Exploring the role of the circadian clock in modulating gut microbiota and susceptibility to bacterial oral infections in Drosophila melanogaster

The Earth’s rotation around its axis leads to daily fluctuations in environmental cues such as light, temperature, and humidity. To anticipate and prepare to these changes, almost all organisms have evolved the circadian clock, an internal timekeeping mechanism that regulates physiological and behavioral activities. These daily variations in biological processes, known as circadian rhythms, are synchronized by environmental stimuli and persist even under constant conditions, maintaining a periodicity of about 24 h. Daily variations in the ability to combat pathogens have been observed in various organisms, including mice, nematodes, and fruit flies. However, these studies primarily focused on investigating the time-dependent variations in the response to systemic infections, not considering the potential contribution of natural barriers such as epithelia and gut microbiota. The gut microbiota plays a crucial role in preventing pathogen colonization. Furthermore, daily changes in gut microbiota compositions were detected in mice and humans, suggesting that these variations may impact on daily susceptibility to oral infections. In this context, we investigated whether the gut microbiota in Drosophila exhibits daily fluctuations. To this end, we examined gut microbiota composition and bacterial load in wild-type and arrhythmic flies lacking the clock gene per (per01), under both light: dark cycle (12:12 LD) and in constant darkness (DD). per01 mutants exhibited significant variations in total bacterial load throughout of the day, unlike wild-type individuals. Additionally, the gut microbiota composition differed from wild-type and per01 flies under both 12:12 LD and DD conditions. These findings suggest that the circadian clock plays a role in stabilizing daily gut bacterial load and shaping gut microbiota composition in Drosophila. Daily changes in the gut microbiota composition were detected for both wild-type and per01 mutants, suggesting that these variations are not controlled by the circadian clock. However, some gut bacteria showed 24-h fluctuations in their relative abundance, independent of fly’s circadian clock suggesting the presence of an autonomous time-keeping system among Drosophila gut commensals. We then explored whether Drosophila exhibits a daily sensitivity to oral bacterial infection. When challenged with the Gram-negative bacterium P. entomophila, flies exhibited a time-dependent susceptibility to oral infection, with higher sensitivity at night compared to daytime. In DD conditions, the daily susceptibility to infection was not maintained, indicating that it is mainly influenced by the light cycle rather than the circadian clock. Our data indicate that differences in the daily ability to fight P. entomophila resides, at least in part, in the ability to restrain pathogen proliferation. However, the timing of infection did not result in any observed differences in the activation of pathways related to the humoral immune response, gut repair, or melanization. This indicates that other components of the immune response, or mechanisms of tolerance against pathogens are involved and require further study. We observed that the sensitivity of germ-free flies to oral infection with P. entomophila does not depend on the time in which the challenge occurs. This suggests that the gut microbiota contributes, at least in part, in modulating daily susceptibility to this pathogen. Differently from our expectations, GF flies demonstrated a low sensitivity to P. entomophila, along with the ability to rapidly clear it. Our data indicate that this effectiveness to fight the pathogen may be attributed to the Toll pathway and partially to the JAK/STAT pathways, both of which showed enhanced activation under basal conditions in the absence of gut microbes.