Results

Emergence Probability and In-Hive Behavior. We found no significant differences in the emergence probability among the three temperature groups (32°C, 100%; 34.5°C, 99%; 36°C, 98%). On emergence, none of the young adult bees exhibited obvious morphological defects or slow, uncoordinated movement of the type that have been described in bees raised at more extreme temperatures (2, 12–14). All of the bees raised at 32°C or 36°C introduced into the foster colony in the observation hive behaved apparently normally and started foraging after ≈2 weeks, just as their untreated control nest mates did. However, although the number of 36°C-treated bees did not decrease noticeably, fewer and fewer of those raised at 32°C were seen in the hive as time passed. Individuals treated at 32°C were seen to exit the hive on their normal orientation flights but then disappear. Their bodies were not found within the hive nor at the entrance, suggesting that they had suffered some subtle motor deficiency that prevented them from flying or feeding.

Waggle Dance Performance. Three groups of bees were compared. The first group consisted of five individuals from the foster colony in the observation hive that had been raised under natural conditions. These constituted the control group. The second group consisted of five individuals of the 36°C-treated bees. The third group consisted of individuals of the 32°C-treated bees.

After the bees had made five visits to the feeder, we assumed that they had sufficient experience in terms of the orientation of the feeder and to the hive to convey information through the dance to dance followers.

The three criteria of the dance that were analyzed from the video recordings were the probability to perform waggle dances after a return from the feeder (Fig. 1A), the number of dance circuits (Fig. 1B), and the duration of the waggle phase (Fig. 1C). The three columns in each of the figures depict the performances of the five control bees that were raised under normal conditions (column a), the five bees raised at 36°C (column b), and the four bees that were raised at 32°C (column c), respectively. We could not discern any obvious differences in the behavior of the bees of the three groups in terms of moving in the hive, leaving the hive, or arriving and feeding at the feeders.

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Fig. 1.

(A) Probability (mean ± SD) that bees would dance after a visit to the feeder. Column a, probability for bees raised naturally in a standard hive (25 visits from five foragers); column b, probability for bees that were treated as pupae at 36°C (25 visits from five foragers); column c, those treated at 32°C (20 visits from four foragers). There is no significant difference among the three groups. (B) Number of dance circuits (mean ± SD) performed by the bees after each visit to the feeder. Columns represent the same three groups as described in A. Column a, 23 dances from five foragers; b, 22 dances from five foragers; c, 11 dances from four foragers. There is a significant difference between the bees raised at 32°C (c) and those raised at 36°C (b). (C) Duration of the waggle phase (mean ± SD, ms) in the dances performed by the bees. Columns represent the same three groups as described in A. Column a, 1,127 dance circuits from five foragers; b, 1,224 dance circuits from five foragers; c, 176 dance circuits from four foragers.

In 20 visits by the four foragers raised at 32°C, the probability that they would dance on their return to the hive was ≈60% in comparison with ≈90% in the other two groups. However, these differences were not significant because of a large variance in the 32°C bees, which was significantly larger in the 32°C group compared with the other two groups.

We found a significantly smaller mean number of dance circuits by the bees raised at 32°C compared with those raised at 36°C. Also, the variance in the duration of the waggle phase was larger in 32°C-raised bees compared with 36°C-raised bees.

Learning and Memory Consolidation. Because testing the learning and memory consolidation is far less time consuming than measuring the dancing behavior, we were able to include bees from all three temperature-treated groups, namely those raised at 32°C, 34.5°C, and 36°C. The learning behavior of bees raised under natural conditions has been studied extensively (19), so it was not considered necessary to repeat the experiments here with a group of control animals.

Each bee from each of the three temperature regimes was given one conditioning trial and then was tested for the conditioned response either 1 or 10 min after the conditioning trial. Both time intervals test for nonassociative (sensitization) or for associative learning and for the ability to consolidate memory.

The results of the learning and memory consolidation tests are shown in Fig. 2, and the results of the statistical analysis of these data are given in Table 1. We found that bees raised at 36°C performed significantly better at both test intervals than bees raised at either 32°C or 34.5°C (Fig. 2). The differences in response level between the 36°C group and the two other groups are similar to those found for what have been termed “good” and “bad” learners (ranges given in ref. 20: “bad” learners, 55–70% for 1 min and 50–68% for 10-min interval; “good” learners, 50–70% for 1 min and 72–80% for 10-min intervals). In the 1-min test, bees raised at 36°C showed a response level of 70%, and in the 10-min test, the response level increased to 85%, a performance that is close to the maximum expected in the conditioning of the proboscis extension. There were no significant differences in the response level between the bees reared at 32°C or 34.5°C at both time intervals tested.

