Kristina Sundell

Integrated multitrophic level aquaculture (IMTA) in land-based recirculating aquaculture systems (RAS) has been proposed as an effective nitrogen bioremediation strategy. It can improve the utilization of added nitrogen through further biomass production, which increases the sustainability and efficiency of the farming system. Few studies have evaluated how co-farming in cold-water land-based marine recirculating aquaculture systems affects the nitrogen utilization; here, we quantify the effects of co-farming sea lettuce (Ulva fenestrata) with Atlantic wolffish (Anarhichas lupus) and rainbow trout (Oncorhynchus mykiss) in such a system. Nitrogen budgets were used to evaluate the efficiency of the system by summarizing all processes, both inputs and outputs. In alternating four-week periods for four months, the recirculating system held either only fish or fish together with sea lettuce. Fish biomass held 90–91% of the system nitrogen, while 9–10% was available as dissolved inorganic nitrogen. The nitrogen uptake by the sea lettuce biomass was quantified to 1.0–1.5% of the total nitrogen input. Extrapolations based on these results show that a wet weight ratio of sea lettuce:fish of 0.7 would be optimal for remediation of all added nitrogen.

Introducing novel and sustainable feed ingredients is essential to improve the environmental impact of fish aquaculture. Some species of seaweed, such as the green alga sea lettuce ( Ulva fenestrata ), constitute a promising novel ingredient due to their suitable amino acid profile and favourable growth traits. However, the effects of farmed Ulva in feed for cold‐water salmonids have not yet been studied. Here we show that rainbow trout ( Oncorhynchus mykiss ) growth was improved by including Ulva meal in the feed while maintaining intestinal health and fish welfare. The study lasted for 12 weeks, and experimental diets included a control feed, and two feeds with 3% and 15% Ulva meal, respectively. The growth rate was highest for fish given the 15% Ulva diet during the first 6 weeks and the 3% Ulva diet for the last 6 weeks. This was likely due to an increased feed intake (FI) and improved palatability resulting from the inclusion of Ulva . Analysis of intestinal health parameters, primary barrier function and histomorphology, revealed no impaired barrier or inflammatory responses to any of the diets. Similarly, other primary and secondary stress indicators including plasma cortisol levels and oxidative stress markers in the liver and muscle tissues, were unaffected by dietary treatments. Interestingly, the fillet colour became more yellow with increased Ulva inclusion, which could affect the acceptance of the product to customers. Nevertheless, the results demonstrate that sea‐farmed Ulva can contribute to improving the sustainability of future aquafeeds for rainbow trout.

Recirculating aquaculture systems (RAS) hold significant potential for sustainable aquaculture by providing a stable, controlled environment that supports optimal fish growth and welfare. In RAS, ammonium (NH₄⁺) is biologically converted into nitrate (NO₃⁻) via nitrite (NO₂⁻) by nitrifying bacteria. As a result, NO₃⁻ usually accumulates in RAS and must subsequently be removed through denitrification in full RAS, or by regular water exchanges in partial RAS. The marine anammox bacteria Candidatus Scalindua can directly convert toxic NH₄⁺ and NO₂⁻ into harmless nitrogen gas (N₂) and has previously been identified as a promising alternative to the complex denitrification process or unsustainable frequent water exchanges in marine RAS. In this study, we evaluated the impact of high NO₃⁻ levels typically encountered in RAS on the performance and abundance of Ca. Scalindua in a laboratory-scale bioreactor. The bacterial composition of the granules, including the relative abundance of key nitrogen-cycling taxa, was analyzed along with the functional profile (i.e., NH₄⁺ and NO₂⁻ removal efficiencies). For this purpose, a bioreactor was inoculated and fed a synthetic feed, enriched in NH₄⁺, NO₂⁻, minerals and trace elements until stabilization (Phase 1, 52 days). NO₃⁻ concentrations were then gradually increased to 400 mg·L⁻¹ NO₃⁻-N (Phase 2, 52 days), after which the reactor was followed for another 262 days (Phase 3). The reactor maintained high removal efficiencies; 88.0 ± 8.6% for NH₄⁺ and 97.4 ± 1.7% for NO₂⁻ in Phase 2, and 95.0 ± 6.5% for NH₄⁺ and 98.6 ± 2.7% for NO₂⁻ in Phase 3. The relative abundance of Ca. Scalindua decreased from 22.7% to 10.2% by the end of Phase 3. This was likely due to slower growth of Ca. Scalindua compared to heterotrophic bacteria present in the granule, which could use NO₃⁻ as a nitrogen source. Fluorescence in situ hybridization confirmed the presence of a stable population of Ca. Scalindua, which maintained high and stable NH₄⁺ and NO₂⁻ removal efficiencies. These findings support the potential of Ca. Scalindua as an alternative filtering technology in marine RAS. Future studies should investigate pilot-scale applications under real-world conditions.

