Functional roles of mastigonemes in Ectocarpus gamete swimming revealed by CRISPR-Cas9 mutagenesis.

Mastigonemes on the anterior flagellum (AF) of flagellated Stramenopiles (which includes diverse organisms such as diatoms, brown algae, oomycetes and others) are tripartite tubular structures. We investigated the functions of mastigonemes in gametes of the brown alga Ectocarpus species 7 strain Ec32 using a mas1 mutant generated by CRISPR-Cas9. Loss of mastigonemes in the mas1 mutant gametes could be confirmed by immunofluorescence microscopy using a specific anti-MAS1 antibody and transmission electron microscopy, showing complete loss of mastigonemes from the AF. High-speed video analysis revealed a drastic reduction in swimming speed in the mas1 mutant gametes compared to wild type gametes, despite an increase in the AF beat frequency. Additionally, waveform analysis indicated larger AF double amplitudes in the mas1 mutant gametes. These results suggested that mastigonemes enhance the AF thrust. The mas1 mutant male gametes fertilized female gametes (wild type strain Ec25). A mas1 mutant female strain was established from the heterozygous sporophyte that developed from such a zygote. Both wild type and the mas1 mutant male gametes could fertilize the mas1 mutant female gametes. Mastigonemes are therefore dispensable for gamete recognition and fusion in the brown alga Ectocarpus.