Name: An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response (Video) | JoVE Journal
Uploaded: 2012-05-07T12:00:00.000+00:00
Duration: 13 min 4 s
Description: Learn how to maintain parasitoid wasp cultures on Drosophila hosts and dissect immune tissues including lymph glands and fat bodies. This method enables visualization of gene expression changes using immunocytochemistry and fluorescence microscopy.

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5 minutes

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.

<ol><li>Schlenke, T. A., Morales, J., Govind, S., Clark, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contrasting+infection+strategies+in+generalist+and+specialist+wasp+parasitoids+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">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/pubmed?term=((Identification+and+immuno-electron+microscopy+localization+of+p40%2C+a+protein+component+of+immunosuppressive+virus-like+particles+from+Leptopilina+heterotoma%2C+a+virulent+parasitoid+wasp+of+Drosophila%5BTitle%5D)+AND+%22J.+Gen.+Virol%22%5BJournal%5D)">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/pubmed?term=((VLPs+of+Leptopilina+boulardi+share+biogenesis+and+overall+stellate+morphology+with+VLPs+of+the+heterotoma+clade%5BTitle%5D)+AND+%22Virus+Res%22%5BJournal%5D)">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/pubmed?term=((Role+for+sumoylation+in+systemic+inflammation+and+immune+homeostasis+in+Drosophila+larvae%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">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/pubmed?term=((Cellular+immune+response+to+parasite+infection+in+the+Drosophila+lymph+gland+is+developmentally+regulated%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">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/pubmed?term=((Genetic+analysis+of+contributions+of+dorsal+group+and+JAK-Stat92E+pathway+genes+to+larval+hemocyte+concentration+and+the+egg+encapsulation+response+in+Drosophila%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">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/pubmed?term=((The+Drosophila+lymph+gland+as+a+developmental+model+of+hematopoiesis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Ontogeny+of+the+Drosophila+larval+hematopoietic+organ%2C+hemocyte+homeostasis+and+the+dedicated+cellular+immune+response+to+parasitism%5BTitle%5D)+AND+%22Int.+J.+Dev.+Biol%22%5BJournal%5D)">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/pubmed?term=((The+hematopoietic+stem+cell+and+its+niche%3A+a+comparative+view%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">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/pubmed?term=((The+host+defense+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Annu.+Rev.+Immunol%22%5BJournal%5D)">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/pubmed?term=((Lessons+from+%22lower%22+organisms%3A+what+worms%2C+flies%2C+and+zebrafish+can+teach+us+about+human+energy+metabolism%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">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/pubmed?term=((A+drosomycin-GFP+reporter+transgene+reveals+a+local+immune+response+in+Drosophila+that+is+not+dependent+on+the+Toll+pathway%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">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/pubmed?term=((The+PDGF%2FVEGF+receptor+controls+blood+cell+survival+in+Drosophila%5BTitle%5D)+AND+%22Dev.+Cell%22%5BJournal%5D)">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/pubmed?term=((New+hemocyte-specific+enhancer-reporter+transgenes+for+the+analysis+of+hematopoiesis+in+Drosophila%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+a+lamellocyte+transcriptional+enhancer+located+within+the+misshapen+gene+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Biogenesis%2C+structure%2C+and+immune-suppressive+effects+of+virus-like+particles+of+a+Drosophila+parasitoid%2C+Leptopilina+victoriae%5BTitle%5D)+AND+%22J.+Insect+Physiol%22%5BJournal%5D)">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/pubmed?term=((Hemolymph-dependent+and+-independent+responses+in+Drosophila+immune+tissue%5BTitle%5D)+AND+%22J.+Cell+Biochem%22%5BJournal%5D)">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/pubmed?term=((Developmental+analysis+of+Ganaspis+xanthopoda%2C+a+larval+parasitoid+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22J.+Exp.+Biol%22%5BJournal%5D)">Developmental analysis of Ganaspis xanthopoda, a larval parasitoid of Drosophila melanogaster.</a> J. Exp. Biol. 202, 1885-1896 (1999).</li>
</ol>

No conflicts of interest declared.

