jMorp: Japanese Multi-Omics Reference Panel update report 2023.

Modern medicine is increasingly focused on personalized medicine, and multi-omics data is crucial in understanding biological phenomena and disease mechanisms. Each ethnic group has its unique genetic background with specific genomic variations influencing disease risk and drug response. Therefore, multi-omics data from specific ethnic populations are essential for the effective implementation of personalized medicine. Various prospective cohort studies, such as the UK Biobank, All of Us and Lifelines, have been conducted worldwide. The Tohoku Medical Megabank project was initiated after the Great East Japan Earthquake in 2011. It collects biological specimens and conducts genome and omics analyses to build a basis for personalized medicine. Summary statistical data from these analyses are available in the jMorp web database (https://jmorp.megabank.tohoku.ac.jp), which provides a multidimensional approach to the diversity of the Japanese population. jMorp was launched in 2015 as a public database for plasma metabolome and proteome analyses and has been continuously updated. The current update will significantly expand the scale of the data (metabolome, genome, transcriptome, and metagenome). In addition, the user interface and backend server implementations were rewritten to improve the connectivity between the items stored in jMorp. This paper provides an overview of the new version of the jMorp.

Study Profile of the Tsuruoka Metabolomics Cohort Study (TMCS).

The Tsuruoka Metabolomics Cohort Study (TMCS) is an ongoing population-based cohort study being conducted in the rural area of Yamagata Prefecture, Japan. This study aimed to enhance the precision prevention of multi-factorial, complex diseases, including non-communicable and aging-associated diseases, by improving risk stratification and prediction measures. At baseline, 11,002 participants aged 35-74 years were recruited in Tsuruoka City, Yamagata Prefecture, Japan, between 2012 and 2015, with an ongoing follow-up survey. Participants underwent various measurements, examinations, tests, and questionnaires on their health, lifestyle, and social factors. This study used an integrative approach with deep molecular profiling to identify potential biomarkers linked to phenotypes that underpin disease pathophysiology and provide better mechanistic insights into social health determinants. The TMCS incorporates multi-omics data, including genetic and metabolomic analyses of 10,933 participants and comprehensive data collection ranging from physical, psychological, behavioral, and social to biological data. The metabolome is used as a phenotypic probe because it is sensitive to changes in physiological and external conditions. The TMCS focuses on collecting outcomes for cardiovascular disease, cancer incidence and mortality, disability, functional decline due to aging and disease sequelae, and the variation in health status within the body represented by omics analysis that lies between exposure and disease. It contains several sub-studies on aging, heated tobacco products, and women's health. This study is notable for its robust design, high participation rate (89%), and long-term repeated surveys. Moreover, it contributes to precision prevention in Japan and East Asia as a well-established multi-omics platform.

Evolutionary Tuning of Transient Receptor Potential Ankyrin 1 Underlies the Variation in Heat Avoidance Behaviors among Frog Species Inhabiting Diverse Thermal Niches.

Environmental temperature is a critical factor for all forms of life, and thermal tolerance defines the habitats utilized by a species. Moreover, the evolutionary tuning of thermal perception can also play a key role in habitat selection. Yet, the relative importance of thermal tolerance and perception in environmental adaptation remains poorly understood. Thermal conditions experienced by anuran tadpoles differ among species due to the variation in breeding seasons and water environments selected by parental frogs. In the present study, heat tolerance and avoidance temperatures were compared in tadpoles from five anuran species that spatially and temporally inhabit different thermal niches. These two parameters were positively correlated with each other and were consistent with the thermal conditions of habitats. The species difference in avoidance temperature was 2.6 times larger than that in heat tolerance, suggesting the importance of heat avoidance responses in habitat selection. In addition, the avoidance temperature increased after warm acclimation, especially in the species frequently exposed to heat in their habitats. Characterization of the heat-sensing transient receptor potential ankyrin 1 (TRPA1) ion channel revealed an amphibian-specific alternatively spliced variant containing a single valine insertion relative to the canonical alternative spliced variant of TRPA1, and this novel variant altered the response to thermal stimuli. The two alternatively spliced variants of TRPA1 exhibited different thermal responses in a species-specific manner, which are likely to be associated with a difference in avoidance temperatures among species. Together, our findings suggest that the functional change in TRPA1 plays a crucial role in thermal adaptation processes.

jMorp updates in 2020: large enhancement of multi-omics data resources on the general Japanese population.

