Integrative perspectives on bivalve sex determination: A comparative and evolutionary analysis across phylogeny

Sex determination (SD) in bivalves remains largely unexplored despite their socio-economic importance. This is due to the diversity of mechanisms across species, involving both genetic and environmental factors, and the apparent absence of heteromorphic sex chromosomes, which complicates research. This study integrates comparative genomics, phylogenetic analyses, and in-situ visualization to investigate the molecular basis of SD in bivalves. Key components of gene families known for SD in Metazoa were analyzed, focusing on the Dmrt, Sox, and Fox gene families, which tend to evolve rapidly. Notably, Dmrt-1L and Sox-H were identified as fast-evolving genes in bivalves, supporting their involvement in male SD. To further explore their roles, mRNA in-situ hybridization chain reaction (HCR) was used to assess their transcription patterns in Mytilus galloprovincialis embryos and larvae. Expression patterns of Fox-L2, linked to female SD, and the germline marker Vasa/Vasa were also analyzed. Dmrt-1L and Sox-H were not transcribed during early development (up to 72 hours post-fertilization), while Fox-L2 showed increasing sex-unbiased expression at gastrulation. Vasa-positive cells, marking primordial germ cells, appeared only after larval formation, suggesting SD occurs later in development. This study highlights the value of integrative approaches in researching non-model organisms, providing a foundational reference for SD in bivalves and expanding our understanding of their reproductive biology.