Projection of current and future distribution of adaptive genetic units in an alpine ungulate.

Climate projections predict major changes in alpine environments by the end of the 21st century. To avoid climate-induced maladaptation and extinction, many animal populations will either need to move to more suitable habitats or adapt in situ to novel conditions. Since populations of a species exhibit genetic variation related to local adaptation, it is important to incorporate this variation into predictive models to help assess the ability of the species to survive climate change. Here, we evaluate how the adaptive genetic variation of a mountain ungulate-the Northern chamois (Rupicapra rupicapra)-could be impacted by future global warming. Based on genotype-environment association analyses of 429 chamois using a ddRAD sequencing approach, we identified genetic variation associated with climatic gradients across the European Alps. We then delineated adaptive genetic units and projected the optimal distribution of these adaptive groups in the future. Our results suggest the presence of local adaptation to climate in Northern chamois with similar genetic adaptive responses in geographically distant but climatically similar populations. Furthermore, our results predict that future climatic changes will modify the Northern chamois adaptive landscape considerably, with various degrees of maladaptation risk.

Genetic legacy and adaptive signatures: investigating the history, diversity, and selection signatures in Rendena cattle resilient to eighteenth century rinderpest epidemics.

BACKGROUND: Rendena is a dual-purpose cattle breed, which is primarily found in the Italian Alps and the eastern areas of the Po valley, and recognized for its longevity, fertility, disease resistance and adaptability to steep Alpine pastures. It is categorized as 'vulnerable to extinction' with only 6057 registered animals in 2022, yet no comprehensive analyses of its molecular diversity have been performed to date. The aim of this study was to analyse the origin, genetic diversity, and genomic signatures of selection in Rendena cattle using data from samples collected in 2000 and 2018, and shed light on the breed's evolution and conservation needs. RESULTS: Genetic analysis revealed that the Rendena breed shares genetic components with various Alpine and Po valley breeds, with a marked genetic proximity to the Original Braunvieh breed, reflecting historical restocking efforts across the region. The breed shows signatures of selection related to both milk and meat production, environmental adaptation and immune response, the latter being possibly the result of multiple rinderpest epidemics that swept across the Alps in the eighteenth century. An analysis of the Rendena cattle population spanning 18 years showed an increase in the mean level of inbreeding over time, which is confirmed by the mean number of runs of homozygosity per individual, which was larger in the 2018 sample. CONCLUSIONS: The Rendena breed, while sharing a common origin with Brown Swiss, has developed distinct traits that enable it to thrive in the Alpine environment and make it highly valued by local farmers. Preserving these adaptive features is essential, not only for maintaining genetic diversity and enhancing the ability of this traditional animal husbandry to adapt to changing environments, but also for guaranteeing the resilience and sustainability of both this livestock system and the livelihoods within the Rendena valley.

Mock community as an in situ positive control for amplicon sequencing of microbiotas from the same ecosystem.

Metataxonomy has become the standard for characterizing the diversity and composition of microbial communities associated with multicellular organisms and their environment. Currently available protocols for metataxonomy assume a uniform DNA extraction, amplification and sequencing efficiency for all sample types and taxa. It has been suggested that the addition of a mock community (MC) to biological samples before the DNA extraction step could aid identification of technical biases during processing and support direct comparisons of microbiota composition, but the impact of MC on diversity estimates of samples is unknown. Here, large and small aliquots of pulverized bovine fecal samples were extracted with no, low or high doses of MC, characterized using standard Illumina technology for metataxonomics, and analysed with custom bioinformatic pipelines. We demonstrated that sample diversity estimates were distorted only if MC dose was high compared to sample mass (i.e. when MC > 10% of sample reads). We also showed that MC was an informative in situ positive control, permitting an estimation of the sample 16S copy number, and detecting sample outliers. We tested this approach on a range of sample types from a terrestrial ecosystem, including rhizosphere soil, whole invertebrates, and wild vertebrate fecal samples, and discuss possible clinical applications.

A validated protocol for eDNA-based monitoring of within-species genetic diversity in a pond-breeding amphibian.

In light of the dramatic decline in amphibian biodiversity, new cost-efficient tools to rapidly monitor species abundance and population genetic diversity in space and time are urgently needed. It has been amply demonstrated that the use of environmental DNA (eDNA) for single-species detection and characterization of community composition can increase the precision of amphibian monitoring compared to traditional (observational) approaches. However, it has been suggested that the efficiency and accuracy of the eDNA approach could be further improved by more timely sampling; in addition, the quality of genetic diversity data derived from the same DNA has been confirmed in other vertebrate taxa, but not amphibians. Given the availability of previous tissue-based genetic data, here we use the common frog Rana temporaria Linnaeus, 1758 as our target species and an improved eDNA protocol to: (i) investigate differences in species detection between three developmental stages in various freshwater environments; and (ii) study the diversity of mitochondrial DNA (mtDNA) haplotypes detected in eDNA (water) samples, by amplifying a specific fragment of the COI gene (331 base pairs, bp) commonly used as a barcode. Our protocol proved to be a reliable tool for monitoring population genetic diversity of this species, and could be a valuable addition to amphibian conservation and wetland management.

