Characterization of the microbiota in the guts of Triatoma brasiliensis and Triatoma pseudomaculata infected by Trypanosoma cruzi in natural conditions using culture independent methods

Marcia Gumiel, Fabio Faria da Mota, Vanessa de Sousa Rizzo, Otília Sarquis, Daniele Pereira de Castro, Marli Maria Lima, Eloi de Souza Garcia, Nicolas Carels, Patricia Azambuja

Chagas disease is caused by Trypanosoma cruzi, which is transmitted by triatomine vectors. The northeastern region of Brazil is endemic for Chagas disease and has the largest diversity of triatomine species. T. cruzi development in its triatomine vector depends on diverse factors, including the composition of bacterial gut microbiota. We characterized the triatomines captured in the municipality of Russas (Ceará) by sequencing the cytochrome c oxidase subunit I (COI) gene. The composition of the bacterial community in the gut of peridomestic Triatoma brasiliensis and Triatoma pseudomaculata was investigated using culture independent methods based on the amplification of the 16S rRNA gene by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), DNA fragment cloning, Sanger sequencing and 454 pyrosequencing. Additionally, we identified TcI and TcII types of T. cruzi by sequencing amplicons from the gut metagenomic DNA with primers for the mini-exon gene. Triatomines collected in the peridomestic ecotopes were diagnosed as T. pseudomaculata and T. brasiliensis by comparing their COI sequence with GenBank. The rate of infection by T. cruzi in adult triatomines reached 80% for T. pseudomaculata and 90% for T. brasiliensis. According to the DNA sequences from the DGGE bands, the triatomine gut microbiota was primarily composed of Proteobacteria and Actinobacteria. However, Firmicutes and Bacteroidetes were also detected, although in much lower proportions. Serratia was the main genus, as it was encountered in all samples analyzed by DGGE and 454 pyrosequencing. Members of Corynebacterinae, a suborder of the Actinomycetales, formed the next most important group. The cloning and sequencing of full-length 16S rRNA genes confirmed the presence of Serratia marcescens, Dietzia sp., Gordonia terrae, Corynebacterium stationis and Corynebacterium glutamicum. The study of the bacterial microbiota in the triatomine gut has gained increased attention because of the possible role it may play in the epidemiology of Chagas disease by competing with T. cruzi. Culture independent methods have shown that the bacterial composition of the microbiota in the guts of peridomestic triatomines is made up by only few bacterial species.