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

BACKGROUND: 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. METHODS AND RESULTS: 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 ß-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.

Molecular phylogeny confirms the subspecies delineation of the Malayan Siamang (Symphalangussyndactyluscontinentis) and the Sumatran Siamang (Symphalangussyndactylussyndactylus) based on the hypervariable region of mitochondrial DNA.

Siamangs (Symphalangussyndactylus) are native to Peninsular Malaysia, Sumatra and southern Thailand and their taxonomical classification at subspecies level remains unclear. Morphologically, two subspecies were proposed as early as 1908 by Thomas namely Symphalangus s.syndactylus and Symphalanguss.continentis. Thus, this study aims to clarify the Siamang subspecies status, based on mtDNA D-loop sequences. Faecal samples were collected from wild Siamang populations at different localities in Peninsular Malaysia. A 600-bp sequence of the mitochondrial D-loop region was amplified from faecal DNA extracts and analysed along with GenBank sequences representing Symphalangus sp., Nomascus sp., Hylobates sp., Hoolock sp. and outgroups (Pongopygmaeus, Macacafascicularis and Papiopapio). The molecular phylogenetic analysis in this study revealed two distinct clades formed by S.s.syndactylus and S.s.continentis which supports the previous morphological delineation of the existence of two subspecies. Biogeographical analysis indicated that the Sumatran population lineage was split from the Peninsular Malaysian population lineage and a diversification occurrred in the Pliocene era (~ 3.12 MYA) through southward expansion. This postulation was supported by the molecular clock, which illustrated that the Peninsular Malaysian population (~ 1.92 MYA) diverged earlier than the Sumatran population (~ 1.85 MYA). This is the first study to use a molecular approach to validate the subspecies statuses of S.s.syndactylus and S.s.continentis. This finding will be useful for conservation management, for example, during Siamang translocation and investigations into illegal pet trade and forensics involving Malayan and Sumatran Siamangs.

Habitat shapes the gut microbiome diversity of Malayan tigers (Panthera tigris jacksoni) as revealed through metabarcoding 16S rRNA profiling.

The gut microbiome refers to the microorganism community living within the digestive tract. The environment plays a crucial role in shaping the gut microbiome composition of animals. The gut microbiome influences the health and behavior of animals, including the critically endangered Malayan tiger (Panthera tigris jacksoni). However, the gut microbiome composition of Malayan tigers, especially those living in their natural habitats, remains poorly understood. To address this knowledge gap, we used next-generation sequencing DNA metabarcoding techniques to analyze the gut microbiome of wild Malayan tigers using fecal samples collected from their natural habitats and in captivity. Our aim was to determine the gut microbiota composition of the Malayan tiger, considering the different types of habitat environments. The results revealed a diverse microbial community within the gut microbiome of Malayan tigers. The prominent phyla that were observed included Firmicutes, Proteobacteria, Actinobacteriota, Fusobacteriota and Bacteroidota. Beta diversity analysis revealed significant differences in gut microbiome composition of Malayan tigers that inhabited oil palm plantations, in villages and protected areas. Diversity analysis also revealed significant difference in the gut microbiome between wild and captive Malayan tigers. However, the distinctions of gut microbiome between wild and captive alpha diversity did not yield significant differences. The differences in microbiome diversity resulted from the interplay of dietary intake and environmental factors. This information will facilitate the establishment of focused conservation approaches and enhance our understanding of the effect of microbiome composition on Malayan tiger health.

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

The Malayan tiger (Pantheratigrisjacksoni) 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, Echerichiashigella, 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.

Determining the Dietary Preferences of Wild Asian Elephants (Elephas maximus) in Taman Negara National Park, Malaysia Based on Sex and Age using trnL DNA Metabarcoding Analysis.

