IDENTIFICATION AND REGULATION OF FERRITINS IN THE HYDROTHERMAL VENT MUSSEL BATHYMODIOLUS AZORICUS IN NATURAL AND EXPERIMENTAL POPULATIONS

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Ecotoxicology in aquatic ecosystems

The number of ecotoxicological studies conducted in aquatic ecosystems showed a important growth during the last twenty years principally due to the increasing pollution as a result of an indiscriminate dumping of industrial, urban and agricultural sources into natural aquatic ecosystems. The main contaminants that can be detected in aquatic ecosystems include poly aromatic hydrocarbons (HAPs), pharmaceutical substances, radionuclides and toxic metals. Once contaminants are incorporated into the ecosystem, they can be stored in the sediment, increasing their persistence, be directly incorporated into the organisms and immediately exert their toxic effect like disease, suppression of immune systems, oxidative stress, mutation on DNA or can be bio-accumulated in different tissues and consequently in the food chain.

Ecotoxicology and metals in aquatic systems

Ecotoxicology of trace metal in aquatic systems correspond to raise the question « how does toxic and non-toxic metal vary over space and time in and between aquatic habitats and show how they affect living organisms? The term ‘trace metal’ is used to characterize metals that are present at low (trace) concentrations (sometimes defined as 0.01% dry weight) in the environment, in both physical and biotic components. However, some of those metals are detected at high concentration in organisms. Heavy metals term is mainly used for metals that are above a threshold atomic weight, typically incorporating all transition metals of the periodic table. Other chemical characteristics are also considered such as the similar chemical characteristics that make them biologically relevant. These metals become toxic to biota when present in high bioavailability but many are essential to the metabolism of life, consistently across the eukaryotes with sometimes an excellent but not perfect agreement between eukaryotes and prokaryotes. Numerous biochemical pathways underlying life processes are conserved in all organisms and require the same elements to function. Based on another criteria, the term ‘trace metal’ is restricted to essential metals with essential metabolic function then excluding the non-essential metals that have no metabolic function.
11 For the chemists, a strict definition has been proposed to characterize the term “trace metal”. Nieboer and Richardson (1980) proposed a chemical classification system based on the Lewis acid properties of metal ions. Metals are separated into Class A, Class B or Borderline according to their degree of ‘hardness’ or ‘softness’ as acids and bases. Class A metal ions are Lewis hard acids, readily form cations, and have a ligand affinity order O > N > S. Class B metal ions are Lewis soft acids, ‘more covalent’ and have an affinity order S > N > O. Borderline metal ions present intermediate properties. Metals with Class B or Borderline ions also fit into the category of trace metals. For the non-chemists, the affinity of trace metals for sulphur and nitrogen promotes their binding to molecules in cells especially proteins, and makes some of them essential and all of them toxic link to their ability to bind in the wrong place at the wrong time when available in excess. There are three major categories of trace metal concentration data that are currently measured in order to compare differences in trace metal pollution in aquatic habitats over space and time: 1) trace metal concentrations in water, 2) trace metal concentrations in sediment and 3) trace metal concentrations in resident biota. Evaluation of dissolved trace metal concentration in water is very important to determine if these concentrations are close from those shown to present a toxic effect on organisms using toxicity tests. Dissolved concentrations usually vary over time, particularly for example in estuaries with differential inputs of river and sea water at different states of the tide, and differential river flow according to recent rainfall in the catchment often varying seasonally. Each measurement represents a single time point that may be very different from the dissolved concentration present at that exact location the day before or the day after.

