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Free-living amoebae are rare causes of morbidity and mortality in humans and animals around the globe. Because the route of exposure and clinical progression of disease caused by different species of amoebae may vary in people and animals, determining the species of amoeba present is important. We describe here a fatal infection by the free-living amoeba Balamuthia mandrillaris in a Siberian tiger ( Panthera tigris altaica). The 17-y-old patient had a rapid clinical decline after a peracute onset of severe lethargy, dull mentation, and anorexia. Autopsy did not identify a cause of death. Histology revealed inflammation associated with amoebic trophozoites in the brain, lungs, and iris of one eye. These amoebae were confirmed to be B. mandrillaris based on a PCR assay and sequencing. Although there are subtle morphologic differences between cyst stages of Acanthamoeba spp., B. mandrillaris, and Naegleria fowleri when present and identified on routine staining, other modalities, including PCR, immunofluorescence, electron microscopy, and immunohistochemistry, are typically utilized to confirm the pathogen involved in these cases. We review the reports of balamuthosis in animals.

Tigers ( Panthera tigris ) are flagship big cats and attract extensive public attention due to their charismatic features and endangered status. Despite this, little is known about their prehistoric lineages and detailed evolutionary histories. Through palaeogenomic analyses, we identified a Pleistocene tiger from northeastern China, dated to beyond the limits of radiocarbon dating (greater than 43 500 years ago). We used a simulated dataset and different reads processing pipelines to test the validity of our results and confirmed that, in both mitochondrial and nuclear phylogenies, this ancient individual belongs to a previously unknown lineage that diverged prior to modern tiger diversification. Based on the mitochondrial genome, the divergence time of this ancient lineage was estimated to be approximately 268 ka (95% CI: 187–353 ka), doubling the known age of tigers' maternal ancestor to around 125 ka (95% CI: 88–168 ka). Furthermore, by combining our findings with putative mechanisms underlying the discordant mito-nuclear phylogenetic placement for the South China tigers, we proposed a more complex scenario of tiger evolution that would otherwise be missed using data from modern tigers only. Our study provides the first glimpses of the genetic antiquity of tigers and demonstrates the utility of aDNA-based investigation for further understanding tiger evolution.

OBJECTIVE To determine the utility of blood symmetric dimethylarginine (SDMA) concentration measurement as a diagnostic tool for chronic kidney disease (CKD) in tigers (Panthera tigris) by comparing results for SDMA with those for traditional renal biomarkers and investigating correlations between these biomarkers and histopathologic kidney changes in tigers with CKD. SAMPLE Blood, urine, and kidney samples from 35 tigers with CKD from 2 sanctuaries. PROCEDURES Blood (serum or plasma) and urine samples were collected antemortem. Necropsy, including gross and histologic assessment, was performed for tigers that died or were euthanized for quality-of-life reasons. Results for CKD biomarkers in blood (BUN, creatinine, phosphorus, and SDMA concentrations) and urine (protein concentration, urine protein-to-creatinine ratio, and urine specific gravity) were evaluated for correlation with histologic kidney damage scored with an objective grading scale defined by percentage of inflammation, fibrosis, and tubular atrophy. RESULTS Symmetric dimethylarginine had the strongest significant correlation (ρ = 0.667) with histologic kidney damage score, followed by urine specific gravity (ρ = –0.639), blood creatinine concentration (ρ = 0.624), and BUN (ρ = 0.588). No significant correlation with kidney score was identified for blood phosphorus concentration, urine protein concentration, or the urine protein-to-creatinine ratio. CLINICAL RELEVANCE We recommend SDMA be prioritized as a renal biomarker in tigers, with SDMA results considered in addition to those of other traditional renal biomarkers when assessing kidney function in tigers. Additionally, the grading scale we developed could be replicated across patients and pathologists for more consistent postmortem assessment of CKD in tigers.

