Selection of allozyme genotypes of two species of marine gastropods (genus Littorina) in experiments of environmental stress by nonionic detergent and crude oil-surfactant mixtures

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Selection of allozyme genotypes of two species of marine

gastropods (genus Littorina) in experiments of

environmental stress by nonionic detergent and crude

oil-surfactant mixtures

E. Nevo, B. Lavie

To cite this version:

Original

article

Selection of

_allozyme

_genotypes

of

two

_species

of

marine

_gastropods

_{(genus Littorina)}

in

_experiments

of

environmental

stress

_by

nonionic

detergent

and crude oil-surfactant

mixtures

E. Nevo

B. Lavie

University

of Haifa,

Institute

_of

_Evolution,

Mt

_Carrnel,

_Haifa,

Israel

(received

25

_{January 1989, accepted}

5

May

1989)

Summary - Two marine

gastropods,

Littorina punctata and L. neritoides were

_exposed

in

_{laboratory experiments}

to the controlled environmental stress of

_{pollution by detergent}

and

_by

crude

_{oil-detergent}

mixtures in aqueous solutions. The

allozyme frequencies

of

phosphoglucose

isomerase

_(PGI)

were tested in both

_species

and

_{amino-peptidase}

(AP)

only

in L. neritoides. Our results indicate differential

survivorship

of

_allozyme

genotypes

for both

_species,

both types of

_pollution

and both enzymes observed. These results indicate the

_sensitivity

of

_allozymes

to environmental _stress, reflect the

_adaptive

nature of some

allozymes,

and _support the idea that

_allozymes

could be used as detectors of

_organic

pollutants

in the sea.

selection - _{organic pollution - allozyme polymorphism -} marine _gastropod

Résumé - Sélection de _{génotypes allozymes} chez deux _espèces de _{gastéropodes} marins

(genre Littorina)

dans des _expériences de stress environnemental par un _détergent non

ionique et par ses _mélanges avec des huiles brutes. Deux

_{gastéropodes marins,}

Littorina

punctata et L.

neritoides,

ont été

_exposés

en laboratoire des stress environnementaux

correspondant

à une

pollution

par un

détergent

_{et par} ses

mélanges

avec des huiles brutes en solutions _{acqueuses. Les}

_fréquences

des

_allozymes

de la

_{phosphoglucose-isomérase}

(PGI)

ont été testées chez les deux

_espèces,

et celles de

_{l’amino-peptidase}

_(AP)

chez L. neritoides seulement. Les résultats

_indiquent

une survivance

différentielle

des

différents génotypes,

pour les deux

espèces,

pour les deux types de

_pollution,

et pour les deux enzymes étudiées.

Ces résultats

_indiquent

que les

adloxymes

sont sensibles à des stress de

l’environnement,

ils

reflètent

la nature

_adaptative

de certains

_aldozymes,

et

_renforcent

l’idée que des

allozymes

puissent

être utilisés pour détecter des

polluants organiques

dans la mer.

sélection - _{pollution organique - polymorphisme enzymatique - gastéropode} marin

INTRODUCTION

The

_evolutionary

_significance

and

_dynamics

of the vast amount of

_protein

polymorphisms

in

_nature,

and the relative

_importance

of the deterministic and

this variation is

_largely

_adaptive,

then it is

_exploitable

in

_breeding

_(Nevo

et

_{al., 1982,}

Nevo

_{1986), meaningful}

in conservation

_(Frankel

&

_Soule,

_1981;

Schonewald-Cox

et

al., 1983; Soule,

1987),

and

_exploitable

as a detector and monitor of environmental

quality

(Nevo

1986,

Nevo et

_al.,

_1983).

In the

present

study,

we

_tested,

in controlled

_laboratory

_experiments,

the influence of a nonionic

_detergent

and of crude

_{oil-detergent}

mixtures in aqueous

solutions on

_allozymic

variation in the related

species

of marine

_gastropods

Littorina

punctata

and L. neritoides. Their

_genetic

structure, based on 17 loci in three

Mediterranean

sites

_along

the Israel coast was described

_{by Noy}

et al.

_(1987).

Of

the 17 loci

_{tested, only}

_{phosphoglucose}

isomerase

_{(PGI )}

for both

_species

and amino

peptidase

(AP )

for L. neritoides were found suitable for

_{pollution investigations,}

which

_depend

on the

_{following stringent}

criteria:

- Loci tested for

pollution

must be

_{strongly polymorphic}

_(>10%)

in order to

detect

differential

_mortality

in

_sample

sizes

_involving

several hundreds of animals.

