Depuration kinetics of hexachlorobenzene in the clam, Macoma nasuta

1. The depuration rate constant for [14C]hexachlorobenzene (HCB) in the clam, Macoma nasuta, was determined following a short-term exposure to HCB contaminated seawater. 2. Depuration was not correlated with ventilation volume, nor did the amount of sediment ingested during depuration have a significant effect. 3. The half-life for HCB in M. nasuta was estimated to be 16 days with a bioconcentration factor of 3490 (wet weight basis).     

Biotic interactions revealed by macroborings in arctic bivalve molluscs

The presence of organisms whose bodies have low preservation potential may be deduced by searching for the traces produced by them. The addition of predatory gastropods and soft-bodied epizoans to Quaternary marine faunas dominated by bivalves was facilitated by an examination of borings in bivalve shells. Borings attributed to predatory gastropods (ichnogenus Oichnus ) were observed in shells of Astarte spp., Hiatella arctica and Macoma calcarea. Astarte, Hiatella and Macoma were preyed upon in preference to other members of a diverse suspension-feeding bivalve community. Borings attributed to epizoans (ichnogenus Cautostrepsis ) were observed in bivalve shells (Astarte spp. Hiatella arctica ), calcareous algae and limestone clasts. Biotic interactions revealed by trace fossils are employed, for the first time, to reconstruct the trophic structure of arctic Quaternary marine benthic faunas. ▭ Arctic molluscs, palaeoecology, Oichnus, Caulostrepsis.     

Description of Perkinsus andrewsi n. sp. isolated from the Baltic clam (Macoma balthica) by characterization of the ribosomal RNA locus, and development of a species-specific PCR-based diagnostic assay

A Perkinsus species was isolated from the baltic clam Macoma balthica and an in vitro culture established under conditions described for P. marinus. As reported previously, morphological features remarkable enough to clearly indicate that this isolate is a distinct Perkinsus species were lacking. In this study, regions of the rRNA locus (NTS, 18S, ITS1, 5.8S, and ITS2) of this isolate were cloned, sequenced, and compared by alignment with those available for other Perkinsus species and isolates. Sequence data from the rRNA locus and species-specific PCR assays indicated not only that Perkinsus sp. from M. balthica was not P. marinus, but it was different from P. atlanticus and P. olseni. The degree of difference was comparable to or greater than differences between accepted Perkinsus species. In particular, NTS sequence and length were dramatically different from that of P. marinus and P. atlanticus. Therefore, we formally propose to designate the Perkinsus sp. from M. balthica as a separate species, P. andrewsi n. sp. Primers based on P. andrewsi NTS sequence were used to develop a PCR-based diagnostic assay that was validated for species-specificity and sensitivity. PCR-based assays specific for either P. andrewsi or P. marinus were used to test for their presence in bivalve species sympatric to M. balthica. Although isolated from M. balthica, P. andrewsi was also detected in the oyster Crassostrea virginica and clams Macoma mitchelli and Mercenaria mercenaria, and could coexist with P. marinus in all four bivalve species tested.     

Infaunal hydraulics generate porewater pressure signals

Many activities by infauna, including burrowing and feeding, involve hydraulic mechanisms. We expected these activities to generate low-frequency pressure waves that would propagate through sediments and be detectable at some distance from the source. Pressure sensors in intertidal sediments recorded large-amplitude porewater pressure signals. Laboratory recordings of single individuals allowed us to identify characteristic signals of arenicolid and nereidid polychaetes and tellinid bivalves. In the bivalve Macoma nasuta, these high-amplitude signals were associated with burrowing, expulsion of pseudofeces, and siphon relocation. In the polychaetes Neanthes brandti and Abarenicola pacifica, the high-amplitude pressure signals were associated with burrowing, burrow construction, burrow ventilation, and defecation. These signals were detectable in the field at distances of at least 20 cm. Since the waveforms are species-specific as well as activity-specific, they may provide a mechanism for prey detection, for predator avoidance, for competitor detection, and perhaps even for mate detection.     

