Use of the checkerboard DNA-DNA hybridization technique for bacteria detection in Aedes aegypti (Diptera:Culicidae) (L.)

Abstract Background 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. Findings 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. Conclusions 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.

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).