Metagenomic analysis of taxa associated with Lutzomyia longipalpis, vector of visceral leishmaniasis, using an unbiased high-throughput approach

Abstract

Background: Leishmaniasis is one of the most diverse and complex of all vector-borne diseases worldwide. It is caused by parasites of the genus Leishmania, obligate intramacrophage protists characterised by diversity and complexity. Its most severe form is visceral leishmaniasis (VL), a systemic disease that is fatal if left untreated. In Latin America VL is caused by Leishmania infantum chagasi and transmitted by Lutzomyia longipalpis. This phlebotomine sandfly is only found in the New World, from Mexico to Argentina. In South America, migration and urbanisation have largely contributed to the increase of VL as a public health problem. Moreover, the first VL outbreak was recently reported in Argentina, which has already caused 7 deaths and 83 reported cases.

Methodology/principal findings: An inventory of the microbiota associated with insect vectors, especially of wild specimens, would aid in the development of novel strategies for controlling insect vectors. Given the recent VL outbreak in Argentina and the compelling need to develop appropriate control strategies, this study focused on wild male and female Lu. longipalpis from an Argentine endemic (Posadas, Misiones) and a Brazilian non-endemic (Lapinha Cave, Minas Gerais) VL location. Previous studies on wild and laboratory reared female Lu. longipalpis have described gut bacteria using standard bacteriological methods. In this study, total RNA was extracted from the insects and submitted to high-throughput pyrosequencing. The analysis revealed the presence of sequences from bacteria, fungi, protist parasites, plants and metazoans.

Conclusions/significance: This is the first time an unbiased and comprehensive metagenomic approach has been used to survey taxa associated with an infectious disease vector. The identification of gregarines suggested they are a possible efficient control method under natural conditions. Ongoing studies are determining the significance of the associated taxa found in this study in a greater number of adult male and female Lu. longipalpis samples from endemic and non-endemic locations. A particular emphasis is being given to those species involved in the biological control of this vector and to the etiologic agents of animal and plant diseases.

Figure 1. VL vectors as environmental samples: taxa identified in phlebotomine sandflies considering sampling site conditions.

This figure summarises and associates sampling site characteristics with taxa identified in male and female adult Lu. longipalpis from both locations. In all cases figures are only schematic and not an exact representation of either the sampling sites, phlebotomine sandflies or identified taxa. A) Shows the most significant ecological characteristics of both phlebotomine capture site locations in Argentina and Brazil: Posadas and Lapinha Cave, respectively. Only those animal species confirmed at the time of sampling were represented schematically (Table 1 includes a detailed list of animal and plant species in both locations). (B) Total RNA was extracted from male and female adult Lu. longipalpis specimens and amplified using sequence independent amplification (see Methods section). (C) Shows the different taxa identified by sequence homology in all four samples (bacteria, fungi, protists, metazoans and plants). Taxa are represented schematically and the particular species identified for each taxonomical group are not shown, except in the case of metazoans (see Figure 2 for a detailed list). Grey rectangular boxes group taxa found in female adult Lu. longipalpis. White rectangular boxes group taxa found in male adult Lu. longipalpis. VL: Visceral Leishmaniasis.

Figure 2. Taxa associated with wild adult male and female Lu. longipalpis specimens analysed in this study.

Results for all the samples were integrated and summarised in this figure, which indicates all the taxonomical groups that were identified, the species that were found for each taxon and the number of sequences for each species. Solid black lines group the different taxa (plants, protists, metazoans, bacteria and fungi). For bacteria, the dotted black lines separate and differentiate Gram+ and Gram- rods. The different species that were found are named beneath each column. The number of sequences for each species is indicated on the top part of each cylinder. SS1: adult females from the Endemic VL location (EVL); SS2: adult EVL males; PP1: adult females from the Non-Endemic VL location (NEVL); PP2: adult NEVL males.