Uncovering the magnitude of African pangolin poaching with extensive nanopore DNA genotyping of seized scales.

Trade in pangolins is illegal, and yet tons of their scales and products are seized at various ports. These large seizures are challenging to process and comprehensively genotype for upstream provenance tracing and species identification for prosecution. We implemented a scalable DNA barcoding pipeline in which rapid DNA extraction and MinION sequencing were used to genotype a substantial proportion of pangolin scales subsampled from 2 record shipments seized in Singapore in 2019 (37.5 t). We used reference sequences to match the scales to phylogeographical regions of origin. In total, we identified 2346 cytochrome b (cytb) barcodes of white-bellied (Phataginus tricuspis) (from 1091 scales), black-bellied (Phataginus tetradactyla) (227 scales), and giant (Smutsia gigantea) (1028 scales) pangolins. Haplotype diversity was higher for P. tricuspis scales (121 haplotypes, 66 novel) than that for P. tetradactyla (22 haplotypes, 15 novel) and S. gigantea (25 haplotypes, 21 novel) scales. Of the novel haplotypes, 74.2% were likely from western and west-central Africa, suggesting potential resurgence of poaching and newly exploited populations in these regions. Our results illustrate the utility of extensively subsampling large seizures and outline an efficient molecular approach for rapid genetic screening that should be accessible to most forensic laboratories and enforcement agencies.

Revelación de la magnitud de la caza furtiva del pangolín africano mediante el genotipo extenso de nanoporos de ADN de escamas incautadas Resumen Aunque el mercado de pangolines es ilegal, se incautan toneladas de sus escamas y productos derivados en varios puertos comerciales. Es un reto procesar estas magnas incautaciones y obtener el genotipo completo para usarlo en la trazabilidad logística ascendente e identificación de la especie y así imponer sanciones. Implementamos una canalización escalable del código de barras de ADN en el cual usamos la extracción rápida de ADN y la secuenciación MinION para obtener el genotipo de una proporción sustancial de las escamas de pangolín submuestreadas en dos cargamentos incautados en 2019 en Singapur (37.5 t). Usamos secuencias referenciales para emparejar las escamas con las regiones filogeográficas de origen. Identificamos en total 2,346 códigos de citocromo b (cytb) del pangolín de vientre blanco (Phataginus tricuspis) (de 1,091 escamas), de vientre negro (P. tetradactyla) (227 escamas) y del pangolín gigante (Smutsia gigantea) (1,028 escamas). La diversidad de haplotipos fue mayor en las escamas de P. tricuspis (121 haplotipos, 66 nuevos) que en las de P. tetradactyla (22 haplotipos, 15 nuevos) y S. gigantea (25 haplotipos, 21 nuevos). De los haplotipos nuevos, el 74.2% probablemente provenía del occidente y centro­occidente de África, lo que sugiere un resurgimiento potencial de la caza furtiva y poblaciones recién explotadas en estas regiones. Nuestros resultados demuestran la utilidad de submuestrear extensivamente las grandes incautaciones y esboza una estrategia molecular eficiente para un análisis genético rápido que debería ser accesible para la mayoría de los laboratorios forenses y las autoridades de aplicación.

Isopod mouthpart traits respond to a tropical forest recovery gradient.

Functional trait ecology has the potential to provide generalizable and mechanistic predictions of ecosystem function from data of species distributions and traits. The traits that are selected should both respond to environmental factors and influence ecosystem functioning. Invertebrate mouthpart traits fulfill these criteria, but are seldom collected, lack standardized measurement protocols, and have infrequently been investigated in response to environmental factors. We surveyed isopod species that consume plant detritus, and tree communities in 58 plots across primary and secondary forests in Singapore. We measured body dimensions (body size traits), pereopod and antennae lengths (locomotory traits), dimensions of mandible structures (morphological mouthpart traits), and mechanical advantages generated by mandible shape (mechanical mouthpart traits) for six isopod species found in these plots and investigated if these traits respond to changes in tree community composition, tree diversity, and forest structure. Morphological mouthpart traits responded to a tree compositional gradient reflecting forest recovery degree. Mouthpart features associated with greater consumption of litter (broader but less serrated/rugose lacinia mobilis [an important cutting and chewing structure on the mandible]) were most prevalent in abandoned plantation and young secondary forests containing disturbance-associated tree species. Feeding strategies associated with fungi grazing (narrower and more serrated/rugose lacinia mobilis) were most prevalent in late secondary forests containing later successional tree species. Since morphological mouthpart traits likely also predict consumption and excretion rates of isopods, these traits advance our understanding of environment-trait-ecosystem functioning relationships across contrasting tropical forest plots that vary in composition, disturbance history, and post-disturbance recovery.

