Društvo biologov Slovenije 2025 Vol. 68 | Št. 4 2 Acta Biologica Slovenica, 2025, 68 (4) Acta Biologica Slovenica, 2025, 68 (4) 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 Alenka Gaberščik (SLO), Univerza v Ljubljani, Biotehniška fakulteta 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 Georg A. Janauer (A), University of Vienna 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 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 Mihael Jožef Toman (SLO), Univerza v Ljubljani, Biotehniška fakulteta Miloš Vittori (SLO), Univerza v Ljubljani, Biotehniška fakulteta Oblikovanje/Design Ajda Fortuna Naslovnica/Cover page Hribski urh, avtor/author: Tina Klenovšek 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: 10.14720/abs.68.4 http://journals.uni-lj.si/abs/ Acta Biologica Slovenica je indeksirana v – is indexed in: CAB Abstracts, Web of Science Clarivate 3 Acta Biologica Slovenica, 2025, 68 (4) Original Research Paper 4 The effect of different herbicides and soil moisture regimes on Camelina performance / Vpliv različnih kemičnih herbicidov in režimov vlažnosti tal na uspešnost kameline Asghar Ebadi-Segherloo, Mohsen Janmohammadi, Naser Sabaghnia, Mehdi Mohebodini 17 Umetne vodne akumulacije za zasneževanje na vzhodu Pohorja (Slovenija): mesta razmnoževanja in ekološke pasti za dvoživke / Artificial Water Accumulations for Snowmaking on Eastern Pohorje (Slovenia): Breeding Sites and Ecological Traps for Amphibians Tina Klenovšek, Žiga Tertinek, Ana Skledar 31 Recognising the benefits of large carnivore presence in Slovenia: A focus group study / Prepoznavanje koristi prisotnosti velikih zveri v Sloveniji z metodo fokusnih skupin Irena Kavčič, Manca Dremel, Aleksandra Majić Skrbinšek 45 Distribution of two invasive alien diatom species Achnanthidium delmontii and Achnanthidium druartii in Slovenia / Razširjenost dveh invazivnih tujerodnih vrst diatomej Achnanthidium delmontii in Achnanthidium druartii na območju Slovenije Aleksandra Krivograd Klemenčič, Tadeja Šter 59 Evaluation of selected functional traits in lactic acid bacteria isolated from Algerian Makatia goat milk for food applications / Ocena izbranih funkcionalnih lastnosti mlečnokislinskih bakterij, izoliranih iz alžirskega kozjega mleka Makatia, za uporabo v živilih Ahmed Saci, Samia Gharbi, Fatima Djadouni, Noureddine Karkachi 75 Taxonomical implications of fruit and seed morphology in wild species of Diospyros L. (Ebenaceae) in Odisha, Eastern India / Taksonomske posledice morfologije plodov in semen divjih vrst Diospyros L. (Ebenaceae) v Odishi, vzhodna Indija Sabita Tudu, Jeevan Kumar Jena, Sifan Priyadarshini, Sudhanshu Sekhar Dash, Sudam Charan Sahu 91 Carvacrol Enhances Cell Viability by Reducing Apoptosis in A549 Lung Epithelial Cells Exposed to Elastase / Karvakrol izboljša celično preživetje z zmanjšanjem apoptoze v pljučnih epitelnih celicah A549, izpostavljenih elastazi Khojasteh Hoseininejad, Sabah Mahmoudi Nejad Mounfared, Fereshteh Nejad Dehbashi, Maryam Radan 104 Significant records of plants, algae, fungi, and animals in SE Europe and adjacent regions, 4 / Pomembne zabeležke rastlin, alg, gliv in živali za JV Evropo in sosednje regije, 4 Aljaž Curk, Maj Kastelic, Aljaž Jakob, Alenka Mihorič, Predrag Jakšić, Tina Klenovšek Editors: Simona Strgulc Krajšek, Tina Klenovšek 110 Study of the Effect of Autotrophic and Mixotrophic Culture Media on Heavy Metal Uptake by Fischerella sp. and Desmonostoc alborizicum / Študija vpliva avtotrofnih in miksotrofnih gojišč na absorpcijo težkih kovin s strani Fischerella sp. in Desmonostoc alborizicum Bahareh Nowruzi, Hassan Beiranvand, Roya Rahmani, Seyedeh Sajedeh Al-Tayeb, Amir Aala Zakeralhosseini Khafri Review 126 Continuous Bioprocessing for Recombinant Protein Production in Bacillus subtilis: Opportunities and Challenges / Kontinuirno bioprocesništvo za proizvodnjo rekombinantnih proteinov v Bacillus subtilis: priložnosti in izzivi Andrej Ivanovski, Tomaž Bratkovič News 134 FEMS MICRO Milan 2025 – 11. kongres evropskih mikrobiologov Janez Mulec, Jerneja Ambrožič Avguštin Table of Contents 4 1 Moghan College of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran 2 University of Maragheh, Faculty of Agriculture, Department of Plant Production and Genetics, Maragheh, Iran 3 Department of Horticultural Science, Faculty of Agricultural Science and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran * Corresponding author: E-mail address: mjanmohammadi@maragheh.ac.ir Citation: Ebadi-Segherloo, A., Janmohammadi, M., Sabaghnia, N., Mohebodini, M., (2025). The effect of different herbicides and soil moisture regimes on Camelina performance. Acta Biologica Slovenica 68 (4) Received: 11.06.2025 / Accepted: 15.07.2025 / Published: 18.07.2025 https://doi.org/10.14720/abs.68.4.23011 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research The effect of different herbicides and soil moisture regimes on Camelina performance Asghar Ebadi-Segherloo 1 , Mohsen Janmohammadi 2, * , Naser Sabaghnia 2 , Mehdi Mohebodini 3 Abstract This experiment aimed to evaluate the effect of different herbicides, including H 0 : no application of herbicide, H 1 : trifluralin as a pre-emergence herbicide, H 2 : cycloxydim, H 3 : haloxyfop-R-methyl, and H 4 : pinoxaden as post-emergence herbicides, on weed growth and camelina performance characteristics under well-irrigated (I 1 ) and rainfed (I 2 ) conditions. The response of the studied traits to herbicides varied across moisture regimes. The lowest population density was recorded under conditions of I 1 H 2 (8 plant m-2), I 2 H 3 (7 plant m -2 ), and I 2 H 4 (6.33 plant m -2 ). The lowest weed biomass was obtained under rainfed conditions with the use of cycloxydim. The tallest plants were recorded under I 1 H 2 conditions (110.96 cm), and the shortest plants were recorded under I 2 H 0 conditions (51.87 cm). The highest amount of chlorophyll was observed in plants grown under the conditions of I 1 H 4 (58.83 SPAD unit), I 1 H 2 (56.70), and the lowest amount under the conditions of I 2 H 0 (36.33) and I 2 H 1 (39.00). Seed yield under rainfed conditions decreased by 33% compared to well-irrigated conditions. The highest seed yield was obtained by using cycloxydim (1135 kg ha -1 ) and pinoxaden (1110 kg ha -1 ). Although weed populations were low under rainfed conditions, the effectiveness of herbicides was more prominent under fully irrigated conditions than under rainfed conditions. Keywords Camelina sativa, pinoxaden, post-emergence herbicide, pre-emergence herbicides, haloxyfop-R-methyl 5 Acta Biologica Slovenica, 2025, 68 (4) Vpliv različnih kemičnih herbicidov in režimov vlažnosti tal na uspešnost kameline Izvleček Namen tega poskusa je bil oceniti učinek različnih herbicidov, vključno s H 0 : brez uporabe herbicida, H 1 : trifluralinom kot herbicidom pred vznikom, H 2 : cikloksidimom, H 3 : haloksifop-R-metilom in H 4 : pinoksadenom kot herbicidi po vzniku na rast plevela in proizvodne značilnosti rička v dobro namakanih (I 1 ) in deževnih (I 2 ) pogojih. Odziv preučevanih lastnosti na herbicide se je razlikoval glede na režim vlažnosti. Najnižja gostota populacije je bila zabeležena v pogojih I 1 H 2 (8 rastlin m -2 ), I 2 H 3 (7 rastlin m -2 ) in I 2 H 4 (6,33 rastlin m -2 ). Najnižja biomasa plevela je bila dosežena v deževnih pogojih z uporabo cikloksidima. Najvišje rastline so bile zabeležene v pogojih I 1 H 2 (110,96 cm), najkrajše rastline pa v pogojih I 2 H 0 (51,87 cm). Najvišjo količino klorofila so opazili pri rastlinah, gojenih v pogojih I 1 H 4 (58,83 SPAD enote), I 1 H 2 (56,70), najnižjo pa v pogojih I 2 H 0 (36,33) in I 2 H 1 (39,00). Pridelek semen v deževnici se je v primerjavi z dobro namakanimi pogoji zmanjšal za 33 %. Najvišji pridelek semen je bil dosežen z uporabo cikloksidima (1135 kg ha -1 ) in pinoksadena (1110 kg ha -1 ). Čeprav so bile populacije plevela v deževnici nizke, je bila učinkovitost herbicidov izrazitejša v pogojih polnega namakanja kot v deževnici. Ključne besede Camelina sativa, pinoxaden, herbicid po pojavu plevela, herbicid pred pojavom plevela, haloksifop-R-metil Introduction Water shortage is a major factor limiting crop yields in semi-arid regions. Climate changes in recent decades and a significant increase in air temperature have aggravated the water shortage in these areas (Morante-Carballo et al., 2022). Camelina sativa, an emerging oilseed crop from the Brassicaceae family, has a long history of cultivation but has recently regained attention for its potential agricultural value. Camelina sativa can produce an acceptable yield with low inputs, and it also has high relative resistance against pests, diseases, and adverse environmental condi- tions such as high temperature, drought stress, and salinity (Obour et al., 2015). The main producers of camelina include the United States, Canada, Slovenia, Ukraine, China, Finland, Germany, Austria, and New Zealand (FAOSTAT, 2024). Weeds represent another significant challenge in the cultivation of Camelina. Weeds significantly reduce the growth and quality of crops by competing with crops for light, water, and nutrients. Not controlling weeds in crops of the Brassicaceae family can reduce the crop yield by about 50% (Ullah et al., 2020). Crop production systems have conventionally prioritised plant modification for higher yield over other characteristics, so any procedure of weed conquest must deliberate its net effect on efficiency, given that decreased seed yield in a weed-free environment can be compensated by the yield advantage provided by operative weed control (Ghidoli et al., 2023). Although the area under Camelina cultivation is expanding in semi-arid regions, there is still not much information about the efficacy of herbicides in Camelina production systems. Currently, to control rapeseed weeds, trifluralin as the pre-plant soil incorporated herbicide, clopyralid for control of broadleaf weeds, and cycloxydim, haloxyfop-R-methyl and sethoxy- dim for control of narrow leaf weeds are recommended for the regions of Iran (Shimi et al., 2014). A large portion of Iran is characterised by a semi-arid climate where plant growth is constrained by several environmental and edaphic limitations. These include low annual precipitation, irregular and unpredictable rainfall patterns, and the concentration of precipitation during the cold months, when many plants are in the rosette stage and exhibit minimal growth. Additional challenges include low winter temperatures in high-altitude areas, deficiencies or limited availability of essential nutrients in the rhizosphere, low soil organic matter content, high soil pH, and prolonged periods of dryness and low rainfall during the spring grow- ing season (Modarres and da Silva, 2007; Janmohammadi and Sabaghnia, 2023; Fattahi et al., 2023; Janmohammadi et al., 2024). Although several studies have examined the effects of water deficit on Camelina plants, reporting significant reductions in vegetative growth, seed yield, and 6 Acta Biologica Slovenica, 2025, 68 (4) oil quality (Borzoo et al., 2021; Bukhari et al., 2022; Hazrati et al., 2024), the effect of water deficit conditions on the effectiveness of herbicides has not been well studied yet. Studies investigating the use of herbicides such as quin- clorac, S-metolachlor, dimethenamid-P, pendimethalin, and pyroxasulfone herbicides during the pre-planting stage in the Camelina fields have shown that early Camelina growth is sensitive to these soil-applied chemicals. However, as the plants develop, their sensitivity decreases, and the negative effects of the herbicides are progressively reduced. The application of quinclorac sought the highest safety in the product (Jha and Stougaard, 2013). Propaquizafop and quizalofop-p-ethyl as systemic herbicides can be absorbed by foliage and roots and translocated through the phloem, and finally inhibit fatty acid biosynthesis (inhibitor of Ace- tyl-CoA Carboxylase; ACCase). The use of the mentioned herbicides on various varieties of Camelina indicated the different effects of these herbicides on the chlorophyll flu- orescence and the functioning of photosystem II; however, after 42 days, the adverse effects of these herbicides were removed. However, spray of clopyralid and picloram as selective herbicides for control of broadleaf weeds or woody plants (act as auxin mimics) had little effect on Camelina pho- tosynthetic characteristics (Sobiech et al., 2020). Regarding the effect of water shortage conditions on the effectiveness of herbicides, some reports showed that the effectiveness of clodinafop-propargyl (as an inhibitor of ACCase) and mesosulfuron-methyl + iodosulfuron-methyl sodium (as an inhibitor of amino acid biosynthesis through the inhibition of acetolactate synthase) decreased significantly under drought stress conditions, and this was attributed to the decrease in stomatal conductance and the decrease in the absorption of herbicides (Alizade et al., 2020). Trifluralin is a soil-applied pre-emergent herbicide used to control a wide range of broadleaf and fine-leaf weeds. It inhibits germination or root growth by inhibiting cell division (Li et al., 2022). However, there is no information available on trifluralin's effectiveness in Camelina sativa fields. Addi- tionally, it appears that the function of herbicides and their effects on weeds can be affected by soil moisture regimes. Because under water stress conditions, plants may close the stomata and change the cuticle thickness. Although a few studies have examined the use of certain herbicides in Camelina production systems, the effectiveness of these herbicides under varying soil moisture conditions remains largely unexplored. This study aimed to evaluate the effects of trifluralin as a pre-emergence herbicide, and cycloxydim, haloxyfop-R-methyl, and pinoxaden as post-emergence herbicides, on weed suppression and Camelina seed yield components under both well-irrigated and rainfed condi- tions in the Maragheh region of northwestern Iran. Materials and Methods Crop Growth A field trial was carried out in the Margheh region, East Azarbaijan, Iran, during the 2023-2024 crop season. This area is located in Northwest Iran, on the southeastern slopes of Sahand Mountain at an altitude of 1490 meters. The cli- mate of the studied area is cold semi-arid (with latitude and longitude 37° 23'N; 46° 16'E) with predominant winter rainfall, and the amount of rainfall during the growing season (Sep- tember-July) was estimated at 294 mm. Before conducting the experiment, random samples were taken from the field soil at a depth of 10-30 cm, and some of its physical and chemical properties were examined (Table 1). The studied area is located on the southern slopes of Mount Sahand, where most of its annual precipitation occurs during the cold seasons.. The cultivated area in the present experiment was fallow during the previous cropping season. In mid-October 2023, the farm soil was ploughed using a moldboard plough, and immediately, 10 t ha -1 of cow manure fertilisation was spread on the soil surface and mixed using a sweep to a depth of 15 cm. Final seedbed preparation was carried out through secondary tillage using a rotavator and a furrower in early March 2024. Inter-row spacing was 50 cm. Before planting seeds, 60 kg of phosphorus was consumed through triple super phosphate fertiliser and 100 kg of nitrogen through urea, based on the suggested dose. In the middle of March, the farm was divided into main and sub-plots. Table 1. Soil texture and chemical characteristics of soil samples in the studied field. Tabela 1. Tekstura in kemijske značilnosti vzorcev tal na preučevanem polju. Soil texture Clay (%) Silt (%) Sand (%) N (%) K (mg kg -1 ) P (mg kg -1 ) OC (%) pH EC (ds m -1 ) CaCO₃ (%) silty loam 23 49 28 0.12 282 11 0.24 7.82 0.92 10.8 7 Acta Biologica Slovenica, 2025, 68 (4) Experimental Details The experimental design was a split-plot (2×5) based on a randomised complete block design with three replica- tions. The main factor at two levels included irrigation regimes (I 1 : well irrigated and I 2 : rainfed). In well-irrigated conditions, the field was irrigated 8 times from the time of planting to harvest, and a total of 550 mm of water was consumed. Sub-plots were assigned to chemical herbi- cides. Herbicide treatments were H 0 : no application of herbicide, H 1 : trifluralin as a pre-emergence herbicide, H 2 : cycloxydim, H 3 : haloxyfop-p-methyl, and H 4 : pinoxaden as post-emergence herbicides. Trifluralin herbicide (Treflan, CAS 1582-09-8) is a selective pre-emergent herbicide (EC 48%) obtained from Elixir Agricultural Company, Y azd, Iran. After ploughing and disc operations, and 10 days before seed planting, treflan herbicide was used at the suggested rate of (1.5 L ha -1 ) along with spraying 300-500 litres of water evenly and immediately mixing it with the soil using a light disk. This herbicide disrupts the germination and death of weed seedlings by disrupting mitosis and inhibit- ing microtubule synthesis. Cyclohexidim (Focus, CAS 66-81-9, 10% EC, BASF, Germany) is a systemic and selective herbicide from the cyclohexane oxime group, which is used to control narrow-leaved weeds in canola fields. The selective prop- erties of this herbicide are excellent, and it can be used in all stages of the growth of crops. Focus contains the active ingredient cyclohexadim from the group of cyclohexani- dones, which is used according to the recommendation after germination and after the four-to-five-leaf stage of narrow-leaved weeds in the amount of 2 L ha -1 , with the dysfunction of Acetyl-CoA carboxylase (ACCase) prevent- ing the synthesis of fatty acids and causing the death of weeds. Haloxyfop-R-methyl (CAS Number 72619-32-0, EC 10%) is a systemic herbicide that was produced under the brand name of Super Gallant from Jahan Chemi-Iran. This selective herbicide controls a wide range of one-year and multi-year narrow-leaf weeds in agricultural fields, and it was used at the rate of 1 L ha -1 at the 2-5 leaf stage of the weeds. Haloxyfop belongs to the aryloxyphenoxypropio- nate group of herbicides, which is absorbed by the plants within one hour through the leaves, and by inhibiting the ACCase enzyme, prevents fatty acid biosynthesis and inhibits the growth of weed plants' meristem tissues. Pinoxaden herbicide (Axial, CAS 243973-20-8, and EC 5%) is the only available herbicide from the Pinoxaden group. This poison inhibits the growth of narrow-leafed grasses. This herbicide was used at the stage of 4-6 narrow leaves in the amount of 1.2 L ha -1 . In control conditions or without chemical herbicides, the weeds in the experimen- tal plot were sprayed with distilled water. All the herbicides were sprayed according to the manufacturer's recommen- dations in the vegetative stages of weeds with the help of a motorised backpack sprayer (Solo model 433). Post-emer- gence herbicides were applied to the above-ground parts in the early hours of the day. We tried to cover all the weed shoots with herbicides, but the herbicide drops did not flow on the leaves. The seeds of Camelina sativa "Sohail cv." were obtained from the oil seeds department of the Dryland Agricultural Research Institute, Maragheh, Iran. The mentioned variety has a relatively high adaptability to different weather and soil conditions, and compared to other oilseed plants, it needs less water, fertiliser, and pesticides, and is more resistant to cold (Rostami Ahmadvandi et al., 2021). The seeds were planted manually on top of the ridges at a depth of 1.5 cm. Intra-row spacing was 5 cm. The planting date coincided with the beginning of the rains. The amount of the first rain was enough for seed germination and seed- ling establishment. Data Recorded After applying post-emergence herbicides and in the middle of the main stem elongation stage (BBCH= 35; Martinelli and Galasso, 2011), the surviving weeds between the rows and on the rows were counted using 1 m 2 quadrate from each experimental unit and reported as weed populations density (Hussain et al., 2022), and after cutting them above the ground surface, they were dried in an oven at 70 °C for 48 hours and then weighed (weed biomass). At the early stage of flowering (BBCH=60; First flowers open), the chlorophyll of the upper and developed leaves of Camelina was measured using a manual chlorophyll meter (SPAD 502, Konica Minolta, USA). In the physiological maturity stage of Camelina (BBCH=89; nearly all siliques were ripe, the crop was ready to be harvested), using an arbitrarily 1 m 2 quadrate from each experimental unit, plant height was measured. The lateral growth of the canopy was calculated by measuring the diameter of the canopy from left to right. After harvesting Camelina plants and drying them in the oven, seed yield components such as 8 Acta Biologica Slovenica, 2025, 68 (4) the silique number per plant, number of seeds per silique, weight of 1000 seeds, yield per unit area, biological yield, and harvest index (ratio of grain yield to biological yield) were calculated. Statistical Analysis The experiment was analysed as a split-plot based on a block design. Before performing the ANOVA, the normality of the data was checked and, in some cases, data trans- formation was performed. One-way analysis of variance was performed using SAS software, and comparison of means was performed using LSD. To interpret the similarity between treatment compounds and their effect on the evaluated biological traits, Principal Component Analysis was performed using Minitab software version 19.2. Results Wedd population and biomass The results of the variance analysis indicated that the interaction between irrigation (I) and herbicide (H) treatments had a statistically significant effect on weed population density at the 1% level. The highest geometric mean weed density under well-irrigated conditions without herbicide application (I 1 H 0 ). In contrast, the lowest weed densities were recorded with pinoxaden (6.33) and hal- oxyfop-R-methyl (7.00) under well-irrigated conditions, and with cycloxydim (8.00) under rainfed conditions. Among the herbicides tested, trifluralin demonstrated the least effectiveness in reducing weed population density under both irrigation regimes (Figure 1). Figure 1. Evaluation of weed population density in Camelina sativa field with different herbicide applications (H 0 : no application of herbicide, H 1 : trifluralin as a pre-emergence herbicide, H 2 : cycloxydim, H 3 : haloxyfop-p-methyl, and H 4 : pinoxaden as post-emergence) under well-irri- gated and rainfed conditions in the Maragheh region, northwestern Iran. The dashed line inside the box indicates the mean of the treatment combination, and the bar lines above and below the box indicate the distribution of the data. Boxes with similar letters do not have statisti- cally significant differences at the 5% level. Slika 1. Ocena gostote populacije plevela na polju Camelina sativa z različnimi uporabami herbicidov (H 0 : brez uporabe herbicida, H 1 : triflu- ralin kot herbicid pred vzklitjem, H 2 : cikloksidim, H 3 : haloksifop-p-metil in H 4 : pinoxaden po vzklitju) v dobro namakanih in deževnih razmerah v regiji Maragheh, severozahodno od Irana. Črtkana črta znotraj okvirja označuje povprečje kombinacije obdelave, črte nad in pod okvirjem pa označujejo porazdelitev podatkov. Okvirji z enakimi črkami nimajo statistično pomembnih razlik na ravni 5 %. 9 Acta Biologica Slovenica, 2025, 68 (4) Under well-watered conditions, the application of cycloxydim, haloxyfop-R-methyl and pinoxaden reduced weed population density by 57%, 33%, and 49%, respec- tively, compared to the untreated control. Under rainfed conditions, these herbicides reduced weed density by 22%, 53%, and 48%, respectively, relative to plots without herbi- cide application. Weed population density was reduced by 29% under rainfed conditions compared to well-irrigated conditions. Among the herbicides tested, cycloxydim and pinoxaden were the most effective in reducing weed popu- lation density. The evaluation of the above-ground biomass of weeds showed that the lowest biomass was obtained under rainfed conditions with the application of I 2 H 4 (10.33g) and I 2 H 2 (11.00 g). The highest amount of biomass was recorded under well-irrigated conditions without the use of herbicides (48.33 g) and the use of trifluralin (34.00 g). The most effective herbicides in reducing weed biomass com- pared to herbicide-free conditions were cycloxydim (65%), pinoxaden (55%), haloxyfop-R-methyl (46%) and trifluralin (35%), respectively. Additionally, water deficit under rainfed conditions reduced weed biomass by 45% compared to well-irrigated conditions (Figure 2). Chlorophyll content and growth characteristics of Camelina The interaction effects of irrigation (I) and herbicide (H) treat- ments on the chlorophyll content of Camelina leaves were statistically significant at the 1% level. The lowest amount of chlorophyll was recorded in herbicide-free conditions or in plots under rainfed conditions (Table 2). The highest amount of chlorophyll was recorded under well-watered conditions with the application of cycloxydim (56.70). Water shortage under rainfed conditions resulted in a 21% reduction in chlo- rophyll content compared to well-irrigated conditions. Plots treated with acetyl-CoA carboxylase (ACCase)-inhibiting herbicides exhibited higher chlorophyll content than those subjected to other treatments. Assessment of plant height revealed that the interaction effects of I × H are significant at the 1% level. The tallest plants under well-watered conditions Figure 2. Mean comparison of above-ground biomass of weed under different chemical weed control methods (H 0 : no application of her- bicide, H 1 : trifluralin as a pre-emergence herbicide, H 2 : cycloxydim, H 3 : haloxyfop-p-methyl, and H 4 : pinoxaden as post-emergence) under different soil moisture regimes in the semi-arid region of the Mragheh, Iran. Slika 2. Povprečna primerjava nadzemne biomase plevela pri različnih kemijskih metodah zatiranja plevela (H 0 : brez uporabe herbicida, H 1 : trifluralin kot herbicid pred vzklitjem, H 2 : cikloxidim, H 3 : haloksifop-p-metil in H 4 : pinoxaden po vzklitju) pri različnih vlažnostnih razmerah tal v polsušni regiji Mragheh v Iranu. 10 Acta Biologica Slovenica, 2025, 68 (4) were recorded with the application of cycloxydim (110.96 cm) or the application of haloxyfop-R-methyl (101 cm). The shortest plants were obtained under the rainy conditions without herbicide application (51.82 cm) or trifluralin appli- cation (59.13 cm). Water stress resulting from the absence of irrigation under rainfed conditions reduced plant height by approximately 35% compared to well-irrigated conditions. The effect of the herbicide application on Camelina height was higher under well-irrigated conditions, and the use of trifluralin, cycloxydim, haloxyfop-R-methyl, and pinoxaden increased plant height by 18%, 42%, 31%, and 8%, respec- tively, compared to herbicide-free conditions (Table 2). The highest lateral growth of the canopy was recorded under well-irrigated conditions without herbicide application (67.33 cm) or haloxyfop-R-methyl (64.00 cm) application. However, the lowest canopy width was recorded for plants grown under rainfed conditions without herbicide application (42.33 cm) or with the spray of haloxyfop-R-methyl (47.66 cm). Among Camelina seed yield components, the seed number per silique (NSS) was significantly affected by I×H effects. The highest amount of NSS was obtained under well-watered conditions with cycloxydim (15.95) applica- tion. The lowest NSS value was recorded under rainfed conditions without herbicide application (6.67). The silique number in plants grown under rainfed conditions was 56% lower than in well-irrigated conditions. There was no differ- ence in the number of siliques per plant among the levels of chemical control, but the plants grown under chemical weed control conditions showed a higher number of siliques per plant compared to herbicide-free conditions (Table 2). Irrigations (I) CHL PH LG SNP NSS TSW SY Well-irrigated (I1) 53.85a 93.12a 59.09a 135.13a 15.10a 0.840a 1273.53a Rainfed (I2) 42.86b 60.21b 45.34b 59.72b 8.78b 0.665b 852.07b Herbicides (H) Control (H0) 64.67c 44.25b 46.06c 88.66c 10.65b 0.718b 984.17c Trifluralin (H1) 75.36b 44.94b 49.03c 94.65bc 12.38a 0.718b 1018.33c Cycloxydim (H2) 86.26a 51.35a 53.83b 102.50a 12.48a 0.800a 1110.83a Haloxyfop-R-methyl (H3) 81.56a 49.83a 54.83a 101.66ab 11.75a 0.766a 1065.33b Pinoxaden (H4) 75.48b 51.41a 57.33a 99.66ab 12.38a 0.762a 1135.33a I 1 H 0 51.17b 77.48d 51.80cd 124.33c 14.53b 0.787a 1183.33c I 1 H 1 50.89b 91.60c 52.33c 132.66bc 14.93ab 0.812b 1206.00c I 1 H 2 56.70a 110.96a 60.00b 145.66a 15.93a 0.917a 1308.00b I 1 H 3 51.66b 101.96a 67.33a 139.00ab 14.70ab 0.841b 1281.33b I 1 H 4 58.83a 83.74d 64.00ab 134.00bc 15.43ab 0.845b 1389.00a I 2 H 0 37.33e 51.87g 40.33g 53.00e 6.76d 0.649cd 785.00f I 2 H 1 39.00e 59.13fg 45.73ef 56.63de 9.83e 0.623d 830.67ef I 2 H 2 46.00cd 61.56ef 47.66de 59.33de 9.03c 0.684c 913.67d I 2 H 3 48.00bc 61.29ef 42.33fg 64.33d 8.80c 0.692c 849.33e I 2 H 4 44.00d 67.23e 50.66cd 65.33d 9.47c 0.679cd 881.67de Significance level I 0.05 0.0018 0.008 0.0005 0.009 0.006 0.0026 H <0.0001 <0.0001 <0.0001 0.0002 0.0035 0.0028 <0.0001 I×H 0.003 <0.0001 0.0003 0.157 0.05 0.171 0.011 CV% 4.50 5.76 4.97 6.13 7.49 3.51 4.79 Table 2. Mean comparisons of yield components of Camelina sativa under different irrigation regimes and herbicide utilisation in Northwest Iran, Mragheh. Tabela 2. Povprečne primerjave komponent pridelka Camelina sativa pri različnih režimih namakanja in uporabi herbicidov v severozahodnem Iranu, Mragheh. CV: The coefficient of variation (%), Chl: leaf chlorophyll content (SPAD unit), PH: Plant height (cm), LG: lateral growth of canopy (cm), SNP: silique number per plant, NSS: seed number per silique, TSW: thousand seed weight (g), SY: seed yield (kg ha-1), At the significance level, p values less than 0.05 (p < 0.05) and 0.01 (p < 0.01) are significant at 95% and 99%, respectively. The means with various letters in each trait (column) are statistically different. 11 Acta Biologica Slovenica, 2025, 68 (4) Evaluation of the thousand seed weight revealed that the interaction between irrigation and herbicide treatments was statistically significant at the 5% level. Plants grown under rainfed conditions exhibited a 21% reduction in seed weight compared to those grown under well-irrigated con- ditions. Camelina plants grown under ACCase-inhibiting herbicide conditions (H 2 , H 3 , and H 4 ) had more seed weight compared to other treatments. Assessment of seed yield showed that the mutual effects of irrigation and herbicide on this trait are significant at the 5% level. Plants grown under rainfed conditions had 35% less seed yield com- pared to well-irrigated conditions (Table 2). The highest seed yield (1389 kg ha -1 ) was recorded under well-irrigated conditions with the application of pinoxaden. The lowest seed yield was observed under rainfed conditions without the use of herbicide (785 kg ha -1 ) or with the use of pre- plant herbicide (830 kg ha -1 ). Biological yield The calculation of the biological yield of Camelina indi- cated that the mutual effects of irrigation and herbicide are statistically significant on this trait at the 1% level. The highest biological yield was recorded with the application of cycloxydim under well-irrigated conditions (4249 kg ha -1 ). Haloxyfop-r-methyl and pinoxaden herbicides were used in second place and produced the same biological yield. The lowest biological yield (2592 kg ha -1 ) was recorded under herbicide-free and rainfed conditions (Figure 3). The effect of haloxyfop-r-methyl herbicide on improving biological yield in well-irrigated conditions was more significant than in rainfed conditions. The interaction effects of I×H were significant at the 5% level. The harvest index under rainfed conditions was 3.54% less than under well-irrigated conditions. The Figure 3. Biological yield of Camelina sativa field affected by different herbicides (H 0 : no application of herbicide, H 1 : trifluralin as a pre-emer- gence herbicide, H 2 : cycloxydim, H 3 : haloxyfop-p-methyl, and H 4 : pinoxaden as post-emergence) under well-irrigated and rainfed conditions in Maragheh region, northwestern Iran. Slika 3. Biološki pridelek poljene kamelije (Camelina sativa) pod vplivom različnih herbicidov (H 0 : brez uporabe herbicida, H 1 : trifluralin kot herbicid pred vzklitjem, H 2 : cikloksidim, H 3 : haloksifop-p-metil in H 4 : pinoxaden kot herbicid po vzklitju) v dobro namakanih in deževnih razmerah v regiji Maragheh, severozahodno od Irana. 12 Acta Biologica Slovenica, 2025, 68 (4) highest harvest index was recorded under well-irrigated conditions with the use of pinoxaden (35.27), trifluralin (33.17), and without the use of herbicides (32.62). Under rainfed conditions, the lowest harvest index was obtained with the use of chemical herbicides. This indicates that the effect of herbicides on improving vegetative growth and biological yield was more prominent than seed yield. The herbicide-free treatment under both irrigation conditions caused a significant increase in the harvest index (Figure 4). Cluster analysis and PCA However, under well-irrigated conditions, application of pinoxaden resulted in the highest HI. The clustering of combined treatments according to the similarity in affecting the studied traits showed that the least effective treatments included no use of herbicide and the application of trifluralin under rainfed conditions (cluster I) and no use of herbicide under well-irrigated conditions (cluster III) (Figure 5). Under well-irrigated conditions, Cycloxydim, Haloxyfop-R-methyl, and pinoxaden (H 4 , H 3 , and H 2 ) herbicides were included in cluster II, which were the most effective treatments. However, under rainfed conditions, the effectiveness of all herbicides was almost similar and less than in well-irrigated conditions (cluster IV). The plots obtained from Principal Component Analysis (PCA) showed that the first component was able to differ- entiate the soil moisture conditions from each other. The second component also separated the herbicides in terms of effectiveness (Figure 6). The plot obtained from PCA showed that the effectiveness of herbicides under well-ir- rigated conditions is different from rainfed conditions, and ACCase-inhibiting herbicides were more effective than pre-plant herbicide (Trifluraline). Furthermore, under well-ir- rigated conditions, the effects of Trifluraline on investigated traits were not prominent when compared with control conditions. Pre-plant herbicide could not affect the weed population under well-irrigated conditions. Figure 4. Assessments of harvest index of Camelina under different chemical weed control methods (H 0 : no application of herbicide, H 1 : trifluralin as a pre-emergence herbicide, H 2 : cycloxydim, H 3 : haloxyfop-p-methyl, and H 4 : pinoxaden as post-emergence) under different soil moisture regimes in the semi-arid region of Mragheh, Iran. Slika 4. Ocene indeksa pridelka kamelije pri različnih metodah kemičnega zatiranja plevela (H 0 : brez uporabe herbicida, H 1 : trifluralin kot herbicid pred vzklitjem, H 2 : cikloksidim, H 3 : haloksifop-p-metil in H 4 : pinoxaden kot herbicid po vzklitju) pri različnih vlažnostnih razmerah tal v polsušni regiji Mragheh v Iranu. 13 Acta Biologica Slovenica, 2025, 68 (4) Figure 5. Cluster analysis of combined treatments in terms of similarity in the influence of different evaluated traits of Camelina. I 1 : well-irri- gated condition, I 2 : rainfed condition, H 0 : no application of herbicide, H1: trifluralin as a pre-emergence herbicide, H 2 : cycloxydim, H 3 : haloxy- fop-p-methyl, and H 4 : pinoxaden as post-emergence. Slika 5. Klasterna analiza kombiniranih obdelav glede na podobnost vpliva različnih ocenjenih lastnosti Camelina. I 1 : dobro namakano stanje, I 2 : deževno stanje, H 0 : brez uporabe herbicida, H 1 : trifluralin kot herbicid pred vzklitjem, H 2 : cikloxidim, H 3 : haloksifop-p-metil in H 4 : pinoxaden kot herbicid po vzklitju. Figure 6. The plot was prepared through principal component analysis (PCA) for classification of combined treatments in terms of superiority in influencing the evaluated traits and less effective treatments in the Camelina field. I 1 : well-irrigated condition, I 2 : rainfed condition, H0: no appli- cation of herbicide, H 1 : trifluralin as a pre-emergence herbicide, H 2 : cycloxydim, H 3 : haloxyfop-p-methyl, and H 4 : pinoxaden as post-emergence. Slika 6. Graf, pripravljen z analizo glavnih komponent (PCA) za razvrstitev kombiniranih obdelav glede na vpliv na ocenjene lastnosti in manj učinkovite obdelave na polju kamelije. I 1 : dobro namakano stanje, I 2 : deževno stanje, H 0 : brez uporabe herbicida, H 1 : trifluralin kot herbicid pred vzklitjem, H 2 : cikloksidim, H 3 : haloksifop-p-metil in H 4 : pinoxaden kot herbicid po vzklitju. 14 Acta Biologica Slovenica, 2025, 68 (4) Discussion The results of the experiment indicated that the lack of soil moisture in rainfed conditions reduced biomass and weed density compared to well-irrigated conditions. At the same time, the effectiveness of herbicides also showed a signifi- cant decrease in rainfed conditions. The amount of precipi- tation during the growing season was about 294 mm, while the estimated amount of evaporation and transpiration during the growing season was about 485 mm (Valizadeh 2014). This indicates that if the soil is not accompanied by sufficient amounts of stored moisture in the rainfed system at the beginning of the season, the cultivated plants will face serious water stress. In such conditions, the seed yield components of Camelina can be affected significantly (Neupane et al., 2020). The reduction of weed population under rainfed con- ditions is largely due to insufficient soil moisture. However, some weeds such as field bindweed (Convolvulus arven- sis), wild lettuce (Lactuca virosa), wild radish (Raphanus raphanistrum), hedge mustard (Sisymbrium officinale), shepherd's purse (Capsella bursa-pastoris), whitetop (Lep- idium draba), Baconweed (Chenopodium album), common purslane (Portulaca oleracea), downy brom (Bromus tec- torum), foxtail barley (Hordeum jubatum), and camelthorn (Alhagi maurorum) were able to grow even under water shortage conditions and a major part of the biomass and density was dedicated to these weeds. On the other hand, it seems that under drought stress condition due to the closing of stomata, increased waxing of leaves, morpholog- ical changes in leaves, reduction of leaf surface, reduction of stomatal conductance, and probably the metabolism of the main herbicide molecule inside the weed plant, the effectiveness of the herbicide decreased (Peerzada et al., 2021). Also, for the pre-emergence herbicide (trifluralin), it seems that the low soil moisture content restricted the herbicide molecules' movement in the soil and reduced the possibility of reaching the weed seeds in the seed bank. The results showed that the effect of trifluralin on rainfed conditions was disappointing. The use of ACCase-inhibit- ing herbicides was more effective than trifluralin in both soil moisture environments. This also accords with our earlier observations, which showed that trifluralin was not more effective under well-irrigated conditions (Sabaghnia et al., 2025). Weed control and the reduction of competition for resources such as light, moisture, and food increased Camelina's vegetative growth (plant height and lateral growth) significantly. The use of ACCase-inhibiting herbi- cides, especially pinoxaden and cycloxydim, increased the chlorophyll content in Camelina. This can be attributed to the reduction of weed shading and the increase of light received by the shoots of the Camelina plant, as well as more access to limited water and nutrient resources in the soil (Formisano et al., 2022). Investigating the components of vegetative growth and photosynthesising organs (source size) and seed yield components (sink size) in Camelina indicated that the use of herbicide in the current experiment also affected the source-sink relationships. It seems that the use of herbi- cides, especially ACCase inhibitor herbicides, has increased the production of photoassimilates or their better allocation to reproductive organs through the removal of narrow leaf weeds, reducing competition and increasing access to limited resources. However, in the investigated Camelina variety or the drought stress conditions, it seems that the limitation of the tank was one of the factors limiting the yield. This means that despite the elimination of competition and the increase in vegetative growth and possibly the increase in the supply of photoassimilates, this was not associated with an increase in the number of yield components. In this context, the results of the researchers indicate that in addition to the effect of herbicide use on reducing com- petition, it can affect the relationship between source and reservoir through the manipulation of carbon and nitrogen metabolism. The use of Imazethapyr herbicides (inhibitor of acetolactate synthase) in lentil genotypes caused a signifi- cant change in the synchronisation of carbon and nitrogen metabolism, and this caused a significant increase in the quantity and quality of the seed in some genotypes (Grewal et al., 2023). However, under well-irrigated conditions, both source and sink components showed a significant increase with the application of chemical herbicides. The results indicate that the crop-weed interactions can affect the yield. The presence of weeds through competition can both reduce the size and strength of sources and reduce the size of sinks (Evers and Bastiaans 2016). The obtained results showed that besides improving yield, regular irrigation also improves the efficiency of herbicides. These results confirm the previous reports of Alizadeh et al. (2020). In his research, they found that drought stress reduced the effec- tiveness of benzoylprop-ethyl herbicide (Suffix: Inhibitor of lipid biosynthesis) in the semi-arid region. On the other hand, it seems that weed exposure to long-term drought may lead to herbicide resistance (HR). HR can be inherited 15 Acta Biologica Slovenica, 2025, 68 (4) by the next generations, and it is partially caused by the acti- vation of some defence mechanisms and the scavenging of reactive oxygen species (Mohammad et al., 2022). Among the ACCase inhibitor herbicides, pinoxaden had a greater effect on weed control, which can be attributed to better absorption, greater penetration into the weed shoots, faster translocation and less metabolism inside the weed plant (Pacanoski and Mehmeti, 2023). The reduced effectiveness of ACCase-inhibiting herbicides under certain environmen- tal conditions is primarily attributed to the metabolism and degradation of the herbicides or the conjugation of various compounds with herbicide molecules (Tang et al., 2020). Conclusions The results indicated that regular irrigation in the studied area is essential for Camelina production systems to achieve an acceptable seed yield. The Result revealed that under rainfed conditions, water shortage stress signifi- cantly decreased the seed yield (33%) in comparison with well-irrigated conditions. Also, soil water regimes affected the efficiency of herbicides. Although weed populations were lower under rainfed conditions than under full irriga- tion conditions, the herbicide effect was more noticeable under full irrigation conditions. However, the application of pre-planting herbicides under well-irrigated conditions was not very effective. The highest level of weed control was achieved with the use of ACCase inhibitor herbicides (especially cycloxydim and pinoxaden). However, in rainfed conditions, cycloxydim significantly reduced the biomass and population density of weeds. However, the main weed in the field was broad-leaved (field bindweed), and this may require the investigation of the application of narrow and broad-leaved herbicides for weed control in Camelina fields in the semi-arid region. Author Contributions Conceptualization, A.E. and M.J.; methodology, M.J. and N.S.; software, M.M. and A.E.; validation, A.E. and N.S.; formal analysis, N.S. and M.M.; investigation, M.J.; resources, A.E.; data curation, M.J and A.E.; writing—original draft prepara- tion, M.J.; writing—review and editing, A.E. and M.M; visual- ization, A.E.; supervision, N.S. and A.E.; project administra- tion, M.J.; funding acquisition, A.E and M.J. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship. Acknowledgement The authors would like to thank the Ministry of Science, Research and Technology for providing some of the research necessities. The cooperation, accuracy and assis- tance of local workers who helped during sowing, irriga- tion, and sampling are greatly appreciated. The authors would like to thank the Vice President for Research and Technology. 