Gostaríamos de agradecer a Anthony Weinhold e os inúmeros estudantes que trabalharam connosco ao longo dos anos para suporte técnico, bem como sugestões para melhorar nosso procedimentos de criação de inseto.

1. ovo coleção (Figura 1)
<ol><li>Preparar a incitar a taça, coloque uma das extremidades do pano ~ 500 mL copo cheio de água morna (~ 40 ° C). Sobrepõem-se os lados do copo e fixe com um elástico. Dobre a ponta solta do pano volta por cima da taça.</li>
	<li>Lugar Copa a incitar na gaiola de moscas de 8 – 10 dias de idade para as mulheres Gravid de ~ 2 h. estável voa será ovipositar no pano.</li>
	<li>Remova a incitar a taça de ovos gaiola e enxaguar fora o pano egging em uma panela pequena com lavagem de garrafa.</li>
</ol>2. prepare Pan criação Larval
<ol><li>
Preparar o meio (quantidades para 1 pan de criação)
	<ol>
<li>Combinar o farelo de trigo (500 g), aparas de madeira (200 g) e farinha de peixe (115 g) em panela de prato plástico 10L e misture bem.</li>
		<li>Adicionar o bicarbonato de amónio (50 g) de água (~ 25 ° C, 1600 mL) e misture até dissolver.</li>
		<li>Adicione a solução de água de ingredientes secos e misture para não garantir nenhum material unwetted. Nível médio em panela mas não comprimir.</li>
	</ol>
</li>
	<li>
Adicione os ovos de mosca estáveis
	<ol>
<li>Faça um sulco raso em média o comprimento da bandeja.</li>
		<li>Depósito de 1 mL de ovos de mosca estáveis (~ 8.000 ovos) no sulco com pipeta de ovo (Figura 2). Nota: Pipeta de ovo feita por um corte de uma pipeta graduada plástica no ponto desejado, inserir 100 mesh tela e colagem pipeta junta novamente.</li>
		<li>Cubra os ovos com uma fina camada do meio para evitar a dessecação.</li>
		<li>Cubra a panela com fronha, fechar com uma faixa de borracha e etiqueta. Colocar panelas no quarto larval (23 ± 2 ° C, 30 – 50% RH e 12:12 [L:D] h fotoperíodo). Nota: Desenvolvimento Larval leva de 10 a 14 dias do tempo de oviposição.</li>
	</ol>
</li>
</ol>3. processamento pupa, as larvas se mover para a borda do meio 7 – 9 dias após a oviposição e Pupariate por dias 13 ou 14
<ol><li>Casulos de colher, localizados principalmente em torno das bordas da panela sob a crosta que se forma na superfície do meio, para fora e colocado em uma panela limpa.</li>
	<li>Encha a panela, com casulos, ½ a ¾ cheio de água. Separar grupos de casulos e médio. Componentes de farelo e proteína de trigo do meio de afundam-se enquanto pupários > 1 dia de idade flutuador19.</li>
	<li>Remova materiais flutuantes, casulos e alguns componentes médios com uma peneira e lavagem através de uma série de peneiras (5 #, 7 #, #12, #20) para remover o restante médio. A peneira #12 recolhe o casulo e a #20 evita que resíduos sólidos entrando pelo ralo. Casulos de lavagem entre peneiras com um pulverizador de pia.</li>
	<li>Casulos de enxágue, em peneira #12, em uma panela limpa. Encha a panela com ½ cheio de água. Despeje pupários flutuante em um coador e deixe escorrer o excesso de água.</li>
	<li>Transferência drenado pupários para tela de secagem com o ventilador e deixar até secar (Figura 3).</li>
