The doublesex gene integrates multi-locus complementary sex determination signals in the Japanese ant, Vollenhovia emeryi

Abstract A female diploid, male haploid sex determination system (haplodiploidy) is found in hymenopteran taxa, such as ants, wasps, bees and sawflies. In this system, a single, complementary sex-determination ( sl- CSD) locus functions as the primary sex-determination signal. In the taxa that has evolved this system, females and males are heterozygous and hemi/homozygous at the CSD locus, respectively. While the sl- CSD system enables females to alter sex ratios in the nest, it carries a high cost in terms of inbreeding, as individuals that are homozygous at the CSD locus become sterile diploid males. To counter this risk, some of hymenopteran species have evolved a multi-locus CSD ( ml -CSD) system, which effectively reduces the proportion of sterile males. However, the mechanism by which these multiple primary signals are integrated and how they affect the terminal sex-differentiation signal of the molecular cascade have not yet been clarified. To resolve these questions, we examined the molecular cascade in the Japanese ant Vollenhovia emeryi , which we previously confirmed has two CSD loci. Here, we showed that the sex-determination gene, doublesex ( dsx ), which is highly conserved among phylogenetically distant taxa, is responsible for integrating two CSD signals in V. emeryi . After identifying and characterizing dsx , genotypes containing two CSD loci and splicing patterns of dsx were found to correspond to the sexual phenotype, suggesting that two primary signals are integrated into dsx . These findings will facilitate future molecular and functional studies of the sex determination cascade in V. emeryi , and shed light on the evolution and diversification of sex determination systems in insects.