Hyun-Ki Hong

Flow cytometry studies on the populations and immune parameters of the hemocytes of the Suminoe oyster, Crassostrea ariakensis.

The Suminoe oyster Crassostrea ariakensis has been attempted to be introduced in the Chesapeake Bay, USA, as an alternative to the eastern oyster Crassostrea virginica. Commercial production of Suminoe oysters is currently restricted due to the incomplete understanding of their biological, physiological and immunological nature. Accordingly, understanding immune system of C. ariakensis is crucial to prevent disease associated mortality and subsequent management of the Suminoe oyster. We investigated immunological activities and morphology of hemocytes of the Suminoe oyster using flow cytometry and light microscopy. Three types of hemocytes were identified in the hemolymph including hyalinocyte, granulocyte and blast-like cells. Hyalinocytes were the largest cells and the most abundant, while granulocytes were intermediate-size cell containing numerous granules in the cytoplasm. Blast-like cells were the smallest and least numerous. Flow cytometry revealed that the granulocytes are most active in the cell phagocytosis and spontaneous reactive oxygen species (ROS) production. The hyalinocytes also showed a certain level of the phagocytosis and oxidative activity but in a lesser extent than the granulocytes. In contrast, the blast-like cells did not show any phagocytosis or oxidative activity. The flow cytometry used in this study confirmed that as observed from other marine bivalves, the granulocytes are the main hemocytes involved in the cellular defence in the Suminoe oyster.

First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species, the disk abalone, Haliotis discus discus and the spiny top shell, Turbo cornutus

The disk abalone Haliotis discus discus and the spiny top shell Turbo cornutus are edible gastropod species of high economic value, mainly in Asia. Mortality outbreaks and variations in worldwide stock abundance have been reported and suggested to be associated, at least in part, with pathogenic infections. Ecology, biology and immunology of both species are currently not well documented. The characterisation of the immune systems of these species is necessary to further assess the responses of H. discus discus and T. cornutus to environmental, chemical and disease stresses. In the present study, we investigated the morphology and immune-related activities of hemocytes in both species using light microscopy and flow cytometry. Two types of hemocytes were identified in the disk abalone hemolymph, blast-like cells and hyalinocytes; whereas four main hemocyte types were distinguished in the spiny top shell, blast-like cells, type I and II hyalinocytes, and granulocytes. Flow cytometric analysis also revealed differences between cell types in immune-related activities. Three subsets of hemocytes, defined by differing lysosomal characteristics, were observed in the hemolymph of the spiny top shell, and only one in the disk abalone. Phagocytic activity was higher in H. discus discus hemocytes than in T. cornutus hemocytes, and the kinetics of PMA-stimulated oxidative activity was different between hemocytes of the disk abalone and the spiny top shell. Finally our results suggest for the first time a predominant mitochondrial origin of oxidative activity in gastropod hemocytes.

The known and unknown sources of reactive oxygen and nitrogen species in haemocytes of marine bivalve molluscs.

Reactive oxygen and nitrogen species (ROS and RNS) are naturally produced in all cells and organisms. Modifications of standard conditions alter reactive species generation and may result in oxidative stress. Because of the degradation of marine ecosystems, massive aquaculture productions, global change and pathogenic infections, oxidative stress is highly prevalent in marine bivalve molluscs. Haemocytes of bivalve molluscs produce ROS and RNS as part of their basal metabolism as well as in response to endogenous and exogenous stimuli. However, sources and pathways of reactive species production are currently poorly deciphered in marine bivalves, potentially leading to misinterpretations. Although sources and pathways of ROS and RNS productions are highly conserved between vertebrates and invertebrates, some uncommon pathways seem to only exist in marine bivalves. To understand the biology and pathobiology of ROS and RNS in haemocytes of marine bivalves, it is necessary to characterise their sources and pathways of production. The aims of the present review are to discuss the currently known and unknown intracellular sources of reactive oxygen and nitrogen species in marine bivalve molluscs, in light of terrestrial vertebrates, and to expose principal pitfalls usually encountered.

Assessment of immune parameters of manila clam Ruditapes philippinarum in different physiological conditions using flow cytometry

Cellular and humoral immune parameters are often used as biomarkers to trace environmental and physiological stresses in marine bivalves. In this study, we compared various immune parameters of Manila clams (Ruditapes philippinarum) under normal conditions and under a high level of desiccation, using flow cytometry. The immune parameters analyzed included, total hemocyte count, hemocyte mortality, hemocyte DNA damage, reactive oxygen species (ROS) production, and phagocytosis activity. Total hemocyte count, hemocyte DNA damage, and hemocyte mortality were significantly elevated among clams under high desiccation stress, while phagocytosis activity and spontaneous ROS production were significantly lower compared to those parameters of the control clams (p<0.05). These data suggest that the immune parameters analyzed in this study well reflect the physiological status of clams.

