Culture-Dependent and Culture-Independen

Acta Tropica 115 (2010) 275–281
Contents lists available at ScienceDirect
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

Dillon, 2004). Studies about midgut related bacteria have been
Mosquitoes, in general, are vectors of many infectious diseases carried out from both laboratory-reared and wild populations of
for man, including dengue fever, malaria, yellow fever and filaria- several mosquito species (DeMaio et al., 1996; Pumpuni et al., 1996;
sis, which are a great challenge for public health in many countries. Straif et al., 1998; Gonzalez-Ceron et al., 2003; Pidiyar et al., 2004;
Dengue fever is transmitted by Aedes aegypti a highly anthro- Favia et al., 2007; Rani et al., 2009). However, little is known about
pophilic mosquito and is the most widespread vector-borne virus the role and distribution of the microbiota in the different compart-
disease in the world endemic in more than 100 countries in trop- ments of mosquitoes’ digestive tract. Recently, Gusmão et al. (2007)
ical and sub-tropical regions worldwide (Nene et al., 2007; WHO, showed that A. aegypti gut ventral diverticulum, an impermeable
2009). Studies investigating A. aegypti biology are still of paramount sugar storage organ, harbors microorganisms.
importance to develop new strategies for its control. The objective of this work was to identify A. aegypti microbiota
Many insects harbor large communities of diverse microor- using culture-dependent and culture-independent methods and
ganisms that probably exceed the number of cells in insects also elucidate the dynamics of bacterial colonization in the midgut
themselves. These insect associated microorganisms may have after blood feeding (ABF).
important roles for insect nutrition, reproduction, development,
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 The DNA extraction from bacteria and yeast isolates was adapted
reared in cages, at 27 ◦ C and fed on sterile 10% sucrose solution. from Ausubel et al. (1992). Midguts and ovaries from A. aegypti
A glass container filled with sterile distilled water was kept inside females were obtained under aseptic conditions and DNA extrac-
each cage to maintain humidity. Larvae were fed on a sterile finely tion was performed with the Wizard® Genomic DNA Purification
ground commercial mouse food. Pupae were rinsed and transferred Kit (Promega TM050).
to sterile distilled water and maintained in separate cages until The 16S rRNA gene was amplified using the following univer-
adult emergence. Production of eggs was induced after providing sal primers: 27f (Lane et al., 1985) and 1492r (Delong, 1992). PCR
blood meal (mouse) to the insects. Females laid eggs on moisture fil- reactions were performed with Ready-To-Go kit (Amersham Phar-
ter paper 3 and 4 days after blood meal. After 2 days desiccated eggs macia Biotech), template DNA solution (2 ␮L/100 ng); 27f primer
were transferred to plastic containers filled with deoxygenated dis- (1 ␮L/6 pmol); 1492r primer (1 ␮L/6 pmol); 25 mM MgCl2 (1.5 ␮L)
tilled water for larvae eclosion. and 17.5 ␮L of ultra pure water. Cycling parameters for PCR reac-
All necessary material for handling and dissection of mosquitoes tions included an initial denaturation step at 95 ◦ C for five min,
was autoclaved at 121 ◦ C for 20 min. Some materials, such as stere- followed by 35 cycles of a denaturation step at 95 ◦ C for one min,
omicroscopic and pipettes, were sanitized with 70% ethanol. Five a primer annealing step at 50 ◦ C for 1 min, an extension step at
independent isolation assays were carried out using ten midguts 72 ◦ C for 3 min and a final step of 4 min at 72 ◦ C. The 16S rDNA
for each of the six following samples: midguts from early emerged amplification generated a product of approximately 1500 bp.
females, midguts from 5 days old females fed on sucrose and The divergent D1/D2 domains (nucleotides 63–642 for Saccha-
