Diet-related factors strongly shaped the gut microbiota of Japanese macaques.

Although knowledge of the functions of the gut microbiome has increased greatly over the past few decades, our understanding of the mechanisms governing its ecology and evolution remains obscure. While host genetic distance is a strong predictor of the gut microbiome in large-scale studies and captive settings, its influence has not always been evident at finer taxonomic scales, especially when considering among the recently diverged animals in natural settings. Comparing the gut microbiome of 19 populations of Japanese macaques Macaca fuscata across the Japanese archipelago, we assessed the relative roles of host genetic distance, geographic distance and dietary factors in influencing the macaque gut microbiome. Our results suggested that the macaques may maintain a core gut microbiome, while each population may have acquired some microbes from its specific habitat/diet. Diet-related factors such as season, forest, and reliance on anthropogenic foods played a stronger role in shaping the macaque gut microbiome. Among closely related mammalian hosts, host genetics may have limited effects on the gut microbiome since the hosts generally have smaller physiological differences. This study contributes to our understanding of the relative roles of host phylogeography and dietary factors in shaping the gut microbiome of closely related mammalian hosts.

Mineral acquisition of Japanese macaques: Contents in the foods, digestibility, and sodium-provisioning experiment.

Minerals provide micronutrients that function in various ways in the body, and they are necessary for the survival of animals. In this study, we first compared the mineral content of foods of wild Japanese macaques in lowland Yakushima with that of monkey chow used for many years to feed captive macaques and specifically formulated to obtain good health in captive macaques (National Research Council [NRC] recommendations). Second, we clarified the mineral balance in captive individuals when feeding them monkey chow to clarify the digestibility/bioavailability of the minerals. Third, we investigated the physiological response when we experimentally increased sodium intake. In the lowland of Yakushima, which is in the vicinity (<800 m) of the coast, animals, fungi, and mature leaves had high sodium contents compared with NRC recommendations. The calcium contents of mature leaves and animals were higher than the NRC recommendations. The overall mineral intake in this population was lower than that in the captive animals for calcium, phosphorus, sodium, and iron, while similar for magnesium and higher in potassium. Patterns in the intake and excretion of minerals indicated that excretion was mostly from urine and not from feces, and apparent digestibility was high for sodium. This tendency was opposite for calcium, magnesium, and phosphorus and intermediate for potassium. A sodium-provisioning experiment showed that fecal aldosterone concentration remained low in both control and sodium-provisioning conditions so the macaques do not need reabsorb sodium in the kidneys. Therefore, sodium content in the monkey chow, which is slightly lower than the NRC recommendation, seemed high enough so that the macaques could avoid the need to reabsorb sodium in the kidneys. We advocate similar studies for other primate populations or species to better understand the role of mineral concentrations on food selection and to identify potential mineral deficiencies.

In vitro digestion and fermentation of Japanese macaque (Macaca fuscata) food: The influence of food type and particle size.

Chewing is critical for herbivores to obtain nutrients. Measuring digesta particle size as the outcome of chewing can improve our understanding of the relationship between food and digestion. Previous studies of feeds of domestic animals have shown that smaller digesta particle size leads to more efficient digestion. Increased digesta particle size-either due to animal factors (e.g., a senile dentition) or to feed factors (e.g., fracture resistance) could be a sign of an animal experiencing compromised nutritional intake. However, for some primates that are dietary generalists, digesta particle size has been shown to increase when consuming preferred foods, which raises doubts about the role of chewing in digesting such foods. This uncertainty makes it difficult to understand the connection between diet, chewing, and digestion through digesta particle size in dietary generalists. In this study, using five typical food items from the Japanese macaque (Macaca fuscata) diet, we conducted in vitro digestibility and fermentation assays to explore the effects of particle size on enzymatic and microbial digestion. For the fermentation assays, we used feces from captive Japanese macaques as inoculum. Among the five food items, we found that particle size has a stronger influence on the digestibility of seeds and mature leaves compared to young leaves and pulp. The influence of particle size on the fermentation rate was stronger in pulp and seeds compared to that in leaves. The differences in physical structure, texture, digestion barriers, and soluble components may play important roles in such differences. These results support the hypothesis that reducing food particle size is less important for consuming fruits than for consuming leaves. The limited effects of particle size on digesting fruits suggest that the two fruits examined in this study are cost-effective concerning food processing and chewing.

