Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae)
The endosymbionts of the genus Wolbachia infect numerous arthropods and nematods, and often cause different effects on the reproduction of these hosts. The endosymbiotic bacteria Wolbachia induces the thelytokous mode of reproduction in the egg parasitoids of the genus Trichogramma. The Dot-blot...
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Інститут зоології ім. І.І. Шмальгаузена НАН України
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| Цитувати: | Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae) / C. Pascal, B. Pintureau, C. Katchadourian, S. Grenier, P. Bolland, C. Robin, A. Vallier // Вестн. зоологии. — 2006. — Т. 40, № 5. — С. 417-425. — Бібліогр.: 23 назв. — англ. |
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irk-123456789-94602010-07-02T12:02:54Z Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae) Pascal, C. Pintureau, B. Katchadourian, C. Grenier, S. Bolland, P. Robin, C. Vallier, A. Экология The endosymbionts of the genus Wolbachia infect numerous arthropods and nematods, and often cause different effects on the reproduction of these hosts. The endosymbiotic bacteria Wolbachia induces the thelytokous mode of reproduction in the egg parasitoids of the genus Trichogramma. The Dot-blot technique was performed to compare the symbiont Wolbachia density using the wsp gene of Wolbachia and the 18S gene of Trichogramma. It was established that Wolbachia density is not different in two host species, Trichogramma cordubensis Vargas et Cabello and T. evanescens Haliday. Эндосимбиотические бактерии рода Wolbachia заражают различных артропод и нематод, оказывая различный эффект на их репродуктивные особенности. Бактерии рода Wolbachia вызывают телитокию у яйцеедов рода Trichogramma. Использована техника Dot-blot для сравнения плотности симбионта Wolbachia, с помощью выделения гена wsp у симбионта Wolbachia и гена 18S у Trichogramma. Экспериментально установлено, что плотность Wolbachia у двух видов, Trichogramma cordubensis Vargas et Cabello и T. evanescens Haliday, одинаковая. 2006 Article Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae) / C. Pascal, B. Pintureau, C. Katchadourian, S. Grenier, P. Bolland, C. Robin, A. Vallier // Вестн. зоологии. — 2006. — Т. 40, № 5. — С. 417-425. — Бібліогр.: 23 назв. — англ. 0084-5604 http://dspace.nbuv.gov.ua/handle/123456789/9460 595.792 en Інститут зоології ім. І.І. Шмальгаузена НАН України |
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| language |
English |
| topic |
Экология Экология |
| spellingShingle |
Экология Экология Pascal, C. Pintureau, B. Katchadourian, C. Grenier, S. Bolland, P. Robin, C. Vallier, A. Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae) |
| description |
The endosymbionts of the genus Wolbachia
infect numerous arthropods and nematods, and often cause different effects on the reproduction of
these hosts. The endosymbiotic bacteria Wolbachia induces the thelytokous mode of reproduction in
the egg parasitoids of the genus Trichogramma. The Dot-blot technique was performed to compare the
symbiont Wolbachia density using the wsp gene of Wolbachia and the 18S gene of Trichogramma. It was
established that Wolbachia density is not different in two host species, Trichogramma cordubensis Vargas
et Cabello and T. evanescens Haliday. |
| format |
Article |
| author |
Pascal, C. Pintureau, B. Katchadourian, C. Grenier, S. Bolland, P. Robin, C. Vallier, A. |
| author_facet |
Pascal, C. Pintureau, B. Katchadourian, C. Grenier, S. Bolland, P. Robin, C. Vallier, A. |
