Studies on the Flavor of Meats

Investigation on the changes of inosinic acid and its related substances in meats (chickens and pigs) revealed that the pathway of the formation and degradation of inosinic acid in meats was as follows: ATP→ADP→AMP→IMP→Inosine→Hypoxanthine. The time and the amount reaching maximum content of inosinic acid in meats were varied with the species of animals and slaughter methods.     

A comparison of the cholinesterases of an oyster (Crassostrea virginica) and a clam (Macrocallista nimbosa).

Cholinesterase activities in the hearts and ganglia of an oyster (Crassostrea virginica) and a venerid clam (Macrocallista nimbosa) were measured and compared. Tissue extracts were partially purified by ammonium sulfate fractionation followed by gel column chromatography. Enzymatic activity was assayed spectrophotometrically; substrates were acetyl-, butyryl-, and propionylthiocholine (ATC, BTC, PTC). Kinetic constants characterizing each enzyme were derived. At all substrate concentrations, the hydrolysis rates of both clam enzymes were in the order: BTC greater than PTC greater than ATC. With oyster enzymes the ranking was ATC greater than or equal to PTC greater BTC. The specific activities of oyster heart and ganglion enzymes were similar. In contrast, clam ganglion extracts were 75-100 times more active than clam heart extracts and, with any substrate, had greater activity than either oyster enzyme. All enzyme preparations proved to be homogeneous on the bases of constant substrate activity ratios in successive column fractions, and of intermediate velocities with mixed substrates. Six cholinesterase inhibitors were tested. The specific acetylcholinesterase antagonist, B.W. 62C47, WAS MUCH MORE EFFECTIVE AGAINST OYSTER ENZYMES, WHILE THE SPECIFIC ANTIBUTYRYLCHOLINESTERASE, ISO-OMPA, almost totally inhibited calm enzyme activity, but had little effect on oyster. Eserine was the most effective inhibitor of both enzymes. In conclusion, the enzymes in oyster tissues are acetylcholinesterases, while clam enzymes are butyrylcholinesterases. Nevertheless, clam ganglion esterase is sifficiently active to hydrolyze the physiological substrate, acetylcholine. These results explain the long-observed differences in isolated heart pharmacology between ostreid and venerid bivalves.     

Clams (Corbicula fluminea) as bioindicators of fecal contamination with Cryptosporidium and Giardia spp. in freshwater ecosystems in California

This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank exposure experiments to evaluate clams that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting Cryptosporidium parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.     

The diet of the mud clam Geloina coaxans (Mollusca, Bivalvia) as indicated by fatty acid markers in a subtropical mangrove forest of Okinawa, Japan

Fatty acid compositions in the tissues of the clam Geloina coaxans collected from Oura mangal, Okinawa, Japan, during the cold and warm seasons (January and July 2001, respectively) were compared with those in suspended materials (SM) in order to assess the clams' diet. In both seasons, the suspended mangrove detritus at the sediment-water interface was high as indicated by the mean percentage of even-numbered long-chain fatty acids in SM (12.8-18.4%). The contribution of this marker in the clam tissues, especially during the cold season (3.9%), indicates the consumption of mangrove detritus in considerable amounts by the clams. The occurrence of the fatty acids 16:1ω7, 18:1ω9, 18:2ω6 and 18:3ω3 in SM was most likely due to the mangrove detritus sources, whereas in the SM they together constituted 12.9% and 23.9% of total fatty acid contents during the cold and warm seasons, respectively. As a result, their contribution in the clam tissues was high in the cold (15.4%) and warm seasons (19.0%). These results indicate that mangrove detritus play a significant role in the clams' diet. The mean percentages of bacterial markers (odd-numbered branched fatty acids and vaccenic acid, 18:1ω7) in the SM and tissues during both seasons ranged from 8.1% to 9.5%. This indicates that the clam diet is also dependent on the attached bacteria on the partially decomposed leaf detritus suspended at the sediment-water interface. The relative contribution by microalgae markers (18:4ω3, 20:5ω3 and 22:6ω3) in clam tissues ranged from 4.3% to 7.6%, suggesting considerable microalgae sources in the diets.     

