RESUMEN

SGTs (small glutamine-rich TPR-containing proteins) are dimeric proteins that belong to the class of co-chaperones characterized by the presence of TPR domains (containing tetratricopeptide repeats). Human (SGTA) and yeast (Sgt2) SGTs are characterized by three distinct domains: an N-terminal dimerization domain, a central TPR-domain important for binding to other proteins (chaperones included) and a C-terminal domain involved in hydrophobic interactions. Both these SGTs are involved in the cellular PQC (protein quality control) system, as they interact with chaperones and have functions that aid stress recovery. However, there are differences between them, such as structural features and binding specificities, that could be better understood if other orthologous proteins were studied. Therefore, we produced and characterized a putative SGT protein, designated AaSGT, from the mosquito Aedes aegypti, which is a vector of several diseases, such as dengue and Zika. The protein was produced as a folded dimer which was stable up to 40 °C and was capable of binding to AaHsp90 and fully protecting a model protein, α-synuclein, from aggregation. The conformation of AaSGT was investigated by biophysical tools and small angle X-ray scattering, which showed that the protein had an elongated conformation and that its C-terminal domain was mainly disordered. The results with a C-terminal deletion mutant supported these observations. Altogether, these results are consistent with those from other functional SGT proteins and add to the understanding of the PQC system in Aedes aegypti, an important aim that may help to develop inhibitory strategies against this vector of neglected diseases.

Asunto(s)

Aedes/química , Proteínas de Insectos/química , Chaperonas Moleculares/química , Multimerización de Proteína , Aedes/genética , Aedes/metabolismo , Animales , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

The mosquito Aedes aegypti L. is a vector transmitting diseases such as dengue, chikungunya and Zika virus fever. The water-soluble lectin from Moringa oleifera Lam. seeds (WSMoL) is larvicidal, ovicidal and can stimulate oviposition in A. aegypti. This study aimed to investigate whether WSMoL could bind to membrane proteins from A. aegypti legs. Initially, proteins from the legs were extracted using sodium deoxycholate, digitonin, dodecyl sodium sulfate (SDS) or Triton X-100. The protein concentration was found to be higher in the extract obtained using Triton X-100, which was applied to a WSMoL-Sepharose column. The adsorbed proteins were evaluated using gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE) in presence of SDS. The similarity in the sequences of adsorbed proteins with those available in databases was determined. The proteins adsorbed on the matrix were eluted forming a single peak. Gel filtration chromatography and SDS-PAGE revealed the presence of proteins with molecular masses of approximately 20 kDa and polypeptide bands of 17.0 and 23.7 kDa, respectively. MS/MS analysis indicated similarity between these proteins and ABC carriers, which are expressed in the legs of mosquitos. WSMoL could bind to membrane proteins in the legs of A. aegypti females and induce oviposition through these interactions.

Asunto(s)

Aedes , Proteínas de Insectos/química , Proteínas de la Membrana/química , Moringa oleifera/química , Oviposición/efectos de los fármacos , Lectinas de Plantas , Aedes/anatomía & histología , Aedes/química , Animales , Proteínas de Insectos/metabolismo , Proteínas de la Membrana/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/farmacología

Asunto(s)

Resistencia a los Insecticidas/efectos de los fármacos , Aedes/química

RESUMEN

Blood-feeding exoparasites are rich sources of protease inhibitors, and the mosquito Aedes aegypti, which is a vector of Dengue virus, Yellow fever virus, Chikungunya virus and Zika virus, is no exception. AaTI is a single-domain, noncanonical Kazal-type serine proteinase inhibitor from A. aegypti that recognizes both digestive trypsin-like serine proteinases and the central protease in blood clotting, thrombin, albeit with an affinity that is three orders of magnitude lower. Here, the 1.4 Šresolution crystal structure of AaTI is reported from extremely tightly packed crystals (∼22% solvent content), revealing the structural determinants for the observed inhibitory profile of this molecule.

