Internal transcribed spacer (ITS) barcoding reveals species differentiation and evolutionary lineages of Trypanorhyncha (Cestoda) in teleosts imported to Jordan from the Mediterranean and Red Seas.

Fish-borne trypanorhynch cestodes are socio-economically important marine parasites with veterinary and public health relevance, yet integrated molecular data from the eastern Mediterranean-Red Sea trans-boundary trading corridors remain scarce. This study utilized internal transcribed spacer (ITS) rDNA barcoding to characterize trypanorhynch larvae infecting commercial teleosts imported to Jordan from Egyptian marine fisheries. Larvae were isolated from 36 examined fish spanning five species: Solea aegyptiaca Chabanaud, 1927, Thunnus spp. South, 1845, and Xiphias gladius Linnaeus, 1758 from the Mediterranean Sea, alongside Plectropomus areolatus (Rüppell, 1830) and Epinephelus tauvina (Forsskål, 1775) from the Red Sea. The core ribosomal ITS cassette was PCR-amplified and sequenced, yielding 20 high-quality operational sequences that were evaluated alongside 101 global GenBank reference entries. Evolutionary and structural dynamics were resolved using distance-based Neighbor-Joining phylogeny, alignment-free 4-mer composition analysis, hierarchical Ward's clustering, and SHAP-based machine-learning classification. Seventeen plerocercoid isolates demonstrated clear genetic homology (>96.3% identity) to global Callitetrarhynchus gracilis references across both marine basins. Six isolates recovered from X. gladius displayed host-driven sub-clustering in the hierarchical dendrogram, positioning close to reference Molicola sequences while verified as C. gracilis via identity metrics. This result highlighting structural population patterns dictated by apex predatory hosts. Crucially, three highly divergent isolates (PV917196-PV917198) derived exclusively from S. aegyptiaca yielded a maximum BLASTN identity of only (~81.5%) to any public sequence, resolving as an independent basal group near the order Lecanicephalidea. Alignment-free machine-learning frameworks achieved perfect binary discrimination (1.000) accuracy) for these three isolates, unmasking systematic oligonucleotide shifts including significant enrichments in TGTG (1.87×) and CTGT (2.72×) motifs, confirming their status as a putative novel species. These findings demonstrate that combining ITS barcoding with alignment-free compositional profiling reliably unmasks host-driven population structures and flags cryptic lines within the Eucestoda, while providing new host and geographical distribution records for the region.