Gill epithelial cell line ASG-10 from Atlantic salmon as a new research tool for solving water quality challenges in aquaculture.

Here we evaluated the gill epithelial cell line ASG-10 from Atlantic salmon, as an in vitro model for research on known water quality challenges in aquaculture. Ammonia/ammonium (NH3/NH4+), a recognized challenge in water-intensive recirculating aquaculture systems (RAS), induced lysosomal vacuolization, reduced protein degradation and cell migration of the ASG-10 cells. Aluminium (Aln+), another challenge in freshwater aquaculture facilities had only minor effects. Next, we investigated the tolerance for direct water exposure of ASG-10. The cells tolerated water with osmolarity between 169 and 419 mOsmol/kg for 24 h. However, cells exposed for 3 h to water at 863 mOsmol/kg changed cellular morphology and induced gene expression related to stress (gpx1, casp3, hsp70), and after 24 h exposure cellular viability was severely reduced. Nevertheless, when the cells were grown in transwell inserts, they tolerated 863 mOsmol/kg for 3 h and induction of stress response associated genes was considerably reduced. Lastly, the ASG-10 cells were exposed to water samples, with no known quality issues, from different aquaculture facilities. The cells showed no differences in viability or morphology compared to their representative control. In conclusion, the ASG-10 cell line is a promising in vitro model to study water quality challenges and whole water samples.

Ecotoxicological assessment of Cu-rich acid mine drainage of Sulitjelma mine using zebrafish larvae as an animal model.

Acid mine drainage (AMD) is widely acknowledged as a substantial threat to the biodiversity of aquatic ecosystems. The present study aimed to study the toxicological effects of Cu-rich AMD from the Sulitjelma mine in zebrafish larvae. The AMD from this mine was found to contain elevated levels of dissolved metals including Mg (46.7 mg/L), Al (20.2 mg/L), Cu (18.3 mg/L), Fe (19.8 mg/L) and Zn (10.6 mg/L). To investigate the toxicological effects, the study commenced by exposing zebrafish embryos to various concentrations of AMD (ranging from 0.75% to 9%) to determine the median lethal concentration (LC50). Results showed that 96 h LC50 for zebrafish larvae following AMD exposure was 2.86% (95% CI: 2.32-3.52%). Based on acute toxicity results, zebrafish embryos (<2 hpf) were exposed to 0.1% AMD (Cu: 21.7 µg/L) and 0.45% AMD (Cu: 85.7 µg/L) for 96 h to assess development, swimming behaviour, heart rate, respiration and transcriptional responses at 116 hpf. Light microscopy results showed that both 0.1% and 0.45% AMD reduced the body length, eye size and swim bladder area of zebrafish larvae and caused phenotypic abnormalities. Swimming behaviour results showed that 0.45% AMD significantly decreased the locomotion of zebrafish larvae. Heart rate was not affected by AMD exposure. Furthermore, exposure caused a significant increase in oxygen consumption indicating vascular stress in developing larvae. Taken altogether, the study shows that even heavily diluted AMD with environmentally relevant levels of Cu caused toxicity in zebrafish larvae.

The complete mitochondrial genome of the Atlantic spiny lumpsucker Eumicrotremus spinosus (Fabricius, 1776).

The complete mitogenome of the Atlantic spiny lumpsucker (Eumicrotremus spinosus) was generated using the PacBio Sequel II HiFi sequencing platform. The mitogenome assembly has a length of 19,281 bp and contains 13 protein-coding sequences, 22 tRNA genes, 2 rRNA genes, one control region containing the D-loop (2383 bp) and a duplicate control region (1133 bp) Phylogenetic analysis using maximum likelihood revealed that E. spinosus is closely related to the Siberian lumpsucker (E. asperrimus). The mitogenome of the spiny lumpsucker will be useful in population genomics and systematic studies of Cyclopteridae, Liparidae, and Cottidae.

Distinct polymer-dependent sorption of persistent pollutants associated with Atlantic salmon farming to microplastics.

