Regulation of fructose-2,6-bisphosphate content in mantle tissue of the sea musselMytilus galloprovincialis

6-phosphofructo-2-kinase (PFK-2) from the mantle of the sea musselMytilus galloprovincialis Lmk, collected from the Ría de Arosa (NW Spain) in 1990, was purified 550-fold by extraction and sequential affinity chromatography on Affi-gel Blue and ATP-agarose columns. The enzyme was a dimer with a native molecular weight of 100 kilodaltons (KDa) and a subunitM _r of 53 KDa. PFK-2 activity is dependent on the presence of P_i. At physiological P_i concentrations, the enzyme exhibits hyperbolic kinetics with both ATP and Fru-6-P, withK _m values of 0.62 and 0.37 mM respectively. In vivo, PFK-2 activity is limited by the concentration of Fru-6-P which is low in comparison with theK _m for this substrate. Citrate and PEP inhibited PFK-2 activity.     

Seasonal cycle of lysosomal enzyme activities in the mantle tissue and isolated cells from the musselMytilus edulis

InMytilus edulis L., gametogenesis takes place in the mantle at the expense of the connective storage tissue. There are two main types of storage cells: vesicular (VC) cells storing large amounts of glycogen and adipogranular (ADG) cells containing large numbers of protein granules, lipid droplets and lesser amounts of glycogen. One of the ways in which stored reserves can be mobilized for gamete formation is by controlled autophagy, in which the cellular constituents are degraded by lysosomes. Mussels were collected from the Menai Strait, North Wales, and monthly measurements made, over two years (1984–1986), of the activities of lysosomal acid hydrolases (acid phosphatase,-N-acetyl-D-glucosaminidase and-glucuronidase) and Cathepsins B and L in the mantle tissue, isolated ADG cells, low-density cells and, during spawning, in the mature oocytes of female mussels. The lysosomal proteinases, Cathepsins B, L and H, were further characterised by activation with thiol compounds and inhibition with thiol blockers and by leupeptin. Because of the low activity in the mantle tissue ofM. edulis, Cathepsin H was not assayed on a seasonal basis. There was a general increase in lysosomal enzyme activity during the winter, which can be related to increased autolysis in the storage cells and to the process of maturation in the developing oocytes. The activity of Cathepsin B was highest in the ADG and low-density cells, implying an important role in proteolysis within the ADG cells. By contrast, Cathepsin L displayed the highest activity in the mature oocytes, suggesting a major function of Cathepsin L in the development and maturation of the oocytes. Two different-glucosidase activities were measured in the monthly assays, one with a pH optimum of 4.5 (acid) and the other at pH 7.5 (neutral). Highest activities of the acid-glycosidase were found in the low-density cells, but there were no significant seasonal changes in the mantle tissue as a whole. Activities of the neutral-glucosidase were low in the ADG cells and mature oocytes, but showed high activities in the mantle tissue, with marked seasonal changes that corresponded to the mobilization of glycogen reserves in the VC cells.     

Effect of fructose 2,6-bisphosphate and ammonium ions on AMP-mediated activation of phosphofructokinase from the posterior adductor muscle of the sea musselMytilus galloprovincialis

The effects of different modulators on the phosphofructokinase (PFK) activity from the posterior adductor muscle of the sea musselMytilus galloprovincialis Lmk. were studied in mussels collected from N.W. Spain in spring/summer, 1988. Adenosine monophosphate (AMP), fructose 2,6-bisphosphate (Fru 2,6-P₂) and ammonium ions individually activated PFK. AMP and Fru 2,6-P₂ reduced ATP-mediated inhibition and the value ofS _0.5 [concentration of fructose 6-phosphate (Fru 6-P) for half-maximum velocity], whereas ammonium ions increasedV _max (the rate at the saturating concentrations of Fru 6-P). Fru 2,6-P₂ and ammonium ions both enhanced the effect of AMP, but in different ways: Fru 2,6-P₂ reduced apparentK _m for AMP (concentration of AMP for half-maximum activation) without affectingK _m , whereas ammonium ions only altered the latter. The sharp increases in the values ofS _0.5 for Fru 6-P and of apparentK _m for AMP that are caused by a drop in pH were reduced by the presence of Fru 2,6-P₂, but not by ammonium ions. The results shows that Fru 2,6-P₂ may contribute to maintain the PFK activity during hypoxia periods, whereas ammonium ions (in the presence of high AMP concentrations) may activate the enzyme during the first few hours of post-hypoxia re-immersion.     

