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.

Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of, , and mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10 enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

The habit of blood feeding evolved independently in many insect orders of families. Sand flies and mosquitoes belong to separate lineages of blood-feeding Diptera and are thus considered to have evolved the trait independently. Because of this, sand fly salivary proteins differ structurally from those of mosquitoes, and orthologous groups are nearly impossible to define. An exception is the long-form D7-like proteins that show conservation with their mosquito counterparts of numerous residues associated with the N-terminal domain binding pocket. In mosquitoes, this pocket is responsible for the scavenging of proinflammatory cysteinyl leukotrienes and thromboxanes at the feeding site. Here we show that long-form D7 proteins AGE83092 and ABI15936 from the sand fly species, Phlebotomus papatasi and P . duboscqi , respectively, inhibit the activation of platelets by collagen and the thromboxane A 2 analog U46619. Using isothermal titration calorimetry, we also demonstrate direct binding of U46619 and cysteinyl leukotrienes C 4 , D 4 and E 4 to the P . papatasi protein. The crystal structure of P . duboscqi ABI15936 was determined and found to contain two domains oriented similarly to those of the mosquito proteins. The N-terminal domain contains an apparent eicosanoid binding pocket. The C-terminal domain is smaller in overall size than in the mosquito D7s and is missing some helical elements. Consequently, it does not contain an obvious internal binding pocket for small-molecule ligands that bind to many mosquito D7s. Structural similarities indicate that mosquito and sand fly D7 proteins have evolved from similar progenitors, but phylogenetics and differences in intron/exon structure suggest that they may have acquired the ability to bind vertebrate eicosanoids independently, indicating a convergent evolution scenario.