Linking gut microbiome with the feeding behavior of the Arunachal macaque (Macaca munzala)

Exploring the gut microbiome is an emerging tool for monitoring wildlife health and physiological conditions which often sustained under the variety of stresses and challenges. We analyzed gut microbiome of Arunachal macaque (Macaca munzala) of two disjunct populations from Arunachal Pradesh, India, to validate whether the geography or the feeding habits plays a principal role in shaping the gut microbiome in natural populations. We observed geography has a mere effect but feeding habits (i.e. feeding upon the leftover food and crop-raiding) significantly influenced the gut microbiome composition. The phylum Proteobacteria found to be enriched in leftover feeding group while phylum Bacteroidetes was differentially abundant in crop-raiding group. We observed predominant phyla Firmicutes followed by Proteobacteria and Bacteroidetes with the dominant classes represented by the Clostridia. Interestingly, one individual with known diarrheal/metabolic disorder exhibited complete dominance of the order Bacillales and showed 100% sequence similarity with genus Solibacillus. We raise concern that shift in diet of macaques may compel them to expose for various human diseases as two macaques feeding upon the leftover food exhibited dysbiotic gut microbiome. The present study provides the pragmatic evidences of how the alteration of food resources can harm the physiological condition of the macaques in wild and raises alarm to the forest officials/managers in strategise planting of natural food resources and monitor anthropogenic activities in the distribution of Arunachal macaques.

www.nature.com/scientificreports/ We on the pilot attempts displayed an altered gut bacterial composition in Arunachal macaque which surprisingly resembled with humans indicating nature of anthropogenic food provisioning 26 . Based on our field observations of Arunachal macaques, we found two types of feeding behavior, one group encountered to feed upon the left over human food at Tawang and West Kameng, while the second group was crop-raiding and mostly encountered to feed upon the cultured vegetables at West Kameng. Thus, we testify the hypothesis which factor-the geographical origin or the feeding behavior (diet type) influence the gut microbiome of Arunachal macaques in Tawang and West Kameng (Fig. 1).

Methods
Sample collection. We collected ten fresh fecal samples of M. munzala which were defecated in front of the research team and stored immediately in 95% ethanol following Song et al. 27 . All fecal samples were collected during December 2019 and we presumed that gut microbiome might not be influenced due to the seasonal availability/variability of food resources. Based on the geographical origin, these samples represented two wild populations, Tawang (Group A of two samples) and West Kameng (Group B and C of four samples each). Two individuals (MT1398 and MT2182) from Tawang belong to two different troops often observed to feed upon the human leftover food from dumping sites. Four individuals from West Kameng (group B) belong to a resident troops living near a monastery and had similar feeding behavior like group A, while the other four individuals of group C were the crop raiding troops. In addition, we also included data from our previous study 26 in group A. Interestingly, we also monitored a macaque from West Kameng (group B; #MT2173) that seemed to experience diarrheal/metabolic disorder (Fig. S1). We extracted DNA from the inner and outer part of the fecal samples following QIAamp DNA Stool Mini Kit (Qiagen, Germany) for identifying species, individuals and gut microbiome analysis.

