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During the recent yellow fever (YF) epidemics in Brazil, human cases were attributed to spillover infections via sylvatic mosquito transmission. Despite YF virus (YFV) transmission in major urban centers with insufficient vaccination coverage and abundant populations of the domestic vector, Aedes aegypti, there was no evidence of human-amplified transmission. Furthermore, the historic absence of YF in Asia, despite abundant Ae. aegypti and an immunologically naive human population, is unexplained. We tested the hypothesis that pre-existing, heterologous flavivirus immunity, specifically from dengue (DENV) and Zika (ZIKV) viruses, limits YFV viremia and transmission by Ae. aegypti. We infected cynomolgus macaques with DENV or ZIKV, then challenged them 6-9 months later with YFV. We then measured viremia and disease and allowed Ae. aegypti mosquitoes to feed during peak macaque viremia. Although prior heterologous immunity had variable effects on disease, DENV and ZIKV immunity consistently suppressed YFV viremia. Despite no statistical difference due to a small sample size, the suppression in viremia led to a significant reduction in Ae. aegypti infection and a lack of transmission potential. These results support the hypothesis that, in DENV- and ZIKV-endemic regions such as South America and Asia, human flavivirus immunity suppresses YFV human amplification potential, reducing the risk of urban outbreaks., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Despite the critical role of the Anopheles innate immune system in defending against Plasmodium infection, there is still limited information about the key immune mechanisms in Anopheles. This review assesses recent findings on the expression characteristics of immune-related genes in Anopheles following exposure to Plasmodium. A literature review, unrestricted by publication date, was conducted to evaluate immune-related gene expression in different organs of Anopheles after Plasmodium infection. Mosquito immune responses in the midgut are essential for reducing parasite populations. Additionally, innate immune responses in the salivary glands and hemocytes circulating in the hemocoel play key roles in defense against the parasite. Transcriptomic analysis of the mosquito's innate immune response to Plasmodium infection provides valuable insights into key immune mechanisms in mosquito defense. A deeper understanding of immune mechanisms in different organs of Anopheles following Plasmodium infection will aid in discovering critical targets for designing novel control strategies., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The World Health Organization recently declared a global initiative to control arboviral diseases. These are mainly caused by pathogenic flaviviruses (such as dengue, yellow fever and Zika viruses) and alphaviruses (such as chikungunya and Venezuelan equine encephalitis viruses). Vaccines represent key interventions for these viruses, with licensed human and/or veterinary vaccines being available for several members of both genera. However, a hurdle for the licensing of new vaccines is the epidemic nature of many arboviruses, which presents logistical challenges for phase III efficacy trials. Furthermore, our ability to predict or measure the post-vaccination immune responses that are sufficient for subclinical outcomes post-infection is limited. Given that arboviruses are also subject to control by the immune system of their insect vectors, several approaches are now emerging that aim to augment antiviral immunity in mosquitoes, including Wolbachia infection, transgenic mosquitoes, insect-specific viruses and paratransgenesis. In this Review, we discuss recent advances, current challenges and future prospects in exploiting both vertebrate and invertebrate immune systems for the control of flaviviral and alphaviral diseases., (© 2024. Springer Nature Limited.)

The Aedes aegypti mosquito is a vector of many infectious agents, including flaviviruses such as Zika virus. Components of mosquito saliva have pleomorphic effects on the vertebrate host to enhance blood feeding, and these changes also create a favorable niche for pathogen replication and dissemination. Here, we demonstrate that human CD47, which is known to be involved in various immune processes, interacts with a 34-kilodalton mosquito salivary protein named Nest1. Nest1 is up-regulated in blood-fed female A. aegypti and facilitates Zika virus dissemination in human skin explants. Nest1 has a stronger affinity for CD47 than its natural ligand, signal regulatory protein α, competing for binding at the same interface. The interaction between Nest1 with CD47 suppresses phagocytosis by human macrophages and inhibits proinflammatory responses by white blood cells, thereby suppressing antiviral responses in the skin. This interaction elucidates how an arthropod protein alters the human response to promote arbovirus infectivity.

The Dengue virus (DENV), primarily spread by Aedes aegypti and also by Aedes albopictus in some regions, poses significant global health risks. Alternative techniques are urgently needed because the current control mechanisms are insufficient to reduce the transmission of DENV. Introducing Wolbachia pipientis into Ae. aegypti inhibits DENV transmission, however, the underlying mechanisms are still poorly understood. Innate immune effector upregulation, the regulation of autophagy, and intracellular competition between Wolbachia and DENV for lipids are among the theories for the mechanism of inhibition. Furthermore, mainly three immune pathways Toll, IMD, and JAK/STAT are involved in the host for the suppression of the virus. These pathways are activated by Wolbachia and DENV in the host and are responsible for the upregulation and downregulation of many genes in mosquitoes, which ultimately reduces the titer of the DENV in the host. The functioning of these immune pathways depends upon the Wolbachia , host, and virus interaction. Here, we summarize the current understanding of DENV recognition by the Ae. aegypti 's immune system, aiming to create a comprehensive picture of our knowledge. Additionally, we investigated how Wolbachia regulates the activation of multiple genes associated with immune priming for the reduction of DENV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mushtaq, Sarwar and Munzoor.)

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae . Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei . In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zeineddine, Jaber, Saab, Nakhleh, Dimopoulos and Osta.)

