Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs.

Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

Japanese Planocerid Flatworms: Difference in Composition of Tetrodotoxin and Its Analogs and the Effects of Ingestion by Toxin-Bearing Fishes in the Ryukyu Islands, Japan.

Tetrodotoxin (TTX), known as pufferfish toxin, is a potent neurotoxin blocking sodium channels in muscle and nerve tissues. TTX has been detected in various taxa other than pufferfish, including marine polyclad flatworms, suggesting that pufferfish toxin accumulates in fish bodies via food webs. The composition of TTX and its analogs in the flatworm Planocera multitentaculata was identical to those in wild grass puffer Takifugu alboplumbeus. Previously, Planocera sp. from Okinawa Island, Japan, were reported to possess high level of TTX, but no information was available on TTX analogs in this species. Here we identified TTX and analogs in the planocerid flatworm using high-resolution liquid chromatography-mass spectrometry, and compared the composition of TTX and analogs with those of another toxic and non-toxic planocerid species. We show that the composition of TTX and several analogs, such as 5,6,11-trideoxyTTX, dideoxyTTXs, deoxyTTXs, and 11-norTTX-6(S)-ol, of Planocera sp. was identical to those of toxic species, but not to its non-toxic counterpart. The difference in the toxin composition was reflected in the phylogenetic relationship based on the mitochondrial genome sequence. A toxification experiment using predatory fish and egg plates of P. multitentaculata demonstrated that the composition of TTX and analogs in wild T. alboplumbeus juveniles was reproduced in artificially toxified pufferfish. Additionally, feeding on the flatworm egg plates enhanced the signal intensities of all TTX compounds in Chelonodon patoca and that of deoxyTTXs in Yongeichthys criniger.

Transcriptome analysis of Edwardsiella piscicida during intracellular infection reveals excludons are involved with the activation of a mitochondrion-like energy generation program.

Phylogenetic evidence suggests a shared ancestry between mitochondria and modern Proteobacteria, a phylum including several genera of intracellular pathogens. Studying these diverse pathogens, particularly during intracellular infection of their hosts, can reveal characteristics potentially representative of the mitochondrial-Proteobacterial ancestor by identifying traits shared with mitochondria. While transcriptomic approaches can provide global insights into intracellular acclimatization by pathogens, they are often limited by excess host RNAs in extracts. Here, we developed a method employing magnetic nanoparticles to enrich RNA from an intracellular Gammaproteobacterium, Edwardsiella piscicida, within zebrafish, Danio rerio, fin fibroblasts, enabling comprehensive exploration of the bacterial transcriptome. Our findings revealed that the intracellular E. piscicida transcriptome reflects a mitochondrion-like energy generation program characterized by the suppression of glycolysis and sugar transport, coupled with upregulation of the tricarboxylic acid (TCA) cycle and alternative import of simple organic acids that directly flux into TCA cycle intermediates or electron transport chain donors. Additionally, genes predicted to be members of excludons, loci of gene pairs antagonistically co-regulated by overlapping antisense transcription, are significantly enriched in the set of all genes with perturbed sense and antisense transcription, suggesting a general but important involvement of excludons with intracellular acclimatization. Notably, genes involved with the activation of the mitochondrion-like energy generation program, specifically with metabolite import and glycolysis, are also members of predicted excludons. Other intracellular Proteobacterial pathogens appear to employ a similar mitochondrion-like energy generation program, suggesting a potentially conserved mechanism for optimized energy acquisition from hosts centered around the TCA cycle.IMPORTANCEPhylogenetic evidence suggests that mitochondria and Proteobacteria, a phylum encompassing various intracellular pathogens, share a common ancestral lineage. In this study, we developed a novel method employing magnetic nanoparticles to explore the transcriptome of an aquatic Gammaproteobacterium, Edwardsiella piscicida, during intracellular infection of host cells. We show that the strategy E. piscicida uses to generate energy strikingly mirrors the function of mitochondria-energy generators devoid of glycolytic processes. Notably, several implicated genes are members of excludons-gene pairs antagonistically co-regulated by overlapping antisense transcription. Other intracellular Proteobacterial pathogens appear to adopt a similar mitochondrion-like energy generation program, indicating a possibly conserved strategy for optimized energy acquisition from hosts centered around the tricarboxylic acid cycle.

