Siamo grati al professor Todd Schlenke per Trichopria drosophilae, Prof. Tony Ip per i ceppi transgenici mosca, e il Prof. Carl Hashimoto per gli anticorpi anti-Spätzle. Ringraziamo i membri presenti e passati del laboratorio per il loro contributo a questa presentazione. Questo lavoro è stato supportato da i seguenti aiuti: dal NIH (S06 GM08168, RISE 41399-009 e G12-RR03060), USDA (NRI / USDA CSREES 2006-03817 e 2009-35302-05277) e PSC-CUNY.

<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>

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L&#39;interesse per le vespe parassite di Drosophila è in aumento come tecniche molecolari per decodificare genomi interi diventare efficiente e conveniente. Tuttavia, rispetto ai loro eccezionalmente ben studiati padroni di casa, molti aspetti affascinanti della biologia vespa restano oscuri. Questi includono le questioni relative ad ospitare gamma, soppressione immunitaria, superparasitism, e il comportamento. L&#39;obiettivo di questa presentazione è stato quello di dimostrare gli effetti di contagio sui t...

<table border="1"><tbody><tr><td> Materiale </td><td> Tipo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Materiali per la manutenzione della cultura insetti </td><td></td><td></td><td></td></tr><tr><td> Lievito </td><td> Secco attivo </td><td> Fisher Scientific </td><td> S802453 </td></tr><tr><td> Vola alimentare </td><td> Farina di mais, zucchero </td><td></td><td> Ricetta standard </td></tr><tr><td> Miele </td><td> Trifoglio </td><td> Olandese Oro </td><td></td></tr><tr><td> Fiale </td><td> Polipropilene shell vial (stretto) </td><td> Fisher Scientific </td><td> AS514 </td></tr><tr><td> Vial chiusura </td><td> Cotton spina </td><td> Fisher Scientific </td><td> AS212 </td></tr><tr><td> Vial chiusura </td><td> Buzz spina </td><td&gt; Genesee Scientific </td><td> AS273 </td></tr><tr><td> Incubatore refrigerato </td><td> Precision 815 </td><td> Thermo Scientific </td><td> 3721 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Materiali per la preparazione del campione </td><td></td><td></td><td></td></tr><tr><td> CO 2 serbatoio </td><td> Bone grade a secco </td><td> TW Smith </td><td> UN1013 </td></tr><tr><td> Spatola </td><td> Spatola Micro (14 cm) </td><td> Fisher Scientific </td><td> 21-401-15 </td></tr><tr><td> Pyrex piastre di prova in loco </td><td> 9-ben dissezione piastra 85 mm x 100 mm </td><td> Thomas Scientific </td><td> 7812G17 </td></tr><tr><td> Pipette Pasteur </td><td> Soda lime </td><td> J &amp; H Berge </td><td> 71-5200-05 </td></tr><tr><td> Forcipe </td><td> Style # 5 </td><td> Sigma </td><td> T-4662 </td></tr><tr><td> Etanolo </td><td> 190 prova USP </td><td> Fisher