Biology subjects

Shibata, N.

Publications and source records attributed to Shibata, N..

12 recordsLinked to original sources

bioRxiv2025

High-resolution insights into the geographic differentiation and hybridisation of Odontobutis gobies through integrated eDNA and SNP analyses

Understanding fine-scale population genetic structure is essential for biodiversity conservation and evolutionary research, but conventional phylogeographic studies often face labour and financial cost constraints. This study proposes a two-step survey strategy that integrates environmental DNA (eDNA) analysis and PCR-based genome-wide SNP genotyping, aiming to evaluate its effectiveness by comprehensively characterising the population structure of widely distributed species. As a model system, we selected the odontobutid gobies, Odontobutis obscurus and O. hikimius, which occur in western Japan. Initially, water samples were collected from 335 sites across western Japan. Subsequently, tissue sampling for SNP analysis was conducted at 49 sites representing the regional groups and species. The eDNA analysis revealed two major mitochondrial clades within O. obscurus, each comprising multiple geographically distinct groups. Subsequently, tissue sampling and SNP analysis were conducted at representative sites of each regional group and species. Nuclear genomic SNP data (661 loci) corroborated the deep divergence between the two clades of O. obscurus and, unexpectedly, indicated that O. hikimius, whose range lies at their boundary, originated through hybridisation between them. Geographic patterns of the regional groups inferred from both mitochondrial and nuclear data were largely explained by historical geological events such as mountain uplift and ancient river system dynamics, and provide unprecedentedly detailed insight into the population structuring of the focal species. This study demonstrates that the integration of eDNA and SNP analyses provides a cost-effective and scalable approach for high-resolution phylogeographic surveys.

molecular biology↗

bioRxiv2025

A Practical and Cost-Effective Approach to Long-Fragment eDNA Sequencing for High-Resolution Genetic Diversity Assessment

Environmental DNA (eDNA) analysis is increasingly recognised as a valuable method for assessing genetic diversity. However, its resolution and applicability are limited by the short length of sequences that can be analysed (typically < 400 bp) and high analytical costs. This study developed a practical, low-cost long-fragment eDNA analysis method using commercial full-length plasmid sequencing via a nanopore platform and evaluated its effectiveness in assessing population genetic structure. 1 L of surface water was collected from 52 sites across Hokkaido, Japan, targeting Barbatula oreas. Two mitochondrial regions (ND5 and cyt b; approximately 1,000 bp each) were species-specifically amplified, circularised, and sequenced. Library preparation took 2.5 hours, with a total cost per sample of 4,390 JPY ({approx}25.55 EUR, {approx}29.87 USD). High-quality reads were obtained from 34 samples, allowing for the reconstruction of multiple haplotypes per region through haplotype phasing. The eDNA concentration required to achieve a 50% sequencing success was within a range easily attainable for common species. Phylogenetic analysis using 62 concatenated haplotypes (1,968 bp) obtained from each sample identified two clades and multiple regional subgroups, providing higher-resolution phylogeographic information than the previous study. Furthermore, the differentiation of each clade and group was suggested to reflect geological and climatic events. These results demonstrate the feasibility and utility of long-fragment eDNA analysis for evaluating genetic diversity, and its broad application is anticipated in ecological research, conservation management, and environmental policy formulation.

molecular biology↗

bioRxiv2023

Identification of fish spawning based on size-selected sampling and diurnal concentration changes of environmental DNA resulting from sperm-release

Spawning is one of the most significant aspects of fish life history, so understanding spawning is critical for the conservation and management of species. Recently, an efficient and non-invasive spawning detection method for spawning behaviour has been proposed, utilising the increased environmental DNA (eDNA) concentration and nuclear/mitochondrial eDNA ratio associated with fish spawning. However, little is known about the characteristics and dynamics of sperm-derived eDNA, which is key to detection. This study focused on changes in eDNA particle size distribution (PSD) and concentrations pre-, post-, and during spawning of Ayu, Plecoglossus altivelis. Firstly, PSD changes between pre- and post-spawning were investigated by comparing concentrations and proportions of eDNA obtained from filters with different pore sizes. Secondly, the diurnal changes in eDNA concentration were monitored at the peak of the spawning season by collecting river water every one or three hours. Results showed that eDNA related to sperm-head size increased at post-spawning, and eDNA concentrations had significant diurnal changes with a peak during the spawning time window. These findings suggest that semi-selective recovery of sperm-derived eDNA based on particle size and/or sampling during the spawning time window with increased concentrations can improve the detection sensitivity of eDNA-based spawning surveys. This study provides essential basic information for advancing eDNA-based spawning surveys and contributes to their further development.

