YBRBI 1507 No.

of Pages 9, Model 5G
7 January 2010
ARTICLE IN PRESS

Brain, Behavior, and Immunity xxx (2010) xxx–xxx

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Contents lists available at ScienceDirect

Brain, Behavior, and Immunity

journal homepage: www.elsevier.com/locate/ybrbi

Photoperiod modulates gut bacteria composition in male Siberian hamsters

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2

3 (Phodopus sungorus)

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4 Michael T. Bailey a,b,*,1, James C. Walton c,1, Scot E. Dowd d, Zachary M. Weil c,2, Randy J. Nelson b,c
5 a
Section of Oral Biology, College of Dentistry, The Ohio State University, Columbus, OH 43210, USA
6 b
Institute for Behavioral Medicine Research, College of Medicine, The Ohio State University, Columbus, OH 43210, USA
7 c
Department of Neuroscience, The Ohio State University, Columbus, OH 43210, USA
8 d
Research and Testing Laboratory and Medical Biofilm Research Institute, Lubbock, TX 79407, USA

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9
a r t i c l e i n f o a b s t r a c t
1
2 1
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12 Article history: Seasonal changes in day length (i.e., photoperiod) provide animals with a reliable environmental cue to 25
13 Received 1 November 2009 determine time of year, and many physiological changes occur in laboratory animals simply by extending 26
14 Received in revised form 21 December 2009 or shortening day length. Male Siberian hamsters (Phodopus sungorus) housed in long summer-like day
D 27
15 Accepted 27 December 2009
lengths have significantly elevated body and fat masses compared to short-day hamsters. Because others 28
16 Available online xxxx
have demonstrated that the intestinal microbiota of humans and rodents promotes host adiposity, we 29
hypothesized that photoperiod-induced changes in body and fat masses could be associated with changes 30
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17 Keywords:
in the microbial composition in the intestines. We used bacterial tag-encoded FLX amplicon pyrose- 31
18 Seasonality
19 Day length
quencing (bTEFAP) to assess microbial diversity in the cecal contents of hamsters; long days significantly 32
20 Siberian hamsters increased the relative abundance of bacteria in the phylum Proteobacteria. This effect was primarily due 33
21 Metabolism to a significant increase in the abundance of the genus Citrobacter, with both the abundance of Proteobac- 34
22 Gut efficiency teria and Citrobacter spp. significantly correlated with body mass and with inguinal fat mass. In general, 35
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23 the abundance of the Firmicutes phylum was inversely associated with body mass. These data indicate 36
that the intestinal microbiota are responsive to changes in photoperiod and suggest that these changes 37
may in part influence photoperiodic changes in body and fat masses. 38
Ó 2010 Published by Elsevier Inc. 39

40
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41
42 1. Introduction is secreted in proportion to night length; thus, relatively long dura- 56
tion of melatonin signals short day lengths. Short days inhibit repro- 57
43 Individuals of nontropical animal species are exposed to seasonal duction, bolster certain features of immune function, increase gut 58
44 changes in food availability and energy expenditures. Reduced food efficiency, reduce metabolic rate, and alter body mass (Bartness 59
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45 availability often coincides with energetic requirements for thermo- and Wade, 1984; Dark and Zucker, 1984; Drazen et al., 2001; Mercer 60
46 genesis and therefore winter is a particularly difficult time for small et al., 2000). 61
47 animals to breed and survive. To cope with this energetic challenge, It is common for many species to gain body fat prior to the win- 62
48 several seasonal adaptations have evolved including adjustments in ter and then slowly utilize those stored fuels while environmental 63
49 reproduction, immune function, and metabolism (Atcha et al., 2000; conditions are harsh, however, other species such as the Siberian 64
50 Bartness, 2002; Nelson, 2004; Wade and Bartness, 1984). The timing hamsters (Phodopus sungorus), utilize a different strategy. These 65
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51 of the onset of many seasonal adaptations is provoked by the annual animals gain body mass to support the energetically costly pro- 66
52 change in daily photoperiod (day length) (Goldman, 2001). In mam- cesses associated with reproduction in long days and then reduce 67
53 mals, day length information is transduced from an environmental body mass and particularly body fat prior to winter and run their 68
54 factor into a physiological signal by the nightly duration of elevated metabolic systems at a much lower rate (Dark and Zucker, 1984; 69
55 melatonin secretion (Lincoln et al., 2006; Lincoln, 2006). Melatonin Heldmaier et al., 1981; Wade and Bartness, 1984). Several central 70
and peripheral mechanisms coordinate this autumnal transition 71
into a lean phenotype including alterations in neuropeptide sys- 72
* Corresponding author. Address: Section of Oral Biology, College of Dentistry, tems such as leptin and agouti-related peptide that regulate feed- 73
The Ohio State University, 305W. 12th AVE., Columbus, OH 43210, USA. ing and alterations in autonomic regulation of adipocyte lipolysis 74
E-mail address: bailey.494@osu.edu (M.T. Bailey). (Bowers et al., 2005; Demas and Bartness, 2001; Mercer et al., 75
1
Both authors contributed equally to this work.
2
2000; Weil et al., 2009). Additionally, some studies have reported 76
Present address: Laboratory of Neurobiology and Behavior and Laboratory of
Neuroendocrinology, Rockefeller University, 1230 York Avenue, New York, NY 10021,
short day-induced reductions in food intake in Siberian hamsters, 77
USA. whereas others have reported that the weight loss is independent 78