Infections pose a challenge for the fast growing aquaculture sector. Glycosphingolipids are cell membrane components that pathogens utilize for attachment to the host to initiate infection. Here, we characterized rainbow trout glycosphingolipids from five mucosal tissues using mass spectrometry and nuclear magnetic resonance and investigated binding of radiolabeled Aeromonas salmonicida to the glycosphingolipids on thin-layer chromatograms. 12 neutral and 14 acidic glycosphingolipids were identified. The glycosphingolipids isolated from the stomach and intestine were mainly neutral, whereas glycosphingolipids isolated from the skin, gills and pyloric caeca were largely acidic. Many of the acidic structures were poly-sialylated with shorter glycan structures in the skin compared to the other tissues. The sialic acids found were Neu5Ac and Neu5Gc. Most of the glycosphingolipids had isoglobo and ganglio core chains, or a combination of these. The epitopes on the rainbow trout glycosphingolipid glycans differed between epithelial sites leading to differences in pathogen binding. A major terminal epitope was fucose, that occurred attached to GalNAc in a α1-3 linkage but also in the form of HexNAc-(Fuc-)HexNAc-R. A. salmonicida were shown to bind to neutral glycosphingolipids from the gill and intestine. This study is the first to do a comprehensive investigation of the rainbow trout glycosphingolipids and analyze binding of A. salmonicida to glycosphingolipids. The structural information paves the way for identification of ways of interfering in pathogen colonization processes to protect against infections in aquaculture and contributes towards understanding A. salmonicida infection mechanisms.

The objective of the present study was to investigate the optimal dietary protein requirement and the effect of varying protein levels on the growth and health of juvenile, wild‐caught Atlantic wolffish, Anarhichas lupus, a promising candidate for cold‐water aquaculture diversification. Six iso‐energetic (ca. 18.3 MJ kg⁻¹), fish meal‐based experimental diets were formulated with crude protein levels ranging from 35% to 60%, with graded increments of 5% in a 12‐week feeding trial in a recirculating aquaculture system (RAS). Weight gain, specific growth rate (SGR), and condition factor (K) were evaluated in response to dietary protein levels. Liver, muscle, and blood parameters were assessed for possible changes in protein and lipid metabolism and welfare. Overall growth was highly variable throughout the experiment on all diets, as expected for a wild population. The feed with highest in protein (60%) inclusion resulted in the highest growth rates, with an average weight gain of 37.4% ± 33.8% and an SGR of 0.31% ± 0.2% day⁻¹. This was closely followed by feeds with 55% and 50% protein inclusion with an average weight gain of 22.9% ± 34.8% and 28.5% ± 38.3%, respectively, and an SGR of 0.18% ± 0.3% day⁻¹ and 0.22% ± 0.3% day⁻¹, respectively. Fish fed the high protein diets generally had increased hepatic lipid deposition (17%–18%) and reduced free fatty acid levels (3.1–6.8 μmol L⁻¹) in the plasma relative to fish that were fed the lower protein diets (35%–45%). No effects of diet were found on plasma protein levels or muscle protein content. Furthermore, stress parameters such as plasma cortisol and glucose levels were unaffected by diet, as were plasma ghrelin levels. Overall, these results suggest that a high protein inclusion in the diet for Atlantic wolffish is required to sustain growth with a minimum protein level of 50%.

A nutritional bottleneck in salmonid aquaculture is the procurement of marine‐derived compounds, such as essential amino and fatty acids, including omega‐3 fatty acids, lysine, and methionine. Therefore, insects containing these compounds are highly promising as feed ingredients. The present study evaluates larvae of a “marine” insect (Coelopa frigida, the bristly‐legged seaweed fly larvae, SWFL) reared on brown algae side streams as a feed ingredient for rainbow trout (Oncorhynchus mykiss). SWFL contained, on a dry matter basis, 60% crude protein (CP), 3.5% lysine, and 1.5% methionine, as well as 17% lipids, including 4% eicosapentaenoic acid and docosahexaenoic acid. Four isoenergetic (ca. 23 MJ kg⁻¹ gross energy) and isoproteic (ca. 45% CP) feeds were evaluated in a 10‐week feeding trial. The diets included a control containing 25% fish meal, a commercial reference, and two diets substituting 40% fish meal with either SWFL or partially defatted black soldier fly larvae (BSFL) meal (Hermetia illucens). SWFL‐fed fish displayed higher specific growth rates (SGR) compared to BSFL‐fed fish and similar SGR compared to control and reference diet‐fed fish. Feed intake in SWFL‐fed fish was higher than for the control and BSFL diets and similar to the reference diet. The feed conversion ratio of fish fed the BSFL and SWFL diets was higher compared to the control, suggesting lower bioavailability of both insect meals compared to fish meals. No difference in intestinal health parameters was noted between the insect diets and the control diet, indicating good intestinal health across all treatments. However, changes in electrogenic intestinal transport were observed between the SWFL and BSFL diets, illustrating the heterogeneous effect of different insect products. Overall, SWFL meal is a promising alternative marine feed ingredient, compatible with circular production systems, as it can be efficiently cultivated using marine side streams.

