• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.3
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• pp.303-309
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• 1999

Mass mortality of the Manila clam, Ruditapes philippinarum has been reported all along the west and south coast of Korea for the past several years. As a pathogenic agent, Perkinsus sp., an endoparasitic protozoan has been identified in this study and believed to be responsible for the mass mortalities. Prevalence and infection intensity of Perkinsus sp. was investigated from a Manila clam population inhabiting at Komsoe Bay in the west coast where mass mortality of the clam has been reported. A total of 142 Manila clam, 50 oyster, Crassostrea gigas, 10 ark shell, Scapharca broughtonii, and 5 predatory gastropada, Rapana venosa were examined for the presence and the quantity of Perkinsus sp. Ray's fluid thioglycollate medium method (FTM method) with modified Mackin's infection intensity scale and Choi's quantitative method were used in detecting and quantifying the parasite. All individuals of R. philippinarum examined in this study were infected with Perkinsus sp., indicating $100\%$ prevalence while none of the oysters and the gastropods exhibited the parasite. Six to ten individual hypnospores of Perkinsus sp. were counted from the ark shells. The number of hypnospores in the clam tissues varied from 16,667 to 4,091,667, with a mean number of 1,077,628. Average infection intensity according to Mackin's was 2.87, indicating a moderate infection. A negative correlation was observed between the number of Perkinsus sp. in the tissue and the condition index, a ratio tissue wet weight to shell cavity volume. The clam size and the infection intensity in terms of total number of parasites were positively correlated; the bigger clam, the heavier infection. Such high number of Perkinsus sp. counted in the clams could be enough to cause physiological disturbance of clams, such as retarded growth and reproduction. It is also believed that such a high infection leads mortality of the clam via continuous draining of the energy by metabolic activities and reproduction of the parasites. Correlation between the condition index and the infection intensity observed in this study supports this hypothesis.

• The Korean Journal of Malacology
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• v.28 no.1
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• pp.65-71
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• 2012

Protozoan parasites belonging to the genus Perkinsus elicit severe inflammatory responses and are associated with mass mortality of commercially important marine shellfish worldwide. In the present study, we examined the external features of P. olseni zoospores in detail using light and scanning electron microscopy. Our study showed that the zoospores have an oval body with a long anterior flagellum and a short posterior flagellum. The anterior flagellum has a unilateral array of mastigonemes. Mean body dimensions were $3.37{\pm}0.33{\mu}m{\times}1.72{\pm}0.22{\mu}m$. The average length of the anterior and posterior flagella was $16.34{\pm}1.52{\mu}m$ and $8.25{\pm}1.39{\mu}m$, respectively. Zoospores of P. olseni found in Korean waters have shorter and narrower bodies, longer anterior flagella, and shorter posterior flagella than zoospores of Perkinsus spp. found in the mollusks of North America and Europe.

• Journal of fish pathology
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• v.23 no.2
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• pp.145-153
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• 2010

Prevalence of a protozoan parasite Perkinsus olseni in Manila clam Ruditapes philippinarum was surveyed from July to December 2009 on the west and south coast of Korea. P. olseni infection was diagnosed using two primer sets, P.olseni NTS Forward/P.olseni NTS Reverse set and PolsITS-140F/PolsITS-600R set in polymerase chain reaction(PCR). The results using PolsITS-140F and PolsITS-600R primer set was retained up to 60% at all stations from July to December, except for Padori. Especially, Goheung showed 100% prevalence from October to December. The results about comparison of the 4 station's DNA sequences which were analyzed from PCR products(457bp) using PolsITS-140F and PolsITS-600R primer set, there were only 2base differences at Sunjedo.

• Fisheries and Aquatic Sciences
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• v.3 no.3_4
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• pp.205-212
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• 2000

Light and electron microscopical characteristics of Perkinsus sp. parasitizing in Manila clam, Ruditapes philippinarum, in Korea were investigated. Trophozoite within the tissue was spherical or ovoid and ranged $2.5-10.5\mu m$ $(mean = 6.2\mu m)$ in diameter. Trophozoite had a nucleus with a prominent nucleolus and a large cytoplasmic vacuole within the cytoplasm. Single trophozoite was phagocytozed by host hemocyte and cluster cells were encapusulated by hemocytes aggregation within the host tissues. Hypnospores incubated in thioglycollate medium (FTM) for 1 to 15 days were also spherical or ovoid and ranged $10-132\mu m$ $(mean\pm S.D.\;:\;44.25\pm 7.91\mu m)$ in diameter. Zoospores were spherical or ovoid, had a nucleus and two flagella. Zoospores contained apical complex, which consisted of conoid, subpellicular microtubules, rhoptries and rectilinear micronemes.