0889-1591/$ - see front matter Ó 2010 Published by Elsevier Inc.

doi:10.1016/j.bbi.2009.12.010

Please cite this article in press as: Bailey, M.T., et al. Photoperiod modulates gut bacteria composition in male Siberian hamsters (Phodopus sungorus). Brain
Behav. Immun. (2010), doi:10.1016/j.bbi.2009.12.010
 YBRBI 1507 No. of Pages 9, Model 5G
7 January 2010
ARTICLE IN PRESS

2 M.T. Bailey et al. / Brain, Behavior, and Immunity xxx (2010) xxx–xxx

79 of food intake (Jethwa et al., 2009; McElroy et al., 1986) or the experiment. All procedures were approved by the Ohio State Uni- 132
80 weight loss precedes changes in food intake (Wade and Bartness, versity Institutional Animal Care and Use Committee and are in 133
81 1984). compliance with guidelines established by the National Institutes 134
82 Siberian hamsters, in common with all vertebrates exist in com- of Health published in Guide for the Care and Use of Laboratory Ani- 135
83 mensal harmony with many microbial organisms in residence in mals (1996). 136
84 their guts. The presence of these microbes benefits the host spe-
85 cies, at least in part, because these organisms express glycoside 2.2. Food intake 137
86 hydrolases and polysaccharide lysases, that are not expressed in
87 mammals, and thus allow the energetic utilization of otherwise After 8 weeks in photoperiod, each animal was weighed and to- 138
88 indigestible polysaccharides (Sonnenburg et al., 2005; Xu et al., tal available food in the cage was weighed. Food was weighed daily 139
89 2003). Therefore the composition and activity of gut microbial 3 h prior to the dark phase for 7 days. 140
90 populations can alter the energetic yield of a diet. Indeed, mount-

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91 ing evidence suggests that the relative abundance of different 2.3. Sample collection 141
92 microbial genera is associated with obese phenotypes in humans

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93 and animal models and the existence of extensive coevolution be- After 9 weeks in a long or short photoperiod, 3 h prior to the on- 142
94 tween gut microbes and their hosts (Ley et al., 2006). Germ-free set of the dark phase, hamsters were deeply anesthetized with iso- 143
95 mice are resistant to diet induced obesity and when inoculated flurane vapors and killed by rapid decapitation. Reproductive 144
96 with a normal gut microbiome increase body mass rapidly without tissues, abdominal fat stores, spleen, and adrenals were collected 145
97 increasing food intake (Backhed et al., 2004; Backhed et al., 2007). and weighed. Using sterile technique, the cecum was removed 146
98 Leptin deficient mice (OB/OB) also exhibit a shift in the ratio of Fer- and the contents were flash frozen in sterile microfuge tubes on 147

