RESUMO
The midgut of anopheline mosquito is the entry of Plasmodium, the causative agent of malaria.When the mosquito feeds on parasite infected host, Plasmodium parasites reach the midgut and must confront digestive enzymes, the innate immune response and go across the peritrophic matrix (PM), a thick extracellular sheath secreted by the mosquito midgut epithelial cells. Then, to continue its development, the parasite must reach the salivary glands to achieve transmission to a vertebrate host. We report here the morphological and biochemical descriptions of the midgut changes after a blood meal in Anopheles albimanus. Before blood feeding, midgut epithelial cells contained numerous electrondense vesicles distributed in the central to apical side. These vesicles were secreted to the luminal side of the midgut after a blood meal. At early times after blood ingest, the PM is formed near microvilli as a granulous amorphous material and after it consolidates forming a highly organized fibrillar structure, constituted by layers of electrondense and electronlucent regions. Proteomic comparative analysis of sugar and blood fed midguts showed several molecules that modify their abundance after blood intake; these include innate immunity, cytoskeletal, stress response, signaling, and digestive, detoxifying and metabolism enzymes. Biological significance In the midgut of mosquitoes during bloodfeeding, many simultaneous processes occur, including digestion, innate immune activities, cytoskeleton modifications, construction of a peritrophic matrix and hormone production, between others. Mechanical forces are very intense during bloodfeeding and epithelial and muscular cells must resist the stress, modifying the actin cytoskeleton and coordinating intracellular responses by signaling. Microorganisms present in midgut contents reproduce and interact with epithelial cells triggering innate immune response. When infectious agents are present in the blood meal they must traverse the peritrophic matrix, an envelope formed from secretion products of epithelial cells, and evade the immune system in order to reach the epithelium and continue their journey towards salivary glands, in preparation for the transmission to the new hosts. During all these processes, proteins of mosquitoes are modified in order to deal with mechanical and biological challenges, and the aim of this work is to study these changes.
Assuntos
Anopheles/metabolismo , Sistema Digestório/metabolismo , Proteoma , Animais , Anopheles/parasitologia , Citoesqueleto/metabolismo , Eletroforese em Gel Bidimensional , Células Epiteliais/parasitologia , Feminino , Interações Hospedeiro-Parasita , Humanos , Imunidade Inata , Insetos Vetores/metabolismo , Insetos Vetores/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Plasmodium/metabolismo , Proteômica , Serpinas/química , Transdução de Sinais , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
Salivary glands of female mosquitoes produce proteins, not completely described yet, that participate in carbohydrate and blood feeding. Here, we report an acidic glycoprotein of 35 kDa (GP35 ANOAL) secreted in the saliva of the malaria vector mosquito Anopheles albimanus. GP35 ANOAL is produced exclusively in the distal lateral lobes of adult female salivary glands, it has a pI of 4.45 and is negatively stained by regular silver stain. An 888 bp cDNA clone encoding a predicted product of 240 amino acids has a signal peptide, potential post-translational modification sites, and a disintegrin signature RGD. The GP35 ANOAL sequence depicts high similarities with the 30 kDa saliva allergen of Aedes aegypti, 30 kDa allergen-like hypothetical proteins, and GE-rich proteins present in several Anopheles species, as well as in Ae. albopictus and Culex pipiens quinquefasciatus. The function of this protein family is still unknown.
Assuntos
Anopheles/metabolismo , Glicoproteínas/metabolismo , Proteínas de Insetos/metabolismo , Sequência de Aminoácidos , Animais , Anopheles/genética , Anopheles/crescimento & desenvolvimento , Sequência de Bases , Feminino , Glicoproteínas/genética , Proteínas de Insetos/genética , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Glândulas Salivares/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
Anopheles albimanus and An. pseudopunctipennis differ in their susceptibilities to Plasmodium vivax circumsporozoite phenotypes. An. pseudopunctipennis is susceptible to phenotype VK247 but almost refractory to VK210. In contrast, An. albimanus is almost refractory to VK247 but susceptible to VK210. To investigate the site in the mosquito and the parasite stage at which resistance mechanisms affect VK247 development in An. albimanus, parasite development was followed in a series of experiments in which both mosquitoes species were simultaneously infected with blood from patients. Parasite phenotype was determined in mature oocysts and salivary gland sporozoites by use of immunofluorescence and Western blot assays and/or gene identification. Ookinete maturation and their densities within the bloodmeal bolus were similar in both mosquito species. Ookinete densities on the internal midgut surface of An. albimanus were 4.7 times higher than those in An. pseudopunctipennis; however, the densities of developing oocysts on the external midgut surface were 6.12 times higher in the latter species. Electron microscopy observation of ookinetes in An. albimanus midgut epithelium indicated severe parasite damage. These results indicate that P. vivax VK247 parasites are destroyed at different parasite stages during migration in An. albimanus midguts. A portion, accumulated on the internal midgut surface, is probably destroyed by the mosquito's digestive enzymes and another portion is most likely destroyed by mosquito defense molecules within the midgut epithelium. A third group, reaching the external midgut surface, initiates oocyst development, but over 90% of them interrupt their development and die. The identification of mechanisms that participate in parasite destruction could provide new elements to construct transgenic mosquitoes resistant to malaria parasites.
