Single-cell atlas of prawn gonads identifies NLRP2 regulated mitochondrial dysfunction in estrogen induced sex reversal

Sex reversal is widely used in biological breeding, but its cellular and molecular consequences in crustaceans are poorly understood. Here, we combine estrogen induced feminization, single cell RNA sequencing, histology and RNA interference to dissect gonadal development in the oriental river prawn ( Macrobrachium nipponense ). Feeding post-larval males with 17β-estradiol generated neo-females with ovary like gonads. Single cell profiling of testis, ovaries and neo-female gonads produced a gonadal atlas in which, using newly validated markers, we resolved major oogenic and spermatogenic cell types. Cell-type composition and pseudotime analyses showed that neo-female gonads are dominated by early germ cells, whereas mature oocytes and spermatozoa are strongly depleted; histology confirmed this developmental arrest. Stage resolved differential expression and KEGG enrichment consistently highlighted oxidative phosphorylation as the most perturbed pathway across key germ-cell stages. Among the affected genes, MnNLRP2 displayed primary spermatocyte specific and testis enriched expression and was conserved among decapods. RNA interference of MnNLRP2 in PL10 prawns shifted phenotypic sex ratios toward females and altered sex steroid levels, reducing MT in males while elevating E ₂ in females; knockdown in adults caused spermatogenic arrest and down-regulation of oxidative phosphorylation components. These findings provide unprecedented insights into crustacean germ cell dynamics and the metabolic and transcriptional networks governing incomplete sex reversal, thereby offering critical resources for reproductive biology and aquaculture research.