RESUMO
In French Guiana, the malaria, a parasitic infection transmitted by Anopheline mosquitoes, remains a disease of public health importance. To prevent malaria transmission, the main effective way remains Anopheles control. For an effective control, accurate Anopheles species identification is indispensable to distinguish malaria vectors from non-vectors. Although, morphological and molecular methods are largely used, an innovative tool, based on protein pattern comparisons, the Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) profiling, emerged this last decade for arthropod identification. However, the limited mosquito fauna diversity of reference MS spectra remains one of the main drawback for its large usage. The aim of the present study was then to create and to share reference MS spectra for the identification of French Guiana Anopheline species. A total of eight distinct Anopheles species, among which four are malaria vectors, were collected in 6 areas. To improve Anopheles identification, two body parts, legs and thoraxes, were independently submitted to MS for the creation of respective reference MS spectra database (DB). This study underlined that double checking by MS enhanced the Anopheles identification confidence and rate of reliable classification. The sharing of this reference MS spectra DB should make easier Anopheles species monitoring in endemic malaria area to help malaria vector control or elimination programs.
Assuntos
Anopheles/classificação , Mosquitos Vetores/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Anopheles/química , Guiana Francesa , Malária/classificação , Malária/transmissão , Especificidade da Espécie , TóraxRESUMO
BACKGROUND: Saliva of mosquitoes contains anti-platelet, anti-clotting, vasodilatory, anti-complement and anti-inflammatory substances that help the blood feeding process. The salivary polypeptides are at a fast pace of evolution possibly due to their relative lack of structural constraint and possibly also by positive selection on their genes leading to evasion of host immune pressure. RESULTS: In this study, we used deep mRNA sequence to uncover for the first time the sialomes of four Amazonian anophelines species (Anopheles braziliensis, A. marajorara, A. nuneztovari and A. triannulatus) and extend the knowledge of the A. darlingi sialome. Two libraries were generated from A. darlingi mosquitoes, sampled from two localities separated ~ 1100 km apart. A total of 60,016 sequences were submitted to GenBank, which will help discovery of novel pharmacologically active polypeptides and the design of specific immunological markers of mosquito exposure. Additionally, in these analyses we identified and characterized novel phasmaviruses and anpheviruses associated to the sialomes of A. triannulatus, A. marajorara and A. darlingi species. CONCLUSIONS: Besides their pharmacological properties, which may be exploited for the development of new drugs (e.g. anti-thrombotics), salivary proteins of blood feeding arthropods may be turned into tools to prevent and/or better control vector borne diseases; for example, through the development of vaccines or biomarkers to evaluate human exposure to vector bites. The sialotranscriptome study reported here provided novel data on four New World anopheline species and allowed to extend our knowledge on the salivary repertoire of A. darlingi. Additionally, we discovered novel viruses following analysis of the transcriptomes, a procedure that should become standard within future RNAseq studies.
Assuntos
Anopheles/genética , Peptídeos/genética , Saliva/química , Proteínas e Peptídeos Salivares/genética , Sequência de Aminoácidos/genética , Animais , Anopheles/química , Brasil , Humanos , Insetos Vetores/química , Insetos Vetores/genética , Mosquitos Vetores/genética , Ácido N-Acetilneuramínico/química , Peptídeos/química , RNA Mensageiro/genética , Proteínas e Peptídeos Salivares/química , Seleção Genética/genéticaRESUMO
INTRODUCTION: The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of specific IgG antibodies in the host, which can be used as specific biomarkers of exposure to Anopheles sundaicus . The objective of this study was to identify immunogenic proteins from the salivary glands of Anopheles sundaicus by reaction with sera from individuals living in malaria-endemic areas who are thus exposed to Anopheles mosquitoes. METHODS: IgG antibodies targeting salivary gland proteins in serum samples from individuals living in malaria-endemic areas were measured by enzyme-linked immunosorbent assay (ELISA). Sera from healthy individuals living in non-endemic areas were used as negative controls. Determination of the presence of salivary gland immunogenic proteins was carried out by western blotting. RESULTS: Sixteen bands appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with molecule weights ranging from 22 to 144kDa. Among the exposed individuals, IgG responses to salivary gland proteins were variable. Protein bands with molecular weights of 46, 41, 33, and 31kDa were the most immunogenic. These immunogenic proteins were consistently recognized by pooled serum and individual samples from people living in malaria-endemic areas but not by negative controls. CONCLUSIONS: These results support the potential use of immunogenic proteins from the salivary glands of Anopheles as candidate markers of bite exposure or in malaria vaccines.
