During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ligand binding specificity and physiological relevance of two D7 long proteins from Culex quinquefasciatus mosquito, the vector of filaria parasites or West Nile viruses. CxD7L2 binds biogenic amines and eicosanoids. CxD7L1 exhibits high affinity for ADP and ATP, a binding capacity not reported in any D7. We solve the crystal structure of CxD7L1 in complex with ADP to 1.97 Å resolution. The binding pocket lies between the two protein domains, whereas all known D7s bind ligands either within the Nor the C-terminal domains. We demonstrate that these proteins inhibit hemostasis in ex vivo and in vivo experiments. Our results suggest that the ADP-binding function acquired by CxD7L1 evolved to enhance blood-feeding in mammals, where ADP plays a key role in platelet aggregation.

DAY and BWA conceived of and designed the project. ASP and SKB carried out mosquito husbandry, viral propagation, molecular work and collected project data. CLD performed the nutrient analyses and mosquito wing length measurements. DAY analyzed and interpreted the data with BWA.

9Adult female mosquitoes require a vertebrate blood meal to develop eggs and continue their life 10 cycle. During blood feeding, mosquito saliva is injected at the bite site to facilitate blood meal 11 acquisition through anti-hemostatic compounds that counteract blood clotting, platelet 12 aggregation, vasoconstriction and host immune responses. D7 proteins are among the most 13 abundant components of the salivary glands of several blood feeding insects. They are members 14 of a family of proteins that have evolved through gene duplication events to encode D7 proteins 15 of several lengths. Here, we examine the ligand binding specificity and physiological relevance 16 of two D7 long proteins, CxD7L1 and CxD7L2, from Culex quinquefasciatus mosquitoes, the 17 vector of medical and veterinary diseases such as filariasis, avian malaria, and West Nile virus 18 infections. CxD7L1 and CxD7L2 were assayed by microcalorimetry for binding of potential host 19 ligands involved in hemostasis, including bioactive lipids, biogenic amines, and 20 nucleotides/nucleosides. CxD7L2 binds serotonin, histamine, and epinephrine with high affinity 21 as well as the thromboxane A2 analog U-46619 and several cysteinyl leukotrienes, as previously 22 described for other D7 proteins. CxD7L1 does not bind any of the ligands that are bound by 23 CxD7L2. Unexpectedly, CxD7L1 exhibited high affinity for adenine nucleotides and 24 nucleosides, a binding capacity not reported in any D7 family member. We solved the crystal 25 structure of CxD7L1 in complex with bound ADP to 1.97 Å resolution. The binding pocket for 26 ADP is located between the two domains of CxD7L1, whereas all known D7s bind ligands either 27 within the N-terminal or the C-terminal domains. We demonstrated that these two CxD7 long 28 proteins inhibit human platelet aggregation in ex vivo experiments. CxD7L1 and CxD7L2 help 29 blood feeding in mosquitoes by scavenging host molecules that promote vasoconstriction, 30 platelet aggregation, itch, and pain at the bite site. The novel ADP-binding function acquired by CxD7L1 evolved to enhance blood feeding in mammals where ADP plays a key role in platelet 32 aggregation. 33 Culex quinquefasciatus (Diptera: Culicidae) commonly known as the southern house mosquito, 35 is a vector of medical and veterinary importance of filaria parasites, including Wuchereria 36 bancrofti and Dirofilaria immitis 1, 2 and avian malaria parasites (Plasmodium relictum) 3 . They 37 also can transmit several arboviruses including Rift Valley fever, West Nile, St. Louis or 38 Western equine encephalitis viruses 4, 5 . Adult female mosquitoes need to acquire vertebrate 39 blood for egg development. During blood feeding, mosquito saliva is injected at the bite site and 40 facilitates blood meal acquisition through anti-hemostatic compounds that prevent blood clotting, 41 platelet aggregation and vasoconstriction as well as host immune responses 6 . 42D7 proteins are among the most abundant components in the salivary glands of several blood 43 feeding ar...