The evolutionary history of Drosophila buzzatii. XVII. Double mating and sperm predominance

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The evolutionary history of Drosophila buzzatii. XVII.

Double mating and sperm predominance

A Barbadilla, Je Quezada-Díaz, A Ruiz, M Santos, A Fontdevila

To cite this version:

Original

article

The

_evolutionary

_history

of

_Drosophila

buzzatii.

XVII.

Double

_mating

and

_sperm

predominance

A

Barbadilla

JE

_{Quezada-Díaz,}

A Ruiz

M

_Santos,

A Fontdevila

Universidad Autdnoma de Barcelona,

De

P

artamento

de Genetica y Microbiologia,

08193

_{Bellaterra, Barcelona, Spain}

(Received

22

_January

_1990;

_accepted

30

_January

₁₉₉₁₎

Summary -

Sperm predominance

in males and double

mating

in females have been studied

in 2 stocks of the

cactophilic species Drosophila

buzzatii. The

_relationship

between double

mating

and total

_productivity

of females was also ascertained. Our results show

_high

values

of _sperm

_predominance

and double

_{mating. Moreover,}

female

_productivity

is increased with a second mate. These results are discussed in relation to the

_mating

strategy of this

species.

Drosophila buzzatti

_/

sperm predominance

/

double mating

/

mate _{strategy /} total

productivity

Résumé - Histoire évolutive de Drosophila 6uzzatü. XVII. Accouplement double et

prédominance du sperme. On a étudié la

_{prédominance}

du _sperme chez les mâles et le double

accouplement

chez les

femelles

dans 2 souches de

_{l’espèce cactophile Drosophila}

buzzatii. La relation entre le double

_accouplement

et la

productivité

totale de.s

_femelles

a été aussi recherchée. Nos résultats montrent des ’valeurs élevées pour la

prédominance

du sperme et pour le double

accouplement.

De

plus,

on _{constate que} la

_{productivité}

des

femelles

est

_augmentée

_par un deuxième

accouplement.

Ces résultats sont discutés par

rapport à la

stratégie d’accouplement

de cette

espèce.

Drosophila buzzatü

_/

_{prédominance du sperme}

_/

_accouplement double

_/

_stratégie

d’accouplement

/

productivité totale

INTRODUCTION

Multiple mating

is a

_widespread

_phenomenon

_{among insect} females

(Thornhill

and

Alcock, 1983; Smith,

1984;

Ridley,

1988).

If the _sperm of the first male is not

exhausted before female

_remating,

then sperm

competition

occurs in the

_storage

organs of the female

between the sperms of different

origin

(Parker,

1970,

1984).

Sperm predominance, usually

that of the last mated

_male,

is the

_general

result of

*

this

_competition.

When there are

_genetic

differences in the

_degree

of

_{predominance,}

sperm

predominance

may result in sexual selection. Prout and

Bundgaard

(1977)

showed

_{theoretically}

how this

_type

of selection could maintain a

_population

in stable

equilibrium

for 2 alleles. The

_{mating strategies}

of many

species

are determined

_by

the

importance

of _sperm

_predominance

in males and

_{multiple mating}

in females

(Smith, 1984).

Several

_experimental

studies have been carried out to ascertain the

_degree

of sperm

predominance

in

Drosophila

(Gromko

et

al,

1984).

In the

present

work,

we

have studied sperm

predominance

in the

_cactophilic

_species

_Drosophila

buzzatii.

In

_addition,

the

_frequency

of double

_mating

and its influence on the total female

productivity

were determined. D buzzatii

_belongs

to the

_repleta

_group of

_Drosophila

and several

_aspects

of its

_ecology

and

_mating

behaviour have been

_extensively

studied in our

laboratory

(Ruiz

et

al, 1986,

Santos et

al, 1988,

1989).