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99 micutes to Bacteroidetes as assessed by shotgun sequencing and dry ice and held at 80 °C for microbial diversity sequencing. 148
100 transplantation of the gut microbiome from OB/OB mice to lean
101 wildtype conspecifics leads to the development of an obese pheno- 2.4. Microbial diversity assessment with bacterial tag-encoded FLX 149
102 type (Ley et al., 2005; Turnbaugh et al., 2006). In humans, obesity is amplicon pyrosequencing (bTEFAP) 150
103 characterized by a reduction in bacterial diversity, altered repre-
104 sentation of bacterial metabolism related genes, and the same shift D 2.4.1. DNA extraction 151
105 in Fermicutes to Bacteroidetes ratio (Ley et al., 2008). Total genomic DNA was extracted from the cecal samples as de- 152
106 Siberian hamsters undergo seasonal changes in body mass. Be- scribed previously (Dowd et al., 2008) using a Tissuelyser and 153
107 cause gut microbiota affect body mass, we hypothesized that pho- using a QIAmp stool DNA mini kit per manufacturer directions 154
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108 toperiod might affect body mass by adjusting the composition of (Qiagen, Velencia, CA). DNA samples were quantified using a Nano- 155
109 the gut bacterial contents. If true, we predicted that the commu- drop spectrophotometer (Nyxor Biotech, Paris, France). 156
110 nity structure of the intestinal microbiota would be significantly
111 different as a function of photoperiod, and that microbiota abun- 2.4.2. PCR to create tag encoded amplicons 157
112 dance would be associated with body and fat mass. We used bac- A 100 ng aliquot of each sample’s DNA was used for a 50 ll step 1 158
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113 terial tag-encoded FLX amplicon pyrosequencing (bTEFAP) to PCR reaction. Bacterial tag-encoded FLX amplicon pyrosequencing 159
114 characterize the microbiota in the cecal contents of hamsters to (bTEFAP) was performed as described previously (Dowd et al., 160
115 test the hypothesis that photoperiod changes the composition of 2008) at the Research and Testing Laboratory (Lubbock, TX). The 161
116 the intestinal microbiota. new bacterial tag-encoded FLX-Titanium amplicon pyrosequencing 162
(bTEFAP) approach is based upon similar principles but utilizes Tita- 163
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117 2. Methods and materials nium reagents and Titanium procedures including a one-step PCR, 164
mixture of Hot Start and HotStar high fidelity taq polymerases, 165
118 2.1. Animals and amplicons originating from the 27F region numbered in relation 166
to E. coli rRNA. 167
119 Siberian hamsters (Phodopus sungorus) were bred in our colony
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120 maintained at the Ohio State University. Twenty male hamsters 2.4.3. Bacterial diversity data analysis 168
121 were weaned at 21 days of age and singe housed in polycarbonate Following sequencing, all failed sequence reads, low quality se- 169
122 cages (28  17  12 cm) on free-standing racks until they reached quence ends and tags were removed and sequences were depleted 170
123 sexual maturity (>60 days). Six weeks post-weaning, animals were of any non-bacteria ribosome sequences and chimeras using cus- 171
124 randomly assigned to either long-day (n = 10, LD; 16L:8D) or short- tom software described previously (Dowd et al., 2008) and the 172
125 day (n = 10, SD; 8L:16D) conditions (light intensity 600 lux) (see Black Box Chimera Check software B2C2 (described and freely 173
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126 Table 1 for experimental timeline). All animals were maintained at available at http://www.researchandtesting.com/B2C2.html). Se- 174
127 constant temperature (21 ± 4 °C), relative humidity (50 ± 5%), and quences less than 350 bp were removed. To determine the identity 175
128 given ad libitum access to filtered tap water and food (Harlan Tek- of bacteria in the remaining sequences, sequences were first que- 176
129 lad 8640, Indianapolis, IN, USA), and were cared for by The Ohio ried using a distributed BLASTn.NET algorithm (Dowd et al., 177
130 State University Laboratory Animal Resource staff in accordance 2005) against a database of high quality 16s bacterial sequences 178
131 with USDA animal welfare regulations for the duration of the derived from NCBI. Database sequences were characterized as high 179

Table 1
Timeline of experimental procedures.

Weaning In photoperiod Food intake Samples taken

3 weeks 9 weeks 17 weeks 18 weeks

Experimental manipulation (above) with corresponding age of each animal at time of manipulation (below).

Please cite this article in press as: Bailey, M.T., et al. Photoperiod modulates gut bacteria composition in male Siberian hamsters (Phodopus sungorus). Brain
Behav. Immun. (2010), doi:10.1016/j.bbi.2009.12.010
 YBRBI 1507 No. of Pages 9, Model 5G
7 January 2010
ARTICLE IN PRESS

M.T. Bailey et al. / Brain, Behavior, and Immunity xxx (2010) xxx–xxx 3

180 quality based upon the criteria of RDP ver 9 (Cole et al., 2005). of bacteria in the phylum Proteobacteria compared to the abun- 240
181 Using a .NET and C# analysis pipeline, the resulting BLASTn out- dance found in the hamsters exposed to long day lengths 241
182 puts were compiled, validated using taxonomic distance methods, (t(18) = 3.13, p < 0.01; Fig. 3B). This effect of photoperiod did not 242
183 and data reduction analysis performed as described previously appear to be due to a significant extinction or bloom of specific 243
184 (Acosta-Martinez et al., 2008). Rarefaction, ace, and Chao1 to esti- species of bacteria because evaluation of diversity and richness 244
185 mate mathematically predicted diversity and richness in the treat- estimates based upon rarefaction, chao1, and ace methods, as well 245
186 ments using 350 bp trimmed, non-ribosomal sequenced depleted, as the use of curve fitting equations to predict maximum potential 246
187 chimera depleted, high quality reads was performed as described operational taxonomic units (Acosta-Martinez et al., 2008) did not 247
188 previously (Acosta-Martinez et al., 2008). find any significant differences between samples from long- and 248
short-day hamsters (Table 2). Although the change in the Proteo- 249