The applicability of the specific human IgG antibody response to Anopheles gambiae salivary Gland Protein-6 peptide 1 (gSG6-P1 salivary peptide) as a biomarker able to distinguish the level of exposure to mosquito bites according to seasonal variations has not yet been evaluated in Central African regions. The study aimed to provide the first reliable data on the IgG anti-gSG6-P1 response in rural area in Cameroon according to the dry- and rainy-season. Between May and December 2020, dry blood samples were collected from people living in the Bankeng village in the forest area of the Centre region of Cameroon. Malaria infection was determined by thick-blood smear microscopy and multiplex PCR. The level of IgG anti-gSG6-P1 response, was assessed by enzyme-linked immunosorbent assay. Anopheles density and aggressiveness were assessed using human landing catches. The prevalence of malaria infection remains significantly higher in the rainy season than in the dry season (77.57% vs 61.44%; p = 0.0001). The specific anti-gSG6-P1 IgG response could be detected in individuals exposed to few mosquito bites and showed inter-individual heterogeneity even when living in the same exposure area. In both seasons, the level of anti-gSG6-P1 IgG response was not significantly different between Plasmodium infected and non-infected individuals. Mosquito bites were more aggressive in the rainy season compared to the dry season (human biting rate-HBR of 15.05 b/p/n vs 1.5 b/p/n) where mosquito density was very low. Infected mosquitoes were found only during the rainy season (sporozoite rate = 10.63% and entomological inoculation rate-EIR = 1.42 ib/p/n). The level of IgG anti-gSG6-P1 response was significantly higher in the rainy season and correlated with HBR (p ˂ 0.0001). This study highlights the high heterogeneity of individual's exposure to the Anopheles gambiae s.l vector bites depending on the transmission season in the same area. These findings reinforce the usefulness of the anti-gSG6-P1 IgG response as an accurate immunological biomarker for detecting individual exposure to Anopheles gambiae s.l. bites during the low risk period of malaria transmission in rural areas and for the differentiating the level of exposure to mosquitoes., (© 2024. The Author(s).)

Resistance to clinical malaria takes years to develop even in hyperendemic regions and sterilizing immunity has rarely been observed. To evaluate the maturation of the host response against controlled repeat exposures to P. falciparum (Pf) NF54 strain-infected mosquitoes, we systematically monitored malaria-naïve participants through an initial exposure to uninfected mosquitoes and 4 subsequent homologous exposures to Pf-infected mosquitoes over 21 months (n = 8 males) (ClinicalTrials.gov# NCT03014258). The primary outcome was to determine whether protective immunity against parasite infection develops following repeat CHMI and the secondary outcomes were to track the clinical signs and symptoms of malaria and anti-Pf antibody development following repeat CHMI. After two exposures, time to blood stage patency increases significantly and the number of reported symptoms decreases indicating the development of clinical tolerance. The time to patency correlates positively with both anti-Pf circumsporozoite protein (CSP) IgG and CD8 + CD69+ effector memory T cell levels consistent with partial pre-erythrocytic immunity. IFNγ levels decrease significantly during the participants' second exposure to high blood stage parasitemia and could contribute to the decrease in symptoms. In contrast, CD4-CD8 + T cells expressing CXCR5 and the inhibitory receptor, PD-1, increase significantly after subsequent Pf exposures, possibly dampening the memory response and interfering with the generation of robust sterilizing immunity., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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., Competing Interests: Z.X. is affiliated with Guangzhou Wolbaki Biotech Co., Ltd. The other authors have declared that no competing interests exist., (Copyright: © 2024 She et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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/β 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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Chea, Willen, Nhek, Ly, Tang, Oristian, Salas-Carrillo, Ponce, Leon, Kong, Ly, Sath, Lon, Leang, Huy, Yek, Valenzuela, Calvo, Manning and Oliveira.)

Wolbachia, a maternally transmitted symbiotic bacterium of insects, can suppress a variety of human pathogens in mosquitoes, including malaria-causing Plasmodium in the Anopheles vector. However, the mechanistic basis of Wolbachia-mediated Plasmodium suppression in mosquitoes is not well understood. In this study, we compared the midgut and carcass transcriptomes of stably infected Anopheles stephensi with Wolbachia wAlbB to uninfected mosquitoes in order to discover Wolbachia infection-responsive immune genes that may play a role in Wolbachia-mediated anti-Plasmodium activity. We show that wAlbB infection upregulates 10 putative immune genes and downregulates 14 in midguts, while it upregulates 31 putative immune genes and downregulates 15 in carcasses at 24 h after blood-fed feeding, the time at which the Plasmodium ookinetes are traversing the midgut tissue. Only a few of these regulated immune genes were also significantly differentially expressed between Wolbachia-infected and non-infected midguts and carcasses of sugar-fed mosquitoes. Silencing of the Wolbachia infection-responsive immune genes TEP 4, TEP 15, lysozyme C2, CLIPB2, CLIPB4, PGRP-LD and two novel genes (a peritrophin-44-like gene and a macro domain-encoding gene) resulted in a significantly greater permissiveness to P. falciparum infection. These results indicate that Wolbachia infection modulates mosquito immunity and other processes that are likely to decrease Anopheles permissiveness to Plasmodium infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Vandana et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Malaria continues to remain a major global health problem with nearly a quarter of a billion clinical cases and more than 600,000 deaths in 2022. There has been significant progress toward vaccine development, however, poor efficacy of approved vaccines requiring multiple immunizing doses emphasizes the need for continued efforts toward improved vaccines. Progress to date, nonetheless, has provided impetus for malaria elimination., Areas Covered: In this review we will focus on diverse immune mechanisms targeting gametocytes in the human host and gametocyte-mediated malaria transmission via the mosquito vector., Expert Opinion: To march toward the goal of malaria elimination it will be critical to target the process of malaria transmission by mosquitoes, mediated exclusively by the sexual stages, i.e. male, and female gametocytes, ingested from infected vertebrate host. Studies over several decades have established antigens in the parasite sexual stages developing in the mosquito midgut as attractive targets for the development of transmission blocking vaccines (TBVs). Immune clearance of gametocytes in the vertebrate host can synergize with TBVs and directly aid in maintaining effective transmission reducing immune potential.

