Wolbachia mediates crosstalk between miRNA and Toll pathways to enhance resistance to dengue virus in Aedes aegypti.

The obligate endosymbiont Wolbachia induces pathogen interference in the primary disease vector Aedes aegypti, facilitating the utilization of Wolbachia-based mosquito control for arbovirus prevention, particularly against dengue virus (DENV). However, the mechanisms underlying Wolbachia-mediated virus blockade have not been fully elucidated. Here, we report that Wolbachia activates the host cytoplasmic miRNA biogenesis pathway to suppress DENV infection. Through the suppression of the long noncoding RNA aae-lnc-2268 by Wolbachia wAlbB, aae-miR-34-3p, a miRNA upregulated by the Wolbachia strains wAlbB and wMelPop, promoted the expression of the antiviral effector defensin and cecropin genes through the Toll pathway regulator MyD88. Notably, anti-DENV resistance induced by Wolbachia can be further enhanced, with the potential to achieve complete virus blockade by increasing the expression of aae-miR-34-3p in Ae. aegypti. Furthermore, the downregulation of aae-miR-34-3p compromised Wolbachia-mediated virus blockade. These findings reveal a novel mechanism by which Wolbachia establishes crosstalk between the cytoplasmic miRNA pathway and the Toll pathway via aae-miR-34-3p to strengthen antiviral immune responses against DENV. Our results will aid in the advancement of Wolbachia for arbovirus control by enhancing its virus-blocking efficiency.

Immunometabolic crosstalk in Aedes fluviatilis and Wolbachia pipientis symbiosis.

Wolbachia pipientis is a maternally transmitted symbiotic bacterium that mainly colonizes arthropods, potentially affecting different aspects of the host's physiology, e.g., reproduction, immunity, and metabolism. It has been shown that Wolbachia modulates glycogen metabolism in mosquito Aedes fluviatilis (Ae. fluviatilis). Glycogen synthesis is controlled by the enzyme GSK3, which is also involved in immune responses in both vertebrate and invertebrate organisms. Here we investigated the mechanisms behind immune changes mediated by glycogen synthase kinase ß (GSK3ß) in the symbiosis between Ae. fluviatilis and W. pipientis using a GSK3ß inhibitor or RNAi-mediated gene silencing. GSK3ß inhibition or knockdown increased glycogen content and Wolbachia population, together with a reduction in Relish2 and gambicin transcripts. Furthermore, knockdown of Relish2 or Caspar revealed that the immunodeficiency pathway acts to control Wolbachia numbers in the host. In conclusion, we describe for the first time the involvement of GSK3ß in Ae. fluviatilis immune response, acting to control the Wolbachia endosymbiotic population.

Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs.

The exogenous small interfering RNA (exo-siRNA) pathway is a key antiviral mechanism in the Aedes aegypti mosquito, a widely distributed vector of human-pathogenic arboviruses. This pathway is induced by virus-derived double-stranded RNAs (dsRNA) that are cleaved by the ribonuclease Dicer 2 (Dcr2) into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are used by the effector protein Argonaute 2 within the RNA-induced silencing complex to cleave target viral RNA. Dcr2 contains several domains crucial for its activities, including helicase and RNase III domains. In Drosophila melanogaster Dcr2, the helicase domain has been associated with binding to dsRNA with blunt-ended termini and a processive siRNA production mechanism, while the platform-PAZ domains bind dsRNA with 3' overhangs and subsequent distributive siRNA production. Here we analyzed the contributions of the helicase and RNase III domains in Ae. aegypti Dcr2 to antiviral activity and to the exo-siRNA pathway. Conserved amino acids in the helicase and RNase III domains were identified to investigate Dcr2 antiviral activity in an Ae. aegypti-derived Dcr2 knockout cell line by reporter assays and infection with mosquito-borne Semliki Forest virus (Togaviridae, Alphavirus). Functionally relevant amino acids were found to be conserved in haplotype Dcr2 sequences from field-derived Ae. aegypti across different continents. The helicase and RNase III domains were critical for silencing activity and 21 nt vsiRNA production, with RNase III domain activity alone determined to be insufficient for antiviral activity. Analysis of 21 nt vsiRNA sequences (produced by functional Dcr2) to assess the distribution and phasing along the viral genome revealed diverse yet highly consistent vsiRNA pools, with predominantly short or long sequence overlaps including 19 nt overlaps (the latter representing most likely true Dcr2 cleavage products). Combined with the importance of the Dcr2 helicase domain, this suggests that the majority of 21 nt vsiRNAs originate by processive cleavage. This study sheds new light on Ae. aegypti Dcr2 functions and properties in this important arbovirus vector species.

Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti.

As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, this has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been explored. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vector Aedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-infected blood meal further protects females against infection with arboviruses from different families. Sugar feeding blocks arbovirus initial infection and dissemination from the gut and lowers infection prevalence and intensity, thereby decreasing the transmission potential of female mosquitoes. Finally, we show that the antiviral role of sugar is mediated by sugar-induced immunity. Overall, our findings uncover a crucial role of sugar feeding in mosquito antiviral immunity which in turn decreases vector competence for arboviruses. Since Ae. aegypti almost exclusively feed on blood in some natural settings, our findings suggest that this lack of sugar intake could increase the spread of mosquito-borne arboviral diseases.

Activation of mosquito immunity blocks the development of transmission-stage filarial nematodes.

Mosquito-borne helminth infections are responsible for a significant worldwide disease burden in both humans and animals. Accordingly, development of novel strategies to reduce disease transmission by targeting these pathogens in the vector are of paramount importance. We found that a strain of Aedes aegypti that is refractory to infection by Dirofilaria immitis, the agent of canine heartworm disease, mounts a stronger immune response during infection than does a susceptible strain. Moreover, activation of the Toll immune signaling pathway in the susceptible strain arrests larval development of the parasite, thereby decreasing the number of transmission-stage larvae. Notably, this strategy also blocks transmission-stage Brugia malayi, an agent of human lymphatic filariasis. Our data show that mosquito immunity can play a pivotal role in restricting filarial nematode development and suggest that genetically engineering mosquitoes with enhanced immunity will help reduce pathogen transmission.

A mosquito juvenile hormone binding protein (mJHBP) regulates the activation of innate immune defenses and hemocyte development.

Insects rely on the innate immune system for defense against pathogens, some aspects of which are under hormonal control. Here we provide direct experimental evidence showing that the juvenile hormone-binding protein (mJHBP) of Aedes aegypti is required for the regulation of innate immune responses and the development of mosquito blood cells (hemocytes). Using an mJHBP-deficient mosquito line generated by means of CRISPR-Cas9 gene editing technology we uncovered a mutant phenotype characterized by immunosuppression at the humoral and cellular levels, which profoundly affected susceptibility to bacterial infection. Bacteria-challenged mosquitoes exhibited significantly higher levels of septicemia and mortality relative to the wild type (WT) strain, delayed expression of antimicrobial peptides (AMPs), severe developmental dysregulation of embryonic and larval hemocytes (reduction in the total number of hemocytes) and increased differentiation of the granulocyte lineage. Interestingly, injection of recombinant wild type mJHBP protein into adult females three-days before infection was sufficient to restore normal immune function. Similarly, injection of mJHBP into fourth-instar larvae fully restored normal larval/pupal hemocyte populations in emerging adults. More importantly, the recovery of normal immuno-activation and hemocyte development requires the capability of mJHBP to bind JH III. These results strongly suggest that JH III functions in mosquito immunity and hemocyte development in a manner that is perhaps independent of canonical JH signaling, given the lack of developmental and reproductive abnormalities. Because of the prominent role of hemocytes as regulators of mosquito immunity, this novel discovery may have broader implications for the understanding of vector endocrinology, hemocyte development, vector competence and disease transmission.

JNK pathway restricts DENV2, ZIKV and CHIKV infection by activating complement and apoptosis in mosquito salivary glands.

