Institute of Zoology and Department of Life Science, National Taiwan. University ... Animals. One Formosan field mouse (Apodemus semotus) and one Formosan.
MICROBIAL DIVERSITY IN INTESTINAL TRACTS OF TWO SPECIES OF RODENTS IN MONTANE AREAS IN TAIWAN Hsiao-Pei Lu and Hon-Tsen Yu Institute of Zoology and Department of Life Science, National Taiwan University, Taipei, Taiwan, ROC.
Abstract Microorganisms living in mammalian guts have important effects on immune function, nutrient processing and other host activities. We analyzed the bacterial communities in three sections of the digestive tracts, including small intestines, caeca and large intestines of two montane species of rodents (Apodemus semotus and Eothenomys melanogaster) from the central mountain ranges in Taiwan. We constructed 16S rDNA clone libraries and identified the sequences through database searches. At the phylum level, the intestinal microflorae of A. semotus were composed of Firmicutes (43.26%), Proteobacteria (42.23%), Deinococcus-Thermus (9.84%), Verrucomicrobia (2.33%), Cyanobacteria (1.81%) and Actinobacteria (0.52%). The intestinal microflorae of E. melanogaster were composed of Proteobacteria (40.68%), Firmicutes
(32.70%),
Bacteroidetes
(22.05%),
Spirochaetes
(1.90%),
Actinobacteria (1.52%), TM7 (0.76%) and Deferribacteres (0.38%). We also found that the bacterial composition percentages between three gut sections of the same individual differed from one another. Moreover, while the SPF (specific pathogen free) mouse and human microflorae were dominated by Firmicutes and Bacteroidetes, the mouse and the vole in this study, respectively, have a large group of Proteobacteria which comprises a huge number of environmental bacteria. This discrepancy suggests that wild rodents have specific immune tolerance for some of Proteobacteria and these microbes may own unique metabolic traits to colonize the gut. This research offers an opportunity to know the commensal host-bacterial relationships in the guts of wild rodents.
1. Introduction The animal gut is the one of the most densely populated ecosystems on Earth (Savage, 1977). There are up to 1014 bacteria in the human intestinal tract, which is more than the total number of tissue cells in the entire body. Microorganisms have been traditionally identified through characterization of their morphological and physiological traits (Savage, 1986). Although the endogenous gastrointestinal microflorae have been studied in great detail by culture techniques, most of them cannot be cultured with available media. Microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated (Suau et al., 1999). The endogenous gastrointestinal microflorae play an important role in health and disease, yet this ecosystem remains mostly uncharacterized and its diversity poorly defined (Ramakrishna, 2007). Because only a limited number of species could be identified by using traditional culturing methods, investigators have begun to explore this ecosystem by using molecular techniques, such as polymerase chain reaction (PCR) and sequence analysis of cloned microbial small-subunit ribosomal RNA genes (16S rDNA) (Wilson & Blitchington, 1996; Wintzingerode et al., 1997). Remarkably, microorganisms living in mammalian guts have important effects on immune function, nutrient processing and other host activities. Studying the normal gut microflorae can improve our understanding of hostmicrobe interactions. Most uncultivable species become detectable when we use molecular techniques to investigate intestinal microbial populations (Zoetendal et al., 2006). However, a comprehensive enumeration of the gut microflorae has not yet been reported for wild mice or voles, even though rodents provide a good model for exploring the intestinal microbial ecology. This research offers an opportunity to know the commensal host-bacterial relationships in the guts of wild rodents.
2. Materials and methods Animals One Formosan field mouse (Apodemus semotus) and one Formosan black-bellied vole (Eothenomys melanogaster) from the central mountain ranges in Taiwan were employed for the study. The mouse was collected from Mt. Ho-Huan and the vole was collected from Mt. Ali. The animals were sacrificed one day after trapped and gut samples were frozen immediately in liquid nitrogen. DNA extraction Total DNA was extracted from small intestine, large intestine and caecum of each individual, respectively. Tissue samples (ca. 1 g) were placed in Eppendorf tubes with 500 μl lysozyme buffer (25 mM tris-HCl, 10 mM EDTA, 50 mM glucose, pH 8) and 50 μg lysozyme (20000 units/mg), followed by incubation at 370C with occasional agitation for 3 hr. At the beginning of the third hour, 100 μg proteinase K (60 units/mg), 100 μg RNase A (60 units/mg) and 500 μl lysis buffer (50 mM tris-HCl, 10 mM EDTA, 1% SDS, 100 mM NaCl, pH 8) were added to each tube, followed by gentle inversion. Freezethaw cycles of incubation at -800C for 60 min followed by 600C for 30 min were performed three times to release microbial DNA. The cell lysate was extracted with equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), rotated for 10 min to obtain the well-mixed milky solution and centrifuged (12000 rpm) for 10 min at room temperature. The top aqueous layer was placed in a new Eppendorf tube and 1 ml cold 100% ethanol (EtOH) was added. The tube was inverted to precipitate DNA and centrifuged for 5 min at 12000 rpm to pellet DNA. The pellet was re-suspended with 1 ml cold 70% EtOH and centrifuged for 2 min at 12000 rpm. Repeat again and pour off the supernatant. The pellet was air dried overnight or vacuum dried for 5 min with a MicroVacTM MV-100. The DNA was dissolved in 50 μl distilled water and stored at -200C.
