The aim of this dissertation was to identify and characterise haemolymph clotting factors in insects. Starting from an almost exclusively morphological description of the haemolymph coagulum, several putative clot proteins of two insect species, Galleria mellonella and Drosophila melanogaster have been studied.
A functional involvement of some molecules like Drosophila hemomucin remains largely unclear and has to be further investigated. The data collected in the present study underline earlier assumptions of broad biological functions for this protein in immunity (microparticle formation, pathogen entrapment in the clot) and development (e. g. possibly trachea formation). Therefore the approach chosen here to knock down hemomucin in vivo at different developmental time stages and in specific organs by RNA interference still seems valuable. Since the synthesis of a DNA-construct for in vivo RNAi of hemomucin was not successful, one can still only speculate about the specific roles of this protein.
Anti-clot antiserum and pull-out assay independently led to the identification of lipophorin and prophenoloxidase as clot components. Together with the enzyme transglutaminase these proteins might represent more conserved constituents in insect haemolymph coagulation. Even arylphorins of both species, are candidate clotting factors with analogous counterparts in D. melanogaster and G. mellonella, including the Galleria gallysin, Drosophila larval serum protein 1c (lsp1c) and fat body protein 1 (fbp1). In contrast, relatively high variability may be expected for transglutaminase substrates in insects. In agreement with this idea, two species-specific putative substrates of the enzyme, the a-crystallin-like Galleria protein and the Drosophila CG 15825 gene product, evolved independently. In summary, the evolution of insect clotting proteins seems to be characterised by both conservative traits (e. g. vWF D-like domains, transglutaminase) and quick development of new species-specific elements by evolutionary tinkering (glutamine residues in e. g. CG 15825; a-crystallin).
The factors mentioned in the previous paragraph allow only vague assumptions for the molecular mechanisms underlying the initial steps of haemolymph coagulation. Self-aggregation of proteins due to certain sequence characteristics such as vWF domains and glutamine residues, seems to be a likely explanation for quick clot formation. Drosophila hemolectin is a good candidate for one of the first clot proteins attaching to a wound and its domain structure suggests interactions with various additional factors. An in vivo RNAi knock-down of hemolectin led to a clearly affected clotting process. Drosophila tiggrin and other RGD-containing proteins in the coagulum possibly represent the link to the haemocytes that assist in closing the wound and stimulate formation of the new epithelium.
In addition, it becomes increasingly clear that both transglutaminase and phenoloxidase are involved in later steps of clot formation and strengthen the primary coagulum by cross-linking of proteins. The interplay of these enzymatic activities is still largely unexplored and an investigation of their crosstalk may provide insights into their probably dissimilar roles in clot formation.
Drosophila represents an ideal model organism to study insect haemolymph clotting, since genetic tools and a huge number of available mutants will facilitate a detailed description of the coagulation process and its role in the immune system of the fly. Simultaneously evolutionary questions may rather be successfully addressed by comparing clotting proteins of different insect orders and species. In the near future the putative clotting factors described here may be investigated more specifically for their functions as well as for their interactions with each other. A redundancy of multiple possible ligands is already commonly accepted for components of the vertebrate coagulum (Furlan, 2002), but remains to be shown for insects. Similarly many clotting defects in insects may turn out to be of a quite moderate nature due to overlapping enzymatic cross-linking activities and possibly even flexibility in the spectrum of covalently linked substrate proteins.
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