Društvo biologov Slovenije 2026 Special Issue 13th International Symposium on Wild Fauna Man, Nature and Ecosystem Services 12–14 June 2025 • Vienna • Austria © WAVES & SYNYO GmbH 2 Acta Biologica Slovenica, 2026, Special issue Acta Biologica Slovenica, 2026, Special Issue Založila/Published by Založba Univerze v Ljubljani / University in Ljubljana Press Društvo biologov Slovenije / Slovenian biological society Za založbo/For the publisher Gregor Majdič, rektor Univerze v Ljubljani / the Rector of the University of Ljubljana Anita Jemec Kokalj, predsednica Društva biologov Slovenije / Chairman of Slovenian Biological Society Izdala/Issued by Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za biologijo / University of Ljubljana, Biotehnical Faculty, Department of Biology Za izdajatelja/For the Issuer Marina Pintar, dekanja Biotehniške fakultete UL / Dean of Biotechnical Faculty Naslov uredništva/Editorial Office Address Univerza v Ljubljani, Biotehniška fakulteta, Acta Biologica Slovenica, Večna pot 111, 1000 Ljubljana, Slovenija Glavni urednik/Editor-in-chief Matevž Likar, Slovenija / Slovenia, matevz.likar@bf.uni-lj.si Odgovorna urednica/Managing editor Anita Jemec Kokalj, Slovenija / Slovenia, anita.jemec@bf.uni-lj.si Uredniški odbor/Editorial Board Gregor Belušič (SLO), Univerza v Ljubljani, Biotehniška fakulteta Tina Eleršek (SLO), Nacionalni inštitut za biologijo Božo Frajman (A), Univerza v Innsbrucku Király Gergely (HU), University of Sopron, Faculty of Forestry Gordana Glavan (SLO), Univerza v Ljubljani, Biotehniška fakulteta Aleksandra Golob, Univerza v Ljubljani, Biotehniška fakulteta Cene Gostinčar, Univerza v Ljubljani, Biotehniška fakulteta Katarina Hančević (HR), Institute for Adriatic Crops and Karst Reclamation Margit Heinlaan (EST), National Institute of Chemical Physics and Biophysics Vida Jojić (SRB), Univerzitet u Beogradu, Institut za biološka istraživanja „Siniša Stanković” Tina Klenovšek (SLO), Univerza v Mariboru, Fakulteta za naravoslovje in matematiko Dana Kühnel (GER), Helmholtz Centre for Environmental Research GmbH - UFZ Alenka Malej (SLO), Nacionalni inštitut za biologijo Nataša Mori (SLO), Nacionalni inštitut za biologijo Polona Mrak (SLO), Univerza v Ljubljani, Biotehniška fakulteta Maria Mueller (A), University of Salzburg Siniša Ozimec (HR), Univerza Josipa Juraja Strossmayerja Anastasija Panevska (SLO), Univerza v Ljubljani, Biotehniška fakulteta Hubert Potočnik (SLO), Univerza v Ljubljani, Biotehniška fakulteta Tomislav Radić (HR), Institute for Adriatic Crops and Karst Reclamation Tomaž Skrbinšek, Univerza v Ljubljani, Biotehniška fakulteta Simona Strgulc Krajšek (SLO), Univerza v Ljubljani, Biotehniška fakulteta Miloš Vittori (SLO), Univerza v Ljubljani, Biotehniška fakulteta Anja Klančnik (SLO), Univerza v Ljubljani, Biotehniška fakulteta Oblikovanje/Design Ajda Fortuna To delo je ponujeno pod licenco Creative Commons Priznanje avtorstva-Deljenje pod enakimi pogoji 4.0 Mednarodna licenca (izjema so fotografije). / This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License (except photographies). Izdajanje revije sofinancira Javna agencija za znanstvenoraziskovalno in inovacijsko dejavnost Republike Slovenije (ARIS) The journal is co-financed by Slovenian Research and Innovation Agency (ARIS) Publication is free of charge. ISSN 1854-3073 (spletna verzija/online version) UDK 57(497.4) DOI: https://doi.org/10.14720/abs.69.Special%20issue http://journals.uni-lj.si/abs/ Acta Biologica Slovenica je indeksirana v – is indexed in: CAB Abstracts, Web of Science Clarivate Naslovnica/Cover page Cervus nippon, avtor/author: Volodymyr Burdiak, Shutterstock Inc. (extended license, order no. 2461580967). All rights reserved. 3 Acta Biologica Slovenica, 2026, Special issue 5 Introduction 7 Free-Living Birds as Reservoirs of Enterococcus spp.: A Potential Environmental Health Concern / Prostoživeče ptice kot rezervoarji bakterij vrste Enterococcus: potencialna nevarnost za okoljsko zdravje Sinem Arslan, Rossana Schena, Nadia Piscopo, Marcello Stefanucci, Daniela Hildisch, Marcos Dias, Ana Raquel Calapez, Jaime A. Ramos, Maria João Feio, Peter Goethals, Marie-Anne Forio, Luisa De Martino, Ana Cláudia Norte, Francesca Paola Nocera 15 Radioactivity in aquatic ecosystems: implications for biodiversity / Radioaktivnost v vodnih ekosistemih: posledice za biotsko raznovrstnost Pierluigi Casolaro 22 Landscape approaches for risk mitigation from wildlife: The case of wild boar road accidents in Avellino Province, Southern Italy / Krajinski pristopi za zmanjševanje tveganj, povezanih z divjadjo: primer prometnih nesreč z divjimi prašiči v provinci Avellino na jugu Italije Elena Cervelli, Stefania Pindozzi, Ester Scotto di Perta, Antonio Raffaele, Roberto Della Sala, Gianpiero Sepe, Nicola Boscia, Luigi Esposito, Nadia Piscopo 30 Radiographic study of multiple chronic traumatic lesions of the forelimbs in a wolf (Canis lupus italicus) from the National Park of Abruzzo, Lazio and Molise / Rentgenska preiskava večkratnih kroničnih travmatskih poškodb sprednjih okončin pri volku (Canis lupus italicus) iz Narodnega parka Abruzzo, Lazio in Molise Dario Costanza, Nadia Piscopo, Maria Pia Pasolini, Mariaelena de Chiara, Serena Panetta, Vincenza di Pirro, Leonardo Gentile, Luciano Sammarone, Micheletino Matarazzo, Adelaide Greco, Leonardo Meomartino 38 A complex network: wild boar, water and biodiversity / Kompleksne interakcije: divji prašiči, voda in biotska raznovrstnost Luigi Esposito, Mariaelena de Chiara, Marcello Stefanucci, Massimo Mazzone, Camillo Campolongo, Ivan Maraviglia, Nadia Piscopo 46 Prevalence of hindgut pathogens Brachyspira hyodysenteriae and Salmonella enterica in wild boars in northwest Spain / Razširjenost patogenov v zadnjem delu črevesja Brachyspira hyodysenteriae in Salmonella enterica pri divjih prašičih v severozahodni Španiji Fabiana Esposito, Samuel Gómez-Martínez, Lucía Pérez-Pérez, Javier Merino, Héctor Argüello, Ana Carvajal, Vicente González-Eguren 51 Lawsonia intracellularis infection in wild boars in northwest Spain / Okužba z Lawsonia intracellularis pri divjih prašičih v severozahodni Španiji Fabiana Esposito, Samuel Gómez-Martínez, Lucía Pérez-Pérez, Javier Merino, Vicente González-Eguren, Héctor Argüello, Ana Carvajal 57 Using camera traps to assess spatial and temporal interactions between cattle and red deer in a Mediterranean silvo-pastoral system / Uporaba fotopasti za oceno prostorskih in časovnih interakcij med govedom in jelenjadjo v sredozemskem gozdno-pastirskem sistemu Pedro Girotti, Paolo Viola, Gloria Bernabucci, Carlo M. Rossi, Luigi Esposito, Nadia Piscopo, Loredana Basiricò, Bruno Ronchi, Riccardo Primi Table of Contents 4 Acta Biologica Slovenica, 2026, Special issue 64 Natura 2000 Sites (SCI/SAC - SPA) wildlife populations estimation and comparison between Abruzzo (IT) and Castilla y León (ES) regions / Ocena populacij prostoživečih živali na območjih Natura 2000 (SIC/ZSC – ZPS) ter primerjava med regijama Abruzzo (IT) in Castilla y León (ES) Martina Iovino, Vicente González Eguren, Pablo Santos Redin, Leonardo Gentile, Alice Martinelli, Jesús Palacios Alberti, Elena Cervelli 71 The State of Wildlife Heritage within Natura 2000 Regional Sites in Campania (Italy) and Norte (Portugal) / Stanje naravne dediščine v regionalnih območjih Natura 2000 v Kampaniji (Italija) in Norte (Portugalska) Adriana Mangini, Luigi Esposito, Aurora C. Monzon Capapé 79 Shedding new light on bioluminescence / Novo razumevanje bioluminiscence Alessandra Pelagalli, Alessandro Pezzella, Andrea Becchimanzi, Raffaele Marrone, Caterina Squillacioti, Francesco Caso, Luciana Cozzolino, Pierluigi Casolaro 90 Ecosystem services and disservices related to the presence of wild boar in Avellino province, Italy / Ekosistemske storitve in negativni vplivi, povezani s prisotnostjo divjih prašičev v provinci Avellino v Italiji Nadia Piscopo, Luigi Esposito, Fabian Capitanio, Antonio Raffaele, Alessia Lombardi, Aurora C. Monzon Capapé 100 Management of the wolf in the transition of its condition from "highly protected" to "protected". The case of Italy / Upravljanje populacije volkov v obdobju prehoda njihovega statusa iz »strogo zaščitenih« v »zaščitene«. Primer Italije Nadia Piscopo 106 Forensic approach to livestock predation / Forenzični pristop k napadom plenilcev na živino Marina Siano, Giuseppe Piegari, Orlando Paciello, Nadia Piscopo 111 Ecological status assessment of watercourses in Benevento, Southern Italy / Ocena ekološkega stanja vodotokov v Beneventu v južni Italiji Maria Tafuro, Roberta Brunetti, Maria João Feio, Simona Tafuri, Maria Mandato, Nadia Piscopo, Maria Ottaiano, Loredana Baldi, Pasquale Gallo, Mauro Esposito 5 Acta Biologica Slovenica, 2026, Special issue Our President has written this brief introductory note. We look forward to your feedback. The 13th International Wildlife Symposium, that has been held June 12-14, 2025, at the prestigious Natural History Museum in Vienna, is a great opportunity for dialogue and exchange of experiences between experts in terrestrial and aquatic ecosystems with multidisciplinary expertise. The new vision of the holistic approach includes the natural environment and agroecosystems in which wild and domesticated biodiversity coexist, requiring the identification of appropriate management systems to ensure their optimal health. The objectives of the 13th ISoWiF remain those outlined since the first edition in 1998, but have been adapted to modern needs, summarized in the motto: "Humans, Nature, and Ecosystem Services." These themes were developed in the three working sessions: 1) Terrestrial Ecosystems, emphasizing the importance of research on management, conservation, hunting, and wildlife regulations, in order to ensure, through the exchange of scientific contributions, both human control for environmental protection and the connections between human actions and wildlife. 2) Aquatic ecosystems, emphasizing the importance of research on management, conservation, fishing, and regulations regarding fish fauna, in order to ensure, through the exchange of scientific contributions, the protection of the aquatic environment but also, and above all, the valorization of the ecosystem services associated with these environments, which are too often undervalued. 3) A One Health approach dedicated to veterinary sciences, in order to ensure, through the complex study and interaction of different living species, the protection of environments and ecosystems, sup- porting the needs identified in the first two themes above. I thank those who ensured the longevity of Waves and contributed to the realization of this conference, both scientifically and technically, in such a splendid setting as the city of Vienna, and in particular the Natural History Museum. I thank those who have always believed in the growth of our association, such as the founding members, including the Honorary Presidents who continue to support us. I thank the universities and various institutions that have collaborated and will continue to collaborate with us. I thank the OneAquaHealth project, which contributed distinguished speakers and the initial data from fieldwork. I thank all those who participated in the conference, contributing to its success, and all those who will read the articles in this special issue of the journal "Acta Biologica Slovenica." A special thanks goes to the editorial staff of ABS, whose belief in this project from the very beginning and rigorously promoted the quality of the submissions. Indeed, of the 61 papers submitted as oral or poster presentations, a selection of 15 passed peer review, meeting the criteria required for an indexed journal, in the form of short notes. The holistic vision, which has always been a driving force behind WAVES activities, is perfectly cap- tured by the papers selected for this special publication. A group of four papers (Tafuro et al.; Casolaro, Esposito L. et al., Piscopo et al.) report emerging data on the health of minor river waters in an inland Introduction 6 Acta Biologica Slovenica, 2026, Special issue Italian area, stressing the need to understand water components, including radioactive isotopes, while not forgetting that the biodiversity present in these complex ecosystems provides ecosystem services and disservices. A second group of papers (Iovino et al., Mangini et al., Pelagalli et al.) emphasizes the close connection with the findings presented by the previous group and the difficulty of working with the multitude of living organisms, including some insects that are indicators of ecological well-being, that populate sites important to the European community but not always controllable with orthodox scientific methodologies. One health is often understood as a diagnostic tool for potential pathogens for animals and humans. However, the third group of papers presented in the special issue (Arslan et al., Esposito F. et al.a, Esposito F. et al.b, Costanza et al.) identifies a series of microorganisms that, while considered pathogenic, do not appear to carry disease in the animals from which they were isolated. Here too, the presence of these organisms must be placed within the complexity of the ecosystems in which they occur. Conversely, clinical diagnosis of a single animal, while ethically under- standable, is not useful for the economics of ecosystems, which, instead, benefit from management efforts conducted on a local scale. The latest group of selected works (Piscopo, Siano et al., Cervelli et al., Girotti et al.) demonstrates that knowing who frequents a given environment, whether wild or domestic, allows us to propose innovative process systems that, even when wildlife management regulations change, can ensure the coexistence of humans and animals and, more importantly, the conservation of all living species. We are confident that the message conveyed by the Symposium participants will be amplified by this publication, which will be evaluated by the scientific community, from which we expect stimulating enrichments for the near future. — Nadia Piscopo President of WAVES International 7 1 Department of Veterinary Medicine and Animal Productions - University of Naples Federico II, Italy 2 Italian League for Bird Protection (LIPU), Benevento, Italy 3 Marine and Environmental Science Centre, University of Coimbra, Portugal 4 Department of Animal Science and Aquatic Ecology, University of Ghent, Belgium * Corresponding author: E-mail address: francescapaola.nocera@unina.it § Authors share senior authorship, with equal contribution Citation: Arslan, S., Schena, R., Piscopo, N., Stefanucci, M., Hildisch, D., Dias. M., Calapez, A. R., Ramos, J., João Feio, M., Goethals, P., Forio, M., De Martino, L., Norte, A. C., Nocera, F. P. (2026). Free-Living Birds as Reservoirs of Enterococcus spp.: A Potential Environmental Health Concern. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.28988 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Free-Living Birds as Reservoirs of Enterococcus spp.: A Potential Environmental Health Concern Sinem Arslan 1 , Rossana Schena 1 , Nadia Piscopo 1 , Marcello Stefanucci 2 , Daniela Hildisch 2 , Marcos Dias 3 , Ana Raquel Calapez 3 , Jaime A. Ramos 3 , Maria João Feio 3 , Peter Goethals 4 , Marie-Anne Forio 4 , Luisa De Martino 1 , Ana Cláudia Norte 3 * § , Francesca Paola Nocera 1 * § Abstract This study investigated the occurrence of Enterococcus species, known for their worrying antibiotic resistance profiles, in free-living birds, due to their role as potential reservoirs of these bacteria. Cloacal swabs were collected from 94 wild birds, including Eurasian blackcap, blackbird, house sparrow, and common kingfisher, captured around urban streams in Coimbra, Portugal. Faecal samples from birds in Benevento, Italy, and Ghent, Belgium, were also analysed. After isolation, bacteria were identified using MALDI-TOF MS. Enterococcus faecalis was the predominant species across all bird species sampled in Coimbra, with high occurrence in common kingfishers (66.7%), house sparrows (31.6%), blackbirds (19.4%), and Eurasian blackcaps (16.7%). Enterococcus faecium was found only in house sparrows (26.3%) and blackbirds (5.6%) in Coimbra. E. faecalis was also detected in faecal samples from Benevento and Ghent in blackbirds, house sparrows, and common kingfishers, while E. Faecium was not isolated from samples of these two geographical areas. The findings obtained confirm the presence of E. faecalis and E. faecium in avian fauna inhabiting aquatic environments. The occurrence in aquatic birds like the common kingfisher highlights the potential link between water quality, biodiversity, and the spread of opportunistic pathogens, emphasising the importance of a One Health approach. Keywords Enterococcus faecalis, Enterococcus faecium, reservoirs, free-living birds 8 Acta Biologica Slovenica, 2026, Special issue Introduction Free-living birds can act as reservoirs and disseminators of various bacteria, including Enterococcus species, which are known for their intrinsic and acquired antibiotic resistance. Enterococci encompass both pathogenic and commensal microorganisms widely distributed in diverse environments. Indeed, they can be isolated from soil, water, plants, wild animals, birds, and insects, frequently colonis- ing the gut as symbionts (Ramos et al., 2020). From a complex perspective and with a One Health approach, there appears to be no scientific evidence demonstrating the passage from one living organism to another. However, it is possible that birds closely associated with aquatic environments could contaminate water with their excreta and transmit Enterococcus spp. to biodiversity (Li et al., 2024); aquatic ecosystems (fish, amphibians, reptiles, molluscs, mammals). Wild birds are increasingly recognised as reservoirs of enterococci (genus Enterococcus). For any ecological, epidemiological, or One Health study of enterococci in avifauna, it is important to reliably identify the isolates to the species level (Radhouani et al, 2021). MALDI-TOF MS has emerged as a powerful tool for bacterial identification, and its application to wild-bird isolates of Enterococcus has been described. In one study from central Europe (Poland), 54 isolates of Enterococcus spp. obtained from 25 species of wild birds were analysed by MALDI-TOF MS, and subsequently via rpoA gene sequencing for confirmation. Using MALDI-TOF MS: all 54 isolates (100 %) were correctly assigned to the genus Enterococcus; 51 (94.4 %) were identified to species level with log(score) ≥ 2.0; the remaining three (5.6 %) were identified to genus or probable species (log(score) 1.88- 1.92 (Stępień-Pyśniak et al., 2017). The rpoA sequencing confirmed the identifications. Among the identified spe- cies, E. faecalis was the most frequent (~50 %), followed by E. faecium (~33.3 %), E. hirae (~9.3 %), E. durans (~3.7 %) and E. casseliflavus (~3.7 %) (Stępień-Pyśniak et al., 2017). Environmental/wildlife isolates may include fewer common species or strains, so misidentifications or lower log values are possible. Therefore, the first essential prerequisite for culture-based isolation is the use of MAL- DI-TOF MS to grow pure colonies. Culture quality (purity, growth conditions) influences the spectra. MALDI-TOF mass spectroscopy enables rapid and reliable species-level identification of Enterococcus spp. in wild birds, enabling ecological and surveillance studies to accurately charac- Prostoživeče ptice kot rezervoarji bakterij vrste Enterococcus: potencialna nevarnost za okoljsko zdravje Izvleček V naši študiji smo preučevali pojavnost vrst Enterococcus, znanih po zaskrbljujočih profilih odpornosti proti antibiotikom, pri prostoživečih pticah, ki predstavljajo potencialne rezervoarje teh bakterij. Vzorci iz kloake so bili odvzeti pri 94 prostoživečih pticah, vključno z evrazijsko črnoglavko, kosom, hišnim vrabcem in navadnim kraljevskim ribičem, ujetih v bližini mestnih potokov v Coimbri na Portugalskem. Analizirani so bili tudi vzorci iz iztrebkov ptic iz Beneventa v Italiji in Genta v Belgiji. Po izolaciji so bakterije identificirali z metodo MALDI-TOF MS. Enterococcus faecalis je bila prevladujoča vrsta pri vseh vrstah ptic, vzorčenih v Coimbri, z visoko pogostostjo pri kraljevih ribičih (66,7 %), hišnih vrabcih (31,6 %), kosih (19,4 %) in evrazijskih črnoglavkah (16,7 %). Enterococcus faecium je bil v Coimbri ugotovljen le pri hišnih vrabcih (26,3 %) in kosih (5,6 %). E. faecalis je bil odkrit tudi v vzorcih iztrebkov iz Beneventa in Genta pri kosih, hišnih vrabcih in navadnih ledarjih, medtem ko E. faecium ni bil izoliran iz vzorcev iz teh dveh geografskih območij. Ugotovitve potrjujejo prisotnost E. faecalis in E. faecium v ptičji favni, ki naseljuje vodna okolja. Pojavnost pri vodnih pticah, kot je navaden ledar, poudarja potencialno povezavo med kakovostjo vode, biotsko raznovrstnostjo in širjenjem oportunističnih patogenov, kar izpostavlja pomen pristopa »One Health«. Ključne besede Enterococcus faecalis, Enterococcus faecium, rezervoarji, prostoživeče ptice 9 Acta Biologica Slovenica, 2026, Special issue terise species distribution in avian reservoirs and prevent potential zoonotic risks. Notably, Enterococcus faecalis (E. faecalis) and Entero- coccus faecium (E. faecium) are two species of clinical rel- evance, as clinical isolates of both bacterial species exhibit resistance to numerous commonly used antimicrobial agents, including ampicillin and vancomycin (Van Harten et al., 2017). This study investigated the occurrence of Entero- coccus spp. isolated from wild bird populations in three geographically distinct European areas: Coimbra, Portugal; Benevento, Italy; Ghent, Belgium. The identification of the isolated strains was performed by MALDI-TOF MS, which allows rapid and reliable species-level identification of Enterococcus spp. from wild birds, enabling ecological and surveillance studies to accurately characterise species distributions in avian reservoirs. Materials and Methods Sample collection Bird feces were sampled using the OneAquaHealth project sampling protocol available at Zenodo (https://doi. org/10.5281/zenodo.21300693). Captures were carried out after official authorisation from the respective competent authorities. On days without wind or rain, mist nets were deployed 5 hours before sunrise and 5 hours before sunset and kept active until darkness. The operators moved approxi- mately 500 meters away and waited for approximately 40 minutes. The birds captured by the nets were identified by species, age, and sex. Biometric data (wing length, tarsus length, body mass) were collected for each bird, and they were then placed in paper bags for approximately 10 minutes in the shade, while waiting for defecation (Figure 1). The faeces collected from the bags were placed in 1.5 ml microtubes, stored in a portable refrigerator, and, once they reached the laboratory, plated on selective culture plates. From the captured birds, four species present at all three sampling sites were selected. Fecal samples from blackcaps (Sylvia atricapilla), blackbirds (Turdus merula), house sparrows (Passer domesticus), and common king- fishers (Alcedo atthis) collected near urban waterways in Coimbra (Portugal), Benevento (Italy) were analyzed for pathogenic microorganisms (Figure 2). While in Ghent (Bel- gium), tissue traps placed under riparian vegetation were used to collect faecal samples. Figure 1. Birds were placed in paper bags waiting for defecation. Slika 1. Ptice so bile postavljene v papirnate vrečke, kjer so čakale na iztrebljanje. 10 Acta Biologica Slovenica, 2026, Special issue Bacterial isolation and identification Once delivered to the Bacteriology Laboratory of the Department of Veterinary Medicine and Animal Production, University of Naples “Federico II” (Naples, Italy), specimens were seeded on solid and differential culture media. In particular, for Enterococcus spp isolation, Columbia CNA supplemented with 5% sheep blood (Liofilchem, Teramo, Italy), a selective medium for Gram-positive bacteria, was used. After overnight incubation at 37 °C, colonies from pure cultures, suspected to belong to Enterococcus spp , were subjected to rapid screening tests encompassing colony morphology on Columbia CNA plates, cellular morphology determined by Gram staining, and catalase and oxidase tests (bioMérieux, Marcy L'Etoile, France). Bacterial identification was performed by using Matrix-As- sisted Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (MALDI-TOF MS) (Bruker Daltonics GmbH, Bremen, Germany), according to the manufacturer's guide- lines. For identification, all samples were tested in triplicate, and Enterococcus faecalis ATCC® 29212 ™ was used as quality control strain. Identifications with scores below 1.7 were considered unreliable. Scores between 1.7 and 1.99 suggested a probable genus identification. A score of ≥2.0 indicated a certain genus identification and a highly probable species identification. Sampling A total of 125 faecal samples were obtained in three sampling research sites of Europe: 94 in Coimbra, 12 in Benevento, and 19 in Ghent (Table 1). The number of faecal samples was divided among the four bird species chosen for the study. Faecal samples from 20 common kingfishers, 28 house sparrows, 47 blackbirds, and 30 Eurasian black- caps were analysed. Figure 2. Sampled free-living birds in Coimbra (Portugal), Benevento (Italy), Ghent (Belgium): Alcedo atthis (a); Passer domesticus (b); Turdus merula (c); Sylvia atricapilla (d). Slika 2. Vzorčene prostoživeče ptice v Coimbri (Portugalska), Beneventu (Italija) in Gentu (Belgija): Alcedo atthis (a); Passer domesticus (b); Turdus merula (c); Sylvia atricapilla (d). Variable Alcedo atthis (common kingfisher) Passer domesticus (house sparrow) Turdus merula (blackbird) Sylvia atricapilla (Eurasian blackcap) All feces analyzed Coimbra 9 19 36 30 94 Benevento 5 3 4 0 12 Ghent 6 6 7 0 19 All 20 28 47 30 125 Table 1. Number of bird faeces samples analysed at the three OneAquaHealth research sites. Tabela 1. Število vzorcev ptičjih iztrebkov, analiziranih na treh raziskovalnih lokacijah OneAquaHealth. 11 Acta Biologica Slovenica, 2026, Special issue Results Enterococcus spp. isolation and identification From the 125 faecal samples analysed, 48 strains belong- ing to Enterococcus spp. were isolated in monoculture from Columbia CNA agar plates. All strains were identified with a log score ≥2.0, suggesting a highly probable species identification. In particular, E. faecalis was the predominant bacterium in all bird species sampled at the three research sites, with occurrence values of 50.00% (10 strains/20 samples) in the common kingfisher, 28.57% (8 strains/28 samples) in the house sparrow, and 21.28% (10 strains/47 samples) in the blackbird. In the Portuguese city, the black- cap showed an occurrence of 16.67% (5 strains/30 swabs). The statistical comparison of E. faecalis occurrences between the three European cities shows significant differ- ences for the kingfisher in Coimbra versus Benevento and Ghent (66.67 vs 40.00 and 33.33; P<0.01) and for the house sparrow in Ghent versus Benevento and Coimbra (16.67 vs 33.33 and 31.58; P<0.01). No significant differences were recorded for the blackbird. Positive results in the blackcap were recorded only in Coimbra, while they were absent in the other two European cities. E. faecium was identified only in Coimbra, in house sparrows (31.58%; 6 strains/19 samples) and blackbirds (5.56%; 2 strains/36 samples). The two occurrences were significantly different (P<0.01). In the Portuguese city, blackbird faecal samples tested positive for E. mundtii (11.11%; 4 strains/36 samples), E. casseliflavus (2.78%; 1 strain/36 samples), and E. marmotae (2.78%; 1 strain/36 samples). E. hirae was identified in house sparrows (5.26%; 1 strain/19 samples). Discussion Free-living birds are crucial components of ecosystems, but their mobility also positions them as potential reservoirs and disseminators of various bacteria, including Entero- coccus species (Kobuszewska and Wysok, 2024). This is particularly relevant, given the clinical significance of both intrinsic and acquired antibiotic resistance in Enterococci, a phenomenon driven by their ability to develop resistance to various antibiotics (Cebeci, 2024). Enterococci are ubiquitous microorganisms, serving as both pathogens and commensals across diverse environments. Their pres- ence has been documented in different kind of habitats, including soil, water, plants, wild animals, birds, insects, and as gut symbionts (Nocera et al., 2021; Freitas et al, 2024; Ferrara et al., 2024). Notably, E. faecalis and E. faecium are the two species of greatest particular relevance, due to their worrying antibiotic resistance profiles and being often Figure 3. Enterococcus faecalis (a) and Enterococcus faecium (b) grow on Columbia CNA agar plates. Slika 3. Rast bakterij Enterococcus faecalis (a) in Enterococcus faecium (b) na agarskih ploščah Columbia CNA. 12 Acta Biologica Slovenica, 2026, Special issue site n/% E. faecalis E. faecium E. mundtii E. casseliflavus E. marmotae E. hirae Coimbra n 6 0 0 0 0 0 % 66.67 0.00 0.00 0.00 0.00 0.00 Benevento n 2 0 0 0 0 0 % 40.00 0.00 0.00 0.00 0.00 0.00 Ghent n 2 0 0 0 0 0 % 33.33 0.00 0.00 0.00 0.00 0.00 Coimbra n 6 6 0 0 0 0 % 31.58 31.58 0.00 0.00 0.00 0.00 Benevento n 1 0 0 0 0 1 % 33.33 0.00 0.00 0.00 0.00 5.26 Ghent n 1 0 0 0 0 0 % 16.67 0.00 0.00 0.00 0.00 0.00 Coimbra n 7 2 4 1 1 0 % 19.44 5.56 11.11 2.78 2.78 0.00 Benevento n 1 0 0 0 0 0 % 25.00 0.00 0.00 0.00 0.00 0.00 Ghent n 2 0 0 0 0 0 % 28.57 0.00 0.00 0.00 0.00 0.00 Coimbra n 5 0 0 0 0 0 % 16.67 0.00 0.00 0.00 0.00 0.00 Benevento n 0 0 0 0 0 0 % 0.00 0.00 0.00 0.00 0.00 0.00 Ghent n 0 0 0 0 0 0 % 0-00 0.00 0.00 0.00 0.00 0.00 Table 2. Occurrence of Enterococcus faecalis, Enterococcus faecium and other Enterococci in bird faeces sampled in Coimbra (PT), Benevento (IT), and Ghent (BE). Tabela 2. Prisotnost Enterococcus faecalis, Enterococcus faecium in drugih vrst rodu Enterococcus v ptičjih iztrebkih, odvzetih v Coimbri (PT), Beneventu (IT) in Gentu (BE). Alcedo atthis (Common kingfisher) Passer domesticus (house sparrow) Turdus merula (blackbird) Sylvia atricapilla (Eurasian blackcap) associated with both severe healthcare infections and outbreaks worldwide (Schell et al., 2020). This study aimed to investigate, by MALDI-TOF MS identification, the occur- rence of Enterococcus species in free-living birds across three distinct European geographical regions: Coimbra, Portugal; Benevento, Italy; and Ghent, Belgium. MALDI-TOF MS has proven to be a highly effective, rapid and reliable tool for the identification of Enterococ- cus species isolated from wild birds. Numerous studies have demonstrated that the technique can accurately assign isolations to both genus and species levels, with success rates exceeding 90% when compared to molec- ular confirmation methods. Its high throughput capability, minimal sample preparation, and cost-effectiveness make it particularly suitable for large-scale ecological and epidemiological studies of avian reservoirs. By enabling precise species identification, MALDI-TOF MS supports a better understanding of the distribution, diversity, and antimicrobial resistance patterns of Enterococcus spp. in wild bird populations. However, continued expansion of spectral reference databases and integration with molec- ular methods are recommended to enhance accuracy for less common or environmental strains. Overall, MALDI-TOF MS represents a valuable tool in One Health surveillance, strengthening the link between wildlife microbiology and public-health monitoring of antimicrobial resistance. 13 Acta Biologica Slovenica, 2026, Special issue The sampling carried out in Coimbra underlined the highest occurrence of Enterococci among the studied bird species: Sylvia atricapilla (Eurasian blackcap), Turdus merula (blackbird), Passer domesticus (house sparrow) and Alcedo atthis (common kingfisher). However, in this study, E. faecalis turned out to be the most frequently isolated Enterococcus species across all Portuguese, Italian and Belgian collected bird samples. In particular , the widespread presence of E. faecalis across all three geographically dis- tinct regions and these diverse bird species underscores its adaptability and potential for broad dissemination within avian hosts across Europe. In contrast to E. faecalis, E. faecium, despite its clinical importance, showed a more restricted distribution among bird specimens from Coimbra and a total absence in samples from Benevento and Ghent. This interesting result suggests potential regional or host-specific factors influencing the distribution and colonisation dynamics of E. faecium in free-living birds. Moreover, the different occurrence between E. faecalis and E. faecium among the investigated samples aligns with previous studies that often report E. faecalis as the more frequently isolated enterococcal species from animal sources (Tan et al., 2018; Nocera et al., 2021; Gołaś-Prądzyńska et al., 2022). Beyond these two dominant species, other Enterococcus species were also detected in the Portuguese samples, even though with lower frequencies. Notably, blackbirds exhib- ited the highest variety of identified Enterococcus species, including E. mundtii, E. casseliflavus, and E. marmotae. This greater enterococcal diversity in blackbirds could suggest a broader exposure to varied environmental sources or a more permissive gut environment for Enterococcus spp colonisation compared to the other bird species sampled. Conclusions This study confirms the effectiveness and rapidity of MAL- DI-TOF MS for the accurate identification of Enterococcus spp. Furthermore, the findings of this study contribute to the understanding of E. faecalis and E. faecium occurrence in free-living birds, emphasising their role as potential envi- ronmental carriers of these opportunistic pathogens, which have been recognised as a significant cause of nosocomial infections in both humans and domestic animals. Additionally, among the birds tested, the kingfisher was the sole piscivorous species, making it more directly dependent on the quality and biodiversity of watercourses. Investigating the food chain helps us understand the role of each living being in the ecology of a given environment, and describing the presence of opportunistic pathogens is essential from a One Health perspective. Author Contributions Conceptualization, F.P.N., L.d.M., N.P., A.C.N., M.J.F., P.G.; methodology, F.P.N., L.d.M., N.P., A.C.N., A.R.C.; software, S.A., F.P.N., R.S.; validation, A.C.N., M.J.F., P.G.; formal anal- ysis, F.P.N., L.d.M., A.C.N., M.A.F.; investigation, S.A., R.S., N.P., M.D., A.R.C., J.A.R., M.A.F., M.S., D.H.; resources, M.J.F., P.G., N.P.; data curation, F.P.N., R.S., A.C.N., A.R.C., N.P.; writ- ing—original draft preparation, F.P.N., L.d.M., A.C.N., N.P.; visualization, S.A., M.S., D.H., M.D., A.R.C., J.A.R., M.A.F.; supervision, L.d.M., A.C.N., P.G., N.P.; project administration, N.P., M.J.F., P.G. All authors have read and agreed to the published version of the manuscript. Acknowledgement We acknowledge the volunteers of the Local Allies of the OneAquaHealth project (Campania Region, MoGAE, CeR- VEnE, IZSM, LIPU, WWF, FIPSAS, ATC Benevento) who contributed to the field sampling, as well as the Bioelec- tronics Task Force of the University of Naples Federico II, which allowed us to use its Pathogen Readiness Platform for CERIC-ERIC (prp) Research Infrastructure funded by the Italian National Recovery and Resilience Plan (PNRR) to study human, animal, and plant pathogens with Maldi-Tof technology. Funding This research was funded by the European Union Horizon- CL6-2022-GOVERNANCE-01-09, grant number 101086521 – Protecting Urban Aquatic Ecosystems to Promote One Health OneAquaHealth. Data Availability Statement The research data are deposited at Zenodo (https://doi. org/10.5281/zenodo.21300693). Conflicts of Interest The authors declare no conflict of interest. The study was conducted independently by researchers of the Univer- sity; consequently, no external parties influenced the study design, data collection, analysis, manuscript preparation, or the decision to publish the results. 14 Acta Biologica Slovenica, 2026, Special issue References Cebeci, T. (2024). Species prevalence, virulence genes, and antibiotic resistance of enterococci from food-producing animals at a slaughterhouse in Turkey. Scientific Reports, 14, 13191. https://doi.org/10.1038/s41598-024-63984-y Ferrara, G., Piscopo, N., Pagnini, U., Esposito, L., & Montagnaro, S. (2024). Detection of selected pathogens in reproductive tissues of wild boars in the Campania region, southern Italy. Acta Veterinaria Scandinavica, 66(1), 9. https://doi.org/10.1186/s13028-024-00731-3 Freitas, A. A. R., Faria, A. R., Mendes, L. T., Merquior, V. L. C., Neves, D. M., Pires, J. R., & Teixeira, L. M. (2024). 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Trends in Microbiology, 25(6), 467–479. https://doi.org/10.1016/j.tim.2017.01.004 15 1 Department of Physics “Ettore Pancini”, Università degli Studi di Napoli Federico II, Complesso Univ. Monte S. Angelo, Napoli, I-80126, Italy 2 INFN Sezione di Napoli, Complesso Univ. Monte S. Angelo, Napoli, I-80126, Italy * Corresponding author: E-mail address: pierluigi.casolaro@unina.it Citation: Casolaro, P. (2026). Radioactivity in aquatic ecosystems: implications for biodiversity. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.24167 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Radioactivity in aquatic ecosystems: implications for biodiversity Pierluigi Casolaro 1,2 * Abstract This work reviews the impacts of natural and anthropogenic radioactivity, focusing on both abiotic and biotic matrices in aquatic ecosystems. Several nuclear accidents, such as those at Chornobyl (Ukraine, 1986) and Fukushima (Japan, 2011), have introduced substantial radionuclide loads, turning these sites into long-term case studies for understanding radiological stress on biodiversity. Wildlife indicators, such as wild boars and sentinel aquatic species, play an important role in revealing how radiocontamination moves within the ecosystems, supporting the need for multidisciplinary strategies to safeguard biodiversity. Radioactivity poses significant challenges to ecosystems, and understanding key case studies provides insights into how radionuclide contamination impacts biodiversity. Keywords Radioactivity; biodiversity; radiological risk 16 Acta Biologica Slovenica, 2026, Special issue Radioactivity Unstable atomic nuclei decay by emitting radiation that can be harmful to humans, animals, and ecosystems. This radiation originates from the nucleus itself and may occur through different decay modes, including α (emission of 4 He nuclei), β (emission of electrons, β¯ positrons β + ), and γ (emission of high-energy photons). The half-lives T 1/2 (namely, the time by which a radioactive substance reduces by one half) of such nuclear decays span more than 50 orders of magnitude, ranging from about 10 -24 s to 10 30 s, as illustrated in Figure 1. Biological effects of radiation α, β and γ radiations are ionising, therefore they can damage living cells by inducing single- or double-strand breaks into the DNA (Nikjoo et al., 2016). Today’s accepted model for radiological risk is the linear no-threshold (LNT), according to which any arbitrary small radiation dose can induce cell damage. However, the LNT application at low doses is controversial and still debated (Hooker et al., 2004; Weber & Zanzonico, 2017). The United Nations Scientific Com- mittee on the Effects of Atomic Radiation (UNSCEAR) has emphasised the uncertainty in estimating health risks from low doses of ionising radiation (Pradhan, 2013; Siegel et al., 2017). On the other hand, recent findings suggest that the scientific evidence available does not conflict with the use of the LNT model for evaluating radiation-related cancer risks in radiological protection, and no alternative dose-risk relationship is currently considered more suitable (Laurier et al., 2023). Stochastic effects are characterised by having no threshold: their probability rises with dose, and they include cancer and genetic mutations. Deterministic radi- ation effects, by contrast, are defined by the existence of a threshold dose, beyond which both incidence and severity rise. These effects are primarily driven by radiation-induced cell death (with the exception of cataracts) and may present as either early or late tissue damage, with evidence from atomic-bomb survivors indicating their occurrence at doses as low as 0.5 Sv (Fry, 2001), where 1 Sv = 1 J/Kg describes the biological effect of the radiation energy absorbed by the human tissue. Historically, international radiation pro- tection standards focused mainly on safeguarding humans, under the assumption that this approach would indirectly protect the environment in a broader sense. Nowadays, the International Atomic Energy Agency (IAEA) explicitly includes the protection of ecosystems in its standards, aiming to prevent impacts on biodiversity, habitat integrity, and ecosystems (Beauquier et al., 2023). Natural and anthropogenic radioactivity Overall, natural radioactivity yields a background annual average dose per person of 3 mSv (UNSCEAR, 2016). How- ever, geological differences on Earth’s surface cause large variations in radiation exposure. As an example, average radiation exposures of about 250 mSv/y are measured at Radioaktivnost v vodnih ekosistemih: posledice za biotsko raznovrstnost Izvleček Naša študija pregleduje vplive naravne in antropogene radioaktivnosti, pri čemer se osredotoča tako na abiotične kot na biotične matrice v vodnih ekosistemih. Hude jedrske nesreče, kot sta bili nesreči v Černobilu (Ukrajina, 1986) in Fukušimi (Japonska, 2011), so povzročile znatno obremenitev z radionuklidi, zaradi česar so ta območja postala dolgoročne študije primerov za razumevanje radiološkega stresa na biotsko raznovrstnost. Kazalniki prostoživečih živali, kot so divji prašiči in vodne vrste, ki delujejo kot indikatorji, igrajo pomembno vlogo pri razkrivanju, kako se radioaktivna onesnaženost širi znotraj ekosistemov, kar podpira potrebo po multidisciplinarnih strategijah za zaščito biotske raznovrstnosti. Radioaktivnost predstavlja pomembne izzive za ekosisteme, razumevanje ključnih primerov pa ponuja vpogled v to, kako onesnaženost z radionuklidi vpliva na biotsko raznovrstnost. Ključne besede Radioactivity; biodiversity; radiological risk 17 Acta Biologica Slovenica, 2026, Special issue Pocos de Caldas in Brazil. Guarapari Beach, in Brazil, is renowned for its elevated natural radioactivity, as its sands are primarily composed of monazite, a mineral rich in tho- rium and rare earth elements (Silveira et al., 2016). Dose levels in Ramsar, Iran, have been found to be as high as 400 mSv/y, due to springs rich in radium (Shahbazi-Gahrouei et al., 2013). Natural variation in background radioactivity has been shown to have biological effects for several living organisms, including humans, animals, plants, and microbes (Møller & Mousseau, 2013), and even low-dose exposure can affect mutation rates, reproductive success, immune function, and population densities. Anthropogenic radionuclides are a major source of environmental radio- activity, originating primarily from nuclear weapons testing (Prăvălie, 2014), large-scale nuclear accidents such as Chernobyl (Saenko et al., 2011) and Fukushima (Koo et al., 2014), and releases from nuclear facilities, especitally into aquatic systems such as the Yenisey and Techa rivers in Siberia and Urali mountains, respectively (Trapeznikov et al., 1993; Vakulovsky et al., 1995). Additional contributions to anthropogenic radioactivity arise from the use of radio- isotopes in medicine, research, and industry (Schmor, 2011). Environmental monitoring of radioactivity is crucial for assessing the impact of both natural and human-made sources of ionising radiation, e.g. in aquatic environments near nuclear power plants (Caridi et al., 2021). Studies in the Ebro river basin revealed that the radioactivity present in the river waters originates predominantly from naturally occurring radionuclides, with contributions enhanced by agricultural practices and industrial activities. Importantly, contamination from artificial radionuclides related to nuclear facilities was found to be minimal, underscoring the dominance of natural background radiation and technolog- ically enhanced naturally occurring radioactive materials in the region (Pujol & Sanchez-Cabeza, 2000). A systematic study of natural radioactivity levels in the riverine environ- ments of three major Indian rivers, Kali, Sharavathi, and Netravathi, revealed significant variations in radioactivity among the rivers. While some sediment and rock samples exhibited elevated radiological hazard indices, the majority of the values remained within or close to internationally accepted regulatory safety limits (Narayana et al., 2007). Figure 1. Table of nuclides by ground state half-life. Reproduced from Kondev et al. (2021) under CC BY 3.0 (http://creativecommons.org/ licenses/by/3.0/). Slika 1. Tabela nuklidov po razpolovnem času v osnovnem stanju. Prevzeto iz Kondev in sod. (2021) pod licenco CC BY 3.0 (http://creative- commons.org/licenses/by/3.0/). 18 Acta Biologica Slovenica, 2026, Special issue Radiation effects on living organisms Catastrophic events, such as those discussed above, serve as long-term case studies for understanding the impacts of radiological stress on biodiversity. The effects of radiation on living organisms have been extensively studied in con- taminated areas using specific bioindicators. Radiocontamination in water bodies Among the used bioindicators, fluctuating asymmetry in aquatic invertebrates and plants has been employed to assess developmental instability in water bodies near Chornobyl contaminated with 90 Sr and 137 Cs. According to (Barescut et al., 2009), radiation may be considered determinative in affecting the significance of asymmetry in zebra mussels (Dreissena polymorpha Pall.), indicating their sensitivity to radiological stress. In contrast, the study suggested that floating pondweed (Potamogeton natans L.) was less responsive, and therefore less suitable for fluctuating asymmetry analysis in this context. While acute radiation effects are well reported, long-term impacts of chronic exposure on organisms remain poorly understood and controversial, both for biotic and abiotic matrices in the Chornobyl region. Fish are known to be among the most radiosensitive aquatic organisms (Sazykina & Kryshev, 2003), yet studies on populations from contaminated lakes near Chornobyl have reported no chromosomal damage in blood cells and generally good physiological and repro- ductive health of populations of perch (Perca fluviatilis) and roach (Rutilus rutilus) (Lerebours et al., 2018). Although concentrations of 137 Cs and 90 Sr exceeded EU food safety limits, dose rates in these contaminated lakes are close to the ecosystem protection threshold of 10 μGy/h. Fish abun- dance was comparable between lakes, with no significant effects on biodiversity observed. A similar pattern was found for aquatic invertebrates (Murphy et al., 2011): no evidence suggested that the ecological status of lake macroinverte- brate communities was influenced by direct contamination from the Chornobyl accident. In fact, the most contaminated lake, Glubokoye, exhibited the highest richness of aquatic invertebrate species (Murphy et al., 2011). The Yenisei River floodplain has been contaminated over the years of nuclear activity at the nearby Min- ing-and-Chemical Combine (MCC) (Bolsunovsky et al., 2017; Bolsunovsky and Melgunov, 2019; Bolsunovsky et al., 2020). The radionuclides exhibit varied composition and mobility, posing long-term ecological risks and making the site a critical case study for understanding freshwater contamination. Aquatic plants are biological indicators of aquatic ecosystems, as they are abundant in water bodies and can build up radionuclides. In the contaminated area of the Yenisei River, where the last reactor was shut down in 2010, more than thirty artificial radionuclides were ini- tially detected in plant biomass (Bolsunovsky et al., 2020). Remarkably, eleven radionuclides were still present two years after the shutdown, and five were detectable after five years, highlighting the persistence of contamination in the ecosystem even after reactor shutdown. Laboratory experiments showed that radioactive bottom sediments of the Yenisei River were toxic to aquatic plants, Elodea canadensis in particular (Zotina et al., 2015), for which high frequencies of chromosomal aberrations were observed (Bolsunovsky et al., 2020). Radiation exposure following radioactive waste releases from the Mayak plutonium production facility into the Techa River during 1949-1956 led to contamination and population exposure. Dose reconstruction showed mean red bone marrow doses around 0.4 Gy, with about 5% of individuals receiving doses to the red bone marrow over 1 Gy (Degteva et al., 1994). Significant amounts of 90 Sr and 137 Cs, and Pu isotopes remain in river sediments and floodplain soils, with contamination concentrated near the discharge site. The estimates underscore continuing envi- ronmental presence and potential transport of radioactivity into larger river systems and the Arctic Ocean (Trapeznikov et al., 1993). Fish species in the upper Techa River were exposed to very high radiation dose rates during the peak contamination period, exceeding safety screening levels. Despite these high historical doses, current fish size and growth are not significantly different from control areas (Shishkina et al., 2019). Radiocontamination in wild boars Among wildlife bioindicators, wild boar (Sus scrofa) and earthworms are considered particularly effective for tracking the transfer and distribution of radiocontamination across ecosystems (Barrios-Garcia & Ballari, 2012; Nemoto et al., 2018; Steinhauser & Saey, 2016). Their diets, rich in fungi, plants, and insects, facilitate the uptake of bioavail- able radionuclides. Notably, wild boars can accumulate 19 Acta Biologica Slovenica, 2026, Special issue radionuclides with a resorption rate approaching 100%. Following the Chornobyl accident, most forest species exhibited elevated levels of 137 Cs (β and γ emitter, T 1/2 = 30 y), which declined over time. However, wild boars have shown little to no decrease in contamination levels since 1986. In some regions, the reduction has even progressed more slowly than the physical half-life of 137 Cs, an unex- pected trend known as the "wild boar paradox". In parts of Europe, particularly Bavaria, wild boar still frequently exceed regulatory limits for radioactive contamination. A recent study (Stäger et al., 2023) identified two primary sources of caesium in wild boar tissues: 1) atmospheric nuclear weapons testing and 2) the Chornobyl accident. 135 Cs (T 1/2 = 2.3 My) originates from the decay of 135 Xe, which has a high cross-section for thermal neutron capture and is thus suppressed in nuclear reactors. By analysing the ratio of 135 Cs to 137 Cs, the authors of this study were able to distinguish and quantify the relative contributions of each source, still finding the presence of caesium from atmospheric nuclear weapons testing in wild boars (Stäger et al., 2023). Discussion and conclusion Radioactivity presents complex and enduring challenges to ecosystems, affecting both abiotic components such as water, sediment, soil, and biotic elements, including microbes, plants, invertebrates, fish, and wildlife. The case studies discussed, from Chornobyl and Fukushima to the Techa and Yenisei rivers, demonstrate how radionuclide contamination can lead to long-term ecological disruptions, but also highlight the heterogeneity of responses across taxa and ecosystems. While certain organisms, such as some fish and invertebrate species, appear to maintain physiological and reproductive functions despite chronic exposure, others exhibit signs of radiological stress, including developmental instability and persistent contam- ination levels after initial release. The observed variability in ecological outcomes can be attributed to several factors, including the species-specific radiosensitivity, ecosystem structure, and the bioavailability of radionuclides. In some cases, some radionuclides, such as 137 Cs, continue to circu- late within food webs. The example of wild boars maintain- ing high levels of contamination years after the Chornobyl accident exemplifies how ecological and behavioural traits (such as diet and habitat use) can influence long-term exposure, giving rise to paradoxical contamination trends. These findings underscore the importance of long-term biomonitoring. Such efforts are essential not only for understanding ecological consequences but also for distinguishing between background radiation and anthro- pogenic sources. Furthermore, wildlife bioindicators, such as wild boars, aquatic plants, and invertebrates, play a crit- ical role in revealing how radiocontamination propagates through ecosystems. Finally, a multidisciplinary approach is necessary to interpret radiological impacts on biodiversity and to design appropriate conservation and management strategies. Acknowledgement The author would like to acknowledge the OneAquaHealth project for the access to reports and information. Funding This work was supported by the Italian Research Infrastruc- ture "PRP@CERIC-Pathogen Readiness Platform for CERIC ERIC upgrade" (IR0000028) and by the PRIN 2022 PNRR - OPTICS, both funded by EU - Next Generation EU. Data Availability The research data is deposited at Zenodo (https://doi. org/10.5281/zenodo.21301260). Conflicts of Interest The author declares no conflict of interest. 20 Acta Biologica Slovenica, 2026, Special issue References Barescut, J., Y avnyuk, A., Efremova, N., Protsenko, O., Gudkov, D., & Nazarov, A. (2009). Fluctuating asymmetry of zebra mussel (Dreissena polymorpha Pall.) and floating pondweed (Potamogeton natans L.) in water bodies within the Chornobyl accident exclusion zone. Radioprotection, 44(5), 475–479. Barrios-Garcia, M. N., & Ballari, S. A. (2012). 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Landscape approaches for risk mitigation from wildlife: The case of wild boar road accidents in Avellino Province, Southern Italy. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.24159 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Landscape approaches for risk mitigation from wildlife: The case of wild boar road accidents in Avellino Province, Southern Italy Elena Cervelli 1 * , Stefania Pindozzi 1 * , Ester Scotto di Perta 1 , Antonio Raffaele 2 , Roberto Della Sala 2 , Gianpiero Sepe 2 , Nicola Boscia 2 , Luigi Esposito 3 , Nadia Piscopo 3 Abstract Wildlife-vehicle collisions (WVCs) are an increasing concern at the intersection of biodiversity conservation and public safety. Among the species most frequently involved, the wild boar (Sus scrofa) has shown a notable rise in road-related incidents across Europe. This study adopts a landscape-based approach to examine the spatial dynamics of wild boar collisions in the Province of Avellino, Southern Italy. Using Geographic Information System (GIS) tools, roadkill data collected over several years were geolocated and analyzed in relation to environmental and anthropogenic variables, including protected areas, the hydrographic network, road infrastructure, and land use patterns (Corine Land Cover 2018). Results reveal two main hotspots: the Guardia Lombardi– Sant’Angelo dei Lombardi–Conza corridor, overlapping with protected natural areas and regional roads; and the Avellino Nord–Pratola Serra axis, corresponding to a high-traffic inter-provincial route. A strong spatial correlation with river systems and fragmented landscapes—especially mosaics of forest, farmland, and transitional cover—was also observed. These findings highlight the need to integrate spatial planning, ecological connectivity, and road safety measures in mitigation strategies. The study contributes to the development of GIS-based methodologies for wildlife management and offers practical insights for regional planning and conservation policy. Keywords Wild boar; wildlife-vehicle collisions; GIS analysis; Kernel density; landscape management 23 Acta Biologica Slovenica, 2026, Special issue Introduction In recent years, wild boar (Sus scrofa) populations have expanded significantly across Europe, raising growing concerns among ecologists, land managers, policymakers, and public safety authorities. This expansion extends across a range of environments—including urban, peri-ur- ban, agricultural, and forested areas—demonstrating the species' exceptional adaptability and its growing influence on human-wildlife interactions (Massei et al., 2015; Piscopo et al., 2023). A combination of ecological and anthropogenic factors lies behind this trend. Warmer winters driven by climate change have reduced seasonal mortality and extended feeding periods, boosting survival rates (Morelle et al., 2015). At the same time, access to abundant human-related food sources, from crops to urban waste, has led to increased reproductive success (Massei et al., 2015). The decline in hunting activity and the lack of natural predators have further contributed to population growth (Fernández-Gil et al., 2018; Piscopo et al., 2021). The consequences are wide range. Ecologically, wild boars disturb habitats through rooting, wallowing, and seed consumption, which can hinder vegetation recovery and reduce biodiversity (Lombardini et al., 2017). From a public health perspective, they play a role in spreading zoonotic diseases, such as African Swine Fever, which poses a serious risk to pig farming and has significant eco- nomic implications (Pittiglio et al., 2018). Among the most immediate and visible effects, however, is the increase in wildlife-vehicle collisions (WVCs), which pose dangers to both people and animals (Lange et al., 2016). Italy offers a particularly striking example. Wild boar populations have spread widely, especially throughout the central and south- ern Apennines, including regions such as Lazio, Abruzzo, Molise, Campania, and Umbria (Benfatto et al., 2023). These areas, with their blend of forests, farmland, and fragmented infrastructure, create ideal conditions for boar movement and proliferation. As consequence, more than 10,000 wildlife-vehicle collisions are recorded each year, with wild boars responsible for a substantial proportion. Their tendency to move in groups further increases the risk and severity of such incidents (Thurfjell et al., 2015). Rural depopulation and land abandonment have only accelerated this process, encouraging rewilding (Navarro & Pereira, 2012). Within this broader national context, the Province of Avellino in Campania, with its varied landscape, dense road Krajinski pristopi za zmanjševanje tveganj, povezanih z divjadjo: primer prometnih nesreč z divjimi prašiči v provinci Avellino na jugu Italije Izvleček Trki med divjadjo in vozili (WVC) predstavljajo vse večjo skrb na stičišču ohranjanja biotske raznovrstnosti in javne varnosti. Med vrstami, ki so najpogosteje vpletene v te trke, je bilo opazno povečanje pri celotni Evropi zaznati pri divjem prašiču (Sus scrofa). Ta študija uporablja pristop, ki temelji na krajinski analizi, za preučitev prostorske dinamike trkov z divjimi prašiči v provinci Avellino na jugu Italije. Z uporabo orodij geografskega informacijskega sistema (GIS) so bili podatki o poginulih živalih na cestah, zbrani v več letih, geolokalizirani in analizirani v povezavi z okoljskimi in antropogenimi spremenljivkami, vključno z zaščitenimi območji, hidrografsko mrežo, cestno infrastrukturo in vzorci rabe tal (Corine Land Cover 2018). Rezultati kažejo na dve glavni kritični točki: koridor Guardia Lombardi–Sant’Angelo dei Lombardi–Conza, ki se prekriva z zaščitenimi naravnimi območji in regionalnimi cestami; ter os Avellino Nord– Pratola Serra, ki ustreza prometni medprovincialni cesti z visokim prometom. Ugotovljena je bila tudi močna prostorska korelacija z rečnimi sistemi in fragmentiranimi pokrajinami – zlasti z mozaiki gozdov, kmetijskih površin in prehodnih pokritosti. Ti izsledki poudarjajo potrebo po vključitvi prostorskega načrtovanja, ekološke povezanosti in ukrepov za varnost v cestnem prometu v strategije za zmanjševanje tveganj. Študija prispeva k razvoju metodologij za upravljanje divjadi, ki temeljijo na GIS, ter ponuja praktične vpoglede za regionalno načrtovanje in politiko ohranjanja narave. Ključne besede Divji prašič; trki med divjadjo in vozili; analiza GIS; gostota jedra; upravljanje krajine 24 Acta Biologica Slovenica, 2026, Special issue system, and numerous Natura 2000 protected areas, has become a hotspot for WVCs—particularly during dawn and dusk hours (Amici et al., 2018; Rosell et al., 2008). Efforts to reduce these risks—such as fencing, wildlife crossings, and vehicle-based detection systems—must be carefully targeted to areas of highest vulnerability. In this regard, Geographic Information Systems (GIS) have become essential tools for identifying and prioritizing intervention zones (Belda et al., 2012). This study uses Kernel Density Estimation and spatial analysis to examine WVC patterns in Avellino, with the aim of supporting proactive, data-driven approaches to road safety and landscape management. Materials and Methods Workflow Overview, Study Area and Data Sources and Collection This study investigates the spatial distribution of wild boar (Sus scrofa)–vehicle collisions in the Province of Avellino, located in Southern Italy, using Geographic Information Sys- tems (GIS) as the primary analytical tool. The research fol- lowed a structured workflow comprising: (1) data collection and validation, (2) spatial preprocessing, (3) geostatistical and cartographic analysis, and (4) the integration of environ- mental and infrastructural data to identify high-risk collision zones. All analyses were conducted using ArcGIS 10.0 (ESRI). Avellino covers approximately 2,800 km² of ecologically diverse landscapes, including mountain ranges, woodlands, agricultural areas, and a dense river network. Elevations in the province range from around 200 to over 1,800 masl. Several rivers serve as natural corridors for wild boar move- ment, facilitating their dispersal across the territory. The area also hosts numerous protected zones, including sev- eral Natura 2000 sites (Sites of Community Importance and Special Protection Areas), which support biodiversity but also increase the likelihood of wildlife-vehicle collisions— particularly along roads adjacent to sensitive habitats. The province’s road infrastructure includes national highways, regional routes, and a dense grid of municipal roads, many of which intersect natural and semi-natural areas. Data on wild boar collisions (from 2015 to 2020) were sourced from the Avellino Designated Hunting Area (ATC) and reported at the municipal level. Although precise GPS coordinates were not available for all collisions, spatial patterns were inferred via thematic classification maps and kernel density analysis. Additional spatial data sets, including roads, rivers, protected areas, and administrative boundaries—were obtained from ISPRA and the Campania Region’s geoportal. All layers were standardized and reprojected to UTM Zone 33N (WGS84) to ensure consistency across the analysis. Data Preprocessing The data preprocessing phase was aimed to ensure the accuracy, consistency, and suitability of all datasets, for spa- tial analysis. The first task was to check the completeness of the attribute data for each municipality. Next, municipal boundaries were standardized using the official shapefiles from the Campania Region, ensuring alignment and consis- tency across the study area. Collision data, originally pro- vided in tabular format, were then georeferenced and con- verted into spatial polygon features. These were linked to their corresponding municipalities, allowing the calculation of the total number of wild boar–vehicle collisions (WVCs), between 2015 and 2020, reported in each area. This value was then used to produce a classification map illustrating how collision frequency varies across the province. Kernel Density Estimation and Hotspot Identification To explore spatial clustering of WVCs, Kernel Density Estimation (KDE) was applied using ArcGIS 10.0 with the Spatial Analyst extension. Since precise coordinates were not always available, the analysis focused on municipalities where ten or more collisions had been reported and where spatial locations could either be georeferenced directly or inferred from recurring incident sites in the official records. KDE settings were adjusted to match the geography of the study area: a 500-meter cell size was chosen to provide a detailed output, while a 5,000-meter search radius offered a balance between detection sensitivity and the spatial scale of the road network. The result was a continuous raster heatmap showing collision density. To make the findings accessible to non-specialist audiences, the raster was classified using the Equal Interval method. High-den- sity areas—those falling within the upper quartile—were extracted as vector hotspot polygons. These polygons were then overlaid with key spatial layers, including major and minor road categories, rivers and streams, and protected areas such as Natura 2000 sites and the Monti Picentini Regional Park. 25 Acta Biologica Slovenica, 2026, Special issue Figure 1. Wildlife-vehicle collisions study area in Avellino Province (Italy). Slika 1. Območje raziskave trkov med divjadjo in vozili v provinci Avellino (Italija). Figure 2. Spatial distribution of Wildlife-vehicle collisions in Avellino Province (Italy). Slika 2. Prostorska porazdelitev trkov med divjadjo in vozili v provinci Avellino (Italija). 26 Acta Biologica Slovenica, 2026, Special issue Results Incident Mapping by Municipality The spatial distribution of wild boar–vehicle collisions in the Province of Avellino (Figure 2) shows clear geographic variation in incident frequency. Based on municipal-level data, the choropleth map highlights Conza della Campania as the area with the highest number of reported collisions between 2015 and 2020. Other municipalities with notable counts include Sant’Angelo dei Lombardi, Guardia dei Lombardi, and Pratola Serra. These areas are located near ecologically sensitive zones, such as Sites of Community Importance (SCIs) and Special Protection Areas (SPAs), and are intersected by major roads like the SS7 dir C. The clus- tering of high-incident municipalities along the east-west axis of the southern Apennines points to a recurring pat- tern: key roadways often cross wildlife movement corridors, especially in areas where habitat connectivity remains rela- tively intact (Figure 3). In contrast, municipalities with fewer reported collisions tend to be more urbanized or located in areas where natural habitats are more fragmented and ecological corridors are limited. Hotspot Identification via Kernel Density Estimation (KDE) The Kernel Density Estimation (KDE) analysis, classified using the Equal Interval method, identified prominent col- lision hotspots in Conza della Campania, with extensions into Sant’Andrea di Conza and Sant’Angelo dei Lombardi. These clusters are closely linked to road segments near Natura 2000 sites and ecotonal areas where forest meets farmland. A clear spatial correlation also emerged with major watercourses, particularly the Ofanto and Sabato rivers, underscoring the importance of riparian corridors in facilitating wild boar movement. The SS7 dir C stood out as a key conflict route, intersecting several of the identified hotspot zones. Discussion This study addresses a key aspect of spatial planning, the management and mitigation of wildlife–vehicle collisions (WVCs) risk, which requires close integration between wildlife ecology and land-use management, and which is gaining increasing importance across many parts of Europe, particularly within the Mediterranean basin. By combining municipality-level collision data with hotspot mapping through Kernel Density Estimation (KDE), it refines existing approaches to WVC risk assessment and offers a spatially explicit framework for prioritizing mitigation mea- sures. Methodologically, the integration of thematic maps and KDE enables analysis at multiple scales: choropleth maps reveal broader municipal patterns, while KDE high- lights more localized collision hotspots. This multi-scale perspective aligns with previous GIS-based studies on WVCs involving species such as deer, moose, and wild boar (Bíl et al., 2025). The KDE parameters used here are consistent with those adopted in similar European studies (Diggle, 1985), ensuring both methodological rigor and comparability. Ecologically, the results reinforce well-docu- mented patterns in wild boar behavior. As noted by Morelle et al. (2015) and Thurfjell et al. (2015), wild boars often rely on riparian corridors, forest edges, and human-modified landscapes for movement. The overlap between WVC hotspots and the Ofanto and Sabato rivers confirms the role of watercourses in shaping their dispersal routes. These findings echo earlier work by Rosell et al. (2008) and Pittiglio et al. (2018), which emphasize the risks posed by roads cutting through ecological corridors—especially in rural and peri-urban areas. The Province of Avellino presents a particularly complex scenario. Its fragmented road network, coupled with the proximity of settlements to protected areas, creates conditions that are especially prone to human–wildlife conflict. Similar challenges have been observed in other Italian regions, such as Tuscany (Ferretti et al., 2021). However, the steep terrain and exten- sive coverage of Natura 2000 sites in Avellino complicate the deployment of conventional mitigation strategies like continuous fencing. This highlights the need for targeted, ecologically informed interventions that are sensitive to the landscape context. One of the key contributions of this research is its application of KDE to wild boar collisions in southern Italy—a region still underrepresented in the WVC literature. The identification of the SS7 dir C as a recurrent conflict corridor provides valuable, actionable insight for targeted mitigation. More broadly, the study’s integration of ecological and infrastructural variables within a spatial modeling framework reflects best practices in road ecol- ogy and conservation planning (Ascensão et al., 2017). All spatial analyses were carried out using both proprietary and open-source GIS platforms to ensure transparency 27 Acta Biologica Slovenica, 2026, Special issue Figure 4. Kernel density estimation via Equal Interval classification, in relation to natural protected areas and rivers network of Avellino Province (Italy). Slika 4. Ocena gostote jedra z uporabo klasifikacije z enakimi intervali v povezavi z naravnimi zaščitenimi območji in rečnim omrežjem v provinci Avellino (Italija). Figure 3. Spatial distribution of wildlife-vehicle collisions, in relation to the land area and the length of road networks in each municipality of Avellino Province (Italy). Slika 3. Prostorska porazdelitev trkov med divjadjo in vozili v povezavi s površino ozemlja in dolžino cestnega omrežja v vsaki občini v provinci Avellino (Italija). 28 Acta Biologica Slovenica, 2026, Special issue and replicability. Nonetheless, some limitations must be acknowledged. The absence of GPS-tagged collision data restricted spatial precision, and the likely underreporting of minor incidents may have affected the completeness of the dataset. Future research should incorporate GPS telemetry, camera traps, and remote sensing to improve spatial accu- racy. In addition, socio-ecological variables such as traffic volume, driver behavior, and land-use dynamics were not included in this study, though they are crucial for building more accurate predictive models. Ultimately, this study provides a replicable GIS-based approach for identifying and addressing WVCs in ecologically complex regions. It supports the development of integrated strategies that bring together ecologists, transportation engineers, land- use planners, and local authorities to reduce collision risks and improve landscape connectivity. Conclusions This study demonstrates the utility of a GIS-based landscape approach in identifying and analyzing wild boar-vehicle collision hotspots in the Province of Avellino. By mapping incident frequency and applying KDE with the Equal Interval method, we have delineated high-risk zones where wildlife management and road safety interventions can be focused. The overlap of hotspots with protected areas and hydrographic corridors highlights the ecological dimensions of WVCs. Future work should aim to integrate traffic flow data and improve spatial resolution of incident reporting to refine hotspot detection and guide targeted mitigation efforts. Author Contributions Conceptualization, E.C., N.P., L.E.; methodology, E.C., S.P., N.P., L.E.; software, E.C.; validation, E.C., N.P., L.E.; formal analysis, E.C., S.P., E.S.d.P.; investigation, E.C., N.P., L.E., A.R., R.d.S., G.S., N.B.; resources, N.P., L.E., A.R., R.d.S., G.S., N.B.; data curation, E.C.; writing—original draft preparation, E.C., N.P., L.E.; visualization, E.C.; supervision, E.C., S.P., N.P., L.E.; project administration, N.P., L.E. All authors have read and agreed to the published version of the manuscript. Acknowledgement We acknowledge the offices of the Provincial T erritorial Ser- vice of Avellino - Campania Region Administration (STP-AV) for their technical support and the provision of official data. Funding This research was funded by Ambito Territoriale di Caccia della Provincia di Avellino, grant number 9013 – Con- sulenza Tecnico Scientifica per la Redazione/Rielaborazi- one del Piano Faunistico Venatorio della Provincia di Avel- lino 2019-2023. Data Availability Statement Research data is available at Zenodo (https://doi. org/10.5281/zenodo.21301296). Conflicts of Interest The authors declare no conflict of interest. The study was conducted independently by researchers of University, consequently, no external parties influenced the study design, data collection, analysis, manuscript preparation, or the decision to publish the results. References Amici, A., Serrani, F., Rossi, C. M., & Primi, R., 2018. Wildlife–vehicle collisions in a Mediterranean area: Distribution and identification of hot spots. Environmental Management, 62(3), 558–567. https://doi.org/10.1007/s00267-018-1057-5 Ascensão, F., Desbiez, A.L.J., Medici, E.P., Bager, A., 2017. 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Radiographic study of multiple chronic traumatic lesions of the forelimbs in a wolf (Canis lupus italicus) from the National Park of Abruzzo, Lazio and Molise. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.23771 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Radiographic study of multiple chronic traumatic lesions of the forelimbs in a wolf (Canis lupus italicus) from the National Park of Abruzzo, Lazio and Molise Dario Costanza 1 * , Nadia Piscopo 2 , Maria Pia Pasolini 2 , Mariaelena de Chiara 2 , Serena Panetta 3 , Vincenza di Pirro 4 , Leonardo Gentile 4 , Luciano Sammarone 4 , Micheletino Matarazzo 1 , Adelaide Greco 1 , Leonardo Meomartino 1 Abstract In November 2023, a 5-month-old female Apennine wolf (Canis lupus italicus) was recovered from the Umbria region and transferred to the wildlife area of the Abruzzo, Lazio and Molise National Park. After an initial acclimatisation period in isolation, she was introduced into the common enclosure with the resident pack. In August 2024, she developed severe lameness in the left forelimb. A radiographic examination performed in October of the same year revealed a transverse diaphyseal fracture of the left radius and ulna, accompanied by marked periosteal reaction, moderate bone resorption, and surrounding soft tissue swelling. Additionally, a luxation of the third digit’s metacarpophalangeal joint in the right manus was observed. These findings were consistent with chronic traumatic injuries, fracture and luxation, associated with periostitis and osteomyelitis, likely caused by bites from other wolves. Despite the severity, the fractures were stable, and the right manus lesion did not impair weight-bearing. A conservative treatment approach was chosen. The wolf has since made a full clinical recovery but has not yet been reintegrated into the pack. Keywords Wildlife management; intraspecific aggression; diagnostic imaging; captive carnivores; conservation medicine 31 Acta Biologica Slovenica, 2026, Special issue Introduction Wolves are highly social animals that live in packs, extended family groups typically composed of a breeding "alpha" pair and their subordinate offspring, including pups from one or more years (Packard 2012). In recent years, wolf populations in Europe have grown significantly, nearly doubling from 11,193 individuals in 2012 to 20,300 in 2023 (La Morgia et al., 2022). This demographic expansion has led to more fre- quent interactions and, in some cases, conflicts with human activities, including livestock farming and land use, thereby complicating conservation and management efforts. Until recently, the wolf was listed under Annex IV of the EU Habitats Directive (92/43/EEC; European Council, 1992), which designates species of "strict protection." This classifi- cation prohibits any deliberate killing, disturbance, capture, or destruction of breeding sites and resting places, and imposes strong limitations on management interventions, even when population control or damage prevention may be necessary. However, on December 3, 2024, the Council of Europe’s Bern Convention Standing Committee proposed downlisting the wolf from “strictly protected” (Appendix II) to “protected” (Appendix III) (Council of Europe, 2024). Subsequently, on March 7, 2025, the European Parliament officially reclassified the species from Annex IV to Annex V of the Habitats Directive (FACE, 2025). Annex V includes species of community interest whose taking in the wild and exploitation may be subject to management measures. This shift allows member states greater flexibility in implement- ing population control, damage mitigation, and coexistence strategies, provided that such actions are sustainable and compatible with maintaining populations at a favourable conservation status. Within wildlife parks, wolves are often kept in semi-nat- ural enclosures that allow for the expression of social behaviours like those observed in the wild. However, close and prolonged contact within confined spaces can lead to episodes of intraspecific aggression, especially during the establishment or reorganisation of dominance hierar- chies. These encounters may result in injuries such as bite wounds, fractures, and joint damage. Continuous monitor- ing by trained personnel enables timely detection of such interactions, facilitating prompt veterinary assessment and appropriate intervention to ensure animal welfare and group stability (Piscopo et al., 2021). Bone fractures in wolves can be caused by natural causes (ex. prey-inflicted injuries), and mainly by human-re- lated activities (ex. traffic accidents, illegal trapping) or captures for scientific purposes (Mech & Boitani 2003, Arnemo et al. 2006, Frame & Meier 2007), which could result in death or impaired locomotor function, which can hamper the wolf's biological requirements for covering Rentgenska preiskava večkratnih kroničnih travmatskih poškodb sprednjih okončin pri volku (Canis lupus italicus) iz Narodnega parka Abruzzo, Lazio in Molise Izvleček Novembra 2023 so v regiji Umbrija našli petmesečno samico apeninskega volka (Canis lupus italicus) in jo premestili v območje za prostoživeče živali v Narodnem parku Abruzzo, Lazio in Molise. Po začetnem obdobju privajanja v izolaciji so jo vključili v skupno ogrado s tamkajšnjim volčjim tropom. Avgusta 2024 se je pri njej pojavila huda šepavost v levi sprednji okončini. Rentgenska preiskava, opravljena oktobra istega leta, je pokazala prečni diafizealni zlom levega radiusa in ulne, ki ga je spremljala izrazita periostalna reakcija, zmerna kostna resorpcija in oteklina okoliškega mehkega tkiva. Poleg tega je bila ugotovljena luksacija metakarpofalangealnega sklepa tretjega prsta na desni sprednji okončini. Ti izvidi so bili skladni s kroničnimi travmatskimi poškodbami, zlomom in luksacijo, povezanimi s periostitisom in osteomielitisom, ki so jih verjetno povzročili ugrizi drugih volkov. Kljub resnosti so bili zlomi stabilni, poškodba na desni sprednji nogi pa ni ovirala nosilnosti. Izbrali so konzervativni pristop k zdravljenju. Volkulja je popolnoma klinično okrevala, vendar še ni bila ponovno vključena v trop. Ključne besede Upravljanje divjih živali; medvrstna agresija; diagnostično slikanje; mesojedci v ujetništvu; ohranitvena medicina 32 Acta Biologica Slovenica, 2026, Special issue wide ranges and hunting large prey. However, there are studies based on osteological collections from free-rang- ing wolves demonstrating successful ossification of old fractures (Wobeser 1992, Kreeger 2003). As there are few data published concerning traumatic skeletal lesions and their treatment in wolves (Silva, 2016), in this case report, a thoracic limb fracture repair in a free-ranging Iberian Wolf is presented with its successful rehabilitation and release into the wild. Thanks to the availability of portable digital radiography equipment, it is now possible to perform diagnostic imaging directly in the field. This avoids the need to transport ani- mals to specialised facilities, thereby reducing stress and allowing for more efficient on-site management of injuries when feasible. The aim of this case report is to describe the radiographic findings and clinical management of a trauma- tised wolf (Canis lupus italicus) from the Parco Nazionale di Abruzzo, Lazio and Molise, Italy. Materials and Methods In November 2023, a 5-month-old female Apennine wolf (Canis lupus italicus) was recovered from the Umbria region and transferred to the “Area faunistica” of the Parco Nazionale d’Abruzzo, Lazio e Molise in Civitella Alfedena (Abruzzo, Italy), a fenced enclosure of approximately 4 hectares housing a resident pack of 13 wolves. During the initial acclimatisation period, the young wolf was kept separated from the pack in a dedicated, enclosed area. After this initial period, she was introduced into the resident group. At the end of August 2024, she developed severe lameness in the left forelimb and was once again isolated in the separate enclosure for monitoring. On October 29th, 2024, a radiographic examination was carried out on site to investigate the reason for the lameness. Sedation was achieved via teleanesthesia, using remote darting with a CO₂-powered rifle to administer intramuscularly 0.06 mg/kg of medetomidine (Medetor, Virbac Italia, Milano, Italy) and 4.5 mg/kg of ketamine (Ketavet 100, MSD Italia, Roma, Italy). Following sedation, a thorough physical examination was performed. Radiographic imaging was then performed on the sedated animal using a portable, wireless X-ray device (Econet MeX+20 BT, Medical Econet, Oberhausen, Ger- many) coupled with a digital radiography detector (AGFA Dura-line XD 14, AGFA HealthCare, Mortsel, Belgium). Radiographic studies of both the left and right antebrachii and manii were performed in mediolateral (ML) and cranio- caudal/dorsopalmar (CrCa/DPa) projections (Fig.1). Additional radiographic projections of the skull, thorax, abdomen, pelvis, and hindlimbs were also obtained to rule out other potential causes of lameness, as well as the presence of other lesions that were not evident on visual inspection and/ or clinical examination. For all the examined regions, the focus-to-cassette distance was approximately 100 cm. The X-ray device was operated by one of the authors (L.M.), a veterinary radiologist with over 25 years of experience. All standard radiation safety personal protective equipment— including lead apron, protective gown, thyroid shield, leaded eyewear, and a personal dosimeter—was appropriately employed to ensure operator safety and minimise radiation exposure. The acquired radiographs were reviewed on-site by the same operator immediately after acquisition using a 14-inch MacBook Pro (Apple Inc., Cupertino, CA, USA). Results The clinical examination revealed significant soft tissue swelling in the left antebrachium and right manus, although no abnormalities in joint mobility or range of motion were detected. The radiographic study revealed a minimally displaced transverse diaphyseal fracture of the distal third of the left radius and ulna, with marked periosteal reaction, moder- ate bone resorption at the fracture line, and surrounding soft tissue swelling (Fig. 2). The distal fracture fragments showed mild cranial displacement (procurvatum). Although the fracture margins appeared partially apposed, no radiographic evidence of bridging callus formation was observed, but the fracture ends were still open. Addition- ally, a marked, mixed periosteal reaction—comprising both solid and spiculated components—was present along the adjacent cortices, accompanied by pronounced sclerosis of the cancellous bone and significant locoregional soft tissue swelling, particularly surrounding the fracture site. In the right manus (Fig. 3), a luxation of the metacar- pophalangeal joint of the third digit was observed, accom- panied by a pronounced periosteal reaction along the first phalanx, irregular mineralisation, soft tissue swelling, and a mild increase in medullary canal opacity in the correspond- ing metacarpal bone. No additional radiographic lesions were detected in the remaining examined body regions. 