, Volume 59, Issue 5, pp 423–436 | Cite as

Improving the standards for gut microbiome analysis of fecal samples: insights from the field biology of Japanese macaques on Yakushima Island

  • Takashi Hayakawa
  • Akiko Sawada
  • Akifumi S. Tanabe
  • Shinji Fukuda
  • Takushi Kishida
  • Yosuke Kurihara
  • Kei Matsushima
  • Jie Liu
  • Etienne-Francois Akomo-Okoue
  • Waleska Gravena
  • Makoto Kashima
  • Mariko Suzuki
  • Kohmei Kadowaki
  • Takafumi Suzumura
  • Eiji Inoue
  • Hideki Sugiura
  • Goro Hanya
  • Kiyokazu Agata
Original Article


Fecal DNA-based 16S ribosomal RNA (rRNA) gene sequencing using next-generation sequencers allows us to understand the dynamic gut microbiome adaptation of animals to their specific habitats. Conventional techniques of fecal microbiome analysis have been developed within the broad contexts defined by human biology; hence, many of these techniques are not immediately applicable to wild nonhuman primates. In order to establish a standard experimental protocol for the analysis of the gut microbiomes of wild animals, we selected the Japanese macaques (Macaca fuscata yakui) on Yakushima Island. We tested different protocols for each stage of fecal sample processing: storage, DNA extraction, and choice of the sequencing region in the bacterial 16S rRNA gene. We also analyzed the gut microbiome of captive Japanese macaques as the control. The comparison of samples obtained from identical macaques but subjected to different protocols showed that the tested storage methods (RNAlater and lysis buffer) produced effectively the same composition of bacterial operational taxonomic units (OTUs) as the standard frozen storage method, although the relative abundance of each OTU was quantitatively affected. Taxonomic assignment of the detected bacterial groups was also significantly affected by the region being sequenced, indicating that sequencing regions and the corresponding polymerase chain reaction (PCR) primer pairs for the 16S rRNA gene should be carefully selected. This study improves the current standard methods for microbiome analysis in wild nonhuman primates. Japanese macaques were shown to be a suitable model for understanding microbiome adaptation to various environments.


Japanese macaque Gut microbiome Field biology Fecal collection Next-generation sequencing 16S rRNA gene 



We would like to thank our friends and colleagues in Yakushima. Experiment 3 of this study was performed in the Yakushima Field and Genome Science Courses of Kyoto University in spring 2014 and we thank the fellow students and lectures who joined in the courses and the administration staff for making these courses possible behind the scene. We are grateful to Dr. Hirohisa Hirai, Dr. Hiroo Imai, Dr. Nami Suzuki-Hashido, and other members of the Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University for valuable discussions. We also thank members of the Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, particularly Mr. Takayoshi Natsume for collecting samples from captive animals. The study was financed by the Leading Graduate Program of Primatology and Wildlife Science (PWS) of Kyoto University, MEXT/JSPS KAKENHI (#12J04270 and #16K18630 to TH, #17H01911 to TH and GH, #25840170 to AS, and #25291100 to GH) and Fund for the Promotion of Joint International Research (#15KK0256 to GH).

Supplementary material

10329_2018_671_MOESM1_ESM.docx (169 kb)
Supplementary material 1 (DOCX 168 kb)
10329_2018_671_MOESM2_ESM.csv (1.1 mb)
Supplementary material 2 Dataset S2: Operational taxonomic units (OTUs), assigned taxa, nucleotide sequences, and number of sequencing reads based on the 16S V3–V4 region. (CSV 1082 kb)
10329_2018_671_MOESM3_ESM.csv (356 kb)
Supplementary material 3 Dataset S1: Operational taxonomic units (OTUs), assigned taxa, nucleotide sequences, and number of sequencing reads based on the 16S V1–V2 region. (CSV 356 kb)


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Copyright information

© Japan Monkey Centre and Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Takashi Hayakawa
    • 1
    • 2
  • Akiko Sawada
    • 3
    • 4
  • Akifumi S. Tanabe
    • 5
  • Shinji Fukuda
    • 6
    • 7
    • 8
    • 9
  • Takushi Kishida
    • 10
  • Yosuke Kurihara
    • 4
    • 11
  • Kei Matsushima
    • 10
  • Jie Liu
    • 10
  • Etienne-Francois Akomo-Okoue
    • 12
  • Waleska Gravena
    • 13
    • 14
  • Makoto Kashima
    • 15
  • Mariko Suzuki
    • 16
  • Kohmei Kadowaki
    • 17
  • Takafumi Suzumura
    • 10
  • Eiji Inoue
    • 18
  • Hideki Sugiura
    • 10
  • Goro Hanya
    • 11
  • Kiyokazu Agata
    • 10
    • 19
  1. 1.Department of Wildlife Science (Nagoya Railroad Co., Ltd.)Primate Research Institute, Kyoto UniversityInuyamaJapan
  2. 2.Japan Monkey CentreInuyamaJapan
  3. 3.Graduate School of Life and Environmental SciencesKyoto Prefectural UniversityKyotoJapan
  4. 4.Japan Society for the Promotion of ScienceTokyoJapan
  5. 5.Department of Environmental Solution Technology, Faculty of Science and TechnologyRyukoku UniversityOtsuJapan
  6. 6.Institute for Advanced BiosciencesKeio UniversityTsuruokaJapan
  7. 7.Intestinal Microbiota ProjectKanagawa Institute of Industrial Science and TechnologyKawasakiJapan
  8. 8.Transborder Medical Research CenterUniversity of TsukubaTsukubaJapan
  9. 9.PRESTO, Japan Science and Technology AgencyKawaguchiJapan
  10. 10.Wildlife Research CenterKyoto UniversityKyotoJapan
  11. 11.Ecology and Conservation SectionPrimate Research Institute, Kyoto UniversityInuyamaJapan
  12. 12.Research Institute in Tropical Ecology (IRET-CENAREST)LibrevilleGabon
  13. 13.Aquatic Mammals LaboratoryNational Institute of Amazonian ResearchManausBrazil
  14. 14.Health and Biotechnology InstituteAmazonas Federal UniversityCoariBrazil
  15. 15.Research Institute for Food and AgricultureRyukoku UniversityOtsuJapan
  16. 16.International Center for Island Studies Amami StationKagoshima UniversityAmamiJapan
  17. 17.Center for the Promotion of Interdisciplinary Education and Research, Education and Research Unit for Studies on Connectivity of Hills, Humans and Oceans (CoHHO)Kyoto UniversityKyotoJapan
  18. 18.Faculty of ScienceToho UniversityFunabashiJapan
  19. 19.Department of Life Science, Faculty of Science, Graduate Course in Life Science, Graduate School of ScienceGakushuin UniversityTokyoJapan

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