Toxic effects of mercury on the hard clam,Meretrix lusoria,in various salinities

1. The 96-hr lc₅₀ values for juvenile hard clams, Meretrix lusoria, were 328, 392 and 194 μg/l Hg in 10, 20 and 30 ppt salinities at 25 ± 1°C, respectively; for adult hard clams 341 and 140 μg/l Hg in 20 and 30 ppt salinities, respectively.2. Acclimatizing the adult clams to low salinity of 10 ppt lessened the toxicity of mercury. However, juvenile animals appeared to be more sensitive to mercury poisoning after 96 hr exposure in 10 ppt salinity.3. All embryos exposed to 40 μg/l Hg and above died within 30 hr. In the control, 44% of hatched embryos had developed into D-stage larvae, while those exposed to 20 μg/l Hg were still in the trochophore stage. Most of the retarded larvae developed into abnormal forms within 30 hr at 28°C in 15 ppt salinity.4. In order to maintain water quality and protect natural resources, the recommended safe level of mercury is 0.046 (0.039–0.053) μg/l Hg, based on the estimated 30-hr EC₅₀ for the clam embryos, with an application factor of 0.01.     

Purification and kinetic characteristics of strombine dehydrogenase from the foot muscle of the hard clam (Meretrix lusoria)

Strombine dehydrogenase (SDH, EC 1.5.1.22) from the foot of the hard clam Meretrix lusoria was purified over 470-fold to apparent homogeneity. It has a monomeric structure with a relative molecular mass of 46,000. Two isoenzymes were identified with isoelectric points of 6.83 and 6.88. SDH is heat labile, and has pH and temperature optima of 7.4–7.6 and 45–46 °C, respectively. l-Alanine, glycine, and pyruvate are the preferred substrates. l-Serine is the third preferred amino acid. Iminodiacetate with the lowest K_i of SDH at both pH 6.5 and 7.5 was the strongest inhibitor among succinate, acetate, iminodiacetate, oxaloacetate, and l-/d-lactate. The inhibitory activities of succinate at pH 6.5, and iminodiacetate and oxaloacetate at pH 7.5 on the SDH were higher. These inhibitors are either competitive or mixed-competitive inhibitors. Half of the enzymatic activity of SDH was inhibited by 0.2 mM Fe³⁺ and 0.6 mM Zn²⁺.     

A comparative study of alkaline phosphatases among human placenta, bovine milk, hepatopancreases of shrimp Penaeus monodon (Crustacea: Decapoda) and clam Meretrix lusoria (Bivalvia: Veneidae): to obtain an alkaline phosphatase with improved characteristics as a reporter

1. Alkaline phosphatases were purified from human placenta, bovine milk, shrimp and clam with a final spec. act. of 67,000, 32,000, 22,000 and 15,000 U/mg of protein respectively. 2. The alkaline phosphatase from Meretrix lusoria is unique with its thermostability at 65 degrees C for 30 min; whereas the remaining enzymes studied, including the human placental alkaline phosphatase, are inactivated and have negligible activities. 3. The alkaline phosphatase from Penaeus monodon can be differentiated by its pH optimum at 9.0; the remaining enzymes studied have their optimal pH at 10.0. 4. The alkaline phosphatases from shrimp and clam are proposed to be applied as "reporters" in the study of mammalian cells.     

Simultaneous flow cytometric assessment for cellular types and phagocytic abilities of the haemocytes of the hard clam, Meretrix lusoria

The aim of this study was to devise a simple protocol for flow cytometric analysis to separate various haemocytic populations of the hard clam Meretrix lusoria based on the mitochondrial membrane potential diversity detected by the fluorescence probe 3,3-dihexyloxacarbocyanine iodide (DiOC6). Compared with the traditional technique for separation of haemocytic populations, continuous Percoll gradient centrifugation, our novel method was more efficient and yielded a higher ratio in separating the clams' haemocytic populations. Based on fluorescence 1 (FL-1) and side scatter (SSC) analysis for haemocytes stained with various fluorescent densities of DiOC6 using flow cytometer, the data showed that there were three obvious cell regions R1, R2, and R3, identified by hyalinocytes, small granulocytes and large granulocytes, respectively. At the same time our results showed that the percentages of haemocytes in R1, R2, and R3 were 49.71+/-0.65%, 19.35+/-00.74%, 30.94+/-0.69%, respectively. After classifying the haemocytic populations, phagocytic activity of the haemocytes was simultaneously analysed with phycoerythrin (PE)-labeled Vibrio vulnificus and detected by flow cytometry. Our results demonstrated that there were higher percentages of large granulocytes compared with hyalinocytes and the percentage of small granulocytes was related to the mitochondrial membrane potential and phagocytic activities.     

