High-throughput DNA metabarcoding for determining the gut microbiome of captive critically endangered Malayan tiger (Panthera tigris jacksoni) during fasting

The Malayan tiger (Panthera tigris jacksoni) is a critically endangered species native to the Malaysian Peninsula. To imitate wild conditions where tigers do not hunt every day, numerous wildlife sanctuaries do not feed their tigers daily. However, the effects of fasting on the gut microbiota of captive Malayan tigers remains unknown. This study aimed to characterise the gut microbiota of captive Malayan tigers by comparing their microbial communities during fasting versus normal feeding conditions. This study was conducted at the Melaka Zoo, Malaysian Peninsula and involved Malayan tigers fasted every Monday. In total, ten faecal samples of Malayan tiger, two of Bengal tiger (outgroup) and four of lion (outgroup) were collected and analysed for metabarcoding targeting the 16S rRNA V3–V4 region. In total, we determined 14 phyla, 87 families, 167 genera and 53 species of gut microbiome across Malayan tiger samples. The potentially harmful bacterial genera found in this study included Fusobacterium, Bacteroides, Clostridium sensu stricto 1,Solobacterium, Echerichia shigella, Ignatzschineria and Negativibacillus. The microbiome in the fasting phase had a higher composition and was more diverse than in the feeding phase. The present findings indicate a balanced ratio in the dominant phyla, reflecting a resetting of the imbalanced gut microbiota due to fasting. These findings can help authorities in how to best maintain and improve the husbandry and health of Malayan tigers in captivity and be used for monitoring in ex-situ veterinary care unit

Partial mtDNA sequencing data of vulnerable Cephalopachus bancanus from the Malaysian Borneo

Tarsier is an endangered nocturnal primate in the family Tarsiidae and is an endemic to Sundaic islands of Philippine (Carlito syrichta), Sulawesi (Tarsius tarsier-complex) and Borneo (Cephalopachus bancanus). Recent records indicated that most molecular studies were done on the Eastern Tarsier and little information for the other group of tarsiers. Here, we present a partial cytochrome b data set of C. bancanus in Sarawak, Malaysian Borneo. Standard mist nets were deployed at strategic locations in various habitat types. A total of 18 individuals were caught, measured and weighed. Approximately, 2 × 2 mm of tissue samples were taken and preserved in molecular grade alcohol. Out of 18, only 11 samples were screened with partial mtDNA (cytochrome b) and the DNA sequences were registered in the GenBank (accession numbers: KY794797-KY794807). Phylogenetic trees were constructed with 20 additional mtDNA sequences downloaded from GenBank. The data are valuable for the management authorities to regulate the type of management units for the metapopulation to sustain population genetics integrity of tarsiers in the range countries across the Sunda Shelf

Alteration of the gut microbial composition of critically endangered Malayan tigers (Panthera tigris jacksoni) in captivity during enrichment phase

Enrichment activities may influence the microbiomes of captive tigers’, affecting their health, digestion, and behavior. Currently, there are few studies that address the impact of enrichment activity on tigers’ health. This study aimed to determine the diversity of the gut microbiome in captive Malayan tigers at Zoo Melaka and Night Safari during the environmental enrichment phase using a metabarcoding approach.This study utilized different enrichment activities which catered for food, sensory, and cognitive enrichment. Eleven fresh fecal samples from captive Malayan tigers at Zoo Melaka and Night Safari were collected under different conditions. All samples were extracted and 16S rRNA V3–V4 region amplicon sequencing was used to characterize the gut microbiome of captive Malayan tigers subjected to various enrichment activities. Firmicutes, Actinobacteriota, and Fusobacteriota were the dominant phyla observed in the gut microbiome of captive Malayan tigers during enrichment activities. This study revealed β-diversity significantly varied between normal and enrichment phase, however no significant differences were observed in α-diversity. This study demonstrates that environmental enrichment improves the gut microbiome of Malayan tigers because gut microbes such as Lachnoclostridium, which has anti-inflammatory effects and helps maintain homeostasis, and Romboutsia, which has a probiotic effect on the gut microbiome. Conclusions This study provides valuable insights into the effects of enrichment activities on the gut microbiome of captive Malayan tigers, offering guidance for enhancing captive management practices aimed at promoting the health and well-being of Malayan tiger in captivity

DNA forensic case study: species identification from suspected crocodile penis

Department of Wildlife and National Parks (PERHILITAN) began developing the capacity on wildlife DNA forensic since 2009 to assist in law enforcement activities. Most of the forensic cases require DNA species identification of animal parts where key morphological characters are missing. Among the cases frequently confiscated are from traditional Chinese medicine (TCM), which often claim to use animal parts such as reproductive organs. Dried crocodile penises, in particular, are believed to have medicinal benefits and are highly demanded in TCM industries since millennials ago. In this case study, we analysed four enforcement cases comprising of 44 exhibits which resemble crocodile penis using the partial cytochrome b gene of the mitochondrial DNA. Sequence similarity searches were conducted using both the BLAST search engines of GenBank and also PERHILITAN’s MyWILDNA database to identify the species. Out of 44 exhibits, 22 items produced DNA sequences in which three were found to be derived from Crocodylus porosus while the remaining was identified as Bos taurus, Bos javanicus, and Bos indicus. This case study showed that most of TCM which claimed to be derived from crocodile penis turned out to be counterfeit products

