Cloning of cDNAs and hybridization analysis of lysozymes from two oyster species, Crassostrea gigas and Ostrea edulis

In bivalve molluscs including oysters, lysozymes play an important role in the host defense mechanisms against invading microbes. However, it remains unclear in which sites/cells the lysozyme genes are expressed and which subsequently produced the enzyme. This study cloned lysozyme cDNAs from the digestive organs of Pacific oyster Crassostrea gigas and European flat oyster Ostrea edulis. Both complete sequences of two oysters' lysozymes were composed of 137 amino acids. Two translated proteins present a high content in cysteine residues. Phylogenetic analyses showed that these oysters' lysozymes clustered with the invertebrate-type lysozymes of other bivalve species. In the Pacific oyster, lysozyme mRNA was expressed in all tissues except for those of the adductor muscle. In situ hybridization analyses revealed that lysozyme mRNA was expressed strongly in basophil cells in the digestive gland tubule of C. gigas, but not in digestive cells. Results indicated that the basophil cells of the oyster digestive gland are the sites of lysozyme synthesis.     

A PLANIMETRIC STUDY OF MORPHOLOGICAL VARIABILITY IN THE DIGESTIVE DIVERTICULA OF LITTORINA LITTOREA (LINNAEUS) AND MYTILUS EDULIS LINNAEUS

The inter-and intratubular morphological variability in thedigestive diverticula of Littorina littorea and Mytilus edulishas been investigated by planimetric procedures. Five parametershave been measured: mean epithelial thickness (MET), mean diverticularradius [MDR], mean luminal radius (MLR), MLR/MET ratio and MET/MDRratio. The results indicate that irrespective of the patternof tubule organization within the digestive diverticula (whethertubule types are clustered or not), variability between individualsis greater than that between zones of the digestive gland. Thishas implications for the design of sampling strategies in investigationsof the morphology of digestive diverticula in physiologicaland pathological studies. The variability in the epithelialthickness within diverticular sections is of minor relevancein assessing the overall condition of the digestive gland inthese studies, because variability in epithelial thickness betweentubules is significantly greater than within tubules in bothspecies. (Received 10 March 1989; accepted 16 October 1989)     

Changes of Blue Mussels Mytilus edulis L. Lipid Composition Under Cadmium and Copper Toxic Effect

The lipid and fatty acid composition of the blue mussels Mytilus edulis L. gills and digestive glands was evaluated after 24 and 72 h of cadmium (Cd) and copper (Cu) exposure. Mussels were exposed to different cadmium (10, 100, and 500 μg/L) and copper (5, 50, and 250 μg/L) concentrations. Similar stress response of predominant membrane phospholipids level as well as polyenoic and non-methylene interrupted (NMI) fatty acids content was observed in mussel gills under both cadmium and copper effects. Increased NMI fatty acids level after 24 h, the metal ions treatment suggests that these acids contribute to the protective response to the membrane oxidative stress caused by accumulation of the metals. The content of cholesterol, some minor membrane phospholipids, and storage lipids (triacylglycerols, TAG) in the mussels’ organs alter significantly under the cadmium and copper effect. A two-step response at the digestive glands TAG level depends on the duration of the cadmium and copper treatments (24 and 72 h) on the mussels. The results demonstrate that Cd and Cu impact has adverse effects on gills and digestive glands lipid and fatty acids composition. The type of observed effects varies with the nature and concentration of the metal ions and depends on the role of the metals in the mussels’ life activity.     

Biochemical studies of aminopeptidase polymorphism in Mytilus edulis. I. Dependence of enzyme activity on season,tissue, and genotype

Aminopeptidase-I is polymorphic in the marine bivalve Mytilus edulis and catalyzes the liberation of neutral and aromatic N-terminal amino acids from oligopeptides. The enzyme is abundant in the digestive gland, where it is lysosomal, but is present in several other tissues. Temporal variation in enzyme activity was monitored for 2.5 years in two natural populations. The temporal pattern of variation was similar in gill, mantle, and digestive gland tissues; variations occurred over both short and long time periods. Enzyme activity under ambient temperature conditions was seasonally related to temperature in gill and digestive gland, but varied with reproductive cycle in mantle tissue. In the last, maximum activity corresponded to the postreproductive period in each population. Enzyme activity varies in response to tissue-specific metabolic demands. Population differences in enzyme activity are due to both genotype-dependent enzyme activity, since allele frequencies differ between populations, and environmental salinity. High salinity induces high activity, which is a response to the need for higher intracellular concentrations of free amino acids for cell volume regulation. Salinity has comparable effects on enzyme activity in natural and experimental populations. Genotype-dependent specific activities are a consequence of both differing kinetic properties among genotypes [Koehn, R. K., and Siebenaller, J. S. (1981). Biochem. Genet. 19:1143] and genotype-specific concentrations of enzyme protein that change in response to environmental salinity.     

