The aim of this research program is to investigate the anti-hemostatic and anti-inflammatory compounds in the saliva of blood-feeding insects and ticks which allow efficient blood feeding and enhancement of pathogen transmission. Anti-hemostatic compounds of interest include anti-clotting, anti- platelet and vasodilators. Anti-inflammatory compounds include immunomodulatory compounds as well as compounds that modify effector arms of the immune response, such as anti-complement activity found in the saliva of some ticks, sand flies and mosquitoes. While the vector attempts to modify the feeding site to enhance success of blood feeding, such site becomes locally compromised in its ability to react to injury and becomes an easier site for pathogen invasion. On the other hand, when the vertebrate mounts an immune response to these salivary compounds, pathogen transmission may be compromised. Novel pharmaceuticals and novel targets for vaccine development will be ultimate benefits of this program. The work involves obtaining saliva or salivary glands of the insects and ticks under study, bioassay of their biological activity using both in vivo and in vitro tests, purification of the individual activities using chromatographic and electrophoretic techniques, microsequencing of the peptides and proteins, and final identification of the total primary sequence by molecular biology methods. Confirmation of the clones is found by expression in bacterial or eukaryotic cell lines. We have initiated a reverse approach where salivary cDNA libraries from blood-feeding insects and ticks are being mass sequenced and expressed recombinantly, providing new insights in the discovery of novel compounds. Summary Vector arthropods, such as mosquitoes, triatomine bugs and ticks, salivate while they puncture our skin in their search of blood. This saliva contains dozens to hundreds of compounds that have anti-clotting, anti-platelet, vasodilatory, anti-inflammatory, and immunomodulatory functions. While helping the vector to feed, it also modifies the site where pathogens are injected and, in many cases, facilitates the infection process. For this reason, salivary proteins of vectors can be used as vaccine targets for the diseases they transmit. Salivary proteins can also be used as immuno-epidemiological markers of vector exposure, and in themselves can have potent and novel pharmacological activities. Because the saliva of hematophagous animals is under attack of their host's immune system, their constituents are under a rapid evolutionary pressure in an arms' race scenario with their hosts, causing an enormous variety of unique protein families even in closely related organisms. The section of vector biology aims at uncovering the biodiversity of salivary proteins in the near 500 genera of blood sucking arthropods, and to discover the function of the novel protein families that we encounter. Accordingly, we have developed a two pronged approach focusing in sialotranscriptome discovery projects and functional sialomic studies. In addition to these core studies on the saliva of vector arthropods, the section also collaborated with other members of the LMVR, LPD and other extramural scientists lending its expertise in bioinformatics, structural biology and vascular biology. In the current fiscal year, members of the Section of Vector Biology contributed to a total of 13 papers as detailed below: Sialotranscriptome discovery and evolution projects: Because host hemostasis (the physiological process that prevents blood loss, consisting of platelet aggregation, blood clotting and vasoconstriction) is a complex and redundant phenomenon, the salivary glands of blood sucking arthropods consist of a magic potion with diverse chemicals that in a redundant way counteract host mechanisms to prevent blood loss, allowing the fast acquisition of a meal. Salivary transcriptome made in the past few years indicate that the magic potion consists of 70-100 different proteins in the case of mosquitoes, for example, to over 1,000 in the case of ticks (Ticks feed for several days and have to disarm host immune reactions, in addition to the hemostatic system). Transcriptome studies also show that the salivary proteins of blood sucking arthropods are at a very fast pace of evolution, perhaps explaining why every genus studied so far has several unique protein families. Indeed, there are unique proteins found at the subgenus level. Given we can now describe in detail the sialotranscriptome (from the Greek word sialo = saliva) of a single organism, we can ask now what the universe of salivary proteins is associated to blood feeding, the so called sialoverse. There are near 19,000 species of blood sucking arthropods in 500 different genera. If we find (minimally) 5 novel protein families per genus (within the 70-500 proteins in each sialome), there are at least 2,500 novel proteins to be discovered, each one with an interesting