Camera dfc 420 c

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)}.

To obtain worker bees of a defined age, single frames of late-stage capped brood (Binder, Tuttlingen, Germany) were brought to the laboratory and incubated in the dark at 32 °C, with humidity provided by open water jars. The frames with worker brood were collected from two full-sized colonies, which were regularly inspected for symptoms of disease and tested for Chronic bee paralysis virus, Deformed wing virus, Acute bee paralysis virus, and Sac brood virus^{65 (link)}. Newly-emerged bees (≤ 24 h) were collected, color marked, and transferred to the experimental colonies on July 28 (15 marked bees per colony). During the second week of exposure, the marked bees (Suppl. Table 2) were removed from the colonies after 12 days in the hive and immobilized on ice. The HPGs were dissected in ice-cold phosphate-buffered saline (PBS, pH 7.4). The specimens were fixed in formaldehyde (4% in PBS, Carl Roth), rinsed three times in PBS, and mounted in Aquapolymount (Polysciences, Eppelheim, Germany). Three pictures of each gland (only one gland per bee) were photographed at 400x magnification using a phase contrast/fluorescence microscope (Leica DMIL, Leica camera DFC 420C) and LAS v4.4 image-capturing software (Leica Microsystems, Wetzlar, Germany). To measure the size of each gland, the diameters of 30 acini per bee were measured using ImageJ v1.49o (http://rsb.info.nih.gov/ij/index.html).