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Gut microbial communities are highly dynamic and respond to factors such as diet and host metabolism. However, there is limited information about how these responses can generate community dynamics at different time scales, such as circadian rhythms. The aim of the present study was to characterize variations along a daily cycle in the gut microbiota of fish on different feeding regimes. In addition, an estimation of the potential contribution of the gut bacterial community to the digestive process of the fish was assessed. The greater amberjack (Seriola dumerili) was used as experimental species, a promising species for Mediterranean aquaculture. Fish were randomly distributed in two different feeding regimes, in triplicate rearing tanks. Feeding regimes were continuous feeding and time-restricted feeding (three meals per day). Fish feces for RNA-based amplicon sequencing and bacterial proteolytic capacity were sampled along a 24h cycle. Time-restricted fed fish exhibited changes in the composition of the active bacterial community promoted by feeding, thus revealing two types of bacterial community profiles: “pre-feeding” and “active-feeder” profiles. Continuous-fed fish maintained an “active-feeder” community profile throughout the whole day. Bray-Curtis and Sørensen similarity indicated that differences in the composition between the two community profiles were mainly driven by the absence/presence of infrequent ASVs (amplicon variant sequences). The potential proteolytic bacterial activity tended to be higher before feeding. Thus, gut microbiota could be a key factor to understanding fish digestion, playing a role in interactions with host metabolism. In conclusion, our results suggest that feeding pattern and time of sampling are important parameters when assessing the microbiome’s contribution to host metabolism.

Abstract Excessive usage of antibiotics threatens the bacterial diversity in the microbiota of animals. An alternative to antibiotics that has been suggested to not disturb the microbiota is (bacterio)phage therapy. In this study, we challenged germ-free and microbially colonized yolk sac fry of Atlantic salmon with Flavobacterium columnare and observed that the mere presence of a microbiota protected the fish against lethal infection. We then investigated the effect of phage- or oxytetracycline treatment on fish survival and rearing water bacterial community characteristics using 16S rRNA gene amplicon sequencing. Phage treatment led to an increased survival of F. columnare-challenged fish and reduced the relative amounts of the pathogen in the water microbiota. In the absence of F. columnare, phage treatment did not affect the composition or the α-diversity of the rearing water microbiota. In the presence of the phage’s host, phage treatment induced minor changes to the bacterial community composition, without affecting the α-diversity. Surprisingly, oxytetracycline treatment had no observable effect on the water microbiota and did not reduce the relative abundance of F. columnare in the water. In conclusion, we showed that phage treatment prevents mortality while not negatively affecting the rearing water microbiota, thus suggesting that phage treatment may be a suitable alternative to antibiotics. We also demonstrated a protective effect of the microbiota in Atlantic salmon yolk sac fry.

Abstract Phage treatment is suggested as an alternative to antibiotics; however, there is limited knowledge of how phage treatment impacts resident bacterial community structure. When phages induce bacterial lysis, resources become available to the resident community. Therefore, the density of the target bacterium is essential to consider when investigating the effect of phage treatment. This has never been studied. Thus, we invaded microcosms containing a lake-derived community with Flavobacterium columnare strain Fc7 at no, low or high densities, and treated them with either the bacteriophage FCL-2, the antibiotic Penicillin or kept them untreated (3 × 3 factorial design). The communities were sampled over the course of one week, and bacterial community composition and density were examined by 16S rDNA amplicon sequencing and flow cytometry. We show that phage treatment had minor impacts on the resident community when the host F. columnare Fc7 of the phage was present, as it caused no significant differences in bacterial density α- and β-diversity, successional patterns, and community assembly. However, a significant change was observed in community composition when the phage host was absent, mainly driven by a substantial increase in Aquirufa. In contrast, antibiotics induced significant changes in all community characteristics investigated. The most crucial finding was a bloom of γ-proteobacteria and a shift from selection to ecological drift dominating community assembly. This study investigated whether the amount of a bacterial host impacted the effect of phage treatment on community structure. We conclude that phage treatment did not significantly affect the diversity or composition of the bacterial communities when the phage host was present, but introduced changes when the host was absent. In contrast, antibiotic treatment was highly disturbing to community structure. Moreover, higher amounts of the bacterial host of the phage increased the contribution of stochastic community assembly and resulted in a feast-famine like response in bacterial density in all treatment groups. This finding emphasises that the invader density used in bacterial invasion studies impacts the experimental reproducibility. Overall, this study supports that phage treatment is substantially less disturbing to bacterial communities than antibiotic treatments.

