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Somatic cells in the gonadal area of male and female deep-sea clams, Calyptogena pacifica, were examined using light and transmission electron microscopy. Acini both at the pre-spawning stage and at the stage of active spermatogenesis were observed to be simultaneously present in sections through a male gonad. Oocytes of various degrees of maturity were simultaneously present in female acini. No storage tissue and cells similar to adipogranular cells or vesicular connective tissue of other mollusks were found in the gonadal area of C. pacifica. Instead, in both males and females, numerous hemocytes surround the acini. Among hemocytes, two types of granulocytes and erythrocytes were identified. Bundles of muscle cells were also found in the interacinar space. Male intraacinar accessory cells were rather large, glycogen-rich, with lipid inclusions, and phagosomes contained spermatogenic cells. Female accessory cells had well-developed endoplasmic reticulum, but they did not form any follicles around oocytes, being in their basal part, closer to basal lamina. Such a specific pattern of gonad organization can presumably be explained by both symbiosis with sulfide-oxidizing bacteria and phylogenetic aspects that should be further studied. Some evidence for continuous gametogenesis are discussed., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In the present study, we have investigated the ultrastructures of the mature gonadal spermatozoa of R. variegata and T. literatus and presented comparisons with the Manila clam, R. philippinarum, sperm ultrastructure examined. Spermatozoa of R. variegata consist of (in anterior to posterior sequence): an elongate conical, deeply invaginated, acrosomal vesicle (length 1.58 ± 0.06 μm; width 0.99 ± 0.07 μm; invagination occupied by a granular subacrosomal material); a barrel-shaped nucleus (length 1.82 ± 0.06 μm; width 1.50 ± 0.03 μm); a midpiece consisting of two orthogonally arranged centrioles, surrounded by four spherical mitochondria; nine satellite fibers connecting the distal centriole to the plasma membrane; and a flagellum originating from the distal centriole. Contents of the acrosomal vesicle of R. variegata are differentiated into a very electron-dense basal ring and a less electron-dense zone (with seven dense transverse layers structure) on the anterior region of the acrosome. Spermatozoa of T. literatus differ from those of R. variegata and are characterized by a rounded-conical invaginated, acrosomal vesicle (length 0.88 ± 0.08 μm; width 0.77 ± 0.06 μm), with a basal ring; and an anteriorly-tapered, barrel-shaped nucleus (length 1.57 ± 0.04 μm; width 1.60 ± 0.09 μm); a midpiece composed of four mitochondria. Centriolar and flagellar details are essential as for R. variegata. Sperm morphology separating R. variegate, R. philippinarum, and T. literatus in different clades. The anterior region of the acrosomal vesicle in R. variegata sperm had the transverse bands structure whereas the apex of the acrosomal vesicle of T. literatus sperm had no such structure. This difference advocated that acrosomal feature could be an important character for taxonomic distinction. Our data supported the previous studies that the ultrastructure of bivalve sperm is species-specific. This advocates that the phyletic relationships of Tapetinae, commonly based on shell morphology, should also add additional and newer approaches., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The process of spermatogenesis and spermatozoon morphology was characterized from a deep-sea bivalve, Calyptogena pacifica (Vesicomyidae, Pliocardiinae), a member of the superfamily Glossoidea, using light and electron microscopy. Spermatogenesis in C. pacifica is generally similar to that in shallow-water bivalves but, the development of spermatogenic cells in this species has also some distinguishing features. First proacrosomal vesicles are observed in early spermatocytes I. Although, early appearance of proacrosomal vesicles is well known for bivalves, in C. pacifica, these vesicles are associated with electron-dense material, which is located outside the limiting membrane of the proacrosomal vesicles and disappears in late spermatids. Another feature of spermatogenesis in C. pacifica is the localization of the axoneme and flagellum development. Early spermatogenic cells lack typical flagellum, while in spermatogonia, spermatocytes, and early spermatids, the axoneme is observed in the cytoplasm. In late spermatids, the axoneme is located along the nucleus, and the flagellum is oriented anteriorly. During sperm maturation, the bent flagellum is transformed into the typical posteriorly oriented tail. Spermatozoa of C. pacifica are of ect-aqua sperm type with a bullet-like head of about 5.8 μm in length and 1.8 μm in width, consisting of a well-developed dome-shaped acrosomal complex, an elongated barrel-shaped nucleus filled with granular chromatin, and a midpiece with mainly four rounded mitochondria. A comparative analysis has shown a number of common traits in C. pacifica and Neotrapezium sublaevigatum., (© 2020 Wiley Periodicals LLC.)

Spermatozoan ultrastructure and complete mitochondrial genome in the marine bivalve mollusk Meretrix sp. (Taiwan) from Taiwan are described and contrasted with other bivalves, especially within Meretrix. We have examined the features of the mature gonadal spermatozoa of Meretrix sp. (Taiwan) and provided comparisons with the other four Meretrix species (M. petechialis, M. meretrix, M. lyrata, and M. lamarckii). The morphological characteristics of these spermatozoa are diagnostic for each of the species studied here. The most marked interspecific difference was found in the acrosome. Meretrix sp. (Taiwan) is genetically distinct and is a different species from M. petechialis and M. lusoria (Japan) based on complete mitochondrial genome data. Sperm data for Meretrix are limited but show remarkable congruence with the molecular results. We suggest use Meretrix formosa Gwo and Hsu as the scientific name for Taiwanese hard clams, Meretrix sp. (Taiwan). Additional species, particularly the Japanese hard clam (M. lusoria) require examination before this tentative conclusion can be verified., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Freshwater mussels are a species-rich group with biodiversity patterns strongly shaped by a life history strategy that includes an obligate parasitic larval stage. In this study, we set out to reconstruct the life history evolution and systematics in a clade of freshwater mussels adapted to parasitizing a molluscivorous host fish. Anchored hybrid enrichment and ancestral character reconstruction revealed a complex pattern of life history evolution with host switching and multiple instances of convergence, including reduction in size of larvae, increased fecundity, and growth during encapsulation. Our phylogenomic analyses also recovered non-monophyly of taxa exhibiting multiple traits used as the basis for previous taxonomic hypotheses. Taxa with axe-head shaped glochidia were resolved as paraphyletic, but our results strongly suggest the complex morphology is an adaptation to reduce larval size, with reduction in size further accentuated in taxa previously assigned to Leptodea. To more accurately reflect the evolutionary history of this group, we make multiple systematic changes, including the description of a new genus, Atlanticoncha gen. nov., and the synonymy of the genus Leptodea under Potamilus. Our findings contribute to the growing body of literature showing that cladistic hypotheses based solely on morphological characters, including larval morphology, can be flawed in freshwater mussels., (© The Willi Hennig Society 2020.)