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Fig. 2.

Proboscis-extension reflex response 1 or 10 min after one-trial odor conditioning in bees, the pupae of which were reared at 32°C, 34.5°C, and 36°C. Columns represent the percentage of responders after 1 min (55 bees raised at 32°C, 43 raised at 34.5°C, and 40 raised at 36°C) or 10 min (58 bees raised at 32°C, 54 raised at 34.5°C, and 23 raised at 36°C).

Discussion

Brood Warming and Hive Energy Partition. The members of many social insect colonies cooperate with one another to regulate the aspects of the environment within their nests (21), but the honey bees appear to be the only group that have achieved a high degree of homeothermy in their nests, keeping the brood combs at temperatures which, although never constant, vary within a relatively narrow window of ≈3°C, despite outside temperatures that range from below freezing to above the melting point of the wax of the combs. The results of the experiments that we have reported in this paper show that the temperature at which the pupae are reared influences their dance communication and odor-learning abilities, the consequences of which are explored below.

The energy required to maintain the relatively stabile brood temperature is obtained from the sugar contained in the nectar brought into the hive by the foragers. “Heater” bees transform the energy in the nectar to heat by rapidly contracting the antagonistic flight muscles in the thorax (6, 7, 22) and then transmitting this warmth to the pupal cells (ref. 23; M. Kleinhenz, B. Bujok, S. Fuchs, and J.T., unpublished data).

The partition of energy within a bee hive can be estimated from the following information, which has been taken from publications. (i) The number of foragers produced by one colony (based on the egg-laying rate of a queen) per year is ≈200,000 (3). (ii) The number of foraging days per bee is ≈10 (3, 4). (iii) The mean number of trips per forager per day is around five (24, 25). (iv) The average energy brought back to the hive per trip is ≈500 J (4, 26). Thus, the total energy collected in a summer season per honey bee colony is ≈5,000,000 kJ.

This energy is used for (i) basic metabolism and heating of the winter cluster during winter (1,600,000 kJ; calculation based on refs. 27–29) and (ii) basic metabolism of all bees, energy for comb building, walking in the hive, and all outdoor trips (but not brood heating) during summer (1,500,000 kJ; calculation based on refs. 30 and 31); leaving (iii) ≈2,000,000 kJ per year for heating of the brood nest, which is ≈40% of the total energy brought back into the nest in 1 year.

Two aspects are relevant for the present study. First, the 40% investment in maintaining the brood temperature is a significant portion of the energy budget and attests to the importance of this activity. Second, the total energy required for the colony is collected with surprisingly little effort. On average, a forager will make only five trips in a day and will forage only for 10 days; hence, it will make a total of only 50 trips (25). Given that some bees have been recorded to make many more than five foraging trips per day, it would seem that others are working far less hard than popularly supposed.

It has been known for many years that honey bees hatch and progress through a number of stages within the hive during which they will carry out various tasks ranging through cell cleaning, nursing, comb building, entrance guarding, and foraging. It is also known that although cell cleaning and foraging are the specialties of the youngest and the oldest bees, the bees between these extremes carry out a number of different tasks and will frequently switch from one task to another in a short space of time (3). Different individuals also will spend different amounts of time on in-hive activities compared with outside activities whereas others may never forage at all (4). The reason for these differences between bees has been the subject of speculation, and we would suggest here that the temperature regime to which they were subjected as pupae may play an important role.

Bees raised artificially at constant temperatures on the lower end of the naturally occurring brood nest temperature range perform less well in dance communication and scent learning. Under natural conditions, this would lead to “bad dancers,” as von Frisch termed forager bees with a low probability to dance and those performing only few dance circuits compared with “good dancers” (unpublished protocols by K. von Frisch, available from J.T.). Such behavior will lead to less nectar inflow into the colony (32, 33) because no dances or short dances with only few circuits will attract no or few recruits (3). Also, a highly variable waggle phase should guide recruited bees to areas scattered around the goal because the feeder distance is coded by the duration of the waggle phase (34).