Developmental stability is not disrupted by extensive hybridization and introgression among populations of the marine bivalve molluscs Mytilus edulis (L.) and M. galloprovincialis (Lmk.) from south-west England

The effects of hybridization and introzgression were assessed among two naturally hybridizing bivalve molluscs (the mussels Mytilus edulis and M. galloprovincialis) from western Europe to estimate how disruptive these processes are to developmental stability (measured in terms of morphological variability). Ten shell traits were measured for 392 mussels from four allopatric populations (two each of At. edulis and At. galloprovincialis) and two hybrid populations. An index of variability (defined as Z_i= |y_i–y_i where y_; is the population mean of the length-standardized trait, and y_i is the individualcar;s length-standardized trait value) was constructed for each trait, and for the sum of the traits. The hybrid populations did not exhibit greater mean variability than the allopatric populations for any of the indices. Upon pooling, the hybrid populations had significantly lower variability than the pooled M. edulis populations and the pooled M. galloprovincialis populations in two analyses, and had similar means in the remaining nine analyses. Where significant differences existed, the pooled M. galloprovincialis had lower levels of mean trait variability than the pooled M. edulis. Among the two hybrid populations, the putative Fl hybrids and backcross individuals exhibited means of trait variability which were similar to those of the parental types. Thus, extensive hybridization and introgression do not adversely affect developmental stability among these mussel populations. There was a strong significant correlation between the ranking of indices (based on the amount of variability) across all six populations, indicating that a large genetic component determines the measured morphological variability. It is concluded that the genes or gene complexes which control morphological development in M. edulis and M. galloprovincialis arc very similar, providing further evidence of the close evolutionary relatedness of these mussel taxa.     

Antioxidant response of the bivalve Pinna nobilis colonised by invasive red macroalgae Lophocladia lallemandii

Invasive species represent a risk to natural ecosystems and a biodiversity hazard. The present work aims to determine the antioxidant enzyme response – superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), the phase II detoxifying enzyme – glutathione S-transferase (GST) – and markers of oxidative damage – thioredoxin reductase (TR) and malondialdehyde (MDA) – in gills and digestive gland of Pinna nobilis and to study the antioxidant response effects in the bivalve colonised by the invasive macroalgae Lophocladia lallemandii. Colonised specimens were collected in a control area without L. lallemandii and another area completely colonised by L. lallemandii. All enzyme activities were found to be present in gills and digestive gland, with some tissue differences. CAT and SOD activities were higher in gills than digestive gland, whereas GST activity and MDA levels were higher in digestive gland. The presence of L. lallemandii induced a significant increase in the activities of antioxidant enzymes in both gills and digestive gland, except for CAT activity in gills. GST and TR activities were also increased in both tissues, as well as the MDA concentration. We can conclude that the presence of L. lallemandii colonising P. nobilis induces a biological stress and oxidative damage to the fan mussel.     

Observations on the ultrastructure of large cells associated with putative neoplastic disorders of mussels,Mytilus edulis,from Yaquina Bay,Oregon

Large cells, generally thought to be associated with neoplastic disorders in bivalve mollusks, were studied with the electron microscope. The atypical cells had an average diameter of 15 μm and a nuclear to cytoplasmic ratio of about 1–1.5. Nuclei were extensively pleomorphic and bizarre shapes were common. Nucleoli were prominent and often multiple. In the cytoplasm of the large cells, there was a wide range of variability in anomalous organelles. Two cell types were originally thought to exist; however, it is now thought that the two cell types represent the two extreme morphological expressions of a single cell line. Their varying appearance is correlated with the density of ribosomes and abundance of cellular organelles. The large Mytilus edulis cells possess many ultrastructural properties that are characteristic of certain malignant vertebrate cells. However, alternative explanations for their structure and function are also possible.     

Effects of ageing on nuclear DNA integrity and metabolism in mussel cells (Mytilus edulis L.)

