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The Pliocene lacustrine infill of Camp dels Ninots maar (Girona, Spain) hosts an exceptional record of complete articulated skeletons. Here we present the taphonomical description of 22 fossil water frogs recovered from the Pit 7/8 of Can Argilera Sector. Description of their skeletal taphonomy is done on the basis of their distribution, completeness, articulation and limb position, and by establishing comparisons with the late Miocene site of Libros (Spain). Microscopic examination shows no evidence of digestion and absence of weathering. Mass mortality or any environmental stress-induced factors cannot be ruled out, but natural death seems to be the most probable. Limited time of decay prior burial seems to be the main pattern explaining the relatively high degree of articulation and completeness. Floatation is not to be disregard for some more (proximally) disarticulated specimens. Taphonomical patterns document two types of sub-environments inside a cool and deep lake, suggesting a more complex depositional scenario for Camp dels Ninots than the ones described in others similar Konservat-Lagerstätten. [ABSTRACT FROM AUTHOR]

Water frogs of the genus Pelophylax have been widely traded by humans across all European countries for decades, and so far they have been shown to be invasive in most of them. The spread of alien Pelophylax threatens the persistence of native populations mainly via competition and hybridization. Particularly, the latter is worrying for the persistence of the native hybridogenetic systems, as it may induce hybrid swarms whose final outcome is hard to foresee. Alien water frogs have been already detected across several Italian regions, but the extension of the invasion is still unknown, in terms of both species composition and distribution. Here, we carried out the most extensive molecular survey of alien water frog populations across northern Italy, the main invaded area, and obtained a worrying scenario of multiple introduced alien taxa, often co-occurring at the same site. Frogs carrying the native mitochondrial haplotypes were found mainly at the edge of the invaded range. The most widespread taxa turned out to be P. ridibundus and P. kurtmuelleri, but members of the highly diversified P. bedriagae species complex also occurred in the western (P. bedriagae) and the eastern sector (P. cf. bedriagae sensu stricto and P. cf. bedriagae "Cilician West") of the range. We inferred the geographic origin of alien taxa according to mitochondrial haplotype variation and provide the fact-finding background to investigate the impact of alien introductions on native populations, in order to evaluate effective and reliable conservation strategies for native hybridogenetic systems. [ABSTRACT FROM AUTHOR]

The Karpathos water frog (Pelophylax cerigensis) is classified as Critically Endangered according to the IUCN Red List and its distribution is restricted on the island of Karpathos, South Aegean Sea. Utilizing a next generation sequencing approach, we obtained whole mitochondrial genomes of P. cerigensis to determine the species phylogenetic position within the genus Pelophylax and to clarify the phylogenetic relationship between the species' population on Karpathos Island and the population of P. cf. bedriagae from the neighboring Rhodes Island. High-throughput sequencing generated mean ± SD = 227 945 ± 18 306 reads averaged per sample. High quality reads were assembled resulting in the complete mitogenome of P. cerigensis of a total size of 17 922 bp. Mitogenome organization was similar to other Pelophylax species, comprising of 13 Protein Coding Genes (PCGs), 22 tRNAs, two rRNAs and one Control Region. Overall mean genetic distance for the 13 PCGs, within the Ranidae family, ranged from 8.01% (COIII) to 11.6% (ATP8), while ratios of synonymous and non-synonymous substitutions were <1 in all the 13 PCGs, indicating purifying selection. Maximum Likelihood and Bayesian Inference analyses based on the concatenated sequences of 13 PCGs and two rRNAs (12S and 16S), showed that individuals from Karpathos and Rhodes form a well-supported monophyletic group with low genetic distance between them (p-distance = 0.1%). Hence, the Rhodes populations seem to belong to P. cerigensis and not to P. bedriagae as formerly thought, highlighting the need for revision of P. cerigensis current conservation status as well as further examination of the P. bedriagae species group. [ABSTRACT FROM AUTHOR]

Geographic peculiarities of population structure and hemicloning reproduction of the water frog Pelophylax esculentus complex of 904 samples within southern East European Plain were analyzed. The proportion of populations of P. ridibundus is 0.667 against those 0.042 of P. lessonae. The RE-type populations are the most common among mixed ones (0.153). The colonies of the two parental species and P. kl. esculentus (REL-type), as well as those of P. lessonae with P. kl. esculentus (LE-type), have the frequencies 0.072 and 0.046, respectively. All-hybrid populations (E-type) are not numerous across the region (0.017). In the populations of Central, Northern and Western Ukraine, the P. lessonae genome is eliminated during gametogenesis within hybrids while in the Lower Dnieper and Lower Danube drainages, genome of P. ridibundus is eliminated. In the Eastern Ukraine populations, hybrids usually produce diploid gametes or haploid gametes with the P. ridibundus genome, less often with the chromosome set of P. lessonae only, and even more rarely a mixture of different types of gametes. The predominance of P. ridibundus in hybrid communities and the elimination of the P. lessonae genome in hybrids inevitably leads to the transformation of hybrid populations into "pure" R-type populations. This circumstance makes hybridization with P. ridibundus a factor in the shrinking of P. lessonae populations. That trend is especially expressed in the Forest steppe zone, where hybridization proceeds most intensively, the P. lessonae genome is eliminated in hybrids, and unstable populations with a numerical superiority of P. ridibundus predominate. [ABSTRACT FROM AUTHOR]

