• Korean Journal of Environmental Biology
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• v.32 no.3
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• pp.225-233
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• 2014

In this study, we investigated the changes in the physiological and histological traits of a sea squirt (Halocynthia roretzi) with the emergence of the soft tunic syndrome induced by the water temperature control (6, 9, 12, 15, 18, 21, 24 and $27^{\circ}C$). It was observed that the induction rate of the soft tunic syndrome was highest at $15^{\circ}C$, but lowest at $24^{\circ}C$. Based on the tunic color condition and contraction strength, the whole process were classified into 4 stages as S0, S1, S2 and S3. Interestingly, there were significant differences in oxygen consumption and filtration rate were observed during S0-S3. The most distinctive aspects were change of blood cell composition at stage S3, whereas multi-vacuole cell ratio was decreased by 1/2 and morula cell ratio expanded about 10 times during S0-S3. Further, change of organ structure started following the syndrome such as degeneration of epithelial cells, microfilaments, increment in hemocytes and damage in muscle fiber have been detected in tunic, siphon, branchial sac, body wall musculature and pyloric gland. Briefly, our study results indicated that the normal physiological functions of the sea squirt can be affected due to the soft tunic syndrome induced by water temperature.

• Journal of fish pathology
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• v.34 no.1
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• pp.63-70
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• 2021

As a part of research to elucidate the pathogenesis of so called Soft Tunic Syndrome(STS), that caused mass mortalities in the cultured sea squirts, Halocynthia roretzi, the epidemiological and pathological analysis were done to both clinically normal and diseased groups of the farms of Tongyoung and Geoje coastal areas in southeast sea from February to July, 2008. In the histological finding of the tunic, most of individuals showed tunic softness syndromes that included the disarrangement and destruction of tunic fiber with the simultaneous presence of flagellates-like cells, recently suspected as main agents of tunic softness syndromes. Simultaneously, the intensive degenerative changes of the skeletal muscle of diseased sea squirts were recognized. The changes were characterized with the hyalinization and condensation of muscle fibril and hemocytic infiltration in the muscle fibers. Those were thought to be a kind of typical Zenker's necrosis as in the skeletal muscle of higher vertebrates. Besides of the diseased sea squirts, Zenker's necrosis of skeletal muscles were seen in the normal ones. Epidemiological inquiry for diseased groups revealed that the higher incidences of tunic softness syndrome were recorded in the fast growing groups and in the sites presuming the organic pollution. And Higher malondialadehyde(MDA) and glutathione peroxidase(GPx) activity were detected in the groups showing STS. Those results suggested that Zenker's necrosis of body muscles was a kind of"nutritional myopathy" by oxidative stress. Conclusively, it was considered that Zenker's necrosis of body muscles gives an important clue for elucidating pathogenesis of STS of cultured squirts. And it seems that the necrosis were caused by the oxidative stress to body muscle during abnormal rapid growth of sea squirts.

• Journal of fish pathology
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• v.24 no.3
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• pp.197-204
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• 2011

The edible ascidian Halocynthia roretzi is a commercially important fisheries resource in Korea. However, for the last several years, outbreaks of mass mortalities of the species have been occurring along the south and east coasts of Korea, where most ascidians are produced. Although it is known that tunic-softness syndrome is associated with these mortality events, the agent causing the syndrome has not yet been confirmed. To determine the agent causing tunic-softness syndrome, healthy and diseased ascidians were collected in March 2011 from Tongyeong, on the south coast of Korea, and were used for biological and pathological investigations. The results showed that diseased ascidians exhibited remarkably reduced body fluid, fatness index, and tunic index compared with healthy specimens. Interestingly, bi-flagellated protozoans were observed specifically in the tissue imprints and tunic cultures of diseased ascidians at an occurrence rate of 97.5%. Histological observation showed that the thickness of the tunics of diseased ascidians was reduced by half, and irregular structure and breakdown of the tunic fiber bundles were observed. In particular, flagellate-like cells were observed in the diseased ascidians. Our study clearly shows that bi-flagellated protists are present only in the softened ascidians, suggesting that the flagellates are partly or entirely associated with soft-tunic syndrome. Accordingly, further investigations to verify the effects of the flagellates found in the present study on soft-tunic syndrome should be conducted.