Seasonal responses and host uniqueness of gut microbiome of Japanese macaques in lowland Yakushima.

BACKGROUND: Changes in the gut microbial composition is an important response to cope with the seasonal fluctuations in the environment such as food availability. We examined the bacterial gut microbiome of the wild nonhuman primate, Japanese macaque (Macaca fuscata) in Yakushima over 13 months by noninvasive continuous sampling from three identified adult females. RESULTS: Dietary composition varied considerably over the study period and displayed marked shifts with the seasons. Feeding of leaves, fruits, and invertebrates were their main foods for at least one month. Diet had a significant influence on the gut microbiome. We also confirmed significant effect of host uniqueness in the gut microbiome among the three macaques. Leaf-dominated diet shaped unique gut microbiome structures where the macaques had the highest alpha diversity and their gut microbiome was enriched with Spirochaetes and Tenericutes. Diet-related differences in the putative function were detected, such as a differentially abundant urea cycle during the leaf-feeding season. CONCLUSION: Both diet and host individuality exerted similar amounts of effect on gut microbe community composition. Major bacterial taxa showed a similar response to monthly fluctuations of fruit and invertebrate feeding, which was largely opposite to that of leaf feeding. The main constituents of fruits and invertebrates are both digestible with the enzyme of the host animals, but that of leaves is not available as an energy source without the aid of the fermentation of the gut microbiome.

Temporal activity patterns suggesting niche partitioning of sympatric carnivores in Borneo, Malaysia.

To propose proper conservation measures and to elucidate coexistence mechanisms of sympatric carnivore species, we assessed temporal activity patterns of the sympatric carnivore species using 37,379 photos collected for more than 3 years at three study sites in Borneo. We categorized activity patterns of nine carnivore species (one bear, three civets, two felids, one skunk, one mustelid, one linsang) by calculating the photo-capturing proportions at each time period (day, night, twilight). We then evaluated temporal activity overlaps by calculating the overlap coefficients. We identified six nocturnal (three civets, one felid, one skunk, one linsang), two diurnal (one felid, one mustelid), and one cathemeral (bear) species. Temporal activity overlaps were high among the nocturnal species. The two felid species possessing morphological and ecological similarities exhibited clear temporal niche segregation, but the three civet species with similar morphology and ecology did not. Broad dietary breadth may compensate for the high temporal niche overlaps among the nocturnal species. Despite the high species richness of Bornean carnivores, almost half are threatened with extinction. By comparing individual radio-tracking and our data, we propose that a long-term study of at least 2 or 3 years is necessary to understand animals' temporal activity patterns, especially for sun bears and civets, by camera-trapping and to establish effective protection measures.

Effects of diet and age-sex class on the fecal particle size of wild Japanese macaques (Macaca fuscata yakui).

Fecal particle size provides important information on the feeding and digestion of herbivores. Understanding the effects of the potential proximate determinants on fecal particle size helps us interpret this widely used measurement. In folivores, previous studies found that diet composition, dietary toughness, and age-sex-related factors, such as body size and tooth wear, influenced fecal particle size. However, the role of these factors remains unknown in frugivorous and omnivorous primates. This study aims to clarify how age-sex class and diet influence fecal particle size in omnivorous Japanese macaques in Yakushima. We expected that their variable diet and differences among age-sex classes would cause variations in fecal particle size. We simultaneously documented Japanese macaques' diet, dietary toughness, and fecal particle size in the lowland area of Yakushima in the period from March 2018 to April 2019. Unexpectedly, fecal particle size showed limited differences across months and no difference among age-sex classes. Dietary toughness showed no effects on fecal particle size, while the consumption of fruits showed only a marginally significant negative effect. Our data indicate that the results of chewing were not affected by dietary toughness in our study subjects, while age-sex classes showed no difference in food comminution. This lack of variation might derive from a diet with low dietary toughness. We also found that the physical structure of preferred foods played an important role in fecal particle size variations. These results suggest that food comminution is less variable in frugivorous and omnivorous primates compared to highly specialized species (e.g., geladas). Factors other than what we examined in this study, such as food physical structure and chewing behavior, should also be taken into consideration.

