RESUMO
Wolbachia pipientis is a maternally transmitted symbiotic bacterium that mainly colonizes arthropods, potentially affecting different aspects of the host's physiology, e.g., reproduction, immunity, and metabolism. It has been shown that Wolbachia modulates glycogen metabolism in mosquito Aedes fluviatilis (Ae. fluviatilis). Glycogen synthesis is controlled by the enzyme GSK3, which is also involved in immune responses in both vertebrate and invertebrate organisms. Here we investigated the mechanisms behind immune changes mediated by glycogen synthase kinase ß (GSK3ß) in the symbiosis between Ae. fluviatilis and W. pipientis using a GSK3ß inhibitor or RNAi-mediated gene silencing. GSK3ß inhibition or knockdown increased glycogen content and Wolbachia population, together with a reduction in Relish2 and gambicin transcripts. Furthermore, knockdown of Relish2 or Caspar revealed that the immunodeficiency pathway acts to control Wolbachia numbers in the host. In conclusion, we describe for the first time the involvement of GSK3ß in Ae. fluviatilis immune response, acting to control the Wolbachia endosymbiotic population.
Assuntos
Aedes , Simbiose , Wolbachia , Wolbachia/fisiologia , Wolbachia/metabolismo , Aedes/microbiologia , Aedes/imunologia , Aedes/metabolismo , Animais , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Glicogênio/metabolismoRESUMO
The sialotranscriptomes of Aedes aegypti revealed a transcript overexpressed in female salivary glands that codes a mature 7.8 kDa peptide. The peptide, specific to the Aedes genus, has a unique sequence, presents a putative secretory nature and its function is unknown. Here, we confirmed that the peptide is highly expressed in the salivary glands of female mosquitoes when compared to the salivary glands of males, and its secretion in mosquito saliva is able to sensitize the vertebrate host by inducing the production of specific antibodies. The synthetic version of the peptide downmodulated nitric oxide production by activated peritoneal murine macrophages. The fractionation of a Ae. aegypti salivary preparation revealed that the fractions containing the naturally secreted peptide reproduced the nitric oxide downmodulation. The synthetic peptide also selectively interfered with cytokine production by murine macrophages, inhibiting the production of IL-6, IL-12p40 and CCL2 without affecting TNF-α or IL-10 production. Likewise, intracellular proteins associated with macrophage activation were also distinctively modulated: while iNOS and NF-κB p65 expression were diminished, IκBα and p38 MAPK expression did not change in the presence of the peptide. The anti-inflammatory properties of the synthetic peptide were tested in vivo on a dextran sulfate sodium-induced colitis model. The therapeutic administration of the Ae. aegypti peptide reduced the leukocytosis, macrophage activity and nitric oxide levels in the gut, as well as the expression of cytokines associated with the disease, resulting in amelioration of its clinical signs. Given its biological properties in vitro and in vivo, the molecule was termed Aedes-specific MOdulatory PEptide (AeMOPE-1). Thus, AeMOPE-1 is a novel mosquito-derived immunobiologic with potential to treat immune-mediated disorders.
Assuntos
Aedes/imunologia , Colite/etiologia , Colite/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Proteínas e Peptídeos Salivares/imunologia , Sequência de Aminoácidos , Animais , Biomarcadores , Colite/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Imunomodulação , Ativação Linfocitária/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Proteínas e Peptídeos Salivares/química , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Prostaglandins (PGs) are immuno-active lipids that mediate the immune response in invertebrates and vertebrates. In insects, PGs play a role on different physiological processes such as reproduction, ion transport and regulation of cellular immunity. However, it is unclear whether PGs play a role in invertebrate's humoral immunity, and, if so, which immune signaling pathways would be modulated by PGs. Here, we show that Aedes aegypti gut microbiota and Gram-negative bacteria challenge induces prostaglandin production sensitive to an irreversible inhibitor of the vertebrate cyclooxygenase, acetylsalicylic acid (ASA). ASA treatment reduced PG synthesis and is associated with decreased expression of components of the Toll and IMD immune pathways, thereby rendering mosquitoes more susceptible to both bacterial and viral infections. We also shown that a cytosolic phospholipase (PLAc), one of the upstream regulators of PG synthesis, is induced by the microbiota in the midgut after blood feeding. The knockdown of the PLAc decreased prostaglandin production and enhanced the replication of Dengue in the midgut. We conclude that in Ae. aegypti, PGs control the amplitude of the immune response to guarantee an efficient pathogen clearance.
