Morphological, Ultrastructural, and Immunocytochemical Characterization and Assessment of Puma (Puma concolor Linnaeus, 1771) Cell Lines After Extended Culture and Cryopreservation.

Cell lines are valuable tools to safeguard genetic material from species threatened with extinction that is mainly due to human action. In this scenario, the puma constitutes a species whose population is being rapidly reduced in the ecosystems it inhabits. For the first time, we characterized puma skin-derived cell lines and assessed these cells after extended culture (experiment 1) and cryopreservation (experiment 2). Initially, we identified and characterized four dermal fibroblast lines using morphology, ultrastructure, and immunofluorescence assays. Moreover, we evaluated the effects of culture time (1st, 3rd, and 10th passages) and cryopreservation on their morphology, ultrastructure, viability, metabolism, proliferative activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis. The cells showed a typical spindle-shaped morphology with centrally located oval nuclei. The cells were identified as fibroblasts by staining for vimentin. In vitro culture after the 1st, 3rd, and 10th passages did not alter most of the evaluated parameters. Cells in the 3rd and 10th passages showed a reduction in ROS levels (p < 0.05). The ultrastructure revealed morphological damage in the prolongments, and nuclei of cells derived from the 3rd and 10th passages. Moreover, cryopreservation resulted in a reduction in ΔΨm compared with that of noncryopreserved cells, suggesting that the optimization of cryopreservation methods for puma fibroblasts is essential. In conclusion, we found that viable fibroblasts could be obtained from puma skin, with slight changes after the 10th passage in in vitro culture and cryopreservation. This is the first report on the development of cell lines derived from pumas.

The differential genetic signatures related to climatic landscapes for jaguars and pumas on a continental scale.

Modern and paleoclimate changes may have altered species dynamics by shifting species' niche suitability over space and time. We analyze whether the current genetic structure and isolation of the two large American felids, jaguar (Panthera onca) and puma (Puma concolor), are mediated by changes in climatic suitability and connection routes over modern and paleoclimatic landscapes. We estimate species distribution under 5 climatic landscapes (modern, Holocene, last maximum glaciations [LMG], average suitability, and climatic instability) and correlate them with individuals' genetic isolation through causal modeling on a resemblance matrix. Both species exhibit genetic isolation patterns correlated with LMG climatic suitability, suggesting that these areas may have worked as "allele refuges." However, the jaguar showed higher vulnerability to climate changes, responding to modern climatic suitability and connection routes, whereas the puma showed a continuous and gradual transition of genetic variation. Despite differential responsiveness to climate change, both species are subjected to the climatic effects on genetic configuration, which may make them susceptible to future climatic changes, since these are progressing faster and with higher intensity than changes in the paleoclimate. Thus, the effects of climatic changes should be considered in the design of conservation strategies to ensure evolutionary and demographic processes mediated by gene flow for both species.

Heterologous microsatellite primers are informative for paca (Cuniculus paca), a large rodent with economic and ecological importance.

OBJECTIVE: This study was designed to facilitate genetic studies that would allow information on population structure and genetic diversity of natural or captive stocks of paca (Cuniculus paca), a species of ecological and socioeconomic importance, by testing cross-amplification of 20 heterologous microsatellite primer pairs developed for guinea pigs (Cavia porcellus) and capybara (Hydrochoerus hydrochaeris). RESULTS: Those primers that showed the best amplification profile in blood samples were subsequently applied to scats and saliva samples, to evaluate their efficiency. Of the 13 microsatellite pairs that amplified in blood, one-third (32%) were successfully amplified in saliva and scat samples. This initial work demonstrates successful cross-amplification in paca providing a solid and promising foundation for future genetic studies with this species. The ability to quantify genetic diversity using noninvasive samples from free-ranging paca is essential to developing applied management strategies for this large neotropical rodent that is not only a prey favored by wide-ranging carnivores [e.g., jaguar (Panthera onca), puma (Puma concolor)], but is also a species targeted by illegal hunting and wildlife trade.

