Biology subjects

Yoshitake, K.

Publications and source records attributed to Yoshitake, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Omitting end preparation reduces index misassignment in Nanopore-based DNA metabarcoding: application to a decadal coastal time series in the Sea of Okhotsk

Environmental DNA (eDNA) metabarcoding enables sensitive, non-invasive assessment of fish communities, but highly multiplexed analyses using Oxford Nanopore Technologies (ONT) platforms require stringent control of sample-index misassignment and sequencing errors. We developed a MiFish experimental workflow combining unique dual indexes, a library protocol that omitted end preparation and used 5'-phosphorylated primers, BLAST-based demultiplexing, quality-dependent clustering, consensus generation, and haplotype partitioning by SNP/INDEL patterns. Omitting pooled end-prep reduced the mean index-chimera rate from 0.0674% to 0.000420%, a 160-fold reduction. We applied the workflow to an archive of seawater samples collected weekly off Monbetsu, Hokkaido, Japan, from 2012 to 2022. Relative read abundance (RRA) data were obtained for 274 samples, and eight taxa showed significant seasonality. For six of these taxa, the three-year mean RRA peaks coincided with reported spawning periods in Hokkaido. The workflow substantially reduced index misassignment and enabled cost-efficient, highly multiplexed analysis of a decadal fish eDNA time series.

ecology↗

bioRxiv2026

Chromosome-scale genome assembly of Cycas revoluta provides insights into cycad diversification and species diversity

Cycads are an ancient group of seed plants. Despite their ancient origin, many extant cycad genera exhibit high species diversity. The specialized reproductive traits in cycads, dioecy governed by the XY sex-determination system and the elaborate co-evolutionary synergy with insect pollinators, may facilitate lineage diversification. Here, we present a chromosome-scale genome sequence of Cycas revoluta, the species in which plant spermatozoids were discovered in 1896. The genome sequence spanned 11.6 Gb, 98.4% of which were anchored onto the 11 cycad chromosomes. Repetitive sequences occupied 9.8 Gb, and 31,481 genes were predicted. Based on this genome assembly, the X- and Y-associated genomic regions were characterized, and candidate genes for sex determination were identified. In addition, the genomic positions of genes for sex-related traits were determined. Subsequently, we analyzed transcriptomes for thermogenesis responsible for attracting insect pollinators. Through these comprehensive analyses, we provide new insights into the genomic basis of cycad diversification and species diversity.

genomics↗

bioRxiv2026

Gluconeogenesis and glycogen metabolism in the epidermis and endoderm of Xenopus tropicalis embryos and larvae.

In many oviparous animals, egg yolk is the sole source of nutrition until feeding begins, and carbohydrates are present in only small amounts in the yolk. Glucose plays an important role in the developmental processes of various animals. In addition, gluconeogenesis has been reported to occur in the yolk syncytial layer (YSL) of cartilaginous fish and teleosts. In contrast, the role of gluconeogenesis in tetrapods remains unclear. In this study, we used Xenopus tropicalis, an anuran amphibian, which lacks YSL, and therefore provide an opportunity to examine the evolutionary conservation of gluconeogenic mechanisms among vertebrates. In X. tropicalis, liquid chromatography/mass spectrometry revealed that glucose levels increased before liver formation. Subsequent tracer experiments using 13C-labeled metabolic substrates detected gluconeogenesis activity from glycerol and lactate. Expression analyses showed that gluconeogenic genes are expressed in the epidermis and endoderm. Consistently, generation 0 (G0) knockout of fbp1, a key gluconeogenic gene, resulted in a significant reduction in glucose levels, affecting brain development. These findings first demonstrate that gluconeogenesis supports development of X. tropicalis. To the best of our knowledge, gluconeogenesis in developing epidermis has not been reported, highlighting previously unrecognized diversity in tissue-specific metabolism during vertebrate development. Comparative analyses across species will provide further insights into the evolution and functional significance of embryonic gluconeogenesis and nutrient metabolism.

