Department of Experimental Limnology, Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), 12587, Berlin, Germany.
Leibniz Institute for Baltic Sea Research (IOW), Seestrasse 15, 18119, Rostock, Germany.
Microb Ecol. 2023 Jan;85(1):9-23. doi: 10.1007/s00248-021-01893-7. Epub 2021 Dec 2.
Fungal microparasites (here chytrids) are widely distributed and yet, they are often overlooked in aquatic environments. To facilitate the detection of microparasites, we revisited the applicability of two fungal cell wall markers, Calcofluor White (CFW) and wheat germ agglutinin (WGA), for the direct visualization of chytrid infections on phytoplankton in laboratory-maintained isolates and field-sampled communities. Using a comprehensive set of chytrid-phytoplankton model pathosystems, we verified the staining pattern on diverse morphological structures of chytrids via fluorescence microscopy. Empty sporangia were stained most effectively, followed by encysted zoospores and im-/mature sporangia, while the staining success was more variable for rhizoids, stalks, and resting spores. In a few instances, the staining was unsuccessful (mostly with WGA), presumably due to insufficient cell fixation, gelatinous cell coatings, and multilayered cell walls. CFW and WGA staining could be done in Utermöhl chambers or on polycarbonate filters, but CFW staining on filters seemed less advisable due to high background fluorescence. To visualize chytrids, 1 µg dye mL was sufficient (but 5 µg mL are recommended). Using a dual CFW-WGA staining protocol, we detected multiple, mostly undescribed chytrids in two natural systems (freshwater and coastal), while falsely positive or negative stained cells were well detectable. As a proof-of-concept, we moreover conducted imaging flow cytometry, as a potential high-throughput technology for quantifying chytrid infections. Our guidelines and recommendations are expected to facilitate the detection of chytrid epidemics and to unveil their ecological and economical imprint in natural and engineered aquatic systems.
真菌微寄生虫（这里是壶菌）广泛分布，但在水生环境中往往被忽视。为了便于检测微寄生虫，我们重新研究了两种真菌细胞壁标记物——Calcofluor White（CFW）和麦胚凝集素（WGA）在实验室维持的分离物和野外采样群落中直接观察浮游植物上壶菌感染的适用性。使用一套全面的壶菌-浮游植物模式病理系统，我们通过荧光显微镜验证了不同形态结构的壶菌的染色模式。空孢子囊染色效果最佳，其次是包囊游动孢子和未成熟/成熟孢子囊，而对于根状菌、柄和休眠孢子，染色成功率则更为多变。在少数情况下，染色不成功（主要是 WGA），可能是由于细胞固定不足、胶状细胞涂层和多层细胞壁。CFW 和 WGA 染色可以在 Utermöhl 室或聚碳酸酯过滤器上进行，但由于背景荧光较高，CFW 染色在过滤器上似乎不太可取。为了可视化壶菌，1μg 染料 mL 就足够了（但推荐使用 5μg mL）。使用双重 CFW-WGA 染色方案，我们在两个自然系统（淡水和沿海）中检测到了多种，主要是未描述的壶菌，而假阳性或假阴性染色的细胞也很容易被检测到。作为一个概念验证，我们还进行了成像流式细胞术，作为一种潜在的高通量技术来定量壶菌感染。我们的指南和建议有望促进壶菌流行的检测，并揭示它们在自然和工程水生系统中的生态和经济影响。
