Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection

Abstract

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.

Keywords: Chagas disease; Rhodnius prolixus; genome; hematophagy; immunity.

Fig. 1.

Rhodnius genome. (A) Genome overview. All scaffolds are represented in layer I and are organized clockwise from the longest to the shortest, starting at the arrowhead. The genic (layer II, red) and TEs (layer III, blue) showed opposite densities until the asterisk (*) and were similarly low from this point until the end (shorter scaffolds). The Wolbachia sp. insertions (layer IV, orange) were observed throughout the genome without a trend. The kernel picture illustrates an adult R. prolixus. (B) Gene clustering. The Venn diagram partitions 15,439 OrthoMCL gene clusters according to their species compositions for R. prolixus and three other Hemimetabola (blue), four Diptera (yellow), four other Holometabola (green), and four noninsect outgroup species (pink). The 6,993 R. prolixus genes show widespread orthology (white circle, and bars, Bottom Left): these are part of the 7,115 clusters that have representatives from each of the four species sets, of which a conserved core of 2,253 clusters have orthologs in all 16 species. The 5,498 R. prolixus genes show no confident orthology (bars, Bottom Right), but most of these are homologous (e-value < 1e-05) to genes from other animals or to genes in its own genome. (C) TE distribution. The inner chart represents the three main classes of TEs (LTRs, non-LTRs, and class II), and the outer shows the distribution of TE superfamilies within each class. The charts are based on the total base pairs occupied by TE-related sequences longer than 0.5 Kb, as shown in SI Appendix, Table A1.

Fig. 2.

Characterization of IMD pathway and control of T. cruzi replication. Although several proteins from the IMD pathway are missing from the R. prolixus genome, the pathway is still active. (A) The rpRelish protein contains a Rel homology domain (RHD), an Ig-like fold, Plexins, a transcription factor domain (IPT), and several ankyrin (Ank) domains. (B and C) In response to the growth of the native microbiota following a blood meal, rpRelish expression is increased at 24 and 72 h postblood meal in both the midgut (B) and fat body (C). Fas: fasting. When rpRelish expression is knocked down (pink) in the gut using RNAi (SI Appendix, Fig. A6B), the expression of defensin A (Def) is greatly reduced, and the expression of lysozyme A (LizA) is increased (D). Upon knockdown of rpRelish expression (pink), the bacteria load in the anterior midgut (AM) (E) and posterior midgut (PM) (F) is increased, with the same trend observed in the rectum (R) (G). dsMal control was shown in gray, *P < 0.05. Upon knockdown of rpDorsal or rpRelish expressions (SI Appendix, Fig. A6), T. cruzi levels are not altered in any section of the digestive tract 14 d after infection (H). dsMal control (gray), dsRelish (pink) and dsDorsal (blue). Median values are at the graphic top. (I) Representation of the IMD R. prolixus immune pathways depicting the absence of key IMD pathway components. In R. prolixus, although the Toll and Jak/STAT pathways are usually highly conserved, members of IMD pathway are missing (dashed lines shapes with red “X”), including the IMD, Fadd, and Dredd genes and the negative modulators Pirk (poor IMD response upon knock-in) and Caspar. However, the pathway receptors, PGRPs and a homolog of the transcription factor Relish (rpRelish) are present, suggesting that the activity of the pathway is exerted through a noncanonical mechanism.

Fig. 3.

Thioredoxin reductases and nitrophorins trees. (A) The tree shows the phylogenetic relationship of thioredoxin reductases (TR) in nine Paraneoptera genomes. Colored balls indicate the amino acid aligned to the Sec position: green (U) selenocysteine, red (C) cysteine, and gray (–) unknown/unaligned. One of the two Cys-TR in R. prolixus (RPRC014349) clusters with the Sec-TR proteins, implying a Sec to Cys conversion. (B) Nitrophorins branch of lipocalins tree exemplify a Rhodnius enriched cluster. Sequences clustered by OrthoMCL on group 691 together with unclustered sequences similar to lipocalins were aligned and a tree constructed (SI Appendix, Fig. A15). Black dots mark new Rhodnius sequences. TRIDI, Triatoma dimidiata; TRIMA, Triatoma matogrossensis. Support values based on bootstrap are included at nodes.