- The

enzyme tested must also remain active in the dead animals so that the

distribution of

_genotypes

can be

compared directly

between dead and live animals.

Thus,

no

_sampling

error is

added

that would bias the results.

- A

high

resolution is

imperative

when

_scoring

the

_{electrophoretic results,}

because

the difference in

_{allozymic frequencies}

between live and dead animals may be small.

Our results indicate differential tolerance of

_allozymes

to both

_{pollutants tested,}

as was found in our

_previous

studies

_(reviewed

in

_Nevo,

_{1986), implying}

that at

least some of the

_{genetic diversity}

found in natural

_populations

may be

exploitable

in the service of man as a

_genetic

monitor of marine

_pollution.

MATERIAL AND METHODS

Species

tested and

_{experimental design}

The

present

study

involves two related marine mollusc

_species:

Littorina

_punctata

(Gemelin)

and L. neritoides

_(L.)

whose

_ecology

in Israel was studied

_by

Palant & Fishelson

_(1968).

Several hundreds of individuals from each

_species

were collected from the

_rocky

shores of the Haifa

_region

and introduced in batches of 50-100

individuals into

_partially

filled 80 L

_aquaria

₍₇₀

x 30 x 40

_cm)

at the Institute

of

Evolution,

University

of

_Haifa,

Israel. Fresh sea water was

pumped

from a 30m

depth

at the Shikmona National Institute of

_Oceanography

and

_transported

to the

laboratory.

The two Littorina

_species

tend to crawl on the

_aquaria

_walls,

above the

water level. To avoid

this,

they

were

placed

in

quadrangular

cages

(25

x 25 x 5

_cm)

made of _{perspex. These cages} were subdivided into smaller interconnected cells

(5 x 5 x 5 cm)

by

a

_plastic

net so that water currents _{could pass}

_{freely through}

all cells. Each cell held two animals. Conditions in all

aquaria

were identical

(22

°C,

pH

= 8.1;

constant aeration and no food was

_provided

_throughout

_testing).

All tests

were conducted

_{simultaneously}

and were matched with a control. Survival in the controls was

_always

100%.

The

_experimental

_organisms

were observed

_daily;

dead animals were removed and frozen

_(-80

°C).

When

_mortality

reached

50%

(LD

so

),

the test was terminated and all survivors were frozen

(-80 °C).

The duration of the

test

_{(2-10 days)}

_{varied, depending}

_upon the concentrations of

_pollutants

_(Tables

_I,

II).

Similar trends have been observed in all

_{concentrations,}

therefore the results

The

_pollutants

The nonionic

_detergent

used was the

_commercially

available

_Marlophen

89

_(Hulls,

West

_Germany).

This

_product

is a

nonylphenol ethoxylate

having

an

approx-imate molecular

_weight

of

643,

the molar ratio between the

_ethylene

oxide and the

_{nonylphenol being}

9.55:1

_{respectively.}

This nonionic

_detergent

is of the

&dquo;hard&dquo;

_type,

that

is,

resistant to

_{biological degradation.}

The crude oil used

(&dquo;Sonol

2&dquo;,

obtained from the Israeli Oil Refineries

_Ltd,

in

_Haifa),

had the

fol-lowing specifications: density

at 15 °C: 0.9152. Distillation

_Range

_(%

_wt):

to

150°C,

8.3%;

150-250

_°C,

_17.7%;

250-400

_°C,

_21.2%;

Res. >

_400,

51.9%.

Kine-matic

_Viscosity:

at 122

_f,

15.8 cP. Total Sulfur

_{(% wt):}

1.4. Salt PTB: 11.

Water

_{(% V):}

0.05. Carbon Residue

_{(% wt):}

_5.3;

_Asphaltenes

_(%

_wt):

0.8.

Electrophoretic analysis

For the

_{electrophoretic analysis,}

whole frozen animals were

homogenized

and studied

_by

horizontal starch

_{gel electrophoresis}

_(Selander

et

al.,

1971).

For the

preparation

of the buffers used for the assay, see

_Noy

et al.

_(1987).

The alleles were recorded

_according

to their

place

on the

_{electrophoretic gel:}

F was the fastest

(most

anodal)

and S- was the slowest allele. The PGIlocus in both

species

had 3 alleles

(S,

M,

F);

the AP locus had 5 alleles

_{(S-, SMM}

₊

_,

_F).

