Acta Tropica 115 (2010) 275–281

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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

Culture-dependent and culture-independent characterization of microorganisms

associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial
colonization in the midgut
Desiely S. Gusmão a , Adão V. Santos b , Danyelle C. Marini c , Mauricio Bacci Jr. d ,
Marília A. Berbert-Molina b , Francisco José A. Lemos b,∗
a
Instituto Federal de Educação, Ciência e Tecnologia Fluminense-IFF, 28.030-130, Campos dos Goytacazes, RJ, Brazil
b
Laboratório de Biotecnologia, Universidade Estadual do Norte Fluminense-UENF, Av. Alberto Lamego, 2000, 28.013-602, Campos dos Goytacazes, RJ, Brazil
c
Faculdades Integradas Maria Imaculada, 13.840-040, Mogi-Guaçu, SP, Brazil
d
Laboratório de Evolução Molecular, Universidade Estadual Paulista-UNESP, 13.506-900, Rio Claro, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: In this work we show that the lumen of Aedes aegypti midgut is highly colonized by bacteria that were
Received 3 August 2009 identified by culture-dependent and culture-independent methods. rDNA sequences obtained were com-
Received in revised form 16 April 2010 pared with those from GenBank and the main bacterial genera identified were: Serratia, Klebsiella, Asaia,
Accepted 21 April 2010
Bacillus, Enterococcus, Enterobacter, Kluyvera and Pantoea. All genera were identified in midgut except
Available online 29 April 2010
Enterobacter that was observed only in eggs. Asaia and Pantoea were also identified in eggs and ovary,
respectively. In addition two yeast genera were observed in A. aegypti: Pichia isolated from midgut and
Keywords:
Candida identified in midgut and ovary. The genus Serratia was dominant in all isolation assays represent-
Aedes aegypti
Midgut
ing 54.5% of the total of microorganisms. Thirty-nine and 24 bacterial clones were successfully obtained
Bacterial colonization from midguts 24 and 48 h after blood feeding (ABF), respectively. The majority of clones obtained were
Serratia from Serratia sp. (48.7% and 50% for 24 and 48 h ABF, respectively). Light microscopy showed that bac-
teria were located preferentially in the posterior midgut, around the blood meal and associated with
peritrophic matrix. Scanning electron microscopy images showed a high number of bacteria in midgut
during blood digestion and the peak of bacterial enumeration was reached 48 h ABF, stage in which lumen
was almost totally occupied by bacteria that were also interacting with epithelial microvilli. Our results
show the dynamics of microbial colonization and their distribution in midgut during blood digestion.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction behavior and resistance to pathogen colonization (Dillon and

Mosquitoes, in general, are vectors of many infectious diseases
for man, including dengue fever, malaria, yellow fever and filaria-
sis, which are a great challenge for public health in many countries.
Dengue fever is transmitted by Aedes aegypti a highly anthro-
pophilic mosquito and is the most widespread vector-borne virus
disease in the world endemic in more than 100 countries in trop-
ical and sub-tropical regions worldwide (Nene et al., 2007; WHO,
2009). Studies investigating A. aegypti biology are still of paramount
importance to develop new strategies for its control.
Many insects harbor large communities of diverse microor-
ganisms that probably exceed the number of cells in insects
themselves. These insect associated microorganisms may have
important roles for insect nutrition, reproduction, development,
Dillon, 2004). Studies about midgut related bacteria have been
carried out from both laboratory-reared and wild populations of
several mosquito species (DeMaio et al., 1996; Pumpuni et al., 1996;
Straif et al., 1998; Gonzalez-Ceron et al., 2003; Pidiyar et al., 2004;
Favia et al., 2007; Rani et al., 2009). However, little is known about
the role and distribution of the microbiota in the different compart-
ments of mosquitoes’ digestive tract. Recently, Gusmão et al. (2007)
showed that A. aegypti gut ventral diverticulum, an impermeable
sugar storage organ, harbors microorganisms.
The objective of this work was to identify A. aegypti microbiota
using culture-dependent and culture-independent methods and
also elucidate the dynamics of bacterial colonization in the midgut
after blood feeding (ABF).

