Aragonitic dendritic prismatic shell microstructure in Thracia (Bivalvia,Anomalodesmata)

The shells of most anomalodesmatan bivalves are composed of an outer aragonitic layer of either granular or columnar prismatic microstructure, and an inner layer of nacre. The Thraciidae is one of the few anomalodesmatan families whose members lack nacreous layers. In particular, shells of members of the genus Thracia are exceptional in their possession of a very distinctive but previously unreported microstructure, which we term herein “dendritic prisms.” Dendritic prisms consist of slender fibers of aragonite which radiate perpendicular to, and which stack along, the axis of the prism. Here we used scanning and transmission electron microscopical investigation of the periostracum, mantle, and shells of three species of Thracia to reconstruct the mode of shell calcification and to unravel the crystallography of the dendritic units. The periostracum is composed of an outer dark layer and an inner translucent layer. During the free periostracum phase the dark layer grows at the expense of the translucent layer, but at the position of the shell edge, the translucent layer mineralizes with the units typical of the dendritic prismatic layer. Within each unit, the c‐axis is oriented along the prismatic axis, whereas the a‐axis of aragonite runs parallel to the long axis of the fibers. The six‐rayed alignment of the latter implies that prisms are formed by {110} polycyclically twinned crystals. We conclude that, despite its distinctive appearance, the dendritic prismatic layer of the shell of Thracia spp. is homologous to the outer granular prismatic or prismatic layer of other anomalodesmatans, while the nacreous layer present in most anomalodesmatans has been suppressed.     

On the unique isotropic aragonitic tube microstructure of some serpulids (Polychaeta,Annelida)

A Scanning electron microscopy study of peculiar serpulid tube microstructures was carried out to determine whether this structure is unique to serpulids and to understand its formation and evolution. The isotropic aragonitic tube microstructure of some serpulids, termed spherulitic irregularly oriented prismatic structure (SIOP), is unknown in other animal skeletons. After nucleation, initial spherulitic prismatic aragonite crystals presumably grow in length and width at one end, collide with the surrounding growing crystal fans, and then continue intertwined growth until the secreted carbonate‐charged mucus is completely crystallized. Spherulitic irregularly oriented prismati structure presumably evolved either from the IOP or homogeneous rounded crystal microstructures. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Biomineral ultrastructure, elemental constitution and genomic analysis of biomineralization-related proteins in hemichordates

Here, we report the discovery and characterization of biominerals in the acorn worms Saccoglossus bromophenolosus and Ptychodera flava galapagos (Phylum: Hemichordata). Using electron microscopy, X-ray microprobe analyses and confocal Raman spectroscopy, we show that hemichordate biominerals are small CaCO(3) aragonitic elements restricted to specialized epidermal structures, and in S. bromophenolosus, are apparently secreted by sclerocytes. Investigation of urchin biomineralizing proteins in the translated genome and expressed sequence tag (EST) libraries of Saccoglossus kowalevskii indicates that three members of the urchin MSP-130 family, a carbonic anhydrase and a matrix metaloprotease are present and transcribed during the development of S. kowalevskii. The SM family of proteins is absent from the hemichordate genome. These results increase the number of phyla known to biomineralize and suggest that some of the gene-regulatory 'toolkit', if not mineralized tissue themselves, may have been present in the common ancestor to hemichordates and echinoderms.     

Discovery of the mineral brucite (magnesium hydroxide) in the tropical calcifying alga Polystrata dura (Peyssonneliales,Rhodophyta)

Red algae of the family Peyssonneliaceae typically form thin crusts impregnated with aragonite. Here, we report the first discovery of brucite in a thick red algal crust (~1 cm) formed by the peyssonnelioid species Polystrata dura from Papua New Guinea. Cells of P. dura were found to be infilled by the magnesium‐rich mineral brucite [Mg(OH)₂]; minor amounts of magnesite and calcite were also detected. We propose that cell infill may be associated with the development of thick (> ~5 mm) calcified red algal crusts, integral components of tropical biotic reefs. If brucite infill within the P. dura crust enhances resistance to dissolution similarly to crustose coralline algae that infill with dolomite, then these crusts would be more resilient to future ocean acidification than crusts without infill.     