Background: Entomological surveillance for malaria is inherently resource-intensive and produces crude population-level measures of vector exposure which are insensitive in low-transmission settings. Antibodies against Anopheles salivary proteins measured at the individual level may serve as proxy biomarkers for vector exposure and malaria transmission, but their relationship is yet to be quantified., Methods: A systematic review of studies measuring antibodies against Anopheles salivary antigens (PROSPERO: CRD42020185449). Multilevel modelling (to account for multiple study-specific observations [level 1], nested within study [level 2], and study nested within country [level 3]) estimated associations between seroprevalence with Anopheles human biting rate (HBR) and malaria transmission measures., Results: From 3981 studies identified in literature searches, 42 studies across 16 countries were included contributing 393 study-specific observations of anti- Anopheles salivary antibodies determined in 42,764 samples. A positive association between HBR (log transformed) and seroprevalence was found; overall a twofold (100% relative) increase in HBR was associated with a 23% increase in odds of seropositivity (OR: 1.23, 95% CI: 1.10-1.37; p<0.001). The association between HBR and Anopheles salivary antibodies was strongest with concordant, rather than discordant, Anopheles species. Seroprevalence was also significantly positively associated with established epidemiological measures of malaria transmission: entomological inoculation rate, Plasmodium spp. prevalence, and malarial endemicity class., Conclusions: Anopheles salivary antibody biomarkers can serve as a proxy measure for HBR and malaria transmission, and could monitor malaria receptivity of a population to sustain malaria transmission. Validation of Anopheles species-specific biomarkers is important given the global heterogeneity in the distribution of Anopheles species. Salivary biomarkers have the potential to transform surveillance by replacing impractical, inaccurate entomological investigations, especially in areas progressing towards malaria elimination., Funding: Australian National Health and Medical Research Council, Wellcome Trust., Competing Interests: EK, PA, VC, JC, JS, FF No competing interests declared, (© 2021, Kearney et al.)

Malaria is caused when Plasmodium sporozoites are injected along with saliva by an anopheline mosquito into the dermis of a vertebrate host. Arthropod saliva has pleiotropic effects that can influence local host responses, pathogen transmission, and exacerbation of the disease. A mass spectrometry screen identified mosquito salivary proteins that are associated with Plasmodium sporozoites during saliva secretions. In this study, we demonstrate that one of these salivary antigens, Anopheles gambiae sporozoite-associated protein (AgSAP), interacts directly with Plasmodium falciparum and Plasmodium berghei sporozoites. AgSAP binds to heparan sulfate and inhibits local inflammatory responses in the skin. The silencing of AgSAP in mosquitoes reduces their ability to effectively transmit sporozoites to mice. Moreover, immunization with AgSAP decreases the Plasmodium burden in mice that are bitten by Plasmodium -infected mosquitoes. These data suggest that AgSAP facilitates early Plasmodium infection in the vertebrate host and serves as a target for the prevention of malaria. IMPORTANCE Malaria is a vector-borne disease caused by Plasmodium sporozoites. When an anopheline mosquito bites its host, it releases Plasmodium sporozoites as well as saliva components. Mosquito proteins have the potential to serve as antigens to prevent or influence malaria without directly targeting the pathogen. This may help set a new paradigm for vaccine development. In this study, we have elucidated the role of a novel salivary antigen, named Anopheles gambiae sporozoite-associated protein (AgSAP). The results presented here show that AgSAP interacts with Plasmodium falciparum and Plasmodium berghei sporozoites and modulates local inflammatory responses in the skin. Furthermore, our results show that AgSAP is a novel mosquito salivary antigen that influences the early stages of Plasmodium infection in the vertebrate host. Individuals living in countries where malaria is endemic generate antibodies against AgSAP, which indicates that AgSAP can serve as a biomarker for disease prevalence and epidemiological analysis.

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., (© 2021. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist.

Background: A number of zoonotic mosquito-borne viruses have emerged in Europe in recent decades. Batai virus (BATV), a member of the genus Orthobunyavirus, is one example of a relatively newly emerged mosquito-borne virus, having been detected in mosquitoes and livestock. We conducted vector competency studies on three mosquito species at a low temperature to assess whether Aedes and Culex mosquito species are susceptible to infection with BATV., Methods: Colonised lines of Aedes aegypti and Culex pipiens and a wild-caught species, Aedes detritus, were orally inoculated with BATV strain 53.2, originally isolated from mosquitoes trapped in Germany in 2009. Groups of blood-fed female mosquitoes were maintained at 20 °C for 7 or 14 days. Individual mosquitoes were screened for the presence of BATV in body, leg and saliva samples for evidence of infection, dissemination and transmission, respectively. BATV RNA was detected by reverse transcription-PCR, and positive results confirmed by virus isolation in Vero cells., Results: Aedes detritus was highly susceptible to BATV, with an infection prevalence of ≥ 80% at both measurement time points. Disseminated infections were recorded in 30.7-41.6% of Ae. detritus, and evidence of virus transmission with BATV in saliva samples (n = 1, days post-infection: 14) was observed. Relatively lower rates of infection for Ae. aegypti and Cx. pipiens were observed, with no evidence of virus dissemination or transmission at either time point., Conclusions: This study shows that Ae. detritus may be a competent vector for BATV at 20 °C, whereas Ae. aegypti and Cx. pipiens were not competent. Critically, the extrinsic incubation period appears to be ≤  7 days for Ae. detritus, which may increase the onward transmissibility potential of BATV in these populations., (© 2021. The Author(s).)

Mosquito-borne viruses of the Flavivirus genus ( Flaviviridae family) pose an ongoing threat to global public health. For example, dengue, Japanese encephalitis, West Nile, yellow fever, and Zika viruses are transmitted by infected mosquitoes and cause severe and fatal diseases in humans. The means by which mosquito-borne flaviviruses establish persistent infection in mosquitoes and cause disease in humans are complex and depend upon a myriad of virus-host interactions, such as those of the innate immune system, which are the main focus of our review. This review also covers the different strategies utilized by mosquito-borne flaviviruses to antagonize the innate immune response in humans and mosquitoes. Given the lack of antiviral therapeutics for mosquito-borne flaviviruses, improving our understanding of these virus-immune interactions could lead to new antiviral therapies and strategies for developing refractory vectors incapable of transmitting these viruses, and can also provide insights into determinants of viral tropism that influence virus emergence into new species.