Species and individual identification.
Since, other sympatric macaque species were also present in the study area, we first identified species of macaques by sequencing the partial fragment of mitochondrial d-loop 28 . Maximum likelihood (ML) phylogenetic tree was reconstructed in MEGA X 29 with 1000 bootstrap runs using Hasegawa-Kishino-Yano model and gamma distribution of substitution rate (HKY + G) to confirm the species of origin. Although, the fecal samples were defecated in front of research team, we still genotyped each sample thrice with nine polymorphic microsatellite loci for individual identification to avoid the same sample being analyzed for gut microbiome analyses 26,30 . The obtained genotypes were analyzed to determine unique individuals using GenAlEx v6 31 and genealogical relationship among individuals was determined using ML-RELATE 32 .
Library preparation and amplicon sequencing. We  Data analyses. The obtained sequences were processed using Mothur MiSeq SOP pipeline for analysis of 16S rRNA sequences 37,38 . We allowed maximum of 8 homopolymers in a sequence and removed all sequences containing N. Further, the chimeric sequences were searched using the VSEARCH algorithm 39 and the taxonomic assignment of the sequences made using Bayesian classifier with 80% bootstrap support with Silva v138.1 reference data 40 . The sequences of mitochondria and chloroplast were removed. Sequences with simi- www.nature.com/scientificreports/ larities > 97% considered as single OTU. We visualized Mothur output using Phyloseq package of R 41 . The significance of the differences in Alpha diversity indices i.e., ACE, Observed OTUs, Chao1, Shannon, Simpson and Inverse Simpson were estimated using microbial R package 42 . Top 50 OTUs were selected on the basis of abundance in the samples to visualize bacterial composition within the samples. The beta diversity estimations were performed through Principal Coordinate (PCA) and Cannonical Correlation Analysis (CCA) using weighted UniFrac distances 43 . We compared the fecal microbiome between macaque groups with different feeding behaviour (leftover feeding and crop-raiding) and geographical origin (Tawang and West Kameng) through Permutational Multivariate Analysis of Variance (PERMANOVA) test implemented in vegan R package using Bray-Curtis distance. The effect of feeding habits on gut microbiome was analysed between the crop-raiding and leftover feeding groups after exclusion of MT1398 from group A and MT2173 from group B as they showed dysbiosis. We identified differentially abundant taxa between the study groups using LeFSe test. Metagenomic function prediction was performed using Tax4Fun2 R package. Differentially abundant features were identified at level 2 and level 3 KEGG Orthology groups (KOs) through LEfSe analysis in Galaxy server of Huttenhower lab 44 . Metabolic pathways were compared between crop-raiding and leftover feeding groups and between dysbiotic (MT2173and MT1398) and non-dysbiotic (other samples).

Ethics declarations.
This was exempted as all samples were collected non-invasively without disturbing the macaques. Necessary permission was obtained from the State Forest Department Arunachal macaque.

Results
Species identification and assigning genealogical relationship. The obtained sequences clustered with known sequences of M. munzala in ML phylogenetic tree with high bootstrap support (Fig. S2). We observed that all analysed individuals were unrelated except two individuals-MT 2175 and MT2180, which were full-siblings and belonging to the group-B and group-C, respectively.
Gut microbiome composition and alpha diversity. We obtained a total of 662 OTUs in 11 samples and the Firmicutes was the principal phylum followed by Proteobacteria and Bacteroidetes (Fig. 2). The Clostridia was found to be the dominant class in all samples except sample-'MT2173'and 'MT1398' . The sample, 'MT2173' represented complete dominance (~ 95%) of the order Bacillales, contributed by a single OTU (OTU1) and showed 100% sequence similarity with genus Solibacillus. We did not find any significant difference between sample groups in any of the alpha diversity measures i.e. Observed OTUs, Shannon, Simpson, Chao1 and ACE (Fig. 3) while the 'MT2173' appeared as outlier in Shannon, Simpson, Chao1 and ACE indices. Another sample, 'MT1398' from group-A showed a lower level of dysbiosis with excess of Lentisphaerae. www.nature.com/scientificreports/ Beta diversity and differential abundance. The cluster dendrogram using weighted UniFrac distance also showed that MT2173 had distinct composition of bacterial communities which is not related with other samples (Fig. 4a). The PCA using weighted UniFrac distance showed PC1-30.5%, PC2-19.9% and PC3-14.5% variation among the samples (Fig. 4b). The CCA showed CA1-22%, CA2-15.2% and CA3-13.8% variation among the samples and the CA1 segregated the sample MT2173 from remaining samples (Fig. 5). Furthermore, we found that three of the four samples belonging to group-C formed distinct clustered in PCoA, CCA and cluster dendrogram. However, upon PERMANOVA test, we did not observe significant difference between leftover-feeding (group A and B) and crop-raiding (group c) (p = 0.166: Fig. 6a). One sample-MT2181 from group C clustered with other samples of group A and B that assumed to feed on leftover despite belonging to crop raiding group (group C). Therefore, we performed PERMANOVA test again after removing the odd sample-MT2181. The results showed significant compositional difference between two groups with different feeding habit (p = 0.022: Fig. 6b). In contrast, we did not see geography has any role in shaping the gut microbiome (p = 0.475: Fig. S3). Further, we found that phylum Proteobacteria is enriched in leftover feeding group while phylum Bacteroidetes is differentially abundant in crop-raiding group (Fig. 7).