Interactions of microplastics and persistent organic pollutants (POPs) associated with Atlantic salmon farming were studied to assess the potential role of microplastics in relation to the environmental impact of aquaculture. HDPE, PP, PET and PVC microplastics placed for 3 months near fish farms sorbed POPs from aquafeeds. PET and PVC sorbed significantly higher levels of dioxins and PCBs compared to HDPE, while the levels sorbed to PP were intermediate and did not differ statistically from PET, PVC or HDPE. In addition, the composition of dioxins accumulated in caged blue mussels did not reflect the patterns observed on the microplastics, probably due to polymer-specific affinity of POPs. In conclusion, the results of this study show that microplastics occurring near fish farms can sorb aquafeed-associated POPs and, therefore, microplastics could potentially be vectors of such chemicals in the marine environment and increase the environmental impact of fish farming.

Cohabitation With Atlantic Salmon (Salmo salar) Affects Brain Neuromodulators But Not Welfare Indicators in Lumpfish (Cyclopterus lumpus).

Lumpfish are utilized to combat ectoparasitic epidemics in salmon farming. Research gaps on both cleaning behavior and client preferences in a natural environment, emphasizes the need to investigate the physiological impacts on lumpfish during cohabitation with piscivorous Atlantic salmon. Lumpfish (39.9 g, S.D ± 8.98) were arranged in duplicate tanks (n = 40 per treatment) and exposed to Live Atlantic salmon (245.7 g, S.D ± 25.05), salmon Olfaction or lifelike salmon Models for 6 weeks. Growth and health scores were measured every second week. In addition, the final sampling included measurements of neuromodulators, body color, and plasma cortisol. A stimulation and suppression test of the hypothalamic-pituitary-interrenal (HPI) axis was used for chronic stress assessment. Results showed that growth, health scores, and body color remained unaffected by treatments. Significant reductions in levels of brain dopamine and norepinephrine were observed in Live compared to Control. Plasma cortisol was low in all treatments, while the stimulation and suppression test of the HPI axis revealed no indications of chronic stress. This study presents novel findings on the impact on neuromodulators from Atlantic salmon interaction in the lumpfish brain. We argue that the downregulation of dopamine and norepinephrine indicate plastic adjustments to cohabitation with no negative effect on the species. This is in accordance with no observed deviations in welfare measurements, including growth, health scores, body color, and stress. We conclude that exposure to salmon or salmon cues did not impact the welfare of the species in our laboratory setup, and that neuromodulators are affected by heterospecific interaction.

Exposure to low environmental copper concentrations does not affect survival and development in Atlantic cod (Gadus morhua) early life stages.

In this study we investigated potential impacts of Cu exposure at low, environmentally relevant, concentrations on early live stages of Atlantic cod (Gadus morhua). Cod embryos and larvae were exposed to 0.5 µg/L (low), 2 µg/L (medium), and 6 µg/L (high) Cu from 4 to 17 days post fertilisation (dpf). Hatching success, mortality, oxygen consumption, biometric traits, and malformations were determined. A dynamic energy budget (DEB) model was applied to identify potential impacts on bioenergetics. A positive correlation was found between Cu exposure concentrations and Cu body burden in eggs, but not in larvae. The tested concentrations did not increase mortality in neither embryos nor larvae, or larvae deformations. Further, the DEB model did not indicate effects of the tested Cu concentrations.

Effects of marine mine tailing exposure on the development, growth, and lipid accumulation in Calanus finmarchicus.

Marine tailing disposal (MTD) is sometimes practiced as an alternative to traditional mine tailing deposition on land. Environmental challenges connected to MTD include spreading of fine particulate matter in the water column and the potential release of metals and processing chemicals. This study investigated if tailing exposure affects the marine copepod Calanus finmarchicus, and whether effects are related to exposure to mineral particles or the presence of metals and/or processing chemicals in the tailings. We investigated the impacts of three different tailing compositions: calcium carbonate particles with and without processing chemicals and fine-grained tailings from a copper ore. Early life stages of C. finmarchicus were exposed over several developmental stages to low and high suspension concentrations for 15 days, and their development, oxygen consumption and biometry determined. The data was fitted in a dynamic energy budget (DEB) model to determine mechanisms underlying responses and to understand the primary modes of action related to mine tailing exposure. Results show that copepods exposed to tailings generally exhibited slower growth and accumulated less lipids. The presence of metals and processing chemicals did not influence these responses, suggesting that uptake of mineral particles was responsible for the observed effects. This was further supported by the applied DEB model, confirming that ingestion of tailing particles while feeding can result in less energy being available for growth and development.