molecular biology↗

bioRxiv2022

Environmental DNA phylogeography: successful reconstruction of phylogeographic patterns of multiple fish species from a cup of water

Phylogeography is an integrative field of science linking micro- and macro-evolutionary processes, contributing to the inference of vicariance, dispersal, speciation, and other population-level processes. Phylogeographic surveys usually require considerable effort and time to obtain numerous samples from many geographical sites covering the distribution range of target species; this associated high cost limits their application. Recently, environmental DNA (eDNA) analysis has been useful not only for detecting species but also for assessing genetic diversity; hence, there has been growing interest in its application to phylogeography. As the first step of eDNA-based phylogeography, we examined (1) data screening procedures suitable for phylogeography and (2) whether the results obtained from eDNA analysis accurately reflect known phylogeographic patterns. For these purposes, we performed quantitative eDNA metabarcoding using group-specific primer sets in five freshwater fish species belonging to two taxonomic groups from a total of 94 water samples collected from western Japan. As a result, three-step data screening based on the DNA copy number of each haplotype detected successfully eliminated suspected false positive haplotypes. Furthermore, eDNA analysis could almost perfectly reconstruct the phylogenetic and phylogeographic patterns obtained for all target species with the conventional method. Despite existing limitations and future challenges, eDNA-based phylogeography can significantly reduce survey time and effort and is applicable for simultaneous analysis of multiple species in single water samples. eDNA-based phylogeography has the potential to revolutionise phylogeography.

molecular biology↗

bioRxiv2022

Differences in the genetic structure between and within two landlocked Ayu groups with different migration patterns in Lake Biwa revealed by environmental DNA analysis

Ayu, Plecoglossus altivelis, is largely an annual amphidromous fish, although a landlocked population lives in Lake Biwa, the largest lake in Japan. The landlocked population comprises two migrant groups, spring migrants and autumn migrants, which run to inlet rivers from the lake at different seasons. We used environmental DNA (eDNA) analysis, which is reported to be more sensitive and cost-effective than capture surveys, to clarify the genetic structure of this landlocked Ayu population with different migration patterns in Lake Biwa. We took water samples in 11 inlet rivers in the spring and autumn for two years in a row and quantitatively detected a total of 265 haplotypes of the mitochondrial D-loop region. The pairwise fixation index (FST) value and haplotype diversity indicated that there were genetic differences between the two migrant groups in their respective rivers, and the FST values were negatively related to latitude and the presence of artificial fish release. Additionally, the isolation by distance within each migrant group was observed when the lake was divided into the east and west sides. These findings show that the landlocked Ayu population in Lake Biwa has the genetic structure associated with migration patterns and geographical distance. This study demonstrates that the eDNA approach will be effective for conducting a large-scale investigation of genetic structure beyond simple presence/absence tests.

ecology↗

bioRxiv2021

Raman microspectroscopy imaging analysis of extracellular vesicles (EVs) biogenesis by filamentous fungus Penicilium chrysogenum

Mechanism of production of extracellular vesicles (EVs) and their molecular contents are of great interest owing to their diverse roles in biological systems and are far from being completely understood. Even though, cellular cargo release mediated by EVs have been demonstrated in several cases, their role in secondary metabolite production and release remains elusive. In this study we investigate this aspect in detail using Raman micro-spectroscopic imaging. We provide considerable evidence to suggest that the release of antibiotic penicillin by filamentous fungus Penicillium chrysogenum involves EVs. Morphological modifications of the fungal body during biogenesis, changes in cell composition at the locus of biogenesis, and major molecular contents of the released EVs are also revealed in this study. ImportanceExtracellular vesicles (EVs) play a key role in cellular communications. EVs role in functioning of fungi are relatively less explored. Here we show selective enrichment of chemical contents at certain locations of mycelium of P. chrysogenum forming protruding regions. Secondary metabolite penicillin is excessively localized in them. We provide evidence to show that EVs are released from these protrusions. Raman imaging has been applied for molecular profiling of the mycelium and for characterizing chemical contents of the EVs. Our study suggests a possible general role of EVs in the release of antibiotics from the producing organisms.

cell biology↗

bioRxiv2021

High-throughput screening and selection of PCB-bioelectrocholeaching, electrogenic microbial communities using single chamber microbial fuel cells based on 96-well plate array.

