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Hemolymph coagulation and phenoloxidase in Drosophila larvae
Stockolms universitet.
Södertörn University, School of Life Sciences.
Stockholms universitet.
Södertörn University, School of Life Sciences.
2005 (English)In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 29, no 8, 669-679 p.Article in journal (Refereed) Published
Abstract [en]

Hemolymph coagulation is a first response to wounding in insects. Although studies have been performed in large-bodied insects such as the moth Galleria mellonella, less is known about clotting in Drosophila melanogaster, the insect most useful for genetic and molecular analyses of innate immunity. Here we show the similarities between clots in Drosophila and Galleria by light- and electron microscopy. Phenoloxidase changes the Drosophila clot's physical properties through cross-linking and melanization, but it is not necessary for preliminary soft clot formation. Bacteria associate with the clot, but this alone does not necessarily kill them. The stage is now set for rapid advances in our understanding of insect hemolymph coagulation, its roles in immune defense and wound healing, and for a more comprehensive grasp of the insect immune system in general.

Place, publisher, year, edition, pages
2005. Vol. 29, no 8, 669-679 p.
National Category
Immunology Zoology
Identifiers
URN: urn:nbn:se:sh:diva-14400DOI: 10.1016/j.dci.2004.11.007ISI: 000229159200001PubMedID: 15854679ScopusID: 2-s2.0-18044376633OAI: oai:DiVA.org:sh-14400DiVA: diva2:468847
Available from: 2011-12-21 Created: 2011-12-21 Last updated: 2016-12-01Bibliographically approved
In thesis
1. Molecular and functional characterization of the insect hemolymph clot
Open this publication in new window or tab >>Molecular and functional characterization of the insect hemolymph clot
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

All metazoans possess an epithelial barrier that protects them from their environment and prevents loss off body fluid. Insects, which have an open circulatory system, depend on fast mechanism to seal wounds to avoid excessive loss of body fluids. As in vertebrates, and non-insect arthropods such as horseshoe crab and crustaceans, insects form a clot as the first response to tissue damage. Insect hemolymph coagulation has not been characterized extensively at the molecular level before, and the aim of my studies was to gain more knowledge on this topic. Morphological characterization of the Drosophila hemolymph clot showed that it resembles the clots previously described in other larger bodied insects, such as Galleria mellonella. The Drosophila clot is a fibrous network of cross-linked proteins and incorporated blood cells. The proteins building up the clot are soluble in the hemolymph or released from hemocytes upon activation. Since bacteria are caught in the clot matrix and thereby prevented from spreading it is likely that the clot serves as a first line of defense against microbial intruders. The bacteria are not killed by the clot. What actually kills the bacteria is not known at this point, although the phenoloxidase cascade does not seem to be of major importance since bacteria died in the absence of phenoloxidase. We identified and characterized a new clot protein which we named gp150 (Eig71Ee). Eig71Ee is an ecdysone-regulated mucin-like protein that is expressed in salivary glands, the perithophic membrane of the gut and in hemocytes, and can be labeled with the lectin peanut agglutinin (PNA). Eig71Ee was found to interact with another clot protein (Fondue), and the reaction was catalyzed by the enzyme transglutaminase. This is the first direct functional confirmation that transglutaminase acts in Drosophila coagulation. A protein fusion construct containing Fondue tagged with GFP was created. The fusion construct labeled the cuticle and the clot, and will be a valuable tool in future studies. Functional characterization of the previously identified clotting factor Hemolectin (Hml) revealed redundancy in the clotting mechanism. Loss of Hml had strong effects on larval hemolymph clotting ex vivo, but only minor effects, such as larges scabs, in vivo when larvae were wounded. An immunological role of Hml was demonstrated only after sensitizing the genetic background of Hml mutant flies confirming the difficulty of studying such processes in a living system. Hemolectin was previously considered to contain C-type lectin domains. We reassessed the domain structure and did not find any Ctype lectin domains; instead we found two discoidin domains which we propose are responsible for the protein’s lectin activity. We also showed that lepidopterans, such as Galleria mellonella and Ephestia kuehniella, use silk proteins to form clots. This finding suggests that the formation of a clot matrix evolved in insects by the co-option of proteins already participated in the formation of extracellular formations.

Place, publisher, year, edition, pages
Stockholm: Stockholms universitet, 2008. 48 p.
Keyword
Innate immunity, hemolymph coagulation, transglutaminase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-31269 (URN)978-91-7155-563-2 (ISBN)
Public defence
2008-02-15, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 8 C, 10:00 (English)
Opponent
Supervisors
Available from: 2016-12-01 Created: 2016-12-01 Last updated: 2016-12-01Bibliographically approved

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