4.6.4. Finalization of the analysis.

The data evaluation is based on a developmental described by the following pattern: 1111222333444444444444, with the digits representing the expected developmental stage on consecutive days during larval development, i.e. the first four digits (1) correspond to days 0 to 3 with egg stage, the fifth to the seventh digits (2) correspond to days 4 to 6 with young larva stage, etc. If necessary, the user has the possibility to change this pattern and/or to assign a maximum of two days of tolerance for either delayed or accelerated development. Once all images of a frame have been processed the analysis is finalized by pressing F6 or choosing the menu “Make gallery”. The program will then run the analyses.

1. The program creates a folder with the name “AAAAAA_BB_CCD Results 
     yymmdd_hhmmss”, where all results files of the evaluation are saved to (“yymmdd_ 
     hhmmss” corresponds to a date and time of the analysis). Copies of all ROI files used 
     for the analysis are saved into this folder.

2. The ROI data from subsequent days of the same frame are pooled into one tab-
    delimited file and saved as AAAAAA_BB_CCD_RawData.xls.

3. The program populates the classification data of each individual cell from the different
    observation days as numeric values (data of one cell are in one row; data of the same
    day are in columns “BFDnn”; e.g. BFD05 for the fifth brood fixing day).

4. The program rates the development as normal or terminated by comparing the set
    developmental pattern to the developmental stage expected for that cell on that day.
    These data are populated to the others in columns “BTRnn” (BTR = Brood  
    Termination Rate). Brood termination-rate: Based on the brood termination-rate the
    failure of individual eggs or larvae to develop is quantitatively assessed. For the
    calculation of the brood termination-rate the observed cells are split into 2 categories:
a. The bee brood in the observed cell reached the expected brood stage at the
    different assessments days or was found empty or containing an egg after hatch    
    of the adult bee on BFD +22 = successful development.
b. The bee brood in the observed cell did not reach the expected brood stage at
     one of the assessment days or food was stored in the cell during BFD +5 to 
     +16 termination of the bee brood development.

For the final calculation the number of cells, where a termination of the bee brood development was recorded, is summed up for each treatment and colony, is multiplied by 100 and divided by the number of cells observed in order to obtain of the brood termination-rate in %.

5.  The program determines the brood index (BI) for each cell and each day and populates
     the data to the previous ones in the columns “BInn”.
Brood Index:
The brood-index is an indicator of the bee brood development and facilitates a comparison between different treatments. The brood-index is calculated for each assessment day and colony. Therefore the brood development in each cell will be checked starting from BFD 0 up to BFD +22. The cells are classified from 1 to 5 (1: egg stage, 2: young larvae (L1 – L2), 3: old larvae (L3 – L5), 4: pupal stage (capped cell), 5: empty after hatching or again filled with brood (eggs and small larvae) if the cells contain the expected brood stage at the different assessment days. If a cell does not contain the expected brood stage or food is stored in the cell during BFD +5 to +16 (see Table 17) the cell has to be counted 0 at that assessment day and also on the following days, irrespective whether the cell is filled again with brood. For the final calculation the values of all individual cells in each treatment, assessed at the same day, are summed up and divided by the number of observed cells in order to obtain the average brood-index.

6. The program determines the compensation index (CI) for each cell on each observation
    day and populates the results to the previous ones in the columns “CInn”.
Compensation index:
The compensation-index is an indicator for recovery of the colony and will also be calculated for each assessment day and colony. The cells are classified from 1 to 5 (see brood index), solely based on the identified growth stage on the assessment days. By that the compensation of bee brood losses will be included in the calculation of the indices. For the final calculation the values of all individual cells in each treatment, assessed at the same day, are summed up and divided by the number of observed cells in order to obtain the average compensation-index.

7. The program does a frequency analysis for each day and parameter and populates the
    results below all the other data.

8. The program summarises all data by calculating the brood termination rate (BTR), BI
    and CI for each observation day and populates the results below the other data.

9. Finally, the developmental pattern and the tolerances used in the analysis are written at
    the end of this file. This file is saved as a tab-delimited file under the name “AAAAAA  
   _BB_CCD_FinalData.xls”.

10. If on BFD00 other than egg-containing cells are selected, then the program separates
     the cells based on their developmental stage or content and performs the above 
     analyses (steps 5.6. to 5.9.) for each of the developmental stage or content separately
    and creates additional files with the names according to the following examples:
“AAAAAA_BB_CCD_StartAge_00_03.xls” for cells containing egg on BDF00,“AAAAAA_BB_CCD_StartAge_10_21.xls” for capped cells on BFD00,“AAAAAA_BB_CCD_StartContent_empty.xls” for cells empty on BFD00.11. The program creates a gallery, where the images of the individual cells on the different observation days are assembled together side-by-side (similarly to a stamp-collection). This allows the user to have a visual verification of the assessment at a glance. This file is saved as a multi-page TIF file und the name “AAAAAA_BB_CCD_gallery.tif”.

The temporal resolution of a standard study (observation on BFD00 followed by observation on BFD05, etc.) is four days, because the egg stage is four days-long. Insertion of an additional observation day before the end of the egg-stage allows the refinement of the calculated age of the eggs. This can enhance the temporal resolution of the study by a maximum of four fold. If an additional observation before BFD04 was inserted (e.g. on BFD02), than the program separates the egg containing cells according to their expected age into separate files and performs the above analyses (steps 4 to 9) for each age separately and creates additional tab-delimited files with the names according to the following examples:
“AAAAAA_BB_CCD_StartAge_00_01.xls” for egg containing cells, where the eggs had a calculated age of 0 to 1 days.