Tiger Samples Research Articles

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.

METHOD COMPARISON FOR MEASUREMENT OF SYMMETRIC DIMETHYLARGININE IN TIGERS (PANTHERA TIGRIS).

Renal disease is well documented in nondomestic felids and is monitored and diagnosed by serum concentration of blood urea nitrogen, creatinine, and phosphorous. Symmetric dimethylarginine (SDMA) has proven to be an earlier and more sensitive biomarker for the assessment of glomerular filtration rate. Although SDMA is commonly measured in nondomestic felids, information concerning the validity of the assay is lacking. The purpose of the study was to perform a method comparison between high-throughput immunoassay and the reference method, liquid chromatography-tandem mass spectrometry (LC-MS/MS), to quantify SDMA concentrations in tiger blood samples. Concentrations of SDMA were measured for 81 individual tiger samples. The SDMA immunoassay demonstrated excellent correlation to the LC-MS/MS reference method. A Passing and Bablok linear regression analysis had a slope of 1.03 (95% CI, 0.99-1.11), an intercept of 1.64 (95% CI, 0.46-2.34), and a Pearson R= 0.99. The mean bias was 1.53 µg/dl (95% CI, 0.63-2.42 µg/dl), and the limit of agreement was ±7.96 µg/dl. The degree of bias is within established acceptance criteria of 1-3 µg/dl for the immunoassay. Although this study provides good evidence of the utility of the immunoassay to measure SDMA in tiger serum and plasma, further assay validation is recommended.

Phylogeography of Panthera tigris in the mangrove forest of the Sundarbans

Tigers Panthera tigris in the Sundarbans represent the only population adapted to living in mangrove forest habitat. Several studies, based on limited morphological and genetic data, have described the population as being differentiated from the Bengal tiger subspecies P. tigris tigris. The phylogenetic ancestry of the Sundarbans population has also remained poorly understood. We generated 1263 bp of mtDNA sequences across 4 mtDNA genes for 33 tiger samples from the Bangladesh Sundarbans and compared these with 33 mtDNA haplotypes known from all subspecies of extant tigers. We detected 3 haplotypes within the Sundarbans tigers, of which one is unique to this population and the remaining 2 are shared with tiger populations inhabiting central Indian landscapes. Phylogenetic analyses using maximum likelihood and Bayesian inferences supported the Sundarbans tigers as being paraphyletic, indicating a close phylogenetic relationship with other populations of Bengal tigers, from which the Sundarbans population diverged around 26000 yr ago. Our phylogenetic analyses, together with evidence of ecological adaptation to the unique mangrove habitat, indicate that the Sundarbans population should be recognised as a separate management unit. We recommend that conservation management must focus on sustaining this representative tiger population adapted to mangrove habitat while at the same time recognising that trans-boundary conservation efforts through reintroduction or exchange of individuals, to enhance genetic diversity, might be needed in the future as a last resort for population recovery.

Effects of faecal inorganic content variability on quantifying glucocorticoid and thyroid hormone metabolites in large felines: Implications for physiological assessments in free-ranging animals

Faecal glucocorticoid (GC) and triiodothyronine (T3) metabolites and their interactions are increasingly used to monitor perceived stress and nutritional challenges in free-ranging animals. However, a number of extrinsic and intrinsic factors including hormone-inert dietary materials, inorganic matters etc. are known to affect reliable hormone metabolite quantifications. In this study, the impacts of inorganic matter (IOM) on faecal GC (fGCMs) and T3 (fT3Ms) metabolite measure were addressed in wild tiger (n = 193 from Terai Arc landscape, India) and captive lion (n = 120 from Sakkarbaug Zoological Garden, Gujarat, India) and possible corrective measures were evaluated. The wild tiger samples contained highly variable IOM content (9–98%, mostly with > 40% IOM) compared to captive Asiatic lion (17–57%, majority with < 40% IOM). Significant correlations were observed between IOM content and tiger fGCM (r = -0.46, p = 0.000), fT3M (r = -0.58, p = 0.000) and lion fT3M measures (r = -0.43, p = 0.003). Two corrective measures viz. removing samples with ≥ 80% IOM and subsequently expressing concentrations as per gram of organic dry matter (instead of total dry matter) reduced IOM influence on tiger fGCM, fT3M and lion fT3M, without affecting lion fGCM measures. The corrective measures changed the interpretations of fT3M data of field-collected tiger samples with no significant changes in fGCM (both tiger and lion) and fT3M (lion) data. As faecal IOM content is common in many wild species, the results emphasize the need to reduce IOM-driven hormone data variation for ecologically relevant interpretations towards species conservation.

