Persistent Wolbachia and Cultivable Bacteria Infection in
the Reproductive and Somatic Tissues of the Mosquito
Vector Aedes albopictus
Karima Zouache1,2, Denis Voronin1,2, Van Tran-Van1,2, Laurence Mousson3, Anna-Bella Failloux3, Patrick
1Universite ´ de Lyon, Lyon, France, 2Universite ´ Lyon 1, Villeurbanne, CNRS, UMR5557, Ecologie Microbienne, Lyon, France, 3Institut Pasteur, Ge ´ne ´tique mole ´culaire des
Bunyavirus, Paris, France
Background: Commensal and symbiotic microbes have a considerable impact on the behavior of many arthropod hosts,
including hematophagous species that transmit pathogens causing infectious diseases to human and animals. Little is
known about the bacteria associated with mosquitoes other than the vectorized pathogens. This study investigated
Wolbachia and cultivable bacteria that persist through generations in Ae. albopictus organs known to host transmitted
arboviruses, such as dengue and chikungunya.
Methodology/Principal Findings: We used culturing, diagnostic and quantitative PCR, as well as in situ hybridization, to
detect and locate bacteria in whole individual mosquitoes and in dissected tissues. Wolbachia, cultivable bacteria of the
genera Acinetobacter, Comamonas, Delftia and Pseudomonas co-occurred and persisted in the bodies of both males and
females of Ae. albopictus initially collected in La Re ´union during the chikungunya outbreak, and maintained as colonies in
insectaries. In dissected tissues, Wolbachia and the cultivable Acinetobacter can be detected in the salivary glands. The other
bacteria are commonly found in the gut. Quantitative PCR estimates suggest that Wolbachia densities are highest in ovaries,
lower than those of Acinetobacter in the gut, and approximately equal to those of Acinetobacter in the salivary glands.
Hybridization using specific fluorescent probes successfully localized Wolbachia in all germ cells, including the oocytes, and
in the salivary glands, whereas the Acinetobacter hybridizing signal was mostly located in the foregut and in the anterior
Conclusions/Significance: Our results show that Proteobacteria are distributed in the somatic and reproductive tissues of
mosquito where transmissible pathogens reside and replicate. This location may portend the coexistence of symbionts and
pathogens, and thus the possibility that competition or cooperation phenomena may occur in the mosquito vector Ae.
albopictus. Improved understanding of the vectorial system, including the role of bacteria in the vector’s biology and
competence, could have major implications for understanding viral emergences and for disease control.
Citation: Zouache K, Voronin D, Tran-Van V, Mousson L, Failloux A-B, et al. (2009) Persistent Wolbachia and Cultivable Bacteria Infection in the Reproductive and
Somatic Tissues of the Mosquito Vector Aedes albopictus. PLoS ONE 4(7): e6388. doi:10.1371/journal.pone.0006388
Editor: Niyaz Ahmed, University of Hyderabad, India
Received April 13, 2009; Accepted June 25, 2009; Published July 27, 2009
Copyright: ? 2009 Zouache et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: KZ was supported by a PhD fellowship from the French Ministe `re de l’Education Nationale, de la Recherche et des Nouvelles Technologies. This work
was funded by the CNRS and the Agence Nationale de Recherche (ChikVendoM ANR-06-SEST07). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Mosquitoes are medically important arthropod vectors of
vertebrate pathogens. For instance, Aedes albopictus, and its sister
taxon Aedes aegypti, are vectors of a large number of arboviruses,
notably dengue and chikungunya . Since 2005, La Re ´union
and neighboring islands in the Indian Ocean have experienced
severe epidemics of chikungunya involving high incidences in the
population [http://www.invs.sante.[fr, 2]]. The isolation and
sequencing of the chikungunya virus from patients in La Re ´union
during a massive disease outbreak have revealed a prevalence of
clinical isolates harboring nucleotide changes in both structural
and non-structural loci; one particular mutation was found in
glycoprotein E1, in a region predicted to interact with the target
membrane . Entomological field surveys [4,5] and vector
competence assays in the laboratory  have demonstrated that
Ae. albopictus, which is much more anthropophilic than Ae. aegypti in
La Re ´union, is a very efficient vector.
