Deep-sea mining: using hyperbaric conditions to study the impact of sediment plumes in the subtidal clam Spisula solida.

With the growing interest to exploit mineral resources in the deep-sea, there is the need to establish guidelines and frameworks to support hazard and risk assessment schemes. The present study used a subtidal species of filter-feeding bivalve, the clam Spisula solida, as a proxy to better understand the impacts of sediment plumes in marine organisms under hyperbaric conditions. Four concentrations of suspended sediments (0 g/L, 1 g/L, 2 g/L, and 4 g/L) were used in a mixture with different grain sizes at 4 Bar for 96 h. Functional (filtration rate-FR) and biochemical endpoints (catalase-CAT, glutathione s-transferase-GST, and lipid peroxidation-LPO) were analyzed in the gonads, digestive gland, and gills of S. solida after a 96-h exposure at 4 Bar (the natural limit of the species vertical distribution). The FR showed a decreasing trend with the increasing sediment concentrations (significant effects at 2 and 4 g/L). Additionally, significant changes were observed for some of the tested oxidative stress biomarkers, which were concentration and tissue-dependent, i.e., CAT activity was significantly elevated in gills (1 g/L treatment), and GST was decreased in digestive gland (1 g/L treatment). Overall, the results show that suspended sediments, at 2 and 4 g/L, have negative functional impacts in the bivalve S. solida providing additional insights to improve hazard assessment of deep-sea mining. These findings represent a step forward to ensure the mitigation of the potential negative effects of deep-sea resource exploitation.

Structural and dynamic characterization of the hexa-coordinated globin from Spisula solidissima.

High energy consumption in the nervous system requires a continuous supply of O2. This role is assisted by proteins from the globin super-family in the nerve cells of invertebrates, where 'nerve hemoglobins' (nHbs) are mainly present at mM concentrations and exhibit oxygen affinities comparable to those of vertebrate myoglobins. To gain insight into the structural bases of this function, we report the crystal structure of nHb from the Atlantic surf clam Spisula solidissima (SsHb), previously suggested to display a bis-histidyl hexa-coordinated heme in the deoxy state, high O2 affinity, and ligand binding cooperativity when assayed in situ. The crystallized protein forms a dimer through packing of a 4-helix bundle involving helices E and F of each subunit. The SsHb 'classic' globin fold displays bis-histidyl (His71(E7) and His103(F8)) hexa-coordination of the heme-Fe atom, with structural and dynamics variations found in the inter-helix hinge regions. Molecular Dynamics simulations of both monomeric and dimeric species in the bis-histidyl hexa-coordinated, deoxy penta-coordinated, and O2-bound hexa-coordinated states reveal distinct structural rearrangements at the interface between subunits in the dimer; these would affect the magnitude of the conformational fluctuations observed between monomer and dimer, and the topology of cavities within the protein matrix and at the interface. These results point to a distal site opening mechanism allowing access of the exogenous ligand to the heme and cast hypotheses on the dimer interface structural and dynamic properties that may support ligand binding cooperativity in dimeric SsHb.

The influence of temperature stress on the physiology of the Atlantic surfclam, Spisula solidissima.

Atlantic surfclam populations have significantly declined in state and federal waters from the south shore of Long Island, New York to the Delmarva Peninsula since the early 2000s. Previous studies have demonstrated that surfclams in this geographic range show signs of physiological stress, suggested to be a result of increasing ocean temperatures. In this study, we examined the effect of 2 temperature regimes (19 °C and 23 °C) on surfclam physiology. These temperatures were chosen because they represent maximal (23 °C) and minimal (19 °C) temperatures prevailing in New York clamming areas during summer. Results demonstrated enhanced energy metabolism and significant reductions in filtration rate, scope for growth, and immune functions in clams exposed to the warmer temperature treatment. Although net energy gains remained positive in both treatments under our experimental conditions, the findings suggest that temperature stress is involved in the recent observations of surfclams in poor condition. The impact of elevated temperatures on phytoplankton quantity/quality and other environmental variables in combination with the direct impact on surfclam filtration and metabolic rates could lead to a negative energy balance. While some uncertainties remain about population-scale impacts of overall warming trends, we fear that future increases in temperature may lead to the collapse of the Atlantic surfclam between New York and Virginia, especially within inshore regions.

