4.9. Feeding of diets
- Warm diet to 34.5°C in the incubator
- Place rearing plates on pre-incubated thermoblocks
- Check mortality à Section 5.12. Assessing survival
- Remove dead larvae
- Place food by
using a pipette next to the head of each larvae
Feedings can either be administered every day (for 6 days) in different amounts or, if allowed by the study design, the first two portions (10 + 10µl) can be administered the first day. This option, recommended by Aupinel et al. (2005), gives one day off from the lab and it is assumed that this does not affect mortality or the quality of the test. The total amount of diet given to each larva is 160µl and should be reported if altered. When feeding 160µl of diets in total, honey bee larvae will consume all the food administered: therefore the cleaning of plastic cups or transfer to new cups is not necessary, as each manipulation increases mortality. Prior to feeding, the diet is carefully heated on a magnetic stirrer or in incubators to 34.5°C. During the feeding procedure, the pipette should be placed on the inner side of the plastic cup and the food drop should be placed next to the mouth of the larvae. Drowning of the larvae should be avoided. If drowning occurs occasionally, lift the larvae with a sterile paintbrush; if it occurs systematically, check method (e.g. humidity inside the desiccator or water content of diet).
When larvae are reared following a protocol implying feeding ad libitum, each well is filled with an appropriate volume of diet depending on the volume of the well (e.g. 500µl per well of a 24-well plate), and larvae are then added to the well by placing them carefully on top of the larval diet. Larvae are transferred to newly filled wells every day and the number of larvae per well is adjusted to accommodate the size of the growing larvae. While ten first instar larvae can easily be placed into one well of a 24-well plate, each engorged larva needs a separate well (Fig. 2b). When using this rearing protocol, it is essential to remove any dead larva from the wells and to not transfer dead larvae into newly filled wells. Shortly before pupation, i.e. shortly before or after defecation, larvae are gently cleaned from adhering food and faeces by carefully rolling them over tissue paper (Kimwipes) and then transferred into pupation wells lined with filter paper (Peng, 1992; Genersch et al., 2006). If defecation continues in the pupation-well, developing larvae are placed in new wells lined with clean filter paper.
It is important to pay attention to temperature during feeding. Thermoblocks (pre-warmed in incubators at 34.5°C) underneath the rearing plates can be used to stabilize the temperature of the larvae.