Isolation and Characterization of Hemolymph Clotting Factors in Drosophila melanogaster by a Pullout Method

Abstract

Clotting is critical in limiting loss of hemolymph and initiating wound healing in insects as well as in vertebrates. Clotting is also an important immune defense, quickly forming a secondary barrier to infection, thereby immobilizing, and possibly killing bacteria directly. Here, we describe methods to assess clotting and to extract the clot from Drosophila larval hemolymph by using aggregation of paramagnetic beads. The validity of the assay was demonstrated by characterization of mutants. We show that clotting occurs in the absence of phenoloxidase and that the Drosophila clot binds bacteria. We also describe a pullout assay to purify the clot as a whole, free from entrapped hemocytes and cellular debris. Proteins subsequently identified by mass spectrometry include both predicted and novel clot proteins. Immune induction has been shown for three of the latter, namely Tiggrin and two unknown proteins (GC15825 and CG15293) that we now propose function in hemolymph clotting. The most abundant clot protein is Hemolectin, and we confirm that hemolectin mutant larvae show clotting defects.

The Host Defense of Drosophila melanogaster

“…In Drosophila larvae, a clot composed of fibers trapping hemocytes is rapidly generated at the site of injury (Figure 7a,c). This reaction is independent of melanization because it still occurs in proPO-deficient mutants (see below) (203). It is, however, assumed that crosslinking enzymes, including proPO itself and transglutaminase, may be involved in hardening of clots (204,205).…”

“…Subsequent steps in wound closure include melanization and epithelial movements (Figure 7c) (92,93). One plasmatocyte-specific gene, hemolectin, has been demonstrated to be required for efficient clot formation in Drosophila (197,203). Hemolectin is a large protein with several domains that are also present in other clotting factors (Figure 7b).…”

Drosophila14-3-3ε has a crucial role in anti-microbial peptide secretion and innate immunity

“…After 4 hours of challenge, the level of GFP fluorescence in larvae was checked under the fluorescent dissection microscope (Olympus, Japan) and if detected, haemolymph was collected from each larvae by gently puncturing with a 26-gauge needle to let the haemolymph bleed onto a coverslip. The exposure to air induces a rapid and extensive coagulation of insect haemolymph, entrapping haemocytes within the clot (Bidla et al, 2005;Karlsson et al, 2004;Scherfer et al, 2004). The distinct composition of the insect clot (Theopold et al, 2002) makes it insensitive to detergent and vertebrate anti-coagulation reagents.…”

Crystal cell rupture after injury in Drosophila requires the JNK pathway, small GTPases and the TNF homolog Eiger

“…For photometric measurement of PO activity, 30 l hemolymph from each strain was pooled on ice by quickly bleeding 10-12 larvae and withdrawing 6 l hemolymph each time into 10 l of insect Ringer with EDTA instead of Ca 2+ (anticoagulant) (Scherfer et al, 2004). After pipette mixing, 8 l of each hemolymph was aliquoted and activated at 25°C for 10 minutes.…”