The world's largest terrestrial mammal, Asian elephants, are known to have enormous feeding needs. Several factors such as season, sex, age, and daily activities influence the amount of food required by an individual. Generally, captive elephants have a limited choice of food on a daily basis compared with that of elephants in the wild. Elephants in captivity are fed according to a prepared feeding schedule, whereas wild elephants are free to choose the type of plants that they consume in their natural habitat. In the past, ecological observations have been widely used to determine the diet of wild elephants. However, the molecular approach has never been carried out. In the present study, we aimed to; 1) identify the plant diet of wild Asian elephants in Taman Negara National Park (TNNP) according to their sex and age using high-throughput DNA metabarcoding; and 2) determine the dietary formulation of captive elephants based on the generated plant metabarcoding database. DNA was extracted from 24 individual fecal samples collected using noninvasive sampling techniques from TNNP and the National Elephant Conservation Centre (NECC) Kuala Gandah. Seven pooled samples from male adult, female adult, male subadult, female subadult, male juvenile, female juvenile, and captive elephants were amplified and sequenced targeting the trnL region (50-150 base pairs). The CLC Genomic Workbench and PAST 4.02 software were used for data analysis. In total, 24 orders, 41 families, 233 genera, and 306 species of plants were successfully detected in the diet of the Asian elephants. The most abundant plant genera consumed were Sporobolus (21.88%), Musa (21.48%), and Ficus (10.80%). Plant variation was lower in samples from male elephants than in those from female elephants. The plant species identified were correlated with the nutrient benefits required by elephants. Adults and subadults consumed more plant species than were consumed by juvenile elephants. However, there was no significant difference between ages and sexes. The findings of this study can be used as guidance by the Department of Wildlife and National Parks for the management of captive elephants, especially in NECC Kuala Gandah.

Metabarcoding data analysis revealed the plant dietary variation of long-tailed macaque Macacafascicularis (Cercopithecidae, Cercopithecinae) living in disturbed habitats in Peninsular Malaysia.

The long-tailed macaque (Macacafascicularis) has a wide range in both Peninsular Malaysia and Borneo. Although the primates are especially vulnerable to habitat alterations, this primate lives in disturbed habitats due to human-induced land-use. Thus, this study presents a faecal metabarcoding approach to clarify the plant diet of long-tailed macaques from five locations in Peninsular Malaysia to represent fragmented forest, forest edge, island and recreational park habitats. We extracted genomic DNA from 53 long-tailed macaque faecal samples. We found 47 orders, 126 families, 609 genera and 818 species across these five localities. A total of 113 plant families were consumed by long-tailed macaques in Universiti Kebangsaan Malaysia, 61 in the Malaysia Genome and Vaccine Institute, 33 in Langkawi Island, 53 in Redang Island and 44 in the Cenderawasih Cave. Moraceae (33.24%) and Fabaceae (13.63%) were the most common families consumed by long-tailed macaques from the study localities. We found that habitat type impacted diet composition, indicating the flexibility of foraging activities. This research findings provide an understanding of plant dietary diversity and the adaptability of this macaque with the current alteration level that applies to long-tailed macaque conservation management interest in the future.

Determining the diet of wild Asian elephants (Elephasmaximus) 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.

De novo sequencing, assembly and analysis of eight different transcriptomes from the Malayan pangolin.

Pangolins are scale-covered mammals, containing eight endangered species. Maintaining pangolins in captivity is a significant challenge, in part because little is known about their genetics. Here we provide the first large-scale sequencing of the critically endangered Manis javanica transcriptomes from eight different organs using Illumina HiSeq technology, yielding ~75 Giga bases and 89,754 unigenes. We found some unigenes involved in the insect hormone biosynthesis pathway and also 747 lipids metabolism-related unigenes that may be insightful to understand the lipid metabolism system in pangolins. Comparative analysis between M. javanica and other mammals revealed many pangolin-specific genes significantly over-represented in stress-related processes, cell proliferation and external stimulus, probably reflecting the traits and adaptations of the analyzed pregnant female M. javanica. Our study provides an invaluable resource for future functional works that may be highly relevant for the conservation of pangolins.

Pangolin genomes and the evolution of mammalian scales and immunity.

Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.