Stomach and colonic microbiome of wild Japanese macaques.

Within the gastrointestinal tract, the physiochemical microenvironments are highly diversified among the different stages of food digestion. Accordingly, gut microbiome composition and function vary at different gut sites. In this study, we examine and compare the compositional and functional potential between the stomach and colonic microbiome of wild Japanese macaques (Macaca fuscata yakui) living in the evergreen forest of Yakushima Island. We find a significantly lower microbial diversity in the stomach than in the colon, possibly due to the stomach's acidic and aerobic environment, which is suboptimal for microbial survival. According to past studies, the microbial taxa enriched in the stomach are aero- and acid-tolerant. By functional prediction through PICRUSt2, we reveal that the stomach microbiome is more enriched in pathways relating to the metabolism of simple sugars. On the contrary, the colonic microbiota is more enriched with fiber-degrading microbes, such as those from Lachnospiracea, Ruminococcaceae, and Prevotella. Our study shows a clear difference in the microbiome between the stomach and colon of Japanese macaques in both composition and function. This study provides a preliminary look at the alpha diversity and taxonomic composition within the stomach microbiome of Japanese macaques, a hindgut-fermenting nonhuman primate.

Factors influencing riverine utilization patterns in two sympatric macaques.

Many species of terrestrial animals, including primates, live in varied association with the aquatic (e.g., riverine or coastal) environment. However, the benefits that each species receive from the aquatic environment are thought to vary depending on their social and ecological characteristics, and thus, elucidating those benefits to each species is important for understanding the principles of wild animal behaviour. In the present study, to gain a more complete picture of aquatic environment use, including social and ecological factors in primates, factors affecting riverine habitat utilization of two macaque species (Macaca nemestrina and M. fascicularis) were identified and qualitative comparisons were made with sympatric proboscis monkeys (Nasalis larvatus), which have different social and ecological characteristics. Temporal variation in sighting frequency of macaques at the riverbanks was positively related to the fruit availability of a dominant riparian plant species and negatively related to the river water level which affects the extent of predation pressure. Riverine utilization of macaques was greatly influenced by distribution and abundance of food (especially fruit) resources, possibly in association with predation pressure. Additionally, qualitative ecological comparisons with sympatric proboscis monkeys suggest that the drivers of riverine utilization depend on the feeding niches of the species, and different anti-predator strategies resulting from their differing social structures.

Ranging patterns of Japanese macaques in the coniferous forest of Yakushima: Home range shift and travel rate.

Ranging is one of the most important behavioral adaptations for coping with seasonally fluctuating food and thermal conditions. We studied the ranging patterns, in particular home range shift and travel rate of Japanese macaques (Macaca fuscata) in the coniferous forest of Yakushima by tracking a group for 17 months. We also supplemented our data with records collected every August over a 5-year annual census. The macaque group used the eastern part of their home range from May to September and the western part during the rest of the year. The eastern part of the home range was largely primary forest in the national park, and the altitude was higher than in the western part. When they used the western part, the macaques ate more herbs and fruits from small-sized trees, the availability of which was higher in the logged forest. This east-west home range shift occurred repeatedly over multiple years. A neighboring group occupied the western part of the focal group's home range in summer, which the focal group did not use in that season. Both temperature and diet affected seasonal changes in the monthly average travel rate. Animals need more energy for thermoregulation when the temperature is low, so the macaques decreased their ranging efforts to save energy at times of low temperature. They increased their ranging distance to eat fungi, since their encounters with this food would increase with the total distance walked. They also increased their travel rate when eating flowers, which had lower food patch (tree) density than other foods such as fruits. The data supported the hypothesis that the macaques capitalize on habitat heterogeneity in a seasonally fluctuating habitat by shifting their home range and modifying their travel rate.