As the

_{electrophoretic analysis}

is much more

_expensive

and time

_consuming

than the

_viability

_survey,

_only

_part

of the

_participants

in these

_experiments

were

_{analyzed electrophoretically}

_(Table

_I,

II).

RESULTS

Table I summarizes all the data obtained on

_{allozyme frequencies}

under treatment with nonionic

_detergent

and crude

_{oil-detergent}

mixtures in aqueous solutions at

the PGI

_locus,

for Littorina

_punctata.

When the

_pollutant

used was the nonionic

detergent by

itself,

selection acted

_against

the

_genotype

FF and the allele F in

general

and favored the

_genotype

MM. The fact that the statistical results also show a

_{higher frequency}

of the allele M among the live animals than among the

dead,

is a _consequence of the

_huge

_proportion

of the

_genotype

MM _among the live

animals;

other M

_bearing

_genotypes

were not favoured.

’

When the

_pollutant

used was crude oil and non-ionic

_detergent

_mixture,

a

reversion in the

_viability

fitness could be observed: In

_general,

the FF

genotype

and F allele were favoured

_by

the selection which acted

_against

the SS

_genotype

and the S allele.

Table II summarizes all the data obtained on

_{allozyme frequencies}

under treat-ment with nonionic

_detergent

and crude

_{oil-detergent}

mixtures in _aqueous solutions

at the PGI and AP loci for Littorina neritoides. The trends were similar in both

types

of

_pollution,

but more accentuated in the combined

_pollution.

_{Heterozygotes,}

in

_general,

showed

_higher

fitness

_viability

_(especially

F1VI in the

_{PGI system}

in oil

pollution.

In the AP

_system,

the

FIBr

+

in

_{detergent pollution}

and

M

+

S

in oil

pollu-tion). Considering

homozygotic

genotypes,

the

_{detergent applied by itself}

favoured the FF

_genotype

of

_PGI,

and the combined

_pollution

acted

_against

the MM

Notably,

while the trend of

_{higher frequency}

of

_{heterozygotes}

_among the

sur-vivors of oil

_pollution

was not

_significant

for Littorina

_punctata

_alone,

the combined

probabilities

for both

species

was P <

0.001; x! =

25.4346

_(Sokal

&

Rohlf,

1969).

DISCUSSION

The effects of

_detergents

on the marine biota were

_{reported by}

several studies:

Man-well & Baker

₍₁₉₆₇₎

_performed

starch

_{gel electrophoresis}

on a

_variety

of _enzymes

and other

_proteins

that had been

_exposed

to

_{detergent pollution. They}

worked on

in vitro

tissues,

so

_they

were unable to observe the differential

_survivorship

of

vari-ous

_genotypes.

They

discussed

only

the

general

influence of

detergent

on

solubility,

activity

and

_{electrophoretic}

_mobility

of

proteins.

The in vivo effects of

_detergents

on the marine biota were

_reported

in several

studies;

on the chemical senses of

fish

_(Bardach

et

al.,

1965)

on marine

_gastropods

_{(Bryan, 1969)}

and on crustacea

(Kaim-Malka, 1972).

But these studies did not deal with the influence of

_detergent

pollution

on the

_genetic

_patterns

of

allozyme

polymorphisms.

Oil

_pollution

of the sea has received a

_great

deal of attention from research

be shown in

_laboratory

_experiments

to be harmful to marine

_organisms.

Effects of oil

_pollution

on coral reef communities has been

_critically

reviewed

_{by Loya}

& Rinkevitch

_(1980).

_They

reported

a

syndrome

of oil

effects, including complete

lack of colonization

by hermatypic

corals in reef areas

_chronically

_polluted

_{by oil,}

decrease in

_{colony viability, damage}

to the

_reproductive

_systems

of

_corals,

lower life

expectancy

of

_planulae,

and abnormal behavioral _responses of

_planulae

and corals.

For the

_{herring gull}

chick

_{(Larus argentatus),}

Miller et al.

₍₁₉₇₈₎

_reported

that a

single

small oral dose of Kuwait or South Louisiana crude

_oil,

caused cessation of

growth, osmoregulatory

impairment,

and

_hypertrophy

of

_hepatic

adrenal and nasal

gland

tissue. The

_biological

effects of oil

_pollution

on littoral communities were

described

_by

Beer

_(1968),

and the acute toxicities of crude oils and oil

_dispersant

mixtures on marine fish and inverterbrates were

_{reported by}

Eisler

_(1975).