The bacterial colonization of newly hatched fish is important for the larval development and health. Still, little is known about the ontogeny of the early microbiota of fish. Here, we conducted two independent experiments with yolk sac fry of Atlantic salmon that were (1) either reared conventionally, with the eggs as the only source for bacteria (egg-derived microbiota; EDM) or (2) hatched germ-free and re-colonized using lake water (lake-derived microbiota; LDM). First, we characterized the gut and skin microbiota at 6, 9, and 13 weeks post hatching based on extracted RNA. In the second experiment, we exposed fry to high doses of either a fish pathogen or a commensal bacterial isolate and sampled the microbiota based on extracted DNA. The fish microbiota differed strongly between EDM and LDM treatments. The phyla Proteobacteria, Bacteroidetes, and Actinobacteria dominated the fry microbiota, which was found temporarily dynamic. Interestingly, the microbiota of EDM fry was more stable, both between replicate rearing flasks, and over time. Although similar, the skin and gut microbiota started to differentiate during the yolk sac stage, several weeks before the yolk was consumed. Addition of high doses of bacterial isolates to fish flasks had only minor effects on the microbiota.

Gnotobiotic models have had a crucial role in studying the effect that commensal microbiota has on the health of their animal hosts. Despite their physiological and ecological diversity, teleost fishes are still underrepresented in gnotobiotic research. Moreover, a better understanding of host-microbe interactions in farmed fish has the potential to contribute to sustainable global food supply. We have developed a novel gnotobiotic experimental system that includes the derivation of fertilized eggs of farmed and wild Atlantic salmon, and gnotobiotic husbandry of fry during the yolk sac stage. We used a microscopy-based approach to estimate the barrier function of the skin mucus layer and used this measurement to select the derivation procedure that minimized adverse effects on the skin mucosa. We also used this method to demonstrate that the mucus barrier was reduced in germ-free fry when compared to fry colonized with two different bacterial communities. This alteration in the mucus barrier was preceded by an increase in the number of cells containing neutral mucosubstances in the anterior segment of the body, but without changes in the number of cells containing acidic substances in any of the other segments studied along the body axis. In addition, we showed how the microbial status of the fry temporarily affected body size and the utilization of internal yolk stores during the yolk sac stage. Finally, we showed that the presence of bacterial communities associated with the fry, as well as their composition, affected the size of adipose tissue. Fry colonized with water from a lake had a larger visceral adipose tissue depot than both conventionally raised and germ-free fry. Together, our results show that this novel gnotobiotic experimental system is a useful tool for the study of host-microbe interactions in this species of aquacultural importance.

