Name: an experimental and bioinformatics protocol for rna-seq analyses of photoperiodic diapause in the asian tiger mosquito, aedes albopictus
Uploaded: 2014-11-30T15:00:00.000+00:00
Duration: 12 min 10 s
Description: Watch this Scientific Journal Video about An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus at JoVE.com

This work was supported by the National Institutes of Health grant 5R21AI081041-02 and Georgetown University.

1. larval criação de duas A. Grupos albopictus à Idade Adulta <ol><li> Definir dois armários de fotoperíodo com iluminação programável a 21 ° C para a expressão diapause óptima 16 e cerca de 80% de umidade relativa. <ol><li> Programa de um gabinete para a 16L: luz 8D: ciclo escuro (a não diapausa induzindo LD fotoperíodo). Defina o segundo armário para um 8L: 16D luz: ciclo escuro (diapausa induzindo) 13. </li><li> &#39;Luzes sobre&#39; programa ao mesmo tempo em ambos os armários para sincronizar o tempo circadiano entre fotoperíodo. </li></ol></li><li> Calcular a quantidade de ovos necessários para a realização da experiência. Apontar para 300-500 ovos por gaiola. Pelo menos três réplicas gaiolas diapausa e três réplicas de diapausa gaiolas não são necessários para a produção de ARN. Isso totaliza até cerca de 1,800-3,000 ovos por experimento. <ol><li> Chocar os ovos, submergindo papéis de ovos em cerca de 500 ml de H 2 O. deionizada </li><li> Adicionar &lt;em&gt; ca. 1 ml suspensão alimento que consiste em alimentos para cães chão e camarão de água salgada, como descrito anteriormente 24. Cubra o recipiente com malha, e manter a malha no lugar com um elástico. </li><li> Coloque no gabinete LD fotoperíodo durante cerca de 24 horas. </li></ol></li><li> Transferência larvas eclodidas de 10 x 10 x 2 cm, de Petri pratos cheios com cerca de 90 ml de água desionizada H 2 O. <ol><li> Manter cerca de 30 larvas por prato. Larvas de limpar pratos a cada 48-72 hr transferir, por exemplo, todas as segundas, quartas e sexta-feira (MWF) 24. </li><li> Alimentação de cerca de 1 ml suspensão alimento que consiste em alimentos para cães chão e camarão de água salgada em água deionizada cada MWF como descrito anteriormente 24. </li></ol></li><li> Configurar 3-4 gaiolas adultos para cada tratamento fotoperíodo, onde cada gaiola compreende uma réplica biológica. <ol><li> A partir de lados opostos de 9,5 L baldes, cortar um 10-a-14 centímetros buraco, e um outro furo, com 15 cm de diâmetro. CoVer o primeiro com malha. Cortar aproximadamente um comprimento de um pé da meia ortopédica, e cola uma extremidade em torno do interior do outro furo. </li><li> Corte a ponta do pé da meia off e nó fechado - aberto apenas quando o acesso ao interior da gaiola é necessário. Para a tampa gaiola, cortar todo o interior, deixando apenas o aro, e substituir o plástico interior com malha 24. </li><li> Observe o fotoperíodo, replicar número, gaiola data de início, e outras informações relevantes para o experimento com marcador permanente no lado da gaiola. </li></ol></li><li> Alinhar a parte inferior das gaiolas adultos com papel de filtro molhado. Humedecer o papel de filtro com água desionizada suficiente H2O para aumentar a humidade local na gaiola, mas evite a água de pé, que pode estimular a oviposição sobre o papel de filtro 24. Verifique o filtro de papel por dia, durante a secagem, re-molhado quando necessário. </li><li> Para produzir ovos suficientes para uma biblioteca de RNA, incluir, pelo menos 100 mulheres / 9.5 L cage, com não mais de 500 mosquitos por gaiola. </li><li> Recolha MWF pupas e coloque em um copo pequeno de H 2 O limpo a uma densidade de não mais de 50 pupas por 25 ml de H 2 O. Transfira o copo pupas de uma gaiola de adulto. Coloque as gaiolas no respectivo gabinete de fotoperíodo - A. albopictus pupas são fotossensíveis 15. </li><li> Certifique-se de diário que H 2 O em copos é limpo e claro, e remover pupas morto, porque a acumulação de pupas mortos pode causar mortalidade em massa. Retirar H 2 O copos depois de todos pupas emergir. </li><li> Coloque passas orgânicas na parte superior da gaiola de malha para fornecer açúcar para adultos emergiram. Monitorar passas, e alterá-las a cada 3-5 dias para evitar o acúmulo de mofo. </li></ol> 2. Manutenção de Adultos para Permitir acasalamento e produção de ovos <ol><li> Manter gaiolas com alta umidade (aprox. 80%), forrando o fundo da gaiola com um papel de filtro umedecido, e fornecer acesso a passas não mofados, como descrito acima (Sections 1,5 e 1,9). </li></ol> 3. Sangue-feeding <ol><li> Prepare-se para as fêmeas de sangue de alimentação entre dois a seis dias após a eclosão de garantir que as fêmeas foram expostos a pelo menos oito dias curtos inequívocas antes oviposição começa por quase 100% dos ovos de diapausa 14. </li><li> Prepare o sistema de alimentação Hemotek membrana. Ligue as unidades de alimentação à fonte de alimentação. Ajustar a temperatura de cada unidade a 37 ° C, utilizando o parafuso de ajustamento. Usar um termómetro electrónico e sonda para medir a temperatura da unidade de alimentação durante a calibração. </li><li> Prepare o reservatório de alimentação. Estique uma praça de alimentação membrana de colágeno sobre a abertura do reservatório de alimentação e fixe-a com um