The role of temperature on the development of circadian rhythms in honey bee workers

Abstract

Circadian rhythms in honey bees are involved in various processes that impact colony survival. For example, young nurses take care of the brood constantly throughout the day and lack circadian rhythms. At the same time, foragers use the circadian clock to remember and predict food availability in subsequent days. Previous studies exploring the ontogeny of circadian rhythms of workers showed that the onset of rhythms is faster in the colony environment (~2 days) than if workers were immediately isolated after eclosion (7-9 days). However, which specific environmental factors influenced the early development of worker circadian rhythms remained unknown. We hypothesized that brood nest temperature plays a key role in the development of circadian rhythmicity in young workers. Our results show that young workers kept at brood nest-like temperatures (33-35 °C) in the laboratory develop circadian rhythms faster and in greater proportion than bees kept at lower temperatures (24-26 °C). In addition, we examined if the effect of colony temperature during the first 48 h after emergence is sufficient to increase the rate and proportion of development of circadian rhythmicity. We observed that twice as many individuals exposed to 35 °C during the first 48 h developed circadian rhythms compared to individuals kept at 25 °C, suggesting a critical developmental period where brood nest temperatures are important for the development of the circadian system. Together, our findings show that temperature, which is socially regulated inside the hive, is a key factor that influences the ontogeny of circadian rhythmicity of workers.

Keywords: Circadian rhythms; Development; Honey bees; Temperature; Workers.

Figure 1. The rate and proportion of young workers developing circadian rhythms are greater at high (33–35 °C) than at lower (24–26 °C) temperatures.

Cumulative distributions of rhythmic young workers at low and high temperatures in constant darkness for two colonies: (A) Colony 1, (B) Colony 2. Dotted line across each plot represents 0.5 proportion of rhythmic bees. At high temperatures (33–35 °C), the rate of development and the proportion of bees developing strong circadian rhythms were significantly higher than at lower temperatures, as shown by generalized estimating equations.

Figure 2. Locomotor activity patterns of young honey bee workers under high or low-temperature constant darkness.

Double-plotted actograms of representative workers at (A) low and (B) high temperatures in constant darkness. Autocorrelation plots were used to determine the rhythmicity of locomotor activity and calculate the endogenous period length (p), rhythm index (RI), and rhythm strength (RS) from days 1–5 and 6–10 for each individual. (C and D) The mean free-running period for rhythmic individuals at high temperatures (24.5 ± 0.13 h SEM) was closer to 24 h and significantly different from that measured at low temperatures for both colonies (23.10 ± 0.29 h SEM) (unpaired t-test Colony 1: t(40) = 3.835, p = 0.0004***) (unpaired t-test Colony 2: t(81) = 4.964, p < 0.0001****).

Figure 3. Survival of isolated young workers decreases at lower temperatures and in arrhythmic individuals.

Survival plot of 1-day-old honey bee cohorts of (A) Colony 1 and (B) Colony 2 at low (solid line) and high (intermittent line) temperature conditions. Statistical comparisons of the cohorts revealed that the survival of individuals was higher in the high-temperature cohort (Gehan-Breslow-Wilcoxon, p < 0.0001****). Panels (C and D) show bar graphs of mean survival and standard error for arrhythmic and rhythmic individuals separated by experimental cohort (low or high). Asterisks indicate significant differences in Šídák’s multiple comparisons tests (Adjusted p < 0.05*).

Figure 4. Temperature (35 °C) during the first 48 h after emergence increases the rate and number of rhythmic workers.

(A) Cumulative distribution of rhythmic young workers exposed to 35 °C during the first 48 h after emergence and afterward placed at 25 °C for the remainder of the experiment (dashed line) compared to that of bees placed at 25 °C (solid line) after emergence. Generalized Estimating Equations revealed significant differences between the rate and proportion of individuals developing rhythmic behavior under these conditions (Time p < 0.001***, Temperature p < 0.001***, Interaction p < 0.001***). (B) Survival plot of 1-day-old honey bee cohorts at 25 °C (solid line) and bees exposed to 35 °C for the first 48 h after emergence (intermittent line). Individuals in the 35–25 °C cohort presented significantly better survival rates than bees placed at 25 °C since the beginning of the experiment (Gehan-Breslow-Wilcoxon, n = 256, p < 0.001***).