In the Tohoku Medical Megabank project, genome and omics analyses of participants in two cohort studies were performed. A part of the data is available at the Japanese Multi Omics Reference Panel (jMorp; https://jmorp.megabank.tohoku.ac.jp) as a web-based database, as reported in our previous manuscript published in Nucleic Acid Research in 2018. At that time, jMorp mainly consisted of metabolome data; however, now genome, methylome, and transcriptome data have been integrated in addition to the enhancement of the number of samples for the metabolome data. For genomic data, jMorp provides a Japanese reference sequence obtained using de novo assembly of sequences from three Japanese individuals and allele frequencies obtained using whole-genome sequencing of 8,380 Japanese individuals. In addition, the omics data include methylome and transcriptome data from ∼300 samples and distribution of concentrations of more than 755 metabolites obtained using high-throughput nuclear magnetic resonance and high-sensitivity mass spectrometry. In summary, jMorp now provides four different kinds of omics data (genome, methylome, transcriptome, and metabolome), with a user-friendly web interface. This will be a useful scientific data resource on the general population for the discovery of disease biomarkers and personalized disease prevention and early diagnosis.

Evaluation of clinical formalin-fixed paraffin-embedded tissue quality for targeted-bisulfite sequencing.

Formalin-fixed paraffin-embedded (FFPE) tissues are promising biological resources for genetic research. Recent improvements in DNA extraction from FFPE samples allowed the use of these tissues for multiple sequencing methods. However, fundamental research addressing the application of FFPE-derived DNA for targeted-bisulfite sequencing (TB-seq) is lacking. Here, we evaluated the suitability of FFPE-derived DNA for TB-seq. We conducted TB-seq using FFPE-derived DNA and corresponding fresh frozen (FF) tissues of patients with kidney cancer and compared the quality of DNA, libraries, and TB-seq statistics between the two preservation methods. The approximately 600-bp average fragment size of the FFPE-derived DNA was significantly shorter than that of the FF-derived DNA. The sequencing libraries constructed using FFPE-derived DNA and the mapping ratio were approximately 10 times and 10% lower, respectively, than those constructed using FF-derived DNA. In the mapped data of FFPE-derived DNA, duplicated reads accounted for > 60% of the obtained sequence reads, with lower mean on-target coverage. Therefore, the standard TB-seq protocol is inadequate for obtaining high-quality data for epigenetic analysis from FFPE-derived DNA, and technical improvements are necessary for enabling the use of archived FFPE resources.

Characterisation of major histocompatibility complex class I genes in Japanese Ranidae frogs.

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.

Microsatellite marker development by multiplex ion torrent PGM sequencing: a case study of the endangered Odorrana narina complex of frogs.

The endangered Ryukyu tip-nosed frog Odorrana narina and its related species, Odorrana amamiensis, Odorrana supranarina, and Odorrana utsunomiyaorum, belong to the family Ranidae and are endemically distributed in Okinawa (O. narina), Amami and Tokunoshima (O. amamiensis), and Ishigaki and Iriomote (O. supranarina and O. utsunomiyaorum) Islands. Because of varying distribution patterns, this species complex is an intrinsic model for speciation and adaptation. For effective conservation and molecular ecological studies, further genetic information is needed. For rapid, cost-effective development of several microsatellite markers for these and 2 other species, we used next-generation sequencing technology of Ion Torrent PGM™. Distribution patterns of repeat motifs of microsatellite loci in these modern frog species (Neobatrachia) were similarly skewed. We isolated and characterized 20 new microsatellite loci of O. narina and validated cross-amplification in the three-related species. Seventeen, 16, and 13 loci were cross-amplified in O. amamiensis, O. supranarina, and O. utsunomiyaorum, respectively, reflecting close genetic relationships between them. Mean number of alleles and expected heterozygosity of newly isolated loci varied depending on the size of each inhabited island. Our findings suggested the suitability of Ion Torrent PGM™ for microsatellite marker development. The new markers developed for the O. narina complex will be applicable in conservation genetics and molecular ecological studies.