Assuntos
Anopheles/imunologia , Proteínas de Insetos/imunologia , Glândulas Salivares/imunologia , Adulto , Animais , Anopheles/química , Biomarcadores/análise , Estudos de Casos e Controles , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Proteínas de Insetos/análise , Proteínas e Peptídeos Salivares/análiseRESUMO
The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of specific IgG antibodies in the host, which can be used as specific biomarkers of exposure to
IgG antibodies targeting salivary gland proteins in serum samples from individuals living in malaria-endemic areas were measured by enzyme-linked immunosorbent assay (ELISA). Sera from healthy individuals living in non-endemic areas were used as negative controls. Determination of the presence of salivary gland immunogenic proteins was carried out by western blotting.
Sixteen bands appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with molecule weights ranging from 22 to 144kDa. Among the exposed individuals, IgG responses to salivary gland proteins were variable. Protein bands with molecular weights of 46, 41, 33, and 31kDa were the most immunogenic. These immunogenic proteins were consistently recognized by pooled serum and individual samples from people living in malaria-endemic areas but not by negative controls.
These results support the potential use of immunogenic proteins from the salivary glands of
Assuntos
Adulto , Animais , Feminino , Humanos , Anopheles/imunologia , Proteínas de Insetos/imunologia , Glândulas Salivares/imunologia , Anopheles/química , Biomarcadores/análise , Estudos de Casos e Controles , Eletroforese em Gel de Poliacrilamida , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Proteínas de Insetos/análise , Proteínas e Peptídeos Salivares/análiseRESUMO
The main vector for transmission of malaria in Mexico is the Anopheles albimanus mosquito. The midgut of disease-transmitting mosquitoes carries out a variety of functions that are related to blood feeding. We analyzed the midgut of A. albimanus infected with Plasmodium berghei (resistant mosquito) using a proteomic approach to identify putative short peptides that are enriched in the midgut after blood feeding. Mosquito midguts were analyzed by two-dimensional electrophoresis to determine the changes in protein profiles. We identified 21 spot proteins that are differentially expressed in the blood of mosquitoes during the immune challenge. Molecular weight of the spots varied from 13 to 36 kDa, with a broad isoelectric point range of 3.92-8.90. We identified the differentially expressed proteins using mass spectrometry and constructed a proteomic data base of the A. albimanus midgut with diverse functions, some of them proteins with digestive and immunologic functions. Identification of these proteins may have important implications for understanding the blood meal digestion process, as well as developing novel vector control strategies and understanding parasite vector interactions.
Assuntos
Anopheles/genética , Anopheles/parasitologia , Expressão Gênica , Proteínas de Insetos/genética , Insetos Vetores/genética , Insetos Vetores/parasitologia , Plasmodium berghei/fisiologia , Sequência de Aminoácidos , Animais , Anopheles/química , Anopheles/metabolismo , Sistema Digestório/parasitologia , Eletroforese em Gel Bidimensional , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Insetos Vetores/química , Insetos Vetores/metabolismo , Anotação de Sequência MolecularRESUMO
En Colombia, el uso de mosquiteros es promovido por el Programa nacional de prevención y control de enfermedades transmitidas por vectores en áreas endémicas para malaria. A pesar de esto, son pocos los estudios realizados para evaluar su efectividad y en particular el grado de aceptabilidad por los usuarios lo cual es fundamental para mantener la adherencia a esta medida de control. En el presente estudio se evaluó, a pequeña escala, la aceptación de mosquiteros impregnados con insecticida de larga duración (Olyset®) y su efecto biológico en mosquitos, en una localidad de la región pacífica colombiana. Se instalaron 100 mosquiteros Olyset® en 30 viviendas con la recomendación, basados en una encuesta previa, de lavarlos mensualmente. Cuatro, ocho y doce meses después, se aplicó una encuesta en donde se determinó la aceptación teniendo en cuenta: el porcentaje de mosquiteros Olyset® en uso y la percepción de protección de los habitantes contra las picaduras de los mosquitos. Además, en la primera encuesta se indagó sobre los posibles efectos secundarios en la salud de los usuarios por el uso de estos mosquiteros. Grupos de hembras criadas en laboratorio de Anopheles albimanus Wiedemann y Culex quinquefasciatus Say fueron expuestos a los mosquiteros cada cuatro meses para determinar el efecto letal.