| author_sort |
Pascal, C. |
| title |
Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae) |
| title_short |
Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae) |
| title_full |
Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae) |
| title_fullStr |
Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae) |
| title_full_unstemmed |
Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae) |
| title_sort |
comparison of wolbachia bacterial density in females of four thelythokous strains of trichogramma cordubensis and t. evanescens (hymenoptera, trichogrammatidae) |
| publisher |
Інститут зоології ім. І.І. Шмальгаузена НАН України |
| publishDate |
2006 |
| topic_facet |
Экология |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/9460 |
| citation_txt |
Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae) / C. Pascal, B. Pintureau, C. Katchadourian, S. Grenier, P. Bolland, C. Robin, A. Vallier // Вестн. зоологии. — 2006. — Т. 40, № 5. — С. 417-425. — Бібліогр.: 23 назв. — англ. |
| work_keys_str_mv |
AT pascalc comparisonofwolbachiabacterialdensityinfemalesoffourthelythokousstrainsoftrichogrammacordubensisandtevanescenshymenopteratrichogrammatidae AT pintureaub comparisonofwolbachiabacterialdensityinfemalesoffourthelythokousstrainsoftrichogrammacordubensisandtevanescenshymenopteratrichogrammatidae AT katchadourianc comparisonofwolbachiabacterialdensityinfemalesoffourthelythokousstrainsoftrichogrammacordubensisandtevanescenshymenopteratrichogrammatidae AT greniers comparisonofwolbachiabacterialdensityinfemalesoffourthelythokousstrainsoftrichogrammacordubensisandtevanescenshymenopteratrichogrammatidae AT bollandp comparisonofwolbachiabacterialdensityinfemalesoffourthelythokousstrainsoftrichogrammacordubensisandtevanescenshymenopteratrichogrammatidae AT robinc comparisonofwolbachiabacterialdensityinfemalesoffourthelythokousstrainsoftrichogrammacordubensisandtevanescenshymenopteratrichogrammatidae AT valliera comparisonofwolbachiabacterialdensityinfemalesoffourthelythokousstrainsoftrichogrammacordubensisandtevanescenshymenopteratrichogrammatidae |
| first_indexed |
2025-07-02T12:46:59Z |
| last_indexed |
2025-07-02T12:46:59Z |
| _version_ |
1836539361408057344 |
| fulltext |
UDC 595.792
COMPARISON OF WOLBACHIA BACTERIAL DENSITY
IN FEMALES OF FOUR THELYTHOKOUS STRAINS
OF TRICHOGRAMMA CORDUBENSIS AND T. EVANESCENS
(HYMENOPTERA, TRICHOGRAMMATIDAE)
C. Pascal, B. Pintureau, C. Katchadourian,
S. Grenier, P. Bolland, C. Robin, A. Vallier
Biologie Fonctionnelle, Insectes et Interactions-UMR INRA/INSA de Lyon,
INSA Bâtiment L. Pasteur, 69621-Villeurbanne-cedex, France
E-mail: bernard.pintureau@jouy.inra.fr
Accepted 14 March 2006
Ñðàâíåíèå ïëîòíîñòè áàêòåðèé Wolbachia ó ñàìîê ÷åòûðåõ òåëèòîêè÷íûõ ëèíèé Trichogramma cor-
dubensis è T. evanescens (Hymenoptera, Trichogrammatidae). Ïàñêàëü Ê., Ïèíòþðî Á., Kaò÷àäîðè-
àí Ê., Ãðåíüå Ñ., Áîëëàí Ï., Ðoáåí Ê., Âàëëüå A. — Ýíäîñèìáèîòè÷åñêèå áàêòåðèè ðîäà Wolba-
chia çàðàæàþò ðàçëè÷íûõ àðòðîïîä è íåìàòîä, îêàçûâàÿ ðàçëè÷íûé ýôôåêò íà èõ ðåïðîäóêòèâ-
íûå îñîáåííîñòè. Áàêòåðèè ðîäà Wolbachia âûçûâàþò òåëèòîêèþ ó ÿéöååäîâ ðîäà Trichogramma.
Èñïîëüçîâàíà òåõíèêà Dot-blot äëÿ ñðàâíåíèÿ ïëîòíîñòè ñèìáèîíòà Wolbachia, ñ ïîìîùüþ
âûäåëåíèÿ ãåíà wsp ó ñèìáèîíòà Wolbachia è ãåíà 18S ó Trichogramma. Ýêñïåðèìåíòàëüíî óñòà-
íîâëåíî, ÷òî ïëîòíîñòü Wolbachia ó äâóõ âèäîâ, Trichogramma cordubensis Vargas et Cabello è
T. evanescens Haliday, îäèíàêîâàÿ.
Êëþ÷åâûå ñ ëîâ à: Hymenoptera, Trichogramma, ÿéöååäû, Bacteria, ïëîòíîñòü Wolbachia, äîò-
áëîòòèíãîâûé àíàëèç, ýíäîñèìáèîíòû, ãåí wsp, ãåí 18S, òåëèòîêèÿ.
