Nosema ceranae Infection in Honey Bees

Brood for the experiment was taken from a healthy Apis mellifera colony belonging to the experimental apiary of the Faculty of Veterinary Medicine, University of Belgrade. The colony was Nosema-free, as confirmed by PCR, using methodology described in Stevanovic et al. [9 ], and without any signs of other infections (bacterial, viral, protozoan or fungal) in the past two years. The presence of viruses was checked according to symptoms described earlier [52 ] and Varroa infestation was kept at a low level. Frames with sealed brood were incubated at 34°C ± 1°C and newly emerged worker bees were taken, confined to six cage groups containing 40 bees in each and kept in the incubator [53 ]. In order to provide absolutely equal conditions for all bees from same group, and exclude the impact of all external factors (position in incubator, humidity, temperature, food amount etc.) some modifications (Fig 1) of cages presented in Williams et al. [53 ] were made. There were 40 individuals in each cage, needed for each treatment group (5 replicates for gene expression analyses and 5 for Nosema spore counting for each of the three collection times, plus 10 for mortality recording). Two independent series of experiments with essentially similar results were conducted, so the data were merged. The bees were fed ad libitum with a solution of sucrose (50% w/w). One control group was experimentally infected with N. ceranae spores (I group), the other was not (NI group), but both were fed on pure sugar syrup (without supplement). The remaining four groups were fed with sugar syrup enriched with supplement starting from day 1 (I-BW1 group), 3 (I-BW3 group), 6 (I-BW6 group) and 9 (I-BW9 group) after emerging (Table 2). All groups, except NI, were infected with N. ceranae spores. Small petri dishes (Fig 1) with the same volume of food (12 ml) were replaced daily in all cages. We have monitored the intake and noticed that the whole quantities were consumed. The supplemented sugar solutions were consumed as readily as the non-supplemented. Bees did not regurgitate the food. Dead bees were removed daily and their numbers recorded. In a preliminary investigation in both laboratory and field conditions no obvious harmful effects on bees have been observed.