220.127.116.11.2.1 Preparation of the honey stomachs
The forager bees collected as described above will be stored deep frozen (£-18 °C) in separate containers for each treatment group until preparation in house of the honey stomachs. In principle, it is possible to determine the nectar source of single bees with pollen analysis of the honey stomach content. Successful foragers could be identified by their body weight before the preparation process.
The preparation of the honey stomachs from forager bees will be done as follows (see Carreck et al., 2013 for more information):
1) All bees of one sample will be allowed to thaw for a few minutes.
2) Bees will be fixed at their thorax and their abdomens will be stretched flat with a pair of tweezers.
3) The abdomens or the tergite plates will be removed, so that the honey stomachs will be free.
4) The honey stomach will be held at the lowest part of the oesophagus (see Carreck et al., 2013).
5) The main front part of the oesophagus should be removed.
6) The honey stomach will be held with a pair of tweezers at the small remaining part of the oesophagus.
7) The total weight of the honey stomachs will be determined.
8) The honey stomach contents from one sampling time, treatment and replicate hive will be pooled to get at least 0.2g per sample. The number of prepared bees per sampling time, treatment and replicate, will be recorded. The nectar sample will be transferred into the freezer immediately after the preparation of one forager bee sample.
9) Bees from the control sampling will be processed first. Once this task has been completed, the process will be started with the last sampling.
10) After preparation, the contents of the honey stomachs will be stored separately for each sample at ≤ -18 °C.