Azumiobodo Hoyamushi Research Articles

Regeneration of tunic cuticle is suppressed in edible ascidian Halocynthia roretzi contracting soft tunic syndrome.

Soft tunic syndrome is an infectious disease caused by the flagellate Azumiobodo hoyamushi, which severely damages the aquaculture of the edible ascidian Halocynthia roretzi. Tunic is a cellulosic extracellular matrix entirely covering the body in ascidians and other tunicates, and its dense cuticle layer covers the tunic surface as a physical barrier against microorganisms. When the tunic of intact H. roretzi individuals was cut into strips, electron-dense fibers (DFs) appeared on the cut surface of the tunic matrix and aggregated to regenerate a new cuticular layer in seawater within a few days. DF formation was partially or completely inhibited in individuals with soft tunic syndrome, and DF formation was also inhibited by the presence of some proteases, indicating the involvement of proteolysis in the process of tunic softening as well as cuticle regeneration. Using pure cultures of the causative flagellate A. hoyamushi, the expression of protease genes and secretion of some proteases were confirmed by RNA-seq analysis and a 4-methylcoumaryl-7-amide substrate assay. Some of these proteases may degrade proteins in the tunic matrix. These findings suggest that the proteases of A. hoyamushi is the key to understanding the mechanisms of cuticular regeneration inhibition and tunic softening.

Innate immunity in the edible ascidian Halocynthia roretzi developing soft tunic syndrome: Hemolymph can eliminate the causative flagellates and discriminate allogeneic hemocytes

The infection of the kinetoplastid flagellate Azumiobodo hoyamushi causes soft tunic syndrome that often results in mass mortality in the aquaculture of the edible ascidian Halocynthia roretzi. In the diseased ascidian individuals, the flagellates are exclusively found in the tunic matrix that entirely cover the epidermis, and never invade into internal tissues, such as a mantle. The present study for the first time demonstrated that the ascidian blood plasma and hemolymph have an activity to agglutinate and disintegrate the flagellates, suggesting the innate immunity protects the internal tissue from the invasion of A. hoyamushi. This activity is indifferent between the healthy and the diseased individuals. Allo-specific recognition and cytotoxic reaction among ascidian hemocytes, so-called contact reaction, occur among the individuals of healthy–healthy, healthy–diseased, and diseased–diseased combination, and therefore, the hemocytes from diseased individuals still retain the allo-reactivity. Moreover, the allo-reactive combinations are not changed under the presence of the flagellates, indicating the flagellates neither suppress nor induce the effector system of the contact reaction. These results suggest that the infection of A. hoyamushi does not impair the innate immunity in the ascidian hemolymph.

Measurement of Tunic Hardness in an Edible Ascidian, Halocynthia roretzi, with Remarks on Soft Tunic Syndrome.

The infection caused by a kinetoplastid flagellate, Azumiobodo hoyamushi, in an ascidian, Halocynthia roretzi, results in softening of the tunic, and finally death. This disease is usually recognized using palpation of the softening tunic, and A. hoyamushi infection is detectable using microscopy or PCR amplification of specific gene fragments. The present study is the first quantitative evaluation of the symptoms of soft tunic syndrome by measuring the amount of bending (bending) and the peak force required to pierce the tunic (force). There was a strong correlation between bending and force. Correlation analyses among other parameters (ascidian total weight, tunic thickness, and tunic water content) indicated that larger ascidians had harder and thicker tunics with a higher water content. As compared to the tunic of healthy individuals, softened tunic was thinner and had lower water content. Infected tunics thus possibly lose water and become softer and thinner. Mechanisms for maintaining the appropriate water level content may be crucial for preventing tunic softening.

Tunic extract of the host ascidian attracts the causal agent of soft tunic syndrome, Azumiobodo hoyamushi (Kinetoplastea: Neobodonida).

