8.3. Preparation and treatment of bees
Forager-aged bees are needed for flight tests because they will most likely perform long and persisting flights. Care should be taken to use same-aged bees both in the treatment and control groups (see the section on ‘Obtaining brood and adults of known age’ in the BEEBOOK paper on miscellaneous methods (Human et al., 2013)). When bees are specially treated or manipulated at earlier age, they should be marked and introduced into a nucleus or observation hive until experiments, to allow the ontogeny from hive bees to foragers including orientation and defecation flights. Otherwise, bees from differentially treated colonies can be readily used at foraging age (see the BEEBOOK paper on standard methods for maintaining adult Apis mellifera in cages under in vitro laboratory conditions (Williams et al., 2013)). Caged bees may not fly as well as those from colonies. You should prepare the bees for the further experiment like this:
1. Collect the bees.
2. Immobilize them with needles on balsa wood (or similar material).
3. Glue the connecting tube (about 4 mm long, ca. 1.5 mm in diameter) to the thorax with Pattex (Henkel); super glue is not suitable because bees will die quickly.
4. Allow a few minutes for glue drying before using the bees for flight experiments.
Alternatively, they may also be kept in hoarding cages until usage. The inexperienced experimenter should keep the bees in cages, because the longer the connecting tube is fixed to the bees, the more naturally the bees behave, and the more likely they will fly successfully in the roundabout.
5. Attach the bees to the spur of the arm of the roundabout without prior feeding.
6. Perform an "emptying" flight to deplete all of their energy reserves.
6.1. Stimulate flight by removing the small ball of paper or styrofoam that the bee holds with its legs (Fig. 18B) and which prevents the bee from flying.
6.2. "Restart" the bees several times, in case they cease flying after a few rounds. Stimulate them again to completely deplete their reserves until the movements of the wings are very weak, and the "state of 'apparent exhaustion' cannot be prolonged but leads quickly to the state of complete exhaustion" (Sotavalta, 1954). During experimental flights with defined feeding, the arm must be prevented manually from swinging when the bee stops flying to avoid false counts of revolutions; we recommend implementing an acoustic signal at every rotation of the arm for aural control of flight (a metronome-like frequency confirms constant flight). Once the bees have started flying, they will continue to fly until they are exhausted (depending on their sugar reserves in the honey crop; they will first become slower and then stop when they have no more sugar solution in their honey crops and hardly any sugar reserves in their haemolymph).
7. Weigh the bees.
8. Feed them 10 µl of 1 M or 2 M glucose solution with a pipette within few minutes after the emptying flight, bees are now ready for experiments.
9. Weigh the bees once more.
Weighing before and after feeding allows controlling for complete ingestion of food (Brodschneider et al., 2009). Ingestion of 10 µl of glucose solution corresponds to an increase of about 10 mg of weight.
10. Attach the bees to the roundabout.
11. Place a small paper ball between their legs.
12. Allow exactly 5 minutes of rest after feeding before starting experimental flights; to prevent them from flying during this time, you should give them a ball again. An acoustic signal helps monitoring that bees do not drop the ball and fly. If they do so, they should be stopped immediately and given a ball again.
It takes some experience to find a good flight position which allows optimal power transmission of the bees to the roundabout and hence maximum speed.
According to the construction of the flight mill, bees fed with 10 µl of a 1 M glucose solution fly for about 15 minutes (Brodschneider et al., 2009). The flight is characterized by an increase in flight speed in the first three minutes followed by flight with constant speed and a decrease in flight speed, leading to the total exhaustion of the bee. At this point, further flight experiments with feeding of e.g. 2 M glucose solution can be conducted, because bees quickly recover when provided with energy. Flight time, distance covered (the perimeter of one round is known), maximum speed, average speed and other parameters can be calculated and compared among groups (Gmeinbauer and Crailsheim, 1993). A good verification of the method is to demonstrate that feeding bees with 2 M glucose solutions increases their maximum flight speed compared to that of bees fed with 1 M glucose solution (Gmeinbauer and Crailsheim, 1993; Brodschneider et al., 2009).
Testing the flight performance of honey bees has been applied to compare sexes, castes, the value of energetic substrates and to identify physiological deficiencies such as poor larval nutrition (Jungmann et al., 1989; Gmeinbauer and Crailsheim, 1993; Hrassnig et al., 2005; Brodschneider et al., 2009). Studying the effect of physiological deficiencies on flight performance requires the comparison of treated versus untreated groups in defined conditions as described above. Other applications might include testing the capability of bees of a certain age or treatment to perform long and persisting flights within a given period (e.g. 20 minutes). However, the rate of successful flights should be reported for both types of experiments and can be compared among groups using chi2 tests.