Organic matrices in metazoan calcium carbonate skeletons: Composition, functions, evolution.

Calcium carbonate skeletal tissues in metazoans comprise a small quantity of occluded organic macromolecules, mostly proteins and polysaccharides that constitute the skeletal matrix. Because its functions in modulating the biomineralization process are well known, the skeletal matrix has been extensively studied, successively via classical biochemical approaches, via molecular biology and, in recent years, via transcriptomics and proteomics. The optimistic view that the deposition of calcium carbonate minerals requires a limited number of macromolecules has been challenged, in the last decade, by high-throughput approaches. Such approaches have made possible the rapid identification of large sets of mineral-associated proteins, i.e., 'skeletal repertoires' or 'skeletomes', in several calcifying animal models, ranging from sponges to echinoderms. One of the consequences of this expanding set of data is that a simple definition of the skeletal matrix is no longer possible. This increase in available data, however, makes it easier to compare skeletal repertoires, shedding light on the fundamental evolutionary mechanisms affecting matrix components.

Impact evaluation of the industrial activities in the Bay of Bakar (Adriatic Sea, Croatia): recent benthic foraminifera and heavy metals.

The Bay of Bakar is one of the most heavily polluted bays at the Eastern Adriatic. Three major industrial companies potentially endanger the bay. The concentration of major, minor and trace elements in surface sediments from thirteen stations was discussed in relation to the sediment type and foraminiferal assemblages. The distribution of major elements in the bay is influenced by geological nature of surroundings. Heavy metal distribution depends on pollution sources and on amount of mud fraction: fine-grained sediments are enriched by them in comparison with coarse-grained ones. Different sediment quality criteria complicate the pollution assessment in the bay. Heavy metal concentrations generally fall into allowed depositional values for marine environments; only area in front of the coke plant and the City of Bakar harbor is heavily polluted. Stress-tolerant foraminiferal species dominate at stations with higher concentrations of heavy metals and coarse-grained sediments consist of larger number of epifaunal taxa.

Silver nanoparticle toxicity in sea urchin Paracentrotus lividus.

Silver nanoparticles (AgNPS) are an important model system for studying potential environmental risks posed by the use of nanomaterials. So far there is no consensus as to whether toxicity is due to AgNPs themselves or Ag(+) ions leaching from their surfaces. In sea urchin Paracentrotus lividus, AgNPs cause dose dependent developmental defects such as delayed development, bodily asymmetry and shortened or irregular arms, as well as behavioural changes, particularly in swimming patterns, at concentration ∼0.3 mg/L AgNPs. It has been observed that AgNPs are more toxic than their equivalent Ag(+) ion dose.

The shell matrix of the pulmonate land snail Helix aspersa maxima.

In mollusks, the shell mineralization process is controlled by an array of proteins, glycoproteins and polysaccharides that collectively constitute the shell matrix. In spite of numerous researches, the shell protein content of a limited number of model species has been investigated. This paper presents biochemical data on the common edible land snail Helix aspersa maxima, a model organism for ecotoxicological purposes, which has however been poorly investigated from a biomineralization viewpoint. The shell matrix of this species was extracted and analyzed biochemically for functional in vitro inhibition assay, for amino acid and monosaccharides compositions. The matrix was further analyzed on 1 and 2D gels and short partial protein sequences were obtained from 2D gel spots. Serological comparisons were established with a set of heterologous antibodies, two of which were subsequently used for subsequent immunogold localization of matrix components. Our data suggest that the shell matrix of Helix aspersa maxima may differ widely from the shell secretory repertoire of the marine mollusks studied so far, such as the gastropod Haliotis or the pearl oyster Pinctada. In particular, most of the biochemical properties generally attributed to soluble shell matrices, such as calcium-binding capability, or the capacity to interfere in vitro with the precipitation of calcium carbonate or to inhibit the precipitation of calcium carbonate, were not recorded with this matrix. This drastic change in the biochemical properties of the landsnail shell matrix puts into question the existence of a unique molecular model for molluscan shell formation, and may be related to terrestrialisation.