Comparative study on the hemocytes of subtropical oysters Saccostrea kegaki (Torigoe & Inaba, 1981), Ostrea circumpicta (Pilsbry, 1904), and Hyotissa hyotis (Linnaeus, 1758) in Jeju Island, Korea: morphology and functional aspects.

We first characterized the morphology and immune-related activities of hemocytes in the subtropical oysters Saccostrea kegaki, Ostrea circumpicta, and Hyotissa hyotis using light microscopy and flow cytometry. Hemocytes of these three oyster species were classified into three main types: 1) granulocytes containing numerous granules in the cytoplasm, 2) hyalinocytes with no or fewer granules, and 3) blast-like cells characterized by the smallest size and very thin cytoplasm. The percentage of each hemocyte population was similar in all species; hyalinocytes were the most abundant cell in the hemolymph accounting for more than 59%, followed by granulocytes (23-31%) and blast-like cells (3-5%). The size of granulocytes of S. kegaki was smaller (P < 0.05) than those of O. circumpicta and H. hyotis. Light microscopy also allowed the description of vacuolated cells characterized by large vacuoles in the cytoplasm. Flow cytometry analysis confirmed that the granulocytes of the three oyster species were the major hemocytes engaged in cellular defense with the largest lysosome content, and the most active phagocytosis activity and oxidative activity, as was previously reported in several marine bivalves. Phagocytic activity was the lowest in S. kegaki hemocytes, and PMA-stimulated oxidative activity was the lowest in H. hyotis hemocytes. Our results provide the basic information of hemocytes population of three subtropical oysters for further investigations associated with various environmental disease stresses.

Population dynamics of a high-latitude coral Alveopora japonica Eguchi from Jeju Island, off the southern coast of Korea

Although coral reefs are facing severe challenges from a variety of natural and anthropogenic stresses, there is anecdotal evidence that the high-latitude coral species Alveopora japonica Eguchi, 1968, has increased its population over the past two decades around Jeju Island, off the southern coast of Korea. The present study provides the first ecological data on this species. Alveopora japonica is opportunistically occupying the empty space left vacant following the recent kelp-forest decline. Colony abundance, age- and size-frequency distributions, lifespan, growth rates and biological characteristics such as surface area, weight and volume of two A. japonica populations in Jeju Island were investigated. Alveopora japonica around Jeju Island is characterised by a mean colony size of 30cm2, a slow growth rate (4.8mm year–1), and a short lifespan of 12–13 years, as determined by X-radiographic measurements. Alveopora japonica presented a dense population of 120 colonies m–2 on average. Population-age and -size structures at both sites reflected a healthy status and indicated a local stability, with a stationary size structure allowing population maintenance over time. The present study provided data to develop population-dynamics models to predict the potential outcomes of A. japonica populations to alternative management scenarios in Jeju Island.

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.

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.

Substantial changes in hemocyte parameters of Manila clam Ruditapes philippinarum two years after the Hebei Spirit oil spill off the west coast of Korea

Two years after the Hebei Spirit oil spill occurred off the west coast of Korea, we determined sub-lethal effects of the spilled oil on hemocyte parameters of Ruditapes philippinarum in the damaged areas. Clams in the spilled sites displayed unusually high proportion of granulocytes, which may result in higher phagocytosis capacity and reactive oxygen species production. Hemocytes in clams from the polluted sites also displayed less DNA damage and mortality than in the control site, possibly due to a faster phagocytosis of the impaired cells. Glycogen, the major energetic reserve, was depleted in clams from the spilled sites, potentially due to energetic consumption for maintenance of a large pool of granulocytes, detoxification processes and oxidative stress. Modified hemocyte parameters in clams in the spilled area, may reflect sub-lethal physiological stresses caused by the residual oils in the sediment, in conjunction with environmental modifications such as food availability and pathogens pattern.

Assessment of the fitness of the mussel Mytilus galloprovincialis two years after the Hebei Spirit oil spill

In December 2007, >150km of the West coast of Korea were heavily polluted by crude oil leaked from the oil tanker Hebei Spirit, leading to mass mortality of bivalve mollusks on the intertidal areas. Two years after, mussels Mytilus galloprovincialis were collected from two impacted sites to investigate sub-lethal effects of the oil spill. Tissue content in polycyclic aromatic hydrocarbons (PAHs), hemocyte parameters, reproductive status and energetic reserves were analyzed. PAHs in tissues of mussels as well as hemocyte parameters were not different between impacted and control sites. Energetic reserves were altered in mussels from the impacted sites. Glycogen content remained low at polluted sites, whatever the season. Two years after the Hebei Spirit oil spill, mussels then presented altered energetic metabolism. Further investigations are thus warranted to monitor the sustainability of mussel populations on the oil spilled West coast of Korea.