Hyun-Sung Yang

Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a protozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decomposing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseni antibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2kDa. Immunofluorescent assay indicated that the rabbit anti-P. olseni IgG was specific to all life stages, including the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where P. olseni-associated clam mortality has recurred for the past decade. Purified cells were positively stained with the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time the presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1-2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes.

Growth, Reproductive Condition, and Digestive Tubule Atrophy of Pacific Oyster Crassostrea gigas in Gamakman Bay off the Southern Coast of Korea

ABSTRACT Spat of Pacific oysters (Crassostrea gigas) were collected from Gamakman Bay, Korea, and raised in a spat hardening facility located in the low intertidal zone of the bay for a “hardening/stunting” period of 10 mo. Seasonal changes in growth, reproductive condition, and digestive tubule atrophy (DTA) of these “hardened/stunted” oysters were monitored for more than a year after transplanting to a suspended longline system in a grow-out area in the bay. After transplantation, the hardened/stunted oysters showed a logarithmic increase in shell size for the first 4 mo, from June to October, and growth remained stable from late fall to early spring. During the 12 mo of the grow-out, the shell size of the hardened/stunted oysters increased from 15.4–74.2 mm, and tissue weight increased from 0.49–12.85 g. Histological analysis revealed that gametogenesis of hardened/stunted oysters commenced as early as February when water temperature remained at 10°C, and spawning occurred from July to September when water temperature reached 25–27°C. DTA assessed from histological analysis was higher from September to February, when the chlorophyll a level in the bay was lower. These data suggest that seasonal fluctuations in water temperature and food availability in the water column are the 2 main environmental parameters governing reproduction and growth of oyster in Gamakman Bay, and DTA could be a useful biomarker for monitoring the nutritional condition of oysters.

Seasonal Changes in Reproduction and Biochemical Composition of the Cockle, Fulvia mutica Reeve (1884), in Cheonsu Bay off the West Coast of Korea

ABSTRACT This is the first study of seasonal changes in gonad development and tissue biochemical composition of the cockle Fulvia mutica in Cheonsu Bay off the west coast of Korea. Gametogenesis commenced in December when the surface water temperature was 9.1°C. In May, the first discharge of oocytes was observed, and spawning continued until October. The condition index (CI), a ratio of tissue dry weight to shell dry weight, increased gradually from late May to July and declined dramatically in August and September, indicating that active discharge of gametes occurred in the bay during this period. Histology and the CI suggested that F. mutica spawning in Cheonsu Bay is continuous from early summer to early fall, with two spawning peaks in June and July, and September and October. All cockles examined in November were sexually inactive. The relatively low CI recorded in late fall to mid winter (November to January) coincided with high levels of ash and low levels of lipids and carbohydrates in the tissue, whereas protein levels remained unchanged. The observed spawning pattern of F. mutica in Cheonsu Bay suggests that the currently established timing for fishing closure, from July to September, should be expanded from June to October to protect earlyspawning broodstocks. This change may secure recruitment and lead to a subsequent increase of the wild cockle population in the bay.

Temporal Variation of Perkinsus olseni Infection Intensity in the Manila Clam Ruditapes philippinarum in Gomso Bay, Off the West Coast of Korea

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 Rays fluid thioglycollate medium assay and Chois 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.

FIRST REPORT ON THE ANNUAL GAMETOGENESIS OF HETEROMACOMA IRUS (HANLEY, 1845) IN A ROCKY INTERTIDAL AREA, NORTHERN JEJU ISLAND, KOREA

ABSTRACT The annual reproductive cycle of the Indo-Pacific tellinid clam Heteromacoma irus (Hanley, 1845) was investigated, using histology, for the first time in this study. The sex ratio of H. irus collected from a rocky intertidal area on the northern coast of Jeju Island, Korea, was found to be 1:1, indicating that H. irus is gonochoric. In January, most clams were in the resting phase, although a small number of females showed small oogonia on their follicular epithelium. From February to May, oocyte diameter increased rapidly, and fully mature eggs (44.8–56.8 &mgr;m) appeared in May. The first spawning males and females were observed in July, when the water temperature reached 18.0°C, and spawning activity continued until the end of August. During September and December, most clams were sexually in the resting stage. H. irus is considered to be a summer spawner, and annual gametogenesis was closely associated with the seasonal variation of the surface water temperature. Despite warm water temperatures in Jeju, the resting phase of female and male clams at the study site was prolonged from October to March, suggesting that food availability at this site could be poor and may act as a limiting factor in gametogenesis.

First report on the annual reproductive cycle of Burchardi's cockle, Acrosterigma (=Vasticardium) burchardi Dunker 1877 (Bivalvia: Cardiidae) on a subtidal sand flat off southern Jeju Island, Korea

Abstract We report for the first time on the annual gametogenesis of the Burchardis cockle, Acrosterigma (=Vasticardium) burchardi (Dunker, 1877) on a subtidal sand flat off southern Jeju Island, Korea. Seasonal changes in gamete development and oocyte size were investigated using histology. Intermingled ovaries and testis in the gonads located in the foot indicated that, like other cockles, A. burchardi is a functional hermaphrodite. In August, early developing oocytes and spermatogonia were observed in gonads suggesting that gametogenesis commenced when the water temperature reached 22.7–28.5EC. Early vitellogenic oocytes 8–10 μm in diameter appeared in gonads from September to November when water temperatures ranged from 16.9 to 22.2°C. Oocyte size increased exponentially from November (8 μm) to April (53 μm). Condition index dropped dramatically from May (0.51) to June (0.38), indicating that the Burchardis cockles off southern Jeju Island spawned during this period. A. burchardi spawning appeared to be synchronous, with the release of oocytes and sperm being observed only in May and June (16.4–22.8°C). The annual reproductive cycle of A. burchardi in Jeju Island could be summarized as (1) resting and early gametogenesis from August to January, (2) late gametogenesis from February to April, (3) ripe from March to May, (4) spawning in May and June, and (5) spent/resorbing in July and August.