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><tbody><tr><td>Yeast</td><td>Fisher Scientific</td><td>S80245-3</td><td>Active Dry</td></tr><tr><td>Fly Food </td><td>Home made</td><td>Corn meal, sugar</td><td>Standard</td></tr><tr><td>Honey</td><td>Dutch Gold</td><td>From the store</td><td>Clover</td></tr><tr><td>Vials</td><td>Fisher Scientific</td><td>AS514</td><td></td></tr><tr><td>Cotton plug/Foam stopper</td><td>Fisher Scientific</td><td>14-127-40A</td><td></td></tr><tr><td>Spatula</td><td>Fisher Scientific</td><td>21-401-15</td><td></td></tr><tr><td>Pyrex dissecting dish</td><td>Fisher Scientific</td><td>50-930-382</td><td>9-well</td></tr><tr><td>Microscope slides pre-cleaned </td><td>Fisher Scientific</td><td>7101</td><td>25.4 mm x 76.2 mm</td></tr><tr><td>Glass coverslips</td><td>Pearl</td><td>D12544</td><td>22 x 50</td></tr><tr><td>Glass coverslips</td><td>Pearl</td><td>G12544</td><td>22 x 60</td></tr><tr><td>Pasteur Pipette</td><td>J H Berge</td><td>71-5200-05</td><td></td></tr><tr><td>100 mm Tweezers</td><td>Sigma-Aldrich</td><td>T-4662</td><td>Style # 5</td></tr><tr><td>Wash Bottle</td><td>Fisher Scientific</td><td>03-409-22A</td><td>Fisherbrand</td></tr><tr><td>Kim Wipess</td><td>Fisher Scientific</td><td>34155</td><td>Kimberly Clark</td></tr><tr><td>Paper Towel</td><td>Fisher Scientific</td><td>Fisher X-101-C</td><td>1/8 x 13 1/8 in.</td></tr><tr><td>Leica stereomicroscope </td><td>Empire Imaging Systems, INC.</td><td>10446208</td><td>MZFLIII</td></tr><tr><td>Zeiss Stereomicroscope </td><td>Carl Zeiss, Inc.</td><td>000000-1006-126</td><td>&quot;Stemi&quot; 1000 or 2000-C</td></tr><tr><td>Light Source -LED Gooseneck illuminator</td><td>Fisher Scientific</td><td>12563501</td><td></td></tr><tr><td>Stage</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>Zeiss Laser 510 Scanning Confocal Microscope</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>Incubator</td><td>Thermo Fisher Scientific, Inc.</td><td>815</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Salt Solution 1X </td><td>Fisher Scientific</td><td>MT-21-030-CM Mediatech</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>64-17-5</td><td>99.5 %</td></tr><tr><td>Formaldehyde (37% w/w)</td><td>Fisher Scientific</td><td>F79-1</td><td></td></tr><tr><td>H&amp;#8211;chst 33258</td><td>Molecular Probes, Life Technologies</td><td>H-1398</td><td>0.2 &amp;#x03BC;g/ml Excitation 352 nm; Emission 461 nm</td></tr><tr><td>Rhodamine phalloidin</td><td>Molecular Probes, Life Technologies</td><td>R415</td><td>200 units/ml (6.6 &amp;#x03BC;M) Excitation 546 nm; Emission 565 nm</td></tr><tr><td>Alexa Fluor 488 phalloidin </td><td>Molecular Probes, Life Technologies</td><td>A22289</td><td>Excitation 495 nm; Emission 518 nm 300 units/ml</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; tank</td><td>TW Smith</td><td></td><td></td></tr><tr><td>Anti-Spz antibody</td><td>Gift Dr. C. Hashimoto (Yale)</td><td></td><td></td></tr><tr><td>Secondary antibody TRITC-conjugated donkey anti-rabbit, concentration 1:50. </td><td>Jackson ImmunoResearch</td><td>711-165-152</td><td>Detected at Excitation 546 nm; Emission 565 nm</td></tr><tr><td>Antifade </td><td>MP Biomedicals</td><td>ICN10274790</td><td>0.08 mg of N-propyl gallate in 20 ml of 50% glycerol in 1X PBS</td></tr><tr><td>&lt;strong&gt;Wasp Strains&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Fly Strains&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;L. victoriae&lt;/em&gt;&lt;sup&gt;16&lt;/sup&gt;</td><td></td><td></td><td>&lt;em&gt;y w&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;L. boulardi 17&lt;/em&gt;&lt;sup&gt;1&lt;/sup&gt;</td><td></td><td></td><td>&lt;em&gt;UAS-GFP-Dorsal&lt;/em&gt;&lt;sup&gt;17&lt;/sup&gt;</td></tr><tr><td>&lt;em&gt;L. heterotoma&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt;</td><td></td><td></td><td>&lt;em&gt;SerpentHemoGal4&lt;/em&gt;&lt;sup&gt;13&lt;/sup&gt;</td></tr><tr><td>&lt;em&gt;L. heterotoma 14&lt;/em&gt;&lt;sup&gt;1&lt;/sup&gt;</td><td></td><td></td><td>&lt;em&gt;MSNF9-moCherry&lt;/em&gt;&lt;sup&gt;14&lt;/sup&gt;</td></tr><tr><td>&lt;em&gt;Trichopria drosophilae &lt;/em&gt;</td><td></td><td></td><td>&lt;em&gt;MSNF-GFP&lt;/em&gt;&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>&lt;em&gt;G. xanthopoda&lt;/em&gt;&lt;sup&gt;18&lt;/sup&gt;</td><td></td><td></td><td>&lt;em&gt;y w Serpent-Gal4 UAS GFP-Dorsal/Basc&lt;/em&gt;&lt;sup&gt;4&lt;/sup&gt;</td></tr><tr><td colspan="4">&lt;em&gt;y w ; Drosomycin-GFP/CyO y+&lt;/em&gt;&lt;sup&gt;12&lt;/sup&gt;</td></tr></tbody></table>

