Am. J. Trop. Med. Hyg., 70(6), 2004, pp.

597603
Copyright 2004 by The American Society of Tropical Medicine and Hygiene

STUDIES ON CULTURED AND UNCULTURED MICROBIOTA OF WILD CULEX

QUINQUEFASCIATUS MOSQUITO MIDGUT BASED ON 16S RIBOSOMAL RNA
GENE ANALYSIS
VYANKATESH J. PIDIYAR,* KAMLESH JANGID,* MILIND S. PATOLE, AND YOGESH S. SHOUCHE
Molecular Biology Unit, National Centre for Cell Science, University of Pune, Pune, India

Abstract. To describe the midgut microbial diversity and the candidate bacteria for the genetic manipulation for the
generation of transgenic mosquitoes refractory to transmission of diseases, the microbiota of wild Culex quinquefasciatus
mosquito midgut was studied using a conventional culture technique and analysis of a 16S ribosomal RNA (rRNA) gene
sequence library. The culturable microbiota was identified as Acinetobacter junii, Ac. calcoaceticus, Aeromonas culicicola, Bacillus thuringiensis, Microbacterium oxydans, Pantoea agglomerans, Pseudomonas aeruginosa, Staphylococcus
epidermidis, Stenotrophomonas maltophila and an unidentified bacterium from the host Drosophila paulistorium. The
16S rRNA gene library was composed of 46% unidentified and uncultured bacteria, 41% Acinetobacter spp., and 13%
Lactococcus spp. The coverage calculated for the 150 clones was 83.3%. Thus, the probability of the next cloned
sequence falling in a novel operational taxonomic unit (not yet observed) was 16.7%. The majority of the cultured
isolates and the 16S rRNA gene library clones belonged to the -proteobacteria class. Most of the bacteria have been
previously reported to inhabit the midgut of different mosquito species. Therefore, the results of this study indicate that
different mosquito species harbor common representatives of the microbiota that may be the potential candidates for
genetic manipulation to control the disease transmission capabilities of the host.
teractions between midgut bacteria and malaria parasites in
wild mosquito populations could explain how the vector potential for malaria parasite transmission is modulated by environmental factors such as acquisition of different types of
bacteria.
Little is known about the normal midgut microbiota of
mosquitoes. In early 1960s, a few studies were carried out on
midgut microbiota of laboratory-bred species of Culex.810
The presence of oxidase-positive bacteria from the midgut of
anopheline mosquitoes has been reported.11,12 The successful
colonization of Serratia marcescens in laboratory-bred
Anopheles stephensii has also been reported.13 A study of
wild Aedes triseriatus, Cx. pipiens, and Psorophora columbiae
using routine laboratory bacteriologic techniques indicated
the presence of S. marcescens, Klebsiella ozonae, Pseudomonas aeruginosa, and Enterobacter agglomerans.6 A study of
the midgut of Cx. quinquefasciatus larvae indicated the presence of bacteria represented by Bacillus spp., Staphylococcus
spp., and Pseudomonas spp., while Aspergillus and Streptomyces spp. represented the fungal and actinomycete inhabitants, respectively.9,14 The available conventional culture techniques limit the isolation and identification of all the components of the microbiota inhabiting any niche or any
environmental sample since it is not possible to simulate the
conditions for their growth under laboratory conditions.15 Ribosomal RNA (rRNA)based sequence studies of environmental organisms can identify the abundant organisms in the
environments studied. All necessitate the need to apply
rRNA-based analyses for this purpose.
We previously reported the isolation and taxonomic characterization of a new species, Aeromonas culicicola Microbial
Type Culture Collection (MTCC) 3249T (CIP 107763T) from
the midgut of Cx. quinquefasciatus, and two strains of A.
culicicola (SH and SLH) from Aedes aegypti during the same
study,16 indicating that different mosquito species in the same
environment may harbor common representatives of the microbiota. In this report, we studied the midgut microbiota of
wild Cx. quinquefasciatus using both culture-dependent and
culture-independent techniques.