Proteomics and multilocus sequence analysis confirm intraspecific variability of Vibrio tapetis

Vibrio tapetis is the etiological agent of brown ring disease (BRD) in clams. Phenotypic, antigenic and genetic variability have been demonstrated, with three groups being established associated with host origin. In this work we analyze the variability of representative strains of these three groups, CECT 4600(T) and GR0202RD, isolated from Manila clam and carpet-shell clam, respectively, and HH6087, isolated from halibut, on the basis of the whole proteome analysis by 2D-PAGE and multilocus sequence analysis (MLSA). A quantitative analysis of the proteome match coefficient showed a similarity of 79% between the clam isolates, whereas fish isolate showed similarities lower than 70%. A preliminary mass spectrometry (MS) assay allowed the identification of 27 proteins including 50S ribosomal protein L9, riboflavin synthase β subunit, ribose-phosphate pyrophosphokinase and succinyl-CoA synthase α subunit. The MLSA approach gave similar results, showing a 99.4% similarity of the clam isolates, which was higher than that observed between the fish isolate and either clam strain (98.2%). The topology of the maximum parsimony tree, obtained from 2D-PAGE analysis, and the phylogenetic tree, constructed with the maximum likelihood algorithm from concatenated sequences of 16S rRNA gene and five housekeeping genes (atpA, pyrH, recA, rpoA and rpoD), was very similar, confirming the closer relationship between the two clam isolates.     

Molecular characterization of a glutathione peroxidase gene and its expression in the selected Vibrio-resistant population of the clam Meretrix meretrix

Glutathione peroxidase (GPx) is an important member of cellular enzymatic antioxidant system, which may be involved in pathogen defense of host. In the present study, a selenium-dependent glutathione peroxidase (MmeGPx) gene from clam Meretrix meretrix was cloned and analyzed. The MmeGPx gene was composed of two introns of 723 bp and 238 bp and an open reading frame (ORF) of 711 bp. The ORF encodes a protein of 237 amino acids with a putative selenocysteine residue encoded by an unusual stop codon. MmeGPx shares a higher level of similarity with human GPx 3 than with other human GPx isozymes. The level of MmeGPx mRNA roughly paralleled GPx enzyme activity in different tissues except in gills, with the highest mRNA expression and enzyme activity occurring in hepatopancreas. MmeGPx mRNA expressions were detected in different larval stages and the results showed that MmeGPx mRNA increased significantly in pediveliger stage, which may be a response to oxidative stress. After challenge of clam with a Vibrio parahaemolyticus-related bacterium (MM21), the expression of MmeGPx was significantly up-regulated at 6 h and 12 h in hepatopancreas, which suggested that MmeGPx may be involved in the immune response to MM21 infection. To better understand its role in the immunity of clam, the expression of MmeGPx in hepatopancreas was compared between a selected Vibrio-resistant population and a control population after immersion challenge with MM21. Early up-regulation of MmeGPx was observed in the resistant population. These results suggested that MmeGPx might be involved in maintaining the redox state of immune system, and the early immune response to pathogen infection may help the clam against pathogen infection.     

Movement and aggregation in the fluted giant clam (Tridacna squamosa L.)