2. Material and methods

∗ Corresponding author. Tel.: +55 22 27397031/90; fax: +55 22 27397031. Insects were obtained from colonies of A. aegypti (Rockfeller
E-mail address: franze@uenf.br (F.J.A. Lemos). strain), maintained in the insectary of the Laboratory of Biotech-

0001-706X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2010.04.011
276 D.S. Gusmão et al. / Acta Tropica 115 (2010) 275–281
nology (UENF, Campos dos Goytacazes, Brazil). Mosquitoes were
reared in cages, at 27 ◦ C and fed on sterile 10% sucrose solution.
A glass container filled with sterile distilled water was kept inside
each cage to maintain humidity. Larvae were fed on a sterile finely
ground commercial mouse food. Pupae were rinsed and transferred
to sterile distilled water and maintained in separate cages until
adult emergence. Production of eggs was induced after providing
blood meal (mouse) to the insects. Females laid eggs on moisture fil-
ter paper 3 and 4 days after blood meal. After 2 days desiccated eggs
were transferred to plastic containers filled with deoxygenated dis-
tilled water for larvae eclosion.
All necessary material for handling and dissection of mosquitoes
was autoclaved at 121 ◦ C for 20 min. Some materials, such as stere-
omicroscopic and pipettes, were sanitized with 70% ethanol. Five
independent isolation assays were carried out using ten midguts
for each of the six following samples: midguts from early emerged
females, midguts from 5 days old females fed on sucrose and
midguts from females dissected 6, 12, 24 and 48 h ABF.
Mosquitoes were surface sterilized by rinsing in the following
solutions for 1 min: 1% sodium hypochlorite; sterile phosphate-
buffered saline (PBS) [81 mM Na2 HPO4 , 19 mM NaH2 PO4 , 150 mM
NaCl, pH 7.4] and 70% ethanol. Finally, insects were rinsed three
times in sterile PBS for 1 min. Aliquots of 100 ␮L from the last PBS
washes were plated in brain heart infusion (BHI) agar as control
groups of the insect surface sterilization process. Mosquitoes were
dissected under a stereomicroscope, in a double cavity glass slide
containing sterile PBS. Midgut was carefully removed from insect
abdomen, rinsed in sterile PBS and transferred into a 1.5 mL tube
containing 100 ␮L of PBS. Tube content was mixed thoroughly with
a pestle and serially diluted (10−1 through 10−7 ) and an aliquot
of 100 ␮L of each one was transferred to Petri dishes contain-
ing BHI agar. Plates were incubated at 28 ◦ C for 24–48 h. Bacterial
isolates were maintained at −70 ◦ C in a 15% glycerol solution for
further identification. The described procedure was also employed
for microbial enumeration that was done in triplicates by count-
ing the colony forming units (CFU), using one midgut for each
plate.
Microorganisms were also isolated from eggs as follows: three
sets of 100 eggs were transferred to 1.5 mL tubes containing 500 ␮L
of 1% sodium hypochlorite. After 3 min the sedimented eggs were
rinsed three times in 1 mL sterile PBS and homogenized in 50 ␮L PBS
with a pestle. After sedimentation supernatant was transferred to
Petri dishes containing BHI agar and incubated at 28 ◦ C for 24–48 h.
Microorganisms were first screened based on (a) colony char-
acteristics (color, size, shape, opacity, margin, elevation and
viscosity); (b) morphology and arrangement of isolates, Gram stain-
ing and motility.
Table 1
Phylogenetic affiliations of culturable microorganisms from Aedes aegypti midgut and ovary based on 16S and 28S rRNA gene analysis.
Isolate GenBank accession number
I5a FJ372760
I8a FJ372761
I11a FJ372762
I12a FJ372763
I17a FJ372764