Biochemical identification and numerical taxonomy of Aeromonas spp. isolated from environmental and clinical samples in Spain

AIMS: To study the phenotypic characteristics of Aeromonas spp. from environmental and clinical samples in Spain and to cluster these strains by numerical taxonomy. METHODS AND RESULTS: A collection of 202 Aeromonas strains isolated from bivalve molluscs, water and clinical samples was tested for 64 phenotypic properties; 91% of these isolates were identified at species level. Aeromonas caviae was predominant in bivalve molluscs and Aerom. bestiarum in freshwater samples. Cluster analyses revealed eight different phena: three containing more than one DNA-DNA hybridization group but including strains that belong to the same phenospecies complex (Aerom. hydrophila, Aerom. sobria and Aerom. caviae), Aerom. encheleia, Aerom. trota and three containing unidentified Aeromonas strains isolated from bivalve molluscs. CONCLUSIONS:Aeromonas spp. are widely distributed in environmental and clinical sources. A selection of 16 of the phenotypical tests chosen allowed the identification of most isolates (91%), although some strains remain unidentified, mainly isolates from bivalve molluscs, suggesting the presence of new Aeromonas species. Numerical taxonomy was not in total concordance with the identification of the studied strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Numerical taxonomy of Aeromonas strains isolated from different sources revealed the presence of potentially pathogenic Aeromonas spp., especially in bivalve molluscs, and phena with unidentified strains that suggest new Aeromonas species.     

Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum

In many bivalve molluscs, lectins are present in the hemolymph and are thought to be important for internal host defense mechanisms. For this study, we purified a novel isoform of the Manila clam lectin (designated MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum, using affinity chromatography and gel filtration. Native PAGE results showed that the MCL-4 consisted of 70 kDa protein. MCL-4 was found to be composed of 58-kDa and 43-kDa bands when examined using SDS-PAGE under reducing and non-reducing conditions. The native MCL-4 was revealed as a 147 kDa molecular mass protein by gel filtration. The purified MCL-4 agglutinates calcium-dependently in the erythrocytes of sheep and rabbit, but not in cells of the three species of marine bacteria tested. However, the phagocytic ability of the R. philippinarum hemocytes for the MCL-4-opsonized Vibrio tubiashii cells was significantly greater than that for the BSS-treated bacterial cells. Addition of purified MCL-4 markedly suppressed Alteromonas haloplanktis growth. These results suggest that MCL-4, because of its opsonizing and bacteriostatic properties, might contribute to the host defense mechanisms against invading microorganisms in R. philippinarum.     

Significance of new records of Tridacna squamosa Lamarck, 1819, in the Tuamotu and Gambier Archipelagos (French Polynesia)

The giant clam subfamily Tridacninae (family Cardiidae) is an important group of bivalve molluscs found throughout the Red Sea and Indo-Pacific, from East Africa to the Eastern Pacific biogeographic region. The Tridacna genus is currently revised with numerous cryptic species identified with molecular markers. New Tridacna records from the fringe of the known distribution areas are extremely useful to identify genetically unique species, geographic ranges, and to examine processes associated with species differentiation. While Tridacna maxima is abundant in French Polynesia (Central South Pacific Ocean) the larger fluted giant clam Tridacna squamosa was formerly reported only in the Austral Islands in the south. Following a recent survey that spanned 23 islands and atolls of the Society, Tuamotu and Gambier Archipelagos, the presence of T. squamosa between the Cook Islands and Pitcairn Islands is confirmed using both morphological and molecular information, suggesting a relic distribution across the Central Pacific Ocean. Tridacna squamosa is rare, but present throughout Tuamotu and Gambier. However, it remained undetected from the Society Islands, probably due to historical over-fishing. This species is valued by local inhabitants, and is sought after mainly as gifts and also for a limited local shell trade. The rarity of T. squamosa may call for conservation measures in the near future.     