Aquaculture has been the traditional focus of tridacnid giant clam research whereas their ecology and behaviour have received much less attention. This study was based on the observation that juvenile fluted giant clams (Tridacna squamosa), when evenly distributed in a tank, will move and aggregate over time. We observed movement in clams ranging from 10 to 313 mm in shell length and ‘climbing’ up the sides of tanks was noted for clams with lengths between 10 and 22 mm. Locomotion also occurred after byssal attachment to the substrate; there was a highly significant association between type of movement (i.e. translation, rotation and no movement) and presence of attachment. Tests for phototaxis were negative. Aggregation was examined by placing clams in regular patterns on grids. After three days in the aquarium and 24 h in the field, their positions were analysed to obtain a statistical parameter for ‘clumpiness’. This was found to be greater in the live clam runs than both random walk and random distribution simulations, suggesting that clams were attracted to conspecifics. The latter was tested by recording clam movement with respect to five types of fixed ‘targets’ (i.e. live clam, fouled clam shell, foul-free clam shell, random inanimate object and none). The test clams moved, non-significantly, towards live clam targets and displayed higher mobility compared to tests with other target objects; a negative correlation between mobility and clam length was also observed. A choice experiment using bidirectional water inflow with clam effluent as one source resulted in clams moving toward the effluent, offering the first direct support for positive chemotaxis among conspecifics in Bivalvia. Together, our results indicate the presence of chemical signalling among clams, leading to movement toward one another and clumping. Aggregation could serve several ecological functions, such as defence against predation, physical stabilisation and facilitation of reproduction. With worldwide decline in natural giant clam densities, the opportunity for conspecific clumping is reduced, and local stocks could be facing increased vulnerability to Allee affects.     

Development of a real time quantitative PCR assay for the hard clam pathogen Quahog Parasite Unknown (QPX)

Quahog Parasite Unknown (QPX) is a thraustochytrid pathogen responsible for catastrophic mortalities of the northern quahog (hard clam) Mercenaria mercenaria. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to assist research efforts on QPX ecology and pathology. Sensitivity of the assay was evaluated with serial dilutions of QPX-cultured cells to determine the lowest concentration of DNA that remained detectable in both the presence and absence of extraneous environmental substances. QPX cells were quantified before DNA extraction to calibrate standard curves to cell counts. Based on our results, the qPCR assay is able to quantify QPX within the range of 1 to several thousand organisms per reaction. Specificity of the assay was assessed by testing 29 thraustochytrid-like protists isolated from suspension-feeding bivalves from China, Oregon, Maryland, and Virginia. Application of the assay was demonstrated with positive qPCR results from naturally contaminated environmental samples including marine aggregates (i.e. marine snow), clam pseudofeces, and inflammatory nodules from infected clams. This quantitative assay for QPX will provide a valuable tool for characterizing QPX parasite abundances in coastal environments and for improving clam disease diagnostics.     

The effects of water depth and density on the growth of a unionid clam*

SUMMARY. 1. Unionid clams from Narrow Lake, Alberta, were collected to quantify the natural variation in growth, to assess the natural variation in abundance, age and size distribution, and growth with water depth in the lake, and to conduct in situ experiments to directly test the effects of water depth (temperature) and clam abundance on clam growth. 2. The unionid clam, Anodonta grandis simpsoniana, showed wide variation in length at a given age. There were no significant differences in growth between clams collected at 1,3, 5, and 7m depths in the lake despite marked differences in water temperature. The wide variation in clam biomass within each depth zone may have masked possible effects of water depth. 3. The effect of water depth and variation in clam density on clam growth was tested directly by stocking clams into small enclosures at densities equivalent to 50, 150, 250, 350 and 450g m-2 (live weight) at each of 1, 3, 5 and 7 m depths in Narrow Lake (each depth and abundance treatment in triplicate). A uniform sandy substrate was used in all enclosures to eliminate any possible effect of substrate type on growth. 4. Mortality was negligible (0.9%) during the experiment. Clam density had no significant effect on clam growth which suggests that clam growth was not food limited in the lake. 5. Clams reared at 7 m grew more slowly than clams reared at 1, 3 and 5 m. Clams reared at 5 m grew more slowly than clams reared at 1 and 3m. Growth of clams reared at 1 and 3m did not differ. These differences in growth were strongly correlated with the measured differences in water temperature between depths. 6. Migration between depths probably accounts for the lack of a depth effect on clams growing in the natural habitat.     

