Occurrence and seasonality of Gyrodactylus salaris and G. salmonis (Monogenea) on Arctic char (Salvelinus alpinus (L.)) in the Fustvatnet lake, Northern Norway.

Gyrodactylus spp. (Monogenea) were found on 16.9% (233 out of 1376) Arctic char, Salvelinus alpinus (L.), sampled from September 2010 to October 2011 in the Fustvatnet lake, Northern Norway. Two species were identified: G. salaris Malmberg, 1957, and G. salmonis Yin & Sproston, 1948. Gyrodactylus salaris was only found on Arctic char larger than 28 cm and only in samples obtained in the autumn (September and October). Gyrodactylus salmonis was found on Arctic char of all sizes (11-47 cm) and throughout the year, with a small peak in abundance in the late autumn (November). Gyrodactylus salaris was found to prefer the tail and dorsal fin. Based on the results, we recommend that surveys of Arctic char for the presence of G. salaris are based on the examination of the fins of large fish sampled during the spawning season (autumn).

Recurrent oxidant treatment induces dysregulation in the brain transcriptome of Atlantic salmon (Salmo salar) smolts.

Peracetic acid (PAA) is an organic peroxide that produces free radicals, which contribute to its potent disinfection power. At therapeutic doses, PAA is considered a mild stressor that can trigger transient local and systemic oxidative stress in fish, but the resulting consequences in the brain have yet to be identified. Therefore, we report the brain transcriptome of Atlantic salmon (Salmo salar) smolts that have been periodically exposed to PAA. Fish were treated three times (every 15 days) with PAA with either short (15 min) or long (30 min) exposure periods. After the third treatment, the whole brain was collected and subjected to biochemical and transcriptomic analyses. The level of reactive oxygen species in the brain was not significantly affected by recurrent PAA treatments. Microarray analysis was performed on the whole brain and revealed 205 differentially expressed genes (DEGs), regardless of the duration of the treatment. The short exposure duration had a more considerable impact on the brain transcriptome, correlating with 70% more DEGs than the long exposure. Strikingly, the brain transcriptome was characterised by the downregulation of gene expression, especially in the short exposure group, and around 82% of the identified DEGs were downregulated. Some of the highly affected genes were key molecules of the vasotocinergic and isotocinergic systems and the corticotropin-releasing factor signalling system, indicating interference of the stress axis but could also suggest an anxiolytic effect. In addition, there were alterations in genes involved in cellular metabolism and processing, signalling and trafficking, and innate immunity, which underscores the physiological changes in the brain following recurrent PAA treatment. Overall, the transcriptomic data reveal that recurrent oxidant treatment could influence brain functions, and although the magnitude was marginal, the alterations suggested neurological adaptations of fish to PAA as a potential chemical stressor. The results identify the risks of PAA, which would be valuable in drafting a framework for its empirically driven use in fish farming.

Mucosal immune and stress responses of Neoparamoeba perurans-infected Atlantic salmon (Salmo salar) treated with peracetic acid shed light on the host-parasite-oxidant interactions.

Treatment development for parasitic infestation is often limited to disease resolution as an endpoint response, and physiological and immunological consequences are not thoroughly considered. Here, we report the impact of exposing Atlantic salmon affected with amoebic gill disease (AGD) to peracetic acid (PAA), an oxidative chemotherapeutic. AGD-affected fish were treated with PAA either by exposing them to 5 ppm for 30 min or 10 ppm for 15 min. Unexposed fish from both infected and uninfected groups were also included. Samples for molecular, biochemical, and histological evaluations were collected at 24 h, 2 weeks, and 4 weeks post-treatment. Behavioral changes were observed during PAA exposure, and post-treatment mortality was higher in the infected and PAA treated groups, especially in 10 ppm for 15 min. Plasma indicators showed that liver health was affected by AGD, though PAA treatment did not exacerbate the infection-related changes. Transcriptome profiling in the gills showed significant changes, triggered by AGD and PAA treatments, and the effects of PAA were more notable 24 h after treatment. Genes related to immune pathways of B- and T- cells and protein synthesis and metabolism were downregulated, where the magnitude was more remarkable in 10 ppm for 15 min group. Even though treatment did not fully resolve the pathologies associated with AGD, 5 ppm for 30 min group showed lower parasite load at 4 weeks post-treatment. Mucous cell parameters (i.e., size and density) increased within 24 h post-treatment and were significantly higher at termination, especially in AGD-affected fish, with some treatment effects influenced by the dose of PAA. Infection and treatments resulted in oxidative stress-in the early phase in the gill mucosa, while systemic reactive oxygen species (ROS) dysregulation was evident at the later stage. Infected fish responded to elevated circulating ROS by increasing antioxidant production. Exposing the fish to a crowding stress revealed the interference in the post-stress responses. Lower cortisol response was displayed by AGD-affected groups. Collectively, the study established that PAA, within the evaluated treatment protocols, could not provide a convincing treatment resolution and, thus, requires further optimization. Nonetheless, PAA treatment altered the mucosal immune and stress responses of AGD-affected Atlantic salmon, shedding light on the host-parasite-treatment interactions. .

