Flavobacterium psychrophilum can infect rainbow trout Oncorhynchus mykiss and ayu Plecoglossus altivelis, detrimentally affecting global aquaculture. ​This study aimed to understand the infectivity and host specificity of two F. psychrophilum strains, SG950607 and PH0424, which were isolated from the kidneys of rainbow trout and ayu, respectively. ​Rainbow trout (fry) and ayu (adult and fry) were infected via two methods: bath immersion and intraperitoneal injection. ​We investigated the ability of each F. psychrophilum strain to survive in the serum of non-original host fish species. ​Rainbow trout died upon being infected by PH0424. ​In addition, the complement of the rainbow trout did not show a bactericidal effect against PH0424. ​On the other hand, ayu infected with SG950607 survived, and their complement eliminated this strain. ​The difference in mortality rates is likely associated with the susceptibility of each strain to the complement activity possessed by the host fish.

A direct relationship exists between diverse corals and fish farming in Keten Bay, Amami-Oshima, Japan. The release of coral mucus has a significant impact on the microbial activity of surrounding seawater. To obtain a more detailed understanding of biogeochemical cycles in this environment, the effects of coral mucus on the community structure and function of bacteria in surrounding seawater need to be elucidated. We herein used a bromodeoxyuridine approach to investigate the structures and functions of bacterial communities growing close to mucus derived from two different Acropora corals, AC1 and AC2. The alpha diversities of actively growing bacteria (AGB) were lower in mucus-containing seawater than in control seawater and their community structures significantly differed, suggesting that the growth of specific bacteria was modulated by coral mucus. Rhodobacteraceae and Cryomorphaceae species were the most dominant AGB in response to the mucus of Acropora AC1 and AC2, respectively. In contrast, the growth of Actinomarinaceae, Alteromonadaceae, Flavobacteriaceae, and SAR86 clade bacteria was inhibited by coral mucus. The results of a Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) ana-lysis suggested that the predicted functions of AGB in mucus-containing seawater differed from those in seawater. These functions were related to the biosynthesis and degradation of the constituents of coral mucus, such as polysaccharides, sugar acids, and aromatic compounds. The present study demonstrated that complex bacterial community structures and functions may be shaped by coral mucus, suggesting that corals foster diverse bacterial communities that enhance the ecological resilience of this fish farming area.

We investigated the succession process of intestinal bacteria during seed production in full-cycle aquaculture of Pacific bluefin tuna (Thunnus orientalis). During the seed production, eggs, healthy fish, rearing water, and feeds from three experimental rounds in 2012 and 2013 were collected before transferring to offshore net cages and subjected to a fragment analysis of the bacterial community structure. We identified a clear succession of intestinal bacteria in bluefin tuna during seed production. While community structures of intestinal bacteria in the early stage of larvae were relatively similar to those of rearing water and feed, the bacterial community structures seen 17 days after hatching were different. Moreover, although intestinal bacteria in the late stage of larvae were less diverse than those in the early stage of larvae, the specific bacteria were predominant, suggesting that the developed intestinal environment of the host puts selection pressure on the bacteria in the late stage. The specific bacteria in the late stage of larvae, which likely composed ‘core microbiota’, were also found on the egg surface. The present study highlights that proper management of the seed production process, including the preparation of rearing water, feeds, and fish eggs, is important for the aquaculture of healthy fish.

Flavobacterium psychrophilum can be divided into three genotypes, G-C type (ayu type), A-C type (multi-fish type), and A-T type (salmon-trout type), by two single-nucleotide polymorphisms in the DNA gyrase subunit A (gyrA) gene. We isolated F. psychrophilum from various samples collected from the lower basin of a river flowing into Lake Biwa, as a model for bacterial survey, in Shiga Prefecture in June, September, and December from 2010 to 2013 and investigated their gyrA genotypes. All three types of F. psychrophilum were isolated in June when ayu went up the river from the lake. In September, ayu gathered in the lower river basin for spawning and became high in density, almost all of the isolates were of the G-C genotype and the A-T genotype was never isolated. In December, only the A-T type was isolated from the river samples, when Biwa trout Oncorhynchus masou rhodurus came to the river to spawn and were present in the sampling area. In accordance with the seasonal change of the host fish species in the river, the genotype of F. psychrophilum isolated from the environment seems to have changed as well.

The possible impacts of resuspension of low-density bottom sediments on the microbial decomposition process of organic matter were investigated at a coastal fish farming area. Hydrolysis and mineralization rates were much higher in sinking particles, resuspendable particles, and bottom sediments than in seawater. The cell-specific mineralization rate of free-living bacteria in seawater was several times higher than that of particle-associated bacteria at the other three sites. Conversely, no significant difference was observed in the cell-specific ecto-enzymatic hydrolysis rate. These results indicated different strategies in the utilization of organic matter: free-living bacteria actively respire low-molecular-weight compounds produced from the high-molecular-weight compounds resulting from extracellular enzyme activity of particle-associated bacteria. Both hydrolysis and mineralization were higher in sinking particles than in the other three sites. Hydrolysis rates were higher in resuspendable particles than in bottom sediments. Furthermore, leucine aminopeptidase and mineralization rates tended to be highest during winter in all four sites within the water column. These results suggest that the microbial decomposition of organic matter is stimulated by the resuspension of bottom sediments, especially during winter, when vertical mixing is relatively strong.

 We explored the impact of combined sewer overflow (CSO) on the water quality of an urban river. We characterized the bacterial community of the Higashiyokobori-gawa waterway using next-generation DNA sequencing (Illumina Miseq). We examined bacterial 16S rRNA gene amplicons during rainfall periods. When rainfall exceeded 4 mm/h at our experimental site on the waterway, the bacterial community composition in the river water exhibited transient changes attributable to the CSO. The bacterial community composition in the waterway differed greatly from those in sewage, suggesting that the CSO had been diluted with bulk rainwater prior to discharge into the river. After a newly constructed stormwater storage pipe called Heisei-no-Taikougesui was commissioned, we found no changes in the bacterial community in river water even when rainfall exceeded 8 mm/h. This indicates that the storage pipe effectively prevented CSO discharge into the waterway. We suggest that bacterial community composition analysis is useful when evaluating/monitoring water quality.

A major concern in larvae production is a mass mortality caused by fish diseases. In larvae production, pumped-up natural seawater filtered through a sand filter system is used for fish rearing, and microalgae and rotifer cultures. Here, we investigated the community structures of eukaryotic microbes, as well as total bacteria and vibrios, in various processed waters' used in a larvae production site. We observed that ultraviolet irradiation of seawater was effective to reduce not only total bacteria and vibrios but also eukaryotic microbes. Moreover, the community structures of total bacteria and vibrios in rearing waters for fish larvae were different from those in rotifer cultures fed with Chlorella, but rather similar to those in natural seawater and microalgae cultures. These results suggest that the bacterial community in rearing waters may originate mainly from natural seawater and then be selected by microalgae in rearing water. Overall, this study provides useful information for avoiding the risk of fish disease outbreaks in a larvae production site.

We investigated the influence of solar ultraviolet radiation (UVR) on microbial mineralization activity in three tidal flats located in Osaka Bay, Japan. Within each sampling site, two experimental areas were defined: one area was covered with a transparent acrylic resin board (area without solar UVR) and the other was set as the control area (not manipulated, area with solar UVR). At low tide, UVR and sediment surface temperature were measured in situ and the top 10 mm of the sediment (oxic layer) was core sampled in summer, autumn, and winter in 2003, 2004, and 2005. Each core sample was then examined for potential microbial mineralization activity using C-14-labeled glucose. In the summer, mineralization rates in the areas without solar UVR were significantly higher (by a factor of 1.6-2.2) than those in the areas with solar UVR (t-test, p < 0.05). A reduction in the mineralization activity caused by solar UVR was either not observed or not significant (t-test, p > 0.05) in autumn and winter. Our results could contribute to revealing the potential effects of solar UVR on biogeochemical cycles.

