Isolation of Bacteria from Freeze-Dried Samples and the Functional Characterization of Species-Specific Lactic Acid Bacteria with a Comparison of Wild and Captive Proboscis Monkeys.

Previously, we isolated a novel lactic acid bacteria species (Lactobacillus nasalidis) from the fresh forestomach contents of a captive proboscis monkey (Nasalis larvatus) in a Japanese zoo. In this study, we isolated two strains of L. nasalidis from the freeze-dried forestomach contents of a wild proboscis monkey inhabiting a riverine forest in Malaysia. The samples had been stored for more than six years. Phenotypic analysis showed that strains isolated from the wild individual had more diverse sugar utilization and lower salt tolerance than strains previously isolated from the captive counterpart. These phenotypic differences are most likely induced by feeding conditions; wild individuals consume a wide variety of natural food, unlike their zoo-raised counterparts that consume formula feed with sodium sufficiency. Since 16s rRNA sequences of L. nasalidis were detected in the previously created 16S rRNA libraries of wild, provisioned, and captive proboscis monkeys in Malaysia and Japan, L. nasalidis may be an essential bacterium of the foregut microbial community of the proboscis monkey. The currently established method for the isolation of gut bacteria from freeze-dried samples under storage will be applicable to many already-stored precious samples.

External environmental conditions impact nocturnal activity levels in proboscis monkeys (Nasalis larvatus) living in Sabah, Malaysia.

Recently, several diurnal nonhuman anthropoids have been identified displaying varying degrees of nocturnal activity, which can be influenced by activity "masking effects"-external events or conditions that suppress or trigger activity, temporarily altering normal activity patterns. Environmental masking characteristics include nocturnal temperature, rainfall, cloud cover, and moon brightness. Similarly, other ecological characteristics, including proximity to humans and predators and daytime activity, may also trigger or suppress nocturnal activity. Understanding the effects of external conditions on activity patterns is pertinent to effective species conservation. We investigated the presence of nocturnal activity and the influence of masking effects on the level of nocturnal activity displayed by wild proboscis monkeys (Nasalis larvatus) in Sabah, Malaysian Borneo. Dual-axis accelerometers were attached by collar to six male proboscis monkeys from different one-male, multi-female groups to record activity continuously (165-401 days each). We measured the monkeys' nocturnal and diurnal activity levels and investigated the effects of seven potential masking effects. Nocturnal activity was much lower than diurnal activity. Still, proboscis monkeys did display varying levels of nocturnal activity. Generalized linear mixed models identified higher nocturnal activity in the study individuals during nights with cooler temperatures, higher rainfall, and after higher diurnal activity. These three masking effects affected nocturnal activity levels during the observation period that informed our model, although they did not predict nocturnal activity outside of this period. While the generalizability of these results remains uncertain, this study highlights the utility of accelerometers in identifying activity patterns and masking effects that create variability in these patterns.

Parasite community structure in sympatric Bornean primates.

Parasites are important components of ecosystems, influencing trophic networks, competitive interactions and biodiversity patterns. Nonetheless, we are not nearly close to disentangling their complex roles in natural systems. Southeast Asia falls within global areas targeted as most likely to source parasites with zoonotic potential, where high rates of land conversion and fragmentation have altered the circulation of wildlife species and their parasites, potentially resulting in altered host-parasite systems. Although the overall biodiversity in the region predicts equally high, or even higher, parasite diversity, we know surprisingly little about wild primate parasites, even though this constitutes the first step towards a more comprehensive understanding of parasite transmission processes. Here, we characterise the gastrointestinal helminth parasite assemblages of a community of Bornean primates living along the Kinabatangan floodplain in Sabah (Malaysian Borneo), including two species endemic to the island. Through parasitological analyses, and by using several measures of parasite infection as proxies for parasite diversity and distribution, we show that (i) most parasite taxonomic groups are not limited to a single host, suggesting a greater flexibility for habitat disturbance, (ii) parasite infracommunities of nocturnal primates differ from their diurnal counterparts, reflecting both phylogenetic and ecological constraints, and (iii) soil-transmitted helminths such as whipworm, threadworm and nodule worm are widespread across the primate community. This study also provides new parasite records for southern pig-tailed macaques (Macaca nemestrina), silvered langurs (Trachypithecus cristatus) and Western tarsiers (Cephalopachus bancanus) in the wild, while adding to the limited records for the other primate species in the community. Given the information gap regarding primate-parasite associations in the region, the information presented here should prove relevant for future studies of parasite biodiversity and infectious disease ecology in Asia and elsewhere.