Comparison of Wolbachia Bacterial Density in Females of Four Thelythokous Strains of Trichogramma
cordubensis and T. evanescens (Hymenoptera, Trichogrammatidae). Pascal C., Pintureau B., Katchadou-
rian C., Grenier S., Bolland P., Robin C., Vallier A. — The endosymbionts of the genus Wolbachia
infect numerous arthropods and nematods, and often cause different effects on the reproduction of
these hosts. The endosymbiotic bacteria Wolbachia induces the thelytokous mode of reproduction in
the egg parasitoids of the genus Trichogramma. The Dot-blot technique was performed to compare the
symbiont Wolbachia density using the wsp gene of Wolbachia and the 18S gene of Trichogramma. It was
established that Wolbachia density is not different in two host species, Trichogramma cordubensis Vargas
et Cabello and T. evanescens Haliday.
Ke y wo r d s: Hymenoptera, Trichogramma, egg parasitoids, Bacteria, Wolbachia density, dot-blot
analysis, endosymbionts, wsp gene, 18S gene, thelytoky.
Introduction
The micro-wasps of the genus Trichogramma Westwood are egg parasitoids of various insect species, used
in the biological control of many lepidopterous pests of crops (Smith, 1996). They show bisexual reproduction
(a non-fertilized egg develops into a haploid male and a fertilized egg develops into a diploid female) or, more
rarely, a thelytokous mode of reproduction. In this case, a non-mated female produces only daughters. Such
a phenomenon is induced in most of the Trichogramma species by the presence of Bacteria of the genus
Wolbachia (Pintureau et al., 2001) which stops chromosome segregation during anaphase of the first mitotic
division and causes diploidisation leading to the development of females (Stouthamer, Kazmer, 1994).
Wolbachia are endosymbiotic bacteria belonging to the Rickettsiaceae family of the subdivision of
Proteobacteria (Williams et al., 1991). These bacteria are mostly present in the reproductive tissues of their
hosts (arthropods and filarial nematodes) and also in the somatic tissues (Dobson et al., 1999). They are
vertically transmitted from mothers to daughters but, occasionally, can also be horizontally transmitted
between individuals, even those belonging to different species. According to the first mode of transmission,
Vestnik zoologii, 40(5): 417–425, 2006
© C. Pascal, B. Pintureau, C. Katchadourian,
S. Grenier, P. Bolland, C. Robin, A. Vallier, 2006
Ýêîëîãèÿ
which is the most frequently observed, the bacteria are exchanged from one generation to the next by the
oocytes (Louis et al., 1993). The second mode of transmission was indicated by the lack of congruence
between the phylogenies of the Wolbachia and their hosts (Johanowicz, Hoy, 1996). Wolbachia induce
different reproductive alterations in Arthropods: cytoplasmic incompatibility, thelytokous parthenogenesis,
male feminization, male killing (O’Neill et al., 1997) and an increase in fecundity (Girin, Boule´treau, 1995).
These alterations further the spread of the symbiont over generations.
Nevertheless, the intensity of an effect induced by Wolbachia in a host may be variable, being notably
influenced by the bacterial density, the host genotype and the degree of co-adaptation between the host and
its symbiont (Boyle et al., 1993; Bourtzis et al., 1996). For example, old thelytokous females of Trichogramma
produce more and more males (Jardak et al., 1979), probably in response to a decrease in the density of
Wolbachia.
The aim of the present study was to improve our understanding of the relationships between the inten-
sity of the thelytokous mode of reproduction and the Wolbachia density in Trichogramma. More precisely,
the objective was to compare the bacterial density in two species showing different Wolbachia prevalence,
using four populations from different origins that all show 100% thelytoky. Differences in density would
indicate that host genomes have variable abilities in regulating the symbiont population and, possibly, the
level of thelytokous reproduction. This work was carried out with the Dot-blot technique (four Dot-blots were
achieved) enabling us to make an estimation of the bacterial density within the whole Trichogramma body.
Material and methods
B io l o g i c a l ma t e r i a l s
Four thelytokous strains including only females and belonging to two Trichogramma species were
studied (tabl. 1). The Grey strain of T. cordubensis, including individuals with the «dark body» mutation (db)
(i. e. body with grey and black zones instead of yellow and dark zones in the wild form, black eyes instead
of red), was selected from the MB35 strain.
