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Emerging ecological functions and relationships of Alpine ungulates amid wildlife recovery and anthropogenic influences: a multi-disciplinary approach
No full textHuman-induced environmental changes are placing unprecedented pressures on ecosystems worldwide, driving biodiversity losses that threaten ecosystem functioning and resilience. Among the most critical yet understudied consequences of these losses is the disruption or rapid modification of ecological interactions among species. However, investigating such interactions requires disentangling multiple co-occurring and interrelated factors within complex pathways that often cannot be addressed with a single approach. In this PhD dissertation, I explore how emerging ecological interactions and functions shape a large herbivore community in the Alps — including red deer (Cervus elaphus), roe deer (Capreolus capreolus), and Alpine chamois (Rupicapra rupicapra) — amidst the ongoing natural recolonisation by wolves (Canis lupus) and decades of diverse anthropogenic influences, such as hunting and outdoor recreation. To address these complexities, I designed a comprehensive field study using a quasi-experimental approach along a temporal gradient since wolf recolonisation across 350 km2 in the Italian Alps. This study combines complementary techniques — including camera-trapping, animal movement tracking, stable isotope analysis, genetic diet metabarcoding, and gut microbiota metataxonomics — each offering unique but interconnected insights into ecological processes. Together, these methods reveal patterns spanning spatial and temporal scales, biological organisation levels (e.g. population, community, ecosystem), ecological pathways (e.g. horizontal and vertical, top-down and bottom-up) and biological mechanisms (e.g. trophic, behavioural). The dissertation opens with an overview of the study context, objectives and thesis structure. Particular attention is then given to field sampling, presenting a synthetic version of the study design (detailed in the Field Protocol, Appendix A) and the descriptive results. The core of the thesis follows in Chapters 1 through 5, each exploring specific ecological dimensions and analytical perspectives. Chapters 1 and 2 focus on vertical functions of ungulates, examining their antipredator behaviours in response to wolves and human hunters, while also considering the influences of vegetation and topography. Chapters 3 and 4 delve into horizontal relationships, analysing environmental, behavioural and trophic mechanisms of herbivore coexistence, considering isotopic niche hypervolumes, multi-species co-occurrence patterns and intraspecific grouping behaviour. Chapter 5 outlines ongoing analyses and preliminary results expanding on these findings. Finally, all these results are synthesised and discussed in an integrated framework. In Chapter 1, I examine diel activity responses in a community of sympatric ungulates along the ongoing wolf recolonisation in the Central-Eastern Alps, considering the pervasive presence of human activity. Camera-trap data from 158 locations across four sites representing a temporal gradient of wolf recolonisation (with reproductive pairs established in 2017, 2019, 2021 and 2025) were used to analyse ungulate activity during two six-month periods (May–October, 2022–2023). In summer, red deer showed higher diurnality in sites with a longer history of wolf presence (a 7% increase over five years, on average) and progressively reduced nocturnality within sites as local wolf establishment advanced (a 5% decrease per year, on average), also heightening activity overlap with humans. This ‘diel shield effect’ disappeared when human hunting occurred. In contrast, roe deer adjusted diel activity only in response to hunting, while chamois responded solely to the spatial distribution of outdoor recreation. These findings highlight that wolf recovery can induce detectable diel activity shifts in large herbivores over relatively short timescales, yet responses depend on species biology and behavioural plasticity. Importantly, human risk and disturbance can offset or override these behavioural adjustments, potentially altering the ecosystemic effects of returning large carnivores. In Chapter 2, I explore the scale-specific mechanisms of emerging antipredator behaviours by examining how proaction and reaction interact to shape the movements of ungulates in response to hunter-related lethal risk. Using GPS-collared red deer and high-resolution hunting data as a case study, I applied movement metric models and integrated step selection functions that incorporated high-resolution canopy cover data (LiDAR) and individual site familiarity (derived from movementbased utilisation distributions), within a spatiotemporally dynamic landscape of risk. Red deer either proactively avoided areas of chronic risk, or they selected canopy cover where and when risk was predictably