Recirculating aquaculture systems (RAS) are promising candidates for the sustainable development of the aquaculture industry. A current limitation of RAS is the production and potential accumulation of nitrogenous wastes, ammonium (NH₄⁺), nitrite (NO₂⁻) and nitrate (NO₃⁻), which could affect fish health and welfare. In a previous experiment, we have demonstrated that the marine anammox bacteria Candidatus Scalindua was a promising candidate to treat the wastewater (WW) of marine, cold-water RAS. However, the activity of the bacteria was negatively impacted after a direct exposure to RAS WW. In the current study, we have further investigated the potential of Ca. Scalindua to treat marine RAS WW in a three-phase experiment. In the first phase (control, 83 days), Ca. Scalindua was fed a synthetic feed, enriched in NH₄⁺, NO₂⁻ and trace element (TE) mix. Removal rates of 98.9% and 99.6% for NH₄⁺ and NO₂⁻, respectively, were achieved. In the second phase (116 days), we gradually increased the exposure of Ca. Scalindua to nitrogen-enriched RAS WW over a period of about 80 days. In the last phase (79 days), we investigated the needs of TE supplementation for the Ca. Scalindua after they were fully acclimated to 100% RAS WW. Our results show that the gradual exposure of Ca. Scalindua resulted in a successful acclimation to 100% RAS WW, with maintained high removal rates of both NH₄⁺ and NO₂⁻ throughout the experiment. Despite a slight decrease in relative abundance (from 21.4% to 16.7%), Ca. Scalindua remained the dominant species in the granules throughout the whole experiment. We conclude that Ca. Scalindua can be successfully used to treat marine RAS WW, without the addition of TE, once given enough time to acclimate to its new substrate. Future studies need to determine the specific needs for optimal RAS WW treatment by Ca. Scalindua at pilot scale.

With laboratory zebrafish (Danio rerio) being an established and popular research model, there is a need for universal, research-based husbandry guidelines for this species, since guidelines can help promote good welfare through providing appropriate care. Despite the widespread use of zebrafish in research, it remains unclear how holding densities affect their welfare. Previous studies have mainly evaluated the effects of holding densities on a single parameter, such as growth, reproductive output, or social interactions, rather than looking at multiple welfare parameters simultaneously. Here we investigated how chronic (nine weeks) exposure to five different holding densities (1, 4, 8, 12, and 16 fish/L) affected multiple welfare indicators. We found that fish in the 1 fish/L density treatment had higher free water cortisol concentrations per fish, increased vertical distribution, and displayed aggressive behaviour more frequently than fish held at higher densities. On the other hand, density treatments had no effect on anxiety behaviour, whole-brain neurotransmitter levels, egg volume, or the proportion of fertilised eggs. Our results demonstrate that zebrafish can be held at densities between 4 and 16 fish/L without compromising their welfare. However, housing zebrafish in the density of 1 fish/L increased their stress level and aggressive behaviour.

One of the most important bacterial diseases in salmonid aquaculture is furunculosis, caused by Aeromonas salmonicida. Bacterial communication through secreted autoinducer signals, quorum sensing, takes part in the regulation of gene expression in bacteria, influencing growth and virulence. The skin and mucosal surfaces, covered by a mucus layer, are the first point of contact between fish and bacteria. Mucins are highly glycosylated and are the main components of mucus. Here, we validate the Vibrio harveyi BB170 bioreporter assay for quantifying A. salmonicida quorum sensing and study the effects of Atlantic salmon mucins as well as mono- and disaccharides on the AI-2 levels of A. salmonicida. Atlantic salmon mucins from skin, pyloric ceca, proximal and distal intestine reduced A. salmonicida AI-2 levels. Among the saccharides abundant on mucins, fucose, N-acetylneuraminic acid and GlcNAcβ1-3Gal inhibited AI-2 A. salmonicida secretion. Removal of N-acetylneuraminic acid, which is the most abundant terminal residue on mucin glycans on Atlantic salmon mucins, attenuated the inhibitory effects on AI-2 levels of A. salmonicida. Deletion of A. salmonicida luxS abolished AI-2 production. In conclusion, Atlantic salmon mucins regulate A. salmonicida quorum sensing in a luxS and N-acetylneuraminic acid-dependent manner.

Recirculating aquaculture systems (RAS) are good candidates for the sustainable development of the aquaculture sector. A current limitation of RAS is the production and accumulation of nitrogenous waste, which could affect fish health. We investigated the potential of the anaerobic ammonia oxidation (anammox) process to treat marine wastewater from a cold-water RAS. We show that the marine anammox bacteria Candidatus Scalindua is a promising candidate. However, its activity was affected by unknown compounds in the RAS wastewater and/or the sub-optimum content of essential trace elements (TEs). Anammox activity dropped to 2% and 13% in NH₄⁺ and NO₂⁻ removal, respectively, when NO₃-rich RAS wastewater was used as a medium in the absence of TE supplementation. A TE supplementation was added to the RAS wastewater in a subsequent phase, and a recovery in anammox activity was shown (25% and 24% in NH₄⁺ and NO₂⁻ removal, respectively). Future studies need to identify the unknown factor and determine the specific needs regarding TE for optimal RAS wastewater treatment by Candidatus Scalindua.