Marsh frog, Pelophylax bedriagae tadpoles habituate in temporary wetlands where intraspecific aggression has been observed. In this study, we examined the independent and interactive effects of predator cues, water level and density on head width and head, tail and whole-body cannibalism of larval P. bedriagae. We designed a 2×2×3 factorial experiment, by crossing two levels of predatory cues (presence/absence), two levels of density (low/ high) and three levels of water (low/ high/ decreasing) that were carried out within 29 weeks. Despite using conditions that might facilitate cannibalism (i.e., the presence of predator cues, low water level, and high density), a low rate of cannibalism was observed in all treatments. Independently, the highest percentage of total cannibalism was indicated in the presence of predator cues (2.26 %), high water level (2.04 %) and high density (2.24 %). Interactively, the highest rate of total cannibalism was recorded for larvae reared in the high density/absence of predator cues/low water level treatment (3.55%). In contrast, the lowest rate of total cannibalism was observed in the both the low density/presence of predator cues/decreasing water level and low-density/presence of predator cues/high water level treatments (0.44%). The results of the MANOVA indicated that predator cues, water level and density independently and interactively had not significant effect on rate of total cannibalism. At the end of experiment, the maximum size of head width was measured in low-density/absence of predator cues/high water level treatment (9.60±0.39mm), and there was no significant relationship between the maximum head width and the highest rate of cannibalism. According to the results of this experiment, it seems that P. bedriagae larvae may function to avoid the risk of cannibalism by conspecific. [ABSTRACT FROM AUTHOR]

Climate conditions during the Pliocene Climate Optimum (3.2 - 2.5 Ma) are significant due to their proposed correspondence with future climate. There are various difficulties involved in obtaining terrestrial temperatures for this period, so new sources of information are therefore important. This work proposes a new approach based on frog body size, facilitating a palaeoclimate reconstruction of the palaeontological site of Camp dels Ninots (Caldes de Malavella, NE Spain), a Pliocene Konservat-Lagerstatte, dated at around 3.2 Ma. For this purpose, an extensive database of current populations of the genus Pelophylax from across Eurasia has been generated making it possible to establish a correlation between frog body size and eco-climate parameters such as temperature, precipitation rates and real evapotranspiration. Eco-climate values were obtained using OLS regression models and compared with previous palaeoclimate reconstructions from other archaeo-palaeontological sites of interest (Barranco Leon and Fuente Nueva 3, SE Spain). Our new proxy results in a palaeotemperature of 14.3 ± 2.6°C and a palaeoprecipitation rate of 846.8 ± 165.4 mm; according to primary productivity values, a 'Summer-green broad-leaved forest'-like biome is proposed for Camp dels Ninots, in line with previous palaeobotanical studies. The new values generated for the other sites follow the same trends as previous reconstructions, although with lower values in most cases, especially in terms of temperature. This method is a promising new approach to complement previous palaeoclimate reconstructions and could also generate new data. [ABSTRACT FROM AUTHOR]

Vocalization in anurans is mainly studied in the context of male-specific advertisement calls, while release calls – characteristic of both sexes – usually attract less attention. Here we compare release calls of both sexes of hybrid Pelophylax esculentus (considering triploid hybrids) and its parental species P. ridibundus in Ukrainian population systems. The principal component analysis resulted in 83.3% of total variance explained by the first three components, while discriminant analysis correctly differentiated 69.6% of frogs only. Triploid male hybrids had intermediate positions between diploid hybrids and parental species. Triploid females had intermediate positions only in terms of temporal parameters because frequency parameters mainly depended on their body size. A significant correlation between peak frequency and body size was shown for females of both taxonomical groups and P. ridibundus males. Although we identified differences between the studied groups, these differences were not unambiguous, with overlapping frequency parameters in males. Including more triploids and the other parental species is important for further analysis. [ABSTRACT FROM AUTHOR]

In this paper, we present the distribution of the Balkan endemic and vulnerable water frog Pelophylax shqipericus in Montenegro based on our field work and literature data. The dataset includes 129 records, 47% of which have not been published before. The new data contribute significantly to a better understanding of the distribution of Pelophylax shqipericus in the Skadar Lake basin and to the delimitation of the northern margin of the species' range in the Bjelopavlići Plain. The data presented provide important basic information for the study of spatial patterns and conservation measures for this range-restricted species. [ABSTRACT FROM AUTHOR]

Hyaloklossia Labbé, 1896 (Apicomplexa: Sarcocystidae) is a renal coccidium that infects anuran species. The genus consists of two species: H. lieberkuehni, recorded from Pelophylax kl. esculentus, Pelophylax ridibundus, and Rana temporaria in Europe; and H. kasumiensis, recorded from Pelophylax porosus porosus in Japan. However, there have been no reports of Hyaloklossia in the other anurans in Japan. On June 2021, we examined a total of 58 adult frogs comprising 2 P. p. porosus, 23 Pelophylax nigromaculatus, 8 Rana japonica, 3 Glandirana rugosa (Ranidae), 13 Fejervarya kawamurai (Dicroglossidae), and 9 Buergeria buergeri (Rhacophoridae) for infection by Hyaloklossia. Microscopic examination of kidney tissues revealed a high infection incidence of 47.8% (11/23) in P. nigromaculatus, but the other frog species were negative for Hyaloklossia. Morphological and molecular analyses using nuclear ribosomal and mitochondrial genes confirmed the infective species as H. kasumiensis. This is a new host record for H. kasumiensis.

Icosiella neglecta (Diesing, 1851) is a filarial nematode infecting anuran amphibians in the western Palaearctic. In the present survey, we found I. neglecta in 27 out of 28 localities in eight regions of Ukraine. In those localities, 56.5 % of sampled green frogs (Pelophylax esculentus complex) were infected. The infection prevalence was similar in the two parental species, P. ridibundus (Pallas) and P. lessonae (Camerano), and their hybrid form, P. esculentus (Linnaeus), while the infection intensity was comparatively higher in P. ridibundus, as well as in the samples including both P. ridibundus and P. esculentus. Morphological examination of 40 females and 80 males of I. neglecta confirmed the consistency of main qualitative characters, whereas most metrical characters appeared to be rather variable and related to body length. Muscular oesophagus dimensions and spicule length in males were found to be the least variable. In the studied sample of males, 54 % had a longer spicule on the left side and a shorter one on the right side; the other 46 % had a longer spicule on the right side. Our molecular study using mitochondrial COI gene sequences confirmed the strong genetic similarity of I. neglecta populations in Ukraine and most other parts of its distribution area. A single nuclear 18S sequence generated was identical to one previously deposited in GenBank, originating from Ukraine.