Calcium sensitivy of foot muscle myosin from clam (Meretrix lusoria).

1. It was found that Mg-ATPase of clam foot myosin is strongly activated by calcium or strontium ions and is as sensitive to those divalent cations as the Mg-ATPase and superprecipitation of rabbit skeletal acto-clam foot myosin are. 2. It was also found that desensitization and resensitization of clam foot myosin result in the loss of superprecipitation activity with acto-desensitized myosin and in its recovery with acto-resensitized myosin, respectively. However, the ATP-ASE activity in the absence of calcium ions rises with acto-desensitized myosin and falls again with acto-resensitized myosin. 3. It is thus proposed that the primary role of the EDTA-light chain component in calcium regulation is to inhibit myosin-ATPase rather than to inhibit the actin-myosin interaction.     

Ostrincola koe (Copepoda,Myicolidae) and mass mortality of cultured hard clam (Meretrix meretrix) in China

We discuss the primary cause of mass mortalities of cultured hard clam (Meretrix meretrix) which occurred in 1988 and 1989 in southern Jiansu, China. Based on a re-examination of the old samples and analysis of information from Japan and Korea, it is concluded that the population explosion of the parasitic copepod, Ostrincola koe Tanaka, in the clam mantle cavity, combined with an acute vibriosis in the intestine, were responsible for the mass mortality. Other incidences of hard clam mass mortalities in China, Korea and Taiwan in the 1970s and 1980s are also discussed.     

The clam Meretrix lamarckii (Bivalvia: Veneridae) is a rich repository of marine lactic acid bacterial strains

Lactic acid bacteria (LAB) were isolated from the intestinal tract of the wild clam Meretrix lamarckii caught from the coastal waters of Kashima, Ibaraki, Japan. As many as 415 isolates were obtained using the culture method, of which 70 were considered presumptive LAB strains based on phenotypic tests. Phylogenetic analysis of these presumptive isolates of LAB based on the sequence of the 16S rRNA gene demonstrated that the species belonged to several genera of Lactobacillus, Lactococcus and Pediococcus. Interestingly, however, the species composition was different between the samples in July and October 2010. Further analyses based on the fermentation profiles revealed that the LAB from the clam caught in July 2010 were identified to be Lactobacillus curvatus, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris and Pediococcus pentosaceus, whereas those in October 2010 were identified to be Lactobacillus plantarum, Lactococcus lactis subsp. lactis and P. pentosaceus. The diversity of LAB in the intestinal tract of the clam suggests that the filter feeder bivalves such as M. lamarckii are a rich repository of marine isolates of LAB.     

Immobilized Isochrysis galbana (Haptophyta) for long-term storage and applications for feed and water quality control in clam (Meretrix lusoria) cultures

The marine microalga Isochrysisgalbana was cultivated and entrapped inalginate beads for long-term storage. Theentrapped cells were alive and maintainedtheir physiological activities after oneyear of storage in absolute darkness at4 ^°C without a liquid medium. Thenumber of cells in the beads increased morethan 32 times when they were subsequentlyre-cultured in an aqueous medium for fiveweeks, showing that they had remained aliveduring storage. TEM observations showedthat the entrapped cells reduced their cellcovering and pyrenoid size compared withthe normal free-living cells afterlong-term storage. The algal beads werealso applied to feed and water qualitycontrol in clam cultures' leading to amarked decrease in ammonium concentrations.Algal cells escaped from the beads provideda food source for the clams. This mightreduce the cost of clam culture compared totraditional culture methods. Therefore,immobilized I. galbana can be usedfor long-term preservation of algal stockin the laboratory and applied practicallyto clam cultures.     