Discussion
During monitoring of macaques in field, we recorded that in Tawang, macaques often fed upon the leaves as well as on the human leftover food remains while macaques in West Kameng were observed to forage upon the cultivated crops as well as on the left over food. Here, we observed clustering of three out of four samples of the crop raiding group that indicates feeding habits is the principal driver for shaping the composition of the gut microbiome instead the geography. Further, the result from PERMANOVA test showed significant differences between crop-raiding group and leftover feeding group (p = 0.022) after exclusion of two dysbiotic ('MT2173' and 'MT1398') and one odd sample (MT2181). Thus, we found that feeding habits played a key role in shaping the gut microbiome in Arunachal macaque as revealed by the compositional shift in the gut microbiome between two groups. Further, we did not obtain any difference in the level 2 KO pathways possibly due to the limited characterization of bacterial species from environmental samples. A few studies identified dysbiosis of gut microbiome with low alpha diversity in captive primates caused by increased stress, shift in diet and other external factors 2,16,17 but dysbiosis in wild primates is not well reported. In the present study, we monitored an individual (MT2173) which had diarrhoea and seemed to be physically weak. Interestingly, this individual had distinct composition of bacterial community (complete dominance of Solibacillus sp.) and grouped distantly from others in cluster dendrogram. A previous study on rumen acidosis in dairy cows demonstrated positive correlation of Solibacillus with valerate, a branched chain volatile fatty acid [BCVFA] 45 , though the underlying mechanism behind the association is not clear yet. Interestingly, all the dysbiotic samples belong to leftover feeding group. This shift in diet prevails risk of various diseases which may become a threat for long term survival of this endangered macaque. Further, dysbiotic gut microbiota is reported to be harmful in several cases 46,47 and so we raise our concern to the shift in the feeding habits of Arunachal macaques in Arunachal Pradesh. It is noteworthy that West Kameng district of Arunachal Pradesh possesses www.nature.com/scientificreports/ high human-macaque conflict due to crop-raiding by macaques and retaliatory killings of macaques by locals 23,48 .
During the present study, we encountered group C macaques to forage upon the cultivated crops like cabbage, maize and tomato which plausibly shaped distinct gut microbial composition. Notably, the macaque group (A & B) that feed upon the leftover food showed substantial fractions of shared bacterial taxa than the group C macaques having crop-raiding feeding habits. This is evident to prove that feeding habits have the principal role in shaping gut microbiome in Arunachal macaques. Further, the Tawang and West Kameng represent notable differences in the forest types. Though, Tawang has relatively large proportion of high elevation scrublands (4000-5250 m asl.), the habitats of Arunachal macaques in both districts comprise of temperate broadleaf (up to 3000 m) and mixed conifer and broadleaf forests (3000-4200 m) 48,49 . Monpa is the principal community in Tawang and they do not hunt due to their cultural beliefs 50 but members of other tribes practice hunting 48 . While, a few forested habitats in Tawang are represented by community reserves where hunting is prohibited 51 . We observed macaques to retain an inherent behaviour of adaptation and they are often sighted nearby villages. Based on our field experience, we found their movements are largely governed towards the sites of ease availability of food resources. Thus, feeding habits have profound impact in shaping gut microbiome in Arunachal macaque over the geography. Since, we obtained significant differences in the bacterial taxa between macaques having different feeding habits, we also believe associating feeding behavior, nutrient characterization of the diet and the gut microbiome can provide valuable information in the context of linking bacterial taxa and with the various metabolic pathways or in digestion of the different types of food materials. The present study identified dysbiotic microbiome in M. munzala and provides empirical evidences for immediate conservation needs. Therefore, leftover-feeding may impose major threat by changing the natural diet of the macaques and increases the possibility of transfer of pathogen from human to macaques and vice-versa. Therefore, we propose that restoring the natural habitat of M. munzala by removal of waste, fencing the croplands and plantation of their natural food plants may help in conservation of this endangered macaque which in turn also minimize the level of human-macaque conflicts.