के परजीवी wasps के लिए एक परिचय<em> ड्रोसोफिला</em> और Antiparasite इम्यून रिस्पांस
Summary
Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.
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
Discussion
Interest in parasitic wasps of Drosophila is surging as molecular techniques to decode whole genomes become efficient and cost-effective. However, relative to their exceptionally well-studied hosts, many fascinating aspects of wasp biology remain obscure. These include issues related to host range, immune suppression, superparasitism, and behavior. The focus of this presentation was to demonstrate the effects of infection on the fly’s immune tissues. The dissection techniques demonstrated here can be used for th…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We are grateful to Prof. Todd Schlenke for Trichopria drosophilae, Prof. Tony Ip for transgenic fly strains, and Prof. Carl Hashimoto for anti-Spätzle antibodies. We thank present and past members of the lab for their contributions to this presentation. This work was supported by the following grants: from NIH (S06 GM08168, RISE 41399-009, and G12-RR03060), USDA (NRI/USDA CSREES 2006-03817 and 2009-35302-05277) and PSC-CUNY.
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 |
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