Lipid-derived signaling molecules known as eicosanoids have integral roles in mediating immune and inflammatory processes across metazoans. This includes the function of prostaglandins and their cognate G protein-coupled receptors (GPCRs) to employ their immunological actions. In insects, prostaglandins have been implicated in the regulation of both cellular and humoral immune responses, yet in arthropods of medical importance, studies have been limited. Here, we describe a prostaglandin E2 receptor ( Ag PGE2R) in the mosquito Anopheles gambiae and demonstrate that its expression is most abundant in oenocytoid immune cell populations. Through the administration of prostaglandin E2 (PGE2) and AgPGE2R- silencing, we demonstrate that prostaglandin E2 signaling regulates a subset of prophenoloxidases (PPOs) and antimicrobial peptides (AMPs) that are strongly expressed in populations of oenocytoids. We demonstrate that PGE2 signaling via the Ag PGE2R significantly limits both bacterial replication and Plasmodium oocyst survival. Additional experiments establish that PGE2 treatment increases phenoloxidase (PO) activity through the increased expression of PPO1 and PPO3 , genes essential to anti- Plasmodium immune responses that promote oocyst killing. We also provide evidence that the mechanisms of PGE2 signaling are concentration-dependent, where high concentrations of PGE2 promote oenocytoid lysis, negating the protective effects of lower concentrations of PGE2 on anti- Plasmodium immunity. Taken together, our results provide new insights into the role of PGE2 signaling on immune cell function and its contributions to mosquito innate immunity that promote pathogen killing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kwon, Hall and Smith.)

Mosquito immune cells, known as hemocytes, are integral to cellular and humoral responses that limit pathogen survival and mediate immune priming. However, without reliable cell markers and genetic tools, studies of mosquito immune cells have been limited to morphological observations, leaving several aspects of their biology uncharacterized. Here, we use single-cell RNA sequencing (scRNA-seq) to characterize mosquito immune cells, demonstrating an increased complexity to previously defined prohemocyte, oenocytoid, and granulocyte subtypes. Through functional assays relying on phagocytosis, phagocyte depletion, and RNA-FISH experiments, we define markers to accurately distinguish immune cell subtypes and provide evidence for immune cell maturation and differentiation. In addition, gene-silencing experiments demonstrate the importance of lozenge in defining the mosquito oenocytoid cell fate. Together, our scRNA-seq analysis provides an important foundation for future studies of mosquito immune cell biology and a valuable resource for comparative invertebrate immunology., Competing Interests: HK, MM, OF, JA, RS No competing interests declared, (© 2021, Kwon et al.)

The genetic basis of antiviral immunity in dipteran insects is extensively studied in Drosophila melanogaster and advanced technologies for genetic manipulation allow a better characterization of immune responses also in non-model insect species. Especially, immunity in vector mosquitoes is recently in the spotlight, due to the medical impact that these insects have by transmitting viruses and other pathogens. Here, we review the current state of experimental evidence that supports antiviral functions for immune genes acting in different cellular pathways. We discuss the well-characterized RNA interference mechanism along with the less well-defined JAK-STAT, Toll, and IMD signaling pathways. Furthermore, we highlight the initial evidence for antiviral activity observed for the autophagy pathway, transcriptional pausing, as well as piRNA production from endogenous viral elements. We focus our review on studies from Drosophila and mosquito species from the lineages Aedes, Culex, and Anopheles, which contain major vector species responsible for virus transmission., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The stalling global progress in the fight against malaria prompts the urgent need to develop new intervention strategies. Whilst engineered symbiotic bacteria have been shown to confer mosquito resistance to parasite infection, a major challenge for field implementation is to address regulatory concerns. Here, we report the identification of a Plasmodium-blocking symbiotic bacterium, Serratia ureilytica Su&#95;YN1, isolated from the midgut of wild Anopheles sinensis in China that inhibits malaria parasites via secretion of an antimalarial lipase. Analysis of Plasmodium vivax epidemic data indicates that local malaria cases in Tengchong (Yunnan province, China) are significantly lower than imported cases and importantly, that the local vector A. sinensis is more resistant to infection by P. vivax than A. sinensis from other regions. Analysis of the gut symbiotic bacteria of mosquitoes from Yunnan province led to the identification of S. ureilytica Su&#95;YN1. This bacterium renders mosquitoes resistant to infection by the human parasite Plasmodium falciparum or the rodent parasite Plasmodium berghei via secretion of a lipase that selectively kills parasites at various stages. Importantly, Su&#95;YN1 rapidly disseminates through mosquito populations by vertical and horizontal transmission, providing a potential tool for blocking malaria transmission in the field.

Mosquito-borne viral infections are responsible for a significant degree of morbidity and mortality across the globe due to the severe diseases these infections cause, and they continue to increase each year. These viruses are dependent on the mosquito vector as the primary means of transmission to new vertebrate hosts including avian, livestock, and human populations. Due to the dynamic host environments that mosquito-borne viruses pass through as they are transmitted between vector and vertebrate hosts, there are various host factors that control the response to infection over the course of the pathogen's life cycle. In this review, we discuss these host factors that are present in either vector or vertebrate models during infection, how they vary or are conserved between hosts, and their implications in future research pertaining to disease prevention and treatment.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cheng, Liu, Tsou, Li, Chien, Su, Liu, Huang, Wu, Tsai, Hsieh and Chen.)

Mosquitoes are vectors of a large number of viral pathogens. In recent years, increased urbanization and climate change has expanded the range of many vector mosquitoes. The lack of effective medical interventions has made the control of mosquito-borne viral diseases very difficult. Understanding the interactions between the mosquito immune system and viruses is critical if we are to develop effective control strategies against these diseases. Mosquitoes harbor multiple conserved immune pathways that curb invading viral pathogens. Despite the conservation of these pathways, the activation and intensity of the mosquito immune response varies with the mosquito species, tissue, and the infecting virus. This article reviews major conserved antiviral immune pathways in vector mosquitoes, their interactions with invading viral pathogens, and how these interactions restrict or promote infection of these medically important viruses., (Copyright © 2020. Published by Elsevier Ltd.)

Repeated blood meals provide essential nutrients for mosquito egg development and routes for pathogen transmission. The target of rapamycin, the TOR pathway, is essential for vitellogenesis. However, its influence on pathogen transmission remains to be elucidated. Here, we show that rapamycin, an inhibitor of the TOR pathway, effectively suppresses Plasmodium berghei infection in Anopheles stephensi. An. stephensi injected with rapamycin or feeding on rapamycin-treated mice showed increased resistance to P. berghei infection. Exposing An. stephensi to a rapamycin-coated surface not only decreased the numbers of both oocysts and sporozoites but also impaired mosquito survival and fecundity. Transcriptome analysis revealed that the inhibitory effect of rapamycin on parasite infection was through the enhanced activation of immune responses, especially the NF-κB transcription factor REL2, a regulator of the immune pathway and complement system. Knockdown of REL2 in rapamycin-treated mosquitoes abrogated the induction of the complement-like proteins TEP1 and SPCLIP1 and abolished rapamycin-mediated refractoriness to Plasmodium infection. Together, these findings demonstrate a key role of the TOR pathway in regulating mosquito immune responses, thereby influencing vector competence., Competing Interests: The authors have declared that no competing interests exist.

