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.     

Bioaccumulation and distribution of mercury in the hard clam,Meretrix lusoria (Bivalvia:Veneidae)

1. Total mercury concentration in whole soft parts of the hard clam, Meretrix lusoria, increased significantly with increasing exposure concentrations. It reached 4.247–7.084 and 9.956–13.643 μg T-Hg/g dry weight in 5 and 50 μg/l Hg mercury solution, respectively, against a background level of 1.824–0.577μg T-Hg/g dry weight.2. Bioconcentration factor was 4–6 times higher in 5 μg/l Hg than that of 50 μg/l Hg. An estimated biological half-life of 20.2 and 18.4 days in 5 and 50 μg/l Hg, respectively, were also obtained after 14 days uptake.3. Accumulation of mercury in tissues of the clams was greater in the gills and viscera than in the mantle, adductor, foot, or hemolymph.4. The amount of mercury present in the gills is related to be a linear relationship with the mercury content of viscera.     

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⁺.     

Simultaneous flow cytometric detection of cellular c-myc protein, incorporated bromodeoxyuridine, and DNA

We describe a multivariate flow cytometric technique for simultaneous analysis of specific nuclear protein, bromodeoxyuridine (BrdUrd) incorporated into DNA and DNA content in single cells in suspension. The procedure involves fixation of BrdUrd-exposed cells with paraformaldehyde, heat denaturation of cellular DNA, followed by sequential immunochemical reactions to label incorporated BrdUrd and nuclear protein, and finally staining of total DNA with propidium iodide. The cells are analyzed flow cytometrically and multivariate data acquired in list mode to facilitate analyses of heterogeneous subpopulations. We applied this technique to measure c-myc protein, incorporated BrdUrd, and DNA content in subpopulations present in a recombinant Chinese hamster ovary (CHO) cell line carrying approximately 800 copies of murine c-myc sequences under control of an inducible heat shock promoter.     

Quantitative assessment of phagocytic activity of hemocytes in the prawn, Penaeus merguiensis, by flow cytometric analysis

BACKGROUND: The blood cells of crustaceans are involved in phagocytosis of invading microorganisms, contributing to their defense mechanisms. In this study, phagocytic activity of hemocytes of the prawn, Penaeus merguiensis, was quantitated by means of flow cytometric analysis. METHOD: This study was done in vitro. Hemolymph, which was extracted from prawns, was mixed with an equal volume of anticoagulant. Heat-killed Escherichia coli prestained with propidium iodide (PI) was then added. Hemocytes were fixed at various time intervals for flow cytometric analysis. This study was supplemented with electron micrographs using transmission electron microscopy (TEM), which showed three populations of hemocytes. RESULTS: It was observed that those hemocytes that were more active engulfed and digested bacteria readily, thus having higher red fluorescence intensity. The phagocytic activity was expressed as fluorescence unit or engulfed E. coli number per hemocyte. CONCLUSIONS: With this approach, the phagocytic and cellular activity of individual hemocyte populations could be studied quantitatively.     

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.     

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.     

In vitro toxicity of nitrite on haemocytes of the tiger shrimp, Penaeus monodon, using flow cytometric analysis

This study investigated the in vitro effects of nitrite on reactive oxygen species (ROS) production, NO production, esterase activity and cell apoptosis of Penaeus monodon haemocytes. Haemocytes were in vitro exposed to different dose of nitrite (0, 0.1, 0.5, 1, 5 and 10 μM). Cellular responses of nitrite-treated haemocytes were determined by flow cytometry. The results revealed that haemocytes treated by nitrite in vitro showed conspicuous time- and dose-dependent decreases in ROS and NO production as well as esterase activity. Additionally, 0.1 and 0.5 μM nitrite did not affect the apoptotic cell ratio during the 3h experimental time, while significant increases in apoptotic cells were observed after haemocyte exposure to nitrite at 1 μM for 3h, and at 5 or 10 μM for 1h. These results indicated that nitrite suppresses cellular functions, including production of ROS and NO, and activity of esterase. Cell apoptosis of haemocytes would be induced by extracellular nitrite as doses exceed 1 μM.     