Annual reproductive cycle of the chiton Acanthopleura japonica

Annual reproductive cycle of the chiton Acanthopleura japonica (Lischke) was studied by observing both gonad index and histological features from October 1980 to October 1981. No significant changes in each factor were observed from October to April. Increased values of gonad index and spermatogenesis or vitellogenesis in gondas were observed from May to September. In the same time, gonad index decreased and sperms or oocytes in gonalds disappeared in the days around spring tide, and they were recovered around neap tide. Suggested spawning around spring tide from May to September is also supported by the occurrence of released eggs in the days around the new and full moons.     

Antioxidant enzymes in the digestive gland of the common musselMytilus edulis

Antioxidant enzymes function to remove deleterious reactive oxygen species, including the superoxide anion radical and H₂O₂. Subcellular distributions and optimal and other properties of catalase (EC. 1.11.1.6), superoxide dismutase (SOD; EC. 1.15.1.1), selenium-dependent glutathione peroxidase (Se-GPX; EC. 1.11.1.9) and total glutathione peroxidase (GPX) activities were determined in the digestive gland of the common musselMytilus edulis L. by spectrophotometric and cytochemical/electron microscopic (catalase) techniques. Assay conditions for Se-GPX and total GPX activities were determined which optimized the difference between the non-enzymic and enzymic rates of reaction. General peroxidase activity (guaiacol as substrate) (EC. 1.11.1.7) was not detectable in any subcellular fraction. Catalase was largely, if not totally, peroxisomal, whereas SOD and GPX activities were mainly cytosolic. Distinct mitochondrial (Mn-SOD) and cytosolic (CuZn-SOD) SOD forms were indicated. Catalase properties were consistent with a catalase, rather than a catalase-peroxidase. The pH-dependence and temperature-dependence of GPX activity were different with H₂O₂ or CHP as substrate, and these and other observations indicate the existence of a distinct Se-GPX. Under saturating or optimal (GPX) assay conditions, the apparent Michaelis constantsK _m (mM) were: catalase, 48 to 68 (substrate, H₂O₂); Se-GPX, 0.11 (H₂O₂) and 2.0 (glutathione); and total GPX, 2.2 (eumene hydroperoxide) and 1.2 (glutathione). Calculated catalase activity was 2 to 4 orders of magnitude greater than Se-GPX activity over an [H₂O₂] of 1 to 1000 M. The results are discussed in relation to theoretical calculations of in vivo oxyradical production and phylogenetic differences in antioxidant enzyme activities.     

Particle capture in the musselMytilus edulis: The role of latero-frontal cirri

Microscope video graphs of particle paths near one-filament-thick mussel gill preparations, stimulated with a nerve transmitter (10^–6 M serotonin which restores normal ciliary activity), were used to disclose the capture of 6 m algal cells. Suspended algal cells carried with the water were stopped for a while at the entrance to the interfilament gap by the action of the latero-frontal cirri (Ifc), and transferred to the frontal side of the filament to be transported towards the marginal food groove. The event of transfer took place during approximately a time interval of 1150 to 1/25 s. To gain a better understanding of the capture mechanism and retention efficiency versus particle size, the flow through and around the Ifc was theoretically estimated. Normally beating Ifc create periodic, unsteady, three-dimensional flows at the entrance to the interfilament canal. During the active beat most of the water is deflected to flow around the branching cilia of the Ifc while some of the water is strained by these. Large particles (> 4 m) are stopped and transferred to the frontal current, whereas smaller particles either follow the flow around the Ifc and escape or they are stopped by the branching cilia.     

