Novel tools for conservation genetics in marine fish: population structure and evolution of the European hake (Merluccius merluccius) inferred by SNP variation and applications to traceability

The research presented in my PhD thesis is part of a wider European project, FishPopTrace, focused on traceability of fish populations and products. My work was aimed at developing and analyzing novel genetic tools for a widely distributed marine fish species, the European hake (Merluccius merluccius), in order to investigate population genetic structure and explore potential applications to traceability scenarios. A total of 395 SNPs (Single Nucleotide Polymorphisms) were discovered from a massive collection of Expressed Sequence Tags, obtained by high-throughput sequencing, and validated on 19 geographic samples from Atlantic and Mediterranean. Genome-scan approaches were applied to identify polymorphisms on genes potentially under divergent selection (outlier SNPs), showing higher genetic differentiation among populations respect to the average observed across loci. Comparative analysis on population structure were carried out on putative neutral and outlier loci at wide (Atlantic and Mediterranean samples) and regional (samples within each basin) spatial scales, to disentangle the effects of demographic and adaptive evolutionary forces on European hake populations genetic structure. Results demonstrated the potential of outlier loci to unveil fine scale genetic structure, possibly identifying locally adapted populations, despite the weak signal showed from putative neutral SNPs. The application of outlier SNPs within the framework of fishery resources management was also explored. A minimum panel of SNP markers showing maximum discriminatory power was selected and applied to a traceability scenario aiming at identifying the basin (and hence the stock) of origin, Atlantic or Mediterranean, of individual fish. This case study illustrates how molecular analytical technologies have operational potential in real-world contexts, and more specifically, potential to support fisheries control and enforcement and fish and fish product traceability.

Preparation and functional characterisation of recombinant silicatein-alpha from the sponge Suberites domuncula

The marine world is an immense source of biodiversity that provides substances with striking potentials in medicinal chemistry and biotechnology. Sponges (Porifera) are marine animals that represent the most impressive example of organisms possessing the ability to metabolise silica through a family of enzymes known as silicateins. Complex skeletal structures (spicules) made of pure biogenic silica (biosilica) are produced under physiological conditions. Biosilica is a natural material comprising inorganic and organic components with unique mechanical, optical, and physico-chemical properties, including promising potential to be used for development of therapeutic agents in regenerative medicine. Unravelling the intimate physiological mechanisms occurring in sponges during the construction of their siliceous spicules is an on-going project, and several questions have been addressed by the studies proposed by our working group. In this doctoral work, the recombinant DNA technology is exploited for functional and structural characterisation of silicatein. Its precursors are produced as fusion proteins with a chaperone tag (named TF-Ps), and a robust method for the overexpression of native soluble proteins in high concentrations has been developed. In addition, it is observed and proven experimentally that the maturation of silicatein is an autocatalytic event that: (i) can be modulated by rational use of protease inhibitors; (ii) is influenced by the temperature of the environment; (iii) only slightly depends on the pH. In the same experimental framework, observations on the dynamics in the maturation of silicateins allow a better understanding of how the axial filaments form during the early stages of spicule construction. In addition, the definition of new distinct properties of silicatein (termed “structure-guiding” and “structure-forming”) is introduced. By homology models and through comparisons with similar proteins (the cathepsins), domains with significant surface hydrophobicity are identified as potential self-assembly mediators. Moreover, a high-throughput screening showed that TF-Ps could generate crystals under certain conditions, becoming promising for further structural studies. With the goal of optimise the properties of the recombinant silicatein, implementation of new production systems are tried for the first time. Success in the expression of silicatein-type proteins in insect and yeast cells, constitute a promising basis for further development, towards the establishment of an efficient method for the production of a high-value pure and soluble protein.

Tracing the origin of Arctic driftwood

Arctic environments, where surface temperatures increase and sea ice cover and permafrost depth decrease, are very sensitive to even slight climatic variations. Placing recent environmental change of the high-northern latitudes in a long-term context is, however, complicated by too short meteorological observations and too few proxy records. Driftwood may represent a unique cross-disciplinary archive at the interface of marine and terrestrial processes. Here, we introduce 1445 driftwood remains from coastal East Greenland and Svalbard. Macroscopy and microscopy were applied for wood anatomical classification; a multi-species subset was used for detecting fungi; and information on boreal vegetation patterns, circumpolar river systems, and ocean current dynamics was reviewed and evaluated. Four conifer (Pinus, Larix, Picea, and Abies) and three deciduous (Populus, Salix, and Betula) genera were differentiated. Species-specific identification also separated Pinus sylvestris and Pinus sibirica, which account for ~40% of all driftwood and predominantly originate from western and central Siberia. Larch and spruce from Siberia or North America represents ~26% and ~18% of all materials, respectively. Fungal colonization caused different levels of driftwood staining and/or decay. Our results demonstrate the importance of combining wood anatomical knowledge with insight on boreal forest composition for successfully tracing the origin of Arctic driftwood. To ultimately reconstruct spatiotemporal variations in ocean currents, and to better quantify postglacial uplift rates, we recommend consideration of dendrochronologically dated material from many more circumpolar sites.

