4.6.1. Introduction

The development of the bee brood is assessed in individually-marked brood cells of all colonies within a study. At the assessment before the application of a treatment (BFD = Brood Fixing Day), one or more brood combs are taken out of each colony and identified with the study code, treatment group, hive number, comb number, comb side and BFD date. The frames are photographed with a high-quality digital photo camera (full frame CMOS chip with a resolution of 20 megapixels or more) controlled via a laptop computer. In the laboratory, all photos are transferred to a personal computer and cells to be analysed, are chosen on the screen. Cells with any type of cell content can be selected, although for a typical evaluation according to Oomen et al. (1992), only egg-containing cells would be selected. The exact position of the markers and of each cell and its content are 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 (see Table 17). Thus, the development of each individually marked cell can be determined throughout the duration of the study (pre-imaginal development period of worker honey bees typically averages 21 days). For studies focussing on specific brood development stages e.g. young / old larvae, the BFD may start at this stage and the assessment days must be adjusted according to the expected development time of the specific brood stage. Following the OECD guideline 75, the brood development is checked 5 times: start with eggs, five days later these eggs have turned from young to old larvae, sixteen days after the start the brood in the marked cells are in the pupal stage and 22 days later the cells should be empty or contain again eggs. The program can cope with any number of observation days, meaning that frames, if necessary in the scope of the study, can be analysed each day. All data evaluation and files (with results), are adapted automatically. The program will generate additional result files for each of the starting stages (or starting contents). Depending on the study objective it is possible to start with other brood stages and with more frequent check dates. On brood fixing day 0 (BFD 00) cells with any brood stage can be selected. In this case, in addition to the standard data evaluation, egg-containing cells are selected and images of BFD 01 or BFD 02 or BFD 03 are analysed, the presumed age of the egg is calculated accordingly and the time resolution of the study is increased.

When photographing the brood containing frames, the bees of the frames to be checked need to be brushed gently from the combs. The combs should not be shaken since too harsh handling might disturb the brood. In order to prevent drying out of the brood and so disturbing the normal development, the frames are taken from the colony, photographed immediately and transferred back to the colony as quickly as possible. Using fixed apparatuses for taking the photograph requires only minutes.

For the European honey bee, the egg stage varies and is approximately 3 days (70 – 76 h). The larval stage (unsealed stage is considered as the larval stage) can varies between 5 and 6 days , with an average of 5.5 days and the pupal stage (capped cells) is 12 days (Winston, 1987; Jean-Prost and Médori, 1994; refer also to the section 2.5 on obtaining adults and brood of known age in this paper). Working with, for instance African honey bees, the assessment days must be adjusted to the duration of the development stages of the brood (see Fletcher 1978).

Table 17. Assessment of the development of the bee brood starting with the brood stage “egg”.

Assessment days

Determination brood stage in marked cells

BFD

egg

 

 

Assessment days

Expected brood stage in marked/selected cells

+   5 days (± 1 day) after BFD

Young to old larvae

+ 10 days (± 1 day) after BFD

Capped brood

+ 16 days (± 1 day) after BFD

Capped brood shortly before hatch

+ 22 days (± 1 day) after BFD

Empty cells or egg containing cells