Preliminary Research on Identification of Tetraselmis Parasite Unknown (TPX) in Tetraselmis sp.  

Tetraselmis sp. is a phytoplankton that is widely used as live feed for marine bivalves. Infection by an unidentified parasite-like organism (TPX) causes mass mortality in this phytoplankton species. We aimed to investigate whether TPX is a contagious pathogen. For this, 4 Tetraselmis groups were cultured for 10 days at 20°C under sunlight at an 18:6 h light:dark ratio. The cultured groups were as follows: uninfected Tetraselmis sp.; Tetraselmis sp. inoculated with formalin killed TPX; Tetraselmis sp. and TPX inoculated (positive control); and Tetraselmis sp. inoculated with frozen TPX. The cultured Tetraselmis sp. was monitored every 2 days for TPX infection by using an inverted microscope. To examine the infection rate, 10 photos were randomly taken to monitor changes in the number of normal cells except TPX cells. To identify the TPX, PCR analysis was performed on DNAs extracted from the uninfected Tetraselmis sp. control group, TPX infected Tetraselmis sp., and TPX uninfected Dunaliella tertiolecta (LB999). A protozoan-specific universal primer set (P-SSU-342: 5′-GCC TCT GTG GTT TGC TCC TT-3′; Medline B: 5′-TAC TGG GCG GCT TGG ATC TCG T-3′) and fungal universal primer set (nu-SSU 0817: 5′-TTA GCA TGG AAT AAT RRA ATA GGA-3′; nu-SSU 1536: 5′-ATT GCA ATG CYC TAT CCC CA-3′) were used for the PCR analysis. The PCR products were subjected to 1% agarose gel electrophoresis and expected amplified products of 1360 bp and 719 bp, respectively were confirmed. From the inoculation results, the uninfected and formalin-killed groups maintained 100% non-infected cells during the 10-day study period. However, in the frozen or live TPX infected groups, the number of normal cells gradually decreased from 100% to 48.43 ± 18.15% and 45.95 ± 3.29%, respectively. The 18S rRNA sequence was PCR amplified by only the universal fungal primer set in TPX infected Tetraselmis sp. and no DNA amplification by protozoan-specific universal primer set was noted. Consequently, we assumed that TPX found in Tetraselmis sp. is a contagious pathogen and, and the TPX detected in Tetraselmis sp. is presumed to be a fungus belonging to the phylum Chytridiomycota.