Effects of mine tailing exposure on early life stages of cod (Gadus morhua) and haddock (Melanogrammus aeglefinus).

Mining and processing of minerals produce large quantities of tailings as waste. Some countries, including Norway, allow disposal of mine tailings in the sea. In this study we investigated the impacts of tailings from a calcium carbonate (CaCO3) processing plant on early live stages of haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua). Fish eggs (3 days post fertilisation; dpf) were exposed for 48 h to three concentrations of tailings, nominally 1 mg L-1 (low, L); 10 mg L-1 (medium, M) and 100 mg L-1 (high, H); with L and M representing concentrations occurring at tailing release points. Results show that tailings rapidly adhered to eggs of both species, causing negative buoyancy (sinking of eggs) in M and H exposures. While tailings remained on egg surfaces in both species also after exposure termination, adhesion seemed more pronounced in cod, leading to larger impacts on buoyancy even after exposure. Tailing exposure further induced early hatching and significantly reduced survival in M and H exposed embryos in both fish species, and in cod from the L exposure group. Moreover, tailing exposure caused reduced survival and malformations in larvae, potentially related to premature hatching. This study shows that mineral particles adhere to haddock and cod eggs, affecting egg buoyancy, survival and development.

Synchrony and multimodality in the timing of Atlantic salmon smolt migration in two Norwegian fjords.

The timing of the smolt migration of Atlantic salmon (Salmo salar) is a phenological trait increasingly important to the fitness of this species. Understanding when and how smolts migrate to the sea is crucial to understanding how salmon populations will be affected by both climate change and the elevated salmon lice concentrations produced by salmon farms. Here, acoustic telemetry was used to monitor the fjord migration of wild post-smolts from four rivers across two fjord systems in western Norway. Smolts began their migration throughout the month of May in all populations. Within-population, the timing of migration was multimodal with peaks in migration determined by the timing of spring floods. As a result, migrations were synchronized across populations with similar hydrology. There was little indication that the timing of migration had an impact on survival from the river mouth to the outer fjord. However, populations with longer fjord migrations experienced lower survival rates and had higher variance in fjord residency times. Explicit consideration of the multimodality inherent to the timing of smolt migration in these populations may help predict when smolts are in the fjord, as these modes seem predictable from available environmental data.

Compromised thermal tolerance of cardiovascular capacity in upstream migrating Arctic char and brown trout-are hot summers threatening migrating salmonids?

Heat waves are threatening fish around the world, leading sometimes to mass mortality events. One crucial function of fish failing in high temperatures is oxygen delivery capacity, i.e. cardiovascular function. For anadromous salmonids, increased temperature could be especially detrimental during upstream migration since they need efficiently working oxygen delivery system in order to cross the river rapids to reach upstream areas. The migration also occurs during summer and early autumn exposing salmonids to peak water temperatures, and in shallow rivers there is little availability for thermal refuges as compared to thermally stratified coastal and lake habitats. In order to shed light on the mechanisms underpinning the capacity of migrating fish to face high environmental temperatures, we applied a physiological and molecular approach measuring cardiovascular capacities of migrating and resident Arctic char (Salvelinus alpinus) and brown trout (Salmo trutta) in Northern Norway. The maximum cardiovascular capacity of migrating fish was significantly lower compared to the resident conspecifics. The onset of cardiac impairment started only 2°C higher than river temperature, meaning that even a small increase in water temperature may already compromise cardiac function. The migrating fish were also under significant cellular stress, expressing increased level of cardiac heat shock proteins. We consider these findings highly valuable when addressing climate change effect on migrating fish and encourage taking action in riverine habitat conservation policies. The significant differences in upper thermal tolerance of resident and migrating fish could also lead changes in population dynamics, which should be taken into account in future conservation plans.

Effects of Agricultural Pesticides in Aquafeeds on Wild Fish Feeding on Leftover Pellets Near Fish Farms.