Digitaria digitaria, a small astartid usually less than 10 mm in length, has a non-brooding behaviour in spite of its limited space for gonad development. This species lives in highly unstable environments with strong currents, which represent a challenge for fertilization and larval settlement. The studied population of D. digitaria from the Strait of Gibraltar area was dioecious, with significant predominance of females and sexual dimorphism, where females are larger than males. The reproductive cycle is asynchronous throughout the year, without a resting period, but with successive partial spawning events. The presence of stored sperm in the suprabranchial chamber and inside the gonad of some females, together with the release of eggs along the dorsal axis of both gills, points to internal oocyte fertilization. Bacteriocytes were found in the female and male follicle walls, but no bacteria were observed inside any of the gametes. Digitaria digitaria could represent a "missing link" between spermcast mating bivalves with brooded offspring and bivalves with broadcast release of eggs and sperm. The small size, limiting the oocyte production, together with the unstable environment could represent evolutionary pressures towards sperm uptake in D. digitaria.

Tentacles are remarkable anatomical structures in invertebrates for their diversity of form and function. In bivalves, tentacular organs are commonly associated with protective, secretory, and sensory roles. However, anatomical details are available for only a few species, rendering the diversity and evolution of bivalve tentacles still obscure. In Pteriomorphia, a clade including oysters, scallops, pearl oysters, and relatives, tentacles are abundant and diverse. We investigated tentacle anatomy in the group to understand variation, infer functions, and investigate patterns in tentacle diversity. Six species from four pteriomorphian families (Ostreidae, Pinnidae, Pteriidae, and Spondylidae) were collected and thoroughly investigated with integrative microscopy techniques, including histology, scanning electron microscopy, and confocal microscopy. Tentacles can be classified as middle fold tentacles (MFT) and inner fold tentacles (IFT) according to their position with respect to the folds of the mantle margin. While MFT morphology indicates intense secretion of mucosubstances, no evidence for secretory activity was found for IFT. However, both tentacle types have appropriate ciliary distribution and length to promote mucus transportation for cleaning and lubrication. Protective and sensory functions are discussed based on different lines of evidence, including secretion, cilia distribution, musculature, and innervation. Our results support the homology of MFT and IFT only for Pterioidea and Ostreoidea, considering their morphology, the presence of ciliated receptors at the tips, and branched innervation pattern. This is in accordance with recent phylogenetic hypotheses that support the close relationship between these superfamilies. In contrast, major structural differences indicate that MFT and IFT are probably not homologous across all pteriomorphians. By applying integrative microscopy, we were able to reveal anatomical elements that are essential for the understanding of homology and function when dealing with such superficially similar structures., (© 2019 Wiley Periodicals, Inc.)

Spermatogenesis in the freshwater pearl mussel Margaritifera laevis was investigated using light and electron microscopy. The testes of M. laevis are composed of numerous acini. We observed type A spermatogonia, large cells of irregular shape, solely near the acinus basal lamina. Type A spermatogonia proliferate and become type B spermatogonia, which are also irregular in shape and form clusters of germ cells of the same developmental stage. The numerous clusters differ with respect to developmental stage and are arranged randomly along the acinus periphery. The central region of the acinus was observed to contain only mature spermatozoa. This germ cell arrangement contrasts that of other bivalvians and may be characteristic of Margaritiferidae and Unionidae. We noted that each germ cell cluster is entirely covered throughout spermatogenesis by Sertoli cells that are loosely bound together. This report is the first to describe the involvement of Sertoli cells in Unionoidea spermatogenesis. Mature spermatozoa of M. laevis are of the primitive sperm type, having a cylindrical head with a discoidal acrosome and a midpiece with five spherical mitochondria., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Fine structural features of Mizuhopecten yessoensis in different embryonic and larval developmental stages from oocyte to early veliger have been studied. Spermatozoa have a conical head, consisting of a short acrosome, nucleus, and 4 mitochondria in the middle piece. The flagellum is approximately 40 μm in length. Oocyte cytoplasm contains numerous yolk granules, which are gradually expended during the larval development and disappear at the early veliger stage. Gastrula has two invaginations: shell gland anlage on the dorsal side and ventral side archenteron. Prototroch of a trochophore consists of two ciliary rings. Telotroch is absent. Myoblasts containing bundles of myofilaments are found in the blastocoel of trochophore. Velum and shell begin forming at the late trochophore stage. Cells of gut epithelium form numerous microvilli, organized into a brush border structure on the surface of enterocytes. A suggestion is made that larvae at the late trochophore stage are capable of digesting food and feeding. The structure of a M. yessoensis veliger is typical for pectinids. The velar retractor muscles have cross striation with a period of 1 μm. The digestive system of an 8-day-old veliger consists of the esophagus, stomach (with subdivided gastric shield and style sac), and short intestine. The digestive gland (with no discernible loops at this stage) is a part of the stomach epithelium with two types of digestive enzyme-producing cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The effects of high hydrostatic pressure (HHP) treatments (200, 300, and 400 MPa for 1, 3, 5 and 10 min) on the shelling efficacy (the rate of shelling, the rate of integrity and yield of razor clam meat) and the physicochemical (drip loss, water-holding capacity, pH, conductivity, lipid oxidation, Ca 2+ -ATPase activity, myofibrillar protein content), microbiological (total viable counts) and microstructural properties of fresh razor clam (Sinonovacula constricta) were investigated. HHP treatments significantly (P < 0.05) increased shelling efficiency, water-holding capacity, pH, conductivity, and lipid oxidation, and HHP-treated razor clam showed lower levels of microorganisms and drip loss than untreated razor clam. Levels of thiobarbituric acid reacting substances (TBA) in HHP-treated razor clam were greatly increased (up to 0.93 ± 0.09 mg MDA/kg at 400 MPa for 10 min) which was caused by the formation of hydroperoxides during HHP treatment. All HHP treatments were found to have adverse effects on the activity of Ca 2+ -ATPase and the content of myofibrillar protein (MP), which might be due to the substantial damage to the tertiary structure of proteins at high pressure. Moreover, scanning electron microscopy (SEM) revealed the compaction of the muscle fibers and a decrease in the extracellular space with increasing pressure and holding time. This phenomenon was mainly correlated with the compaction of muscle fibers and denaturation, aggregation, and gelation of muscle protein triggered by high pressure. In general, HHP could be applied as a safe and effective nonthermal technology to produce high-quality shelled razor clam., Practical Application: High hydrostatic pressure (HHP) is now well known as a nonthermal processing technology and becoming increasingly acknowledged. However, it has not been widely applied to shell seafood due to its uncertain influence on its quality and shelling property. This study could provide valuable information regarding the shelling efficacy, physicochemical properties, and microstructure of razor clam treated by HHP. And it demonstrated that HHP showed a positive impact on quality of razor clam treated by HHP., (© 2018 Institute of Food Technologists®.)