Origin and evolution of reactive and noble gases dissolved in matrix pore water

Reactive and noble gases dissolved in matrix pore water of low permeable crystalline bedrock were successfully extracted and characterized for the fist time based on drillcore samples from the Olkiluoto investigation site (SW Finland). Interaction between matrix pore water and fracture groundwater occurs predominately by diffusion. Changes in the chemical and isotopic composition of gases dissolved in fracture groundwater are transmitted and preserved in the pore water. Absolute concentrations, their ratios and the stable carbon isotope signature of hydrocarbon gases dissolved in pore water give valuable indications about the evolution of these gases in the nearby-flowing fracture groundwaters. Inert noble gases dissolved in matrix pore water and their isotopes combined with their in-situ production and accumulation rates deliver information about the residence time of pore water.

Climatic impact on Al, K, Sc and Ti in marine sediments: Evidence from ODP Site 1144, South China Sea

Al, K, Sc and Ti concentrations of the terrestrial material-dominant sediments from ODP site 1144 were reported. Comparison between the bulk and the acid-leached sediments indicates that about 20~30% of the Al, K and Sc in the bulk sediments are not hosted in terrestrial detritus, rather they are of authigenic origin. However, authigenic Ti is negligible. The results indicate that Ti rather than Al is the best proxy for terrestrial materials. Significant climate controls are displayed in the Al/Ti, K/Ti and Sc/Ti variation patterns both for the bulk and the acid leached sediments. Such variation patterns can be mainly accounted for in terms of climate change in their provenance areas in South China. Elevated Al/Ti, K/Ti and Sc/Ti ratios during interglacial periods indicate that chemical weathering then was stronger than during glacial periods, which might be related to a more humid climate in interglacial periods.

Sedimentology and origin of lower Cretaceous pelagic carbonates and redeposited clastics at DSDP Hole 76-534A

Drilling at Site 534 in the Blake-Bahama Basin recovered 268 m of Lower Cretaceous, Berriasian to Hauterivian, pelagic carbonates, together with volumetrically minor intercalations of claystone, black shales, and terrigenous and calcareous elastics. Radiolarian nannofossil pelagic carbonates accumulated in water depths of about 3300 to 3650 m, below the ACD (aragonite compensation depth) but close to the CCD (calcite compensation depth). Radiolarian abundance points to a relatively fertile ocean. In the Hauterivian and Barremian, during times of warm, humid climate and rising sea level, turbiditic influxes of both terrigenous and calcareous sediments, and minor debris flows were derived from the adjacent Blake Plateau. The claystones and black shales accumulated on the continental rise, then were redeposited onto the abyssal plain by turbidity currents. Dark organic-rich and pale organic-poor couplets are attributed to climatic variations on land, which controlled the input of terrigenous organic matter. Highly persistent, fine, parallel lamination in the pelagic chalks is explained by repeated algal "blooms." During early diagenesis, organic-poor carbonates remained oxygenated and were cemented early, whereas organic-rich intervals, devoid of burrowing organisms, continued to compact later in diagenesis. Interstitial dissolved-oxygen levels fluctuated repeatedly, but bottom waters were never static nor anoxic. The central western Atlantic in the Lower Cretaceous was thus a relatively fertile and wellmixed ocean basin.

Distribution, origin, and hydrocarbon-potential of organic matter in sediments from DSDP Leg 66 Holes

As part of an ongoing program of organic geochemical studies of sediments recovered by the Deep Sea Drilling Project, we have analyzed the types, amounts, and thermal alteration indices of organic matter collected from the Pacific continental margin of southern Mexico on Leg 66. The samples were pieces of core frozen aboard ship. Some of them were analyzed by pyrolysis, heavy C15+ hydrocarbons, and nonhydrocarbons to help determine their origin and hydrocarbon potential. Our main objectives were to find out how much organic matter was being deposited; to establish whether it derived from marine or terrestrial sources; to determine the controls of deposition of organic matter; to estimate the hydrocarbon potential of the drilled section; and to compare and contrast organic sedimentation here with that on other margins.