Interspecific hybridization can disrupt canonical gametogenic pathways, leading to the emergence of clonal and hemiclonal organisms. Such gametogenic alterations usually include genome endoreplication and/or premeiotic elimination of one of the parental genomes. The hybrid frog Pelophylax esculentus exploits genome endoreplication and genome elimination to produce haploid gametes with chromosomes of only one parental species. To reproduce, hybrids coexist with one of the parental species and form specific population systems. Here, we investigated the mechanism of spermatogenesis in diploid P. esculentus from sympatric populations of P. ridibundus using fluorescent in situ hybridization. We found that the genome composition and ploidy of germ cells, meiotic cells, and spermatids vary among P. esculentus individuals. The spermatogenic patterns observed in various hybrid males suggest the occurrence of at least six diverse germ cell populations, each with a specific premeiotic genome elimination and endoreplication pathway. Besides co-occurring aberrant cells detected during meiosis and gamete aneuploidy, alterations in genome duplication and endoreplication have led to either haploid or diploid sperm production. Diploid P. esculentus males from mixed populations of P. ridibundus rarely follow classical hybridogenesis. Instead, hybrid males simultaneously produce gametes with different genome compositions and ploidy levels. The persistence of the studied mixed populations highly relies on gametes containing a genome of the other parental species, P. lessonae. [ABSTRACT FROM AUTHOR]

Interspecific hybridization can disrupt canonical gametogenic pathways, leading to the emergence of clonal and hemiclonal organisms. Such gametogenic alterations usually include genome endoreplication and/or premeiotic elimination of one of the parental genomes. The hybrid frog Pelophylax esculentus exploits genome endoreplication and genome elimination to produce haploid gametes with chromosomes of only one parental species. To reproduce, hybrids coexist with one of the parental species and form specific population systems. Here, we investigated the mechanism of spermatogenesis in diploid P. esculentus from sympatric populations of P. ridibundus using fluorescent in situ hybridization. We found that the genome composition and ploidy of germ cells, meiotic cells, and spermatids vary among P. esculentus individuals. The spermatogenic patterns observed in various hybrid males suggest the occurrence of at least six diverse germ cell populations, each with a specific premeiotic genome elimination and endoreplication pathway. Besides co-occurring aberrant cells detected during meiosis and gamete aneuploidy, alterations in genome duplication and endoreplication have led to either haploid or diploid sperm production. Diploid P. esculentus males from mixed populations of P. ridibundus rarely follow classical hybridogenesis. Instead, hybrid males simultaneously produce gametes with different genome compositions and ploidy levels. The persistence of the studied mixed populations highly relies on gametes containing a genome of the other parental species, P. lessonae.

We present nine observations of abnormal amplexus in anurans, reported for the first time from Latvia. In total, nine types of abnormal amplexi are documented for four anuran species: Bombina bombina (Bombinatoridae), Bufo bufo (Bufonidae), Pelophylax lessonae and Rana temporaria (Ranidae). According to the one of our observations, abnormal amplexus resulted in the extirpation of a competing anuran species from a breeding pond for two seasons. [ABSTRACT FROM AUTHOR]

We propose a modified and updated protocol to obtain mitotic chromosomes from the regenerated tissue of Pelophylax tadpole tail tips. Chromosomal preparations from regenerated tissue results in high-quality and clean slides suitable for further staining and study. Tadpoles remain alive, undergo minimum suffering, and can be grown to adulthood for further investigation. The method could be used for other groups of Anura and modified for other species with the ability to regenerate their tissues. [ABSTRACT FROM AUTHOR]

Hyaloklossia Labbé, 1896 (Apicomplexa: Sarcocystidae) is a renal coccidium that infects anuran species. The genus consists of two species: H. lieberkuehni , recorded from Pelophylax kl. esculentus, Pelophylax ridibundus, and Rana temporaria in Europe; and H. kasumiensis , recorded from Pelophylax porosus porosus in Japan. However, there have been no reports of Hyaloklossia in the other anurans in Japan. On June 2021, we examined a total of 58 adult frogs comprising 2 P. p. porosus, 23 Pelophylax nigromaculatus , 8 Rana japonica , 3 Glandirana rugosa (Ranidae), 13 Fejervarya kawamurai (Dicroglossidae), and 9 Buergeria buergeri (Rhacophoridae) for infection by Hyaloklossia. Microscopic examination of kidney tissues revealed a high infection incidence of 47.8% (11/23) in P. nigromaculatus, but the other frog species were negative for Hyaloklossia. Morphological and molecular analyses using nuclear ribosomal and mitochondrial genes confirmed the infective species as H. kasumiensis. This is a new host record for H. kasumiensis. [Display omitted] • Hyaloklossia genus contains two parasitic coccidian species that infect anurans. • A total of 58 frogs of 3 families, 5 genera, and 6 species were examined for renal coccidian parasites in Japan. • We found that 47.8% of Pelophylax nigromaculatus hosted Hyaloklossia kasumiensis. • All other frog species examined did not harbour Hyaloklossia infections. • This is the first description of H. kasumiensis in Pelophylax nigromaculatus. [ABSTRACT FROM AUTHOR]

Pelophylax cretensis is an endemic Cretan frog, whose biology has not still been studied in detail. Here we present some details regarding the hypsometric distribution and habitat requirements of P. cretensis that indicate this frog is an adaptable species, and can inhabit different types of water bodies up to 1330 m a. s. l. [ABSTRACT FROM AUTHOR]

