Autores gostariam de agradecer a John Frey e Ben Peterson para a criação de mosquito. Agradecemos também a Christine e Gary Davis Radice para ajudar com micrografias hemócitos. Esta pesquisa foi financiada pela Universidade de Richmond Artes e comunhão de verão Ciência para AA Qayum e conceder faculdade de A. Telang.

1. Preparação antes da coleta de hemócitos <ol><li> Prepare uma solução diluente hemolinfa que consiste em 60% Medium Schneider, 10% de soro fetal bovino (FBS), e tampão citrato 30% (anticoagulante; 98 mM NaOH, 186 mM NaCl, 1,7 mM EDTA, 41 mM de ácido cítrico, pH 4,5) 11. Faça alíquotas 1,0 mL que podem ser armazenadas a -20 ° C. Use cada alíquota apenas uma vez (veja Materiais). </li><li> Prepare lâminas de vidro de microscópio (75 x 25 x 1 mm), marcando um círculo 1 cm de diâmetro em uma extremidade usando uma ferramenta de gravação de vidro. Alternativamente, lâminas de microscópio com dois pré-gravado círculos podem ser adquiridos (por exemplo, Fisher Scientific ou fontes EMS). Uma lâmina de vidro por mosquito serão necessários. </li><li> Preparar um conjunto de agulhas de vidro (veja Materiais) puxou de acordo com as seguintes configurações sugeridas (Sutter Instruments, modelo P-87): <table border="1"><tbody><tr><td> Rampa de calor 5 </td></tr><tr><td> Pull: 45 </td></tr><tr><td> Vel: 75 </td></tr><tr><td> Tempo: 175 </td></tr><tr><td> Pressão 580 </td></tr></tbody></table> Usando as configurações sugeridas nesta extrator, construímos agulhas que são em torno de 500 mm de comprimento com comprimentos de haste de cerca de 3 mm. Pré-puxado agulhas também podem ser adquiridos (por exemplo, Tritech Research). </li></ol> 2. Preparação de microinjetor antes da coleta de hemócitos <ol><li> Inserir uma agulha puxado para dentro do porta-agulha microinjetor de acordo com instruções do fabricante. Aterrar a microinjetor tubulação (veja Materiais), seringa e puxou agulha de vidro com óleo mineral de acordo com instruções do fabricante. </li></ol> 3. Preparação de mosquitos fêmeas antes da coleta de hemócitos <ol><li> Aspirar 15 fêmeas adultas em pequenos recipientes gaiola sólido que pode estar em contato com gelo. Mergulhe gaiola com os mosquitos no gelo a uma profundidade suficiente para que a altura total da gaiola e todos os lados estão em contato com gelo. Isso irá garantir que todos os mosquitos serão expostos ao frio. Mosquitos frio anestesiar por cerca de 8 minutos ou até que eles não são mais móveis. </li><li> Coloque um mosquito em uma corrediça de depressão. Este slide servirá como local para injectáveis. </li></ol> 4. Coleta de hemócitos <ol><li> Definir o microinjetor para assumir 12 mL de diluente hemolinfa em agulha de vidro puxado e injectar a solução para o mosquito entre o sétimo e oitavo segmentos abdominais. Cerca de 3 mosquitos pode ser injetado com 3-3,5 mL do volume total de 12 mL retomada. O mosquito deve ser cuidadosamente mantidos no lugar com uma pinça, enquanto a injeção é realizada. Um pequeno volume de diluente deve permanecer na ponta da agulha, de modo a evitar que o óleo mineral de ser injetado. Após a injeção do abdômen deve olhar &quot;cheio&quot; ou inchado. </li><li> Lugar injetado mosquitos em um recipiente separado limpa no gelo para recuperar por 5 min. A incubação da solução diluente anticoagulante dentro do mosquito irá ajudar a expelir hemócitos aderindo ao tecido interno. Em média 3-5 mosquitos fresco pode ser injetado em cada período de recuperação de 5 min. </li><li> Após 5 minutos de snip de recuperação, as pernas, asas, ea ponta do abdômen (no oitavo segmento) de cada um mosquito em um tempo usando uma tesoura micro. Isto deve ser feito no copo de recuperação colocados no gelo. Cortar as pernas e as asas reduz a probabilidade de escalas de ser transferido para slides coleção. Nota: não corte as pernas e asas muito perto de locais de fixação torácica ou você corre o risco de criar outras aberturas para a hemolinfa para escapar. Hemolinfa só deve ser recolhida através da abertura criada na ponta do abdome. </li><li> Lavar um copo lâmina de microscópio gravado com etanol 70% e seque com um kimwipe. Uma posição e recuperou mosquito injetado na lâmina na borda externa do círculo gravado. Definir microinjetor pegar 12 mL de diluente hemolinfa fresca e injetar este volume para o mosquito na lateral de sua mesotórax. Entregar a maior parte mas não todo o volume do solvente (aproximadamente 8 - 10 ml), de modo a evitar que o óleo mineral de ser injetado. Um pequeno volume de diluente deve permanecer na ponta da agulha. </li><li> Posição do mosquito para que a abertura do corte abdominal está na borda do círculo de 1 cm. Após a injeção da hemolinfa diluída deve ser coletado dentro da área marcada. Uma vez que a agulha é removida do local da injeção mesothoracic, retomar segurando o mosquito entre os dois ramos da pinça e esprema delicadamente o abdômen com os fórceps para dirigir a hemolinfa para a área do círculo gravado. </li><li> Permitir hemolinfa diluída para secar no slide por aproximadamente 10 minutos ou até que seja visivelmente seca. </li></ol> 5. Hemócitos fixação e coloração <ol><li> Mancha cada slide separadamente com HEMA3 7 (ver Materiais): Dip slide em fixador 5 vezes por 1 segundo a cada vez; Dipslide em solução I 3 vezes por 1 segundo cada e, finalmente, mergulhar slide na solução II 3 vezes por 1 segundo cada. Lavar a parte de trás da lâmina com água DI. A frente do slide não deve ser lavado. Blot-seco na frente do slide fora do círculo gravado contendo tecido manchado. </li><li> Aplique meio de montagem (por exemplo, Shur / Mount ou Polymount) em torno do centro gravado e coloque uma lamínula sobre ele. Pressione levemente para a lamínula (25 x 25 mm), para que a montagem spreads médios para toda a área coberta pela lamínula (incluindo círculo gravado contendo tecido manchado). Permitir que as lâminas secar por no mínimo 3 horas ou durante a noite. Hemócitos deve ser visto dentro de 2 semanas após a colheita para a visualização ideal e de imagem. </li></ol> 6. Resultados representante Exemplos de fixo e HEMA3 hemócitos manchada coletados de uma fêmea Aedes aegypti adulto são mostrados na Figura 1A-C. Figura 1A mostra um hemócitos que identificamos como um prohemocyte com base em seu pequeno tamanho, forma esférica-esferoidal e alta nuclear a relação citoplasma (550x de ampliação) 11. A Figura 1B mostra um hemócitos classificado como um oenocytoid com base em sua forma esferoidal e citoplasma elevada relação nuclear (550x de ampliação). Por fim, a Figura 1C mostra uma hemócitos de granulócitos-tipo (550x de ampliação). Granulócitos são mais granular na natureza e tendem a ser mais amoeboid em forma e comportamento como aderem a superfícies de vidro. Usando critérios publicados anteriormente para a digitação hemócitos e recuperação de 11, o nosso método de coleta rendeu um número similar de hemócitos total e nós também descobriram que os granulócitos constituem a maior porcentagem de hemócitos total de 11 (Fig. 2). <img src="/files/ftp_upload/2772/2772fig1.jpg" alt="Figura 1" /> Figura 1. Imagens de microscópio de luz de um representante HEMA3 prohemocyte, fixo coradas (A), oenocytoid (B) e granulócitos (C) de um Ae. aegypti fêmea adulta. C = citoplasma, N = G = núcleo grânulos. <img src="/files/ftp_upload/2772/2772fig2.jpg" alt="Figura 2"/> Figura 2. Contagens totais ± SE de todos os hemócitos e granulócitos obtidos por mosquito fêmea pelos nossos diluição hemolinfa e método de coleta.