Crystalline arrangement and nanostructure of aragonitic crossed lamellar layers of the Meretrix lusoria shell

The crystallographic microstructure of Meretrix lusoria shells was investigated using scanning electron microscopy (SEM), X-ray diffractometry (XRD), and transmission electron microscopy (TEM). Crystallite sizes were determined by XRD analysis as 72 nm, which was quite similar to the 70 nm as measured by SEM. The shell comprised aggregates of hexagonal plates of aragonite (500 nm wide, 70 nm high) and organic matter. These plates were fourth-order units of an aragonitic crossed order lamellar structure. Subsequent TEM images showed the hexagonal plates’ nanostructure. The electron diffraction pattern of the fourth-order units revealed a consistent orientation of the hexagonal plates. The fourth-order lamellae (hexagonal crystallites) were piled up in the [0 0 1] direction to produce slender prisms (third-order lamellae), arranged mutually parallel, thereby forming a broad tablet (second-order lamellae). The second-order lamellae were piled up in different directions to form the first-order lamellae. The orientation level obtained from XRD and SEM images showed that the crossed lamellar layer was piled up curvilinearly, forming semi-circular growth lines. X-ray diffraction patterns of the cross-sections of the middle layer (vertical and parallel to the growth line) showed that the c axes of aragonite have a disposition of about 20° to the growth direction.     

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)     

Morphological and cytoenzymatic characterization of haemocytes of the venus clam Chamelea gallina

A morphological and enzymatic characterization of Chamelea gallina haemocytes was carried out as a prerequisite for further studies on venus clam immunobiology. Two main types of circulating haemocytes were identified (1) hyalinocytes (79.2%), agranular cells with a central nucleus surrounded by a little cytoplasm, and (2) granulocytes (16.5%), smaller granular cells with smaller nuclei. Small cells with a strongly basophilic nucleus and a thin layer of peripheral cytoplasm, probably undifferentiated blast cells (4.3%), were also observed. Both granulocytes and hyalinocytes can assume a spreading or round morphology. The enzymatic activities of haemocytes were also investigated. Some of the granulocytes and hyalinocytes were positive for hydrolytic enzymes, suggesting a role for these cells in phagocytosis; no oxidative enzymes were detected in C. gallina haemocytes. Granulocytes and hyalinocytes can easily adhere to the substratum and exhibit a low phagocytosis activity towards foreign particles (6.3%), whereas the fraction of cells containing ingested material significantly increased after pre-incubation of test particles with cell-free haemolymph, which suggests the presence of opsonin(s) in the haemolymph.     

A flow cytometric approach to the study of crustacean cellular immunity

Responses of hemocytes from the crayfish Procambarus zonangulus to stimulation by fungal cell walls (zymosan A) were measured by flow cytometry. Changes in hemocyte physical characteristics were assessed flow cytometrically using forward- and side-scatter light parameters, and viability was measured by two-color fluorescent staining with calcein-AM and ethidium homodimer 1. The main effects of zymosan A on crayfish hemocytes were reduction in cell size and viability compared to control mixtures (hemocytes in buffer only). Adding diethyldithiocarbamic acid, an inhibitor of phenoloxidase, to hemocyte and zymosan mixtures delayed the time course of cell size reduction and cell death compared to zymosan-positive controls. The inclusion of trypsin inhibitor in reaction mixtures further delayed the reduction in hemocyte size and cell death, thereby indicating that a proteolytic cascade, along with prophenoloxidase activation, played a key role in generating signal molecules which mediate these cellular responses. In addition to traditional methods such as microscopy and protein chemistry, flow cytometry can provide a simple, reproducible, and sensitive method for evaluating invertebrate hemocyte responses to immunological stimuli.     

Morphologic, cytometric and functional characterisation of abalone (Haliotis tuberculata) haemocytes

This work presents the first detailed microscopic and functional analysis of the haemocytes of an abalone; the European Haliotis tuberculata. It is shown that in contrast to the situation in bivalves, only very few basophilic "granulocytes" could be found and exclusively with a histological stain. Neither flow cytometry, phase contrast observation nor transmission electron microscopy were able to detect any granular cells. The large majority of cells was constituted of "hyalinocytes", which could be sorted by flow cytometry, for the first time, into small (blast-like) and large cells. This permits a detailed analysis of haemocytes and especially of the lowly represented blast-like cells. The differences in haemolymph cell composition between bivalves and gastropods is reviewed in depth and discussed in view of the new data we present. Most of the abalone haemocytes analysed harbour many vacuoles, large glycogen deposits, lipid inclusions and acidic compartments. However, although the number of these "inclusions" was rather variable in between individual hyalinocytes, these experiments did not allow to discern subpopulations using these criteria, and the population appears more as a "differentiation continuum". Haemocytes adhere very rapidly and are immunologically active as they quickly phagocytose latex beads and zymozan particles. This study is the first step towards understanding the H. tuberculata immune system by adapting new tools to gastropods and in providing a first detailed morpho-functional study of their haemocytes.     