MATERIALS AND METHODS

Two stocks were used in this

_{experiment. One,}

the wild

type

stock,

was derived

from a natural

population

collected at

_Carboneras,

Almeria

_{(SE Spain),}

in

_May

1986. The other stock was

_homozygous

for the sex-linked recessive white mutant

which arose

_{spontaneously}

and was

_subsequently

isolated in our

_laboratory

in

_April

1983. Since no

_attempt

was made to randomize the

_genetic

_background

of the 2

_{stocks, they}

_might

differ at _many loci and the mutant white was

_merely

a

_genetic

marker.

The

_{experimental procedure}

was similar to that of Turner and Anderson

_(1984).

The crosses

_performed

are shown in table I. The

w/+

females and the

w/Y

males

were the

_{hybrid offspring}

from the 2

_parental

stocks. The

_experiment

_began

with the first cross. Five to 6-d old

_virgin

females were crossed

individually

with 2 males

of the same _age. The crosses were carried out in 2 x 8 cm vials with ca 8

cm

3

of

All the individuals were _grown at

_{nearly optimal density}

_(4-5

larvae _per

cm

3

of

medium).

A modified formula of David’s

_{killed-yeast Drosophila}

medium

_(David,

1962)

was used as food. The flies were

_kept

at 23°C. The

_offspring

of each female was classified

_by

sex and

phenotype.

Statistical

_analysis

were conducted with the BMDP Statistical Software which was

_implemented

on the VAX

Operating System.

RESULTS

The results are

_presented

in table II. We have estimated some

_population

parame-ters that characterize sperm

predominance

in double

matings.

P

2

is the

_proportion

of second male

_offspring

after

_remating

_(Boorman

and

Parker,

1976). P

2

w

is the

weighted

mean of

P

2

,

equivalent

to the mean _{of progeny}

_proportions

_per female

weighted by

the female’s total

_{productivity.}

_P

_I

is the fraction of

_offspring

sired

by

the first male and P’ is the

_proportion

of the first male’s _sperm that is used

by

a female before she remates

_(Gromko

et

_al,

_{1984). P’}

was estimated from the

control crosses as the

_{weighted proportion}

of

_lst-3-day

offspring

over total female

productivity.

Both

_P,

and

P

Z

were estimated from the female

offspring,

since the

male

_offspring

did not allow ascertainment of the sperm

origin.

The estimates of

P

2

’

have been corrected for

viability

differences between the

_offspring

of the 2 male

genotypes

(detected

by

the

_{3-way ANOVA;}

see

below).

In all _cases, the values of

P

Z

were

_{high. Groups}

1b and 2b

(2nd

male

w/Y)

showed the lowest

values,

0.91 and 0.92

_{respectively.}

The other 2 values

_(groups

1a and

_2a)

were close to 1. More-over,

only

on the 1st d after the 2nd cross did we find

offspring

of the 1st male.

On the other

hand,

some of these descendants

_might

have been

_produced

prior

to

the time when the 2nd

_mating

occurred. This would indicate that the actual

P

2

values _may be

_larger.

The values of

_P’,

the fraction of _sperm

_effectively

used

_by

the female before she

remates,

indicate the presence of at least

25-50%

of _sperm of the 1st male when the 2nd cross occurs. These values are

_{underestimates,}

since

after the 2nd cross was started females may

lay

eggs before

remating.

Given the

high

values of

_P

₂

_,

the bias of these underestimates is

_{insignificant.}

The

_large

values of

P

2

suggest

that the

_remaining

sperm is not used in the

_following

fertilizations.

Since the

_P

_z

_’s

variances are not

_equal,

we used the

Brown-Forsythe

test

_(Dixon,

1985)

to _compare the

P

2

values. The

P

2

angular

mean was used as the

_dependent

variable. Differences were

_{statistically significant}

between _{groups la,} 2a and

_1b,

2b

(change

in the order of

males,

P <

_0.001),

but not between _{groups la,} 1b and

_2a,

2b

_(change

in the female

_genotype,

P =

0.33).

So the variation in

_P

₂

is _a function

of the male

_genotype.

We

_{find, therefore,}

a

_{high degree}

of

_predominance

of the last mated

male,

as well as

possible

selective differences in this

_component.

