Shell Microstructure of Protobranchia (Mollusca: Bivalvia): Diversity, New Microstructures and Systematic Implications

<p>Bivalves offer outstanding potential as environmental archives. However, vital effects exert a strong control on the incorporation of many trace and minor elements into the shell so that their use as environmental proxies is currently limited. Furthermore, Sr and Mg show a strong relationship to the micrometer-sized shell architecture (shell microstructure), i.e., near growth lines, which are typically dominated by irregular simple/spherulitic prismatic microstructures, the concentrations of these elements are significantly higher than in portions between growth lines (= growth increments, which are microstructurally more complex). In contrast, Ba is uncoupled from the prevailing shell microstructure. To shed more light on these issues, we conducted a combined element chemical (in-situ analysis by means of LA-ICP-MS) and microstructural analyses (using SEM) of shells of <em>Arctica islandica</em> collected alive in NE Iceland.</p><p>According to our findings, (1) contemporaneous shell portions in the hinge and ventral margin (both belonging to the outer shell layer) within individual specimens showed nearly identical Sr/Ca and Mg/Ca values, but Ba/Ca was 1.5 – 2.5 times higher in the ventral margin than in the hinge. (2) In agreement with previous studies, Sr and Mg were strongly elevated near annual growth lines. (3) Along an isochronous transect from the inner portion of the outer shell layer near the myostracum toward the outer shell surface (in the ventral margin), Si/Ca values increased, on average, by 75% ± 11%, whereas Na/Ca values decreased by 7% ± 1%. Along this transect, the shell microstructure gradually changed from crossed-acicular to homogeneous suggesting that Si and Na are linked to the prevailing nanometer-sized shell architecture or underlying physicochemical processes controlling their formation. (4) In the hinge, Ba/Ca, Sr/Ca, Mn/Ca and Mg/Ca attained highest values along the axis of maximum growth, but gradually decreased in slower growing (contemporaneous) shell portions away from that axis. (5) In contemporaneous shell portions (in either the hinge or the ventral margin), the concentration of some elements varied significantly among specimens, whereas others showed little variability. For example, in similar and contemporaneous shell portions of different specimens, Na/Ca values exhibited only little variation (17.4 – 23.7 mmol/mol), whereas Sr/Ca and B/Ca differed more severely (0.3 – 1.6 mmol/mol and 0.04 – 0.07 mmol/mol, respectively; both within growth increments). Despite these inter-specimen chemical differences, the shell microstructure remained largely invariant.</p><p>Our findings firstly suggest that the extrapallial fluid, if it exists at all, is chemically inhomogeneous. This could result from differences in the efficiency of transmembrane ion transport or to differences in shell formation rate along the growing margin (e.g., faster growth in the outer portion of the outer shell layer than in portions closer to the myostracum). Secondly, chemical differences among specimens may be attributed to physiological differences. Thirdly, some elements such as Ba are uncoupled to microstructural properties, but co-vary strongly among specimens suggesting an environmental control on the uptake and incorporation of this element into the shell.</p>

