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Application of vaccines and dietary supplements in aquaculture: possibilities and challenges

The development of vaccines has proven essential for the development of a successful finfish aquaculture industry by preventing the occurrence of
diseases like furunculosis and vibriosis in industrialised finfish farming. Further developments, like DNA vaccines, will aid in controlling even more diseases in the future. There are however many diseases where it
is difficult to produce effective vaccines. Furthermore, many disease outbreaks may occur due to impaired animal welfare. Identifying factors associated with disease and optimizing health and welfare through
biotechnological developments is likely to be an important research area in the future. The fact that dietary manipulation can affect fish gut microbiota thus improving disease resistance is well known from
mammalian science, and is slowly gaining ground in finfish research. Both prebiotic and probiotic approaches have been used in fish, with particular
focus on lactic acid bacteria. Positive effects include enhanced growth and feed efficiency, improved immunity and disease resistance. The synbiotic concept (using a combination of probiotics and prebiotics) is particularly promising and is gaining increased interest within the research community. Immunostimulants may also improve disease resistence via increase humoral and cellular immune responses. The most promising immunostimulants at present are b-glucans, alginate and Ergosan. Additionally, medical plant extracts and their products are receiving increased attention as immune modulators, but further studies are needed. There are also great expectations or the future usage of microalgae to control microbiota and optimize fish health.

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Novel Highly Thermostable Endolysin from Thermus scotoductus MAT2119 Bacteriophage Ph2119 with Amino Acid Sequence Similarity to Eukaryotic Peptidoglycan Recognition Proteins

In this study, we present the discovery and characterization of a highly thermostable endolysin from bacteriophage Ph2119 infecting Thermus strain MAT2119 isolated from geothermal areas in Iceland. Nucleotide sequence analysis of the 16S rRNA gene affiliated the strain with the species Thermus scotoductus. Bioinformatics analysis has allowed identification in the genome of phage 2119 of an open reading frame (468 bp in length) coding for a 155-amino-acid basic protein with an Mr of 17,555. Ph2119 endolysin does not resemble any known thermophilic phage lytic enzymes. Instead, it has conserved amino acid residues (His30, Tyr58, His132, and Cys140) that form a Zn2+ binding site characteristic of T3 and T7 lysozymes, as well as eukaryotic peptidoglycan recognition proteins, which directly bind to, but also may destroy, bacterial peptidoglycan. The purified enzyme shows high lytic activity toward thermophiles, i.e., T. scotoductus (100%), Thermus thermophilus (100%), and Thermus flavus (99%), and also, to a lesser extent, toward mesophilic Gram-negative bacteria, i.e., Escherichia coli (34%), Serratia marcescens (28%), Pseudomonas fluorescens (13%), and Salmonella enterica serovar Panama (10%). The enzyme has shown no activity against a number of Gram-positive bacteria analyzed, with the exception of Deinococcus radiodurans (25%) and Bacillus cereus (15%). Ph2119 endolysin was found to be highly thermostable: it retains approximately 87% of its lytic activity after 6 h of incubation at 95°C. The optimum temperature range for the enzyme activity is 50°C to 78°C. The enzyme exhibits lytic activity in the pH range of 6 to 10 (maximum at pH 7.5 to 8.0) and is also active in the presence of up to 500 mM NaCl.

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Genetic structure of the lumpfish Cyclopterus lumpus across the North Atlantic

Lumpfish, or lumpsucker, Cyclopterus lumpus (Linnaeus, 1758) is widely distributed in the North Atlantic Ocean. It has a considerable economic value and substantial fisheries occur in several North Atlantic regions owing to the use of its fully ripe internal egg masses in the ovaries as an alternative to sturgeon caviar. Despite being intensively fished in several locations, biological knowledge is limited and no genetic structure information is available. In this study, the stock structure of C. lumpus was investigated across the North Atlantic using ten microsatellite loci. Out of ten loci, two exhibited higher level of differentiation but their inclusion/exclusion from the analyses did not drastically change the observed genetic pattern. A total of three distinct genetic groups were detected: Maine–Canada–Greenland, Iceland–Norway and Baltic Sea. These results, discussed in terms of origin of differentiation, gene flow, and selection, showed that gene flow was rather limited among the detected groups, and also between Greenland and Maine–Canada.