Assessing Red Fox (Vulpes vulpes) Demographics to Monitor Wildlife Diseases: A Spotlight on Echinococcus multilocularis.

The assessment of red fox population density is considered relevant to the surveillance of zoonotic agents vectored by this species. However, density is difficult to estimate reliably, since the ecological plasticity and elusive behavior of this carnivore hinder classic methods of inference. In this study, red fox population density was estimated using a non-invasive molecular spatial capture-recapture (SCR) approach in two study areas: one in a known hotspot of the zoonotic cestode Echinococcus multilocularis, and another naïve to the parasite. Parasitological investigations on collected samples confirmed the presence of the parasite exclusively in the former area; the SCR results indicated a higher fox population density in the control area than in the hotspot, suggesting either that the relationship between fox density and parasite prevalence is not linear and/or the existence of other latent factors supporting the parasitic cycle in the known focus. In addition, fox spotlight count data for the two study areas were used to estimate the index of kilometric abundance (IKA). Although this method is cheaper and less time-consuming than SCR, IKA values were the highest in the areas with the lower molecular SCR density estimates, confirming that IKA should be regarded as a relative index only.

Mapping the geographic origin of captive and confiscated Hermann's tortoises: A genetic toolkit for conservation and forensic analyses.

The illegal trade has been threatening tortoise populations worldwide for decades. Nowadays, however, DNA typing and forensic genetic approaches allow us to investigate the geographic origin of confiscated animals and to relocate them into the wild, providing that suitable molecular tools and reference data are available. Here we assess the suitability of a small panel of microsatellite markers to investigate patterns of illegal translocations and to assist forensic genetic applications in the endangered Mediterranean land tortoise Testudo hermanni hermanni. Specific allelic ladders were created for each locus and tested on several reference samples. We used the microsatellite panel to (i) increase our understanding of the population genetic structure in wild populations with new data from previously unsampled geographic areas (overall 461 wild individuals from 28 sampling sites); (ii) detect the presence of non-native individuals in wild populations; and (iii) identify the most likely geographic area of origin of 458 confiscated individuals hosted in Italian seizure and recovery centers. Our analysis initially identified six major genetic clusters corresponding to different geographic macro-areas along the Mediterranean range. Long-distance migrants among wild populations, due to translocations, were found and removed from the reference database. Assignment tests allowed us to allocate approximately 70 % of confiscated individuals of unknown origin to one of the six Mediterranean macro-areas. Most of the assigned tortoises belonged to the genetic cluster corresponding to the area where the respective captivity center was located. However, we also found evidence of long-distance origins of confiscated individuals, especially in centers along the Adriatic coast and facing the Balkan regions, a well-known source of illegally traded individuals. Our results clearly show that the microsatellite panel and the reference dataset can play a beneficial role in reintroduction and repatriation projects when confiscated individuals need to be re-assigned to their respective macro-area of origin before release, and can assist future forensic genetic applications in detecting the illegal trade and possession of Testudo hermanni individuals.

Changes in host densities and co-feeding pattern efficiently predict tick-borne encephalitis hazard in an endemic focus in northern Italy.

Tick-borne encephalitis is an important zoonosis in many parts of north-western, central and eastern Europe, Russia and the Far East, with considerable altitudinal and latitudinal shifts described during recent decades. The reported routes of transmission for TBE virus include the saliva-activated non-viraemic transmission between co-feeding ticks taking place on rodent hosts. During the period 2001-2014, a population of the yellow-necked mouse (Apodemus flavicollis), which is considered among the most efficient TBE competent host, especially in central and western Europe, was intensively live-trapped in a known TBE focus in the Province of Trento, Italy. Individual live-trapped mice were checked for the number and position of feeding ticks and serologically screened for TBEv antibodies. A combined effect of climatic conditions and density of both roe deer and mice on the number of co-feeding tick groups was observed. Specifically, the occurrence of co-feeding ticks on mice during the questing season was affected by autumnal cooling in the previous season. On the other hand, co-feeding occurrence was also positively associated with roe deer abundance, while mouse density showed a hump-shaped pattern. Individual features of A. flavicollis such as weight and sex also affected co-feeding occurrence with the heaviest (breeding adult) males carrying more co-feeding ticks. We also found that the overall number of co-feeding ticks on mice positively affected TBEv antibody detection in this species the following year. In conclusion, a specific combination of climatic conditions in conjunction with certain rodent and roe deer densities are the principal determinants of the number of co-feeding ticks on A. flavicollis and, consequently, TBEv circulation. These variables can be used to provide an early warning signal for a TBE hazard, thus representing a useful tool for Public Health authorities to prepare action for prevention and control within TBEv circulation areas.

