Mitsuru Eguchi

Disinfection of seawater for hatchery aquaculture systems using electrolytic water treatment

A recently marketed electrolytic water treatment system (Hoshizaki) was evaluated for disinfection of seawater used in disease-prone high-intensity aquaculture systems. Bacterial plate counts (CFU), direct bacterial total counts using 4V,6V diamidino-2-phenylindole (DAPI) staining, and viable bacterial total counts using 6-carboxy fluorescein diacetate (6CFDA) showed complete inactivation of bacterial populations at an intensity of z1.3 amp (z2.13 mg Cl l 1 ). This included disinfection of seawater experimentally dosed with the known scallop pathogen Vibrio anguillarum. Experimental use of electrolysis between 1.0 and 4.0 A was able to disinfect cultures of the (food) microalga Isochrysis galbana without deleterious effects on its growth rate. When this technique was applied on a commercial scale in a scallop hatchery, higher microalgal growth rates were achieved compared to those of traditionally autoclaved seawater, or seawater treated with germicidal ultraviolet light (UV). Results suggested that disinfection of hatchery culture waters could be achieved using electrolytic release of very low levels of active Cl ion, providing an effective and economically attractive alternative to currently used methods in these culture systems. D 2002 Elsevier Science B.V. All rights reserved.

The starvation-stress response of Vibrio (Listoneila) anguillarum

Summary: 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 exposal to the latter three stresses after initiation of starvation was also examined. Results of these experiments indicated that when starved for carbon, nitrogeand 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 [35S]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 V. anguillarum differs significantly from those starvation responses reported for other bacteria, including responses displayed by other Vibrio species.

The phytoplankton Nannochloropsis oculata enhances the ability of Roseobacter clade bacteria to inhibit the growth of fish pathogen Vibrio 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 (109 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 14C- photosynthetic metabolites (14C-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.

Association between bacterial community structures and mortality of fish larvae in intensive rearing systems

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, γ-proteobacteria became dominant (∼70% of total bacteria) on day 13, there after heavy larval mortalities occurred. In Tank 1, however, α-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., γ-proteobacteria increased during rearing. In rearing water with Nannochloropsis sp., α-prote obacteria and the Cytophaga-Flavobacterium cluster were predominant at the beginning of the experiments and the relative abundance of γ-proteobacteria was maintained at a lower level throughout the experiments. The predominance of α-proteobacteria and the Cytophaga-Flavobacterium cluster appears to be a good indicator of successful larval production.

Population structure of the fish pathogen Flavobacterium psychrophilum at whole-country and model river levels in Japan

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-1). 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.

Analysis of bacterial communities in Nannochloropsis sp. cultures used for larval fish production

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 7.72×105-2.39×106 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 α-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.

Benefits of live phytoplankton, Chlorella vulgaris, as a biocontrol agent against fish pathogen Vibrio anguillarum

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.

Transcriptional regulation of the Na+-NADH:quinone oxidoreductase gene, nqr, in Vibrio anguillarum, a fish pathogen, in the stationary phase

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.

Erratum to: Microbial decomposition process of organic matter in sinking particles, resuspendable particles, and bottom sediments at a coastal fish farming area

Erratum to: Fish Sci (2017) 83:635–647 DOI 10.1007/s12562-017-1098-9.