Molecular analysis of scats revealed diet and prey choice of grey wolves and Eurasian lynx in the contact zone between the Dinaric Mountains and the Alps.

A comprehensive understanding of the dietary habits of carnivores is essential to get ecological insights into their role in the ecosystem, potential competition with other carnivorous species, and their effect on prey populations. Genetic analysis of non-invasive samples, such as scats, can supplement behavioural or microscopic diet investigations. The objective of this study was to employ DNA metabarcoding to accurately determine the prey species in grey wolf (Canis lupus) and Eurasian lynx (Lynx lynx) scat samples collected in the Julian Alps and the Dinaric Mountains, Slovenia. The primary prey of wolves were red deer (Cervus elaphus) (detected in 96% scat samples), European roe deer (Capreolus capreolus) (68%), and wild boar (Sus scrofa) (45%). A smaller portion of their diet consisted of mesocarnivores, small mammals, and domestic animals. In contrast, the lynx diet mostly consisted of European roe deer (82%) and red deer (64%). However, small mammals and domestic animals were also present in lynx diet, albeit to a lesser extent. Our findings indicate that the dietary habits of wolves and lynx are influenced by geographical location. Snapshot dietary analyses using metabarcoding are valuable for comprehending the behaviour and ecology of predators, and for devising conservation measures aimed at sustainable management of both their natural habitats and prey populations. However, to gain a more detailed understanding of wolf and lynx dietary habits and ecological impact, it would be essential to conduct long-term genetic monitoring of their diet.

Determination of linearized pDNA template in mRNA production process using HPLC.

The recent clinical success of messenger RNA (mRNA) technology in managing the Covid pandemic has triggered an unprecedented innovation in production and analytical technologies for this therapeutic modality. mRNA is produced by enzymatic transcription of plasmid DNA (pDNA) using polymerase in a cell-free process of in vitro transcription. After transcription, the pDNA is considered a process-related impurity and is removed from the mRNA product enzymatically, chromatographically, or by precipitation. Regulatory requirements are currently set at 10 ng of template pDNA per single human dose, which typically ranges between 30 and 100 µg. Here, we report the development of a generic procedure based on enzymatic digestion and chromatographic separation for the determination of residual pDNA in mRNA samples, with a limit of quantification of 2.3 ng and a limit of detection of less than 0.1 ng. The procedure is based on enzymatic degradation of mRNA and anion exchange HPLC separation, followed by quantification of residual pDNA with a chromatographic method that is already widely adopted for pDNA quality analytics. The procedure has been successfully applied for in-process monitoring of three model mRNAs and a self-amplifying RNA (saRNA) and can be considered a generic substitution for qPCR in mRNA in-process control analytical strategy, with added benefits that it is more cost-efficient, faster, and sequence agnostic.

Microsatellite based assignment reveals history of extirpated mountain ungulate.

During the early 1900s, Northern chamois (Rupicapra rupicapra) populations in the northern Dinaric Mountains were extirpated. During the 1960s and 1970s there were several reintroductions of individuals from two Northern chamois subspecies (Alpine chamois, R. r. rupicapra and Balkan chamois, R. r. balcanica) from neighbouring areas in the attempt to re-establish the population. Accurate taxonomic classification, at subspecies level, of the autochthonous extirpated population was not known. To clarify which subspecies was present before reintroduction, we genotyped four male chamois skulls originating from Velebit Mountain, collected around 25 years before the population local extinction. DNA was successfully extracted from middle layer and outer sheath of horns. Assignment based on microsatellite loci, using both Bayesian clustering in STRUCTURE (with q values between 0.55 and 0.73) and DAPC (with individual membership probabilities of 0.99 and 1.00) indicated higher assessed likelihood for the Alpine subspecies.

Neutral and Selective Processes Shape MHC Diversity in Roe Deer in Slovenia.

Disease control and containment in free-ranging populations is one of the greatest challenges in wildlife management. Despite the importance of major histocompatibility complex (MHC) genes for immune response, an assessment of the diversity and occurrence of these genes is still rare in European roe deer, the most abundant and widespread large mammal in Europe. Therefore, we examined immunogenetic variation in roe deer in Slovenia to identify species adaptation by comparing the genetic diversity of the MHC genes with the data on neutral microsatellites. We found ten MHC DRB alleles, three of which are novel. Evidence for historical positive selection on the MHC was found using the maximum likelihood codon method. Patterns of MHC allelic distribution were not congruent with neutral population genetic findings. The lack of population genetic differentiation in MHC genes compared to existing structure in neutral markers suggests that MHC polymorphism was influenced primarily by balancing selection and, to a lesser extent, by neutral processes such as genetic drift, with no clear evidence of local adaptation. Selection analyses indicated that approx. 10% of amino acids encoded under episodic positive selection. This study represents one of the first steps towards establishing an immunogenetic map of roe deer populations across Europe, aiming to better support science-based management of this important game species.