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Acta Biologica Slovenica 68 (4) Received: 03.03.2025 / Accepted: 14.07.2025 / Published: 21.07.2025 https://doi.org/10.14720/abs.68.4.21999 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Umetne vodne akumulacije za zasneževanje na vzhodu Pohorja (Slovenija): mesta razmnoževanja in ekološke pasti za dvoživke Tina Klenovšek 1, * , Žiga Tertinek 2 , Ana Skledar 2 Izvleček Umetni vodni habitati so lahko za dvoživke mesta za razmnoževanje kot tudi ekološke pasti. Vodne akumulacije za umetno zasneževanje niso optimalni habitati za dvoživke in o njihovem razmnoževanju v teh vodnih telesih je malo znanega. V raziskavi smo dvoživke vzorčili v dveh akumulacijah za umetno zasneževanje na območju Mariborskega Pohorja. Ena se nahaja ob vznožju Pohorja (312 m nmv) v urbanem okolju, druga pa na pobočju Pohorja (560 m nmv), obdana z gozdom, ki je del Natura 2000 območja Pohorje. V akumulacijah smo skupno potrdili razmnoževanje sedmih vrst dvoživk: velikega pupka (Triturus carnifex), planinskega pupka (Ichthyosaura alpestris), navadnega pupka (Lissotriton vulgaris), hribskega urha (Bombina variegata), navadne krastače (Bufo bufo), sekulje (Rana temporaria) in rosnice (Rana dalmatina). Hribski urh in veliki pupek spadata med kvalifikacijske vrste za Natura 2000 območje Pohorje. V akumulaciji ob vznožju Pohorja, v kateri so bile tudi ribe, razmnoževanje dvoživk ni bilo uspešno. V akumulaciji na pobočju Pohorja, kjer rib ni bilo, sta strma brežina (30–60°) iz nepokrite geomembrane in nizka gladina vode povzročili pogin odraslih hribskih urhov in številnih (> 500) mladih osebkov rjavih žab. Upad gladine vode je povzročil tudi propad jajc in zarodkov velikega pupka. Akumulaciji za zasneževanje sta iz več vidikov neprimerni za dvoživke, vendar jih te kljub temu uporabljajo za razmnoževanje. Za zmanjšanje poginov dvoživk bi bili potrebni naslednji ukrepi: ohranjanje maksimalnega vodostaja v času razmnoževanja (marec–julij), prekritje geomembrane z nedrsečim materialom in zmanjšanje naklona brežine akumulacije, kar bi dvoživkam olajšalo izhod iz akumulacije, odstranitev rib z jesenskim praznjenjem akumulacije ter preprečevanje povozov na cestah ob akumulaciji. Keywords Bombina variegata, dvoživke, ekološka past, Natura 2000, Triturus carnifex, umetni habitat, vodna zajetja, zadrževalniki za umetno zasneževanje 18 Acta Biologica Slovenica, 2025, 68 (4) Artificial Water Accumulations for Snowmaking on Eastern Pohorje (Slovenia): Breeding Sites and Ecological Traps for Amphibians Abstract Artificial water habitats can be breeding sites for amphibians, but may also function as ecological traps. Water accumulations for artificial snowmaking are not optimal habitats for amphibians, and little is known about their reproduction in these water bodies. In this study, we sampled amphibians in two accumulations for snowmaking in the Mariborsko Pohorje area. One is located at the foothills of Pohorje (312 m a.s.l.) in an urban environment, and the other on the slopes of Pohorje (560 m a.s.l.), surrounded by forest that is part of the Natura 2000 Pohorje. In total, we confirmed the reproduction of seven amphibian species in these accumulations: Italian Crested Newt (Triturus carnifex), Alpine Newt (Ichthyosaura alpestris), Smooth Newt (Lissotriton vulgaris), Yellow-bellied Toad (Bombina variegata), Common Toad (Bufo bufo), Common Frog (Rana temporaria), and Agile Frog (Rana dalmatina). The Yellow-bellied Toad and the Italian Crested Newt are qualifying species for the Natura 2000 Pohorje site. In the accumulation at the foothill of Pohorje, where fish were present, amphibian reproduction was unsuccessful. In the accumulation on the slope of Pohorje, where there were no fish, the steep banks (30–60°) with uncovered geomembrane and the low water level led to the mortality of adult Y ellow-bellied T oads and numerous (>500) juvenile Rana sp. frogs. The drop in water level also resulted in the stranding of eggs and embryos of the Italian Crested Newt. Snowmaking reservoirs are unsuitable for amphibians in several respects, yet they still use them for breeding. To reduce amphibian mortality, the following measures should be implemented: maintaining maximum water levels during the breeding season (March–July), covering the geomembrane with non-slip material and reducing the bank steepness to facilitate amphibian exit, removing fish through autumn reservoir drainage, and preventing roadkill on roads adjacent to the reservoirs. Keywords Amphibians, artificial habitat, Bombina variegata, ecological trap, Natura 2000, snowmaking ponds, Triturus carnifex, water reservoirs Uvod Dvoživke za svoj življenjski cikel potrebujejo tako kopen- ske kot vodne habitate. Prav habitati celinskih voda so med najbolj ogroženimi v Evropi (Janssen in sod., 2016). Degradacija in izguba habitatov sta med glavnimi razlogi za upad populacij dvoživk, tako je po podatkih IUCN v Evropi ogrožena skoraj četrtina dvoživk (Temple in Cox, 2009; IUCN, 2025). V Sloveniji živi 21 vrst dvoživk (Stanković in sod., 2015; Strah in sod., 2024), kar jo uvršča med bolj pestra območja Evrope glede na število vrst dvoživk (Temple in Cox, 2009). Dvoživke v Sloveniji ogrožajo predvsem izguba kakovostnih habitatov, ki jo povzročajo nenadzorovana in razpršena urbanizacije, regulacija in hidromorfološke spre- membe vodotokov in obvodnih zemljišč, onesnaževanje z organskimi snovmi in gnojili, vnos tujerodnih rib, izginjanje mlak ter izsuševanja mokrišč (Stanković in sod., 2015). Dvoživke za razmnoževanje uporabljajo tudi vodna telesa umetnega nastanka (Clevenot in sod., 2018; Valdez in sod., 2021), tj. telesa površinskih vod, ki so nastala zaradi posega v prostor (Ur. l. RS., 2002). Takšna vodna telesa so za dvoživke sekundarni vodni habitati, kjer je večinoma manjša biotska pestrost kot v primerljivih naravnih vodnih habitatih (Zamora- Marín in sod. 2021). Kljub temu lahko na zelo prepustnih tleh, sušnih ali močno spremenjenih območ- jih prevladujejo in imajo ključno vlogo za razmnoževanje ter obstoj populacij dvoživk (Brand in Snodgrass, 2010; Buono in sod., 2019; Lešnik, 2021; Zamora- Marín in sod., 2021; Caballero-Díaz in sod., 2022). Umetni vodni habitati lahko zaradi svojih lastnosti in namenske rabe delujejo tudi kot ekološke pasti (Dwernychuk in Boag, 1972), tj. kadar jih živali na podlagi okoljskih signalov ocenijo kot ustrezne in prednostno izberejo, čeprav imajo v primerjavi z drugimi razpoložljivimi habitati v njih manjše možnosti 19 Acta Biologica Slovenica, 2025, 68 (4) za preživetje ter uspešno razmnoževanje (Robertson in Hutto, 2006; Clevenot in sod., 2018). Umetni vodni habi- tat lahko predstavlja ekološko past tudi, kadar povzroča neposredno smrtnost osebkov ali če kot razmnoževalno mesto ne omogoča zadostnega razmnoževalnega uspeha za ohranjanje stabilne ali rastoče populacije brez priselje- vanja (Battin, 2004). Primer umetnih vodnih habitatov za razmnoževanje dvoživk so vodne akumulacije namenjene umetnemu zasneževanju smučišč. Zaradi namenske rabe imajo te akumulacije podobne značilnosti. Med drugim, da so umetnega nastanka, značilne oblike, s strmimi in ravnimi brežinami prekritimi s hidroizolacijsko folijo, povsem brez vodne vegetacije, raven vode pa v njih hitro in močno niha (Fait in sod., 2020; Gerfand in sod., 2024). Zadrževalniki voda niso optimalni umetni vodni habitati za dvoživke, a so lahko edini vodni habitati, ki zadržujejo vodo dovolj dolgo, da ličinke zaključijo razvoj (Brand in Snodgrass, 2010). Redno vzdrževanje, tj. letno praznjenje in čiščenje akumulacij ob ustreznem času, preprečuje obstoj rib, kar je prav tako ugodno za dvoživke (McCarthy in Lathrop, 2011; Zamora- Marín in sod., 2021). Za razliko od zadrževalnikov meteornih voda v urbanih okoljih ali ob avtocestah (pre- gled v Clevenot in sod., 2018), je podatkov o dvoživkah v akumulacijah za zasneževanje zelo malo, četudi število takšnih akumulacij raste (Gerfand in sod., 2024). Gerfand in sod. (2024) so ocenili ekološko kakovost 28 akumulacij za umetno zasneževanje v francoskih Alpah. Dvoživke so našli v 68 % vseh akumulacij in akumulacije zaradi zasnove ter načina uporabe ocenili kot potencialne ekološke pasti. Akumulacije za umetno zasneževanje so tudi na delih Pohorja, kjer je razvit smučarski turizem. Pohorje je predalpsko hribovje, ki se razteza 50 km v dolžino v smeri vzhod-zahod. Na vzhodu prehaja v Dravsko dolino in mesto Maribor. Na Mariborskem Pohorju sta dve vodni zajetji za zasneževanje smučišč. Akumulacija Arena je ob vznožju in akumulacija Trikotna jasa na pobočju Pohorja. Za namen predvidene razširitve in posodobitve zimsko šport- nega centra Pohorje so na vplivnem območju Govedič in sod. (2009) popisali 130 lokacij in potrdili 5 vrst dvoživk, od do takrat znanih 10 vrst dvoživk za Pohorje in vznožje Mariborskega Pohorja med Radvanjem in Razvanjem (Vogrin, 1998; Poboljšaj, 1998; Poboljšaj in sod., 2001; Cipot, 2007). V akumulaciji Trikotna jasa so bili takrat naj- deni samo paglavci sekulje (Rana temporaria), medtem ko v akumulaciji Arena dvoživk niso zabeležili. V akumulaciji Arena je bilo kasneje potrjeno razmnoževanje navadne krastače (Bufo bufo) in sekulje (Tertinek in Skledar, 2022; Tertinek, 2024). Akumulacija Trikotna jasa je na ekološko pomembnem območju Pohorje (ID 41200). Strnjen gozd južno in vzhodno od akumulacije pa je del Natura 2000 območja Pohorje (ID 3000270), v katerem sta med kvalifik- acijskimi vrstami dvoživk hribski urh (Bombina variegata) in veliki pupek (Triturus carnifex) (Ur. l. RS 49/2004, Ur. l. RS 110/2004, Ur. l. RS 47/18). Vrsti sta zaradi svojega pomena relativno dobro raziskani, monitoring se je izvajal tudi na Pohorju (Poboljšaj in sod., 2011; Cipot in sod., 2011; Grudnik in Triglav Brežnik, 2015). Na območju akumulacij za zasneževanje na Mari- borskem Pohorju se stikajo strnjen gozd, spremenjeno površje za namen športa in turizma ter urbano naselje. Na pobočjih Pohorja so naravne stoječe vode redke, potoki pa imajo hudourniški značaj, zato so umetne stoječe vode, kot so akumulacije za zasneževanje, lahko pomemben sekundarni habitati za razmnoževanje dvoživk. Vendar pa lahko akumulacije za zasneževanje, zaradi svojih značilnosti in rabe, dvoživkam hkrati predstavljajo grožnjo za preživetje in razmnoževalni uspeh. V letu 2024 je bil na območju akumulacije Arena izveden monitoring pom- ladanskih selitev dvoživk po naročilu Mestne občine Mari- bor (Tertinek, 2024). Neodvisno so bili istega leta izvedeni tudi popisi dvoživk v akumulaciji na Trikotni jasi. Namen raziskave akumulacij za zasneževanje na Mariborskem Pohorju je bil (1) ugotoviti, katere vrste dvoživke so prisotne v akumulacijah in ali se tam tudi razmnožujejo, (2) opisati značilnosti akumulacij, ki lahko vplivajo na preživetje in razmnoževanje dvoživk v vodnih telesih, (3) oceniti vpliv teh značilnosti akumulacij na dvoživke in njihov potencial, da delujejo kot ekološke pasti, ter (4) predlagati ukrepe za izboljšanje značilnosti akumulacij z vidika vpliva na preživetje in razmnoževanje dvoživk. Pričakujemo, da bomo v akumulacijah našli več vrst dvoživk, kot jih je bilo zabeleženih do sedaj (do sedaj sta bili zabeleženi le navadna krastača in sekulja), ter da bomo potrdili njihovo razmnoževanje. Ker pa akumulacije niso optimalni habitati za dvoživke in globoke stoječe vode brez vegetacije ne ustrezajo vsem vrstam, pričakujemo, da bo število vrst kljub temu majhno. Glede na specifične značilnosti in namensko rabo, predvidevamo, da so akumulacije potencialne ekološke pasti za dvoživke, a lahko z ukrepi za izboljšanje postanejo ustrezni sekundarni habitati za razmnoževanje dvoživk. Raziskava je tudi prispevek k boljšemu razume- vanju vloge umetnih vodnih teles pri ohranjanju dvoživk v preoblikovanih gozdnih ekosistemih. 20 Acta Biologica Slovenica, 2025, 68 (4) Material in metode Akumulaciji za zasneževanje na Mariborskem Pohorju Akumulacija Arena in Trikotna jasa (Sl. 1) sta nastali s posegom v prostor z namenom zajemanja vode za umetno zasneževanje smučišč na Mariborskem Pohorju. Akumulacija Arena je umeščena ob vznožju Pohorja v Zgornjem Radvanju, na nadmorski višini 312 m, medtem ko se akumulacija ob Trikotni jasi nahaja na pobočju Pohorja na nadmorski višini 560 m. Med akumulacijama je 1000 m zračne razdalje. Večji stalni stoječi vodi v bližini akumulacij sta Radvanjski ribnik in zajezitev Radvanjskega potoka, obe na podobni nadmorski višini kot akumulacija Arena (Sl. 1). Radvanjski ribnik je od akumulacije Arena oddaljen 780 m in od akumulacije Trikotna jasa 1350 m zračne razdalje, zajezitev Radvanjskega potoka pa 420 m oziroma 1000 m zračne razdalje. Na podlagi podatkov o povprečni letni količini padavin za obdobje 1981–2010 leži akumulacija Arena na območju s 1100–1200 mm padavin letno, akumulacija Trikotna jasa pa na območju s 1200–1300 mm padavin letno. Obe vodni telesi pripadata območju s povprečno letno temperaturo 8–10 °C, prav tako merjeno za obdobje 1981–2010 (ARSO, 2021). V gradbeni dokumentaciji (Akumulacija za zasneževanje Arena (št.2998/07), 2007) je kot leto izgradnje akumulacije Arena navedeno leto 1989. Od takrat je bila akumulacija vsaj štirikrat povečana in obnovljena. Njena globina znaša 5,9 m, površina ojezeritve 5400 m², prostornina pa 21.455 m³. Vtok v akumulacijo Arena je iz Radvanjskega potoka. Naklon brežine je 1:2 ali 26,6˚. Iz te akumulacije se voda prek cevovoda prečrpava v akumulacijo Trikotna jasa ali se iz akumulacije direktno zasnežuje območje snežnega stadiona. Gradnja akumulacije ob Trikotni jasi se je začela leta 1992 in zaključila 1994 (Sanacija zadrževalnika ob Trikotni jasi (št. 18/95), 1995). Njena globina znaša 5,1 m, Slika 1. Lokacija akumulacij za umetno zasneževanje Arena in Trikotna jasa na Mariborskem Pohorju z bližnjimi vodnimi telesi ter naravovarst- venimi območji (levo). Desno: Lega akumulacij v severovzhodni Sloveniji (rdeča točka) in na območju vzhodnega Pohorja z mestom Maribor (rdeč okvir). (Vir: ARSO, 2021; DRSV, 2024. Oblikovanje in kartografija: Žiga Tertinek) Figure 1. Location of snowmaking accumulations, Arena and Trikotna jasa on Mariborsko Pohorje with proximal water bodies and nature conservation areas (left). Right: The location of the accumulations in northeastern Slovenia (red dot) and within the area of eastern Pohorje with the city of Maribor (red frame). (Source: ARSO, 2021; DRSV, 2024. Design and cartography: Žiga Tertinek) 21 Acta Biologica Slovenica, 2025, 68 (4) površina ojezeritve 2450 m² (ARSO, 2021), prostornina pa 5643 m³. Vtok v akumulacijo je iz Mrzlega potoka na zahodni in potoka na vzhodni strani. Naklon brežine je od 1,9 do 1:1,5 ali 27,7˚-33,7˚. Akumulacijski bazen obeh vodnih teles je tesnjen s hrapavo polietilensko hidroizolacijsko folijo (geomembrano) iz PEHD, ki je položena na geotekstil in popolnoma neprepustna za vodo (Sanacija zadrževal- nika ob Trikotni jasi (št. 18/95), 1995; Akumulacija za zas- neževanje Arena (št. 2998/07), 2007). Geomembrana je črne barve in ni prekrita (z npr. kamni, gramozom, peskom, rastlinami). Ker je geomembrana drseča, so vzdolž brežin obeh akumulacijskih bazenov nameščene lestve iz gumijas- tih obročev, namenjene izhodu divjadi. V akumulaciji Arena sta nameščeni dve lestvi in v akumulaciji Trikotna jasa pet. Splošne značilnosti akumulacij so povzete v Tab. 2. Akumulacija Trikotna jasa z okolico je del ekološko pomembnega območja Pohorje (EPO Pohorje ID 41200). Strnjen gozd južno in vzhodno je Natura 2000 območje Pohorje (ID 3000270) (Sl. 1). Zaradi bližine Natura 2000 (18 m) je akumulacija del vplivnega območja za kvalifikacijski vrsti hribski urh in veliki pupek (ZRSVN, 2021; Ur. l. RS 49/2004 in 110/2004). Popisi dvoživk Terensko delo je vključevalo popis dvoživk v akumulacijah Arena in Trikotna jasa. V akumulaciji Arena so bili marca in maja 2024 izvedeni štirje popisi dvoživk v okviru mon- itoringa pomladanske selitve dvoživk po naročilu Mestne občine Maribor (Tertinek, 2024). V akumulaciji Trikotna jasa je bilo med marcem in oktobrom 2024 izvedenih devet popisov. Datumi in ure terenskih obiskov so navedeni v Tab. S1. Dvoživke smo v akumulacijah iskali predvsem z opazovanjem med počasno hojo po nasipu zajetja. Zaradi strmih in gladkih brežin, prekritih z geomembrano, je bil dostop do vode mogoč le na mestih, kjer so bile vzdolž brežine nameščene lestve iz gum ali pa je gladina segala do travnatega dela brežine. Na teh mestih smo lovili z vod- nimi mrežami ali z roko. Urhe smo beležili tudi s poslušan- jem oglašajočih se samcev. S predvajanjem oglašanja smo izzivali samce zelene rege. Dokumentirali smo prisotnost posameznih vrst ali taksonov. Za določanje smo uporabili dihotomni ključ Veenvliet in Kus Veenvliet (2008) ter slikovni ključ Speybroeck in sod. (2016). Ujetim osebkom smo določili spol in jih razvrstili v starostne skupine: odrasli oz. adultni osebki, spolno nezreli oz. subadultni osebki (starejši od enega leta), mladi oz. juvenilni osebki (v letu po preobrazbi) in ličinke. Paglavce in ličinke smo določali s pomočjo ročnih lup že na terenu. Mreste žab in jajca pupkov smo določili do najnižjega možnega taksona. Število oseb- kov ali jajc posameznih vrst oziroma taksonov dvoživk smo na dan popisa ocenili z vizualnim pregledom in štetjem med obhodom akumulacije. Zaradi odsotnosti vegetacije je bilo mreste in dvoživke ob dobri vidljivosti razmeroma enos- tavno opaziti. Razmnoževanje v vodnih akumulacijah smo za posamezne vrste potrdili s prisotnostjo ampleksusa, jajc, mrestov, ličink ali pravkar preobraženih osebkov. Načrtno nismo ugotavljali uspešnosti razmnoževanja z iskanjem juvenilnih in subadultnih osebkov na kopnem, zabeležili smo le preobražene osebke v vodi ali neposredno ob vodi, tj. na brežini akumulacije. Po opravljenih meritvah in določitvi vrst smo vse ujete osebke, brez poškodb, vrnili na prvotno mesto najdbe. Vse ugotovitve smo dokumentirali, vključno s fotografijami ali video posnetki dvoživk kot tudi obeh akumulacij. Terensko delo je potekalo z minimalno invazivnimi postopki in v skladu z dovoljenjem št. 35606-5/2023-2550-5 z dne 28. 3. 2023 izdanim s strani Ministrstva za naravne vire in prostor. Kopnega izven nasipa akumulacije nismo načrtno pregledovali. Zabeležili smo prisotnost drugih vretenčarjev v akumulaciji. Vodnih nevretenčarjev nismo vzorčili, a smo zabeležili prisotnost vrst, ki smo jih opazili s prostim očesom. Značilnosti akumulacij za zasneževanje, pomembne za dvoživke V letu 2024 smo za obe akumulaciji popisali dejavnike ozi- roma značilnosti (Tab. 2), ki lahko vplivajo na preživetje in razmnoževanje dvoživk v vodnih telesih. Te smo povzeli iz splošne literature o dvoživkah (Veenvliet in Kus Veenvliet, 2008; Speybroeck in sod., 2016), iz raziskav o dvoživkah v umetnih akumulacijah, predvsem v zadrževalnikih mete- ornih vod in akumulacijah za zasneževanje (Brand in Sno- dgrass, 2010; McCarthy in Lathrop, 2011; Clevenot in sod., 2018; Fait in sod., 2020; Mathwin in sod., 2021; Gerfand in sod., 2024) ter druge literature o dejavnikih ogrožanja dvoživk (Stanković in sod., 2015; Poboljšaj in sod., 2018). Pri popisu dejavnikov se nismo osredotočili na specifične vrste dvoživk, četudi lahko isti dejavniki na različne vrste vplivajo različno (Clevenot in sod., 2018). Podatke o aku- mulacijah, upravljanju in okolici smo pridobili iz gradbene dokumentacije akumulacij, od upravljalca (Marprom, ustno), iz Naravovarstvenega atlasa (ZRSVN, 2021), Atlasa voda (DRSV, 2024) in meritev oziroma opazovanj na terenu med 22 Acta Biologica Slovenica, 2025, 68 (4) popisi dvoživk. Med terenskimi obiski smo v akumulaciji pre- verjali prisotnost/odsotnost: vode, plitvin (delov z globino vode do 30 cm), vodne vegetacije in drugih naravnih (npr. kamni, veje) ali umetnih (npr. cevi, večji odpadki) skrivališč v vodi, osončenosti gladine ter drugih vretenčarjev. Pregle- dali smo brežino in nasip akumulacije za prisotnost lesne vegetacije. Pregledali smo neposredno okolico akumulacije (do 50 m oddaljenosti) in zabeležili vodne habitate (stoječe vode, tekoče vode), kopenske habitate (travnik, gozd) ter ceste. Ob maksimalni napolnjenosti bazena, ki je določena z iztokom iz akumulacije, smo določili maksimalno višino vode. Na dneve popisov, ko so bile v akumulaciji prisotne dvoživke, smo z merilnim trakom (± 1 cm) izmerili morebiten upad vode od maksimalne višine vode in tako dobili oceno o nihanju gladine. Vsakič smo pregledali tudi celotno brežino akumulacije in preverili morebitno prisotnost suhega pasu geomembrane, tj. geomembrane, ki je nad gladino vode. Morebiten suh pas geomembrane smo na več mestih izmerili z merilnim trakom (± 1 cm) in tako dobili oceno širine suhega pasu geomembrane, t. razdaljo med gladino vode v akumulaciji in med travnatim delom brežine. Vsakič smo s kotomerom na več mestih izmerili tudi naklon brežine v območju suhega pasu geomembrane. Podatke o letnem praznjenju in čiščenju akumulacije smo dobili od upravljalca. Najkrajšo zračno oddaljenost cest, gozda in drugih naravnih habitatov od najbližjega roba akumulacije smo izmerili na zemljevidu območja (ZRSVN, 2021). Rezultati Dvoživke v akumulacijah V letu 2024 smo v dveh akumulacijah na Mariborskem Pohorju popisali osem vrst dvoživk: navadnega močerada (Salamandra salamandra), velikega pupka (Triturus carnifex), planinskega pupka (Ichthyosaura alpestris), navadnega pupka (Lissotriton vulgaris), hribskega urha (Bombina var- iegata), navadno krastačo (Bufo bufo), sekuljo (Rana tem- poraria) in rosnico (Rana dalmatina) (Tab. 1). V akumulaciji Arena so bile dvoživke zabeležene samo na enem od štirih izvedenih popisov (Tab. S1). Najdeni so bili mresti sekulje in mresti ter paglavci navadne krastače. V maju v akumulaciji ni bilo ličink ali v neposredni bližini preobraženih osebkov, zato uspešnega razmnoževanja nismo potrdili. V akumu- laciji Trikotna jasa so bile dvoživke zabeležene na vseh popisih od marca do oktobra (Tab. S1). Razmnoževanje je bilo potrjeno za velikega pupka, planinskega pupka, navad- nega pupka, hribskega urha, sekuljo in rosnico. Močerad in pupki so bili najdeni zgolj v akumulaciji Trikotna jasa (Tab. 1, Tab. S1). Navadni močerad je bil zaznan le kot kadaver v oktobrskem popisu. Odrasle pupke smo opazili med dviganjem proti gladini po zrak in ležanjem na podlagi v plitvi vodi. Zaradi omejene dostopnosti in slabše vidljivosti, večine nismo identificirali do vrste oziroma spola. Potrdili smo samo oba spola velikega pupka. Od jajc Tabela 1. Dvoživke v akumulacijah Arena in Trikotna jasa na Mariborskem Pohorju. Največje število primerkov (glede na takson in razvojni stadij) na posamezen popis: * < 5 primerkov, ** 5–10 primerkov, *** > 10 in < 30 primerkov, **** > 30 primerkov Table 1. Amphibians in the snowmaking accumulations, Arena and Trikotna jasa at Mariborsko Pohorje. Maximum number of specimens (by taxon and developmental stage) per visit: * < 5 specimens, ** 5–10 specimens, *** > 10 and < 30 specimens, **** > 30 specimens Akumulacija Arena Akumulacija Trikotna jasa Name of the Bacterial species mrest ličinke jajca/mrest ličinke mladi odrasli navadni močerad, Salamandra salamandra * veliki pupek, Triturus carnifex *** ** planinski pupek, Ichthyosaura alpestris **** navadni pupek, Lissotriton vulgaris * pupki nedoločeni **** *** hribski urh, Bombina variegata * navadna krastača, Bufo bufo **** **** sekulja, Rana temporaria *** **** **** **** rosnica, Rana dalmatina ** * Rana sp. nedoločeni **** **** **** **** 23 Acta Biologica Slovenica, 2025, 68 (4) pupkov smo opazili samo jajca in nekaj dni stare zarodke velikega pupka, ki so bili prilepljeni ter zaviti v odpadnem kosu papirja, ki je ležal v vodi med gumami (Sl. 2). Do naslednjega popisa je gladina vode toliko upadla, da je papir z jajci ostal na suhem. Ličink velikega pupka nismo našli. Ličinke planinskega pupka so bile številčne in v juliju potrjene na vseh popisih, medtem ko so bile ličinke navad- nega pupka najdene samo na enem popisu (Tab. 1, Tab. S1). Navadna krastača je bila najdena samo v akumulaciji Arena, kjer smo marca zabeležili mreste in ličinke. Hribski urh je bil najden samo v akumulaciji Trikotna jasa. Najdeni so bili samo odrasli osebki in to v juniju ter juliju (Tab. S1). Opaženi so bili samci, ki so lebdeli na sredini akumulacije in se oglašali (največ 2 na posamezen popis). V začetku julija je bil, razen posameznih samcev, pod gumami v vodi najden tudi ampleksus. Teden kasneje so bili v vodi tik ob brežini, na treh različnih mestih, hkrati najdeni 3 mrtvi odrasli hribski urhi (Sl. 3). Med popisi v bližini akumulacije nismo opazili manjših začasnih stoječih vod (v jarkih, kolesnicah) ali luž, v katerih bi se lahko zadrževali. Marca so bili v aku- mulaciji Arena mresti sekulje (Tab. S1), vendar paglavcev ali preobraženih osebkov ni bilo najdenih v nobenem od popisov. V akumulaciji Trikotna jasa so bili marca prisotni mresti sekulje in rosnice kot tudi odrasli osebki obeh vrst, pri čemer so mresti sekulje prevladovali. Konec junija so bili najdeni tako paglavci kot pravkar preobraženi osebki rjavih žab (Tab. S1). V začetku julija so množično migrirali na kopno, pri tem pa se prilepili na geomembrano in poginili. Pojav smo opazovali kljub dopoldanski uri vzorčenja (8–10h). Na dan vzorčenja 5. 7. 2024 smo na geomembrani brežine prešteli okoli 500 mrtvih pravkar preobraženih osebkov (Sl. 3). Nekaj kadavrov mladih osebkov je bilo tudi v vodi (< 5). Marca in oktobra so bili ob robu v vodi tudi posamezni kadavri odraslih rjavih žab. Značilnosti akumulacij za zasneževanje, pomembne za dvoživke Za akumulaciji Arena in Trikotna jasa smo v letu 2024 popisali dejavnike oziroma značilnosti vodnih teles (Tab. 2), ki so glede na literaturo pomembne za preživetje in razmnoževanje dvoživk v vodnih telesih. Slika 2. Odpadni papir v gumah v akumulaciji Trikotna jasa na Mariborskem Pohorju (levo), v katerega so bila zavita jajca velikega pupka (Triturus carnifex) (desno). (Fotografiji: Tina Klenovšek (levo) in Ana Skledar (desno)). Figure 2. Waste paper within tires in the Trikotna jasa accumulation at Mariborsko Pohorje (left), in which the eggs of the Italian Crested Newt (Triturus carnifex) were wrapped (right). (Photos: Tina Klenovšek (left), Ana Skledar (right)) 24 Acta Biologica Slovenica, 2025, 68 (4) Akumulacija Arena Ob akumulaciji Arena se nahajajo naselje, smučarski center in v manjši meri naravni habitati (Sl. 1). V neposredni bližini akumulacije so športni objekt, parkirišče in dve povezovalni cesti. Vzhodno od akumulacije so raznoliki habitati, med njimi nižinski travniki, mejice, zelenice, fragmenti gozda in mokrotni travniki z majhnimi nestalnimi vodami. V bližini je kanaliziran Radvanjski potok, ki napaja akumulacijo, vendar je med njima cesta. Večji strnjen gozd je oddaljen več kot 250 m, najkrajša migracijska pot do gozda pa je prekinjana z objekti in cestami. Nasip okoli akumulacije je bil v letu 2024 poraščen s travo, košen in brez lesne vegetacije. Ob mak- simalni napolnjenosti akumulacije je gladina vode segala do roba trave. Na prehodu med travnatim delom nasipa in akumulacijskim bazenom, ki je tesnjen z geomembrano, je bil približno 1 m širok pas iz neprekrite geomembrane, ki je bil ob maksimalni napolnjenosti akumulacije zalit z vodo Splošne značilnosti akumulacij Akumulacija Arena Akumulacija Trikotna jasa lokacija 46.53396, 15.60361 46.52448, 15.60344 nadmorska višina 312 m 560 m leto izgradnje 1989 1994 globina 5,9 m 5,1 m površina ojezeritve 5400 m² 2450 m² prostornina 21.455 m³ 5643 m³ naklon brežine 1:2 ali 26,6˚ 1:1,9–1:1,5 ali 27,7˚–33,7˚ material obloge ojezeritve geomembrana PEHD geomembrana PEHD Značilnosti akumulacij pomembnih za dvoživke stalna voda v času razmnoževanja da da nihanje gladine vode do 30 cm do 100 cm plitvine (< 30 cm globine) da da vodna vegetacija ne ne druga naravna skrivališča v vodi ne ne umetna skrivališča v vodi lestvi iz gum lestve iz gum osončenost vode (senčenje) da (brez senčenja) da (senčenje bližnjega gozda) ribe sončni ostriži, krapovci ne drugi plenilci (vretenčarji) v/na vodi želve belouške, mlakarice letno praznjenje in čiščenje akumulacije ne da (jeseni) suh del brežine ob maksimalni gladini vode travnat travnat, pas geomembrane širina suhega pasu geomembrane ob maksimalni gladini vode 0 cm 20-170 cm širina suhega pasu geomembrane ob minimalni izmerjeni gladini vode do 50 cm 200-450 cm naklon brežine iz suhe geomembrane (izmerjen) do 30˚ 30˚-60˚ lesna vegetacija na brežini/nasipu akumulacije ne ne druge stalne stoječe vode do 1000 m oddaljenosti (število in zračna razdalja) 3 (420 m, 780 m, 1000 m) 2 (obe 1000 m) neposredna bližina (< 50 m) tekočih voda 1 kanaliziran potok 2 naravna potoka neposredna bližina (< 50 m) manjših nestalnih stoječih voda da zelo redko, občasno oddaljenost strnjenega gozda > 250 m < 50 m neposredna bližina (< 50 m) travnikov da da lega znotraj naravovarstvenega območja ne da (EPO Pohorje) neposredna bližina (< 50 m) ceste 2 asfaltni cesti 1 gozdna cesta urbano okolje da ne Tabela 2. Značilnosti akumulacij za umetno zasneževanje Arena in Trikotna jasa na Mariborskem Pohorju. Table 2. Characteristics of the snowmaking accumulations Arena and Trikotna jasa at Mariborsko Pohorje. 25 Acta Biologica Slovenica, 2025, 68 (4) in tvoril plitvino. Akumulacija je bila povsem brez vodne vegetacije. Na nasipu ali v vodi ni bilo naravnih skrivališč za dvoživke. Edina nedrseča podlaga za odlaganje mrestov sta bili lestvi iz gumijastih obročev, na katerih so bili najdeni mresti sekulje in navadne krastače. V vodi so bili sončni ostriži (Lepomis sp.), krapovci (Cyprinidae) in invazivna tuje- rodna vrsta želve (Trachemys sp.). Akumulacije se trenutno ne čisti. Med vsemi popisi je bila v akumulaciji voda. Na popisu v marcu, ko so bili v akumulaciji mresti in paglavci, je bila gladina 30 cm nižja od maksimalne. Med travnatim delom brežine in vodo je bil zato suh pas geomembrane, širine do 50 cm. Akumulacija Trikotna jasa Akumulacijo Trikotna jasa obdaja 5–20 m širok košen travnat pas, v katerem se nahajata nasip akumulacije in gozdna cesta. Pas obdaja obsežno območje strnjenega gozda (kolinska kisloljubna bukovja), ki ga na zahodni strani prekinja smučišče s suhimi travišči na silikatih in ruderal- nimi združbami. Ob akumulaciji sta dva potoka z manjšimi tolmuni (do 1 m 2 ), v bližini katerih so bili med popisi posa- mezni spolno nezreli osebki rjavih žab. Na vseh popisih je bila v akumulaciji voda, a je gladina nihala do 100 cm (Sl. 3). Voda v akumulaciji se je poleti uporabljala za zalivanje površin v dolini. Za razliko od akumulacije Arena, voda, niti ob maksimalni napolnjenosti akumulacije, na nobenem delu ni segala do travnatega dela nasipa. Pas nepokrite geomembrane, ki je bil tudi ob maksimalnem vodostaju suh, je bil okrog in okrog akumulacije neprekinjen ter širok od 20 cm na najožjem ter do 170 cm na najširšem delu. Ob najnižjem izmerjenem vodostaju (100 cm pod maksimalno gladino) je bil pas suhe geomembrane na najožjem delu širok 200 cm in na najširšem 450 cm (Sl. 3). Tudi ob najniž- jem vodostaju so lestve iz gum segale do vode. Naklon brežine iz suhe geomembrane je bil povsod strm, od 30˚ do 60˚. Pas iz geomembrane je bil ovira na migracijski poti dvoživk iz vode na kopno. Na suhi geomembrani okrog akumulacije smo v enem popisu prešteli več kot 500 mrtvih mladih osebkov rjavih žab. Ob najnižjem vodostaju Slika 3. Pogini dvoživk v akumulaciji za umetno zasneževanje Trikotna jasa na Mariborskem Pohorju. Levo: Gladina vode v akumulaciji je bila med popisom v juliju 75 cm nižja od maksimalne. Zaradi nižje gladine je bil pas iz suhe geomembrane širok od 170 do 360 cm. Suha, gladka in strma brežina iz geomembrane je bila za dvoživke ovira med migracijo iz vode na travnat nasip. Desno zgoraj: Mrtev hribski urh v vodi ob robu brežine. Desno spodaj: Mrtvi, pravkar preobraženi mladi osebki rjavih žab, ki so se med migracijo iz vode prilepili na geomembrano in posušili. (Fotografije: Tina Klenovšek) Figure 3. Amphibian mortalities in the Trikotna jasa snowmaking accumulation. Left: The water level in the accumulation was 75 cm below the maximum during sampling in July. Due to the lower water level, the strip of dry geomembrane was 170 to 360 cm wide. The dry, smooth, and steep geomembrane bank was an obstacle for amphibians migrating from the water to the grassy embankment. Top right: Dead Yel- low-bellied Toad in the water at the edge of the bank. Bottom right: Dead, newly metamorphosed juvenile brown frogs that adhered to the geomembrane and dried out during migration from the water. (Photos: Tina Klenovšek) 26 Acta Biologica Slovenica, 2025, 68 (4) so bili v vodi akumulacije tik ob brežini najdeni 3 kadavri odraslih hribskih urhov (Sl. 3). Podobno so bili ob različnih popisih najdeni tudi kadavri posameznih odraslih rjavih žab, navadnega močerada, kuščaric in kopenskih žuželk. Zaradi upada gladine vode so bili na suho naplavljeni jajca in zarodki velikega pupka. V akumulaciji ni bilo vodne vegetacije, obvodne lesne vegetacije ali drugih naravnih skrivališč. Dno in stranice bazena akumulacije so bile iz neprekrite geomembrane in zato zelo gladke. Edina skrivališča in podlaga za odlaganje mrestov so bile v vodo spuščene lestve iz trdih gumijastih obročev oziroma avtomobilskih gum. Tam so rjave žabe odlagale mreste. Med popisom v oktobru so bile žabe in pupki opaženi izključno v in pod avtomobilskimi gumami. Rib v akumulaciji nismo zaznali. Akumulacijo se vsako leto izprazni in očisti. Na vseh poletnih popisih so bile v vodi ali na travnatem nabrežju akumulacije opažene belouške (Natrix natrix), največ 3 hkrati. Opazili smo, da na geomem- brani nimajo dovolj oprijema in lahko iz akumulacije, ob nizki vodi, splezajo samo po ali tik ob lestvi iz gum. Na travnatem obrežju je bil tudi slepec (Anguis fragilis) in v vodi en par rac mlakaric (Anas platyrhinchos). Na vseh popisih so bile v vodi številne hrbtoplovke (Notonectidae), medtem ko drugih večjih nevretenčarjev nismo opazili. Diskusija Raziskava prisotnosti dvoživk v akumulacijah za zas- neževanje na Mariborskem Pohorju je potrdila, da so lahko umetni vodni habitati za dvoživke mesta za razmnoževanje kot tudi potencialne ekološke pasti. V eni sezoni smo v dveh akumulacijah skupno popisali osem vrst dvoživk, od tega sedem v akumulaciji Trikotna jasa in dve v akumulaciji Arena. Pri vseh, razen navadnem močeradu, smo potrdili razmnoževanje v akumulacijah. Zabeležili smo tudi propad jajc in pogine dvoživk, ki so bili neposredno povezani s specifičnimi značilnostmi in namensko rabo akumulacij za zasneževanje. V akumulaciji Arena smo zabeležili le sekulje in navadne krastače, četudi smo glede na nizko nadmorsko višino in število zabeleženih vrst dvoživk v okolici akumu- lacije (Poboljšaj in sod., 2001; Tertinek, 2024) pričakovali več vrst. Glavni razlog za odsotnost drugih vrst je verjetno prisotnost rib, saj so akumulacijo Arena prav zaradi velikega števila rib Govedič in sodelavci že leta 2009 označili kot neprimerno za dvoživke. Druge vrste dvoživk lahko v bližini najdejo tudi druga ustreznejša vodna telesa, saj akumulacija leži na ravnem terenu, kjer ilovnata tla dobro zadržujejo vodo. Četudi so mokriščni habitati v večji meri degradirani, so v bližini manjša vodna telesa, v katerih so bile najdene dvoživke (Tertinek, 2024). Navadni krastači in sekulji akumulacije za zasneževanje ustrezajo, saj so v študiji iz francoskih Alp v kar 68 % od skupno 28 akumulacij za zasneževanje potrdili razmnoževanje prav teh dveh vrst (Gerfand in sod., 2024). Na razmnoževanje sekulje in navadne krastače v akumulaciji Arena je, razen rib, močno vplivalo tudi nihanje gladine vode, saj so mreste v glavnem odložile na lestve iz gum ali v plitvine, kjer so se le-ti ob spustu gladine ujeli ali prilepili na geomembrano ter posušili. Podoben množičen propad mrestov je bil v akumulaciji Arena zabeležen že v letu 2022 (Tertinek in Skledar, 2022). Negativno na preživetje odraslih dvoživk med selitvijo na mrestišče vpliva tudi lega akumulacije Arena. Predvsem navadne krastače se v akumulacijo selijo iz strnjenega gozda na pobočju Pohorja, pri čemer morajo prečkati vsaj dve cesti in jih je veliko povoženih (Tertinek in Skledar, 2022, druga lastna opažanja). Navadna krastača je vrsta, ki za razmnoževanje izbira velike, globoke stoječe vode (Veenvliet in Kus Veenvliet, 2008), kar so tudi glavne značilnost vodnih akumulaciji. Če živali takšno vodo ocenijo kot ustrezno in jo prednostno izberejo, kljub temu da imajo v njej manjše možnosti za preživetje ter razmnoževanje kot v drugih razpoložljivih habitatih, to ustreza definiciji ekološke pasti (Robertson in Hutto, 2006). V letu 2024 v bližnjih sto- ječih vodah (pregledana ni bila edino zajezitev Radvanjs- kega potoka), ni bilo najdenih mrestov navadne krastače (ta raziskava, Tertinek 2024). Navadna krastača je torej, kljub drugim razpoložljivim habitatom, za razmnoževanje izbrala akumulacijo Arena, četudi je izbira vodila v propad mrestov in paglavcev. Ocenjujemo, da je akumulacija Arena za navadno krastačo potencialna ekološka past, saj menimo, da trenutno ne omogoča zadostnega razmnoževalnega uspeha za vzdrževanje stabilne ali rastoče populacije brez priseljevanja (Battin 2004). Za potrditev akumulacije Arena kot ekološke pasti za navadno krastačo bi bilo potrebno bolj sistematično spremljanje razmnoževanja in razvoja osebkov tako v akumulaciji kot drugih razpoložljivih vodnih habitatih skozi celo sezono. V akumulaciji Trikotna jasa smo popisali sedem vrst dvoživk in od tega za šest potrdili razmnoževanje. Največ je bilo rjavih žab, predvsem sekulj, ki so tudi sicer najpogoste- jša in splošno razširjena vrsta dvoživk na Mariborskem Pohorju (Govedič in sod., 2009). Z naravovarstvenega 27 Acta Biologica Slovenica, 2025, 68 (4) vidika sta bili najpomembnejši najdbi hribskega urha in velikega pupka. Hribske urhe smo v akumulaciji zabeležili na več zaporednih popisih, dvakrat po tri odrasle osebke hkrati. Samostojne stoječe vode, kot je akumulacija, šte- jejo kot vode z veliko gostoto hribskih urhov, če v njih ob enkratnem ogledu opazimo več kot štiri odrasle osebke (Poboljšaj in sod., 2011). Na enem od ogledov smo našli tudi tri mrtve odrasle osebke. Gladina vode je bila takrat nizka, suha brežina iz geomembrane pa široka. Geomembrana se je čez dan tudi močno segrela. Lahko so poginili od izčr- panosti, ko so poskušali zapustiti akumulacijo, saj običajno naseljujejo manjše plitke vode in niso dobri plezalci. V sezoni razmnoževanja se selijo med vodnimi in kopenskimi habitati. Daljše zadrževanje v akumulaciji pa je lahko neu- godno zaradi pomanjkanja hrane, saj v vodi razen hrbtop- lovk nismo opazili vodnih nevretenčarjev ali njihovih ličink. Hribski urh je na Mariborskem Pohorju zaradi pomanjkanja primernih razmnoževalnih habitatov redek (Govedič in sod., 2024; Poboljšaj in sod., 2011). V bližini akumulacije Trikotna jasa je bil v preteklosti že zabeležen v manjši stoječi vodi v kolovozu (Govedič in sod. 2009), kar je sicer zanj značilen sekundarni habitat. Med popisi v letu 2024 v bližini akumu- lacije nismo opazili podobnih manjših stoječih vod. Druga pomembna vrsta je bil veliki pupek. V akumulaciji Trikotna jasa smo zabeležili odrasle osebke in jajca kot tudi propad jajc in zarodkov, vendar za razliko od rjavih žab in hribskih urhov, v vodi na nobenem od popisov nismo našli mrtvih odraslih pupkov. V letu 2009 Govedič in sodelavci, kljub intenzivnemu vzorčenju na Mariborskem Pohorju, velikega pupka niso našli. Akumulaciji Arena in Trikotna jasa so, zaradi stalnosti in velikosti vode, prepoznali kot najbolj primerni mrestišči za velikega pupka. Predvidevali so, da ga v akumulaciji Trikotna jasa ni zaradi popolne odsotno- sti vodne vegetacije. Veliki pupek je v Sloveniji splošno razširjen, a razmeroma redek (Veenvliet in Kus Veenvliet, 2008). Za ohranitev viabilne populacije je potrebna mreža ustreznih mrestišč, tj. stalnih ali začasnih stoječih vod, lahko tudi antropogenih vodnih habitatov, kjer je pogosto najden (Cipot in sod., 2011; Lešnik, 2021). Akumulacija Trikotna jasa je lahko eno od mrestišč v mreži vodnih habitatov, ki vključuje tudi območja v Radvanju in Razvanju, oddaljena približno 1300 m, s katerimi jo povezuje strnjen gozd, ki je del Natura 2000 območja. Takšna mreža potencialnih mrestišč je pomembna tako za velikega pupka kot tudi za druge dvoživke. V akumulaciji Trikotna jasa je prišlo tudi do pogina velikega števila (> 500) mladih, pravkar preobraženih rjavih žab. Pogin je bil v celoti posledica suhega pasu geomem- brane, na katero so se osebki prilepili med migracijo iz vode ali nazaj. Najdeni so bili na celotnem pasu suhe geomem- brane, kljub različnim naklonom in širini pasu, in niso izbrali določene smeri ali mesta migracije. Manjši naklon je sicer za dvoživke bolj ugoden, a je bila pot v tem primeru iz vode do trave daljša (do 450 cm; Tab. 2). Glavna razloga za pogin juvenilnih osebkov rjavih žab sta bila nepokritost suhega pasu geomembrane in strm naklon brežine (30° in 60°, Tab. 2). Za primerjavo, Gerfand in sod. (2024) so ugotovili, da se smrtnost dvoživk in drugih živali v akumulacijah za zasneževanje sistematično pojavlja z nakloni brežine več- jimi od 16,5°, pri čemer je bilo 73 % opaženih mrtvih živali najdenih v akumulacijah brez ali s samo delno prekritostjo geomembrane (npr. s kamni, gramozom). Pestrost dvoživk, tako po številu vrst kot številu oseb- kov, je bila v akumulaciji Trikotna jasa nepričakovano visoka. Manjšo pestrost smo pričakovali iz več razlogov. Prvič, v tej akumulaciji je bila do sedaj zabeležena samo sekulja (Gov- edič in sod., 2009). Drugič, akumulacije za zasneževanje so za dvoživke z več vidikov neugodni sekundarni habitati, ki lahko delujejo kot ekološke pasti in zmanjšujejo njihovo šte- vilčnost ali celo povzročijo lokalno izumrtje populacij (Battin, 2004). In tretjič, globoke stoječe vode, ali vode popolnoma brez vegetacije, niso značilen vodni habitat za večino od najdenih vrst (Veenvliet in Kus Veenvliet, 2008), zato ne bi pričakovali, da jih bodo izbrale za razmnoževanje. Značilno- sti akumulacije Trikotna jasa, ki verjetno vplivajo na visoko pestrost dvoživk so odsotnost rib, pomanjkanje ali oddal- jenost alternativnih vodnih habitatov in bližina ustreznih kopenskih habitatov. Razpoložljivost in povezanost s kopenskimi habitati pa je pomemben napovednik pestrosti dvoživk v akumulacijah (McCarthy in Lathrop, 2011). V aku- mulaciji smo pričakovali še navadno krastačo. Za vznožje Pohorja so znane še zelena rega (Hyla arborea) in zelene žabe (Pelophylax kl. esculentus), a jih, verjetno zaradi višje nadmorske višine in popolne odsotnosti vodne vegetacije, v akumulaciji Trikotna jasa nismo potrdili. Akumulacija Trikotna jasa ima zaradi strme brežine iz nepokrite suhe geomembrane, ki je drseča in se na soncu močno segreje, elemente ekološke pasti. Nekaterim dvoživkam namreč onemogoča izhod iz akumulacije, zaradi česar te v vodi ali na brežini poginejo. Učinek je še posebej izrazit ob nizkem vodostaju, ko je brežina iz suhe geomembrane široka več metrov. Vendar učinek ni za vse vrste ali razvojne stadije dvoživk enak. Najbolj so ogroženi hribski urh in mladi osebki rjavih žab, medtem ko vpliva na 28 Acta Biologica Slovenica, 2025, 68 (4) odrasle pupke nismo opazili. Za natančno oceno in potrd- itev učinka ekološke pasti bi morali za vsako vrsto dvoživke posebej, podobno kot za navadno krastačo v akumulaciji Arena, raziskati razmnoževalni uspeh tako v akumulaciji kot v bližnjih alternativnih habitatih. Druge omejitve te raziskave so, da vzorčenje ni bilo kontinuirano skozi celotno sezono raziskave, zato smo lahko katero od vrst dvoživk spregle- dali. Prav tako smo lahko spregledali vplive, ki jih imajo posamezne značilnosti akumulacij na dvoživke. Dodatno bi lahko popisali še povezljivost akumulacije z drugimi vod- nimi habitati, temperaturo vode in morebitno onesnaženje vode. Zaradi razlik med trajanjem vzorčenja, rezultati med akumulacijama tudi niso neposredno primerljivi. Iz rezultatov povzemamo, da akumulacije za zas- neževanje niso optimalni vodni habitati za dvoživke, a jih te kljub temu uporabljajo za razmnoževanje. Skupne značil- nosti akumulacij za zasneževanje so predvsem odsotnost vodne vegetacije, stalna prisotnost vode v času sezone razmnoževanja dvoživk in strme brežine. Med akumulaci- jami so lahko precejšnje razlike v vrstni sestavi dvoživk, ki akumulacijo uporabljajo za razmnoževanje. Na to med drugim vplivajo prisotnost rib, lega in okolica akumulacij. Dvoživke v akumulacijah najbolj ogrožajo strme brežine iz nepokrite suhe geomembrane in nihanje gladine vode, vendar ti dejavniki na različne vrste dvoživk vplivajo različno. Naša raziskava dopolnjuje ugotovitve, da lahko umetni vodni habitati na dvoživke delujejo kot ekološke pasti (Brand in Snodgrass, 2010; Clevenot in sod., 2018; Lešnik, 2021; Caballero-Díaz in sod., 2022; Gerfand in sod., 2024). S primernim upravljanjem in načrtovanjem akumulacij za zasneževanje lahko preprečimo, da bi te zaradi svojih lastnosti in namenske rabe za dvoživke učinkovale kot pasti. Osnovni ukrep za uspešnost razmnoževanja v akumulaciji Arena bi bilo letno pozno jesensko praznjenje akumulacije in odstranjevane usedlin, kar bi preprečilo obstoj rib (Mathwin in sod., 2021). V obeh akumulacija bi bilo potrebno geomembrano prekriti z materialom, ki bi dvoživkam omogočil, da v vseh vodostajih varno zapustijo akumulacijo. V času razmnoževanja dvoživk (marec–julij) se iz akumulacij ne bi smela spuščati voda, saj nihanje gladine na suho naplavi jajca oziroma mreste. Nove akumulacije bi morale razen naštetega imeti položne brežine (do 16,5°), predele z vodno vegetacijo in morebitne cestne podhode za dvoživke. Supplementary Material Dod. Mat. Tabela S1. Prisotnost dvoživk (z razvojnim sta- dijem in spolom) v akumulacijah Arena in Trikotna jasa na Mariborskem Pohorju glede na datum popisa. Supp. Mat. Table S1. Presence of amphibians (with devel- opmental stage and sex) in the Arena and Trikotna jasa accumulations on Mariborsko Pohorje according to sam- pling date. Author Contributions Koncept, metodologija, validacija, urejanje podatkov in pisanje prispevka T.K., Ž.T. in A.S. Vsi avtorji so prebrali objavljeno različico rokopisa in se z njo strinjali. Acknowledgement Zahvaljujemo se Skupni občinski upravi Maribor, Javnemu podjetju Marprom in Društvu študentov naravoslovja (DŠN) za sodelovanje in podporo pri raziskavi. Za pomoč na terenu se zahvaljujemo Primožu Kovačiču, Lari Kovačič in Timu Skudniku. Za strokovno pomoč se zahvaljujemo Anji Bolčina in Pii Golob. Funding Zbiranje podatkov v akumulaciji Arena v Zgornjem Rad- vanju je bilo izvedeno v okviru monitoringa pomladanskih selitev dvoživk v Radvanju po naročilu Skupne občinske uprave Maribor. Delo je nastalo tudi s podporo P1-0403 Računsko intenzivni kompleksni sistemi (financer: ARIS). Conflicts of Interest Avtorji ne navajajo navzkrižja interesov. 29 Acta Biologica Slovenica, 2025, 68 (4) Viri Akumulacija za zasneževanje Arena (št.2998/07), 2007. Vodnogospodarski biro Maribor d.d. ARSO, 2021. Atlas okolja. Ljubljana, Ministrstvo za naravne vire in prostor. Agencija RS za okolje. Battin, J., 2004. 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Zavod Republike Slovenije za varstvo narave. 31 1 Biotehnical Faculty, University of Ljubljana, Jamnikarjeva ulica 101, 1000 Ljubljana, Slovenia 2 Urban Planning Institute of the Republic of Slovenia, Trnovski pristan 2, 1000 Ljubljana 3 DivjaLabs d.o.o., Stegne 21c, 1000 Ljubljana, Slovenia * Corresponding author: E-mail address: irena.kavcic@bf.uni-lj.si Citation: Kavčič, I., Dremel, M., Majić Skrbinšek, A., (2025). Recognising the benefits of large carnivore presence in Slovenia: A focus group study. Acta Biologica Slovenica 68 (4) Received: 05.05.2025 / Accepted: 25.07.2025 / Published: 01.08.2025 https://doi.org/10.14720/abs.68.4.22661 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Recognising the benefits of large carnivore presence in Slovenia: A focus group study Irena Kavčič 1, * , Manca Dremel 2 , Aleksandra Majić Skrbinšek 1,3 Abstract In recent decades, populations of the brown bear, grey wolf, and Eurasian lynx in Europe have made a significant recovery. This success is largely attributable to effective legislation, an increase in prey species, and the expansion of forested areas. As a result, large carnivores are gradually returning to regions from which they had long been absent. However, the return of these predators also brings challenges for local communities. While much research emphasises conflicts and damages, this study focuses on the often-overlooked positive perspectives held by key stakeholder groups in Slovenia. Through focus group interviews with hunters, farmers, tourism professionals, and artists, the study explores the benefits of large carnivore presence using the framework of Cultural Ecosystem Services (CES), based on the CICES classification. Present research has found that tourism professionals perceive the presence of large carnivores as a key advantage for the development of tourism. For hunters, encounters with these animals provide opportunities to deepen their ecological knowledge, develop tracking skills, and share this expertise with pride within their communities. Some farmers, despite experiencing livestock losses, acknowledge the role of predators in regulating populations of wild herbivores. Artists experience large carnivores as a source of inspiration and as an opportunity to foster better interpersonal relationships and social dialogue. Recognising the benefits and values that different stakeholder groups attribute to large carnivores can support the development of more inclusive conservation strategies, strengthen dialogue with stakeholders, and shift the focus from conflict mitigation toward fostering coexistence. Keywords Large carnivores; coexistence; cultural ecosystem services; benefits. 32 Acta Biologica Slovenica, 2025, 68 (4) Prepoznavanje koristi prisotnosti velikih zveri v Sloveniji z metodo fokusnih skupin Izvleček V zadnjih desetletjih so si populacije rjavega medveda, volka in evrazijskega risa v Evropi občutno opomogle. Ta uspeh gre pripisati učinkoviti zakonodaji, porastu številčnosti plenskih vrst in povečanju gozdnih površin. Velike zveri se tako ponovno vračajo na območja, kjer so bile dolgo časa odsotne. Vrnitev velikih zveri pa hkrati prinaša tudi izzive za lokalno prebivalstvo. Medtem ko večina dosedanjih raziskav poudarja predvsem konflikte med ljudmi in velikimi zvermi, se pričujoča študija osredotoča na pogosto prezrte pozitivne perspektive ključnih deležnikov v Sloveniji. S pomočjo fokusnih skupin z lovci, kmeti, turističnimi delavci in umetniki smo raziskali koristi prisotnosti velikih zveri v okviru kulturnih ekosistemskih storitev, na podlagi mednarodne klasifikacije CICES. Turistični delavci prisotnost velikih zveri vidijo kot ključno prednost za razvoj turizma. Lovcem srečanja z velikimi zvermi omogočajo pridobivanje znanja o ekologiji vrst, razvijanje veščin sledenja, svoje znanje pa pogosto delijo znotraj širše skupnosti. Nekateri kmetje, kljub izgubi pašnih živali, prepoznavajo vlogo plenilcev pri uravnavanju številčnosti prakljarjev v naravi. Umetniki velike zveri doživljajo kot vir navdiha in priložnost za izboljšanje medsebojnih odnosov in družbenega dialoga. Študija razkriva bogato paleto vrednot, ki jih različne deležniške skupine pripisujejo velikim zverem. Razumevanje teh raznolikih perspektiv lahko prispeva k oblikovanju bolj vključujočih naravovarstvenih strategij, krepitvi dialoga z deležniki ter preusmeritvi pozornosti od konflikta k iskanju trajnostnih oblik sobivanja. Ključne besede velike zveri; sobivanje; kulturne ekosistemske storitve, koristi. Introduction Large carnivores are returning to many parts of Europe from which they were previously eradicated due to human per- secution. This conservation success story, driven by legal protection, rewilding efforts, and changing socio-political landscapes, is accompanied by growing societal tensions. As people hold divergent views on how large carnivore populations should be managed, their return often leads to polarisation and conflict (Redpath et al., 2017; Lute et al., 2018; Lute et al., 2020). Humans often determine where large carnivores will exist, and our ability to conserve them in the long term will ultimately depend upon our tolerance for their existence (Bruskotter and Wilson, 2014). Despite the growing attention towards the socio-eco- logical aspects of human-carnivore relationships in recent decades, the existing literature is skewed and predominantly centred on conflicts rather than on ecosys- tem services (Rode et al., 2021; Giergiczny et al., 2022; Palacios-Pacheco et al., 2024). Conflicts related to the presence of large carnivores attract media and fuel heated debates, especially in human-dominated landscapes of Europe. Even academic literature is biased towards investigations of negative economic impacts and public safety issues, while the positive impact of large carnivore presence and broader socio-economic impacts are often underrepresented (Rode et al., 2021). Moreover, recent research highlights that people's tolerance is also shaped by their perception of the benefits these species provide (Bruskotter and Wilson, 2014). To fill this gap, our investigation engaged with diverse stakeholder groups to reflect upon benefits and opportuni- ties arising from the presence of large carnivores. We can expect that different groups of people will have different views on these benefits, and it is important to communicate these diverse local perspectives around the presence of large carnivores in shared landscapes (Linnell and Immer- zeel, 2023). Broad, generalised messaging often fails—or even backfires—when it clashes with group-specific norms and values (Toomey et al., 2023). Therefore, we looked into benefits as seen by four stakeholder groups: farmers, hunters, tourism professionals, and artists. These groups were chosen based on their distinct relationships with large carnivores, which we expected would reveal a diverse range of practical and symbolic benefits associated with carnivore presence. 33 Acta Biologica Slovenica, 2025, 68 (4) To investigate the benefits provided by large carnivore presence in Slovenia, we have used the Cultural ecosystem services (CES) framework provided by the International Classification of Ecosystem Services (CICES), which offers several categories and supports a comprehensive capture of benefits (Daněk et al., 2023). CES are primarily regarded as "the physical settings, locations or situations that give rise to changes in the physical or mental states of people, and whose character is fundamentally dependent on living processes" (Haines-Young and Potschin, 2013). They are somewhat unique as these are regarded as entirely linked to human societies and to a human' appreciation' of biodiversity (Horgan et al., 2021). Although often intangible and hard to measure (Slovák et al., 2023), there has been a growing recognition of the value of cultural services in driving actions and determining the success or failure of conservation programs (Horgan et al., 2021). We have used the focus group method, which has proven effective for exploring CES, to gather in-depth insights from the selected stakeholders (Slovák et al., 2023). In this article, we present the benefits of large car- nivore presence as perceived by each stakeholder group investigated. Our findings underscore the importance of recognising a broader set of motivations behind tolerance and conservation support. Identified benefits can help to support the development of more targeted communication strategies that foster coexistence and broaden public sup- port for conservation. Materials and Methods Conceptual framing To frame the identified benefits provided by large car- nivores, we used the CICES framework (Haines-Young and Potschin, 2018) as it offers several categories and supports a comprehensive capture of benefits from large sets of qualitative data (Daněk et al., 2023). Moreover, CICES also allows translation between different ecosystem service classification systems, such as those used by the Millennium Ecosystem Assessment (MA) and The Econom- ics of Ecosystems and Biodiversity (TEEB). 