Biology subjects

Watanabe, K.

Publications and source records attributed to Watanabe, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Harness engineering enables a multi-agent LLM system to generate cross-domain hypotheses beyond single-investigator reach

Large language models (LLMs) are increasingly used to generate scientific hypotheses, and a more capable model is widely assumed to drive output quality. We tested this with a biology-focused multi-agent system coupling an LLM to five Model Context Protocol servers exposing 66 external tools. Upgrading the Synthesizer and Critic from Claude Sonnet 4.6 to Opus 4.8 initially appeared to improve quality, count, and reliability. Under a fully matched harness (identical prompts, schema, hypothesis count, and queries), the differences vanished: scores were indistinguishable on an iPSC-reprogramming task and favoured Sonnet 4.6 on a CHO cell-line-engineering task (18.4 vs 17.4; Welch t = 2.89, p < 0.05). The dominant, controllable driver of quality is therefore the prompting harness, not the model tier. We then introduce the ABCD-Stack (contract-gated prompting, diversity-first generation, adversarial refinement, and cross-family evaluation), which builds these properties in as architecture and shifts outputs toward explicitly falsifiable, causally framed hypotheses beyond the reach of a single-domain investigator. The hypotheses are falsifiable by construction but not yet experimentally validated.

bioinformatics↗

bioRxiv2026

Detection of Japanese Encephalitis Virus in Mosquitoes Collected from Selected Areas of Laguna, Philippines Using an Optimized One-Step RT-PCR Protocol

Japanese encephalitis virus is a leading cause of encephalitis in humans and agricultural animals across Southern and Eastern Asia. It is primarily transmitted by mosquitoes of the Culex genus, notably Culex tritaeniorhynchus. In the Philippines, JEV is reported to account for 7% to 18% of clinical cases of meningitis and encephalitis. Therefore, updated and comprehensive data on circulating JEV strains in the country are crucial for developing effective mitigation strategies. This study aimed to optimize an assay for JEV detection using one-step reverse transcription polymerase chain reaction (RT-PCR). Additionally, the presence of JEV in mosquitoes collected from eleven cities and municipalities in Laguna was assessed, and the mosquito carriers were identified based on morphological characteristics. Results demonstrated that the optimized one-step RT-PCR assay successfully detected JEV at various dilutions, with a limit of detection (LoD) of 0.115 ng/L. The assay exhibited 100% specificity, with no amplification observed for any viruses other than JEV. Sequence analysis of the positive control, JEV SA14-14-2 RNA, revealed 96.96% to 98.41% similarity to JEV reference sequences in the NCBI database. Among the 5,522 female mosquitoes collected, Culex tritaeniorhynchus and Culex gelidus were the most prevalent species. The highest mosquito species diversity and the most even distribution were found in the three barangays of Paete, but Siniloan, Laguna had the highest vector density for the three Culex species, Culex tritaeniorhynchus, Culex gelidus, and Culex fuscocephala. RT-PCR analysis of 189 pooled mosquito samples revealed no detectable JEV genetic material. This study is likely the first investigation of JEV presence in mosquito samples from Laguna, Philippines. These findings contribute to proactive JEV surveillance, enhancing our understanding of the virus's presence and distribution. The results underscore the importance of targeted prevention and control measures to mitigate JEV transmission in high-risk areas.

molecular biology↗

bioRxiv2026

RIFT-VAE: grammar-conditioned pretraining and latent-space optimization for RNA inverse folding

