Polymorphism of the hereditary rhabdovirus sigma in wild populations of its host, Drosophila melanogaster

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Polymorphism of the hereditary rhabdovirus sigma in

wild populations of its host, Drosophila melanogaster

A Fleuriet

To cite this version:

Original

article

Polymorphism

of

the

_hereditary

rhabdovirus

_sigma

in wild

_populations

of its

_{host, Drosophila melanogaster}

A Fleuriet

Université de Clermont Ferrand

_II,

Laboratoire de

_Génétique

63177 Aubière

Cedex,

France

(Received

6

_{July 1990; accepted}

22 November

₁₉₉₀₎

Summary — In natural

populations

of

Drosophila melanogaster throughout

the world a

minority

of flies are infected

by

a

rhabdovirus, sigma,

which is not

_contagious

but is transmitted

_through

gametes. Transmission of the virus

_by

males is a cornerstone for

its maintenance in

_populations.

The

_{experiments reported}

in this paper show that in the

wild

_European

_populations

examined the

_efficiency

of transmission

by

males is determined

mainly by

viral _genotype. In African

_{populations, genetic coadaptation}

of both _partners

can lead to a _{very low transmission} of the virus

by

males. Evidence is also

_given

of the coexistence in

_populations

of different _genotypes of the virus. The situation

_reported

is thus another

_example

of the

_{genetic polymorphism displayed by}

the

_sigma

virus in the wild.

Drosophila melanogaster

/

sigma virus

_/

_polymorphism

_/

vertical transmission

Résumé — _{Polymorphisme} du virus héréditaire _sigma dans les populations

na-turelles de son _{hôte, Drosophila melanogaster.} Dans les

populations

naturelles de

Drosophila melanogaster quelle

que soit leur

origine géographique,

un

_{rhabdovirus, sigma,}

est habituellement

présent

dans un

_petit

nombre

d’individus;

ce virus n’est _pas

_contagieux

mais

_uniquement

transmis _par les

_gamètes.

Pour sa

_{perpétuation}

dans les

_populations,

le

virus est

dépendant

de sa transmission _par les mâles. Les

expériences présentées

ici

mon-trent que dans les

_{populations européennes examinées, l’efficacité}

de transmission par les

mâles

_dépend

surtout du

_génotype

viral. Dans les

_{populations africaines}

le virus est très _peu transmis par les

mâles,

ce _{qui peut} être dû à une

coadaptation génétique

des 2

partenaires.

Différents génotypes

du virus coexistent dans les

_populations.

La situation

_présentée

ici constitue donc un autre

_exemple

du

_{polymorphisme génétique}

du virus

_sigma.

Drosophila

melanogaster

/ sigma

/ polymorphisme

/ transmission

verticale

INTRODUCTION

A

_{rhabdovirus, sigma,}

is

_regularly

found in natural

populations

of

_Drosophila

melanogaster

around the world

_{(Fleuriet, 1988).}

_Sigma

virus is not

_contagious

but

is transmitted

_{only through}

_gametes;

it is not

_integrated

in the

_fly

_chromosomes,

but remains in the

_cytoplasm.

_Analysis

of the

_{Drosophila-sigma}

_system

is facilitated

The fact that

_sigma

is not

_contagious

should be

_stressed,

as its maintenance in

populations

is then

_comparable

to that of other

_genetic

elements which are more

efficiently

transmitted than a Mendelian allele.

Fly populations

are also

_polymorphic

for 2

_alleles,

0 and

P,

of a _{gene for} resistance to the

virus,

the

_ref(2)P

locus

_{(Gay, 1978).}

The P

_allele,

which is in

the

_minority

in the

_wild,

interferes with viral

_{multiplication}

and transmission. Two

viral

types

coexist in

_populations:

_type

_I,

which is _very sensitive to the P allele and

type

II,

which is more resistant

(Fleuriet, 1988).

One

_important

characteristic of the

_sigma

virus is that it is

_vertically

transmitted not

_{only by}

females but also

_by

males;

some level of male

transmission,

in addition to the very efficient transmission

through

the female

gametes

is the cornerstone for its maintenance in

_populations.

This

_parameter

has been shown to _vary over

space

(Fleuriet, 1986)

and time

(Fleuriet, 1990).

The

experiments reported

in

this paper were aimed at

_establishing

whether the value of this

_parameter

was

mainly

determined

_by

_fly

or virus

_genotype

(or both).

