Gut microbiome dynamics associated with dietary shifts
Gut bacterial community diversity increased when transitioning from the OMD to OBD diets of pandas, while richness exhibited an opposite trend. Gut bacterial community richness has been observed as higher in pandas with lower fiber diets compared to those with higher fiber diets (Guo et al 2018, Wu et al 2017). These results may be explained by the variable structural complexity of fiber and the relatively low richness of bacteria that can use fiber as a growth substrate (Lynd et al 2002). However, high fiber diets can increase the diversity of GM in humans, while high fat diets are associated with lower diversity (Carlotta et al 2010, Tap et al 2016, Zhernakova et al 2016). Competitive interactions among bacteria are ubiquitous in natural systems, although many studies have shown that lignocellulose is a complex substrate that promotes positive interactions and synergistic growth of bacterial populations compared to labile substrates like glucose and fat (Haruta et al 2002, Sarunyou et al 2012, Deng et al2016). Indeed, lignocellulose is a cross-linked structure that is difficult to degrade. Thus, bacteria may need to form consortia to synergistically achieve lignocellulose degradation (Deng et al 2016, Perez et al 2002). Thus, fiber content could be an important factor underlying variation in richness and diversity of gut microbial populations among the pandas in the OMD, MBD, and OBD groups. Consequently, our results support that high fiber diets could increase the diversity, but decrease the richness of gut bacterial communities in giant pandas.
Among all sampled gut communities, the Proteobacteria and Firmicutes dominated, which is consistent with previous studies of giant panda GMs (Yang et al 2018, Zhang et al 2018). Proteobacteria may be more dominant in the guts of herbivores with low metabolic rates (Dill-McFarland et al 2016), which is consistent with the low expenditure and physical activity of giant pandas (Yonggang et al 2015). Firmicutes are typically dominant in the guts of mammalian herbivores and play critical roles in fiber digestion (Dill-McFarland et al 2016, Nelson et al 2010). Interestingly, Firmicutes abundances exhibited a gradient in the transition from OMD to OBD group communities. Firmicutes abundances have also been positively associated with fiber content in human guts (Carlotta et al 2010), in addition to significantly associated with supplemented dietary fiber in dogs (Costa et al 2012). The positive association of Firmicutes abundances with lignocellulose ingestion in giant pandas could suggest that they are important for digesting high fiber bamboo foods into more labile nutritional components.
At the genus level, Streptococcus (Firmicutes phylum) abundances have been shown to significantly increase upon introduction of a bamboo diet (Ouwehand et al 2010). Moreover,Streptococcus are associated with giant panda gut mucus (Williams et al 2016) that is critical for dietary conversions of giant pandas from low diet to high fiber diets. Mucus helps protect guts from injuries due to high fiber contents and aids the movement of high fiber components through the gut (Montagne et al 2003). We observed the presence of the gene encoding Protein-Npi-phosphohistidine-cellobiose phosphotransferase (EC 2.7.1.205, celB ) in the Streptococcus MAG, which is important for cellulose digestion (Lai et al 1997), thus indicating the potential for cellobiose utilization by the Streptococcus in these panda gut communities. In addition, genes encoding beta-glucosidase (EC 3.2.1.21, bglB ) and 6-phospho-beta-glucosidase (EC 3.2.1.86 celF ) were identified in the Streptococcus MAG, as inferred from comparison to the KEGG databases. Beta-glucosidase (EC 3.2.1.21) and 6-phospho-beta-glucosidase (EC 3.2.1.86) are both involved in cellulose digestion (Ghorai et al , Rytioja et al 2014). In particular, GHs are often associated with digestion of cellulose and hemicellulose (Stewart et al 2018), and were accordingly identified in the Streptococcus MAG via comparison to the CAZy databases. It should be noted that cellulose and hemicellulose are cross-linked with lignin, and the removal of lignin is the first step in digesting cellulose and hemicellulose (Rytioja et al 2014). Accordingly, several enzyme-encoding genes involved in hemicellulose and lignin degradation were present in the Streptococcus MAG including CE1, CE3, CE4, CE5, AA3, AA4, AA6, and AA7 group genes (Zhang et al 2018, Zhen and Jr 2016). Moreover, several other cellulose, hemicellulose, and lignin degradation associated genes were also observed in the Pseudomonas , Enterococcus, Lactococcus, and Acinetobacter MAGs including cellulase (EC 3.2.1.4) and 1,4-beta-cellobiosidase (EC 3.2.1.91). The combined activities of cellulase and 1,4-beta-cellobiosidase can convert cellulose into cellobiose, and cellobiose is a key intermediate in the conversion of cellulose to D-glucose (Lifeng et al 2011). Consistent with these genomic predictions, Pseudomonas, Clostridium,Lactobacillus, Enterococcus, Lactococcus and Acinetobacterabundances exhibited a gradient of increase when transitioning from the OMD to OBD group pandas. Clostridium and Enterococcus have been positively correlated with crude fiber digestibility, whileLactobacillus , Enterococcus, andPseudomonas have been positively associated with acid detergent fiber digestibility (Niu et al 2015). Moreover, the involvement of Pseudomonas andAcinetobacter in the degradation of lignin has been previously demonstrated (Jiménez et al 2015). Thus, our results indicate that Streptococcus , Pseudomonas ,Enterococcus, Lactococcus, Acinetobacter, andClostridium may contribute to the utilization of cellulose and hemicellulose from bamboo, thereby providing energy and nutrients for their giant panda hosts.
Interestingly, gut bacterial communities of giant pandas were more similar to those of carnivores than herbivores in a previous comparison of human GM and 59 other mammalian species (Ley et al 2008). Likewise, Xue et al. observed that the composition of gut bacterial communities in giant pandas were more similar to those of bears and entirely distinct to those of herbivores via comparison among 57 mammalian species including giant pandas, its close relatives, typical carnivores, and distantly related herbivores (Xue et al 2015). We therefore hypothesize that the gut bacterial communities of bears and even carnivores have the potential to metabolize fiber or otherwise that these bacterial communities have evolved in concert with giant panda evolution. Nevertheless, additional research is needed to evaluate the above hypothesis.
No significant differences were observed in the richness and diversity of fungal communities among the three dietary groups. Interestingly, Basidiomycota abundances significantly increased in the transition from the OMD to OBD diets, suggested that they may play a role in the utilization of bamboo by giant pandas. Ascomycota and Basidiomycota have been previously shown to dominate the fungal gut communities of giant pandas (Tun et al 2014, Zhang et al 2018b), which coincides with their dominance in soil (Xu et al 2012) and bamboo (Zhou et al 2017) fungal communities. These observations have led to the hypothesis that giant panda gut microbiomes may originate from their food sources or even from soils (Hannula et al 2019, Nina et al 2013). Candida was the dominant fungal genus in the OMD communities and significantly declined in abundance in the transition from the OMD to OBD diets. Candidaabundances in gut fungal communities have been strongly associated with the consumption of carbohydrates (Christian et al 2013, Iannotti et al 1973). Milk has higher carbohydrate contents than bamboo (Mainka et al 1989), suggesting thatCandida may be involved in milk metabolism in the guts of newborn giant pandas.Cystofilobasidium ,Guehomyces,and Gibberella abundances increased markedly in the transition from the OMD to OBD diets (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA>4). Therefore, we also hypothesized thatCystofilobasidium , Guehomyces, and Gibberella may contribute to the ability of giant pandas to digest bamboo.