Extrapallial Space Research Articles

Deciphering the molecular toolkit: regulatory elements governing shell biomineralization in marine molluscs.

The intricate process of shell biomineralization in marine molluscs is governed by a complex interplay of regulatory elements, encompassing secretomes, transporters, and noncoding RNA. This review delves into recent advancements in understanding these regulatory mechanisms, emphasizing their significance in elucidating the functions and evolutionary dynamics of the molluscan shell biomineralization process. Central to this intricate orchestration are secretomes with diverse functional domains, selectively exported to the extrapallial space, which directly regulate crystal growth and morphology. Transporters are crucial for substrate transportation in the calcification and maintenance of cellular homeostasis. Beyond proteins and transporters, noncoding RNA molecules are integral components influencing shell biomineralization. This review underscores the nonnegligible roles played by these genetic elements at the molecular level. To comprehend the complexity of biomineralization in mollusc, we explore the origin and evolutionary history of regulatory elements, primarily secretomes. While some elements have recently evolved, others are ancient genes that have been co-opted into the biomineralization toolkit. These elements undergo structural and functional evolution through rapidly evolving repetitive low-complexity domains and domain gain/loss/rearrangements, ultimately shaping a distinctive set of secretomes characterized by both conserved features and evolutionary innovations. This comprehensive review enhances our understanding of molluscan biomineralization at the molecular and genetic levels.

Skeletal microstructures of cheilostome bryozoans (phylum Bryozoa, class Gymnolaemata): crystallography and secretion patterns

Gymnolaemata bryozoans produce CaCO3 skeletons of either calcite, aragonite, or both. Despite extensive research, their crystallography and biomineralization patterns remain unclear. We present a detailed study of the microstructures, mineralogy, and crystallography of eight extant cheilostome species using scanning electron microscopy, electron backscatter diffraction, atomic force microscopy, and micro-computed tomography. We distinguished five basic microstructures, three calcitic (tabular, irregularly platy, and granular), and two aragonitic (granular-platy and fibrous). The calcitic microstructures consist of crystal aggregates that transition from tabular or irregularly platy to granular assemblies. Fibrous aragonite consists of fibers arranged into spherulites. In all cases, the crystallographic textures are axial, and stronger in aragonite than in calcite, with the c-axis as the fiber axis. We reconstruct the biomineralization sequence in the different species by considering the distribution and morphology of the growth fronts of crystals and the location of the secretory epithelium. In bimineralic species, calcite formation always predates aragonite formation. In interior compound walls, growth proceeds from the cuticle toward the zooecium interior. We conclude that, with the exception of tabular calcite, biomineralization is remote and occurs within a relatively wide extrapallial space, which is consistent with the inorganic-like appearance of the microstructures. This biomineralization mode is rare among invertebrates.

Parvatrema spp. (Digenea, Gymnophallidae) with parthenogenetic metacercariae: diversity, distribution and host specificity in the palaearctic

There are several species of gymnophallid digeneans in the genus Parvatrema that are unique in developing metacercariae that reproduce by parthenogenesis in the second intermediate host. Transmission of these digeneans takes place in coastal ecosystems of the North Pacific and North Atlantic seas. The first intermediate hosts are bivalves, the second ones are gastropods, and the definitive hosts are migratory birds. We integrated data accumulated over 25 years of research and differentiated a complex of five closely related species. They differ in the molluscan second intermediate hosts, distribution ranges, and life cycles patterns. The type I life cycle includes two generations of parthenogenetic metacercariae, followed by development of metacercariae which are invasive for the definitive host. In the type II life cycle, the number of generations of parthenogenetic metacercariae is unlimited, and they can also produce cercariae. These cercariae emerge into the environment and can infect new individuals of the second intermediate host. We conclude that the type I life cycle is a derived option that has evolved as a better fit to transmission in the unstable conditions in the intertidal zone. Another evolutionary trend in Parvatrema is transition from inhabiting the extrapallial space of the gastropod second intermediate host to endoparasitism in its mantle and internal organs. rDNA sequence analysis highlighted that Parvatrema spp. with parthenogenetic metacercariae form a monophyletic clade and suggested the Pacific origin of the group, with two transfers to the North Atlantic and colonisation of new second intermediate host species. Apparently the group formed in the late Pliocene-Pleistocene and diversified as a result of recurrent isolation in inshore refugia during glacial periods. We argue that parthenogenetic metacercariae in Parvatrema may serve as a model for early digenean evolution, demonstrating the first steps of adopting the molluscan first intermediate host and becoming tissue parasites.

