We are grateful to Prof. Todd Schlenke for Trichopria drosophilae, Prof. Tony Ip for transgenic fly strains, and Prof. Carl Hashimoto for anti-Sp&auml;tzle 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.

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 ...

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

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...

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

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.

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. ...

an-introduction-to-parasitic-wasps-drosophila-antiparasite-immune

Research

JoVE Journal

Immunology and Infection

18.3K vistas.  The City College of New York, CUNY. Parasitoide (parásitos) avispas constituyen una clase importante de enemigos naturales de muchos insectos, incluyendo Drosophila melanogaster. Vamos a introducir las técnicas para propagar estos parásitos en Drosophila Spp. y demostrar la forma de analizar sus efectos sobre los tejidos inmunes de Drosophila Larvas.