33 Acta Biologica Slovenica, 2026, Special issue Figure 1. Mediolateral projection of the right forelimb. The wolf was positioned in right lateral recumbency on a soft cloth, with the radio- graphic detector plate placed directly beneath the right forelimb and centred over the area of interest. The contralateral (left) forelimb was pulled caudally and held with a band to prevent superimposition of skeletal structures during image acquisition. Slika 1. Mediolateralna projekcija desne sprednje okončine. Volk je ležal v desnem bočnem položaju na mehki tkanini, rentgenska detek- torska plošča pa je bila nameščena neposredno pod desno sprednjo okončino in poravnana s preučevanim območjem. Kontralateralno (levo) sprednjo okončino so potegnili v kaudalno smer in jo pritrdili z trakom, da bi preprečili prekrivanje skeletnih struktur med zajemanjem slike. 34 Acta Biologica Slovenica, 2026, Special issue Figure 2. (A) Mediolateral and (B) craniocaudal projections of the left antebrachium. The distal fracture fragments show mild cranial displace- ment (procurvatum). Although the fracture margins are partially apposed, there is no radiographic evidence of bridging callus formation, but the fracture ends appear still open. A marked, mixed periosteal reaction—both solid and spiculated—is present along the adjacent cortices, accompanied by pronounced sclerosis of the underlying cancellous bone and significant locoregional soft tissue swelling. Abbreviation L, left. Slika 2. (A) Mediolateralna in (B) kraniokaudalna projekcija levega predlaktišča. Distalni fragmenti zloma kažejo blago kranialno premikanje (procurvatum). Čeprav so robovi zloma delno stisnjeni skupaj, na rentgenski sliki ni vidnih znakov nastajanja mostičastega kalusa, konca zloma pa se zdita še vedno odprta. Ob sosednjih kortikalnih plasteh je prisotna izrazita, mešana periostalna reakcija – tako trdna kot špi- casta –, ki jo spremlja izrazita skleroza pod njo ležeče spongiozne kosti ter znatno lokoregionalno otekanje mehkih tkiv. Okrajšava L, levo. 35 Acta Biologica Slovenica, 2026, Special issue Figure 3. (A) Mediolateral and (B) dorsopalmar projections of the right manus. Luxation of the metacarpophalangeal joint of the third digit is evident. There is associated periosteal reaction along the first phalanx, mild increase in opacity of the medullary canal in the corresponding metacarpal bone and locoregional soft tissue swelling. Abbreviation R, right. Slika 3. (A) Mediolateralna in (B) dorsopalmarna projekcija desne roke. Vidna je luksacija metakarpofalangealnega sklepa tretjega prsta. Opazna je spremljajoča periostalna reakcija vzdolž prve falange, blago povečano motnost kostnega mozga v ustreznem metakarpalnem kosti ter lokoregionalni otek mehkih tkiv. Okrajšava R pomeni desno. 36 Acta Biologica Slovenica, 2026, Special issue Discussion Radiographic findings were consistent with chronic, delayed-union fractures of the left radius and ulna (Henry and Cole, 2018), along with a chronic luxation of the third metacarpophalangeal joint of the right forelimb. Both con- ditions were associated with periostitis and osteomyelitis, most likely resulting from intraspecific aggression, specifi- cally bite injuries inflicted by other wolves in the pack. The use of on-site radiographic imaging proved to be particu- larly advantageous in this case, as it allowed for a prompt and accurate diagnosis without the need to transport the animal outside of its familiar environment. Performing the examination in situ reduced the risks associated with stress and handling, which are particularly relevant in wild species such as wolves. The availability of portable digital radiographic equipment represents a valuable diagnostic tool in wildlife medicine, especially in cases where clinical conditions do not warrant hospitalisation or referral to specialised veterinary facilities. This approach facilitates decision-making while respecting animal welfare and mini- mising the potential for additional trauma or stress. Despite the severity of the lesions, particularly the radial-ulnar fracture, the fracture site exhibited discrete stability at the time of evaluation. Additionally, although the lesion of the right manus was advanced and involved the third digit, it did not appear to compromise weight-bear- ing or basic locomotion. Given the chronic nature of the injuries, the absence of systemic clinical signs, the relative mechanical stability of the fractures, and the challenges involved in surgically managing a wild animal, especially in a semi-captive environment, a conservative, non-surgical treatment approach was deemed the most appropriate course of action. This decision was further supported by the animal’s overall clinical condition, as well as the feasibility of continued monitoring within the controlled environment of the wildlife enclosure. At the time of writing this manuscript, the wolf has made a full clinical recovery, but she has not yet been reintegrated into the pack. Moreover, this case highlights the importance of closely monitoring individuals introduced into established wolf packs within managed or semi-natural enclosures. Wolves are highly territorial and socially structured animals; the introduction of a new member, particularly one not born within the group, can lead to social tension and aggression (Fox et al., 1974). Individuals who are not fully integrated or are perceived as subordinate or foreign by the dominant members may become targets of repeated attacks. For this reason, it is essential to implement rigorous behavioural observation protocols during and after the introduction phase. Early identification of incompatibilities or signs of conflict can help prevent serious injuries and improve long- term outcomes for both the introduced individual and the stability of the resident pack. Conclusions This case underscores the clinical and welfare benefits of portable radiography in the management of wildlife species, allowing for accurate, low-stress diagnosis and informed decision-making directly in the field. Conser- vative management proved effective in ensuring clinical recovery despite the severity of the lesions, highlighting its suitability in semi-captive wolves when surgical inter- vention is impractical. Finally, the case emphasises the critical need for careful behavioural monitoring during pack introductions to mitigate the risk of intraspecific aggression and safeguard both individual and group welfare. Author Contributions Conceptualization, L.M; D.C.; N.P. and A.G.; methodology, D.C.; L.M; investigation, L.M.; N.P; A.G.; M.P.P.; M.d.C; S.P.; V.d.P; L.G.; L.S.; M.M.; resources, L.M,; M.P.P; S.P.; data cura- tion, D.C.; L.M.; writing—original draft preparation, D.C. and L.M; writing—review and editing, L.M.; N.P; A.G.; M.P.P.; M.d.C; S.P.; V.d.P; L.G.; L.S.; M.M.; visualization, D.C.; super- vision, L.M.; A.G. and M.P.P project administration, L.M. and N.P.; funding acquisition, L.M. and A.G. All authors have read and agreed to the published version of the manuscript. Acknowledgement We acknowledge the National Park of Abruzzo, Lazio and Molise. Funding This research received no external funding. Data Availability Statement The research data is deposited at Zenodo (https://doi. org/10.5281/zenodo.21301361). Conflicts of Interest The authors declare no conflict of interest. 37 Acta Biologica Slovenica, 2026, Special issue References Arnemo, J. M., Ahlqvist, P., Andersen, R., Berntsen, F., Ericsson, G., Odden, J., ... Swenson, J. E. (2006). Risk of capture-related mortality in large free-ranging mammals: Experiences from Scandinavia. Wildlife Biology, 12(2), 109–113. https://doi.org/10.2981/0909-6396(2006)12[109:ROCMIL]2.0.CO;2 European Council. (2024, December 3). Bern Convention Standing Committee approves EU proposal to modify wolf protection. https://www.coe.int/en/web/ portal/-/bern-convention-standing-committee-approves-eu-proposal-to-modify-wolf-protection Federation of Associations for Hunting and Conservation of the EU. (2025, June 13). 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Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.29131 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note A complex network: wild boar, water and biodiversity Luigi Esposito 1 * , Mariaelena de Chiara 1 , Marcello Stefanucci 2 , Massimo Mazzone 2 , Camillo Campolongo 3 , Ivan Maraviglia 3 , Nadia Piscopo 1 Abstract The study was conducted in the city of Benevento (Italy), a research site for the Horizon "OneAquaHealth" project (www.oneaquahealth). Camera traps were installed in 18 streams within the urban ecosystem to identify the presence and family structure of wild boars. Images were analysed from January to July 2023, and the results allow us to identify the presence of wild boar family groups or solitary animals. The number of wild boars counted in the study area shows an increasing trend from January to June. From a one health perspective, the study aimed to demonstrate that each wild boar is accompanied by numerous internal and external parasites that it will spread throughout the area and to other living organisms. Based on the literature, the organisms associated with wild boars include ecto and endoparasites, bacteria, and viruses. Currently, 26 ectoparasites (10 types of ticks, 11 types of mosquitoes, 3 types of flies, 1 flea, 1 louse, 1 mite) and 10 endoparasites (1 nematode, 4 trematodes, 2 tapeworms, 3 protozoa) can be described. Potential causative agents of disease in wild boar include 29 bacteria and 6 viruses. No fungal skin infections have been described. Finally, a microbiome analysis indicates the presence of 65 bacteria in the large intestine. Keywords One Health, Urban aquatic ecosystems, ecological net, Sus scrofa, biological index 39 Acta Biologica Slovenica, 2026, Special issue Introduction The interest that European society and politics have paid to large wild animals in the 20th century has focused mainly on the conflict with man (Chapron et al., 2014; Piscopo et al., 2021). Numerous descriptions have been provided regarding the patterns and quantification of livestock predation by wolves (Canis lupus), bears (Ursus arctos) and lynxes (Lynx lynx) (Amici et al., 2012), but even more attention has been paid to species such as wild boars (Sus scrofa). For this one, despite no reliable data on population size (Piscopo et al., 2023; 2025; Verde et al., 2025), the EU reports a growing population (Saez-Royuela & Telleriia, 1986; Massei et al., 2015; Boronyak et al., 2022). The negative pressure exerted by wild boars on ecosystems, agriculture and urban environments (Chapron et al., 2014; Piscopo et al., 2021) has led to the inclusion of wild boars in the blacklist of the 100 most harmful invasive species in the world (Lowe et al., 2000). Most studies on wild boars concern their association with damage to agriculture (Amici et al., 2012; Laznik et al., 2014), road accidents (Primi et al., 2009; Kruuse et al., 2016) and urban invasions (Castillo-Contreras et al., 2021). Due to their ability to occupy any type of habitat (Apollonio, Randi & T oso, 1988), wild boars are accompanied by numerous inter- nal (Pilarczyk et al., 2024) and external (Piscopo et al., 2025) parasites, including 26 ectoparasites (10 types of ticks, 11 types of mosquitoes, 3 types of flies, 1 flea, 1 louse, 1 mite) (Fila & Woźniakowski, 2020; Guardone et al., 2023; Matei et al., 2023; Piscopo et al., 2025) and 10 endoparasites (1 nem- atode, 4 trematodes, 2 tapeworms, 3 protozoans) (Mansouri et al., 2016; Ozolina et al., 2020; de-la-Rosa-Arana, 2021; Power et al., 2023); 29 bacteria and 6 viruses (Ruiz-Fons et al., 2008; Perrat et al., 2022; Radulski et al., 2022; Ferrara et al., 2024). Three fungal skin infections have been described (Mancianti et al., 1997). Finally, a microbiome analysis indi- cates the presence of 65 bacteria in the large intestine (Wei et al., 2022; Rahman et al., 2024). This work is part of the Horizon 2020 project "OneA- quaHealth," whose objectives are: i) to identify as many living species as possible associated with urban aquatic ecosystems; ii) to describe, from a One Health perspective, the interconnections between biodiversity, aquatic ecosys- tems, and citizens. The study was conducted in the city of Benevento (Italy), one of the project's five sampling sites (www.oneaquahealth), and aimed to describe most of the organisms that coexist with wild boars in the waterways within the urban context of Benevento. Kompleksne interakcije: divji prašiči, voda in biotska raznovrstnost Izvleček Raziskava je potekala v mestu Benevento (Italija), ki je raziskovalno območje v okviru projekta Horizon »OneAquaHealth« (www.oneaquahealth). V 18 potokih znotraj mestnega ekosistema so bile nameščene fotopasti, s katerimi se je želelo ugotoviti prisotnost divjih prašičev in strukturo njihovih družin. Slike so bile analizirane v obdobju od januarja do julija 2023, rezultati pa omogočajo ugotavljanje prisotnosti družinskih skupin divjih prašičev ali posameznih živali. Število divjih prašičev, preštetih na območju študije, kaže naraščajoči trend od januarja do junija. Cilj študije »One Health« je bil pokazati, da vsakega divjega prašiča spremljajo številni notranji in zunanji zajedalci, ki se bodo širili po celotnem območju in na druge žive organizme. Glede na literaturo med organizme, povezane z divjimi prašiči, spadajo ektoparaziti in endoparaziti, bakterije ter virusi. Trenutno je mogoče opisati 26 ektoparazitov (10 vrst klopov, 11 vrst komarjev, 3 vrste muh, 1 bolha, 1 uš, 1 pršica) in 10 endoparazitov (1 nematoda, 4 trematode, 2 trakulje, 3 protozoji). Med potencialnimi povzročitelji bolezni pri divjih prašičih je 29 bakterij in 6 virusov. Glivičnih okužb kože ni bilo opisano. Nazadnje analiza mikrobioma kaže na prisotnost 65 bakterij v debelem črevesu. Ključne besede One Health, mestni vodni ekosistemi, ekološka mreža, Sus scrofa, biološki indeks 40 Acta Biologica Slovenica, 2026, Special issue Materials and Methods Study Area The study area considered the urban characteristics of the city of Benevento and the entire province of Sannio. The latter comprises 78 municipalities of varying demographic size and urban structure (only three municipalities exceed 10,000 inhabitants, including Benevento with 55,319 (as of June 30, 2025); the remaining municipalities have popula- tions ranging from 5,000 to less than 2,000 inhabitants). A significant portion of the population lives in rural areas, isolated hamlets, and scattered homes, a condition that gives the urban system of the Benevento area a distinctly unique character (Bencardino, 2009). The perimeter of the Benevento urban system has a radial shape, starting from the capital and branching out toward the other municipalities in the province. To define and characterise the Benevento urban system, several indicators were chosen, including areas of building continuum, population density, and the distribution of socioeconomic activities. The intersection of these indicators allows us to define a complex urban system characterised by continuous housing, a relatively high popu- lation density with the presence of communication structures, and portions of territory with homogeneous socioeconomic characteristics. The urban system thus defined consists of a higher-level urban area, comprising the city of Benevento, three main development routes (the Caudina route, the Benevento-Ceppaloni-Arpaise route, and the Medio Calore route), and a diffused development area (called the Territorial Area of Diffused Settlement Development) to the southeast of the capital. Complemented by two peripheral systems, definable based on their geographic location, the Northwest Peripheral System and the Northeast Peripheral System. Therefore, due to the characteristics identified, the system presents itself as a "radial or constellation-like urban structure" that to varying degrees involves 20 other municipal areas (Bencardino, 2009). The urban structure of Benevento thus encompasses citizens and residents of the historic centre of Benevento, the Madonna delle Grazie area, Viale Mellusi, parts of the Rione Ferrovia and Rione Libertà districts, and the Pacevecchia and Capodimonte neighbourhoods. As one moves toward the outskirts of the capital, in the area south of Benevento, along the Medio Calore road, in the municipalities of Apice, Torrecuso, and the Tufara area, one encounters tertiary sector workers, manufacturing, and commercial construction companies, which foster a system undergoing suburbanization. Industrial and tertiary activities appear to be fairly recent in the areas of Ponte Valentino and Paduli, Contrada Olivola and Contrada Tor- repalazzo, straddling the municipalities of Benevento and Paduli, and the Caudina road, where various commercial and manufacturing businesses are located. Furthermore, the percentage of people employed in agriculture is sub- stantially lower than the provincial average in almost all the municipalities that are part of the Benevento urban system. Data Sources, Collection and Preprocessing Between January 1, 2023, and December 31, 2024, in col- laboration with local allies in the OneAquaHealth project (LIPU, WWF, and ATC Benevento), 32 camera traps (one per bank) were installed on 16 waterways (approximately 25,000 hectares). 25,038 minutes of time and 50,076 images were analysed, recorded on transects of 50 linear meters each. All images captured by the camera traps allowed the identification of the animals that passed along the sample streams. Within the area studied, nine wild boar hunting teams were contacted through the responsible veterinarians (Figure 2); hunters provided the various samples on which the surveys were conducted. All data were archived in Excel files and then processed cumulatively over the two years of observation. Qualitative observations were subjected to the chi-square test. Results Estimated wild boar population A comparison between the two study years shows a popu- lation growth trend from January to August (Figure 3), with an increase of approximately 248 animals. Calvings begin in January and peak in May and June. At the end of summer, a slight decrease is observed, estimated at approximately 7%, due to increased mortality due to climatic factors (excessive heat) and the reduction of trophic components. The numerical decline is significantly more marked (47%) in the subsequent months of October, November, and December, when wild boar hunting is permitted in Italy. With respect to age classes, in both years, the highest percentage calculated on the total number of animals 41 Acta Biologica Slovenica, 2026, Special issue Figure 1. The eighteen watercourses surveyed at the Benevento OAH research site (https://www.oneaquahealth.eu/). Slika 1. Osemnajst vodotokov, ki so bili raziskani na raziskovalnem območju OAH v Beneventu (https://www.oneaquahealth.eu/). Figure 2. Special Wild Boar Hunting Areas in the OneAquaHealth Study Area (Fonte https://www.campaniacaccia.it/ modificato OAH). Slika 2. Posebna območja za lov na divje prašiče na območju študije OneAquaHealth (vir: https://www.campaniacaccia.it/, prilagojeno s strani OAH). 42 Acta Biologica Slovenica, 2026, Special issue reported in the study area is attributable to striped (40.02%), followed by adult females (33.63%) and subadults (23.80%). Adult males represent only 2.55% (Table 1). The percentage of adult males is significantly different (P<0.01) from the other three age classes, which, on the whole, do not differ from each other. The age-class analysis shows a negative correlation (-0.63) between the number of adult females (R 2 =0.70) and the striped wild boar piglets (R 2 =0.39). A positive correla- tion (0.94) is found between adult females and subadult individuals (R 2 =0.46). Finally, the correlation is still negative (-0.43) between the striated individuals and subadult individuals (Figure 4). Figure 4 shows the distribution of the different age groups by month. It is clear that striped bears are most present between January and September, peaking in May, June, and July. They are completely absent in October, November, and December. Along with the decline in striped bear numbers, there is an increase in the number of adult females, benefiting from the subadults reaching puberty. These, if spared from hunting, contribute to the fertility of the groups and the persistence of the population into the following year. The presence of adult males is almost constant throughout the year and decreases during the months when hunting is open. Figure 3. Increasing trend of the wild boar population from months (2023 and 2024). Slika 3. Naraščajoči trend populacije divjih prašičev v posameznih mesecih (2023 in 2024). 2023 2024 Total Age classes Average n. ± SD % Average n. ± SD % Average n. ± SD % Striped 60.83±42.18 38.85 Aa 123.58±90.69 40.62 A 184.42±132.27 40.02 A Adult female 52.00±19.23 33.21 A 103.00±58.64 33.85 AB 155.00±75.21 33.63 A Subadult 39.33±17.37 25.12 Ab 70.33±11.54 23.12 B 109.67±24.50 23.80 A Adult male 4.42±1.78 2.82 B 7.33±3.89 2.41 C 11.75±5.61 2.55 B All 156.58±38.20 304.30±74.38 460.83±112.43 Table 1. The number of wild boars counted with camera trapping in the years 2023 and 2024, divided by age classes. Tabela 1. Število divjih prašičev, preštetih s foto-pastmi v letih 2023 in 2024, razdeljeno po starostnih razredih. 43 Acta Biologica Slovenica, 2026, Special issue Estimate of wild boar hosts During the 2023-2024 hunting season (October-Decem- ber 2023), 65 wild boars (22 males and 45 females) were analysed, while in the following 2024-2025 hunting season (October-December 2024), 70 wild boars (22 males and 48 females) were analysed. Sampling of the 135 wild boars killed allowed the identification of the living organisms associated with the ungulate. The results identified: Five types of ectoparasites: Ticks 2 types (Dermacentor reticulatus, Dermacentor marginatus); Fly 1 type (Hippobosca equina); Louse 1 type (Haematopinus suis); Mite 1 type (Sarcoptes scabiei). Five types of endoparasites: Nematodes 7 types (Eucoleus garfiai, Ascaris suum, Oesophagostomum dentatum, Hyostrongylus rubidus, Strongyloides ransomi, Metastrongylus apri, Trichuris suis); Protozoan 1 type (Sarcocystis miescheriana). Coccidia 3 species (Eimeria spinosa, Eimeria scabra, Isos- pora suis). Intestinal Bacteria 65 Genera (Acidaminococcus, Actinobacil- lus, Akkermansia, Anaeroplasma, Anaerostipes, Anaerovib- rio, Anaerovorax, Bacillus, Bacteroidales, Bacteroides, Bifidobacterium, Bilophila, Blautia, Bulleidia, Campylobacter, Clostridium, Collinsella, Coprobacillus, Coprococcus, Corio- bacteriaceae, Dehalobacterium, Desulfovibrio, Dialister, Dorea, Elusimicrobiaceae, Enterococcus, Epulopiscium, Eubacterium, Faecalibacterium, Fibrobacter, Fusobacterium, Lachnobacterium, Lachnospira, Lactobacillus, Megaspaera, Methylobacterium, Mitsuokella, Mycoplasma, Oscillospira, Paludibacter, Parabacteroides, Paraprevotella, Paraprevotel- laceae, Peptostrptococcus, Phascolarctobacterium, Plano- coccaceae, Prevotella, Pseudobutyrivibrio, Pyramidobacter, Roseburia, Ruminococcus, Sharpea, Slackia, Solibacillus, Sphaerochaeta, Sporobacter, Sporosarcina, Streptococcus, Succinivibrio, Sutterella, Syntrophomonas, Treponema, Turicibacter, vadin CA 11, Veillonella). Discussion The study demonstrates that wild boars are accompanied throughout their lives and in their habitat by numerous internal and external hosts that can spread throughout the area and migrate to other living organisms depending on their life cycles. Although sampling was limited to the autumn-winter months (October-December), from a One Health perspective, the information provided by wild boars, used as indicators of the health of aquatic ecosystems, has allowed us to describe ectoparasites, endoparasites, and Figure 4. Distribution of age groups by month (2023–2024). Slika 4. Porazdelitev starostnih skupin po posameznih mesecih v letu (2023–2024). R ² = 0.3936 R ² = 0.6963 R ² = 0.4601 0 50 100 150 200 250 300 350 St ri ped Females Subadults 44 Acta Biologica Slovenica, 2026, Special issue bacterial vectors or potential causative agents of diseases in wild boars and humans. This study addresses a key aspect of the complexity of life in urban aquatic environments and aims to highlight how the presence of wildlife in peri-urban or urban envi- ronments can help us understand the close interactions between ecosystems and human land-use management. Given the recent increasing importance of the presence and distribution of wild boar in many parts of Europe, particularly in the Mediterranean basin, the Benevento model proposes wild boar as an indicator of urban aquatic ecosystem health. The study refines existing approaches to identifying wild boar commensals and, by combining data on encountered biodiversity, offers a framework that allows us to prioritise disease prevention from a One Health perspective. Conclusions The model proposed in this study does not aim to quantify the wild boar's diverse commensals but rather highlights a complexity that requires a prospective, multi-scale and multidisciplinary study of the diverse life forms hosted by the mammal. The parameters used by the OneAquaHealth project are based on methodological rigour and compara- bility with similar studies. Our results reinforce well-docu- mented patterns in wild boar behaviour, which often use stream banks as riparian corridors for their movements. The survey conducted on the 16 streams surrounding and within the city of Benevento confirms the role of waterways in the choice of travel routes used by animals to obtain food and water or to escape hunting. The experiences gained by animals that survive hunting and the reduction of living spaces push them increasingly towards rural and peri-urban areas, increasing the health risks linked to the proximity and exchange of possible pathogens from the environment to humans and vice versa. Author Contributions Conceptualization, N.P., L.E.; methodology, N.P., L.E.; soft- ware, M.d.C.; validation, N.P., L.E.; formal analysis, N.P., M.d.C.; investigation, N.P., L.E., M.d.C., M.S., M.M., C.C., I.M.; resources, N.P., L.E.; data curation, N.P., M.d.C.; writing— original draft preparation, N.P., L.E.; visualization, M.d.C., M.S., M.M., C.C., I.M.; supervision, N.P., L.E.; project admin- istration, N.P., L.E. All authors have read and agreed to the published version of the manuscript. Acknowledgement We acknowledge the volunteers of the Local Allies of the OneAquaHealth project (MoGAE, LIPU, WWF, ATC Benevento) who contributed to the field sampling. Funding “This research was funded by the European Union Horizon- CL6-2022-GOVERNANCE-01-09, grant number 101086521 – Protecting Urban Aquatic Ecosystems to Promote One Health -OneAquaHealth”. Data Availability Statement The research data is deposited at Zenodo (https://doi. org/10.5281/zenodo.21310798). 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Comparison of changes in gut microbiota in wild boars and domestic pigs using 16S rRNA gene and metagenomics sequencing technologies. Animals, 12(17), 2270. https://doi.org/10.3390/ani12172270 46 1 Veterinary Clinical Hospital, University of León, Spain 2 Department of Animal Health, University of León, Spain 3 INDEGSAL, University of León, Spain 4 Department of Biomedical Sciences, University of León, Spain 5 Department of Animal Production, University of León, Spain * Corresponding author: E-mail address: fabianaesposito.vet@gmail.com Citation: Esposito, F., Gómez-Martínez, S., Pérez-Pérez, L., Merino, J., Argüello, H., Carvajal, A., González-Eguren, V. (2026). Prevalence of hindgut pathogens Brachyspira hyodysenteriae and Salmonella enterica in wild boars in northwest Spain. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.29132 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Prevalence of hindgut pathogens Brachyspira hyodysenteriae and Salmonella enterica in wild boars in northwest Spain Fabiana Esposito 1 * , Samuel Gómez-Martínez 2,3 , Lucía Pérez-Pérez 2,3 , Javier Merino 4 , Héctor Argüello 2,3 , Ana Carvajal 2,3 , Vicente González-Eguren 5 Abstract Enteric infections in wild boars pose a risk to pig production, public health, and food safety, as some of these pathogens are capable of crossing the species barrier, infecting other suids, other animals, and even humans. The main objective of this research was to determine the prevalence of two relevant hindgut pathogens of swine, Brachyspira hyodysenteriae and Salmonella enterica, in wild boars from hunting reserves in the provinces of Zamora and Valladolid, Spain and to investigate the role of wild boar as a potential reservoir. Our results showed a low prevalence of faecal shedders for both bacteria (3.3 %), and allow us to conclude that wild boars shot in northwestern Spain do not currently play a relevant role as reservoirs for B. hyoddysenteriae and S. enterica. Keywords Swine dysentery; Swine salmonellosis; Spill-over; Domestic-Wildlife interface 47 Acta Biologica Slovenica, 2026, Special issue Introduction The increasing interaction between wild and domestic animal populations poses significant challenges to animal health, food security, and public health. In particular, the ecological adaptability, high reproductive rate, and expand- ing range of wild pigs contribute to their rising population densities in many regions (Massei et al., 2015), increasing the likelihood of direct and indirect contact with domestic swine, particularly in areas where extensive or outdoor pig farming is practiced. Accordingly, the transmission of pathogens between wild pigs (Sus scrofa), including feral swine and wild boar, to domestic pigs represents a growing concern globally. Transmission routes between wild and domestic pigs include direct physical contact, environmental contam- ination, shared water sources, and fomites (Gortázar et al., 2007). Wild pigs can be infected or even become reservoirs for a wide range of viral, bacterial, and parasitic pathogens that can finally spill over into domestic pig pop- ulations (Gortázar et al., 2007; Meng et al., 2009). A clear example is the pivotal role of wild boar in the maintenance and spread of the African swine virus genotype II in the Eurasian pig population (EFSA, 2025). On the other hand, wild pigs can also serve as sentinels in the surveillance of health hazards in the environment (Halliday et al., 2007). Enteric pathogens affecting the large intestine of suids reduce welfare and cause severe diarrhoea or even death in non-treated animals. Highly beta-hemolytic intestinal spirochetes causing swine dysentery, mainly Brachyspira hyodysenteriae, as well as Salmonella enterica subsp. Enterica serovar Typhimurium (S. Typhimurium) are, without a doubt, the most relevant hindgut pathogen among pigs in Spain and Europe. Although there are a number of studies on the prevalence of these pathogens in pigs, little infor- mation is available regarding their relevance in wild pigs. The aim of this study was to investigate the prevalence of B. hyodysenteriae and S. enterica in wild boars hunted in a pig-producing area in northwest Spain. Materials and Methods Sampling area Sampling was carried out in five locations in Castilla y León, in northwest Spain, the largest autonomous region in Spain by area, covering 94,222 km 2 with a population density below 30 inhabitants/km 2 . Castilla y León has an increasing population of wild boars as well as an important porcine production, with more than 4,000 farms housing almost 5,000,000 pigs and a relevant extensive pig production, including free-range farms. The five locations were distrib- uted in two provinces, Zamora (4) and Valladolid (1). Sample collection A total of 30 wild boars shot during hunting activities in Razširjenost patogenov v zadnjem delu črevesja Brachyspira hyodysenteriae in Salmonella enterica pri divjih prašičih v severozahodni Španiji Izvleček Črevesne okužbe pri divjih prašičih predstavljajo tveganje za prašičerejo, javno zdravje in varnost hrane, saj so nekateri od teh povzročiteljev bolezni sposobni prečkati vrstno pregrado ter okužiti druge svinje, druge živali in celo ljudi. Glavni cilj te raziskave je bil ugotoviti razširjenost dveh pomembnih patogenov v zadnjem delu črevesja prašičev, Brachyspira hyodysenteriae in Salmonella enterica, pri divjih prašičih iz lovnih rezervatov v provincah Zamora in Valladolid v Španiji ter preučiti vlogo divjih prašičev kot potencialnega rezervoarja. Naši rezultati so pokazali nizko razširjenost izločanja obeh bakterij s fekalijami (3,3 %) in nam omogočajo sklep, da divji prašiči, ustreljeni v severozahodni Španiji, trenutno ne igrajo pomembne vloge kot rezervoarji za B. hyodysenteriae in S. enterica. Ključne besede Prašičja dizenterija; Prašičja salmoneloza; Prenos okužbe; Stik med domačimi in divjimi živalmi 48 Acta Biologica Slovenica, 2026, Special issue the studied locations were sampled (10 in the province of Valladolid and 20 in the province of Zamora). From each animal, colonic content (CC) was collected by cutting the colon 5 cm distal from the ileocecal junction (Image 1). Sam- ples were collected in zip-lock bags, identified, immediately refrigerated during transport and stored at -80°C at the facilities of the Infectious Diseases Unit of the University of León until processing. Information on sex, approximate weight, and geographic location was collected. DNA extraction and B. hyodysenteriae and S. Enterica detection and quantification A commercial kit (QIAamp® PowerFecal® Pro DNA Kit) was used for DNA extraction, starting with 200 mg of colonic content and following the recommendations of the manufacturer. DNA concentrations were assessed using a NanoDrop ND-1000 v.3.1. spectrophotometer. Detection and quantification of B. hyodysenteriae and S. enterica were carried out using qPCR in a QuantStudio 1 thermal cycler (Applied Biosystems) as previously described (Table 1). An in-house standard curve for each pathogen was used to extrapolate cycle threshold (Ct) values to bacteria/g of CC. Standard curves were prepared using ten-fold serial dilutions of a B. hyodysenteriae (reference strain B204, ATCC31212) or S. Typhimurium pure cultures (range 10 10 - 10 2 ). The detection limit was defined by the linear portion of the standard curve and was set at 10 3 bacteria/mL. Statistical analysis The open-access software Epi Info 7.2.6.0 was used for statistical analysis. Prevalence rates and 95% confidence intervals (CI) were calculated. Figure 1. Study area (in red) and sample collection (colon contents). Slika 1. Območje raziskave (v rdeči barvi) in odvzem vzorcev (vsebina debelega črevesa). Primers Probe Target Reference B. hyodysenteriae Forward 5´- TTG CTA CTG GTT CTT GGC CTG-3´ FAM-5’-CGA AGG CTT AAA ACA AGA AGG A-3’-BHQ NADH oxidase gene Song & Hampson, 2009 Reverse 5´-GAA TGC TTC TAT AAG TTC AAC ACC TAT -3´ S. enterica Forward 5´AGCGTACTGGAAAGGGAAAG-3´ SYBR-GREEN invA gene Kasturi & Drgon, 2017 Reverse 5´ATACCGCCAATAAAGTTCACAAAG-3 Table 1. qPCRs used for the detection of B. hyodysenteriae and S. enterica. Tabela 1. qPCR-metode, uporabljene za odkrivanje B. hyodysenteriae in S. enterica. 