1Biology Department, The City College of New York, CUNY, 2The Graduate Center, The City University of New York
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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).
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).
The entire protocol for the experiment is divided into four steps (Fig. 9). (1) Culturing wasps on fly larvae; (2) Setting up infections and preparing animals for dissection; (3) Isolating and fixing host/parasite structures; (4) Analyzing immune tissues.
1. Culturing Wasps on Drosophila Larvae
Maintenance of wasp cultures requires careful planning. Relative to growing flies, it is fairly labor intensive. We maintain our wasp colonies in the laboratory on larvae or pupae of the y w strain of Drosophila at 24 °C. Culturing wasps involves keeping a continuous source of hosts at the right stage and clearing the "infected" vials of flies that emerge before the wasps do. Wasps follow the haplodiploid method of sex determination and it is therefore important that the females are mated before they infect the hosts.
2. Setting up Infections and Preparing Infected Animals for Dissection
Before you start, have a couple of clean glass or plastic Petri dishes, a Pyrex dissecting dish with 9 depressions, Kimwipes, distilled water (in a squirt bottle), 70% ethanol (in a squirt bottle), 1X PBS (in a squirt bottle), microscope slides, and a small, clean spatula handy.
3. Isolating and Fixing Host/parasite Structures
The larval lymph gland is a small hematopoietic organ7. At the third larval instar, the lymph gland contains a large pair of anterior lobes that flank the dorsal vessel (Fig. 3A, 6B-C, 7A-D). The anterior lobes are further divided into specialized regions with unique cell properties7. Progenitors of three cell types, plasmatocytes, lamellocytes, and crystal cells reside in the anterior lobes. Pericardial cells separate the anterior lobes from the smaller posterior lobes. The Drosophila lymph gland is a model for insect and mammalian hematopoiesis8,9.
Fat body is functionally similar to the mammalian liver. The humoral response is triggered in the fat body following microbial or wasp infection1,4,10. As a result, a unique combination of antimicrobial peptide genes are activated and the peptides are secreted into the hemolymph1. The fat body is also the primary tissue for glycogen and triglyceride production and storage11. The larval fat body occupies substantial volume of the hemocoel and, so unlike the lymph gland, it is easy to locate. We will now demonstrate how to dissect the lymph gland and the fat body from third instar larvae.
Before you start
Need fine forceps (tweezers) and ethanol-cleaned microscope slides.
Larval lymph gland dissection
Note: A properly dissected lymph gland will have one pair of anterior lobes and two sets of posterior lobes along the dorsal vessel (Fig. 6B). The lobes can be damaged easily or can fall off from the dorsal vessel. The samples should therefore be handled with great care. The gland also has a tendency to shrivel or contract. Gently straightening out the organ at its posterior end allows for all parts and cells to be presented well. This is especially necessary for the immunostaining protocols.
Fay body dissection
Note 1: The fat body has only one cell layer and it is therefore important to have all cells flattened in the same plane on the glass slide. Cells are endopolyploid and easy to visualize. A well-dissected fat body sample should maintain normal cell contacts, and should have minimal fat globules around the dissected sample (Fig. 6I, J).
Note 2: If wasp eggs remain unaffected by the host immune system, they will initiate development almost immediately. Early developmental stages of the parasite (from the host larva) are easily accessible from the dissected hemocoel (Fig. 8). Parasite eggs or larvae either adhere to the fat body or other organs, or simply slip onto the glass slide during the dissection.
4. Analyzing Immune Tissues
5. Representative Results
Figure 1. Different wasp species and oviposition of wasp egg. All images were obtained using a Leica stereomicroscope. A Trichopria drosophilae female wasp oviposits egg into a D. melanogaster pupa. B Magnification of oviposition of Trichopria drosophilae into host pupa. C Melanized spots indicate wound healing at the site of oviposition. D L. boulardi male. E G. xanthopoda female. F L. victoriae female. G L. heterotoma male (left) and female.
Figure 2. Life cycles showing the fly/wasp arms race. Oviposition results in one of two outcomes. Either host immune reactions succeed to block the development of the wasp (left), or the wasp thwarts the host's immune responses and succeeds (right). Modified from Melk and Govind, 199918.