O19b FJ372765
I22a FJ372766
I23a FJ372767
I24c FJ372768
I26a FJ372769
O27b FJ372770
I28c FJ372771
I34a FJ372772
a
Midgut isolates.
b
Egg isolates.
c
Isolates of midguts from early emerged females.
The DNA extraction from bacteria and yeast isolates was adapted
from Ausubel et al. (1992). Midguts and ovaries from A. aegypti
females were obtained under aseptic conditions and DNA extrac-
tion was performed with the Wizard® Genomic DNA Purification
Kit (Promega TM050).
The 16S rRNA gene was amplified using the following univer-
sal primers: 27f (Lane et al., 1985) and 1492r (Delong, 1992). PCR
reactions were performed with Ready-To-Go kit (Amersham Phar-
macia Biotech), template DNA solution (2 ␮L/100 ng); 27f primer
(1 ␮L/6 pmol); 1492r primer (1 ␮L/6 pmol); 25 mM MgCl2 (1.5 ␮L)
and 17.5 ␮L of ultra pure water. Cycling parameters for PCR reac-
tions included an initial denaturation step at 95 ◦ C for five min,
followed by 35 cycles of a denaturation step at 95 ◦ C for one min,
a primer annealing step at 50 ◦ C for 1 min, an extension step at
72 ◦ C for 3 min and a final step of 4 min at 72 ◦ C. The 16S rDNA
amplification generated a product of approximately 1500 bp.
The divergent D1/D2 domains (nucleotides 63–642 for Saccha-
romyces cerevisiae) at the 5 -end of the large-subunit 28S rRNA gene
was symmetrically amplified with primers NL1 and NL4 (O’Donnell,
1993). Each PCR was performed with Ready-To-Go kit (Amersham
Pharmacia Biotech) adding a solution containing 1 ␮L DNA; NL-1
primer (1.6 ␮L/6 pmol); NL-4 primer (1.1 ␮L/6 pmol); 1.5 ␮L 25 mM
MgCl2 and 17.8 ␮L of ultra pure water.
Sequencing reaction was prepared as follows: 2 ␮L Big Dye Ter-
minator Kit (Applied Biosystems, USA), 2 ␮L “Save Money” buffer
[200 mM Tris–HCl, pH 9.0; 5 mM MgCl], forward T7 or reverse SP6
promoters (1 ␮L/5.0 pmol), DNA (2 ␮L/100 ng) and 2 ␮L ultra pure
water. PCR conditions were 96 ◦ C for 10 min, 50 ◦ C for 5 min and four
min at 60 ◦ C, 40 cycles. Sequencing of the 16S rRNA gene was done
using a 27f primer (5 -end; 500 nucleotide region) and NL1 primer
for bacterial and yeast clones, respectively. Sequencing was carried
out on an ABI Prism 377 DNA sequencer (Applied Biosystems, USA).
Obtained sequences were initially compared to those from Gen-
Bank database using BLAST (http://www.ncbi.nlm.nih.gov/BLAST).
Generated sequences and their homologs were aligned using
CLUSTAL W 1.4 program (Thompson et al., 1994). Dendrograms
were obtained by “neighbor-joining” (PAUP program, Swofford,
2000). Sequences obtained were deposited in the GenBank
database.
Midgut samples were fixed in 2.5% glutaraldehyde, 0.1 M
sodium cacodylate, pH 7.2, for 12 h at room temperature. Tissues
were dehydrated in acetone, embedded in Epon and sections of
0.6 ␮M were cut with diamond knife. Samples were stained by tolu-
idine blue and examined under a light microscope (Zeiss, Axioplan).
Midguts were fixed as described above and post-fixed with 0.8%
K3 Fe(CN)6 /0.5% OsO4 in 0.1 M sodium cacodylate for 1 h at room
temperature. Samples were dehydrated through a graded acetone
Closest relative according to Blast (% identity)
Klebsiella pneumoniae AF453251 (97)
Asaia sp. AB025932 (98)
Pichia ohmeri AY267821 (100)
Klebsiella pneumoniae Y17657 (99)
Serratia marcescens AY514433 (100)
Enterobacter asburiae AB004744 (99)
Serratia sp. EF032328 (99)
Serratia sp. EF032328 (98)
Bacillus subtilis AY881636 (100)
Serratia sp. EF032328 (98)
Asaia krungthepensis AB102953 (99)
Bacillus sp. AF326365 (96)
Enterococcus caccae AY943820 (98)
 D.S. Gusmão et al. / Acta Tropica 115 (2010) 275–281 277

