2023 Fiscal Year Final Research Report

Mechanisms of tunic softening in Halocynthia roretzi infected with a pathogenic flagellate, Azumiobodo hoyamushi

Research Project

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Project/Area Number	20H03074
Research Category	Grant-in-Aid for Scientific Research (B)
Allocation Type	Single-year Grants
Section	一般
Review Section	Basic Section 40040:Aquatic life science-related
Research Institution	Ehime University
Principal Investigator	KITAMURA Shin-Ichi 愛媛大学, 沿岸環境科学研究センター, 准教授 (40448379)
Co-Investigator(Kenkyū-buntansha)	広瀬裕一琉球大学, 理学部, 教授 (30241772) 柳田哲矢山口大学, 共同獣医学部, 准教授 (40431837) 仲山慶愛媛大学, 沿岸環境科学研究センター, 講師 (80380286) 飯田貴次岡山理科大学, 獣医学部, 教授 (70159557)
Project Period (FY)	2020-04-01 – 2023-03-31
Keywords	マボヤ / Azumiobodo hoyamushi / 被嚢軟化症
Outline of Final Research Achievements	We hypothesized that the mechanism of tunic softening in an ascidian Halocynthia roretzi is caused by the extracellular proteases of Azumiobodo hoyamushi degrading tunic proteins. To test this hypothesis, we conducted proteome analysis of the softened and healthy tunics and detection of extracellular proteases from Azumiobodo hoyamushi. As a result of the proteome analysis, four decreased proteins were identified in softening tunic. We raised a polyclonal antibody against one of these proteins, dynein. Immunohistochemical assay showed a specific reaction in the hemocytes within the tunic, but no difference was observed between diseased and healthy individuals. Regarding the extracellular protease from the parasite, using 20 substrates, we successfully detected proteases that degraded three substrates mainly those degraded by cathepsins.
Free Research Field	魚病学
Academic Significance and Societal Importance of the Research Achievements	蛍光標識二次元ディファレンスゲル電気泳動解析、ショットガン解析、レーザーマイクロダイセクション法で得たサンプルのショットガン解析と、3つの方法でプロテオーム解析したが、被嚢軟化に関わるタンパク質は同定できなかった。しかしながら、世界で初めて、モデル生物の一つであるホヤ類の被嚢中にどのようなタンパク質が存在しているのかを明らかにすることができた。また、ホヤムシが他の寄生虫で病原性因子として報告されているカテプシン様のプロテアーゼを分泌していることも明らかにされ、魚病学、寄生虫学に新規知見をもたらすことでできた。