</ol>4. pupa prateleira, um método alternativo para a coleta de pupários é com uma prateleira Pupal18
Nota: A prateleira é feita de um pedaço de plástico corte da extremidade de uma panela de prato de 10 L (10,2 cm de altura x 10,2 cm de largura x 31,9 cm de comprimento).
<ol><li>Prepare o suporte conforme descrito no ponto 2.1. Cone médio em panela de ~2.5 cm no fundo uma ponta de ~7.5 cm profundo no outro.</li>
	<li>
Coloque a prateleira no meio na parte rasa da criação pan e fita para evitar larvas se rastejando ao lado da prateleira (Figura 4).
	<ol>
<li>Saturar a esponja (~14.5 x 9.0 x 4.5 cm3, celulose regenerada) e cobrir com um pano embebido de água (30.5 x 42 cm2, algodão) e lugar na prateleira de cerca de meia polegada do meio.</li>
	</ol>
</li>
	<li>
Adicione os ovos, conforme descrito no ponto 2.2 e proceder como anteriormente.
	<ol>
<li>Verifique panelas diariamente para garantir que a esponja permaneça úmida.</li>
	</ol>
</li>
	<li>Casulos de enxaguamento da plataforma, esponja e pano em um prato limpo panela e despeje no coador, 14 dias após a oviposição.</li>
</ol>5. controle de qualidade
<ol><li>Pese todos os casulos produzidos no pan (peso total)..</li>
	<li>Isolar 100 casulos, pesar e colocado em uma placa de Petri de 9 cm.</li>
	<li>Contagem e sexo adultos ~ 5 a 8 dias mais tarde para determinar a taxa de surgimento e proporção sexual. Loja de placas de Petri com adultos surgiu em um freezer para a contagem em uma data posterior, se necessário. Moscas são sexadas pela forma de seus olhos e a largura da placa fronto-orbital20 ou genitália em pequeno aumento.</li>
	<li>Peso total de registro, peso de 100 casulos e número de surgiu adultos machos e fêmeas.</li>
</ol>6. preparação de sangue
<ol><li>Colete o sangue fresco de bovino de um matadouro local em baldes de 19 L contendo 70 g de citrato de sódio tribásico dihidrato em 500 mL de água. Mexa vigorosamente por 5-10 min de sangue e voltou para o laboratório.</li>
	<li>Sangue de tensão através de uma peneira para remover os coágulos e particionado em recipientes de 2 L, etiqueta com a data de colheita e armazenamento em um freezer (-20 ° C).</li>
	<li>Precisava de 2 dias antes do recipiente retire do congelador e coloque na geladeira. Sangue pode ser usado por ~ 2 semanas uma vez descongelados e armazenados no freezer por até 1 ano.</li>
</ol>7. adulta manutenção
<ol><li>Prepare as gaiolas, fundo de gaiola linha com papel pardo para facilitar a limpeza.</li>
	<li>Coloque 50 g de pupas de mosca secas estáveis (~ 3.500) em cada gaiola. Eclose adultos algumas horas a alguns dias após o processamento pupal.</li>
	<li>Lugar um sangue fresco embebido guardanapo feminino em cima de cada gaiola para alimentação. Começam a alimentar-se dentro de 24 h de adulto eclosão e repetir diariamente, até um dia antes da oviposição (9-10 dias após a emergência).</li>
	<li>Após a oviposição (secção 1), coloque a gaiola no congelador para 4 – 8 h e depois limpar, removendo o papel, enxaguando Ivo dos lados e andar com água quente e esfregar todas as superfícies com detergente e cloro. Lave as gaiolas com água quente e deixe para secar.</li>
</ol>