Arbovirus infection of Aedes aegypti salivary glands (SGs) determines transmission. However, there is a dearth of knowledge on SG immunity. Here, we characterized SG immune response to dengue, Zika and chikungunya viruses using high-throughput transcriptomics. We also describe a transcriptomic response associated to apoptosis, blood-feeding and lipid metabolism. The three viruses differentially regulate components of Toll, Immune deficiency (IMD) and c-Jun N- terminal Kinase (JNK) pathways. However, silencing of the Toll and IMD pathway components showed variable effects on SG infection by each virus. In contrast, regulation of the JNK pathway produced consistent responses in both SGs and midgut. Infection by the three viruses increased with depletion of the activator Kayak and decreased with depletion of the negative regulator Puckered. Virus-induced JNK pathway regulates the complement factor, Thioester containing protein-20 (TEP20), and the apoptosis activator, Dronc, in SGs. Individual and co-silencing of these genes demonstrate their antiviral effects and that both may function together. Co-silencing either TEP20 or Dronc with Puckered annihilates JNK pathway antiviral effect. Upon infection in SGs, TEP20 induces antimicrobial peptides (AMPs), while Dronc is required for apoptosis independently of TEP20. In conclusion, we revealed the broad antiviral function of JNK pathway in SGs and showed that it is mediated by a TEP20 complement and Dronc-induced apoptosis response. These results expand our understanding of the immune arsenal that blocks arbovirus transmission.

Subgenomic flavivirus RNA binds the mosquito DEAD/H-box helicase ME31B and determines Zika virus transmission by Aedes aegypti.

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

Aedes aegypti NeSt1 Protein Enhances Zika Virus Pathogenesis by Activating Neutrophils.

Saliva from the mosquito vector of flaviviruses is capable of changing the local immune environment, leading to an increase in flavivirus-susceptible cells at the infected bite site. In addition, an antibody response to specific salivary gland (SG) components changes the pathogenesis of flaviviruses in human populations. To investigate whether antigenic SG proteins are capable of enhancing infection with Zika virus (ZIKV), a reemerging flavivirus primarily transmitted by the Aedes aegypti mosquito, we screened for antigenic SG proteins using a yeast display library and demonstrate that a previously undescribed SG protein we term neutrophil stimulating factor 1 (NeSt1) activates primary mouse neutrophils ex vivo Passive immunization against NeSt1 decreases pro-interleukin-1ß and CXCL2 expression, prevents macrophages from infiltrating the bite site, protects susceptible IFNAR-/- IFNGR-/- (AG129) mice from early ZIKV replication, and ameliorates virus-induced pathogenesis. These findings indicate that NeSt1 stimulates neutrophils at the mosquito bite site to change the immune microenvironment, allowing a higher level of early viral replication and enhancing ZIKV pathogenesis.IMPORTANCE When a Zika virus-infected mosquito bites a person, mosquito saliva is injected into the skin along with the virus. Molecules in this saliva can make virus infection more severe by changing the immune system to make the skin a better place for the virus to replicate. We identified a molecule that activates immune cells, called neutrophils, to recruit other immune cells, called macrophages, that the virus can infect. We named this molecule neutrophil-stimulating factor 1 (NeSt1). When we used antibodies to block NeSt1 in mice and then allowed Zika virus-infected mosquitoes to feed on these mice, they survived much better than mice that do not have antibodies against NeSt1. These findings give us more information about how mosquito saliva enhances virus infection, and it is possible that a vaccine against NeSt1 might protect people against severe Zika virus infection.

Wolbachia-mediated virus blocking in mosquito cells is dependent on XRN1-mediated viral RNA degradation and influenced by viral replication rate.