11 categories from CICES version 4.3 were used: experiential use, physi- cal use, scientific, educational, heritage/cultural, aesthetic, entertainment, symbolic, sacred and/or religious, existence and bequest (Table 1). Data collection In this study, we employed the focus group methodology, augmented with a structured protocol designed to eluci- date benefits in the frame of CES as a potent tool for uncov- ering the positive facets of large carnivore coexistence. Unlike quantitatively driven surveys, this approach allows for a nuanced exploration of the values and relationships between local communities and their ecosystems. In the first phase, we prepared a protocol for conduct- ing focus groups to identify and document the range of different CES provided by large carnivores (Table S1). We modelled our approach after the elicitation method for nonmaterial values described by Gould et al. (2014), which combines various qualitative data collection techniques. In the protocol, we defined themes based on the classes of the CES outline in the CICES. A set of questions was developed to encourage participants in all focus groups to express their opinions, feelings, and values, covering 11 classes of CES presented in Table 1. Based on their responses, we asked additional customised sub-ques- tions during the interviews. We did not use the term "cul- tural ecosystem services" during the interviews. Instead, we addressed questions related to the topics associated with large carnivores: personal experiences with large carnivores, benefits, and disservices of large carnivores for people, ecotourism, recreation and hunting, spiritual, artistic value, heritage, local and traditional knowledge, educational value, and intergenerational value. To stimu- late the sharing of thoughts and feelings, we also showed participants 14 photographs of large carnivores in differ- ent situations (in their natural habitat, causing damage, interacting with people). We designed the protocol to obtain as many diverse benefits of large carnivores and their significance for the participants themselves and the society they live in as possible. We encouraged interac- tion among participants and a free flow of thoughts while also allowing for real-time clarification of the researcher's understanding. In the next phase, we defined stakeholder groups that we wanted to interview. Following Litosseliti's (2003) guideline that focus groups typically include 4 to 6 par- ticipants to ensure diverse perspectives without limiting individual contributions, our focus groups were composed of participants from the same interest groups who shared relevant experiences with the studied topic. Stakeholders associated with large carnivores are usually individuals 34 Acta Biologica Slovenica, 2025, 68 (4) influenced by their presence, individuals who can influ- ence their population or have other interests in large carnivores and their populations (Linnell, 2013; Grossmann et al., 2020). We have chosen four stakeholder groups: farmers (beekeepers, livestock breeders from large carnivore area), hunters from two hunting families active in the large carnivore area and involved in bear viewing activities, tourism professionals engaged in wildlife tour- ism and artists. Artists participated in a lynx-themed art residency organised by the LIFE Lynx project (LIFE16 NAT/ SI/000634), where they engaged with lynx conservation topics through presentations and field trips, and created lynx-related artworks during the residency. Farmers were selected because of the impacts large carnivore presence has on their livelihoods, hunters are an important stake- holder group because they can influence large carnivore populations, while tourism professionals and artists were selected due to their interest in large carnivore popula- tions. We initially contacted stakeholders based on our previous cooperation with them through other projects related to large carnivores in Slovenia. After that, we fol- lowed a snowballing process to identify additional relevant people for our focus group interviews. Focus groups were conducted between October and December 2019. They lasted between 2 and 4 hours and took place in the local setting of the focus groups: for farmers at the tourist farm, for hunters at the hunting lodge, for artists at the place of their art residency and for tourism professionals at the tourism centre. Altogether, we interviewed 29 participants (five farmers, eight hunters, nine tourism workers and seven artists). Each focus group was internally homogeneous in terms of participants' pro- fessional or interest background, consisting exclusively of farmers, hunters, tourism professionals, or artists. Group sizes were intentionally kept small to facilitate open discus- sion and ensure that diverse individual perspectives could be expressed. The final number of participants in each group was also influenced by participants' availability and willingness to engage in the study. Sessions were led by a facilitator who used a semi-struc- tured interview approach that relies on participants' responses (Gould et al., 2014; Table S1). The aim for the discussions was that they were enjoyable, without partic- ipants feeling pressured to speak or seek consensus, but rather to feel encouraged to express different viewpoints. The focus groups were recorded on a voice recorder (dict- aphone) with the consent of the stakeholders. Data analysis and interpretation Digital recordings were manually transcribed for analysis. From each transcript, we extracted statements (sentences or coherent sequences of sentences) related to the CES of large carnivores and entered them into a spreadsheet. Each statement was placed in its own row, and the speakers of the statements remained anonymous. For each recorded statement, we also noted the corresponding stakeholder group (farmers, hunters, tourism professionals, or artists). We collected all statements reflecting participants' sub- jective attitudes or personal experiences regarding large carnivores, whether positive or negative. We assigned nominal variables to each analysed statement: the type of large carnivore the statement referred to (bear, wolf, lynx, or all three large carnivores) and one of the 11 classes of CES according to CICES (experiential use, physical use, scientific, educational, heritage-cultural, entertainment, aesthetic, symbolic, sacred and/or religious, existence, and bequest). The category "other" was used when we were unable to fit the statement into the existing categories. Many times, statements were difficult to categorise within the CICES framework, and the categorisation process is subjective. For that reason, the statements were reviewed by two researchers to minimise the degree of subjectivity. Next, the collected statements were categorised according to the framework proposed by Rode et al. (2021), which defines specific large carnivore-related impacts within three overarching categories: economic impacts, health and well-being impacts, and social and cultural impacts. Each of these categories includes both positive and negative impacts. Following this structure, we assigned each statement in our dataset to the appropriate positive impact category, enabling us to identify and extract statements referring specifically to the positive contribu- tions, benefits, and cultural ecosystem services associated with the presence of large carnivores. Results In all focus groups combined, we identified a total of 476 statements that discuss the positive contributions or benefits of large carnivores for individuals and society. The number of statements related to a specific class of CES for each interest group is presented in Fig. 1. Most statements were classified as having experiential or educational value. 35 Acta Biologica Slovenica, 2025, 68 (4) Class Description and example of goods or benefits Experiential use In situ, large carnivore watching and recreation are in the area of large carnivore presence. Physical use Hunting of large carnivores. Scientific Large carnivores are a subject matter for research both on location and via other media, and knowledge about large carnivores and their habitat is important. Educational Large carnivores are a subject matter of education, both on location and via other media, and they provide skills and knowledge about large carnivore management and their habitat. Heritage, cultural Large carnivore resonance in culture or heritage, history, local identity, historic records of a place, and preserved cultural heritage. Aesthetic Large carnivores and their environment are a source of artistic inspiration and representation, aesthetic experiences, appreciation of the beauty of large carnivores, and a sense of place. Entertainment Ex situ experience large carnivores and their environment via different media. Symbolic Symbolic meaning of large carnivores, large carnivores as emblems, signifiers. Sacred and/or religious Large carnivores as a spiritual or ritual identity, holy places, and sacred animals. Existence Non-utilitarian qualities of the presence of large carnivores, such as the enjoyment/philosophical perspective provided by the knowledge of their existence. Bequest Moral/ethical perspective or belief regarding large carnivores, willingness to preserve large carnivore and their environment for the experience and use of future generations. Table 1. Categories of CES from CICES version 4.3 and their descriptions were adapted to the purpose of the present research. Tabela 1. Kategorije kulturnih ekosistemskih storitev glede na CICES različico 4.3 in njihovi opisi prilagojeni glede na namen te raziskave. Figure 1. The number of statements related to a specific class of CES for each interest group. Slika 1. Število izjav v posamezni kategoriji kulturnih ekosistemskih storitev za vsako interesno skupino. 36 Acta Biologica Slovenica, 2025, 68 (4) Table 2 displays various keywords and terms that we identified in the statements from focus groups. We do not evaluate them by importance, as it is impossible to do so solely based on the transcript analysis. Keywords from CES classes are listed below in order to reflect the number of statements we have placed in each class. There were no statements within the sacred/religious class; therefore, only 10 CES classes are shown. Class Other was estab- lished for statements that couldn't be attributed to any of the CES classes. Below, we present all CICES categories, listed in order based on the number of statements attributed to each cate- gory (Fig. 1). For each CICES category, we provide narrative examples drawn from stakeholder group discussions. Experiential values The experiential class of Cultural Ecosystem Services (CES) encompasses non-intrusive, sensory and emotional engagements with land- and seascapes, such as wildlife observation, that provide personal enrichment and enjoy- ment (Czúcz et al., 2018). Respondents most often mention direct observations of animals, but also express enjoyment when encountering their signs of presence, or because of the mere awareness of the existence of these animals. Among focus group participants, especially tourism professionals and hunters, watching/encountering large carnivores and related activities are a benefit. Observing the animals in the sense of organised guiding mainly referred to bears. Participants noted that this tourism, in turn, stimulates the local economy, with ancillary benefits to hospitality, retail, and other service sectors: "Those who come to see the bears also go to restaurants, accommodation providers, shops and other tourist attractions." They recognise the value of bears and, to a lesser extent, the value of wolves and lynx in increasing the attractiveness of the local area and transforming previously overlooked areas into destinations with distinctive appeal: "What was previously 'no name’ has now become ‘The land of bears’". Hunters within our focus group highlighted the economic benefits of wildlife tourism, particularly bear watching, which they increasingly view as an important supplementary source of income alongside traditional hunting activities. As one participant noted: "We didn't hunt in the summer, because photographic tourism is our main source of income." Beside direct observation, the benefits of the sole presence of large carnivores in the Class Keywords and terms Experiential encounters with large carnivores, photo hunt, photo tourism, socialising, tourism, watching animals Educational Bear-friendly label, best practice farm, better coexistence, interpretation centre, interpretation, learning for life, learning through the media, museum, natural science centre, natural science day in school, nature trail, self-upbringing/ education, tourism, understanding nature Heritage, cultural cultural landscape, folk tradition, hunting tradition, interpretation of cultural heritage, literature, metaphors, monuments, proper names, toponyms, tourism, tradition of coexistence, traditional methods of protection against damage Aesthetic aesthetic experiences, beauty, creation, imagination, inspiration, photos, the spirit of space Existence autochthony, diversity, inspiration and ideas, interest, part of the culture and system, rejection to shoot, respect, tolerance, understanding nature Bequest economic benefit, emotional and social enrichment, ethics and morals, national interest, organisations for the conservation and growth of the population, population management, species conservation projects Symbolic authenticity, autochthony, coat-of-arms, logos, metaphors, personification of species, proverbs, recognisability, symbol of preserved nature Physical use hunting equipment, hunting tourism, hunting, population management, sale of (bear) meat and fur, stuffed animals, trophies Scientific better coexistence and management, lynx reintroduction, photo traps, recording tracks, research, technology development, telemetry collars Other agreement, balance, partnership Table 2. Generated keywords and terms mentioned during focus groups, classified for different CES classes. Tabela 2. Ključne besede in izrazi, omenjeni med fokusnimi skupinami, razvrščeni po različnih razredih kulturnih ekosistemskih storitev. 37 Acta Biologica Slovenica, 2025, 68 (4) area was recognised as well: "An Englishman once told me that they don't need to see the bear, just show them a footprint. You must tell a story. And then you win." Others described deep emotional and even spiritual connections formed through rare, unpredictable wildlife encounters: "Even though you know they are there, you cannot predict the meeting. And that's always a fantastic moment because it's unexpected." Some statements reflect the awe-inspiring nature of large carnivores, suggesting that their presence limits human overuse of natural areas: "If it weren't for the large carnivores in our forests, people would walk all over these areas, everywhere". Finally, the bear also emerged as a socialising factor for locals and tourists alike, bringing people together through shared experiences like group hikes due to safety concerns: "The bear has become a socialising moment, because those of us who are regular hikers don't go alone, we go together." Educational values Educational class refers to the contributions of living systems to education, training, and the development of skills and knowledge (Potschin and Haines-Young, 2018). Large car- nivores, as keystone species, offer powerful opportunities for learning. Their presence can serve as a focal point for understanding ecological relationships, the importance of biodiversity, and the interconnectedness of natural systems. In our study, hunters most frequently highlighted the educational value of large carnivores. They described a deep observational engagement with these animals, often inspired by admiration for their behaviour and hunting abil- ities: "I have seen wolves chasing a deer, how they ambush it. A couple of them go ahead, some are behind… how they intercept it. I analysed how they caught a deer." Some are challenged by their secretive nature: "To see a lynx, you had to be able to move properly, know the location, its specific behaviour." Through these encounters, they learn about their basic ecology, gain tracking skills and recognise their value for obtaining a healthy forest ecosystem. They want to pass on their knowledge and respectful attitude to the younger generation by designing nature education visitor centres and trails. Additionally, they work with older members of the community through creative programs such as art workshops: "We talk while we create, we bring books about nature, and we try to eradicate stereotypes about the wolf in the minds of the older people who have formed stereotypes about the wolf." Though more reserved in their expressions, farmers show their understanding of natural processes and pred- ator-prey relationships. They recognised the role of large carnivores in maintaining a healthy forest ecosystem: "If large carnivores were not in our environment, we would be worse off as far as fields are concerned. The wolf and the bear are also scavengers, and they also maintain the deer." Artists conveyed additional, more indirect educational values, as people gain awareness and interconnectedness of nature and their own behaviour and develop ethical values and empathy: "The presence of large carnivores can also build a relationship with our fellow man. Just as we behave towards animals, we can behave towards humans." Their presence provoked reflection on human actions: »We can slaughter an animal and it's OK. If a wolf eats the animal, it is horrible. The outcome is the same." Although tourism professionals discussed educational value to a lesser extent, they recognise the opportunity of their presence for interpretation and interpretation of nature in different ways. They offer guided tours where they teach visitors about the biology of large carnivores and the nature of the area through interpretation and the search for animal tracks, often including cultural heritage in the interpretation. Heritage, cultural values Heritage/cultural class refers to elements of nature that hold cultural or historical significance, helping people iden- tify with the history or culture of where they live or come from (Potschin and Haines-Young, 2018). Among all stakeholder groups, tourism professionals most strongly emphasised the cultural importance of large carnivores. For them, these animals are integral to the region's identity: "Bears and other large carnivores are part of our culture, part of our system. If they disappeared now, we would become very poor and empty". Several partici- pants reflected on historical practices like shepherding and protection of livestock, underscoring the effort required in preventing conflicts with large carnivores and preserving traditional ways of life: "In our youth, there were no sheep slaughtered by wolves ... No livestock was left outside, there was always a shepherd present, and there were a couple of hundred sheep in the flock." Hunters expressed sense of pride in local efforts to live harmoniously with large carnivores: "Large carnivores give a good picture of Slove- nia ... In the museum in Paris, our place was presented as 38 Acta Biologica Slovenica, 2025, 68 (4) an example of good practice of coexistence between bears and humans." Same sense of pride is mentioned by artists, seeing the presence of large carnivores as a conservation success: "For us, the presence of large carnivores means the conservation of the species and an opportunity to show that coexistence is possible, which is also of interest to other countries". Farmers mostly mentioned different surnames, names of villages that are derived from animal names or associated with large carnivores, reflecting a long-standing cultural imprint of large carnivores on everyday life. Aesthetic value Aesthetic classes are elements of nature that enable aesthetic experiences and are appreciated for their beauty, which can become a source of artistic inspiration (Potschin and Haines-Young, 2018). In this context, artists most vividly expressed the aesthetic and inspirational power of these animals: "Large carnivores are an immense source of ideas and inspiration for creation, for enriching life, including spiritual life. They do not have a concrete impact on us, but an indirect one. It is almost impossible to live without it." Hunters described visual encounters with large carnivores as special, unique, unforgettable aesthetic experiences that have an enduring value in shaping personal memories and contributing to the overall aesthetic appreciation of nature: "I saw lynx when it was just walking along nicely in the shade. It was a full moon... It was a picture you never forget." Even farmers acknowledged their beauty, but with a sense of caution due to the potential impacts on their livelihoods:" A picture of a wolf and her cubs is beautiful, idyllic, cute, as long as they are in their natural habitat, as long as they are not under my feet, or doing harm." Existence value The existence value refers to the intrinsic worth of these species simply by virtue of their presence in nature— independent of any direct human use (Potschin and Haines-Young, 2018). Mostly the value was recognised by artists, who emphasised that the mere presence of large carnivores contributes to overall diversity: "Our country is more colourful, more diverse and more fulfilled because of the large carnivores". This value was also clearly articulated by hunters who emphasized the importance of large car- nivores simply being part of the landscape, regardless of direct human benefit: "Every animal, every individual has its function in nature." Participants from various interest groups expressed their deep appreciation for the untamed nature of large carnivores, reminding us that some things in nature still operate beyond our influence." The wolf pack expresses something primal, wild; they have a life of their own". Bequest value Bequest value expresses the willingness of individuals to preserve animals and ecosystems to ensure that future generations can also enjoy and benefit from the resource (Potschin and Haines-Y oung, 2018). The desire to conserve large carnivore populations for present and future genera- tions was highlighted in all focus groups. Artists expressed their concern for the potential ecolog- ical consequences of the absence of large carnivores: "The absence of large carnivores would be the bleakest progno- sis for life because if it started with the umbrella species, it could lead to the decline of bees, and that is the worst stage of the apocalypse, affecting all of us, even if we don't have a personal connection to these animals or their habitats." Participants of the focus groups advocate for the conser- vation of the population on ethical and moral grounds, as well as the economic benefits that large carnivores bring to the area through tourism and other activities. They oppose unplanned and inappropriate culling and stress that the long-term conservation of large carnivores also requires appropriate management in several areas. Hunters and farmers are mentioning appropriate management in terms of population size: "The aim is to conserve large carnivores, but only in certain numbers." Farmers specifically acknowl- edged their interconnection with nature, and the well-being of the environment: "Farmers are connected to nature, and we do not want to kill or destroy a population", they believe large carnivores belong to Slovenia: "Large carnivores are autochthonous to Slovenia and should therefore be pre- served, while foreign species that lack natural predators (e.g., jackals) should not be introduced." Entertainment Entertainment class includes characteristics or qualities of species or ecosystems that provide material or subject matter that can be communicated to others via different media. (e.g. films or books) (Potschin and Haines-Young, 2018). Several statements recall childhood memories that 39 Acta Biologica Slovenica, 2025, 68 (4) involve stories and folklore from that time, including fairy tales and folktales like Red Riding Hood and popular movies. In modern times, social media has changed the way the public receives and transmits news, where sensationalistic reports receive a higher number of shares than reports presenting facts more objectively, which can generate unwarranted fear and prejudice against predators (Nanni et al., 2020). Some participants in our focus groups mention that large carnivores are a useful topic for sensationalist media and are used to further polarise society:" I would add that this is a media campaign. These are presentations of catastrophic situations as if politics is bloodthirsty. Before it was migrants, they needed this victim." Symbolic Symbolic class includes elements of nature that hold sym- bolic meaning, help shape a sense of place, and serve as emblems of identity, character, or meaning (Potschin and Haines-Young, 2018). Participants' statements reflect a complex and diverse set of symbolic values associated with large carnivores, ranging from their role in local identity and authenticity to their portrayal in stories: "Large carnivores are part of the authenticity of these places." The presence of a bear in the municipal emblem signifies its significance as a symbol of the area's identity and values. Participants also projected human-like qualities onto these species. One hunter regards bears "as another human being"; this suggests a deep connection and perhaps a sense of kinship with bears. Another statement portrayed the wolf as "professors who study and observe humans", adding depth to their symbolism, while the lynx is portrayed as "a princely figure" because of its unique behaviours, such as covering its prey with leaves: "The lynx is a prince to me because it covers its prey with leaves and returns to eat". For tourism professionals' large carnivores are not only subjects of ecological interest, but integral to their personal identity: "For me as interpreter of nature, my personal life would change a lot in the absence of large carnivores, because I don't know what I would be looking at in the puddles, whose footprints I would be following…" Physical use Physical use of large carnivores refers mainly to hunting and related activities; therefore, it was mostly recognised by hunters: "Deer hunting or wolf hunting is indeed hunt- ing. It takes hours and hours. Yes, I hunt. That is why I am a hunter. I hunt." Scientific value The scientific class includes elements of living systems that enable scientific investigation or the creation of traditional ecological knowledge. Focus group respondents rec- ognised the importance of scientists and experts research- ing these animals, especially for better coexistence and management:" When defining the acceptable number of large carnivores in Slovenia, the experts must take into account the results of technology (cameras, collars), where and how many animals are currently present." Other Some statements could not be classified into any of the classes, but nevertheless, they express the important opportunities offered by the presence of large carnivores, mostly related to recognising and respecting diverse perspectives and opportunities for mutual agreements and partnerships. An artist shared the following statement, reflecting the viewpoints of people directly affected by large carnivore presence: "Personally, I think there are too many large carnivores in many areas of Slovenia. Farmers say that three wolf packs would be enough for Slovenia, and around 200–500 bears at most. That's the number, according to those who live with nature, that would be sustainable for a small country like Slovenia." Discussion Public attitude studies in Slovenia indicate that people gen- erally support the conservation of large carnivores (Majić Skrbinšek et al., 2019; Bele et al., 2022; Krofel et al., 2025), with the presence of bears perceived as beneficial for local communities (Kavčič and Majić Skrbinšek, 2023). How- ever, the fact that humans were once able to significantly reduce or eliminate carnivore populations suggests that successful conservation requires a deeper understanding of the factors driving human tolerance towards these spe- cies (Bruskotter and Wilson, 2014). Previous studies have identified knowledge of species biology and behaviour as a key determinant of attitudes and conservation beliefs 40 Acta Biologica Slovenica, 2025, 68 (4) (Majić Skrbinšek et al., 2019; Oražem at. al, 2019; Oražem et al., 2021). In addition, tolerance towards large carnivores is also influenced by people's perception of the benefits these species provide (Slagle et al., 2013; Bruskotter and Wilson, 2014). Bruskotter and Wilson (2014) argue that com- munication should not only address risks but also highlight benefits and provide guidance for mitigating potential conflicts. Marino et al. (2020) found a significant positive relationship between perceptions of intangible benefits and tolerance towards bears and wolves, suggesting that enhancing recognition of these intangible benefits could serve as an important management tool to foster tolerance. A recent review of the academic literature identified a clear research gap regarding the positive, especially non-ma- terial, impacts of large carnivores (Rode et al., 2021). Our study sought to address this gap by applying the cultural ecosystem services framework to identify and explore the benefits associated with large carnivore presence in Slove- nia. Through focus groups conducted with four stakeholder groups, farmers, hunters, tourism professionals and artists, we provide insights into both the evident and less obvious benefits linked to large carnivores. Tourism professionals emphasised both economic and cultural benefits of large carnivores. This is reflected in the way large carnivores are used to design tourism programmes, while cultural heritage arising from the long tradition of coexistence is used as part of storytelling. They also noted that the presence of these animals stim- ulates the local economy and supports a wide range of businesses, generating indirect but meaningful economic benefits across the community. However, the value of large carnivores extends beyond economics. For some tourism professionals, these animals have become a defining element of personal identity, contributing directly to their well-being and sense of purpose. As Buijs et al. (2020) note, happiness generated through the positive emotions induced by wildlife encounters can foster greater empathy and increase tolerance toward controversial species like large carnivores. Marino et al. (2020) similarly reported that perceptions of benefits, combined with exposure to positive meaningful experiences, like unexpected encounters that elicit positive emotions, can enhance tolerance towards bears and wolves in the Abruzzo region in Italy. They sug- gest that organised wildlife viewing could generate similar emotional benefits across the wider community. Likewise, Oražem et al. (2022) highlighted that direct experiences with animals serve as effective precursors of positive attitudes. While our study did not directly assess tourists' emotional responses, the insights from tourism professionals suggest that positive emotional connections with large carnivores— whether experienced by visitors engaged in wildlife tourism or by professionals themselves—represent a potential for promoting tolerance. These could be extended to the wider community through organised nature treks (Marino et al., 2020) or educational encounters in informal learning settings, like zoos (Oražem et al., 2022). Hunters offered a different perspective grounded in personal experience, ecological knowledge, and their role in wildlife management. They expressed deep interest in the biology and behaviour of large carnivores. Particularly capti- vated by the animals' secretive nature, they are challenged to gain the ability to track or hunt them. They most often encounter large carnivores in the wild, and their encounters are described as unique, special, and unforgettable. These immersive experiences with wildlife may contribute to long- term well-being (Buijs et al., 2020). Slovenian hunters are often involved in wildlife monitoring; they provide hunting bag data and collect biological samples. Their voluntary engagement in wildlife management suggests that such activities may be emotionally rewarding and reinforce stewardship practices (Buijs et al., 2020). Their statements show that they are feeling proud of their contributions; similar narratives were collected in other participatory pro- cesses where hunters wanted to be respected as legitimate managers and stewards of their land (Salvatori et al., 2021). Hunters also play an active role in community education, engaging with children and the elderly, organising guided walks, and designing educational trails and visitor centres. They often express pride in local efforts to live harmoniously with large carnivores, which is recognised internationally. Some mentioned the economic benefits of bear watching, saying that a bear is worth more alive than dead. In certain instances, they adapted hunting practices to accommodate photo tourism by avoiding hunting in specific areas. In contrast to hunters and tourism professionals, farm- ers offered a more cautious and critical view, shaped by the direct impacts of large carnivore presence. Farmers found it hardest to articulate the benefits associated with large carnivores, as their experiences are often shaped by livestock losses and threats to livelihoods. For farmers, large carnivores are not only an economic threat but also a challenge to values closely tied to their way of life (Larsson et al., 2022). Research among Slovenian vocational stu- dents revealed that those in agricultural programmes held 41 Acta Biologica Slovenica, 2025, 68 (4) more negative attitudes and had less knowledge about large carnivores compared to their peers in environmental and veterinary programmes, highlighting the influence of early informal learning experiences on attitude formation (Oražem and Tomažič, 2018). Nevertheless, even farmers in our focus group showed their understanding of natural processes and predator-prey relations, but they believe that wolves have an important ecological role and regulate prey populations. This aligns with findings from Sweden, where farmers cited wolves' regulatory role in prey populations as a rationale for their conservation (Karlsson and Sjöström, 2008). Despite concerns, farmers in our study expressed a strong connection to nature and emphasised the need to balance conservation goals with viable farming. They sup- ported keeping carnivore numbers at "reasonable" levels, as defined by experts, that would still allow sustainable livestock production. This perspective reflects the findings of Piscopo et al. (2021), who stressed that successful coexistence depends on collaboration between scientists, farmers, and policymakers. Stakeholders most directly affected by large carnivores, like farmers and hunters, tend to hold the most negative attitudes (Dressel et al., 2014; Franchini et al., 2021). Con- sidering that negative perceptions often outweigh positive ones in shaping attitudes (Kansky et al., 2014), we cannot expect communication alone to change their views. How- ever, engagement with these groups remains essential, as it can help anticipate potential areas of social conflict over species protection (Lischka et al., 2019). It is essential to approach communication strategies with stakeholder groups like farmers and hunters with sensitivity and a rec- ognition of the complexities involved. Acknowledging the additional efforts these stakeholders have made to protect their livelihoods (farmers) or to contribute to large carnivore research (hunters) can foster a more constructive dialogue and minimise tensions. Together, the perspectives of tourism professionals, hunters, and farmers illustrate the wide range of tangible and intangible benefits and challenges associated with large carnivores. A group of artists offered a distinct view- point, describing these animals as a source of inspiration and appreciating their aesthetic value. They also conveyed additional, less obvious benefits, believing that the presence of large carnivores teaches us about respect and patience, enables us to build a better relationship with other people and promotes mutual understanding. Large-carnivore con- flicts may, in fact, represent an opportunity for wider social learning and for improved stakeholder relationships (Hov- ardas, 2020), leading to better mutual understanding and collaboration among stakeholders. Participatory processes over large carnivore management are being used increas- ingly to share different views and increase knowledge of other points of view and recognition of the legitimacy of stakeholder positions (Salvatori et al., 2021). To promote coexistence between large carnivores and humans in Europe, greater attention must be paid to under- standing how specific stakeholder groups perceive not only the detrimental but also the beneficial aspects of living alongside these species (Marino et al., 2020; Palacios-Pa- checo et al., 2024). Recognising and highlighting the ben- efits of local practices aimed at fostering coexistence may help reduce conflict (Salvatori et al., 2021; Pettersson et al., 2022). Emerging communication models should focus on creating spaces for dialogue and debate, engaging emotions, and embracing diverse perspectives (Toomey, 2023). Additionally, promoting direct experiences, such as observing animals (Oražem et al., 2021, 2022), as well as increasing positive experiences and implementing damage prevention measures (Marino et al., 2020), can help shift attitudes. Communication strategies should also place greater emphasis on the benefits large carnivores provide (Bruskotter and Wilson, 2014; Rode et al., 2021). Integrating these stakeholder perspectives into policy design could support more inclusive and adaptive management strate- gies, tailored to the diverse values and experiences across affected communities. Within this study, we applied the cultural ecosystem services lens to identify a wide range of benefits, as perceived by key stakeholder groups in Slo- venia. Our findings contribute to the growing recognition that intangible benefits are important drivers of tolerance towards large carnivores and should be more prominently integrated into conservation strategies. Conclusions Our engagement with stakeholders through focused dia- logue sessions facilitated the discovery of diverse benefits and a multitude of values associated with large carnivores, each varying across stakeholder groups. Recognising and understanding different social groups' values and attitudes relating to wildlife is important for fostering trust and facili- tating fruitful dialogue and communication. Our study pro- vides insights that illuminate the origins of both opposition 42 Acta Biologica Slovenica, 2025, 68 (4) and agreement concerning large carnivore management, making one step forward in navigating the complex world of the plurality of values. Supplementary Materials Table S1. Interview protocol Author Contributions Conceptualisation, I.K. and A.M.S.; methodology, I.K. and A.M.S.; formal analysis, M.D. and I.K.; investigation, I.K., M.D. and A. M. S.; writing—original draft preparation, I.K.; writing—review and editing, I.K., M.D., A.M.S. All authors have read and agreed to the published version of the man- uscript. Acknowledgement We express our sincere appreciation to all participants who generously contributed their time, experiences, and insights to this study. We thank the LIFE Lynx project for enabling access to focus group participants, including artists involved in its lynx-themed residency. The authors acknowledge the AI language model ChatGPT (OpenAI) was used in order to improve text (language). After using this tool, we reviewed and edited the content and take full responsibility for the content of the publication. Funding We acknowledge financial support provided by Interreg VA Slovenia-Croatia programme. The research was conducted as part of the CARNIVORA DINARICA project. The authors acknowledge the project Thinking Animals, J6 3129, finan- cially supported by the Slovenian Research Agency for writing of the manuscript. Conflicts of Interest The authors declare no conflict of interest. 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Biological Conservation, 278, 109886. https://doi.org/10.1016/j.biocon.2022.109886 45 1 Slovenian Environment Agency, Vojkova 1b, SI-1000 Ljubljana, Slovenia 2 Faculty of Civil and Geodetic Engineering, University of Ljubljana, Hajdrihova 28, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail address: aleksandrakrivograd@gmail.com Citation: Krivograd Klemenčič, A., Šter, T., (2025). Distribution of two invasive alien diatom species Achnanthidium delmontii and Achnanthidium druartii in Slovenia. Acta Biologica Slovenica 68 (4) Received: 26.05.2025 / Accepted: 01.08.2025 / Published: 08.08.2025 https://doi.org/10.14720/abs.68.4.22822 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Distribution of two invasive alien diatom species Achnanthidium delmontii and Achnanthidium druartii in Slovenia Aleksandra Krivograd Klemenčič 1,2, * , Tadeja Šter 1 Abstract The distribution of two invasive diatom species in Slovenia, Achnanthidium delmontii (ADMO) and Achnanthidium druartii (ADRU), was investigated in this study. Data from 87 rivers and 11 lakes collected between 2019 and 2024 in the frame of national monitoring of the ecological status of surface waters were included. ADMO was present in 40 rivers (46%) and was dominant (>5% relative abundance) in 27 rivers (31%), with the highest abundance reaching 77% in the Bolska River. It was rare in lakes, detected in only three lakes (27%). ADRU was detected in 12 rivers (14%) and was dominant in four rivers (5%), with the highest abundance reaching 27% in the Drava River. In contrast, ADRU was common in lakes, with a presence in eight lakes (73%), dominating Lake Slivnica and Lake Pernica. ADMO presence was associated with reduced diatom species diversity and evenness, although no direct ecological impact was observed. ADMO was present mainly in upland river sections, while ADRU was more frequent in lowland river sections and lakes. The results of this study confirmed the invasive character of ADMO, whereas ADRU did not affect species diversity and evenness of the diatom assemblages, and thus its invasive character could not be confirmed. Keywords Achnanthidium delmontii; Achnanthidium druartii; alien invasive species; diatoms; Slovenia 46 Acta Biologica Slovenica, 2025, 68 (4) Razširjenost dveh invazivnih tujerodnih vrst diatomej Achnanthidium delmontii in Achnanthidium druartii na območju Slovenije Izvleček V raziskavi smo proučevali razširjenost dveh invazivnih tujerodnih vrst diatomej v Sloveniji, Achnanthidium delmontii (ADMO) in Achnanthidium druartii (ADRU). V raziskavo smo vključili podatke iz 87 rek in 11 jezer zbranih med letoma 2019 in 2024 v okviru nacionalnega monitoringa ekološkega stanja površinskih voda. ADMO je bil prisoten v 40 rekah (46 %), dominantna vrsta (>5 % relativne abundance) je bil v 27 rekah (31 %), pri čemer je s 77 % v reki Bolski dosegel najvišjo zastopanost. ADMO je bil v jezerih redek, zaznan je bil le v treh jezerih (27 %). ADRU je bil prisoten v 12 rekah (14 %), dominantna vrsta je bil v štirih rekah (5 %), z največjo zastopanostjo 27 % v reki Dravi. V jezerih je bil ADRU pogost, prisoten je bil v osmih jezerih (73 %), prevladoval je v Slivniškem in Perniškem jezeru. Prisotnost ADMO je bila povezana z zmanjšano vrstno pestrostjo in enakomernostjo združb diatomej, čeprav neposrednega negativnega ekološkega vpliva nismo zaznali. ADMO se je večinoma pojavljal v povirnih delih rek, medtem ko je bil ADRU pogostejši v nižinskih delih rek in jezerih. Rezultati potrjujejo invazivni značaj ADMO, medtem ko invazivnost ADRU ni bila potrjena, saj njegova prisotnost ni pomembno vplivala na sestavo združb diatomej. Ključne besede Achnanthidium delmontii; Achnanthidium druartii; invazivne vodne vrste; diatomeje; Slovenija Introduction Primary producers in rivers and lakes form the basis of the food web, and as such, any change in their quantity or in the composition of their communities can result in a disturbed ecological balance in waterbodies, with a domino effect on higher trophic levels such as benthic macroinvertebrates and fish (Buczkó et al., 2022). Among invasive benthic diatoms, the most attention in the past has been paid to one of the largest freshwater diatom species, Didymosphenia geminata (Lyngbye) W.M. Schmidt, which is native to Europe and North America but highly invasive and aggressive in New Zealand. It causes a lot of problems in freshwater systems, such as the forma- tion of large and extensive mats that impact fish, aquatic plants, and insects, resulting in severe disturbances in food webs (Blanco and Ector, 2009). However, according to Taylor and Bothwell (2014), D. geminata blooms in New Zealand are not caused solely by the introduction of its cells in new areas, as similar, nearly synchronous blooms have occurred in areas where D. geminata is native, such as North America and Europe. Next to large-celled diatoms such as D. geminata, also small-celled diatoms (<25 µm), such as Achnanthidium delmontii Pérès, Le Cohu & Bar- thès (ADMO), and Achnanthidium druartii Rimet & Couté (ADRU), are considered invasive or potentially invasive species (e.g., Buczkó et al., 2022; Falasco et al., 2023; Ivanov, 2018). ADMO was discovered in 2007 and formally described in 2012, based on specimens from a French river (Pérès et al., 2012), while ADRU was first discovered in 2004 in the Rhone River in France and formally described in 2010 as a new species invading the rivers in France and Spain (Rimet et al., 2010). Since then, ADMO has been reported from several countries in Europe (France, Germany, Hungary, Italy, Neth- erlands, Switzerland) and Asia (China) (Guiry and Guiry, 2022), while ADRU has been reported from several coun- tries in Europe (Bulgaria, France, Germany, Netherlands, Serbia and Spain), North and South America, the Middle East, and Asia (Guiry and Guiry, 2022). ADMO has been reported in all Slovenian neighbouring countries, namely Hungary, Italy, Austria, and Croatia. Buczkó et al. (2022) reported the presence of ADMO in Hungary from 2015 onward and in Austria in several sections of the Danube River from 2013 onward. Falasco et al. (2023) reported on the first findings of ADMO in Italy in 2013 in the rivers of Liguria (NW-Italy). In Croatia, there are records of ADMO in the Drava River and the Danube River from 2019, which 47 Acta Biologica Slovenica, 2025, 68 (4) were collected in the framework of Joint Danube Survey 4 (JDS4) (ICPDR, 2019). Information on the presence of ADRU in Slovenia’s neighbouring countries is scarce. There is a record of ADRU from the Danube River in Austria from 2019, collected in the framework of JDS4 (ICPDR, 2019). However, according to our information, there is no available information on the presence of ADRU in Croatia, Hungary, or Italy. In this research, the distribution of two potentially invasive, alien benthic diatom species, namely ADMO and ADRU, in the territory of Slovenia is presented for the first time. Moreover, for the sampling sites where ADMO and ADRU were identified, nutrient and organic matter load- ing, as well as species diversity and evenness, are also reported. Materials and Methods Phytobenthos sampling Sampling of the phytobenthos was carried out in 2019–2024 as part of the national monitoring of surface water quality according to the Water Framework Directive (WFD) (Directive 2000/60/EC). Altogether, 247 phytobenthos samples were collected at 175 sampling sites in rivers, and 35 samples were collected at 32 sampling sites in lakes and reservoirs (hereinafter referred to as lakes) across Slovenia according to the national programmes for monitoring chemical and ecological status of surface waters (ARSO, 2017, 2022). In total, 87 rivers and 11 lakes were included in this research. According to the Slovenian national methodologies for ecological status assessment of rivers and lakes using phytobenthos and macrophytes (MOP, 2016a, 2016b), river samples were collected once per year from June to September, up to a depth of 60 cm and at a distance of at least 1 m from the riverbanks, or in cases of smaller rivers, at least 10% of the river’s width from the banks. A multi-habitat approach was used, meaning that the sam- ples were collected from a range of habitats (e.g., gravel, mud, riffle, and pool) differing in substrate type, depth, current velocity, and shadiness. For lakes, the national methodology applies only to natural lakes (Lake Bled and Lake Bohinj). Multi-habitat sampling was also performed in these and other lakes. Phytobenthos was collected along a 50 m shoreline stretch that included various habitats characterised by varying depth, substrate, and shading conditions. In each lake, phytobenthos was sampled once per year, typically between June and September, and usually at three different sampling sites representing the dominant substrate type. An exception was Lake Vogršček, where only two sites were sampled. Sampling in Lake Pernica deviated from the recommended timeframe, as it was conducted in October, outside the June-September window defined by national methodology for natural lakes. In both rivers and lakes, phytobenthos was removed from the substrate (stones, pebbles, wood, macrophytes, etc.) using a toothbrush in a tray containing a small amount of river or lake water. The material was homogenised and poured into a wide-necked plastic bottle. Each sample was fixed with ethanol to a final concentration of 70%. Laboratory analyses The phytobenthos samples were transferred to the labo- ratory and treated with concentrated nitric acid (HNO 3 ) to remove cell contents and other organic matter, following the standard procedure (SIST EN 14407, 2014) and the instructions of the Slovenian national methodologies for ecological status assessment of rivers and lakes using phy- tobenthos and macrophytes (MOP, 2016a, 2016b). Cleaned samples were mounted in Naphrax® (Brunel Microscopes, Chippenham, Wiltshire, UK), a medium with a high refrac- tive index, for permanent slide preparation. Permanent slides were examined using a light microscope (Leica DM RB, Germany) at 1000× magnification. Diatom identification and enumeration were performed according to the stan- dard procedure (SIST EN 14407, 2014) and the Slovenian national methodologies (MOP, 2016a, 2016b). For each permanent slide, at least 500 valves were counted and identified to the species or lower taxonomic level. Diatom identification and nomenclature followed the identification monograph by Lange-Bertalot et al. (2017). Scanning elec- tron microscope (SEM) images were obtained using a JEOL JSM-7500F microscope and sample preparation according to Hasle and Fryxell (1970). Statistical analyses The abundance of diatom species was expressed as relative counts (in %). The levels of nutrient and organic loading in the investigated rivers were evaluated using the Trophic Index (TI) (Rott et al., 1999) and Saprobic Index (SI) (Rott et al., 1997), respectively. For the investigated lakes, 48 Acta Biologica Slovenica, 2025, 68 (4) nutrient loading levels were evaluated using the TI (Rott et al., 1999). Both indices were calculated solely based on diatom data, followed by an assessment of the ecological trophic status (for both rivers and lakes) and the ecological saprobic status (for rivers only), in accordance with the Slovenian national methodologies for ecological status assessment of rivers and lakes using phytobenthos and macrophytes (MOP, 2016a, 2016b). Species diversity was assessed using the Shannon-Wie- ner diversity index (SW), and species evenness was assessed using the Evenness index (E). Both indices were calculated using OMNIDIA 6.0.9, a software tool for the calculation of 18 diatom water quality indices. Correlations between the abundance of target species, species diversity, and species evenness were calculated using the Pearson correlation coefficient and Microsoft Excel software. Results and Discussion Altogether, 403 diatom taxa were identified in river samples and 238 diatom taxa in lake samples. In Slovenia, benthic diatoms are used, together with macrophytes, phytoplank- ton, fish, benthic macroinvertebrates, and macroalgae, as a biological quality element for the assessment of the ecological status of surface waters, namely the quality of the structure and functioning of aquatic ecosystems, as defined by the WFD (Directive 2000/60/EC). Two diatom indices, the Trophic index (Rott et al., 1999) and the Sapro- bic index (Rott et al., 1997), are used in Slovenia to assess nutrient and organic matter loading in rivers, respectively. For natural lakes in Slovenia (Lake Bled and Lake Bohinj), the trophic state is assessed using benthic diatoms and the Trophic index (Rott et al., 1999), in combination with phytoplankton, in accordance with national methodology (MOP, 2016b). For other lakes, where there is no national methodology, benthic diatoms and the Trophic Index (Rott et al., 1999) may still be used as an additional or indicative assessment tool, but are not formally part of the ecological status assessment under national monitoring. Morphology of Achnanthidium delmontii and Achnanthidium druartii The morphology of ADMO is described in detail in the work of Pérès et al. (2012) and that of ADRU in the work of Rimet et al. (2010), in which both species were first formally described. ADMO and ADRU both belong to the group of taxa related to Achnanthidium pyrenaicum (Hustedt) Kobayasi based on the stria density, which is around 20 μm, and the linear-lanceolate valve shape. According to Pérès et al. (2012), the main morpho- logical characteristics of ADMO (Figure 1) are as follows: Valves are linear with rounded apices, becoming elliptical in smaller individuals; valve length ranges from 7.3 to 21.4 μm, and valve width from 3.3 to 5.1 μm. On the raphe valve, the axial area is narrow, and the central area is irregular, typically forming a rectangular fascia, but a shortened stria may be present on one of the margins. The raphe is filiform and straight, with distinct central pores. Striae are slightly radial, numbering 20–26 in 10 μm in the central part of the valve, and up to 35 in 10 μm near the apices. On the rapheless valve, the axial area is acicular. Striae are parallel to slightly radiate near the apices; in most cases, two striae in the central part are slightly more widely spaced. Striae are slightly radial, numbering 18–22 in 10 μm in the central part and up to 25 in 10 μm at the apices. Under light microscopy, ADMO can be distinguished from similar taxa by a rectangular fascia on the raphe valve and a typical irregular cell shape. The main morphological characteristics of ADRU (Figure 2) according to Rimet et al. (2010) are: Valves are lanceolate with slightly subrostrate ends, never capitate; valve length ranges from 12 to 29 μm, and valve width from 3.9 to 5.8 μm. The raphe sternum is larger in the middle of the valve than in the extremities. The rapheless valve is convex, with a narrow, straight sternum that is only slightly enlarged in the centre. Striae are very weakly radiate throughout on both valves. Occasionally, short striae are inserted near the middle of the valve. For both valves, stria density is 15-22 in 10 μm in the central part of the valve and approximately 40-50 in 10 μm near the apices. Under light microscopy, ADRU can be distinguished from similar taxa, namely A. convergens (Kobayasi) Kobayasi, A. deflexum (Reimer) Kingston, A. japonicum (Kobayasi) Kobayasi, A. latecephalum Kobayasi, A. pyrena- icum (Hustedt) Kobayasi and A. rivulare Potapova & Pon- ader, by its generally wider and longer valve dimensions. However, in many cases, the size may overlap (Rimet et al., 2010). According to current knowledge, only A. pyrenaicum and A. rivulare have been recorded in Slovenia among the above-listed taxa. Another interesting characteristic that differentiates ADRU is that it shows an important stria density difference between the centre and the apices. 