Coral mucus influences the composition and abundance of bacteria in the surrounding seawater. In this study, the phylogenetic affiliations of actively growing bacteria (AGB) in seawater supplemented with the mucus of Acropora sp. during a 24-h incubation period were determined. For this purpose, bromodeoxyuridine magnetic-beads immunocapture and PCR-DGGE (BUMP-DGGE) analysis was used. The coral mucus contained higher concentrations of particulate organic carbon and nitrogen and exhibited higher bacterial abundance than seawater did, and the organic matter and bacteria varied dramatically during the incubation. BUMP-DGGE analysis showed that the AGB stimulated by the coral mucus also varied during the incubation. The bulk of the active growers originated from seawater and not the mucus. However, not all of the bacterial phylotypes in seawater could use the coral mucus as a suitable growth substrate. Cluster analysis revealed that the AGB community structures identified across the incubation period were considerably different in seawater supplemented with coral mucus as compared with those in coral mucus. In total, 12 phylotypes of AGB were identified, and these belonged to Alphaproteobacteria (Rhodobacterales 5 phylotypes), Gammaproteobacteria (Oceanospirillales 3 phylotypes, Vibrionales 1 phylotype, Alteromonadales 1 phylotype), and Bacteroidetes (Flavobacteriales 2 phylotypes). In the coral mucus, phylotypes belonging to Oceanospirillales appeared only at the beginning of the incubation, whereas those belonging to Rhodobacterales appeared only in the late phase of the incubation. Thus, coral mucus can alter the AGB community structure and create an organic matter flux that is specific to a reef environment. (C) 2015 Elsevier B.V. All rights reserved.

Coral mucus influences the abundance and composition of microbes in the surrounding seawater. Here, the hypothesis that the mucus of the coral genera Acropora, Favia, Favites, and Goniastrea would influence bacterial growth (production) in seawater (measured using 3H-thymidine and 3H-leucine incorporation methods) was tested. The bacterial production in mucus-supplemented seawater was significantly greater than that in unsupplemented seawater: production in the mucus-supplemented seawater ranged from 108.2 to 4111 mu g C.L-1.d(-1) when measured by thymidine incorporation and from 72.9 to 115.9 mu g C.L-1.d(-1) when measured by leucine incorporation. This study showed that the enhanced production may be due to bacteria originating from seawater and not from coral mucus. Nonetheless, the effect of mucus on bacterial production was different among the coral genera: the mucus of Favia did not always enhance bacterial production. Additionally, even a very small amount of coral mucus, only similar to 300 mu L. of mucus per liter of seawater, was sufficient to enhance bacterial production by approximately 2-fold compared to unsupplemented seawater. These results suggest that coral mucus has a significant impact on the marine biogeochemical cycle around coral reefs. (C) 2014 Elsevier B.V. All rights reserved.

We assessed the role of planktonic processes, in comparison to allochthonous input from fish cages and sedimentary loss, in the organic carbon (OC) budget of the water column in a semi-enclosed fish-culturing area (culturing red sea bream Pagrus major and yellow tail Seriola quinqueradiata). The sedimentation rate of particulate organic carbon (POC) at the fish-cage station was an average of 1.5 times that at non-cage stations. There was no significant difference in photosynthesis or respiration rates between fish-cage and non-cage stations. Annual allochthonous OC input in the form of leftover feed and fish feces was estimated to be 5 or 10 times that of autochthonous OC input by planktonic photosynthesis. In contrast, POC derived from phytoplankton accounted for a significant part (8 to 61 %) of total POC sedimentation. As to sinks of OC in the water column, annual planktonic respiration was twice as high as sedimentary loss at the fish-cage station. The plankton community tended to act as a source of OC in spring and summer and as an OC sink in fall and winter. The present study shows that a significant part of allochthonous and autochthonous OC input is respired by plankton and that the remaining OC input is deposited on the seafloor of fish-culturing areas.

The bacterium Flavobacterium psychrophilum is a serious problem for salmonid farming worldwide. This study investigates by multilocus sequence typing (MLST) the population structure of this pathogen in Japan where it is also a major concern for ayu, a popular game fish related to salmoniforms. A total of 34 isolates collected across the country and 80 isolates sampled in a single model river by electrofishing were genotyped. The data accounting for 15 fish species allowed identifying 35 distinct sequence types (ST) in Japan. These ST are distinct from those reported elsewhere, except for some ST found in rainbow trout and coho salmon, two fish that have been the subject of intensive international trade. The pattern of polymorphism is, however, strikingly similar across geographical scales (model river, Japan, world) in terms of the fraction of molecular variance linked to the fish host (∼50%) and of pairwise nucleotide diversity between ST (∼5 Kbp§ssup§-1§esup§). These observations go against the hypothesis of a recent introduction of F. psychrophilum in Japan. Two findings were made that are important for disease control: 1) at least two independent F. psychrophilum lineages infect ayu and 2) co-infections of the same individual fish by different strains occur. © 2013 Fujiwara-Nagata et al. licensee BioMed Central Ltd.

Microbial mineralization rates in sinking particles, bottom sediments and seawater were determined in a coastal fish (red sea bream Pagrus major) culturing area to clarify the mineralization process of organic matter (OM) in the entire water column. The mineralization rates of C-14-labelled glutamate and glucose per unit volume were highest in the sinking particles and were 131572 and 749 times higher than those of the seawater and bottom sediments respectively. The turnover time of glutamate tended to be shorter than that of glucose at all three sites of the water column. Bacteria appeared to prefer amino acids to monosaccharides because amino acids could be utilized as both energy and nitrogen sources. The sedimentation rate of particulate organic carbon (POC) derived from phytoplankton accounted for 961% of the total POC, and it was particularly high in early summer (61% and 50% at fish cage and non-cage stations respectively). The present study clearly shows that sinking particles serve as an important site of microbial mineralization process of OM within the water column, and that phytoplankton production was another serious cause of organic pollution of the seafloor in addition to the organic wastes directly discharged from fish farms.

We developed a simple genotyping method for Flavobacterium psychrophilum for analysing two single nucleotide polymorphisms (SNPs) in the gyrA gene and to distinguish between isolates that are virulent and avirulent to ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel). The genotyping method is an on/off switch assay and is based on the polymerase chain reaction technique with phosphorothioated primers. We classified 232 isolates from four families of fish (i.e. Plecoglossidae, Osmeridae, Cyprinidae and Salmonidae) into four genotypes (G-C, A-T, A-C and G-T). The G-C type isolates exhibited strong pathogenicity to ayu, whereas the A-T and G-T types did not show any pathogenicity to this species. The A-C type exhibited no or weak pathogenicity to ayu. These results indicate that genotyping F.similar to psychrophilum isolates with two SNPs from gyrA can clearly distinguish between isolates potentially harmful to ayu (G-C type) and those that are potentially not harmful or less harmful (A-C, A-T and G-T type). The on/off switch assay provides a quick, simple, and very powerful DNA genotyping technique for F.similar to psychrophilum isolates.

