Summary

寄生蜂简介<em>果蝇</em>和Antiparasite的免疫反应的

Published: May 07, 2012
doi:

Summary

寄生蜂(寄生)构成了许多昆虫的天敌,包括各大类<em>果蝇</em>。我们将引进的技术,这些寄生虫传播<em>果蝇</em>属。并演示如何分析免疫组织的影响<em>果蝇</em>幼虫。

Abstract

最知名的寄生蜂攻击的果蝇幼虫或蛹的阶段。虽然Trichopria果蝇感染主机( 图1A-C的 ),蛹的阶段,女性的,属Leptopilina( 图1D,1F,1G)Ganaspis( 图1E)攻击的幼虫。我们使用这些寄生虫,一个种族的生物武器研究的分子基础。寄生蜂有防菌的巨大价值。他们大多携带的毒力和其他因素,修改主机的生理和免疫力。分析果蝇黄蜂提供特定物种的相互作用如何塑造自然群落的遗传结构的见解。这些研究还为了解主机的免疫生理以及如何协调免疫反应,这一类寄生虫挫败模型。

幼虫/蛹的表皮,作为第一线的夏德芬SE。蜂产卵器是一个尖锐的针状结构,可有效提供主机hemocoel的鸡蛋。其次是在表皮伤口愈合反应( 图1C,箭头)产卵。有些黄蜂可以插入同一台主机上的两个或两个以上的鸡蛋,虽然只有一个鸡蛋的开发成功。的编外鸡蛋或发展幼虫被淘汰一个尚未了解的过程。这些小蜂,因此被称为为孤寄生。

根据动态应变和蜂种,蜂卵有两种命运。它可以封装,以便其发展受阻(主机出现; 图2左);或黄蜂卵孵化,开发,molts,生长到成年(黄蜂出现图2)。L。 heterotoma果蝇寄生蜂的最好研究的物种之一。这是一个“多面手”,这意味着它可以利用大多数果蝇小号的pecies作为主机1 与heterotomaL。 victoriae是姐妹物种和它们产生的病毒样颗粒封装响应2,积极干预。与L。 heterotoma,L. boulardi是一个专业的寄生虫和果蝇的范围,它采用的是相对有限的1。L。 boulardi也产生病毒样颗粒3,虽然他们在自己的能力成功地 D显着不同 1。一些这些研究boulardi株是很难成长, D动态主机1 果蝇作为经常成功地封装它们的卵。因此,重要的是要在特定的实验协议双方的合作伙伴的知识。

此外屏障组织(角质层,肠道和气管), 果蝇幼虫有全身的细胞免疫和体液免疫反应,产生fROM功能的血细胞和脂肪体,分别。产卵由L. boulardi同时激活免疫武器1,4。流通中的血细胞被发现,在柄的人群,分段角质层下和淋巴腺。淋巴腺是一个小的背侧幼虫的造血器官。造血干细胞群,称为叶,成对排列分段沿背船只沿着动物的前后轴( 图3A)运行。脂肪体是一个大型的多功能器官( 图3B)。它分泌抗菌肽微生物和后生动物感染。

黄蜂感染激活免疫信号( 4)4。在细胞水平上,它触发的血细胞的分裂和分化。在自卫,聚集和胶囊开发感染动物hemocoel( 5)5,6。 ACtivated血细胞迁移向蜂卵(或黄蜂幼虫)和它周围开始形成一个胶囊( 图5A – F)。一些血细胞聚集形成结节( 图5G – H)。仔细分析发现,黄蜂感染引起淋巴腺叶最前,在其外围分散( 图6C,D)的。

我们提出用电话信号转导通路元件背和Spätzle( 图4,5,7),其目标Drosomycin( 图6)具有代表性的数据,来说明如何在淋巴腺和hemocoel的具体变化,可以研究黄蜂感染后。这里描述的剥离协议,也能产生从主机淋巴黄蜂( 图8)鸡蛋(或发展的黄蜂阶段)。

Protocol

整个实验的协议分为四个步骤( 图9)。 (1)蝇蛆养殖黄蜂(2)建立感染和准备解剖动物;(3)隔离和修复主机/寄生虫结构;(4)免疫组织分析。 1。对果蝇幼虫培养黄蜂维护黄蜂文化需要仔细规划。相对于日益增长的苍蝇,这是相当劳力密集。我们维持我们在实验室YW应变果蝇幼虫或蛹的蜂殖民地在24°C培养黄蜂保持在合…