49 Acta Biologica Slovenica, 2025, 68 (4) Figure 1. Scanning electron microscope (SEM) images of Achnanthidium delmontii (ADMO). SEM: external view of the raphe valve with a rectangular fascia (marked with an arrow) (A), external view of the rapheless valve (B), external girdle view of the raphe valve (C), internal view of the rapheless valve (D). One of the main characteristics of ADMO is irregular cell shape (B, C, D). Slika 1. Fotografije z vrstičnim elektronskim mikroskopom (SEM) Achnanthidium delmontii (ADMO). SEM: pogled na zunanji del valve z rafo in dobro vidnim pravokotnim praznim prostorom (puščica) (A), pogled na zunanji del valve brez rafe (B), pogled na zunanji del valve z rafo z bočne strani (C), pogled na notranji del valve brez rafe (D). Ena glavnih značilnosti ADMO je nepravilna oblika celic (B, C, D). Figure 2. Scanning electron microscope (SEM) images of Achnanthidium druartii (ADRU). SEM: external views of the raphe valve (A, D), external view of the rapheless valve (B), internal view of the raphe valve (C). Slika 2. Fotografije z vrstičnim elektronskim mikroskopom (SEM) Achnanthidium druartii (ADRU). SEM: pogled na zunanji del valve z rafo (A, D), pogled na zunanji del valve brez rafe (B), pogled na notranji del valve z rafo (C). 50 Acta Biologica Slovenica, 2025, 68 (4) Occurrence of Achnanthidium delmontii and Achnanthidium druartii in Slovenia Table 1 provides an overview of the sampling sites where ADMO and ADRU were detected, including national sampling site codes, date of sampling, and the relative abundance of both species expressed as a percentage of counted valves. The table also includes information on nutrient and organic matter loading at each site, as well as measures of species diversity and evenness, expressed as the Shannon-Wiener diversity index and the Evenness index, respectively. Regarding rivers, a total of 247 phytobenthos samples collected at 175 sampling sites located on 87 rivers were included in the study. Among all investigated rivers, ADMO was present in 100 samples (41%) collected at 74 sampling sites (42%) from 40 rivers (46%) (Table 1). ADMO was a dominant species (more than 5% in relative abundance) in 56 phytobenthos samples (23%) collected at 45 sampling sites (26%) from 27 rivers (31%). The highest abundance of ADMO was recorded in the Bolska River (Figure 3), specif- ically at sampling sites Dolenja vas and Čeplje in August 2022, with relative abundances of 77% and 64%, respec- tively. Regarding ADMO representation, the Bolska River was followed by the Mirna River, where at the sampling site Dolenji Boštanj, the ADMO relative abundance reached 69%, also in August 2022. ADRU was present in 32 river samples (13%) collected at 22 sampling sites (13%) from 12 rivers (14%) (Table 1). ADRU was a dominant species (more than 5% in relative abun- dance) in 11 phytobenthos samples (5%) collected at eight sampling sites (5%) from 4 rivers (5%). The highest abun- dance of ADRU was detected in the Drava River (Figure 4), specifically at sampling site Ruše in September 2021, with a relative abundance of 27%, followed by sampling site Ranca in August 2024 with 20%, and sampling site Tribej in September 2021 with a relative abundance of 19%. In Slovenian lakes, 35 phytobenthos samples were collected at 32 sampling sites across 11 lakes. Among the two investigated species, ADMO was relatively rare, being detected in only five samples (14%) across three lakes. It was never dominant in any of the samples, with the highest recorded abundance reaching just 0.38% in the Gajševsko Lake (GaFB03, June 2023). In contrast, ADRU was more widespread and frequently encountered, being present in 15 samples (43%) in 8 of the 11 studied lakes. It showed dominance (relative abundance >5%) in 3 samples from two lakes. The highest relative abundance of ADRU was observed in Lake Slivnica (10% at SlFB04, May 2023), followed by Lake Pernica (6.6% at P2FB05, October 2024). River/Lake Sampling site Date of sampling ADMO (%) ADRU (%) TI SI EQR TI EQR SI SW E Mura Bad Radkersburg 21.01.2021 33.84 2.63 2.03 0.71 0.60 3.45 0.77 Mura Bad Radkersburg 30.08.2024 17.15 1.92 1.69 1.00 1.00 2.68 0.62 Mura Ceršak 21.01.2021 11.04 2.90 2.09 0.61 0.58 3.96 0.82 Mura Ceršak 30.08.2024 4.16 + 2.28 1.87 0.85 0.81 3.35 0.73 Mura Mele 21.01.2021 6.13 2.93 2.07 0.61 0.58 3.84 0.78 Mura Mele 30.08.2024 11.30 1.93 1.71 1.00 1.00 3.02 0.66 Mura Mota 14.02.2023 0.78 2.71 1.87 0.69 0.80 3.85 0.78 Mura Gibina 14.02.2023 5.86 3.15 2.16 0.47 0.55 3.75 0.77 Ledava Domajinci 7.03.2019 2.39 2.72 1.89 0.75 0.77 4.07 0.75 Table 1. List of sampling sites where invasive diatoms Achnanthidium delmontii (ADMO) and Achnanthidium druartii (ADRU) were detected, including national sampling site codes, date of sampling, relative abundance of both species expressed in percentage of counted valves, Trophic index (TI), Sap- robic index (SI), ecological trophic status (EQR TI), ecological saprobic status (EQR SI), Shannon-Wiener diversity index (SW), and Evenness index (E). + indicates that the species was observed during the qualitative examination of the sample but not during valve counting. / indicates that the ecological status assessment methodology has not been developed. Blue colour indicates high ecological status, green indicates good ecological status, and yellow indicates moderate ecological status. Tabela 1. Seznam vzorčnih mest z ugotovljeno prisotnostjo invazivnih diatomej Achnanthidium delmontii (ADMO) in Achnanthidium druartii (ADRU) z navedenimi nacionalnimi šiframi vzorčnih mest, datumom vzorčenja, relativno pogostostjo obeh vrst izraženo v odstotkih od preštetih polovic lupinic, trofičnim indeksom (TI), saprobnim indeksom (SI), ekološkim trofičnim (EQR TI) in ekološkim saprobnim (EQR SI) stanjem, Shannon-Wiennerjevim diver- zitetnim indeksom (SW) in indeksom Evenness (E). Z znakom + je označeno, da je bila vrsta zaznana le med kvalitativnim pregledom vzorca in ne med štetjem polovic lupinic. Z znakom / je označeno, da metodologija vrednotenja ekološkega stanja ni razvita. Modra barva označuje zelo dobro ekološko stanje, zelena označuje dobro ekološko stanje in rumena zmerno ekološko stanje. 51 Acta Biologica Slovenica, 2025, 68 (4) Drava Tribej 22.09.2021 18.70 2.52 1.94 0.75 0.71 4.4 0.82 Drava Tribej 25.09.2024 17.81 2.34 1.89 0.83 0.77 3.85 0.74 Drava Ruše 22.09.2021 12.08 26.53 2.28 1.79 0.85 0.93 3.86 0.75 Drava Ruše 21.08.2024 3.28 13.68 2.19 1.76 0.89 0.96 3.96 0.77 Drava Krčevina pri Ptuju 22.09.2021 21.97 1.98 2.04 1.66 0.96 1.00 3.12 0.67 Ptujsko jezero Ranca 16.09.2021 0.39 13.80 1.83 1.51 1.00 1.00 3.87 0.76 Drava Ranca 21.08.2024 19.68 2.55 1.69 0.74 1.00 4 0.75 Drava Prepolje 6.08.2019 0.19 5.78 2.88 2.03 0.62 0.60 4.53 0.79 Drava Prepolje 24.08.2022 1.93 2.80 1.90 0.65 0.76 4.53 0.85 Drava Gorišnica 6.08.2019 0.77 4.44 2.67 1.90 0.70 0.76 4.37 0.82 Drava Gorišnica 24.08.2022 5.08 2.83 1.98 0.64 0.66 4.54 0.83 Drava Borl 16.09.2021 55.00 3.14 2.57 2.06 0.73 0.59 2.49 0.57 Drava Borl 23.08.2024 12.28 1.56 2.52 1.71 0.75 1.00 3.84 0.75 Drava Ormož 6.08.2019 4.79 1.80 2.63 1.98 0.71 0.66 5.01 0.83 Drava Ormož 23.12.2020 4.23 0.38 2.54 1.99 0.75 0.64 4.41 0.79 Drava Ormož 4.08.2022 2.47 0.38 2.95 1.94 0.59 0.70 4.61 0.83 Drava Ormož 23.08.2024 5.81 6.97 2.54 1.77 0.75 0.95 4.39 0.79 Drava Grabe 15.09.2021 26.21 + 1.50 1.48 1.00 1.00 2.03 0.55 Meža Topla 17.07.2024 + 1.93 1.58 0.94 1.00 2.87 0.63 Mislinja Mala vas 17.07.2024 0.39 1.85 1.53 1.00 1.00 2.33 0.57 Mislinja Otiški vrh 17.07.2024 0.78 2.15 1.67 0.98 0.92 2.72 0.59 Dravinja Videm pri Ptuju 6.08.2024 15.5 1.84 1.50 1.00 1.00 2.54 0.59 Pesnica Zamušani 21.09.2023 1.89 2.28 1.98 0.96 0.74 3.98 0.85 Sava Dolinka Zelenci 11.07.2023 1.75 1.43 1.41 1.00 1.00 3.07 0.61 Sava Bohinjka nad izlivom Jezernice 29.08.2022 1.37 2.14 1.65 1.00 1.00 3.91 0.82 Sava Bohinjka Bodešče 29.08.2022 4.89 2.54 2.02 1.00 0.75 2.66 0.58 Sava Struževo 28.07.2021 6.9 1.39 1.51 1.00 1.00 2.72 0.72 Sava Dragočajna 21.06.2022 1.36 0.39 1.78 1.65 1.00 1.00 3.71 0.71 Sava Dragočajna 14.08.2024 9.23 2.45 1.79 0.70 0.77 4.68 0.87 Sava Medno 14.08.2024 12.08 1.70 1.53 1.00 1.00 2.57 0.58 Sava Šentjakob 26.07.2019 0.78 1.84 1.78 1.00 0.78 3.06 0.6 Sava Kresnice 12.07.2023 4.9 1.82 1.56 1.00 1.00 2.53 0.58 Sava Podkraj 16.08.2024 9.76 2.00 1.55 0.95 1.00 2.86 0.59 Sava Vrhovo 20.08.2021 8.57 2.66 1.98 0.70 0.66 4.31 0.83 Sava Vrhovo 23.06.2023 0.39 2.34 1.94 0.82 0.71 4.08 0.74 Sava Brestanica 20.08.2021 1.86 2.81 2.03 0.65 0.59 4.04 0.74 Sava Podgračeno 14.09.2021 0.77 2.53 1.99 0.75 0.64 3.95 0.76 Sava Jesenice na Dolenjskem 21.08.2019 50.65 2.68 1.92 0.69 0.74 2.95 0.56 Sava Jesenice na Dolenjskem 11.08.2020 1.36 2.23 1.59 0.88 1.00 3.1 0.6 Sava Jesenice na Dolenjskem 14.09.2021 6.81 2.62 2.07 0.72 0.58 3.93 0.78 Sava Jesenice na Dolenjskem 17.08.2022 4.64 0.77 2.85 2.01 0.63 0.61 3.98 0.79 Sava Jesenice na Dolenjskem 9.10.2023 9.3 2.03 1.75 0.97 0.98 3.61 0.74 Sava Jesenice na Dolenjskem 14.08.2024 16.01 1.61 1.48 1.00 1.00 1.93 0.49 Kokra Kranj 26.07.2021 28.85 1.89 1.61 1.00 1.00 3.29 0.71 Sora Medvode 22.06.2021 31.52 1.44 1.45 1.00 1.00 1.67 0.42 River/Lake Sampling site Date of sampling ADMO (%) ADRU (%) TI SI EQR TI EQR SI SW E 52 Acta Biologica Slovenica, 2025, 68 (4) Poljanska Sora Na Dobravi 1.07.2019 1.32 1.90 1.72 1.00 1.00 3.21 0.67 Poljanska Sora Na Dobravi 22.06.2021 26.21 1.48 1.48 1.00 1.00 2.27 0.58 Selška Sora Vešter 22.06.2021 6.83 1.76 1.59 1.00 1.00 3.31 0.78 Kamniška Bistrica Ihan 26.08.2024 28.37 2.34 1.70 1.00 1.00 3.19 0.64 Kamniška Bistrica Beričevo 26.08.2024 3.1 2.46 1.88 1.00 0.87 2.38 0.49 Pšata Bišče 1.07.2021 13.23 2.74 1.99 0.52 0.73 4.09 0.79 Pšata Bišče 11.07.2024 21.79 2.74 1.97 0.52 0.74 4.18 0.77 Mirna Dolenji Boštanj 17.06.2019 0.39 2.60 2.06 0.58 0.71 4.05 0.76 Mirna Dolenji Boštanj 1.08.2022 68.95 0.30 2.47 1.87 0.64 0.78 1.99 0.42 Sotla Rigonce 14.09.2021 0.39 2.64 1.92 1.00 1.00 3.44 0.69 Bistrica Zagaj 17.06.2019 25.58 2.06 1.58 1.00 1.00 3.48 0.73 Kolpa Osilnica 4.07.2024 6.99 1.42 1.55 1.00 1.00 3.14 0.71 Kolpa Radenci 14.06.2021 44.53 1.51 1.53 1.00 1.00 2.75 0.62 Kolpa Radoviči (Metlika) - Bubnjarci 4.08.2021 24.43 0.38 2.11 1.60 0.68 1.00 4.03 0.76 Kolpa Radoviči (Metlika) - Bubnjarci 4.07.2024 14.45 0.40 2.29 1.68 0.61 1.00 4.37 0.86 Lahinja Geršiči 12.09.2023 + 5.56 2.32 1.67 0.76 1.00 4.23 0.81 Gruberjev prekop Ljubljana 25.07.2022 0.39 2.73 2.02 0.56 0.75 4.26 0.81 Gruberjev prekop Ljubljana 13.09.2023 5.09 2.76 1.99 0.55 0.76 3.83 0.79 Iščica Ižanska cesta 17.08.2022 + 2.54 1.95 0.61 0.72 3.7 0.78 Iščica Ižanska cesta 11.07.2024 0.78 2.56 1.86 0.60 0.75 3.21 0.69 Mali Graben Dolgi most 21.06.2022 1.15 2.40 1.73 0.67 1.00 3.16 0.64 Gradaščica Dvor 21.06.2022 15.59 2.15 1.73 1.00 1.00 3.29 0.71 Savinja Grušovlje 16.07.2024 1.94 2.05 1.52 1.00 1.00 2.34 0.54 Savinja Medlog 4.09.2024 5.03 1.98 1.57 1.00 1.00 2.46 0.57 Savinja Veliko Širje 4.09.2024 17.58 2.05 1.63 1.00 1.00 3.03 0.65 Dreta Spodnje Kraše 10.09.2021 15.67 2.16 1.70 1.00 1.00 3.48 0.7 Paka Ločan 10.09.2021 8.43 2.60 1.87 0.77 1.00 3.82 0.75 Paka Šoštanj 16.07.2024 2.73 2.47 1.93 1.00 1.00 3.23 0.67 Paka Slatina 16.07.2024 10.4 3.05 2.54 0.69 0.60 3.06 0.73 Bolska Čeplje 2.08.2022 63.99 2.51 1.95 1.00 0.92 2.12 0.45 Bolska Dolenja vas 2.08.2022 76.67 2.03 1.81 1.00 1.00 1.59 0.33 Gračnica Gračnica 25.08.2023 8.54 1.87 1.64 1.00 1.00 2.59 0.61 Krka Soteska 20.08.2020 0.19 2.38 1.94 0.68 0.78 3.31 0.64 Krka Krška vas 20.08.2020 1.74 2.50 1.86 0.92 1.00 4.32 0.8 Radulja Grič pri Klevevžu 25.08.2023 3.50 1.85 1.65 1.00 1.00 2.97 0.64 Soča Solkanski jez 30.08.2019 4.14 1.73 1.37 1.00 1.00 3.43 0.7 Soča Solkanski jez 26.08.2020 4.82 1.77 1.47 0.99 1.00 3.12 0.69 Idrijca nad Divjim jezerom 15.06.2021 3.88 1.41 1.49 0.91 1.00 3.09 0.7 Idrijca Hotešk 18.09.2020 59.72 2.22 1.90 0.64 0.77 2.36 0.53 Trebuščica Most pri Sovi 5.10.2023 8.15 1.71 1.59 0.74 1.00 3.02 0.72 Bača Grapa 5.10.2023 8.81 1.55 1.44 1.00 1.00 1.39 0.37 Vipava Velike Žablje 27.08.2020 1.51 2.18 1.88 0.83 0.87 2.91 0.63 Vipava Velike Žablje 18.07.2023 0.77 2.39 1.82 0.73 1.00 2.2 0.45 Vipava Miren 27.08.2020 5.71 2.66 1.90 0.43 0.73 3.92 0.76 Vipava Miren 18.07.2023 5.87 2.63 1.94 0.44 0.71 4.05 0.8 River/Lake Sampling site Date of sampling ADMO (%) ADRU (%) TI SI EQR TI EQR SI SW E 53 Acta Biologica Slovenica, 2025, 68 (4) Hubelj Ajdovščina 5.03.2021 + 1.69 1.67 1.00 1.00 3.17 0.67 Nadiža Robič 3.07.2024 3.14 0.39 1.65 1.65 0.98 1.00 3.18 0.76 Reka Podgraje 10.03.2021 2.09 1.75 1.78 1.00 0.78 3.56 0.78 Reka Topolc 21.06.2021 27.72 1.94 1.63 1.00 1.00 2.86 0.6 Reka Topolc 7.09.2023 25.48 2.48 1.77 0.75 1.00 3.33 0.72 Reka Cerkvenikov mlin 8.03.2021 6.73 2.39 1.98 1.00 0.82 4.21 0.83 Klivnik Brid 25.02.2020 1.17 1.54 1.48 1.00 1.00 2.28 0.48 Molja Zarečica 13.09.2023 6.65 2.36 1.81 0.81 0.77 4.33 0.8 Rižana Dekani nad pregrado 17.06.2022 12.4 2.34 1.81 0.75 1.00 3.43 0.72 Rižana Dekani nad pregrado 7.09.2023 14.85 1.60 1.53 1.00 1.00 3.25 0.72 Dragonja Planjave 8.03.2021 4.31 1.37 1.46 1.00 0.80 3.29 0.71 Dragonja Podkaštel 8.03.2021 5.34 1.65 1.71 0.98 0.72 3.74 0.74 Blejsko jezero BlFB08 12.08.2019 0.20 2.41 0.43 3.52 0.68 Bohinjsko jezero BOFB05 21.06.2022 + 1.32 0.75 3.87 0.74 Bohinjsko jezero BOFB08 21.06.2022 + 1.25 0.77 3.77 0.74 Šmartinsko jezero SmFB04 26.05.2023 1.23 2.02 4.64 0.87 Šmartinsko jezero SmFB05 26.05.2023 0.97 1.59 3.04 0.61 Šmartinsko jezero SmFB06 26.05.2023 1.95 1.57 3.77 0.77 Slivniško jezero SlFB02 29.05.2023 5.34 2.12 3.8 0.76 Slivniško jezero SlFB04 29.05.2023 10.02 2.30 3.77 0.75 Slivniško jezero SlFB05 29.05.2023 4.30 2.43 3.91 0.76 Gajševsko jezero GaFB02 14.06.2023 + 2.67 3.85 0.75 Gajševsko jezero GaFB03 14.06.2023 0.38 + 2.64 4.05 0.77 Mola MoFB02 1.08.2024 0.38 1.80 2.23 0.54 Vogršček V2FB04 7.08.2024 0.60 1.62 2.65 0.65 Vogršček V2FB06 7.08.2024 0.38 1.61 3.45 0.68 Klivnik KLFB03 8.08.2024 + + 1.68 2.62 0.63 Perniško jezero 2 P2FB03 23.10.2024 2.94 3.17 4.36 0.8 Perniško jezero 2 P2FB05 23.10.2024 6.61 2.98 2.3 0.54 Perniško jezero 2 P2FB06 23.10.2024 + 3.06 2.85 0.64 River/Lake Sampling site Date of sampling ADMO (%) ADRU (%) TI SI EQR TI EQR SI SW E For the purpose of this study, SW and E of the samples with ADMO and/or ADRU were recorded (Table 1). More- over, the correlations between ADMO and ADRU abun- dance and SW and E were calculated using the Pearson correlation coefficient. The results showed moderate neg- ative correlations of -0.46 and -0.50 between ADMO abun- dance and SW, and ADMO abundance and E, respectively. These results indicate that the presence of ADMO in the samples is associated with lower diatom species diversity and lower diatom species evenness, which is most evident in the samples with the highest abundance of ADMO (Table 1). High abundance of ADMO in the samples, reaching up to 77%, is most probably due to its invasive character. According to Buczkó et al. (2022), AD, MO has a wide eco- logical range, which serves to confirm its potential invasive behaviour. High biodiversity and high species evenness are often associated with balanced environmental condi- tions and low anthropogenic pressure, which reflect good or high ecological status of water bodies. However, in the dataset used in this research, the river sections in which ADMO abundance was the highest (more than 60%) and diatom species diversity and evenness were the lowest are associated with good or high ecological saprobic and eco- logical trophic status (Table 1). Moreover, ADMO was pres- ent mainly in the upland river sections (Figure 5), which are, in most cases, hydromorphologically undisturbed, which 54 Acta Biologica Slovenica, 2025, 68 (4) corresponds with the findings of Falasco et al. (2023). We can conclude that even if ADMO is very abundant, the consequent disturbance of the aquatic environment is not noticed, at least as far as the physical appearance of river sections (Figure 3) and ecological saprobic and trophic status assessment are concerned. These findings are in accordance with the findings from the literature (Buczkó et. al., 2022). In the lakes, ADMO was detected mainly during the qualitative inspection of the samples (Lake Bohinj, Gajševsko Lake and Lake Klivnik). With a slightly higher abundance of 0.4% ADMO was detected only in one sample in Gajševsko Lake in June 2023 (Table 1). The results of the correlations between ADRU abun- dance and SW and E were the opposite compared with ADMO, showing a weak positive correlation of 0.25 and 0.23 between ADRU abundance and SW, and between ADRU abundance and E, respectively. Rimet et al. (2010) considered ADRU as an invasive species; however, in the dataset used in this research, ADRU was present in much lower proportions (up to 27%) compared to ADMO (up to 77%) and did not affect the species diversity and evenness of the diatom assemblages in the samples. These findings are in accordance with other studies in which the invasive- ness of ADRU could not be confirmed (Ivanov, 2018). In the dataset used in this research, ADRU was present mainly in the lowland river sections, lakes and reservoirs (Figure 6), which corresponds with the findings of Ivanov (2018). ADRU reached the highest abundance in the Drava River, a river with a high number of reservoirs above the dams of Hydro-Power Plants, namely at the sampling sites Ranca, Ruše, and Tribej. In the dataset used in this research, the river sections in which ADRU abundance was the highest (>5%) are associated with good or high ecological saprobic and ecological trophic status (Table 1). Among the 11 lakes included in this study, ADRU was confirmed in eight, with the highest abundance of 10% recorded in Lake Slivnica in May 2023. In Slovenia, there are two natural lakes, namely the subalpine Lake Bled and the alpine Lake Bohinj. ADRU was found in Lake Bled in August 2019 with a low abundance of 0.2%; however, in Lake Bohinj, which is considered one of the most ecologically pristine lakes in Slovenia (good ecological status; Table 1), it was not found. Moreover, Lake Bled was included in the dataset used in this research in the years 2019 and 2022; however, ADRU was found only in one sample in 2019 and was not detected again in 2022. The distribution of ADMO and ADRU in Slovenian rivers and lakes in the years 2019–2024, accompanied by the rel- ative abundance of both species expressed in percentage, is presented in Figures 5 and 6. Buczkó et al. (2022) found that ADMO started to spread in the Danube River from the source downstream. In 2013, it was found in Germany and Austria, and three years later, it was found in the Hungarian section of the Danube River. However, this pattern was not confirmed in Slovenian rivers. Figure 3. River Bolska has the highest occurrence of invasive benthic diatom Achnanthidium delmontii (ADMO) in Slovenia. A-Bolska-Čeplje and B-Bolska-Dolenja vas in August 2022. Photo: archive ARSO. Slika 3. Reka Bolska z najvišjo številčnostjo invazivne bentoške diatomeje Achnanthidium delmontii (ADMO) v Sloveniji. A-Bolska-Čeplje in B-Bolska-Dolenja vas avgusta 2022. Foto: arhiv ARSO. 55 Acta Biologica Slovenica, 2025, 68 (4) Figure 4. River Drava has the highest abundance of invasive alien diatom species, Achnanthidium druartii (ADRU), in Slovenia. A-Drava-Ruše and B-Drava-Ranca in August 2024. Photo: archive ARSO. Slika 4. Reka Drava z najvišjo številčnostjo invazivne tujerodne vrste diatomeje Achnanthidium druartii (ADRU) v Sloveniji. A-Drava-Ruše in B-Drava-Ranca avgusta 2022. Foto: arhiv ARSO. Figure 5. Distribution and relative abundance (in percentage) of Achnanthidium delmontii (ADMO) in Slovenia between 2019 and 2024. Slika 5. Porazdelitev in relativna abundanca (v odstotkih) vrste Achnanthidium delmontii (ADMO) v Sloveniji med leti 2019 in 2024. 56 Acta Biologica Slovenica, 2025, 68 (4) The first record of ADMO and ADRU in Slovenia is from the national monitoring of surface waters in 2019. The most probable reason is that in 2019, the Slovenian Environment Agency switched from the diatom taxonomy of Süßwasser- flora von Mitteleuropa (Krammer and Lange-Bertalot, 1986, 1988, 1991a, 1991b) to the taxonomy of Freshwater Benthic Diatoms of Central Europe (Lange-Bertalot et al., 2017). In the monograph of Lange-Bertalot et al. (2017), both ADMO and ADRU were already included, while in older mono- graphs, they are missing since both species were only for- mally described as new diatom species in 2012 (ADMO) and 2010 (ADRU). Although ADMO was formally first observed in Slovenia in 2019, it was most likely present in Slovenian freshwaters several years earlier and had been counted under Achnanthes biasolettiana Grunow, which is currently regarded as a synonym of Achnanthidium pyrenaicum (Hustedt) H. Kobayasi. One reason to assume the earlier occurrence of ADMO in Slovenia is that it had already been reported in the freshwaters of neighbouring countries: as early as 2013 in Austria (Buczkó et al., 2022) and Italy (Falasco et al., 2023), and in 2015 in Hungary (Buczkó et al., 2022). Buczkó et al. (2022) reported that in 2013, when ADMO was first recorded in German and Austrian sections of the Danube River, it was present in low abundances (<3%); however, by 2019, ADMO had become one of the most abundant and frequent diatom species found in the Danube River. Given that ADMO, when first recorded in Slovenian samples in 2019, was already present with very high abundances, reaching up to 51% in Sava - Jesenice na Dolenjskem (Table 1, Figure 5), we can conclude that it had already been present in Slovenia well before 2019. In con- trast, according to our information, there are no records of ADRU occurring in neighbouring countries before 2019, and thus it may not have been present in Slovenian waters earlier. Furthermore, ADRU, when first recorded in Slovenian samples in 2019, showed a relative abundance Figure 6 Distribution and relative abundance (in percentage) of Achnanthidium druartii (ADRU) in Slovenia between 2019 and 2024. Slika 6. Porazdelitev in relativna abundanca (v odstotkih) vrste Achnanthidium druartii (ADRU) v Sloveniji med leti 2019 in 2024. 57 Acta Biologica Slovenica, 2025, 68 (4) of up to 6% in Drava - Prepolje, which is 8.5 times lower than that of ADMO. There is a possibility that ADRU will be more widely distributed in Slovenian waters with a higher relative abundance after a certain amount of time, as was the case with ADMO. The data presented in this study show only the present state, which might be temporally biased. Although there are no records of ADRU in Croatia, Hungary, and Italy, we can conclude from its presence in Slovenian rivers near the border of the above-mentioned countries, such as the Kolpa, Sotla, Dragonja, Ledava, and Nadiža (Table 1, Figure 6), that ADRU is most likely present in the territory of those countries. Although diatoms are useful indicators of various pres- sures such as nutrient and organic loading, acidification, or salinity in running and standing surface waters, ADMO and ADRU are not yet considered as indicator organisms. However, ongoing research is exploring their potential as indicator organisms, which would be reasonable given their increasing prevalence across Europe. Conclusions Altogether 247 phytobenthos samples collected at 87 rivers and 35 phytobenthos samples collected at 11 lakes in the frame of the national monitoring of surface water quality from 2019 to 2024 were included in this study. Achnanthidium delmontii (ADMO) was present in 100 river samples (41%) from 40 rivers (46%) and was a dominant species in 56 phytobenthos samples (23%) from 27 rivers (31%). The highest abundance of ADMO was recorded in the Bolska River at sampling sites Dolenja vas and Čeplje with relative abundances of 77% and 64%, respectively. Achnanthidium druartii (ADRU) was present in 32 river sam- ples (13%) from 12 rivers (14%) and was a dominant species in 11 phytobenthos samples (5%) from 4 rivers (5%). The highest abundance of ADRU was detected in the Drava River at sampling site Ruše with a relative abundance of 27%. In lakes, ADMO was relatively rare, being detected in only five samples (14%) across three lakes and was never dominant in any of the lake samples. ADRU was present in 15 samples (43%) from 8 lakes and was a dominant species in 3 samples from two lakes. The highest relative abundance of ADRU was observed in Lake Slivnica with a relative abundance of 10%. The results of this study show that the presence of ADMO in the samples is associated with lower diatom species diversity and evenness, indicating, together with very high relative abundances, its invasive character. How- ever, the river sections in which ADMO abundance was the highest and diatom species diversity and evenness were the lowest are associated with good or high ecological sap- robic and ecological trophic status. Moreover, ADMO was present mainly in the upland river sections, which are, in most cases, hydromorphologically undisturbed. Although ADMO was present in the investigated rivers in very high numbers, the consequent disturbance of the aquatic environment was not noticed, at least as far as the physical appearance of river sections and ecological saprobic and trophic status assessment are concerned. ADRU, which was present mainly in the lowland river sections and lakes, did not affect the species diversity and evenness of the diatom assemblages in the samples, and thus its invasive character could not be confirmed. Author Contributions Conceptualization, A.K.K.; methodology, A.K.K. and T.Š.; investigation, A.K.K. and T.Š.; resources, A.K.K.; data cura- tion, A.K.K. and T.Š.; writing—original draft preparation, A.K.K.; writing—review and editing, A.K.K. and T.Š.; funding acquisition, A.K.K. All authors have read and agreed to the published version of the manuscript. Acknowledgement Special thanks are extended to Mr. Matej Cunder from the Slovenian Environment Agency for creating the distribution maps used in this study. Funding The authors acknowledge the financial support from the Slovenian Environment Agency and the Slovenian Research Agency (research core funding No. P2-0180). Conflicts of Interest The authors declare no conflict of interest. 58 Acta Biologica Slovenica, 2025, 68 (4) References ARSO, 2017. Program monitoringa kemijskega in ekološkega stanja voda, program za obdobje 2016 do 2021. Agencija Republike Slovenije za okolje, Ministrstvo za okolje in prostor, Ljubljana. https://www.gov.si/assets/organi-v-sestavi/ARSO/Vode/Stanje-voda/Program-monitoringa-kemijskega-in-ekoloskega-stanja- voda-za-obdobje-2016-do-2021.pdf (accessed 20.4.2025) ARSO, 2022. Program monitoringa kemijskega in ekološkega stanja voda, program za obdobje 2022 do 2027. 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BioScience, 64(6), 531–538. https://doi.org/10.1093/biosci/biu060 59 1 Department of Biology, Faculty of Life and Nature Sciences, University of Oran 1 Ahmed BenBella, Oran 31100, Algeria. 2 Department of Biotechnology, Faculty of Life and Nature Sciences, University of Science and Technology of Oran, Mohamed-Boudiaf USTOMB, Oran 31000, Algeria. 3 Department of Biology, Faculty of Life and Nature Sciences, University of Mustapha Stambouli, Mascara 29000, Algeria. 4 Laboratory of Applied Microbiology, Department of Biology, Faculty of Life and Nature Sciences, University of Oran 1 Ahmed BenBella, Oran 31100, Algeria. * Corresponding author: E-mail address: saci.ahmed@edu.univ-oran1.dz Citation: Saci, A., Gharbi, S., Djadouni, F., Karkachi, N., (2025). Evaluation of selected functional traits in lactic acid bacteria isolated from Algerian Makatia goat milk for food applications. Acta Biologica Slovenica 68 (4) Received: 29.01.2025 / Accepted: 13.08.2025 / Published: 14.08.2025 https://doi.org/10.14720/abs.68.4.21735 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Evaluation of selected functional traits in lactic acid bacteria isolated from Algerian Makatia goat milk for food applications Ahmed Saci 1, * , Samia Gharbi 2 , Fatima Djadouni 3 , Noureddine Karkachi 4 Abstract While studies on the health aspects of lactic acid bacteria (LAB) may receive more attention from the scientific community, the focus on the technological aspects of these bacteria has also become equally important, as these bacteria possess physiological properties that qualify them for use in various food productions. This study examined some of these traits for 10 LAB isolates from Makatia goat milk in the Wilaya of Mascara in western Algeria. Tests were conducted to evaluate acidifying activity, enzymatic activity, growth capability at different pH levels, in high salt environments, different temperatures, exopolysaccharide (EPS) production, high-temperature tolerance, antagonistic activity, and antibiotic sensitivity. All isolates lacked amylolytic activity, while proteolytic activity varied between 14.33 and 32.67 mm of lysis zones, and lipolytic activity varied between 8.43 and 12.53 mm. Thermotolerance at 63 °C for 30 minutes was revealed for all 10 isolates, along with variable growth capacities under different conditions (high, low pH, salted environment, and different temperatures). Significant antagonistic activity ranged between 11.5 and 40.66 mm of inhibition zones against the tested indicator strains. All 10 LAB isolates from goat milk exhibited remarkable technological properties that may or may not be helpful in food technology. For example, they are desirable in the fermentation industry and undesirable in some pasteurisation-sensitive products or some meat food industries. Keywords Lactic acid bacteria, Technological properties, Goat milk, Food preservation, Food spoilage, Fermentation, Antimicrobial activity 60 Acta Biologica Slovenica, 2025, 68 (4) Ocena izbranih funkcionalnih lastnosti mlečnokislinskih bakterij, izoliranih iz alžirskega kozjega mleka Makatia, za uporabo v živilih Izvleček Medtem ko študije o zdravstvenih vidikih mlečnokislinskih bakterij (LAB) morda pritegnejo več pozornosti znanstvene skupnosti, je enako pomembno tudi osredotočanje na tehnološke vidike teh bakterij, saj imajo fiziološke lastnosti, ki jih kvalificirajo za uporabo v različnih živilskih proizvodnjah. Ta študija je preučila nekatere od teh lastnosti za 10 izolatov LAB iz kozjega mleka Makatia v Wilaya v Mascari v zahodni Alžiriji. Opravili so se testi za oceno kisle aktivnosti, encimske aktivnosti, sposobnosti rasti pri različnih vrednostih pH, v okoljih z visoko vsebnostjo soli, pri različnih temperaturah, proizvodnje eksopolisaharidov (EPS), tolerance na visoke temperature, antagonistične aktivnosti in občutljivosti na antibiotike. Vsi izolati so bili brez amilolitične aktivnosti, proteolitična aktivnost pa je bila med 14,33 in 32,67 mm liznih con, lipolitična aktivnost pa med 8,43 in 12,53 mm. Vsi 10 izolati so pokazali termotoleranco pri 63 °C za 30 minut, skupaj z različnimi sposobnostmi rasti v različnih pogojih (visoka, nizka pH, slano okolje in različne temperature). Pomembna antagonistična aktivnost je bila med 11,5 in 40,66 mm inhibicijskih con proti testiranim indikatorjem. Vseh 10 izolatov LAB iz kozjega mleka je pokazalo izjemne tehnološke lastnosti, ki so lahko koristne v prehrambni industriji. Na primer, so zaželeni v fermentacijski industriji, vendar nezaželeni v nekaterih proizvodih, občutljivih na pasterizacijo, ali v nekaterih mesnih industrijah. Ključne besede Mlečnokislinske bakterije, tehnološke lastnosti, kozje mleko, konzerviranje živil, pokvarljivost živil, fermentacija, protimikrobna aktivnost Introduction LAB are considered the most interesting microbial group due to their extensive use as probiotics, bio-preservatives, or fermentation starter cultures (Tidona et al., 2020). Due to their enzymatic potential, LAB play a key role in various food productions. These enzymes directly contribute to developing organoleptic properties (flavour, consistency, texture, etc.) (García-Cano et al., 2019). In addition to influencing organoleptic properties, lactic acid bacteria possess important antioxidant and antimicrobial properties (Baureder and Hederstedt, 2013). Regardless the number of technological characteristics of LAB, their ability to survive in different physicochemical conditions, such as storage and preservation conditions at different pH values, at different salt concentrations, at different heat treatments, and different storage tempera- tures, is considered as the key factor in their selection as potential sources for food preservation and processing (Sionek et al., 2024). A lot of studies focus on the positive aspects of LAB, such as their probiotic and technological capabilities. The latter also plays an equally important role in food spoilage, particularly in those foods where manufacturing treatments do not target specific types of LAB. These foods are often subjected to spontaneous fermentation processes by present autochthonous microbiota or infected by undesir- able contamination by LAB (uncontrolled manufacturing processes) (Ağagündüz et al., 2022; Peng et al., 2020; Vinderola, 2019). Several raw materials for isolating LAB are available. Among these materials, goat milk occupies the smallest portion of the various scientific studies. Compared to other types of milk, goat milk has many health benefits for humans, such as high calcium content, low allergenic potential, and easy digestibility of its fat in the presence of bile (Nayik et al., 2021). In the present study, we selected 10 isolates of LAB from 4 samples of milk from the Algerian Makatia goat breed. The Makatia goat is a purebred Algerian breed, native to the Saharan Atlas region, specifically the provinces of Laghouat and Djelfa. It is classified as a dual-purpose breed (meat and milk) and is renowned for its remarkable adaptability to harsh steppe environments. Intending to 61 Acta Biologica Slovenica, 2025, 68 (4) contribute to detecting some technologically essential properties, such as their ability to grow under different conditions (pH, NaCl, temperature), enzymatic activity, and antimicrobial properties. Materials and methods Sample collection and preparation Raw Makatia goat milk samples were collected from four healthy animals in the region of Mascara, western Algeria (Figure 1), during March 2024. The samples (50 mL) were collected aseptically into sterile containers and transported to the laboratory at 4 °C within 4 hours for immediate processing. The pH of the goat milk samples was determined using a pH meter (Adwa AD1030, Szeged, Hungary) previously calibrated with a standard buffer (Webster, 2003). Isolation and identification of lactic acid bacteria Twenty-three isolates were obtained from four samples of raw goat milk from the Mascara province. Only 10 Gram-pos- itive, catalase-negative, non-sporulating, and non-motile isolates were selected. 10 mL of each goat milk sample was diluted 10 times in sterile ¼ strength Ringer solution. 0.1 mL of each dilution was inoculated on the surface of an MRS agar (Liofilchem S.r.l., Italy) and then incubated at 30 °C for 48 hours under anaerobic conditions (2.5-litre anaerobic jar). Then, successive streaks were performed on MRS agar until pure cultures were obtained. These isolates were kept at -20 °C in MRS broth containing 25 % glycerol for further analysis (Haghshenas et al., 2017). The morphology of the colonies, the Gram type, and the cellular morphology of the bacteria were determined for all 10 isolates Smith and Hussey, 2005. Motility was examined by inoculation in semi-solid agar (Patricia and Laura, 2011). Figure 1. Geographic location of the sampling sites. The map displays the three communes (Sehaillia, Hachem, and Ain Farrah) within the Wilaya of Mascara, western Algeria, from which raw milk samples of the native Makatia goat breed were collected. The inset on the upper left provides a broader geographical context by showing the location of the Wilaya of Mascara within Algeria. The legend indicates the specific area of each commune. Map data is adapted from Google Maps. Slika 1. Geografska lega mest vzorčenja. Zemljevid prikazuje tri občine (Sehaillia, Hachem in Ain Farrah) v provinci Mascara v zahodni Alžiriji, iz katerih so bili odvzeti vzorci surovega mleka avtohtone pasme koz Makatia. Vstavek v zgornjem levem kotu prikazuje širši geografski kontekst z lokacijo province Mascara v Alžiriji. Legenda označuje posebno območje vsake občine. Podatki zemljevida so prilagojeni iz Google Maps. 62 Acta Biologica Slovenica, 2025, 68 (4) The catalase and oxidase tests were examined using 3 % hydrogen peroxide and oxidase strips (Merck Bactident oxidase 181, Darmstadt, Germany), respectively. Staphy- lococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as positive and negative controls for Gram type test, cata- lase activity and motility (Khadija et al. 2022). Bacterial genomic DNA was extracted from isolates using the GF-1 Nucleic Acid Extraction Kit (Vivantis Tech- nologies Sdn Bhd, Selangor, DE, Malaysia) according to the manufacturer's protocol (Vivantis Technologies Sdn Bhd, 2010). The integrity and concentration of the extracted DNA were measured using a NanoDrop™ One Spectropho- tometer (Thermo Fisher Scientific, USA). The 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal primers 27F (5'-AGA GTT TGA TCG TCC TGG CTC AG-3') and 1492R (5'-CCG TCA ATT CCT TTG AGT TT-3') (Edwards et al., 1989). PCR was conducted in a 50 µL reaction volume containing 1.25 U of Hot Start Taq DNA Polymerase (Solis Biodyne, Estonia), 1X reaction buffer with 1.5 mM MgCl₂, 0.3 µM of each primer, 25–50 ng of template DNA, and nuclease-free water. Amplification was performed in an iCycler thermocycler (Bio-Rad, USA) under the following conditions: an initial denaturation step at 94 °C for 12 minutes; followed by 30 cycles of denaturation at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds, and extension at 72 °C for 1 minute and 40 seconds; and a final extension step at 72 °C for 7 minutes. The presence and size of the PCR amplicons were verified by electrophoresis on a 1.5 % agarose gel stained with Midori Green Advance (Nippon Genetics, Japan) and visualised under UV light. The purified PCR products were sent for bidirectional sequencing to Sarl GENE LIFE SCIENCES (Sidi Bel Abbès, Algeria). The sequencing reaction was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit on a 3130 Genetic Analyser (Applied Biosystems, USA), which corresponds to the Sanger sequencing method (Sanger et al., 1977). The resulting forward and reverse sequence chromatograms for each isolate were assembled and edited to generate a final consensus sequence using Bio- numerics v3.5 software (Applied Maths, Belgium). The qual- ity of sequences was visually assessed, and poor-quality regions were manually edited or trimmed. The consensus sequences were then identified by performing a homology search against the GenBank database using the National Centre for Biotechnology Information (NCBI) BLASTn tool. Technological profiling The acidifying activity of 10 isolates was examined over a time interval of 6 and 48 hours, using a rapid screening method to evaluate the acidifying activity of LAB (colouri- metric assay), described by Ribeiro et al. (2021). The α-amylase activity was examined by inoculating 10 µL of each fresh bacterial culture onto sterile filter paper discs placed on MRS agar devoid of glucose and supple- mented with 0.3 % starch. After incubation at 30 °C for 48 hours, the Petri dishes were flooded with Lugol solution to detect α-amylase activity (The appearance of transparent yellow halos around bacterial cultures indicates starch decomposition) (Amapu et al., 2016). The proteolytic activity was evaluated by inoculating 10 µL of a fresh culture of each isolate onto a sterile filter paper disc placed on the surface of an MRS agar plate sup- plemented with 1 % skimmed milk. After incubation at 37 °C for 48 hours, the diameters of the transparent zones (halos indicating positive proteolytic activity) were measured (Islam et al., 2021). The lipolytic activity was assessed by inoculating 10 µL of a fresh culture of each isolate onto a sterile filter paper disc on an MRS agar plate surface enriched with 1 % Tween 80. After incubation at 37 °C for 48 hours, the diameters of the transparent zones (halos indicating positive lipolytic activity) were measured (Tanasupawat et al., 2015). The ability of the isolates to produce EPS was examined by streaking pure and fresh cultures on Mayeux Sandine and Elliker (MSE) agar. The appearance of slimy and colloi- dal colonies after incubation at 37 °C for 48 hours indicates positive EPS production (Fguiri et al., 2016). The ability of the isolates to grow in NaCl (weight/ volume) solutions at 4, 6, and 10 % was evaluated by inocu- lating fresh culture colonies of each isolate into MRS broths supplemented with 4, 6, and 10 % NaCl (weight/volume) and bromocresol purple indicator. After incubation at 37 °C for 48 hours, the colour change from purple to yellow was evidence of cell growth (Ruiz et al., 2024). The ability of the isolates to grow at different pH levels was tested by inoculating fresh colonies into several tubes containing MRS broths adjusted to different pH levels: 2.2, 4.5, 6.2, and 9.6. The appearance of turbidity after incubation at 37 °C for 48 hours indicates the cell viability of the isolates. The growth of the isolates at different temperatures was examined by inoculating 50 µL of fresh cultures of each isolate into tubes containing 7 mL of MRS broth with bro- 63 Acta Biologica Slovenica, 2025, 68 (4) mocresol purple indicator, four tubes for each isolate. Each series of these tubes was incubated at temperatures of 4, 10, 15, and 45 °C for 72 hours. Bacterial growth was observed in the colour change from purple to yellow (Kim et al., 2019). The ability of the isolates to tolerate high temperatures was examined according to the method described by Hur- tado-Bautista et al. (2021), with modifications (adaptation of the technique with LAB isolates and modification of the temperature exposure time). 100 µL of each bacterial isolate was inoculated into tubes containing 7 mL of MRS broth supplemented with bromocresol purple indicator. The tubes were immersed in a water bath previously set to 63 °C for 30 minutes. Then, they were incubated at 37 °C for 48 hours. The pH indicator's colour change indicated the bacterial cells' survival. The antimicrobial activity of isolates against indicator strains (Staphylococcus (S.) aureus ATCC 25923, Pseudomo- nas (P.) aeruginosa ATCC 27853, Escherichia (E.) coli ATCC 25922, Bacillus (B.) cereus ATCC 10987, and Candida (C.) albicans ATCC 10231) was evaluated using the direct spot test. The goat milk isolates were inoculated as 0.5 mm diam- eter spots on an appropriate number of Petri dishes pre-filled with soft MRS agar (0.8 % MRS agar). Melted soft MRS agar (45 °C) in tubes was inoculated with 100 µL of each indica- tor strain. Then, the contents of each of these tubes were poured into the appropriate Petri dishes already inoculated with LAB isolates. The size of inhibition zones was measured after incubation at 37 °C for 24 hours (Akman et al., 2021). The examination of the fermentation profile (sugar fermentation) was carried out using an API 50 CHL system according to the manufacturer's instructions (BioMérieux, Marcy-l'Étoile, France), aimed at identifying Lactobacillus and related genera (Ghanbari et al., 2009). Briefly, four fresh and pure colonies of each goat milk isolate were inoc- ulated into API 50 CHL medium (7 mL). This inoculum was then inoculated into the gallery of wells (49 tests plus one control) up to the marker line, followed by the addition of 3 to 4 drops of sterile paraffin oil. The system was incubated at 37 °C for 24-48 hours. The colour change indicates a positive fermentation of carbohydrates. Antibiotic resistance The sensitivity of LAB isolates to antibiotics was evaluated against 10 antibiotics (Liofilchem S.r.l., Italy): penicillin (P) 10 µg, augmentin (AUG) 30 µg, ampicillin (AMP) 30 µg, cefepime (FEP) 30 µg, cefoxitin (FOX) 30 µg, ofloxacin (OFX) 5 µg, nalidixic acid (NA) 30 µg, chloramphenicol (C) 30 µg, erythromycin (E) 15 µg, and tetracycline (TE) 30 µg according to the Kirby-Bauer method (Hudzicki, 2009). According to Clinical and Laboratory Standards Institute guidelines (CLSI, 2020), the sensitivity data were categorized as resistant (R, zone diameter <14 mm), moderately susceptible (I, zone diameter between and including 14 to 20 mm), or suscepti- ble (S, zone diameter >20 mm) (Meradji et al., 2023). Statistical analysis The results were expressed as mean ± standard deviation of three repetitions (n = 3). Statistical significance was demonstrated by Two-way ANOVA, accompanied by Duncan's post hoc test for the examination of acidifying capacity and antimicrobial activity, and One-way ANOVA, accompanied by Tukey's post hoc test for the assessment of proteolytic activity and lipolytic activity using GraphPad Prism 9.5.1 (733) software. The data for which the P value is less than 0.05 were statistically significant. Results Isolation and identification of lactic acid bacteria The 10 isolates were examined based on their macroscopic, microscopic, and physiological characteristics. As expected for LAB, Gram staining revealed them as Gram-positive and rod-shaped. The motility, catalase, and oxidase tests were negative. The morphology of the colonies varied from one isolate to another (Table 1). The 10 isolates named GL.01, GL.02, GL.03, GL.04, GL.05, GL.06, GL.07, GL.08, GL.09, and GL.10 were identified using 16S rRNA sequencing. Sequences obtained were subjected to NCBI blast tool in NCBI GenBank (https:// www. ncbi. nlm. nih.gov/genbank/) and were found to be Lactiplantibacillus plantarum (isolates GL.01 and GL.09), Lactobacillus planta- rum (isolates GL.02 and GL.10), Lactiplantibacillus pentosus (isolate GL.07), Lactobacillus fermentum (isolate GL.03) and Lactobacillus sakei (isolate GL.06), Lactiplantibacillus sp (isolate GL.04, GL.05 and GL.08) These last three isolates showed a similarity percentage lower than the limit allowed for species identification (98.7 %). The % of similarity to the closest bacterial strains, together with their accession numbers, is shown in Table 2. 