Application of loop-mediated isothermal amplification (LAMP) for rapid detection of Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus) and haddock (Melanogrammus aeglefinus).

Codfish is a commercially important species of sea fish and plays an important role in the world fishery. In our study, two loop-mediated isothermal amplification (LAMP) assays (real-time fluorescence LAMP and visual LAMP) were established for the identification of three cod species in Gadidae (Gadus morhua, Gadus macrocephalus and Melanogrammus aeglefinus). 12S rDNA gene was used to design primers to distinguish the Gadidae and non-Gadidae species, and the mitochondrial Cytb gene was selected for discrimination of three cod species. After optimization, the 12S rDNA system and species-specific systems performed well, and target cod DNA could be detected in single or mixed samples. In the species-specific systems, the absolute limit of detection (LODa) of three cod species were 285, 37 and 197 pg/µL, and the relative limit of detection (LODr) reached to 1%, 0.1% and 1%, respectively. In the 12S rDNA system, the LODa of three cod species were 28.5, 37 and 19.7 pg/µL, respectively, and the LODr reached to 0.1%. Through the detection of 13 commercial cod products, the LAMP systems can detect cod contents in raw materials and deep-processed products as well. It indicated that the methods developed in this study have strong practicability and can meet the needs of routine testing.

Boreal marine fauna from the Barents Sea disperse to Arctic Northeast Greenland.

As a result of ocean warming, the species composition of the Arctic seas has begun to shift in a boreal direction. One ecosystem prone to fauna shifts is the Northeast Greenland shelf. The dispersal route taken by boreal fauna to this area is, however, not known. This knowledge is essential to predict to what extent boreal biota will colonise Arctic habitats. Using population genetics, we show that Atlantic cod (Gadus morhua), beaked redfish (Sebastes mentella), and deep-sea shrimp (Pandalus borealis) recently found on the Northeast Greenland shelf originate from the Barents Sea, and suggest that pelagic offspring were dispersed via advection across the Fram Strait. Our results indicate that boreal invasions of Arctic habitats can be driven by advection, and that the fauna of the Barents Sea can project into adjacent habitats with the potential to colonise putatively isolated Arctic ecosystems such as Northeast Greenland.

Automatic grunt detector and recognizer for Atlantic cod (Gadus morhua).

Northwest Atlantic cod (Gadus morhua) have been heavily overfished in recent years and have not yet recovered. Passive acoustic technology offers a new approach to identify the spatial location of spawning fish, as well as their seasonal and long term persistence in an area. To date, the lack of a species-specific detector has made searching for Atlantic cod grunts in large amounts of passive acoustic data cumbersome. To address this problem, an automatic grunt detection and recognition algorithm that processes yearlong passive acoustic data recordings was designed. The proposed technique is a two-stage hypothesis testing algorithm that includes detecting and recognizing all grunt-like sounds. Test results demonstrated that the algorithm provided a detection probability of 0.93 for grunts with a signal-to-noise ratio (SNR) higher than 10 dB, and a detection probability of 0.8 for grunts with the SNR ranging from 3 to 10 dB. This detector is being used to identify cod in current and historical data from U.S. waters. Its use has significantly reduced the time required to find and validate the presence of cod grunts.

Identification of Atlantic cod (Gadus morhua), ling (Molva molva), and Alaska pollock (Gadus chalcogrammus) by PCR-ELISA using duplex PCR.

Species-specific PCR-ELISA assays for the identification of Atlantic cod (Gadus morhua), Alaska pollock (Gadus chalcogrammus), and ling (Molva molva) in food products have been developed. The method, comprising a set of primers common to the first two species, a set of primers for M. molva, and a probe for each species, was designed using ND4 and cytochrome b genes as molecular markers. The sensitivity and selectivity were then determined for each assay. These assays were afterward used to analyze DNA extracted from commercial fish products. The presence of the target species was successfully detected in all analyzed samples, demonstrating the applicability of this method to the analysis of food products.

Historical DNA reveals the demographic history of Atlantic cod (Gadus morhua) in medieval and early modern Iceland.