Multiomics Provide Insights into the Key Molecules and Pathways Involved in the Physiological Adaptation of Atlantic Salmon (Salmo salar) to Chemotherapeutic-Induced Oxidative Stress.

Although chemotherapeutics are used to treat infections in farmed fish, knowledge on how they alter host physiology is limited. Here, we elucidated the physiological consequences of repeated exposure to the potent oxidative chemotherapeutic peracetic acid (PAA) in Atlantic salmon (Salmo salar) smolts. Fish were exposed to the oxidant for 15 (short exposure) or 30 (long exposure) minutes every 15 days over 45 days. Unexposed fish served as the control. Thereafter, the ability of the remaining fish to handle a secondary stressor was investigated. Periodic chemotherapeutic exposure did not affect production performance, though survival was lower in the PAA-treated groups than in the control. Increased ventilation, erratic swimming, and a loss of balance were common behavioural manifestations during the oxidant exposure. The plasma reactive oxygen species levels increased in the PAA-treated groups, particularly after the third exposure, suggesting an alteration in the systemic oxidative stress status. Plasma indicators for internal organ health were affected to a certain degree, with the changes mainly observed after the second and third exposures. Metabolomics disclosed that the oxidant altered several circulating metabolites. Inosine and guanosine were the two metabolites significantly affected by the oxidative stressor, regardless of exposure time. A microarray analysis revealed that the gills and liver were more responsive to the oxidant than the skin, with the gills being the most sensitive. Moreover, the magnitude of the transcriptomic modifications depended on the exposure duration. A functional analysis showed that genes involved in immunity and ribosomal functions were significantly affected in the gills. In contrast, genes crucial for the oxidation-reduction process were mainly targeted in the liver. Skin mucus proteomics uncovered that the changes in the mucosal proteome were dependent on exposure duration and that the oxidant interfered with ribosome-related processes. Mucosal mapping revealed gill mucous cell hypertrophy after the second and third exposures, although the skin morphological parameters remained unaltered. Lastly, repeated oxidant exposures did not impede the ability of the fish to mount a response to a secondary stressor. This study provides insights into how a chemical oxidative stressor alters salmon physiology at both the systemic and mucosal levels. This knowledge will be pivotal in developing an evidence-driven approach to the use of oxidative therapeutics in fish, with some of the molecules and pathways identified as potential biomarkers and targets for assessing the physiological cost of these treatments.

Catching the fish with the worm: a case study on eDNA detection of the monogenean parasite Gyrodactylus salaris and two of its hosts, Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Environmental DNA (eDNA) monitoring is growing increasingly popular in aquatic systems as a valuable complementary method to conventional monitoring. However, such tools have not yet been extensively applied for metazoan fish parasite monitoring. The fish ectoparasite Gyrodactylus salaris, introduced into Norway in 1975, has caused severe damage to Atlantic salmon populations and fisheries. Successful eradication of the parasite has been carried out in several river systems in Norway, and Atlantic salmon remain infected in only seven rivers, including three in the Drammen region. In this particular infection region, a prerequisite for treatment is to establish whether G. salaris is also present on rainbow trout upstream of the salmon migration barrier. Here, we developed and tested eDNA approaches to complement conventional surveillance methods. METHODS: Water samples (2 × 5 l) were filtered on-site through glass fibre filters from nine locations in the Drammen watercourse, and DNA was extracted with a CTAB protocol. We developed a qPCR assay for G. salaris targeting the nuclear ribosomal ITS1 region, and we implemented published assays targeting the mitochondrial cytochrome-b and NADH-regions for Atlantic salmon and rainbow trout, respectively. All assays were transferred successfully to droplet digital PCR (ddPCR). RESULTS: All qPCR/ddPCR assays performed well both on tissue samples and on field samples, demonstrating the applicability of eDNA detection for G. salaris, rainbow trout and Atlantic salmon in natural water systems. With ddPCR we eliminated a low cross-amplification of Gyrodactylus derjavinoides observed using qPCR, thus increasing specificity and sensitivity substantially. Duplex ddPCR for G. salaris and Atlantic salmon was successfully implemented and can be used as a method in future surveillance programs. The presence of G. salaris eDNA in the infected River Lierelva was documented, while not elsewhere. Rainbow trout eDNA was only detected at localities where the positives could be attributed to eDNA release from upstream land-based rainbow trout farms. Electrofishing supported the absence of rainbow trout in all of the localities. CONCLUSIONS: We provide a reliable field and laboratory protocol for eDNA detection of G. salaris, Atlantic salmon and rainbow trout, that can complement conventional surveillance programs and substantially reduce the sacrifice of live fish. We also show that ddPCR outperforms qPCR with respect to the specific detection of G. salaris.

