Bacterial diversity of the American sand fly Lutzomyia intermedia using high-throughput metagenomic sequencing

Abstract

BackgroundParasites of the genus Leishmania cause a broad spectrum of diseases, collectively known as leishmaniasis, in humans worldwide. American cutaneous leishmaniasis is a neglected disease transmitted by sand fly vectors including Lutzomyia intermedia, a proven vector. The female sand fly can acquire or deliver Leishmania spp. parasites while feeding on a blood meal, which is required for nutrition, egg development and survival. The microbiota composition and abundance varies by food source, life stages and physiological conditions. The sand fly microbiota can affect parasite life-cycle in the vector.MethodsWe performed a metagenomic analysis for microbiota composition and abundance in Lu. intermedia, from an endemic area in Brazil. The adult insects were collected using CDC light traps, morphologically identified, carefully sterilized, dissected under a microscope and the females separated into groups according to their physiological condition: (i) absence of blood meal (unfed = UN); (ii) presence of blood meal (blood-fed = BF); and (iii) presence of developed ovaries (gravid = GR). Then, they were processed for metagenomics with Illumina Hiseq Sequencing in order to be sequence analyzed and to obtain the taxonomic profiles of the microbiota.ResultsBacterial metagenomic analysis revealed differences in microbiota composition based upon the distinct physiological stages of the adult insect. Sequence identification revealed two phyla (Proteobacteria and Actinobacteria), 11 families and 15 genera; 87 % of the bacteria were Gram-negative, while only one family and two genera were identified as Gram-positive. The genera Ochrobactrum, Bradyrhizobium and Pseudomonas were found across all of the groups.ConclusionsThe metagenomic analysis revealed that the microbiota of the Lu. intermedia female sand flies are distinct under specific physiological conditions and consist of 15 bacterial genera. The Ochrobactrum, Bradyrhizobium and Pseudomonas were the common genera. Our results detailing the constituents of Lu. intermedia native microbiota contribute to the knowledge regarding the bacterial community in an important sand fly vector and allow for further studies to better understand how the microbiota interacts with vectors of human parasites and to develop tools for biological control.

The Gut Microbiome of the Vector Lutzomyia longipalpis Is Essential for Survival of Leishmania infantum

“…It must be emphasized that our study was performed on colony-reared sand flies fed on sterile blood or sucrose or an infected blood meal. It is very likely that midguts from sand flies feeding on wild plants and mammals would differ phylogenetically from those in our study, although similarities between our findings and those of Monteiro et al suggest that at least some genera may be common (22). In future studies, it will be essential to determine by sequence analysis the similarity between the midgut microbiomes of laboratory-reared sand flies and wild-caught sand flies from neighborhoods where L. infantum infection is endemic and whether some bacteria can substitute for others in supporting the growth and differentiation of Leishmania spp.…”

“…Another commonality was Pseudomonas (phylum Proteobacteria ), which was prominent in Lu. intermedia and present in all of our samples (22). Pseudomonas was actually one of the most variable genera between conditions in our study, with levels diminishing as the levels of both the family Acetobacteraceae and the L. infantum infection increased.…”

“…One study performed 16S ribosomal DNA (rDNA) sequencing on genomic DNA extracted from whole sand flies, including the exoskeleton and other body parts, and did not distinguish midgut from surface microbiota (21). A more comprehensive 16S rDNA sequencing approach was taken by a group studying uninfected flies (22). These findings are all valid, but they do not provide a view of the complexity of the midgut microbiome surrounding the developing parasite.…”

Comparative analysis of microbial community in the whole body and midgut from fully engorged and unfed female adult Melophagus ovinus

“…This trend is similar to that reported in Culex quinquefasciatus (Telang & Skinner, ). Although these findings are in contrast with the results reported for sand flies (Monteiro et al ., ) and Aedes aegypti (Frentiu et al ., ), Wolbachia can still be considered an obligately intracellular bacterium for which the relative abundance decreases under oxidative stress conditions (Berticat et al ., ; Pan et al ., ).…”

Reciprocal interactions between Leishmania and their microenvironments during infection in the sand fly gut and human macrophages

“…Most insects largely possess a microbiome dominated by Gram (-) bacteria (mostly Enterobacteriaceae) with low overall diversity [29,30,45,50,51]. Specifically in sand flies, a variety of methods have been previously used to characterize the gut microbiome, many with non-sequence based methods including Denaturing Gradient Gel Electrophoresis (DGGE) and bacterial culture, or 16S rDNA/total high throughput sequencing (HTS), the latter is the preferred method because many bacterial species cannot be isolated in culture with current culture capabilities [52][53][54]. Furthermore, some studies have targeted the midgut microbiota or the midgut associated with the entire insect body in either colony-reared or endemic sand flies [53,55,56].…”