The taxonomy of Hysterothylacium genus in Mediterranean waters remains incomplete and unresolved. The aim of the current study was to investigate the morphological and molecular identification of selected species of Hysterothylacium larvae in marine fish from the Tunisian Mediterranean coasts. A total of 192 marine fish samples were examined. In total, thirty-seven third-stage larvae of Hysterothylacium were morphologically identified as Hysterothylacium type V. In the present study, representatives of this type from the Mediterranean Sea were genetically characterized for the first time by sequencing the rDNA ITS (ITS1-5.8S-ITS2) regions and mtDNA cox2 gene. This study represents the first report of Hysterothylacium type V from the Mediterranean Sea. We also report Mullus barbatus, M. surmuletus, and Pagellus erythrinus as new hosts for this larval type. Based upon molecular and phylogenetic analyses considering the rDNA ITS regions, the Hysterothylacium type V described here was classified as a new genotype, named Genotype B. The valid genetic data of the described Hysterothylacium type V in the present study can be used to establish the phylogenetic relationships among Hysterothylacium species from the Mediterranean Sea and worldwide for future research.
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