Assuntos
Aedes/virologia , Vírus da Dengue/fisiologia , Imunidade Humoral , Prostaglandinas/metabolismo , Aedes/imunologia , Animais , Linhagem Celular , Vírus da Dengue/imunologia , Feminino , Regulação Enzimológica da Expressão Gênica , Interações Hospedeiro-Patógeno , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Prostaglandinas/genéticaRESUMO
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 WolbachiawMel 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.
Assuntos
Aedes/microbiologia , Aedes/virologia , Alphavirus/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Wolbachia/fisiologia , Aedes/imunologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/virologia , Animais , Feminino , Humanos , Imunidade Inata , Mosquitos Vetores/imunologia , Mosquitos Vetores/microbiologia , Mosquitos Vetores/virologia , Wolbachia/imunologiaRESUMO
Several studies have observed that the immune response in insects can be conserved, a phenomenon known as immune priming, which has been mostly tested in adult stages. However, it is unknown if induction of immune priming in larval stages protects against dengue virus (DENV) infections in adult mosquitoes. In this work, we primed larval instar 3rd of Aedes aegypti with inactive dengue virus, producing adult mosquitoes with i) an enhanced antiviral-immune response; ii) a reduction in the load and replication of RNA of dengue virus (DENV); iii) a decline in viral infective particles production. Adult mosquitoes previously primed during larval stages over-expressed RNA interference (RNAi) markers Argonaute-2 (AGO-2) and Dicer-2 (DCR-2). We also observed inter-individual variations of DENV infection in adult mosquitoes, indicating a heterogeneous response to DENV infection in the same mosquito strain. However, mosquitoes primed during larval stages appear to control the infection, reducing the viral load. The over-expression of interferon-like factors (VAGO) and AGO-2 in the pupa stage suggests a fast activation of antiviral mechanisms after immune priming in larvae, creating a condition in which adult mosquitoes are resistant to the pathogen in the posterior exposure.
Assuntos
Aedes/imunologia , Aedes/virologia , Envelhecimento/fisiologia , Vírus da Dengue/imunologia , Dengue/prevenção & controle , Dengue/virologia , Animais , Feminino , Tolerância Imunológica , Proteínas de Insetos/metabolismo , Larva/imunologia , Larva/virologia , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Carga Viral/imunologia , Vírion/metabolismoRESUMO
Mayaro (MAYV) is an emerging arthropod-borne virus belonging to the Alphavirus genus of the Togaviridae family. Although forest-dwelling Haemagogus mosquitoes have been considered as its main vector, the virus has also been detected in circulating Aedes ssp mosquitoes. Here we assess the susceptibility of Aedes aegypti and Aedes albopictus to infection with MAYV and their innate immune response at an early stage of infection. Aedes albopictus was more susceptible to infection with MAYV than Ae. aegypti. Analysis of transcript levels of twenty immunity-related genes by real-time PCR in the midgut of both mosquitoes infected with MAYV revealed increased expression of several immune genes, including CLIP-domain serine proteases, the anti-microbial peptides defensin A, E, cecropin E, and the virus inducible gene. The regulation of certain genes appeared to be Aedes species-dependent. Infection of Ae. aegypti with MAYV resulted in increased levels of myeloid differentiation2-related lipid recognition protein (ML26A) transcripts, as compared to Ae. albopictus. Increased expression levels of thio-ester-containing protein 22 (TEP22) and Niemann-Pick type C1 (NPC1) gene transcripts were observed in infected Ae. albopictus, but not Ae. aegypti. The differences in these gene expression levels during MAYV infection could explain the variation in susceptibility observed in both mosquito species.
