Amphibian roadkill mitigation has primarily targeted adult individuals; however, it is important to also focus on juveniles to restore populations and to understand the actual effects of roadkill on other species traits, such as demographic or metapopulation dynamics. This study aimed to determine the effects of different life stages on the occurrence of two frog species with different ecological characteristics on paved roads. We conducted transect surveys in the Shonai region of northeastern Japan to search for adult and juvenile Pelophylax nigromaculatus (Hallowell, 1861) and Rana tagoi tagoi Okada, 1928 on paved roads. We analyzed the effects of the surrounding landscape within various radius buffers and weather conditions on the number of adult and juvenile individuals. The juveniles of both species were dependent on neighboring paddy fields or forest areas, whereas the effects of forest edges differ between life stages, and juveniles were less likely to occur on roads with complex landscapes. Additionally, the optimal spatial scale for juveniles was narrower than that for adults in both species. Our results suggest that ignoring life stages may lead to incorrect estimates when assessing roadkill risk for frogs and that future roadkill mitigation measures should consider juveniles as well as adults.

Background/Objective: Amphibian skin is a valuable source of host defense peptides (HDPs). This study aimed to identify HDPs with novel amino acid sequences from the skin of Rana tagoi yakushimensis and analyze their functions. Methods: cDNAs encoding HDP precursors were cloned and sequenced using RT-PCR and 3'-RACE. The novel HDPs were synthesized to evaluate their antimicrobial activity, antioxidant activity, and cytotoxicity. Antimicrobial activity was evaluated by way of broth microdilution and endotoxin- and β-glucan-binding capacity using an enzyme-linked endotoxin binding assay (ELEBA) and a modified ELEBA, respectively. Results: Nine cDNAs encoding precursors for various HDP families, including temporin, ranatuerin-2, brevinin-1, amurin-9, and a novel yakushimin peptide, were identified. Brevinin-1TYa exhibited antibacterial activity against Staphylococcus aureus, and brevinin-1TYa and amurin-9TYa induced morphological changes in Escherichia coli and S. aureus. Yakushimin-TYa, amurin-9TYa, and brevinin-1TYa showed concentration-dependent antibacterial effects against the plant pathogens Xanthomonas oryzae pv. oryzae and Clavibacter michiganensis subsp. michiganensis. Amurin-9TYa demonstrated strong binding affinity to lipopolysaccharide, lipoteichoic acid, and β-glucan, exhibited antioxidant activity, and lacked cytotoxicity, making it a promising therapeutic candidate. Moreover, brevinin-1TYa showed strong cytotoxicity, whereas yakushimin-TYa exhibited weak cytotoxicity. Conclusions: These findings highlight the potential of these peptides, particularly amurin-9TYa, for future applications as antimicrobial and therapeutic agents.

Sex chromosome turnover is the transition between sex chromosomes and autosomes. Although many cases have been reported in poikilothermic vertebrates, their evolutionary causes and genetic mechanisms remain unclear. In this study, we report multiple transitions between the Y chromosome and autosome in the Japanese Tago’s brown frog complex. Using chromosome banding and molecular analyses (sex-linked and autosomal single nucleotide polymorphisms, SNPs, from the nuclear genome), we investigated the frogs of geographic populations ranging from northern to southern Japan of two species, Rana tagoi and Rana sakuraii (2n = 26). Particularly, the Chiba populations of East Japan and Akita populations of North Japan in R. tagoi have been, for the first time, investigated here. As a result, we identified three different sex chromosomes, namely chromosomes 3, 7, and 13, in the populations of the two species. Furthermore, we found that the transition between the Y chromosome (chromosome 7) and autosome was repeated through hybridization between two or three different populations belonging to the two species, followed by restricted chromosome introgression. These dynamic sex chromosome turnovers represent the first such findings in vertebrates and imply that speciation associated with inter- or intraspecific hybridization plays an important role in sex chromosome turnover in frogs.

Recent molecular analyses cast doubt on the subspecific relationships of Japanese brown frogs Rana tagoi tagoi, R. t. okiensis, and R. t. yakushimensis. Many samples of R. tagoi from three main islands, i.e., Honshu, Shikoku, and Kyushu tend to form a genetic unit with rich variation. On the other hand, R. t. okiensis, R. t. yakushimensis, and some insular samples are clearly isolated from R. t. tagoi and each other. Thus, we propose to treat each subspecies of R. tagoi as full species and describe part of the Goto Islands populations as Rana matsuoi sp. nov. The new species and R. tagoi show a complicated distributional pattern within the Goto Islands but the former is distinguishable from the latter by their smaller male body size, relatively longer limbs, more developed toe webs, nuptial pad formation in breeding male, and advertisement call with repetition of short notes.