Bacterial 16S rDNA amplification A pair of primers 516 F (5' TGCCAGCAGCCGCGGTA 3') and 985 R (5' GTAAGGTTCTTCGCGTT 3') were used to amplify the bacterial 16S rDNA fragments (Nagashima et al., 2003). The PCR reaction mixture contained 100 ng DNA, 2.0 mM MgCl2, 0.4 mM dNTP, 0.4 µM each primer, 1X Ex TaqTM buffer and 1 U of Ex TaqTM polymerase (Takara). The thermal cycling conditions were initial denaturation at 940C for 3 min; followed by 25 cycles at 940C for 30 s, 560C for 30 s and 720C for 1 min; and final extension at 720C for 10 min. The concentration of amplified DNA was determined by electrophoresis on the 1% agarose gel (1 g agarose + 100 ml tris-Borate-EDTA buffer). Amplified products of the expected size (ca. 450 bp) were confirmed and excised from the gel to be recovered with QIAquick Gel Extraction Kits (QIAGEN). Cloning & Sequencing The purified products were ligated into the yT&A vector of the TA Cloning Kits (Yeastern) according to the manufacturer’s instructions. Ligated DNA was transformed into ECOS101 competent cells in the kits by heat shock at 42 0C for 45 s. The resulting clones were selected on Luria-Bertani medium with ampicillin (50 μg/ml), IPTG (isopropyl-β-D-thiogalactopyranoside) (0.1 M) and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (50 mg/ml). White colonies were picked from each clone libraries and screened for inserts by performing direct PCR using M13 forward and M13 reverse primers. Each insert DNA was obtained for sequencing with the TempliPhi DNA Amplification Kits (Amersham) following the manufacturer’s protocols. The partial fragments of 16S rDNA were sequenced with DY Enamic ET Dye Terminator Cycle Sequencing Kit (GE) and 109 aF primer. All reactions were done by a MegaBACE 1000 automated sequencer (GE).
Phylogenetic analysis Sequences were adjusted and trimmed with Sequencher program (Gene Code Corporation) to obtain sequences with high equal quality. Moreover, sequences were tested for possible chimeras with two online analyses, Bellerophon server (http://foo.maths.uq.edu.au/%7Ehuber/bellerophon.pl) and Chimera Check (http://35.8.164.52/cgis/chimera.cgi?su=SSU). Confirmed chimeric sequences were excluded from further analysis. Sequences were compared with those in the GenBank database by BLASTTM searches. Sequences with 99% similarity ( < 1% diversity ) were grouped into operational taxonomic units (OTUs). All sequences and their closest relatives were aligned with Clustal X program (Jeanmougin et al., 1998). Phylogenetic analysis was carried out using PAUP* 4.0 program (Swofford, 1993). The pairwise distances were analyzed with Kimura’s twoparameter correction (Kimura, 1980). The tree was generated from distance matrices by the neighbor-joining method. The stability of branches was checks by bootstrapping.
3. Results Phylogenetic analysis of the 16S rDNA sequences We obtained 386 sequences (89 sequences from the small intestine, 131 sequences from the caecum, and 166 sequences from the large intestine) from A. semotus and 263 sequences (93 sequences from the small intestine, 96 sequences from the caecum, and 74 sequences from the large intestine) from E. melanogaster in the final analysis. We used 99% similarity as a threshold to determine OTUs (Drancourt et al., 2000). In mouse, a total of 386 sequences were grouped into 67 OTUs and assigned to 6 phyla (subdivided into 9 classes) (Table 1). In vole, a total of 263 sequences were grouped into 98 OTUs and assigned to 7 phyla (subdivided into 13 classes) (Table 2). The reference sequence information and clone number of each OTU were also given in Table 1 and Table 2.
Figure 1 showed the percentage of each group from three parts of gastrointestinal tracts of A. semotus. The overall intestinal microflorae were composed of Firmicutes (43.26%), Proteobacteria (42.23%), DeinococcusThermus (9.84%), Verrucomicrobia (2.33%), Cyanobacteria (1.81%) and Actinobacteria (0.52%). The phylum of Firmicutes was divided into 3 classes:Mollicutes (0.26%), Bacilli (3.11%), and Clostridia (39.89%). The phylum of Proteobacteria was divided into 2 classes:Gammaproteobacteria (17.88%) and Deltaproteobacteria (24.35%). Figure 2 showed the percentage of each group from three parts of gastrointestinal tracts of E. melanogaster. The intestinal microflorae were composed of Proteobacteria (40.68%), Firmicutes (32.70%), Bacteroidetes (22.05%), Spirochaetes (1.90%), Actinobacteria (1.52%), TM7 (0.76%) and Deferribacteres (0.38%). The phylum of Firmicutes was divided into 3 classes:Mollicutes (5.70%), Bacilli (0.76%), and Clostridia (26.24%). The phylum of Proteobacteria was divided into 5 classes:Alphaproteobacteria (7.98%),
Betaproteobacteria
(0.38%),
Gammaproteobacteria
(20.53%),
Deltaproteobacteria (10.65%), and Epsilonproteobacteria (1.14%). The phylogeny trees of 67 OTUs from A. semotus and 98 OTUs from E. melanogaster were given in Figure 3 and Figure 4, respectively. In mouse, most OTUs belonged to Clostridia (42 OTUs). The next dominant group was Deltaproteobacteria, containing 10 OTUs. The remaining 15 OTUs belonged to 7 classes, and each class contained only 1 to 4 OTUs, respectively (Figure 3). In vole, most OTUs also belonged to Clostridia (40 OTUs). But the next dominant group was Bacteroidetes, containing 28 OTUs. The remaining 30 OTUs belonged to 11 classes, and each class only contained 1 to 4 OTUs, respectively (Figure 4).
