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To search immune defense proteins in skin mucus of Japanese flounder fed with a diet containing high concentration of ascorbic acid, we carried out 2D-PAGE and compared the resolved pattern of proteins between control group that fed commercial diet and ascorbic acid supplemented group (AsA group) fed a diet supplemented with high concentration of ascorbic acid (2,000 mg/kg) for 7 days. The results revealed that there were many proteins exhibited distinct increase in AsA group. Among them, 6 regions that showed a dramatic elevation were chosen for protein identification using LC-MS/MS analysis and Mascot database search. Six proteins were identified, i.e. serotransferrin (Sero), transferrin (Trans), warm temperature acclimation-related 65 kDa protein (Wap65), complement component c3 (C3), hemoglobin beta-A chain (Hbs) and apolipoprotein A-1 (Apo). Quantitative RT-PCR analysis showed that the mRNA level of Hbs in epidermis of AsA group gave much higher increase (11.6 folds) than control group; the levels of Sero/Trans, Wap65, C3 and Apo showed no apparent difference between the two groups. The mRNA levels of wap65 and c3 in the liver and Apo in the kidney of AsA group exhibited significant increase in comparison to control group. In the case of secreted immunoglobulin M (IgM) and lysozyme (lyz), no difference of the mRNA levels of IgM in epidermis, gill, kidney, spleen and intestine, and lyz in epidermis, gill, spleen and intestine, was observed. The results of in situ hybridization confirmed the elevation of Hbs mRNA level in the epidermis tissue of AsA group. Our present study provided additional evidence showing the effectiveness of AsA in activating innate immune defense system in skin mucosal tissue of fish.

A new combination and a new species of onchobothriid tapeworm are described from triakid sharks. We found Platybothrium xiamenensis Wang & Yang, 2001 and Erudituncus musteli (Yamaguti, 1952) from Hemitriakis japanica (Muller & Henle). Based on the morphology of the hooks P. xiamenensis is transferred to the genus Erudituncus Healy, Scholz & Caira, 2001. The specimens studied by us differ from the original description in the number of proglottids and testes and in the size of the cirrus-sac. However, we consider them conspecific with E. xiamenensis due to the consistent hook morphology and laciniations in both descriptions and believe the differences reflect intraspecific variation. The type-host of E. xiamenensis was reported as Mustelus griseus Pietschmann. However, in the present study, this parasite was found only in H. japanica and never in M. griseus although many specimens of the latter host were examined. This suggests that the type-host in the original description has probably been misidentified. We found another undescribed species in M. griseus, Calliobothrium shirozame n. sp., which is distinguished from the congeners by having a unique combination of the number of laciniations: four in the cephalic peduncle, six in the immature proglottids and four in the mature proglottids.

In situvisualization of microbial communities within their natural habitats provides a powerful approach to explore complex interactions between microorganisms and their macroscopic hosts. Specifically, the application of fluorescence in situ hybridization (FISH) to simultaneously identify and visualize diverse microbial taxa associated with coral hosts, including symbiotic algae (Symbiodinium), Bacteria, Archaea, Fungi and protists, could help untangle the structure and function of these diverse taxa within the coral holobiont. However, the application of FISH approaches to coral samples is constrained by non-specific binding of targeted rRNA probes to cellular structures within the coral animal tissues (including nematocysts, spirocysts, granular gland cells within the gastrodermis and cnidoglandular bands of mesenterial filaments). This issue, combined with high auto-fluorescence of both host tissues and endosymbiotic dinoflagellates (Symbiodinium), make FISH approaches for analyses of coral tissues challenging. Here we outline the major pitfalls associated with applying FISH to coral samples and describe approaches to overcome these challenges.

Histological observations represent an important application to directly characterize the structure of tissues and locate host-associated and invasive microorganisms. Histological approaches have been widely employed in studies of the physiology, pathology and ecology of corals. However, the conventional preparation of histological sections requires decalcification of coral samples to remove the hard skeletal structure in a series of acid or EDTA solutions over a weeks’ period, causing potential disturbances in coral structures on prepared slides. In this study, we present an alternative method to prepare thin sections from undecalcified frozen coral tissues, which has been developed for retaining the structural integrity of histological sections including skeleton. Cross sections of normal and diseased (rapid tissue necrosis and black band disease) coral specimens prepared with this method displayed the complete structure of the coral fragments including soft tissues and skeleton structure at microscopic scales, thereby clearly showing the orientation of damaged tissues and associated microorganisms in relation to skeletal structures. This study demonstrates that the present cryosectioning method without the need for decalcification is a highly effective tool for in-depth understanding of coral tissue/skeleton interactions.