Assuntos
Aedes/virologia , Infecções por Alphavirus/transmissão , Alphavirus/imunologia , Imunidade Inata , Aedes/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Expressão Gênica/imunologia , Perfilação da Expressão Gênica , Imunidade Inata/genética , Mosquitos Vetores/virologia , Reação em Cadeia da Polimerase em Tempo Real , Serina Proteases/genética , Serina Proteases/metabolismoRESUMO
During probing and blood feeding, haematophagous mosquitoes inoculate a mixture of salivary molecules into their vertebrate hosts' skin. In addition to the anti-haemostatic and immunomodulatory activities, mosquito saliva also triggers acute inflammatory reactions, especially in sensitized hosts. Here, we characterize the oedema and the cellular infiltrate following Aedes aegypti mosquito bites in the skin of sensitized and non-sensitized BALB/c mice by flow cytometry. Ae. aegypti bites induced an increased oedema in the ears of both non-sensitized and salivary gland extract- (SGE-)sensitized mice, peaking at 6 hr and 24 hr after exposure, respectively. The quantification of the total cell number in the ears revealed that the cellular recruitment was more robust in SGE-sensitized mice than in non-sensitized mice, and the histological evaluation confirmed these findings. The immunophenotyping performed by flow cytometry revealed that mosquito bites were able to produce complex changes in cell populations present in the ears of non-sensitized and SGE-sensitized mice. When compared with steady-state ears, the leucocyte populations significantly recruited to the skin after mosquito bites in non-sensitized and sensitized mice were eosinophils, neutrophils, monocytes, inflammatory monocytes, mast cells, B-cells and CD4+ T-cells, each one with its specific kinetics. The changes in the absolute number of cells suggested two cell recruitment profiles: (i) a saliva-dependent migration; and (ii) a migration dependent on the immune status of the host. These findings suggest that mosquito bites influence the skin microenvironment by inducing differential cell migration, which is dependent on the degree of host sensitization to salivary molecules.
Assuntos
Aedes/imunologia , Quimiotaxia de Leucócito , Edema/imunologia , Mordeduras e Picadas de Insetos/imunologia , Leucócitos/imunologia , Mastócitos/imunologia , Saliva/imunologia , Pele/imunologia , Animais , Microambiente Celular , Modelos Animais de Doenças , Feminino , Cinética , Masculino , Camundongos Endogâmicos BALB C , Infiltração de NeutrófilosRESUMO
Objetivos: Determinar la resistencia de una cepa de Aedes. aegypti de Orotina a insecticidas organofosforados (temefós y malatión), y a piretroides (deltametrina, lambda cialotrina y cipermetrina). Métodos: La evaluación de la resistencia se efectuó mediante bioensayos larvarios. A partir de cada uno de los insecticidas se calculó la concentración letal 50% (CL50). También se calculó el factor de resistencia 50% (FR50) con respecto a la cepa Rockefeller, que sirvió como control susceptible. En casos de resistencia, la evaluación se repitió exponiendo previamente las larvas a butóxido de piperonilo (BP), S, S, S tributilfosforotritionato (DEF) y ácido etacrínico (AE) para establecer los mecanismos de detoxificación asociados con la resistencia. En cada caso se calculó un factor de sinergismo 50% (FS50). Resultados: La cepa Orotina mostró susceptibilidad a temefós, malatión, deltametrina y lambda cialotrina, pero mostró resistencia incipiente a cipermetrina (CL50= 0,01103 mg/L, FR50 = 5,32). Sólo el BP revertió la resistencia a este insecticida (FS50 =10,92), lo que representa un mecanismo de detoxificación asociado con el sistema citrocromo P450 monooxigenasa. Discusión: Aunque la cepa de Ae. aegyptide Orotina mostró resistencia a cipermetrina, existen otros insecticidas para los cuales fue susceptible, que brindan opciones a las autoridades de salud para su implementación en el control químico del vector. Conclusiones: El monitoreo de la resistencia es requerido para asegurar la efectividad de los insecticidas que se utilizan en el control químico.
Abstract Objectives: To determine the resistance to organophosphate (temephos and malathion) and pyrethroid (deltamethrin, lambda cyhalothrin, and cypermethrin) insecticides in a strain of Aedes aegypti from Orotina. Methods: The evaluation of the resistance was carried out by larval bioassays. Lethal concentration 50% (LC50) was calculated for each insecticide. A factor of resistance 50% (FR50) was also calculated with respect to the Rockefeller strain, which served as susceptible control. In cases of resistance, the evaluation was replicated by exposing the larvae to piperonyl butoxide (PB), S, S, S tributylphosphorotritionate (DEF), and ethacrynic acid (AE), in order to establish the detoxification mechanisms associated with the resistance. In these cases, a factor of synergism 50% (FS50) was also calculated. Results: The Orotina strain of Ae. aegyptiwas susceptible to temephos, malathion, deltamethrin and lambda cyhalothrin, but showed incipient resistance to cypermethrin (LC50 = 0.01103 mg/L, FR50 = 5.32). Only PB reversed the state of resistance (FS50 = 10.92), which suggests a detoxification mechanism associated with the citochrome P450 monooxygenase system. Discussion: Although the Ae. aegyptistrain from Orotina showed resistance to cypermethrin, it was susceptible to other insecticides, which can be used as alternative options for chemical control of the vector. Conclusions: Monitoring of resistance is required to ensure the effectiveness of insecticides used in chemical control.
