The genetic control of ovariole number in Sitophilus oryzae L (Coleoptera, Curculionidae) is temperature sensitive

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The genetic control of ovariole number in Sitophilus

oryzae L (Coleoptera, Curculionidae) is temperature

sensitive

Am Grenier, P Nardon

To cite this version:

Original

article

The

_genetic

control of

ovariole

number

in

_Sitophilus

_oryzae

L

(Coleoptera,

Curculionidae)

is

_temperature

sensitive

AM

_Grenier,

P

Nardon

INSA,

UA INRA

_227,

Laboratoire de

_{Biologie Appliquée,}

Bât

₄

_06,

20,

avenue

_A-Einstein,

69621 Villeurbanne

cedex,

France

(Received

3

_May

1993;

accepted

29

April

1994)

Summary - The influence of

_genetical

and

_epigenetical

factors on the determinism of the ovariole number was studied in

Sitophilus

_oryzae. This weevil

normally

_possesses 4 ovarioles with

apical

bacteriomes

_{harbouring symbiotes. By selecting}

females with a reduced number of

ovarioles,

the

_genetic

control of this trait was demonstrated with

regard

to the easy

selection of this character and its similar transmission to the progeny

by

females and

males. Several genes could be

implicated

and a weak maternal effect cannot be

_rejected.

The character is _temperature sensitive: at

_30°C,

abnormal females with 2 or 3 ovarioles

prevail,

and

_conversely,

normal females appear at 20°C. The reduction of the number

of ovarioles is correlated with a decrease in fitness

_(reduced

progeny and

lighter body

weight

of

_females).

Only

ovary formation seems to be

affected,

because a

_higher

_progeny per ovariole in abnormal females can be indicative of a normal

yolk production

in the

fat

body. Counting

bacteria in ovaries shows that the

_regulation

of

symbiote

number per

female occurs at the ovariole itself and not within the whole insect.

Sitophilus oryzae

/

ovariole number / selection / genetic control /

temperature-sensitive character

Résumé - Le contrôle _génétique du nombre des ovarioles chez _Sitophilus oryzae L

(Coleoptera, Curculionidae) est sensible à la température.

L’influence

de

_facteurs

génétiques

et

épigénétiques

sur le déterminisme du nombre des ovarioles a été étudiée

chez

Sitophilus

oryzae. Ce

charançon possède normalement

4 ovarioles

terminés par

un bactériome

apical hébergeant

des

symbiotes.

En sélectionnant les

femelles

ayant un

nombre réduit d’ovarioles, nous avons _pu démontrer le contrôle

génétique

de ce caractère : .’

sélection aisée et transmission à la descendance aussi bien _par les

femelles

que par les

mâles. Plusieurs

_gènes

pourraient être

_impliqués

et un

_{léger effet}

maternel se

_manifeste

par ailleurs. Le caractère est thermo-sensible : à

_30°C,

les

_femelles

anormales à 2 ou 3 ovarioles sont

_{majoritaires,}

alors que, à

20°C,

le

phénotype

normal

prédomine.

(descendance

réduite et

_{poids corporel}

des

_{femelles plus}

_faible).

Seule la

_formation

de l’ovaire semble être

_affectée,

car une

_{fertilité plus grande}

_par ovariole chez les

femelles

anormales

_indiquerait

une

_production

normale de vitellus dans le _corps

_adipeux.

Les dénombrements de bactéries dans les ovaires ont montré que la

régulation

du nombre

de

_{symbiotes par femelle}

intervient au niveau de l’ovariole

_{lui-même, plutôt qu’au}

niveau

de l’insecte entier.

Sitophilus oryzae

/

nombre d’ovarioles

_/

sélection

_/

contrôle génétique

/

caractère thermosensible

INTRODUCTION

Sitophilus

oryzae, the rice

weevil,

is a

_major

cereal

_pest.

The factors

affecting

the

biotic

potential

have been studied

_by

several authors

_(Birch,

_1945;

_Segrove,

_1951;

Nardon,

1978a),

but little attention was focused on

_{genetic factors,}

_particularly

those

_{affecting reproduction.}

The

_present

_{study completes}

_previous

observations

concerning

the

_variability

of the structure of the female

_genital

tract and the influence of various factors

_(Nardon

and

_Grenier,

_1983).

S _oryzae females have 2

_ovaries,

each divided into 2 ovarioles. This structure

is

_typical

of the

_Sitophilus

genus, as well as of the

majority

of

_{Rhynchophorinae}

(Murray

and

_{Tiegs, 1935;}

_{Vernier, 1970;}

_{Ganesalingam,}

_1974).

At the anterior

_tip

of each

_ovariole,

a bacteriome

containing

intracellular bacteria is formed

(Mansour,

1930; Nardon,

1971).