In the absence of effective vaccines, arbovirus transmission can
only be reduced by limiting mosquito densities by the mechanical
reduction of breeding sites and by the application of insecticides.
Unfortunately, insecticides impact non-target insects as well, and
most mosquito species have developed resistance . There is
increasing interest in the use of microbes associated with
arthropod vectors to interfere with the transmission of pathogens
with a view to overcoming these difficulties by sustainable
approaches . Indeed, if on the one hand, microbial symbionts
can confer a fitness gain on their arthropod hosts, including better
PLoS ONE | www.plosone.org1July 2009 | Volume 4 | Issue 7 | e6388
nutrition , heat tolerance [10,11], and resistance to pathogens
[12,13,14], on the other hand, arthropod microbiota can be
pathogenic for the host vector [15,16], or can have deleterious
effects on host reproduction [17,18]. Finally, microbes associated
with arthropods can either enhance or weaken vector competence
[19,20,21]. Consequently, interference with one or more of these
aspects of host behavior by natural or transgenic microbes could
be exploited to manage arthropod vector-borne diseases by an
approach known as ‘‘paratransgenesis’’ [22,23].
Despite the importance of microbes in the ecology and behavior
of many arthropods , including hematophagous vectors such
as ticks , tsetse flies  and lice , little is known about the
mosquito-associated microbiota. Most of the few studies that
have investigated the bacterial communities of Culex and Anopheles
mosquitoes have focused on the midgut compartment [28,
29,30,31,32,34,35,36]. Very little is known about Aedes-associated
bacteria. DeMaio and co-workers  were the first to report
the midgut bacterial flora of wild Aedes triseriatus. Recently,
members of the Bacillus and Serratia genera have been identified
in the larval gut , and adult ventral diverticulum  of Ae.
aegypti, respectively. Attempts have been made to use the gut-
inhabiting bacteria to interfere with parasite transmission in
mosquitoes [40,41,42]. In Ae. albopictus, the obligate intracellular
bacterium Wolbachia has mainly been looked for in laboratory
colonies and field-caught individuals [43,44]. This bacterium
induces cytoplasmic incompatibility [45,46,47,48] that causes
embryogenic death, a feature that could be exploited to control
insect pests . More studies are needed to make a complete
inventory of the microbial communities of Ae. albopictus, and
identify taxa that could be manipulated for paratransgenesis
purposes. In this study, we investigated the presence and location
of Wolbachia and of cultivable bacteria in a colony of Ae. albopictus,
collected during the explosive chikungunya epidemics in La
Re ´union, and maintained under laboratory conditions since 2006.
Culturing and PCR-based techniques coupled with in situ
hybridization were used to detect Wolbachia and cultivable
bacterial genera differentially distributed in somatic and repro-
Characteristics of the dominant Proteobacteria
A total of 3 to 100 CFU were found per early emerging Ae.
albopictus mosquito. Eight colony types were obtained in the two
media used; notably two types from male mosquitoes and six types
from females. Two representatives of each colony type were used
for genomic DNA extraction and PCR amplification of the rrs
gene using universal primers (Table 1). Amplified rDNA
restriction analysis (ARDRA) of the amplified rrs genes revealed
five distinct patterns (not shown), the corresponding PCR products
of which were fully sequenced. Blastn analysis (Table 2) identified
two nearly complete rrs gene sequences as being closely related to
uncultured Comamonas spp. (99% similarity), and the other three
were affiliated to three species, Acinetobacter calcoaceticus (99%
similarity), Delftia sp. (99% similarity), and Pseudomonas alcaligenes
(99% similarity). Isolates of the genera Comamonas, Delftia and
Pseudomonas were recovered from females, whereas Acinetobacter
isolates were obtained from males.