Assuntos
Anopheles/parasitologia , Insetos Vetores/parasitologia , Plasmodium vivax/fisiologia , Proteínas de Protozoários/fisiologia , Animais , Anopheles/imunologia , Feminino , Insetos Vetores/imunologia , Microscopia Eletrônica , Fenótipo , Plasmodium vivax/crescimento & desenvolvimento , Plasmodium vivax/imunologia , Plasmodium vivax/ultraestrutura , Proteínas de Protozoários/química , Proteínas de Protozoários/genéticaRESUMO
The geographic distribution of Plasmodium vivax circumsporozoite protein phenotypes from patient blood used to infect colonized Anopheles albimanus and An. pseudopunctipennis was investigated in southern Mexico. Parasite phenotype types were determined in blood samples by a polymerase chain reaction and oligoprobe hybridization or by immunofluorescent assay of sporozoites. The proportion of infected mosquitoes and the number of oocysts per mosquito confirmed previous in vitro observations indicating that An. albimanus is more susceptible to VK210 and that An. pseudopunctipennis is more susceptible to VK247. All patients living on the coast were infected with VK210 and most patients living above 170 meters above sea level had VK247. Both phenotypes infected patients from intermediate altitudes. These results concur with the distribution of the anophelines, indicating that An. albimanus is the main vector of the phenotype VK210, but that An. pseudopunctipennis transmits both phenotypes. These conditions have direct implications on parasite transmission rates and malaria epidemiology in Mexico.
Assuntos
Anopheles/parasitologia , Insetos Vetores/parasitologia , Malária Vivax/epidemiologia , Plasmodium vivax/classificação , Altitude , Animais , Anticorpos Monoclonais , Anticorpos Antiprotozoários/análise , Antimaláricos/uso terapêutico , Cloroquina/uso terapêutico , Primers do DNA/química , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Feminino , Fluorimunoensaio , Humanos , Malária Vivax/sangue , Malária Vivax/parasitologia , Masculino , México/epidemiologia , Hibridização de Ácido Nucleico , Fenótipo , Plasmodium vivax/química , Plasmodium vivax/genética , Reação em Cadeia da Polimerase , Prevalência , Primaquina/uso terapêutico , Recidiva , Análise de RegressãoRESUMO
A novel peptide, scorpine, was isolated from the venom of the scorpion Pandinus imperator, with anti-bacterial activity and a potent inhibitory effect on the ookinete (ED(50) 0.7 microM) and gamete (ED(50) 10 microM) stages of Plasmodium berghei development. It has 75 amino acids, three disulfide bridges with a molecular mass of 8350 Da. Scorpine has a unique amino acid sequence, similar only to some cecropins in its N-terminal segment and to some defensins in its C-terminal region. Its gene was cloned from a cDNA library.
Assuntos
Antibacterianos/isolamento & purificação , Antimaláricos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos , Proteínas/isolamento & purificação , Proteínas/farmacologia , Venenos de Escorpião/química , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antimaláricos/química , Antimaláricos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Sequência de Bases , Clonagem Molecular , Defensinas , Dissulfetos/metabolismo , Relação Dose-Resposta a Droga , Gametogênese/efeitos dos fármacos , Células Germinativas/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peso Molecular , Peptídeos/química , Peptídeos/farmacologia , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/fisiologia , Proteínas/química , Proteínas/genética , Venenos de Escorpião/genética , Venenos de Escorpião/farmacologia , Escorpiões/química , Escorpiões/genética , Homologia de Sequência de AminoácidosRESUMO
The susceptibility to two coindigenous Plasmodium vivax Grassi & Feletti phenotypes VK210 and VK247 of three colonized Anopheles albimanus Wiedemann strains (white-striped, green and brown) from southern Mexico was investigated. Mosquitoes of the three strains were simultaneously fed with P. vivax-infected patient blood and examined 1 wk later for the presence of oocysts. The circumsporozoite protein phenotype type (VK210 and VK247) was determined by immunoflorescence of salivary gland sporozoites using monoclonal antibodies. The proportions of specimens infected and the number of oocyst per mosquito indicated that all mosquito strains were more susceptible to the phenotype VK210 than to VK247, but the white-striped strain was more susceptible to both parasite phenotypes than the other two strains.
