Summary

Введение в паразитических наездников<em> Drosophila</em> И противопаразитарной иммунного ответа

Published: May 07, 2012
doi:

Summary

Паразитоид (паразитарных) ос представляют собой основной класс естественных врагов многих насекомых, включая<em> DROSOPHILA MELANOGASTER</em>. Введем методы для распространения этих паразитов в<em> Drosophila</em> SPP. и продемонстрировать, как анализировать их влияние на иммунную ткани<em> Drosophila</em> Личинок.

Abstract

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 Spätzle (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).

Protocol

Весь протокол эксперимента разделили на четыре этапа (рис. 9). (1) Культивирование осы на личинки мух (2) Установка инфекции и подготовка животных для вскрытия (3) изоляции и крепления хост / паразита структур; (4) Анализ иммунного тканей. 1. Культивирование Осы на личин…

Discussion

Интерес к паразитической осы дрозофилы приливает в качестве молекулярных методов для расшифровки полного генома стать эффективным и рентабельным. Тем не менее, по сравнению с их исключительно хорошо изученных хозяев, много интересных аспектов осы биология остаются неясными. К н?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Мы благодарим профессора Тодда Schlenke для Trichopria drosophilae, профессор Тони Ip для трансгенных линий мух, и профессор Карл Хасимото по борьбе с Spätzle антител. Мы выражаем благодарность настоящим и прошлым членам лаборатории за их вклад в эту презентацию. Эта работа была поддержана грантами следующее: из NIH (S06 GM08168, RISE 41399-009 и G12-RR03060), USDA (NRI / USDA CSREES 2006-03817 и 2009-35302-05277) и ОАО Нью-Йоркского университета.

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. Genetics. 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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Cite This 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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