As the age of a cell increases, so does the potential for DNA damage. Recent theories on ageing suggest accumulative DNA damage is the primary cause of cellular senescence, possibly due to the decreased ability of DNA to act as a template for gene expression.In this paper we investigate the effects of ageing on the level of nuclear DNA damage in tissues of wild mussels of three different age groups; 2–4 years (group I), 6–8 years (group II) and 10 years (group III). In the digestive gland and haemolymph cells, a significant age-dependent increase of DNA damage was observed, as evaluated by the fluorimetric alkaline DNA unwinding technique, which is able to detect both direct single strand DNA breaks as well as alkali-labile apurinic sites.In addition, the rate of DNA polymerase activity was studied in order to determine whether DNA damage was dependent on DNA alteration, or because of a reduced rate of DNA repair. Unscheduled DNA repair synthesis in isolated nuclei of digestive gland cells in older mussels, was significantly decreased in comparison to younger mussels (−42% in group II and −37% in group III, p<0.01). In the digestive gland, salt extraction gives a slight, but significant, decrease of aphidicolin-sensitive DNA polymerase activity in age group III of −25%, p<0.05.Finally, we looked at the age variation in relation to oxidative stress. This was evaluated by measuring malondialdehyde accumulation in mussel cells. Digestive gland cells of group III, showed a significant age-related increase in malondialdehyde content of 170%, p<0.01, indicative of enhanced peroxidative processes.Taken together, these data suggest that the accumulation of DNA damage in group II is mainly dependent on the impairment of DNA repair systems. This is contrary to group III DNA damage, where a possible relationship between oxidative stress and alteration of nuclear DNA metabolism is found, probably deriving from an antioxidant defence decline.     

Biodeposit production by the mussel Mytilus edulis L. on rocky shores

Seasonal changes in the amount of biodeposit (faeces and pseudofaeces) produced by the mussel Mytilus edulis L., which is one of the representative suspension-feeders in the rocky intertidal and shallow subtidal regions of Mutsu Bay, were studied in the laboratory. The effects of water temperature, light, food concentration, flow rate, body size, age, and spawning on biodeposit production were investigated. More biodeposit was produced in summer than in other seasons. Throughout the year, the amount of biodeposit was positively correlated with body size. Relatively more biodeposit was produced by smaller than by larger individuals. A M. edulis population living in one square meter was estimated to produce 9.20 kg of faeces and 2.71 kg of pseudofaeces per year (dry wt). More biodeposit was produced at water temperatures of 17.6–20.2° C than at 4.5–7.6° C and 25.2–26.0° C. The optimum temperature for biodeposit production was found to be ≈ 20.0 °C. When kept in the dark, M. edulis produced more biodeposit than in the light. When food concentration is increased, more psuedofaeces are produced; the amount of faeces, however, remains constant. With increasing flow rate, the amount o f biodeposit per h increased but the biodeposition rate decreased. Larger amounts of faeces and smaller amounts of pseudofaeces were produced by younger mussels than by older ones of a similar size. Spawning also affected biodeposit production.     

Use of microstereology and quantitative cytochemistry to determine the effects of crude oil-derived aromatic hydrocarbons on lysosomal structure and function in a marine bivalve mollusc, Mytilus edulis

Summary The marine bivalve mollusc,Mytilus edulis (blue mussel), is a noted accumulator of many environmental pollutants and is increasingly used for the chemical and biological assessment of environmental impact. The toxic effects of crude oil-derived aromatic hydrocarbons (30 g/l total hydrocarbons) on the lysosomal-vacuolar system of the digestive cells have been investigated in cryostat sections of hexane-frozen digestive glands. Exposure to aromatic hydrocarbons reduced the cytochemically determined latency of lysosomal -N-acetylhexosaminidase; lysosomal volume density and surface density increased while the numerical density decreased. Experimental exposure resulted in the formation of very large lysosomes which are believed to be largely autophagic in function and these results indicate a significant structural and functional disturbance of digestive cell lysosomes in response to hydrocarbons.     