Vanadium transfer in the mussel Mytilus galloprovincialis

Radiotracers were used to study processes controlling the accumulation and elimination of vanadium in the Mediterranean mussel Mytilus galloprovincialis. Vanadium uptake rates varied inversely with both salinity and vanadium concentration in water, but were independent of temperature. After a 3 wk exposure to ⁴⁸V, the highest concentration factors were found in the byssus (1900) with much lower values computed for shell ( 70) and soft tissues (5). More than 90% of the total ⁴⁸V accumulated was fixed to shell, suggesting that uptake is primarily a result of surface sorption processes. Much of the vanadium in shell was firmly bound to the periostracum and was not easily removed by acid leaching. Food-chain experiments indicated that the assimilation coefficient for ingested vanadium is low (7%) and that the assimilated fraction is rapidly excreted from the mussel. These findings coupled with knowledge of in situ and experimentally-derived vanadium concentration-factors have allowed a preliminary assessment of the relative importance of the food and water pathways in the contamination of mussels under conditions of acute and chronic exposure. Contaminated mussels transferred to clean sea water lost ⁴⁸V at rates that depended upon temperature but were largely unaffected by either salinity or by vanadium levels in mussel tissues. Total vanadium depuration was slow and was governed by loss from a slowly-exchanging compartment with a characteristic half-time of about 100 d. Individual mussel tissues were analyzed for stable vanadium and the possibility of using these tissues, particularly the byssus, as bioindicators of ambient vanadium levels in the marine environment is also discussed.     

Chemical changes accompanying maturation of the connective tissue skeletons of gorgonian and antipatharian corals

The skeletons of 3 gorgonian and 2 antipatharian corals were subjected to quantitative chemical analysis. Protein values ranged from 70.4 to 93.6%; ash from 3.0 to 19.4%; lipid from 0 to 8.4%; carbohydrate from 1.24 to 3.94% and halogen from 4.2 to 24.9% of the dry skeletal weight. Hydroxyproline and phenolic values were 0 to 3.0% and 11.6 to 25.0% of the protein, respectively. Lipid, present in 2 gorgonian species and 1 antipatharian, significantly decreased with age in all three cases. Glucose and galactose accounted for over 90%, and sialic acids for an additional 1 to 10%, of the carbohydrate in the gorgonian skeletons studied; the glucose content of the gorgonian skeleton decreased with age. The antipatharian skeletons possessed no glucose or galactose, but contained significant levels of amino sugars; the presence of chitin is confirmed. In the gorgonians, bromine and iodine, the predominant halogens, increased with skeletal age and were present in nearly equal amounts. Small amounts of bromine were found in the antipatharian skeletons, but very large amounts of iodine were found in older parts of the skeleton. The basal regions of both antipatharian species contained >23% by weight of iodine, the highest content of iodine yet recorded for any organism.     

Lysosomal,tissue and cellular alterations in the gills,palps and intestine of the mussels Mytilus galloprovincialis,in relation to pollution

Lysosomal, tissue and cellular alterations of the gill, the palp and the intestine epithelium of the mussel Mytilus galloprovincialis (L.) collected from six stations along a closed estuarine system (Thermaikos Gulf, northern Greece) were monitored in May, August and December. Semi-quantitative evaluation of histopathological alterations was performed under the light microscope, while certain of the latter alterations were confirmed electron microscopically. In addition, morphometrical evaluation of the volume density of the lysosomes under the electron microscope was conducted. The observed alterations, as the detachment of gill epithelial cells from the basement membranes and the dilated extracellular spaces formed either between the lateral plasma membranes or between the infoldings of the basal plasma membrane in the palps and the intestine are possibly related to the degree of pollution at the examined stations. The particularly expanded extracellular spaces formed by basal plasma membrane infoldings in the intestine in spring may be attributable to red tide algal infection. On the other hand, differences in morphometric parameters of lysosomal structures indicated no direct relationships with pollution levels.Communicated by O. Kinne, Oldendorf/Luhe     

Decomposition of faecal matter and somatic tissue of Mytilus galloprovincialis: changes in organic matter composition and microbial succession