A comparative evaluation of thermal camera and visual counting methods for primate census in a riparian forest at the Lower Kinabatangan Wildlife Sanctuary (LKWS), Malaysian Borneo.

A number of primate census techniques have been developed over the past half-century, each of which have advantages and disadvantages in terms of resources required by researchers (e.g., time and costs), availability of technologies, and effectiveness in different habitat types. This study aims to explore the effectiveness of a thermal imaging technique to estimate the group size of different primate species populations in a degraded riparian forest in the Lower Kinabatangan Wildlife Sanctuary (LKWS), Sabah. We compared this survey technique to the conventional visual counting method along the riverbank. For 38 days, a total of 138 primate groups were observed by thermal camera and visually throughout the study. Optimal conditions for the thermal camera were clear weather, not more than 100 m distance from the observer to the targeted area, boat speed ranging between 5 and 12 km/h, and early morning between 04:30 and 05:30 am. The limitations of the thermal cameras include the inability to identify individual species, sexes, age classes, and also to discern between animals closely aggregated (i.e., mothers with attached infants). Despite these limitations with the thermal camera technique, 1.78 times more primates were detected than counting by eye (p < 0.001), showing the potential benefit of using thermal cameras as an important tool in primate surveys. Nevertheless, ground truthing must be conducted immediately after, or simultaneously during, the thermal survey to verify the species of animals observed on the thermal imagery.

Large male proboscis monkeys have larger noses but smaller canines.

The uniquely enlarged noses of male proboscis monkeys are prominent adornments, and a sexually selected male trait. A recent study showed significant correlations among nose, body, and testis sizes and clear associations between nose size and the number of females in a male's harem. However, to date, the analyses of other common male traits, i.e., canines, are lacking. Whereas male nose size had a positive correlation with body size, we unexpectedly found a negative correlation between body and canine sizes. We explain this by an interaction between sexual and natural selection. Larger noses in males may interfere with the use of canines, thereby reducing their effectiveness as weapons. Additionally, longer canines are opposed by natural selection because the larger gape it imposes upon its bearer reduces foraging efficiency, particularly in folivores. This unique case of decoupling of body and canine size reveals that large canines carry an ecological cost.

First description of male worms of Enterobius (Colobenterobius) serratus (Nematoda: Oxyuridae), the pinworm parasite of proboscis monkeys.

Males of Enterobius (Colobenterobius) serratus Hasegawa et al., 2003 (Nematoda: Oxyuridae) are described for the first time based on six individuals collected from the feces of proboscis monkeys, Nasalis larvatus, in the Lower Kinabatangan Wildlife Sanctuary, Sabah, Malaysian Borneo. The males show identical cephalic morphology to females, being readily distinguishable from their congeners by the serrated inner margins of the lips. The bicolored esophageal corpus, long thin spicule and developed spicular pouch with paired muscular bands are also remarkable characteristics, presumably shared by other Asian members of the subgenus.

Correction to: Review of GPS collar deployments and performance on nonhuman primates.

In the original publication of the article, figure 1 was wrongly published as a duplication.

Review of GPS collar deployments and performance on nonhuman primates.

