Mosquito cellular immunity at single-cell resolution.

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.

Efecto de la temperatura y salinidad en la eclosión y supervivencia de Aedes aegypti (L) (Diptera: Culicidae) procedentes del occidente de México / Effect of temperature and salinity on the eclosion and survival of Aedes aegypti (L) (Diptera: Culicidae) from Western Mexico

Introducción: Aedes aegypti (L) (Diptera: Culicidae), es una especie cosmopolita y vector de arbovirosis. Las variaciones de la temperatura y salinidad del agua influyen en la eclosión y supervivencia de fases larvales. Objetivo: Evaluar el efecto de diferentes temperaturas y salinidades en la eclosión de huevos y la supervivencia de larvas, pupas y adultos bajo condiciones de laboratorio. Métodos: Se colectaron larvas de Ae. aegypti, de reservorios artificiales en la zona periurbana de Puerto Vallarta, Jalisco, México, y se mantuvieron hasta la fase adulta. Los huevos obtenidos se sometieron a ocho temperaturas (15, 17, 20, 25, 27, 30, 32 y 35 °C). Se colocaron 15 huevos por quintuplicado y se evaluó la eclosión durante 96 h. Se colocaron 100 huevos con agua ajustada a 0.3, 2, 5, 10, 15,18 y 22 ups y se evaluó la eclosión hasta las 96 h. Adicionalmente se utilizaron larvas del estadio IV, por quintuplicado, sometiéndose a las mismas salinidades y se evaluó la supervivencia hasta las 48 h. El efecto de la salinidad en la ovoposición de las hembras se llevó a cabo introduciendo recipientes con las mismas concentraciones salinas, dentro en las jaulas entomológicas. Resultados: Se registró el 100 por ciento de eclosión a las 24 y 48 h; la temperatura de 35° C no registró eclosión. Las salinidades de 22 y 18 ups, provocaron mortalidad del 100 por ciento a las 24 h. En la salinidad de 15 ups, sobrevivió el 50 por ciento. Las concentraciones de 2, 5 y 10 ups demostraron 100 por ciento de supervivencia hasta la fase de adulto. La supervivencia de larvas del estadio IV en los tratamientos 2, 5 y 10 fue del 100 por ciento y en 15,18 y 22 ups disminuyó a 50, 80 y 100 por ciento, respectivamente (p˂ 0,05). Las diferentes concentraciones salinas no afectaron significativamente la ovoposición. La eclosión solo se presentó en las concentraciones de 0,3; 2; 5 y 10 ups. Los huevos ovopositados en concentraciones de 15, 18 y 22 ups no eclosionaron hasta que fueron transferidos a agua dulce con porcentajes de eclosión de entre el 80 y 90 por ciento. Conclusiones: Los embriones de Ae. aegypti poseen una amplia plasticidad para soportar cambios drásticos de temperatura y salinidad. El control efectivo de sus poblaciones debe incluir la revisión de charcas o reservorios que contengan aguas salobres hasta 18 ups(AU)

