Measurement of adenosine 3',5'-cyclic monophosphate and guanosine 3', 5'-cyclic monophosphate in mussel (Mytilus galloprovincialis lmk.) by high-performance liquid chromatography with diode array detection

Two fast and sensitive methods for the determination of cAMP and cGMP levels in mantle tissue of the sea mussel Mytilus galloprovincialis Lmk. are described. Both methods use ion-pair high-performance liquid chromatography with diode array detection. The use of the diode array detector permitted the simultaneous detection of the absorbance at two different wavelengths and the obtaining of the UV absorption spectrum for each detected peak, confirming peak purity and identity. Method precision was good. The detection limit for both nucleotides was 2.5 pmol (signal-to-noise ratio = 4 at 254 nm). Previous to the injection onto the chromatograph, both nucleotides were purified by precipitation of the adenine and guanine 5'-ribonucleotides with ZnSO(4)-Na(2)CO(3). The supernatant obtained was subsequently passed over an anion-exchange column (AG l-X8 formate form resin). Early results seem to indicate that there is a seasonal variation in the contents of both cyclic nucleotides in mantle tissue. Such variation is probably related to the annual gametogenic cycle.     

Zonal variability and seasonal changes of the content of glycogen and glucose in the Mytilus mantle

Glycogen and free-glucose content in the ventral, central and dorsal parts, as well as glucose-6-phosphate phosphatase activity in mantle of Mytilus galloprovincialis Lmk. were examined. The glycogen content of mantle did not manifest asymmetrical distribution among the three parts. In the period studied, the typical glycogen content profile variation was found, being maximum in July. The tissue free-glucose content was similar in each part, and the obtained seasonal variation profile was opposite to the glycogen content, reaching the minimum in July. For every part of mantle, free-glucose/glycogen ratio showed similar monthly profiles. In each part the 50% point was found in July. Glucose-6-phosphate phosphatase activity was not found in the mantle tissue.     

Two forms of alpha-amylase in mantle tissue of Mytilus galloprovincialis: purification and molecular properties of form II

alpha-Amylase activity has been shown for the first time in a non-digestive tissue from Mytilus galloprovincialis. alpha-amylase from mussel mantle tissue has been purified by affinity chromatography on insoluble starch, followed by gel-filtration chromatography on Superdex-200. The chromatographic and electrophoretic behaviour of M. galloprovincialis alpha-amylase and stability characteristics suggest two forms of this enzyme: one form forming stable aggregates (form I) and a monomeric form (form II) that is more abundant, active and unstable. Both forms show an inverse quantitative variation. Purified form II was highly unstable and the molecular mass was estimated to be 66 kDa by sodium dodecyl sulphate (SDS)-gel electrophoresis. Maximum activity was noted at pH 6.5 and 35 degrees C.     

The activity of antioxidant defence enzymes in the mussel Mytilus galloprovincialis from the Adriatic Sea

The activity of the antioxidant defence enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione peroxidase (GSH-Px, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2) and the phase II biotransformation enzyme glutathione-S-transferase (GST, EC 2.5.1.18) in whole mussels (Mytilus galloprovincialis) were studied. The mussels were collected in winter and in spring at two localities in the Adriatic Sea: Bar Port and Tivat Bay. Our results show that the activities of SOD, GSH-Px and GST were seasonally dependent with higher activities in winter. GR activity was also higher in winter, but only in mussels from Bar Port. In mussels from Tivat Bay, GR activity was lower in winter compared to spring. In addition, a decrease in CAT activity in mussels from Bar Port compared to those from Tivat Bay was found. It can be concluded that seasonal variations should be incorporated into interpretation of biomonitoring studies in mussels.     

Nutrient storage cells isolation from mantle tissue of Mytilus galloprovincialis: glucose release and glycogen content

A method for obtaining isolated mantle nutrient storage cells and purifying vesicular (VC) and adipogranular (ADG) cells from mantle tissue of Mytilus galloprovincialis is reported. Tissue digestion is partly mechanical (stirring) and partly enzymatic (collagenase + dispase). Purification is carried out through continuous and discontinuous Percoll gradients. VC appears in fraction 3 (d = 1.05-1.08 g/ml) and ADG in fraction 2 (d = 1.09 g/ml). Intracellular glycogen and free-glucose content in September-April period is studied. When glycogen is detectable it is always accompanied by intracellular free-glucose pool in a concentration relationship glycogen/glucose 10:1. Furthermore, a glucose releasing activity elicited by the Ca2(+)-ionophore A23187 was found in isolated cells, which reproduce the former behaviour found with mantle tissue fragments in our laboratory.     

