Cytopathological effects of Bacillus sphaericus Cry48Aa/Cry49Aa toxin on binary toxin-susceptible and -resistant Culex quinquefasciatus larvae.

The Cry48Aa/Cry49Aa mosquitocidal two-component toxin was recently characterized from Bacillus sphaericus strain IAB59 and is uniquely composed of a three-domain Cry protein toxin (Cry48Aa) and a binary (Bin) toxin-like protein (Cry49Aa). Its mode of action has not been elucidated, but a remarkable feature of this protein is the high toxicity against species from the Culex complex, besides its capacity to overcome Culex resistance to the Bin toxin, the major insecticidal factor in B. sphaericus-based larvicides. The goal of this work was to investigate the ultrastructural effects of Cry48Aa/Cry49Aa on midgut cells of Bin-toxin-susceptible and -resistant Culex quinquefasciatus larvae. The major cytopathological effects observed after Cry48Aa/Cry49Aa treatment were intense mitochondrial vacuolation, breakdown of endoplasmic reticulum, production of cytoplasmic vacuoles, and microvillus disruption. These effects were similar in Bin-toxin-susceptible and -resistant larvae and demonstrated that Cry48Aa/Cry49Aa toxin interacts with and displays toxic effects on cells lacking receptors for the Bin toxin, while B. sphaericus IAB59-resistant larvae did not show mortality after treatment with Cry48Aa/Cry49Aa toxin. The cytopathological alterations in Bin-toxin-resistant larvae provoked by Cry48Aa/Cry49Aa treatment were similar to those observed when larvae were exposed to a synergistic mixture of Bin/Cry11Aa toxins. Such effects seemed to result from a combined action of Cry-like and Bin-like toxins. The complex effects caused by Cry48Aa/Cry49Aa provide evidence for the potential of these toxins as active ingredients of a new generation of biolarvicides that conjugate insecticidal factors with distinct sites of action, in order to manage mosquito resistance.

Ultrastructural analysis of midgut cells from Culex quinquefasciatus (Diptera: Culicidae) larvae resistant to Bacillus sphaericus.

The larvicidal action of the entomopathogen Bacillus sphaericus towards Culex quinquefasciatus is due to the binary (Bin) toxin present in crystals, which are produced during bacterial sporulation. The Bin toxin needs to recognize and bind specifically to a single class of receptors, named Cqm1, which are 60-kDa alpha-glucosidases attached to the apical membrane of midgut cells by a glycosylphosphatidylinositol anchor. C. quinquefasciatus resistance to B. sphaericus has been often associated with the absence of the alpha-glucosidase Cqm1 in larvae midgut microvilli. In this work, we aimed to investigate, at the ultrastructural level, the midgut cells from C. quinquefasciatus larvae whose resistance relies on the lack of the Cqm1 receptor. The morphological analysis showed that midgut columnar cells from the resistant larvae are characterized by a pronounced production of lipid inclusions, throughout the 4th instar. At the end of this stage, resistant larvae had an increased size and number of these inclusions in the midgut cells, while only a small number were observed in the cells from susceptible larvae. The morphological differences in the midgut cells of resistant larvae found in this work suggested that the lack of the Cqm1 receptor, which also has a physiological role as being an alpha-glucosidase, can be related to changes in the cell metabolism. The ultrastructural effects of Bin toxin on midgut epithelial cells from susceptible and resistant larvae were also investigated. The cytopathological alterations observed in susceptible larvae treated with a lethal concentration of toxin included breakdown of the endoplasmic reticulum, mitochondrial swelling, microvillar disruption and vacuolization. Some effects were observed in cells from resistant larvae, although those alterations did not lead to larval death, indicating that the receptor Cqm1 is essential to mediate the larvicidal action of the toxin. This is the first ultrastructural study to show differences in the cell morphology of resistant larvae and further investigation is needed to understand the impact of the lack of expression of midgut enzymes on the physiology of resistant insects.

