Temporal variation of Perkinsus olseni infection intensity in the Manila clam Ruditapes philippinarum in Gomso Bay, off the West Coast of Korea.
Article Type: Report
Subject: Clams (Diseases)
Alveolates (Research)
Authors: Yang, Hyun-Sung
Park, Kyung-Il
Donaghy, Ludovic
Adhya, Mausumi
Choi, Kwang-Sik
Pub Date: 08/01/2012
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: August, 2012 Source Volume: 31 Source Issue: 3
Topic: Event Code: 310 Science & research
Product: Product Code: 0913030 Clams NAICS Code: 114112 Shellfish Fishing SIC Code: 0913 Shellfish
Geographic: Geographic Scope: South Korea Geographic Code: 9SOUT South Korea
Accession Number: 303011391
Full Text: ABSTRACT Interannual variation of Perkinsus olseni infection intensity in the adult Manila clam Ruditapes philippinarum in Gomso Bay, off the west coast of Korea, was monitored from 1999 to 2000. Infection intensity of P. olseni (i.e., total number of P. olseni cells in unit tissue weight) was determined using Ray's fluid thioglycollate medium assay and Choi's 2-M NaOH digestion. In Gomso Bay, P. olseni monthly infection prevalence ranged from 83 (April 2000)--100%. It was remarkable that of the 18 mo of sampling, the prevalence remained at 100% in 12 sampling months. Infection intensity of P. olseni in Manila clam ranged 366,001 (July 1999)-2,235,325 cells/g wet tissue (October 1999). The infection intensity recorded in 1999 was significantly higher than the level measured in 2000, suggesting an interannual variation in the intensity (P < 0.05). A very high level of infection intensity observed during the fall (September, October, and November) coincided with a relatively low condition index and mass morality of clams in the bay. Our data suggest that the mass mortality of clams observed during late summer to mid fall in Gomso Bay could be, in part, explained by the high level of Perkinsus infection coupled with the poor physiological condition of clams during the postspawning season.

KEY WORDS: Manila clam, Perkblsus olseni, Ruditapes philippinarum, seasonality, infection intensity and prevalence, RFTM


Numerous studies have reported deleterious effects of Perkinsus sp. infection on some commercially important marine shellfish, including oyster, clam, and abalone (see reviews by Soniat (1996) and Villalba et al. (2004)). A high level of Perkinsus sp. infection often results in pathological symptoms such as tissue inflammation and necrosis, and mass mortality of the host organisms (Mackin 1962, Da Ros & Canzonier 1985, Chagot et al. 1987, Choi et al. 1989, Choi et al. 1994, Park & Choi 2004, Park et al. 2008). Heavy infection with Perkinsus was also reported to be responsible for retarded growth and gonad development, reduced reproductive effort, and depression of cellular defense activities (Dittman 1993, Choi et al. 1994, Ford & Tripp 1996, Paynter 1996, Ordas et al. 1999, Park et al. 2006a).

Since the first report of the occurrence of Perkinsus in Asian waters, Perkinsus infection has been reported from various locations with different

host animals, such as Manila clam Ruditapes philippinarum populations along the coastal Yellow Sea (Choi & Park 1997, Hamaguchi et al. 1998, Liang et al. 2001, Park & Choi 2001, Choi et al. 2002, Ngo & Choi 2004, Dungan & Reece 2006, Moss et al. 2008, Yang et al. 2010, Wu et al. 2011) and the Gulf of Thailand (Leethochavalit et al. 2003, Leethochavalit et al. 2004). In Korea, a Perkinsus--like pathogen was first identified in Manila clams on the west and south coast (Choi & Park 1997, Park & Choi 2001), and later the pathogen was confirmed as Perkinsus olseni (Park et al. 2005), and was also identified in another venerid clam, Protothaca jedoensis, in a small bay on the south coast (Park et al. 2006b). Although several studies have reported P. olseni infection in Manila clams, the investigations are limited to a certain season or month (Park et al. 1999, Ahn & Kim. 2001, Park & Choi 2001, Park et al. 2008). Accordingly, little is known about the seasonal variation of P. olseni infection in Manila clam, and about the influence of seasonal variation of environmental parameters in the progression and decline of the infection (Park et al. 2006a).