high. However, when risk was encountered anyway, canopy cover was no longer selected, and instead only modulated a reactive response along a remain-to-leave continuum. This reaction was even more evident when the environment was unfamiliar, underlining the importance of memory in such reaction patterns. I describe how proaction and reaction fuse in an antipredator sequence of interconnected movement decisions in a large herbivore, and discuss how this result may help disentangle the ecological consequences of behavioural responses to predation. Moving toward horizontal relationships within the mammal community, Chapter 3 examines the niche-based mechanisms enabling coexistence among sympatric ungulates and the functional community structure, using stable isotope ratios (δ13C, δ15N, δ34S, δ18O, δ2H) in hair. Understanding these mechanisms and structure is essential to anticipate how ecological communities may respond to ongoing environmental change and shifting population dynamics. I applied a Bayesian framework to quantify species-specific n-dimensional niche hypervolumes, and estimate niche region and overlaps, alongside univariate and multivariate analyses to detect niche segregation among sympatric ungulates. Red deer exhibited the broadest niche, consistent with their generalist ecology and large spatial range. Results indicated distinct niche segregation among red deer, roe deer, and chamois, with mean pairwise isotopic niche overlap under 40%. Three primary axes of differentiation emerged: water sourcing (δ18O, δ2H), diet quality (δ15N), and habitat openness (δ13C). Specifically, chamois appeared to derive more water from plant intake rather than direct drinking, consume a higher-quality diet than Cervids, and rely more heavily on open habitats for resource use compared to red deer. Additional isotopic differences between red deer and roe deer may stem from fine-scale abiotic conditions like microclimate and topography. Together, these patterns suggest functional differentiation critical for promoting coexistence in this ungulate community. In Chapter 4, I investigate patterns of co-occurrence among sympatric ungulates, as well as intraspecific grouping behaviour, to highlight behavioural dimensions of coexistence under rapid change. Using camera-trap data, I tested how environmental, predator, and anthropogenic factors shape ungulate co-occurrence and grouping during summer. Overall, red deer, roe deer and chamois neither actively avoided nor preferentially associated with one another. However, with advancing wolf recolonisation, roe deer increasingly occupied sites where red deer were absent, indicating potential unidirectional avoidance. Moreover, red deer formed larger groups as time since wolf establishment increased, particularly in open habitats — a likely response to perceived predation risk. Anthropogenic pressures did not significantly influence interspecific co-occurrence but modulated intraspecific grouping behaviour through species- and habitat-specific responses: red deer formed larger groups near human settlements and under higher recreational activity in forests, whereas chamois grouped more in forested areas close to settlements but less where recreational use was high. Notably, both chamois and red deer individually preferred steep terrain, yet avoided both conspecifics and heterospecifics under such conditions, revealing shared environmental constraints influencing both inter- and intraspecific interactions. Chapter 5 outlines ongoing genetic and isotopic analyses aimed at uncovering mechanisms behind observed niche segregation. These include studies of dietary composition through DNA metabarcoding, the role of gut microbiota in digestion and host-environment interactions, nutrient pathways, spatial foraging strategies, and isotopic calibration via a controlled experiment. Overall, this dissertation advances our understanding of the structure of ecological networks involving Alpine large mammals and how these networks reorganise under the combined pressures of carnivore recolonisation and pervasive human influence. By integrating behavioural, spatial, and trophic data across horizontal and vertical ecological processes, my findings show that simultaneous ecological differentiation and overlap support coexistence in this community. At the same time, ungulate species adjust their behaviours, and intra- and interspecific associations in response to emerging risks, reshaping the network structure through behavioural dynamics. This multi-disciplinary approach underscores that ecosystems cannot be understood through simplified, single-species perspectives, highlighting the crucial role of both predators and ungulates as active agents shaping ecological networks. Such insights contribute to a deeper understanding of species coexistence and future ecosystem dynamics in increasingly human-dominated landscape
Evolution of Brettanomyces bruxellensis during secondary fermentation of sparkling wines and counteraction strategies
