Comparative study on intestinal microbiome composition and function in young and adult Hainan gibbons (Nomascus hainanus)

Introduction

The Hainan gibbon (Nomascus hainanus) is a species endemic to China, belonging to the Primates order, Hylobatidae family, and Nomascus genus. Historically, Hainan gibbons have been widely distributed in tropical rain forests on Hainan Island. Since the 1950s, due to indiscriminate poaching and a reduction in natural habitat areas, the population of Hainan gibbons has decreased rapidly. At present, they are only distributed in the Hainan Tropical Rain Forest National Park, with a total of 35 individuals in five family groups (Zhang, 2021), making it one of the least prevalent primates in the world. The Hainan gibbon is listed as a Class I key protected wild species in China, and the International Union for Conservation of Nature has assessed the status of this species as ‘critically endangered’ (Geissmann & Bleisch, 2020). Since the 1970s, researchers have carried out a series of studies on Hainan gibbons, focusing on habitat range (Liu & Tan, 1990; Tang & Jin, 2021), population size (Fellowes et al., 2008), behaviour (Zhou, Chen & Wei, 2008), genetic diversity (Li, Wei & Zhou, 2010; Han, 2019), and feeding habits (Tang, Bi & Jin, 2021). However, little research has been conducted on the intestinal microbiome of Hainan gibbons.

The bacteria in the host intestine are variable, and their composition is affected by host genotype (Kovacs et al., 2011), diet (Li et al., 2017; Singh et al., 2017), age (Odamaki et al., 2016), habitat (Dulski, Kozowski & Ciesielski, 2020), disease (Owyang & Wu, 2014), and other factors. A large number of studies have confirmed that the dynamic balance of the intestinal microbiome is closely related to host immunity, development, nutrient absorption, and energy metabolism (Bäckhed et al., 2004; Round & Mazmanians, 2009; Myer et al., 2017), and its potential significance in maintaining host health is becoming increasingly prominent. Therefore, studies on the intestinal microbiome have attracted increasing attention in the field of endangered species protection. Non-human primates have closely related to humans; thus, the study of intestinal microbiome in non-human primates is of significance. To date, intestinal microbiome composition of a variety of non-human primates, including lemurs and lorises (McKenney, Rodrigo & Yoder, 2015), New World monkeys and Old World monkeys (Amato et al., 2013; Sun et al., 2016), and apes (Ochman et al., 2010) has been reported. In general, the intestinal microbiome of non-human primates is composed of bacteria from 12 phyla such as Bacteroidetes, Firmicutes, Proteobacteria, Tenericutes, Actinobacteria.

In this study, we examined the intestinal microbiome of young and adult Hainan gibbons in the dry season in order to explore and compare the composition and functional differences in the intestinal microorganisms of Hainan gibbons at different ages, in order to provide a theoretical basis for the protection of this extremely endangered species.

Materials and Methods

Results

Intestinal microbiome composition of young and adult Hainan gibbon

At the phylum level (Figs. 1A and 1B), Firmicutes (Y: 0.21 ± 0.07%; A: 0.56 ± 0.09%), and Bacteroidetes (Y: 0.69 ± 0.10%; A: 0.33 ± 0.12%) were the main dominant phyla in young and adult Hainan gibbons, followed by Actinobacteria (Y: 0.01 ± 0.01%; A: 0.05 ± 0.03%), Fibrobacteres (Y: 0.03 ± 0.03%; A: 0.02 ± 0.01%), and Spirochaetes (Y: 0.02 ± 0.01%; A: 0.01 ± 0.01%). At the genus level (Figs. 1C and 1D), Prevotella (Y: 0.52 ± 0.09%; A: 0.23 ± 0.10%) was the main dominant genera, followed by Bacteroides (Y: 0.06 ± 0.01%; A: 0.03 ± 0.01%), Clostridium (Y: 0.02 ± 0.01%; A: 0.04 ± 0.01%), and Eubacterium (Y: 0.01 ± 0.01%; A: 0.03 ± 0.01%).

Lefse analysis

LEfSe analysis identified 22 taxa that showed significantly different abundances between young and adult Hainan gibbons (Fig. 2). At the phylum level, the relative abundance of Firmicutes was significantly higher in adults than in young, whereas Bacteroidetes and Proteobacteria were significantly higher in young than in adults. At the genus level, Ruminococcus, Clostridium, and Butyrivibrio were significantly higher in adults than in young, whereas Prevotella and Bacteroides were significantly higher in young than in adults.

