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.

Extracellular proteases are released by ciliates in defined seawater microcosms.

The biodegradation of proteins in seawater requires various proteases which are commonly thought to be mainly derived from heterotrophic bacteria. We, however, found that protists showed a high protease activity and continuously produced trypsin-type enzymes. The free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium was isolated and used for microcosm incubation with different concentrations of killed bacteria as food for 10 days. The results showed that the co-existence of the ciliate with its associated bacterium produced a significant protease activity in both cell-associated and cell-free fractions while that in the associated bacterium only microcosm was negligible. The protease profiles are different between cell-associated and cell-free fractions, and a trypsin-type enzyme hydrolyzing Boc-Val-Leu-Lys-MCA was detected throughout the period in the presence of ciliates. This suggests that ciliates release proteases into the surrounding environment which could play a role in protein digestion outside cells. It has been previously suggested that bacteria are the major transformers in seawater. We here present additional data which indicates that protists, or at least ciliates with their specific enzymes, are a potential player in organic matter degradation in water columns.

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.