Wolbachia is currently being developed as a novel tool to block the transmission of dengue viruses (DENV) by Aedes aegypti. A number of mechanisms have been proposed to explain the DENV-blocking phenotype in mosquitoes, including competition for fatty acids like cholesterol, manipulation of host miRNAs and upregulation of innate immune pathways in the mosquito. We examined the various stages in the DENV infection process to better understand the mechanism of Wolbachia-mediated virus blocking (WMVB). Our results suggest that infection with Wolbachia does not inhibit DENV binding or cell entry, but reduces virus replication. In contrast to a previous report, we also observed a similar reduction in replication of West Nile virus (WNV). This reduced replication is associated with rapid viral RNA degradation in the cytoplasm. We didn't find a role for host miRNAs in WMVB. Further analysis showed that the 3' end of the virus subgenomic RNA was protected and accumulated over time suggesting that the degradation is XRN1-mediated. We also found that sub genomic flavivirus RNA accumulation inactivated XRN1 in mosquito cells in the absence of Wolbachia and led to enhancement of RNA degradation in its presence. Depletion of XRN1 decreased WMVB which was associated with a significant increase in DENV RNA. We also observed that WMVB is influenced by virus MOI and rate of virus replication. A comparatively elevated blocking was observed for slowly replicating DENV, compared to WNV. Similar results were obtained while analysing different DENV serotypes.

The spread of Wolbachia through mosquito populations.

In many regions of the world, mosquito-borne viruses pose a growing threat to human health. As an alternative to traditional control measures, the bacterial symbiont Wolbachia has been transferred from Drosophila into the mosquito Aedes aegypti, where it can block the transmission of dengue and Zika viruses. A recent paper has reported large-scale releases of Wolbachia-infected Ae. aegypti in the city of Cairns, Australia. Wolbachia, which is maternally transmitted, invaded and spread through the populations due to a sperm-egg incompatibility called cytoplasmic incompatibility. Over a period of 2 years, a wave of Wolbachia infection slowly spread out from 2 release sites, demonstrating that it will be possible to deploy this strategy in large urban areas. In line with theoretical predictions, Wolbachia infection at a third, smaller release site collapsed due to the immigration of Wolbachia-free mosquitoes from surrounding areas. This remarkable field experiment has both validated theoretical models of Wolbachia population dynamics and demonstrated that this is a viable strategy to modify mosquito populations.

Aedes (Stegomyia) aegypti mosquito bite hypersensitivity in a dog: a case report.

BACKGROUND: Mosquitoes are vectors of several pathogens of considerable importance to humans and companion animals, including nematode helminths such as Dirofilaria immitis and Dirofilaria repens that cause heartworm disease and subcutaneous dirofilariosis, respectively. In addition to mosquito-borne pathogen transmission, mosquito bites can cause discomfort and irritation in pets, and even lead to severe hypersensitivity reactions. In the present study, we report an acute local hypersensitivity reaction in a dog following experimental exposure to Aedes (Stegomyia) aegypti. CASE PRESENTATION: A healthy six-year-old male beagle was included in an efficacy study in which dogs (n = 28) were exposed to Ae. aegypti mosquitoes. On Day - 6, the dog was allocated to one of the study groups, consisting of seven dogs to be treated on Day 0 with an imidacloprid/flumethrin collar. After sedation, animals were exposed to approximately 50 females of Ae. aegypti for 60 (± 5) minutes on Days - 6, 1, 7, 14, 21, 28, 55, and 83. On Day - 6, no allergic reaction to the mosquito bites was observed. However, on Day 1, corresponding to the second challenge, the dog demonstrated an acute allergic reaction characterized by swelling of the face (especially in the base of the muzzle and around the eyes), redness of the eyes, and conjunctival edema of the right eye was also observed. The dog was immediately treated with an intramuscular injection of a commercially available antihistamine treatment, Pen-Hista-Strep® containing a suspension of benzylpenicillin, chlorphenamine, dexamethasone, dihydrostreptomycin, and procaine at a dosage of 1 mL per 10 kg. A few hours after treatment, the dog showed noticeable improvement. CONCLUSIONS: This case provides the first evidence of canine acute local hypersensitivity reaction to mosquito bites under laboratory conditions. This observation suggests that invasive mosquito species such as Aedes spp. may affect the health and comfort of our companion animals, especially for pets with outdoor access without individual protective measures against insect bites.