INTRODUCTION
Mosquitoes are medically important arthropod vectors.
Culex quinquefasciatus is a mosquito vector for Wuchereria
bancrofti, the filarial worm that causes filariasis, which is one
of the six important tropical diseases. Approximately 80 million people are infected globally, and of these approximately
30 million chronic cases manifest as typical elephantiasis.1
The disease is highly prevalent in developing countries such
as India. Moreover, Japanese encephalitis virus has been isolated from field-collected Cx. quinquefasciatus. This makes
Cx. quinquefasciatus an important vector species.
Mosquito control still remains the primary strategy for controlling mosquito-borne diseases. Insecticide resistance of
mosquitoes, drug resistance of parasites, cost of new drug
development, limitations of vaccines, and environmental hazards of pesticide application all necessitate the need for the
development of novel disease control strategies. There have
been attempts to generate transgenic mosquitoes refractory
to transmission of diseases, but the genetic mechanisms of
refractoriness are poorly understood and are at times multigenic.26 An alternative approach to manipulating the mosquito genome might be to use the normal bacterial symbionts
of the mosquito midgut. Genetic manipulation could be done
in these species for which well-studied gene transfer mechanisms are available. Cecropin expressed in the endosymbiont
bacterium Rhodococcus rhodnii by genetic manipulation was
found to block transmission of Trypanosoma in sand flies.1
For such strategies to be successful, an understanding of the
microbial community structure of the mosquito midgut is necessary, which will enable us to identify the organisms that
participate significantly in the maintenance of the communities and are predicted to play significant roles in the environmental chemistry. It was previously reported that a reduction
in the normal bacterial flora in the mosquito midgut increased
Plasmodium falciparum infection rates in experimentally infected Anopheles mosquitoes.7 A better understanding of in* These authors contributed equally to this report.

597

598

PIDIYAR AND OTHERS

MATERIALS AND METHODS

Collection of mosquito species and isolation of bacterial
flora from midgut. Wild Cx. quinquefasciatus were collected
from surroundings of the National Centre for Cell Science
(Pune, Maharashtra, India) and brought live to the laboratory
in cages. Only the females were selected for further studies.
The species identity of the collected mosquitoes was confirmed using sequencing of the mitochondrial 12S and 16S
rRNA genes as previously described.17 Those females containing blood bolus within the midgut were considered to
have taken a blood meal prior to collection because the blood
bolus is visible for hours after a blood meal. During three
independent collections, 2030 mosquitoes were collected
each time. Mosquitoes were fed (time not exceeding 23
hours) on a sterile glucose solution until dissected. The dissection was done under sterile conditions after surface sterilization with 70% ethanol as previously reported.6 The midgut
contents were suspended in 500 L of 0.85% (w/v) NaCl. A
100-L aliquot of these contents was serially diluted up to
106 and plated onto blood agar base (HiMedia, Mumbai,
India) with 5% (v/v) human blood and incubated at 30C for
1824 hours. The sterility of all reagents was checked during
the entire procedure.
Amplification and cloning of 16S rRNA gene. Chromosomal DNA was isolated from the remaining midgut contents
by the standard phenol/chloroform extraction method.18 The
1.5-kilobase partial sequence of the 16S rRNA gene was amplified from the pooled chromosomal DNA representing all
the samples using a polymerase chain reaction (PCR) and
universal Eubacteria-specific primers 16F27 (5-CCA GAG
TTT GAT CMT GGC TCA G-3) and 16R1525XP (5-TTC
TGC AGT CTA GAA GGA GGT GWT CCA GCC-3).16
The PCR conditions used were an initial denaturation at 94C
for two minutes, followed by 35 cycles of denaturation at 95C
for one minute, annealing at 55C for one minute, and extension at 72C for one minute, and a final extension at 72C for
10 minutes.
The amplified PCR product was a mixture of 16S rRNA
genes from all the Eubacteria present in the midgut of the
mosquito. To study these genes individually, a 16S rRNA
gene library was constructed using pGEM-T Easy vector
(Promega, Madison, WI) in Escherichia coli DH5. Plasmid DNA preparations from all the clones positive for the
1.5-kilobase insert were made as previously described19 and
sequenced on an ABI310 automated DNA sequencer using
the Big Dye terminator kit (Applied Biosystems, Inc., Foster
City, CA) as previously described.20 The identification of the
isolates from blood agar plates was done by 16S rRNA gene
sequencing after preliminary biochemical identification. The
amplified 16S rRNA gene PCR products from these isolates
were directly sequenced after purification by precipitation
with polyethylene glycol and NaCl. The primers used to obtain the complete sequence of 16S rRNA gene of the isolates
were the same as for PCR amplification (16F27N and
16R1625XP). An internal primer (16F536, 5-GTG CCA
GCA GCC GCG GTR ATA-3) was also used in addition to
the other primers. The sequencing of the 16S rRNA gene
insert from the clones was done using the 16F27N primer. The
5-terminus, a 500-nucleotide region of the 16S rRNA gene,
contains variable regions (positions approximately 100-200)
that not only discriminate between closely related species, but