midguts from females dissected 6, 12, 24 and 48 h ABF. romyces cerevisiae) at the 5 -end of the large-subunit 28S rRNA gene
Mosquitoes were surface sterilized by rinsing in the following was symmetrically amplified with primers NL1 and NL4 (O’Donnell,
solutions for 1 min: 1% sodium hypochlorite; sterile phosphate- 1993). Each PCR was performed with Ready-To-Go kit (Amersham
buffered saline (PBS) [81 mM Na2 HPO4 , 19 mM NaH2 PO4 , 150 mM Pharmacia Biotech) adding a solution containing 1 ␮L DNA; NL-1
NaCl, pH 7.4] and 70% ethanol. Finally, insects were rinsed three primer (1.6 ␮L/6 pmol); NL-4 primer (1.1 ␮L/6 pmol); 1.5 ␮L 25 mM
times in sterile PBS for 1 min. Aliquots of 100 ␮L from the last PBS MgCl2 and 17.8 ␮L of ultra pure water.
washes were plated in brain heart infusion (BHI) agar as control Sequencing reaction was prepared as follows: 2 ␮L Big Dye Ter-
groups of the insect surface sterilization process. Mosquitoes were minator Kit (Applied Biosystems, USA), 2 ␮L “Save Money” buffer
dissected under a stereomicroscope, in a double cavity glass slide [200 mM Tris–HCl, pH 9.0; 5 mM MgCl], forward T7 or reverse SP6
containing sterile PBS. Midgut was carefully removed from insect promoters (1 ␮L/5.0 pmol), DNA (2 ␮L/100 ng) and 2 ␮L ultra pure
abdomen, rinsed in sterile PBS and transferred into a 1.5 mL tube water. PCR conditions were 96 ◦ C for 10 min, 50 ◦ C for 5 min and four
containing 100 ␮L of PBS. Tube content was mixed thoroughly with min at 60 ◦ C, 40 cycles. Sequencing of the 16S rRNA gene was done
a pestle and serially diluted (10−1 through 10−7 ) and an aliquot using a 27f primer (5 -end; 500 nucleotide region) and NL1 primer
of 100 ␮L of each one was transferred to Petri dishes contain- for bacterial and yeast clones, respectively. Sequencing was carried
ing BHI agar. Plates were incubated at 28 ◦ C for 24–48 h. Bacterial out on an ABI Prism 377 DNA sequencer (Applied Biosystems, USA).
isolates were maintained at −70 ◦ C in a 15% glycerol solution for Obtained sequences were initially compared to those from Gen-
further identification. The described procedure was also employed Bank database using BLAST (http://www.ncbi.nlm.nih.gov/BLAST).
for microbial enumeration that was done in triplicates by count- Generated sequences and their homologs were aligned using
ing the colony forming units (CFU), using one midgut for each CLUSTAL W 1.4 program (Thompson et al., 1994). Dendrograms
plate. were obtained by “neighbor-joining” (PAUP program, Swofford,
Microorganisms were also isolated from eggs as follows: three 2000). Sequences obtained were deposited in the GenBank
sets of 100 eggs were transferred to 1.5 mL tubes containing 500 ␮L database.
of 1% sodium hypochlorite. After 3 min the sedimented eggs were Midgut samples were fixed in 2.5% glutaraldehyde, 0.1 M
rinsed three times in 1 mL sterile PBS and homogenized in 50 ␮L PBS sodium cacodylate, pH 7.2, for 12 h at room temperature. Tissues
with a pestle. After sedimentation supernatant was transferred to were dehydrated in acetone, embedded in Epon and sections of
Petri dishes containing BHI agar and incubated at 28 ◦ C for 24–48 h. 0.6 ␮M were cut with diamond knife. Samples were stained by tolu-
Microorganisms were first screened based on (a) colony char- idine blue and examined under a light microscope (Zeiss, Axioplan).
acteristics (color, size, shape, opacity, margin, elevation and Midguts were fixed as described above and post-fixed with 0.8%
viscosity); (b) morphology and arrangement of isolates, Gram stain- K3 Fe(CN)6 /0.5% OsO4 in 0.1 M sodium cacodylate for 1 h at room
ing and motility. temperature. Samples were dehydrated through a graded acetone
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 Closest relative according to Blast (% identity)
I5a FJ372760 Klebsiella pneumoniae AF453251 (97)