Abstract Background Fish skin represents an ancient vertebrate mucosal surface, sharing characteristics with other mucosal surfaces including those of the intestine. The skin mucosa is continuously exposed to microbes in the surrounding water and is therefore important in the first line defense against environmental pathogens by preventing bacteria from accessing the underlying surfaces. Understanding the microbe-host interactions at the fish skin mucosa is highly relevant in order to understand and control infection, commensalism, colonization, persistence, infection, and disease. Here we investigate the interactions between the pathogenic bacteria Aeromonas salmonicida (A. salmonicida) and Yersinia ruckeri (Y. ruckeri), respectively, and the skin mucosal surface of Atlantic salmon fry using AFM force spectroscopy. Results The results obtained revealed that when retracting probes functionalized with bacteria from surfaces coated with immobilized mucins, isolated from salmon mucosal surfaces, rupture events reflecting the disruption of adhesive interactions were observed, with rupture strengths centered around 200 pN. However, when retracting probes functionalized with bacteria from the intact mucosal surface of salmon fish fry no adhesive interactions could be detected. Furthermore, rheological measurements revealed a near fluid-like behavior for the fish fry skin mucus. Taken together, the experimental data indicate that the adhesion between the mucin molecules within the mucous layer may be significantly weaker than the interaction between the bacteria and the mucin molecules. The bacteria, immobilized on the AFM probe, do bind to individual mucins in the mucosal layer, but are released from the near fluid mucus with little resistance upon retraction of the AFM probe, to which they are immobilized. Conclusion The data provided in the current paper reveal that A. salmonicida and Y. ruckeri do bind to the immobilized mucins. However, when retracting the bacteria from intact mucosal surfaces, no adhesive interactions are detected. These observations suggest a mechanism underlying the protective function of the mucosal surface based on the clearing of potential threats by adhering them to loosely attached mucus that is subsequently released from the fish skin.

Abstract In this study, nitrification before and after abrupt cross-transfer in salinity was investigated in two moving bed biofilm reactors inoculated with nitrifying cultures that had adaptation to freshwater (FR) and seawater salinities (SR). FR and SR MBRRs were exposed to short and long term cross-transfer in salinity, and the functional capacity of nitrifying microbial communities was quantified by the estimation of ammonia and nitrite oxidation rates. Salinity induced successions were evaluated before and after salinity change by deep sequencing of 16S rRNA gene amplicons and statistical analysis. The bacterial community structure was characterized and Venn diagrams were included. The results indicated that after salinity cross-transfer, the FR was not significantly recovered at seawater salinity whereas SR showed high resistance to stress caused by low-salt. Succession and physiological plasticity were the main mechanisms of the long-term adaption of the nitrifying communities exposed to abrupt salinity changes. Independently of salinity, some nitrifiers presented high physiological plasticity towards salinity and were very successful at both zero and full seawater salinity. SR culture is robust and suitable inoculum for ammonium removal from recirculating aquaculture systems and industrial wastewaters with variable and fast salinity changes. Our findings contradict the current perspective of the significance of salinity on the structure of nitrifying communities.

Decline in fish oil and fish meal availability has forced the aquaculture sector to investigate alternative and sustainable aquafeed ingredients. Despite that several studies have evaluated the effect of fish oil replacement in aquaculture fish species, there is a knowledge gap on the effects of alternative dietary lipid sources on the gut microbiota in early life stages of Salmo salar. The present study evaluated the influence of dietary administration of two different lipid sources (fish oil and vegetable oil) on the intestinal microbiota of first feeding Atlantic salmon (S. salar) up to 93 days post first feeding (dpff). The two diets used in this study, FD (fish oil diet) and VD (blend of rapeseed, linseed and palm oils diet), were formulated to cover the fish nutritional requirements. Apart from the lipid source, the rest of the feed components were identical in the two diets. Hindgut samples were collected at 0, 35, 65, and 93 dpff. Moreover, fertilized eggs, yolk sac larvae, rearing water and feed were also collected in order to assess a possible contribution of their microbiota to the colonization and bacterial succession of the fish intestines. To analyze the bacterial communities, amplicon sequencing was used targeting the V3–V4 region of the 16S rRNA gene. The findings indicate that feeding on either fish oil or vegetable oil-based diet, fish growth variables (mean wet weight and total length) did not differ significantly during the experiment (p > 0.05). No significant differences were also found between the two dietary groups, regarding their gut bacteria composition, after the analysis of the 16S rRNA sequencing data. Instead, gut microbiota changed with age, and each stage was characterized by different dominant bacteria. These operational taxonomic units (OTUs) were related to species that provide different functions and have been isolated from a variety of environments. The results also show little OTUs overlap between the host and rearing environment microbiota. Overall, this study revealed the occurrence of a core microbiota in early life of Atlantic salmon independent of the feed-contained oil origin.

