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'Nurse' honeybees tend brood around the clock with attenuated or no circadian rhythms, but the brood signals inducing this behavior remain elusive. We first tested the hypothesis that worker circadian rhythms are regulated by brood pheromones. We monitored locomotor activity of individually isolated nurse bees that were exposed to either various doses of larval extract or synthetic brood ester pheromone (BEP). Bees orally treated with larval extract showed attenuated circadian rhythms in one of four tested colonies; a similar but statistically non-significant trendwas seen in two additional colonies. Nurse bees treated with synthetic BEP showed rhythm attenuation in one of three tested colonies. Next, we tested the hypothesis that capped brood, which does not require feeding, nevertheless induces around-the-clock activity in nurses. By combining a new protocol that enables brood care by individually isolated nurse bees, detailed behavioral observations and automatic high-resolution monitoring of locomotor activity, we found that isolated nurses tended capped brood around the clock with attenuated circadian rhythms. Bees individually isolated in similar cages but without brood showed strong circadian rhythms in locomotor activity and rest. This study shows for the first time that the need to feed hungry larvae is not the only factor accounting for around-the-clock activity in nurse bees. Our results further suggest that the transition between activity with and without circadian rhythms is not a simple switch triggered by brood pheromones. Around-theclock tending may enhance brood development and health in multiple ways that include improved larval feeding, thermoregulation or hygienic behavior. [ABSTRACT FROM AUTHOR]