Bee Hemolymph Extraction and Analysis

Bees were anesthetized on ice, and hemolymph was extracted by inserting a microinjection needle (Hartenstein, Würzburg, Germany) into the proximal abdomen between the 3^rd and 4^th tergum^{25 (link),26 (link)}. Hemolymph (1 µl) was transferred to PCR-tubes (Biozym, Hessisch Oldendorf, Germany) containing 1 µl of DAPI-staining solution (4’,6-diamidino-2-phenylindole; 1:100 dilution, lifetechnologies, Carlsbad, California, USA) and 3 µl PBS (pH 7.4; Sigma Aldrich, St. Louis, USA). Hemocytes were counted in a counting chamber (Bürker, Carl Roth, Karlsruhe, Germany) under a phase contrast/fluorescent microscope (Leica DMIL, Leica camera DFC 420 C)^{25 (link),26 (link)}.