Non-invasive sexing of the long-spined sea urchin, Diadema setosum

Sea urchins are highly valued due to their commercial significance and essential roles in marine ecosystems. However, traditional methods for sexing sea urchins, such as spawning induction or gonad histology, are typically invasive, season-dependent, time-consuming, and labour-intensive. This study investigated non-invasive sexing techniques for Diadema setosum by assessing various morphological traits, performing a genome wide association study (GWAS), and testing PCR markers previously developed for Mesocentrotus nudus . Of the 16 morphological traits examined, only genital papillae exhibited sexual dimorphism, achieving 82.5 % accuracy for sex identification in a blinded experiment. Importantly, the sexual divergence of this trait was independent of the urchins’ reproductive status, making it a reliable year-round sexing indicator. The PCR markers from M. nudus did not prove applicable for D. setosum . However, a genomic region with 22 male-heterozygous SNPs on Scaffold 2 was identified, spanning just 1154 bp. This region holds potential for development into a PCR- or sequencing-based sexing barcode for D. setosum . These findings present a promising avenue for non-invasive, season-independent sex identification in this species, with potential to enhance the efficiency and sustainability of sea urchin aquaculture. • A range of non-invasive sexing characteristics for Diadema setosum were tested. • The genital papillae opening is a reliable, season-independent, sex marker. • A ∼1 kb region on Scaffold 2 containing 22 male-heterozygous SNPs was identified. • There is potential for image- or genetic-based in-situ sexing methods for D. setosum. • Non-invasive sexing promotes aquaculture efficiency and aids sea urchin research.