A hairy business—Periostracal hair formation in two species of helicoid snails (Gastropoda,Stylommatophora, Helicoidea)

In molluscs, the calcareous shell is covered externally by a thin organic layer, the periostracum. The periostracum of some pulmonate species is of special taxonomic interest because it bears distinct microscale architectures. Where and how these structures are formed is as yet unknown. Using histological sections through their shells, gelatin cuts, and live observations I studied the pattern by which the periostracal hair‐like projections in two helicoid land snail species are secreted and evenly arranged on the shell. The results indicate a complex mechanism: a hair is formed in the periostracal groove independently of the periostracum, after which it is attached to the edge of the shell, drawn out of the tissue, and finally swivelled to the upper side of the periostracum. Upon further growth of the periostracum, the hairs are finally fixed upright on the shell. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

Environmental contaminants influencing immunefunction in marine bivalve molluscs

The increased observation of pollution induced disease conditions in marine organisms has led to a growing interest on the effects of environmental contaminants on the immune system. Most studies on modulation of the immune system in bivalves by pollutants have concentrated on the effects of heavy metals and polycyclic aromatic hydrocarbons (PAHs). The current literature on contaminant effects on specific components of the bivalve immune system is reviewed together with the effects on susceptibility to infection. Data are presented showing the effects on immune parameters of exposure to Vibrio tubiashi following pre-exposure to copper or cadmium. Mussels exposed to cadmium for 7 days followed by 7 days exposure to V. tubiashi demonstrated significantly higher numbers of circulating haemocytes compared with non-Vibrio-exposed groups. Similar experiments conducted with copper exposure for both 7 days and 7 weeks followed by V. tubiashi for 7 days demonstrated a significant decrease in the percentage of circulating eosinophils compared with basophilic cells for both short and long term exposures. The intracellular release of superoxide (NBT reduction) by haemocytes was stimulated in Vibrio-challenged mussels with no copper pre-exposure but was significantly reduced in mussels pre-exposed to 0·2 ppm of copper for 7 weeks. The mortalities for the copper experiments showed increased levels with increasing copper concentration and were consistently higher in the V. tubiashi challenged mussels which had also been exposed to copper.     

Electron diffraction of mollusc shell organic matrices and their relationship to the mineral phase

Electron diffraction patterns showing orientation of the chitin and protein constituents of the insoluble organic matrix of mollusc shell nacreous layers have been obtained, using low dose conditions and samples cooled to −100°C. Diffraction patterns of the aragonite crystals were also observed. In a gastropod and a bivalve the spatial relationship between the organic matrix constituents and the aragonite crystallographic axes were shown to be the same as was previously observed for a cephalopod using X-ray diffraction, supporting the notion that mineral crystal growth occurs epitaxially upon a matrix template.     

Multilocus genetic analysis of Cryptosporidium in naturally contaminated bivalve molluscs

AIMS: To evaluate the application of discriminatory multilocus PCR procedures for the characterization of Cryptosporidium in samples of naturally contaminated bivalve molluscan shellfish. METHODS AND RESULTS: Nucleic acid was extracted from 22 shellfish previously identified as contaminated with Cryptosporidium spp. and subjected to PCR-based analysis for two independent fragments of the Cryptosporidium oocyst wall protein (COWP) gene, three microsatellite markers (ML 1, GP 15 and MS 5) and an extra-chromosomal small double-stranded RNA (dsRNA). Overall, at least one COWP gene fragment was amplified from all 22 samples, 21 amplified the dsRNA and 14 at least one of the three microsatellite loci. More than one dsRNA or microsatellite allele was detected in 50% of samples. The majority of samples were contaminated with Cryptosporidium parvum types circulating in both humans and livestock. A novel dsRNA element was identified in one sample, which did not amplify any of the three microsatellite loci investigated. CONCLUSIONS: Multilocus analysis of Cryptosporidium can be applied to DNA extracted from naturally contaminated shellfish. SIGNIFICANCE AND IMPACT OF STUDY: This multilocus genetic analysis highlights that filter feeder molluscs are a potential source of cryptosporidial oocysts, which may be infectious to humans.     

Effects of GABA and epinephrine on the settlement and metamorphosis of the larvae of four species of bivalve molluscs

Competent larvae of different marine bivalve species were treated with GABA and epinephrine at different concentrations and times of exposure to test the ability of the drugs to induce settlement and metamorphosis. GABA induced both settlement and metamorphosis in the mussel Mytilus galloprovincialis, the clams Venerupis pullastra and Ruditapes philippinarum and the oyster Ostrea edulis. Maximum induction of settlement (>39%) was achieved after exposure of V. pullastra larvae to 10⁻⁴ M GABA; this concentration of GABA also induced the highest percentages of metamorphosis in the four species studied. Epinephrine was identified as an active inducer of settlement and metamorphosis in bivalve molluscs. Exposure to 10⁻⁵ M epinephrine induced significant levels of settlement in Mytilus, Venerupis and Ostrea. In contrast, epinephrine failed to induce settlement behaviour in Ruditapes. Maximum induction of metamorphosis was produced by 10⁻⁵ M epinephrine in mussels, clams and oysters; Ruditapes showed the highest percentage of metamorphosis (>78%). This is the first report in which the involvement of GABA in the settlement and metamorphosis of bivalve molluscan larvae is demonstrated. It was also recognised that epinephrine plays a role not only in inducing metamorphosis but also in initiating settlement.     