Serum Protein Gel Agarose Electrophoresis in Captive Tigers.

Simple SummaryThe tiger is the largest of the wild cats. There are fewer than 4000 wild tigers (Panthera tigris) worldwide and all subspecies of tigers are globally endangered. Due to pressures from poaching and retaliatory killings, this carnivore had lost an estimated 93% of its historic range. Given the critical situation of these wild felines, the health of each individual is of prime importance and laboratory blood testing, as well as evaluation of their physical condition, is important in their health assessment. Protein concentrations in the blood can be altered by malnutrition and dehydration as well as by disease. Serum electrophoresis allows the identification of the different protein fractions present in the blood and represents a useful tool in the diagnosis and monitoring of a number of diseases. Due to the nature of wild Panthera tigris, it is extremely difficult to obtain biological samples from free-living subjects, and therefore the values obtained from captive tigers provide very useful data. This study reports serum protein electrophoresis in 11 adult captive individuals. These results will be useful for the evaluation of physiological and pathological alterations in wild and captive tigers and populations.Given the endangered status of tigers (Panthera tigris), the health of each individual is important and any data on blood chemistry values can provide valuable information alongside the assessment of physical condition. The nature of tigers in the wild makes it is extremely difficult to obtain biological samples from free-living subjects, therefore the values obtained from captive tigers provide very useful data. Serum protein electrophoresis is a useful tool in the diagnosis and monitoring of a number of diseases. In this study, we evaluated agarose gel serum protein electrophoresis on samples from 11 healthy captive tigers. Serum electrophoresis on all 11 tiger samples successfully separated proteins into albumin, α1, α2, β1, β2 and γ globulin fractions as in other mammals. Electrophoretic patterns were comparable in all tigers. Mean± standard deviation or median and range values obtained for each protein fraction in healthy tigers were, respectively: 3.6 ± 0.2, 0.21 (0.2–0.23), 1.2 ± 0.2, 10.7 ± 0.2, 0.4 (0.3–0.6), 1.2 (1–1.8) gr/dL. The results of this preliminary study provide the first data on serum electrophoretic patterns in tigers and may be a useful diagnostic tool in the health assessment of this endangered species.

Empowering conservation practice with efficient and economical genotyping from poor quality samples.

Moderate- to high-density genotyping (100 + SNPs) is widely used to determine and measure individual identity, relatedness, fitness, population structure and migration in wild populations.However, these important tools are difficult to apply when high-quality genetic material is unavailable. Most genomic tools are developed for high-quality DNA sources from laboratory or medical settings. As a result, most genetic data from market or field settings is limited to easily amplified mitochondrial DNA or a few microsatellites.To enable genotyping in conservation contexts, we used next-generation sequencing of multiplex PCR products from very low-quality DNA extracted from faeces, hair and cooked samples. We demonstrated utility and wide-ranging potential application in endangered wild tigers and tracking commercial trade in Caribbean queen conch.We genotyped 100 SNPs from degraded tiger samples to identify individuals, discern close relatives and detect population differentiation. Co-occurring carnivores do not amplify (e.g. Indian wild dog/dhole) or are monomorphic (e.g. leopard). Sixty-two SNPs from conch fritters and field-collected samples were used to test relatedness and detect population structure.We provide proof of concept for a rapid, simple, cost-effective and scalable method (for both samples and number of loci), a framework that can be applied to other conservation scenarios previously limited by low-quality DNA samples. These approaches provide a critical advance for wildlife monitoring and forensics, open the door to field-ready testing, and will strengthen the use of science in policy decisions and wildlife trade.

Species, sex and geo-location identification of seized tiger (Panthera tigris tigris) parts in Nepal-A molecular forensic approach.