The structure of pristine preserved shells of Mesozoic ammonoids was examined in order to identify whether there are homologous or homoplasic traits in the internal structure of the shell wall that can be used for phylogeny and taxonomy, particularly in regard to episodic growth interruptions and the dorsal shell wall. Different sculptural elements that are usually associated with an interruption in shell growth, like varices, constrictions, flares, parabolae and radial lirae, were analysed to determine whether these have a general ground plan in internal structure or not. Varices and constrictions shows no interruptions in shell growth but have characteristic internal thickenings formed by the nacreous layer. Owing to the general structural conformity of varices and constrictions in the shells of Jurassic and Cretaceous taxa, they are probably homologous traits. Flares, parabolae and radial lirae are characterized by an interruption in growth, they mark the succession of two different shell generations. Parabolae and flares mark an interruption of shell growth caused by the formation of a frill-like, outwardly bent temporary aperture (but differ in their general shape). The outer prismatic layer, the nacreous layer and an apertural prismatic coating (i.e. indicates a mantle cover) build their aperture. Radial lirae are formed by imbrication of two successive sigmoid shell lamellae, formed either by the organic periostracum or the outer prismatic layer. Each lira has a characteristic adorally-projecting, scythe-like appendage arising from its crest. Owing to the general structural and morphological conformity of flares and parabolae it is likely that both represent different expressions of a homologous structure. Morphological transitions imply that parabolae and radial lirae are probably homologous as well. Mesozoic Ammonoidea had different types of the dorsal shell wall. The vast majority of planispirally taxa forms a prismatic reduced dorsal shell wall, consisting of an outer organic component (e.g. wrinkle layer) and the subsequent formed dorsal inner prismatic layer. The latter is only secreted in the rear of the living chamber. Reduced dorsal shell wall construction of Ammonoidea is generally homologous and exists in the early ontogenetic stages of all taxa. Rather few ammonoid taxa reinforce the dorsal shell wall. Some planispirally taxa form a nacreous reduced dorsal shell wall in the rear part of the living chamber, which is reinforced by secondary pair of nacreous and inner prismatic layers. Heteromorph taxa and some planispirally taxa secreted a complete dorsal shell wall at the aperture, i.e. the ventral, lateral and dorsal shell wall form a continum aperturally and adapically. The dorsal shell layers are identical with the ventral shell wall. The broad phylogenetic distribution of dorsally secreted nacre indicates a plesiomorphic relic trait of the orthocone ammonoid ancestors.

Environmental control of strontium and magnesium in Mytilus

An endocochliate embryo model for the Mesozoic Ammonitida is proposed based on scanning electron microscopy of the ammonitella (= embryonic shell) stage of well‐preserved Japanese Cretaceous specimens belonging to nine species of five superfamilies. As in other specimens described previously, the ammonitella wall succeeding from the initial chamber ("protoconch") in the species examined consists of the inner prismatic, middle subprismatic and outer prismatic layers, with minute tubercles resting on the outer. Developmental patterns of these structures and their comparison with primary shell formation in modern Nautilus and Spirula suggest that the outer thin prismatic layer with microtubercles was secreted by the exterior epithelium after the completion of the main ammonitella wall by the interior shell gland. Thus, the early ammonite embryo might have had an endocochliate structural plan as in coleoids, and at the time of hatching the overlying mantle epithelium had shifted anteriorly to become an ectocochliate condition.

Mollusc shells are composite structures built of calcium carbonate crystals (calcite and/or aragonite) and biopolymers (protein, polysaccharides and lipids). These two classes of materials create a great variety of microstructures characterised by complex architectures and unique material properties. The formation of shell microstructures is controlled by environmental and physiological factors and the variety of microstructures is believed to be of phylogenetic and adaptive biomechanical significance. Here, through SEM and XRD analyses, we provide for the first time a detailed characterisation, description and illustration of the shell microstructure and mineralogy of three mollusc species: the bivalves Anadara uropigimelana (Bory de Saint-Vincent, 1827) and Tivela stefaninii (Nardini, 1933), and the gastropod Oliva bulbosa (Röding, 1798). The specimens were collected in the Upper Holocene HAS1 settlement and in a shell midden in the Khor Rori Archaeological Park (Oman). These species frequently occur in archaeological assemblages and show clear growth lines and increments in their shells, making them excellent tools to be used for high resolution palaeoclimatic and palaeoenvironmental studies. However, data on their shell microstructure and mineralogy, that are fundamental for fossil shell preservation analyses, are lacking. Our results show that shells of Anadara uropigimelana are aragonitic with an outer crossed lamellar layer, an inner complex crossed lamellar layer and an irregular simple prismatic pallial myostracum; periodic bands of dendritic nondenticular composite prisms occur in the outer part of the outer layer, reflecting seasonal changes in water temperatures and growth rates. Tivela stefaninii shells are aragonitic and show an outer composite prismatic layer, a middle crossed lamellar layer, and an inner complex crossed lamellar layer. Shells of Oliva bulbosa are composed of an irregular alternation of aragonitic crossed lamellar layers; a transitional layer characterised by the occurrence of tidally controlled growth lines, a crossed lamellar callus and a myostracal layer are also described in Oliva bulbosa specimens. With this analysis, we are able to provide novel microstructural and mineralogical data on three poorly known mollusc species, which are useful for crystallographic, phylogenetic, evolutionary and palaeoenvironmental studies.