Characterization of mesenchymal stem cells derived from adipose tissue of a cougar (Puma concolor)

Adipose derived mesenchymal stem cells (AMSCs) have been isolated from domestic and wild cats. For wild cats, the isolation of AMSCs has been reported in the black-footed cats (Felis nigripes) and guigna (Leopardus guigna). Stromal vascular fraction (SVF) isolated from cougar adipose tissue have been used to restore elbow functionality in the cougar (Puma concolor) but multipotent characteristics of these cells have not been described. The present study describes for the first time the isolation and characterization of mesenchymal stem cells derived from adipose tissue of cougar. AMSCs and fibroblasts from six months female cougar were isolated and cultured in DMEM/F12, supplemented with FBS 10% + 1% Antibiotic/Antifungal + 2.4 mM L-Glutamine + 2.4 mM pyruvate up to passage 5. Expression of pluripotent and surface marker genes was evaluated at mRNA level. Mesodermal differentiation (adipogenic, osteogenic and chondrogenic) was described. AMSCs expressed mRNA of pluripotent genes Oct4, Nanog, Sox2 and Klf4 and surface markers Cd44, Cd90, Cd105 and MHCII. Fibroblasts showed similar mRNA expression with the exception of Sox2. AMSCs obtained from cougar exhibit multipotency features similar to domestic cats MSC, nevertheless, other analyses are required. AMSCs from cougar could be a source of interest for treatment of individuals that remain in captivity or arrive to wildlife rehabilitation centers.

Characterization of mesenchymal stem cells derived from adipose tissue of a cougar (Puma concolor)

Adipose derived mesenchymal stem cells (AMSCs) have been isolated from domestic and wild cats. For wild cats, the isolation of AMSCs has been reported in the black-footed cats (Felis nigripes) and guigna (Leopardus guigna). Stromal vascular fraction (SVF) isolated from cougar adipose tissue have been used to restore elbow functionality in the cougar (Puma concolor) but multipotent characteristics of these cells have not been described. The present study describes for the first time the isolation and characterization of mesenchymal stem cells derived from adipose tissue of cougar. AMSCs and fibroblasts from six months female cougar were isolated and cultured in DMEM/F12, supplemented with FBS 10% + 1% Antibiotic/Antifungal + 2.4 mM L-Glutamine + 2.4 mM pyruvate up to passage 5. Expression of pluripotent and surface marker genes was evaluated at mRNA level. Mesodermal differentiation (adipogenic, osteogenic and chondrogenic) was described. AMSCs expressed mRNA of pluripotent genes Oct4, Nanog, Sox2 and Klf4 and surface markers Cd44, Cd90, Cd105 and MHCII. Fibroblasts showed similar mRNA expression with the exception of Sox2. AMSCs obtained from cougar exhibit multipotency features similar to domestic cats MSC, nevertheless, other analyses are required. AMSCs from cougar could be a source of interest for treatment of individuals that remain in captivity or arrive to wildlife rehabilitation centers.(AU)

Puma genomes from North and South America provide insights into the genomic consequences of inbreeding.

Pumas are the most widely distributed felid in the Western Hemisphere. Increasingly, however, human persecution and habitat loss are isolating puma populations. To explore the genomic consequences of this isolation, we assemble a draft puma genome and a geographically broad panel of resequenced individuals. We estimate that the lineage leading to present-day North American pumas diverged from South American lineages 300-100 thousand years ago. We find signatures of close inbreeding in geographically isolated North American populations, but also that tracts of homozygosity are rarely shared among these populations, suggesting that assisted gene flow would restore local genetic diversity. The genome of a Florida panther descended from translocated Central American individuals has long tracts of homozygosity despite recent outbreeding. This suggests that while translocations may introduce diversity, sustaining diversity in small and isolated populations will require either repeated translocations or restoration of landscape connectivity. Our approach provides a framework for genome-wide analyses that can be applied to the management of similarly small and isolated populations.

