Isolation, characterization and virulence of Metanophrys sp. (Ciliophora: Scuticociliatida) from large yellow croaker (Larimichthys crocea) in China

In recent years, an outbreak of large yellow croaker (Larimichthys crocea) disease caused by unknown ciliate infections has occurred along the southeast coast of China, resulting in substantial economic losses. To investigate the taxonomic status and pathogenic characteristics of the ciliate, we isolated the ciliate from the brain tissue of naturally diseased L. crocea larvae, established in vitro monoclonal culture, carried out molecular and morphological studies, and observed the host histopathology. In the present study, we characterized three linked genes (ITS1-5.8S-ITS2, LSU rDNA, SSU rDNA) of the ciliate. The mitochondrial cytochrome oxidase subunit I (mt-COI rDNA) and mitochondrial small subunit ribosome DNA (mt-SSU rDNA) were also applied. The phylogenetic analysis based on ITS1-5.8S-ITS2, LSU, SSU, mt-COI, and mt-SSU rDNA molecular markers showed that the ciliate had the closest genetic relationship with Metanophrys sinensis, and the sequence similarity was 89.95%, 98.22%, 98.74%, 94.63%, and 94.08%, respectively. It was also clustered in a branch with M. sinensis on the phylogenetic tree. Morphological observation showed that the ciliate was melon seed shaped, with a slightly sharp front end and a blunt rear end; a tail cilium, approximately 10 um long; and an oval macronucleus. There were 10-11 somatic kineties, and mitochondria were distributed beneath the cell surface membrane, forming a mitochondrial system that conforms to the morphological characteristics of Metanophrys. After artificially infecting the healthy L. crocea with the ciliate, a large number of ciliates with identical molecular markers and morphology were isolated from the infected L. crocea. The fins and skin of infected fish were partially ulcerated, and the tail or fin base was white, which is the same as the symptoms of the above-mentioned naturally diseased L. crocea. In addition, the results of histopathological observation showed that the ciliate could cause local necrosis and dissolution of L. crocea muscle fibers, infiltration of inflammatory cells in muscle spaces, increased swelling cells in gills and skin, and vacuolation of liver and spleen cells. Therefore, the ciliate that causes the above disease can be classified into the genus Metanophrys, which has been isolated and identified for the first time.