Unraveling the Genetic Diversity of Lernaea Parasites Infecting Carp Through 18 S rRNA Gene Analysis.

Abstract

BACKGROUND: Lernaea spp., commonly referred to as anchor worms, are widespread ectoparasitic copepods known for their broad host range and high morphological variability, which complicates accurate taxonomic identification. PURPOSE: The hypothesis of this study was to accurately identified lernaea and differentiate at the species level using 18S rRNA gene sequencing, thereby overcoming the limitations and ambiguities of conventional morphological identification. METHODS: In this study, approximately 200 Lernaea specimens were collected from infected carps at fish farms in the Kasur district of Pakistan. Parasites were detached from hosts using potassium permanganate (KMnO₄) treatment and preserved in 70% ethanol for both morphological and molecular analyses. Initial morphological assessment was conducted under a stereomicroscope, focusing on key diagnostic features such as the structure of the anchor-shaped holdfast. For molecular characterization, genomic DNA was extracted using the Thermo Scientific GeneJET Genomic DNA Purification Kit. DNA quality and concentration were evaluated with a NanoDrop spectrophotometer. The partial 18S rRNA gene was amplified via polymerase chain reaction (PCR), and the products were visualized through agarose gel electrophoresis before sequencing. Sequence data were edited using BioEdit v7.0 and subjected to BLAST analysis against the NCBI GenBank database. RESULTS: Phylogenetic relationships were inferred using the neighbor-joining method, revealing the presence of three Lernaea species: L. cyprinacea, L. polymorpha, and L. ctenopharyngodonis. CONCLUSION: The findings underscore the effectiveness of molecular tools particularly 18S rRNA gene analysis in resolving morphological ambiguities and provide novel sequence data that helps to explain Lernaea diversity in Pakistan.