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...

Across diverse insect taxa, the behavior and physiology of females dramatically changes after mating – processes largely triggered by the transfer of seminal proteins from their mates. In the vinegar flyDrosophila melanogaster, the seminal protein sex peptide (SP) decreases the likelihood of female flies remating and causes additional behavioral and physiological changes that promote fertility including increasing egg production. Although SP is only found in theDrosophilagenus, its receptor, sex peptide receptor (SPR), is the widely-conserved myoinhibitory peptide (MIP) receptor. To test the functional role of SPR in mediating post-mating responses in a non-Drosophiladipteran, we generated two independentSpr-knockout alleles in the yellow fever mosquitoAedes aegypti. Although SPR is needed for post-mating responses inDrosophilaand the cotton bollwormHelicoverpa armigera,SprmutantAe. aegyptishow completely normal post-mating decreases in remating propensity and increases in egg laying. In addition, injection of synthetic SP or accessory gland homogenate fromD. melanogasterinto virgin female mosquitoes did not elicit these post-mating responses. Our results indicate thatSpris not required for these canonical post-mating responses inAe. aegypti, indicating that unknown signaling pathways are likely responsible for these behavioral switches in this disease vector.

BackgroundSalivary glands from blood-feeding arthropods secrete several molecules that inhibit mammalian hemostasis and facilitate blood feeding and pathogen transmission. The salivary functions from Simulium guianense, the main vector of Onchocerciasis in South America, remain largely understudied. Here, we have characterized a salivary protease inhibitor (Guianensin) from the blackfly Simulium guianense.Materials and methodsA combination of bioinformatic and biophysical analyses, recombinant protein production, in vitro and in vivo experiments were utilized to characterize the molecula mechanism of action of Guianensin. Kinetics of Guianensin interaction with proteases involved in vertebrate inflammation and coagulation were carried out by surface plasmon resonance and isothermal titration calorimetry. Plasma recalcification and coagulometry and tail bleeding assays were performed to understand the role of Guianensin in coagulation.ResultsGuianensin was identified in the sialotranscriptome of adult S. guianense flies and belongs to the Kunitz domain of protease inhibitors. It targets various serine proteases involved in hemostasis and inflammation. Binding to these enzymes is highly specific to the catalytic site and is not detectable for their zymogens, the catalytic site-blocked human coagulation factor Xa (FXa), or thrombin. Accordingly, Guianensin significantly increased both PT (Prothrombin time) and aPTT (Activated partial thromboplastin time) in human plasma and consequently increased blood clotting time ex vivo. Guianensin also inhibited prothrombinase activity on endothelial cells. We show that Guianensin acts as a potent anti-inflammatory molecule on FXa-induced paw edema formation in mice.ConclusionThe information generated by this work highlights the biological functionality of Guianensin as an antithrombotic and anti-inflammatory protein that may play significant roles in blood feeding and pathogen transmission.

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Food quality and quantity serve as the basis for cycling of key chemical elements in trophic interactions, yet the role of nutrient stoichiometry in shaping host-parasite interactions is under appreciated. Most of the emergent mosquito-borne viruses affecting human health are transmitted by mosquitoes that inhabit container systems during their immature stages, where allochthonous input of detritus serves as the basal nutrients. Quantity and type of detritus (animal and plant) were manipulated in microcosms containing newly hatched Aedes aegypti mosquito larvae. Adult mosquitoes derived from these microcosms were allowed to ingest Zika virus infected blood and then tested for disseminated infection, transmission, and total nutrients (percent carbon, percent nitrogen, ratio of carbon to nitrogen). Treatments lacking high quality animal (insect) detritus significantly delayed development. Survivorship to adulthood was closely associated with the amount of insect detritus present. Insect detritus was positively correlated with percent nitrogen, which affected Zika virus infection. Disseminated infection and transmission decreased with increasing insect detritus and percent nitrogen. We provide the first definitive evidence linking nutrient stoichiometry to arbovirus infection and transmission in a mosquito using a model system of invasive Ae. aegypti and emergent Zika virus.

U7 snRNP is a multi-subunit endonuclease required for 3’ end processing of metazoan replication-dependent histone pre-mRNAs. In contrast to the spliceosomal snRNPs, U7 snRNP lacks the Sm subunits D1 and D2 and instead contains two related proteins, Lsm10 and Lsm11. The remaining five subunits of the U7 heptameric Sm ring, SmE, F, G, B and D3, are shared with the spliceosomal snRNPs. The pathway that assembles the unique ring of U7 snRNP is unknown. Here, we show that a heterodimer of Lsm10 and Lsm11 tightly interacts with the methylosome, a complex of the arginine methyltransferase PRMT5, MEP50 and pICln known to methylate arginines in the C-terminal regions of the Sm proteins B, D1 and D3 during the spliceosomal Sm ring assembly. Both biochemical and Cryo-EM structural studies demonstrate that the interaction is mediated by PRMT5, which binds and methylates two arginine residues in the N-terminal region of Lsm11. Surprisingly, PRMT5 also methylates an N-terminal arginine in SmE, a subunit that does not undergo this type of modification during the biogenesis of the spliceosomal snRNPs. An intriguing possibility is that the unique methylation pattern of Lsm11 and SmE plays a vital role in the assembly of the U7 snRNP.