All the strains were reared on UV-irradiated Ephestia kuehniella Zeller (Lep.: Pyralidae) eggs previously
glued with arabic gum solution onto cardboard strips (4.8 x 0.8 cm). These strips were placed in glass tubes
(8 cm in length x 1 cm in diameter) where Trichogramma adults were present, together with a diluted honey
drop as food. The rearing was performed at 23°C (70 ± 5% RH, 16: 8h L: D) allowing a development
duration of about 14 days.
Adults, used to extract the DNA, were collected by phototactic migration to guarantee the fresh
condition of the material and to avoid Wolbachia contamination by the E. kuehniella eggs which are also
infected. Several successive migrations were performed from the same rearing tube to collect the maximum
of individuals.
DNA e x t r a c t i o n
The DNA extraction is difficult in very minute insects, such as Trichogramma. The classical technique
using phenol, chloroform and isoamylic alcohol does not allow the extraction of good quality DNA, probably
due to the high concentration of red pigments from Trichogramma eyes. We thus used the Promega “Genomic
DNA Purification kit” for vegetal DNA, following the manufacturer’s specifications but with some
modifications to optimize the technique. Modifications concerned the incubation time (more than 12 hours
instead of 15 min) and the incubation method (with stirring instead of no stirring). The extraction was
performed on the homogenate of about 300 Trichogramma adults stored at –80°C (about 2.4 mg). The DNA
samples were maintained at 4°C for 24 hours prior to storage at –20°C.
The Dot-blot technique requires good quality DNA and in sufficient quantities. An estimation of the
quality and quantity was thus carried out by means of an electrophoretic migration on a 1% agarose gel in
TAE buffer (40 mM Tris-acetate; 1 mM EDTA) followed by comparison with a control of known size and
concentration (DNA Ladder 1Kb, Gibco BRL). This method allows the differentiation of the samples and
was used to prepare different DNA quantities for the Dot-blot 1. Nevertheless, afterwards, such an estimation
of the DNA quantity appeared not to be very reliable. We then attempted to use the spectrometry technique
Ta b l e 1. Thelytokous Trichogramma strains in which Dot-blots were performed
Òà á ëèö à 1. Ëèíèè òåëèòîêè÷åñêèõ Trichogramma, â êîòîðûõ áûë ïðîâåäåí äîò-áëîò àêíàëèç
418 C. Pascal, B. Pintureau, C. Katchadourian et al.
Species Strain Geographic origin
Date
of collection
Host
T. cordubensis Vargas &
Cabello
MB35 Mora, Alentejo, Portugal 1992 Noctuidae
Grey Mutant obtained from MB35 Obtention in
1994
1032 Sa¯o Jorge, Azores, Portugal June 1992 ?
T. evanescens Westwood M36 Cagnes-sur-mer, Alpes-Maritimes,
France
August 1982 Noctuidae
but it did not provide good results from our samples, probably because of the presence of proteins and red
pigments from Trichogramma eyes. This is why, for the Dot-blots 2, 3 and 4, we decided to analyse four
dilutions of the raw solution of total DNA extracted.
E s t ima t i o n o f t h e r e l a t i v e d en s i t y o f Wo l b a ch i a u s i n g Do t - b l o t
The Dot-blot technique allows a rough estimation of the relative frequency of one DNA sequence
among a heterogeneous population of DNA sequences. The DNA extracted from each sample was diluted in
400 l of SSC 6X buffer (SSC 20X: 0.3 M Na-citrate; 3 M NaCl). It was then denatured at 100°C for 10 min,
and quickly cooled on ice. The samples were transferred to the Dot-blot device, in a line on nylon membranes
(Hybond, Amersham) based on two sheets of Watman 3M paper previously moistened in the buffer SSC 6X.
After the vacuum was created, the samples took on a disc shape of about 7 mm in diameter. The nylon
membrane was then treated with a denaturing solution (0.4 N NaOH; 1 M NaCl) for 10 min, and with a
neutralisation solution (0.5 M Tris-HCl pH 7.5; 1.5 M NaCl) for 5 min. Finally, it was deposited on dry
filter paper and heated at 80°C for 2 hours to fix the DNA. The membranes can be stored at room
temperature, or at 4°C when longer storage is required.
