220.127.116.11. Honey bees
Traditionally, the effects of pesticides are investigated in honey bee foragers that are the individuals first exposed to pesticides. Considering the contamination of pollen and honey by systemic insecticides, all individuals may be potentially exposed by ingestion of a contaminated food. Thus, the exploration of the toxico-pathological interactions has also been studied in cohorts of young isolated bees of known age, which represent a relatively homogeneous biological material. A sufficient amount of honey bee colonies not infected by Nosema, as confirmed by PCR and using primers previously described (Martin-Hernandez et al., 2007), must be selected in order to obtain the desired number of emerging bees. To make the collection of emerging bees easier, queens can be isolated 20 days before the start of the experiment, using a queen excluder grid during 24 hours.
To fully sustain their physiological maturation after emergence, bees ingest pollen during the first days of their life. Pollen is the natural source of proteins for bees but the risk of contamination by pesticides cannot be ruled out (Chauzat et al., 2006; Mullin et al., 2010). A chemical analysis should normally yield information on the pesticide residues present in the pollen. However, the limit of detection of pesticides achieved with multi-residue methods are above 2 µg/kg for a large number of substances. Thus, a substance may be not detected but might still induce toxicity below its limit of detection. In addition, pathogens, notably Nosema and viruses, can be found in the pollen (Higes et al., 2008; Singh et al., 2010). For this reason, pollen is replaced by yeast extracts for protein supply. Commercial protein supplies can be used.
The day before starting the study, frames of sealed brood are sampled from colonies, put in boxes and placed in an incubator in the dark at 34°C with 80% relative humidity.
The day of the study, emerging honey bees (0-1 day) present in the boxes are collected, confined to laboratory cages (e.g. Pain type, 10.5x7.5x11.5 cm) in groups of 30-50 (see Williams et al., 2013), and maintained in the incubator for different periods of time at 30-32°C and 70-80% relative humidity. To mimic the hive environment, a little piece of wax and a Beeboost® (Pherotech; Delta, BC, Canada) releasing one queen-equivalent of queen mandibular pheromone per day, are placed in each cage.