Asunto(s)

Aedes/química , Proteínas de Insectos/química , Insectos Vectores/química , Inhibidores de Serinpeptidasas Tipo Kazal/química , Trombina/química , Aedes/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Insectos Vectores/metabolismo , Simulación del Acoplamiento Molecular , Pichia/genética , Pichia/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Inhibidores de Serinpeptidasas Tipo Kazal/genética , Inhibidores de Serinpeptidasas Tipo Kazal/metabolismo , Trombina/antagonistas & inhibidores , Trombina/genética , Trombina/metabolismo

RESUMEN

Criminal traces commonly found at crime scenes may present mixtures from two or more individuals. The scene of the crime is important for the collection of various types of traces in order to find the perpetrator of the crime. Thus, we propose that hematophagous mosquitoes found at crime scenes can be used to perform genetic testing of human blood and aid in suspect investigation. The aim of the study was to obtain a single Aedes aegypti mosquito profile from a human DNA mixture containing genetic materials of four individuals. We also determined the effect of blood acquisition time by setting time intervals of 24, 48, and 72 h after the blood meal. STR loci and amelogenin were analyzed, and the results showed that human DNA profiles could be obtained from hematophagous mosquitos at 24 h following the blood meal. It is possible that hematophagous mosquitoes can be used as biological remains at the scene of the crime, and can be used to detect human DNA profiles of up to four individuals.

Asunto(s)

Aedes/química , Dermatoglifia del ADN/métodos , ADN/aislamiento & purificación , Genética Forense/métodos , Aedes/fisiología , Animales , Mordeduras y Picaduras/sangre , Células Sanguíneas/química , Crimen , ADN/genética , Femenino , Pruebas Genéticas/métodos , Voluntarios Sanos , Humanos , Masculino , Repeticiones de Microsatélite

RESUMEN

The coordination of physiological processes requires precise communication between cells. Cellular interactions allow cells to be functionally related, facilitating the maintaining of homeostasis. Neuropeptides functioning as intercellular signals are widely distributed in Metazoa. It is assumed that neuropeptides were the first intercellular transmitters, appearing early during the evolution. In Cnidarians, neuropeptides are mainly involved in neurotransmission, acting directly or indirectly on epithelial muscle cells, and thereby controlling coordinated movements. Allatostatins are a group of chemically unrelated neuropeptides that were originally characterized based on their ability to inhibit juvenil hormone synthesis in insects. Allatostatin-C has pleiotropic functions, acting as myoregulator in several insects. In these studies, we analyzed the myoregulatory effect of Aedes aegypti Allatostatin-C in Hydra sp., a member of the phylum Cnidaria. Allatostatin-C peptide conjugated with Qdots revealed specifically distributed cell populations that respond to the peptide in different regions of hydroids. In vivo physiological assays using Allatostatin-C showed that the peptide induced changes in shape and length in tentacles, peduncle and gastrovascular cavity. The observed changes were dose and time dependent suggesting the physiological nature of the response. Furthermore, at highest doses, Allatostatin-C induced peristaltic movements of the gastrovascular cavity resembling those that occur during feeding. In silico search of putative Allatostatin-C receptors in Cnidaria showed that genomes predict the existence of proteins of the somatostatin/Allatostatin-C receptors family. Altogether, these results suggest that Allatostatin-C has myoregulatory activity in Hydra sp, playing a role in the control of coordinated movements during feeding, indicating that Allatostatin-C/Somatostatin based signaling might be an ancestral mechanism.

Asunto(s)

Evolución Molecular , Neuropéptidos/metabolismo , Somatostatina/metabolismo , Aedes/química , Animales , Hydra/efectos de los fármacos , Hydra/crecimiento & desarrollo , Neuropéptidos/química , Neuropéptidos/genética , Neuropéptidos/farmacología , Transducción de Señal , Somatostatina/genética , Somatostatina/farmacología