This

_apical

bacteriome

_disappears

in the absence

_{of symbiotes}

(Nardon, 1973).

On account of their economic

_importance,

these weevils have been

extensively

studied and seem to _possess

_great

_genetic

_stability

with

_regard

to the few

morpho-logical

anomalies which have been observed. In S

_granarius,

a

_wing

malformation was noticed

_{by Strong}

(1959),

and in S _{oryzae 2} mutations have been described: one

_affecting

antenna and called ’fused antennae’

_{(Campbell-Brown}

and

_Champ,

1971)

and the other

_modifying

rostrum and

_{elytra morphology}

_(Nardon

and

Nar-don,

1983).

In another

_{Curculionidae,}

_Hypera

_postica,

some females with 5 ovarioles were observed in a natural

population by

Hower

(1971)

but with a _very low

fre-quency

(0.1-0.2%).

The same observation has been made for S _oryzae

_(Nardon

and

Grenier,

1983).

However,

after

irradiation,

different kinds of ovarial anomalies could be obtained: either of functional

_(absence

of

_oogenesis)

or structural

_origin

(variable

ovariole

number) (Nardon,

1978b;

Nardon and

_Grenier,

_1983).

Structural anomalies were

the most

_frequent

_{when young} larvae were irradiated

during

_ovary formation since

irradiation acts

_directly

on the differentiation _process. The

_persistence

of these

anomalies in the next

_generations,

as well as the _appearance of abnormal females

in the _progeny of irradiated fathers

_suggested

a

_genetic

determinism.

In

_{Drosophila, genetic}

studies have shown that the ovariole number varies from

1 strain to another often with a

_geographical

_pattern

and seems to be under the control of a

_polygenic

_system

_(De

_{Scheemaeker-Louis, 1970, 1971; Thomas-Orillard,}

1975;

Capy

et

_al,

_1993).

In some

Scarabaeinae,

Pluot

(1979)

observed reduction of

to

_only

1

_definitively

formed ovariole. This

_phenomenon

is

_genetically

controlled and is the result of a

_regressive

phylogenic

evolution.

In S _oryzae, a

_newly

introduced

_laboratory

strain was found to _possess some

females with

_only

2 or 3 ovarioles. We

thought

that this would be a

good

_opportunity

to start selection on the reduction of the ovariole number with this

_strain,

in order

to

_study

the

genetic

control

_(chromosomal

and

_{epigenetical)}

in the determinism of the ovariole number in

_Sitophilus.

With such a

_strain,

which

_normally

_presents

a reduced number of

_ovarioles,

we had the

advantage, contrarily

to structural anomalies

_{previously artificially}

obtained in the

_laboratory,

of

_rejecting

additional effects of irradiation treatment. It was also

_interesting

to

_study

the influence of the reduction of ovariole number on

_fertility

and female

_{body weight,}

as well as on the

regulation

of the number of ovarial

_symbiotes.

_During

_experiments

on

_selection,

we

found that a decrease in

_temperature

modified ovariole

_{distributions,}

so we studied

the effect of

_temperature

on this character more

carefully.

MATERIALS AND METHODS

Breeding

and test for

_temperature

influence

The S _oryzae strain used for selection

_(called

the W

_strain)

was found on wheat in a _grocery in

_Lyon

and

_breeding

was conducted on

_wheat,

in latticed

_plastic

boxes

kept

in ventilated incubators at 27.5°C and

75%

RH

_(Laviolette

and

Nardon,

1963).

For

_temperature

effects on the ovariole

number,

4

_temperatures

were tested:

_20,

23.5, 27.5

and 30°C. In order to limit the ’female

_effect’,

we tested

homogeneity

of

3 series of females which we first allowed to

_lay

_eggs at 27.5° C for 4

d,

before each

was transferred to another

_temperature

for a 1-week

egg-laying period.

Selection

_procedure

and

_crossings

At its arrival in the

_laboratory,

the W strain

_presented

5 females out of 197 with structural ovarial anomalies: 4 females with 3 ovarioles and 1 with 2 ovarioles. The selection was conducted from the _progeny of these abnormal females. At the

beginning

of the

_{selection, only}

females with

_1,

2 and 3 ovarioles were allowed to

give

progeny for the next

generation

(Go

to

_Gg).

After this

_time,

selection was

only applied

in

_G

₁₂

_,

_G

₃₅

and

G

36

,

when the

percentage

of abnormal females was

decreasing

(see

table

_I).

For

_{genetic experiments, virgin}

adults were obtained

_{by isolating oviposited}

wheat kernels in cavities bored in a

polyurethane plate

and covered with a

glass

sheet.

_{Egg-laying density}

was chosen so as to have

_only

1

_imago

_emerging

from a

kernel. After sex determination and before

_crossings,

weevils were

_kept

_separated

on wheat until sexual

_maturity.

