Influence of DNA extraction kits on freshwater fungal DNA metabarcoding

[Background] Environmental DNA (eDNA) metabarcoding is a common technique for efficient biodiversity monitoring, especially of microbes. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes. [Methods] DNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extractmicrobial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results; i.e., operational taxonomic unit (OTU) number and composition of the extracted samples. [Results] No PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals

Degree of high phenotypic plasticity in wild populations of Daphnia in early spring

Inducible defenses have been empirically known as defensive phenotypes that are triggered by predator kairomones. We hypothesized that morphological defense of wild Daphnia varies not only with predator density, but also with the predator regime in the field. We observed how the morphological defenses of two Daphnia species (D. ambigua and D. pulex) changed according to the population density in the water column at daytime of predatory insect larvae Chaoborus flavicans in Lake Fukami-ike, Japan, from February to July. In both Daphnia species, the inducible defense morphology was highly expressed in March and April. Its degree of expression decreased, and did not change with increases in predator density from May to July. These seasons are generally considered as when the number of the larvae and fish increases, and predation becomes more active due to their growth and breeding. We suggest that the degree of inducible defense of Daphnia is higher at the end of the overwintering season, when Chaoborus larvae began to inhabit the water column during the daytime and is constant in other seasons regardless of predators regime changes. Field observation of wild populations is important for understanding the seasonal changes in the morphology, and to provide more realistic explanations of phenomena in inducible defense

Variety of phenotypic plasticity in defensive traits of freshwater zooplankton

学位の種別: 課程博士審査委員会委員 : （主査）東京大学准教授 吉田 丈人, 東京大学教授 嶋田 正和, 東京大学教授 伊藤 元己, 滋賀県立大学教授 伴 修平, 東北大学教授 占部 城太郎University of Tokyo(東京大学

Stable C and N isotope abundances in water-extractable organic matter from air-dried soils as potential indices of microbially utilized organic matter

Stable carbon (C) and nitrogen (N) isotopes (13C and 15N) in water-extractable organic matter (WEOM) derived from air-dried soils may be applicable to elucidate the microbial decomposition of soil organic matter (SOM), which is crucial in terrestrial C cycles. A total of 40 soil samples were collected from a depth of 0–6 cm from a temperate broadleaved forest in Japan with vegetation succession from grassland approximately 150 years ago. Those soil samples were air-dried before the water extraction process and organic matter analysis. The C and N concentrations of WEOM were <3.6% of those of the bulk soil and were positively correlated with those of the bulk soil at a p-value of < 0.01. A positive correlation between the two fractions (i.e., WEOM and bulk soils) was also found for natural 13C and 15N abundances (δ13C and δ15N; p < 0.01). However, the C/N ratio of WEOM was slightly correlated with that of bulk soils, exhibiting a narrow range of values of ~10. Thus, those features of the WEOM were similar to the well-known features of microbial biomass. The δ13C and δ15N enrichments in WEOM relative to bulk soil, the difference in stable isotope abundances between bulk SOM and WEOM were negatively and positively correlated, respectively, with the concentrations of organo-mineral complexes and short-range order minerals (non-crystalline oxyhydroxides of aluminum and iron, allophane, imogolite, and allophane-like constituents), which play significant roles in SOM stabilization in soils. These relationships suggest that the stable isotopic enrichments in WEOM can be a good indicator of the microbial utilization of soil C and N under different substrate availabilities, which are crucial to SOM decomposition and decomposability substantially varying from local to global scales

Structure of MSPL–inhibitor complex

Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R↓ sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae. 1. Internal Structure of the Biofilm

The Gram-negative bacterium Flavobacterium johnsoniae employs gliding motility to move rapidly over solid surfaces. Gliding involves the movement of the adhesin SprB along the cell surface. F. johnsoniae spreads on nutrient-poor 1% agar-PY2, forming a thin film-like colony. We used electron microscopy and time-lapse fluorescence microscopy to investigate the structure of colonies formed by wild-type (WT) F. johnsoniae and by the sprB mutant (∆sprB). In both cases, the bacteria were buried in the extracellular polymeric matrix (EPM) covering the top of the colony. In the spreading WT colonies, the EPM included a thick fiber framework and vesicles, revealing the formation of a biofilm, which is probably required for the spreading movement. Specific paths that were followed by bacterial clusters were observed at the leading edge of colonies, and abundant vesicle secretion and subsequent matrix formation were suggested. EPM-free channels were formed in upward biofilm protrusions, probably for cell migration. In the nonspreading ∆sprB colonies, cells were tightly packed in layers and the intercellular space was occupied by less matrix, indicating immature biofilm. This result suggests that SprB is not necessary for biofilm formation. We conclude that F. johnsoniae cells use gliding motility to spread and maturate biofilms

DESIGN APPROACH TO URBAN SPACE TRANSFORMATION BASED ON OPEN SPACE TYPOLOGY

　社会・産業の構造的変化と都市空間の変容が同時に進むときに、ドイツや日本などの脱産業化が進む地域においては、持続可能な開発が求められている。このとき、オープンスペースは、地域や広域の生活環境をより豊かにしていくための要になる。人口減少などによる地域の縮退を前提としつつ環境再生を実現するには、開発管理や計画調整においてオープンスペースの機能を積極的に活かしていく必要がある。　近年の研究やケーススタディにもとづき、都市の空間変容におけるオープンスペースの質とは何かを議論するワークショップを行い、デザインとプランニングの観点からオープンスペースの可能性を検討し、新たな研究の枠組みを構築することをめざした。　ワークショップでの議論を通じて、オープンスペースからの計画デザイン論を展開するために、以下の3つの論点を見いだした：1）地域性（Eigenart）、自然や生き物との共生システムの持続、相互包括性（Inclusion）が、オープンスペースの質をとらえるときの分析・評価の軸となりうること。2）動的な都市変容の過程におけるオープンスペースの多機能性。3）計画デザインにおける2つのスケール（身近な地域空間と広域性）の相互作用。In the process of the spatial transformation and structural change due to social and industrial alteration, the sustainable developments is commonly required in both post-industrial regions of Germany and Japan. In this context, open space becomes one of the keystones of the common interests to enhance both local and regional living quality, and thus should be given more positive functions to manage the development for the smart shrinking with environmental regeneration