CASE DESCRIPTION A 12-year-old sexually intact male zoo-managed Sumatran tiger (Panthera tigris sumatrae) was evaluated for a 3-day history of vomiting, hyporexia, and lethargy. Radiographs were supportive of gastrointestinal obstruction, and an exploratory laparotomy was performed. CLINICAL FINDINGS Diffuse tan foci were present on the liver parenchyma, and the tiger became icteric throughout the procedure. Hepatic histopathology and immunohistochemistry resulted in a diagnosis of leptospirosis. Serum microagglutination testing for Leptospira spp antibody titers were positive for L kirschneri serovar Grippotyphosa, rising from 1:400 to 1:3,200 in 2 days. TREATMENT AND OUTCOME The tiger was treated with antimicrobials, ursodiol, and mirtazapine, and increased biosecurity measures were instituted. Free-ranging wildlife on grounds were trapped, euthanized, and submitted for necropsy to screen for disease vectors. The tiger’s urine was intermittently opportunistically collected from the enclosure and remained PCR assay negative for Leptospira spp until being positive once again on day 595. Although the tiger was without clinical signs at that time, antimicrobial therapy and increased biosecurity protocols were instituted a second time until urinary Leptospira shedding was confirmed to have stopped. By 1,071 days after initial presentation, the tiger remained nonclinical, with no additional urinary shedding episodes. CLINICAL RELEVANCE While domestic and nondomestic free-ranging felids have been reported as subclinical Leptospira spp carriers, this report indicates the clinical importance of leptospirosis when a tiger presents with generalized gastrointestinal signs and icterus. Due to the zoonotic potential, biosecurity measures are necessary. This patient had a clinically successful outcome with antimicrobial therapy and supportive care.

Dissection reports of large cats (family Felidae) have been published since the late 19th century. These reports generally describe the findings in words, show drawings of the dissection, and usually include some masses of muscles, but often neglect to provide muscle maps showing the precise location of bony origins and insertions. Although these early reports can be highly useful, the absence of visual depictions of muscle attachment sites makes it difficult to compare muscle origins and insertions in living taxa and especially to reconstruct muscle attachments in fossil taxa. Recently, more muscle maps have been published in the primary literature, but those for large cats are still limited. Here, we describe the muscular anatomy of the forelimb of the tiger (Panthera tigris), and compare muscle origins, insertions, and relative muscle masses to other felids to identify differences that may reflect functional adaptations. Our results reiterate the conservative nature of felid anatomy across body sizes and behavioral categories. We find that pantherines have relatively smaller shoulder muscle masses, and relatively larger muscles of the caudal brachium, pronators, and supinators than felines. The muscular anatomy of the tiger shows several modifications that may reflect an adaptation to terrestrial locomotion and a preference for large prey. These include in general a relatively large m. supraspinatus (shoulder flexion), an expanded origin for m. triceps brachii caput longum, and relatively large m. triceps brachii caput laterale (elbow extension), as well as relatively large mm. brachioradialis, abductor digiti I longus, and abductor digiti V. Muscle groups that are well developed in scansorial taxa are not well developed in the tiger, including muscles of the cranial compartment of the brachium and antebrachium, and m. anconeus. Overall, the musculature of the tiger strongly resembles that of the lion (Panthera leo), another large-bodied terrestrial large-prey specialist.