Crosses were effected between the abnormal line selected for a reduced number

of ovarioles

_(called

A

_line)

and the

_original

normal line with 4 ovarioles

_(called

N control line and bred without

_selection).

These crosses were conducted at 27.5°C with

_generation

_G

₆

_,

at the

_beginning

of the

_selection,

and with

_G

₅₀

_,

when the character was well established in the

_population.

For each

_experiment

we studied

phenotype

could be determined

_easily

in females

_{by dissecting}

_ovaries,

but in

_males,

we named the brothers of females of N line with 4 ovarioles ’normal’

_males,

and those of females with 1-3 ovarioles ’abnormal’ males .

Determination of

_biological

_parameters

Fertility

The whole _progeny of a female was difficult to measure because the total

_egg-laying

time was

longer

than 30 weeks.

_Therefore,

the

_fertility

was estimated

by

the mean

Sex ratio

Total female and male numbers were noted

throughout

the

_experiments,

but the sex ratio was not taken into account in the

_results,

because it remained stable

_(close

to 1).

Weight

’

Adult females were

_{individually weighed}

on an

_{electromagnetic}

balance

_(Setaram

- y21)

with a

10-

2

_mg

_precision.

Ovariole number

After

_weighing,

females were dissected in a

_Yeager

solution

(pH

6.8, pO

400 mosm;

Nardon and

Grenier,

1983).

The last

_tergite

was lifted with small

_forceps

to extract

the

_genital

_apparatus

in toto. In the ’abnormal’

_line,

ovaries could show from 0 to

4 ovarioles.

Determination

_{of symbiote}

number in ovaries

Symbiotes

are

_rod-shaped

and more or less flexuous bacteria

_{(Nardon, 1971).}

They

are located in a bacteriome at ovariole

_tips

in

_trophocytes

and

oocytes.

Their number was estimated

_{by counting}

in a Thoma’s cell under a

_{phase-contrast}

microscope.

For each

female,

ovaries were crushed in 0.1 ml

Yeager

solution. At the same

_time,

the

_morphology

of the ovariole

_tip

(single

or

double)

was also recorded.

RESULTS

Selection of the ’reduced number of ovarioles’ character at 27.5°C

Selection of the abnormal A line was studied on 50 successive

_generations.

Dissec-tions of females were effected on each

_generation

at the

_beginning

of the

selection,

but

_only

on some of them at the end. Results are

_reported

in table

_I,

while the

rel-ative evolutions of ovariole mean numbers in both A line and N control line _appear

in

_figure

1.

_{Heterogeneous}

numbers of dissected females in successive

_generations

were due to 2 different

_{strategies: genetic experiments}

with numerous dissections

and

_simple

records of selection with few females.

At

Go,

only

4 females with 3 ovarioles and 1 with 2 ovarioles were observed

in the total

_population

of 197 females

_(ovariole

mean number 7 =

3.97).

By

selecting

abnormal females for egg

laying,

we obtained

33%

of abnormal females in

G

2

(x

=

3.60)

and ₆

_generations

after

(G

8

),

_we

got

a line with

_nearly

97%

of females

having

a reduced ovariole number

_(x

=

2.24).

After this

_time,

every female of the progeny contributed to the next

_generation

without selection of females with a low

number of ovarioles and

_percentages

of abnormal females remained around

87%.

With

_just

a new selection of abnormal females at

_G

₁₂

_,

the character stabilized

near

95%

anomalies without selection until

G

34

.

At

G

35

and

G

36

,

selection was

time no more selection was

applied,

and at the end of the

_experiment

_(G

_5o

₎

a

well-established character was obtained with

96.8%

ovarial anomalies and an ovariole

mean number of 2.21. A minimum of 2 ovarioles seemed to be an

_asymptotic

value.

The effect of selection could also be observed on the _progeny of females

_showing

a normal

_phenotype

with 4 ovarioles

_{(table II).}

The mean number of ovarioles

of females with 4 ovarioles were

_analyzed

in this case. We found that mothers with

4 ovarioles _gave abnormal

_daughters

and

_anomaly

_percentages

increased

_throughout

selection. It was evident that the male

_genotype

has been modified

by

selection;

nevertheless,

in

_G

_SO

_,

the

_high

_percentage

of abnormal

_daughters

cannot be

_explained

by

genotypic

modification of males

_only.

At this

_generation,

crosses between females

with 4 ovarioles and abnormal males would

_give

_{progeny similar} to Fl

_hybrids

_(see

AN cross in table IV

_below)

with

54%

anomalies,

but we obtained

98%

anomalies,

like in crosses between abnormal females and males.