Biological significance of metals

Living organisms store and transport various transition metals to provide appropriate concentrations for basic metabolism (mainly enzyme cofactors). The normal concentration range varies according to metals and is difficult to determine in biological systems. More, metal deficiencies and excesses both contribute to generate pathological changes. The complexity of cell types in multicellular organisms also participates to the dynamics of metals distribution; the storage and the transport of transition metals being not carried by all cells but specialized ones. The form of the metals remains always ionic but the oxidation state may vary based on cell biological needs. The main heavy metals in environmental monitoring studies are mercury, cadmium, lead and copper.
12 Mercury (Hg) is a heavy metal present under three chemical forms: elemental (Hg without any additional atoms attached to it), organic, and inorganic that are interconvertible, and can all produce systemic toxicity (Graeme and Pollack, 1998). Origin of Hg in the environment are both natural and from anthropogenic sources like mining, fossil fuels combustion, incineration, emission from smelters, fungicides and catalyst activities. Although mainly present in the atmosphere, a large part of Hg returns into the coastal sea as precipitates. Hg is also present at high concentrations in sediments of aquatic environment since both inorganic and organic Hg are linked to particles, colloids and high molecular weight organic matter (Schiff, 2000). Inorganic Hg can be converted by specific bacteria into methylmercury which represent the most toxic chemical species able to provoke deleterious effects to the central nervous system, deficiencies in the immune system and development [Harada et al., 1998). Dissolved methylmercury is easily bioavailable and can bioaccumulated and biomagnified into the marine food chains to reach very high concentrations in upper levels of the chain. Toxicity of Hg has been studied in several marine invertebrates species such as the clam Ruditapes philippinarum, (Liu et al., 2011), the crustacean Ligia italic, in which ultrastructural alterations in the hepatopancreas epithelium have been observed (Longo et al., 2013) or Scylla serrata in which modifications of several immune related parameters (total haemocyte count, lysosomal membrane stability, phenoloxidase, superoxide generation and phagocytosis) have been reported (Singaran et al., 2013). In embryos of the sea urchin Strongylocentrotus purpuratus exposed to mercury, the inhibition of specific molecular transporters increases intracellular accumulation of inorganic Hg but had no effect on accumulation of organic Hg. These results illustrated the existence of a specific elimination of inorganic Hg and a differential accumulation and potency of the two major forms of Hg found in marine environments (Bosnjak et al., 2009). Accumulation of Hg in the soft tissues of the oysters Saccostrea cucullata (Shimeshan et al., 2012) and Crassostrea angulata illustrated the interest of such organism for monitoring of Hg mercury in the aquatic system. Proteomic analysis conducted on gonads of oysters following food-chain contamination with HgCl evidenced different proteins such as 14 – 3 – 3 protein, GTP binding protein, arginine kinase and 71 kDa heat shock cognate protein) as good candidate biomarkers for environmental Hg contamination (Zhang et al., 2013). Cadmium (Cd), is a heavy metal released both from natural sources and anthropogenic activities resulting from its large utilization in some industrial and agricultural activities (e.g. pigments, nickel-cadmium batteries, smelting and refining of metals and many 13 other sources). Cd is a highly toxic environmental pollutant and potent cell poison which induce different types of damage including cell death. Cd toxicity is amplified in organisms as a consequence of the metal’s long biological half-life which range from 15 to 30 years according to species and tissues. Cd easily penetrates the cells, via transport mechanisms normally used for other purposes, but is eliminated very slowly (Jarup et al., 1998). Since Cd is a non-essential metal which present no physiological function that is irreversibly accumulated into cells and strongly interact with various cellular components and molecular targets. Cd may enter cells via divalent