New data on the distribution of Pallas's spadefoot toad ( (Pallas, 1771)) and fire-bellied toad ( L., 1761) (Anura, Amphibia) on the territory of the Saratov region and adjacent territories

New data on the geographical distribution of Pelobates vespertinus and Bombina bombina in the Saratov region and adjacent districts of the Volgograd, Voronezh, Penza and Ulyanovsk regions are presented. The habitation of B. bombina and P. vespertinus populations in 25 and 22 localities, respectively, was established. Most modern populations of these species of anuran amphibians have been recorded near water bodies in river valleys.

An overview of helminths of the European fire-bellied toad Bombina bombina (Amphi-bia, Anura) in the Volga River Basin

The helminthofauna of Bombina bombina (Linnaeus, 1761) has been studied to an unequal degree in different parts of the habitat. Thus, it has been studied in more detail in the west of its range (in the countries of central and eastern Europe) and in less detail in the center (in Belarus and Ukraine). There were few data on helminths of this host in the east of its range (in Russia). For the first time, an inventory of the helminthofauna in B. bombina was carried out for populations in the Volga River Basin. The results of our own research are presented and supplemented with information from other authors. We summarized scattered data on helminths from 390 specimens of amphibians collected over more than 40 years in the territory of five regions: Kaluga and Samara regions, the Republics of Mordovia, Tatarstan and Bashkortostan. The helminthofauna includes 21 species from three classes: Trematoda (15), Chromadorea (5) and Clitellata (1). For each species, we give the systematic position, localization, places of detection, geographical distribution and characteristics of the life cycle. The leech Helobdella stagnalis (Linnaeus, 1758) was first recorded in the European fire-bellied toad in Europe. Four species of trematodes are new to this amphibian species in Russia: Haematoloechus abbreviatus (Bychowsky, 1932), Paralepoderma cloacicola (Luhe, 1909), larvae, Tylodelphys excavata (Rudolphi, 1803), larvae and Astiotrema monticelli (Stossich, 1904), larvae. Another species of trematode – Strigea strigis (Schrank, 1788), larvae – was first recorded in this host within the boundaries of the Volga Basin. A specific parasite is the trematode Haematoloechus abbreviatus (Bychowsky, 1932). The number and composition of the species of helminths of the European fire-bellied toad vary in different regions; the structure of the helminth fauna is generally stable and includes three groups of species: adult and larval stages of trematodes, adult nematodes-geohelminths. The results of the study create a database for further population studies and contribute to the development of ideas about the distribution and formation of the amphibian helminth fauna in Europe, Russia and the Volga Basin.

Amphibian diversity of the Dnipro river within the limits of the Dnipro city agglomeration

Amphibians play an important role in the formation of biodiversity and functioning in most terrestrial and freshwater ecosystems. Urbanization threatens to survive many species due to the destruction of natural ecosystems whose remains become fragmented, isolated, and modified by urbanization processes. The floodplain and coastal ecosystems along both banks of the Dnipro River within the limits of the Dnipro city agglomeration are investigated. The current diversity of amphibians in this territory is represented by six species, which is 55% of the total amount of amphibian fauna of the Dnipropetrovsk region. They are Lissotriton vulgaris, Pelophylаx ridibundus, Pelobates vespertinus, Bombina bombina, Hyla orientalis, and Bufotes viridis. The largest diversity of amphibians was found on the site near the Samara Gulf, where small urbanization and anthropogenic influence are observed. The smallest diversity of amphibians was found for the site of residential community "Pobeda", where only two species of amphibians were revealed. The assessment of the similarity of amphibian communities shows that their diversity is the most similar for ecosystems of the right bank of the Dnipro River near the residential communities "Chervony Kamin", "Pokrovsky", and "Parus" and the ecosystems on the left bank near the Samara Gulf. The most numerous and widespread species is the marsh frog P. ridibundus, which lives along the coastline of the Dnieper River. Its populations are relatively stable according to their high number and distribution. The populations of L. vulgaris, B. bombina, and H. orientalis are under the threat of declining, as evidenced by their low number and dependence on small water bodies. Therefore, the reconstruction of the coastline and any earthwork and building activity should be accompanied by the measures on the preservation of small reservoirs along the Dnieper River. The obtained results develop our knowledge about the actual state of biodiversity and species wealth abundance of the Dnieper River within the northern steppe subzone of Ukraine and may be used for planning of the environmental protection measures.

Male-male amplexus between two amphibian families

The amplexus between two different anuran males is observed very rarely. Therefore, here we provide the first documented observation of a long-lasting male-male amplexus between Bombina bombina (Bombinatoridae) and Hyla arborea (Hylidae) together with an overview of the data published in literature. The observed mating pattern is reported from Southern Moravia, the Czech Republic. The possible reasons for its occurrence during the mass breeding season are discussed.

A dense linkage map for a large repetitive genome: discovery of the sex-determining region in hybridising fire-bellied toads (Bombina bombina and B. variegata)

Genomic analysis of hybrid zones offers unique insights into emerging reproductive isolation and the dynamics of introgression. Because hybrid genomes consist of blocks inherited from one or the other parental taxon, linkage information is essential. In most cases, the spectrum of local ancestry tracts can be efficiently uncovered from dense linkage maps. Here we report the development of such a map for the hybridising toads, Bombina bombina and B. variegata (Anura: Bombinatoridae). Faced with the challenge of a large (7-10 Gb), repetitive genome, we set out to identify a large number of Mendelian markers in the non-repetitive portion of the genome that report B. bombina vs. B. variegata ancestry with appropriately quantified statistical support. Bait sequences for targeted enrichment were selected from a draft genome assembly, after filtering highly repetitive sequences. We developed a novel approach to infer the most likely diplotype per sample and locus from the raw read mapping data, which is robust to over-merging and obviates arbitrary filtering thresholds. Validation of the resulting map with 4,755 markers underscored the large-scale synteny between Bombina and Xenopus tropicalis. By assessing the sex of late-stage F2 tadpoles from histological sections, we identified the sex-determining region in the Bombina genome to 7 cM on LG5, which is homologous to X. tropicalis chromosome 5, and inferred male heterogamety. Interestingly, chromosome 5 has been repeatedly recruited as a sex chromosome in anurans with XY sex determination.

The gastrin-releasing peptide/bombesin system revisited by a reverse-evolutionary study considering Xenopus

Bombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (Xenopus tropicalis), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand the derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in Xenopus. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of Xenopus. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the ‘gut-brain peptide’ systems.

An abnormally early hibernation ending of the Red-bellied toad (Bombina bombina) (Discoglossidae, Anura) in the populations of the Medveditsa river valley (Saratov region)

In the course of our long-term studies of the phenology of spawning migrations of anuran amphibians, which began in the Medveditsa river valley (near the Uritskoye village, Lysogorsky district, Saratov region) in 2009, an abnormally early end of the wintering period was recorded for the red-bellied toad (Bombina bombina) in the spring of 2020, namely, March 13. The anomaly was longer than a month in relation to the long-term average date of arrival of this species to its spawning grounds (April 18). The water temperature in the shallow waters of the spawning reservoir on this day was 10.2°С to a depth of 0.1 m, and 4.8°С at a depth of 0.5 m, and did not differ significantly from the average values typical for this phase of the B. bombina annual cycle in adjacent populations. The possible significance of this anomaly for the reproduction of local populations of B. bombina is discussed.

Revisiting the Gastrin-releasing Peptide/bombesin System: A Reverse-evolutionary Study Considering Xenopus

Bombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (Xenopus tropicalis), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand its derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in Xenopus. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of Xenopus. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the ‘gut-brain peptide’ systems.