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

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.