Kwang-Sik Choi

Spatial distribution of the protozoan parasite Perkinsus sp. found in the Manila clams, Ruditapes philippinarum, in Korea

Abstract Epizootic mortalities of Manila clams, Ruditapes philippinarum , have been reported along the west and south coast of Korea for the past several years. This is the first report on the spatial distribution of the infection, and infection intensity of Perkinsus sp. in Korean waters. Rays fluid thioglycollate medium technique (RFTM) and Chois 2 M NaOH digestion technique were used in detection and quantification of Perkinsus sp. Perkinsus sp. was detected in clams collected from 18 of 22 sites investigated along the west and the south coast of Korea. Perkinsus sp. cells were found mostly in connective tissues of gills, mantle, gonad and digestive gland while they were rarely observed in the foot and siphon tissues. The trophozoites were often enclosed in amorphous granules resulting from the cellular defense reaction of the clams. White nodules were often observed on the surface of heavily infected clam tissues as a result of inflammatory responses. Among the infected clams, infection prevalence ranged from 23% to 100%. Average infection intensity at each sampling site varied from 12 to 3,924,309 Perkinsus cells/individual or 6 to 873,000 cells/g tissue. Infection intensity and prevalence was much higher among the clams collected from commercial clam beds located on tidal flats along the west and south coast. In contrast, clams collected from sand beaches on the east coast and Weido and Kimnyong in Cheju Island were not infected by Perkinsus . It was believed that spatial distribution of Perkinsus sp. is in some way controlled by temperature, salinity and substrate type.

Enzyme-linked immunosorbent assay (ELISA) used in quantification of reproductive output in the pacific oyster, Crassostrea gigas, in Korea

Abstract Polyclonal antibody developed from egg proteins of the Pacific oysters, Crassostrea gigas , was used in quantification of the eggs. Quantity of the egg protein was measured using ELISA and a weight-normalized gonadosomatic index (GSI) was then established. Histology indicated that the oysters collected from Goseong Bay, Korea, in 2000 initiated gametogenesis as early as late February and spawned mostly in late June to early July when water temperature reached 23–25 °C. A second spawning peak was also observed in late August and the spawning activity continued to late October. Oyster egg proteins could be detected by ELISA in all months except March and November; a few oysters collected during December and January contained a measurable amount of eggs. The highest monthly mean GSI, 0.423, was observed in mid-June when the oysters were ready to spawn. The maximum GSI recorded in this study was 0.667. A positive correlation was found between oyster size and fecundity; the number of eggs increased as oyster biomass increased ( r 2 =0.7497). Our data suggest that oysters in Goseong Bay produce 3–196 million eggs during the spawning season. The immunological method developed in this study was sensitive enough to measure a small quantity of eggs and considered to be method of choice for studying bivalve reproduction.

Induction of G2/M arrest, endoreduplication, and apoptosis by actin depolymerization agent pextenotoxin-2 in human leukemia cells, involving activation of ERK and JNK

Pectenotoxin-2 (PTX-2) is a natural compound from marine sponges and has been known to inhibit cytokinesis through the depolymerization of actin filaments. To investigate the role of actin dysfunction by PTX-2 in human leukemia cells, we analyzed the effect of PTX-2 on the cell cycle and apoptosis. Cell cycle analysis showed that the depolymerization of actin with PTX-2 induces G2/M phase arrest at 12 h and endoreduplication at 24 h. Analysis of the cell cycle regulatory proteins demonstrated that PTX-2 increases phosphorylation of cdc25c and decreases the protein levels of cdc2 and cyclin B1. The M phase specific marker protein, phospho-histone 3, was also increased by PTX-2. Furthermore, p21 and CDK2, which are associated with the induction of endoreduplication, were also upregulated. PTX-2 also inhibited the growth of leukemia cells and caused a marked increase in apoptosis, as characterized by annexin-V+ cells and caspase-3 activity. Interestingly, we found that induction of G2/M phase arrest, endoreduplication, and apoptosis by PTX-2 is regulated by the extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) pathway. Inhibitors of ERK and JNK more increased the phosphorylation of cdc25c expression at G2/M arrest stages, and decreased p21 and CDK2 expression at endoreduplication stages and Bax expression at apoptotic stages in the presence of PTX-2. These molecular mechanisms provide that PTX-2 induces G2/M phase arrest, endoreduplication, and apoptosis through the ERK and JNK signal pathway via actin depolymerization.