To estimate the ratio of Wolbachia DNA to host DNA, the same membrane was successively hybridized
with two radioactive probes, wsp for Wolbachia and 18S for Trichogramma. Each radioactive probe was
directly labelled by PCR (50 Ci of radioactive dCTP 32P) and purified. PCR were performed on the total
DNA of the Grey strain of T. cordubensis with the specific primers wsp: 81F 5’ TGG–TCC–AAT–
AAG–TGA–TGA–AGA–AAC–3’ and 691R 5’–AAA–AAT–TAA–ACG–CTA– CTC–CA–3’, or 18S:
185F 5’–ATG–CTT–GTC–TCA–AAG–ATT–AAG–C–3’ and 185R 5’–GGA–GCT–GGA–ATT–
ACC–GCG–G. The purification was carried out on a Sephadex G50 column.
To saturate the membrane, a pre-hybridization at 50°C for 6 hours was performed in a solution
including DNA from herring and salmon sperm denatured at 95°C (1% solution), 10% Denhardt 50X, 25%
SSC 20X, 0.2% SDS and 50% formamide. The hybridization with the wsp probe (radioactive and denatured)
was then performed overnight at 50°C in the same solution. To eliminate the non-specific interactions,
washings were carried out in different baths of SSC (three concentrations: 2X, 1X, 0.1X) containing 0.1%
SDS, for 15 min at 55°C for each concentration. In each Dot-blot, a non-symbiotic species (T. brassicae
Bezdenko, B strain) was used as a negative control of the wsp probe activity.
The estimation of the radioactive DNA quantity was carried out with a Storm apparatus (Molecular
Dynamics, USA) which allows rapid radioactive detection and linear quantification on five orders of
magnitude. The data collection and analysis were performed using the ImageQuant software.
Before hybridizing the radioactive probe 18S, the membrane was de-hybridized by two successive
washings in 0.2 M NaOH solution, at 42°C for 10 min, and by one washing in SSC 2X solution for 15 min.
The estimation of the quantity of DNA was performed with the same method from the 18S gene of
Trichogramma and from the wsp gene of Wolbachia. Each replicate from each sample was then characterized
by its ratio of intensities wsp/18S.
A data filtration test was performed in order to eliminate the values of low quality. Hence, the
correlation was calculated between three (Dot-blot 1) or four (other Dot-blots) intensities of wsp spots and
three or four intensities of corresponding 18S spots. Dot-blots were thus performed using three or four
quantities of total DNA from each sample: three quantities of DNA (0.50, 0.75 and 1 μg in Dot-blot 1) or
four volumes of DNA solution (3, 6, 9 and 12 μl in Dot-blot 2; 1, 3, 6 and 9 μl in Dot-blot 3; 5, 7, 10 and
20 μl in Dot-blot 4). Samples associated with non-correlated (p > 0.05) intensities were rejected because the
absence of correlation indicates technical problems (pipette handling, signal saturation, …). The different
ratios of intensities obtained in each validated sample were considered as «replicates» to calculate the means,
although variability shown by these replicates does not reflect biological variability since only one DNA
extraction was possible from one sample.
Results
The DNA quantity in the samples, estimated on agarose gel to be about 2.5 μg in
50 μl (i. e. 0.05 μg/μl), was sufficient to provide very clear spots.
Do t - b l o t 1
The Dot-blot was performed using three DNA quantities (0.50, 0.75 and 1 μg) of
each sample (strain 1032 of T. cordubensis and strain M36 of T. evanescens). The two
coefficients of correlation between the wsp and 18S spot intensities were significant
(p < 0.05) (tabl. 2) and all the data were thus kept to compare the strains.
The «t» test showed that the two Trichogramma strains studied are not infected by
different Wolbachia densities (fig. 1). Therefore, the bacterial density does not appear
to be different in the two species T. cordubensis and T. evanescens. Moreover, it does
419Comparison of Wolbachia Density in Females…
not seem to have any relation to the group of species, since T. cordubensis belongs to
the minutum group and T. evanescens to the evanescens group (Pintureau, 1994), two
groups, however, closely related and merged in the exiguum section by J. D. Pinto
(1998). Finally, the Wolbachia density does not seem to have any relation to the preva-
lence of infection in the species, since T. cordubensis is completely infected (all the
examined populations and individuals are infected) whereas T. evanescens is only par-
tially and rarely infected (only some populations and individuals are infected). An inter-
mediate prevalence is observed in other Trichogramma species when the infection is par-
tial but frequent (most examined populations and individuals are infected) (Pintureau
et al., 2002). Nevertheless, the present comparison is based on a low number of repli-
cates and can only provide preliminary conclusions requiring further confirmation in
the subsequent Dot-blots.
