Differential gene expression analysis and cytological evidence reveal a sexual stage of an amoeba with multiparental cellular and nuclear fusion.

Sex is a hallmark of eukaryotes but its evolution in microbial eukaryotes is poorly elucidated. Recent genomic studies revealed microbial eukaryotes possess a genetic toolkit necessary for sexual reproduction. However, the mechanism of sexual development in a majority of microbial eukaryotes including amoebozoans is poorly characterized. The major hurdle in studying sex in microbial eukaryotes is a lack of observational evidence, primarily due to its cryptic nature. In this study, we used a tractable fusing amoeba, Cochliopodium, to investigate sexual development using stage-specific Differential Gene Expression (DGE) and cytological analyses. Both DGE and cytological results showed that most of the meiosis and sex-related genes are upregulated in Cochliopodium undergoing fusion in laboratory culture. Relative gene ontology (GO) category representations in unfused and fused cells revealed a functional skew of the fused transcriptome toward DNA metabolism, nucleus and ligases that are suggestive of a commitment to sexual development. However, the GO categories of unfused cells were dominated by metabolic pathways and other processes indicative of a vegetative phase. Our study provides strong evidence that the fused cells represent a sexual stage in Cochliopodium. Our findings have further implications in understanding the evolution and mechanism of inheritance involving multiparents in other eukaryotes with a similar reproductive strategy.