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 breakdown of Symbiodiniaceae-host symbiosis endangers these ecosystems, necessitating urgent study of the 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. A meticulous histological exploration of the nudibranch Pteraeolidia semperi, employing optical, fluorescence, and electron microscopy, has revealed fine tubules extending to the body surface, with associated epithelial cells having been shown to adeptly encapsulate Symbiodiniaceae intracellularly. By tracing the stages of the "bleaching" in nudibranchs, it was inferred that algal cells, translocated via the digestive gland, are directly phagocytosed and expelled by these epithelial cells. Collectively, these insights contribute substantially to the scholarly discourse on critical marine symbiotic associations.

Optical coherence tomography findings of the peripheral retina in patients with congenital X-linked retinoschisis.

Introduction: Congenital X-linked retinoschisis (XLRS) presents as macular retinoschisis/degeneration in almost all patients and as peripheral retinoschisis in half the patients. Although the optical coherence tomography (OCT) findings of macular retinoschisis have been well investigated, those of peripheral retinoschisis have rarely been reported. This study aimed to report the ultra-widefield OCT findings of the peripheral retina in patients with XLRS. Methods: Medical records of 10 Japanese patients (19 eyes) with clinically and/or genetically diagnosed XLRS were retrospectively reviewed. Funduscopic, electroretinographic, and OCT findings were reviewed and evaluated. Some were also genetically evaluated for the RS1 gene. Results: OCT of the macula revealed schises and/or cystoid changes in the inner nuclear layer (INL) and outer nuclear layer. In contrast, OCT of the peripheral retina revealed schises and/or cystoid changes in the INL in eight eyes (44%), and/or splitting in the ganglion cell layer (GCL) in 10 (56%) of the 18 eyes with clear OCT images. No schisis or cystoid changes were found in the peripheral OCT images of eight eyes (44%). A 16-year-old boy presented with retinal splitting of the GCL and INL of the inferior retina, although he had no ophthalmoscopic peripheral retinoschisis. Genetic examinations were performed on three patients, all of whom had reported missense mutations in the RS1 gene. Conclusion: In XLRS, peripheral bullous retinoschisis results from GCL splitting in the retina. One of the 10 patients with XLRS showed intraretinal retinoschisis in the GCL in the inferior periphery, which was unremarkable on ophthalmoscopy (occult retinoschisis). Although both peripheral bullous retinoschisis and occult retinoschisis showed splitting/cystic changes in the GCL, further studies are needed to determine whether occult retinoschisis progresses to bullous retinoschisis.

Structure Determination of Kahalalide Analogues Based on Metagenomic Analysis of a Bryopsis sp. Marine Green Alga.

Two kahalalide analogues were isolated from a Bryopsis sp. marine green alga. Even though our initial structure determination of the peptides by NMR and MS identified them as kahalalide Z1 (KZ1; 3) and Z2 (KZ2; 4), the absolute configuration of the Thr residues by Marfey's analysis was different from those found in kahalalide F (KF), 3, and 4. To ascertain the absolute configuration of the amino acid residues genetically, we conducted a metagenomic analysis for symbiotic bacteria in the alga, leading to the biosynthetic gene cluster (BGC) responsible for producing the kahalalides named kahalalides Z3 (KZ3; 1) and Z4 (KZ4; 2). The identification of amino acid residues based on the A-domain suggested these peptides possess the amino acid sequence d-allo-Thr-l-Val-l-Val-d-Val residues at the N-terminus, instead of the d-Val-l-Thr-l-Val-d-Val residues found in KF, 3, and 4. The N-terminal amino acid sequence including absolute configuration was unambiguously determined by a comparison of LCMS data of synthetic tetrapeptides and the hydrolysates derived from 1 and 2. This structural difference is caused by swapping the substrate specificities of the first two A-domains.

Complete genome sequence of Edwardsiella sp. NBRC12716 isolated in 1962 from the liver of diseased eel.

We report the complete genome sequence of Edwardsiella sp. NBRC12716 isolated from a diseased eel in 1962. The genome consists of a single, circular chromosome 3,771,060 bp in length with 59.74% GC content and encodes 25 rRNA, 96 tRNA, and 3,182 protein-coding genes.

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.

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 sequences (spanning 161.0 Mb) and a 76 kb circular mitochondrial genome. All the 13 sequences were supported with telomeric repeats at the ends. GC-rich regions are located at the middle of the sequences and are enriched with long interspersed nuclear elements (LINEs). Repetitive sequences including long-terminal repeats (LTRs) and LINEs occupy 71.6% of the genome. A total of 21,887 potential protein-coding genes were predicted. The genomic data reported in this study served not only matsutake gene sequences but also genome structures and intergenic sequences. The information gained would be a great reference for exploring the genetics, genomics, and evolutionary study of matsutake in the future, and ultimately facilitate the conservation of this vulnerable genetic resource.