Origin and evolution of Cycladophora davisiana (Radiolaria) in DSDP Hole 19-192, Northwest Pacific

This paper documents the evolutionary history of Cycladophora davisiana Ehrenberg from an uppermost Miocene to Pleistocene sedimentary record in the high-latitude Northwest Pacific. It apparently evolved from C. sakaii Motoyama through a series of intermediates. C. sakaii has a relatively large shell with an external spongy layer. The evolutionary transition is characterized by a relatively rapid decrease in thorax size with a reduction of the spongy appendage. This change occurred during about 0.4 m.y. from 2.8 to 2.4 Ma without cladogenesis. Following this interval, a decrease in thorax size continued gradually up to the Recent, resulting in a very small morphology. Although the population of C. davisiana first appeared at about 2.5 Ma, some morphotypic specimens may occur in earlier periods as indistinguishable very small endmembers in the C. sakaii populations. Timing of the first appearance events both of morphotypic specimens and of a population of C. davisiana in Site 192 and previously reported cores does not disprove the idea that C. davisiana evolved first in the Northwest Pacific region, and later migrated into other regions of the world ocean. Biometrics clearly indicate no direct phylogenetic relationships between C. davisiana and C. cornutoides Kling in the studied core. Thus, the latter species, which was originally described as a variation and later elevated to a subspecies of the former species, is separated from the former species and raised to the species rank.

Distribution of marine litter at bathyal and abyssal depths in the Mediterranean Sea

The distribution, type and quantity of marine litter accumulated on the bathyal and abyssal Mediterranean seafloor has been studied in the framework of the Spanish national projects PROMETEO and DOS MARES and the ESF-EuroDEEP project BIOFUN. Litter was collected with an otter trawl and Agassiz trawl while sampling for megafauna on the Blanes canyon and adjacent slope (Catalan margin, north-western Mediterranean) between 900 and 2700 m depth, and on the western, central and eastern Mediterranean basins at 1200, 2000 and 3000 m depth. All litter was sorted into 8 categories (hard plastic, soft plastic, glass, metal, clinker, fabric, longlines and fishing nets) and weighed. The distribution of litter was analysed in relation to depth, geographic area and natural (bathymetry, currents and rivers) and anthropogenic (population density and shipping routes) processes. The most abundant litter types were plastic, glass, metal and clinker. Lost or discarded fishing gear was also commonly found. On the Catalan margin, although the data indicated an accumulation of litter with increasing depth, mean weight was not significantly different between depths or between the open slope and the canyon. We propose that litter accumulated in the canyon, with high proportions of plastics, has predominantly a coastal origin, while litter collected on the open slope, dominated by heavy litter, is mostly ship-originated, especially at sites under major shipping routes. Along the trans-Mediterranean transect, although a higher amount of litter seemed to be found on the Western Mediterranean, differences of mean weight were not significant between the 3 geographic areas and the 3 depths. Here, the shallower sites, also closer to the coast, had a higher proportion of plastics than the deeper sites, which had a higher proportion of heavy litter and were often affected by shipping routes. The weight of litter was also compared to biomass of megafauna from the same samples. On the Blanes slope, the biomass of megafauna was significantly higher than the weight of litter between 900 and 2000 m depth and no significant differences were found at 2250 and 2700 m depth. Along the trans-Mediterranean transect, no significant differences were found between biomass and litter weight at all sites except in two sites: the Central Mediterranean at 1200 m depth, where biomass was higher than litter weight, and the Eastern Mediterranean at 1200 m depth, where litter weight was higher than biomass. The results are discussed in the framework of knowledge on marine litter accumulation, its potential impact on the habitat and fauna and the legislation addressing these issues.

Major, trace element and XRF data for sediments and fish tooth samples, from a shallow marine setting with varying redox conditions (Site U1356, Wilkes Land Margin, E.Antarctica)