Get PDFThe scope of this work is the study of the evolution of Brettanomyces bruxellensis, the main wine spoilage yeast, during bottle fermentation of sparkling wines. Lambrusco (Emilia, Italy) was considered as a model wine, for its high concentration of precursors for B. bruxellensis activity, especially cinnamic acids. Five Lambrusco base wines furnished by a cooperative winery were inoculated with a 3-log concentration of B. bruxellensis and then underwent secondary fermentation in the bottle. Two strategies of contrast to B. bruxellensis, already successfully applied in red winemaking, were tested here for the first time in bottle fermentation: chitosan and a yeast proposed as a biocontrol agent. Bottle fermentation was monitored from a chemical and microbiological perspective. The resulting sparkling wines were analyzed by GC and HPLC–MS/MS to verify the presence of the key molecules indicating B. bruxellensis activity—biogenic amines, volatile phenols, and pyridines. Sensory analysis was also performed to establish the effects of the treatments on the overall wine profile. The results demonstrate that B. bruxellensis is capable of growing up to 5-log units, causing severe alterations of the wines, both from a chemical and sensorial point of view. The addition of chitosan at the beginning of bottle fermentation effectively mitigated the effects of B. bruxellensis, resulting in the wines being similar to the uncontaminated control. The effectiveness of the biocontrol agent under these conditions was lower and requires further investigatio
Investigating the link between antioxidant capacity and atypical aging in white wines: orac as a predictive tool
No full textTransgene-free CRISPR-Cas and cisgenesis approaches for resistance to powdery and downy mildew in grapevine
Get PDFThe development of disease-resistant tree crops is essential to sustainable agriculture, particularly under increasing biotic stresses driven by climate change. This project explores a dual strategy to confer resistance to powdery mildew (PM) and downy mildew (DM) in grapevine employing (i) a transgene-free CRISPR-Cas genome editing platform based on ribonucleoprotein (RNP) delivery, and (ii) a cisgenic transformation approach utilizing native resistance (R) genes. Both approaches rely on efficient production of embryogernic callus and regeneration of modified grapevine plants. Our CRISPR-Cas RNP system enables precise, transient gene editing without the integration of foreign DNA, targeting key susceptibility (S) genes known to be involved in PM and DM pathogenesis–specifically, MLO and DMR6 genes, respectively. In parallel, we are implementing a cisgenic strategy to introduce naturally occurring R genes from sexually compatible and disease-resistant donor genotypes. This strategy uses a binary vector system carrying a heat-inducible recombination cassette. Following selection, regenerated plants containing the cisgenic construct and the desired R gene are subjected to controlled heat shocks to activate the recombinase, leading to the excision of unwanted transgenic sequences. The resulting plants retain only the native R gene sequence. We present initial results demonstrating successful simultaneous editing of one MLO and two DMR6 genes. In cisgenic lines, we report stable expression of the Resistance to Plasmopara viticola1 (RPV1) gene. Both approaches are designed to align with emerging regulatory frameworks and enable the preservation of the genetic backrounds of elite grapevine cultivars such as Chardonnay and Merlot, which are disrupted through conventional breeding. All resulting lines from both cisgenic and gene editing strategies will undergo sequencing to assess and ensure the absence of unwanted genomic modifications
La selezione dei batteri lattici per l’enologia
No full textIn virtù del suo impatto sensoriale, la fermentazione malolattica è una tappa importante della vinificazione, anche per i vini con basse concentrazioni di acido L-malico. Le colture starter di batteri malolattici sono ormai un fattore della qualita
Aquaporin translation tunes plant water transport to external conditions in grapevine
Get PDFWater stress challenges global crop productivity, particularly for perennial species such as grapevines, where effective water management is crucial for berry quality and yield. Aquaporins, a family of water channel proteins, play a key role in regulating water transport within plant cells, affecting water uptake and redistribution. Although the transcriptional response of aquaporin genes to water stress in grapevines has been documented, their translational regulation remains less explored. This study investigates the transcriptional and translational dynamics of three Plasma Membrane Intrinsic Proteins and three Tonoplast Intrinsic Proteins in leaves and roots of a grafted ‘Pinot Noir’ on ‘Kober 5BB’ rootstock during water deficit conditions and recovery. Aquaporin translation analyzed by polysome