Table 2
Phylogenetic affiliations of non-culturable microorganisms from Aedes aegypti midgut and ovary based on 16S and 28S rRNA gene sequencing.

Clone GenBank accession number Closest relative according to Blast (% identity)

a
C1BI24 FJ372773 Serratia marcescens EF208030 (98%)
C2BI24a FJ372774 Uncultured bacterium AF529356 (98%)
C3BI24a FJ372775 Serratia marcescensAY946291 (99%)
C4BI24a FJ372776 Uncultured bacterium AY700617 (99%)
C5BI24a FJ372777 Serratia marcescens AY551938 (99%)
C6BI24a FJ372778 Serratia marcescens AY946291 (98%)
C8BI24a FJ372779 Serratia marcescens AY730005 (97%)
C10BI24a FJ372780 Klebsiella pneumoniae DQ122369 (98%)
C11BI24a FJ372781 Enterobacter aerogenes FJ360760 (99%)
C12BI24a FJ372782 Serratia marcescens AY498856 (100%)
C13BI24a FJ372783 Serratia sp. EU693548 (100%)
C14BI24a FJ372784 Klebsiella pneumoniae AY838331 (96%)
C17BI24a FJ372785 Uncultured bacterium AF371852 (98%)
C18BI24a FJ372786 Uncultured bacterium AY510235 (99%)
C19BI24a FJ372787 Pantoea agglomerans* DQ122347 (96%)
C23BI24a FJ372788 Enterobacter sp. AY689045 (98%)
C24BI24a FJ372789 Bactéria endofítica AY842147 (99%)
C27BI24a FJ372790 Serratia marcescens AJ550467 (99%)
C28BI24a FJ372791 Serratia marcescens AY498856 (98%)
C29BI24a FJ372792 Serratia marcescens AY498856 (98%)
C31BI24a FJ372793 Enterobacter sp. AY689062 (97%)
C33BI24a FJ372794 Serratia marcescens AY498856 (98%)
C35BI24a FJ372795 Uncultured bacterium AY700617 (98%)
C38BI24a FJ372796 Citrobacter freundii DQ133536 (99%)
C40BI24a FJ372797 Uncultured bacterium AY700604 (96%)
C41BI24a FJ372798 Enterobacter sp. AY689045 (98%)
C42BI24a FJ372799 Serratia marcescens AY946291 (98%)
C43BI24a FJ372800 Serratia marcescens AY498856 (97%)
C45BI24a FJ372801 Uncultured bacterium AY700617 (99%)
C46BI24a FJ372802 Serratia marcescens AY498856 (99%)
C50BI24a FJ372803 Uncultured bacterium DQ068885 (96%)
C52BI24a FJ372804 Serratia marcescens AY498856 (98%)
C53BI24a FJ372805 Uncultured bacterium AY700604 (99%)
C54BI24a FJ372806 Serratia marcescens AY395011 (99%)
C55BI24a FJ372807 Uncultured bacterium AY510238 (99%)
C56BI24a FJ372808 Serratia marcescens AY498856 (97%)
C57BI24a FJ372809 Serratia marcescens EF208030 (99%)
C58BI24a FJ372810 Serratia marcescens DQ357510 (91%)
C59BI24a FJ372811 Enterobacter sp. EF175731 (97%)
C2BI48b FJ372812 Uncultured bacterium EU775650 (98%)
C3BI48b FJ372813 Klebsiella pneumoniae AY905691 (99%)
C5BI48b FJ372814 Serratia marcescens AY498856 (99%)
C6BI48b FJ372815 Uncultured bacterium DQ906059 (99%)
C7BI48b FJ372816 Cedecea davisae AF493976 (100%)
C8BI48b FJ372817 Serratia marcescens AY946291 (94%)
C9BI48b FJ372818 Enterobacter sp. AB098582 (96%)
C11BI48b FJ372819 Pantoea sp. AY941839 (99%)
C13BI48b FJ372820 Serratia marcescens AY498856 (98%)
C14BI48b FJ372821 Pantoea sp. DQ083475 (94%)
C15BI48b FJ372822 Uncultured bacterium EU236240 (100%)
C16BI48b FJ372823 Serratia sp. EF600986 (100%)
C17BI48b FJ372824 Uncultured bacterium DQ068894 (97%)
C19BI48b FJ372825 Serratia marcescens EU233275 (99%)
C21BI48b FJ372826 Serratia marcescens EU221361 (99%)
C23BI48b FJ372827 Enterobacter sp. AB098582 (98%)
C24BI48b FJ372828 Serratia marcescens AY946291 (98%)
C25BI48b FJ372829 Serratia marcescens AY395011 (96%)
C27BI48b FJ372830 Kluyvera ascorbata AF176567 (99%)
C30BI48b FJ372831 Pantoea agglomerans DQ122348 (95%)
C31BI48b FJ372832 Serratia marcescens AF124042
C32BI48b FJ372833 Serratia marcescens EF208030 (97%)
C33BI48b FJ372834 Serratia marcescens EU233275 (98%)
C34BI48b FJ372835 Serratia sp. FJ360761 (98%)
C1LI48c FJ372836 Pichia guilliermondii AY497698 (95%)
C12LI48c FJ372837 Candida nodaensis U45726 (100%)
C17LI48c FJ372838 Candida fermentati AY894826 (98%)
C19LI48c FJ372839 Candida nodaensis U45726 (98%)
C28LI48c FJ372840 Pichia guilliermondii AY497698 (99%)
C30LI48c FJ372841 Pichia guilliermondii AY497698 (97%)
C31LI48c FJ372842 Candida nodaensis U45726 (99%)
C32LI48c FJ372843 Candida fermentati AY187283 (97%)
C44LI48c FJ372844 Candida nodaensis U45726 (97%)
278 D.S. Gusmão et al. / Acta Tropica 115 (2010) 275–281