polymorphs, calcite, aragonite, and vaterite, of which the former two are most commonly observed as biological polymorphs in marine mollusks. The pearl oyster P. fucata can generate these two polymorphs in vivo; calcite crystals in the outer prismatic layer and aragonite crystals in the inner nacreous layer of the shell, under the same physical conditions. These phenomena are a key to solving the enigma of bio-mineralization. The early research on shells of mollusks was mainly carried out by electron-microscopic observation [2–6]. Shells are mainly composed of two distinct layers. The outer prismatic layer shows the honeycomb-like network structure of a polygonal prism. The inner nacreous layer shows a cemented brick or veneer-like multilayer structure. Figure 1 shows a schematic representation of the inner nacreous layer. In this case, part of the brick corresponds to the aragonite crystal and part of the cement corresponds to the organic matrix. The organic matrix was first discovered in 1855 by Fremy [7], and is designated as “conchiolin”. Biochemical research on the organic matrix has recently developed, and now it has become clear that the organic matrix is mainly composed of proteins. Some parts of the organic matrix are soluble in water containing EDTA, and are called the water-soluble organic matrix (WSM). Most parts of the organic matrix are insoluble in water and are called the water-insoluble organic matrix (WISM). The group of Morse and colleagues [8–12] and that of Weiner and colleagues [13– 17] reported that shell proteins existing in the WSM mainly controlled the polymorphs of calcium carbonate crystals. In contrast, the early work of Watabe and Wilbur [18] and Wilbur and Watabe [19] and more recent reports by Wheeler [20] and by Samata et al. [21] showed that WISM proteins were mainly involved in polymorphic control. At present, it is difficult to determine which view is correct, because the groups with opposing views used different materials. The groups of Morse and colleagues [8–12] and Weiner and colleagues

Limpets of the family Nacellidae construct their shells with an outer prismatic layer, underlain by an acicular-foliated layer, and occasionally an internal crossed-lamellar layer. The outer layer consists of prisms oriented approximately perpendicular to the shell growth surface. At the transition to the acicular-foliated layer, these prisms subdivide into segments that become incorporated into the underlying folia. The folia are extensive, radially oriented, and further subdivided into transverse acicles, hence the term "acicular-foliated". Crystallographically, the outer prisms are remarkable in having their c-axes oriented at a high angle to their elongation and aligned toward the shell margin, with one a-axis typically perpendicular to the growth surface. The prisms exhibit a high degree of co-orientation, with textures ranging from diffuse to well-organized sheet textures. When considering rhombohedral face orientations, the texture may appear either single- or double-crystal-like. This crystallographic arrangement is inherited by the acicular-foliated layer, where the texture invariably becomes stronger. Such an unusual orientation likely results from oriented nucleation on an organic template, possibly the mantle surface, combined with crystal competition in the direction toward the shell margin. From both morphological and crystallographic perspectives, the prismatic-acicular-foliated microstructural complex of nacellids is unparalleled among molluscs.

