and acetic-acid tolerance could be

adaptively

maintained

by spatially varying fermenting

habitats

along

the north-south

axis of the Indian sub-continent.

ethanol tolerance

_/

acetic-acid tolerance

_/

clinal variation

_/

_{geographical population}

_/

Drosophila immigrans

Résumé - Tolérance à l’éthanol et à l’acide acétique de _{populations géographiques}

de _{Drosophila immigrans.} Des

populations géographiques

de

Drosophila immigrans

ont

révélé une absence de variation

_allozymique

au locus de la

déshydrogénase alcoolique.

Des

clines latitudinaux de tolérance à l’éthanol

_(de

1,5 à

_.!,2%J

et à l’acide

_acétique

_(de

_2,9

à

4,9%)

ont été observés chez les adultes

_de

₄

_{populations géographiques}

de D

_immigrans.

La tolérance à l’éthanol et à l’acide

_acétique

décroît

_quand

on se

_rapproche

de

_{l’équateur.}

Les

modes d’utilisation de l’éthanol et de l’acide

acétique

sont

parallèles

et semblent liés aux concentrations de ces 2 métabolites dans les ressources alimentaires naturelles. Ainsi les

différences intraspécifiques

de tolérance à ces substances

pourraient-elles

être maintenues

sous

l’e,f,!’et

de la sélection naturelle dans des conditions de

_fermentation

soumises à des

variations

_spatiales

le

_long

d’un axe nord-sud du sous-continent indien.

tolérance à l’éthanol et à l’acide acétique

/

variation elinale

_/

populations

géographi-ques / Drosophila immigrans

*

Correspondence

and

_reprints:

_{446/23 (Near}

_Park),

DLF

_{Colony, Rohtak, 124001,}

INTRODUCTION

Colonising

species

populations

offer most suitable material for

_{micro-evolutionary}

studies

_(Endler,

_1977,

_1986).

_Eight

_Drosophila

_species

have been known as

cos-mopolitan

while 21

_drosophilids

have been

_designated

as

wide-spread

(David

and

Tsacas, 1981;

Lemeunier et

al,

1986).

Many

species

of the

_{drosophilidae family}

feed on diverse

_types

of

_fermenting

and

_rotting

_{fruits, vegetables,}

_cacti,

flowers and

decaying

organic

food materials

_(Carson,

_1971;

Atkinson and

Shorrocks,

1977).

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.

Recently

acetic acid has been

found to be a resource

_parallel

to that of ethanol

_(Chakir

et

_al,

_1993).

The

_phenomenon

of ethanol tolerance has been studied from the

_ecological,

physiological

and

_genetic

view

_points

in D

_melanogaster

_(McKenzie

and

_Parsons,

1972; Parsons,

1983).

Alcohol

_{dehydrogenase}

_(ADH)

is known to be involved in the utilisation and detoxification of exogenous alcohols. The fermentation

byproducts

produced

in the environment

_depend

on the

_type

of microflora

(yeasts

and other

microbes)

involved and the

types

of

_organic

matter

_{undergoing decomposition}

(Parsons, 1983).

Thus,

it can be

_predicted

that diverse

_types

of

_drosophilids

could

reflect

_{interspecific}

differences in tolerance to different alcoholic resources. David

and Van

_Herrewege

₍₁₉₈₃₎

revealed D

_melanogaster

and D lebanonsis as

highly

ethanol tolerant while many

species

were found to be ethanol sensitive.

_Except

for

a

_{single preliminary study}

on a D

_immigrans

population

from Australia

(Parsons

and

_Spence,

_1981),

information on D

_immigrans

_populations

from

_temperate

and

sub-tropical

parts

of the world is still

_lacking.

D

_melanogaster

_populations

_living

in wine cellars

_{(ethanol rich)}

and in the

surroundings

(with

low ethanol

_{concentration)}

have revealed microdifferentiation

in the alcohol tolerance as well as in

Adh

F

frequency

(Alonso-Moraga

et

al,

1988).

However,

such information on

_microspatial

differentiation in other

drosophilids

is

_lacking.

D

_immigrans

_populations

were found to

_exploit

man-made alcoholic

fermentation in _sugar mills in

India,

and therefore it was considered worthwhile to

_analyse

the

_possibility

of

_microspatial

differentiation in ethanol tolerance in

D

_immigrans

_populations

from

_sugar-mills

versus fruit stalls. The

objectives

of the

present

study

were to

_analyse

acetic-acid and ethanol utilisation in 4

_geographical

populations

of D

_immigrans

as well as to

_{analyse microspatial}

differentiation in

ethanol and acetic-acid utilisation in local

_sugar-mill

versus fruit-stall

populations

of this

_species.