Table 2 ( Continued ).

Clone GenBank accession number Closest relative according to Blast (% identity)

d
C7BO FJ372845 Pantoea agglomerans AY435402 (97%)
C1LOe FJ372846 Candida sp. AM420306 (99%)
C2LOe FJ372847 Candida sp. AM420306 (99%)
a
Bacterial clones – midgut 24 h after blood feeding (ABF).
b
Bacterial clones – midgut 48 h ABF.
c
Yeast clones – midgut 48 h ABF.
d
Bacterial clones – ovary 48 h ABF.
e
Yeast clones – ovary 48 h ABF.

series and embedded in Epon. Longitudinal sections of 70 nm were from midguts 48 h ABF: Serratia sp. (50%), uncultured bacteria
cut with an ultramicrotome (Reichert, Ultracut S), stained with 1% (16.7%), Pantoea (12.5%), Enterobacter sp. (8.3%), Klebsiella sp. (4.2%),
uranyl acetate and lead citrate, and observed at 80 kV on a transmis- Kluyvera (4.2%) and Cedecea (4.2%) (Table 2). Pantoea agglomerans
sion electron microscope (Zeiss, EM 900). Midguts were dehydrated was the only bacterial clone originated from ovaries. Nine yeast
in acetone and embedded in Epon. clones and two Candida sp. clones were obtained from midguts 48 h
Oblique cuts obtained from dissected midguts were fixed in ABF and from ovaries, respectively (Table 2).
2% formaldehyde, 2% glutaraldehyde, 0.1 M sodium cacodylate,
pH 7.2 for 1 h at room temperature. Subsequently, the samples
were washed two times in 0.1 M sodium cacodylate buffer, pH
7.2, pos-fixed for 1 h in 1% osmium tetroxide, rewashed in sodium
cacodylate and dehydrated through a graded ethanol series, critical
point dried in CO2 and sputter-coated with gold before examination
in a ZEISS 964 scanning electron microscope at 5 kV accelerating
voltage.

