7.2.4. Sensitization to pesticides by a previous exposure to pathogens
1) Bees are distributed in different experimental groups:
- uninfected controls,
- infected with the pathogen only (e.g. N. ceranae),
- uninfected and chronically exposed to the pesticide at different doses 10 days post-infection (d.p.i.),
- infected with the pathogen and chronically exposed to the pesticide at different doses 10 d.p.i.
2) Honey bees are first individually infected with the pathogen (see section 7.2.3.). If studies are conducted on emerging bees, go to step 3. If studies are performed on aged bees, go to step 5.
3) Studies on emerging bees. Honey bees are individually infected by feeding with 3 µl of a freshly prepared 50% (w/v) sucrose solution containing the appropriate inoculum of pathogen. Emerging honey bees are then fed during 10 days with 50% (w/v) sucrose syrup supplemented with 1% (w/v) protein 10 h per day and thereafter with candy and water ad libitum 14 h per day. Each day, feeders are replaced and the daily sucrose consumption is quantified.
4) Ten days after infection, honey bees are chronically exposed for 10 days to the pesticide by ingesting ad libitum, 10 h per day, 50% (w/v) sucrose syrup containing 1% proteins, the pesticide at the appropriate concentration and 0.1% DMSO. Honey bees not exposed to insecticides are fed ad libitum with sucrose syrup containing 1% proteins and 0.1% DMSO. Then, bees are fed with candy and water ad libitum 14 h per day.
5) Studies on aged bees. At a given post-emergence time, caged bees are CO2-anaesthetized, put individually in infection boxes consisting of ventilated compartments (3.5x4x2 cm) and starved for 2 h. Each compartment is supplied with a tip containing the appropriate inoculum of pathogen in 3 µL of sucrose syrup (non-infected bees are similarly treated with sucrose syrup devoid of pathogen).
6) Infection boxes are placed in the incubator and 1 h later, bees that have consumed the total pathogen solution are again encaged (50 bees per cage). Bees are then fed during 10 days with 50% (w/v) sucrose syrup supplemented with 1% (w/v) proteins 10 h per day and thereafter with candy and water ad libitum 14 h per day. Each day, feeders are replaced and the daily sucrose consumption is quantified.
7) Ten days after infection, honey bees are then exposed for 10 days to the pesticide (see step 4 above).
8) Throughout both types of experiments, each cage is checked every morning and dead honey bees 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.
9) At the end of the experiment (20 d.p.i.), surviving honey bees can be subjected to investigations or may be quickly frozen and set aside for subsequent analysis.