For this _purpose, viral clones

differing

in the

_efficiency

with which

_they

were

_originally

transmitted

by

males

were transferred into flies of identical

_genotype.

Measurement of male transmission

would then indicate which was the main

_component

of its value. These data also illustrate the

_polymorphism

of wild

_sigma

virus clones and

_give

another

_example

of

coadapted

genotypes

in a host

parasite

system

(Carton, 1986).

MATERIALS AND METHODS

Culture conditions

Flies were maintained on axenic food

_{(David, 1959)}

at 20°C under natural

_light

conditions.

Frequency

of infected flies

The

_C0

₂

test was used to measure the

_frequency

of infected flies as described

_by

Plus

_(1954).

Standard strains

B2’ was a wild strain derived from a

sample

collected in

Brittany

in 1972. The

XM

S

B/Y, IIM

S/

Cy,

IIIMS/DcxF

males used in each

experiment

were the

progeny of a cross between

_X/Y,

Cy/Pm, DcxF 1 Sb

males and

M

5

B

Birmingham

females. These 2 strains carried a wild

_type

fourth chromosome. The

XM

S

B,

IICy

and IIIDcxF chromosomes were

used

to _suppress

_crossing

over because of the inversions

_they

carried.

_0/0

and

_P/P

standard strains were also used

(Fleuriet,

1980).

Wild

_{populations: origin}

of viral clones

1986

_(Fleuriet

et

_al,

_1990).

In the second

experiment

carried out in

_1988,

infected

lines derived from

_samples

collected in

_September

1987 at Biziat

_{(northeastern}

France),

Tfbingen

(Germany,

Pr

_Sperlich),

Andasib6 and Mandraka

_(Madagascar,

Pr

_David).

In the third

experiment

carried out in

_1989,

infected lines derived from

samples

collected in

_September

1988 at

_Biziat,

M6n6tr6ol

_{(central France)}

and

Gilroy

(California,

Pr

_Ayala).

Andasib6 1987 was

_again

used.

Measurement of the transmission of the virus

_by

males

The tested lines were &dquo;stabilized&dquo; lines isolated from

samples

collected in the wild

_(Fleuriet,

_1990).

Each line was assumed to _carry one viral clone

only

(since

most _germ line cells are infected

by

one viral

_particle

only).

In a stabilized

line,

each female transmits the virus and the stabilized condition to its whole _progeny

(Fleuriet, 1988).

Each male transmits the virus to

_only

a

_proportion

of its progeny.

The &dquo;valence&dquo; of a stabilized male

corresponds

to the

_frequency

of infected flies

in its

_offspring

_{(Fleuriet, 1988).}

In these

_experiments,

valences were measured in the _progeny of individual males crossed with uninfected

_0/0

females of a reference

strain.

_(Valences

were also measured in the progeny of males crossed with uninfected

P/P

females for determination of the viral

type

(Fleuriet, 1988),

but the results will not be

_presented

in detail in this

_paper.)

Protocols

Experiment

1

The

_protocol

used in this

_experiment

is as described in

_figure

_1,

and is based on the fact that stabilized females transmit the virus and the stabilized condition to their entire progeny. The

experiment

was carried out until

_generation

4

_only.

Experiment

2

The

_protocol

is

_presented

in

_figure

_1,

and is

_exactly

the same as in

_{Experiment I,}

with 2 additional

_generations,

which resulted in each viral clone

_again

_being

in the

original

genotype

of the

_{corresponding}

line.

Experiment

3

The

_protocol

for this

_experiment

is

_given

in

_figure

5.

RESULTS AND DISCUSSION

Experiment

1

The valence of a male is the

_frequency

of infected flies in its progeny. The

average value of valences in a line is characteristic of that line and is transmitted over

_generations.

Previous observations have shown

that,

in natural

_populations,

valences can _vary over _space

(Fleuriet, 1986)

and time

(Fleuriet,

1990).

This

virus was transmitted

_by

males in a line

depended

mainly

on

fly

or virus

_genotype

(or both).

For this purpose, the viral clones

perpetuated

in different lines with

high

or low valences were transferred into

isogenic

flies. If valence was

mainly

determined

by

fly

genotype,

it would then become identical for all the viral

_clones,

whatever its initial value may have been. If valence was

_mainly

of viral

_origin,

the differences observed between lines would

persist,

even after standardization of

fly

_genotype.