RESUMEN

Current therapies for inflammatory bowel disease (IBD) are not totally effective, resulting in persistent and recurrent disease for many patients. Mosquito saliva contains immunomodulatory molecules and therein could represent a novel therapy for IBD. Here, we demonstrated the therapeutic activity of salivary gland extract (SGE) of Aedes aegypti on dextran sulfate sodium (DSS)-induced colitis. For this purpose, C57BL/6 male mice were exposed to 3% DSS in drinking water and treated with SGE at early (days 3-5) or late (days 5-8) time points, followed by euthanasia on days 6 and 9, respectively, for sample collection. The results showed an improvement in clinical disease outcome and postmortem scores after SGE treatment, accompanied by the systemic reduction in peripheral blood lymphocytes, with no impact on bone marrow and mesenteric lymph nodes cellularity or macrophages toxicity. Moreover, a local diminishment of IFN-γ, TNF-α, IL-1ß and IL-5 cytokines together with a reduction in the inflammatory area were observed in the colon of SGE-treated mice. Strikingly, early treatment with SGE led to mice protection from a late DSS re-challenging, as observed by decreased clinical and postmortem scores, besides reduced circulating lymphocytes, indicating that the mosquito saliva may present components able to prevent disease relapse. Indeed, high performance liquid chromatography (HPLC) experiments pointed to a major SGE pool fraction (F3) able to ameliorate disease signs. In conclusion, SGE and its components might represent a source of important immunomodulatory molecules with promising therapeutic activity for IBD.

Asunto(s)

Aedes/química , Factores Inmunológicos/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Glándulas Salivales/química , Extractos de Tejidos/uso terapéutico , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Colon/efectos de los fármacos , Colon/inmunología , Colon/patología , Citocinas/análisis , Sulfato de Dextran/administración & dosificación , Sulfato de Dextran/farmacología , Modelos Animales de Enfermedad , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/efectos adversos , Factores Inmunológicos/inmunología , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Extractos de Tejidos/administración & dosificación , Extractos de Tejidos/efectos adversos , Extractos de Tejidos/inmunología

RESUMEN

Girgensohnine alkaloid was used as a natural model in the design and generation of new alkaloid-like α-aminonitrile series that was completed by the use of SSA-catalyzed Strecker reaction between commercial and inexpensive substituted benzaldehydes, piperidine (pyrrolidine, morpholine and N-methylpiperazine) and acetone cyanohydrin. Calculated ADMETox parameters of the designed analogs revealed their good pharmacokinetic profiles indicating lipophilic characteristics. In vitro AChE enzyme test showed that obtained α-aminonitriles could be considered as AChEIs with micromolar IC50 values ranging from 42.0 to 478.0 µM (10.3-124.0 µg/mL). Among this series, the best AChE inhibitor was the pyrrolidine α-aminonitrile 3 (IC50 = 42 µM), followed by the piperidine α-aminonitriles 2 and 6 (IC50 = 45 µM and IC50 = 51 µM, respectively), and the compound 7 (IC50 = 51 µM). In vivo insecticidal activity of more active AChEIs against Aedes aegypti larvae was also performed showing a good larvicidal activity at concentrations less than 140 ppm, highlighting products 2 and 7 that could serve as lead compounds to develop new potent and selective insecticides.

Asunto(s)

Acetilcolinesterasa/metabolismo , Aedes/química , Inhibidores de la Colinesterasa/farmacología , Dengue/tratamiento farmacológico , Diseño de Fármacos , Insectos Vectores/química , Insecticidas/farmacología , Nitrilos/farmacología , Pirrolidinas/farmacología , Animales , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/química , Relación Dosis-Respuesta a Droga , Insecticidas/síntesis química , Insecticidas/química , Larva/efectos de los fármacos , Estructura Molecular , Nitrilos/síntesis química , Nitrilos/química , Pirrolidinas/síntesis química , Pirrolidinas/química , Relación Estructura-Actividad

RESUMEN

Norte de Santander is a region in Colombia with a high incidence of dengue virus (DENV). In this study, we examined the serum concentration of anti-Aedes salivary gland extract (SGE) antibodies as a biomarker of DENV infection and transmission, and assessed the duration of anti-SGE antibody concentration after exposure to the vector ceased. We also determined whether SGE antibody concentration could differentiate between positive and negative DENV infected individuals and whether there are differences in exposure for each DENV serotype. We observed a significant decrease in the concentration of IgG antibodies at least 40 days after returning to an "Ae. aegypti-free" area. In addition, we found significantly higher anti-SGE IgG concentrations in DENV positive patients with some difference in exposure to mosquito bites among DENV serotypes. We conclude that the concentration of IgG antibodies against SGE is an accurate indicator of risk of dengue virus transmission and disease presence.

