Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World

Abstract

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.

Fig 1. Lutzomyia longipalpis site locations for copulation songs and pheromone types.

Samples were collected from three allopatric populations: Marajó (Pará State; 0°56’S 49°38’W), Jacobina (Bahia State; 11⁰ 10’S 40⁰ 31’W), and Lapinha Cave (Minas Gerais State; 19⁰ 33’S 43⁰ 57’W); and two sympatric populations from Sobral (Ceará State; 3⁰ 41’S 40⁰ 21’W). Copulation songs: Burst-type (B) and Pulse-types (P1, P2, and P3). Pheromone types: sobralene (SOB), (S)-9-methylgermacrene-B (9MGB), and 3-methyl-α-himachalene (3MαH). For each location, the number of SNPs identified in each population with respect to the reference genome (VectorBase, LlonJ1) is indicated. A total of 1,937,819 SNPs were identified among all the populations. Main map source: World Imagery (Source: Esri, Maxar, Earthstar Geographics, and the GIS User Community; http://goto.arcgisonline.com/maps/World_Imagery). Inset map source: World Dark Gray Canvas Base (Esri, HERE, Garmin, OpenStreetMap contributors, and the GIS user community; http://goto.arcgisonline.com/maps/Canvas/World_Dark_Gray_Base).

Fig 2. Molecular species phylogeny and ortholog sharing.

(A) The quantitative maximum likelihood species phylogeny computed from the concatenated superalignment of 1,627 orthologous protein-coding genes places the sand flies (Psychodomorpha) as a sister group to the mosquitoes (Culicomorpha) rather than the flies (Muscomorpha), with all branches showing 100% bootstrap support. The Culicomorpha are represented by four Anopheles mosquito species and Culex quinquefasciatus and the Muscomorpha include four Drosophila fruit fly species and the tsetse fly, G. morsitans. Outgroup species represent Lepidoptera (Bombyx mori), Coleoptera (T. castaneum), Hymenoptera (Apis mellifera), and the phylogeny is rooted with the phthirapteran human body louse, Pe. humanus. The inset boxplots show that single-copy (1:1) and multi-copy (X:X) ortholog amino acid percent identity is higher between each sand fly (Ll, Lu. longipalpis; Pp, Ph. papatasi) and An. gambiae (Ag) than D. melanogaster (Dm). Boxplots show median values with boxes extending to the first and third quartiles of the distributions. (B) The Venn diagram summarizes the numbers of orthologous groups and mean number of genes per species (in parentheses) shared among the two sand flies (L. lon., Lu. longipalpis; P. pap., Ph. papatasi) and/or the Culicomorpha and/or the Muscomorpha. Analysis of ortholog sharing shows that the sand flies share more than three times as many orthologous groups exclusively with the Culicomorpha (Anopheles and Culex) compared to the Muscomorpha (Drosophila, Glossina) (subsets highlighted with thin and thick dashed lines). Numbers of unique genes are in italics. Colors in panel A and panel B match species and sets of species analyzed.

Fig 3. Lutzomyia longipalpis population structure.

Inferred population structure of Lu. longipalpis individuals collected from Marajó (MAR; pink), Lapinha (LAP; blue), from Jacobina (JAC; red), and Sobral, including Sobral 1S (S1S; orange) and 16 Sobral 2S (S2S; green). (A) Rooted neighbor joining (NJ) radial tree. We included both N. intermedia (INT; yellow) and M. migonei (MIG; purple) and used M. migonei to root the trees. Bootstrap values represent the percentage of 1,000 replicates. (B) Principal component analysis (PCA). Individuals were plotted according to their coordinates on the first two principal components (PC1 and PC2). (C) Admixture analysis. Ancestry proportions for Admixture models from K = 2 to K = 7 ancestral populations. Each individual is represented by a thin vertical line, partitioned into K coloured segments representing the individual’s estimated membership fractions to the K clusters. These data are the average of the major q-matrix clusters derived by CLUMPAK analysis.

Fig 4. Genomic regions with high pairwise F_ST between the different populations of Lutzomyia longipalpis.

(A) Venn diagram depicting the number of 1 kb non-overlapping genomic windows having F_ST values in the top 2.5% quantile (outlier) among the different population comparisons. (B) Box plots of outlier F_ST windows shared with another population comparison (blue) or unique to a population comparison (pink). Box plots show the medians (lines) and interquartile ranges (boxes); the whiskers extend out from the box plots to 1.5 times the interquartile range, and values outside this limit are represented by dots. Mean F_ST values are represented by open diamonds.

Fig 5. Measures of divergence in 1 kb non-overlapping genomic windows between the different populations of Lutzomyia longipalpis.

(A) Box plots of D_xy and F_ST values in the top 2.5% quantile (outlier) of each population comparison. (B) Box plots of D_xy and F_ST values for windows having both high D_xy and high F_ST (differentiation islands). (C) Box plots of Tajimas’ D values for the differentiation islands. (D) Box plots of FLK values for sites within differentiation islands. Box plots show the medians (lines) and interquartile ranges (boxes); the whiskers extend out from the box plots to 1.5 times the interquartile range, and values outside this limit are represented by dots. Mean values are represented by open diamonds.