Shell microstructure of the early bivalve Pojetaia and the independent origin of nacre within the mollusca

Abstract: Pojetaia and Fordilla are the oldest bivalve molluscs, occurring in roughly co‐eval rocks from the Tommotian, and are the only undisputed, well‐known bivalves from the Cambrian. New specimens reveal that Pojetaia had a laminar inner shell microstructure reminiscent of the foliated aragonite of modern monoplacophorans, and the same is true for Fordilla. A similar shell microstructure is seen in Anabarella and Watsonella, providing support for the hypothesis that they are the ancestors of bivalves. Foliated aragonite shares many similarities with nacre, and it may have been the precursor to nacre in bivalves. No cases of undisputed nacre occur in the Cambrian, in spite of much shell microstructure data from molluscs of this time period. Thus, although considered by many to be homologous among molluscs, we conclude that nacre convergently evolved in monoplacophorans, gastropods, bivalves, and cephalopods. This independent origin of nacre appears to have taken place during, or just prior to, the Great Ordovician Biodiversification Event and represents a significant step in the arms race between predators and molluscan prey.     

Anaerobic metabolism of bivalve molluscs during exposure to air

The anaerobic metabolism of Mytilus edulis, Cardium edule, Scrobicularia plana and Macoma balthica was investigated. On exposure to the atmosphere, all of these species were found to be able to utilise ¹⁴CO₂. This suggests that these species gape during exposure to the atmosphere. A comparative study on the pattern of ¹⁴C incorporation suggests that there is a similarity between M. edulis and S. plana in the extent of the utilization of the succinate pathway during exposure to air. However C. edule and M. balthica were also similar in the extent of utilizing the succinate pathway, even though there were significant species differences between the similar animals. It is suggested that exposure in S. plana represents a stressful situation and that this species might react to this stress by utilizing the succinate pathway. A higher incorporation of radiocarbon into alanine by M. balthica could be due to high activity of the enzymes that control the reactions leading to production of radioactive pyruvate.     

Structure,molting, and mineralization of the dorsal ossicle complex in the gastric mill of the blue crab,Callinectes sapidus

This study examined the mesocardiac and urocardiac ossicles in the gastric mill of the blue crab to describe its structure, mineralization, and dynamics throughout the molt cycle, and to assess its possible utility in age determination. Morphologically, the mineralized ossicles are similar to the calcified dorsal carapace having a lamellate structure comprised of sheets of chitin/protein fibrils. Staining with acridine orange showed the same arrangement of an epicuticle, exocuticle, and endocuticle. In much of the mesocardiac and urocardiac ossicles, the endocuticle is very reduced, with the exocuticle predominating; the reverse of the dimensions of the exoskeleton. The lamellate structure of the ossicles was confirmed with scanning electron microscopy; however, elemental mapping by energy‐dispersive analysis of X‐rays revealed that the ossicles are mineralized with calcium phosphate, in contrast to the calcium carbonate biomineral of the exoskeleton. The medial tooth of the urocardiac ossicle is not calcified, but the epicuticle is highly elaborated and impregnated with silica. Histological examination of the ossicles demonstrated that they are molted during ecdysis, so despite the appearance of bands in the mesocardiac ossicle, it is difficult to hypothesize how the bands could represent a record of chronological age. J. Morphol. 276:1358–1367, 2015. © 2015 Wiley Periodicals, Inc.     

Effects of temperature stress on NO-synthase and tyrosine hydroxylase activities in the central nervous system of bivalve molluscs

Effects of temperature stress on activities of NO-synthase (NOS) and tyrosine hydroxylase (TH) in the CNS of two species of bivalve molluscs, Mizuchopecten yessoensis and Chlamys farreri nipponensis (Pectinidae) were studied using NADPH-diaphorase histochemistry and immunocytochemistry. General and specific peculiarities in distribution and relative proportion of TH- and NO-containing neurons in the CNS nerve ganglia were revealed in norm and under stress at 30°C for 10, 30, and 60 min. The initial stress stage (for 10 min) has been found to be accompanied by an increase of the relative content of TH-positive neurons in some CNS areas of both mollusc species. In intact Chlamys farreri nipponensis, the presence of NOS in the CNS and its significant activation under temperature stress might have possibly been an important neuroprotective component of stress reaction in some mollusc species.     