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Entomol. 4 (1), 1-11 (1987).</li><li>Lysyk, T., Kalischuk-Tymensen, L., Selinger, L., Lancaster, R., Wever, L., Cheng, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+stable+fly+larvae+(Diptera:+Muscidae)+on+an+egg+yolk+medium.">Rearing stable fly larvae (Diptera: Muscidae) on an egg yolk medium.</a> J. Med. Entomol. 38, 382-388 (1999).</li><li>Romero, A., Broce, A., Zurek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+bacteria+in+the+oviposition+behaviour+and+larval+development+of+stable+flies.">Role of bacteria in the oviposition behaviour and larval development of stable flies.</a> Med. Vet. Entomol. 20, 115-121 (2006).</li><li>Glaser, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+flies+for+experimental+purposes+with+biological+notes.">Rearing flies for experimental purposes with biological notes.</a> J. Econ. Entomol. 17 (4), 486-496 (1924).</li><li>Melvin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+studies+on+the+effect+of+flies+and+fly+sprays+on+cattle.">Physiological studies on the effect of flies and fly sprays on cattle.</a> J. Econ. Entomol. 25 (6), 1151-1164 (1932).</li><li>Doty, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convenient+method+of+rearing+the+stable+fly.">Convenient method of rearing the stable fly.</a> J. Econ. Entomol. 30 (2), 367-369 (1937).</li><li>Bridges, A. C., Spates, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+medium+for+the+stable+fly+Stomoxys+calcitrans+(L.).">Larval medium for the stable fly Stomoxys calcitrans (L.).</a> Southwest. Entomol. 8 (1), 6-10 (1983).</li><li>Hogsette, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+diets+for+production+of+house+flies+and+stable+flies+(Diptera:+Muscidae)+in+the+laboratory.">New diets for production of house flies and stable flies (Diptera: Muscidae) in the laboratory.</a> J. Econ. Entomol. 85 (6), 2291-2294 (1992).</li><li>McGregor, W. S., Dreiss, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+stable+flies+in+the+laboratory.">Rearing stable flies in the laboratory.</a> J. Econ. Entomol. 48 (3), 327-328 (1955).</li><li>Goodhue, L. D., Cantrel, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+vermiculite+in+medium+for+stable+fly+larvae.">The use of vermiculite in medium for stable fly larvae.</a> J. Econ. Entomol. 51 (2), 250 (1958).</li><li>Friesen, K., Berkebile, D. R., Zhu, J. J., Taylor, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmenting+laboratory+rearing+of+stable+fly+(Diptera:+Muscidae)+larvae+with+ammoniacal+salts.">Augmenting laboratory rearing of stable fly (Diptera: Muscidae) larvae with ammoniacal salts.</a> J. Insect Sci. 17 (1), 1-6 (2017).</li><li>Berkebile, D. R., Weinhold, A. P., Taylor, D. 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+new+method+for+collecting+clean+stable+fly+(Diptera:Muscidae)+pupae+of+known+age.">A new method for collecting clean stable fly (Diptera:Muscidae) pupae of known age.</a> Southwest. Entomol. 34 (4), 469-476 (2009).</li><li>Champlain, R. A., Fisk, F. W., Dowdy, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+improvements+in+rearing+stable+flies.">Some improvements in rearing stable flies.</a> J. Econ. Entomol. 47 (5), 940-941 (1954).</li><li>Zumpt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The stomoxyine biting flies of the world. , (1973).</li><li>Wienhold, B. J., Taylor, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+properties+of+stable+fly+(Diptera:+Muscidae)+developmental+sites+associated+with+round+bale+hay+feeding+sites+in+eastern+Nebraska.">Substrate properties of stable fly (Diptera: Muscidae) developmental sites associated with round bale hay feeding sites in eastern Nebraska.</a> Environ. Entomol. 41 (2), 213-221 (2012).</li><li>Friesen, K., Berkebile, D. R., Wienhold, B. J., Durso, L., Zhu, J., Taylor, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+parameters+associated+with+stable+fly+(Diptera:+Muscidae)+development+at+hay+feeding+sites.">Environmental parameters associated with stable fly (Diptera: Muscidae) development at hay feeding sites.</a> Environ. Entomol. 45 (3), 570-576 (2016).</li><li>Albuquerque, T. A., Zurek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+changes+in+the+bacterial+community+of+animal+feces+and+their+correlation+with+stable+fly+oviposition,+larval+development,+and+adult+fitness.">Temporal changes in the bacterial community of animal feces and their correlation with stable fly oviposition, larval development, and adult fitness.</a> Front. Microbiol. 5 (590), 1-9 (2014).</li><li>Bailey, D. L., Whitfield, T. L., LaBrecque, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+biology+and+techniques+for+mass+producing+the+stable+fly,+Stomoxys+calcitrans+(L.)+(Diptera:+Muscidae).">Laboratory biology and techniques for mass producing the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae).</a> J. Med. Entomol. 12 (2), 189-193 (1975).</li><li>Smith, J. 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Os autores não têm nada para divulgar.

