Essential and toxic elements in sustainable and underutilized seafood species and derived semi-industrial ready-to-eat products.

Blue whiting (Micromesistius poutassou), pouting (Trisopterus luscus) and common dab (Limanda limanda) are underutilized fish species attractive in terms of sustainability. However, there is limited information about their nutritional characteristics as well as potential presence of environmental contaminants. Specimens caught in European waters were characterised for their content of essential and toxic elements. The three species, along with common carp and blue mussels, relevant for sustainable production too, were employed as raw materials for the development of semi-industrial ready-to-eat products. Calcium, copper, iodine, iron, selenium, zinc, cadmium, lead, mercury, and nickel were determined by ICP-MS, whereas methylmercury was determined by HPLC-ICP-MS. These two techniques were also used to determine arsenic and inorganic arsenic, respectively, in blue mussel and derived products. Differences in element contents were related to the biology and ecology of the examined species. Intake of nutrients and exposure to contaminants were assessed in relation to the relevant DRVs and HBGVs, respectively. All the species were found to be valuable dietary sources of selenium. Pouting was rich in iodine and mussels were good sources of iodine and iron. These two species had comparatively higher levels of mercury and lead, respectively. However, the levels of contaminants were generally of no concern in both raw materials and products. Iodine bioaccessibility was studied in blue whiting, a species with an intermediate iodine content, and found to be 98%. Selenium:mercury molar ratios were assessed and found to be favourable. The semi-industrial products were found to be good sources of selenium and many of them provided appreciable amounts of calcium, iron, copper and zinc.

Phylogenetic prospecting for cryptic species of the genus Merluccius (Actinopterygii: Merlucciidae).

Hakes of the genus Merluccius include 11 valid species as well a number of rare morphotypes suspected to be "cryptic species". Concatenated nucDNA ITS1-rDNA and mtDNA cyt b sequences plus nested ITS1Nes sequences allowed to ascribe 14 specimens of nine rare morphotypes from the South Pacific and the South Atlantic to the phylogenetic backbone of this genus. Bayesian analyses pointed to M. bilinearis and M. albidus as the oldest species of the genus and the New World cluster, respectively. The phylogenetic status of M. angustimanus from the upper Gulf of California suggests its hybrid origin between M. gayi and M. productus from about 0.25 MYA, although an ever since confinement of a subset of those species cannot be ruled out. The molecular phylodiagnostic test suggests a common origin of all rare morphotypes and the absence of cryptic hake species in the Southern Cone. The molecular background of the morphotypes distributed between the Western Pacific South of New Zealand and the western Atlantic South of Argentina is compatible with their hybrid origin between M. gayi and both, M. australis or M. hubbsi, respectively.

Molecular mechanism and history of non-sense to sense evolution of antifreeze glycoprotein gene in northern gadids.

A fundamental question in evolutionary biology is how genetic novelty arises. De novo gene birth is a recently recognized mechanism, but the evolutionary process and function of putative de novo genes remain largely obscure. With a clear life-saving function, the diverse antifreeze proteins of polar fishes are exemplary adaptive innovations and models for investigating new gene evolution. Here, we report clear evidence and a detailed molecular mechanism for the de novo formation of the northern gadid (codfish) antifreeze glycoprotein (AFGP) gene from a minimal noncoding sequence. We constructed genomic DNA libraries for AFGP-bearing and AFGP-lacking species across the gadid phylogeny and performed fine-scale comparative analyses of the AFGP genomic loci and homologs. We identified the noncoding founder region and a nine-nucleotide (9-nt) element therein that supplied the codons for one Thr-Ala-Ala unit from which the extant repetitive AFGP-coding sequence (cds) arose through tandem duplications. The latent signal peptide (SP)-coding exons were fortuitous noncoding DNA sequence immediately upstream of the 9-nt element, which, when spliced, supplied a typical secretory signal. Through a 1-nt frameshift mutation, these two parts formed a single read-through open reading frame (ORF). It became functionalized when a putative translocation event conferred the essential cis promoter for transcriptional initiation. We experimentally proved that all genic components of the extant gadid AFGP originated from entirely nongenic DNA. The gadid AFGP evolutionary process also represents a rare example of the proto-ORF model of de novo gene birth where a fully formed ORF existed before the regulatory element to activate transcription was acquired.

Long-read sequence capture of the haemoglobin gene clusters across codfish species.