developmental biology↗

bioRxiv2026

Oribatid Mites Supply Tetrodotoxin to Poisonous Newts

Tetrodotoxin (TTX) is a potent neurotoxin widely distributed among marine and terrestrial animals, yet how terrestrial vertebrates acquire this toxin has been debated for decades. Here, we identify oribatid mites as the primary dietary sources of TTX for the Japanese fire-bellied newt, Cynops pyrrhogaster. We detect TTX and its analogs in the mites Scheloribates processus and Galumna sp. KM1, which are preferentially consumed by terrestrial newts. In a controlled toxification experiment, juvenile newts accumulated TTX after consuming TTX-bearing mites, directly demonstrating dietary sequestration. Our results demonstrate that the early terrestrial stage is critical for lifelong TTX acquisition in newts. Notably, we also detect pumiliotoxin, a poison-frog alkaloid, in S. processus, suggesting that newts and poison frogs have convergently evolved to exploit toxin-bearing microarthropods for chemical defense. We propose a "terrestrial toxic web" hypothesis in which oribatid mites act as a shared environmental reservoir linking putative microbial toxin production to vertebrate chemical defense, highlighting microarthropods as key mediators in terrestrial toxin flow.

ecology↗

bioRxiv2025

Environmental exosomes: Evidence of extracellular RNA release by aquatic organisms

Aquatic organisms continuously interact with surrounding water, yet whether they release extracellular vesicles remains unknown. We hypothesized that pearl oysters (Pinctada fucata) release exosomes/small extracellular vesicles (sEVs) into the aquatic environment. To this end, we collected exosomes/sEV-sized components by ultrafiltration from tank water and open-sea culture areas. Microscopy revealed abundant vesicles consistent with exosome/sEV size, and RNA sequencing identified oyster-specific piRNAs that matched sequences previously detected in hemolymph exosomes. These findings demonstrated that pearl oysters actively released exosomes containing species-specific nucleic acids into surrounding water. We propose referring to these vesicles as environmental exosomes/environmental sEVs (eExosomes/esEVs). This finding suggests that aquatic exosomes serve as carriers of RNA and may contribute to inter-organismal communication networks. Beyond their functional role, eExosomes/esEVs also hold promise as novel targets for environmental DNA/RNA (eDNA/eRNA) analysis, offering new opportunities for ecological monitoring and biodiversity research.

ecology↗

bioRxiv2025

Chromosome-scale genome assembly of Malcolmia littorea using long-read sequencing and single-pollen genotyping technologies

Malcolmia littorea, a member of the family Brassicaceae, is adapted to coastal and sandy environments and has become a model in studies of reproductive barriers. However, genomic resources for the species are limited. Here, with the aim of understanding the molecular mechanisms underlying key traits in M. littorea, including its survival under harsh conditions, we present a de novo genome assembly consisting of 10 chromosome-scale sequences. We employed a high-fidelity long-read sequencing technology for genome assembly. To anchor the sequences to chromosomes, we developed a single-pollen genotyping method to construct a genetic linkage map based on SNPs derived from transcriptomes of pollen grains, possessing recombinant haploid genomes. We built a genome assembly consisting of 10 chromosome-scale sequences (214 Mb in total) for M. littorea containing 30,861 predicted genes. A comparative genome analysis and gene prediction indicated that the genome of M. littorea is double the size of the Arabidopsis thaliana genome, consistent with a whole-genome duplication followed by gene subfunctionalization and/or neofunctionalization in M. littorea. This study provides a basis for research on M. littorea, an understudied species with ecological and evolutionary significance.

genomics↗

bioRxiv2025

The minimal endosymbiosis between Spurilla braziliana (Aeolidiidae, Nudibranchia) and Symbiodiniaceae

Symbiotic relationships between dinoflagellates of the family Symbiodiniaceae and marine invertebrates underpin the functioning of certain shallow-water marine ecosystems. Although the Symbiodiniaceae-Cladobranchia nudibranch association has been proposed as a promising model for symbiosis research, interspecific variation in the extent of this association remains poorly resolved. Here, we assessed the algal symbiotic characteristics of the nudibranch Spurilla braziliana. Histological analyses revealed limited branching of the digestive gland and the presence of intact Symbiodiniaceae cells within lysosome-rich epithelial digestive cells. Metagenomic profiling further showed a complete absence of Endozoicomonas--bacteria typically linked to Symbiodiniaceae symbioses--in this species. These findings indicate that S. braziliana can harbor Symbiodiniaceae but exhibits only primitive morphological and microbial adaptations to the symbiotic state. Collectively, these results provide new insights into the evolutionary and structural diversity of nudibranch-algal symbioses.