We investigated the induction of protective immunity against bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum by warmed water treatment in ayu (Plecoglossus altivelis). Fish were immersed in a live bacterial suspension (10(7) CFU mL(-1)) for 30 min and placed in 700 L concrete tanks. The 28 degrees C warmed water treatment lasted 3 days and began 1, 6, and 24 h after immersion in the live bacterial suspension. A naive control fish group was immersed in a sterilized modified Cytophaga (MCY) broth instead of the bacterial suspension. Fourteen days after the immersion, agglutination antibody titers against E psychrophilum were measured by using micro-titer methods. Fish were then exposed to a bacterial bath to infect them with live E psychrophilum, and cumulative mortality was monitored. Fish treated with warmed water at 1, 6, and 24 h after immersion in the live bacterial suspension had cumulative mortalities of 36%, 30%, and 18%, respectively, all of which were significantly lower than the cumulative mortality of the naive control fish (90%). Treated fish also showed high antibody titers against E psychrophilum in agglutination tests. These results demonstrate that warmed water treatment could not only cure BCWD but also immunize the fish against the causative agent E psychrophilum. (C) 2011 Elsevier Ltd. All rights reserved.

The ability of the probiotic bacterium Sulfitobacter to inhibit the growth of two virulent strains (psh-9019 and ATCC 43306) of the fish pathogenic bacterium Vibrio anguillarum was tested. Probiotic and pathogenic bacteria were inoculated individually or together in three different types of media. Two of the phytoplankton media tested were filtrates of phytoplankton Chlorella vulgaris cultures, either the live phytoplankton (live-CV) or a condensed phytoplankton (condensed-CV). Phytoplankton culture medium, ESM, was used as a control medium without phytoplankton. In ESM, Sulfitobacter decreased the viable cell counts of both V. anguillarum strains by tenfold. In the live-CV filtrate, V. anguillarum was eradicated by Sulfitobacter within one week. Although colony counts of strain ATCC 43306 declined during the two-week co-incubation with Sulfitobacter, its growth was not fully inhibited; however, the counts were tenfold lower than that in control ESM medium. Neither of the pathogenic V. anguillarum strains were inhibited nor eradicated by Sulfitobacter in the condensed-CV filtrate medium. Our study indicates that commercially available condensed phytoplankton can enhance the growth of V. anguillarum. Thus, the addition of live phytoplankton, including the introduction of Roseobacter clade bacteria to fish larvae tanks, leads to better biocontrol of the fish pathogen V. anguillarum.

High- and low-virulence serotypes of the fish pathogen Vibrio anguillarum were challenged with a beneficial bacterium (Sulfitobacter sp. RO3) in the presence of the phytoplankton Nannochloropsis oculata. The inhibitory activity of Sulfitobacter sp. against these V. anguillarum serotypes were observed in (1) phytoplankton culturing control medium (ESM) and (2) NCF, a filtrate prepared from N. oculata cultured in ESM. The presence of Sulfitobacter sp. in ESM significantly reduced the number of viable cells of all V. anguillarum serotypes tested (O2, O3, O4, O9 and O10). In NCF, Sulfitobacter sp. totally eradicated the O2 and O3 serotypes. The viabilities of the low-virulence serotypes O4, O9 and O10 when cultured in NCF in the presence of Sulfitobacter sp. were not significantly different from those cultured in ESM. We conclude, therefore, that phytoplankton cultured in the presence of naturally occurring Sulfitobacter sp. provide an essential tool for inhibiting the growth of the economically significant high-virulence fish pathogen V. anguillarum.

Background: Phytoplankton cultures are widely used in aquaculture for a variety of applications, especially as feed for fish larvae. Phytoplankton cultures are usually grown in outdoor tanks using natural seawater and contain probiotic or potentially pathogenic bacteria. Some Roseobacter clade isolates suppress growth of the fish pathogen Vibrio anguillarum. However, most published information concerns interactions between probiotic and pathogenic bacteria, and little information is available regarding the importance of phytoplankton in these interactions. The objectives of this study, therefore, were to identify probiotic Roseobacter clade members in phytoplankton cultures used for rearing fish larvae and to investigate their inhibitory activity towards bacterial fish pathogens in the presence of the phytoplankton Nannochloropsis oculata. Methodology/Principal Findings: The fish pathogen V. anguillarum, was challenged with 6 Roseobacter clade isolates (Sulfitobacter sp. (2 strains), Thalassobius sp., Stappia sp., Rhodobacter sp., and Antarctobacter sp.) from phytoplankton cultures under 3 different nutritional conditions. In an organic nutrient-rich medium (VNSS), 6 Roseobacter clade isolates, as well as V. anguillarum, grew well (10(9) CFU/ml), even when cocultured. In contrast, in a phytoplankton culture medium (ESM) based on artificial seawater, coculture with the 6 isolates decreased the viability of V. anguillarum by approximately more than 10-fold. Excreted substances in media conditioned by growth of the phytoplankton N. oculata (NCF medium) resulted in the complete eradication of V. anguillarum when cocultured with the roseobacters. Autoclaved NCF had the same inhibitory effect. Furthermore, Sulfitobacter sp. much more efficiently incorporated (14)C-photosynthetic metabolites ((14)C-EPM) excreted by N. oculata than did V. anguillarum. Conclusion/Significance: Cocultures of a phytoplankton species and Roseobacter clade members exhibited a greater antibacterial effect against an important fish pathogen (V. anguillarum) than roseobacters alone. Thus, cooperation of N. oculata, and perhaps other phytoplankton species, with certain roseobacters might provide a powerful tool for eliminating fish pathogens from fish-rearing tanks.

We developed a quantitative PCR assay for detecting the parasitic ciliate Cryptocaryon irritans, which causes "white spot disease" in marine fishes, from the natural environment. A specific primer set for C. irritans was designed and its high specificity was confirmed in silico: almost all of the sequences deposited in the GenBank nucleotide database were covered, 22/23 for the forward primer and 7/7 for the reverse primer. We estimated that there were 3,415.9 rRNA gene copies per genome of C. irritans. In artificial mixture experiments to validate whether the qPCR assay is applicable to natural samples, the estimated copy numbers showed significantly positive correlations with the number of theronts added (p < 0.001). When we applied this qPCR assay to natural samples collected bimonthly from surface and bottom seawaters at an aquaculture site (water depth, 10 m) from May 2009 to March 2010, we only detected C. irritans (112.0 +/- A 6.3 cells/l) in the surface seawater sample in November. This qPCR assay is a useful tool for detecting C. irritans rapidly and quantitatively in natural environments; it could also help advance our understanding of the ecology of C. irritans, as well as facilitate the diagnosis of the disease.

仔魚飼育水を模した、天然海水とナンノクロロプシスを添加したマイクロコスム実験を行い、ビブリオ属細菌の動態を13日間モニターした。クロロフィルa濃度は、タンク1で8.71～235μg／l、タンク2で10.9～255μg／l の範囲で変動し、両タンクとも培養6日目にピークを迎えた。培養可能なビブリオ属細菌数および定量PCR法で推定したビブリオ属細菌数は、クロロフィルa濃度の増加とともに減少した。面白いことに、クロロフィルa濃度がピークを迎えた後は、培養可能なビブリオ属細菌は検出されず、かつ定量PCR法においてもわずかな数しか検出されなかった。ビブリオ属細菌の群集構造は、実験開始時は非常に多様であったが、クロロフィルa濃度が増加するにつれ、単純化した。これらの結果は、仔魚飼育水のナンノクロロプシスは、ビブリオ属細菌の増殖を制御しており、仔魚の生残や成長の鍵を握る要因の一つとなることを示唆する。

Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum leads to heavy mortality of ayu Plecoglossus altivelis in Japan. Previously, a 28 degrees C warmed water treatment was shown to be an effective treatment for BCWD, since F psychrophilum can not grow and maintain colonies at this temperature. However, it was unclear whether the bacteria might resuscitate after the treatment was over. Therefore, we investigated colony formation, membrane potential and pathogenicity of F psychrophilum at various temperatures (15 degrees C, 23 degrees C, 28 degrees C and 33 degrees C) in sterilized underground water. Within 2 days at 28 degrees C, F psychrophilum completely lost their colony-forming abilities but still maintained their membrane potentials. It seemed that these cells entered into viable but non-culturable (VBNC) state. However, experimental ayu infection revealed that VBNC F psychrophilum cells were unable to cause BCWD, suggesting that the cells were progressing towards death at 28 degrees C.