Combining high-throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short-read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long-read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom-made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage-specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long-read capture as a versatile approach for comparative genomic studies by generation of a cross-species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes.

Diversity of the CO1 Gene of Mitochondrial DNA in Representatives of Genus Antimora (Moridae, Gadiformes) in the World Oceans.

The frequency of occurrence of the COI gene of mitochondrial DNA in the Pacific flatnose Antimora microlepis and blue antimora Antimora rostrata (Moridae, Gadiformes) was analyzed in samples collected in different areas of the World Ocean. The revealed maximum haplotype diversity of COI in the blue antimora in the North Atlantics may indicate that this species emerged in this region, from which it widely distributed in the World Ocean. The Pacific flatnose Antimora microlepis originated from Antimora rostrata. Antimora might penetrate into the North Pacific by several routes: through the Panama Strait, along the coast of Antarctica, or through the Indian Ocean along the coast of Australia.

DNA barcoding coupled to HRM analysis as a new and simple tool for the authentication of Gadidae fish species.

This work aimed to exploit the use of DNA mini-barcodes combined with high resolution melting (HRM) for the authentication of gadoid species: Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), Alaska pollock (Theragra chalcogramma) and saithe (Pollachius virens). Two DNA barcode regions, namely cytochrome c oxidase subunit I (COI) and cytochrome b (cytb), were analysed in silico to identify genetic variability among the four species and used, subsequently, to develop a real-time PCR method coupled with HRM analysis. The cytb mini-barcode enabled best discrimination of the target species with a high level of confidence (99.3%). The approach was applied successfully to identify gadoid species in 30 fish-containing foods, 30% of which were not as declared on the label. Herein, a novel approach for rapid, simple and cost-effective discrimination/clustering, as a tool to authenticate Gadidae fish species, according to their genetic relationship, is proposed.

Determination of the Geographical Origin of All Commercial Hake Species by Stable Isotope Ratio (SIR) Analysis.

The determination of the geographical origin of food products is relevant to comply with the legal regulations of traceability, to avoid food fraud, and to guarantee food quality and safety to the consumers. For these reasons, stable isotope ratio (SIR) analysis using an isotope ratio mass spectrometry (IRMS) instrument is one of the most useful techniques for evaluating food traceability and authenticity. The present study was aimed to determine, for the first time, the geographical origin for all commercial fish species belonging to the Merlucciidae family using SIR analysis of carbon (δ13C) and nitrogen (δ15N). The specific results enabled their clear classification according to the FAO (Food and Agriculture Organization of the United Nations) fishing areas, latitude, and geographical origin in the following six different clusters: European, North African, South African, North American, South American, and Australian hake species.

Depth as a driver of evolution in the deep sea: Insights from grenadiers (Gadiformes: Macrouridae) of the genus Coryphaenoides.

Here we consider the role of depth as a driver of evolution in a genus of deep-sea fishes. We provide a phylogeny for the genus Coryphaenoides (Gadiformes: Macrouridae) that represents the breadth of habitat use and distributions for these species. In our consensus phylogeny species found at abyssal depths (>4000m) form a well-supported lineage, which interestingly also includes two non-abyssal species, C. striaturus and C. murrayi, diverging from the basal node of that lineage. Biogeographic analyses suggest the genus may have originated in the Southern and Pacific Oceans where contemporary species diversity is highest. The abyssal lineage seems to have arisen secondarily and likely originated in the Southern/Pacific Oceans but diversification of this lineage occurred in the Northern Atlantic Ocean. All abyssal species are found in the North Atlantic with the exception of C. yaquinae in the North Pacific and C. filicauda in the Southern Ocean. Abyssal species tend to have broad depth ranges and wide distributions, indicating that the stability of the deep oceans and the ability to live across wide depths may promote population connectivity and facilitate large ranges. We also confirm that morphologically defined subgenera do not agree with our phylogeny and that the Giant grenadier (formerly Albatrossia pectoralis) belongs to Coryphaenoides, indicating that a taxonomic revision of the genus is needed. We discuss the implications of our findings for understanding the radiation and diversification of this genus, and the likely role of adaptation to the abyss.

Parasite fauna of Antarctic Macrourus whitsoni (Gadiformes: Macrouridae) in comparison with closely related macrourids.