Establishment of a Captive Cave Nectar Bat (Eonycteris spelaea) Breeding Colony in Singapore.

Bats are known natural reservoirs of several highly pathogenic zoonotic viruses, including Hendra virus, Nipah virus, rabies virus, SARS-like coronaviruses, and suspected ancestral reservoirs of SARS-CoV-2 responsible for the ongoing COVID-19 pandemic. The capacity to survive infections of highly pathogenic agents without severe disease, together with many other unique features, makes bats an ideal animal model for studying the regulation of infection, cancer, and longevity, which is likely to translate into human health outcomes. A key factor that limits bat research is lack of breeding bat colonies. To address this need, a captive bat colony was established in Singapore from 19 wild-caught local cave nectar bats. The bats were screened for specific pathogens before the start of captive breeding. Custom-made cages and an optimized diet inclusive of Wombaroo dietary formula, liquid diet, and supplement of fruits enabled the bats to breed prolifically in our facility. Cages are washed daily and disinfected once every fortnight. Bats are observed daily to detect any sick bat or abnormal behavior. In addition, bats undergo a thorough health check once every 3 to 4 mo to check on their overall wellbeing, perform sampling, and document any potential pregnancy. The current colony houses over 80 bats that are successfully breeding, providing a valuable resource for research in Singapore and overseas.

Host specificity of Hepatocystis infection in short-nosed fruit bats (Cynopterus brachyotis) in Singapore.

Haemosporidians infect a wide diversity of bat genera and species, yet little is known about their transmission cycles or epidemiology. Though several recent studies have focused on the genus Hepatocystis, an Old World parasite primarily infecting bats, monkeys, and squirrels, this group is still understudied with little known about its transmission and molecular ecology. These parasites lack an asexual erythrocytic stage, making them unique from the Plasmodium vertebrate life cycle. In this study, we detected a prevalence of 31% of Hepatocystis in short-nosed fruit bats (Cynopterus brachyotis) in Singapore. Phylogenetic reconstruction with a partial cytochrome b sequence revealed a monophyletic group of Hepatocystis from C. brachyotis in Malaysia, Singapore, and Thailand. There was no relationship with infection and bat age, sex, location, body condition or monsoon season. The absence of this parasite in the five other bat species sampled in Singapore indicates this Hepatocystis species may be host restricted.

Urbanization minimizes the effects of plant traits on soil provisioned ecosystem services across climatic regions.

An increasingly urbanized world is one of the most prominent examples of global environmental change. Across the globe, urban parks are designed and managed in a similar way, resulting in visually pleasing expansions of lawn interspersed with individually planted trees of varying appearances and functional traits. These large urban greenspaces have the capacity to provide various ecosystem services, including those associated with soil physicochemical properties. Our aim was to explore whether soil properties in urban parks diverge underneath vegetation producing labile or recalcitrant litter, and whether the impact is affected by climatic zone (from a boreal to temperate to tropical city). We also compared these properties to those in (semi)natural forests outside the cities to assess the influence of urbanization on plant-trait effects. We showed that vegetation type affected percentage soil organic matter (OM), total carbon (C) and total nitrogen (N), but inconsistently across climatic zones. Plant-trait effects were particularly weak in old parks in the boreal and temperate zones, whereas in young parks in these zones, soils underneath the two tree types accumulated significantly more OM, C and N compared to lawns. Within climatic zones, anthropogenic drivers dominated natural ones, with consistently lower values of organic-matter-related soil properties under trees producing labile or recalcitrant litter in parks compared to forests. The dominating effect of urbanization is also reflected in its ability to homogenize soil properties in parks across the three cities, especially in lawn soils and soils under trees irrespective of functional trait. Our study demonstrates that soil functions that relate to carbon and nitrogen dynamics-even in old urban greenspaces where plant-soil interactions have a long history-clearly diverged from those in natural ecosystems, implying a long-lasting influence of anthropogenic drivers on soil ecosystem services.

Emergence of the zoonotic tick Dermacentor (Indocentor) auratus Supino, 1897 (Acari: Ixodidae) in Singapore.

Though ticks pose a significant public health risk, until recently, little research had focused on the diversity of ticks and tick-borne diseases in Singapore. To date, only fourteen tick species in five genera have been recorded there. For the first time, Dermacentor auratus is recorded from Singapore from a range of hosts, including humans. DNA sequences are provided at 2 loci, for D. auratus, the cytochrome c oxidase I (COI) for DNA barcoding and the 16S large subunit ribosomal RNA (16S lsu rRNA). The health risk posed by D. auratus in Singapore is discussed.

