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1.
Methods Mol Biol ; 2276: 67-85, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34060033

RESUMEN

Respirometry analysis is an effective technique to assess mitochondrial physiology. Insects are valuable biochemical models to understand metabolism and human diseases. Insect flight muscle and brain have been extensively used to explore mitochondrial function due to dissection feasibility and the low sample effort to allow oxygen consumption measurements. However, adequate plasma membrane permeabilization is required for substrates/modulators to reach mitochondria. Here, we describe a new method for study of mitochondrial physiology in insect tissues based on mechanical permeabilization as a fast and reliable method that do not require the use of detergents for chemical permeabilization of plasma membrane, while preserves mitochondrial integrity.


Asunto(s)
Aedes/fisiología , Drosophila/fisiología , Mitocondrias/fisiología , Aedes/ultraestructura , Animales , Respiración de la Célula/fisiología , Drosophila/ultraestructura , Mitocondrias Musculares/fisiología , Consumo de Oxígeno/fisiología , Permeabilidad
2.
Mem Inst Oswaldo Cruz ; 111(6): 411-3, 2016 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-27276186

RESUMEN

In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.


Asunto(s)
Aedes/ultraestructura , Virus del Dengue/ultraestructura , Microscopía Electrónica de Transmisión/métodos , Aedes/virología , Animales , Técnicas de Cultivo de Célula , Centrifugación/métodos , Chlorocebus aethiops , Fijadores , Indicadores y Reactivos , Células Vero/ultraestructura
3.
Mem. Inst. Oswaldo Cruz ; 111(6): 411-413, June 2016. graf
Artículo en Inglés | LILACS | ID: lil-784251

RESUMEN

In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.


Asunto(s)
Animales , Aedes/ultraestructura , Virus del Dengue/ultraestructura , Microscopía Electrónica de Transmisión/métodos , Aedes/virología , Técnicas de Cultivo de Célula , Centrifugación/métodos , Chlorocebus aethiops , Fijadores , Indicadores y Reactivos , Células Vero/ultraestructura
4.
Mem Inst Oswaldo Cruz ; 107(6): 705-12, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22990957

RESUMEN

The vectorial capacity of Aedes aegypti is directly influenced by its high reproductive output. Nevertheless, females are restricted to a single mating event, sufficient to acquire enough sperm to fertilize a lifetime supply of eggs. How Ae. aegypti is able to maintain viable spermatozoa remains a mystery. Male spermatozoa are stored within either of two spermathecae that in Ae. aegypti consist of one large and two smaller organs each. In addition, each organ is divided into reservoir, duct and glandular portions. Many aspects of the morphology of the spermatheca in virgin and inseminated Ae. aegypti were investigated here using a combination of light, confocal, electron and scanning microscopes, as well as histochemistry. The abundance of mitochondria and microvilli in spermathecal gland cells is suggestive of a secretory role and results obtained from periodic acid Schiff assays of cell apexes and lumens indicate that gland cells produce and secrete neutral polysaccharides probably related to maintenance of spermatozoa. These new data contribute to our understanding of gamete maintenance in the spermathecae of Ae. aegypti and to an improved general understanding of mosquito reproductive biology.


Asunto(s)
Aedes/ultraestructura , Glándulas Exocrinas/ultraestructura , Inseminación/fisiología , Espermatozoides/fisiología , Aedes/fisiología , Animales , Glándulas Exocrinas/metabolismo , Glándulas Exocrinas/fisiología , Femenino , Histocitoquímica , Masculino , Microscopía Electrónica , Oviductos/anatomía & histología , Transporte Espermático
5.
Mem. Inst. Oswaldo Cruz ; 107(6): 705-712, set. 2012.
Artículo en Portugués | LILACS | ID: lil-649483