The occurrence of glycosphingolipids containing mannose in the sea-water bivalve, Meretrix lusoria (Hamaguri)

Total neutral and acidic glycosphingolipids were prepared from whole tissues of the sea-water bivalve, Meretrix lusoria, and the former preparation was further fractionated into subgroups by silicic acid column chromatography. The fractions obtained as mono-(ceramide monosaccharide, CMS), di-(CDS) and triglycosylceramides (CTS) were characterized by thin-layer chromatography, partial hydrolysis with exoglycosidases, methylation studies, CrO3 oxidation, and GLC analysis of the component sugars, fatty acids and long-chain bases. The following structures are proposed: Gal-Cer and Glc-Cer for CMS, Gal(beta 1----4)Glc-Cer and Man(beta 1----4)Glc-Cer (MlOse2Cer) for CDS, Man(alpha 1----3)Man(beta 1----4)Glc-Cer (MlOse3Cer) and Gal(alpha 1----3)Man(beta 1----4)Glc-Cer (II3 alpha Gal-MlOse2Cer) for CTS. To our knowledge II3 alpha Gal-MlOse2Cer has not previously been reported. The fatty acid composition of CMS, CDS, and CTS consisted almost entirely of saturated C16-C24 acids with large amounts of 2-hydroxypalmitic acid and 2-hydroxystearic acid. The long-chain bases consisted of 4-sphingenine and 4,8-sphingadienine. More complex neutral glycolipids than CTS, as well as an acidic glycolipid, were examined by TLC and GLC of the constituent sugars, and an immunochemical technique.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reproductive cycle and parasitism in the clam Anomalocardia brasiliana (Bivalvia: Veneridae)

The reproductive cycle and parasitism in the clam Anomalocardia brasiliana were studied in two different areas, an intermediate beach (Cidade) and a tidal flat (Araçá), in Southeast Brazil. Four gametogenic stages were described for males and females in both areas. Mature and spawning individuals were present throughout the period of study at Cidade and Araçá; however, major temporal differences were recorded between sites. Whereas only a few individuals in the resting stage were recorded at Cidade throughout the study period, resting stage individuals were frequent at Araçá. Moreover, a shorter period of gametogenesis was observed at Araçá than at Cidade. Oocyte number was the most effective parameter to differentiate gametogenic stages. An unidentified digenetic trematode was the only parasite infecting A. brasiliana at the study sites, and caused castration of all hosts. Parasite prevalence (proportion of infected hosts) was similar and low (ca 7.5%) at Cidade and Araçá and therefore is not expected to compromise the reproductive output of these populations. Comparison with data from previous studies suggests a latitudinal pattern for the reproductive biology of A. brasiliana, with resting stages occurring only at two high-latitude sites; however, no evidence was found for a latitudinal pattern of trematode prevalence in this species.     

Reproductive strategy of the semelparous clam Gaimardia bahamondei (Bivalvia,Gaimardiidae)

Abstract. Semelparity is one of the most drastic reproductive strategies found among marine invertebrates. It is frequently found in species whose members have small adult sizes and brood embryos internally. In this study, we describe the reproductive strategy of the bivalve Gaimardia bahamondei to explore the possible causes of the association between semelparity and internal brooding. Males of this species exhibit continuous gonadal activity throughout the breeding season. Apparently continuous spawning of sperm is associated with an abundance of captured sperm in the adfrontal region of the gill filaments of both males and females. Females are capable of brooding three cohorts of embryos simultaneously while also producing three new cohorts of oocytes. This suggests that females are able to generate at least six cohorts of embryos during the breeding season. Embryos are brooded in the suprabranchial chamber and are individually surrounded by a membrane with a projecting peduncle; embryos are anchored by this peduncle to the adfrontal region of the gill filaments. Members of the youngest cohort differ in size, color, and shell ornamentation from members of the two older cohorts. There is no difference between members of the two older cohorts in size, but there is with respect to coloring and shell ornamentation. The importance of the embryonic cohorts in terms of their percentage of the total number of embryos varied among brooding females, suggesting among‐female variation in the timing of release of the oldest cohort of embryos. Members of this cohort break loose from the gills, lose their surrounding membrane, and fall into the ventral region of the suprabranchial chamber, from which they are evacuated to the exterior. Continuous sperm production in males and the production of at least six cohorts of embryos in females suggest that the costs of reproduction are high, which may partly explain the semelparity identified in this species.     

