Animals develop sex-specific morphological structures that are diverse between organisms. However, understanding the developmental and evolutionary mechanisms governing these traits is still limited and largely restricted to DM domain genes, which are conserved, sex-specific developmental regulators identified in genetic models. Here, we report a sex-specific developmental regulator gene, glubschauge (glu) that selectively regulates sexually dimorphic eye differentiation in honeybees. We found that the sex determination gene feminizer (fem) controls sex-specific splicing of glu transcripts, establishing a genetic switch in which Glu proteins with a zinc finger (ZnF) domain are only expressed in females. We showed that female coding sequence was essential and sufficient for partial feminization. Comparative sequence and functional studies revealed that the evolutionary origination of the genetic switch was followed by the mutational origin of the essential ZnF domain. Our results demonstrate that glu is a newly evolved sex-specific genetic switch for region-specific regulation of a dimorphic character.

Functional genetic studies in honeybees have been limited to transposon mediated transformation and site directed mutagenesis tools. However, site- and sequence-specific manipulations that insert DNA fragments or replace sequences at specific target sites are lacking. Such tools would enable the tagging of proteins, the expression of reporters and site-specific amino acid changes, which are all gold standard manipulations for physiological, organismal and genetic studies. However, such manipulations must be very efficient in honeybees since screening and crossing procedures are laborious due to their social organization. Here, we report an accurate and remarkably efficient site-specific integration of DNA-sequences into the honeybee genome using CRISPR/Cas9 mediated HDR (homology-directed repair). We employed early embryonic injections and selected a highly efficient sgRNA in order to insert 294 bp and 729 bp long DNA sequences into a specific locus at the dsx gene. These sequences were locus-specifically integrated in 57% and 59% of injected bees. Most importantly 21% and 25% of the individuals lacked the wildtype sequence demonstrating that we generated homozygous mutants in which all cells are affected (no mosaicism). The highly efficient, locus-specific insertions of nucleotide sequences generating homozygous mutants demonstrate that systematic molecular studies for honeybees are in hand that allow somatic mutation approaches via workers or studies in the next generation using queens with their worker progeny. The employment of early embryonic injections and screenings of highly efficient sgRNAs may offer the prospect of highly successful sequence- and locus-specific mutations also in other organisms.

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