The

_protocol

used in this

_experiment

is described in

_figure

1. Thirteen lines were tested each of them

_bringing

its viral clone. At the end of the

experiment

(gener-ation

_4),

the 13 viral clones

_perpetuated

in these lines were carried

by

13 lines whose

_genotype

had been made identical. The

_genotype

chosen was that of a strain of wild

_{origin kept}

in the

_laboratory

since 1972

_(B

_2’

_strain).

It would of course have been easier to

_inject

viral clones into flies of the chosen

genotype.

This was not

_done,

since it is well known that the viral

types

selected for are not the same after

injection

or

_hereditary

transmission

(unpublished results).

The intention was to remain as close as

possible

to viral

_types

found in wild

populations.

Viral clones were thus

_only

transferred

through

maternal transmission. This was also the reason

why

each

experiment

was

performed

on

recently

collected viral

_samples

_(collected

.less than 6 months

_ago).

Valences were measured on

G

4

males in which the

B

2’

_genotype

had been

reconstituted.

They

were also measured on

G

3

males of the same

_genotype

as those used to

_produce

_generation

4

_{(fig 1).}

The reason

_why

these

G

3

males were also examined was that it was not

_certain,

a

_priori,

that

enough G

4

males of the chosen

genotype

would be obtained at the end of the

_experiment,

and that _many of them would not be sterile.

_G

₃

males did not

_present

the entire

_B2!

_genotype,

but

_only

half of it for the 3 main chromosomes

_(the

fourth

_chromosome,

which carries very

few _genes, was not controlled in these

_{experiments).}

The

_{important point}

is that

they

were nevertheless of identical

_genotype.

Results are

_presented

in

_figure

2. In many cases, data were too scarce to allow

precise quantitative

comparison.

Some

_unambiguous

conclusions can nevertheless be drawn from a

_qualitative

_analysis

of the results. Three series of measurements were

performed

on each line

_(see

_{fig 2).}

Lines were distributed

according

to the

original

value of valence. It appears

clearly

that in

graphs

c, where

genotypes

have been standardized for all the infected

lines,

valences are not identical in the different

lines;

when valence is

_high

in the line

_{(graph a),}

it remains

_high

in

_B

_2’

_genotype

(J,

K, L,

M).

When it is weak or

heterogeneous,

(A,

B, C, D, E, F, G, H,

I),

it

remains so in the

_{B2! genotype.}

This indicates that the valence value observed in

a line is

_mainly

of viral

_origin

since it

_keeps

its

_original

value even after the

fly

genotype

has been made uniform.

But another observation confirms what has

_long

been known

_(unpublished

results):

some

_fly

_genotypes

can

_modify

valence. In

_graph

b,

(ie

on

_G

₃

males of

M

S/

CyDcxF

genotype),

for lines

_presenting

weak

_values,

valences are

systemati-cally

higher

than in

_graphs

a and c. It is clear that in this

particular

_genotype,

viral clones are better transmitted than in the

_original

_genotype

of the line. It is to be noted that this

_genotype

is artificial and does not exist in the

_{wild, contrarily}

to

original

or B2’

_genotypes,

all of wild

_origin.

It indicates

that,

on the average, these

restricted in the wild

_by

their host

_genotype.

These observations are true for

_type

I

(with

reference to the P

_allele)

viral clones

_(A,

B, C, D,

J),

as for

_type

II viral clones

_(E,

F,

G, H,

I,

K, L,

M).

Measuring

in each case valences with a

P/P

reference strain also

_shows,

as was

expected,

that

_modifying

the

_{fly genetic background}

does not

_change

the

_sensitivity

of viral clones to the

_ref(2)P

locus,

which is a viral characteristic

(data

not

_presented

here).

Experiment

2

The aim was the same as that of first

_experiment,

with an additional control. An

might

explain

the difference observed between

_graphs

a and c in

figure

2G for

example.

As a

_control,

the

_original

_genotype

of each line was thus reconstituted at the end of the

_experiment

and male valences were then measured

again.

The

protocol

is

_presented

in

_figure

1. It is

_exactly

the same as in

_{Experiment 1,}

with 2

additional

_generations,

which result in each viral clone

_again

_being

in the

_original

genotype

of the

_{corresponding}

line. Four measurements of valences are

_performed.