Dimension reduction of microbiome data linked Bifidobacterium and Prevotella to allergic rhinitis.

Dimension reduction has been used to visualise the distribution of multidimensional microbiome data, but the composite variables calculated by the dimension reduction methods have not been widely used to investigate the relationship of the human gut microbiome with lifestyle and disease. In the present study, we applied several dimension reduction methods, including principal component analysis, principal coordinate analysis (PCoA), non-metric multidimensional scaling (NMDS), and non-negative matrix factorization, to a microbiome dataset from 186 subjects with symptoms of  allergic rhinitis (AR) and 106 controls. All the dimension reduction methods supported that the distribution of microbial data points appeared to be continuous rather than discrete. Comparison of the composite variables calculated from the different dimension reduction methods showed that the characteristics of the composite variables differed depending on the distance matrices and the dimension reduction methods. The first composite variables calculated from PCoA and NMDS with the UniFrac distance were strongly associated with AR (FDR adjusted P = 2.4 × 10-4 for PCoA and P = 2.8 × 10-4 for NMDS), and also with the relative abundance of Bifidobacterium and Prevotella. The abundance of Bifidobacterium was also linked to intake of several nutrients, including carbohydrate, saturated fat, and alcohol via composite variables. Notably, the association between the composite variables and AR was much stronger than the association between the relative abundance of individual genera and AR. Our results highlight the usefulness of the dimension reduction methods for investigating the association of microbial composition with lifestyle and disease in clinical research.

Integrated analysis of human DNA methylation, gene expression, and genomic variation in iMETHYL database using kernel tensor decomposition-based unsupervised feature extraction.

Integrating gene expression, DNA methylation, and genomic variants simultaneously without location coincidence (i.e., irrespective of distance from each other) or pairwise coincidence (i.e., direct identification of triplets of gene expression, DNA methylation, and genomic variants, and not integration of pairwise coincidences) is difficult. In this study, we integrated gene expression, DNA methylation, and genome variants from the iMETHYL database using the recently proposed kernel tensor decomposition-based unsupervised feature extraction method with limited computational resources (i.e., short CPU time and small memory requirements). Our methods do not require prior knowledge of the subjects because they are fully unsupervised in that unsupervised tensor decomposition is used. The selected genes and genomic variants were significantly targeted by transcription factors that were biologically enriched in KEGG pathway terms as well as in the intra-related regulatory network. The proposed method is promising for integrated analyses of gene expression, methylation, and genomic variants with limited computational resources.

Epigenetic profile of Japanese supercentenarians: a cross-sectional study.

BACKGROUND: Centenarians and supercentenarians with exceptional longevity are excellent models for research towards improvements of healthy life expectancy. Extensive research regarding the maintenance and reduction of epigenetic age has provided insights into increasing healthy longevity. To this end, we explored the epigenetic signatures reflecting hallmarks of exceptional healthy longevity, including avoidance of age-related diseases and cognitive functional decline. METHODS: In this cross-sectional study, we enrolled Japanese non-centenarians (eligible participants aged 20-80 years) from the Tohoku Medical Megabank Community-Based Cohort Study and centenarians and supercentenarians (aged 101-115 years) from the Tokyo Centenarian Study and the Japanese Semi-supercentenarian Study. We assessed participants' whole-blood DNA methylation profiles and then developed sex-specific and non-specific first-generation epigenetic clocks by elastic net regression, calculated individuals' epigenetic ages, and assessed their age acceleration. We also screened for age-related CpG sites in non-centenarians by epigenome-wide linear regression analyses and ANOVA. We subsequently investigated which CpG sites in centenarians and supercentenarians had DNA methylation patterns following the age-related findings obtained from non-centenarians and which did not. We further characterised CpG sites with hypermethylation or hypomethylation in the centenarians and supercentenarians using enrichment and protein-protein interaction network analyses. FINDINGS: We enrolled 421 non-centenarians (231 [55%] women and 190 [45%] men; age range 20-78 years), recruited between May 20, 2013, and March 31, 2016, and 94 centenarians and supercentenarians (66 women [70%] and 28 [30%] men; age range 101-115 years), recruited between Jan 20, 2001, and April 17, 2018. Non-sex-specific epigenetic clock showed the highest accuracy (r=0·96) based on which centenarians and supercentenarians had negative epigenetic age acceleration. Epigenome-wide association analyses further showed that centenarians and supercentenarians had younger-than-expected epigenetic states (DNA methylation profiles similar to those of non-centenarians) for 557 CpG sites enriched in cancer-related and neuropsychiatric-related genes, whereas these individuals had advanced (or older) epigenetic states for 163 CpG sites represented by genes related to TGF-ß signalling, which is involved in anti-inflammatory responses and known to contribute to healthy ageing. INTERPRETATION: These results indicate that exceptionally healthy longevity depends not only on maintaining young epigenetic states but also on advanced states of specific epigenetic regions. FUNDING: The Japan Agency for Medical Research and Development, KDDI Research, and Keio University. TRANSLATION: For the Japanese translation of the abstract see Supplementary Materials section.