OBJECTIVE To calculate the necessary pseudophakic intraocular lens (IOL) power to approximate emmetropia in adult tigers. ANIMALS 17 clinically normal adult tigers. PROCEDURES 33 eyes of 17 clinically normal adult tigers underwent routine ophthalmic examination and B-scan ultrasonography while anesthetized for unrelated procedures. Specific ultrasound data (globe measurements and corneal curvature) and estimated postoperative IOL positions were utilized to calculate predicted IOL power by use of Retzlaff and Binkhorst theoretical formulas. Applanation tonometry and refraction were also performed. RESULTS Mean ± SD axial globe length was 29.36 ± 0.82 mm, preoperative anterior chamber depth was 7.00 ± 0.74 mm, and crystalline lens thickness was 8.72 ± 0.56 mm. Mean net refractive error (n = 33 eyes) was +0.27 ± 0.30 diopters (D). By use of the Retzlaff formula, mean predicted IOL power for the postoperative anterior chamber depth (PACD), PACD – 2 mm, and PACD + 2 mm was 43.72 ± 4.84 D, 37.62 ± 4.19 D, and 51.57 ± 5.72 D, respectively. By use of the Binkhorst equation, these values were 45.11 ± 4.91 D, 38.84 ± 4.25 D, and 53.18 ± 5.81 D, respectively. Mean intraocular pressure for all eyes was 14.7 ± 2.69 mm Hg. CLINICAL RELEVANCE The calculated tiger IOL was lower than reported values for adult domestic felids. Further studies evaluating actual PACD and pseudophakic refraction would help determine the appropriate IOL power to achieve emmetropia in this species.

A male Malayan tiger cub developed well-circumscribed, erythematous, alopecic lesions on the face, torso, and paws when 1-wk-old. Biopsies of a torso lesion and a right front paw lesion at 1-mo-old confirmed cutaneous mast cell tumors (MCTs). MCTs on the paws grew into pendulous masses up to 6.5 cm in diameter by 3-mo-old, but those on the face and torso regressed. Fine-needle aspiration of the spleen at 3-mo-old revealed marked mast cell infiltration. The spleen and the right paw cutaneous MCT were removed; the paw MCT recurred within 7 d. A 12-bp tandem duplication, suggesting a somatic mutation, was identified in exon 8 of c-KIT in DNA extracted from the cutaneous MCT on the right paw and from one over the torso, but not from the spleen. Remaining MCTs on the paws regressed slowly following splenectomy and had completely regressed by 1-y-old. At 7-y-old, there was no recurrence of any mast cell disease. Mast cell disease in this tiger cub is similar to a report in a domestic kitten and to pediatric mastocytosis in humans, which commonly begins in infancy, improves by adolescence, and is associated with somatic c-kit mutations. To our knowledge, mastocytosis has not been reported previously in a juvenile exotic felid.

Identification of genetic structure within wildlife populations have implications in their conservation and management. Accurately inferring population genetic structure requires whole-genome data across the geographical range of the species, which can be resource-intensive. A cheaper strategy is to employ a subset of markers that can efficiently recapitulate the population genetic structure inferred by the whole genome data. Such ancestry informative markers (AIMs), have rarely been developed for endangered species such as tigers utilizing single nucleotide polymorphisms (SNPs). Here, we first identify the population structure of the Indian tiger using whole-genome sequences and then develop an AIMs panel with a minimum number of SNPs that can recapitulate this structure. We identified four population clusters of Indian tigers with North-East, North-West, and South Indian tigers forming three separate groups, and Terai and Central Indian tigers forming a single cluster. To evaluate the robustness of our AIMs, we applied it to a separate dataset of tigers from across India. Out of 92 SNPs present in our AIMs panel, 49 were present in the new dataset. These 49 SNPs were sufficient to recapitulate the population genetic structure obtained from the whole genome data. To the best of our knowledge, this is the first-ever SNP-based AIMs panel for big cats, which can be used as a cost-effective alternative to whole-genome sequencing for detecting the biogeographical origin of Indian tigers. Our study can be used as a guideline for developing an AIMs panel for the management of other endangered species where obtaining whole genome sequences are difficult.

