BackgroundMosquito salivary proteins play a crucial role in blood meal acquisition and are known to disrupt host immune responses and homeostasis. Previous studies have identified salivary gland extract from the prominent arbovirus vector, Aedes aegypti, that contains pharmacologically active proteins which can induce cell death in splenic lymphocyte populations in mice. However, it has remained unclear until now whether this effect is unique to A. aegypti or conserved across other Aedes species.MethodsHere, we characterize the immunomodulatory properties of the salivary gland extracts from Aedes triseriatus, the primary vector of La Crosse virus (LACV). Murine, guinea pig, and human lymphocytes were exposed to salivary gland extracts at varying concentrations and time points. Lymphocyte proliferation was evaluated using colorimetric metabolic assays and flow cytometry analysis to characterize apoptotic mechanisms and define affected lymphocyte subsets.ResultsWe found that Ae. triseriatus salivary gland extract inhibited splenocyte proliferation in both mice and guinea pigs through the induction of apoptosis and suppression of cytokine expression. We identified a strong dose-dependent apoptotic phenotype present in CD4+, CD8+, and CD19+ lymphocytes. However, primary human lymphocytes and human lymphoid cell lines did not exhibit reduced proliferation after exposure to salivary gland extracts.ConclusionTogether, these discoveries suggest that Aedes species saliva contains evolutionarily adapted immunomodulatory proteins that could help facilitate arboviral persistence in rodent reservoirs.

Salivary secretions from blood-feeding arthropods are enriched with bioactive molecules that counteract host hemostatic and immune defenses, including blood coagulation, platelet aggregation, complement activation, and vasoconstriction. Although numerous salivary components have been identified and functionally characterized across hematophagous arthropods, their specific roles in complement inhibition in black flies remain insufficiently defined. Previously, it was discovered that Simukunin, a salivary protein from Simulium vittatum, inhibits procoagulant activity. Here, using surface plasmon resonance and enzymatic assays, we found that Simukunin also acts as a specific inhibitor of the complement lectin pathway (LP). Simukunin binds with high affinity to mannose-associated serine protease 2 (MASP-2), a serine protease crucial for LP activation, preferentially blocking cleavage of C2 over C4, resulting in reduced downstream membrane attack complex deposition. No interaction with other downstream complement proteins was found. Simukunin also inhibits the procoagulant activity of MASP-2 in vitro. Notably, pretreatment with Simukunin significantly improved survival rates in a murine model of lipopolysaccharide-induced sepsis. Overall, Simukunin emerges as a novel complement inhibitor and a potential candidate for therapeutic applications.

Abstract Background Aedes triseriatus is an endemic North American mosquito and the primary vector of La Crosse virus, the etiologic agent of La Crosse encephalitis. This neuroinvasive disease is the leading cause of arboviral encephalitis in the United States and disproportionately affects pediatric populations. Despite its medical importance, the genomic architecture and vectorial capacity of A. triseriatus remain incompletely understood. Results We generated a chromosome-scale genome assembly and developmental transcriptome for A. triseriatus using an integrated approach combining PacBio long-read sequencing, Hi-C scaffolding, and Illumina sequencing technologies. The final assembly spans approximately 2.4 Gb, representing the largest mosquito genome reported to date, and consists of chromosome-sized scaffolds supported by Hi-C data and manual curation. Transcriptome profiling across larvae, pupae, and adult males and females revealed dynamic, stage-specific gene expression patterns associated with distinct physiological processes. Conclusions These genomic and transcriptomic resources provide a comprehensive foundation for investigating the molecular basis of La Crosse virus transmission, mosquito development, and vector–host interactions in A. triseriatus. The chromosome-scale assembly establishes an essential framework for future functional, evolutionary, and vector biology studies in this medically important species.

We report a global survey of viral small RNAs (vsmRNAs) from >200 Aedes aegypti samples to identify many mosquito viruses that actively infect this prominent arboviral vector. Ae. aegypti viruses in the Americas are abundant, with some displaying geographical boundaries. Viruses infecting Asian Ae. aegypti are similar to those in the Americas and reveal the first wild example of dengue vsmRNAs. African Ae. aegypti display vsmRNAs from viruses unique to these African strains. Academic lab colonies generally lack viruses, yet two commercial strains are deeply infected by a tombus-like virus that is related to plant viruses. Comparing matched viral long RNAs to vsmRNAs reveal viral transcripts evading the mosquito RNA interference (RNAi) pathway. By infecting mosquito cells with Ae. aegypti homogenates, we generate stably infected cell lines which produce vsmRNAs that were comparable to native mosquito vsmRNA patterns. Lastly, we demonstrate that these stably infected mosquito cells producing vsmRNAs can exert gene silencing of reporters bearing viral sequence segments, providing a potential explanation for how Ae. aegypti can resist viral infections. This vsmRNA genomics approach in Ae. aegypti can add to existing vector surveillance approaches by discovering new viruses that persist in mosquito populations.

