Evolutionary, comparative, and functional analyses of STATs and regulation of the JAK-STAT pathway in lumpfish upon bacterial and poly(I:C) exposure.

Background: The Janus kinase/signal transducers and activators of transcription (JAK-STAT) system regulates several biological processes by affecting transcription of genes as a response to cytokines and growth factors. In the present study, we have characterized the STAT genes in lumpfish (Cyclopterus lumpus L.), belonging to the order Perciformes, and investigated regulation of the JAK-STAT signaling pathway upon exposure to bacteria (Vibrio anguillarum) and poly(I:C), the latter mimicking antiviral responses. Methods: Characterization and evolutionary analyses of the STATs were performed by phylogeny, protein domain, homology similarity and synteny analyses. Antibacterial and antiviral responses were investigated by performing KEGG pathway analysis. Results: We observed that lumpfish have stat1a, 2, 3, 4, 5a, 5b, and 6. Transcriptome-wide analyses showed that most components of the JAK-STAT pathway were present in lumpfish. il-6, il-10, il-21, iκBα and stat3 were upregulated 6 hours post exposure (hpe) against bacteria while type I interferons (IFNs), irf1, irf3, irf10, stat1 and 2 were upregulated 24 hpe against poly(I:C). Conclusions: Our findings shed light on the diversity and evolution of the STATs and the data show that the STAT genes are highly conserved among fish, including lumpfish. The transcriptome-wide analyses lay the groundwork for future research into the functional significance of these genes in regulating critical biological processes and make an important basis for development of prophylactic measure such as vaccination, which is highly needed for lumpfish since it is vulnerable for both bacterial and viral diseases.

Effects of Exposure Timing on cyp1a Expression, PAH Elimination, and Lipid Utilization in Lumpfish Embryos Exposed to Produced Water.

Intentional discharges of produced water from oil production platforms to the marine environment contain a complex mixture of toxicants, including polycyclic aromatic hydrocarbons (PAHs). Early life stages of fish are highly sensitive to petrogenic exposure, and short-term exposure during critical periods of embryonic development may have detrimental effects on larvae health and survival. However, why different periods are more sensitive to exposure than others are not fully understood. Three identical exposure experiments (48 h, approx. 30 µg/L tPAH, sum 42 PAHs) on lumpfish (Cyclopterus lumpus) embryos were conducted where only exposure timing was varied: 0-48 h post fertilization (hpf, starting before chorion hardening), 36-84 hpf (starting after chorion hardening), and 240-288 hpf (during organogenesis). Total PAH (tPAH) uptake at the end of exposure was 5× higher when exposed during fertilization than when exposed late (during organogenesis). The first evidence of cyp1a induction in lumpfish during embryogenesis was observed after 84 hpf. Early exposure affected lipid droplet coagulation, indicating altered lipid utilization during embryogenesis. Although no significant impacts of exposure were observed on hatching success, hatching was delayed when exposed at the latest time point. This study shows that chorion properties, lipid content, biotransformation potential, and timing of produced water exposure during lumpfish embryogenesis affected PAH uptake and elimination.

In situ biomonitoring using caged lumpfish (Cyclopterus lumpus) eggs reveal plastic and rubber associated chemicals in a harbour area in Central Norway.

Plastics- and rubber-derived chemicals are given increasing focus due to their migration into the environment and potential for causing detrimental effects. The current study demonstrates the use of a novel biomonitoring platform using caged fertilized eggs of lumpfish (Cyclopterus lumpus) in combination with gas chromatography tandem mass spectrometry analysis of a selection of target chemicals extracted from the lumpfish eggs after deployment. A monitoring campaign in the Trondheim harbor and off the coast of Trøndelag in Norway was executed using the described system. Here we found accumulation of UV stabilizers (benzophenone and benzothiazoles), plasticizers (n-butylbenzenesulfonamide), reagents, and polymer synthesis precursors (bisphenol A, acetophenone, phthalide, and phthalimide) in deployed eggs. Several of the compounds were detected in concentrations above previously quantified legacy contaminants in the same study areas.

Magnetic fields generated by submarine power cables have a negligible effect on the swimming behavior of Atlantic lumpfish (Cyclopterus lumpus) juveniles.