profiling and co-sedimentation analysis of their transcripts highlighted that water stress had a general negative effect, although significant only for VviTIP1-3. Conversely, recovery measured at 6 h after rewatering was characterized by a boost of translation reactivation for all but one aquaporins. Transcriptional profiling of the same aquaporins revealed significant down-regulation at prolonged stress in roots, highlighting the contribution of aquaporins to osmoregulation and drought tolerance. Moreover, transcriptional modulation resembles a long-term adaptative response to limit water loss. In the leaf, only two specific genes, VviPIP2-5 and VviTIP2-1, were modulated during water deficit and even more during recovery and positively correlated with stomatal conductance and leaf water potential. They represent important regulators of water homeostasis and good candidates for breeding programs. This study uncovered an additional level of aquaporin post-transcriptional control finely tuning vines to changing external conditions
Comparative volatilome profiling of milk kefir and cereal-based kefir analogues fermented with milk and water kefir grains
Get PDFMilk and water kefir are traditional fermented beverages attracting considerable scientific and commercial interest, due to their microbial diversity, health-enhancing properties and consumer preferences. They are linked to tradition, meet the tastes of global consumers, and are model microbiomes/fermented products of considerable relevance. In this study, a Direct Injection Mass Spectrometry (DIMS) technique, i.e., Proton Transfer Reaction Time of Flight Mass Spectrometry (PTR-ToF-MS), was exploited for the rapid and non-invasive profiling of volatile organic compounds (VOCs) during the fermentation of i) milk, ii) oat, iii) corn, and iv) barley, using both milk kefir grains and water kefir grains as fermentation microbiomes. The impact of the supplementary inoculation of a strain of Lactiplantibacillus plantarum M5MA1-B2 of biotechnological relevance, in association with kefir microbiomes, has also been evaluated. The intensity 62 ions, corresponding to volatile compounds belonging to the chemical classes of acids, aldehydes, ketones, esters, alcohols, lactones and sulfur compounds, was monitored at 13 different times, representative of the different phases of the overall fermentation time of 48 h, offering a time-scale omics approach in microbiomes study (i.e. kefir microbiomes in milk and in cereal flours). The addition of L. plantarum M5MA1-B2 to kefir grains serves as a suitable model for demonstrating the perturbation of a single-strain starter culture when inoculated in matrices fermented by complex microbiomes. A complex modulation was detected, with an overall intensity-reducing effect for many VOCs, including acetoin, diacetyl, acetaldehyde, and ethanol. Heptanal/2-heptanone (in cereal-based matrices) 2-nonanone (in all the matrices) are good markers of strain addition with respect to milk kefir grains. This study also contributed to extending the volatilome of milk kefir, describing for the first time 19 VOCs associated with this matrix (e.g. propenoic acid, methanethiol, hexenal, 2,4-heptadienal
Monitoring the effect of raw milk refrigeration on milk kefir fermentation: implications for quality and food safety
No full textA deep analytical investigation of the aroma chemistry of incrocio Bruni 54 and its differentiation from Italian white varieties
Get PDFIncrocio Bruni 54 is a little-known white grape variety developed in the Marche region (Italy) from a cross between Verdicchio and Sauvignon Blanc to combine aromatic freshness with structure. In light of the growing interest in minor and autochthonous cultivars, this study provides the first comprehensive chemical characterization of the aroma profile of Incrocio Bruni 54 wines. Seventeen commercial wines were analyzed for varietal compounds, such as terpenes, norisoprenoids, volatile thiols, methyl salicylate and its glycosides, and fermentative compounds, including esters, alcohols, acids, phenols, aldehydes, and ketones, using GC-MS/MS and LC-MS/MS. Odor activity value (OAV) calculations revealed an aroma profile dominated by ethyl esters, such as ethyl caproate and isopentyl acetate, β-damascenone, 4-vinylguaiacol, TDN, and the volatile thiols 3MH and 4MMP, imparting fruity, floral, spicy, and tropical notes. Comparison with datasets of 246 Italian monovarietal white wines and related sub-datasets composed of Verdicchio and Lugana showed significantly higher concentrations of 3MH and free methyl salicylate in Incrocio Bruni 54, but markedly lower levels of glycosylated methyl salicylate forms, suggesting a greater expression of this odorant in young wines balanced by a lower potential over aging. These findings highlight the distinctive aromatic fingerprint of Incrocio Bruni 54, combining parental traits with unique sensory potential, and support its knowledge and valorization in wine production