Estável de moscas encontram-se desenvolvendo em uma ampla variedade de substratos na natureza e podem ser criadas em muitos tipos de mídia no laboratório. Nós usamos aparas de madeira e vermiculita como agentes de volume. Vermiculita funcionou bem, mas fez separação pupários de criação meio difícil e era caro (~$0.60/pan). Possivelmente por causa do rigor adicionado de separar pupários do meio, eclosão também foi menor com vermiculita, 57% vs. 75% de aparas de madeira. Aparas de Cottonwood foram comparáveis...

Estável de moscas, Stomoxys calcitrans (L.), são hematófagos moscas cujas mordidas dolorosas interrompem o comportamento de pastoreio de gado, causam dor e sofrimento para animais de companhia e interromper atividades recreativas humanas em todo o mundo. Imaturas estáveis moscas desenvolvem-se em fermentação vegetativa passa, muitas vezes contaminada com resíduos animais. Culturas e práticas agronômicas mudanças produziram graves surtos de moscas estáveis em resíduos de culturas, legumes em Austrália1, cana-de-açúcar no Brasil2e abacaxi em Costa Rica3. Embora apenas 14 estáveis moscas por animal são consideradas o limite económico4, foram feitas observações de mais de 2.000 moscas por animal durante recentes surtos5. Esses níveis de infestação reduzem a produtividade de anfitrião para perto de zero e podem causar mortalidade6. Como resultado de surtos associados agronomicamente, moscas estáveis recebendo renovado interesse e demanda por colônias de laboratório tem aumentado dramaticamente.
Quanto a todos os insetos holometamorphic, moscas estáveis obter todos os nutrientes necessários para o crescimento durante o estágio larval ou imaturo. Portanto, um importante componente de um sistema de criação é a dieta larval ou substrato. Foram observadas larvas de mosca estáveis desenvolvimento em uma ampla gama de substratos no campo7 e eles são dependentes da comunidade microbiana do substrato8,9. Substratos larvas naturais são compostos principalmente de decomposição ou fermentação vegetativas materiais frequentemente contaminados com resíduos nitrogenados.
Para a criação de laboratório, substratos larvas moscas estáveis são geralmente compostos por um material vegetativo e uma fonte de nitrogênio adicionado. Vários materiais foram usados para dietas larvas moscas estáveis. As dietas larvas primeiras imitaram substratos naturais e incluíam da fermentação, palha de aveia e de cavalo ou vaca estrume10,11. Fontes de carboidratos incluem trigo farelo12,13,14, alfafa refeição12,13,14 e uma formulação comercial desenvolvido por especialidades químicas Fabricantes Association (CSMA, 33% de farelo de trigo, farinha de alfafa de 27%, levedura de cerveja 40% grânulos)13,14,15,16. Fontes de nitrogênio incluem levedura suspensão12, farinha de peixe e de bicarbonato de amónio17. Volume de materiais inertes são frequentemente incluídos nas dietas, incluindo aveia cascos12, bagaço13, vermiculita16, lascas de madeira13,18 e peletizadas hulls do amendoim14.
Um objetivo principal do laboratório de criação é de produzir um produto que é como fisiologicamente semelhante ao "tipo selvagem" quanto possível, a fim de que experimentos de laboratório produzirá resultados refletindo de populações do campo. Isto requer que na criação e seleção ser minimizados para manter genético diversidade e recursos nutricionais ser comparáveis no campo. Objetivos secundários são para minimizar o trabalho e despesas. Um componente importante de minimizar despesas é o uso de componentes de dieta disponíveis localmente. A mosca de estábulo criação sistema apresentado foi desenvolvida para atender a estes objectivos.

<table>
	<tbody>
		<tr>
			<td>Diamalt</td>
			<td>Premier Malt Products, Inc., Saddle Brook, NJ</td>
			<td>2540</td>
			<td></td>
		</tr>
		<tr>
			<td>CSMA Fly media</td>
			<td>Purina Animal Nutrition, Arden Hills, MN</td>
			<td>5S6Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin Maxi Pad</td>
			<td>The Tranzonic Co., Cleveland, OH, USA</td>
			<td>5001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Calf Manna</td>
			<td>MannaPro, Chesterfield, MO, USA</td>
			<td>Manna Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate</td>
			<td>Spectrum Chemical Manufacturing Corp, Gardena, CA</td>
			<td>A1125</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat bran, Coarse</td>
			<td>Siemer Milling Company, Teutopolis, IL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wood shavings</td>
			<td>Tractor Supply Company, Brentwood, TN</td>
			<td>502770699</td>
			<td></td>
		</tr>
		<tr>
			<td>Fishmeal</td>
			<td>Consumer Supply Distributing, North Sioux City, SD</td>
			<td>F1550</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult cages</td>
			<td>All Aluminum Window Company, Lincoln, Ne</td>
			<td>Custom</td>
			<td>45 &#215; 45 &#215; 45 cm, 18 &#215; 16 mesh aluminum screen, stockinette access</td>
		</tr>
		<tr>
			<td>9 &#215; 28 cm black cotton cloth</td>
			<td>Robert Kaufman Fabrics, Los Angeles, CA</td>
			<td>K040-114</td>
			<td>Egging cloth</td>
		</tr>
		<tr>
			<td>10 liter plastic dish pans</td>
			<td>Rubbermaid, Saratoga Springs, NY</td>
			<td>FG2951ARWHT</td>
			<td>Larval pans</td>
		</tr>
		<tr>
			<td>Stockinette, Cotton, 12&#34; X 25 yd roll</td>
			<td>Tex-Care Medical Company, Burlington, NC</td>
			<td>91311-225</td>
			<td></td>
		</tr>
	</tbody>
</table>