Distributional change and epidemic introgression in overlapping areas of Japanese pond frog species over 30 years.

Pelophylax nigromaculatus, P. porosus porosus, and P. p. brevipoda are three pond frog species distributed in Japan. Their distributions overlap at two basins in central Japan (P. nigromaculatus and P. p. porosus in the Matsumoto basin, and P. nigromaculatus and P. p. brevipoda in the Ina basin), and hybrid descendants have been found in these areas. To clarify the distribution areas and hybrid zones of the frogs, and to understand the mode of introgressive hybridization and its impact on the frog populations, we conducted exhaustive sampling at each basin and performed allozyme and mtDNA analyses of 233 individuals. Analysis using genetic markers clearly detected nine F1 hybrids and 94 hybrid descendants of P. nigromaculatus and P. porosus from the overlapping areas of both basins. Allozyme and mtDNA data suggest directional hybridization between female P. p. porosus and male P. nigromaculatus in the Matsumoto basin. Over the past 30 years, the distribution of P. p. porosus has been narrowed and fragmented by the invasion of P. nigromaculatus, seemingly because of directional hybridization in the Matsumoto basin. In the Ina basin, the "pure" P. p. brevipoda (n = 8) population was extremely reduced by gene introgression from P. nigromaculatus, yet its distribution was barely changed compared to the Matsumoto basin. Consequently, this study shows that P. porosus populations are threatened by interspecific hybridization with P. nigromaculatus, and that introgressive hybridization damaged P. porosus populations by different means in each basin.

DNA Methylation Abnormalities and Altered Whole Transcriptome Profiles after Switching from Combustible Tobacco Smoking to Heated Tobacco Products.

BACKGROUND: The use of heated tobacco products (HTP) has increased exponentially in Japan since 2016; however, their effects on health remain a major concern. METHODS: Tsuruoka Metabolome Cohort Study participants (n = 11,002) were grouped on the basis of their smoking habits as never smokers (NS), past smokers (PS), combustible tobacco smokers (CS), and HTP users for <2 years. Peripheral blood mononuclear cells were collected from 52 participants per group matched to HTP users using propensity scores, and DNA and RNA were purified from the samples. DNA methylation (DNAm) analysis of the 17 smoking-associated DNAm biomarker genes (such as AHRR, F2RL3, LRRN3, and GPR15), as well as whole transcriptome analysis, was performed. RESULTS: Ten of the 17 genes were significantly hypomethylated in CS and HTP users compared with NS, among which AHRR, F2RL3, and RARA showed intermediate characteristics between CS and NS; nonetheless, AHRR expression was significantly higher in CS than in the other three groups. Conversely, LRRN3 and GPR15 were more hypomethylated in HTP users than in NS, and GPR15 expression was markedly upregulated in all the groups when compared with that in NS. CONCLUSIONS: HTP users (switched from CS <2 years) display abnormal DNAm and transcriptome profiles, albeit to a lesser extent than the CS. However, because the molecular genetic effects of long-term HTP use are still unknown, long-term molecular epidemiologic studies are needed. IMPACT: This study provides new insights into the molecular genetic effects on DNAm and transcriptome profiles in HTP users who switched from CS.