The effect of season and location on phosphoadenylate concentrations and adenylate energy charge in two species of freshwater clams

Summary Concentrations of phosphoadenylate nucleotides and the adenylate energy charge ((ATP+1/2ADP)/(ATP+ADP+AMP)) have been suggested as sensitive integrating measures of the energy state of organisms. This synoptic study investigated the seasonal and spatial variation of phosphoadenylate concentrations and AEC in two freshwater bivalve molluscs, the paper-shell clam, Anodonta imbecillis and the asian clam, Corbicula fluminea. Concentrations of all three adenylates, as well as the total adenylate concentration and adenylate energy charge of both species varied seasonally. These fluctuations were closely related to reproductive periods in both species. Total adenylate concentrations and ATP concentrations were slightly negatively correlated with shell length in A. imbecillis but the ADP and AMP concentrations and AEC were not significantly correlated with shell length. In C. fluminea the AEC was negatively correlated were positively correlated with shell length. Neither species exhibited significant differences in AEC between two collection locations. When C. fluminea collected from the Savannah River were acclimated and fed in the laboratory their AEC increased significantly.     

Soft-Shell Clam, Mya arenaria, a Convenient Laboratory Animal for Screening Pathogens of Bivalve Mollusks

Attempts to introduce infectious or foreign material into oysters and other bivalve mollusks usually involve force or trauma because of immediate, prolonged adduction of the tightly closing valves. The soft-shell clam, Mya arenaria, is unable to seal its valves completely and relaxes readily, exposing soft tissue and a large siphon. This species is free from fouling organisms and is readily available at all seasons in the New England and mid-Atlantic areas. Suspensions of five strains of Vibrio sp. that cause bacillary necrosis in larval and juvenile bivalve mollusks were injected into the heart, siphon tissue, and the incurrent and excurrent siphon lumina of soft-shell clams. All vibrio strains caused significant mortality, usually within 2 days. Heaviest losses resulted from heart and excurrent siphon injections. No mortality occurred in control clams injected with seawater, broth, Serratia sp., and Escherichia coli. The soft-shell clam appears to be a useful animal for testing the pathogenicity of marine microorganisms for bivalve mollusks.     

In vitro interactions between several species of harmful algae and haemocytes of bivalve molluscs

Harmful algal blooms (HABs) can have both lethal and sublethal impacts on shellfish. To understand the possible roles of haemocytes in bivalve immune responses to HABs and how the algae are affected by these cells (haemocytes), in vitro tests between cultured harmful algal species and haemocytes of the northern quahog (= hard clam) Mercenaria mercenaria, the soft-shell clam Mya arenaria, the eastern and Pacific oysters Crassostrea virginica and Crassostrea gigas and the Manila clam Ruditapes philippinarum were carried out. Within their respective ranges of distribution, these shellfish species can experience blooms of several HAB species, including Prorocentrum minimum, Heterosigma akashiwo, Alexandrium fundyense, Alexandrium minutum and Karenia spp.; thus, these algal species were chosen for testing. Possible differences in haemocyte variables attributable to harmful algae and also effects of haemolymph and haemocytes on the algae themselves were measured. Using microscopic and flow cytometric observations, changes were measured in haemocytes, including cell morphology, mortality, phagocytosis, adhesion and reactive oxygen species (ROS) production, as well as changes in the physiology and the characteristics of the algal cells, including mortality, size, internal complexity and chlorophyll fluorescence. These experiments suggest different effects of the several species of harmful algae upon bivalve haemocytes. Some harmful algae act as immunostimulants, whereas others are immunosuppressive. P. minimum appears to activate haemocytes, but the other harmful algal species tested seem to cause a suppression of immune functions, generally consisting of decreases in phagocytosis, production of ROS and cell adhesion and besides cause an increase in the percentage of dead haemocytes, which could be attributable to the action of chemical toxins. Microalgal cells exposed to shellfish haemolymph generally showed evidence of algal degradation, e.g. loss of chlorophyll fluorescence and modification of cell shape. Thus, in vitro tests allow a better understanding of the role of the haemocytes and the haemolymph in the defence mechanisms protecting molluscan shellfish from harmful algal cells and could also be further developed to estimate the effects of HABs on bivalve molluscs in vivo.     

