Bannon Catherine C, Campbell Douglas A
Biology Department, Mount Allison University, Sackville, NB, Canada.
PLoS One. 2017 Oct 3;12(10):e0185166. doi: 10.1371/journal.pone.0185166. eCollection 2017.
Diatoms are marine primary producers that sink in part due to the density of their silica frustules. Sinking of these phytoplankters is crucial for both the biological pump that sequesters carbon to the deep ocean and for the life strategy of the organism. Sinking rates have been previously measured through settling columns, or with fluorimeters or video microscopy arranged perpendicularly to the direction of sinking. These side-view techniques require large volumes of culture, specialized equipment and are difficult to scale up to multiple simultaneous measures for screening. We established a method for parallel, large scale analysis of multiple phytoplankton sinking rates through top-view monitoring of chlorophyll a fluorescence in microtitre well plates. We verified the method through experimental analysis of known factors that influence sinking rates, including exponential versus stationary growth phase in species of different cell sizes; Thalassiosira pseudonana CCMP1335, chain-forming Skeletonema marinoi RO5A and Coscinodiscus radiatus CCMP312. We fit decay curves to an algebraic transform of the decrease in fluorescence signal as cells sank away from the fluorometer detector, and then used minimal mechanistic assumptions to extract a sinking rate (m d-1) using an RStudio script, SinkWORX. We thereby detected significant differences in sinking rates as larger diatom cells sank faster than smaller cells, and cultures in stationary phase sank faster than those in exponential phase. Our sinking rate estimates accord well with literature values from previously established methods. This well plate-based method can operate as a high throughput integrative phenotypic screen for factors that influence sinking rates including macromolecular allocations, nutrient availability or uptake rates, chain-length or cell size, degree of silification and progression through growth stages. Alternately the approach can be used to phenomically screen libraries of mutants.
硅藻是海洋初级生产者，部分由于其硅质壳的密度而沉降。这些浮游植物的沉降对于将碳隔离到深海的生物泵以及生物体的生存策略都至关重要。沉降速率先前已通过沉降柱、或与沉降方向垂直排列的荧光计或视频显微镜进行测量。这些侧视技术需要大量培养物、专门设备，并且难以扩大规模以进行多个同时测量用于筛选。我们建立了一种通过在微量滴定板中顶视监测叶绿素a荧光来对多种浮游植物沉降速率进行平行、大规模分析的方法。我们通过对影响沉降速率的已知因素进行实验分析来验证该方法，这些因素包括不同细胞大小物种的指数生长期与稳定生长期；假微型海链藻CCMP1335、链状的海洋骨条藻RO5A和辐射圆筛藻CCMP312。当细胞从荧光计探测器下沉时，我们将衰减曲线拟合到荧光信号降低的代数变换，然后使用最少的机制假设，通过RStudio脚本SinkWORX提取沉降速率（米/天）。由此我们检测到沉降速率的显著差异，因为较大的硅藻细胞比较小的细胞下沉得更快，并且稳定期的培养物比指数期的培养物下沉得更快。我们的沉降速率估计值与先前建立的方法的文献值非常吻合。这种基于微孔板的方法可以作为一种高通量综合表型筛选方法，用于筛选影响沉降速率的因素，包括大分子分配、养分可用性或吸收速率、链长或细胞大小、硅化程度以及生长阶段的进展。或者，该方法可用于对突变体文库进行表型筛选。
