The embryo-oil drop assembly: the timing and morphology of a critical event for fish early-life history survival.

Egg specific gravity is of relevance for fish recruitment since the ability to float influences egg and larvae development, dispersal and connectivity between fishing grounds. Using zootechnics, histological approaches, optical and electronic transmission microscopy, this study describes the morphogenetic mechanism of adhesion of the oil-drop covering layer (OCL) to the oil droplet (OD) in embryos of Merluccius merluccius under physical conditions reflecting the marine environment. The herein described primordial (p)OCL is a substructure of the inner yolk syncytial layer which contains egg organella aimed to mobilize lipidic reserves from the oil drop (OD) towards the embryo blood. It is shown that the timely OD-OCL assembly is a critical morphogenetic process for embryo and larvae survival. Such assembly depends on egg buoyance because of its influence on the embryo capacity to rotate within the perivitelline space. Therefore, oil droplet adhesion (ODA) eggs are capable to complete their development while oil droplet non-adhesion eggs (ODNA) dye soon after hatching. We show that gravity-dependent egg buoyance categories exhibit different ODA/ODNA ratios (0-77%) and that relationship diminishes under incubation systems such as sprayers, that do not assure a dynamic seawater surface mixing to avoid egg desiccation. As an adaptive trait, egg gravity strongly depends on oceanic properties such as current dynamics, turbulence, oxygen, rainfall, and salinity, whose rapid changes would likely challenge the sustainability of fisheries recruitment.

Influence of Pediveliger Larvae Stocking Density on Settlement Efficiency and Seed Production in Captivity of Mytilus galloprovincialis in Amsa Bay, Tetouan.

In mussel hatchery systems, the settlement process is a crucial element influencing seed yield. The current study assayed the influence of five densities of competent pediveliger larvae on settlement success and post-larvae production. We showed an inverse relationship between density and settlement efficiency, e.g., an attachment success of 99.4% at the lowest density (35 larvae/cm2) but only 9% at the highest density (210 larvae/cm2). However, post-larvae production was higher at intermediate larvae densities (70 larvae/cm2). The reimplementation of treatments upon post-larvae density after 6 weeks post settlement showed that the lowest-density groups bore both the highest post-larvae growth rate (22.24 ± 4.60 µm/day) and the largest head batch (48% of the size distribution), as compared to the higher-post-larvae-density groups. These results highlight the importance of optimizing both pediveliger larvae density and post-larvae density, to maximize high-quality seed yield in local hatcheries. Current rearing technologies would assure a timely commercial seed production to protect natural sea rocky beds in Alboran Sea coasts.

Population Genetic Divergence among Worldwide Gene Pools of the Mediterranean Mussel Mytilus galloprovincialis.

The Mediterranean mussel Mytilus galloprovincialis is distributed in both hemispheres either natively or introduced. The updated population genetic distribution of this species provides a useful knowledge against which future distribution shifts could be assessed. This study, performed with seven microsatellite markers and three reference species (M. edulis, M. chilensis and M. trossulus), aimed to determine the scenario of genetic divergence between 15 samples of M. galloprovincialis from 10 localities in Europe, Africa, Asia, Australia, North America and South America. In agreement with previous data, M. trossulus was the most divergent taxon of the genus, but M. chilensis appeared as an intermediate taxon between M. edulis and M. galloprovincialis, though closer to this latter. M. galloprovincialis from the Atlantic Northeast appears as the most likely source of worldwide exotic settlements instead of the previously thought Mediterranean population. The successful worldwide establishment of M. galloprovincialis suggests it is a flexible evolutionary species (FES), i.e., a species or population whose genetic background allows it to rapidly adapt to changing environments. This natural endowed plastic adaptation makes it a candidate resilient species amidst the ongoing climatic change.

Genetic Divergence and Connectivity among Gene Pools of Polyprion americanus.