A

_possible

source of error in the estimation of the

_P

₂

values must be now

considered. If

_during

the time in which the 2nd cross occurs a female remates

more than _once, then our

P

2

values will be

_spurious,

since

they

will

_correspond

to

P2,3,...,n,!

where n is the number of times a female has remated from the

_begining

of the

_experiment.

Patterson and Stone

₍₁₉₅₂₎

found that the time between

_matings

Wheeler

₍₁₉₄₇₎

found that D buzzatii

_presents

a

_{high degree}

of

_expression

of the insemination reaction. Patterson and Stone

₍₁₉₅₂₎

and Markow

₍₁₉₈₅₎

_suggest

that the insemination reaction works as a mechanism to

_preclude

_remating

in females.

_Accordingly,

D buzzatii females would

_present

a

_long

refractory

_period

before

_{period remating,}

which would diminish the chances of a

_remating

_during

the

_period

of the 2nd cross. In this _sense, D buzzatii would be

_analogous

to D

_mojavensis,

which

_delays

additional

_matings

_{(Markow, 1985).}

On the other

hand,

we have estimated the

P

2

values in the case of females

_being

remated 2 or 3 times. We have

supposed

that the

_proportion

between

Pi

and

P

i

_

1

is 1:2. This

_assumption

is based on the values of

P

2

and

P

3

found in D

hydei by

Markow

(1985).

For females that remate twice the estimated values of

P

2

for each group are:

_la

_,

₂

_P

=

0.94,

P

16

,

2

=

0.84,

2

P

,

2a

= 0.98 and

P

26

,

2

= 0.94. For 3 times remated

females

_they

are:

_P

_la

_,

₂

=

_0.93,

₂

_,

_lb

_P

=

0.81,

!,2 !

0.98 and

Pz

b

,z

= 0.83. These

values are still

_{large. So,}

we think that the

_P

₂

values may be biased

_upwards,

but not so much as to invalidate the conclusion that sperm

predominance

is

high.

However,

the differences found in

_P

₂

between male’s

_{genotypes may}

be due to differential

mating

success rather than to selection for sperm

predominance.

Additionally,

we

_performed

a

_{3-way analysis}

of variance to confirm the

_previous

comparisons

(table III).

The

_log

_{of the progeny number sired}

_by

the first male was used as a

_dependent

variable. The

_goal

of this

analysis

was to test:

_a),

the

effect of the female

_remating;

_b),

the effect of the female’s

genotype;

and

_c),

the

effect of the male’s

_genotype

on the

offspring

of the 1st male. We found

_significant

fixed effects for each

_factor,

but no _{interaction among them.}

_First,

the number of

progeny sired

by

a male is

significantly

reduced if his

_partner

_subsequently

remates

(F

=

_9.42,

P _<

0.01).

From this _we deduce

Second,

the female’s

_genotype

also influences

_productivity

_(F

=

182.32,

P <

_0.001)

very

significantly.

These differences are

_probably

due to the different

_genetic

background

of both stocks and not to the

_genetic

marker itself. From the third

factor,

we find

_genetic

differences _among male

_genotypes

_(F

=

10.95,

P _<

0.01),

but these cannot be attributed to selective differences in sperm

predominance

since the mate x male interaction is

_{non-significant}

_(F

=

_0.50,

P =

0.48).

The absence

of interaction indicates that the variance of this factor is

_negligible

in relation to

the

variance

due to other

_components,

such as

_viability.

It would _appear,

_therefore,

that selection for sperm

predominance,

if it

_exists,

is not as

_important

as the other

components

in these

_experiments.

Except

for groups 1b and

3b, statistically significant

differences were found for

the total progeny between the

single

and double mated females. Similar results have also been obtained in D

_{pseudoobscura}

_(Turner

and

_Anderson,

₁₉₈₃₎

and D

_Tnojavensis

_{(Markow, 1982),}

_although

_contrary

results have been

_reported

for D

_melanogaster

_(cf

Boorman and

_{Parker, 1976;}

Prout and

_Bundgaard,

_1977;

Gromko and

_Pyle,

_1978;

Alvarez and

_Fontdevila,

_1981).