We demonstrate a single chamber, 96-well plate based Microbial Fuel Cell (MFC). This invention is aimed at robust selection of electrogenic microbial community under specific conditions, (pH, external resistance, inoculum) that can be altered within the 96 well plate array. Using this device, we selected and multiplicated electrogenic microbial communities fed with acetate and lactate that can operate under different pH and produce current densities up to 19.4 A/m3 (0.6 A/m2) within 5 days past inoculation. Moreover, studies shown that Cu mobilization through PCB bioleaching occurred, thus each community was able to withstand presence of Cu2+ ions up to 600 mg/L. Metagenome analysis reveals high abundance of Dietzia spp., previously characterized in MFCs, but not reported to grow at pH 4, as well as novel species, closely related to Actinotalea ferrariae, not yet associated with electrogenicity. Microscopic observations (combined SEM and EDS) reveal that some of the species present in the anodic biofilm were adsorbing copper on their surface, probably due to the presence of metalloprotein complexes on their outer membranes. Taxonomy analysis indicated that similar consortia populate anodes, cathodes and OCP controls, although total abundances of aforementioned species are different among those groups. Annotated metagenomes showed high presence of multicopper oxidases and Cu-resistance genes, as well as genes encoding aliphatic and aromatic hydrocarbon-degrading enzymes. Comparison between annotated and binned metagenomes from pH 4 and 7 anodes, as well as their OCP controls revealed unique genes present in all of them, with majority of unique genes present in pH 7 anode, where novel Actinotalea spp. was present.

bioengineering↗

bioRxiv2021

Strategy for detecting off-target sites in genome-edited rice

Genome-editing using the CRISPR-Cas9 system can substantially accelerate crop breeding. Because off-target editing is the main problem with this system, a reliable method for comprehensively detecting off-target sites is required for the editing of food crop genomes. However, a method that accurately predicts off-target sites has not been established. In this study, we performed a SITE-Seq analysis to predict potential off-target sites. SITE-Seq is an unbiased method applicable for the in vitro detection of double-strand breaks (DSBs). To analyze SITE-Seq data, we developed a novel Galaxy system, which can perform simple and reproducible analyses without a command line operation. We conducted a SITE-Seq analysis of a rice genome modified by OsFH15 gRNA-Cas9, and identified 41 DSB sites in the annotated regions. Amplicon-sequencing revealed mutations at one off-target site in the genome-edited rice. The presence of an uncommon protospacer adjacent motif (NTG PAM) likely makes this off-target site difficult to identify using in silico methods. Of the six tested programs, only CRISPRdirect predicted this off-target site, but it also predicted 6,080 off-target sites in total. These results suggest the SITE-Seq method presented herein can efficiently predict off-target sites and is useful for assessing the safety of genome-edited food.

bioinformatics↗

bioRxiv2020

PLZF is a new substrate of CRBN with thalidomide and 5-hydroxythalidomide

Thalidomide induces cereblon (CRBN)-dependent degradation of proteins. Human cytochrome P450s are thought to provide two monohydroxylated metabolites from thalidomide, and the metabolites are also considered to be involved in thalidomide effects. However, it remains unclear. We report that human PLZF/ZBTB16 is a target protein of CRBN with thalidomide and its derivatives, and that 5-hydroxythalidomide has high potential for degrading PLZF. Using a human transcription factor protein array produced by a wheat cell-free protein synthesis system, PLZF was found to bind to CRBN with thalidomide. PLZF is degraded by the CRL4CRBN complex with thalidomide and its derivatives. Mutagenesis analysis revealed that both 1st and 3rd zinc finger domains conserved in vertebrates are recognized for thalidomide-dependent binding and degradation by CRBN. In chicken limbs, knockdown of Plzf induced skeletal abnormalities, and Plzf was degraded after thalidomide or 5-hydroxythalidomide treatment. Our findings suggest that PLZF is a pivotal substrate involving thalidomide-induced teratogenesis.