Introduction: Aedes aegypti (L) (Diptera: Culicidae) is a cosmopolitan species and a vector of arboviruses. Variations in the temperature and salinity of the water affect eclosion and survival during the larval stages. Objective: Evaluate the effect of different temperatures and salinities on the eclosion of eggs and the survival of larvae, pupae and adults in laboratory conditions. Methods: Ae. aegypti larvae were collected from artificial reservoirs in a peri-urban area of Puerto Vallarta, Jalisco, Mexico, and maintained until the adult stage. The eggs obtained were subjected to eight temperatures (15, 17, 20, 25, 27, 30, 32 and 35 °C). Fifteen eggs were placed in quintuplicate and eclosion was evaluated for 96 h. One hundred eggs were placed with water adjusted to 0.3, 2, 5, 10, 15, 18 and 22 psu and eclosion was evaluated until 96 h. Additionally, stage IV larvae were used in quintuplicate, subjecting them to the same salinities and evaluating survival until 48 h. The effect of salinity on oviposition by females was determined by introducing containers with the same salinity into the entomological cages. Results: 100 percent eclosion was recorded at 24 and 48 h, whereas no eclosion occurred at a temperature of 35 °C. Salinities of 22 and 18 psu caused 100 percent mortality at 24 h, whereas 50 percent survived at a salinity of 15 psu. At concentrations of 2, 5 and 10 psu 100 percent of the larvae survived until the adult stage. Survival of stage IV larvae in treatments 2, 5 and 10 was 100%, whereas in 15, 18 and 22 psu it fell to 50, 80 and 100 percent, respectively (p˂ 0.05). The different salinities did not affect oviposition significantly. Eclosion only occurred at concentrations of 0.3, 2, 5 and 10 psu. Oviposited eggs at concentrations of 15, 18 and 22 psu did not eclose until they were transferred to fresh water, where eclosion percentages ranged between 80 percent and 90 percent. Conclusions: Ae. aegypti embryos have great plasticity to endure drastic changes in temperature and salinity. Effective control of their populations should include inspection of ponds and reservoirs containing brackish water of up to 18 psu(AU)

Chemical depletion of phagocytic immune cells in Anopheles gambiae reveals dual roles of mosquito hemocytes in anti-Plasmodium immunity.

Mosquito immunity is composed of both cellular and humoral factors that provide protection from invading pathogens. Immune cells known as hemocytes, have been intricately associated with phagocytosis and innate immune signaling. However, the lack of genetic tools has limited hemocyte study despite their importance in mosquito anti-Plasmodium immunity. To address these limitations, we employ the use of a chemical-based treatment to deplete phagocytic immune cells in Anopheles gambiae, demonstrating the role of phagocytes in complement recognition and prophenoloxidase production that limit the ookinete and oocyst stages of malaria parasite development, respectively. Through these experiments, we also define specific subtypes of phagocytic immune cells in An. gambiae, providing insights beyond the morphological characteristics that traditionally define mosquito hemocyte populations. Together, this study represents a significant advancement in our understanding of the roles of mosquito phagocytes in mosquito vector competence and demonstrates the utility of clodronate liposomes as an important tool in the study of invertebrate immunity.

A Vaccine Based on a Modified Vaccinia Virus Ankara Vector Expressing Zika Virus Structural Proteins Controls Zika Virus Replication in Mice.

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that affects humans and can cause severe neurological complications, including Guillain-Barré syndrome and microcephaly. Since 2007 there have been three large outbreaks; the last and larger spread in the Americas in 2015. Actually, ZIKV is circulating in the Americas, Southeast Asia, and the Pacific Islands, and represents a potential pandemic threat. Given the rapid ZIKV dissemination and the severe neurological and teratogenic sequelae associated with ZIKV infection, the development of a safe and efficacious vaccine is critical. In this study, we have developed and characterized the immunogenicity and efficacy of a novel ZIKV vaccine based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the ZIKV prM and E structural genes (termed MVA-ZIKV). MVA-ZIKV expressed efficiently the ZIKV structural proteins, assembled in virus-like particles (VLPs) and was genetically stable upon nine passages in cell culture. Immunization of mice with MVA-ZIKV elicited antibodies that were able to neutralize ZIKV and induced potent and polyfunctional ZIKV-specific CD8+ T cell responses that were mainly of an effector memory phenotype. Moreover, a single dose of MVA-ZIKV reduced significantly the viremia in susceptible immunocompromised mice challenged with live ZIKV. These findings support the use of MVA-ZIKV as a potential vaccine against ZIKV.

Salivary factor LTRIN from Aedes aegypti facilitates the transmission of Zika virus by interfering with the lymphotoxin-ß receptor.