Over the past 20 years, GPS collars have emerged as powerful tools for the study of nonhuman primate (hereafter, "primate") movement ecology. As the size and cost of GPS collars have decreased and performance has improved, it is timely to review the use and success of GPS collar deployments on primates to date. Here we compile data on deployments and performance of GPS collars by brand and examine how these relate to characteristics of the primate species and field contexts in which they were deployed. The compiled results of 179 GPS collar deployments across 17 species by 16 research teams show these technologies can provide advantages, particularly in adding to the quality, quantity, and temporal span of data collection. However, aspects of this technology still require substantial improvement in order to make deployment on many primate species pragmatic economically. In particular, current limitations regarding battery lifespan relative to collar weight, the efficacy of remote drop-off mechanisms, and the ability to remotely retrieve data need to be addressed before the technology is likely to be widely adopted. Moreover, despite the increasing utility of GPS collars in the field, they remain substantially more expensive than VHF collars and tracking via handheld GPS units, and cost considerations of GPS collars may limit sample sizes and thereby the strength of inferences. Still, the overall high quality and quantity of data obtained, combined with the reduced need for on-the-ground tracking by field personnel, may help defray the high equipment cost. We argue that primatologists armed with the information in this review have much to gain from the recent, substantial improvements in GPS collar technology.

Seed dispersal by proboscis monkeys: the case of Nauclea spp.

Frugivorous vertebrates such as primates are important dispersal agents in tropical forests, although the role of folivorous colobines is generally not considered. However, recent studies reported seed dispersal by endo- and epizoochory in colobine primates, including the proboscis monkey (Nasalis larvatus), suggesting that the role colobines play in seed dispersal might have been underestimated. In the Lower Kinabatangan Floodplain in Sabah, Malaysian Borneo, we investigated whether seeds were still able to germinate after being ingested by proboscis monkeys. Faecal samples (n = 201) from proboscis monkeys were collected between 2015 and 2017. Intact seeds belonging to eight plant species were found in 77% of the faecal samples. Nauclea spp. were the most abundant plant species, accounting for 98% of all intact seeds. This study is the first to conduct germination trials on seeds defecated by proboscis monkeys. Higher germination success was recorded in ingested Nauclea spp. seeds than in control seeds, from both ripe and unripe Nauclea orientalis fruits (P < 0.001). Therefore, we suggest that proboscis monkeys play a role in seed dispersal by enhancing the germination success of defecated seeds for at least some plant species. Similar to other colobines, although proboscis monkeys may provide a lower contribution to seed dispersal (low seed diversity over short distances) than other sympatric frugivores, this study emphasises that proboscis monkeys do contribute to the dispersal of intact seeds, such as Nauclea spp., in potentially suitable riverine habitats.

Molecular characterization of nodule worm in a community of Bornean primates.

Strongyles are commonly reported parasites in studies of primate parasite biodiversity. Among them, nodule worm species are often overlooked as a serious concern despite having been observed to cause serious disease in nonhuman primates and humans. In this study, we investigated whether strongyles found in Bornean primates are the nodule worm Oesophagostomum spp., and to what extent these parasites are shared among members of the community. To test this, we propose two hypotheses that use the parasite genetic structure to infer transmission processes within the community. In the first scenario, the absence of parasite genetic substructuring would reflect high levels of parasite transmission among primate hosts, as primates' home ranges overlap in the study area. In the second scenario, the presence of parasite substructuring would suggest cryptic diversity within the parasite genus and the existence of phylogenetic barriers to cross-species transmission. By using molecular markers, we identify strongyles infecting this primate community as O. aculeatum, the only species of nodule worm currently known to infect Asian nonhuman primates. Furthermore, the little to no genetic substructuring supports a scenario with no phylogenetic barriers to transmission and where host movements across the landscape would enable gene flow between host populations. This work shows that the parasite's high adaptability could act as a buffer against local parasite extinctions. Surveys targeting human populations living in close proximity to nonhuman primates could help clarify whether this species of nodule worm presents the zoonotic potential found in the other two species infecting African nonhuman primates.

Long-Tailed Macaque Response to Deforestation in a Plasmodium knowlesi-Endemic Area.

Land-use changes can impact infectious disease transmission by increasing spatial overlap between people and wildlife disease reservoirs. In Malaysian Borneo, increases in human infections by the zoonotic malaria Plasmodium knowlesi are hypothesised to be due to increasing contact between people and macaques due to deforestation. To explore how macaque responses to environmental change impact disease risks, we analysed movement of a GPS-collared long-tailed macaque in a knowlesi-endemic area in Sabah, Malaysia, during a deforestation event. Land-cover maps were derived from satellite-based and aerial remote sensing data and models of macaque occurrence were developed to evaluate how macaque habitat use was influenced by land-use change. During deforestation, changes were observed in macaque troop home range size, movement speeds and use of different habitat types. Results of models were consistent with the hypothesis that macaque ranging behaviour is disturbed by deforestation events but begins to equilibrate after seeking and occupying a new habitat, potentially impacting human disease risks. Further research is required to explore how these changes in macaque movement affect knowlesi epidemiology on a wider spatial scale.

