3.1. Classical objective mode

One of the recurring pitfalls of honey bee field research is large experimental error which handicaps the investigator’s attempts to discriminate statistical differences among effects of interest. One of the best ways to minimize this problem is to begin experiments with colonies as uniform as possible in regard to comb space, food resources, and populations of adult bees and brood. It is the job of the investigator to distribute these resources equitably among experimental colonies.

The following synthesis draws from methods pioneered by John Harbo who was interested chiefly in reducing environmental variation in honey bee breeding programs (Harbo, 1983, 1986, 1988, 1993; Delaplane and Harbo, 1987) and adapted later by workers who recognized the utility of these methods for field research on Varroa destructor (Delaplane and Hood, 1997, 1999; Ellis et al., 2001; Strange and Sheppard, 2001; Berry et al., 2010) and colony growth (Berry and Delaplane, 2001).

1. Field colonies are equalized with regard to bees, brood, mites, and food resources within units of higher-order experimental replication, i.e. blocks or whole plots, usually based on geography.

2. Empty hives are pre-stocked with brood, empty combs, syrup feeders, and a caged queen in advance of receiving worker bees (Fig. 1). Bottom boards and hive bodies are stapled together to prepare them for moving. Hive entrances are screened to temporarily trap bees; this is done for two reasons: 1. experimental colonies often need to be moved to a permanent site and away from the source colonies from which workers are collected and; 2. a period of in-hive confinement, usually overnight, seems to help bees orient to their new hive and queen.

3. Brood for incipient experimental colonies can be collected from the same source colonies used to collect adults. A near-equal quantity of brood is then assigned to experimental colonies without regard to source. We do not prescribe “random” brood assignment because the investigator should place a higher priority on equalizing quantity of brood over concerns of non-random assignment of brood. Efforts should be made to equalize the relative quantity of sealed versus open brood. A measure of beginning quantity of brood is done by overlaying on each side of every brood comb a grid pre-marked in cm2 and visually summing the area of brood (Fig. 2). The area (cm2) of brood can be converted to cells of brood by multiplying cm2 by the average cells per cm2. This value varies by geography and bee genetics (Table 2), or the investigator can determine local average cell density per cm2 by counting the number of cells directly in a square equaling one cm2 and using the mean of at least ten measurements.

4. In a similar fashion the investigator can derive and equalize the beginning number of cells of honey or pollen or even cells that are empty. Depending on one’s standards for strict uniformity, it may be simpler to provide nothing but brood or empty cells and to provide uniform nutrition across the experiment by use of sugar syrup and protein supplements.

5. Variation due to bee genetics is minimized by providing each colony a sister queen reared from the same mother and open-mated in the same vicinity. A more robust option is to instrumentally inseminate sister queens (see the BEEBOOK paper on instrumental insemination of queens (Cobey et al., 2013)) with the same pool of mixed drone semen.

6. Adult bees are collected for experimental set-up by shaking workers from a diversity of source colonies into one large, common, ventilated cage, allowing workers (and diseases and parasites) to freely mix. With African subspecies, it helps minimize loss from flight to first spray bees on the comb with water mist. The cage is maintained in cool conditions to prevent bee death from over-heating for at least 24 hours to allow thorough admixing of bees, resulting in a uniformly heterogeneous mixture. The weight of bees collected (kg) should exceed the target weight of bees needed for the study by at least 2 kg, or at least by a third in the case of African subspecies, to account for bee loss through death or flight. Bee survival in the cage is greatly improved if the investigator designs it to accommodate 5-6 Langstroth sized brood combs to provide clustering surface (Fig. 3).

7. In order to equalize initial colony populations, it is preferable to make colony-specific caged cohorts. Empty screened cages, ideally made to fit on top of an empty hive, are each pre-weighed or tared with a balance in the field. The large common cage is opened, the bees sprayed with water to reduce flight, then bees transferred from the common cage into the smaller colony-specific cages with the aid of cups or scoops (Fig. 4). Bees are added or removed from each colony-cage until the target weight is achieved and recorded, preferably ≥2 kg.

