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

Sex determination in honey bees (<i>Apis mellifera</i>) is controlled by the complementary sex determiner (<i>csd</i>) gene, which directs female- or male-specific splicing of the downstream feminizer (<i>fem</i>) transcript. Previous studies have reported contradictory data on the expression of <i>fem</i> 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 (<i>fem^F</i>) and male-specific (<i>fem^M</i>) 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 <i>fem^F</i> is highly specific for females, with approximately 100-fold higher expression in females than in males, whereas <i>fem^M</i> is less sex-specific, with only ~10-fold higher expression in males even at early developmental stages. Notably, <i>fem^F</i> transcripts are detectable in males, and <i>fem^M</i> expression increases in females during later pupal stages. Quantitative comparison indicates that <i>fem^M</i> expression in males is similar to <i>fem^F</i> 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 <i>fem</i> transcript dynamics and has important implications for interpreting sex-determination mechanisms in honey bees.