Biotin-avidin sandwich elisa with specific human isotypes IgG1 and IgG4 for Culicidae mosquito blood meal identification from an epizootic yellow fever area in Brazil

With a view toward investigating the feeding behavior of Culicidae mosquitoes from an area of epizootic yellow fever transmission in the municipalities of Garruchos and Santo Antônio das Missões, Rio Grande do Sul State, Brazil, specimens were collected by aspiration from September 2005 to April 2007. The engorged females were submitted to blood meal identification by enzyme-linked immunosorbent assay (ELISA). A total of 142 blood-engorged samples were examined for human or monkey blood through species-specific IgG. Additional tests for specificity utilizing isotypes IgG1 and IgG4 of human monoclonal antibodies showed that only anti-human IgG1 was effective in recognizing blood meals of human origin. The results indicated a significant difference (p = 0.027) in detection patterns in samples of Haemagogus leucocelaenus recorded from human blood meals at Santo Antônio das Missões, which suggests some degree of exposure, since it was an area where epizootic outbreaks have been reported.

Biotin/avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns.

Observations on Haemagogus janthinomys Dyar (Diptera: Culicidae) and other mosquito populations within tree holes in a gallery forest in the northwestern region of Sao Paulo state, Brazil

In 2000, an outbreak of sylvatic yellow fever possibly occurred in gallery forests of the Grande river in the Paraná basin in the northwestern region of São Paulo state. The aim of this study was to obtain information on the bionomics of Haemagogus and other mosquitoes inside tree holes in that area. Eighteen open tree holes were sampled for immature specimens. Adults were collected twice a month in the forest in Santa Albertina county from July 2000 to June 2001. The seasonal frequency of fourth instars was obtained by the Williams geometric mean (Mw), while the adult frequency was estimated either by hourly arithmetic or the Williams' means. Cole's index was applied to evaluate larval inter-specific associations. Among the ten mosquito species identified, the most abundant was Aedes terrens Walker followed by Sabethes tridentatus Cerqueira and Haemagogus janthinomys Dyar. Larval and adult abundance of these species was higher in summer than in winter. Although larval abundance of Hg. janthinomys peaked in the rainy season, correlation with rainfall was not significant. Six groups of larval associations were distinguished, one of which the most positively stable. The Hg. janthinomys and Ae. terrens association was significant, and Limatus durhamii Theobald was the species with most negative associations.

Biotin/Avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns

The Function and Three-Dimensional Structure of a Thromboxane A(2)/Cysteinyl Leukotriene-Binding Protein from the Saliva of a Mosquito Vector of the Malaria Parasite

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD(7). In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the omega-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.

Structure of an Odorant-Binding Protein from the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive ""Lid""

Background: The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission. Methodology: Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP) from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 angstrom resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date. Conclusion: The structure of AaegOBP1 (= AaegOBP39) shares the common fold of insect OBPs with six alpha-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG) was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors). A C-terminal loop covers the binding cavity and this ""lid"" may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.

Coloured Petri nets and graphical simulation for the validation of a robotic cell in aircraft industry

This paper proposes a mixed validation approach based on coloured Petri nets and 3D graphic simulation for the design of supervisory systems in manufacturing cells with multiple robots. The coloured Petri net is used to model the cell behaviour at a high level of abstraction. It models the activities of each cell component and its coordination by a supervisory system. The graphical simulation is used to analyse and validate the cell behaviour in a 3D environment, allowing the detection of collisions and the calculation of process times. The motivation for this work comes from the aeronautic industry. The automation of a fuselage assembly process requires the integration of robots with other cell components such as metrological or vision systems. In this cell, the robot trajectories are defined by the supervisory system and results from the coordination of the cell components. The paper presents the application of the approach for an aircraft assembly cell under integration in Brazil. This case study shows the feasibility of the approach and supports the discussion of its main advantages and limits. (C) 2011 Elsevier Ltd. All rights reserved.