Habitat use by a primate community in a lowland dipterocarp forest in Danum Valley, Borneo.

Knowledge of niche partitioning with respect to habitat is indispensable to understand the mechanism of coexistence of multiple species. Among primates, however, data are still deficient because repeated survey for a sufficiently long time, covering seasonal changes over a large area, is the only way to clarify habitat segregation within a seasonally fluctuating environment. Southeast Asia is particularly interesting because of the supra-annual, highly unpredictable seasonality in fruiting known as mast fruiting. We conducted repeated route census, habitat monitoring, and group tracking for 25 months in two study sites (ca. 10 km apart) in the largely primary lowland dipterocarp forest of the Danum Valley Conservation Area, eastern Sabah, northern Borneo, Malaysia. The five species of diurnal primates (Bornean orangutan Pongo pygmaeus, Müeller's gibbon Hylobates muelleri, red leaf monkey Presbytis rubicunda, long-tailed macaque Macaca fascicularis, and southern pig-tailed macaque M. nemestrina) did not show horizontal spatial segregation. Red leaf monkeys showed preferences for places with short tree height, but their distribution was not confined to such places. In response to the fruiting peak observed once during the study period, orangutans increased their numbers simultaneously in the two study sites. The average tree height used by the five species was different, but their range overlapped substantially. Compared with other primate communities, the lack of horizontal spatial segregation and the suggested long-distance movement of orangutans seem to be unique characteristics in Borneo, although the use of different forest strata is a widespread phenomenon among primate communities throughout the world.

Fermentation Ability of Gut Microbiota of Wild Japanese Macaques in the Highland and Lowland Yakushima: In Vitro Fermentation Assay and Genetic Analyses.

Wild Japanese macaques (Macaca fuscata Blyth) living in the highland and lowland areas of Yakushima are known to have different diets, with highland individuals consuming more leaves. We aim to clarify whether and how these differences in diet are also reflected by gut microbial composition and fermentation ability. Therefore, we conduct an in vitro fermentation assay using fresh feces from macaques as inoculum and dry leaf powder of Eurya japonica Thunb. as a substrate. Fermentation activity was higher for feces collected in the highland, as evidenced by higher gas and butyric acid production and lower pH. Genetic analysis indicated separation of highland and lowland in terms of both community structure and function of the gut microbiota. Comparison of feces and suspension after fermentation indicated that the community structure changed during fermentation, and the change was larger for lowland samples. Analysis of the 16S rRNA V3-V4 barcoding region of the gut microbiota showed that community structure was clearly clustered between the two areas. Furthermore, metagenomic analysis indicated separation by gene and pathway abundance patterns. Two pathways (glycogen biosynthesis I and D-galacturonate degradation I) were enriched in lowland samples, possibly related to the fruit-eating lifestyle in the lowland. Overall, we demonstrated that the more leaf-eating highland Japanese macaques harbor gut microbiota with higher leaf fermentation ability compared with the more fruit-eating lowland ones. Broad, non-specific taxonomic and functional gut microbiome differences suggest that this pattern may be driven by a complex interplay between many taxa and pathways rather than single functional traits.

Seasonal variation in energy balance of wild Japanese macaques (Macaca fucata yakui) in a warm-temperate forest: a preliminary assessment in the coastal forest of Yakushima.

