Percentage of Primnoella chilensis polyps activity states under sediment exposure of 15 days, observation period 21 h, Control, Punta Gruesa@en

To determine behavioural stress, polyp activity states of Primnoella chilensis colonies were observed for 21 hours after eleven days under turbid seawater exposure in an experimental setup (Treatment: equals sedimentation rate of 450 mm/y; control group: ambient seawater). Image analysis was conducted by drawing a segmented selection line along the centre of the colonies' internal axis using the image-processing program ImageJ. This line was moved to the side by 175 % of the internal axis' width measured in pixels and 250 % respectively, resulting in two lines that were named selection A and B. These distances were chosen because fully expanded polyps showed a horizontal spread of about two times the internal axis' diameter and fully closed polyps less than one time respectively. For both lines, a plot profile was created showing grey values of all pixels under the segmented selection line. Lighter pixels of the extended polyps in comparison to the black background resulted in countable peaks. Therefore, the plot was divided by the highest and lowest grey values and bisected horizontally using the vector graphics editor Adobe Illustrator. The number of peaks in the upper part of the divided plot-image was then counted in ImageJ using the function 'analyze particles'. Lastly, the number of polyps of a category was calculated: category 0 (closed): = total polyps observed minus counted peaks for selection line A; category 1 (semi-open): = counted peaks for selection line A minus counted peaks for selection line B; category 2: = counted peaks for selection B.为量化行为应激水平，本研究在实验装置中设置两组对照：处理组的海水沉积速率为450 mm/年，对照组采用自然海水；将智利原柳珊瑚（Primnoella chilensis）群落置于浑浊海水环境中暴露11天后，连续21小时观测其水螅体的活动状态。图像分析流程如下：使用图像处理软件ImageJ(ImageJ)沿群落内部轴的中心绘制分段选线，随后分别将该选线向两侧平移至内部轴像素宽度的175%与250%处，得到两条选线并分别命名为选线A与选线B。选择该平移距离的依据为：完全伸展的水螅体水平扩散范围约为内部轴直径的2倍，而完全闭合的水螅体水平扩散范围则小于1倍。随后为两条选线分别生成灰度分布曲线，展示分段选线下方所有像素的灰度值；相较于黑色背景，伸展的水螅体像素亮度更高，可形成可被计数的峰值。将该曲线的灰度值归一化至最高与最低灰度值区间后，使用矢量图形编辑软件Adobe Illustrator(Adobe Illustrator)对其进行水平二等分。利用ImageJ的「分析粒子」功能，统计分割后的曲线图像上半部分的峰值数量。最后计算各分类的水螅体数量：分类0（闭合型）= 观测总水螅体数 - 选线A统计得到的峰值数；分类1（半开放型）= 选线A统计峰值数 - 选线B统计峰值数；分类2= 选线B统计峰值数。