Asunto(s)

Aedes/química , Anticuerpos/inmunología , Mezclas Complejas/química , Virus del Dengue , Inmunoglobulina G/inmunología , Mordeduras y Picaduras de Insectos/inmunología , Insectos Vectores/química , Glándulas Salivales/química , Adulto , Aedes/inmunología , Animales , Anticuerpos/sangre , Colombia/epidemiología , Mezclas Complejas/inmunología , Dengue/sangre , Dengue/inmunología , Dengue/transmisión , Femenino , Humanos , Inmunoglobulina G/sangre , Mordeduras y Picaduras de Insectos/sangre , Mordeduras y Picaduras de Insectos/epidemiología , Insectos Vectores/inmunología , Masculino , Factores de Riesgo , Glándulas Salivales/inmunología

RESUMEN

BACKGROUND: Aedes albopictus is a vector for several fatal arboviruses in tropical and sub-tropical regions of the world. The midgut of the mosquito is the first barrier that pathogens must overcome to establish infection and represents one of the main immunologically active sites of the insect. Nevertheless, little is known about the proteins involved in the defense against pathogens, and even in the processing of food, and the detoxification of metabolites. The identification of proteins exclusively expressed in the midgut is the first step in understanding the complex physiology of this tissue and can provide insight into the mechanisms of pathogen-vector interaction. However, identification of the locally expressed proteins presents a challenge because the Ae. albopictus genome has not been sequenced. METHODS: In this study, two-dimensional electrophoresis (2DE) was combined with liquid chromatography in line with tandem mass spectrometry (LC-MS/MS) and data mining to identify the major proteins in the midgut of sugar-fed Ae. albopictus females. RESULTS: Fifty-six proteins were identified by sequence similarity to entries from the Ae. aegypti genome. In addition, two hypothetical proteins were experimentally confirmed. According to the gene ontology analysis, the identified proteins were classified into 16 clusters of biological processes. Use of the STRING database to investigate protein functional associations revealed five functional networks among the identified proteins, including a network for carbohydrate and amino acid metabolism, a group associated with ATP production and a network of proteins that interact during detoxification of toxic free radicals, among others. This analysis allowed the assignment of a potential role for proteins with unknown function based on their functional association with other characterized proteins. CONCLUSION: Our findings represent the first proteome map of the Ae. albopictus midgut and denotes the first steps towards the description of a comprehensive proteome map of this vector. In addition, the data contributes to the functional annotation of Aedes spp. genomes using mass spectrometry-based proteomics data combined with complementary gene prediction methods.

Asunto(s)

Aedes/química , Carbohidratos/administración & dosificación , Dieta/métodos , Proteínas de Insectos/biosíntesis , Proteoma/análisis , Animales , Cromatografía Liquida , Biología Computacional , Electroforesis en Gel Bidimensional , Femenino , Tracto Gastrointestinal/química , Espectrometría de Masas en Tándem

RESUMEN

Juvenile hormones (JHs) play key roles in regulating metamorphosis and reproduction in insects. The last two steps of JH synthesis diverge depending on the insect order. In Lepidoptera, epoxidation by a P450 monooxygenase precedes esterification by a juvenile hormone acid methyltransferase (JHAMT). In Orthoptera, Dictyoptera, Coleoptera and Diptera epoxidation follows methylation. The aim of our study was to gain insight into the structural basis of JHAMT's substrate recognition as a means to understand the divergence of these pathways. Homology modeling was used to build the structure of Aedes aegypti JHAMT. The substrate binding site was identified, as well as the residues that interact with the methyl donor (S-adenosylmethionine) and the carboxylic acid of the substrate methyl acceptors, farnesoic acid (FA) and juvenile hormone acid (JHA). To gain further insight we generated the structures of Anopheles gambiae, Bombyx mori, Drosophila melanogaster and Tribolium castaneum JHAMTs. The modeling results were compared with previous experimental studies using recombinant proteins, whole insects, corpora allata or tissue extracts. The computational study helps explain the selectivity toward the (10R)-JHA isomer and the reduced activity for palmitic and lauric acids. The analysis of our results supports the hypothesis that all insect JHAMTs are able to recognize both FA and JHA as substrates. Therefore, the order of the methylation/epoxidation reactions may be primarily imposed by the epoxidase's substrate specificity. In Lepidoptera, epoxidase might have higher affinity than JHAMT for FA, so epoxidation precedes methylation, while in most other insects there is no epoxidation of FA, but esterification of FA to form MF, followed by epoxidation to JH III.