3. Results

Gram-negative rods were the major group of bacteria found in

midgut during the blood digestion, represented by 25 isolates (out
of 33) obtained by culturable-dependent techniques. Gram positive
rods and coccus, Gram-negative coccus and one yeast were also
identified.
Isolates were subjected to 16S rRNA gene sequence analysis and
the following bacterial genera were identified: Asaia, Bacillus, Enter-
obacter, Enterococcus, Klebsiella and Serratia (Table 1). The genus
Serratia was dominant in all isolation assays representing 54.5% of
total microorganisms, followed by Klebsiella (15.2%), Asaia (12.9%),
Bacillus (9.0%), Enterococcus (6.0%) and Enterobacter (3.0%). Serra-
tia and Klebsiella were continuously isolated after sucrose feeding
and during the whole blood digestion. Asaia was detected in eggs,
sucrose-fed mosquitoes and at the beginning of blood digestion
(6 h ABF). Enterococcus was isolated after sucrose feeding and 24 h
ABF while Bacillus occurred only in midguts from early emerged
females. Enterobacter sp. was observed only in eggs and the yeast
Pichia sp. was isolated only from sugar-fed mosquitoes.
Bacteria identified by culture-independent techniques cor-
responded to 39 bacterial clones obtained from midguts 24 h
ABF: Serratia (48.7%), uncultured bacteria (25.6%), Enterobacter
(12.8%), Klebsiella (5.1%), endophytic bacteria (2.6%), Pantoea (2.6%)
and Citrobacter (2.6%). Twenty-four bacterial clones were derived

Table 3
Average number of microorganisms present in midgut of Aedes aegypti females at
different times after blood feeding.

Midguts origin Average number of SDb

microorganisms (CFUa )