Azumiobodo hoyamushi, a kinetoplastid flagellate, is the causative agent of soft tunic syndrome, an infectious disease of the edible ascidian Halocynthia roretzi. The flagellate is thought to invade the tunic matrix via a damaged area of the tunic on the siphon wall. We hypothesized that the flagellate locates the tunic entry site by a chemotactic response to soluble substances diffused from the host ascidians. To investigate this hypothesis, we examined whether the flagellate shows a chemotactic response to tissue extracts (tunic and other tissues) from the host ascidian H. roretzi. We tested extracts from 5 tissues as well as hemolymph. Only the tunic extract showed significant positive chemotactic activity, and the activity decreased with increasing dilution. Furthermore, autoclaved tunic extract, extracts from diseased individuals, and extract from the styelid ascidian Styela clava also had chemotactic activity, although the activities were lower than that of tunic extract from healthy H. roretzi. Ultrafiltration of the tunic extract through a 3 kDa cutoff membrane completely abrogated the activity; the ultrafiltration retentate still showed activity. Thus, the soluble factors that attract the flagellate are present exclusively in the tunic extract, and the chemotactic factors are larger than 3 kDa. Our experiments also suggested that the tunic extract contains both heat-stable and heat-labile factors. We conclude that the flagellate locates the tunic entry site by chemotaxis toward soluble factors that diffuse from a damaged area of the tunic on the siphon wall.

Biochemical changes and drug residues in ascidian Halocynthia roretzi after formalin\u2013hydrogen peroxide treatment regimen designed against soft tunic syndrome

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 (H2O2) 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 H2O2 were detectable 1 h post-bathing; however, the compounds were completely undetectable after 48 h of bathing. Formalin–H2O2 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.

Global Kinetoplastea phylogeny inferred from a large-scale multigene alignment including parasitic species for better understanding transitions from a free-living to a parasitic lifestyle.

All members of the order Trypanosomatida known to date are parasites that are most likely descendants of a free-living ancestor. Trypanosomatids are an excellent model to assess the transition from a free-living to a parasitic lifestyle, because a large amount of experimental data has been accumulated for well-studied members that are harmful to humans and livestock (Trypanosoma spp. and Leishmania spp.). However, recent advances in our understanding of the diversity of trypanosomatids and their close relatives (i.e., members of the class Kinetoplastea) have suggested that the change in lifestyle took place multiple times independently from that which gave rise to the extant trypanosomatid parasites. In the current study, transcriptomic data of two parasitic kinetoplastids belonging to orders other than Trypanosomatida, namely Azumiobodo hoyamushi (Neobodonida) and Trypanoplasma borreli (Parabodonida), were generated. We re-examined the transition from a free-living to a parasitic lifestyle in the evolution of kinetoplastids by combining (i) the relationship among the five orders in Kinetoplastea and (ii) that among free-living and parasitic species within the individual orders. The former relationship was inferred from a large-scale multigene alignment including the newly generated data from Azumiobodo and Trypanoplasma, as well as the data from another parasitic kinetoplastid, Perkinsela sp., deposited in GenBank; and the latter was inferred from a taxon-rich small subunit ribosomal DNA alignment. Finally, we discuss the potential value of parasitic kinetoplastids identified in Parabodonida and Neobodonida for studying the evolutionary process that turned a free-living species into a parasite.

Methods to eradicate soft tunic syndrome (STS)-causing protozoa Azumiobodo hoyamushi, the highly infectious parasite from the edible ascidian (Halocynthia roretzi)

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 (H2O2) 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-H2O2 (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) + H2O2(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.

Disinfection of fertilized eggs of the edible ascidian Halocynthia roretzi for prevention of soft tunic syndrome.

Azumiobodo hoyamushi, the causative agent of soft tunic syndrome, was likely introduced to farming sites of the edible ascidian Halocynthia roretzi via ascidian spat. The source of infection is thought to be cysts of A. hoyamushi that reside in the substrates on which the ascidian spat are attached, but not the spat themselves. Thus, there is a need to develop methods to prevent contamination of the substrates with A. hoyamushi during seed production of the ascidian. We evaluated the protozoacidal effects of sodium hypochlorite and povidone-iodine against the flagellate and temporary cyst forms of A. hoyamushi. Additionally, we evaluated the effects of these disinfectants on the development of fertilized ascidian eggs. The flagellate form of A. hoyamushi was completely inactivated by povidone-iodine (5 ppm, 1 min) and sodium hypochlorite (1 ppm, 1 min). The temporary cysts of A. hoyamushi were completely inactivated by both disinfectants (5 ppm, 1 min). Disinfection with 50 ppm povidone-iodine for 15 min or 5 ppm sodium hypochlorite for 15 min had no effect on ascidian embryogenesis. Thus, horizontal transmission of A. hoyamushi via the substrates can be efficiently prevented by disinfecting ascidian eggs or tools used for spawning with povidone-iodine baths ranging from 5 ppm for 1 min to 50 ppm for 15 min without any side effects.

