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.

Altered thermal preferences of infected or immune-challenged Aedes aegypti and Aedes japonicus mosquitoes.

Temperature is a critical factor shaping physiology, life cycle, and behaviour of ectothermic vector insects, as well as the development and multiplication of pathogens within them. However, the influence of pathogen infections on thermal preferences (behavioural thermoregulation) is not well-understood. The present study examined the thermal preferences of mosquitoes (Aedes aegypti and Ae. japonicus) infected with either Sindbis virus (SINV) or Dirofilaria immitis over 12 days post exposure (p.e.) or injected with a non-pathogenic Sephadex bead over 24 h in a thermal gradient (15-30 °C). SINV-infected Ae. aegypti preferred 5 °C warmer temperatures than non-infected ones at day 6 p.e., probably the time of highest innate immune response. In contrast, D. immitis-infected Ae. japonicus preferred 4 °C cooler temperatures than non-infected ones at day 9 p.e., presumably a stress response during the migration of third instar larvae from their development site to the proboscis. Sephadex bead injection also induced a cold preference in the mosquitoes but to a level that did not differ from control-injections. The cold preference thus might be a strategy to escape the risk of desiccation caused by the wound created by piercing the thorax. Further research is needed to uncover the genetic and physiological mechanisms underlying these behaviours.

Mechanisms of Flavivirus Cross-Protection against Yellow Fever in a Mouse Model.

The complete lack of yellow fever virus (YFV) in Asia, and the lack of urban YFV transmission in South America, despite the abundance of the peridomestic mosquito vector Aedes (Stegomyia.) aegypti is an enigma. An immunologically naïve population of over 2 billion resides in Asia, with most regions infested with the urban YF vector. One hypothesis for the lack of Asian YF, and absence of urban YF in the Americas for over 80 years, is that prior immunity to related flaviviruses like dengue (DENV) or Zika virus (ZIKV) modulates YFV infection and transmission dynamics. Here we utilized an interferon α/ß receptor knock-out mouse model to determine the role of pre-existing dengue-2 (DENV-2) and Zika virus (ZIKV) immunity in YF virus infection, and to determine mechanisms of cross-protection. We utilized African and Brazilian YF strains and found that DENV-2 and ZIKV immunity significantly suppresses YFV viremia in mice, but may or may not protect relative to disease outcomes. Cross-protection appears to be mediated mainly by humoral immune responses. These studies underscore the importance of re-assessing the risks associated with YF outbreak while accounting for prior immunity from flaviviruses that are endemic.

Antibodies to Aedes aegypti D7L salivary proteins as a new serological tool to estimate human exposure to Aedes mosquitoes.

Introduction: Aedes spp. are the most prolific mosquito vectors in the world. Found on every continent, they can effectively transmit various arboviruses, including the dengue virus which continues to cause outbreaks worldwide and is spreading into previously non-endemic areas. The lack of widely available dengue vaccines accentuates the importance of targeted vector control strategies to reduce the dengue burden. High-throughput tools to estimate human-mosquito contact and evaluate vector control interventions are lacking. We propose a novel serological tool that allows rapid screening of human cohorts for exposure to potentially infectious mosquitoes. Methods: We tested 563 serum samples from a longitudinal pediatric cohort study previously conducted in Cambodia. Children enrolled in the study were dengue-naive at baseline and were followed biannually for dengue incidence for two years. We used Western blotting and enzyme-linked immunosorbent assays to identify immunogenic Aedes aegypti salivary proteins and measure total anti-Ae. aegypti IgG. Results: We found a correlation (rs=0.86) between IgG responses against AeD7L1 and AeD7L2 recombinant proteins and those to whole salivary gland homogenate. We observed seasonal fluctuations of AeD7L1+2 IgG responses and no cross-reactivity with Culex quinquefasciatus and Anopheles dirus mosquitoes. The baseline median AeD7L1+2 IgG responses for young children were higher in those who developed asymptomatic versus symptomatic dengue. Discussion: The IgG response against AeD7L1+2 recombinant proteins is a highly sensitive and Aedes specific marker of human exposure to Aedes bites that can facilitate standardization of future serosurveys and epidemiological studies by its ability to provide a robust estimation of human-mosquito contact in a high-throughput fashion.