Emerging Rodent-Borne Viral Zoonoses in Trento, Italy.

Rodent-borne hanta- and arenaviruses are an emerging public health threat in Europe; however, their circulation in human populations is usually underestimated since most infections are asymptomatic. Compared to other European countries, Italy is considered 'low risk' for these viruses, yet in the Province of Trento, two pathogenic hantaviruses (Puumala and Dobrava-Belgrade virus) and one arenavirus (Lymphocytic Choriomeningitis Virus) are known to circulate in rodent reservoirs. In this paper, we performed a follow-up serological screening in humans to detect variation in the prevalence of these three viruses compared to previous analyses carried out in 2002. We also used a statistical model to link seropositivity to risk factors such as occupational exposure, cutting firewood, hunting, collecting mushrooms, having a garden and owning a woodshed, a dog or a companion rodent. We demonstrate a significant increase in the seroprevalence of all three target viruses between 2002 and 2015, but no risk factors that we considered were significantly correlated with this increase. We conclude that the general exposure of residents in the Alps to these viruses has probably increased during the last decade. These results provide an early warning to public health authorities, and we suggest more detailed diagnostic and clinical investigations on suspected cases.

Effect of Climate and Land Use on the Spatio-Temporal Variability of Tick-Borne Bacteria in Europe.

The incidence of tick-borne diseases caused by Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp. has been rising in Europe in recent decades. Early pre-assessment of acarological hazard still represents a complex challenge. The aim of this study was to model Ixodes ricinus questing nymph density and its infection rate with B. burgdorferi s.l., A. phagocytophilum and Rickettsia spp. in five European countries (Italy, Germany, Czech Republic, Slovakia, Hungary) in various land cover types differing in use and anthropisation (agricultural, urban and natural) with climatic and environmental factors (Normalized Difference Vegetation Index (NDVI), Normalized Difference Water Index (NDWI), Land Surface Temperature (LST) and precipitation). We show that the relative abundance of questing nymphs was significantly associated with climatic conditions, such as higher values of NDVI recorded in the sampling period, while no differences were observed among land use categories. However, the density of infected nymphs (DIN) also depended on the pathogen considered and land use. These results contribute to a better understanding of the variation in acarological hazard for Ixodes ricinus transmitted pathogens in Central Europe and provide the basis for more focused ecological studies aimed at assessing the effect of land use in different sites on tick-host pathogens interaction.

Prevalence and genetic variability of Anaplasma phagocytophilum in wild rodents from the Italian alps.

BACKGROUND: Human granulocytic anaplasmosis is a zoonotic bacterial disease with increasing relevance for public health in Europe. The understanding of its sylvatic cycle and identification of competent reservoir hosts are essential for improving disease risk models and planning preventative measures. RESULTS: In 2012 we collected single ear biopsy punches from 964 live-trapped rodents in the Province of Trento, Italy. Genetic screening for Anaplasma phagocytophilum (AP) was carried out by PCR amplification of a fragment of the 16S rRNA gene. Fifty-two (5.4%) samples tested positive: 49/245 (20%) from the bank vole (Myodes glareolus) and 3/685 (0.4%) samples collected from the yellow-necked mouse (Apodemus flavicollis). From these 52 positive samples, we generated 38 groEL and 39 msp4 sequences. Phylogenetic analysis confirmed the existence of a distinct rodent strain of AP. CONCLUSIONS: Our results confirm the circulation of a specific strain of AP in rodents in our study area; moreover, they provide further evidence of the marginal role of A. flavicollis compared to M. glareolus as a reservoir host for this pathogen.

Morphological and molecular analyses of epikarstic Parastenocarididae (Copepoda: Harpacticoida) from two Sicilian caves, with description of a new Stammericaris.

We describe Stammericaris destillans sp. nov., and re-describe Stammericaris trinacriae (Pesce, Galassi and Cottarelli 1988) based on new material. The two species were collected from epikarstic drips and pools on the floor of two different caves: a karstic (Molara Cave) and a gypsum (Entella Cave) cave, respectively, both located in Sicily, Italy. We also report the presence of previously undescribed structures for Stammericaris amyclaea (Cottarelli 1969) and Stammericaris orcina (Chappuis 1938). Phylogenetic analyses of the mitochondrial COI and ribosomal 18S genes attributed the new species to Stammericaris Jakobi 1972, therefore the structure of the male P4 endopod of S. destillans is interpreted as an autapomorphy; other morphological features (structure of male antennule and P3, of female P3; inner ornamentation of P1 basis, armature of caudal rami and shape and armature of P5 of both sexes) correspond to those typical of the genus. Hence, we slithgly amended the generic diagnosis. [zoobank.org:pub:4CC84A0C-C511-4388-9728-41647E58097A].

