Influence of size and spatial competition on the bioactivity of coral reef sponges from Torres Strait (MTSRF Project)

Size frequency surveys of the sponges Coscinoderma matthewsi, Hyrtios erecta and Ianthella basta (yellow color morph) were conducted at Masig Island, central Torres Strait, in March 2007. At each of eight sites, separated by at least 200 m, three randomly positioned 30 x 1 m transects were surveyed, with each transect separated by at least 20 m. All transects were located on sloping reef at between 10 and 12 m depth.The greatest dimension of each sponge was measured for Coscinoderma matthewsi and Ianthella basta, and used as an approximation of overall sponge size. Due to the branching morphology of Hyrtios erecta, the length of each individual branch was included to give an overall measurement. Sponges were also assigned a competition class based on the percentage of their perimeter in contact or within 1 cm of surrounding or encroaching organisms.Three tissue samples, each 1-2 cm³ were collected from 20 different individuals of each species from a single site to examine the influence of sponge size on bioactivity. Prior to sampling, sponges were also measured for size, photographed and assigned a competition class. The sponges sampled encompassed the full size range of each respective species, except for very small sponges, which did have sufficient tissue to obtain three independent tissue samples. Sponges displaying signs of disease were not sampled. Samples were frozen at -20°C within one hour of collection and kept frozen until freeze dried.Freeze-dried sponge tissue samples were weighed and extracted 3 times with methanol (MeOH). Crude extracts were made up into a 100 mg/ml concentration in MeOH based on individual extract weights. 100 µl of each sample was then aliquoted to microtitre plates and dried down, yielding 10 mg of dry extract per sample for bioactivity screening.The production of bioactive metabolites and overall bioactivity of sponges was examined using the sulforhodamine B (SRB) assay. Crude extracts were re-suspended in dimethyl sulfoxide (DMSO) and added to 96 well plates containing 100 µl of pre-incubated cell suspension. Several concentrations were tested for each species, with the concentration showing the greatest fine-scale variation in toxicity between individual samples selected for the analyses: 100 µg/ml for Coscinoderma matthewsi; 0.1875 µg/ml for Hyrtios erecta; and 100 µg/ml for Ianthella basta. One replicate plate was made for each sample plate and for each concentration, with appropriate controls. After 48 hours of incubation, plates were fixed with trichloroacetic acid and stained using 0.4% SRB. Plates were read using the Wallac Victor plate reader at an absorbance of 490 nm, and % cell growth values were calculated. This study was undertaken to examine the influence of individual size and spatial competition on the bioactivity of three coral reef sponges of differing morphologies, occurring in Torres Strait: Coscinoderma matthewsi (a massive, hemispherical sponge), Hyrtios erecta (a club-shaped branching sponge) and Ianthella basta (a fan-like sponge).

2007年3月，在托雷斯海峡中部的马西格岛对海绵Coscinoderma matthewsi、Hyrtios erecta及Ianthella basta（黄色形态）开展了大小频率调查。在8个间距至少200米的站点中，每个站点随机设置3条30×1米的样带（样带间距至少20米）进行调查；所有样带均位于10-12米水深的倾斜礁体上。对于Coscinoderma matthewsi和Ianthella basta，测量其最大维度以近似表征整体大小；针对Hyrtios erecta的分支形态，则通过累加各分支长度获得整体尺寸。此外，根据海绵周长与周围生物接触或相距≤1cm的比例，为其划分竞争等级。

从单一站点选取每种海绵的20个个体，各采集3份1-2 cm³的组织样本，以探究海绵大小对生物活性的影响。采样前，对海绵进行大小测量、拍照及竞争等级划分；采样个体涵盖各物种的全尺寸范围，但排除无法获取3份独立组织样本的极小个体，且不采集患病个体。样本于采集后1小时内冷冻至-20℃，并保持冷冻状态直至冻干处理。

冻干海绵组织样本经称重后，用甲醇（methanol，MeOH）提取3次；基于各提取物重量，将粗提物配制成100 mg/ml的甲醇溶液。取100 μl样本溶液分装至微孔板并干燥，每个样本获得10 mg干提取物用于生物活性筛选。

采用磺酰罗丹明B（sulforhodamine B，SRB）测定法分析海绵生物活性代谢产物的产生及整体生物活性：将粗提物重悬于二甲基亚砜（dimethyl sulfoxide，DMSO）中，加入含100 μl预培养细胞悬液的96孔板。对每个物种测试多个浓度，选择个体样本间毒性差异最显著的浓度进行分析——Coscinoderma matthewsi为100 μg/ml、Hyrtios erecta为0.1875 μg/ml、Ianthella basta为100 μg/ml。每个样本板及浓度设置1个重复板，并加入适当对照。培养48小时后，用三氯乙酸固定平板，再以0.4% SRB染色；通过Wallac Victor酶标仪在490 nm波长下读取吸光度值，计算细胞生长百分比。

本研究旨在考察个体大小与空间竞争对托雷斯海峡三种不同形态珊瑚礁海绵生物活性的影响，三种海绵分别为：Coscinoderma matthewsi（块状半球形）、Hyrtios erecta（棒状分支形）及Ianthella basta（扇状）。