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ABSTRACT Macrobenthic communities in a lake are affected by the type of bottom cover such as macrophytes or algae. In the southern basin of Lake Biwa, mats of the benthic cyanobacteria (BC) Microseira wollei widely cover the lake bottom and are interspersed with submerged macrophytes (SMs). Because different macrobenthos species appear to occur at those bottoms, we investigated the composition of the communities. Furthermore, as M. wollei is supposed to be inedible to consumer organisms owing to its hard sheath and toxins, the food possibility of the cyanobacteria and macrophytes for macrobenthos was analyzed. In this study, macrobenthos were collected, identified molecularly, counted in the BC and SM zones, and analyzed for carbon and nitrogen stable‐isotopic compositions. In the BC zone, asellids and amphipods dominated the macrobenthic community, while chironomids dominated the SM zone. The stable‐isotope analysis revealed that M. wollei was a minor food source for macrobenthos and other higher‐level consumers, with some macrophytes, particulate organic matter and bottom sediment potentially being a major source. The dominance of crustacean macrobenthos in M. wollei mats suggested that they provided a refuge from predation for crustaceans, whereas SMs were not sufficiently abundant to achieve this. Although different macrobenthic communities in the BC and SM zones were likely supported by common food sources, with the exeption of M. wollei, the present study was conducted over a short period and lacked advanced methods for gut content analysis. Therefore, further monitoring and food web analysis are required.

Water flea Ceriodaphnia dubia has been widely used for risk assessments of chemicals and environmental contamination. In this study, the complete mitochondrial genome (mitogenome) of this species NIES strain was determined using short-read high throughput and long-read sequencing technologies. The mitogenome of C. dubia was 15,170 bp in length and consisted of 13 protein-coding genes (PCGs), 2 ribosomal RNAs (rRNAs), and 22 transfer RNAs (tRNAs). The gene order was identical to the pattern conserved across crustaceans. The complete mitogenome of the NIES strain will serve as genetical reference in ecological risk assessments in Japan, as well as resources for future phylogenetical studies using cladocerans.

Abstract Ozone is a phytotoxic air pollutant that has various damaging effects on plants, including chlorosis and growth inhibition. Although various physiological and genetic studies have elucidated some of the mechanisms underlying plant ozone sensitivity and lesion development, our understanding of plant response to this gas remains incomplete. Here, we show evidence for the involvement of certain apoplastic proteins called phytocyanins, such as AtUC5, that protect against ozone damage. Two representative ozone-inducible responses, chlorosis and stomatal closure, were suppressed in AtUC5-overexpressing plants. Analysis of transgenic plants expressing a chimeric protein composed of AtUC5 fused to green fluorescent protein indicated that this fusion protein localises to the apoplast of plant cells where it appears to suppress early responses to ozone damage such as generation or signalling of reactive oxygen species. Moreover, yeast two-hybrid analyses suggest that AtUC5 may physically interact with stress-related proteins such as copper amine oxidase and late embryogenesis abundant protein-like protein. In addition to AtUC5, other examined phytocyanins such as AtUC6 and AtSC3 could confer ozone tolerance to plants when overexpressed in A. thaliana, suggesting that these proteins act together to protect plants against oxidative stress factors.

iPSCs from three endangered avian species (including Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl) are developed and characterized as a potential resource for their conservation.

There is still much room for development in pluripotent stem cell research on avian species compared to human stem cell studies. Neural cells are useful for the evaluation of risk assessment of infectious diseases since several avian species die of encephalitis derived from infectious diseases. In this study, we attempted to develop induced pluripotent stem cells (iPSCs) technology for avian species by forming organoids containing neural-like cells. In our previous study, we established two types iPSCs from chicken somatic cells, the first is iPSCs with PB-R6F reprogramming vector and the second is iPSCs with PB-TAD-7F reprogramming vector. In this study, we first compared the nature of these two cell types using RNA-seq analysis. The total gene expression of iPSCs with PB-TAD-7F was closer to that of chicken ESCs than that of iPSCs with PB-R6F; therefore, we used iPSCs with PB-TAD-7F to form organoids containing neural-like cells. We successfully established organoids containing neural-like cells from iPSCs using PB-TAD-7F. Furthermore, our organoids responded to poly:IC through the RIG-I-like receptor (RLR) family. In this study, we developed iPSCs technology for avian species via organoid formation. In the future, organoids containing neural-like cells from avian iPSCs can develop as a new evaluation tool for infectious disease risk in avian species, including endangered avian species.

