Experiment II: Effect of temperature and salinity on survival of cyprids (Fig. 3a)@en

Experiment II - cypris settlement Newly released nauplius larvae were held at a density of 0.5 larvae ml-1 in filtered (0.2 µm) seawater in 20 l containers at 25°C and a salinity of 30 (\"standard\" culture conditions at the time of collection, (see Berntsson et al. for culture details 31)). Containers were gently aerated and provided with a 1:1 mixture of the unicellular algae, T. pseudonana and S. marinoi, at a concentration of 2 × 105 cells ml-1. Every third day, the water in each container was carefully replaced by filtered seawater and fresh food was added. When cyprids appeared in the cultures (usually after 6 to 7 days), the cultures were sieved (200 µm) and cyprids were collected. Ten cyprids were incubated under continuous light in 10 ml filtered (0.2 µm) seawater in each well of the six-well plates at the temperature and salinity combinations outlined above. The transition from initial to target temperature and salinity conditions was achieved gradually (2 salinity units h-1, 1°C h-1) to prevent acute shock to the cyprids. Every second day, the water in each well was carefully replaced with fresh filtered seawater at the respective temperature and salinity. Settlement and survival of cyprids were monitored daily over 26 days. From these data we calculated: i) survival and ii) settlement of cyprids.实验二：腺介幼虫（cypris）附着实验。刚孵化的无节幼体（nauplius larvae）以0.5只/mL的密度，置于经0.2 µm过滤的海水中，于20升容器内培养，培养温度为25℃，盐度为30，该条件为样本采集时的标准培养流程（培养细节详见Berntsson等的相关研究，文献31）。容器持续轻柔曝气，并投喂按1:1比例混合的单细胞藻类——假微型海链藻（T. pseudonana）与S. marinoi，投喂浓度为2×10⁵细胞/mL。每三日，小心更换各容器内的过滤海水，并补充新鲜饵料。当培养体系中出现腺介幼虫时（通常在6~7天后），将培养液通过200 µm孔径的筛网过滤，收集腺介幼虫。取10只腺介幼虫，置于六孔板的每个孔中，每孔加入10 mL经0.2 µm过滤的海水，在上述温度与盐度组合条件下以持续光照方式培养。为避免腺介幼虫遭受急性应激，从初始培养条件过渡至目标温度与盐度的过程需逐步完成：盐度变化速率为2单位/小时，温度变化速率为1℃/小时。每两日，小心更换每孔内的海水，更换为对应温度与盐度的新鲜过滤海水。在26天的培养周期内，每日监测腺介幼虫的附着与存活情况。基于上述实验数据，我们计算得到两项核心指标：i）腺介幼虫存活率；ii）腺介幼虫附着率。