Investigation of the bacterial communities associated with females of Lutzomyia sand fly species from South America

Abstract

Phlebotomine sand flies are vectors of Leishmania that are acquired by the female sand fly during blood feeding on an infected mammal. Leishmania parasites develop exclusively in the gut lumen during their residence in the insect before transmission to a suitable host during the next blood feed. Female phlebotomine sand flies are blood feeding insects but their life style of visiting plants as well as animals, and the propensity for larvae to feed on detritus including animal faeces means that the insect host and parasite are exposed to a range of microorganisms. Thus, the sand fly microbiota may interact with the developing Leishmania population in the gut. The aim of the study was to investigate and identify the bacterial diversity associated with wild adult female Lutzomyia sand flies from different geographical locations in the New World. The bacterial phylotypes recovered from 16S rRNA gene clone libraries obtained from wild caught adult female Lutzomyia sand flies were estimated from direct band sequencing after denaturing gradient gel electrophoresis of bacterial 16 rRNA gene fragments. These results confirm that the Lutzomyia sand flies contain a limited array of bacterial phylotypes across several divisions. Several potential plant-related bacterial sequences were detected including Erwinia sp. and putative Ralstonia sp. from two sand fly species sampled from 3 geographically separated regions in Brazil. Identification of putative human pathogens also demonstrated the potential for sand flies to act as vectors of bacterial pathogens of medical importance in addition to their role in Leishmania transmission.

Figure 1. Neighbour-joining phylogram of bacterial 16S sequences obtained from field collected Lutzomyia species.

Numbering refers to clones described in Table 1. Bacterial species from Lapinha (Minas Gerais-Brazil) are presented in purple; species identified from sand flies collected in Alagoas (Brazil) are presented in green; species identified in Corumbá (Brazil) are presented in pink and red represents bacterial species identified from Lu. longipalpis collected in Callejón (Colombia). Bacterial sequences extracted from Genbank for comparison are presented in black.

Figure 2. Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified bacterial gene fragments derived from field-collected individual female Lu. longipalpis.

Lanes L1 to L10 corresponds to DGGE profiles of Lu. longipalpis collected from Lapinha cave-Minas Gerais; A1 to A5 correspond to DGGE profiles of Lu. longipalpis collected from Alagoas state/Brazil. Lanes labelled S correspond to standard DGGE markers prepared from a selection of bacterial 16S rRNA gene products to enable gel to gel comparison.

Figure 3. Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified bacterial gene fragments derived from field-collected individual female Lu. longipalpis.

Lanes A6 to A12 corresponds to DGGE profiles of Lu. longipalpis collected from Alagoas state/Brazil; C1 to C8 correspond to DGGE profiles of Lu. cruzi collected from Corumbá-Mato Grosso-Brazil. Lanes labelled S corresponds to DGGE markers prepared from a selection of bacterial 16S rRNA gene products to enable gel-to-gel comparison.