Scientific </td><td> 04-355-221 </td></tr><tr><td> Formaldeide </td><td> 37% w / w </td><td> Fisher Scientific </td><td> F79-1 </td></tr><tr><td> Anticorpo secondario Cy3 AffiniPure asino anti-IgG di coniglio (H + L) </td><td> 01:50 eccitazione 546 nm, emissione 565 nm </td><td> Jackson Immuno Research Laboratories, Inc. </td><td> 711-165-152 </td></tr><tr><td> Antifade (N-propil gallato) </td><td> 4 microgrammi / ml in 50% glicerolo in 1X PBS </td><td> MP Biomedicals </td><td> 10274790 </td></tr><tr><td> Glicerina </td><td></td><td> Fisher Scientific </td><td> G33-1 </td></tr><tr><td> Hoechst 33258 </td><td> 0,2 ug / ml di eccitazione 352 nm, emissione 461 nm </td><td> Molecular Probes </td><td> H-1398 </td></tr><tr><td> Rhodamine phalloidin </td><td> 200 unità / ml (6,6pM) di eccitazione 540 nm, emissione 565 nm </td><td> Molecular Probes </td><td> R415 </td></tr><tr><td> Alexa Fluor 488 phalloidin </td><td> 300 unità / ml di eccitazione 495 nm, emissione 518 nm </td><td> Molecular Probes </td><td> A12379 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Disposables </td><td></td><td></td><td></td></tr><tr><td> Bottiglia di lavaggio </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> Tovagliolo di carta </td><td> 1 strato C-Fold </td><td> Penna </td><td> 901-7CFTB2400 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Microscopia </td><td> &amp;nbsp; </td><td></td><td></td></tr><tr><td> Stereomicroscopio Leica </td><td> MZFLIII </td><td> Empire Imaging Systems, Inc. </td><td> 10446208 </td></tr><tr><td> Stereomicroscopio Zeiss </td><td> Stemi 1000 o 2000-C </td><td> Carl Zeiss </td><td> 000000-1006-126 </td></tr><tr><td> Sorgente luminosa - LED </td><td> Gooseneck illuminatore </td><td> Fisher Scientific </td><td> 12563501 </td></tr><tr><td> Fase </td><td> Trasmesso light box con piastra </td><td> Carl Zeiss </td><td> 455137000 </td></tr><tr><td> Zeiss microscopio confocale a scansione laser </td><td> LSM 510 </td><td> Carl Zeiss </td><td></td></tr><tr><td> Zeiss microscopio composto </td><td> Axioplan 2 montante </td><td> Carl Zeiss </td><td></td></tr></tbody></table><table border="1"><tbody><tr><td> Wasp Ceppi </td><td> Vola Strains </td></tr><tr><td> Leptopilina victoriae 16 </td><td> yw </td></tr><tr><td> Leptopilina Boulardi 17 1 </td><td> UAS-GFP-dorsale 17 </td></tr><tr><td> Leptopilina heterotoma 2 </td><td> SerpentHemoGal4 13 </td></tr><tr><td> Leptopilina heterotoma 14 1 </td><td> MSNF9 moCherry-14 </td></tr><tr><td> Trichopria drosophilae </td><td> MSNF GFP-15 </td></tr><tr><td> Ganaspis xanthopoda 18 </td><td> yw Serpent-Gal4 UAS GFP-Dorsal/Basc 4 </td></tr><tr><td></td><td> yw; 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