Bacterial cold-water disease (BCWD) causes heavy mortality of ayu Plecoglossus altivelis in Japan. To control BCWD of ayu, warmed water treatment at 28 degrees C has been used. However, the mechanism of this treatment is unknown. Hence, we investigated the growth/survival of Flavobacterium psychrophilum at 15-28 degrees C under two nutritional conditions: in modified cytophaga (MCY) broth and sterilized underground water. Within 2 days at 28 C in both the conditions, F psychrophilum totally lost its colony-forming ability. We also studied distributions of F psychrophilum in infected fish organs and in rearing water after various treatments. For bacteria detection, the colony-counting method and loop-mediated isothermal amplification (LAMP) method were used. For warmed water treatment, the rearing water was warmed from 18 degrees C to 23 degrees C or 28 degrees C for 3 days. The treatments were started 1 day after immersion infection. In the experimentally infected fish, F psychrophilum was detected neither in any fish organs nor in the rearing water after the 28 degrees C treatment for 3 days. These results indicate the effectiveness of the warmed water treatment at 28 degrees C against BCWD of ayu.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout fry syndrome of salmonids. The pathogen has been reported from all regions in the world involved in salmonid aquaculture, but also from natural fresh-water environments. We established a quantitative loop-mediated isothermal amplification of DNA (LAMP) method to estimate quantities of F. psychrophilum. LAMP primers were designed based on the sequence of the DNA topoisomerase IV subunit B gene, parE, of F. psychrophilum. parE LAMP exhibited a high specificity for the parE gene of F. psychrophilum but not for other related species. parE LAMP detected the gene in a wide range of concentrations from 2.0 x 101 to 2.0 x 109 copies/reaction within 70 min and revealed a good correlation between threshold times and gene copy number.

マーブルゴビーの初期飼育水における原生動物による微細藻類の捕食について

The influence of phytoplankton photosynthesis (PP) on dissolved oxygen (DO) dynamics was evaluated in a red sea bream (Pagrus major) aquaculture site. The surface PP was monitored continuously by a natural fluorescence sensor for 2-3 weeks during all seasons, except spring. In summer, when the daily PP was low during cloudy weather, DO at the surface markedly decreased below the critical level (5.7 mg O-2 L-1), which is needed for normal fish growth. However, on the subsequent clear day, active PP restored the DO back to above the critical level. In fall, under cloudy skies, the surface DO also decreased below the critical level and remained there, even when the subsequent days were clear. In winter, DO remained at high levels throughout the water column due to an active supply from the air and vertical mixing. Besides seasonal changes, surface DO showed a die] cycle with a minimum early in the morning and maximum in the evening. This diel cycle was also regulated by planktonic photosynthesis. This was most obvious and its amplitude was largest in summer, as PP was very high and thermal stratification prevented diffusion of DO from the surface to deeper water. The present study is the first detailed report on the close coupling of DO with PP in coastal waters, even under non-bloom conditions. Cloudy weather and early mornings were found to be the most critical periods in oxygen supply to cultured fish and other pelagic organisms in a fish culture site and other eutrophic coastal area. (c) 2007 Elsevier Ltd. All rights reserved.

Bacterial community structures were analyzed in water used for rearing fish larvae by fluorescence in situ hybridization. In Experiment 1, red sea bream Pagrus major larvae were reared in two commercial seed production tanks. The survival rate in Tank 1 was higher than in Tank 2, even though phytoplankton, Nannochloropsis sp., was added to both tanks. In Tank 2, gamma-proteobacteria became dominant (similar to 70% of total bacteria) on day 13, thereafter heavy larval mortalities occurred. In Tank 1, however, alpha-proteobacteria and the Cytophaga-Flavobacterium cluster were predominant from day -1 until day 13; no significant mortality was recorded. In Experiment 2, marble goby Oxyeleotris marmoratus larvae were cultured with or without Nannochloropsis sp. At the end of the experiment, larval survival rates in aquaria with Nannochloropsis sp. were significantly (P < 0.05) higher than those without. In rearing water without Nannochloropsis sp., gamma-proteobacteria increased during rearing. In rearing water with Nannochloropsis sp., alpha-proteobacteria and the Cytophaga-Flavobacterium cluster were predominant at the beginning of the experiments and the relative abundance of gamma-proteobacteria was maintained at a lower level throughout the experiments. The predominance of alpha-proteobacteria and the Cytophaga-Flavobacterium cluster appears to be a good indicator of successful larval production.

Prey-predator interactions among planktonic organisms in larval tanks of bluefin tuna and red sea bream were investigated quantitatively, and carbon flow models were constructed from the results of feeding experiments. Two different carbon pathways were uncovered in the rearing water of both fish species. One was from autotrophic nano-plankton (ANP) to rotifers to fish larvae (artificial food chain) and the other was from bacteria to heterotrophic nano-plankton (HNP) to heterotrophic micro-plankton (HMP) (a microbial loop). Surprisingly, bluefin tuna selectively consumed HMP, especially dinoflagellates, throughout the experiment. In the bluefin tuna tank, the microbial loop was linked to the artificial food chain. On the other hand, red sea bream larvae did not consume HMP and the pathway in the red sea bream tank was more straightforward than in the bluefin tuna tank. Energy and nutrients transferred to red sea bream larvae via the artificial food chain only, except for 3 days after hatching. Biomass of micro-protozoa ware high (3.2-158.4 ng C ml(-1)) in the bluefin tuna tank with higher water temperature (26.0-27.6 degrees C and low (3.0-4.9 ng C ml(-1)) in the red sea brearn tank with lower temperature (19.8-20.1 degrees C. The study suggests that difference in water temperature is a significant factor for the microbial loop in rearing tanks of fish larvae. This is the first report suggesting that a microbial loop, established naturally, contributes energy and nutrient gain to bluefin tuna larvae reared in an artificially controlled environment. (C) 2007 Elsevier B.V. All rights reserved.

Phytoplankton used in fish hatcheries is mass-cultured in the open air and usually contains large numbers of bacteria. In commercial fish production, the phytoplankton cultures are usually added into the larval rearing tanks; however, the numbers and types of bacteria introduced into the rearing tanks simultaneously are unknown. In this study, the bacterial community structures in Nannochloropsis sp. cultures were analyzed by using fluorescence in situ hybridization (FISH). A direct viable count (DVC)-FISH analysis was also performed as DVC is useful for the detection of actively growing cells. Total numbers of bacteria in Nannochloropsis sp. cultures ranged from 7.72 x 10(5)-2.39 x 10(6) cells/mL. High proportions of the total bacteria (31.6-53.6%) in the Nannochloropsis sp. cultures showed growth potential. DVC-FISH analysis revealed that alpha-proteobacteria and the Cytophaga-Flavobacterium cluster were abundant in the bacterial community of actively growing cells. Thus, the high growth potentials of the distinct bacterial communities in Nannochloropsis sp. culture must influence the bacterial communities in larval rearing tanks.