Submarine power cables carry electricity over long distances. Their geographic distribution, number, and areal coverage are increasing rapidly with the development of, for example, offshore wind facilities. The flow of current passing through these cables creates a magnetic field (MF) that can potentially affect marine organisms, particularly those that are magnetosensitive. The lumpfish (Cyclopterus lumpus) is a migratory species that is widely distributed in the North Atlantic Ocean and Barents Sea. It migrates between coastal spawning grounds and pelagic offshore feeding areas. We tested whether lumpfish respond to MFs of the same intensity as those emitted by high voltage direct current (HVDC) submarine power cables. Laboratory experiments were conducted by placing juvenile lumpfish in an artificial MF gradient generated by a Helmholtz coil system. The intensity of the artificial MF used (230 µT) corresponded to the field at 1 m from a high-power submarine cable. The fish were filmed for 30 min with the coil either on or off. Swimming speeds, and presence in the different parts of a raceway, were extracted from the videos and analyzed. Juvenile lumpfish activity, defined as the time that the fish spent swimming relative to stationary pauses (attached to the substrate), and the distance travelled, were unaffected by exposure to the artificial MF. The swimming speed of juvenile lumpfish was reduced (by 16%) when the coil was on indicating that the fish could either sense the MF or the induced electric field created by the movement of the fish through the magnetic field. However, it seems unlikely that a 16% decrease in swimming speed occurring within 1 m of HVDC cables would significantly affect Atlantic lumpfish migration or homing.

Monitoring ocean water quality by deployment of lumpfish (Cyclopterus lumpus) eggs: In situ bioaccumulation and toxicity in embryos.

Fish embryos can bioaccumulate and are particularly sensitive to a wide range of contaminants, which makes them suitable sentinels for environmental biomonitoring. However, fish embryos are very rarely utilized in environmental monitoring surveys, possibly due to their fragility and seasonality. In the present work, we assessed the applicability of caged lumpfish (Cyclopterus lumpus) eggs for in situ biomonitoring of exposure and effects of organic contaminants focusing on polyaromatic hydrocarbons and phenolic compounds. Fertilized eggs (1 dpf) were transplanted for 17-19 days at different locations that differed in terms of contaminant load, depths and weather conditions, namely at three stations close to the city of Trondheim (two harbour areas and a one in the Fjord) and three stations at a coastal aquaculture facility. High survival upon retrieval after deployment showed that lumpfish eggs are relatively robust and survive encaging in different environments. Bioaccumulation of organic contaminants (PAHs and phenolic compounds) was measured and potential effects on hatching, development, survival and larvae morphometry were determined. Chemical analyses showed that especially PAHs were effectively accumulated in eggs in contaminated sites, with concentrations of Æ©PAHs being 15 - 25 times higher in harbour areas compared to those at the aquaculture facility. A higher incidence of embryonic deformations was observed in the most polluted deployment location, but larvae morphometry revealed no evidence of toxicity related to pollutant body burden. In conclusion, the in-situ exposure method was proven to work well, making it attractive for implementations in environmental monitoring programs.

Genomic Analysis of Pasteurella atlantica Provides Insight on Its Virulence Factors and Phylogeny and Highlights the Potential of Reverse Vaccinology in Aquaculture.

Pasteurellosis in farmed lumpsuckers, Cyclopterus lumpus, has emerged as a serious disease in Norwegian aquaculture in recent years. Genomic characterization of the causative agent is essential in understanding the biology of the bacteria involved and in devising an efficient preventive strategy. The genomes of two clinical Pasteurella atlantica isolates were sequenced (≈2.3 Mbp), and phylogenetic analysis confirmed their position as a novel species within the Pasteurellaceae. In silico analyses revealed 11 genomic islands and 5 prophages, highlighting the potential of mobile elements as driving forces in the evolution of this species. The previously documented pathogenicity of P. atlantica is strongly supported by the current study, and 17 target genes were recognized as putative primary drivers of pathogenicity. The expression level of a predicted vaccine target, an uncharacterized adhesin protein, was significantly increased in both broth culture and following the exposure of P. atlantica to lumpsucker head kidney leucocytes. Based on in silico and functional analyses, the strongest gene target candidates will be prioritized in future vaccine development efforts to prevent future pasteurellosis outbreaks.

Interleukin-1 Ligands and Receptors in Lumpfish (Cyclopterus lumpus L.): Molecular Characterization, Phylogeny, Gene Expression, and Transcriptome Analyses.