in most cases will also underestimate the degree of sequence

similarity of nearly complete sequences by 112% for strains
belonging to different phyla.21
Sequence analysis. An equal portion (approximately 500
basepairs) of the 16S rRNA gene (E. coli positions 67 to 572,
Accession number J01859), was used for sequence analysis at
the Ribosomal Database Project (RDP II; Michigan State
University, East Lansing, MI) and the National Center for
Biotechnology Information (Bethesda, MD) (http//:www.
ncbi.nlm.nih.gov/BLAST). The similarity matrix was prepared using the similarity matrix calculator at the RDP II site.
The presence of chimeric sequences was checked with the
RDP Chimera Check program and by comparing independently the alignments at the beginning of each sequence and
at the end of each sequence and the alignments of the entire
sequence. The alignment of all the 16S rRNA gene sequences
was done using CLUSTAL W program at the European Bio
Informatics server (European Bioinformatics Institute,
Wellcome Trust Genome Campus, Hinxton, Cambridge,
United Kingdom) (http//:www.ebi.ac.uk/clustalw) to determine similarity value and distance matrix. The phylogenetic
tree was constructed using 500 basepair aligned sequences by
the neighbor joining method using Kimura 2 parameter distances in MEGA 2.1 software.22 It was reported earlier that
phylogenetic trees based on partial sequences have the same
topologies as those based on a complete sequence with the
established groups being identical but some deep branches
differing slightly.23,24 Furthermore, sequences of 500 nucleotides are sufficient for placement if some longer sequence is
closely related (> 90% identity in homologous nucleotides) to
the query sequence.25
An operational taxonomic unit (OTU) or molecular species, as used here, consisted of all sequences with less than 2%
divergence from the aligned homologous nucleotides. This
threshold was based on earlier conclusion that the sequence
divergence of clones belonging to the same OTU [is] generally low (between 2 and 0%) and was also generally consistent with the results of the comparison of 16S rRNA similarity
and DNA-DNA reassociation values.26,27 Coverage was calculated by Goods method, according to which the percentage
of coverage was calculated with the formula [1 (n/N)] 100,
where n is the number of molecular species represented by
one clone (single-clone OTUs) and N is the total number of
sequences.28 The sequences of the isolates from the culturedependent study were deposited in GenBank with accession
numbers AF170914, AF417863, AF417866, AF417868,
AF417870, AF417872, AF417874, AF417876, AF417877, and
AY057450, whereas sequences of clones from the library
were deposited with accession numbers AY119288 to
AY119440, and AY332749 to AY332756.
RESULTS
Isolation and 16S rRNA sequence analysis of strains. Direct plating of the mosquito midgut contents was used for the
isolation of the culturable microbiota. The colonies on blood
agar were selected on the basis of conventional bacteriological techniques, and initially those with minor variations were
also picked for further isolation. The initial number of 32
isolates was reduced to 10 after a first round of screening
based on colony characteristics (involving colony size, shape,
color, margin, opacity, elevation, and consistency) and the

599

MIDGUT MICROBIOTA OF CX. QUINQUEFASCIATUS

morphology of isolates studied by Gram staining and motility

by the hanging drop technique. To determine the phylogenetic relatedness of the strains, the isolates were subjected to
analysis with the complete 16S rRNA gene sequence.29
Analysis with the 16S rRNA gene sequence identified the
10 different bacterial isolates as Acinetobacter junii, Ac. calcoaceticus, Bacillus thuringiensis, Microbacterium oxydans,
Pantoea agglomerans, Pseudomonas aeruginosa, Staphylococcus epidermidis, Stenotrophomonas maltophila, and an unidentified bacterium of the host Drosophila paulistorium, the
details of which are shown in Table 1. The tenth isolate, which
showed a 98.2% similarity with close relatedness to A. jandaei
and an increased count (2,000-fold) after a blood meal, was
found to be a novel species of the genus Aeromonas and was
characterized in detail in a separate study as A. culicicola
MTCC 3249T.16
The majority of these isolates have been previously reported to inhabit the midgut of Cx. pipiens and Psorophora
columbiae.6 Furthermore, seven of the 10 isolates belonged to
the -proteobacteria class, emphasizing that the majority of
the mosquito midgut microbiota are composed of bacteria
belonging to this class. The percent similarity between the
isolated sequences and the type strains in the database was
mostly greater than 97%, except for a strain related to Pantoea agglomerans JCM 1236 (95.8%). Phylogenetic grouping
of the isolates was tested with the respective representatives
of the clone library and is shown in Figures 13.
Analysis of the 16S rRNA gene clone library. A total of 233
clones were found positive for the insert and were partially
sequenced, 153 of which were found to contain the amplified
16S rRNA gene. Of these, three were shown to be chimeras
and were therefore not included for further analysis. The phylogenetic analysis of the remaining 150 clones (Table 2) was
done using 500-basepair aligned homologous nucleotide sequences. The percentage distribution of the clones from the
16S rRNA gene library representing the microbiota of the
midgut of Cx. quinquefasciatus was determined and is shown
in Table 2. On the basis of sequence similarity to the existing
GenBank database entries, the clones were clustered together
to form three major groups: the Acinetobacter group, the Lactococcus group, and the unidentified and uncultured bacteria
group. The last group included all the uncharacterized and as
yet uncultured bacteria. The coverage calculated for the 150
clones was 83.3%. Thus, the probability of the next cloned

