Inês Martins

Enrichment in trace metals (Al, Mn, Co, Cu, Mo, Cd, Fe, Zn, Pb and Hg) of macro-invertebrate habitats at hydrothermal vents along the Mid Atlantic Ridge

The present study describes several features of the aquatic environment with the emphasis on the total vs. filter-passing fraction (FP) of heavy metals in microhabitats of two typical deep-sea vent organisms: the filter-feeder, symbiont-bearing Bathymodiolus and the grazer shrimps Rimicaris/Mirocaris from the Mid-Atlantic Ridge (MAR). The concentration of 10 trace elements: Al, Mn, Co, Cu, Mo, Cd, Fe, Zn, Pb and Hg was explored highlighting common and distinctive features among the five hydrothermal vent sites of the MAR: Menez Gwen, Lucky Strike, Rainbow, Saldanha, and Menez Hom that are all geo-chemically different when looking at the undiluted hydrothermal fluid composition. The drop off in the percentage of FP from total metal concentration in mussel and/or shrimp inhabited water samples (in mussel beds at Rainbow, for instance, FP fraction of Fe was below 23%, Zn 24 %, Al 65%, Cu 70%, and Mn 89%) as compared to non-inhabited areas (where 94% of the Fe, 90% of the Zn, 100% of the other metals was in the FP fraction) may indicate an influence of vent organisms on their habitat’s chemistry, which in turn may determine adaptational strategies to elevated levels of toxic heavy metals. Predominance of particulate fraction over the soluble metals, jointly with the morphological structure and elemental composition of typical particles in these vent habitats suggest a more limited metal bioavailability to vent organisms as previously thought. In addition, it is evoked that vent invertebrates may have developed highly efficient metal-handling strategies targeting particulate phase of various metals present in the mixing zones that enables their survival under these extreme conditions.

Spatial variation of metal bioaccumulation in the hydrothermal vent mussel Bathymodiolus azoricus

The variability of the bioaccumulation of metals (Ag, Cd, Cu, Fe, Mn and Zn) was extensively studied in the mussel Bathymodiolus azoricus from five hydrothermal vent sites inside three main vent fields of increasing depth along the Mid-Atlantic Ridge: Menez Gwen, Lucky Strike and Rainbow. Metal bioaccumulation varied greatly between vent fields and even between sites inside a vent field with B. azoricus showing a great capacity to accumulate metals. The bioaccumulation of these metals also varied significantly among tissues. The main target was the gills where metals were mainly associated with soluble compounds whereas in the digestive gland they were mainly associated with insoluble compounds. Storage of metals under insoluble forms in B. azoricus seems to be a major pathway for the detoxification of both essential and non-essential metals. Mussels from the studied fields can be discriminated following their metallic load but the segregation relies partially on the composition of the metal-enriched fluids.

Mercury concentrations in invertebrates from Mid-Atlantic Ridge hydrothermal vent fields

Mercury determinations were carried out in mussels ( Bathymodiolus azoricus ) from three Mid-Atlantic Ridge hydrothermal vents (Lucky Strike, Menez Gwen and Rainbow) and shrimps ( Rimicaris exoculata and Mirocaris fortunata ) from Rainbow. Among the three hydrothermal vents, mussels of Menez Gwen show the highest levels of total Hg and comparing mussels and shrimps from Rainbow the former show more Hg than shrimps. Mussels from different hydrothermal vents are exposed to different kinds of environment which may result in distinct bioaccumulation processes. Detoxification processes in shrimps are related to the low concentrations found. When compared with coastal species from unpolluted sites, mussels show higher concentrations of total Hg and shrimps lower levels. The methyl-mercury concentrations found were very low, not exceeding the detection limit of the technique.

Variation in physiological indicators in Bathymodiolus azoricus (Bivalvia: Mytilidae) at the Menez Gwen Mid-Atlantic Ridge deep-sea hydrothermal vent site within a year

Bathymodiolus azoricus, thriving at Mid-Atlantic Ridge deep vents, benefits from a symbiosis with methane- and sulphide-oxidising (MOX and SOX) bacteria, and feeds on particulate and dissolved organic matter. To investigate the temporal evolution in their nutrition adult mussels were collected from one location at the Menez Gwen vent site (817 m depth) on four occasions between 2006 and 2007 and studied using different techniques, including stable isotope analyses and FISH. Gill and mantle tissues delta13C and delta15N signatures varied by 2-3 per thousand during the year and these variations were linked to fluctuations in tissue condition index, C and N contents and SOX/MOX volume ratios as quantified by 3D-FISH. October and January mussels presented a particularly poor condition, possibly related with the prolonged summer period of low sea-surface primary production and/or with the stress of the transplant to acoustically retrievable cages for the October mussels, and with their reproductive state in January mussels, since they were spawning. Our results point to the possibility that May mussels benefited from a pulse of sinking sea-surface plankton material. Results underline the dependency of stable isotopic signatures on the physiological state of the mussel at the time of collection, and on the type of tissue analyzed.