Dart formation in Helix aspersa has been investigated by SEM of isolated darts at progressive stages in their development, and by histology of dart sacs at the same times. Dart formation begins at the tip of a tubercle where a small group of epithelial cells secrete an organic material filling a small CaCO3 cone that is the first mineralized part of the shaft. Subsequent secretory activity by an increasing area of the tubercle epithelium results in an increase in the diameter and anterior lengthening of the shaft. Continued secretion by the tubercle and dart sac epithelium produces the flare and finally the corona. A pattern of deposition is also evident in the fine structure of the mineral. In the shaft and vanes there is an inner layer of spherulitic prismatic structure which is covered by a layer of irregular patches of simple prismatic structure. The outermost layer of the shaft and vanes has a continuous simple prismatic structure. Two layers are present in the flare, an inner granular amorphous layer and an outer spherulitic prismatic layer. The corona consists of a single rarefied prismatic layer. A mechanism of dart formation is suggested that involves two types of organic matrix, calcifying and non-calcifying. Measurements of the calcium content of darts, dart sacs, and collars indicate that the hemolymph is the probable source of calcium for the dart.

Mussel Mytilus galloprovincialis (Lamarck, 1819) is widespread in the north-western part of the Black Sea, it is the active filter feeders sea water, is an important component of various zoocenoses. Mollusk is the object of fisheries and mariculture. Three phenotypes of mussel are separated on features of distribution of a violet pigment in outer prismatic shell layer: Fa – in a prismatic layer of purple pigment absent; Fb – pigment paints the whole of prismatic layer; Fc – pigment is localized in the form of radial strips alternating with unpigmented zones. The first two forms are interpreted homozygotes, last – heterozygotes.The purpose of the work is to study the distribution of mussels different phenotypic groups on the depths of the Odessa coast.The material for the work were the mussels collected in may 2012 at two stations – on the beach Langeron (depth 3,5 m) and in the area of hydrobiological station of Odessa National Mechnikov University (at a depth of 1 m breakwater; 6 m – fouling stones). Samples were collected with the frame area of 0,01 m2. Total collected and analyzed 459 exemplars of mussels, which were divided into the size groups with an interval of 10 mm.Index deficiency of heterozygotes (D) was calculated according to the equation:D = (Hо– Hс)/Нс ,Hо – the number of heterozygotes observed in the population; Hс – the number of heterozygotes expected by Hardy-Weinberg equation. A positive value means a deficit of heterozygotes D and negative – of their surplus.At a depth of 3,5 m represented only mussels of three size groups. About 52% of the total numbers of mollusks are size class of 20-29 mm, for other two classes remaining accounted to 24%.Among the small mollusks provided only at a depth of 6 m, the dominant were mussels of phenotype Fb (60%); half mussel of phenotype Fa (30%) and 10% – mussels of phenotype Fc. In large size class at a depth of 1 m dominated mussels of phenotype Fb – up to 100%.Mussels of phenotype Fa represent a smaller part of mollusks in almost all researched depths (1, 3,5, and 6 m), respectively, 23, 18 and 25% of the total number.At a depth of 1 m the number of mussels of phenotypes Fb and Fc is almost the same – 40 and 37%. At a depth of 3,5 m the number of these mussels of phenotypes differ slightly – 35 and 47%. Mussels of phenotype Fb, on the contrary, is dominated at a depth of 6 m, they accounted for more than half of the total number of animals – 55%. Analysis of the conformity of actual frequencies of phenotypes with theoretical data calculated by the relation of the Hardy-Weinberg equilibrium using the criterion c2 showed that at depths of 1 and 3,5 m obtained facts were not statistically significant.At a depth of 1,0 and 3,5 m observed a deficit of heterozygotes, and at the 6-meter depth – their surplus.