We have qualitative evidence that exposing the pupae of bees to a temperature of 32°C may have a more far-reaching effect than producing poor dancers. Of the 80 bees that were treated at 32°C and introduced to the observation hive, only very few remained at the end of the 2-week period. The others apparently left the hive and never returned. One could speculate that these animals, seemingly perfectly able to carry out the in-hive nest duties, had trouble finding their way back to the hive after undertaking their orientation flights, and, indeed, some of these bees were found in hives in the neighborhood of their own. The learning and memory consolidation tests (see below) confirm that not only are the 32°C-raised bees poor dancers, they also are bad learners. Learning and memory consolidation, therefore, do appear to be critically important for foraging.

A characteristic of bee-training experiments is that only a small number of bees can be trained to the feeder compared with the large number of individuals that will fly past a feeder placed next to the entrance to a hive. Although there is no reason not to select the best bees in such experiments for the study of the foraging process, the broader picture of a diversity of foraging ability, or even the ability to forage at all, has been obscured. Our results indicate that the existence of different brood-temperature regimes creates diversity in the abilities of the bees that emerge and even a limitation of the numbers of bees that forage successfully. This suggests that there will be an effect on the balance between the bees that carry out in-hive activities and those that forage. We propose that when outside temperatures are high, fewer heater bees will be needed and a larger number of successful foragers will be produced by the higher temperature in the brood. A large amount of nectar, potential energy, will be stored in the combs. However, during colder weather, fewer foragers will be produced, more heater bees will be available to raise the brood temperature, and so the number of successful foragers required for the next round of warmer weather will rise.

The bees that were selected arbitrarily from the colony and for which we did not know the raising conditions showed a significantly larger variance in the number of dance circuits. The dance circuit number was significantly different between the “low-” and “high-temperature” bee groups; this makes sense because it can be expected that bees from a certain range of raising conditions were summed under “normal bees.”

Learning and Memory Consolidation. Although it is possible that the facilities of learning and memory are necessary for bees to carry out in-hive activities, there is little doubt of the need for these in foraging bees, and we find that bees raised at different temperatures during pupal development show differences in learning performance. The measurement of memory consolidation, first established by Pelz et al. (35), allows different, important features of learning and memory formation to be tested. This test is a condensed version of the original experiment on memory dynamics in honey bees that demonstrated that, after one learning trial, bees exhibit a biphasic memory curve (36, 37). In a series of experiments, Menzel and coworkers (19, 37) revealed that the biphasic memory dynamic is a result of two different and separate processes. The high response level to an odor stimulus within the first minute after the conditioning trial is a result of a nonassociative sensitization induced by the sugar-water stimulation. The associative response to the conditioned odor, also induced by sugar-water stimulation, develops with time. After 10 min, the learned response reaches its maximum response level (38, 39).

In our experiments, the bees reared at 36°C during pupal development performed significantly better than those reared at 32°C and 34.5°C, and this was the case at both test intervals, 1 and 10 min after the conditioning trial. The higher response level must be a result of an increased sensitization and reward function of the sugar stimulus and, to a lesser extent, to an increased odor-stimulus strength (36, 38, 39).

Regarding behavioral consequences of the biphasic memory dynamic, it was suggested that both time intervals, (i) <1 min and (ii) in the range of up to 10 min after the learning experience, are important time windows for the foraging honey bee. Immediately after the flower visit, an increased arousal state will heighten the perception and recognition of any important flower feature (odor, color, and shape). About 10 min pass before the bee returns from the hive to the feeding place, and, to recognize the flower from which it had successfully collected nectar, the associative memory should be high. Both processes increase the bee’s ability to respond appropriately and efficiently to flower signals and, thus, reduce searching as well as handling time. Reduction of searching and handling time should have direct consequences for the colony’s food collecting.

Temperature and the Development of the Nervous System. In holometabolous insects like the honey bee, the larval nervous system becomes completely remodeled during pupal metamorphosis to accommodate the changes in sensory equipment and motor systems, which are associated with the change from the worm-like larva to the adult insect (40). Because of these extensive changes in the CNS taking place during the pupal phase, this period may be expected to be very sensitive to environmental changes such as temperature. In the moth Manduca sexta, for example, temperature can be used to desynchronize axonal or dendritic outgrowth, or both, and related defects during the development of a functional circuitry in the olfactory system (41).

The differences that we found in waggle dance performance and odor-learning abilities in temperature-treated honey bees may well be caused by temperature-mediated influences on anatomical and physiological characters of the developing CNS, the hormonal system, or both. An investigation of these effects presently is underway in our laboratories.