Variations in the biochemical composition of pseudo-faeces and faeces egested by Mytilus galloprovincialis (Lamarck) and in detritus derived from the somatic tissue of mussels during the decomposition process were investigated by means of two intensive experiments. During the degradation process, the biochemical composition of pseudo-faeces and faeces showed a clear increase in protein content related to the microbial colonization. Changes also occurred in the biochemical composition of particulate organic matter (POM) in the surrounding water due to faecal matter decomposition, heterotrophic utilization and conversion of particulate carbohydrates and proteins to the dissolved pool. The study of production and heterotrophic utilization of the POM derived from the somatic tissue of M. galloprovincialis collected in the Gulf of Tigullio, Italy in 1990 indicates that this kind of material is rapidly decomposable and largely available for benthic consumers. Bacteria utilized selectively the different compounds, and proteins proved to be the most suitable substrate for bacterial growth. The input of organic detritus into the experimental system resulted in an enhancement of bacterial activity and consequently of the RNA/DNA ratio. Bacterial DNA accounted on average for 17% of particulate DNA. During decomposition processes, nutrient release was about ten times higher than the value calculated from individual excretion rates, indicating that mussel beds may be important sites for nutrient regeneration. Carbon conversion efficiency for bacteria growing on faecal matter was, on average, 17.2%. The potential importance of faecal output and bacterial production as a carbon resource for benthic communities near mussel culture areas is discussed.     

Annual cycle of biomass of a threatened population of the intertidal seagrass Zostera japonica in Hong Kong

The phenology and primary productivity of a population of Zostera japonica (Aschers. & Graebn.) threatened by the construction of Hong Kong's new international airport were studied over a 12-month period. The need to conserve the population, and the small leaf size of Z. japonica rendered traditional destructive or marking techniques inapplicable for percentage cover and biomass estimation. A nondestructive method based on image analysis techniques was therefore devised for repeated estimation of percentage cover, biomass and leaf area index. This technique, which involved random quadrat sampling, photographic recording and image analysis, was able to provide data on the three parameters with acceptable precision and was cost-effective in the field. Z. japonica demonstrated a strongly seasonal cycle of vegetative growth, with different patterns for leaf density (peak in March) and overall bed area (peak in June). Total (above- and below-ground) net primary productivity was estimated at between 344 and 688 g AFDW m⁻² yr⁻¹. Percentage cover of Z. japonica was negatively correlated with total suspended solids (TSS) in the water column while total bed area was negatively correlated with water salinity. Increased sedimentation associated with the new airport project was identified as one important factor affecting the growth of the seagrass, as TSS reached the high level of ≈1 g DW l⁻¹ during the first half of the study period. Sediment traps set in the beds also recorded potential sedimentation rates at between 2.89 and 14.5 mg cm⁻² d⁻¹. This high turbidity resulted in a sharp decrease in the density of Clithon spp., the dominant grazers of epiphytic algae on Z. japonica. Effects of sedimentation and shading on growth of Z. japonica were investigated by field manipulative experiments. Experimental increase of sedimentation rate and shade both resulted in larger decreases in percentage cover and above-ground AFDW compared with the control. Received: 3 March 1997 / Accepted: 14 March 1997     

Metabolic fate of aromatic amines in the mussel Mytilus galloprovincialis

The postmitochondrial fraction of the digestive gland from the marine mussel Mytilus galloprovincialis possesses FAD-containing monooxygenase (EC 1.14.38) but lacks cytochrome P-450 dependent benzo(a)pyrene monooxygenase (EC 11.4.14.1). This is also evidenced by the ability of the mussel preparation to activate carcinogenic aromatic amines, but not carcinogenic benzo(a)pyrene, to Salmonella typhimurium TA 98 mutagens. This metabolic activity is NADPH dependent. Mussel digestive gland postmitochondrial fraction also possesses the enzymes needed for the detoxicating part of the aromatic amine metabolism: UDP-glucuronyl transferase (EC 2.4.1.17) and -glucuronidase (EC 3.2.1.31). Under the experimental conditions used here, this aromatic amine metabolic pathway converts up to 8% of 2-acetylamino(9-¹⁴C)fluorene, but not (G-³H)benzo(a)pyrene, to water soluble glucuronides. Glucuronic acid stimulates the formation of these glucuronides. The metabolites liberated from these glucuronides by the -glucuronidase treatment could be converted to TA 98 strain mutagens by the carp liver postmitochondrial fraction, but not by the mussel's digestive gland preparation. The presence of such a selective potential for the bioactivation and detoxication of aromatic amines, and not polycyclic aromatic hydrocarbons, in the marine invertebrate(s) may bring new insight to our understanding of the effects and the fate of carcinogens in the marine environment.     