MATERIALS AND METHODS

Mass bred

_populations

of D

_immigrans

from north India

_(Manali,

32° 0’N and

Rohtak,

28°

_54’N)

and

south

India

_(Pune,

18° 35’N and

_Bangalore,

12°

_58’N)

were used for ethanol and acetic-acid utilisation as well as ADH

polymorphism.

Homogenates

of

_single

individuals were

subjected

to

_{electrophoresis}

at 250 V and 25 mA at 4°C for 4 h. The

_gel

slices were stained for the ADH _gene-enzyme

ethanol and acetic-acid tolerance

_patterns

were assessed

_following

the

_procedures

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 transferred with the

_help

of an

_aspirator

to

_air-tight

_plastic

vials. The flies were not etherised for all the

_experiments.

The control vials contained 2 ml

of

2%

sucrose solution absorbed on cellulose wool in order to

_prevent

_starvation,

while treatment vials were

_supplemented

with various concentrations of ethanol

or acetic acid. 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

difference 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 assessed from the number of flies alive after various time

intervals and

_LT

₅₀

values were

_expressed

as the number of hours at which

50%

of the flies died and were estimated

_by

linear

interpolation.

The ethanol and

acetic-acid threshold values were used as

_indices,

ie if the vapours were utilised as resources

then

LT

50

maximum/LT

50

control was found to be more than

_1;

if this ratio was

less than

_1,

then it acted as

_stress,

and the threshold values were determined when

LT

50

maximum/LT

50

control = 1.

RESULTS

The ADH

_{electrophoretic phenotypes}

revealed 2 banded

_patterns

of faster

_mobility.

Fifty

to 60 individuals of each

_population

did not reveal any variation in the

num-ber as well as

mobility

value of ADH

phenotypes.

These 2 banded

_patterns

were

found to be

_quite

close in

_mobility

to that of D

_melanogaster

individuals

homozy-gous for the fast

electromorph

(Adh

F

).

Thus,

D

immigrans

populations

possess the

fast

_electromorph

_(Adh

_F

_).

The Adh locus was found to be

_monomorphic

in all the

populations

of D

_immigrans.

The northern and southern

_populations

of D immi-grans revealed no

_genetic

divergence

at the Adh locus. The

_longevity

data revealed that south Indian

_populations

of

_Bangalore

had a

_longevity

of 74 h

_compared

with

162 h for the north Indian

_populations

of Manali

_{(table I).}

The data on

_LT

₅₀

maximum/LT

50

control

_(which

constitute a measure of resource versus

stress)

for

the 4 D

_immigrans

_populations

are shown in

_figure

1. The adult ethanol threshold

values were found _{to vary}

_clinally

in the _range of 1.3 to

3.9%

among 4

popula-tions of D

_immigrans

from south to north localities of the Indian sub-continent

(table I).

Thus,

ethanol concentration in the _range of 3.3 to

3.9%

served as a re-sources for north Indian

_populations

while

_{significantly}

lower ethanol concentrations

(1.3-1.7%)

could be utilised

_by

south Indian

_populations

of D

_immigrans.

Since all 4 Indian

_populations

of D

_immigrans

could utilise both ethanol

and acetic acid as a resource _up to

_3%,

_comparative

_longevity

effects at

3%

revealed

_{parallel interpopulational divergence,}

ie under

_experimental

conditions,

longevity

for northern

_populations

was found to be 10 d as

compared

with

5 d in southern

_populations.

LC

50

ethanol concentrations were calculated from

mortality

data of adults on the 3rd

day

of ethanol treatment and these

LC

50

values

populations

(figure 2a).

Thus,

the maximum

_mortality

was observed at

2%

ethanol

Interestingly,

adult

stages

of all 4

_populations

of D

_immigrans

could utilise acetic acid in a

_parallel

_way to that of ethanol. The

_longevity

data revealed

parallel

but lesser effect of acetic-acid utilisation. The data on acetic-acid threshold

values,

LC

50

values,

mortality

and

_longevity

responses further

supported

the

conclusion that the acetic acid was utilised as a resource in all 4

populations

of D

_immigrans

_(fig

2b and table

_I).

In order to determine the extent of

variance test was

performed

for 2

randomly

selected isofemale strains of each of

4

_populations

of D

_immigrans.

_{Significant inter-populational heterogeneity}

was

observed for different acetic-acid concentrations. The correlation was found to be

significant

between latitude and adult acetic-acid tolerance

_(r

=

0.97).