To obtain an overview of the total bacterial community, PCR-
DGGE fingerprints of samples from whole insects and from dissected
tissues were produced using specific rrs -gene primers and the
corresponding hypervariable V3 regions (Figure S1). Bands were gel-
excised and re-amplified. Direct sequencing of the PCR product
generated in some cases double sequences, indicating the presence of
more than one V3 in a particular excised band. These were excluded
an uncultivable bacterium, as well as the genera Mesorhizobium and
Stenotrophomonas (Table 2). The presence of sequences affiliated with
Wolbachia and with the four cultivable genera (Acinetobacter, Comamonas,
Delftia and Pseudomonas) was also found.
Amplification with specific primers was performed to further
explore Wolbachia and the dominant cultivable bacteria in the
insect tissues. Positive PCR signals corresponding to Wolbachia
strains wAlbA and wAlbB were obtained for all three of the organs
tested (salivary glands, ovaries, and gut), as well as in the eggs, for
four generations (Table 3), confirming the ‘‘invasive behavior’’ of
this vertically-transmitted bacterial genus. The genus Acinetobacter
was detected in the gut and salivary glands, whereas PCR products
corresponding to Comamonas, Delftia and Pseudomonas were obtained
only in the gut. Sequencing the amplified fragments confirmed the
identity of each targeted bacterium (not shown). In the subsequent
experiments, we focused on Wolbachia and Acinetobacter, which were
detected in both gut and salivary glands.
Densities of Wolbachia and Acinetobacter in mosquito
The numbers of the bacterial cells varied depending on
the genus and on the targeted organs (Table 4). The relative
density (number of wsp gene per host actin gene) of Wolbachia was
higher (P,0.005) in ovaries than in the gut and salivary glands. No
differences (P.0.05) were found between the two Wolbachia wAlbA
and wAlbB strains in all the three organs. The highest density of
Acinetobacter was found in the gut (P,0.001), outnumbering
Acinetobacter were not significantly different in the salivary glands
Localization of bacteria in dissected tissues.
the bacteria in mosquito tissues, FISH genus-specific probes
available for Acinetobacter and Wolbachia were used. To do this,
FISH probes were first tested using Acinetobacter calcoaceticus isolate
KZ-OAlM cultured in rich medium, and Wolbachia strain wAlbB
hosted in Ae. albopictus cell line Aa23. Specific signals were detected
for both Acinetobacter (Fig. 1A) and Wolbachia (not shown). The
probes were then hybridized against the dissected tissues using
three independent biological samples. Confocal microscopic
observations of somatic tissues showed hybridizing signals for
Acinetobacter in the inner surface of epithelial cells and in lumen
space of the foregut and the anterior midgut (Fig. 1C). These
signals were observed in all 10 of the dissected guts of females from
generations F2 to F5. Acinetobacter could not be detected in the cell
cytoplasm or basal or ventral parts of the epithelial cells, suggesting
that this bacterial genus is mainly located in the intervillous space.
No significant Acinetobacter signal was detected in the central part of
midgut or hindgut. Wolbachia probes detected signals in the
cytoplasm of salivary gland cells (Fig. 2). The medium lobe
displayed relatively low signal intensity (Fig. 1B) compared to high
hybridizing dots found in the lateral lobes (Fig. 2C and D). In
contrast to the positive PCR results (see above), no significant
fluorescent signals were observed in the gut for Wolbachia, nor in
the ovary for Acinetobacter (not shown).
To monitor the bacteria in female reproductive tissues, the
ovaries were dissected before vitellogenesis. Confocal images of the
germarium and egg chambers revealed Wolbachia in all types of
ovarian cells, including follicular and nurse cells, as well as in the
future oocytes (Fig. 3). The highest density of bacteria was found in
the future oocyte confirming a common feature of Wolbachia,
which is to transfer from nurse cells into the oocyte through
cytoplasmic dumping as has been shown in Drosophila . As
expected from the PCR results, no significant signal for Acinetobacter
was found in the ovaries (not shown).
