Sequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques.

Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton-Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.

Comparative ecological and behavioral study of Macaca assamensis and M. mulatta in Shivapuri Nagarjun National Park, Nepal.

Resource partitioning reduces the competition between different species within the same habitat, promoting their coexistence. To understand how such species co-adapt to reduce conflicts, we examined the behavior of two primates, Assamese macaque (Macaca assamensis) and rhesus macaque (Macaca mulatta), from April 2017 to March 2018 in Sivapuri Nagarjun National Park (SNNP), Kathmandu Valley, Nepal. We performed 1580 and 1261 scan sessions on wild multi-male/multi-female groups of Assamese and rhesus macaques, respectively, at 15-min sampling intervals. Assamese macaques consumed fewer plant species (38 species) than rhesus macaques (88 species). Overlapping food sources between the macaque species resulted in a Pianka index of 0.5. Assamese macaques consumed more items of tree, climber, and vine species, whereas rhesus macaques fed on more shrub, herb, and grass species. The proportions of plant parts consumed by the two species differed-more leaves, fruits and cones were used by Assamese macaques than rhesus macaques, whereas more flowers, seeds, and pods were consumed by rhesus macaques than Assamese macaques. Assamese macaques had a smaller home range (0.55 km2) than rhesus macaques (4.23 km2), and Assamese macaques had a shorter daily moving distance (1.6 km) than rhesus macaques (4.0 km). Although feeding time did not differ between the two macaque species, less time was devoted to social activities by Assamese macaques (16.0%) than by rhesus macaques (33.7%). Assamese macaques were generally arboreal, with 94.0% of their activities in trees, whereas rhesus macaques were largely terrestrial, with 58.5% of their activities on the ground. These differences in food selection, home-range size, ranging and activity patterns, and habitat use suggest that Assamese and rhesus macaques reduce resource competition through resource partitioning to coexist in a landscape matrix.

MRSA Strains in Nepalese Rhesus Macaques (Macaca mulatta) and Their Environment.

This study looked at 227 saliva samples from Rhesus macaques (Macaca mulatta) and 218 samples from the surrounding environments. From these samples, MRSA isolates were collected from Rhesus saliva samples (n = 13) and environmental samples (n = 19) near temple areas in Kathmandu, Nepal. For comparison, selected MRSA isolates (n = 5) were obtained from patients with wound infections from a Kathmandu hospital. All isolates were characterized using Abbott StaphyType® DNA microarrays. Eighteen isolates (62%) from monkeys (n = 4; 31%) and environmental samples (n = 14; 74%), were CC22-MRSA-IV. Most (n = 16) of them carried both, the PVL locus and toxic shock toxin gene (tst1), an unusual combination which is the same as in previously characterized strain from Nepalese macaques and pigs. The five human isolates also belonged to that strain type. Eight monkey MRSA isolates were CC361-MRSA-IV. One MRSA from a monkey and one from an environmental sample, were CC88-MRSA-V. Other environmental MRSA included one each, CC121-MRSA-VT, and CC772 -MRSA-V. Two were CC779-MRSA-VT, potentially a novel clone. All MRSA carried the blaZ gene. The aacA-aphD, dfrA, and erm (C) genes were very common in isolates from all sources. One macaque MRSA carried the resistance genes aphA3 and sat, neither previously identified in primate MRSA isolates. This current study suggests that humans could be a potential source of the MRSA in the macaques/environment and transmission may be linked to humans feeding the primates and/or living in close proximity to each other.

The function of geophagy in Nepal gray langurs: Sodium acquisition rather than detoxification or prevention of acidosis.