64 Acta Biologica Slovenica, 2025, 68 (4) Macroscopic appearance Microscopic appearance Physiological tests Isolate Animal milk sample sources Sample pH Form of colony Size (mm) of colony Colony margin Colony color Gram-Stain Form Catalase Oxidase Motility GL.01 01 6.42 ± 0.8 C 01 E W + Rods - - - GL.02 C 01 E W + Rods - - - GL.03 C 01 E W + Rods - - - GL.04 02 6.63 ± 1.4 C 01 E Y. W + Rods - - - GL.05 C 01 E W + Rods - - - GL.06 03 6.76 ± 0.6 C 01 E W + Rods - - - GL.07 04 6.15 ± 1.2 C 01 E W + Rods - - - GL.08 C 01 E W + Rods - - - GL.09 C 01 E Y. W + Rods - - - GL.10 C 01 E W + Rods - - - Molecular identification Isolates Species Strain Similarity (%) Accession number GL.01 Lactiplantibacillus plantarum 2546 99.82 MT611578 GL.02 Lactobacillus plantarum B_16LAB 99.83 MF405177 GL.03 Lactobacillus fermentum KLAB15 99.36 KM485578 GL.04* Lactiplantibacillus sp 1583 90.69 MT597488 GL.05* Lactiplantibacillus sp HBMSS8 96.48 MF662593 GL.06 Lactobacillus sakei HBMSS7 99.32 MF662592 GL.07 Lactiplantibacillus pentosus MYSDV3 98.92 MT152901 GL.08* Lactiplantibacillus sp HBMSS4 97.46 MF662589 GL.09 Lactiplantibacillus plantarum MYSDV5 99.80 MT131268 GL.10 Lactobacillus plantarum B_16LAB 100.00 MF405177 Table 1. Macroscopic, microscopic, and physiological characteristics of ten lactic acid bacteria isolates (GL.01-GL.10) collected from raw milk of Makatia goat breed samples, along with the pH values of the original samples. Tabela 1. Makroskopske, mikroskopske in fiziološke lastnosti desetih izolatov mlečnokislinskih bakterij (GL.01-GL.10), zbranih iz surovega mleka vzorcev koz pasme Makatia, skupaj z vrednostmi pH izvirnih vzorcev. Table 2. Genotypic identification of lactic acid bacteria isolates from raw milk of the Algerian Makatia goat breed, based on 16S rRNA. Tabela 2. Genotipska identifikacija izolatov mlečnokislinskih bakterij iz surovega mleka alžirske koze pasme Makatia na podlagi 16S rRNA. The isolates were obtained from four Makatia goat milks collected from the western region of Algeria. Macroscopic appearance: C: Circular; E: Entire; W: White; Y.W: Yellowish white. Microscopic appearance and physiological tests: (+): Positive reaction (Gram-positive); (-): Negative reaction (for Catalase, Oxidase, and Motility tests). All isolates were identified as rod-shaped bacteria. Identification was performed by comparing the 16S rRNA gene sequences of the isolates against the NCBI GenBank database using BLAST. The table shows the closest species match, the strain designation of that match, the percentage sequence similarity, and the corresponding GenBank accession number. (*) Isolates were identified to the genus level because their 16S rRNA gene sequence similarity was below the 98.7% threshold recommended for species delineation. 65 Acta Biologica Slovenica, 2025, 68 (4) Technological profiling The 10 isolates showed variable acidifying activities within the 6 to 8-hour incubation time range (Table 3). The acidi- fying activity values ranged from 5.00 to 6.11 after 6 hours of incubation and from 3.24 to 4.47 after 48 hours. After 6 hours of incubation, the isolate GL.03 showed the lowest pH value, while after 48 hours, the isolate GL.08 had the lowest pH value. A Two-way ANOVA comparison test, followed by a post hoc test (Sidak's multiple comparisons test), showed significant differences with P < 0.5. A complete absence of starch hydrolysis activity appeared for the 10 isolates, represented by the absence of transparent or yellowish halos around the inoculated spots after adding Lugol's iodine (Table 3). A varied proteolytic activity was revealed for the 10 iso- lates. Clear proteolysis zones ranged from 14.33 to 32.67 mm (Table 3). The isolate GL.02 exhibited the highest pro- teolytic activity with a proteolysis zone of 32.67 mm, while the isolate GL.07 showed the lowest activity with a proteol- ysis zone of 14.33 mm. A One-way ANOVA comparison test, followed by a post hoc test (Tukey's multiple comparisons test), showed significant differences with P < 0.5. The 10 isolates showed lipolytic activities ranging from 8.43 to 12.53 mm of lipolytic zones (Table 3). In comparison with proteolytic activity, lipolytic activity was relatively modest. Isolate GL.02 showed the highest activity with a lipolysis zone of 12.53 mm, followed by isolates GL.10 and GL.06 with lipolysis zones of 12.33 mm and 11.17 mm, respectively, while the isolate GL.03 exhibited the lowest activity with a lipolysis zone of 8.43 mm. A One- way ANOVA comparison test, followed by a post hoc test (Tukey's multiple comparisons test), showed significant differences with P < 0.5. Six isolates were revealed to be EPS production posi- tive (GL.01, GL.04, GL.05, GL.06, GL.08, and GL.09), while four isolates were revealed to be EPS production negative (GL.02, GL.03, GL.07, and GL.10) (Table 3). Acidifying activity Amylolytic activity Proteolytic activity Lipolytic activity EPS production pH Halos (mm) Isolates 06 h 48 h GL.01 5.21 ± 0.6 a 3.55 ± 0.3b 0.0 22.50 ± 0.5 f 9.12 ± 0.5 lm + GL.02 6.11 ± 0.1 a 4.21 ± 0.7b 0.0 32.67 ± 1.3 c 12.53 ± 0.1 j - GL.03 5.00 ± 1.3 a 3.28 ± 1.6b 0.0 26.50 ± 0.5 e 8.43 ± 1.5 m - GL.04 5.12 ± 0.7 a 3.49 ± 0.5b 0.0 25.33 ± 0.5 e 9.36 ± 0.5 lm + GL.05 5.36 ± 0.1 a 3.41 ± 0.4b 0.0 22.33 ± 1.0 fg 8.90 ± 1.2 lm + GL.06 6.10 ± 1.2 a 4.38 ± 0.3b 0.0 17.00 ± 0.5 h 11.17 ± 0.5 jk + GL.07 5.33 ± 0.4 a 3.64 ± 1.0b 0.0 14.33 ± 0.8 i 9.73 ± 0.5 k - GL.08 5.06 ± 1.5 a 3.24 ± 0.2b 0.0 20.33 ± 0.5 g 9.03 ± 1.4 lm + GL.09 5.56 ± 1.2 a 3.82 ± 0.6b 0.0 21.00 ± 0.5 fg 9.63 ± 1.0 l + GL.10 6.09 ± 0.3 a 4.47 ± 0.5b 0.0 30.17 ± 1.0 d 12.33 ± 0.5 j - Table 3. T echnological properties of lactic acid bacteria isolates from raw milk of the Algerian Makatia goat breed, including acidifying, enzymatic, and exopolysaccharide (EPS) production capabilities. Tabela 3. Tehnološke lastnosti izolatov mlečnokislinskih bakterij iz surovega mleka alžirske koze pasme Makatia, vključno z zmožnostjo kislega delo- vanja, encimsko aktivnostjo in proizvodnjo eksopolisaharidov (EPS). Values are presented as mean ± standard deviation of three replicates. EPS: exopolysaccharide, (+): positive production, (-): negative production. For acidifying activity, within each row (isolate), values with different superscripts (a, b) are significantly different (Student's t-test, p < 0.05). For proteo- lytic and lipolytic activities, within each column, values followed by different superscripts (c-m) are significantly different (one-way ANOVA followed by Tukey's post-hoc test, p < 0.05). All isolates showed no amylolytic activity. 66 Acta Biologica Slovenica, 2025, 68 (4) Eight out of 10 isolates demonstrated the ability to grow under different percentages of NaCl, 4, 6, and 10 %, for 48 hours (Table 4). As an exception, the isolates GL.02 and GL.10 (both identified as Lactobacillus plantarum) could not grow at 10% NaCl. The 10 isolates revealed an ability to grow under differ- ent pH levels of 4.5, 6.2, and 9.6 and showed a complete absence of growth in highly acidic media (pH 2.2) (Table 4). All the isolates were grown in slightly acidic and alkaline media within a pH range of 4.5 to 9.6. In a 72-hour incubation period, the 10 isolates could grow at 10, 15, and 45 °C (Table 4). In contrast, at a tem- perature of 4 °C, only the isolates GL.02 and GL.10 (Lacto- bacillus plantarum), and GL.03 (Lactobacillus fermentum) could grow. After exposing all 10 bacterial isolates to 63 °C for 30 minutes, they all were able to grow (incubated at 37 °C for 48 hours) (Table 4). A significant inhibition of indicator strains was revealed for all 10 isolates. Variably, E. coli and P. aeruginosa were the most sensitive to the antagonistic activity of 10 isolates, while S. aureus was the least sensitive. B. cereus was the most resistant strain against the antagonism of isolates GL.05 and GL.07. All 10 isolates exhibited satisfactory antagonistic effects against all the tested indicator strains, ranging from 11.5 to 40.66 mm (Figure 2). A Two-way ANOVA comparison test, followed by a post hoc test (Tukey's multiple compari- sons test), showed significant differences with P < 0.5. All 10 isolates showed distinctive fermentative profiles of various carbohydrates. In general, they have been able to ferment carbohydrates widespread in foods (Table 5). The antibiotic sensitivity test showed different levels of resistance and sensitivity to 10 antibiotics. In short, all 10 isolates were entirely resistant to FEP (30 μg), moderately sensitive to AUG (30 μg), and AM (10 μg). For the rest of the antibiotics, the sensitivity varied (Table 6). Growth under different conditions NaCl (%) (48 h) pH (48 h) Temperature (°C) (72 h) High-temperature tolerance (63 °C for 30 min) Isolates 04 06 10 2.2 4.5 6.2 9.6 04 10 15 45 GL.01 + + + - + + + - + + + + GL.02 + + - - + + + + + + + + GL.03 + + + - + + + + + + + + GL.04 + + + - + + + - + + + + GL.05 + + + - + + + - + + + + GL.06 + + + - + + + - + + + + GL.07 + + + - + + + - + + + + GL.08 + + + - + + + - + + + + GL.09 + + + - + + + - + + + + GL.10 + + - - + + + + + + + + Table 4. Growth profiles and thermotolerance of lactic acid bacteria isolates from raw milk of the Algerian Makatia goat breed under various stress conditions. Tabela 4. Profili rasti in toplotna toleranca izolatov mlečnokislinskih bakterij iz surovega mleka alžirske koze pasme Makatia v različnih stresnih pogojih. Growth was assessed in MRS broth. (+): growth observed; (-): no growth observed. Standard incubation conditions were 37 °C for 48 h in MRS broth with a pH of 6.2. For NaCl tolerance, growth was tested in MRS broth with 4 %, 6 %, and 10 % (w/v) NaCl at 37 °C. For pH tolerance, growth was tested in MRS broth adjusted to pH 2.2, 4.5, 6.2, and 9.6, with incubation at 37 °C. For optimal growth temperature, growth was tested in standard MRS broth at 4 °C, 10 °C, 15 °C, and 45 °C. For the high-temperature (thermo) tolerance, isolates were exposed to 63 °C for 30 min in MRS broth and then incubated under standard conditions to check for survival. 67 Acta Biologica Slovenica, 2025, 68 (4) Figure 2. Antagonistic activity of ten lactic acid bacteria isolates against five pathogenic indicator strains. The LAB isolates were obtained from raw Algerian Makatia goat milk. Activity was measured as the diameter of the inhibition zone (mm) using a spot-on-lawn assay. Bars represent the mean of three independent replicates (n=3), and the error bars indicate the standard deviation. The indicator strains tested were Staphylococcus (S.) aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Bacillus cereus ATCC 10987, and Candida albicans ATCC 10231. Slika 2. Antagonistična aktivnost desetih izolatov mlečnokislinskih bakterij proti petim patogenim indikatorjem. Izolati LAB so bili pridobljeni iz surovega alžirskega kozjega mleka Makatia. Aktivnost je bila izmerjena kot premer inhibicijske cone (mm) z uporabo testa spot-on-lawn. Stolpci predstavljajo povprečje treh neodvisnih ponovitev (n = 3), napake pa so prikazane s standardnim odklonom. Testirani indikatorji so bili Staphylococcus (S.) aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Bacillus cereus ATCC 10987 in Candida albicans ATCC 10231. Carbohydrates Isolates GL.01 GL.02 GL.03 GL.04 GL.05 GL.06 GL.07 GL.08 GL.09 GL.10 Control - - - - - - - - - - Glycerol - - - - - - + - - - Erythrol - - - - - - - - - - D-arabinose - - - - - - - - - - L-arabinose + + + + - + + + + + Ribose + + + + - - + + + + Table 5. Carbohydrate fermentation profiles of ten lactic acid bacteria isolates from raw Algerian Makatia goat milk were determined using the API 50 CHL system. Tabela 5. Profili fermentacije ogljikovih hidratov desetih izolatov mlečnokislinskih bakterij iz surovega alžirskega kozjega mleka Makatia, določeni z uporabo sistema API 50 CHL. 68 Acta Biologica Slovenica, 2025, 68 (4) D-xylose - - + - - - + - - - L-xilose - - + - - - - - - - Adonitol - - + - - - - - - - Β-Methyl-D-Xyloside - - + - + - - - - - Galactose + + + + + + + + + + Glucose + + + + + + + + + + Fructose + + + + + + + + + + Mannose + + + + + + + + + + Sorbose - - - - + + - - - - Rhamnose - + - - - - W - W + Dulcitol - - - - - - - - - - Inositol - - - - - - - - - - Mannitol + + - + + - + + + + Sorbitol + + - + - - + + + + 1-methyl-d-mannoside + + - + - - - + + + 1-methyl-d-glucoside - + - W - - + - - + N-acetyl-glucosamine + + - + + + + + + + Amygdaline + + - + + - + + + + Arbutin + + - + + - + + + + Esculin + + - + + + + + + + Salicin + + - + W - + + + + Cellobiose + + - + + - + + + + Maltose + + + + + - + + + + Lactose + + + + + - + + + + Melibiose + - + + + - + + + - Saccharose + W - + + + + + + - Trehalose + - + W + - + + + - Inulin - - - - - - - - - - Melezitose + - - + + - + W W - Raffinose + - + + + - + - W - Amidon - - - - - - - - - - Glycogen - - - - - - - - - - Xylitol - - - - - - - - - - Gentiobiose + + - + W - + + + w D-turanose + - - + - - W + + - D-lyxose - - - - - - - - - - D-tagatose - - - - - - - - - - D-fucose - - - - - - - - - - L-fucose - - - - - - - - - - D-arabitol + + - + - - - + + + L-arabitol - - - - - - - - - - Gluconate - + + - + W W - - + 2-keto-gluconate - - - - - - - W - - 5-keto-gluconate - - - - - W - W - - Fermentation profiles were determined using the API 50 CHL system (bioMérieux, Marcy-l'Étoile, France). Strips were inoculated and incubated at 37 °C for 48 hours according to the manufacturer's instructions. Acid production, resulting in a colour change of the pH indicator, indicated fermen- tation. (+): positive fermentation; (-): negative fermentation; (W): weak positive fermentation. The control well (00) contains the basal medium without any carbohydrate and is a negative control. 69 Acta Biologica Slovenica, 2025, 68 (4) Discussion This study analysed some technological traits of 10 differ- ent LAB isolates from 4 samples of Makatia goat milk in the Wilaya of Mascara (west of Algeria) that might be favour- able for the food and agricultural industries' applications. Most important technological properties have been tested such as ability to convert/ferment raw material to novel food with new characteristics and keep it fresh, withstand harsh growth conditions (the ability to grow in a range of unfavorable temperature, pH, and NaCl presence), their ability to withstand heat treatment with high temperatures (63 °C/30 min), which is similar to the pasteurization pro- cess, and their hydrolytic ability. Acidifying activity is one of the primary properties of starter microorganisms, particularly in dairy, meat, vegeta- ble industry, and silage production (González et al., 2015). The ability to increase the acidity through the fermentation of LAB is one of the options to preserve foods and, at the same time, is one of the most widely exploited technolog- ical properties (Hitendra et al., 2016). On the other hand, this technological property is undesirable in some technol- ogies due to the development of sour/untypical flavours, particularly in vinification and beer production (Wang et al., 2021), and is more considered as spoilage. The 10 isolates of LAB, analysed in our study, revealed a remarkable ability to reduce the pH value within 6 to 48 hours. After six hours of incubation, tested LAB were able to drop pH to 5.00 - 6.11, and after 48 h of incubation, the pH drop was even more evident, to 3.24 - 4.47. Abarquero et al. (2022) reported that the strains of 8 LAB species showed different acidifying capacities after 8 and 24 hours of incu- bation in milk, respectively, with the strains of Lactococcus lactis showing the most remarkable acidifying capacity with pH and total acidity values of 4.44. González et al. (2015) found that five strains/20 tested reduced pH values to less than six during 6 hours of incubation. Although different species of LAB possess different acidifying abilities, this trait is species-specific. This could be due to the diverse enzymatic potential that helps bacteria to specifically break down the carbon and nitrogen sources in the medium (Badis et al., 2004). Isolates Antibiotics P (10 µg) AUG (30 µg) AM (10 µg) FEP (30 µg) FOX (30 µg) OFX (5 µg) NA (30 µg) C (30 µg) E (15 µg) TE (30 µg) GL.01 S S S R I R I S R I GL.02 I S I R R R R S S I GL.03 S S S R I I R S S S GL.04 S S S R S I R S S S GL.05 S S S R S R R S S S GL.06 S S S S I R R I S S GL.07 S I S R R S R S S R GL.08 I S S R I I R S I S GL.09 I S S R I S R S I S GL.10 R S S R I R R S I R Table 6. Antibiotic susceptibility profiles of lactic acid bacteria isolates from raw Algerian Makatia goat milk, determined by the Kirby-Bauer disk diffusion method. Tabela 6. Profili občutljivosti na antibiotike izolatov mlečnokislinskih bakterij iz surovega alžirskega kozjega mleka Makatia, določeni z metodo difuzije diskov Kirby-Bauer. Susceptibility was tested on Mueller-Hinton agar using the Kirby-Bauer disk diffusion method. R, resistant (zone size <14 mm), I, intermediate (14 mm ≥ zone size ≤ 20 mm), S, sensitive (zone size > 20 mm). (CLSI). P: Penicillin (10 µg); AUG: Amoxicillin/Clavulanic acid (30 µg); AM: Ampicillin (10 µg); FEP: Cefepime (30 µg); FOX: Cefoxitin (30 µg); OFX: Ofloxacin (5 µg); NA: Nalidixic acid (30 µg); C: Chloramphenicol (30 µg); E: Erythromycin (15 µg); TE: Tetracycline (30 µg). 70 Acta Biologica Slovenica, 2025, 68 (4) Among LAB, the most critical technological characteris- tics are their enzymatic activities. These characteristics are essential in fermentation and food processing (ripening and flavour development). Enzymatic activity is not necessarily linked to the fact that the bacterium is cultivable, as it can leave a group of metabolites and enzymes behind during autolysis. This trait is crucial for food preservation via fer- mentation (Luigi-Sierra et al., 2024; Yüce et al., 2017; Zam- bonelli et al., 2002). Although amylolytic activity can be a desirable trait in microorganisms, used as starters, LAB are known not to possess the necessary enzymatic systems (Meena et al., 2022; Owusu-Kwarteng et al., 2015). On the contrary, proteolytic activity is much more typical in LAB, as this mechanism contributes to the development of flavours and the nutritional quality of fermented products, particu- larly dairy products. Thanks to the production of peptides and biologically active amino acids, LAB also prevent the allergic effect of milk by producing non-epitopic peptides (Lopez-Kleine and Monnet, 2011; Pescuma et al., 2009). Our study revealed significant proteolytic activity with lysis zones as big as 14.33 to 32.67 mm. These results are even more promising compared to the results of Fguiri et al. (2017), who reported analysis of 10 isolates with lysis zones between 15 and 21 mm. Islam et al. (2021) reported that nine strains of LAB out of 11 possessed proteolytic activity, while Pediococcus pentosaceus and Enterococcus faecalis showed no proteolytic activity. In the study conducted by Abarquero et al. (2022), similar low proteolytic activity was revealed among the groups of LAB (P > 0.05), where the strains of Lactococcus lactis showed the highest values (close to 1 mmol Gly L− 1 milk). Further, due to their industrial potential, lipases are an important group of enzymes. They are widely used across diverse biotechnological domains, including food, pharmaceutical, detergent, agrochemical, oleochemical, textile, and cosmetic industries (Dinçer and Kıvanç, 2018). On the other hand, microorganisms that pos- sess lipolytic potential are considered highly undesirable, particularly in the food industry. They cause food spoilage, especially those rich in fat, such as meat, fish, butter, and other high-fat dairy products. They degrade fat as a carbon source, directly or indirectly causing what is known as the rancidity phenomenon, which negatively alters the organoleptic properties of foods (Alford and Pierce, 1961; Anand, 2011). In our study, all 10 isolates exerted lipolytic activity, with lysis zones ranging from 8.43 to 12.53 mm. Isolates GL.02 and GL.10 (Lactobacillus plantarum) had the highest lipolytic activity, 12.33 and 12.53 mm, respectively. Other studies have also reported low lipolytic activities in several genera of LAB (Fguiri et al., 2017; Islam et al., 2021; Monfredini et al., 2012). The ability of LAB to produce EPS continues to gener- ate significant interest, particularly in the food industry. EPS are considered as ural thickeners capable of improving the rheological properties of foods. They also have health and nutritional benefits, such as regulating blood sugar, having antioxidant properties, and improving intestinal absorption and microbiome quality (Jurášková et al., 2022). In our study, six isolates (Lactobacillus and Lactiplantibacillus genera) out of 10 tested appeared as mucoid colloidal colonies, indicating their ability to produce EPS. Moreover, Islam et al. (2021) isolated and identified altogether 50 LAB strains from 18 goat milk samples. Among these 50 isolates, they selected 11 representative strains, which were further tested for technological and probiotic traits. Genus and species identification served as the basis for the selection of these 11 strains. One of the technological traits tested was also the ability to produce EPS. All 11 strains of tested LAB produced EPS, varied between 20 and 93 mg/L, where Lactobacillus delbrueckii subsp. Bulgaricus and Streptococcus thermophilus exerted the highest levels of EPS production, namely 93.0 and 90.7 mg/L, respectively. Chun-lei (2014) reported that EPS production in 11 strains of LAB (Leuconostoc, Streptococcus, and Pediococcus genera) was as high as 536.90 mg/L. LABs are considered the primary spoilage agents in vacuum-packed meats and refrigerated foods because they produce undesirable metabolites that affect sensory and market quality (Silva et al., 2018). However, their ability to grow under different conditions, such as a wide range of temperatures and pH, high salinity, plays a crucial role in fer- mented food preservation, mainly due to the inability of the majority of spoilage microorganisms to grow under these hostile conditions and LAB inhibitory activity (Dillon, 2014). From a health and nutritional perspective, the ability of LAB to grow under unfavourable conditions, such as very low temperatures, like refrigeration temperature, gives them a long shelf life and, thus, significant probiotic potential (Fan et al., 2017). Generally, if we summarise the results of our study, 10 isolates could grow in the presence of high salt concentration, namely in a range of 4 to 10 % NaCl, except for the isolates GL.02 and GL.10 (Lactobacillus plantarum), which could not grow at 10 % NaCl. The 10 isolates were also able to grow at different temperatures, ranging from 10 to 45 °C, while only the isolates GL.02, GL.10, and GL.03 71 Acta Biologica Slovenica, 2025, 68 (4) (Lactobacillus species) could grow at 4 °C as well. Our iso- lates were also able to grow at a wide range of pH levels (from 4.5 to 9.6) but not at pH 2.2. Several studies have shown that various LAB species can grow under different hostile conditions, such as unfavourable acidity, alkalinity, salinity, storage, and preservation temperatures (Fguiri et al., 2017; Islam et al., 2021; Johanningsmeier et al., 2012). In some industries, LAB are essential food spoilage agents (cooked and vacuum-packed meat, varieties of thermally treated foodstuffs) (Franz, 1996). After being exposed to 63 °C for 30 minutes, all 10 isolates retained cultivability (at 37 °C/48h). Fossi and Ndjouenkeu (2017) found that two isolates of LAB (Lactobacillus plantarum and Lactobacillus acidophilus) were resistant to heat treatment (60 °C/60 minutes). Among LAB isolated from commercial sausages, four strains (Lactobacillus plantarum, Pediococ- cus acidilacti, Lactobacillus curvatus, and Pediococcus pentosaceus) survived heat treatment at 70 °C for 60 min- utes (Pérez-Chabela et al., 2008). According to Kulkarni et al. (2018), the Lactobacillus acidophilus strain only survived 40 minutes at 65 °C. It is well known that the antagonistic activity of LAB plays a crucial role as a selection factor for probiotic strains. This activity is essential not only for the putative probiotic potential but also for the technological and industrial potential. It is known that LAB produce a wide variety of metabolites, such as organic acids, bacteriocins, diacetyl, hydrogen peroxide, etc. (Dillon, 2014; Ibrahim et al., 2021) that have antimicrobial potential. So, our strains of LAB were no exception. P. aeruginosa and E. coli were the most sensitive indicator strains for the antagonism of the 10 isolates, with inhibition zones ranging from 20.33 to 40 mm and 20.33 to 40.66 mm, respectively. In comparison, S. aureus was the most resistant strain to the antagonism of the 10 isolates, with inhibition zones ranging from 11.5 to 27.33. In general, the antagonistic activity of 10 isolates varied between 11.5 and 40.66 mm. Owusu-Kwarteng et al. (2015) published that some L. fermentum strains' antago- nistic activities against S. aureus ATCC 1448 were insuffi- cient, with inhibition zones smaller than 4 mm. This may be because antimicrobial capacity is a highly strain-dependent property and not a constant characteristic of the entire bac- terial species. The problem may not be only the weakness of L. fermentum but also the strength and resistance of S. aureus. Islam et al. (2021) found antagonistic activities of some LAB, particularly L. plantarum, with inhibition zones reaching up to 20 mm against five pathogens. The broad spectrum of activity against a wide range of pathogens is because L. plantarum produces a diverse and potent group of antimicrobial compounds, the most well-known of which are bacteriocins such as plantaricin. These compounds are often highly effective even at low concentrations. In a study of Divyashree et al. (2024), the antimicrobial activity of 6 Lactobacillus strains was tested against some enteric pathogens, such as Salmonella typhi, Escherichia coli, Listeria monocytogenes, Salmonella paratyphi, Klebsiella pneumonia, Staphylococcus aureus, and Pseudomonas aeruginosa. Therefore, they prepared cell-free superna- tants (CFS) of 6 lactobacilli strains that were used in the microdilution method in 96 96-well microtiter plate. All six lactobacilli CFS exhibited antagonistic properties against pathogens, achieving over 90 % inhibition, which was calculated according to the formula from the publication. The different genera of LAB can metabolise a wide range of carbohydrates, including polysaccharides, which determines their industrial applications, whether in food or non-food sectors (Wang et al., 2021). The API 50 CHL system was used to examine the fermentation profile. The 10 isolates fermented various carbohydrates, including galactose, glucose, fructose, mannose, mannitol, sorbitol, and more. Also, none of the isolates metabolised the sugars numbered 16, 33, 36-38, 41-44, 46, and 48 (Table 5). Several studies have confirmed the ability of LAB to metabolise a wide range of carbohydrates and use them as energy sources (Gunkova et al., 2021; Islam et al., 2021; Ni et al., 2015; Ruiz et al., 2024). Regarding the importance of health safety, evaluating antibiotic resistance is one of the selection criteria for interesting bacteria, either probiotic or technological. Before using a starter culture or a probiotic product, it is necessary to check that the bacterial strains involved do not possess high resistance or antibiotic resistance genes (Fguiri et al., 2017). The 10 isolates revealed varying levels of sensitivity and resistance depending on the types of antibiotics tested. Generally, they showed apparent resistance to FEP (30 µg) and NA (30 µg), while they were either sensitive or moderately sensitive to the other tested antibiotics. Many studies have examined the resistance of different genera of LAB to antibiotics. The results have always varied from one type to another, and according to the classes of antibiotics. However, it is important that, to date, no LAB have been reported to exhibit multiple antibi- otic resistance (Amenu and Bacha, 2023; Choi et al., 2018; Meradji et al., 2023). 72 Acta Biologica Slovenica, 2025, 68 (4) Conclusion In this study, 10 LAB strains were isolated from 4 milk samples of Makatia breed goats in the Wilaya of Mascara (western Algeria). 4 isolates were identified as Lactiplan- tibacillus plantarum, two were identified as Lactobacillus plantarum, and 1 isolate each as Lactobacillus fermentum, Lactiplantibacillus paraplantarum, Lactobacillus sakei, and Lactiplantibacillus pentosus. The isolates exhibited hydro- lytic activities, the ability to grow under different storage conditions, remarkable thermotolerance, and antimicrobial activity against pathogens. At the same time, all 10 isolates were not reported to possess multidrug resistance. These characteristics make 10 isolates useful in the food industry as starter cultures and natural alternatives to chemical preservatives. In certain specific sectors, the same char- acteristics may also serve as important factors to avoid the possibility of certain undesirable interactions. Therefore, more specialized and targeted studies are still necessary to determine the relevance of exploiting the technological properties of LAB with various food manufacturing or processing methods. Author Contributions Conceptualization, A.S. and S.G.; methodology, all authors; software, A.S.; validation, all authors; formal analysis, A.S. and S.G.; investigation, A.S. and S.G.; resources, Scientific Research Laboratories of the Universities of Oran 1 and Mascara, Algeria; data curation, all authors; writing—origi- nal draft preparation, all authors; writing—review and edit- ing, all authors; visualization, A.S. and S.G.; supervision, S.G.; project administration, S.G. All authors have read and agreed to the published version of the manuscript. Acknowledgments The authors would like to thank Prof. Madani Benyoucef, Director of the Laboratory of Geomatics, Ecology and Envi- ronment (LGEO2E), University of Mascara, Algeria, for his cooperation in providing the necessary research facilities to carry out this study. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 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Food Technology and Biotechnology, 40(4), 347–351. 75 1 Biosystematics Laboratory, Department of Botany, Maharaja Sriram Chandra Bhanja Deo University, Baripada-757003, Odisha, India 2 Botanical Survey of India, CGO Complex, Sector -1, Salt Lake, Kolkata, West Bengal, India * Corresponding author: E-mail address: sudamsahu.bdk@gmail.com Citation: Tudu, S., Jena, J. K., Priyadarshini, S., Dash, S. S., Sahu, S. C., (2025). Taxonomical implications of fruit and seed morphology in wild species of Diospyros L. (Ebenaceae) in Odisha, Eastern India. Acta Biologica Slovenica 68 (4) Received: 19.04.2025 / Accepted: 18.08.2025 / Published: 20.08.2025 https://doi.org/10.14720/abs.68.4.22558 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Taxonomical implications of fruit and seed morphology in wild species of Diospyros L. (Ebenaceae) in Odisha, Eastern India Sabita Tudu 1 , Jeevan Kumar Jena 1 , Sifan Priyadarshini 1 , Sudhanshu Sekhar Dash 2 and Sudam Charan Sahu 1, * Abstract The family Ebenaceae exhibits a diverse array of characteristics and evolutionary histories. The members of the genus Diospyros L. are mostly dioecious and exhibit a great variation in their leaf, flower, fruit and seed morphology. These morphological variations often lead to ambiguity in species delimitation and misidentification in the genus Diospyros. As a pilot study, a detailed morphological and morphometric assessment was carried out in ten species of Diospyros growing wild in Odisha, Eastern India. Twenty (20) taxonomically important morphological characters of fruits and seeds were selected to study the similarity and variations among the taxa. The phenetic clustering divided the studied taxa of Diospyros into three groups at a cophenetic correlation coefficient (r) of 0.89, which provided insights into species differentiation and their closest taxonomic relationship. Moreover, the principal component analysis (PCA) indicated that the length of the calyx lobe and the size of the seed were significant in explaining the total variation among the studied taxa. The study came up with a dichotomous key and revealed the distinct morphological characters of fruits and seeds, contributing to a refined understanding of the Diospyros species complex. To clarify the taxonomic ambiguity, fruit and seed morphological characteristics provide an important tool for accurate species identification and enriching our understanding of the systematics of these specific Ebenaceae members. Keywords Diospyros L., Ebenaceae, morphology, phenetic clustering, Principal Component Analysis (PCA), Odisha 76 Acta Biologica Slovenica, 2025, 68 (4) Taksonomske posledice morfologije plodov in semen divjih vrst Diospyros L. (Ebenaceae) v Odishi, vzhodna Indija Izvleček Družina Ebenaceae ima raznolike značilnosti in evolucijsko zgodovino. Člani rodu Diospyros L. so večinoma dvodomni in se med seboj zelo razlikujejo po morfologiji listov, cvetov, plodov in semen. Te morfološke razlike pogosto vodijo do nejasnosti pri razmejevanju vrst in napačni identifikaciji v rodu Diospyros. Kot pilotna študija je bila izvedena podrobna morfološka in morfometrična ocena desetih vrst Diospyros, ki rastejo v divji naravi v Odishi v vzhodni Indiji. Za preučevanje podobnosti in razlik med taksoni je bilo izbranih dvajset (20) taksonomsko pomembnih morfoloških značilnosti plodov in semen. Fenetično združevanje je preučevane taksone Diospyros razdelilo v tri skupine s kophenetičnim korelacijskim koeficientom (r) 0,89, kar je omogočilo vpogled v razlikovanje vrst in njihovo najbližjo taksonomsko sorodnost. Poleg tega je analiza glavnih komponent (PCA) pokazala, da sta dolžina listne čaše in velikost semena pomembna za pojasnitev skupnih razlik med preučevanimi taksoni. Študija je prinesla dihotomni ključ in razkrila različne morfološke značilnosti plodov in semen, kar je prispevalo k boljšemu razumevanju kompleksa vrst Diospyros. Morfološke značilnosti plodov in semen so pomembno orodje za natančno določanje vrst in bogatijo naše razumevanje sistematike teh posebnih članov družine Ebenaceae. Ključne besede Diospyros L., Ebenaceae, morfologija, fenetično združevanje, analiza glavnih komponent (PCA), Odisha Introduction The taxonomically diverse family Ebenaceae exhibits a wide range of variation in vegetative and reproductive characteristics, leading to challenges in classification and identification. Due to the rich species diversity and dioe- cious habit, the family was subjected to several variable classifications with respect to the status of genera from time to time by various taxonomists (Bentham and Hooker, 1876; Clarke, 1882; Bakhuizen van den Brink, 1933; White, 1988; Singh, 2005). Recent studies reveal that the family Ebena- ceae represents three genera, such as Diospyros L., Euclea L. and Lissocarpa Benth, distributed mostly in pantropical regions of the world (Utteridge and Jennings, 2021; WFO, 2025). Among these genera, Diospyros L. is the largest genus, consisting of about 787 species globally (Govaerts, 2021). Members of this genus are distributed pantropically in the warmer regions of Southeast Asia, including China, Japan, Korea, India, and Pakistan, as well as parts of the Middle East and Africa. Diospyros species generally prefer warmer climates, whereas a few can also be found in temperate regions (Shashwathi and Krishnamurthy, 2024). These species exhibit a wide range of diversity, thriving in ecosystems such as dry forests, rainforests, mixed forests, lowland forests, and dry deciduous forests (Turner et al., 2013; Rauf et al., 2017). In India, the genus Diospyros comprises about 66 taxa (Singh, 2005; POWO, 2025; WFO, 2025). It is estimated that 17 taxa are endemic to India, and their distribution is mostly concentrated in the peninsular region, North-Eastern region and Andaman and Nicobar Islands (Gamble, 1922; Haines, 1922; Singh, 2005; POWO, 2025; WFO, 2025). Members of this genus are taxonomically characterised by tree or shrub, dioecious, evergreen or semi-evergreen; simple, alternate, coriaceous leaves; axillary cyme or pistillate flower solitary; fleshy with persistent calyx; 1-16 seeded fruit (Singh, 2005; Abd El Halim et al., 2014; Schatz and Lowry, 2018; Samuel et al., 2019). In addition to the taxonomic diversity, the spe- cies of Diospyros are also recognised for their economic value, providing high-quality timber, medicinal compounds and edible fruits (Nematollahi et al., 2012; Butt et al., 2015; Ullah et al., 2015; Ribeiro et al., 2023). The state of Odisha, located on the eastern coast of India, is home to a rich diversity of flora, including several species of Diospyros L. The varied climatic conditions and ecosystems ranging from coastal plains to dense tropical evergreen forests support this diversity. The work carried out by Saxena and Brahmam (1995) was the last effort in 77 Acta Biologica Slovenica, 2025, 68 (4) the field of conventional taxonomy on the genus Diospyros from Odisha. Subsequently, significant contributions to the taxonomy of the genus have been made, such as the mono- graph of Indian Diospyros (Singh, 2005); the work of Schatz and Lowry (2020) for the Malagasy region; Rindyastuti et al. (2021) for Indonesian Diospyros L. These studies have advanced our understanding of the genus through detailed evaluation of morphological traits, including leaves, flowers, and fruits. However, morphological inconsistencies, particu- larly in vegetative and floral characters, continue to compli- cate species identification. Consequently, fruit morphology has emerged as a more consistent and reliable diagnostic feature for distinguishing among Diospyros species (Schatz and Lowry, 2018; Sandratriniaina et al., 2023). Moreover, seed morphology has been investigated in terms of shape, size, germination, and the results showed that seed char- acteristics clearly have taxonomic relevance (Ahmed and Qaiser, 1989; Corral et al., 1989; Karaismailoglu and Erol, 2018; Abdel et al., 2021; Ozbek and Uzunhisarcıklı, 2023). The numerical taxonomy employs statistical and computational techniques to analyse morphological data, providing an objective framework for species delimitation (Sneath, 2001). By utilising multivariate analysis, this approach facilitates the reconstruction of more precise phylogenetic relationships, offering clear insights into evo- lutionary connections. In the case of Diospyros, numerical taxonomy can significantly contribute to identifying key morphological characteristics that are vital for distinguish- ing species and resolving confusion in species groups. Accordingly, this study was carried out to improve the accuracy in species identification based on comparative key characters and statistical measures of fruit and seed morphology. The present study aimed to a) analyse the fruit and seed morphological characteristics of Diospyros L. taxa found in Odisha; b) determine the closest relationship among the studied taxa, and c) find out the key characters used for delimitation of Diospyros species complex. Materials and Methods Study area The present study was conducted in Odisha, located on the eastern coast of India (17°49' to 22°34' N, 81°27' to 87°29' E) (Figure 1). Odisha accounts for 4.87% (1,55,707 km²) of India's total geographical area. The state features four distinct physiographic regions: the Northern Plateau, Eastern Ghats, Central Table Land, and Coastal Plains (Government of Odisha, 2025). Nearly one-third of Odisha is forested, covering 37.34% of the state's total land area. The forest cover extends over 58,136.869 km² (ISFR, 2023). Odisha experiences a tropical monsoon climate, with hot summers and substantial monsoonal rainfall. The average annual temperature ranges between 25°C and 28°C, with rainfall varying from 1,200 mm to 1,600 mm. Soil types include mixed red and black soils in the Central Table Land, and red, yellow, and fertile alluvial soils in the Coastal Plains. These climatic and soil variations support diverse vegetation, including tropical dry deciduous, mixed, moist deciduous, and semi-evergreen forests (Champion and Seth, 1968; Saxena and Brahmam, 1994). Field survey and Specimen collection Several field tours were carried out across different regions of the state during the period from January 2022 to Sep- tember 2024. Fresh plant samples were collected for all ten Diospyros species from 13 distinct localities across the state of Odisha. For each species, a minimum of 5 to 10 individuals were sampled per locality. The location coordinates of each specimen were recorded using a handheld Global Positioning System (GPS) with World Geodetic System 1984 (a geographic coordinate system) (Table 1). The preliminary identification was made through detailed observation of the collected specimen, along with a survey of the regional and national herbaria (CSIR-IMMT, Bhubaneswar; Central National Herbarium, Howrah (CAL), regional floras (Saxena and Brahmam, 1995; Haines, 1922), monograph and available literature. Voucher specimens for all collected taxa were prepared following the standard protocol (Kottapalli et al., 2016) and deposited in the Herbarium at the Department of Botany, MSCB University, Baripada (MSCBUB). These vouchers are available for future reference and taxonomic validation. During field surveys, the habitat characteristics of each Diospyros species were recorded in situ at the point of specimen collection. Data included associated vegetation and forest types (i.e., moist deciduous, dry deciduous or coastal forest) following Champion and Seth's (1968) forest type classification. Further, the information regarding local names, growth form, fruiting phenology, and local uses of Diospyros species was obtained through informal inter- views with local communities residing near the sampling 78 Acta Biologica Slovenica, 2025, 68 (4) sites. A total of 25 informants (aged between 30 and 70 years), including forest-dependent villagers and traditional knowledge holders, were interviewed using a purposive sampling approach. The key questions included: (i) What is the local name of this tree? (ii) Is the tree known for any particular use (e.g., fruit, timber, medicine)? (iii) During which months does it bear fruit? and (iv) What is its local significance or perceived abundance? Responses were documented through note-taking during direct conver- sations in the Odia and Santali languages. Additionally, the threat status and distribution data were presented by referring to the IUCN Red List data and Plants of the World Online (POWO, 2025), respectively. Morphological analysis Morphological characteristics of fruits and seeds were ana- lysed and described according to the Kew Plant Glossary (Beentje, 2016). The key characters that were examined include infructescence pedicel; shape, colour, surface of fruit; calyx format, number of calyx-lobe, calyx-lobe format, margin, aestivation of fruiting calyx; quantity, format, colour of seeds and endosperm (Table 2). Endosperm type: "Rumi- nate" indicates irregular intrusions of the seed coat into the endosperm, while "uniform" indicates a uniform and uninterrupted endosperm. Colour of fruits and seeds was determined visually in daylight and compared against the Royal Horticultural Society (RHS) Colour Chart to ensure consistency and reduce subjectivity. Morphometric analysis For morphometric analysis, fruit length, fruit diameter, length and width of fruiting calyx, seed length and width were measured using a digital calliper. Fruit length was measured from the base of the fruit to the apex, excluding the calyx; fruit diameter was taken at the widest cross-sec- tion. Calyx length and width were measured from the base to the tip of the lobes and across the widest span of the calyx, respectively. Seed length and width were measured along the longest and widest axes, respectively. All charac- ters were measured on mature fruits and were expressed as the mean of 10 measurements. "Thin" pericarp refers to a thickness of <2 mm; "Thick" is >2 mm and rigid. Addition- ally, the number of seeds per fruit and the number of calyx lobes were documented through direct observation. Figure 1. Map of the study area with collection localities. (Source: Map generated using QGIS v3.34). Slika 1. Zemljevid območja raziskave z lokacijami zbiranja. (Vir: zemljevid, izdelan z uporabo QGIS v3.34). 79 Acta Biologica Slovenica, 2025, 68 (4) Statistical analysis The phenetic relationship and the key characters for morphological variation in Diospyros were investigated using two statistical analyses. Firstly, the Cluster Analysis (CA) was conducted using the Unweighted Pair Group Method (UPGMA) to establish the phenetic relationship that explains the closest relationship among the studied Diospyros species. Secondly, principal components analy- sis (PCA) was performed to identify the effects of selected characters on the delimitation of the Diospyros species complex. In total, 20 taxonomically important characters with 34 morphological attributes and eight morphometric attributes were selected and codified numerically (Table 2). The cluster analysis was performed using statistical soft- ware PAST (Paleontological Statistics) ver. 4.03. whereas PCA was plotted by ggbiplot in the R environment. Results Species Diversity The present study documented a total of ten wild-growing taxa belonging to the genus Diospyros L. in Odisha, East- ern India (Table 3). This diversity encompasses a range of trees and shrubs, and was found to be adopted in different habitats. The species D. melanoxylon Roxb. was a notable species within the studied taxa, which primarily thrives in mixed deciduous forests. Likewise, the species D. ebenum Koenig., D. montana Roxb. and D. sylvatica Roxb. were found to share the same habitat across the moist deciduous forest type of Odisha. The species D. malabarica (Desr.) Kostel is typically distributed near streams, where it coexists with other tree species. In contrast, the two representative shrub species were D. ferrea (Willd.) Bakh. and D. chloroxylon Species Name Coordinates Locality Accession No. D. candolleana Wight 18°57′51.92"N 84°22′22.41"E Mahendragiri, Gajapati MSCBUB/01510 D. chloroxylon Roxb. 20°18'00.08"N 85°43'59.08" E Chandaka Wildlife Sanctuary MSCBUB/01511 20°03'34.66"N 85°12'43.77"E Beguniapatana, Nayagarh MSCBUB/01512 D. ebenum Koenig. 20°36'13.96"N 85°01'43.95"E Satkosia, Angul MSCBUB/01513 19°57'12.98"N 85°07'29.72"E Similijhar, Nayagarh MSCBUB/01514 D. exculpta Buch. -Hum. 19°00′58.94"N 84°14′47.52"E Raikaguma, Gajapati – D. ferrea (Willd.) Bakh. 20°18'00.92"N 85°43'00.46" E Chandaka Wildlife Sanctuary MSCBUB/01515 19°57'45.00"N 86°15'39.71"E Astaranga, Puri MSCBUB/01516 D. malabarica (Desr.) Kostel. 21°17'24.03"N 86°14'39.91"E Pitanau, Keonjhar MSCBUB/01517 21°55'33.78"N 86°27'15.97"E Similipal BR, Mayurbhanj – 20°36'02.26"N 85°01'40.92"E Satkosia WS, Angul MSCBUB/01518 D. melanoxylon Roxb. 20°35'20.04"N 84°46'48.27"E Padmadala, Angul – 22°15'41.04"N 86°26'43.33"E Argarghati, Mayurbhanj MSCBUB/01519 21°50'15.05"N 86°35'08.35"E Dukura, Mayurbhanj MSCBUB/01520 20°16'17.90"N 85°09'44.42"E Khandapada, Nayagarh – D. montana Roxb. 21°52'13.35"N 86°30'33.86"E Similipal BR, Mayurbhanj MSCBUB/01521 22°15'55.10"N 86°26'30.36"E Argarghati, Mayurbhanj – 21°50'52.39" N 86°33'30.34"E Dukura, Mayurbhanj MSCBUB/01522 19°57'26.43"N 85°07'27.19"E Similijhar, Nayagarh – D. ovalifolia Wight. 