an introduction to parasitic wasps of drosophila and the antiparasite immune response

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).

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.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

The overall goals of this procedure are to show how to maintain cultures of parasitoid wasps on drosophila hosts and how to dissect lymph gland and fat body. This is accomplished by first culturing wasps on drosophila larvae. Animals are then dissected to isolate either the larva lymph glands, which involves peeling off the cuticle and removing the fat bodies and other finer structures to isolate the lymph glands.Likewise, the fat bodies themselves can be isolated using a similar procedure using immuno cyto chemistry and fluorescence microscopy. Gene expression and cell specific changes are visualized in the immune tissues. After wasp infestation, We study a unique system of infection and immunity that utilizes drosophila hose and its parasitic wasp.Our lab is one of few where life cultures of drosophila parasitoids are maintained. Thus, we are often asked to share our techniques. Parasitoids are natural enemies of many insects.The species we study attack the larval stages of many drosophila species. In the lab, we use drosophila melan gastro to understand how the immune system of insects are eff affected by parasitoid attacked. Secondly, we want to discover the strategies that are used by wass to overcome host defense.Generally, individuals who are new to dissections for fat body and lymph gland struggle because while oph larvae are easy to grow, they are fairly small in size, and it takes considerable familiarity to routinely dissect these laval organs with ease. After discovering that constitutive activation of NF kappa B signaling leads to an autoimmune like encapsulation of cell tissues, we became curious about how this reaction would occur in oph fla mutants. So we hypothesized that was induced encapsulation is its natural counterpart and develop these standard protocols that we will demonstrate Today, I will now demonstrate how to prepare and maintain wasp cultures.Begin by preparing a fresh fly food vial with a small dab of freshly prepared yeast paste. Transfer 50 to 60 young wild type flies to the vial to lay eggs for 48 hours at 24 degrees Celsius. After two days of egg laying, remove the flies from the vial and add a drop of honey to the buzz plug.And finally, recap the vial. Lower the flow of carbon dioxide prior to anesthetizing wasps because they're more sensitive to the gas than flies. Once ready to anesthetize wasps sort out six to eight female and six to eight male wasps and add them to the vial.With the drosophila eggs and larvae. Gently place the vial on its side until the wasps wake up. Then place the vial in a 24 degrees Celsius incubator.Unaffected gilo host and hosts that successfully defend themselves will pup paint at the normal development time point. They can be removed prior to the wasps e explosion. Wasps usually AC close around 25 to 30 days to prepare wasps at the same stage of development.Rear the wasps with host eggs laid over a two to six hour period instead of a 48 hour period to harvest infected larvae for dissections of immune tissues or parasites. Transfer the contents of the fly vials to a small glass or plastic Petri dish. Then using a stereo microscope and find forceps carefully pick six to 10 larvae.Place the larvae into a well with one XPBS first, then transfer them into water, 70%ethanol water and one XPBS successively. The purpose of these steps is to free the animals of the fly food and thoroughly clean and sterilize their surface. On the dissecting stage of a Zes 1000 dissecting microscope, place a light box that allows incident light to be transmitted through the sample.Using fine forceps, select a cleaned wandering third instar larvae from the PBS. Place the larvae onto a microscope slide. Using forceps position the animal with the ventral side up.Tiled the light source away from the sample so that the organism appears transparent and the internal organs are visible. As you transfer the larvae onto the slide, you want to make sure that there is no excess PBS as this can sometimes make the section more difficult. Using two pairs of forceps, hold the animal at either side of the posterior end and gently pull the cuticle to introduce a small superficial tear in the cuticle.The hemo lymph containing he cytes gut and fat bodies will begin to escape from the body cavity. The hemolymph can be taken up with a 10 microliter pipette man to make smears if needed, placing both forceps to the left side of the animal just beneath the mouth hooks carefully. Make a gentle tear in the cuticle.Move both forceps on the right side of the animal just beneath the mouth hooks and carefully make another small tear. Now while holding the mouth hooks of the animal, gently peel back the cuticle towards the posterior end of the animal. As the cuticle is pulled back, the brain fat body imaginal discs, salivary glands and proven ventricular will become exposed.The detached cuticle at the posterior end has the