Genetic characterisation of wild ungulates: successful isolation and analysis of DNA from widely available bones can be cheap, fast and easy.

Genetic characterisation of wild ungulates can be a useful tool in wildlife management and in obtaining a greater understanding of their biological and ecological roles in a wider spatiotemporal context. Different ways of optimising methodologies and reducing the costs of genetic analyses using widely available bone tissues collected within regular hunting allocations were examined. Successful isolation and analysis of DNA from widely available bones can be cheap, fast and easy. In particular, this study explored the possibility of using bones for extracting high quality nuclear DNA for microsatellite analysis. The utility of applying a modified demineralisation process using two commercially available DNA isolation kits, which differ significantly in price, was evaluated. The sample sets included bones and, for comparison, muscle tissues from four wild ungulate species: chamois (Rupicapra rupicapra), roe deer (Capreolus capreolus), wild boar (Sus scrofa), and Alpine ibex (Capra ibex). For the recent bones, these results confirmed that the DNA concentrations and microsatellite amplification were sufficiently high, even when using low-cost kits, after prior demineralisation. For old bones, prior demineralisation and use of a specially designed isolation kit led to a more successful extraction of DNA. Besides reducing kit-related costs, low-cost kits are much faster and therefore make genetic analysis more efficient.

First Insights into the Population Genetic Structure and Heterozygosity-Fitness Relationship in Roe Deer Inhabiting the Area between the Alps and Dinaric Mountains.

Across its pan-European distribution, the European roe deer (Capreolus capreolus) faces a wide diversity of environmental and climatic conditions; therefore, several factors, including intrinsic ones, shape life-history traits and cause significant variability in parameters of fitness. By utilizing microsatellite variations in 214 roe deer females collected throughout Slovenia, Central Europe, we determined the genetic variability and population structure of this species in the contact zone between the Alps and the Dinaric Mountains, i.e., over a wider area where data on the genetic outlook for this-the most common and widespread European wild ungulate-have been completely lacking so far. Throughout the country, we found moderate microsatellite diversity (Ho = 0.57-0.65) in relation to the observed heterozygosity reported for other roe deer populations in Europe. Spatial differences in genetic diversity of the species in Slovenia can be explained by population history linked to varying approaches to population management and/or different connectivity among subpopulations in topographically differentiated habitats. A country-wide pattern of genetic structure is clearly defined by separation of the populations into three groups present in the following regions: (i) Southern sub-Mediterranean and Karst regions, (ii) Central Slovenia, and (iii) the Sub-Pannonian Region in the north-east. This is also confirmed by evidencing a moderate isolation by distance, especially by separating southern samples (coastal Slovenia) from others. Levels of genetic differentiation vary among populations, which can be explained by the effect of natural geographical barriers or the presence of anthropogenic barriers such as urban areas and highways. In the subset of 172 yearling females, we analyzed the influence of genetic advantage (individual heterozygosity) and other genetic data (reflected in the structuring of the population) on body mass and reproductive ability. We found evidence that genetic factors influence the body mass of roe deer yearling females (explaining altogether 18.8% of body mass variance), and the level of individual heterozygosity alone also positively affected body mass, which is in accordance with the theory that heterozygosity is commonly positively correlated with fitness in wild populations. However, we did not uncover any effect of heterozygosity on two parameters of reproductive ability (fertility and potential reproductive outcome), indicating that several other factors, especially environmental ones, have a predominant effect on the parameters of fitness in roe deer.

Prevalence and genotype identification of Toxoplasma gondii in suburban rodents collected at waste disposal sites.

To assess the prevalence of Toxoplasma gondii infection in native and commensal rodents as indicators of environmental pollution, we analyzed brain tissue from small mammals collected on legal and illegal waste sites in the Slovenian and Croatian parts of Istria. A total of 136 animals and five species of the family Muridae were analyzed: black rat (Rattus rattus), domestic mouse (Mus musculus), wood mouse (Apodemus sylvaticus), striped field mouse (Apodemus agrarius), and yellow-necked mouse (Apodemus flavicollis). Using quantitative Polymerase Chain Reaction (qPCR), T. gondii DNA was detected in four homogenized brain tissue samples (2.94%), from all of the analyzed species, except black rat. Out of these, two samples, domestic mouse (Mus musculus) and wood mouse (Apodemus sylvaticus) had sufficient DNA for genotyping of T. gondii isolates in which we demonstrated the presence of clonal type II using RFLP PCR with four markers (SAG1, SAG2, GRA6 and GRA7). Three of four infected animals (75%) were collected on dumpsites.