19°57'11.25"N 85°07'29.05"E Simili jhar, Nayagarh MSCBUB/01523 D. sylvatica Roxb. 21°55'55.95"N 86°32'25.63"E Similipal BR, Mayurbhanj MSCBUB/01524 22°15'55.23"N 86°26'21.86"E Argarghati, Mayurbhanj – 21°51'09.25"N 86°32'56.80"E Dukura, Mayurbhanj – 19°57'38.10"N 85°07'32.88"E Similijhar, Nayagarh MSCBUB/01525 Table 1. Details of coordinates (World Geodetic System 1984), specimen collection locality, and accession number were deposited for Diospyros L. Tabela 1. Podrobnosti o koordinatah (Svetovni geodetski sistem 1984), kraju zbiranja vzorcev in številki dostopa so bile deponirane za Diospyros L. 80 Acta Biologica Slovenica, 2025, 68 (4) Roxb., mostly found in coastal forest areas. The diversity of the remaining three species, such as D. candolleana Wight., D. exculpta Buch. -Hum. and D. ovalifolia Wight, was found to be confined within a small geographical area (Figure 2). Phenology and IUCN status The baseline information on local names, growth form, phe- nology, threat status, local importance, and distribution of the studied taxa is presented in Table 3. Regarding phenological events, the present study documented the fruiting period for all taxa. The fruits of the Diospyros species developed after flowering and ripened at varying times based on species and local climate. However, the study observed that peak fruiting maturity for most species occurred between April and May. In case of the two shrub species, D. chloroxylon Roxb. and D. ferrea (Willd.) Bakh., the fruiting period peaked from September to October. Additionally, seed germination begins between June and July, following fruit maturation. As per the distribution, the collected Diospyros taxa were mostly found to be distributed in India and Sri Lanka. Nota- bly, D. candolleana and D. exculpta are endemic to India. Concerning the threat status, D. candolleana has been cat- egorised as Vulnerable (VU) based on criteria A2cd of the IUCN Red List. In contrast, the IUCN status of the remaining species indicated that D. chloroxylon, D. exculpta, D. ferrea, D. malabarica, D. melanoxylon, D. montana, D. ovalifolia, and D. sylvatica were listed as Not Evaluated (NE), while D. ebenum was categorised as Data Deficient (DD). Economic Importance The documentation on local importance indicated that Diospyros species were traditionally valued for their edible fruit, timber, medicinal properties, and fodder. Timber from species such as D. candolleana, D. malabarica, and the well-known ebony D. ebenum was employed in building materials and artisanal craftsmanship. Certain species, including D. malabarica, D. melanoxylon and D. montana, were recognised for their medicinal properties used for treating stomach ailments. The foliage of D. melanoxylon, known as bidi leaf (used for smoking purposes), contrib- Characters Structure analysed States/measurements Morphological attributes Fruit Pedicel Sessile (1), subsessile (2) shape Globose (1), ovoid (2) colour Pale green (1), olive red (2), yellow (3), red (4), black (5), orange (6), salmon (7) surface glabrous (1), pubescent (2) Fruiting calyx calyx format Flat (1), campanulate (2), bowl-shaped (3) calyx lobe format Triangular (1), rounded (2), ovate (3) margin Entire (1), rolled (2), wavy (3) aestivation Valvate (1), contorted (2) texture of calyx Thin leafy (1), thick leafy (2), woody (3) Seed seed format Oblong (1), rounded (2), elliptic (3) colour Brown (1), black (2) endosperm Ruminate (1), uniform (2) Morphometric attributes Fruit length cm diameter cm Fruiting calyx no. of calyx lobe 3 to 5 length cm diameter cm Seed seed quantity 1 to 8 length cm diameter cm Table 2. List of morphological and morphometric attributes selected for the present study. Tabela 2. Seznam morfoloških in morfometričnih lastnosti, izbranih za to študijo. 81 Acta Biologica Slovenica, 2025, 68 (4) uted to local livelihoods through its use in bidi production. Additionally, species like D. ferrea and D. ovalifolia were utilised as fodder for livestock, underscoring their signifi- cance in local agriculture. Morphological characterisation The present morphological analysis showed remarkable differences in fruit, fruiting calyx and seeds (Table 4). All the fruits were fleshy and indehiscent. There was also a variation observed in fruit shape that varies from globose to ovoid. The pre-mature fruits were green, but a gradual variation was found with yellow, orange, red, olive red, black and salmon colour. The fruit surface was mostly glabrous in the young and mature states of all taxa. However, the fruit surface of D. melanoxylon and D. ovalifolia was pubescent in young fruit. The analyses of fruiting calyx showed significant taxonomical differences in format, aestivation and texture of the calyx lobe. As per the observation, four major types of calyx format were observed: flat, campanulate, flattened triangularly, and bowl-shaped, among the ten species. Of these, about seven species showed flat calyx format, while D. malabarica was flattened triangularly. Besides, D. chloroxylon and D. ferrea showed campanulate-type calyx format, and D. ebenum showed bowl-shaped calyx format. Ovate, rounded, triangular calyx lobes were observed in these species, with the exception of D. malabarica, which showed an ovate-triangular calyx lobe. Among the seed characters, endosperm showed poten- tial distinguishing tools for the delimitation of taxa. The majority of the species had uniform endosperm, while the four species, i.e., D. candolleana, D. exculpta, D. melanoxy- lon and D. sylvatica, were characterised by ruminate endo- sperm. The observed seed format was rounded, elliptic and oblong. Among the ten species, nine had brown-coloured seeds, except D. montana, which had black-coloured seeds. Morphometric analysis The fruit, calyx and seeds of ten wild-growing Diospyros taxa were critically examined for the analysis of the morphomet- ric variations. The analysis included length and diameter of fruit, number of calyx lobes, length and width of calyx lobes, seed quantity, seed length, and seed width, which were represented in Table 5. The study came up with a variation in fruit size of 2.46±1.57 × 1.95±1.41 cm (length × diameter), where D. ferrea (0.98±0.04 cm) was the smallest fruit and D. malabarica (6.06±0.08 cm) was found to be the largest one. A significant difference in seed quantity per fruit was observed within the individual species of the studied taxa. The seed number varies from 1 (D. ferrea; D. ovalifolia) to 8 (D. malabarica), and the remaining species have an average of 4 seeds within the taxa. A negligible variation was found in the number of calyx lobes per fruit within the same taxa. However, the species like D. melanoxylon and D. sylvatica exhibit minimum variation, where the number of calyx lobes ranges from 5 to 6 and 3 to 4, respectively. Phenetic relationship The dendrogram, generated by cluster analysis of mor- phological characters, clustered the studied taxa into three groups (Figure 3). The first group comprises three taxa, namely D. ovalifolia, D. chloroxylon and D. ferrea, at 0.3 taxonomic distance. In the second group, D. exsculpta, D. candolleana and D. melanoxylon were clustered at 0.2 tax- onomic distance. In the last group, clustering of D. ebenum, D. malabarica, D. montana and D. sylvatica was found at about 0.29 taxonomic distance. This dendrogram exhibited a cophenetic correlation coefficient (r) of 0.89, which was considered a good fit for the observed data. Principal component analysis (PCA) The plotted result of Principal component analysis shows the key diagnostic traits and patterns of variation that assisted in distinguishing among the species of Diospyros (Figure 4). The analysis resulted in the separation of variables along PC1 and PC2, explained with a variance of 38% and 29%, respectively. The PC1 was found to have a weighted contrast between the length of the calyx lobe and the seed width. Both the characters exhibited higher influence in variation with a positive coefficient (loading coefficient=0.37). On the other hand, the number of calyx lobes, texture of calyx lobes, endosperm, calyx lobe margin and fruit surface are found to show inverse relationships with principal compo- nents. The second Principal component was dominated by calyx lobe format, fruit shape, aestivation of calyx and seed colour, with the highest positive loading. Moreover, there was an overlapping variable, i.e., fruit diameter and length of seed, which shows more similarity among the original variables. The ellipses in the PCA biplot represented two distinct clusters of species, providing independent support for morphological groupings based on trait loadings. 82 Acta Biologica Slovenica, 2025, 68 (4) Figure 2. Field photographs of Diospyros species in the fruiting stage: a) D. mala- barica, b) D. montana, c) D. ebenum, d) D. chloroxylon, e) D. ovalifolia, f) D. ferrea, g) D. sylvatica, h) D. melanoxylon, and i) D. candolleana. Slika 2. Terenske fotografije vrst Diospyros v plodnem obdobju: a) D. malabarica, b) D. montana, c) D. ebenum, d) D. chloroxylon, e) D. ovalifolia, f) D. ferrea, g) D. sylvatica, h) D. Melanoxylon in i) D. candolleana. Species Name Local name Growth form Fruiting Phenology Threat Status Local importance Distribution D. candolleana Wight. Kendu Tree March-June VU Timber, fodder India D. chloroxylon Roxb. Kosai kendu Shrub Oct.-Dec. NE Edible fruit India, Bangladesh D. ebenum Koenig. Kendu Tree June DD Timber India, Sri Lanka D. exculpta Buch. -Hum. Kendu Tree Oct.-Dec. NE Timber India D. ferrea (Willd.) Bakh. Guakoli Shrub Sep. NE Edible fruit, fodder India, Sri Lanka, Myanmar, Malaysia, Indonesia, Australia, Africa D. malabarica (Desr.) Kostel. Mankada kendu Tree June NE Edible fruit, timber, medicine Indo-Malesia D. melanoxylon Roxb. Kendu Tree May NE Edible fruit, foliage, timber, medicine India, Sri Lanka D. montana Roxb. Halada Tree Apr. NE Fish poisoning, medicine Indo-Malesia D. ovalifolia Wight. Kendu Small tree Apr. NE Fodder India, Sri Lanka, Bangladesh D. sylvatica Roxb. Kalucha Tree March NE Edible fruit, timber, medicine India, Sri Lanka Table 3. Details on the baseline information of Diospyros species in Odisha, Eastern India. Tabela 3. Podrobnosti o osnovnih podatkih vrst Diospyros v Odishi, vzhodna Indija. [Abbr.: VU- Vulnerable; DD- Data Deficient; NE- Not evaluated. Distribution data were compiled from Plants of the World Online (POWO, 2025), and threat status was referenced from the IUCN Red List (2024).] 83 Acta Biologica Slovenica, 2025, 68 (4) Fruit Species Name Pedicel Fruit colour (mature) Fruit shape Surface (young fruit) Surface (mature fruit) D. candolleana Wight. sessile pale yellow ovoid glabrous glabrous D. chloroxylon Roxb. sessile olive red globose glabrous glabrous D. ebenum Koenig. subsessile yellow ovoid glabrous glabrous D. exculpta Buch. -Hum. sessile yellow ovoid glabrous glabrous D. ferrea (Willd.) Bakh. sessile red globose glabrous glabrous D. malabarica (Desr.) Kostel. subsessile salmon ovoid Red scurf glabrous D. melanoxylon Roxb. sessile yellow ovoid pubescent glabrous D. montana Roxb. subsessile black globose glabrous glabrous D. ovalifolia Wight. subsessile orange globose pubescent glabrous D. sylvatica Roxb. subsessile black globose glabrous glabrous Fruiting calyx Calyx format Calyx lobe format Calyx lobe margin Aestivation Texture of calyx D. candolleana Wight. sessile pale yellow ovoid glabrous glabrous D. chloroxylon Roxb. sessile olive red globose glabrous glabrous D. ebenum Koenig. subsessile yellow ovoid glabrous glabrous D. exculpta Buch. -Hum. sessile yellow ovoid glabrous glabrous D. ferrea (Willd.) Bakh. sessile red globose glabrous glabrous D. malabarica (Desr.) Kostel. subsessile salmon ovoid Red scurf glabrous D. melanoxylon Roxb. sessile yellow ovoid pubescent glabrous D. montana Roxb. subsessile black globose glabrous glabrous D. ovalifolia Wight. subsessile orange globose pubescent glabrous D. sylvatica Roxb. subsessile black globose glabrous glabrous Seed (mature) Seed format Seed colour Endosperm D. candolleana Wight. oblong brown ruminate D. chloroxylon Roxb. rounded brown uniform D. ebenum Koenig. elliptic brown uniform D. exculpta Buch. -Hum. elliptic brown ruminate D. ferrea (Willd.) Bakh. rounded brown uniform D. malabarica (Desr.) Kostel. elliptic brown uniform D. melanoxylon Roxb. elliptic brown ruminate D. montana Roxb. elliptic black uniform D. ovalifolia Wight. rounded brown uniform D. sylvatica Roxb. oblong brown ruminate Table 4. Fruit morphological characters of Diospyros L. in Odisha, Eastern India. Tabela 4. Morfološke značilnosti plodov Diospyros L. v Odishi, vzhodna Indija. 84 Acta Biologica Slovenica, 2025, 68 (4) Species Name Fruit length (cm) Fruit diam. (cm) No. of calyx-lobe Length of calyx lobe (cm) Width of calyx-lobe (cm) Seed quantity Length of seed (cm) Width of seed (cm) D. candolleana 2.7±0.22 1.8±0.1 5 1.05±0.07 0.67±0.05 4 1.2±0.05 0.8±1.3 D. chloroxylon 1.03±0.05 0.94±0.12 4 0.36±0.05 0.47±0.05 3 0.7±0.05 0.5±0.1 D. ebenum 2.03±0.05 1.9±0.09 4 0.95±0.12 0.62±0.05 4 1.22±0.05 0.6±0.07 D. exculpta 3.5±0.23 2.5±0.57 5 2.45±0.07 0.8±0.09 5 1.07±0.09 0.72±0.05 D. ferrea 0.98±0.04 0.7±0.11 3 0.42±0.05 0.38±0.12 1–4 0.7±0.09 0.62±0.04 D. malabarica 6.06±0.08 5.5±0.4 4 3±0.07 2.5±0.57 8 2.5±0.08 1.2±0.05 D. melanoxylon 3.4±0.21 2.42±0.22 5 2.5±0.05 0.9±0.14 4–6 1.5±0.05 0.91±0.14 D. montana 2.4±0.06 1.99±0.09 4 1.5±0.08 0.87±0.13 6 1.06±0.08 0.6±0.09 D. ovalifolia 1.04±0.11 0.92±0.15 4 0.5±0.05 0.45±0.05 1–2 0.91±0.09 0.45±0.07 D. sylvatica 1.5±0.07 0.9±0.12 4 0.8±0.03 0.85±0.15 6 1±0.05 0.75±0.05 Table 5. Fruit morphometric characters of Diospyros L. in Odisha, Eastern India. Tabela 5. Morfometrične značilnosti plodov Diospyros L. v Odishi, vzhodna Indija. Figure 3. A dendrogram was produced through cluster analysis of the examined characters. Slika 3. Dendrogram je bil izdelan s pomočjo skupinske analize preučevanih značilnosti. 85 Acta Biologica Slovenica, 2025, 68 (4) Key to the species of the genus Diospyros L. Here, an identification key was prepared based on constant diagnostic characters of fruit and seed morphology for more natural and practical segregation of taxa. Further, a photo plate was also provided to support the morpho-taxo- nomical information on collected Diospyros species (Figure 5; Figure 6). 1. Fruiting calyx lobe 3 to 4 ……………………………………………... 2 1. Fruiting calyx lobe 5……………………..………………………………. 8 2. Calyx-lobe contorted …………….…….....………. D. chloroxylon 2. Calyx-lobe valvate …………….……………………………..…………… 3 3. Fruiting calyx bowl-shaped ……….………….……... D. ebenum 3. Fruiting calyx flat or campanulate …...…….…….……………... 4 4. Fruiting calyx woody ……….…….…………………... D. ovalifolia 4. Fruiting calyx thin to thick leafy …...……….…………………... 5 5. Endosperm ruminate ………………………….………... D. sylvatica 5. Endosperm uniform ……………….……………………………………... 6 6. Size of mature fruit up to 1 × 1 cm; calyx lobe format rounded ………..……...................................................… D. ferrea 6. Size of mature fruit more than 1 × 1 cm; calyx lobe format ovate-triangular ……...............................................…..........…... 7 7. Matured fruit black in colour …………….……….… D. montana 7. Matured fruit salmon in colour ….……………. D. malabarica 8. Young fruit surface glabrous; calyx lobe margin reflexed ………………….................................................................….………... 9 8. Young fruit surface pubescent; calyx lobe margin wavy ……………............................................................. D. melanoxylon 9. Seeds oblong, shining, margin obscure.............................. ………….………...…...............................…............... D. candolleana 9. Seeds elliptic, compressed, margin prominent .................. .………...….……….……...........................................…... D. exculpta Figure 4. Loading plot showing the contribution of fruit and seed mor- phological characters in the first two Principal components. The ellipses represent 95% confidence intervals for the individual groups of samples. [Calyx format (CF); Calyx lobe format (CLF); Calyx lobe margin (CLM); Texture of calyx (TC); Fruit shape (FS); Fruit Surface (SY); Seed colour (SC); Seed format (SF); Fruit diameter (FD); Number of calyx lobe (NC); length of calyx lobe (LC); width of calyx lobe (WC); seed quantity (SQ); seed length (LS) and seed width (WS)]. Slika 4. Diagram prikazuje prispe- vek morfoloških značilnosti plodov in semen v prvih dveh glavnih komponentah. Elipse predstavljajo 95-odstotne interval zaupanja za posamezne skupine vzorcev. [Oblika keliha (CF); Oblika kelihovega lista (CLF); Rob kelihovega lista (CLM); Tek- stura keliha (TC); Oblika ploda (FS); Površina ploda (SY); Barva semena (SC); Oblika semena (SF); Premer ploda (FD); Število cvetnih listov (NC); Dolžina cvetnega lista (LC); Širina cvetnega lista (WC); Količina semen (SQ); Dolžina semena (LS) in širina semena (WS)] 86 Acta Biologica Slovenica, 2025, 68 (4) Figure 5. Comparative fruit morphology of Diospyros L. species: a) D. malabarica, b) D. montana, c) D. ebenum, d) D. sylvatica, e) D. chloroxylon, f) D. ovalifolia, g) D. ferrea, h) D. exculpta, i) D. melanoxylon, and j) D. candolleana. All fruits were photographed at the premature stage, preserving the diagnostic characters used for species identification. Slika 5. Primerjalna morfologija plodov vrst Diospyros: a) D. malabarica, b) D. montana, c) D. ebenum, d) D. sylvatica, e) D. chloroxylon, f) D. ovalifolia, g) D. ferrea, h) D. exculpta, i) D. melanoxylon, in j) D. candolleana. Vsi plodovi so bili fotogra- firani v nezrelem stanju, da so ohranili diagnostične značilnosti, ki se uporabljajo za identifikacijo vrst. Figure 6. Comparative seed morphology of Diospyros L. species: a) D. malabarica, b) D. montana, c) D. ebenum, d) D. sylvat- ica, e) D. chloroxylon, f) D. ovalifolia, g) D. ferrea, h) D. exculpta, i) D. melanoxylon, and j) D. candolleana. Seed images were captured from mature fruits, except D. candolleana, which was photographed at the immature stage. Slika 6. Primerjalna morfologija semen vrst Diospyros L.: a) D. malabarica, b) D. montana, c) D. ebenum, d) D. sylvatica, e) D. chloroxylon, f) D. ovalifolia, g) D. ferrea, h) D. exculpta, i) D. melanoxylon, in j) D. candolleana. Slike semen so bile posnete z zrelih plodov, razen D. candolleana, ki je bila fotografirana v nezrelem stanju. 87 Acta Biologica Slovenica, 2025, 68 (4) Discussion The genus Diospyros L. exhibits considerable species richness in India, with most taxa having highly localised distributions and minimal geographic overlap (Bakhuizen van den Brink, 1933; Singh, 2005). The present study documented the occurrence of ten wild-growing Diospy- ros taxa in Odisha, predominantly of Indian origin. Their occurrence patterns generally correspond with previous floristic accounts for the state (Gamble, 1922; Haines, 1922; Saxena and Brahmam, 1995; Singh, 2005), indicating broad stability in distribution over time. The majority of taxa are concentrated in the forested uplands of the Eastern Ghats, while a few, such as D. ferrea and D. chloroxylon, also occur in lowland coastal forests. Notably, this study documented new distributional localities for D. chloroxylon, D. ebenum, D. exculpta, and D. ovalifolia (Table 1), providing updated field confirmations that strengthen and extend earlier reports for the region. The surveyed Diospyros species in Odisha show diverse local uses, largely centred on timber, edible fruits, fodder, and medicinal applications. Timber-yielding taxa such as D. candolleana, D. ebenum, and D. malabarica are valued for their durability and fine grain, echoing earlier reports from southern and central India (Gamble, 1881; Troup, 1986). Edible fruits from species like D. chloroxylon, D. ferrea, D. melanoxylon and D. sylvatica are consumed seasonally and occasionally sold in local markets, a pattern also noted by Mahapatra and Panda (2009). Certain spe- cies, including D. montana and D. malabarica, are employed in traditional medicine (Ahmed, 1993; Joshi, 1993; Singh, 1993; Jayaraman, 1996; Achiwa et al., 1997; Prajapati et al., 2003), while D. montana is additionally used as a fish poison, a practice recorded in parts of the Western Ghats (Narayanan et al., 2011). In addition to these known uses, this survey recorded two novel observations. Ripe fruits of D. candolleana were observed being consumed by the giant squirrel (Ratufa indica) and bonnet macaque (Macaca radiata), suggesting a potential role for these mammals in seed dispersal. Furthermore, D. montana was cited in local ethnomedicinal practice as a treatment for gastric ulcers, a use not previously reported in the literature. Although the uses recorded are generally consistent with regional ethnobotanical patterns, the present survey expands both the geographic and functional scope of existing records and confirms the persistence of traditional knowledge in rural communities. Such information, when integrated with conservation assessments, may help prioritise species that are both ecologically significant and culturally valued. Previous taxonomic studies of Diospyros have largely emphasised vegetative and floral traits for species iden- tification (Putri and Chikmawati, 2015; Schatz and Lowry, 2020; Gnonlonfin et al., 2022; Meeprom et al., 2024). In contrast, the present work demonstrates that fruit and seed morphology can serve as a reliable and distinct diag- nostic tool for species delimitation, offering advantages where vegetative or floral material is unavailable. Similar approaches have been successfully applied in other plant families, including Araceae (Camelo et al., 2023), Apocyna- ceae (Sarikaya and Guven, 2024), Rosa L. (Zarkovic et al., 2024), and Cistus L. (Gokmen et al., 2024). The addition of morphometric analysis further strengthened the dis- criminatory power of fruit and seed traits, as also reported in other taxa (Sonibare et al., 2004; Abdulrahaman and Oladele, 2005; Nandini et al., 2015). A notable contribution of this study is the resolution of morphological overlap between D. montana and D. sylvatica. While both species share similarities in fruit and calyx form, morphometric evaluation revealed significant differences in fruit length, fruit diameter, and calyx lobe length, providing key traits for accurate identification. However, seed size was found to be less diagnostic. This clarification addresses an area of uncertainty in the regional taxonomy of the genus. The clustering analysis revealed phenetic groupings that in many cases align with known morphological or phyloge- netic relationships within Diospyros. The close association between D. melanoxylon, D. exculpta and D. candolleana was found to be consistent with shared fruit and calyx traits previously noted in regional floras (Saxena and Brahmam, 1995), and may reflect a relatively recent divergence within Indian lineages. Similarly, the grouping of D. ebenum, D. malabarica, D. montana, and D. sylvatica parallels patterns observed in molecular phylogenetic studies from South- east Asia, where these taxa cluster within the same clade despite ecological differences (Duangjai et al., 2006). The separation of D. ferrea and D. chloroxylon from D. ovalifolia supports earlier morphological classifications based on fruit size and calyx structure (Singh, 2005), suggesting that these traits are evolutionarily conserved within certain subgroups. Moreover, the PCA shows the most significant char- acter that explains the total variation among the studied taxa. This analysis indicated that the length of the calyx, seed length, seed width, fruit diameter, and seed quantity are the most significant features with the highest relative 88 Acta Biologica Slovenica, 2025, 68 (4) variation rate, which can be useful in the delimitation of the Diospyros species complex. These traits likely hold strong taxonomic value because they are linked to reproductive structures, which tend to be more genetically constrained and less influenced by local environmental variation than vegetative traits (Sonibare et al., 2004). The present study confirms that fruit and seed morphology, when analysed qualitatively and quantitatively, offers a reliable and effec- tive tool for taxonomic delimitation (Camelo et al., 2023; Belo et al., 2024), and they provide a regional baseline for integrating morphometric data into broader systematic and phylogenetic frameworks. While these findings advance our understanding of Diospyros taxonomy in the region, the study acknowledges certain limitations. Phenetic groupings based solely on morphological data should be validated using molecular techniques such as DNA barcoding or phylogenetic analysis to strengthen species delimitation. Cytological studies, including chromosome counts and palynological analyses, could provide additional insights into evolution- ary relationships and genetic diversity. Integrating these complementary approaches would offer a more robust, multidimensional framework for resolving species bound- aries in Diospyros. Conclusion The present study provided more comprehensive diagnos- tic characters of fruits and seed morphology of Diospyros at the species level. The analysis of the shape, size, colour, and texture of the calyx lobe, fruit, and seed shows a clear understanding of the diversity of the Diospyros species of Odisha. Moreover, the result obtained from statistical mea- sures indicated that the length of the calyx lobe and size of the seed were significant in explaining the total variation among the studied taxa. The resulting phenetic clustering provided insights into species differentiation and their clos- est taxonomic relationship, which can aid in more accurate species delimitation. Although this study is important in understanding the diversity and relationships of species within Diospyros, there is growing recognition of the value of integrating diverse methods, including molecular and ecological studies. As such, further studies are necessary to resolve taxonomic issues within this genus. Besides, the study documented some taxa of Diospyros that had a high demand for edible fruit and timber. Conservation assessment of such valuable species should be of prime importance. Therefore, the baseline information on distri- bution, phenology, economic importance and taxonomic key will boost the conservation manager to take further steps towards their conservation and sustainable utilisation for the benefit of the local people. Author Contributions Conceptualization, S.C.S., S.S.D., and S.T.; methodology, S.C.S., S.S.D., S.T., and S.P.; software S.T., J.K.J.and S.P.; vali- dation, S.T., S.P., J.K.J., and S.C.S.; formal analysis, ST; inves- tigation, S.T., J.K.J., and S.P.; resources, S.C.S.; data cura- tion, S.T., J.K.J.and S.P.; writing—original draft preparation, S.T.; writing—review and editing, S.C.S., S.S.D., and J.K.J.; visualization, S.T. and S.C.S.; supervision, S.C.S. and S.S.D.; project administration, S.C.S.; funding acquisition, S.C.S.. All authors have read and agreed to the published version of the manuscript. Acknowledgements The authors acknowledge Ministry of Environment, Forest and Climate Change (MoEFandCC), Government of India for financial assistance (Sanction Order No. BSI- 298/01/29/2024/ABG-Tech/278/5778). Thanks are due to the PCCF (Wildlife) and Chief Wildlife Warden, Odisha for granting permission to carry out the study. We are also thankful the forest department staff for their cooperation during field work. Funding The study was supported by Ministry of Environment, Forest and Climate Change (MoEFandCC), Government of India under Sanction Order No. BSI-298/01/29/2024/ABG- Tech/278/5778. Conflicts of Interests The authors have no competing interests to declare that are relevant to the content of this study. 89 Acta Biologica Slovenica, 2025, 68 (4) References Abd El Halim, A.M., Hafeez, R.H., Safwat, A.A., 2014. Taxonomic Revision of Ebenaceae in Egypt. Current Science International, 3(4), 414–425. Abdel, K.K., Al-Ruzayza, S., Farid, A., 2021. Taxonomic significances of seed morphology in some tribes of subfamily Malvoideae (Malvaceae) in Saudi Arabia. Aust. J. Crop Sci., 15(8), 1204–1216. https://doi.org/10.21475/ ajcs.21.15.08p3360. Abdulrahaman, A.A., Oladele, F.A., 2005. Stamata, trichomes and epidermal cells as diagnostic features in six species of genus Ocimum L. (Lamiaceae). 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Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, 158(4), 823–835. http://doi.org/10.1080/11263504.2024.2370269 91 1 Cellular and Molecular Research Centre, Persian Gulf Physiology Research Centre, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 2 School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 3 Cellular and Molecular Research Centre, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. * Corresponding author: E-mail address: radan.maryam@yahoo.com Citation: Hoseininejad, K., Mahmoudi Nejad Mounfared, S., Nejad Dehbashi, F. , Radan, M., (2025). Carvacrol Enhances Cell Viability by Reducing Apoptosis in A549 Lung Epithelial Cells Exposed to Elastase. Acta Biologica Slovenica 68 (4) Received: 13.05.2025 / Accepted: 16.07.2025 / Published: 01.10.2025 https://doi.org/10.14720/abs.68.4.22698 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Carvacrol Enhances Cell Viability by Reducing Apoptosis in A549 Lung Epithelial Cells Exposed to Elastase Khojasteh Hoseininejad 1 , Sabah Mahmoudi Nejad Mounfared 2 , Fereshteh Nejad Dehbashi 3 , Maryam Radan 1, * Abstract Chronic obstructive pulmonary disease (COPD) represents a significant global health issue characterised by mechanisms such as inflammation, apoptosis, and oxidative stress. Carvacrol, a phenolic compound known for its antioxidant and an- ti-inflammatory effects, may mitigate damage caused by oxidative stress and apop- tosis. This research aimed to explore the protective effects of carvacrol against elastase-induced apoptosis and oxidative stress in A549 lung epithelial cells. A549 cells were cultured and treated with elastase (60 mU/ml) along with varying con- centrations of carvacrol (0.1, 0.2, and 0.4 mmol/L) for 24 hours. Cytotoxicity was evaluated using the MTT assay. Malondialdehyde (MDA) levels were measured as a marker of oxidative stress, while total antioxidant capacity (TAC) and the expression of apoptotic genes BAX and BCL2 were assessed via RT-PCR. Both H₂O₂ and elas- tase significantly reduced cell viability, increased MDA levels and BAX expression, and decreased TAC and BCL2 expression. Treatment with carvacrol in combination with elastase led to a dose-dependent increase in cell viability, a reduction in MDA levels and BAX expression, and an enhancement in TAC and BCL2 expression, with the strongest protective effect observed at 0.4 mmol/L. Pearson's correlation analysis revealed a positive correlation between TNF-α and BAX expression (r = 0.43, P = 0.002), a negative correlation between TNF-α and BCL2 expression (r = -0.62, P = 0.0031), and a negative correlation between BAX and BCL2 expres- sion (r = -0.56, P = 0.007). Carvacrol demonstrated significant protective effects on pulmonary epithelial cells exposed to elastase-induced oxidative stress, reducing apoptosis and enhancing antioxidant capacity. While our in vitro results suggest potential mechanisms that could be targeted in COPD, we acknowledge that these findings are preliminary. Further studies involving in vivo models and clinical trials are essential to validate the therapeutic relevance of these observations. Keywords A549 Cell, Apoptosis, Carvacrol, Elastase, Oxidative Stress 92 Acta Biologica Slovenica, 2025, 68 (4) Karvakrol izboljša celično preživetje z zmanjšanjem apoptoze v pljučnih epitelnih celicah A549, izpostavljenih elastazi Izvleček Kronična obstruktivna pljučna bolezen (KOPB) je pomemben globalni zdravstveni problem, za katerega so značilni mehanizmi, kot so vnetje, apoptoza in oksidativni stres. Karvakrol, fenolna spojina, znana po svojih antioksidativnih in protivnetnih učinkih, lahko ublaži poškodbe, povzročene z oksidativnim stresom in apoptozo. Namen te raziskave je bil proučiti zaščitne učinke karvakrola proti apoptozi, povzročeni z elastazo, in oksidativnemu stresu v pljučnih epitelnih celicah A549. Celice A549 so bile gojene in obdelane z elastazo (60 mU/ml) skupaj z različnimi koncentracijami karvakrola (0,1, 0,2 in 0,4 mmol/L) v 24 urah. Citotoksičnost je bila ocenjena z testom MTT. Ravni malondialdehida (MDA) so bile izmerjene kot marker oksidativnega stresa, medtem ko so bila skupna antioksidativna sposobnost (TAC) in izražanje apoptotičnih genov BAX in BCL2 ocenjena z RT-PCR. Tako H₂O₂, kot elastaza sta znatno zmanjšala celično vitalnost, povečala ravni MDA in izražanje BAX ter zmanjšala TAC in izražanje BCL2. Zdravljenje s karvakrolom v kombinaciji z elastazo je povzročilo odvisno od odmerka povečanje celične vitalnosti, zmanjšanje ravni MDA in izražanja BAX ter povečanje TAC in izražanja BCL2, pri čemer je bil najmočnejši zaščitni učinek opazen pri 0,4 mmol/L. Pearsonova korelacijska analiza je pokazala pozitivno korelacijo med TNF-α in izražanjem BAX (r = 0,43, P = 0,002), negativno korelacijo med TNF-α in izražanjem BCL2 (r = -0,62, P = 0,0031) in negativno korelacijo med BAX in ekspresijo BCL2 (r = -0,56, P = 0,007). Karvakrol je pokazal pomembne zaščitne učinke na pljučne epitelne celice, izpostavljene oksidativnemu stresu, povzročenemu z elastazo, saj je zmanjšal apoptozo in povečal antioksidativno sposobnost. Čeprav naši rezultati in vitro kažejo na možne mehanizme, na katere bi se lahko osredotočili pri KOPB, se zavedamo, da so ti rezultati le predhodni. Za potrditev terapevtske pomembnosti teh ugotovitev so nujne nadaljnje študije z in vivo modeli in kliničnimi študijami. Ključne besede A549 celica, apoptoza, karvakrol, elastaza, oksidativni stres Introduction Chronic obstructive pulmonary diseases (COPDs) encom- pass conditions such as emphysema, characterised by the destruction and enlargement of alveoli; chronic bronchitis, marked by persistent cough and increased mucus pro- duction; and small airway disease, involving heightened resistance in the smaller bronchioles. The underlying mechanisms include inflammation, apoptosis, cellular dysfunction, extracellular matrix degradation, and oxidative stress. The lungs are continuously exposed to oxidising agents, and elevated oxidative stress is considered a major contributor to the pathogenesis of pulmonary disorders (Barnes, 2022). Elastase, a protease, disrupts the protease–antipro- tease balance and is widely used in animal models to induce COPD and emphysema. Studies have shown that elastase promotes apoptosis in pulmonary epithelial cells, increases mucin secretion, and elevates inflammatory mediators such as interleukin-8 (IL-8) in bronchial epithelial cells. These mediators, in turn, stimulate neutrophil recruit- ment, exacerbating inflammation (Voynow and Shinbashi, 2021; Chakraborty and Bhattacharyya, 2013). Elastase also enhances the expression of cathepsin B and matrix metalloproteinase-2 (MMP-2) in macrophages, contributing to tissue remodelling and impaired mucociliary clearance (Greenlee et al. 2007; McKelvey et al. 2021). Furthermore, elastase induces oxidative stress and free radical formation, which upregulate MUC5AC expression — a gene promoting excessive mucin secretion and airway obstruction (Shao and Nadel, 2005; Li and Y e, 2020). A free radical is defined as a molecule with an unpaired electron, making it highly reactive. These molecules initiate chain reactions by interacting with lipids, proteins, and DNA, amplifying oxidative damage. Antioxidants can interrupt this process by scavenging free radicals (Andrade et al. 2005). 93 Acta Biologica Slovenica, 2025, 68 (4) Among phytochemicals, polyphenols represent a major group found in plant-based foods and are known for their antioxidant properties. Diets rich in polyphenols have been associated with reduced oxidative stress and improved cellular defence mechanisms through modulation of intra- cellular signalling pathways (Rana et al. 2022). Carvacrol, a monoterpenoid phenol (C₁₀H₁₄O), has a boiling point of ~238 °C and a density of 0.976 g/cm³ at 20 °C and 0.975 g/cm³ at 25 °C. It is a small, lipophilic mol- ecule capable of crossing the blood–brain barrier. Both in vitro and in vivo studies have shown that carvacrol exhibits a broad range of pharmacological effects, including antioxi- dant, antibacterial, antifungal, anticancer, anti-inflammatory, hepatoprotective, spasmolytic, and vasodilatory activities (Naghdi Badi et al. 2017; Suntres et al. 2015). Carvacrol neutralises various reactive species such as peroxyl, superoxide, hydrogen peroxide, and nitric oxide radicals—mainly due to its hydroxylated aromatic ring. Its mild acidity enhances reactivity with free radicals. Addi- tionally, Carvacrol has been shown to modulate immune responses and reduce inflammation, thereby attenuating cell death. Phenolic compounds like carvacrol may decrease pro-inflammatory cytokines (IL-4, TGF-β, IL-17) and increase anti-inflammatory cytokines such as IFN-γ and FOXP3 (Imran et al. 2022). Given elastase's established role in inducing oxidative damage in pulmonary epithelial cells and the limited research on carvacrol's protective effects in this context, the present study aims to evaluate the potential of car- vacrol in mitigating elastase-induced oxidative stress and apoptosis in human A549 lung epithelial cells. Material and Methods In this study, various parameters of pulmonary epithelial cells in cell culture medium were investigated using the A549 cell line. All experiments were performed in three independent biological replicates (n = 3) unless otherwise stated. Each biological replicate was conducted on separate days using independently cultured cells. Technical triplicates were also included for each condition in assays such as MTT, ELISA, and RT-PCR to ensure measurement accuracy. Grouping: 1. Negative control group cultured under standard con- ditions (37 °C, 5% CO₂, and humidified atmosphere) in RPMI-1640 medium without any treatment for 24 hours. 2. Positive control group receiving H 2 O 2 (a well-known oxi- dant) 100 μM for 24 hours (Vilema-Enriquez et al., 2016). 3. Group receiving elastase 60 mU/mL for 24 hours (Hou et al., 2014). 4. Group receiving elastase 60 mU/mL plus Carvacrol 0.1 mmol/L for 24 hours 5. Group receiving elastase 60 mU/mL plus Carvacrol 0.2 mmol/L for 24 hours 6. Group receiving elastase 60 mU/mL plus Carvacrol 0.4 mmol/L for 24 hours (Yin et al., 2012). Lung epithelial cell culture The A549 cell line, derived from lung epithelial cells, was obtained from the Pasteur Institute of Iran. These cells were cultured at a density of 1.06 × 10 6 cells in 100 mm dishes using RPMI-1640 medium supplemented with 10% FBS, with a total volume of 10 ml. The cultures were incu- bated at 37°C in a 5% CO2 environment, and the medium was refreshed every two days. Once the flasks reached 80-90% confluency, the cells were passaged. Following the establishment of the required number of flasks and cells, the experimental groups were organised, ensuring an equal distribution of cells across each group before initiating the treatment phase. After the designated study period, the cells were taken out of the incubator, and the necessary measurements were conducted in accordance with established protocols. Cell treatment Human neutrophil elastase (HNE) was obtained from Sigma-Aldrich (USA; Catalogue No. E8140). The enzyme had a specific activity of ≥4 units/mg protein, as provided by the manufacturer. The CAS number used in this study was 9004-06-2. A549 cells were treated with elastase at a concentration of 60 mU/mL for a duration of 24 hours, based on preliminary dose–response testing to determine sub-cytotoxic oxidative stress induction. Also, A549 cells were treated with Carvacrol (co-treatment) for 24 hours. Carvacrol was added at final concentrations of 0.1, 0.2, and 0.4 mmol/L concurrently with the oxidative agents (elastase or H₂O₂) directly into the culture medium. This co-treatment approach was used to evaluate the pro- tective effects of carvacrol against oxidative stress and apoptotic responses. 94 Acta Biologica Slovenica, 2025, 68 (4) Evaluation of cytotoxicity based on MTT analysis The MTT assay was employed to assess the metabolic activity of A549 cells following treatment with elastase and Carvacrol, serving as an indirect indicator of cell viability. The experiment consisted of three main phases: cell culture, treatment, and MTT analysis. After 24 hours of incubation, the medium was replaced with 200 μl of fresh medium con- taining MTT solution (0.5 mg/ml; Sigma-Aldrich, USA), and the plates were incubated at 37 °C in a 5% CO₂ atmosphere for 2–4 hours. During this period, mitochondrial succinate dehydrogenase in metabolically active cells reduced MTT to purple formazan crystals, which were visible under a microscope. Because these crystals are insoluble in aque- ous solution, they were dissolved using dimethyl sulfoxide (DMSO) prior to colourimetric measurement. Absorbance was read using a microplate reader, and metabolic activity was expressed as a percentage relative to the control group (untreated cells). A reduction in metabolic activity was interpreted as an indicator of compound-induced cyto- toxicity. Cells were treated with Carvacrol at concentrations of 0.1, 0.2, and 0.4 mmol/L for 24 hours at 37 °C. Measurement of Total Antioxidant Capacity (TAC) and Malondialdehyde (MDA) in Lung Epithelial Cells At the end of the treatment period, the culture superna- tants were collected and stored at −70 °C until analysis. TAC and MDA levels were measured using commercial assay kits (Kiazist, Iran) according to the manufacturer's instructions. T otal antioxidant capacity was assessed based on the ability of antioxidants in the sample to reduce ferric ions (Fe 3+ ) to ferrous ions (Fe 2+ ). The reaction was mea- sured colourimetrically at a wavelength of 593 nm. Trolox was used as the calibration standard, and results were expressed in Trolox equivalents (pg/mL). MDA, a marker of lipid peroxidation, was quantified using the thiobarbituric acid reactive substances (TBARS) method. Absorbance was recorded at 532 nm. The standard curve was pre- pared using 1,1,3,3-tetramethoxypropane (TMP), and MDA concentrations were reported in pg/mL. RT-PCR analysis for apoptosis gene expression (BAX- BCL2) in lung epithelial cells Total RNA was extracted from A549 cells using the RNA extraction kit and reverse-transcribed into cDNA using the cDNA synthesis kit, following the manufacturer's protocol. Quantitative real-time PCR was performed using SYBR Green Master Mix on a PCR system. Gene expression was normalised to GAPDH using the 2-ΔΔCt method. GAPDH was selected based on its stable expression across all treatment conditions. Reactions were performed in tripli- cate. Melt curve analysis was conducted to ensure primer specificity. Data Analysis The data obtained were analysed using SPSS version 22 software. Prior to analysis, the assumptions of normality and homogeneity of variances were assessed using the Shapiro-Wilk test and Levene's test, respectively. Data meeting these assumptions were subjected to one-way analysis of variance (ANOVA), followed by Tukey's post hoc test for multiple comparisons. The correlation between dif- ferent variables was calculated using Pearson's correlation coefficient. Statistical significance was set at p < 0.05. Gene Forward Primer (5′→3′) Reverse Primer (5′→3′) Annealing Temp (°C) Efficiency (%) BAX TTTGCTTCAGGGTTTCATCC CAGTTGAAGTTGCCGTCAGA 60 97.8 BCL2 GGTGAACTGGGGGAGGATTGT CAGGCTGGAAGGAGAAGATGC 60 98.2 TNF-α CCAGGGACCTCTCTCTAATC ATGGGCTACAGGCTTGTCACT 58 96.7 GAPDH GAAGGTGAAGGTCGGAGT GAAGATGGTGATGGGATTTC 58 98.5 Table 1. Primer sequences, annealing temperatures, and amplification efficiencies used for RT-PCR analysis. Tabela 1. Sekvence primerjev, temperature annealinga in učinkovitost amplifikacije, uporabljene za analizo RT-PCR. 95 Acta Biologica Slovenica, 2025, 68 (4) Results Carvacrol reduces oxidative stress marker levels in A549 cells exposed to elastase Table 1 presents the metabolic activity across various experimental groups, with the control group serving as the reference point at 100%. In the H₂O₂ group, a notable reduction in metabolic activity of 28.24% was recorded, highlighting the detrimental impact of oxidative stress induced by H₂O₂. Conversely, the elastase (ELS) group demonstrated partial mitigation of the harmful effects of H₂O₂, raising metabolic activity to 48.20%. Furthermore, in the groups receiving both ELS and Carvacrol (CR), a dose-dependent enhancement in metabolic activity was observed. Specifically, the combination of ELS and CR at a dose of 0.1 mmol/L resulted in metabolic activity increasing to 64.73%. As the CR dosage was escalated to 0.2 and 0.4 mmol/L, metabolic activity further improved to 72.99% and 78.29%, respectively. This pattern suggests that higher doses of CR confer a more pronounced protective effect (Figure 1). The optical density (OD) values from the MTT assay, measured spectrophotometrically, were used to calculate the percentage of metabolic activity relative to the control group. Carvacrol reduces oxidative stress marker levels in A549 cells exposed to elastase The concentration of malondialdehyde (MDA), a well-es- tablished marker of oxidative stress, was measured across experimental groups. The control group exhibited an MDA level of 3.10 pg/mL, reflecting normal cellular conditions. Exposure to H₂O₂ significantly increased MDA concen- tration to 8.16 pg/mL, indicating elevated oxidative stress. Treatment with elastase (ELS) reduced MDA levels to 6.23 pg/mL, suggesting partial mitigation of H₂O₂-induced oxidative damage (p < 0.01 compared to the H₂O₂ group). Moreover, co-treatment with ELS and Carvacrol (CR) further decreased MDA levels in a dose-dependent manner. Spe- cifically, at CR doses of 0.1, 0.2, and 0.4 mmol/L, MDA levels were reduced to 6.11, 5.36, and 4.17 pg/mL, respectively, Figure 1. Measurement and comparison of cell viability in all study groups, including control, H 2 O 2 , Elastase (ELS), Elastase plus 0.1 mmol/L Carvacrol, Elastase plus 0.2 mmol/L Carvacrol and Elastase plus 0.4 mmol/L Carvacrol. The y-axis represents cell viability (%). Data are mean ± SEM (n = 3). One-way ANOVA: F (5,12) = 41.65, P < 0.001. Tukey's post hoc test. **p < 0.01, ***p < 0.001 Vs control group and #p < 0.05, ##p < 0. 01 Vs ELS group. Slika 1. Merjenje in primerjava celične vitalnosti v vseh študijskih skupinah, vključno s kontrolno skupino, H 2 O 2 , elastazo (ELS), elastazo plus 0,1 mmol/L karvakrol, elastazo plus 0,2 mmol/L karvakrol in elastazo plus 0,4 mmol/L karvakrol. Os y predstavlja celično vitalnost (%). Podatki so povprečje ± SEM (n = 3). Enosmerna ANOVA: F (5,12) = 41,65, P < 0,001. Tukeyjev post hoc test. **p < 0,01, ***p < 0,001 v primerjavi s kon- trolno skupino in #p < 0,05, ##p < 0,01 v primerjavi s skupino ELS. 96 Acta Biologica Slovenica, 2025, 68 (4) demonstrating a progressive reduction in oxidative stress. These findings highlight the protective effect of Carvacrol against oxidative stress-induced lipid peroxidation in A549 cells (Figure 2). Carvacrol improves total antioxidant capacity in A549 cells exposed to elastase As demonstrated in Figure 3, the control group exhibited the highest TAC value at 10.28 pg/mL, reflecting a healthy and normal antioxidant status. In contrast, both the H 2 O 2 and ELS groups demonstrated a marked reduction in TAC levels, with the H 2 O 2 group measuring 4.96 pg/mL and the ELS group at 5.19 pg/mL, both significantly lower than the control group, highlighting the detrimental impact of oxidative stress on total antioxidant capacity. Conversely, in groups receiving a combination of ELS and CR at varying doses, there was a progressive increase in TAC levels. The ELS+CR 0.1 group recorded a TAC value of 5.71 pg/mL, indi- cating a relative enhancement compared to the H 2 O 2 and ELS groups. The TAC level further rose to 6.24 pg/mL in the ELS+CR 0.2 group, marked by a significant difference from other groups. Ultimately, the ELS+CR 0.4 group achieved the highest TAC value of 7.81 pg/mL, representing the most substantial improvement among the treatment groups. Carvacrol reduces BAX gene expression in A549 cells exposed to elastase. Figure 4 illustrates the expression level of the BAX (pro-apoptosis) gene across different groups. The control group shows the lowest BAX gene expression, indicating a normal physiological state. The H₂O₂ group demonstrates a significant increase, attributed to the induc- tion of oxidative stress and apoptosis. Similarly, the ELS group shows a significant increase in BAX expression. In the ELS+CR groups with different doses, a gradual reduction in BAX gene expression is observed. The ELS+CR 0.1 group shows a relative decrease. In the ELS+CR 0.2 group, BAX expression is significantly reduced, and in the ELS+CR 0.4 group, it reaches the lowest level among the treatment groups. These results indicate that oxidative stress increases BAX gene expression; however, treatment with CR in com- bination with ELS leads to a significant, dose-dependent reduction in gene expression and apoptotic effects. Figure 2. Oxidative stress index (MDA) in different groups, including control, H 2 O 2 , Elastase (ELS), Elastase plus 0.1 mmol L Carvacrol, Elas- tase plus 0.2 mmol L Carvacrol and Elastase plus 0.4 mmol L Carvacrol. The y-axis represents MDA (pg/mL). Data are mean ± SEM (n = 3). One-way ANOVA: F (5,12) = 31.707, P < 0.001. Tukey's post hoc test. **p < 0.01, ***p < 0.001 Vs control group and #p < 0.05, ##p < 0. 01 Vs ELS group. Slika 2. Indeks oksidativnega stresa (MDA) v različnih skupinah, vključno s kontrolno skupino, H 2 O 2 , elastazo (ELS), elastazo plus 0,1 mmol L karvakrola, elastazo plus 0,2 mmol L karvakrola in elastazo plus 0,4 mmol L karvakrola. Os y predstavlja MDA (pg/mL). Podatki so povprečje ± SEM (n = 3). Enosmerna ANOVA: F (5,12) = 31,707, P < 0,001. Tukeyjev post hoc test. **p < 0,01, ***p < 0,001 v primerjavi s kontrolno skupino in #p < 0,05, ##p < 0,01 v primerjavi s skupino ELS. 97 Acta Biologica Slovenica, 2025, 68 (4) Figure 4. Comparison of BAX gene expression in different groups, including control, H 2 O 2 , Elastase (ELS), Elastase plus 0.1 mmol/L Carvacrol, Elastase plus 0.2 mmol/L Carvacrol and Elastase plus 0.4 mmol/L Carvacrol. The y-axis represents relative gene expression to GAPDH. Data are mean ± SEM (n = 3). One-way ANOVA: F (5,12) = 53.708, P < 0.001. Tukey's post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 Vs control group and ##p < 0.01, ###p < 0. 001 Vs ELS group. Slika 4. Primerjava izražanja gena BAX v različnih skupinah, vključno s kontrolno skupino, H 2 O 2 , elastazo (ELS), elastazo plus 0,1 mmol/L kar- vakrol, elastazo plus 0,2 mmol/L karvakrol in elastazo plus 0,4 mmol/L karvakrol. Os y predstavlja relativno izražanje gena glede na GAPDH. Podatki so povprečje ± SEM (n = 3). Enosmerna ANOVA: F (5,12) = 53,708, P < 0,001. Tukeyjev post hoc test. *p < 0,05, **p < 0,01, ***p < 0,001 v primerjavi s kontrolno skupino in ##p < 0,01, ###p < 0,001 v primerjavi s skupino ELS. Figure 3. Total antioxidant capacity (TAC) in different groups, including control, H 2 O 2 , Elastase (ELS), Elastase plus 0.1 mmol L Carvacrol, Elastase plus 0.2 mmol L Carvacrol and Elastase plus 0.4 mmol L Carvacrol. The y-axis represents TAC (pg/ml). Data are mean ± SEM (n = 3). One-way ANOVA: F (5,12) = 51.37, P < 0.001. Tukey's post hoc test. **p < 0.01 Vs control group and #p < 0.05, ##p < 0. 