A pinworm's tale: The evolutionary history of Lemuricola (Protenterobius) nycticebi.

Lemuricola (Protenterobius) nycticebi is the only pinworm species known to infect strepsirrhine primates outside Africa, and the only pinworm species yet described in slow lorises. Here, we provided a detailed morphological comparison of female and male worms, and a first description of fourth-stage larvae collected from free-living slow lorises (Nycticebus menagensis) in Sabah, Malaysian Borneo. Using mitochondrial and nuclear markers, we also reconstructed the species' phylogenetic relationship with other pinworms infecting primates. Both morphological and molecular results indicated a distinct association between L. (P.) nycticebi and its host. However, while taxonomy identified this species as a member of the Lemuricola clade and grouped pinworms infecting lemurs and slow lorises together, phylogenetic reconstruction split them, placing L. (P.) nycticebi within the Enterobius clade. Our results suggest that L. (P.) nycticebi may represent a different taxon altogether, and that it is more closely related to pinworm species infecting Old World primates outside Madagascar. Pongobius pongoi (Foitová et al., 2008) n. comb. is also proposed.

Faecal Particle Size in Free-Ranging Proboscis Monkeys, Nasalis larvatus: Variation between Seasons.

Reducing the size of food particles is crucial for herbivores. Seasonal dietary changes are known to influence animals' chewing efficiency. Proboscis monkeys (Nasalis larvatus) are foregut fermenters, with a high chewing efficiency allowing them to achieve very fine faecal particles. In this study, we investigated how proboscis monkeys' chewing efficiency varies between wet and dry seasons, hypothesising differences possibly related to diet change. Faecal particle size analysis is an established approach to estimate chewing efficiency in mammalian herbivores. We analysed 113 proboscis monkey faecal samples collected in the Lower Kinabatangan Wildlife Sanctuary, between 2015 and 2017. By following standard sieve analysis protocols, we measured a mean particle size MPS0.025-8 of 0.45 ± 0.14 mm, and confirmed a previous result that proboscis monkeys have a very low faecal MPS. This study highlights a seasonal influence on proboscis monkeys' chewing efficiency, with smaller MPS (better chewing efficiency) during the wet season. During that time of the year, individuals may potentially change their diet, as all faecal samples contained intact seeds. Whether the seasonal MPS difference in proboscis monkeys is smaller than in other colobines due to their "rumination" strategy remains to be investigated.

Lurking in the dark: Cryptic Strongyloides in a Bornean slow loris.

Within host communities, related species are more likely to share common parasitic agents, and as a result, morphological similarities have led researchers to conclude that parasites infecting closely related hosts within a community represent a single species. However, genetic diversity within parasite genera and host range remain poorly investigated in most systems. Strongyloides is a genus of soil-transmitted nematode that has been reported from several primate species in Africa and Asia, and has been estimated to infect hundreds of millions of people worldwide, although no precise estimates are available. Here we describe a case of infection with a cryptic species of Strongyloides in a Bornean (Philippine) slow loris (Nycticebus menagensis) living within a diverse community of several primate species in the Lower Kinabatangan Wildlife Sanctuary, Malaysian Borneo. Fresh fecal samples were collected from five primate species and nematode larvae cultured from these samples were selected for phylogenetic analyses. Sequences obtained for most larvae were identified as S. fuelleborni, grouping into three different clusters and showing no aggregation within specific hosts or geographic location. In contrast, a set of parasite sequences obtained from a slow loris clustered closely with S. stercoralis into a different group, being genetically distinct to sequences reported from other primate hosts, humans included. Our results suggest that although S. fuelleborni infects all haplorrhines sampled in this primate community, a different species might be infecting the slow loris, the only strepsirrhine in Borneo and one of the least studied primates in the region. Although more data are needed to support this conclusion, we propose that Strongyloides species in primates might be more diverse than previously thought, with potential implications for ecological and evolutionary host-parasite associations, as well as epidemiological dynamics.