Analysis of functional gene difference of intestinal microbiome in young and adult Hainan gibbon

Heatmaps were constructed to explore the differences in the abundance of genes related to various KEGG, COG (Fig. 3; Tables S2 and S3) and CAZy (Fig. 4) categories between young and adult Hainan gibbons. Based on KEGG annotation for adult Hainan gibbons, the abundance of the carbohydrate metabolism, nucleotide metabolism, membrane transport, energy metabolism, Cellular community—prokaryotes, signal transduction, biosynthesis of other secondary metabolites, metabolism of terpenoids and polyketides and Xenobiotics biodegradation and metabolism categories was significantly higher than in young Hainan gibbons. Based on COG annotation for adult Hainan gibbons, the abundance of the general function prediction only, carbohydrate transport and metabolism, amino acid transport and metabolism, transcription, energy production and conversion, Inorganic ion transport and metabolism, defense mechanisms, and signal transduction mechanisms categories was significantly higher than in young Hainan gibbons. For young Hainan gibbons, the abundance of the posttranslational modification, protein turnover, chaperones category was significantly higher than in adult Hainan gibbons.

Based on CAZy annotation, the database divides carbohydrate active enzymes into the following six classes: auxiliary activities (AAs), glycosyltransferases (GTs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycoside hydrolases (GHs), and carbohydrate-binding modules (CBMs). The results showed that the sequences were enriched in 109 GH families, 73 GT families, 72 CBM families, 18 PL families, 15 CE families, and 9 AA families. The content of GHs in adult and young Hainan Gibbons was the highest, accounting for 43.31% and 41.81% of the total carbohydrate active enzymes, respectively, followed by GTs (32.42%, 32.28%), CBMs (9.94%, 11.26%), CEs (10.62%, 10.74%) and PLs (2.27%, 2.70%), whereas, the abundance of AAs was the lowest, accounting for only 1.44% and 1.19% of the total carbohydrate active enzymes, respectively. GHs hydrolyse glycosidic bonds between carbohydrate molecules. GHs involved in the degradation of cellulose, hemicellulose and oligosaccharide were abundant in the intestinal tract of the two groups of Hainan gibbons. Among the 20 most abundant carbohydrate enzymes, GH5 is the predominant enzyme involved in cellulose degradation, GH2 and GH3 play a major role in oligosaccharide degradation, and GH28 plays an important role in hemicellulose degradation. The results showed that the abundance of enzyme involved in cellulose and hemicellulose degradation in the adult Hainan gibbon group was higher than that in the young Hainan gibbon group, whereas, the abundance of enzymes involved in oligosaccharide degradation in the young Hainan gibbon group was higher than that in the adult Hainan gibbon group.

Discussion

In this study, metagenome sequencing was used to analyse the composition and functional differences in the intestinal microbiome between young and adult Hainan gibbons, with the aim to explore the influence of age-related factors on the host intestinal microbiome.

Firmicutes and Bacteroidetes were the dominant phyla in the intestines of young and adult Hainan gibbons. Previous studies have reported the composition of intestinal microbiome in a variety of non-human primates such as Macaca thibetana (Sun et al., 2016), Alouatta pigra (Amato et al., 2014), Gorilla gorilla gorilla (Pafčo et al., 2019), and Rhinopithecus bieti (Xu et al., 2015). The proportion of Bacteroidetes and Firmicutes is the highest in the intestinal microbiome of most non-human primates. In this study, the proportion of Firmicutes increased with age and that of Bacteroidetes decreased with age. This may be because the immune system of young Hainan gibbons is not mature yet and the composition of the intestinal microbiome is not stable. Bacteroides can promote immune system development to enhance host immunity (Hooper, 2004) and maintain intestinal microbial ecological balance (Hooper et al., 2001; Sears, 2005); therefore, it is highly abundant in the intestines of young Hainan gibbons. In addition, the intestine of Hainan gibbon contains Fibrobacteres members, which mainly exist in the rumen of ruminants, where cellulase is present in the periplasm; Fibrobacteres members can decompose cellulose and enable hosts to absorb it (Jewell et al., 2013). Zhou et al. (2014) also confirmed that the intestinal microbiome composition of golden monkeys is similar to that of ruminants, such as cattle, and this is related to their similar dietary structure. Prevotella, Bacteroides, and Clostridium were the dominant bacteria at the genus level. These bacteria are generally closely related to host food digestion, and they can help leaf-eating non-human primates degrade structural carbohydrates such as cellulose in leaves (Amato et al., 2014), whereas Bacteroides members can help decompose non-structural carbohydrates such as fructose in fruits.