Figure 3. Third instar larvae showing the organs of interest prior to dissection. All images were obtained using a Leica stereomicroscope. A Hml>GFP larva with GFP (green) fluorescence (arrow) in individual cells of the lymph gland indicates the general location of the lymph gland in an intact animal. The larva was imaged with fluorescence and bright field optics. Arrowheads point to locations important for the lymph gland dissection protocol described in the text. B Cg>GFP larva with GFP expression in the fat body to indicate the location of the organ in an intact animal. Arrowheads point to locations important for the fat body dissection protocol described in the text.
Figure 4. Immuno-genetic circuit of host immune response against parasitoid attack. Core components of the Toll pathway are shown. Spätzle (Spz) is activated by the protease Spätzle processing enzyme, SPE. Activated Spz serves as the ligand for Toll. Intracellular signaling leads to the activation of NF-κB transcription factors, Dorsal and Dif. Cactus serves as the IκB inhibitor of the pathway. Activation of Drosomycin, a canonical Toll pathway target gene, can be monitored using transgenic fly strains with the GFP reporter (see Fig. 6I, J).
Figure 5. Encapsulation in response to L. victoriae infestation in Serpent>GFP-dl Drosophila hosts. An enhancer in the Serpent gene is expressed in blood cells13. When this enhancer drives the expression of the GFP-Dorsal fusion protein in a transgene, Dorsal is detected via the green fluorescence of GFP in some plasmatocytes and lamellocytes that make up the capsules and aggregates. All images were obtained using a Zeiss LSM confocal microscope. A Egg of L. victoriae. B-D Lamellocytes (white arrows) at the posterior end of the egg express GFP-Dorsal. C-D Higher magnifications of the sample in panel B. E-H Samples are counterstained with rhodamine-labeled phalloidin to visualize filamentous actin (F-actin, red) and with Hoechst 33258 to visualize DNA (blue). E Encapsulated egg of L. victoriae. F Melanized capsule of L. victoriae. G-H L. victoriae infection induces blood cell (plasmatocyte and lamellocyte) aggregation.
Figure 6. Immune responses against wasp infection. Images in panels A and B were obtained using a Zeiss Axio Scope. Images in panels C-J were obtained using a Zeiss LSM confocal microscope. A Host larva showing melanized encapsulated wasp egg (arrowhead) through the transparent cuticle. B Representative third instar larval lymph gland stained with Hoechst 33258 reveals cells of all lobes and interspersed pericardial cells. Scale bar represents 100 μ. C-J All samples were counterstained with Hoechst 33258 (to label nuclei) and rhodamine phalloidin (to label F-actin). C-D Anterior lymph gland lobes from control (C) and L. victoriae-infected (D) MSNF9-GFP animals. In infected animals, MSNF enhancer expression, specific to lamellocytes14, is detected with a nuclear GFP reporter. E Plasmatocytes isolated from uninfected animals do not express GFP. These animals have very few if any lamellocytes. F Plasmatocytes (GFP-negative, short arrow) and newly-differentiated, larger lamellocytes (long arrows) with weak nuclear GFP expression from L. victoriae-infected MSNF9-GFP larva. G Free and aggregated plasmatocytes and lamellocytes from the hemocoel of a L. victoriae-infected Drs-GFP larva. Expression of the Drs-GFP reporter is activated in some plasmatocytes (short arrow), but not in lamellocytes (long arrows) post-infection. H Encapsulated and melanized wasp larva from L. boulardi-infected MSNF9-GFP host larva. Numerous MSNF9-GFP-positive lamelloctyes surround the wasp larva (dark structure; thick arrow) and exhibit strong nuclear GFP expression (long arrows). This panel previously published in PLoS One15. I-J Fat body cells dissected from uninfected (I) and L. victoriae-infected (J) Drs-GFP larva. GFP is nuclear and cytoplasmic in fat body cells of infected animals. Click here to view larger figure.
Figure 7. Spätzle expression after parasitoid wasp infection. A-J Samples were counterstained with Hoechst 33258 to visualize DNA. All images were obtained using a Zeiss LSM confocal microscope. A-F Anterior lobes dissected from uninfected y w larvae. Samples were processed for indirect immunostaining without primary antibody (A and B) or with anti-Spz antibody (red; C, D, E, F) and counterstained with Alexa Flour-phalloidin to label F-actin in cells (green; B, D, F). G-J Anterior lobes dissected from infected y w larvae and stained with anti-Spz antibody (red; G, H, I, J) and Alexa Fluor-phalloidin (green; panels H, J). E-F Higher magnifications of samples in C and D, respectively. I-J Higher magnifications of the samples in G and H, respectively. Scale bars in panels A-D, G, H represent 50 μm.