Discussion

在对果蝇的寄生蜂的兴趣正在涌动解码整个基因组的分子生物学技术成为高效率和成本效益。然而,相对于他们非常出色的研究主机,蜂生物学的许多有趣的方面仍模糊不清。这些措施包括申办范围,免疫抑制,过寄生和行为有关的问题。这次演讲的重点是要证明在果蝇的免疫组织感染的影响。这里展示的解剖技术可以用于基因表达分析,RNA水平( 原位杂交),或在提取核酸芯片或…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢的托德Schlenke教授,教授托尼IP转基因果蝇株,为Trichopria果蝇和教授卡尔桥本反Spätzle抗体。此演示实验室的贡献,我们感谢过去和现在的成员。支持这项工作是由下列补助:(S06 GM08168,高层41399-009,和G12-RR03060),,从国立卫生研究院,美国农业部(野村综合研究所/农业部CSREES 2006-03817和2009-35302-05277)和PSC-纽约市立大学。

Materials

Materials Type Company Catalog number
Materials for insect culture maintenance      
Yeast Active dry Fisher Scientific S802453
Fly food Corn meal, sugar   Standard recipe
Honey Clover Dutch Gold  
Vials Polypropylene shell vials (narrow) Fisher Scientific AS514
Vial closures Cotton plug Fisher Scientific AS212
Vial closures Buzz plug Genesee Scientific AS273
Refrigerated incubator Precision 815 Thermo Scientific 3721
       
Materials for sample preparation      
CO2 tank Bone dry grade TW Smith UN1013
Spatula Micro spatula (14 cm) Fisher Scientific 21-401-15
Pyrex spot test plates 9-well dissecting plate 85 mm X 100 mm Thomas Scientific 7812G17
Pasteur Pipettes Soda lime J & H Berge 71-5200-05
Forceps Style # 5 Sigma T-4662
Ethanol 190 proof USP Fisher Scientific 04-355-221
Formaldehyde 37% w/w Fisher Scientific F79-1
Secondary antibody Cy3 AffiniPure donkey anti-rabbit IgG (H + L) 1:50 Excitation 546 nm; Emission 565 nm Jackson Immuno Research Laboratories, Inc. 711-165-152
Antifade (N-propyl gallate) 4 μg/ml in 50% glycerol in 1X PBS MP Biomedicals 10274790
Glycerol   Fisher Scientific G33-1
Hoechst 33258 0.2 μg/ml Excitation 352 nm; Emission 461 nm Molecular Probes H-1398
Rhodamine phalloidin 200 units/ml (6.6 μM) Excitation 540 nm; Emission 565 nm Molecular Probes R415
Alexa Fluor 488 phalloidin 300 units/ml Excitation 495 nm; Emission 518 nm Molecular Probes A12379
       
Disposables      
Wash bottle Fisherbrand Fisher Scientific 03-409-22A
Kimwipes Kimberly Clark Fisher Scientific 06-666A
Paper Towel 1 ply C-Fold Quill 901-7CFTB2400
       
Microscopy      
Leica stereomicroscope MZFLIII Empire Imaging Systems, Inc. 10446208
Zeiss Stereomicroscope Stemi 1000 or 2000-C Carl Zeiss 000000-1006-126
Light Source – LED Gooseneck illuminator Fisher Scientific 12563501
Stage Transmitted light box with plate Carl Zeiss 455137000
Zeiss laser scanning confocal microscope LSM 510 Carl Zeiss  
Zeiss compound microscope Axioplan 2 upright Carl Zeiss  
Wasp Strains Fly Strains
Leptopilina victoriae16 y w
Leptopilina boulardi 171 UAS-GFP-Dorsal17
Leptopilina heterotoma2 SerpentHemoGal413
Leptopilina heterotoma 141 MSNF9-moCherry14
Trichopria drosophilae MSNF-GFP15
Ganaspis xanthopoda18 y w Serpent-Gal4 UAS GFP-Dorsal/Basc4
  y w ; Drosomycin-GFP/CyO y+12