Non-fed females 2.1 × 102 1.2 × 102

Sucrose-fed females 2.3 × 103 6.6 × 102
Females dissected 3 h ABFc 1.4 × 104 9.4 × 103
Females dissected 24 h ABF 1.5 × 107 7.9 × 103
Females dissected 48 h ABF 1.2 × 108 4.1 × 107
Females dissected 67 h ABF 2.3 × 107 4.3 × 103 Fig. 1. Distribution of microorganisms in midguts of A. aegypti females dissected
24 h after blood feeding. Tissue sections stained by Toluidine blue were observed
a
Colony forming unit. under light microscopic. (A) Midgut anterior portion. (B) Midgut middle portion. (C)
b
Standard deviation. Midgut posterior portion. Many bacteria () are observed at the periphery of the
c
After blood feeding. food bolus. EP: epithelium; N: nucleus; PM: peritrophic matrix.
 D.S. Gusmão et al. / Acta Tropica 115 (2010) 275–281
Fig. 2. Scanning electron microscopy of A. aegypti midgut. (A) Central region of food bolus 24 h after blood feeding, showing intact erythrocytes () in midgut lumen. (B) Food
bolus 24 h after blood feeding showing bacteria (arrows) at periphery. (C) Central and (D) peripheric regions of food bolus 48 h after blood feeding, showing a high number
of bacteria (arrows). EP: epithelium.
About 200 bacteria were found in midgut lumen of non-fed
mosquitoes (Table 3), while sucrose-fed mosquitoes showed a
slight increase in bacterial number (2.3 × 103 CFU). After blood
feeding, the average number of bacteria increased progressively,
reaching the peak of 1.2 × 108 CFU (Table 3). Approximately twenty
million bacteria remained in midgut 67 h ABF, representing a five-
fold reduction in microbial population.
Light microscopy observations of females’ midguts 24 h ABF
revealed that bacteria were located preferably in the posterior
region, specifically between PM and food bolus (Fig. 1). It was possi-
ble to observe, through scanning electron microscopy, that midgut
lumen of A. aegypti dissected 24 h ABF contained blood cells repre-
sented mainly by intact erythrocytes (Fig. 2A). During this stage
of digestion bacteria were observed in the peripheral region of
the food bolus whereas none was viewed in the lumen central
region (Fig. 2B). Bacteria entirely occupied midgut lumen 48 h ABF
, stage in which all erythrocytes had already been lysed (Fig. 2C
and D). Some bacteria were also seen embedded in PM 24 h ABF
(Fig. 3A and B) and interacting with epithelial microvilli 48 h ABF
(Fig. 3C).
4. Discussion
Our findings provide comprehensive information on the com-
position of the A. aegypti microbiota and its distribution in the
midgut along blood digestion. We used two different approaches
(culture-dependent and culture-independent assays) to screen the
microorganisms associated with A. aegypti. Similar to previous
studies, the majority of bacterial isolates were Gram-negative rods,
mainly from Enterobacteriaceae family.
The genera Enterobacter, Enterococcus, Pantoea, Klebsiella,
Kluyvera and Serratia (Enterobacteriaceae), Asaia (Acetobacter-
aceae) and Bacillus (Bacillaceae) identified in this work had already
279
been described for some mosquito species (DeMaio et al., 1996;
Pumpuni et al., 1996; Straif et al., 1998; Fouda et al., 2001;
Gonzalez-Ceron et al., 2003; Lindh et al., 2005; Favia et al., 2007;
Terenius et al., 2008; Crotti et al., 2009; Dong et al., 2009; Rani et
al., 2009). These findings show that such bacteria are widespread
in mosquitoes, suggesting that they are capable of maintaining a
stable association with these insects, as described for Asaia sp. This
bacterium is a true symbiont of Anopheles sp. and a candidate to be
used in paratransgenic approaches (Favia et al., 2007). This is the
first work to identify yeasts in the midgut and ovary of A. aegypti
to date. Yeasts are usually associated with wood and bark beetles
(Grünwald et al., 2010) and adults of chrysopids (Woolfolk and
Inglis, 2004). Like mosquitoes, these insects are nectar and hon-
eydew feeders and the symbiotic yeasts provide the hosts with
essential amino acids absent in their diet (Hagen et al., 1970).
Serratia marcescens was the predominant bacterium found in A.
aegypti midgut suggesting that it may possess some competitive
advantages over the other bacteria. Similarly, Serratia sp. was also
one of the dominant species found in five generations of Anopheles
gambiae (Dong et al., 2009). The genus Serratia is widely distributed
in the environment and some species are described as insects’ sym-
bionts. Serratia sp. is described as symbiont of the sugar beet root
maggot, Tetanops myopaeformis, and may play a role in host nutri-
tion and chitin degradation of the insect puparium (Iverson et al.,
1984).
Asaia sp. and Enterobacter sp. are probably transovarially trans-
mitted to offspring since they were successfully identified in A.