Abstract In upflow anaerobic sludge bed (UASB) reactors, biomass present as granules allows for long solids retention time. Here, granules from a process treating pulp and paper industrial wastewater were successfully applied as inoculum in UASB reactors treating pig manure supernatant, despite high particle content and high ammonium concentrations in the influent. We did a detailed characterization of archaeal and bacterial communities associated with the inoculum and with the aggregated and dispersed fractions of the influent and the reactors after one year of operation. The granular communities underwent major changes and adapted to the highly distinct conditions without disintegration of the granules. Although the granules persisted in the reactors, non-granular aggregates accumulated, and partly replaced the granules. Particles introduced to the reactors by the pig manure influent apparently contributed both as food and biofilm growth support. Archaeal communities in the dispersed reactor phase were similar to those dispersed in the influents, implying successful retention and little loss of archaeal biomass due to detachment or disintegration of granules and other aggregates. Unique bacterial communities developed in the dispersed fraction of the reactors despite of low hydraulic retention times. They probably consisted of fast growing organisms consuming readily degradable organic matter.

The roles of host-associated bacteria have gained attention lately, and we now recognise that the microbiota is essential in processes such as digestion, development of the immune system and gut function. In this study, Atlantic cod larvae were reared under germ-free, gnotobiotic and conventional conditions. Water and fish microbiota were characterised by 16S rRNA gene analyses. The cod larvae’s transcriptional responses to the different microbial conditions were analysed by a custom Agilent 44 k oligo microarray. Gut development was assessed by transmission electron microscopy (TEM). Water and fish microbiota differed significantly in the conventional treatment and were dominated by different fast-growing bacteria. Our study indicates that components of the innate immune system of cod larvae are downregulated by the presence of non-pathogenic bacteria, and thus may be turned on by default in the early larval stages. We see indications of decreased nutrient uptake in the absence of bacteria. The bacteria also influence the gut morphology, reflected in shorter microvilli with higher density in the conventional larvae than in the germ-free larvae. The fact that the microbiota alters innate immune responses and gut morphology demonstrates its important role in marine larval development.

Abstract Little information is available on the link between host development (growth rate and ontogeny) and the composition of the microbiota in fish larvae. This study was carried out to examine potential correlations of microbiota composition with age and growth rate of Atlantic cod larvae. Small and large cod larvae of the same age, representing slow and fast growing individuals, were sampled 10 times during a period of 42 days post hatching (dph), and the composition of the larval microbiota was investigated using a PCR/DGGE (Denaturing Gradient Gel Electrophoresis) strategy. We found significant differences in the intestinal microbiota of small and large larvae of the same age for 4 of the 10 age stages studied. We further found that the variation in the composition of the larval microbiota was more strongly correlated to age than to growth rate for larvae up to 28 dph, whereas for the older larvae growth rate and age was equally correlated to the composition of the microbiota. These results indicate that larval development may structure the microbiota through a change in selection pressure due to host-microbe and microbe-microbe interactions, and that the composition of the microbiota may influence larval development through improved energy gain.