RESUMEN

The vectorial capacity of Aedes aegypti is directly influenced by its high reproductive output. Nevertheless, females are restricted to a single mating event, sufficient to acquire enough sperm to fertilize a lifetime supply of eggs. How Ae. aegypti is able to maintain viable spermatozoa remains a mystery. Male spermatozoa are stored within either of two spermathecae that in Ae. aegypti consist of one large and two smaller organs each. In addition, each organ is divided into reservoir, duct and glandular portions. Many aspects of the morphology of the spermatheca in virgin and inseminated Ae. aegypti were investigated here using a combination of light, confocal, electron and scanning microscopes, as well as histochemistry. The abundance of mitochondria and microvilli in spermathecal gland cells is suggestive of a secretory role and results obtained from periodic acid Schiff assays of cell apexes and lumens indicate that gland cells produce and secrete neutral polysaccharides probably related to maintenance of spermatozoa. These new data contribute to our understanding of gamete maintenance in the spermathecae of Ae. aegypti and to an improved general understanding of mosquito reproductive biology.


Asunto(s)
Animales , Femenino , Masculino , Aedes/ultraestructura , Glándulas Exocrinas/ultraestructura , Inseminación/fisiología , Espermatozoides/fisiología , Aedes/fisiología , Glándulas Exocrinas/fisiología , Glándulas Exocrinas , Histocitoquímica , Microscopía Electrónica , Oviductos/anatomía & histología , Transporte Espermático
6.
Rev. patol. trop ; 41(2): 222-232, abr.-jun. 2012. ilus
Artículo en Portugués | LILACS | ID: lil-653353

RESUMEN

O dengue é uma doença viral transmitida por Aedes aegypti em mais de 100 países na faixa intertropical do mundo e, até o momento, as principais formas de controle são as ações antivetoriais. Neste trabalho, são apresentadas as alterações ultraestruturais provocadas pelo diflubenzuron (DFB) nas larvas de Ae. aegypti. Os experimentos foram realizados com larvas de terceiro estádio de Ae. aegypti com DFB nas concentrações de 0,1 e de 1miug/mL. Após 24 horas de exposição, as larvasforam coletadas, fixadas, desidratadas, emblocadas, cortadas, contrastadas com acetato de uranila a 3por cento e citrato de chumbo e analisadas em microscópio eletrônico. As alterações ultraestruturaisforam observadas na cutícula e no mesêntero dessas larvas. Por meio de microscopia de varredura, observou-se o aumento do número das cerdas, que se apresentaram mais delgadas e mais longas do que o controle e exibiram um padrão de enrolamento nos sulcos intersegmentares. As análises nomicroscópio eletrônico de transmissão revelaram que as epicutículas antigas se desprenderam quase que totalmente da nova epicutícula e não possuíam pontos de reforço comumente encontrados no controle. As células do mesêntero de larvas expostas ao DFB apresentaram um arcabouço esponjosoe nas secções ultrafinas se apresentaram danificadas e vacuolizadas, mas com a presença de vesículas de secreção e integridade mitocondrial. Este estudo mostrou que o DFB interfere no processo da ecdise e impede a liberação da cutícula velha que se acumula nos espaços intersegmentares estrangulando as porções segmentares num processo sucessivo e acumulativo, também bloqueia a muda e provoca a morte da larva. Este é o mecanismo de ação larvicida do DFB sobre Ae. aegypti,entretanto o produto age também no mesêntero destruindo as células.


Asunto(s)
Aedes/ultraestructura , Control Biológico de Vectores , Dengue , Diflubenzurón
7.
Rev Soc Bras Med Trop ; 44(2): 194-200, 2011.
Artículo en Portugués | MEDLINE | ID: mdl-21468474