Introduction : The development of effective vaccines remains a major health priority to combat the global burden of malaria, a life-threatening disease caused by Plasmodium parasites. Transmission-blocking vaccines (TBVs) elicit antibodies that neutralize the sexual stages of the parasite in blood meals ingested by the Anopheles mosquito, interrupting parasite development in the vector host and preventing disease spread to other individuals. Areas covered : The P. falciparum gametocyte surface antigens Pfs230, Pfs48/45, and Pfs47, the parasite ookinete surface protein Pfs25, and the male gametocyte specific protein PfHAP2 are leading TBV candidates, some of which are in clinical development. The recent expansion of methodology to study monoclonal antibodies isolated directly from humans and animal models, coupled with effective measures for parasite neutralization, has provided unprecedented insight into TBV efficacy and development. Expert opinion : Available structural and functional data on antigen-monoclonal antibody (Ag-mAb) complexes, as well as epitope classification studies, have identified neutralizing epitopes that may aid vaccine development and improve protection. Here, we review the clinical prospects of TBV candidates, progress in the development of novel vaccine strategies for TBVs, and the impact of structural vaccinology in TBV design.

Introduction: Transmission-blocking vaccines (TBV) prevent community spread of malaria by targeting mosquito sexual stage parasites, a life-cycle bottleneck, and will be used in elimination programs. TBV rely on herd immunity to reduce mosquito infections and thereby new infections in both vaccine recipients and non-recipients, but do not provide protection once an individual receives an infectious mosquito bite which complicates clinical development., Areas Covered: Here, we describe the concept and biology behind TBV, and we provide an update on clinical development of the leading vaccine candidate antigens. Search terms 'malaria vaccine,' 'sexual stages,' 'transmission blocking vaccine,' 'VIMT' and 'SSM-VIMT' were used for PubMed queries to identify relevant literature., Expert Opinion: Candidates targeting P. falciparum zygote surface antigen Pfs25, and its P. vivax orthologue Pvs25, induced functional activity in humans that reduced mosquito infection in surrogate assays, but require increased durability to be useful in the field. Candidates targeting gamete surface antigens Pfs230 and Pfs48/45, respectively, are in or nearing clinical trials. Nanoparticle platforms and adjuvants are being explored to enhance immunogenicity. Efficacy trials require special considerations, such as cluster-randomized designs to measure herd immunity that reduces human and mosquito infection rates, while addressing human and mosquito movements as confounding factors.

Inter-host transmission of pathogenic arboviruses such as dengue virus (DENV) and Zika virus (ZIKV) requires systemic infection of the mosquito vector. Successful systemic infection requires initial viral entry and proliferation in the midgut cells of the mosquito followed by dissemination to secondary tissues and eventual entry into salivary glands 1 . Lack of arbovirus proliferation in midgut cells has been observed in several Aedes aegypti strains 2 , but the midgut antiviral responses underlying this phenomenon are not yet fully understood. We report here that there is a rapid induction of apoptosis (RIA) in the Aedes aegypti midgut epithelium within 2 hours of infection with DENV-2 or ZIKV in both in vivo blood-feeding and ex vivo midgut infection models. Inhibition of RIA led to increased virus proliferation in the midgut, implicating RIA as an innate immune mechanism mediating midgut infection in this mosquito vector.

The principal vector of dengue, Zika and chikungunya viruses is the mosquito Aedes aegypti , with its ability to transmit pathogens influenced by ambient temperature. We use chikungunya virus (CHIKV) to understand how the mosquito transcriptome responds to arbovirus infection at different ambient temperatures. We exposed CHIKV-infected mosquitoes to 18, 28 and 32°C, and found that higher temperature correlated with higher virus levels, particularly at 3 days post infection, but lower temperature resulted in reduced virus levels. RNAseq analysis indicated significantly altered gene expression levels in CHIKV infection. The highest number of significantly differentially expressed genes was observed at 28°C, with a more muted effect at the other temperatures. At the higher temperature, the expression of many classical immune genes, including Dicer-2 , was not substantially altered in response to CHIKV. The upregulation of Toll, IMD and JAK-STAT pathways was only observed at 28°C. Functional annotations suggested that genes in immune response and metabolic pathways related to energy supply and DNA replication were involved in temperature-dependent changes. Time post infection also led to substantially different gene expression profiles, and this varied with temperature. In conclusion, temperature significantly modulates mosquito gene expression in response to infection, potentially leading to impairment of immune defences at higher temperatures.

The trade-off between reproduction and immunity has been established for a number of insect species. However, the regulatory mechanisms governing this event is not well understood. In the mosquito Aedes aegypti, the vector of dangerous human arboviral diseases, juvenile hormone (JH) is required for the female post-eclosion development and reproductive maturation. In this study, we have revealed the JH negative effect on the expression of immunity-related genes, such as antimicrobial peptides (AMPs), during the post-eclosion phase of the female mosquito gonadotrophic reproductive cycle. Mosquitoes treated with JH became more sensitive to microbial infection. Mosquitoes subjected to the RNA interference knockdown (RNAi) of the JH receptor, Methoprene-tolerant (Met), showed increased expression of several AMP genes. Met binds to the E-box-like recognition motifs in the regulatory region of the diptericin (Dpt) gene, indicating that JH can suppress the Dpt gene expression through its receptor Met. Hence, JH is involved in the modulation of immune responses during the post-eclosion phase of reproduction. The RNAi knockdown of the peptidoglycan recognition protein (PGRP-LC) led to a significant reduction of the Dpt transcript level, indicating the PGRP-LC activating role on this AMP gene. Thus, Dpt appeared to be under the dual regulation of both the JH and the immune deficiency (IMD) signaling pathways. Our study provides a better understanding of how JH regulates insect immunity in adult mosquitoes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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.