The global decline of freshwater mussels can be partially attributed to their complex life cycle. Their survival from glochidium to adulthood is like a long obstacle race, with juvenile mortality as a key critical point. Mass mortality shortly after entering into a juvenile state has been reported in both wild and captive populations, thus weakening the effective bivalve population. A similar phenomenon occurs during metamorphosis in natural and hatchery populations of juvenile marine bivalves. Based on a morphological analysis using scanning electron microscopy of newly formed juveniles of the freshwater species Margaritifera margaritifera (L.) (Margaritiferidae) and Unio mancus Lamarck (Unionidae), we show that a second metamorphosis, consisting of drastic morphological changes, occurs that leads to suspension feeding in place of deposit feeding by the ciliated foot. We hypothesize that suspension feeding in these two species improves due to a gradual development of several morphological features including the contact between cilia of the inner gill posterior filaments, the inner gill reflection, the appearance of the ctenidial ventral groove and the formation of the pedal palps. Regardless of the presence of available food, a suspension feeding mode replaces deposit feeding, and juveniles unable to successfully transition morphologically or adapt to the feeding changes likely perish., (© 2017 Wiley Periodicals, Inc.)

The byssus of Pinna nobilis, the largest bivalve mollusc in the Mediterranean Sea, was investigated by histochemistry, immunohistochemistry, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). At low magnification, the byssus threads appeared distinctively elliptical in cross-section, with a typical size approaching 50 x 25 micron and a featureless glassy appearance. Histochemical and immunohistochemical techniques confirmed the presence of elastic domains but the absence of collagen, which is known to be the main component in other molluscs. Ultrastructural analysis by TEM revealed the presence of at least two components within the thread, and an inner arrangement of straight, tightly packed longitudinal streaks. SEM observations while confirming the inner packing of straight, parallel subfibrils, suggested in the fracture surfaces the presence of unidentified substance which cemented together the same subfibrils and which was removed by exposure to extreme pH values. AFM micrographs added further evidence for the tight packing of subfibrils and provided some evidence of orthogonal, barely visible connecting structures. Finally, HCl or NaOH treatment left the subfibrils clean and free from any other component.Â.

Environmental stressors impact marine larval growth rates, quality and sizes. Larvae of the Antarctic bivalve, Laternula elliptica, were raised to the D-larvae stage under temperature and pH conditions representing ambient and end of century projections (-1.6°C to +0.4°C and pH 7.98 to 7.65). Previous observations using light microscopy suggested pH had no influence on larval abnormalities in this species. Detailed analysis of the shell using SEM showed that reduced pH is in fact a major stressor during development for this species, producing D-larvae with abnormal shapes, deformed shell edges and irregular hinges, cracked shell surfaces and even uncalcified larvae. Additionally, reduced pH increased pitting and cracking on shell surfaces. Thus, apparently normal larvae may be compromised at the ultrastructural level and these larvae would be in poor condition at settlement, reducing juvenile recruitment and overall survival. Elevated temperatures increased prodissoconch II sizes. However, the overall impacts on larval shell quality and integrity with concurrent ocean acidification would likely overshadow any beneficial results from warmer temperatures, limiting populations of this prevalent Antarctic species.

Several bivalve species produce byssus threads to provide attachment to substrates, with mechanical properties highly variable among species. Here, we examined the distal section of byssal threads produced by a range of bivalve species ( Mytilus edulis , Mytilus trossulus , Mytilus galloprovincialis , Mytilus californianus , Pinna nobilis , Perna perna , Xenostrobus securis , Brachidontes solisianus and Isognomon bicolor ) collected from different nearshore environments. Morphological and mechanical properties were measured, and biochemical analyses were performed. Multivariate redundancy analyses on mechanical properties revealed that byssal threads of M. californianus , M. galloprovincialis and P. nobilis have very distinct mechanical behaviours compared with the remaining species. Extensibility, strength and force were the main variables separating these species groups, which were highest for M. californianus and lowest for P. nobilis Furthermore, the analysis of the amino acid composition revealed that I. bicolor and P. nobilis threads are significantly different from the other species, suggesting a different underlying structural strategy. Determination of metal contents showed that the individual concentration of inorganic elements varies, but that the dominant elements are conserved between species. Altogether, this bivalve species comparison suggests some molecular bases for the biomechanical characteristics of byssal fibres that may reflect phylogenetic limitations., (© 2017. Published by The Company of Biologists Ltd.)

Multiple groups of bivalve molluscs produce calcitic shell layers, many of these broadly classified as "prismatic." Various pteriomorphian bivalves (such as oysters, pterioids, and mussels) secrete prismatic microstructures with high organic content and clear, strong biological control. However, we present the results of a detailed analysis by scanning electron microscopy (SEM), thermogravimetric analysis, and electron backscatter diffraction to characterize the calcitic prisms in two different clades within the euheterodont bivalves: the extant Chama arcana and the extinct rudists. These results show that the form of prisms constructed is both closely similar between the two taxa and significantly different from those of the pteriomorph bivalves. Most notably, C. arcana and the extinct rudists lack the clear organic outer envelopes and uniform polygonal, cross-sectional appearance. Instead, they form interdigitating crystals of very varied diameters, with some crystals encapsulating others. We advocate retaining the term "fibrillar prisms" to classify these euheterodont microstructures. These fibrillar prisms are more closely similar to abiotic speleothem deposits than to the calcitic prisms of pteriomorph bivalves. We argue that calcite prism growth in euheterodonts is dominated by abiotic constraints whereas, in pteriomorphs (such as oysters, pterioids, and mussels), it is under strong biological control.