anel-O. Retire cuidadosamente os cantos para remover rugas; aparar o excesso de membrana com uma tesoura. NOTA: membrana de colagénio pode não funcionar bem para todas as espécies de mosquitos, e pode ser necessário tentar vários tipos para encontrar a membrana óptima se trabalhar com umexceto A. espécies albopictus. Parafilm funciona bem com o Culex pipiens. <ol><li> Se o sangue é armazenado congelado, descongelar à temperatura ambiente durante pelo menos 1 hr antes de usar. </li><li> Mantenha o reservatório de modo a membrana está voltada para baixo, sem suporte e os orifícios de enchimento estão voltados para cima. Usar uma pipeta ou uma seringa para encher o reservatório com cerca de 3 ml de sangue total a partir de galinhas que tem o citrato de sódio como anti-coagulante. Selar orifícios de enchimento com tampões de plástico. </li></ol></li><li> Anexar o reservatório preparado para o alimentador rodando no pino na placa de transferência de calor na parte inferior do alimentador. Inverta o alimentador e coloque-o em cima da gaiola, lado membrana para baixo, de modo que os mosquitos podem se alimentar através da malha da gaiola. Mantenha o alimentador na gaiola durante cerca de 45 minutos para maximizar a alimentação. </li></ol> 4. Estimular a oviposição <ol><li> Quatro a cinco dias após a farinha de sangue, equipar cada gaiola com uma cor escura 50 ml c-se forrada com papel não branqueado a germinação das sementes (papel de ovo) ou com texturas toalha não-branqueada de papel e encha até a metade com água deionizada 9. Se mais de 250 mosquitos estão em uma gaiola, use dois copos. NOTA: Para pequenas gaiolas ou frascos single-fêmea &quot;, infusão de feno&quot;, em recipientes de oviposição pode aumentar oviposição devido ao odor da flora microbiana 25. </li></ol> 5. coletar e armazenar ovos <ol><li> Começar a coleta de ovos dentro de 4-5 dias de alimentação de sangue, porque a produção de ovos tipicamente pico cerca de cinco dias após a alimentação de sangue, e depois desaparece ao longo da próxima semana. </li><li> Varie freqüência de coleta de ovos em necessidades do experimento. Para fins gerais, recolher papéis de ovos em uma programação MWF. Remova papéis de ovos de cada gaiola e substituir por papel novo. Coloque papéis removido recentemente em placas de Petri e guardar no armário fotoperíodo SD para evitar efeitos de confusão de armazenamento de ovos. </li><li> Permitir papéis de ovo para re<html> principal molhado por 2 dias pós-oviposição para permitir a formação da cutícula serosa, o que aumenta a resistência à dessecação ovo 26. </li><li> Posto de Arrecadação aproximadamente 48 hr, ovos secar ao ar livre. Seca-se o papel de tal forma que é mole e ligeiramente húmido ao toque, mas não de modo que o papel molhado é escuro a partir de H 2 O ou estimula a eclosão dos ovos. NOTA: A &quot;x 4&quot; papel 6.5 pode demorar cerca de 3,5 horas para secar. Tenha cuidado para não sobre-seco papéis de ovos, pois isso irá resultar em ovo dessecação 27. </li><li> Reservar ovos adicionais, tanto LD e SD fotoperíodo para avaliar a diapausa e interpretar o efeito photoperiodic (ver medição diapausa, Seção 6). </li><li> Para armazenamento a longo prazo, tenha papéis de ovos a 21 ° C e cerca de 80% de humidade em placas de Petri. Manter pratos de Petri em um recipiente de armazenamento Tupperware com um frasco de água para manter a humidade local como o desenvolvimento embrionário leva quatro a cinco dias a 21 ° C. </li></ol>ove_title &quot;&gt; 6. Meça diapausa Incidência <ol><li> Use embriões reservados adicionais (ver Estimular oviposição, secção 4), que são 7-20 dias de idade para quantificar a resposta diapausa. </li><li> Anote o número de ovos presentes em cada papel ovo. </li><li> Estimular os ovos para chocar, submergindo completamente papéis individuais de ovos em um prato de 90 ml Petri com aproximadamente 80 ml ​​de H 2 O. deionizada Adicionar aproximadamente 0,25 ml de suspensão de alimentação. </li><li> Após 24 h contar o número de larvas eclodidas primeiro instar. Colocar a placa de Petri sobre uma superfície preta para visualizar larvas e colocar uma fonte de luz de um lado do prato. As larvas irão mover-se para longe da fonte de luz, o que permite uma contagem de larvas claro indivíduo. Remover larvas indivíduo com uma pipeta, contando para evitar recontando larvas individual. </li><li> Papers lugar de ovos em uma nova placa de Petri e re-seco. Re-ovos eclodem depois de ~ 1 semana e novamente concordância ovos eclodidos usando o método acima. </li><li> Papers lugar de ovo com o restante u<html> ovos n-eclodidos em novos 90 ml placas de Petri com solução de branqueamento de aproximadamente 80 ml ​​28. Certifique-se de que os papéis de ovos estão completamente submerso na solução de branqueamento e deixe durante a noite sob uma coifa para evitar o odor de água sanitária. NOTA: Branqueamento solução pode ser armazenada durante ~ 1 semana a 4 ° C, mas de outro modo deve ser feita. </li><li> Inspecione ovos usando um microscópio de luz como o branqueamento irá limpar o chorion e permitir a visualização de embrionados, ovos un-chocados. Se o ovo é embrionados, o ovo terá uma cor off-white com os olhos aparecem como dois pequenos pontos pretos opostos um ao outro na