The influence of nutritional conditions on metal uptake by the mixotrophic dual symbiosis harboring vent mussel Bathymodiolus azoricus.

The vent mussel Bathymodiolus azoricus, host thioautotrophic and methanotrophic bacteria, in their gills and complementary, is able to digest suspended organic matter. But the involvement of nutritional status in metal uptake and storage remains unclear. The influence of B. azoricus physiological condition on its response to the exposure of a mixture of metals in solution is addressed. Mussels from the Menez Gwen field were exposed to 50 μgL(-1) Cd, plus 25 μgL(-1) Cu and 100 μgL(-1) Zn for 24 days. Four conditions were tested: (i) mussels harboring both bacteria but not feed, (ii) harboring only methanotrophic bacteria, (iii) without bacteria but fed during exposure and (iv) without bacteria during starvation. Unexposed mussels under the same conditions were used as controls. Eventual seasonal variations were assessed. Metal levels were quantified in subcellular fractions in gills and digestive gland. Metallothionein levels and condition indices were also quantified. Gill sections were used for fluorescence in situ hybridization (FISH) to assess the temporal distribution of symbiotic associations. Starvation damages metal homeostasis mechanisms and increase the intracellular Zn and MT levels function. There is a clear metallic competition for soluble and insoluble intracellular ligands at each condition. Seasonal variations were observed at metal uptake and storage.

Identifying Toxic Impacts of Metals Potentially Released during Deep-Sea Mining—A Synthesis of the Challenges to Quantifying Risk

In January 2017, the International Seabed Authority released a discussion paper on the development of Environmental Regulations for deep-sea mining within the Area Beyond National Jurisdiction (the ‘Area’). With the release of this paper, the prospect for commercial mining in the Area within the next decade has become very real. Moreover, within nations’ Exclusive Economic Zones, the exploitation of deep-sea mineral ore resources could take place on very much shorter time scales and, indeed, may have already started. However, potentially toxic metal mixtures may be released at sea during different stages of the mining process and in different physical phases (dissolved or particulate). As toxicants, metals can disrupt organism physiology and performance, and therefore may impact whole populations, leading to ecosystem scale effects. A challenge to the prediction of toxicity is that deep-sea ore deposits include complex mixtures of minerals, including potentially toxic metals such as copper, cadmium, zinc, and lead, as well as rare earth elements. Whereas the individual toxicity of some of these dissolved metals has been established in laboratory studies, the complex and variable mineral composition of seabed resources makes the a priori prediction of the toxic risk of deep-sea mining extremely challenging. Furthermore, although extensive data quantify the toxicity of metals in solution in shallow-water organisms, these may not be representative of the toxicity in deep-sea organisms, which may differ biochemically and physiologically and which will experience those toxicants under conditions of low temperature, high hydrostatic pressure, and potentially altered pH. In this synthesis, we present a summation of recent advances in our understanding of the potential toxic impacts of metal exposure to deep-sea meio- to megafauna at low temperature and high pressure, and consider the limitation of deriving lethal limits based on the paradigm of exposure to single metals in solution. We consider the potential for long-term and far-field impacts to key benthic invertebrates, including the very real prospect of sub-lethal impacts and behavioural perturbation of exposed species. In conclusion, we advocate the adoption of an existing practical framework for characterising bulk resource toxicity in advance of exploitation.

Physiological impacts of acute Cu exposure on deep-sea vent mussel Bathymodiolus azoricus under a deep-sea mining activity scenario

Over the past years, several studies have been dedicated to understanding the physiological ability of the vent mussel Bathymodiolus azoricus to overcome the high metal concentrations present in their surrounding hydrothermal environment. Potential deep-sea mining activities at Azores Triple junction hydrothermal vent deposits would inevitably lead to the emergence of new fluid sources close to mussel beds, with consequent emission of high metal concentrations and potential resolubilization of Cu from minerals formed during the active phase of the vent field. Copper is an essential metal playing a key role in the activation of metalloenzymes and metalloproteins responsible for important cellular metabolic processes and tissue homeostasis. However, excessive intracellular amounts of reactive Cu ions may cause irreversible damages triggering possible cell apoptosis. In the present study, B. azoricus was exposed to increasing concentrations of Cu for 96h in conditions of temperature and hydrostatic pressure similar to those experienced at the Lucky Strike hydrothermal vent field. Specimens were kept in 1L flasks, exposed to four Cu concentrations: 0μg/L (control), 300, 800 and 1600μg/L and pressurized to 1750bar. We addressed the question of how increased Cu concentration would affect the function of antioxidant defense proteins and expression of antioxidant and immune-related genes in B. azoricus. Both antioxidant enzymatic activities and gene expression were examined in gills, mantle and digestive gland tissues of exposed vent mussels. Our study reveals that stressful short-term Cu exposure has a strong effect on molecular metabolism of the hydrothermal vent mussel, especially in gill tissue. Initially, both the stress caused by unpressurization or by Cu exposure was associated with high antioxidant enzyme activities and tissue-specific transcriptional up-regulation. However, mussels exposed to increased Cu concentrations showed both antioxidant and immune-related gene suppression. Under a mining activity scenario, the release of an excess of dissolved Cu to the vent environment may cause serious changes in cellular defense mechanisms of B. azoricus. This outcome, while adding to our knowledge of Cu toxicity, highlights the potentially deleterious impacts of mining activities on the physiology of deep-sea organisms.