Mantle tissue in the pearl oyster Pinctada fucata secretes immune components via vesicle transportation

Molluscan bivalves secrete shell matrices into the extrapallial space (EPS) to guide the precipitation of rigid shells. Meanwhile, immune components are present in the EPS and shell matrices, which are pivotal in resistant to invaded pathogens, thus ensuring the shell formation process. However, the origin of these components remains unclear. In this study, we revealed numerous vesicles were secreted from the outer mantle epithelial cells by using light and electron microscopes. The secreted vesicles were isolated by gradient centrifugation and confirmed by transmission electron microscopy. Proteomics analysis showed that the secreted vesicles were composed of cytoplasmic and immune components, most of which do not have signal peptides, indicating that they were secreted by a non-classical pathway. Moreover, real-time PCR revealed that some immune components were highly expressed in the mantle tissue, compared to the hemocytes. FTIR analysis verified the presence of lipids in the shell matrices, indicating that the vesicles have integrated into the shell layers. Taken together, our results suggested that mantle epithelial cells secreted some important immune components into the EPS via secreted vesicle transportation, thus cooperating with the hemocytes to play a vital role in immunity during shell formation.

A Bivalve Biomineralization Toolbox.

Mollusc shells are a result of the deposition of crystalline and amorphous calcite catalyzed by enzymes and shell matrix proteins (SMP). Developing a detailed understanding of bivalve mollusc biomineralization pathways is complicated not only by the multiplicity of shell forms and microstructures in this class, but also by the evolution of associated proteins by domain co-option and domain shuffling. In spite of this, a minimal biomineralization toolbox comprising proteins and protein domains critical for shell production across species has been identified. Using a matched pair design to reduce experimental noise from inter-individual variation, combined with damage-repair experiments and a database of biomineralization SMPs derived from published works, proteins were identified that are likely to be involved in shell calcification. Eighteen new, shared proteins likely to be involved in the processes related to the calcification of shells were identified by the analysis of genes expressed during repair in Crassostrea gigas, Mytilus edulis, and Pecten maximus. Genes involved in ion transport were also identified as potentially involved in calcification either via the maintenance of cell acid–base balance or transport of critical ions to the extrapallial space, the site of shell assembly. These data expand the number of candidate biomineralization proteins in bivalve molluscs for future functional studies and define a minimal functional protein domain set required to produce solid microstructures from soluble calcium carbonate. This is important for understanding molluscan shell evolution, the likely impacts of environmental change on biomineralization processes, materials science, and biomimicry research.

Shell-adductor muscle attachment and Ca2+ transport in the bivalves Ostrea stentina and Anomia ephippium

Among bivalve muscles, the adductors are particularly important for animal survival because they control valve closure. Most studies have addressed the type and morphology of this muscle in bivalves but few have focused on the mechanism that anchors it to the shell myostracum layer. Moreover, the possible calcium transport mechanism through the adductor muscle cells to the myostracum shell layer, which is necessary for bivalve biomineralisation, has never been addressed. Our results indicate that the muscle cell-shell attachment is mediated by the outer mantle epithelial cell layer, here termed tendon cells. These cells are modified at the muscle scar zone by the presence of actin cytoskeletal bundles, which anchor cells to the extracellular matrix via focal adhesion (or focal contact) junctions at the basal side and to extrapallial matrix at the apical side, both rich in collagen. From apical focal adhesions, bundles of collagen-rich fibres cross the extrapallial space and penetrate the myostracum shell layer. The latter constitutes one of the strongest anchoring structures among invertebrates. Numerous vesicles protrude from the tendon cells into the extrapallial space. TEM-EDX analysis reveals the presence of Ca2+ inside some of these vesicles both in tendon cells and in the extrapallial space. This suggests a potential mechanism for calcium transport from cells to the myostracum. Statement of SignificanceThe interfaces between bivalve shells and muscular attachments are unique and of special interest as adhesive functional biomaterials, being one of the strongest invertebrate anchoring structures. We present an updated ultrastructural model of the adductor muscle-shell attachment. Muscle cells connect with the shell through epithelial `tendon cells`, which have a cytoskeleton of actin microfilaments that connect to the extracellular matrix via focal adhesions. Collagen-rich fibres arise from apical focal adhesions, cross the nanometric extrapallial space and penetrate the myostracum where they form an organic network. Calcium is present inside vesicles that are released into the extrapallial space. The lack of direct cellular control on secretion restricts the myostracal microstructure to prismatic aragonitic similar to its inorganic counterpart.