The interleukin (IL)-1 family play a fundamental role as immune system modulators. Our previous transcriptome-analyses of leukocytes from lumpfish (Cyclopterus lumpus L.) showed that IL-1ß was among the most highly upregulated genes following bacterial exposure. In the present study, we characterized IL-1 signaling pathways, identified and characterized four ligands of the IL-1 family in lumpfish; IL-1ß type I and type II, IL-18, and the novel IL-1 family members (nIL-1F), both at mRNA and gene levels. The two IL-1ß in lumpfish is termed IL-1ß1 (type II) and IL-1ß2 (type I). Furthermore, a comprehensive phylogenetic analysis of 277 IL-1 ligands showed that nIL-1F, in common with IL-1ß, likely represents an ancestral gene, as representatives for nIL-1F were found in cartilaginous and lobe-finned fish, in addition to teleosts. This shows that nIL-1F is not exclusively present in teleosts as previously suggested. Our analyses of exon-intron structures, intron phases, phylogeny and synteny clearly show the separation of IL-1ß into groups; type I and type II, which likely is a result of the third whole genome duplication (3R WGD). The phylogenetic analysis shows that most teleosts have both type I and type II. Furthermore, we have determined transcription levels of the IL-1 ligands in leukocytes and 16 different tissues, and their responses upon in vitro stimulation with seven different ligands. In addition, we have identified the IL-1 receptors IL-1R1, IL-1R2, IL-1R4 (ST2/IL-33 receptor/IL-1RL), IL-1R5 (IL-18R1), and partial sequences of DIGIRR and IL-1R3 (IL-RAcP). Identification of immune molecules and description of innate responses in lumpfish is interesting for comparative and evolutionary studies and our study constitutes a solid basis for further functional analyses of IL-1 ligands and receptors in lumpfish. Furthermore, since lumpfish are now farmed in large numbers to be used as cleaner fish for removal of sea lice on farmed salmon, in-depth knowledge of key immune molecules, signaling pathways and innate immune responses is needed, as the basis for design of efficient immune prophylactic measures such as vaccination.

The proinflammatory cytokines TNF-α and IL-6 in lumpfish (Cyclopterus lumpus L.) -identification, molecular characterization, phylogeny and gene expression analyses.

The proinflammatory cytokines TNF-α and IL-6 are important mediators of inflammatory reactions and orchestrators of the immune system in vertebrate. In this study, we have identified TNF-α and IL-6 in lumpfish, molecular characterized them at mRNA and gene level, performed homology modelling and measured their gene expression in different tissues and upon in vitro stimulation. A comprehensive phylogenetic analysis of TNF-α teleost sequences give novel insight into the TNF -α biology. Interestingly, we identified two isoforms of luIL-6. In normal tissue and leukocyte, the level of luTNF-α transcripts was higher than luIL-6. The expression pattern were parallel, except for brain, eye and gonad, and they displayed a similar induction pattern upon exposure to PAMPs, being most highly upregulated by flagellin. This is the first in-depth characterization of TNF and IL-6 in lumpfish. In recent years, lumpfish has become an important species for the aquaculture industry and establishment of qPCR-assays of luTNF-α and luIL-6 provide a valuable tool to measure effect of immune modulation, such as vaccination, microbiological disease and physiological trials. Lumpfish is also interesting for comparative studies as it represent a phylogenetic group that is poorly described immunologically.

Protection and antibody reactivity in lumpsucker (Cyclopterus lumpus L.) following vaccination against Pasteurella sp.

Two monovalent vaccines against pasteurellosis were developed and tested for efficacy using a previously established bath challenge model. High levels of specific antibodies were detected following vaccination. While the vaccine efficacy trial indicated that some level of protection was obtained, high mortality was still observed. qPCR analysis of head kidney tissues from surviving fish post challenge showed no difference in bacterial numbers in vaccinated and non-vaccinated fish. Clinical symptoms observed in moribund and diseased fish included white spots on the skin and around the eyes, frayed fins and redness around the mouth and fin bases. Despite production of specific antibodies, the protection against experimental challenge was relatively weak. A reason for this could potentially be that the specific antibodies produced are not alone enough to provide complete protection against pasteurellosis in lumpsuckers. Confocal and scanning electron microscopy of head kidney leucocytes exposed to Pasteurella sp. in vitro gave indications of the interactions between the pathogen and leucocytes. The results indicate that parts of the immune system other than humoral antibodies could be important for protection against pasteurellosis. Our combined results highlight the need for further work on host-pathogen interaction between Pasteurella sp. and lumpsuckers.