멍게 물렁증의 원인충인 Azumiobodo hoyamushi에 대한 유기산의 살충효과 연구

멍게 물렁증의 원인충인 Azumiobodo hoyamushi에 대한 유기산의 살충효과 연구

Seasonal variation in Azumiobodo hoyamushi infection among benthic organisms in the southern coast of Korea.

BackgroundRecent studies have reported that soft tunic syndrome (STS) in the edible ascidian Halocynthia roretzi is caused by the kinetoplastid parasite Azumiobodo hoyamushi. In this study, we attempted to detect and quantify the pathogen in benthic animals.MethodsFour species of ascidians, three species of echinoderms, two species of bivalves, one species each of sponge and algae, as well as seawater, were collected in 2014 and 2015 from an ascidian farm on the southern coast of Korea by SCUBA diving. Samples were collected from ascidian hanging culture ropes or the sea bottom. Inhalent siphons were excised for the analysis of ascidians, and soft body tissues were excised from the other species. Membrane filters were used to filter collected seawater. Tissues and membrane filters were analysed using culture testing, PCR testing, and qPCR diagnoses.ResultsOnly organisms belonging to Ascidiacea are susceptible to A. hoyamushi infection. The infection rate (% infected of the total number collected) and infection intensity (number of cells infected/g tissue wet weight) varied depending on the seasonal variation in seawater temperatures. Most ascidians examined were infected with A. hoyamushi and showed higher infection intensity in cold water seasons (April 2014 and February 2015), followed by a dramatic drop during warm water seasons (August and November, 2014). In addition, infection intensity of A. hoyamushi during the warm water period was higher in ascidians from the sea bottom than those from the hanging culture rope.ConclusionsAmong benthic organisms that inhabit the southern coast of Korea, most ascidians are susceptible to A. hoyamushi infection. Seasonal cycle of infection rates and intensities of the pathogen correspond well with the STS disappearance and onset cycle observed in ascidian farms. The high intensity of A. hoyamushi infection in the ascidians on the sea bottom of ascidian farms during summer suggest further studies on the role of the pathogen in resumption of STS occurrence in late fall or early winter in the southern coast of Korea.

Cellulose is not degraded in the tunic of the edible ascidian Halocynthia roretzi contracting soft tunic syndrome.

Soft tunic syndrome is a fatal disease in the edible ascidian Halocynthia roretzi, causing serious damage to ascidian aquaculture in Korea and Japan. In diseased individuals, the tunic, an integumentary extracellular matrix of ascidians, softens and eventually tears. This is an infectious disease caused by the kinetoplastid flagellate Azumiobodo hoyamushi. However, the mechanism of tunic softening remains unknown. Because cellulose fibrils are the main component of the tunic, we compared the contents and structures of cellulose in healthy and diseased tunics by means of biochemical quantification and X-ray diffractometry. Unexpectedly, the cellulose contents and structures of cellulose microfibrils were almost the same regardless of the presence or absence of the disease. Therefore, it is unlikely that thinning of the microfibrils occurred in the softened tunic, because digestion should have resulted in decreases in crystallinity index and crystallite size. Moreover, cellulase was not detected in pure cultures of A. hoyamushi in biochemical and expressed sequence tag analyses. These results indicate that cellulose degradation does not occur in the softened tunic.

Encystment and excystment of kinetoplastid Azumiobodo hoyamushi, causal agent of soft tunic syndrome in ascidian aquaculture.

Soft tunic syndrome in the edible ascidian Halocynthia roretzi is caused by the kinetoplastid flagellate Azumiobodo hoyamushi, which was found to assume a fusiform cell form with 2 flagella in axenic, pure culture. When the flagellate form was incubated in sterilized artificial seawater (pH 8.4), some of the cells became cyst-like and adhered to the bottom of the culture plate. The cyst-like forms were spherical or cuboidal, and each had 2 flagella encapsulated in its cytoplasm. Encystment was also induced in culture medium alkalified to the pH of seawater (8.4) but not in unmodified (pH 7.2) or acidified media (pH 6.4). More than 95% of the cyst-like cells converted to the flagellate form within 1 d following transfer to seawater containing ascidian tunic extracts from host ascidians. The cyst-like cells were able to survive in seawater with no added nutrients for up to 2 wk at 20°C and for a few months at 5 to 15°C. The survival period in seawater depended on temperature: some cyst-like cells survived 3 mo at 10°C, and ca. 95% of these converted to flagellate forms in seawater containing tunic extracts. Thus, A. hoyamushi is able to persist under adverse conditions in a cyst-like form able to adhere to organic and inorganic substrata for protracted periods of time.