In our _{case, it is} clear that a

_single

insemination is not sufficient to fertilize all the _eggs a female can

normally lay.

This is

_against

one

_assumption

of the model of Prout and

_Bungaard

(1977),

which claims that female

_output

is

_independent

of the number of

_matings.

DISCUSSION

In the face of _sperm

_competition,

the male

_strategies

can lead to 2

_different,

not

necessarily exclusive,

ways of

adaptation.

One which increases

_P

_l

_,

the fraction of

offspring

sired

_by

the first

male,

either

by

an increase of P’

(ie, through

an increase in the time before female

_remating)

or

_{by &dquo;resisting&dquo;}

the

_predominance

of the

second male. The other _way is to increase

P

a

.

Our results seem to

_suggest

that the

male

_mating

_strategy

of D buzzatii is to maximize

_P

₂

_(ie

the &dquo;remator&dquo;

_strategy)

as well as P’

(see

Gwynne,

_1984;

_p 141 for a

_possible

selective

explanation),

whereas

that of the female is

_{multiple mating.}

In order to confirm this conclusion it would

time of _{the second cross, since}

_P

₂

could

_depend

both on the stock

_background

and on the

_remating

time

_(Boormer

and

_Parker,

_1976).

Gwynne

(1984)

believes that the males of those

_species

with

_{higher predominance}

supply

food to the female or to his

_offspring.

Markow and

_Ankney

_{(1984, 1988)}

found in D

_mojavensis

that males transfer nutrients to females. We have indirect

evidence that D buzzatii males transfer

_yeast

to females

_{during mating}

_(Starmer

et

_al,

_1988).

_Thus,

transmission of nutrients

_might

_help

to

_explain

the increases in female

_productivity

with the number of

_matings.

A more

likely

possibility

to account for female

_{multiple mating}

is in relation with the

_high

total

_productivity

found in this

_species

_(Barker

and

_{Fredline, 1985; Barbadilla,}

_1986).

_Ridley

₍₁₉₈₈₎

reviews extensive evidence

_showing

that

_species

with

_higher

_{productivities}

are less

likely

to receive sufficient sperms at one

_mating

than

_species

with low

_{productivity,}

whereby

in

_species

with a

_{high productivity}

a

_multiple

mated female will leave more

offspring

than a

_single

one.

The

_strong

sperm

predominance

found in D buzzatii has a considerable technical interest. Natural

_populations

of this

_species

show a

_{moderately high}

inversion

polymorphism

in 2 autosomes. The

_karyotype

of wild males can be ascertained

by crossing

them with

_virgin

females from a

_laboratory

stock

_homozygous

for certain chromosome

_{arrangements,}

and

_analyzing

the

_{salivary gland}

chromosomes

of a number of larvae from each _progeny. On the other

_hand,

we seldom find out the

karyotype

of wild

females,

for

_they

are

_{usually already}

inseminated when collected in the field. One _way to overcome this

_difficulty

could be to remove _sperm from the

females

by

means of a low

_temperature

treatment

_prior

to their

_mating

with the

laboratory

stock males

_(Anderson

et

_al,

_1979).

This

_{method, however,}

has _proven to

be

_completelly

ineffective in D buzzatii

_{(unpublished results).}

The

_high

values of

_P

₂

suggest

a

_simple

solution to this

problem.

If wild females

_brought

to the

_laboratory

are crossed with males of known

_karyotypes

and then transferred _every other

_day

to

a new vial with fresh

food,

we

_expect

that after a few

days

almost all the progeny will be sired

_by

the

_{laboratory males,}

_allowing

the correct identification of the

female’s

_karyotype.

In a recent

_study

carried out in the

_population

of Carboneras

(Ruiz

et

_al,

_submitted)

this

_prediction

has

_proved

to be

_essentially

correct.

ACKNOWLEDGMENTS

We wish to thank _{2 anonymous} referees for their constructive comments on this

manuscript.

This work has been

_{supported by}

_grant No PB85-0071 from the Direccion General de

_{Investigaci6n}

Cientifica _y T6cnica

_{(DGICYT, Spain),}

awarded to AF.

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