BACKGROUND: The extreme, isolated environment within the Antarctic Convergence has fuelled the evolution of a highly endemic fauna with unique adaptations. One species known from this area is the Whitson's grenadier Macrourus whitsoni (Regan, 1913). While closely related species occurring in the Northern Hemisphere were targets of a variety of studies, knowledge on M. whitsoni is scarce, including not only its ecology but also its parasite fauna. Parasites, an often overlooked but important component of every ecosystem, can provide important insights into host ecology, including feeding habits, food web interactions and distribution patterns. The aim of our study was to increase the currently limited knowledge on the ecology of M. whitsoni and its parasite life-cycles. METHODS: In this study, parasite fauna and stomach content of 50 specimens of M. whitsoni were sampled off Elephant and King George Islands. Fish samples were morphological, food ecological and parasitological examined and parasites morphological and partly molecular identified. To evaluate the findings, results were compared with other macrourid species. RESULTS: The parasite fauna of M. whitsoni revealed 9 genera and 17 species. Stomach content analysis indicated Amphipoda and Mysida as the primary food source. Considering the parasites of M. whitsoni, the highest diversity was found within the Digenea, while prevalence was highest for the Acanthocephala and Nematoda. The diverse parasite fauna of M. whitsoni together with the stomach content analysis, suggests a benthopelagic mode of life. Furthermore, an extensive evaluation of the parasite fauna of species of the Macrourinae was conducted, which is probably the most thorough one yet, to compare the findings with closely related host fish species. A similarity analysis revealed a strong connection between the parasite fauna composition and geographical distribution, with a clear separation between the parasite faunas in fishes sampled in the Pacific and the Atlantic Oceans. CONCLUSIONS: Due to the isolated habitat within the Antarctic Conversion, the parasite fauna of M. whitsoni differs clearly from those of closely related and closely occurring species of the genus Macrourus. Our study revealed an endemically dominated parasite fauna, with parasites often host-specific to M. whitsoni. The comparison with the faunas of other species of the Macrourinae revealed a largely endemic parasite fauna, which emphasizes again the isolated character of the Antarctic shelf regions.

Redescription of Urophycis brasiliensis (Kaup 1858), a senior synonym of Urophycis latus Miranda Ribeiro 1903 (Gadiformes: Phycidae).

Urophycis Gill 1864 is a genus of demersal fish composed by eight valid species with anti-tropical distributions in the western Atlantic. Only two species occur in the South Atlantic: U. brasiliensis (Kaup 1858) and U. mystacea Miranda Ribeiro 1903. These species have similar ranges from southeastern Brazil to northern Argentina, but U. brasiliensis occurs in more shallow waters than U. mystacea. Both species are important fishery resources in southern Brazil and Uruguay. Herein, we redescribe U. brasiliensis based on comparison of types and additional specimens, including the description of the Sagitta otolith, formerly place U. latus Miranda Ribeiro 1903 as a junior synonym of U. brasiliensis, and provide an updated map of distribution.

New records of larval stages of the eel cod genus Muraenolepis Günther 1880 (Gadiformes: Muraenolepididae) from the western Antarctic Peninsula.

Three newly discovered larval specimens of the genus Muraenolepis collected from the waters of the western Antarctic Peninsula are described. Knowledge of their natural history is sparse and information about their early life history is based on only a few larval stages. Here, the available literature on larval eel cods is reviewed, and the specimens placed in context.

netview p: a network visualization tool to unravel complex population structure using genome-wide SNPs.

Network-based approaches are emerging as valuable tools for the analysis of complex genetic structure in wild and captive populations. netview p combines data quality control with the construction of population networks through mutual k-nearest neighbours thresholds applied to genome-wide SNPs. The program is cross-platform compatible, open-source and efficiently operates on data ranging from hundreds to hundreds of thousands of SNPs. The pipeline was used for the analysis of pedigree data from simulated (n = 750, SNPs = 1279) and captive silver-lipped pearl oysters (n = 415, SNPs = 1107), wild populations of the European hake from the Atlantic and Mediterranean (n = 834, SNPs = 380) and grey wolves from North America (n = 239, SNPs = 78 255). The population networks effectively visualize large- and fine-scale genetic structure within and between populations, including family-level structure and relationships. netview p comprises a network-based addition to other population analysis tools and provides user-friendly access to a complex network analysis pipeline through implementation in python.