Unlabelled: We show with laboratory experiments that self-assembled mineral tube formation involving precipitation around a templating jet of fluid - a mechanism well-known in the physical sciences from the tubular growth of so-called chemical gardens - functions with carbonates, and we analyse the microstructures and compositions of the precipitates. We propose that there should exist biological examples of fluid-flow-templated tubes formed from carbonates. We present observational and theoretical modelling evidence that the complex structure of biomineral calcium carbonate tubules that forms the 'rose' of the watering-pot shells, Clavagelloidea, may be an instance of this mechanism in biomineralization. We suggest that this is an example of self-organization and self-assembly processes in biomineralization, and that such a mechanism is of interest for the production of tubes as a synthetic biomaterial., Statement of Significance: The work discussed in the manuscript concerns the self-assembly of calcium carbonate micro-tubes and nano-tubes under conditions of fluid flow together with chemical reaction. We present the results of laboratory experiments on tube self-assembly together with theoretical calculations. We show how nature may already be making use of this process in molluscan biomineralization of the so-called watering-pot shells, and we propose that we may be able to take advantage of the formation mechanism to produce synthetic biocompatible micro- and nano-tubes., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The fan mussel Pinna nobilis (Linnaeus, 1758) is one of the biggest bivalves worldwide. Currently, no updated information is available in the literature concerning the morpho-functional aspects of haemocytes from this bivalve species. Consequently, in this study, we characterised P. nobilis haemocytes from both a morphological and functional point of view. The mean number of haemocytes was about 5 (×10(5)) cells mL haemolymph(-1), and the cell viability was about 92-100%. Two haemocyte types were distinguished under the light microscope: granulocytes (51.6%), with evident cytoplasmic granules, and hyalinocytes (48.4%), with a few granules. The granules of the granulocytes were mainly lysosomes, as indicated by the in vivo staining with Neutral Red. Haemocytes were further distinguished in basophils (83.75%), acidophils (14.75%) and neutrophils (1.5%). After adhesion to slides and fixation, the cell diameter was approximately 10 μm for granulocytes and 7 μm for hyalinocytes. The granulocytes and hyalinocytes were both positive to the Periodic Acid-Schiff reaction for carbohydrates. Only granulocytes were able to phagocytise yeast cells. The phagocytic index (6%) increased significantly up to twofold after preincubation of yeast in cell-free haemolymph, suggesting that haemolymph has opsonising properties. In addition, haemocytes produce superoxide anion and acid and alkaline phosphatases. Summarising, this preliminary study indicates that both the granulocytes and hyalinocytes circulate in the haemolymph of P. nobilis and that they are active immunocytes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Isognomon spathulatus (Reeve, 1858) is redescribed based on type material and original collections from Kungkrabaen Bay, Thailand. The species agrees with previously described isognomonids in most conchological and anatomical features, but possesses a suite of diagnostic characters, including a comma-shaped outline of the nacreous border, an uncoiled ventral diverticulum of the stomach, and the thickened mantle lobes with granulated cells. This study is the most comprehensive morphological analysis to date for any species of Isognomonidae Woodring, 1925 (1828). It describes and illustrates a number of previously unrecognized or underutilized anatomical characters of potential phylogenetic significance: the morphology of the byssal threads (cross-sectional shape, plumate rootlets, and the shape of adhesive disks), the presence and extent of the interdemibranchial buttresses, the presence of secretory cells in the central zone of the mantle, the shape of the ventral diverticulum of the gastric chamber, the presence of the typhlosolar guard ridge, and the position of the renal pore. A comparison is made between I. spathulatus and morphologically similar Isognomon ephippium (Linnaeus, 1758) with which it has been previously synonymized. Pearls of both species are described and illustrated. Individuals of I. spathulatus inhabit mangroves, where they attach by byssus to prop roots, typically in parapatry with individuals of I. ephippium that occupy adjacent mudflats. The spacial distribution and diverging adaptive strategies (pertaining to physical stabilization and response to predation) displayed by the two isognomonid species are considered in the light of the ecological speciation theory.

The Atlantic jackknife clam, Ensis directus, is currently being researched as a potential species for aquaculture operations in Maine. The goal of this study was to describe the hemocytes of this species for the first time and provide a morphological classification scheme. We viewed hemocytes under light microscopy (using Hemacolor, neutral red, and Pappenheim's stains) as well as transmission electron microscopy (TEM). The 2 main types of hemocytes found were granulocytes and hyalinocytes (agranular cells). The granulocytes were subdivided into large and small granulocytes while the hyalinocytes were subdivided into large and small hyalinocytes. The large hemocytes had both a larger diameter and smaller nucleus to cell diameter ratio than their smaller counterparts. A rare cell type, the vesicular cell, was also observed and it possessed many vesicles but few or no granules. Using TEM, granulocytes were found to contain both electron-lucent and electron-dense granules of various sizes. These numerous granules were the only structures that took up the neutral red stain. Hyalinocytes had few of these granules relative to granulocytes. Large hyalinocytes had both various organelles and large vesicles in their abundant cytoplasm while small hyalinocytes had little room for organelles in their scant cytoplasm. Total hemocyte counts averaged 1.96×10(6) cells mL(-1) while differential hemocyte counts averaged 11% for small hyalinocytes, 12% for large hyalinocytes, 59% for small granulocytes, and 18% for large granulocytes. The results of this study provide a starting point for future studies on E. directus immune function., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The effects of temperature and food addition on particle mixing in the deposit-feeding bivalve Abra alba were assessed using an experimental approach allowing for the tracking of individual fluorescent particle (luminophore) displacements. This allowed for the computations of vertical profiles of a set of parameters describing particle mixing. The frequency of luminophore displacements (jumps) was assessed through the measurement of both waiting times (i.e., the time lapses between two consecutive jumps of the same luminophore) and normalized numbers of jumps (i.e., the numbers of jumps detected in a given area divided by the number of luminophores in this area). Jump characteristics included the direction, duration and length of each jump. Particle tracking biodiffusion coefficients (Db) were also computed. Data originated from 32 experiments carried out under 4 combinations of 2 temperature (Te) and 2 food addition (Fo) levels. For each of these treatments, parameters were computed for 5 experimental durations (Ed). The effects of Se, Fo and Ed were assessed using PERmutational Multivariate ANalyses Of VAriance (PERMANOVAs) carried out on vertical depth profiles of each particle mixing parameter. Inversed waiting times significantly decreased with Ed whereas the normalized number of jumps did not, thereby suggesting that it constitutes a better proxy of jump frequency when assessing particle mixing based on the measure of individual particle displacements. Particle mixing was low during autumn temperature experiments and not affected by Fo, which was attributed to the dominant effect of low temperature. Conversely, particle mixing was high during summer temperature experiments and transitory inhibited by food addition. This last result is coherent with the functional responses (both in terms of activity and particle mixing) already measured for individual of the closely related clam A. ovata originating from temperate populations. It also partly resulted from a transitory switch between deposit- and suspension-feeding caused by the high concentration of suspended particulate organic matter immediately following food addition.

Protozoan parasites of the genus Marteilia have been detected in marine bivalves and other invertebrates around the world, associated in some cases with mass mortalities. The present paper reports the characterization of the Marteilia sp. protozoan infecting the digestive gland of the Grooved Razor Shell clam (Solen marginatus) from Galicia (NW Spain), proposing a novel species in the genus: Marteilia octospora n. sp. Morphological and molecular techniques were used for the description of this parasite. Tissue imprints were essential in the study to confirm the presence of 8 spores per sporangium, a number never reported in other species from this genus. An ultrastructural study revealed that the size and number of dense granules, free in the mature sporangia, were quite different from granules in other Marteilia spp. Another morphological difference is the absence of a layer of concentric membranes found surrounding the mature spore in other species. In addition, concentric membranous structures observed in the different stages of the parasite have never been mentioned in other species of genus Marteilia. Moreover, molecular analysis of the rDNA intergenic spacer (IGS) and the internal transcribed spacer (ITS-1) showed differences with the sequences available for other Marteilia spp., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This paper reports on the acute inflammatory and cellular process in the yellow clam, Mesodesma mactroides, induced by injection of India ink into the muscular foot. Histological observations with optical and electronic microscopy were made at 24 and 48 h after injection. The induced cellular inflammatory response consisted of a general hemocyte infiltration without necrosis and apoptotic activity. Migration of ink-laden phagocytes across the intestinal epithelium was recorded. It appeared that the yellow clam "excreted" ink particles through the gill and kidney. The positive staining for apoptosis was observed in the digestive gland. Electronic microscopy revealed ultrastructural changes of endoplasmic reticulum stress and apoptotic bodies in the digestive gland. The mechanism by which the India ink particles induce apoptosis remains unknown, but might possibly be associated with the endoplasmic reticulum stress. This work has highlighted features that require further discussion in the restricted field the inflammatory responses of mollusks.