The Okinawa rail is endemic to Okinawa Island and is categorized as an endangered animal. In this study, we focused on innate immunity because it is the first line of host defense. In particular, signals recognizing foreign RNA (e.g., viruses) are important for host defense because they activate the host immune system. The retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) families (RIG-I, MDA5, and LGP2) are sensors that activate innate immunity. Therefore, we analyzed these functions in the Okinawa rail using genomic and cellular analyses of fibroblasts. Fibroblasts can be obtained from dead individuals, allowing these cells to be obtained from dead individuals, which is particularly useful for endangered species. The MDA5 gene of Okinawa rail was sequenced using the Sanger method following PCR amplification and extraction of the amplified sequence from agarose gel. Additionally, mRNA expression analysis of cultured fibroblasts exposed to poly I:C was done. The MDA5 gene was found to be a mutated nonfunctional gene in the Okinawa rail. The mRNA expression rates of inflammatory cytokine genes type I IFN, and Mx1 were slower in Okinawa rail than in chicken cultured fibroblasts. Similar to the mRNA expression results, cell number and live cell ratio also slowly decreased in the Okinawa rail compared with chicken cultured fibroblasts, indicating that the innate immune reaction differs between chicken and the Okinawa rail. To the best of our knowledge, this is the first experimental evaluation of the loss of function of the Okinawa rail innate immune genes. In conclusion, our results provide a basis for conservation strategies for the endangered Okinawa rail.

Total DNA extracts from the intestinal contents of 60 flying red-crowned cranes (juveniles, subadults and adults) found dead in 2006–2021, and the feces of 25 chicks collected in June and July of 2016–2018, were used for PCR reactions with primers specific for 16 crops, followed by high-throughput sequencing. The most predominant crop detected was corn in adult and subadult cranes (61.7%). Other grains (barley, wheat, soybean) (5.0–8.3%) and vegetables (tomatoes, Chinese cabbage, etc.) (1.7–6.7%) were also detected in flying cranes. Surprisingly, some of the detected crops were not grown in the Kushiro and Nemuro regions. There was no significant difference in crop intake status in winter and that in other seasons for most of the crops. Corn (28.0%), soybeans (8.0%), wheat and beet (4.0%) were detected in crane chicks in summer, though the detection rates were generally lower than those in flying cranes. Alfalfa, which is not grown in eastern Hokkaido but is used in some cattle feed, was detected in some cranes. Rice, buckwheat, adzuki beans, common beans, potatoes and carrots were not detected at any life stage, indicating the preferences of red-crowned cranes. The results suggest that red-crowned cranes in Hokkaido are dependent on dairy farmers for their feed supply.

The complete mitochondrial genomes of Notomastus sp. (15,776 bp) (Annelida: Capitellidae) and Armandia sp. (18,538 bp) (Annelida: Opheliidae) were assembled for the first time. A group II intron (303 bp) was found in cox1 of Notomastus sp. A phylogenetic analysis revealed that Notomastus sp. and Armandia sp. were monophyletic, and this clade was clustered with echiurans, although the possibility of the effect of long-branch attraction should be considered.

The nearly complete mitochondrial genome of the threatened tideland snail Pirenella pupiformis (Mollusca: Cerithioidea: Potamididae) was determined by shotgun next-generation sequencing. The mitogenome is comprised of 13 protein-coding genes (PCGs), two ribosomal RNA (12S and 16S) genes, and 22 transfer RNA genes (tRNAs). This gene order is consistent with the previously published mitochondrial genomes of other species belonging to the family Potamididae. The family Potamididae including P. pupiformis was recovered as a monophyletic group in the superfamily Cerithioidea.