Black flies (Diptera: Simuliidae) are important vectors of pathogens affecting human and animal health, yet the absence of a chromosome-level nuclear reference genome has constrained molecular and evolutionary studies of the family. Here, we present the first chromosome-level nuclear genome and a developmental transcriptomic resource for the long-established IS-7 laboratory lineage of Simulium vittatum. Combining Oxford Nanopore long-read sequencing with Hi-C scaffolding, we assembled a 340.4-Mb genome, with 99.1% of the assembly resolved into three chromosome-length scaffolds (N50=104.8 Mb, BUSCO completeness 93.9%), consistent with the known 2n = 6 karyotype. Using the historically mapped molecular landmarks SVAT and SVEP, we assigned the two arms of chromosome III as IIIS and IIIL, respectively, linking sequence coordinates to the classical polytene chromosome map. Repetitive DNA comprises 46.48% of the assembly, including 29.68% unclassified repeats, and annotation identified 14,732 protein-coding genes and 16,417 transcripts. Integration with larval RNA-seq recovered the previously characterized silk gland proteins SGP-1-SGP-3 and resolved the expressed SGP-2 sequence despite a remaining discrepancy with the genomic sequence. This reference genome connects classical black fly cytogenetics with sequence-level analyses of genome organization, structural variation, and gene content, addressing a major genomic gap within Culicomorpha and providing a foundation for comparative studies of chromosome evolution, hematophagy, and vector biology across Simuliidae.

Abstract Introduction Complement inhibition is a key strategy used by many blood-feeding arthropods to ensure successful feeding. Mosquitoes, ticks, sand flies, and other hematophagous species secrete salivary proteins that block different steps of the complement cascade. These inhibitors prevent complement-mediated lysis and inflammation at the feeding site, allowing the arthropod to feed efficiently while also creating a more permissive environment for pathogen transmission Methods Here, we characterize the anti-complement activity of Sicpin, a salivary protein from the blackfly Simulium nigrimanum. Sicpin was found to inhibit all three pathways of complement activation. Surface plasmon resonance analysis revealed a strong and specific interaction with C3, with no detectable binding to other complement components common to the pathways. Results The binding induces a conformational change in C3 that blocks the interaction with the C3 convertase, consequently inhibiting the deposition of downstream complement proteins. Additionally, in both acute lung injury and sepsis models, Sicpin enhances survival and exhibits anti-inflammatory activity. Conclusion These findings support that Sicpin modulates the complement system, with potential implications for pathogen transmission, blood feeding, and therapeutic development Funding Source NIH Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Salivary secretions from blood-feeding arthropods are rich in bioactive compounds that counteract blood clotting, platelet aggregation, complement activation, and vasoconstriction. Despite the identification and characterization of various salivary components in blood-feeding arthropods, their role in complement inhibition, particularly in black flies, remains underexplored. Here, we found that Guianensin, a salivary protein from Simulium guianense, is a specific inhibitor of the lectin pathway of the complement. Guianensin targets MASP-2, a serine protease crucial for LP activation resulting in reduced C4 and C5b-9 complex deposition. Guianensin also inhibits the procoagulant activity of MASP-2 in vitro. No interaction with C1s, C1r, or other downstream complement proteins was found. Using a lipopolysaccharide-induced murine lung injury model, Guianensin significantly reduced neutrophil numbers and inflammatory cytokines, demonstrating its strong anti-inflammatory activity in vivo. Guianensin emerges as a novel complement inhibitor and a potential candidate for therapeutic applications.