Pilot study of a comprehensive resource estimation method from environmental DNA using universal D-loop amplification primers.

Many studies have investigated the ability of environmental DNA (eDNA) to identify the species. However, when individual species are to be identified, accurate estimation of their abundance using traditional eDNA analyses is still difficult. We previously developed a novel analytical method called HaCeD-Seq (haplotype count from eDNA by sequencing), which focuses on the mitochondrial D-loop sequence for eels and tuna. In this study, universal D-loop primers were designed to enable the comprehensive detection of multiple fish species by a single sequence. To sequence the full-length D-loop with high accuracy, we performed nanopore sequencing with unique molecular identifiers (UMI). In addition, to determine the D-loop reference sequence, whole genome sequencing was performed with thin coverage, and complete mitochondrial genomes were determined. We developed a UMI-based Nanopore D-loop sequencing analysis pipeline and released it as open-source software. We detected 5 out of 15 species (33%) and 10 haplotypes out of 35 individuals (29%) among the detected species. This study demonstrates the possibility of comprehensively obtaining information related to population size from eDNA. In the future, this method can be used to improve the accuracy of fish resource estimation, which is currently highly dependent on fishing catches.

Draft genome sequencing and assembly of Risso's dolphin, Grampus griseus.

The Risso's dolphin (Grampus griseus) is one of the migratory marine mammals and they have commonly dispersed in tropical and temperate seas. It is a least concerned species in the IUCN red list of threatened species. However, their population size and factors affecting their population structure are unknown. Due to the wide distribution of this species, their populations might be genetically stable and less structured. To support genetic studies on dolphins and other marine mammals, we assembled the draft genome of Risso's dolphin that was found in Japan. The tissue samples were used to extract high molecular DNA and subjected to sequencing by Illumina HiSeq X, Oxford Nanopore MinION, and Bionano Saphyr. The assembled hybrid genome was 75.9% of complete eukaryotic BUSCOs and the genome size was 2.256 Gb with 2.042 Mb of scaffold N50. De novo assembly of this genome by Bionano Saphyr recovered 2.036 Gb total genome map length and structural variations. The gene structures of this draft genome were identified by BRAKER2, and 9947 genes were recovered. The data will be useful for future studies of cetaceans.

Genetic characterization of 1210 Japanese pedigrees with inherited retinal diseases by whole-exome sequencing.

Inherited retinal diseases (IRDs) comprise a phenotypically and genetically heterogeneous group of ocular disorders that cause visual loss via progressive retinal degeneration. Here, we report the genetic characterization of 1210 IRD pedigrees enrolled through the Japan Eye Genetic Consortium and analyzed by whole exome sequencing. The most common phenotype was retinitis pigmentosa (RP, 43%), followed by macular dystrophy/cone- or cone-rod dystrophy (MD/CORD, 13%). In total, 67 causal genes were identified in 37% (448/1210) of the pedigrees. The first and second most frequently mutated genes were EYS and RP1, associated primarily with autosomal recessive (ar) RP, and RP and arMD/CORD, respectively. Examinations of variant frequency in total and by phenotype showed high accountability of a frequent EYS missense variant (c.2528G>A). In addition to the two known EYS founder mutations (c.4957dupA and c.8805C>G) of arRP, we observed a frequent RP1 variant (c.5797C>T) in patients with arMD/CORD.

Environmental DNA study on aquatic ecosystem monitoring and management: Recent advances and prospects.

Environmental DNA (eDNA) is organismal DNA that can be detected in the environment and is derived from cellular material of organisms shed into aquatic or terrestrial environments. It can be sampled and monitored using molecular methods, which is important for the early detection of invasive and native species as well as the discovery of rare and cryptic species. While few reviews have summarized the latest findings on eDNA for most aquatic animal categories in the aquatic ecosystem, especially for aquatic eDNA processing and application. In the present review, we first performed a bibliometric network analysis of eDNA studies on aquatic animals. Subsequently, we summarized the abiotic and biotic factors affecting aquatic eDNA occurrence. We also systematically discussed the relevant experiments and analyses of aquatic eDNA from various aquatic organisms, including fish, molluscans, crustaceans, amphibians, and reptiles. Subsequently, we discussed the major achievements of eDNA application in studies on the aquatic ecosystem and environment. The application of eDNA will provide an entirely new paradigm for biodiversity conservation, environment monitoring, and aquatic species management at a global scale.