The methodology termed scanning transmission electron microscopy in scanning electron microscopy (STEM-in-SEM) has been used in this work to study the uptake of citrate stabilized gold nanoparticles (AuNPs) (average particle sizes of 23.5 ± 4.0 nm) into tissue samples upon in vitro exposure of the dissected gills of the Ruditapes philippinarum marine bivalve to the nanoparticle suspensions. The STEM-in-SEM methodology has been optimized for achieving optimum resolution under SEM low voltage operating conditions (20-30 kV). Based on scanning microscope assessments and resolution testing (SMART), resolutions well below 10 nm were appropriately achieved by working at magnifications over 100k×, with experimental sample thickness between 300 and 200 nm. These relatively thick slices appear to be stable under the beam and help avoid NP displacement during cutting. We herein show that both localizing of the internalized nanoparticles and imaging of ultrastructural disturbances in gill tissues are strongly accessible due to the improved resolution, even at sample thicknesses higher than those normally employed in standard TEM techniques at higher voltages. Ultrastructural imaging of bio-nano features in bioaccumulation experiments have been demonstrated in this study.

Symbiotic bivalves at hydrothermal vents and cold seeps host chemosynthetic bacteria intracellularly in gill cells. In bivalves, the gills grow continuously throughout their lifetime by forming new filaments. We examined how newly developed gill tissues are colonized in bivalves with horizontal and vertical symbiont transmission (Bathymodiolus mussels versus a vesicoymid clam) using fluorescence in situ hybridization and transmission electron microscopy. Symbiont colonization was similar in mussels and clams and was independent of the transmission modes. Symbionts were absent in the growth zones of the gills, indicating that symbionts colonize newly formed gill filaments de novo after they are formed and that gill colonization is a continuous process throughout the host's lifetime. Symbiont abundance and distribution suggested that colonization is shaped by the developmental stage of host cells. Self-infection, in which new gill cells are colonized by symbionts from ontogenetically older gill tissues, may also play a role. In mussels, symbiont infection led to changes in gill cell structure similar to those described from other epithelial cells infected by intracellular pathogens, such as the loss of microvilli. A better understanding of the factors that affect symbiont colonization of bivalve gills could provide new insights into interactions between intracellular bacteria and epithelial tissues., (© 2014 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Bacteria containing magnetosomes (protein-bound nanoparticles of magnetite or greigite) are common to many sedimentary habitats, but have never been found before to live within another organism. Here, we show that octahedral inclusions in the extracellular symbionts of the marine bivalve Thyasira cf. gouldi contain iron, can exhibit magnetic contrast and are most likely magnetosomes. Based on 16S rRNA sequence analysis, T. cf. gouldi symbionts group with symbiotic and free-living sulfur-oxidizing, chemolithoautotrophic gammaproteobacteria, including the symbionts of other thyasirids. T. cf. gouldi symbionts occur both among the microvilli of gill epithelial cells and in sediments surrounding the bivalves, and are therefore facultative. We propose that free-living T. cf. gouldi symbionts use magnetotaxis as a means of locating the oxic-anoxic interface, an optimal microhabitat for chemolithoautotrophy. T. cf. gouldi could acquire their symbionts from near-burrow sediments (where oxic-anoxic interfaces likely develop due to the host's bioirrigating behavior) using their superextensile feet, which could transfer symbionts to gill surfaces upon retraction into the mantle cavity. Once associated with their host, however, symbionts need not maintain structures for magnetotaxis as the host makes oxygen and reduced sulfur available via bioirrigation and sulfur-mining behaviors. Indeed, we show that within the host, symbionts lose the integrity of their magnetosome chain (and possibly their flagellum). Symbionts are eventually endocytosed and digested in host epithelial cells, and magnetosomes accumulate in host cytoplasm. Both host and symbiont behaviors appear important to symbiosis establishment in thyasirids.

Photobleaching of the fluorophore fluorescein in an aqueous solution is dependent on the oxygen concentration. Therefore, the time-dependent bleaching behavior can be used to measure of dissolved oxygen concentrations. The method can be combined with epi-fluorescence microscopy. The molecular states of the fluorophore can be expressed by a three-state energy model. This leads to a set of differential equations which describe the photobleaching behavior of fluorescein. The numerical solution of these equations shows that in a conventional wide-field fluorescence microscope, the fluorescence of fluorescein will fade out faster at low than at high oxygen concentration. Further simulation showed that a simple ratio function of different time-points during a fluorescence decay recorded during photobleaching could be used to describe oxygen concentrations in an aqueous solution. By careful choice of dye concentration and excitation light intensity the sensitivity in the oxygen concentration range of interest can be optimized. In the simulations, the estimation of oxygen concentration by the ratio function was very little affected by the pH value in the range of pH 6.5-8.5. Filming the fluorescence decay by a charge-coupled-device (ccd) camera mounted on a fluorescence microscope allowed a pixelwise estimation of the ratio function in a microscopic image. Use of a microsensor and oxygen-consuming bacteria in a sample chamber enabled the calibration of the system for quantification of absolute oxygen concentrations. The method was demonstrated on nitrifying biofilms growing on snail and mussel shells, showing clear effects of metabolic activity on oxygen concentrations., (Copyright © 2014 Wiley Periodicals, Inc.)

Bacterial symbiont transmission is a key step in the renewal of the symbiotic interaction at each host generation, and different modes of transmission can be distinguished. Vesicomyidae are chemosynthetic bivalves from reducing habitats that rely on symbiosis with sulfur-oxidizing bacteria, in which two studies suggesting vertical transmission of symbionts have been published, both limited by the imaging techniques used. Using fluorescence in situ hybridization and transmission electron microscopy, we demonstrate that bacterial symbionts of Isorropodon bigoti, a gonochoristic Vesicomyidae from the Guiness cold seep site, occur intracellularly within female gametes at all stages of gametogenesis from germ cells to mature oocytes and in early postlarval stage. Symbionts are completely absent from the male gonad and gametes. This study confirms the transovarial transmission of symbionts in Vesicomyidae and extends it to the smaller species for which no data were previously available.