Mytilus galloprovincialis as a bioindicator of environmental conditions: the case of the eastern coast of the Adriatic Sea.

The marine mussel Mytilus galloprovincialis lives attached to the surface of hard substrata, where its exposure and relative immobility allow it to record changes in ambient seawater. It is also found along the eastern coast of the Adriatic Sea. Oxygen and carbon isotopes were analysed for calcite and aragonite in separate shell layers, while major, minor and trace elements in the bulk shell were analysed to evaluate environmental conditions such as the temperature of carbonate deposition, freshwater influence and locations of anthropogenic pollution. We found that, on average, aragonite is enriched by 1.1‰ in (13)C and by 0.2‰ in (18)O compared with calcite. The calculated temperatures for M. galloprovincialis shell growth from the investigated area range from 13.4 to 20.9 °C for calcite and from 16.6 to 23.1 °C for aragonite. According to the δ(18)O and δ(13)C values of shell layers, we can separate the investigated area into three locations: those with more influence of freshwater, those with less influence of freshwater and those with marine environments. The highest concentrations of manganese, barium, boron, arsenic, nickel and chromium were observed in shells from Omis, Bacvice and Zablace (Central Adriatic) and Sv. Ivan (South Adriatic), where chemical and heavy industries are located and where sewage is known to be discharged into coastal areas. The highest concentrations of zinc, lead and copper were measured in samples from Pula, Rijeka and Gruz, where there are also ports in addition to industry.

Nacre calcification in the freshwater mussel Unio pictorum: carbonic anhydrase activity and purification of a 95 kDa calcium-binding glycoprotein.

The formation of the molluscan shell is finely tuned by macromolecules of the shell organic matrix. Previous results have shown that the acid-soluble fraction of the nacre matrix of the freshwater paleoheterodont bivalve Unio pictorum shell displays a number of remarkable properties, such as calcium-binding activity, the presence of extensive glycosylations and the capacity to interfere at low concentration with in vitro calcium carbonate precipitation. Here we have found that the nacre-soluble matrix exhibits a carbonic anhydrase activity, an important function in calcification processes. This matrix is composed of three main proteinaceous discrete fractions. The one with the highest apparent molecular weight is a 95 kDa glycoprotein that is specific to the nacreous layer. P95, as it is provisionally named, is enriched in Gly, Glx and Asx and exhibits an apparent pI value of approximately 4, or approximately 7 when chemically deglycosylated. Furthermore, its glycosyl moiety, consisting of sulfated polysaccharides, is involved in calcium binding. Purified fractions of the three main proteins were digested with trypsin, and the resulting peptides were analysed by mass spectrometry. Our results suggest that identical peptides are constitutive domains of the different proteins. Partial primary structures were obtained by de novo sequencing and compared with known sequences from other mollusc shell proteins. Our results are discussed from an evolutionary viewpoint.

Effect of hypoosmotic stress by low salinity acclimation of Mediterranean mussels Mytilus galloprovincialis on biological parameters used for pollution assessment.