Foamy oysters: vesicular microstructure production in the Gryphaeidae via emulsification.

The vesicular microstructure is a very distinctive arrangement of calcite, consisting of hollow cavities (vesicles) of diverse sizes and shapes, usually elongated in the direction of shell thickening. It is uniquely found among living bivalves in a single oyster family, Gryphaeidae. The vesicles are distributed in lenses interleaved with compact foliated layers. We have studied the morphology and distribution of vesicles within the lenses using optical and electron microscopy, and micro-computed tomography. At a small scale, vesicles do not follow a classical von Neumann-Mullins route typical of ideal foams. At a larger scale, the initiation and evolution of a vesicular layer statistically proceed like a foam, with vesicles becoming more numerous, larger and more even in size. In summary, the vesicular material follows a foam-like coarsening to reduce the number of energetically costly interfaces. However, a steady state is never reached because the animal permanently introduces energy in the system by creating new vesicles. The fabrication of the vesicular material is mediated by the production of an emulsion between the extrapallial fluid and the precursor PILP of the calcitic walls within the thin extrapallial space. For this mechanism to proceed, the mantle cells must perform highly sophisticated behaviours of contact recognition and secretion. Accordingly, the vesicular material is under mixed physical-biological control.

A potential zoonotic parasite, the digenean Gymnophalloides nacellae, on the Magellanic coast in the Southwestern Atlantic Ocean: its life cycle and geographical distribution

This is an integrative study of a potential zoonotic digenean from the Magellanic Southwestern Atlantic coast. The life cycle of the gymnophallid Gymnophalloides nacellae Cremonte, Pina, Gilardoni, Rodrigues, Chai and Ituarte, 2013 (Digenea) at the type locality, Puerto Deseado (47° 45′ S, 65° 51′ W), Santa Cruz province, was elucidated. This digenean uses the upper subtidal clam Gaimardia trapesina (Lamarck) (Gaimardiidae), which lives on the fronds of the giant kelp, as first intermediate host. A very short-stem furcocercous cercaria emerges and enters the limpets, Nacella magellanica (Gmelin) and N. deaurata (Gmelin) (Nacellidae), which live in the lower rocky intertidal zone. The unencysted metacercariae inhabit the extrapallial space of the limpet at high prevalences and intensities of infection. When the black oystercatcher Haematopus ater Vieillot and Oudart (Charadriidae) preys upon it, it becomes infected, acting as the definitive host. This parasite seems to exhibit a high specificity for their first and second intermediate hosts. Its geographical distribution is from 47 to 55° S in Patagonia, and it is restricted to those sites where the giant kelp reaches to lower intertidal and upper subtidal zones where the limpets are present. Gymnophalloides seoi Lee, Chai and Hong, 1993, a parasite of the Pacific oyster Magallana gigas (Thunberg) (Ostreidae), causes a zoonotic disease in Korea; thus, G. nacellae represent a risk of being a zoonotic parasite if infected limpets are consumed.

Terebratulide brachiopod shell biomineralization by mantle epithelial cells

To understand mineral transport pathways for shell secretion and to assess differences in cellular activity during mineralization, we imaged with TEM and FE-SEM ultrastructural characteristics of outer mantle epithelium (OME) cells. Imaging was carried out on Magellania venosa shells embedded/etched, chemically fixed/decalcified and high-pressure frozen/freeze-substituted samples from the commissure, central shell portions and from puncta. Imaging results are complemented with morphometric evaluations of volume fractions of membrane-bound organelles.At the commissure the OME consists of several layers of cells. These cells form oblique extensions that, in cross-section, are round below the primary layer and flat underneath fibres. At the commissure the OME is multi-cell layered, in central shell regions it is single-cell layered. When actively secreting shell carbonate extrapallial space is lacking, because OME cells are in direct contact with the calcite of the forming fibres. Upon termination of secretion, OME cells attach via apical hemidesmosomes to extracellular matrix membranes that line the proximal surface of fibres. At the commissure volume fractions for vesicles, mitochondria and lysosomes are higher relative to single-cell layered regions, whereas for endoplasmic-reticulum and Golgi apparatus there is no difference.FE-SEM, TEM imaging reveals the lack of extrapallial space between OME cells and developing fibres. In addition, there is no indication for an amorphous precursor within fibres when these are in active secretion mode. Accordingly, our results do not support transport of minerals by vesicles from cells to sites of mineralization, rather by transfer of carbonate ions via transport mechanisms associated with OME cell membranes.

