- Short report
- Open Access
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).
- Gusmao DS, Santos AV, Marini DC, Bacci M, Berbert-Molina MA, Lemos FJ: Culture-dependent and culture-independent characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut. Acta trop. 2010, 115: 275-281. 10.1016/j.actatropica.2010.04.011.View ArticlePubMedGoogle Scholar
- Gusmão DS, Santos AV, Marini DC, Russo ES, Peixoto AMD, Bacci MJ, Berbert-Molina MA, Lemos FJA: First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association. Mem Inst Oswaldo Cruz. 2007, 102: 919-924. 10.1590/S0074-02762007000800005.View ArticlePubMedGoogle Scholar
- Terenius O, de Oliveira CD, Pinheiro WD, Tadei WP, James AA, Marinotti O: 16S rRNA gene sequences from bacteria associated with adult Anopheles darlingi (Diptera: Culicidae) mosquitoes. J Med Entomol. 2008, 45: 172-175. 10.1603/0022-2585(2008)45[172:SRGSFB]2.0.CO;2.View ArticlePubMedGoogle Scholar
- Spratt DA: Significance of bacterial identification by molecular biology methods. Endodont Top. 2004, 9: 5-14. 10.1111/j.1601-1546.2004.00106.x.View ArticleGoogle Scholar
- do Nascimento C, Santos Barbosa RE, Mardegan Issa JP, Watanabe E, Yoko Ito I, Monesi N, Albuquerque Junior RF: The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method. Microbiol Res. 2008, 163: 403-407. 10.1016/j.micres.2006.11.020.View ArticlePubMedGoogle Scholar
- Papaioannou W, Gizani S, Haffajee AD, Quirynen M, Mamai-Homata E, Papagiannoulis L: The microbiota on different oral surfaces in healthy children. Oral Microbiol Immunol. 2009, 24: 183-189. 10.1111/j.1399-302X.2008.00493.x.View ArticlePubMedGoogle Scholar
- Sakamoto M, Siqueira JF, Rocas IN, Benno Y: Diversity of spirochetes in endodontic infections. J Clin Microbiol. 2009, 47: 1352-1357. 10.1128/JCM.02016-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst FE, Levin AE: "Checkerboard" DNA-DNA hybridization. Biotechniques. 1994, 17: 788-792.PubMedGoogle Scholar
- do Nascimento C, de Albuquerque RF, Monesi N, Candido-Silva JA: Alternative method for direct DNA probe labeling and detection using the checkerboard hybridization format. J Clin Microbiol. 2010, 48: 3039-3040. 10.1128/JCM.00390-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Crotti E, Damiani C, Pajoro M, Gonella E, Rizzi A, Ricci I, Negri I, Scuppa P, Rossi P, Ballarini P: Asaia, a versatile acetic acid bacterial symbiont, capable of cross-colonizing insects of phylogenetically distant genera and orders. Environm Microbiol. 2009, 11: 3252-3264. 10.1111/j.1462-2920.2009.02048.x.View ArticleGoogle Scholar
- Gaio Ade O, Gusmao DS, Santos AV, Berbert-Molina MA, Pimenta PF, Lemos FJ: Contribution of midgut bacteria to blood digestion and egg production in Aedes aegypti (diptera: culicidae) (L.). Parasit Vectors. 2011, 4: 105-10.1186/1756-3305-4-105.View ArticlePubMedGoogle Scholar
- Moll RM, Romoser WS, Modrzakowski MC, Moncayo AC, Lerdthusnee K: Meconial peritrophic membranes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis. J Med Entomol. 2001, 38: 29-32. 10.1603/0022-2585-38.1.29.View ArticlePubMedGoogle Scholar
- Dong Y, Manfredini F, Dimopoulos G: Implication of the mosquito midgut microbiota in the defense against malaria parasites. PLoS Pathog. 2009, 5: e1000423-10.1371/journal.ppat.1000423.PubMed CentralView ArticlePubMedGoogle Scholar
- Favia G, Ricci I, Damiani C, Raddadi N, Crotti E, Marzorati M, Rizzi A, Urso R, Brusetti L, Borin S: Bacteria of the genus Asaia stably associate with Anopheles stephensi, an Asian malarial mosquito vector. PNAS. 2007, 104: 9047-9051. 10.1073/pnas.0610451104.PubMed CentralView ArticlePubMedGoogle Scholar
- Carina Audisio M, Torres MJ, Sabate DC, Ibarguren C, Apella MC: Properties of different lactic acid bacteria isolated from Apis mellifera L. bee-gut. Microbiol Res. 2011, 166: 1-13. 10.1016/j.micres.2010.01.003.View ArticlePubMedGoogle Scholar
- Cox CR, Gilmore MS: Native microbial colonization of Drosophila melanogaster and its use as a model of Enterococcus faecalis pathogenesis. Infect Immun. 2007, 75: 1565-1576. 10.1128/IAI.01496-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Gouveia C, Asensi MD, Zahner V, Rangel EF, Oliveira SM: Study on the bacterial midgut microbiota associated to different Brazilian populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae). Neotrop Entomol. 2008, 37: 597-601. 10.1590/S1519-566X2008000500016.View ArticlePubMedGoogle Scholar
- Mrázek J, Strosová L, Fliegerová K, Kott T, Kopecný J: Diversity of insect intestinal microflora. Folia Microbiol. 2008, 53: 229-233. 10.1007/s12223-008-0032-z.View ArticleGoogle Scholar
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