Taxonomic relationship of two forms of Rana tagoi, a brown frog endemic to Japan, is assessed. Based on morphological and bioacoustical comparisons, as well as molecular analysis, we consider the large-form-lineage as true R. tagoi, and describe the small-form-lineage as Rana kyoto sp. nov. The new species and R. tagoi occur sympatrically in many places around the Kinki District, although the former can be distinguished from the latter by smaller body size, more developed dark marking on ventral side of legs, less developed toe webs, and advertisement call with fewer notes and lower dominant frequency. Rana kyoto sp. nov. sympatric with R. tagoi show smaller body size than the allopatric ones, whereas R. tagoi sympatric with the new species show larger body size than the allopatric ones, suggesting that the character displacement of body size occurs between the two species around the type locality of Rana kyoto sp. nov.

In the Kitayama-region of Kyoto city, two types of the brown frog Rana tagoi occur syntopically. They are populations of small and large frogs that do not overlap in adult body size and proved to be heterospecific genetically. In order to clarify the cause and significance of this body size difference, we studied the growth pattern in the two populations. For comparison, we also examined an allopatric population of R. tagoi from Shishiga-tani, Kyoto, which is genetically very close to the large type, and occurs without co-inhabiting different sized individuals. We examined sections of periosteal bones, and counted the number of LAGs in the bone to determine ages of individuals. We used logistic model and estimated instantaneous growth rate and asymptotic size, to describe growth pattern. Each of the two types from Kitayama exhibited unique growth pattern, and the Shishiga-tani population showed a growth pattern intermediate between those of the two types in asymptotic size. Difference in size of two types is discussed with relation to the character displacement.

Viral infections represent a serious threat to the world population and are becoming more frequent. The search and identification of broad-spectrum antiviral molecules is necessary to ensure new therapeutic options, since there is a limited availability of effective antiviral drugs able to eradicate viral infections, and consequently due to the increase of strains that are resistant to the most used drugs. Recently, several studies on antimicrobial peptides identified them as promising antiviral agents. In detail, amphibian skin secretions serve as a rich source of natural antimicrobial peptides. Their antibacterial and antifungal activities have been widely reported, but their exploitation as potential antiviral agents have yet to be fully investigated. In the present study, the antiviral activity of the peptide derived from the secretion of Rana tagoi, named AR-23, was evaluated against both DNA and RNA viruses, with or without envelope. Different assays were performed to identify in which step of the infectious cycle the peptide could act. AR-23 exhibited a greater inhibitory activity in the early stages of infection against both DNA (HSV-1) and RNA (MeV, HPIV-2, HCoV-229E, and SARS-CoV-2) enveloped viruses and, on the contrary, it was inactive against naked viruses (PV-1). Altogether, the results indicated AR-23 as a peptide with potential therapeutic effects against a wide variety of human viruses.

The phylogeographic puzzle of Pseudoacanthocephalus toshimai, an amphibian acanthocephalan in northern Japan.

Two species of frogs endemic to Japan, Rana neba and R. tagoi, have previously been separated based on differences in the number of chromosomes and the dominant frequencies of their advertisement calls. However, through a comparison of advertisement calls around the type localities of both species, we found that interspecific differences are instead apparent in the fundamental frequencies, not in the dominant frequencies. Additionally, the calls of R. neba are more easily distinguished by their unique first notes, which can be differentiated by the strong frequency modulation, longer and fewer pulses, and lower pulse rate. Compared with R. neba, the calls of R. tagoi are composed of notes with similar acoustic structure, although the last note is longer in duration, contains more pulses, and has longer inter-pulse intervals than in the other notes in the call. Based on these results, we present a new acoustic diagnosis for R. neba and R. tagoi.