Symbionts of Aedes albopictus
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In recent years attempts have been made to investigate the
possible use of native or genetically modified microbes to control
pest arthropods and vector-borne diseases. The outcomes have
varied considerably . Greater knowledge about the behavior,
persistence, and tissue tropism of microbes associated with vectors
is essential to enhance the efficiency of paratransgenesis. Here, we
investigated Wolbachia and the dominant cultivable bacteria in a
colony of Ae. albopictus caught in La Re ´union island during the
2005–2006 chikungunya epidemics. We found that the Ae.
albopictus colony was infected by Wolbachia strains wAlbA and
wAlbB. This is in accordance with what had been reported in
studies of field-caught Ae. albopictus, where the prevalence of
double infection by Wolbachia is commonly over 96% [43,44]. We
also report here for the first time the presence of cultivable bacteria
of the genera Acinetobacter, Comamonas, Delftia and Pseudomonas
in the whole bodies of Ae. albopictus individuals. In addition,
GGE analysis yielded sequences closely related to the Mesorhizo-
bium and Stenotrophomonas genera, as well as an uncultivable
Genus-specific diagnostic PCR demonstrated the co-occurrence
of the four cultivable genera (Acinetobacter, Comamonas, Delftia and
Pseudomonas) together with Wolbachia, throughout four generations
in both males and females, indicating persistent infections. The
cultivable bacteria found here are widespread in nature, and can
be found in water, soil and living organisms, including Drosophila
. Among the few data reported for mosquitoes, members of
the Pseudomonas genus have been recorded in Anopheles [34,36],
Aedes triseriatus and Culex pipiens . The genus Acinetobacter has
been detected in the midgut of wild Culex quinquefasciatus .
When dissected tissues of mosquito females were subjected to
diagnostic PCR detection, signals were found in the gut of all the
cultivable bacteria. In situ hybridization with the specific
oligonucleotide probes available made it possible to detect
Acinetobacter in the lumen of the foregut and the anterior midgut
of Ae. albopictus females. Interestingly, the PCR signal of Acinetobacter
was also detected in the salivary glands, indicating the ability of
this bacterium to spread throughout the insect body. This is
consistent with the reported presence of Acinetobacter in the
hemolymph of the glassy-winged Sharpshooter or Homalodisca
vitripennis . No cultivable bacteria were detected in the oocytes
Table 1. Primers used in this study.
GroupGene Primer namePrimer sequence (59–39) Amplicon size/TmReferences
Eubacteriarrs pA59 AGAGTTTGATCCTGGCTCAG 39
pH59 AAGGAGGTGATCCAGCCGCA 39
rrs16S (V3) 338F59 GCCGCCCGCCGCGCGCGGCGGGCGGGGCGGGG-
16S (V3) 520R59ATTACCGCGGCTGCTGG 39
Wolbachia rrs99F59 TTGTAGCCTGCTATGGTATAACT 39
1994R59 GAATAGGTATGATTTTCATGT 39
wsp81F59 TGGTCCAATAAGTGTATGAAGAAAC 39
183F59 AAGGAACCGAAGTTCATG 39
328F59 CCAGCAGATACTATTGCG 39
691R59 AAAGGGGACTGATGATGT 39
Comamonas rrsCom199F59 CCTTGTGCTACTAGAGC 39
433/53 This study
Com614R59 GCAGTCACAATGGCAGTT 39
Delftia rrs Delf63F59 TAACAGGTCTTCGGACGC 39
Delf440R59 CCCCTGTATTAGAAGAAGCT 39
Pseudomonasrrs Ps For59 GGTCTGAGAGGATGATCAGT 39
Ps Rev59 TTAGCTCCACCTCGCGGC 39
Acinetobacterrrs Acine159 ACTTTAAGCGAGGAGGAGGCT 39
Ac59 GCGCCACTAAAGCCTCAAAGGCC 39
pQuantAlb wsp wAlbAQADir159 GGGTTGATGTTGAAGGAG 39
QArev259 CACCAGCTTTTACTTGACC 39
wsp wAlbB 183F59 AAGGAACCGAAGTTCATG 39
QBrev259 AGTTGTGAGTAAAGTCCC 39
actinActAlb-dir59 GCAAACGTGGTATCCTGAC 39
ActAlb-rev59 GTCAGGAGAACTGGGTGCT 39
TOPO 2.1rrs AcinetobacterACA59 TAGAGTGTGGGAGAGGAT 39
Ac59 GCGCCACTAAAGCCTCAAAGGCC 39
Symbionts of Aedes albopictus
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of the Ae. albopictus colony, ruling out the possibility of transovarial
transmission. Canonical transovarial transmission of cultivable
bacteria is in fact not a common event. In the mosquito, only the
cultivable bacterium Asaia has been reported to be transmitted via
the eggs of Anopheles stephensi, under the laboratory conditions .