Effects of cnidarian biofouling on salmon gill health and development of amoebic gill disease.

This study examines the potential implications of biofouling management on the development of an infectious disease in Norwegian farmed salmon. The hydroid Ectopleura larynx frequently colonises cage nets at high densities (thousands of colonies per m2) and is released into the water during regular in-situ net cleaning. Contact with the hydroids' nematocysts has the potential to cause irritation and pathological damage to salmon gills. Amoebic gill disease (AGD), caused by the amoeba Paramoeba perurans, is an increasingly international health challenge in Atlantic salmon farming. AGD often occurs concomitantly with other agents of gill disease. This study used laboratory challenge trials to: (1) characterise the gill pathology resulting from the exposure of salmon to hydroids, and (2) investigate if such exposure can predispose the fish to secondary infections-using P. perurans as an example. Salmon in tanks were exposed either to freshly 'shredded' hydroids resembling waste material from net cleaning, or to authentic concentrations of free-living P. perurans, or first to 'shredded' hydroids and then to P. perurans. Gill health (AGD gill scores, non-specific gill scores, lamellar thrombi, epithelial hyperplasia) was monitored over 5 weeks and compared to an untreated control group. Nematocysts of E. larynx contained in cleaning waste remained active following high-pressure cleaning, resulting in higher non-specific gill scores in salmon up to 1 day after exposure to hydroids. Higher average numbers of gill lamellar thrombi occurred in fish up to 7 days after exposure to hydroids. However, gill lesions caused by hydroids did not affect the infection rates of P. perurans or the disease progression of AGD. This study discusses the negative impacts hydroids and current net cleaning practices can have on gill health and welfare of farmed salmon, highlights existing knowledge gaps and reiterates the need for alternative approaches to net cleaning.

Identification of genetic markers associated with Gyrodactylus salaris resistance in Atlantic salmon Salmo salar.

Gyrodactylus salaris Malmberg, 1957 is a freshwater monogenean ectoparasite of salmonids, first recorded in Norway in 1975 and responsible for extensive epizootics in wild Atlantic salmon Salmo salar L. The susceptibility of different populations of Atlantic salmon to G. salaris infection differs markedly, with fish from the Baltic being characterised as relatively resistant whereas those from Norway or Scotland are known to be (extremely) susceptible. Resistance to Gyrodactylus infection in salmonids has been found to be heritable and a polygenic mechanism of control has been hypothesised. The current study utilises a 'Quantitative trait loci' (QTL) screening approach in order to identify molecular markers linked to QTL influencing G. salaris resistance in B1 backcrosses of Baltic and Scottish salmon. Infection patterns in these fish exhibited 3 distinct types; susceptible (exponential parasite growth), responding (parasite load builds before dropping) and resistant (parasite load never increases). B1 backcross fish were screened at 39 microsatellite markers and single marker-trait associations were examined using general linear modelling. We identified 10 genomic regions associated with heterogeneity in both innate and acquired resistance, explaining up to 27.3% of the total variation in parasite loads. We found that both innate and acquired parasite resistance in Atlantic salmon are under polygenic control, and that salmon would be well suited to a selection programme designed to quickly increase resistance to G. salaris in wild or farmed stocks.

The effect of aluminium in Atlantic salmon (Salmo salar) with special emphasis on alkaline water.

