Counting the number of beetles and defining their spatial distribution in winter clusters killed by dipping in liquid nitrogen

This method is modified from Schäfer et al. (2011). It is an accurate method for counting the number and determining the spatial distribution of adult SHB inside honey bee winter clusters. One can, presumably, find 100% of the beetles inhabiting the colony. However, the method is fatal to the colony and is useful only under certain circumstances. Furthermore, it requires manipulation of the hives and the availability of liquid nitrogen and a cool room that can accommodate the clustering colonies.

  1. Place the colonies in hives without entrances and with screened bottom and lid (mesh width, 1 mm).
  2. Transfer the colonies into a cold room at -5°C and constant darkness, to allow the formation of the clusters.
  3. Kill the colonies by dipping the whole hives into liquid nitrogen (see section, immediately after taking them out of the cool room.
    This will fix the spatial distribution of adult A. tumida inside the clusters and keep the structure of the cluster.
  4. Leave the colonies inside the liquid nitrogen long enough to kill all the bees and SHBs (1 min for a cluster of ~4,000 bees).
  5. Store the colonies in a cold room until they are carefully dissected.
  6. Record the position of the cluster (e.g. by taking pictures) and of each SHB (all bees inside cells have to be removed to investigate the cell bottoms).
    The positions of SHB can be recorded as follows: core (= central area of the cluster), periphery (= inside cluster, except core area) and outside clusters or elsewhere inside the colony.

 Note: it is very important to follow the safety regulations for handling liquid nitrogen.