Phage therapy minimally affects the water microbiota in an Atlantic salmon (Salmo salar) rearing system while still preventing infection.

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.

The impact of phage treatment on bacterial community structure is minor compared to antibiotics.

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 ß-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.

Aquaculture rearing systems induce no legacy effects in Atlantic cod larvae or their rearing water bacterial communities.

The microbial rearing quality influences the survival of marine larvae. Microbially matured water treatment systems (MMS) provide a more favourable rearing water microbiome than flow-through systems (FTS). It has previously been hypothesised, but not investigated, that initial rearing in MMS leaves a protective legacy effect in Atlantic cod larvae (Gadus morhua). We tested this hypothesis through a crossover 2 × 2 factorial experiment varying the rearing water treatment system (MMS vs FTS) and the microbial carrying capacity (+ /- added organic matter). At 9 days post-hatching, we switched the rearing water treatment system. By comparing switched and unswitched rearing tanks, we evaluated if legacy effects had been established in the larvae or their surrounding rearing water bacterial community. We analysed the bacterial communities with flow cytometry and 16S rRNA gene sequencing. We found no evidence that the initial rearing condition left a legacy effect in the communities by evaluating the bacterial community diversity and structure. Instead, the present rearing condition was the most important driver for differences in the rearing water microbiota. Furthermore, we found that MMS with high microbial carrying capacity appeared to seed a stable bacterial community to the rearing tanks. This finding highlights the importance of keeping a similar carrying capacity between the inlet and rearing water. Moreover, we reject the hypothesis that the initial rearing condition leaves a protective legacy effect in larvae, as the larval survival and robustness were linked to the present rearing condition. In conclusion, our results highlight the importance of maintaining a beneficial microbial rearing environment from hatching and throughout the larval rearing period.

Robust bacterial co-occurence community structures are independent of r- and K-selection history.

Selection for bacteria which are K-strategists instead of r-strategists has been shown to improve fish health and survival in aquaculture. We considered an experiment where microcosms were inoculated with natural seawater and the selection regime was switched from K-selection (by continuous feeding) to r-selection (by pulse feeding) and vice versa. We found the networks of significant co-occurrences to contain clusters of taxonomically related bacteria having positive associations. Comparing this with the time dynamics, we found that the clusters most likely were results of similar niche preferences of the involved bacteria. In particular, the distinction between r- or K-strategists was evident. Each selection regime seemed to give rise to a specific pattern, to which the community converges regardless of its prehistory. Furthermore, the results proved robust to parameter choices in the analysis, such as the filtering threshold, level of random noise, replacing absolute abundances with relative abundances, and the choice of similarity measure. Even though our data and approaches cannot directly predict ecological interactions, our approach provides insights on how the selection regime affects the composition of the microbial community, providing a basis for aquaculture experiments targeted at eliminating opportunistic fish pathogens.

The effect of periodic disturbances and carrying capacity on the significance of selection and drift in complex bacterial communities.

Understanding how periodical disturbances affect the community assembly processes is vital for predicting temporal dynamics in microbial communities. However, the effect of dilutions as disturbances are poorly understood. We used a marine bacterial community to investigate the effect of disturbance (+/-) and carrying capacity (high/low) over 50 days in a dispersal-limited 2 × 2 factorial study in triplicates, with a crossover in the disturbance regime between microcosms halfway in the experiment. We modelled the rate of change in community composition between replicates and used this rate to quantify selection and ecological drift. The disturbed communities increased in Bray-Curtis similarity with 0.011 ± 0.0045 (Period 1) and 0.0092 ± 0.0080 day-1 (Period 2), indicating that selection dominated community assembly. The undisturbed communities decreased in similarity at a rate of -0.015 ± 0.0038 day-1 in Period 1 and were stable in Period 2 at 0.00050 ± 0.0040 day-1, suggesting drift structured community assembly. Interestingly, carrying capacity had minor effects on community dynamics. This study is the first to show that stochastic effects are suppressed by periodical disturbances resulting in exponential growth periods due to density-independent biomass loss and resource input. The increased contribution of selection as a response to disturbances implies that ecosystem prediction is achievable.