Vibrio anguillarum kills various kinds of fish over a range of salinities from sea water to fresh water, and causes serious damage to aquaculture systems. In this study, the transcriptional regulation of the Na+-NADH:quinone oxidoreductase (Na+-NQR) operon in V. anguillarum from the logarithmic to stationary phases was investigated. Cloning of the Na+-NQR operon revealed a 7 kb nucleotide sequence composed of six open reading frames with amino acid sequence identity of more than 80% with other Vibrio species. Two promoters, nqrP1 and nqrP2, were identified in the region upstream of the nqrA gene using an S1 nuclease assay. The nqrP1 promoter was constitutively activated throughout the logarithmic to stationary phases and possessed -10 (5'-TAGACT-3') and -35 (5'-ATGGCA-3') sequences, which were similar to the consensus sequence of Escherichia coli. On the other hand, the nqrP2 promoter was activated only at the stationary phase and had only a -10 (5'-CATACT-3') and not a -35 sequence. These results suggest that nqrP2, which works specifically in the stationary phase, contributes to starvation-survival in V. anguillarum.

The safety of electrolyzed seawater was evaluated by measuring the production rate of organic halogen compounds and the occurrence of reverse mutations. Aquaculture feedwater and wastewater were collected from a fish-culturing facility, and available chlorine of approximately 1.0 mg/L was generated to ensure a disinfectant effect. More than 90% of the generated organic halogen compounds were bromoform. The amount of bromoform was far less than the reference values for drinking water standards in Japan and the U.S., provided that the electrolyzation was performed within the range of normal use. The reverse mutation assay of electrolyzed seawater showed no mutagenicity. Electrolyzed seawater with available chlorine at an adequate level for disinfection can be used safely and effectively in various aspects of aquaculture. (c) 2006 Elsevier B.V. All rights reserved.

Vibrio anguillarum kills various kinds of fish over a range of salinities from sea water to fresh water, and causes serious damage to aquaculture systems. In this study, the transcriptional regulation of the Na+-NADH:quinone oxidoreductase (Na+-NQR) operon in V. anguillarum from the logarithmic to stationary phases was investigated. Cloning of the Na+-NQR operon revealed a 7 kb nucleotide sequence composed of six open reading frames with amino acid sequence identity of more than 80% with other Vibrio species. Two promoters, nqrP1 and nqrP2, were identified in the region upstream of the nqrA gene using an S1 nuclease assay. The nqrP1 promoter was constitutively activated throughout the logarithmic to stationary phases and possessed -10 (5'-TAGACT-3') and -35 (5'-ATGGCA-3') sequences, which were similar to the consensus sequence of Escherichia coli. On the other hand, the nqrP2 promoter was activated only at the stationary phase and had only a -10 (5'-CATACT-3') and not a -35 sequence. These results suggest that nqrP2, which works specifically in the stationary phase, contributes to starvation-survival in V. anguillarum.

環境水におけるアユ冷水病菌Flavobacterium psychrophilumの定量的モニタリング．

ノロウイルスの代替としてネコカリシウイルスを使用した電解水による厨房内の二次汚染防止に関する検討．

The activity of membrane-bound NADH oxidase of Vibrio anguillarum M93 (serotype J-O-1), which causes vibriosis in freshwater area was activated by Na+ in the same manner as other marine Vibrios. However, in addition to Na+, K+ was also found to positively enhance the NADH oxidase activity of strain M93. This tendency has not been recognized in other marine Vibrios. Furthermore, the Na+-dependent NADH oxidase of strain M93 required less Na+ (0.1 M) for its maximum activity than those of other Vibrios such as Vibrio alginolyticus and 'Vibrio angustum' S14, which were in the range of 0.4 M NaCl, similar as seawater. Destruction of H+ motive force by a proton conductor carbonylcyanide m-chlorophenylhydrazone (CCCP) revealed high dependency of V anguillarum on the primary H+ pump. Even at pH 8.5, V anguillarum strains other than serotype O-4 could not grow well with the addition of CCCP. In contrast, marine-type Vibrios such as V. alginolyticus and V. angustum S14 can grow well at pH 8.5 even with the addition of CCCP. The lower requirement for Na+ in V anguillarum probably reflects the salinity of their original habitats. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Vibriosis caused by Vibrio anguillarum serotype J-O-1 seriously affects the freshwater fish ayu (Plecoglossus altivelis) in Lake Biwa, Japan. Survival patterns of V. anguillarum were investigated in aged lake water (ALW) supplemented with or without 0.75% NaCl. It was found that 0.1-1.0% of V. anguillarum cells maintained the ability to form colonies even after 6 weeks in 0.75% NaCl-ALW. Under the same starved conditions, MPN counts with liquid medium were 100 times higher than CFU counts. When exposed to sterilized aged lake water without NaCl, V. anguillarum entered a non-culturable state within half a day. As the mineralization activity of non-culturable cells was still 0.1-5.4% of that in the culturable phase, this physiological state can be described as “viable but non-culturable”. However, all attempts to return to a culturable state including re-infection were un-successful. The non-culturable cells in ALW lost all pathogenicity in fish. Since a longer exposure to ALW resulted in less mineralization activity, the non-culturable state of V. anguillarum cells in freshwater seems to reflect a phase of decay leading to cell death. However, certain environmental factors such as coldness and microaerobiosis seem to help the pathogen to survive longer in freshwater without a loss of culturability. © 2003, Japanese Society of Microbial Ecology & The Japanese Society of Soil Microbiology. All rights reserved.

In May 2000, an experimental artificial tidal flat, based on dredged bottom mud and covered with sand, was constructed in Osaka Bay, Japan. We measured dissolved oxygen consumption by bottom sediments and some other environmental indicators such as sulfate reducing bacteria (SRB) to estimate the water-purifying potential of the tidal flat. Viable counts of SRB in bottom sediments of the tidal flat area reduced from 10(6) cells/g wet mud (June) to 10(2) cells/g wet mud (September and December). In September, biological oxygen consumption rate (BOCR) early in the evening, just before a high tide (17:40) was more than 40 times higher than that early in the morning, just after a high tide (6:10), whereas no difference was observed in chemical oxygen consumption rates (COCR). The biological respiratory activity in tidal flat seemed to change hourly. In December, COCR was not detected, however BOCR early in the evening kept the same level of that in September.

ABSTRACT Numerous studies have established the importance of picoplankton (microorganisms of ≤2 μm in length) in energy flow and nutrient cycling in marine oligotrophic environments, and significant effort has been directed at identifying and isolating heterotrophic picoplankton from the world's oceans. Using a method of diluting natural seawater to extinction followed by monthly subculturing for 12 months, a bacterium was isolated that was able to form colonies on solid medium. The strain was isolated from a 10 ⁵ dilution of seawater where the standing bacterial count was 3.1 × 10 ⁵ cells ml ⁻¹ . This indicated that the isolate was representative of the most abundant bacteria at the sampling site, 1.5 km from Cape Muroto, Japan. The bacterium was characterized and found to be ultramicrosized (less than 0.1 μm ³ ), and the size varied to only a small degree when the cells were starved or grown in rich media. A detailed molecular (16S rRNA sequence, DNA-DNA hybridization, G+C mol%, genome size), chemotaxonomic (lipid analysis, morphology), and physiological (resistance to hydrogen peroxide, heat, and ethanol) characterization of the bacterium revealed that it was a strain of Sphingomonas alaskensis . The type strain, RB2256, was previously isolated from Resurrection Bay, Alaska, and similar isolates have been obtained from the North Sea. The isolation of this species over an extended period, its high abundance at the time of sampling, and its geographical distribution indicate that it has the capacity to proliferate in ocean waters and is therefore likely to be an important contributor in terms of biomass and nutrient cycling in marine environments.