RESUMEN

INTRODUCTION: Dengue is an important public health problem in many countries and its main vector Aedes aegypti, is the mosquito most adapted to urban areas. For the first time, the mechanism of action of labdane diterpenoid extracted from Copaifera reticulata and the fraction enriched of catechin tannins extracted from Magonia pubescens is demonstrated through ultrastructural alterations of Aedes aegypti larvae. METHODS: Experiments were performed using a 0.9 ppm solution of diterpenoid and 3.7 ppm of the fraction as the main catechin tannin of molecular mass 846 Da. The compounds were obtained by thin layer chromatography and identified by nuclear magnetic resonance of hydrogen and mass spectrometry. Larvae that achieved lethargic state were collected and dissected. Next, they were contrasted with 1% uranyl acetate, dehydrated, embedded and polymerized. Ultrathin sections were made, mixed with 3% uranyl acetate and lead citrate and placed in an electron microscope. RESULTS: The main ultrastructural alterations caused by the diterpenoid and by tanins in larvae of Aedes aegypti were: cytoplasmic vacuolation, alteration of microvilli, cellular aging, cell disruption and degeneration, formation of secretion vesicles and structural changes in microvilli, irregular nuclei and displacement of cells in the basal lamina. CONCLUSIONS: The fraction containing tannins and the diterpenoid caused the death of Aedes aegypti larvae by cell destruction in the midgut.


Asunto(s)
Aedes/efectos de los fármacos , Diterpenos/farmacología , Fabaceae/química , Insecticidas , Extractos Vegetales/farmacología , Sapindaceae/química , Taninos/farmacología , Aedes/crecimiento & desarrollo , Aedes/ultraestructura , Animales , Diterpenos/aislamiento & purificación , Insecticidas/aislamiento & purificación , Intestinos/diagnóstico por imagen , Intestinos/efectos de los fármacos , Larva/efectos de los fármacos , Larva/ultraestructura , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Taninos/aislamiento & purificación , Ultrasonografía
8.
Rev. Soc. Bras. Med. Trop ; Rev. Soc. Bras. Med. Trop;44(2): 194-200, Mar.-Apr. 2011. ilus
Artículo en Portugués | LILACS | ID: lil-586107

RESUMEN

INTRODUÇÃO: Dengue é um importante problema de saúde pública, em vários países, e tem como principal vetor o Aedes aegypti, mosquito mais adaptado às áreas urbanizadas. Apresenta-se, pela primeira vez, as alterações ultraestruturais em larvas de 3º estádio, desse mosquito, causadas pelos larvicidas naturais, um diterpeno labdano, extraído de Copaifera reticulata, e uma fração rica em taninos catéquicos, extraída de Magonia pubescens, evidenciando o mecanismo de ação dessas substâncias. MÉTODOS: Os experimentos foram realizados com larvas de 3º estádio em solução de 0,9ppm, do diterpeno (3-β-acetoxylabdan-8(17)-13-dien-15-óico) e de 3,7ppm, da fração majoritária de tanino catéquico de massa molecular 864Da. Obtiveram-se as substâncias através de fracionamentos cromatográficos sucessivos, identificadas por ressonância magnética nuclear de hidrogênio e espectrometria de massas. As larvas que atingiram estado letárgico foram coletadas e dissecadas e seus tubos digestórios fixados, desidratados, emblocados e polimerizados. Cortes ultrafinos foram feitos e contrastados com acetato de uranila 3 por cento e citrato de chumbo, posteriormente, levados ao microscópio eletrônico. RESULTADOS: As principais alterações ultraestruturais provocadas pelos diterpeno e tanino sobre larvas de Aedes aegypti foram vacuolização citoplasmática, desorganização e degeneração celular, mudança estrutural dos microvilos e deslocamento das células da lâmina basal. CONCLUSÕES: O diterpeno e a fração rica em taninos catéquicos provocaram a morte das larvas de Aedes aegypti através da destruição celular no intestino médio.


