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. 2020 Jul 17;10:11930. doi: 10.1038/s41598-020-68717-5

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Detailed phenotyping of the exflagellation defect in NF54/MAP-2GFPDD parasites. (a) Ploidy of GFP-positive microgametocytes 0 and 10 min after activation of gametogenesis under protein-stabilizing (+ Shield-1) and -degrading (− Shield-1) conditions assessed by flow cytometry. Parasites were split (± Shield-1) 24 h before analysis. Values show the means ± SD of three biological replicates. (b) Number of male gametocytes that form axonemes 15 min after activation of gametogenesis, determined from anti-α-tubulin IFAs. Protein-stabilizing (+ Shield-1) and -degrading (− Shield-1) culturing conditions are compared. Parasites were split (± Shield-1) 24 h before analysis. Values show the means ± SD of five biological replicates with individual data points represented by open symbols. ns, not significant (paired two-tailed Student’s t test). (c) Representative images of anti-α-tubulin IFAs showing axoneme formation 15 min after activation of gametogenesis. Protein-stabilizing (+ Shield-1) and -degrading (− Shield-1) culturing conditions are compared. White arrowheads indicate incorporation of DNA into newly forming microgametes exclusively in parasites cultured under protein-stabilizing (+ Shield-1) conditions. Parasites were split (± Shield-1) 24 h before probing. Scale bar = 2 µm.