Glucose release in mantle tissue of Mytilus: regulation by calcium ions

Glucose release activity in mantle tissue of Mytilus galloprovincialis was studied. Mantle tissue shows a basal glucose releasing activity. The external Ca2+ absence increases 2 to 3-fold the basal glucose release, and when A23187 (10 microM) was simultaneously present the release doubled that obtained in Ca2(+)-absence. EGTA (2 mM), chlorpromazine (200 microM) and lanthanum (3 mM) decreased the glucose release promoted by external Ca2+ absence. This and other data suggest that glucose release activity in mantle tissue might be controlled by Ca2+ ions.     

Effect of temporal and geographical factors on fatty acid composition of M. galloprovincialis from the Adriatic sea

Soft body tissue and gill and mantle microsomal membranes of Mytilus galloprovincialis, were analyzed to assess geographical and temporal effects on fatty acid distribution with special focus on n-3 polyunsaturated fatty acid (PUFA) content. Mussels and plankton samples were collected in April and in October from three locations of the North Adriatic Sea. Plankton fatty acid composition apparently depended both on the sampling site and time and differences referable to temporal features prevailed on the geographical ones. Accordingly, also mussel fatty acid composition appeared to be more effectively modulated by sampling time rather than by sampling location. Membrane fatty acids showed high homeostatic capabilities to reduce effects of exogenous input on cellular organization. Selective capabilities apparently enable mussels to modulate their fatty acid composition combining plankton input, environmental effects on lipid metabolism and physiological requirements. The constantly higher n-3/n-6 ratio in April than in October, shared by fatty acids of plankton, soft tissues and microsomal membranes, confirms the influence of temporal factors in fatty acid modulation. On these bases, late spring is suggested to be the more suitable period for collecting mussels destined for healthy diet of humans or other animals.     

In vitro effects of lead ions on peripheral benzodiazepine receptors and adenylyl cyclase activity in the mantle of Mytilus galloprovincialis

As an extension of our previous work, where the density of peripheral benzodiazepine receptors (PBR) increased in mantle mitochondria of the marine mollusk Mytilus galloprovincialis Lmk. under chronic exposure to lead, the present study investigates the in vitro effects of an exogenous source of lead ions on PBR and on adenylyl cyclase (AC) complex in mantle membranes of mussels collected from a non-polluted coastal area. PBR binding experiments used the specific isoquinoline carboxamide derivative [3H]PK 11195, and AC activity was measured using a modified procedure adapted to M. galloprovincialis. Lead ions (Pb2+) dose-dependently decreased either the [3H]PK 11195 specific binding in mitochondria or basal AC velocity in plasma membranes of mussel mantle. The IC50 values for lead ions were 10 microM with [3H]PK 11195 binding and 25 microM with AC activity, with maximal inhibition values of 60% and 70%, respectively. Moreover, lead behaved as a non-competitive inhibitor on [3H]PK 11195 binding and as a 'mixed' inhibitor on AC activity. The present results suggest that some of the early effects induced by lead in mussel cell metabolism consist in significant changes of the PBR density and cyclic AMP production in the mantle of M. galloprovincialis.     

Regulatory properties of p105: a novel PKC isoenzyme in mantle tissue from marine mussels.

Previous results suggested operative similarities between Apl II from nervous cells of Aplysia californica, epsilonPKC from brain of vertebrates, and p105 from mantle tissue of Mytilus galloprovincialis Lmk., all of them belonging to the nPKC family. The optimal substrate for Apl II and p105 from mussel is protamine sulfate. In contrast, Ca2+ inhibits p105 but does not affect Apl II. As occurs in other epsilonPKC, p105 is autophosphorylable; however, in Apl II, no P-Tyr residues are detected in the most phosphorylated form. The presence of p105 in all the tissues of M. galloprovincialis studied, proves the important, yet unknown, physiological role that this enzyme must play.     

Regulation of fructose-2,6-bisphosphate content in mantle tissue of the sea mussel Mytilus galloprovincialis Lmk. Regulation of fructose-2,6-bisphosphatase activity.

Fructose-2,6-bisphosphatase (FBPase-2) from the mantle tissue of the mussel Mytilus galloprovincialis shows a hyperbolic kinetic with a Km value (0.40 mM) for its substrate, that suggest that the "in vivo" Fru-2,6-P2 concentration is not a limiting factor for activity. The enzyme possesses an optimum pH for activity between 6 and 7 units, similar to the reached in mussel mantle during physiological hypoxia. The modulation of activity by the pH, and in addition, the positive effect of ATP are in keeping with the little decrease in concentration of the Fru-2,6-P2 that occurs during the first hour of hypoxia due to the valve closure.     