189 2.4.4. Bacterial identification bacteria was the only difference that was evident at the phylum 250
level, Fig. 3D provides a summary of 6 genera that differed be- 251

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190 Based upon the above BLASTn derived sequence identity (per-
191 cent of total length query sequence which aligns with a given data- tween the long- and short-day hamsters. The clustering analysis 252

192 base sequence) and validation using taxonomic distance methods, indicated that overall, microbial populations within 8 of the 10 253

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193 the bacteria were classified at the appropriate taxonomic levels short-day hamsters were more similar to each other than they 254

194 based upon the following criteria. Sequences with identity scores were to microbial populations found in the long-day hamsters. 255

195 to known or well characterized 16S sequences greater than 96.5% The primary population leading to this clustering was Citrobacter 256

196 identity (i.e., <3% dissimilarity) were resolved at the species level sp., which was found to be significantly lower in the short-day 257

197 when possible, between 94.5% and 96.4% at the genus level, be- hamsters compared to the long-day hamsters (Fig. 3C). 258

198 tween 89.5% and 94.4% at the family and between 80% and 89.4% The relative abundance of bacteria in the Proteobacteria phy- 259

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199 at the order level. After resolving based upon these parameters, lum were positively correlated with hamster body mass 260

200 the percentage of each bacterial identification was individually ana- (r(18) = 0.55, p < .05; Fig. 4A);. This association was most likely 261

201 lyzed for each cecal sample providing relative abundance informa- due to the positive association between Proteobacteria and ingui- 262

202 tion within and among the samples based upon relative numbers nal fat mass (r(18) = 0.509, p < .05; Fig. 4B). The effects at the 263

203 of reads within a given sample. When multiple identifications were phylum level could be traced down to the genus level where it 264

204 found (e.g., Bacteroides caccae and Bacteroides stercoris) such identi- was evident that the relative abundance of Citrobacter was also 265

205
206
fications were resolved arbitrarily to the top hit. Evaluations
presented at a given taxonomic level, except species level, represent
Dassociated with body mass (r(18) = 0.468, p < .05; Fig. 4C), but was
only moderately associated with inguinal fat mass (r(18) = 0.415,
266
267

207 all sequences resolved to their primary genera identification or their p = .08, Fig. 4D). 268
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208 closest relative (where indicated). Although photoperiod did not significantly change the relative 269
abundance of bacteria in the phylum Firmicutes (t(18) = 1.42, 270
p = .17; see Fig. 3A), the relative abundance of Firmicutes was in- 271
209 2.5. Statistical analyses
versely associated with body mass (r(18) = 0.564, p < .01, Fig. 4E) 272
and with inguinal fat mass (r(18) = 0.489, p < .05; Fig. 4F). In this 273
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210 Independent-samples t tests were used to compare food intake,

case, animals that had higher levels of Firmicutes tended to have 274
211 reproductive tissues, spleen, and adrenal mass between treatment
a lower body mass. There was a trend for the relative abundance 275
212 groups. Spleen, paired adrenal mass, and food intake data were
of bacteria within the genus Lachnobacterium, which belongs to 276
213 corrected for body mass. Clustering methods based upon weighted
the Firmicutes phylum, to be increased in the hamsters exposed 277
214 pairs, Manhattan distances and standard average deviation scaling
to short day length (t(18) = 1.78, p = .09; data not shown). The 278
215 were performed as described previously (Dowd et al., 2008). Inde-
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relative abundance of Lachnobacterium sp. was low (0.12 ± 0.03% 279

216 pendent-samples t tests were also used to compare the relative
for LD hamsters and 0.20 ± 0.03% for SD hamsters), but the inverse 280
217 abundance of specific phyla, families, and genera of bacteria be-
association between the Firmicutes phylum and body mass could 281
218 tween the long day and short-day hamsters. All mean differences
be traced to this genus of bacteria. The relative abundance of 282
219 were considered statistically significant at p < 0.05.
Lachnobacterium sp. was inversely associated with body mass 283
(r(18) = 0.583, p < .01; Fig. 4G) and with inguinal fat mass (r(18) = 284
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220 3. Results 0.521, p < .05; Fig. 4H). 285