Coastal acidification impacts on shell mineral structure of bivalve mollusks

Ocean acidification is occurring globally through increasing CO₂ absorption into the oceans creating particular concern for calcifying species. In addition to ocean acidification, near shore marine habitats are exposed to the deleterious effects of runoff from acid sulfate soils which also decreases environmental pH. This coastal acidification is being exacerbated by climate change‐driven sea‐level rise and catchment‐driven flooding. In response to reduction in habitat pH by ocean and coastal acidification, mollusks are predicted to produce thinner shells of lower structural integrity and reduced mechanical properties threatening mollusk aquaculture. Here, we present the first study to examine oyster biomineralization under acid sulfate soil acidification in a region where growth of commercial bivalve species has declined in recent decades. Examination of the crystallography of the shells of the Sydney rock oyster, Saccostrea glomerata, by electron back scatter diffraction analyses revealed that the signal of environmental acidification is evident in the structure of the biomineral. Saccostrea glomerata, shows phenotypic plasticity, as evident in the disruption of crystallographic control over biomineralization in populations living in coastal acidification sites. Our results indicate that reduced sizes of these oysters for commercial sale may be due to the limited capacity of oysters to biomineralize under acidification conditions. As the impact of this catchment source acidification will continue to be exacerbated by climate change with likely effects on coastal aquaculture in many places across the globe, management strategies will be required to maintain the sustainable culture of these key resources.     

Carbonate and Phosphate Precipitation by Saline Soil Bacteria in a Monitored Culture Medium

Carbonate and phosphate precipitation by bacteria isolated from a saline soil was studied in vitro in a liquid culture medium over 45 days. Physicochemical parameters of this medium were continuously monitored using both selective electrodes (continuous monitoring, CM) and individual measurements by other techniques on days 5, 10, 15, 20, 25, 35 and 45 (discontinuous monitoring, DM). In DM, the precipitated minerals were studied (XRD and SEM-EDX) and the saturation index of the mineral phases was analyzed (PHREEQC program). Using the CM and DM data it was possible to distinguish several temporary stages in which both the medium and the mineralogy changed: 1) 0 to 10 days: pH reaches 8.4; significant loss of Mg²⁺ (incorporated into the bacterial biomass) and Ca²⁺ (through mineral precipitation); formation of crystals, although not in sufficient quantity to be studied until day 10. 2) 10 to 25 days: pH decreases but remains above 8; appreciable loss of Mg²⁺ and Ca²⁺ due to formation of spherical carbonate bioliths with traces of phosphates occluded within these carbonates. 3) After 25 days: biomineralization slow down; pH returns to initial values and struvite is formed (idiomorphic prismatic crystals). These trends are in agreement with the findings of other workers, although with some peculiarities regarding stages and types of mineral precipitated. In some cases the struvite contained small quantities of K and Ca, possibly because these are intermediate mineral species between typic-struvite, K-struvite and Ca-struvite. The bacteria-mediated precipitation of carbonates of Ca and/or Mg and phosphates (struvite) by the bacteria from a saline soil is demonstrated. However, struvite was not found in the soils of origin of the bacteria, possibly because it is a metastable mineral in most soils.     

Formation of the prismatic layer in the freshwater bivalve Hyriopsis cumingii: the feedback of crystal growth on organic matrix

The squeezing hypothesis and the organic frameworks preformation hypothesis propose two different mechanisms to explain the interaction between organic frameworks and crystals during biomineralization of the prismatic layer of the mollusk shell. In this study, we began to study Hyriopsis cumingii shell formation and discover that this species seemed to follow the squeezing hypothesis. During the formation of the aragonite prismatic layer in the freshwater bivalve H. cumingii, we found that crystal growth was involved in controlling initiation of formation of the interprismatic organic membranes. First, newly formed crystals were embedded in the periostracum. Next, the interprismatic organic membranes of the prismatic layer were produced via squeezing between neighboring crystals. The organic matrix secreted by the mantle continuously self‐assembled into the interprismatic organic membranes as the crystals grew. In the mature stage, the interprismatic organic membranes were shaped by crystal growth. These findings provide evidence to support the squeezing hypothesis and add to the existing knowledge about interactions that occur at the organic–inorganic interfaces during mollusk shell biomineralization.     