• Proceedings of the Korean Aquaculture Society Conference
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• 2004.05a
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• pp.513-513
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• 2004

Manila clam, Ruditapes philippinarum, is commercially and ecologically important marine bivalve in Korea and japan. However, clam landings in the two countries have dramatically declined since the 1980-1990's. In the present study, the protozoan parasite, Perkinsus sp., lectin (host's defense-related glycoprotein) and histopathological features were investigated in Manila clams collected from Gomso Bay in Korea and Ariake Bay in japan (one of the largest clam beds in each country) during summer and fall, 2002-2003. DNA sequences of non-transcribe spacer (NTS), internal transcribed space. (ITS) and 5.85 rRNA of Perkinsus sp. were identical to those of P. atlanticus that was reported in Europe and Korea. For diagnosis of Perkinsus, the fluid thioglycollate medium (FTM) and the 2 M NaOH lysis methods were used. Prevalence of the parasite varied from 92.5-98.7% in Gomso Bay and 35.5-37.9% in Ariake Bay. Infection intensity, in terms of the number of Perkinsuscells per gram tissue wet weight, in the clams of Gomso Bay in fall 2002 averaged 1,010,077-470,937 recording approximately100 times higher than that of Ariake Bay, and these were twice higher than those of summer samples in each location. Mean hemagglutination titer of the clams from Gomso Bay was approximately 60-folds higher than that of clams from Ariake Bay in 2002. In histological preparation of the clams from Gomso Bay in 2002, trophozoites of P. atlanticus were in groups and resulted in severe inflammatory response of host clam. Prevalence of the trematod, Cercaria tapes-like in the clams of Gomso Bay and Ariake Bay were 8.8 % and 10.5% respectively. In conclusion, the clams from Gomso Bay showed more severe pathologic symptoms and higher immune response than those of the clams from Ariake Bay.

• The Korean Journal of Malacology
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• v.26 no.3
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• pp.211-215
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• 2010

The genus Perkinsus are parasitic protozoans that cause massive inflammatory responses in infected marine shellfish worldwide. This ultimately leads to great economic losses. This study examined the effects of water temperature and salinity on the formation of prezoosporangia and zoosporangia in order to understand the ecology of the pathogens. The induction of prezoosporangia from trophozoites occurred readily at higher water temperatures (20 and $30^{\circ}C$) and they had larger diameters than those incubated at lower temperatures (4 and $10^{\circ}C$). The formation of zoospores in prezoosporangia was also strongly influenced by water temperature and salinity; prezoosporangia exposed to water temperatures of 20 and $30^{\circ}C$ and salinities of 20 and 30 ppt had high rates of zoosporulation, while no or very low rates of zoosporulation were observed at temperatures below $10^{\circ}C$ or salinity below 10 ppt. Our data will be useful for the development of strategies to counter P. olseni proliferation in Korean waters.

• Proceedings of the Korea Society of Environmental Biology Conference
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• 2000.11a
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• pp.11-11
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• 2000

• Proceedings of the Korean Aquaculture Society Conference
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• 2006.05a
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• pp.434-436
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• 2006

• Proceedings of the Malacological Society of Korea Conference
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• 1999.11a
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• pp.12-12
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• 1999

• The Korean Journal of Malacology
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• v.27 no.4
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• pp.387-393
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• 2011

The objective of this study was to develop a real-time PCR method for the rapid detection and quantification of the protozoan pathogen Perkinsus olseni using a TaqMan probe. For the standard, genomic DNA was extracted from $10^5$ in vitro-cultured P. olseni trophozoites, and then 10-fold serial dilutions to the level of a single cell were prepared. To test the reliability of the technique, triplicates of genomic DNA were extracted from $5{\times}10^4$ cells and 10-fold serial dilutions to the level of 5 cells were prepared. The standards and samples were analyzed in duplicate using an $Exicycler^{TM}$ 96 real-time quantitative thermal block. For quantification, the threshold cycle ($C_T$) values of samples were compared with those obtained from standard dilutions. There was a strong linear relationship between the $C_T$ value and the log concentration of cells in the standard ($r^2$ = 0.996). Detection of DNA at a concentration as low as the equivalent of a single cell showed that the assay was sensitive enough to detect a single cell of P. olseni. The estimated number of P. olseni cells was similar to the original cell concentrations, indicating the reliability of P. olseni quantification by real-time PCR. Accordingly, the designed primers and probe may be used for the rapid detection and quantification of P. olseni from clam tissue, environmental water, and sediment samples.