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.

An Aedes-Anopheles Vaccine Candidate Supplemented with BCG Epitopes Against the Aedes and Anopheles Genera to Overcome Hypersensitivity to Mosquito Bites.

BACKGROUND: Skeeter syndrome is a severe local allergic response to mosquito bites that is accompanied by considerable inflammation and, in some cases, a systemic response like fever. People with the syndrome develop serious allergies, ranging from rashes to anaphylaxis or shock. The few available studies on mosquito venom immunotherapy have utilized whole-body preparations and small sample sizes. Still, owing to their little success, vaccination remains a promising alternative as well as a permanent solution for infections like Skeeter's. METHODS: This study, therefore, illustrated the construction of an epitope-based vaccine candidate against Skeeter Syndrome using established immunoinformatic techniques. We selected three species of mosquitoes, Anopheles melas, Anopheles funestus, and Aedes aegypti, to derive salivary antigens usually found in mosquito bites. Our construct was also supplemented with bacterial epitopes known to elicit a strong TH1 response and suppress TH2 stimulation that is predicted to reduce hypersensitivity against the bites. RESULTS: A quality factor of 98.9496, instability index of 38.55, aliphatic index of 79.42, solubility of 0.934747, and GRAVY score of -0.02 indicated the structural (tertiary and secondary) stability, thermostability, solubility, and hydrophilicity of the construct, respectively. The designed Aedes-Anopheles vaccine (AAV) candidate was predicted to be flexible and less prone to deformability with an eigenvalue of 1.5911e-9 and perfected the human immune response against Skeeter (hypersensitivity) and many mosquito-associated diseases as we noted the production of 30,000 Th1 cells per mm3 with little (insignificant production of Th2 cells. The designed vaccine also revealed stable interactions with the pattern recognition receptors of the host. The TLR2/vaccine complex interacted with a free energy of - 1069.2 kcal/mol with 26 interactions, whereas the NLRP3/vaccine complex interacted with a free energy of - 1081.2 kcal/mol with 16 molecular interactions. CONCLUSION: Although being a pure in-silico study, the in-depth analysis performed herein speaks volumes of the potency of the designed vaccine candidate predicting that the proposition can withstand rigorous in-vitro and in-vivo clinical trials and may proceed to become the first preventative immunotherapy against mosquito bite allergy.

OTU7B Modulates the Mosquito Immune Response to Beauveria bassiana Infection via Deubiquitination of the Toll Adaptor TRAF4.

The Aedes aegypti mosquito transmits devastating flaviviruses, such as Zika, dengue, and yellow fever viruses. For more effective control of the vector, the pathogenicity of Beauveria bassiana, a fungus commonly used for biological control of pest insects, may be enhanced based on in-depth knowledge of molecular interactions between the pathogen and its host. Here, we identified a mechanism employed by B. bassiana, which efficiently blocks the Ae. aegypti antifungal immune response by a protease that contains an ovarian tumor (OTU) domain. RNA-sequencing analysis showed that the depletion of OTU7B significantly upregulates the mRNA level of immunity-related genes after a challenge of the fungus. CRISPR-Cas9 knockout of OTU7B conferred a higher resistance of mosquitoes to the fungus B. bassiana. OTU7B suppressed activation of the immune response by preventing nuclear translocation of the NF-κB transcription factor Rel1, a mosquito orthologue of Drosophila Dorsal. Further studies identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as an interacting protein of OTU7B. TRAF4-deficient mosquitoes were more sensitive to fungal infection, indicating TRAF4 to be the adaptor protein that activates the Toll pathway. TRAF4 is K63-link polyubiquitinated at K338 residue upon immune challenge. However, OTU7B inhibited the immune signaling by enzymatically removing the polyubiquitin chains of mosquito TRAF4. Thus, this study has uncovered a novel mechanism of fungal action against the host innate immunity, providing a platform for further improvement of fungal pathogen effectiveness. IMPORTANCE Insects use innate immunity to defend against microbial infection. The Toll pathway is a major immune signaling pathway that is associated with the antifungal immune response in mosquitoes. Our study identified a fungal-induced deubiquitinase, OTU7B, which, when knocked out, promotes the translocation of the NF-κB factor Rel1 into the nucleus and confers enhanced resistance to fungal infection. We further found the counterpart of OTU7B, TRAF4, which is a component of the Toll pathway and acts as an adaptor protein. OTU7B enzymatically removes K63-linked polyubiquitin chains from TRAF4. The immune response is suppressed, and mosquitoes become much more sensitive to the Beauveria bassiana infection. Our findings reveal a novel mechanism of fungal action against the host innate immunity.

