Wolbachia are intracellular alpha proteobacteria that are maternally transmitted to offspring and are widespread in arthropods1,2,3. Our laboratory Drosophila colony is composed of multiple fly strains from all over the world, including those from Bloomington Drosophila Stock Center (Indiana University, USA), and the Vienna Drosophila Resource Center (Austria). Approximately half of the clock mutant fly stains randomly tested in our laboratory colony were infected with Wolbachia (Fig. S2A). The presence of Wolbachia in laboratory fly colonies has also been reported by other groups30,31,32. These frequent infections are not surprising because more than 60% of insect species, including D. melanogaster, are infected by Wolbachia2. Thus, we speculate that many D. melanogaster studies, even those performed using in a closed laboratory environment, are under the influence of Wolbachia symbionts.

The most dramatic behavioral phenotype observed in this study was a significant increase in nighttime activities in uninfected flies regardless of ambient temperature levels. At 29 °C under LD cycles, average nighttime activities were beyond the daytime levels, resulting in a loss of diurnal locomotor rhythms in these flies. To determine the specific influence of Wolbachia symbionts, we analyzed locomotor activities in F1 offspring of uninfected females and infected males. Because most of the other microorganisms including the gut-associated microenvironment are restored in the F1 offspring33, we compared the phenotypes with those of fully sterilized flies. The observed increased nighttime activities in F1 offspring were similar to those seen in uninfected strain flies, suggesting the importance of Wolbachia symbionts for the manifestation of apparent diurnal rhythms in flies. To further explore these mechanisms, we analyzed the temporal distribution of locomotor activities under DD, revealing increases in locomotor activities during subjective nighttime in uninfected strain flies and F1 offspring. Thus, the influence of Wolbachia on nighttime activities was not the result of “masking effects” which are not mediated by circadian clock oscillations.

Heterogeneity of peripheral circadian clocks has been reported in D. melanogaster34: endocrine oscillators in the prothoracic gland are strongly regulated by central pacemakers22 whereas MTs contain independent oscillators19. The present study demonstrated damped per transcriptional oscillations in the peripheral circadian clocks of uninfected flies. Moreover, hsp60 and PER immunostaining assays revealed that Wolbachia symbionts have little effect on central circadian pacemaker neurons, making unique unbalanced clock oscillations within uninfected fly bodies. Because free-running rhythms and photic entrainment of circadian rhythms are indistinguishable between infected- and uninfected flies, a reduction in peripheral clock oscillations may have limited effect on basic clock movements. However, we suggest that daily activity patterns in flies are under the strong influence of peripheral clock movements and Wolbachia symbionts.

Wolbachia infection has previously been documented in the Drosophila brain, but the extent of infection was dependent on the Wolbachia strain, host Drosophila strain, and brain region35. Indeed, the Wolbachia strains wRiv and wPop showed higher titers in the brain than wMel, while higher Wolbachia titers were seen in D. simulans than in D. melanogaster35. Correlation analyses suggested that Wolbachia infection influenced sleep time in flies35. Furthermore, Bi et al.8 recently reported that wMel infection increased nighttime sleep quantity under LD cycles in D. melanogaster. Thus, the present results of an increase in nocturnal activities in uninfected flies may be caused by the reflective effects of sleep disturbance. Bi et al.8 found that Wolbachia may affect dopaminergic activities because two essential genes involved in dopamine synthesis, Pale and Ddc, were significantly upregulated in Wolbachia-infected flies. In the present study, we also observed partial enhancement of PER-ir rhythms in s-LN v s in uninfected flies. However, wMel infections were barely detectable in the brain of D. melanogaster, including in PDF-positive pacemaker neurons. This result is consistent with the findings of Albertson et al.35. Thus, the actions of wMel on central neurons could be the result of indirect influences on peripheral systems.

The effect of Wolbachia symbionts may influence diverse host cellular activities. Of these, one of the most common phenotype induced by Wolbachia is sperm–egg cytoplasmic incompatibility (CI), a form of male-derived zygotic lethality1,5,36. Additionally, a series of studies demonstrated that Wolbachia infections elevated reactive oxygen species and caused changes in the redox homeostasis of host cells37,38. Furthermore, a microarray assay for filarial nematodes demonstrated that diverse genes regulating translation, transcription, protein folding/sorting, metabolic control, and intracellular signaling are under the control of Wolbachia symbionts25. The synthesis of heme in filarial nematodes is dependent on Wolbachia, so heme-dependent energy metabolisms, which can be compensated for by mitochondrial gene expression, are under the strong influence of Wolbachia symbionts25. This indicates that metabolic oscillators, as supportive components of clock gene transcription rhythms39, could also be under the influence of Wolbachia symbionts.

Oxidation and reduction cycles of peroxiredoxin represent a circadian periodicity in a diverse range of cells from human red blood cells to bacteria, so the redox cycle may be an evolutionarily conserved, nuclear-independent circadian oscillator40,41,42. Thus, we speculated that oxidation and reduction cycles may be primary candidates for Wolbachia-dependent circadian oscillators. Based on this hypothesis, we examined the effects of Wolbachia symbionts on the levels of oxidized peroxiredoxin in headless fly bodies. However, oxidation rhythms were observed both in infected and uninfected flies with shifts in their circadian phases (Fig. S4). Therefore, further studies are required to uncover the intracellular mechanisms regulating Wolbachia-dependent per oscillations in peripheral circadian clocks.

Here, we demonstrated that a ubiquitous Wolbachia endosymbiont could influence peripheral clock oscillations and circadian activity patterns in Drosophila models. Notably, in 2017, the US Environmental Protection Agency approved the utilization of Wolbachia to kill wild mosquitoes that transmit viruses such as dengue, yellow fever, and Zika43. Based on our findings, we suggest that future work with Wolbachia, including other Wolbachia strains, and other native endosymbionts should consider whether they modulate the circadian behavioral patterns of insect species. Such a question is beyond the scope of the current study but is an important concern.