Apart from transovarial transmission, other possible mechanisms
of symbiont diffusion include proctophagy, the deposition of
capsule containing microbes, and environmental acquisition
[9,53,54,55]. The presence of Acinetobacter in the gut and salivary
glands, two organs where viruses are known to replicate, implies
that the virus and the bacterium may share the same space
or co-localize. Interestingly, a recent study has shown that
Acinetobacter sp. strain KNF2022 was able to produce an antiviral
compound with inhibitory effects on the tobacco mosaic virus .
The role that the cultivable bacteria found here may play in the
bio-ecology or vectorial competence of Ae. albopictus needs to be
Like the cultivable bacteria, Wolbachia was found to be associated
with both female and male Ae. albopictus. Wolbachia are obligate
intracellular symbionts, and are generally passed transovarially from
the female to her offspring during the early stages of oogenesis or
embryogenesis. Consequently, reproductive tissues have been
reported to be the main targets of Wolbachia infection in both
arthropods and nematodes [57,58,59]. In Ae. albopictus, the
development of diagnostic PCR revealed two Wolbachia strains,
named wAlbA and wAlbB, that occur either separately or
concomitantly in natural Asian populations [43,60]. These two
Wolbachia strains are transovarially transmitted, and induce cytoplas-
mic incompatibility (CI) in both native Ae. albopictus [45,46,47,48,61]
and trans-infected Ae. aegypti, which is naturally devoid of Wolbachia
. Theoretical modeling has predicted that CI-inducing Wolbachia
could be used to control the spread of mosquitoes [63,64], this was
achieved byempirical research in the medfly.Here we show that
the Ae. albopictus colony from La Re ´union also harbored Wolbachia
strains wAlbA and wAlbB, which are clearly transmitted during
oogenesis, as high levels of specific in situ hybridization signals were
found in ovarian cells. Indeed,Wolbachia was present in the cytoplasm
of germ cells, and in that of all the cells in egg chambers, notably
follicular cells, nurse cells and future oocytes. The high density of
Wolbachia in the ovaries also supports these assumptions.
It has been established that Wolbachia can also infect somatic
tissues [65,66]. Dobson and co-workers  detected the WSP
protein of Wolbachia in ovaries and testes, but also in heads,
thoracic muscles, midguts, and Malpighian tubules. Genes
encoding this protein could be present in Wolbachia per se, or could
be part of a DNA fragment inserted into the host genome
[68,69,70]. Recently, electron microscopic images of Wolbachia
were reportedly detected in the salivary glands of the mosquito
Armigeres subalbatus [71,72]. Using specific oligonucleotide probes,
we report here for the first time the detection of Wolbachia in the
cell cytoplasm of the three lobes from salivary glands of Ae.
albopictus females. This is a finding of major importance, as the
Table 2. Bacterial community of Aedes albopictus.
Name of clone/
Band number Size (bp)
betaproteobacteria Uncultured Comamonas sp.
betaproteobacteriaDelftia sp. 332 EU888308.11524/1525 (99)
1529FJ688378GammaproteobacteriaPseudomonas alcaligenes strain
1529FJ688379Gammaproteobacteria Acinetobacter calcoaceticus type
strain NCCB 22016
AJ888983.1 1513/1515 (99)
[1; 2; 14; 15]a, b, c, d, e
169GQ290053 AlphaproteobacteriaWolbachia sp. wRi, complete
[3; 4; 16]a, b, d, e
betaproteobacteriaUncultured Comamonas sp.