face dorsal. Contar o número de un-chocado, ovos embrionados 13. </li><li> Determinar a diapausa com a seguinte fórmula:% diapausa = nenhum. ovos embrionados un-nascidos / (no. ovos eclodidos + não. ovos un-eclodidas embrionados) x 100 13. </li></ol> 7. Extração de RNA a partir de ovos / larvas pharate ontent &quot;&gt; NOTA: Use Trizol em uma capela de fluxo laminar. <ol><li> Ovos do mosquito da escova contendo embriões em desenvolvimento ou larvas pharate em momentos distintos do desenvolvimento de trabalhos de ovos para moedores de vidro usando um pincel de pêlo de camelo. Triture os ovos em Trizol (1 ml por 50-100 mg de tecido) até que esteja completamente pulverizada. Utilize pelo menos 400 ovos per biblioteca para produzir RNA suficiente. <ol><li> Alternativamente, encaixe ovos congelados em azoto líquido e armazenado a -80 ° C em tubos de microcentrífuga de até um mês antes da moagem em Trizol. </li></ol></li><li> Realizar a extracção de ARN em Trizol seguido por precipitação com isopropanol de acordo com as instruções do fabricante. </li><li> Tratar o banco com solução RNase descontaminação ou outros agentes para remover quaisquer nucleases residuais para evitar a degradação do RNA. <ol><li> Tratar o RNA extraído com DNase. De acordo com as instruções do fabricante, incubar as amostras de ARN com DNase durante 30 min a 37 ° C. Use 1 &amp; #181; l de ADNase para até 10 ug de RNA numa reacção de 50 ul. Aumentar a quantidade de ADNase, se houver mais do que 10 ug de RNA em uma reacção. </li><li> Inactivar ADNase por adição de 5 ul de reagente de inactivação de DNase suspenso. Incubar 5 min à temperatura ambiente, a mistura três vezes durante o período de incubação (vortex suave). </li><li> Centrifugar a 10.000 xg por 1,5 min. Transferir o sobrenadante contendo as amostras de RNA tratada para tubos frescos para as etapas subsequentes. </li></ol></li><li> Avaliar a qualidade das amostras de ARN totais por fluorometria. Enviar as amostras para uma instalação de especialidade com o instrumento adequado para esta tarefa. A facilidade vai efectuar a electroforese em gel em chips, para determinar os tamanhos de espécies de RNA presentes na amostra, visualizadas por coloração fluorescente instilada no chip. Os resultados serão retornados como um electroferograma. <ol><li> Determinar a integridade das amostras de ARN total por a presença ou ausência de produtos de degradação, tal como evidenciado pela presença of os picos entre 18S e 5S picos de ARN ribossomal no electroferograma resultante (Figura 1B). </li></ol></li></ol> 8. RNA Sequencing <ol><li> Enviar amostras de ARN totais com qualidade suficientemente elevada (Figura 1A) e da quantidade (geralmente&gt; 3 ug por biblioteca) para um centro de sequenciação comercial para a construção de bibliotecas enriquecidas emparelhado-finais de ARNm e a sequenciação de ARNm, seguindo protocolos padrão. </li><li> Se mais de uma faixa está sendo usado para sequenciar um único experimento, dividir bibliotecas individuais em duas pistas de sequenciamento para explicar variação técnica entre pistas durante o seqüenciamento. </li></ol> 9. lllumina Leia Limpeza NOTA: A Figura 2 resume a porção bioinformática deste protocolo. Para obter uma lista completa de todos os programas e recursos usados ​​na seção bioinformática deste protocolo, consulte a Tabela 1. EmAdicionalmente, Suplementar Arquivo 1 contém exemplos de linha de comando para cada uma das seguintes etapas do protocolo de bioinformática. <ol><li> Use ssaha2 29 (Tabela 1) para identificar as correspondências com 95% de identidade ou superior para a base de dados do NCBI UniVec núcleo (Tabela 1), A. albopictus sequência rRNA (GenBank # L22060.1) e adaptadores de seqüenciamento (exemplos detalhados de linha de comando fornecidas em Suplementar Arquivo 1). Remover pares de leitura com jogos usando Perl ou uma ferramenta de scripting semelhante, por exemplo, adaptando o script Perl fornecida (Suplementar Arquivo 2). </li><li> Limpe restante lê com o pacote SolexaQA 30 (Tabela 1; Suplementar Arquivo 1): aparar as regiões com uma pontuação equivalente Phred inferior a 20 usando as configurações padrão de DynamicTrim.pl. <ol><li> Remover lê mais curto do que 25 bp com LengthSort.pl em ambos frente e verso lê simultaneamente. Avaliar a qualidade dos arquivos limpos fastq com FastQC (Tabela 1 </strong&gt;) - em particular, verificar se a qualidade sequência per-base e os índices de qualidade per-seqüência estão acima de 20. </li></ol></li></ol> 10. Digital Normalização <ol><li> Execute uma rodada de normalização digital no limpos lê usando a ferramenta khmer 31 (Tabela 1; Suplementar Arquivo 1), especificamente normalize-by-median.py (usando tamanho k-mer 20, uma cobertura de corte de 20, e x = 1e10). </li><li> Como alternativa, se uma máquina com alta RAM disponível (centenas de GBs), usar o script normalize_by_kmer_coverage.pl de Trinity (Tabela 1). </li></ol> 11. De Assembléia transcriptoma Novo <ol><li> Obter acesso a um grupo de computadores ou computador com até 256 Gb de RAM e CPUs 24, dependendo do tamanho do conjunto. </li><li> Use Trinity 32 (Tabela 1; Suplementar Arquivo 1) para montar a leitura digital normalizado definido em contigs. Para reduziruso de memória, use --min_kmer_cov 