In the present study, we investigated the progressive acclimation of the mussel Mytilus galloprovincialis to different reduced seawater (SW) salinities and its effect on several biochemical markers and biotests. Mussels were purchased from a local mariculture facility during summer (SW temperature 27 degrees C, salinity 37.5 psu) and winter (13 degrees C, 37 psu) seasons, and transferred to the laboratory for acclimation to reduced SW salinities (37, 28, 18.5 and 11 psu). At the beginning and at the end of acclimation processes tests of mussel survival in air were provided. After 14 days of acclimation the DNA integrity, p38-MAPK activation, metallothionein induction, oxygen consumption rate, and condition index were measured. Survival in air (SOS test), as a physiological index of mussel's health and vitality, had significantly lower LT50 values (11 psu) in the summer than in the winter, and it seems to be negatively affected by acclimation in comparison to controls (37 psu and mariculture). Condition indexes (CIs) were not significantly different, but mussel's acclimation resulted in decline (i.e., a negative trend), especially of CI-2 and CI-3 calculated on the basis of mussel tissue weight and shell sizes. Oxygen consumption rate (VO2) of M. galloprovincialis acclimated to reduced salinities was a concentration-dependent process and increased considerably to about 51 and 65% in lower SW concentrations (28 and 18 psu) compared to control mussels (37 psu). DNA integrity, determined by Fast Micromethod, was negatively impacted by salinity acclimation and corresponding physiological stress as well. Some differences in 1D protein expression patterns between control groups and mussels acclimated to 28, 18.5 and 11 psu (SW) were established. Reduced SW salinities (18.5 and 11 psu) resulted in significantly higher p38-MAPK phosphorylation, whereas the SW salinity of 28 psu decreased p-p38 significantly compared to control (37 psu). The concentration of metallothioneins in mussels' gills was reduced at 28 and 18.5 psu, while it was significantly higher at 11 psu. Results indicated that SW salinity variation (i.e., hypoosmotic stress) in the marine environment can affect all investigated parameters. This investigation expands our understanding of multifactorial effects of the physical marine environment on the specificity of investigated biomarkers and biotests, providing insight into the acclimation, adaptive and stress response processes of mussels. Effects of environmental factors have to be considered in sampling strategies for monitoring programmes to prevent false interpretation of results.

Molluscan shell proteins: primary structure, origin, and evolution.

In the last few years, the field of molluscan biomineralization has known a tremendous mutation, regarding fundamental concepts on biomineralization regulation as well as regarding the methods of investigation. The most recent advances deal more particularly with the structure of shell biominerals at nanoscale and the identification of an increasing number of shell matrix protein components. Although the matrix is quantitatively a minor constituent in the shell of mollusks (less than 5% w/w), it is, however, the major component that controls different aspects of the shell formation processes: synthesis of transient amorphous minerals and evolution to crystalline phases, choice of the calcium carbonate polymorph (calcite vs aragonite), organization of crystallites in complex shell textures (microstructures). Until recently, the classical paradigm in molluscan shell biomineralization was to consider that the control of shell synthesis was performed primarily by two antagonistic mechanisms: crystal nucleation and growth inhibition. New concepts and emerging models try now to translate a more complex reality, which is remarkably illustrated by the wide variety of shell proteins, characterized since the mid-1990s, and described in this chapter. These proteins cover a broad spectrum of pI, from very acidic to very basic. The primary structure of a number of them is composed of different modules, suggesting that these proteins are multifunctional. Some of them exhibit enzymatic activities. Others may be involved in cell signaling. The oldness of shell proteins is discussed, in relation with the Cambrian appearance of the mollusks as a mineralizing phylum and with the Phanerozoic evolution of this group. Nowadays, the extracellular calcifying shell matrix appears as a whole integrated system, which regulates protein-mineral and protein-protein interactions as well as feedback interactions between the biominerals and the calcifying epithelium that synthesized them. Consequently, the molluscan shell matrix may be a source of bioactive molecules that would offer interesting perspectives in biomaterials and biomedical fields.

Mineralogy of shells from two freshwater snails Belgrandiella fontinalis and B kuesteri.

X-ray powder diffraction (XRD) was used to study the mineral composition of shells of snails Belgrandiella fontinalis and Belgrandiella kuesteri collected from three freshwater springs in northeastern Slovenia. The fractions of aragonite, calcite, dolomite and quartz in particular shells were determined. The analysed shells consisted of two or more distinct inorganic layers. The outer shell layer for both species and all sampling localities contained aragonite. The outer layer of B. fontinalis collected at one locality, also contained a small fraction of calcite ( approximately 1 molar%) besides the dominant aragonite. Calcite was identified in the inner layer(s) of both species (2 to 3 molar%), while quartz was found only in B. kuesteri (5-7 molar%). However, both species sampled at one locality showed the presence of dolomite (approx. 20 molar%) in the inner layer(s). The presence of dolomite in the shells of adult gastropods and even molluscs is unusual. A possible formation mechanism and specific ecological factor that could influence the precipitation of dolomite in the shells of different Belgrandiella species is discussed.