Atlantic salmon (Salmo salar) parr were exposed to aluminium under both steady state and non-steady state chemical conditions in alkaline water. Under alkaline (pH 9.5) steady state conditions, approximately 350 microg Al l(-1) (predominantly aluminate, Al(OH)(4)(-)) had no acute toxic effect on the salmon. The fish, however, showed a physiological response after 3 weeks of exposure ( approximately 300% increase in blood glucose concentration, about 30% increase in blood haematocrit, and about 15% decrease in plasma Cl(-) concentration). No increase in toxicity was evident under non-steady state conditions, i.e. lowering Al solubility as pH was lowered from 9.5 to 7.5. The results indicate that the toxicity of the aluminate ion (Al(OH)(4)(-)) is low, and particularly lower than the corresponding toxicity of cationic Al hydroxides. The effects observed in fish exposed to Al-rich water at pH 9.5 were counteracted as Al solubility was decreased by lowering pH to 7.5. This is contrary to previous observations where Al solubility has been lowered by increasing pH from 5.0 to 6.5.

Reservoir hosts for Gyrodactylus salaris may play a more significant role in epidemics than previously thought.

BACKGROUND: Gyrodactylus salaris Malmberg, 1957 has had a devastating impact on wild Norwegian stocks of Atlantic salmon Salmo salar L., and it is the only Office International des Epizooties (OIE) listed parasitic pathogen of fish. The UK is presently recognised as G. salaris-free, and management plans for its containment and control are currently based on Scandinavian studies. The current study investigates the susceptibility of British salmonids to G. salaris, and determines whether, given the host isolation since the last glaciation and potential genetic differences, the populations under test would exhibit different levels of susceptibility, as illustrated by the parasite infection trajectory over time, from their Scandinavian counterparts. METHODS: Populations of S. salar, brown trout Salmo trutta L., and grayling Thymallus thymallus (L.), raised from wild stock in UK government hatcheries, were flown to Norway and experimentally challenged with a known pathogenic strain of G. salaris. Each fish was lightly anaesthetised and marked with a unique tattoo for individual parasite counting. A single Norwegian population of S. salar from the River Lærdalselva was used as a control. Parasite numbers were assessed every seven days until day 48 and then every 14 days. RESULTS: Gyrodactylus salaris regularly leads to high mortalities on infected juveniles S. salar. The number of G. salaris on British S. salar rose exponentially until the experiment was terminated at 33 days due to fish welfare concerns. The numbers of parasites on S. trutta and T. thymallus increased sharply, reaching a peak of infection on days 12 and 19 post-infection respectively, before declining to a constant low level of infection until the termination of the experiment at 110 days. CONCLUSIONS: The ability of S. trutta and T. thymallus to carry an infection for long periods increases the window of exposure for these two hosts and the potential transfer of G. salaris to other susceptible hosts. This study demonstrates that G. salaris can persist on S. trutta for longer periods than previously thought, and that the role that S. trutta could play in disseminating G. salaris needs to be considered carefully and factored into management plans and epidemics across Europe.

Molecular responses to toxicological stressors: profiling microRNAs in wild Atlantic salmon (Salmo salar) exposed to acidic aluminum-rich water.

Atlantic salmon (Salmo salar) is among the most sensitive organisms toward acidic, aluminum exposure. Main documented responses to this type of stress are a combination of hypoxia and loss of blood plasma ions. Physiological responses to stress in fish are often grouped into primary, secondary and tertiary responses, where the above mentioned effects are secondary responses, while primary responses include endocrine changes as measurable levels of catecholamines and corticosteroids. In this study we have exposed young (14 months) Atlantic salmon to acid/Al water (pH ≈ 5.6, Al(i) ≈ 80 µg L⁻¹) for 3 days, and obtained clear and consistent decrease of Na⁺ and Cl⁻ ions, and increases of glucose in blood plasma, hematocrit and P(CO2) in blood. We did not measure plasma cortisol (primary response compound), but analyzed effects on microRNA level (miRNA) in muscle tissue, as this may represent initial markers of primary stress responses. miRNAs regulate diverse biological processes, many are evolutionarily conserved, and hundreds have been identified in various animals, although only in a few fish species. We used a novel high-throughput sequencing (RNA-Seq) method to identify miRNAs in Atlantic salmon and specific miRNAs as potential early markers for stress. A total of 18 miRNAs were significantly differentially expressed (FDR<0.1) in exposed compared to control fish, four down-regulated and 14 up-regulated. An unsupervised hierarchical clustering of significant miRNAs revealed two clusters representing exposed and non-exposed individuals. Utilizing the genome of the zebrafish and bioinformatic tools, we identified 224 unique miRNAs in the Atlantic salmon samples sequenced. Additional laboratory studies focusing on function, stress dose-responses and temporal expression of the identified miRNAs will facilitate their use as initial markers for stress responses.