Callinectes sapidus — coast to coast: Integrating stable isotope analysis and shotgun metagenomics to unravel trophic dynamics and microlitter ingestion across two Mediterranean sites

The increasing presence of microlitter in the marine environment poses a growing threat to aquatic organisms. This study investigates microlitter ingestion and trophic ecology of Callinectes sapidus from two populations of the Mediterranean basin: the Adriatic and Tyrrhenian Seas. To disentangle potential differences in feeding strategies between the populations, we adopted an integrated framework combining stable isotope analysis with shotgun metagenomic analysis of gastrointestinal contents, thus providing a complementary view of long-term trophic position and short-term dietary composition. Gastrointestinal analysis revealed microlitter ingestion in 39% of Adriatic and 50% of Tyrrhenian individuals, with 123 particles retrieved. Fibres dominated (94.3%), though composition varied regionally: Adriatic individuals ingested mainly cellulose-based microlitter (62.5%), whereas Tyrrhenian ones mostly synthetic polymers (61.4%). Eight chemical types were identified, with cellulose, polyethylene terephthalate, and resin-based polymers most abundant. Stable isotope analysis (δ¹⁵N and δ¹³C) indicated distinct trophic patterns: Adriatic population had higher δ¹⁵N (mean ± sd: 11.50 ± 2.27‰) and less depleted δ¹³C (-16.20 ± 1.52‰) compared to the Tyrrhenian one (δ¹⁵N: 9.01 ± 2.27‰; δ¹³C: -18.57 ± 0.88‰), suggesting region-specific feeding strategies. Shotgun metagenomics provided complementary information on prey composition, helping to characterise the opportunistic diet of C. sapidus. Overall, these findings highlight spatial differences in microlitter exposure and trophic dynamics, likely shaped by environmental availability and feeding behaviour. By integrating microlitter ingestion, stable isotope analysis, and metagenomics, this study provides insight into how C. sapidus interacts with anthropogenic and natural resources, emphasizing the feeding flexibility underlying its invasive success in Mediterranean Sea.