For

_populations

of

_{European origin}

_{(8 lines),}

results are

_presented

in

_figure

3. As in

figure

2,

lines are distributed

_according

to their

valence,

weak or

_strong.

Most of the results are

_quite

similar to those of

_Experiment

1.

_{Unfortunately, only}

one viral clone

among those available was _very

_efficiently

transmitted

_{(graph H).}

Nevertheless,

a

_comparison

of

_graphs

_He,

Ac and

Be,

for

_example,

for which

_original

valences were

_{unambiguously different, clearly}

shows that valences remain different once

_fly

genotype

has been standardized.

may be

explained by

a variation of viral

_genotype:

the values observed on

_d,

after

reconstitution of the

_original

_genotype

also differ from a, but in each case, c and d

are _very close to each other. For

_F,

on the _contrary, the results observed in c are much more similar to those in a than those observed in

_d,

2

_{generations later,}

when the

_original

_genotype

has been reconstituted.

The results of

_Experiment

2 confirm those of

_Experiment

1: male valence in these lines is not determined

_by

_fly

_genotype

but is

_mainly

of viral

_origin.

This conclusion

might

be of

_{general significance,}

since the

European

populations

examined in

Experiment

2 differ from those of

_Experiment

1 in their

_geographical

_origin

and also their evolution in the wild

_(Fleuriet,

_1990).

These results confirm

_previous

observations made on natural

populations

(Fleuriet

et

al,

1990).

As in

Exper-iment

_1,

viral clones are better transmitted

_by

males of

M

5/

CyDczf

_genotype

(graphs

b).

The results observed with 2

_populations

of African

_origin

are

_presented

_in

figure

4. It has

_previously

been shown

_{(Fleuriet, 1986)}

that in African

_populations,

valences are _very low and the

_sigma

virus is

_practically

not transmitted

_by

males

(<

10%

of their

_progeny).

The purpose of this

analysis

was to determine whether this low transmission is of viral or

_{fly origin.}

The

_protocol

used was that

_presented

genotype

(graphs

b and

_c)

_they

are much better transmitted. When

_put

back into

the

_original

_genotype

_{(graph d),}

valence

_regains

its

_original

_value,

which excludes selection of more efficient viral

types

during

the

experiment.

It thus appears that in these

populations,

transmission of the virus is

_strongly

restricted

by

the host. The clone collected at Andasib6 can be _very

_efficiently

transmitted

_by

males of another

_genotype.

_{The progeny of the clone collected} at Mandraka _appears to be

heterogeneous:

some clones are _very

efficiently

transmitted,

while others are

_only

a little better transmitted in

_{B2! genotype.}

It is not

_possible

to determine

_clearly

whether this reflects a

_segregation

of viral or

_B

_2’

_genes. A few other

examples

are known where transmission of viruses is restricted in their host vectors

_(Hardy

et

_aL,

1983).

In the presence of the P

allele,

transmission of the virus

_by

males remains nil

as it was in the

_original

_genotype,

since Andasib6 and Mandraka clones are

_type

I clones

_(data

not

_presented

_here).

The

_genetic

determinism of transmission restriction in flies is not known. The

protocol

used would not discriminate between the effect of one locus or of various

loci on the 3 main chromosomes. This effect cannot be due to the

_ref(2)P

_P

allele:

firstly,

its effect _upon viral clones is not the same and

secondly,

P allele

frequency

in these

_populations

_{is very} low

_{(below 0.05)}

as it is in other African

_populations

(Fleuriet, 1986).

It was of course verified at the end of these

_experiments

that no

accidental fixation of the P allele had occurred in the Andasib6 strain while it was

kept

in the

_laboratory.

It is assumed that in other African

_populations

a

comparable

_system

is also

effective, responsible

for the _very low transmission

_by

males observed in all

the

_populations

examined and very different from that

prevailing

in

European

populations

(Fleuriet, 1986)

It seemed of interest to determine whether this restriction of transmission was

specifically

directed

_against

viral clones

perpetuated

in these

_populations

or whether

any viral

genotype

would be affected. The purpose of

Experiment

3 was to

_try

to answer this

_question.

Experiment

3

This

_experiment

was aimed at

_determining

whether the host

_genetic

mechanism

lowering

viral transmission

_by

males in the Andasib6

_population

was effective on

any viral clone or on the Andasib6 virus

_only.