sequence falling in a novel OTU (not yet observed) was

16.7%. This value gives an estimation of how well the clones
analyzed account for the biodiversity within the original
sample by the present methodology (i.e., with our PCR conditions and primer set).28
Sixty-two clones (41%) belonged to Acinetobacter group
and all except three clones had similarity values of more than
98%. Of these, 60 clones showed highest similarity to a single
Acinetobacter strain (AJ410290), whereas the remaining two
were found related to another Acinetobacter strain
(AY055373). Among the clones that showed similarity to the
cultured and identified bacteria, this was the largest group.
These results correlated with the culture studies since two
species of Acinetobacter were isolated from the midgut. There
were only three one-clone OTUs within the Acinetobacter
group; thus, the percentage of coverage in this group was high
(95.2%). The phylogenetic analysis of the 62 clones along
with the two Acinetobacter isolates from the culture study and
the nearest neighbor database entries is shown in Figure 1.
The unidentified and the uncultured group comprised 46%
(69 clones) of the total 16S rRNA gene library with similarity
values ranging from 84.8% to 100% with the sequences in the
database. An initial taxonomic analysis of all the clones that
belonged to this group showed their affiliation to the family
Proteobacteria of the domain bacteria with higher relatedness
to the -proteobacteria class. Four different clusters, depending upon the nearest neighbor in the database, represented
the group. The majority of the clones showed highest similarities with the isolates from the deepest sea mud of the
Mariana Trench.30 Thirty-two (22%) clones showed similarities to different isolates of unidentified -proteobacteria with
similarities between 88% and 100%. Twenty (13%) clones
showed similarity to unidentified bacteria from activated
sludge and 12 of these clones had more than 98% similarity,
whereas for the remaining ones, the values were below 95%.
Seventeen (11%) clones showed similarities to sequences representing uncultured bacteria. Eleven of the 17 clones showed
maximum similarity to a clone from the marine environment.31,32 Of these 11, six clones showed more than 98%
sequence similarity and the remaining five clones showed
similarities between 97% and 89%. The remaining six clones
of the uncultured bacteria group showed similarities to uncultured soil bacteria.33 In all, there were 21 single-clone
OTUs; therefore the coverage for the group was less (70%).

TABLE 1
Phylogenetic affiliation of isolates obtained from the culture-based techniques based on complete 16S ribosomal RNA (rRNA) gene sequence*
Midgut isolate

Blast analysis

Isolate
name

Accession
number

Nearest phylogenetic
neighbor

Accession
number

%
Similarity

A
E
F
G
H
P
1a
6
7c
9

AF417863
AF417877
AY057450
AF417866
AF170914
AF417868
AF417870
AF417872
AF417874
AF417876

Acinetobacter junii
Unidentified bacterium
Staphylococcus epidermidis
Stenotrophomonas maltophila
Aeromonas jandaei
Microbacterium oxydans
Pantoea agglomerans
Acinetobacter calcoaceticus
Bacillus thuringiensis
Pseudomonas aeruginosa

X81664
U20277
D83362
AJ293470
AF099025
Y17227
AB004691
AF159045
AF155955
AF094713

99.3
99.0
99.3
97.0
98.2
98.6
95.8
97.8
98.7
98.9

* The sequence analysis was done at the Ribosomal Database Project (Michigan State University, East Lansing, MI) and the National Center for Biotechnology Information (Bethesda, MD)
(http//:www.ncbi.nlm.nih.gov/BLAST) and is based on the complete 16S rRNA gene sequence.
Isolate H was completely characterized as Aeromonas culicicola Microbial Type Culture Collection 3249T in a separate study.16

600

PIDIYAR AND OTHERS

FIGURE 1. Phylogenetic affiliation of the members of the Acinetobacter group from the midgut of Culex quinquefasciatus based on a
partial sequence of the 16S ribosomal RNA gene. The sequence alignment was performed using the CLUSTAL W program and the tree was
generated using the neighbor joining method with Kimura 2 parameter
distances in MEGA 2.1 software. Only positions 67-572 (Escherichia coli
numbering) were considered for the alignment. Numbers at nodes indicate percent bootstrap values above 50 (1,000 replicates). The bar indicates the Jukes-Cantor evolutionary distance. Names in bold represent
isolated strains and the nearest neighbor obtained in Blast analysis.

