Population genetics and microbial analysis of the Dictyoceratida sponge, Coscinoderma matthewsi, from central and eastern Torres Strait, Australia (MTSRF Project 1.3.2)

During the central and eastern Torres Strait survey in November 2006, tissue samples of 10 individuals of the sponge Coscinoderma matthewsi were collected from 5 island groups: Ugar (Stephen Island) and Erub (Darnley Island) in eastern Torres Strait; and the Masig group (Kodall Island and Keats Island), Poruma (Coconut Island) and Warraber (Sue Island) in central Torres Strait. These island groups are on average, 66 km apart. All sponge samples were placed in separate cryo-tubes and preserved in liquid nitrogen until they could be stored at -80°C. Approximately 2 g of tissue from each sample was homogenised in liquid nitrogen and 750 µl of lysis buffer [100 mM Tris pH 9, 100 mM EDTA, 1% SDS, 100 mM NaCl, 0.5 mg/ml Proteinase K], and subsequently incubated at 65°C for 1 hour with gentle agitation. KoAc was added to a final concentration of 1 M, followed by incubation on ice for 30 minutes. The samples were centrifuged at 8000 rpm for 15 minutes, and the supernatant was reserved for DNA precipitation with isopropanol using the standard protocol.A fragment of nuclear DNA containing part of the 28S rRNA gene was amplified for all individuals using RD3A (5'-GACCCGTCTTGAAACACGA) and RD5B2 (5'- ACACACTCCTTAGCGGA) primers. Recombinant Pfu Polymerase (Fermentas) was used for the PCR. A total of 50 µl of reaction mixture was prepared for each sample according to the protocol. PCR was performed under the following conditions: initial denaturation at 95°C for 3 minutes; 35 cycles of 95°C for 30 seconds, 50°C for 20 seconds, 72°C for 1 minute; a final extension step of 72°C for 10 minutes. Products were purified with QIAquick (Qiagen) columns according to protocol. Sequencing was performed at Macrogen Inc. with a 3730xl DNA analyser using both forward and reverse primers.For microbial analysis, a DNA fingerprinting technique (denaturing gradient gel electropohoresis - DGGE) was used to determine the stability of bacterial associations within Coscinoderma matthewsi across wide spatial scales.Four replicate sponges were analysed from Keats Island and Kodall Island and three replicate sponges were analysed from Erub, Ugar, Poruma and Warraber. DNA was extracted from individual sponges by homogenising approx 1g of tissue from each individual in 0.5 ml of grinding buffer (2 ml 1 M Tris, 4 ml 0.5M EDTA, 2 ml 10% SDS, 400 µl 5 M NaCl and 11.6 ml distilled water). Tubes were immersed in liquid nitrogen and ground with plastic pestles. Samples were incubated at 65ºC for 60 min prior to addition of 187 µl 5 M potassium acetate. Samples were incubated on ice for 30 min and centrifuged at 8000 x g for 15 min. The supernatants were transferred to fresh tubes and DNA was precipitated with 0.8 vol of isopropanol.The 16S rDNA from each sample was amplified by PCR with universal bacterial primers 1055f: 5'-ATG GCT GTC GTC AGC T-3' and 1406r: 5'-ACG GGC GGT GTG TAC-3'. The reverse primer was modified to incorporate a 40 bp GC clamp. Primers 1055f and 1406r match over 56,000 and 62,800 sequences respectively in the Ribosomal Database Project. Products from triplicate PCR reactions were combined and 15 µl applied to duplicate 40% wt/vol polacrylamide (37:5:1) gels containing a 50-70% denaturing gradient of formamide and urea. Gels were electrophoresed at 60ºC for 17 h in 1 x TAE buffer at 50V using the Ingeny D-Code system. Gels were stained with 1 x Sybr Gold for 30 min, visualised under UV illumination and photographed.
This project was undertaken to determine connections between sponge populations in central and eastern Torres Strait and to assess whether there would be any risks associated with translocation of sponges across large areas. Risks investigated were the possibility of transfers between genetically distinct populations, which would result in a decrease in the genetic diversity of wild populations or the potential to introduce new sponge-associated microbe types into a region.