BackgroundRNA inverse folding designs nucleotide sequences expected to adopt a prescribed secondary structure. Search-based solvers can optimize folding-model objectives effectively, but difficult targets can require extensive sampling, and structural optimization alone does not explicitly preserve the sequence distributions or conserved motifs of natural RNA families. MethodsWe developed RIFT-VAE, a Transformer-based conditional variational autoencoder that receives a context-free grammar parse-tree representation of a target secondary structure and generates nucleotide labels on the corresponding tree. The framework combines progressively richer grammar rules, self-refinement learning from generated structure-sequence pairs, and cross-entropy-method optimization in the learned latent space. We evaluated RNAfold minimum-free-energy agreement on an RNAcentral-derived test set and the EteRNA100 benchmark, compared the method with four search-based solvers under matched total time budgets, and examined GC-content control and covariance-model family annotation. ResultsThe complete pipeline achieved RNAfold-Correct/RNAfold-MCC values of 0.833/0.994 on the RNAcentral-derived test set and 0.760/0.977 on EteRNA100. Latent-space optimization accounted for the largest increase in exact structural recovery. Under a 3,600-s total budget on EteRNA100, sequences generated by RIFT-VAE improved the exact-match rate of every tested downstream search method when used as warm starts; the largest change was observed for RNAInverse (Correct, 0.297 to 0.803; MCC, 0.505 to 0.985). The pretrained model also produced sequences with measurable correct-family covariance-model hits and supported explicit GC-content conditioning. ConclusionsRIFT-VAE is best interpreted as a hybrid generative-search framework: pretraining supplies a structure- and family-informed proposal distribution, whereas latent optimization concentrates evaluations in high-scoring regions. The reported structural scores are specific to RNAfold minimum-free-energy validation and do not establish biochemical function. Orthogonal folding predictors, stricter homology-controlled splits, diversity-aware evaluation, architecture-matched dot-bracket ablations, and experimental assays remain priorities for validation.

bioinformatics↗

bioRxiv2026

Hydration Energetics Shape Antibody Discrimination between Sulfotyrosine and Phosphotyrosine

Chemically similar post-translational modifications can mediate distinct biological functions, but how proteins distinguish between them remains unclear. Sulfotyrosine (sTyr) and phosphotyrosine (pTyr) exemplify this problem because they have similar sizes, local geometries, and electrostatic properties but function in different biological contexts. Here, we used the monoclonal antibody PSG2, which recognizes sTyr independently of the surrounding peptide sequence, to examine how a protein distinguishes these modifications. The crystal structure of PSG2 bound to an sTyr-containing peptide revealed a deep electropositive pocket with no modeled water molecules in direct contact with the sulfate group. Gas-phase density functional theory calculations favored pTyr over sTyr, showing that direct protein-ligand interactions alone are insufficient to explain PSG2 selectivity. Explicit first-shell hydration calculations showed that pTyr has a larger desolvation penalty than sTyr, and accounting for this difference reversed the calculated energetic order. Isothermal titration calorimetry showed favorable enthalpic and entropic contributions to sTyr binding, whereas no detectable heat signal was observed for pTyr. These results show that PSG2 distinguishes sTyr from pTyr through the balance between direct protein-ligand interactions and ligand desolvation.

biophysics↗

bioRxiv2026

Selective depletion of AMH-expressing granulosa cells in vivo impairs follicular development and fertility in female mice

In mammals, ovarian follicle development is a highly coordinated process that underlies female fertility. Granulosa cells expressing anti-Mullerian hormone (AMH) are widely used as a marker of growing follicles. However, the in vivo roles of granulosa cells in follicular development and female fertility remain unclear. Here, we analyzed AMH-toxin receptor-mediated cell knockout (AMH-TRECK) transgenic (Tg) mice on a NOG background, in which AMH-expressing granulosa cells are specifically depleted by diphtheria toxin (DT). We first found that, after a single DT injection into postnatal AMH-TRECK Tg females, AMH-expressing granulosa cells in primary and secondary follicles exhibited cleaved caspase-3 signals 1 day later and were depleted 4 days later. Second, after repeated DT injections weekly from 1 to 7 weeks of age in AMH-TRECK Tg females, antral follicles and corpora lutea were rarely observed, and the numbers of primordial, primary, and secondary follicles were decreased. Following PMSG and hCG stimulation, repeated DT-injected Tg females exhibited a reduced number of ovulated oocytes with a low proportion of mature oocytes, resulting in reduced IVF rates and fertility. Further, after a cessation of repeated DT treatment, ovarian weight and follicular development recovered: the numbers of primary, secondary, and antral follicles were recovered, whereas the primordial follicle pool remains reduced. We conclude that selective depletion of AMH-expressing granulosa cells in vivo impairs follicular development and fertility. Our model enables assessment of the in vivo effects of granulosa cell depletion and may provide a useful platform for future transplantation-based studies to understand complex follicular dynamics.