8. A sample of ca. 300 workers is collected from each incipient colony into a pre-weighed or -tared screw-top container, weighed fresh, then the number of bees counted in the lab to derive a colony-specific measure of average fresh weight of individuals (mg per bee). To count bees it is necessary to first immobilize them, either by freezing them or non-sacrificially with CO2 narcosis. Dividing initial colony cohort size (kg, from step 5) by average fresh weight of individuals (mg) gives initial bee population for the colony.

9. A variation of steps 7 and 8 is available if the investigator is using nucleus hives small enough to weigh entire in the field (Fig. 5). In these cases, the intermediate step of a colony-specific cage is not necessary and the investigator can scoop bees from the common cage directly into the pre-weighed or tared hive. The net weight (kg) of bees is recorded, then initial population determined the same way as given in step 8.

10. If initial measures reveal outliers in the amount of bees, brood, honey, pollen, and empty cells, corrective action should be taken. In general, corrections aimed at minimizing experimental error are permissible until the point at which treatments are begun.

11. After bees and all resources are placed inside hives (with entrances screened, but without sugar syrup which can spill in transit), hives are then moved to their permanent apiary site. Over-heating is a risk, and hives must be kept as cool as possible. There is a special advantage to setting up colonies late in the day and moving hives to the experimental apiary at night. Not only is it cooler, but once hives are unloaded and entrances opened, the bees do not fly because of the darkness and this protracted period inside the hive seems to help them orientate to the new queen and reduces drifting. Colonies can be given sugar syrup after they are unloaded or 24 hours later after bees have settled down.

12. Apiaries should be arranged to limit worker drift between colonies. This can be done by “complicating” the visual field of bees with orientating landmarks near their nest entrances. This can be as simple as using rocks or trees or more deliberate such as painting varying geometric shapes on hive fronts. Arranging hives in a strongly linear arrangement is not good because hives at the ends tend to accumulate bees. For this reason some investigators place hives in a circle.

13. Colony maintenance should include control of non-target diseases and disorders, queen conservation, swarm prevention, and feeding as necessary. Of these, queen loss and swarming tend to be the most disruptive to colony populations. Cutting out queen cells, adding honey supers, marking queens, and regular inspections reduce these problems. If honey supers are added, it is best to add them above a queen excluder to limit the range of the queen’s egg-laying activity. The goal of these manipulations is to decrease experimental residual error.

Table 2. Surface area of some regionally common frame types and expected bee density when frame is fully occupied by worker bees.

Region

Local frame type

Number bees per fully-occupied side

Surface (cm2) per side of frame

Bees / cm2

Ref

Worker cells / cm2

North America

Deep Langstroth

1215

880

1.38

a

3.7c-3.9d

North America

3/4s

910

655

1.39

a

 

North America

Western

785

565

1.39

a

 

North America

Shallow

640

461

1.39

a

 

Europe

Swiss

1200

930

1.29

b

4.0e

Europe

Dadant

1400

1130

1.24

b

4.0e

Europe

German normal

900

720

1.25

b

4.0e

Europe

Langstroth

1100

880

1.25

b

4.0e

Europe

Zander

1000

810

1.23

b

4.0e

South and Central America

Jumbo for brood chamber (modified Dadant)

1980

1130

1.75

f

4.1-4.7g

South and Central America

Jumbo for super (modified Dadant)

920

520

1.77

f

4.1-4.7g



Fig. 1. Investigators pre-stocking experimental hives with equal numbers of brood combs and honey combs and caged queens in preparation for receiving worker bees from a common cage.

Figure 1

Fig. 2. A transparent plastic grid etched in square centimetres is used to visually sum the surface area of brood.

 Figure 2

Fig. 3. A ventilated cage made to hold a large common heterogeneous mixture of bees for starting experiments.

 Figure 3

Fig. 4. Bees are transferred from the common cage to hive-specific cohort cages by use of cups or scoops.

 Figure 4

Fig. 5. Nucleus colonies are small enough to be weighed directly in the field, bypassing the need for intermediate hive-specific cohort cages.

 Figure 5