Microhabitat and microtrophic differentiation of ecological niches of green frogs (Ranidae: Pelophylax esculentus complex) in riparian areas of South Banat, Serbia

elene žabe iz Pelophylax esculentus kompleksa su akvatične, sintopijske i gregarne žabe širokog rasprostranjenja sa izraženom ulogom u ekosistemima koje naseljavaju. Iako se na teritoriji Srbije i susednih zemalja zelene žabe istražuju u kontinuitetu već više od sto godina, saznanja o njihovoj ekologiji i prostornoj integraciji populacija su malobrojna posebno u delu areala kompleksa gde se javljaju populacioni sistemi sa sva tri taksona u sintopiji. U okviru ove teze urađena je parametrizacija mikrostanišne i mikrotrofičke niše zelenih žaba iz Pelophylax esculentus kompleksa na području Južnog Banata kao i analiza prostorne integracije populacija sa analizom rizika izumiranja. Dodatno, analizirane su epibiontske zajednice kože žaba i kondicioni status sva tri taksona na svakom od lokaliteta. Na kraju, urađena je sintetska reevaluacija lokalnog konzervacionog statusa na istraživanom području. Jedinke su sakupljane na tri lokaliteta u Južnom Banatu koji se razlikuju po svojim hidrološkim, orografskim i stanišnim karakteristikama ali i po stepenu antropogenog uticaja. Jedinke su identifikovane na osnovu standardnih mera za analizu varijabilnosti morfoloških karaktera (6 kvalitativnih i 8 kvantitativnih) uz adekvatnu statističku obradu i validaciju nezavisnom grupom uzoraka identifikovanih genetičkim markerima. Na taj način je od ukupno 317 analiziranih žaba identifikovano 63 jedinki P. ridibundus, 29 jedinki P. lessonae i 225 jedinki P. esculentus pri čemu je konstatovano da se korišćenjem morfoloških karaktera može na brz i ekonommičan način uraditi dovoljno pouzdana identifikacija taksona unutar Pelophylax esculentus kompleksa. Kondicioni status jedinki je procenjen korišćenjem rezidualnog kondicionog indeksa koji predstavlja uobičajenu metodu u batrahologiji. Parentalne vrste su imale bolju telesnu kondiciju u odnosu na hibridni takson a sva tri taksona su generalno lošiju telesnu kondiciju imale na lokalitetu koji je pod najvećim antropogenim pritiskom. Materijal za trofičku analizu je prikupljen neinvazivnom tehnikom ispiranja želuca a obrasci trofije su utvrđeni multivarijantnim statističkim metodama i testirani permutacionim analizama prema različitim scenarijima trofičke diferencijacije. Rezultati su pokazali da zelene žabe istraživanog područja imaju široke, nespecijalizovane trofičke niše čiji se položaj razlikuje u zavisnosti od lokaliteta što ukazuje da je ishrana u većoj meri definisana dostupnošću plena i karakteristikama lokaliteta a manje takson-specifičnim karakteristikama životne forme...Green frogs from the Pelophylax esculentus complex are aquatic, syntopic and gregarious frogs of wide distribution with a distinct role in the ecosystems they inhabit. Although green frogs have been continuously studied on the territory of Serbia and neighbouring countries for more than a hundred years, knowledge about their ecology and spatial integration of populations is scarce, especially in the part of the area of the complex where population systems with all three taxa occur in syntopy. In this work, the parameterisation of the microhabitat and microtrophic niche of green frogs from the Pelophylax esculentus complex in South Banat and the analysis of the spatial integration of populations with the analysis of extinction risk were carried out. In addition, frog skin epibiont communities and body condition of all three taxa at each site were analysed. Finally, a synthetic reassessment of the local conservation status in the studied area was made. Specimens were collected from three sites in southern Banat, differing in their hydrological, orographic and habitat-typical characteristics, but also in the degree of anthropogenic pressure. Individuals were identified using standard measures for the analysis of variability of morphological traits (6 qualitative and 8 quantitative) with appropriate statistical processing and cross-validation by an independent group of samples identified by genetic markers. In this way, out of 317 frogs analysed, 63 individuals of P. ridibundus, 29 individuals of P. lessonae and 225 individuals of P. esculentus were identified and it was concluded that the use of morphological traits allows sufficiently reliable taxon identification within the Pelophylax esculentus complex in a rapid and cost-effective manner. The condition status of the individuals was assessed using the residual condition index, a method frequently used in amphibian research. The parental species had better body condition than the hybrid taxon, and all three taxa generally had lower body condition at the site with the greatest anthropogenic pressure. Material for trophic analysis was collected using the non-invasive stomach-flushing method, and trophic niche patterns were identified using multivariate statistical methods and tested by permutation analyses according to different scenarios of trophic differentiation. The results showed that green frogs in the study area have broad, non-specialised trophic niches whose position varies with location, suggesting that diet is determined to a greater extent by prey availability and site characteristics than by taxon-specific life form characteristics..