4. Discussion Compare the shared dominant groups in the clone libraries In this study, there were three phyla (Actinobacteria, Firmicutes, and Proteobacteria) shared between A. semotus and E. melanogaster. Only a few of sequences belonged to Actinobacteria and they just occurred in the mouse small intestine and the vole caecum. Both in mouse and in vole, Firmicutes and Proteobacteria were the dominant groups in the clone libraries; however, the bacterial composition of three gut sections of the same individual differed from one another. In mouse, over 60% of clone sequences from the small intestinal sample belonged to Proteobacteria, but less than 25% of clone sequences were identified as the members of Firmicutes. From the caecal sample, the percentage of Firmicutes and Proteobacteria were almost equal. By contrast, from the large intestinal sample, there were only 28% of clone sequences belonging to Proteobacteria, but more than 54% of clone sequences belonging to Firmicutes (Figure 1). In vole, there were 67% of clone sequences from the small intestinal sample belonging to Proteobacteria, but only 15% of clone sequences were identified as the members of Firmicutes. By contrast, from the caecal sample, there were only 21% of clone sequences belonging to Proteobacteria, but almost 50% of clone sequences belonging to Firmicutes. From the large intestinal sample, the percentage of Firmicutes and Proteobacteria were the same (Figure 2). Compare the clone sequences between mouse and vole The sequences belonging to Deinococcus-Thermus, Verrucomicrobia, and Cyanobacteria occurred only in the mouse. Although three parts of gut samples contained some sequences classified as Deinococcus-Thermus, the number of each sample was notable different. Verrucomicrobia occurred in all
gut samples, while Cyanobacteria were only detected in the caecal and large intestinal samples (Table 1). Compared with mouse, the phyla Bacteroidetes, Spirochaetes, TM7 and Deferribacteres occurred only in the vole. The clone libraries of the small intestine, caecum and the large intestine contained 18%, 20%, and 30% of sequences belonging to Bacteroidetes, respectively. Spirochaetes were both detected in the caecal and large intestinal samples, while TM7 only occurred in caecal sample and Deferribacteres only occurred in large intestinal sample (Table 2). Generally speaking, the intestinal microbial diversity of E. melanogaster was higher than A. semotus. In vole, we analyzed 263 sequences and got 98 OTUs. But in mouse, we analyzed 386 sequences and only got 67 OTUs. The most difference between the microflorae of these two species of rodents was the existence of Bacteroidetes or not. In vole, there were total 22% of 16S rDNA clones belonging to Bacteroidetes which contained 28 OTUs. But in mouse, there was none in this group. Besides, although both rodents had more than 40% of bacterial clones in Proteobacteria, the OTUs affiliated with Alphaproteobacteria, Betaproteobacteria, and Epsilonproteobacteria were only present in vole. However, the biggest OTU of mouse or vole intestinal microflora belonged to the same group:Gammaproteobacteria. Compared with the SPF mouse and human microflorae Ley et al have analyzed 5,088 bacterial 16S rDNA sequences from the distal intestinal microflorae of genetically obese ob/ob mice, lean ob/+ and wild-type siblings, and their ob/+ mothers. Although the majority species of each mouse are unique, their microflorae are dominated by Firmicutes and Bacteroidetes (Ley et al., 2005). In the specific pathogen free (SPF) mice, the majority of bacterial rDNA clones from the murine caecal and large intestines were affiliated with the taxa Bacteroidetes and Firmicutes, too (Kibe et al., 2004; Scupham et al., 2006).
Eckburg et al. have analyzed 13,355 prokaryotic 16S rDNA sequences from multiple colonic mucosal sites and faces of healthy humans. A total of 395 bacterial phylotypes were identified. A majority of the bacterial sequences corresponded to uncultivated species and novel microorganisms. Most of the inferred organisms were members of the Firmicutes and Bacteroidetes phyla (Eckburg et al., 2005). Other studies also indicate that about 75% of fecal bacteria can be characterized, and belong to the group Clostridium and the group Bacteroides (Ramakrishna, 2007). Based on the previous studies, the mouse and human microflorae are similar at the division lever, with Firmicutes and Bacteroidetes dominating. However, the gut samples from the mouse and the vole in this study, respectively, have a large of 16S rDNA clones belonging to Proteobacteria which comprises a huge number of environmental bacteria. This dissimilarity suggests that wild rodents have specific immune tolerance for some members of Proteobacteria and these microbes may own unique metabolic traits to colonize the gut (Schiffrin & Blum, 2002).