INTRODUCTION: Dengue is an important public health problem in many countries and its main vector Aedes aegypti, is the mosquito most adapted to urban areas. For the first time, the mechanism of action of labdane diterpenoid extracted from Copaifera reticulata and the fraction enriched of catechin tannins extracted from Magonia pubescens is demonstrated through ultrastructural alterations of Aedes aegypti larvae. METHODS: Experiments were performed using a 0.9ppm solution of diterpenoid and 3.7ppm of the fraction as the main catechin tannin of molecular mass 846Da. The compounds were obtained by thin layer chromatography and identified by nuclear magnetic resonance of hydrogen and mass spectrometry. Larvae that achieved lethargic state were collected and dissected. Next, they were contrasted with 1 percent uranyl acetate, dehydrated, embedded and polymerized. Ultrathin sections were made, mixed with 3 percent uranyl acetate and lead citrate and placed in an electron microscope. RESULTS: The main ultrastructural alterations caused by the diterpenoid and by tanins in larvae of Aedes aegypti were: cytoplasmic vacuolation, alteration of microvilli, cellular aging, cell disruption and degeneration, formation of secretion vesicles and structural changes in microvilli, irregular nuclei and displacement of cells in the basal lamina. CONCLUSIONS: The fraction containing tannins and the diterpenoid caused the death of Aedes aegypti larvae by cell destruction in the midgut.


Asunto(s)
Animales , Aedes/efectos de los fármacos , Diterpenos/farmacología , Fabaceae/química , Insecticidas , Extractos Vegetales/farmacología , Sapindaceae/química , Taninos/farmacología , Aedes/crecimiento & desarrollo , Aedes/ultraestructura , Diterpenos/aislamiento & purificación , Insecticidas/aislamiento & purificación , Intestinos/efectos de los fármacos , Intestinos , Larva/efectos de los fármacos , Larva/ultraestructura , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Taninos/aislamiento & purificación
9.
J Am Mosq Control Assoc ; 26(2): 205-9, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20649130

RESUMEN

The eggs of Aedes scapularis analyzed by scanning electron microscopy are black and elliptical in outline, measuring approximately 620.4 +/- 16.74 microm long and 163.7 +/- 16.90 microm (n = 10) wide, with an egg index (length/width ratio) of 3.79. The anterior extremity tapered abruptly from a width of 51.6 microm, while such tapering was more gradual at the posterior extremity, from a width of 61.4 microm. The ventral surface of the chorionic coating presented cells with a tubular aspect containing tubercles in rows at a density of 5 to 9 per cell with 2 different sizes, the largest measuring 7.23 +/- 0.98 microm in a longitudinal diameter and the smallest 4.15 +/- 0.53 microm (n = 30). In the dorsal region, the external chorionic reticulum had a porous appearance, and its thickness ranged from 2.5 to 4.1 microm. Isolated tubercles presented wide variation per cell. In the central region of some chorionic cells were tubercles of greater diameter, characterized as central tubercles of 8.45 +/- 0.67 microm, and around them 3 to 5 smaller tubercles measuring 2.57 +/- 0.26 microm. The micropylar apparatus presented a collar with a very evident molding and edges with defined margins for the transition area and a thickness of around 11.1 microm. The micropyle disc margins were raised, measuring around 17.8 microm in diameter and 229 microm in circumference. The micropyle orifice was very evident, with a diameter of 1.41 microm.


Asunto(s)
Aedes/ultraestructura , Óvulo/ultraestructura , Animales , Argentina , Brasil , Femenino , Microscopía Electrónica de Rastreo
10.
Proc Natl Acad Sci U S A ; 106(28): 11530-4, 2009 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-19561295

RESUMEN

Vector control is a key means of combating mosquito-borne diseases and the only tool available for tackling the transmission of dengue, a disease for which no vaccine, prophylaxis, or therapeutant currently exists. The most effective mosquito control methods include a variety of insecticidal tools that target adults or juveniles. Their successful implementation depends on impacting the largest proportion of the vector population possible. We demonstrate a control strategy that dramatically improves the efficiency with which high coverage of aquatic mosquito habitats can be achieved. The method exploits adult mosquitoes as vehicles of insecticide transfer by harnessing their fundamental behaviors to disseminate a juvenile hormone analogue (JHA) between resting and oviposition sites. A series of field trials undertaken in an Amazon city (Iquitos, Peru) showed that the placement of JHA dissemination stations in just 3-5% of the available resting area resulted in almost complete coverage of sentinel aquatic habitats. More than control mortality occurred in 95-100% of the larval cohorts of Aedes aegypti developing at those sites. Overall reductions in adult emergence of 42-98% were achieved during the trials. A deterministic simulation model predicts amplifications in coverage consistent with our observations and highlights the importance of the residual activity of the insecticide for this technique.