The

_present

study provided

a

_quantitative

assessment of

_{intra-specific}

_patterns

of resource

utilisation in 4

_{geographical populations}

of D

_immigrans

which differed in adult threshold values for ethanol and acetic-acid utilisation.

Microspatial

differentiation

In order to know the level of ethanol

_tolerance,

the

_populations

were

exposed

to

different concentrations

_(1-7%)

of ethanol and acetic acid and the data on ethanol

tolerance indices

_(LT

₅₀

_{h, LT}

_5o

₅₀

_LT

_X/

_a

_M

_control,

adult threshold values and

_LC

_SO

₎

are

_given

in table II. The maximum ethanol tolerance in fruit-stall

_populations

of D

_immigrans

was found to be 154 h while

_sugar-mill

_populations

revealed a

significantly higher

value of 212 h

_{(table II).}

The ethanol threshold concentration

was

4.1%

in the case of

_{sugar-mill populations}

versus

3.3%

for fruit-stall

populations

of D

_immigrans

_{(fig 3).}

The survival data at

3%

ethanol revealed that

_sugar-mill

populations

survived for a

_{significantly longer}

duration than fruit-stall

_populations

(fig 4a).

The

_mortality

data at 4 d ethanol treatment also revealed a

_significant

difference in

_LC

₅₀

_values,

ie

3.2%

for fruit-stall

_populations

and

4.5%

for _sugar mill

_populations

_(fig

_4b).

_{Interestingly,}

the adult

_stages

of both

_populations

_(from

sugar mills and fruit

stalls)

could utilise acetic acid in a

_parallel

_{way to} ethanol.

The increased

_longevity

data revealed a

_parallel

but lesser effect of acetic-acid

utilisation. The data on

intraspecific

variation in acetic-acid threshold

values, LC

50

values,

mortality

and

_longevity

_response revealed

_parallel

_patterns

to that of ethanol utilisation

_{(table II).}

DISCUSSION

The Adh locus has been found to be

_monomorphic

in D

_immigrans

_populations.

Since ethanol tolerance had been

_suggested

to be

_genotype

_dependent,

the observed

intraspecific

difference in ethanol tolerance in D

_immigrans

could be due to

regula-tory

genetic

mechanisms rather than to the Adh locus. The northern and southern

populations

of D

_immigrans

from India revealed

_significant

_genetic

_divergence

in their

_potential

to utilise both ethanol and acetic acid. The lower threshold values of

ethanol

_(1.3-1.7%)

and acetic-acid

_(1.0-1.3%)

utilisation in southern

_populations

of D

_immigrans

seem to be correlated with the lower levels of alcohols in diverse

_types

of the fermented sweet fruits in the

tropical

parts

of the Indian sub-continent. D

im-migrans

populations

utilised ethanol and acetic-acid resources in

_parallel

_ways. The acetic-acid threshold values and

LC

50

values were found to be

_slightly

lower than the ethanol utilisation indices. D

_immigrans

revealed a lack of effective

_allozymic

polymorphism

at the Adh locus and lower ethanol and acetic-acid threshold indices

than D

_{melanogaster.}

It has been

_argued

that

_{neighbouring populations}

can

_undergo

_genetic

differen-tiation in response to environmental

_{heterogeneity,}

ie if the selection pressure is

against

the uniform pressure of

dispersal

and gene flow over short distances

(Slatkin,

1987;

Pecsenye, 1989;

Vouidibio et

_al,

_1989).

_Contrary

to the earlier views of random

mating

populations

of various

_drosophilids

in a

_region,

some cases of sub-divided or

_{meta-populations}

under

_ecological

or behavioural conditions have been

docu-mented

_(Spiess,

_1989).

The

_analysis

of ethanol tolerance of D

_!mrn!m!

flies from

sugar-mill

and fruit-stall areas revealed

significant divergence,

ie the

sugar-mill

pop-ulation was found to be characterised as a subdivided

_population

(metapopulation)

with a

_{distinctly higher potential}

of ethanol tolerance.

Thus,

the

_present

results

agree with the

arguments

of

_Hickey

and McLean

₍₁₉₈₀₎

that

_populations

located

3 km

_apart

or more reveal

_significant

differences in ethanol tolerance. The

_present

data on ethanol tolerance lend further

_support

to the occurrence of

microspatial

ACKNOWLEDGMENTS

The financial assistance from

_CSIR,

New

_{Delhi, is gratefully acknowledged..}

We are

_grateful

to the reviewers for

_helpful

comments and to M Weber for

drawing

some of the

figures.

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