Quantitative Analysis of Sex-Specific Feminizer (fem) Transcripts During Honey Bee (Apis mellifera) Development

Sex determination in honey bees (Apis mellifera) is controlled by the complementary sex determiner (csd) gene, which directs female- or male-specific splicing of the downstream feminizer (fem) transcript. Previous studies have reported contradictory data on the expression of fem transcripts in both sexes, but no rigorous quantitative analysis across developmental stages had been performed. Here, we optimized Real-Time PCR conditions to reliably detect and quantify both female-specific (fem^F) and male-specific (fem^M) transcripts, addressing challenges posed by AT-rich sequences, repeated regions, and cDNA instability. Using these methods, we analyzed transcript levels in eggs, larvae, and pupae of both sexes. Our results show that fem^F is highly specific for females, with approximately 100-fold higher expression in females than in males, whereas fem^M is less sex-specific, with only ~10-fold higher expression in males even at early developmental stages. Notably, fem^F transcripts are detectable in males, and fem^M expression increases in females during later pupal stages. Quantitative comparison indicates that fem^M expression in males is similar to fem^F expression in females, indicating that despite the presence of the premature stop codon in the male transcript, this transcript is not degraded through the mRNA surveillance mechanism. Our study provides a framework for evaluating fem transcript dynamics and has important implications for interpreting sex-determination mechanisms in honey bees.