Microscopic anatomy of gonadal area in the deep-sea clam Calyptogena pacifica (Bivalvia: Vesicomyidae) with emphasis on somatic cells.

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.

Sperm ultrastructure of Ruditapes variegata and Tapes literatus (Mollusca, Bivalvia, Veneridae, Tapetinae) from Pescadores, Taiwan.

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.

Ultrastructural aspects of spermatogenesis in Calyptogena pacifica Dall 1891 (Vesicomyidae; Bivalvia).

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.

Ultrastructure of sperm and complete mitochondrial genome in Meretrix sp. (Bivalvia: Veneridae) from Taiwan.

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.

Spermcast mating with release of zygotes in the small dioecious bivalve Digitaria digitaria.

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.

Comparative phylogenomics reveal complex evolution of life history strategies in a clade of bivalves with parasitic larvae (Bivalvia: Unionoida: Ambleminae).

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.

Form and function of tentacles in pteriomorphian bivalves.

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.

Spermatogenesis of the freshwater pearl mussel Margaritifera laevis (Bivalvia: Margaritiferidae): A histological and ultrastructural study.

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.

The ultrastructural features of embryonic and early larval development in Yesso scallop, Mizuhopecten yessoensis.

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.

Impact of High Hydrostatic Pressure on the Shelling Efficacy, Physicochemical Properties, and Microstructure of Fresh Razor Clam (Sinonovacula constricta).

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, Ca2+ -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 Ca2+ -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.

Who wins in the weaning process? Juvenile feeding morphology of two freshwater mussel species.

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.

Physical versus Biological Control in Bivalve Calcite Prisms: Comparison of Euheterodonts and Pteriomorphs.

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.

High resolution microscopy reveals significant impacts of ocean acidification and warming on larval shell development in Laternula elliptica.

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.

Interspecies comparison of the mechanical properties and biochemical composition of byssal threads.

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.

The byssus threads of Pinna nobilis: A histochemical and ultrastructural study.

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.

Fluid-flow-templated self-assembly of calcium carbonate tubes in the laboratory and in biomineralization: The tubules of the watering-pot shells, Clavagelloidea.

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.

Morphology and taxonomy of Isognomon spathulatus (Reeve, 1858), a cryptic bivalve from the mangroves of Thailand.

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.

Pinna nobilis: A big bivalve with big haemocytes?

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.

Experimental Assessment of the Effects of Temperature and Food Availability on Particle Mixing by the Bivalve Abra alba Using New Image Analysis Techniques.

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.

Morphological characterization via light and electron microscopy of Atlantic jackknife clam (Ensis directus) hemocytes.

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.