[Proteic profile and antigenic recognition of extracts from Wuchereria bancrofti L3 infective larvae]. / Perfil protéico e reconhecimento antigênico de extratos de larvas infectantes (L3) de Wuchereria bancrofti.

A study of protein characterization and recognition of the antigenic profile was accomplished in extracts of infective larvae (L3) from Wuchereria bancrofti. Two proteins of relative molecular weight of 49 and 55 kDa were recognized as antigenic in all extracts by the tested sera. The secretory/excretory antigen presented the largest number of recognized bands (105, 100, 76, 55, 49, 39 and 32 kDa) followed by the somatic antigen (100, 76, 55 and 49 kDa) when incubated with pools of sera from healthy individuals resident in endemic areas (normal endemics). Human sera and parasitized blood used to infect mosquitoes in order to obtain L3, were collected from microfilaraemic individuals living in a filariasis endemic area. From 792 persons screened with the thick smear technique, 87 (11%) were positive. No statistical significance was observed between genders. The group between 11 and 19 years of age presented higher percentage of infection (36.8%).

Sustained reduction of the dengue vector population resulting from an integrated control strategy applied in two Brazilian cities.

Aedes aegypti has developed evolution-driven adaptations for surviving in the domestic human habitat. Several trap models have been designed considering these strategies and tested for monitoring this efficient vector of Dengue. Here, we report a real-scale evaluation of a system for monitoring and controlling mosquito populations based on egg sampling coupled with geographic information systems technology. The SMCP-Aedes, a system based on open technology and open data standards, was set up from March/2008 to October/2011 as a pilot trial in two sites of Pernambuco -Brazil: Ipojuca (10,000 residents) and Santa Cruz (83,000), in a joint effort of health authorities and staff, and a network of scientists providing scientific support. A widespread infestation by Aedes was found in both sites in 2008-2009, with 96.8%-100% trap positivity. Egg densities were markedly higher in SCC than in Ipojuca. A 90% decrease in egg density was recorded in SCC after two years of sustained control pressure imposed by suppression of >7,500,000 eggs and >3,200 adults, plus larval control by adding fishes to cisterns. In Ipojuca, 1.1 million mosquito eggs were suppressed and a 77% reduction in egg density was achieved. This study aimed at assessing the applicability of a system using GIS and spatial statistic analysis tools for quantitative assessment of mosquito populations. It also provided useful information on the requirements for reducing well-established mosquito populations. Results from two cities led us to conclude that the success in markedly reducing an Aedes population required the appropriate choice of control measures for sustained mass elimination guided by a user-friendly mosquito surveillance system. The system was able to support interventional decisions and to assess the program's success. Additionally, it created a stimulating environment for health staff and residents, which had a positive impact on their commitment to the dengue control program.

Developing new approaches for detecting and preventing Aedes aegypti population outbreaks: basis for surveillance, alert and control system.

A new approach to dengue vector surveillance based on permanent egg-collection using a modified ovitrap and Bacillus thuringiensis israelensis(Bti) was evaluated in different urban landscapes in Recife, Northeast Brazil. From April 2004 to April 2005, 13 egg-collection cycles of four weeks were carried out. Geo-referenced ovitraps containing grass infusion, Bti and three paddles were placed at fixed sampling stations distributed over five selected sites. Continuous egg-collections yielded more than four million eggs laid into 464 sentinel-ovitraps over one year. The overall positive ovitrap index was 98.5% (over 5,616 trap observations). The egg density index ranged from 100 to 2,500 eggs per trap-cycle, indicating a wide spread and high density of Aedes aegypti (Diptera: Culicidae) breeding populations in all sites. Fluctuations in population density over time were observed, particularly a marked increase from January on, or later, according to site. Massive egg-collection carried out at one of the sites prevented such a population outbreak. At intra-site level, egg counts made it possible to identify spots where the vector population is consistently concentrated over the time, pinpointing areas that should be considered high priority for control activities. The results indicate that these could be promising strategies for detecting and preventing Ae. aegypti population outbreaks.

