Morphological and molecular characterizations for the developmental stages of Hysterothylacium species infecting Argyrops spinifer

Hysterothylacium species are perhaps the most abundant and diverse group of marine ascaridoids; however, their life cycle and specific identification in larval stages are difficult. In this study, three members of the genus Hysterothylacium (Ward and Magath, 1917) are morphologically described from the digestive tract of Argyrops spinifer, genetically characterized and their relationship with related taxa are compared and discussed. The former species (Hysterothylacium reliquens Norris and Overstreet, 1975) (p = 10%) is mainly characterized by its large body (male 30.89, females 34.15 mm long), the shape of lips, the presence of lateral alae, a short caecum, and a long ventricular appendix, length of spicules, number and distribution of genital papillae, and tail tip with numerous spines. The other species [fourth (L4) and third (L3) larval stages] (p = 46.66, 26.66%, respectively) are mainly characterized by body length (18.21 and 6.20 mm long, respectively), distinct three lips for L4 and poorly developed for L3, a shorter caecum than the ventricular appendix, tail tip lacks any distinct cuticular projections. Molecular tools were done via sequencing and analyzing target regions of ribosomal [internal transcribed spacer (ITS) and small ribosomal DNA (18S)] and mitochondrial [cytochrome c oxidase subunit 1 (COX1)]. These sequences demonstrate discrimination at the species level and confirm the validity of species determined by morphological identification. All sequenced parasite samples were identified as H. reliquens based on nucleotide sequence comparisons with other ascaridoids. In these analyses, Raphidascarididae formed a sister group to Anisakidae with strong nodal support. On MP trees, a genus-specific cluster with a well-support value was delineated in genera Raphidascaris, Contracaecum, and Hysterothylacium. This study demonstrated that selected gene regions of H. reliquens yielded unique sequences (gbl MZ148786.1, MZ148788.1, and MZ148789.1) that confirmed its taxonomic position in Raphidascarididae. Therefore, morphological and molecular tools are important for accurate diagnosis of genus and species of ascaridoids.