A broad transition zone between an inner Baltic hybrid swarm and a pure North Sea subspecies of Macoma balthica (Mollusca, Bivalvia)

The populations of the bivalve clam Macoma balthica in the low-salinity Northern Baltic Sea represent an admixture of two strongly diverged genomic origins, the Pacific Macoma balthica balthica (approx. 60% genomic contribution) and Atlantic Macoma balthica rubra (40%). Using allozyme and mtDNA characters, we describe the broad transition from this hybrid swarm to the pure M. b. rubra in the saline North Sea waters, spanning hundreds of kilometre distance. The zone is centred in the strong salinity gradient of the narrow Öresund strait and in the adjacent Western Baltic. Yet the multilocus clines show no simple and smoothly monotonic gradation: they involve local reversals and strong differences between neighbouring populations. The transitions in different characters are not strictly coincident, and the extent of introgression varies among loci. The Atlantic influence extends further into the Baltic in samples from the southern and eastern Baltic coasts than on the western coast, and further in deeper bottoms than at shallow (< 1 m) sites. This fits with the counterclockwise net circulation pattern and with a presumably weaker salinity barrier for invading Atlantic type larvae in saline deeper water, and corresponding facilitation of outwards drift of Baltic larvae in diluted surface waters. Genotypic disequilibria were strong particularly in the shallow-water samples of the steepest transition zone. This suggests larval mixing from different sources and limited interbreeding in that area, which makes a stark contrast to the evidence of thorough amalgamation of the distinct genomic origins in the inner Baltic hybrid swarm of equilibrium structure.     

Characterization of aspartate transcarbamylase activity from gonads of the soft shell clam,Mya arenaria

Aspartate transcarbamylase (ATCase, EC 2.1.3.2) has been shown to be a good index of the reproductive cycle in marine molluscs. However, this enzyme has never been studied in the soft shell clam Mya arenaria. The characteristics of gonadal ATCase of the soft shell clam, Mya arenaria were therefore determined since we need powerful tools to assess the degree of effects of endocrine disruptors in this species at risk. Enzyme kinetic values observed at pH 8.3 were significantly lower than those measured at pH 9.4. The optimal conditions for the enzyme assays were reached in the presence of a 10 mM of substrate concentration and at pH 9.2 for 60 min at 37 °C. We have found that the enzyme was heat sensitive, markedly activated by DMSO and DMF, but no effect was observed with ethanol, ATP or CTP. However, clam ATCase activity was partly inhibited by the addition of CuSO₄ and PHMB to the medium, an inhibition that could be attributed to the presence of SH sites in cysteine residues localized in the catalytic site of this enzyme. All these results will be very useful in the near future to study the gametogenetic process of Mya arenaria, since little is known about the factors that control the physiological process of reproduction in this bivalve of ecological and economic importance. Studies of variations of the activity of aspartate transcarbamylase will also be useful as a potential biomarker to evaluate the disruption of gametogenesis in clams exposed to endocrine disruptors in situ.     

Influence of low-temperature processing of the brackish-water bivalve, Corbicula japonica, on the ornithine content of its extract

The ornithine content of an extract of the brackish-water bivalve, Corbicula japonica, increased when the bivalve was frozen. It was not influenced by the period of freezing. This phenomenon was not apparent in the scallop, little-neck clam, or hard clam. We applied various low-temperature conditions for processing the bivalve from 4 degrees C to -10 degrees C and measured the ornithine content of each extract. The ornithine content was maximized by processing at - 4 degrees C. The increase in this ornithine content was reduced when the bivalve was stored at 5 degrees C or 15 degrees C after processing at - 4 degrees C, this decrease being reversed when the bivalve was again processed at - 4 degrees C after warming. Low-temperature processing of the brackish-water bivalve therefore increased the ornithine content of the extract.     

