Rapid molecular sex identification in Ruditapes philippinarum using a male-specific mitochondrial marker

Many bivalves including Manila clam Ruditapes philippinarum lack reliable visual sexual dimorphism, making sex determination difficult outside the spawning season using conventional histology. This limitation poses a major bottleneck for selective breeding and population studies. In this study, we successfully addressed this challenge by developing and validating the first molecular sexing methods for this species, based on the mitochondrial Doubly Uniparental Inheritance (DUI) system. By comparative analysis of F- and M-type mitochondrial genes, we identified nad2 gene as a marker with substantial genetic distance and male-specific amplification. Consequently, we developed both a conventional PCR and a rapid loop-mediated isothermal amplification assay (LAMP) targeting the M-type nad2 gene. Both methods demonstrated complete concordance with microscopic gonadal examination in all 67 sexually mature clams examined, with 26 identified as male. We further explored the applicability of these markers for undifferentiated or spent gonads, where histological examination was inconclusive. PCR and LAMP showed consistent M-type nad2 amplification patterns in these putative male samples, paralleling those seen in adult males. We then applied the same markers to three additional geographically distinct populations, yielding concordant results across all samples. This work provides a powerful and versatile toolkit for bivalve sex diagnosis, establishes a generalizable framework for sex identification, and has the potential to accelerate breeding programs and improve population management strategies in DUI bivalves.