Hierarchical composite materials design in biological exoskeletons achieves penetration resistance through a variety of energy-dissipating mechanisms while simultaneously balancing the need for damage localization to avoid compromising the mechanical integrity of the entire structure and to maintain multi-hit capability. Here, we show that the shell of the bivalve Placuna placenta (~99 wt% calcite), which possesses the unique optical property of ~80% total transmission of visible light, simultaneously achieves penetration resistance and deformation localization via increasing energy dissipation density (0.290 ± 0.072 nJ μm(-3)) by approximately an order of magnitude relative to single-crystal geological calcite (0.034 ± 0.013 nJ μm(-3)). P. placenta, which is composed of a layered assembly of elongated diamond-shaped calcite crystals, undergoes pervasive nanoscale deformation twinning (width ~50 nm) surrounding the penetration zone, which catalyses a series of additional inelastic energy dissipating mechanisms such as interfacial and intracrystalline nanocracking, viscoplastic stretching of interfacial organic material, and nanograin formation and reorientation.

The glochidium larvae of freshwater mussels of the family Unionidae need to find suitable hosts to attach themselves and metamorphose into free-living juveniles. The specificity of the host-parasite relationship was investigated for the Iberian Unio tumidiformis Castro, 1885 by means of experimental infections and also by analyzing naturally infected fish. The process of encapsulation of glochidia was studied using scanning electron microscopy. Unio tumidiformis has proven to be an unusual host-specific unionid mussel, apparently parasitizing only fish of the genus Squalius Bonaparte, 1837. Successful encapsulation or complete metamorphosis was observed in five fish taxa: S. aradensis (Coelho, Bogutskaya, Rodrigues et Collares-Pereira), S. caroliterti (Doadrio), S. pyrenaicus (Günther), S. torgalensis (Coelho, Bogutskaya, Rodrigues et Collares-Pereira) and S. alburnoides (Steindachner) complex (only for the nuclear hybrids with at least one copy of the S. pyrenaicus genome). Complete metamorphose was achieved in 6 to 14 days at mean temperatures ranging from 21.8 to 26.1 degrees C. The current study provides support for cell migration being the main force of cyst formation and shows the influence of potential host's genome in response to the infection process to determine the success of the metamorphosis.

The arenophilic system of anomalodesmatan clams comprises multicellular glands, muscular papillae, and radial lines of adhesive secretion, which glue sand grains and other extraneous particles to the external surface of the shell. The presence and morphology of these organs have been established in an increasing number of taxa, but arenophilic glands were not previously known to occur in the deep-sea, carnivorous family Cuspidariidae. Dissections, histological sections and ultrastructural examination of members of the cuspidariid genera Cuspidaria and Cardiomya revealed a functional arenophilic system positioned around the siphonal apparatus. Glands are contained in muscular papillae at the posterior tip of the siphonal sheath, from where they deposit their adhesive secretion onto the shell rostrum. Discovery of an arenophilic system in cuspidariid clams adds support to the idea that the glands are a synapomorphy of Anomalodesmata, secondarily absent in only one of the major component branches (the clade joining Thraciidae, Cleidothaeridae, and Myochamidae). Moreover, similarity between the arenophilic papillae we observed on the siphonal sheath of Cuspidaria, Cardiomya, and Myonera, and literature reports on the crown of tentacles of Multitentacula is puzzling and highlights the need for further studies of the latter genus., (Copyright © 2013 Wiley Periodicals, Inc.)