Assuntos
Humanos , Resistência a Inseticidas , Aedes/imunologia , Vírus Chikungunya , Saúde Pública , Costa Rica , Dengue , Zika virusRESUMO
BACKGROUND: During the feeding process, the mouthparts of hematophagous mosquitoes break the skin barrier and probe the host tissue to find the blood. The saliva inoculated in this microenvironment modulates host hemostasis, inflammation and adaptive immune responses. However, the mechanisms involved in these biological activities remain poorly understood and few studies explored the potential roles of mosquito saliva on the individual cellular components of the immune system. Here, we report the immunomodulatory activities of Aedes aegypti salivary cocktail on murine peritoneal macrophages. RESULTS: The salivary gland extract (SGE) of Ae. aegypti inhibited the production of nitric oxide and inflammatory cytokines such as interleukin-6 (IL-6) and IL-12, as well as the expression of inducible nitric oxide synthase and NF-κB by murine macrophages stimulated by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). The spare respiratory capacity, the phagocytic and microbicidal activities of these macrophages were also reduced by Ae. aegypti SGE. These phenotypic changes are consistent with SGE suppressing the proinflammatory program of M1 macrophages. On the other hand, Ae. aegypti SGE did not influence M2-associated markers (urea production, arginase-1 and mannose receptor-1 expression), either in macrophages alternatively activated by IL-4 or in those classically activated by LPS plus IFN-γ. In addition, Ae. aegypti SGE did not display any cytokine-binding activity, nor did it affect macrophage viability, thus excluding supposed experimental artifacts. CONCLUSIONS: Given the importance of macrophages in a number of biological processes, our findings help to enlighten how vector saliva modulates vertebrate host immunity.
Assuntos
Aedes/imunologia , Diferenciação Celular , Inflamação , Macrófagos Peritoneais/imunologia , Saliva/imunologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Feminino , Fatores Imunológicos , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mosquitos Vetores/imunologia , Glândulas Salivares/química , Extratos de Tecidos/farmacologiaRESUMO
Dengue virus (DENV) is an arbovirus responsible for a significant number of deaths in Latin America. This virus is transmitted through the bite of Aedes aegypti, the main mosquito vector, and Ae. albopictus. During blood uptake, the mosquito injects its saliva into the host to facilitate the feeding process. Mosquito saliva contains potent immunogens capable of inducing antibody production directly related to mosquito bite exposure intensity and disease risk. In this study, we first determined the DENV infection status by two different DENV non-structural protein 1 (NS1) based rapid tests and qRT-PCR, then measured the levels of IgG1 and IgG4 antibodies against salivary proteins of Ae. aegypti female mosquitoes in volunteers living in a dengue endemic area. Our results show that people with a positive DENV diagnosis present higher levels of IgG4 antibodies than people with a negative diagnostic test, and that these antibody levels were higher in people with secondary DENV infections. With this study, we show that detection of IgG4 antibodies against mosquito saliva may be a reliable method to evaluate the risk of dengue infection.