2. </li></ol> 12. Avaliação Assembleia <ol><li> Execute assemblathon_stats.pl do projeto Assemblathon2 33 na saída contig Trindade. Este script executa cálculos básicos relevantes para a avaliação da qualidade de montagem, tais como número de andaimes, N50, composição de montagem, e mais (Tabela 1; Suplementar arquivo 1). </li></ol> 13. Anotação do transcriptoma montado <ol><li> Execute Blastx (Tabela 1) do conjunto contra um conjunto de proteínas de referência; para os mosquitos, Drosophila melanogaster, Anopheles gambiae, Culex pipiens, e Aedes aegypti são referências adequadas. Especificamente, formatar o arquivo fasta proteína de referência para a explosão, seguida de blastx (Suplementar Arquivo 1). </li></ol> 14. Mapa Lê à Assembléia Usando RSEM 34 (Tabela 1) <ol><li> Criar um arquivo &#39;transcrição-to-gene-map&#39;, em que oprimeira coluna contém os IDs de genes de referência, e a segunda coluna os IDs contig. Em um editor de planilhas, trocar as primeira e segunda colunas a partir da saída Blastx, e escrever estas colunas em um arquivo .txt. Use o programa LineBreak para converter as quebras de linha no arquivo .txt resultante para formato Unix. </li><li> Criar um conjunto de dados de referência a partir do arquivo fasta transcriptoma usando o script rsem-prepare-referência, fornecido no pacote RSEM (Suplementar Arquivo 1). </li><li> Calcular os valores de expressão separadamente para cada biblioteca usando o comando rsem-calcular-expressão, fornecido no pacote RSEM (Suplementar Arquivo 1). Como lê, utilizar os ficheiros emparelhados fastq resultantes do passo de limpeza de leitura (passo 9.2). <ol><li> Se RNA a partir de uma réplica biológica foi dividida em duas pistas para o seqüenciamento, incluem ambos os arquivos fastq no cálculo expressão para gerar um único arquivo. </li></ol></li><li> Converter os resultados de expressão de cada biblioteca a uma matriz facilmente processados ​​por outro pROGRAMAS usando o rsem-gerar-data-matriz do script fornecido, fornecido no pacote RSEM (Suplementar Arquivo 1). </li></ol> 15. Análise da expressão diferencial <ol><li> Instale R e aparador (Tabela 1). </li><li> Use read.delim para carregar os resultados RSEM da etapa 14.3 (Suplementar Arquivo 1). Se necessário, completar a contagem para o número inteiro mais próximo. NOTA: O guia facetadora recomenda limitar o conjunto de dados de genes com expressão elevada o suficiente para detectar significado. </li><li> Para formatar os dados para facetadora, gerar um objeto DGEList do arquivo de dados carregado (Suplementar Arquivo 1). Em seguida, normalizar os dados usando TMM normalização (Suplementar Arquivo 1). Estimar as dispersões comuns e tagwise dos dados (Suplementar arquivo 1). </li><li> Identificar genes diferencialmente expressos com um Benjamini-Hochberg corrigido p &lt;0,05 (File Suplementar 1). Traça-se a distribuição de log-fold-change vs. abundância (Suplementar Arquivo 1). </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+adaptive+evolution+of+photoperiodic+response+during+invasion+and+range+expansion+across+a+climatic+gradient.">Rapid adaptive evolution of photoperiodic response during invasion and range expansion across a climatic gradient.</a> Am. Nat. 179, 490-500 (2012).</li><li>Lounibos, L. P., Escher, R. L., Lourenco-de-Oliveria, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+evolution+of+photoperiodic+diapause+in+temperate+and+tropical+invasive+populations+of+Aedes+albopictus+(Diptera+Culicidae).">Asymmetric evolution of photoperiodic diapause in temperate and tropical invasive populations of Aedes albopictus (Diptera Culicidae).</a> Ann. Entomol. Soc. Am. 96, 512-518 (2003).</li><li>Mori, A., Oda, T., Wada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Studies on the egg diapause and overwintering of Aedes albopictus in Nagasaki. Trop. Med. 23, 79-90 (1981).</li><li>Pumpuni, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Factors influencing photoperiodic control of egg diapause in Aedes albopictus [dissertation]. , (1989).</li><li>Wang, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+influence+of+photoperiod+on+egg+diapause+in+Aedes+albopictus+Skuse.">Observations on the influence of photoperiod on egg diapause in Aedes albopictus Skuse.</a> Acta Entomol. Sinica. 15, 75-77 (1966).</li><li>Sota, T., Mogi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival-time+and+resistance+to+desiccation+of+diapause+and+non-diapause+eggs+of+temperate+Aedes+(Stegomyia)+mosquitoes.">Survival-time and resistance to desiccation of diapause and non-diapause eggs of temperate Aedes (Stegomyia) mosquitoes.</a> Entomol. Exp. Appl. 63, 155-161 (1992).</li><li>Reynolds, J. A., Poelchau, M. F., Rahman, Z., Armbruster, P. A., Denlinger, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+profiling+reveals+mechanisms+for+lipid+conservation+during+diapause+in+the+mosquito,+Aedes+albopictus.">Transcript profiling reveals mechanisms for lipid conservation during diapause in the mosquito, Aedes albopictus.</a> J. Insect Physiol. 58, 966-973 (2012).</li><li>Andrewartha, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diapause+in+relation+to+the+ecology+of+insects.">Diapause in relation to the ecology of insects.</a> Biol. Rev. 27, 50-107 (1952).