ion transporters, such as zinc transporters (Kingsley and Frazier, 1979), can cross the plasma membrane as divalent ions, exerting an agonistic role against calcium ionic channels (Foulkes, 2000). Toxicity of Cd is also modulated by various abiotic factors (De Lisle and Roberts, 1988). Among Cd effects, cases of teratogenesis and carcinogenesis, due to cytotoxic concentrations of the ion, have been reported both in invertebrates and in higher organisms. Accumulated evidence has also shown that Cd increased not only cellular ROS levels, but also lipid peroxidation and alteration in glutathione (GSH) levels in various cell types, suggesting that Cd-induced apoptosis may be connected with oxidative stress (Rana, 2008). In invertebrates it up-regulates the expression of antioxidant enzymes, metallothioneins and heat shock proteins (HSPs) and down-regulates the expression of digestive enzymes, esterases and phospholipase A2. Cd also interferes with tissue organization, immune responses and cell cycles by inducing apoptosis (Sokolova et al., 2004). Due to its high level of resilience, Cd is a contaminant which accumulate in the food-chain involving that for many aquatic predators, Cd comes largely from food and the ease with which Cd penetrate tissues mainly depends on the form in which this metal is bound in prey cells (Cd present in the cytosol being more available than Cd associated with insoluble prey components (Dubois and Hare, 2009). High concentrations of Cd has been reported in aqueous organisms including invertebrates, such as sponges, mollusks, crustaceans, echinoderms with very often the existence of significant differences in Cd concentration in tissues in differently contaminated sites. Various effects of Cd have been reported in different species. For example, modifications in cell morphology and cell aggregation in Scopalina lophyropoda, by enhancing pseudopodia/filopodia formation which promotes cell movement have been described (Cebrian and Uriz, 2007). High concentrations of Cd are detected in the gills and digestive gland of the mussel Mytilus galloprovincialis associated with alteration in the physiology of respiration and feeding processes (Viarengo et al., 1994), in the digestive gland and kidney of mussel Crenomytilus grayanus (Podgurskaya and Kavun, 2006), in the renal tissue of Antarctic bivalve Laternula elliptica (Rodrigues et al., 2009) or in the 14 hepatopancreas of Mytilus edulis and body wall of echinoderms such as Asterias rubens. . Similar observations have been made in few species of Antarctic molluscs highlighting the importance of food in the primary pathway for Cd bio-accumulation (Nigro et al., 1997). Data on the effect of Cd on cellular and molecular defense strategies such as apoptosis, autophagy, metal detoxication and stress proteins have been obtained in Paracentrotus lividus embryos (Rochherri and Matranga, 2010). Lead (Pb) is a heavy metal naturally present in the environment that becomes highly toxic when ingested and cause severe damages to the nervous system and causing different disorders. Pb compounds exist in two main oxidation states, +2 and +4 (Nava-Ruiz et al., 2012). Pb is a bio-persistent pollutant mainly originating from human activities that accumulates at the top of the food chain. In marine invertebrates, the Pb toxicity varies according to species but also to their life stage in a dose and time dependent manner. A characterization of the cytosolic distribution of Pb was carried out in the digestive gland of Mytilus galloprovincialis, Pb is present in molecule with high molecular weight but when Pb concentrations are elevated, Pb is also present in low molecular weight biomolecules, illustrating suitability of the distribution of selected metals among different cytosolic ligands as potential indicator for metal exposure (Strizak et al., 2014). Sea urchin embryos of the sea urchin Paracentrotus lividus exposed to Pb exhibit alterations of morphology at gastrula and pluteus stages (Geraci et al., 2004) and a reduction of calcium accumulation is observed in embryos of Strongylocentrotus purpuratus (Tellis et al., 2014). In the mussel Perna viridis exposed to environmental Pb concentrations, several enzymes classically used in toxicology (catalase, reduced glutathione, glutathione S-transferase, and lipid peroxides) showed differences in activities (Hariharan et al., 2014).