221 In response to short days, hamsters reduced body mass

222 (t(18) = 4.71, p < 0.001; Fig. 1A) and reduced inguinal fat pad mass 4. Discussion 286
223 (t(18) = 8.67, p < 0.001; Fig. 1B). Additionally, hamsters maintained
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224 in short days showed regression in mass of the reproductive tis- As previously demonstrated, photoperiod resulted in several 287
225 sues; paired testes (t(18) = 15.84, p < 0.001; Fig. 1C), seminal vesi- phenotypic changes (Navara et al., 2007; Weil et al., 2007). In com- 288
226 cles (t(18) = 6.55, p < 0.001; Fig. 1D), and epididymides (t(18) = 7.96, parison to hamsters in the long day photoperiod, the body, inguinal 289
227 p < 0.001; Fig. 1D). Exposure to short days had no effect on cor- fat, epididymides, seminal vesicles, and testes masses were signif- 290
228 rected paired adrenal mass (t(18) = 0.07, p = 0.987; Fig. 1E) or cor- icantly reduced by a short photoperiod. This reduction in mass was 291
229 rected spleen mass (t(18) = 0.49, p = 0.627; Fig. 1F). Short days organ specific and not widespread across the body because the 292
230 had no effect on food intake (t(18) = 0.88, p = 0.39; not shown) or mass of the spleen and the adrenal glands were not significantly 293
231 food intake corrected for body mass (t(18) = 1.57, p = 0.14; Fig. 2). affected by photoperiod. Moreover, the reduction in body and fat 294
232 The microbiota in the cecal contents of hamsters was comprised masses were not due to a change in diet, because long- and 295
233 of bacteria from 7 phyla, with the highest proportion of bacteria short-day hamsters consumed the same amount of standard labo- 296
234 belonging in the Firmicutes phylum, followed by the Bacteroidetes, ratory chow. Thus, it is evident that the enhanced masses were due 297
235 and Proteobacteria (Fig. 3A). Less than 1% of the bacteria belonged to a difference in the ability of the hamsters to harvest energy from 298
236 in the phyla Actinobacteria, Tenericutes, Verrucomicrobia, and the consumed food. 299
237 Spirochaetes (Fig. 3A). We did not detect any bacteria in the phy- Several studies have now demonstrated that different commu- 300
238 lum Deferribacteres or Fusobacterium. Exposure to short day nity profiles of intestinal microbiota also have differing capacities 301
239 lengths caused a significant reduction in the relative abundance to harvest energy from food. This appears to be due to an increase 302

Please cite this article in press as: Bailey, M.T., et al. Photoperiod modulates gut bacteria composition in male Siberian hamsters (Phodopus sungorus). Brain
Behav. Immun. (2010), doi:10.1016/j.bbi.2009.12.010
 YBRBI 1507 No. of Pages 9, Model 5G
7 January 2010
ARTICLE IN PRESS

4 M.T. Bailey et al. / Brain, Behavior, and Immunity xxx (2010) xxx–xxx

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Fig. 1. Photoperiodic responses to short day lengths in Siberian hamsters. Short days reduced overall body mass (A), inguinal fat mass (B), paired testes mass (C), paired
epididymal, and paired seminal vesicle mass (D). Short day lengths had no effect on corrected paired adrenal mass (E) or corrected spleen mass (F). All graphs reported as
mean ± 1 standard error of the mean (SEM); LD = long-day (n = 10), SD = short-day (n = 10), *p < 0.05.

hamsters housed in long day lengths having a higher abundance 308

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than hamsters housed in short day lengths. This increase was asso- 309
ciated with body mass; hamsters with more Proteobacteria had 310
elevated body and fat masses. Most studies in humans and in ro- 311
dents report that increased body mass is associated with a signifi- 312
cant increase in the relative abundance of bacteria in the phylum 313
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Firmicutes, with a concomitant decrease in the relative abundance 314

of Bacteroidetes (Ley et al., 2005, 2006; Turnbaugh et al., 2006; 315
Turnbaugh et al., 2009). However, other studies have reported a 316
significant increase in the relative abundance of the Phylum Prote- 317
obacteria. For example, mice fed a high fat diet to induce obesity 318
significantly increased Proteobacteria and Actinobacteria and in- 319
Fig. 2. Photoperiodic changes in food consumption in Siberian hamsters. Short days creased Firmicutes and decreased Bacteroidetes (Hildebrandt 320
had no effect on food consumption corrected for body mass after 8 weeks in et al., 2009). Our finding of an increased abundance of Proteobac- 321
photoperiod. Mean ± 1 standard error of the mean; LD = long-day (n = 10), teria as a consequence of long day length, and a significant corre- 322
SD = short-day (n = 10).
lation with body and fat masses, suggests that the Proteobacteria 323
are involved with photoperiod-induced increases in body mass. 324
303 in factors such as glucose absorption and fatty acid production and It was unexpected that photoperiod did not significantly affect 325
304 absorption (Backhed et al., 2004). In our study, photoperiod re- the relative abundance of the Firmicutes, because previous reports 326
305 sulted in significant differences in the composition of the intestinal have linked the relative abundance of the Firmicutes to obesity in 327
306 microbiota. This was primarily manifest as a difference in the rel- humans and in rodents (Ley et al., 2005; Ley et al., 2006; Turnb- 328
307 ative abundance of bacteria in the Phylum Proteobacteria, with augh et al., 2006; Turnbaugh et al., 2009). Changes in Proteobacte- 329