For this _purpose 7 viral clones were

chosen, differing

in their

_genetic

characteristics

_(eg

M6n6tr6ol and

_{Biziat; Fleuriet,}

1990)

or their

_geographic

_origin

_{(France, USA);}

some were of viral

_type

_I,

_eg the

Andasib6

_virus,

others of viral

_type

II. All were transferred

_by

maternal transmission

into flies of Andasib6

_genotype

_(chromosome

4 was not

_controlled),

and valence of males measured. The

_original

_genotype

was then reconstituted in each line and

valence of males measured

_again,

to _{check any}

_possible

variation of viral

_genotype

(see

protocol

in

_figure

_5).

As a

_control,

the same _process was used with the Andasib6

infected line in order to ensure that the

genetic

effect detected in

_Experiment

2 was

The measurements made on the Andasib6 line

_{(graph 7H)}

show that the

_genetic

determinism of restriction was still

_present

in the Andasib6

_genotype

(compare

graphs

b and

_c)

even if it was somewhat less efficient than

_originally

_(graphs

a and

b).

This difference shows that

_fly

_and/or

virus

_genotypes

evolved

_slightly

in the

laboratory

since their collection 2 _yr before.

Of the 7 lines

_examined,

4 were almost unaffected

_by

the Andasib6

_genotype

_(A,

B,

C,

D).

All had been collected in

_France;

_among

_them,

one was

_type

I

(C),

the other 3 were

_type

II. A

_strong

effect was observed on the E viral clone. It was a viral

_type

_II,

from the same

_population

_(M6n6tr6ol)

as the D viral clone.

Some effect was observed on the 2 lines from the same American

_population

(Gilroy),

both of viral

_type

I: a weak effect on the F

_clone,

but a _very

_strong

one on the G clone. The effect was thus not

_specific

to the viral

_type

_(I

or

II)

nor to

the

_{geographic origin.}

2 _{yr in} the

_laboratory.

But one cannot exclude the

_possibility,

if

_only

one locus is

involved,

of fixation of the allele for resistance in the Andasib6 strain.

It thus appears from this

experiment

that in

populations

of various

geographic

origins,

some viral clones can be found which are sensitive to the

_genetic

restriction

present

in the Andasibe

_{population. They}

_might

be in the

_minority

₍₂

clones as

strongly

affected as in the

_original

_population,

out of 7

_examined).

_They

can

_coexist,

as another

example

of the viral

polymorphism,

with viral clones resistant to that

_fly

genotype

(as

observed in the Menetreol

_population).

A

_parallel

_might

be established with the situation at the

_ref(2)P

locus: 2 viral

_types,

one _very sensitive to the P allele

_{(type I)}

the other more resistant

(type II),

coexist in

_populations

in _presence of the P allele

_{(Fleuriet, 1988).}

The difference is that in the

_population

_(M6n6tr6ol)

where the 2

_types

of clones have been

_found,

there is no indication that the Andasib6

alleles(s)

might

be found

_(and

this observation is another

_example

of mutation

randomness). Conversely,

in the African

_populations

where these restriction alleles are

_found,

no indication of a viral

_polymorphism

has as

_yet

been observed.

The

_{interesting point}

is that viral clones resistant to these alleles can thus be found in wild

_populations,

but none has

_yet

been collected in Africa. This

_might

be

_interpreted

as a local

_coadaptation

of both

_partners,

the virus and the

_fly;

it would lead to a low

_frequency

of infected flies in these

_populations

where for unknown _reasons, the P allele

_frequency

_{is very} low

_{(Fleuriet, 1986).}

A similar

effect,

preventing

the virus from

_invading

host

_populations

thus _appears to take

_place

in

European

(Fleuriet,

1988)

and African

populations

through

different mechanisms. It may be assumed that other such

systems

of

_coadapted

_genotypes

would be found

in

_samples

collected around the world. ACKNOWLEDGMENTS

I would like to thank F

_Ayala,

J David and D

_Sperlich

for

_collecting

flies and JC

Bregliano

for

_{critically reading}

the

_manuscript.

This work was

_{supported by}

_grants

from the Centre National de la Recherche

_Scientifique

_(UA

360, GRECO

₄₄₎

and the Fondation pour la Recherche Medicate

Franqaise.

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