FIGURE 2. Phylogenetic affiliation of the members of the -proteobacteria group from the midgut of Culex quinquefasciatus based
on a partial sequence of the 16S ribosomal RNA gene. The sequence
alignment was performed using the CLUSTAL W program and the
tree was generated using the neighbor joining method with Kimura 2
parameter distances in MEGA 2.1 software. Only positions 67-572
(Escherichia coli numbering) were considered for the alignment.
Numbers at nodes indicate percent bootstrap values above 50 (1,000
replicates). The bar indicates the Jukes-Cantor evolutionary distance.
Names in bold represent isolated strains and the nearest neighbor
obtained in Blast analysis. Aeromonas culicicola is isolate H from the
present study reported elsewhere.16

601

MIDGUT MICROBIOTA OF CX. QUINQUEFASCIATUS

FIGURE 3. Phylogenetic affiliation of the members of the Firmicutes family from the midgut of Culex quinquefasciatus based on a partial
sequence of the 16S ribosomal RNA gene. The sequence alignment was performed using the CLUSTAL W program and the tree was generated
using the neighbor joining method with Kimura 2 parameter distances in MEGA 2.1 software. Only positions 67-572 (Escherichia coli numbering)
were considered for the alignment. Numbers at nodes indicate percent bootstrap values above 50 (1,000 replicates). The bar indicates the
Jukes-Cantor evolutionary distance. Names in bold represent isolated strains and the nearest neighbor obtained in Blast analysis.

The phylogenetic analysis of the group along with the isolates

from the culture-based study belonging to the -proteobacteria class is shown in Figure 2.
The third group of the 16S rRNA gene library along with
the remaining three isolates from the culture studies belonged
to the Firmicutes group (Gram-positive bacteria) of the domain bacteria with higher relatedness to the family Bacilli.
Nineteen (13%) clones belonged to the Lactococcus group,
with a majority of them showing 100% similarity with two
different L. garvieae 16S rRNA sequences in the database.
Seven of the 19 clones showed similarities greater than 98%
to Enterococcus seriolicida (AF061005). There was only one
single-clone OTU in the group; thus, the coverage was very
high (94.7%). The isolates F, P, and 7c from the culture-based
study showed the highest similarity to members of the same

class to which these 19 clones belonged. The phylogenetic

analysis of these clones is shown in Figure 3.
DISCUSSION
Members of the genera Aeromonas, Acinetobacter, and
Pseudomonas have been reported in the mosquito midgut in
previous studies and our results are consistent with those of
these earlier reports.6,89 One isolate had a similarity to an
unidentified bacterium isolated from a semi-species comprising the D. paulistorum complex, and it was reported to be
infectious in lepidopterans when introduced by inoculation.34
The tenth isolate, which was characterized as A. culicicola,
showed an increased count after a blood meal. A 7016,000fold increase in bacterial counts 24 hours after a blood meal

602

PIDIYAR AND OTHERS

TABLE 2
Phylogenetic affiliation of 16S ribosomal RNA (rRNA) gene clones in different clusters of domain bacteria based on a partial 16S rRNA
gene sequence*
Strain
no.

-proteobacteria cluster
1.
2.
2a.

2b.
2c.
2d.

Firmicutes cluster
3.

No. of
clones

16S rRNA %
similarity

%
distribution

Acinetobacter group
Acinetobacter sp. EVA 14 (AJ410290)
Acinetobacter sp. aerobic (AY055373)
Unidentified and Uncultured bacteria group
Unidentified proteobacteria group
Isolate HTA527 (AB002657)
Isolate HTA554 (AB002656)
Isolate HTA580 (AB002659)
Isolate HTA608 (AB002658)
Unidentified bacteria group (Z93992)
Uncultured bacteria clone CD3D6 (AY038379)
Uncultured soil bacteria
Clone 114-1 (AF423229)
Clone 431-1 (AF423262)
Clone 816-1 (AF423294)

62
60
2
69
32
3
18
9
2
20
11
6
2
1
3

88.4999.4
91.4899.4
88.4994.27
84.8100
88.1100
95.7100
91.4100
94100
9299.1
90.2100
89100
95.199.8
99.399.6
99.8
96.998.9

41.3

Lactococcus group
Lactococcus garvieae strain FLG12 (AF352166)
Lactococcus garvieae (AF283499)
Enterococcus seriolicida (AF061005)