Aeromonas veronii is a Gram-negative opportunistic pathogen in fish. We have previously investigated the intestinal microbiota of carp Cyprinus carpio and revealed that most bacterial isolates with high mucus-binding capability were A. veronii. With regard to infection, mucosal adhesion of bacteria is regarded as a prerequisite for colonization and invasion. In the present study, we selected three A. veronii strains with different mucus-binding capability and pathogenicity, and compared the colonization capability and localization of these strains in the intestinal tract of carp, using an in vivo imaging system. We showed that only a pathogenic strain was mainly visible around the intestinal bulb and rectum of the intestinal tract at 48 h after administration. Frozen sections with fluorescence signals revealed that the administered bacteria were localized on the mucosal surfaces and in the lamina propria of the intestinal bulb, whereas they were mainly detected in the remaining intestinal contents in the rectum. These results suggest that the intestinal bulb is the primary region for A. veronii to invade and proliferate in healthy carp.

Fish genomes possess three type II interferon (IFN) genes, ifnγ1, ifnγ2 and ifnγ-related (ifnγrel). The IFNγ-dependent STAT signalling pathway found in humans and mice had not been characterized in fish previously. To identify the antiviral functions and signalling pathways of the type II IFN system in fish, we purified the ifnγ1, ifnγ2 and ifnγrel proteins of ginbuna crucian carp expressed in bacteria and found them to elicit high antiviral activities against crucian carp hematopoietic necrosis virus. We also cloned two distinct ifnγ receptor alpha chain (ifngr1) isoforms, 1 and 2, and stably expressed them in HeLa cells by transfecting the cells with ifngr1-1 or ifngr1-2 cDNA. When receptor transfectants were treated with the ligands in a one-ligand-one-receptor manner (ifnγ1 and ifngr1-2 or ifnγ2 and ifngr1-1), the stat1 protein was phosphorylated at both serine-727 and tyrosine-701 residues. Gel shift mobility analysis and reporter assay clearly showed that the specific ligand-receptor interaction resulted in the binding of the stat1 protein to the GAS element and enhanced transcription. Therefore, the actions of ifnγ1 and ifnγ2 were found to be mediated by a specific receptor for each signalling pathway via a stat1-dependent mechanism.

The Japanese eight-barbel loach Lefua echigonia, which is a freshwater fish native to Japan, is distributed from the Tohoku to Kinki districts and is divided into six regional populations according to mtDNA analysis. In this study, we investigated L. echigonia collected from several locations in Yamagata Prefecture and neighboring prefectures using mtDNA control region sequences and confirmed the spatial distribution pattern among the new regional population (Yamagata population). The new population was limited to the Mogami river system in the inland area of Yamagata Prefecture and is distinguished from other regional populations by high sequence divergences.

Aims: In the present study, we focused on one of the Aeromonas veronii isolates that exhibited marked adhesion onto carp intestine and studied its membrane-associated proteins for their possible involvement in mucosal adhesion. Methods and Results: We isolated a strain of Aer. veronii (CWP11) that exhibited a high degree of temperature-dependent adhesion activity onto carp intestinal tract and studied its adhesion factor. A proteomic analysis of the membrane-associated fraction showed the presence of multiple proteins that were specifically expressed in CWP11 cells cultured at 25°C. Of these, a 30 kDa protein was identified to be OmpA by a mass fingerprint analysis. Cloning and nucleotide sequencing of the ompA region of CWP11 revealed the presence of two tandem ompA homologues (ompAI-ompAII). Escherichia coli that expressed either OmpAI or OmpAII exhibited marked adhesion onto carp intestinal surface. Disruption of ompAI by a homologous recombination technique resulted in marked reduction of the adhesion activity in CWP11. Conclusion: The OmpA homologue plays an important role in the adhesion of the Aer. veronii strain onto the surface of intestinal tract. Significance and Impact of the Study: We successfully identified an OmpA homologue to be an adhesion factor of Aer. veronii, an optimistic pathogen that habituates in carp intestinal tract.