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.

Dengue NS1 antigen kit shows high sensitivity for detection of recombinant dengue virus-2 NS1 antigen spiked with Aedes aegypti mosquitoes.

Dengue, caused by the dengue virus (DENV) is a significant vector-borne disease. In absence of a specific treatment and vaccine, dengue is becoming a rising threat to public health. Currently, control of dengue mainly focuses on the surveillance of the mosquito vectors. Improved surveillance methods for DENV in mosquito populations would be highly beneficial to the public health. However, current methods of DENV detection in mosquitoes requires specialized equipment and expensive reagents and highly trained personnel. As an alternative, commercially available dengue NS1 antigen ELISA kits could be used for detection of DENV infection in Aedes aegypti mosquitoes. In this study, we explored the utility of commercially available Dengue NS1 antigen kit (J. Mitra & Co. Pvt. Ltd) for the detection of recombinant dengue virus-2 (rDENV-2) NS1 protein and serum of dengue infected patient spiked with Ae. aegypti mosquito pools. The kit was found to be highly sensitive and specific towards detection of all serotypes of DENV. Further, it could detect as low as 750 femto gram rDENV-2 NS1 protein. It was also observed that rDENV-2 NS1 antigen spiked with blood-fed and unfed mosquito pools could be detected. In addition, the kit also detected dengue infected patient serum spiked with Ae. aegypti mosquito pools. Overall, the Dengue NS1 antigen kit displayed high sensitivity towards detection of recombinant as well as serum NS1 protein spiked with Ae. aegypti mosquito pools and could be considered for the dengue virus surveillance after a field evaluation in Ae. aegypti mosquitoes.

Impacts of fungal entomopathogens on survival and immune responses of Aedes albopictus and Culex pipiens mosquitoes in the context of native Wolbachia infections.

Microbial control of mosquitoes via the use of symbiotic or pathogenic microbes, such as Wolbachia and entomopathogenic fungi, are promising alternatives to synthetic insecticides to tackle the rapid increase in insecticide resistance and vector-borne disease outbreaks. This study evaluated the susceptibility and host responses of two important mosquito vectors, Ae. albopictus and Cx. pipiens, that naturally carry Wolbachia, to infections by entomopathogenic fungi. Our study indicated that while Wolbachia presence did not provide a protective advantage against entomopathogenic fungal infection, it nevertheless influenced the bacterial / fungal load and the expression of select anti-microbial effectors and phenoloxidase cascade genes in mosquitoes. Furthermore, although host responses from Ae. albopictus and Cx. pipiens were mostly similar, we observed contrasting phenotypes with regards to susceptibility and immune responses to fungal entomopathogenic infection in these two mosquitoes. This study provides new insights into the intricate multipartite interaction between the mosquito host, its native symbiont and pathogenic microbes that might be employed to control mosquito populations.

Functional characterization of two clip domain serine proteases in innate immune responses of Aedes aegypti.