Tiger (Panthera tigris) populations are in danger across their entire range due to habitat loss, poaching and the demand for tiger parts. The Bengal tiger (Panthera tigris tigris) is an endangered apex predator with a population size estimated to be less than 200 in Nepal. In spite of strict wildlife protection laws, illegal trade of tiger parts is increasing; and Nepal has become one of the major sources and transit routes for poached wildlife parts. Identification of wildlife parts is often challenging for law enforcement officials due to inadequate training and lack of available tools. Here, we describe a molecular forensic approach to gain insight into illegally trafficked tiger parts seized across Nepal. We created Nepal’s first comprehensive reference genetic database of wild tigers through the Nepal Tiger Genome Project (2011–2013). This database has nuclear DNA microsatellite genotype and sex profiles, including geo-spatial information, of over 60% (n = 120) of the wild tigers of Nepal. We analyzed 15 putative cases of confiscated poached tiger parts and all were confirmed to be of tiger. Ten samples were identified as male and five were female. We determined probable geo-source location for 9 of the 14 samples with 6–8 nuclear DNA microsatellite loci using inferences from four different statistical assignment methods. Six samples were assigned to Bardia National Park and one of these was an exact match to a female tiger previously profiled in our fecal DNA reference database. Two tiger samples were assigned to Shuklaphanta Wildlife Reserve and one to Chitwan National Park. We are unable to definitively assign five tiger samples which could be offspring dispersers or might have come from tiger population outside of Nepal. Our study revealed that the western region, particularly Bardia National Park, is a poaching hotspot for illegal tiger trade in Nepal. We present feasibility of using molecular forensic based evidence to incriminate criminals in a court of law in the fight against wildlife crime.

Do rivers influence fine-scale population genetic structure of tigers in the Sundarbans?

Global tiger Panthera tigris populations mostly survive within the geographically fragmented forest patches, thereby limited genetic exchange between isolated populations. Assessing the genetic status of these populations can reveal the effects of dispersal barriers and provide critical insights to guide future conservation actions. Using non-invasively collected biological samples, we investigated fine-scale genetic structure of tigers in the Sundarbans mangrove forests intersected by the complex river systems, and which holds one of the largest global tiger populations. We genotyped 52 tiger samples at 10 polymorphic microsatellite loci, and sequenced 33 of them for a total of 1263 base-pairs at four mitochondrial gene fragments. Microsatellite analyses exhibit a signature of fine-scale genetic structure, which might have been the consequence of limited tiger dispersal due to wide rivers across the Sundarbans. Similarly, mitochondrial data show a historic pattern of population isolation that might be due to wider rivers across the entire Sundarbans shared by Bangladesh and India. Given the intrinsic nature of the mangrove habitat embedded with numerous rivers, increased commercial traffic and human activities may further impede tiger dispersal across wide rivers, escalating further genetic isolation of the Sundarbans tigers.

Using non-invasively collected genetic data to estimate density and population size of tigers in the Bangladesh Sundarbans

Population density is a key parameter to monitor endangered carnivores in the wild. The photographic capture-recapture method has been widely used for decades to monitor tigers, Panthera tigris, however the application of this method in the Sundarbans tiger landscape is challenging due to logistical difficulties. Therefore, we carried out molecular analyses of DNA contained in non-invasively collected genetic samples to assess the tiger population in the Bangladesh Sundarbans within a spatially explicit capture-recapture (SECR) framework. By surveying four representative sample areas totalling 1994 km2 of the Bangladesh Sundarbans, we collected 440 suspected tiger scat and hair samples. Genetic screening of these samples provided 233 authenticated tiger samples, which we attempted to amplify at 10 highly polymorphic microsatellite loci. Of these, 105 samples were successfully amplified, representing 45 unique genotype profiles. The capture-recapture analyses of these unique genotypes within the SECR model provided a density estimate of 2.85 ± SE 0.44 tigers/100 km2 (95% CI: 1.99–3.71 tigers/100 km2) for the area sampled, and an estimate of 121 tigers (95% CI: 84–158 tigers) for the total area of the Bangladesh Sundarbans. We demonstrate that this non-invasive genetic surveillance can be an additional approach for monitoring tiger populations in a landscape where camera-trapping is challenging.

Fine-scale population genetic structure of the Bengal tiger (Panthera tigris tigris) in a human-dominated western Terai Arc Landscape, India.