Salivary secretions from blood feeding arthropods are rich in bioactive compounds that counteract blood clotting, platelet aggregation, complement activation, and vasoconstriction. Despite the identification and characterization of various salivary components in blood feeding arthropods, their role in complement inhibition, particularly in black flies, remains underexplored. In this study, we found that Guianensin, a salivary protein from Simulium guianense, is a specific inhibitor of the lectin pathway (LP) of complement. Guianensin belongs to the Kunitz domain of protease inhibitors and targets various serine proteases involved in hemostasis and inflammation, including MASP-2, a serine protease crucial for LP activation resulting in reduced C4 and C5b-9 complex deposition. Guianensin also inhibits the procoagulant activity MASP-2 in vitro. Surface plasmon resonance analysis confirmed its specific binding to MASP-2, with high affinity. No interaction with C1s, C1r, or other downstream complement proteins was found. These results underscore the potential of Guianensin for investigating the role of the complement inhibition in blood feeding and pathogen transmission by Simulium spp., offering new insights into the adaptive functions of salivary components in these vectors. Guianensin emerges as a novel complement inhibitor and a possible candidate for therapeutic applications. This research was supported by the Division of Intramural Research Program of the NIH/NIAID (AI001246) Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Olfactory receptors (Olfr) are G protein–coupled receptors that are normally expressed on olfactory sensory neurons to detect volatile chemicals or odorants. Interestingly, many Olfrs are also expressed in diverse tissues and function in cell–cell recognition, migration, and proliferation as well as immune responses and disease processes. Here, we showed that many Olfr genes were expressed in the mouse spleen, linked to Plasmodium yoelii genetic loci significantly, and/or had genome-wide patterns of LOD scores (GPLSs) similar to those of host Toll-like receptor genes. Expression of specific Olfr genes such as Olfr1386 in HEK293T cells significantly increased luciferase signals driven by IFN-β and NF-κB promoters, with elevated levels of phosphorylated TBK1, IRF3, P38, and JNK. Mice without Olfr1386 were generated using the CRISPR/Cas9 method, and the Olfr1386 −/− mice showed significantly lower IFN-α/β levels and longer survival than wild-type (WT) littermates after infection with P. yoelii YM parasites. Inhibition of G protein signaling and P38 activity could affect cyclic AMP-responsive element promoter-driven luciferase signals and IFN-β mRNA levels in HEK293T cells expressing the Olfr1386 gene, respectively. Screening of malaria parasite metabolites identified nicotinamide adenine dinucleotide (NAD) as a potential ligand for Olfr1386, and NAD could stimulate IFN-β responses and phosphorylation of TBK1 and STAT1/2 in RAW264.7 cells. Additionally, parasite RNA (pRNA) could significantly increase Olfr1386 mRNA levels. This study links multiple Olfrs to host immune response pathways, identifies a candidate ligand for Olfr1386, and demonstrates the important roles of Olfr1386 in regulating type I interferon (IFN-I) responses during malaria parasite infections.

The discovery that Africans were resistant to infection by Plasmodium vivax (P. vivax) led to the conclusion that P. vivax invasion relied on the P. vivax Duffy Binding Protein (PvDBP) interacting with the Duffy Antigen Receptor for Chemokines (DARC) expressed on erythrocytes. However, the recent reporting of P. vivax infections in DARC-negative Africans suggests that the parasite might use an alternate invasion pathway to infect DARC-negative reticulocytes. To identify the parasite ligands and erythrocyte receptors that enable P. vivax invasion of both DARC-positive and -negative erythrocytes, we expressed region II containing the Duffy Binding-Like (DBL) domain of P. vivax erythrocyte binding protein (PvEBP-RII) and verified that the DBL domain binds to both DARC-positive and -negative erythrocytes. Furthermore, an AVidity-based EXtracelluar Interaction Screening (AVEXIS) was used to identify the receptor for PvEBP among over 750 human cell surface receptor proteins, and this approach identified only Complement Receptor 1 (CR1, CD35, or C3b/C4b receptor) as a PvEBP receptor. CR1 is a well-known receptor for P. falciparum Reticulocyte binding protein Homology 4 (PfRh4) and is present on the surfaces of both reticulocytes and normocytes, but its expression decreases as erythrocytes age. Indeed, PvEBP-RII bound to a subpopulation of both reticulocytes and normocytes, and this binding was blocked by the addition of soluble CR1 recombinant protein, indicating that CR1 is the receptor of PvEBP. In addition, we found that the Long Homology Repeat A (LHR-A) subdomain of CR1 is the only subdomain responsible for mediating the interaction with PvEBP-RII.

