Measuring individual locomotor rhythms in honey bees, paper wasps and other similar-sized insects

Abstract

Circadian rhythms in social insects are highly plastic and are modulated by multiple factors. In addition, complex behaviors such as sun-compass orientation and time learning are clearly regulated by the circadian system in these organisms. Despite these unique features of social insect clocks, the mechanisms as well as the functional and evolutionary relevance of these traits remain largely unknown. Here we show a modification of the Drosophila activity monitoring (DAM) system that allowed us to measure locomotor rhythms of the honey bee, Apis mellifera (three variants; gAHB, carnica and caucasica), and two paper wasps (Polistes crinitus and Mischocyttarus phthisicus). A side-by-side comparison of the endogenous period under constant darkness (free-running period) led us to the realization that these social insects exhibit significant deviations from the Earth's 24 h rotational period as well as a large degree of inter-individual variation compared with Drosophila. Experiments at different temperatures, using honey bees as a model, revealed that testing the endogenous rhythm at 35°C, which is the hive's core temperature, results in average periods closer to 24 h compared with 25°C (23.8 h at 35°C versus 22.7 h at 25°C). This finding suggests that the degree of tuning of circadian temperature compensation varies among different organisms. We expect that the commercial availability, cost-effectiveness and integrated nature of this monitoring system will facilitate the growth of the circadian field in these social insects and catalyze our understanding of the mechanisms as well as the functional and evolutionary relevance of circadian rhythms.

Keywords: Apis mellifera; Circadian rhythms; Honey bees; Locomotor activity; Mischocyttarus; Polistes; Temperature compensation; Wasps.

Fig. 1.

Locomotor activity monitoring system. (A) Locomotor activity monitor (LAM16) as sold by Trikinetics Inc. The LAM16 monitor contains 32 channels. Each channel contains three infrared beams and their corresponding receptor. The wiring of the internal board of the monitors is arranged such that the interruption of one or more beams on each channel counts as a single activity event. (B) Water system assembly ensures that every individual has a constant supply of water. This assembly minimizes handling and refilling time compared with individual water supplies. A detailed description of this system has been provided in the Materials and methods. (C) Sampling tubes and honey candy. The sampling tube is a standard 15 ml centrifuge tube with holes for air and water system connection. The honey candy is first placed directly into the tube cap and then a piece of cheese-cloth is placed on top of the food. The cloth prevents spillage and spreading of the food throughout the tube, and thus prevents individuals from becoming stuck. (D) Fully assembled LAM system including the water system assembly and the sampling tubes.

Fig. 2.

Honey bee foragers exhibit short period phenotype under constant darkness (<24 h endogenous circadian rhythm). (i) Double-plotted actograms showing the locomotor activity pattern of representative individuals of the different Apis mellifera groups sampled: (A) gAHB, (B) carnica and (C) caucasica. Each row contains the locomotor activity (counts per 30 min) of two consecutive days and the last day is repeated such that the second day is always the beginning of the next row. The x-axis shows the time of day under constant darkness expressed as circadian time (CT). (ii) Autocorrelation plots used to determine the period (p), rhythm index (RI) and rhythm strength (RS), as described previously (Levine et al., 2002b). In general, the oscillation of this function shows periodicity. The asterisk shown on the third peak of the autocorrelation plot indicates the specific time point used for the determination of the rhythm parameters. (iii) The maximum entropy spectral analysis (MESA) plot is an independent algorithm used to determine period (t) (Levine et al., 2002b).

Fig. 3.

Female wasps exhibit long period phenotype under constant darkness (>24 h endogenous circadian rhythm). (i) Double-plotted actograms showing the locomotor activity pattern of representative individuals of (A) Mischocyttarus phthisicus and (B) Polistes crinitus. (ii) Autocorrelation plots used to determine the period (p), rhythm index (RI) and rhythm strength (RS). (iii) MESA plot.

Fig. 4.

The period of honey bees and wasps significantly deviates from 24 h and exhibits a large degree of inter-individual variation. Box plots of the endogenous period distribution in constant darkness for individuals of the A. mellifera subspecies carnica (n=11) and caucasica (n=13) and the hybrid gAHB (n=58); Drosophila melanogaster (n=32); Polistes crinitus (n=13); and Mischocyttarus phthisicus (n=24). The dashed lines represent the calculated minimum and maximum values for circadian period measured from D. melanogaster individuals. A Kruskal–Wallis test revealed significant differences across all of the sampled groups (F₅=94.80, P<0.0001). Box plots with different letters are significantly different at P<0.05 level in a post hoc Tukey's HSD tests.

Fig. 5.

Testing the endogenous rhythm at 35°C decreases period deviations from 24 h. (A) Box plots of the endogenous rhythm distribution under constant darkness of honey bee foragers at 25°C (white; n=51) and 35°C (shaded; n=54) reveal significant differences (Kruskal–Wallis test, F₅=26.41, P<0.01). (B) Examining the endogenous rhythm at different temperatures of foragers in six different colonies reveals significant differences of the period distributions at different temperatures for colonies 1 (P=0.04), 3 (P<0.01) and 6 (P=0.04), while comparison of these conditions in colonies 2, 4 and 5 did not yield significant differences.