Peroxisomal proteomic approach for protein profiling in blue mussels (Mytilus edulis) exposed to crude oil

Peroxisomal proteomic protein profiles of exposure to marine pollution have been recently introduced in biomonitoring experiments. However, laboratory experiments to study the independent effect of common pollutants are needed to define a minimal protein expression signature (PES) of exposure to a specific pollutant. The aim of this study was to obtain PESs in blue mussels (Mytilus edulis) exposed to two different crude oil mixtures for future application in biomonitoring areas affected by oil spills. In the study, peroxisome-enriched fractions from digestive gland of M. edulis (L., 1758) were analysed by two-dimensional fluorescence difference electrophoresis (DIGE) and mass spectrometry (MS) after 3 weeks of exposure to crude oil mixtures: crude oil or crude oil spiked with alkylated phenols (AP) and extra polycyclic aromatic hydrocarbons (PAH) in a laboratory flow-through system. A minimal PES composed by 13 protein spots and unique PESs of exposure to the two different mixtures were identified. A total of 22 spots from the two-dimensional maps that had shown a significant increase or decrease in abundance in each of the exposed groups exposed were analysed. The hierarchical clustering analysis succeeded in discriminating the exposed groups from the control groups based on the unique PES. The PESs obtained were consistent with protein patterns obtained in previous field experiments. The results suggest that the protein profiles obtained by peroxisomal proteomics could be used to assess oil exposure in marine pollution assessments.     

Arylsulphatase activity associated with phenanthrene induced digestive cell deletion in the marine mussel Mytilus edulis

Summary The acid hydrolase arylsulphatase has been localized at the ultrastructural level in digestive cells of the marine musselMytilus edulis for control and phenanthrene-treated (200µg/l) animals. In untreated mussels the activity was generally restricted to the lysosomal—vacuolar system and the Golgi apparatus. It was associated with all types of vesicle, although not all individual vesicles were reactive. In heterolysosomes which were filled with precipitate the reaction product was most densely associated with the limiting membranes. Lipid inclusions commonly occurred in the digestive cells; these sometimes showed limited reaction for enzyme activity. The striking difference between normal and phenanthrene-treated samples was the presence in all treated animals of reaction product in the inter-cellular spaces and varying degrees of cytoplasmic activity in a number of digestive cells. This is interpreted as a sign of impending cell deletion. Sections for morphological examination showed evidence of increased digestive cell deletion in phenanthrene-treated mussels. The process results in release of membrane-bound bodies into the tubule lumen.     

Trophic transfer of trace metals: Subcellular compartmentalisation in bivalve prey and comparative assimilation efficiencies of two invertebrate predators

We test the hypothesis that accumulated metal in prey that is trophically available to one predator is not necessarily equally trophically available to another predator feeding on the same prey, given the variability between invertebrate digestive systems. We provided two predators, the neogastropod mollusc Hinia reticulata and the palaemonid decapod crustacean Palaemonetes varians, with the digestive glands and adductor muscles of four bivalves radiolabelled with Zn, Cd or Ag. The bivalves (the mussel Mytilus edulis, the clam Ruditapes philippinarum, the scallop Aequipecten opercularis, the oyster Crassostrea gigas) have different metal accumulation patterns with differential dependence on soluble and insoluble detoxification, as confirmed by fractionation of the prey tissues. We found no consistent significant difference between the AE of the two predators for the three trace metals accumulated in the same prey tissues. There were no significant correlations for either predator between percentages of metal in soluble form (or soluble form with organelle-associated metal) and percentage AE for any of the three metals, allowing the conclusion that both predators are assimilating each metal from more than the soluble and organelle-associated metal fractions. For neither predator did an increased percentage of Zn in the form of metal rich granules (MRG) affect its Zn AE, but increases in the percentages of both Cd and Ag bound to MRG decreased the AE of the relevant metal in P. varians but not H. reticulata. Thus the Cd and Ag in some Cd-rich and Ag-rich granules in the bivalve tissues are not as trophically available to P. varians as they are to H. reticulata. This interspecific difference confirms that the neogastropod has the stronger digestive and assimilative powers involving Cd and Ag bound in prey than the palaemonid decapod.     