Please cite this article in press as: Bailey, M.T., et al. Photoperiod modulates gut bacteria composition in male Siberian hamsters (Phodopus sungorus). Brain
Behav. Immun. (2010), doi:10.1016/j.bbi.2009.12.010
 YBRBI 1507 No. of Pages 9, Model 5G
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M.T. Bailey et al. / Brain, Behavior, and Immunity xxx (2010) xxx–xxx 5

A. Phyla B. Proteobacteria C. Citrobacter sp.

100 12 LD n=10 10 LD n=10
SD n=10
95 10
8
*

% of Sequences
% of Sequences

% of Sequences
*
90 Firmicutes
Bacteoidetes 8
85
Proteobacteria 6
Actinobacteria
Tenericutes 6
80 Verrucomicrobia
4
Spirochaetes
4
75
2
10 2
5
0 0 0

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Long Day Short Day Long Day Short Day Long Day Short Day

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D. Double Clustering Dendogram at the Genus Level
Relative Abundance (%
of Total Sequences) Sample Similarity

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Parabacteroides sp.

Candidatus sp.
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Oribacterium sp.
Genus

Citrobacter sp.
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Clostridium sp.
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Papillibacter sp.

Animal Identifcation
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(LD = Long Day, SD = Short Day)

Fig. 3. Photoperiodic changes in the composition of the cecal microbiota. (A) Exposure to short day lengths changed the relative abundance of bacteria in specific phyla
presented as a percentage of total sequences derived from the cecal contents of n = 10 long-day and n = 10 short- day hamsters. (B) Exposure to short day lengths resulted in a
significant reduction in the relative abundance of bacteria within the phylum Proteobacteria. Data are depicted as the mean ± SEM of the % of total sequences identified.
*p < .05. (C) Exposure to short day lengths resulted in a significant reduction in the relative abundance of bacteria with the genus Citrobacter. Data are the mean ± SEM of the %
of total sequences identified. *p < .05. (D) A double dendogram showing genera of bacteria that differ between long-day and short-day hamsters. The heat map shows the
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relative abundance of the given genera within each sample with a color legend and scale provided; distance of the individual samples is based upon weighted pair linkage and
Manhattan distance methods. The clustering analysis indicated that overall, microbial populations within short-day hamsters were more similar to each other than they were
to microbial populations found in long-day hamsters. The bacterial genera and the associated clustering are provided along the Y-axis and their associated distance scores
indicated. Data are from the cecal contents of n = 10 short-day and n = 10 long-day hamsters.

330 ria, however, may be more relevant for seasonal changes in ham- diet (Hildebrandt et al., 2009), thus an increase in Proteobacteria 340
331 sters in natural settings, because during summer months, hamsters would be expected to occur during the summer when high fat 341
332 prefer to eat seeds that are high in fat and protein content (Fine seeds are available. How photoperiodic changes in Proteobacteria 342
333 and Bartness, 1996). This preference for eating high fat seeds occur in hamsters ingesting comparable amounts of a standardized 343
334 may not be simply due to the fact that seeds are more abundant diet remains unspecified, but may involve changes to gastrointes- 344
335 in the summer, because hamsters housed in long summer-like tinal functioning. 345
336 day lengths in the laboratory also show an increased preference Gastrointestinal functioning changes in many mammalian spe- 346
337 for high fat diets in comparison to hamsters housed in short win- cies during different seasons, presumably to maintain energy 347
338 ter-like day lengths (Fine and Bartness, 1996). Blooms of bacteria requirements in the face of seasonal variations in food abundance 348
339 in the phylum Proteobacteria occur when mice are fed a high fat and quality. In general, many species, including Cavies (Microcavia 349

Please cite this article in press as: Bailey, M.T., et al. Photoperiod modulates gut bacteria composition in male Siberian hamsters (Phodopus sungorus). Brain
Behav. Immun. (2010), doi:10.1016/j.bbi.2009.12.010
 YBRBI 1507 No. of Pages 9, Model 5G
7 January 2010
ARTICLE IN PRESS