</li><li>Danks, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+Dormancy:+An+Ecological+Perspective.">Insect Dormancy: An Ecological Perspective.</a> Biological Survey of Canada (Terrestrial Arthropods). , (1987).</li><li>Denlinger, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+diapause.">Regulation of diapause.</a> Ann. Rev. Entomol. 47, 93-122 (2002).</li><li>Rev Entomol, A. n. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 59, 93-122 (2014).</li><li>Armbruster, P. A., Conn, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+variation+of+larval+growth+in+North+American+Aedes+albopictus+(Diptera).">Geographic variation of larval growth in North American Aedes albopictus (Diptera).</a> Culicidae). Ann. Entomol. Soc. Am. 99, 1234-1243 (2006).</li><li>Reiter, P., Amador, M. A., Colon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+CDC+ovitrap+with+hay+infusions+for+daily+monitoring+of+Aedes+aegypti+populations.">Enhancement of the CDC ovitrap with hay infusions for daily monitoring of Aedes aegypti populations.</a> J. Am. Mosquito Contr. Association. 7 (1), 52 (1991).</li><li>Rezende, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+desiccation+resistance+in+Aedes+aegypti:+presumptive+role+of+the+chitinized+serosal+cuticle.">Embryonic desiccation resistance in Aedes aegypti: presumptive role of the chitinized serosal cuticle.</a> BMC Dev. Biol. 8, 182 (2008).</li><li>Munstermann, L., Crampton, J., Beard, C., C, L. o. u. i. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Care+and+maintenance+of+Aedes+mosquito+colonies.">Care and maintenance of Aedes mosquito colonies.</a> The Molecular Biology of Insect Disease Vectors. , 13-20 (1997).</li><li>Trpis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+bleaching+and+decalcifying+method+for+general+use+in+zoology.">A new bleaching and decalcifying method for general use in zoology.</a> Can. J. Zoolog. 48, 892-893 (1970).</li><li>Ning, Z. M., Cox, A. J., Mullikin, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SSAHA:+A+fast+search+method+for+large+DNA+databases.">SSAHA: A fast search method for large DNA databases.</a> Genome Res. 11 (10), 1725-1729 (2001).</li><li>Cox, M. P., Peterson, D. A., Biggs, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SolexaQA:+At-a-glance+quality+assessment+of+Illumina+second-generation+sequencing+data.">SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data.</a> BMC Bioinformatics. 11, 485 (2010).</li><li>Grabherr, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-length+transcriptome+assembly+from+RNA-Seq+data+without+a+reference+genome.">Full-length transcriptome assembly from RNA-Seq data without a reference genome.</a> Nat. Biotechnol. 29 (7), 644-652 (2011).</li><li>Bradnam, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assemblathon+2:+evaluating+de+novo+methods+of+genome+assembly+in+three+vertebrate+species.">Assemblathon 2: evaluating de novo methods of genome assembly in three vertebrate species.</a> GigaScience. 2, 10 (2013).</li><li>Li, B., Dewey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RSEM:+accurate+transcript+quantification+from+RNA-Seq+data+with+or+without+a+reference+genome.">RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.</a> BMC Bioinformatics. 12, 323 (2011).</li><li>White, B. N., De Lucca, F. L., Turner, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+analysis+of+RNA.">Preparation and analysis of RNA.</a> Analytical Biochemistry of Insects. , (1977).</li><li>Hawley, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+Aedes+albopictus.">The biology of Aedes albopictus.</a> J. Am. Mosq. Contr. Assoc. 4, 1-39 (1988).</li><li>Dowling, Z., Ladeau, S. L., Armbruster, P., Biehler, D., Leisnham, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Socioeconomic+status+affect+mosquito+(Diptera:Culicidae)+larval+habitat+type+availability+and+infestation+level.">Socioeconomic status affect mosquito (Diptera:Culicidae) larval habitat type availability and infestation level.</a> J. Med Entomol. 50, 764-772 (2013).</li><li>Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2,4,10.+Bloodfeeding:+Membrane+apparatuses+and+animals.">Chapter 2,4,10. Bloodfeeding: Membrane apparatuses and animals.</a> Methods in Anopheles Research. , (2010).</li><li>Das, S., Garver, S., Ramirez, J. R., Xi, Z., Dimopolous, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dengue+infections+in+mosquitoes+(A.+aegypti)+and+infection+phenotype+determination.">Protocol for dengue infections in mosquitoes (A. aegypti) and infection phenotype determination.</a> J. Vis. Exp. 5 (220), (2007).</li><li>Goff, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iPlant+collaborative:+cyberinfrastructure+for+plant+biology.">The iPlant collaborative: cyberinfrastructure for plant biology.</a> Plant Sci. 2 (34), (2011).</li><li>Robinson, M. D., McCarthy, D. J., Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=edgeR:+a+Bioconductor+package+for+differential+expression+analysis+of+digital+gene+expression+data.">edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.</a> Bioinformatics. 26, 139-140 (2010).</li><li>Edgar, R. C., Haas, B. J., Clemente, J. 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The authors have nothing to disclose.

