First comparative transcriptomic analysis of wild adult male and female Lutzomyia longipalpis, vector of visceral leishmaniasis

Abstract

Leishmaniasis is a vector-borne disease with a complex epidemiology and ecology. Visceral leishmaniasis (VL) is its most severe clinical form as it results in death if not treated. In Latin America VL is caused by the protist parasite Leishmania infantum (syn. chagasi) and transmitted by Lutzomyia longipalpis. This phlebotomine sand fly is only found in the New World, from Mexico to Argentina. However, due to deforestation, migration and urbanisation, among others, VL in Latin America is undergoing an evident geographic expansion as well as dramatic changes in its transmission patterns. In this context, the first VL outbreak was recently reported in Argentina, which has already caused 7 deaths and 83 reported cases. Insect vector transcriptomic analyses enable the identification of molecules involved in the insect's biology and vector-parasite interaction. Previous studies on laboratory reared Lu. longipalpis have provided a descriptive repertoire of gene expression in the whole insect, midgut, salivary gland and male reproductive organs. Nevertheless, the study of wild specimens would contribute a unique insight into the development of novel bioinsecticides. 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. In this study, total RNA was extracted from the sand flies, submitted to sequence independent amplification and high-throughput pyrosequencing. This is the first time an unbiased and comprehensive transcriptomic approach has been used to analyse an infectious disease vector in its natural environment. Transcripts identified in the sand flies showed characteristic profiles which correlated with the environment of origin and with taxa previously identified in these same specimens. Among these, various genes represented putative targets for vector control via RNA interference (RNAi).

Figure 1. Sequence analysis workflow.

This figure shows an overview of the rationale supporting the analysis of the reads (transcripts) obtained in this study and summarises the different steps that were followed. 1) Indicates the first stage of analysis, in which all the reads were blasted against DB:Llon_contigs+nt+16S. Results were classified in three datasets: reads that mapped to Llon_contigs by sequence homology (mapped-reads); reads that showed homology to nt16S (read-nt16S); and reads that returned no significant hits (no hits) (see text for details). 2) Indicates the second stage of analysis, in which the mapped-reads dataset was blasted against DB:nt16S (see text for details). 3) Indicates the third stage of analysis, in which selected reads (mapped-reads and read-nt16S) were separately blasted against three databases (est-others, nr and uniprotKB) and annotated using Blast2GO (see text for details). The ‘selected mapped-reads (4910)’ excluded mapped-reads 15379, 23694, 25834, 27903, 27904 and 9281, which showed homology to insect rDNA after BLASTN against DB:nt16S, and contig 31202, with unknown function after BLASTN against DB:nt16S (see text for details). The ‘selected read-nt16S (70)’ included read-nt16S that did not show homology to either insect rDNA or taxa other than insects (see text for details).

Figure 2. Sand flies as environmental samples: expression profiles in EVL and NEVL sand flies correlated with environmental conditions and taxa previously identified in these samples.

This figure integrates data from the function categories identified in these wild EVL and NEVL sand flies with sampling site characteristics and taxa previously found in these same samples . Figures are only schematic and not an exact representation of either the sampling sites, phlebotomine sand flies or identified taxa. A) Shows the function categories that the transcripts were assigned to in all the samples and the number of transcripts assigned to each function category for each sample. Values are expressed on a logarithmic scale and indicated for each sample on the corresponding bar. Significant differences in the number of transcripts in each category between samples (Fisher's Exact Test; p<0.05) are indicated as: a, significantly overrepresented with respect to PP1; b, significantly overrepresented with respect to PP2; c, significantly overrepresented with respect to SS1; and d, significantly overrepresented with respect to SS2. B) The top part shows a schematic of the sandflies (female or male) from both locations and of the taxa we previously identified in all four samples . Barrels group the taxa found in each sample. Previously identified taxa in SS1: bacteria, protists, metazoans (human) and plants; SS2: protists, metazoans (human and chicken) and plants; PP1: bacteria, fungi, metazoans (human, chicken and lizard) and plants; and PP2: bacteria, fungi, metazoans (human) and plants. Taxa are represented schematically and the particular species identified for each taxonomical group are not shown, except in the case of metazoans. The bottom part shows the most significant ecological characteristics of both capture site locations in Argentina and Brazil, Posadas and Lapinha Cave, respectively. Only those animal species confirmed in the sampling sites in both locations at the time of sampling were represented schematically. EVL sampling site (Posadas, Argentina): human, dog, cat and chicken; NEVL sampling site (Lapinha Cave, Brazil): human and chicken. SS1 (indicated in yellow): EVL adult female Lu. longipalpis (Posadas, Argentina); SS2 (indicated in orange): EVL adult male Lu. longipalpis (Posadas, Argentina); PP1 (indicated in green): NEVL adult female Lu. longipalpis (Lapinha Cave, Brazil); PP2 (indicated in pale blue): NEVL adult male Lu. longipalpis (Lapinha Cave, Brazil).