Age-Related Dynamics of Fecal Microbiota in the Captive Chimpanzee (Pan troglodytes )
Categoria dell'articolo: Original Paper
Pubblicato online: 16 set 2025
Pagine: 363 - 373
Ricevuto: 22 apr 2025
Accettato: 06 ago 2025
DOI: https://doi.org/10.33073/pjm-2025-031
Parole chiave
© 2025 JUAN LIU et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
The developmental process of chimpanzees includes three distinct ontogenetic stages: juvenile (1–5 years), adolescent (5–10 years), and adult (> 10 years) stages (Goodall 1986; Pusey et al. 2005), each characterized by specific physiological and behavioral transitions that shape the gut microbiome assembly. The juvenile stage encompasses weaning and maternal dependency dissolution, during which the microbiome transitions from the abundance of milk-adapted taxa to a broader fermentative capacity. Adolescence is associated with social independence and sexual maturation, which drive microbiome restructuring via hormonal shifts and dietary exploration. Microbial communities stabilize by adulthood to support full somatic and reproductive functions (Moeller et al. 2016; Reese et al. 2021; Amato et al. 2025). In wild populations, fiber-degrading specialists essential for metabolizing diverse plant substrates are dominant (Nishida and Ochman 2019; Reese et al. 2021; Baniel et al. 2022). Under captive conditions, dietary regimes fundamentally reconfigure the nutritional landscape relative to wild ecosystems, driving divergent microbiome trajectories and cascading effects on host physiology, behavior, and conservation viability. The wild chimpanzees consume about 174 plant species annually, with seasonal shifts in frugivory and folivory promoting microbiome plasticity essential for nutrient extraction and immune resilience (Tutin and Fernandez 1993; Reese et al. 2021). Contrastingly, the captive diets rich in cultivated fruits and vegetables (e.g., apples and leafy greens), which include limited browse (elm/willow), exhibit 40–60% reductions in insoluble fiber and a near-absence of secondary plant metabolites (Campbell et al. 2020; Narat et al. 2020). A study towards the captive apes in Wildlife Reserves Singapore and Longleat Safari and Adventure Park showed that the diet with reduced water-soluble carbohydrates neutral and increased detergent fibre significantly increased ‘travelling ’, ‘foraging ’ and ‘social affiliative ’ behaviors, and decreased ‘inactivity’ and ‘abnormal behavior patterns’, such as ‘regurgitation and reingestion’. They pointed out that great apes in captivity have been affected by a variety of conditions, including obesity, heart, gastrointestinal and dental diseases, and diabetes, all of which are at least influenced by an inappropriate diet (Cabana et al. 2017). Zoos in Southeast Asia and North America demonstrated the convergence toward a “captive enterotype ” dominated by
Although the chimpanzee husbandry protocol followed across Chinese institutions adheres to the EAZA Best Practice Guidelines for Great Apes Taxon Advisory Group Chimpanzees (
Chimpanzee husbandry protocol adheres to the EAZA Best Practice Guidelines (Carlsen et al. 2022) and the Chimpanzee (
The detail information of grouping.
| Age group | Group | Sample size |
|---|---|---|
| 2 < years old < 5 | juvenile group A | 7 |
| 5 < years old < 10 | adolescent group B | 7 |
| > 15 years old | adult group C | 7 |
Total genomic DNA was extracted from fecal samples using the CTAB/SDS method. The DNA concentration and purity were analyzed using a 1% agarose gel. Subsequently, DNA was diluted to 1 ng/μl using sterile water, and all DNA samples were stored at -20°C for further PCR amplification.
For PCR reactions, a mix (30μl) was prepared using 15μl of Phusion™High-Fidelity PCR Master Mix (New England Biolabs, USA), 0.2μM of forward and reverse primers, and about 10 ng template DNA. The thermal cycling process included an initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10s, annealing at 50°C for 30s, elongation at 72°C for 60s, and a final step at 72°C for 5 min. The PCR products were purified from a 2% agarose gel using a Gel Extraction Kit. Sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, USA) following the manufacturer’s recommendations. After the assessment was performed using a Qubit® 2.0 Fluorometer (Invitrogen™; Thermo Fisher Scientific Inc., USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, Inc., USA), the library was sequenced on an Illumina®NovaSeq™ 6000 platform (Illumina, Inc., USA), and 250 bp paired-end reads were generated. The 341F (5′CCTAYGGGRBGCASCAG3′) and 806R (5′GGACTACNNGGGTATCTAAT3′) primers were used to amplify the V3–V4 region of the 16S rRNA gene. The sequencing data has been deposited in NCBI (PRJNA1290099).