Assuntos
Aedes/imunologia , Dengue/epidemiologia , Imunoglobulina G/imunologia , Proteínas e Peptídeos Salivares/imunologia , Adolescente , Adulto , Animais , Formação de Anticorpos/imunologia , Antígenos Virais/imunologia , Criança , Pré-Escolar , Colômbia/epidemiologia , Feminino , Humanos , Lactente , Pessoa de Meia-Idade , Fatores de Risco , Glândulas Salivares/metabolismo , Proteínas não Estruturais Virais/metabolismo , Adulto JovemRESUMO
Aedes (Stegomyia) aegypti, the principal global vector of dengue viruses, has differences in its susceptibility to dengue virus infection. We compared the global expression of genes in the midguts of Colombian Ae. aegypti dengue-susceptible (Cali-S) and dengue-refractory (Cali-MIB) field derived strains after ingesting either a sugarmeal, a bloodmeal, or a bloodmeal containing dengue virus serotype 2 (DENV-2). Microarray-based transcriptome analysis among treatments indicated a total of 4725 transcripts with differential expression between the two strains. Eleven genes were selected from different functional groups based on their significant up or down expression levels as well as reports in the literature suggesting they are associated with dengue virus elimination. We measured mRNA abundance of these 11 genes at 0, 8, 24, and 36 h postinfection using quantitative real time PCR (qPCR) to confirm the microarray results and assess any temporal patterns. Four genes were selected (Gram-negative binding protein-GNBP [AAEL009176], Niemann Pick Type-C2-NPC2 [AAEL015136], Keratinocyte lectin [AAEL009842], and Cathepsin-b [AAEL007585]) for knockdown experiments using RNA interference (RNAi) methodology to determine the phenotype (DENV-2 susceptible or refractory). Silencing GNBP, Cathepsin-b and Keratinocyte lectin reduced the percentage of mosquitoes with disseminated virus in the Cali-S strain to 8%, 20%, and 12% respectively compared with 96% in the controls. Silencing of NPC2 increased the percentage of mosquitos with disseminated virus infections in Cali-MIB to 66% compared with 35% in the controls. This study provides insight into genes that may contribute to the Cali-S susceptible and Cali-MIB refractory phenotypes in Ae. aegypti.
Assuntos
Aedes/genética , Vírus da Dengue/imunologia , Interações Hospedeiro-Patógeno/genética , Proteínas de Insetos/fisiologia , Mosquitos Vetores/genética , Aedes/imunologia , Aedes/virologia , Animais , Feminino , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno/imunologia , Mosquitos Vetores/imunologia , Mosquitos Vetores/virologiaRESUMO
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 chromatinprofiling 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. CONCLUSIONS: 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 infectioninduced 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.
Assuntos
Aedes/genética , Vírus da Dengue/fisiologia , Genes de Insetos , Proteínas de Insetos/genética , Mosquitos Vetores/genética , Aedes/imunologia , Animais , Montagem e Desmontagem da Cromatina , Feminino , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Imunidade Inata , Intestinos/virologia , Mosquitos Vetores/imunologia , Análise de Sequência de DNARESUMO
Priming is the conceptual term defining memory phenomenon in innate immune response. Numerous examples of enhanced secondary immune response have been described in diverse taxa of invertebrates; which naturally lacks memory response. In mosquitoes, a previous non-lethal challenge with some specific pathogens modify their immune response against the same microorganism; developing an improved antimicrobial reaction. In this work, we explore the ability of Aedes aegypti to mount a higher antiviral response upon a second oral DENV challenge. When previously challenged with inactive virus, we observed that the posterior infection showed a diminished number of DENV infectious particles in midguts and carcasses. In challenged tissues, we detected higher de novo midgut DNA synthesis than control group, as determined by DNA incorporation of 5-bromo-2-deoxyuridine. We demonstrated that inactive DENV particle are capable to induce DNA synthesis levels comparable to infective DENV. We considered the Drosophila melanogaster hindsight and Delta-Notch mosquitoes orthologues as potential de novo DNA synthesis pathway components (as observed in fly oocyte development and midgut tissue renewal). We showed that Aedes aegypti hindsight transcript relative expression levels were higher than control during DENV infection and inactive DENV particle alimentation. Also, Aedes aegypti second challenge with active DENV induced higher hindsight, Delta and Notch transcriptions in the primed mosquitoes (compared with the primary infection levels). Considering that the mosquito de novo DNA synthesis is concomitant to viral particle reduction, this finding opens a new perspective on the mechanisms underlying the vector antiviral immune response and the effector molecules involved.
Assuntos
Aedes/imunologia , Vírus da Dengue/fisiologia , Dengue/imunologia , Intestinos/virologia , Imunidade Adaptativa , Aedes/virologia , Animais , DNA , Humanos , Imunização Secundária , Memória Imunológica , Mosquitos Vetores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Carga Viral , Vírion/metabolismoRESUMO
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.