aegypti eggs. Crotti et al. (2009) using PCR methods detected Asaia
sp. in A. aegypti larvae, pupae and adults, showing that this bac-
terium efficiently colonized guts, male and female reproductive
systems and salivary glands. Our findings along with those from
Crotti et al. (2009) indicate that Asaia sp. is symbiotically associ-
ated with A. aegypti, as reported for A. stephensi reared in laboratory
280 D.S. Gusmão et al. / Acta Tropica 115 (2010) 275–281
Fig. 3. Micrographs showing bacteria in A. aegypti midgut compartments. (A) Light
microscopy showing bacteria (arrows) within the PM. (B) Enlarged detail of A. (C)
Transmission electron microscopy showing interaction of bacteria (arrows) with
epithelial microvilli. EP: epithelium; ER: erythrocytes; MV: microvilli; PM: per-
itrophic matrix; N: nucleus.
(Favia et al., 2007) and in Scaphoideus titanus originated from the
field (Crotti et al., 2008).
The high number of bacteria observed in A. aegypti midgut
48 h ABF was similar to the results found in A. triseriatus, C. pipi-
ens, Psorophora columbiae (DeMaio et al., 1996), some Anopheline
species (Pumpuni et al., 1996) and Phlebotomus duboscqi (Volf et al.,
2002). The intense bacterial growth after blood feeding was prob-
ably enhanced by the high concentration of iron and protein in the
blood meal (Azambuja et al., 2004).
Blood digestion observed in A. aegypti followed the pattern
described by Akov (1965) and Briegel and Lea (1975), beginning
at the periphery of the food bolus and continuing from periphery
inwards with progressive lyse of erythrocytes. Twenty-four hours
after blood meal intact erythrocytes could still be observed in the
center of the food bolus and digestion was completed 48 h ABF.
Bacteria were dynamically present in whole A. aegypti digestion
process. At 24 h ABF, bacteria were not distributed uniformly along
the midgut but predominated in the posterior region of the abdom-
inal midgut, along the periphery of the food bolus. This bacterial
distribution coincides with trypsin activity, the major A. aegypti
endopeptidase, during blood digestion (Graf et al., 1986). At the
end of blood digestion bacteria occupied the entire midgut lumen
(48 h ABF). These observations suggest that bacteria may be either
obtaining benefits from the nutrients released in these highly diges-
tive regions or contributing for blood digestion.
Bacteria were also found associated with PM 24 h ABF and were
present in high number in midgut lumen after PM degradation 48 h
ABF. Interaction between bacteria and PM was also observed in
other insect species (Peters et al., 1983; Mead et al., 1988; Murphy
et al., 1994; Nayduch et al., 2005). S. marcescens is known to produce
chitinolytic enzymes and is a very effective bacterium for degra-
dation of chitin (Brurberg et al., 1996; Vaaje-Kolstad et al., 2005).
These enzymes may also be used to interact and digest mosquito
PM. We still observed some bacteria intrinsically associated with A.
aegypti epithelial microvilli. Bacteria-midgut interaction may also
be responsible for triggering insect immune system against micro-
bial infections (Rakoff-Nahoum et al., 2004; Ryu et al., 2008; Xi et
al., 2008; Dong et al., 2009). This intimate interaction in midgut is
advantageous for bacteria that will be protected from elimination
in the excretion process. As observed by Gusmão et al. (2007), bac-
teria are also found in A. aegypti gut ventral diverticulum where
they are protected from elimination at the end of blood digestion.
Gram-negative bacteria have been associated with inhibitory
activity of sporogonic development of Plasmodium parasites in
midgut of both laboratory-reared and field mosquitoes (Micks and
Ferguson, 1961; Pumpuni et al., 1993; Dong et al., 2009). This was
not investigated in this work but is also important for future works
that intend to develop new control strategies for insect vectors.
Many studies are still necessary to understand the interaction
of midgut microbiota and their role in insect physiology. This work
demonstrated that A. aegypti host a dense population of rod-shaped
Gram-negative bacteria in midgut along the blood digestion pro-
cess. The role of these associated bacteria to the viability of the
insect is still under investigation. However, their occurrence and
dynamics along blood digestion suggest a nutritional interdepen-
dence with the host.
Acknowledgements
This study was supported by grants from FAPERJ and CNPq.
Sequence reactions were done at the Center for the Study of Social
Insects, UNESP, Rio Claro, São Paulo, Brazil. The authors wish to
thank Ms Rívea Cristina Custódio Rodrigues and Telma Ferreira
Costa Aguiar for technical assistance.
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