Background: Vector control with Bacillus sphaericus (Bs) is an effective way to block the transmission of malaria. However, in practical application of Bs agents, a sublethal dose effect was often caused by insufficient dosing, and it is little known whether the Bs exposure would affect the surviving mosquitoes' vector capacity to malaria., Methods: A sublethal dose of the Bs 2362 strain was administrated to the early fourth-instar larvae of Anopheles dirus to simulate shortage use of Bs in field circumstance. To determine vector competence, mosquitoes were dissected and the oocysts in the midguts were examined on day 9-11 post-infection with Plasmodium yoelii. Meanwhile, a SYBR quantitative PCR assay was conducted to examine the transcriptional level of the key immune molecules of mosquitoes, and RNA interference was utilized to validate the role of key immune effector molecule TEP1., Results: The sublethal dose of Bs treatment significantly reduced susceptibility of An. dirus to P. yoelii, with the decrease of P. yoelii infection intensity and rate. Although there existed a melanization response of adult An. dirus following challenge with P. yoelii, it was not involved in the decrease of vector competence as no significant difference of melanization rates and densities between the control and Bs groups was found. Further studies showed that Bs treatment significantly increased TEP1 expression in the fourth-instar larvae (L4), pupae (Pu), 48 h post-infection (hpi) and 72 hpi (P < 0.001). Further, gene-silencing of TEP1 resulted in disappearance of the Bs impact on vector competence of An. dirus to P. yoelii. Moreover, the transcriptional level of PGRP-LC and Rel2 were significantly elevated by Bs treatment with decreased expression of the negative regulator Caspar at 48 hpi, which implied that the Imd signaling pathway was upregulated by Bs exposure., Conclusions: Bs exposure can reduce the vector competence of An. dirus to malaria parasites through upregulating Imd signaling pathway and enhancing the expression of TEP1. The data could not only help us to understand the impact and mechanism of Bs exposure on Anopheles' vector competence to malaria but also provide us with novel clues for wiping out malaria using vector control.

Hemocytes limit the capacity of mosquitoes to transmit human pathogens. Here we profile the transcriptomes of 8506 hemocytes of Anopheles gambiae and Aedes aegypti mosquito vectors. Our data reveal the functional diversity of hemocytes, with different subtypes of granulocytes expressing distinct and evolutionarily conserved subsets of effector genes. A previously unidentified cell type in An. gambiae , which we term "megacyte," is defined by a specific transmembrane protein marker (TM7318) and high expression of lipopolysaccharide-induced tumor necrosis factor-α transcription factor 3 (LL3). Knockdown experiments indicate that LL3 mediates hemocyte differentiation during immune priming. We identify and validate two main hemocyte lineages and find evidence of proliferating granulocyte populations. This atlas of medically relevant invertebrate immune cells at single-cell resolution identifies cellular events that underpin mosquito immunity to malaria infection., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictus genome is essential to develop new approaches that involve genetic manipulation of mosquitoes., Results: We use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictus genome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes., Conclusion: The AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictus genome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.

Mayaro virus (MAYV), a sylvatic arbovirus belonging to the Togaviridae family and Alphavirus genus, is responsible for an increasing number of outbreaks in several countries of Central and South America. Despite Haemagogus janthinomys being identified as the main vector of MAYV, laboratory studies have already demonstrated the competence of Aedes aegypti to transmit MAYV. It has also been demonstrated that the Wolbachia w Mel strain is able to impair the replication and transmission of MAYV in Ae. aegypti . In Ae. aegypti , the small interfering RNA (siRNA) pathway is an important antiviral mechanism; however, it remains unclear whether siRNA pathway acts against MAYV infection in Ae. aegypti . The main objective of this study was to determine the contribution of the siRNA pathway in the control of MAYV infection. Thus, we silenced the expression of AGO2, an essential component of the siRNA pathway, by injecting dsRNA-targeting AGO2 (dsAGO2). Our results showed that AGO2 is required to control MAYV replication upon oral infection in Wolbachia -free Ae. aegypti . On the other hand, we found that Wolbachia -induced resistance to MAYV in Ae. aegypti is independent of the siRNA pathway. Our study brought new information regarding the mechanism of viral protection, as well as on Wolbachia mediated interference.

Mayaro virus is a mosquito-borne Alphavirus endemic to forests of tropical South America with a sylvatic cycle involving non-human primates and Haemagogus mosquitoes. Human infection with Mayaro virus causes a febrile illness and long-lasting arthralgia and cases are often associated with exposure to tropical forest habitats. Human movement between tropical forest habitats and urban settings may allow for imported cases and subsequent local transmission by domestic mosquito Aedes aegypti . The relative importance of Ae. aegypti as a vector of Mayaro virus may depend on the pathogenic effects of the virus on fitness correlates, especially those entomological parameters that relate to vectorial capacity. We performed mosquito infection studies and compared adult survival and fecundity of females from Brazilian and Floridian populations of Ae. aegypti following oral ingestion of uninfectious (control) and Mayaro virus infectious blood. Mayaro virus infected and refractory mosquitoes had similar or 30-50% lower fecundity than control (unexposed) mosquitoes, suggesting a reproductive cost to mounting an immune response or phenotypic expression of refractoriness. Survival of adult female mosquitoes and targeted gene expression in the Toll and IMD pathways were not altered by Mayaro virus infection. Adult lifespan and fecundity estimates were independent of measured viral titer in the bodies of mosquitoes. The lack of adverse effects of infection status on female survival suggests that Mayaro virus will not alter vectorial capacity mediated by changes in this parameter.