Hidden dwellers on green frog’s skin (Pelophylax esculenta complex)

Recent research of frog skin microbiota were mainly focused on presence of pathogens, especially Batrachochytrium dendrobatidis, as well as on describing the bacterial communities on their skin. e specialized amphibian lifestyle where frogs’ skin is in intimate contact with the environment is of primary importance in determining frogs’ resilience to external factors. Cutaneous microbiota is thus an indicator of both frog health as well as of environmental factors acting upon frogs. Many of these factors can be detrimental to frog fitness and it is expected that the first changes in frog fitness could be assessed by identifying the changes in diversity patterns of frog’s skin epibiotic communities and/or isolating specific pathogens. Knowledge of these communities and patterns could contribute to more efficient conservation. Epibiotic communities of the green frog complex (Pelophylax esculenta complex), sampled on three localities in South Banat, Serbia (Stevanove Ravnice, Jaruga canal, DanubeTisaDanube Canal) were investigated using culture depended methods and microscopy techniques. Samples were collected using three sampling methods: 1) scraping by toothbrush for algal sample 2) swabbing with sterile cotton swabs for fungal sample and 3) nonaggressive adhesive tape method for both algal and fungal sample. e aim of this research was describing fungal and algal communities present on frogs’ skin. Culture based methods showed high diversity of airand soilborne fungi (members of genera Acremonium, Alternaria, Aspergillus, Bionectria, Bipolaris, Botrytis, Cladosporim, Epicoccum, Fusarium, Penicillium, Trichoderma, Ulocladium, to name a few), which could be considered transients. However, microscopy techniques revealed spore germination, mycelia formation and sporulation directly on frog skin, of some typical soilborne fungi eg. Fonseceae sp., causative agent of amphibian chromomycoses as well as human phaeohyphomycosis. Microscopy analyses of samples taken or scraped from amphibians’ skin showed the presence of morphological structures belonging to aeroaquatic hyphomycetes (eg. Canalisporium sp.) and stramenopiles, as well as high abundance of diatoms. Presence of c. 40 diatom genera was observed with dominance of mostly ubiquitous eurivalent species found in highly eutrophic waters (members of genera Amphora, Craticula, Cymbella, Cymbopleura, Cymatopleura, Diploneis, Encyonema, Epithemia, Fallacia, Gomphonema, Luticola, Navicula, Neidium, Nitzschia, Pinnularia, Planothidium, Rhopalodia, Staurosira and Surirella). Investigation of microbial communities on frog skin is of great importance due to potential detection of novel amphibian pathogens, and also can lead to better understanding of amphibianmicrobial interactions, which in further researches can be used as effective tool in amphibian protection.Explore and protect the natural beauty of Balkans. International Rufford Small Grants Conference. 27th – 28th September, 2018, Silver Lake, Serbia