[5. 13]a, b, d, e
194 GQ290055 GammaproteobacteriaPseudomonas
[6; 7]a, b, d, e
FJ950659.1 194/194 (100)
8a, b, d
betaproteobacteriaDelftia sp. 332EU888308.1 194/194 (100)
169 GQ290056AlphaproteobacteriaMesorhizobium loti strain U261 DQ310706.1166/169 (98)
10a, b, d
195FJ688379 Gammaproteobacteria Acinetobacter calcoaceticus type
strain NCCB 22016
AJ888983.1 195/195 (100)
194 GQ290058Unknown Uncultured bacterium clone
EF6044192.1 194/194 (100)
Symbionts of Aedes albopictus
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salivary glands are crucial in virus transmission. The epidemio-
logical consequences of this possible co-infection and potential
cellular co-localization calls for careful investigation by arbovirol-
ogists, as it was shown recently that a strain of Wolbachia was able
to reduce the lethal effect of viral pathogens of flies , indicating
that Wolbachia has direct or indirect effects on the virus. Although
the molecular mechanisms involved in this antiviral protection are
still unknown, immunomodulation [73,74,75] and the induction of
a reactive oxygen species burst  by Wolbachia infection could
account for these effects on infectious agents.
In this study, we identified a persistent infection of obligate
intracellular Wolbachia and cultivable bacteria, such as Acinetobacter,
in Ae. Albopictus, a major vector of arboviruses. The bacteria
effectively colonize the ovaries, gut, and salivary glands, organs that
are essential for the replication and transmission of pathogens, such
as arboviruses. Studies of the impact of these multiple infections on
the vectorial competence of the mosquito are in progress.
Materials and Methods
Laboratory-reared Ae. albopictus was obtained from the DRASS
(Direction Re ´gionale des Affaires Sanitaires et Sociales) in La
Re ´union. Aedes albopictus Providence was collected in 2006, and the
F2 to F5 generations were used in these experiments. Colonies
were maintained at 2861uC with a light:dark cycle of 16 h:8 h,
and 80% relative humidity. Larvae were reared in pans containing
1 yeast tablet in 1 liter of tap water. Adults were provided with
10% sucrose solution ad libitum.
Adult mosquitoes were anaesthetized at 4uC, rinsed 3 times in
sterilized water, surface disinfected by dipping in 70% ethanol for
5 min, and then rinsed five times in sterilized water, and once in
sterilized NaCl 0.8%. Three whole mosquitoes were crushed in
250 ml sterilized NaCl 0.8%, two-fold diluted, and 100 ml of the
resulting mix was plated on modified Luria Bertani agar medium
(MLB: Bacto-trypthone 10 g.l21, yeast extract 5 g.l21, NaCl
5 g.l21) and PYC medium (Peptone 5 g.l21, yeast extract 3 g.l21,
CaCl2.2H2O 6 mM, pH 7.0). After incubating at 26uC, single
colonies were streaked in the corresponding medium to check their
purity. Purified isolates were cultured in liquid MLB at 26uC,
stirred, and then stored in 25% glycerol at 280uC, until used.
To recover the various organs, adult females were dissected in
PBS under a binocular microscope using needles. Five whole
individuals or pools of 10 dissected organs were surface disinfected,
as described above. Each whole insectsample was crushed in200 ml
(or 100 ml for the organ samples) of DNA extraction buffer (2%
Hexadecyltrimethyl Ammonium Bromide, 1.4 M NaCl, 0.02 M
EDTA, 0.1 M Tris pH 8, 0.2% 2-b mercaptoethanol) pre-warmed
to 60uC. Homogenateswereincubated for 15 minat 60uC. Proteins
were removed in one volume of chloroform/isoamyl alcohol (24/1).