Screening has revealed that modern-day feeds used in Atlantic salmon aquaculture might contain trace amounts of agricultural pesticides. To reach slaughter size, salmon are produced in open net pens in the sea. Uneaten feed pellets and undigested feces deposited beneath the net pens represent a source of contamination for marine organisms. To examine the impacts of long-term and continuous dietary exposure to an organophosphorus pesticide found in Atlantic salmon feed, we fed juvenile Atlantic cod (Gadus morhua), an abundant species around North Atlantic fish farms, three concentrations (0.5, 4.2, and 23.2 mg/kg) of chlorpyrifos-methyl (CPM) for 30 days. Endpoints included liver and bile bioaccumulation, liver transcriptomics and metabolomics, as well as plasma cholinesterase activity, cortisol, liver 7-ethoxyresor-ufin-O-deethylase activity, and hypoxia tolerance. The results show that Atlantic cod can accumulate relatively high levels of CPM in liver after continuous exposure, which is then metabolized and excreted via the bile. All three exposure concentrations lead to significant inhibition of plasma cholinesterase activity, the primary target of CPM. Transcriptomics profiling pointed to effects on cholesterol and steroid biosynthesis. Metabolite profiling revealed that CPM induced responses reflecting detoxification by glutathione-S-transferase, inhibition of monoacylglycerol lipase, potential inhibition of carboxylesterase, and increased demand for ATP, followed by secondary inflammatory responses. A gradual hypoxia challenge test showed that all groups of exposed fish were less tolerant to low oxygen saturation than the controls. In conclusion, this study suggests that wild fish continuously feeding on leftover pellets near fish farms over time may be vulnerable to organophosphorus pesticides.

Is Habituation Measurable in Lumpfish Cyclopterus lumpus When Used as Cleaner Fish in Atlantic Salmon Salmo salar Aquaculture?

To investigate how lumpfish interact in Atlantic salmon aquaculture, physiological stress responses and changes in behaviour were analysed in experienced and naive lumpfish. Experienced lumpfish (30.2 ± 7.93 g, mean ± SD) coexisted with a commercial scale production unit of Atlantic salmon (1258.5 ± 152.12 g) for 30 to 60 days, while naive lumpfish (38.2 ± 12.37 g) were kept with conspecifics only. Ten trials from each background were tested. For each trial, 10 lumpfish were tagged and transferred to a video monitored experimental tank (2 × 2 × 0.7 m). In each trial, swimming behaviour was mapped for all lumpfish every 60 s in 20 min, 10 min before, and 10 min after the introduction of four Atlantic salmon. Naive lumpfish expressed significantly increased burst swimming activity and maintained longer interspecific distance to Atlantic salmon in comparison with experienced fish. In addition, mean plasma cortisol levels were significantly elevated in naive fish after exposure to Atlantic salmon. We argue that naive lumpfish expressed innate physiological and behavioural stress responses during first encounter with Atlantic salmon, while reduced responses in experienced individuals indicated habituation. The effect from behavioural and physiological stress in newly deployed naive lumpfish-before and during habituation-should be taken account for when lumpfish are introduced in commercial sea cages to improve welfare for the species. In addition, we suggest that habituation could be applicable during the rearing phase to moderate the transition from a simple tank environment with conspecifics only to interspecies interaction with Atlantic salmon in sea cages.

Transcriptional effects of metal-rich acid drainage water from the abandoned Løkken Mine on Atlantic salmon (Salmo salar) smolt.

Runoff of metals represents one of the major environmental challenges related to historic and ongoing mining activity. In this study, transcriptomics (direct RNA sequencing [RNA-seq] and reverse-transcription quantitative polymerase chain reaction [RT-qPCR]) was used to predict toxicity of metal-rich acid mine drainage (AMD) water collected in the abandoned copper (Cu) mine called Løkken Mine on Atlantic salmon liver and kidney, the main target organs of Cu-induced toxicity in fish. Smolts were exposed to control and diluted AMD water, which contains a mixture of metals but is especially enriched with Cu, at 4 concentrations in freshwater (FW) for 96 h, and then were transferred to and kept in seawater (SW) for another 24 h. Significant accumulation of Cu was observed in the gills, but not liver and kidney tissues, after 96 h of exposure. Short-term exposure to metal-rich ADM (high exposure group) significantly upregulated 3201 transcripts and downregulated 3782 transcripts in liver. The strongest effect attributed to exposure was observed on the KEGG pathway "protein processing in endoplasmic reticulum," followed by "steroid biosynthesis." Gene ontology (GO) analysis suggested that exposure predominantly affected "protein folding," possibly by disrupting disulfide bonds as a result of endoplasmic-reticulum-generated stress, and "sterol biosynthetic processes." Transfer to uncontaminated SW for 24 h amended the transcription of several genes, suggesting a transient effect of treatment on some mechanisms. In conclusion, the data show that trace metals in AMD from abandoned pyrite mines might disturb molecular mechanisms linked to protein folding in Atlantic salmon smolt endoplasmic reticulum.