The rapid expansion of hydropower across tropical landscapes has caused extensive habitat loss and degradation, triggering biodiversity loss. Despite known risks to freshwater biodiversity, the flooding of terrestrial habitats caused by dam construction, and associated impacts on terrestrial biota, have been rarely considered. To help fill this knowledge gap, we quantified the habitat loss following inundation of hydropower reservoirs across the range of two iconic species, jaguars and tigers. To do so, we compiled existing and planned dams intersecting the distribution of these apex predators. We found 164 dams intersecting the jaguar range, in total flooding 25,397 km2. For tigers, we identified 421 dams, amounting to 13,750 km2. As hydropower infrastructure is projected to expand in the decades ahead, these values are expected to increase greatly, particularly within the distribution of jaguars where the number of dams will nearly quadruple (429 planned dams). Despite the relatively few dams (41) planned across the range of tigers, most will intersect priority conservation areas for this species. We recommend a more cautious pursuit of hydropower in topographically flat regions, to avoid extensive habitat flooding which has occurred in the Neotropics, and avoiding dam construction in priority conservation landscapes for tigers.

Summary A 15-year-old male white Siberian tiger (Panthera tigris altaica) was evaluated for an elevated, ulcerated, cutaneous mass on the right flank. The mass was removed by excisional biopsy and submitted for histopathology. Based on distinct histological features of intracytoplasmic globular material and positive immunohistochemical staining for Melan-A and vimentin, an amelanotic signet-ring melanoma was diagnosed. While in domestic cats this neoplasm is associated with malignancy and a short survival time, the tiger had no local recurrence or related clinical disease approximately 4 years post surgery. Cutaneous melanocytic tumours are rare in big cats, and the findings in this case suggest that amelanotic signet-ring melanoma is not as malignant as its counterpart in domestic cats.

A 16-year-old, female, captive Bengal tiger (Panthera tigris tigris) had a history of a recurrent subcutaneous mass. After two attempts at surgical removal, euthanasia was elected during the third surgical attempt due to the degree of neoplastic infiltration. At necropsy, a large subcutaneous mass infiltrated the dermis, subcutis and abdominal wall with metastasis to the lungs, spleen and adrenal glands. Microscopically, the neoplasm formed nodules comprising sheets of round cells with large irregular nuclei. Toluidine blue and Giemsa stains were negative. An immunohistochemistry panel revealed membranous and cytoplasmic labelling with ionized calcium-binding adaptor molecule 1 (IBA1), cytoplasmic labelling with vimentin but no labelling with multiple myeloma oncogene 1 (MUM1), CD20 or pancytokeratin markers. Based on the gross distribution and histological and immunohistochemistry features, a diagnosis of disseminated histiocytic sarcoma was made. To the best of our knowledge, this is the first record of disseminated histiocytic sarcoma in a captive Bengal tiger.

The tiger, a poster child for conservation, remains an endangered apex predator. Continued survival and recovery will require a comprehensive understanding of genetic diversity and the use of such information for population management. A high-quality tiger genome assembly will be an important tool for conservation genetics, especially for the Indian tiger, the most abundant subspecies in the wild. Here, we present high-quality near-chromosomal genome assemblies of a female and a male wild Indian tiger (Panthera tigris tigris). Our assemblies had a scaffold N50 of >140 Mb, with 19 scaffolds corresponding to the 19 numbered chromosomes, containing 95% of the genome. Our assemblies also enabled detection of longer stretches of runs of homozygosity compared to previous assemblies, which will help improve estimates of genomic inbreeding. Comprehensive genome annotation identified 26,068 protein-coding genes, including several gene families involved in key morphological features such as the teeth, claws, vision, olfaction, taste, and body stripes. We also identified 301 microRNAs, 365 small nucleolar RNAs, 632 transfer RNAs, and other noncoding RNA elements, several of which are predicted to regulate key biological pathways that likely contribute to the tiger's apex predatory traits. We identify signatures of positive selection in the tiger genome that are consistent with the Panthera lineage. Our high-quality genome will enable use of noninvasive samples for comprehensive assessment of genetic diversity, thus supporting effective conservation and management of wild tiger populations.