OBJECTIVES: Geophagy has several hypothesized functions, among them (1) detoxification of plant secondary compounds, for example, tannins, (2) buffering stomach pH to alleviate acidosis caused by high sugar intake, and (3) supplementing the diet with mineral nutrients. We tested these hypotheses in Nepal gray langurs (Semnopithecus schistaceus), a foregut fermenter for which fruits and leaves dominate the diet at different times of the year. MATERIALS AND METHODS: We collected data on feeding time per food item for 21 unprovisioned adult langurs at Ramnagar, Nepal, for 1 year using instantaneous sampling (773 observation hours). We combined these data with relative sugar and tannin content of food items to estimate diet content. We collected rainfall data to distinguish the wet season (May-September) from the dry season (October-April). We collected soil samples from consumption and control sites to test for pH and sodium, potassium, magnesium, and calcium concentrations. RESULTS: Langurs consumed soil from two sources: termite structures (in almost all months) and river banks and beds (mainly in the dry season). Soil feeding was not positively correlated with dietary tannin or sugar content (GLMM). Soil pH was not high enough to buffer stomach contents. Only sodium was significantly higher in consumed soil. DISCUSSION: The most likely function of geophagy was the acquisition of sodium. This conclusion is consistent with reports for other animals. Buffering stomach pH, an often-suggested function for animals with ruminant-like digestion, was not supported. Detoxification, often proposed for animals with a diet high in secondary compounds, was also not supported.

Mitochondrial DNA analyses and ecological niche modeling reveal post-LGM expansion of the Assam macaque (Macaca assamensis) in the foothills of Nepal Himalaya.

Genetic diversity of a species is influenced by multiple factors, including the Quaternary glacial-interglacial cycles and geophysical barriers. Such factors are not yet well documented for fauna from the southern border of the Himalayan region. This study used mitochondrial DNA (mtDNA) sequences and ecological niche modeling (ENM) to explore how the late Pleistocene climatic fluctuations and complex geography of the Himalayan region have shaped genetic diversity, population genetic structure, and demographic history of the Nepalese population of Assam macaques (Macaca assamensis) in the Himalayan foothills. A total of 277 fecal samples were collected from 39 wild troops over almost the entire distribution of the species in Nepal. The mtDNA fragment encompassing the complete control region (1121 bp) was recovered from 208 samples, thus defining 54 haplotypes. Results showed low nucleotide diversity (0.0075 ± SD 0.0001) but high haplotype diversity (0.965 ± SD 0.004). The mtDNA sequences revealed a shallow population genetic structure with a moderate but statistically significant effect of isolation by distance. Demographic history analyses using mtDNA sequences suggested a post-pleistocene population expansion. Paleodistribution reconstruction projected that the potential habitat of the Assam macaque was confined to the lower elevations of central Nepal during the Last Glacial Maximum. With the onset of the Holocene climatic optimum, the glacial refugia population experienced eastward range expansion to higher elevations. We conclude that the low genetic diversity and shallow population genetic structure of the Assam macaque population in the Nepal Himalaya region are the consequence of recent demographic and spatial expansion.

Low Levels of Fruit Nitrogen as Drivers for the Evolution of Madagascar's Primate Communities.

The uneven representation of frugivorous mammals and birds across tropical regions - high in the New World, low in Madagascar and intermediate in Africa and Asia - represents a long-standing enigma in ecology. Several hypotheses have been proposed to explain these differences but the ultimate drivers remain unclear. Here, we tested the hypothesis that fruits in Madagascar contain insufficient nitrogen to meet primate metabolic requirements, thus constraining the evolution of frugivory. We performed a global analysis of nitrogen in fruits consumed by primates, as collated from 79 studies. Our results showed that average frugivory among lemur communities was lower compared to New World and Asian-African primate communities. Fruits in Madagascar contain lower average nitrogen than those in the New World and Old World. Nitrogen content in the overall diets of primate species did not differ significantly between major taxonomic radiations. There is no relationship between fruit protein and the degree of frugivory among primates either globally or within regions, with the exception of Madagascar. This suggests that low protein availability in fruits influences current lemur communities to select for protein from other sources, whereas in the New World and Old World other factors are more significant in shaping primate communities.

Diet and Activity of Macaca assamensis in Wild and Semi-Provisioned Groups in Shivapuri Nagarjun National Park, Nepal.

Studying the behavioural flexibility and adaptability of macaques to different habitats is one approach to designing a conservation plan. To determine the activity budget and feeding behaviour and evaluate the effects of seasonality in wild and human- altered habitats of Assamese macaques (Macaca assamensis), we conducted this study in the Nagarjun forest of Shivapuri-Nagarjun National Park (SNNP) in central Nepal. We also updated the list of plant food items of Assamese macaques in the SNNP. Using scan and all-occurrence sampling, we recorded the diets and activities of Assamese macaques in 2 social groups, a wild-feeding group (WG) and a semi-provisioned group (SPG), throughout the year from August 2013 to July 2014. Both groups spent most of their time in feeding activities and were quite arboreal, but there were significant differences in the activity budgets and diets between the groups. Human food was the main component of the diet for the SPG, whereas it was fruit for the WG, indicating a normally frugivorous diet. Furthermore, the activity budget and diet composition varied in response to the season. These results indicate that provisioning alters the activity and feeding behaviour of macaques, and can also increase human-macaque conflict and disease transmission.