Three regional gene pools of Polyprion americanus have been described so far, i.e., the North Atlantic, the Southwest Atlantic, and the Indo-Pacific Ocean. However, there is taxonomic uncertainty about the Southeast Atlantic population and there is suspicion on the existence of a third species of Polyprion in that area. Additionally, prior studies have shown a lack of genetic structuring in the Atlantic North. Nonetheless, a more conspicuous characterization of intensity, periodicity, and direction of migration are needed to properly understand the wreckfish connectivity pattern in the North Atlantic population. This study addresses the interspecific concerns highlighted above as well as the intrapopulation structure of P. americanus from the Atlantic North, using the mitochondrial DNA Cytochrome Oxidase I gene and nuclear DNA microsatellite markers on a comprehensive sampling effort. The highly divergent gene pool from South Africa was characterized by the specific Mitochondrial DNA PamCOI.Saf haplotype. Its molecular composition and phylogenetic status were conspicuously intermediate between P. americanus and P. oxygeneios, which suggests its putative hybrid origin between those species. Microsatellite variation exhibited a high differentiation (24%) among four putative Polyprion spp. gene pools which contrasts with the large genetic homogeneity within the Atlantic North stock (FSC = 0.002). The significant migration rates inferred upon Bayesian algorithms suggest a longitudinal bi-directional connectivity pattern which strengthens the migratory hypothesis previously suggested on demographic data in the Atlantic North gene pool.

EST-Microsatellite Types and Structural Scenarios in European Hake Fisheries.

A fishery's structure and connectivity are priors to its effective management. A successful description of such processes depends on both the sampling design and the choice of adequate genetic markers. EST markers are perfusing the studies of marine metapopulations and are believed to provide access to functional polymorphisms. However, the assumed adaptive role of outlier EST loci might not be generalizable. EST-microsatellites represent the upper polymorphic boundary in these regions because of their high mutation rate. We have subclassified the polymorphisms of EST-microsatellites to assess their structural contribution in the European hake, a paradigmatic and highly mobile marine species (HMMS). Because of the counterbalanced forces between directional markers (15%) and balanced markers (23%), the whole marker set offers the same structural situation as the one observed with neutral markers (62%), i.e., k = 2 gene pools. In contrast to outlier EST- microsatellites, neutral EST subsets allow one to measure crucial population phenomena for fisheries' management. The high inter-population divergence of outlier EST-microsatellites is compatible with drifted post-selection genomic regions rather than with ongoing local selective pressures. The structural scenario in hake is explainable by a limited gene flow across the Almería-Oran Front (AOF) and by the within-basin IBD pattern of connectivity plus drift-related demographic events. This study highlights how polymorphic properties of EST-microsatellite types can be useful to address mutually excluding research tasks in fisheries, i.e., to address its evolutionary history (directional markers or FAPS: Fossil Adaptive Polymorphic Systems); to delineate management units (neutral markers or NAPS: Non Adaptive Polymorphic Systems); or to ensure sustainability (balanced markers or APS: Adaptive Polymorphic Systems).

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.

Reconciling differences in natural tags to infer demographic and genetic connectivity in marine fish populations.

Processes regulating population connectivity are complex, ranging from extrinsic environmental factors to intrinsic individual based features, and are a major force shaping the persistence of fish species and population responses to harvesting and environmental change. Here we developed an integrated assessment of demographic and genetic connectivity of European flounder Platichthys flesus in the northeast Atlantic (from the Norwegian to the Portuguese coast) and Baltic Sea. Specifically, we used a Bayesian infinite mixture model to infer the most likely number of natal sources of individuals based on otolith near core chemical composition. Simultaneously, we characterised genetic connectivity via microsatellite DNA markers, and evaluated how the combined use of natural tags informed individual movement and long-term population exchange rates. Individual markers provided different insights on movement, with otolith chemistry delineating Norwegian and Baltic Sea sources, whilst genetic markers showed a latitudinal pattern which distinguished southern peripheral populations along the Iberian coast. Overall, the integrated use of natural tags resulted in outcomes that were not readily anticipated by individual movement or gene flow markers alone. Our ecological and evolutionary approach provided a synergistic view on connectivity, which will be paramount to align biological and management units and safeguard species' biocomplexity.