Abstract Background Cases of polydactyly in natural populations of amphibians have attracted great interest from biologists. At the end of the 1940s, the French biologist Jean Rostand discovered a polymorphic syndrome in some water frog (Anura: Pelophylax) populations that included polydactyly and some severe morphological anomalies (he called it ‘anomaly P’). The cause of this anomaly remains unknown for 70 years. In a previous study, we obtained anomaly P in the laboratory in tadpoles of water frogs that developed together with molluscs Planorbarius corneus (Mollusca: Gastropoda) collected in the field. We thus proposed the ‘trematode hypothesis’, according to which the infectious agent responsible for anomaly P is a trematode species. Methods Metacercariae from tadpoles with anomaly P were identified using ITS2 gene sequencing as Strigea robusta (Trematoda: Strigeidae). To verify teratogenic features of the species, cercariae of S. robusta were tested for the possibility to cause anomalies. Identification of cercariae species was made using morphological and molecular methods (sequencing of ITS2 and 28S rRNA). The tadpoles were exposed to parasites at four doses of cercariae (control, low, medium and high) and divided into two groups: “early” (at 25–27 Gosner stages) and “late” (at 29–34 Gosner stages) exposure. Results A total of 58 (72.5%) tadpoles survived until metamorphosis under the dose-dependent experiment with the trematode S. robusta. Differences in the survival rates were observed between the exposed and unexposed tadpoles both in the group of “early” tadpoles and “late” tadpoles. The exposure of tadpoles to the cercariae of S. robusta induced anomaly P in 82% of surviving tadpoles. The severe forms developed only in “early” stages under all doses of cercariae exposure. Polydactyly predominantly developed in the “late” stages; under a light exposure dose, polydactyly also developed in “early” tadpoles. Laboratory-hatched tadpoles reared together with infected snails had different rates of survival and complexity of deformations associated with the period of coexistence. Conclusions The experiments with direct cercariae exposure provide compelling evidence that S. robusta leads to anomaly P in tadpoles of water frogs. The manifestation of anomaly P turned out to be dependent on the stage of development, cercariae dose, and the location of the cysts.

The genetic variation of the hybrid form Pelophylax esculentus-ridibundus and its parental species (P. ridibundus) of the Tisza River basin was analyzed. The decrease in the level of the inherited genome variation within the hybrid form in comparison with the parental species was demonstrated. A more significant divergence between the samples of the hybrid form than between the samples of marsh frog was found. No correlation between the species composition of the hybrid population and the level of genetic variation of the hybrid form was found. The evolutionary perspectives of clonal and hemiclonal forms of hybrid origin are discussed. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the prevalence and types of antimicrobial resistance among Gram-negative enteric bacteria isolated from Pelophylax sp. Fifty-four frogs were collected from six provinces in the Eastern Black Sea Region of Turkey. In the cloacal swab cultures, bacteria from 160 different colonies were identified by biochemical tests, automated systems, and matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The antimicrobial susceptibility tests were performed by the disk diffusion method. The observed drug resistance rate was the highest to ampicillin and cefazolin, while the lowest against ciprofloxacin and tetracycline. In the molecular assays, blaTEM (8 Citrobacter spp.), blaSHV (2 Escherichia coli, 1 Hafnia alvei, and a Serratia liquefaciens), tetA genes (E. coli and Klebsiella spp.) and a class 1 integron without any gene cassette (E. coli) were detected. Among the strains, no plasmid-mediated quinolone resistance [qnrA, qnrB, qnrS, qepA and aac (6 ′)-Ib-cr] was found. However, two of three quinolone-resistant Klebsiella strains showed the novel amino acid substitution in the gyrA gene resulting in Ser83Asp and Asp87Glu.The clonality between E. coli isolates was also examined by pulsed-field gel electrophoresis. We consider that multidrug-resistant Gram-negative enteric bacteria in the intestinal microbiota of a cosmopolitan frog species might be a reservoir for antibiotic resistance genes. [ABSTRACT FROM AUTHOR]

In this study, 68 specimens of the Levant Green Frog, Pelophylax bedriagae, were collected from agricultural fields around Yasouj and Noor-Abad cities, from August 2018 to May 2019 in southern Iran to investigate the blood cell morphology. Blood specimens were taken from the heart, or abdominal and facial veins. Smears were dried in room temperature, fixed by methanol and stained using Giemsa method. Blood cells (erythrocytes, leucocytes, and thrombocytes) were counted and measured using light microscopy under an optical micrometer. Based on the findings of this study, erythrocytes from the Noor-Abad population has significantly greater values relative to Yasouj population in all of the six metric characters (P < 0.05). RBC's in male frogs are also greater than those in females in all six studied characters (P < 0.05). All studied metric characters in lymphocytes are also different between two localities and sexes (P < 0.05). [ABSTRACT FROM AUTHOR]

Frogs have been harvested from the wild for the last 40 years in Turkey. We analysed the population dynamics of Anatolian water frogs (Pelophylax spp.) in the Seyhan and Ceyhan Deltas during 2013–2015. We marked a total of 13,811 individuals during 3 years, estimated population sizes, simulated the dynamics of a harvested population over 50 years, and collated frog harvest and export statistics from the region and for Turkey as a whole. Our capture estimates indicated a population reduction of c. 20% per year, and our population modelling showed that, if overharvesting continues at current rates, the harvested populations will decline rapidly. Simulations with a model of harvested population dynamics resulted in a risk of extinction of > 90% within 50 years, with extinction likely in c. 2032. Our interviews with harvesters revealed their economic dependence on the frog harvest. However, our results also showed that reducing harvest rates would not only ensure the viability of these frog populations but would also provide a source of income that is sustainable in the long term. Our study provides insights into the position of Turkey in the 'extinction domino' line, in which harvest pressure shifts among countries as frog populations are depleted and harvest bans are effected. We recommend that harvesting of wild frogs should be banned during the mating season, hunting and exporting of frogs < 30 g should be banned, and harvesters should be trained on species knowledge and awareness of regulations. [ABSTRACT FROM AUTHOR]