BACKGROUND: Clip domain serine proteases (CLIPs), a very diverse group of proteolytic enzymes, play a crucial role in the innate immunity of insects. Innate immune responses are the first line of defense in mosquitoes against the invasion of pathogenic microorganisms. The Toll pathway, immunodeficiency (IMD) pathway and melanization are the main processes of innate immunity in Aedes aegypti. CLIPS are classified into five subfamilies-CLIPA, CLIPB, CLIPC, CLIPD, and CLIPE-based on their sequence specificity and phylogenetic relationships. We report the functional characterization of the genes that code for two CLIPs in Ae. aegypti (Ae): Ae-CLIPB15 and Ae-CLIPB22. METHODS: Clustal Omega was used for multiple amino acid sequence alignment of Ae-CLIPB15 and Ae-CLIPB22 with different CLIP genes from other insect species. The spatiotemporal expression profiles of Ae-CLIPB15 and Ae-CLIPB22 were examined. We determined whether Ae-CLIPB15 and Ae-CLIPB22 respond to microbial challenge and tissue injury. RNA interference (RNAi) was used to explore the function of Ae-CLIPB15 and Ae-CLIPB22 in the defense of Ae. aegypti against bacterial and fungal infections. The expression levels of nuclear factor kappa B (NF-κB) transcription factors REL1 and REL2 in the Toll pathway and IMD pathway after bacterial infection were investigated. Finally, the change in phenoloxidase (PO) activity in Ae-CLIPB15 and Ae-CLIPB22 knockdown adults was investigated. RESULTS: We performed spatiotemporal gene expression profiling of Ae-CLIPB15 and Ae-CLIPB22 genes in Ae. aegypti using quantitative real-time polymerase chain reaction. These genes were expressed in different stages and tissues. The messenger RNA (mRNA) levels for both genes were also up-regulated by Gram-negative bacteria Escherichia coli, Gram-positive bacteria Staphylococcus aureus and fungal Beauveria bassiana infections, as well as in the tissue injury experiments. RNAi-mediated knockdown of Ae-CLIPB15 led to a significant decrease of PO activity in the hemolymph of Ae. aegypti, while other RNAi experiments revealed that both Ae-CLIPB15 and Ae-CLIPB22 were involved in immune defense against bacterial and fungal infections. The mRNA expression of NF-κB transcription factors REL1 and REL2 in the Toll pathway and IMD pathway differed between Ae-CLIPB15 and Ae-CLIPB22 knockdown mosquitoes infected with bacteria and wild type mosquitoes infected with bacteria. CONCLUSIONS: Our findings suggest that Ae-CLIPB15 and Ae-CLIPB22 play a critical role in mosquito innate immunity, and that they are involved in immune responses to injury and infection. Their regulation of transcription factors and PO activity indicates that they also play a specific role in the regulation of innate immunity.

Aedes fluviatilis cell lines as new tools to study metabolic and immune interactions in mosquito-Wolbachia symbiosis.

In the present work, we established two novel embryonic cell lines from the mosquito Aedes fluviatilis containing or not the naturally occurring symbiont bacteria Wolbachia, which were called wAflu1 and Aflu2, respectively. We also obtained wAflu1 without Wolbachia after tetracycline treatment, named wAflu1.tet. Morphofunctional characterization was performed to help elucidate the symbiont-host interaction in the context of energy metabolism regulation and molecular mechanisms of the immune responses involved. The presence of Wolbachia pipientis improves energy performance in A. fluviatilis cells; it affects the regulation of key energy sources such as lipids, proteins, and carbohydrates, making the distribution of actin more peripheral and with extensions that come into contact with neighboring cells. Additionally, innate immunity mechanisms were activated, showing that the wAflu1 and wAflu1.tet cells are responsive after the stimulus using Gram negative bacteria. Therefore, this work confirms the natural, mutually co-regulating symbiotic relationship between W. pipientis and A. fluviatilis, modulating the host metabolism and immune pathway activation. The results presented here add important resources to the current knowledge of Wolbachia-arthropod interactions.

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.

AeMOPE-1, a Novel Salivary Peptide From Aedes aegypti, Selectively Modulates Activation of Murine Macrophages and Ameliorates Experimental Colitis.