Effects of mining chemicals on fish: exposure to tailings containing Lilaflot D817M induces CYP1A transcription in Atlantic salmon smolt.

BACKGROUND: Mine tailings, containing metals and production chemicals such as flotation chemicals and flocculants, may pose an environmental threat to aquatic organisms living in downstream ecosystems. The aim of this work was to study to which degree Lilaflot D817M, a flotation chemical extensively used by the mining industry, represents a hazard for migrating salmon in rivers affected by mining activity. Smoltifying Atlantic salmon were exposed to four concentrations of iron-ore mine tailings containing residual Lilaflot D817M [water versus tailing volumes of 0.002 (Low), 0.004 (Medium), 0.013 (High) and 0.04 (Max)]. After 96 h of exposure, gill and liver tissues were harvested for transcriptional responses. Target genes included markers for oxidative stress, detoxification, apoptosis and DNA repair, cell signaling and growth. RESULTS: Of the 16 evaluated markers, significant transcriptional responses of exposure to tailings enriched with Lilaflot D817M were observed for CYP1A, HSP70 and HMOX1 in liver tissue and CYP1A in gill tissue. The significant induction of CYP1A in both liver and gills suggest that the flotation chemical is taken up by the fish and activates cytochrome P450 detoxification via phase I biotransformation in the cells. CONCLUSIONS: The overall weak transcriptional responses to short-term exposure to Lilaflot D817M-containing iron-ore tailings suggest that the mining chemical has relatively low toxic effect on fish. The underlying mechanisms behind the observed CYP1A induction should be studied further.

Cardiac molecular-acclimation mechanisms in response to swimming-induced exercise in Atlantic salmon.

Cardiac muscle is a principal target organ for exercise-induced acclimation mechanisms in fish and mammals, given that sustained aerobic exercise training improves cardiac output. Yet, the molecular mechanisms underlying such cardiac acclimation have been scarcely investigated in teleosts. Consequently, we studied mechanisms related to cardiac growth, contractility, vascularization, energy metabolism and myokine production in Atlantic salmon pre-smolts resulting from 10 weeks exercise-training at three different swimming intensities: 0.32 (control), 0.65 (medium intensity) and 1.31 (high intensity) body lengths s(-1). Cardiac responses were characterized using growth, immunofluorescence and qPCR analysis of a large number of target genes encoding proteins with significant and well-characterized function. The overall stimulatory effect of exercise on cardiac muscle was dependent on training intensity, with changes elicited by high intensity training being of greater magnitude than either medium intensity or control. Higher protein levels of PCNA were indicative of cardiac growth being driven by cardiomyocyte hyperplasia, while elevated cardiac mRNA levels of MEF2C, GATA4 and ACTA1 suggested cardiomyocyte hypertrophy. In addition, up-regulation of EC coupling-related genes suggested that exercised hearts may have improved contractile function, while higher mRNA levels of EPO and VEGF were suggestive of a more efficient oxygen supply network. Furthermore, higher mRNA levels of PPARα, PGC1α and CPT1 all suggested a higher capacity for lipid oxidation, which along with a significant enlargement of mitochondrial size in cardiac myocytes of the compact layer of fish exercised at high intensity, suggested an enhanced energetic support system. Training also elevated transcription of a set of myokines and other gene products related to the inflammatory process, such as TNFα, NFκB, COX2, IL1RA and TNF decoy receptor. This study provides the first characterization of the underlying molecular acclimation mechanisms in the heart of exercise-trained fish, which resemble those reported for mammalian physiological cardiac growth.

Aerobic training stimulates growth and promotes disease resistance in Atlantic salmon (Salmo salar).