Pharmacokinetic Data Show That Oxolinic Acid and Flumequine Are Absorbed and Excreted Rapidly From Plasma and Tissues of Lumpfish.

This study examined the uptake, tissue distribution and elimination of the antibacterial agents oxolinic acid and flumequine in lumpfish (Cyclopterus lumpus L.) by use of LC-MS/MS following a single oral administration of 25 mg/kg fish given in feed. Lumpfish are increasingly used as cleaner fish for removal of sea lice on commercially farmed salmon. The production of lumpfish is successful, but there are challenges with bacterial infections and the number of antibacterial treatments has increased in recent years. As the lumpfish is a novel species to farming, there is a need for pharmacokinetic data and establishment of protocols for efficient antibacterial treatment. The current study describes the pharmacokinetic properties of oxolinic acid and flumequine in lumpfish. Absorption of oxolinic acid was moderate and was characterized by a calculated peak plasma concentration (Cmax) of 2.12 µg/ml after 10.3 h (Tmax) and an elimination half-life (t1/2ß) of 21 h. Area under curve (AUC) and AUC from 0 to 24 h (AUC0-24h) were calculated to be 60.9 and 34.0 h µg/ml, respectively. For flumequine, plasma Cmax was found to be 2.77 µg/ml after 7.7 h (Tmax) with t1/2ß of 22 h. The area under the curve (AUC) and AUC from 0 to 24 h (AUC0-24) were calculated as 104.3 and 50.3 h µg/ml, respectively. Corresponding Cmax values in muscle, liver, and head-kidney for oxolinic acid were 4.01, 3.04, and, 4.68 µg/g, respectively and Tmax of 11.1, 9.2, and 10.0 h, respectively. For flumequine, Cmax values of 4.16, 4.01, and 7.48 µg/g were obtained in muscle, liver, and head kidney, respectively, with corresponding Tmax values of 10.2, 10.3, and 6.0 h. Antimicrobial susceptibility values as determined by minimum inhibitory concentration (MIC) analyses against 28 isolates of Aeromonas salmonicida isolated from diseased lumpfish ranged from 0.06 to 15 µg/ml for oxolinic acid and 0.024 to 6.25 µg/ml for flumequine. Bimodal distributions in susceptibility to both oxolinic acid and flumequine were observed. The combination of pharmacokinetic properties and MIC data make possible calculation of efficient treatment doses, which are needed to improve the welfare of lumpfish and minimize development of antibiotic resistant bacteria.

Sustainable production and use of cleaner fish for the biological control of sea lice: recent advances and current challenges.

Currently, cleaner fish are one of the most widely used sea lice control strategies in Atlantic salmon aquaculture. Two species are currently being farmed in North Atlantic countries, ballan wrasse (Labrus bergylta) and lumpfish (Cyclopterus lumpus), and the sector in most countries is rapidly expanding towards self-sufficiency. The species are very different both in terms of their biology and life histories and, consequently, production and husbandry methods must be tailored to each species. There are numerous health challenges currently experienced in both species, with bacterial and parasitic diseases being the most prevalent, and cohabitation with salmon may increase the risk of disease. Good husbandry and routine health monitoring are essential, although treatment is often required when disease outbreaks occur. Ballan wrasse and lumpfish are both proven to be effective salmon delousers, although delousing efficacy can be variable in farmed fish; the provision of suitable habitat and acclimation to net-pen conditions may encourage natural behaviours, including delousing, and the use of operational welfare indicators can highlight potential welfare issues. Cleaner fish research is progressing rapidly, although much of the basic knowledge regarding the species' biology remains unknown. The simultaneous domestication of two new marine aquaculture species is a significant challenge demanding sustained effort and funding over a prolonged period of time. Research must focus on enhancing the robustness of the farmed stocks and increasing hatchery outputs to meet the urgent demands from the salmon sector and protect wild stocks from overfishing.

An overview of cleaner fish use in Ireland.