01 Vs ELS group. Slika 3. Skupna antioksidativna sposobnost (TAC) v različnih skupinah, vključno s kontrolno skupino, H 2 O 2 , elastazo (ELS), elastazo plus 0,1 mmol L karvakrola, elastazo plus 0,2 mmol L karvakrola in elastazo plus 0,4 mmol L karvakrola. Os y predstavlja TAC (pg/ml). Podatki so povprečje ± SEM (n = 3). Enosmerna ANOVA: F (5,12) = 51,37, P < 0,001. Tukeyjev post hoc test. **p < 0,01 v primerjavi s kontrolno skupino in #p < 0,05, ##p < 0,01 v primerjavi s skupino ELS. 98 Acta Biologica Slovenica, 2025, 68 (4) Carvacrol increases BCL2 gene expression in A549 cells exposed to elastase Figure 5 shows the expression level of the BCL2 (anti-apop- totic) gene across different groups. The control group exhibits the highest BCL2 gene expression, indicating a normal state and strong cellular protection. In the H₂O₂ group, the gene expression level significantly decreases, reflecting a substantial decline due to oxidative stress. The ELS group also showed a similar reduction to that of the control group. In the ELS+CR groups with different doses, a gradual increase in BCL2 gene expression is observed. The ELS+CR 0.1 group shows a relative increase. In the ELS+CR 0.2 group, BCL2 expression significantly increases, and in the ELS+CR 0.4 group, it reaches the highest level among all treatment groups. These results indicate that oxidative stress leads to decreased BCL2 gene expression. However, treatment with CR in combination with ELS results in a dose-depen- dent, significant increase in the anti-apoptotic BCL2 gene expression and improved cellular protection. Carvacrol improves the TNF-α concentration level in A549 cells exposed to elastase Figure 6 shows the TNF-α concentration level in different groups. The control group had the lowest TNF-α level with a value of 50.17 pg/mL, which indicates a normal state and the absence of inflammation. In the H 2 O 2 group, the TNF-α level increased to 208.11 pg/mL, which indicates a severe increase in inflammation caused by oxidative stress. The ELS group also showed a significant increase with a value of 191.26 pg/mL compared to the control group. In the groups treated with the combination of ELS and CR, a gradual decrease in TNF-α levels was observed. The ELS+CR 0.1 group showed a relative decrease with a value Figure 5. Expression level of the BCL2 gene in different groups, including control, H 2 O 2 , Elastase (ELS), Elastase plus 0.1 mmol/L Carvacrol, Elastase plus 0.2 mmol/L Carvacrol and Elastase plus 0.4 mmol/L Carvacrol. The y-axis represents relative gene expression to GAPDH. Data are mean ± SEM (n = 3). One-way ANOVA: F (5,12) = 31.938, P < 0.001. Tukey's post hoc test. **p < 0.01, ***p < 0.001 Vs control group and #p < 0.05, ##p < 0.01, ###p < 0. 001 Vs ELS group. Slika 5. Raven izražanja gena BCL2 v različnih skupinah, vključno s kontrolno skupino, H 2 O 2 , elastazo (ELS), elastazo plus 0,1 mmol/L kar- vakrol, elastazo plus 0,2 mmol/L karvakrol in elastazo plus 0,4 mmol/L karvakrol. Os y predstavlja relativno izražanje gena glede na GAPDH. Podatki so povprečje ± SEM (n = 3). Enostranska ANOVA: F (5,12) = 31,938, P < 0,001. Tukeyjev post hoc test. **p < 0,01, ***p < 0,001 v primerjavi s kontrolno skupino in #p < 0,05, ##p < 0,01, ###p < 0,001 v primerjavi s skupino ELS. 99 Acta Biologica Slovenica, 2025, 68 (4) of 143.18 pg/mL. In the ELS+CR 0.2 group, the TNF-α level decreased to 112.03 pg/mL, and in the ELS+CR 0.4 group, it reached 84.19 pg/mL, which was the lowest among the treatment groups. These results indicate that oxidative stress increases TNF-α gene expression and exacerbates inflammation, but treatment with CR in combination with ELS dose-dependently causes a significant decrease in TNF-α gene expression and improves the inflammatory status (Figure 6). Correlation between TNF-α concentration and BAX/BCL2 Pearson's correlation coefficient results (Table 2) showed that TNF-α concentration was positively correlated with BAX gene expression level (r = 0.43, P = 0.002), while it was negatively correlated with BCL2 expression level (r = −0.62, P = 0.0031). Also, BAX expression had a negative correlation with BCL2 (r = −0.56, P = 0.007). Figure 6. TNF-α concentration level in different groups, including control, H 2 O 2 , Elastase (ELS), Elastase plus 0.1 mmol/L carvacrol, Elastase plus 0.2 mmol/L Carvacrol and Elastase plus 0.4 mmol/L Carvacrol. The y-axis represents TNF-α (pg/mL). Data are mean ± SEM (n = 3). One-way ANOVA: F (5,12) = 51.742, P < 0.001. Tukey's post hoc test. ***p < 0.001 Vs control group and ## p < 0.01, ### p < 0. 001 Vs ELS group. Slika 6. Koncentracija TNF-α v različnih skupinah, vključno s kontrolno skupino, H 2 O 2 , elastazo (ELS), elastazo plus 0,1 mmol/L karvakrol, elastazo plus 0,2 mmol/L karvakrol in elastazo plus 0,4 mmol/L karvakrol. Os y predstavlja TNF-α (pg/mL). Podatki so povprečje ± SEM (n = 3). Enosmerna ANOVA: F (5,12) = 51,742, P < 0,001. Tukeyjev post hoc test. ***p < 0,001 v primerjavi s kontrolno skupino in ## p < 0,01, ### p < 0,001 v primerjavi s skupino ELS. Factors Value Significance BAX 0.56 0.007 BCL2 0.62 0.0031 TNF-α 0.43 0.002 Table 2. Relation between TNF-α, BAX, and BCL2 concentrations. Pearson's correlation coefficient test. Tabela 2. Razmerje med koncentracijami TNF-α, BAX in BCL2. Pearsonov test korelacijskega koeficienta. 100 Acta Biologica Slovenica, 2025, 68 (4) Discussion The present study demonstrates that Carvacrol exhibits a significant, dose-dependent protective effect against elastase- and H₂O₂-induced oxidative stress and apoptosis in A549 lung epithelial cells. Treatment with elastase led to a marked reduction in cell viability, increased levels of malondialdehyde (MDA), and altered apoptotic gene expression, with BAX expression upregulated and BCL2 downregulated. These findings are consistent with those of Hou et al. (2014), who reported that elastase induces apoptosis in A549 cells through activation of the JNK-P38 MAPK signalling pathway. The observed oxidative damage and imbalance in apoptotic signalling further support elastase's pro-apoptotic and cytotoxic role. Moreover, previous research by Koparal et al. (2004) on A549 cells reported that Carvacrol reduced cell numbers and altered cellular morphology, findings which appear to contrast with the protective effects observed in the present study. While our data show that Carvacrol mitigates elastase- and H₂O₂-induced oxidative damage and apoptosis, Koparal et al.'s results suggested a cytotoxic effect at certain concen- trations or conditions. This discrepancy may be attributed to differences in experimental design, such as Carvacrol dosage, exposure duration, and the cellular stress model used. For instance, Koparal and Zeytinoglu examined Carvacrol effects on a non-small cell lung cancer (NSCLC) model under basal conditions, potentially reflecting its antiproliferative and pro-apoptotic properties in cancer cells (Koparal et al. 2003), whereas our study focused on its antioxidant and cytoprotective roles in oxidative stress-induced injury. These divergent findings highlight the dual nature of Carvacrol's biological activity, which may vary depending on cellular context, concentration, and pathological state. Importantly, treatment with Carvacrol significantly counteracted these adverse effects in a concentration-de- pendent manner. Carvacrol improved cell viability, reduced MDA levels, increased total antioxidant capacity (TAC), and favorably regulated the expression of BAX and BCL2, restoring the balance between pro- and anti-apoptotic signals. The most notable protective effect was observed at 0.4 mmol/L, highlighting the compound's dose-related efficacy. These results are in line with previous reports by Yesildag et al. (2021), who showed that Carvacrol reduced MDA levels and increased antioxidant enzyme activity in cadmium-induced toxicity models (Yesildag et al. 2022). Similarly, Shoorei et al. (2019) demonstrated that Carvacrol decreased oxidative stress and apoptosis in testicular tissue by reducing BAX and increasing BCL2 expression and antioxidant enzyme activity. The correlation analysis in our study revealed that TNF-α was positively associated with BAX expression and negatively associated with BCL2, indicating a central role of TNF-α in mediating apoptosis. This observation is supported by Moawadh et al. (2023), who reported similar correlations in blood cancers, and by Setayesh et al. (2021), who found that TNF-α inhibition downregulated BAX and upregulated BCL2, reducing oxidative stress and apoptosis. Additionally, Galeone et al. (2023) noted simi- lar pro-apoptotic effects of TNF-α in the context of heart failure, although tissue-specific differences may influence the magnitude of response. Cruceriu et al. (2020) also con- firmed the involvement of TNF-α in promoting apoptosis and therapeutic resistance in breast cancer. These findings collectively affirm TNF-α's broad pro-apoptotic role across various disease models, including our model of lung epi- thelial injury. Interestingly, the pro-apoptotic effects of Carvacrol observed in other formulations contrast with the present findings. For example, Khan et al. (2018) reported that Car- vacrol nano-emulsions induced reactive oxygen species (ROS) production and apoptosis, contrary to our findings of apoptosis reduction. This discrepancy could be attributed to the nano-formulation's enhanced cellular uptake and sustained release, which might lead to elevated intracellu- lar concentrations and pro-oxidant activity. The current findings are also in agreement with prior in vivo and in vitro studies investigating the cardioprotective and hepatoprotective effects of Carvacrol. Jamhiri et al. (2019) demonstrated reduced apoptosis and MDA levels along with increased antioxidant activity in heart tissue following Carvacrol treatment. Likewise, Sadeghzadeh et al. (2018) reported a decrease in pro-apoptotic gene expression and an increase in anti-apoptotic markers in a cardiac hypertrophy model, while Aristatile et al. (2009) showed that Carvacrol reduced oxidative damage in D-ga- lactosamine-induced hepatotoxicity in rats. These studies corroborate our findings, underscoring Carvacrol's consis- tent antioxidant and anti-apoptotic effects across different organ systems and experimental models. Toxicological considerations are essential when inter- preting Carvacrol's therapeutic effects. While our study utilised concentrations within a safe and effective range, 101 Acta Biologica Slovenica, 2025, 68 (4) prior research has identified potential cytotoxicity and genotoxicity at high doses. For example, a study using the Caco-2 intestinal cell line revealed DNA damage at 460 μM, although similar effects were not seen in other cell types (Nowak et al., 2020). Animal studies have reported spe- cies-specific median lethal doses (LD₅₀), ranging from 73 mg/ kg intraperitoneally in rats to 2,700 mg/kg dermally in rabbits (Maul et al., 2018). These data suggest that while Carvacrol is relatively safe at lower concentrations, caution is warranted in translating in vitro findings to clinical applications. Mechanistically, the results of this study support the hypothesis that Carvacrol exerts its protective effects by interfering with TNF-α-mediated apoptotic pathways and neutralising elastase-induced oxidative stress. TNF-α activates signalling cascades such as NF-κB and MAPK, promoting BAX expression and inhibiting BCL2, leading to mitochondrial dysfunction and activation of caspases (Liu et al. 2004; Aslan et al. 2020). Simultaneously, elastase, released from neutrophils, promotes cellular damage by degrading extracellular matrix components and increasing ROS through JNK-P38 MAPK pathway activation (Preston et al. 2002). Carvacrol appears to counteract both mech- anisms: it reduces ROS generation, enhances TAC, and restores apoptotic homeostasis by downregulating BAX and upregulating BCL2. The synergistic role of elastase and TNF-α in driving epithelial damage has been emphasised in earlier studies, where elastase was shown to stimulate TNF-α release, further amplifying cellular injury. Carvacrol effectively inter- rupts this deleterious feedback loop by modulating TNF-α signalling, reducing lipid peroxidation (MDA), and enhanc- ing overall antioxidant defences. This highlights Carvacrol's potential as a multifunctional protective agent capable of modulating both oxidative and inflammatory pathways. Although the concentrations of Carvacrol used in this study (0.1–0.4 mmol/L) may appear high, they are consistent with concentrations employed in prior in vitro investigations evaluating antioxidant and cytoprotective effects in epithelial and other mammalian cells (Yesildag et al. 2022; Shoorei et al., 2019). It is important to note that in vitro systems often require higher concentrations of bioactive compounds than what is needed in vivo due to limited bioavailability, lack of metabolism, and differences in cellular uptake dynamics. Furthermore, our findings demonstrated a clear dose–response relationship, with protective effects becoming more pronounced as the concentration increased. Nevertheless, future studies are warranted to explore the efficacy of Carvacrol in the low micromolar range and under physiologically relevant conditions, including using in vivo models to evaluate therapeutic safety and dosing thresholds. A limitation of this study is the absence of a Carvac- rol-alone control group, which would have enabled a more precise evaluation of Carvacrol's intrinsic effects on A549 cells under non-stressed conditions. Without this group, we cannot conclusively distinguish whether Carvacrol's observed protective effects in the elastase-treated groups result from direct antioxidant action or interactions specific to the oxidative stress environment. Future studies should include Carvacrol-alone groups to better clarify potential synergistic or antagonistic interactions with elastase. In conclusion, Carvacrol exhibits potent antioxidant and anti-apoptotic properties in A549 lung epithelial cells exposed to elastase and H₂O₂. By attenuating TNF-α-medi- ated apoptosis and suppressing oxidative stress, Carvacrol may hold therapeutic promise in the management of inflammatory lung diseases characterised by excessive neutrophil activity and oxidative damage. Future studies should investigate the clinical relevance of these findings in in vivo models and explore optimal delivery strategies to enhance Carvacrol's therapeutic efficacy. Conclusion In this study, Carvacrol, as an antioxidant and anti-inflam- matory compound, was able to ameliorate the damaging effects of elastase and H 2 O 2 on A549 lung epithelial cells. Carvacrol dose-dependently increased cell viability and reduced oxidative stress. This compound restored the bal- ance between pro-apoptotic and anti-apoptotic pathways by modulating the expression of apoptosis genes. The positive association of TNF-α with BAX and its negative association with BCL2 indicated the key role of these path- ways in cell damage. The findings confirm the effectiveness of Carvacrol in reducing elastase-induced damage and regulating inflammatory and apoptotic pathways. While our in vitro results suggest potential mechanisms that could be targeted in COPD, we acknowledge that these findings are preliminary. Further studies involving in vivo models and clinical trials are essential to validate the therapeutic rel- evance of these observations. Therefore, our current data serve as a foundation for future research rather than direct evidence for clinical application. 102 Acta Biologica Slovenica, 2025, 68 (4) Author Contributions Conceptualization, K.H. and M.R.; methodology, F.N.D.; soft- ware, S.M.N.M.; validation, M.R.; formal analysis, S.M.N.M.; investigation, S.M.N.M.; resources, K.H.; data curation, M.R.; writing—original draft preparation, K.H.; writing—review and editing, M.R.; visualization, F.N.D.; supervision, K.H. and M.R.; project administration, M.R.; funding acquisition, M.R.. All authors have read and agreed to the published version of the manuscript. 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For correspondence, see the individual chapters. Citation: Curk, A., Kastelic, M., Jakob, A., Mihorič, A., Jakšić, P., Klenovšek, T., (2025). Significant records of plants, algae, fungi, and animals in SE Europe and adjacent regions, 4. Acta Biologica Slovenica 68 (4) https://doi.org/10.14720/abs.68.4.24300 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Significant records of plants, algae, fungi, and animals in SE Europe and adjacent regions, 4 Aljaž Curk 1 , Maj Kastelic 1 , Aljaž Jakob 2 , Alenka Mihorič 3 , Predrag Jakšić 4 , Tina Klenovšek 5 Editors: Simona Strgulc Krajšek 1 and Tina Klenovšek 5 Abstract In this article, we present the first records of the bryophyte Schistostega pennata in Central Slovenia. In addition to its typical occurrence on rocky surfaces, Schistostega pennata was observed on bare loose soil. We also report a rare natural aberration of the male meadow brown butterfly, Maniola jurtina subtus- albida. This represents the first record of this aberration for the Balkan Peninsula. Keywords Schistostega pennata; bryophytes; mosses; Maniola jurtina ab. subtus-albida; Nymphalidae; Slovenia; Montenegro 105 Acta Biologica Slovenica, 2025, 68 (4) Pomembne zabeležke rastlin, alg, gliv in živali za JV Evropo in sosednje regije, 4 Izvleček V tem članku predstavljamo prve zapise o mahovcu Schistostega pennata v osrednji Sloveniji. Poleg tipičnega pojavljanja na skalnatih površinah je bil Schistostega pennata opažen tudi na goli, rahli prsti. Poročamo tudi o redki naravni aberaciji samca metulja Maniola jurtina subtus-albida. To je prvi zapis te aberacije na Balkanskem polotoku. Ključne besede Schistostega pennata; briofiti; mahovi; Maniola jurtina ab. subtus-albida; Nymphalidae; Slovenija; Črna gora Author(s) Aljaž Curk, Maj Kastelic, Aljaž Jakob, Alenka Mihorič Corresponding author Aljaž Jakob (aljaz.jakob@zrc-sazu.si) Leg. Aljaž Curk, Maj Kastelic, Aljaž Jakob, Alenka Mihorič Country Slovenia Statement of significance First finds of the species in Central Slovenia Locality description Slovenia, Ljubljana, Golovec, along a forestry road between the observatory and the new Codelli bridge, 200 m NW of the observatory, 370 m a. s. l. Slovenia, Ljubljana, Tivoli, Rožnik and Šiška Hill Landscape Park, near a trail in the forest, 200 m SW of the Petra Držaja hospital, 305 m a. s. l. Slovenia, Ljubljana, Rožnik, along a walking path from the parking lot of ZOO Ljubljana to Cankarjev vrh, 140 m NE of the main entrance to the ZOO, 316 m. a. s. l. Slovenia, Gorenjska, Lukovica, Mali Jelnik, shale hollow next to a forest path, 490 m a. s. l. Habitat A small depression in the clay on the bank of the forestry road A crevice cut horizontally into the slope of the hill, comprised of loose soil and plant roots Loose soil in a horizontal crevice under an overhang of clay, caused by erosion On shale between roots in the road cut bank in a mixed forest, SE exposition Date of observation 2024-04-26 2024-03-16 2024-10-24 2024-11-01 Geographical coordinates N 46.0454291°, E 14.53018952° N 46.066693°, E 14.485182° N 46.0536539°, E 14.4733961° N 46.17844°, E 14.80125° Voucher Photo documented Schistostega pennata, (Hedw.) F. Weber & D. Mohr fam. Schistostegaceae On site no. 1 Schistostega pennata grew in small depres- sions in a steep bank above a forestry road. Only protonema was present. On site no. 2, it was located in a crevice cut horizontally into the slope, where it grew on unconsoli- dated soil sediment intertwined with roots. The habitat was shaded, protonema inhabited the bottom of this crevice. On site no. 3 the plant grew above a walking path in a few metres-long horizontal crevice under a soil overhang. The habitat was shaded; other moss species grew in lighter parts of the crevice. Protonema and gametophores were 106 Acta Biologica Slovenica, 2025, 68 (4) Figure 1. Gametophytes of Schistostega pennata growing on soil on site no. 3. (photo: M. Kastelic). Slika 1. Gametofiti Schistostega pennata, ki rastejo na tleh na lokaciji št. 3. (foto: M. Kastelic). present (Figure 1). On site no. 4 the plant was growing on shale in the roadcut directly on the rock, exposed to the morning sun. Protonema and gametophytes were present (Figure 2). The moss is recognisable by its reflective protonema. This is caused by enlarged vacuoles of protonemal cells, acting like lenses (Atherton et al., 2010; Robla et al., 2023). Shoots are 1.5cm tall, with short, nerveless and decurrent green leaves arranged in 2 ranks. It is found in Europe from Spain to Russia (Ignatov & Ignatova, 2001; Hodgetts & Lockhart, 2020). It is considered vulnerable in Finland and Spain and near-threatened in Portugal, but least-concern on the Slovenian and European Red Lists (Hodgetts & Lockhart, 2020; Martinčič 2023). In Slovenia, the recent finds are in the Alpine parts of the country in the Julian Alps, Karawanks, Pohorje, Kozjak-mountain and Meža-Mislinja valley, and additionally before 1959 in the Kamnik-Savinja Alps. The observations published in this paper are the first from Central Slovenia, in the pre-Alpine part of the country. Schistostega pennata grows on rocky surfaces such as siliceous and other non-calcareous rocks, overhangs and walls of excavations (Ochyra et. al., 1988). An exam- ple of such a site is depicted in Figure 2. Similar habitats are described from Poland (Ochyra et. al., 1988), Norway (Lye, 1972), Japan (Kanda, 1971), North America (Crum & Anderson, 1981) and Russia (Ignatov & Ignatova, 2001). It does not tolerate competition from other plants (Atherton et al., 2010). Habitats that we found in Central Slovenia on localities 1-3 differ from the habitats described above; here, it is found on bare loose soil (Figure 1). Similar habitats are reported from central Russia (Ignatov & Ignatova 2001) and Poland (Ochyra et. al., 1988), where the plant is found on bare soil on the upturnings of fallen trees. In our cases, the soil is exposed by roadcuts and erosion. The synanthropic nature of all our new localities suggests that this species may benefit from human activity in an area where natural processes providing appropriate habitat are largely absent. Funding This research was funded by the Slovenian Research and Innovation Agency (ARIS), grant numbers MR-55879 and P1-0236. 107 Acta Biologica Slovenica, 2025, 68 (4) Figure 2. Reflection of green light from the protonema of Schistostega pennata on site no. 4. (photo: A. Mihorič). Slika 2. Odboj zelene svetlobe od protoneme Schistostega pennata na lokaciji št. 4. (foto: A. Mihorič). Author(s) Predrag Jakšić, Tina Klenovšek Corresponding author Predrag Jakšić (jaksicpredrag@gmail.com) Leg. Predrag Jakšić Country Montenegro Statement of significance Rare natural aberration of the meadow brown, Maniola jurtina. First record for the Balkan Peninsula. Locality description Montenegro, Čakor, Karamanov krš, 1134 m a. s. l. Habitat Rocky, limestone-dominated grassland with shallow soil and xerophytic grassland species. Date of observation 2022-07-05 Geographical coordinates N 42.68056°, E 19.92417° Voucher Author’s (P.J.) personal collection Maniola jurtina (Linnaeus, 1758) ab. subtus-albida Silbernagel, 1943, fam. Nymphalidae (animal) 108 Acta Biologica Slovenica, 2025, 68 (4) The meadow brown, Maniola jurtina (Linnaeus, 1758), is a butterfly from the family Nymphalidae distributed across the Palearctic region. Its habitats include grasslands, forest edges, shrublands, and woodland clearings. Its altitudinal range spans from sea level up to approximately 2000 m, and it is active from May to September (Scott, 1990). The wings of Maniola jurtina are dark brown, with a distinctive large eyespot located in the upper third of the forewings. The underside of the hindwings displays a pattern of small eyespots. The number of the small eyespots is variable, typically numbering two, though the total can range from zero to six (Scott, 1990). An aberration is an unusual variation in the wing pattern, shape or colouration, or other body structures, within a particular species. Variations of the usual form of a species can result from genetic (Rivera-Colón et al., 2020) or environmental factors (Mowbray et al., 2024) or a com- bination of both. Some aberrant forms, although rare, can occur on a relatively regular basis and were, in the past, documented as new forms of a butterfly species. Many forms, aberrations, and varieties were also described in M. jurtina (Spuler, 1908; Rebel & Zerny, 1931; Russwurm, 1978). Nowadays, they no longer hold taxonomic significance. The aberration of male M. jurtina subtus-albida was first described by Silbernagel (1943). The underside of the fore- wings of which, in typical individuals, is rich ochre yellow with a wide dark border, in this form is whitish with a faint yellowish tinge. The dark border is preserved. The upper- side of both wings is normal. Only the dusting of the fore- wings is slightly translucent due to the light background. This aberration has only been documented in specimens collected in the Czech Republic (Silbernagel, 1943) and Great Britain (Russwurm, 1978; Barrington, 1987, 1991). Now it is also reported for Montenegro (Figure 3), where it was collected by the first author (P.J.). Compared to the speci- men presented in Russwurm (1978), the black eyespots on the underside of the forewings of the specimen collected in Montenegro are not centred with a white spot (pupilled). Figure 3. Maniola jurtina ab. subtus-albida. Left: Rare natural aberration of a male meadow brown, Maniola jurtina ab. subtus-albida col- lected on Karamanov krš in Montenegro (underside view) (photo: Predrag Jakšić). Right: Eyespot on the underside of the right forewing (photo: Miloš Jović). Slika 3. Maniola jurtina ab. subtus-albida. Levo: Redka naravna aberacija samca navadnega lešnikarja, Maniola jurtina ab. subtus-albida, najdena na Karamanovem kršu v Črni gori (pogled od spodaj) (foto: Predrag Jakšić). Desno: Očesna lisa na spodnji strani desnega spredn- jega krila (foto: Miloš Jović). 109 Acta Biologica Slovenica, 2025, 68 (4) Acknowledgement We thank Miloš Jović for the photos of the eyespot. Funding T.K. was supported by the Slovenian Research and Innova- tion Agency (ARIS) and research programme Computation- ally Intensive Complex Systems (P1-0403). References Atherton, I., Bosanquet, S.D., Lawley, M., 2010. Mosses and liverworts of Britain and Ireland: a field guide. British Bryological Society, Plymouth, Great Britain, p. 419. Barrington, R.D.G., 1987. Further notes on variation in a north Dorset colony of the meadow brown butterfly Maniola jurtina L. Entomologist’s Record and Journal of Variation, 99, 97–102. Barrington, R.D.G., 1991. Continued notes on a north Dorset colony of the meadow brown butterfly Maniola jurtina L. Entomologist’s Record and Journal of Variation, 103, 7–15. Crum, H.A., Anderson, L.E., 1981. Mosses of the Eastern North America. Columbia Univ. Press, New Y ork, USA, p. 1328. Hodgetts, N., Lockhart, N., 2020. Checklist and country status of European bryophytes - update 2020. Irish Wildlife Manuals, No. 123. 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Atlas Rozmieszczenia Roslin Zarodnikowich w Polsce. Ser. 5 Mchy (Musci), 4, 15–17. Rebel, H., Zerny, H., 1931. Die Lepidopterenfauna Albaniens. Denkschriften der Akademie der Wissenschaften in Wien. Math. Nat. Klasse. Vienna. Rivera-Colón, A.G., Westerman, E.L., Van Belleghem, S.M., Monteiro, A., Papa, R., 2020. Multiple loci control eyespot number variation on the hindwings of Bicyclus anynana butterflies. Genetics, 214 (4), 1059–1078. https://doi.org/10.1534/genetics.120.303059 Robla, J., González-García, V., Santamarina, S., Artazkoz, M., 2023. Unravelling the ecological drivers of Schistostega pennata (Hedw.) F. Weber & D. Mohr on the Iberian Peninsula: distribution and conservation. Journal of Bryology, 45(3), 192–207. Russwurm, A.D.A., 1978. Aberrations of British Butterflies. EW Classey, Farrington. Scott J., 1990. Adult structure and function. In: Kudrna O. (ed.). Butterflies of Europe, Volume 2 Introduction to Lepidopterology. AULA–Verlag, 108–151. Silbernagel, A., 1943. Nekolik novych forem macrolepidopter z Čech. Časopis české společnosti entomologické, 40, 1–7. Spuler A., 1908. Die Schmetterlinge Europas. E. Schweizerbartsche Verlagsbuchhandlung, Stuttgart. 110 1 Department of Biology, SR.C., Islamic Azad University, Tehran, Iran 2 Department of Biology, SR.C., Islamic Azad University, Tehran, Iran * Corresponding author: E-mail address: baharehnowruzi77@iau.ac.ir Citation: Nowruzi, B., Beiranvand, H., Rahmani, R., Al-Tayeb, S. S., Zakeralhosseini Khafri, A. A., (2025). Study of the Effect of Autotrophic and Mixotrophic Culture Media on Heavy Metal Uptake by Fischerella sp. and Desmonostoc alborizicum. Acta Biologica Slovenica 68 (4) Received: 10.06.2025 / Accepted: 16.09.2025 / Published: 07.10.2025 https://doi.org/10.14720/abs.68.4.23013 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Study of the Effect of Autotrophic and Mixotrophic Culture Media on Heavy Metal Uptake by Fischerella sp. and Desmonostoc alborizicum Bahareh Nowruzi 1, * , Hassan Beiranvand 2 , Roya Rahmani 2 , Seyedeh Sajedeh Al-Tayeb 2 , Amir Aala Zakeralhosseini Khafri 2 Abstract Heavy metals, even at low concentrations, can have detrimental effects on human health and the environment. The use of biological methods for heavy metal removal is considered a suitable alternative to conventional physicochemical methods due to lower costs and environmental compatibility. Among these methods, exopolysaccharides produced by cyanobacteria play a significant role in the adsorption of metal ions. This study aimed to evaluate the capability of two native Iranian cyanobacteria, Fischerella sp. and Desmonostoc alborizicum, in removing the heavy metals chromium, nickel, and copper. The cyanobacterial strains Fischerella sp. and Desmonostoc alborizicum were cultured in mixotrophic media containing glucose, maltose, lactose, and sucrose (10 g/L), as well as in a control medium (carbohydrate- free). Dry cell weight (6–48 hours), exopolysaccharide content (at 48 hours), and heavy metal removal (copper, chromium, and nickel, 10–90 minutes) were measured. Carbohydrate and protein contents were determined using the phenol-sulfuric acid method and the Lowry method, respectively. Additionally, volatile compounds produced by Fischerella sp. in the presence of copper were analysed using gas chromatography-mass spectrometry (GC-MS). The results demonstrated that Fischerella sp. exhibited superior performance in maltose- enriched culture media compared to Desmonostoc alborizicum. This strain led to a 1.3-fold increase in exopolysaccharide production, a 1.02-fold increase in copper removal, and higher dry cell weight relative to Desmonostoc alborizicum. The removal efficiency of chromium and nickel ranked second and third, respectively, after copper in maltose-enriched media. Quantitative comparison of the compounds identified by GC-MS in Fischerella sp. under control conditions and in the presence of maltose and copper revealed an increase in the levels of 3-methylbutane, 2-methylbutane, methyl acetate, 2-butanone, acetic acid, 111 Acta Biologica Slovenica, 2025, 68 (4) Študija vpliva avtotrofnih in miksotrofnih gojišč na absorpcijo težkih kovin s strani Fischerella sp. in Desmonostoc alborizicum Izvleček T ežke kovine lahko že v majhnih koncentracijah škodljivo vplivajo na zdravje ljudi in okolje. Uporaba bioloških metod za odstranjevanje težkih kovin se šteje za ustrezno alternativo konvencionalnim fizikalno-kemijskim metodam zaradi nižjih stroškov in okoljske združljivosti. Med temi metodami imajo eksopolisaharidi, ki jih proizvajajo cianobakterije, pomembno vlogo pri adsorpciji kovinskih ionov. Cilj te študije je bil oceniti sposobnost dveh avtohtonih iranskih cianobakterij, Fischerella sp. in Desmonostoc alborizicum, za odstranjevanje težkih kovin kroma, niklja in bakra. Cianobakterijski sevi Fischerella sp. in Desmonostoc alborizicum so bili gojeni v mixotrofnih medijih, ki so vsebovali glukozo, maltozo, laktozo in saharozo (10 g/l), ter v kontrolnem mediju (brez ogljikovih hidratov). Merili so suho maso celic (6–48 ur), vsebnost eksopolisaharidov (po 48 urah) in odstranjevanje težkih kovin (baker, krom in nikelj, 10–90 minut). Vsebnost ogljikovih hidratov in beljakovin je bila določena z metodo fenol-žveplove kisline oziroma metodo Lowry. Poleg tega so bile hlapne spojine, ki jih je proizvedla Fischerella sp. v prisotnosti bakra, analizirane z uporabo plinske kromatografije-masne spektrometrije (GC-MS). Rezultati so pokazali, da je Fischerella sp. v primerjavi z Desmonostoc alborizicum pokazala boljšo učinkovitost v gojiščih, obogatenih z maltozo. Ta sev je v primerjavi z Desmonostoc alborizicum povzročil 1,3-kratno povečanje proizvodnje eksopolisaharidov, 1,02-kratno povečanje odstranjevanja bakra in višjo suho maso celic. Učinkovitost odstranjevanja kroma in niklja je bila na drugem in tretjem mestu, takoj za bakrom v mediju, obogatenem z maltozo. Kvantitativna primerjava spojin, identificiranih z GC-MS v Fischerella sp. v kontrolnih pogojih in v prisotnosti maltoze in bakra, je pokazala povečanje ravni 3-metilbutana, 2-metilbutana, metilacetata, 2-butanona, ocetne kisline, 3-metilbutilacetata, 1-butanola, 3-metil-1-butanola, 3-hidroksi-2-butanona, 2-feniletanola in 3-okten-2-ola. Ta študija je pokazala, da je Fischerella sp. pokazala visoko učinkovitost pri odstranjevanju težkih kovin, proizvodnji eksopolisaharidov in beljakovin v mediju, obogatenem z maltozo. Zato se ta sev lahko šteje za okolju prijazen kandidat za zmanjšanje onesnaževanja okolja. Ključne besede Odstranjevanje težkih kovin, Cianobakterije, Eksopolisaharidi, Mešano trofično gojišče, Avtotrofno gojišče 3-methylbutyl acetate, 1-butanol, 3-methyl-1-butanol, 3-hydroxy-2-butanone, 2-phenylethanol, and 3-octen-2-ol. This study demonstrated that Fischerella sp. exhibited high efficiency in heavy metal removal, exopolysaccharide, and protein production in maltose-enriched media. Therefore, this strain can be considered an eco-friendly candidate for reducing environmental pollution. Keywords Heavy metal removal, Cyanobacteria, Exopolysaccharides, Mixotrophic culture medium, Autotrophic culture medium 112 Acta Biologica Slovenica, 2025, 68 (4) Introduction Heavy metals, even in extremely low concentrations, can be hazardous to many biological processes. Due to the large- scale industrial applications of pharmaceuticals, pesticides, plastics, slaughterhouses, rubber, organic chemicals, and wood products, the free forms of heavy metals in soil and aquatic systems are continuously increasing. Furthermore, the non-degradable nature of heavy metals prolongs their persistence in the environment. Their high solubility in water leads to bioaccumulation, ultimately resulting in severe and irreversible damage, including potential carcinogenicity, even at very low concentrations of approximately 1 mg/L (Pierre et al., 2016). Reducing the bioavailability or mobility of heavy metal ions is crucial to mitigate the risks associated with their absorption and accumulation. Therefore, removing these contaminants from wastewater before their release into the environment is imperative (Pereira et al., 2013). Various physicochemical methods, such as adsorption, chemical precipitation, solvent extraction, and ion exchange techniques, may be employed for heavy metal removal. How- ever, these methods have notable disadvantages, including incomplete metal removal, the requirement for expensive equipment and monitoring systems, high energy consump- tion, and the generation of contaminated residues that must be disposed of properly (Pereira et al., 2016). Additionally, these methods are often ineffective when metal ion concen- trations range between 10 and 100 mg/L. These limitations have increased interest in developing environmentally friendly and cost-effective biological treatment methods. Initially, it was believed that metals only exert toxic effects on microbial systems; however, it was later discovered that microorganisms can develop metal resistance mechanisms. Microorganisms actively bind to heavy metal ions, thereby reducing their toxic impact on the environment. For many years, the isolation and identification of cya- nobacterial strains with potential bioremediation activity have highlighted their significance as valuable organisms in the wastewater treatment industry. Cyanobacteria are particularly promising candidates for heavy metal removal, as they eliminate metal ions through biosorption or bioac- cumulation, and in many cases, via both mechanisms. The advantages of using microorganisms in heavy metal biore- mediation include their reliance on natural and renewable resources, cost-effectiveness, rapid metal removal effi- ciency, the ability to eliminate metal ions even at low con- centrations, autonomous purification of contaminated water containing various metal ions, and the recovery of valuable metals through biosorption (Liu et al., 2003; Knowles and Castenholz, 2008). The exopolysaccharides extracted from cyanobacteria provide protection against extreme condi- tions, including high pH and temperature. The net anionic charge of exopolysaccharides facilitates the adsorption of positively charged heavy metal ions, while the presence of negatively charged uronic acids plays a crucial role in cation binding. Consequently, the exopolysaccharides produced by cyanobacteria could serve as a viable alternative to conventional chemical and physicochemical methods for removing metal cations from contaminated water sources (Pierre et al., 2016; Pereira et al., 2009). Additionally, enriching the culture medium and increas- ing exopolysaccharide production can further highlight the adaptability of cyanobacteria to mixotrophic environments. In fact, optimising the culture medium enhances biomass and exopolysaccharide production. The chemical charac- teristics and abundance of extracellular polysaccharide structures depend on the specific cyanobacterial strain and the environmental or culture conditions. Therefore, in this study, mixotrophic media enriched with glucose, sucrose, maltose, and lactose were utilised (Ghorbani et al., 2022). The freshwater wetlands and qanats of Golestan Prov- ince serve as active ecosystems, providing habitats for vari- ous aquatic species and native birds. Given the presence of environmental pollutants and the significant influx of waste- water into these areas, as well as the seepage of contami- nants into wetlands and freshwater qanats, this study aims to evaluate and compare the heavy metal removal capabilities of two native Iranian cyanobacterial strains, Fischerella sp. and Desmonostoc alborizicum, isolated from the wetlands and qanats of Golestan Province. By investigating their biosorption effectiveness under mixotrophic conditions, this research seeks to identify the most efficient strain for biore- mediation and to contribute novel insights into the applica- tion of native cyanobacteria for environmentally sustainable heavy metal removal in polluted aquatic ecosystems. Materials and methods Cultivation of cyanobacterial strains The cyanobacterial strains Fischerella sp. and Desmono- stoc alborizicum used in this study were obtained from the cyanobacteria culture collection of the Alborz Herbarium, 113 Acta Biologica Slovenica, 2025, 68 (4) Islamic Azad University, Science and Research Branch. These strains were selected due to their native origin from wetlands and qanats of Golestan Province in Iran, repre- senting locally adapted biota with potential environmental significance. The strains were cultured in standard Z8 liquid medium and maintained in a growth chamber at 28°C under continuous fluorescent illumination at an intensity of 300 μE/m²/s for 30 days, following established protocols (Nowruzi and Lorenzi, 2021; Nowruzi et al., 2023). Design and preparation of different culture media To evaluate the effects of carbon sources on growth and heavy metal uptake, four mixotrophic culture media were prepared by supplementing Z8 medium with 10 g/L of one of the following carbohydrates: glucose, maltose, lactose, or sucrose. A control medium consisted of Z8 without any added carbohydrates to represent autotrophic conditions. All media were sterilised, cooled, and the pH was adjusted to approximately 7.2 prior to inoculation with cyanobacte- rial strains (Figure 1) (Ghorbani et al., 2022). This cultivation approach is widely used in cyanobacterial research to enhance biomass and metabolite production, facilitating subsequent bioremediation assessments. Determination of dry cell weight To determine the dry cell weight, samples were collected at 6, 12, 24, and 48 hours and dried at 100°C (Ghorbani et al., 2022). Determination of extracellular polysaccharides Extraction of exopolysaccharides (EPS) was performed using a modified method of Ozturk and Aslim. Cells were separated by centrifugation at room temperature, and the resulting pellet was boiled in distilled water. The final solu- tion was washed with ethanol and freeze-dried to obtain purified EPS (Ozturk and Aslim, 2010). Estimation of heavy metal removal Dialysis bags containing the culture medium were treated with HCl to remove metal ions and subsequently dialysed with water. The treated cultures were exposed to a metal solution containing Cu(II), Cr(III), and Ni(II) at 30°C for 24 hours. Biomass was separated from the solution by centrifugation and filtration. The final metal content in the supernatant was measured using atomic absorption spectrophotometry at Figure 1. Cultivation of (A) Fischerella sp., and a microscopic image. (B) Desmonostoc alborizicum, and a microscopic image. Slika 1. Gojenje (A) Fischerella sp. in mikroskopska slika. (B) Desmonostoc alborizicum in mikroskopska slika. 114 Acta Biologica Slovenica, 2025, 68 (4) wavelengths of 232 nm for copper, 359.9 nm for chromium, and 324.7 nm for nickel. The metal removal efficiency was determined by measuring the difference in metal concentra- tion before and after exposure to the cyanobacterial culture and was compared to a blank control. All experiments were performed in triplicate, and data were analysed as mean ± standard deviation. The amount of metal removed (q) was expressed as milligrams of metal removed per gram of dry weight using the equation: q (mg g -1 ) = V (C₁ - C₂) / m Where: V = sample volume (L), C₁ = initial metal concentration (mg/L), C₂ = final metal concentration (mg/L), m = dry biomass weight (g). The dry biomass weight (g/L) was determined by filtering the dialysed cultures and drying the filters at 100°C (Rossi and De Philippis, 2015). Quality assessment methods Carbohydrates were quantified using the phenol-sulfuric acid method, with glucose as the standard, at an absor- bance wavelength of 490 nm. Polysaccharide solutions were mixed with sulfuric acid and phenol, incubated at 25°C, and their absorbance was measured at 490 nm. A standard glucose curve was prepared using diluted glucose solutions, and absorbance values were recorded with a spectrophotometer. Proteins were quantified using a modified Lowry method at a wavelength of 750 nm (DuBois et al., 1956; Olson and Markwell, 2007). Identification of volatile compounds using GC-MS First, 50 mL of the sample was centrifuged, and the super- natant was mixed with a methanolic solution of 2M HCl for 4 hours at 100°C. The solution was then centrifuged at 8000 rpm for 10 minutes, and the supernatant was transferred to a round-bottom flask connected to a rotary evaporator. After rotary evaporation, 2 mL of hexane was added to the flask contents. The mixture was filtered through a PTFE membrane filter with a pore size of 0.45 μm. The resulting solution was centrifuged again at 8000 rpm for 10 minutes, and the supernatant was collected for GC-MS analysis. A 2-μL aliquot of the supernatant was injected into the GC-MS instrument using a specialised syringe, and the spectrum was obtained (Guo et al., 2024). Methods and data analysis tools The experimental data were analysed using one-way anal- ysis of variance (One-way ANOVA). Statistical tests were conducted using SPSS software, version 26. A significance level of p ≤ 0.05 was considered for all data comparisons (Parikh and Madamwar, 2006). Results Results of dry cell weight measurement The results of dry cell weight measurements (g/l) in culture media containing maltose, lactose, glucose, sucrose, and control showed that this value was significantly higher in the Fischerella sp. strain compared to the Desmonostoc alborizicum strain. In both strains, the dry cell weight sig- nificantly increased until the end of 48 hours (Figure 2). Results of extracellular polysaccharide measurement The results of exopolysaccharide measurements in culture media containing maltose, lactose, glucose, sucrose, and control indicated that the extracellular exopolysaccharide produced by the Fischerella sp. strain in the maltose-con- taining medium was approximately 1.30 times higher than that of the Desmonostoc alborizicum strain (Figure 3). Results of heavy metal removal The results of heavy metal removal measurements in the control culture medium showed no significant difference in heavy metal removal between the two strains. However, the analysis of heavy metal removal in media containing maltose, lactose, glucose, and sucrose demonstrated that the highest heavy metal removal occurred in the Fischerella sp. strain in the maltose-containing medium with copper. At time T10 (the first 10 minutes), the amount was 360 mg/g, which was 1.02 times higher than that of the Desmonostoc alborizicum strain. Following copper, the highest levels of heavy metal removal were observed for chromium and nickel (Figure 4). 115 Acta Biologica Slovenica, 2025, 68 (4) Figure 2. Results of dry cell weight measurements (g/l) in cyanobacterial strains (a) Fischerella sp., (b) Desmonostoc alborizicum in culture media containing (1) maltose, (2) lactose, (3) glucose, (4) sucrose, and (5) control. Slika 2. Rezultati meritev suhe teže celic (g/l) v sevih cianobakterij (a) Fischerella sp., (b) Desmonostoc alborizicum v gojiščih, ki vsebujejo (1) maltozo, (2) laktozo, (3) glukozo, (4) saharozo in (5) kontrolno. 116 Acta Biologica Slovenica, 2025, 68 (4) Figure 3. Results of extracellular polysaccharide (µg/ml) measurements in cyanobacterial strains (a) Fischerella sp., (b) Desmonostoc alborizicum in culture media containing (1) maltose, (2) lactose, (3) glucose, (4) sucrose, and (5) control. Slika 3. Rezultati meritev ekstracelularnih polisaharidov (µg/ml) v sevih cianobakterij (a) Fischerella sp., (b) Desmonostoc alborizicum v gojiščih, ki vsebujejo (1) maltozo, (2) laktozo, (3) glukozo, (4) saharozo in (5) kontrolno. 117 Acta Biologica Slovenica, 2025, 68 (4) Figure 4. Results of heavy metal removal measurements in cyanobacterial strains (a) Fischerella sp., (b) Desmonostoc alborizicum in culture media containing (1) maltose, (2) lactose, (3) glucose, (4) sucrose, and (5) control. Slika 4. Rezultati meritev odstranjevanja težkih kovin v sevih cianobakterij (a) Fischerella sp., (b) Desmonostoc alborizicum v gojiščih, ki vse- bujejo (1) maltozo, (2) laktozo, (3) glukozo, (4) saharozo in (5) kontrolno skupino. 118 Acta Biologica Slovenica, 2025, 68 (4) Results of exopolysaccharide measurement in the presence of heavy metals The results of exopolysaccharide measurements (µg/l) showed that the highest exopolysaccharide levels were produced by the Fischerella sp. strain in media containing copper and maltose (14 µg/L), which was 1.16 times higher than that of the Desmonostoc alborizicum strain (Figure 5). Results of cellular protein measurement The results of cellular protein measurements in the control culture medium showed that the protein level in the Fischerella sp. strain was significantly higher than in the Desmonostoc alborizicum strain. In both strains, the cellular protein content significantly increased until the end of 48 hours. However, the protein levels produced by the Fischerella sp. strain in the control, maltose, and lactose-containing media were approximately 8.88, 1.08, and 1.03 times higher, respectively, than those in the Des- monostoc alborizicum strain. Additionally, the results of cellular protein measure- ments in glucose- and sucrose-containing media indicated that in both strains, protein levels significantly decreased until the end of 48 hours, with no significant difference observed between the two strains (Figure 6). Identification of volatile compounds using GC-MS Since the results of heavy metal removal demonstrated that Fischerella sp. exhibited the highest efficiency in removing copper in the maltose-containing culture medium, the anal- ysis of volatile compounds in this medium was conducted using Fischerella sp. The analysis of volatile compounds in Fischerella sp. under control conditions using GC-MS revealed the presence of 18 compounds, including furan, aldehyde, ester, ketone, benzene, acid, alkyne alcohol, and alcohol. According to the results, the most abundant compounds were 3-methylbutanal and 2-methylbutanal (12.210%), followed by 2-methyl-L-propanol (11.056%). The least abundant compound identified was 2-phenylethanol (2.003%). The analysis of volatile compounds in Fischerella sp. cultured in maltose-containing medium with copper using GC-MS identified similar classes of compounds, including furan, aldehyde, ester, ketone, benzene, acid, alkyne alcohol, and alcohol. The most abundant compounds were 3-methylbutanal and 2-methylbutanal (18.718%), followed by 3-methyl-1-propanol (12.566%). Eth- anol (1.763%) was the least abundant compound. A quan- titative comparison of volatile compounds in Fischerella sp. under control conditions and in the maltose-containing medium with copper revealed a decrease in 2-methylfuran, ethyl acetate, 2-pentanone, ethanol, 1,3-dimethylbenzene, 2-methyl-L-propanol, acetic acid, benzaldehyde, and 1-octen-3-ol compared to the control. Conversely, an increase was observed in 3-methylbutane, 2-methylbutane, methyl acetate, 2-butanone, acetic acid, 3-methylbutyl ester, 1-butanol, 3-methyl-1-butanol, 3-hydroxy-2-butanone, 2-phenylethanol, and 3-octen-2-ol (Table 1). Discussion Heavy metal contamination is one of the major environ- mental challenges, particularly in aquatic ecosystems, where it has extensive negative impacts. The use of bio- logical methods for removing these pollutants, including the application of cyanobacteria as natural and efficient agents, has garnered significant attention. Cyanobacteria are capable of adsorbing and removing heavy metals from aquatic environments, with the production of EPS playing a key role in this process. In this context, the present study investigates the impact of autotrophic and mixotrophic culture conditions on heavy metal adsorption by two cyanobacterial species, Fischerella sp. and Desmonostoc alborizicum, isolated from wetlands and qanats in Golestan Province. This study represents a novel approach aimed at optimising heavy metal removal and enhancing the bioremediation efficiency of these species. The findings of this study indicated that Fischerella sp. exhibited a greater ability to produce dry cell biomass com- pared to Desmonostoc alborizicum. This advantage may be attributed to factors such as higher efficiency in nutrient uptake and metabolism, particularly in sugar utilisation, in Fischerella sp. The significant increase in dry cell weight in both strains up to 48 hours suggests their adaptation to the culture conditions and effective utilisation of available resources, particularly maltose as a carbon and energy source for growth and proliferation. A study by Xie et al. (2011) examined the effect of glucose on the growth of the cyanobacterium Synechocystis PCC 6714. Their results showed that glucose supplementation not only enhanced growth but also led to glucose consumption and a reduc- 119 Acta Biologica Slovenica, 2025, 68 (4) Figure 5. Results of exopolysaccharide measurements in cyanobacterial strains (a) Fischerella sp., (b) Desmonostoc alborizicum in culture media containing heavy metals and (1) maltose, (2) lactose, (3) glucose, (4) sucrose, and (5) control. Slika 5. Rezultati meritev eksopolisaharidov v cianobakterijskih sevih (a) Fischerella sp., (b) Desmonostoc alborizicum v gojiščih, ki vsebujejo težke kovine in (1) maltozo, (2) laktozo, (3) glukozo, (4) saharozo in (5) kontrolno skupino. 