Occurrence and phylogenetic analysis of Candidatus Mycoplasma haemominutum in wild felines from Paraná, Brazil / Ocorrência e análise filogenética de Candidatus Mycoplasma haemominutum em felinos selvagens no Paraná, Brasil

Hemoplasma infections are emerging and wild fauna can represent an important reservoir of these pathogens. However, there are very few epidemiological studies about the occurrence of hemoplasmas in wild cats around the world. The purpose of this study is twofold: (1) evaluate the occurrence and phylogeny of hemoplasmas in captive wild felines at a zoo in the state of Paraná, Brazil, and (2) verify the correlation between subpopulations of these bacteria and the hematological and biochemical parameters of the animals. PCR was used to detect hemoplasmas in the blood of three cougars (Puma concolor), a jaguar (Panthera onca), a tiger (Panthera tigris) and a lion (Panthera leo), followed by sequencing and phylogenetic analysis. The cougars and jaguar were found to be hemoplasma-positive by PCR. The phylogenetic analysis of the 16S rRNA gene sequences enabled the identification of genotypes of Candidatus Mycoplasma haemominutum circulating in this zoo. The identified sequences were closely related to hemoplasma sequences originating from domestic cats and other wild cats, but the infected cougars and jaguar were healthy and showed no hematological or biochemical changes. It was concluded that P. concolor and P. onca are exposed to Candidatus Mycoplasma haemominutum in Paraná, but further research is suggested to assess the resistance of wild cats to different hemoplasma subpopulations.

Infecções por hemoplasmas são emergentes e os animais selvagens podem representar um importante reservatório desses patógenos. Entretanto, são escassos os estudos epidemiológicos sobre a ocorrência de hemoplasmas em felinos selvagens em todo o mundo. O objetivo deste estudo foi (1) avaliar a ocorrência e a filogenia de hemoplasmas em felinos selvagens cativos em um zoológico do Estado do Paraná, Brasil, (2) e verificar a correlação entre as subpopulações destas bactérias e os parâmetros hematológicos e bioquímicos dos animais. A PCR foi utilizada para detecção de hemoplasmas em sangue de três pumas (Puma concolor), uma onça (Panthera onca), um tigre (Panthera tigris) e uma leoa (Panthera leo), seguida de sequenciamento e análise filogenética. Constatou-se que os pumas e a onça foram PCR-positivos para hemoplasma. A análise filogenética das sequências do gene 16S rRNA permitiu identificar genótipos de Candidatus Mycoplasma haemominutum circulando neste zoo. As sequências identificadas apresentaram relações estreitas com sequências de hemoplasmas procedentes de gatos domésticos e de outros felinos selvagens; contudo, os pumas e a onça infectados apresentaram-se saudáveis e sem alterações hematológicas ou bioquímicas. Conclui-se que P. concolor e P. onca são expostos à Candidatus Mycoplasma haemominutum no Paraná, porém sugere-se a realização de pesquisas futuras para avaliar a resistência dos felinos selvagens frente à diferentes subpopulações dehemoplasmas.

Occurrence and phylogenetic analysis of Candidatus Mycoplasma haemominutum in wild felines from Paraná, Brazil / Ocorrência e análise filogenética de Candidatus Mycoplasma haemominutum em felinos selvagens no Paraná, Brasil

Hemoplasma infections are emerging and wild fauna can represent an important reservoir of these pathogens. However, there are very few epidemiological studies about the occurrence of hemoplasmas in wild cats around the world. The purpose of this study is twofold: (1) evaluate the occurrence and phylogeny of hemoplasmas in captive wild felines at a zoo in the state of Paraná, Brazil, and (2) verify the correlation between subpopulations of these bacteria and the hematological and biochemical parameters of the animals. PCR was used to detect hemoplasmas in the blood of three cougars (Puma concolor), a jaguar (Panthera onca), a tiger (Panthera tigris) and a lion (Panthera leo), followed by sequencing and phylogenetic analysis. The cougars and jaguar were found to be hemoplasma-positive by PCR. The phylogenetic analysis of the 16S rRNA gene sequences enabled the identification of genotypes of Candidatus Mycoplasma haemominutum circulating in this zoo. The identified sequences were closely related to hemoplasma sequences originating from domestic cats and other wild cats, but the infected cougars and jaguar were healthy and showed no hematological or biochemical changes. It was concluded that P. concolor and P. onca are exposed to Candidatus Mycoplasma haemominutum in Paraná, but further research is suggested to assess the resistance of wild cats to different hemoplasma subpopulations.(AU)