Pairedend reads generated from the original DNA fragments were merged using FLASH (Magoč and Salzberg 2011), designed to merge paired-end reads when at least some of the reads overlapped with reads generated from the opposite end of the same DNA fragment. Paired-end reads were assigned to each sample based on unique barcodes. Sequence analysis was performed using the UPARSE software package with UPARSE-OUT (Operational Taxonomic Units) and UPARSE-OUT ref algorithms (Edgar 2013). In-house Perl scripts were used to analyze alpha (within samples) and beta (among samples) diversities. Sequences exhibiting ≥ 97% similarity were assigned to the same OTUs. We selected representative sequences for each OTU and used the RDP classifier to annotate the taxonomic information for each representative sequence. To assess the alpha diversity, we rarified the OTU table and assessed the species richness and diversity of chimpanzees based on the following indices: Chao1 index and Observed Species (to assess species richness), Shannon and Simpson indices (to assess the diversity of species). The dilution curve of the corresponding index was generated to assess the saturation of the overall detection of the experimental microbial community. Based on a Venn diagram, we compared the species composition between different samples. The species composition was compared between different samples or groups using dimensionality reduction analysis with NMDS. Significant differences in the microbial community structure between groups were analyzed using ANOSIM. Linear discriminant analysis (LDA) coupled with effect size (LEfSe) was used to evaluate differentially abundant taxa, focusing on both statistical significance and biological relevance.
After processing the raw data, a total of 1,986,038 (47,188–74,191/each) high-quality reads were generated through the 16S rRNA sequencing of 21 fecal samples (seven juveniles, seven adolescents, and seven adults). The valid tags, rates, and quality scores Q20% and Q30% are presented in Table SI. While analyzing the diversity of species composition in the samples, the UCLUST in QIIME v1.8.0 (Caporaso et al. 2010) was used to cluster the clean reads of all samples. Next, the clean reads were first subjected to dechimerism processing, then the repeated sequences were clustered into Operational Taxonomic Units (OTUs) based on 97% homology. Representative sequences of OTUs were annotated with species using the annotation database Silva (Quast et al. 2013) (Table SII). Fig. 1A shows each curve representing a different sample. The sequencing depth first increased as the number of features increased, followed by a plateau, indicating that a small number of new features were detected with the increase in the sequencing depth. Therefore, this trend suggested that the sequenced samples were sufficient and reasonable. A total of 1,310, 2,452, and 1,185 features were detected in the juvenile, adolescent, and adult groups, respectively. The Venn diagram (Fig. 1B) revealed that the three tested groups shared 915 common features, suggesting a common effect on chimpanzee gastrointestinal microbiota at the three different developmental stages. There were 75, 1,139, and 108 unique features in the juvenile (Group A), adolescent (Group B), and adult (Group C) groups, respectively. The large number of unique features in adolescents indicates that the chimpanzee gut microbiota played an essential role at this developmental stage.
Gut Microbiotah Composition of
A) The rarefaction curve of the microbiota of all samples. Each curve representing a different sample. The curves E3, E5, E6, E7, E8, E9, and F1 indicate the juvenile group; the curves C1, C2, C3, C4, D1, D2, and D3 indicate the adolescent group; the curves A1, A2, A3, B2, B3, B5, and B6 indicate the adult group. The x axis showed qualitative sequence of random number; The y axis showed OTU number. B) Venn plots show the OTU distribution based Venn plots between juvenile (A) adolescent (B) and adult (C) groups. C) The microbial community composition of
A total of 31 bacterial phyla were identified, including Firmicutes, Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Tenericutes, Euryarchaeota, Spirochaetes, Patesobacteria, and Acidobacteria, which were the top 10 phyla in all samples (Fig. 1C). Firmicutes and Bacteroidetes presented the most abundant phyla. Firmicutes accounted for 54.39% ± 6.00%, 44.92% ± 9.77%, and 53.57% ± 11.89% in the juvenile, adolescent, and adult groups, respectively, whereas Bacteroidetes accounted for 36.12% ± 7.76%, 43.92% ± 7.90%, and 31.42% ± 5.23%, respectively, in these groups. The ratio of Firmicutes/Bacteroidetes (F/B) was 1.51 in the juvenile group, which decreased to 1.02 in the adolescent group, and increased to 1.70 in the adult group (Fig. 1D).