Este protocolo apresenta métodos para descobrir genes diferencialmente expressos, devido à photoperiodically diapausa induzida em A. albopictus. O protocolo é significativo na medida em que combina de forma única de criação do mosquito e técnicas de bioinformática para fazer todos os aspectos experimentais de um programa de fisiologia molecular acessível a usuários novatos - em especial para as que incidam sobre a resposta ao fotoperíodo diapausa. Os métodos existentes, a nosso conhecimento, não fo...

Rapid advances in next-generation sequencing (NGS) technologies are providing exciting opportunities to probe the molecular underpinnings of a wide range of genetically complex ecological adaptations in a broad diversity of non-model organisms1&#8211;3. This approach is extremely powerful because it establishes a basis for population and functional genomics studies of organisms with an especially interesting and/or well-described ecology or evolutionary history, as well as organisms of practical concern, such as agricultural pests and disease vectors. Thus, NGS technologies are leading to rapid advances in the fields of ecology and have the potential to address problems such as understanding the mechanistic bases of biological responses to rapid contemporary climate change4, the spread of invasive species5, and host-pathogen interactions6,7.
The extraordinary potential of NGS technologies for addressing basic and applied questions in ecology and evolutionary biology is in part due to the fact that these approaches can be applied to any organism at a moderate cost that is feasible for most research laboratories. Furthermore, these approaches provide genome-wide information without the requirement of a priori genetic resources such as a microarray chip or complete genome sequence. Nevertheless, to maximize the productivity of NGS experiments requires careful consideration of experimental design including issues such as the developmental timing and tissue-specificity of RNA sampling. Furthermore, the technical skills required to analyze the massive amounts of data produced by these experiments, often up to several hundred million DNA sequence reads, has been a particular challenge and has limited the widespread implementation of NGS approaches.
Recent RNA-Seq studies on the transcriptional bases of diapause in the invasive and medically important mosquito Aedes albopictus provide a useful example of some of the experimental protocols that can be employed to successfully apply NGS technology to studying the molecular basis of a complex ecological adaptation in a non-model organism8&#8211;10. A. albopictus is a highly invasive species that is native to Asia but has recently invaded North America, South America, Europe, and Africa11,12. Like many temperate insects, temperate populations of A. albopictus survive through winter by entering a type of dormancy referred to as photoperiodic diapause. In A. albopictus, exposure of pupal and adult females to short (autumnal) day lengths leads to the production of diapause eggs in which embryological development is completed, but the pharate larva inside the chorion of the egg enters a developmental arrest that renders the egg refractory to hatching stimulus15&#8211;17. Diapause eggs are more desiccation resistant5,18 and contain more total lipids19 than non-diapause eggs. Photoperiodic diapause in A. albopictus is thus a maternally controlled, adaptive phenotypic plasticity that is essential for surviving the harsh conditions of winter in temperate environments. Despite the well-understood ecological significance of photoperiodic diapause in a wide range of insects20,21, the molecular basis of this crucial adaptation is not well characterized in any insect22. In organisms such as A. albopictus that undergo an embryonic diapause at the pharate larval stage, it remains a particularly compelling challenge to understand how the photoperiodic signal received by the mother is passed to the offspring and persists through the course of embryonic development to cause arrest at the pharate larval stage.
This protocol describes mosquito rearing, experimental design and bioinformatics analyses for NGS experiments (transcriptome sequencing) performed to elucidate transcriptional components of photoperiodic diapause in A. albopictus. This protocol can be used for additional studies of diapause in A. albopictus, can be adapted to investigate diapause in other closely related species such as other aedine mosquitoes that undergo egg diapause23, and is also more generally relevant to employing NGS approaches to study the transcriptional bases of any complex adaptation in any insect.