Vibriosis caused by Vibrio anguillarum seriously damages freshwater fish (Salmoniforms) almost every year in Lake Biwa, Japan. This pathogen requires NaC1 for its growth. When the pathogen was exposed to sterilized aged lake water, it lost its culturability without loosing respiratory activity. Although this physiological state appears to be 'viable but nonculturable' (VBNC), the cells could not be resuscitated even in host fish bodies. As these cells totally lost their pathogenicity, the VBNC-like state is probably on the pathway to cell death. Environmental factors such as darkness, coldness, and anaerobiosis helped the pathogen to survive in freshwater without reaching a nonculturable state.

The starvation-stress response of Vibrio (Listonella) anguillarum was investigated and characterized with regard to changes in cell morphology and the ability of V. anguillarum to survive starvation, heat shock, exposure to H2O2 and exposure to ethanol. The ability of V. anguillarum to survive exposure to the latter three stresses after initiation of starvation was also examined. Results of these experiments indicated that when starved for carbon, nitrogen and phosphorus, the c.f.u. of V. anguillarum declined by about one order of magnitude over the first 5-7 d of starvation; starvation for an additional 3-4 weeks resulted in a gradual decline in c.f.u. by another order of magnitude. Examination of starved cells by electron microscopy revealed that while most cells formed spherical ultramicrocells during starvation, some of the cells elongated to form short spirals. While cross-protection against other stresses such as oxidative stress (exposure to H2O2) and exposure to ethanol developed, only a small degree of resistance to heat shock developed. Moreover, in all cases these resistances disappeared during prolonged starvation (usually > 5 d). Additionally, the rate of protein synthesis per c.f.u., measured by [S-35]methionine incorporation, declined during the initial 6 h of starvation and increased to over 70% of the rate measured in exponentially growing cells by 5 d of starvation. It was concluded that the starvation-stress response of Il. anguillarum differs significantly from those starvation responses reported for other bacteria, including responses displayed by other Vibrio species.

Low osmotic stress responses of the fish pathogen, Vibrio anguillarum, were investigated and characterized with regard to changes in cell density and cation type in natural lake water (aged lake water, ALW) and MilliQ water (MQW). This pathogen showed higher survival (% CFU) in ALW than in MQW at all the examined cell densities (10(6)-10(9) CFU ml(-1)). Higher resistance in ALW was partially due to divalent cations such as Ca2+ and Mg2+ present in ALW. It was also observed that addition of the supernatant, which was obtained from the pre-suspension of V. anguillarum cells in ALW, significantly increased the survival of the pathogen in ALW. This indicated that the extracellular substance(s) released from the pathogen also played an effective role(s) for survival in ALW. Thus, divalent cations and cell density assist the pathogen, V. anguillarum, to overcome the low osmotic stress in natural freshwater environments.

The distribution of Vibrio anguillarum serotype J-O-1, a causative agent of vibriosis of ayu Plecoglossus altivelis in Lake Biwa, was investigated by both fluorescence antibody method using specific monoclonal antibody against V. anguillarum J-O-1 (FA-DAPI method) and a conventional cultural method. There were 3.2 x 10(4) cells/cm(3) of V. anguillarum J-O-1 deposited on the bottom sediment surface (0 similar to 0.1 cm) under the net cages of an ayu culturing farm in Lake Biwa. In ayu culturing ponds, which are concrete tanks on the lakeshore, we also detected this pathogen (1.6 x 10(5) cells/cm(3)) in the bottom sediments collected from the drains for outflow of pond water into Lake Biwa. In non-culturing areas of the lake, V. anguillarum J-O-1 cells were not found in any water, bottom sediment nor plankton samples. In the same samples from which V. anguillarum J-O-1 cells were detected by the FA-DAPI method, no cells were recognized by the conventional cultural method. This is the first report for direct detection of V. anguillarum J-O-1 from freshwater environments.

Sphingomonas sp. strain RB2256 was isolated from Resurrection Bay in Alaska and possibly represents the dominant bacterial species in some oligotrophic marine environments. Strain RB2256 has a high-affinity nutrient uptake system when growing under nutrient-limiting conditions, and growing cells are very small (<0.08 (mu)m(sup3)). These characteristics indicate that RB2256 is highly evolved for withstanding nutrient limitations and grazing pressure by heterotrophic nanoflagellates. In this study, strain RB2256 was subjected to nutrient starvation and other stresses (high temperature, ethanol, and hydrogen peroxide). It was found that growing cells were remarkably resistant, being able to survive at a temperature of 56(deg)C, in 25 mM hydrogen peroxide, or in 20% ethanol. In addition, growing cells were generally as resistant as starved cells. The fact that vegetative cells of this strain are inherently resistant to such high levels of stress-inducing agents indicates that they possess stress resistance mechanisms which are different from those of other nondifferentiating bacteria. Only minor changes in cell volume (0.03 to 0.07 (mu)m(sup3)) and maximum specific growth rate (0.13 to 0.16 h(sup-1)) were obtained for cells growing in media with different organic carbon concentrations (0.8 to 800 mg of C per liter). Furthermore, when glucose-limited, chemostat-grown cultures or multiple-nutrient-starved batch cultures were suddenly subjected to excess glucose, maximum growth rates were reached immediately. This immediate response to nutrient upshift suggests that the protein-synthesizing machinery is constitutively regulated. In total, these results are strong evidence that strain RB2256 possesses novel physiological and molecular strategies that allow it to predominant in natural seawater.

The distribution and abundance of photoautotrophic picoplankton (PPP, Synechococcus group) in the aphotic bottom sediments of Lake Biwa were investigated by direct counting and viable counting (most probable number, MPN) methods. In the surface layer of bottom sediments (0-1 cm), where large PPP blooms occurred in the past 5 years, >10(5) cells cm(-3) of PPP were found to be viable throughout the year. Furthermore, the density of PPP deposited on the sediment surface (0-0.1 cm) was one order of magnitude higher (MPN = 1.3 x 10(6) cells cm(-3), direct count = 9.9 x 10(6) cells cm(-3)) than that of bulked surface sediments (0-1 cm). Even in the deeper layer (13-14 cm) of bottom mud, viable PPP were still found (10(1) cells cm(-3)). In winter, viable PPP in the aphotic bottom sediments were 10(4)-10(5) times greater per unit volume than those in the euphotic lake water. Since the aphotic bottom sediments have high levels of PPP, as well as high growth potential (high ratio of viable count/total direct count), they are likely to seed PPP blooms in the North Basin of Lake Biwa.

Five monoclonal antibodies (MAbs) against cell surface antigens of Vibrio anguillarum serotype J-O-1 were developed by a hybridoma technique. Three of the MAbs, VA19-1, VA19-2, and VA19-4, reacted only with serotype J-O-1. The remaining two MAbs, VA07-1 and VA07-2, showed cross-reaction with some other Vibrio species. MAbs VA19-1, VA19-2, and VA19-4 could react with heat treated (100 degrees C for 1 h) cells of V. anguillarum J-O-1. This indicated that the antigens recognized by these MAbs were stable molecules such as lipopolysaccharide. As almost all strains of V. anguillarum isolated from diseased ayu Plecoglossus altivelis in freshwater belong to serotype J-O-1, MAbs VA19-1, VA19-2, and VA19-4 are useful for rapid identification and direct detection of V. anguillarum J-O-1 in natural freshwater environments. Furthermore, MAbs VA19-1, VA19-2, and VA19-4 specifically reacted with V. anguillarum serotype O2 among ten Danish serotypes (O1 similar to O10) and did not react with V. ordalii. Thus, these MAbs are also useful for distinction between closely related species, V. anguillarum O2 and V. ordalii.