Seasonal variation in haematological parameters in male and female Tinca tinca

This study was designed to investigate any seasonal (spring, summer, autumn and winter) changes in haematological parameters in the blood of Tinca tinca measuring the number of red blood cells (RBC), haematocrit, white blood cells, and total plasma proteins.The results show significant changes in RBC and haematocrit in males comparing spring and summer with autumn and winter, whereas in females the RBC remained constant for all 4 seasons but the haematocrit decreased in autumn and winter compared to spring and summer. The white blood cells of male and female animals were significantly lower in spring and winter compared to summer and autumn. In male fish total protein contents significantly decreased in autumn and winter compared to spring and summer, whereas in females protein output significantly decreased in winter compared to the other seasons. The results indicate marked seasonal variation in the blood of male and female Tinca tinca. This variation may play a important protective role for the survival of the animals.     

Geographical variation in heading traits in wild emmer wheat, Triticum dicoccoides. I. Variation in vernalization response and ecological differentiation

 Geographical variation in vernalization response and narrow-sense earliness was investigated for accessions of wild emmer wheat, Triticum dicoccoides, collected in Israel. Wide variation between and within populations was observed in both characters. The analysis of vernalization response showed that 2 accessions from Tabigha were of a strong spring growth habit, and thus wild emmer wheat was classified into four types, i.e., strongly spring type, moderately spring type, moderately winter type, and strongly winter type, according to their vernalization response. Whereas winter types were frequently found in most populations except that of Tabigha, the distribution of spring types was sporadic and restricted to warmer areas. It was thus suggested that spring type in T. dicoccoides might have evolved from a winter prototype as an adaptation to warmer conditions. Within moderately winter and moderately spring types, quantitative differences in vernalization response, measured as Dof₇₀/Dof₂₀ and Dof₂₀/Dof₀, were observed between populations. Inter- and intra-population variation in vernalization response could be explained to some extent by the difference in growing conditions at each habitat. It was clearly indicated that environmental heterogeneity caused ecogenetic differentiation in wild emmer wheat in Israel. Wild emmer wheat also varied considerably for narrow-sense earliness, ranging from 32.9 days to 69.5 days among accessions. However, it was difficult to explain its geographical variation simply by a linear relationship with environmental factors, and a nonlinear relationship and/or unknown microgeographic heterogeneity may be responsible. Received: 18 March 1996/Accepted: 13 December 1996     

Implication of PKC in the seasonal variation of the immune response of the hemocytes of Mytilus galloprovincialis Lmk. and its role in interleukin-2-induced nitric oxide synthesis

The hemocytes are the cells responsible for the immune response in marine mollusks. The role of NO in processes related to the activation of the hemocytes has turned out evident over the late years. In the case of the mussel Mytilus galloprovincialis Lmk., hemocyte NO basal production varies throughout the year, showing a maximum in summer and a minimum in winter. IL-2 reverts the low winter NO basal production through a process mediated by cAMP-dependent protein kinase and by an apparent side effect of protein kinase C. The seasonal variation of NO production in the presence of the PKC inhibitor bisindolylmaleimide (BSM) allows suggesting a model in which PKC would modulate the activity of the enzymes responsible for nitric oxide production.     

Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis

Several isoforms of the cAMP-dependent protein kinase catalytic subunit (C-subunit) were separated from the posterior adductor muscle and the mantle tissues of the sea mussel Mytilus galloprovincialis by cation exchange chromatography, and identified by: (a) protein kinase activity; (b) antibody recognition; and (c) peptide mass fingerprinting. Some of the isozymes seemed to be tissue-specific, and all them were phosphorylated at serine and threonine residues and showed slight but significant differences in their apparent molecular mass values, which ranged from 41.3 to 44.5 kDa. The results from the MS analysis suggest that at least some of the mussel C-subunit isoforms arise as a result of alternative splicing events. Furthermore, several peptide sequences from mussel C-subunits, determined by de novo sequencing, showed a high degree of homology with the mammalian Calpha-isoform, and contained some structural motifs that are essential for catalytic function. On the other hand, no significant differences were observed in the kinetic parameters of C-subunit isoforms, determined by using synthetic peptides as substrate and inhibitor. However, the C-subunit isoforms separated from the mantle tissue differed in their ability to phosphorylate in vitro some proteins present in a mantle extract.     

Cadmium binding and zinc variations in Mytilus galloprovincialis

The accumulation of Cadmium in Mytilus galloprovincialis during a 18 days exposure period was studied. Cadmium content of the mantle, viscera, gills increased throughout the time. The study of the distribution of Cd in homogenates of mussels chronically intoxicated has shown that the metal is principally bound to low mol. wt. proteins similar to metallothioneins. Mytilus galloprovincialis metallothioneins are also comparable to the vertebrate proteins, suggesting a biological function ubiquitous in the living world.     