Food scarcity is a major challenge for primates living in temperate forests, where food availability varies markedly among seasons. In Japanese macaques, which are exclusively distributed in temperate zones, the fat accumulation ability has been highlighted as an adaptation for survival during the lean season and for reproductive success. However, the knowledge of energetic strategies of Japanese macaques has been mainly derived from data on cool-temperate forests, where fallback foods comprise winter buds and bark. Data on Japanese macaques in warm-temperate forests where fallback foods comprise mature leaves are still lacking. We aimed to identify seasonal variations in energy balance and the relative importance of ingestion rates (dry matter intake per feeding time), energy content of the food, and feeding time in energy intake of Japanese macaques in the coastal forest of Yakushima. We estimated energy balance of 6-12 adult females from October 2012 to October 2013. We estimated energy intake based on the data on feeding behavior and energy content of the diet and calculated energy expenditure based on the previously established relationship between body mass and total energy expenditure. We also quantified urinary C-peptide, which is a non-invasive biomarker of energetic conditions. We demonstrated that energy balance was more positive in the fruit/seed-feeding period than in the mature-leaf-feeding and fruit/fungi-feeding periods and that ingestion rates were the most important component of energy intake. The present study suggests that the fat accumulation ability is an essential adaptation in Japanese macaques even in warm-temperate forests.

Spatial position-associated mating strategies employed by male Japanese macaques (Macaca fuscata yakui) in Yakushima.

In bisexual groups, dominant males occupy the central part of the group, which is advantageous because it is reproductively beneficial. However, high-ranking males do not necessarily monopolize reproductive success, which indicates that low-ranking males increase their reproductive success through alternative mating strategies. To reveal the effectiveness and cost of these strategies employed by group males, it is necessary to clarify the spatial configuration of males and their group, and show how males combine strategies in different situations. This study demonstrates the spatial configuration of male Japanese macaques (Macaca fuscata yakui) and their group when males adopt three mating strategies: mate guarding, sneak mating, and cross-boundary mating (mating with females of another group). High-ranking males tended to perform mate guarding, but they also mated with females in other groups when there were few sexually active females in their group. Low-ranking males performed sneak mating; they remained in the central part of the group to monitor sexually active females and moved toward the periphery for copulation but never completely left the group. In addition, males individually ventured outside the group's ranging area and succeeded in mating with females of other groups. The cross-boundary mating strategy has the advantage of increasing the number of potential mating partners, which is not present in the other two strategies. However, because of considerable costs and low contribution to mating frequency, this strategy is complementary and is employed when the expectation of mating success in their group is low.

Gut microbiota composition of Japanese macaques associates with extent of human encroachment.

In recent decades, human-wildlife interaction and associated anthropogenic food provisioning has been increasing and becoming more severe due to fast population growth and urban development. Noting the role of the gut microbiome in host physiology like nutrition and health, it is thus essential to understand how human-wildlife interactions and availability of anthropogenic food in habitats can affect an animal's gut microbiome. This study, therefore, set out to examine the gut microbiota of Japanese macaques (Macaca fuscata) with varying accessibility to anthropogenic food and the possibility of using gut microbiota as indicator for macaques' reliance on anthropogenic food. Using 16S ribosomal RNA gene sequencing, we described the microbial composition of Japanese macaques experiencing different types of human disturbance and anthropogenic food availability-captive, provisioned, crop-raiding, and wild. In terms of alpha diversity, our results showed that observed richness of gut microbiota did not differ significantly between disturbance types but among collection sites, whereas Shannon diversity index differed by both disturbance types and sites. In terms of beta diversity, captive populations harbored the most distinctive gut microbial composition, and had the greatest difference compared with wild populations. Whereas for provisioned and crop-raiding groups, the macaques exhibited intermediate microbiota between wild and captive. We identified several potential bacterial taxa at different taxonomic ranks whose abundance potentially could help in assessing macaques' accessibility to anthropogenic food. This study revealed the flexibility of the gut microbiome of Japanese macaques and provided possible indices based on the gut microbiome profile in assessing macaques' accessibility to/reliance on anthropogenic foods.

Within-Population Variations in Home Range Use and Food Patch Use of Japanese Macaques: A Perspective of Intergroup Hostility.