A high level of P. olseni infection in Manila clam has been reported in Gomso Bay, located off the west coast of Korea, during late summer. According to Park et al. (1999), the infection intensity of P. olseni in 2-3-y-old adult Manila clams was 16,777-4,091,667 cells/g tissue (mean, 1,077,628 cells/g tissue). Such a heavy infection was also observed in some clam culture grounds on the west and south coast of Korea (Choi & Park 1997, Park & Choi 2001), although temporal variation of the infection intensity and prevalence is poorly investigated (Park et al. 2006a).

In an attempt to understand seasonal dynamics of P. olseni infection in the Manila clam, we surveyed the infection intensity of P. olseni in the Manila clam for a 2 y using Ray's fluid thioglycollate medium assay (RFTM) and the 2-M NaOH digestion technique. The number of P. olseni cells in whole clam tissue was determined using RFTM and the 2-M NaOH assay, and the current study reports the annual variation of P. olseni infection intensity and the prevalence in Manila clam in Gomso Bay.


Gomso Bay, located on the west coast of Korea (Fig. 1), is one of the biggest commercial clam beds in Korea. It is characterized by a well-developed sand-mud tidal flat. For the analysis, 30-40 clams were collected monthly in 1999 and bimonthly in 2000. On arrival at the laboratory, clams were kept in a seawater tank for 24 h to depurate sediments in the stomach. Shell length (i.e., longest axis of the shell) and the wet tissue weight of each clam were recorded. Clams with a shell length more than 27 mm (i.e., age, >2 y) were included exclusively in the analysis because Perkinsus infection in the Manila clam is often size dependent (Park & Choi 2001, Villalba et al. 2004). The condition index (CI) was calculated as a ratio of wet tissue weight to shell length (Park & Choi 2004).


To determine the infection intensity, 10 mL fluid thioglycollate medium (FTM) was placed in a 15-mL plastic conical tube and sterilized. After cooling, 200 U mycostatin (nystatin) and 2 mg chloromycetin (chloramphenicol) dissolved in 50 [micro]L distilled water was added to each tube to depress bacterial growth during incubation. Whole tissue of each clam was then added to the test tube and incubated at room temperature (20-25[degrees]C) for a week in the dark (Ray 1952, Ray 1966). After incubation, the tubes were centrifuged at 760g for 10 min to discard FTM. Ten milliliters 2-M NaOH was then added to each tube and incubated at 50[degrees]C for 2 h to digest the tissue (Choi et al. 1989). The digested clam tissues were washed several times with phosphate-buffered saline (pH 7.3) by centrifuging at 800g for 10 min. The total number of P. olseni cells in each clam was then determined by counting the prezoosporangia in a 1/10 or 1/100 diluted clam subsample using a hemocytometer. The infection intensity was then expressed as the number of P. olseni cells per gram wet tissue.

To test seasonality in the infection intensity, the sampling months were grouped as spring (March, April, and May), summer (June, July, and August), fall (September, October, and November), and winter (December, January, and February). Analysis of variance (ANOVA) with Duncan's multiple range test was performed using SAS statistical analysis software to test the seasonality, and the test was run by year to determine interannual variation.


A total of 510 clams with a shell length that ranged from 30.0 (August 1999)-36.6 mm (July 1999), and a wet tissue weight of 1.052 (August 1999)-2.988 g (July 1999) were used in the analysis (Table 1). The monthly mean CI ranged from 3.435 (October 2000)-7.809 (July 1999; Fig. 2). CI increased gradually from February to July, then decreased dramatically in August in 1999 as a result of a massive clam spawning during this period.

Monthly prevalence (i.e., percentage of infected clam) of P. olseni infection in Manila clam ranged from 83 (April 2000)-100%, with a mean of 97%. The prevalence was remarkably high (100%) during most of the sampling periods. Because of the high prevalence, no clear seasonal pattern was observed in the prevalence during the 2 y of sampling.


Figure 3 shows seasonal changes in infection intensity, which ranged from 366,001 (July 1999)-2,235,325 P. olseni cells/g tissue (October 1999). ANOVA with Duncan's multiple range test showed that, in 1999, the infection intensity recorded in fall and spring was significantly higher than the value recorded in spring and summer (P < 0.001). In year 2000, the infection intensity measured in fall was significantly higher compared with other seasons, whereas P. olseni cells per gram tissue recorded in summer was significantly lower than the value recorded in any other season (P < 0.001; Table 2).