Correlation between the morpho-cytohistochemistry of the outer mantle epithelium of Anodonta cygnea with seasonal variations and following pollutant exposure

Seasonal and experimental conditions induce morphological and cytochemical variations in the outer mantle epithelium (OME) of the freshwater bivalve Anodonta cygnea, probably influencing the shell calcification mechanism. In this study, OME samples were taken from untreated animals in autumn, winter, spring and summer as well as from animals exposed to divalent metals (cadmium, chromium, lead, copper and zinc) and pesticides (setoxidim and dimethoate) and observed by light microscopy. The present results showed that OME cells have larger cell volumes and increased amounts of secreted macromolecules during spring and summer than in autumn and winter. This correlates with higher shell calcification rates in spring and summer and lower shell calcification rates in autumn and winter. The experiments showed that incubation with pollutants for 8 months dramatically reduced the cellular volume so that the density of cytoplasmic material appeared higher that in the control samples. The pronounced changes in OME cells suggest a significant decrease in secretory activity following exposure to toxic agents and this has implications for the shell calcification process.     

Correlation between the morpho-cytohistochemistry of the outer mantle epithelium of Anodonta cygnea with seasonal variations and following pollutant exposure

Seasonal and experimental conditions induce morphological and cytochemical variations in the outer mantle epithelium (OME) of the freshwater bivalve Anodonta cygnea, probably influencing the shell calcification mechanism. In this study, OME samples were taken from untreated animals in autumn, winter, spring and summer as well as from animals exposed to divalent metals (cadmium, chromium, lead, copper and zinc) and pesticides (setoxidim and dimethoate) and observed by light microscopy. The present results showed that OME cells have larger cell volumes and increased amounts of secreted macromolecules during spring and summer than in autumn and winter. This correlates with higher shell calcification rates in spring and summer and lower shell calcification rates in autumn and winter. The experiments showed that incubation with pollutants for 8 months dramatically reduced the cellular volume so that the density of cytoplasmic material appeared higher that in the control samples. The pronounced changes in OME cells suggest a significant decrease in secretory activity following exposure to toxic agents and this has implications for the shell calcification process.     

Seasonal patterns of metabolism and the heat shock response (HSR) in farmed mussels Mytilus galloprovincialis

Mussels (Mytilus galloprovincialis) are the main bivalves cultured in the Mediterranean Sea and in the Thermaikos gulf North of Greece. Global warming might affect these cultures since increases in the sea temperatures especially during summertime and may cause mass mortalities. Thus projections of the effects of global warming and the knowledge of the mechanisms of thermal limitation and adaptation are considered important in this context. In the present work we studied the seasonal expression of Hsp70 and Hsp90 of aquacultured mussels in nature in an effort to examine whether mussels are seasonally thermally stressed. Moreover we determined the activity of the key glycolytic enzyme PK in order to examine seasonal changes in the metabolic profile. The present data showed a biphasic seasonal pattern in the expression of Hsps in the mantle and PAM of M. galloprovincialis. The expression of inducible Hsp72 and Hsp90 increased between February and mid April, and remained constant or decreased slightly by the end of May. Hsps expression started again in early June leading to a gradual increase by mid July and remained by the end of August. The pattern of the HSR indicates increased protein turnover or protein damage in the tissues of aquacultured M. galloprovincialis between winter and summer seasons. The induction temperature is probably limited, and beyond some upper limits there is no further change in the HSR induction temperatures and the HSR sets in. PK activity in the mantle was significantly higher in winter than in spring. Early May saw a reactivation of PK to higher than spring values. Similar to mantle PK, PK activity in PAM was significantly higher in winter than in spring. A slight reactivation was observed by the end of August.     

Zinc and 17beta-estradiol induce modifications in Na+/H+ exchanger and pyruvate kinase activity through protein kinase C in isolated mantle/gonad cells of Mytilus galloprovincialis

We investigated the transduction pathway mediated by Zn and 17beta-estradiol in isolated mantle/gonad cells of the mussel Mytilus galloprovincialis. Both the essential metal Zn, and the estrogen 17beta-estradiol, caused an increase in intracellular pH (pHi) of isolated mantle/gonad cells of the mussel M. galloprovincialis, thus indicating the activation of the Na+/H+ exchanger (NHE). The observed effect was inhibited by EIPA (20 nM), a specific NHE inhibitor, thus verifying NHE activation. Protein kinase C (PKC) also seemed to play an activating role in zinc and 17beta-estradiol effects on NHE and PK activity. In addition, the glycolytic enzyme pyruvate kinase (PK) was increased after zinc, while it was decreased after 17beta-estradiol treatment. It is noteworthy that, both the latter effects were reversed in the presence of EIPA, indicating the involvement of NHE in the signaling mechanism. cAMP seems to participate in the signaling mechanism induced by Zn but not to that induced by 17beta-estradiol. The potential implication of the heavy metal and 17beta-estradiol on the reproductive activity of the marine animals is discussed.