_Moreover,

we could

_verify

with

hybrids

between abnormal selected females in

_G

_SO

and males from the normal

_line,

that at this

time,

the

_anomaly

_percentage

is intermediate between

parents

(44%).

Therefore,

the

_genotype

of these females was

_modified,

even if their

_phenotype

was

normal.

At each

_generation,

we could also observe a

relationship

between the

_proportion

of females with 3 ovarioles and the

proportion

of females with 4 and 2 ovarioles. This relation could be

_{expressed by}

the

_following

formula:

where the

_frequency

of 3 ovariole females is about the _square root of the

_product

of

_frequencies

of females with 2 and 4 ovarioles

_{(geometrical mean).}

These results

_proceeded

from observed ratios. At the

_beginning

of the

selection,

females with

_4,

3 and 2 ovarioles were in the ratio 9: 3: 1 in

_G

₃

(

X

2

=

_1.77,

2

_{df ),}

4: 2: 1 in

_G

₄

₍

_X

₂

=

_0.74,

2

_{df ),}

1:1: 1 in

_G

_S

_(x

₂

=

0.68,

2

df)

and after this

_time,

the

_proportions

were inverted. For

_example,

_they

were 1: 2:

4 (X2

=

_0.61,

2

df)

in

G

9

,

1: 3:

_{9 (}

_X

₂

=

1.20,

2

df)

in

G

12

,

1: 4: 16

(

X

Z

=

0.57,

2

df )

in

G

18

and 1: 5: 25

(

X

2

=

_0.36,

2

df )

in

G

SO

.

2

The

X

_tests calculated _on 23

generations

(observations

on 15 562

females)

showed that the

_experimental

ratios were

_compatible

with the

theoretical ones: with 2

_{df ,}

_X

₂

= 5.99 at the

5%

significance

level.

Crosses between normal and reduced ovariole number lines at 27.5°C

Crosses at the

G

6

generation

Crosses between N and A lines were conducted at

_27.5°C,

and results appear in

table III. At the 6th

_generation,

the

_anomaly

percentage

was

_only

about

61%

in

the

parental

A line

_(x

=

3.11).

In Fl

hybrids,

distributions of ovarioles in both

reciprocal

crosses were not

_{significantly}

different at the

5%

level

_(x2

=

_4.09;

2

_df)

but

_they

were _very different from the

_parental

A

_population:

X

2

= 187.7 for the

9

A x

(f

N

(AN)

cross

and

X

2

= 346.7 for the

9

N x

d’

A

_(NA)

cross, with 2

df.

Therefore,

the ovariole mean numbers were both _very close

(3.75

in AN and 3.80 in

_NA)

but

_higher

than arithmetical

_(x

=

3.54)

or

_geometrical

(g

=

3.51)

parental

means.

Therefore,

from this

_experiment,

it could be

thought

that neither sex-linked

effects nor

_epigenetical

factors were involved.

As

_egg-laying

Fl females had

_{heterogeneous phenotypes}

_(4,

3 or 2

_ovarioles),

the F2 obtained was difficult to

_analyse.

We have thus

_reported

in table III

_only

the

composition

of the F2

_{produced by}

Fl females with 4 ovarioles in order to

_appreciate

the

_{’disjunction’}

of the character. It seemed more

_important

in AN

(hybrids

which

cytoplasm).

Therefore,

the smaller number

_of

ovarioles in AN

_(x

=

3.45)

than in

NA

_(x

=

3.81)

may

suggest

a weak maternal effect.

Crosses at the

G

50

generation

These results _{appear in} table IV. At the 50th

_generation

of

selection,

the abnormal line

_(A)

reached

95%

anomalies and the normal line

_{(simultaneously}

bred but without

_selection)

had

_only

2.8%

anomalies. It could be noted that the

_original

line N did not _vary, because the ovariole mean number

_stayed

_unchanged

between

In

_Fl,

AN and NA

_{reciprocal hybrids}

showed

_anomaly

_percentages

and ovariole

mean numbers which were intermediate between those of

parents

(arithmetical

mean x = 3.13 and

geometrical

mean g

=

3.02).

It is

noteworthy

that the

2 distributions were

_{significantly}

different

(

X

Z

=

9.87;

2

df,

p <

_0.01),

as well as their mean numbers of ovarioles

_(e

=

2.47).

The Fl

hybrids

have abnormal

cytoplasm

(AN)

and showed more anomalies than the

_reciprocal

(perhaps

due to

a maternal effect

?).

The mean number of ovarioles of

9

AN x

(f

AN _{F2 progeny}

(obtained

from a random

sample

of Fl females with

_4,

3 or 2

_ovarioles)

was not

significantly

different from Fl values

₍

_E

= 1.20 for AN Fl and _e = 0.12 for NA

F1).