Behavioral mechanisms of intergroup feeding competition remain unclear, despite its importance as a benefit of group living. Japanese macaques in the coastal and highland forests of Yakushima, Japan, are ideal study subjects because the intensity of intergroup feeding competition differs without phylogenetic effects. We aimed to test whether macaques modify home range use and food patch use in response to the location-specific risk of intergroup encounters. Using behavioral data from 3 groups, we examined the border avoidance and the effects of food patch location on food patch use. All 3 groups used the border less frequently than expected from its area. In the coastal forest characterized by frequent intergroup aggression, the smaller subordinate group, not the larger dominant group, increased the number of co-feeding individuals along the border. This response might reduce the potential costs of intergroup aggression. Feeding duration in one patch, visual scanning, and co-feeding with adult males did not depend on food patch location for the 2 groups. In contrast, the highland group did not modify food patch use along the border owing to the low risk of intergroup encounters. Our results highlight the importance of intergroup hostility as a social factor affecting animal behavior.

Seasonal variation of energy expenditure in Japanese macaques (Macaca fuscata).

Animals living in seasonal environments must adapt to a wide variation of temperature changes which requires flexible adjustments of time budget and metabolic processes for efficient thermoregulation. The Japanese macaque (Macaca fuscata) is one of only a handful of nonhuman primate species that experience seasonal climates over a wide temperature range. We used behavior observations, accelerometer sensors and the doubly-labelled water (DLW) method to measure activity and total daily energy expenditure (TDEE) of M. fuscata housed in captivity but exposed to natural seasonal variations at day lengths ranging from 10 to 12 h and temperature ranging from 0° to 32°C. Although overall activity was significantly lower in winter compared to summer and autumn, we found no effect of temperature on day-time activity. However nocturnal inactivity and mean length of sleeping bouts significantly increased along a gradient of decreasing temperatures from summer through winter, suggesting the importance of adaptive behavioral thermoregulation in this species. Energy expenditure that was unaccounted for by Basal Metabolic Rate (BMR) and physical activity i.e. expended through diet-induced thermogenesis or thermoregulation was between 14% and 32%. This residual energy expenditure differed between summer/autumn and winter and was relatively consistent across individuals (approximately 5-8% higher in winter). The percentage of body fat and residual energy expenditure were negatively correlated, supporting that fat storage was higher when less energy was required for thermoregulation. Our results suggest that physiological mechanisms like behavioral and autonomic thermoregulation enable M. fuscata to adapt to wide fluctuations in environmental conditions which provide insights into the evolutionary adaptations of nonhuman primates in seasonal climate.

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

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.

Activity of wild Japanese macaques in Yakushima revealed by camera trapping: Patterns with respect to season, daily period and rainfall.

Animals are subject to various scales of temporal environmental fluctuations, among which daily and seasonal variations are two of the most widespread and significant ones. Many biotic and abiotic factors change temporally, and climatic factors are particularly important because they directly affect the cost of thermoregulation. The purpose of the present study was to determine the activity patterns of wild Japanese macaques (Macaca fuscata) with a special emphasis on the effect of thermal conditions. We set 30 camera traps in the coniferous forest of Yakushima and monitored them for a total of 8658 camera-days between July 2014 and July 2015. Over the one-year period, temperature had a positive effect, and rainfall had a negative effect on the activity of macaques during the day. Capture rate was significantly higher during the time period of one hour after sunrise and during midday. During winter days, macaques concentrated their activity around noon, and activity shifted from the morning toward the afternoon. This could be interpreted as macaques shifting their activity to warmer time periods within a single day. Japanese macaques decreased their activity during the time before sunrise in seasons with lower temperatures. It was beneficial for macaques to be less active during cooler time periods in a cold season. Even small amounts of rainfall negatively affected the activity of Japanese macaques, with capture rates decreasing significantly even when rainfall was only 0.5-1 mm/min. In conclusion, thermal conditions significantly affected the activity of wild Japanese macaques at various time scales.