To elucidate the degradation process of the posterior silk gland (PSG) during metamorphosis of the silkworm Bombyx mori, tissues collected on the 6th day after entering the 5th instar (V6), prior to spinning (PS), during spinning (SP), and after cocoon formation (CO) were used to analyze macroautophagy, chaperon-mediated autophagy (CMA), and the adenosine triphosphate (ATP)-dependent ubiquitin-proteasome. Immediately after entering the metamorphosis stage at PS, the levels of ATP and phosphorylated p70S6 kinase protein decreased spontaneously and continued to decline at SP, followed by a notable restoration at CO. In contrast, phosphorylated AMP-activated protein kinase α (AMPKα) showed increases at SP and CO. Most of the Atg8 protein was converted to form-II at all stages. The levels of ubiquitinated-proteins were high at SP and CO, and low at PS. The proteasome activity was high at V6 and PS but low at SP and CO. In the isolated lysosome fractions, levels of Hsc70/Hsp70 protein began to increase at PS and continued to rise at SP and CO. The lysosomal cathepsin B/L activity showed a dramatic increase at CO. Our results clearly demonstrate that macroautophagy occurs before entering the metamorphosis stage and strongly suggest that the CMA pathway may play an important role in the histolysis of the PSG during metamorphosis.

Fish, like mammals, have several stages of wound healing, including inflammation, reepithelialization, proliferation, organization, and differentiation. However, little is known about the molecular mechanisms of fish skin wound healing. In the present study, 23 polymerase chain reaction (PCR) bands were identified which appeared to be specifically expressed during wound healing by a simple differential display (DD) method. Sequences of cDNAs from these 14 bands were highly homologous to known genes. However, reverse transcriptase PCR (RT-PCR) analyses of these 14 bands revealed that only one of them, the gene for matrix metalloproteinase 9 (MMP-9), was significantly expressed in wounded skin, and that the others were probably false positives. MMPs are known to have gelatinolytic activity, and film in situ zymography detected strong gelatinolytic activity in the surface and dermis of wounded skin of flounder.

To study the direct cause of liver enlargement in the Japanese flounder Paralichthys olivaceus infected with Edwardsiella tarda, the fish were challenged with E. tarda and reared without feeding. The liver of fish exposed to the bacteria was markedly enlarged compared to that of the controls while no severe histopathological change appeared in the organ during the experiments. No notable difference was observed in the crude fat, glycogen, and water content of the liver between challenged and control fish. The size of liver cells and nuclei of the challenged fish was apparently larger than that of the controls. Analysis of crude DNA in the liver suggested that the number of liver cells of starved control fish significantly decreased during the experiment while that of the challenged fish was maintained at a level of the initial control. RNA/DNA ratio of the liver of challenged fish clearly increased while it decreased in the control fish during the experiment. These observations suggest that liver enlargement of flounder infected with E. tarda, at least in the early stage of infection, is not a result of any readily observable histopathological changes and that E. tarda infection causes hypertrophy of the cells, as well as preventing decrease in liver cell number.

Flavobacterium columnare の実験感染がウナギの lgM 産生能に及ぼす影響について検討した結果, 血清総 lgM 量, 血清総タンパク質量に占める血清総 lgM 量の割合および lgM 産生細胞数が有意に減少した。 また, 当菌の主要病原因子と推定される菌体外産生物（ECP）を腹腔内接種した場合でも, ウナギの lgM 産生能は低下した。 ECP は, in vitro においてもウナギリンパ球に対して細胞毒性を示したことから, ECP がウナギの lgM 産生能の低下に重要な役割を果たしていると考えられた。

ニホンウナギの皮膚および飼育水から分離した282菌株の細菌を用いて, ウナギ皮膚粘液抽出物に対する凝集反応試験を行った。その結果, 皮膚分離細菌は, 飼育水分離細菌に比べ被凝集菌株が少なく, 凝集力価も低い傾向が認められた。また, ウナギ皮膚粘液レクチン（SML）に特異性をもつラクトースを用いて凝集阻害試験を行った結果, 両分離細菌ともに60%以上の菌株が阻害されなかった。これは, SMLとは別の細菌凝集素がウナギ皮膚粘液中に存在することを示している。