Developing new approaches for detecting and preventing Aedes aegypti population outbreaks: basis for surveillance, alert and control system

A new approach to dengue vector surveillance based on permanent egg-collection using a modified ovitrap and Bacillus thuringiensis israelensis(Bti) was evaluated in different urban landscapes in Recife, Northeast Brazil. From April 2004 to April 2005, 13 egg-collection cycles of four weeks were carried out. Geo-referenced ovitraps containing grass infusion, Bti and three paddles were placed at fixed sampling stations distributed over five selected sites. Continuous egg-collections yielded more than four million eggs laid into 464 sentinel-ovitraps over one year. The overall positive ovitrap index was 98.5 percent (over 5,616 trap observations). The egg density index ranged from 100 to 2,500 eggs per trap-cycle, indicating a wide spread and high density of Aedes aegypti (Diptera: Culicidae) breeding populations in all sites. Fluctuations in population density over time were observed, particularly a marked increase from January on, or later, according to site. Massive egg-collection carried out at one of the sites prevented such a population outbreak. At intra-site level, egg counts made it possible to identify spots where the vector population is consistently concentrated over the time, pinpointing areas that should be considered high priority for control activities. The results indicate that these could be promising strategies for detecting and preventing Ae. aegypti population outbreaks.

Perfil protéico e reconhecimento antigênico de extratos de larvas infectantes (L3) de Wuchereria bancrofti / Proteic profile and antigenic recognition of extracts from Wuchereria bancrofti L3 infective larvae

A caracterização protéica dos extratos de larvas infectantes (L3) de Wuchereria bancrofti foi realizada por eletroforese em gel de poliacrilamida, em presença de dodecil sulfato de sódio (SDS-PAGE) e o reconhecimento antigênico das proteínas por Western-blot. O maior número de bandas protéicas reconhecidas foi evidenciado nos extratos AgSE (105, 100, 76, 55, 49, 39 e 32 kDa) e AgS (100, 76, 55, e 49 kDa) na presença de soros de indivíduos endêmicos normais. As bandas de 49 e 55 kDa foram reconhecidas pelos soros dos microfilarêmicos, endêmicos normais (residentes de área endêmica livres de infecção filarial) e portadores da forma crônica da doença. As larvas infectantes foram obtidas pela dissecção de mosquitos Culex quinquefasciatus infectados com sangue microfilarêmico de voluntários portadores de microfilaremia, residentes do Município de Olinda-PE. Nos 792 indivíduos investigados pela técnica da gota espessa mensurada (60æl de sangue) 87 foram positivos (11 por cento). A diferenca da positividade entre homens e mulheres não foi significativa e a faixa etária de 11 a 19 anos foi a de maior prevalência.

Perspectivas da municipalizaçäo do controle da filariose linfática na regiäo metropolitana do Recife / Views on the municipalization of lymphatic filariasis control in greater metropolitan Recife

A filariose linfática resulta da infecçäo por três nematódeos: Brugia malayi, Brugia timori e Wuchereria bancrofti. Atingindo países considerados em desenvolvimento, é mais distribuída em áreas urbanas. Estima-se que quatro bilhöes de pessoas vivem em áreas de risco. No Brasil, a endemia é causada por W. bancrofti e foi documentada em 1878; na cidade do Recife, foi detectada em 1952. Atualmente, apenas na regiäo metropolitana do Recife e Belém, esta endemia é considerada um problema de saúde pública. Objetiva-se abordar e discutir a questäo da epidemiologia e controle da filariose linfática e revisar o histórico de seu controle desde sua notificaçäo em 1952 por Rachou, até nossos dias. Analisa-se o modelo campanhista do program institucional da Sucam/FNS e os métodos alternativos atuais propostos como inovadores. Apresenta estratégias viáveis para o controle da filariose, através da rede primária de saúde, da municipalizaçäo e do Programa de Agentes Comunitários de Saúde (Pacs).