Infecções por hemoplasmas são emergentes e os animais selvagens podem representar um importante reservatório desses patógenos. Entretanto, são escassos os estudos epidemiológicos sobre a ocorrência de hemoplasmas em felinos selvagens em todo o mundo. O objetivo deste estudo foi (1) avaliar a ocorrência e a filogenia de hemoplasmas em felinos selvagens cativos em um zoológico do Estado do Paraná, Brasil, (2) e verificar a correlação entre as subpopulações destas bactérias e os parâmetros hematológicos e bioquímicos dos animais. A PCR foi utilizada para detecção de hemoplasmas em sangue de três pumas (Puma concolor), uma onça (Panthera onca), um tigre (Panthera tigris) e uma leoa (Panthera leo), seguida de sequenciamento e análise filogenética. Constatou-se que os pumas e a onça foram PCR-positivos para hemoplasma. A análise filogenética das sequências do gene 16S rRNA permitiu identificar genótipos de Candidatus Mycoplasma haemominutum circulando neste zoo. As sequências identificadas apresentaram relações estreitas com sequências de hemoplasmas procedentes de gatos domésticos e de outros felinos selvagens; contudo, os pumas e a onça infectados apresentaram-se saudáveis e sem alterações hematológicas ou bioquímicas. Conclui-se que P. concolor e P. onca são expostos à Candidatus Mycoplasma haemominutum no Paraná, porém sugere-se a realização de pesquisas futuras para avaliar a resistência dos felinos selvagens frente à diferentes subpopulações dehemoplasmas.(AU)

Molecular and morphological evidence of Taenia omissa in pumas (Puma concolor) in the Peruvian Highlands / Evidência molecular e morfológica de Taenia omissa em onça-pardas (Puma concolor) dos Andes Peruanos

Abstract A total of 41 cestodes were collected during necropsy examination on 2 pumas (Puma concolor) that were found in 2 communities in Canchis province, Cuzco region, Peru, at 4500 meters above sea level (Peruvian Andes). The cestodes were evaluated morphologically and molecularly. A fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) was used as a genetic marker. All the cestodes were identified as Taenia omissa. In the present report, we give a brief description by molecular and morphological diagnosis of the cestodes and compare nucleotide sequences with previous isolates from GenBank. Upon comparison, the sequences showed a difference in the cox1 gene of 5.1 to 5.3% with other teniids sequences. This finding constitutes the first report of T. omissa in Peru and expands the geographic distribution of this parasite.(AU)

Resumo Um total de quarenta e um cestóides foram coletados durante a necropsia de duas onça-pardas (Puma concolor) encontradas em duas comunidades na província de Canchis, em Cuzco, a 4500 metros acima do nível do mar, nos Andes peruanos. Os cestóides foram avaliados morfologicamente e molecularmente. Um fragmento do gene citocromo C oxidase subunidade 1 (cox1) foi utilizado como marcador genético. Todos os cestóides foram identificados como Taenia omissa. No presente relato, dá-se uma breve descrição dos cestóides e compara-se sequências de nucleotídeos com isolados anteriores presentes no GenBank. Após a comparação, as sequências mostraram uma diferença de 5,1-5,3% entre o gene cox1 e outras sequências de tênias. Esse achado constitui o primeiro relato de T. omissa no Peru e amplia a informação sobre a distribuição geográfica deste parasita.(AU)

A Comparative Analysis of Genetic Diversity and Structure in Jaguars (Panthera onca), Pumas (Puma concolor), and Ocelots (Leopardus pardalis) in Fragmented Landscapes of a Critical Mesoamerican Linkage Zone.

