Morphological and Molecular Identification of Fish Lice ( <i>Argulus coregoni, A. follicaeus, A. japonicus</i> ) in Ornamental Freshwater Fish

Introduction: Fish lice of the genus Argulus are among the most important ectoparasites affecting freshwater ornamental fish, causing economic losses and threatening fish health. The present study aimed to identify and characterize three species of fish lice (Argulus coregoni, A. foliaceus, and A. japonicus) collected from ornamental freshwater fish in local markets. Methods: A total of 442 ornamental fish, including goldfish (Carassius auratus), koi carp (Cyprinus carpio), Black Moor, and other varieties, were examined for parasitic infestation. Morphological observations were carried out using a light microscope equipped with a Lusida digital camera at 40×magnification. Diagnostic features such as carapace shape, appendage arrangement, and oral structures allowed the identification of A. japonicus and A. foliaceus, whereas A. coregoni could not be reliably distinguished based on morphology alone. Molecular analysis was performed using PCR amplification of ribosomal DNA markers. The 18S rRNA gene (850 bp) was successfully amplified in A. coregoni and A. foliaceus, while the 28S rDNA gene (400 bp) was used for A. japonicus. Sequencing confirmed the species identity, and all obtained sequences were deposited in the NCBI GenBank with accession numbers. Results: BLAST analysis demonstrated high similarity between the local isolates and reference sequences from Iran, Indonesia, and India. Phylogenetic trees revealed that A. coregoni and A. foliaceus clustered closely with international isolates, while A. japonicus formed a separate branch, reflecting nucleotide variations in its 28S region. Conclusion: The integration of morphological and molecular approaches provided accurate identification of the studied Argulus species. The molecular data, supported by phylogenetic analysis and sequence registration in GenBank, not only confirmed species identity but also highlighted genetic relationships with isolates from other geographical regions. These findings contribute to the understanding of fish lice diversity in ornamental freshwater fish and provide a valuable reference for future parasitological and molecular studies.