zoology↗

bioRxiv2025

Development of a Comprehensive Analytical Workflow for eDNA Analysis of Vertebrates, Arthropods, and Mollusks

Environmental DNA (eDNA) analysis is a powerful tool for biodiversity monitoring, but conventional methods are often limited to specific taxonomic groups and short-read sequencing. This study aimed to enhance species detection using Oxford Nanopore long-read sequencing and 16Sar/br primers. Our results showed that MiFish primers effectively detected fish species, while COI primers primarily detected bacterial DNA, limiting eukaryotic identification. In contrast, 16Sar/br primers enabled the detection of diverse taxa, including fish, mollusks, and crustaceans. To improve accuracy, we expanded the mitochondrial 16S database, increasing its size from 4.7 GB to 7.0 GB and the number of sequences from 1.69M to 2.15M, leading to improved mollusk detection. Additionally, an automated analysis pipeline was developed, streamlining data processing. These findings highlight the potential of long-read sequencing and database enhancement for more comprehensive eDNA-based biodiversity assessments.

genomics↗

bioRxiv2025

The Greenland shark genome: insights into deep-sea ecology and lifespan extremes

The Greenland shark (Somniosus microcephalus) is known for its slow metabolism and deep-sea habitat. It is considered the longest-lived vertebrate on Earth, with an estimated lifespan of 392{+/-}120 years. Despite its remarkable longevity and lifestyle, there have been no genomic studies on this species. Here, we report the first, chromosome-level assembly of the Greenland shark genome, which is 5.9 Gb in size with an N50 length of 233 Mb, and contains 37,125 predicted genes with a completeness score of 86.5%. We found that the copy numbers of three gene families (TNF, TLR, LRRFIP), which are involved in activating the NF-{kappa}B signaling pathway, are significantly increased in the Greenland shark compared to short-lived shark species. In the rhodopsin of this deep-sea dweller, we detected amino acid substitutions that result in spectral tuning for the so-called blue shift, suggesting adaptive evolution to dim-light vision. We also elucidate the dynamics of the effective population size (Ne) of the Greenland shark and its close relative, the Pacific sleeper shark (Somniosus pacificus). These genomic analyses offer new insights into the molecular basis of the exceptional longevity of the Greenland shark and highlight potential genetic mechanisms that could inform future research into longevity.

genomics↗

bioRxiv2024

Symbiotic Bacterial Partners for Chemical Defense are Vertically Transmitted in a Marine Sponge

Marine sponges host diverse symbiotic bacteria, some of which are essential for chemical defense. However, the mechanisms governing the inheritance of these defensive symbionts remain elusive. Here we report the first case of a transmission mechanism for symbionts producing cytotoxins in the sponge Mycale nullarosette, for which we established the full life-cycle cultivation method. The identification of Candidatus Synechomycale tutelaris as the cytotoxins producer enabled us to unveil their vertical transmission to offspring via bacteriocyte-like structures from the embryonic stage. This study demonstrates that sponges have evolved a sophisticated system to ensure the inheritance of significant bacterial partners crucial for host defense, which is likely one of the reasons that sponges have survived throughout the long history of multicellular organisms.

microbiology↗

bioRxiv2024

Metagenomic Insights Reveal Unrecognized Diversity of Entotheonella in Japanese Theonella Sponges

Numerous biologically active natural products have been discovered from marine sponges, particularly from Theonella swinhoei, which is known to be a prolific source of natural products such as polyketides and peptides. Recent studies have revealed that many of these natural products are biosynthesized by Candidatus Entotheonella phylotypes, which are uncultivated symbionts within T. swinhoei. Consequently, Entotheonella is considered an untapped biochemical resource. In this study, we conducted metagenomic analyses to assess the diversity of Entotheonella in four T. swinhoei individuals (two each of chemotypes Y and W), after separating filamentous bacteria using density gradient centrifugation. We obtained five Entotheonella genomes from filamentous bacteria-enriched fractions. Notably, one of these genomes is significantly different from previously reported Entotheonella variants. Additionally, we identified closely related Entotheonella members present across different chemotypes of T. swinhoei. Thus, our metagenomic insights reveal that the diversity of Entotheonella within Theonella sponges is greater than previously recognized.