Fossil fish teeth from pelagic open ocean settings are considered a robust archive for preserving the neodymium (Nd) isotopic composition of ancient seawater. However, using fossil fish teeth as an archive to reconstruct seawater Nd isotopic compositions in different sedimentary redox environments and in terrigenous-dominated, shallow marine settings is less proven. To address these uncertainties, fish tooth and sediment samples from a middle Eocene section deposited proximal to the East Antarctic margin at Integrated Ocean Drilling Program Site U1356 were analyzed for major and trace element geochemistry, and Nd isotopes. Major and trace element analyses of the sediments reveal changing redox conditions throughout deposition in a shallow marine environment. However, variations in the Nd isotopic composition and rare earth element (REE) patterns of the associated fish teeth do not correspond to redox changes in the sediments. REE patterns in fish teeth at Site U1356 carry a typical mid-REE-enriched signature. However, a consistently positive Ce anomaly marks a deviation from a pure authigenic origin of REEs to the fish tooth. Neodymium isotopic compositions of cleaned and uncleaned fish teeth fall between modern seawater and local sediments and hence could be authigenic in nature, but could also be influenced by sedimentary fluxes. We conclude that the fossil fish tooth Nd isotope proxy is not sensitive to moderate changes in pore water oxygenation. However, combined studies on sediments, pore waters, fish teeth and seawater are needed to fully understand processes driving the reconstructed signature from shallow marine sections in proximity to continental sources. This article is protected by copyright. All rights reserved.

NMR spectrum of lindgomycin from a Marine Fungus of the Lindgomycetaceae

An unusual polyketide with a new carbon skeleton, lindgomycin (1), and the recently described ascosetin (2) were extracted from mycelia and culture broth of different Lindgomycetaceae strains, which were isolated from a sponge of the Kiel Fjord in the Baltic Sea (Germany) and from the Antarctic. Their structures were established by spectroscopic means. In the new polyketide, two distinct domains, a bicyclic hydrocarbon and a tetramic acid, are connected by a bridging carbonyl. The tetramic acid substructure of compound 1 was proved to possess a unique 5-benzylpyrrolidine-2,4-dione unit. The combination of 5-benzylpyrrolidine-2,4-dione of compound 1 in its tetramic acid half and 3-methylbut-3-enoic acid pendant in its decalin half allow the assignment of a new carbon skeleton. The new compound 1 and ascosetin showed antibiotic activities with IC50 value of 5.1 (±0.2) µM and 3.2 (±0.4) µM, respectively, against methicillin-resistant Staphylococcus aureus.

Distribution of biological, anthropogenic, and volcanic constituents in the San Francisco Bay Coastal System

Although conventional sediment parameters (mean grain size, sorting, and skewness) and provenance have typically been used to infer sediment transport pathways, most freshwater, brackish, and marine environments are also characterized by abundant sediment constituents of biological, and possibly anthropogenic and volcanic, origin that can provide additional insight into local sedimentary processes. The biota will be spatially distributed according to its response to environmental parameters such as water temperature, salinity, dissolved oxygen, organic carbon content, grain size, and intensity of currents and tidal flow, whereas the presence of anthropogenic and volcanic constituents will reflect proximity to source areas and whether they are fluvially- or aerially-transported. Because each of these constituents have a unique environmental signature, they are a more precise proxy for that source area than the conventional sedimentary process indicators. This San Francisco Bay Coastal System study demonstrates that by applying a multi-proxy approach, the primary sites of sediment transport can be identified. Many of these sites are far from where the constituents originated, showing that sediment transport is widespread in the region. Although not often used, identifying and interpreting the distribution of naturally-occurring and allochthonous biologic, anthropogenic, and volcanic sediment constituents is a powerful tool to aid in the investigation of sediment transport pathways in other coastal systems.

Effects of ocean acidification on sponge communities

The effects of ocean acidification on lower invertebrates such as sponges may be pronounced because of their low capacity for acid-base regulation. However, so far, most studies have focused on calcifiers. We present the first study of the effects of ocean acidification on the Porifera. Sponge species composition and cover along pH gradients at CO2 vents off Ischia (Tyrrhenian Sea, Italy) was measured at sites with normal pH (8.1-8.2), lowered pH (mean 7.8-7.9, min 7.4-7.5) and extremely low pH (6.6). There was a strong correlation between pH and both sponge cover and species composition. Crambe crambe was the only species present in any abundance in the areas with mean pH 6.6, seven species were present at mean pH 7.8-7.9 and four species (Phorbas tenacior, Petrosia ficiformis, Chondrilla nucula and Hemimycale columella) were restricted to sites with normal pH. Sponge percentage cover decreased significantly from normal to acidified sites. No significant effect of increasing CO2 levels and decreasing pH was found on spicule form in Crambe crambe. This study indicates that increasing CO2 concentrations will likely affect sponge community composition as some demosponge species appear to be more vulnerable than others. Further research into the mechanisms by which acidification affects sponges would be useful in predicting likely effects on sessile marine communities.