Modeling of Distributed Control Systems in Intelligent Building based on Colored Petri Nets

Distributed control systems consist of sensors, actuators and controllers, interconnected by communication networks and are characterized by a high number of concurrent process. This work presents a proposal for a procedure to model and analyze communication networks for distributed control systems in intelligent building. The approach considered for this purpose is based on the characterization of the control system as a discrete event system and application of coloured Petri net as a formal method for specification, analysis and verification of control solutions. With this approach, we develop the models that compose the communication networks for the control systems of intelligent building, which are considered the relationships between the various buildings systems. This procedure provides a structured development of models, facilitating the process of specifying the control algorithm. An application example is presented in order to illustrate the main features of this approach.

Modeling extended Petri nets compatible with GHENeSys IEC61131 for industrial automation

Petri net (PN) modeling is one of the most used formal methods in the automation applications field, together with programmable logic controllers (PLCs). Therefore, the creation of a modeling methodology for PNs compatible with the IEC61131 standard is a necessity of automation specialists. Different works dealing with this subject have been carried out; they are presented in the first part of this paper [Frey (2000a, 2000b); Peng and Zhou (IEEE Trans Syst Man Cybern, Part C Appl Rev 34(4):523-531, 2004); Uzam and Jones (Int J Adv Manuf Technol 14(10):716-728, 1998)], but they do not present a completely compatible methodology with this standard. At the same time, they do not maintain the simplicity required for such applications, nor the use of all-graphical and all-mathematical ordinary Petri net (OPN) tools to facilitate model verification and validation. The proposal presented here completes these requirements. Educational applications at the USP and UEA (Brazil) and the UO (Cuba), as well as industrial applications in Brazil and Cuba, have already been carried out with good results.

THE TEMPO AND MODE OF EVOLUTION OF TRANSPOSABLE ELEMENTS AS REVEALED BY MOLECULAR PHYLOGENIES RECONSTRUCTED FROM MOSQUITO GENOMES

Although many mathematical models exist predicting the dynamics of transposable elements (TEs), there is a lack of available empirical data to validate these models and inherent assumptions. Genomes can provide a snapshot of several TE families in a single organism, and these could have their demographics inferred by coalescent analysis, allowing for the testing of theories on TE amplification dynamics. Using the available genomes of the mosquitoes Aedes aegypti and Anopheles gambiae, we indicate that such an approach is feasible. Our analysis follows four steps: (1) mining the two mosquito genomes currently available in search of TE families; (2) fitting, to selected families found in (1), a phylogeny tree under the general time-reversible (GTR) nucleotide substitution model with an uncorrelated lognormal (UCLN) relaxed clock and a nonparametric demographic model; (3) fitting a nonparametric coalescent model to the tree generated in (2); and (4) fitting parametric models motivated by ecological theories to the curve generated in (3).

Mosquito (Diptera: Culicidae) Diversity of a Forest-Fragment Mosaic in the Amazon Rain Forest

To study the impact of Amazonian forest fragmentation on the mosquito fauna, an inventory of Culicidae was conducted in the upland forest research areas of the Biological Dynamics of Forest Fragments Project located 60 km north of Manaus, Amazonas, Brazil. The culicid community was sampled monthly between February 2002 and May 2003. CDC light traps, flight interception traps, manual aspiration, and net sweeping were used to capture adult specimens along the edges and within forest fragments of different sizes (1, 10, and 100 ha), in second-growth areas surrounding the fragments and around camps. We collected 5,204 specimens, distributed in 18 genera and 160 species level taxa. A list of mosquito taxa is presented with 145 species found in the survey, including seven new records for Brazil, 16 new records for the state of Amazonas, along with the 15 morphotypes that probably represent undescribed species. No exotic species [Aedes aegypti (L.) and Aedes albopictus (Skuse)] were found within the sampled areas. Several species collected are potential vectors of Plasmodium causing human malaria and of various arboviruses. The epidemiological and ecological implications of mosquito species found are discussed, and the results are compared with other mosquito inventories from the Amazon region.

Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) similar to 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.

A assembléia de abelhas (Hymenoptera, Apidae) de uma área restrita de campos naturais do Parque Estadual de Vila Velha, Paraná e comparações com áreas de campos e cerrado

A assembléia de abelhas (Hymenoptera, Apidae) de uma área restrita de campos naturais do Parque Estadual de Vila Velha, Paraná e comparações com áreas de campos e cerrado. Foram realizadas coletas sistemáticas de abelhas em uma área restrita no Parque Estadual de Vila Velha, Paraná, no período de outubro de 2003 a setembro de 2004. Essa amostragem visou gerar subsídios para comparações entre áreas de campo dentro do Parque e com outras áreas de vegetação aberta brasileiras. A área de estudo possui cerca de quatro hectares e foi subdividida em quatro subáreas contínuas e com o mesmo perímetro. As abelhas em flores e em vôo foram capturadas com auxílio de rede entomológica. Foram coletados 1.459 espécimes pertencentes a 122 espécies de abelhas. Estas espécies estão distribuídas em 51 gêneros, 20 tribos e cinco subfamílias. As plantas visitadas correspondem a 93 espécies, pertencentes a 62 gêneros e 29 famílias. Neste estudo e em outro estudo realizado anteriormente em outra área do Parque foram coletadas 222 espécies sendo 74 espécies em comum as duas áreas e uma soma de 148 espécies restritas a uma ou outra área. Ainda, registros históricos de abelhas para Vila Velha elevam a riqueza conhecida para 261 espécies. O índice de similaridade de Morisita entre as áreas foi de 0,50 quando utilizadas as plantas como variáveis e 0,88 quando utilizados gêneros e espécies de abelhas, revelando uma notável heterogeneidade. Comparações entre as áreas do Parque Estadual de Vila Velha e áreas de campos e cerrado foram realizadas através do índice de similaridade e da análise de correspondência. Foram observados dois agrupamentos fortemente evidentes, referentes aos campos e ao cerrado. A análise de correspondência sugere que alguns gêneros podem ser relacionados a determinadas formações vegetais. Esse estudo indica que a fauna de abelhas de Vila Velha é composta principalmente por gêneros relacionados às áreas de campos, porém com presença de elementos de cerrado.

Dimorfismo sexual alar em Aedes scapularis (Diptera: Culicidae)

A detecção do sexo de mosquitos da família Culicidae é importante em estudos faunísticos e epidemiológicos, pois somente as fêmeas possuem competência vetora para patógenos. O dimorfismo sexual de genitália e de apêndices cefálicos é, em geral, facilmente visível em culicídeos. As asas também podem ser dimórficas e assim poderiam complementar o procedimento de sexagem. No entanto, tal distinção não é facilmente notável à observação direta. Visando descrever formalmente o dimorfismo sexual alar em Aedes scapularis, um culicídeo vetorialmente competente para arbovírus e filárias, asas de machos e fêmeas foram comparadas usando-se métodos de morfometria geométrica e análise estatística multivariada. Nestas análises, populações dos municípios São Paulo e Pariquera-Açu (Estado de São Paulo) foram amostradas. A forma das asas mostrou evidente dimorfismo sexual, o que permitiu um índice de acurácia de 100% em testes-cegos de reclassificação, independentemente da origem geográfica. Já o tamanho alar foi sexualmente dimórfico apenas na população de São Paulo. Aparentemente, a forma alar é evolutivamente mais estável que o tamanho, interpretação que está de acordo com a teoria de Dujardin (2008b), de que a forma alar de insetos seria composta por caracteres genéticos quantitativos e pouco influenciada por fatores não-genéticos, enquanto que o tamanho alar seria predominantemente determinado por plasticidade decorrente de influências ambientais.