Sleep patterns, daytime predation, and the evolution of diurnal sleep site selection in lorisiforms.

OBJECTIVES: Synthesize information on sleep patterns, sleep site use, and daytime predation at sleep sites in lorisiforms of Asia and Africa (10 genera, 36 species), and infer patterns of evolution of sleep site selection. MATERIALS AND METHODS: We conducted fieldwork in 12 African and six Asian countries, collecting data on sleep sites, timing of sleep and predation during daytime. We obtained additional information from literature and through correspondence. Using a phylogenetic approach, we established ancestral states of sleep site selection in lorisiforms and traced their evolution. RESULTS: The ancestral lorisiform was a fur-clinger and used dense tangles and branches/forks as sleep sites. Use of tree holes and nests as sleep sites emerged ∼22 Mya (range 17-26 Mya) in Africa, and use of bamboo emerged ∼11 (7-14) Mya in Asia and later in Africa. Fur clinging and some sleep sites (e.g., tree holes, nests, but not bamboo or dense tangles) show strong phylogenetic signal. Nests are used by Galagoides, Paragalago, Galago and Otolemur; tree holes by Galago, Paragalago, Sciurocheirus and Perodicticus; tangles by Nycticebus, Loris, Galagoides, Galago, Euoticus, Otolemur, Perodicticus and Arctocebus; all but Sciurocheirus and Otolemur additionally sleep on branches/forks. Daytime predation may affect sleep site selection and sleep patterns in some species of Nycticebus, Galago, Galagoides, Otolemur and Perodicticus. Most lorisiforms enter their sleep sites around sunrise and leave around sunset; several are active during twilight or, briefly, during daytime. CONCLUSION: Variations in sleep behavior, sleep patterns and vulnerability to daytime predation provide a window into the variation that was present in sleep in early primates. Overall, lorisiforms use the daytime for sleeping and no species can be classified as cathemeral or polycyclic.

First report of foregut microbial community in proboscis monkeys: are diverse forests a reservoir for diverse microbiomes?

Foregut fermentation is well known to occur in a wide range of mammalian species and in a single bird species. Yet, the foregut microbial community of free-ranging, foregut-fermenting monkeys, that is, colobines, has not been investigated so far. We analysed the foregut microbiomes in four free-ranging proboscis monkeys (Nasalis larvatus) from two different tropical habitats with varying plant diversity (mangrove and riverine forests), in an individual from a semi-free-ranging setting with supplemental feeding, and in an individual from captivity, using high-throughput sequencing based on 16S ribosomal RNA genes. We found a decrease in foregut microbial diversity from a diverse natural habitat (riverine forest) to a low diverse natural habitat (mangrove forest), to human-related environments. Of a total of 2700 bacterial operational taxonomic units (OTUs) detected in all environments, only 153 OTUs were shared across all individuals, suggesting that they were not influenced by diet or habitat. These OTUs were dominated by Firmicutes and Proteobacteria. The relative abundance of the habitat-specific microbial communities showed a wide range of differences among living environments, although such bacterial communities appeared to be dominated by Firmicutes and Bacteroidetes, suggesting that those phyla are key to understanding the adaptive strategy in proboscis monkeys living in different habitats.

Nasalization by Nasalis larvatus: Larger noses audiovisually advertise conspecifics in proboscis monkeys.

Male proboscis monkeys have uniquely enlarged noses that are prominent adornments, which may have evolved through their sexually competitive harem group social system. Nevertheless, the ecological roles of the signals encoded by enlarged noses remain unclear. We found significant correlations among nose, body, and testis sizes and a clear link between nose size and number of harem females. Therefore, there is evidence supporting both male-male competition and female choice as causal factors in the evolution of enlarged male noses. We also observed that nasal enlargement systematically modifies the resonance properties of male vocalizations, which probably encode male quality. Our results indicate that the audiovisual contributions of enlarged male noses serve as advertisements to females in their mate selection. This is the first primate research to evaluate the evolutionary processes involved in linking morphology, acoustics, and socioecology with unique masculine characteristics.