Juvenile hormone III and nutrition effects on spermatogenesis in the 4th instar nymphs of Panstrongylus megistus (Hemiptera: Reduviidae)

When 4th instar nymphs of Panstrongylus megistus are fed eith a saturant blood meal, there is an intense proliferation of the spermatogonia. At the end of the intermoult, the older spermatogonial cysts differentiate into 1st primary spermatocyte cysts. In the nymphs deprived of the blood meal this evolution is not observed, but a small growth of the testicular follicles occurs, due to a few mitotic divisions. This growth is observed at least, until 25 days after ecdysis. Since day 15, an autolytic process starts in the older spermatogonial cysts. The presence of exogenous juvenile hormone III (JH III) does not promote the development of the germ cells in the fasting insects. There is only a small growth of the testicular follicles and the autolytic process is also observed. In the precocious adults obtained by allatectomy or prcocene II treatment, germ cells are observed in all development stages, except packed and elongated spermatozoa bundels

Effects of gamma irradiation on the reproductive capacity and the longevity of Panstrongylus (Hemiptera: Reduviidae)

The effects of radiation on the reproductive capacity and the longevity of Panstrongylus magistus were studied. An indirect correlation between longevity and radiation doses was observed. Males were more affected than females. Longevity of females submitted to 10 Gy was not different when compared to controls. Some of the irradiated males copulated and in these cases semen was transferred to females, but only few eggs were laid. Fertility was seriously affected in all irradiated groups, decreasing with increasing intensity of dose. The dose of 80 Gy induced sterility in males and females. Gelatinous spermatophores that were expelled by females irradiated with 20 and 40 Gy, may be a consequence of irradiation that induced modification in the bursa copulatrix pH

Intestinal helminthiasis and anaemia in youngsters from Matriz da Luz, district of Säo Lourenço da Mata, State of Pernambuco, Brazil

A group of youngsters (4-18 years old) in northeast Brazil was studied to stablish the prevalence of anaemia and intestinal parasitism, as well as to analyse the correlation between them. Two criteria were used to determine the state of anaemia, the level of haemoglobin and the mean of corpuscular volume. The first was considered a single criterion and the second an associated criteria, used in an attempt to correlate anaemia with iron deficiency. The prevalence of intestinal parasitism was 93 per cent while the prevalence of anaemia was 43.1 per cent and 16.1 per cent according to the criteria employed (single or associated respectively). Anaemia was significantly associated with both sex and age. No significant statistical difference was observed when the association was made between each parasite and anaemia even with those more related to anaemia.

Controle integrado do vetor da filariose com participaçäo comunitária, em uma área urbana do Recife, Brasil / Integrated control of the filariasis vector with community participation in an urban area of Recife, Pernambuco, Brazil

Um projeto piloto para o controle da filariose bancroftiana foi desenvolvido em duas áreas do Recife, com índices iniciais de prevalência de microfilaremia de 10 por cento. Nas duas áreas, foi aplicado um tratamento em massa com doses baixas de dietilcarbamazina. Em uma das áreas, esse tratamento foi associado ao controle do vetor, integrando medidas físicas, como o uso de camadas flutuantes de esferas de poliestireno, ao tratamento periódico dos criadouros com a bactéria entomopatógena Bacillus sphaericus. Como resultado, a densidade populacional do vetor, de ordem de sessenta a 120 Culex/quarto/noite antes das intervençöes, foi drasticamente reduzida, mantendo-se, durante dois anos, em valores entre quatro e 16 Culex/quarto/noite. Neste contexto, as açöes para envolver a comunidade escolar local no controle do vetor resultaram na realizaçäo de atividades curriculares e extraclasses, culminando com a participaçäo efetiva de grupos de alunos ("Vigilantes de vetores") na aplicaçäo das medidas de controle e na divulgaçäo de conhecimentos básicos sobre a biologia do Culex quinquefasciatus. O entusiástico envolvimento de professores e alunos mostrou ser a escola um espaço aberto à participaçäo em programas desta natureza.