Despite massive global conservation strategies, tiger populations continued to decline until recently, mainly due to habitat loss, human-animal conflicts, and poaching. These factors are known to affect the genetic characteristics of tiger populations and decrease local effective population sizes. The Terai Arc Landscape (TAL) at the foothills of the Himalaya is one of the 42 source sites of tigers around the globe. Therefore, information on how landscape features and anthropogenic factors affect the fine-scale spatial genetic structure and variation of tigers in TAL is needed to develop proper management strategies for achieving long-term conservation goals. We document, for the first time, the genetic characteristics of this tiger population by genotyping 71 tiger samples using 13 microsatellite markers from the western region of TAL (WTAL) of 1800 km2. Specifically, we aimed to estimate the genetic variability, population structure, and gene flow. The microsatellite markers indicated that the levels of allelic diversity (MNA = 6.6) and genetic variation (Ho = 0.50, HE = 0.64) were slightly lower than those reported previously in other Bengal tiger populations. We observed moderate gene flow and significant genetic differentiation (FST= 0.060) and identified the presence of cryptic genetic structure using Bayesian and non-Bayesian approaches. There was low and significantly asymmetric migration between the two main subpopulations of the Rajaji Tiger Reserve and the Corbett Tiger Reserve in WTAL. Sibship relationships indicated that the functionality of the corridor between these subpopulations may be retained if the quality of the habitat does not deteriorate. However, we found that gene flow is not adequate in view of changing land use matrices. We discuss the need to maintain connectivity by implementing the measures that have been suggested previously to minimize the level of human disturbance, including relocation of villages and industries, prevention of encroachment, and banning sand and boulder mining in the corridors.

The development and application of a multiplex short tandem repeat (STR) system for identifying subspecies, individuals and sex in tigers.

Poaching and trans-boundary trafficking of tigers and body parts are threatening the world's last remaining wild tigers. Development of an efficient molecular genetic assay for tracing the origins of confiscated specimens will assist in law enforcement and wildlife forensics for this iconic flagship species. We developed a multiplex genotyping system "tigrisPlex" to simultaneously assess 22 short tandem repeat (STR, or microsatellite) loci and a gender-identifying SRY gene, all amplified in 4 reactions using as little as 1 ng of template DNA. With DNA samples used for between-run calibration, the system generates STR genotypes that are directly compatible with voucher tiger subspecies genetic profiles, hence making it possible to identify subspecies via bi-parentally inherited markers. We applied "tigrisPlex" to 12 confiscated specimens from Russia and identified 6 individuals (3 females and 3 males), each represented by duplicated samples and all designated as Amur tigers (Panthera tigris altaica) with high confidence. This STR multiplex system can serve as an effective and versatile approach for genetic profiling of both wild and captive tigers as well as confiscated tiger products, fulfilling various conservation needs for identifying the origins of tiger samples.

The complete mitochondrial genome analysis of the tiger (Panthera tigris)

The complete mitochondrial genomes of five tiger samples from three subspecies (P. t. sumatrae, P. t. altica, and P. t. tigris) were successfully obtained by using 26 specifically designed Panthera-specific primer sets. The genome organization and gene arrangement of the five tiger samples were similar to each other; however polymorphic tandem repeat sequences were observed in the control region (CR). This led to a difference in the genome lengths obtained from these five samples with an average size of 16,994bp for the five tiger mitochondrial genomes. The nucleotide base composition was on average as follows: A, 31.8%; T, 27.0%; C, 26.6%; G, 14.6% and exhibited compositional asymmetry. Most of tiger mitochondrial genome characteristics are similar to those of other common vertebrate species; however, some distinctive features were observed in the CR. First, the repetitive sequence 2 (RS 2) contained two repeat units of 80bp and the first 15bp of what would be the third repeat motif. The repetitive sequence 3 (RS 3) contained 47-50 repeat motifs of a shorter 8bp (ACGTAYAC)(n). Second, length heteroplasmy polycystosine (poly-C) stretches was observed at the end of the HV I locus in all tiger samples.

