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One of the main concerns in industrialized countries is represented by per- and poly-fluoroalkyl substances (PFAS), persistent contaminants hardly to be dealt with by conventional wastewater treatment processes. Phyco-remediation was proposed as a green alternative method to treat wastewater. Synechocystis sp. PCC6803 is a unicellular photosynthetic organism candidate for bioremediation approaches based on synthetic biology, as it is able to survive in a wide range of polluted waters. In this work, we assessed the possibility of applying Synechocystis in PFAS-enriched waters, which was never reported in the previous literature. Respirometry was applied to evaluate short-term toxicity of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), which did not affect growth up to 0.5 and 4 mg L−1, respectively. Continuous and batch systems were used to assess the long-term effects, and no toxicity was highlighted for both compounds at quite high concentration (1 mg L−1). A partial removal was observed for PFOS and PFOA, (88% and 37%, with removal rates of about 0.15 and 0.36 mg L−1 d−1, respectively). Measurements in fractionated biomass suggested a role for Synechocystis in the sequestration of PFAS: PFOS is mainly internalized in the cell, while PFOA is somehow transformed by still unknown pathways. A preliminary bioinformatic search gave hints on transporters and enzymes possibly involved in such sequestration/transformation processes, opening the route to metabolic engineering in the perspective application of this cyanobacterium as a new phyco-remediation tool, based on synthetic biology.

Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E . multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E . multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E . multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

Background Echinococcus multilocularis is a small tapeworm affecting wild and domestic carnivores and voles in a typical prey-predator life cycle. In Italy, there has been a focus of E. multilocularis since 1997 in the northern Italian Alps, later confirmed in red foxes collected from 2001 to 2005. In this study, we report the results of seven years of monitoring on E. multilocularis and other cestodes in foxes and describe the changes that occurred over time and among areas (eco-regions) showing different environmental and ecological features on a large scale. Methods Eggs of cestodes were isolated from feces of 2872 foxes with a sedimentation/filtration technique. The cestode species was determined through multiplex PCR, targeting and sequencing ND1 and 12S genes. Analyses were aimed to highlight variations among different eco-regions and trends in prevalence across the study years. Results Out of 2872 foxes, 217 (7.55%) samples resulted positive for cestode eggs at coproscopy, with differences of prevalence according to year, sampling area and age class. Eight species of cestodes were identified, with Taenia crassiceps (2.65%), Taenia polyacantha (1.98%) and E. multilocularis (1.04%) as the most represented. The other species, Mesocestoides litteratus, Taenia krabbei, T. serialis, T. taeniaeformis and Dipylidium caninum , accounted for < 1% altogether. Echinococcus multilocularis was identified in foxes from two out of six eco-regions, in 30 fecal samples, accounting for 1.04% within the cestode positives at coproscopy. All E. multilocularis isolates came from Bolzano province. Prevalence of cestodes, both collectively and for each of the three most represented species ( T. crassiceps, T. polyacantha and E. multilocularis ), varied based on the sampling year, and for E. multilocularis an apparent increasing trend across the last few years was evidenced. Conclusions Our study confirms the presence of a focus of E. multilocularis in red foxes of northeast Italy. Although this focus seems still spatially limited, given its persistence and apparent increasing prevalence through the years, we recommend research to be conducted in the future on the ecological factors that, on a smaller scale, allow this zoonotic species to persist. On the same scale, we recommend a health education campaign to inform on the measures to prevent this zoonosis, targeted at people living in the area, especially hunters, dog owners, forestry workers and other potentially exposed categories.

Background Prey species can display antipredator movement behaviours to reduce predation risk, including proactive responses to chronic or predictable risk, and reactive responses to acute or unpredictable risk. Thus, at any given time, prey movement choice may reflect a trade-off between proaction and reaction. In previous studies, proaction and reaction have generally been considered separately, which neglects their potentially simultaneous influence on animal movement decisions and overall space use. Methods In this study, we analysed how proaction and reaction interact to shape the movements of GPS-collared red deer ( Cervus elaphus ) in response to hunting by humans. Using an exhaustive inventory of red deer hunting events and very high-resolution canopy cover density (LiDAR), we combined movement metric (displacement and path length) models and integrated step selection functions to investigate antipredator movement responses to lethal risk on various spatiotemporal scales, considering a dynamic landscape of risk. Results Our results show that 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, but 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. Conclusions We 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. Finally, we lay the foundations for further investigations into the origins of similarities and differences between proactive and reactive movement responses.

Humans are often perceived as predators by free‐living animals, and thus, even non‐consumptive human activities such as outdoor recreation may trigger behavioural and physiological responses, often with negative consequences on individual fitness and population persistence. Nonetheless, there is growing evidence that wildlife can also have remarkable behavioural tolerance, but no clear picture has yet emerged regarding the mechanisms explaining different responses to humans. We investigated the effect of different types of human activity – hunting and outdoor recreation – on behavioural tolerance to humans in Alpine marmots Marmota marmota. Marmots were studied in areas with contrasting protection regimes and under different levels of outdoor recreation in northern Italy over three seasons (2021–2023). Flight initiation distance (i.e. the distance at which an animal escapes from an approaching person) was used as a proxy of tolerance to human disturbance and tested against levels of outdoor recreation and hunting using linear mixed modelling. Marmots were more sensitive to human disturbance in hunted as compared to protected areas, whereas we did not find evidence for changes in behavioural tolerance when exposed to varying levels of outdoor recreation. In turn, our study suggests that hunting, by reducing behavioural tolerance to humans, could exacerbate the negative effects of non‐lethal human activities on wildlife. This has implications for conservation and management strategies aimed at promoting coexistence in human‐altered landscapes.

Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E. multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E. multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E. multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

In the last two centuries, persecution and deforestation caused grey wolf Canis lupus populations in Europe to decline. Recently, their numbers started to recover although most populations still remain isolated from one another. This study presents the first documented evidence of the successful reconnection of the Dinaric-Balkan and the Alpine wolf populations via long-distance dispersal and subsequent reproduction. A young male wolf radiocollared in the Dinaric Mountains in July 2011 travelled through Slovenia and Austria to the Italian Alps, where he settled in March 2012. During the 98 days of dispersal period, the wolf has travelled a cumulative line distance of 1176 km crossing multiple anthropogenic and natural barriers, and successfully hunting wild prey until he settled 233 km straight line distance from its natal territory. Camera trapping, snow tracking and genetic evidence in the new territory confirmed pairing with a female wolf from the neighbouring Alpine population. In the following year, the pair has produced a first documented “mixed” litter between wolves from the Dinaric-Balkan and the Alpine wolf populations. This case study demonstrates the potential for the future merging of European wolf populations even in human-dominated landscapes and highlights the importance of transboundary cooperation in wolf research and management.

This paper describes the modifications applied to EPANET, a public-domain water distribution system modelling software package, that does not correctly compute the hydraulics of a water distribution network (WDN) with variable tank heads in (slow) unsteady flow conditions. Firstly the methodology adopted to extend the Global Gradient Algorithm (GGA) implemented in the original EPANET source code to the Extended Period Simulation-GGA (EPS-GGA) is described. Then the convergence and stability conditions of the theta method, used for the discretisation in time of the set of differential equations describing the hydraulic behaviour of a WDN, are discussed. The reasons for EPS-GGA numerical stability are demonstrated and a fully implicit discretisation of differential equations (i.e. theta = 1) is suggested as the optimal choice as implicitly proposed in Giustolisi et al. but without theoretical justification. Both the modified and original versions of EPANET are applied to a particularly severe test case of a WDN. Moreover, the procedures for the correct numerical representation of the tanks' maximum and minimum level boundary conditions are developed and compared with previously proposed procedures. The modified version of EPANET source code does not show the significant instabilities which are evident in the original version, nor the lack of consistency due to the improper maximum and minimum level boundary condition schematisations formerly proposed in the scientific literature.

Prey species can display antipredation movement behaviours to reduce predation risk including proactive responses to chronic or predictable risk, and reactive responses to acute or unpredictable risk. Thus, at any given time, prey movement choice may reflect the trade-off between proaction and reaction. In previous studies, proaction and reaction have generally been considered separately, which neglects their simultaneous influence on animal decisions. In this study, we analysed how proaction and reaction interact to shape the movements of GPS-collared red deer (Cervus elaphus), in response to human hunting of conspecifics. Our results show that red deer proactively selected canopy cover where and when risk was predictably high. However, when they were unable to avoid risk, canopy cover was no longer selected, but only modulated a reactive response along a freeze-to-escape continuum. This reaction was even more evident when the environment was unfamiliar, underlining the importance of memory in such reaction patterns. Therefore, to our knowledge, for the first time, we describe how proaction and reaction fuse in an antipredation sequence of interconnected movement decisions in a large herbivore, and we lay the foundations for further investigations into the evolutionary origins of similarities and differences between proactive and reactive behaviours.

Surveillance of E. multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence and burden in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. We reassessed the prevalence of E. multilocularis at the southern border of its distribution in Alto Adige (Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared results from the diagnostic test currently implemented for surveillance (based on Coproscopy+Multiplex PCR - CMPCR), against a real-time quantitative PCR (qPCR) for 235 fox faeces collected in 2019-2020. The performances of the two tests were estimated using a scraping technique (SFCT) as the gold standard applied to the small intestines of a subsample (n=123) of the same hosts. True prevalence was calculated and sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E. multilocularis in foxes (14.3%) was definitely higher than reported in the last decade (never >5% from 2012 to 2018). The qPCR also had a higher sensitivity (83%) compared to CMPCR (21%). Agreement with the gold standard was far higher for qPCR (0.816) than CMPCR (0.298) as well, determining a smaller sample size required to detect the disease. Alto Adige should be considered a highly endemic area. Surveillance at the edges of E. multilocularis distribution should adopt qPCR diagnostics on definitive hosts on a small geographic scale. Echinococcus multilocularis is an intestinal flatworm, whose adult stage in Europe is harboured mainly by the red fox (Vulpes vulpes), which spreads parasite’s eggs by faeces. This parasite is the agent of a severe and potentially lethal zoonosis, the alveolar echinococcosis, affecting humans after accidental ingestion of parasite’s eggs. In the Italian Alpine area, which represents the southernmost border of E. multilocularis European range, surveillance is hindered by a fragmented distributional pattern, where presence in foxes has been consistently reported only in few isolated foci in Alto Adige (Bolzano province – Italy) of low prevalence. In order to improve the efficiency of monitoring efforts, we tested the performances of two diagnostic protocols on fox faeces (sedimentation, filtration, counting technique followed by standard PCR, and whole stool real-time PCR) against a benchmark technique on fox intestines (scraping, filtration, counting technique - considered as the gold standard). This allowed not only to determine qPCR as a far more sensitive and sample-efficient diagnostic tool for E. multilocularis detection in marginally affected areas, but also to re-assess its prevalence in Alto Adige, which should be considered a highly endemic area. Consequent actions in the field and modifications in the surveillance strategy should be therefore considered.