developmental biology↗

bioRxiv2026

Non-retroviral Endogenous Viral Element Acts as a Stable Antiviral Regulator Across Mosquito Life Stages and Generations

Endogenous viral elements (EVEs) from non-retroviral RNA viruses are widespread in host genomes, yet their functional significance in vivo remains poorly understood. In mosquitoes, EVEs have been shown to suppress cognate viral replication through the PIWI-interacting RNA (piRNA) pathway. However, how this antiviral activity shapes virus-host dynamics beyond reducing viral replication remains unclear. Here, we established Aedes aegypti mosquito lines naturally infected with cell-fusing agent virus (CFAV) that differ in the presence or absence of a CFAV-derived EVE (CFAV-EVE1). Using this natural virus-host system, we showed that CFAV-EVE1 suppressed viral replication across sexes, tissues, developmental stages, and generations. This antiviral effect is initiated by EVE-derived primary piRNAs, as demonstrated in ovaries and heads. Despite reducing viral load, CFAV-EVE1 conferred no measurable benefits on mosquito survival or reproduction, nor did it eliminate CFAV from the naturally infected population under standard laboratory conditions. However, vertical transmission rates were reduced in EVE-containing mosquitoes and prolonged egg storage further reduced viral prevalence, suggesting that environmental stress may reveal EVE-mediated viral suppression. Together, these findings demonstrate that EVEs function as stable, heritable regulators of persistent virus-host interactions and highlight that ecological context may be critical for fully appreciating their functional significance.

microbiology↗

bioRxiv2026

Conjugation dynamics and persistence of a carbapenem resistance gene blaOXA-72 from Acinetobacter pittii to Acinetobacter baumannii

Carbapenem-resistant Acinetobacter (CRA) has been associated with increased morbidity and mortality in clinical settings. In this study, we explored the transfer potential of a mobilizable plasmid-harboring blaOXA-72 gene between Acinetobacter species originating from patient, municipal wastewater, and pig farm wastewater. PCR-based evidence suggested putative transfer of blaOXA-72 from Acinetobacter pittii to Acinetobacter baumannii. In this pair, the apparent frequency of PCR-marker-positive putative transconjugants varied depending on temperature and meropenem supplementation, with higher number observed at 27{degrees}C compared to 17{degrees}C and 37{degrees}C. Likewise, the presence of antibiotic pressure yields to higher apparent conjugation frequency, however this observation was limited to a singled donor-recipient pair. Further, we revealed a phenotypic conversion in terms of meropenem susceptibility and a fitness cost in the putative transconjugants. While whole genome sequencing did not conclusively verify the presence of blaOXA-72 or fully resolved plasmid configuration, Oxford Nanopore read mapping consistently detected the chromosomal strA gene in all isolates. In contrast, only a limited number of reads aligned with blaOXA-72 gene, traC, or the complete plasmid sequences. Comparative analyses further revealed variations in the surface-associated factors and defense systems composition of the recipient strains, which could be considered as barriers in conjugation. Lastly, the persistence of PCR-detectable marker genes in putative transconjugants was variable and generally unstable over a 30-day period. Overall, these findings provide preliminary insights into the factors that may influence horizontal gene transfer and short-term maintenance of blaOXA-72. IMPORTANCECarbapenem-resistant Acinetobacter (CRA) is a serious clinical concern linked to elevated morbidity and mortality. This study shed light on how the carbapenemase gene blaOXA_72 spreads in the environment via horizontal gene transfer, Using isolates of Acinetobacter pittii and Acinetobacter baumannii from municipal wastewater treatment plant, we show that conjugation efficiency depends on temperature and antibiotic exposure. The process also incurs fitness costs, and the transferred gene persists only briefly in recipient cells. These results emphasize the importance of environmental sources as reservoirs for antimicrobial resistance gene exchange. Additionally, variations in surface structures and defense systems may act as potential barriers to conjugation. Overall, the work improves our understanding of AMR movement and supports the need for better integrated surveillance under a One Health approach.

microbiology↗

bioRxiv2026

Trem2 R47H mutation shows mild, but functionally divergent alterations in microglial phenotypes compared to Trem2 deficiency in aged AppNL-F knock-in mice

