Molecular and histological identification of the protozoan parasite Perkinsus olseni in the blood cockle Anadara kagoshimensis (Tokunaga, 1906) occurring on the south coast of Korea

Abstract

A recent report on co-infection by two Perkinsus species in mud arks Anadara trapezia (Bivalvia Arcidae) in Australia implies possible Perkinsus infection in other Arcidae species. Blood cockle Anadara kagoshimensis has been exploited since the 1960s on the south coast of Korea, and the landings have decreased due to mass mortality in recent years. During a histopathology survey of blood cockle on the south coast, we identified a Perkinsus-like pathogen in the visceral mass. Accordingly, we applied Perkinsus species-specific PCR and subsequent sequencing to identify the pathogen to species level using internal transcribed spacer (ITS) rDNA. Ray's fluid thioglycollate medium (RFTM) and immunofluorescence assays (IFA) were also used to quantify and visualize the infection in this novel host. Perkinsus-like organisms were mainly found in the hemal space of visceral mass connective tissue. The Bayesian inference tree based on ITS rDNA sequences is strongly affiliated with P. olseni, suggesting that the Perkinsus-like organism in A. kagoshimensis is P. olseni, commonly found in Manila clams in Korean waters. RFTM and subsequent 2 M NaOH digestion assay revealed that the infection intensity ranged from 8000 to 1,481,000 cells/g gill, with a mean of 1.2 × 105 ± 2.5 × 105, and the prevalence was higher in September (fall) with 13.5% (101/750) than in June (spring) (0.4%, 3/750). Histology revealed that heavily infected clams exhibited tissue inflammation suggesting that P. olseni infection could damage the structure and physiological function of A. kagoshimensis; although we could not find a significant difference in condition index between the infected and uninfected individuals.