The hemiclonal structure of the hybrid form Pelophylax esculentus-ridibundus (Amphibia: Ranidae) within the Dnieper and Desna rivers' basins was analyzed. The presence of polyclonality in almost all populations was demonstrated. The monoclonality of the studied hybrid form was revealed in only 2 of 17 populations studied. Wide fluctuations in the level of genetic variability within P. esculentus-ridibundus in the study area were shown. The decrease in the genetic variation level was caused by the loss of rare hemiclones. A specific feature of P. esculentus-ridibundus, detected in the studied populations in the Dnieper and Desna basins, is the recombination of parental genomes. This leads to the emergence of new hemiclones and increases the genetic variation level of the inherited genome within this hybrid form. [ABSTRACT FROM AUTHOR]

Hybridogenesis is a reproductive tool for sexual parasitism. Hybridogenetic hybrids use gametes from their sexual host for their own reproduction, but sexual species gain no benefit from such matings as their genome is later eliminated. Here, we examine the presence of sexual parasitism in water frogs through crossing experiments and genome-wide data. We specifically focus on the famous Central-European populations where Pelophylax esculentus males (hybrids of P. ridibundus and P. lessonae) live with P. ridibundus. We identified a system where the hybrids commonly produce two types of clonal gametes (hybrid amphispermy). The haploid lessonae genome is clonally inherited from generation to generation and assures the maintenance of hybrids through a process, in which lessonae sperm fertilize P. ridibundus eggs. The haploid ridibundus genome in hybrids received from P. ridibundus a generation ago, is perpetuated as clonal ridibundus sperm and used to fertilize P. ridibundus eggs, yielding female P. ridibundus progeny. These results imply animal reproduction in which hybridogenetic taxa are not only sexual parasites, but also participate in the formation of a sexual taxon in a remarkable way. This occurs through a process by which sexual gametes are being captured, converted to clones, and returned to sexual populations in one generation. [ABSTRACT FROM AUTHOR]

Alaria alata is known as a trematode with a complex life cycle. The trematode Alaria alata infects amphibians as second intermediate hosts. In the present study, we examined 390 amphibians—European water frogs Pelophylax esculentus complex (n = 335), common frogs Rana temporaria (n = 19), moor frogs Rana arvalis (n = 3), and common toads Bufo bufo (n = 30) collected from randomly selected wetland habitats in Latvia. Out of all examined specimens, 80 were tadpoles and 310 were adult amphibians. Mesocercariae of A. alata was detected in 108 specimens from all examined amphibian species, except the common toad, reaching the overall prevalence of 27.7%. Tadpoles were found to be more frequently infected with A. alata, when compared with adults, 58.8% and 22.4%, respectively. The results showed that mesocercariae accumulate in visceral membranes, different internal organs, and muscles in the head area. This is a comprehensive study to identify A. alata mesocercariae predilection sites in amphibians. [ABSTRACT FROM AUTHOR]

Abstract Background Uranotaenia unguiculata Edwards, 1913 is a species of mosquito (Diptera: Culicidae) native to central Europe. Recently a novel lineage of the West Nile virus (WNV-lineage 4c) was identified in pools of adult female Ur. unguiculata. To increase the body of knowledge about this species, various trapping methods were evaluated to determine the most efficient method for capturing adult female Ur. unguiculata. Results Sound traps collected equivalent numbers of female Ur. unguiculata as low-hanging light-baited downdraft traps. Hosts were identified as Pelophylax lessonae and P. ridibunda (Anura: Ranidae) species group frogs from the blood found in engorged females. In addition to confirming infection by WNV-lin. 4c, a potentially integrated flavivirus sequence was detected in male mosquitoes. A novel Alphamesonivirus 1 (Nidovirales: Mesoniviridae) was found to be widespread in the Ur. unguiculata population and is herein described. Conclusions Efficient collection methods for Ur. unguiculata for arbovirus surveillance reflect mosquito questing behavior. Uranotaenia unguiculata targets frog species which call from the water, and it is likely that the novel WNV-lin. 4c is maintained in a frog-mosquito transmission cycle. The improved trapping methods listed here will assist future studies of the vector status of Ur. unguiculata for WNV and other arboviruses.

Abstract Background Sexual parasites offer unique insights into the reproduction of unisexual and sexual populations. Because unisexuality is almost exclusively linked to the female sex, most studies addressed host-parasite dynamics in populations where sperm-dependent females dominate. Pelophylax water frogs from Central Europe include hybrids of both sexes, collectively named P. esculentus. They live syntopically with their parental species P. lessonae and/or P. ridibundus. Some hybrid lineages consist of all males providing a chance to understand the origin and perpetuation of a host-parasite (egg-dependent) system compared to sperm-dependent parthenogenesis. Methods We focused on P. ridibundus-P. esculentus populations where P. ridibundus of both sexes lives together with only diploid P. esculentus males. Based on 17 microsatellite markers and six allozyme loci, we analyzed (i) the variability of individual genomes, (ii) the reproductive mode(s) of all-male hybrids, and (iii) the genealogical relationships between the hybrid and parental genomes. Results Our microsatellite data revealed that P. esculentus males bear Mendelian-inherited ridibundus genomes while the lessonae genome represents a single clone. Our data indicate that this clone did not recently originate from adjacent P. lessonae populations, suggesting an older in situ or ex situ origin. Conclusions Our results confirm that also males can perpetuate over many generations as the unisexual lineage and successfully compete with P. ridibundus males for eggs provided by P. ridibundus females. Natural persistence of such sex-specific hybrid populations allows to studying the similarities and differences between male and female reproductive parasitism in many biological settings.