The big cats (genus Panthera) represent some of the most popular and charismatic species on the planet. Although some reference genomes are available for this clade, few are at the chromosome level, inhibiting high-resolution genomic studies. We assembled genomes from 3 members of the genus, the tiger (Panthera tigris), the snow leopard (Panthera uncia), and the African leopard (Panthera pardus pardus), at chromosome or near-chromosome level. We used a combination of short- and long-read technologies, as well as proximity ligation data from Hi-C technology, to achieve high continuity and contiguity for each individual. We hope that these genomes will aid in further evolutionary and conservation research of this iconic group of mammals.

Diarrhea-predominant irritable bowel syndrome (IBS-D) is common among the captive South China tigers in zoos. 16S rRNA gene sequencing was performed to demonstrate the compositions and structures of the gastrointestinal microbiota of this species with IBS-D. Their healthy (F1) and mushy (F2) feces were allocated into two groups. A total of 21 and 31 fecal bacterial communities of major phyla and genera were detected, respectively. The F1 and F2 groups had five common microbiotas at the phylum level (Firmicutes, Proteobacteria, Fusobacteria, Actinobacteria, and Bacteroidetes). Among the five phyla, the abundance of Bacteroidetes in the F2 group was significantly lower than that in the F1 group. The diversity level of fecal microbiota within the mild-diarrhea stool was also significantly lower than that of the healthy counterpart. Thirty-two metabolites were correlated to four genus-level bacteria ( Bacteroides, Pseudoclavibacter, Streptococcus, and Ruminococcaceae-UCG-005). Due to its normal role in protein degradation and metabolism, we hypothesized that the lower abundance of Bacteroides within the F2 group could be associated with the IBS-D symptoms. Therefore, this work implied that ameliorating the daily diet with a supplement of probiotics, such as Bacteroides, could improve the gut health of this species.

Deforestation and agricultural intensification have resulted in an alarming change in the global land cover over the past 300 years, posing a threat to species conservation. Dhole is a monophyletic, social canid and, being an endangered and highly forest-dependent species, is more prone to the loss of favorable habitat in the Anthropocene. We determined the genetic differentiation and demographic history of dhole across the tiger reserves of Maharashtra using the microsatellite data of 305 individuals. Simulation-based analyses revealed a 77–85% decline in the major dhole sub-populations. Protected areas have provided refuge to the historically declining dhole population resulting in clustering with strong genetic structure in the remnant dhole population. The historical population decline coincides with the extreme events in the landscape over the past 300 years. The study highlights the pattern of genetic differentiation and diversity of a highly forest-dependent species which can be associated with the loss of forest cover outside tiger reserves. It also warrants attention to develop conservation plans for the remnant surviving population of dholes in India.

A three-year-old male South China tiger died in the tiger enclosure of the China Tiger Park in the Meihua Mountains on December 2018 after being bitten by a tick. This tiger presented clinical symptoms like whole-body severe jaundice, hepatosplenomegaly, kidney, and lymph node hemorrhages. The Colpodella sp.-specific 18S rRNA gene was detected using nested PCR. Interestingly, the DNA isolated from the blood of the tiger was found to be 100% similar to that of the tick by NCBI BLAST analysis. However, the DNA fragments isolated from the tiger's blood were 90.1% similar to the Colpodella sp. strain human erythrocyte parasite (HEP, MH208621) and 90.4% similar to the Colpodella sp. strain Heilongjiang (HLJ, KT364261). To investigate the species of ticks and ticks-carried Colpodella parasites in this region, the species of ticks obtained from the grasses outside the tiger enclosure and the species of Colpodella carried by ticks were identified. The DNA from ticks as well as that from the tick-borne Colpodella sp. were amplified from each tick using PCR followed by amplicon sequencing. In total 402 adult ticks samples were collected, among which 22 were positive for Colpodella sp. (5.5%), and the species were further determined by morphology, DNA sequencing and phylogenetic analyses. Interestingly, one Colpodella sp. was found to have 94.2% sequence similarities to the Colpodella sp. strain HEP (MH208621). This strain was previously reported to infect a woman in Yunnan, China. In addition, three Colpodella sp. showed 87-91% sequence similarities to the Colpodella sp. strain HLJ (KT364261), which was previously reported to infect human in Heilongjiang, China. This study disclosed the possibility of zoonotic transmission of Colpodella sp. by ticks in China. Finally, it provides a basis for urgently determining and monitoring the repertoire of ticks-borne piroplasmid pathogens, with the ultimate aim of strategic control.