<ol>
	<li>Enayati, A., Hemingway, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+management:+past,+present,+and+future.">Malaria management: past, present, and future.</a> Annu. Rev. Entomol. 55, 569-569 (2010).</li><li>Medlock, J., Luz, P. M., Struchiner, C. J., Galvani, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+transgenic+mosquitoes+on+dengue+virulence+to+humans+and+mosquitoes.">The impact of transgenic mosquitoes on dengue virulence to humans and mosquitoes.</a> The American Naturalist. 174, 565-565 (2009).</li><li>Baton, L. A., Garver, L., Xi, Z., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+studies+on+the+innate+immunity+of+disease+vectors.">Functional genomics studies on the innate immunity of disease vectors.</a> Insect Sci. 15, 15-15 (2008).</li><li>Thomas, R. E., Wu, W. K., Verleye, D., Rai, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midgut+basal+lamina+thickness+and+dengue-1+virus+dissemination+rates+in+laboratory+strains+of+Aedes+albopictus+(Diptera:+Culicidae).">Midgut basal lamina thickness and dengue-1 virus dissemination rates in laboratory strains of Aedes albopictus (Diptera: Culicidae).</a> J. Med. Entomol. 30, 326-326 (1993).</li><li>Bartholomay, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+infection+responses+in+the+haemocytes+of+the+mosquito,+Aedes+aegypti.">Profiling infection responses in the haemocytes of the mosquito, Aedes aegypti.</a> Insect Mol. Biol. 16, 761-761 (2007).</li><li>Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insect+cellular+immune+response.">The insect cellular immune response.</a> Insect Sci. 15, 1-1 (2008).</li><li>Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+mortality+in+immune+challenged+mosquitoes+(Aedes+aegypti)+correlates+with+a+decrease+in+haemocyte+numbers.">Age-associated mortality in immune challenged mosquitoes (Aedes aegypti) correlates with a decrease in haemocyte numbers.</a> Cell. Microbiol. 7, 39-39 (2005).</li><li>Chen, C. C., Laurence, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+on+humoral+encapsulation+of+microfilariae:+establishment+of+technique+and+description+of+reaction.">In vitro study on humoral encapsulation of microfilariae: establishment of technique and description of reaction.</a> International Journal for Parasitology. 17, 781-781 (1987).</li><li>Beerntsen, B. T., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dirofilaria+immitis:+effects+on+hemolymph+polypeptide+synthesis+in+Aedes+aegypti+during+melanotic+encapsulation+reactions+against+Microfilariae.">Dirofilaria immitis: effects on hemolymph polypeptide synthesis in Aedes aegypti during melanotic encapsulation reactions against Microfilariae.</a> Exp. Parasitol. 71, 406-406 (1990).</li><li>Hillyer, J. F., Schmidt, S. L., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibacterial+innate+immune+response+by+the+mosquito+Aedes+aegypti+is+mediated+by+hemocytes+and+independent+of+Gram+type+and+pathogenicity.">The antibacterial innate immune response by the mosquito Aedes aegypti is mediated by hemocytes and independent of Gram type and pathogenicity.</a> Microb. Infect. 6, 448-448 (2004).</li><li>Castillo, J. C., Robertson, A. E., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+hemocytes+from+the+mosquitoes+Anopheles+gambiae+and+Aedes+aegypti.">Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.</a> Insect Biochem. Mol. Biol. 36, 891-891 (2006).</li></ol>