Nacre tablets of mollusks develop two kinds of features when either the calcium carbonate or the organic portions are removed: (1) parallel lineations (vermiculations) formed by elongated carbonate rods, and (2) hourglass patterns, which appear in high relief when etched or in low relief if bleached. In untreated tablets, SEM and AFM data show that vermiculations correspond to aligned and fused aragonite nanogloblules, which are partly surrounded by thin organic pellicles. EBSD mapping of the surfaces of tablets indicates that the vermiculations are invariably parallel to the crystallographic a-axis of aragonite and that the triangles are aligned with the b-axis and correspond to the advance of the {010} faces during the growth of the tablet. According to our interpretation, the vermiculations appear because organic molecules during growth are expelled from the a-axis, where the Ca-CO3 bonds are the shortest. In this way, the subunits forming nacre merge uninterruptedly, forming chains parallel to the a-axis, whereas the organic molecules are expelled to the sides of these chains. Hourglass patterns would be produced by preferential adsorption of organic molecules along the {010}, as compared to the {100} faces. A model is presented for the nanostructure of nacre tablets. SEM and EBSD data also show the existence within the tablets of nanocrystalline units, which are twinned on {110} with the rest of the tablet. Our study shows that the growth dynamics of nacre tablets (and bioaragonite in general) results from the interaction at two different and mutually related levels: tablets and nanogranules., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The study aimed to assess morphological, structural and compositional alterations in Scrobicularia plana nacre environmentally exposed to mercury in order to seek out the possibility of the assessed alterations as a monitoring tool to handle complexity and interactions of metals in the environment involving a non-invasive methodology. Bivalves were collected from a mercury contaminated site (Laranjo basin - Ria de Aveiro, Portugal) and a reference site in the same aquatic system. The combination of scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS) technique depicted a sheet like morphology of bivalve nacre collected from the reference site. Moreover, EDS plot exhibited the presence of potassium, oxygen, calcium, and carbon elements. Shells collected from the contaminated area depicted lamellar patches like structures with particle like morphology composition. SEM images corresponding to the elemental analysis by EDS plot clearly denoted the presence of mercury. SEM images from the other locations of the contaminated shells depicted large surface area, a broken or ruptured symmetry of organic matrix as well as crack-like gaps. The influence of environmental mercury affecting the surface morphology of S. plana nacre showed dimple like morphology (as proved by transmission electron microscopy, TEM). The possible explanation may be the replacement of calcium elements with other elements or alloys from the nacre composite collected from contaminated region. Therefore, the nacre fingerprint may be useful as innovative knowledge and applicable tool aiming at risk reduction from noxious mercury present in the environment. Overall results suggested the use of shell as an indelible fingerprint of metal exposure., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The degree of aggregation and/or coalescence of Au-citrate nanoparticles (AuNPs, mean size 21.5 ± 2.9 nm), after delivery in simulated seawater, are shown to be concentration-dependent. At low concentrations no coalescence and only limited aggregation of primary particles were found. Experiments were performed in which the marine bivalve (Ruditapes philippinarum) was exposed to AuNPs or dissolved Au and subsequently, bivalve tissues were studied by Scanning and Transmission Electron Microscopy and chemical analyses. We show that the bivalve accumulates gold in both cases within either the digestive gland or gill tissues, in different concentrations (including values of predicted environmental relevance). After 28 days of exposure, electron-dense deposits (corresponding to AuNPs, as proven by X-ray microanalysis) were observed in the heterolysosomes of the digestive gland cells. Although non-measurable solubility of AuNPs in seawater was found, evidence is presented of the toxicity produced by Au(3+) dissolved species (chloroauric acid solutions) and its relevance is discussed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This study reports evidence of a neoplastic disorder in the clam Venerupis aurea. In the first stage of the disease, masses of neoplastic cells were mainly observed in connective tissue of gills. These masses of neoplastic cells appeared more compact than in cases of disseminated neoplasia of other bivalve molluscs. As disease progresses, masses of abnormal cells were also observed in the connective tissue of all organs, thus alteration of vital functions in late stage is likely. The neoplastic cells had aneuploid DNA content, ranging from 1.6n to 7.8 n. The ploidy level increased with the severity of the disease. A comparison of three light microscopy diagnostic procedures was performed. Histology was the most sensitive diagnostic test, whereas two different haematological techniques assayed presented unsatisfactory low levels of sensitivity. Specificity was high for the three assayed tests., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In this research, four types of waste seashells, including short-necked clam, green mussel, oyster, and cockle, were investigated experimentally to develop a cement product for masonry and plastering. The parameters studied included water demand, setting time, compressive strength, drying shrinkage and thermal conductivity of the mortars. These properties were compared with those of a control mortar that was made of a conventional Portland cement. The main parameter of this study was the proportion of ground seashells used as cement replacement (5%, 10%, 15%, or 20% by weight). Incorporation of ground seashells resulted in reduced water demand and extended setting times of the mortars, which are advantages for rendering and plastering in hot climates. All mortars containing ground seashells yielded adequate strength, less shrinkage with drying and lower thermal conductivity compared to the conventional cement. The results indicate that ground seashells can be applied as a cement replacement in mortar mixes and may improve the workability of rendering and plastering mortar., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Electromechanical coupling is a nearly universal property of biomaterials, and may play an important role in many physiological and functional phenomena. The intrinsic or externally-generated electric field within biomaterials may also contribute to their predominant mechanical properties. Mollusc shells are well known for their outstanding mechanical properties, which are generally believed to originate from their hierarchical structures in multi-levels. This paper is therefore focused on the studies of the hierarchical structures and electromechanical coupling behaviors of clamshell from micro- to nano-levels, and in particular, the biopolymer concentrated regions. Detailed studies are performed to characterize the piezoelectric and ferroelectric properties of clamshell. It was found that the piezoresponse of clamshell is originated from the biopolymers between the mineral grains, as well as those intercalated within the mineral crystalline structure after the biomineralization process. Local ferroelectric hysteresis loops of clamshell have also been observed and analyzed on the samples with different orientations, biopolymer contents, or moisture contents. It is believed that the overall functioning of the clamshell or even other mollusc shells may incorporate many mechanisms interacting together, rather than originate from the hierarchical structure alone. This study of the electromechanical coupling effects of clamshell can be a path to have more comprehensive understandings of the properties and behaviors of mollusc shells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In marine invertebrates that acquire their symbionts from the environment, these are generally only taken up during early developmental stages. In the symbiosis between lucinid clams and their intracellular sulfur-oxidizing bacteria, it has been shown that the juveniles acquire their symbionts from an environmental stock of free-living symbiont forms, but it is not known if adult clams are still competent to take up symbiotic bacteria from the environment. In this study, we investigated symbiont acquisition in adult specimens of the lucinid clam Codakia orbiculata, using transmission electron microscopy, fluorescence in situ hybridization, immunohistochemistry and PCR. We show here that adults that had no detectable symbionts after starvation in aquaria for 6 months, rapidly reacquired symbionts within days after being returned to their natural environments in the field. Control specimens that were starved and then exposed to seawater aquaria with sulfide did not reacquire symbionts. This indicates that the reacquisition of symbionts in the starved clams returned to the field was not caused by high division rates of a small pool of remaining symbionts that we were not able to detect with the methods used here. Immunohistochemistry with an antibody against actin, a protein involved in the phagocytosis of intracellular bacteria, showed that actin was expressed at the apical ends of the gill cells that took up symbionts, providing further evidence that the symbionts were acquired from the environment. Interestingly, actin expression was also observed in symbiont-containing cells of untreated lucinids freshly collected from the environment, indicating that symbiont acquisition from the environment occurs continuously in these clams throughout their lifetime., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Brazilian endemic species Diplodon expansus (Küster, 1856) is found in freshwater bodies in the country's southeast, in large anthropogenic influence regions especially with an extensive agriculture emphasis. One of the main pesticides used in the species occurrence region is the atrazine herbicide, which has a great contamination potential in the aquatic environment. Therefore, several studies into its toxicity in aquatic systems have been developed. However, the tested concentrations are usually very high and rarely found in the environment and the short-term exposure responses in other aquatic organisms such as native bivalves are still scarce. Thus, this study sought to consider the potential effects of environmentally realistic concentrations of atrazine herbicide on the surface morphology of gill filaments of the bivalve D. expansus under laboratory-controlled conditions after short-term exposure. None of the animals died before the end of the experiment. The main alterations were observed on the frontal surface of filaments, which include mucus accumulation, cilia loss, and disruption. Mucus increased secretion and accumulation in the frontal filaments region preceded as a protective mechanism. Cilia loss and disruption on the frontal surface of the gill filament indicated that ciliated frontal cells were more sensitive to atrazine exposure and these alterations may cause gills functional damages, compromising the uptake of food particles and respiration. Therefore, higher sublethal concentrations of atrazine may compromise the survival and consequently the population of D. expansus in agriculture areas after a longer period of continuous exposure., (Copyright © 2011 Wiley Periodicals, Inc.)

It is widely accepted that the structure of bivalve ligament contains two layers: outer organic lamellar and inner aragonite fibrous layer. However, by using scanning electron microscopy (SEM), X-ray diffraction (XRD), and infrared spectroscopy (FTIR), we found an additional protein fibrous layer in the Solen grandis ligament. The ligament consists of three layers: an outer layer (OL), middle layer (ML), and inner layer (IL). OL consists of lamellar protein, oriented normal to dorsal edge. ML has unique spring-like protein fibers (ca. 120 nm in diameter) structure, stretching continuously from the left to right valve. Interestingly, IL comprises of aragonite fibers (ca. 100 nm in diameter) oriented posteriorly and inclined at an angle about 26° to dorsal edge at dorsal part; then they turn gradually to ventral part, showing striking zigzag structure vertical to ventral surface (VS). The ligament presents a new three-layered structural model different from previous reports; it may inspire not only in materials synthesis and mechanical design, but also in bivalve classification and evolution., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