The sialotranscriptomes of Aedes aegypti revealed a transcript overexpressed in female salivary glands that codes a mature 7.8 kDa peptide. The peptide, specific to the Aedes genus, has a unique sequence, presents a putative secretory nature and its function is unknown. Here, we confirmed that the peptide is highly expressed in the salivary glands of female mosquitoes when compared to the salivary glands of males, and its secretion in mosquito saliva is able to sensitize the vertebrate host by inducing the production of specific antibodies. The synthetic version of the peptide downmodulated nitric oxide production by activated peritoneal murine macrophages. The fractionation of a Ae. aegypti salivary preparation revealed that the fractions containing the naturally secreted peptide reproduced the nitric oxide downmodulation. The synthetic peptide also selectively interfered with cytokine production by murine macrophages, inhibiting the production of IL-6, IL-12p40 and CCL2 without affecting TNF-α or IL-10 production. Likewise, intracellular proteins associated with macrophage activation were also distinctively modulated: while iNOS and NF-κB p65 expression were diminished, IκBα and p38 MAPK expression did not change in the presence of the peptide. The anti-inflammatory properties of the synthetic peptide were tested in vivo on a dextran sulfate sodium-induced colitis model. The therapeutic administration of the Ae. aegypti peptide reduced the leukocytosis, macrophage activity and nitric oxide levels in the gut, as well as the expression of cytokines associated with the disease, resulting in amelioration of its clinical signs. Given its biological properties in vitro and in vivo, the molecule was termed Aedes-specific MOdulatory PEptide (AeMOPE-1). Thus, AeMOPE-1 is a novel mosquito-derived immunobiologic with potential to treat immune-mediated disorders.

Evidence of Adaptive Evolution in Wolbachia-Regulated Gene DNMT2 and Its Role in the Dipteran Immune Response and Pathogen Blocking.

Eukaryotic nucleic acid methyltransferase (MTase) proteins are essential mediators of epigenetic and epitranscriptomic regulation. DNMT2 belongs to a large, conserved family of DNA MTases found in many organisms, including holometabolous insects such as fruit flies and mosquitoes, where it is the lone MTase. Interestingly, despite its nomenclature, DNMT2 is not a DNA MTase, but instead targets and methylates RNA species. A growing body of literature suggests that DNMT2 mediates the host immune response against a wide range of pathogens, including RNA viruses. Curiously, although DNMT2 is antiviral in Drosophila, its expression promotes virus replication in mosquito species. We, therefore, sought to understand the divergent regulation, function, and evolution of these orthologs. We describe the role of the Drosophila-specific host protein IPOD in regulating the expression and function of fruit fly DNMT2. Heterologous expression of these orthologs suggests that DNMT2's role as an antiviral is host-dependent, indicating a requirement for additional host-specific factors. Finally, we identify and describe potential evidence of positive selection at different times throughout DNMT2 evolution within dipteran insects. We identify specific codons within each ortholog that are under positive selection and find that they are restricted to four distinct protein domains, which likely influence substrate binding, target recognition, and adaptation of unique intermolecular interactions. Collectively, our findings highlight the evolution of DNMT2 in Dipteran insects and point to structural, regulatory, and functional differences between mosquito and fruit fly homologs.

Type I hypersensitivity promotes Aedes aegypti blood feeding.

Mosquitoes play a major role in human disease by serving as vectors of pathogenic microorganisms. Mosquitoes inject saliva into host skin during the probing process. Mosquito saliva contains a number of proteins that facilitate blood feeding by preventing hemostasis. Mosquito saliva also contains potent allergens that induce type I hypersensitivity reactions in some individuals. Type I hypersensitivity reactions in skin involve IgE-mediated degranulation of mast cells, which leads to vasodilation and an itch sensation. We hypothesized that hypersensitivity to mosquito saliva influences blood feeding. To test this hypothesis, we recruited human subjects who consented to Aedes aegypti bites. We measured their first sensation of itch, the strength of their itch sensation, the number of times mosquitoes attempted to feed, the number of times mosquitoes probed their skin, feeding time, engorgement status, and wheal diameter. Here we show that hypersensitive subjects had a stronger itch sensation, and that the time to first itch sensation was inversely correlated with wheal diameter; however, mosquitoes tended to probe less and engorge more on these subjects. Follow-up experiments testing the impact of oral antihistamine treatment on mosquito feeding parameters failed to reveal a statistically significant result. Histamine also failed to promote blood feeding on an artificial membrane feeder. This study suggests that mosquito saliva-induced type I hypersensitivity promotes blood feeding but that this may be independent from histamine or histamine signaling.

Sex, age, and parental harmonic convergence behavior affect the immune performance of Aedes aegypti offspring.

