Rhythmicity of the intestinal microbiota is regulated by gender and the host circadian clock Xue Lianga, Frederic D. Bushmanb, and Garret A. FitzGeralda,1 aDepartment of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, PA 19104; and bDepartment of Microbiology, Perelman School of Medicine, University of Pennsylvania, PA 19104 Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved July 8, 2015 (received for review January 20, 2015) In mammals, multiple physiological, metabolic, and behavioral is conditioned by the sex of the host, being more pronounced in processes are subject to circadian rhythms, adapting to changing femalesthaninmales. light in the environment. Here we analyzed circadian rhythms in the fecal microbiota of mice using deep sequencing, and found Results that the absolute amount of fecal bacteria and the abundance of Diurnal Oscillation of the Microbiota in C57BL/6 Mice. The circadian Bacteroidetes exhibited circadian rhythmicity, which was more behavior in the fecal microbiota of wild-type mice was assessed pronounced in female mice. Disruption of the host circadian clock by sampling fecal pellets from each mouse every 4 h for 48 h. by deletion of Bmal1, a gene encoding a core molecular clock com- Circadian rhythmicity of the fecal microbiota was tested by the ponent, abolished rhythmicity in the fecal microbiota composition JTK_CYCLE algorithm (21). The fecal bacterial load, as measured in both genders. Bmal1 deletion also induced alterations in bacte- by the total 16S rRNA gene copy numbers, oscillated diurnally rial abundances in feces, with differential effects based on sex. during the light–dark cycle both in the group as a whole (Fig. 1A) Thus, although host behavior, such as time of feeding, is of recog- andwhensegregatedbysex(Fig.1B). The bacterial load peaked nized importance, here we show that sex interacts with the host around 11:00 PM and gradually decreased toward the late dark circadian clock, and they collectively shape the circadian rhythmicity phase until the lowest level was noted around 7:00 AM when the and composition of the fecal microbiota in mice. light was on. Male mice had more bacteria overall in feces than female mice; however, female mice showed more significant MICROBIOLOGY microbiome | Bmal1 gene | circadian rhythm | gender differences diurnal oscillation (P = 2.8E-06) than male mice (P = 0.032). The relative abundances of Bacteroidetes and Firmicutes, the two most he composition of intestinal microbiota is influenced by host abundant components of mammalian microbiota, varied during the Tgenetics (1), aging (2), antibiotic exposure (3), lifestyle (4), light–dark cycle (Fig. 1C). The average abundance of Bacteroidetes diet (5), pet ownership (6), and concomitant disease (7, 8). The washigherat11:00PM(66%)and11:00AM(60%)andlowerat impact of diet in shaping the composition of the microbiota has other times, whereas that of Firmicutes was higher at 3:00 AM been well established in both humans and mice (9, 10). The type (45%) and 7:00 AM (45%) and lowest at 11:00 PM (29%). of food consumed and also the feeding behavior of the host in- The microbiota composition can be analyzed by relative fluence the microbiota. For example, a 24-h fast increases the abundance or by absolute abundance. The former is commonly abundance of Bacteroidetes and reduces that of Firmicutes in used, yet it may exaggerate or mask the actual microbial be- mouse cecum, without altering the communal microbial diversity havior. For these reasons, we present analysis of both. We ob- (11). Bacteroidetes are also dominant in the microbiota of the served diurnal oscillations in fecal microbial composition at the fasted Burmese python, whereas ingestion of a meal shifts the phylum level (Fig. 1 D–I). Both the relative abundance and the intestinal composition toward Firmicutes (12). inferred absolute abundance of Bacteroidetes oscillate during The rotation of the earth results in the oscillation of light during the light–dark cycle in mice of combined genders (Fig. 1D), as the 24-h cycle. Organisms adapted to this cycle by developing a circadian rhythm, an endogenous and entrainable mechanism that Significance times daily events such as feeding, temperature, sleep-wakeful- ness, hormone secretion, and metabolic homeostasis (13, 14). In Recent studies of the murine microbiome have revealed circadian