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Fin whale MDH-1 and MPI allozyme variation is not reflected in the corresponding DNA sequences

The appeal of genetic inference methods to assess population genetic structure and guide management efforts is grounded in the correlation between the genetic similarity and gene flow among populations. Effects of such gene flow are typically genomewide; however, some loci may appear as outliers, displaying above or below average genetic divergence relative to the genomewide level. Above average population, genetic divergence may be due to divergent selection as a result of local adaptation. Consequently, substantial efforts have been directed toward such outlying loci in order to identify traits subject to local adaptation. Here, we report the results of an investigation into the molecular basis of the substantial degree of genetic divergence previously reported at allozyme loci among North Atlantic fin whale (Balaenoptera physalus) populations. We sequenced the exons encoding for the two most divergent allozyme loci (MDH-1 and MPI) and failed to detect any nonsynonymous substitutions. Following extensive error checking and analysis of additional bioinformatic and morphological data, we hypothesize that the observed allozyme polymorphisms may reflect phenotypic plasticity at the cellular level, perhaps as a response to nutritional stress. While such plasticity is intriguing in itself, and of fundamental evolutionary interest, our key finding is that the observed allozyme variation does not appear to be a result of genetic drift, migration, or selection on the MDH-1 and MPI exons themselves, stressing the importance of interpreting allozyme data with caution. As for North Atlantic fin whale population structure, our findings support the low levels of differentiation found in previous analyses of DNA nucleotide loci.

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Present-Day Genetic Structure of Atlantic Salmon (Salmo salar) in Icelandic Rivers and Ice-Cap Retreat Models

Due to an improved understanding of past climatological conditions, it has now become possible to study the potential concordance between former climatological models and present-day genetic structure. Genetic variability was assessed in 26 samples from different rivers of Atlantic salmon in Iceland (total of 2,352 individuals), using 15 microsatellite loci. F-statistics revealed significant differences between the majority of the populations that were sampled. Bayesian cluster analyses using both prior information and no prior information on sampling location revealed the presence of two distinguishable genetic pools – namely, the Northern (Group 1) and Southern (Group 2) regions of Iceland. Furthermore, the random permutation of different allele sizes among allelic states revealed a significant mutational component to the genetic differentiation at four microsatellite loci (SsaD144, Ssa171, SSsp2201 and SsaF3), and supported the proposition of a historical origin behind the observed variation. The estimated time of divergence, using two different ABC methods, suggested that the observed genetic pattern originated from between the Last Glacial Maximum to the Younger Dryas, which serves as additional evidence of the relative immaturity of Icelandic fish populations, on account of the re-colonisation of this young environment following the Last Glacial Maximum. Additional analyses suggested the presence of several genetic entities which were likely to originate from the original groups detected.

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Outlier SNP markers reveal fine-scale genetic structure across European hake (Merluccius merluccius) populations

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome-wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large- and fine-scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (FCT = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (FCT range 0.275–0.705) and fine-scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries.

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PFAAs in Fish and Other Seafood Products from Icelandic Waters

Perfluorinatedalkyl acids (PFAAs) are of growing concern due to possible health effects on humans. Exposure assessments indicate that fish consumption is one of the major sources of perfluorooctane sulfonate (PFOS) exposure to humans, one of the major PFASs, whereas concerns of overestimation of this exposure source have been raised. Therefore, PFAAs concentrations in fish from the North Atlantic (Icelandic fishing grounds) in the flesh of different fish species were investigated along with more detailed analyses of tissue concentrations in cod (Gadus morhua) and lumpfish (Cyclopterus lumpus). Further, fish feed was investigated as a possible source of PFAAs in aquaculture by examining fish meal as feed ingredient. No PFAAs were detected in the edible part of all fish samples, except for PFOS in pollock (Pollachius virens, 0,05 ng/g wet weight). PFOS was the only PFAA detected in the fish meal samples with the exception of PFOSA in blue whiting (Micromesistius poutassou) meal (0,45 ng/g dry weight (d.w.)), where the PFOS concentration was 1,3–13 ng/g d.w. in the capelin (Mallotus villosus) and mackerel (Scomber scombrus) meal samples. The conclusions of the study are that fish commonly consumed from the Icelandic fishing grounds are unlikely to be an important source of PFAAs exposure.