カツオ卵巣内の漿膜から卵巣腔側に至る結合組織間に Uvitex2B 染色で蛍光発色するほぼ円形の微胞子虫様寄生体の集塊が食細胞に貪食された状態で認められた。寄生体は胞子様構造の大きさが平均 1.22 μm のほぼ球形を呈し，微胞子虫類の一種と考えられた。529 個体中 518 個体に寄生がみられ，調査した 20 ヶ月間における採集月別の寄生率は 88～100% となり，寄生率に月別の規則性はみられなかった。

Nonspecific responses of Japanese eels to environmental stress were monitored by assaying various lytic activities in eel epidermal extract. In fish maintained at 10 and 30 °C for up to 10 days, epidermal proteolytic activities due to serine protease and aminopeptidase and hemolytic activity varied within a 2-fold value range. Other proteolytic activities, due to cathepsins B and L, in the fish at 30 °C increased for up to 8 days and were 3.4 and 2.9-fold over those in fish maintained at 10 °C, respectively. This was accompanied by a 3.0-fold increase in bacteriolytic activity. Other forms of stress were exerted on the fishes by immersing them in a suspension of Flavobacterium columnare or giving them intraperitoneal injections of Edwardsiella tarda over 72 h. Although serine protease and aminopeptidase activities and hemolytic activity in the fishes exposed to F. columnare changed marginally, and were similar to those in the control fish, cathepsins B and L activities in the infected fishes increased more than 1.5-fold over their initial values over a 48 h period, along with a 4.5-fold increase in bacteriolytic activity. No marked change was detected in any of the lytic activities of the fishes exposed to E. tarda. These findings indicate that epidermal cathepsins B and L probably participate in bacteriolysis associated with Japanese eel skin and that their activities are elicited by environmental stimuli and may be an important nonspecific response of eels. Abbreviations: Cbz – carbobenzoxy; MCA – 4-methylcoumaryl-7-amide.

Histochemical localization of proteolytic activities in the dorsal epidermis of Japanese eel was demonstrated by fluorescent microscopy utilizing 4-methoxy-2-naphthylamide (4M$\beta$NA) derivatives as substrates and 5-nitrosalicylaldehyde as a trapping agent. Carbobenzoxy-L-phenylalanyl-L-arginyl-4M$\beta$NA (Cbz-Phe-Arg-4M$\beta$NA) and Cbz-Arg-Arg-4M$\beta$NA were used for direct detection of cathepsins L and B activities, respectively, in fresh frozen sections and unfixed cells of the eel epidermis. The fluorescing areas, where Cbz-Phe-Arg-4M$\beta$NA was hydrolyzed by cathepsin L, were shown in mucus secretory cells and club cells and broadly around skin surface. The fluorescing areas due to Cbz-Arg-Arg-4M$\beta$NA hydrolysis by cathepsin B were localized similarly in these tissues. The fluorescing intensity for both catheptic activities in mucus secretory cells was higher than that in club cells, where small fluorescing granules were distributed. These results indicate that eel cathepsins L and B are stored in epidermal secretory cells at different levels and probably serve as defense factors before or after secretion by these cells. Abbreviations: Cbz – carbobenzoxy; 4M$\beta$NA – 4-methoxy-2-naphthylamide; NSA – 5-nitrosalicylaldehyde.