What is a vector?

Many important and rapidly emerging pathogens of humans, livestock and wildlife are 'vector-borne'. However, the term 'vector' has been applied to diverse agents in a broad range of epidemiological systems. In this perspective, we briefly review some common definitions, identify the strengths and weaknesses of each and consider the functional differences between vectors and other hosts from a range of ecological, evolutionary and public health perspectives. We then consider how the use of designations can afford insights into our understanding of epidemiological and evolutionary processes that are not otherwise apparent. We conclude that from a medical and veterinary perspective, a combination of the 'haematophagous arthropod' and 'mobility' definitions is most useful because it offers important insights into contact structure and control and emphasizes the opportunities for pathogen shifts among taxonomically similar species with similar feeding modes and internal environments. From a population dynamics and evolutionary perspective, we suggest that a combination of the 'micropredator' and 'sequential' definition is most appropriate because it captures the key aspects of transmission biology and fitness consequences for the pathogen and vector itself. However, we explicitly recognize that the value of a definition always depends on the research question under study.This article is part of the themed issue 'Opening the black box: re-examining the ecology and evolution of parasite transmission'.

Identification of Ixodes ricinus blood meals using an automated protocol with high resolution melting analysis (HRMA) reveals the importance of domestic dogs as larval tick hosts in Italian alpine forests.

BACKGROUND: In Europe, Ixodes ricinus L. is the main vector of a variety of zoonotic pathogens, acquired through blood meals taken once per stage from a vertebrate host. Defining the main tick hosts in a given area is important for planning public health interventions; however, until recently, no robust molecular methods existed for blood meal identification from questing ticks. Here we improved the time- and cost-effectiveness of an HRMA protocol for blood meal analysis and used it to identify blood meal sources of sheep tick larvae from Italian alpine forests. METHODS: Nine hundred questing nymphs were collected using blanket-dragging in 18 extensive forests and 12 forest patches close to rural villages in the Province of Trento. Total DNA was either extracted manually, with the QIAamp DNA Investigator kit, or automatically using the KingFisher™ Flex Magnetic Particle Processors (KingFisher Cell and Tissue DNA Kit). Host DNA was amplified with six independent host group real-time PCR reactions and identified by means of HRMA. Statistical analyses were performed in R to assess the variables important for achieving successful identification and to compare host use in the two types of forest. RESULTS: Automating DNA extraction improved time- and cost-effectiveness of the HRMA protocol, but identification success fell to 22.4% (KingFisher™) from 55.1% (QIAamp), with larval hosts identified in 215 of 848 questing nymphs; 23 mixed blood meals were noted. However, the list of hosts targeted by our primer sets was extended, improving the potential of the method. Host identification to species or genus level was possible for 137 and 102 blood meals, respectively. The most common hosts were Rodentia (28.9%) and, unexpectedly, Carnivora (28.4%), with domestic dogs accounting for 21.3% of all larval blood meals. Overall, Cetartiodactyla species fed 17.2% of larvae. Passeriformes (14.6%) fed a significantly higher proportion of larvae in forest patches (22.3%) than in extensive forest (9.6%), while Soricomorpha (10.9%) were more important hosts in extensive forest (15.2%) than in forest patches (4.3%). CONCLUSIONS: The HRMA protocol for blood meal analysis is a valuable tool in the study of feeding ecology of sheep ticks, especially with the cost- and time- reductions introduced here. To our knowledge, we show for the first time that domestic dogs are important larval hosts in the Alps, which may have possible implications for tick-borne disease cycles in urbanized areas.

Habitat fragmentation is associated to gut microbiota diversity of an endangered primate: implications for conservation.

The expansion of agriculture is shrinking pristine forest areas worldwide, jeopardizing the persistence of their wild inhabitants. The Udzungwa red colobus monkey (Procolobus gordonorum) is among the most threatened primate species in Africa. Primarily arboreal and highly sensitive to hunting and habitat destruction, they provide a critical model to understanding whether anthropogenic disturbance impacts gut microbiota diversity. We sampled seven social groups inhabiting two forests (disturbed vs. undisturbed) in the Udzungwa Mountains of Tanzania. While Ruminococcaceae and Lachnospiraceae dominated in all individuals, reflecting their role in extracting energy from folivorous diets, analysis of genus composition showed a marked diversification across habitats, with gut microbiota α-diversity significantly higher in the undisturbed forest. Functional analysis suggests that such variation may be associated with food plant diversity in natural versus human-modified habitats, requiring metabolic pathways to digest xenobiotics. Thus, the effects of changes in gut microbiota should not be ignored to conserve endangered populations.