Asunto(s)
Aedes/efectos de los fármacos , Dengue/prevención & control , Ecosistema , Insectos Vectores/efectos de los fármacos , Hormonas Juveniles/toxicidad , Metamorfosis Biológica/efectos de los fármacos , Control de Mosquitos/métodos , Aedes/ultraestructura , Animales , Simulación por Computador , Insecticidas , Microscopía Electrónica de Rastreo , Modelos Biológicos , Perú
11.
J Med Entomol ; 45(6): 1102-7, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19058635

RESUMEN

The fat body is the intermediary metabolism organ of insects and the main source of hemolymph components. In the current study, the microanatomy of Aedes aegypti (L., 1762) fat body was studied through scanning electron microscopy to observe the effects of blood feeding and aging. Three groups of female mosquitoes were used: newly emerged females, 18-d-old sugar-fed females, and 18-d-old blood-fed females. In Ae. aegypti, the fat body is located beneath the integument, and it is subdivided into dorsal, ventral, and lateral lobes, with the latter two being larger than the dorsal lobes. The lobes projected into the body cavity, and they were covered externally by a basal lamina with rounded cells beneath it. In 18-d-old sugar-fed females, the ventral and dorsal fat bodies seemed more developed than in newly emerged mosquitoes. The fat body hypertrophy caused by aging in the sugar-fed mosquito was probably associated with lipid accumulation due to the sugar diet. The blood-fed 18-d-old mosquitoes showed flattened fat bodies in all locations. The fat body modifications after the blood ingestion may be associated with midgut expansion after blood feeding, followed by ovary hypertrophy that mechanically compresses the fat body against the body wall. The structural changes in the fat body after a bloodmeal may be important for midgut extension to maximize blood storage and subsequent ovary enlargement, leading to the organ's reorganization in the body cavity. In addition, the depletion of fat body content during vitellogenesis could be responsible for the shrinking and flattening of the fat body lobes.


Asunto(s)
Aedes/ultraestructura , Envejecimiento/fisiología , Cuerpo Adiposo/ultraestructura , Aedes/fisiología , Animales , Cuerpo Adiposo/fisiología , Conducta Alimentaria , Femenino , Microscopía Electrónica de Rastreo
12.
Microsc Res Tech ; 71(9): 663-8, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18567013

RESUMEN

Histological and ultrastrucutural alterations in the midgut of Aedes albopictus larvae infected with Bacillus thuringiensis var. israelensis (Bti) were observed by light and transmission electron microscopy (TEM). Two formulations of Bti were used: granulated and powder, with 0.2% active ingredient in 90 larvae of Ae. albopictus distributed in three containers containing 30 larvae each (one control group and two test groups). The midgut epithelium of the control group presented flattened and elongated cells with mace-shape with a narrow base. Midgut epithelium cells' surface was convex and had a large circular nucleus located in the median-apical portion of the cell. These cells also presented a basal lamina with a small accumulation of extracellular fibrous matrix, thus characterizing a basal membrane, with a muscle layer and a peritoneal membrane more externally. After Bti ingestion, the larvae stopped/slowed their natural movements down in 5 min. After 30 min approximately, the swimming movements stopped completely. Internally, the intestinal cells showed a disorganization of the basal processes, dilatation and fragmentation of the rough endoplasmic reticulum, with intense cytoplasmic vacuolization. There were concentric dense laminas accumulated in the cytoplasm, and these residual membranous bodies were seen greatly increased in size after 60 min. Mitochondria, fragments of rough endoplasmic reticulum and other remainder organelles were surrounded and segregated from the cytoplasm by exocytosis. This article reports the histopathological alterations in the midgut of Ae. albopictus after infection with Bti and contributes to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against mosquito larvae.