Angiotensin III as well as angiotensin II regulates water flow through aquaporins in a clam worm

Angiotensin III has been reported to exist in various animals and tissues. The physiological role, however, is still unclear except that brain angiotensin III is a central regulator of vasopressin release. In this study, angiotensin III as well as angiotensin II enhanced an increase in body weight of clam worms of Perinereis sp. under a hypo-osmotic condition and suppressed a decrease in body weight under a hyper-osmotic condition. When clam worms were treated with tetrachloroaurate (III) after angiotensin-treatment, these enhancing and suppressive effects of the angiotensins under hypo- and hyper-osmotic conditions were inhibited. In contrast, when clam worms were pretreated with tetrachloroaurate (III) before angiotensin-treatment, these effects of angiotensins were not inhibited. Since tetrachloroaurate (III) is a representative blocker of aquaporins, these results indicate that angiotensin III as well as angiotensin II regulates water flow through aquaporins in clam worms.     

生态因子对红螯相手蟹捕食毛蚶苗种的影响

敌害生物的捕食在控制海洋底栖生物群落的丰度和组成中起着关键性的作用。以红螯相手蟹(Sesarma haematocheir)和毛蚶(Scapharca subcrenata)为试验对象,研究了红螯相手蟹的密度、规格、性别,以及毛蚶的密度、规格、海水温度和底质条件对毛蚶苗种存活的影响。结果表明,蟹表现出了第二种类型的功能反应,高密度底播毛蚶苗种可以显著提高成活率;当毛蚶苗种壳长达到20mm以上时,蟹的摄食速率显著下降;随着蟹个体的增大,其摄食速率显著增加,毛蚶的存活率下降;当蟹的密度逐渐增加的时候,同种个体之间的干扰竞争显著提高了毛蚶存活率;雄蟹凭借强有力的螯导致了更多毛蚶苗种的死亡;海水温度较低的春季和秋季底播毛蚶苗种可以显著提高成活率;底质条件的复杂性和异质性为毛蚶的存活提供了"庇护空间",从而减少了敌害生物捕食所带来的损失。     

Age related properties of the Adriatic clam Chamelea gallina (L. 1758) hemocytes

The clam Chamelea gallina (L 1758) represents an important shellfish resource along Mediterranean coasts and its progressive depletion has been ascribed both to the overexploitation of stocks and to environmental or anthropic stressors. In this context, the investigation on immune parameters could represent a valid approach to measure the clam homeostasis condition and its possible influence on population dynamics. On this basis, the innate immune system, mainly represented by hemocyte phagocytosis, was investigated in organisms of different size. The results indicated a better phagocytic response in larger clams, strictly related to a greater concentration of granulocytes. A such variation in hemolymph composition appeared not dependent on environmental or endogenous factors, but rather on clam aging.     

Structure and antithrombin-binding properties of heparin isolated from the clams Anomalocardia brasiliana and Tivela mactroides

Heparin with high anticoagulant activity was isolated from the two marine clam species Anomalocardia brasiliana and Tivela mactroides. A large portion of the polysaccharide chains of both preparations bound with high affinity to immobilized antithrombin. Titrations monitored by tryptophan fluorescence showed that clam polysaccharide chains with Mr approximately 22,500 contained up to three binding sites for antithrombin and that the binding constants for the interaction of these chains with antithrombin were higher than those reported for mammalian heparin of comparable size. Structural analysis of clam heparin fractions and subfractions of clam heparin with differing affinity for immobilized antithrombin revealed the presence of large amounts (up to 25-30% of the total disaccharide units) of the 3-O-sulfated saccharide sequences (-GlcNSO3)-GlcA-GlcNSO3(3-OSO3)- and (-GlcNSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-, previously identified as unique markers for the antithrombin-binding region of heparin. The content of these saccharide sequences was found to increase with increasing affinity of the parent polysaccharide for antithrombin. Structural analysis of the clam heparins also demonstrated the occurrence of a novel saccharide sequence, tentatively identified as (-GlcNSO3)-IdA-GlcNSO3(3,6-di-OSO3)-, that has not previously been found in heparin or related polysaccharides. The contents of this latter sequence, at most 3-4% of the total disaccharide units, showed no correlation with the affinity for antithrombin.     