The TREM2 R47H variant increases the risk of Alzheimers disease (AD), yet its functional impact in aged mouse models remains incompletely understood. We generated a humanized Trem2 R47H knock-in (KI) line on the AppNL-F background and compared it with a Trem2 knockout (KO) line to assess the degree of TREM2 functional impairment. Accumulation of amyloid {beta} 42 and formation of dystrophic neurites were increased in Trem2 KO mice but not in Trem2 R47H KI mice at 18 or 24 months. qPCR and transcriptomic analyses revealed Trem2 KO mice showed deficits in upregulation of microglial genes while Trem2 R47H KI mice showed a response similar to control mice. Differential gene expression analysis identified altered expressions of genes responsible for ER stress/unfolded protein response and intracellular signalling in Trem2 R47H KI mice. Among the differentially expressed genes, Pmel and Gpnmb were or tended to be downregulated in Trem2 R47H KI as well as in Trem2 KO mice indicating their involvement in AD pathogenesis. These results clearly indicate that the TREM2 R47H variant confers a mild, rather than null, effect on microglial alterations during AD development and that Trem2 R47H KI mice should be used to understand pathological mechanism elicited by TREM2. Further identification and characterization of genes differentially expressed in Trem2 R47H KI mice will provide important insights into how the TREM2 risk variant modulates Alzheimers disease-related pathology. HighlightsO_LIExon2-humanized Trem2 R47H knock-in mice are established, which will serve as a platform to study the role of TREM2 in Alzheimers disease development. C_LIO_LITrem2 knockout mice exhibit deficits in clearance of highly aggregated A{beta}42, suppression of dystrophic neurites and regulation of microglial genes in AppNL-F mice, whereas Trem2 R47H knock-in mice do not. C_LIO_LIRNA-seq reveals transcriptional profiles of Trem2 R47H knock-in mice C_LIO_LIqPCR confirms that Gpnmb and Pmel are or tended to be downregulated in Trem2 R47H knock-in mice. C_LIO_LIFindings demonstrate that TREM2 R47H is hypomorphic rather than loss of function. C_LI

neuroscience↗

bioRxiv2026

MyD88 deficiency modestly attenuates disease in a Leigh syndrome mouse model while enrofloxacin accelerates disease

Primary genetic mitochondrial diseases (GMDs) are a clinically and genetically diverse group of diseases estimated to impact over 1 in 4,000 individuals. Leigh syndrome (LS) is the most common pediatric presentation of GMD. LS typically presents within the first years of life and is a severe progressive multi-system disorder. Symmetric progressive inflammatory brain lesions are a defining feature of the disease. Patients can also present with seizures, metabolic dysfunction, muscle weakness, and other symptoms. No effective clinical treatments currently exist. Recent data from the Ndufs4(-/-) mouse model shows that peripheral macrophages contribute to brain lesions in LS, that disease is causally driven by innate immune populations, and that depletion of innate immune cells prevents LS disease. However, the precise mechanisms underlying immune activation remain unknown. Certain mitochondrial macromolecules retain bacterial signatures and can act as potent agonists for innate immune pathways. For example, cytoplasmic mitochondrial RNA and mitochondrial DNA are detected by Toll-like receptors (TLRs) 7 and 9, respectively, at the endosome. Accordingly, these are considered strong candidates for mediating innate immune activation in LS. Here, we generated TLR signaling deficient Ndufs4(-/-)/MyD88(-/-) animals to assess whether TLR signaling plays a role in disease onset or progression in LS. Loss of MyD88 in Ndufs4(-/-) animals statistically significantly increased survival and delayed the onset of some symptoms, but the benefits were modest compared to CSF1R inhibition from prior work. We conclude that Myd88-mediated immune signaling is not a primary driver of LS. Notably, prophylactic enrofloxacin treatment, which was necessary for production of innate immune deficient MyD88(-/-) animals, modestly decreased survival and accelerated disease. The impact of enrofloxacin and similar drugs in the context of mitochondrial disease warrants further investigation.