3.外洋環境における低栄養細菌の特性と役割、4.海洋におけるVBNC（生きているが培養できない）細菌の存在

和歌川河口干潟の微生物調査を行い、海水と間隙と底砂では存在する細菌群集構造が異なること、呼吸によって無機化活性も異なることを明らかにした。

淡水にさらすと死滅する魚病細菌Vibrio anguillarum をバイオフィルム状態で淡水へ入れると、1 ヵ月以上生残することを明らかにした。

大阪湾のなぎさ環境を形成する微生物群の働きについて、大阪南港野鳥公園の人工干潟や岸和田市地先の阪南2区人工干潟における調査結果をもとに講演した。

大阪湾に造成された実験用の人工干潟において、干潟の生物群（特に微生物群集）による呼吸活性（無機化能力、水質浄化能力）を測定し、呼吸活性が激しく時間変動していることを明らかにした。

大阪湾に造成された人工干潟において、 生物的溶存酸素消費速度 （呼吸活性） を測定した。 干潟の呼吸活性が季節変動することは周知であるが、 呼吸活性が数倍から数 10 倍の幅で大きく時間変動することを明らかにした。

大阪湾岸和田市地先に造成された人工干潟における底質の生物的 （呼吸活性） 及び科学的酸素消費速度の昼夜観測を行った。 潮汐に合わせたように、 底質の呼吸活性が時間変動することが明らかになった。

呼吸鎖に一次ナトリウムポンプを保有する Vibrio 属細菌の増殖と飢餓生残 （エネルギー欠乏） における、 外部環境の pH と各種カチオンの影響を明らかにした。

人工干潟の海水浄化能力を底質の溶存酸素消費速度から評価した。 （英文）

魚病細菌 Vibrio anguillarum の Na＋ 依存型呼吸鎖が、 他の海洋性 Vibrio 属細菌のものとは質的・量的に異なることを明らかにした。 （英文）

電解水処理により、 養殖魚の初期減耗が有意に低下することを示し、 養殖現場での電解水処理の有効性を明らかにした。 （英文）

日本近海の深層水から限界希釈法により分離した超微小低栄養性海洋細菌の生理学的・分類学的諸特性を明らかにした。 （英文）

海洋の環境特性は高い Na＋ 濃度と弱アルカリ性である。 増殖状態と異なり飢餓状態では、 Vibrio 属細菌の場合、 この環境特性を有効に利用した Na＋ ポンプの存在が極めて重要になることを示した。 （英文）

増殖には必ずしも Na＋ を要求しないビブリオ属の魚病細菌が､ 飢餓生残には Na＋ を要求し､ この Na＋ が特に呼吸鎖において重要であることを示した｡

養殖漁場では、水域生態系に負荷される有機物量が生簀がない場合に比べて大きい。この有機物負荷が当該水域の持つ自浄能力を超えると、有機汚濁、富栄養化、底層の貧酸素化といった自家汚染を起こす。養殖漁場の持つ自浄能力を評価することは難しい。有効な評価指標の一つが、当該水域における高分子有機物（例えばタンパク質）を低分子有機物（例えばアミノ酸）に加水分解する酵素活性の測定である。この高分子有機物が細菌群が利用可能な低分子有機物に加水分解されるプロセスが水域における有機物フローの制限要因となり、これを"gate keeper"と呼ぶ。 この有機物の流れを主に担う海洋細菌群の加水分解活性は、水温、塩分、溶存酸素などの環境要因により変化する。今までの研究から、水域が成層し、海底が貧酸素化しやすい夏季には、この加水分解活性が特に底層で停滞しがちであり、強風により鉛直混合が起こる冬季に活性が上がることを確認している。本研究課題では、加水分解活性が停滞しやすい夏季底層に強いインパクトを与える台風の影響を評価しようとしている。 2021年の台風発生数は例年よりも少なく、2020年に引き続き台風が暴風域を伴ったまま上陸することは皆無であった。野外調査で台風の影響を評価することが出来なかったが、室内実験により興味深い成果を得ることができた。それは、雨水の影響である。降雨により表層海水の塩分が低下するとペプチドの末端からアミノ酸を一つずつ切り離していくエキソペプチダーゼの活性が有意に高くなるのである。タンパク質の内部のペプチド結合を切るエンドペプチダーゼや糖類の加水分解酵素ではそのような傾向は確認できなかった。雨水により加水分解活性が上昇する現象を室内実験で確認したことは新規な知見と言える。

「水作り」という言葉がある。養殖魚類の受精卵を収容する飼育水の水質環境を微細藻類により整えることを指す。本研究では微細藻類による飼育水細菌群の制御機構の解明を目的とし、微細藻類の培養液に多く存在する善玉細菌(例: Roseobacter属細菌)の働きと微生物ループの生態学的機能に注目した。善玉細菌は常にビブリオ属魚病細菌の増殖を抑制したが、微細藻類が共存するとその抑制効果が飛躍的に向上した。また、飼育水温の高いクロマグロ仔魚はマダイ仔魚などと異なり、微生物ループを経由した栄養を積極的に利用していた。これは経験的に行われてきた「水作り」を、科学的に検証した初めてのケースといえる。

The purpose of this study is to develop the method for the early recovery of the biodiversity in the oligotropical coastal area. It is of importance in the recovery of the biodiversity to make kelp forest grow in the concerned area. In order for it, sufficient nutrient is required as well as the proper seedbed. Hence, in this study, granulated fertilizer, which contains nutrient, such as nitrogen, phosphorus and iron, is coated by the special polymer, and then, is mixed into the porous concrete in order to provide seedbed and nutrient simultaneously. The physical and chemical properties of porous concrete with granulated fertilizer are discussed. Then brief note is appended to describe the method to select the place of implantation and to control the implantation with the aid of the artificial satellite to measure the chlorophyll concentration on sea surface. Research results from 2001 to 2005 are as follows. 1)Manufacturing method and rheological properties of binder and also segregation of fresh porous concrete (POC.) 2)Physical and chemical properties of porous concrete (POG)., and clarified the durability of freezing and thawing, washout test by water and abrasion by grain in sea 3)Properties of POC mixed with fertilizer and converter slag in seawater. 4)Seaweed adhesion onto POC mixed fertilizer and converter slag. 5)Water purification performance of POC. 6)Properties and seaweed adhesion of POC using the recycled material. 7)Chlorophyll concentration of sea surface and consistency of data measured from satellite observation.

Benthic fauna and budgets of materials such as nitrogen, suspended solids and chlorophyll a were studied from June 2000 to September 2003 at the north pond of Nanko Bird Sanctuary and at an artificial tidal flat of Hannan Second District, Osaka Bay, Japan. Species and numbers od benthic organisms increased with time course at the artificial tidal flat of Hannan Second District, and its fauna became equivalent or rich compared to other artificial and/or natural tidal flats of the eutrphicated estuary of Japan. At the north pond of Nanko Bird Sanctuary, the output of suspended solids to coastal water exceeded its input to the pond, while chlorophyll a was intercepted within the pond through the tidal exchange. Dissolved inorganic nitrogen imported into the pond was temporally incorporated into the biomass and was then exported in the form of dissolved organic nitrogen. Particulate nitrogen was abundant as a resource of nitrogenous nutrients to coastal water in the summer of 2001. These phenomena are thought to be correlated with biological activities such as feeding, fixation and decomposition by benthic animals, seaweeds and microalgae. At the young artificial tidal flat, Hannan Second District, the net exchange of nitrogen significantly changed between 2000 and 2003. In September 2000 only five months after its construction, nitrogen was released from the artificial tidal flat to the adjacent waters at a rate of 5.39 kg N/day. In contrast, the artificial tidal flat functioned as a site of sink for nitrogen in relation to the growth of seaweeds in September of 2001 and 2002, but played as a source of nitrogen in accordance with seaweed decrese in October 2003. As a result, nitrogen uptake by the dominant species of seaweeds in Septembers 2001 and 2002 reached 4.8 and 3.3 kgN/day which accounted for 102 and 92% of the total nitrogen fixed at the artificial tidal flat within one day.