Atlantic cod (Gadus morhua) vertebrae from archaeological sites were used to study the history of the Icelandic Atlantic cod population in the time period of 1500-1990. Specifically, we used coalescence modelling to estimate population size and fluctuations from the sequence diversity at the cytochrome b (cytb) and Pantophysin I (PanI) loci. The models are consistent with an expanding population during the warm medieval period, large historical effective population size (NE), a marked bottleneck event at 1400-1500 and a decrease in NE in early modern times. The model results are corroborated by the reduction of haplotype and nucleotide variation over time and pairwise population distance as a significant portion of nucleotide variation partitioned across the 1550 time mark. The mean age of the historical fished stock is high in medieval times with a truncation in age in early modern times. The population size crash coincides with a period of known cooling in the North Atlantic, and we conclude that the collapse may be related to climate or climate-induced ecosystem change.

The use of archived tags in retrospective genetic analysis of fish.

Collections of historical tissue samples from fish (e.g. scales and otoliths) stored in museums and fisheries institutions are precious sources of DNA for conducting retrospective genetic analysis. However, in some cases, only external tags used for documentation of spatial dynamics of fish populations have been preserved. Here, we test the usefulness of fish tags as a source of DNA for genetic analysis. We extract DNA from historical tags from cod collected in Greenlandic waters between 1950 and 1968. We show that the quantity and quality of DNA recovered from tags is comparable to DNA from archived otoliths from the same individuals. Surprisingly, levels of cross-contamination do not seem to be significantly higher in DNA from external (tag) than internal (otolith) sources. Our study therefore demonstrates that historical tags can be a highly valuable source of DNA for retrospective genetic analysis of fish.

Identification of stock components using morphological markers.

This study investigated the development of a quantitative method for distinguishing stock components of Icelandic cod Gadus morhua based on visual examination of morphology. The stock is known to be structured into genetically distinct geographic components (north and south of Iceland) and behavioural types that spawn sympatrically. Differences in morphology were tested between locations, genotypes (a proxy for behaviour) and sexes. Results show morphological markers on the head, fins and body of G. morhua that are correlated with the sex, genotype of the fish at the pantophysin (pan-I) locus and the location at which the fish were caught. Females were found to have relatively deep bodies, and the pan-I(BB) genotype (associated with deep-water feeding behaviour) have greater gaps between their fins. Overall, morphology is more useful for distinguishing sympatric genotypes but less powerful at identifying genetically distinct geographic sub-populations, perhaps because counter-gradient evolution reduces phenotypic differences even with an underlying genetic cause.

Mass spectrometric analyses of microsomal cytochrome P450 isozymes isolated from ß-naphthoflavone-treated Atlantic cod (Gadus morhua) liver reveal insights into the cod CYPome.

Four cytochrome P450 (CYP) isozymes were purified earlier from liver microsomes of Atlantic cod (Gadus morhua) exposed to ß-naphthoflavone. These isozymes were named P-450a-d and described with regard to optical properties, enzymatic activity, molecular weight and immunological reactivity. Subsequent analyses of the individual CYP fractions have shown that P-450c corresponds to a CYP1A isozyme, and immunochemical analyses have indicated that P-450b belongs to the CYP3A subfamily. However, no sequence data has been obtained to confirm these results, and the identities of the P-450a and P-450d have also remained unknown. The sequencing effort of the cod genome and expanding cod EST-databases (www.codgenome.no) have substantially increased the number of protein sequences available from cod. Mass spectrometric techniques were therefore applied to further characterize the proteins in historically archived samples of P-450a-d fractions. These analyses revealed large heterogeneities of CYPs within the purified samples. The most prominent CYP isozymes present include members of the CYP1A, CYP1C, CYP3A and CYP4V families. In total, 29 unique CYPs belonging to 9 CYP gene families and 15 subfamilies were identified with mass spectrometry. This analysis was also accompanied with genomic mining as the first step to unveil the full suite of cod CYP genes. In total, 55 CYP genes were predicted from the cod genome, distributed among 16 CYP gene families that are also present in other fish as well as mammals. Importantly, the majority of the CYPs revealed in this study have not previously been reported from cod, and represents the first proteomic survey to uncover the expressed complement of CYPs in any non-mammalian species.