Aquaculture has the potential to become a major food supplier in a world with an increasing human population, and increased consumption of fish will likely have positive health implications. For marine aquaculture, the production of high quality juveniles is a bottleneck. Survival until the juvenile stage is typically as low as 10–15% for many species, which indicates suboptimal rearing conditions. Substantial evidence indicates that the poor performance and viability of larvae is largely due to detrimental larvae-microbiota interactions. This emphasises the need for microbial management strategies in the cultivation of marine fish larvae. Disinfection and probiotics are the most studied microbial management methods so far. However, most studies on these methods overlooked the role of mutualistic relationships between microbes and hosts, and have not proposed or examined methods steering toward such relationships. Based on ecological theory and a number of experiments, we find support for the hypothesis that current practise in aquaculture generally selects for r-strategic, opportunistic microbes, which results in detrimental host–microbiota interactions. Thus, the challenge is to develop technology and methods for microbial management at the ecosystem level that creates a K-selected microbial community, and by this mean select against r-strategic opportunists. Here we summarise experiments done during 25 years and with marine larvae of five different species showing that: (1) K-selection strategies result in different water microbiota with less opportunists, (2) this influences the microbiota of the fish larvae, and (3) the larvae cultivated in water inhabited by a K-selected microbiota perform better. Improved performance of larvae includes improved appetite, earlier onset of and faster growth, increased survival, and increased robustness to stress. K-selection as a method for management of the microbial community is a robust approach that allows steering of host–microbiota interactions in larviculture toward mutualism. It could also be applicable for young stages of other domesticated animals. Our review illustrates that a change from a “beat-them” to a “join-them” strategy for microbial management in larval rearing can lead to a more sustainable aquaculture industry.

The availability of high-quality juveniles is a bottleneck in the farming of many marine fish species. Detrimental larvae-microbe interactions are a main reason for poor viability and quality in larval rearing. In this review, we explore the microbial community of fish larvae from an ecological and eco-physiological perspective, with the aim to develop the knowledge basis for microbial management. The larvae are exposed to a huge number of microbes from external and internal sources in intensive aquaculture, but their relative importance depend on the rearing technology used (especially flow-through vs. recirculating systems) and the retention time of the water in the fish tanks. Generally, focus has been on microbes entering the system, but microbes from growth within the system is normally a substantial part of the microbes encountered by larvae. Culture independent methods have revealed an unexpected high richness of bacterial species associated with larvae, with 100–250 operational taxonomic units associated with one individual. The microbiota of larvae changes rapidly until metamorphosis, most likely due to changes in the selection pressure in the digestive tract caused by changes in host-microbe and microbe-microbe interactions. Even though the microbiota of larvae is distinctly different from the microbiota of the water and the live food, the microbiota of the water strongly affects the microbiota of the larvae. We are in the early phase of understanding larvae-microbe interactions in vivo, but some studies with other animals than fish emphasize that we so far have underestimated the complexity of these interactions. We present examples demonstrating the diversity of these interactions. A large variety of microbial management methods exist, focusing on non-selective reduction of microbes, selective enhancement of microbes, and on improvement of the resistance of larvae against microbes. However, relatively few methods have been studied extensively. We believe that there is a lot to gain by increasing the diversity of approaches for microbial management. As many microbial management methods are perturbations of the microbial community, we argue that ecological theory is needed to foresee and test for longer term consequences in microbe-microbe and microbe-larvae interactions. We finally make some recommendations for future research and development.

We have previously shown that K-selection and microbial stability in the rearing water increases survival and growth of Atlantic cod (Gadus morhua) larvae, and that recirculating aquaculture systems (RAS) are compatible with this. Here, we have assessed how water treatment influenced the larval microbiota and host responses at the gene expression level. Cod larvae were reared with two different rearing water systems: a RAS and a flow-through system (FTS). The water microbiota was examined using a 16S rDNA PCR/DGGE strategy. RNA extracted from larvae at 8, 13, and 17 days post hatching was used for microbiota and microarray gene expression analysis. Bacterial cDNA was synthesized and used for 16S rRNA amplicon 454 pyrosequencing of larval microbiota. Both water and larval microbiota differed significantly between the systems, and the larval microbiota appeared to become more dissimilar between systems with time. In total 4 phyla were identified for all larvae: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The most profound difference in larval microbiota was a high abundance of Arcobacter (Epsilonproteobacteria) in FTS larvae (34 ± 9% of total reads). Arcobacter includes several species that are known pathogens for humans and animals. Cod larval transcriptome responses were investigated using an oligonucleotide gene expression microarray covering approximately 24,000 genes. Interestingly, FTS larvae transcriptional profiles revealed an overrepresentation of upregulated transcripts associated with responses to pathogens and infections, such as c1ql3-like, pglyrp-2-like and zg16, compared to RAS larvae. In conclusion, distinct water treatment systems induced differences in the larval microbiota. FTS larvae showed up-regulation of transcripts associated with responses to microbial stress. These results are consistent with the hypothesis that RAS promotes K-selection and microbial stability by maintaining a microbial load close to the carrying capacity of the system, and ensuring long retention times for both bacteria and water in the system.

