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ABSTRACT Following the increased mass mortality of Pinna nobilis populations in the Mediterranean, reliable protocols for the transport, maintenance, and controlled reproduction of this highly endangered species were drawn up within the European Life Pinna project. To test these protocols, the large Pinnidae Atrina fragilis, which shares similar habits to P. nobilis, has been used. In December 2022, a transport trial of nine specimens of A. fragilis from Trieste (NE Italy) to Camogli (NW Italy) was carried out. Two positioning (vertical and horizontal) of the specimens were tested inside the transport box. In the laboratory, after acclimatization, the specimens were placed inside three tanks and fed three times a week with a mix of live microalgae and artificial feed. The transport and maintenance protocols tested on A. fragilis were then applied to 11 P. nobilis collected in the Venice lagoon (NE Italy) and transported to the laboratory in June 2023. Due to the possible infection with Haplosporidium pinnae, considered one of the main etiological agents of mass mortality, P. nobilis individuals were evaluated through molecular analyses during their stay in the tank. Furthermore, these specimens were used as breeders: They spontaneously released already fertilized eggs, as a consequence of transport stress. Rapid larval development stopped at the early veliger stage, and the larvae were fed three times a week with a mixture of microalgae. After the reproduction period, the four specimens that survived 6 months in the laboratory, and constantly tested negative for H. pinnae, were transplanted to the Capo Mortola Marine Protected Area (Liguria, Italy) and monitored monthly.

Pinna nobilis, commonly known as the noble pen shell, is a marine bivalve endemic to the Mediterranean Sea. Unfortunately, due to a multifactorial disease that began affecting its populations in 2016, the species is currently facing the threat of extinction. To gain insights into the evolutionary history of P. nobilis before the mass mortality event (MME), and to obtain a comprehensive understanding of how evolutionary processes led to the adaptation of the species into the Mediterranean Sea, phylogenetic and phylogeographic analyses were carried out. The dataset analysed includes 469 sequences of COI gene fragment both from GenBank and the present study (100). The analysis performed evidenced that P. nobilis diverged about 2.5 mya, after the entrance of its ancestor into the Mediterranean Sea following the Zanclean flood (5.33 mya). Moreover, our results suggest that the starting point of colonisation was the central part of the western Mediterranean basin, with the eastern basin being populated subsequently. From a conservational viewpoint, these results provide important hints for present and future restocking plans, helping to reconstruct the pre-existing genetic variability in sites where the species became extinct.