【公開日：2024.07.25】【最終更新日：2024.06.29】
課題データ / Project Data
課題番号 / Project Issue Number
23UT0323
利用課題名 / Title
Temperature-Dependent Microstructural Phenotypes in the Pinctada fucata Nacre
利用した実施機関 / Support Institute
東京大学 / Tokyo Univ.
機関外・機関内の利用 / External or Internal Use
内部利用（ARIM事業参画者以外）/Internal Use (by non ARIM members)
技術領域 / Technology Area
【横断技術領域 / Cross-Technology Area】（主 / Main）計測・分析/Advanced Characterization（副 / Sub）-
【重要技術領域 / Important Technology Area】（主 / Main）次世代バイオマテリアル/Next-generation biomaterials（副 / Sub）次世代ナノスケールマテリアル/Next-generation nanoscale materials
キーワード / Keywords
bivalves, thermal and/or pH stress, various microstructural phenotypes of nacre,電子顕微鏡/ Electronic microscope
利用者と利用形態 / User and Support Type
利用者名（課題申請者）/ User Name (Project Applicant)
Siu Sienna
所属名 / Affiliation
東京大学 大学院理学系研究科
共同利用者氏名 / Names of Collaborators in Other Institutes Than Hub and Spoke Institutes
ARIM実施機関支援担当者 / Names of Collaborators in The Hub and Spoke Institutes
利用形態 / Support Type
（主 / Main）機器利用/Equipment Utilization（副 / Sub）-
利用した主な設備 / Equipment Used in This Project
報告書データ / Report
概要（目的・用途・実施内容）/ Abstract (Aim, Use Applications and Contents)
Marine bivalves produce calcareous shells of spectacular morphological diversity through biomineralization, during which a complex protein matrix supports and, to some extent, determines the orientation and growth of calcium crystals (Marin et al., 2016). These crystals become increasingly disorganised and disoriented as temperature increases (e.g. Olson et al., 2012), but can show signs of adaptation to prolonged stress (Hahn et al., 2012), thus their biological mediators warrant more attention, and this attribute provides fertile ground for exploring the links between gene expression, epigenetics, and phenotypes, including those exhibiting plasticity. Molecular studies on phenotypic plasticity necessitates establishing causality between a particular genetic or epigenetic mechanism and an easily observable phenotype. Bivalve shells with nacreous layers are suitable candidates, as the variations in the nacre microstructures count as easily observable phenotypes. Aragonitic nacres are functionally superior in many respects but very costly to secrete, and are the first mineralized structures to be compromised under unfavorable conditions. Certain lineages within the subclass Protobranchia, for example, replaced nacreous shells with a less costly homogeneous structure in the Paleozoic and Mesozoic, and the yet unclear mechanism underlying nacre microstructural plasticity is postulated to have driven this trajectory (Sato et al., 2022). My project seeks to characterize the various microstructural phenotypes of nacre exhibited when bivalves experience thermal and/or pH stress, in order to lay the groundwork for my next study on the genetic and epigenetic mechanisms that enabled the expression of plastic microstructural traits, with an aim at gaining insights into their evolutionary implications. The species Pinctada fucata serves as a good candidate for my purposes for three reasons: it is known to exhibit microstructural plasticity (e.g. Sato et al., 2022), are easy to obtain, and have a wealth of genomic resources (e.g. Takeuchi et al., 2012, 2016, 2022) for me to draw upon for further molecular investigations after completing the proposed project.
実験 / Experimental
Last year, I have constructed a rearing setup, kept 60 individuals under different temperatures and pH for 30 days, and measured the average nacre tablet thickness in their shells using FE-SEM. Linear mixed model analyses revealed statistically significant (p<0.05), temperature-induced differences in nacre tablet thickness between individuals.The next part of my project is to obtain orientation maps using electron backscatter diffraction, for which I am using ARIM's equipment. The samples prepared for FE-SEM, which have been embedded in epoxy resin, radially sectioned, polished, and etched with dilute hydrochloric acid, are reprocessed for EBSD by colloidal silica polishing, then recoated with carbon. I will be measuring the crystal orientation by generating orientation maps with three Euler angles using the data obtained from EBSD. The results will provide a source of new phenotypic traits as well as a basis to verify the orientations of the tablets used for the thickness measurements.
結果と考察 / Results and Discussion
Towards the end of last year, I reprocessed some of the samples from batch 1 for EBSD analyses by polishing them with colloidal silica, and recoating them with a thinner layer of carbon. However, no diffracted signals could be detected, despite recoating the samples with osmium as well. Per suggestions by technicians in the engineering department, my next goal is to test out several slight adjustments to how my samples are etched, as well as using ion beam polishing instead of colloidal silica. To find out the optimum acid concentration and duration for etching, 15 spare shells will embedded, sectioned, polished, and etched using hydrochloric acid of 5 different concentrations for 10-30 seconds. The blocks will then be polished using ion beam, coated with osmium, then examined under an electron microscope to test for diffracted signals. The concentration and duration will be altered further if none of the combinations work. This should be completed by the end of April, 2024. Upon confirming the optimum sample preparation procedure, the specimens that have been analyzed under FE-SEM will be reprocessed accordingly. The data obtained from EBSD will be used to generate orientation maps with three Euler angles to assess tablet layer uniformity. EBSD analyses for batches 1 and 2 (80 specimens), and SEM analyses for batch 2 (40 specimens) should be completed by the end of July, 2024.
図・表・数式 / Figures, Tables and Equations
その他・特記事項（参考文献・謝辞等） / Remarks(References and Acknowledgements)
Financial support: Kenneth E. & Annie Caster Award, Paleontological Society; Academic Grant, Conchologists of America
成果発表・成果利用 / Publication and Patents
論文・プロシーディング（DOIのあるもの） / DOI (Publication and Proceedings)
口頭発表、ポスター発表および、その他の論文 / Oral Presentations etc.
特許 / Patents
特許出願件数 / Number of Patent Applications：0件
特許登録件数 / Number of Registered Patents：0件