neuroscience↗

bioRxiv2026

Feeding ecology and ecological risks of the invasive fish Coreoperca herzi revealed by gut content DNA and environmental DNA metabarcoding

Understanding the dietary patterns of introduced predators is essential for assessing their impacts on freshwater ecosystems. Here, we investigated the feeding ecology of the invasive Korean perch (Coreoperca herzi) introduced to the Oyodo River system, Japan, by integrating gut content DNA metabarcoding and environmental DNA (eDNA) metabarcoding. Fifty specimens were collected, and prey taxa were identified using metabarcoding targeting fish, aquatic insects, and crustaceans. In parallel, eDNA metabarcoding of habitat water samples was used to assess prey availability and selectivity. The results revealed that the Korean perch prey extensively on aquatic insects and fish. Aquatic insect prey were dominated by epilithic clinger taxa inhabiting stone surfaces, particularly mayflies, suggesting visual-mediated prey selection. Fish predation was frequently detected even in small individuals (<100 mm SL), in contrast to previous studies based on conventional methods, indicating that piscivory begins early and ontogenetic dietary shifts are not pronounced. Furthermore, quantitative fish eDNA analysis showed a positive relationship between eDNA concentrations of prey species and predation frequency, indicating opportunistic feeding on abundant, size-accessible prey. By applying two metabarcoding approaches, this study provides an integrated assessment of prey utilisation and environmental context, highlighting ecological risks posed by the Korean perch to freshwater communities in Japan.

ecology↗

bioRxiv2026

Lack of Period1 accelerates colorectal tumorigenesis in ApcMin/+ mice

The circadian clock coordinates physiology and behavior through [~]24-h rhythms, and disruption of core clock genes has been implicated in tumorigenesis. However, the impact of Per1, a major clock gene, on colorectal tumor development remains unclear. Here, we investigated how Per1 deletion influences intestinal tumorigenesis using the ApcMin/+ mouse model and ApcMin/+Per1-/-mice generated by crossing ApcMin/+ and Per1-/- lines (C57BL/6J background). Mice were maintained under controlled light-dark conditions, and we assessed survival, intestinal polyp burden, histopathology using Swiss-roll sections, {beta}-catenin protein abundance (immunofluorescence and western blotting), Ctnnb1 mRNA expression (RT-qPCR), and crypt proliferation (5-bromo-2-deoxyuridine (BrdU) immunohistochemistry). Per1 deletion did not significantly alter overall survival in ApcMin/+ mice but increased inter-individual variability. In contrast, polyp number was markedly increased by Per1 deletion, affecting both small (<2 mm) and large ([≥]2 mm) polyps across intestinal segments. Histology confirmed aberrant crypt foci and polyps in both ApcMin/+ genotypes. {beta}-Catenin protein levels in the whole intestine were significantly increased by Per1 deficiency and Apc mutation (two-way ANOVA), whereas Ctnnb1 mRNA was largely unchanged across regions. BrdU-based crypt proliferation was increased by the Apc mutation but not by Per1 deletion. These results indicate that Per1 loss exacerbates intestinal polyp formation and elevates {beta}-catenin predominantly through non-transcriptional mechanisms, supporting a tumor-suppressive role of Per1 in colorectal tumorigenesis.

cancer biology↗

bioRxiv2026

Dietary lipids attenuate IGF-1-Akt and injure epithelial-endothelial injury program that accelerates obstructive lung disease