Pathogens have co-evolved with mosquitoes to optimize transmission to hosts. Mosquito salivary-gland extract is known to modulate host immune responses and facilitate pathogen transmission, but the underlying molecular mechanisms of this have remained unknown. In this study, we identified and characterized a prominent 15-kilodalton protein, LTRIN, obtained from the salivary glands of the mosquito Aedes aegypti. LTRIN expression was upregulated in blood-fed mosquitoes, and LTRIN facilitated the transmission of Zika virus (ZIKV) and exacerbated its pathogenicity by interfering with signaling through the lymphotoxin-ß receptor (LTßR). Mechanically, LTRIN bound to LTßR and 'preferentially' inhibited signaling via the transcription factor NF-κB and the production of inflammatory cytokines by interfering with the dimerization of LTßR during infection with ZIKV. Furthermore, treatment with antibody to LTRIN inhibited mosquito-mediated infection with ZIKV, and abolishing LTßR potentiated the infectivity of ZIKV both in vitro and in vivo. This study provides deeper insight into the transmission of mosquito-borne diseases in nature and supports the therapeutic potential of inhibiting the action of LTRIN to disrupt ZIKV transmission.

Dengue virus infection induces chromatin remodeling at locus AAEL006536 in the midgut of Aedes aegypti / La infección por virus dengue induce remodelación de la cromatina en el locus AAEL006536 en el intestino medio de Aedes aegypti

Abstract: Objective: To identify and characterize Aedes aegypti's AAEL006536 gene proximal upstream cis-regulatory sequences activated by dengue virus infection. Materials and methods: A. aegypti Rockefeller strain mosquitoes were blood fed or infected with dengue virus 2. Open chromatin profiling was then carried out in pools of midguts from each group of mosquitoes. Results: The proximal upstream region does not contain open chromatin sites in the midguts of blood-fed mosquitoes as detected by FAIRE-qPCR. In contrast, two cis-regulatory sites were identified in the same upstream region of dengue virus-infected mosquito midguts. The distal sequence contains STAT-, REL- and C/EBP-type transcription factor binding sites. Conclusion: The activation of two proximal cis-regulatory sequences, induced by dengue virus infection, is mediated by chromatin remodeling mechanisms. Binding sites suggest a dengue virus infection-induced participation of immunity transcription factors in the up-regulation of this gene. This suggests the participation of the AAEL006536 gene in the mosquito's antiviral innate immune response.

Resumen: Objetivo: Identificar y caracterizar las secuencias reguladoras activadas por la infección por virus dengue en la región proximal del gen AAEL006536 de Aedes aegypti. Material y métodos: Mosquitos de la cepa Rockefeller de A. aegypti se infectaron con virus dengue o se alimentaron con sangre. Se obtuvieron los perfiles de cromatina abierta del locus en los intestinos de cada uno de los grupos. Resultados: Se identificaron dos sitios reguladores solo en los intestinos de mosquitos infectados por virus dengue. El sitio distal contiene sitios de unión a factores de transcripción tipo REL, STAT y C/EBP. Conclusiones: La activación de dos sitios reguladores proximales está mediada por la remodelación de la cromatina. Los sitios de unión a factores de transcripción en el sitio regulador distal sugieren la participación de las vías de inmunidad en la regulación del gen. Esto sugiere la participación de este gen en la respuesta inmune del mosquito frente a la infección viral.

Assessment of the ability of V920 recombinant vesicular stomatitis-Zaire ebolavirus vaccine to replicate in relevant arthropod cell cultures and vector species.

V920, rVSVΔG-ZEBOV-GP, is a recombinant vesicular stomatitis-Zaire ebolavirus vaccine which has shown an acceptable safety profile and provides a protective immune response against Ebola virus disease (EVD) induced by Zaire ebolavirus in humans. The purpose of this study was to determine whether the V920 vaccine is capable of replicating in arthropod cell cultures of relevant vector species and of replicating in live mosquitoes. While the V920 vaccine replicated well in Vero cells, no replication was observed in Anopheles or Aedes mosquito, Culicoides biting midge, or Lutzomyia sand fly cells, nor in live Culex or Aedes mosquitoes following exposure through intrathoracic inoculation or feeding on a high-titer infectious blood meal. The insect taxa selected for use in this study represent actual and potential epidemic vectors of VSV. V920 vaccine inoculated into Cx. quinquefasciatus and Ae. aegypti mosquitoes demonstrated persistence of replication-competent virus following inoculation, consistent with the recognized biological stability of the vaccine, but no evidence for active virus replication in live mosquitoes was observed. Following administration of an infectious blood meal to Ae. aegypti and Cx. quinquefasciatus mosquitoes at a titer several log10 PFU more concentrated than would be observed in vaccinated individuals, no infection or dissemination of V920 was observed in either mosquito species. In vitro and in vivo data gathered during this study support minimal risk of the vector-borne potential of the V920 vaccine.