mammals, this rhythm is controlled by a master clock that resides behavior and linked it to host feeding time, but the mechanism in the suprachiasmatic nucleus of the hypothalamus. It responds to responsible for rhythmicity has not been fully clarified. Here we the changing light cycle and signals this information to peripheral report that both the host circadian system and host gender in- clocks in most tissues (15). The core mammalian clock is com- fluence the rhythmicity of the total load and taxonomic abun- prised of activators BMAL1 and CLOCK as well as repressors dances in the fecal microbiota, and that regulation by the host PERIOD (PER) and CRYPTOCHROME (CRY), forming an clock is dominant. Disruption of the host circadian clock by de- Bmal1 interlocked regulatory loop (14). letion of altered the fecal microbial composition in a Circadian rhythms also exist in fungi and cyanobacteria (16). gender-dependent fashion. Our analyses suggest the need to consider circadian factors and host gender in the design of For example, a pacemaker in cyanobacteria transduces the oscil- microbiome studies, and highlight the importance of analyzing lating daylight signal to regulate gene expression and to time cell absolute abundance in understanding the microbiota. division (17, 18). Hence, the synchronization of endogenous cir- cadian rhythms with the environment is crucial for the survival of Author contributions: X.L., F.D.B., and G.A.F. designed research; X.L. performed research; the bacteria as well as metazoa. X.L., F.D.B., and G.A.F. analyzed data; and X.L. and G.A.F. wrote the paper. Recent studies show that the intestinal microbiota undergo di- The authors declare no conflict of interest. urnal oscillation under the control of host feeding time, and that This article is a PNAS Direct Submission. ablation of the host molecular clock Per genescausesdysbiosis(19, Freely available online through the PNAS open access option. 20). Here, we report that microbial composition and its oscillation 1To whom correspondence should be addressed. Email: [email protected]. Bmal1 are influenced by the host clock, including the -dependent This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. forward limb of the signaling pathway.Wealsofindthatrhythmicity 1073/pnas.1501305112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1501305112 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 ABFecal Bacterial Load Fecal Bacterial Load C 100% Actinobacteria 10 13 10 13 29% 45% 45% 35% 37% 36% Bacteroidetes 80% Cyanobacteria 60% Deferribacteres 10 12 10 12 Firmicutes 40% 66% 50% 49% 60% 59% 59% Fusobacteria Proteobacteria 20% 10 11 10 11 M F TM7 16S Copies ( /g feces) m m m m m 16S Copies ( /g feces) pm a pm p a p m m m m m 0% Tenericutes 1 7 3 1 7 3 1p p am p p a pm p 1 1 1 1 7 3 1 7 3 1 11pm 3am 7am 11am 3pm 7pm 1 1 1 Other Inferred Absolute Abundance Inferred Absolute Abundance Bacteroidetes Bacteroidetes (16S copies/g feces) D (16S copies/g feces) E 100 10 13 100 10 13 80 80 60 60 10 12 10 12 40 40 20 20 0 10 11 0 M F M F 10 11 Relative Abundance (%) Relative Abundance (%) m m m m m m m m m m m m m m m p am pm p am pm p p am pm a p p a pm a p p am p a pm 1 7 3 1 7 3 1 1 7 3 1p 7 3 1 1p 7 3 1pm 7 3 1pm 1 7 3 1pm 7 3 1pm 1 1 1 1 1 1 1 1 1 1 1 1 Inferred Absolute Abundance Inferred Absolute Abundance Firmicutes Firmicutes (16S copies/g feces) F (16S copies/g feces) G 80 10 13 80 10 13 60 60 40 10 12 40 10 12 20 20 0 10 11 0 M F M F 10 11 Relative Abundance (%) Relative Abundance (%) m m m m m m m m m m m m pm am p p am p pm p am p a p pm am pm pm am pm am pm p a p p 1 7 3 1 7 3 1 1 7 3 1p 7 3 1pm 1 7 3 1 7 3 1pm 1pm 7 3 1 7 3 1 1 1 1 1 1 1 1 1 1 1 1 1 Inferred Absolute Abundance Inferred Absolute Abundance (16S copies/g feces) H Proteobacteria (16S copies/g feces) I Proteobacteria 4 10 11 8 10 12 M F M F 3 6 10 11 2 10 10 4 10 10 1 2 0 10 9 0 10 9 Relative Abundance (%) Relative Abundance (%) m m m m m m m m m m m m m m m m m m m m am pm p a p pm p a p p am p pm a p p a p p p am p p a p pm 1pm 7 3 1 7 3 1 1 7 3 1 7 3 1 1p 7 3 1 7 3 1 1 7 3 1 7 3 1 1 1 1 1 1 1 1 1 1 1 1 1 Fig. 1. Diurnal oscillation of intestinal microbiota composition in C57BL/6 mice. (A and B) Fecal bacterial load oscillates diurnally in mice both when combined (A)and separated by sex (B), as indicated by 16S rRNA copy numbers normalized to sample weight.