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An improved method for nematode infection assays in Drosophila larvae
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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2012 (English)In: Fly, ISSN 1933-6934, Vol. 6, no 2, 75-79 p.Article in journal (Refereed) Published
Abstract [en]

The infective juveniles (IJs) of entomopathogenic nematodes (EPNs) seek out host insects and release their symbiotic bacteria into their body cavity causing septicaemia, which eventually leads to host death. The interaction between EPNs and their hosts are only partially understood, in particular the host immune responses appears to involve pathways other than phagocytosis and the canonical transcriptional induction pathways. These pathways are genetically tractable and include for example clotting factors and lipid mediators. The aim of this study was to optimize the nematode infections in

Drosophila melanogaster larvae, a well-studied and genetically tractable model organism. Here we show that two nematode species namely Steinernema feltiae and Heterorhabditis bacteriophora display different infectivity towards Drosophila larvae with the latter being less pathogenic. The effects of supporting media and IJ dosage on the mortality of the hosts were assessed and optimized. Using optimum conditions, a faster and efficient setup for nematode infections was developed. This newly established infection model in Drosophila larvae will be applicable in large scale screens aimed at identifying novel genes/pathways involved in innate immune responses.

Place, publisher, year, edition, pages
2012. Vol. 6, no 2, 75-79 p.
Keyword [en]
Drosophila melanogaster, Galleria mellonella, entomopathogenic nematodes, Steinernema feltiae, Heterorhabditis bacteriophora
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-75211DOI: 10.4161/fly.19553ISI: 000305965500002OAI: oai:DiVA.org:su-75211DiVA: diva2:515015
Available from: 2012-04-11 Created: 2012-04-11 Last updated: 2012-10-11Bibliographically approved
In thesis
1. Functional study of hemolymph coagulation in Drosophila larvae
Open this publication in new window or tab >>Functional study of hemolymph coagulation in Drosophila larvae
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many pathogen infections in nature are accompanied by injury and subsequent coagulation. Despite the contribution of hemolymph coagulation to wound sealing, little is known about its immune function. Based on the molecular knowledge of Drosophila innate immunity, this thesis investigated the immune function of clot both in vitro and in vivo, the immune relevant genes involved in a natural infection model, involving entomopathgenic nematodes (EPN) and the factors leading to crystal cell activation. Transglutaminase (TG) and its substrate Fondue (Fon) have been identified as bona fide clot components in Drosophila larvae. By knocking down TG or Fon via RNAi, we observed an increased susceptibility to EPN in larvae. In addition, this increased susceptibility was associated with an impaired ability of hemolymph clots to entrap bacteria. Immunostaining revealed that both clot components (Fon and TG) were able to target microbial surfaces. All these data suggest an immune function for the Drosophila hemolymph clot. Strikingly, similar results were obtained when we ran parallel experiments with human FXIIIa, an ortholog of Drosophila TG, indicating a functional conservation. We also found evidence for the regulation on both clot and immunity by eicosanoids in Drosophila larvae. The combination of EPN infection with the Drosophila model system allowed us to discover an immune function for TEP3 and Glutactin. However the molecular mechanism underlying the involvement of these two proteins in this particular host-pathogen interaction remains to be elucidated. Prophenoloxidase, the proform of enzyme involved in hardening the clot matrix, has been shown to be released by rupture of crystal cells. This cell rupture is dependent on activation of the JNK pathway, Rho GTPases and Eiger. Our work further identified the cytoskeletal component, Moesin, and the cytoskeletal regulator Rac2 as mediators of cell rupture. Despite the possible role of caspases in crystal cell activation, such cell rupture was turned out to be different from apoptosis. The implication of Rab5 in this process indicated that proper endocytosis is required for cell activation and subsequent melanization. Our findings furthered not only our understanding of the release of proPO via cell rupture but also our knowledge on different paths of immune cell activation.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University, 2012. 48 p.
Keyword
innate immunity, hemolymph coagulation, clot components, Drosophila larvae
National Category
Biological Sciences Immunology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-75179 (URN)978-91-7447-501-2 (ISBN)
Public defence
2012-05-11, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
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Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: In press. Paper 4: Manuscript.

Available from: 2012-04-19 Created: 2012-04-11 Last updated: 2012-04-25Bibliographically approved

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