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Life history of turbot in Icelandic waters: intra- and inter-population genetic diversity and otolith tracking of environmental temperatures

The stock structure of turbot was investigated between samples from S-Norway, the Irish Sea and the Kattegat, using 12 microsatellite loci and compared to the turbot caught in Icelandic waters. Highly significant genetic differentiation was observed between samples from Kattegat and other areas. Significant genetic differentiation was also observed between the Irish Sea sample on one hand and Iceland and S-Norway on the other hand. No significant genetic differentiation was observed between Iceland and S-Norway. Otoliths of 25 turbot, age ranging from 3 to 19 years, were subjected to nearly 300 mass spectrometry determinations of stable oxygen and carbon isotopes. Oxygen isotope composition (δ18O) in the otolith samples was used to estimate ambient temperature at time of otolith accretion, and yielded estimated temperatures experienced by the turbot ranging from 3 to 15 °C. Overall, the genetic analysis indicates panmixia between turbot in Icelandic and Norwegian waters. While the extensive migration of larvae between Norway and Iceland is unlikely, passive drift of turbot larva from other areas (e.g. Ireland) cannot be ruled out.

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Isolation, growth and genome of the Rhodothermus RM378 thermophilic bacteriophage

Several bacteriophages that infect different strains of the thermophilic bacterium Rhodothermus marinus were isolated and their infection pattern was studied. One phage, named RM378 was cultivated and characterized. The RM378 genome was also sequenced and analyzed. The phage was grouped as a member of the Myoviridae family with A2 morphology. It had a moderately elongated head, with dimensions of 85 and 95 nm between opposite apices and a 150 nm long tail, attached with a connector to the head. RM378 showed a virulent behavior that followed a lytic cycle of infection. It routinely gave lysates with 1011 pfu/ml, and sometimes reached titers as high as 1013 pfu/ml. The titer remained stable up to 65 °C but the phage lost viability when incubated at higher temperatures. Heating for 30 min at 96 °C lowered the titer by 104. The RM378 genome consisted of ds DNA of 129.908 bp with a GC ratio of 42.0 % and contained about 120 ORFs. A few structural proteins, such as the major head protein corresponding to the gp23 in T4, could be identified. Only 29 gene products as probable homologs to other proteins of known function could be predicted, with most showing only low similarity to known proteins in other bacteriophages. These and other studies based on sequence analysis of a large number of phage genomes showed RM378 to be distantly related to all other known T4-like phages.

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Isothermal DNA amplification by a novel and non-ubiquitous Thermus polymerase A

A novel and non-ubiquitous thermostable DNA polymerase in Thermus antranikianii was expressed in E. coli, isolated and biochemically characterized. The enzyme here referred to as Thermophi, has a C-terminal polymerase domain and a proofreading 3′→5′ exonuclease domain, but lack the 5′→3′ exonuclease domain. The corresponding gene is apparently only found in some but not all Thermus strains. The initial rate of specific activity of this polymerase on nicked DNA was about 360,000 U/mg protein. The optimum activity was found at 55 °C, pH 8.5 and 1.5 mM Mg+2. The polymerase was stable at 70 °C and lost 50% of its activity after 5 min at 85 °C, but could be stabilized above 80 °C by addition of 0.5 M L-proline. A pronounced strand-displacement activity was indicated by the large amount of DNA produced by the enzyme after an overnight, isothermal incubation in presence of hexamer primers. Both single and double stranded DNA was isothermally amplified by the enzyme. The amplified DNA was large and apparently highly branched material and composed of both single and double stranded DNA. The produced material could be partly digested by T7 enonuclease I but it was difficult to cut with common restriction enzymes. Amplification of selected genes from dilute samples was successfully demonstrated with the human β-actin gene. Good amplification was also found with 5 microsatellite markers from salmon DNA. Thermophi amplifies DNA by orders of magnitude but upon extended reaction time the DNA becomes very large and highly branched. It is composed of both single and double strands and then correctly amplified sequences only represent about 10-20% of the total DNA, and long stretches of TATATA repeats frequently occur in the amplified DNA.

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