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

Fish exhibit different muscle structures and growth characteristics compared with mammals. We used a spatial transcriptomics approach and examined myotomal muscle sections from zebrafish. Adult muscles were divided into eight regions according to spatial gene expression characteristics. Slow muscle was located in the wedge-shaped region near the lateral line and at the base of the dorsal fin, intermediate muscle was located in a ribbon-shaped region adjacent to slow muscle, and fast muscle was located in the deep region of the trunk, surrounded by intermediate muscle; the interior of fast muscle was further divided into 6 parts by their transcriptomic features. Combined analysis of adult and larval data revealed that adult muscles contain specific regions similar to larval muscles. These regions showed active myogenesis and a high expression of genes associated with muscle hyperplasia. This is the first study to apply spatial transcriptomics to fish myotomal muscle structure and growth.

Age-Associated Different Transcriptome Profiling in Zebrafish and Rats: an Insight into the Diversity of Vertebrate Aging.

Most mammals, including humans, show obvious aging phenotypes, for example, loss of tissue plasticity and sarcopenia. In this regard, fish can be attractive models to study senescence because of their unique aging characteristics. The lifespan of fish varies widely, and several species can live for over 200 years. Moreover, some fish show anti-aging features and indeterminate growth throughout their life. Therefore, exploring the aging mechanism in fish could provide new insights into vertebrate aging. To this end, we conducted RNA sequencing (RNA-seq) assays for various organs and growth stages of zebrafish and compared the data with previously published RNA-seq data of rats. Age-associated differentially expressed genes (DEGs) for all zebrafish tissue samples reveal the upregulation of circadian genes and downregulation of hmgb3a. On one hand, a comparative analysis of DEG profiles associated with aging between zebrafish and rats identifies upregulation of circadian genes and downregulation of collagen genes as conserved transcriptome changes. On the other hand, in zebrafish, upregulation of autophagy-related genes in muscles and AP-1 transcription factor genes in various tissues is observed, which may imply fish-specific anti-aging characteristics. Consistent with our knowledge of mammalian aging, DEG profiles related to tissue senescence are observed in rats. We also detect age-associated downregulation of muscle homeostasis and differentiation-related genes in zebrafish gills, indicating a fish-specific senescence phenotype. Our results indicate both common and different aging profiles between fish and mammals, which could be used for future translational research.

Evolution of nacre- and prisms-related shell matrix proteins in the pen shell, Atrina pectinata.

The molluscan shell is a good model for understanding the mechanisms underlying biomineralization. It is composed of calcium carbonate crystals and many types of organic molecules, such as the matrix proteins, polysaccharides, and lipids. The pen shell Atrina pectinata (Pterioida, Pinnidae) has two shell microstructures: an outer prismatic layer and an inner nacreous layer. Similar microstructures are well known in pearl oysters (Pteriidae), such as Pinctada fucata, and many kinds of shell matrix proteins (SMPs) have been identified from their shells. However, the members of SMPs that consist of the nacreous and prismatic layers of Pinnidae bivalves remain unclear. In this study, we identified 114 SMPs in the nacreous and prismatic layers of A. pectinata, of which only seven were found in both microstructures. 54 of them were found to bind calcium carbonate. Comparative analysis of nine molluscan shell proteomes showed that 69 of 114 SMPs of A. pectinata were found to have sequential similarity with at least one or more SMPs of other molluscan species. For instance, nacrein, tyrosinase, Pif/BMSP-like, chitinase (CN), chitin-binding proteins, CD109, and Kunitz-type serine proteinase inhibitors are widely shared among bivalves and gastropods. Our results provide new insights for understanding the complex evolution of SMPs related to nacreous and prismatic layer formation in the pteriomorph bivalves.

METTL23 mutation alters histone H3R17 methylation in normal-tension glaucoma.