Jumping in the Night: An Investigation of the Leaping Activity of the Western Tarsier (Cephalopachus bancanus borneanus) Using Accelerometers.

Accelerometers enable scientists to quantify the activity of free-living animals whose direct observation is difficult or demanding due to their elusive nature or nocturnal habits. However, the deployment of accelerometers on small-bodied animals and, in particular, on primates has been little explored. Here we show the first application of accelerometers on the western tarsier (Cephalopachus bancanus borneanus), a nocturnal, small-bodied primate endemic to the forests of Borneo. The fieldwork was carried out in the Lower Kinabatangan Wildlife Sanctuary, Sabah, Malaysian Borneo. We provide guidelines for the deployment of accelerometers on tarsiers that might also be applied to other primate species. Our collected data on 2 females show levels of leaping activity comparable to those previously described using direct observation of wild or captive individuals. The 2 females showed different patterns of leaping activity, which calls for work to explore individual differences further. Our work demonstrates that accelerometers can be deployed on small primates to acquire body motion data that would otherwise be demanding to collect using classic field observations. Future work will be focused on using accelerometer data to discriminate in more detail the different behaviours tarsiers can display and to address the causes and consequences of individual variations in activity.

Evaluating methods for estimating home ranges using GPS collars: A comparison using proboscis monkeys (Nasalis larvatus).

The development of GPS tags for tracking wildlife has revolutionised the study of home ranges, habitat use and behaviour. Concomitantly, there have been rapid developments in methods for estimating habitat use from GPS data. In combination, these changes can cause challenges in choosing the best methods for estimating home ranges. In primatology, this issue has received little attention, as there have been few GPS collar-based studies to date. However, as advancing technology is making collaring studies more feasible, there is a need for the analysis to advance alongside the technology. Here, using a high quality GPS collaring data set from 10 proboscis monkeys (Nasalis larvatus), we aimed to: 1) compare home range estimates from the most commonly used method in primatology, the grid-cell method, with three recent methods designed for large and/or temporally correlated GPS data sets; 2) evaluate how well these methods identify known physical barriers (e.g. rivers); and 3) test the robustness of the different methods to data containing either less frequent or random losses of GPS fixes. Biased random bridges had the best overall performance, combining a high level of agreement between the raw data and estimated utilisation distribution with a relatively low sensitivity to reduced fixed frequency or loss of data. It estimated the home range of proboscis monkeys to be 24-165 ha (mean 80.89 ha). The grid-cell method and approaches based on local convex hulls had some advantages including simplicity and excellent barrier identification, respectively, but lower overall performance. With the most suitable model, or combination of models, it is possible to understand more fully the patterns, causes, and potential consequences that disturbances could have on an animal, and accordingly be used to assist in the management and restoration of degraded landscapes.

Use and Selection of Sleeping Sites by Proboscis Monkeys, Nasalislarvatus, along the Kinabatangan River, Sabah, Malaysia.

The choice of a sleeping site is crucial for primates and may influence their survival. In this study, we investigated several tree characteristics influencing the sleeping site selection by proboscis monkeys (Nasalis larvatus) along Kinabatangan River, in Sabah, Malaysia. We identified 81 sleeping trees used by one-male and all-male social groups from November 2011 to January 2012. We recorded 15 variables for each tree. Within sleeping sites, sleeping trees were taller, had a larger trunk, with larger and higher first branches than surrounding trees. The crown contained more mature leaves, ripe and unripe fruits but had vines less often than surrounding trees. In addition, in this study, we also focused on a larger scale, considering sleeping and non-sleeping sites. Multivariate analyses highlighted a combination of 6 variables that revealed the significance of sleeping trees as well as surrounding trees in the selection process. During our boat surveys, we observed that adult females and young individuals stayed higher in the canopy than adult males. This pattern may be driven by their increased vulnerability to predation. Finally, we suggest that the selection of particular sleeping tree features (i.e. tall, high first branch) by proboscis monkeys is mostly influenced by antipredation strategies.