Asunto(s)

Aedes/enzimología , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Hormonas Juveniles/biosíntesis , Metiltransferasas/química , Metiltransferasas/metabolismo , Aedes/química , Aedes/genética , Aedes/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Ácidos Grasos Insaturados/metabolismo , Proteínas de Insectos/genética , Insectos/química , Insectos/enzimología , Insectos/genética , Isomerismo , Hormonas Juveniles/química , Metiltransferasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Alineación de Secuencia , Especificidad por Sustrato

RESUMEN

O controle de larvas e adultos de Aedes aegypti é a forma disponível para controlar a Dengue. O uso de temefós é importante para o controle de larvas. O controle de adultos é realizado por nebulização de malathion nos imóveis, apresentando riscos ocupacionais aos aplicadores. Objetivou-se avaliar o período residual do temefós em recipientes de diferentes materiais pela validação de método analítico; propor um nível de aceitabilidade de controle de larvas; determinar o período residual de controle (PRC) em recipientes de borracha (pneus); propor um procedimento para monitorar o PRC em Pontos Estratégicos (PEs); avaliar as exposições dérmicas (EDs) e respiratórias (ERs); calcular a necessidade de controle das exposições potenciais (EPs); comparar a eficiência de duas vestimentas de proteção; classificar a segurança das condições de trabalho sem e com o uso das vestimentas; calcular a necessidade de controle das exposições e o tempo de trabalho seguro (TTS) proporcionado pelas vestimentas. O método validado é adequado para determinar a concentração do temefós na água. O material do recipiente afeta a dissipação do temefós. O controle de larvas nos pneus é aceitável (90%) por 22 dias após a aplicação. O procedimento com pneus amostradores é adequado para determinar o PRC de larvas em PEs. As EDs foram muito superiores às ERs. É necessário controlar 91,34 % das EPs. A vestimenta Agro Light é mais eficiente que a da SUCEN e a única em que a atividade foi classificada como segura. A atividade executada com a vestimenta da SUCEN se torna segura se for aumentado o controle da exposição em 11,4 %, ou a redução do tempo de trabalho ao TTS de 1,4 horas...

Asunto(s)

Animales , Aedes/crecimiento & desarrollo , Aedes/metabolismo , Aedes/patogenicidad , Aedes/química

RESUMEN

BACKGROUND: Microsatellite markers have proven useful in genetic studies in many organisms, yet microsatellite-based studies of the dengue and yellow fever vector mosquito Aedes aegypti have been limited by the number of assayable and polymorphic loci available, despite multiple independent efforts to identify them. Here we present strategies for efficient identification and development of useful microsatellites with broad coverage across the Aedes aegypti genome, development of multiplex-ready PCR groups of microsatellite loci, and validation of their utility for population analysis with field collections from Haiti. RESULTS: From 79 putative microsatellite loci representing 31 motifs identified in 42 whole genome sequence supercontig assemblies in the Aedes aegypti genome, 33 microsatellites providing genome-wide coverage amplified as single copy sequences in four lab strains, with a range of 2-6 alleles per locus. The tri-nucleotide motifs represented the majority (51%) of the polymorphic single copy loci, and none of these was located within a putative open reading frame. Seven groups of 4-5 microsatellite loci each were developed for multiplex-ready PCR. Four multiplex-ready groups were used to investigate population genetics of Aedes aegypti populations sampled in Haiti. Of the 23 loci represented in these groups, 20 were polymorphic with a range of 3-24 alleles per locus (mean = 8.75). Allelic polymorphic information content varied from 0.171 to 0.867 (mean = 0.545). Most loci met Hardy-Weinberg expectations across populations and pairwise FST comparisons identified significant genetic differentiation between some populations. No evidence for genetic isolation by distance was observed. CONCLUSION: Despite limited success in previous reports, we demonstrate that the Aedes aegypti genome is well-populated with single copy, polymorphic microsatellite loci that can be uncovered using the strategy developed here for rapid and efficient screening of genome supercontig assemblies. These loci are suitable for genetic and population studies using multiplex-PCR.