Assuntos
Animais , Feminino , Genes de Insetos , Proteínas de Insetos/genética , Aedes/genética , Vírus da Dengue/fisiologia , Mosquitos Vetores/genética , Regulação Viral da Expressão Gênica , Análise de Sequência de DNA , Aedes/imunologia , Montagem e Desmontagem da Cromatina , Interações Hospedeiro-Patógeno , Mosquitos Vetores/imunologia , Imunidade Inata , Intestinos/virologiaRESUMO
BACKGROUND: Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens. The ubiquitously expressed 14-3-3 protein family is evolutionarily conserved from yeast to mammals, and at least two isoforms of 14-3-3, the ε and ζ, have been identified in insects. These proteins have been shown to participate in both humoral and cellular immune responses in Drosophila. As mosquitoes of the genus Aedes are the primary vectors for arboviruses, causing several diseases such as dengue fever, yellow fever, Zika and chikungunya fevers, cell lines derived from these mosquitoes, Aag-2 from Aedes aegypti and C6/36 HT from Aedes albopictus, are currently used to study the insect immune system. Here, we investigated the role of 14-3-3 proteins (ε and ζ isoform) in phagocytosis, the main cellular immune responses executed by the insects, using Aedes spp. cell lines. RESULTS: We evaluated the mRNA and protein expression of 14-3-3ε and 14-3-3ζ in C6/36 HT and Aag-2 cells, and demonstrated that both proteins were localised in the cytoplasm. Further, in C6/36 HT cells treated with a 14-3-3 specific inhibitor we observed a notable modification of cell morphology with filopodia-like structure caused through cytoskeleton reorganisation (co-localization of 14-3-3 proteins with F-actin), more importantly the decrease in Salmonella typhimurium, Staphylococcus aureus and E. coli phagocytosis and reduction in phagolysosome formation. Additionally, silencing of 14-3-3ε and 14-3-3ζ expression by mean of specific DsiRNA confirmed the decreased phagocytosis and phagolysosome formation of pHrodo labelled E. coli and S. aureus bacteria by Aag-2 cells. CONCLUSION: The 14-3-3ε and 14-3-3ζ proteins modulate cytoskeletal remodelling, and are essential for phagocytosis of Gram-positive and Gram-negative bacteria in Aedes spp. cell lines.
Assuntos
Proteínas 14-3-3/metabolismo , Aedes/imunologia , Imunidade Celular , Proteínas de Insetos/metabolismo , Mosquitos Vetores/imunologia , Fagocitose , Proteínas 14-3-3/deficiência , Proteínas 14-3-3/genética , Actinas/metabolismo , Aedes/citologia , Animais , Linhagem Celular , Citoplasma/química , Citoesqueleto/fisiologia , Escherichia coli/imunologia , Inativação Gênica , Proteínas de Insetos/deficiência , Proteínas de Insetos/genética , Mosquitos Vetores/citologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Staphylococcus aureus/imunologiaRESUMO
Control of dengue virus (DenV) transmission, primarily based on strategies to reduce populations of the principle vector Stegomya aegypti (= Aedes aegypti) (Diptera: Culicidae), is difficult to sustain over time. Other potential strategies aim to manipulate characteristics such as vector competence (VC), the innate capacity of the vector to transmit the virus. Previous studies have identified genetic factors, including differential expression of apoptosis-related genes, associated with the refractory and susceptible phenotypes in selected strains of S. aegypti from Cali, Colombia. The present study was designed to evaluate the variability of VC in selected strains against different DenV serotypes and to determine whether field-collected mosquitoes respond similarly to selected laboratory strains in terms of enhanced or reduced expression of apoptosis-related genes. Vector competence differed between strains, but did not differ in response to different DenV serotypes. Differences in VC were observed among mosquitoes collected from different localities in Cali. The overexpression of the pro-apoptosis genes, caspase 16 and Aedronc, was conserved in field-collected refractory mosquitoes and the selected laboratory refractory strain. The results suggest that the apoptosis response is conserved among all refractory mosquitoes to inhibit the development of all DenV serotypes.