There was no clear seasonality in P. olseni prevalence in Gomso Bays as a result of the exceptionally high prevalence year-round, as was also reported in previous studies. From March 1999 to February 2000, Park et al. (2006a) investigated monthly prevalence and infection intensity of P. olseni in the Manila clam in Gomso Bay using RFTM and the 2-M NaOH digestion technique. In their study, the prevalence and infection intensity were assessed from the gill tissue, because other parts of the tissues were used in other biochemical analysis. Of the 14 mo of sampling, the monthly prevalence was 100% for 13 sampling periods, except December 1999 (70%) (Park et al. 2006a). Leite et al. (2004) also reported no obvious seasonal variation of P. olseni infection prevalence in the carpet shell clam Ruditapes decussatus along the Portuguese coast, where prevalence ranged from 20-100%. In contrast, Villalba et al. (2005) observed a clear seasonality in prevalence: high in spring and summer when the surface water temperature remained warm. Recently, Uddin et al. (2010) also observed a seasonal difference in the prevalence of P. olseni in the Manila clam in Incheon Bay, approximately 200 km to the north of Gomso Bay. In Incheon Bay, prevalence ranged from 38-97% annually, and was significantly higher in fall than in spring and summer, when most clam are in the postspawning stage.


Contrary to prevalence, infection intensity did show a clear seasonality. The infection intensity recorded in 1999 did show a different seasonal pattern, and the intensity recorded during the fall and winter was significantly greater than the values recorded during the spring and summer. In 2000, the different seasonal pattern was observed again, with infection intensity highest in the fall and lowest during the summer. It is remarkable that the infection intensity increased dramatically from August (486,827 cells/g tissue) to September (1,254,707 cells/g tissue) in 1999 (Fig. 3). Such a dramatic increase in intensity could be attributable to the warn: water temperatures during August and September, and to the poor physiological condition of the clams. The CI of clams in the fall is much lower than spring and early summer, and this difference is a result of the spawning activity of the clam (Fig. 2). According to Park and Choi (2004), the Manila clam in Gomso Bay spawns from late May to September, with a strong spawning pulse in late July. In the fall, most clams are spent reproductively or are in a resting phase (Park & Choi 2004). Several studies have reported mass mortalities or poor health conditions in marine bivalves during postspawning periods (Cheney et al. 2000, Costil et al. 2005, Royer et al. 2008). Using flow cytometry, Hong (2010) first investigated the immunological condition of the Manila clam inhabiting the west coast of Korea during the postspawning period. In October 2010, Manila clams collected from Goheung, off the south coast of Korea, were mostly spent (60%) or partially spawned (25%). Flow cytometry results indicated that hemocyte viability and the phagocytosis activity of clams that were spent or in the resting stage were poorer than those of clams in the ripe (or prespawning) stage. Therefore, it is believed that the dramatic increase of P. olseni infection intensity in the Manila clam from late summer to fall in Gomso Bay could be associated with the poor immunological condition of the clams during postspawning period, which might enhance proliferation of the parasites in clam bodies. It is also postulated that the extremely high number of P. olseni observed during the fall in Gomso Bay could be responsible for the autumn mass mortality of clams recurring in Gomso Bay.

The recurring mortality of clams during the fall in Gomso Bay is believed to be the result of complex interactions among the physiological conditions of the clam, P. olseni infection, and the environmental conditions in the bay. Quantity and quality of food available to the clan: have drawn little attention in relation to clam parasite interactions, although several studies have demonstrated that the food condition modulates a defense-related mechanism in the clam (Chu & La Peyre 1993, Chu et al. 1993, Hegaret et al. 2004, Delaporte et al. 2003, Delaporte et al. 2006a, Delaporte et al. 2006b, Delaporte et al. 2007). Choi et al. (1989) first estimated the energy consumption of Perkinsus marinus in the Eastern oyster in Galveston Bay and concluded that P. marinus in heavily infected oysters requires more energy than the oyster would have available after meeting its own metabolic demand. Casas (2002) also estimated the energy demand of P. olseni in the market-sized carpet shell clam R. decussatus. In Galicia Spain, the energy demand of the high level of P. olseni infection in the carpet shell clam exceeded the energy available to the clam for its own growth, especially under conditions of warm temperatures and low food availability. The negative energy balance caused by the progression of Perkinsus infection in the hosts could help to explain the lethal and sublethal effects (Villalba et al. 2004).