Background: Cases of polydactyly in natural populations of amphibians have attracted great interest from biologists. At the end of the 1940s, the French biologist Jean Rostand discovered a polymorphic syndrome in some water frog (Anura: Pelophylax) populations that included polydactyly and some severe morphological anomalies (he called it 'anomaly P'). The cause of this anomaly remains unknown for 70 years. In a previous study, we obtained anomaly P in the laboratory in tadpoles of water frogs that developed together with molluscs Planorbarius corneus (Mollusca: Gastropoda) collected in the field. We thus proposed the 'trematode hypothesis', according to which the infectious agent responsible for anomaly P is a trematode species. Methods: Metacercariae from tadpoles with anomaly P were identified using ITS2 gene sequencing as Strigea robusta (Trematoda: Strigeidae). To verify teratogenic features of the species, cercariae of S. robusta were tested for the possibility to cause anomalies. Identification of cercariae species was made using morphological and molecular methods (sequencing of ITS2 and 28S rRNA). The tadpoles were exposed to parasites at four doses of cercariae (control, low, medium and high) and divided into two groups: "early" (at 25–27 Gosner stages) and "late" (at 29–34 Gosner stages) exposure. Results: A total of 58 (72.5%) tadpoles survived until metamorphosis under the dose-dependent experiment with the trematode S. robusta. Differences in the survival rates were observed between the exposed and unexposed tadpoles both in the group of "early" tadpoles and "late" tadpoles. The exposure of tadpoles to the cercariae of S. robusta induced anomaly P in 82% of surviving tadpoles. The severe forms developed only in "early" stages under all doses of cercariae exposure. Polydactyly predominantly developed in the "late" stages; under a light exposure dose, polydactyly also developed in "early" tadpoles. Laboratory-hatched tadpoles reared together with infected snails had different rates of survival and complexity of deformations associated with the period of coexistence. Conclusions: The experiments with direct cercariae exposure provide compelling evidence that S. robusta leads to anomaly P in tadpoles of water frogs. The manifestation of anomaly P turned out to be dependent on the stage of development, cercariae dose, and the location of the cysts. [ABSTRACT FROM AUTHOR]

The lungs and skin are important respiratory organs in Anura, but the pulmonary structure of amphibians remains unclear due to the lack of a suitable procedure. This study improved the procedure used for fixing lungs tissues and used light microscopy, transmission electron microscopy and scanning electron microscopy to reveal the differences in the lung and skin morphologies between Pelophylax nigromaculatus (P. nigromaculatus) and Bufo gargarizans (B. gargarizans). In P. nigromaculatus and B. gargarizans, the cystic lungs comprise a continuous outer pulmonary wall on which primary, secondary, and tertiary septa attach, and a number of regular lattices form from raised capillaries and the pulmonary epithelium on the surfaces of the pulmonary wall and septa. Each lattice in P. nigromaculatus consists of several elliptical sheets and flat bottom, and the septa are distributed with denser sheets and have a larger stretching range than the pulmonary wall. The lattice in B. gargarizans consists of thick folds and an uneven bottom with several thin folds, and the septa have more developed thick and thin folds than the pulmonary wall. However, the density of the pulmonary microvilli, the area of a single capillary, the thicknesses of the blood-air barrier, pulmonary wall and septum, and the lung/body weight percentage obtained for B. gargarizans were higher than those found for P. nigromaculatus. In P. nigromaculatus, the dorsal skin has dense capillaries and a ring surface structure with mucus layer on the stratum corneum, and the ventral skin is slightly keratinized. In B. gargarizans, the stratum corneum in both the dorsal and ventral skins is completely keratinized. A fine ultrastructure analysis of P. nigromaculatus and B. gargarizans revealed that the pulmonary septa are more developed than the pulmonary walls, which means that the septa have a stronger respiratory function. The more developed lungs are helpful for the adaptation of B. gargarizans to drought environments, whereas P. nigromaculatus has to rely on more vigorous skin respiration to adapt to a humid environment. [ABSTRACT FROM AUTHOR]

The morphological features of pigment-containing cells and phagocytic cells were observed on the smears of the liver of the lake frogs. Based on an analysis of more than 1500 cells, it was concluded that cells that have pigment granules in the cytoplasm do not have phagocytic inclusions. On the other hand, phagocytic cells can have pigment inclusions only in the composition of phagocytic vacuoles. Thus, in this organ pigment cells and macrophages are different lines of cell differentiation. For two types of cells, the morphological features characterizing young, functionally active and aging cells are described. [ABSTRACT FROM AUTHOR]

Purpose: The study aims to characterise and compare the helminth assemblages and helminth infracommunities in the marsh frog, Pelophylax ridibundus and the edible frog, P. esculentus collected in the northern part of Ukraine. Methods: Occurrence and abundance of the helminths were analysed by calculating the prevalence, intensity, and mean abundance of infection; similarities between the infracommunities were estimated by the Bray–Curtis index and visualised using nMDS plots. Dissimilarities were estimated using the ANOSIM and SIMPER routines. Results: In total, 27 helminth species were found in 143 frogs. Pelophylax ridibundus (n = 86) harboured 20 species of helminths, 24 species were found in P. esculentus (n = 57), and 17 species were shared by the two hosts. Oswaldocruzia bialata and larval Strigea sp. were absent in P. ridibundus, while they reached the prevalence of 30% and 10%, respectively, in P. esculentus. Cosmocerca ornata, Diplodiscus subclavatus, Opisthioglyphe ranae, and Codonocephalus urniger had significantly larger prevalence in P. ridibundus, whereas Haematoloechus asper was found to be more prevalent in P. esculentus. Acanthocephalus ranae, Icosiella neglecta, Haematoloechus variegatus, Pleurogenes claviger, Pleurogenoides medians, and Prosotocus confusus were equally common in both hosts. Helminth infracommunities in the two hosts had identical species richness (1–10 species, 4 on average); abundance was significantly higher in P. ridibundus. Conclusions: Helminth assemblages of the two hosts in northern Ukraine are rather similar; however, small but significant differences were found in their species composition, parameters of infection in some species, and structure of helminth infracommunities. [ABSTRACT FROM AUTHOR]