5. Conclusion We constructed the bacterial 16S rDNA clone libraries from three parts of gastrointestinal tracts of two species of rodents and compared the microbial composition between different samples. This research offers an opportunity to further our understanding of the intestinal microflorae of wild rodents. Those kinds of researches provide a solid scientific basis for the effective use and development of probiotics (Marchesi & Shanahan, 2007). In the future, studies are needed to characterize the rules controlling microbial diversity in the gastrointestinal tracts. Multiple factors regulate the population number of these bacteria, including gastric acidity, intestinal transit, dietary factors, antibiotic use, and bacterial interactions with other bacteria and with the host epithelium (Ramakrishna, 2007). Much more extensive work would be required if the microflorae diversity reflect differences in diet, age, or genotype of each host individual (Bennegadi-Laurent et al., 2003).
6. Reference Bennegadi-Laurent N., Fonty G., Liliane M., Thierry G., and Dominique L. (2003). Effects of age and dietary fibre level on caecal microbial communities of conventional and specific pathogen-free rabbits. Microbial Ecology in Health & Disease 15: 23-32. Drancourt M., Bollet C., Carlioz A., Martelin R., Gayral J. P., and Raoult D. (2000). 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. Journal of Clinical Microbiology 38: 3623-3630. Eckburg P. B., Bik E. M., Bernstein C. N., Purdom E., Dethlefsen L., Sargent M., Gill S. R., Nelson K. E., and Relman D. A. (2005). Diversity of the human intestinal microbial flora. Science 308: 1635-1638. Jeanmougin F., Thompson J. D., Gouy M., Higgins D. G., and Gibson T. J. (1998). Multiple sequence alignment with Clustal x. Trends in Biochemical Sciences 23: 403-405. Kibe R., Sakamoto M., Hayashi H., Yokota H., and Benno Y. (2004). Maturation of the murine cecal microbiota as revealed by terminal restriction fragment length polymorphism and 16S rRNA gene clone libraries. FEMS Microbiology Letters 235: 139-146. Kimura M. (1980). A Simple Method for Estimating Evolutionary Rates of Base Substitutions through Comparative Studies of NucleotideSequences. Journal of Molecular Evolution 16: 111-120. Ley R. E., Backhed F., Turnbaugh P., Lozupone C. A., Knight R. D., and Gordon J. I. (2005). Obesity alters gut microbial ecology. Proceedings of the National Academy of Sciences of the United States of America 102: 11070-11075. Marchesi J., and Shanahan F. (2007). The normal intestinal microbiota. Current Opinion in Infectious Diseases 20: 508-513. Nagashima K., Hisada T., Sato M., and Mochizuki J. (2003). Application of new primer-enzyme combinations to terminal restriction fragment
length polymorphism profiling of bacterial Populations in human Feces. Applied and Environmental Microbiology 69: 1251-1262. Ramakrishna B. S. (2007). The normal bacterial flora of the human intestine and its regulation. Journal of Clinical Gastroenterology 41: S2-S6. Savage D. C. (1977). Microbial Ecology of Gastrointestinal-Tract. Annual Review of Microbiology 31: 107-133. Savage D. C. (1986). Gastrointestinal Microflora in Mammalian Nutrition. Annual Review of Nutrition 6: 155-178. Schiffrin E. J., and Blum S. (2002). Interactions between the microbiota and the intestinal mucosa. European Journal of Clinical Nutrition 56: S60S64. Scupham A. J., Presley L. L., Wei B., Bent E., Griffith N., McPherson M., Zhu F. L., Oluwadara O., Rao N., Braun J., and Borneman J. (2006). Abundant and diverse fungal microbiota in the murine intestine. Applied and Environmental Microbiology 72: 793-801. Suau A., Bonnet R., Sutren M., Godon J. J., Gibson G. R., Collins M. D., and Dore J. (1999). Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Applied and Environmental Microbiology 65: 4799-4807. Swofford D. L. (1993). Paup - a Computer-Program for Phylogenetic Inference Using Maximum Parsimony. Journal of General Physiology 102: A9-A9. Wilson K. H., and Blitchington R. B. (1996). Human colonic biota studied by ribosomal DNA sequence analysis. Applied and Environmental Microbiology 62: 2273-2278. Wintzingerode F. V., Gobel U. B., and Stackebrandt E. (1997). Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. Fems Microbiology Reviews 21: 213-229. Zoetendal E. G., Vaughan E. E., and de Vos W. M. (2006). A microbial world within us. Molecular Microbiology 59: 1639-1650.
Table 1. The intestinal microflorae of Apodemus semotus Phylum
Class
Group
Libraries
Reference sequence
Accession no. Similarity % Clones no.