Transcriptome of the Aedes aegypti Mosquito in Response to Human Complement Proteins.

Aedes aegypti is the primary mosquito vector of several human arboviruses, including the dengue virus (DENV). Vector control is the principal intervention to decrease the transmission of these viruses. The characterization of molecules involved in the mosquito physiological responses to blood-feeding may help identify novel targets useful in designing effective control strategies. In this study, we evaluated the in vivo effect of feeding adult female mosquitoes with human red blood cells reconstituted with either heat-inactivated (IB) or normal plasma (NB). The RNA-seq based transcript expression of IB and NB mosquitoes was compared against sugar-fed (SF) mosquitoes. In in vitro experiments, we treated Aag2 cells with a recombinant version of complement proteins (hC3 or hC5a) and compared transcript expression to untreated control cells after 24 h. The transcript expression analysis revealed that human complement proteins modulate approximately 2300 transcripts involved in multiple biological functions, including immunity. We also found 161 upregulated and 168 downregulated transcripts differentially expressed when human complement protein C3 (hC3) and human complement protein C5a (hC5a) treated cells were compared to the control untreated cells. We conclude that active human complement induces significant changes to the transcriptome of Ae. aegypti mosquitoes, which may influence the physiology of these arthropods.

Aedes albopictus Autophagy-Related Gene 8 (AaAtg8) Is Required to Confer Anti-Bacterial Gut Immunity.

Autophagy is an important process by which pathogens and damaged or unused organelles are eliminated. The role of autophagy in development and the immune response to pathogens is well established. Autophagy-related protein 8 (Atg8) is involved in the formation of the autophagosome and, with the help of the serine protease Atg4, mediates the delivery of both vesicles and the autophagosome to the vacuole. Here, we cloned the Aedes albopictus autophagy-related protein 8 (AaAtg8) gene and characterized its role in the innate immunity of the mosquito against microbial infections. AaAtg8 is comprised of an open reading frame (ORF) region of 357 bp encoding a polypeptide of 118 amino acid residues. A domain analysis of AaAtg8 revealed an Atg8 ubiquitin-like domain, Atg7/Atg4 interaction sites, and peptide binding sites. The AaAtg8 mRNA expression was high in the Malpighian tubules and heads of both sugar-fed and blood-fed adult female mosquitoes. The expression level of AaAtg8 mRNA increased in the midgut and abdominal carcass following being challenged with Listeria monocytogenes. To investigate the role of AaAtg8 in the innate immune responses of Ae. albopictus, AaAtg8 gene-silenced adult mosquitoes were challenged by injection or by being fed microorganisms in blood. High mortality rates were observed in mosquitoes in which AaAtg8 was silenced after challenges of microorganisms to the host by blood feeding. This suggests that Atg8-autophagy plays a critical role in the gut immunity in Ae. albopictus.

Spatial Assessment of Contact Between Humans and Anopheles and Aedes Mosquitoes in a Medium-Sized African Urban Setting, Using Salivary Antibody-Based Biomarkers.

BACKGROUND: Anarchic and poorly controlled urbanization led to an increased risk of mosquito-borne diseases (MBD) in many African cities. Here, we evaluate the spatial heterogeneity of human exposure to malaria and arboviral disease vectors in an urban area of northern Senegal, using antibody-based biomarkers of exposure to Anopheles and Aedes mosquito bites. METHODS: A cross-sectional study was undertaken during the rainy season of 2014 in 4 neighborhoods of Saint-Louis, a city in northern Senegal. Among children aged 6-59 months in each neighborhood, the dried blood spot technique was used to evaluate immunoglobulin G (IgG) responses to both gSG6-P1 (Anopheles) and Nterm-34-kDa (Aedes) salivary peptides as validated biomarkers of respective mosquito bite exposure. RESULTS: IgG response levels to gSG6-P1 and Nterm-34-kDa salivary peptides varied significantly between the 4 neighborhoods (P < .0001). The level of exposure to Aedes bites also varied according to household access to sanitation services (P = .027), whereas that of exposure to Anopheles bites varied according to insecticide-treated bed net use (P = .006). In addition, spatial clusters of high contact between humans and mosquitoes were identified inside 3 neighborhoods. CONCLUSIONS: Antibody-based biomarkers of exposure to Anopheles and Aedes mosquito bites could be helpful tools for evaluating the heterogeneity of exposure to malaria and arboviral disease vectors by national control programs.