Do t - b l o t 2
The Dot-blot was performed using four volumes of DNA solution (3, 6, 9 and
12 μl) of each sample (strains MB35, Grey and 1032 of T. cordubensis, and strain M36
of T. evanescens). Among the 9 coefficients of correlation between the wsp and 18S spot
intensities, 8 were significant (p < 0.05) (tabl. 3) and an obvious majority of the data
was thus kept to compare the strains. Among all the 4 Dot-blots performed, the Dot-
blot 2 included the highest number of replicates.
According to the ANOVA, no differences exist between three of the four
Trichogramma strains studied (fig. 1). Only the strain Grey of T. cordubensis differs from
the other strains by a higher bacterial density. Therefore, this result confirms the
absence of any difference in Wolbachia density between the two species T. cordubensis
and T. evanescens, between the minutum and the evanescens groups of Trichogramma,
and between completely infected species and rarely infected species. The recorded dif-
ference between strains of T. cordubensis requires confirmation in the subsequent Dot-
blots. Such variability in the bacterial density would not be in relation to the geograph-
ic origin since the two different strains Grey and MB35 come from the same location
(Alentejo, Portugal), and the two similar strains MB35 and 1032 come from Alentejo
and Azores, respectively.
Do t - b l o t 3
This Dot-blot was performed using four volumes of DNA solution (1, 3, 6 and
9 μl) of each sample (strains MB35, Grey and 1032 of T. cordubensis, and strain M36
of T. evanescens). All 4 coefficients of correlation between the wsp and 18S spot inten-
sities were significant (p < 0.05) (tabl. 4) and all the data were thus kept to compare
the strains. The ratio of intensities wsp/18S is obviously higher in this Dot-blot than in
420 C. Pascal, B. Pintureau, C. Katchadourian et al.
Ta b l e 2. Analysis of the Dot-blot 1 performed with one thelytokous strain of T. cordubensis and one thelytok-
ous strain of T. evanescens
Òàáëèö à 2. Äîò-áëîò àíàëèç 1, ïðîâåäåííûé ñ îäíîé òåëèòîêè÷åñêîé ëèíèåé T. cordubensis è îäíîé
òåëèòîêè÷åñêîé ëèíèåé T. evanescens
* p < 0.05
T. cordubensis 1032 0.50 82810 123 566 0.99* 0.67
0.75 107364 208 140 0.52
1.00 143768 290 815 0.49
T. evanescens M36 0.50 65803 128 832 0.99* 0.51
0.75 85836 163 951 0.52
1.00 93765 189 143 0.50
Species Strain
Quantity (μg)
of DNA
Spot intensity Coefficient
of correlation
wsp-18S
Ratio
of intensities
wsp/18Swsp 18S
the preceding ones. The reason is a lengthening of the hybridization period with the wsp
probe but not with the 18S probe.
According to the ANOVA, no differences exist between three of the four
Trichogramma strains studied (fig. 1). Only the strain 1032 of T. cordubensis differed
from the other strains with a higher bacterial density. Therefore, like the Dot-blot 2,
this result confirms the absence of any difference in Wolbachia density between the two
species studied, belonging to two different groups of Trichogramma and to two differ-
ent categories of symbiont prevalence. On the other hand, the difference between
strains of T. cordubensis recorded in Dot-blot 2 is not the same as the difference record-
ed in Dot-blot 3, where a logical explanation appears in relation to the geographic dis-
tribution (Azores vs. continental Portugal): Grey > 1032 = MB35 in Dot-blot 2 vs.
421Comparison of Wolbachia Density in Females…
Fig. 1. Estimation of the Wolbachia density in three strains of Trichogramma cordubensis (MB35, Grey and
1032) and one strain of T. evanescens (M36). n — number of spots per strain in each Dot-blot performed.
Means were compared by t-tests (Dot-blots 1 and 4; p = 0.422 and 0.524, respectively) or ANOVAs (Dot-
blots 2 and 3; p = 0.003 and 0.002, respectively). In Dot-blots 2 and 3, means followed by the same letter
are not significantly different, according to the Fisher PLSD test (p > 0.05).