We determined the complete mitochondrial sequences of female-transmitted (F) mitogenomes of six unionid specimens from the Lake Biwa system, Japan. Their gene contents and orders agreed with those of the typical F mitogenome of freshwater mussels. Molecular phylogenetic analysis using fifteen previously identified partial COI and 12 (six previously identified and six newly determined) whole mitogenome sequences revealed that five of the six mitogenomes (LC592401, LC592402, LC592403, LC592408, and LC592410) were those of Beringiana fukuharai, while the remaining one (LC592406) was Sinanodonta tumens.

The Indian rhinoceros (Rhinoceros unicornis) is a large herbivore found in northern India and southern Nepal. It is a critically endangered species, with an estimated population of approximately 3,600 in the wild. Genetic factors, such as the loss of genetic diversity and the accumulation of deleterious variations, are critical risk factors for the extinction of endangered species, such as the Indian rhinoceros. To support the conservation efforts of the Indian rhinoceros, we assembled its draft genome. The new genomic data will enable the study of functional genes associated with the ecological and physiological characteristics of Indian rhinoceros and help us establish more effective conservation measures. The muscles of an Indian rhinoceros that died from prostration at a zoo were collected, and the samples were stored at the National Institute for Environmental Studies (Tsukuba, Japan). Sequence data were obtained using an Illumina NovaSeq 6000 platform for short reads and an Oxford Nanopore Technologies PromethION for long reads. We generated approximately 235.2 Gbp of data. From these sequences, we assembled a 2,375,051,758 bp genome consisting of 7,615 contigs. The genome data are available from the National Center Biotechnology Information BioProject database under accession number BOSQ00000000.

We determined the complete mitochondrial sequences of female-transmitted (F) mitogenomes of two unionid specimens from the Lake Biwa system, Japan. Their gene contents and orders agreed with those of the typical F mitogenome of freshwater mussels. Molecular phylogenetic analysis using 20 previously identified partial COI and seven (five previously identified and two newly determined) whole mitogenome sequences revealed that one of the two mitogenomes was that of Nodularia douglasiae, while the other was N. nipponensis.

The mitochondrial genome of the neritid snail Nerita (Heminerita) japonica (Mollusca: Neritimorpha) from Kumamoto, Japan was determined by whole-genome sequencing. This mitogenome is comprised of 13 protein-coding genes, 2 ribosomal RNA (12S and 16S) genes, and 22 transfer RNA genes, with the same gene order as in the other species of the family Neritidae. A likelihood-based phylogenetic reconstruction recovered the subgenus Heminerita (including N. japonica as its type and N. yoldii from China) as monophyletic and sister to a clade with four species of the subgenera Nerita and Theliostyla.

Strain SCT is an iodate-reducing bacterium isolated from marine sediment in Kanagawa Prefecture, Japan. In this study, we determined the draft genome sequence of strain SCT and compared it to complete genome sequences of other closely related bacteria, including Pseudomonas stutzeri. A phylogeny inferred from concatenation of core genes revealed that strain SCT was closely related to marine isolates of P. stutzeri. Genes present in the SCT genome but absent from the other analyzed P. stutzeri genomes comprised clusters corresponding to putative prophage regions and possible operons. They included pil genes, which encode type IV pili for natural transformation; the mer operon, which encodes resistance systems for mercury; and the pst operon, which encodes a Pi-specific transport system for phosphate uptake. We found that strain SCT had more prophage-like genes than the other P. stutzeri strains and that the majority (70%) of them were SCT strain-specific. These genes, encoded on distinct prophage regions, may have been acquired after branching from a common ancestor following independent phage transfer events. Thus, the genome sequence of Pseudomonas sp. strain SCT can provide detailed insights into its metabolic potential and the evolution of genetic elements associated with its unique phenotype.

Red-crowned crane Grus japonensis is an endangered species in two separate populations: the mainland population in the Eurasian continent and the island population in eastern Hokkaido, Japan. We found 11 insertion/deletion (InDel) markers in the genome of the red-crowned crane and designed primer sets across these InDels that can be analyzed with conventional agarose gel electrophoresis. Sixty-six samples of whole blood and skeletal muscle obtained from red-crowned cranes, including 12 families in eastern Hokkaido from 1994 to 2021, showed different patterns in gel images of 11 InDel PCR reactions except for two pairs. The combined non-exclusion probability of the 11 markers indicates that individuals can be determined with a probability of 99.9%. In 39 non-relative chicks, the expected heterozygosity (He) was 0.316, suggesting low genetic diversity. This might not be caused by high levels of inbreeding since the average FIS was not significantly different from zero (0.095, p = 0.075). The results suggest that the 11 InDel primer sets can be used for fairly accurate individual identification as well as genetic population analyses in red-crowned cranes in the island population.