Não há conflitos de interesse declarados.

O método de coleta de hemócitos aqui descrita é modificada a partir de métodos publicados anteriormente e permite isolar os preparativos limpa de hemócitos de Aedes mosquitos com menos etapas. Enquanto o nosso interesse particular reside na caracterização de populações de hemócitos em mosquitos Aedes, acreditamos que esta técnica pode ser aplicada a grupos mosquito outros após testes iniciais são realizados para determinar volumes de injeção adequada. Nosso protocolo utiliza buffers anti...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Médio Schneider </td><td> Sigma-Aldrich </td><td> S0146 </td><td></td></tr><tr><td> Soro fetal bovino </td><td> Sigma-Aldrich </td><td> F0643 </td><td></td></tr><tr><td> Hema3 kit mancha </td><td> Pescador </td><td> 123-869 </td><td></td></tr><tr><td> Agulhas de vidro (Borosilicato com filamento) </td><td> Instrumento Sutter </td><td> BF100-78-10 </td><td> 1,0 milímetros e 0,78 milímetros OD ID </td></tr><tr><td> Extrator de agulhas </td><td> Sutter Instruments </td><td> Modelo P-87 </td><td></td></tr><tr><td> Microinjetor </td><td> Tritech Research </td><td> Minj-PD </td><td></td></tr><tr><td> Porta agulha </td><td> Tritech Research </td><td> Minj-4 </td><td></td></tr></tbody></table>

a protocol for collecting and staining hemocytes from the yellow fever mosquito aedes aegypti