Morphology and Immune-related activities of hemocytes of the mussel Mytilus coruscus (Gould, 1861) from East Sea of Korea

Mytilus coruscus is a cold water mussel commonly occurring on shallow subtidal rocky substrata along the east coast of Korea. Despite its abundance, there have only been a few studies conducted reporting on its biology and ecology. In this study, we investigated the immune-related activities of hemocytes of M. coruscus using light microscopy and flow cytometry. Based on the morphology and cellular activities, 3 types of hemocytes were identified, as granulocytes, hyalinocytes, and blast-like cells. Granulocytes, containing numerous intra-cytoplasmic granules, were intermediate-size (11.8μm) and the most abundant cells (70.2%), while hyalinocytes were the largest cells (16.0μm), with no or few granules in their cytoplasm. The granulocytes exhibited higher lysosomal content, phagocytosis activity and oxidative capacities than the hyalinocytes. In contrast, the blast-like cells were the smallest cells (6.4μm) and displayed a lack of phagocytosis and oxidative capacities. In M. coruscus, the granulocytes appear as the major cell type involved in cellular defense, whereas the blast-like cells may not be directly involved in cell-mediated immune response.

Spatial variation in the annual reproductive cycle of Turbo (Batillus) cornutus (Gastropoda: Trochidae) at Jeju Island, Korea

The annual reproductive cycle of the top shell Turbo cornutus from Jeju Island was investigated in two populations using histology. In the northern population, gametogenesis commenced in January as the surface water temperature reached 14 °C, while in the southern population gametogenesis began a month earlier, as the water temperature remained at 17 °C. Ripe top shells first appeared in June and spawning continued from June to October when water temperatures were between 20 and 24 °C. Histology indicated that the spawning period of the southern population was a month earlier and lasted longer (June–October) than in the northern population (July–September). The percentage gonad area of animals in the southern population in March and April was significantly higher than in the northern population (p < 0.05), suggesting that the gonads of the southern population matured earlier. The observed earlier maturation and the subsequent spawning of the top shells from the south coast of Jeju Island could be associated with the warmer water temperature and more favorable environmental conditions. The major spawning period of top shells observed in this study from July to September suggested that the currently implemented period of the fishing closure from June to August needs to be re-evaluated to protect more spawning adults in late summer.

Quantitative assessment of reproductive effort of the Manila clam Ruditapes philippinarum in a lagoon on Jeju Island (Korea) using enzyme-linked immunosorbent assay

We investigated gonad development and reproductive effort (RE) of the Manila clam Ruditapes philippinarum at Jeju Island, Korea. Gonad maturation and RE were determined using histology and an indirect enzyme-linked immunosorbent assay (ELISA). In June 2006, most of the clams (80%) in the lagoon were in the resting stage. Spawning clams first appeared in late July, and most clams spawned from early August to mid-September. The condition index increased gradually from early July to late August, then declined from early to mid-September, suggesting that spawning occurred during this period. The gonadosomatic index assessed by ELISA also increased dramatically from June (0.9), peaked in early August (19.7) then declined from late August to mid-September, indicating that clams at the study site had only one spawning pulse during the spawning period. Spawning at Jeju Island was one month later than Manila clams on the west coast of Korea. The delayed spawning and low RE of the clams could be in part, be explained by lower food availability, as the level of chlorophyll-a recorded in this study was much lower than that found in water from the west and south coast.

First report on the occurrence of the comb pen shell, Atrina pectinata (Linnaeus, 1767) (Bivalvia: Pinnidae) in Ulleungdo Island in the East Sea: Ecology and molecular identification of the species using COI gene sequence

Pen shell is one of the largest marine bivalves inhabiting shallow subtidal soft bottoms in the west Pacific and Indian Oceans. In Korea, the comb pen shell Atrina pectinata fisheries has been established on the south and west coasts. Recently, a pen shell population has been discovered from a subtidal sand flat (25–30 m depth) in Ulleungdo Island located in the East Sea of Korea, suggesting a potential shellfish resource in this area. In the present study, we first surveyed the population density and size of the unique pen shell using SCUBA, and identified the pen shell to species level using mitochondrial cytochrome oxidase I gene (COI) sequence. An underwater survey carried out from July to September 2013 revealed that populations of pen shell patched on subtidal sand flat at a depth of 20–25 m. Grain size analysis indicated that sand particles accounted for 99% of the 600 × 700 m sand flat. The underwater survey also indicated that density of the pen shell ranged between 6–19 ind/m2, with a mean of 11 ind/m2. Shell height (i.e. longest axis of the shell) of the pen shell on the sand flat varied between 17.2 cm to 28.8 cm, with a mean of 25.1 cm, and the age was estimated to range between 1.5–7.5 yrs, with a mean of 5 yr. COI DNA sequence obtained from the pen shell in this study showed 98.9–99.2% similarity to Atrina pectinata (Linnaeus 1767) reported from Japan. In the cluster analysis, the COI DNA sequence of the pen shells from Ulleungdo Island was grouped with A. pectinata reported from Japan and China, indicating that the pen shell discovered in this study was A. pectinata, commonly distributed on the west and south coasts of Korea.