Seventy-nine classes were identified, which included 33 classes identified in all three groups. Among these, Spirochaetia, Actinobacteria, Bacilli, Alphaproteobacteria, Oxyphotobacteria, Gammaproteobacteria, Erysipelotrichia, Negativicutes, Clostridia, and Bacteroidia presented the top 10 classes (Fig. 1E), with Clostridia and Bacteroidia exhibiting the highest abundance. Clostridia accounted for 37.83% ± 6.47%, 36.33% ± 7.93%, and 38.46% ± 11.27% in the juvenile, adolescent, and adult groups, respectively, whereas Bacteroidia accounted for 36.12% ± 7.76%, 43.92% ± 7.90%, and 31.41% ± 5.23%, respectively, in these groups. The proportion of Bacteroidia first increased transiently, and subsequently, decreased with age. Notably, the higher abundance of Bacilli was detected in the adult group than in the juvenile and adolescent groups. Interestingly, Bacteroidia and Bacilli belong to Bacteroidetes and Firmicutes, respectively; hence, these distribution patterns were consistent with the phylum-level data.
Among the 511 identified genera, 215 genera were identified in all three groups.
Next, the sequences were used in a serial estimation of and diversity to evaluate the fecal bacterial diversity in the juvenile, adolescent, and adult chimpanzees. Significant differences in the Ace (A:905.87 ± 45.52 vs. B:1128.06 ± 357.01 vs. C:792.09 ± 5 3.08;
Diversity analysis of fecal microbiota of
A–D) Box plot of the α-diversity index of Ace (A) Chao1 (B) Observed species (C) and Goods-covrage (D). E) The Non-Metric Multi-Dimensional Scaling (NMDS) illustrating that the microbial community differences between chimpanzees in groups A, B and C was statistically significant at the Stress level of 1 × 10−4.
The ANOSIM analysis was used to test the significance of differences in community structure between groups.
| Groups | ||
|---|---|---|
| A vs. B | 0.0068027 | 0.383 |
| A vs. C | 0.1574344 | 0.082 |
| B vs. C | 0.1977648 | 0.064 |
| A vs. B vs. C | 0.1209373 | 0.045 |
| C vs. B vs. A | 0.1209373 | 0.045 |
A linear discriminant analysis was used to identify the discriminative keystone taxa between the juvenile, adolescent, and adult groups (
The keystone taxa of
A) Histogram of the linear discriminant analysis (LDA) scores computed for features differentially abundant. Red green and blue bars represented bacterial conmmunities of juvenile adolescent and adult groups respectively. The criteria was set as
This comprehensive study demonstrates the comparative fecal microbiota structure in captive chimpanzees at different developmental stages. We observed dynamic changes in the abundance of the gut microbial community at the phylum, class, and genus levels in the juvenile, adolescent, and adult stages. The chimpanzees at all stages were maintained using the same husbandry protocol and the standard recipes. However, the unique features reflected in the results strongly suggest that age critically regulates the gut microbiota structure in captive chimpanzees.
LEfSe analysis identified the family
Previously, the gut microbial communities within chimpanzees were reported to show a decrease in the α diversity (Shanon index) from infant to elderly (Degnan et al. 2012). Similarly, we found a declining trend in the gut microbial communities in captive chimpanzees. Additionally, we demonstrated that α diversity indices, such as Chao1, ACE, Observed species, decreased from the juvenile to the adult. Moreover, a transient increase of the α diversity index was found in the adolescent stage; however, no significant change was detected. This transient climax of the gut microbiome may be attributed to intensified host physiological demands, particularly somatic growth, immune maturation, and metabolic adaptation during adolescence. However, further analysis to elucidate the interplay of the gut microbial community structure throughout the development of chimpanzees will be required in our future research.
We, as well as other researchers (Szekely et al. 2010; Degnan et al. 2012; Moeller and Ochman 2013), have reported Firmicutes and Bacteroidetes to be the two dominant phyla. The Firmicutes phylum enables the host to extract substantial energy from dietary substrates (Sun et al. 2023; Dias et al. 2025), whereas Bacteroidetes exhibits comparatively limited efficiency in converting nutrients into available energy (Wexler and Goodman 2017; Pan et al. 2023). Therefore, the F/B ratio is widely accepted to regulate body weight crucially (Koliada, Syzenko et al. 2017; Aragón-Vela et al. 2021; Mohamed Qadir and Assafi 2021; Komodromou et al. 2024). In an extensive ape study, the F/B ratio was approximately 3.34 in wild adult chimpanzees (Degnan et al. 2012). In this study, the F/B ratio was lower than that of the wild species. Interestingly, a relatively low F/B ratio in captive chimpanzees compared to that in wild species was also reported previously (Narat et al. 2020). We hypothesized that captive chimpanzees require less biological energy to sustain vital functions than their wild counterparts owing to the reduced ecological demands, which is substantiated by the reduced body weights (Table SIII) observed in captive individuals than in the corresponding wild conspecifics at comparable age (Pusey et al. 2005). Interestingly, we found that the abundance of
Collectively, this study, for the first time, delineates the ontogenetic trajectory of the gut microbiome assembly in captive chimpanzees (