La proporción de mosquiteros Olyset® en uso fue disminuyendo en cada evaluación: 77,5%, 51,1% y 46,6% a los cuatro, ocho y doce meses, respectivamente. La misma tendencia fue observada en la percepción de protección contra la picadura de los mosquitos: 82,7%, 62% y 46%, respectivamente. La mortalidad de A. albimanus fue de 100%, 77,8%, 78,6% y 43,7% y la de C. quinquefasciatus de 100%, 42,3%, 74,1% y 5,7% a los cero, cuatro, ocho y doce meses de uso de los mosquiteros, respectivamente. Sólo un 5% de los usuarios manifestó irritación de la piel en los primeros días de uso de estos mosquiteros. En conclusión, durante el año de seguimiento se evidenció una disminución gradual en la aceptación de los mosquiteros Olyset®. Esto probablemente debido a que la notable reducción del efecto letal de los mosquiteros permitió un aumento en las tasas de picadura de los mosquitos que ocasionó la deserción de varios de los usuarios. Sin embargo, no se puede descartar que otras causas hayan contribuido a la reducción en la aceptación.
The use of bednets is promoted by the National Program of Promotion and Prevention of Diseases Transmitted by Vectors in malaria endemic areas of Colombia. This study estimated the acceptance and retention of long-lasting insecticide-treated nets (Olyset®). The study also measured the efficacy of the bednets after being washed once a month. Thirty dwellings were chosen in the village of Beté (Chocó department) to receive bednets. Interviews were administered in four month intervals to determine the frequency of bednet use and to assess the health outcomes. Among the recipients, nets were readily accepted and used at the beginning. However, usage began to decline: 77.5%, 51.1% and 46.6% after 4, 8 and 12 months of use respectively. In addition, 5% of recipients reported skin irritation after 15 days of continuous use. Anopheles albimanus and Culex quinquefasciatus females were exposed to used bednets every four months to estimate mortality and knock down rates. Bioassays revealed a decrease in efficacy over time. A. albimanus mortality was 77.8%, 78.6% and 43.7% after 4, 8 and 12 months of use, respectively. Mortality for C. quinquefasciatus was variable. A. albimanus mean knock down percent was above 80% at each sampling period. C. quinquefasciatus mean knock down percent was at a similar >80% at the 4 and 8 month sampling times, however, declined to 5.7% after 12 months of use. One year of use and monthly washings of bednets results in significantly less acceptance among recipients and a decrease in efficacy.
Assuntos
Humanos , Masculino , Feminino , Anopheles/química , Controle de Pragas/métodos , Inseticidas/toxicidade , Malária/prevenção & controle , Doenças Transmissíveis , Saúde PúblicaRESUMO
Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3--10 days was approximately 1.08 +/- 0.04 microg/female and 0.1 +/- 0.05 microg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.
Assuntos
Anopheles/química , Proteínas de Insetos/análise , Insetos Vetores/química , Glândulas Salivares/química , Animais , Anopheles/anatomia & histologia , Anopheles/enzimologia , Apirase/metabolismo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Feminino , Insetos Vetores/anatomia & histologia , Insetos Vetores/enzimologia , Malária/transmissão , Masculino , Glândulas Salivares/anatomia & histologia , Glândulas Salivares/enzimologia , alfa-Glucosidases/metabolismoRESUMO
Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3 - 10 days was approximately 1.08 ± 0.04 æg/female and 0.1 ± 0.05 æg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.