In certain domains, people represent some of an individual's properties (e.g., a tiger's ferocity), but not others (e.g., a tiger's being in the zoo), as stemming from the assumed "essence" of the individual's category. How do children identify which properties of an individual are essentialized and which are not? Here, we examine whether formal explanations-that is, explanations that appeal to category membership (e.g., "That's ferocious

Jintiange capsule is composed of bionic tiger bone powder and has similar ingredients to natural tiger bone.To characterize the subacute toxicities of Jintiange capsule in rats and beagle dogs for preclinical safety assessment.Suspensions of Jintiange capsule were given via gastric lavage over a 26-week period at low (500 mg/kg), mid (1500 mg/kg) and high doses (4000 mg/kg) in SD rats. Beagles were given by gastric lavage of suspensions of Jintiange capsule once daily for 6 days per week for 39 weeks at low (300 mg/kg), mid (900 mg/kg) or high dose (2000 mg/kg).Repeated gastric lavages of suspensions of Jintiange capsule at doses from 500 to 4000 mg/kg over 26 weeks caused no significant toxicity (No Observed Adverse Effect Level, NOAEL) in rats. In addition, repeated gastric lavages of suspensions of Jintiange capsule at doses from 300 to 2000 mg/kg over 39 weeks caused NOAEL in beagles.Jintiange capsule was safe in rats at a dose 66.7 times the clinically recommended dose and in beagles at 33.3 times the clinically recommended dose. Our subacute toxicity studies in rats and beagles demonstrated no apparent overall toxicities including haematotoxicities, hepatotoxicities and renal toxicities.

Endometrial hyperplasia (EH) is a pathologic condition of the uterus with increased endometrial gland to stroma ratio compared to normal cyclic uterine proliferation. In domestic animals, EH often involves cystic distension of proliferating endometrial glands and may be concurrent with pyometra. In large captive nondomestic felids, an association between EH and pyometra is common; however, detailed species differences between the histological uterine findings in lions (

Currently, Tigers (the top predator of an ecosystem) are on the list of endangered species. Thus the need is to understand the tiger's population genomics to design their conservation strategies. We analyzed the molecular evolution of tiger diversity using NADH dehydrogenase subunit 4 (ND4), a significant electron transport chain component. We have analyzed nucleotide composition and distribution pattern of ND genes, molecular evolution, evolutionary conservation pattern and conserved blocks of NADH, phylogenomics of ND4, and estimating species divergence, etc., using different bioinformatics tools and software, and MATLAB programming and computing environment. The nucleotide composition and distribution pattern of ND genes in the tiger genome demonstrated an increase in the number of adenine (A) and a lower trend of A+T content in some place of the distribution analysis. However, the observed distributions were not significant (P > 0.05). Evolutionary conservation analysis showed three highly align blocks (186 to 198, 406 to 416, and 527 to 545). On mapping the molecular evolution of ND4 among model species (n = 30), we observed its presence in a broader range of species. ND4 based molecular evolution of tiger diversity and time divergence for a tiger (20 different other species) shows that genus Panthera originated more or less at a similar time. The nucleotide composition and nucleotide distribution pattern of tiger ND genes showed the evolutionary pattern and origin of tiger and Panthera lineage concerning the molecular clock, which will help to understand their adaptive evolution.