I8a FJ372761 Asaia sp. AB025932 (98)
I11a FJ372762 Pichia ohmeri AY267821 (100)
I12a FJ372763 Klebsiella pneumoniae Y17657 (99)
I17a FJ372764 Serratia marcescens AY514433 (100)
O19b FJ372765 Enterobacter asburiae AB004744 (99)
I22a FJ372766 Serratia sp. EF032328 (99)
I24c FJ372768 Bacillus subtilis AY881636 (100)
O27b FJ372770 Asaia krungthepensis AB102953 (99)
I28c FJ372771 Bacillus sp. AF326365 (96)
I34a FJ372772 Enterococcus caccae AY943820 (98)
a
Midgut isolates.
b
Egg isolates.
c
Isolates of midguts from early emerged females.
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%)
C10BI24a FJ372780 Klebsiella pneumoniae DQ122369 (98%)
C11BI24a FJ372781 Enterobacter aerogenes FJ360760 (99%)
C13BI24a FJ372783 Serratia sp. EU693548 (100%)
C14BI24a FJ372784 Klebsiella pneumoniae AY838331 (96%)
C17BI24a FJ372785 Uncultured bacterium AF371852 (98%)
C19BI24a FJ372787 Pantoea agglomerans* DQ122347 (96%)
C23BI24a FJ372788 Enterobacter sp. AY689045 (98%)
C24BI24a FJ372789 Bactéria endofítica AY842147 (99%)
C27BI24a FJ372790 Serratia marcescens AJ550467 (99%)
C38BI24a FJ372796 Citrobacter freundii DQ133536 (99%)
C50BI24a FJ372803 Uncultured bacterium DQ068885 (96%)
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%)
C9BI48b FJ372818 Enterobacter sp. AB098582 (96%)
C11BI48b FJ372819 Pantoea sp. AY941839 (99%)
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%)
C23BI48b FJ372827 Enterobacter sp. AB098582 (98%)
C27BI48b FJ372830 Kluyvera ascorbata AF176567 (99%)
C30BI48b FJ372831 Pantoea agglomerans DQ122348 (95%)
C31BI48b FJ372832 Serratia marcescens AF124042
C32BI48b FJ372833 Serratia marcescens EF208030 (97%)
C34BI48b FJ372835 Serratia sp. FJ360761 (98%)
C1LI48c FJ372836 Pichia guilliermondii AY497698 (95%)
C12LI48c FJ372837 Candida nodaensis U45726 (100%)
C17LI48c FJ372838 Candida fermentati AY894826 (98%)
C32LI48c FJ372843 Candida fermentati AY187283 (97%)
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.
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 been described for some mosquito species (DeMaio et al., 1996;
mosquitoes (Table 3), while sucrose-fed mosquitoes showed a Pumpuni et al., 1996; Straif et al., 1998; Fouda et al., 2001;
slight increase in bacterial number (2.3 × 103 CFU). After blood Gonzalez-Ceron et al., 2003; Lindh et al., 2005; Favia et al., 2007;
feeding, the average number of bacteria increased progressively, Terenius et al., 2008; Crotti et al., 2009; Dong et al., 2009; Rani et
reaching the peak of 1.2 × 108 CFU (Table 3). Approximately twenty al., 2009). These findings show that such bacteria are widespread
million bacteria remained in midgut 67 h ABF, representing a five- in mosquitoes, suggesting that they are capable of maintaining a
fold reduction in microbial population. stable association with these insects, as described for Asaia sp. This
Light microscopy observations of females’ midguts 24 h ABF bacterium is a true symbiont of Anopheles sp. and a candidate to be
revealed that bacteria were located preferably in the posterior used in paratransgenic approaches (Favia et al., 2007). This is the
region, specifically between PM and food bolus (Fig. 1). It was possi- first work to identify yeasts in the midgut and ovary of A. aegypti
ble to observe, through scanning electron microscopy, that midgut to date. Yeasts are usually associated with wood and bark beetles
lumen of A. aegypti dissected 24 h ABF contained blood cells repre- (Grünwald et al., 2010) and adults of chrysopids (Woolfolk and
sented mainly by intact erythrocytes (Fig. 2A). During this stage Inglis, 2004). Like mosquitoes, these insects are nectar and hon-