Proteínas das glândulas salivares do Anopheles dirus B (Diptera: Culicidae), vetor da malária humana foram determinadas e analisadas. A quantidade de proteínas das glândulas salivares em mosquitos com três a 10 dias de idade foi de aproximadamente 1,08 ± 0,04 æg/ fêmea e de 0,1 ± 0,05 æg/macho. As glândulas salivares de ambos os sexos mostraram organização morfológica semelhante à de outros mosquitos anofelinos. Em fêmeas, apirase acumula-se nas regiões distais, enquanto alfa-glucosidase foi encontrada na região proximal dos lóbulos laterais. Esta distribuição diferencial das enzimas analisadas reflete a especialização de diferentes regiões para alimentação de açucares e sangue. Análise SDS-PAGE revelou que pelo menos sete proteínas foram encontradas nas glândulas salivares de fêmeas, das quais cada região morfológica continha diferentes proteínas principais. Perfis eletroforéticos de proteínas semelhantes foram detectados comparando-se mosquitos não alimentados e alimentados por sangue, sugerindo que não existe proteína específica induzida pelo mesmo. Análise por gel poliacrilamida bi-dimensional mostrou a mais abundante proteína de glândulas salivares com aproximadamente 35 kilodaltons de massa molecular e ponto isoelétrico de aproximadamente 4,0. Estes resultados dão informações básicas que levariam a estudos adicionais sobre o papel das proteínas salivares do An. dirus B na transmissão da doença e hematofagia.
Assuntos
Animais , Masculino , Feminino , Anopheles/química , Proteínas de Insetos/análise , Insetos Vetores/química , Glândulas Salivares/química , Anopheles/anatomia & histologia , Anopheles/enzimologia , Apirase/metabolismo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Insetos Vetores/anatomia & histologia , Insetos Vetores/enzimologia , Malária/transmissão , Glândulas Salivares/anatomia & histologia , Glândulas Salivares/enzimologia , alfa-Glucosidases/metabolismoRESUMO
Biopterin, isoxanthopterin and 6-pterincarboxylic acid were identified in the head of the malaria vector mosquito Anopheles albimanus Weidemann (Diptera: Culicidae) by HPLC. Total pteridine concentrations (TPC) were estimated in heads, body parts (BP: abdomen, legs and wings) and whole bodies of insectary-reared and field-collected females, by spectrofluorometry, to investigate whether they could be used for age determination. Pteridine concentrations diminished with age in both mosquito groups. TPC correlated with chronological age in insectary-reared sugar-fed females (heads: r2 = 0.35, BP: r2 = 0.34, P < 0.001), but lower correlation occurred in blood-fed females (heads: r2 = 0.22, BP: r2 = 0.27). TPC differed among females of the same age fed with blood at different times (P < 0.05), indicating that bloodmeals modify the diminution rate of pteridines with age. Nevertheless, a polynomial significant correlation was documented for TPC and the number of ovipositions (heads: r2 = 0.24, BP: r2 = 0.27, whole body: r2 = 0.52, P < 0.001) in insectary-reared mosquitoes. This correlation was lower in field-collected mosquitoes (heads: r2 = 0.14, BP: r2 = 0.10, P < 0.05), which showed a remarkable pteridine increase in one-parous females. The correlation of TPC in whole body with physiological age was much less (r2 = 0.03). These observations indicate that TPC determination by spectrofluorometry is not a reliable method to estimate the age of An. albimanus females from the feral population.
Assuntos
Envelhecimento/fisiologia , Anopheles/química , Anopheles/fisiologia , Pteridinas/análise , Abdome , Criação de Animais Domésticos , Animais , Animais Selvagens , Constituição Corporal , Cromatografia Líquida de Alta Pressão , Dieta , Extremidades , Feminino , Cabeça , Insetos Vetores/química , Insetos Vetores/fisiologia , Malária , Ovário/fisiologia , Oviposição/fisiologia , Pteridinas/química , Asas de Animais/químicaRESUMO
The salivary gland proteins of adult female Anopheles darlingi were fractionated by reverse-phase HPLC and the five major peaks were submitted for amino-terminal sequencing using automated Edman degradation. The amino acid sequence of one of the purified salivary gland proteins showed similarity with the D7r3 protein of An. gambiae. Cloning and sequencing of two cDNAs allowed the prediction of the complete sequence of the An. darlingi D7 protein. The D7r3 protein is present specifically in adult female salivary glands of An. darlingi and despite being one of the major salivary gland proteins its function is not known. Predictions of secondary and tertiary structures revealed the similarity of the An. darlingi D7 protein to insect odorant binding proteins. This suggests that D7 proteins may act as carriers of hydrophobic molecules in mosquito saliva.