The Tsushima leopard cat (Prionailurus bengalensis euptilurus) (TLC) is a regional population of the Amur leopard cat (P. bengalensis euptilurus) that lives only on the Tsushima Island in Japan and is threatened with extinction. Because the TLC population is small, genetic management is important. In this study, we obtained the draft genome of the TLC and identified single-nucleotide polymorphism (SNP) markers using a genotyping by random amplicon sequencing-direct (GRAS-Di) analysis. We genotyped 31 captive individuals and 50 wild individuals, of which 48 were from a previous study. The identified SNPs were used to clarify the genetic diversity and genetic structure of the wild and captive populations of the TLC. The size of the genome was estimated to be about 2.42 Gb. The number of SNP markers developed was 139, and although PID and probability of exclusion obtained using these SNP markers were not as high as those reported in the studies of other wild species, these SNP markers could be used to identify individuals and parentage. Moreover, the genetic diversity indices of the captive population were similar to those of the wild population. These SNP markers will be useful for understanding the ecology of the TLC and planning conservation strategies.

A non-biting midge Chironomus yoshimatsui has been widely used in ecotoxicology and chemical risk assessments. In this study, the complete mitochondrial genome (mitogenome) sequence of C. yoshimatsui was determined using short-read next-generation sequencing technologies. The mitogenome was 15,734 bp in length and consisted of 13 protein coding genes, 2 ribosomal RNAs, and 22 transfer RNAs. The A + T content was 77.8%. The gene order was identical to the pattern conserved across Diptera. The mitocgenome sequence obtained in this study provides a useful resource for further evolutionary and ecological studies.

We aimed to reveal the dispersal and gene flow of the local wild boar (Sus scrofa) population and find their genetic boundary in Fukushima Prefecture. After the nuclear incident in 2011, the land was considered a difficult-to-return zone, and the increase in the number of wild boars was pronounced. To provide an effective management strategy for the wild boar population, we used multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) and clarified the genetic structure of wild boars. We obtained 328 single-nucleotide polymorphisms from 179 samples. STRUCTURE analysis showed that the most likely number of population cluster was K = 2. Molecular analysis of variance showed significant genetic differences between groups of wild boars inhabiting in the east and west across the Abukuma River. The migration rate from the eastern population to the western population is higher than in the reverse case based on BayesAss analysis. Our study indicates that both the Abukuma River and anthropogenic urbanization along the river may affect the migration of wild boars and the population in western was established mainly by the migration from other neighboring prefectures.

The complete mitochondrial genome sequence of an estuarine amphipod Grandidierella osakaensis was determined. The mitochondrial genome was 14,658 bp in length with 37 mitochondrial genes (13 protein-coding genes [PCGs], 2 ribosomal RNAs [rRNAs], and 22 transfer RNAs [tRNAs]). The order of PCGs of G. osakaensis was identical to those of other two Grandidierella species. A maximum likelihood-based phylogenetic analysis showed that G. osakaensis formed a monophyletic clade with the other two Grandidierella species within the infraorder Corophiida. The mitochondrial genome sequence obtained in this study provides useful information for further phylogenetic and ecological studies.

We determined the complete mitochondrial DNA sequence of Silurus lithophilus, a catfish endemic to Lake Biwa, Japan. The mitogenome was 16,524 bp in length and the gene contents and order agreed with those of typical vertebrates. A molecular phylogenetic analysis based on 12 published Silurus mitogenomes showed that the mitogenome of S. lithophilus (LC520058) was closest to that of Silurus asotus from Japan (AP012022). These two mitogenomes were identical in the short (ca. 170 bp) gene region widely used in metabarcoding for environmental DNA analysis of fishes.