By comparing

every ovariole mean number in the different crosses, we

_got

significant

differences as follows: ! P AA <

_{x BC}

_NAxA

<

_!(FlAN

= F2 =

Fl

N

p)

<

!BCANxN

< !PNN.

This relation is

_typical

of a

polygenic

character,

but nevertheless back-crosses

had ovarial

_anomaly

_percentages

which were intermediate between those of the Fl

hybrid

and those of the

_parent

used in the cross

(27.85%

anomalies in

_Q

AN x d’ N

and

76.21%

in 9

NA x

d’

_A).

Theoretical ovariole mean numbers in back-crosses

could be estimated

_by

the means between Fl

_hybrids

and

_parents

and were _very

close to the observed values:

- in

9 AN

x

d’

NN: x = 3.61

_{or g}

= 3.59 _versus 3.62

observed;

- in

Q NA

x

c3’

AA: x = 2.78

_or

₌

2.74 _versus 2.70 observed.

In the same

_analysis,

the F2 distribution can be treated as a mixture with

1/4

N

_genotype,

_1/4

A

_genotype

and

_1/2

Fl

_genotype.

Temperature

influence on ovariole number

The effect of

_temperature

on the ’reduced ovariole number’ character was studied

at 2 different times of selection: in the 12th and 23rd

_generations.

In

G

12

,

the selected character was well established

(anomaly

_percentage

higher

than

_90%)

and the ovariole distribution was stable for several

_generations

_(see

table

_I).

After egg

laying,

grains

were

kept

at 2 different

temperatures:

27.5°C

(normal

temperature

for

_laboratory

weevil

_breeding)

and 23°C. These results

appear in table V.

When the

_temperature

decreased from 27.5 to

_23°C,

the distribution of ovarioles

in the _progeny was modified:

_percentage

of normal females increased from 5.9 to

24.7%

while the

_percentage

of 2 ovarioles was halved

(74.5

to

_35.6%)

and that of

3 ovarioles doubled

_(17.3

to

_39.6%).

The mean ovariole number increased from 2.27

to 2.89 ovarioles _per female. This

_study

was carried out on 4 successive

_generations

by selecting

progeny of females with 2 and 3 ovarioles. Ovariole mean numbers

observed in Fl

_stayed

_nearly

_unchanged

in F2 and

F4,

with

_only

the same decrease in ovariole mean numbers at the 2

_{temperatures,}

due to the selection pressure.

In

_G

₂₃

_,

for a better

study

of the effects of

_temperature

on the character

expression,

egg

laying

and larval

_development

were

performed

at

_20,

_23.5,

27.5 and 30°C

_{(temperatures}

_compatible

with insect

_{development).}

To avoid ’female

effects’, progenies

of the same females were

compared

at 27.5°C and at another

temperature.

The 3 series were

_homogeneous

at 27.5°C

₍

_X

₂

= 4.26 with 4

_{df ),}

and

so the results were

_grouped.

These results are

_reported

in table VI.

Distributions were

clearly

different as a function of

_temperature

(x

z

= 540 with

character

_(55.93%

ovarial anomalies at 20°C versus

99.28%

at

_30°C)

and modified the ovariole distribution

_(26.78%

females with 2 ovarioles at 20°C versus

94.96%

at

30°C,

for

_example).

There was a

_negative

linear correlation between ovariole mean

number and

_temperature

_(r

=

-0.992).

Relationships

between reduced number of ovarioles and

_biological

pa-rameters

Fertility

For both A and N

_lines,

we

_represented

the mean number of the _progeny obtained

per female and per

d,

at the time of maximal

_fertility,

as a function of the ovariole

number in females. Different selection times were

_{considered, C}

₁₂

_,

_G

₂₉

and

_G

₅

_o

(table VII).

Selection on the A line also seemed to reduce the

_fertility

because the observed values in the A line were

_always

much lower than in the unselected N line.

and

_experiments

were conducted in the same abiotic conditions. We noted a linear

correlation between the progeny number per female and the number of ovarioles of

the mother

_(all

correlation coefficients were close to

_1).

In the abnormal

_line,

we

also observed that for the same ovariole

_number,

there was a

_tendency

to a

_fertility

restoration across

_generations

which

_might

be due to natural selection. Female

_{body weight}

To

_investigate

the effect of ovariole number on female

_weight,

in the

_G

₄₂

_generation,

we

_weighed

and then dissected 2 female

_samples

_(A

and N

_lines).

The results _appear

in table VIII.

In the A

_line,

mean

_weights

of the 3 series

_(4,

3 and 2

_ovarioles)

were not

significantly

different from each other at the

5%

level

_(t

<

_0.58),

but were _very

different from the N line

_(t

from 4.6 to

_12.10).