Merluccius tasmanicus Matallanas & Lloris 2006 is a junior synonym of M. australis (Hutton 1872) (Gadiformes: Merluciidae) based on morphological and molecular data.

The high intraspecific variation among and the conservative external morphology of Merluccius spp. have resulted in serious identification difficulties. Four hundred and twenty fresh and preserved specimens of Merluccius were analyzed, including the type series of Merluccius australis, M. tasmanicus and M. hubbsi; specimens of M. hubbsi from Argentina, Brazil and Uruguay, and individuals of M. australis from Argentina and New Zealand were examined. The nomenclatural status of the type specimens of M. australis is discussed and the designation of a lectotype and a paralectotype is proposed. The comparative study of morphology, meristic, traditional and landmark-based morphometry, both external and internal, and through DNA-based Barcoding molecular tools demonstrates that Merluccius tasmanicus is a junior synonym of Merluccius australis. Meristic and morphometric characters of types of M. tasmanicus completely overlap those of M. australis, whereas M. hubbsi show fewer scales along the lateral line, total vertebrae, second dorsal and anal-fin rays. A trend of a longer snout and wider head in M. australis and M. tasmanicus, and larger eyes and longer pelvic fins, in M. hubbsi was observed. While discriminant characters were found in the internal elements (hyomandibula, urohyal and sagitta otolith) between M. hubbsi and M. australis, none were observed between M. australis and those reported for M. tasmanicus. DNA barcoding analyses found no evidence of the existence of other species of Merluccius besides M. hubbsi and M. australis.

Characterization of mitochondrial control region in Merlucciidae: sequence variation and molecular phylogeny.

In order to describe the structure and evolution of Merlucciidae and related Gadiformes mitochondrial control region we analysed 470 bp of 31 taxa belonging to 28 different species. The general structure and conserved sequence blocks observed in Gadiformes mitochondrial control region are similar to those present in other teleost fishes. The length of this segment is variable among related species due to the presence of numerous indels at domain I. Domain II is the most conserved region with a high G content. The GTGGG-box is absent in all Merluccius and seven other Gadidae species. Several methods of phylogenetic analyses has revealed the monophyly of Gadiformes, Gadinae and Merlucciidae. Merlucciidae is most closely related to Gadidae. Within Merlucciidae, American and Euroafrican clades show similar levels of differentiation to those within Gadinae where Trisopterus and Micromesistius are sister taxa. Genetic distance values for Merluccius subspecies pairs are less than half of those between species, comparable to intra specific differentiation levels in marine fish species.

New tools (DNA barcoding), old hypothesis: the case of the taxonomic identity of the Argentine hakes (Actinopterygii: Merluccius).

The present study evaluated the possible occurrence of cryptic species among Merluccidae from Argentina by examining sequences of cytochrome c oxidase subunit I (coI) mtDNA. This approach can discriminate Merluccius hubbsi and Merluccius australis; specimens with morphological diagnostic characters of Merluccius patagonicus formed a cohesive cluster with M. hubbsi specimens. BIN analysis confirmed the effectiveness of barcoding within a global context.

Material properties of Pacific hake, Humboldt squid, and two species of myctophids in the California Current.

Material properties of the flesh from three fish species (Merluccius productus, Symbolophorus californiensis, and Diaphus theta), and several body parts of the Humboldt squid (Dosidicus gigas) collected from the California Current ecosystem were measured. The density contrast relative to seawater varied within and among taxa for fish flesh (0.9919-1.036), squid soft body parts (mantle, arms, tentacle, braincase, eyes; 1.009-1.057), and squid hard body parts (beak and pen; 1.085-1.459). Effects of animal length and environmental conditions on nekton density contrast were investigated. The sound speed contrast relative to seawater varied within and among taxa for fish flesh (0.986-1.027) and Humboldt squid mantle and braincase (0.937-1.028). Material properties in this study are similar to values from previous studies on species with similar life histories. In general, the sound speed and density of soft body parts of fish and squid were 1%-3% and 1%-6%, respectively, greater than the surrounding seawater. Hard parts of the squid were significantly more dense (6%-46%) than seawater. The material properties reported here can be used to improve target strength estimates from acoustic scattering models, which could increase the accuracy of biomass estimates from acoustic surveys for these nekton.

Evaluating the resolution power of new microsatellites for species identification and stock delimitation in the Cape hakes Merluccius paradoxus and Merluccius capensis (Teleostei: Merlucciidae).

