Two Doublesex1 mutants revealed a tunable gene network underlying intersexuality in Daphnia magna

Purpose: To justify whether sex is strictly binary or variable along a spectrum, exhaustive investigation into intersexuality is necessary. In this study, by mutating the sex determining locus Doublesex1, we generated two Daphnia magna strains from which two different forms of feminized males could be obtained. mRNA sequencing of these intersex males was carried out to explore the transcritomic shift responsible for the changes in sexual phenotype. Methods: Two Doublesex1 mutants, namely Line A and Line B, were generated by microinection of designed TALENs into Daphnia magna eggs. Line A males showed severe, while Line B males showed minor bodywide feminization. Whole body mRNA profiles of 40 hour post oviposition embryos were generated for wildtype female, Line A male, Line B male and wildtype male using Illumina sequencing (mRNA sequencing service provided by Novogene). Data analysis was done using CLC Genomics Workbench software. Results: Among 731 sex-biased genes identified in this dataset, more than half (449 genes) showed non-binary expression profile in which their expression in the mutant males was at an intermediate level between wildtype female and wildtype male. Conclusions: Downstream genes of Doublesex1 cascade may fluctuate in activity and give rise to a spectrum of sexual phenotypes in Daphnia magna. Overall design: mRNA profile comparison of four groups: wildtype female, Line A male, Line B male, and wildtype male. Triplicates for each group (total 12 samples). Same age (40 hour post oviposition).