In the selected A

_line,

the ovariole number of females did not appear to have any influence on

weight,

but the

body

Ovarial

_symbiote

number _per female

The ovarial

_bacteriome,

which is located at the

_tip

of each

ovariole,

is filled with

symbiotes.

This bacteriome

_generally

_appears as a

_single

structure. In the A

_line,

when the ovary could not differentiate into 2 entire

_ovarioles,

the

_apical

bacteriome could sometimes be

_divided,

and the

_unique

ovariole showed 2

_apical

more or less

well-separated

bacteriomes.

_{Morphological}

studies of these

_phenomena

were made

by

Nardon and Grenier

_(1983).

The

_{symbiote density}

_per

_female,

as well as the

apical

bacteriome

_aspect,

were

studied as a function of ovariole number at the

G

50

generation,

and the results are

reported

in table IX.

When

_apical

bacteriomes were normal

(single),

the

female

had a

symbiote

number

_nearly

_proportional

to the ovariole number. The correlation coefficient was

0.998 and the

_regression

line of

_symbiote

number

_per

female

_(y)

as a function of

the ovariole number

_(x)

was of the form: &dquo;’ .

In females with 2

_ovarioles,

but when the bacteriome was

_divided,

even

_partially,

the

_symbiote

number per ovariole was twice as

high.

DISCUSSION

Genetic control of the ovariole number

In

_Drosophila,

the

_study

of the influence of irradiation on

_genesis

of the female

gen-ital

_apparatus

_(Geigy,

_1931;

_Aboim,

₁₉₄₅₎

showed that its differentiation is

_largely

independent

of the germ cells.

Nevertheless,

in the absence of germ

cells,

the

ovari-oles never

develop.

Therefore,

according

to

_Abo

_ï

_m,

the

complete

morphogenesis

of

ovaries results from an interaction between _germ cells and mesodermal cells. In S _oryzae, the

_genesis

of ovaries was described

by Murray

and

_Tiegs

(1935)

and

Tiegs

and

_Murray

_(1938).

In the

_{early embryo,}

the _germ cells arise

_by

_migration

associated with

_symbiotic

bacteria. The future

_{bacteriocytes}

of ovaries also

_develop

very

early and,

when the germ band is

formed,

they

become associated with the germ cells.

At

the end of the 2nd

day,

the germ cells come in close contact with the

_adjacent

coelomic sacs and their mass divides into

_right

and left halves. The

investing

sheath of the

_gonad

arises from the

_splanehnic

wall of the

_posterior

coelomic sac. The

genital

ducts

develop

on the 4th

day

from the mesodermal cells

that ensheath the

_{gonad. Shortly}

before the larva _emerges, a

_pair

of solid stalks are formed at the base of the 9th

_segment

where

_{they impinge}

on the

epidermis.

Therefore,

in the _young

larva,

rudimentary

ovaries appear as 2

_spherical

bodies

embedded in the fat

_body.

These _{ovaries grow} and divide into 2

_pear-shaped

bodies which

_{rapidly expand during}

the larval

_stage.

In the

_early

prepupa, the

development

proceeds

more

_rapidly,

the stalks

_lengthen

and a lumen is formed. The

_imaginal

disk differentiates into

_vagina

and oviduct.

_During

_pupation,

the ovarial tubules

elongate

and bacteriomes

_containing

intracellular

_symbiotes

are formed at the

_tip

of each ovariole

_(Mansour,

_{1930; Nardon,}

_1971).

_{They disappear}

in the absence of

symbiotes

(Nardon, 1973).

The ovary divides from the apex to the

oviduct,

and

all intermediate structures can be found from 4 to 2

_typical

ovarioles

_(Nardon

and

Grenier,

1983).

This

_suggests

a factor of division

_acting

at the apex of

ovaries,

perhaps

on the terminal

filament,

as

proposed by King

et al

₍₁₉₆₈₎

and Eiche

(1972)

in

_Drosophila,

_where,

_during

ovarial

_{morphogenesis,}

the terminal filament

seems to determine of the ovariole number. Such a

phenomenon probably

occurs in

Sitophilus.

From this

_description,

it _appears that the formation of ovaries results from 3 main

events. In the absence of _germ

_cells,

it is

_probable

that the rudiment of ovaries is not

formed in the _egg, and this would

_explain

the fact that some females are

_completely

devoid of ovarioles

_{(table I).}

This is the first level of control. The second level is the division of the mass of _germ cells. In the absence of this

_division,

we obtain females

with

_only

1 ovariole

_{(table I).}

The third

_level,

and the more

_frequently

_affected,

is in _{the larva and the young pupa,} where the

_single

ovaries may divide. When the 2 rudimental ovaries

_divide,

4 ovarioles are

obtained,

when

only

one

divides,

3 ovarioles are

_formed,

and

_only

2 ovarioles occur in the absence of division. In the S _oryzae

_female,

our results

_{clearly show,}

as

_previously

_expected

_(Nardon

and

_Grenier,

_1983),

the _presence of a

_genetic

determinism of the number of ovarioles.

