Håkon Dahle

Persistence of Infectious Salmon Anaemia Virus (ISAV) and the effect of practical biosecurity interventions was evaluated in small-scale moving-bed RAS units and bioreactors. ISAV HPRΔ infected Atlantic salmon (~ 50 g) were stocked to three RAS units containing 15 ppt brackish water and removed after 14 days. After removal of shedder fish, the systems were drained, lightly scrubbed, refilled and fallowed for a variable period before restocking with naïve fish. In parallel, biomedia from each RAS unit were transferred to triplicate bioreactors and exposed to three field-relevant biosecurity treatments i.e. no treatment, water replacement, or ozonation. In the RAS units, detectable ISAV RNA levels increased in water, swab, and biomedia samples during the shedding period, peaking on the day when shedder fish were removed (day 0). Thereafter, detection declined rapidly, with water and swab samples consistently indicating higher ISAV RNA levels than biomedia (p = 0.004). The virus could not be detected 17 days post-removal of shedder fish. All naïve fish were ISAV-negative at the end of study. ISAV RNA detection levels varied according to treatment and sample matrix, with no detection in the ozonated reactors and sporadic detection in the bioreactors with the two other treatments up to 14 days after onset of the biosecurity intervention. The findings indicate that ISAV may persist transiently in the RAS environment with uncertain infectivity post fallowing and may be influenced by several factors such as environmental conditions, host-pathogen interactions and support the use of ozone as an effective disinfection method of water and biomedia.

Persistence of Infectious Salmon Anaemia Virus (ISAV) and the effect of practical biosecurity interventions was evaluated in small-scale moving-bed RAS units and bioreactors. ISAV HPRΔ infected Atlantic salmon (~ 50 g) were stocked to three RAS units containing 15 ppt brackish water and removed after 14 days. After removal of shedder fish, the systems were drained, lightly scrubbed, refilled and fallowed for a variable period before restocking with naïve fish. In parallel, biomedia from each RAS unit were transferred to triplicate bioreactors and exposed to three field-relevant biosecurity treatments i.e. no treatment, water replacement, or ozonation. In the RAS units, detectable ISAV RNA levels increased in water, swab, and biomedia samples during the shedding period, peaking on the day when shedder fish were removed (day 0). Thereafter, detection declined rapidly, with water and swab samples consistently indicating higher ISAV RNA levels than biomedia (p = 0.004). The virus could not be detected 17 days post-removal of shedder fish. All naïve fish were ISAV-negative at the end of study. ISAV RNA detection levels varied according to treatment and sample matrix, with no detection in the ozonated reactors and sporadic detection in the bioreactors with the two other treatments up to 14 days after onset of the biosecurity intervention. The findings indicate that ISAV may persist transiently in the RAS environment with uncertain infectivity post fallowing and may be influenced by several factors such as environmental conditions, host-pathogen interactions and support the use of ozone as an effective disinfection method of water and biomedia.

Molecular hydrogen (H2) is a widespread, energetically efficient reductant supporting microbial metabolism across most known ecosystems. Although seafloor hydrothermal vents are major energy providers for H2-oxidizing microorganisms, the diversity of H2 oxidation potential in H2-rich systems remains poorly constrained. Here, we use a metagenomic approach to, for the first time, assess the genome-resolved microbial energy conservation potential within hydrothermal deposits and sediments from the ice-covered, extraordinarily H2-rich Aurora Vent Field in the Arctic Ocean. Community-wide analysis revealed broad taxonomic representation of microorganisms with the potential to consume H₂ for energy conservation. Notably, we report the first genome belonging to the cosmopolitan Zetaproteobacteria genus Mariprofundus encoding the capacity for H2 oxidation. Additionally, novel, highly abundant Aquificota at Aurora encode uptake hydrogenases not previously characterized as central to H2 oxidation at deep sea vents. The encoded gene content of abundant taxa points to a preference for flexible rather than obligate lithotrophic energy metabolism. A substantial fraction of inferred H₂-oxidizing potential is associated with presumed heterotrophs, potentially enhancing carbon transfer efficiency within the Aurora microbial food web. Overall, this study sheds new light on the importance of H2 availability for shaping microbial communities in hydrothermal systems.