Ðèñ. 1. Ïëîòíîñòü Wolbachia â òðåõ ëèíèÿõ Trichogramma cordubensis (MB35, Grey and 1032) è îäíîé
ëèíèè T. evanescens (M36). n — êîëè÷åñòâî ïÿòåí íà ëèíèþ â êàæäîì äîò-áëîòå. Ñðåäíèå çíà÷åíèÿ
ñðàâíèâàëèñü ñ ïðèìåíåíèåì t-òåñòîâ (äîò-áëîòû 1 è 4; p = 0,422 è 0,524 ñîîòâåòñòâåííî) èëè
ANOVA (äîò-áëîòû 2 è 3; p = 0,003 è 0,002 ñîîòâåòñòâåííî).  äîò-áëîòàõ 2 è 3, ñðåäíèå çíà÷åíèÿ,
ñëåäóþùèå çà ñîîòâåòñòâóþùåé áóêâîé, ñóùåñòâåííî íå îòëè÷àþòñÿ, ñîãëàñíî òåñòó PLSD Ôèøåðà
(p > 0,05).
1032 > Grey = MB35 in Dot-blot 3. Such an inconsistency does not lead to an obvi-
ous conclusion, and it is preferable to temporarily consider that the bacterial density
can only be occasionally higher than the density recorded in the strain MB35, for some
unknown reason.
Do t - b l o t 4
The Dot-blot was performed using four volumes of DNA solution (5, 7, 10 and
20 μl) of each sample (strain MB35 of T. cordubensis, and strain M36 of T. evanescens).
Half of the 4 coefficients of correlation between the wsp and 18S spot intensities were
significant (p < 0.05) (tabl. 5) and half of the data were thus kept to compare the strains.
422 C. Pascal, B. Pintureau, C. Katchadourian et al.
Ta b l e 3. Analysis of the Dot-blot 2 performed with three thelytokous strains of T. cordubensis and one the-
lytokous strain of T. evanescens
Òàáëèö à 3. Äîò-áëîò àíàëèç 2, ïðîâåäåííûé ñ òðåìÿ òåëèòîêè÷åñêèìè ëèíèÿìè T. cordubensis è îäíîé
òåëèòîêè÷åñêîé ëèíèåé T. evanescens
Species Strain
Volume (μl)
of DNA solution
Spot intensity Coefficient
of correlation
wsp-18S
Ratio
of intensities
wsp/18Swsp 18S
* p < 0.05
T. cordubensis MB35 3 47037 99330 0.92* 0.47
6 46636 153048 0.30
9 49756 213568 0.23
12 51896 259104 0.20
MB35 3 29146 76783 0.96* 0.38
6 32037 112400 0.29
9 43274 145794 0.30
12 45966 177374 0.26
MB35 3 35936 68600 0,64
6 53016 173623
9 40230 187874
12 46977 208520
MB35 3 15415 30148 0.98* 0.51
6 22329 40884 0.55
9 33733 74505 0.45
12 38034 99226 0.38
Grey 3 63973 131459 0.99* 0.49
6 77375 179641 0.43
9 96214 247896 0.39
12 118076 305095 0.39
Grey 3 39635 73615 0.92* 0.54
6 65807 128562 0.51
9 81689 142302 0.57
12 80118 181742 0.44
Grey 3 50854 68911 0.97* 0.74
6 56514 118953 0.48
9 64070 134461 0.48
12 70030 171283 0.41
1032 3 40173 107586 0.89* 0.37
6 50777 186547 0.27
9 83680 228310 0.37
12 79407 287685 0.28
T. evanescens M36 3 33588 89735 0.99* 0.37
6 40284 123948 0.33
9 51711 185929 0.28
12 58112 214594 0.27
According to the «t» test, no differences exist between the two Trichogramma
strains studied (fig. 1). This result confirms the absence of any difference in Wolbachia
density between the two species T. cordubensis and T. evanescens, and between the
strains MB35 and M36 (Dot-blots 2 and 3).
Discussion and conclusions
The Wolbachia density does not seem to be different in T. cordubensis and
T. evanescens. Therefore, this density is not a species specific character or even a species
group specific character. In T. evanescens, where the infection prevalence is clearly
lower than in T. cordubensis, the restricted proportion of infected individuals is thus not
caused by a poor installation of Wolbachia in the infected individuals. The restriction
of prevalence could therefore come from other factors, such as the existence of more
or less resistant host genotypes as shown in Culex pipiens L. (Berticat et al., 2002) or
Drosophila sp. (McGraw et al., 2002). The host genome is also known to regulate the
Wolbachia effect, as shown with the level of cytoplasmic incompatibility in Nasonia sp.