Quantitative assessment of Azumiobodo hoyamushi distribution in the tunic of soft tunic syndrome-affected ascidian Halocynthia roretzi using real-time polymerase chain reaction.

BackgroundThe kinetoplastid parasite, Azumiobodo hoyamushi, is the causative agent of soft tunic syndrome (STS) in ascidians and leads to their mass mortality in Korean waters. This study was conducted to quantify A. hoyamushi density during the development of STS in the tunics of ascidians (Halocynthia roretzi) using real-time polymerase chain reaction (qPCR).FindingsThe infection intensity of A. hoyamushi, as measured by qPCR, varied depending on the part of the tunic analyzed, as well as the stage of STS development. The highest infection intensity was recorded in the tunics of the siphons. The infection intensity of A. hoyamushi in the siphons was only 2.9 cell/tunic (area, 0.25 cm2) or 106.0 cell/gram tunic (GT) in the early phase of STS, but this value increased dramatically to 16,066 cells/tunic (0.25 cm2) or 617,004 cell/GT at the time of death. The number of A. hoyamushi parasites increased gradually and their distribution spread from the siphons to the other parts of the tunics.ConclusionsqPCR enabled the quantitation of A. hoyamushi and the results revealed that parasite density increased as STS progressed. In addition, our results suggested that the siphons might function as the portal of entry for A. hoyamushi during infection.

Azumiobodo hoyamushi, the kinetoplastid causing soft tunic syndrome in ascidians, may invade through the siphon wall.

The infectious kinetoplastid Azumiobodo hoyamushi causes 'soft tunic syndrome', a serious problem in aquaculture of the edible ascidian Halocynthia roretzi. Infection tests using diseased tunics demonstrated that juvenile (0.8 yr old) individuals never developed soft tunic syndrome, but all individuals in the other age groups (1.8, 2.8, and 3.8 yr old) showed the disease symptoms. In the infection tests, tunic softening was first observed at the tunic around siphons. Based on ultrastructural observation of the inner wall of the branchial siphon, the tunic lining the inner wall in juveniles (0.5 yr old) was completely covered with cuticle, which had a dense structure to prevent bacterial and protist invasion. In contrast, the tunic was often partly damaged and not covered with cuticle in healthy adults (≥2.5 yr old). The damaged tunic in the siphon wall could be an entrance for A. hoyamushi into the tunic of adult hosts.

Quantitative assessment of Azumiobodo hoyamushi distribution in the tunic of soft tunic syndrome¿affected ascidian Halocynthia roretzi using real-time polymerase chain reaction

Quantitative assessment of Azumiobodo hoyamushi distribution in the tunic of soft tunic syndrome¿affected ascidian Halocynthia roretzi using real-time polymerase chain reaction

The kinetoplastid parasite Azumiobodo hoyamushi, the causative agent of soft tunic syndrome of the sea squirt Halocynthia roretzi, resides in the East Sea of Korea

Mass mortality of the edible sea squirt Halocynthia roretzi since the 1990s in the southern and eastern seas of Korea has caused large economic losses. The disease is characterized by symptoms of initially softened and thinned tunics that eventually rupture. Thus, the disease is called soft tunic syndrome (STS); however, the causative agent in these regions is unknown. In the present study, two kinetoplastid organisms were isolated from STS sea squirts collected from culture farms in Tongyeong located in the East Sea of Korea. Phylogenetic analysis of 18S rRNA sequences identified these organisms as Azumiobodo hoyamushi and Procryptobia sorokini. These kinetoplastids were injected into healthy sea squirts and cultured at 15°C for 13days. Sea squirts injected with A. hoyamushi showed 100% STS whereas, P. sorokini did not induce disease, thereby confirming A. hoyamushi as the causative agent of STS. A. hoyamushi flourishes in vitro at 10–15°C, and dies at temperatures below 5°C or above 20°C. The optimum salinity level for growth is 30–35psu, and death occurs below 25psu. These optima coincide with marine temperature and salinity levels between March and June on the southern coasts of Korea, the period when the syndrome occurs at the highest frequency. The identification here of A. hoyamushi as the causative agent of STS and our findings regarding its optimum growth conditions should lead to methods for reducing the incidence of STS.