of digestion bacteria were observed in the peripheral region of eydew feeders and the symbiotic yeasts provide the hosts with
the food bolus whereas none was viewed in the lumen central essential amino acids absent in their diet (Hagen et al., 1970).
region (Fig. 2B). Bacteria entirely occupied midgut lumen 48 h ABF Serratia marcescens was the predominant bacterium found in A.
, stage in which all erythrocytes had already been lysed (Fig. 2C aegypti midgut suggesting that it may possess some competitive
and D). Some bacteria were also seen embedded in PM 24 h ABF advantages over the other bacteria. Similarly, Serratia sp. was also
(Fig. 3A and B) and interacting with epithelial microvilli 48 h ABF one of the dominant species found in five generations of Anopheles
(Fig. 3C). gambiae (Dong et al., 2009). The genus Serratia is widely distributed
in the environment and some species are described as insects’ sym-
4. Discussion bionts. Serratia sp. is described as symbiont of the sugar beet root
maggot, Tetanops myopaeformis, and may play a role in host nutri-
Our findings provide comprehensive information on the com- tion and chitin degradation of the insect puparium (Iverson et al.,
position of the A. aegypti microbiota and its distribution in the 1984).
midgut along blood digestion. We used two different approaches Asaia sp. and Enterobacter sp. are probably transovarially trans-
(culture-dependent and culture-independent assays) to screen the mitted to offspring since they were successfully identified in A.
microorganisms associated with A. aegypti. Similar to previous aegypti eggs. Crotti et al. (2009) using PCR methods detected Asaia
studies, the majority of bacterial isolates were Gram-negative rods, sp. in A. aegypti larvae, pupae and adults, showing that this bac-
mainly from Enterobacteriaceae family. terium efficiently colonized guts, male and female reproductive
The genera Enterobacter, Enterococcus, Pantoea, Klebsiella, systems and salivary glands. Our findings along with those from
Kluyvera and Serratia (Enterobacteriaceae), Asaia (Acetobacter- Crotti et al. (2009) indicate that Asaia sp. is symbiotically associ-
aceae) and Bacillus (Bacillaceae) identified in this work had already ated with A. aegypti, as reported for A. stephensi reared in laboratory
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.

Fig. 3. Micrographs showing bacteria in A. aegypti midgut compartments. (A) Light Sequence reactions were done at the Center for the Study of Social
microscopy showing bacteria (arrows) within the PM. (B) Enlarged detail of A. (C) Insects, UNESP, Rio Claro, São Paulo, Brazil. The authors wish to
Transmission electron microscopy showing interaction of bacteria (arrows) with thank Ms Rívea Cristina Custódio Rodrigues and Telma Ferreira
epithelial microvilli. EP: epithelium; ER: erythrocytes; MV: microvilli; PM: per-
Costa Aguiar for technical assistance.
itrophic matrix; N: nucleus.
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Acta Tropica 115 (2010) 275–281

Contents lists available at ScienceDirect

Culture-dependent and culture-independent characterization of microorganisms

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

2. Material and methods

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

I5a FJ372760 Klebsiella pneumoniae AF453251 (97)

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

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

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

Midguts origin Average number of SDb

Non-fed females 2.1 × 102 1.2 × 102

Blood digestion observed in A. aegypti followed the pattern

This study was supported by grants from FAPERJ and CNPq.

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