The utility of 15 new and 17 previously published microsatellite markers was evaluated for species identification and stock delimitation in the deep-water hake Merluccius paradoxus and the shallow-water hake Merluccius capensis. A total of 14 microsatellites were polymorphic in M. paradoxus and 10 in M. capensis. Two markers could individually discriminate the species using Bayesian clustering methods and a statistical power analysis showed that the set of markers for each species is likely to detect subtle genetic differentiation (FST < 0·006) that will be valuable to delimit and characterize genetic stocks.

Applying population genetics for authentication of marine fish: the case of saithe (Pollachius virens).

The number of fishery products with a quite detailed description of the origin is increasing. This trend is driven by the interest of consumers and the fight against illegal unregulated and unreported fisheries. Unfortunately, there is a lack of methods to prove this information experimentally besides the document-based traceability assessments. For marine fish population genetics is a promising strategy, but research is concentrated only on a few species. Saithe is a commercially important fish species, despite the fact that genetic knowledge is scarce regarding the specification of populations. For a comparative study cost- and time-effective strategies were tested: We found RAPD-PCR to be a useful method for low-budget research or prestudies. Adoption of microsatellites from closely related species turned out to be possible with limited success quota. Our results suggest a clustered structure of populations within the Northeast Atlantic, probably overlapping in the northern North Sea.

Head and otolith morphology of the genera Hymenocephalus, Hymenogadus and Spicomacrurus (Macrouridae), with the description of three new species.

The fishes of the genus Hymenocephalus live over continental slope terrain, chiefly between 300 and 1000 m water depths, in all tropical oceans, except the eastern Pacific. They are characterized by an elongated light organ with two lenses, striations on jugular and thorax, and by an extraordinary development of sensory reception organs: strongly enlarged eyes, exceptionally large and specialized sagittal otoliths and extremely wide and deep head canals resembling caverns and housing the cephalic sensory organ for motion reception (lateral line system).        The purpose of this study is to evaluate the potential that a detailed analysis of the head and otolith morphology can offer for distinguishing the various species and assessment of their interrelationships. About 500 specimens were investigated, representing all 22 nominal species of the genus Hymenocephalus, except for H. barbatulus (specimens of which could not be located), the two species of the related genus Hymenogadus and three of the four species of Spicomacrurus.  Because of the delicate and thin nature of the head bones and head skin typical for the fishes of the genus Hymenocephalus and the deteriorating effects of formalin to the aragonitic otoliths, only a fraction of the studied specimens actually contributed useful data, although that fraction represented all species studied.        Otoliths in particular and aspects of the cephalic canal system were found to contribute additional characters that help to verify the status of certain controversial species such as H. heterolepis, H. nascens and species within the H. striatissimus and H. grimaldii Groups. Hymenocephalus longiceps is revised to represent a junior synonym of H. longibarbis. Eight species groups are defined within the genus Hymenocephalus. Three new species are being described in the course of this review: H. iwamotoi from off northwestern Australia, H. sazonovi from the Sala y Gomez and Nazca Ridges, and H. punt from northern Somalia and the Gulf of Aden, now raising the count of valid species in the genus to a total of 24.        The specializations of the sensory reception organs show a variety of developments with well-expressed phylogenetic polarities that are discussed in the context of their evolution and interrelationships. A well-documented case of polarity reversal of certain characters in the H. aterrimus Group is interpreted as a functional adaptation to migration of these fishes into a deeper water environment that favors different specializations of the sensory reception.

A cladistic re-analysis of the Gadiformes (Teleostei, Paracanthopterygii) using three-item analysis.

In the present paper, we describe LisBeth, a newly published phylogenetic program. LisBeth implements the cladistic three-item analysis for systematics and biogeography. We show how LisBeth handles character representation, character analysis, exact search functions, reconstruction of the intersection tree and other features, such as retention index, completeness index, character history and link with other programs such as PAUP*, version 4.0b10, TNT and Xper2. Using LisBeth, we reanalyze the phylogeny based on morphological characters of the order Gadiformes (Teleostei, Paracanthopterygii) published by Endo (2002) and compare our results. We also describe the generation of hypotheses of homology following Endo's guidelines versus 3ia representation schemes. We compare the topologies of all results and show the implications of the interpretation of character history.