Verrucomicrobia Actinobacteria
Verrucomicrobiae Actinobacteria
OTU_10
S2C4L3
Uncultured bacterium
AY986278
100
9
OTU_50
S1
Corynebacterium pseudogenitalium
AJ439348
100
1
OTU_52
S1
Human intestinal bacterium
AY310748
93
1
OTU_14
C5L2
Uncultured cyanobacterium
AY304018
95
7
OTU_02
S10C1L26
Meiothermus silvanus
Y13599
100
37
OTU_51
S1
Meiothermus silvanus
Y13599
100
1
OTU_65
L1
uncultured Mollicutes bacterium
AB218347
96
1
OTU_15
S4L3
Streptococcus uberis
AB023576
96
7
OTU_37
S3
Anoxybacillus flavithermus
AY672762
100
3
OTU_66
S1
Streptococcus uberis
AB023576
96
1
OTU_67
L1
Lactobacillus intestinalis
AJ306299
100
1
OTU_16
L6
Uncultured Clostridiales bacterium
AB234467
90
6
OTU_49
L2
Uncultured Clostridiales bacterium
AB234467
90
2
OTU_63
L1
Unidentified rumen bacterium
AB009208
94
1
OTU_64
S1
Candidatus Arthromitus
D86305
98
1
OTU_09
C8L3
Uncultured bacterium
DQ014910
99
11
OTU_11
C2L5
Uncultured bacterium
DQ014992
98
7
OTU_12
C1L6
Uncultured bacterium
DQ015401
99
7
OTU_20
C3L2
Clostridium orbiscindens
AY730665
97
5
OTU_23
C4
Clostridium orbiscindens
AY730665
97
4
OTU_26
C2L2
Uncultured bacterium
DQ015401
98
4
Cyanobacteria Deinococcus-Thermus Firmicutes
Cyanobacteria Deinococci Mollicutes Bacilli
Clostridia
OTU_27
L4
Uncultured bacterium
DQ015402
98
4
OTU_28
L3
Uncultured bacterium
DQ014825
99
3
OTU_29
C1L2
Clostridium orbiscindens
AY730665
97
3
OTU_36
C3
Ruminococcus bromii
X85099
96
3
OTU_40
L2
Uncultured bacterium
DQ014992
97
2
OTU_41
L2
Uncultured bacterium
DQ015401
98
2
OTU_48
L2
Uncultured bacterium
DQ015614
99
2
OTU_59
C1
Uncultured bacterium
DQ014588
99
1
OTU_60
C1
Uncultured bacterium
AY992487
99
1
OTU_61
L1
Anaerotruncus colihominis
DQ002932
95
1
OTU_62
L1
Uncultured bacterium
AY916378
99
1
OTU_06
C8L6
Uncultured bacterium
AY992805
97
14
OTU_07
C6L8
Uncultured bacterium
AY994018
98
14
OTU_13
S2C2L3
Clostridium sp.
AF157053
98
7
OTU_17
C1L5
Uncultured bacterium
AY991865
98
6
OTU_19
S5L1
Clostridium sp.
AF157053
97
6
OTU_22
C3L2
Uncultured bacterium
AY993232
98
5
OTU_24
C4
Uncultured bacterium
AY993337
100
4
OTU_25
L4
Clostridium sp.
AY960574
94
4
OTU_34
S2L1
Uncultured bacterium
AY993706
99
3
OTU_35
L3
Uncultured bacterium
DQ015606
99
3
OTU_38
C1L1
Butyrate-producing bacterium
AJ270488
96
2
OTU_39
L2
Uncultured bacterium
DQ015316
96
2
OTU_42
C1L1
Uncultured bacterium
DQ015511
98
2
Proteobacteria
Deltaproteobacteria
Gammaproteobacteria
OTU_43
C1L1
Uncultured bacterium
AY991749
98
2
OTU_44
S1C1
Uncultured bacterium
AY993686
97
2
OTU_45
S1C1
Uncultured bacterium
AY992550
98
2
OTU_54
C1
Butyrate-producing bacterium
AY305314
95
1
OTU_55
C1
Butyrate-producing bacterium
AY305315
95
1
OTU_56
C1
Uncultured bacterium
AY991749
97
1
OTU_57
L1
Uncultured bacterium
AY991750
97
1
OTU_58
L1
Uncultured bacterium
DQ014892
98
1
OTU_03
S8C13L6
Uncultured delta proteobacterium
AB192287
93
27
OTU_04
S1C14L6
Desulfovibrio sp.
AJ251630
96
21
OTU_05
S1C13L1
Uncultured delta proteobacterium
AB192287
92
15
OTU_08
S1C4L8
Desulfovibrio desulfuricans
DQ092636
96
13
OTU_18
C4L2
Desulfovibrio desulfuricans
DQ092636
93
6
OTU_30
C1L2
Bilophila wadsworthia
L35148
94
3
OTU_31
C3
Desulfovibrio fairfieldensis
U42221
95
3
OTU_32
S1C1L1
Desulfomonas pigra
AF192152
92
3
OTU_46
S1C1
Desulfovibrio sp.
AJ251630
94
2
OTU_53
L1
Bilophila wadsworthia
L35148
91
1
OTU_01
S37C7L15
Shigella boydii
AY696681
100
59
OTU_21
C2L3
Uncultured bacterium
AY591501
91
5
OTU_33
S2L1
Shigella boydii
AY696681
99
3
OTU_47
S2
Yersinia aleksiciae
AJ627597
100
2
Table 2. The intestinal microflorae of Eothenomys melanogaster Phylum
Class
Group
Actinobacteria
Actinobacteria
OTU_14
C2
subsurface water clone EV818CFSSAHH49
DQ336995
100
2
OTU_48
C1
mouse cecum clone SWPT15_aaa04f07
EF098514
91
1
OTU_49
C1
mouse cecum clone SWPT15_aaa04f07
EF098514
92
1
AB192001
84
7
Bacteroidetes
Bacteroidetes
OTU_28
Libraries Reference sequence
S3C1L3 termite gut homogenate clone M1PL1-23
Accession no. Similarity % clone no.