Rediscovery of nycticebus coucang insularis Robinson, 1917 (Primates: Lorisidae) at Tioman Island and its mitochondrial genetic assessment

Slow lorises (Nycticebus) consist of eight species native to Southeast Asia while three species are recognised in Malaysia - N. coucang, N. menagensis and N. kayan. This study reports on the rediscovery of the subspecies N. coucang insularis Robinson, 1917 in Tioman Island and the genetic assessment of its mitochondrial DNA variation. Morphological measurements conform the specimen as the putative N. coucang but with distinct colour and markings. Two mitochondrial DNA segments (cytochrome b and control region) were produced from the subspecies representing their first registered sequences in GenBank. Genetically, the subspecies showed 99% of nucleotide similarity to N. coucang species type for both the DNA segments and constitute its own unique haplotype. Phylogenetic trees constructed using three methods (neighbour joining, maximum likelihood and Bayesian inference) showed two major groups within Nycticebus; the basal group was formed by N. pygmaeus while the second group consisted of the remaining Nycticebus species. The phylogenetic position of the subspecies, however, remains unresolved due to the observed mixing between N. coucang and N. bengalensis. Several reasons could lead to this condition including the lack of well documented voucher specimens and the short DNA fragments used. In addition, the possibility of hybridisation event between N. coucang and N. bengalensis could not be excluded as a possible explanation since both species occur sympatrically at the Isthmus of Kra region until the Thailand-Malaysia border. The rediscovery of this subspecies displays the unique faunal diversity that justifies the importance of Tioman Island as a protected area

Interspecific variation in the diet of Symphalangus syndactylus and Macaca nemestrina at Genting Highlands, Pahang, Peninsular Malaysia

Primate communities in the Genting Highlands consist of a single species of Hylobatidae and four species of Cercopithecidae, which are known to exhibit social interaction behaviour. Thus, a study on the diets of Symphalangus syndactylus (siamang; family Hylobatidae) and Macaca nemestrina (pig-tailed macaque; family Cercopithecidae) was carried out at Genting Highlands, in order to compare the dietary preferences and interspecific competition between the two primate families. A DNA metabarcoding approach was used to analyse diet intake using non-invasive samples based on the trnL region. Based on the 140 amplicon sequence variants (ASVs) generated, 26 plant orders, 46 different families, 60 genera and 49 species were identified from 23 different plant classes. Fabaceae and Moraceae were classified as the most preferred plants at the family level for S. syndactylus; meanwhile, Piperaceae and Arecaceae were classified as the most preferred for M. nemestrina. Only six out of the 60 different plant genera classified in this study, were found to be consumed by both species. Therefore, the low similarity of preferred plants in the diets between the two families suggests that there is little interspecific competition. These findings are important for future conservation management of highland primates, especially in the Genting Highlands

Natural Plasmodium infection in wild macaques of three states in peninsular Malaysia

Zoonotic cases of Plasmodium knowlesi account for most malaria cases in Malaysia, and humans infected with P. cynomolgi, another parasite of macaques have recently been reported in Sarawak. To date the epidemiology of malaria in its natural Macaca reservoir hosts remains little investigated. In this study we surveyed the prevalence of simian malaria in wild macaques of three states in Peninsular Malaysia, namely Pahang, Perak and Johor using blood samples from 103 wild macaques (collected by the Department of Wildlife and National Parks Peninsular Malaysia) subjected to microscopic examination and nested PCR targeting the Plasmodium small subunit ribosomal RNA gene. As expected, PCR analysis yielded significantly higher prevalence (64/103) as compared to microscopic examination (27/103). No relationship between the age and/or sex of the macaques with the parasitaemia and the Plasmodium species infecting the macaques could be identified. Wild macaques in Pahang had the highest prevalence of Plasmodium parasites (89.7%), followed by those of Perak (69.2%) and Johor (28.9%). Plasmodium inui and P. cynomolgi were the two most prevalent species infecting the macaques from all three states. Half of the macaques (33/64) harboured two or more Plasmodium species. These data provide a baseline survey, which should be extended by further longitudinal investigations that should be associated with studies on the bionomics of the anopheline vectors. This information will allow an accurate evaluation of the risk of zoonotic transmission to humans, and to elaborate effective strategies to control simian malaria

Determining the diet of wild Asian elephants (Elephas maximus) at human–elephant conflict areas in Peninsular Malaysia using DNA metabarcoding