The complete mitochondrial genome sequences of Grandidierella rubroantennata and G. fasciata were determined in this study. The mitochondrial genomes were 14,469 bp and 14,656 bp in length and consisted of 37 mitochondrial genes (13 PCGs, 2 rRNAs, and 22 tRNAs). The phylogenetic analyses showed that both Grandidierella species formed one clade and were grouped with species belonging to the infraorder Corophiida, which is consistent with morphology-based classification. The results obtained in this study provide a useful reference for further phylogenetic and ecological studies.

Rapeseed (Brassica napus L.) is one of the most economically important oilseed crops worldwide. In Japan, it has been cultivated for more than a century and has formed many feral populations. The aim of this study was to elucidate the genetic diversity of feral rapeseeds by genotyping 537 individuals (among which 130 were determined to be genetically modified) sampled from various regions in Japan. Analysis of 30 microsatellite markers amplified 334 alleles and indicated moderate genetic diversity and high inbreeding (expected heterozygosity, 0.50; observed heterozygosity, 0.16; inbreeding coefficient within individuals, 0.68) within the feral populations. The Mantel test showed only an insignificant weak positive correlation between geographic distance and genetic distance. Analysis of molecular variance showed a greater genetic diversity among individuals than between populations. These results are in accordance with population structure assessed by using principal coordinate analysis and the program STRUCTURE, which showed that the 537 individuals could be assigned to 8 genetic clusters with very large genetic differences among individuals within the same geographic population, and that among feral individuals, many are closely related to rapeseed accessions in the NARO Genebank but some have unknown origins. These unique feral rapeseeds are likely to be affected by strong selection pressure. The results for genetically modified individuals also suggest that they have two different sources and have a considerable degree of diversity, which might be explained by hybridization with nearby individuals and separation of hybrid cultivars. The information obtained in this study could help improve the management of feral rapeseed plants in Japan.

The Tsushima leopard cat (Prionailurus bengalensis euptilurus) lives on Tsushima Island in Japan and is a regional population of the Amur leopard cat; it is threatened with extinction. Its genetic management is important because of the small population. We used genotyping by random amplicon sequencing-direct (GRAS-Di) to develop a draft genome and explore single-nucleotide polymorphism (SNP) markers. The SNPs were analyzed using three genotyping methods (mapping de novo, to the Tsushima leopard cat draft genome, and to the domestic cat genome). We examined the genetic diversity and genetic structure of the Tsushima leopard cat. The genome size was approximately 2.435 Gb. The number of SNPs identified was 133–158. The power of these markers was sufficient for individual and parentage identifications. These SNPs can provide useful information about the life of the Tsushima leopard cat and the pairings and for the introduction of founders to conserve genetic diversity with ex situ conservation. We identified that there are no subpopulations of the Tsushima leopard cat. The identifying units will allow for a concentration of efforts for conservation. SNPs can be applied to the analysis of the leopard cat in other regions, making them useful for comparisons among populations and conservation in other small populations.

We determined two complete mitochondrial sequences of female-transmitted (F) mitogenomes of two Hyriopsis schlegelii specimens from this species’ original habitat, Lake Biwa. The mitogenomes were both 15,951 bp in length, and the gene contents and orders agreed with those of the typical F mitogenome of Hyriopsis. Molecular phylogenetic analysis based on the previously identified 13 partial sequences confirmed that the two mitogenomes both belonged to H. schlegelii and not to a closely related Chinese species, Hyriopsis cumingii. The same analysis revealed that two mitogenomes (HQ641406 and FJ529186) previously published as H. schlegelii and H. cumingii might be misidentified.

In this study, Babesia screening was conducted in 55 rodents and 160 tick samples collected from primary forests and an oil palm plantation in Sarawak, Malaysian Borneo. PCR targeting the 18S ribosomal DNA revealed the presence of Babesia spp. DNA detected in two questing male Haemaphysalis shimoga ticks collected from the oil palm plantation. Sequence analysis revealed that both sequences were identical and had 98.6% identity to a Babesia macropus sequence obtained from Eastern grey kangaroos (Macropus giganteus) in Australia. Phylogenetic tree revealed clustering with marsupial-associated Babesia spp. in the Babesia sensu stricto clade. Whether or not H. shimoga is the competent vector and the importance of the Babesia sp. detected in this study warrants more investigation. [ABSTRACT FROM AUTHOR]