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

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

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

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 area

Spatial and temporal dynamics of lymphocytic choriomeningitis virus in wild rodents, Northern Italy

We determined the prevalence of infection with lymphocytic choriomeningitis virus (LCMV) among small mammals in northern Italy and analyzed long-term dynamics of LCMV in a rodent population in the province of Trento. LCMV is circulating among the most widespread and common wild rodent species in this area (Apodemus flavicollis, Myodes glareolus, and Microtus arvalis); overall prevalence is 6.8%. During 2000–2006, intensive monitoring of LCMV in a population of yellow-necked mice (A. flavicollis) showed a positive correlation between prevalence of infection and rodent density. At the individual level, weight and sex appeared to correlate with antibody prevalence, which suggests that horizontal transmission of LCMV occurs principally among heavier, older males and occurs during fighting. Isolation and genetic characterization of this virus will be the crucial next steps for a better understanding of its ecology

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

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Assessing Red Fox (<i>Vulpes vulpes</i>) Demographics to Monitor Wildlife Diseases: A Spotlight on <i>Echinococcus multilocularis</i>

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

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

Raw reads are available under the NCBI bioproject “PRJNA813419” (Accession number for BioSamples: SAMN26501511 - SAMN26502071 and SRA: SRR18252263-SRR18252823).International audienceClimate 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

Dispersal and habitat dynamics shape the genetic structure of the Northern chamois in the Alps

International audienceAim: Understanding the drivers of species distribution ranges and population genetic structure can help predict species' responses to global change, while mitigating threats to biodiversity through effective conservation measures. Here, we combined species habitat suitability through time with process-based models and genomic data to investigate the role of landscape features and functional connectivity in shaping the population genetic structure of Northern chamois. Location: European Alps. Taxon: Northern chamois (Rupicapra rupicapra)