pharmacological property. We have so far explored less than 20 genera of blood sucking arthropods, and it is our goal to extend sialotranscriptome discovery to map this pharmacological mine for future studies, and in the process learn the paths taken by genomes in their evolution to blood feeding and identify proteins with pharmacological and vaccine potential. In the current fiscal year, we produced three papers related to sialotranscriptome and proteome discovery and evolution of hematophagous arthropods, including the first disclosure of the sialome of the kissing bug Panstrongylus lignarius (1), a deep sequencing of male and female salivary transcriptomes of the mosquito vector of West Nile virus, Culex tarsalis (2) and the first sialotranscriptome of ticks using single salivary gland libraries that showed fast shifting of sialome transcription on the Lyme disease vector, Ixodes ricinus (3). Functional studies: We advanced our knowledge regarding the function of vector salivary proteins as reported in four publications, describing for the first time the plasminogen activating peptide of a tick vector of Lyme disease (4), the possible use of salivary hyaluronidase as a vaccine target to prevent leishmaniasis (5), the characterization of a salivary anti-complement activity of a sand fly vector of leishmaniasis (6) and in understanding the anti-tick immune response of natural hosts of the Lyme disease vector, Ixodes scapularis (7). Bioinformatic collaborations: Dr. Ribeiro collaborated with intra and extra mural investigators lending his expertise in bioinformatics in six publications related to vectors of human disease or nematode parasites. We continued our collaboration with Dr. Thomas Nutman from LPD, collaborating on studies of Onchocerca volvulus population biology through multilocus immunophenotyping (8), with Brazilian investigators of the Sao Paulo University investigating the transcriptional response of tick midguts following rickettsial infections (9), with Dr. Bruno Aca from the University of Rome on deciphering the olfactory repertoire of the tiger mosquito, Aedes albopictus (10), with Dr. Louis Miller from LMVR on plasmodium transcriptomes leading to understanding of the function of parasite protein kinases (11), with Dr. Sanjay Desai from LMVR by creating an annotated catalog of over 680 thousand guideRNA sequences from Plasmodium falciparum that will help scientists to harvest the benefits of CRISPER-Cas9 transfections (12), and with investigators from the U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland, USA, we helped the annotation of proteomic results resulting from the interaction of the Sindbis virus with it mammalian and mosquito host cells (13).

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Schuchman, Ryan; Kilianski, Andy; Piper, Amanda et al. (2018) Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virion and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication. J Virol 92:
Perner, Jan; Kropá?ková, Sára; Kopá?ek, Petr et al. (2018) Sialome diversity of ticks revealed by RNAseq of single tick salivary glands. PLoS Negl Trop Dis 12:e0006410
Ribeiro, José M C; Martin-Martin, Ines; Moreira, Fernando R et al. (2018) A deep insight into the male and female sialotranscriptome of adult Culex tarsalis mosquitoes. Insect Biochem Mol Biol 95:1-9
Assumpção, Teresa C; Mizurini, Daniella M; Ma, Dongying et al. (2018) Ixonnexin from Tick Saliva Promotes Fibrinolysis by Interacting with Plasminogen and Tissue-Type Plasminogen Activator, and Prevents Arterial Thrombosis. Sci Rep 8:4806
Martin-Martin, Ines; Chagas, Andrezza Campos; Guimaraes-Costa, Anderson B et al. (2018) Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection. PLoS Pathog 14:e1007006
Ribeiro, Jose M; Garriga, Meera; Potchen, Nicole et al. (2018) Guide RNA selection for CRISPR-Cas9 transfections in Plasmodium falciparum. Int J Parasitol 48:825-832
Bansal, Abhisheka; Molina-Cruz, Alvaro; Brzostowski, Joseph et al. (2018) PfCDPK1 is critical for malaria parasite gametogenesis and mosquito infection. Proc Natl Acad Sci U S A 115:774-779
Nevoa, Jessica C; Mendes, Maria T; da Silva, Marcos V et al. (2018) An insight into the salivary gland and fat body transcriptome of Panstrongylus lignarius (Hemiptera: Heteroptera), the main vector of Chagas disease in Peru. PLoS Negl Trop Dis 12:e0006243
Martins, Larissa A; Galletti, Maria F B de Melo; Ribeiro, José M et al. (2017) The Distinct Transcriptional Response of the Midgut of Amblyomma sculptum and Amblyomma aureolatum Ticks to Rickettsia rickettsii Correlates to Their Differences in Susceptibility to Infection. Front Cell Infect Microbiol 7:129
Maruyama, Sandra R; Garcia, Gustavo R; Teixeira, Felipe R et al. (2017) Mining a differential sialotranscriptome of Rhipicephalus microplus guides antigen discovery to formulate a vaccine that reduces tick infestations. Parasit Vectors 10:206

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