For more than two decades, the Manila clam Ruditapes philippinarum has been regularly affected by Brown Ring Disease (BRD), an epizootic event caused by the bacterium Vibrio tapetis and characterized by the development of a brown deposit on the inner face of valves. Although BRD infection is often lethal, some clams recover by mineralizing a new repair shell layer, which covers the brown deposit and fully isolates it from living tissues. In order to understand this specific shell repair process, the microstructures of repaired zones were compared to those of shells unaffected by BRD. In addition, the organic matrix associated with unaffected shells and to repair patches were extracted and compared by biochemical and immunological techniques. Our results show that the repaired zones exhibit microstructures that resemble the so-called homogeneous microstructure of the internal layer, with some marked differences, like the development of crossed-acicular crystals, which form chevron-like patterns. In the three tested batches of repaired layers, the matrices exhibit certain heterogeneity, i.e., they are partially to widely different from the ones of shells unaffected by BRD, as illustrated by SDS-PAGE and by serological comparisons. Our results strongly suggest a modification of the secretory regime of calcifying mantle cells during the shell repair process. Polyclonal antibodies, which were developed against specific protein fractions of the shell, represent relevant tools for localizing by immunohistology the cells responsible for the repair., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Shells of adult individuals from two different bivalve families, Hyriopsis cumingii and Diplodon chilensis patagonicus, were studied by Micro-Raman spectroscopy and Focussed Ion Beam-assisted TEM. The shells contain amorphous calcium carbonate in a zone at the interface between the periostracum and the prismatic layer. In this area, the initial prism structures protrude from the inner periostracum layer and it is demonstrated that these structures systematically consist of highly disordered and amorphous calcium carbonate. Within this zone, ordered and disordered areas are intermingled discounting the existence of a crystallization front and favouring models of domainal crystallization processes via so-called mesocrystals. These observations are the first documentation of the use of amorphous calcium carbonate as a precursor phase by adult mollusc species and lend further support to hypotheses postulating widespread use of amorphous phases as building material of skeletal tissue in biology., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The phases of embryonic development of Anodontites trapesialis lasidia are described for the first time. Adult specimens were obtained from two fish farms located in Londrina, Paraná, Brazil. The internal demibranchs of 120 individuals were studied using a routine histological technique; 70 of these carried eggs and/or larvae in the marsupium and were utilized for the description of the phases of embryonic development. The demibranchs of five specimens were evaluated by scanning electron microscopy to detail the morphology of the larvae. Five phases of development were established: phase I, corresponding to the initial stage of cleavage with the formation of apical cells; phase II, including the stages of the morula and blastula; phase III, where the gastrula forms; phase IV, where the larva formed is still inside the egg envelope; and phase V, where the lasidium can still be identified immediately after eclosion.

The mineral phase of shell repair in the Manila clam Venerupis philippinarum affected by brown ring disease (BRD) was characterised at various scales and at various stages of shell repair by confocal Raman microspectrometry and scanning electron microscopy. Spherulitic and quadrangular aragonite microstructures associated with polyene pigments were clearly observed. Von Kossa staining showed that at the beginning of shell repair, hemocytes are filled with insoluble calcium carbonate salts in all fluids and then are transported toward the extrapallial fluids and the repair sites. Our analyses suggest that after a Vibrio tapetis attack and BRD deposit some clams rapidly cover the deposit, resulting in a modification in the microstructure, which could be produced by the participation of both the mantle and hemocytes.

The first studies of the 16S rRNA gene diversity of the bacterial symbionts found in lucinid clams did not clarify how symbiotic associations had evolved in this group. Indeed, although species-specific associations deriving from a putative ancestral symbiotic association have been described (coevolution scenario), associations between the same bacterial species and various host species (opportunistic scenario) have also been described. Here, we carried out a comparative molecular analysis of hosts, based on 18S and 28S rRNA gene sequences, and of symbionts, based on 16S rRNA gene sequences, to determine as to which evolutionary scenario led to modern lucinid/symbiont associations. For all sequences analyzed, we found only three bacterial symbiont species, two of which are harbored by lucinids colonizing mangrove swamps. The last symbiont is the most common and was found to be independent of biotope or depth. Another interesting feature is the similarity of ctenidial organization of lucinids from the Philippines to those described previously, with the exception that two bacterial morphotypes were observed in two different species (Gloverina rectangularis and Myrtea flabelliformis). Thus, there is apparently no specific association between Lucinidae and their symbionts, the association taking place according to which bacterial species is present in the environment., (FEMS Microbiology Ecology © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original French government works.)

Annually resolved growth increments in the shell of the bivalve mollusc Arctica islandica have previously been used in combination with geochemical measurements to successfully construct high-resolution proxy records of past marine environmental conditions. However, to ensure the accuracy of these paleoenvironmental reconstructions it is essential that the annual growth series of increments within the examined shells are reliably identified, and can be distinguished from spurious lines caused by nonannual perturbations such as those resulting from storm disturbance. The current methods used for identifying the growth increment series are sometimes compromised because of ambiguity that results from the employed preparation methods. Here it is shown that backscattered electron imaging of polished shell cross sections may be used to clearly discriminate between the two compositionally and structurally distinct increments that comprise 1 year of outer shell growth. This method, involving minimal specimen preparation, is likely to be primarily useful as a validation technique of particular value in cases where increment identification using existing methods is difficult or ambiguous., (© 2010 The Authors Journal of Microscopy © 2010 The Royal Microscopical Society.)

Background: Doubly Uniparental Inheritance (DUI) of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI). DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos., Methodology/principal Findings: We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow., Conclusions/significance: In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown, they could be a variation of the mechanism regulating the mitochondrial bottleneck in all metazoans.

Using immunocytochemistry combined with light and electron microscopy, the distribution and ultrastructure of tyrosine hydroxylase (TH)-immunoreactive neurons in the CNS of bivalve mollusc, Megangulus venulosus, have been studied under the influence of increased temperature and hypoxia. It has been established, that the stress causes changes in the amount of TH and in the structure of TH-immunopositive neurons in all ganglia. The most essential changes in CNS of M. venulosus were revealed after 60 min exposure to increased temperature and hypoxia; degenerative changes in large neurons, reduction of the synapses and reduction of TH-immunoreactivity in neurons and neuropil.

The formation of calcite prism architecture in the prismatic layer of the mollusk shell involves the participation of a number of different proteins. One protein family, Asprich, has been identified as a participant in amorphous calcium carbonate stabilization and calcite architecture in the prismatic layer of the mollusk, Atrina rigida . However, the functional role(s) of this protein family are not fully understood due to the fact that insufficient quantities of these proteins are available for experimentation. To overcome this problem, we employed stepwise solid-phase synthesis to recreate one of the 10 members of the Asprich family, the 61 AA single chain protein, Asprich "3". We find that the Asprich "3" protein inhibits the formation of rhombohedral calcite crystals and induces the formation of round calcium carbonate deposits in vitro that contain calcite and amorphous calcium carbonate (ACC). This mineralization behavior does not occur under control conditions, and the formation of ACC and calcite is similar to that reported for the recombinant form of the Asprich "g" protein. Circular dichroism studies reveal that Asprich "3" is an intrinsically disordered protein, predominantly random coil (66%), with 20-30% β-strand content, a small percentage of β-turn, and little if any α-helical content. This protein is not extrinsically stabilized by Ca(II) ions but can be stabilized by 2,2,2-trifluoroethanol to form a structure consisting of turn-like and random coil characteristics. This finding suggests that Asprich "3" may require other extrinsic interactions (i.e., with mineral or ionic clusters or other macromolecules) to achieve folding. In conclusion, Asprich "3" possesses in vitro functional and structural qualities that are similar to other reported for other Asprich protein sequences.