Genome analysis of a major urban malaria vector mosquito, Anopheles stephensi

Abstract

Background: Anopheles stephensi is the key vector of malaria throughout the Indian subcontinent and Middle East and an emerging model for molecular and genetic studies of mosquito-parasite interactions. The type form of the species is responsible for the majority of urban malaria transmission across its range.

Results: Here, we report the genome sequence and annotation of the Indian strain of the type form of An. stephensi. The 221 Mb genome assembly represents more than 92% of the entire genome and was produced using a combination of 454, Illumina, and PacBio sequencing. Physical mapping assigned 62% of the genome onto chromosomes, enabling chromosome-based analysis. Comparisons between An. stephensi and An. gambiae reveal that the rate of gene order reshuffling on the X chromosome was three times higher than that on the autosomes. An. stephensi has more heterochromatin in pericentric regions but less repetitive DNA in chromosome arms than An. gambiae. We also identify a number of Y-chromosome contigs and BACs. Interspersed repeats constitute 7.1% of the assembled genome while LTR retrotransposons alone comprise more than 49% of the Y contigs. RNA-seq analyses provide new insights into mosquito innate immunity, development, and sexual dimorphism.

Conclusions: The genome analysis described in this manuscript provides a resource and platform for fundamental and translational research into a major urban malaria vector. Chromosome-based investigations provide unique perspectives on Anopheles chromosome evolution. RNA-seq analysis and studies of immunity genes offer new insights into mosquito biology and mosquito-parasite interactions.

Figure 1

Physical map. A physical map of the An. stephensi genome was created from FISH on polytene chromosomes comprising 227 probes and 86 scaffolds. These 86 scaffolds comprise 137.14 Mb or 62% of the An. stephensi genome. Orientation was assigned to 32 of the 86 scaffolds. The physical map includes 28 of the 30 largest scaffolds.

Figure 2

Molecular species phylogeny and orthology. (A) The maximum likelihood molecular species phylogeny estimated from universal single-copy orthologs supports the recognized species relationships with An. stephensi and An. gambiae in subgenus Cellia within the genus Anopheles. (B) Comparative analysis of orthologs from An. stephensi, An. gambiae, Ae. aegypti, and D. melanogaster. Orthologous genes were retrieved from OrthoDB. A total of 7,305 genes were shared among all four species, 1,297 genes were specific to An. stephensi, 653 genes were Anopheles-specific, and 1,863 genes were mosquito-specific.

Figure 3

Gene clustering according to expression profile. Twenty groups of genes were clustered by expression profile. The expression profiles used for grouping were generated using 11 RNA-seq samples spanning developmental time points including: 0 to 1, 2 to 4, 4 to 8, and 8 to 12 h embryos, larva, pupa, adult males, adult females, non-blood-fed ovaries, blood-fed ovaries, and 24 h post-blood-fed female carcass without ovaries. Male stage are colored blue, female stages are colored green, ovary samples are colored yellow, embryo samples are colored red, larva samples are colored pink, and pupa samples are colored purple. Many of these clusters correspond to either a specific developmental stage or specific sex.

Figure 4

Genome landscape. Density of genes (black vertical lines), transposable elements (TEs; green vertical lines), and short tandem repeats (STRs; red vertical lines) in 100 kb windows of mapped scaffolds. Based on the physical map, scaffolds were ordered and oriented respective to their position in the chromosomes and then 100 kb non-overlapping windows were generated for each scaffold (X-axis). The density of genes and TEs (Y-axis) was determined using coverageBed. Satellite sequences were identified using TandemRepeatFinder. The short tandem repeats track is a combination of the number of microsatellites, minisatellites, and satellites per 100 kb window.

Figure 5

Average density/100 kb/ARM. A comparison of the average density per 100 kb of genes, TEs, S/MARS, microsatellites, minisatellites, and satellites between chromosome arms.

Figure 6

FISH with Aste72A, rDNA, and DAPI on mitotic chromosomes. The pattern of hybridization for satellite DNA Aste72A on mitotic sex chromosomes of An. stephensi. Aste72A hybridizes to pericentric heterochromatin in both X and Y chromosomes while ribosomal DNA locus maps next to the heterochromatin band in sex chromosomes.

Figure 7

Synteny. Synteny between An. stephensi and An. gambiae based on 6,448 single-copy orthologs. Orthologs with the same orientation in An. stephensi and An. gambiae are connected with red lines and orthologs with the opposite orientation are connected with blue lines. Orthologous genes from An. stephensi and An. gambiae were retrieved from OrthoDB. The physical map was used to identify the relative locations of genes on the An. stephensi chromosomes. The relationship of the position between the An. stephensi and An. gambiae orthologs were plotted with GenoPlotR. 66 syntenic blocks were identified on the X chromosome. A total of 104 and 64 syntenic blocks were identified on 2R and 2L (3L in An. stephensi). A total of 104 and 42 syntenic blocks were identified on 3R and 3L (2L in An. stephensi). Therefore, the X chromosome has undergone the most rearrangements per megabase.

Figure 8

Chromosome evolution in Anopheles and Drosophila . (A) Higher rates of rearrangement on the X chromosome compared to autosomes between An. stephensi and An. gambiae. Arm designations for the figure are according to An. stephensi. (B) The ratio of the X chromosome evolution rate to the total rate of rearrangement is higher in Anopheles than in Drosophila.