Background: In animal models, immunity to mosquito salivary proteins protects animals against mosquito-borne disease. These findings provide a rationale to vaccinate against mosquito saliva instead of the pathogen itself. To our knowledge, no vector salivary protein-based vaccine has been tested for safety and immunogenicity in humans. We aimed to assess the safety and immunogenicity of Anopheles gambiae saliva vaccine (AGS-v), a peptide-based vaccine derived from four A gambiae salivary proteins, in humans., Methods: In this randomised, placebo-controlled, double-blind, phase 1 trial, participants were enrolled at the National Institutes of Health Clinical Center in Bethesda, MD, USA. Participants were eligible if they were healthy adults, aged 18-50 years with no history of severe allergic reactions to mosquito bites. Participants were randomly assigned (1:1:1), using block randomisation and a computer-generated randomisation sequence, to treatment with either 200 nmol of AGS-v vaccine alone, 200 nmol of AGS-v with adjuvant (Montanide ISA 51), or sterile water as placebo. Participants and clinicians were masked to treatment assignment. Participants were given a subcutaneous injection of their allocated treatment at day 0 and day 21, followed by exposure to feeding by an uninfected Aedes aegypti mosquito at day 42 to assess subsequent risk to mosquito bites in a controlled setting. The primary endpoints were safety and immunogenicity at day 42 after the first immunisation. Participants who were given at least one dose of assigned treatment were assessed for the primary endpoints and analysis was by intention to treat. The trial was registered with ClinicalTrials.gov, NCT03055000, and is closed for accrual., Findings: Between Feb 15 and Sept 10, 2017, we enrolled and randomly assigned 49 healthy adult participants to the adjuvanted vaccine (n=17), vaccine alone (n=16), or placebo group (n=16). Five participants did not complete the two-injection regimen with mosquito feeding at day 42, but were included in the safety analyses. No systemic safety concerns were identified; however, one participant in the adjuvanted vaccine group developed a grade 3 erythematous rash at the injection site. Pain, swelling, erythema, and itching were the most commonly reported local symptoms and were significantly increased in the adjuvanted vaccine group compared with both other treatment groups (nine [53%] of 17 participants in the adjuvanted vaccine group, two [13%] of 16 in the vaccine only group, and one [6%] of 16 in the placebo group; p=0·004). By day 42, participants who were given the adjuvanted vaccine had a significant increase in vaccine-specific total IgG antibodies compared with at baseline than did participants who were give vaccine only (absolute difference of log 10 -fold change of 0·64 [95% CI 0·39 to 0·89]; p=0·0002) and who were given placebo (0·62 [0·34 to 0·91]; p=0·0001). We saw a significant increase in IFN-γ production by peripheral blood mononuclear cells at day 42 in the adjuvanted vaccine group compared with in the placebo group (absolute difference of log 10 ratio of vaccine peptide-stimulated vs negative control 0·17 [95% CI 0·061 to 0·27]; p=0·009) but we saw no difference between the IFN-γ production in the vaccine only group compared with the placebo group (0·022 [-0·072 to 0·116]; p=0·63)., Interpretation: AGS-v was well tolerated, and, when adjuvanted, immunogenic. These findings suggest that vector-targeted vaccine administration in humans is safe and could be a viable option for the increasing burden of vector-borne disease., Funding: Office of the Director and the Division of Intramural Research at the National Institute of Allergy and Infectious Diseases, and National Institutes of Health., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dirofilaria immitis is the globally distributed agent of heartworm disease. Infection in canines causes debilitating disease that can be fatal if left untreated. Macrocyclic lactones can prevent heartworm disease in dogs, cats and ferrets by killing larvae before they develop into adult worms in the pulmonary artery. However, administration of prophylactic drugs to wild canids to prevent D. immitis infection is not feasible. Furthermore, a vaccine against heartworm is currently unavailable and drug resistant D. immitis have been identified, highlighting the need for new strategies to prevent parasite transmission. We recently established a method to block development of emerging third-stage larvae (eL3) from the mosquito Aedes aegypti by over-activating the Toll pathway, one of the major innate immune signaling pathways in mosquitoes. Our previous study used a drug-sensitive strain of D. immitis and it remains unknown if the strategy is effective against different D. immitis genotypes and, more importantly, if it would work against drug-resistant genotypes. The purpose of this study was to determine whether Toll pathway activation is capable of blocking eL3 development of D. immitis strains that are resistant to macrocyclic lactones. We infected mosquitoes with two independent strains of D. immitis previously confirmed as being resistant to macrocyclic lactones, and then activated Toll signaling by RNAi-mediated silencing of the pathway inhibitor, IκB/Cactus, and quantitatively measured eL3 development. Similar to the drug-sensitive strain, eL3 were strongly reduced by Toll activation in both drug-resistant strains. Furthermore, similar to the drug-sensitive strain, the reduction of eL3 in both drug-resistant strains suggests a defect in larval invasion of, or development in, the Malpighian tubules - the organ in the mosquito to which microfilariae migrate after ingestion and where the larvae undergo several developmental molts. In summary, Toll pathway activation blocks the development of three distinct D. immitis genotypes, including two different drug-resistant genotypes. If this strategy can be applied to heartworm vectors in the field, it may help reduce the spread of disease and is not predicted to favor the spread of drug resistance., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Malaria, caused by the protozoan parasite Plasmodium and transmitted by Anopheles mosquitoes, represents a major threat to human health. Plasmodium's infection cycle in the Anopheles vector is critical for transmission of the parasite between humans. The midgut-stage bottleneck of infection is largely imposed by the mosquito's innate immune system. microRNAs (miRNAs, small noncoding RNAs that bind to target RNAs to regulate gene expression) are also involved in regulating immunity and the anti-Plasmodium defense in mosquitoes. Here, we characterized the mosquito's miRNA responses to Plasmodium infection using an improved crosslinking and immunoprecipitation (CLIP) method, termed covalent ligation of endogenous Argonaute-bound RNAs (CLEAR)-CLIP. Three candidate miRNAs' influence on P. falciparum infection and midgut microbiota was studied through transgenically expressed miRNA sponges (miR-SPs) in midgut and fat body tissues. MiR-SPs mediated conditional depletion of aga-miR-14 or aga-miR-305, but not aga-miR-8, increased mosquito resistance to both P. falciparum and P. berghei infection, and enhanced the mosquitoes' antibacterial defenses. Transcriptome analysis revealed that depletion of aga-miR-14 or aga-miR-305 resulted in an increased expression of multiple immunity-related and anti-Plasmodium genes in mosquito midguts. The overall fitness cost of conditionally expressed miR-SPs was low, with only one of eight fitness parameters being adversely affected. Taken together, our results demonstrate that targeting mosquito miRNA by conditional expression of miR-SPs may have potential for the development of malaria control through genetically engineered mosquitoes., Competing Interests: The authors have declared that no competing interests exist.