6 M.T. Bailey et al. / Brain, Behavior, and Immunity xxx (2010) xxx–xxx

Table 2 Physical activity influences microbiota composition in both hu- 370

Photoperiod does not impact microbial diversity or richness. mans and laboratory animals (Matsumoto et al., 2008; Santacruz 371
Rarefaction OTU et al., 2009). Although we did not measure physical activity in 372

5% 3% 1% 5% 3% 1% the current study, previous studies in our laboratory have demon- 373
strated that photoperiod influences locomotor activity in Siberian 374
Long day 328 ± 8 415 ± 10 587 ± 15 334 ± 8 424 ± 11 601 ± 15
Short 323 ± 8 412 ± 13 584 ± 23 326 ± 7 417 ± 12 592 ± 21
hamsters (Weil et al., 2009). In general, hamsters housed under 375
day long summer-like day lengths are more active than hamsters 376
ACE Chao housed in short summer-like day lengths. These differences are 377
5% 3% 1% 5% 3% 1% the most pronounced during the early and late periods of the dark 378

Long day 395 ± 13 543 ± 20 883 ± 31 400 ± 13 551 ± 20 875 ± 26

cycle, with no differences in locomotor activity observed during 379

Short 397 ± 9 550 ± 18 884 ± 41 399 ± 9 540 ± 17 864 ± 39 the light cycle (Weil et al., 2009). Although the mechanisms 380
day through which physical activity influences the microbiota are not 381

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known, it is possible that the association involves the autonomic 382
Mathematical evaluation of diversity and richness estimates based upon rarefac-
tion, chao, ace, and operational taxonomic units methodology indicated that pho- nervous system which is affected by photoperiod (Weil et al., 383

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toperiod did not affect any estimate of diversity or richness. The data show the 2009). 384
calculated richness and diversity from all the samples pooled together, thus it is not Many endocrine systems are affected by alterations in photope- 385
possible to calculate variability.
riod, with the most widely studied hormone being melatonin. This 386
hormone is produced by the pineal gland, which receives photope- 387
350 australis) (Sassi et al., 2007), marmots (Marmota marmota) (Hume riodic information from the retina through the actions of the 388
351 et al., 2002), and prairie voles (Microtos ochrogaster) (Gross et al., suprachiasmatic nucleus. Because melatonin production occurs 389

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352 1985) have significantly increased masses of their digestive organs during the dark, melatonin levels are higher in short days than dur- 390
353 during periods of scarce food or low quality food to maximize the ing long days. Melatonin is also produced in the gastrointestinal 391
354 intestinal surface area on which digestion and/or fermentation of tract in levels that far exceed levels produced by the pineal gland 392
355 foods and absorption of nutrients occurs (Green and Millar, (Bubenik, 2002). Although a direct impact of melatonin on bacte- 393
356 1987; Toloza et al., 1991). But, these changes in gastrointestinal rial growth has not been clearly demonstrated in vitro, it is possible 394
357 functioning or morphology are also likely to influence intestinal that photoperiodic changes in other hormones directly affect 395
358 microbial profiles. For example, in hibernating species, such as
359 13-lined ground squirrels (Spermophilus tridecemlineatus), signifi-
D
microbial populations. There is growing evidence that bacteria 396
can sense and respond to neurotransmitters and hormones in their 397
360 cant changes in gastrointestinal tract morphology occur during immediate environment (Clarke and Sperandio, 2005; Lyte, 2004; 398
TE
361 hibernation with a concomitant reduction in viable microbiota Lyte and Bailey, 1997). The most well-studied neuroendocrine-bac- 399
362 (Barnes, 1970; Barnes and Burton, 1970). A single study in Soay terial interactions involve growth enhancement by exposure to 400
363 sheep has investigated the impact of photoperiod on the microbi- catecholamine hormones, namely epinephrine, norepinephrine, 401
364 ota; short chain fatty acids and overall microbial diversity changed and dopamine, through iron-dependent and independent mecha- 402
365 as a function of photoperiod. However, this effect could reflect nisms (Freestone et al., 2007; Hughes et al., 2009; Pacheco and 403
EC

366 changes in food consumption because sheep exposed to long day Sperandio, 2009; Sandrini et al., 2009). Moreover, there is a grow- 404
367 lengths consumed more food (McEwan et al., 2005). In our study, ing appreciation that many different species of bacteria produce a 405
368 the microbiota were affected by photoperiod even though the wide range of molecules with striking similarity to mammalian 406
369 hamsters consumed the same amounts of chow. hormones, such as estrogen, thyroid stimulating hormone, insulin, 407
RR