The microorganisms inhabiting the gastrointestinal tract play important roles in many host physiological processes, including the absorption and metabolism of nutrients and immune function. The Amur tiger (Panthera tigris altaica) is listed by the International Union for the Conservation of Nature (IUCN) as a threatened species. Efforts are underway to breed Amur tigers under artificial settings to preserve this rare species. To maximize the imitation of the diet that this species consumes in the wild, the diet in the present study was composed of a variety of raw meats and was administered with regular fasting. In view of the important roles that the microbiota play in the host, in the present study, the microbiota of Amur tigers at three different ages were investigated. The results showed that the microbial diversity and richness decreased with age. Principal coordinate analysis showed significant differences among the three age groups. Linear discriminant analysis (LDA) of effect size (LEfSe) demonstrated the enrichment of the genus unclassified_f__Ruminococcaceae, genus Coprococcus_1, genus Ruminococcus__gauvreauii_group, family unclassified_o__Clostridiales and genus unclassified_o__Clostridiales in the JB group (1- year old) and the enrichment of the genus Catenisphaera in the AB group (over 4-year old). The results of the present study demonstrated the adaptation of the microbiota in captive Amur tigers to a diet similar to the one they consume in the wild. Furthermore, these results may reflect the microbiota of wild Amur tigers to a certain extent.

The zoos manage small populations of endangered big cat species like tiger, lion, and leopard for display, research, and conservation breeding. Genetic management of these populations is essential to ensure long term survival and conservation utility. Here we propose a simple and cost effective microsatellite based protocol for the genetic management of captive big cats. We sampled 36 big cat individuals from Seoul Grand Park Zoo (Republic of Korea) and amplified 33 published microsatellite loci. Overall, allelic richness and gene diversity was found highest for leopards, followed by lions and tigers. Twelve of the thirty-three markers showed a high degree of polymorphism across all target species. These microsatellites provide a high degree of discrimination for tiger (1.45 × 10−8), lion (1.54 × 10−10), and leopard (1.88 × 10−12) and thus can be adopted for the genetic characterization of big cats in accredited zoos globally. During captive breeding, zoo authorities rely on pedigree records maintained in studbooks to ensure mating of genetically fit unrelated individuals. Several studies have reported errors in studbook records of big cat species. Microsatellites are simple and cost effective tool for DNA fingerprinting, estimation of genetic diversity, and paternity assessment. Our unified microsatellite panel (12-plex) for big cats is efficient and can easily be adopted by zoo authorities for regular population management.

Accurate species and sex identification of non-invasive and forensic samples of the tiger and leopard is still confusing when using the allele-specific methods. We designed allele-specific methods with penultimate nucleotide mismatch in a nested manner for the exact identification and double-checking of forensic samples. The mismatch design is a novel concept in species and sex identification, making the allele-specific targeting precise. We developed three sets of markers, a 365 bp outer and a 98 bp inner marker for nested tiger species identification assay, 136 bp leopard specific marker, and carnivore sex identification markers. We validated the method with tissue/blood forensic samples of various felids and herbivorous available in our lab and on known fecal samples from Vandalur Zoo. We also collected 37 scat samples at diverse stages of deterioration from the Mudumalai Tiger Reserve, Tamil Nadu, India. The 365 bp targeted markers resulted in 70.2% (n = 22; 22/37) amplification success, while the 98 bp FAM-labelled marker amplified 89% (n = 33; 33/37) scat samples independently. The 136 bp leopard markers answered four scat samples (11%) unrequited by the tiger specific markers. We evaluated species and the sex identification with these markers in another 190 non-invasive samples provided by the Mudumalai Tiger Reserve authorities. Among which 56.3% (n = 107) of samples were recognized as tiger (64 male and 43 female) and 38.9% (n = 74) as leopard (41 male and 33 female). The method supersedes any other previous methods in this regard by its high accuracy and simplicity.