References

  1. Schlenke, T. A., Morales, J., Govind, S., Clark, A. G. Contrasting infection strategies in generalist and specialist wasp parasitoids of Drosophila melanogaster. PLoS Pathog. 3, 1486-1501 (2007).
  2. Chiu, H., Morales, J., Govind, S. Identification and immuno-electron microscopy localization of p40, a protein component of immunosuppressive virus-like particles from Leptopilina heterotoma, a virulent parasitoid wasp of Drosophila. J. Gen. Virol. 87, 461-470 (2006).
  3. Gueguen, G., Rajwani, R., Paddibhatla, I., Morales, J., Govind, S. VLPs of Leptopilina boulardi share biogenesis and overall stellate morphology with VLPs of the heterotoma clade. Virus Res. 160, 159-165 (2011).
  4. Paddibhatla, I., Lee, M. J., Kalamarz, M. E., Ferrarese, R., Govind, S. Role for sumoylation in systemic inflammation and immune homeostasis in Drosophila larvae. PLoS Pathog. 6, e1001234 (2010).
  5. Sorrentino, R. P., Carton, Y., Govind, S. Cellular immune response to parasite infection in the Drosophila lymph gland is developmentally regulated. Dev. Biol. , 243-265 (2002).
  6. Sorrentino, R. P., Melk, J. P., Govind, S. Genetic analysis of contributions of dorsal group and JAK-Stat92E pathway genes to larval hemocyte concentration and the egg encapsulation response in Drosophila. Génétique. 166, 1343-1356 (2004).
  7. Jung, S. H., Evans, C. J., Uemura, C., Banerjee, U. The Drosophila lymph gland as a developmental model of hematopoiesis. Development. 132, 2521-2533 (2005).
  8. Krzemien, J., Crozatier, M., Vincent, A. Ontogeny of the Drosophila larval hematopoietic organ, hemocyte homeostasis and the dedicated cellular immune response to parasitism. Int. J. Dev. Biol. 54, 1117-1125 (2010).
  9. Martinez-Agosto, J. A., Mikkola, H. K., Hartenstein, V., Banerjee, U. The hematopoietic stem cell and its niche: a comparative view. Genes Dev. 21, 3044-3060 (2007).
  10. Lemaitre, B., Hoffmann, J. The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 25, 697-743 (2007).
  11. Schlegel, A., Stainier, D. Y. Lessons from “lower” organisms: what worms, flies, and zebrafish can teach us about human energy metabolism. PLoS Genet. 3, e199 (2007).
  12. Ferrandon, D., Jung, A. C., Criqui, M., Lemaitre, B., Uttenweiler-Joseph, S., Michaut, L., Reichhart, J., Hoffmann, J. A. A drosomycin-GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway. EMBO J. 17, 1217-1227 (1998).
  13. Bruckner, K., Kockel, L., Duchek, P., Luque, C. M., Rorth, P., Perrimon, N. The PDGF/VEGF receptor controls blood cell survival in Drosophila. Dev. Cell. 7, 73-84 (2004).
  14. Tokusumi, T., Shoue, D. A., Tokusumi, Y., Stoller, J. R., Schulz, R. A. New hemocyte-specific enhancer-reporter transgenes for the analysis of hematopoiesis in Drosophila. Genesis. 47, 771-774 (2009).
  15. Tokusumi, T., Sorrentino, R. P., Russell, M., Ferrarese, R., Govind, S., Schulz, R. A. Characterization of a lamellocyte transcriptional enhancer located within the misshapen gene of Drosophila melanogaster. PLoS One. 4, e6429 (2009).
  16. Morales, J., Chiu, H., Oo, T., Plaza, R., Hoskins, S., Govind, S. Biogenesis, structure, and immune-suppressive effects of virus-like particles of a Drosophila parasitoid, Leptopilina victoriae. J. Insect Physiol. 51, 181-195 (2005).
  17. Bettencourt, R., Asha, H., Dearolf, C., Ip, Y. T. Hemolymph-dependent and -independent responses in Drosophila immune tissue. J. Cell Biochem. 92, 849-863 (2004).
  18. Melk, J. P., Govind, S. Developmental analysis of Ganaspis xanthopoda, a larval parasitoid of Drosophila melanogaster. J. Exp. Biol. 202, 1885-1896 (1999).

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Citer Cet Article
Small, C., Paddibhatla, I., Rajwani, R., Govind, S. An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response. J. Vis. Exp. (63), e3347, doi:10.3791/3347 (2012).

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