Historic and modern genomes unveil a domestic introgression gradient in a wild red junglefowl population.

The red junglefowl Gallus gallus is the ancestor of the domestic chicken and arguably the most important bird species on Earth. Continual gene flow between domestic and wild populations has compromised its gene pool, especially since the last century when human encroachment and habitat loss would have led to increased contact opportunities. We present the first combined genomic and morphological admixture assessment of a native population of red junglefowl, sampled from recolonized parts of its former range in Singapore, partly using whole genomes resequenced from dozens of individuals. Crucially, this population was genomically anchored to museum samples from adjacent Peninsular Malaysia collected ~110-150 years ago to infer the magnitude of modern domestic introgression across individuals. We detected a strong feral-wild genomic continuum with varying levels of domestic introgression in different subpopulations across Singapore. Using a trait scoring scheme, we determined morphological thresholds that can be used by conservation managers to successfully identify individuals with low levels of domestic introgression, and selected traits that were particularly useful for predicting domesticity in genomic profiles. Our study underscores the utility of combined genomic and morphological approaches in population management and suggests a way forward to safeguard the allelic integrity of wild red junglefowl in perpetuity.

Ecology of bat flies in Singapore: A study on the diversity, infestation bias and host specificity (Diptera: Nycteribiidae).

Bat flies are highly-specialized, hematophagous arthropods that are globally ubiquitous. There is little published research on bat flies (Diptera: Nycteribiidae) in Singapore and understanding the diversity of nycteribiids, host association and infestation rates can provide insight into this host-ectoparasite relationship. Nycteribiids were collected from bats trapped in Singapore (2011-2016) and identified using morphological keys. Host-ectoparasite relationships were investigated with logistic regression and Bayesian poisson regression. Nycteribiids were found to be monoxenously associated with their host bat species and host age, sex, species, and BBCI appear to contribute to differences in prevalence and intensity. Differences in host specificity between bat fly species in Singapore and their conspecifics in less disturbed habitats with higher bat biodiversity, such as Malaysia, Philippines and Thailand, suggest that the high host specificity in Singapore derives from the paucity of suitable hosts and abundance of single species roosts and not from their coevolved restrictions to them.

Serologic Evidence of Fruit Bat Exposure to Filoviruses, Singapore, 2011-2016.

To determine whether fruit bats in Singapore have been exposed to filoviruses, we screened 409 serum samples from bats of 3 species by using a multiplex assay that detects antibodies against filoviruses. Positive samples reacted with glycoproteins from Bundibugyo, Ebola, and Sudan viruses, indicating filovirus circulation among bats in Southeast Asia.

Influence of age and body condition on astrovirus infection of bats in Singapore: An evolutionary and epidemiological analysis.

Bats are unique mammals that are reservoirs of high levels of virus diversity. Although several of these viruses are zoonotic, the majority are not. Astroviruses, transmitted fecal-orally, are commonly detected in a wide diversity of bat species, are prevalent at high rates and are not thought to directly infect humans. These features make astroviruses useful in examining virus evolutionary history, epidemiology in the host, and temporal shedding trends. Our study screened for the presence of astroviruses in bats in Singapore, reconstructed the phylogenetic relations of the polymerase genes and tested for population characteristics associated with infection. Of the seven species screened, astroviruses were detected in Rhinolophus lepidus and Eonycteris spelaea. The R. lepidus sequences grouped with other Rhinolophus astrovirus sequences from China and Laos, while the Eoncyteris sequences formed a distinct clade with astroviruses from Rousettus spp. in Laos and Pteropus giganteus in Bangladesh, but not with other E. spelaea sequences. Longitudinal collections of Eonycteris feces demonstrated variable shedding. Juvenile status of bats was a risk factor for astroviruses. This study highlights the diversity of astroviruses in nectivorous and insectivorous bats in Singapore and provides a predictive framework for understanding astrovirus infection in these bats. It also suggests that in addition to host phylogenetic relatedness, host ecology, such as roosting behavior, may drive co-infections, virus maintenance and spillover.

Survey of Treponemal Infections in Free-Ranging and Captive Macaques, 1999-2012.

Survey results showed treponemal infection among pet macaques in Southeast Asia, a region with a high prevalence of human yaws. This finding, along with studies showing treponemal infection in nonhuman primates in Africa, should encourage a One Health approach to yaws eradication and surveillance activities, possibly including monitoring of nonhuman primates in yaws-endemic regions.

Urban primate ranging patterns: GPS-collar deployments for Macaca fascicularis and M. sylvanus.