Asunto(s)

Aedes/química , Genoma de los Insectos , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Aedes/genética , Animales , Dosificación de Gen , Genética de Población , Haití

RESUMEN

Dengue is a vector-borne disease transmitted by the mosquito Aedes aegypti. The incidence of dengue disease shows a clear dependence on seasonal variation. How does the temperature affect the incidence? We addressed this question indirectly by estimating the size of the A. aegypti population for different temperatures applying population dynamics theory. In order to achieve this objective we designed temperature-controlled experiments to assess the entomological parameters regarding the mosquito's life-cycle at different temperatures. By obtaining the mortality, transition and oviposition rates for different stages of the life-cycle of the mosquito we were able to calculate the basic offspring number Q0, which is the capacity of vector reproduction and ultimately gives the size of the vector population...

Asunto(s)

Animales , Aedes , Aedes/crecimiento & desarrollo , Aedes/química

RESUMEN

The incidence of dengue infection, a vector-borne disease transmitted by the mosquito Aedes aegypti, shows clear dependence on seasonal variation. Based on the quantification method that furnishes the size of the A. aegypti population in terms of the estimated entomological parameters for different temperatures, we assessed the risk of dengue outbreaks. The persistence and severity of epidemics can be assessed by the basic reproduction number R0, which varies with temperature. The expression for R0 obtained from ‘true’ and ‘pseudo’ mass action laws for dengue infection is discussed...

Asunto(s)

Animales , Aedes , Aedes/crecimiento & desarrollo , Aedes/metabolismo , Aedes/química

RESUMEN

BACKGROUND: One of the major problems concerning dengue transmission is that embryos of its main vector, the mosquito Aedes aegypti, resist desiccation, surviving several months under dry conditions. The serosal cuticle (SC) contributes to mosquito egg desiccation resistance, but the kinetics of SC secretion during embryogenesis is unknown. It has been argued that mosquito SC contains chitin as one of its components, however conclusive evidence is still missing. RESULTS: We observed an abrupt acquisition of desiccation resistance during Ae. aegypti embryogenesis associated with serosal cuticle secretion, occurring at complete germ band extension, between 11 and 13 hours after egglaying. After SC formation embryos are viable on dry for at least several days. The presence of chitin as one of the SC constituents was confirmed through Calcofluor and WGA labeling and chitin quantitation. The Ae. aegypti Chitin Synthase A gene (AaCHS1) possesses two alternatively spliced variants, AaCHS1a and AaCHS1b, differentially expressed during Ae. aegypti embryonic development. It was verified that at the moment of serosal cuticle formation, AaCHS1a is the sole variant specifically expressed. CONCLUSION: In addition to the peritrophic matrix and exoskeleton, these findings confirm chitin is also present in the mosquito serosal cuticle. They also point to the role of the chitinized SC in the desiccation resistance of Ae. aegypti eggs. AaCHS1a expression would be responsible for SC chitin synthesis. With this embryological approach we expect to shed new light regarding this important physiological process related to the Ae. aegypti life cycle.

Asunto(s)

Aedes/embriología , Quitina/fisiología , Desecación , Óvulo/crecimiento & desarrollo , Aedes/química , Aedes/metabolismo , Secuencia de Aminoácidos , Animales , Quitina/química , Quitina Sintasa/biosíntesis , Quitina Sintasa/genética , Dengue/transmisión , Proteínas del Huevo/química , Proteínas del Huevo/genética , Femenino , Proteínas de Insectos/química , Proteínas de Insectos/genética , Datos de Secuencia Molecular , Óvulo/química , Empalme del ARN , Factores de Tiempo