With increasing anthropogenic impact and landscape change, terrestrial carnivore populations are becoming more fragmented. Thus, it is crucial to genetically monitor wild carnivores and quantify changes in genetic diversity and gene flow in response to these threats. This study combined the use of scat detector dogs and molecular scatology to conduct the first genetic study on wild populations of multiple Neotropical felids coexisting across a fragmented landscape in Belize, Central America. We analyzed data from 14 polymorphic microsatellite loci in 1053 scat samples collected from wild jaguars (Panthera onca), pumas (Puma concolor), and ocelots (Leopardus pardalis). We assessed levels of genetic diversity, defined potential genetic clusters, and examined gene flow for the three target species on a countrywide scale using a combination of individual- and population-based analyses. Wild felids in Belize showed moderate levels of genetic variation, with jaguars having the lowest diversity estimates (HE = 0.57 ± 0.02; AR = 3.36 ± 0.09), followed by pumas (HE = 0.57 ± 0.08; AR = 4.20 ± 0.16), and ocelots (HE = 0.63 ± 0.03; AR = 4.16 ± 0.08). We observed low to moderate levels of genetic differentiation for all three target species, with jaguars showing the lowest degree of genetic subdivision across the country, followed by ocelots and pumas. Although levels of genetic diversity and gene flow were still fairly high, we detected evidence of fine-scale genetic subdivision, indicating that levels of genetic connectivity for wild felids in Belize are likely to decrease if habitat loss and fragmentation continue at the current rate. Our study demonstrates the value of understanding fine-scale patterns of gene flow in multiple co-occurring felid species of conservation concern, which is vital for wildlife movement corridor planning and prioritizing future conservation and management efforts within human-impacted landscapes.

Noninvasive individual and species identification of jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) in Belize, Central America using cross-species microsatellites and faecal DNA.

There is a great need to develop efficient, noninvasive genetic sampling methods to study wild populations of multiple, co-occurring, threatened felids. This is especially important for molecular scatology studies occurring in challenging tropical environments where DNA degrades quickly and the quality of faecal samples varies greatly. We optimized 14 polymorphic microsatellite loci for jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) and assessed their utility for cross-species amplification. Additionally, we tested their reliability for species and individual identification using DNA from faeces of wild felids detected by a scat detector dog across Belize in Central America. All microsatellite loci were successfully amplified in the three target species, were polymorphic with average expected heterozygosities of HE = 0.60 ± 0.18 (SD) for jaguars, HE = 0.65 ± 0.21 (SD) for pumas and HE = 0.70 ± 0.13 (SD) for ocelots and had an overall PCR amplification success of 61%. We used this nuclear DNA primer set to successfully identify species and individuals from 49% of 1053 field-collected scat samples. This set of optimized microsatellite multiplexes represents a powerful tool for future efforts to conduct noninvasive studies on multiple, wild Neotropical felids.

Molecular characterization of SINEs integrated in endogenous retrovirus sequences from Leopardus geoffroyi and Puma concolor