19
11
1
7

92.8100
98.1100
94.5
98.6100

Clusters

46
21.3

13.3
7.3
4

12.7

* The sequence analysis was done at the Ribosomal Database Project, Michigan State University, East Lansing, MI) and the National Center for Biotechnology Information (Bethesda, MD)
(http//:www.ncbi.nlm.nih.gov/BLAST) and is based on partial 16S rRNA gene sequence (Escherichia coli positions 67572). Sequence showing more than 98% similarity were considered to be
a single operational taxonomic unit (OTU) or molecular species based on the conclusion that the sequence divergence of clones belonging to the same OTU [is] generally low (between 2 and
0%).26

has been previously reported.6 It was suggested that the rapid

growth of midgut bacteria after a blood meal is fueled by the
iron and protein-rich bolus of blood. The -hemolytic character of A. culicicola might help in blood digestion in the
midgut and might play a major role in symbiotic association.
In the 16S rRNA gene library, the majority of the clones
showed sequence similarities to either the cultured or the
uncultured members of -proteobacteria group, which correlates with the culturable bacterial flora in which seven of 10
isolates belonged to this group. Moreover, two isolates were
of the genus Acinetobacter and 40% of the clones showed the
highest similarity to this genus, indicating that -proteobacteria, especially those related to Acinetobacter, may form a
significant proportion of the mosquito midgut microbiota. We
were unable to isolate any clones related to A. culicicola. In
the earlier studies on human gut microbiota, investigators
failed to identify any rRNA sequences corresponding to the
most commonly observed bacteria in the human gut.3537 If
one considers breeding water to be the source of bacteria in
the midgut, the presence of sequences related to activated
sludge or soil clones was not surprising.31,33 Forty-three
(28%) clones showed a maximum similarity to sequences
from marine environments such as deep-sea mud and diseased corals, which probably reflects the metabolic diversity
of -proteobacteria.30,32
To the best of our knowledge, this is the first attempt to
study the midgut microbiota of a medically important insect
such as the Cx. quinquefasciatus using culture-independent
methods. Earlier results and the isolation of A. culicicola from
the midgut of both Cx. quinquefasciatus and Ae. aegypti indicate that at least a fraction of mosquito midgut inhabitants
could be common for different mosquito species inhabiting
the same environment.6,16 Mosquitoes are known to illicit a
specific immune response against parasites, Gram-positive
bacteria, and Gram-negative bacteria.38 Some of these im-

mune responsive genes are expressed in response to both

protozoa and bacteria, and this raises the possibility that the
presence of specific bacteria in the gut may have an effect on
the efficacy at which a pathogen is transmitted by a vector
mosquito.39 The present study assumes importance in the
light of earlier studies39 and our own observations that the
composition of midgut microbiota has a significant effect on
the survival of pathogens in the gut lumen.40
Furthermore, studies involving the effect of the isolates of
the midgut bacterial flora from this study on the susceptibility
of Cx. quinquefasciatus to Japanese encephalitis virus have
already been undertaken. Our results indicate that the incorporation of the Pseudomonas and Acinetobacter isolates in
the mosquito blood meal resulted in an increased susceptibility of Cx. quinquefasciatus to this virus.41 Isolates affiliated
with the genus Acinetobacter form a major part of the midgut
microbiota of various mosquito species, as have been reported in this study and many of the earlier reports.6,89
Moreover, the availability of reliable expression systems and
transposable elements for the genus Acinetobacter provides
an easy tool for the manipulation of the bacteria to produce
anti-parasitic proteins. Once established in the mosquito midgut using artificial means, these modified bacteria can thus be
used for the generation of transgenic mosquitoes refractory to
transmission of diseases. Thus, -proteobacteria, especially
those related to Acinetobacter could be considered as the candidate bacteria for the genetic manipulation of mosquitoes.
Received October 6, 2003. Accepted for publication November 3,
2003.
Authors address: Vyankatesh J. Pidiyar, Kamlesh Jangid, Milind S.
Patole, and Yogesh S. Shouche, Molecular Biology Unit, National
Centre for Cell Science, Pune University Campus, Ganeshkhind,
Pune-411007, Maharashtra, India, Telephone: 91-20-569-0922, Fax:
91-20-569-2259, E-mails: vpidiyar@hotmail.com, jangidk@nccs.res.in,
milindpatole@hotmail.con, and yogesh@nccs.res.in.