• Journal of fish pathology
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• v.22 no.3
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• pp.229-237
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• 2009

The causative agent for the tunic softness syndrome of the cultured ascidian Halocynthia roretzi from Jan 1999 to Feb 2009 was identified using virus isolation and polymerase chain reaction (PCR). The pathogenicity of the isolated virus MABV UR-1 strain was determined by experimental infection trials. The cytopathic effects was observed in CHSE-214 cell line at a level 5.1% (4/78) in normal ascidian and 1.8% in abnormal ascidian showing tunic softness syndrome signs. MABV gene was detected in 16.8% (18/107) of normal and 13.1% (5/38) of abnormal organisms by PCR. The ratio of MABV isolation and gene detection was similar level in normal and soft tunic diseased ascidian. Based on the VP2/NS junction region sequences, eight strains of virus isolated from ascidian, were included in the same genogroup with MABV which is originally isolated in wide ranges of marine fish and shellfish species. The UR-1 strain caused 60% mortality (36.5% mortality in control group) by immersion infection and 37% mortality (same mortality in control group) in injection infection indicating no significant differences in infected and control groups. These results suggest that ascidian can act as reservoir of the MABV, and this virus is not directly related with the ascidian mortality.

• Journal of fish pathology
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• v.28 no.3
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• pp.117-123
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• 2015

Economic loss by soft tunic syndrome of edible ascidian, Halocynthia roretzi has become a serious problem. Recently, it has discovered that the cause of this syndrome is infection by a protozoan parasite Azumiobodo hoyamushi. However, only a few studies have been conducted to control this parasitic disease. In a previous research, non-specific disinfectants have been found to be effective in controling the causative parasite. In an attempt to eradicate this causative parasite, organic acids were tested in this study to evaluate their in vitro and in vivo efficacy. In vitro tests showed that 8 different organic acids used in this study were moderately or highly effective with protozoan-killing effects ($EC_{50}=153{\sim}275{\mu}g/ml$). Despite weak in vivo penetration of organic acids into the tunic tissues, treatment with high concentration reduced the mortality of ascidian caused by infection the parasite, indicating that we might be able to develop a disinfection method using environmentally-friendly organic acids.

• Journal of fish pathology
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• v.26 no.1
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• pp.31-38
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• 2013

The edible ascidian, Halocynthia roretzi is a commercially important fisheries resource in Korea. However, there have been outbreaks of mass mortality due to soft tunic syndrome. It was discovered recently that the cause of death is infection by a protozoan parasite Azumiobodo hoyamushi. Alamar blue assay and microscopic counting were used to estimate anti-protozoal effects of 20 drugs having different action mechanisms. Through comparison of alamar blue assay and microscopic counting, 6 drugs were found to be potential in protozoan-killing effects: amphotericin B, formalin, hydrogen peroxide, bithionol, benzalkonium chloride, bronopol (24hr-$EC_{50}{\leq}20{\mu}g/ml$). The preliminary data can be used as a basis to develop anti-protozoal agents against A. hoyamushi.

• Fisheries and Aquatic Sciences
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• v.20 no.7
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• pp.12.1-12.7
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• 2017