Background: Short interspersed nuclear elements (SINEs) are transposable elements which are transcribed by RNA polymerase III and widespread in mammalian genomes. Can-SINE is a family of SINE sequences specific to carnivores, predominant in their genomes and present in high copy numbers. The aim of this study was to characterize sequences of Can-SINEs integrated into sequences of endogenous retroviruses (ERVs) from Brazilian wild cats Puma concolor and Leopardus geoffroyi. Additionally, these sequences are considered from some perspectives of their evolution. Material, Methods and Results: By using PCR and sequencing to screen for ERVs within the genomes of L. geoffroyi and P. concolor, two new ERV sequences were amplified with an insertion around 220 nucleotides long, similar to published carnivore SINEs. The sequences were further identified and characterized using a combination of BLAST, BLAT searches and phylogenetic analyses. The results showed that SINE sequences integrated into the ERV from P. concolor (SINE_Pco) and L. geoffroyi (SINE_Lg) are lysine-tRNA derived. These sequences presented a typical RNA polymerase III-specific internal promoter sequence followed by a microsatellite region (TC)n and by an A/T-rich tail with the polyadenylation signal AATAAA. BLAST searches using the whole sequence of L. geoffroyi clone as query (ERV plus SINE) detected two sequences which were highly similar to the cougar (P. concolor) and the domestic cat. However, the SINE from Leopardus geoffroyi is not present in these related sequences. On the other hand, during searches using the whole sequence of the P. concolor clone as query, we found the same SINE insertion in a very similar ERV from domestic cat. All insertions occurred in the RT domain, but SINE_Lg was integrated in a distinct site when compared to SINE_Pco. Another interesting difference between these SINE sequences was that the statistics reported in BLAST searches recovered a much higher number of hits from the domestic cat genome using SINE_Lg as seed than in searches for sequences related to SINE_Pco. The phylogenetic tree based on the SINE fragment grouped these new SINE sequences with Can-SINEs from felids. Within this major clade SINE_Lg and SINE_Pco are related to different lineages of felids Can-SINEs. Discussion: In this study we showed that two different sequences from felid endogenous retrovirus harbor Can-SINE sequences. These insertions are not surprising taking account that ~11% of domestic cat genome is composed of SINE sequences and they are ubiquitous in felid genomes. Furthermore, the insertions of SINEs into the ERV sequences reported here are not unique events. However, they are curious insertions representing genomic fossils and a little piece of felid history. Based on the results of the phylogenetic analyses and position of the integration sites, we suggest that SINE_Lg and SINE_Pco represent independent integration events originated by derived copies from different progenitors. We hypothesized that SINE_Lg is a "young" integration due to the absence of highly similar ERVs from Puma concolor and Felis catus. This lineage may be recently active in felid genomes given that we found very similar MegaBLAST hits at EST database from domestic cat. Instead, SINE_Pco seems to be "old", sharing an identical insertion site to ERVs from domestic cat and its lineage could be inactive in felids considering that any MegaBLAST hits resulted from EST database searches. The latter suggests an integration event in an ancestor species at least 6.7 million years ago, which represents the split between puma and domestic cat lineages.

Genetic structure and conservation of Mountain Lions in the South-Brazilian Atlantic Rain Forest

The Brazilian Atlantic Rain Forest, one of the most endangered ecosystems worldwide, is also among the most important hotspots as regards biodiversity. Through intensive logging, the initial area has been reduced to around 12 percent of its original size. In this study we investigated the genetic variability and structure of the mountain lion, Puma concolor. Using 18 microsatellite loci we analyzed evidence of allele dropout, null alleles and stuttering, calculated the number of allele/locus, PIC, observed and expected heterozygosity, linkage disequilibrium, Hardy-Weinberg equilibrium, F IS, effective population size and genetic structure (MICROCHECKER, CERVUS, GENEPOP, FSTAT, ARLEQUIN, ONESAMP, LDNe, PCAGEN, GENECLASS software),we also determine whether there was evidence of a bottleneck (HYBRIDLAB, BOTTLENECK software) that might influence the future viability of the population in south Brazil. 106 alleles were identified, with the number of alleles/locus ranging from 2 to 11. Mean observed heterozygosity, mean number of alleles and polymorphism information content were 0.609, 5.89, and 0.6255, respectively. This population presented evidence of a recent bottleneck and loss of genetic variation. Persistent regional poaching constitutes an increasing in the extinction risk.

Development and testing of an optimized method for DNA-based identification of jaguar (Panthera onca) and puma (Puma concolor) faecal samples for use in ecological and genetic studies.

The elusive nature and endangered status of most carnivore species imply that efficient approaches for their non-invasive sampling are required to allow for genetic and ecological studies. Faecal samples are a major potential source of information, and reliable approaches are needed to foster their application in this field, particularly in areas where few studies have been conducted. A major obstacle to the reliable use of faecal samples is their uncertain species-level identification in the field, an issue that can be addressed with DNA-based assays. In this study we describe a sequence-based approach that efficiently distinguishes jaguar versus puma scats, and that presents several desirable properties: (1) considerably high amplification and sequencing rates; (2) multiple diagnostic sites reliably differentiating the two focal species; (3) high information content that allows for future application in other carnivores; (4) no evidence of amplification of prey DNA; and (5) no evidence of amplification of a nuclear mitochondrial DNA insertion known to occur in the jaguar. We demonstrate the reliability and usefulness of this approach by evaluating 55 field-collected samples from four locations in the highly fragmented Atlantic Forest biome of Brazil and Argentina, and document the presence of one or both of these endangered felids in each of these areas.