Detecting Callinectes sapidus expansion through eDNA: a case study from inland waters of Friuli Venezia Giulia

Callinectes sapidus (Rathbun, 1896), native to the western Atlantic and Gulf of Mexico, has become a prominent invasive species in the Mediterranean over the past decade. Due to its adaptability and aggressive predatory behavior, it is considered a highly invasive species. Since the early 20th century, it has spread into the Mediterranean Sea, raising concerns about its potential ecological and economic impacts. The blue crab thrives in both marine and brackish environments, with a life cycle strongly influenced by fluctuations in salinity and temperature. Juveniles and adults typically inhabit estuarine and coastal waters, where salinity conditions are variable. For reproduction, females migrate to high-salinity areas to spawn, and larvae develop in these saltier environments before dispersing into lower-salinity estuaries. The ANNOTATE project monitors C.sapidus through environmental DNA (eDNA) in river tributaries flowing into the Marano and Grado Lagoon (16,364 ha, Northern Adriatic). While the species has been established in the lagoon since the early 2000s, this study investigates its ongoing expansion into the connected freshwater environments, with sampling conducted at 48 sites. Surface water from six rivers was sampled monthly during summer and fall at two sites each, and its chemical parameters (temperature, salinity, conductivity, dissolved oxygen, and pH) were measured. eDNA was extracted from the water samples and analyzed using a species-specific COI barcoding assay based on hydrolysis probe chemistry in qPCR to detect the presence/absence of C.sapidus. Initial results showed a variation of temperature and salinity across both rivers and along sampling points. Mean values ranged from 17.7°C to 21.5°C and from 3.5 × 10⁻⁶ ppm to 238 ppm, respectively, with a ΔT of 3.8 °C and a ΔS of 3.5 ppt. C.sapidus was detected in all surveyed tributaries, including the northernmost sampling points, located approximately 20km from the river mouths. Overall, these findings confirm the ability of C.sapidus to tolerate a wide range of environmental conditions, effectively expanding its ecological niche. In addition, this study highlighted the effectiveness of eDNA as an innovative method for detecting the distribution of invasive species. This study was carried out within the Interconnected Nord-Est Innovation Ecosystem (iNEST) and received funding from the European Union Next-GenerationEU (PNRR–MISSIONE 4 COMP. 2, INV. 1.5–D.D. 1058 23/06/2022, ECS00000043).