This character is

_easily

affected

_by

selection

_and,

at

_27.5°C,

_only

8

_generations

were

necessary to decrease the mean number of ovarioles from 3.97 to

_2.24,

with no more

reliable _progress afterwards. The

_genetic

control can be demonstrated

_by

crosses

in the

G

6

and

C

50

generations,

since males are as able as females to transmit the

genetic

factor(s)

to their

_daughters.

No sex-linked effect is detectable. The

_question

arises as to the nature of the

_genetic

_system

of control involved.

In

G

so

,

the Fl values are

significantly

different from each other at the

5%

significance

level,

and

_therefore,

it is

_possible

that a maternal effect also occurs.

intervention of several _genes, which could be reinforced

_by

the observation of the

following relationship

between means,

evoking

a

polygenic

_system:

In

_{Drosophila melanogaster,}

numerous studies have dealt with the influence

of the genome on the ovariole number

_(Robertson,

_{1957; Teissier, 1958; Melou,}

1961).

David

₍₁₉₆₁₎

showed that the ovariole number character was

rapidly

and

completely

transmitted

_by

males and

_depended

on nuclear

_{polygenic hereditary}

factors. De Scheemaeker-Louis

₍₁₉₇₀₎

described a

_polygenic

_system

where the estimated number of genes varied between 4 and

12,

12

_being

the most

_probable

value. Thoma,s-Orillard

₍₁₉₇₅₎

located the _genes on chromosomes II and III. These

genes have additive effects with interactions of dominance

_type,

as shown

_by

a

positive

correlation between

_high

number of ovarioles and numerous dominant

genes

(Thomas-Orillard, 1982).

A maternal effect associated with the _presence of

a

_picornavirus

also occurs

(Thomas-Orillard,

_1984;

Thomas-Orillard and

_Jeune,

1985)

which increases the number of

ovarioles,

and at the same time reduces the

mean

_development

time and

_gives

heavier females.

Evidence for

_temperature

_sensitivity

of the character

In

_Sitophilus,

the ’reduced ovariole number’ character was enhanced

_by

a

tem-perature

increase

_(remaining

in

_biological

_conditions),

_leading

to

_{a negative}

linear correlation between the mean of ovariole number and

_temperature.

Penetrance and

expressivity

of the character varied as a function of

_temperature:

at

_30°C,

the

pene-trance was almost

_complete

_(95%)

with most of the females with 2

_ovarioles,

while

at 20°C the

_penetrance

_only

reached

56%

with females

_having

2 or 3 ovarioles in

nearly equal

quantity.

The different

_expressivity

₍₂

or 3

ovarioles)

_depends

on the

moment when the division is elicited or

_suppressed.

This

_{temperature-sensitive}

effect can be

_compared

with Wool and

_{Mendlinger’s}

observations

₍₁₉₇₃₎

on a

_spontaneous

_{morphological}

mutation in Tribolium

casta-neum larva and _pupa: 3 or 4

urogomphi

could be seen on the _{terminal abdominal}

segments

in mutants instead of 2 in normal insects. The

penetrance

of the gene was

variable:

_incomplete

at 30°C and full at 25°C.

Many

temperature-sensitive

mutations have been described in

_Drosophila:

in

mutations

_affecting

_pteridine

concentration

_(lethal

mutation at 29°C and viable

mutation at

_22°C,

_Grigliatti

and

_Suzuki,

_1970);

_{in eye} facet

_arrangement

(hetero-zygous females are of wild

_type

at 29°C and mutant

_type

at

_21°C,

Foster and

Suzuki,

1970);

and in a homeotic mutation

_affecting

_{imaginal discs, giving}

normal arista

_segment

of the antennal

_complex

at 29°C and a tarsus at

_17°C,

with all the intermediate forms

_(Grigliatti

and

Suzuki,

1971).

The variation in thoracic bristle number described

_by

Louis et al

₍₁₉₈₈₎

in

D simulans is also

_temperature

sensitive. The

_expressivity

of this trait was

me-diated

_by

a

hereditary

virus

_(DSV).

At extreme

_temperatures

₍₁₇

and

_28°C)

wild

phenotypes

were

observed,

whereas the abnormal character was

expressed

between

using

antibiotics or infestation

_experiments.

It must be recalled that the

symbi-otic bacteria of S _oryzae seem to enhance the rate of ovarial

_anomalies,

which are

significantly

reduced in

_aposymbiotic

weevils

_(Nardon

and

_Grenier,

_1983).

