Abstract
Abstract The PRIN 2022 project “Unraveling Sex Determination in Bivalves: An Integrative Single-Cell Analysis during the Early Embryo Development of Mytilus galloprovincialis” was financed by the European Union - NextGenerationEU, Mission 4 Component 2. The project was coordinated by Prof. Liliana Milani (University of Bologna), in collaboration with the Stazione Zoologica “Anton Dohrn” of Naples. The project addressed a key question in developmental and evolutionary biology: how sex determination and germline specification occur in bivalve molluscs. These mechanisms are still poorly understood in many animal groups, although they are crucial for explaining the evolution of reproductive strategies. Bivalves represent a particularly valuable model because they show remarkable biological and ecological diversity and include species of major economic and nutritional importance. The project focused on the Mediterranean mussel, Mytilus galloprovincialis, a species especially suitable for investigating these processes. In this species, offspring can show strong deviations from the expected 50:50 sex ratio, with some females producing predominantly male or predominantly female progenies. This suggests that sex-inducing molecular factors are deposited in the egg during oogenesis. Moreover, M. galloprovincialis shows Doubly Uniparental Inheritance of mitochondria, a system in which paternal mitochondria follow a sex-specific distribution from the earliest embryonic divisions, making it possible to infer embryo sex at very early stages. The main aims of the project were to identify maternal molecular factors potentially involved in sex determination, to clarify the mechanisms of early germline specification, and to investigate the molecular processes underlying differential mitochondrial segregation. To achieve these goals, the project combined controlled fertilization experiments, fluorescence microscopy, RNA sequencing of eggs producing male- or female-biased progenies, comparative genomics, molecular evolutionary analyses, and innovative single-cell RNA sequencing of early embryos. The expected results included the identification of candidate genes and regulatory molecules involved in sex determination and mitochondrial inheritance, the development of the first single-cell transcriptomic characterization of early embryonic development in M. galloprovincialis, and the creation of new knowledge useful both for basic science and for future applications in aquaculture and environmental monitoring.
Results achieved
The project successfully achieved its planned scientific objectives. During the first phase, the research team analyzed RNA extracted from eggs predicted to produce either male- or female-biased offspring. By comparing transcriptomic profiles, the project identified 203 protein-coding genes and 57 long non-coding RNAs that were differentially transcribed between the two types of eggs. Many of these genes were associated with mitochondrial dynamics, cytoskeletal organization, and vesicular trafficking. The integration of transcriptomic data with comparative and evolutionary analyses made it possible to identify candidate molecular networks potentially involved in mitochondrial segregation and sex determination. In particular, the project highlighted factors such as RALA and CDK1, together with long non-coding RNAs, that may contribute to organelle selection and mitochondrial transmission across generations. The first major outcome of this work was published in BMC Biology in 2025. During the second phase, the project successfully applied single-cell RNA sequencing to early embryos of M. galloprovincialis with different sex biases. This was a major technical achievement, as single-cell approaches are still rarely applied to non-model marine organisms and required specific protocol optimization for mussel embryos. Embryos were sampled at 3 and 6 hours post-fertilization, corresponding to early cleavage and blastula stages. The project generated the first single-cell transcriptomic dataset for early mussel embryogenesis. The analysis identified distinct cell clusters and two putative germline-associated clusters, recognized through the expression of known germline markers such as vasa and spPHI. These results provide a new high-resolution framework for understanding cell lineage specification during early bivalve development and offer candidate genes for future functional studies. The team is currently completing complementary validation analyses through in situ hybridization and Hybridization Chain Reaction. In parallel, the project carried out a comparative genomic study of sex-determination-related gene families, including dmrt, sox, and fox genes, across more than 40 bivalve species. This work revealed shared evolutionary patterns between bivalves and mammals and contributed to the construction of a broader comparative framework for the study of sex determination in animals. The study was published in Molecular Ecology in 2025. The project also opened new methodological perspectives through the preliminary application of Raman spectroscopy to bivalve germ cells and embryos. This label-free technique allows the biochemical characterization of cellular compartments and may support future investigations into components of germ cells and mitochondria. Scientific results were disseminated through publications, national and international conferences, workshops, and training activities. The project team presented findings at several scientific meetings in Europe and beyond, contributed to international networking in mollusc genomics and evolutionary developmental biology, and helped organize the MytEED Winter School on Mytilus galloprovincialis as an emerging model for eco-evo-devo studies. Overall, the project advanced knowledge of sex determination, germline specification, and mitochondrial inheritance in bivalves. It produced new molecular resources for M. galloprovincialis, strengthened collaboration between the University of Bologna and the Stazione Zoologica “Anton Dohrn”, and created a foundation for future research with potential relevance for marine biology, aquaculture sustainability, and the conservation of economically and ecologically important bivalve species. Data and scientific outputs are being made available in line with Open Access principles.Dettagli del progetto
Responsabile scientifico: Liliana Milani
Strutture Unibo coinvolte:
Dipartimento di Scienze Biologiche, Geologiche e Ambientali
Coordinatore:
ALMA MATER STUDIORUM - Università di Bologna(Italy)
Contributo totale di progetto: Euro (EUR) 187.500,00
Contributo totale Unibo: Euro (EUR) 139.587,00
Durata del progetto in mesi: 24
Data di inizio
28/09/2023
Data di fine:
28/02/2026