MIDGUT MICROBIOTA OF CX. QUINQUEFASCIATUS

REFERENCES
1. Durvasula RV, Gumbs A, Panackal A, Kruglov O, Aksoy S,
Merrifield RB, Richards FF, Beard CB, 1997. Prevention of
insect borne diseases: an approach using transgenic symbiotic
bacteria. Proc Natl Acad Sci USA 94: 32743278.
2. Carlson J, 1996. Genetic manipulation of mosquitoes: an approach to controlling disease. Trends Biotechnol 1: 447448.
3. Ghosh AK, Ribolla PEM, Jacobs-Lorena M, 2001. Targeting
Plasmodium ligands on mosquito salivary glands and midgut
with a phage display peptide library. Proc Natl Acad Sci USA
98: 1327813281.
4. Johnson BW, Olson KE, Allen-Miura T, Rayms-Keller A, Carlson JO, Cates CJ, Jasinskiene N, James AA, Beaty BJ, Higgs
S, 1999. Inhibition of luciferase expression in transgenic Aedes
aegypti mosquitoes by Sindbis virus expression of antisense
luciferase RNA. Proc Natl Acad Sci USA 96: 1339913403.
5. Olson KE, Higgs S, Gaines PJ, Powers AM, Davis BS, Kamrud
KI, Carlson JO, Blair CD, Beaty BJ, 1996. Genetically engineered resistance to dengue-2 virus transmission in mosquitoes. Science 272: 884886.
6. DeMaio J, Pumpuni CB, Kent M, Beier JC, 1996. The midgut
bacterial flora of wild Aedes triseriatus, Culex pipiens and Psorophora columbiae mosquitoes. Am J Trop Med Hyg 54: 219223.
7. Beier MS, Pumpuni CB, Bizio JC, Davis JR, 1994. Effect of paraaminobenzenoic acid, insulin and gentamicin on Plasmodium
falciparum development in Anopheline mosquitoes (Diptera:
Culicidae). J Med Entomol 31: 561565.
8. Chao J, Wistreich GA, 1959. Microbial isolations from the midgut
of Culex tarsalis Coquillett. J Insect Pathol 1: 311318.
9. Chao J, Wistreich GA, 1960. Microorganisms from the midgut of
larval and adult Culex quinquefasciatus Say. J Insect Pathol 2:
220224.
10. Ferguson MJ, Micks DW, 1961. Microorganisms associated with
mosquitoes: bacteria isolated from adult Culex fratigans
Wiedemann. J Insect Pathol 3: 112119.
11. Jadin J, 1965. Les bacteries photosynthetiques pourpres peuventelles jouer un role dans la sporogonie des Plasmodium? Bull
Acad Natl Med 149: 470472.
12. Jadin J, Vincke IH, Dunjie A, Delville JP, Wery M, Bafort J,
Scheepers-Diva M, 1966. Role des Pseudomonas dans la
sporogonie de Phematozoarie du paludisme chez le moustique.
Bull Soc Pathol Exot 59: 514525.
13. Seitz HM, Maier WA, Rottok M, Becker-Feldmann H, 1987.
Concomitant infections of Anopheles stephensi with Plasmodium berghei and Serratia marcescens: additive detrimental effects. Zentralbl Bakteriol Hyg 266: 155166.
14. Vasanthi V, Hoti SL, 1992. Microbial flora in gut of Culex quinquefasciatus breeding in cess pits. Southeast Asian J Trop Med
Public Health 2: 312317.
15. Olson GJ, Woese CR, Overbeek R, 1994. The winds of (evolutionary) change: breathing new life into microbiology. J Bacteriol 176: 16.
16. Pidiyar VJ, Kaznowski A, Badri Narayan N, Patole MS, Shouche
YS, 2002. Aeromonas culicicola sp. nov., from the midgut of
Culex quinquefasciatus. Int J Syst Evol Microbiol 52: 17231728.
17. Pidiyar VJ, Jangid K, Patole MS, Shouche YS, 2003. Detection
and phylogenetic affiliation of Wolbachia sp. from Indian mosquitoes Culex quinquefasciatus and Aedes albopictus. Curr Sci
84: 11361139.
18. Sambrook J, Fritsch EF, Maniatis T, 1989. Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory Press.
19. Lee SY, Rasheed S, 1990. A simple procedure for maximum yield
of high quality plasmid DNA. Biotechniques 9: 676679.
20. Hauben L, Vauterin L, Swings J, Moore ERB, 1997. Comparison
of 16S ribosomal DNA sequences of all Xanthomonas species.
Int J Syst Bacteriol 47: 328335.
21. Stackebrandt E, Rainey FA, 1995. Partial and complete 16S
rDNA sequences, their use in generation of 16S rDNA phylogenetic trees and their implications in molecular ecological
studies. Akkermans ADL, van Elsas JD, de Bruijn FJ, eds.
Molecular Microbial Ecology Manual. Volume 3.1.1. Dordrecht, The Netherlands: Kluwer Academic Publishers, 117.
22. Kumar S, Tamura K, Jakobsen IB, Nei M, 2001. MEGA2: Mo-

23.
24.
25.
26.