Background Altered lipid metabolism is increasingly implicated in chronic obstructive pulmonary disease (COPD), but it remains unclear how a pre-existing obstructive lung state modifies the response to systemic lipid excess. We investigated whether high-fat diet (HFD) amplifies COPD-relevant lung injury and examined epithelial and vascular programs associated with this response. Methods Male wild-type (WT) and beta-epithelial sodium channel-transgenic ({beta}ENaC-Tg) mice were fed control diet or HFD for 10-11 weeks. Lung structure and function, whole-lung transcriptomes, Akt-FOXO1 signaling, apoptosis-related responses, and pulmonary vascular profiles were assessed. Streptozotocin-induced insulin-deficient diabetes and pharmacological IGF-1 receptor inhibition were used as mechanistic comparators. Palmitate responses were examined in human bronchial epithelial cells, ENaC-hyperactive epithelial cells, and endothelial cells, including conditioned-medium transfer. HFD preconditioning was also evaluated in an elastase-induced emphysema model. Statistical analyses included unpaired two-tailed Student's t tests, one-way ANOVA with Tukey-Kramer or Dunnett multiple-comparison testing, Pearson correlation, and Benjamini-Hochberg correction for RNA-sequencing analyses. Results HFD produced similar increases in body weight, glycemia, and adiposity in WT and {beta}ENaC-Tg mice, while further increasing distal-airspace enlargement and reducing FEV0.1/FVC in {beta}ENaC-Tg mice. Lung transcriptomics revealed coordinated remodeling of lipid metabolic, PI3K-Akt, and vascular programs. HFD reduced Akt phosphorylation, increased FOXO1 and Fasl, and increased TUNEL-positive cells in epithelial regions. Palmitate attenuated IGF-1-induced Akt activation in bronchial epithelial cells, whereas IGF-1 receptor inhibition reproduced Akt suppression and apoptosis-related responses without fully reproducing the HFD phenotype. HFD preconditioning also increased elastase-induced airspace enlargement, accompanied by parallel upregulation of FOXO1 and TUNEL positivity. HFD reduced pulmonary CD34-positive vascular profiles, and palmitate activated endothelial cells directly and through conditioned media from ENaC-hyperactive epithelium. In men with airflow obstruction, hepatic steatosis coincided with lower percent-predicted FEV1. Conclusions Dietary lipid stress amplifies obstructive lung injury and engages complementary epithelial and vascular responses. Impaired epithelial IGF-1-Akt signaling and epithelial-endothelial crosstalk provide a mechanistic framework linking systemic metabolic stress to reduced resilience of the obstructive lung.

molecular biology↗

bioRxiv2026

The significance of sukumo (composted indigo leaves) as a microbial source for traditional Japanese indigo dyeing

A critical process in traditional Japanese indigo dyeing is the microbial reduction of indigo within the dye suspension, which consists solely of sukumo (fermented indigo leaves), wood ash lye, and microbial nutrients such as wheat bran. Although sukumo has long been recognized as a potential microbial source, the microbial community dynamics during its production is still largely unexplored, and its contribution to dyeing performance via microbial supply remains poorly characterized. In this study, we investigated the significance of sukumo as a microbial source, in addition to its established role as a pigment source. We conducted a time-series analysis of microbial communities throughout the four-month fermentation process of sukumo, using weekly samples collected from two geographically distinct indigo dyeing studios. Microbial profiling revealed similar successional patterns between the two sites. Notably, in the later stages of fermentation, known indigo-reducing bacteria, such as the genus Oceanobacillus and the family Tissierellaceae, emerged at both locations. Laboratory-scale dyeing experiments using immature sukumo demonstrated that supplementation with a small amount of mature sukumo restored dyeing activity and increased the abundance of Oceanobacillus and Tissierellaceae. Furthermore, the addition of the indigo-reducing isolate Tissierellaceae strain TU-1 to the immature sukumo-based dye suspension led to a marked enhancement in dyeing performance. These findings highlight the critical role of sukumo as a microbial source in traditional indigo dyeing and suggest that prolonged fermentation is essential for nurturing functional indigo-reducing bacteria. This insight provides a foundation for improving dye suspension performance through targeted microbial community management.

microbiology↗

bioRxiv2026

Haplotype-level metabarcoding revealed the relationship between species occurrence and population genetic structure at the catchment scale in aquatic insects

A longstanding question in ecology and evolutionary biology is understanding the evolutionary mechanisms whereby some species appear frequently while others do not. Current research examined this phenomenon through computer simulations and empirical studies involving a few species, while community-wide investigations of this relationship in natural settings remain largely unexplored. To address this, we employed haplotype-level bulk DNA metabarcoding to infer haplotypes from many aquatic insect species with varying occurrence in a catchment. We identified 891 haplotypes belonging to 200 taxonomically identified Operational Taxonomic Units (OTUs). Results showed that species with higher occurrence in the catchment had greater {gamma}-genetic diversity, a wider range of environmental conditions, and an increased probability of {beta}-species-genetic diversity correlations (SGDC). We found negative -SGDC in species with lower occurrence in the catchment. Dispersal abilities and environmental factors may influence the relationship among species occurrence, population genetic structure, and SGDC. Our findings present a novel approach to address the association of species occurrence and population genetic structure, as well as SGDC, from a community-level perspective. This has important implications for addressing biodiversity loss driven by rapid environmental changes from human activities and climate change.