49 Acta Biologica Slovenica, 2026, Special issue Results Prevalence of faecal shedders of B. hyodysenteriae was 3.3% (CI 95% 0.08%-17.22%) with only one positive individ- ual out of 30 investigated. A positive sample was collected from a female with an estimated weight of 50 kg, hunted in Pereruela, Zamora. The estimated concentration of B. hyodysenteriae was 2.8 × 10 3 bacteria per gram of CC. Similarly, a single S. Enterica-positive animal was detected, a female weighing 65 kg (approx.), hunted in Gallegos del Río, Zamora. The estimated concentration of S. enterica in the positive sample was 9.0 × 10 3 bacteria per gram of CC. Discussion B. hyodysenteriae is currently a highly relevant pathogen in both intensive and extensive pig production in Spain and Europe. Despite being a strict anaerobic bacterium, B. hyodysenteriae has a significant capacity to remain viable under humid conditions, in the presence of faeces, or in contaminated ponds or soils (Álvarez-Ordóñez et al., 2013). Rodents, ducks, and other hosts such as dogs and/or cats can also participate in the maintenance and spread of the infection both within and between pig farms (Álvarez-Or- dóñez et al., 2013). Among the scarce information in the literature, we can highlight that infection in boars raised under intensive conditions in Spain was described in 2017, but not in free-living adults in southwestern Spain (Vadillo et al., 2017) or in Sweden (Jacobson et al., 2005). However, the prevalence of faecal shedders was similar to our study in free-ranging wild boars hunted in Germany (2.4%) (Reiner et al., 2011) or Australia (8.1%) (Phillips et al., 2009). The concentration of B. hyodysenteriae in the only positive sample detected in our study was less than 10 4 bacteria/g of faeces, a value that, in the case of domestic pigs, has been reported as not associated with inflammatory lesions in the colon or diarrhoea (Pérez-Pérez et al., 2024). In contrast, S. enterica is a ubiquitous bacterium found in a wide variety of environments and animal species. It is a multi-host pathogen with great economic relevance, a significant public and animal health problem, as well as an excellent indicator of the interaction between wild and domestic animals (Navarro-González et al., 2012). Our results are consistent with the prevalence determined in two recent studies in Spain; in 2019, a study on more than 1,000 wild boars shot in Spain determined 2.9% Salmonella faecal shedders (Gil Molino et al., 2019), while a study of wild boars captured in peri-urban areas showed 3% of positive results in faecal samples (Castillo-Contreras et al., 2022). Regarding the concentration of S. Enterica, we can highlight that it was relatively low, indicating with high probability that it corresponds to a carrier animal. Conclusions Apart from deleterious effects due to inflammatory intes- tinal disease, wild boars harbouring hindgut pathogens may play an important role as potential sources of infection for domestic pigs and other susceptible animals. The prevalence of faecal shedders of B. hyodysenteriae and S. enterica found in this investigation is clearly lower than that observed among domestic pigs on swine farms of the investigated area. These results rule out wild boars in the region as an infectious source or reservoir of these patho- gens at the present time and allow us to propose that both infections are most likely spreading to these wild animals from domestic pigs, and not the other way round. Author Contributions Conceptualisation, V.G., H.A. and A.C.; methodology, A.C., H.A., L.P. and S.G.; sampling and laboratory analysis, F.E., Sex Geographical location Female Male Villar de Ciervos (ZA) Montamarta (ZA) Gallegos del Río (ZA) Pereruela (ZA) Villalba de los Alcores (VA) N. wild boars shooted 20 10 2 8 5 5 10 B. hyodysenteriae 5% - - - - 20% - S. enterica 5% - - - 20% - - Table 2. Prevalence of B. hyodysenteriae and S. enterica in colon content (CC) in the studied population of wild boars. Tabela 2. Razširjenost bakterij B. hyodysenteriae in S. enterica v vsebini debelega črevesa (CC) v preučevani populaciji divjih prašičev. ZA: Zamora; VA: Valladolid 50 Acta Biologica Slovenica, 2026, Special issue J.M. and S.G.; writing—original draft preparation, F.E.; writ- ing—review and editing, V.G, H.A., and A.C. All authors have read and agreed to the published version of the manuscript. Acknowledgement We thank the staff in the Infectious Diseases Unit, particu- larly Diana Molina, and the staff at Cárnicas La Culebra for their help and support. Funding Part of this research was performed under the framework of the project SDyMiHO funded by the Spanish Ministe- rio de Ciencia e Innovación (ref PID2019-110662RB-I00). Samuel Gómez-Martínez is granted by the Junta de Castilla y Leon pre-doctoral programme (ORDEN EDU/1868/2022), and Lucía Pérez-Pérez (PRE2020-093762) is supported by the Spanish Ministry of Education. Data Availability Research data was deposited at Zenodo (https://doi. org/10.5281/zenodo.21310872). Conflicts of Interest The authors declare no conflict of interest. References Álvarez-Ordóñez, A., Martínez-Lobo, F. J., Argüello, H., Carvajal, A., & Rubio, P. (2013). Swine dysentery: Aetiology, pathogenicity, determinants of transmission and the fight against the disease. International Journal of Environmental Research and Public Health, 10(5), 1927–1947. https://doi.org/10.3390/ijerph10051927 Castillo-Contreras, R., Marín, M., López-Olvera, J. R., Ayats, T., Fernandez Aguilar, X., Lavín, S., ... & Cerdà-Cuéllar, M. (2022). Zoonotic Campylobacter spp. and Salmonella spp. carried by wild boars in a metropolitan area: Occurrence, antimicrobial susceptibility and public health relevance. Science of the Total Environment, 822, 153444. https://doi.org/10.1016/j.scitotenv.2022.153444 EFSA (European Food Safety Authority), Ståhl, K., Boklund, A. 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Veterinary Record, 181, 145. https://doi.org/10.1136/vr.104348 51 1 Veterinary Clinical Hospital, University of León, Spain 2 Department of Animal Health, University of León, Spain 3 INDEGSAL, University of León, Spain 4 Department of Biomedical Sciences, University of León, Spain 5 Department of Animal Production, University of León, Spain * Corresponding author: E-mail address: fabianaesposito.vet@gmail.com Citation: Esposito, F., Gómez-Martínez, S., Pérez-Pérez, L., Merino, J., González-Eguren, V., Argüello, H., Carvajal, A. (2026). Lawsonia intracellularis infection in wild boars in northwest Spain. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.29133 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Lawsonia intracellularis infection in wild boars in northwest Spain Fabiana Esposito 1 * , Samuel Gómez-Martínez 2,3 , Lucía Pérez-Pérez 2,3 , Javier Merino 4 , Vicente González-Eguren 5 , Héctor Argüello 2,3 , Ana Carvajal 2,3 Abstract Lawsonia intracellularis infection in wild boars poses a risk to swine production and other animals, as this bacterium can cross species barriers, infecting other animals and even causing disease in some. This research aimed to determine the prevalence of faecal shedders among wild boars from game reserves in the provinces of Zamora and Valladolid, Spain, to explore their potential as sources of infection or even reservoirs of L. intracellularis. Colon content samples were collected from hunted wild boars and processed for the extraction of DNA and detection and quantification of L. intracellularis using qPCR. Up to 40% of the animals were faecal shedders, with a higher prevalence among females (55%) than males (10%). According to these results, wild boars culled in northwestern Spain are a significant source of L. intracellularis infection for other suids and other animal species. Keywords Ileitis; Porcine proliferative enteropathy; Spill-over; Domestic-wildlife interface 52 Acta Biologica Slovenica, 2026, Special issue Introduction Lawsonia intracellularis is an obligate intracellular bacte- rium in the family Desulfovibrionaceae that causes porcine proliferative enteropathy in pigs. Clinically affected pigs show anorexia and chronic diarrhoea due to malabsorp- tion and present pathognomonic microscopic lesions of crypt hypertrophy in the distal ileum (Lawson & Gebhart, 2000; Karuppannan & Opriessnig, 2018). Its widespread distribution, with more than 90% of farms worldwide testing positive, together with its negative impact on productive performance, makes L. intracellularis one of the most rele- vant pathogens on swine farms nowadays. While initially described and extensively studied in pigs, L. intracellularis has demonstrated its capacity to infect a variety of non-swine species, including horses, rodents, lagomorphs, non-human primates, and a range of wildlife, highlighting its broad host range (Vannucci & Gebhart, 2014). Among non-swine species, equine proliferative enteropathy in foals has emerged as the most clinically significant manifestation, with affected animals presenting lethargy, weight loss, peripheral oedema, diarrhoea, and hypoproteinemia (Page et al., 2014). The pathogenesis in horses closely mirrors that observed in swine, involving colonisation of crypt enterocytes and disruption of intes- tinal homeostasis. Similar lesions and disease have been documented in experimental models using hamsters (Karuppannan & Opriessnig, 2018), while sporadic cases of proliferative enteropathy have been reported in other domestic species such as rabbits, guinea pigs, dogs, sheep, ratite birds, and broiler chickens (Ohta et al., 2017). Accordingly, reports of L. intracellularis infection in wildlife are increasing, both in zoological collections and free-ranging species. Hence, L. intracellularis infection has been reported in canids such as foxes, wolves, coyotes (Tomanová et al., 2003; Pusterla et al., 2008) or raccoon dogs (Hossain et al., 2016), cervids as red deer (Tomanová et al., 2003), fallow deer (Dezorzova-T omabova et al., 2006), roe deer or water deer (Hossain et al., 2016), leporids as rabbits and hares (Pusterla et al., 2008), mephitids as striped skunks (Pusterla et al., 2008) or wild rodents (Backhans et al., 2013; Francois-Brazier et al., 2021), among others. Although the predicted probability of exposure of domestic pigs to different pathogens is higher for rodents or even birds, such as starlings (Pearson et al., 2015), wild boars are also hosts for L. intracellularis and can have a role in the transmission and environmental persistence of this pathogen. The aim of this research was to determine the prevalence and infectious burden of L. intracellularis in wild boars from hunting reserves of Castilla y León, Spain, allowing for the investigation of their role as an infectious source and potential reservoir of this pathogen. Okužba z Lawsonia intracellularis pri divjih prašičih v severozahodni Španiji Izvleček Okužba z Lawsonia intracellularis pri divjih prašičih predstavlja tveganje za prašičerejo ter tudi za druge živali, saj ta bakterija lahko prečka medvrstne ovire, okuži različne živalske vrste in pri nekaterih med njimi celo povzroči bolezen. Cilj te raziskave je bil ugotoviti prevalenco izločevalcev bakterije v iztrebkih pri divjih prašičih iz lovskih rezervatov v provincah Zamora in Valladolid v Španiji, da bi raziskali njihov potencial kot vir okužbe ali celo kot rezervoarje L. intracellularis. Vzorci vsebine debelega črevesa so bili odvzeti od uplenjenih divjih prašičev in obdelani za izolacijo DNK ter odkrivanje in kvantifikacijo L. intracellularis s pomočjo qPCR. Ugotovljeno je bilo, da je do 40 % živali izločalo bakterijo s fekalijami, pri čemer je bila prevalenca višja pri samicah (55 %) v primerjavi s samci (10 %). Glede na te rezultate so divji prašiči, ki so bili izločeni v severozahodni Španiji, pomemben vir okužbe z L. intracellularis za druge svinje ter tudi za druge živalske vrste. Ključne besede ileitis; proliferativna enteropatija prašičev; širjenje okužbe; stik med domačimi in prostoživečimi živalmi 53 Acta Biologica Slovenica, 2026, Special issue Materials and Methods Sampling area Wild boars were shot during hunting activities in the prov- inces of Zamora and Valladolid, located in Castilla y León, in northwest Spain. The animals were distributed within five different geographical locations, as shown in Figure 1. According to the most recent data, wild boar is widely represented and rapidly evolving throughout the Castilla y León region (JCYL, 2023); reported captures in hunting reserves (Figure 2A) exceeded 53,000 animals in the 2020-21 season, with a clear upward trend over the last ten years. Furthermore, population controls associated with damage caused by this species in the region included a total of 11,923 interventions, a clearly higher figure than the 3,994 recorded previously (2016-2017). Furthermore, Castilla y León has an important porcine production with more than 3,300,000 sows (Figure 2B), being the third most important region within the Spanish pig sector. It also has a relevant extensive pig production with more than 1,100,000 Iberian pigs, some of them in free-range piggeries, which increases the risk of spillover/spillback events sharing pathogens with different wildlife populations. Figure 1. Sampling area: five spots in the provinces of Valladolid (1) and Zamora (4) in Castilla y León, Spain. Slika 1. Območje vzorčenja: pet lokacij v pokrajinah Valladolid (1) in Zamora (4) v regiji Kastilija in León, Španija. Figure 2. Geographic distribution of wild boar captures (A) and breeding sows (B) in Castilla y León in 2022 (data and images from Junta de Castilla y León, 2023). Slika 2. Geografska porazdelitev ulovov divjih prašičev (A) in plemenitih svinj (B) v Kastilji in Leónu leta 2022 (podatki in slike: Junta de Castilla y León, 2023). 54 Acta Biologica Slovenica, 2026, Special issue Sample collection For each animal, the complete digestive tract was obtained, collecting proximal colon contents by making a cut in the colon (approximately 5 cm from the ileocecal junction). Colon contents were collected in zip-lock bags, which were identified and immediately refrigerated until arrival at the laboratory. Once at the Infectious Diseases Unit of the Animal Health Department, the samples were stored at -80°C until processing for DNA extraction. Information on sex, approximate weight, and geographic location was collected. DNA extraction and L. intracellularis detection and quantification DNA was extracted from 200 mg of each colon content sample using a commercial kit, QIAamp® PowerFecal® Pro DNA Kit Handbook (QIAgen) and following the man- ufacturer's instructions. The DNA concentrations were measured on NanoDrop ND-1000 v.3.1. spectrophotometer. A previously described TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S ribosomal gene was used for L. Intracellularis detection (Richter et al., 2010). Each reaction mixture contained 10 µL of Maxima Probe real-time PCR Master Mix 2X (Thermo Scientific), 0.3 µL of 10 µM of each primer, 0.2 µL of 10 µM Taq-Man probe, 0.12 µL of Rox (diluted 1:10 in nuclease-free water, Thermo Scientific), 2 µL of the template DNA and nuclease-free water (up to 20 µL). The qPRCs were run in a QuantStudio 1 thermal cycler (Applied Biosystems), and the concentration of L. intracellularis in each sample was estimated using an in-house standard curve, which was prepared by ten- fold serial dilutions of a L. intracellularis preparation with approximately 5 log 10 L. intracellularis/mL (range 2 log 10 to 5 log 10 bacteria/mL) as previously described (Rodríguez et al., 2024). Results were expressed as the number of L. intracellularis/g of intestinal content. Statistical analysis The open-access software Epi Info 7.2.6.0 was used for statistical analysis. Prevalence rates and 95% confidence intervals (CI) were calculated. Chi-square test was used to study the statistical association between infection (positive/ negative) and sex or municipality, while the statistical asso- ciation between bacterial load between different categories was done using ANOVA or Kruskal-Wallis on the depen- dence of normality and variance homogeneity (Bartlett test). Results Infection by L. intracellularis was prevalent in the studied population, with 12 positive samples out of 30 investi- gated (40%, 95% CI 22.7%-59.4%). The distribution of positive individuals according to sex and geographic location is shown in Table 1. Statistically significant differ- ences were reported between municipalities (Chi 2 = 13.75; p = 0.008). Moreover, the prevalence of L. intracellularis shedders was different depending on the sex (Chi 2 = 3.91; p = 0.048) and based on the estimated odds ratio (OR), females in the sampled area had 11-fold higher risk of being faecal shedders of L. intracellularis compared to males (OR=11; 95% CI 1.16–104). In contrast, no differences were observed in the risk of faecal shedding based on weight (F=1.62; p=0.213). The concentration of L. intracellularis varied between 3x10 4 and 5.8x10 5 bacteria/g of colon content, being similar between males and females (H=1.70; p=0.192), as well as between the different municipalities investigated (H=6.01; p=0.112). Sex Geographical location Female Male Villar de Ciervos (Zamora) Montamarta (Zamora) Gallegos del Rio (Zamora) Pereruela (Zamora) Villalba de los Alcores (Valladolid) N. wild boars shooted 20 10 2F 6F 2F 1F+1M 0 Prevalence 55% 10% 100% 75% 40% 40% 0% Table 1. Prevalence of L. intracellularis in the studied population of wild boars according to sex (F= Female; M= Male) and geographic location. Tabela 1. Razširjenost L. intracellularis v preučevani populaciji divjih prašičev po spolu (F = samica; M = samec) in geografski lokaciji. 55 Acta Biologica Slovenica, 2026, Special issue Discussion Wild boars have been reported as a significant source of infection or even a reservoir for various infections affecting domestic animals, particularly pigs or humans. The Castilla y León region combines a high density of wild boar with significant densities of pigs and even the presence of pigs raised in free-range or extensive conditions, making it a suitable area for exploring interactions between domestic and wild suids. The prevalence of L. intracelluaris infected wild boars found in our research was slightly higher than previously described in wild boars from Germany (20.6%) (Reiner et al., 2011), the Czech Republic (29.6%) (Tomanova et al., 2002) or Australia (18.9%) (Phillips et al., 2009). However, and in agreement with our results, some studies have shown the presence of specific antibodies in the serum of 23-93% of wild boars in Korea, Canada or the USA (Yeh, 2014; McGregor et al., 2015; Baroch et al., 2015; Cleveland et al., 2017). Differences observed in prevalence according to geographical location could be a consequence of differ- ences in the probability of contact with infected animals, including pigs and other domestic, wild or synantropic animals. On the other hand, differences between males and females have not been reported previously to the best of our knowledge and could be related to the time of sampling, in the first months of the year when the highest percentage of births occurs among wild boar populations in Spain (Herrero et al., 2008). Finally, the infectious burden of L. intracellularis was higher than 10 5 bacteria/g of colon content in 30% of the positive animals (4/12). Taking into account previous reports in pigs (Pedersen et al., 2012), at least part of the studied wild boars would have suffered from diarrhoea and hyperplastic lesions in the distal ileon due to intestinal multiplication of L. intracellularis. However, further studies to evaluate the clinical and lesional impact of the infection should be carried out in order to conclude on the disease linked to L. intracellularis infection in wild boars. Conclusions These results allow us to conclude that L. intracellularis infection is widespread among wild boars in northwest Spain, reaching data similar to or higher than those described in pig populations worldwide. Accordingly, there is a moderate to high risk of L. intracellularis transmission to other animal species, including both wildlife and domestic animals, such as pigs or foals. Robust biosecurity, including fencing against wildlife or effective rodent control, is a crucial preventive strategy to minimise the risk of spill-over events. Author Contributions Conceptualisation, V.G., H.A. and A.C.; methodology, A.C., H.A., L.P. and S.G.; sampling and laboratory analysis, F.E., J.M. and S.G.; writing—original draft preparation, F.E.; writ- ing—review and editing, V.G, H.A. and A.C. All authors have read and agreed to the published version of the manu- script. Acknowledgement We thank the staff in the Infectious Diseases Unit, particu- larly Diana Molina, and the staff at Cárnicas La Culebra for their help and support. Funding This research received no external funding. Samuel Gómez-Martínez is granted by the Junta de Castilla y Leon pre-doctoral programme (ORDEN EDU/1868/2022), and Lucía Pérez-Pérez (PRE2020-093762) is supported by the Spanish Ministry of Education. Data Availability Research data was uploaded to Zenodo (https://doi. org/10.5281/zenodo.21310931). Conflicts of Interest The authors declare no conflict of interest. 56 Acta Biologica Slovenica, 2026, Special issue References Backhans, A., Jacobson, M., Hansson, I., Lebbad, M., Thisted Lambertz, S. T., Gammelgård, E., ... Fellström, C. (2013). Occurrence of pathogens in wild rodents caught on Swedish pig and chicken farms. Epidemiology and Infection, 141(9), 1885–1891. https://doi.org/10.1017/S0950268812002609 Baroch, J. A., Gagnon, C. A., Lacouture, S., & Gottschalk, M. 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Lawsonia intracellularis in wild mammals in the Slovak Carpathians. Journal of Wildlife Diseases, 39(2), 407–411. https://doi.org/10.7589/0090-3558-39.2.407 Vannucci, F.A., Gebhart, C.J. (2014). Recent Advances in Understanding the Pathogenesis of Lawsonia intracellularis Infections. Veterinary Pathology, 51(2), 465-477. doi:10.1177/0300985813520249. Y eh, J.Y. (2014). Seroprevalence of porcine proliferative enteropathy among wild boars in the Republic of Korea. BMC Veterinary Research, 10, 5. doi: 10.1186/1746- 6148-10-5. 57 1 Department of Agriculture and Forest Sciences, University of Tuscia, Viterbo, Italy 2 Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Napoli, Italy * Corresponding author: E-mail address: gloria.bernabucci@unitus.it Citation: Girotti, P., Viola, P., Bernabucci, G., Rossi, C., Esposito, L., Piscopo, N., Basiricò, L., Ronchi, B., Primi, R. (2026). Using camera traps to assess spatial and temporal interactions between cattle and red deer in a Mediterranean silvo-pastoral system. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.29134 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Using camera traps to assess spatial and temporal interactions between cattle and red deer in a Mediterranean silvo-pastoral system Pedro Girotti 1 , Paolo Viola 1 , Gloria Bernabucci 1 * , Carlo M. Rossi 1 , Luigi Esposito 2 , Nadia Piscopo 2 , Loredana Basiricò 1 , Bruno Ronchi 1 , Riccardo Primi 1 Abstract This study investigated spatial and temporal interactions between cattle (Bos taurus) and red deer (Cervus elaphus) in a Mediterranean silvo-pastoral system using non-invasive camera trapping. The aim was to assess coexistence patterns and potential competition or complementarity in habitat use during the winter season (January), with implications for sustainable grazing management. The research was conducted in a 2330-ha farm in central Italy (Lazio region), comprising 565 ha of pasture and 1765 ha of woodland, hosting 430 Maremmana cattle and wild ungulates. Seventeen camera traps were systematically distributed across five silvo-pastoral sections (totalling 271 ha). Relative abundance was calculated as Trapping Rate (TR), and spatial use was analysed through spline analysis in ArcGIS Pro. T emporal activity and overlap were examined in R using the “activity” and “overlap” packages. Cattle showed irregular diurnal activity, while red deer were more fragmented but shared an evening peak. The temporal overlap coefficient was 0.74, suggesting moderate coexistence. Spatially, red deer were more frequent in areas with low cattle TR, although co-occurrence was significant (Z = 2.670, p = 0.007), especially near fences. These results highlight the usefulness of camera trapping in informing adaptive management in silvo- pastoral systems, promoting coexistence between livestock and wildlife. Keywords Camera trapping; livestock; wildlife 58 Acta Biologica Slovenica, 2026, Special issue Introduction Extensive farming systems are multifunctional landscapes that support the coexistence of domestic livestock and wild herbivores. These systems play a crucial role in preserving biodiversity, maintaining ecosystem functions, and supporting rural livelihoods, particularly in marginal or harsh environments (Nori et al., 2005; Battini et al., 2021). Understanding the interactions between livestock and wildlife within these shared landscapes is fundamental to ensuring the sustainable use of natural resources and to guide adaptive management strategies that prevent land degradation and biodiversity loss (Spencer et al., 2024). Red deer (Cervus elaphus) frequently overlaps in range and resource use with domestic cattle (Bos taurus) (Cooper et al., 2008; Lindgren and Sullivan, 2014) and their coexis- tence can influence grazing pressure, spatial heterogeneity of vegetation, plant community structure and composition as well as the behaviour of sympatric species (Holechek et al., 2006; Köhler et al., 2016), potentially leading to competition or, in some cases, complementary habitat use. Traditional methods based on visual observation have long been used to study such interactions, but they are often limited by the discontinuous monitoring, scarce visibility, and bias linked to observers’ presence (Animal Behaviour Society, 2012; Smith et al., 2022). In this context, recent advances in Precision Livestock Extensive Farming (PLEF) have highlighted the potential of sensor-based and remote monitoring technologies to optimise management decisions in extensive systems, including wildlife-livestock interactions (Bernabucci et al., 2025). Among these tech- nologies, camera traps (CT) have emerged as a powerful, non-invasive tool for monitoring wildlife (Delisle et al., 2021) and livestock activity simultaneously across large areas and over extended periods. Camera traps provide time- stamped data that allow for detailed analyses of spatial and temporal patterns of habitat use without influencing animal behaviour. This study used CT to investigate the spatial and temporal overlap between free-ranging Maremmana cattle and red deer in a Mediterranean silvo-pastoral system. Focusing on the winter season (January), the aim was to evaluate patterns of coexistence and to assess potential competition or complementarity in habitat use. These insights may inform sustainable grazing management and land-use planning, particularly under seasonal resource constraints and in the context of biodiversity conservation. Uporaba fotopasti za oceno prostorskih in časovnih interakcij med govedom in jelenjadjo v sredozemskem gozdno-pastirskem sistemu Izvleček Ta študija je preučevala prostorske in časovne interakcije med govedom (Bos taurus) in jelenom (Cervus elaphus) v mediteranskem gozdno-pastirskem sistemu z uporabo fotopasti. Namen je bil oceniti vzorce sobivanja ter morebitno konkurenco ali komplementarnost pri rabi habitata v zimskem obdobju (januar), z vidika trajnostnega upravljanja paše. Raziskava je potekala na 2330 ha veliki kmetiji v osrednji Italiji (dežela Lacij), ki vključuje 565 ha pašnikov in 1765 ha gozdov, z okoli 430 glavami goveda pasme Maremmana in prostoživečimi parkljarji. Sedemnajst fotopasti je bilo sistematično nameščenih v petih gozdno-pastirskih enotah (skupaj 271 ha). Relativna številčnost je bila izračunana kot stopnja zaznavanja (TR – Trapping Rate), prostorska raba pa analizirana s pomočjo spline interpolacije v programu ArcGIS Pro. Časovna aktivnost in prekrivanje sta bila analizirana v R z uporabo paketov »activity« in »overlap«. Govedo je kazalo nepravilen dnevni vzorec aktivnosti, jelen pa bolj razpršeno aktivnost z večernim vrhom. Koeficient časovnega prekrivanja je znašal 0,74, kar kaže na zmerno sobivanje. Prostorsko so bili jeleni pogostejši v območjih z nizko TR goveda, čeprav je bila prostorska sočasnost statistično značilna (Z = 2,670, p = 0,007), zlasti v bližini ograj. Rezultati poudarjajo uporabnost fotopastnega monitoringa pri prilagodljivem upravljanju gozdno-pastirskih sistemov, ki spodbuja sobivanje domačih in divjih živali. Ključne besede Fotopasti; domače živali; prostoživeče živali 59 Acta Biologica Slovenica, 2026, Special issue Materials and Methods Study area and camera traps deployment The study was conducted in a 2330-ha game management area in central Italy (Lazio region, 42°29'16"N 11°38'37" E), consisting of 565 ha of pasture and 1765 ha of woodland. The area hosts 430 Maremmana cattle, a native breed that is managed under an extensive free-range system. Feed supplementation (i.e., hay and concentrates) is provided during critical periods of the year. This practice is necessary due to the Mediterranean climate, characterised by mild, wet winters and hot, dry summers (Lionello et al., 2006). Such conditions strongly limit forage growth and availabil- ity, especially during summer and winter months, making supplemental feeding essential to maintain animal health and productivity. In addition to the Maremmana cattle, the study area supports a diverse community of wild ungulates, notably including red deer. To monitor spatial and temporal activity patterns of domestic and wild ungulates during winter, seventeen CT (Browning Spec Ops Elite HP5) were deployed across five silvo-pastoral sections, covering a total of 271 hectares within the study area. The place- ment of CT followed a systematic grid design consisting of 17 square cells, each measuring 450x450 m (Figure 1) (Triguero-Ocaña et al., 2020), allowing for adequate spatial coverage of the study area. Each CT was placed at the cell centroid, or within a 15 m radius when necessary to ensure an adequate field of view or better representation of habitat heterogeneity. The camera traps worked from 5 to 31 January, during which period the sun rose on average at 7:31 a.m. and set on average at 05:07 p.m. The devices operated continuously 24 hours per day using passive infrared sensor (PIR) motion detection and were set to video mode, recording 30-second clips upon activation, with a minimum delay of 1 second between videos. Statistical analysis Data obtained from the CT were used to estimate the relative abundance of both domestic and wild species, expressed as Trapping Rate (TR), calculated as the number of captures per unit of time. Spatial patterns of habitat use were mod- elled using Spline interpolation analysis in ArcGIS Pro 3.2.0 (Esri Inc.), enabling a continuous surface visualisation of the species' relative abundance across the study area. Tempo- ral activity and overlap between Maremmana cattle and red deer were analysed in R (R Core Team) using the "activity" and "overlap" packages (Ridout and Linkie, 2009; Rowcliffe et al., 2014; Palencia et al., 2023). To investigate environ- mental or anthropogenic factors (e.g., distance from fences) affecting species-specific distribution and abundance patterns, Kendall’s Tau correlation was computed. The significance of association was tested using the Z-score, which expresses how strongly a variable is associated with species presence: positive values suggest avoidance (i.e., presence decreases with proximity), while negative values indicate preference (i.e., presence increases with proximity). Figure 1. Systematic fishnet grid of 17 square cells (450 m side, 20.25 ha each). Camera traps were placed at each cell’s centre, with orientation based on site conditions. In purple are the wooded pasture sections. Slika 1. Sistematična mreža iz 17 kvadratnih celic (stranica 450 m, vsaka s površino 20,25 ha). Fotoaparati s senzorjem so bili nameščeni v središču vsake celice, njihova usmeritev pa je bila prilagojena razmeram na terenu. V vijolični barvi so označeni gozdnati deli pašnikov. 60 Acta Biologica Slovenica, 2026, Special issue Results and Discussion Activity pattern and temporal overlap The time activity analysis revealed an irregular daily pattern in Maremmana cattle. Their activity was low during the early morning hours, from 12:00 to 6:00 a.m., followed by a moderate peak starting around 10:00 a.m. The highest activity was recorded in the late afternoon, from 4:00 to 7:00 p.m., followed by a gradual decline (Figure 2A). A simi- lar irregular pattern was found for red deer, with low activity during early night hours, from 12:00 to 4:00 a.m. However, unlike cattle, red deer exhibited several smaller peaks dis- tributed throughout the central hours of the day, in addition to their main activity peak in the late afternoon, from 6:00 to 7:00 p.m. (Figure 2B). The time overlap analysis revealed a moderate coexistence between the two species, with a coefficient of temporal overlap of 0.74, indicating partially shared activity patterns, particularly during the late after- noon hours (Figure 3). Figure 3. Temporal overlap in activity patterns between Maremmana cattle and red deer. The shaded area represents the coefficient of overlap across the 24-hour cycle. Slika 3. Časovno prekrivanje vzorcev aktivnosti med govedom pasme Maremmana in rdečim jelenom. Senčeno območje predstavlja koefi- cient prekrivanja v 24-urnem ciklu. Figure 2. A (left). Time activity pattern of Maremmana cattle illustrating the distribution of activity throughout the day and night. B (right). Time activity pattern of red deer illustrating the distribution of activity throughout the day and night. Slika 2. A (levo). Vzorec časovne aktivnosti goveda pasme Maremmana, ki prikazuje porazdelitev aktivnosti skozi dan in noč. B (desno). Vzorec časovne aktivnosti rjavega jelena, ki prikazuje porazdelitev aktivnosti skozi dan in noč. 61 Acta Biologica Slovenica, 2026, Special issue Spatial distribution analysis The analysis of spatial distribution, based on TR values, revealed distinct habitat use patterns between Marem- mana cattle and red deer. Trapping rate, expressed as the number of detections per unit of time for each camera trap, is summarised in Table 1 for both species. These values were used as the basis for the spline inter- polation analysis and subsequent spatial modelling (Figure 4). The results indicated that 8.88% of the monitored area displayed no presence of either species, suggesting either limited accessibility or unsuitable habitat conditions. Cattle and red deer overlapped in approximately 55% of the area, indicating a moderate spatial overlap. However, a clear inverse relationship between the two species emerged: red deer were more frequently detected in zones with low or null cattle activity, particularly in areas where cattle TR ranged from 0 to 0.2, which accounted for 10.82% of the study area. Overall, as cattle TR increased, red deer detec- tions decreased, suggesting spatial segregation likely driven by differences in foraging strategies, interspecific competition, or avoidance behaviour. These patterns align with previous findings on habitat partitioning between wild and domestic herbivores (Loft et al., 1991; Stewart et al., 2002; Cooper et al., 2008), and support the hypothesis of distinct spatial preferences influenced by species-specific ecological requirements. Kendall's Tau correlation Despite the overall pattern of spatial segregation between cattle and red deer previously highlighted, the Kendall’s Tau correlation analysis revealed a significant positive spatial association between their abundance (Z = 2.670, p = 0.007). This apparent contradiction is resolved by consider- ing that the two species overlapped only in small but rele- vant hotspots (3.20% of the study area). In these sites, both red deer and cattle reached their highest TR, suggesting that particularly attractive areas may override the general tendency for spatial segregation. These hotspots may be driven by shared preferences for specific habitat features that require further site-specific investigations. Additionally, a significant negative association with distance from fences was observed (Z = –2.131, p = 0.033), indicating that red deer were more frequently detected near fenced areas. This may reflect behavioural responses to anthropogenic structures or landscape features. CT Latitude Longitude TR TR Maremmana cattle Red deer A 42°29’33’’ N 11°37’37’’ E 0.00 0.00 B 42° 29'35" N 11°37'56" E 0.00 0.00 C 42°29'36" N 11°38'15" E 0.00 0.00 D 42°29'37" N 11°38'35" E 0.00 0.00 E 42°29'37" N 11°38'53" E 0.27 0.10 F 42°29'20" N 11°37'37" E 0.23 0.10 G 42°29'21" N 11°37'57" E 0.00 0.00 H 42°29'21" N 11°38'15" E ---- ---- I 42°29'22" N 11°38'36" E 0.33 0.00 L 42°29'23" N 11°38'55" E 0.63 0.10 M 42°29'06" N 11°37'38" E 0.14 0.00 N 42°29'06" N 11°37'59" E 0.00 0.07 O 42°29'07" N 11°38'18" E 0.07 0.00 P 42°29'08" N 11°38'33" E 0.07 0.03 Q 42°28'52" N 11°37'40" E 0.10 0.10 R 42°28'52" N 11°37'58" E 0.00 0.07 S 42°28'53" N 11°38'15" E 0.00 0.00 Table 1. Geographic coordinates (latitude and longitude) and trapping rate of Maremmana cattle and red deer, for each camera trap. Tabela 1. Geografske koordinate (zemljepisna širina in dolžina) ter pogostost zajetja goveda pasme Maremmana in rjavega jelena za vsako foto past. CT= camera trap, TR = Trapping rate 62 Acta Biologica Slovenica, 2026, Special issue Conclusions Camera trapping revealed that red deer and cattle coexist temporally but exhibit a general pattern of spatial segre- gation, with small but relevant hotspots of overlap where both species reached the highest activity levels. These hotspots, likely driven by shared habitat preferences, sug- gest that while spatial segregation may reduce competition, attractive areas can concentrate both wild and domestic herbivores, potentially amplifying interactions. This dual dynamic underscores the complexity of coexistence mechanisms and highlights the importance of site-specific investigations to identify and manage key areas of overlap. These findings underscore the value of passive, continuous monitoring in capturing spatio-temporal dynamics, which are often overlooked in traditional surveys. By providing evidence-based insights, such approaches can inform adaptive management strategies in silvo-pastoral systems, aiming to balance livestock production with biodiversity conservation and overall ecosystem health. Given that over- or under-grazing areas can develop, studies like this are crucial to better plan livestock and wildlife carrying capacities, optimising grazing pressure to maintain eco- system balance. Future research should extend monitoring across different seasons to capture potential temporal shifts in activity patterns and interactions. Expanding the temporal and spatial scale of investigation will be essen- tial to fully understand the dynamics of coexistence and to support more resilient and multifunctional land-use strategies. Additionally, the use of automated field devices capable of directly detecting and counting animals would provide valuable support by reducing manual workload and improving data collection efficiency, thereby enhanc- ing monitoring capabilities. Author Contributions Conceptualisation, P.V. and R.P.; methodology, P.G., P.V., C.M.R., R.P.; software, P.G., C.M.R., G.B.; validation, P.V., B.R. and R.P.; formal analysis, P.G., P.V. and R.P.; investigation, P.G., P.V., and R.P.; resources, B.R. and R.P.; data curation, P.G., P.V. and C.M.R.; writing—original draft preparation, G.B.; writing—review and editing, P.G., P.V., L.E., N.P., L.B., B.R. and R.P.; visualization, P.V. and R.P.; supervision, B.R.; project administration, B.R. and R. P.; funding acquisition, B.R and R.P. All authors have read and agreed to the pub- lished version of the manuscript.” Figure 4. Spatial interpolation of trapping rate values for Maremmana cattle and red deer using spline analysis in ArcGIS Pro. Warmer colours indicate higher activity levels. Slika 4. Prostorska interpolacija vrednosti stopnje ujetja za govedo pasme Maremmana in rjavega jelena z uporabo splyne analize v pro- gramu ArcGIS Pro. Toplejše barve označujejo višjo raven aktivnosti. 63 Acta Biologica Slovenica, 2026, Special issue Acknowledgement We would like to thank Francesco Spada and Silvia Com- pagnucci for their invaluable assistance in data collection. We also express our gratitude to the Società Agricola di Musignano for their collaboration and logistical support during the study. Funding This work was conducted within the AGRITECH project funded by the European Union NextGeneration EU (Grant number J83C22000830005). Data Availability Research data was deposited at Zenodo (https://doi. org/10.5281/zenodo.21311098). Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analy- ses, or interpretation of data; in the writing of the manu- script; or in the decision to publish the results. References Animal Behaviour Society. (2012). Guidelines for the treatment of animals in behavioural research and teaching. Animal Behaviour, 83(1), 301–309. https://doi. org/10.1016/j.anbehav.2011.10.031 Battini, M., Renna, M., Giammarino, M., Battaglini, L., & Mattiello, S. (2021). Feasibility and reliability of the AWIN welfare assessment protocol for dairy goats in semi-extensive farming conditions. Frontiers in Veterinary Science, 8, Article 731927. https://doi.org/10.3389/fvets.2021.731927 Cooper, S. M., Perotto-Baldivieso, H. L., Owens, M. K., Meek, M. G., & Figueroa-Pagan, M. (2008). Distribution and interaction of white-tailed deer and cattle in a semi-arid grazing system. Agriculture, Ecosystems & Environment, 127, 85–92. https://doi.org/10.1016/j.agee.2008.03.004 Delisle, Z. 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Journal of Range Management, 46(3), 245–250. 64 1 Department of Veterinary Medicine and Animal Productions - University of Naples Federico II, Italy 2 Department of Animal Production, Veterinary Faculty - University of León, Spain 3 Territorial Environmental Service of Zamora, Spain 4 Doctor of Veterinary Medicine – National Park of Abruzzo, Lazio and Molise, Italy 5 Forestry Engineers - Outdoor Centre Gole del Sangro, Civitella Alfedena, Italy 6 Forestry Engineers, Spain 7 Department of Agriculture - University of Naples Federico II, Italy * Corresponding author: E-mail address: martinaiovino45@gmail.com Citation: Iovino, M., Eguren, V. G., Redin, P. S., Gentile, L., Martinelli, A., Alberti, J. P., Cervelli, E.. (2026). Natura 2000 Sites (SCI/SAC - SPA) wildlife populations estimation and comparison between Abruzzo (IT) and Castilla y León (ES) regions. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.29135 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Natura 2000 Sites (SCI/SAC - SPA) wildlife populations estimation and comparison between Abruzzo (IT) and Castilla y León (ES) regions Martina Iovino 1 * , Vicente González Eguren 2 , Pablo Santos Redin 3 , Leonardo Gentile 4 , Alice Martinelli 5 , Jesús Palacios Alberti 6 , Elena Cervelli 7 Abstract This study aims to demonstrate that the Natura 2000 Network is the appropriate tool adopted by the European Union for biodiversity conservation. Twenty-five years after its establishment, conservation policies and economic and social practices have not yet been integrated. A comparison of the Standard Data Forms of the regions of Abruzzo (Italy) and Castile and León (Spain) highlights differences in biodiversity management and monitoring. The results highlight critical issues ranging from the lack of management plans in Italy to limited knowledge of the number of individuals among the species described as present in the 174 sites compared. Due to the lack of reliable data, it is impossible to determine whether the various species are experiencing drastic population declines or territorial rarities. The study suggests future conservation strategies that take due account of climate change and agricultural sustainability. The comparison between the two European regions and the analysis of official data lay the foundation for a broader debate on the need to improve management practices in protected areas, thus contributing to more effective biodiversity conservation. Keywords Biodiversity; Natura 2000 Network; Management plans; Conservation status. 65 Acta Biologica Slovenica, 2026, Special issue Introduction The evolution and continuous adaptation of living organ- isms to the changing conditions of the biosphere have led to the current state of biodiversity. Biodiversity comprises three main components: living species, genetic variation within species, and ecosystem diversity. To develop concrete conservation plans, it is necessary to consider the dynamic nature of systems composed of interacting communities of plants, animals, and microorganisms, and their abiotic environments, which form functional ecolog- ical units. The 21st century has heightened scientific attention to the inappropriate exploitation of natural resources and the simultaneous need to adapt to the rapid development of new technologies. There is a growing awareness that human activities are the main causes of environmental degradation and that natural resources are insufficient to support uncontrolled human development. At the 1992 Earth Summit (Bell, 1993; Decision 93/626/EEC, 1993; Bowman and Redgwell, 1995), the Convention on Biolog- ical Diversity established three fundamental objectives for conservation (protection of biological diversity, sustainable use of its components, and fair and equitable sharing of benefits arising from the use of resources). The European Union implements conservation strat- egies through the adoption of Directive 92/43/EEC of 21 May 1992 (the Habitats Directive). Its primary objective is to safeguard biodiversity by ensuring the conservation of natural habitats and wild species of flora and fauna within the territories of the Member States. To achieve this goal, the directive establishes specific measures to maintain or restore, at a favourable conservation status, the habitats and species of Community interest listed in its annexes (Council Directive 92/43/EEC). Complementary to the Habitats Directive is Directive 79/409/EEC, codified and updated as Directive 2009/147/ EC, known as the Birds Directive. Together, these two directives form the foundation of the EU's biodiversity conservation policy and provide the legal basis for the establishment and management of the Natura 2000 net- work, a coherent ecological network of protected areas. Within the designated sites, Natura 2000 intends to apply conservation measures for the habitats and species listed in Annexes I and II, as well as outlining protection regimes for the species listed in Annexes IV and V (Council Directive 79/409/EEC; Directive 2009/147/EC). The directive also includes provisions on site manage- ment and impact assessments (Article 6), financial support mechanisms (Article 8), national monitoring and reporting Ocena populacij prostoživečih živali na območjih Natura 2000 (SIC/ZSC – ZPS) ter primerjava med regijama Abruzzo (IT) in Castilla y León (ES) Izvleček Namen te študije je pokazati, da je omrežje Natura 2000 ustrezno orodje, ki ga je Evropska unija sprejela za ohranjanje biotske raznovrstnosti. Petindvajset let po njeni ustanovitvi politike ohranjanja ter gospodarske in družbene prakse še niso bile usklajene. Primerjava standardnih obrazcev za poročanje v regijah Abruzzo (Italija), Kastilija in León (Španija) izpostavlja razlike v upravljanju in spremljanju biotske raznovrstnosti. Rezultati poudarjajo ključna vprašanja, ki segajo od pomanjkanja načrtov upravljanja v Italiji do omejenega znanja o številu osebkov med vrstami, opisanimi kot prisotne na 174 primerjanih območjih. Zaradi pomanjkanja zanesljivih podatkov ni mogoče ugotoviti, ali različne vrste doživljajo drastičen upad populacij ali so le redke na določenih območjih. Študija predlaga prihodnje strategije ohranjanja, ki ustrezno upoštevajo podnebne spremembe in trajnost kmetijstva. Primerjava med tema dvema evropskima regijama in analiza uradnih podatkov postavljata temelje za širšo razpravo o potrebi po izboljšanju praks upravljanja v zavarovanih območjih, s čimer prispevata k učinkovitejšemu ohranjanju biotske raznovrstnosti. Ključne besede Biotska raznovrstnost; omrežje Natura 2000; načrti upravljanja; ohranitveno stanje. 66 Acta Biologica Slovenica, 2026, Special issue (Articles 11 and 17), and conditions for derogations (Article 16). It also emphasises the ecological role of landscape elements that provide essential connectivity for wild fauna and flora (Article 10). To ensure the conservation of Natura 2000 sites, the Habitats Directive identifies the Management Plan as a key planning tool (Article 6). This tool integrates ecological, socioeconomic, and administrative dimensions to safe- guard the specific characteristics of each site. A distinctive feature of Natura 2000 management plans is that they are not always mandatory, but once adopted, they must take into account the specific characteristics of the site and all planned activities. These plans can be developed as stand- alone documents or integrated into broader development or land-use plans. Although optional, the management plan is considered a priority among conservation measures due to its tailored approach. It must also be harmonised with other spatial planning tools, including Landscape Plans, Forest Management Plans, Wildlife and Hunting Plans, Urban Development Plans, and Protected Area Plans, where rel- evant. Each management plan includes an assessment of ecological value, identification of threats and critical issues, definition of conservation objectives and strategies, and operational guidelines for site management. Within the complex system for biodiversity conser- vation, this work analyses the management plans of the Natura 2000 areas of the Abruzzo Region (Italy) and Cas- tilla y Leon (Spain). Materials and Methods Data Sources and Collection The work began with official data and documentation pro- vided by the technical-scientific bodies of the Italian and Spanish Ministries (ISPRA, 2025; MiTECO, 2025) relating to biodiversity conservation under the EU Habitats and Birds Directives. The currently available databases refer to the third reporting cycle (2007-2012) and the fourth reporting cycle (2013-2018). The focus was on a comparative analysis between the two regions based on the data provided in section 3.2 of the Standard Data Module (SDF): Species referred to in Article 4 of Directive 2009/147/EC and listed in Annex II of Directive 92/43/EEC and related site assessment. Specifically (Commission EU, 2013; Genovesi et al., 2014; Angelini et al., 2016), the following data fields were analysed: (i) Species; (ii) Type; (iii) Minimum quantity; (iv) Maximum quantity; (v) Data quality (field census data). The results were evaluated considering the surface area of each site and the threat level of each species, based on the IUCN Red List. Results Surfaces and management plans The analysis of the official Standard Data Forms (SDFs) shows that, out of the 54 Special Areas of Conservation (SACs) in the Abruzzo region, only one is equipped with a management plan, while none of the 16 Special Protection Areas (SPAs) has one. In contrast, all 120 SACs and 70 SPAs in the Castilla y León region (Spain) are provided with man- agement plans (Table 1). Areas dedicated to Natura 2000 are similar or overlapping in percentage terms. Species referred to in Article 4 of Directive 2009/147/EC (Annex II of Directive 92/43/EEC) Out of the 306 “Species referred to in Article 4 of Directive 2009/147/EC and listed in Annex II of Directive 92/43/EEC and site evaluation for them” analysed within the SACs, 132 species were identified in Abruzzo and 248 in Castilla y León. A total of 74 species is found in both regions (Table 2). In the same Table 2, it is possible to see that of the 324 “Species referred to in Article 4 of Directive 2009/147/ EC and listed in Annex II of Directive 92/43/EEC and site evaluation for them” analysed within the SPAs, 75 species were identified in Abruzzo and 316 in Castilla y León. A total of 67 species is shared between the two regions. Among the 206 bird species reported as present in the SACs of the two regions examined, average abun- dances—calculated from the minimum and maximum values recorded in official censuses in Abruzzo and Castilla y León—are available for only 91 bird species (Figure 1). The bird species censused in Natura 2000 sites of the Abruzzo region amount to 69 out of 83 recorded species. Among the censused fauna, some species are represented by a single individual (e.g., Lesser Grey Shrike, Caspian T ern, Short-toed Eagle), while others reach up to 1,490 individuals (Collared Flycatcher). In Castilla y León, 60 bird species were 67 Acta Biologica Slovenica, 2026, Special issue censused out of 177 present; some species are represented by a single individual (e.g., Iberian Imperial Eagle, Caspian Tern, Common Tern, Bearded Vulture, Chestnut-vented Warbler), with a maximum count of 1,179 individuals (Par- tridge). The mean abundance values, ordered from highest to lowest, are for the 91 bird species with official numerical data available in both regions studied. Among the amphibian, reptile, and fish species listed in the Italian and Spanish SPAs, with the exception of Triturus carnifex and Vipera ursinii, for which average counts of 7.5 and 40 individuals, respectively, were reported in Italy, no census numbers are available. Additionally, data quality, assessed at individual Natura 2000 sites (SACs), is classi- fied as “Data Deficient” for 189 species in Italy and 470 in Spain; only two species (Triturus carnifex and Vipera ursinii) show “Good” data quality, and only in Italy. Six amphibian species are reported in Natura 2000 sites in Abruzzo, and three in Castilla y León, with only Discoglossus jeanneae and Discoglossus galganoi shared between the two regions. The presence of region-specific species is also evident among reptiles, with four species in Abruzzo and three in Castilla y León; only Emys orbicularis occurs in both. No fish species are shared between the two regions, with Abruzzo hosting 10 fish species and Castilla y León 9. Among invertebrates, except for Margaritifera margari- tifera, which has an average of 200 individuals reported in Castilla y León, no numerical data are available in either country. Data quality for invertebrates at individual Natura 2000 sites is classified as “Data Deficient” for 45 species in Italy and 280 in Spain. Nine invertebrate species occur in Abruzzo’s Natura 2000 sites and 18 in Castilla y León, with eight species common to both regions. The presence of species closely tied to regional habitats is particularly evident among plants. Data quality is classified as “Moderate” in Spain, based on partial data with some extrapolation, while in Italy, it is “Data Deficient” for its seven plant species. RTS ha Nat n Nat ha Nat/RTS % MP n Abruzzo 1,082,889 SAC 54 256,000 23.64 1 SPA 16 363,710 33.59 0 Castilla y León 9,422,300 SAC 120 1,896,215 20.12 120 SPA 70 2,001,886 21.25 70 Class Abruzzo Castilla y León Present in both regions Abruzzo Castilla y León Present in both regions Species SACs SPAs Birds 83 177 54 37 224 33 Mammals 14 17 12 12 19 12 Invertebrates 9 17 7 6 19 6 Plants 7 21 - 5 27 5 Fishes 10 9 - 7 16 7 Reptiles 5 4 1 4 7 3 Amphibians 4 3 - 4 4 1 Total 132 248 74 75 316 67 Table 1. Regional T otal Surface (RTS ha), Natura 2000 number (Nat n), Natura 2000 surface (Nat ha), percentage of Natura 2000/RTS (NAT/RTS %) and number of Management Plans (MP) of Special Areas of Conservation (SAC) and Special Protection Areas (SPA) of Abruzzo Region (Italy) and Castilla y León Region (Spain). Tabela 1. Skupna površina območja (RTS ha), število območij Natura 2000 (Nat n), površina območij Natura 2000 (Nat ha), odstotek območij Natura 2000 glede na skupno površino regije (NAT/RTS %) ter število načrtov upravljanja (MP) posebnih ohranitvenih območij (SAC) in posebnih zaščitenih območij (SPA) v regiji Abruzzo (Italija) in regiji Castilla y León (Španija). Table 2. Number of Species per Class described in the Special Areas of Conservation (SAC) and Special Protection Areas (SPA) of the Regions of Abruzzo (Italy) and Castilla y León (Spain). Tabela 2. Število vrst po razredih, opisanih v posebnih ohranitvenih območjih (SAC) in posebnih zaščitnih območjih (SPA) v regijah Abruzzo (Italija) in Castilla y León (Španija). 68 Acta Biologica Slovenica, 2026, Special issue Figure 2. Average number of individuals of mammal species in Italian and Spanish SACs (with species absence indicated in red). Slika 2. Povprečno število osebkov sesalskih vrst v italijanskih in španskih posebnih območjih ohranjanja (SAC) (vrste, ki na območju niso prisotne, so označene z rdečo barvo). Figure 1. Average number of individuals of a bird species recorded in Italian and Spanish SCIs. Slika 1. Povprečno število osebkov ptic po vrstah, zabeleženih v italijanskih in španskih območjih posebnega pomena (SCI). MAMMALS ES IT MAMMALS ES IT Miniopterus schreibersii 77,093 208 Rupicapre pyrenaica ornata 60 325 Myotis myotis 13,525 750 Ursus arctos 57 7 Myotis emarginatus 1,615 0 Mustela lutreola 1 Myotis blythii 1,125 312 Canis lupus 0 115 Myotis bechsteinii 0 0 Lutra lutra 0 2 Rhinolophus ferrumequinum 3,806 24 Tursiops truncatus 0 Rhinolophus hipposiderus 606 7 Galemys pyrenaicus 0 Rhinolophus euryale 540 25 Lynx pardina 0 Rhinolophus mehelyi 96 0 Microtus cabrerae 0 Barbastella barbastellus 0 300 Figure 2 lists the mammal species present in the Natura 2000 sites (SACs) of Italy and Spain. In the Abruzzo region, 15 species are recorded. Among these, some species report a minimum census count of two individuals (Otter) and seven individuals (Lesser horseshoe bat and Brown bear), up to a maximum of 750 individuals (Greater mouse-eared bat). In the Castilla y León region, species with a minimum of one recorded individual (European mink) are reported, up to a maximum of 77,093 individuals (Common bent-wing bat). No census data are reported for Myotis bechsteinii in either Italy or Spain. Census data are also missing for six mammal species in Spain (Wolf, Barbastelle bat, Otter, Pyrenean desman, Iberian lynx, Cabrera vole) and three species in Italy (Schreibers’ bat, Mehely’s horseshoe bat, Bottlenose dolphin). The species Mustela lutreola, Gale- mys pyrenaica, Lynx pardina, and Microtus cabrerae are not present in Abruzzo, while Tursiops truncatus is absent in Castilla y León. Data quality, assessed at individual Natura 2000 sites (SACs), is classified as “Good” for five species in Italy and ten in Spain; “Data Deficient” for 28 species in Italy and 336 in Spain. 69 Acta Biologica Slovenica, 2026, Special issue Discussion The international collaboration agreement between the University of Naples Federico II (Italy) and the University of León (Spain) has enabled the sharing of a methodology and data collection plan for the biodiversity described as present in the Natura 2000 sites of the Abruzzo and Castile-León regions. The validation of the data processing was scientifically and technically supported by the aca- demic staff involved, under the patronage of the Abruzzo, Lazio, and Molise National Park. The Mediterranean biogeographical region is defined by the influence of the Mediterranean basin and, despite its lack of physical and geological geographic uniformity, is one of the best-defined regions from a phytogeographical and zoogeographical perspective. Originally limited to the land areas of the narrowly defined Mediterranean zone, it has been extended to its current boundaries, which include mountainous northwest Africa (Morocco, Algeria, and Tunisia), Spain, the southern coasts of France, and the Tyrrhenian coasts of Italy. A comparison of data from the regions of Abruzzo and Castile and León showed that 306 species (animals and plants) can benefit from the protection measures guaranteed by the Natura 2000 network. However, the results also reveal a series of critical issues that threaten their favourable conservation status. The first issue concerns the lack of management plans in Italy (only 1 out of 54 sites has one), and although all Natura 2000 sites in Spain (120 out of 120) have manage- ment plans, inadequate verification of their actual conser- vation status is lacking. The second critical point concerns the very limited numerical knowledge of the individuals recorded among the species described as present in the 174 sites compared. Without reliable data, it is impossible to determine, as stated in the Fourth National Report, whether widespread or common species have suffered or are suf- fering drastic population declines or range contraction. The Fourth National Report itself emphasises that to counteract numerical declines, it is necessary to investigate the main causes and create the conditions to improve the conserva- tion status of threatened species, countering threats with the most appropriate and species-specific actions. Our investigation shows that official information is based on decidedly unsatisfactory data quality: 3,191 Of the 3,566 records (89.48%) are data-deficient, compared to only 375 of 3,566 (10.52%) classified as good. It is unclear how specific conservation strategies can be prioritised in the absence of reliable or at least trustworthy information. One of the key conclusions drawn from the analysis of the 174 Natura 2000 sites is that any strategy aimed at halting biodiversity loss cannot propose management plans that ignore climate change, ecosystem changes in agroecosystems, and the potential of biodiversity as a tool for defining ecosystem services. Current agricultural environments have become extremely simplified, and agroecosystems are unable to support food chains, exacerbating the decline of grasslands and other natural features. The situation is exacerbated by the widespread use of pesticides, mowing practices that fail to adequately address the ecological needs of species, the reduction of traditional agricultural practices, and the abandonment of mountain meadows and pastures. A viable strategy to mitigate biodiversity loss is to encourage farmers to adopt environmentally sustainable agricultural practices. It should also be noted that hunting is permitted in Natura 2000 sites not included in protected areas, and while hunting is limited to certain species, it significantly impacts warm- blooded fauna (both hunting and non-hunting) and the entire communities that comprise the habitats. The com- plexity of identifying the appropriate approach to correctly formulate management plans for Natura 2000 sites is the most tangible result highlighted by this work. Conclusions Pursuant to Article 11 of the Habitats Directive, Member States are required to monitor the conservation status of habitats (listed in Annex I) and species (listed in Annexes II, IV, and V) of Community interest throughout their national territory. Monitoring results are transmitted to the European Commission pursuant to Article 17 of the Habitats Directive (DG Environment., 2017), which requires the preparation of a National Report every six years. Data must be reported for each habitat and species at the biogeographical level, using the format provided by the European Commission, which includes a general section with information on the imple- mentation of the Directive and a section on the assessment of the conservation status of habitats and species. Monitoring the conservation status is also essential for assessing the achievement of Article 2 of the Birds Directive, which aims to ensure that all bird species reach an adequate level of conservation (Saunders, Hobbs & 70 Acta Biologica Slovenica, 2026, Special issue Margules, 1991). The 4th National Report under Article 17 of the Habitats Directive, covering the period 2013-2018, is currently available (Ercole et al., 2021). The National Reports under the Habitats and Birds Directives provide the most accurate and up-to-date overview of the conservation status of habitats and species of Community interest in the EU Member States. They are therefore strategically import- ant not only for assessing the successful implementation of the EU Biodiversity Directives, but also as a reference for identifying priorities and critical issues for the next financial programming period 2021-2027 and for measuring the achievement of the objectives established by both EU poli- cies and national legislation, with reference to the National and European Biodiversity Strategies. The Birds and Habitats Directives have proven, and continue to prove, crucial for the improvement and main- tenance of numerous populations, including through the implementation of the Natura 2000 network. Author Contributions Conceptualization, M.I., E.C.; methodology, P.S.R., J.P.A., E.C.; software, E.C., A.M.; validation, V.G.E., J.P.A., E.C.; formal analysis, E.C., P.S.R., L.G., J.P.A.; investigation, M.I., V.G.E., P.S.R., L.G., A.M., J.P.A., E.C.; resources, M.I., E.C.; data curation, M.I., E.C.; writing—original draft preparation, M.I., E.C.; visualization, V.G.E., P.S.R., L.G., A.M., E.C.; super- vision, V.G.E., J.P.A., E.C.; project administration, E.C. All authors have read and agreed to the published version of the manuscript. Acknowledgement We acknowledge the offices of the National Park of Abru- zzo, Lazio, and Molise; Junta de Castilla y León; Universi- dad de León; University of Naples Federico II for their tech- nical support. Funding This research received no external funding. Data Availability Statement Research data was uploaded to Zenodo (https://doi. org/10.5281/zenodo.21311137). Conflicts of Interest The authors declare no conflict of interest. The study was conducted independently by researchers of the Univer- sity; consequently, no external parties influenced the study design, data collection, analysis, manuscript preparation, or the decision to publish the results. References Angelini, P., Casella, L., Grignetti, A., & Genovesi, P., 2016. Manuali per il monitoraggio di specie e habitat di interesse comunitario (Direttiva 92/43/CEE) in Italia: Habitat. ISPRA, Serie Manuali e Linee Guida, 142. Bell, D. E., 1993. The 1992 Convention on Biological Diversity: The Continuing Significance of U.S. Objection at the Earth Summit. The George Washington Journal of International Law and Economics. Bowman, M.-Redgwell, C., 1995. International Law and the Conservation of Biological Diversity, London-The Hague-Boston, 5-31. Commission of the European Community., 2013. Interpretation manual of European Union habitats (EUR 28). DG Environment., 2017. Reporting under Article 17 of the Habitats Directive: Explanatory notes and guidelines for the period 2013–2018. Brussels, Belgium. Ercole, S., Angelini, P., Carnevali, L., Casella, L., Giacanelli, V., Grignetti, A., La Mesa, G., Nardelli, R., Serra, L., Stoch, F., Tunesi, L., & Genovesi, P. (Eds.), 2021. Rapporti Direttive Natura (2013–2018). Sintesi dello stato di conservazione delle specie e degli habitat di interesse comunitario e delle azioni di contrasto alle specie esotiche di rilevanza unionale in Italia. ISPRA, Serie Rapporti, 349. Genovesi, P., Angelini, P., Bianchi, E., Dupré, E., Ercole, S., Giacanelli, V., Ronchi, F., & Stoch, F. (Eds.). (2014). Specie e habitat di interesse comunitario in Italia: Distribuzione, stato di conservazione e trend (pp. 17–69). ISPRA, Serie Rapporti, 194. Saunders, D. A., Hobbs, R. J., & Margules, C. R., 1991. Biological consequences of ecosystem fragmentation. Council Directive 79/409/EEC of 2 April 1979 on the conservation of wild birds. (1979). Official Journal of the European Communities, L 103, 1–18. Council Directive 92/43/EEC of 21 May 1992 on the conservation of natural habitats and of wild fauna and flora. (1992). Official Journal of the European Communities, L 206, 7–50 Council of the European Union., 1993. Council Decision 93/626/EEC of 25 October 1993 concerning the conclusion of the Convention on Biological Diversity. Official Journal of the European Communities, L 309, 1–20. Directive 2009/147/EC of the European Parliament and of the Council of 30 November 2009 on the conservation of wild birds (codified version). (2009). Official Journal of the European Union, L 20, 7–25. Instituto para la Conservación de la Naturaleza y los Bosques / Ministerio para la Transición Ecológica y el Reto Demográfico (MiTECO). (2025). Natura 2000 database and Standard Data Forms. Ministry for the Ecological Transition and the Demographic Challenge, Government of Spain. https://www.miteco.gob.es Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA). (2025). Natura 2000 database and Standard Data Forms. ISPRA. https://www.isprambiente.gov.it 71 1 Doctor of Agronomy, for Department of Veterinary Medicine and Animal Production - University of Naples Federico II, via Delpino, 1, Italy 2 Department of Veterinary Medicine and Animal Production - University of Naples Federico II, via Delpino, 1, Italy 3 Department of Forestry Sciences and Landscape Architecture - University of Trás- os-Montes e Alto Douro, Quinta de Prados, 5000-801 Vila Real, Portugal. * Corresponding author: E-mail address: ad.mangini@studenti.unina.it Citation: Mangini, A., Esposito, L., Monzon Capapé, A. (2026). The State of Wildlife Heritage within Natura 2000 Regional Sites in Campania (Italy) and Norte (Portugal). Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.29137 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note The State of Wildlife Heritage within Natura 2000 Regional Sites in Campania (Italy) and Norte (Portugal) Adriana Mangini 1 * , Luigi Esposito 2 , Aurora C. Monzon Capapé 3 Abstract The main instrument for biodiversity conservation in the EU is the Natura 2000 network (Habitats Directive 92/43/EEC and Birds Directive 2009/147/EC). This vast ecological network, established across the entire EU since 1995 with the proposal for the Sites of Community Importance and the Special Protection Areas, has developed to the present day with the recognition of the areas containing significant biodiversity (animals, plants, and ecosystems) as Special Areas of Conservation. The official Natura 2000 datasets and the standard data modules for all SACs/SCIs in the regions of Campania (RC), Italy, and Tras-os- Montes e Alto Douro (TAD), Portugal, were compared. Natura 2000 sites cover 19% of the Italian and 13% of the Portuguese territory. 76% (RC) and 61% (TAD) of Natura 2000 areas are SACs/SCIs, while 10% (RC) and 36% (TAD) are SPAs. 14% (RC) and 3% (TAD) are areas that include both conditions. Animal biodiversity is made up of 248 species (205 birds) in the Italian region and 121 (85 birds) in the Portuguese region. In 2024, Campania has equipped itself with management plans, while the Tras-os-Montes e Alto Douro Region has not yet prepared them. Keywords Natura 2000 sites, biodiversity, biogeographic regions, and management plans. 72 Acta Biologica Slovenica, 2026, Special issue Introduction Since 1992, European environmental policies have pro- posed to apply the "Habitats" Directive 92/43/EEC (C.D., 1992) and the "Birds" Directive 2009/147/EC (E.C., 2009). The Natura 2000 network (EEA, 2024a) has become the main instrument of the European Union relating to policies for the conservation of biodiversity. In Italy, it is regulated by the Presidential Decree n. 357/1997 (DPR n. 357, 1997), and in Portugal by the Legislative Decree/140-1999 (MAE, 1999). What has happened since the Convention on Biological Diversity (CBD) can be chronologically summarised in the following list: • In 2010, the EU Council (COM, 2015) determined its position on biodiversity loss and set the goal of restoring it by 2020, which is linked to the decline of ecosystem services. Subsequently, the areas requiring greater attention in order to achieve the objectives were highlighted. • In 2016, the UN Conference of the Parties (UN, 2016) highlighted the need for international action, and in 2018, a strategic plan to protect biodiversity beyond 2020 was proposed (UN, 2021). In Europe, the Council called on the European Commission to develop a credi- ble and realistic EU strategy to be implemented by 2030 to halt the loss of biodiversity. • In 2020, the European Commission published the new strategy within the framework of the European Green Deal (EC, 2020). • In 2022, COP 15 proposed a post-2020 global biodiver- sity framework (UN, 2022) with short-term (2030) and long-term (2050) objectives. “The Kunming-Montreal Agreement is the first comprehensive global agreement to ensure the stability of ecosystem services essential for human security, economic development, nature conservation (at least 30% of protected areas by 2030) and the fight against climate change. It is a new barrier against the global environmental crisis.” • In 2023, in order to fulfil the Kunming-Montreal com- mitments, an agreement was reached that provides for the restoration of ecosystems in the EU by 2050, with the restoration of at least 20% of terrestrial and marine areas by 2030. To meet the objectives of the European Stanje naravne dediščine v regionalnih območjih Natura 2000 v Kampaniji (Italija) in Norte (Portugalska) Izvleček Glavni instrument za ohranjanje biotske raznovrstnosti v EU je omrežje Natura 2000 (Direktiva o habitatih 92/43/ EGS in Direktiva o pticah 2009/147/ES). Ta obsežna ekološka mreža, ki se je od leta 1995 vzpostavljala po celotni EU s predlogom za območja skupnostnega pomena in posebna zaščitna območja, se je do danes razvila s priznanjem območij z znatno biotsko raznovrstnostjo (živali, rastline in ekosistemi) kot posebnih ohranitvenih območij. Primerjali smo uradne podatkovne nize Natura 2000 in standardne podatkovne module za vsa posebna ohranitvena območja (SAC) in območja skupnega pomena (SCI) v regijah Kampanija (RC) v Italiji ter Tras-os-Montes e Alto Douro (TAD) na Portugalskem. Območja Natura 2000 pokrivajo 19 % italijanskega in 13 % portugalskega ozemlja. 76 % (RC) in 61 % (TAD) območij Natura 2000 so posebna ohranitvena območja (SAC)/območja posebnega pomena (SCI), medtem ko je 10 % (RC) in 36 % (TAD) območij posebnega pomena za ptice (SPA). 14 % (RC) in 3 % (TAD) so območja, ki izpolnjujejo pogoje za oba statusa. Biotska raznovrstnost živali obsega 248 vrst (205 ptic) v italijanski regiji in 121 vrst (85 ptic) v portugalski regiji. Leta 2024 je Kampanija sprejela načrte upravljanja, medtem ko jih regija Tras-os-Montes e Alto Douro še ni pripravila. Ključne besede območja Natura 2000, biotska raznovrstnost, biogeografske regije, načrti upravljanja 73 Acta Biologica Slovenica, 2026, Special issue Biodiversity Strategy for 2030, Italy (MASE, 2020) proposes the "National Biodiversity Strategy for 2030" and the Lusitanian Country (INCF, 2018) proposes the «Portugal's National Strategy for Nature Conservation and Biodiversity 2030». National Strategies align with the EU's Biodiversity Strategy and are being revised to address national challenges in eight intervention sectors (Protected Areas; Species, Habitats and Ecosys- tems; Food, Agricultural Systems and Livestock; Forests; Urban Greenery; Inland Waters; Sea; Soil). The Strategies are based on two objectives to be achieved by 2050: 1. “Build a coherent network of terrestrial and marine Protected Areas” with the achievement of the targets of 30% of protected areas to be established on land and at sea, and 10% of strictly protected areas. 2. “Restore terrestrial and marine ecosystems,” achieving the target of 30% restoration of the conservation status of habitats and species, particularly through activities conducted at the regional scale relating to the objec- tives and conservation measures of the Natura 2000 Network sites.” Aware that achieving the two main objectives of the SNB requires a multidisciplinary approach, in order to actively participate in the conservation of biodiversity in the third millennium, we decided to deepen our knowledge by comparing two European situations: Italy in the Mediter- ranean Biogeographic Region and Portugal in the Atlantic Biogeographic Region (EEA, 2024b). The vast ecological network established across the European Union, starting in 1995 with the proposal for Sites of Community Importance (SCIs) and Special Protection Areas (SPAs), has developed to the present day with the recognition of areas containing significant biodiversity (ani- mals, plants, ecosystems) as Special Areas of Conservation (SACs). To ensure the long-term maintenance of natural habitats and threatened or rare flora and fauna species at the EU level, specific management plans are required for each site (Bowman & Redgwell, 1995). Each site is officially described in a Standard Form containing all information deemed sensitive and worthy of conservation and protec- tion. The objective of this research was to analyse animal biodiversity records in Italian and Portuguese SCIs/SACs and SPAs of two regions, highlighting key discrepancies for approaching 2030 conservation objectives. Materials and Methods Data Sources and Collection The work began with consultation of official data and documentation provided by the technical-scientific bodies of the Italian (ISPRA, 2025) and Portuguese (MATE, 2025; MiTECO, 2025) ministries relating to biodiversity conserva- tion under the EU Habitats and Birds Directives. The cur- rently available databases refer to the third reporting cycle (2007-2012) and the fourth reporting cycle (2013-2018). For the study, the following were used: • Datasets published on Natura Viewer (https:// natura2000.eea.europa.eu). • Natura 2000 standard data form of all the sites analysed linked to Natura Viewer (EEA, 2024a); • https://natura2000.eea.europa.eu/Natura2000/SDF. aspx?site=PT... • The data was archived and sorted with the support of Mic- rosoft Excel software, ArcGIS 10.8.2 and ArcMap 10.8.2. • The SHP files were retrieved from official sources or from sites for users of mapping programs (ICNF, Portu- gal; Dati Open, Italy). Study Areas Two European regions were compared (Figure 1): 1) “Norte”, which represents 8.51% of the Portuguese surface area (7,898 km 2 /92,256 km 2 ), and 2) the “Cam- pania”, which represents 4.53% of the Italian surface area (13,671 km 2 /302,069 km 2 ). The Campania region is entirely included in the Med- iterranean Biogeographic Region (ITM), while the Norte region, which represents the westernmost tip of Europe, includes part of both the Mediterranean Biogeographic Region (PTM) and the Atlantic Biogeographic Region (PTA) (EEA, 2024b). Experimental plan The data for each Region of the two regions compared were: • Divided, as reported by Natura Viewer, into SCI/SAC and SPA. • Quantified terrestrial sites and removed marine ones. • Evaluated based on the characteristics of the different bioregions. 74 Acta Biologica Slovenica, 2026, Special issue • Quantified based on the biodiversity components of the two regions being compared. • Used to construct thematic comparison maps. • Extrapolated from the data reported in the Standard Data Forms for the following information: 1. Species. 2. Site assessment. 3. Management’s plan. The information relating to the "Species" concerns their unique identification within Europe. For experimental pur- poses, data relating to the following entries were selected and acquired: "group membership"; "unique identification code"; and "scientific name." All this information has allowed us to develop statistics demonstrating the quality of the information regarding species of community interest. Results Surfaces and management plans Out of 302,073 km 2 in a territorial extension of Italy, there are 0.76 sites/100 km 2 , which guarantee the Natura 2000 network with 57,394 km 2 or 19% of the national surface area. The same criteria highlight that, in Portugal, on a surface area of 92,391 km 2 , there are 0.13 sites/100 km 2 for a total of 19,424 km 2 , which guarantee 21% of the national surface to the Natura 2000 network. With the Habitats Directive (C.D., 1992) and the Birds Directive (C.D. 1979; E.C., 2009), the Council of the European Communities (now the Council of the European Union) set the goal of ensuring the long- term conservation of habitats and the species that live there, declaring them Sites and Species of Community interest. To reconcile environmental and local socioeconomic needs, the directives indicate management plans for Natura 2000 sites as an implementation tool. Most of the Italian Natura 2000 sites were proposed between 1992 and 1995 and were implemented after national implementation (DPR n. 357, 1997) until the SCIs were transformed into SACs (MASE, 2019). In Portugal, the process of establishing SCIs and SPAs began in 1997 (Decree law, 1999) and was intended to result in the establishment of 62 Special Areas of Conservation (SACs) and 42 Special Protection Areas (SPAs). Despite pres- sure from EU bodies, more than 30 years after the creation of Natura 2000 sites, Portugal continues to experience delays in finalising management measures for Natura 2000 areas. Indeed, the Norte region has not yet completed manage- ment plans for its 23 Natura 2000 sites. On the other hand, the Campania Region has only recently implemented 57 management plans for 93 SCIs/SACs (RCR, 2024) (Table 1). Figure 1. Number of Natura 2000 sites in Portugal and Italy. In circle the regions studied Slika 1. Število območij Natura 2000 na Portugalskem in v Italiji. V krogu so označene preučevane regije. 75 Acta Biologica Slovenica, 2026, Special issue Species referred to in Article 4 of Directive 2009/147/EC (Annex II of Directive 92/43/ EEC) & Other important species of flora and fauna The comparison between the two countries (Table 1) indicates that, respectively in Italy and Portugal, 76% and 74% of the Natura 2000 areas are SACs/SCIs, and that 12% and 26% are SPAs. Furthermore, in Italy, 12% are areas that include both conditions (SACs/SPAs). Of the 561 "Species referred to in Article 4 of Directive 2009/147/EC listed in Annex II of Directive 92/43/EEC & Other important species of flora and fauna" identified within the Natura 2000 areas of the two regions compared (Cam- pania, Italy, and Northern Portugal), our attention focused on animal biodiversity, consisting of 419 different species. Table 2 shows the number of animals present, broken down by class (Amphibians, Birds, Fish, Invertebrates, Mammals, and Reptiles), as described in the official Stan- dard Data Forms of the two countries. 377 species were identified in Campania, Italy, which falls entirely within the Mediterranean Biogeographic Region (ITM), and 162 in the Northern Region, Portugal. Of these, 34 species occur only in the Mediterranean Biogeographic Region (PTM), 38 only in the Atlantic Biogeographic Region (PTA), and 90 species are shared between the two r Lusitanian regions (PTM & PTA). In the two Portuguese biogeographic profiles, 16 and 7 species, respectively, are found only in the Mediterranean (PTM) or Atlantic (PTA). Portugal shares 19 species described in both the Mediterranean and Atlan- tic regions (PTM & PTA), while 71 species are found in all three (ITM, PTM & PTA). In the Mediterranean region (ITM & PTM) alone, the two countries share 18 species, while the Italian Mediterranean region shares 31 species with the Portuguese Atlantic region (ITM & PTA). Regardless of biogeographical region, the regions of the two European countries share 120 animal species. From Table 2, it can be deduced that among all species, birds are the most represented (61%), both in Italy (55%) and in Portugal (29%). Invertebrates and mammals are the most represented classes after birds, 12% and 10% in Italy, 3% and 3.6% in Portugal. Fish, reptiles, and amphibians each represent 4% of the species described in Italy. Lower percentages are described in Portugal (fish 2%, reptiles 0.7%, and amphibi- ans 0.5%). Amphibians and reptiles are the classes of the animal kingdom that most characterise habitats. In fact, only two amphibian species have been reported in Portuguese Natura 2000 sites (both Mediterranean and Atlantic), and 17 species have been described only in Italy. A similar situ- ation occurs for reptiles, with the exception of Emys orbicu- laris, which is described in all three situations. Fifteen fish species are described as occurring either only in Campania or only in the Norte. Conversely, the Italian region shares one fish species with the Portuguese Mediterranean region and two with the Atlantic. Invertebrate species are even more closely linked to habitat: only six species are shared across the three condi- tions, while 45 species are described only in Campania and Administrative Biogeographic RTS Natura 2000 Nat Nat Nat/RTS MP Region Region (ha) Type (n) (ha) (%) (n) Campania Mediterranean 1,361,928 SACs 93 323,111.69 23.72 44 Italy SPAs 15 178,766.23 13.13 9 SACs/SPAs 15 40,633.36 2.98 4 Norte Mediterranean 2,127,800 SACs 12 339,405.56 15.95 0 Portugal SPAs 4 212,662.30 9.99 0 Atlantic SACs 5 104,765.54 4.92 0 SPAs 2 66,824.72 3.14 0 Table 1. Regional Total Surface (RTS ha), Natura 2000 number (Nat n), Natura 2000 surface (Nat ha), percentage of Natura 2000/RTS (NAT/RTS %) and number of Management Plan (MP) of Special Areas of Conservation (SACs) and Special Protection Areas (SPAs) of Campania Region (Italy) and Norte Region (Portugal), within their respective Biogeographic Regions. Tabela 1. Skupna površina regije (RTS ha), število območij Natura 2000 (Nat n), površina območij Natura 2000 (Nat ha), odstotek območij Natura 2000 glede na skupno površino regije (NAT/RTS %) ter število načrtov upravljanja (MP) posebnih ohranitvenih območij (SAC) in posebnih zaščitnih območij (SPA) v regiji Kampanija (Italija) in regiji Norte (Portugalska) znotraj njihovih zadevnih biogeografskih regij. 