Differential expression and biological activity of two piscidin paralogues and a novel splice variant in Atlantic cod (Gadus morhua L.).

The piscidin (pis) family of potent antimicrobial peptides with broad-spectrum activity has an important role in innate host defence. We have identified and characterized two pis paralogues in Atlantic cod (pis1 and pis2), as well as a novel splice variant of pis2, termed pis2-ß. Pis1 and pis2 genes have most likely originated from a recent duplication event, since they share the same four-exon structure with up to 91% identity at the intron level. The alternative transcript pis2-ß is derived from intron retention and even if not translated it may regulate pis expression through nonsense mediated decay. In spite of their overall conservation, pis genes are being shaped by positive selection and pis1, pis2 and pis2-ß code for structurally diverse mature peptides, which have different functional properties. Synthetic Pis1 displays antibacterial activity in the micromolar range against Gram-(+) and Gram-(-) bacteria, including the fish pathogens Vibrio anguillarum and Yersinia ruckeri. In contrast, synthetic Pis2 and Pis2-ß have limited or no antibacterial activity, respectively, but exhibit more potent antiparasitic activity against Tetrahymena pyriformis. In adult cod, pis1 and pis2-ß are constitutively expressed in immune-related organs, whereas pis2 is constitutively expressed in all tissues examined. Differential expression is also observed during embryonic development. In particular, pis2 and pis2-ß are maternally inherited but pis1 transcripts are only present from gastrulation onwards. It was found that antigenic challenge with attenuated V. anguillarum induces a general down-regulation of all pis in head kidney, spleen and distal intestine, suggesting that they may be used as health indicators. Taken together, our data indicate that pis is an important component of the cod innate immune system. Moreover, the two pis paralogues have undergone structural diversification and it is likely that they play multifunctional roles in Atlantic cod.

Identification and analysis of differentially expressed genes in immune tissues of Atlantic cod stimulated with formalin-killed, atypical Aeromonas salmonicida.

Physiological changes, elicited in animal immune tissues by exposure to pathogens, may be studied using functional genomics approaches. We created and characterized reciprocal suppression subtractive hybridization (SSH) cDNA libraries to identify differentially expressed genes in spleen and head kidney tissues of Atlantic cod (Gadus morhua) challenged with intraperitoneal injections of formalin-killed, atypical Aeromonas salmonicida. Of 4,154 ESTs from four cDNA libraries, 10 genes with immune-relevant functional annotations were selected for QPCR studies using individual fish templates to assess biological variability. Genes confirmed by QPCR as upregulated by A. salmonicida included interleukin-1 beta, interleukin-8, a small inducible cytokine, interferon regulatory factor 1 (IRF1), ferritin heavy subunit, cathelicidin, and hepcidin. This study is the first large-scale discovery of bacteria-responsive genes in cod and the first to demonstrate upregulation of IRF1 in fish immune tissues as a result of bacterial antigen stimulation. Given the importance of IRF1 in vertebrate immune responses to viral and bacterial pathogens, the full-length cDNA sequence of Atlantic cod IRF1 was obtained and compared with putative orthologous sequences from other organisms. Functional annotations of assembled SSH library ESTs showed that bacterial antigen stimulation caused changes in many biological processes including chemotaxis, regulation of apoptosis, antimicrobial peptide production, and iron homeostasis. Moreover, differences in spleen and head kidney gene expression responses to the bacterial antigens pointed to a potential role for the cod spleen in blood-borne pathogen clearance. Our data show that Atlantic cod immune tissue responses to bacterial antigens are similar to those seen in other fish species and higher vertebrates.

Validation of a tRNA-Glu-cytochrome b key for the molecular identification of 12 hake species (Merluccius spp.) and Atlantic Cod (Gadus morhua) using PCR-RFLPs, FINS, and BLAST.

