Gut microbiota is associated with circadian rhythms and clock gene expression in honeybees.

To accommodate daily environmental changes, insects exhibit species-specific circadian rhythms in behavior and physiology. Compared to non-social insects, honeybees display robust, socially regulated circadian rhythms aligned with colony tasks, while bumblebees show more flexible rhythms linked to simpler social structures. The gut microbiota has been proposed to influence circadian rhythms in mammals through microbial metabolites, hormone regulation, and gene expression. However, its role in honeybee and bumblebee circadian rhythms remains unclear. In this study, we generated gnotobiotic honeybees and bumblebees to explore microbiota-driven circadian modulation through behavioral and transcriptional analyses. Behavioral assays showed that gut microbiota had no significant effect on bumblebee locomotor activity, rhythmicity strength, or free-running period under the tested conditions. In contrast, microbiota colonization in honeybees led to increased daily activity, enhanced rhythmicity strength, and stronger circadian oscillations without affecting the intrinsic free-running period. Notably, qPCR analysis revealed that, at T5, colonized bees exhibited higher per expression together with reduced cwo and serotonin receptor 5-ht1 expression in the honeybee brain. These transcriptional changes indicate potential links between the gut microbiota and honeybee circadian rhythms via the gut-brain axis, possibly involving altered serotonergic signaling. Overall, our results indicate an association between gut microbiota and circadian rhythmicity in honeybees, whereas bumblebees showed little responsiveness, suggesting potential species-specific interactions between microbial signals and host circadian systems. This study advances understanding of microbiota-driven behavioral regulation in eusocial insects and lays the groundwork for future mechanistic investigations.