Age, diet, and season do not affect longevity-related differences in peroxidation index between Spisula solidissima and Arctica islandica.

The susceptibility of membrane lipids to peroxidation (peroxidation index [PI]) increases with the double bond content of fatty acids and is inversely correlated to longevity in mammals, birds, and bivalve molluscs. In molluscs, membrane polyunsaturated fatty acids content can be affected by temperature, nutrition, and the individual's age. In this study, we evaluated how these three parameters may alter correlations between PI and longevity. We determined the fatty acid and dimethyl acetal compositions of phospholipids from gill mitochondrial and nonmitochondrial preparations from the short-lived Spisula solidissima (maximum longevity = 37 years) and the long-lived Arctica islandica (maximum longevity = 507 years) exposed to diet abundance and temperature (season) treatments. We also evaluated the effect of individual age on PI in S. solidissima (from 6 to 23 years). The temperature increase from winter to summer (2 to 12°C) coincided with decreases in values of PI, proportions of eicosapentaenoic acid, and dimethyl acetals. Higher microalgae supplementation increased polyunsaturated fatty acids and PI and decreased dimethyl acetals; age did not affect the PI in S. solidissima. Our finding that the PI of A. islandica remained significantly lower than that of S. solidissima in corresponding fractions throughout treatments suggests that longevity-related differences in PI are resilient to environmental conditions.

Low hydrogen peroxide production in mitochondria of the long-lived Arctica islandica: underlying mechanisms for slow aging.

The observation of an inverse relationship between lifespan and mitochondrial H2O2 production rate would represent strong evidence for the disputed oxidative stress theory of aging. Studies on this subject using invertebrates are surprisingly lacking, despite their significance in both taxonomic richness and biomass. Bivalve mollusks represent an interesting taxonomic group to challenge this relationship. They are exposed to environmental constraints such as microbial H2S, anoxia/reoxygenation, and temperature variations known to elicit oxidative stress. Their mitochondrial electron transport system is also connected to an alternative oxidase that might improve their ability to modulate reactive oxygen species (ROS) yield. Here, we compared H2O2 production rates in isolated mantle mitochondria between the longest-living metazoan--the bivalve Arctica islandica--and two taxonomically related species of comparable size. In an attempt to test mechanisms previously proposed to account for a reduction of ROS production in long-lived species, we compared oxygen consumption of isolated mitochondria and enzymatic activity of different complexes of the electron transport system in the two species with the greatest difference in longevity. We found that A. islandica mitochondria produced significantly less H2O2 than those of the two short-lived species in nearly all conditions of mitochondrial respiration tested, including forward, reverse, and convergent electron flow. Alternative oxidase activity does not seem to explain these differences. However, our data suggest that reduced complex I and III activity can contribute to the lower ROS production of A. islandica mitochondria, in accordance with previous studies. We further propose that a lower complex II activity could also be involved.

Localization of rRNA transcribed spacer domains in the nucleolinus and maternal procentrosomes of surf clam (Spisula) oocytes.

The nucleolinus is a nuclear subcompartment long ago posited to play a role in cell division. In a recent study using surf clam oocytes, cytoplasmic foci containing a nucleolinar protein were shown to later recruit γ-tubulin, identifying them as centrosomal precursors. (1) We now demonstrate the presence of structural RNAs from the nucleolinus in these procentrosomes. They include the well-known but poorly understood rRNA-transcribed spacer regions. In situ hybridization revealed a specific and dynamic association of these structural RNAs with the cell division apparatus that extends through the early stages of meiosis. In addition to their bearing on the debate over the nature of centrosome- and spindle-associated RNAs, the observations also suggest that rRNA spacer regions are not simply waste products to be discarded immediately, but may be functional byproducts that play a role in formation of the cell division apparatus.