Iron availability affects West Nile virus infection in its mosquito vector.

BACKGROUND: Mosquitoes are responsible for transmission of viruses, including dengue, West Nile and chikungunya viruses. Female mosquitoes are infected when they blood-feed on vertebrates, a required step for oogenesis. During this process, mosquitoes encounter high iron loads. Since iron is an essential nutrient for most organisms, including pathogens, one of the defense mechanisms for the host includes sequestration of iron away from the invading pathogen. Here, we determine whether iron availability affects viral replication in mosquitoes. METHODS: To elucidate effect of iron availability on mosquito cells during infection, Culex cells were treated with either ferric ammonium citrate (FAC) or the iron chelator, deferoxamine (DFX). Real time RT-PCR was performed using ferritin (heavy chain) and NRAMP as a measure of iron homeostasis in cells. To determine iron requirement for viral replication, Culex cells were knocked down for NRAMP using dsRNA. Finally, the results were validated in Culex mosquito-infection model, by treating infected mosquitoes with DFX to reduce iron levels. RESULTS: Our results show that infection of Culex cells led to induction in levels of ferritin (heavy chain) and NRAMP mRNAs in time-dependent manner. Results also showed that treatment of cells with FAC, reduced expression of NRAMP (iron transporter) and increase levels of ferritin (heavy chain). Interestingly, increasing iron levels increased viral titers; while reducing intracellular iron levels, either by NRAMP knock-down or using DFX, reduced viral titers. The results from Culex mosquito infection showed that mosquitoes treated with DFX had reduced viral titers compared with untreated controls in midgut as well as carcass 8 days pi. Saliva from mosquitoes treated with DFX also showed reduced viral titers compared with untreated controls, indicating low viral transmission capacity. CONCLUSIONS: Our results indicate that iron is required for viral replication in mosquito cells. Mosquitoes respond to viral infection, by inducing expression of heavy chain ferritin, which sequesters available iron, reducing its availability to virus infected cells. The data indicates that heavy chain ferritin may be part of an immune mechanism of mosquitoes in response to viral infections.

The human immune system's response to carcinogenic and other infectious agents transmitted by mosquito vectors.

It has been hypothesised that mosquitoes [Diptera: Culicidae] may play more of a role in certain cancers than is currently appreciated. Research links 33 infectious agents to cancer, 27 of which have a presence in mosquitoes, and that, in addition, mosquito saliva downregulates the immune system. The objective of this paper is to review the literature on the immune system and cancer-causing infectious agents, particularly those present in mosquitoes, with a view to establishing whether such infectious agents can, in the long run, defeat the immune system or be defeated by it. Many of the viruses, bacteria and parasites recognised by the International Agency for Research on Cancer (IARC) as carcinogenic and suspected by others as being involved in cancer have evolved numerous complex ways of avoiding, suppressing or altering the immune system's responses. These features, coupled with the multiplicity and variety of serious infectious agents carried by some species of mosquitoes and the adverse effects on the immune system of mosquito saliva, suggest that post-mosquito bite the immune system is likely to be overwhelmed. In such a situation, immunisation strategies offer little chance of cancer prevention, unless a single or limited number of critical infectious agents can be isolated from the 'mosquito' cocktail. If that proves to be impossible cancer prevention will, therefore, if the hypothesis proves to be correct, rest on the twin strategies of environmentally controlling the mosquito population and humans avoiding being bitten. The latter strategy will involve determining the factors that demark those being bitten from those that are not.