microbiology↗

bioRxiv2023

A novel monocarboxylate transporter involved in 3-hydroxykynurenine transport for ommochrome coloration

Ommochromes are widespread pigments in invertebrates utilized for screening pigments in compound eyes and for reddish coloration in epidermis and wings. Ommochromes are derived from 3-hydroxykynurenine (3OHK), which is incorporated into cells from hemolymph or synthesized from tryptophan within cells. While the synthetic pathway from tryptophan to 3OHK has been well characterized, the gene responsible for cellular uptake of 3OHK has been poorly understood. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of ommochrome pigments. By using positional cloning method, we found that a novel monocarboxylate transporter, 3-hydroxykynurenine transporter (3OHKT), is responsible for the recessive mutant maternal brown of Tsujita (b-t) of B. mori. In b-t mutant, the color of the eggs is light brown, whereas the color of the compound eyes is normal, and we identified a 2-kb deletion in 3OHKT gene. TALEN-mediated knockout of 3OHKT gene produced the same coloration phenotype as b-t mutant, and the complementation test between b-t mutant and 3OHKT knockout strain proved that 3OHKT is responsible for b-t phenotype. 3OHKT protein was localized in the cellular membrane, and LC-MS analysis indicated that the uptake of 3OHK from hemolymph into the ovary was suppressed in the b-t mutant. Moreover, we confirmed that 3OHKT gene is specifically expressed at the reddish region and the time of pigmentation in the pupal wing of nymphalid butterflies. RNA interference of 3OHKT prevented reddish pigmentation in wings, highlighting its general involvement in ommochrome-based pigmentation other than compound eyes. SignificanceOmmochromes are widely distributed pigments in invertebrates and are synthesized from intracellular tryptophan or 3-hydroxykynurenine (3OHK). Ommochrome-based red markings on butterfly wings are often used for sexual selection, warning colors and mimicry. Most genes involved in the ommochrome synthesis pathway have been elucidated from analyses of eye color mutants in Drosophila. However, this study reveals that the ommochrome synthesis pathway has a different genetic repertoire depending on the tissues, and that the novel monocarboxylate transporter identified in this study has a major role in ommochrome pigmentation other than in compound eyes. In particular, our results suggest that classical ommochrome-related genes are rarely involved in the wing pigmentation of the nymphalid butterflies.

genetics↗

bioRxiv2023

The highly developed symbiotic system between the solar-powered nudibranch Pteraeolidia semperi and Symbiodiniacean algae

The intricate coexistence of Symbiodiniacean algae with a diverse range of marine invertebrates underpins the flourishing biodiversity observed within coral reef ecosystems. However, the phenomenon of coral bleaching--precipitated by the dissolution of symbiotic relationships with Symbiodiniaceae--poses a significant threat to these ecosystems, thereby necessitating an urgent investigation into the underlying symbiotic mechanisms. The symbiosis between nudibranchs and Symbiodiniaceae has been identified as an efficacious model for examining these mechanisms, yet a comprehensive understanding of their histological structures and cellular processes remains elusive. In this study, we concentrate on the nudibranch host Pteraeolidia semperi, renowned for its exceptional symbiotic capabilities, and we elucidate its sophisticated symbiotic architecture. Furthermore, we delineate the bleaching process within the nudibranch, uncovering the associated cellular processes. Collectively, these findings furnish invaluable insights into the intricate relationship between nudibranchs and Symbiodiniaceae, thereby contributing to our understanding of the symbiosis that sustains these critical marine ecosystems.

cell biology↗

bioRxiv2023

The complete mitochondrial genome of Spurilla braziliana MacFarland 1909 (Nudibranchia, Aeolidiidae).