Sea lice infestations represent one of the most significant challenges facing the salmon farming industry, giving rise to lost production, additional costs of treatment and potential negative interactions with wild stocks. At present, cleaner fish, which actively remove lice from salmon, are an effective, biological, long-term option which has been adopted by many countries. In Ireland, several key studies were conducted in the 1990s on the use of wild-caught wrasse (corkwing, goldsinny and rock cook) as cleaner fish in experimental and commercial scale trials. More recently, the National University of Ireland Galway (NUIG), at their marine research facility in Carna (CRS), has undertaken applied research on ballan wrasse and lumpsucker. Currently, CRS is providing lumpsucker juveniles and research and development for the Irish salmon industry with support from BIM (Ireland's Seafood Development Agency) and Marine Harvest Ireland. There is a large amount of research currently being carried out in this area in all countries that are utilizing cleaner fish technology. The current focus in Ireland is the development of a native lumpsucker broodstock to facilitate its sustainable production. The aim of this article was to provide an overview of the research, challenges and use of cleaner fish in Ireland.

Skin mucus proteins of lumpsucker (Cyclopterus lumpus).

Fish skin mucus serves as a first line of defense against pathogens and external stressors. In this study the proteomic profile of lumpsucker skin mucus was characterized using 2D gels coupled with tandem mass spectrometry. Mucosal proteins were identified by homology searches across the databases SwissProt, NCBInr and vertebrate EST. The identified proteins were clustered into ten groups based on their gene ontology biological process in PANTHER (www.patherdb.org). Calmodulin, cystatin-B, histone H2B, peroxiredoxin1, apolipoprotein A1, natterin-2, 14-3-3 protein, alfa enolase, pentraxin, warm temperature acclimation 65 kDa (WAP65kDa) and heat shock proteins were identified. Several of the proteins are known to be involved in immune and/or stress responses. Proteomic profile established in this study could be a benchmark for differential proteomics studies.

Genetic structure of West Greenland populations of lumpfish Cyclopterus lumpus.

In this study, 11 microsatellite markers were used to determine the structure of West Greenlandic lumpfish Cyclopterus lumpus populations across six spawning locations spanning >1500 km and compared with neighbouring populations in Canada and Iceland. To evaluate whether data allow for identification of origin of C. lumpus in Greenlandic waters, genetic assignment analysis was performed for 86 C. lumpus sampled on a feeding migration. Significant structuring with isolation by distance was observed in the West Greenland samples and two major subpopulations, north and south, were suggested. Based on FST values, closer relationships were observed between Greenland and Canada, than Greenland and Iceland. Surprisingly, the North Greenland population showed more similarities with Canadian samples, than did the geographically closer south-west Greenland population. Origin could be assigned for a high proportion of non-spawning fish and demonstrated a marked east-west spatial separation of fish of Greenlandic and Icelandic genotypes.

Inter-decadal patterns of population and dietary change in sea otters at Amchitka Island, Alaska.

After having been hunted to near-extinction in the Pacific maritime fur trade, the sea otter population at Amchitka Island, Alaska increased from very low numbers in the early 1900s to near equilibrium density by the 1940s. The population persisted at or near equilibrium through the 1980s, but declined sharply in the 1990s in apparent response to increased killer whale predation. Sea otter diet and foraging behavior were studied at Amchitka from August 1992 to March 1994 and the data compared with similar information obtained during several earlier periods. In contrast with dietary patterns in the 1960s and 1970s, when the sea otter population was at or near equilibrium density and kelp-forest fishes were the dietary mainstay, these fishes were rarely eaten in the 1990s. Benthic invertebrates, particularly sea urchins, dominated the otter's diet from early summer to mid-winter, then decreased in importance during late winter and spring when numerous Pacific smooth lumpsuckers (a large and easily captured oceanic fish) were eaten. The occurrence of spawning lumpsuckers in coastal waters apparently is episodic on a scale of years to decades. The otters' recent dietary shift away from kelp-forest fishes is probably a response to the increased availability of lumpsuckers and sea urchins (both high-preference prey). Additionally, increased urchin densities have reduced kelp beds, thus further reducing the availability of kelp-forest fishes. Our findings suggest that dietary patterns reflect changes in population status and show how an ecosystem normally under top-down control and limited by coastal zone processes can be significantly perturbed by exogenous events.