7.2.3. Joint action of pathogens and pesticides
1) The day of the study, emerging honey bees (0-1 day) present in the boxes are collected and distributed in different experimental groups: (i) uninfected controls, (ii) infected with the pathogen only (e.g. N. ceranae), (iii) uninfected and chronically exposed to the pesticide at different doses, and (iv) infected with the pathogen and chronically exposed to the pesticide at different doses. Emerging bees can be handled relatively easily because they are quiet and neither sting or fly.
2) Honey bees are first individually infected by feeding with 3 µl of a freshly prepared 50% (w/v) sucrose solution containing the appropriate inoculum of the pathogen. Feeding is performed by holding each bee with its mouthparts touching the sucrose droplet at the tip of a micropipette (Malone and Gatehouse, 1998). This induces the extension of the proboscis and allows the bees consuming the entire droplet. Non-infected bees are similarly treated with the sucrose solution devoid of pathogen.
3) Bee are then confined to laboratory cages in groups of 30-50, and maintained in the incubator at 30-32°C and 80 % relative humidity.
4) Honey bees are chronically exposed to pesticides for different periods of time by ingesting ad libitum, 10 h per day, 50% sucrose syrup containing, 1% (w/v) proteins, the pesticide at the appropriate concentration and 0.1% DMSO. The remaining 14 h, bees are fed with Candy and water ad libitum.
5) During the experiment, each cage is checked every morning and dead honey bees are removed and counted. The food, containing or not the pesticide, is freshly prepared and renewed daily. The actual insecticide consumption is quantified by measuring the daily amount of sucrose syrup consumed per bee.