A

_post

hoc

_approach

to the

_problem

of selection pressure on

specific

_genotypes

of marine

_{organisms by}

oil

_pollution

was

_{reported by Battaglia}

et al.

_(1980),

who

compared

the

_genotypic

_pattern

of Tisbe holothuriae and

_{Mytilus galloprovincialis}

at sites with various

_degrees

of oil

_pollution.

They

found

_significant

differences in

viability, especially

for

genotypes

of the locus

PGI

In an

_early

_study

with

_detergent

and with crude

_{oil-detergent pollution,}

we have found in

laboratory

controlled

experiments,

differential

_viability

of PGI

_genotypes

of the marine

_gastropods

Monodonta turbinata and M.

_turbiformis

_(Lavie

et

_al.,

₁₉₈₄₎

and of six loci tested in the marine

_gastropod

Cerithivrn

_scabridvrra,

either

_singly

or in a two-locus

_genetic

structure

_(Nevo

&

_Lavie,

_1989).

The present

study

reports

the selective nature of the

_genotypes

in an a

_priori

designed

experiment,

with

_precise

concentrations of

_specific

_pollutants,

_avoiding

any effects of additional unknown

pollutants.

The

pollutant

concentration needed in order to obtain

_LD

₅₀

_was, in all _cases, much

_higher

than its

_{concentration,}

even in the

_polluted

sea environment.

_Yet,

while

_pollution

_{effects may be} small

in open sea

_situations,

effects are

certainly

appreciable locally

in estuaries and

coastal waters. The

_{laboratory experiments}

considered

_{only survivorship}

because

they

involved

species

that do not

_{reproduce successfully}

in

_laboratory

conditions.

Yet,

in

_nature,

tolerant

_genotypes

may not

_only

live

_longer,

but

_presumably

also

reproduce successfully

in the

_polluted

environment.

Like

_Battaglia

et al.

_(1980),

we found that oil

pollution

favoured the

heterozy-gotes.

As in our

_previous

studies on

_detergent

and oil

pollution

(Lavie

et

al., 1984;

Nevo et

_al.,

_1988),

we found for L.

_neritoides,

similar trends in

_detergent

pollu-tion

_by

itself and in the

_{pollution by}

oil-surfactant mixtures. This may have been

influenced

_by

the fact that the surfactant used in the oil-surfactant

_mixtures,

was the

_detergent

in case. Yet for L.

punctata,

the

synergism

of the two

_pollutants

pro-duced results

_{completely opposed}

to the

_{pollution by detergent}

alone. This is in

good

accord with our

_{previous experiments}

on the

_synergetic

effects of

pollutants

(cadmium

and mercury

pollution).

We found that the combined

pollution

had a

unique

influence on the distribution

of allozyme frequences

for Cerithium scabridum

(Lavie

&

_Nevo,

_1988);

for L.

_punctata

and L. neritoides

_(Lavie

&

_Nevo,

_1987),

and for Palaemon

_elegans

_(Ben-Shlomo

&

_Nevo,

_1988).

The biochemical mechanisms

involved in differential

_viability,

and the

_specific

_targets

of selection need future

We conclude that the

_present

_study

_supports

_previous

ones

(reviewed

in

Nevo,

1986),

suggesting

that at least some

_allozyme

_{polymorphisms}

are

_subject

to natural selection.

_Actually,

_during

our _{research program in} controlled environments

₍₁₀

types

of

_pollutants

and six

_species

of marine

_organisms),

_only

a

_single

case of no differential

_survivorship

due to

_pollution

could be detected

_(e.g.

in Monodonta

turbifor!ais

in zinc

_pollution)

_[Nevo

et

_al.,

_1983!.

The

_sensitivity

of

_change

in allele

frequencies

of

_allozymes

to environmental stress makes them

appropriate

as

_genetic

detectors and monitors of

_{organic pollutants.}

Such direct

_genetic

indicators can

complement

other methods of bio- and chemical

_monitoring.

The merit of

_genetic

monitoring

is that it alerts us to both the short-and

long-term genetic

changes

that

populations undergo,

before their extermination

_resulting

from environmental stress

pollution.

ACKNOWLEDGEMENTS

We thank Shimon Simson for field and

_laboratory

assistance,

and wish to extend our

_{deep gratitude}

for financial

_support,

to

_FAO/UNEP,

to the Israel Discount Bank Chair of

_Evolutionary

_Biology

and to the Ancell-Teicher Research Foundation

for Genetics and Molecular

_Evolution,

established

by

Florence and Theodore

Baumritter of New York.

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