An Insightful Model to Study Innate Immunity and Stress Response in Deep‐Sea Vent Animals: Profiling the Mussel Bathymodiolus azoricus

Deep‐sea environments are, in some cases, largely unexplored ecosystems, where life thrives driven by the geochemical features of each location. Among these environments, chemosynthesis‐based ecosystems, in the Mid Atlantic Ridge, have an exclusive combination of high depth, high sulfur, and high methane concentrations. This is believed to modulate the biological composition of vent communities and influence the overall vent animal transcriptional activity of genes involved in adaptation processes to extreme environments. This opens, thus, the possibility of finding gene expression signatures specific to a given hydrothermal vent field. Regardless of the extreme physicochemical conditions that characterize deep‐sea hydrothermal vents, the animals dwelling around the vent sites exhibit high productivity and thus must cope with toxic nature of vent surrounding, seemingly deleterious to the animals, while developing surprisingly successful strategies to withstand adverse environmental conditions, including environmental microbes and mechanical stress whether ensuing from animal predation or venting activity. The deep-sea vent mussel Bathymodiolus azoricus has adapted well to deep‐sea extreme environments and represents the dominating faunal community from hydrothermal vent sites in the Mid‐Atlantic Ridge, owing its successful adaptation and high biomasses to specialized exploitation of methane and sulfide sources from venting activity. Its extraordinary capabilities of adapting and thriving in chemosynthesis‐ based environments, largely devoid of photosynthetic primary production and characterized by rapid geochemical regime changes are due to symbiotic associations with chemosynthetic bacteria within its large gills. In an attempt to understand physiological reactions in animals normally set to endure extreme deep‐sea environments, our laboratory has undertaken, for thelast few years, a series of investigations, aimed at characterizing molecular indicators of adaptation processes of which components of the host defense system has received most attention. This study reviews recent advances on the characterization of molecules and genes participating in immune reactions, using in vivo and ex vivo models, to elucidate cellular and humoral defense mechanisms in vent mussels and the strategies they have adopted to survive under extreme environments.

Elemental composition of two ecologically contrasting seamount fishes, the bluemouth (Helicolenus dactylopterus) and blackspot seabream (Pagellus bogaraveo).

Concentrations of V, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Cd and Pb were determined in muscle, liver and gonads of two ecologically contrasting fishes, Helicolenus dactylopterus (benthic) and Pagellus bogaraveo (benthopelagic). Elevated concentrations of As, Se and Cd found in tissues of both species appear to mirror the contribution of volcanic activity to the natural inputs of elements to Azorean waters. Results showed different element accumulation between the two species. Whereas higher concentrations were found in the liver of P. bogaraveo, elevated values were observed in the muscle of H. dactylopterus. Differences in accumulation are most likely related to metabolic rates, diet specificities and habitat. Concentrations in gonads varied up to four orders of magnitude, being higher and more variable in P. bogaraveo than H. dactylopterus. Elevated values of Cd were detected in gonads of both species despite its non-essential role on metabolic functions, presumably related to elimination.

Assessment of Cu sub-lethal toxicity (LC50) in the cold-water gorgonian Dentomuricea meteor under a deep-sea mining activity scenario

Previous aquaria-based experiments have shown dissolution and leaching of metals, especially copper (Cu), from the simulated sediment plumes generated during mining activities resulting in a pronounced increase of Cu contamination in the surrounding seawater. Metals are bioavailable to corals with food, through ingestion (particulate phase) and through tissue-facilitated transport (passive diffusion). With corals being particularly vulnerable to metal contamination, resuspension of metal-bearing sediments during mining activities represents an important ecological threat. This study was undertaken to evaluate the impact of acute copper exposure (LC50;96 h) on the survival of the cold-water octocoral Dentomuricea aff. meteor. The experimental design was divided in two stages. In stage one, a Cu range-finding toxicity test was performed using Cu dilutions in filtered seawater with concentrations of 0 (control); 60; 150; 250; 450; 600 μg/L. Coral mortality was investigated visually based on the percent surface area of tissue changing from natural yellow colour to black colour indicative of tissue necrosis and death. In stage two, we used the results obtained in the range-finding experiment, to define sub-lethal Cu exposure treatments and exposed D. meteor to Cu concentration of 0 (control); 50; 100; 150; 200; 250 μg/L for 96 h. The corals physical conditions were inspected daily and seawater conditions recorded. Corals were considered dead when all of their tissue turned black. The LC50 value was calculated with regression analysis following Probits methodology. Our results indicate that Cu LC50;96 h for the octocoral D. meteor is 137 μg/L.