(Bordenstein, Werren, 1998), or the induction of the thelytokous mode of reproduc-
tion in Trichogramma sp. (Pintureau et al., 2000).
In T. cordubensis, some differences between the strains were recorded, but these
differences were inconsistent since, according to the experiment, they concerned either
the Grey strain or the 1032 strain and the other strains. Moreover, the Grey strain is
derived from the MB35 strain and so the difference recorded between them is very sur-
prising. It is thus difficult to state that actual differences in Wolbachia density exist in
natural populations of this species. Similarly, M. E. Clark and T. L. Karr (2002) did
not record differences in symbiont density between two Drosophila simulans Sturtevant
strains, although S. P. Sinkins et al. (1995) recorded differences between Aedes albopic-
tus (Skuse) strains.
In species with a high symbiont prevalence, a co-adaptation between Wolbachia
and Trichogramma is expected, leading to a lower physiological cost and density of bac-
teria. Our results did not confirm this hypothesis, but they could be preliminary.
423Comparison of Wolbachia Density in Females…
Ta b l e 4. Analysis of the Dot-blot 3 performed with three thelytokous strains of T. cordubensis and one the-
lytokous strain of T. evanescens
Òàáëèö à 4. Äîò-áëîò àíàëèç 3, ïðîâåäåííûé ñ òðåìÿ òåëèòîêè÷åñêèìè ëèíèÿìè T. cordubensis è îäíîé
òåëèòîêè÷åñêîé ëèíèåé T. evanescens
Species Strain
Volume (μl)
of DNA solution
Spot intensity Coefficient
of correlation
wsp-18S
Ratio
of intensities
wsp/18Swsp 18S
* p < 0.05
T. cordubensis MB35 1 51851 21152 0.99* 2.45
3 92991 37809 2.46
6 102816 39169 2.62
9 124085 50878 2.44
Grey 1 97128 52498 0.99* 1.85
3 143824 65776 2.19
6 212246 87504 2.43
9 264105 116641 2.26
1032 1 74107 23158 0.99* 3.20
3 104929 38133 2.75
6 223513 77667 2.88
9 192490 62433 3.08
T. evanescens M36 1 88848 46175 1.00* 1.92
3 194440 87279 2.23
6 346697 130286 2.66
9 295819 118852 2.49
Indeed, to better describe the variability of Wolbachia density in the Trichogramma
genus, or to confirm the absence of variability, other species and populations have to
be analysed. In addition, the methods used in the work to measure this density need to
be improved, and other methods such as quantitative PCR and confocal microscopy
techniques will probably be required for better reliability (Sinkins et al., 1995; Noda et
al., 2001; Berticat et al., 2002; Clark, Karr, 2002; Kondo et al., 2002).
F. Gressent provided important advice for using the Storm apparatus. Strain 1032 was provided by
P. Garcia (Univ. Azores, Portugal), strains M36 by J. Pizzol (INRA-Antibes, France) and strain MB35 by
I. Silva (Univ. Wageningen, The Netherlands). L. Neto (Univ. Algarve, Portugal) obtained the mutant strain
Grey. All the strains were reared by A. Clavel (INRA/INSA de Lyon).
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424 C. Pascal, B. Pintureau, C. Katchadourian et al.
Ta b l e 5. Analysis of the Dot-blot 4 performed with one thelytokous strain of T. cordubensis and one thelytok-
ous strain of T. evanescens
Òàáëèö à 5. Äîò-áëîò àíàëèç 4, ïðîâåäåííûé ñ îäíîé òåëèòîêè÷åñêîé ëèíèåé T. cordubensis è îäíîé
òåëèòîêè÷åñêîé ëèíèåé T. evanescens
Species Strain
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wsp-18S
Ratio
of intensities
wsp/18Swsp 18S
* p < 0.05
T. cordubensis MB35 5 32016 118837 0.43
7 25595 183513
10 29090 137165
20 43320 201647
MB35 5 45294 132107 0.92* 0.34
7 26583 89406 0.30
10 31017 133735 0.23
20 61385 282810 0.22
T. evanescens M36 5 34763 94109 0.99* 0.37
7 35377 115363 0.31
10 36862 125343 0.29
20 52879 229319 0.23
M36 5 31919 81222 0.83
7 33205 80864
10 45121 141516
20 40697 175020
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