新立體影像技術量化萼形柱珊瑚之共棲微菌聚生體特徵

Abstract

珊瑚能在清澈卻貧瘠的海域中生長，除了倚靠共生藻提供的光合能量，還有賴各種共棲於珊瑚周遭 (體內 )的微生物，包含細菌、古菌、真菌、病毒等，綜合珊瑚自身、共生藻、共棲微生物，整個生物群體稱作珊瑚共生體 (Coral holobiont)。珊瑚共生體中關於細菌與珊瑚的互動仍未被闡明，部分的珊瑚共棲細菌常以聚生體的形式 (Coral-associated microbial aggregate, CAMA)出現在多種珊瑚的組織間， CAMA對珊瑚組織並無負面損害，且已知能組成 CAMA的內 生桿菌 (Endozoicomonas)也被認為與珊瑚的健康有正相關，然而因觀察技術的限制，針對其如數量、大小等資訊都未曾被清楚地描述或比較。本實驗使用台灣墾丁與日本沖繩的萼柱珊瑚(Stylophora pistillata)，透過組織透化 (Tissue clearing)技術與螢光原位雜合連鎖反應(DNA-HCR)標定，首度以層光顯微鏡 (Lightsheet microscope)原位觀察到單一珊瑚蟲體內的 CAMA，並經由分析影像軟體 Imaris建立出的珊瑚蟲與 CAMA立體影像，獲得珊瑚蟲體內 CAMA的分布、數量、預估體積。另外，我們也在珊瑚組織切片上以 DNA-HCR和 DAPI標定 CAMA內部的細菌，透過共軛焦顯微鏡 (Confocal microscope)建構出 CAMA內部菌體的高解析度立體影像，並藉此計算出 CAMA內的平均細菌密度 (cell/μm3)。 結合上述 CAMA的數量、體積、細菌密度我們將可 以針對每個 CAMA、每個珊瑚蟲，甚至珊瑚群落內所含有的平均 CAMA細菌生物量進行估算。比較墾丁與沖繩的 CAMA特徵，我們發現兩樣點的 CAMA皆分布在觸手的位置，平均數量約為三個左右；而在 CAMA的平均體積和細菌密度上，墾丁樣本則顯著地高於沖繩樣本，平均每個珊瑚蟲體內的 CAMA生物量，墾丁樣本約是沖繩的三倍之多。分布和數量的相似可能表示宿主的內部調控較環境影響大；體積與密度的相異則代表著特定特徵仍受環境因子所作用，但不能排除珊瑚個體差異存在。本實驗以立體影像技術來檢視CAMA的各項特徵，使 CAMA研究不再侷限於組織切片觀察，而菌體立體影像的計數也能再做更多元的應用，希望藉由這些新穎的研究方法能為將來與 CAMA相關的實驗帶來更多不同角度的理解。

Corals harbor a wide variety of microbes including symbiotic algae, bacteria, archaea, fungi, and viruses, the whole entity is named “Coral holobiont”. Inside coral holobiont, the relation between coral and their associated bacterium remain unclear. Some of the coral-associated bacteria favored to form aggregates inside coral tissue, named coral-associated microbial aggregate (CAMA). Studies have suggested that CAMA cannot be harmful to corals, and one of the CAMA forming bacteria, Endozoicomonas, is a potential probiotic bacteria candidate. However, due to the limitation on observation, few of CAMA characters were described and compared. In this study, Stylophora pistillata coral branches were collected from Kenting and Okinawa for comparative analysis. First, we conducted the first whole coral polyp fluorescence in situ hybridization chain reaction (DNA-HCR) experiment coupled with tissue clearing methods to reconstruct the three-dimensional (3D) single polyp and CAMA model by Lightsheet microscopy. The 3D model provided us the information of CAMA distribution, numbers, and volume estimation. Second, by DNA-HCR and DAPI staining to thick coral tissue sections, we rebuild the high-resolution 3D cell model inside CAMA by Confocal microscopy. By manual cell counting, we were able to quantify the cell number within a defined volume, in terms of cell density (cell/μm3). Compiling CAMA’s numbers, volume estimation, and cell density data, we can estimate the total biomass of CAMA inside a single polyp. After data analysis, we found that most CAMAs were distributed in tentacles across two sample sites with average 3 CAMAs inside each polyp. The average CAMA volume and cell density were significantly different between locations. Kenting samples had a three times greater biomass than Okinawa samples. The similarity of CAMA distribution and number indicated that regulation from host may be the major factors, however environment difference still affected some certain CAMA characters, like volume. Our study using 3D-imaging technique advances CAMA research from limited 2D observation, providing us more understanding about CAMA. Moreover, 3D cell counting can also be applied to different study fields to gain a better estimation on cell density. Further research for revealing the interaction between CAMA, coral, and environment will be facilitated by our novel experiment result.