Membrane associated nonmuscle myosin II functions as a motor for actin-based vesicle transport in clam oocyte extracts.

Nonmuscle myosin II (Myo2) has been shown to associate with membranes of the trans-Golgi network and to be involved in Golgi to ER retrograde protein transport. Here, we provide evidence that Myo2 not only associates with membranes but functions to transport vesicles on actin filaments (AFs). We used extracts from unactivated clam oocytes for these studies. AFs assembled spontaneously in these extracts and myosin-dependent vesicle transport was observed upon activation. In addition, actin bundles formed and moved relative to each other at an average speed of 0.30 microm/s. Motion analysis revealed that vesicles moved on the spontaneously assembled AFs at speeds greater than 1 microm/s. The motor on these vesicles was identified as a member of the nonmuscle Myo2 family based on sequence determination by Edman chemistry. Vesicles in these extracts were purified by sucrose gradient centrifugation and movement was reconstituted in vitro using skeletal muscle actin coated coverslips. When peripheral membrane proteins of vesicles including Myo2 were removed by salt stripping or when extracts were treated with an antibody specific to clam oocyte nonmuscle Myo2, vesicle movement was inhibited. Blebbistatin, a Myo2 specific inhibitor, also blocked vesicle movement. Myo2 light chain kinase activity was found to be essential for vesicle movement and sliding of actin bundles. Together, our data provide direct evidence that nonmuscle Myo2 is involved in actin-dependent vesicle transport in clam oocytes.

Cyclin E/Cdk2 is required for sperm maturation, but not DNA replication, in early sea urchin embryos.

The cell cycle is driven by the activity of cyclin/cdk complexes. In somatic cells, cyclin E/cdk2 oscillates throughout the cell cycle and has been shown to promote S-phase entry and initiation of DNA replication. In contrast, cyclin E/cdk2 activity remains constant throughout the early embryonic development of the sea urchin and localizes to the sperm nucleus following fertilization. We now show that cyclin E localization to the sperm nucleus following fertilization is not unique to the sea urchin, but also occurs in the surf clam, and inhibition of cyclin E/cdk2 activity by roscovitine inhibits the morphological changes indicative of male pronuclear maturation in sea urchin zygotes. Finally, we show that inhibition of cyclin E/cdk2 activity does not block DNA replication in the early cleavage cycles of the sea urchin. We conclude that cyclin E/cdk2 activity is required for male pronuclear maturation, but not for initiation of DNA replication in early sea urchin development.

The nerve hemoglobin of the bivalve mollusc Spisula solidissima: molecular cloning, ligand binding studies, and phylogenetic analysis.

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (microM). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mM), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P(50) approximately 0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons.

Protamines and other basic proteins from spermatozoa of molluscs.

The basic proteins obtained from spermatozoa of different species of the phylum Mollusca have been extracted and fractionated. The amino acid analysis and electrophoretic mobility of these proteins show a considerable variation in the types and relative amounts of the components present in different species. In some case-(Gibbula, Haliotis, Loligo, Octopus) the main components are similar to the protamines found in the salmonid fishes, although they appear to be larger in size (40-80 amino acids) and show significant differences in amino acid composition. In other cases (Mytilus, Chiton) a complex mixture of proteins is present, which including somatic-like histones and proteins intermediate in size and composition betweeln protamines and histones. Other molluscs (Ostrea, Spisula, Patella) also contain proteins intermediate in composition between protamines and histones, but their molecular weight appears to be larger than in histones. In Eledone a complex mixture of proteins containing cystine is obtained, with some components rich in arginine. In most species, somatic-like histones are also present. Their type and relative amount are different in each species. The significance of these results towards an understanding of the evolutionary history of these proteins is discussed. It is suggested that these proteins evolved from histone precursors.