laboratory rearing of stable flies and other muscoid diptera

Estável de moscas, Stomoxys calcitrans, são pragas sérias de gado, seres humanos, animais de companhia e animais selvagens em todo o mundo. Durante os últimos 20 + anos, mudanças nas práticas agronômicas resultaram em graves surtos de moscas estáveis em vários países. Estes focos interrompeu a produção animal e recreação humana resultando em demandas públicas para aumentar os esforços de pesquisa e de gestão para esta praga. É apresentado um procedimento simples e de baixo custo para a criação de moscas estáveis para estudos de laboratório. O procedimento utiliza suprimentos, equipamentos e componentes da dieta disponíveis localmente. O procedimento pode ser adaptado para a criação de outras moscas de muscoid incluindo a mosca doméstica (Musca domestica), mosca do chifre (Haematobia irritans) e mosca do rosto (Musca autumnalis). O procedimento produz pupários mosca estável, com média de 12,5 mg e ~ 35% ovo para a sobrevivência de adulto. Cerca de 3000 moscas são produzidas em cada panela.

As larvas pupariate 10 a 14 dias e adultos emergem 14 – 16 dias após a oviposição. Tempo de geração, ovo a ovo, é ~ 24 dias. Dados de criação para maio de 2013 para de 2017 de Janeiro com três diferentes agentes de volume e duas colónias são apresentados na Figura 5. Cottonwood deu o melhor rendimento, 3867 ±1442 (<img alt="Equation" src="/files/ftp_upload/57341/57341eq1.jpg"> </img> ± desvio padrão) pupas pesando 12,5 mg ±1.6 com 74 ±19% ec...

Watch this Scientific Journal Video about Laboratory Rearing of Stable Flies and Other Muscoid Diptera at JoVE.com

Laboratory Rearing of Stable Flies and Other Muscoid Diptera

Um procedimento para a criação de moscas estáveis (Stomoxys calcitrans) é apresentado. O procedimento usa materiais localmente disponíveis para suprimentos, equipamentos e componentes de dieta.

Laboratório de criação de moscas estáveis e outros Muscoid Diptera

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in ...

laboratory-rearing-of-stable-flies-and-other-muscoid-diptera

Research

JoVE Journal

Biology

8.5K visualizações.  ARS-USDA. Um procedimento para a criação de moscas estáveis (Stomoxys calcitrans) é apresentado. O procedimento usa materiais localmente disponíveis para suprimentos, equipamentos e componentes de dieta.

Vídeo: Laboratory Rearing of Stable Flies and Other Muscoid Diptera

Microbial Communities in Nature and Laboratory - Interview

Comunidades microbianas na Natureza e Laboratório - Entrevista

Biologia

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

Protocolo para a Criação Mosquito (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

Colheita e Preparação Drosophila Os embriões para Gravação eletrofisiológicos e outros procedimentos

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA-Stable Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Um protocolo Optimizado para Criação Fopius arisanus, Um parasitóide de Frutas Tephritid Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Aseptic Laboratory Techniques: Plating Methods

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: 
 
&#9679; Perform plating procedures without contaminating media. 
&#9679; Isolate single bacterial colonies by the streak-plating method. 
&#9679; Use pour-plating and spread-plating methods to determine the concentration of bacteria. 
&#9679; Perform soft agar overlays when working with phage. 
&#9679; Transfer bacterial cells from one plate to another using the replica-plating procedure. 
&#9679; Given an experimental task, select the appropriate plating method.

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

Assépticas Técnicas de Laboratório: Métodos Plating

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

Simple Homemade Tools to Handle Fruit Flies&#8212;Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as adult flies fly. Demonstrated here is how to make some simple and cost-effective tools to address difficult issues in the handling of Drosophila. Holes in foam stoppers are made and pipette tips or funnels are inserted into the holes. Flies then move only in one direction into the pipette tip/funnel assemblage, allowing efficient control of the transfer of adult Drosophila into or out of a vial. Existing protocols have been modified for cool-anesthetizing flies by chilling in crushed ice and transferring them onto a cold, hard icepack surface. The icepack is covered with a piece of medical gauze that keeps immobilized flies from the condensed water when examined under a stereomicroscope. The flies are finally euthanized for counting and sorting or discarded by microwaving. A bottle-shaped cage has also been developed for collecting eggs, as well as a labor-saving device and accompanying protocol for cleaning glass culture vials.

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling Drosophila.

Ferramentas caseiras simples para lidar com moscas de frutas —Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

<meta charset="utf8"></head><body>Dissecção do saco endolinfático de camundongos

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

Dissecção do saco endolinfático de camundongos

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Isolamento escalável e purificação de vesículas extracelulares de Escherichia coli e outras bactérias

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

Laboratório de criação de moscas estáveis e outros Muscoid Diptera

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