The importance of protein in leaf selection of folivorous primates.

Protein limitation has been considered a key factor in hypotheses on the evolution of life history and animal communities, suggesting that animals should prioritize protein in their food choice. This contrasts with the limited support that food selection studies have provided for such a priority in nonhuman primates, particularly for folivores. Here, we suggest that this discrepancy can be resolved if folivores only need to select for high protein leaves when average protein concentration in the habitat is low. To test the prediction, we applied meta-analyses to analyze published and unpublished results of food selection for protein and fiber concentrations from 24 studies (some with multiple species) of folivorous primates. To counter potential methodological flaws, we differentiated between methods analyzing total nitrogen and soluble protein concentrations. We used a meta-analysis to test for the effect of protein on food selection by primates and found a significant effect of soluble protein concentrations, but a non-significant effect for total nitrogen. Furthermore, selection for soluble protein was reinforced in forests where protein was less available. Selection for low fiber content was significant but unrelated to the fiber concentrations in representative leaf samples of a given forest. There was no relationship (either negative or positive) between the concentration of protein and fiber in the food or in representative samples of leaves. Overall our study suggests that protein selection is influenced by the protein availability in the environment, explaining the sometimes contradictory results in previous studies on protein selection. Am. J. Primatol. 79:e22550, 2017. © 2016 Wiley Periodicals, Inc.

Synanthropic primates in Asia: potential sentinels for environmental toxins.

Macaques are similar to humans both physiologically and behaviorally. In South and Southeast Asia they are also synanthropic, ecologically associated with humans. Synanthropy with humans raises the possibility that macaques come into contact with anthropogenic toxicants, such as lead and mercury, and might be appropriate sentinels for human exposures to certain toxic materials. We measured lead (Pb) and mercury (Hg) levels and characterized the stable isotopic compositions of delta(15)N and delta(13)C in hair from three groups of free-ranging macaques at the Swoyambhu temple in Kathmandhu, Nepal, an urban population that has abundant contact with humans. Hair lead levels were significantly higher among young macaques and differed among the three groups of macaques that were sampled. Hair Hg levels were low. No statistical association was found between stable isotopic compositions (delta(15)N and delta(13)C) and Pb and Hg levels. Our data did not find evidence that lead levels were associated with diet. We conclude that, in this population of macaques, behavioral and/or physiologic factors may play a significant role in determining exposure to lead. Chemical analysis of hair is a promising, noninvasive technique for determining exposure to toxic elements in free-ranging nonhuman primates.

Diverse contexts of zoonotic transmission of simian foamy viruses in Asia.

In Asia, contact between persons and nonhuman primates is widespread in multiple occupational and nonoccupational contexts. Simian foamy viruses (SFVs) are retroviruses that are prevalent in all species of nonhuman primates. To determine SFV prevalence in humans, we tested 305 persons who lived or worked around nonhuman primates in several South and Southeast Asian countries; 8 (2.6%) were confirmed SFV positive by Western blot and, for some, by PCR. The interspecies interactions that likely resulted in virus transmission were diverse; 5 macaque taxa were implicated as a potential source of infection. Phylogenetic analysis showed that SFV from 3 infected persons was similar to that from the nonhuman primate populations with which the infected persons reported contact. Thus, SFV infections are likely to be prevalent among persons who live or work near nonhuman primates in Asia.

The importance of ingestion rates for estimating food quality and energy intake.