Genomic tools like RAD-sequencing can provide the resolution to understand the complex genetic patterns underlying cryptic biological invasions. Here we use this method to clarify the genetic consequences of a rampant invasion by the Italian pool frog (Pelophylax bergeri) north of the Alps, threatening the native P. lessonae and its hybridogenetic hybrid P. esculentus. Patterns of genetic structure were consistent with massive genetic introgression, except for an isolated mountain valley in Switzerland (Joux). Moreover, alien alleles also reached the hybridogens, which should be considered non-native as well. Potentially initiated during antic times, this hybrid swarm likely extends all over W-Europe and rises among the largest amphibian invasions documented on the old continent. Invasion genomics thus hold great promises to reliably inform conservationists, especially in animal groups for which conventional genetic markers may not always be diagnostic between closely-related, morphologically similar species. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine the histological structure of the kidney of Pelophylax bedriagae and the distribution of hyaluronic acid (HA) in the kidney tissue. The kidneys of P. bedriagae are long, brown, ribbon-like structures covered with connective tissue. Nephrons, the functional units of the kidney, consist of the renal corpuscle, proximal and distal tubules, collecting tubule, and collecting duct. The renal corpuscle is composed of the glomerulus and Bowman's capsule. There is no structure similar to Henle's loop, which is found in higher vertebrates. The visceral layer of Bowman's capsule is composed of podocytes surrounding the glomerular capillaries. The parietal layer of Bowman's capsule is lined with a simple squamous epithelium. The proximal tubule is formed by cubic epithelial cells with a brush border, and the distal tubule is covered with a simple cubic epithelium without a brush border. The collecting ducts consist of columnar or cubic cells, and they are larger than the proximal and distal tubules. Many melanomacrophage centers were observed in the kidney parenchyma. The localization of HA was determined to be in the interstitium surrounding the collecting ducts. HA probably plays a significant role in renal water handling and electrolyte balance due to its ability to retain water and bind cations. [ABSTRACT FROM AUTHOR]

The distribution of Alaria-spp.-mesocercariae within the host is relevant for the examination via Alaria spp. mesocercariae migration technique (AMT) regarding predilection sites and may indicate an interaction between parasite and host. Naturally Alaria-exposed frogs of Pelophylax species (n = 13) were examined for systemic distribution and localization-specific parasite density of Alaria spp. mesocercariae. The frogs were necropsied and their body was divided into the following localizations: inner organs, head, torso, forelimbs, and hind limbs. The localizations were analyzed individually and in toto using Alaria spp. mesocercariae migration technique. Our results showed neither statistical differences concerning the number of mesocercariae in the different localizations nor in respect of the rate of positive localizations. Therefore, an accumulation in a particular predilection site seems unlikely. Further research on a representative sample is necessary before final conclusions can be drawn. [ABSTRACT FROM AUTHOR]

The hemiclonal structure of the Pelophylax esculentus-ridibundus hybrid form from the Pripyat, Dniester, and Southern Boug river basins is analyzed. The interbasin and interpopulation differences in the inherited genome variation level are demonstrated. The genetic diversity level of the P. esculentus-ridibundus inherited genome in the Pripyat basin is significantly lower than in the other two basins. Monoclonal populations of this hybrid form have been identified only in the Pripyat basin. There were no significant differences in the genetic variability level of this hybrid form between the populations of the Dniester and Southern Boug basins. The relationship between the variability level of the inherited genome of the hybrid form and the potentially possible multiple hybridization of parental species is analyzed. [ABSTRACT FROM AUTHOR]

Invasive species are characterized by their ability to establish and spread in a new environment. In alien populations of anurans, dispersal and fitness-related traits such as endurance, burst performance and metabolism are key to their success. However, few studies have investigated inter-individual variation in these traits and more specifically have attempted to understand the drivers of variation in these traits. Associations of anatomical features may be excellent predictors of variation in performance and could be targets for selection or subject to trade-offs during invasions. In this study, we used marsh frogs (Pelophylax ridibundus), a species that has been introduced in many places outside its native range and which is now colonizing large areas of Western Europe. We first measured the inter-individual variation in resting metabolism, the time and distance they were able to jump until exhaustion, and their peak jump force, and then measured the mass of specific organs and lengths of body parts suspected to play a role in locomotion and metabolism. Among the 5000 bootstrap replicates on body size-corrected variables, our statistical models most often selected the stomach (75.42%), gonads (71.46%) and the kidneys (67.26%) as predictors of inter-individual variation in metabolism, and the gluteus maximus muscle (97.24%) mass was the most frequently selected predictor of jump force. However, endurance was poorly associated with the anatomical traits (R2distance=0.42, R2time=0.37). These findings suggest that selection on these predictors may lead to physiological changes that may affect the colonization, establishment and dispersal of these frogs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Dispersers are often assumed to have the mean phenotype observed across the entire metapopulation, despite growing evidence of dispersal–phenotype correlations. We examined three dispersal–phenotype correlations in Green Frogs (Rana clamitans Latreille, 1801 = Lithobates clamitans (Latreille, 1801)). Two were in traits that have been previously tied to fitness (body size and body condition), while a third (relative hindlimb length) has been linked to movement performance. We constructed a spatially dispersed array of experimental ponds in close proximity to source ponds known to support Green Frog breeding populations. Over the course of two breeding seasons (four sampling periods), we measured phenotypes of all Green Frogs that had colonized the experimental ponds and a sample of individuals from the source ponds. After only 1 month, a positive correlation was detected between dispersal and body size within the population of dispersers occupying the experimental ponds. After a 2nd month, this positive dispersal – body size correlation was also present when comparing the population of dispersers to the population of nondispersers remaining at the source ponds. Even if generated solely by plasticity, a positive correlation between dispersal and body size (a trait tightly linked to fitness) has the ability to alter metapopulation capacity and thus the probability of regional species persistence. [ABSTRACT FROM AUTHOR]

Skeletochronology is a reliable tool for assessing several parameters in amphibian populations. We used skeletochronology to determine the age structure, growth rate, age at first reproduction, and longevity of Levantine frog Pelophylax bedriagae populations from different localities in Denizli, Turkey. All examined individuals (N = 161) exhibited Lines of Arrested Growth in the bone cross-sections. Age structure and age at first reproduction were similar among localities and sexes, while longevity and growth rates showed significant variation among localities. Obtained results were compared with literature data on age-related and grow parameters in Pelophylax bedriagae and cognate species. [ABSTRACT FROM AUTHOR]