Quantitative assessment of reproductive condition of the Antarctic clam, Laternula elliptica (King & Broderip), using image analysis

Summary We quantitatively assessed the reproductive state of the Antarctic clam, L. elliptica, collected from a small cove on King George Island in the spawning season. Using computer-based image analysis, percentage gonad area (PGA), percentage egg area within a follicle (FI), and mean oocyte diameter (MOD) were determined from gonadal cross-sections prepared for histological studies. These indices were then compared on the basis of scores from 1 to 6 on a conventional maturity index (MI), which is scored using subjective criteria determined by microscopic examination. Clams collected during two different spawning periods were analyzed. The FI and MOD values differed significantly between the two groups, but MI and PGA values did not, suggesting that FI and MOD are the more reliable and sensitive indicators for differentiating reproductive condition. This study also showed that a planimetric technique using computer-based image analysis is fast, convenient to use, and better than the conventional MI at providing reliable quantitative information about L. elliptica reproduction. We also investigated whether these indices varied with body size. The FI and MOD values peaked in clams of 76 to 85 mm shell length, indicating that clams of this size have the highest reproductive output at spawning time. Therefore, animals of a standard size (76 to 85 mm) should be monitored seasonally or over a longer term.

Lipid content and composition of the Antarctic lamellibranch, Laternula elliptica (King & Broderip) (Anomalodesmata : Laternulidae), in King George Island during an austral summer

Abstract Total lipid content, lipid classes and fatty acid composition were studied in various tissues of the Antarctic clam Laternula elliptica in an early austral summer. A histological examination of the gonads revealed that most of the clams examined were spawning or ready to spawn. Lipid content was highest in gills (14.9% of tissue dry weight), followed by gonads (10.9%) and digestive glands (9.9%), and averaged 8.2% for the soft tissues. The overall lipid contents were relatively low compared to temperate bivalves at a similar reproductive stage. Lipid class composition in the total lipid of L. elliptica was quite similar to those of most marine bivalves at lower latitudes, being dominated by triacylglycerols (19.3–41.4% of total lipids) and phospholipids (18.9–28.3%) in most of the organs. Large amounts of triacylglycerol deposits in non-reproductive tissues, particularly in siphon and gill, indicate a potential role of lipid as maintenance energy reserve, although the low lipid contents suggest that lipid may not serve as an energy reserve for any food-limited periods. Fatty acid composition in L. elliptica was also typical of marine bivalves with predominance of 16:0 (26%) and 20:5n-3 (18%) acids. Total fatty acids from the soft tissues showed a moderate level of unsaturation (50.6%), and about 35% of the total fatty acids were polyunsaturated. These values were not significantly different from, or even lower than those of marine bivalves in warmer waters. However, the content of 20:5n-3 (18.2% of total fatty acids), which dominated n-3 polyunsaturated fatty acids, was similar to those reported for other marine bivalve species in temperate waters. The fatty acid composition of L. elliptica reflected dietary input of some microalgal species. The nanoflagellates Cryptomonas spp., which were reportedly rich in 16:0, 18:3n-3 and 20:5n-3, predominated in the water column during the present investigation.

DEVELOPMENT OF A SAXIDOMUS PURPURATUS (MOLLUSCA: BIVALVIA) EGG-SPECIFIC ANTIBODY FOR THE QUANTIFICATION OF EGGS USING AN ENZYME-LINKED IMMUNOSORBENT ASSAY

Abstract We developed a polyclonal antibody from egg proteins of the butter clam, Saxidomus purpuratus, to quantify eggs using an enzyme-linked immunosorbent assay (ELISA). SDS-PAGE showed that the egg protein was composed of several peptides with molecular weights of 247, 200, 99, 91, 54 and 47 kDa. An immunoblotting assay indicated that the egg-specific antibodies were developed from the 200 and 99 kDa peptides. The rabbit anticlam egg IgG was able to detect as little as 0.078 μg/mL of S. purpuratus egg protein by ELISA. A weight-normalized gonadosomatic index (GSI, mg dry egg/mg dry tissue) was determined to follow the monthly changes in egg production of the clams. Clam-egg protein could be detected by ELISA during the entire sampling period (December to July), and GSI ranged from 0.035 (December) to 0.156 (February). The fecundity of the clams was measured from two spawning peaks in February and May and was estimated to be 22.6 million eggs (GSI of 0.16) in February and 16 million eggs in May (GSI of 0.15). The indirect ELISA used in this study was rapid, affordable and sensitive enough to assess minute amounts of egg protein, which is difficult to measure using traditional methods such as induced spawning or histology.