本研究は磯焼け現象で藻場が消失した海域や貧栄養化した海岸域に無機質の栄養素を多孔質材料(ポーラスコンクリート)内に挿入して沿岸海域に沈漬し、それらの栄養源を徐々に海域に溶出させ、藻場を再生または造成する方法を提案した。室内試験では無機系の栄養塩類を特殊なポリマーで2層構造としてシールし、コンクリートの製造時においても破壊されない状態として挿入し、肥料成分がコンクリートの強度や耐久性に与える影響について検討した。さらに実海域にこの種の多孔質複合材料を沈漬し、それらの機械的性質や藻類をはじめ各種生物の付着状態を照査した。これらの調査結果から、この手法は海岸水域の環境創造用材料としての利用が可能なることを示唆した。 具体的には次に示す内容の研究を実施し、それぞれ以下の研究成果を得た。 1)表面処理した粒状肥料が多孔質コンクリートの製造と硬化後の物性に与える影響につき調査した。 肥料成分をシールした粒状物を多孔質コンクリート内に混合挿入する手法を示した。また、硬化体の機械的性質として材令と圧縮強度をはじめ各種強度の関係を明らかにした。化学的性質としては、多孔質硬化体からの肥料成分の溶出状況を把握すると共に溶出量の制御方法を検討した。 2)自然海域に沈漬した肥料を混入した多孔質複合材料の諸性質 自然の海水中に上記の複合材料を沈漬した場合、強度等の機械的性質と肥料成分の溶出特性を経時的に調査した。その結果、骨材空隙への結合材量とその強度が複合材の耐久性と肥料成分の溶出に大きく影響することを明らかにした。 3)肥料を混入した多孔質複合材料への各種海洋生物の付着性状を検討した。 ポリマーで表面処理した肥料成分と鉄成分を多量に含有する鉄鉱石や転炉スラグを骨材として連続した空隙を有する多孔質コンクリートの硬化体への海藻類や小動物類の付着性状を調査した。 4)平成11年度から平成12年度に実施した研究結果を取り纏めた。

The purpose of this study was to develop the method for the early recovery of the biodiversity in the oligotrophical coastal area. It is important in the recovery of the biodiversity to make kelp forest grow in the concerned area. In order for it, sufficient nutrient is required as weii as the proper seedbed. Hence, in this study, granulated fertilizer, which contains nutrient, such as nitrogen, phosphorus and iron, is coated by the special polymer, and then, is mixed into the porous concrete in order to provide seedbed and nutrent simultaneously. The physical and chemical properties, such as voids ratio, water permeability, compressive strength and elution of nutrient from test specimens of porous concrete with granulated fertilizer are discussed. When this type of concrete is immersed shallowly into seawater, not only its rough surface, but also its continuous interior voids are fully exposed to water and rapidly neutralized. This will them lead to the attachment and growth of marine microbes and eventually to the formation of a layer of biotic membranes. As the results, decomposition and mineralization of the marine organic matters and the growth of algae attached. Especially, attachment of large algae and many kinds of animal are accelerated, thereby providing the water area with a better biological environments. Then brief note is appended to describe the method to create the biodiversity in sea area.

Vibriosis caused by Vibrio anguillarum seriously damages freshwater fish (Salmoniforms) almost every year in Lake Biwa, Japan. This pathogen requires NaCl for its growth. When the pathogen was exposed to sterilized aged lake water, it lost its culturability without loosing respiratory activity. Although this physiological state looks like "VBNC", they could not resuscitate even in host fish bodies. As these cells totally lost their pathogenicity, the VBNC-like state is probably on the way to death. Environmental factors such as darkness, coldness and anaerobiosis helped the pathogen to survive in freshwater without getting into nonculturable state. Culturable V. anguillarum cells were detected from planktonic samples such as freshwater amphipods (Jesogammarus annandalei), which migrate vertically. This indicates that freshwater amphipods have roles as intermediate hosts in the infectious process of V. anguillarum, which were deposited on the bottom sediments, to the final host, ayu.

1987年、カナダ東海岸で養殖ムラサキガイによる記憶喪失性中毒症が発生し、死者がでた。原因はムラサキガイの消化管に蓄積されていた羽状珪藻Psudonitzschia sp.の産生するドウモイ酸である。本藻は極めてありふれた珪藻類であり、わが国の沿岸域にも常在するが、その生活環には不明な点が多く残されていた。本研究では本藻の生活環を明らかにすることを目的とし,特に従属栄養的な増殖過程に注目して実験を行った。 本藻を光条件下、20℃において,無機栄養培地で培養した場合,最大増殖速度定数(μ_)は,1.0d^<-1>であった。次に,ペプトンと酵母エキスを主成分とする有機栄養培地において本藻を暗条件で培養したところ,培養開始直後から細胞は増殖し始め(μ_=0.4d^<-1>),培養開始4〜5日後から細胞が徐々に肥大化し,10日後には短径が元の2〜3倍となった。有機栄養培地にグルコースを添加した培地,ならびに無機栄養培地にグルコースまたはグルタミン酸を添加した培地における本藻の暗所における増殖を調べると,いずれの培地においても本藻は増殖し,μ_は,それぞれ1.0d^<-1>,1.4d^<-1>,0.4d^<-1>であった。この場合,グルコースが添加されていると増殖速度は大幅に増大し,しかも細胞の肥大化は起こらなかったが,グルタミン酸をエネルギー源とした場合,細胞が肥大化した。従属栄養的な増殖過程において肥大化した細胞を再び光条件下の無機栄養培地へ移したところ,それらの細胞は光合成独立栄養的に増殖し,通常の細長い舟形の形状に戻った。 従来の珪藻類の従属栄養増殖に関する研究では,利用可能な有機物の種類などに関する報告はあるものの,本研究で明らかにしているような,従属栄養増殖と形態変化に関する報告は皆無であり,本研究で得られた結果は本藻類の生活環に関する新しい知見といえる。

One of the most undesirable environmental conditions infish-culturing inland Bays might be the formation of oxygen deficient layer in bottom water in summer saeason.Toward an aid of the solution of the problem,wetried to elucidate the mechanism of organic polution of bottom sediment in fish-farm inland bays,comparin with that in usual eutrophicated water regions. From the vertical distributions of plant pigments,organic carbon and nitrogen,organic acids,aminoacid and sugar composition of organic substances in the bottom sediment,etc.in the two kinds of organic poluted water regions,the following inference can be drawn from the results obtained in this study.Namely,even though the sea bottom of fish-culturing inland bays are subjected to heavy loads of organic suspended matter with high nitrogen content which is derived from remaining bait and fish excretion,organic polution of the bottom sediment in the water region may proceed by th processes 1) sedimentaion of organic suspended matter on the bottom surface 2) decomposition and mineralization in the water-bottom surface layer 3) eutrophication of sea water j) growth of phytoplanktion 5) sedimentation of dead bodies of phytoplanktion 6) organic polution of bottom sediment.