The goal of this study was to develop a diagnostic key for hake meat to solve the limitations of previous identification methodologies, mainly related to the high degradation of the DNA recovered from processed foods. We describe the development of two molecular tools based on polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphisms of the cytochrome b gene, respectively, to identify DNA from 12 hake species in commercial products. The first assay is an exclusion test consisting of the PCR amplification of a 122 bp fragment using nested primers interspecifically conserved in Merluccius spp. and in Gadus morhua. This 122 bp amplicon, being the shortest one so far designed for hake DNA, is a useful traceability tool for highly degraded samples because its sequence contains enough interspecific diagnostic variation to identify 10 hake species and cod and has been successfully amplified from most commercial products so far tested. The second identification key follows a positive outcome of the exclusion test and consists of the PCR amplification of a 464-465 bp fragment and its digestion with three restriction enzymes whose targets map at interspecifically nonconserved sites of the cytochrome b. The key presented here has passed through a rigorous methodological calibration including its testing for genus specificity, its validation on a large number of authenticated sample types from each species range, and its implementation with a maximum likelihood method for the assignment of unknown samples. Together, these two procedures constitute the most complete molecular key so far developed for Merluccius spp., which is optimal for routine identification of hakes in large commercial samples at a reasonable cost-time ratio.

Random forests, a novel approach for discrimination of fish populations using parasites as biological tags.

Due to the complexity of host-parasite relationships, discrimination between fish populations using parasites as biological tags is difficult. This study introduces, to our knowledge for the first time, random forests (RF) as a new modelling technique in the application of parasite community data as biological markers for population assignment of fish. This novel approach is applied to a dataset with a complex structure comprising 763 parasite infracommunities in population samples of Atlantic cod, Gadus morhua, from the spawning/feeding areas in five regions in the North East Atlantic (Baltic, Celtic, Irish and North seas and Icelandic waters). The learning behaviour of RF is evaluated in comparison with two other algorithms applied to class assignment problems, the linear discriminant function analysis (LDA) and artificial neural networks (ANN). The three algorithms are used to develop predictive models applying three cross-validation procedures in a series of experiments (252 models in total). The comparative approach to RF, LDA and ANN algorithms applied to the same datasets demonstrates the competitive potential of RF for developing predictive models since RF exhibited better accuracy of prediction and outperformed LDA and ANN in the assignment of fish to their regions of sampling using parasite community data. The comparative analyses and the validation experiment with a 'blind' sample confirmed that RF models performed more effectively with a large and diverse training set and a large number of variables. The discrimination results obtained for a migratory fish species with largely overlapping parasite communities reflects the high potential of RF for developing predictive models using data that are both complex and noisy, and indicates that it is a promising tool for parasite tag studies. Our results suggest that parasite community data can be used successfully to discriminate individual cod from the five different regions of the North East Atlantic studied using RF.

Phylogeographic analysis of complete mtDNA genomes from Walleye Pollock (Gadus chalcogrammus Pallas, 1811) shows an ancient origin of genetic biodiversity.

Ursvik et al. compared the complete mitochondrial DNA (mtDNA) genome sequences of Walleye Pollock (Gadus ( = Theragra) chalcogrammus) from the Pacific Ocean with a pair of fish from an isolated population of Norwegian pollock in the Barents Sea. They concluded that the Norwegian population was recently introduced from the Pacific. We test this hypothesis within a temporal framework provided by a phylogeographic analysis of complete genomes from the pollocks' sister species, Atlantic Cod (Gadus morhua), and their divergence 3.5 mya. Pollock have a coalescent ancestor 189 +/- 25 kya. The two Norwegian fish have a common ancestor 87 +/- 7 kya, which suggests an ancient origin rather than a recent human-mediated introduction. Mitochondrial genomic biodiversity in pollock antedates the most recent glacial cycle. The clade structure of the whole-genome tree indicates that previously described single-locus mtDNA haplotypes and haplogroups are typically paraphyletic.