The global increase in urbanization is leading to heavier interface between humans and wildlife. Within these anthropogenic landscapes, little is known about ranging patterns, particularly with regard to urban primates. Here we present the results of the first long-term deployment of multiple GPS collars on two species of macaques to investigate the impacts of urbanization on urban primate ranging patterns in Singapore and Gibraltar. Collars data acquisition were excellent with respect to the amount, quality, and accuracy of data collected; however, remote connectivity and drop-off functionality was poor across all deployments. Analyses highlighted high variability in ranging patterns between individuals within each species that aligned with access to human food resources and patterns of tourism. Individuals from troops with less access to human food had much larger home, core, and day ranges relative to those with regular provisioning or raiding opportunities. Almost no temporal range overlap was observed between any focal individuals at either site and spatial overlap was low for all but two troops at each site. We found no relationship between anthropogenic schedules and changes in ranging patterns. Significant seasonal variation existed for daily path length and day range size for both the Singapore long-tailed and the Gibraltar Barbary macaques, with long-tailed macaques increasing their range during the equatorial monsoon season and Barbary macaques increasing their range during drier, summer months. This study highlights how the behavioral plasticity found within the genus Macaca is reflected in ranging pattern variability within urban environments.

The identity of Vespertilio oreias Temminck, 1840-solving a taxonomic puzzle.

Vespertilio oreias (generally known as Myotis oreias) has long been considered an endemic bat species to Singapore but its taxonomic status has been in doubt, and no specimens have been found since its description in 1840. Temminck formally described it based on a mounted skin (now in poor condition and accompanied by some skull fragments). The holotype was re-examined and we found it to be a composite, consisting of two separate individuals representing two distinct genera, the skin belonging to a Kerivoula whereas the skull fragments are of a Myotis. The mounted skin is accepted herewith as the name-bearing type, as the skull fragments were taken out after Temminck had published his description. Unfortunately, neither the skin nor the dental remains show enough anatomical details to identify the species unambiguously. Hence, the name Vespertilio oreias is considered a nomen dubium and the name oreias should be referred to the genus Kerivoula.

Citizen Science and the Urban Ecology of Birds and Butterflies - A Systematic Review.

Citizen science has gained widespread currency as a tool for ecological research over the past decade. However, in the discipline of urban ecology, the existing contributions and future potential of citizen science engagement, specifically in terms of knowledge gain, have not yet been comprehensively explored. Here, we present a systematic review of published work on the urban ecology of birds and butterflies in relation to their use of citizen science data between 2005 and 2014. We compared the number of studies that used citizen science data to the number of studies that could potentially have employed data derived from citizen science. The take-up rates of citizen science data were 21% and 26% for birds and butterflies respectively. Most studies that employed citizen science used volunteer-derived data as primary data, and adopted Collegial, Collaborative and Contributional engagement modes to the exclusion of Contractual and Co-created arrangements. There was no evidence that citizen science studies investigated a different organismal scale (community vs. species) compared to the urban ecology literature. For both taxa, citizen science contributions were lower than expected compared to their representation in the urban ecology literature for studies on species-environment relationships at landscape and micro-environment scales, as well as behavioural ecology in general. Other research topics that could benefit from further citizen science involvement include breeding studies and guild analyses for birds, and multi-taxa studies for butterflies. Promising models of citizen science engagement for urban ecology are highlighted in relation to their thematic foci and methodological detail, and a number of research questions that could be productively addressed using citizen science are identified. The dynamics of contemporary engagement between citizen science and urban ecology described by this review could inform the design and refinement of urban ecology-citizen science programmes in order to optimise their scientific contributions.

Variation in hair δ(13)C and δ (15)N values in long-tailed macaques (Macaca fascicularis) from Singapore.

Much of the primatology literature on stable isotope ratios of carbon (δ(13)C) and nitrogen (δ(15)N) has focused on African and New World species, with comparatively little research published on Asian primates. Here we present hair δ(13)C and δ(15)N isotope values for a sample of 33 long-tailed macaques from Singapore. We evaluate the suggestion by a previous researcher that forest degradation and biodiversity loss in Singapore have led to a decline in macaque trophic level. The results of our analysis indicated significant spatial variability in δ(13)C but not δ(15)N. The range of variation in δ(13)C was consistent with a diet based on C3 resources, with one group exhibiting low values consistent with a closed canopy environment. Relative to other macaque species from Europe and Asia, the macaques from Singapore exhibited a low mean δ(13)C value but mid-range mean δ(15)N value. Previous research suggesting a decline in macaque trophic level is not supported by the results of our study.

Influenza virus infection in nonhuman primates.