Annual life cycle of Chattonella spp., causative flagellates of noxious red tides in the Inland Sea of Japan

Effects of storage temperature on the dormancy and maturation of dormant cells of Chattonella spp. were investigated by the extinction dilution method using sediment samples collected in 1984 after the blooming in Harima-Nada. Seasonality of germinability in fresh sediments was also studied in 1985. A storage period of more than 4 months with a low temperature (11°C) was essential for the maturation to induce the germination capacity of dormant cells. The optimal temperature for germination (22°C) had no effects on the maturation. Using fresh Suo-Nada sediments, a marked seasonality of germinability was confirmed. It was weak from autumn to early winter, then strengthened gradually up to a high level, which was maintained between spring and early summer, and again decreased rapidly during summer. The annual life cycle of Chattonella spp., including vetetative and dormant phases, was summarized as follows: (1) vegetative cells in early summer originate from germination of dormant cells in sediments; (2) they form overwintering dormant cells during the summer season; (3) dormant cells spend a period of spontaneous dormancy until next spring; (4) the duration of post dormancy, an enforced one due to low temperatures, follows until early summer. The life cycle of Chattonella spp. is therefore well adapted to the temperature regime in temperate seas such as the Seto Inland Sea of Japan.     

Oil pollution monitoring in the Lagoon of Venice using the mussel Mytilus galloprovincialis

Gas chromatographic analyses of mussels, Mytilus galloprovincialis, from different areas of the Lagoon of Venice show that these organisms contain a very complex mixture of hydrocarbons attributable to fuel oil contamination. The measured amounts normally range from 0.8 to 8.7 mg/100 g wet weight, but values as high as 22.0 mg/100 g have been recorded. This high value indicates a saturation limit for these organisms which is considerably higher than those values normally found in mussels from the lagoon. The aliphatic hydrocarbon levels in mussels are related to distance from pollution sources and to the degree of exchange between the sea and the area sampled. On the basis of this relationship between overall hydrocarbon pollution load and level of contamination of M. galloprovincialis, it appears that this bivalve might be effectively utilized as a self-integrating monitoring index of oil pollution in the wasters of the lagoon.     

Effect of titanium dioxide nanoparticles on the expression of apoptotic markers in mouse blastocysts

Oocyte maturation, embryo development and expression of apoptotic-specific genes were evaluated in blastocysts of mice treated with titanium dioxide nanoparticles. Female mice received 0, 50 or 100?mg/kg/day titanium dioxide intraperitoneally for five consecutive days. After the last injection, pregnant mare serum gonadotropin and, 48?h later, human chorionic gonadotropin were administered intraperitoneally for induction of ovulation. After 14?h, mice were sacrificed and oocytes were collected. The number of mature oocytes was evaluated and then fertilization was carried out in vitro and the numbers of fertilized and cleaved oocytes and of blastocysts and the expression of Bax, caspase 3, and Bcl-xL genes in blastocysts were evaluated. The number of mature, fertilized and cleaved oocytes and of blastocyst embryos in the experimental groups were not different from control. The expression of Bax and caspase 3 genes was significantly elevated in the experimental groups compared to control, while expression of the Bcl-xL gene was significantly lower in the high dose group. Uptake of titanium dioxide nanoparticles at daily doses of 50?mg/kg and more may affect embryo development by alteration of pro-apoptotic and anti-apoptotic gene expression.     

Annual cycle,species diversity and succession of phytoplankton in lower Saronicos Bay,Aegean Sea

Phytoplankton cycles of lower Saronicos Bay, Aegean Sea, are described on the basis of net samples collected during 1967. The annual cycle is largely due to changes in diatom concentrations (cells/liter). The diatom summer poverty is due mainly to high light intensities, phosphate depletion and extensive grazing. The species succession and diversity have been examined, and the results are discussed.     