Evaluation of short-term epigenetic age fluctuation.

Considerable effort has been spent on lowering and maintaining the epigenetic age. However, the extent to which epigenetic age fluctuates under normal conditions is poorly understood. Therefore, we analyzed methylation data from monocytes and peripheral blood mononuclear cells collected from two Japanese men. The ranges of the Pan-tissue, Skin and blood, and DNAm PhenoAge epigenetic age during 3 months were ≥ 5.62, ≥ 3.04, and ≥ 8.23 years, and the maximum daily changes were 5.21, 3.20, and 6.53 years, respectively. These fluctuations were not suppressed by correcting for cell-type composition. Although the underlying biological mechanism remains unclear, there was a nonnegligible degree of age fluctuation which should inform personalized clinical applications.

Low MICA Gene Expression Confers an Increased Risk of Graves' Disease: A Mendelian Randomization Study.

Background: Expression of natural killer group 2 member D (NKG2D) ligand (NKG2DL) plays a major role as a "danger signal" on stressed cells to promote removal of the latter by NKG2D-expressing cytotoxic lymphocytes. NKG2DL expression has been found in peripheral immune cells as well, such as in macrophages; however, the effect of this expression is yet to be determined. Methods: We determined instrumental variables (IVs; R2 <0.01 in linkage disequilibrium), explaining the major variance in major histocompatibility complex class I chain-related protein A (MICA) and B (MICB) gene expression levels from the expression-quantitative trait locus (eQTL) of NKG2DLs based on the RNA-seq analysis of peripheral blood mononuclear cells (PBMCs) from 381 Japanese. Simultaneously, the target outcomes were filtered by PheWAS from 58 health risks, using a community-based cohort study composed of 44,739 Japanese residents. Finally, we estimated the causal effect of gene expression levels on the outcomes using the Mendelian randomization approach. Results: We determined nine and four IVs, explaining 87.6% and 33.0% of MICA and MICB gene expression levels, respectively. In the association test, we identified 10 or 13 significant outcomes associated with the MICA or MICB eQTLs, respectively, as well as the causal effect of MICA expression on Graves' disease (GD) (p = 4.2 × 10-3; odds ratio per 1 S.D. difference in the expression: 0.983 [confidence interval: 0.971-0.995]), using the weighted median estimator, without significant pleiotropy (p > 0.05), and the results were consistent across the sensitivity analyses. Conclusions: Our study provide novel evidence associating NKG2DL expression with GD, an autoimmune thyroiditis; direction of the effect indicated the immunoregulatory role of MICA expression in PBMCs, suggesting the importance of further functional assays in inflammatory diseases.

Resistance to chemical carcinogenesis induction via a dampened inflammatory response in naked mole-rats.

Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted studies on the responsible mechanisms to provide clues for human cancer prevention. However, it remains unknown whether and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration. NMRs harbour loss-of-function mutations in RIPK3 and MLKL genes, which are essential for necroptosis, a type of necrotic cell death that activates strong inflammation. In mice, disruption of Ripk3 reduced immune cell infiltration and delayed carcinogenesis. Therefore, necroptosis deficiency may serve as a cancer resistance mechanism via attenuating the inflammatory response in NMRs. Our study sheds light on the importance of a dampened inflammatory response as a non-cell-autonomous cancer resistance mechanism in NMRs.

Longitudinal DNA methylation dynamics as a practical indicator in clinical epigenetics.

BACKGROUND: One of the fundamental assumptions of DNA methylation in clinical epigenetics is that DNA methylation status can change over time with or without interplay with environmental and clinical conditions. However, little is known about how DNA methylation status changes over time under ordinary environmental and clinical conditions. In this study, we revisited the high frequency longitudinal DNA methylation data of two Japanese males (24 time-points within three months) and characterized the longitudinal dynamics. RESULTS: The results showed that the majority of CpGs on Illumina HumanMethylation450 BeadChip probe set were longitudinally stable over the time period of three months. Focusing on dynamic and stable CpGs extracted from datasets, dynamic CpGs were more likely to be reported as epigenome-wide association study (EWAS) markers of various traits, especially those of immune- and inflammatory-related traits; meanwhile, the stable CpGs were enriched in metabolism-related genes and were less likely to be EWAS markers, indicating that the stable CpGs are stable both in the short-term within individuals and under various environmental and clinical conditions. CONCLUSIONS: This study indicates that CpGs with different stabilities are involved in different functions and traits, and thus, they are potential indicators that can be applied for clinical epigenetic studies to outline underlying mechanisms.