Relationships

with other

_biological

characters

Fertility

and female

_{body weight}

In S _oryzae, female

_fertility

is correlated with the ovariole

number,

both in normal

(N)

and in abnormal

_(A)

_lines,

but selection reduced

_fertility

of A lines

_by

more than

50%.

The same

_phenomenon

was also observed in

_Drosophila

_(De

Scheemaeker-Louis,

1970).

It

_might

be

_thought

that whatever the

_genetical

determinism

is,

it acts

_essentially

_{(or exclusively)}

on _{ovary formation} and does not

_{modify yolk}

production

in the fat

_body.

A

_compensation

can take

_{place during vitellogenesis.}

As a matter of

_fact,

_regulation

of

_{vitellogenesis}

seems to occur at the individual

level,

because a female with 2 ovarioles

_gives

more _progeny per ovariole than a

female with 4 ovarioles:

- in the N line: 2.5

progeny/ovariole

with 2 ov mothers versus 1.55 with 4 ov

mothers;

- in the A line:

1.46

_{progeny/ovariole}

with 2 ov mothers versus 0.90 with 4 ov

mothers.

Conversely,

female

_{body weight}

is not correlated with ovariole number

_(females

of the abnormal line have similar

_body

_weights),

but abnormal line females were

significantly lighter

than those of the normal line. Several

_hypotheses

may

explain

these decreases in fitness

_(fertility

and

_weight).

1)

The

_decreasing

number of ovarioles

_by

itself should have diminished the female

body weight,

but this

_hypothesis

can be

_rejected

because a variation from 2 to

4 ovarioles in the abnormal line has no effect on the

body

weight.

2)

A

_{consanguinity}

effect _may have occurred because selection started with

a small female number and selection pressure is

high. Therefore,

an increase in

homozygosity might

be

_{directly responsible}

for the reduction in the

_reproductive

fitness of selected strains

_(Lerner,

_1954,

in

_Pyle,

_1976).

3)

Selection can lead to the

_mortality

of a

_part

of the

_{population by}

the

_expression

of lethal genes when

homozygous.

4)

An

_epigenetical

factor may

interfere,

such as the

maternally

inherited DSV virus

_(Louis

et

al,

1988)

in D

_simulans,

which can

_modify

the thoracic bristle number

but also decreases

_fertility

and

_viability

in its host

_(Comendador

et

_al,

_1986).

5)

The lower

_weights

are

possibly

not the result of

selection,

but rather the

consequence of a faster stabilization of

_{body weight}

in the

_laboratory.

Indeed,

we

used to observe that

_{laboratory breeding}

in

_optimal

conditions and without selection

always

induces an increase in

body weight.

When the W strain was introduced

in the

_laboratory,

adult

_{body weights}

were _very

_light

_(Q

= 1.23 ::!: 0.07 mg and

d = 1.16 t 0.07

_mg).

_Forty-two

_{generations later,}

female

_weights

_{(1.66 !}

0.04

_mg)

were a third

_higher

than the

_weights

at arrival. On the other

_hand,

when selection

was

_applied,

the

_{body weight}

became stable faster but at a lower level. This

phenomenon

can be

_{explained by}

the coselection of _genes

influencing

the

body

weight, by

a faster selection of recessive

_homozygotes

or

_perhaps

more

_simply

_by

6)

A

_positive

_genetical

correlation between ovariole number and

_fertility

on one

hand,

and ovariole number and

_{body weight}

on the other

_might

_explain

the selection

response

observed,

when

selecting

on the ’ovariole number’ criterion. Ovarial

_symbiote

number _per female

Despite

the fact that in

_Sitophilus

ovaries

_symbiotes

are harboured in not

only

apical

bacteriomes but also

_oocytes

and

_trophocytes,

when the

_apical

bacteriomes

are normal

_(single)

the

_symbiote

number is

_{nearly proportional}

to the ovariole number. On the

_contrary,

when the ovariole

_tip

is divided

_{(even partially),}

the

symbiote

number per ovariole is doubled.

_Therefore,

the

_regulation

of the

_symbiote

number seems to

_happen

at the ovary itself and not in the whole

_organism.

The observation of a double

apical

bacteriome could

_suggest

an effect on the

division of the terminal filament of the

_ovariole,

as described above.

CONCLUSIONS

To

conclude,

our results

suggest

that the number of ovarioles in the S oryzae weevil

can be controlled

_by

several _genes

_acting

in the larva and _{young pupa,} to allow the division of the rudimental ovaries.

_Epistatic

interactions are also

_acting

with

the

_{embryo morphogenesis.}

The

_expression

of this

_{morphogenetic}

_system

is

_highly

sensitive to

_temperature.

The selection for a lower number of ovarioles affects the

fitness of the weevil and

_particularly

reduces the

_fertility

_(despite

a

_compensating

effect)

and the female

_{body weight.}

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