Development of the bee brood in single cells

The time schedule of the brood assessment days was chosen in order to check the bee brood at different expected stages during the development as mentioned in the Table 7.

1) The application in the tunnels should be performed shortly after BFD (within 2 days afterwards).

2) In contrast to the method described in the OECD Guidance Document 75, it is now common to use the digital photo method (Jeker et al., 2011 but see section 5.2.3. of the present manuscript) to follow the development from an egg to the adult honey bee. In the following text, this method will be used to describe the system.

3) The development of bee brood is assessed in individual marked brood cells of all colonies within a study. At the assessment before the application (BFD) one or more brood combs should be taken out of each colony, marked with the study code, treatment group, hive number, comb number, comb side and BFD date, and photographed with a digital camera. In the laboratory, all photos are transferred to a personal computer and areas with at least 100 cells containing eggs are marked on the screen. The exact position of the markers and of each cell and its content should be stored in a computer file that serves as a template for later assessments. The same cells are assessed on each of the following assessment dates (Table 7). Thus, the development of each individually marked cell throughout the duration of the study can be determined (pre-imaginal development period of worker honey bees typically averages 21 days).

4) For the evaluation of the different brood stages of single marked cells, the recorded growth stages are transformed into values counting from 0 to 5 as listed below:

   - 0: termination/breakup of the development (e.g. nectar or pollen found in a cell, if in the previous assessments the presence of brood was recorded)
   - 1: egg stage
   - 2: young larvae (L1 or L2)
   - 3: old larvae (L3 to L5)
   - 4: pupal stage (capped cell)
   - 5: empty after hatching or again filled with brood (eggs and small larvae)
   - N: cell containing nectar
   - P: cell containing pollen

Cells filled with nectar and pollen after the termination of brood development in the respective cell (counted 0) may be identified by an “N” and “P” in the following assessments; the respective cells have to be excluded from further calculations, but should be included in the overall evaluation in the end.

5) Based on the numbering described above, mean values (indices) can be calculated for each colony and assessment day.

6) Assuming that at the first assessment only eggs will be marked, the index is one. An increase of the brood index during the following assessment can be observed, if a normal development of the brood is presumed. This increase is caused by the development from eggs to larval stages, from larvae to pupae and from pupae to adults. Details of the evaluation of the results are presented by Schur et al. (2003).

Table 7. Time schedule of the brood assessment in semi-field brood tests
BFD = brood area fixing day. *Assessments will be performed outside the tunnels at the monitoring location.


Determined brood stage in marked cells

BFD (1-2 days before application)



Expected brood stage in marked cells

5(±1) days after BFD

Young to old larvae

10(±1) days after BFD*

Capped cells

16(±1) days after BFD*

Capped cells shortly before hatch

22(±1) days after BFD*

Empty cells or cells containing eggs, young larvae, nectar or pollen