Iron-oxidizing Betaproteobacteria and Zetaproteobacteria are generally associated with freshwater and marine environments, respectively. Despite repeated cross-environment observations of these taxa, there has been no focused exploration of the genomes of marine Gallionella (Betaproteobacteria) to understand transitions between freshwater and marine habitats. Consequently, their roles in these environments remain uncertain. Here, we present strong evidence for the co-occurrence of Gallionella and Zetaproteobacteria at deep-sea hydrothermal vents at the Arctic Mid-Ocean Ridges through metagenomic analyses. Phylogenomic analysis of Gallionella metagenome-assembled genomes (MAGs) suggests that seawater adaptation is an evolutionary event that occurred multiple times in distinct lineages. Similarly, several distinct evolutionary events for freshwater and terrestrial Mariprofundus and other Zetaproteobacteria are predicted. The presence of c -type cytochrome cyc2 iron oxidation genes in co-occurring marine Betaproteobacteria and Zetaproteobacteria implies an overlap in niches of these iron-oxidizers. Functional enrichment analyses reveal genetic differences between marine MAGs of both iron-oxidizing groups and their terrestrial aquatic counterparts linked to salinity adaptation. Though scanning electron microscopy confirms the presence of Fe(III) oxyhydroxide stalks where Gallionella and Mariprofundus co-occur, Gallionella MAGs from hydrothermal vents lack evidence of putative stalk formation genes. Mariprofundus is therefore the likely sole stalk-producing iron-oxidizer in this environment. Conversely, the discovery of putative stalk formation genes in Mariprofundus MAGs across the marine-freshwater barrier suggests that Fe(III) oxyhydroxide stalks might not be an exclusive signature for single iron-oxidizing taxa in marine and freshwater environments. Our research provides novel insights into the iron-oxidizing capacities, stalk production, environmental adaptation, and evolutionary transitions between marine and freshwater habitats for Gallionella and Zetaproteobacteria. IMPORTANCE: Iron-oxidizing bacteria (FeOB) play an important role in the global cycling of iron, carbon, and other metals. While it has previously been assumed that bacterial evolution does not frequently involve crossing the salinity barrier, recent studies indicate that such occurrences are more common than previously thought. Our study offers strong evidence that this also happens among FeOB, with new insights into how these bacteria adapt to the new environment, including hydrothermal vents and freshwater habitats. In addition, we emphasize the importance of accurate iron-oxidizing taxa identification through sequencing, rather than relying solely on the morphology of Fe(III) oxyhydroxides and environment. On a larger scale, microorganisms within established communities need to respond to changes in salinity due to events like seawater intrusion in coastal aquifers, and thus, our findings underscore the importance of knowledge of transitions across habitat types with different salt concentrations.

Sugar kelp (Saccharina latissima) is an ecologically and increasingly economically important kelp, distributed from temperate to Arctic rocky shores. However, S. latissima is presently threatened by ongoing climate changes. Genetic variations have previously been identified across S. latissima populations. However, little is known regarding the genetic basis for adaptation and acclimation to different environmental conditions. In this study, a common garden experiment was performed with sporophytes originated from North‐Norway (NN), Mid‐Norway (MN), and South‐Norway (SN), representing areas with highly different temperatures and photoperiods. Transcriptomic analyses revealed significant variation in the gene expression of cultures from North‐Norway, associated with low temperature and long photoperiods, compared to Mid‐ and South‐Norway. Differentially expressed genes included genes linked to photosynthesis, chlorophyll biosynthesis, and heat response, suggesting that they are directly involved in temperature and light adaptation. In addition, genes related to growth, metabolism, protein synthesis, and translation were upregulated in the NN genotype, providing evidence that the NN genotype is better adapted to low temperatures than the SN and MN genotypes. Significant variation in gene expression among populations found in this study is influenced by the environment, but genetic differentiation by origin seems to play a role as responses were population specific. This study provides a baseline for deeper insight into the local adaptation potential of S. latissima populations along the Norwegian Coast with implications for the conservation of natural populations.

Sugar kelp (Saccharina latissima) is an ecologically and increasingly economically important kelp, distributed from temperate to Arctic rocky shores. However, S. latissima is presently threatened by ongoing climate changes. Genetic variations have previously been identified across S. latissima populations. However, little is known regarding the genetic basis for adaptation and acclimation to different environmental conditions. In this study, a common garden experiment was performed with sporophytes originated from North-Norway (NN), Mid-Norway (MN) and South-Norway (SN), representing areas with highly different temperatures and photoperiods. Transcriptomic analyses revealed significant variation in the gene expression of cultures from North-Norway, associated with low temperature and long photoperiods, compared to Mid- and South-Norway. Differentially expressed genes included genes linked to photosynthesis, chlorophyll biosynthesis, and heat response exhibited differential expression, suggesting that they are directly involvement in temperature and light adaptation. In addition, genes related to growth, metabolism, protein synthesis, and translation were upregulated in the NN genotype proving evidence that the NN genotype is better adapted to low temperatures than the SN and MN genotypes. Significant variation in gene expression among populations found in this study is influenced by the environment, but genetic differentiation by origin seems to play a role as responses were population specific. This study provides a baseline for deeper insight into the local adaptation potential of S. latissima populations along the Norwegian Coast with implications for the conservation of natural populations.