A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro

Cell-free extracts prepared from activated clam oocytes contain factors which induce phosphorylation of the single 67-kD lamin (L67), disassemble clam oocyte nuclei, and cause chromosome condensation in vitro (Dessev, G., R. Palazzo, L. Rebhun, and R. Goldman. 1989. Dev. Biol. 131:469-504). To identify these factors, we have fractionated the oocyte extracts. The nuclear lamina disassembly (NLD) activity, together with a protein kinase activity specific for L67, appear as a single peak throughout a number of purification steps. This peak also contains p34cdc2, cyclin B, and histone H1-kinase activity, which are components of the M-phase promoting factor (MPF). The NLD/L67-kinase activity is depleted by exposure of this purified material to Sepharose conjugated to p13suc1, and is restored upon addition of a p34cdc2/p62 complex from HeLa cells. The latter complex phosphorylates L67 and induces NLD in the absence of other clam oocyte proteins. Our results suggest that a single protein kinase activity (p34cdc2-H1 kinase, identical with MPF) phosphorylates the lamin and is involved in the meiotic breakdown of the nuclear envelope in clam oocytes.     

Concentration and detection of hepatitis A virus and rotavirus from shellfish by hybridization tests.

A modified polyethylene glycol precipitation method for concentration of virus followed by a new method to recover nucleic acid was used to detect hepatitis A virus (HAV) and rotavirus (SA11) in shellfish (oysters and hard-shell clams) by hybridization tests. Infectious virus, seeded into relatively large quantities of shellfish, was recovered consistently, with greater than 90% efficiency as measured by either in situ hybridization (HAV) or plaque assay (rotavirus SA11). Viral nucleic acid for dot blot hybridization assays was extracted and purified from virus-containing polyethylene glycol concentrates. Separation of shellfish polysaccharides from nucleic acid was necessary before viral RNA could be detected by dot blot hybridization. Removal of shellfish polysaccharides was accomplished by using the cationic detergent cetyltrimethylammonium bromide (CTAB). Use of CTAB reduced background interference with hybridization signals, which resulted in increased hybridization test sensitivity. After polysaccharide removal, dot blot hybridization assays could detect approximately 10(6) physical particles (corresponding to approximately 10(3) infectious particles) of HAV and 10(4) PFU of SA11 rotavirus present in 20-g samples of oyster and clam meats. These studies show continuing promise for the development of uniform methods to directly detect human viral pathogens in different types of shellfish. However, practical applications of such methods to detect noncultivatable human viral pathogens of public health interest will require additional improvements in test sensitivity.     

Effects of tributyltin on the MFO system of the clam Ruditapes decussata: a laboratory and field approach

The in vivo interaction of tributyltin (TBT) with the microsomal monooxygenase (MFO) system of the clam Ruditapes decussata was studied. For this purpose, two experiments were designed: (1) a laboratory exposure to increasing nominal doses of TBT (90, 454 and 2268 ng l⁻¹) for 1 week and (2) a clam transplant from a clean area to an organotin polluted marina for periods of up to 5 weeks. Chemical analysis of organotins in clam tissue was used to relate TBT body burden to the MFO response. Neither the laboratory nor the field transplant experiment showed any significant TBT effect on the clam’s digestive gland MFO components (cytochrome P450 and cytochrome b₅). However, a significant elevation in the NADPH cytochrome (P450) reductases at the low and medium TBT doses in the laboratory and a significant decrease in NADH cytochrome (b₅) reductases, 1 week after the field transplant, was observed with further recovery to control levels thereafter.