Name: an introduction to parasitic wasps of drosophila and the antiparasite immune response
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Description: Watch this Scientific Journal Video about An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response at JoVE.com

Somos gratos ao Prof Todd Schlenke para Trichopria drosófilas, Prof Ip Tony para linhagens de moscas transgénicas, e Carl Prof Hashimoto para anti-Spätzle anticorpos. Agradecemos membros presentes e passadas de laboratório para suas contribuições para esta apresentação. Este trabalho foi financiado pelas subvenções seguintes: a partir de NIH (S06 GM08168, RISE 41399-009, e-RR03060 G12), USDA (NRI / USDA CSREES 2006-03817 e 2009-35302-05277) e PSC-CUNY.

Todo o protocolo para a experiência é dividido em quatro passos (Fig. 9). (1) Cultivar vespas em larvas de mosca, (2) Configurando infecções e preparar o animal para dissecção, (3) Isolamento e fixação hospedeiro / agente estruturas; (4) Analisando os tecidos imunes. 1. Vespas cultura sobre larvas de Drosophila Manutenção de culturas de vespas requer um planejamento cuidadoso. Em relação ao moscas em crescimento, é bastante tr...

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Interesse em vespas parasitas de Drosophila está surgindo como técnicas moleculares para decodificar genomas inteiros se tornar eficiente e rentável. No entanto, em relação aos seus excepcionalmente bem estudadas anfitriões, muitos aspectos fascinantes da biologia vespa permanecem obscuros. Estes incluem questões relacionadas com a gama de hospedeiros, imunossupressão, superparasitismo e comportamento. O foco desta apresentação foi demonstrar os efeitos da infecção nos tecidos imunes a mosca. As té...