Evaluation of inflammatory skin infiltrate following Aedes aegypti bites in sensitized and non-sensitized mice reveals saliva-dependent and immune-dependent phenotypes.

During probing and blood feeding, haematophagous mosquitoes inoculate a mixture of salivary molecules into their vertebrate hosts' skin. In addition to the anti-haemostatic and immunomodulatory activities, mosquito saliva also triggers acute inflammatory reactions, especially in sensitized hosts. Here, we characterize the oedema and the cellular infiltrate following Aedes aegypti mosquito bites in the skin of sensitized and non-sensitized BALB/c mice by flow cytometry. Ae. aegypti bites induced an increased oedema in the ears of both non-sensitized and salivary gland extract- (SGE-)sensitized mice, peaking at 6 hr and 24 hr after exposure, respectively. The quantification of the total cell number in the ears revealed that the cellular recruitment was more robust in SGE-sensitized mice than in non-sensitized mice, and the histological evaluation confirmed these findings. The immunophenotyping performed by flow cytometry revealed that mosquito bites were able to produce complex changes in cell populations present in the ears of non-sensitized and SGE-sensitized mice. When compared with steady-state ears, the leucocyte populations significantly recruited to the skin after mosquito bites in non-sensitized and sensitized mice were eosinophils, neutrophils, monocytes, inflammatory monocytes, mast cells, B-cells and CD4+ T-cells, each one with its specific kinetics. The changes in the absolute number of cells suggested two cell recruitment profiles: (i) a saliva-dependent migration; and (ii) a migration dependent on the immune status of the host. These findings suggest that mosquito bites influence the skin microenvironment by inducing differential cell migration, which is dependent on the degree of host sensitization to salivary molecules.

Dengue subgenomic flaviviral RNA disrupts immunity in mosquito salivary glands to increase virus transmission.

Globally re-emerging dengue viruses are transmitted from human-to-human by Aedes mosquitoes. While viral determinants of human pathogenicity have been defined, there is a lack of knowledge of how dengue viruses influence mosquito transmission. Identification of viral determinants of transmission can help identify isolates with high epidemiological potential. Additionally, mechanistic understanding of transmission will lead to better understanding of how dengue viruses harness evolution to cycle between the two hosts. Here, we identified viral determinants of transmission and characterized mechanisms that enhance production of infectious saliva by inhibiting immunity specifically in salivary glands. Combining oral infection of Aedes aegypti mosquitoes and reverse genetics, we identified two 3' UTR substitutions in epidemic isolates that increased subgenomic flaviviral RNA (sfRNA) quantity, infectious particles in salivary glands and infection rate of saliva, which represents a measure of transmission. We also demonstrated that various 3'UTR modifications similarly affect sfRNA quantity in both whole mosquitoes and human cells, suggesting a shared determinism of sfRNA quantity. Furthermore, higher relative quantity of sfRNA in salivary glands compared to midgut and carcass pointed to sfRNA function in salivary glands. We showed that the Toll innate immune response was preferentially inhibited in salivary glands by viruses with the 3'UTR substitutions associated to high epidemiological fitness and high sfRNA quantity, pointing to a mechanism for higher saliva infection rate. By determining that sfRNA is an immune suppressor in a tissue relevant to mosquito transmission, we propose that 3'UTR/sfRNA sequence evolution shapes dengue epidemiology not only by influencing human pathogenicity but also by increasing mosquito transmission, thereby revealing a viral determinant of epidemiological fitness that is shared between the two hosts.

Distinct melanization pathways in the mosquito Aedes aegypti.

