Multiple Blastocystis subtypes in Mediterranean marine turtles and cetaceans by amplicon-based NGS

Blastocystis is a genetically diverse enteric protist commonly found in humans and a wide range of vertebrate hosts. Although its prevalence and subtype (ST) distribution have been extensively studied in terrestrial ecosystems, its occurrence in marine organisms remains less known. In this study, we performed amplicon-based next-generation sequencing (NGS) to investigate, for the first time, the presence of Blastocystis in loggerhead sea turtles (Caretta caretta) and to expand existing data on ST diversity in cetaceans, stranded along the Italian Mediterranean coast. A total of 97 faecal samples were collected from 69 individuals of loggerhead sea turtles and 28 ceta- ceans. Blastocystis was detected in 44 % of the samples by real-time PCR—specifically in 39 loggerhead sea turtles and 4 fin whales (Balaenoptera physalus)—and further characterized by NGS. Ten STs were identified in loggerhead sea turtles and six in fin whales, with mixed infections frequently observed, particularly in turtles. Among the 18 STs detected, several represented new host records for marine organisms. ST4 was the most prevalent, especially in loggerhead sea turtles from the Tyrrhenian coast, and it exhibited a high degree of intra-subtype genetic varia- tion. Comparison of ST4 sequences from this study with those of terrestrial origin revealed a certain level of substructuring; however, the most common haplotypes were shared between marine and terrestrial sources, supporting the hypothesis of a terrestrial origin for the marine STs. These findings highlight the potential use of Blastocystis STs occurring in marine megafauna as ecological indicators of faecal pollution from terrestrial origin in coastal marine environment. Moreover, they underscore the importance of applying a One Health framework, supported by NGS technologies, to elucidate the transmission dynamics of Blastocystis STs among humans, terrestrial, and marine hosts.