Shell repair and the potential microbial causal in a shell disease of the pearl oyster Pinctada fucata

The pearl oyster Pinctada fucata is famous for producing luxurious pearls. As filter feeders, they are confronted with various infectious microorganisms. Despite a long history of aquaculture, diseases in P. fucata are not well studied, which limits the development of the pearl industry. We report here a shell disease in P. fucata and a study of the shell repair processes. Scanning electron microscopy (SEM) revealed that the nacreous layer gradually recovered from disordered CaCO3 deposition, accompanied by a polymorphic transition from a calcite-aragonite mixture to an aragonite-dominant composition, as revealed by X-ray diffraction analysis. SEM also showed that numerous microbes were embedded in the abnormal shell layers. Similar indications were induced by a high concentration of microbes injected into the extrapallial space, suggesting the potential pathogenic effect of uncontrolled microbes. Furthermore, hemocytes were found to participate in pathogens resistance and might promote shell repair. These results further our understanding of pathogen-host interactions in pearl oysters and have implications for biotic control in pearl aquaculture.

Physical and Biological Determinants of the Fabrication of Molluscan Shell Microstructures

Molluscs are grand masters in the fabrication of shells, because these are composed of the largest variety of microstructures found among invertebrates. Molluscan microstructures are highly-ordered aggregates of either calcite or aragonite crystals with varied morphologies and three-dimensional arrangements. Classically, every aspect of the fabrication of microstructural aggregates is attributed to the action of proteins. There was, however, only direct evidence that the mineral phase, and indirect evidence that nucleation and the crystal shape, are determined by the types of soluble proteins. Some authors imply that crystal competition may also play a role. In addition, the fabrication of intergranular organic matrices typical of some microstructures (nacre, columnar prismatic) cannot have a protein-based explanation. Over the last decade I and collaborators have been applying a holistic view, based on analyzing and interpreting the features of both the organic (mantle, extrapallial space, periostracum, organic matrices) and inorganic (crystallite morphology, arrangement and crystallography) components of the biomineralization system. By interpreting them on biophysical principles, we have accumulated evidence that, in addition to the activity of proteins, other mechanisms contribute in an essential way to the organization of molluscan microstructures. In particular, we have identified processes such as: (1) crystal nucleation on preformed membranes, (2) nucleation and growth of crystals between and within self-organized membranes, (3) active subcellular processes of contact recognition and deposition. In summary, besides the activity of organic macromolecules, physical (crystal competition, self-organization) and /or biological (direct cellular activity) processes may operate in the fabrication of microstructures. The balance between the physical and biological varies among microstructures, with some being based exclusively on either physical or biological processes, and others having a mixed nature. Other calcifying invertebrates (e.g. corals, cirripeds, serpulids) secrete microstructures that are very similar to inorganic crystal aggregates, and only some brachiopods and, to a lesser extent, bryozoans may have secretory abilities comparable to those of molluscs. Here I provide a new perspective, which may allow microstructures to be understood in terms of evolutionary constraints, to compare the secretional abilities among taxa, and even to evaluate the probability of mimicking microstructures for the production of functional synthetic materials.

