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. 2010 Nov;76(22):7444-50.
doi: 10.1128/AEM.01747-10. Epub 2010 Sep 17.

Molecular evidence for multiple infections as revealed by typing of Asaia bacterial symbionts of four mosquito species

Bessem Chouaia  1 , Paolo Rossi, Matteo Montagna, Irene Ricci, Elena Crotti, Claudia Damiani, Sara Epis, Ingrid Faye, N'fale Sagnon, Alberto Alma, Guido Favia, Daniele Daffonchio, Claudio Bandi
Affiliations

Molecular evidence for multiple infections as revealed by typing of Asaia bacterial symbionts of four mosquito species

Bessem Chouaia et al. Appl Environ Microbiol. 2010 Nov.

Abstract

The recent increased detection of acetic acid bacteria (AAB) of the genus Asaia as symbionts of mosquitoes, such as Anopheles spp. and Aedes spp., prompted us to investigate the diversity of these symbionts and their relationships in different mosquito species and populations. Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with four mosquito species, Anopheles stephensi, Anopheles gambiae, Aedes aegypti, and Aedes albopictus, which are important vectors of human and/or animal pathogens. Denaturing gradient gel electrophoresis (DGGE) analysis based on the 16S rRNA gene revealed the presence of several bacterial taxa, among which Asaia sequences were among the dominant in most of the samples. A collection of 281 Asaia isolates in cell-free media was established from individuals belonging to the four species. The isolates were typed by internal transcribed spacer (ITS)-PCR, tRNA-PCR, BOX-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR, revealing that different Asaia strains are present in different mosquito populations, and even in single individuals.

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Figures

FIG. 1.
FIG. 1.
DGGE profiles of 16S rRNA gene fragments amplified by PCR using general primers (357f and 907r) from whole-mosquito DNA templates. (A) DGGE profiles from mosquitoes of the genus Anopheles. Bands showing sequence identity with Asaia, Burkholderia, Elizabethkingia, and Bacillus spp. are indicated with I, II, III, and V, respectively. (B) DGGE profiles from mosquitoes of the genus Aedes. Bands with sequence identity with Asaia and Burkholderia spp. are indicated with I and II, respectively. IV indicates a band that was not identified, due to unreadable sequences. The numbers (40% and 60%) indicate the percentage of denaturating condition at the respective gel positions. Anopheles gambiae strain G3 indicates a laboratory strain reared in Stockholm, Sweden. Aedes aegypti (Camerino) and Aedes aegypti (Milano) indicate individuals reared in the laboratories in Camerino and Milan. Asterisks on the left of the bands indicate the bands that were cut and sequenced.
FIG. 2.
FIG. 2.
Similarity tree between the various profile patterns of Asaia spp. isolated from the mosquitoes. The tree, generated with the UPGMA methods using the Jaccard coefficient, allowed us to cluster the different profiles in a tree, presenting a similarity higher than 50% among all of the patterns. We can distinguish 8 groups (from I to VIII) that share a similarity higher than 75%.
FIG. 3.
FIG. 3.
Phylogenetic positions of the strains of Asaia indicated in Table 3, based on 16S rRNA gene sequences (neighbor-joining method; Kimura correction; all positions containing gaps and missing data were eliminated from the data set). Numbers at each node represent the bootstrap percentages of replications calculated from 2,000 replicated trees. The scale bar represents sequence divergence. Group 1 includes in the same cluster Asaia strains AG2.7 Aβ (FN814277), AA7.5 Aα (FN814292), AS1.7 Aα (FN814296), AS2.6 Aα (FN814297), and AS3.6Aα (FN814299). Group 2 includes in the same cluster Asaia strains: AG3.7 Aα (FN814279), AG6.6 Aα (FN814281), AG7.6 Aα (FN814283), AA1.5 Aβ (FN814286), AA1.6 Aβ (FN814287), AA3.7 Aα (FN814288), AA5.6 Aα (FN814290), AA 6.7 Aα (FN814291), AA 8.6Aβ (FN814293), AA8.7 Aβ (FN814294), and AA9.6 Aβ (FN814295).
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