Risk assessment of human activities on landscape fragmentation in nature reserves can effectively balance the conflict between wildlife conservation and human development. However, previous studies had been unable to quantitatively assess the risk of human activities on landscape fragmentation. Thus, we constructed a risk assessment methodology to quantitatively assess the risk of different human activities on the Landscape Fragmentation Composite Index (LFCI) in the Northeast China Tiger and Leopard National Park (NCTLNP). First, we fitted the relationship curve between LFCI and different human activity factors based on the Generalized Additive Model (GAM) to determine the impact patterns of each factor on LFCI. Secondly, we identified impact risk areas of each human activity factor on LFCI by the location of threshold points in the curve and analyzed their spatiotemporal variation characteristics from 2015 to 2020. The results show that the relationship between LFCI and Land Use Intensity (LUI) showed an inverted "U" shape, the relationship with Population Density (POPD) showed a "rising-flat-rising" trend, and the relationship with Traffic Accessibility (TA) and Industrial and Mining Activity (IMA) showed a positive correlation after a flat interval. In addition, we found that the LUI and IMA impact risk areas were widely distributed and remained stable for five years. But the POPD impact risk area was mainly distributed around settlements and expanded by 6.6 % from 2015 to 2020. The TA impact risk area was distributed in strips and expanded by 16.38 % from 2015 to 2017 due to the construction of the G331 national road. And the joint impact risk area of these four factors expanded by 1.55 times in five years. Our research can provide a reference for ecological risk assessment under the impact of human activities on other nature reserves in the world.

The temperate oak tasar silkworm, Antheraea proylei, is frequently infested with Antheraea proylei nucleopolyhedrovirus (AnprNPV) causing tiger band disease. This disease is one of the key factors that obstructs production and productivity of oak tasar sericulture. The current study aimed to investigate the pathogenicity of AnprNPV, its mode of transmission, and detection of AnprNPV in different tissues. Transmission electron micrographs of AnprNPV showed single rod-shaped bodies and occlusion derived virus (ODV) enclosed within multiple envelopes. The infecting AnprNPV displayed tissue tropism with higher copy numbers detected in the insect fat body and ovary. The virus was observed to multiply in all developmental stages of the silkworm such as egg, larva, pupa, and moth, confirming its ability to spread throughout the silkworm lifecycle. Baculovirus isolated from infected A. proylei showed cross-infectivity in other Saturniidae wild silkworm species such as Antheraea pernyi, A. frithi, and Samia ricini, widening their probable host range for infection. Baculoviruses generally display a horizontal mode of transmission, mainly through ingestion of occlusion bodies (OBs); however, the present study revealed a trans-ovum vertical mode of transmission in addition to a horizontal mode. The observations made in this study aid a detailed understanding of the tiger band disease and its causative pathogen AnprNPV, which will support future studies and disease management in oak tasar sericulture.

Unprecedented advances in sequencing technology in the past decade allow a better understanding of genetic variation and its partitioning in natural populations. Such inference is critical to conservation: to understand species biology and identify isolated populations. We review empirical population genetics studies of Endangered Bengal tigers within India, where 60–70% of wild tigers live. We assess how changes in marker type and sampling strategy have impacted inferences by reviewing past studies, and presenting three novel analyses including a single-nucleotide polymorphism (SNP) panel, genome-wide SNP markers, and a whole-mitochondrial genome network. At a broad spatial scale, less than 100 SNPs revealed the same patterns of population clustering as whole genomes (with the exception of one additional population sampled only in the SNP panel). Mitochondrial DNA indicates a strong structure between the northeast and other regions. Two studies with more populations sampled revealed further substructure within Central India. Overall, the comparison of studies with varied marker types and sample sets allows more rigorous inference of population structure. Yet sampling of some populations is limited across all studies, and these should be the focus of future sampling efforts. We discuss challenges in our understanding of population structure, and how to further address relevant questions in conservation genetics. This article is part of the theme issue ‘Celebrating 50 years since Lewontin's apportionment of human diversity’.