Hemocytes in the extrapallial space of Pinctada fucata are involved in immunity and biomineralization

In bivalves, the mantle tissue secretes organic matrix and inorganic ions into the extrapallial space (EPS) to form the shells. In addition, more and more evidences indicate the participation of hemocytes in shell mineralization, but no direct evidence has been reported that verifies the presence of hemocytes in the EPS, and their exact roles in biomineralization remain uncertain. Here, we identified hemocytes from the EPS of Pinctada fucata. Numerous components involved in cellular and humoral immunity were identified by proteome analysis, together with several proteins involved in calcium metabolism. The hemocytes exerted active phagocytosis and significantly upregulated the expression of immune genes after immune stimulation. A group of granulocytes were found to contain numerous calcium-rich vesicles and crystals, which serve as a calcium pool. During shell regeneration, some genes involved in calcium metabolism are upregulated. Strikingly, most of the shell matrix proteins were absent in the hemocytes, suggesting that they might not be solely responsible for directing the growth of the shell. Taken together, our results provided comprehensive information about the function of hemocytes in immunity and shell formation.

Characterization of hemocytes from different body fluids of the eastern oyster Crassostrea virginica

Bivalve hemocytes are involved in a variety of physiological and immunological functions. Circulating hemocytes in the hemolymph represent the main component of the internal self-defense system while hemocytes present in the extrapallial space (between the mantle and the shell) are actively involved in biomineralization and shell formation. This study focused on the characterization of hemocytes from different body fluids of the eastern oyster Crassostrea virginica. Hemocytes present in the hemolymph were compared to those contained in the extrapallial fluid. Hemocytes associated with the mucus layer covering pallial organs (mantle, gills, body wall) were also investigated because of their potential role as sentinel cells. Hemocytes were characterized using flow cytometry in conjunction with fluorescent epitope markers (clusters of differentiation, lectins) as well as functional assays (i.e. phagocytosis and reactive oxygen species -ROS). Compared with the hemolymph, there was a significantly greater percentage of granulocytes and agranulocytes among extrapallial and pallial hemocytes, respectively. Accounting for the different percentages of hemocyte sub-populations, significant differences in surface carbohydrate and clusters of differentiation signatures were also revealed between the different fluids. Most informative epitope markers included concanavalin A, peanut agglutinin, soybean agglutinin, CD11b and CD14. Functional assays revealed significant differences in phagocytic activity and ROS production between hemocytes from the extrapallial fluid and hemolymph; however, less robust differences were observed between hemolymph cells and hemocytes associated with the pallial mucus. Findings from this study suggest that there are markedly different hemocyte populations in the three body fluids. The role of peripheral cells, particularly those associated with the pallial mucus, requires further investigations.

MSP22.8 is a protease inhibitor-like protein involved in shell mineralization in the edible mussel Mytilus galloprovincialis.

The mussel shell protein 22.8 (MSP22.8) is recognized by a monoclonal antibody (M22.8) directed against larvae of the mussel Mytilus galloprovincialis. After being secreted by cells of the mantle‐edge epithelium into the extrapallial (EP) space (the gap between the mantle and the shell), the protein is detected in the extrapallial fluid (EPF) and EP hemocytes and finally becomes part of the shell matrix framework in adult specimens of M. galloprovincialis. In the work described here, we show how MSP22.8 is detected in EPF samples from different species of mussels (M. galloprovincialis, Mytilus edulis, and Xenostrobus securis), and also as a shell matrix protein in M. galloprovincialis, Mytilus chilensis, and Perna canaliculus. A multistep purification strategy was employed to isolate the protein from the EPF, which was then analyzed by mass spectrometry in order to identify it. The results indicate that MSP22.8 is a serpin‐like protein that has great similarity with the protease inhibitor‐like protein‐B1, reported previously for Mytilus coruscus. We suggest that MSP22.8 is part of a system offering protection from proteolysis during biomineralization and is also part of the innate immune system in mussels.

Gastropod-derived haemocyte extracellular traps entrap metastrongyloid larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior

BackgroundPhagocyte-derived extracellular traps (ETs) were recently demonstrated mainly in vertebrate hosts as an important effector mechanism against invading parasites. In the present study we aimed to characterize gastropod-derived invertebrate extracellular phagocyte trap (InEPT) formation in response to larval stages of important canine and feline metastrongyloid lungworms. Gastropod haemocytes were isolated from the slug species Arion lusitanicus and Limax maximus, and the snail Achatina fulica, and exposed to larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior and investigated for gastropod-derived InEPT formation.ResultsPhase contrast as well as scanning electron microscopy (SEM) analyses of lungworm larvae-exposed haemocytes revealed ET-like structures to be extruded by haemocytes thereby contacting and ensnaring the parasites. Co-localization studies of haemocyte-derived extracellular DNA with histones and myeloperoxidase in larvae-entrapping structures confirmed classical characteristics of ETs. In vivo exposure of slugs to A. vasorum larvae resulted in InEPTs being extruded from haemocytes in the slug mucous extrapallial space emphasizing the pivotal role of this effector mechanism against invasive larvae. Functional larval entrapment assays demonstrated that almost half of the haemocyte-exposed larvae were contacted or even immobilized by released InEPTs. Overall, as reported for mammalian-derived ETs, different types of InEPTs were here observed, i.e. aggregated, spread and diffused InEPTs.ConclusionsTo our knowledge, this study represents the first report on metastrongyloid lungworm-triggered ETosis in gastropods thereby providing evidence of early mollusc host innate immune reactions against invading larvae. These findings will contribute to the better understanding on complex parasite-intermediate host interactions since different gastropod species bear different transmitting capacities for metastrongyloid infections.

Characterization of a Monoclonal Antibody Directed against Mytilus spp Larvae Reveals an Antigen Involved in Shell Biomineralization.

The M22.8 monoclonal antibody (mAb) developed against an antigen expressed at the mussel larval and postlarval stages of Mytilus galloprovincialis was studied on adult samples. Antigenic characterization by Western blot showed that the antigen MSP22.8 has a restricted distribution that includes mantle edge tissue, extrapallial fluid, extrapallial fluid hemocytes, and the shell organic matrix of adult samples. Other tissues such as central mantle, gonadal tissue, digestive gland, labial palps, foot, and byssal retractor muscle did not express the antigen. Immunohistochemistry assays identified MSP22.8 in cells located in the outer fold epithelium of the mantle edge up to the pallial line. Flow cytometry analysis showed that hemocytes from the extrapallial fluid also contain the antigen intracellularly. Furthermore, hemocytes from hemolymph have the ability to internalize the antigen when exposed to a cell-free extrapallial fluid solution. Our findings indicate that hemocytes could play an important role in the biomineralization process and, as a consequence, they have been included in a model of shell formation. This is the first report concerning a protein secreted by the mantle edge into the extrapallial space and how it becomes part of the shell matrix framework in M. galloprovincialis mussels.

Transcriptional changes in Manila clam (Ruditapes philippinarum) in response to Brown Ring Disease

Brown Ring Disease (BRD) is a bacterial infection affecting the economically-important clam Ruditapes philippinarum. The disease is caused by a bacterium, Vibrio tapetis, that colonizes the edge of the mantle, altering the biomineralization process and normal shell growth. Altered organic shell matrices accumulate on the inner face of the shell leading to the formation of the typical brown ring in the extrapallial space (between the mantle and the shell). Even though structural and functional changes have been described in solid (mantle) and fluid (hemolymph and extrapallial fluids) tissues from infected clams, the underlying molecular alterations and responses remain largely unknown. This study was designed to gather information on clam molecular responses to the disease and to compare focal responses at the site of the infection (mantle and extrapallial fluid) with systemic (hemolymph) responses. To do so, we designed and produced a Manila clam expression oligoarray (15K Agilent) using transcriptomic data available in public databases and used this platform to comparatively assess transcriptomic changes in mantle, hemolymph and extrapallial fluid of infected clams. Results showed significant regulation in diseased clams of molecules involved in pathogen recognition (e.g. lectins, C1q domain-containing proteins) and killing (defensin), apoptosis regulation (death-associated protein, bcl-2) and in biomineralization (shell matrix proteins, perlucin, galaxin, chitin- and calcium-binding proteins). While most changes in response to the disease were tissue-specific, systemic alterations included co-regulation in all 3 tested tissues of molecules involved in microbe recognition and killing (complement-related factors, defensin). These results provide a first glance at molecular alterations and responses caused by BRD and identify targets for future functional investigations.