<table><tbody><tr><td>Incubator - Model 818</td><td>Thermo-Scientific</td><td>3751</td><td>120 V</td></tr><tr><td>Controlled environment room</td><td>Thermax Scientific</td><td>N/A</td><td>Walk-in controlled environment room built to custom specifications by Thermax Scientific Products. A larger alternative to an incubator. http://thermmax.com/</td></tr><tr><td>Cool Fluorescent bulb</td><td>Philips</td><td>392183</td><td>4 W</td></tr><tr><td>Petri Dish 100 mm x 20 mm</td><td>Fisher</td><td>08-772-E</td><td></td></tr><tr><td>Filter Paper 20.5 cm</td><td>Fisher</td><td>09-803-6J</td><td></td></tr><tr><td>9.5 L Bucket</td><td>Plastican</td><td>Bway Products</td><td>http://www.bwayproducts.com/sites/portal/plastic-products/plastic-open-head-pails/117</td></tr><tr><td>Utility Fabric-Mosquito Netting White</td><td>Joann</td><td>10173292</td><td>http://www.joann.com/utility-fabric-mosquito-netting-white/10173292.html</td></tr><tr><td>Orthopedic stockings</td><td>Albahealth</td><td>23650-040</td><td>product no. 081420</td></tr><tr><td>Organic Raisins</td><td>Newman&#039;s Own</td><td>UPC: 884284040255</td><td></td></tr><tr><td>Oviposition cups (brown)</td><td>Fisher Scientific</td><td>03-007-52</td><td>The product is actually an amber 125 ml bottle that we saw the top off of.</td></tr><tr><td>Recycled Paper Towels</td><td>Seventh Generation</td><td>30BPT120</td><td></td></tr><tr><td>Modular Mates Square Tupperware Set</td><td>Tupperware</td><td></td><td>http://order.tupperware.com/pls/htprod_www/coe$www.add_items</td></tr><tr><td>Glass Grinder</td><td>Corning Incorporated</td><td>7727-2</td><td>These Tenbroeck tissue grinders break the eggs and release RNA into the TRI Reagent.</td></tr><tr><td>TRI Reagent</td><td>Sigma Aldrich</td><td>T9424</td><td>Apply 1 ml TRI Reagent per 50-100 mg of tissue. Caution &amp;#8212; this reagent is toxic.</td></tr><tr><td>TURBO DNA-free</td><td>Ambion/Life Technologies</td><td>AM1907</td><td>This kit generates greater yield than traditional DNase treatment followed by phenol/chloroform cleanup, and it is simpler to use.</td></tr><tr><td>RNaseZap</td><td>Ambion/Life Technologies</td><td>AM9782</td><td>Apply liberally on the bench surfaces and any equipment that might be in contact with the RNA samples. The solution is slightly alkaline/corrosive, can cause irritation and is harmful when swallowed.</td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2939AA</td><td>Place up to 12 RNA samples on one chip.</td></tr><tr><td>Hemotek Membrane Feeder</td><td>Hemotek&amp;nbsp;</td><td>5W1</td><td>This system&amp;nbsp; provides 5 feeding stations that can be used simultaneously. Includes PS5 Power Unit and Power cord; 5 FUI Feeders + Meal Reservoirs and O-rings; Plastic Plugs, Hemotek collagen feeding membrane; Temperature setting tool; and Plug extracting tool. The company&#039;s mailing address is: Hemotek Ltd; Unit 5 Union Court; Alan Ramsbottom Way; Great Harwood; Lancashire, UK; BB6 7FD; tel: +44 1254 889 307.</td></tr><tr><td>Digital Thermometer and Probe</td><td>Hemotek&amp;nbsp;</td><td>MT3KFU</td><td>MicroT3 thermometer and KFU probe. This is used to set the temperature of each FUI feeding unit.</td></tr><tr><td>Chicken Whole Blood, non-sterile with Sodium Citrate</td><td>Pel-Freez Biologicals</td><td>33130-1</td><td>The 500 ml of blood were frozen and stored in 20 ml aliquots at -80 &amp;#176;C for up to 1 year. Thaw blood at room temperature for at least 1 hr before using.</td></tr></tbody></table>