DNA was precipitated at room temperature for 10 min with one
volumeofisopropylalcohol.DNApelletwaswashedonce with 70%
ethanol, air dried, and then dissolved in 30 ml of sterilized water. To
extract the genomic DNA from the mosquito eggs, 30 to 70 mg of
sterilized water, surface disinfected in 70% ethanol for 5 min or
dechorionized in 2.6% hypochlorite, before being rinsed twice in
sterilized water. DNA extraction was then carried out as described
above. For bacterial genomic extraction, an overnight culture was
centrifuged at 12,000 x g, and the pelleted bacterial cells were
following the Manufacturer’s instructions (QIAGEN, Courtaboeuf,
France). For Plasmid DNA extraction, the QIAprep spin miniprep
kit was used following the Manufacturer’s instructions (QIAGEN,
Courtaboeuf, France). All DNA samples were stored at 220uC
Diagnostic and quantitative PCR
The oligonucleotide primers used were synthesized by Invitro-
gen, and are listed in Table 1. PCR amplification of rrs genes using
mosquito genomic DNA (60 ng) was performed in 25 ml of the
reaction mixture in 1X polymerase reaction buffer (Roche),
200 mM of each deoxynucleoside triphosphate, 500 nM of each
Table 3. Genus-specific PCRaamplifications in whole body
Whole body male
glands Ovaries Eggs
aPrimers used are listed in Table 1. Identity of products was confirmed by
bPools of 10 organs from females were tested in three biological replicates.
Table 4. Bacterial density in female Ae. albopictus organs.
OrgansNo. of Wolbachia (102) per 10 organs
No. of Wolbachia wsp gene/
No. of Ae. albopictus actin gene
No. of Acinetobacter
(102) per 10 organs
No. of Acinetobacter rrs/
No. Ae. albopictus actin gene
Statistical analysis was performed on log-transformed values. The dependent t-test was used to compare two means. Since multiple and non-independent tests were
performed, the exact risk of rejecting a true null hypothesis is hard. For safety, we chose to reject H0 at P,0.005. Mean values6SE marked with the same letter are not
significantly different (P.0.005).
Symbionts of Aedes albopictus
PLoS ONE | www.plosone.org5July 2009 | Volume 4 | Issue 7 | e6388
primer, 0.025 mg.ml21of T4 gene protein 32 (Roche), and
0.25 U of Expand DNA polymerase (Roche, France). Wolbachia
was detected using specific primers targeting the 16S rDNA and
wsp loci (Table 1) under the following conditions: 25 ml of the
reaction mixture containing 60 ng of DNA template in 1X
polymerase reaction buffer (Invitrogen), 1.5 mM MgCl2, 0.2 mM
of each deoxynucleoside triphosphate, and 0.5 U of Taq
polymerase (Invitrogen). Diagnostic PCR reactions were per-
formed in a T gradient thermocycler (Biometra, France). Real-
time quantitative PCR was performed using the LightCycler
LC480 apparatus (Roche). The 20 ml reaction mixture contained
1X LightCycler DNA master SYBR green I (Roche), 300 nM of
each primer, and 10 ng of template DNA. Amplifications
consisted of 10 minutes at 95uC, followed by 40 cycles of 15 s at
95uC, 1 min at 60uC or 63uC for the wsp and rrs amplifications
respectively, and a final elongation at 72uC for 30 s. Standard
curves were drawn on DNA plasmids pQuantAlb  and TOPO
2.1-Acin, a TOPO 2.1 vector in which we have cloned a 280 bp-
rrs gene fragment from Acinetobacter calcoaceticus (Table 1).
Ingeny PhorU (Apollo Instruments, Compie `gne, France) system
was used for DGGE analysis of the V3 PCR products as published
. Briefly, the 6% acrylamide gel contained a linear chemical
gradient of urea and formamide from 35% to 65% (100%=7 M
urea and 40% [v/v] deionized formamide). PCR products (5 mg per
well) were run in TAE buffer (40 mM Tris [pH 8.0], 20 mM acetic
acid, 1 mM EDTA) at 60uC for 17 h at 100 V. After electrophoresis,
the gels were immersed in SYBR green for 30 min at 4uC, rinsed in
sterilized water, and then photographed under a UV lamp. Bands
were excised, transferred to Eppendorftubes, and washed three times
with sterilized water. After all trace of liquid had been eliminated,
30 ml of water was added to the tubes, whichwere heated to 60uC for
30 min, and kept overnight at 4uC. Two ml of eluate were used for
amplification.Products were purified (MinElute PCR purification kit,
Invitrogen), and then direct sequenced using primers from the rrs V3
region (Genoscreen, Lille, France).