Microbial community of green frog’s skin

Amphibian populations have been declining throughout the world in the last several decades. Although numerous factors, including habitat destruction, pollution, introduced species, and global environmental changes contribute the most to amphibian population decline, yet many amphibians are dying of infectious diseases. Recent studies of amphibian skin infections were mainly focused on the presence of chytridiomycosis neglecting other members of the frogs’ skin communities. Samples for mycological and algological analyses were collected from 100 individuals belonging to Pelophylax esculenta complex on three localities in South Banat, Serbia using non-aggressive, rapid and inexpensive “adhesive tape” method for microbial biofilm observation. In analysed samples variety of fungal reproductive structures, and in few samples spore germination, and mycelia formation were detected. Sporulation of some typical soil-borne fungi and the causative agent of amphibian chromomycosis as well as human phaeohyphomycosis, Fonseceae sp. was detected along with water mold, Aphanomyces sp. the pathogen responsible for amphibian egg mortality and saprolegniasis of mostly salamanders and tadpoles. For both pathogens, these were the first reports for green frogs in Serbia. Microscopy analysis showed the presence of aeroaquatic hyphomycetes (eg. Canalisporium sp.). Besides mycobiota, the frog's skin has proved to be a good medium for the survival of diatoms (detected in high diversity and abundance), Cyanobacteria and Chlorophyta. Adhesive tape method proved itself useful as a consequential diagnostic tool for preliminary observation of the microbial community on the skin of amphibians including potential pathogens and symbionts. It provides minimal stress to the studied animal and can be easily applicable in various conditions of field research.20th European Congress of Herpetology, Milan, 02-06.09.2019. [http://seh-congress-2019.unipv.it/

Diatoms on the green frogs skin (Pelophylax esculentus and P. ridibundus)

It is known that diatoms may be present on the skin of various animals (whales, dolphins, manatees, turtles…) (Frankovich et al. 2015, Wetzel et al. 2012). In our study diatoms were collected from the skin of two green frogs’ species: Pelophylax esculentus and P. ridibundus. Studied frogs were captured from the Special Nature Reserve “Deliblatska Pescara” at the locality Stevanove ravnice (Vojvodina province, Serbia). Two sampling methods were performed: (1) nonaggressive adhesive tape method (Urzì and de Leo 2001) and (2) scraping by toothbrush. In laboratory conditions, samples taken by the first method were stained with a drop of Lactophenol Cotton Blue and put on slides to be analyzed. Samples taken by the second method were treated by hot HCl and KMnO4 (Taylor et al. 2005) in order to obtain permanent slides. Light microscope observations and micrographs were made by Zeiss AxioImagerM.1 microscope with DIC optics (x1000 and x1600 magnification) and AxioVision 4.8 software. The present study describes diversity of diatoms from the green frog skin. According to literature data no studies have been published on diatoms living on the frog skin. Analysis of adhesive tape samples confirmed the presence of live diatom cells on the frog skin. Among them the most abundant were taxa from the genera Cocconeis, Epithemia, Gomphonema, Navicula and Rhopalodia. Presence of c. 30 diatom genera, such as Amphora, Craticula, Cymbella, Cymbopleura, Cymatopleura, Diploneis, Encyonema, Epithemia, Fallacia, Gomphonema, Luticola, Navicula, Neidium, Nitzschia, Pinnularia, Planothidium, Rhopalodia, Staurosira, Surirella, etc., was observed on permanent slides. The most dominant taxa were: Geissleria decussis sensu lato, Placoneis sp., Lemnicola hungarica, Cocconeis placentula var. lineate and Sellaphora bacillum. We consider that the great diversity and abundance of diatoms is consequence of their transfer from the environment (mud and macrophytes from eutrophic ponds) onto mucous frog skin. Highly mucous skin is an excellent environment which enables primary conditions for the survival of diatoms. Future research, especially under laboratory conditions, could show whether the frog skin is permanent or temporary diatom habitat