The existence of fish-specific isoforms of interferon (IFN)γ, known as IFNγ-related (IFNγrel), has been reported in several fish species. However, comparisons with deduced amino acid sequences of known IFNγrels among several fish species have indicated significant differences at the C-terminus basic amino acid continuous sequences, which indicate the existence of multiple IFNγrel isoforms. Two distinct cDNAs, encoding two IFNγrels, ifngrel 1 and ifngrel 2, were cloned from ginbuna crucian carp (Carassius auratus langsdorfii). Recombinant IFNγrel 1 and IFNγrel 2 have shown high antiviral activities against the lethal crucian carp hematopoietic necrosis virus. Both ligands exhibit biological activity as monomers despite the fact that the functional conformation of IFNγ is a homodimer. Both interferons have a high degree of sequence similarity, but differ in the C-terminus region. In this region, IFNγrel 1 contains a functional nuclear localization sequence which induces the translocation of green fluorescent protein from the cytoplasm to the nucleus. IFNγrel 2 lacks this sequence. These results indicate that IFNγrel 1 and IFNγrel 2 are functional antiviral cytokines. These structurally related ligands play distinct antiviral roles through different intracellular translocation mechanisms. Thus, IFNγrels form a novel, distinct subtype included in type II IFNs. The cyprinid fish IFNγ subtype currently consists of four members, including two IFNγ isoforms and two distinct additional IFNγrel isoforms specific to the fish.

C.columnaris ホルマリン死菌ワクチンを用いて, 浸漬法および注射法によるウナギに対する免疫処理の有効性を検討した. 体表をアルコール綿で損傷させた免疫魚を菌浴により攻撃した. その結果, 最終生残率は浸漬免疫区60％, 注射免疫区20％, 対照区0％であった. 浸漬免疫魚は感染の進行が遅く, 感染部位の皮膚にはその他の実験区の魚に比べて多数の白血球が観察された. また, C.columnaris に対する皮膚の付着阻害活性も浸漬免疫魚が最も高い値を示した.

To know whether caspases are involved in virus-induced apoptosis in fish cells, CHSE-214 cells were infected with viral deformity virus (VDV), which is a member of Birnaviridae. Some of the infected cells exhibited morphological features that are typical of apoptosis, although the ratio of these cells was less than 10% throughout the experiment. Caspase activities, especially that of caspase 6, were detected in infected cells, but not in uninfected cells. Activity for caspases 3 or 7 peaked later than those for the other caspases. These observations suggest the involvement of caspase cascade in virus-induced apoptosis in fish cells and functional differentiation among caspases.

Aims: The aims of the present study are to characterize the intestinal microbial community displaying a high-adhesive capability in fish, and to evaluate the relationship between mucosal adhesion of intestinal bacteria and fish health and disease. Methods and Results: A total of 707 aerobic bacteria isolated from carp intestine that were maintained under either feeding (feeding group) or no-feeding (no-feeding group) conditions and were performed adhesive assay. Isolates were divided into three categories on the basis of adhesive capability: high-, medium-, and low- adhesive capabilities. The average proportions of isolates with high-adhesive capability in the feeding and no-feeding groups were 30% and 32%, respectively. A phylogenetic analysis using a partial 16S rRNA gene demonstrated that most isolates with high-adhesive capability in both groups were classified as belonging to an Aeromonas group, and populations of isolates within high- and low-adhesive categories were markedly different. Conclusions: Intestinal bacteria with a high-adhesive capability in relation to intestinal mucous always colonize on the surface of intestinal mucosa and grow in the intestinal tract of feeding carp. The adhesive capability of intestinal bacteria is essential for colonization and growth in the intestinal tract of fish. Significance and Impact of the Study: Our results indicate that members of the Aeromonas group with adhesive capability always colonize on the surface of intestinal mucosa.

Japanese eel immunoglobulin M (IgM) was purified from the sera of Anguilla japonica immunized with Edwardsiella tarda FPU 347 and characterized. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions revealed that the eel IgM was a tetrameric protein with a molecular weight of 790 000; it contained an equimolar heavy chain and light chain with molecular weights of 72 000 and 25 000, respectively. While the N-terminal sequence of the heavy chain, VELTQPGSMVLKPGQSLTI, showed similarity to the variable regions of those of teleost fishes Igs, the N-terminal sequence of the light chain, DIVLTQSPAVQSVQLGDT, was similar to the variable regions of chondrostei and mammalian kappa chains. Lectin-binding assays showed that the binding of concanavalin A (Con A) to the Japanese eel IgM heavy chain was competitively inhibited by d -mannose and could be abolished by α-mannosidase treatment indicating the presence on the heavy chain of oligosaccharides, whose terminal were a bound mannoses. The average IgM concentration in the sera of the healthy eels was 3.4 mg ml−1; it amounted to 10.3% of the total serum protein.