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Response to heavy metals in marine invertebrates. 

Exposure to different heavy metals and bioaccumulation in natural population has been reported in many bivalves (Marigómez et al., 2002), showing the high tolerance of these species and given great potential for assess the status of chemical pollutants in aquatic ecosystems (biomarkers of toxicity). Heavy metals have been described as one of the main selective pressure acting in marine invertebrates, affecting embryonic development, stress protein induction, immune system, DNA, RNA and lipid damages with subsequent cellular apoptosis (Chiarelli and Roccheri, 2014). The degree of sensitivity of species depends on the metal type and its concentration exposure, which strongly vary between species. For example, exposure to copper in limpet (Patella vulgata), crabs (Carcinus maenas) and mussels (Mytilus edulis) reveals that at high concentration, metals interfere with different physiological parameters such as survival ability, heart rate, proteins levels in hemolymph, lysosomal stability, neurotoxic effect (acetylcholinesterase activity) and specific biomarkers of metal exposure (metallothionein) and a gradient in sensitivity is observed where limpet is more sensitive than crab and mussel (Brown et al., 2004). Bivalves species have been chosen as model organisms especially in marine environments to understand the mechanisms involved in the response of metal exposure, because they can accumulate different types of essential and non essential metals in high levels and the major models used are oysters, scallops, clams and mussels (Chandurvelan et al., 2015; Lavradas et al., 2016; Paul-Pont et al., 2012; Sakellari et al., 2013; Varotto et al., 2013; Wang et al., 2009). In bivalves used as metal bioindicators of aquatic contamination different methodologies have been developed to localize and quantify metal concentrations, from histochemistry, spectrophotometric techniques and electronic microscopy, allowing understanding the uptake pathways from the ambient water (Sakellari et al 2013; Chandurvelan et al, 2015).
Once the metal is acumulated in the organism have the potential to be incorporated in different physiological pathways (normally for the essencial metals, Cu, Zn) but when his concentration exeed the levels of tolerance or are toxic (non essencial metals, Cd, Pb, As, Hg) metals can be stored in compartimentalizated vacuoles or can be begin detoxification mechanism of manner temporaly or permanently, however both process require specific metal binding enzymes or proteins that permit the transport internally to a particular organ for be excreted from the organism (Rainbow, 1997).
At molecular level different specific and non specific biomarkers of metal pollution has benn described, showing a conserved mechanis across different taxas but that vary in the level of efficiency and network complexity.

Hydrothermal ecosystems as natural laboratory for heavy metal adaptation

Deep-sea hydrothermal vents ecosystems are distributed among all the oceans, principally along mid-ocean ridges where new oceanic crust is generated. Vent fluid is formed by infiltration of deep-sea water into fractures of the oceanic plate and chemical transformation at high temperature deep in the crust, under the influence of the underlying magmatic chamber. The vent fluid returns to the ocean through diffuse or focused venting, with typically high temperature (up to 400°C depending on sites), loaded with heavy metals and reduced gases such as carbon dioxide (CO2), sulfide (H2S), hydrogen (H2) and methane (CH4) (Figure 10). When mixing with cold deep-sea water the hydrothermal flux is diluted, its 29 high temperature is reduced, the pH becomes more basic, some metals precipitate forming the classical chimney structures and some remain in the water column. However these characteristics are very variable for each vent site, providing a biotope with different environmental conditions (Demina and Galkin, 2016; Von Damm, 1995).

Table of contents :

CHAPTER I 3

INTRODUCTION
1. BIOMARKERS
2. ECOTOXICOLOGY IN AQUATIC ECOSYSTEMS
3. ECOTOXICOLOGY AND METALS IN AQUATIC SYSTEMS
4. RESPONSE TO HEAVY METALS IN MARINE INVERTEBRATES.
5. OMICS ERA IN POLLUTION RESPONSE
6. HYDROTHERMAL ECOSYSTEMS AS NATURAL LABORATORY FOR HEAVY METAL ADAPTATION
7. OBJECTIVES
CHAPTER II METAL ACCUMULATION AND REGULATION OF METAL RELATED GENE EXPRESSION IN THE HYDROTHERMAL VENT MUSSEL BATHYMODIOLUS AZORICUS AS A SIGNATURE OF ENVIRONMENTAL CONTAMINATION
CHAPTER III DIFFERENTIAL EXPRESSION OF SUPEROXIDE DISMUTASE ISOFORMS AS INDICATOR OF OXIDATIVE STRESS IN RESPONSE TO METALS IN THE HYDROTHERMAL MUSSEL BATHYMODIOLUS AZORICUS: APPLICATION TO FIELD AND EXPERIMENTAL POPULATIONS.
CHAPTER IV IDENTIFICATION AND REGULATION OF FERRITINS IN THE HYDROTHERMAL VENT MUSSEL BATHYMODIOLUS AZORICUS IN NATURAL AND EXPERIMENTAL POPULATIONS
CHAPTER V LARGE SCALE TRANSCRIPTOMIC ANALYSIS OF BATHYMODIOLUS AZORICUS DISCUSSION, CONCLUSION, PERSPECTIVES 
BIBLIOGRAPHY 

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