BackgroundReducing health inequalities is a challenge for policymakers and civil society. A multisectoral and multilevel approach is most promising to reduce those inequalities. Previous research showed what key elements of Zwolle Healthy City, an integrated community-based approach aimed at reducing socioeconomic health inequalities, are. To fully understand approaches that are complex and context dependent, questions as 'how does the intervention work' and 'in what context does it work' are just as important as 'what works'. The current study aimed to identify mechanisms and contextual factors associated with the key elements of Zwolle Healthy City, using a realist evaluation perspective.MethodsTranscripts of semi-structured interviews with a wide range of local professionals were used (n = 29). Following realist evaluation logic in the analysis of this primary data, context-mechanism-outcome configurations were identified and thereafter discussed with experts (n = 5).ResultsHow mechanisms (M) in certain contexts (C) were of influence on the key elements (O) of the Zwolle Healthy City approach are described. For example, how, in the context of the responsible aldermen embracing the approach (C), regular meetings with the aldermen (M) increased support for the approach among involved professionals (O). Or, how, in the context of available financial resources (C), assigning a program manager (M) contributed positively to coordination and communication (O). All 36 context-mechanism-outcome configurations can be found in the repository.ConclusionThis study showed what mechanisms and contextual factors are associated with the key elements of Zwolle Healthy City. By applying realist evaluation logic in the analysis of primary qualitative data we were able to disentangle the complexity of processes of this whole system approach and show this complexity in a structured manner. Also, by describing the context in which the Zwolle Healthy City approach is implemented, we contribute to the transferability of this approach across different contexts.

BackgroundSince 2010, the Zwolle Healthy City approach, an integrated community-based approach, has been implemented in the Dutch municipality of Zwolle. This approach is proven successful in reducing health inequalities. However, the key elements of this approach are not clear. The current study aimed to identify key elements of this successful local community-based approach, according to the perspectives of various stakeholders.MethodsSemi-structured interviews were carried out with 29 professionals who were involved in the approach in the period 2010-2018 and have occupations at the strategic (n = 4), tactical (n = 17) and operational level (n = 8). Data was analyzed using the thematic analysis approach.ResultsWe identified nine perceived key elements that contributed to the success of the approach aimed at reducing socioeconomic health inequalities. The respondents indicated the following key elements: (1) collaboration between a variety of local organizations that want to have impact on the health of citizens; (2) support for the approach on the strategic, tactical and operational level of involved organizations; (3) proper communication and coordination, both for the network and within the organizations; (4) embeddedness in organizations' policies and processes and (5) collaboration with private organizations is of added value, although there is no "one size fits all". Other key elements are (6) collaboration with citizens, (7) profiling the approach like a brand and (8) moving along with and taking advantage of opportunities. Finally, (9) continuous monitoring and evaluating goals and processes, and learning from the results, is important.ConclusionNine key elements were identified that, according to various stakeholders, contributed to the success of the Zwolle Healthy City approach. These insights are important to further strengthen the Zwolle Healthy City approach but may also help and inspire other local integrated community-based approaches aimed at reducing socioeconomic health inequalities, to improve and adapt the approach within their specific local and dynamic context.