Mollusc shells are composite structures made of calcite and/or aragonite crystals and biopolymers, arranged in a great variety of microstructures. The formation of shell microstructures is affected by environmental and physiological factors and differences among microstructural types are believed to be of phylogenetic and adaptive biomechanical significance. Here, we characterise and illustrate for the first time, through SEM and XRD analyses, the shell microstructure and mineralogy of specimens of the bivalves Anadara uropigimelana (Bory de Saint-Vincent, 1827) and Tivela stefaninii (Nardini, 1933), and of the gastropod Oliva bulbosa (Röding, 1798), collected in the Upper Holocene HAS1 settlement and in a shell midden in the Khor Rori Archaeological Park (Oman). Anadara uropigimelana shows an aragonitic shell with an outer crossed lamellar layer, an inner complex crossed lamellar layer and an irregular simple prismatic pallial myostracum; periodic bands of dendritic nondenticular composite prisms occur in the outer part of the outer layer, reflecting seasonal changes in water temperatures and growth rates. Shells of Tivela stefaninii are aragonitic with an outer composite prismatic layer, a middle crossed lamellar layer and an inner complex crossed lamellar layer, whereas those of Oliva bulbosa are characterised by an irregular alternation of aragonitic crossed lamellar layers; a transitional layer defined by the occurrence of tidally controlled growth lines, a crossed lamellar callus and a myostracal layer are also described are also described in Oliva bulbosa. With this investigation, we provide novel microstructural and mineralogical data on these poorly known mollusc species, providing useful characters for phylogenetic, evolutionary, crystallographic, and palaeoenvironmental studies.

Growth performance of the Antarctic bivalve Laternula elliptica was examined both by shell microstructural observation and by applying a fluorescent substance, tetracycline, as a shell growth marker. The shell was composed of two calcareous layers: the thick outer layer was homogeneous or granular in structure and the thin inner layer was nacreous. The architecture of Antarctic L. elliptica was different from that of temperate L. marilina, and the ratio of thickness between the outer and inner layers appeared to be different. The growth rate of the nacreous layer was analyzed to be very low. High correlations were found between the major axis of chondrophore and both shell length and shell dry weight, respectively. It is suggested that the chondrophore is an appropriate growth indicator, and combining the information of growth increments with the fluorescent method may be useful in estimating the bivalve growth performance in the Antarctic sea.

Abstract. Marine bivalve mollusk shells can offer valuable insights into past oceanographic variability and seasonality. Given its ecological and archeological significance, Mytilus californianus (California mussel) presents the opportunity to examine seasonal and decadal changes recorded in its shell over centuries to millennia. While dark–light growth bands in M. californianus shells could be advantageous for reconstructing past environments, uncertainties remain regarding shell structure, environmental controls of dark–light-band formation, and the amount of time represented by a dark–light pair. By analyzing a suite of M. californianus shells collected in 2002, 2003, 2019, and 2020 from Bodega Bay, California, we describe the mineralogical composition; establish relationships among the growth band pattern, micro-environment, and collection season; and compare shell structure and growth band expression between the archival (2002–2003) and modern (2019–2020) shells. We identified three mineralogical layers in M. californianus: an outer prismatic calcite layer, a middle aragonite layer, and a secondary inner prismatic calcite layer, which makes M. californianus the only Mytilus species to precipitate a secondary calcite layer. Within the inner calcite layer, light bands are strongly correlated with winter collection months and could be used to reconstruct periods with moderate, stable temperatures and minimal upwelling. Additionally, modern shells have significantly thinner inner calcite layers and more poorly expressed growth bands than the archival shells, although we also show that growth band contrast is strongly influenced by the micro-environment. Mytilus californianus from northern California is calcifying differently, and apparently more slowly, than it was 20 years ago.