76 Acta Biologica Slovenica, 2026, Special issue five in both Portuguese regions. Mammals have the highest percentage of species shared across the three conditions (24%), as well as the highest characterisation only in the Italian region (67%). For all species, it is not possible to estimate the quality of the population data (almost 100% are classified as "Data Deficient" or “Present”). The presence of species closely linked to regional hab- itats is particularly evident in Campania, where 68 of the species are described exclusively. In Portugal, the link to habitat appears less strong, with 13% of the Mediterranean species and 5.5% of the Atlantic species affected. The higher percentage of species appears to benefit from the transition from one biogeographical region to another. Discussion The international collaboration agreement between the University of Naples Federico II (Italy) and the University of Trás-os-Montes e Alto Douro (Portugal) has enabled the comparison of data regarding the presence of biodiver- sity in the Natura 2000 sites of the Campania and Norte regions, using the same methodology. The Mediterranean biogeographical region extends from the North African coast of the Maghreb to the south- ern border of the Norte region of Portugal. This situation prompted a comparison of the animal biodiversity present in one of the most characteristic habitats of southern Italy with that described in the Natura 2000 areas of the Portuguese region. However, since it was not possible to ignore the presence of habitats attributable to the Atlantic biogeographical region in the Norte region itself, the animal biodiversity present there was also taken into account. The most striking result of this comparison is the greater richness, expressed in the number of species described as present (+215), in Campania. Nonetheless, the 419 animal species described all deserve to benefit from the protection measures offered by the European Natura 2000 network. The second result, far more significant than the first, is the critical issues related to: 1) the insufficient presence (Campania) and total absence (Portugal) of management plans, a technical and admin- istrative tool that ensures the measures to be used to limit or eliminate threats to their favourable conservation status; 2) The limited or very limited knowledge of the number of individuals that make up the populations of the animal species described as present. Conclusions In conclusion, the analysis of the 146 Natura 2000 sites in Campania and the North indicates that it is impossible to choose any one strategy to halt biodiversity loss, ignoring much of the information regarding individual species described as present, but about which too little is known to support any management plan. It is also impossible to state with the data officially produced by the two regions that any animal species has suffered or is suffering drastic population declines or increases. Class Campania Norte Present in both regions Total (n) (n) (n) (n) Birds 133 25 98 256 Invertebrates 45 5 6 56 Mammals 30 3 12 45 Fishes 15 5 3 23 Reptiles 17 2 1 20 Amphibians 17 2 0 19 Total 257 42 120 419 Table 2. Number of Species per Class described in the Special Areas of Conservation (SACs) and Special Protection Areas (SPAs) of the Campania Region (Italy) and the Norte Region (Portugal). Tabela 2. Število vrst po razredih, opisanih v posebnih ohranitvenih območjih (SAC) in posebnih zaščitnih območjih (SPA) v regiji Kampanija (Italija) in regiji Norte (Portugalska). 77 Acta Biologica Slovenica, 2026, Special issue In the absence of reliable data, it is not possible to identify data quality information such as Type (individuals, pairs, or other units according to the standard list of pop- ulation units and codes in accordance with Articles 12 and 17); information on population size or Abundance (common, rare, very rare, present, data deficient); and Data Quality (Good, Moderate, Poor, Very Poor). Indeed, closer attention must be devoted to investi- gating the territorial shifts driven by climate change within the ecosystems and agroecosystems of the two regions. Furthermore, it is essential to highlight that the Natura 2000 sites in Campania and Norte serve as a catalyst for biodiversity-related initiatives, capable of mobilising a broad spectrum of ecosystem services. Author Contributions Conceptualisation, L.E.; methodology, A.C.M.C., L.E.; soft- ware, A.M.; validation, L.E., A.C.M.C.; formal analysis, L.E., A.C.M.C., A.M.; investigation, A.M., L.E.; resources, L.E.; data curation, L.E., A.M.; writing—original draft preparation, L.E., A.C.M.C., A.M.; visualisation, A.C.M.C.; supervision, L.E.; project administration, L.E., A.M. All authors have read and agreed to the published version of the manuscript. Acknowledgement The authors acknowledge the international agreement between the University of Naples Federico II (IT) and the University of Tras-os-Montes and Alto Douro (PT). Funding This research received no external funding. Data Availability Statement Research data was uploaded to Zenodo (https://doi. org/10.5281/zenodo.21312977). Conflicts of Interest The authors declare no conflict of interest. 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Kunming-Montreal Global Biodiversity Framework. https://www.unep.org/resources/kunming-montreal-global-biodiversity-framework 79 1 Department of Advanced Biomedical Sciences -University of Napoli Federico II, Italy, alessandra.pelagalli@unina.it 2 Institute of Biostructures and Bioimages, National Research Council, Naples, Italy 3 Department of Physics "Ettore Pancini" - University of Napoli Federico II, Italy, alessandro.pezzella@unina.it, pierluigi.casolaro@unina.it 4 Department of Veterinary Medicine and Animal Productions - University of Napoli Federico II, Italy, raffaele.marrone@unina.it, caterina.squillacioti@unina.it 5 Department of Agricultural Sciences (general and applied entomology) - University of Napoli Federico II, Italy, andrea.becchimanzi@unina.it 6 Master Innovative Systems for the Conservation of Euro-Mediterranean Fauna, Italy, Luciana.cozzolinovet@gmail.com, francesco.caso.ext@lozoodinapoli.it * Corresponding author: E-mail address: alessandra.pelagalli@unina.it; francesco.caso.ext@lozoodinapoli.it Citation: Pelagalli, A., Pezzella, A., Becchimanzi, A., Marrone, R., Squillacioti, C., Caso, F., Cozzolino, L., Pierluigi Casolaro, P. (2026). Shedding new light on bioluminescence. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.29138 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Shedding new light on bioluminescence Alessandra Pelagalli 1,2 * , Alessandro Pezzella 3 , Andrea Becchimanzi 4 , Raffaele Marrone 5 , Caterina Squillacioti 5 , Francesco Caso 6 * , Luciana Cozzolino 6 , Pierluigi Casolaro 3 Abstract Bioluminescence is an essential trait of several organisms, prevalent in marine environments but rare on land and almost nonexistent in freshwater. This often poses a challenge to scientific research studying the phenomenon of bioluminescence. Specifically, one of the main unknowns concerns the specific mechanism and variations of bioluminescence among different species, as well as the factors controlling its intensity and colour . We review the advances in molecular biology, biochemistry, genetics and quantum mechanics which provide new tools for exploring these phenomena. Within the project 'OneAquaHealth' (https://www. oneaquahealth.eu/), sampling of arthropod species producing bioluminescence was carried out along the banks of the streams chosen as sampling sites for biodiversity (WP2) in the urban area of Benevento. During sampling, one species of arthropod (Lampyris fuscata) was encountered, and it is of particular interest in consideration of the specific environment of the investigated area, rich in biodiversity and of significant naturalistic value. These results associated with the project aim stimulate further research towards other terrestrial animalia showing the biochemical phenomenon of luminescence. The "Bioelectronics Task Force" of the University of Federico II works with a multidisciplinary research approach that evaluates the interactions and connections between biological subjects and electronic objects, and promoted this study. Keywords Bioluminescence; Lampyridae; excited electronic state; luciferin; real-time imaging of molecular events; energy emission 80 Acta Biologica Slovenica, 2026, Special issue Introduction Bioluminescence is a process that generates light through chemical reactions and is largely diffused in a wide range of organisms, ranging from bacteria to invertebrates. Since its discovery as a chemical process based on an enzymatic reaction of luciferase, which uses luciferin as a substrate and a light-emitting molecule by Raphaël Dubois in the 1880s (Dubois, 1885; Shimomura, 2006), extensive research studies have been focused on analysing differ- ences in the organisms producing bioluminescence, which is more prevalent in marine environments than on land. The evolution of the process in many different threads has suggested that it adapts easily to the environment (McKenna et al., 2015; 2019). In fact, from an evolutionary point of view, bioluminescence in several luminous organ- isms has several roles, including defensive and offensive functions such as repelling an attacker or attacking prey, camouflage through counter illumination, and inter- and intra-specific communication (Carlson and Copeland, 1985; Haddock et al., 2010; Zarubin et al., 2012; Ramesh and Mohanraju, 2019). Yet, other biological functions are still often unknown (Mallefet and Shimomura, 1995). Moreover, the specific evolutionary pathway that has driven the diversification of bioluminescence, as well as the specific mechanisms deployed by different species and how they adapt their light sources in response to the environment, are not fully understood and recently represent an area of active research. However, it is also important to emphasise that the discovery of “bioluminescence proteins” fostered a stream of research in the non-invasive method for biolu- minescence imaging (BLI) for monitoring and collection of qualitative and quantitative data of biological processes at the cellular and molecular levels in a living organism, such as an animal model of disease. This technology has evolved over several years, becoming an important research tool, improving the knowledge of biological processes in vivo and in real-time. BLI is widely performed with preclinical rodent models to understand disease processes and eval- uate potential cell-or gene-based therapy. Apart from these considerations for using biolumines- cence in several research and medical approaches, a devel- oping interest is the characterisation of several light-emitting systems in land organisms (particularly insects), also in rela- tion to the environment where they live. Insects represent one of the most diverse orders of organisms in terms of biochemistry (Viviani V., 2002), and among the Coleoptera order, fireflies (Lampyridae) are characterised by typical yel- low-green flashes produced from ventral lanterns of females for mating purposes (Viviani, 2002; Wood, 1995). Differently, the adult males of fireflies do not emit luminescence or, in a weak form, while in the larval stage and larviform, female luminescence is intense (Viviani, 2002; Wood, 1995). The specific reaction of bioluminescence implies that the mol- Novo razumevanje bioluminiscence Izvleček Bioluminiscenca je izjemno zanimiv pojav v živalskem kraljestvu, saj je ta specifičen biološki mehanizem številnim živalskim vrstam omogočil prilagoditev na različna naravna okolja, zlasti na vodne ekosisteme. Ta morfološko- funkcionalna prilagoditev je igrala ključno vlogo v evolucijskih procesih. Namen pričujoče raziskave je poglobljeno preučiti ta pojav in razširiti znanstveno razumevanje, s posebnim poudarkom na študiju luciferaze—encima, ki je bistven za oksidacijo luciferina in uravnava emisijo energije v obliki specifičnih valovnih dolžin prek eksergonskih reakcij. Energija, sproščena v tem biokemičnem procesu, lahko predstavlja osnovo za nadaljnje raziskave mehanizmov, ki nadzorujejo ta naravni pojav, z možnimi prihodnjimi aplikacijami na okoljskem, zdravstvenem in farmacevtskem področju. Poleg tega spremljanje ciljne vrste v tej študiji (Lampyris fuscata) predstavlja pomembno orodje za ocenjevanje kakovosti okolja prek opazovanja teh izjemnih organizmov. Ključne besede Bioluminiscenca; kresnička; vzbujeno elektronsko stanje; luciferin; slikanje molekularnih dogodkov v realnem času; emisija energije 81 Acta Biologica Slovenica, 2026, Special issue ecule d-luciferin is first activated by ATP and magnesium ions as cofactors, to be then oxidised by molecular oxygen; carbon dioxide, AMP, pyrophosphate and oxyluciferin in an electronically excited state are released (Figure 1). Based on this reaction, the firefly luciferase has been used for methods involving ATP measurements applied to monitor biomass, as well as to analyse biological fluid microbial contamination or to assess cell viability (Camp- bell, 1988). However, at the time, several questions remain, including the Lampyridae geographical distribution in Italy as well as the specific habitat where they live and the impact of environmental change and light pollution (Viviani et al., 2010; Owens et al., 2022). Fireflies are increasingly recognised as valuable bioin- dicators of environmental quality, extending their relevance beyond insect conservation to a broader One Health framework that integrates ecosystem, animal, and human well-being. Many firefly species are tightly associated with wetlands, riparian zones, and moist environments at the interface between terrestrial and aquatic ecosystems, habitats that are particularly sensitive to urbanisation and hydrological alteration (Kazama et al. 2007). The depen- dence of firefly life stages on consistently moist conditions around streams, rivers, wetlands, and lakes makes their presence indicative of intact hydrologic regimes and suitable microclimatic conditions. Importantly, larval stages develop in soils and sediments adjacent to freshwater bodies and are therefore exposed to changes in water and soil physicochemical properties, allowing firefly popula- tions to reflect local water quality and ecosystem integrity (Abdullah et al. 2025, Ghosh and Chakraborty 2026). The study was conducted as part of the larger "OneA- quaHealth" (OAH) project, whose primary objective is to apply the One Health concept to improve the sustainability, integrity, and restoration potential of aquatic ecosystems, particularly in urban settings (https://www.oneaquahealth. eu/). Collecting information and sampling aquatic eco- system biodiversity allows us to use living organisms as biological models and bioindicators. Specifically, this study aims to collect data on the pos- sible presence of Lampyridae along stream banks (OAH sampling sites) in the urban area of Benevento. In this regard, a part of the project (designed as WP2) was focused on the detection of arthropod species (includ- ing Lampyris fuscata) (Audisio et al., 1995; De Jong, 2014) whose presence has already been reported in the most sheltered riparian zones of the urban area of Benevento (Pignataro and Vicidomini, 2005). By combining repeated firefly activity surveys across sites and years, we assessed spatial and temporal determinants of firefly abundance in riparian ecosystems. These data allow for a consideration of the health status of this natural environment as well as the advances in molecular biology, biochemistry, genetics and quantum mechanics relative to the bioluminescence, which provide new tools for exploring these phenomena. Figure 1. Mechanism of bioluminescence in insects (Coleoptera). Slika 1. Mehanizem bioluminiscence pri žuželkah (Coleoptera). 82 Acta Biologica Slovenica, 2026, Special issue Materials and Methods Investigation area The study was conducted as part of the larger "OneA- quaHealth" project, which investigates urban aquatic ecosystems at the European level. In conjunction with the assessment of faunal biodiversity, the presence of Lampyr- idae was studied along the banks of four different streams in the province of Benevento (Southern Italy): San Nicola (41.126785 N - 14.803334 E); Corvo-Serretelle (41.069210 N - 14.709592 E); Lossauro (41.132210 N - 14.7127 E); and Jenga (41.15126 N - 14.709361 E) (Figure 2). These sites cover an area of approximately 40 km² and are characterised by a typically Mediterranean climate, with hot, dry summers, humid autumns, and winters with long rainy periods. December is the wettest and humidest month, while July is the driest. The average annual rainfall is approximately 920 mm (Magliulo et al., 2022). Field Equipment and Sampling Procedures Sample collection was carried out during the survey period (February 2023-September 2024). The preliminary phase involved characterising the sampling sites by measuring var- ious parameters (e.g., GPS geolocation, air and water tem- perature of watercourses, identification of certain sources of pollutants, macrophytes and riparian vegetation, etc.). After this preliminary data collection, the OAH project envisioned sampling the biodiversity of urban aquatic ecosystems through the application of specific and shared protocols for determining diatoms, the epilithic and water microbiome, benthic macroinvertebrates, fish, amphibians, insects, ticks, spiders, and birds. Adult fireflies (Lampyridae) were collected in the selected riparian habitats using entomological nets during daylight hours (Fig. 3). Captured individuals were immedi- ately transferred to plastic tubes and subsequently iden- tified to species level based on morphological diagnostic characters, following the species description for Lampyris fuscata. Sampling during daylight was qualitative and aimed at species identification rather than abundance estimation. At twilight, firefly abundance was estimated through visual surveys of light signals (Cratsley and Lewis 2005, Picchi et al. 2013). Two linear transects (100 × 50 m each) were established along the left and right banks of the river, for a total of approximately 10,000 square meters for each sampling site. Two teams of two operators each walked the transect and noted the individual light signals in a notebook. The operation was repeated three times over a two-hour period, approximately 20 minutes apart (Figure 3). Because light emissions could not be unambiguously attributed to L. fuscata, these observations were used as a proxy for overall firefly activity rather than species-specific abundance. Figure 2. Localisation of the sampling sites (yellow solid dots) for analysis of fauna biodiversity and the presence of Lampyridae. Five site samples of the Benevento Research site of the OneAquaHealth project are reported in the table. Slika 2. Lokacije mest vzorčenja (rumene polne pike) za analizo biotske raznovrstnosti favne in prisotnosti družine Lampyridae. V tabeli so navedeni vzorci s petih lokacij raziskovalnega območja Benevento v okviru projekta OneAquaHealth. 83 Acta Biologica Slovenica, 2026, Special issue During the OneAquaHealth sampling, several parame- ters related to the aquatic habitats involved in the urban context of Benevento were monitored. Five sampling sites were chosen to assess the presence of fireflies as a com- ponent of natural biodiversity despite the anthropogenic pressure exerted by urbanisation (Figure 4). Once back in the laboratory, a dataset of repeated measurements was compiled to: (i) assess the coverage and any biases in the available firefly distribution data at the study sites; (ii) calculate the density per hectare at each site and during the different sampling periods. Statistical Analyses All analyses were conducted in R (version ≥ 4.0.0). Firefly abundance data consisted of count observations and were analysed using models assuming a negative binomial error distribution to account for overdispersion. Seasonal pat- terns in abundance were analysed using generalised linear mixed models (GLMMs) fitted with the glmer.nb function in the lme4 package. Year, Month, and their interaction were included as fixed effects, while Stream was included as a random intercept to account for repeated measurements within localities and to model between-stream variability in baseline abundance. To test for differences in firefly abundance among streams, we additionally fitted generalised linear models (GLMs) using the glm.nb function from the MASS package, treating Stream, Year, and Month as fixed effects. Pairwise comparisons among streams were performed using esti- mated marginal means with the emmeans package, and con- trasts were expressed as abundance ratios on the response scale. Statistical significance was assessed at p < 0.05. Results The results of our study indicated that the Lampyridae population of the five sampled riparian sites of Benevento province is dominated by L. fuscata, with 100% specimen collected belonging to this species. However, light signals emitted during twilight were used as a proxy for fireflies' abundance in general. Using a GLMM with Year and Month (including polyno- mial terms) as fixed effects and Stream as a random effect, we found a significant quadratic effect of Month on firefly abundance, indicating peak activity in mid-season (Fig. 5). Indeed, firefly abundance exhibited a pronounced seasonal pattern characterised by a strong quadratic trend (p < 0.001). No significant differences were detected between years (p = 0.87), nor were year-by-month interactions signif- icant (p = 0.20), suggesting stable phenology across years. Substantial variation among localities (variance = 0.442, SD = 0.665) indicates that streams differ consistently in their overall abundance levels, even after accounting for year and seasonal trends. Figure 3. Five sites’ samples of the Benevento Research site of the OneAquaHealth project were investigated for firefly detection. Slika 3. V okviru projekta OneA- quaHealth so bili za odkrivanje svetlikavcev preiskani vzorci s petih lokacij raziskovalnega območja Benevento. 84 Acta Biologica Slovenica, 2026, Special issue Figure 4. Five of the twenty sampling sites in Benevento were used to evaluate the presence of fireflies as a component of the faunal bio- diversity of the OneAquaHealth project. Slika 4. Pet od dvajsetih mest vzorčenja v Beneventu je bilo uporabljenih za oceno prisotnosti kresničk kot sestavnega dela biotske raz- novrstnosti v okviru projekta OneAquaHealth. Figure 5. Seasonal phenology of firefly abundance at OAH sampling sites in the Benevento area. Lines represent mean monthly abundance (number of light signals per hectare) recorded from April to September in 2023 and 2024. Slika 5. Sezonska fenologija številčnosti svetlikavcev na mestih vzorčenja OAH v okolici Beneventa. Črte prikazujejo povprečno mesečno številčnost (število svetlobnih signalov na hektar), zabeleženo od aprila do septembra v letih 2023 in 2024. 85 Acta Biologica Slovenica, 2026, Special issue These results were confirmed by the GLM, which revealed a strong effect of Month on firefly abundance (χ² = 816.64, df = 5, P < 0.001), reflecting pronounced sea- sonal variation in counts. The main effect of Year was not significant (χ² = 0.49, df = 1, P = 0.48), indicating comparable overall abundance levels between 2023 and 2024. The Month × Year interaction was also not significant (χ² = 3.44, df = 5, P = 0.63), suggesting that seasonal phenology was broadly consistent across years, with no detectable shifts in the timing or shape of abundance patterns. Firefly abun- dance differed significantly among streams (χ² = 326.40, df = 4, P < 0.001), indicating substantial spatial heterogeneity. To further explore these spatial differences, post-hoc pairwise comparisons of estimated marginal means were conducted (Table 1). Lossauro consistently showed the highest abundance, differing significantly from all other sites, whereas Cretarossa and Ponticelli generally sup- ported lower abundance levels. In contrast, no significant difference was detected between Corvo Tre Santi and Jenga, or between Cretarossa and Ponticelli. For each stream and year, we derived descriptive ecological metrics including total abundance, timing and magnitude of peak activity, season length, and seasonality indices based on monthly mean counts (Table 2). Firefly activity peaked in June in 2023 at all streams, whereas in 2024, peak abundance shifted to July at most sites (Table 2). Activity duration ranged between four and five months, with only minor interannual variation, while seasonality indices indicated a more pronounced peak at Lossauro and a more diffuse activity pattern at Ponticelli. Notably, the lowest mean number of fireflies per hect- are was recorded at the Cretarossa and Ponticelli sites (San Nicola stream), which are located within the urban area of Benevento. Seasonal counts increased from the Corvo Tre Santi site (Corvo Serretelle stream), flowing through an industrial area, toward the two less urbanised sites (Los- sauro and Jenga streams). Discussion Our results reveal pronounced spatial heterogeneity in fire- fly abundance across streams, with consistent differences among sites despite broadly similar seasonal dynamics. Both the generalised linear model and the phenological descriptors indicate that Lossauro and Jenga, the least urbanised streams, supported the highest firefly abun- dances, whereas Cretarossa and Ponticelli, located within the urban area of Benevento, consistently exhibited the lowest abundance levels. These patterns are in line with a growing body of evidence showing that firefly populations are negatively affected by urbanisation and associated Contrast Ratio 1 SE Z ratio P-value 2 Corvo tre santi / Cretarossa 2.304 0.2790 6.890 <0.0001 Corvo tre santi / Jenga 0.788 0.0834 -2.247 0.1626 Corvo tre santi / Lossauro 0.507 0.0518 -6.646 <0.0001 Corvo tre santi / Ponticelli 3.089 0.3950 8.820 <0.0001 Cretarossa / Jenga 0.342 0.0407 -9.008 <0.0001 Cretarossa / Lossauro 0.220 0.0255 -13.046 <0.0001 Cretarossa / Ponticelli 1.341 0.1860 2.115 0.2136 Jenga / Lossauro 0.643 0.0640 -4.438 <0.0001 Jenga / Ponticelli 3.918 0.4930 10.845 <0.0001 Lossauro / Ponticelli 6.097 0.7510 14.678 <0.0001 Table 1. Pairwise contrasts of estimated marginal means of firefly abundance among sampling sites derived from a generalised linear model. Ratios indicate relative differences in abundance between sites. Tabela 1. Parni primerjavi ocenjenih mejnih povprečij številčnosti svetlikavcev med vzorčnimi mesti, izpeljani iz splošnega linearnega modela. Razmerja kažejo relativne razlike v številčnosti med posameznimi mesti. 1 Ratios > 1 indicate higher abundance at the first site listed in the contrast; ratios < 1 indicate higher abundance at the second site; 2 P-values were adjusted for multiple comparisons. 86 Acta Biologica Slovenica, 2026, Special issue Stream Year Seasonal Count 1 Peak Month Max Monthly Abundance Activity Duration Months 2 Seasonality Index 3 Corvo tre santi 2023 253 Jun 94 5 2.23 2024 249 Jul 91 4 2.19 Cretarossa 2023 101 Jun 39 5 2.32 2024 114 Jun 42 4 2.21 Jenga 2023 304 Jun 104 5 2.05 2024 313 Jul 115 5 2.20 Lossauro 2023 555 Jun 241 4 2.60 2024 584 Jul 246 4 2.53 Ponticelli 2023 74 Jun 23 4 1.86 2024 84 Jul 30 5 2.14 Table 2. Phenological and abundance descriptors of firefly populations across streams and years. Tabela 2. Fenološki kazalniki in kazalniki številčnosti populacij svetlikavcev po potokih in letih. 1 Seasonal count represents the cumulative number of individuals recorded from April to September; 2 Activity duration (months) was calculated as the number of months in which mean abundance was at least 10% of the seasonal maximum; 3 The seasonality index was computed as the ratio between maximum and mean monthly abundance, with higher values indicating stronger tempo- ral concentration of activity. habitat alterations, including artificial light at night, channel modification, and reduced riparian vegetation cover (Lewis et al. 2020, Owens and Lewis 2022). Intermediate abundance levels observed at Corvo Tre Santi, a stream flowing through an industrial area, further suggest that firefly populations respond along a gradient of anthropogenic disturbance rather than showing a simple urban–non-urban dichotomy. Similar gradients have been documented in both European and North American firefly species, where habitat quality, moisture availability, and landscape connectivity play key roles in determining local population size (Faust and Faust 2014, Fallon et al. 2021, Lewis et al. 2024). Taken together, these findings support the interpretation that stream-associated habitats with lower urban pressure and greater ecological integrity provide more suitable conditions for sustaining firefly populations. The seasonality index further highlighted differences among streams, with Lossauro showing the highest values, indicative of a more pronounced and synchronised peak, and Ponticelli exhibiting lower values, consistent with a more diffuse temporal distribution of activity. Such differ- ences may arise from variation in larval habitat suitability, hydrological stability, or disturbance regimes, which can influence both developmental synchrony and adult emer- gence patterns (Kazama et al. 2007, Lewis et al. 2020). Indeed, firefly occurrence and abundance correlate with indicators of freshwater health, supporting their use as sentinels of environmental degradation in riparian and wet- land systems (Kazama et al. 2007, Ghosh and Chakraborty 2026). In Southeast Asia, Pteroptyx fireflies have been widely studied as bioindicators, with population dynamics strongly influenced by water quality, soil characteristics, and anthropogenic disturbance in riparian habitats (Abdullah et al. 2025). Because freshwater ecosystem degradation has cascading effects on biodiversity, vector ecology, and human health, fireflies offer a unique opportunity to monitor ecosystem deterioration at the land–water interface. Their sensitivity to hydrological disruption, pollution, and urban encroachment positions them as integrative indicators within a One Health vision, linking habitat quality, biodi- versity conservation, and the sustainable management of freshwater ecosystems. Firefly activity showed a well-defined seasonal pattern, with peak abundance occurring in June-July at all sites, as commonly reported for temperate firefly species (Picchi et al. 2013, Camerini 2025). The strong effect of the month detected by the GLM confirms that seasonality is the dominant driver of variation in firefly counts, reflecting synchronised adult emergence and mating activity. Despite this strong seasonal signal, neither the main effect of year nor the year × month interaction was significant, indicating that overall abundance levels and 87 Acta Biologica Slovenica, 2026, Special issue seasonal dynamics were largely consistent between 2023 and 2024. This temporal stability is further supported by the phenological metrics, which showed comparable seasonal counts and activity durations across years within each stream. Such interannual consistency suggests that, during the study period, environmental conditions were sufficiently similar to maintain stable firefly phenology. From a conservation perspective, our findings reinforce the role of stream-associated habitats as key refugia for fire- fly populations, particularly in landscapes subject to urban expansion. The consistently low abundance recorded at urban streams highlights the vulnerability of fireflies to habitat degradation and supports calls for integrating light pollution management, riparian restoration, and habitat connectivity into urban planning (Lewis et al. 2020). Methodologically, the combined use of GLM-based inference (Owens and Lewis 2022) and simple pheno- logical descriptors proved effective in capturing both broad-scale patterns and fine-scale ecological differences among sites. This approach may be particularly useful for monitoring programs, as it balances statistical robustness with ecological interpretability. The detection of L. fuscata along the riparian sites in the urban area of Benevento revealed interesting aspects of considerable significance for both ecological research and conservation efforts. Historically, L. fuscata was considered a potentially declining species, endemic to Southern Italy, with limited documented occurrences in northern and cen- tral regions (Fanti and Improta 2020, Fanti 2022). Its report in the Benevento area extends the known distribution range of this species and suggests that populations may be more widespread and resilient than previously thought. This study provides new occurrence data, crucial for understanding the biogeographical patterns of Lampyridae in Italy. Conclusions In summary, firefly populations in the Benevento area exhibited strong and consistent seasonal phenology, coupled with marked spatial variation in abundance linked to stream context and degree of urbanisation. While interannual differences were limited over the two-year study period, subtle shifts in peak timing suggest potential sensitivity to environmental variation. Continued long-term monitoring across urban–rural gradients will be essential to better understand the drivers of firefly population dynamics and to inform effective conservation strategies. The presence of this species of arthropod is of partic- ular relevance, and it is evidence of an important adaptive process as a consequence of changed environmental conditions (principally the urbanisation). It supports the potential use of L. fuscata as a bioindicator of environ- mental quality and biological light energy use, contributing to greener, more sustainable production models. The sampling of this arthropod opens new interdisciplinary research avenues, where bioelectronics and neuroscience may intersect with ecology. The experience gained at the OAH research site in Benevento has encouraged the «Bioelectronics Task Force» at the University of Naples Federico II to identify potential model organisms for integrating natural resources into bioelectronic systems and applying the results to experimental neurobiology. The study of microfauna and meiofauna further enables the exploration of other arthropod taxa exhibiting similar bioluminescent or light-associated adaptations, including elaterids, glowworms, mycetophilid flies, centipedes, milli- pedes, and annelids. Certainly, the research on biolumines- cence and organisms producing bioluminescence requires further investigation with the aim of encompassing multiple fields (from the discovery of new light emission mecha- nisms with application in biotechnology to the possible use of this species as a biomarker in the field of stress ecology). It is noteworthy that bio-electronic associated with the development of devices based on bioluminescence is a rapidly expanding field with potential and applications in medicine and in bioanalysis. These new aspects derive from positive efforts using modified or split firefly lucifer- ases developed for the detection of pathogenic bacteria and viruses, for evaluation of protein–protein interactions, assaying of metabolites involved in cell communication and cell signalling (Smirnova and Ugarova, 2017). In addition, taking advantage of the unique properties of bioluminescence (BL), more interesting projects for developing advanced tools and technologies for holistic water monitoring have been launched. BL biosensors can facilitate early detection of water contamination, enabling prompt intervention measures to mitigate risks to the environment and human health (Gregucci et al., 2023). The production of these biosensors can be improved by inte- grating them into wireless sensor networks to monitor the environment and, thus, increase the potential for resource recovery, supporting a circular economy 88 Acta Biologica Slovenica, 2026, Special issue Author Contributions Conceptualisation, A.Pelagalli, A. Pezzella, C.S. and P.C.; methodology, A.Pelagalli, A.B. and C.S.; investigation, F.C. and L.C.; resources, A. Pezzella and P.C; data curation, F.C., R.M. and L. C.; writing—original draft preparation, A.Pelagalli and C.S.; writing—review and editing, A.Pelagalli, A. Pez- zella, A.B., C.S. and P.C.; project administration, A. Pelagalli, R.M.; funding acquisition, A. Pezzella. All authors have read and agreed to the published version of the manuscript. Acknowledgement The author would like to acknowledge the OneAquaHealth project for the access to reports and information as well as the Bioelectronics Task Force of the University of Naples Federico II, which allowed us to use its Pathogen Readi- ness Platform for CERIC-ERIC (prp) Research Infrastructure funded by the Italian National Recovery and Resilience Plan (PNRR) to study human, animal, and plant pathogens with Maldi-Tof technology. Funding This research was funded by the European Union Horizon- CL6-2022-GOVERNANCE-01-09, grant number 101086521 – Protecting Urban Aquatic Ecosystems to Promote One Health -OneAquaHealth. Data Availability Research data was uploaded to Zenodo (https://doi. org/10.5281/zenodo.21313007). Conflicts of Interest The authors declare no conflict of interest. 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Proceedings of the National Academy of Sciences, 109, 853–857. https://doi.org/10.1073/pnas.1116683109 90 1 Department of Veterinary Medicine and Animal Productions - University of Napoli Federico II, Italy. 2 Avellino Hunting District (ATC Avellino), Italy. 3 Department of Forest Sciences and Landscape Architecture-CIFAP/UTAD, University of Trás-os-Montes, Vila Real – Portugal. * Corresponding author: E-mail address: nadia.piscopo@unina.it Citation: Piscopo, N., Esposito, L., Capitanio, F., Raffaele, A., Lombardi, A., Monzon Capapé, A. (2026). Ecosystem services and disservices related to the presence of wild boar in Avellino province, Italy. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.29139 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Ecosystem services and disservices related to the presence of wild boar in Avellino province, Italy Nadia Piscopo 1 * , Luigi Esposito 1 , Fabian Capitanio 1 , Antonio Raffaele 2 , Alessia Lombardi 1 , Aurora C. Monzon Capapé 3 Abstract The study consists of three phases distributed over 4 years of observation, and demonstrates that wild boar not only generates ecosystem disservices but also provides measurable ecosystem services. In the first phase, population size was estimated within a representative study area using integrated census methods. Concurrently, the presence of biodiversity elements listed in the Standard Data Forms of Natura 2000 sites within the study area was verified. The second phase involved the construction of a comprehensive dataset including both negative and positive impacts attributable to wild boar activity. In the third phase, these data were processed and evaluated using SimaPro software to perform a structured ecosystem services and disservices assessment. Ecosystem disservices were quantified as agricultural damage (€429,300) and road traffic accidents (€25,000). Ecosystem services were estimated as hunting-related revenue (€214,639) and cultural ecosystem benefits (€427,554). Overall, the balance between services and disservices indicated a net positive value of €187,893 of ecosystem services, although the habitat and biodiversity component could not be quantified. These findings highlight the importance of adopting a multi-level valuation approach for agroecosystems, demonstrating that wild boar populations generate both costs and benefits that should be jointly considered in sustainable wildlife management and ecosystem service assessment frameworks. Keywords wild boar, ecosystem service, biodiversity, agroecosystem 91 Acta Biologica Slovenica, 2026, Special issue Introduction The "Millennium Ecosystem Assessment" project (Reid et al., 2005) introduced the definition of "Ecosystem Services." An ecosystem can provide "direct" and "indirect" benefits to biodiversity (supply, regulation, cultural, habitat-biodi- versity), which, unlike agri-food products, are not easily converted into monetary value (Esposito et al., 2023). The proper functioning of an agroecosystem depends on the balance established between natural communities and human activities (Caporali et al., 2009). Humans consider the wild boar (Sus scrofa) one of the most invasive species on the planet (Lowe et al., 2000) and attribute negative pressure (ecosystem disturbance) to it (Geisser & Reyer, 2004; Herrero et al., 2006; Amici et al., 2012; Ficetola et al., 2014; Laznik & Trdan, 2014), impacting all existing biodiversity, both that of particular conservation interest (threatened or endangered species) and that considered less significant (species of least concern). Almost all existing literature describes the wild boar as a demographic surplus and a culprit in damage to agriculture, as well as, more recently, road accidents and the invasion of urban centres (Primi et al., 2009; Kruuse, Enno & Oja, 2016; Castillo-Contreras et al., 2021). Residents of the 27 European Union countries express a unanimous opinion, but have never considered the wild boar phenomenon in its entirety (Massei et al., 2014). The first question that remains unanswered is: how many wild boars are present in Italy? Some answers are ineffective: those who say "many" or "too many" don't even deserve attention. However, when the answer comes from national environmental monitoring bodies, it's time to start thinking. ISPRA (2023) reports several 2 million, and even then, this number is derived by making an assumption. Our model suggests starting from an area of known size for a period of no less than three years. It seems clear that future human activities must be closely linked to the compatibility between economic development and environmental protection. It is precisely in this context that "ecosystem services" come into play, for which, however, it is still difficult to find a classification capable of clearly quantifying and economically mapping the services that ecosystems provide to biodiversity, with humanity at the forefront. To standardise and hierarchically classify ecosystem services for their quantification and eco- Ekosistemske storitve in negativni vplivi, povezani s prisotnostjo divjih prašičev v provinci Avellino v Italiji Izvleček Študija obsega tri faze, razporejene v štirih letih opazovanja in dokazuje, da divji prašiči ne povzročajo le negativnih vplivov na ekosistem, temveč zagotavljajo tudi merljive ekosistemske storitve. V prvi fazi je bila na reprezentativnem območju raziskave z uporabo integriranih metod popisa ocenjena velikost populacije. Hkrati je bila preverjena prisotnost elementov biotske raznovrstnosti, navedenih v standardnih obrazcih za podatke območij Natura 2000 znotraj območja raziskave. Druga faza je vključevala oblikovanje obsežnega niza podatkov, ki je zajemal tako negativne kot pozitivne vplive, pripisane dejavnosti divjih prašičev. V tretji fazi so bili ti podatki obdelani in ovrednoteni s programsko opremo SimaPro, da bi izvedli strukturirano oceno ekosistemskih storitev in negativnih vplivov. Negativni vplivi na ekosistem so bili količinsko opredeljeni kot škoda v kmetijstvu (429,300 €) in prometne nesreče (25 000 €). Ekosistemske storitve so bile ocenjene kot prihodki, povezani z lovom (214,639 €), in kulturne koristi ekosistema (427,554 €). Na splošno je bilanca med storitvami in negativnimi učinki pokazala neto pozitivno vrednost ekosistemskih storitev v višini 187,893 €, čeprav komponente habitata in biotske raznovrstnosti ni bilo mogoče količinsko opredeliti. Ti izsledki poudarjajo pomen uporabe večplastnega pristopa k vrednotenju agroekosistemov in kažejo, da populacije divjih prašičev ustvarjajo tako stroške kot koristi, ki jih je treba skupaj upoštevati v okvirih trajnostnega upravljanja divjadi in ocenjevanja ekosistemskih storitev. Ključne besede divji prašič, ekosistemska storitev, biotska raznovrstnost, agroekosistem 92 Acta Biologica Slovenica, 2026, Special issue nomic valuation, the European Environment Agency (EEA, 2010) decided to identify the final services that derive from supporting services and narrowed the categories of trilat- eral services (provisioning, regulation, and maintenance). For the foreseeable future, the European Union has based its environmental policies on the concrete integration of ecosystem services into its efforts for sustainable develop- ment. It is no coincidence that the "EU Biodiversity Strategy 2030," a continuation of the 2020 EU Biodiversity Strategy, is a long-term plan that, through specific actions and commitments, aims to reverse ecosystem degradation and begin the process of recovering Europe's biodiversity by 2030. With the "Mapping and Assessment of Ecosystems and their Services" (MAES, 2020) initiative, the EU asked Member States to map and assess the state of ecosystems and ecosystem services within their respective national territories. The "Common International Classification of Ecosystem Services" (CICES) provides guidelines and indicators for identifying ecosystem types and assessing their status (https://cices.eu). In the above context, in the period 2019-2022, our study began the complex process of identifying Ecosystem Services/Disservices starting from the wild boar species (Sus scrofa) and evaluating the prob- lems related to the conflict between wild boar and humans, as well as the potential benefits associated with it. Materials and Methods Choice of the area of investigation Our investigation focuses on the Mediterranean bio- geographical region, one of the three ecoregions of the Palearctic ecozone represented in Italy. Together with the Alpine and Continental regions, it constitutes one of the most biologically diverse and densely populated areas on Earth outside the tropics. This study was developed on the premise that a key component of the European Union's biodiversity conser- vation strategy is the assessment of forest ecosystem conditions and the services they provide. Research is based on a forest sample site included in the Natura 2000 network and examines a biodiversity component generally regarded as highly detrimental to many habitat types: the wild boar (Sus scrofa). The analysis of data collected over a four-year period has enabled us to provide information and methodological tools for identi- fying ecosystem services associated with the presence of this species, while considering its role within a balanced ecological framework in the study area. The results con- tribute to the implementation of the EU Forest Strategy and suggest potential approaches for assessing and mitigating biodiversity loss. Furthermore, we propose a method for assigning economic value to the wild boar, which may be used in future assessments of the ecosystem services provided by this species. The study area forms part of a larger territory covering approximately 31,800 hectares, identified as “Sistema Territoriale Rurale (STR) 18 – Monte Partenio–Monti di Avella–Pizzo d'Alvano.” This area is characterised by typical pre-Apennine limestone mountain landscapes, including beech forests, mountain grasslands, and karst plateaus, as well as sub-Mediterranean broadleaf forests and xerophilous grasslands on mid- and lower slopes. The slopes are predominantly covered by hazelnut and chestnut groves, while the piedmont plain is almost entirely occupied by hazelnut cultivation. The alluvial plain is char- acterised by arable land and industrial crops interspersed with vineyards and orchards, whereas urban areas account for approximately 6% of the total territory. The survey was conducted within the IT8040013 “Monti di Lauro” Special Area of Conservation (SAC), a site characterised by unique plant and animal biodiversity. This biodiversity is threatened not only by inadequate human management practices but also by the presence of invasive alien species that compete with native flora and fauna, thereby altering ecosystem balance. Among these, the wild boar (Sus scrofa) is considered one of the most problematic species and is included among the 100 worst invasive alien species worldwide (Lowe et al., 2000). Field sampling was concentrated within a smaller area (13,118 ha; 40°50'33.2999'' N, 14°37'40.0001'' E) that is rep- resentative of the vegetation and ecological characteristics of the broader territory, including both the STR 18 area and the IT8040013 SAC. The study area does not fall within a national or regional protected park. However, it is subject to hunting activities regulated by the Territorial Hunting Area (ATC) of the Province of Avellino, in accordance with Campania Regional Law No. 26 of 9 August 2012. Regarding wild boar management, the focal area is located near the ACS04LB (834 ha) and ACS05LB (930 ha) hunting sectors, both situated within the municipality of Quindici and included in Hunting District 10 (“Valle di Lauro”). 93 Acta Biologica Slovenica, 2026, Special issue The ecological impact of the wild boar within the study area was assessed and quantified through the analyses carried out in this research. Wildlife management in the intervention area To properly interpret the study area, it is necessary to understand which institutions are responsible for wildlife management and how management activities are imple- mented within the territory. As throughout the Province of Avellino, wildlife management ultimately falls under the jurisdiction of the Campania Regional Authority. However, two distinct management contexts can be identified: 1. Agroforestry-pastoral areas located within Natura 2000 sites, where wildlife management is carried out by the competent Regional Park Authorities. 2. Agroforestry-pastoral areas where hunting activities are permitted, where wildlife management is entrusted to the Territorial Hunting Area (ATC) of the Province of Avellino. The study area lies within a management framework in which conservation objectives and wildlife management practices coexist, requiring a balance between biodiversity protection and the sustainable regulation of wildlife populations. Methods for the identification of animal biodiversity Between 2019 and 2022, both direct and indirect surveys were conducted to identify and assess the animal species inhabiting representative habitats within the study area (Taylor & Williams, 1956; Laing et al., 2003; Focardi et al., 2020). The monitoring program included the following methods: • Direct surveys along linear transects, aimed at recording species presence and abundance. • Direct surveys within block and strip sampling units, allowing systematic observation of wildlife across differ- ent habitat types. • Indirect surveys based on a modified Capture–Mark– Recapture (CMR) approach are used to estimate popula- tion size and distribution. • Faecal surveys, including pellet and pellet-group counts, estimation of the Faecal Accumulation Rate (FAR), and collection of standing faecal deposits. Decomposition times were assumed to be 125 days in open pasture, 46 days in oak forests, and 19 days in chestnut forests. • Assessment of hunting effort, quantified as the average number of hunting days required to harvest an individ- ual of a given species (Sanders, 1988). Hunting effort was also evaluated using the Capture Per Unit Effort (CPUE) index, defined as the ratio between the number of individuals harvested and the number of hunting days or hours required to obtain them (Roseberry & Woolf, 1991; Hatter, 2001). • Camera-trap monitoring at georeferenced locations. After identifying the main movement corridors used by wild boar family groups, six access and exit points within the study area were selected. A camera trap was installed at each location to monitor animal movements and activity patterns (Piscopo et al., 2023; Trolliet et al., 2014; Verde et al., 2025). Statistical analysis of the densities of wild populations in the study area For the statistical analysis, the wild boar (Sus scrofa) pop- ulation within the study area was first estimated and quan- tified. Subsequently, population densities of all recorded animal species were calculated. Data were processed and evaluated using SimaPro software to perform a structured ecosystem services and disservices assessment. Data collection was conducted over four consecutive and comparable monitoring periods, corresponding to the calendar years 2019, 2020, 2021, and 2022 (January–December of each year). During the same period, the negative impacts associated with wild boar presence on human activities were also quantified, including crop damage and road traffic collisions. All statistical analyses were performed using JMP® Pro 14 software. For each species, differences in population den- sity among the monitoring periods were assessed using a one-sample t-test to determine their statistical significance. Percentage data were analysed using the Chi-square test of independence to evaluate associations between cate- gorical variables. Statistical significance was assessed at the conventional significance level (p < 0.05). Results Biodiversity presence Camera-trap monitoring enabled the detection of the fol- 94 Acta Biologica Slovenica, 2026, Special issue lowing species previously recorded within the study area: Streptopelia turtur, Turdus merula, and Turdus philomelos. In addition, several wild vertebrate species not cur- rently reported in the official records of the IT8040013 “Monti di Lauro” SAC/SCI were documented within the focal area. These included the bird species Athene noctua, Buteo buteo, Columba palumbus, Corvus cornix, Falco tinnunculus, Garrulus glandarius, Phasianus colchicus, Pica pica, Picus viridis, and Strix aluco, as well as the mammal species Canis lupus, Erinaceus europaeus, Lepus euro- paeus, Martes foina, Martes martes, Talpa europaea, and Vulpes vulpes. Camera-trap records also documented the presence of domestic animals, namely Felis catus, Canis lupus familia- ris, and Ovis aries. Wild boars number estimation The data collected during the study were extrapolated to estimate the wild boar population across the entire study region, corresponding to the 31,800-hectare Rural Territo- rial System 18 (STR 18). Although the estimate should be considered approximate, the results suggest a population ranging between 7,018 and 8,214 individuals. This estimated population size provides an important basis for wildlife management and population control planning, supporting the implementation of standardised management strategies aimed at maintaining ecological balance and mitigating potential impacts on biodiversity, agriculture, and human activities (Figure 1). Camera-trap monitoring combined with direct census surveys enabled the identification and quantification of the different age classes of wild boar present within the focal area (Table 1). The estimated number of wild boars present within the study area during the monitoring period is reported in Table 2. The mean total abundance of wild boars (considering all age classes) showed significant monthly variation (P < 0.01), ranging from a peak of 248.25 ± 28.50 individuals in July, corresponding to the end of the reproductive season, to a minimum of 142.75 ± 47.61 individuals in December, following the hunting season. Number of wild boars hunted The study area includes two designated wild boar hunting sectors, where approximately 60 hunters participate annu- ally in population management activities. Hunting is permit- ted from 1 October to 31 December, with a maximum of 34 hunting days per season. Participation requires payment of a hunting concession fee by each hunter (Table 3). At the end of each hunting season, the number of har- vested wild boars is recorded for all age classes, excluding striped piglets. The highest harvest was recorded in 2019, when 155 individuals were removed. In 2020 and 2021, the number of harvested wild boars declined, likely as a consequence of restrictions associated with the COVID-19 pandemic and the resulting reduction in hunting activities (Table 3). T o assess the economic value associated with wild boar hunting, Table 3 also reports the average costs incurred by each hunter during a hunting season. Evaluation of Wild Boar Impacts on Human Activities Table 4 summarises the monetary valuation of both ecosys- tem disservices (road traffic accidents and crop damage) and ecosystem services (provisioning, cultural, and habitat/ biodiversity services) associated with the presence of wild boar (Sus scrofa). In addition to the well-documented damage caused to agricultural crops (Geisser & Reyer, 2004; Herrero et al., 2006; Amici et al., 2012; Ficetola et al., 2014; Laznik & Trdan, 2014), wild boars may also generate high social and economic costs through road traffic collisions (Primi et al., 2009; Kruuse, Enno & Oja, 2016) and, more recently, through their increasing presence in urban environments (Castillo-Contreras et al., 2021). Official records for the study period and area docu- mented ten road traffic accidents involving wild boars. Based on compensation payments awarded by the Campania Regional Authority for accidents attributable to wild boars, the average economic cost per accident was estimated at €25,000. Data provided by the Campania Regional Authority for the Province of Avellino, which encompasses the study area, indicate considerable interannual variation in the damage caused by wild boars to agricultural activities. A similar pattern was observed within the study area, where the municipality of Quindici recorded the highest incidence of crop damage, particularly affecting hazelnut and chest- nut plantations. Damage attributable to wild boars in forest and pasture 95 Acta Biologica Slovenica, 2026, Special issue Period Reproductive period Growth period Hunting period Year - month Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2019 158 189 217 226 242 251 254 240 240 199 131 86 2020 123 144 172 200 225 231 239 234 234 219 196 173 2021 189 216 232 262 284 290 284 270 278 254 216 190 2022 196 196 205 203 204 214 216 216 219 188 150 122 Mean 166.50 186.25 206.50 222.75 238.75 246.50 248.25 240.00 242.75 215 173.25 142.75 SD 33.37 30.40 25.51 28.63 33.93 32.71 28.50 22.45 25.1 28.99 39.46 47.61 Figure 1. Area of intervention and points of dispersion of the wild boar. Slika 1. Območje ukrepanja in točke razširjenosti divjih prašičev. Year 2019 2020 2021 2022 Class of age - month Jan Dec Jan Dec Jan Dec Jan Dec Striped (0-6 mths) 78 0 31 0 61 0 64 0 Red (7-11 mths) 87 21 35 41 15 47 20 19 Young Subadults (12-16 mths) 0 29 0 53 4 71 71 44 Young Adults (17-24 mths) 40 19 38 62 57 49 25 38 Old Adults (>25 mths) 35 17 19 17 52 23 16 21 Total 240 86 123 173 189 190 196 122 Table 1. Estimated distribution of the wild boars’ class of age within the Focal Area. Tabela 1. Ocena porazdelitve starostnih razredov divjih prašičev znotraj območja raziskave. Table 2. Monthly abundance of wild boars (Sus scrofa) recorded during the study period. Tabela 2. Mesečna številčnost divjih prašičev (Sus scrofa), zabeležena v obdobju raziskave. 96 Acta Biologica Slovenica, 2026, Special issue habitats was reported only in 2019. However, the affected areas were limited (4.80 ha and 4.00 ha, respectively), corresponding to incidence rates of 0.03% and 0.58% of the total habitat area. Given their low extent, these impacts were not assigned a monetary value. In contrast, damage to tree crops was more substantial. Wild boar activity affected 64.80 ha of hazelnut groves and 42.12 ha of chestnut orchards, resulting in estimated economic losses of €105,300 and €324,000, respectively. Damage to arable land and coppice forests could not be reliably quantified, while no damage was recorded in vine- yards or olive groves. In addition to the damage caused to agricultural crops and the road traffic accidents previously described, reports from residents of the Irpinia area concerning disturbances associated with the presence of wild boars in urban environments have become increasingly frequent. These reports include sightings in proximity to residential areas, road crossings, incursions into public and private green spaces, and similar events. Many of these occurrences have also been documented by local media outlets. Although such disturbances represent a significant ecosystem disservice and contribute to negative public perceptions of wild boars, their economic impact could not be reliably quantified and, therefore, was excluded from the monetary valuation. To estimate the economic value of ecosystem services associated with the presence of wild boar in the study area, the assessment was completed by quantifying the provisioning services directly attributable to this species. Each wild boar harvested during the hunting season generates an estimated economic value of approximately €2,024.90. This value includes revenues derived from the sale of processed meat products (€1,920.00), teeth (€6.00), and bristles (€98.90). Based on the average annual harvest recorded during the study period (106 individuals), the total value of these provisioning services was estimated at €214,639.40 per year. These results highlight the potential economic contribu- tion of wild boar populations through marketable products, partially offsetting the costs associated with the ecosystem disservices generated by the species. The total expenditure incurred by the 60 hunters autho- rised to participate in wild boar hunting within the desig- nated hunting sectors was estimated at €427,554.50. This amount was attributed to the cultural ecosystem services category, reflecting the recreational, social, and traditional values associated with hunting activities. Overall, the monetary comparison between ecosys- tem disservices and services associated with wild boar presence indicated that the estimated value of ecosystem services exceeded the quantified costs of disservices within the study area. These findings suggest that the role of wild boar should not be evaluated solely in terms of its negative impacts on agriculture, road safety, and human activities. Rather, the species also provides measurable economic and socio-cultural benefits that should be considered in inte- grated wildlife management and conservation strategies. Consequently, the widespread perception of wild boar as exclusively a source of ecological and economic damage may oversimplify its complex role within socio-ecological systems. Year 2019 2020 2021 2022 Number of wild boars killed 155 84 88 97 Cost of hunting concession / year 451 451 451 451 Cost of hunting day / year 2,380 2,380 2,380 2,380 Cost of cartridges / year 350 350 350 350 Cost of dogs / year 1,495 1,495 1,495 1,495 Una tantum costs 2,450 2,450 2,450 2,450 Total costs hunter/killed animal 45.97 84,83 80.98 73.46 Total costs 60 hunters/killed animal 2,758 5,090 4,859 4,408 Total 240 123 189 190 Table 3. Annual number of wild boars harvested, and average costs (€) incurred by a single hunter per year. Tabela 3. Letno število uplenjenih divjih prašičev in povprečni stroški (v evrih), ki jih ima posamezni lovec na leto. 97 Acta Biologica Slovenica, 2026, Special issue Discussion Using a study area located in the Campania Region within the Mediterranean Biogeographical Region, an assessment of ecosystem services and disservices was undertaken through the analysis of the benefits and costs associated with the presence of wild boar. The model adopted in this study focuses on a species traditionally associated exclusively with ecosystem disser- vices, including agricultural damage, road traffic accidents, and incursions into urban areas. However, the comparative assessment of ecosystem services and disservices linked to wild boar indicates that this species is also capable of generating measurable ecosystem services. Although the present investigation remains partial, particularly due to the inability to fully quantify habitat and biodiversity-related values, the results suggest that the overall magnitude of ecosystem services outweighs that of disservices within the study area. Nevertheless, this con- clusion should be interpreted with caution, as it requires further validation through long-term, systematic, and more comprehensive monitoring efforts. Conclusions In conclusion, the present study suggests that the various components of agriculture, together with their associated agroecosystems, can be assigned a quantifiable value that differs from commonly held perceptions, through a detailed multi-level assessment of ecosystem services and disservices. Even when monetary valuation approaches are applied, the use of sustainability frameworks, such as those outlined in the 2030 Agenda for Sustainable Development, appears particularly relevant. In this context, Goal 2 highlights how robust quantification of ecosystem services can support more effective conservation outcomes and promote the sustainable use of natural resources. Innovative and sustainable management of natural resources, starting with wild boar populations, may therefore generate new economic opportunities while also improving control strategies for overabundant wildlife populations. The integration of different perspectives, supported by advances in technological and scientific knowledge, can contribute to addressing more effectively the challenges of reconciling human activities with environ- mental sustainability at the planetary scale. Author Contributions Conceptualisation, N.P. & L.E.; methodology, N.P., L.E. & A.R.; software, A.L. & A.C.M.C.; validation, L.E. & F.C.; formal analysis, A.L. & F.C.; investigation, N.P., L.E. & A.R.; resources, L.E.; data curation, N.P., A.L.; writing—original draft preparation, N.P. & L.E.; visualisation, F.C. & A.C.M.C.; supervision, L.E., F.C. & A.C.M.C.; project administration, N.P., L.E. All authors have read and agreed to the published version of the manuscript. Acknowledgement The author would like to thank “Azienda Agricola Antonio Raffaele” and Regione Campania “For the Natural Regional Park of Monti di Lauro. Funding This research was carried out thanks to European Union funds dedicated to the Campania PSR 2014-2020 - Inter- vention Type 16.1.1 Action 2 "Support for the establishment Disservices Services Difference Damage to agriculture 429,300.00 Road accidents 25,000.00 Provisioning 214,639.40 Cultural 427,554.50 Habitat / Biodiversity ??? ??? 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Sensors, 25(7), 2071. https://doi.org/10.3390/s25072071 100 1 Department of Veterinary Medicine and Animal Productions - University of Napoli Federico II, Italy. * Corresponding author: E-mail address: nadia.piscopo@unina.it Citation: Piscopo, N. (2026). Management of the wolf in the transition of its condition from "highly protected" to "protected". The case of Italy. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.29140 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Management of the wolf in the transition of its condition from "highly protected" to "protected". The case of Italy Nadia Piscopo 1 * Abstract The study examines official data regarding the estimated population of Canis lupus in Europe. Data are compared with those published by the scientific advisory body of the Italian Ministry for the Environment and Energy Security. Following the official change in the wolf's status from "specially protected" to "protected" the latter proposed amending the Habitats Directive to include planned culling as a means of controlling the wolf population. The findings highlight the disparity in wolf population estimates in peninsular Italy (Apennines) between the data reported by the two official sources. The EU data from the latest 2022 census report an Apennine population of 1,366 animals, an 86% increase, while the ISPRA data from the 2022 census report an average number of 2,388 peninsular wolves. Developing the average densities reported by ISPRA for 13 Apennine sample areas across the entire Apennine agricultural-forestry-pastoral area (152,971 km 2 ), the population would be 9,555 wolves. The numerical difference would be considerably different from that reported by the overall estimate (8,189 vs. 2,388 animals). This would result in a density difference (6.25 wolves/100 km 2 vs. 0.89 wolves/100 km 2 ) that is too marked, and currently unprovable, to justify culling for population control purposes in Italy. Keywords wolf, human–large carnivore conflict, population containment, numerical estimate, regulatory update on the wolf 101 Acta Biologica Slovenica, 2026, Special issue Introduction Over the past three centuries, the global human population has grown to an estimated 8.3 billion people (Bradshaw et al., 2026). Current human population density is approxi- mately 30 times higher than the average density expected for an omnivorous animal species of comparable body size (Burger, Weinberger & Marquet, 2017). Humans appropriate nearly 40% of the Earth's net primary production, thereby competing with other species for essential resources such as food, water, and space (Haberl et al., 2007). As a result, anthropogenic activities have profoundly transformed natural landscapes and ecosystems through deforestation, wetland loss, extensive modification of coastal environ- ments, and the conversion of natural habitats into urban and industrial areas. In addition, the mechanization of agricul- ture, the widespread reliance on fossil fuels, and the indus- trialization of the agri-food sector have intensified farming systems, contributing to a dramatic decline in semi-natural grasslands across Europe, with losses exceeding 90% in some regions during the twentieth century (Shipley et al., 2024). Given that the Earth's resources are finite, the estimated 10–50 million non-human species inhabiting the planet are under increasing pressure from human activities. As a result, many species are at risk of extinction, while others are forced to compete with humans for space and essential resources (Niederman et al., 2025). Despite international recognition of the need to halt and reverse biodiversity loss (Convention on Biological Diversity, 1992; United Nations, 2015), achieving this goal remains chal- lenging due to the persistent tension between biodiversity conservation objectives and the socio-economic interests of human societies. This tension arises not only from the frequent spatial overlap between biodiversity-rich regions and areas of high human population density, but also from the perceived trade-off between the growing demand for agricultural land to support food production and the need to preserve natural habitats that are essential for biodiver- sity conservation (Cabernard, Pfister & Hellweg, 2024). Given the recent downgrading of the wolf (Canis lupus) from “strictly protected” to “protected” status in Europe (European Commission, March 7, 2025), this study exam- ines the evolution of official population estimates for the species, from its recovery across Europe to the emergence of conflicts associated with livestock depredation (Piscopo et al., 2021). Upravljanje populacije volkov v obdobju prehoda njihovega statusa iz »strogo zaščitenih« v »zaščitene«. Primer Italije Izvleček Študija preučuje uradne podatke o ocenjeni populaciji volka (Canis lupus) v Evropi. Podatki so primerjani s tistimi, ki jih je objavil znanstveni svetovalni organ italijanskega ministrstva za okolje in energetsko varnost. Po uradni spremembi statusa volka iz »posebej zaščitenega« v »zaščitenega« je slednji predlagal spremembo Direktive o habitatih, da bi se načrtovano iztrebljanje vključilo kot sredstvo za nadzor populacije volkov. Ugotovitve poudarjajo razlike v ocenah populacije volkov na italijanskem polotoku (Apenini) med podatki, ki jih najdemo v uradnih viri. Podatki EU iz zadnjega popisa iz leta 2022 navajajo populacijo v Apeninih v višini 1.366 živali, kar predstavlja 86-odstotno povečanje, medtem ko podatki agencije ISPRA iz popisa iz leta 2022 navajajo povprečno število 2.388 volkov na polotoku. Če bi povprečne gostote, ki jih je poročala agencija ISPRA za 13 vzorčnih območij v Apeninih, ekstrapolirali na celotno kmetijsko-gozdarsko-pastirsko območje Apeninov (152.971 km 2 ), bi populacija znašala 9.555 volkov. Številčna razlika bi bila precej večja od tiste, ki jo navaja splošna ocena (8.189 proti 2.388 živali). To bi povzročilo razliko v gostoti (6,25 volkov/100 km 2 proti 0,89 volkov/100 km 2 ), ki je prevelika in trenutno nedokazljiva, da bi upravičevala odstrel za namene nadzora populacije v Italiji. Ključne besede volk, konflikt med ljudmi in velikimi zvermi, omejevanje populacije, številčna ocena, spremembe predpisov v zvezi z volkom 102 Acta Biologica Slovenica, 2026, Special issue This change in protection status calls for a reassess- ment of management strategies aimed at promoting coexistence between human activities and biodiversity conservation, while ensuring compliance with ecological sustainability principles and existing legislation. Achieving this objective requires reliable population estimates that accurately reflect the true status of the species, providing a sound basis for management decisions that balance the socio-economic interests of human communities with the conservation of wolves and other wildlife species. Materials and Methods Official data on wolf populations in Europe Official data on wolf populations in Europe were analyzed (http://ec.europa.eu/environment/nature/conservation/ species/carnivores/conservation_status.htm) and, subse- quently, EU numbers were compared with those reported, over time, for Italy (lifewolfalps, 2020; Boitani & Salvatori, 2015; La Morgia et al., 2022; Aragno et al., 2022). Since our work intends to generically evaluate the feasibility of proposals for changes in wolf management in Italy on the basis of the change in wolf status in Europe and since at present we do not have the knowledge necessary to correctly calculate the demographic growth of the wolf (theoretically, demographic growth follows a sigmoidal logistic curve conditioned by numerous variables starting from the availability of prey), we used a straight line of demographic growth based on the arithmetic growth rate obtained by dividing the absolute increase between the year 2022 and the year 2012 (2,035) by the number of years that make up the time period considered (10 years) and, for objective reasons, it was not possible to take into account the different numerical size of the populations compared. Results and Discussion Wolf populations in Europe and their numbers Currently, nine officially recognized wolf populations are identified in Europe (Table 1). Data from large carnivore censuses published by the European Commission pro- vided the basis for estimating the size of each population. Particular attention was subsequently given to information released by national authorities and official monitoring bodies, allowing for a more detailed assessment of popula- tion size and distribution in Italy. In Europe (Table 1), wolves have persisted primarily in eastern and southern regions, including Eastern Europe, Greece, the Balkans, the Carpathians, Italy, and the Iberian Peninsula, whereas they were largely eradicated from Western Europe. Population type Population size Census 2012 Census 2016 Census 2022 n n ∆n ∆% n ∆n ∆% Dinaric-Balkan 3,900 2,997 -903 -23.15 2,848 -149 -4.97 Baltic 870-1,400 1,595 195 13.93 2,540 945 59.25 Scandinavian 260-330 3,548 3,218 975.10 3,771 223 6.28 Karelian 150-165 1,888 1,723 104.42 2,893 1,005 53.23 Lowlands-Central Europe 36 packs 1,007 nd nd 3,372 2,365 234.86 Carpathian 3,000 1,553 -1,447 -48.23 2,206 473 30.46 Alpine 280 1,053 773 276.07 1,749 696 66.10 Italian Peninsula 600-800 734 -66 -8.25 1,366 632 86.10 North-West Iberian 2500 (c.2007) 1,508 -992 -39.68 1,639 131 8.69 Sierra Morena 1 pack (c.2008) - - - - - - Estimated total in Europe 12,375 15,883 3,508 28.35 22,204 6,321 39.80 Table 1. European wolf population types: Census counts and changes (number and percentage) among survey years 1 Tabela 1. Vrste evropskih populacij volkov: rezultati popisa in spremembe (v številkah in odstotkih) med posameznimi leti popisa 1 1 http://ec.europa.eu/environment/nature/conservation/species/carnivores/conservation_status.htm 103 Acta Biologica Slovenica, 2026, Special issue The Scandinavian population, distributed across Sweden and Norway, was estimated at approximately 300 individuals in the 2012 census, while the Karelian popula- tion in Finland numbered around 150 wolves. On the Iberian Peninsula (Portugal and Spain), the Northwestern Iberian population comprised approximately 2,500 individuals (2007 census), whereas the Sierra Morena population was reduced to a single pack according to the 2008 census. The Alpine population, spanning France, Italy, Austria, Switzerland, and Slovenia, was estimated at fewer than 300 individuals, while the Peninsular Italian population was estimated at 600–800 individuals. The largest wolf popu- lations occur in the Carpathian and Dinaric–Balkan regions. The Carpathian population, distributed across Poland, the Czech Republic, Slovakia, Hungary, and Romania, was esti- mated at approximately 3,000 wolves. The Dinaric–Balkan population, extending across Bulgaria, Slovenia, Croatia, Greece, Albania, Bosnia and Herzegovina, North Mace- donia, Montenegro, and Serbia, was estimated at around 3,900 individuals. The comparison between the first available census (2012) and the subsequent ones of 2016 and 2022, highlights an increasing trend in the wolf population if we consider the numerical sum of the nine European popula- tions. Conversely, if we consider the individual populations, it is possible to identify 4 in demographic decline between the 2012 and 2016 census: North West Carpathian -48.23%; Iberian -39.68%; Dinaric-Balkan -23.15%; Italian Peninsula -8.25%. In the subsequent census (2022) only the Dinaric-Balkan population maintains a decreasing demographic trend (-4.97%). In this last census all the other eight wolf populations in Europe show a demographic increase measured in a minimum of 6.28% (Scandinavian) to a maximum of 234.86% (Lowlands-Central Europe). Wolf number estimation in Italy The latest ISPRA-MASE census, carried out in the period 2020-2021 and published in 2022 (La Morgia et al., 2022; Aragno et al., 2022) would confirm, although with numer- ical variations between the Apennines and the Alps, the data officially reported by the European Commission (Table 1), respectively 3,307 (2,388 Apennines and 946 Alps), and 3,115 (1,366 Apennines and 1,749 Alps). Using the arithmetic demographic growth line (number of wolves estimated in 2022 vs 2012), a hypothesis is developed (figure 1) to explain the numerical growth officially reported by the EU for Italy (http://ec.europa.eu/ environment/nature/conservation/species/carnivores/ conservation_status.htm). Assuming that a structured wolf population relies solely on the alpha pair for reproductive success, and that each female wolf is capable of giving birth to an average of four pups, the number of wolves reported in 2022 would indicate a positive difference of 2,035 individuals. Dividing this number by the years examined, we estimate an annual increase of approximately 204 animals. To achieve this increase, only 51 alpha wolf females would be needed. Figure 1. Wolf population growth in Italy from 2012 to 2022. Estimated increase based on an assumed population structure. Slika 1. Rast populacije volkov v Italiji v obdobju od 2012 do 2022. Ocenjeno povečanje temelji na predpostavljeni strukturi populacije. 