Recent advancement of environmental DNA (eDNA) methods for surveying species in aquatic ecosystems has been used for various organisms and contributed to monitoring and conservation of species and environments. Amphibians are one of the promising taxa which could be monitored efficiently by applying quantitative PCR (qPCR) or next generation sequencing to eDNA. However, the cost of eDNA detection using these approaches can be quite high and requires instruments that are not usually installed in ecology laboratories. For aiding researchers in starting eDNA studies of amphibians, especially those not specialized in molecular biology, we developed a cost efficient protocol using PCR-RFLP method. We attempted to detect eDNA of three Japanese Rana species (Rana japonica, Rana ornativentris, and Rana tagoi tagoi) in various spatial scales including an area close to the Fukushima nuclear power plant where the environment is recovering after the disaster in 2011. Our PCR-RFLP protocol was successful in detecting Rana species in static water in both laboratory and field; however, it could not detect Rana species in non-static water samples from the field. Even a more sensitive detection method (standard qPCR) was unable to detect frogs in all non-static water samples. We speculate that our new protocol is effective for frogs living in lentic habitats, but not for lotic habitats which may still require the gold standard of field observation for detection approach.

BackgroundIn Japan and East Asia, endemic frogs appear to be tolerant or not susceptible to chytridiomycosis, a deadly amphibian disease caused by the chytrid fungus Batrachochytridium dendrobatidis (Bd). Japanese frogs may have evolved mechanisms of immune resistance to pathogens such as Bd. This study characterizes immune genes expressed in various tissues of healthy Japanese Rana frogs.ResultsWe generated transcriptome data sets of skin, spleen and blood from three adult Japanese Ranidae frogs (Japanese brown frog Rana japonica, the montane brown frog Rana ornativentris, and Tago’s brown frog Rana tagoi tagoi) as well as whole body of R. japonica and R. ornativentris tadpoles. From this, we identified tissue- and stage-specific differentially expressed genes; in particular, the spleen was most enriched for immune-related genes. A specific immune gene, major histocompatibility complex class IIB (MHC-IIB), was further characterized due to its role in pathogen recognition. We identified a total of 33 MHC-IIB variants from the three focal species (n = 7 individuals each), which displayed evolutionary signatures related to increased MHC variation, including balancing selection. Our supertyping analyses of MHC-IIB variants from Japanese frogs and previously studied frog species identified potential physiochemical properties of MHC-II that may be important for recognizing and binding chytrid-related antigens.ConclusionsThis is one of the first studies to generate transcriptomic resources for Japanese frogs, and contributes to further understanding the immunogenetic factors associated with resistance to infectious diseases in amphibians such as chytridiomycosis. Notably, MHC-IIB supertyping analyses identified unique functional properties of specific MHC-IIB alleles that may partially contribute to Bd resistance, and such properties provide a springboard for future experimental validation.

O-198 房総半島に生息するタゴガエルの産卵及び越冬場所に関する地質環境

Abstract: Feeding habits of snakes on Kinkasan Island, where potential food resources for snakes is limited, was investigated based on field surveys of six years. In total 280 snakes, which consisted of five species (Elaphe quadrivirgata, E. climacophora, Euprepiophis conspicillatus, Rhabdophis tigrinus, and Gloydius blomhoffii), were collected. A total of 168 prey items were recovered from the stomach contents of these snakes. The diet of R. tigrinus and G. blomhoffii exclusively consisted of a frog, Rana tagoi, which is the only amphibian species inhabiting Kinkasan Island. Diet of El. quadrivirgata was also predominantly comprised of Ra. tagoi ( 97.7%). O nly e ndothermic animals were found in the stomach contents of El. climacophora and Eu. conspicillatus. Direct observations of feeding behavior and a broad range of body temperature of El. quadrivirgata indicated that during the breeding season of Ra. tagoi, this snake forages for frogs hiding in crevices of rocks immersed in cold running water of mou...

The major histocompatibility complex (MHC) is a key component of adaptive immunity in all jawed vertebrates, and understanding the evolutionary mechanisms that have shaped these genes in amphibians, one of the earliest terrestrial tetrapods, is important. We characterised MHC class I variation in three common Japanese Rana species (Rana japonica, Rana ornativentris and Rana tagoi tagoi) and identified a total of 60 variants from 21 individuals. We also found evolutionary signatures of gene duplication, recombination and balancing selection (including trans-species polymorphism), all of which drive increased MHC diversity. A unique feature of MHC class I from these three Ranidae species includes low synonymous differences per site (d S) within species, which we attribute to a more recent diversification of these sequences or recent gene duplication. The resulting higher d N/d S ratio relative to other anurans studied could be related to stronger selection pressure at peptide binding sites. This is one of the first studies to investigate MHC in Japanese amphibians and permits further exploration of the polygenetic factors associated with resistance to infectious diseases.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-016-0934-x) contains supplementary material, which is available to authorized users.