Asunto(s)
Aedes/anatomía & histología , Sistema Digestivo/microbiología , Sistema Digestivo/ultraestructura , Larva/microbiología , Microscopía Electrónica de Transmisión , Aedes/ultraestructura , Animales , Bacillus thuringiensis/crecimiento & desarrollo , Larva/ultraestructura , Control Biológico de Vectores
13.
Rev. biol. trop ; Rev. biol. trop;56(2): 447-458, jun. 2008. ilus, graf, tab
Artículo en Español | LILACS | ID: lil-637651

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.


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
14.
Virology ; 372(1): 176-86, 2008 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-18023837

RESUMEN

We evaluated infection of Aedes taeniorhynchus mosquitoes, vectors of Venezuelan equine encephalitis virus (VEEV), using radiolabeled virus and replicon particles expressing green (GFP) or cherry fluorescent protein (CFP). More epidemic VEEV bound to and infected mosquito midguts compared to an enzootic strain, and a small number of midgut cells was preferentially infected. Chimeric replicons infected midgut cells at rates comparable to those of the structural gene donor. The numbers of midgut cells infected averaged 28, and many infections were initiated in only 1-5 cells. Infection by a mixture of GFP- and CFP-expressing replicons indicated that only about 100 midgut cells were susceptible. Intrathoracic injections yielded similar patterns of replication with both VEEV strains, suggesting that midgut infection is the primary limitation to transmission. These results indicate that the structural proteins determine initial infection of a small number of midgut cells, and that VEEV undergoes population bottlenecks during vector infection.


Asunto(s)
Aedes/virología , Sistema Digestivo/virología , Virus de la Encefalitis Equina Venezolana/patogenicidad , Encefalomielitis Equina Venezolana/virología , Células Epiteliales/virología , Insectos Vectores/virología , Aedes/ultraestructura , Animales , Línea Celular , Cricetinae , Sistema Digestivo/citología , Sistema Digestivo/ultraestructura , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/crecimiento & desarrollo , Encefalomielitis Equina Venezolana/transmisión , Células Epiteliales/ultraestructura , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Insectos Vectores/ultraestructura , Microscopía Confocal , Replicón
15.
Micron ; 39(2): 184-9, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-17329111

RESUMEN

Mosquitoes have an efficient defence system against infection. Insect blood cells (hemocytes) play an essential role in defense against parasites and other pathogenic organisms that infect insects. We have identified by light and transmission electron microscopy six hemocytes cell types from the hemolymph of Aedes aegypti. They were: prohemocytes (20%), adipohemocytes (29%), granulocytes (16%), plasmatocytes (27%), oenocytoids (7%) and thrombocytoids (0.9%). The prohemocytes were the smallest hemocytes found in the hemolymph. Its cytoplasm occupies only a narrow area around the nucleus. The adipohemocytes were the most abundant cell type presented. These hemocytes exhibited a large lipid like vesicle and mitochondria. In electron micrographs, the granulocytes showed cytoplasm containing dilated rough endoplasmic reticulum (RER) and a round or elongated mitochondria. Electron-dense granules with a proteinaceous material were also present. The plasmatocytes were polymorphic and exhibited plasma membrane with irregular processes, philopodia and pseudopodia. Ultrastructural investigation revealed that the reticular cytoplasm showed a well-developed RER, a Golgi and vacuoles. Oenocytoids showed homogeneous cytoplasm with many mitochondria and ribosomes are scattered throughout the cytoplasm, abundant RER and a small smooth endoplasmic reticulum (SER) present at the cell poles. Thrombocytoids were very fragile and few in number. Similar characteristics were found in oenocytoids, possessing a homogeneous cytoplasm with poorly developed organelles, few mitochondria and granules.