an experimental and bioinformatics protocol for rna-seq analyses of photoperiodic diapause in the asian tiger mosquito, aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

Fluorometria de duas amostras de RNA representativos mostraram duas bandas a aproximadamente 2000 nt (Figura 1A, B). O ARN ribossómico 28S de insectos é composta de duas cadeias polinucleotídicas mantidas juntas por ligações de hidrogénio, que são facilmente rompidas por breve aquecimento ou agentes que perturbam a 35 ligações de hidrogénio. Os dois componentes resultantes são aproximadamente do mesmo tamanho que o ARN ribossómico 18S. A segunda amostra de RNA apresentaram altos n...

Watch this Scientific Journal Video about An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus at JoVE.com

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Uma Experimental e Bioinformática Protocolo de Análises de RNA-seq de fotoperiódicas diapausa no Tigre Asiático Mosquito,<em&gt; Aedes albopictus</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

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Biology

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

13.2K visualizações.  Georgetown University. RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data. 

Vídeo: An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocolo para Ensaios RNAi em mosquitos adultos (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Biologia

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

Reprodutível dsRNA Microinjection e Bioassay de oviposição em mosquitos e moscas domésticas

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

Ambiente

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. 
High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis.

Detecção indel seguindo CRISPR/Cas9 Mutagenesis usando análise de derretimento de alta resolução no mosquito Aedes aegypti

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases ...

Genética

Electroporation-mediated RNA Interference Method in Odonata

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, morphology, and behavior drastically from aquatic larvae to terrestrial/aerial adults without pupal stage. Odonata adults have a well-developed color vision and show a remarkable diversity in body colors and patterns across sexes, stages, and species. While many ecological and behavioral studies on Odonata have been conducted, molecular genetic studies have been scarce mainly due to the difficulty in applying gene functional analysis to Odonata. For instance, RNA interference (RNAi) is less effective in the Odonata, as reported in the Lepidoptera. To overcome this problem, we successfully established an RNAi method combined with in vivo electroporation. Here we provide a detailed protocol including a video of the electroporation-mediated RNAi method as follows: preparation of larvae, species identification, preparation of dsRNA/siRNA solution and injection needles, ice-cold anesthesia of larvae, dsRNA/siRNA injection, in vivo electroporation, and individual rearing until adult emergence. The electroporation-mediated RNAi method is applicable to both damselflies (suborder Zygoptera) and dragonflies (suborder Anisoptera). In this protocol, we present the methods for the blue-tailed damselfly Ischnura senegalensis (Coenagrionidae) as an example of damselfly species and the pied skimmer dragonfly Pseudothemis zonata (Libellulidae) as another example of dragonfly species. As representative examples, we show the results of RNAi targeting the melanin synthesis gene multicopper oxidase 2. This RNAi method will facilitate understanding of various gene functions involved in metamorphosis, morphogenesis, color pattern formation, and other biological features of Odonata. Moreover, this protocol may be generally applicable to non-model organisms in which RNAi is less effective in gene suppression due to the inefficiency and low penetrance.

We provide a detailed protocol for electroporation-mediated RNA interference in insects of the order Odonata (dragonflies and damselflies) using the blue-tailed damselfly (Ischnura senegalensis: Coenagironidae: Zygoptera) and the pied skimmer dragonfly (Pseudothemis zonata: Libellulidae: Anisoptera).

Método de interferência de RNA mediado por eletroporação em Odonata

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, ...

Biologia do Desenvolvimento

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi mediada knockdown do gene e<em&gt; In Vivo</em&gt; Ensaio diurese no Feminino Adulto<em&gt; Aedes aegypti</em&gt; Os mosquitos

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Fisiológicas Recordings e RNA Sequenciação dos gustativas Appendages do Mosquito-febre amarela<em&gt; Aedes aegypti</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi gatilho para o interior<em&gt; Anopheles gambiae</em&gt; As pupas

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

IR-TEx: An Open Source Data Integration Tool for Big Data Transcriptomics Designed for the Malaria Vector Anopheles gambiae

IR-TEx is an application written in Shiny (an R package) that allows exploration of the expression of (as well as assigning functions to) transcripts whose expression is associated with insecticide resistance phenotypes in Anopheles gambiae mosquitoes. The application can be used online or downloaded and used locally by anyone. The local application can be modified to add new insecticide resistance datasets generated from multiple -omics platforms. This guide demonstrates how to add new datasets and handle missing data. Furthermore, IR-TEx can be completely and easily recoded to use-omics datasets from any experimental data, making it a valuable resource to many researchers. The protocol illustrates the utility of IR-TEx in identifying new insecticide resistance candidates using the the microsomal glutathione transferase, GSTMS1, as an example. This transcript is upregulated in multiple pyrethroid resistant populations from C&#244;te D&#39;Ivoire and Burkina Faso. The identification of co-correlated transcripts provides further insight into the putative roles of this gene.

IR-TEx: An Open Source Data Integration Tool for Big Data Transcriptomics Designed for the Malaria Vector Anopheles gambiae

IR-TEx explores insecticide resistance-related transcriptional profiles in the species Anopheles gambiae. Provided here are full instructions for using the application, modifications for exploring multiple transcriptomic datasets, and using the framework to build an interactive database for collections of transcriptomic data from any organism, generated in any platform.

IR-TEx: Uma ferramenta de integração de dados de código aberto para transcrição de big data projetada para o Vector Anopheles gambiae da malária

IR-TEx is an application written in Shiny (an R package) that allows exploration of the expression of (as well as assigning functions to) transcripts ...

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus on one or more gene families that are significant to the organism and study. In this protocol, we outline bioinformatic steps to generate a phylogeny and to quantify the expression of genes of interest. Phylogenetic trees can give insight into how genes are evolving within and between species as well as reveal orthology. These results can be enhanced using RNA-seq data to compare the expression of these genes in different individuals or tissues. Studies of molecular evolution and expression can reveal modes of evolution and conservation of gene function between species. The characterization of a gene family can serve as a springboard for future studies and can highlight an important gene family in a new genome or transcriptome paper.

The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing data.

Um Pipeline bioinforático para investigar a evolução molecular e a expressão genética usando RNA-seq

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

Um protocolo de extração de DNA de mosquito baseado em contas magnéticas para sequenciamento de próxima geração

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

Uma Experimental e Bioinformática Protocolo de Análises de RNA-seq de fotoperiódicas diapausa no Tigre Asiático Mosquito,

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