State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing, China.
University of Chinese Academy of Sciences, Beijing, China.
Microbiol Spectr. 2022 Jun 29;10(3):e0255721. doi: 10.1128/spectrum.02557-21. Epub 2022 May 12.
As the key contributor to plankton biomass and nutrient cycling in aquatic ecosystems, photosynthetic picoeukaryotes (PPEs) have been recently investigated in freshwater ecosystems. However, the limited access to remote areas creates challenges for PPE sample preservation before sorting and counting by flow cytometry (FCM) in the laboratory. Here, we explored the effects of different preservation methods on the PPE community by combining FCM sorting and high-throughput sequencing. Our results showed that dimethyl sulfoxide (DMSO) cryoprotection could destroy the fluorescence and cell structure of the PPEs, making the subsequent FCM analysis and sorting difficult. Aldehyde fixation maintained the PPE fluorescence, and the fixed samples were of sufficient quality for abundance analysis and sorting by FCM. However, the sequencing results showed that, after preservation by aldehydes, the proportion of PPEs dramatically decreased to approximately 10%, in comparison to 90% in the fresh samples, and the sequences of Ascomycota significantly increased. In contrast, preservation with Pluronic F68 (F68) not only could maintain the PPE abundance close to the initial value but also could keep the PPE community similar to that in the fresh samples over a storage time of 6 months. Thus, F68 cryopreservation is a suitable preservation method for PPE communities from freshwater lakes. PPEs contribute significantly to primary productivity in freshwater ecosystems. The combination of FCM sorting and high-throughput sequencing has been shown to be a powerful approach and can largely improve our view of the PPE diversity. However, the water samples could not be counted and sorted immediately after sampling from many lakes due to the inaccessibility of FCM in the field. Thus, the comparison of different preservation methods that allow subsequent analysis of the community structure by high-throughput sequencing is an urgent need. Our results indicated that F68 cryopreservation could maintain the PPE abundance close to the initial value and keep the community similar to that in the fresh samples over a storage time of 6 months.
作为水生生态系统中浮游生物生物量和养分循环的关键贡献者，光合微微体（PPE）最近在淡水生态系统中得到了研究。然而，由于难以进入偏远地区，在实验室通过流式细胞仪（FCM）对 PPE 进行分选和计数之前，对 PPE 样品进行保存存在挑战。在这里，我们通过结合 FCM 分选和高通量测序来探索不同保存方法对 PPE 群落的影响。我们的研究结果表明，二甲基亚砜（DMSO）冷冻保护剂可能会破坏 PPE 的荧光和细胞结构，使得后续的 FCM 分析和分选变得困难。醛固定保持了 PPE 的荧光，固定后的样品质量足以进行丰度分析和 FCM 分选。然而，测序结果表明，醛固定保存后，PPE 的比例急剧下降至约 10%，而新鲜样品中的 PPE 比例约为 90%，并且子囊菌门的序列显著增加。相比之下，使用 Pluronic F68（F68）保存不仅可以使 PPE 的丰度接近初始值，而且可以在 6 个月的储存时间内使 PPE 群落保持与新鲜样品相似。因此，F68 冷冻保存是一种适合保存淡水湖泊中 PPE 群落的方法。PPE 对淡水生态系统的初级生产力有重要贡献。流式细胞仪分选和高通量测序的结合已被证明是一种强大的方法，可以大大提高我们对 PPE 多样性的认识。然而，由于现场无法使用 FCM，许多湖泊的水样无法在采样后立即进行计数和分选。因此，比较不同的保存方法，以便随后通过高通量测序分析群落结构，是当务之急。我们的研究结果表明，F68 冷冻保存可以使 PPE 的丰度接近初始值，并在 6 个月的储存时间内使群落保持与新鲜样品相似。