Microhabitat and microtrophic differentiation of ecological niches of green frogs (Ranidae: Pelophylax esculentus complex) in riparian areas of South Banat, Serbia

elene žabe iz Pelophylax esculentus kompleksa su akvatične, sintopijske i gregarne žabe širokog rasprostranjenja sa izraženom ulogom u ekosistemima koje naseljavaju. Iako se na teritoriji Srbije i susednih zemalja zelene žabe istražuju u kontinuitetu već više od sto godina, saznanja o njihovoj ekologiji i prostornoj integraciji populacija su malobrojna posebno u delu areala kompleksa gde se javljaju populacioni sistemi sa sva tri taksona u sintopiji. U okviru ove teze urađena je parametrizacija mikrostanišne i mikrotrofičke niše zelenih žaba iz Pelophylax esculentus kompleksa na području Južnog Banata kao i analiza prostorne integracije populacija sa analizom rizika izumiranja. Dodatno, analizirane su epibiontske zajednice kože žaba i kondicioni status sva tri taksona na svakom od lokaliteta. Na kraju, urađena je sintetska reevaluacija lokalnog konzervacionog statusa na istraživanom području. Jedinke su sakupljane na tri lokaliteta u Južnom Banatu koji se razlikuju po svojim hidrološkim, orografskim i stanišnim karakteristikama ali i po stepenu antropogenog uticaja. Jedinke su identifikovane na osnovu standardnih mera za analizu varijabilnosti morfoloških karaktera (6 kvalitativnih i 8 kvantitativnih) uz adekvatnu statističku obradu i validaciju nezavisnom grupom uzoraka identifikovanih genetičkim markerima. Na taj način je od ukupno 317 analiziranih žaba identifikovano 63 jedinki P. ridibundus, 29 jedinki P. lessonae i 225 jedinki P. esculentus pri čemu je konstatovano da se korišćenjem morfoloških karaktera može na brz i ekonommičan način uraditi dovoljno pouzdana identifikacija taksona unutar Pelophylax esculentus kompleksa. Kondicioni status jedinki je procenjen korišćenjem rezidualnog kondicionog indeksa koji predstavlja uobičajenu metodu u batrahologiji. Parentalne vrste su imale bolju telesnu kondiciju u odnosu na hibridni takson a sva tri taksona su generalno lošiju telesnu kondiciju imale na lokalitetu koji je pod najvećim antropogenim pritiskom. Materijal za trofičku analizu je prikupljen neinvazivnom tehnikom ispiranja želuca a obrasci trofije su utvrđeni multivarijantnim statističkim metodama i testirani permutacionim analizama prema različitim scenarijima trofičke diferencijacije. Rezultati su pokazali da zelene žabe istraživanog područja imaju široke, nespecijalizovane trofičke niše čiji se položaj razlikuje u zavisnosti od lokaliteta što ukazuje da je ishrana u većoj meri definisana dostupnošću plena i karakteristikama lokaliteta a manje takson-specifičnim karakteristikama životne forme...Green frogs from the Pelophylax esculentus complex are aquatic, syntopic and gregarious frogs of wide distribution with a distinct role in the ecosystems they inhabit. Although green frogs have been continuously studied on the territory of Serbia and neighbouring countries for more than a hundred years, knowledge about their ecology and spatial integration of populations is scarce, especially in the part of the area of the complex where population systems with all three taxa occur in syntopy. In this work, the parameterisation of the microhabitat and microtrophic niche of green frogs from the Pelophylax esculentus complex in South Banat and the analysis of the spatial integration of populations with the analysis of extinction risk were carried out. In addition, frog skin epibiont communities and body condition of all three taxa at each site were analysed. Finally, a synthetic reassessment of the local conservation status in the studied area was made. Specimens were collected from three sites in southern Banat, differing in their hydrological, orographic and habitat-typical characteristics, but also in the degree of anthropogenic pressure. Individuals were identified using standard measures for the analysis of variability of morphological traits (6 qualitative and 8 quantitative) with appropriate statistical processing and cross-validation by an independent group of samples identified by genetic markers. In this way, out of 317 frogs analysed, 63 individuals of P. ridibundus, 29 individuals of P. lessonae and 225 individuals of P. esculentus were identified and it was concluded that the use of morphological traits allows sufficiently reliable taxon identification within the Pelophylax esculentus complex in a rapid and cost-effective manner. The condition status of the individuals was assessed using the residual condition index, a method frequently used in amphibian research. The parental species had better body condition than the hybrid taxon, and all three taxa generally had lower body condition at the site with the greatest anthropogenic pressure. Material for trophic analysis was collected using the non-invasive stomach-flushing method, and trophic niche patterns were identified using multivariate statistical methods and tested by permutation analyses according to different scenarios of trophic differentiation. The results showed that green frogs in the study area have broad, non-specialised trophic niches whose position varies with location, suggesting that diet is determined to a greater extent by prey availability and site characteristics than by taxon-specific life form characteristics..