Maintaining good water quality is essential for successful fish production in land-based recirculating aquaculture systems (RAS). Numerous interdependent factors influence water quality parameters, making it difficult to evaluate which operational strategies are most favorable. Mathematical models and model simulations have proven to be powerful tools to evaluate how RAS design and operation are linked to RAS dynamics, but these models rarely implement pH and carbonate species as dynamic variables. Here, we present a dynamic model for RAS (dynRAS) that combines rates of TAN (total ammonia nitrogen) removal, fish growth, and CO₂ and TAN excretion, with a reaction model for pH and the carbonate system formulated based on the law of mass action. A novel aspect of our approach is the incorporation of a dosage system modelled by a Hill-function, enabling the exploration of diverse dosing strategies for pH and alkalinity management. The model was validated based on empirical data from a pilot scale RAS system operated with a feeding regime involving 12 hours of feeding per day. We found that model simulations could be used to accurately predict diurnal cycling patterns in RAS water quality parameters. Furthermore, we made use of simulations to assess how diurnal cycling varies with changing pH and alkalinity levels. Model results emphasize the complexity of pH and alkalinity control in RAS in relation to overall water quality management. Based on our simulations, we argue that what should be considered as optimal pH and alkalinity in RAS depends on the state of the system. Accordingly, optimal pH and alkalinity thresholds may vary between different RAS units and between different time points of a rearing cycle. More generally, we demonstrate how modelling and model simulations can be an effective way of getting insights into the dynamics of complex RAS interactions and provide a valuable tool to efficiently explore effects of different operational strategies on water quality parameters.

Iron-oxidizing Zetaproteobacteria are well known to colonize deep-sea hydrothermal vent fields around the world where iron-rich fluids are discharged into oxic seawater. How inter-field and intra-field differences in geochemistry influence the diversity of Zetaproteobacteria, however, remains largely unknown. Here, we characterize Zetaproteobacteria phylogenomic diversity, metabolic potential, and morphologies of the iron oxides they form, with a focus on the recently discovered Fåvne vent field. Located along the Mohns ridge in the Arctic, this vent field is a unique study site with vent fluids containing both iron and hydrogen with thick iron microbial mats (Fe mats) covering porously venting high-temperature (227–267°C) black smoker chimneys. Through genome-resolved metagenomics, we demonstrate that Zetaproteobacteria, Ghiorsea spp., likely produce tubular iron oxide sheaths dominating the Fe mats at Fåvne, as observed via microscopy. With these structures, Ghiorsea may provide a surface area for members of other abundant taxa such as Campylobacterota, Gammaproteobacteria, and Alphaproteobacteria. Furthermore, Ghiorsea likely oxidizes both iron and hydrogen present in the fluids, with several Ghiorsea populations co-existing in the same niche. Homologs of Zetaproteobacteria Ni,Fe hydrogenases and iron oxidation gene cyc2 were found in genomes of other community members, suggesting exchange of these genes could have happened in similar environments. Our study provides new insights into Zetaproteobacteria in hydrothermal vents, their diversity, energy metabolism and niche formation. IMPORTANCE: Knowledge on microbial iron oxidation is important for understanding the cycling of iron, carbon, nitrogen, nutrients, and metals. The current study yields important insights into the niche sharing, diversification, and Fe(III) oxyhydroxide morphology of Ghiorsea , an iron- and hydrogen-oxidizing Zetaproteobacteria representative belonging to Zetaproteobacteria operational taxonomic unit 9. The study proposes that Ghiorsea exhibits a more extensive morphology of Fe(III) oxyhydroxide than previously observed. Overall, the results increase our knowledge on potential drivers of Zetaproteobacteria diversity in iron microbial mats and can eventually be used to develop strategies for the cultivation of sheath-forming Zetaproteobacteria.

Marine viral sequence space is immense and presents a promising resource for the discovery of new enzymes interesting for research and biotechnology. However, bottlenecks in the functional annotation of viral genes and soluble heterologous production of proteins hinder access to downstream characterization, subsequently impeding the discovery process. While commonly utilized for the heterologous expression of prokaryotic genes, codon adjustment approaches have not been fully explored for viral genes. Herein, the sequence-based identification of a putative prophage is reported from within the genome of Hypnocyclicus thermotrophus, a Gram-negative, moderately thermophilic bacterium isolated from the Seven Sisters hydrothermal vent field. A prophage-associated gene cluster, consisting of 46 protein coding genes, was identified and given the proposed name Hypnocyclicus thermotrophus phage H1 (HTH1). HTH1 was taxonomically assigned to the viral family Siphoviridae, by lowest common ancestor analysis of its genome and phylogeny analyses based on proteins predicted as holin and DNA polymerase. The gene neighbourhood around the HTH1 lytic cassette was found most similar to viruses infecting Gram-positive bacteria. In the HTH1 lytic cassette, an N-acetylmuramoyl-L-alanine amidase (Amidase_2) with a peptidoglycan binding motif (LysM) was identified. A total of nine genes coding for enzymes putatively related to lysis, nucleic acid modification and of unknown function were subjected to heterologous expression in Escherichia coli. Codon optimization and codon harmonization approaches were applied in parallel to compare their effects on produced proteins. Comparison of protein yields and thermostability demonstrated that codon optimization yielded higher levels of soluble protein, but codon harmonization led to proteins with higher thermostability, implying a higher folding quality. Altogether, our study suggests that both codon optimization and codon harmonization are valuable approaches for successful heterologous expression of viral genes in E. coli, but codon harmonization may be preferable in obtaining recombinant viral proteins of higher folding quality.