Human–elephant conflict (HEC) contributes to the increasing death of Asian elephants due to road accidents, retaliatory killings and fatal infections from being trapped in snares. Understanding the diet of elephants throughout Peninsular Malaysia remains crucial to improve their habitat quality and reduce scenarios of HEC. DNA metabarcoding allows investigating the diet of animals without direct observation, especially in risky conflict areas. The aim of this study was to determine: i) the diet of wild Asian elephants from HEC areas in Peninsular Malaysia using DNA metabarcoding and ii) the influence of distinct environmental parameters at HEC locations on their feeding patterns. DNA was extracted from 39 faecal samples and pooled into 12 groups representing the different sample locations: Kuala Koh, Kenyir, Ulu Muda, Sira Batu, Kupang-Grik, Bumbun Tahan, Belum-Temengor, Grik, Kampung Pagi, Kampung Kuala Balah, Aring 10 and the National Elephant Conservation Centre, which served as a positive control for this study. DNA amplification and sequencing targeted the ribulose-bisphosphate carboxylase gene using the next-generation sequencing Illumina iSeq100 platform. Overall, we identified 35 orders, 88 families, 196 genera and 237 species of plants in the diet of the Asian elephants at HEC hotspots. Ficus (Moraceae), Curcuma (Zingiberaceae), Phoenix (Arecaceae), Maackia (Fabaceae), Garcinia (Clusiaceae) and Dichapetalum (Dichapetalaceae) were the highly abundant dietary plants. The plants successfully identified in this study could be used by the Department of Wildlife and National Parks (PERHILITAN) to create buffer zones by planting the recommended dietary plants around HEC locations and trails of elephants within Central Forest Spine (CFS) landscape

Unveiling prey preferences of endangered wild Malayan tiger, Panthera tigris jacksoni, in Peninsular Malaysia through scat analysis via COI DNA metabarcoding

Understanding the prey preference of Malayan tiger (Panthera tigris jacksoni) in Malaysia is important to guide conservation planning initiatives. The utilisation of DNA metabarcoding provides valuable insights, particularly in the field of carnivora diet research. This technique has been proven to be effective for identifying various species within complex mixtures such as scat materials, where visual identification is challenging. The Cytochrome c oxidase subunit I (COI) locus has been selected as it is a widely used as an effective non-invasive approach for diet studies. Hence, given this advance approach, Malayan tiger scats were collected on the basis of existing records of their presence in two types of habitats, namely, protected areas (PA) and human–tiger conflict (HTC) areas. This study aimed to identify prey species in Peninsular Malaysia, based on Malayan tiger scat samples using DNA metabarcoding. Based on the partial mitochondrial COI region, DNA metabarcoding led to the taxonomic resolution of prey DNA remnants in scats and the identification of prey species consumed by Malayan tiger, which were predominately small-to-medium-sized prey, including livestock. The dominant DNA prey detected belongs to the family Canidae, followed by Bovidae, Vespertilionidae, Homonidae, Felidae, Phasianidae and Muridae. A significant difference (p < 0.05) was observed in alpha and beta diversity using the Shannon index and PERMANOVA with regard to prey richness and evenness in two different habitat groups, namely, PA and HTC. Our finding provides insights into Malayan tiger dietary requirements, which can be used to develop conservation plans and strategies for Malayan tiger, particularly for habitat priorities

Socializing One Health: an innovative strategy to investigate social and behavioral risks of emerging viral threats

In an effort to strengthen global capacity to prevent, detect, and control infectious diseases in animals and people, the United States Agency for International Development’s (USAID) Emerging Pandemic Threats (EPT) PREDICT project funded development of regional, national, and local One Health capacities for early disease detection, rapid response, disease control, and risk reduction. From the outset, the EPT approach was inclusive of social science research methods designed to understand the contexts and behaviors of communities living and working at human-animal-environment interfaces considered high-risk for virus emergence. Using qualitative and quantitative approaches, PREDICT behavioral research aimed to identify and assess a range of socio-cultural behaviors that could be influential in zoonotic disease emergence, amplification, and transmission. This broad approach to behavioral risk characterization enabled us to identify and characterize human activities that could be linked to the transmission dynamics of new and emerging viruses. This paper provides a discussion of implementation of a social science approach within a zoonotic surveillance framework. We conducted in-depth ethnographic interviews and focus groups to better understand the individual- and community-level knowledge, attitudes, and practices that potentially put participants at risk for zoonotic disease transmission from the animals they live and work with, across 6 interface domains. When we asked highly-exposed individuals (ie. bushmeat hunters, wildlife or guano farmers) about the risk they perceived in their occupational activities, most did not perceive it to be risky, whether because it was normalized by years (or generations) of doing such an activity, or due to lack of information about potential risks. Integrating the social sciences allows investigations of the specific human activities that are hypothesized to drive disease emergence, amplification, and transmission, in order to better substantiate behavioral disease drivers, along with the social dimensions of infection and transmission dynamics. Understanding these dynamics is critical to achieving health security--the protection from threats to health-- which requires investments in both collective and individual health security. Involving behavioral sciences into zoonotic disease surveillance allowed us to push toward fuller community integration and engagement and toward dialogue and implementation of recommendations for disease prevention and improved health security