RESUMEN

Morphology and cytochemistry of Aedes aegypti’s cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae). The first cellular line of Aedes aegypti was developed by Grace in 1966; afterwards, other cellular lines of this species have been generated. These have been used for the study of pathogenic organisms like viruses, bacteria and parasites, which demonstrates their importance in biomedical applications. This research describes, for the first time, some cytochemical characteristics of A. aegypti cell cultures, that were infected with (MHOM/CO/87CL412) strain of Leishmania panamensis. A morphological study of the cell culture was also carried out. Maintenance of the cell culture, parasites and infection in vitro were carried out in the Laboratory of Entomology, Cell Biology and Genetics of the Universidad de La Salle. The cell cultures infected with the parasite were maintained in a mixture of mediums Grace/L15, supplemented with 10 % fetal bovine serum (FBS) at pH 6.8 and a temperature of 26 ºC, during 3, 6 and 9 post-infection days. After this, these cell cultures were processed through High Resolution Light Microscopy (HRLM) and Transmission Electron Microscopy (TEM) based on standard protocols defined by the Group of Microscopy and Image Analyses of the Instituto Nacional de Salud. Semi-fine slices of 1 µm colored with toluidine blue were used for the morphological analysis of the culture, and ultra fine cuts of 60 to 90 nm stained with uranyl acetate and lead citrate where used for the ultrastructural study. In addition, PAS and peroxidase staining was carried out in cells fixed with methanol. The morphometric study was analyzed with software ImageJ (NIH). In the semi-fine slices, small cells were observed showing fibroblastic appearance 10.84±2.54 µm in length and 5.31±1.26 µm wide; other cells had epithelial appearance with a great peripheral nucleus, voluminous and vacuolated cytoplasm, 23.04±4,00 µm in length and 13.96±3.70 µm wide. These last ones predominated over the ones with fibroblastic appearance. Regarding the PAS coloration, 7.08 % of the cells presented abundant PAS positive cytoplasmatic granules which indicated polysaccharides presence. The peroxidase test gave a negative result. The greatest percentage of infection (18.90 %) of one total of 101 cells, turned up by day 6. Some cells analyzed by TEM presented a vacuolated aspect cytoplasm; some contained parasites, other fibrillar material and others were empty. The results indicate that A. aegypti cell culture can support the internalization and transformation of the parasite, which demonstrates the capacity that these cell cultures have to be infected with L. panamensis and to maintain the infection for approximately one week. Rev. Biol. Trop. 56 (2): 447-458. Epub 2008 June 30.

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La primera línea celular de Aedes aegypti fue establecida por Grace en 1966 y desde entonces se han utilizado para el estudio de virus, bacterias y parásitos. En el presente trabajo se describen, por primera vez, algunas características citoquímicas de los cultivos celulares de A. aegypti, infectados con la cepa (MHOM/CO/87CL412) de Leishmania panamensis. También se realizó un estudio morfológico de las células del cultivo. Se observaron 30 células pequeñas con apariencia fibrolastoide de 10.84±2.54 µm de largo y 5.31±1.26 µm de ancho; otras 30 presentaron apariencia epitelioide con 23.04±4.00 µm de largo y 13.96±3.70 µm de ancho; éstas últimas predominaron sobre las de apariencia fibroblastoide. De 113 células, un 7.08%, presentaron abundantes gránulos citoplasmáticos positivos con la coloración de PAS, indicando presencia de polisacáridos. La prueba de peroxidasa dio un resultado negativo. El mayor porcentaje de infección (18.90%), de un total de 101 células, se presentó el día 6. Ultraestructuralmente, las células presentaron un citoplasma con aspecto vacuolado; algunas contenían parásitos, otras material fibrilar y otras estaban vacías. Los resultados indican que los cultivos celulares de A. aegypti pueden ser infectados por L. panamensis y mantener dicho proceso por aproximadamente una semana.

Asunto(s)

Animales , Aedes , Leishmania guyanensis/fisiología , Aedes/química , Aedes/citología , Aedes/parasitología , Aedes/ultraestructura , Células Cultivadas , Microscopía Electrónica de Transmisión

RESUMEN

Vector control with larvicides is an important component in dengue control programmes. In Brazil, the extensive use of temephos has led to the evolution of resistance in Aedes aegypti populations in manyparts of the country. One of the strategies proposed for managing temephos resistance is the use of theinsect-growth regulator – pyriproxyfen. This study evaluated the lethal concentration for this product in mosquito populations with different profiles of susceptibility to temephos and semi-field residual responseto a commercial product. The results suggest the possibility of cross-resistance between temephos and pyriproxyfen.