Cytogenetics of the razor clam Solen marginatus (Mollusca: Bivalvia: Solenidae)

The razor clam Solen marginatus has a diploid chromosome number of 38. The karyotype consists of one metacentric/submetacentric, three submetacentric/metacentric, five submetacentric, one submetacentric/subtelocentric, one subtelocentric/submetacentric, six subtelocentric and two telocentric chromosome pairs. Staining with chromomycin A3 revealed bright positive bands subcentromerically in the long arms of one medium-sized subtelocentric pair, while DAPI staining showed uniform fluorescence in all chromosomes of the complement. Fluorescence in situ hybridization using an 18S-5.8S-28S rDNA probe locates these loci at the subcentromeric region of one subtelocentric pair and at the subtelomeric region of another subtelocentric pair.     

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.     

Copepod parasites of a commercial clam (Meretrix meretrix) from Phuket,Thailand

Three species of copepods are reported from hard clams, Meretrix meretrix (L.), obtained from the market in Phuket, Thailand. They are: Conchyliurus bombasticus Reddiah (Clausidiidae), Ostrincola portonoviensis Reddiah (Myicolidae), and Lichomolgus similis Ho & Kim (Lichomolgidae). The first two species are redescribed based on the newly collected material. Conchyliurus fragilis Reddiah is proposed to be relegated to a synonym of C. bombasticus. L. similis is recorded for the first time from the Indian Ocean.     

Larval morphology of the brooding clam Lasaea adansonii (Gmelin, 1791) (Bivalvia, Heterodonta, Galeommatoidea)

The strongly modified mode of development of the small and brooding galeommatoid bivalve Lasaea adansonii (Gmelin, 1791) [syn. Lasaea rubra (Montagu, 1803)] has been studied by means of transmission and scanning electron microscopy and by fluorescent staining of the muscular system and of two neurotracers, FMRFamide and serotonin. In addition, two developmental stages were visualized using computer-aided 3D-reconstruction. All larval stages of L. adansonii lack ciliary rings. The apical organ appears invaginated: the base of the duct contacts the cerebral ganglia and opens on the preoral region. Larval protonephridia are lacking. The adult kidneys develop independently of the pericardial cavity and contain a protonephridial part that enables excretory function until the pericardium is formed. The larval muscular system is composed of smooth muscle fibers; striated fibers are lacking. Posteriorly and immediately below the ligament, a paired cell of unknown function is present that contains serotonin and FMRFamide. In summary, L. adansonii exhibits the direct mode of development. Only few truly larval structures (e.g., the modified apical organ) are elaborated.     

Intracellular Cu/Zn superoxide dismutase (Cu/Zn-SOD) from hard clam Meretrix meretrix: its cDNA cloning, mRNA expression and enzyme activity

Hard clam (Meretrix meretrix) is an economically important bivalve in China. In the present study, a gene coding for an intracellular Cu/Zn-SOD was cloned and characterized from hard clam. The full-length cDNA of this Cu/Zn-SOD (designated as Mm-icCuZn-SOD) consisted of 1,383?bp, with a 462-bp of open reading frame (ORF) encoding 153 amino acids. Several highly conserved motifs, including the Cu/Zn binding sites [H(46), H(48), H(63), and H(119) for Cu binding; H(63), H(71), H(80), and D(83) for Zn binding], an intracellular disulfide bond and two Cu/Zn-SOD signatures were identified in Mm-icCu/Zn-SOD. The deduced amino acid sequence of Mm-icCu/Zn-SOD has a high degree of homology with the Cu/Zn-dependent SODs from other species, indicating that Mm-icCu/Zn-SOD should be a member of the intracellular Cu/Zn-dependent SOD family. Real-time PCR analysis showed that the highest level of Mm-icCu/Zn-SOD expression was in the hepatopancreas, while the lowest level occurred in the hemocytes. Hard clam challenged with Vibrio anguillarum showed a time-dependent increase in Mm-icCu/Zn-SOD expression that reached a maximum level after 6?h. Mm-icCu/Zn-SOD purified as a recombinant protein expressed in E. coli retained a high level of biological activity, 83?% after 10?min incubation at 10–50?°C, and more than 87?% after incubation in buffers with pH values between 2.2 and 10.2. These results indicated that Mm-icCu/Zn-SOD may play an important role in the innate immune system of hard clam.     