Settlement of bivalve spat on artificial collectors in Eyjafjordur, North Iceland

The seasonal pattern of bivalve spat settlement in Eyjafjordur, North Iceland, was investigated using artificial collectors of monofilament netting over 14 months (March 1998–January 2000) at 5, 10 and 15 m depth. SCUBA divers replaced the collectors at 4-weekly intervals. Twelve bivalve species settled on the collectors but only Mytilus edulis and Hiatella arctica were present throughout the year; they were the most abundant bivalve taxa. Of the remaining species, only Chlamys islandica, Heteranomia spp., Arctica islandica, Serripes groenlandicus and Mya spp. were sufficiently abundant to enable statistical analysis. All settled in late summer and autumn. Peak settlement of M. edulis, in September, consisted mainly of primary settlers (0.25–0.5 mm) although secondary settlers (>0.5 mm) were present in all samples. Mytilus edulis settled mostly at 5 m depth, especially larger individuals, possibly reflecting stronger currents at shallower depth and the proximity of mussel beds in the intertidal zone. Primary (<1 mm) and secondary H. arctica settlers (>1 mm) were present in most months, with the former being most numerous in September, 1999; settlement was equally abundant at 5 and 10 m depth. Primary settlement of C. islandica and S. groenlandicus occurred in autumn (mainly in September), and secondary settlers were very scarce and only seen in winter. Arctica islandica, Heteranomia spp. and Mya spp. settled mainly in September 1999 at 10 m depth, except for A. islandica, which was more numerous in August.     

The Ultrastructure of the Digestive Glands in Pinguicula vulgaris L. (Lentibulariaceae) Relative to their Function. II. The Changes on Stimulation

The secretory cells of the digestive glands remain highly activeduring the entire period of prey digestion and absorption ofnutrients. They appear to play a major role in gland activity.A model of the digestive gland's activity on stimulation isproposed. It is very similar to that suggested earlier for Dionaeamuscipula. After the digestion and absorption cycle, destructiveprocesses are initiated in the glands. These appear similarto those observed in the glands of the ageing, unstimulatedleaf and are not associated with feeding. Pinguicula vulgaris L. carnivorous plant, digestive glands, ultrastructure, protein secretion absorption, senescence     

The oxidation of lysine and oxalysine by Mytilus edulis: Identification of the products formed in the presence and the absence of catalase

1. O-(2-Aminoethyl)serine (oxalysine) was shown to be a substrate of the l-amino acid oxidase of the digestive gland of the common mussel, Mytilus edulis. 2. Three atoms of oxygen were consumed per mole of oxalysine oxidized in the presence of catalase; l-lysine under the same conditions consumed only one atom. 3. The products of oxidation of oxalysine in the presence and the absence of catalase were: ethanolamine, N-oxalylethanolamine and 3-morpholone (the oxygen analogue of 2-piperidone). After acid hydrolysis 70% of the oxalysine oxidized was recovered as ethanolamine. 4. In the absence of catalase 2-aminoethoxyacetic acid was also detected. 5. The products identified account quantitatively for the oxalysine oxidized and for the oxygen uptake. 6. N-Oxalylethanolamine and 2-aminoethoxyacetic acid have been synthesized. 7. Treatment of extracts of the digestive gland at pH3·0 completely inactivated the catalase, leaving the l-amino acid oxidase unaffected. 8. The major product of the oxidation of lysine in the absence of catalase was 2-piperidone.     

Species identification of the medicinal plant Tulipa edulis (Liliaceae) by DNA barcode marker

Tulipa edulis (Liliaceae) is the botanical origin of the traditional Chinese medicine (TCM) “Guangcigu”. Due to overexploitation that induced a decline in natural sources, many dried bulbs from other species of Tulipa have been used, adulterating the medicine in recent years. This practice may cause a series of inconsistent therapeutic effects and quality control problems in the herbal medicine industry. Hence, three DNA regions (matK, psbA-trnH and rbcL) were evaluated as barcodes for identifying T. edulis and its adulterants. All candidate DNA barcodes were successfully amplified from leaf samples. Based on the sequence divergences, rbcL and psbA-trnH can assign T. edulis and its adulterants to the correct genus, while matK can accurately differentiate T. edulis and its adulterants. Thus, at the DNA level, the matK intergenic region is a more suitable, accurate and applicable identification of T. edulis and its adulterants than rbcL and psbA-trnH.     

A simple and convenient biochemical method for sex identification in the marine mussel,Mytilus edulis L.

One of two chromophores is formed on heating the mantle tissue of Mytilus edulis with thiobarbituric acid (TBA). Application of the test to male mussels yields a strong yellow colour (λ_max453 and 490 nm), whereas in females, a pink colour (λ_max532 nm) develops. While the latter is characteristic of the products of lipid peroxidation, it appears that the yellow colour may be derived from the 2-deoxyribose moiety of DNA. The TBA reaction can be used for the rapid, accurate sex identification in Mytilus edulis over 9–10 months of the year.