Annual reproductive cycle of the green sea urchin, Strongylocentrotus droebachiensis, in differing habitats in Nova Scotia, Canada

We monitored the reproductive cycle of Strongylocentrotus droebachiensis (OF Müller) between April 1993 and August 1995 in kelp beds, barren grounds and grazing fronts at both a wave-exposed and a sheltered site along the Atlantic coast of Nova Scotia. Gonad index and histological analyses showed that S. droebachiensis has an annual reproductive cycle that is synchronous across sites and habitats, and between females and males. Spawning occurs in March/April of each year but a small proportion of sea urchins in the study populations also spawned in fall 1995. During most of the year, sea urchins in kelp beds and grazing fronts have a higher gonad index than those in barren grounds. Gonad indices also tended to be higher at the wave-exposed than the sheltered site. Interannual variability in peak gonad index was significant in the barren grounds at the wave-exposed site and in the grazing front at the sheltered site. The gametogenic cycle is characterized by six stages based on the abundance of nutritive and germinal/gametic cells. Nutritive phagocytes are abundant after spawning and replaced by increasing numbers of germinal and gametic cells as the gametogenic cycle progresses. The temporal patterns of abundance of each cell type were similar among habitats indicating that the gonads were qualitatively similar despite large differences in gonadal mass. The quantity of gut contents (ratio of food volume to body volume) was similar among habitats, but the quality (percentage of organic material) tended to be higher in kelp beds and grazing fronts than in barren grounds suggesting that differences in gonad index of S. droebachiensis in different habitats are related to differences in diet. The high density of sea urchins in grazing fronts combined with their high fecundity suggests that they make the greatest contribution, per unit area, to the overall larval pool. Received: 20 May 1997 / Accepted: 21 January 1998     

Factors affecting the flux of arsenic through the mussel Mytilus galloprovincialis

Radiotracer experiments were designed to study the effects of certain environmental and biological factors on arsenic accumulation and elimination processes in the Mediterranean mussel Mytilus galloprovincialis. Arsenic (as arsenate) uptake increased with increasing arsenic concentration in the water; however, the response was not proportional, indicating that accumulation was partially suppressed at higher external arsenic concentrations. In general, approximately 80% of the ⁷⁴As taken up was associated with the soft parts, with small mussels concentrating ⁷⁴As to a greater degree than larger individuals. The highest ⁷⁴As concentrations were recorded in the byssus and the digestive gland. Increased temperature enhanced both arsenic uptake and loss. Mussels in sea water at 19 S accumulated approximately three times more ⁷⁴As than those held at 38 S. Arsenic loss was much less affected by salinity, with only a tendency for greater arsenic retention noted at lower salinities. Studies carried out in the laboratory and in situ revealed that arsenic turnover was significantly more rapid in actively growing individuals living under natural conditions. Arsenic-74 loss from the in situ group was essentially biphasic, with biological half-times of approximately 3 and 32 days for the fast and slow compartments, respectively. The active secretion of arsenic in the byssal threads contributed to the total elimination of the element from the mussels.     

Effect of high temperatures on the ultrastructure of Leydig cells in Mytilus galloprovincialis

Individuals of the mussel Mytilus galloprovincialis (L), collected from the Black Sea, were subjected to relatively high temperatures (25°, 28° and 30°C) for 60 h Ultrastructural studies on Leydig cells of the mantle indicated progressive modifications in their structure. Mainly, we established that a continuous decrease of glycogen quantity in the cells took place as the temperature raised, while the lipid content increased, up to 28°C. Between 28° and 30°C the lipid content decreased as a result of a pronounced degradation process. An increase of membrane permeability and the disturbance in the synthesis of mucoproteic secretory granules were observed. It is concluded that for Mytilus galloprovincialis the thermal adaptation zone ranges between 25° and 28°C. 28°C is the upper tolerable limit temperature, while 30°C acts as a lethal temperature.