Spurilla braziliana MacFarland 1909 is a morphologically diverse nudibranch found in the Pacific and Western Atlantic. The complete mitochondrial genome of S. braziliana has been constructed using next-generation sequencing technology. The mitochondrial genome is 14,291 bp and contains 13 protein-coding genes, 2 rRNA genes, and 23 tRNA genes. Molecular phylogenetic analysis using the maximum likelihood method revealed that S. braziliana is included in the superfamily Aeolidioidea and forms a monophyletic group with Berghia stephanieae, a nudibranch of the family Aeolidiidae. This study reinforces existing taxonomic insights and provides a basis for further molecular phylogenetic analysis.

genomics↗

bioRxiv2022

Telomere-to-telomere genome assembly of matsutake (Tricholoma matsutake)

Here, we report the first telomere-to-telomere genome assembly of matsutake (Tricholoma matsutake), which consists of 13 chromosomes (spanning 160.7 Mb) and a 76 kb circular mitochondrial genome. The chromosome sequences were supported with telomeric repeats at the ends. GC-rich regions are located at the middle of the chromosomes and are enriched with long interspersed nuclear elements (LINEs). Repetitive sequences including long-terminal repeats (LTRs) and LINEs occupy 71.7% of the genome. A total of 28,322 potential protein-coding genes and 324 tRNA genes were predicted. Sequence and structure variant analysis revealed 2,322,349 single nucleotide polymorphisms and 102,831 insertions and deletions, 0.6% of which disrupted gene structure and function and were therefore classified as deleterious mutations. As many as 683 copies of the LTR retrotransposon MarY1 were detected in the matsutake genome, 91 of which were inserted in gene sequences. In addition, 187 sequence variations were found in the mitochondrial genome. The genomic data reported in this study would serve as a great reference for exploring the genetics and genomics of matsutake in the future, and the information gained would ultimately facilitate the conservation of this vulnerable genetic resource.

genomics↗

bioRxiv2022

MitoSearch: Construction of fish species composition database with environmental DNA using public databases

Existing fish species distribution databases have the following problems: the possibility of species identification errors due to plasticity of morphological characteristics, the impossibility of quantitative composition, and the difficulty of geographically and time-series integrated interpretation. To solve the above problems, we constructed MitoSearch database. It is a fish species distribution database using 2,160 samples of variable regions of 12S rRNA genes amplified by MiFish primers registered at NCBI/EBI/DDBJ, and visualizes the fish species composition data on a map. By integrating a large amount of data registered in public databases, MitoSearch was aimed to provide the comprehensive understanding of spatial and temporal distribution of organisms on a global scale.

ecology↗

bioRxiv2021

ePat: extended PROVEAN annotation tool

The ePat (extended PROVEAN annotation tool) is a software tool that extends the functionality of PROVEAN: a software tool for predicting whether amino acid substitutions and indels will affect the biological function of proteins. The ePat extends the conventional PROVEAN to enable the following two things, which the conventional PROVEAN could not calculate the pathogenicity of these variants. First is to calculate and score the pathogenicity of indel mutations with frameshift and variants near splice junctions. Second is to use batch processing to calculate the pathogenicity of multiple variants into a variants list (VCF file) in a single step. ePat can help extract variants that affect biological functions by utilizing not only point mutations, and indel mutations that does not cause frameshift, but also frameshift, stop gain, and splice variants. These extended features will increase detection rate and improve diagnostic of inherited diseases or associate specific variant to phenotype.

bioinformatics↗

bioRxiv2021

Zebrafish Danio rerio trunk muscle structure and growth from a spatial transcriptomics perspective

Compared to mammals, some fish exhibit indeterminate growth characteristics, meaning they can continue growing throughout their lives. Zebrafish trunk skeletal muscle can in general be classified into slow, intermediate, and fast based on morphological and physiological characteristics. After hatching, hyperplasia can be observed in the muscles of juvenile zebrafish, and with growth, hyperplasia in the fast muscles gradually decreases until it stagnates, after which fast muscle development relies on hypertrophy. In slow muscle, hyperplasia continues throughout life. Teleost muscle structure and growth has been described mainly by morphological and physiological features based on the expression of a limited number of proteins, transcripts, and metabolites. The details of mechanism remain unclear. Visium Spatial Gene Expression solution was used in this study. On the adult slide, 10 clusters were obtained based on whole gene expression similarities. The spatial expression of myosin heave chains, myosin light chains and myosin-binding proteins was investigated. GO enrichment analysis was also performed on different muscle regions of aged zebrafish. Dorsal and ventral slow muscles share the same processes such as myofibril assembly and muscle tissue development. On the larvae slide, 3 clusters were obtained, GO enrichment analysis suggest active muscle formation in zebrafish larvae.

genomics↗