Testing ecological or socioecological models in primatology often requires estimates of individual energy intake. It is a well established fact that the nutrient content (and hence the energy content) of primate food items is highly variable. The second variable in determining primate energy intake, i.e., the ingestion rate, has often been ignored, and few studies have attempted to estimate the relative importance of the two predictors. In the present study individual ingestion rates were measured in two ecologically very different populations of Hanuman langurs (Semnopithecus entellus) at Jodhpur, India, and Ramnagar, Nepal. Protein and soluble sugar concentrations in 50 and 100 food items. respectively, were measured using standardized methods. Variation in ingestion rates (gram of dry matter per minute) was markedly greater among food items than among langur individuals in both populations, but did not differ systematically among food item categories defined according to plant part and age. General linear models (GLMs) with ingestion rate, protein, and soluble sugar content explained 40-80% of the variation in energy intake rates (kJ/min). The relative importance of ingestion rates was either similar (Ramnagar) or much greater (Jodhpur) than the role of sugar and/or protein content in determining the energy intake rates of different items. These results may impact socioecological studies of variation in individual energy budgets, investigations of food choice in relation to chemical composition or sensory characteristics, and research into habitat preferences that measures habitat quality in terms of abundance of important food sources. We suggest a definition of food quality that includes not only the amount of valuable food contents (energy, vitamins, and minerals) and the digestibility of different foods, but also the rate at which the food can be harvested and processed. Such an extended definition seems necessary because time may constrain primates when feeding competition is intense and foraging is risk-prone.

What Hanuman langur males know about female reproductive status.

In species with a high risk of infanticide, a conflict of interest exists between the sexes over the amount of paternity information that is available to males. While females are expected to keep males unaware of their reproductive status in order to confuse paternity, selection should favor those male traits that enhance the males' assessment of female status and consequently of paternity probability. In Hanuman langurs (Semnopithecus entellus), a species that is extremely vulnerable to infanticide, females have been shown to successfully conceal the exact timing of ovulation from males--perhaps because they exhibit no sexual swelling and mate during all reproductive phases, including gestation. Nevertheless, it remains unclear whether males have hitherto unrecognized information about females' reproductive condition on a broader level that could still enhance male reproductive success. We investigated male assessment of female reproductive states in a population of wild Hanuman langurs as indicated by changes in male behavior, such as rates of copulations, anogenital inspections, and consortships, in relation to different female receptive periods (pregnant, fertile-nonconceptional, and conceptional). Our data indicate that males were able to discern qualitatively distinct reproductive states. Males were more interested in fertile than pregnant females, as indicated by higher copulation rates. Based on consortships, males distinguished fertile from nonfertile phases, as well as fertile, nonconceptional receptive periods from conceptional ones. Hanuman langur males are thus not as unaware of female reproductive condition as previously thought, supporting the idea of an ongoing battle of the sexes over paternity information. However, granting some knowledge while at the same time concealing the exact day of ovulation may also reflect a pure female strategy of balancing paternity concentration with paternity confusion, which is the most likely strategy in this system with high infanticide risk and male defense of infants.

Genetic characterization of rhesus macaques (Macaca mulatta) in Nepal.

Indian-origin rhesus macaques (Macaca mulatta) have long served as an animal model for the study of human disease and behavior. Given the current shortage of Indian-origin rhesus, many researchers have turned to rhesus macaques from China as a substitute. However, a number of studies have identified marked genetic differences between the Chinese and Indian animals. We investigated the genetic characteristics of a third rhesus population, the rhesus macaques of Nepal. Twenty-one rhesus macaques at the Swoyambhu Temple in Kathmandu, Nepal, were compared with more than 300 Indian- and Chinese-origin rhesus macaques. The sequence analyses of two mitochondrial DNA (mtDNA) loci, from the HVS I and 12 S rRNA regions, showed that the Nepali animals were more similar to Indian-origin than to Chinese-origin animals. The distribution of alleles at 24 short tandem repeat (STR) loci distributed across 17 chromosomes also showed greater similarity between the Nepali and Indian-origin animals. Finally, an analysis of seven major histocompatibility complex (MHC) alleles showed that the Nepali animals expressed Class I alleles that are common to Indian-origin animals, including Mamu-A*01. All of these analyses also revealed a low level of genetic diversity within this Nepali rhesus sample. We conclude that the rhesus macaques of Nepal more closely resemble rhesus macaques of Indian origin than those of Chinese origin. As such, the Nepali rhesus may offer an additional resource option for researchers who wish to maintain research protocols with animals that possess key genetic features characteristic of Indian-origin rhesus macaques.