Assuntos
Anopheles/parasitologia , Plasmodium vivax/patogenicidade , Proteínas de Protozoários/análise , Animais , Animais de Laboratório , Anopheles/genética , Antígenos de Protozoários/análise , Antígenos de Protozoários/genética , México , Fenótipo , Proteínas de Protozoários/genéticaRESUMO
Hemocytes of 2- to 3-d-old female Anopheles albimanus Wiedemann are described by morphology, cytochemistry, and functional criteria. Supplemented Grace's insect medium in a modified Foley's perfusion method was used to obtain hemolymph from An. albimanus. Morphological analysis indicated 3 types of hemocytes were present, prohemocytes, plasmatocytes, and granular cells. Prohemocytes were small round cells with a high nuclear/cytoplasmic ratio. Plasmatocytes were the most abundant cell types in the hemolymph, and appeared as small to large and spindle-shaped cells with round or elongate nucleus, variable number of vacuoles, small granules, and pseudopodia. Granular cells were small to large and round with a large number of cytoplasmic granules, vacuoles, and numerous filopodia. Ultrastructurally, prohemocytes were undifferentiated with abundant free ribosomes and with few small electron-dense granules. Plasmatocytes were rich in mitochondria, rough endoplasmic reticulum, free ribosomes, small electron-dense granules, numerous peripheral vacuoles and with an important organelle polarization. Granular cells contained numerous large electron-dense granular inclusions and vacuoles. Cytochemical studies showed that plasmatocytes and granular cells have cationic bactericidal proteins. Only granular cells showed phenoloxidase and probably lysosomal activities. In vitro functional studies demonstrated that both plasmatocytes and granular cells were able to attach to glass slides, and only plasmatocyte had phagocytic activity and motility. These results characterize the hemocytes of An. albimanus and suggest that plasmatocytes and granular cells may have a role in defense responses to foreign organisms.
Assuntos
Anopheles/citologia , Hemócitos , Animais , Feminino , Hemócitos/classificação , Hemócitos/citologia , Hemócitos/imunologia , Hemócitos/metabolismo , Monofenol Mono-Oxigenase/metabolismo , FagocitoseRESUMO
A study was conducted to characterize the risk of Plasmodium vivax infection in the Lacandon forest, southern Mexico. Blood samples and questionnaire data were collected in 1992. Malaria cases (n = 137) were identified by the presence of symptoms and a positive thick blood smear. The control group included individuals with negative antibody titres and no history of malaria (n = 4994). From 7628 individuals studied, 1006 had anti-P. vivax antibodies. Seroprevalence increased with age. Risk factors associated with infection included: place of birth outside the village of residence (odds ratio, OR 11.67; 95% CI 5.21-26.11); no use of medical services (OR 4.69, 95% CI 3.01-7.29), never using bed-nets (OR 3.98, 95 % CI 1.23-12.86) and poor knowledge of malaria transmission, prevention and treatment (OR 2.30, 95 % CI 1.30-4.07). Health education represents the best recommendation for controlling the disease in the area.
Assuntos
Anticorpos Antiprotozoários/sangue , Educação em Saúde , Malária Vivax/epidemiologia , Plasmodium vivax/imunologia , Adolescente , Adulto , Fatores Etários , Animais , Roupas de Cama, Mesa e Banho , Estudos de Casos e Controles , Criança , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Malária Vivax/sangue , Malária Vivax/imunologia , Malária Vivax/prevenção & controle , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Razão de Chances , Plasmodium vivax/isolamento & purificação , Fatores de Risco , Estudos Soroepidemiológicos , Inquéritos e QuestionáriosRESUMO
OBJECTIVE AND METHOD: To compare the utility of an ELISA using 3 recombinant antigens with that of the skin biopsy to estimate incidence of infections in a sentinel cohort of individuals living in an endemic community in southern Mexico during a set of 11 subsequent ivermectin treatments. RESULTS: The apparent community prevalence of infection and microfilarial skin infection before and after 11 treatments with ivermectin plus nodulectomy were 78% and 13%, and 0.68 mf/mg and 0.04 mf/mg, respectively, as measured by skin biopsy. Of a group of 286 individuals participating in all surveys, a sentinel cohort of 42 mf and serologically negative individuals had been followed since 1994. The annual percentage of individuals becoming positive in this cohort was 24% (10/42), 28% (9/33), 0%, and 4.3% (1/23) in 1995, 1996, 1997 and 1998, respectively. Likewise, the incidence in children 5 years and under (n = 13) within this sentinel cohort was 15% (2/13), 18% (2/11), 0% and 11% (1/9), respectively. All individuals became positive to both tests simultaneously, indicating that seroconversion assessed infection incidence as accurately as skin biopsy in the sentinel group. CONCLUSION: Incidence monitoring of a sentinel cohort provides an estimation of the parasite transmission in the community; it is less costly than massive sampling, and a finger prick blood test might be more acceptable in some communities.