Assuntos
Aedes/fisiologia , Aedes/virologia , Dengue/transmissão , Imunidade Inata , Insetos Vetores/fisiologia , Aedes/genética , Aedes/imunologia , Animais , Vírus da Dengue/fisiologia , Feminino , Insetos Vetores/genética , Insetos Vetores/imunologia , Insetos Vetores/virologiaRESUMO
BACKGROUND: Aedes aegypti is the main vector of important arboviruses such as dengue, Zika and chikungunya. During infections mosquitoes can activate the immune pathways Toll, IMD and JAK/STAT to limit pathogen replication. RESULTS: Here, we evaluate the immune response profile of Ae. aegypti against Sindbis virus (SINV). We analyzed gene expression of components of Toll, IMD and JAK/STAT pathways and showed that a blood meal and virus infection upregulated aaREL2 in a microbiota-dependent fashion, since this induction was prevented by antibiotic. The presence of the microbiota activates IMD and impaired the replication of SINV in the midgut. Constitutive activation of the IMD pathway, by Caspar depletion, leads to a decrease in microbiota levels and an increase in SINV loads. CONCLUSION: Together, these results suggest that a blood meal is able to activate innate immune pathways, through a nutrient induced growth of microbiota, leading to upregulation of aaREL2 and IMD activation. Microbiota levels seemed to have a reciprocal interaction, where the proliferation of the microbiota activates IMD pathway that in turn controls bacterial levels, allowing SINV replication in Ae. aegypti mosquitoes. The activation of the IMD pathway seems to have an indirect effect in SINV levels that is induced by the microbiota.
Assuntos
Aedes/virologia , Regulação da Expressão Gênica/imunologia , Microbiota/fisiologia , Sindbis virus/fisiologia , Aedes/imunologia , Animais , Antibacterianos/farmacologia , Interações Hospedeiro-Patógeno , Microbiota/efeitos dos fármacos , Penicilinas/farmacologia , Estreptomicina/farmacologia , TranscriptomaRESUMO
BACKGROUND: Saliva and muscle-derived mosquito allergens have been purified and characterized. However, the complete set of allergens remains to be elucidated. In this study, we identified and characterized IgE-binding proteins from the mosquito species Aedes aegypti. METHODS: Serum was obtained from 15 allergic individuals with asthma and/or rhinitis and sensitized to mosquito. IgE binding was determined by ELISA. Total proteins from freeze-dried bodies of A. aegypti were extracted and IgE-reactive proteins were identified by 2D gel electrophoresis, followed by Western blot with pooled or individual sera. IgE-reactive spots were further characterized by mass spectrometry. RESULTS: Twenty-five IgE-reactive spots were identified, corresponding to 10 different proteins, some of which appeared as different variants or isoforms. Heat-shock cognate 70 (HSC-70) and tropomyosin showed IgE reactivity with 60% of the sera, lysosomal aspartic protease, and "AAEL006070-PA" (Uniprot: Q177P3) with 40% and the other proteins with <33.3% of the sera. Different variants or isoforms of tropomyosin, arginine or creatine kinase, glyceraldehyde-3-phosphate dehydrogenase (GPDH), calcium-binding protein, and phosphoglycerate mutase were also identified. The mixture of three allergens (Aed a 6, Aed a 8, and Aed a 10) seems to identify more than 80% of A. aegypti-sensitized individuals, indicating that these allergens should be considered when designing of improved mosquito allergy diagnostic tools. CONCLUSIONS: The newly identified allergens may play a role in the pathophysiology of mosquito allergy in the tropics, and some of them might be important arthropod-related proteins involved in cross-reactivity between A. aegypti and other allergenic arthropods.