Normal-tension glaucoma (NTG) is a heterogeneous disease characterized by retinal ganglion cell (RGC) death leading to cupping of the optic nerve head and visual field loss at normal intraocular pressure (IOP). The pathogenesis of NTG remains unclear. Here, we describe a single nucleotide mutation in exon 2 of the methyltransferase-like 23 (METTL23) gene identified in 3 generations of a Japanese family with NTG. This mutation caused METTL23 mRNA aberrant splicing, which abolished normal protein production and altered subcellular localization. Mettl23-knock-in (Mettl23+/G and Mettl23G/G) and -knockout (Mettl23+/- and Mettl23-/-) mice developed a glaucoma phenotype without elevated IOP. METTL23 is a histone arginine methyltransferase expressed in murine and macaque RGCs. However, the novel mutation reduced METTL23 expression in RGCs of Mettl23G/G mice, which recapitulated both clinical and biological phenotypes. Moreover, our findings demonstrated that METTL23 catalyzed the dimethylation of H3R17 in the retina and was required for the transcription of pS2, an estrogen receptor α target gene that was critical for RGC homeostasis through the negative regulation of NF-κB-mediated TNF-α and IL-1ß feedback. These findings suggest an etiologic role of METTL23 in NTG with tissue-specific pathology.

Induction of endoplasmic reticulum stress markers in an acromegaly model.

Acromegaly is a growth hormone (GH) excess pathological condition in humans. Acromegaly is associated with somatic disfigurement and a wide range of systemic manifestations such as arthritis, neuropathy, carpal tunnel syndrome, reproductive disorders, metabolic disorders, and gastrointestinal complications. The influence of excess GH on the cellular level could aid in understanding the root causes of acromegaly-related health complications. Previously, we found that GH excess induces DNA damage to somatic cells and reduces the stem cells number and causes premature aging. In this study, an in-depth analysis of the acromegaly RNAseq data revealed the disruption of important biological cellular processes. Gene set enrichment analysis, heatmap, and enrichment analysis of acromegaly RNAseq data revealed induction of endoplasmic reticulum (ER) stress markers in various organs. Interestingly, the induction of ER stress was even more apparent than in aged zebrafish. Splicing of box-binding protein-1 (XBP1) mRNA is a hallmark of ER stress. Therefore, we quantified spliced XBP1 mRNA in different organs of our acromegaly model. Thus, our study emphasizes the importance of ER stress in GH oversecretion, which is important for understanding the health complications of acromegaly.

Exosome-derived small non-coding RNAs reveal immune response upon grafting transplantation in Pinctada fucata (Mollusca).

Exosomes, a subset of small extracellular vesicles, carry various nucleic acids, proteins, lipids, amino acids and metabolites. They function as a mode of intercellular communication and molecular transfer. Exosome cargo molecules, including small non-coding RNAs (sncRNAs), are involved in the immune response in various organisms. However, the role of exosome-derived sncRNAs in immune responses in molluscs remains unclear. Here, we aimed to reveal the sncRNAs involved in the immune response during grafting transplantation by the pearl oyster Pinctada fucata. Exosomes were successfully extracted from the P. fucata haemolymph during graft transplantation. Abundant microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) were simultaneously discovered in P. fucata exosomes by small RNA sequencing. The expression patterns of the miRNAs and piRNAs at the grafting and initial stages were not substantially different, but varied significantly between the initial and later stages. Target prediction and functional analysis indicate that these miRNAs and piRNAs are related to immune response upon grafting transplantation, whereas piRNAs may also be associated with transposon silencing by targeting with genome transposon elements. This work provides the basis for a functional understanding of exosome-derived sncRNAs and helps to gain further insight into the PIWI/piRNA pathway function outside of germline cells in molluscs.

Construction of a chromosome-level Japanese stickleback species genome using ultra-dense linkage analysis with single-cell sperm sequencing.

It is still difficult to construct the genomes of higher organisms as their genome sequences must be extended to the length of the chromosome by linkage analysis. In this study, we attempted to provide an innovative alternative to conventional linkage analysis by devising a method to genotype sperm using 10× Genomics single-cell genome sequencing libraries to generate a linkage map without interbreeding individuals. A genome was assembled using sperm from the Japanese stickleback Gasterosteus nipponicus, with single-cell genotyping yielding 1 864 430 very dense hetero-SNPs and an average coverage per sperm cell of 0.13×. In total, 1665 sperm were used, which is an order of magnitude higher than the number of recombinations used for conventional linkage analysis. We then improved the linkage analysis tool scaffold extender with low depth linkage analysis (SELDLA) to analyze the data according to the characteristics of the single-cell genotyping data. Finally, we were able to determine the chromosomal location (97.1%) and orientation (64.4%) of the contigs in the 456 Mb genome of G. nipponicus, sequenced using nanopores. This method promises to be a useful tool for determining the genomes of non-model organisms for which breeding systems have not yet been established by linkage analysis.