After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 ( Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of malaria transmission blocking., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Background: Wolbachia has been reported to suppress a variety of pathogen infections in mosquitoes, but the mechanism is undefined. Two possibilities have been proposed. One is that Wolbachia activates host immune responses, and the other one is that Wolbachia competes with pathogens for limited nutrients., Methodology/principal Findings: In this study, we compared host immune responses and the densities of two different strains of Wolbachia in naturally occurring parental and artificially created hybrid host genetic backgrounds. No significant difference in Wolbachia density was found between these hosts. We found that Wolbachia could activate host innate immune responses when the host genetic profile was different from that of its natural host. When these hosts were challenged with pathogenic bacteria, mosquitoes in new host-Wolbachia symbioses had a higher survival rate than in old host-Wolbachia symbioses., Conclusions/significance: The presence of Wolbachia per se does not necessarily affect pathogen infections, suggesting that a competition for limited nutrients is not the main reason for Wolbachia-mediated pathogen suppression. Instead, host immune responses are responsible for it. The elucidation of an immunity nature of PI is important to guide future practice: Wolbachia may be genetically engineered to be more immunogenic, it is desired to search and isolate more strains of Wolbachia, and test more host-Wolbachia symbioses for future applications. Our results also suggest Wolbachia-based PI may be applied to naturally Wolbachia-infected mosquito populations, and extend to the control of a broader range of mosquito-borne diseases., Competing Interests: The authors have declared that no competing interests exist.

Mosquito-borne diseases have become an important public health issue of global concern because of their high incidence and transmission rate. As a vector for mosquito-borne diseases, studying the interaction mechanism between mosquitoes and mosquito-borne viruses will help control mosquito-borne diseases. The impaired innate immunity and immune barriers evasion caused by mosquito-borne viruses in mosquitoes pose a potential risk for the persistent infection of the virus in mosquitoes and the outbreak of mosquito-borne diseases. The RNA interference (RNAi) pathway, as a powerful antiviral defense barrier in mosquitoes, can inhibit viral replication and transmission by producing a variety of small RNAs to degrade viral RNA. In this review, we summarize the related studies on the innate immune mechanism against mosquito- borne virus infection in mosquitoes about small interfering RNA (siRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA), aiming to provide a theoretical reference for the prevention and control of mosquito-borne diseases.

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., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20th century in apparent enzootic circulation as well as equine and human disease incidence, provides a unique case study on how reductions in virus activity can be understood by studying evolutionary trends and mechanisms. Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector (house sparrows and Culex tarsalis mosquitoes). We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century., Competing Interests: The authors have declared that no competing interests exist.

The surface protein Pfs47 allows Plasmodium falciparum parasites to survive and be transmitted by making them "undetectable" to the mosquito immune system. P. falciparum parasites express Pfs47 haplotypes compatible with their sympatric vectors, while those with incompatible haplotypes are eliminated by the mosquito. We proposed that Pfs47 serves as a "key" that mediates immune evasion by interacting with a mosquito receptor "the lock," which differs in evolutionarily divergent anopheline mosquitoes. Recombinant Pfs47 (rPfs47) was used to identify the mosquito Pfs47 receptor protein (P47Rec) using far-Western analysis. rPfs47 bound to a single 31-kDa band and the identity of this protein was determined by mass spectrometry. The mosquito P47Rec has two natterin-like domains and binds to Pfs47 with high affinity (17 to 32 nM). P47Rec is a highly conserved protein with submicrovillar localization in midgut cells. It has structural homology to a cytoskeleton-interacting protein and accumulates at the site of ookinete invasion. Silencing P47Rec expression reduced P. falciparum infection, indicating that the interaction of Pfs47 with the receptor is critical for parasite survival. The binding specificity of P47Rec from distant anophelines ( Anopheles gambiae , Anopheles dirus , and Anopheles albimanus ) with Pfs47-Africa (GB4) and Pfs47-South America (7G8) haplotypes was evaluated, and it is in agreement with the previously documented compatibility between P. falciparum parasites expressing different Pfs47 haplotypes and these three anopheline species. Our findings give further support to the role of Pfs47 in the adaptation of P. falciparum to different vectors., Competing Interests: The authors declare no competing interest.

Diseases spread by mosquitoes have killed more people than those spread by any other group of arthropod vectors and remain an important factor in determining global health and economic stability. The mosquito innate immune system can act to either modulate infection with human pathogens or fight off entomopathogens and increase the fitness and longevity of infected mosquitoes. While work remains towards understanding the larval immune system and the development of the mosquito immune system, it has recently become clearer that environmental factors heavily shape the developing mosquito immune system and continue to influence the adult immune system as well. The adult immune system has been well-studied and is known to involve multiple tissues and diverse molecular mechanisms. This review summarizes and synthesizes what is currently understood about the development of the mosquito immune system and includes comparisons of immune components unique to mosquitoes among the blood-feeding arthropods as well as important distinguishing factors between the anopheline and culicine mosquitoes. An explanation is included for how mosquito immunity factors into vector competence and vectorial capacity is presented along with a model for the interrelationships between nutrition, microbiome, pathogen interactions and behavior as they relate to mosquito development, immune status, adult female fitness and ultimately, vectorial capacity. Novel discoveries in the fields of mosquito ecoimmunology, neuroimmunology, and intracellular antiviral responses are highlighted., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The insecticides we use for agriculture and for vector control often arrive in water bodies, where mosquito larvae may be exposed to them. Not only will they then likely affect the development of the larvae, but their effects may carry over to the adults, potentially affecting their capacity at transmitting infectious diseases. Such an impact may be expected to be more severe when mosquitoes are undernourished. In this study, we investigated whether exposing larvae of the mosquito Anopheles gambiae to a sub-lethal dose of permethrin (a pyrethroid) and forcing them to compete for food would affect the immune response of the adults. We found that a low dose of permethrin increased the degree to which individually reared larvae melanised a negatively charged Sephadex bead and slowed the replication of injected Escherichia coli. However, if mosquitoes had been reared in groups of three (and thus had been forced to compete for food) permethrin had less impact on the efficacy of the immune responses. Our results show how larval stressors can affect the immune response of adults, and that the outcome of exposure to insecticides strongly depends on environmental conditions.