Where does this tiger come from?—A robust molecular technique for simultaneous identification of endangered species and subspecies

The Convention on the International Trade in Endangered Species of Wild Fauna and Flora (CITES) monitors the international trade in endangered animal and plant species; a high profile example is the tiger, Panthera tigris. We report on the application of a SNaPshot multiplex technique to simultaneously identify tiger species and subspecies; this test is based on identification of SNPs within the tiger mitochondrial genome. Mitochondrial DNA sequences from four of the five extant putative tiger subspecies were obtained and combined with DNA sequence data from 492 tiger and 349 other mammalian species. A total of 11 SNP loci were identified: five specific for tiger; three specific to Panthera tigris sumatrae and; three specific to P. t. tigris. The multiplex assay was able to reliably identify 15 voucher tiger samples. The sensitivity of the test was 15,000 mitochondrial DNA copies, indicating that it will work on trace amounts of tissue, bone or hair.

The development and validation of a single SNaPshot multiplex for tiger species and subspecies identification—Implications for forensic purposes

The tiger (Panthera tigris) is currently listed on Appendix I of the Convention on the International Trade in Endangered Species of Wild Fauna and Flora; this affords it the highest level of international protection. To aid in the investigation of alleged illegal trade in tiger body parts and derivatives, molecular approaches have been developed to identify biological material as being of tiger in origin. Some countries also require knowledge of the exact tiger subspecies present in order to prosecute anyone alleged to be trading in tiger products. In this study we aimed to develop and validate a reliable single assay to identify tiger species and subspecies simultaneously; this test is based on identification of single nucleotide polymorphisms (SNPs) within the tiger mitochondrial genome. The mitochondrial DNA sequence from four of the five extant putative tiger subspecies that currently exist in the wild were obtained and combined with DNA sequence data from 492 tiger and 349 other mammalian species available on GenBank. From the sequence data a total of 11 SNP loci were identified as suitable for further analyses. Five SNPs were species-specific for tiger and six amplify one of the tiger subspecies-specific SNPs, three of which were specific to P. t. sumatrae and the other three were specific to P. t. tigris. The multiplex assay was able to reliably identify 15 voucher tiger samples. The sensitivity of the test was 15,000 mitochondrial DNA copies (approximately 0.26pg), indicating that it will work on trace amounts of tissue, bone or hair samples. This simple test will add to the DNA-based methods currently being used to identify the presence of tiger within mixed samples.

Diversity and characterization of oxytetracycline-resistant bacteria associated with non-native species, white-leg shrimp (Litopenaeus vannamei), and native species, black tiger shrimp (Penaeus monodon), intensively cultured in Thailand

This study aimed at surveying prevalence of oxytetracycline (OTC)-resistant bacteria in the white-leg shrimp Litopenaeus vannamei, and the black tiger shrimp Penaeus monodon, intensively cultured in Thailand. We investigated the phylogenetic diversity of the bacterial isolates, as well as the minimum inhibitory concentration (MIC) of OTC, the occurrence of major OTC-resistant genes and multiple-antibiotic resistance in the isolates. Shrimps were collected from culture ponds, and the homogenates of whole bodies were plated on tryptic soy agar supplemented with or without OTC. Percentages of OTC-resistant bacteria were 0·3-52·1% in white-leg samples and 0·008-22·3% in black tiger samples. Analyses of 16S rDNA sequences indicated that most OTC-resistant isolates were closely related to Aeromonas spp. and Lactococcus garvieae. MICs of OTC were 4-128 μg ml(-1) in the OTC-resistant aeromonads and 128-256 μg ml(-1) in OTC-resistant L. garvieae. OTC resistance was found to be conferred by the genes tet(A), tet(C), tet(D), tet(E), tet(M) and tet(S), detected either singly or in pairs. No resistance to ceftazidime, imipenem or chloramphenicol was observed in any isolate. Both species of shrimp are associated with OTC-resistant bacteria, occasionally at high densities exceeding 10(6) cfu g(-1). The associated bacteria, predominantly Lactococcus and Aeromonas genera, are potential pathogens and are reservoirs of a variety of OTC-resistant genes. Cultured shrimps can be vehicle to carry OTC-resistant bacteria to domestic and foreign consumers via the food chain. Very low populations of OTC-resistant bacteria observed in the several ponds suggest that levels of the resistant bacteria are artificially high and should be reduced in farmed shrimps.