Expression Changes of MHC and Other Immune Genes in Frog Skin during Ontogeny.

Anuran amphibians undergo major physiological and immunological changes following metamorphosis. Genes of the major histocompatibility complex (MHC) code for receptors important for vertebrate adaptive immunity. We used qPCR to measure skin MHC expression in six different ontological stages of Rana ornativentris (n = 10 per stage); normalized MHC class I and II expression at the mRNA level was significantly higher in stage 28 (mid-larval) compared to stages 24/25 (early-larval) tadpoles. Subsequent transcriptomic analyses of three tadpole (early-, mid-, and late-larval) stages of R. ornativentris and model species Xenopus tropicalis focused on mRNA expression of immune-related genes in the skin. Normalized expression of most MHC class I and II transcripts in both species were significantly higher in mid- and late-larval stages compared to early-larval stage. In addition, gene ontology (GO) analyses of differentially expressed transcripts revealed several immune-related GO terms that were significantly upregulated from the mid-larval stage. Our study provides evidence that both MHC class I and II is expressed during development in both R. ornativentris and X. tropicalis.

ALDH2 genotype modulates the association between alcohol consumption and AST/ALT ratio among middle-aged Japanese men: a genome-wide G × E interaction analysis.

Liver tests (LT), especially to measure AST, ALT and GGT levels, are widely used to evaluate the risk of alcohol-related liver disease (ALD). In this study, we investigated the potential genetic factors that modulate the association between LTs and alcohol consumption. We conducted a genome-wide interaction meta-analysis in 7856 Japanese subjects from Tohoku Medical Megabank Community-Based Cohort (TMM CommCohort) study recruited in 2013, and identified 2 loci (12q24 and 2p16) with genome-wide significance (P > 5 × 10-8). The significant variants in the 12q24 included rs671, a variant associated with alcohol intolerance and located at a coding exon of ALDH2. We found that the amount of alcohol consumption was associated with increased level AST/ALT ratio among the subjects with the rs671 GA genotype. The elevated AST/ALT ratio among subjects with moderate-to-high levels of drinking behavior and the rs671 GA genotype was due to decreased levels of ALT, which was not accompanied with significant differences in AST levels. Although the interaction effect was significant in both men and women, the effect was much larger in men. Our results suggest that the impact of alcohol consumption on LT varies according to the ALDH2 genotype, providing an insight for the accurate screening of ALD in drinkers with the rs671 GA genotype.

Hot spring frogs (Buergeria japonica) prefer cooler water to hot water.

"Hot spring frog" is an informal name used for the Japanese stream tree frog (Buergeria japonica), which is widely distributed in Taiwan and the Ryukyu Archipelago in Japan. Some populations of the species are known to inhabit hot springs. However, water temperature can be extremely high around the sources of hot springs. Thus, it is questionable whether B. japonica selectively inhabits such dangerous environments. To address this question, we conducted a series of observations of water temperature preferences of a hot spring population of B. japonica in Kuchinoshima Island in Japan: (a) a field observation of tadpole density in water pools of different temperatures, (b) a field observation of water temperatures where adult males appear for breeding, and (c) an indoor observation of water temperatures selected by adult females for oviposition. As a result, tadpoles showed a higher density in cooler water. Adult males avoided water pools hotter than 37°C, and adult females selected cooler pools for oviposition. Camera records also showed that adult individuals tend to appear around cooler pools. Thus, we did not find any support for the hypothesis that hot spring frogs prefer hot water. Conversely, they apparently tended to prefer cooler water if it was available. Water temperatures around the sources of the hot spring exceed thermal tolerances of the species and could be a strong selective pressure on the population. Thus, the ability to sense and avoid lethal temperatures may be a key ecological and physiological characteristic for the species that inhabit hot springs.

An application of PCR-RFLP species identification assay for environmental DNA detection.

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.