27.

28.
29.

30.
31.
32.

33.

34.
35.
36.

37.

38.

39.

40.

41.

603

lecular Evolutionary Genetics Analysis software. Bioinformatics 17: 12441245.

Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR, 1985.
Rapid determination of 16S ribosomal RNA sequences for
phylogenetic analyses. Proc Natl Acad Sci USA 82: 69556959.
Schimdt TM, DeLong EF, Pace NR, 1991. Analysis of a marine
picoplankton community by 16S rRNA gene cloning and sequencing. J Bacteriol 173: 43714378.
Hugenholtz P, Goebel BM, Pace NR, 1998. Impact of cultureindependent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180: 47654774.
Godon JJ, Zumstein E, Dabert P, Habouzit F, Moletta R, 1997.
Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol 63: 28022813.
Stackebrandt E, Goebel BM, 1994. Taxonomic note: a place for
DNA-DNA reassociation and 16S rRNA sequence analysis in
the present species definition in bacteriology. Int J Syst Bacteriol 44: 846849.
Good IJ, 1953. The population frequencies of species and the
estimation of population parameters. Biometrica 40: 237264.
Brambilla E, Hippe H, Hagelstein A, Tindall BJ, Stackebrandt E,
2001. 16S rDNA diversity of cultured and uncultured prokaryotes of a mat sample from Lake Fryxell, McMurdo Dry Valleys, Antarctica. Extremophiles 5: 2333.
Takami H, Inoue A, Fuji F, Horikoshi K, 1997. Microbial flora in
the deepest sea mud of the Mariana Trench. FEMS Microbiol
Lett 152: 279285.
Snaidr J, Amann R, Huber I, Ludwig W, Schleifer KH, 1997.
Phylogenetic analysis and in situ identification of bacteria in
activated sludge. Appl Environ Microbiol 63: 28842896.
Frias-Lopez J, Zerkle AL, Bonheyo GT, Fouke BW, 2002. Partitioning of bacterial communities between seawater and
healthy, black band diseased, and dead coral surfaces. Appl
Environ Microbiol 68: 22142228.
Valinsky L, Della Vedova G, Scupham AJ, Alvey S, Figueroa A,
Yin B, Hartin RJ, Chrobak M, Crowley DE, Jiang T, Borneman J, 2002. Analysis of bacterial community composition by
oligonucleotide Fingerprinting of rRNA genes. Appl Environ
Microbiol 68: 32433250.
Miller SG, Campbell BC, Becnel J, Ehrman L, 1995. Bacterial
entomopathogens from the Drosophila paulistorum semispecies complex. J Invertebr Pathol 65: 125131.
Hold GL, Pryde SE, Russell VJ, Furrie E, Flint HJ, 2002. Assessment of microbial diversity in human colonic samples by
16S rDNA sequence analysis. FEMS Microbial Ecol 39: 3339.
Sghir A, Dor J, Mackie RI, 1999. Molecular diversity and phylogeny of human colonic bacteria. Bell CR, Brylinski M, Green
PJ, eds. Microbial Biosystems: New Frontiers. Proceedings of
the Eighth International Symposium on Microbial Ecology.
Halifax, Nova Scotia, Canada. Kentville, Nova Scotia, Canada:
Atlantic Canada Society for Microbial Ecology, Canada.
Suau A, Bonnet R, Sutren M, Godon JJ, Gibson GR, Collins
MD, Dor J, 1999. Direct analysis of genes encoding 16S
rRNA from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol 65:
47994807.
Dimopolos G, Richman A, Muller HM, Kafatos FC, 1997. Molecular immune responses of the mosquito Anopheles gambiae
to bacteria and malaria parasites. Proc Natl Acad Sci USA 94:
1150811513.
Pumpuni CB, Demaio J, Kent M, Davis JR, Beier JC, 1996. Bacterial population dynamics in three anopheline species: the
impact on Plasmodium sporogonic development. Am J Trop
Med Hyg 54: 214218.
Mourya DT, Pidiyar VJ, Patole MS, Gokhale MD, Shouche YS,
2002. Effect of midgut bacterial flora of Aedes aegypti on the
susceptibility of mosquitoes to Dengue viruses. Dengue Bull
26: 190194.
Mourya DT, Gokhale MD, Pidiyar VJ, Barde PV, Patole MS,
Mishra AC, Shouche YS, 2002. Study of the effect of the midgut bacterial flora of Culex quinquefasciatus on the susceptibility of mosquitoes to Japanese Encephalitis virus. Acta Virol
46: 257260.