Detection of the kinetoplastid Azumiobodo hoyamushi, the causative agent of soft tunic syndrome, in wild ascidians Halocynthia roretzi

The occurrence of soft tunic syndrome in wild populations of the ascidian Halocynthia roretzi was monitored by diving at 5 to 6 sites in Miyagi Prefecture in Japan in summer 2010 and 2011. These sites were located at varying distances from farming sites at which the disease had previously been detected. All dead ascidians were collected, and their tunics were examined for Azumiobodo hoyamushi, the causative agent of soft tunic syndrome, using 18S rRNA PCR. In both years, <1% of wild ascidians we observed (18 out of 2100 in 2010, and 30 out of 3100 in 2011) were dead. The flagellates were only detected in 8 out of 18 dead ascidians from 3 sites in 2010, and 4 out of 30 from 2 sites in 2011. Healthy ascidians were successfully experimentally infected with the disease by immersing tunic samples from diseased wild ascidians into the rearing water. When apparently healthy ascidians collected from the wild population were reared for 40 d using pathogen-free water, the tunics of some ascidians became softened. The flagellates were detected in these individuals, which were diagnosed with soft tunic syndrome. Our results suggest that soft tunic syndrome affects the wild population of ascidians in Japan.

In vitro and in vivo efficacy of drugs against the protozoan parasite Azumiobodo hoyamushi that causes soft tunic syndrome in the edible ascidian Halocynthia roretzi (Drasche)

It was discovered recently that infection by a protozoan parasite, Azumiobodo hoyamushi, is the most probable cause for soft tunic syndrome in an edible ascidian, Halocynthia roretzi (Drasche). In an attempt to develop measures to eradicate the causative parasite, various drugs were tested for efficacy in vitro and in vivo. Of the 20 antiprotozoal drugs having different action mechanisms, five were found potent (24-h EC50 <10mgL(-1) ) in their parasite-killing effects: formalin, H2 O2 , bithionol, ClO2 and bronopol. Moderately potent drugs (10<24-h EC50 <100mgL(-1) ) were quinine, fumagillin, amphotericin B, ketoconazole, povidone-iodine, chloramine-T and benzalkonium chloride. Seven compounds, metronidazole, albendazole, paromomycin, nalidixic acid, sulfamonomethoxine, KMnO4 , potassium monopersulphate and citric acid, exhibited EC50 >100mgL(-1) . When ascidians were artificially infected with A.hoyamushi, treated using 40mgL(-1) formalin, bronopol, ClO2 , or H2 O2 for 1h and then monitored for 24h, very low mortality was observed. However, the number of surviving parasite cells in the ascidian tunic tissues was significantly reduced by treating with 40mgL(-1) formalin or ClO2 for 1h. The data suggest that we might be able to develop a disinfection measure using a treatment regimen involving commonly available drugs.

멍게, Halocynthia roretzi 물렁증의 원인충인 Azumiobodo hoyamushi의 살충효과 평가를 위한 현미경계수법과 alamar blue assay 비교

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≤20 ㎍/㎖). The preliminary data can be used as a basis to develop anti-protozoal agents against A. hoyamushi.

Azumiobodo hoyamushi gen. nov. et sp. nov. (Euglenozoa, Kinetoplastea, Neobodonida): a pathogenic kinetoplastid causing the soft tunic syndrome in ascidian aquaculture

We used morphological and genetic analyses to investigate a pathogenic kinetoplastid isolated from a diseased edible ascidian Halocynthia roretzi with soft tunic syndrome. The morphological characteristics of the kinetoplastid are similar to those in the order Neobodonida in the subclass Metakinetoplastida. However, the presence of unique globular bodies distinguishes this kinetoplastid from the other polykinetoplastic genera (i.e. Cruzella, Dimastigella and Rhynchobodo) in this order. These globular bodies are cytoplasmic inclusions without an outer delimiting membrane and are composed of a homologous granular matrix containing electron-dense bands. A phylogenetic tree based on 18S rRNA gene sequences also indicated that the kinetoplastid belongs to the order Neobodonida, although it forms an independent clade in this order. From these results, we propose a new genus in the order Neobodonida, i.e. Azumiobodo gen. nov., and Azumiobodo hoyamushi as the type species for the genus.