Asunto(s)
Aedes/ultraestructura , Hemocitos/ultraestructura , Animales , Núcleo Celular/ultraestructura , Hemocitos/clasificación , Microscopía Electrónica
16.
Rev Biol Trop ; 56(2): 447-58, 2008 Jun.
Artículo en Español | MEDLINE | ID: mdl-19256419

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
17.
Rev. biol. trop ; Rev. biol. trop;54(3): 843-846, sept. 2006. ilus
Artículo en Inglés | LILACS | ID: lil-492308

RESUMEN

Dengue fever is a mosquito-borne viral disease, whose main biological vector is Aedes aegypti. This mosquito colonizes tropical areas where the disease is endemic. The most obvious action against dengue is attacking its vector. Biological control appears to be an alternative approach, using natural enemies of the mosquitoes, such as predatory copepods. Thus, the morphological study of the damage caused by copepods is important to understand its predatory capacity. Twenty-five A. aegypti larvae were exposed to the copepod Mesocyclops thermocyclopoides and the damage caused by the copepods was evaluated using scanning electron microscopy. The larvae showed damage mainly at the anal segment, the siphon and the abdomen; only three attacks to the head were observed. The size of the siphon might be of importance in determining whether or not a copepod will attack a mosquito larva.


El dengue es una enfermedad viral transmitida por mosquitos, cuyo principal vector es Aedes aegypti. Este mosquito coloniza muchas áreas tropicales donde la enfermedad es endémica. La acción más obvia contra el dengue es el ataque a su vector. El control biológico parece una buena alternativa, empleando enemigos naturales de los mosquitos, como los copépodos. Por lo tanto, es importante el estudio morfológico del daño causado por los copépodos para comprender su capacidad depredadora. Veinticinco larvas de A. aegypti fueron expuestas a la actividad depredadora del copépodo Mesocyclops thermocyclopoides. Mediante microscopia electrónica de rastreo se evaluó el daño causado por los copépodos. Éstos atacaron principalmente el segmento anal, el sifón y el abdomen de las larvas; sólo vimos tres ataques a la cabeza. El tamaño del sifón podría ser de importancia para predecir si los copépodos pudiesen atacar larvas de determinado mosquito.


Asunto(s)
Animales , Aedes/ultraestructura , Copépodos/fisiología , Insectos Vectores/ultraestructura , Control Biológico de Vectores/métodos , Aedes/parasitología , Insectos Vectores/parasitología , Larva/parasitología , Larva/ultraestructura , Microscopía Electrónica de Rastreo , Conducta Predatoria
18.
Rev. biol. trop ; Rev. biol. trop;54(3): 847-852, sept. 2006. ilus
Artículo en Inglés | LILACS | ID: lil-492307

RESUMEN

Aedes aegypti is the main insect vector of Dengue fever and dengue hemorrhagic fever/dengue shock syndrome and represents the only vulnerable element in the control of this disease. Therefore, the identification and quantification of this mosquito is an important task; however, the majority of taxonomic keys are based on the 4th larval instar. For that reason, this study describes the four larval instars ofA. aegypti using scanning electron microscopy. Morphological changes during larval development were observed at the pecten, comb scales and the ventral brush of the abdominal segment X; however, the 3rd and 4th instars showed similar structures with only a slight variation. The structures described in this study will be helpful in the identification of the four instars of A. aegypti, a fundamental task for comprehending the natural history of dengue mainly in new territories affected.