A novel flow cytometric protocol for assessment of yeast cell adhesion

Microbial adhesion is a field of recognized relevance and, as such, an impressive array of tools has been developed to understand its molecular mechanisms and ultimately for its quantification. Some of the major limitations found within these methodologies concern the incubation time, the small number of cells analyzed, and the operator's subjectivity. To overcome these aspects, we have developed a quantitative method to measure yeast cells' adhesion through flow cytometry. In this methodology, a suspension of yeast cells is mixed with green fluorescent polystyrene microspheres (uncoated or coated with host proteins). Within 2 h, an adhesion profile is obtained based on two parameters: percentage and cells-microsphere population's distribution pattern. This flow cytometry protocol represents a useful tool to quantify yeast adhesion to different substrata in a large scale, providing manifold data in a speedy and informative manner.     

New fluorochromes, compatible with high wavelength excitation, for flow cytometric analysis of cellular nucleic acids

The spectroscopic properties of three new dyes, EK4, VL772, and LL585, free and bound to nucleic acids, are presented, with particular emphasis on their potential use in flow cytometry. Two of these dyes, EK4 and LL585, exhibit red fluorescence, while dye VL772 exhibits yellow fluorescence. Dye LL585 exhibits specificity for DNA, relative to RNA, and a marked enhancement of fluorescence efficiency upon binding to DNA, needed for a red fluorescent DNA-specific stain for flow cytometry. The dye penetrates live cells, although uniformity of nuclear fluorescence, as evidenced by DNA flow histograms, is better if the cells are first permeabilized with Triton X-100. Dye VL772 exhibits yellow fluorescence and little DNA-RNA discrimination, but may prove useful in conjunction with dye LL585 when simultaneous assay of cellular RNA and DNA is desired. Dye EK4 shares properties of the other two dyes but fluoresces with much less efficiency. Dyes LL585 and VL772, used singly, as a pair, or in combination with blue-fluorescing DNA specific dyes, such as bisbenzimidazole derivatives, should permit new, convenient analyses of the content and organization of cellular nucleic acids.     

Comprehensive quality assessment approach for flow cytometric immunophenotyping of human lymphocytes

Flow cytometric immunophenotypic (IPT) evaluation has become an important adjunct to clinical patient management and epidemiological studies. This has precipitated a need for stringent quality assessment (QA) procedures to ascertain data integrity. We evaluated a QA approach to monitor all elements of the immunophenotyping process, inclusive of blood collection and processing procedures as well as of staining reagent and instrument performance. Central to our approach was preparation each day, in parallel with clinical analytes, of lymphocytes from healthy donors, selected from a 15 donor panel. IPT parameters evaluated over a 19 month period included frequencies of CD3+, CD4+, CD8+, and CD20+ lymphocytes and the ratio of CD4+ to CD8+ lymphocytes. The sensitivity for analytical error detection was reflected by median coefficients of variation of these parameters within individual panel donors, which were 4.1%, 4.5%, 3.9%, 8.2%, and 10.1%, respectively. IPT parameter values were determined each day for two of the panel donors, then averaged and standardized to obtain a quality or Q variate, which was the basis of QA. Error detection sensitivity decreased 0.6-1.7% and the number of false rejections increased 1.2-3.3% when one panel donor rather than two was used daily for QA. This study also illuminated important aspects of what constitutes the norm for longitudinal IPT parameter variation in healthy individuals including: 1) a generally low degree of temporal parameter variation within individual donors, but 2) significant differences between donors with respect to variance estimates for CD3+ and CD8+ lymphocyte frequencies and CD4+/CD8+ lymphocyte ratios, and 3) an apparent seasonal pattern of variation in CD4+ T-cell frequencies.     

