Community Structure and Predicted Functions of Actively Growing Bacteria Responsive to Released Coral Mucus in Surrounding Seawater.

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.

Succession of the intestinal bacterial community in Pacific bluefin tuna (Thunnus orientalis) larvae.

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.

Community structure of actively growing bacteria in a coastal fish-farming area.

In fish-farming areas, copious amounts of organic matter are released into the surrounding environment. Although it is well-known that bacterial community structures and activities are tightly coupled with organic conditions in the environment, actively growing bacteria (AGB) species that are responsible are still largely unknown. Here, we determined seasonal variations in the community structures of free-living and particle-attached AGB in surface and bottom seawater, and also in the easily resuspendable sediment boundary layer. Accordingly, we used bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) analysis. Whereas overall bacterial communities in the resuspendable sediment were quite different from those of the free-living and particle-attached bacteria, the AGB community structures were similar among them. This result suggests that sediment resuspension in aquaculture environments functions as an organic source for bacteria in the water column, and that bacterial species contributing to the environmental capacity and carbon cycle are limited. We identified 25 AGB phylotypes, belonging to Alphaproteobacteria (Roseobacter clade, nine phylotypes), Gammaproteobacteria (five phylotypes), Deltaproteobacteria (one phylotype), Bacteroidetes (seven phylotypes), and Actinobacteria (three phylotypes). Among them, some AGB phylotypes appeared throughout the year with high frequency and were also identified in other coastal environments. This result suggests that these species are responsible for the environmental capacity and carbon cycle, and are key species in this fish-farming area, as well as other coastal environments.

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.

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 (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.

Significance of Na+ in the fish pathogen, Vibrio anguillarum, under energy depleted condition.

Vibrio anguillarum kills various kinds of fish over salinities ranging from seawater to freshwater. In this study, we investigated the role of Na(+) in V. anguillarum, especially under energy-depleted conditions such as in natural seawater. V. angustum S14, which is a typical marine vibrio, was used for comparison. V. anguillarum only required Na(+) for starvation-survival, but in contrast, V. angustum S14 always required Na(+) for both growth and starvation-survival. In marine vibrios, Na(+) is used in the Na(+)-dependent respiratory chain that produces the sodium motive force (SMF) across the cell membrane. It has been considered that marine vibrios always need a SMF produced by Na(+), however in the case of V. anguillarum, the SMF is not required for growth, but becomes more important for starvation-survival.

Characteristics of Na-dependent respiratory chain in Vibrio anguillarum, a fish pathogen, in comparison with other marine Vibrios.

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.

The starvation-stress response of Vibrio (Listoneila) anguillarum.

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.