Harmonic convergence is a potential cue, female mosquitoes use to choose male mates. However, very little is known about the benefits this choice confers to offspring performance. Using Aedes aegypti (an important vector of human disease), we investigated whether offspring of converging parental pairs showed differences in immune competence compared to offspring derived from non-converging parental pairs. Here we show that harmonic convergence, along with several other interacting factors (sex, age, reproductive, and physiological status), significantly shaped offspring immune responses (melanization and response to a bacterial challenge). Harmonic convergence had a stronger effect on the immune response of male offspring than on female offspring. Further, female offspring from converging parental pairs disseminated dengue virus more quickly than offspring derived from non-converging parental pairs. Our results provide insight into a wide range of selective pressures shaping mosquito immune function and could have important implications for disease transmission and control.

A Thioester-Containing Protein Controls Dengue Virus Infection in Aedes aegypti Through Modulating Immune Response.

Complement-like proteins in arthropods defend against invading pathogens in the early phases of infection. Thioester-containing proteins (TEPs), which exhibit high similarity to mammalian complement C3, are thought to play a key role in the innate immunity of arthropods. We identified and characterized anti-dengue virus (DENV) host factors, in particular complement-like proteins, in the mosquito Aedes aegypti. Our results indicate that TEP1 limits DENV infection in Ae. aegypti. We showed that TEP1 transcription is highly induced in mosquitoes following DENV infection. Silencing TEP1 resulted in the up-regulation of viral RNA and proteins. In addition, the production of infectious virus particles increased in the absence of TEP1. We generated a transgenic mosquito line with a TEP1 loss-of-function phenotype under a blood meal-inducible promoter. We showed that viral protein and titers increased in transgenic mosquitoes after an infectious blood meal. Interestingly, expression of transcription factor Rel2 and certain anti-microbial peptides (AMPs) were inhibited in transgenic mosquitoes. Overall, our results suggest that TEP1 regulates the immune response and consequently controls the replication of dengue virus in mosquitoes. This finding provides new insight into the molecular mechanisms of mosquito host factors in the regulation of DENV replication.

Aedes aegypti Toll pathway is induced through dsRNA sensing in endosomes.

Mosquito anti-pathogen immune responses, including those controlling infection with arboviruses are regulated by multiple signal transduction pathways. While the Toll pathway is critical in the defense against arboviruses such as dengue and Zika viruses, the factors and mechanisms involved in virus recognition leading to the activation of the Toll pathway are not fully understood. In this study we evaluated the role of virus-produced double-stranded RNA (dsRNA) intermediates in mosquito immune activation by utilizing the synthetic dsRNA analog polyinosinic-polycytidylic acid (poly I:C). Poly I:C treatment of Aedes aegypti mosquitoes and Aag2 cells reduced DENV infection. Transcriptomic analyses of Aag2 cell responses to poly I:C indicated putative activation of the Toll pathway. We found that poly I:C is translocated to the endosomal compartment of Aag2 cells, and that the A. aegypti Toll 6 receptor is a putative dsRNA recognition receptor. This study elucidates the role of dsRNAs in the immune activation of non-RNAi pathways in mosquitoes.

Transgenic Expression of Human C-Type Lectin Protein CLEC18A Reduces Dengue Virus Type 2 Infectivity in Aedes aegypti.

The C-type lectins, one family of lectins featuring carbohydrate binding domains which participate in a variety of bioprocesses in both humans and mosquitoes, including immune response, are known to target DENV. A human C-type lectin protein CLEC18A in particular shows extensive glycan binding abilities and correlates with type-I interferon expression, making CLEC18A a potential player in innate immune responses to DENV infection; this potential may provide additional regulatory point in improving mosquito immunity. Here, we established for the first time a transgenic Aedes aegypti line that expresses human CLEC18A. This expression enhanced the Toll immune pathway responses to DENV infection. Furthermore, viral genome and virus titers were reduced by 70% in the midgut of transgenic mosquitoes. We found significant changes in the composition of the midgut microbiome in CLEC18A expressing mosquitoes, which may result from the Toll pathway enhancement and contribute to DENV inhibition. Transgenic mosquito lines offer a compelling option for studying DENV pathogenesis, and our analyses indicate that modifying the mosquito immune system via expression of a human immune gene can significantly reduce DENV infection.