104 Acta Biologica Slovenica, 2026, Special issue If we examine the average wolf density estimates reported by the 2020-2022 ISPRA census (Table 2) in thirteen Italian sample areas where non-invasive genetic identification was performed (Aragno et al., 2022), we deduce that 81.2 wolves would be present in 1,300 km 2 . Not taking into account the different densities expressed in the 13 sample areas (2.9/100 km 2 in Campania – 9.6/100 km 2 in Emilia Romagna). The data in Table 2 refer to the Apennine peninsular regions, so the areas relating to the Alpine regions are subtracted from the total Italian area of 302,073 km 2 , corresponding to 274,886 km 2 of Agro-Silvo-Pastoral Area. This operation allows us to identify 192,112 km 2 of Apennine Agro-Silvo-Pastoral Area, from which we must also subtract those relating to the two large islands (Sicily and Sardinia) where the wolf is absent. Finally, we have a surface area of 152,971 km 2 which can be considered the wolf distribu- tion area in the Apennine peninsular Italy. Using the data published by ISPRA (Aragno et al., 2022), the number of wolves in Italy should reach 9,555 animals with a density of 6.25/100 km 2 . Conclusions The results of this work highlight a huge disparity in the numerical evaluation of wolves in Italy. The difference between the numerical size expressed by the European Union (1,366 wolves) for the Italian peninsular population (Apennines) and that calculated by us with the parameters provided by ISPRA (Aragno et al., 2022) would be 8,189 animals, which is too far from reality. It is indisputable that, despite the great efforts made by the scientific community and national and local administra- tive bodies to obtain an estimate of the demographic size of the wolf in Italy, too many uncertainties currently emerge that do not allow for the light-hearted application of the containment actions proposed by the Italian Minister for the Environment and Energy Security following the down- grading of the wolf's status from "particularly protected" to "protected".. In support of the above, there is currently a lack of fundamental information such as: • Do we have sufficient guarantees that the estimates of the increase in wolf numbers in some EU countries are close enough to reality? • Is the flexibility granted to Member States to adopt actions aimed at coexistence with human activities implemented correctly by individual EU countries in order to manage wolf populations while ensuring the appropriate protection of the species? • How many females give birth each year? • How many wolf pups are born each year? • What is the annual neonatal mortality rate? Wolf density per 100 km 2 Sampling Area Italian Region (Province) mean 95% LCI 95% UCI 1 Emilia Romagna (Parma-Piacenza) 9.6 8.6 10.4 2 Toscana (Grosseto-Livorno-Pisa) 5.6 4.8 6.2 3 Marche (Macerata-Ancona) 6.9 5.8 8.7 4 Toscana-Lazio (Grosseto-Viterbo) 4.7 4.0 4.9 5 Umbria-Lazio (Perugia-Terni-Rieti) 6.4 5.3 6.9 6 Abruzzo (L’Aquila-Chieti-Pescara) 7.0 6.1 7.7 7 Molise (Campobasso-Isernia) 6.9 6.0 7.7 8 Puglia (Foggia) 5.3 4.5 5.9 9 Campania (Benevento-Avellino) 2.9 2.3 4.3 10 Puglia-Basilicata (Taranto-Bari-Barletta-Andria-Trani) 5.9 5.0 6.5 11 Calabria (Cosenza) 6.0 5.0 6.5 12 Toscana-Emilia Romagna (Arezzo-Firenze-Forlì-Cesena) 8.6 7.4 9.5 13 Calabria (Reggio Calabria) 5.4 4.5 6.0 Table 2. Estimates of average wolf density in each of 13 areas subjected to non-invasive genetic sampling (Aragno et al., 2022, modified). Tabela 2. Ocene povprečne gostote volkov v vsakem od 13 območij, na katerih je bilo opravljeno neinvazivno genetsko vzorčenje (Aragno et al., 2022, prilagojeno) 105 Acta Biologica Slovenica, 2026, Special issue • What is the real annual mortality rate of the wolf (natural, accidental, illegal)? • How many dispersing wolves are able to form new healthy and structured packs? • What is the true extent of damage caused by wolf preda- tion to domestic livestock? • What is the influence of Canis lupus familiaris on the management of Canis lupus? The management of Canis lupus still requires a lot of infor- mation before proceeding with the suppression of a certain amount of the population. Author Contributions Conceptualization, N.P..; methodology, N.P.; software, N.P.; validation, N.P.; investigation, N.P.; resources, N.P.; data curation, N.P.; writing—original draft preparation, N.P.; visu- alization, N.P.; supervision, N.P.; project administration, N.P. Author have read and agreed to the published version of the manuscript. Acknowledgement The author would like to Regional Councillor Fiorella Zabatta and Department of Biodiversity of Campania Region. Funding “This research received no external funding”. Data Availability Research data is available at Zenodo (https://doi. org/10.5281/zenodo.21313116). Conflicts of Interest The author declares no conflict of interest. References Aragno, P., Salvatori, V., Caniglia, R., De Angelis, D., Fabbri, E., Gervasi, V., ... Velli, E., & Genovesi, P. (2022). La popolazione di lupo nelle regioni dell’Italia peninsulare 2020/2021. ISPRA-MASE. Boitani, L., & Salvatori, V. (2015). Italian draft plan on wolf conservation and management. Unione Zoologica Italiana. Bradshaw, C. J. A., Judge, M. A., Blumstein, D. T., Ehrlich, P. R., Dasgupta, A. N. Z., Wackernagel, M., Weeda, L. J. Z., & Le Souëf, P. N. (2026). Global human population has surpassed Earth's sustainable carrying capacity. Environmental Research Letters, 21(6), 064023. https://doi.org/10.1088/1748-9326/ae51aa Burger, J. R., Weinberger, V. P., & Marquet, P. A. (2017). Extra-metabolic energy use and the rise in human hyper-density. Scientific Reports, 7, Article 43869. https://doi.org/10.1038/srep43869 Cabernard, L., Pfister, S., & Hellweg, S. (2024). Biodiversity impacts of recent land-use change driven by increases in agri-food imports. Nature Sustainability, 7(11), 1512–1524. https://doi.org/10.1038/s41893-024-01433-4 Haberl, H., Erb, K. H., Krausmann, F., Gaube, V., Bondeau, A., Plutzar, C., ... & Fischer-Kowalski, M. (2007). Quantifying and mapping the human appropriation of net primary production in Earth's terrestrial ecosystems. Proceedings of the National Academy of Sciences, 104(31), 12942–12947. https://doi.org/10.1073/ pnas.0704243104 La Morgia, V., Marucco, F., Aragno, P., Salvatori, V., Gervasi, V., De Angelis, D., ... & Genovesi, P. (2022). Stima della distribuzione e consistenza del lupo a scala nazionale 2020/2021. ISPRA-MASE. Life WolfAlps EU. (2020). Coordinated actions to improve wolf-human coexistence at the alpine population level. https://www.lifewolfalps.eu/en/ Niederman, T. E., Aronson, J. N., Gainsbury, A. M., Nunes, L. A., & Dreiss, L. M. (2025). US imperiled species and the five drivers of biodiversity loss. BioScience, 75(7), 524–533. https://doi.org/10.1093/biosci/biaf019 Piscopo, N., Gentile, L., Scioli, E., González Eguren, V., Carvajal Urueña, A. M., Yanes García, T., Palacios Alberti, J., & Esposito, L. (2021). Management models applied to the human-wolf conflict in agro-forestry-pastoral territories of two Italian protected areas and one Spanish game area. Animals, 11(4), 1141. https://doi. org/10.3390/ani11041141 Shipley, J. R., Frei, E. R., Bergamini, A., Boch, S., Schulz, T., Ginzler, C., ... & Rixen, C. (2024). Agricultural practices and biodiversity: Conservation policies for semi-natural grasslands in Europe. Current Biology, 34(16), R753–R761. https://doi.org/10.1016/j.cub.2024.06.062 106 1 Department of Veterinary Medicine and Animal Productions, University of Naples Federico II * Corresponding author: E-mail address: marin.siano@studenti.unina.it Citation: Siano, M., Piegari, G., Paciello, O., Piscopo, N. (2026). Forensic approach to livestock predation. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.29141 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Forensic approach to livestock predation Marina Siano 1 * , Giuseppe Piegari 1 , Orlando Paciello 1 , Nadia Piscopo 1 Abstract The role of forensic veterinarians is becoming increasingly important in protecting the health of animals, humans, and the ecosystem. The following study focuses on the importance of veterinary forensic medicine in distinguishing the differences between predation and necrophagy in a protected area in the Lazio region, the Monti Aurunci Regional Natural Park. The study, conducted from 2002 to 2016, focused on a group of 150 Esperia ponies raised outdoors on a 300-hectare site on Monte Appiolo. Foaling occurs outdoors between February and October each year. To identify the animals present in the study area and their activities on the cadavers, 10 camera traps were placed on natural supports and at strategic points. Mortality, attributed to predation by canids, was absent from 2002 to 2009; it began in 2010 and became significant in 2011 (P<0.01). Mortality increased significantly in 2012 and 2013 (P<0.05, P<0.01). The total percentage of foals killed by predation reached 38.87%. The mortality rate began to increase in April (11.27%), stabilised in May (30.99%), and decreased in June (28.17%), July (13.62%), and August (11.74%). Predation was minimal in September (2.35%), October (0.94%), and November (0.94%). The results indicate a seasonal presence of wolves from April to November. Keywords Canis lupus; Regional Park; Esperia Pony; Sustainable livestock farming; Livestock losses; wolf predation 107 Acta Biologica Slovenica, 2026, Special issue Introduction The presence of wolves in natural environments has always been the subject of considerable attention, both from the scientific community and the public (Boitani et al., 2003). In recent decades, the return of this large predator to many marginal areas has sparked heated debate, especially regarding its incompatibility with human activities (Piscopo et al., 2021). One of the factors complicating this scenario is the presence of feral, abandoned, or stray dogs, the result of human genetic selection to create new dog breeds. Their presence alters the natural balance and makes it difficult to distinguish predation from true wolves, increasing attacks on livestock. It is important, however, to distinguish the predation dynamics of wolves from those of feral canids. Large predators such as wolves, in fact, attack their prey following a highly codified behaviour: they use specific techniques that ensure effective capture, immobilisation, and, ultimately, killing of the prey. This method differs profoundly from the chaotic and often cruel behaviour of stray or hybrid dogs, which can act uncoordinated, playfully or instinctively, inflicting unnecessary suffering on farm animals. The real challenge, therefore, is not just wolf conservation, but also building a balanced coexistence between predator and human activities. It is essential to invest in predation prevention through effective measures, such as the use of guard dogs, electric fences, and proper grazing management (Guadagno et al., 2023). At the same time, it is necessary to implement control and accountability policies to limit dog abandonment and the phenomenon of hybridisation (Caniglia et al., 2020). Ultimately, wolf conser- vation should not be seen as a threat but as an opportunity to rethink our relationship with nature. In a well-organised pastoral context, wolves can coexist with humans, provided the latter are willing to understand ecological dynamics and play their part in protecting a wildlife heritage. Materials and Methods Our study focused on a group of 150 Esperia ponies raised in the wild from 2002 to 2016 to monitor wolf attacks in the study area. Birth generally occurs outdoors and usually with- out human assistance, between February and October each year. To monitor the animals, camera traps were installed to conduct a census of the animals in the study area. The devices were positioned on natural supports, equipped with metal boxes to protect them from the elements, and placed in locations where the most information on the animals could be obtained. The predation investigation followed a Forenzični pristop k napadom plenilcev na živino Izvleček Vloga forenzičnih veterinarjev postaja vse pomembnejša pri varovanju zdravja živali, ljudi in ekosistema. Naša študija se osredotoča na pomen veterinarske forenzike pri razlikovanju med plenjenjem in nekrofagijo v zaščitenem območju v regiji Lazio, in sicer v regionalnem naravnem parku Monti Aurunci. Študija, izvedena v obdobju od leta 2002 do 2016, se je osredotočila na skupino 150 ponijev pasme Esperia, ki so bili vzrejeni na prostem na 300 hektarjev velikem območju na gori Monte Appiolo. Žrebitve potekajo na prostem vsako leto med februarjem in oktobrom. Za identifikacijo živali, prisotnih na območju študije, in njihovih dejavnosti v zvezi s trupli je bilo na naravnih nosilcih in na strateških točkah nameščenih deset fotografski pasti. Smrtnost, pripisana plenjenju s strani psov, med letoma 2002 in 2009 ni bila zabeležena; začela se je leta 2010 in postala statistično pomembna leta 2011 (P < 0,01). Smrtnost se je znatno povečala v letih 2012 in 2013 (P < 0,05, P < 0,01). Skupni odstotek žrebet, ubitih zaradi plenjenja, je dosegel 38,87 %. Stopnja smrtnosti se je začela povečevati v aprilu (11,27 %), se je stabilizirala v maju (30,99 %) ter zmanjšala v juniju (28,17 %), juliju (13,62 %) in avgustu (11,74 %). Plenjenje je bilo minimalno v septembru (2,35 %), oktobru (0,94 %) in novembru (0,94 %). Rezultati kažejo na sezonsko prisotnost volkov od aprila do novembra. Ključne besede Canis lupus; Regionalni park; poniji Esperia; trajnostna živinoreja; izgube živine; plenjenje volkov 108 Acta Biologica Slovenica, 2026, Special issue standardised protocol that involved the collection of gen- eral data, including the characteristics of the area, the type of environment, and the weather conditions at the time the body was found. Data collection also included the number and specific composition of the livestock, the type of farm- ing, and any protection systems adopted. For each animal targeted for predation, the sex, age, microchip number, nutritional status, dehydration status, and the presence of wounds resulting from a predatory attack were recorded. A necropsy was performed to determine the precise cause of death. Analysis of consumed body parts allowed us to estimate the predator's feeding behaviour, classifying the degree of consumption into five percentage levels (0%; 0–25%; 25–50%; 50–75%; 75–100%). Flaying and an inter- nal necropsy examination allowed us to describe the extent and depth of the injuries, as well as evaluate internal tissues and organs to detect any pathologies or deficiencies. The integration of these findings with laboratory analyses allowed us to formulate a reliable etiological diagnosis and assign a scientific cause of death. Results From the year 2002 to 2006, the number of mares increased; consequently, the number of births also increased (min 106, max 125, total average 113±5.48/year). The mortality, ascribed to predation by canids (Figure 1), is absent in the period 2002-2009; it began in 2010 and became significant in 2011 (36/125; P<0.01). The increase in mortality is high in the years 2012 (28.8% vs 42.20%, P<0.05) and 2013 (28.80% vs 63.39%, P<0.01). The total percentage of foals died from predation reach 38.87%. The rate of mortality started to grow from April (11.27%) to become more consistent in May (30.99%), and it decreased in June (28.17%), July (13.62%) and August (11.74%). Preda- tion is residual in September (2.35%), October (0.94%), and November (0.94%). Results (Figure 2) indicate a seasonal presence of wolves or groups in the studied area (from April to November). Discussion Accurately diagnosing the cause of death in domestic livestock is crucial in managing wildlife damage, as access to compensation under current legislation depends on it. However, the availability of public funds has sometimes led to false claims of predation, while in actual cases of attack, a lack of timely response or adequate diagnostic expertise can compromise the identification of the cause of death, generating tensions between farmers and institutions. This conflict, if not properly managed, can lead to the poaching of wolves, a strictly protected species of significant con- servation interest. This study analysed the phenomenon of canid predation on an Esperia Pony farm located in the Monti Aurunci Regional Park in the province of Latina, with the aim of assessing the extent of human-wolf conflict in Figure 1. Esperia Pony, prey of the wolf, into the Monti Aurunci Regional Natural Park. Slika 1. Ponij Esperia, plen volka v regionalnem naravnem parku Monti Aurunci. 109 Acta Biologica Slovenica, 2026, Special issue human-influenced environments. The results highlight that the coexistence of large carnivores and livestock farming continues to be a critical issue, especially in free-ranging and semi-free-ranging farms. This highlights the need to combine wolf conservation with sustainable breeding models and to implement reliable forensic protocols for the scientific verification of predation. Conclusions The conflict between wolves and the livestock sector requires an integrated approach based on proper land man- agement, transparent damage assessment procedures, and collaboration between farmers, conservation agencies, and veterinary professionals. In particular, the intervention of a veterinarian, equipped with health and management skills, is essential to ensure reliable necropsy diagnoses, stray animal control, and the collection of scientifically valid data. It is hoped that protected area management agencies will direct financial resources not only toward compensa- tion, but also toward prevention and improved livestock farming practices, promoting production models compat- ible with the presence of large carnivores. Only through sustainable and legally based management will it be possible to reduce the conflict, ensure wolf protection, and simultaneously safeguard the continuity of traditional livestock farming activities in mountain areas. Author Contributions Conceptualisation, N.P., L.E.; methodology, N.P., L.E., O.P.; software, G.P., M.S.; validation, N.P., O.P.; formal analysis, N.P., M.S.; investigation, N.P., L.E., G.P., M.S., O.P.; resources, N.P., L.E.; data curation, N.P., M.S.; writing—original draft preparation, N.P., L.E., M.S.; visualization, M.S., G.P., O.P.; supervision, N.P., L.E., O.P.; project administration, N.P., L.E. All authors have read and agreed to the published version of the manuscript. Acknowledgement The author would like to thank “Azienda Agricola Mattei” and the “Natural Regional Park of Monti Aurunci”. Funding This research received no external funding. Data Availability Research data are available at Zenodo (https://doi. org/10.5281/zenodo.21313156). Conflicts of Interest The author declares no conflict of interest. Figure 2. Total percentage of foals that died from predation in the Monti Aurunci Regional Natural Park. Slika 2. Skupni odstotek žrebet, ki so poginila zaradi plenilcev v regionalnem naravnem parku Monti Aurunci. 110 Acta Biologica Slovenica, 2026, Special issue References Boitani, L., 2003. Wolf conservation and recovery. In L. D. Mech & L. Boitani (Eds.), Wolves: Behavior, ecology, and conservation (pp. 317–340). University of Chicago Press. Piscopo, N., Gentile, L., Scioli, E., Eguren, V. G., Carvajal Urueña, A. M., García, T. Y., & Esposito, L., 2021. Management models applied to the human-wolf conflict in agro-forestry-pastoral territories of two Italian protected areas and one Spanish game area. Animals, 11(4), 1141. https://doi.org/10.3390/ani11041141 Guadagno, E., Gallizia, A., Galosi, L., Quagliardi, M., Angorini, A., Trenta, F., Ferretti, M., Pennacchioni, G., Roncarati, A., & Morandi, F., 2023. Protection of farms from wolf predation: A field approach. Land, 12(7), 1316. https://doi.org/10.3390/land12071316 Caniglia, R., Fabbri, E., Galaverni, M., Milanesi, P., & Randi, E., 2020. Non-invasive sampling and genetic monitoring of wolves and hybridization with dogs in Europe. Mammal Review, 50(3), 206–219. https://doi.org/10.1111/mam.12183 111 1 National Reference Centre for the analysis and study of correlation among environment, animals and humans- IZS Mezzogiorno, Portici, Italy. 2 University of Coimbra, MARE/ARNET, Coimbra, Portugal 3 Department of Veterinary Medicine and Animal Productions, University of Napoli Federico II, Naples, Italy. * Corresponding author: E-mail address: maria.tafuro@izsmportici.it Citation: Tafuro, M., Brunetti, R., João Feio, M., Tafuri, S., Mandato, M., Piscopo, N., Ottaiano, M., Baldi, L., Gallo, P., Esposito, M. (2026). Ecological status assessment of watercourses in Benevento, Southern Italy. Acta Biologica Slovenica 69 (Special Issue) https://doi.org/10.14720/abs.69.Special issue.29142 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Brief note Ecological status assessment of watercourses in Benevento, Southern Italy Maria Tafuro 1 * , Roberta Brunetti 1 , Maria João Feio 2 , Simona Tafuri 3 , Maria Mandato 1 , Nadia Piscopo 3 , Maria Ottaiano 1 , Loredana Baldi 1 , Pasquale Gallo 1 , Mauro Esposito 1 Abstract The relationship between the quality of watercourses in urban environments and their potential interrelationships with the health of living species, and the water quality of streams flowing through the urban fabric of Benevento, in southern Italy, was characterised. Water analyses were conducted at 20 different sampling points using the Pollution Level procedure for Macrodescriptors (volatile organic compounds, trace elements, anions and cations, and bisphenols). Two sampling points were also tested for organic and inorganic anthropogenic contaminants. Total organic carbon (TOC), dissolved organic carbon (DOC), total phosphorus, NH 4 , N-NO 3 , and N-NO 2 values were determined. The results indicate that the lowest concentrations of DOC, NH 4 , and N-NO 2 were observed at sampling point BN20, while the highest were observed at sampling point BN19. At sampling points BN4 and BN20, wide variability in macroscopic parameters and contaminants was observed, and bisphenols were present in some areas. The study highlighted a wide variability in macroscopic parameters and contaminants, highlighting the presence of bisphenols in some areas. The OAH project highlighted the need to monitor urban waterways. The Benevento results show differences in physicochemical parameters among sampling points, but the presence of bisphenols from microplastics warrants further study. Keywords watercourses, environmental, macro descriptors, contaminants 112 Acta Biologica Slovenica, 2026, Special issue Introduction In recent decades, due to significant human interventions, many national and European waterways are at risk of losing their crucial ecological roles. Despite the requirements of the EU Water Framework Directive (WFD), the ecological status of surface waters in the European Union continues to deteriorate (Directive 2000/60/EC). As reported by the European Environment Agency, only 45.2% of rivers have achieved good status, while 19.8% still have a poor or bad ecological status (https://www.eea.europa.eu/en/ analysis/indicators/ecological-status-of-surface-waters). In view of the 2027 deadline set by the Water Framework Directive, it is necessary to determine the ecological status of watercourses, particularly rivers, so that Member States can urgently implement the measures needed to achieve good ecological status. The human impact has been so severe that many waterways can now be considered threatened ecosystems. On the other hand, maintaining good ecological quality is essential for conserving animal and plant biodiversity. Current projections of rapid expansion of urban areas present fundamental challenges and also opportunities to design more livable, healthy and resilient cities, for example, adaptation to climate change effects (Elmqvist et al., 2015). Moreover, stresses on riverine ecosystems will be exacerbated by steadily rising human demands for water and land, as well as by climate change and seasonal shifts in water availability during periods of high irrigation and ecological demands (Beechie et al., 2010). Inland waterways and rivers provide various human needs, such as fish and other river biota, food sources, irrigation functions, mobility, recreation, drinking, washing, and sanitation (Chabib et al., 2025) The OneAquaHealth project aims to improve the sus- tainability and integrity of freshwater ecosystems in urban environments. For Italy, the choice fell on the rivers that flow through the city of Benevento, in Southern Italy. The goal is to study urban aquatic ecosystems, given that river habitats can be considered valuable ecosystem mosaics formed by different biotic and abiotic elements with the ability to make cities more biodiverse and sustainable (Rodrigues et al., 2025). The EU-WFD criteria require that water quality be assessed by examining a variety of factors, including biological factors (such as the composition and abundance of flora and fauna), hydro-morphological factors (such as flow quantity and dynamics, variations in river depth and width), and additional physicochemical factors (such as temperature, oxygenation, salinity, nutrients, and so on) (Naddeo et al., 2023). Ocena ekološkega stanja vodotokov v Beneventu v južni Italiji Izvleček Na podlagi analize kakovosti vode v potokih, ki tečejo skozi urban del mesta Benevento na jugu Italije, smo analizirali povezavo med kakovostjo vodotokov in morbitnimi mesebojnimi vplivi na zdravje živih vrst. Analize vode so bile izvedene na 20 vzorčnih mestih z uporabo postopka za določanje stopnje onesnaženosti za makroindikatorje (hlapne organske spojine, elemente v sledeh, anione in katione ter bisfenole). Na dveh vzorčnih mestih so bile opravljene tudi analize organskih in anorganskih onesnaževal antropogenega izvora. Določene so bile vrednosti skupnega organskega ogljika (TOC), raztopljenega organskega ogljika (DOC), skupnega fosforja, NH₄, N-NO₃ in N-NO₂. Rezultati kažejo, da so bile najnižje koncentracije DOC, NH₄ in N-NO₂ ugotovljene na mestu vzorčenja BN20, najvišje pa na mestu vzorčenja BN19. Na mestih vzorčenja BN4 in BN20 je bila opazna velika spremenljivost makroskopskih parametrov in onesnaževalcev, na nekaterih območjih pa tudi prisotnost bisfenolov. Študija je poudarila veliko spremenljivost makroskopskih parametrov in onesnaževalcev ter izpostavila prisotnost bisfenolov na nekaterih območjih. Projekt OAH je poudaril potrebo po spremljanju mestnih vodotokov. Rezultati iz Beneventa kažejo razlike v fizikalno-kemijskih parametrih med vzorčnimi točkami, vendar prisotnost bisfenolov iz mikroplastike zahteva nadaljnje raziskave. Ključne besede vodotoki, okolje, makroindikatorji, onesnaževala 113 Acta Biologica Slovenica, 2026, Special issue In this work, we report the study focused on the evalu- ation of the environmental status of the waterways of the city of Benevento in Southern Italy, through the analysis of the Level of Pollution by Macrodescriptors (LIMeco) (DM 260/2010) and the subsequent in-depth analysis through the determination of organic and inorganic contaminants whose presence can be attributed to intense human activity. The indicators we considered in this study to assess the state of aquatic ecosystems should be integrated into future studies to understand the link between ecosystem health and human health from a One Health perspective (De Carvalho et al., 2025). Materials and Methods Study area and sampling sites Benevento is a small city of about 60,000 inhabitants, located in central-southern Italy and is one of the four provincial capitals of the Campania region. It is surrounded by high hills and mountains such as the Taburno Campo- sauro massif, Mount Mutria, and the Dauni Mountains. It is located between the Tyrrhenian Sea and the Adriatic Sea. Over the last 25 years, Benevento has undergone a process of transformation that has affected both the urban centre and the agricultural areas. The town is crossed by the Sabato and Calore rivers, which flow into the northern sector of the urban area, and by an intense network of streams (Ambrosino et al., 2025). The Calore is a tributary of the Volturno river, and the Sabato river flows into the Calore. These rivers have been completely integrated into the urban landscape, and many of their banks have been altered by excessive (mostly illegal) construction in the newer neighbourhoods. In many cases, this situation prevents the separation of rainwater from wastewater, resulting in high levels of water pollution and environmen- tal degradation, which differentiates this research site from others, providing complementary information. In contrast, the numerous streams and minor rivers have rich riparian vegetation with habitats that are home to native, but also invasive, plant and animal species. Water sampling and analysis The study was conducted on 20 different streams located in Benevento city, from which water samples were col- lected between June 2022 and August 2024. The streams were selected based on a grade of urbanisation and the integrity of the riparian vegetation, banks, aquatic habitats, and water pollution. The assessment of the ecological status of water- courses was based mainly on the LIMeco index. The procedure involves calculating a score based on the concentration observed at the site in question of the fol- lowing macro-descriptors: N-NH 4 , N-NO 3 , total phosphorus, and dissolved oxygen (100 - % O 2 saturation). The LIMeco score to be assigned to the site representative of the water body is given by the average of the individual LIMecos. Water quality parameters, including biological oxygen demand, ammonium, nitrite, nitrate, total phosphorus, TOC, Chlorophyll a, Chlorophyll b, Chlorophyll c, were measured following standard procedures. In the two sites where the ecological status of the watercourse was assessed as "poor", an in-depth study was carried out through the determination of volatile organic compounds (VOCs), trace elements, anions, cations, and bisphenols. For the extraction procedure of Volatile Organic Com- pounds, 5 μL of an internal standard solution was added to 50 mL of the sample. The purification and concentration of the sample were carried out by a purge and trap system. The detection and quantification were performed by inject- ing 25 μL of the final extract into the gas chromatograph system coupled with a mass spectrometer. In this study was also determined the occurrence of heavy metals and other trace elements, including arsenic (As), beryllium (Be), cadmium (Cd), cesium (Cs), cobalt (Co), chromium (Cr), iron (Fe), manganese (Mn), lead (Pb), rubid- ium (Rb), thallium (Tl), uranium (U), and vanadium (V). These elements may derive from natural sources, linked to the geological composition of the soil underlying the aquifers, or from human activities such as industrial and agricultural processes. Among the metals analysed, lead (Pb), cad- mium (Cd), and arsenic (As) are recognised as the main environmental risk factors, due to their high persistence and low degradability, which promote accumulation and dispersion in the environment through various pathways, including atmospheric transport. Samples intended for metal determination underwent acid digestion to solubilise the metals present in colloidal and/or organic forms. The trace element content was then quantified by inductively coupled plasma mass spectrometry (ICP-MS). For the chemical characterisation of the watercourses, 114 Acta Biologica Slovenica, 2026, Special issue anions (fluoride, chlorite, bromate, chloride, nitrite, bromide, chlorate, nitrate, sulfate, and phosphate) and cations (lithium, sodium, ammonium, potassium, magnesium, and calcium) were determined. The analyses were performed using ion chromatography with anion- and cation-exchange columns, depending on the ions’ affinity for the stationary phase. The eluent containing the separated analytes passed through a post-column suppressor to reduce background conductivity and enhance the sensitivity of the conductometric detector. Analyte identification was achieved by comparing sample peak retention times with those of reference solutions, while quantification was based on calibration curves obtained from standards of known concentrations. Special attention was given to the determination of endocrine-disrupting compounds (EDCs), particularly bisphenol A (BPA), one of the most widespread and extensively studied chemicals worldwide (Meena et al., 2025). BPA acts as a xenoestrogen by competitively bind- ing to estrogen receptors (ERα, ERβ, and ERβ8), thereby interfering with the regulation of androgenic and thyroid hormones. The qualitative and quantitative analysis of BPA was performed by liquid chromatography coupled with tandem mass spectrometry (LC/ESI-MS/MS) using a QTRAP 6500+ spectrometer (Di Marco Pisciottano et al., 2022). Before analysis, the samples were filtered and centrifuged to ensure matrix purity. Data analyses Descriptive statistics (sum, median, average, standard deviation, and detection frequency) were used to compare oxygen demand, ammonium, and nitrite, nitrate, total phos- phorus, TOC, Chlorophyll a, Chlorophyll b, and Chlorophyll c in 20 sampling sites. The multivariate analyses were performed in RStudio software version 4.4.1. To enable an assessment of the quality status of the water body, the chemical-physical quality index of LIMeco waters was calculated, assigning different scores to the concentration of certain macro-descriptors of water quality according to the indications shown in Italian Ministerial Decree n. 260/2010. Results To enable an assessment of the quality status of the water body, the chemical-physical quality index of LIMeco waters was calculated, assigning different scores to the concentration of certain macro-descriptors of water quality according to the indications shown in Table 1. The average of the scores determines the classification into one of the five quality classes provided for by Ministerial Decree 260/2010, the thresholds for which are shown in Table 2. Of the 20 sites analysed, 16 (80%) have a Limeco status of “High,” 3 sites (15%) have a status of “Poor,” while 1 site (5%) has a status of “Good” (Figure 1). Basic statistics for dissolved oxygen, ammoniacal nitro- gen, nitrate, total phosphorus, TOC, and chlorophyll a, b, and c, which contribute to defining the ecological status of watercourses, were calculated using RStudio software version 4.4.1. Saturation 100-O₂ % shows wide variability (SD = 16.88), but the distribution is balanced (skewness = 0.04), with no obvious outliers. N-NH₄ (ammonia): the median is 0, while the mean is 0.27, and the high asymmetry (1.54) indicates a very unbal- anced distribution with possible high outliers. N-NO₃ (nitrates): the distribution is moderately skewed (skewness = 0.65), with a standard deviation of 1.63 and some high values reaching a maximum of 5.24 mg/L, which could represent outliers. T otal phosphorus: very high variability (SD = 121.1), with a wide range (from 5.00 to 333.4 µg/L), the median is 60.05, so some values are much higher than the average (109.0), indicating possible outliers; TOC: this parameter shows a symmetrical distribution (skewness = 0.21), low standard deviation (0.55), and well-concentrated values. Chlorophyll b: the highly skewed distribution (skewness = 1.35), with a median close to zero (0.01) and a maximum of 0.71 mg/L, indicates the possible presence of anomalous values. Chlorophyll c: a fairly regular distribution (skewness = 0.19, SD = 0.39) does not indicate any anomalies. Based on the initial monitoring results, two points were identified (BN4 corresponding to the Serretelle 4 stream and BN20 corresponding to the Lenta stream) for which a more in-depth analysis of the parameters analysed was carried out (Table 3). The results showed values for trace elements, anions, cations, and VOCs within the maximum limits set by leg- islation, while they highlighted the presence of certain bisphenols at both sampling sites, for which no maximum limits have been established by law. 115 Acta Biologica Slovenica, 2026, Special issue Figure 1. Map of Benevento city showing the 20 sampling points along the rivers and streams (https://www.oneaquahealth.eu/research-cit- ies/benevento/). Slika 1. Zemljevid mesta Benevento, na katerem so prikazane 20 vzorčevalnih točk ob rekah in potokih (https://www.oneaquahealth.eu/ research-cities/benevento/). Variable Mean Std. Dev. Median Min Max Range Asymmetry Curtosis Standard error 100-O₂% 68.7 16.8 66.8 47.1 95.9 48.8 0.04 -1.70 3.8 (N-NH₄, mg/L) 0.27 0.42 0.00 0.00 1.6 1.6 1.5 1.8 0.10 (N-NO₃, mg/ L) 1.8 1.6 1.1 0.00 5.2 5.2 0.65 -1.02 0.37 P tot (µg / L) 109.0 121.1 60.0 5.0 333.4 328.4 0.73 -1.19 27.1 TOC 2.6 0.55 2.6 1.7 3.6 1.8 0.21 -1.24 0.12 Chlorophyll b (mg/ L) 0.14 0.21 0.01 0.00 0.71 0.71 1.3 0.61 0.05 Chlorophyll c (mg/ L) 0.41 0.39 0.40 0.00 1.0 1.0 0.19 -1.63 0.09 Table 1. Basic statistics. Tabela 1. Osnovna statistika. 116 Acta Biologica Slovenica, 2026, Special issue Discussion The study made it possible to assess the quality of Benevento's waterways and analyze the risks to eco- system health resulting from river runoff. Based on the results obtained from the analysis of physical and chemical parameters, the quality of flowing waters was assessed in relation to nutrients and oxygenation, key factors in regulating biological communities in aquatic ecosystems. In fact, plant communities, such as diatoms and aquatic macrophytes, are particularly sensitive to variations in these elements. Low DO levels together with an excess of ammoniacal nitrogen, nitrates, and total phosphorus may suggest the presence of organic substances that consume oxygen during their decomposition. This alters the natural balance, causing algae proliferation (eutrophication) and, consequently, suffocation, stress, and mortality of aquatic organisms. Therefore, the determination of these compounds is essential for assessing pollution from civil, industrial, and agricultural sources. Furthermore, the result obtained from the application of the LIMeco index has made it possible to classify the quality status of almost all the watercourses studied as “High/Good.” The second part of our activities involved determining organic and inorganic contaminants, such as volatile organic compounds and bisphenols. VOCs are compounds that come from water disinfection or human activities such as industrial processes. Continuous and prolonged exposure to VOCs has been linked to long-term negative health effects. Some VOCs are classified as carcinogens and have been linked to an increased risk of developing certain types of cancer, including bladder, kidney, and liver cancer. Bisphenols, on the other hand, are used in combi- nation with other substances in the production of certain plastics and resins. Bisphenol A is a molecule very similar to estrogen. It acts like a hormone, interfering with the normal balance of the endocrine system, and is therefore classified as an endocrine disruptor, binding to the same receptors as estrogen. However, the periodic monitoring required by law does not include the determination of these substances as emerging micropollutants. The study also included the determination of trace elements, anions, and cations that are not required by law but which contributed to the qualitative definition of the watercourses studied. The only critical aspect that should be investigated further is the presence of bisphenols, particularly at point BN20. Analysis of the results obtained shows that all the chemical and physical parameters examined are compatible with river water. With regard to environmental contaminants, the only values exceeding the detection limit are traces of bisphenols. GIS analysis of the BN 20 sampling site revealed the presence of an industrial/commercial settle- ment nearby. This is consistent with the presence of these contaminants. For this reason, it would be interesting to investigate the presence of these contaminants and their effects on the species present with further analysis. Conclusions However, the presence of variability for some chemi- cal-physical parameters suggests the need for further investigation to determine the causes, also in relation to the sampling points and the dynamics of the river flow. Furthermore, the presence, albeit at low concentrations, of organic contaminants such as bisphenols also requires further assessment for their possible harmful effects on the immune system (Lambré, 2023). Author Contributions Conceptualisation, M.T. and M.E.; methodology, M.E.; soft- ware, R.B.; validation, M.T., N.P. and D.S.; formal analysis, M.M.; investigation, M.T.; resources, M.T.; data curation, M.O., M.T.; writing—original draft preparation, M.T.; writing— review and editing, M.E.; supervision, P.G.; funding acqui- sition, N.P., M.E. All authors have read and agreed to the published version of the manuscript. Acknowledgement The authors wish to thank Prof. Luigi Esposito, Department of Veterinary Medicine and Animal Productions, University of Napoli Federico II, Naples, Italy. Funding This research was funded by the OneAquaHealth project. Horizon Europe. HORIZON-CL6-2022- GOVERNANCE-01-093101086521 Data Availability Research data are available at Zenodo (https://doi. org/10.5281/zenodo.21313194). Conflicts of Interest The authors declare no conflict of interest 117 Acta Biologica Slovenica, 2026, Special issue References Ambrosino, M., Palarea-Albaladejo, J., Albanese, S., Lin, X., Ciarcia, S., & Cicchella, D., 2025. Assessing natural background concentrations of chemical elements in urban soils: A case study in Benevento (Italy). Science of the Total Environment, 975, 179298. https://doi.org/10.1016/j.scitotenv.2025.179298 Beechie, T. J., Sear, D. A., Olden, J. D., Pess, G. R., Buffington, J. 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Patterns of pharmaceutical contamination in streams of European cities across urbanisation gradients: Potential impacts on One Health. Journal of Hazardous Materials, Advance online publication, 139946. https:// doi.org/10.1016/j.jhazmat.2025.139946 Acta Biologica Slovenica 2026 Special Issue