Oxidative burst in hard clam (Mercenaria mercenaria) haemocytes

Haemocytes of bivalve molluscs are known to be responsible for many immunological functions, including recognition, phagocytosis, and killing or elimination of invading microorganisms, such as potentially infective bacteria and parasites. In many bivalves, killing of microorganisms engulfed by haemocytes is accomplished by a sudden release of reactive oxygen species (ROS) within the haemocytes; this response is referred to as an oxidative burst. Previous studies have failed to detect oxidative burst in haemocytes of the hard clam (northern quahog), Mercenaria mercenaria. In the present study, we applied a widely used chemical probe for ROS detection in haemocytes, dichlorofluorescin-diacetate (DCFH-DA), to haemocytes from this clam species and used flow cytometry to quantify fluorescence in individual haemocytes. Oxidation of DCFH-DA to the fluorescent product, DCF, within unstimulated haemocytes indicated that ROS were clearly produced in these cells. Two activators of oxidative burst, zymosan and bacterial extracellular products, which have been applied successfully to haemocytes in other species, stimulated large increases in ROS production in hard clam haemocytes. Furthermore, two inhibitors of ROS production, W-13 and diphenylene iodinium (DPI), significantly suppressed ROS production by haemocytes. Nitric oxide synthase inhibitors, NMMA and L-NIO, did not suppress ROS production, indicating that the observed oxidation of DCFH-DA is not mediated by nitric oxide. These results show unequivocally that haemocyte oxidative burst is active in M. mercenaria and, therefore, is a likely mechanism in host response to pathogens and parasites.     

Feeding ecology of Xenoturbella bocki (phylum Xenoturbellida) revealed by genetic barcoding

The benthic marine worm Xenoturbella is frequently contaminated with molluscan DNA, which had earlier caused confusion resulting in a suggested bivalve relationship. In order to find the source of the contaminant, we have used molluscan sequences derived from Xenoturbella and compared them to barcodes obtained from several individuals of the nonmicroscopic molluscs sharing the same environment as Xenoturbella. Using cytochrome oxidase 1, we found the contaminating sequences to be 98% similar to the bivalve Ennucula tenuis. Using the highly variable D1-D2 region of the large ribosomal subunit in Xenoturbella, we found three distinct species of contaminating molluscs, one of which is 99% similar to the bivalve Abra nitida, one of the most abundant bivalves in the Gullmarsfjord where Xenoturbella was found, and another 99% similar to the bivalve Nucula sulcata. These data clearly show that Xenoturbella only contains molluscan DNA originating from bivalves living in the same environment, refuting former hypotheses of a bivalve relationship. In addition, these data suggest that Xenoturbella feeds specifically on bivalve prey from multiple species, possibly in the form of eggs and larvae.     

An unusual cysteine-containing histone H1-like protein and two protamine-like proteins are the major nuclear proteins of the sperm of the bivalve mollusc Macoma nasuta

The sperm-specific protamine-like (PL) components PL-I, PL-II, and PL-III from the sperm of the bent-nose clam Macoma nasuta have been isolated and characterized for the first time. These proteins coexist in the sperm nuclei with a small percentage of a full histone complement. All of them have a very similar amino acid composition, following what seems to be the general composition prototype for the class Bivalvia (Ausió, J. (1986) Comp. Biochem. Physiol. B Comp. Biochem. 85, 439-449). Nevertheless, they have different molecular weights (PL-I = 23,500, PL-II = 15,600, and PL-III = 7,900) as measured by sedimentation equilibrium in the analytical ultracentrifuge. Furthermore, the PL-I component shares common features with the proteins of the histone H1 family. Yet, it is very unusual, for it contains 2 cysteine residues that are located in the trypsin-resistant core of this protein. The protamine-like fraction PL-III exhibits intraspecific microheterogeneity which is reflected by the presence of two protein variants which most probably are the result of an allelic polymorphism.     