dorsal vessel with lymph gland attached to it. The lymph gland is underneath the brain. Cut the proven ventricular and move it away from the lymph gland area.Carefully tease away fat, body, gut, salivary glands, and other structures from the lymph gland. Be careful not to detach the limp gland from the ring gland and dorsal vessel located posterior to the brain. Position the lymph gland flat against the microscope slide and carefully remove all additional tissues until the entire gland unfolds from the anterior lobes to the final set of pericardial cells.The gland is now ready to be fixed and stained on the dissecting stage of a Zes 1000 dissecting microscope. Place a light box that allows incident light to be transmitted through the sample. Position a cleaned third instar larva on a microscope.Slide in 200 microliters of one XPBS. Tilt the light source away from the sample so that the organism appears transparent and the internal organs are visible. Using forceps, position the animal so that the anterior end is away from you.In the next step, it is important to note where you position your forceps to ensure that you tear only the cuticle without touching the internal organs. With one pair of forceps, hold the cuticle of the larvae on left side of the mouth, hooks with the other forceps. Gently tear the cuticle on other side down to the posterior end.Stop before tearing the cuticle away from the body at the posterior end, very gently start removing the gut to one side. Gently remove the brain imaginal discs and the ring gland with the lymph gland running through the dorsal vessel. Finally, gently remove the cuticle on either side of the fat body.Do not remove the salivary glands since there are fat bodies on either side of these glands. Before proceeding with fixing and staining the tissue, be sure to remove the PBS. A well dissected fat body sample should maintain normal cell contacts and should have minimal fat globules around the dissected sample.The effect of wasp infestation on gene expression is visualized in vivo. Using a GFP reporter linked to Theso Mycin promoter in uninfected control animals, GFP expression was not detected. Whereas the GFP signal is clearly detected in the cytoplasm of fat bodies dissected from animals infected with l Victoria.Lymph glands were dissected to visualize the cellular changes induced in the anterior lobes after El Victoria infection. Newly differentiated cytes marked with GFP driven by the misshapen enhancer are seen at the periphery of the lobes. Normally, the glands surround the lobe continuously.However, the integrity of the anterior lobes was disrupted in these glands as the basement membrane was discontinuous after wasp infection from these same dissections. Cytes in the hemolymph were visualized in smears. Some lymphocytes were engaged in the capsule form to block parasite development to detect betzel expression.The dissected lymph glands. The lymph glands were stained with polyclonal anti schpeil antibodies in vivo. Schpeil levels increased in lymph gland cells after wasp infection as compared to uninfected controls.While attempting this procedure, it is important to prepare esophagal cultures at the desired biological age. Treat them gently so that manual handling does not induce immune reactions. Following this procedure, we can use the dissected organs, the fat body, and lymph gland to extract RNA and protein using the extracted RNA or protein.We can perform PCR or Westin analysis to study the gene and protein expression. After watching this video, you should have a good understanding of how to grow. Parasitic was on drosophila hosts and perform dissections of immune tissues for immunohistochemistry and other applications.

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Research

JoVE Journal

Immunology and Infection

Wolbachia Bacterial Infection in Drosophila

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NFκB.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

Drosophila melanogaster Larva Injection Protocol

Ganaspis brasiliensis-a larval parasitoid of Drosophila suzukii (a global invasive fruit crop pest)-has been approved or is considered for introduction into Europe and the United States for biological control of this pest. This article provides protocols for both small-scale and large-scale rearing of this parasitoid.

Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii

The goal of this protocol is to facilitate functional analysis of parasitoid-host interactions in Asobara japonica and Drosophila melanogaster through optimized rearing methods, a single-oviposition infection assay, and RNAi-mediated gene knockdown.

Protocols for Efficient Rearing and Functional Analysis of the Endoparasitoid Wasp Asobara japonica and its Host Drosophila melanogaster

Learn how to maintain parasitoid wasp cultures on Drosophila hosts and dissect immune tissues including lymph glands and fat bodies. This method enables visualization of gene expression changes using immunocytochemistry and fluorescence microscopy.

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

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