Serine protease cascades are involved in blood coagulation and immunity. In arthropods, they regulate melanization, which plays an important role in immune defense and wound healing. However, the mechanisms underlying melanization pathways are not completely characterized. We found that in the mosquito Aedes aegypti, there are two distinct melanization activation pathways carried out by different modules of serine proteases and their specific inhibitors serpins. Immune melanization proteases (IMP-1 and IMP-2) and Serpin-1 mediate hemolymph prophenoloxidase cleavage and immune response against the malaria parasite. Tissue melanization, exemplified by the formation of melanotic tumors, is controlled by tissue melanization protease (CLIPB8), IMP-1, and Serpin-2. In addition, serine proteases CLIPB5 and CLIPB29 are involved in activation of Toll pathway by fungal infection or by infection-independent manner, respectively. Serpin-2 is implicated in the latter activation of Toll pathway. This study revealed the complexity underlying melanization and Toll pathway in mosquitoes.

Identification of a new type of haematopoietic progenitor kinase-interacting protein (HIP-55) in Aedes aegypti mosquito haemocytes and its involvement in immunity-like functions in mosquito: a molecular study.

In this study, we characterize the HIP-55 protein in the mosquito Aedes aegypti for the first time. HIP-55 is a 55-kDa HPK1-interacting protein that is also called SH3P7. HIP-55 constitutively binds HPK1 'via' an HPK1 proline-rich motif 2(PR2) through its C-terminal SH3 domain. HIP-55 critically interacts with ZAP-70, and this interaction was induced by TCR signalling. ZAP-70 phosphorylated HIP-55 at Tyr-334 and Tyr-344 in vitro and in vivo. In our previous findings, AaZAP gene expression strongly proved that AaZAP-70 was involved in immunity-like functions in mosquito. Northern blot analysis of HIP-55 mRNA expression confirmed that it is only expressed in the abdomen and haemocyte tissues; this prediction correlates 100% and a polyclonal antibody also confirmed its localization in haemocytes and the abdomen. We prepared extracts to show the cytoplasmic expression (CE) of this protein. Previous results had proven that this protein is secreted from the cytoplasm; thus, we confirmed here that the protein is a cytoplasmic adaptor protein in mosquitoes and mammalian systems. Furthermore, our polyclonal antibody against HIP-55 also demonstrated that this protein is found in haemocytes and abdomen tissues, which assumes that the protein may be involved in phagocytic-like functions. RNAi (siRNA) silencing studies were used to degrade mosquito HIP-55; however, silencing only slightly affected the HIP-55 sequence and the gene transcriptional level. To characterize this protein, we cloned 609 bp from the 1.6-kb full-length cDNA using a pET28 vector for polyclonal antibody production. Graphical abstract.

Transcriptomic Analysis of Aedes aegypti Innate Immune System in Response to Ingestion of Chikungunya Virus.

Aedes aegypti (L.) is the primary vector of emergent mosquito-borne viruses, including chikungunya, dengue, yellow fever, and Zika viruses. To understand how these viruses interact with their mosquito vectors, an analysis of the innate immune system response was conducted. The innate immune system is a conserved evolutionary defense strategy and is the dominant immune system response found in invertebrates and vertebrates, as well as plants. RNA-sequencing analysis was performed to compare target transcriptomes of two Florida Ae. aegypti strains in response to chikungunya virus infection. We analyzed a strain collected from a field population in Key West, Florida, and a laboratory strain originating from Orlando. A total of 1835 transcripts were significantly expressed at different levels between the two Florida strains of Ae. aegypti. Gene Ontology analysis placed these genes into 12 categories of biological processes, including 856 transcripts (up/down regulated) with more than 1.8-fold (p-adj (p-adjust value) ≤ 0.01). Transcriptomic analysis and q-PCR data indicated that the members of the AaeCECH genes are important for chikungunya infection response in Ae. aegypti. These immune-related enzymes that the chikungunya virus infection induces may inform molecular-based strategies for interruption of arbovirus transmission by mosquitoes.