Prey Preference of Snow Leopard (Panthera uncia) in South Gobi, Mongolia

International audienceAccurate information about the diet of large carnivores that are elusive and inhabit inaccessible terrain, is required to properly design conservation strategies. Predation on livestock and retaliatory killing of predators have become serious issues throughout the range of the snow leopard. Several feeding ecology studies of snow leopards have been conducted using classical approaches. These techniques have inherent limitations in their ability to properly identify both snow leopard feces and prey taxa. To examine the frequency of livestock prey and nearly-threatened argali in the diet of the snow leopard, we employed the recently developed DNA-based diet approach to study a snow leopard population located in the Tost Mountains, South Gobi, Mongolia. After DNA was extracted from the feces, a region of ~100 bp long from mitochondrial 12S rRNA gene was amplified, making use of universal primers for vertebrates and a blocking oligonucleotide specific to snow leopard DNA. The amplicons were then sequenced using a next-generation sequencing platform. We observed a total of five different prey items from 81 fecal samples. Siberian ibex predominated the diet (in 70.4% of the feces), followed by domestic goat (17.3%) and argali sheep (8.6%). The major part of the diet was comprised of large ungulates (in 98.8% of the feces) including wild ungulates (79%) and domestic livestock (19.7%). The findings of the present study will help to understand the feeding ecology of the snow leopard, as well as to address the conservation and management issues pertaining to this wild cat

Induced polyploidy: a tool for forage species improvement

Polyploidy means having more than two basic sets of chromosomes. Polyploid plants may be artificially obtained through chemical, physical and biological (2n gametes) methods. This approach allows an increased gene scope and expression, thus resulting in phenotypic changes such as yield and product quality. Nonetheless, breeding new cultivars through induced polyploidy should overcome deleterious effects that are partly contributed by genome and epigenome instability after polyploidization. Furthermore, shortening the time required from early chromosome set doubling to the final selection of high yielding superior polyploids is a must. Despite these hurdles, plant breeders have successfully obtained polyploid bred-germplasm in broad range of forages after optimizing methods, concentration and time, particularly when using colchicine. These experimental polyploids are a valuable tool for understanding gene expression, which seems to be driven by dosage dependent gene expression, altered gene regulation and epigenetic changes. Isozymes and DNA-based markers facilitated the identification of rare alleles for particular loci when compared with diploids, and also explained their heterozygosity, phenotypic plasticity and adaptability to diverse environments. Experimentally induced polyploid germplasm could enhance fresh herbage yield and quality, e.g., leaf protein content, leaf total soluble solids, water soluble carbohydrates and sucrose content. Offspring of experimentally obtained hybrids should undergo selection for several generations to improve their performance and stability

Genetic diversity and multiplicity of infection in Fasciola gigantica isolates of Pakistani livestock

Fasciola spp. are responsible for over 3 billion US dollars of production loss annually in livestock and cause widespread zoonotic disease. Nevertheless, understating of the emergence and spread of the trematode species is poor. The multiplicity of F. gigantica infection and its spread is potentially influenced by multiple factors, including the abundance of suitable intermediate hosts, climatic conditions favouring the completion of the parasite's lifecycle, and translocation of infected animals, or free-living parasite stages between regions. Here we describe the development of a ‘tremabiome’ metabarcoding sequencing method to explore the numbers of F. gigantica genotypes per infection and patterns of parasite spread, based on genetic characteristics of the mitochondrial NADH dehydrogenase 1 (mt-ND-1) locus. We collected F. gigantica from three abattoirs in the Punjab and Balochistan provinces of Pakistan, and our results show a high level of genetic diversity in 20 F. gigantica populations derived from small and large ruminants consigned to slaughter in both provinces. This implies that F. gigantica can reproduce in its definitive hosts through meiosis involving cross- and self-breeding, as described in the closely related species, Fasciola hepatica. The genetic diversity between the 20 populations derived from different locations also illustrates the impact of animal movements on gene flow. Our results demonstrate the predominance of single haplotypes, consistent with a single introduction of F. gigantica infection in 85% of the hosts from which the parasite populations were derived. This is consistent with clonal reproduction in the intermediate snail hosts.[Display omitted]•To confirm the species identity of recovered Fasciola spp.•To identify the presence of single or multiple genotypes per infection (multiplicity of infection)•Demonstrate the spread of F. gigantica mt-ND-1 haplotype

ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis

Using non-conventional markers, DNA metabarcoding allows biodiversity assessment from complex substrates. In this article, we present ecoPrimers, a software for identifying new barcode markers and their associated PCR primers. ecoPrimers scans whole genomes to find such markers without a priori knowledge. ecoPrimers optimizes two quality indices measuring taxonomical range and discrimination to select the most efficient markers from a set of reference sequences, according to specific experimental constraints such as marker length or specifically targeted taxa. The key step of the algorithm is the identification of conserved regions among reference sequences for anchoring primers. We propose an efficient algorithm based on data mining, that allows the analysis of huge sets of sequences. We evaluate the efficiency of ecoPrimers by running it on three different sequence sets: mitochondrial, chloroplast and bacterial genomes. Identified barcode markers correspond either to barcode regions already in use for plants or animals, or to new potential barcodes. Results from empirical experiments carried out on a promising new barcode for analyzing vertebrate diversity fully agree with expectations based on bioinformatics analysis. These tests demonstrate the efficiency of ecoPrimers for inferring new barcodes fitting with diverse experimental contexts. ecoPrimers is available as an open source project at: http://www.grenoble.prabi.fr/trac/ecoPrimers

Assessment of nematodes in Punjab Urial (Ovis vignei punjabiensis) population in Kalabagh game reserve: development of a DNA barcode approach

Punjab urial (Ovis vignei punjabiensis) is a wild sheep of Pakistan, considered a vulnerable species by IUCN. Major threats to urial populations include habitat loss and poaching, causing severe declines in its population. Nematode infections may also compromise urial survival, but little is known about Punjab urial gastrointestinal nematodes. In this study, a novel DNA barcoding approach was developed using ITS-I as a target region, with a primer pair designed to amplify frequently reported nematode species for small ruminants. The novel primer pair was validated in silico and in vitro and subsequently used to determine the presence of nematodes in Punjab urial samples from Kala Bagh Game Reserve, District Mianwali (Pakistan). DNA barcoding revealed a higher prevalence of Haemonchus contortus (73.91%), Trichuris ovis (16.30%) and Trichostrongylus axei (3.26%) in Punjab urial. This study demonstrates that the novel DNA barcoding approach is a robust tool to detect nematode parasites from faecal samples of Punjab urial. This method can be used to detect nematode infections in wild and domestic hosts for surveillance and population conservation

Assessment of the Food Habits of the Moroccan Dorcas Gazelle in M’Sabih Talaa, West Central Morocco, Using the trnL Approach

Food habits of the Moroccan dorcas gazelle, Gazella dorcas massaesyla, previously investigated in the 1980s using microhistological fecal analysis, in the M’Sabih Talaa Reserve, west central Morocco, were re-evaluated over three seasons (spring, summer and autumn 2009) using the trnL approach to determine the diet composition and its seasonal variation from fecal samples. Taxonomic identification was carried out using the identification originating from the database built from EMBL and the list of plant species within the reserve. The total taxonomic richness in the reserve was 130 instead of 171 species in the 1980s. The diet composition revealed to be much more diversified (71 plant taxa belonging to 57 genus and 29 families) than it was 22 years ago (29 identified taxa). Thirty-four taxa were newly identified in the diet while 13 reported in 1986–87 were not found. Moroccan dorcas gazelle showed a high preference to Acacia gummifera, Anagallis arvensis, Glebionis coronaria, Cladanthus arabicus, Diplotaxis tenuisiliqua, Erodium salzmannii, Limonium thouini, Lotus arenarius and Zizyphus lotus. Seasonal variations occurred in both number (40–41 taxa in spring-summer and 49 taxa in autumn vs. respectively 23–22 and 26 in 1986–1987) and taxonomic type of eaten plant taxa. This dietary diversification could be attributed either to the difference in methods of analysis, trnL approach having a higher taxonomic resolution, or a potential change in nutritional quality of plants over time