pharmacology and toxicology↗

bioRxiv2020

Identifying spawning activity in aquatic species based on environmental DNA spikes

An understanding of the reproductive biology of aquatic organisms is crucial for the efficient conservation and management of species and/or populations. Nevertheless, conventional spawning surveys such as visual- and capture-based monitoring generally require laborious, time-consuming work and are subject to monitoring biases such as observer bias, as well as miscounts due to false spawning. In addition, direct capture often damages eggs or individuals. Thus, an efficient non-invasive approach for monitoring spawning activity on aquatic species would be a valuable tool to understand their reproductive biology and conserving biodiversity. Here, we proposed an environmental DNA (eDNA)-based approach for monitoring and understanding spawning activity by observing spikes in eDNA concentration after spawning activity. We found in cross experiments using two medaka species (Oryzias latipes and O. sakaizumii, 1:1 individual per tank) that an eDNA spike occurred in only male species after spawning activity. In addition, the magnitude of the eDNA spike was dependent on the number of spawning activities with egg and sperm release. In the field survey during the reproductive season, eDNA concentration after spawning were 3-25 times higher than before spawning. On the other hand, there was no increase in eDNA concentration during the non-reproductive season. Therefore, our results demonstrated that spikes in the eDNA concentration are mainly caused by the release of sperm during spawning activity, and it can be used as evidence of spawning in field survey. The presented approach could be a practical tool for studying reproductive biology and provides an opportunity to design effective conservation and environmental management actions.

molecular biology↗

bioRxiv2019

Biosynthesis of β-(1->5)-Galactofuranosyl Chains of Fungal-Type and O-Mannose-Type Galactomannans within the Invasive Pathogen Aspergillus fumigatus

The pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise -(1[->]2)-/-(1[->]6)-mannan and {beta}-(1[->]5)-/{beta}-(1[->]6)-galactofuranosyl chains. We previously revealed that GfsA is a {beta}-galactofuranoside {beta}-(1[->]5)-galactofuranosyltransferase involved in the biosynthesis of {beta}-(1[->]5)-galactofuranosyl chains. Here, we clarified the entire biosynthesis of {beta}-(1[->]5)-galactofuranosyl chains in A. fumigatgus. Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are {beta}-galactofuranoside {beta}-(1[->]5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize {beta}-(1[->]5)-galactofuranosyl oligomers up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from the strains {Delta}gfsB, {Delta}gfsC, {Delta}gfsAC, and {Delta}gfsABC revealed that GfsA and GfsC synthesized all {beta}-(1[->]5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans, and GfsB exhibited limited function in A. fumigatus. The loss of {beta}-(1[->]5)-galactofuranosyl residues decreased the hyphal growth rate and conidia formation ability as well as increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immune-compromised mice.\n\nIMPORTANCE{beta}-(1[->]5)-galactofuranosyl residues are widely distributed in the subphylum Pezisomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of {beta}-(1[->]5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for their understanding. In this study, we show that GfsA and GfsC are responsible for the biosynthesis of all {beta}-(1[->]5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicates that {beta}-(1[->]5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of {beta}-(1[->]5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the subphylum Pezisomycotina.

microbiology↗

bioRxiv2019

Dear Editorial manager PLoS ONEA survey of vaccine-induced measles IgG antibody titer and the verification of changes in temporal differences of measles vaccination in young adults

In Japan, sporadic measles cases increased rapidly in 2019 compared to that in past 6 years. To clarify the persistence of immunity against measles in young adult, this study explored the prevalence of IgG antibody titer against measles virus in 18-to 24-year-old young adult participants. Measles-specific IgG antibody titers determined by enzyme immunoassay in serum samples collected from 506 participants between 18 to 24 years were assessed through statistical analyses. Multiple regression analysis revealed that the distribution of measles IgG antibody titers was significantly correlated with medical history (P < 0.05), while there was no significant difference among the number of vaccinations related to measles IgG titers. Further, measles IgG titers were significantly different, which was determined by the temporal change that elapsed period after last vaccination (P < 0.05). These results indicate that periodic vaccination against measles is required for young and older adults to prevent even sporadic measles infection.

microbiology↗