Aedes aegypti es el principal insecto vector de la fiebre del dengue y del dengue hemorrágico/síndrome del choque por dengue y es el único elemento atacable para el control de esta virosis. La identificación y cuantificación de éste es una tarea importante; no obstante, la mayoría de las llaves taxonómicas se basan en el cuarto estadio larval. Por esta razón, en este trabajo se describen los cuatro estadios larvales de A. aegypti los cuales fueron examinados mediante microscopia electrónica de rastreo. Los cambios morfológicos ocurridos durante el desarrollo larval fueron observados en el pecten, las escamas del peine, el cepillo ventral del décimo segmento. El 3ero y 4to estadios larvales mostraron estructuras similares con sólo ligeras variaciones. Las estructuras descritas en este artículo permiten identificar cualquiera de los cuatro estadios larvales de A. aegypti, lo cual representa una tarea importante en la comprensión de la historia natural del dengue en los nuevos territorios afectados.


Asunto(s)
Animales , Aedes/ultraestructura , Insectos Vectores/ultraestructura , Dengue/transmisión , Larva/ultraestructura , Microscopía Electrónica de Rastreo
19.
J Med Entomol ; 43(1): 68-72, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16506449

RESUMEN

Egg hatching has been studied in Aedes aegypti (L.) through scanning electron microscopy. The first sign of egg hatching is a small protrusion on the eggshell in the anterior pole. The larval movement provokes a crack in the eggshell with the egg buster located in the dorsal head. The egg buster provokes a small transverse fissure in the eggshell that gradually increases in the chorion. Then, the rupture is completed around the eggshell. The separation of the anterior pole occurs, showing the dorsal region of the larva head with the egg buster and the cap. After sequential movements, the larva looses the cap. Finally, the first instar is ready to be free showing details of its body with the egg buster over its head. This structure is a cuticular formation, similar to a cone structure that ends in a very fine tip and emerges from a pear-like depression with high rounded borders. Our results describe the anatomy of the egg hatching process in Ae. aegypti, showing details of the participation of the egg buster.


Asunto(s)
Aedes/fisiología , Aedes/ultraestructura , Insectos Vectores/fisiología , Insectos Vectores/ultraestructura , Aedes/crecimiento & desarrollo , Animales , Femenino , Insectos Vectores/crecimiento & desarrollo , Larva/crecimiento & desarrollo , Larva/fisiología , Larva/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Óvulo/ultraestructura
20.
Parasitol Res ; 99(4): 384-91, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16572337

RESUMEN

Monoxenous trypanosomatids inhabit invertebrate hosts throughout their life cycle. However, there have been cases of HIV-positive patients who have presented opportunistic infections caused by these protozoa, offering new perspectives to the study of interactions between monoxenics and hematophagous insect vectors. Some monoxenous trypanosomatids present a symbiotic bacterium in the cytoplasm, which seems to promote biochemical and morphological changes in the host trypanosomatids, such as alterations in plasma membrane carbohydrates and the reduction of the paraxial rod. In this work, we investigated the colonization of Aedes aegypti with Blastocrithidia culicis, an endosymbiont-bearing trypanosomatid. B. culicis remained in the insect digestive tract for 38 days after feeding. Optical microscopy analysis revealed an infection process characterized by a homogenous distribution of the trypanosomatid along the midgut epithelium; no preferential interaction of protozoa with any cell type was observed. Ultrastructural analysis showed that during the colonization process, trypanosomatids interacted mainly with midgut cells through their flagellum, which penetrates the microvilli preferentially near the tight junctions. Prolonged infections promoted insect midgut degradation, culminating with the arrival of protozoa in the hemocel. By demonstrating B. culicis colonization in a bloodsucking insect, we suggest that vector transmission of monoxenous trypanosomatids to vertebrate host may occur in nature.


Asunto(s)
Aedes/parasitología , Insectos Vectores/parasitología , Intestinos/parasitología , Estadios del Ciclo de Vida/fisiología , Trypanosomatina/fisiología , Aedes/ultraestructura , Animales , Femenino , Interacciones Huésped-Parásitos , Insectos Vectores/ultraestructura , Intestinos/ultraestructura , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Simbiosis , Trypanosomatina/ultraestructura
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