4.4.1.1 Colony establishment

1. Equalize field colonies are in regard to bees, brood, food resources (see section 1.2.2.1 ‘Setting up experimental colonies of uniform strength’ of the BEEBOOK paper on estimating colony strength (Delaplane et al., 2013)) and mites within units of higher-order experimental replication, i.e. blocks or whole plots, usually based on geography.

At least two more source colonies than the target number of experimental colonies should be used to account for bee loss through death or flight.

2. Collect adult bees and phoretic mites for experimental set-up by shaking workers from a diversity of source colonies into one large, common cage, allowing workers (and mites) to freely mix (Harbo, 1993) (Fig. 15).

3. Maintain the cage in cool conditions to prevent bee death from over-heating for at least 24 hours to allow thorough admixing of bees and mites, resulting in a uniformly heterogeneous mixture.

4. Distribute worker cohorts of equal size (preferably by weight, ca. 1 kg) into hive boxes pre-stocked with near-equal amounts of brood, honey, pollen, and empty cells (Fig. 16).

5. Provide each colony a sister queen reared from the same mother and open-mated in the same vicinity to minimize variation due to bee genetics.

6. Collect a sub-sample of ca. 300 workers from each incipient colony.

7. Weigh the samples.

8. Count the number of bees to derive a colony-specific measure of average fresh bee weight (mg).

9. Divide initial cohort size (kg) by average fresh weight of individuals (mg) to obtain initial bee population.

10. Collect a sample of ca. 300 worker bees (can be the same sample as above after it is weighed fresh) to obtain a measure of initial density of phoretic mites.

11. Separate the mites from the bees, see section  4.2.3.1. ‘Infestation rates of adult bees’ (Fig. 17).

12. Count the number of bees and number of mites to derive a colony-specific measure be mite/bee density.

13. Multiply this number by initial bee population to obtain phoretic mite population.

14. Store the samples in alcohol for future reference.

15. Collect brood for incipient experimental colonies from the same source colonies used to collect adults.

16. Assign a near-equal quantity of brood to experimental colonies without regard to source (see section 1.2.2.1.1. ‘Classical objective mode’ of the BEEBOOK paper on estimating colony strength (Delaplane et al., 2013)).

17. Measure the beginning quantity of sealed brood to calculate beginning mite populations.

This is done by overlaying on each side of every brood comb a grid pre-marked in cm2 and visually summing the area of sealed brood (Fig. 18).

18. Convert the area (cm2) of sealed brood to cells of sealed brood by multiplying cm2 by the average cell density per cm2 (see section 1.2.2.1.1. ‘Classical objective mode’ of the BEEBOOK paper on estimating colony strength (Delaplane et al., 2013)).

Cell density per cm2 varies by geography; in the south-eastern USA conversion factors between 3.7 and 3.9 are used (Delaplane and Harbo, 1987; Harbo, 1993). This value should be verified with the honey bee lineage used for the experiment.

19. Measure incipient mite population in brood by opening at least 200 cells of sealed brood per experimental colony and examining under strong light and magnification to see and count sclerotized mites (see section 4.2.3.2. ‘Infestation rates of brood’).

20. Multiply the mite/cell density by cells of sealed brood to obtain the mite population in cells.

21. Sum phoretic mites + mites in cells to obtain the beginning total mite population.

22. Take corrective action should initial measures expose outliers in initial populations of bees, brood, or mites.

In general, corrections aimed at minimizing experimental error are permissible until the point at which treatments are begun.

23. Once colonies are established and queens released and confirmed laying, it is recommended to collect one or more beginning relative mite measures. The most commonly recognized relative measures are mites recovered per 24 h on bottom board sheets (see section 4.2.4. ’Natural mite fall’) and mites per adult bee recovered from samples (as described in section 4.2.3.1.2. ‘Dislodging mites from bees’).

It is to be hoped that these relative measures will correspond closely to real colony mite populations, and the investigator is encouraged to check the validity of the relative measures with regression analyses against total mite population.

24. Colony maintenance should include control of non-target diseases and disorders, swarm prevention, and feeding as necessary.

The goal of these manipulations is to decrease experimental residual error.


Fig. 15. Adult bees with their phoretic mites from several colonies are mixed to homogenise the starting infestation level in the replicates. Photo: Keith Delaplane.

Figure 15

Fig. 16. Boxes are pre-stocked with near-equal amounts of brood, honey, pollen, and empty cells to host the experimental colonies. Photo: Keith Delaplane.

Figure 16
 

Fig. 17. Number of bees and mites are counted to obtain a measure of initial density of phoretic mites. Photo: Keith Delaplane.

Figure 17

Fig. 18. Measuring the beginning quantity of sealed brood. Photo: Keith Delaplane.

Figure 18