DNA-based diet analyses in carnivores

La caractérisation des réseaux trophiques est nécessaire pour comprendre le fonctionnement des écosystèmes et les mécanismes impliqués dans leur stabilité. Il est parfois difficile de déterminer les régimes alimentaires notamment pour des espèces discrètes et difficiles à observer comme les grands carnivores. Cependant, ces espèces jouent un rôle clé dans les écosystèmes dont elles influencent le fonctionnement et la biodiversité. Ainsi, connaitre le régime alimentaire des grands prédateurs avec précision est essentiel pour établir des stratégies de conservation. Diverses méthodes basées sur le monitoring, l'analyse d'échantillons invasifs ou non ont été utilisées pour étudier les régimes alimentaires. Elles sont généralement biaisées ou peu résolutives. Les méthodes basées sur l'identification des fragments d'ADN dans les fèces ont le potentiel de fournir une meilleure information, notamment dans le cadre d'une approche métabarcoding. Il s'agit de caractériser simultanément l'ensemble des espèces dont l'ADN est présent dans un échantillon environnemental, en utilisant les Nouvelles Techniques de Séquençage. Dans ce cas, les amorces universelles nécessaires pour amplifier toutes les proies potentielles amplifient également l'ADN du prédateur s'il y a proximité taxonomique (par exemple mammifères). Ainsi les produits PCR obtenus à partir des fèces sont essentiellement composés d'ADN du prédateur et ne reflètent pas l'ensemble du régime alimentaire. L'utilisation d'un oligonucléotide de blocage limitant spécifiquement l'amplification de l'ADN du prédateur peut résoudre ce problème. Nous avons développé une méthode de ce type basée sur l'utilisation d'amorces universelles pour les vertébrés (amplifiant la région 12SV5) et d'oligonucléotides de blocage. Bien que non quantitative, cette méthode s'est montrée robuste, adaptée à l'étude de prédateurs à très large spectre de proies, et très résolutive pour identifier les proies au niveau du genre et de l'espèce. Nous l'avons appliquée à l'étude du régime alimentaire du chat léopard (Prionailurus bengalensis) qui s'est avéré très diversifié (mammifères, oiseaux, amphibiens et poissons) dans les deux populations du Pakistan étudiées. Avec la même approche, nous avons démontré la réalité du conflit entre l'homme et le léopard commun (Panthera pardus) dont le régime est presque exclusivement composé d'animaux domestiques. Enfin, nous avons pu proposer des actions de conservations pertinentes après avoir montré que le régime de la très menacée panthère des neiges (Panthera uncia) est principalement composé d'ongulés sauvages.Information on food webs is central to understand ecosystem functioning. It also provides information of ecosystem stability by evaluating the resource availability and use. Obtaining information on the diet can be critical especially when dealing with elusive carnivores, which are difficult to observe. However, these large carnivores are keystone species that influence the ecosystem through trophic cascades and maintain biodiversity. Thus, precise knowledge of their diet is a prerequisite for designing conservation strategies of these endangered species. Direct and indirect monitoring as well as invasive and non-invasive approaches that have been used to study the diet are either biased or have a low resolution. The DNA-based analysis of feces is an alternative method that may provide better information. It can be implemented through a metabarcoding approach, which is the simultaneous identification of multiple species from a single environmental sample containing degraded DNA by using Next Generation Sequencing. In this case, the use of universal primers for vertebrates amplifying all potential prey also amplifies the predator DNA when it belongs to a close taxon (e.g. mammals). Thus, the PCR products obtained from feces extracts will mainly consist of predator sequences and may not represent the full diet. The use of oligonucleotides specifically blocking the amplification of the predator DNA may overcome this problem. We have developed such a method based on the concomitant use of a universal primer pair (12SV5, amplifying all vertebrates) and blocking oligonucleotides for identifying the prey DNA fragments from predators feces. Even if the method developed is not quantitative, it is robust and adequate for studying predator with a very large dietary range and has a better resolution than traditional methods for identifying prey at the genus or species level. This methodology has been applied to characterize the highly eclectic diet (mammals, birds, amphibians and fishes) of two Northern-Pakistani populations of leopard cat (Prionailurus bengalensis). With the same approach, we demonstrated the importance of the Human-leopard conflict in Pakistan, due to the almost exclusive consumption of domestic animals by the common leopard (Panthera pardus). We could also highlight relevant conservation issues for the highly endangered and cryptic snow leopard (Panthera uncia), based on the fact that it mainly fed on wild ungulates