Parassitoide (parassita) vespe costituiscono una classe importante di nemici naturali di insetti tra cui<em&gt; Drosophila melanogaster</em&gt;. Introdurremo le tecniche per diffondere questi parassiti in<em&gt; Drosophila</em&gt; Spp. e dimostrare come analizzare i loro effetti sui tessuti del sistema immunitario di<em&gt; Drosophila</em&gt; Larve.

An Introduction to vespe parassite di<em&gt; Drosophila</em&gt; E la risposta immunitaria antiparassitari

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

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Immunology and Infection

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

18.5K Views.  The City College of New York, CUNY. Parassitoide (parassita) vespe costituiscono una classe importante di nemici naturali di insetti tra cui Drosophila melanogaster. Introdurremo le tecniche per diffondere questi parassiti in Drosophila Spp. e dimostrare come analizzare i loro effetti sui tessuti del sistema immunitario di Drosophila Larve.

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

Wolbachia Bacterial Infection in Drosophila

My name is Voia Friedman. I am associate researcher at Princeton University. And today I'm going to tell you about a few protocols that I developed to trans infect, to I initially to isolate, to go back from the heli of infected flies, and later on help to inject the heli feed to unaffected flights.So basically, this is a very simple protocol to isolate him oly for basics. And you will use this device from, from mepo that contains a filter that goes on top of an E.So you basically needs to put you, we start from around a hundred flies. You anize the flies, you put them into the filter device, you can just use a funnel and that will add our other fit very easily on this device and wash it with 70%ethanol.You just take the, take the future away, you can throw away the ethanol, and then after three times your last wash you do with molecule water because you don't want have the bacteria from the, with traces of ethanol. Well, and then you just go to a new weapon door and, and then you spin down on a micro, real micro for two minutes And later on how you can inject that. He only feed do unaffected flies.So basically take a double stick tape and usually these double stick tapes have two side, one side is a sticker than the other. So leave the sticker side up because that's the side that you're gonna glue that flies on top of it. And basically you'll, the gas apparatus, if you have this light on top of it, you'll be no gas flow and the flies you wake up if they're on top of the slide.So you can just direct the flow, making a little hood, a little chamber using tape. Then you direct the gas over the flies. So then you put your, this light here with the tape and you, you have your flies.Stick the flies on the top of the tape, all of them the same orientation, and put their head towards the direction that you'll be bringing the needle from. Because then you can just over the, the wing, you make sure since they fly, you don't want to fly. If they woke up during the procedure, don't want them to go away.So make sure that you take a little tip and pressure against the, the, the wings of the fly on the tail. And it's okay if they wings get damaged. And when you get rid, when you take the flies up the injection, most likely they'll lose their wings anyway, so they'll survive.That can, that can be useful because you're not gonna lose the flies that, so he puts a mineral oil on the micrometer for the back of the needle, then the needle injection polar, and then the, you can just adjust the pressure to bring the material up to the tip of the needle. I, I did to inject Somewhere between 10 to 15 nanoliters, but as, as long, as long as I'm consistents. Okay, so one important detail there is a cushion of this experiment is that the, the fly, once the apply is being injected when you saw the needle needs to come as far as you can through the upper part of the abdomen.Because here, basically when the needle penetrates, you have a, a, a, a, a bigger space to go up and down. And basically if it's close, closer to the cuticle, it's less likely they're gonna penetrate vital organs in the flight. You, it is very likely unlikely that you're gonna punch a hole in your gut, for instance, if that is gonna kill the flight that you are performing the surgery.My name is Voia Friedman. I am associate researcher at Princeton University in the lab of Eric Hels. And today I would like to tell you about this protocol that you just saw, the demonstration.So this protocol was done because we wanted to prove that UBA is capable of crossing tissues. So UBA is one of the most successful intracellular paras on the face of the earth. Infects from 20 to 70%of insects and many particles.And also is, is very important in terms of human diseases because also infect nematodes that transmit diseases like river blindness or s and ssis. And so, and CCA is our obligatory endo in, in these worms. And there's a great, a very big interest in using CCA also as means to control parasitic diseases transmitter by mosquito and Danny and this and by mosquito such as malaria and dengue.So this, this, this protocol basically was developed to show that once CCA inside the abdominal cavity of a fly, they can cross tissues and reach the germ. So basically what you can see here is the oph of the drosophila ovary and go back once present in the he leaf it, it can cross a pink peronial. She that is around the ovary.And then they cross a muscle layer. And actually one of the first places that they, they go inside the ovary is the somatic stem cell image. And then they can reach the germline.And this bacteria is transmitted the same way that mitochondria is transmitted is through, is through the mother transmitted, through the mother cyop plants present in the egg. So of course they needs to be present into the germline. So that's why this protocol is developed.And this protocol is, can be very useful to basically traditionally the way that people would try to trans infect wbaa basically take wbaa and introduce a new insect species. It would be through doing micro injections into the, into the embryo. But those are much more complicated.And basically here we are just taking advantage of the o biology since they are capable to cross tissues and moving into the stem cell tissues from, they can pass to the, through to the germline. So that's make easier taking UBA from certain, from, from certain species of insects that are infected and they can be transmitted to uninfected species. Prob I think it's the injection itself.Basically you need to be careful to mobilize the fly as well as you can. We are using double sided stick tape and you need to penetrate the needle basically as close to the, as parallel to the fly as possible so you don't go to deep. And it's less likely that we're gonna kill the fly after they're performing the surgery.