Improving fish robustness is of utmost relevance to reducing fish losses in farming. Although not previously examined, we hypothesized that aerobic training, as shown for human studies, could strengthen disease resistance in Atlantic salmon (Salmo salar). Thus, we exercised salmon pre-smolts for 6 weeks at two different aerobic training regimes; a continuous intensity training (CT; 0.8bls(-1)) and an interval training (IT; 0.8bl s(-1) 16h and 1.0bl s(-1) 8h) and compared them with untrained controls (C; 0.05bl s(-1)). The effects of endurance training on disease resistance were evaluated using an IPN virus challenge test, while the cardiac immune modulatory effects were characterized by qPCR and microarray gene expression analyses. In addition, swimming performance and growth parameters were investigated. Survival after the IPN challenge was higher for IT (74%) fish than for either CT (64%) or C (61%) fish. While both CT and IT groups showed lower cardiac transcription levels of TNF-α, IL-1ß and IL-6 prior to the IPN challenge test, IT fish showed the strongest regulation of genes involved in immune responses and other processes known to affect disease resistance. Both CT and IT regimes resulted in better growth compared with control fish, with CT fish developing a better swimming efficiency during training. Overall, interval aerobic training improved growth and increased robustness of Atlantic salmon, manifested by better disease resistance, which we found was associated with a modulation of relevant gene classes on the cardiac transcriptome.

Effects of the fungicide azoxystrobin on Atlantic salmon (Salmo salar L.) smolt.

Atlantic salmon smolts were exposed to three doses of the fungicide azoxystrobin for 4 days, and physiological blood parameters and transcriptional effects in liver and muscle were evaluated in search for potential negative effects. Azoxystrobin exposure mediated up-regulation of catalase, MAPK1 and IGFBP1 in liver tissue. Catalase, transferrin, IGFBP1 and TNFR were up-regulated and CYP1A down-regulated in muscle tissue. Blood parameters glucose, hematocrit, pCO(2), HCO(3) and pH grouped together with transcripts levels of MnSOD, MAPK1, IGFBP1, MAP3K7 and GPx4 in liver of fish exposed to the highest azoxystrobin concentration (352 µgL(-1)) using principal component analysis (PCA). In muscle, the blood parameters glucose, hematocrit, pCO(2), HCO(3) and pH grouped together with transcript levels of heme oxygenase, thioredoxin, MnSOD, TNFR and MMP9. These results suggest that the fungicide azoxystrobin affects mitochondrial respiration and mechanisms controlling cell growth and proliferation in fish and may have negative effects on juvenile Atlantic salmon.

Diffusive gradients in thin films sampler predicts stress in brown trout (Salmo trutta L.) exposed to aluminum in acid fresh waters.

Increased levels of aluminum ions released from nutrient-poor soils affected by acid rain have been the primary cause of fish deaths in the acidified watersheds of southern Norway. The complex aluminum chemistry in water requires speciation methods to measure the gill-reactive species imposing toxic effects toward fish. Previously, aluminum speciation has mainly followed the fractionation principles outlined by Barnes/Driscoll, and several analogues of these fractionation principles have been used both in situ and in the laboratory. Due to rapid transformation processes, aluminum speciation in water samples may change even during short storage times. Thus, results obtained by laboratory fractionation methods might be misleading for the assessment of potentially toxic aluminum species in the water. Until now, all in situ field fractionation methods have been time and labor consuming. The DGT technique (diffusive gradients in thin films) is a new in situ sampler collecting a fraction of dissolved metal weighted according to the rate of diffusion and dissociation kinetics. In a field experiment with acid surface water we studied the DGT sampler as a new prediction tool for the gill accumulation of aluminum in trout (Salmo trutta L.) and the induced physiological stress responses measured as changes in blood glucose and plasma chloride. Aluminum determined with DGT (DGT-AI) was higher than labile monomeric aluminum (Ali) determined with a laboratory aluminum fractionation procedure (PCV--a pyrocatechol violet analogue of Barnes/Driscoll), a difference due to collection of a fraction of organically complexed aluminum by DGT and a reduction of the Ali fraction during sample storage. DGT-AI predicted the gill uptake and the aluminum-induced physiological stress responses (increased blood glucose and decreased plasma chloride, r2 from 0.6 to 0.9). The results indicate that DGT-AI is a better predictor for the stress response than laboratory-determined Ali, because the DGT sampler collects a more correct fraction of the gill-reactive aluminum species that induces the stress.