Asunto(s)

Animales , Aedes/crecimiento & desarrollo , Aedes/química , Compuestos Organofosforados

RESUMEN

We adapted the Seliwanoff method to quantify fructose in mosquitoes. This method showed a minimum detection limit of 2.4 microg of fructose, and was more reliable and nearly four times more sensitive than the anthrone test. The Seliwanoffmethod was used to measure the maximum sugar intake by individual mosquitoes and to determine the digestion time of this nutrient by both Aedes aegypti and Aedes albopictus in the laboratory. Sugar intake by Ae. albopictus was up to 1.7 times higher than that of Ae. aegypti. The amount of sugar ingested by females was up to 2.5 times higher than that of males in both species. After 48 h, a fructose meal was not detected any longer in either species. The Seliwanoffmethod was applied to measure fructose content of field-collected Ae. aegypti males and females in Rio de Janeiro. Results showed that even Ae. aegypti females do feed on sugars. The standardized Seliwanoff method proved to be reliable for measuring the sugar content of individual mosquitoes and can be used wherever estimation of small quantities of fructose is needed.

Asunto(s)

Aedes/fisiología , Conducta Alimentaria , Fructosa/metabolismo , Aedes/química , Animales , Femenino , Fructosa/química , Masculino

RESUMEN

Morphology and cytochemistry of Aedes aegypti's cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae). The first cellular line of Aedes aegypti was developed by Grace in 1966; afterwards, other cellular lines of this species have been generated. These have been used for the study of pathogenic organisms like viruses, bacteria and parasites, which demonstrates their importance in biomedical applications. This research describes, for the first time, some cytochemical characteristics of A. aegypti cell cultures, that were infected with (MHOM/CO/87CL412) strain of Leishmania panamensis. A morphological study of the cell culture was also carried out. Maintenance of the cell culture, parasites and infection in vitro were carried out in the Laboratory of Entomology, Cell Biology and Genetics of the Universidad de La Salle. The cell cultures infected with the parasite were maintained in a mixture of mediums Grace/L15, supplemented with 10 % fetal bovine serum (FBS) at pH 6.8 and a temperature of 26 degrees C, during 3, 6 and 9 post-infection days. After this, these cell cultures were processed through High Resolution Light Microscopy (HRLM) and Transmission Electron Microscopy (TEM) based on standard protocols defined by the Group of Microscopy and Image Analyses of the Instituto Nacional de Salud. Semi-fine slices of 1 microm colored with toluidine blue were used for the morphological analysis of the culture, and ultra fine cuts of 60 to 90 nm stained with uranyl acetate and lead citrate where used for the ultrastructural study. In addition, PAS and peroxidase staining was carried out in cells fixed with methanol. The morphometric study was analyzed with software ImageJ (NIH). In the semi-fine slices, small cells were observed showing fibroblastic appearance 10.84 +/- 2.54 microm in length and 5.31 +/- 1.26 microm wide; other cells had epithelial appearance with a great peripheral nucleus, voluminous and vacuolated cytoplasm, 23.04 +/- 4.00 microm in length and 13.96 3.70 microm wide. These last ones predominated over the ones with fibroblastic appearance. Regarding the PAS coloration, 7.08% of the cells presented abundant PAS positive cytoplasmatic granules which indicated polysaccharides presence. The peroxidase test gave a negative result. The greatest percentage of infection (18.90%) of one total of 101 cells, turned up by day 6. Some cells analyzed by TEM presented a vacuolated aspect cytoplasm; some contained parasites, other fibrillar material and others were empty. The results indicate that A. aegypti cell culture can support the internalization and transformation of the parasite, which demonstrates the capacity that these cell cultures have to be infected with L. panamensis and to maintain the infection for approximately one week.

Asunto(s)

Aedes , Leishmania guyanensis/fisiología , Aedes/química , Aedes/citología , Aedes/parasitología , Aedes/ultraestructura , Animales , Células Cultivadas , Microscopía Electrónica de Transmisión