Soft tunic syndrome (STS) is a protozoal disease caused by Azumiobodo hoyamushi in the edible ascidian Halocynthia roretzi. Previous studies have proven that combined formalin-hydrogen peroxide ($H_2O_2$) bath is effective in reducing STS progress and mortality. To secure target animal safety for field applications, toxicity of the treatment needs to be evaluated. Healthy ascidians were bathed for 1 week, 1 h a day at various bathing concentrations. Bathing with 5- and 10-fold optimum concentration caused 100% mortality of ascidians, whereas mortality by 0.5- to 2.0-fold solutions was not different from that of control. Of the oxidative damage parameters, MDA levels did not change after 0.5- and 1.0-fold bathing. However, free radical scavenging ability and reducing power were significantly decreased even with the lower-than-optimal 0.5-fold concentration. Glycogen content tended to increase with 1-fold bathing without statistical significance. All changes induced by the 2-fold bathing were completely or partially restored to control levels 48 h post-bathing. Free amino acid analysis revealed a concentration-dependent decline in aspartic acid and cysteine levels. In contrast, alanine and valine levels increased after the 2-fold bath treatment. These data indicate that the currently established effective disinfectant regimen against the parasitic pathogen is generally safe, and the biochemical changes observed are transient, lasting approximately 48 h at most. Low levels of formalin and $H_2O_2$ were detectable 1 h post-bathing; however, the compounds were completely undetectable after 48 h of bathing. Formalin-$H_2O_2$ bathing is effective against STS; however, reasonable care is required in the treatment to avoid unwanted toxicity. Drug residues do not present a concern for consumer safety.

• Fisheries and Aquatic Sciences
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• v.19 no.1
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• pp.1.1-1.6
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• 2016

Although soft tunic syndrome (STS) in the ascidian is a serious disease, helpful measures have yet not been established. It was examined in this study by applying aniti-parasitic drugs to eradicate the causative protozoa Azumiobodo hoyamushi from infected ascidians. Formalin was synergistic in killing parasites in vitro when co-treated with hydrogen peroxide ($H_2O_2$) or bronopol, but not with chloramine-T or povidone-iodine (PVP-I), when tested with in vitro parasite culture. The synergistic effects did not change when $formalin-H_2O_2$ (or bronopol) ratios were changed. It was found that treatment periods less than 60 min achieved a sub-maximal efficacy. Increasing drug concentration while keeping 30 min period improved anti-parasitic effects. Anti-parasitic effects of $formalin(F)+H_2O_2$(H) were also assessed in an in vivo STS model infected with cultured parasites. It was observed that combined 50 (40F + 10H) and 100 (80F +20H) ppm were effective in partially preventing STS-caused mortality. In horizontally transmitted artificial STS model, significant prevention of ascidian mortality was also observed after 50 ppm. Marked reduction of living parasites were noted after drug treatments in vivo. The results provide a highly useful basis to develop a preventive or treatment measure against the currently uncontrollable STS in the ascidian.

• Parasites, Hosts and Diseases
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• v.52 no.3
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• pp.305-310
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• 2014

Ascidian soft tunic syndrome (AsSTS) caused by Azumiobodo hoyamushi (A. hoyamushi) is a serious aquaculture problem that results in mass mortality of ascidians. Accordingly, the early and accurate detection of A. hoyamushi would contribute substantially to disease management and prevention of transmission. Recently, the loop-mediated isothermal amplification (LAMP) method was adopted for clinical diagnosis of a range of infectious diseases. Here, the authors describe a rapid and efficient LAMP-based method targeting the 18S rDNA gene for detection of A. hoyamushi using ascidian DNA for the diagnosis of AsSTS. A. hoyamushi LAMP assay amplified the DNA of 0.01 parasites per reaction and detected A. hoyamushi in 10 ng of ascidian DNA. To validate A. hoyamushi 18S rDNA LAMP assays, AsSTS-suspected and non-diseased ascidians were examined by microscopy, PCR, and by using the LAMP assay. When PCR was used as a gold standard, the LAMP assay showed good agreement in terms of sensitivity, positive predictive value (PPV), and negative predictive value (NPV). In the present study, a LAMP assay based on directly heat-treated samples was found to be as efficient as DNA extraction using a commercial kit for detecting A. hoyamushi. Taken together, this study shows the devised A. hoyamushi LAMP assay could be used to diagnose AsSTS in a straightforward, sensitive, and specific manner, that it could be used for forecasting, surveillance, and quarantine of AsSTS.