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.

Gene expression patterns in Atlantic salmon (Salmo salar) with severe nephrocalcinosis.

Nephrocalcinosis is a common disorder in farmed Atlantic salmon, but the consequences for the fish physiology are not well understood. We performed a transcriptome study in kidneys of Atlantic salmon (Salmo salar) smolts without and with severe chronic nephrocalcinosis (NC). The study revealed that numerous genes are differentially expressed in fish with NC compared with healthy salmon. The most evident changes in gene expression patterns in the NC group were a massive downregulation of metabolism and energy production, upregulation of signalling pathways important for tissue repair and function maintenance and upregulation of inflammatory responses. Overall, the extensive tissue damage and the gene regulation responses that affect salmon with severe nephrocalcinosis are highly likely to have dramatic consequences on fish survival.

Bacterial community assembly in Atlantic cod larvae (Gadus morhua): contributions of ecological processes and metacommunity structure.

Many studies demonstrate the importance of the commensal microbiomes to animal health and development. However, the initial community assembly process is poorly understood. It is unclear to what extent the hosts select for their commensal microbiota, whether stochastic processes contribute, and how environmental conditions affect the community assembly. We investigated community assembly in Atlantic cod larvae exposed to distinct microbial metacommunities. We aimed to quantify ecological processes influencing community assembly in cod larvae and to elucidate the complex relationship between the bacteria of the environment and the fish. Selection within the fish was the major determinant for community assembly, but drift resulted in inter-individual variation. The environmental bacterial communities were highly dissimilar from those associated with the fish. Still, differences in the environmental bacterial communities strongly influenced the fish communities. The most striking difference was an excessive dominance of a single OTU (Arcobacter) for larvae reared in two of the three systems. These larvae were exposed to environments with higher fractions of opportunistic bacteria, and we hypothesise that detrimental host-microbe interactions might have made the fish susceptible to Arcobacter colonisation. Despite strong selection within the host, this points to a possibility to steer the metacommunity towards mutualistic host-microbe interactions and improved fish health and survival.

K-Selection as Microbial Community Management Strategy: A Method for Improved Viability of Larvae in Aquaculture.

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.

Managing the Microbial Community of Marine Fish Larvae: A Holistic Perspective for Larviculture.

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.

Rearing Water Treatment Induces Microbial Selection Influencing the Microbiota and Pathogen Associated Transcripts of Cod (Gadus morhua) Larvae.

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.

Gene regulation of lipid and phospholipid metabolism in Atlantic cod (Gadus morhua) larvae.

The mechanism of essentiality of dietary phospholipid (PL) for larval fish is not clear. The main objective of the present study was to determine if the PL requirement of Atlantic cod larvae was due to any genetic impairment caused by functional immaturity. Cod larvae were sampled at 1, 3, 8, 13, 17, 18, 30, 42 and 60 days post hatch (dph) for transcriptome analysis using a recently developed microarray. The fatty acid profile and gene expression levels of cod larvae at 17 dph were compared after feeding differently enriched rotifers, which contained different DHA levels in PL. No significant differences (p<0.05) were found for the two rotifer diets in the overall gene expression level of cod larvae, their growth and survival, and their DHA levels in total lipid and PL fraction. The fatty acid data suggested that dietary EPA was elongated to DPA by cod larvae, and a threshold DHA level in PL to maintain membrane fluidity and other functions may exist. There appeared to be no major effect of development on the expression of key genes of PL biosynthesis suggesting no genetic constrain in early developmental stages. Our overall data suggested that besides the possible limited de novo PC synthesis ability in the intestine, other metabolic constraints should also be considered, especially the possible low input of bile PC as a result of immature liver. Further studies are needed to elucidate the gene expression level and enzyme activity in the PL biosynthesis pathways for specific tissue or cells.