A Species-Specific Assay for Salmo cf. trutta and Its Application in eDNA-Based Spawning Activity Monitoring

Understanding salmonid spawning dynamics is critical for conserving cold-water river ecosystems amid increasing human and climate pressures. This study developed and validated a species-specific eDNA (Salmo cf. trutta and Thymallus thymallus) and evaluated its performance for seasonal spawning activity monitoring using droplet digital PCR (ddPCR). Species-specific primers and probes targeting mitochondrial nd5 (S. trutta) and cytb (T. thymallus) genes were designed and optimized as a duplex assay. Performance assessments included in vitro validation, cross-amplification testing, and determining the LOB, LOD, and LOQ. Field validation over a year at two spawning sites in the Gradac River, Serbia, involved seasonal eDNA sampling, filtration, extraction, and ddPCR analysis. Fish community composition was also assessed with electrofishing and metabarcoding. The assay showed high specificity and sensitivity, with LODs of 0.14 cp/µL and LOQs of 0.99 and 1.25 cpµL for S. trutta and T. thymallus. S. trutta eDNA peaked in late autumn during spawning, while T. thymallus remained at or below detection limits, reflecting its lower abundance and different spawning season. Filter type affected filtration efficiency but not eDNA yield. These findings confirm ddPCR-based eDNA as a powerful, non-invasive tool for monitoring salmonid spawning and seasonal changes, supporting adaptive fisheries management and conservation amid environmental changes.