2006年11月开展的托雷斯海峡中东部海域调查期间，研究人员从5个岛屿群采集了10份海绵（*Coscinoderma matthewsi*）个体的组织样本：托雷斯海峡东部的乌加岛（Ugar/Stephen Island）与厄鲁布岛（Erub/Darnley Island）；以及托雷斯海峡中部的马西戈群岛（Masig group，包含科达尔岛Kodall Island与基茨岛Keats Island）、波鲁马岛（Poruma/Coconut Island）与沃拉伯岛（Warraber/Sue Island）。上述岛屿群的平均间距为66千米。

所有海绵样本均置于独立的冻存管（cryo-tube）中，以液氮（liquid nitrogen）临时保存，直至转移至-80℃环境长期储存。从每份样本中取约2g组织，在液氮中研磨匀浆后，加入750μl裂解液[100mM Tris pH 9、100mM EDTA、1% SDS、100mM NaCl、0.5mg/ml蛋白酶K（Proteinase K）]，随后于65℃温和振荡条件下孵育1小时。向体系中加入乙酸钾（KAc）至终浓度1M，之后冰浴30分钟。将样本以8000rpm离心15分钟，收集上清液，按照标准流程采用异丙醇（isopropanol）沉淀DNA。

针对所有个体，利用引物RD3A（5'-GACCCGTCTTGAAACACGA）与RD5B2（5'-ACACACTCCTTAGCGGA）扩增包含部分28S核糖体RNA（rRNA）基因片段的核DNA序列。聚合酶链式反应（PCR）采用重组Pfu DNA聚合酶（Recombinant Pfu Polymerase，Fermentas公司），每份样本按照实验方案配制50μl反应体系。PCR反应程序设置如下：95℃初始变性3分钟；35个循环，每个循环包含95℃变性30秒、50℃退火20秒、72℃延伸1分钟；最后72℃终延伸10分钟。扩增产物采用QIAquick（Qiagen公司）柱式纯化试剂盒按照说明书进行纯化。测序工作由Macrogen Inc.公司完成，使用3730xl型DNA分析仪，同时采用正向与反向引物进行双向测序。

为探究不同空间尺度下海绵（*Coscinoderma matthewsi*）体内细菌共生群落的稳定性，本研究采用DNA指纹技术——变性梯度凝胶电泳（denaturing gradient gel electrophoresis, DGGE）。其中，基茨岛与科达尔岛各取4份重复海绵样本，厄鲁布岛、乌加岛、波鲁马岛与沃拉伯岛各取3份重复海绵样本。

从每份海绵样本中取约1g组织，置于0.5ml研磨缓冲液（配制配方：2ml 1M Tris、4ml 0.5M EDTA、2ml 10% SDS、400μl 5M NaCl与11.6ml蒸馏水）中，将试管浸入液氮后用塑料研杵研磨匀浆。样本于65℃孵育60分钟，随后加入187μl 5M乙酸钾，继续冰浴30分钟，之后以8000×g离心15分钟。将上清液转移至新管，采用0.8倍体积的异丙醇沉淀DNA。

利用通用细菌引物1055f（5'-ATG GCT GTC GTC AGC T-3'）与1406r（5'-ACG GGC GGT GTG TAC-3'）对每份样本的16S核糖体DNA（16S rDNA）进行PCR扩增。其中反向引物经过修饰，引入了40bp的GC夹子。引物1055f与1406r分别可与核糖体数据库项目（Ribosomal Database Project）中超过56000条与62800条序列匹配。将三次重复PCR反应的产物混合后，取15μl上样于两份40%重量/体积的聚丙烯酰胺（37:5:1）凝胶，凝胶包含50%-70%的甲酰胺与尿素变性梯度。采用Ingeny D-Code系统，于60℃、1×TAE缓冲液中以50V电压电泳17小时。凝胶经1×Sybr Gold染色30分钟后，在紫外光照下显影并拍照记录。

本研究旨在明确托雷斯海峡中东部海域海绵种群之间的关联，并评估跨区域海绵移植可能存在的风险。所调查的风险包括：遗传分化种群之间的移植可能导致野生种群遗传多样性下降，或是将新的海绵共生微生物类群引入某一区域。