The brain is among the most energetically costly organs in the vertebrate body. The expensive-tissue hypothesis (ETH) predicts that increasing the size of another costly organ, such as the gut, should compensate for the cost of a small brain. To date, this hypothesis has mainly been tested in homoeothermic animals and in some ectothermic animals (e.g., fishes and anurans). Here, we undertake a test of the ETH by analyzing the relationship between brain size variation and length of the digestive tract in Hylarana guentheri. After controlling for geographical situation and body size, we did not find a correlation between brain mass and the length of the digestive tract in H. guentheri. Our findings suggest that the variation of brain size did not follow general patterns in this species and that the effect of diet quality cannot play a role in the variation of brain. [ABSTRACT FROM AUTHOR]

This study describes the histopathological effects of carbaryl in the spleen of Levantine frog, Pelophylax bedriage. Due to its primary role in immune system, it is important to research the toxic effects of pesticides, which play an important role in environmental pollution, on spleen. To that end, adult frogs were exposed to carbaryl for 96 h. Experimental groups contained low dose, (0.05 mg/g), medium dose (0.1 mg/g) and high dose (0.2 mg/g). After following exposure to carbaryl, the frogs were euthanised and dissected. In low-dose group, there were no important changes in spleen tissue. In medium-dose group, prominence in haemorrhage just below the capsule and an increase in the melanomacrophage number were determined. In high-dose group, in addition to increases in the melanomacrophages, separations in capsule, haemorrhage below capsule and within splenic tissue, sinusoidal enlargement, congestion in dilated sinusoid, hypertrophic plasma cells and fibrosis were determined as important histological lesions in exposed frogs. This study clearly showed that carbaryl caused important histopathological damages in splenic tissue of Pelophylax bedriagae. In view of these findings, it can be said that this insecticide has the capacity to disrupt spleen’ functions. [ABSTRACT FROM AUTHOR]

Previous studies suggest that immune-challenged individuals need to allocate resources to the immune system to combat infection, reducing escape ability and increasing the vulnerability of infected individuals to predators. Such behavioural responses might change in anthropogenic habitats where the balance between predation risk and countering infections can differ from that in natural habitats. We experimentally explored whether fighting an infection has an effect on hiding behaviour in a frog (Pelophylax perezi), and whether frogs from urban ponds respond differently from those from natural forested ponds. Independently of habitat type, we found no evidence for changes in hiding behaviour of male frogs before and after they were immune challenged (injected with lipopolysaccharide) or not (injected with phosphate-buffered saline). We also explored whether immune response (phytohaemagglutinin assay) or morphological traits (head width, body condition, body size and length) were related to variation in hiding time, but we did not find a relationship between the measured traits and behavioural response for males from either habitat. In conclusion, we show that frog anti-predator behaviour is not always affected by an immune challenge and the effect of urban habitats can be weaker than expected, at least in species that could be pre-adapted to the novel environmental conditions. [ABSTRACT FROM AUTHOR]

We explored the population systems of Pelophylax bedriagae (Camerano, 1882) in northwestern, western and southwestern Iran. Statistical significance for a hypothesis of sexual dimorphism of the frogs was tested using t-test and multivariate analysis of variances (MANOVA). Due to lack of sexual differences between the male and female frogs, the data of both sexes were pooled for further analysis. Taxonomic relationships among local populations were investigated using discriminate analysis (by stepwise method) with iterative sample assignment. The multivariate morphometric analyses revealed that the range of P. bedriagae extends to western Iran. The next step was to analyze the advertisement call (five standard parameters: call duration, number of pulse groups per call, duration of pulse groups, interval between pulse groups, and intercall interval) of the water frogs inhabiting three sites in western Iran. Rigorous application of the bioacoustic method removes all doubt that these three sites are inhabited by the same species, which on the basis of priority of nomenclature should be called P. bedriagae. [ABSTRACT FROM AUTHOR]

Model of the our research was the adult male amphibian anura, Pelophylax bergeri, poikilotherm species not considered threatened by the IUCN, sampled in representative sites at different degree. In the first phase, a biochemical characterization of the ADP-ribosylation on the skin of barcoded amphibian anura collected from Matese Lake (clean reference site in CE, Italy) was carried out. Two PARP isoforms were evidence: the first of 66 kDa is localized into nucleus and activated by DNA damage; the second of 150 kDa is in cytoplasm, as demonstrated by biochemical and immunohistochemical analysis. Subsequently, the PARP activity, the quantitative expression of androgen receptor gene, and the levels of arsenic and chromium in skin and testis of frog and soil, water, and sediment collected from sites at different degrees of pollution were measured. A significant variation of PARP activity and androgen receptor expression levels was detected in both tissues of barcoded frogs from Sarno and Scafati, along Sarno River (SA, Italy), suggesting that a PARP activation is correlated to pollution and to steroid-regulated physiology disruption. [ABSTRACT FROM AUTHOR]

Pesticide use in agricultural areas requires the application of numerous chemicals to control target organisms, leaving non-target organisms at risk. The present study evaluates the hepatic metabolomic profile of one group of non-target organisms, amphibians, after exposure to a single pesticide and pesticide mixtures. Five common-use pesticide active ingredients were used in this study, three herbicides (atrazine, metolachlor and 2,4- d ), one insecticide (malathion) and one fungicide (propiconazole). Juvenile green frogs ( Lithobates clamitans ) were reared for 60–90 days post-metamorphosis then exposed to a single pesticide or a combination of pesticides at the labeled application rate on soil. Amphibian livers were excised for metabolomic analysis and pesticides were quantified for whole body homogenates. Based on the current study, metabolomic profiling of livers support both individual and interactive effects where pesticide exposures altered biochemical processes, potentially indicating a different response between active ingredients in pesticide mixtures, among these non-target species. Amphibian metabolomic response is likely dependent on the pesticides present in each mixture and their ability to perturb biochemical networks, thereby confounding efforts with risk assessment. [ABSTRACT FROM AUTHOR]