Depending on the season, the effects of an extremely high level of P. olseni infection observed during the spring and fall could reflect differently on clam physiology. According to Park and Choi (2004), there were late spring to early summer and fall chlorophyll a peaks (9-11 [micro]g/L) in Gomso Bay in 1999. Compared with spring and early summer, the chlorophyll levels were low (24 [micro]g/L) during late fall and winter 1999 and 2000. During spring and early summer 1999, Manila clams could have overcome physiological stresses caused by the heavy infection, because the food availability was high enough to compensate for the energetic cost of the infection. However, during late fall to winter, when food availability is relatively low, P. olseni would probably reclaim most of the available energy, inducing mortality of the most heavily infected clams. It is also likely that the spawning activities of clams during summer and early fall in the bay exhausted the clams, which may aggravate the defense capability of the clan. Consequently, the combined effects of the low level of food supply and the clam's poor physiological condition may facilitate the proliferation of P. olseni in clam tissues during fall and winter.

In summary, infection intensity of P. olseni in the adult Manila clam in Gomso Bay, off the west coast of Korea, was monitored for 2 y (1999 and 2000) using RFTM and the 2-M NaOH digestion technique. Monthly prevalence of P. olseni was mostly 100%, except for a few sampling months, and no clear seasonality was observed in prevalence. Infection intensity was significantly greater in fall and winter, when food availability in the bay was considered to be poor. It is believed that seasonal change in the infection intensity was governed externally by seasonal changes in water temperature and salinity, and internally by the annual reproductive cycle of the clam.


We are grateful to the staff members of the Shellfish Aquaculture and Research Laboratory at Jeju National University for their support in data acquisition. This study was supported by the research grant of Jeju National University (2008-2009) to K. S. C.


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(1) School of Marine Biomedical Science (POST BK-21) and Marine and Environmental Research Institute, Jeju National University, 66 Jejudaehakno, Jeju 690-756, Republic of Korea; (2) East Sea Environment Research Department, East Sea Branch of Korea Ocean Research and Development Institute, Uljin, Republic of Korea; (3) Department of Aquatic Life Medicine, Kunsan National University, Kunsan, Republic of Korea

* Corresponding author. E-mail: skchoi@jejunu.ac.kr

DOI: 10.2983/035.031.0312
Summary of the sampling effort.

Period          n           SL                   TWT

2/20/1999       36   34.1 [+ or -] 3.5   1.831 [+ or -] 0.694
3/20/1999       18   34.7 [+ or -] 2.8   2.329 [+ or -] 0.598
4/17/1999       58   33.5 [+ or -] 2.7   2.250 [+ or -] 1.101
5/15/1999       30   34.4 [+ or -] 4.7   2.622 [+ or -] 1.101
6/30/1999       30   35.8 [+ or -] 3.7   2.449 [+ or -] 0.777
7/18/1999       30   36.6 [+ or -] 5.0   2.980 [+ or -] 1.524
8/18/1999       16   30.0 [+ or -] 1.6   1.052 [+ or -] 0.289
9/1/1999        27   31.3 [+ or -] 3.3   1.565 [+ or -] 0.723
10/4/1999       21   32.7 [+ or -] 2.5   1.158 [+ or -] 0.283
11/23/1999      28   31.4 [+ or -] 3.1   1.380 [+ or -] 0.453
12/23/1999      25   34.1 [+ or -] 5.1   1.691 [+ or -] 0.837
1/15/2000       24   32.6 [+ or -] 3.9   1.490 [+ or -] 0.659
2/23/2000       22   35.8 [+ or -] 4.0   1.855 [+ or -] 1.024
4/20/2000       23   34.0 [+ or -] 4.8   2.604 [+ or -] 1.100
6/5/2000        50   33.3 [+ or -] 3.9   1.957 [+ or -] 0.797
8/3/2000        27   34.2 [+ or -] 4.4   2.160 [+ or -] 0.940
10/2/2000       17   31.2 [+ or -] 2.3   1.082 [+ or -] 0.293
12/16/2000      28   32.8 [+ or -] 3.6   1.523 [+ or -] 0.555

n, number of Manila clams used in the Ray's fluid thioglycollate
medium analysis; SL, shell length (mean [+ or -] SD in
millimeters); TWT, tissue wet weight (mean [+ or -] SD in grams).

Seasonal mean infection intensity measured in Perkinsus
olseni cells per gram tissue.

Year   Season   P. olseni (cells/g tissue)   P < 0.001

1999   Winter           1,225,633                 a
       Spring             857,533                 b
       Summer             582,937                 b
       Fall             1,447,062                 a

2000   Winter             819,541                 a
       Spring             944,529                 a
       Summer             597,784                 b
       Fall             1,330,850                 c

ANOVA and Duncan's multiple range test with significance
level at P < 0.001. Same letters are not significantly
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