The results of the LefSe analysis showed that in adult Hainan gibbons, Ruminococcus, Clostridium, and Butyrivibrio were significantly more abundant than in young Hainan gibbons, whereas Prevotella and Bacteroides were significantly more abundant in young gibbons. This result is similar to the result of high abundance of Prevotella in the intestine of Macaca mulatta (Zhou, 2019). Prevotella plays an important role in the decomposition and utilization of hemicellulose, pectin, starch, and monosaccharides (Flint, 2004); this is related to the consumption on fruits, which contain a large amount of pectin. It is apparent that adult Hainan gibbons are better adapted to digesting structural carbohydrates in leaves (Ding et al., 2001), whereas young Hainan gibbons are better adapted to decomposing non-structural carbohydrates in fruits. Zhou (2019) also assessed the intestinal microbiome of Macaca mulatta of different ages. They found that compared with the young group, the adult group was significantly enriched with Ruminococcaceae members, which have the function of degrading cellulose and hemicellulose, thereby providing an energy source to the host. Lactobacillus is significantly enriched in young individuals, and it usually acts as a probiotic to the host. Eating leaves results in higher digestion costs and lower energy returns than eating fruits, and growth, development, pregnancy, and lactation increase nutritional needs in mammals (Dufour & Sauther, 2002). Similarly, in primates, energy requirements increase with the growth and development of individuals, and young individuals have higher nutritional and energy needs than adult individuals. Similarly, Amato et al. (2014) concluded that young wild black howler monkeys have higher nutritional needs than adult individuals. This suggests that Hainan gibbons of different ages adopt different feeding strategies according to their physiological characteristics. Previous studies have confirmed that primates have a unique microbial community under conditions of no anthropogenic disturbance in the wild. However, under the influence of artificial feeding, the change in food composition changes the original microbial community; it changes along the direction similar to the composition of human microbiota, and finally converges with human intestinal microbiota (Clayton et al., 2016; Manara et al., 2019).

The imputed relative abundance of the COG and KEGG pathways revealed similarities and differences in the functional profiles of the adult and young Hainan gibbons. Compared with the intestinal microbiome of young Hainan gibbons, the intestinal microbiome of adult Hainan gibbons generally harboured a higher abundance of genes related to metabolic processes, such as metabolism of carbohydrates, amino acids, xenobiotics, inorganic ions, nucleotides, terpenoids, and polyketides. This may be due to adaptive advantages in adult Hainan gibbons, such as a stable and mature intestinal microbiome composition that allows them to utilise diverse foods efficiently. The diversity of carbohydrate active enzymes in adult and young Hainan gibbons was similar, but the abundance of carbohydrate active enzymes was different. The results showed that the abundance of GHs was the highest in the two groups, followed by GTs, which reveals that the genes related to carbohydrate transport and metabolism of intestinal microbiome in adult and young Hainan gibbons are the most abundant. This result is similar to that of other studies on animals that mainly eat leaves (Jose et al., 2017; Zhang et al., 2020). In addition, the abundance of enzymes involved in cellulose and hemicellulose degradation in adult Hainan gibbon was higher than that in young Hainan gibbon. Whereas, the abundance of enzymes involved in oligosaccharide degradation in young Hainan gibbon was higher than that in adult Hainan gibbon. This may be because the activities of cellulolytic bacteria and their secreted digestive enzymes dependent on cellulose to some extent. Therefore, the bacteria that degrade fiber, such as Ruminococcus, are the dominant bacteria when eating leaves containing fiber, and they secrete a large amount of cellulose- and hemicellulose-degrading enzymes. When eating more carbohydrate-rich fruits, Prevotella, which degrade non-fibrous plant components such as pectin and starch, are the dominant bacteria; thus, they secrete a large amount of oligosaccharide- and pectin-degrading enzymes.

Conclusions

The Hainan gibbon is one of the most endangered primate species worldwide and the least studied gibbon species in the world. For young and adult Hainan gibbons, different feeding strategies led to differences in the composition and function of the intestinal microbiome. The adult Hainan gibbon intestinal microbiome generally harbours a high abundance of genes related to metabolic processes, such as metabolism of carbohydrates, amino acids, and nucleotides. This may be due to adaptive advantages in adult Hainan gibbons, such as a stable and mature intestinal microbiome composition that allows them to utilise diverse foods efficiently. Therefore, this study helps understand the dynamic changes in the intestinal microbiome of Hainan gibbon during youth and adulthood and plays a key role in the health monitoring and rejuvenation of its population.

Supplemental Information