Assessment of the mechanism of elemental incorporation into bivalve shells (Arctica islandica) based on elemental distribution at the microstructural scale

To address the mechanism of elemental incorporation into shells of the bivalve Arctica islandica, we performed two-dimensional elemental mapping of Sr, Mg, and S at a ∼3μm spatial resolution using electron microprobe. These elemental distributions were precisely compared to the shell microstructure. The Sr distribution was intimately linked to the S concentration and/or shell microstructure. Sr showed a clear annual pattern with higher values at the annual growth lines and lower values in annual growth increments. The Sr and S concentrations were higher near sub-annual growth lines than in the adjacent regions, which were dominated by acicular microstructure (middle shell layer) or homogeneous microstructure (outer shell layer). Furthermore, the Sr concentration was lower in the middle shell layer than in the outer shell layer, but there was no clear difference in the S concentration between the two layers. The observed elemental distribution in the shell can be reasonably explained by the hypothesis that the organic composition at the calcification site, i.e. in the extrapallial space, can facilitate the preferential elemental incorporation into calcium carbonate. The Sr/Ca ratio shows micrometer scale variation within the shell, suggesting that fine spatial resolution, comparable to the shell microstructure, is required for the analysis of elemental distribution to avoid time-averaging and to correctly evaluate the magnitude of the compositional variation in the shell for past environmental reconstruction.

Interacting Constraints and the Problem of Similarity in Gastropod Structure and Function*

Gastropods have served as research subjects for investigating questions that transcend both the taxon and the field of malacology. A major set of questions arises from repeated patterns in the microstructure, sculptural features, and coiling geometry of gastropod shells. The problem of similarity is one of distinguishing among alternative explanations of repetition and close resemblances. What explains the repetition of structural features? Are features similar as a consequence of shared ancestry (homology) or as a consequence of evolutionary convergence on good functional solutions (analogy)? There is increasing evidence for a third alternative that lies outside the evolutionary sphere, invoking the laws of physics and chemistry, building materials, and the assembly rules that apply strongly to the inorganic calcium carbonate of the gastropod shell. Three concepts are introduced to help organize the analysis of recurring similarities in the shells of marine gastropods. They are (1) remote biomineralization, (2) heterotectonic construction and ontogenetic reorganizations of shell secretion, and (3) structural funicity of the shell. Remote biomineralization produces organized structures without influence of the mantle, extrapallial space, and periostracum. Reticulate meshworks of aragonite on the exterior surfaces of larval shells are an example. Heterotectonic construction recognizes repeated instances of change in coiling geometry of the larval shell (protoconch) and the adult shell (teleoconch). Heterostrophy is an example of heterotectonic construction that is specific to the clade Heterobranchia; but there are many constructional changes that recur in other groups that are not closely-related. Examples include the set of changes that thicken larval shells (imbricate thickening) during the metamorphic reorganization of shell secretion and transition to secretion of new calcium carbonate microstructures. Funicity is a property of shell that results in recurring features that record ontogenetic history and ecological events. Funeous details include growth increments as records of periodic environmental rhythms, isotopic records of climate, and records of interaction with other organisms, including non-lethal damage inflicted by predators and modifications of shells by symbionts. Shells also record details of their post-mortem history, including a rich funeous record in deep time on and in shells in the fossil record.

Tissue reaction of Tagelus plebeius (Bivalvia: Psammobiidae) against larval digeneans in mixohaline habitats connected to the south-western Atlantic

This paper describes the organism–organism relationship between a bivalve host and larval digeneans. The studied population of the stout razor clam Tagelus plebeius from the mixohaline Mar Chiquita lagoon (37° 32′S 57° 19′W) showed 100% of prevalence of infection by a larval digenean (metacercaria) of the family Gymnophallidae. The larvae occupied the extrapallial space just below the hinge of the bivalve. The tissue reaction against larval digeneans by T. plebeius consisted of hyperplasia and metaplasia of the outer layer of the mantle epithelium adjacent to parasites, forming an invagination to generate a sac that surrounded gymnophallid larvae. According to the intensity of infection, each sac progressively enlarged to harbour a variable number of larvae. In highly infected clams, a great number of sacs are formed. Eventually, the older sacs containing larvae may become closed, losing their communication with the extrapallial space and sinking into the dorsal part of the visceral mass. Larvae within sacs grow and remain alive until they reach an appropriate definitive bird vertebrate host. Older lesions commonly showed remains of dead larvae which had undergone resorption. As a result, an orange to brownish amorphous material accumulated in the space once occupied by the larvae leaving conspicuous orange marks on the inner surface of the valves. In some cases, calcifications in the form of pearls or blisters were observed.