OTU_29
S1C1
UASB bioreactor wastewater clone E13
AY426457
85
2
OTU_30
S1L2
termite gut homogenate clone MgMjW-42
AB234442
84
3
AB015525
85
8
OTU_31
S3C3L2 deep-sea sediment clone BD1-16
OTU_32
C2
mouse cecum clone SWPT1_aaa04a04
EF098962
96
2
OTU_33
C1L3
mouse cecum clone SWPT14_aaa01b07
EF098075
93
4
OTU_34
S1L1
mouse cecum clone SWPT5_aaa04f10
EF100100
93
2
OTU_35
S4C1
mouse cecum clone SWPT2_aaa01b03
EF099015
93
5
OTU_36
C3L1
rat feces clone rc2-30(4)
AY239409
95
4
OTU_37
C2L1
mouse cecum clone aab22h06
DQ815831
93
3
OTU_43
C1
mouse cecum clone SWPT11_aaa04f05
EF096785
94
1
OTU_46
C1
mouse cecum clone SWPT16_aaa04c08
EF098745
83
1
OTU_47
C1
mouse cecum clone SWPT13_aaa04e03
EF097034
89
1
OTU_52
C1
mouse cecum clone SWPT13_aaa04g02
EF097052
92
1
OTU_60
C1
mouse cecum clone SWPT4_aaa03e02
EF099724
94
1
OTU_66
L1
rat feces clone R-1292
DQ777921
94
1
OTU_67
L1
rat feces clone R-1249
DQ777909
95
1
OTU_68
L1
mouse cecum clone SWPT2_aaa02c04
EF099101
93
1
OTU_69
L1
mouse cecum clone SWPT1_aaa04a04
EF098962
84
1
OTU_72
L1
mouse cecum clone SWPT13_aaa04d06
EF097026
91
1
OTU_73
L1
mouse cecum clone SWPT15_aaa02c06
EF098397
92
1
OTU_74
L1
mouse cecum clone SWPT2_aaa01b03
EF099015
93
1
OTU_77
L1
mouse cecum clone SWPT16_aaa04e11
EF098762
93
1
OTU_78
L1
rat feces clone R-1234
DQ777907
94
1
OTU_82
S1
termite gut homogenate clone BOf6-16
AB288906
88
1
OTU_92
S1
mouse cecum clone aab22h06
DQ815831
93
1
OTU_93
S1
mouse cecum clone SWPT2_aaa01e01
EF099041
92
1
OTU_96
S1
mouse cecum clone SWPT4_aaa02d08
EF099648
92
1
Deferribacteres
Deferribacteres
OTU_76
L1
swine intestine clone p-2881-6C5
AF371927
91
1
Firmicutes
Mollicutes
OTU_16
L15
Mycoplasma penetrans str. GTU-54-6A1
L10839
97
15
Bacilli
OTU_81
L1
mouse cecum clone aab47e08
DQ815398
97
1
Bacilli
OTU_97
S1
mouse cecum clone SWPT4_aaa02f05
EF099666
97
1
Clostridia
OTU_15
C3
mouse cecum clone aab22c03
DQ815781
95
3
Clostridia
OTU_17
C3
mouse cecum clone SWPT20_aaa03a09
EF097743
90
3
Clostridia
OTU_18
C2
mouse cecum clone SWPT16_aaa04e03
EF098757
90
2
Clostridia
OTU_19
C1L1
mouse cecum clone SWPT12_aaa02h08
EF097960
93
2
Clostridia
OTU_20
C1L1
swine intestine clone p-1529-b5
AF371637
98
2
Clostridia
OTU_21
C2
mouse cecum clone aab24f01
DQ815940
97
2
Clostridia
OTU_22
S3
swine intestine clone p-5278-2Wa3
AF371938
96
3
Clostridia
OTU_23
AB198630
89
4
Clostridia
OTU_24
C3
dairy cow rumen clone NED5G11
EF445284
92
3
Clostridia
OTU_25
C2
mouse cecum clone aab41b10
DQ815469
98
2
Clostridia
OTU_26
S1C3L3 mouse cecum clone SWPT15_aaa01g07
EF098372
93
7
Clostridia
OTU_38
C7
mouse cecum clone aab23b10
DQ815849
98
7
Clostridia
OTU_40
C2
mouse cecum clone aab24h11
DQ815966
97
2
S1C2L1 termite gut fluid clone RsC01-059
Clostridia
OTU_41
C1
mouse cecum clone aab23b10
DQ815849
99
1
Clostridia
OTU_42
C1
mouse cecum clone SWPT20_aaa02g11
EF097728
96
1
Clostridia
OTU_44
C1
termite gut wall fraction clone RsW01-038
AB198472
94
1
Clostridia
OTU_45
C1
swine intestine clone p-5459-2Wb5
AF371947
96
1
Clostridia
OTU_50
C1
rat feces clone R-1274(2)
DQ777951
94
1
Clostridia
OTU_51
C1
Selenomonas infelix str. ATCC 43532
AF287802
86
1
Clostridia
OTU_53
C1
mouse cecum clone aab41c10
DQ815479
94
1
Clostridia
OTU_56
C1
mouse cecum clone SWPT14_aaa02e03
EF098164
97
1
Clostridia
OTU_57
C1
termite gut clone Rs-041
AB100480
95
1
Clostridia
