Use of the checkerboard DNA-DNA hybridization technique for bacteria detection in Aedes aegypti (Diptera:Culicidae) (L.)
© Gaio et al; licensee BioMed Central Ltd. 2011
Received: 22 September 2011
Accepted: 20 December 2011
Published: 20 December 2011
Bacteria associated with insects can have a substantial impact on the biology and life cycle of their host. The checkerboard DNA-DNA hybridization technique is a semi-quantitative technique that has been previously employed in odontology to detect and quantify a variety of bacterial species in dental samples. Here we tested the applicability of the checkerboard DNA-DNA hybridization technique to detect the presence of Aedes aegypti-associated bacterial species in larvae, pupae and adults of A. aegypti.
Using the checkerboard DNA-DNA hybridization technique we could detect and estimate the number of four bacterial species in total DNA samples extracted from A. aegypti single whole individuals and midguts. A. aegypti associated bacterial species were also detected in the midgut of four other insect species, Lutzomyia longipalpis, Drosophila melanogaster, Bradysia hygida and Apis mellifera.
Our results demonstrate that the checkerboard DNA-DNA hybridization technique can be employed to study the microbiota composition of mosquitoes. The method has the sensitivity to detect bacteria in single individuals, as well as in a single organ, and therefore can be employed to evaluate the differences in bacterial counts amongst individuals in a given mosquito population. We suggest that the checkerboard DNA-DNA hybridization technique is a straightforward technique that can be widely used for the characterization of the microbiota in mosquito populations.
The identification of bacteria in mosquito guts has relied on both culture-dependent and culture-independent techniques [1–3]. Molecular techniques for bacterial identification have received particular attention because they are more rapid than traditional culture methods and in addition can detect bacteria that cannot be cultured. Culture independent methods have mainly been based on the amplification of the 16S rRNA genes by PCR, followed by the identification of the amplified genes through nucleotide sequence comparisons .
The checkerboard DNA-DNA hybridization technique [5–8] is a semi-quantitative technique that has been extensively employed in odontology to detect and quantify a variety of bacterial species in dental samples and allows the simultaneous analysis of a large number of DNA samples against a range of DNA probes from different bacterial species on a single support membrane . Here we have tested if this technique is suitable to detect and estimate the number of bacteria in total DNA samples extracted from both whole Aedes aegypti and from dissected A. aegypti midguts. In addition, we have also tested if we could detect and estimate the numbers of A. aegypti midgut-associated bacteria species in the midgut of other insect species.
In our experiments we employed a modified version  of the original DNA-DNA hybridization technique  (Additional file 1). As probes we used whole genomic DNA extracted from four bacterial species. Serratia sp. (FJ372764), Asaia sp. (FJ372770) and Klebsiella sp. (FJ372760) were isolated from laboratory-bred A. aegypti[1, 2]. Chryseobacterium sp. (EU169680.1) was isolated from wild-caught A. aegypti.
Estimated numbers of bacterial cells in whole animals and dissected midguts
A. aegypti L1 (w)
5.7 × 105
3.2 × 105
1.8 × 105
A. aegypti L2 (w)
1.1 × 105
A. aegypti P1 (w)
1.6 × 105
1.4 × 105
1.5 × 105
A. aegypti P2 (w)
1.5 × 105
A. aegypti A1 (w)
2.5 × 105
1.2 × 105
1.8 × 105
A. aegypti A2 (w)
3.4 × 105
1.4 × 105
3.1 × 105
A. aegypti L1 (mg)
3.0 × 105
2.8 × 105
2.0 × 105
A. aegypti L2 (mg)
5.1 × 105
4.4 × 105
2.9 × 105
A. aegypti A1 (mg)
A. aegypti A2 (mg)
A. aegypti A3 (mg)
L. longipalpis (mg)
D. melanogaster (mg)
2.1 × 105
B. hygida (mg)
1.4 × 106
8.1 × 105
7.2 × 105
A. mellifera (mg)
3.1 × 105
1.6 × 105
Our results show that the checkerboard DNA-DNA hybridization technique can be employed to detect the presence of bacterial species known to be associated with A. aegypti in A. aegypti samples. This technique reveals differences in the counts of bacteria present in distinct life stages and is sensitive enough to detect differences in the amount of bacterial cells amongst individual samples [for example, Figure 2A, whole larvae (L1 and L2) hybridized to the Asaia sp. probe]. Overall, our results demonstrate that the checkerboard DNA-DNA hybridization is a suitable technique for routine investigation of mosquito samples.
The presence of these four bacterial species was also investigated in midguts dissected from another insect vector, Lutzomyia longipalpis, and from three other insect species Drosophila melanogaster, Bradysia hygida and Apis mellifera (Figure 2B, Table 1). Klebsiella sp. and Serratia sp. were both detected in all four insect species tested. Asaia sp. cells were detected in D. melanogaster, A. mellifera and B. hygida. Chryseobacterium sp. was the only bacterial species not detected in this group of insects. Klebsiella sp. and Serratia sp. have been previously reported in D. melanogaster, A. mellifera and L. longipalpis[15–18]. In addition, our results revealed the presence of A. aegypti midgut-associated bacteria species in the midgut of B. hygida, an insect species in which the indigenous microbiota has not previously been characterized.
The use of the checkerboard DNA-DNA hybridization technique to detect and estimate bacteria from insects is appealing since it can contribute to the characterization of insect microbiota without the need of employing culture dependent methods that are both laborious and time consuming. Sample preparation is simple, which enables the rapid and simultaneous investigation of numerous samples collected from distinct populations. In addition, this method has the sensitivity to detect bacteria in single individuals at different developmental stages (larval, pupal), as well as in a single organ such as the midgut, and therefore, can be employed to determine if there are differences amongst individuals in a single population. Finally, the use of this technique can contribute to the characterization of the microbial ecology associated with mosquitoes, elucidate intrinsic and extrinsic factors that influence bacterial composition and identify the bacteria that are implicated in vectorial capacity differences between mosquito populations.
We thank Dr. Jorge Cury de Almeida for providing B. hygida specimens, Dra. Zilá Luz Paulino Simões for providing A. mellifera specimens, Dra. Maria H. de S. Goldman (FFCLRP-USP) for nucleotide sequencing, Telma Ferreira Costa Aguiar for technical assistance and Dr. Richard J. Ward for helpful comments on the manuscript. This work was funded by the following grants: INCT - Entomologia Molecular and MCT/CNPq (FJAL and PFPP), FAPERJ (FJAL), FAPESP (NM), FAPEMIG and FIOCRUZ (PFPP).
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