Methodological evaluation of DNA-based molecular keys to identify categories of mislabelling in commercial products from genus Merluccius spp.

The large variety of fish formats which are globally commercialized supports use of meta-evaluation studies to test discrimination power among molecular keys available for traceability of highly-degraded and/or chemically-modified DNA material. This paper shows that a combination of DNA identification methods validated for genus Merluccius allows 100% species assignment in hake products and offers higher diagnostic power (97% on products) than individual methods, i.e. Hake-ITS1-RFLP (89%) or Hake-Cytochrome b-RFLP (83%). A global 31% product mislabelling involved 15% of products affected by internal species substitution, as the main cause of mislabelling in ultrafrozen products, and another 16% affected by external species substitution with non-hake species, as the cause of mislabelling in processed and cooked hake-based products. The combination of both DNA keys minimizes the tool-associated rate of diagnostic failure, allowing decoupling of mislabelling categories and maximizing the quantitative adscription of products to species on any kind of hake-based products.

Prey Capture, Ingestion, and Digestion Dynamics of Octopus vulgaris Paralarvae Fed Live Zooplankton.

Octopus vulgaris is a species of great interest in research areas such as neurobiology, ethology, and ecology but also a candidate species for aquaculture as a food resource and for alleviating the fishing pressure on its wild populations. This study aimed to characterize the predatory behavior of O. vulgaris paralarvae and to quantify their digestive activity. Those processes were affordable using the video-recording analysis of 3 days post-hatching (dph), mantle-transparent paralarvae feeding on 18 types of live zooplanktonic prey. We show for the first time in a live cephalopod that octopus paralarvae attack, immobilize, drill, and ingest live cladocerans and copepods with 100% efficiency, which decreases dramatically to 60% on decapod prey (Pisidia longicornis). The majority (85%) of successful attacks targeted the prey cephalothorax while unsuccessful attacks either targeted the dorsal cephalothorax or involved prey defensive strategies (e.g., juvenile crab megalopae) or prey protected by thick carapaces (e.g., gammaridae amphipods). After immobilization, the beak, the buccal mass and the radula were involved in exoskeleton penetration and content ingestion. Ingestion time of prey content was rapid for copepods and cladocerans (73.13 ± 23.34 s) but much slower for decapod zoeae and euphausiids (152.49 ± 29.40 s). Total contact time with prey was always <5 min. Contrary to the conventional view of crop filling dynamics observed in adult O. vulgaris, food accumulated first in the stomach of paralarvae and the crop filled after the stomach volume plateaued. Peristaltic crop contractions (~18/min) moved food into the stomach (contractions ~30/min) from where it passed to the caecum. Pigmented food particles were seen to enter the digestive gland, 312 ± 32 s after the crop reached its maximum volume. Digestive tract contents passed into the terminal intestine by peristalsis (contraction frequency ~50/min) and defaecation was accompanied by an increased frequency of mantle contractions. Current results provide novel insights into both, O. vulgaris paralarvae-live prey capture strategies and the physiological mechanisms following ingestion, providing key information required to develop an effective rearing protocol for O. vulgaris paralarvae.

Disentangling the genetic and morphological structure of Patella candei complex in Macaronesia (NE Atlantic).