OTU_58
C1
swine intestine clone p-30-a5
AF371665
95
1
Clostridia
OTU_59
C1
rat feces clone R-1285
DQ777919
99
1
Clostridia
OTU_61
C1
mouse cecum clone aab42e07
DQ815544
98
1
Clostridia
OTU_62
C1
mouse cecum clone SWPT2_aaa02c11
EF099106
96
1
Clostridia
OTU_63
C1
mouse cecum clone aab42d08
DQ815537
92
1
Clostridia
OTU_64
C1
mouse cecum clone SWPT1_aaa03c05
EF098919
93
1
Clostridia
OTU_65
L1
mouse cecum clone SWPT12_aaa01h07
EF097914
97
1
Clostridia
OTU_71
L1
termite gut homogenate clone RsTz-65
AB192175
90
1
Clostridia
OTU_79
L1
swine intestine clone p-1528-b5
AF371777
89
1
Clostridia
OTU_80
L1
mouse cecum clone aab48c09
DQ815430
97
1
Clostridia
OTU_83
S1
mouse cecum clone SWPT18_aaa01b08
EF097083
91
1
Clostridia
OTU_85
S1
rabbit cecum clone NED2E8
EF445193
96
1
Clostridia
OTU_86
S1
mouse cecum clone SWPT5_aaa02g01
EF099955
96
1
Clostridia
OTU_89
S1
mouse cecum clone obob2_aaa01d07
EF096351
96
1
Clostridia
OTU_90
S1
mouse cecum clone aab48e05
DQ815443
89
1
Clostridia
OTU_91
S1
mouse cecum clone SWPT4_aaa04f09
EF099813
93
1
Clostridia
OTU_94
S1
mouse cecum clone aab22b10
DQ815777
98
1
Clostridia
OTU_98
S1
mouse cecum clone SWPT1_aaa03d10
EF098932
94
1
Genera_incertae_sedis_TM7 Genera_incertae_sedis_TM7 OTU_27 OTU_08 Proteobacteria Alphaproteobacteria
C2
mouse cecum clone SWPT20_aaa01h08
EF097659
100
2
S4
forest soil clone FAC17
DQ451456
83
4
OTU_09
S10
ferromanganous micronodule clone MND8
AF292999
83
10
OTU_10
S6
ferromanganous micronodule clone MND8
AF292999
84
6
OTU_87
S1
carbon tetrachloride contaminated soil clone
DQ248306
87
1
Betaproteobacteria
OTU_55
C1
subsurface water clone EV818CFSSAHH51
DQ336997
96
1
Gammaproteobacteria
OTU_13 S33C2L18 termite gut wall fraction clone RsW01-004
AB198445
91
53
OTU_84
DQ009674.2
89
1
EF098228
91
10
mouse cecum clone SWPT14_aaa03c12
EF098228
90
2
Deltaproteobacteria
Spirochaetes
OTU_01
S1
termite gut homogenate clone RSB1
S3C5S2 mouse cecum clone SWPT14_aaa03c12
OTU_02
C1L1
OTU_03
C2
mouse cecum clone aab48f08
DQ815453
90
2
OTU_04
C2
mouse cecum clone SWPT14_aaa03c12
EF098228
91
2
OTU_05
C1L1
mouse cecum clone aab48f08
DQ815453
91
2
OTU_06
C2
mouse cecum clone SWPT14_aaa03c12
EF098228
90
2
OTU_07
C2L1
mouse cecum clone SWPT14_aaa03c12
EF098228
91
3
OTU_54
C1
mouse cecum clone aab43c05
DQ815586
91
1
OTU_70
L1
U42221
89
1
OTU_75
L1
Desulfovibrio fairfieldensis mouse cecum clone SWPT14_aaa03c12
EF098228
91
1
OTU_88
S1
mouse cecum clone SWPT14_aaa03c12
EF098228
91
1
OTU_95
S1
mouse cecum clone aab48f08
DQ815453
92
1
Epsilonproteobacteria
OTU_11
S2C1
mouse cecum clone SWPT12_aaa02g04
EF097950
96
3
Spirochaetes
OTU_12
C1L1
Treponema zioleckii str. kT
DQ065758
89
2
OTU_39
C3
Brachyspira hyodysenteriae str. 174-92
U14931
97
3
Relative abundance of sequences
Percentage of clones
100%
Class Deltaproteobacteria Gammaproteobacteria
80%
Mollicutes 60%
Clostridia
40%
Bacilli Deinococci
20%
Verrucomicrobiae near Cyanobacteria Actinobacteria
0% S
C
L
Clone libraries of gut samples Figure 1. The percentage of each bacterial class of clone libraries from the small intestine, caecum and large intestine of Apodemus semotus
Relative abundance of sequences Class
Percentage of clones
100%
Genera_incertae_sedis_TM7 Spirochaetes Deferribacteres Bacteroidetes Mollicutes Clostridia Bacilli Epsilonproteobacteria Deltaproteobacteria Gammaproteobacteria Betaproteobacteria Alphaproteobacteria Actinobacteria