<table border="1"><tbody><tr><td> Materiais </td><td> Tipo </td><td> Companhia </td><td> Número de Catálogo </td></tr><tr><td> Materiais para manutenção da cultura de insetos </td><td></td><td></td><td></td></tr><tr><td> Levedura </td><td> Seca ativa </td><td> Fisher Scientific </td><td> S802453 </td></tr><tr><td> Fly alimentos </td><td> O fubá de milho, açúcar </td><td></td><td> Receita padrão </td></tr><tr><td> Mel </td><td> Trevo </td><td> Holandês Ouro </td><td></td></tr><tr><td> Frascos </td><td> Frascos de polipropileno (estreito) </td><td> Fisher Scientific </td><td> AS514 </td></tr><tr><td> Fechamento dos frascos </td><td> Tampão de algodão </td><td> Fisher Scientific </td><td> AS212 </td></tr><tr><td> Fechamento dos frascos </td><td> Zumbido ficha </td><td&gt; Genesee Científico </td><td> AS273 </td></tr><tr><td> Incubadora refrigerada </td><td> Precision 815 </td><td> Thermo Scientific </td><td> 3721 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Materiais para a preparação da amostra </td><td></td><td></td><td></td></tr><tr><td> CO tanque 2 </td><td> Grau seco osso </td><td> TW Smith </td><td> UN1013 </td></tr><tr><td> Espátula </td><td> Espátula Micro (14 cm) </td><td> Fisher Scientific </td><td> 21-401-15 </td></tr><tr><td> Teste Pyrex placas local </td><td> 9-bem dissecar placa de 85 mm x 100 mm </td><td> Thomas Scientific </td><td> 7812G17 </td></tr><tr><td> Pipetas Pasteur </td><td> Cal de soda </td><td> J &amp; H Berge </td><td> 71-5200-05 </td></tr><tr><td> Fórceps </td><td> Estilo # 5 </td><td> Sigma </td><td> T-4662 </td></tr><tr><td> Etanol </td><td> 190 USP prova </td><td> Fisher Scientific </td><td> 04-355-221 </td></tr><tr><td> Formaldeído </td><td> 37% w / w </td><td> Fisher Scientific </td><td> F79-1 </td></tr><tr><td> Anticorpo secundário Cy3 AffiniPure de burro anti-coelho IgG (H + L) </td><td> 1:50 excitação de 546 nm, 565 nm de emissão </td><td> Jackson Immuno Research Laboratories, Inc. </td><td> 711-165-152 </td></tr><tr><td> Antifade (N-propil-galato) </td><td> 4 ug / ml em glicerol a 50% em PBS 1X </td><td> Biomedicals MP </td><td> 10274790 </td></tr><tr><td> Glicerina </td><td></td><td> Fisher Scientific </td><td> G33-1 </td></tr><tr><td> Hoechst 33258 </td><td> 0,2 ug / ml de excitação 352 nm, emissão 461 nm </td><td> Molecular Probes </td><td> H-1398 </td></tr><tr><td> Rodamina faloidina </td><td> 200 unidades / ml (6,6iM de excitação) 540 nm, emissão 565 nm </td><td> Molecular Probes </td><td> R415 </td></tr><tr><td> Alexa Fluor 488 faloidina </td><td> 300 unidades / ml de excitação de 495 nm, emissão 518 nm </td><td> Molecular Probes </td><td> A12379 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Descartáveis </td><td></td><td></td><td></td></tr><tr><td> Esguicho </td><td> Fisherbrand </td><td> Fisher Scientific </td><td> 03-409-22A </td></tr><tr><td> Kimwipes </td><td> Kimberly Clark </td><td> Fisher Scientific </td><td> 06-666A </td></tr><tr><td> Toalha de papel </td><td> 1 ply C-Fold </td><td> Franzir </td><td> 901-7CFTB2400 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Microscopia </td><td> &amp;nbsp; </td><td></td><td></td></tr><tr><td> Estereomicroscópio Leica </td><td> MZFLIII </td><td> Império Imaging Systems, Inc. </td><td> 10446208 </td></tr><tr><td> Stereomicroscope Zeiss </td><td> Stemi 1000 ou 2000-C </td><td> Carl Zeiss </td><td> 000000-1006-126 </td></tr><tr><td> Fonte de Luz - LED </td><td> Iluminador Gooseneck </td><td> Fisher Scientific </td><td> 12563501 </td></tr><tr><td> Etapa </td><td> Transmitido caixa de luz com placa </td><td> Carl Zeiss </td><td> 455137000 </td></tr><tr><td> Zeiss de varredura a laser microscópio confocal </td><td> LSM 510 </td><td> Carl Zeiss </td><td></td></tr><tr><td> Microscópio Zeiss composto </td><td> Axioplan 2 na posição vertical </td><td> Carl Zeiss </td><td></td></tr></tbody></table><table border="1"><tbody><tr><td> Cepas de vespas </td><td> Fly Estirpes </td></tr><tr><td> Leptopilina victoriae 16 </td><td> yw </td></tr><tr><td> Leptopilina boulardi 17 1 </td><td> UAS-GFP Dorsal-17 </td></tr><tr><td> Leptopilina heterotoma 2 </td><td> SerpentHemoGal4 13 </td></tr><tr><td> Leptopilina heterotoma 14 1 </td><td> MSNF9-moCherry 14 </td></tr><tr><td> Trichopria drosófilas </td><td> MSNF-GFP 15 </td></tr><tr><td> Ganaspis xanthopoda 18 </td><td> yw Serpente Gal4 UAS GFP-Dorsal/Basc 4 </td></tr><tr><td></td><td> yw; Drosomycin-GFP/CyO y + 12 </td></tr></tbody></table>

an introduction to parasitic wasps of drosophila and the antiparasite immune response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

Watch this Scientific Journal Video about An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response at JoVE.com

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitóide (parasita) vespas constituem uma classe importante de inimigos naturais de insetos, incluindo muitos<em&gt; Drosophila melanogaster</em&gt;. Vamos introduzir as técnicas para propagar estes parasitas em<em&gt; Drosophila</em&gt; Spp. e demonstrar como analisar seus efeitos sobre os tecidos imunes de<em&gt; Drosophila</em&gt; Larvas.

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