ecology↗

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

PCR-based specific detection of multiple Nigrospora species causing a banana leaf spot disease

The genus Nigrospora has been identified as the primary causative agent of leaf spot disease in banana plantations in Mindanao, the Philippines. The symptoms caused by Nigrospora species are similar to those of black leaf streak disease (black sigatoka), which has made managing the disease challenging. Furthermore, development of rapid and precise molecular diagnostic methodologies for leaf spot disease has remained elusive. In the present study, a polymerase chain reaction (PCR)-based detection system was developed. The system targets a Nigrospora-specific genomic region, which was identified through comparative analysis of the genus Nigrospora and related genera. The newly designed primer set nigF/nigR specifically detected six pathogenic Nigrospora species associated with banana leaf spot disease (N. chinensis, N. lacticolonia, N. nigrocolonia, N. singularis, N. sphaerica, and N. vesicularifera), while no amplicon was observed for 15 non-Nigrospora species, including Pseudocercospora fijiensis isolated from banana leaves. The results indicate that the primers enable clear differentiation from other genera, including the important pathogen P. fijiensis. The primers also detected diverse Nigrospora isolates (N. banbusae, N. chinensis, N. musae, N. oryzae, N. sphaerica, and N. rubi) obtained from different hosts and regions within Japan, demonstrating broad applicability. Sensitivity testing demonstrated the capacity to detect as minimal as 10 pg of genomic DNA. Additionally, a pathogenic Nigrospora isolate was detected successfully from infected banana tissue. This study presents a novel molecular diagnostic tool for Nigrospora species, offering a rapid, specific, and practical approach, which could enhance evidence-based disease management in epidemiological investigations and banana production systems.

molecular biology↗

bioRxiv2025

Outbreak of leaf spot disease of Cavendish banana caused by Nigrospora spp. in Mindanao Island, Philippines

Banana leaf diseases are a significant threat to Cavendish banana production. In the Philippines, the main disease has been diagnosed as Black sigatoka disease caused by Pseudocercospora fijiensis based on symptoms. However, our study showed that the main pathogen in Mindanao island, the largest banana-producing region in the Philippines, belongs to the genus Nigrospora, contradicting previous assumptions. We clarified the phylogenetic positions of 160 Nigrospora isolates based on molecular phylogenetic analyses using ITS, {beta}-tubulin, and tef1 sequences, and compared their morphology with known species. Molecular phylogenetic and morphological analysis revealed that Nigrospora isolates comprised N. chinensis, N. lacticolonia, N. cf. singularis, N. sphaerica, N. vesicularifera, and a novel species, N. nigrocolonia. Pathogenicity tests on banana leaves confirmed that these species are pathogenic. Species other than N. sphaerica were for the first time reported as pathogens of banana leaf. The results of the fungicide sensitivity test using 14 fungicides, including pyrimethanil, spiroxamine, and tebuconazole, for the Sigatoka disease showed 100% inhibition of all isolates at 100 ppm of active ingredients. However, low-sensitivity isolates were observed for the remaining 11 fungicides. Our findings indicated the need for a comprehensive review of banana leaf disease prevention strategies.

molecular biology↗

bioRxiv2025

Nonlinear hippocampal coding of the pair-bonded partner in prairie voles

Neural representation of specific opposite-sex partners remains largely unexplored. Using monogamous prairie voles, we demonstrated that partner preference required the ventral hippocampus, in which 3-4 Hz oscillations were enhanced following partner interaction. We identified ventral hippocampal neurons responding selectively to the pair-bonded partner and stranger, which encoded identity-related information in a nonlinear manner, shedding light upon how mammalian brains create distinct neural representations of romantic opposite-sex partners.

neuroscience↗