Simultaneous flow cytometric analysis of two cell surface markers,telomere length,and DNA content

BACKGROUND: Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate the harsh conditions needed for DNA denaturation during hybridization of the telomere-specific PNA probe. We overcame these problems and developed a method for measuring telomere length in cell subsets characterized by the expression of two surface antigens. METHODS: Alexa Fluor 488 and Alexa Fluor 546 were used for cell surface staining. Antigen-antibody complexes were covalently cross-linked onto the cell membrane before Flow-FISH. Cells were hybridized with a PNA probe conjugated to cyanine 5 (Cy5). Hoechst 33342 (HO342) was added for determination of cellular DNA content. For assay standardization, we added an aliquot of a single batch of 1,301 cells to each sample as an internal control before hybridization with the PNA probe. Samples were prepared in duplicate and analyzed on a standard three-laser BD LSR flow cytometer. For assay validation, the same samples were analyzed in parallel to correlate the percentage of telomere length of the sample versus 1,301 control cells to the mean size of terminal restriction fragments (TRFs) of DNA as determined by Southern gel analysis. RESULTS: The method permitted clear identification of lymphocyte subsets in samples hybridized for Flow-FISH, with subset frequencies comparable to those of untreated samples. At a concentration of 10 nM, the Cy5-labeled telomere-specific PNA probe produced a bright fluorescence signal well separated from background. Addition of HO342 in low concentration did not interfere with Cy5 telomere fluorescence, produced adequate DNA histograms, and permitted clear identification of cell phenotype. The probe concentration of 10 nM also proved optimal for inclusion of 1,301 control cells for assay standardization. Telomere length estimations by the current method correlated highly with TRF calculations by Southern gel hybridization (r(2)= 0.9, P = 0.0003). Application of our protocol to the analysis of human CD8CD28 lymphocyte subsets showed that CD8(+bright)CD28(-) lymphocytes generally exhibit shorter telomeres than CD8(+bright)CD28(+) cells. These data concurred with previous results of telomere shortening in CD8(+)CD28(-) T cells that were obtained by using different techniques. CONCLUSIONS: The multiparameter Flow-FISH protocol permitted rapid determination of differences in telomere length in subpopulations characterized by two surface markers without prior cell separation.     

The complete mitochondrial genome of the hard clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis>

Veneridae is a diverse, commercially important, and cosmopolitan family. Here we present the complete mitochondrial genome of the hard clam Meretrix meretrix (Bivalvia: Veneridae). The entire mitochondrial genome (mitogenome) sequence of M. meretrix is 19,826 bp in length, and contains 37 genes including 12 protein-coding genes, 2 ribosomal RNAs, and 23 tRNAs. All genes are encoded on the heavy strand. In contrast to the typical animal mitochondrial genome, it lacks the protein-coding gene ATP8, and has only one copy of the tRNA^Ser gene, but three duplications of the tRNA^Gln, which is the first report among the present molluscan mtDNAs. We observed that the gene arrangement between M. meretrix and M. petechialis is same except one more tRNAGln gene in M. meretrix., and the sequence similarity is as high as 99%, indicating that M. petechialis and M. meretrix could be treated as a junior synonym of M. meretrix. Maximum Likelihood and Bayeslan analysis of 12 concatenated protein-coding amino acid sequences place the Unionidae as a sister group to other bivalves, which reflects the general opinion that the Unionidae deverged very early in Bivalvia evolution.     

Cloning and characterization of a hsp70 gene from Asiatic hard clam Meretrix meretrix which is involved in the immune response against bacterial infection

In the present study, a 71.43 kDa heat shock protein cDNA was cloned from Asiatic hard clam Meretrix meretrix. The cDNA was 2292 bp, containing an open reading frame (ORF) of 1959 bp, which encodes a protein of 652 amino acids with a theoretical molecular weight of 71.43 kDa and an isoelectric point of 5.32. Based on the amino acid sequence analysis and phylogenetic analysis, this hsp70 cDNA is a member of cytoplasmic hsc70 (constitutive genes) subfamily in the hsp70 family, and is designated as MmeHsc71. Quantitative RT-PCR was carried out to compare the spatial and temporal expression patterns of MmeHsc71 in the mRNA level between control clams and Vibrio parahaemolyticus-infected clams. Spatially, MmeHsc71 mRNA was found in all tested tissues, including foot, hepatopancreas, mantle and gill. MmeHsc71 mRNA expression level in hepatopancreas and gill displayed a significant increase in vibrio-challenged clams at 24h post-infection compared to control clams (P < 0.05). Temporally, there was a significant increase of MmeHsc71 mRNA level in hepathopancreas of vibrio-challenged clams compared to control clams at 6, 12, and 24h post-challenge, respectively. The result of quantitative immunofluorescence also indicated that there was obvious increase of MmeHsc71 in hepatopancreas of vibrio-challenged clams compared to control clams in protein level at 24h post-infection. The results suggested that MmeHsc71 may play an important role in mediating the immune responses of M. meretrix to bacterial challenge.