The oligonucleotides used for in situ hybridization were described previously. Bacteriocytes were visualized

by FISH with oligonucleotide probes Eub338 (5′-GCTGCCTCCCGTAGGAGT-3′) [34], targeting a conserved region of the eubacterial 16S rRNA, and with Bfl172 (5′-CCTATCTGGGTTCATCCAATGGCATAAGGC-3′), targeting a 16S rRNA region specific for B. floridanus [33]. Probes were labelled with the fluorescent dyes Cy3 or FITC at the 5′ end (MWG-BIOTECH AG, Ebersberg, Germany). For protocol process details see STI571 in vitro [2]. The ovaries of three years old queen were dissected, fixed and hybridized like the midguts. The slides were Selleck CDK inhibitor analyzed with a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) and pictures were taken with a RT Slider digital camera (Diagnostic Instruments Inc., Sterling Heights, MI, USA). Evaluation of colony development Colonies collected in 2006 were used to evaluate control colonies versus treated colonies Entospletinib research buy development. Over a period of seven months (including the first three months of antibiotic treatment) the number of brood (larvae and pupae) and workers in each colony were counted each month, during seven months. Encapsulation rate assay Encapsulation followed by melanisation is an efficient innate immune response against

parasites. We can trigger this response by inserting an inert antigen, like nylon filament. To measure the ant immune response, an encapsulation test was performed by inserting a 1.5 mm-long piece of nylon monofilament (0.12 mm diameter) in the pleural membrane between the second and third tergite. This procedure was carried out on three workers from each colony, with a total of 30 workers for each group, based on the procedures adopted by Rantala & Kortet [35]. Baricitinib Twenty four hours after, the implants were removed from the haemocoel and placed on a glass

slide to be mounted into Clarion™ medium. The filament was examined under a light microscope and photographed using a digital camera (Olympus DP50). The mean grey value of the whole implant was measured using the ImageJ 1.37v software. We assumed that the darkest grey received the highest encapsulation rate (total black). The background grey value was subtracted to correct the values of the implants. The midgut of each worker was dissected in sterile PBS (137 mM NaCl-2.7 mM KCl-4.3 mM sodium phosphate-1.4 mM potassium phosphate, pH 7.2) and conserved in tubes independently at -20C° for quantitative PCR. Assessing antibiotic treatment effects Antibiotc treatments effects were assessed by two different and complementary techniques: Real time qPCR and Fluorescent in situ hybridization (Fish).