Wolbachia infezione batterica in Drosophila

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Biologia

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

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, NF&kappa;B.

Misurazione quantitativa della risposta immunitaria e nel sonno<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Immunologia e infezioni

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

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.

Sistemica infezione batterica e immunitario Difesa fenotipi in<em&gt; Drosophila melanogaster</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

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

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

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

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.

Estrazione di emociti da larve di Drosophila melanogaster per infezione microbica e l'analisi

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Istituzione dell'infezione virale e analisi dell'interazione ospite-Virus in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

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

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

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

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

Valutare la risposta immunitaria cellulare del moscerino della frutta, Drosophila melanogaster, utilizzando un test di fagocitosi In Vivo

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Drosophila melanogaster Larva Injection Protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly and raise ethical issues. Unconventional models are notoriously cheap, easy to handle and culture, and do not take much space. They are genetically amenable and possess complete genome sequences, and their use presents no ethical considerations. The fruit fly Drosophila melanogaster, for instance, has provided great insights into a variety of behavior, development, metabolism, and immunity research. More specifically, D. melanogaster adult flies and larvae possess several innate defense reactions that are shared with vertebrate animals. The mechanisms regulating immune responses have been mostly revealed through genetic and molecular studies in the D. melanogaster model. Here a novel larval injection technique is provided, which will further promote investigations of innate immune processes in D. melanogaster larvae and explore the pathogenesis of a wide range of microbial infections.

Drosophila melanogaster Larva Injection Protocol

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 Protocollo di iniezione della larva

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

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.

Infezione batterica orale e spargimento in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

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

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, and parts of Africa over the last decade, becoming a devastating pest of various soft-skinned fruits in its invaded regions. Biological control, especially by means of self-perpetuating and specialized parasitoids, is expected to be a viable option for sustainable area-wide management of this highly mobile and polyphagous pest. Ganaspis brasiliensis Ihering (Hymenoptera: Figitidae) is a larval parasitoid that is widely distributed in East Asia, and has been found to be one of the most effective parasitoids of D. suzukii.
Following rigorous pre-introduction evaluations of its efficacy and potential non-target risks, one of the more host-specific genetic groups of this species (G1 G. brasiliensis) has been approved recently for introduction and field release in the United States and Italy. Another genetic group (G3 G. brasiliensis), which was also commonly found to attack D. suzukii in East Asia, may be considered for introduction in the near future. There is currently enormous interest in rearing G. brasiliensis for research or in mass-production for field release against D. suzukii. This protocol and associated video article describe effective rearing methods for this parasitoid, both on a small scale for research and a large scale for mass-production and field release. These methods may benefit further long-term research and use of this Asian-native parasitoid as a promising biological control agent for this global invasive pest.

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

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.

Metodi per l'allevamento del parassitoide Ganaspis brasiliensis, un promettente agente di controllo biologico per la Drosophila suzukii invasiva

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, ...

An Introduction to Drosophila melanogaster

Drosophila melanogaster, also known as the fruit fly, is a powerful model organism widely used in biological research that has made significant contributions to the greater scientific community over the last century. First, this video introduces the fruit fly as an organism, including its physical characteristics, life cycle, environment, and diet. Next, the reasons why fruit flies make an excellent model organism are discussed. For example, fruit flies are inexpensive to maintain in the laboratory, have simplified genetics, and short generation times allow for quick experiments with high sample numbers. Then, key discoveries and important Drosophila researchers, such as Thomas Hunt Morgan are profiled. Finally, applications of Drosophila research, ranging from genetics to cardiac and neurological development and disease, are provided. This video serves as an overview of the highly-important and influential model organism that is Drosophila melanogaster.

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