Os mosquitos são vetores de uma série de patógenos causadores de doenças como o vírus da febre amarela, malária e parasitas worms filarial. Laboratórios estão investigando anti-patógeno componentes do sistema imune inato na espécie dos vectores de doenças, na esperança de gerar mosquitos transgênicos que são refratários à patógenos, 1, 2. O sistema imune inato de mosquitos consiste de vários mecanismos de defesa 3. Patógenos que conseguem escapar da barreira imposta pelo mosquito epitélio do intestino médio-lined 4 entrar na hemolinfa e encontrar hemócitos circulantes, importantes componentes celulares que sintetizam e patógenos engulf 5, 6. Os pesquisadores não encontraram evidências de tecido hematopoiético em mosquitos e evidência atual sugere que o número de hemócitos é fixada em emergência de adultos e os números podem realmente diminuir com a idade mosquito 7. A habilidade para recolher e identificar hemócitos de insetos de importância médica é um passo essencial para os estudos da imunidade celular. No entanto, o pequeno tamanho dos mosquitos e do volume limitado de hemolinfa representam um desafio para coleta de células do sistema imunológico. Dois métodos estabelecidos para a recolha de hemócitos mosquito incluem a expulsão de hemolinfa a partir de uma tromba de corte 8, e deslocamento de volume (perfusão), em que solução salina é injetada na região membranosa necklike entre a cabeça eo tórax (ie, colo do útero) ea hemolinfa perfundidos é coletada a partir de uma abertura rasgada em uma região distal do abdômen 9, 10. Estas técnicas, no entanto, são limitados pela baixa recuperação de hemócitos e possível contaminação por células de gordura corporal, respectivamente 11. Mais recentemente, um método conhecido como recuperação de injecção / alta recuperação melhorada de imunócitos pelo uso de buffers anticoagulante, reduzindo os níveis de escalas de contaminantes e tecidos internos 11. Enquanto que o método permite um método melhorado de coleta e manutenção hemócitos para cultura primária, envolve uma série de injeção e as etapas de coleta que não são necessárias se o objetivo é coletar a jusante, corrigir e mancha hemócitos para diagnósticos. Aqui, demonstramos o nosso método de coleta de hemolinfa mosquito que combina a simplicidade de perfusão, usando buffers anticoagulante em lugar de solução salina, com a precisão das técnicas de injecção de alta para isolar os preparativos limpa de hemócitos em mosquitos Aedes.

Watch this Scientific Journal Video about A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti at JoVE.com

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplificada ainda método preciso para coletar e mancha hemócitos mosquito é descrito. Nosso método combina a simplicidade de perfusão com a precisão das técnicas de injecção de alta para isolar os preparativos limpa de hemócitos em mosquitos Aedes. Este método facilita os estudos que exigem conhecimento dos tipos de hemócitos e sua abundância.

Um Protocolo de Coleta e coloração Hemócitos do mosquito da febre amarela Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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JoVE Journal

Immunology and Infection

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

15.2K visualizações.  University of Richmond. A simplificada ainda método preciso para coletar e mancha hemócitos mosquito é descrito. Nosso método combina a simplicidade de perfusão com a precisão das técnicas de injecção de alta para isolar os preparativos limpa de hemócitos em mosquitos Aedes. Este método facilita os estudos que exigem conhecimento dos tipos de hemócitos e sua abundância.

Vídeo: A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Protocolo para produção de uma Cruz genética do parasita da malária de roedores

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Imunologia e Infecção

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

Um Ensaio Bioluminescência Cálcio de análise funcional do Mosquito ( Aedes aegypti) E Tick ( Rhipicephalus microplus) G receptores acoplados à proteína

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Um Protocolo de Chelex simples para extração de DNA de<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Um protocolo para hepatite C Analisando replicação de vírus

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

Extração de hemócitos de larvas de Drosophila melanogaster para infecção microbiana e análise

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Co a coloração dos vasos sanguíneos e fibras nervosas no tecido adiposo

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Teste in vitro ELISA para avaliar a potência da vacina antirrábica

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

Análises de competência vetorial sobre mosquitos Aedes aegypti que usam zika vírus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Propagação do Parasita Microsporidiano Edhazardia aedis em Mosquitos Aedes aegypti

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Alimentação e quantificação de sangue derivados de animais e refeições artificiais em mosquitos Aedes aegypti

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...