Molluscan shells are composed of calcium carbonates, with small amounts of extracellular matrices secreted from mantle epithelial cells. Many types of shell matrix proteins (SMPs) have been identified from molluscan shells or mantle cells. The pen shell Atrina pectinata (Pinnidae) has two different shell microstructures, the nacreous and prismatic layers. Nacreous and prismatic layer-specific matrix proteins have been reported in Pteriidae bivalves, but remain unclear in Pinnidae. We performed transcriptome analysis using the mantle cells of A. pectinata to screen the candidate transcripts involved in its prismatic layer formation. We found Asprich and nine highly conserved prismatic layer-specific SMPs encoding transcript in P. fucata, P. margaritifera, and P. maxima (Tyrosinase, Chitinase, EGF-like proteins, Fibronectin, valine-rich proteins, and prismatic uncharacterized shell protein 2 [PUSP2]) using molecular phylogenetic analysis or multiple alignment. We confirmed these genes were expressed in the epithelial cells of the mantle edge (outer surface of the outer fold) and the mantle pallium. Phylogenetic character mapping of these SMPs was used to infer a possible evolutionary scenario of them in Pteriomorphia. EGF-like proteins, Fibronectin, and valine-rich proteins encoding genes each evolved in the linage leading to four Pteriomorphia (Mytilidae, Pinnidae, Ostreidae, and Pteriidae), PUSP2 evolved in the linage leading to three Pteriomorphia families (Pinnidae, Ostreidae, and Pteriidae), and chitinase was independently evolved as SMPs in Mytilidae and in other Pteriomorphia (Pinnidae, Ostreidae, and Pteriidae). Our results provide a new dataset for A. pectinata SMP annotation, and a basis for understanding the evolution of prismatic layer formation in bivalves.

Drawing inspiration from the bionic nacre structure, graphene was incorporated into the epoxy solvent-borne adhesive to construct a laminated architecture. At the same time, ferrocene was employed as a catalyst to induce the in situ growth of carbon nanotubes (CNTs) under high-temperature conditions. This modification endowed the epoxy solvent-borne adhesive with not only high strength in atmospheric environments but also the capability to retain considerable mechanical performance at elevated temperatures. Experimental results demonstrated that when the graphene content in the epoxy solution fell within the range of 3.2–4%, the bonding strength exceeded 3 MPa within the temperature range of 1000–1300 °C. In particular, the adhesive exhibited excellent thermal shock resistance, with no degradation in strength observed after 15 thermal shock cycles at 1300 °C. Such exceptional performance was attributed to the formation of interlaminar CNTs generated after high-temperature treatment. Scanning electron microscopy (SEM) observations clearly revealed the laminated graphene sheets and in situ grown CNTs, confirming the feasibility of the strategy to enhance bonding efficacy by mimicking the nacre structure. This approach represented an innovative breakthrough for further research on the application of the “brick-and-mortar” structure in the bonding layer and the in situ growth of CNTs among lamellar graphene, while also providing detailed supporting data.

Tropical intertidal organisms tolerate large fluctuations in temperature and high desiccation rates when exposed during low tide. In order to withstand the short‐term heat stress, intertidal organisms adopt behavioral responses to maximize their survival. Our previous research showed that tropical littorinids found at the upper and lower intertidal shores in Singapore exhibited different behavioral adaptations during low tide. Most of the upper‐shore Echinolittorina malaccana kept a flat orientation, with the aperture against the substrate and the long axis of the shell towards the sun, whereas a majority of the lower‐shore individuals of Echinolittorina vidua stood with the edge of the aperture perpendicular to the substrate on the rocky shore during low tide. This prompted analyses of the shells of these two species to determine whether the differences in the shell morphometry, microstructure, and thermal conductivity of shells of E. malaccana and E. vidua were associated with their respective behavioral responses to thermal stress. Analyses of shell morphometry and thermal conductivity showed that shells of E. malaccana were more likely to minimize heat gain, despite having a higher thermal conductivity on the outer surface, due to their light‐gray, elongated shell. By contrast, the dark‐colored, globose shells of E. vidua probably gain heat more readily through solar radiation. Scanning electron microscopy images of the shells of both littorinid species further revealed that they have cross‐lamellar structure; however, only individuals of E. vidua showed the presence of disjointed rod layers and a pigmented inner shell surface. Individuals of E. malaccana had a rough outer shell surface with holes that inter‐connect to form water‐trapping channels that probably aid cooling. Individuals of E. vidua, however, had a smooth outer surface with rows of kidney‐shaped depressions as microsculptures which probably help to stabilize shell shape. In both Echinolittorina species, behavioral responses were used to overcome thermal stress during low tide that was associated with shell morphometry and shell thermal conductivity. Such combined adaptations increase survivability of the littorinids at their respective tidal levels.