80% 60%
40% 20%
0 %
S
C
L
Clone libraries of gut samples Figure 2. The percentage of each bacterial class of clone libraries from the small intestine, caecum and large intestine of Eothenomys melanogaster
100
Meiothermus silvanus (Y13599) OTU 02 OTU 51
Deinococci 100
100 100
97
0.1
Uncultured bacterium (AY986278) OTU 10 Uncultured bacterium (AY571501) OTU 21
Uncultured cyanobacterium (AY304018) OTU 14
Yersinia aleksiciae (AJ627597) OTU 47 100 Shigella boydii (AY696681) OTU 01 OTU 33 OTU 53 Uncultured delta proteobacterium (AB192287) OTU 04 OTU 31 OTU 08 OTU 46 OTU 05 100 OTU 32 OTU 03 OTU 18 Desulfomonas pigra (AF192152) Desulfovibrio desulfuricans (DQ092636) Desulfovibrio sp. (AJ251630) Desulfovibrio fairfieldensis (U42221) Bilophila wadsworthia (L35148) OTU 30 Corynebacterium pseudogenitalium (AJ439348) 100 OTU 50 Human intestinal bacterium (AY310748) OTU 52 Streptococcus uberis (AB023576) OTU 15 OTU 66 100 Lactobacillus intestinalis (AJ306299) OTU 67 Anoxybacillus flavithermus (AY672762) 96 OTU 37 Uncultured Mollicutes bacterium (AB218347) OTU 65 Uncultured Clostridiales bacterium (AB234467) OTU 16 100 OTU 49 Candidatus Arthromitus (D86305) OTU 64 Unidentified rumen bacterium (AB009208) OTU 63 Uncultured bacterium (AY916378) OTU 62 Ruminococcus bromii (X85099) OTU 36 Uncultured bacterium (DQ015614) OTU 48 Anaerotruncus colihominis (DQ002932) OTU 61 OTU 29 100 Clostridium orbiscindens (AY730665) OTU 23 OTU 20 Uncultured bacterium (DQ014910) OTU 09 Uncultured bacterium (AY992487) OTU 60 Uncultured bacterium (DQ014992) Uncultured bacterium (DQ015401) OTU 11 OTU 12 OTU 26 OTU 27 OTU 40 OTU 41 91 Uncultured bacterium (DQ014825) OTU 28 Uncultured bacterium (DQ014588) OTU 59 Uncultured bacterium (AY993706) OTU 34 Clostridium sp. (AY960574) OTU 25 Butyrate-producing bacterium (AJ270488) OTU 38 OTU 19 Clostridium sp. (AF157053) OTU 13 100 Uncultured bacterium (AY993337) OTU 24 OTU 54 Butyrate-producing bacterium (AY305314) OTU 55 Uncultured bacterium (DQ015511) OTU 42 Uncultured bacterium (DQ014892) OTU 58 OTU 57 Uncultured bacterium (AY991749) OTU 43 OTU 56 Uncultured bacterium (AY992805) OTU 06 Uncultured bacterium (AY993686) OTU 44 Uncultured bacterium (AY992550) OTU 45 Uncultured bacterium (AY994018) OTU 07 Uncultured bacterium (AY991865) OTU 17 Uncultured bacterium (AY993232) OTU 22 Uncultured bacterium (DQ015316) OTU 39 Uncultured bacterium (DQ015606) OTU 35
Cyanobacteria Verrucomicrobiae Gammaproteobacteria
Deltaproteobacteria
Actinobacteria Bacilli Mollicutes
Clostridia
Figure 3. Neighbor-joining tree of 67 OTUs of Apodemus semotus based on the 16S rDNA sequences.
OTU_27
Genera_incertae_sedis_TM7
Bacteroidetes (total 28 OTUs)
99
OTU_16
50
Mollicutes
OTU_97 99
Bacilli
OTU_81
Clostridia (total 40 OTUs)
50
OTU_76
Deferribacteres
OTU_02 OTU_01 68
OTU_03 OTU_95 OTU_88 OTU_05 100
Deltaproteobacteria
OTU_04 OTU_06 OTU_07 OTU_54 OTU_75 OTU_70 100
OTU_10 OTU_09
62
OTU_08 100
Alphaproteobacteria
OTU_87
OTU_55
Betaproteobacteria
OTU_84 96
OTU_13
Gammaproteobacteria
OTU_11
Epsilonproteobacteria
OTU_14
93
OTU_49 100
Actinobacteria
OTU_48 OTU_12 OTU_39
Spirochaetes
0.05
Figure 4. Neighbor-joining tree of 98 OTUs of Eothenomys melanogaster based on the 16S rDNA sequences.