A. Proteobacteria vs. Body Mass B. Proteobacteria vs. Inguinal Fat Mass C. Citrobactersp. vs. Body Mass D. Citrobactersp. vs. Inguinal Fat Mass
Inguinal Fat Mass (mg)
Inguinal Fat Mass (mg)

45 1400 45 1400
Body Mass (g)

40 1200 40 1200
Body Mass (g)

1000 1000
35 35
800 r =0.259, p<.05 800 r =.17, p=.08
30 30
CO

600 600
25 400 25 400
20 r =0.305, p<.05 200 20 200
r =0.22, p<.05
0 0 0 0
2 4 6 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 2 4 6 8 10 12
% of Sequences % of Sequences % of Sequences % of Sequences
UN

E. Firmicutes vs. Body Mass F. Firmicutes vs. Inguinal Fat Mass G.Lachnobacteriumsp. vs. Body Mass H. Lachnobacteriumsp. vs. Inguinal Fat Mass
Inguinal Fat Mass (mg)
Inguinal Fat Mass (mg)

45 1400 45 1400
Body Mass (g)

1200
Body Mass (g)

40 40 1200
1000 1000
35 800 35 r =.521, p<.05
800
30 600 30 600
400
25 25 400
200
r =.239, p<.05 200
20 r =0.318, p<.01 0 20 r =.340, p<.05
0 0 0
0 5 60 70 80 90 5 60 70 80 90 0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4 0.5
% of Sequences % of Sequences % of Sequences % of Sequences

Fig. 4. The relative abundance of bacterial phyla and genera are significantly correlated with body and fat masses. The relative abundance (expressed as a percentage of total
sequences) of bacteria in the phylum Proteobacteria is positively associated with body mass (A) and with (B) the mass of the inguinal fat pads. The relative abundance of
bacteria in the genus Citrobacter is positively associated with body mass (C). There was a trend of the relative abundance of bacteria in the genus Citrobacter to be positively
associated with inguinal fat mass (p = .08) (D). The relative abundance of bacteria in the phylum Firmicutes was inversely associated with body mass (E) and the mass of the
inguinal fat pads (F). The relative abundance of bacteria found to be in the genus Lachnobacterium was also inversely associated with body mass (G) and the mass of the
inguinal fat pads (H). In all cases, the relative abundance of bacteria in the cecum and the masses were collected from the same animals to allow for Pearson’s correlation. Data
are from n = 10 short-day and n = 10 long-day hamsters.

Please cite this article in press as: Bailey, M.T., et al. Photoperiod modulates gut bacteria composition in male Siberian hamsters (Phodopus sungorus). Brain
Behav. Immun. (2010), doi:10.1016/j.bbi.2009.12.010
 YBRBI 1507 No. of Pages 9, Model 5G
7 January 2010
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408 and b-endorphin (LeRoith et al., 1985; Roth et al., 1985). Bacteria able and predictable ways when housed with different day lengths, 474
409 are also responsive to these hormones, and have been found to this animal model will be a valuable tool in which to study associ- 475
410 contain hormone binding substances that resemble vertebrate- ations between host physiology and commensal gut microbes. 476
411 type receptors (Burshell et al., 1984; Josefsson and Johansson,
412 1979; Loose et al., 1983; Weiss et al., 1983). These hormones are
413 also significantly changed upon photoperiodic cues (Hanon et al., 5. Uncited reference 477

414 2008; Hanon et al., 2009; Helwig et al., 2006; Korhonen et al.,
415 2008; Roberts et al., 1985; Tups et al., 2006), and thus could have Fuentes et al. (2009).. Q1 478

416 direct influences on the microbiota. The likelihood of direct neuro-

417 endocrine-microbiota affects induced by changes in photoperiod Acknowledgments 479
418 will be the topics of future studies.
419 Beyond identifying a link between the microbiota and body

F
We thank Jeffrey Galley for technical assistance and the scien- 480
420 mass, this study has potential implications for other host systems. tists at Research and Testing Laboratory for their stellar efforts. 481
421 Photoperiod has a wide impact on host physiology, and it has been This project was supported by NSF Grants IOS 04-16897 and IOS 482

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422 well documented that immune responses of Siberian hamsters 08-38098. 483
423 (and other mammals) are affected by photoperiod (Nelson, 2004).
424 In general, long day length enhances innate inflammatory re-
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Behav. Immun. (2010), doi:10.1016/j.bbi.2009.12.010
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Please cite this article in press as: Bailey, M.T., et al. Photoperiod modulates gut bacteria composition in male Siberian hamsters (Phodopus sungorus). Brain
Behav. Immun. (2010), doi:10.1016/j.bbi.2009.12.010