A procedure for taxon assessment based on morphological variation in European water frogs (Pelophylax esculentus complex)

Methods for morphological identification of water frogs from the Pelophylax esculentus complex have changed over the decades. Still, without resort to genetic analyses, water frogs remain among the most difficult European amphibians to distinguish due to their hybrid nature. The aim of this study was to identify a reliable set of quantitative and qualitative morphological characters that objectively resolve species’ identification in the Pelophylax esculentus complex and provide a rapid and robust procedure for taxon identification. Our results showed a substantial difference in morphological characteristics among taxa. The shape and size of the metatarsal tubercle along with the coloration of the hind limbs and flanks appear to be the most important diagnostic traits. Linear discriminant analysis with both quantitative and qualitative traits as a rapid method for the assessment of water frog taxa showed satisfactory accuracy and prediction classification for all 3 members of the complex

Green frogs skin harbours microbiota treasure

Cutaneous microbiota of amphibians is considered to be an indicator of the health status of examined individuals, as well as of environmental factors. In that sense the skin of the captured individuals belonging to Pelophylax esculenta complex on the locality Stevanove ravnice within the Special Nature Reserve “Deliblato Sand” (Serbia) was screened for the presence of biofilm-forming cyanobacteria, algae and fungi dwelling directly on frogs’ stratum corneum. Biofilm was sampled using non-aggressive “adhesive tape method” and their constituents were analyzed and characterized microscopically (Carl Zeiss AxioImager.M1 with AxioVision 4.9 software). We detected high diversity and abundance of diatom taxa overall, particularly belonging to genera Navicula, Gomphonema, Geissleria, Placoneis and Nitzschia. Cyanobacteria were less present and with low abundance. Among them chroococcal genera Gloeocapsopsis, Pseudocapsa and Aphanocapsa were recorded. Outside of diatoms and cyanobacteria, genus Chlorella was the only present photoautotrophic biofilm constituent. We consider highly mucous green frogs skin as a favorable environment for the survival of diatoms and cyanobacteria. Light microscopy analyses of biofilm fragments revealed the presence of variety of fungal structures. Documented coenocytic mycelium with zoosporangium, originated presumably from oomycetes, as well as septate melanized hyphae of dematiaceous fungi, suggested colonization of frog skin from both types of environments present, water and surrounding vegetation. A variety of fungal spores (fragmospores, amerospores, dictyospores, didymospores), and in few cases spore germination were recorded on frog skin. Detected biofilm composition was not determinate by species and gender-specific.7th European Phycological Congress, 25-30 August 2019, Zagre

First case report on pathogenic fungus Fonsecaea sp. Negroni from skin of Pelophylax kl. esculentus L. in Serbia

Non-harmful adhesive tape method was applied directly on the skin surface of edible frog (Pelophylax kl. esculentus), captured in vernal ponds on the locality “Stevanove ravnice” within the Special Nature Reserve „Deliblatska peščara“, in order to detect fungal dwellers of frogs’ skin. Light microscopy analyses of Lactophenol Cotton Blue mounted adhesive tape samples taken from frog’s ventrum revealed the presence of melanized septate hyphae, branched conidiophores with chains of single-celled ovoid conidia, arising directly from the skin, which corresponds to morphological features of dematiaceous hyphomycete - Fonsecaea sp. Since members of genus Fonsecaea are frequently cited as causative agents of chromomycosis in amphibians, as well as human phaeohyphomycosis, world­wide, it is of great significance to study the presence of this fungal pathogen on amphibians in Serbia in order to make the basic reference data of the incidence of these pathogens in this region. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173032