The first step in disease pathogenesis for arboviruses is the establishment of infection following vector transmission. For La Crosse virus (LACV), the leading cause of pediatric arboviral encephalitis in North America, and other orthobunyaviruses, the initial course of infection in the skin is not well understood. Using an intradermal (ID) model of LACV infection in mice, we find that the virus infects and replicates nearly exclusively within skin-associated muscle cells of the panniculus carnosus (PC) and not in epidermal or dermal cells like most other arbovirus families. LACV is widely myotropic, infecting distal muscle cells of the peritoneum and heart, with limited infection of draining lymph nodes. Surprisingly, muscle cells are resistant to virus-induced cell death, with long term low levels of virus release progressing through the Golgi apparatus. Thus, skin muscle may be a key cell type for the initial infection and spread of arboviral orthobunyaviruses.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

Mosquito vectors of medical importance both blood and sugar feed, and their saliva contains bioactive molecules that aid in both processes. Although it has been shown that the salivary glands of several mosquito species exhibit α-glucosidase activities, the specific enzymes responsible for sugar digestion remain understudied. We therefore expressed and purified three recombinant salivary α-glucosidases from the mosquito vectors Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus and compared their functions and structures. We found that all three enzymes were expressed in the salivary glands of their respective vectors and were secreted into the saliva. The proteins, as well as mosquito salivary gland extracts, exhibited α-glucosidase activity, and the recombinant enzymes displayed preference for sucrose compared to p-nitrophenyl-α-D-glucopyranoside. Finally, we solved the crystal structure of the Ae. aegypti α-glucosidase bound to two calcium ions at a 2.3 Ångstrom resolution. Molecular docking suggested that the Ae. aegypti α-glucosidase preferred di- or polysaccharides compared to monosaccharides, consistent with enzymatic activity assays. Comparing structural models between the three species revealed a high degree of similarity, suggesting similar functional properties. We conclude that the α-glucosidases studied herein are important enzymes for sugar digestion in three mosquito species.

The evolution of hematophagy involves a series of adaptations that allow blood-feeding insects to access and consume blood efficiently while managing and circumventing the host's hemostatic and immune responses. Mosquito, and other insects, utilize salivary proteins to regulate these responses at the bite site during and after blood feeding. We investigated the function of Anopheles gambiae salivary apyrase (AgApyrase) in regulating hemostasis in the mosquito blood meal and in Plasmodium transmission. Our results demonstrate that salivary apyrase, a known inhibitor of platelet aggregation, interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protease that degrades fibrin and facilitates Plasmodium transmission. We show that mosquitoes ingest a substantial amount of apyrase during blood feeding, which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. AgApyrase significantly enhanced Plasmodium infection in the mosquito midgut, whereas AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammalian host, underscoring the potential for strategies to prevent malaria transmission.

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.

Mosquito borne flaviviruses such as dengue and Zika represent a major public health problem due to globalization and propagation of susceptible vectors worldwide. Vertebrate host responses to dengue and Zika infections include the processing and release of pro-inflammatory cytokines through the activation of inflammasomes, resulting in disease severity and fatality. Mosquito saliva can facilitate pathogen infection by downregulating the host's immune response. However, the role of mosquito saliva in modulating host innate immune responses remains largely unknown. Here, we show that mosquito salivary gland extract (SGE) inhibits dengue and Zika virus-induced inflammasome activation by reducing NLRP3 expression, Caspase-1 activation, and 1L-1β secretion in cultured human and mice macrophages. As a result, we observe that SGE inhibits virus detection in the early phase of infection. This study provides important insights into how mosquito saliva modulates host innate immunity during viral infection.

Hematophagous arthropods are animals that feed on vertebrate blood for egg production. Mosquitoes must pierce the host skin, locate blood vessels, and extract blood without being noticed. Mosquito stylets lacerate host tissues, triggering the activation of the three branches of hemostasis, or stopping of blood flow: vasoconstriction, platelet aggregation, and coagulation. Mosquitoes inject saliva into the host skin during their intradermal search for blood (also called probing), and salivary proteins counteract hemostasis. Blood feeding dynamics have been traditionally described by observational studies, in which researchers using magnifying glasses watched mosquitoes in the act of blood feeding. These studies provided the foundation for protocols to evaluate mosquito blood feeding in a more quantitative manner. Here, we introduce mosquito blood feeding biology with a focus on the feeding steps, which include penetration, probing, and feeding. Understanding blood feeding dynamics is crucial for evaluating probing time and other relevant parameters derived from blood feeding, such as blood meal size, fecundity, and fertility. Other considerations, including the relationship between probing and pathogen transmission and novel technologies to address blood feeding, are also discussed.