The uptake of natural living resources for human consumption has triggered serious changes in the balance of ecosystems. In the archipelagos of Macaronesia (NE Atlantic), limpets have been extensively exploited probably since islands were first colonized. This has led to profound consequences in the dynamics of rocky shore communities. The Patella candei complex includes various subspecies of limpets that are ascribed to a particular archipelago and has been the focus of several taxonomic surveys without much agreement. Under a conservational perspective, we apply morphometric and genetic analyses to test subspecies boundaries in P. candei and to evaluate its current population connectivity throughout Macaronesia (Azores, Madeira, and Canaries). A highly significant genetic break between archipelagos following isolation by distance was detected (FST = 0.369, p < .001). Contrastingly, significant genetic differentiation among islands (i.e., Azores) was absent possibly indicating ongoing gene flow via larval exchange between populations. Significant shell-shape differences among archipelagos were also detected using both distance-based and geometric morphometric analyses. Adaptive processes associated with niche differentiation and strong barriers to gene flow among archipelagos may be the mechanisms underlying P. candei diversification in Macaronesia. Under the very probable assumption that populations of P. candei from each archipelago are geographically and/or ecologically isolated populations, the various subspecies within the P. candei complex may be best thought of as true species using the denomination: P. candei in Selvagens, Patella gomesii in Azores, Patella ordinaria in Madeira, and Patella crenata for Canaries. This would be in agreement with stock delimitation and units of conservation of P. candei sensu latu along Macaronesia.

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.

A microsatellite genetic map of the turbot (Scophthalmus maximus).

A consensus microsatellite-based linkage map of the turbot (Scophthalmus maximus) was constructed from two unrelated families. The mapping panel was derived from a gynogenetic family of 96 haploid embryos and a biparental diploid family of 85 full-sib progeny with known linkage phase. A total of 242 microsatellites were mapped in 26 linkage groups, six markers remaining unlinked. The consensus map length was 1343.2 cM, with an average distance between markers of 6.5 +/- 0.5 cM. Similar length of female and male maps was evidenced. However, the mean recombination at common intervals throughout the genome revealed significant differences between sexes, approximately 1.6 times higher in the female than in the male. The comparison of turbot microsatellite flanking sequences against the Tetraodon nigroviridis genome revealed 55 significant matches, with a mean length of 102 bp and high sequence similarity (81-100%). The comparative mapping revealed significant syntenic regions among fish species. This study represents the first linkage map in the turbot, one of the most important flatfish in European aquaculture. This map will be suitable for QTL identification of productive traits in this species and for further evolutionary studies in fish and vertebrate species.

ITS1-rDNA-based methodology to identify world-wide hake species of the Genus Merluccius.

Species-specific DNA-based tags are valuable tools for the management of both fisheries and commercial fish products. In this study, we have developed a two-step molecular tool to detect the presence of hake DNA (Merluccius spp.) and to identify the exact hake species present in an blind sample. The first test involves PCR amplification of an ITS1-rDNA fragment of 193 bp using nested primers that are interspecifically conserved in Merluccius spp. and Atlantic cod, Gadus morhua. The second test consists of the PCR amplification of a 602-659 bp DNA fragment spanning part of the ribosomal cluster 18S-ITS1-5.8S and digesting it with four restriction enzymes whose targets map at interspecifically nonconserved sites of the ITS1. Alternatively, the identification of hake species can be achieved by FINS or BLAST, using the nucleotide sequence of either the whole ITS1 sequence or its nested fragment of 193 bp. Because of their high reproducibility and ease of execution, these procedures allow for routine analysis and constitute high reliable tools for the rapid identification of 12 species of hake.

Distribution and abundance of microsatellites in the genome of bivalves.

Understanding how microsatellites are distributed in eukaryotic genomes is important to clarify the differential abundance of these repeats under an evolutionary scenario. We have concatenated data from 3165 DNA sequences of 326 Bivalvia species to search for taxonomic patterns of microsatellite distribution in genomic regions of markedly different functionality. Some microsatellite motifs in bivalves showed one of the lowest genomic densities observed among eukaryotes. Contrary to the expectation of a random distribution of microsatellites, they were overrepresented in introns (245 loci/Mb) compared to their frequency in exons (85 loci/Mb). Closely related species showed remarkable differences in microsatellite density suggesting species-specific properties as for mutation/repair efficiency on replication slippage. There was no evidence of a positive correlation between the density of microsatellites in intergenic DNA and the DNA-content. This research is relevant to better understand the forces shaping the distribution of microsatellites in the genome of bivalves.