Effects of variations in carbonate chemistry on the calcification rates of Madracis auretenra (= Madracis mirabilis sensu Wells, 1973): bicarbonate concentrations best predict calcification rates

Physiological data and models of coral calcification indicate that corals utilize a combination of seawater bicarbonate and (mainly) respiratory CO₂ for calcification, not seawater carbonate. However, a number of investigators are attributing observed negative effects of experimental seawater acidification by CO₂ or hydrochloric acid additions to a reduction in seawater carbonate ion concentration and thus aragonite saturation state. Thus, there is a discrepancy between the physiological and geochemical views of coral biomineralization. Furthermore, not all calcifying organisms respond negatively to decreased pH or saturation state. Together, these discrepancies suggest that other physiological mechanisms, such as a direct effect of reduced pH on calcium or bicarbonate ion transport and/or variable ability to regulate internal pH, are responsible for the variability in reported experimental effects of acidification on calcification. To distinguish the effects of pH, carbonate concentration and bicarbonate concentration on coral calcification, incubations were performed with the coral Madracis auretenra (= Madracis mirabilis sensu Wells, 1973) in modified seawater chemistries. Carbonate parameters were manipulated to isolate the effects of each parameter more effectively than in previous studies, with a total of six different chemistries. Among treatment differences were highly significant. The corals responded strongly to variation in bicarbonate concentration, but not consistently to carbonate concentration, aragonite saturation state or pH. Corals calcified at normal or elevated rates under low pH (7.6–7.8) when the seawater bicarbonate concentrations were above 1800 μm . Conversely, corals incubated at normal pH had low calcification rates if the bicarbonate concentration was lowered. These results demonstrate that coral responses to ocean acidification are more diverse than currently thought, and question the reliability of using carbonate concentration or aragonite saturation state as the sole predictor of the effects of ocean acidification on coral calcification.     

Clypeasteroid echinoid tests as benthic islands for gastrochaenid bivalve colonization: evidence from the Middle Miocene of Tarragona,north‐east Spain

Abstract: Middle Miocene tests of Clypeaster from L’Arrabassada (Tarragona, north‐east Spain) show evidence of intense endoskeletozoan colonization, preserved as borings and associated carbonate secretions that allow gastrochaenid bivalves to be identified as the colonizers. Two modes of occurrence have been recognized for these bivalve dwelling cavities; ‘intrastereom clavate borings’ which are restricted to the echinoid stereom, and ‘semi‐endoskeletal dwellings’, which penetrate across the test wall and extend as carbonate crypts into the sediment fill of the internal test cavity. Their size, density and position rule out a syn‐vivo relationship with the echinoids and demonstrate that colonization was post mortem. Because of the endurance of clypeasteroid skeleton and the pronounced bell‐shaped morphology of Clypeaster, the tests of these echinoids provided the most suitable substrates for hard‐bottom colonizers on an otherwise sandy seafloor. The scenario described from Tarragona can be extended to other Neogene and Quaternary strata elsewhere; there is ample evidence for the long‐term utilization of tests of Clypeaster by gastrochaenid bivalves in shoreface palaeoenvironments.     

Chitin helicoids accompany protein helicoids in the periostracum of a whelk, Buccinum

The periostracum of the marine gastropod Buccinum has a helicoidal arrangement of its principal constituent which is a fibrous protein (Hunt and Oates, 1978). Chitin, chemically and physically identified, is present at a concentration of about 6% of the dry weight and can be seen in dispersates of whole periostracum as long fibrils and ribbons between 3 and 14 nm diameter. Deproteinization with hot alkali removes all protein leaving a chitinous 'ghost' of the periostracum. Dispersates, examined negatively stained, show only chitin fibrils and ribbons while sectioned material demonstrates a tenuous, part orthogonal, part helicoidal, architecture based on the chitin residue. The relative roles of the protein and polysaccharide components is speculated upon and comparisons with arthropod cuticle drawn.     

Enzymatic degradation of hyaluronan hydrogels with different capacity for in situ bio‐mineralization

In situ cross‐linked hyaluronan (HA) hydrogels with different capacities for biomineralization were prepared and their enzymatic degradation was monitored. Covalent incorporation of bisphosphonates (BPs) into HA hydrogel results in the increased stiffness of the hydrogel in comparison with the unmodified HA hydrogel of the same cross‐linking density. The rate of enzymatic degradation of HABP hydrogel was significantly lower than the rate of degradation of control HA hydrogel in vitro. This effect is observed only in the presence of calcium ions that strongly bind to the matrix‐anchored BP groups and promote further mineralization of the matrix. The degradation of the hydrogels was followed by noninvasive fluorescence measurements enabled after mild and chemoselective labeling of cross‐linkable HA derivatives with a fluorescent tag.