Recognition of polymorphic Csd proteins determines sex in the honeybee.

Sex in honeybees, Apis mellifera, is genetically determined by heterozygous versus homo/hemizygous genotypes involving numerous alleles at the single complementary sex determination locus. The molecular mechanism of sex determination is however unknown because there are more than 4950 known possible allele combinations, but only two sexes in the species. We show how protein variants expressed from complementary sex determiner (csd) gene determine sex. In females, the amino acid differences between Csd variants at the potential-specifying domain (PSD) direct the selection of a conserved coiled-coil domain for binding and protein complexation. This recognition mechanism activates Csd proteins and, thus, the female pathway. In males, the absence of polymorphisms establishes other binding elements at PSD for binding and complexation of identical Csd proteins. This second recognition mechanism inactivates Csd proteins and commits male development via default pathway. Our results demonstrate that the recognition of different versus identical variants of a single protein is a mechanism to determine sex.

The function and evolution of a genetic switch controlling sexually dimorphic eye differentiation in honeybees.

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.

Sexual diversification of splicing regulation during embryonic development in honeybees (Apis mellifera), A haplodiploid system.

The honeybee is a haplodiploid organism in which sexual development is determined by the complementary sex determiner (csd) gene and realized by sex-specific splicing processes involving the feminizer (fem) gene. We used high throughput transcriptome sequencing (RNA-Seq) to characterize the transcriptional differences between the sexes caused by the fertilization and sex determination processes in honeybee (Apis mellifera) embryos. We identified 758, 372 and 43 differentially expressed genes (DEGs) and 58, 176 and 233 differentially spliced genes (DSGs) in 10-15-h-old, 25-40-h-old and 55-70-h-old female and male embryos, respectively. The early difference in male and female embryos in response to the fertilization and non-fertilization processes resulted mainly in differential expression of genes (758 DEGs vs. 58 DSGs). In the latest sampled embryonic stage, the transcriptional differences between the sexes were dominated by alternative splicing of transcripts (43 DEGs vs. 233 DSGs). Interestingly, differentially spliced transcripts that encode RNA-binding properties were overrepresented in 55-70-h-old embryos, indicating a more diverse regulation via alternative splicing than previous work on the sex determination pathway suggested. These stage- and sex-specific transcriptome data from honeybee embryos provide a comprehensive resource for examining the roles of fertilization and sex determination in developmental programming in a haplodiploid system.

A genetic switch for worker nutrition-mediated traits in honeybees.

Highly social insects are characterized by caste dimorphism, with distinct size differences of reproductive organs between fertile queens and the more or less sterile workers. An abundance of nutrition or instruction via diet-specific compounds has been proposed as explanations for the nutrition-driven queen and worker polyphenism. Here, we further explored these models in the honeybee (Apis mellifera) using worker nutrition rearing and a novel mutational screening approach using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. The worker nutrition-driven size reduction of reproductive organs was restricted to the female sex, suggesting input from the sex determination pathway. Genetic screens on the sex determination genes in genetic females for size polyphenism revealed that doublesex (dsx) mutants display size-reduced reproductive organs irrespective of the sexual morphology of the organ tissue. In contrast, feminizer (fem) mutants lost the response to worker nutrition-driven size control. The first morphological worker mutants in honeybees demonstrate that the response to nutrition relies on a genetic program that is switched "ON" by the fem gene. Thus, the genetic instruction provided by the fem gene provides an entry point to genetically dissect the underlying processes that implement the size polyphenism.