Moitessier's pea clam Pisidium moitessierianum (Bivalvia,Sphaeriidae): a cryptogenic mollusc in the Great Lakes

Pisidium moitessierianum Paladilhe, 1866, a small pea clam native to Europe, was identified for the first time from the lower Great Lakes basin based on an examination of historical collections of Pisidium performed by V. Sterki in 1894 and 1903 and new material collected during 1997 and 1998. During recent surveys, P. moitessierianum individuals were found in the St. Clair River delta, Lake St. Clair and western Lake Erie, but were not detected in the Detroit River or western Lake Ontario. Pisidium moitessierianum was collected on sand, silty sand and mud substrata from water depths ranging between 0.6 and 5.4 m. Populations occurred at an average density of 51 ind. m^–2 and included juveniles and adults. All individuals were less than 2.0 mm in length. We examined the structure of the umbos and hinge, surface sculpture and shape of the shell, and the anatomy of gills, mantle and nephridia in populations from the lower Great Lakes and Ukrainian inland basins (Dnieper River and Lake Beloye). The results indicated that the Great Lakes' pea clams match European specimens of P. moitessierianum in these conchological and anatomical characteristics. As with other nonindigenous sphaeriids in the Great Lakes, P. moitessierianum was likely introduced through shipping activities into the Great Lakes, possibly as early as the 1890s.     

Androgenetic reproduction in a freshwater diploid clam Corbicula fluminea (Bivalvia: Corbiculidae)

Two shell color types of the exotic bivalve Corbicula fluminea were collected in Kyoto city, Japan. DNA microfluorometry revealed that both types were diploids with non-reductional spermatozoa. Maternal chromosomes were found to be extruded as two polar bodies at the first meiosis, and the second meiosis could not be observed. Only the male pronucleus was present in the egg cytoplasm and became metaphase chromosomes at the first mitosis. The present study indicates that the diploid C. fluminea in Japan has the same mode of androgenetic reproduction as the triploid C. leana.     

Embrionary and larval development of the marine clam Tivela mactroides (Bivalvia: Veneridae) in Zulia State, Venezuela

The marine clam, Tivela mactroides, from Ca?o Sagua beach, Venezuela, was spawned and reared under laboratory conditions to monitor its early development. Spawning was spontaneous but in some cases it had to be induced by the additon of eggs and sperm. After fertilization, the embryonic development occurred at 5 hr approximately. Trochophore larvae were observed between eight and ten hours later. Straight-hinged veliger stage appeared 15 hr after fertilization. Transition from veliger stage to the umbo stage occurred about eight days after fertilization. Pediveliger stage was observed 22 days after fertilization. Metamorphosis of T. mactroides was not successful under our laboratory conditions; probably the bacterial contamination and subsequent mortalities were important factors constraining the final phase of the larval cycle. However, in a few cases young individuals were observed. We suspect that this was due to unfavorable conditions (e.g.: bacterial contamination, unsuitable food availability, etc.) and the broad variation in developmental times, suggesting that these stages might be particularly sensitive to environmental changes. These results may not necessarily reflect what happens under natural conditions.     

Ecotoxicological evaluation of tributyltin toxicity to the equilateral venus clam, Gomphina veneriformis (Bivalvia: Veneridae)

Tributyltin (TBT) is the most common pesticide in marine and freshwater environments. To evaluate the potential ecological risk posed by TBT, we measured biological responses such as growth rate, gonad index, sex ratio, the percentage of intersex gonads, filtration rate, and gill abnormalities in the equilateral venus clam (Gomphina veneriformis). Additionally, the biochemical and molecular responses were evaluated in G. veneriformis exposed to various concentrations of TBT. The growth of G. veneriformis was significantly delayed in a dose-dependent manner after exposure to all tested TBT concentrations. After TBT was administered to G. veneriformis, the gonad index decreased and the sex balance was altered. The percentage of intersex gonads also increased significantly in treated females, whereas no intersex gonads were detected in the solvent control group. Additionally, intersex gonads were detected in male G. veneriformis specimens exposed to relatively high TBT concentrations (20 μg L⁻¹). The filtration rate was also reduced in a dose-dependent manner in TBT-exposed G. veneriformis. We also noted abnormal gill morphology in TBT-exposed G. veneriformis. Furthermore, increases in antioxidant enzyme activities were observed in TBT-exposed G. veneriformis clams, regardless of dosage. Vitellogenin gene expression also increased significantly in a dose-dependent manner in G. veneriformis exposed to TBT. These results provide valuable information regarding our understanding of the toxicology of TBT in G. veneriformis. Moreover, the responses of biological and molecular factors could be utilized as information for risk assessments and marine monitoring of TBT toxicity.