120 Acta Biologica Slovenica, 2025, 68 (4) Figure 6. presents the results of protein content measurements (mg/ml) in the cyanobacterial strains (a) Fischerella sp. and (b) Desmonostoc alborizicum in culture media containing (1) maltose, (2) lactose, (3) glucose, (4) sucrose, and (5) control. Slika 6. prikazuje rezultate meritev vsebnosti beljakovin (mg/ml) v cianobakterijskih sevih (a) Fischerella sp. in (b) Desmonostoc alborizicum v gojiščih, ki vsebujejo (1) maltozo, (2) laktozo, (3) glukozo, (4) saharozo in (5) kontrolno skupino. 121 Acta Biologica Slovenica, 2025, 68 (4) tion in its concentration in the culture medium. These find- ings align with those of the present study, highlighting the role of sugars as accessible carbon sources that promote cyanobacterial growth and biomass production. However, it is important to note that Xie et al.'s study utilised glu- cose, whereas the present research focused on maltose. Therefore, it can be inferred that the cyanobacterial strains examined in both studies are capable of utilising glucose, either directly or as part of maltose, for their growth (Xie et al., 2011). The results of EPS measurements in this study revealed that Fischerella sp. produced approximately 1.3 times more exopolysaccharides in the maltose-containing medium compared to Desmonostoc alborizicum. This finding sug- gests that maltose, as a carbon source, has a significant impact on EPS production in these strains, particularly in Fischerella sp. In this regard, Yu et al. (2010) also observed that maltose had the greatest influence on exopolysaccha- ride production in Nostoc flagelliforme (Jia et al., 2010). Yu et al. (2012) demonstrated that adding sugars such as glucose, sucrose, and glycerol to the mixotrophic culture medium of Nostoc flagelliforme leads to an increase in EPS production and biomass. Other studies have also exam- ined the role of different carbon sources in EPS production in cyanobacteria (Yu et al., 2012). Hassan et al. (2012) showed that a maltose-containing medium is suitable for the growth of cyanobacterial species, and treating the strains with sugars such as glucose, sucrose, maltose, and lactose enhances extracellular polysaccharide production, protein content, and biomass (Hassan et al., 2012). Nieves- Morión and Flores (2018) also reported increased growth of Anabaena sp. in the presence of sucrose, fructose, and glucose. Although these studies emphasise the effects of various sugars, the findings of the present research indicate that maltose has a more pronounced impact on EPS production in the studied strains compared to other Peak IDs Molecular Formula RT (time) Nature of Abandnce (%) Increase / Compounds 16 h 24 h Decrease 2-Methylfuran C5H6O 4.57 Furan 7.231 5.181 1.39 % Decrease 3-Methylbutanal, 2-methylbutanal C5H10O 5.14 Aldehyde 12.210 18.718 1.53 % Increase Acetic acid methyl ester C3H6O2 7.02 Eester 5.742 6.518 1.13 % Increase Acetic acid ethyl ester CH3COOC2H5 7.41 Eester 6.574 2.551 2.57 % Decrease 2-Butanone C4H8O 8.33 ketone 4.727 4.838 1.02 % Increase 2-Pentanone C5H10O 8.87 ketone 3.937 3.577 1.10 % Decrease Ethanol CH₃COOH, C9H18O2 9.24 Ester 3.648 1.763 2.06% Decrease Acetic acid, 3-methylbutyl ester CH3(CH2)3OH 13.33 Alcohol 4.141 4.504 1.08 % Increase 1-butanol C6H4(CH3)2 13.82 benzene 2.729 3.024 1.11 % Increase 1,3-dimethylbenzene C4H9OH 14.68 Alcohol 3.982 3.744 1.06 % Decrease 2-Methyl-l-propanol C5H12O 15.07 Alcohol 11.056 7.939 1.39 % Decrease 3-Methyl-1-butanol CH₃COOH, C9H18O2 15.61 Ester 7.986 12.566 1.57% Increase 3-Hydroxy-2-butanone C8H16O4 17.26 ketone 3.105 4.546 1.46% Increase Acetic acid CH3COOH 20.83 Acid 3.615 2.510 1.44% Decrease Benzaldehyde C7H6O 22.85 Aldehyde 8.617 7.477 1.15 % Decrease 2-Phenylethanol C8H10O 28.41 Alcohol 2.003 2.542 1.26% Increase 1-Octen-3-ol C8H16O 28.77 alkenyl alcohol 5.255 4.490 1.17 % Decrease 3-Octen-2-ol C8H16O 29.53 alkenyl alcohol 3.441 3.511 1.02 % Increase Table 1. Comparison of Volatile Compound Analysis in Fischerella sp. Cultured in Maltose-Containing Medium with Copper and Control Medium. Tabela 1. Primerjava analize hlapnih spojin v Fischerella sp., gojeni v gojišču, ki vsebuje maltozo, s bakrom in kontrolnem gojišču. 122 Acta Biologica Slovenica, 2025, 68 (4) examined sugars. In addition to the influence of carbon sources, other factors also play a role in EPS production (Nieves-Morión and Flores, 2018). Pereira et al. (2009), in their review study, highlighted the role of available energy parameters (provided by sugar sources) and the carbon-to-nitrogen (C:N) ratio in EPS pro- duction (Pereira et al., 2009). Finally, the study by Zhang et al. (2019) emphasised the role of maltose in exopolysaccha- ride production in Lactobacillus sanfranciscensis, showing that maltose can serve as an effective carbon source for EPS production in various microorganisms (Zhang et al., 2019). In this study, the results of heavy metal removal indicated that in Fischerella sp., copper removal was higher than that of nickel and chromium. The highest removal efficiency for all three heavy metals was recorded at 10 minutes, after which it decreased at 30 minutes and then reached a relatively stable level at 60 to 90 minutes. Similar results, though with lower efficiency, were observed for the strain Desmonostoc alborizicum. A comparison of heavy metal removal efficiency between Fischerella sp. and Desmono- stoc alborizicum revealed that the strain isolated from the wetlands of Golestan Province exhibited a greater ability to remove heavy metals from the environment within a fixed time period. Mota et al. (2016) conducted an experiment on the unicellular cyanobacterium Cyanothece sp. CCY 0110 to assess the efficiency of removing three pollutants— copper, cadmium, and lead—individually. They reported that released polysaccharides (RPS) from Cyanothece sp. CCY 0110 exhibited higher biosorption efficiency for heavy metal removal (Mota et al., 2016). These studies have demonstrated that cyanobacterial exopolysaccharides can facilitate heavy metal removal from the environment, which aligns with the findings of the present study. Pereira et al. (2011) used exopolysaccharide-producing cyanobacteria for heavy metal removal. In their research, the cyanobacterial species Gloeothece sp. PCC 6909 was employed to study the removal of copper and lead (Pereira et al., 2011). The results showed that both this cyanobacterial species and its mutant strain lacking exopolysaccharides were capable of removing copper and lead from the environment. Fur- thermore, this research group demonstrated that structural changes occurred in both the wild-type and mutant strains when exposed to copper. This study observed that lead was removed more efficiently from the environment than copper. Mohite et al. (2018) investigated heavy metal stress and its role in exopolysaccharide production in Pantoea agglomerans. In this study, the heavy metals mercury, copper, silver, arsenic, lead, chromium, and cadmium were examined. The results indicated that metal-induced stress reduces total protein content (Mohite et al., 2018). Surosz and Palinska (2016) demonstrated that heavy metals such as copper and cadmium significantly decrease total cellular protein content in the cyanobacterium Anabaena flos- aquae. Additionally, this study observed that copper had a greater effect than cadmium in reducing total cellular pro- tein concentration (Surosz and Palinska, 2016). Okmen et al. (2011) evaluated the impact of zinc on chlorophyll a, total carbohydrates, total protein, and biomass. They observed that at a concentration of 10 mg/L (a high concentration of this metal), biomass, total protein, and chlorophyll a levels significantly decreased (Okmen et al., 2011). Shukla et al. (2009) showed that treating the cyanobac- terial species Anabaena doliolum with nickel at concentra- tions of 10 and 50 μmol/L led to an increase in total cellular protein concentration, but increasing the concentration to 100 μmol/L reduced protein content, indicating the toxic effects of this metal at higher concentrations (Shukla et al., 2009). Elsalhin et al. (2016) evaluated the effect of cobalt on total protein and total carbohydrates in the cyanobacterium Spirulina platensis. The results of this study demonstrated that at low concentrations of cobalt, total protein and carbohydrate concentrations increased; however, with increasing concentrations of this heavy metal, these parameters significantly decreased, indicating its toxic effects (Elsalhin et al., 2016). Martínez-Ruiz et al. (2016) investigated the effect of the heavy metal nickel on the total cellular protein, total carbohydrate, and lipid con- tent of the bacterium Microcystis aeruginosa. The results of this study demonstrated that increasing the concentration of nickel up to 1.64 μg/L led to an increase in total cellular protein, carbohydrate, and lipid concentrations. However, at a concentration of 3.7 μg/L, the total carbohydrate content decreased, whereas the total protein and lipid concentrations continued to increase. The findings of previous studies confirm the results of the present study, as we also observed that treatment of Fischerella sp. and Desmonostoc alborizicum species with the heavy metals copper, chromium, and nickel resulted in an increase in total cellular carbohydrate content, with nickel playing a more significant role in enhancing carbohydrate levels (Martínez-Ruiz et al., 2016). In this study, GC-MS was used to analyse the chemi- cal compounds and their relative abundances in both the control sample and samples treated with the heavy 123 Acta Biologica Slovenica, 2025, 68 (4) metal elements chromium, nickel, and copper. The results indicated that treatment with copper led to an increased percentage of the following compounds: 3-methylbutane, 2-methylbutane, methyl acetate ester, 2-butanone, acetic acid, 3-methylbutyl ester, 1-butanol, 3-methyl-1-butanol, 3-hydroxy-2-butanone, 2-phenylethanol, and 3-octan-2-ol. In 2013, researchers demonstrated that the primary fatty acid components in Nostoc and Scenedesmus sp. treated with the heavy metals lead, zinc, copper, cadmium, man- ganese, chromium, and nickel included methyl palmitate, methyl oleate, methyl linoleate, and methyl linolenate. Gheda et al. (2020) reported that the compound cyclotris- iloxane in Oscillatoria acuminata, Oscillatoria amphigranu- lata, and Spirulina platensis exhibited antioxidant activity and mitigated the toxic effects of heavy metals (Gheda et al., 2020). Babu et al. (2010) studied the biodegradation of phthalate esters by cyanobacteria. In this study, the cya- nobacterial species Microcystis aeruginosa and Anabaena flos-aquae G. S. West were examined, and GC-MS results revealed a significant presence of benzenedicarboxylic acid 1,2-, which is consistent with the findings of this study (Babu and Wu, 2010). While this study provides valuable insights into the heavy metal bioremediation potential of Fischerella sp. and Desmonostoc alborizicum under autotrophic and mixotrophic culture conditions, several limitations should be considered. The experiments were conducted under controlled laboratory conditions, which may not fully rep- resent the complex and dynamic conditions of natural eco- systems. Additionally, only a limited range of heavy metals was evaluated, and the long-term stability, regeneration, and reusability of cyanobacterial biomass for practical wastewater treatment were not assessed. Future research should focus on applying these strains in pilot-scale or field studies to validate their heavy metal removal efficiency in real contaminated environments. Furthermore, deeper exploration of the molecular mechanisms involved in exopolysaccharide biosynthesis and heavy metal binding, along with optimisation of culture parameters for scale-up and continuous operation, would facilitate the practical and sustainable application of these native cyanobacterial strains in environmental bioremediation. Conclusion This research demonstrated that native Iranian cyano- bacteria, particularly the Fischerella sp. strain, can serve as effective candidates for the removal of heavy metals from aquatic ecosystems. The increased production of exopolysaccharides in culture media enriched with carbon sources such as maltose played a key role in enhancing metal removal efficiency. Additionally, volatile compound analysis indicated positive metabolic changes under heavy metal treatment. These findings not only introduce high-po- tential cyanobacterial strains for heavy metal removal but also propose an environmentally friendly approach to mitigating metal pollution in water resources. By optimising culture conditions and further investigating the underlying biochemical mechanisms, this study lays a foundation for developing sustainable and scalable bioremediation tech- nologies with practical application potential in wastewater treatment and environmental restoration. Author Contributions Conceptualization, B. N.; methodology, all authors; soft- ware, B.N.; validation, B.N; formal analysis, B.N.; investiga- tion, H.B.; writing—original draft preparation, H.B; writing— review and editing, B.N. All authors have read and agreed to the published version of the manuscript. Acknowledgments Not applicable Funding This research did not receive any specific grant from fund- ing agencies in the public, commercial, or not-for-profit sectors. 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Continuous Bioprocessing for Recombinant Protein Production in Bacillus subtilis: Opportunities and Challenges. Acta Biologica Slovenica 68 (4) Received: 13.05.2025 / Accepted: 14.07.2025 / Published: 21.07.2025 https://doi.org/10.14720/abs.68.4.22623 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Review Continuous Bioprocessing for Recombinant Protein Production in Bacillus subtilis: Opportunities and Challenges Andrej Ivanovski 1, * , Tomaž Bratkovič 2 Abstract The Gram-positive bacterium Bacillus subtilis has long been used for industrial protein production owing to its well-characterised genetic background, capacity for high-density fermentation, and effective protein secretion. With the growing global demand for recombinant proteins across various sectors, there is an increasing need for more efficient and sustainable protein production methods. This review explores the application of continuous bioprocessing for recombinant protein production using B. subtilis, critically evaluating current technologies and discussing their potential for integration into streamlined, integrated biomanufacturing framework. We primarily focus on transgenic B. subtilis, however the solutions for continuous fermentation and protein isolation/purification are the same for both endogenous and heterologous (i.e., recombinant) proteins. Keywords Continuous Bioprocessing; Bacillus subtilis; Upstream Processing; Downstream Processing; Recombinant Protein Production 127 Acta Biologica Slovenica, 2025, 68 (4) Kontinuirno bioprocesništvo za proizvodnjo rekombinantnih proteinov v Bacillus subtilis: priložnosti in izzivi Izvleček Po Gramu pozitivno bakterijo Bacillus subtilis že dolgo uporabljamo v industrijski proizvodnji proteinov. K temu prispevajo njena dobra raziskana genetska zasnova, možnost doseganja visoke biomase med fermentacijo in učinkovito izločanje proteinov. Zaradi naraščajočega svetovnega povpraševanja po rekombinantnih proteinih v različnih sektorjih, se povečuje tudi potreba po učinkovitejših in trajnostnih metodah proizvodnje proteinov. V članku predstavljamo potencial kontinuirnega bioprocesništva pri proizvodnji rekombinantnih proteinov z uporabo B. subtilis. Ponujamo pregled obstoječih tehnologij in njihovega potenciala za vključitev v integrirani koncept učinkovitejše bioproizvodnje rekombinantnih proteinov. Čeprav se prispevek primarno posveča transgenim bakterijam B. subtilis, so rešitve kontinuirne fermentacije in izolacije/čiščenja proteinov enake za endogene in heterologne (tj. rekombinantne) proteine. Ključne besede Kontinuirno bioprocesiranje; Bacillus subtilis; pripravljalni procesi; zaključni postopki; proizvodnja rekombinantnih proteinov Introduction The demand for recombinant proteins is rapidly increas- ing, driven by their expanding applications in healthcare, agricultural and material sector. While the expanding bio- pharmaceutical market remains a key driver, the need for recombinant enzymes, bio-pesticides, bio-stimulants and bio-based materials is also steadily rising, encouraging the development of efficient and scalable production strategies (Evens and Pharm, 2022; Kergaravat et al., 2025; Marrone, 2024). To meet this growing demand, the bioindustry is actively exploring strategies for process intensification. Continuous processing has already demonstrated its ability to boost productivity and reduce turnaround times across various industries. Accordingly, the bioindustry is shifting towards continuous manufacturing to improve productivity and ensure consistent product quality, while also reducing environmental impact (Jungbauer et al., 2024; Peebo and Neubauer, 2018). Importantly, the motivation for adopting continuous biomanufacturing is not solely economic. Regulatory bodies have recognized several advantages of continuous processing that contribute to improved product quality. Maintaining a steady-state operation is crucial for ensuring consistent quality, while the elimination of interruptions between process steps enhances both the reliability and safety of manufacturing (Godawat et al., 2015; Subramanian, 2017; Walther et al., 2015). In the bioindustry, the cultivation of microorganisms is a critical step in protein production (Y ang et al., 2021). In this context, Bacillus species represent an attractive platform due to their high secretion capacity and ability to produce a range of recombinant proteins at high titers, supporting the needs of the expanding market. This paper focuses on Bacillus subtilis, a microorganism long employed for producing diverse proteins, enzymes, and various chemicals (Lopez et al., 2009). The aim is to review approaches in upstream and downstream processing for recombinant protein production using B. subtilis and to explore the potential of continuous biomanufacturing as a solution for process intensification. Bacillus subtilis as a Host for Recombinant Protein Production Escherichia coli remains the most widely used prokaryotic host for recombinant protein production. However, B. subtilis is widely used in industrial homologous protein production and its popularity for recombinant protein expression is increasing (Errington and van der Aa, 2020). Classified as Generally Recognised as Safe (GRAS), B. sub- tilis offers several advantages: a well-characterised genetic 128 Acta Biologica Slovenica, 2025, 68 (4) framework, suitability for high-density fermentation, lack of lipopolysaccharides, and an inherent ability to secrete proteins (Yang et al., 2020, 2021). Numerous strategies, including selection of parental strains and strain engi- neering, have been used to enhance recombinant protein production in B. subtilis. (Zhang et al., 2020a). The initial step is to select a parental strain that shows promising characteristics in terms of its genetic background. This is followed by strain engineering, a process that is essential for meeting the demands of modern recombinant protein production (Zhang et al., 2018). One of the major challenges is that heterologous proteins are generally more suscep- tible to proteolytic degradation than native proteins. This vulnerability arises primarily from slower folding kinetics and an increased tendency for misfolding after membrane translocation (Li et al., 2004). Two main strategies have been proposed to mitigate these issues. The first focuses on improving the secretory protein-folding environment, while the second aims to reduce extracellular proteolytic activity (Zhang et al., 2020a). Regarding the first approach, B. subtilis naturally produces intracellular chaperones that assist in the folding of nascent polypeptides and prevent the formation of intracellular protein aggregates – pro- cesses that have been identified as potential bottlenecks in recombinant protein production. Advances in genome editing technologies have enabled the generation of mul- tiple B. subtilis variants in which secretory pathways and signal peptides have been optimized, and key intracellular chaperones have been characterized and overexpressed (Zhang et al., 2020b). The second approach targets extra- cellular proteolysis. B. subtilis secretes seven extracellular proteases, each capable of degrading proteins at different cleavage sites, often contributing to nutrient recycling. By precisely modulating protease expression levels and activities, researchers have significantly improved the yield of extracellular recombinant proteins (Zhang et al., 2018). Upstream Processing Upstream processing represents a critical phase in each biomanufacturing operation and has specific characteristic when using B. subtilis as a host. Unlike E. coli, B. subtilis naturally secrets many proteins into the medium, avoiding the need for cell lysis (Chen et al., 2024). Generally, three primary modes of upstream processing are distinguished: batch, fed-batch, and continuous (Subramanian, 2017) with various sub-variants, such as repetitive fed-batch processes developed to bridge the gap between tradi- tional fed-batch and fully continuous upstream processing through semi-continuous strategies (Kopp et al., 2020). All these approaches are applicable to B. subtilis. However, their implementation must consider organism-specific chal- lenges, including oxygen transfer rates due to the aerobic nature of B. subtilis, as well as the control of sporulation and protease activity during cultivation (Chen et al., 2024). Despite differences in operation, all three modes share a common initiation process. Each begins with a working cell bank (WCB) aliquots revival carried out in shake flasks, followed by seed expansion in a bioreactor and, ultimately, inoculation of the production bioreactor. The major distinc- tions between batch, fed-batch, and continuous processes emerge during the production phase itself (Subramanian, 2017). A brief comparison of these three operational modes is provided in the following section. Batch mode In batch processing, the bioreactor is filled with growth medium up to the final working volume, inoculated, and processed until completion. The process is monitored online using integrated sensors, and regular sampling is performed for in-process analysis (Subramanian, 2017). For B. subtilis, batch production typically spans 1-2 days (Ji et al., 2015). When the bioprocess is complete, the culture is transferred to downstream processing. Manufacturing is carried out through successive batch operations, with the product accumulated at the end of each cycle. Fed-batch mode In fed-batch processing, the bioreactor is initially filled with growth medium to approximately 65-90% of the final work- ing volume, followed by inoculation. Feeding with nutrient supplements is then initiated based on predefined criteria and continues until the final working volume is reached. As with batch mode, the process is monitored online through integrated sensors, and samples are collected for in-process analysis, including product yield, cell density and biochemical profiling (Subramanian, 2017). For B. sub- tilis, fed-batch production typically lasts between one and four days, depending on the feeding strategy employed (Klausmann et al., 2021). After the bioprocess concludes, the culture is transferred to the downstream processing. 129 Acta Biologica Slovenica, 2025, 68 (4) Like batch mode, production in fed-batch mode is achieved through successive, repeated cycles. Continuous mode In continuous processing, the bioreactor is initially filled to 40-50% of the final working volume and inoculated. Once the target cell density is reached, a continuous dilution process is initiated, involving the steady feeding of fresh medium while simultaneously harvesting culture at the same flow rate (Subramanian, 2017). The system is maintained at a steady state, ensuring a constant cell density and a constant working volume throughout the operation. For B. subtilis, continuous bioprocesses typically run for five to ten days (Kittler et al., 2025). Culture containing the desired product is continuously harvested and sent directly to downstream processing, running in parallel with upstream operations. Continuous Upstream Processing Historically, conventional fed-batch bioprocesses have been the industrial standard for B. subtilis, primarily due to their operational robustness and high space-time product yields (Zhang et al., 2020b). However, technological advances now support continuous processing, and mod- el-based studies suggest that continuous cultivation of microorganisms can achieve similarly high, and sometimes superior production rates. The primary advantage of a continuous bioprocessing lies in the ability to maintain cells in a steady state, enabling sustainable and consis- tent protein production (Kittler et al., 2025; Subramanian, 2017). For example, continuous cultures of B. subtilis have demonstrated the capacity to produce recombinant proteins at stable, high levels over extended periods. One study reported that continuous cultivation-maintained chloramphenicol acetyltransferase production at relatively high level, with stable protein expression observed at a dilution rate of 0.2 h -1 (Vierheller et al., 1995). In contrast, batch and fed-batch processes often experience fluctua- tions in nutrient concentrations and perturbation of cellular metabolic states, leading to variability in both protein yield and quality. While fed-batch processes allow for controlled nutrient addition, they still face challenges such as accu- mulation of inhibitory by-products and undesirable metab- olism shifts. Despite optimization, fed-batch processes for B. subtilis continues to encounter issues related to oxygen limitation and by-product accumulation (Öztürk et al., 2016). Nonetheless, continuous bioprocessing is not without challenges. Prolonged cultivation times increase the risk of contamination, and maintaining genetic stability over extended periods is critical. Loss of plasmids, spontaneous mutations, and genetic drift can lead to significant reduc- tions in productivity (Csörgo et al., 2012; Croughan et al., 2015). Thus, the development of a robust expression system is imperative for successful continuous protein production. Recent studies have further nuanced this discussion. For instance, a study with Bacillus lichenioformis reported that an optimized fed-batch process outperformed a continu- ous process in terms of productivity (Kittler et al., 2025). Therefore, while continuous upstream processing can enhance sustainability and process consistency, it typically demands more sophisticated control systems and may result in lower per-cycle productivity compared to highly optimized fed-batch operations. Downstream Processing Downstream processing (DSP) aims to eliminate impurities and product variants while preserving the integrity of the final product (Jungbauer et al., 2024). When using B. sub- tilis for recombinant protein production, DSP benefits from the organism’s natural capacity to secrete proteins into the extracellular environment, which avoids the need for cell lysis in many cases and simplifies the clarification step (Chen et al., 2024). However, B. subtilis-specific challenges include the removal of secreted host-cell proteins, notably extracellular proteases that can degrade the product. Therefore, the implementation of protease-deficient strains could be critical to preserve product integrity (Zhang et al., 2018). A streamlined biomanufacturing workflow relies on the integration of various technologies, including cell lysis, filtration, chromatography, refolding, precipitation, and extraction (Jungbauer, 2013; Jungbauer et al., 2024). The following section provides a brief general overview of key the technologies used in the context of continuous down- stream protein processing while assessing their relevance for B. subtilis-based recombinant protein production. Cell lysis When the product is expressed intracellularly, the first step of DSP is typically cell lysis, which ruptures the cell membrane to release the intracellular components. High-pressure 130 Acta Biologica Slovenica, 2025, 68 (4) homogenisation is the most widely used industrial method due to its high efficiency and robustness. In this method, a piston pump generates high pressures, leading to slit cavitation around the valve area. The sudden release of the pressure causes effective cell breakage. Modern high-pres- sure homogenisers are capable of operating in continuous mode, enabling integration into continuous biomanufac- turing workflows (Subramanian, 2017). In the production of recombinant proteins with B. subtilis, the product is most often found extracellularly. Therefore, cell lysis is generally bypassed in B. subtilis-based processes unless targeting specific intracellular product (Yang et al., 2021). Centrifugation Centrifugation separates solids from liquids and is a cru- cial step in DSP. In B. subtilis-based recombinant protein production, this operation primarily used for clarification of the fermentation broth by separating the biomass from the extracellularly secreted target proteins (Patel et al., 2019). The most common centrifuge designs are the tubular, chamber and disk-stack centrifuge. Among these, the disk- stack centrifuge is particularly suitable for pseudo-continu- ous operation, allowing the transient removal of liquid and solid phases during processing (Jungbauer, 2013). Filtration Filtration plays a vital in DSP, supporting tasks such as cell-liquid separation, protein concentration, and buffer exchange. Filtration processes are classified into microfil- tration, ultrafiltration and nanofiltration, distinguished by their pore sizes. Microfiltration, with the largest pore size, is used for cell and cell debris separation, whereas ultrafiltra- tion and nanofiltration are commonly employed for protein concentration and buffer exchange. For Bacillus cultures, the high tendency for foaming and the presence of DNA fragments can increase the viscosity of the broth, requiring adapted filtration strategies or precipitation approaches to ensure cell debris and DNA removal, respectively, during continuous processing (Prabakaran and Hoti, 2008). Continuous filtration is an established technique in bio- manufacturing and can be achieved by connecting multiple filtration units in series, where the permeate from one unit feeds into the next enabling uninterrupted flow. Engineer- ing strategies for implementing continuous filtration have been comprehensively reviewed (Jungbauer, 2013). Chromatography Chromatography is often an indispensable step in DSP. Conventional packed-column chromatography operates in batch mode, with sequential loading, washing, and elution steps. However, significant research efforts have focused on developing continuous chromatography methods to improve productivity, purity, cost efficiency, and equipment footprint (Subramanian, 2017; Vogel et al., 2002). Depending on the desired purity of the final product, multiple chromatographic stages (i.e. purification train) may be implemented, utilising various physical and chemical differences between proteins to achieve a higher level of purity (Liu et al., 2010). The simplest form of continuous chromatography involves operating multiple columns in parallel, where columns alternate between loading, washing, elution, regeneration, and re-equili- bration phases. This rapid cycling approach is already widely applied in the bioindustry. Additionally, alternative technologies such as rotating chromatography devices or counter-current chromatography have been developed to minimise equipment needs and streamline continuous operations (Jungbauer, 2013). When applying these strate- gies to B. subtilis, careful optimisation of chromatographic conditions becomes essential due to its high secretion of endogenous proteases and other host proteins, which can co-elute with the target recombinant proteins, requiring tailored purification strategies to achieve the desired purity levels (Shih et al., 2013). Extraction Liquid–liquid extraction (LLE) has emerged as a promising alternative to chromatography-based purification and has been extensively studied over recent decades (Sub- ramanian, 2017). LLE relies on the differential partitioning of target products between the two immiscible aqueous phases. For example, phase-separating mixtures of polyethylene glycol (PEG) or dextran with appropriate salts can be used to achieve selective protein separation. Several continuous LLE methods have been developed and successfully applied for protein separation (Kee et al., 2021; Muniasamy et al., 2022). Since B. subtilis translocate recombinant proteins in the extracellular space, LLE can be applied directly to fermentation broth, enabling in situ or integrated extraction approaches (Kee et al., 2021). 131 Acta Biologica Slovenica, 2025, 68 (4) Integration of Upstream and Downstream Processing Current trends in the bioindustry are increasingly shifting toward integrated continuous bioprocessing. Efforts are focused on developing solutions at every stage of produc- tion to realise the fully integrated continuous bioprocessing concept (Jungbauer et al., 2024), as illustrated in Figure 1. However, literature regarding the continuous production of recombinant proteins using B. subtilis remains limited. Nevertheless, several studies have explored integrative approaches with B. subtilis for enzyme production, providing useful insights into potential strategies for con- tinuous recombinant protein manufacturing. The following paragraph presents various applications of integrative approaches for protein production using B. subtilis. Kee et all (2021) implemented an in situ extractive approach for the production and recovery of B. subtilis xylanase. In their study, crude fermentation broth was directly fed to a bipha- sic (alcohol/salt) flotation unit during the bioprocess. The authors demonstrated that gas bubbles facilitated the par- titioning process, with xylanase being retained in the upper alcohol phase. Muniasamy et all (2022) combined B. subtilis fermentation with in situ liquid-liquid extraction of a secreted fibrinolytic protease (FLP). In this process, shrimp waste hydrolysate served as a sustainable, low-cost substrate, while the FLP enzyme was concurrently extracted in a micel- lar two-phase system. Additionally, gel chromatography was used to quantify the amount of recovered enzyme, followed by final purification through anion exchange chromatogra- phy to achieve maximum purity and enzyme activity. Ng et all (2018) reported the direct extraction of xylanase enzyme from fermentation broth with alcohol/salt aqueous systems. Two earlier studies also highlight that interest in continuous biomanufacturing has existed for several decades. Stre- danský et all (1993) demonstrated the simultaneous produc- tion and purification of B. subtilis α-amylase. Their bioreactor contained an aqueous two-phase system composed of PEG and dextran. During fermentation, culture medium was trans- ferred to an external settler; following phase separation, the dextran-rich bottom phase, containing cells, was returned to the bioreactor, while the PEG-rich upper phase was pumped to an affinity column where the amylase was bound. The PEG flowthrough from the column was then recycled back into the bioreactor. Cooper et all (1981) developed a large- scale continuous bioprocess for the production of surfactin, a cyclic lipopeptide surfactant. In their system, surfactin accumulated in the foamed broth within the bioreactor, and the foam was collected in an external separator for product extraction via acid precipitation. Challenges and Future prospects In the bioindustry, continuous processing is the dominant approach for several applications, including wastewater treatment, composting, and specific bioenergy processes such as biogas and bioethanol production (Brethauer and Wyman, 2010; Matassa et al., 2016; Subramanian, 2017). Figure 1. Fully integrated continuously manufacturing platform concept. Created in BioRender. Slika 1. Koncept popolnoma integrirane platforme za neprekinjeno proizvodnjo. Ustvarjeno v BioRender. 132 Acta Biologica Slovenica, 2025, 68 (4) Nevertheless, most industrial bioprocesses still operate in batch or fed-batch modes, with fed-batch currently being the predominant production strategy. Despite the potential advantages of continuous processing, several organ- ism-specific challenges must be addressed for recombinant protein production in B. subtilis including control of sporu- lation and protease activity during prolonged cultivations, maintenance of genetic stability, and adaptation of oxygen transfer systems due to the aerobic nature of the microor- ganism (Chen et al., 2024). Strategies such as the use of sporulation-deficient strains, targeted protease knockouts, and optimized feeding strategies could help mitigate these limitations while supporting steady-state productivity (K. Zhang et al., 2018, 2020a). Furthermore, the integration of synthetic biology tools for strain stabilization, combined with advanced process monitoring under Quality-by-Design framework, may facilitate the consistent quality and yield of recombinant proteins during continuous production. In DSP, continuous strategies such as counter-current chromatog- raphy, continuous filtration, and integrated LLE systems are emerging to complement continuous upstream operations. These approaches can reduce buffer consumption, equip- ment footprint and overall processing times while increas- ing productivity and cost efficiency. However, implementing continuous DSP for B. subtilis remains challenging due to the complexity of fermentation broth and the secretion of host proteins that may co-elute with target products, requir- ing careful optimisation, as well as real-time monitoring and control. 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Acta Biologica Slovenica 68 (4) https://doi.org/10.14720/abs.68.4.23502 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license News FEMS MICRO Milan 2025 – 11. kongres evropskih mikrobiologov Janez Mulec 1, * , Jerneja Ambrožič Avguštin 1 14. – 17. julij 2025, Milano, Italija FEMS-ov (Federation of European Microbiological Societies) kongres mikrobiologov, ki je bil letos v Milanu v Italiji, je privabil kar 1953 udeležencev iz 95 držav. Z več kot 100 udeleženci iz posamezne države so prišli predstavniki Nemčije, Italije, Južne Koreje, Španije in Združenega kraljestva. Tekom dogodka je bilo v treh dneh na ogled več kot 1400 posterjev. FEMS je tokrat finančno podprl udeležbo kar 300 raziskovalcem na začetku znanstvene kariere, kar se je odrazilo tudi v večjem številu mlajših predavateljev, vključno z doktorskimi študenti in podoktorskimi raziskovalci. Za plenarna predavanja je bil predviden čas eno uro, ostala predavanja so trajala 20, 10 ali 5 minut, zato so se morali predavatelji dobro potruditi, da so svoje znanstvene izsledke predstavili zgoščeno. Tokratni kongres je bil posvečen štirim tematskim sklopom. Prvi sklop, poimenovan »Eko inovacije (Eco Innovations)«, je bil posvečen raziskavam o zapletenem/ prepletenem medsebojnem delovanju med mikrobi in njihovim okoljem, tudi v povezavi s podnebnimi spremembami, urbanizacijo, ekosistemi in onesnaževanjem. Drugi sklop z naslovom »Obzorja zdravja (Health Horizons)« je bil namenjen predstavitvi raziskav o vlogi mikrobov in mikrobiomov, povezanih s človekovim zdravjem, vključno s predstavitvijo novih diagnostičnih in terapevtskih metod ter boja proti mikrobom, odpornih proti protimikrobnim učinkovinam in novo porajajočim patogenov. Prelomne mikrobne tehnologije, od sintetične biologije do trajnostnih rešitev, ki omogočajo preoblikovanje industrije in obravnavajo globalne probleme in izzive, so bile predstavljene v sklopu »Biotehnologija (Biotechnology)«. Zadnji, četrti sklop, z zanimivim naslovom »Vključevanje in rast (Engagement & Growth)«, pa je bil posvečen razvoju ključnih veščin za profesionalnega mikrobiologa, vključno s predstavitvijo možnih kariernih poti, financiranja znanstvenega komuniciranja ter podjetništva. Udeleženci kongresa so imeli možnost izbire prisostvovanja ozko specializiranim znanstvenim sekcijam posameznih sklopov, istočasno jih je potekalo do osem, med celotnim kongresom jih je bilo skupaj na voljo kar 82. 135 Acta Biologica Slovenica, 2025, 68 (4) Slika 1. Iz Slovenije je bilo na kongresu v Milanu s prispevki prijavljenih 35 udeležencev. Aktivno so na kongresu svoje raziskave predstavili na sliki od leve proti desni: Nurainina Binti Ayob (Univerza v Ljubljani), Maša Vodovnik (Univerza v Ljubljani), Marko Verce (Uni- verza v Ljubljani), Ines Mandić Mulec (Univerza v Ljubljani), Janez Mulec (Znanstvenoraziskovalni center SAZU), Jerneja Ambrožič Avguštin (Univerza v Ljubljani), Marjanca Starčič Erjavec (Univerza v Mariboru), Tadeja Vajdič (Univerza v Mariboru), Urša Miklavčič (Nacionalni laboratorij za zdravje, okolje in hrano), Sabina Mlakar (Nacionalni laboratorij za zdravje, okolje in hrano, Univerza v Mariboru). Na kongresu so bila predstavljena številna nova dognanja. Na kratko lahko izpostavimo samo nekatera. Lwoffova nagrajenka za dosežke v mikrobiologiji Carmen Buchrieser je potegnila vzporednico mehanizma vstopa in razmnoževanja bakterije Legionella pneumophila v gos- titeljski celici, ki je okoljska ameba, ali alveolarni makrofag. Pri tem igrajo ključno vlogo številni proteini legionele, ki so podobni evkariontskim, kar kaže na koevolucijo in horizon- talne prenose genov. Če si je kdo predstavljal, da so težave z odpornostjo proti protimikrobnim učinkovinam (AMR – Antimicrobial Resistance) problem samo bogatega sveta in bolnišničnega okolja, temu ni tako. Evolucijsko so se geni za AMR razvili z rekombinacijami in mutacijami in »raznesli« v različne bakterije in ekosisteme s horizontalnimi prenosi. Čeprav so bolnišnice še vedno ene od najpomembnejših vročih točk za razvoj in širjenje AMR v domača okolja in naravo, v tem pogledu ne zaostajajo čistilne naprave, ki omogočajo številne prenose tudi na neklinične seve in povečajo breme teh genov v naravnih ekosistemih. V tem pogledu se raziskuje tudi možen prenos tovrstnih genov na sadje in zelenjavo ter pitno vodo zaradi uporabe vode iz čistilnih naprav v kmetijstvu. Da klasična mikrobiologija »ne izumira« in bo vedno aktualna, dokazuje zavedanje o fenotipski heterogenosti mikrobnih (čistih) kultur in s tem povezano tveganje pri interpretaciji, zlasti kliničnih rezultatov raziskav. Prav tako zanimivo je iskanje novih gojišč in pristopov, s katerimi bi lahko spoznali »mikrobno temno snov (microbial dark matter)«. Izraz, izposojen pri astrofizikih, je bil pogosto uporabljen v različnih sekcijah kongresa in opisuje mikrobe, katerih genetske sledi (sekvenciranje DNA, RNA) poznamo, vendar o njih in njihovi funkciji vemo malo ali nič, ker jih ne moremo gojiti in posledično raziskovati s klasičnimi metodami. Prav tako ne poznamo funkcije veliko genov, tudi kultivabilnih bakterij, vendar je za njihovo karakterizacijo eden, ali več izmed štirih pristopov »omik« (genomika, transkriptomika, proteomika, metabolomika) v veliko pomoč. Takšen celostni pristop je zlasti pomemben za boljše razumevanje mikrobne ekologije, saj ni dovolj vedeti zgolj kateri mikrob zaseda neko nišo, ampak zlasti kaj in koliko tam nečesa počne. Mikrobne interakcije so ključne za razumevanje mikrobne dinamike in fizioloških prilagoditev, kar so npr. izpostavili na primeru interakcije med probiotičnim in sporulirajočim sevom Bacillus subtilis ter patogenom Salmonella typhimurium, ali pa v primeru prevzemanja/ mikrobni kraji sideroforov med Pseudomonas putida, Escherichia coli in Corynebacterium glutamicum. Atmosfera ne predstavlja samo način prenosa mikrobov, ampak tudi njihov habitat in vir energije, svetlobne, pa tudi kemijske v obliki plinov v sledovih, kot so vodik, ogljikov monoksid in metan, ki so sicer vedno prisotni. Atmosferski mikrobi kot rezultat svojih encimatskih reakcij proizvedejo vodo (1 milijon vodnih molekul v minuti v posamezni celici), ki je posebej pomembna za življenje v sušnih področjih, za nekatere mikrobe, ekstremne habitate. Na ekstremne 136 Acta Biologica Slovenica, 2025, 68 (4) razmere za nekatere mikrooorganizme, kot je npr. visoka sla- nost, ne naletimo zgolj v naravnih hiperslanih okoljih, ampak tudi v antropogenih, kot so zanimivo, lahko tudi klobase. Odmevno je bilo uvodno plenarno predavanje z naslo- vom »Dinamika talnih mikroorganizmov v vesolju«. Pred- videva se, da bi lahko talni mikroorganizmi igrali ključno vlogo pri osvajanju zunajzemeljskih okolij, npr. Marsa v smislu ustvarjanja primerne prsti za rast rastlin in užitnih pridelkov. Vendar se je treba zavedati, da nezemeljske razmere, npr. gravitacija, dostopnost vode, pomembno vplivajo na fiziološki odziv mikroorganizmov. V okviru astro(mikro)biologije ostaja še veliko odprtih vprašanj. Veliko obeta koncept oziroma metoda za ocenjevanje geografske porazdelitve in raznolikosti mikrobiomov (npr. Microbiome Geographic Population Structure – mGPS) za mikrobne združbe, ki naseljujejo okolja kot so človeško telo, tla, voda ali zrak. Koncept temelji na predpostavki, da so mikrobiomi geografsko strukturirani. Potencialni primeri uporabe je forenzična mikrobiologija, npr. za določitev lokacije izvora bioloških delcev, ki lahko ključno vplivajo na zdravje in varnost ljudi. Da se svet in varnostne razmere spreminjajo, je dejstvo. Varnost, vključno z biološko var- nostjo, postaja zato vedno bolj cenjena dobrina, kar smo občutili udeleženci kongresa tudi na lastni koži. Naj ome- nimo, da je bilo treba pred vstopom v predavalnice opraviti kar tri varnostne preglede (izkaz udeležbe pred vstopom na območje kongresnega centra, zaznavanje kovinskih pred- metov pred vstopom v kongresno stavbo ter izkaz udeležbe pred vstopom na prizorišče kongresnih aktivnosti). Na kongresu je bil viden trend drobljenja mikrobiologije po posameznih zelo specializiranih področjih, vendar je še vedno ključno ohranjati celosten pregled, medicinska ali okoljska mikrobiologija, mikrobov ne zanima. Vse je »ena mikrobiologija«. FEMS je postal pomembna platforma za ustvarjanje povezav in izmenjavo informacij. Med drugim FEMS izdaja tudi več revij: FEMS Microbiology Letters, FEMS Microbi- ology Reviews, FEMS Microbiology Ecology, FEMS Patho- gens and Disease, FEMS Yeast Research, FEMS Microbes, microLife. Kot spodbudo (mladim) udeležencem in vabilo k objavi rezultatov je vsaka od navedenih revij na koncu kongresa v Milanu podelila nagrado za poster oziroma ustno predstavitev s svojega področja. Kot je bilo napovedano na zaključnem plenarnem zasedanju, bo naslednji, 12. kongres FEMS od 5. do 8. julija 2027 v Ljubljani, ki ga bo gostilo SMD – Slovensko mikro- biološko društvo. To je zelo pomenljivo, saj se skoraj četrt stoletja po prvem kongresu FEMS, le-ta ponovno vrača v mesto, kjer se je vse začelo – Ljubljana je namreč leta 2003 gostovala prvi kongres FEMS. Vabimo k udeležbi na kon- gresu, ki ne omogoča samo predstavitve našega dela širši mikrobiološki skupnosti »v živo«, ampak omogoča mladim raziskovalcem na začetku njihove karierne poti prve korake v svet znanstvenih srečanj, mreženja ter strokovne rasti. Slika 2. Matjaž Ocepek z Veterinarske fakultete Univerze v Ljubljani, slovenski delegat pri FEMS-u, je na zaključni prireditvi sprejel zlati mikroskop, popot- nico naslednjemu prireditelju kongresa, ki bo v Ljubljani od 5. do 8. julija 2027. Na sliki od leve proti desni: Tadeja Vajdič, Jerneja Ambrožič Avguštin, Marjanca Starčič Erjavec, Matjaž Ocepek, Ines Mandić Mulec, Marko Verce. Acta Biologica Slovenica 2025 Vol. 68 | Št. 4