Figure 8. Larval and pupal stages of L. heterotoma and L. boulardi. Individual stages were isolated from larval or pupal fly hosts post infestation, as indicated. All images were obtained using a Zeiss Axio Scope. Top row A-F L. heterotoma stages, 1-6 days after infection. Bottom row A-F Postembryonic L. boulardi stages, 4 to 12 days after infection.
Figure 9. Flow chart of the experimental protocol.
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 the analysis of gene expression at the RNA level (in situ hybridization), or for extraction of nucleic acid for microarrays or PCR, or for Western analyses of proteins. A vast variety of fly strains are available from the stock centers and individual research labs to manipulate and label immune cells. The choice of fly strains is dictated by the experimental questions. These dissection techniques can also be used for the analysis of immune tissues of other Drosophila species.
No conflicts of interest declared.
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.
|Yeast||Fisher Scientific||S80245-3||Active Dry|
|Fly Food||Home made||Corn meal, sugar||Standard|
|Honey||Dutch Gold||From the store||Clover|
|Cotton plug/Foam stopper||Fisher Scientific||14-127-40A|
|Pyrex dissecting dish||Fisher Scientific||50-930-382||9-well|
|Microscope slides pre-cleaned||Fisher Scientific||7101||25.4 mm x 76.2 mm|
|Glass coverslips||Pearl||D12544||22 x 50|
|Glass coverslips||Pearl||G12544||22 x 60|
|Pasteur Pipette||J & H Berge||71-5200-05|
|100 mm Tweezers||Sigma-Aldrich||T-4662||Style # 5|
|Wash Bottle||Fisher Scientific||03-409-22A||Fisherbrand|
|Kim Wipess||Fisher Scientific||34155||Kimberly Clark|
|Paper Towel||Fisher Scientific||Fisher X-101-C||1/8 x 13 1/8 in.|
|Leica stereomicroscope||Empire Imaging Systems, INC.||10446208||MZFLIII|
|Zeiss Stereomicroscope||Carl Zeiss, Inc.||000000-1006-126||"Stemi" 1000 or 2000-C|
|Light Source -LED Gooseneck illuminator||Fisher Scientific||12563501|
|Stage||Carl Zeiss, Inc.|
|Zeiss Laser 510 Scanning Confocal Microscope||Carl Zeiss, Inc.|
|Incubator||Thermo Fisher Scientific, Inc.||815|
|Dulbecco’s Phosphate-Buffered Salt Solution 1X||Fisher Scientific||MT-21-030-CM Mediatech|
|Ethanol||Fisher Scientific||64-17-5||99.5 %|
|Formaldehyde (37% w/w)||Fisher Scientific||F79-1|
|Hoechst 33258||Molecular Probes, Life Technologies||H-1398||0.2 μg/ml Excitation 352 nm; Emission 461 nm|
|Rhodamine phalloidin||Molecular Probes, Life Technologies||R415||200 units/ml (6.6 μM) Excitation 546 nm; Emission 565 nm|
|Alexa Fluor 488 phalloidin||Molecular Probes, Life Technologies||A22289||Excitation 495 nm; Emission 518 nm 300 units/ml|
|CO2 tank||TW Smith|
|Anti-Spz antibody||Gift Dr. C. Hashimoto (Yale)|
|Secondary antibody TRITC-conjugated donkey anti-rabbit, concentration 1:50.||Jackson ImmunoResearch||711-165-152||Detected at Excitation 546 nm; Emission 565 nm|
|Antifade||MP Biomedicals||ICN10274790||0.08 mg of N-propyl gallate in 20 ml of 50% glycerol in 1X PBS|
|Wasp Strains||Fly Strains|
|L. victoriae16||y w|
|L. boulardi 171||UAS-GFP-Dorsal17|
|L. heterotoma 141||MSNF9-moCherry14|
|G. xanthopoda18||y w Serpent-Gal4 UAS GFP-Dorsal/Basc4|
|y w ; Drosomycin-GFP/CyO y+12|