Radiocesium accumulation in the anuran frog, Rana tagoi tagoi, in forest ecosystems after the Fukushima Nuclear Power Plant accident

Amphibian fossils excavated from two Late Pleistocene-Holocene fissures in the southern part of Okinawajima Island in the Ryukyu Archipelago were identified through detailed comparisons with skeletal specimens of extant taxa. The identified species (eight frogs and two newts) were confined to extant elements on the island and were mostly endemic to either the island or the central part of the archipelago including it. Previous Late Pleistocene records of extralimital frogs (two mainland Japanese species [Glandirana rugosa and Rana tagoi] and one Southeast Asian frog currently naturalized in the Ryukyus [Polypedates leucomystax]) from one of the study sites (the Minatogawa Fissure) are therefore considered misidentifications. This may also be the case with Fejervarya kawamurai, a synanthropic frog currently widely distributed in East Asia, including the Central Ryukyus. Of the species obtained from the Late Pleistocene deposits at these sites, five frogs (Limnonectes namiyei, Babina holsti, Odorrana ishikawae, O. narina, and Rana ulma) are forest-dwellers currently confined to the northern forested area of Okinawajima. Additionally, none of these species were detected in the middle Holocene deposits from one of the sites, the Sashiki Fissure. Our findings indicate that the southern limestone area of Okinawajima in the Late Pleistocene harbored a dense and humid forest with diverse lotic habitats, similar to the northern forested area of the island at present, and that these environmental conditions were lost by the midpoint of the Holocene Period.

Cytonuclear discordance and historical demography of two brown frogs, Rana tagoi and R. sakuraii (Amphibia: Ranidae)

Previous studies have suggested that reducing the positive charge of melittin could increase endosomal release activity and improve branched polyethylenimine (BPEI)-mediated transfection. AR-23 is a melittin-related peptide from Rana tagoi, which shows 81% sequence identity with melittin but has less positively-charged residues than melittin. The present study aimed to investigate the mechanistic and functional aspects of the interaction of AR-23 with mammalian cells and thus improve BPEI-mediated gene transfection. AR23 and two AR-23 analogs (AR-20 without positively-charged residues and AR-26 with the same positively-charged residues as melittin) were analyzed. Circular dichroism (CD) spectrometry was used to analyze the secondary structures of the peptides. Peptide-induced depolarization of cell membrane, the membrane-lytic activity of the peptides, and their potency with respect to enhancing the cellular uptake of calcein were evaluated. The physicochemical characters of complexes were measured and the effect of the peptides on BPEI-mediated transfection was determined. The CD spectra results indicated that a positive charge in AR-23 played a crucial role in maintaining the α-helical conformation, whereas an extra positive charge could not increase α-helical formation. AR-23 displayed a similar depolarization ability to melittin. However, AR-23 showed a lower membrane lytic activity under physiological conditions and a higher lytic activity at endosomal pH than melittin and AR-26, which possess more positive charges. Compared to melittin and AR-26, AR-23, with a higher endosomal escaping activity, resulted in a higher enhancement of BPEI-mediated gene transfection, as well as the maintainance of a lower cytotoxicity. We suggest that AR-23 may be considered as a potential enhancer for improving the transfection efficiency of cationic polymers.

The endemic Japanese frog Rana tagoi is unique among Holarctic brown frogs in that it breeds in small subterranean streams. Using mitochondrial 16S ribosomal RNA and NADH dehydrogenase subunit 1 genes, we investigated genealogical relationships among geographic samples of this species together with its relative R. sakuraii, which is also a unique stream breeder. These two species together form a monophyletic group, within which both are reciprocally paraphyletic. Rana tagoi is divided into two major clades (Clade A and B) that are composed of 14 genetic groups. Rana sakuraii is included in Clade A and split into two genetic groups, one of which forms a clade (Subclade A-2) with sympatric R. tagoi. This species-level paraphyly appears to be caused by incomplete taxonomy, in addition to introgressive hybridization and/or incomplete lineage sorting. Rana tagoi strongly differs from other Japanese anurans in its geographic pattern of genetic differentiation, most probably in relation to its unique reproductive habits. Taxonomically, R. tagoi surely includes many cryptic species.

Phase-II conjugation ability for PAH metabolism in amphibians: Characteristics and inter-species differences