To determine whether nonhuman primates are infected with influenza viruses in nature, we conducted serologic and swab studies among macaques from several parts of the world. Our detection of influenza virus and antibodies to influenza virus raises questions about the role of nonhuman primates in the ecology of influenza.

From the mouths of monkeys: detection of Mycobacterium tuberculosis complex DNA from buccal swabs of synanthropic macaques.

Although the Mycobacterium tuberculosis complex (MTBC) infects a third of all humans, little is known regarding the prevalence of mycobacterial infection in nonhuman primates (NHP). For more than a century, tuberculosis has been regarded as a serious infectious threat to NHP species. Advances in the detection of MTBC open new possibilities for investigating the effects of this poorly understood pathogen in diverse populations of NHP. Here, we report results of a cross-sectional study using well-described molecular methods to detect a nucleic acid sequence (IS6110) unique to the MTBC. Sample collection was focused on the oral cavity, the presumed route of transmission of MTBC. Buccal swabs were collected from 263 macaques representing 11 species in four Asian countries and Gibraltar. Contexts of contact with humans included free ranging, pets, performing monkeys, zoos, and monkey temples. Following DNA isolation from buccal swabs, the polymerase chain reaction (PCR) amplified IS6110 from 84 (31.9%) of the macaques. In general, prevalence of MTBC DNA was higher among NHP in countries where the World Health Organization reports higher prevalence of humans infected with MTBC. This is the first demonstration of MTBC DNA in the mouths of macaques. Further research is needed to establish the significance of this finding at both the individual and population levels. PCR of buccal samples holds promise as a method to elucidate the mycobacterial landscape among NHP, particularly macaques that thrive in areas of high human MTBC prevalence.

Lead levels in long-tailed macaque (Macaca fascicularis) hair from Singapore.

Nonhuman primates are potentially good sentinels of environmental toxicants because they share a similar physiology and life history with humans. In this report we present the results of an analysis of lead concentrations in hair from long-tailed macaques in Singapore. We hypothesized that because Singapore is highly urbanized, its macaque population may be exposed to higher levels of lead. The results of our study indicated that Singapore's macaque population has not been exposed to high levels of environmental lead. Compared with previous studies of lead levels in human and nonhuman primate hair, the results of our analysis indicate a low level of exposure of monkeys to environmental lead (n = 27, arithmetic mean = 2.51 ppm, max = 6.45, min = 0.21 ppm). Hair lead concentrations varied both within social groups and by geographic location, with the highest concentrations observed in monkeys residing within an area containing a small-arms firing range and a manufacturing facility. Although lead exposure in this area seems to be low, additional monitoring and possible remediation may be warranted. Our study is among the first to illustrate how primates can serve as potential sentinels of environmental toxicants such as lead. Future research examining the efficacy of primates as sentinels of lead exposure should include monitoring of environmental lead levels, and comparison of hair lead levels with levels measured in blood samples.

Macaque-human interactions and the societal perceptions of macaques in Singapore.

Humans and long-tailed macaques (Macaca fascicularis) interface in several locations in Singapore. We investigated six of these interface zones to assess the level of conflict between the two species. We observed macaque-to-human interactions and distributed questionnaires to residents and visitors of nature reserves. We observed an average of two macaque-to-human interactions per hour at the sites, which included affiliative or submissive behaviors (46.9%), aggression (19.1%), taking food and other items (18.5%) searching bins, cars, and houses (13.4%), and nonaggressive contact (2.1%). Two-thirds of interactions occurred when a human was carrying food or food cues, and one-quarter occurred when a human provoked macaques. Only 8% of interactions occurred without a clear human-triggered context. Our interview showed one-third of respondents experienced nuisance problems from macaques. They had items taken from them (50.5%) and received threats (31.9%). Residents reported more nuisance problems than visitors, and their perceptions toward macaques differed. Residents were more aware of the consequences of food provisioning and that there were regulations against feeding. Residents fed macaques less and held more negative sentiments toward macaques. Nearly half of the interviewed people held neutral attitudes toward macaques and only 26.2% of respondents thought conflict with macaques warranted urgent action. Nearly two-thirds of the respondents supported education programs to ameliorate human-macaque conflict, and less than 15% supported removing or eradicating macaques. 87.6% felt that it is importance to conserve and protect macaques. Our results show that human-macaque conflict exists in Singapore, but that it may not be severe. Human behavior is largely responsible for macaque-to-human interactions, and thus could be lessened with management of human behavior in interface zones (i.e. restrict food carrying and provocation). Moreover, our interviews shows people living in Singapore value macaques, do not wish them entirely removed, prefer education-based solutions, and consider conservation and protection of them important.