Cloning, sequencing and accession numbers
(QIAGEN). ARDRA analysis was performed to screen 16S rDNA
of bacterial isolates in 20 ml-reaction containing 200 ng DNA
sample, 1X Buffer TangoTMand 10 U of each endonuclease RsaI
and HhaI as recommended by the manufacturer (Fermentas,
France). For cloning, selected products were inserted into the
TOPO 2.1 vector, and used to transform the competent TOP10
Escherichia coli cells according to the procedure of the TOPO TA 2.1
cloning kit (Invitrogen).ClonescontainingDNAinsertswerechosen
Figure 1. Microscopic views of Acinetobacter and infected mosquito tissues. FISH with a specific oligonucleotide probe (A) and DAPI (B)
targeting Acinetobacter calcoaceticus grown in a pure culture. (C) Aedes albopictus gut infected with Acinetobacter calcoaceticus (green). Nuclei are
stained with propidium iodide (red). A and B, magnification 100X; C, bar 500 mm.
Symbionts of Aedes albopictus
PLoS ONE | www.plosone.org6 July 2009 | Volume 4 | Issue 7 | e6388
for sequencing. Sequence analyses were performed using the Blastn
program at the NCBI database (http://www.ncbi.nlm.nih.gov).
Sequences have been deposited in the GenBank database (Table 2).
Fluorescence in-situ hybridization (FISH)
Dissected organs (ovaries, salivary glands and guts) were fixed for
20 min in freshly prepared 4% formaldehyde in PBS, and then
an overnight culture of the isolate KZ-OAlM was centrifuged at
10,000 g, then 108pelleted cells were washed with PBS, and fixed as
above. Hybridization was conducted using 200 ng probes in
hybridization buffer [formamide 50%, SSC 5X, dextran sulfate
200 mg.l21, poly(A) 250 mg.ml21, salmon sperm DNA 250 mg.ml21,
tRNA250 mg.ml21,DTT 0.1 M,Denhartdt’ssolution0.5X] at 37uC
overnight. The probes were synthesized by Invitrogen and consisted
of: two Wolbachia probes W2, 59-CTTCTGTGAGTACCGTCAT-
TATC-39  and Wol3, 59-TCCTCTATCCTCTTTCAATC-39
59-ATCCTCTCCCATACTCTA-39  and Ac, 59-GCGCCAC-
TAAAGCCTCAAAGGCC-39  59-labelled with alexa488. Sam-
ples were washed twice in 1X SSC-10 mM DTT and twice in 0.5X
SSC-10 mM DTT at 55uC for 15 min. Finally, samples were rinsed
DAPI (49, 69-diamidino-2-phenylindole) and viewed under a
fluorescent (AXIO Imager.Z1, Zeiss) and a confocal microscope
(LSM510, Zeiss) at the Microscopy Centre of University Lyon I.
albopictus. Females and males from generations F2 to F5 (whole
insect body), dissected ovaries (OV), gut (G), and salivary glands
(SG). wRi, Wolbachia strain purified from Drosophila simulans
Riverside . Numbers correspond to sequenced bands (Table 2).
Found at: doi:10.1371/journal.pone.0006388.s001 (0.37 MB
DGGE profiles of bacterial rrs V3 segments from Aedes
We thank David Fouchet for statistical analysis.
Conceived and designed the experiments: KZ PM. Performed the
experiments: KZ DV VTV LM ABF. Analyzed the data: KZ DV VTV
ABF PM. Contributed reagents/materials/analysis tools: ABF PM. Wrote
the paper: KZ DV PM.
Figure 2. Confocal microscopy of Aedes albopictus salivary glands infected with Wolbachia. (A) General view of salivary glands (SG)
showing cell nuclei stained by SYTOX (green). Wolbachia (red dots) are detected by the rrs specific probe in the cells of the median canal (B) and
lateral lobes (C, D). Nuclei are in green. Bar, 500 mm.
Symbionts of Aedes albopictus
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