Assuntos
Aedes/genética , Genoma de Inseto , Genômica , Aedes/imunologia , Alérgenos/genética , Alérgenos/imunologia , Alérgenos/isolamento & purificação , Animais , Proteínas de Artrópodes/metabolismo , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Genômica/métodos , Humanos , Hipersensibilidade/diagnóstico , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Masculino , Proteômica/métodosRESUMO
Duas tecnologias alternativas para o controle de Aedes aegypti foram avaliadas: a aplicação espacial de larvicida biológico-Bti em potenciais criadouros peridomiciliares, e a liberação de machos estéreis para inviabilização reprodutiva das fêmeas do mosquito. As ações foram realizadas pelos Agentes dos Serviços de Saúde em 15 vilas da Ilha de Fernando de Noronha, e em uma área (900 imóveis) no bairro da Várzea/Recife/Pernambuco. A efetividade dos métodos foi avaliada por indicadores entomológicos,estimados pela presença, quantidade e viabilidade de ovos do mosquito, coletados em armadilhas, e por marcadores genéticos. A aplicação de Bti, com atomizador costal, ocorreu a cada 30 dias em ambas as áreas. Uma redução importante e sustentável da população de A. aegypti,por este método, foi alcançada em 2015/2016 na Várzea e, em 2016, na Ilha, onde a remoção de 18 toneladas de resíduos sólidos em 2015 contribuiu possivelmente para os resultados. Machos esterilizados com radiação gama foram produzidos em massa no laboratório e liberados em uma das vilas da Ilha. A análise espaço-temporal dos indicadores, de dez/2015a ago/2016, revelou redução expressiva da densidade populacional do mosquito e da diversidade genética da população local. Ambas as abordagens parecem ter reduzido o contato homem-vetor e os riscos de transmissão de arboviroses na Ilha, apesar da elevada competência vetorial da população local do mosquito para os vírus Zika e Dengue. Os métodos testados se mostraram eficientes e passíveis de serem integradas às ações do SUS voltadas ao controle de A. aegypti.
Two alternative technologies were evaluated for Aedes aegypti control:the spraying of a biological larvicide (Bti) in potential peridomiciliarybreeding sites and the release of sterile males to promote reproductionblockage in wild females. Actions were carried out by Agents of theHealth Services, in 15 villages of the Fernando de Noronha Island and in 900 properties from the district of Várzea, Recife-PE. The effectiveness of both methods was evaluated by entomological indicators, estimatedby the presence, quantity and viability of eggs from the mosquito collectedin traps and through genetic markers. Bti was delivered by backpacksprayer every 30 days in both areas. A significant and sustainablereduction of the A. aegypti population as a result of this technique wasachieved in 2015/2016 in Várzea and in 2016 in the Island, where it wasstrengthened by the removal of 18 tons of solid waste in 2015. Malessterilized with gamma radiation were mass-produced in the laboratoryand released in one village of the Island. The spatiotemporal analysis ofthe indicators, from Dec/2015 to Aug/2016, revealed a significant reductionin mosquito density, which impacted on the genetic diversity of thelocal population. Both approaches seem to have reduced human-vectorcontact and the risk of arbovirus transmission in the Island, althoughlocal mosquito population presented high vector competence to Zikaand Dengue virus. These methods were efficient and could be integratedinto SUS actions directed to A. aegypti control.
Assuntos
Humanos , Bacillus thuringiensis , Controle de Vetores de Doenças , Aedes/imunologia , Controle Biológico de Vetores , EntomologiaRESUMO
BACKGROUND: The mosquito Aedes aegypti is a potential source of important clinically relevant allergens. However, the allergenicity and cross-reactivity of most of these has not been fully described. METHODS: Natural wild-type mosquito tropomyosin was purified by size exclusion and anionic-exchange chromatography from an A. aegypti extract. Further characterization was accomplished by MALDI-TOF/TOF. Two recombinant variants of tropomyosin were obtained by expression in Escherichia coli. Specific IgE measurement by ELISA and skin tests for mosquito extract were performed in 12 patients with asthma or allergy rhinitis residing on the Caribbean island of Martinique. Cross-reactivity between natural A. aegypti tropomyosin and recombinant tropomyosins from A. aegypti, house dust mite, shrimp and Ascaris lumbricoides was analyzed by ELISA competition. RESULTS: Four variants of natural tropomyosin were purified. A band of 32 kDa in SDS-PAGE representing 2 tropomyosin variants (Aed a 10.0101 and Aed a 10.0201) reacted with specific IgE of 4 of the 12 (33%) allergic patients and with rabbit polyclonal anti-shrimp tropomyosin. A high degree of cross-reactivity (60-70%) was detected between natural mosquito tropomyosin and Blo t 10, Der p 10 and Lit v 1, and a lower degree with Asc l 3 from A. lumbricoides (<30%). rAed a 10.0101 inhibited IgE binding to natural A. aegypti tropomyosin; however, rAed a 10.0201 showed a low inhibitory capacity. CONCLUSION: Tropomyosin is a new IgE-binding protein from A. aegypti. Two of the 4 variants identified showed different degree of cross-reactivity with tropomyosins from other arthropods. The potential allergenic role of each variant should be further investigated.