Comparison of antigenicity among haemocytes of seven bivalve species by monoclonal antibodies against haemocytes of scallop (Chlamys farreri)

In this paper, haemocyte antigenicity of seven bivalve species (scallop (Chlamys farreri), bay scallop (Argoecten irradians), oyster (Crassostrea talienwhanensis), asiatic hard clam (Meretrix meretrix), monila clam (Ruditapes philipinarum), purplish washington clam (Saxidomus purpuratus) and horny ark (Scapharca subcrenta)) were analysed using monoclonal antibodies (MAbs) 1E7, 1F12, 2C6 and 2H5 against haemocytes of C. farreri, employed methods of immuno-dotblotting (IDB), indirect immunofluorescence assay (IIFA) and western-blotting (WB). The four MAbs react with haemocytes of seven bivalve species. As the results for both IDB and IIFA, MAb 1E7 was positive with haemocytes of R. philipinarum, MAb 1F12 with haemocytes of A. irradians, M. meretrix, R. philipinarum and S. purpuratus; MAb 2C6 with haemocytes of the other five bivalve species except for S. purpuratus. MAb 2H5 was negative with haemocytes of the other six bivalve species in IDB, but was positive with haemocytes of R. philipinarum and S. purpuratus in IIFA. Further experiments by WB showed MAb 1F12 was able to recognise the protein of A. irradians haemocyte at molecular weights of 156 and 80 kDa, haemocytes of M. meretris, R. philipinarum, S. purpuratus, at 60, 30, 58 kDa, respectively. MAb 2C6 recognised haemocyte M. meretris proteins at 50 and 37 kDa, A. irradians, C. talienwhanensis, R. philipinarum, S. subcrenta at 40, 38, 38, 45 kDa, respectively. There were no protein bands reacting with MAb 1E7 and MAb 2H5. The results indicate antigenic similarities exist among haemocytes of the seven bivalve species.     

Invasion of Laguncula pulchella (Gastropoda: Naticidae) and predator–prey interactions with bivalves on the Tona coast, Miyagi prefecture, northern Japan

On the Tona coast, Miyagi prefecture, northern Japan, interactions between the alien predator Laguncula pulchella and its bivalve prey were explored using annually collected quadrat samples over a 10 year period, from 2001 to 2010. A single L. pulchella individual was first recorded in 2002, and the density increased 12-fold from 2002 to 2004. In contrast, population densities of Ruditapes philippinarum and Macoma incongrua rapidly decreased during this interval. Based on frequency of predatory drill holes on the dead shells, more than 35 % of Ruditapes philippinarum and 20 % of Macoma incongrua died because of naticid predation after 2004, while Pillucina pisidium was less vulnerable to naticid predation. L. pulchella focused attacks on P. pisidium in 2004, when R. philippinarum and M. incongrua had became scarce due to naticid predation. This species-selective predation affected bivalve community structure, and caused disagreements in taxonomic composition and species’ rank-order abundance between the living bivalve community and the assemblage of dead shells. This approach (live–dead analysis), frequently used in paleoecological research, is a conservative tool to identify impacts of an alien predator on community structure. When sample size is sufficient, frequency of predatory drill holes in preferred prey species is likely to reflect predation intensity.     

An i-type lysozyme from the Asiatic hard clam Meretrix meretrix potentially functioning in host immunity

Lysozymes function in animal immunity. Three types of lysozyme have been identified in animal kingdom and most lysozymes identified from bivalve molluscs belong to the invertebrate (i) type. In this research, we cloned and sequenced a new i-type lysozyme, named MmeLys, from the Asiatic hard clam Meretrix meretrix. MmeLys cDNA was constituted of 552 bp, with a 441 bp open reading frame encoding a 146 amino acid polypeptide. The encoded polypeptide was predicted to have a 15 amino acid signal peptide, and a 131 amino acid mature protein with a theoretical mass of 14601.44 Da and an isoelectric point (pI) of 7.14. MmeLys amino acid sequence bore 64% identity with the Manila clam (Venerupis philippinarum) i-type lysozyme and was grouped with other veneroid i-type lysozymes in a bivalve lysozyme phylogenetic tree predicted using Neighbor-Jointing method. Recombinantly expressed MmeLys showed lysozyme activity and strong antibacterial activity against Gram positive and Gram negative bacteria. MmeLys mRNA and protein were detected to be mainly produced in hepatopancreas and gill by the methods of semi-quantitative RT-PCR and western blotting. In addition, MmeLys gene expression increased following Vibrio parahaemolyticus challenge. Results of this research indicated that MmeLys represents a new i-type lysozyme that likely functions in M. meretrix immunity.