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 cycle of expression of connective tissue polypeptide markers in the mantle of the musselMytilus galloprovincialis

We suggest that gonad development in the mantle tissue of the bivalve molluscMytilus galloprovincialis Lmk. is an example of epithelial/mesenchymal interactions (i.e. soma/germline interactions) and morphogenesis in the adult state. According to this concept, the aim of the present study was to use biochemical and immunochemical methods for identifying and characterizing the mantle cell polypeptide markers whose expression is seasonally and morphogenetically regulated. We showed for the first time thatM. galloprovincialis mantle, of both males and females, contains polypeptides (with an apparent mol. wt of 45 to 53 kDa) specific for connective tissue (mantle connective tissue polypeptides; MCTPs). Electrophoretic, immunoblotting and immunofluorescent experiments demonstrated that MCTPs are primarily localized in the adipogranular (ADG) cells, and their expression in the mantle is seasonally regulated. There is a positive correlation between MCTP expression and connective tissue volume in the mantle. MCTPs are overexpressed during the rest period, when the mantle consists of connective tissue mainly, whereas mature gonads contain only trace amounts of MCTPs. Moreover, there is a temporal correlation between the onset and decrease of MCTP expression and the appearance and disappearance of the ADG cells in the mantle. MCTP localization in the mantle tissue should not be associated with the ADG cells only, because positive immunofluorescence was also detected in follicle membranes (but not in germ cells) and superficial mantle epithelium. Using immunoblotting and immunofluorescence, MCTPs were found in the connective tissue of the mantle, posterior adductor muscle and visceral mass, but not gills, foot or hepatopancreas. Possible mechanisms by which MCTPs could participate in the annual processes of mantle gonad/connective tissue development and involution are discussed.     

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.     

Effects of petroleum hydrocarbons on the cytochrome P450 content of the mollusc bivalveMytilus galloprovincialis

The cytochrome P₄₅₀, identified by the characteristic 450 nm spectral peak of its carbon monoxide complex, was detected in the hepatopancreatic microsomes ofMytilus galloprovincialis, collected from Ponteau (Bay of Fos, France) in 1982 and 1983. When mussels were exposed to a variety of petroleum hydrocarbons, the cytochrome P₄₅₀ content increased. It is shown that aromatic hydrocarbons are more effective than alkanes in inducing a high level of cytochrome P₄₅₀. The possible use of this haemoprotein in hydrocarbon pollutant detection is discussed.     

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.     

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.     

Regulation of spirocyst discharge in the model sea anemone, Nematostella vectensis

Test probes were touched to tentacles to investigate whether discharge of spirocysts likely is regulated by hair bundle mechanoreceptors. Significantly more spirocysts discharge onto test probes in the presence of vibrations at 11–15 Hz as compared to 0 Hz. Adding N-acetylneuraminic acid, NANA, shifts maximal discharge of spirocysts upwards to 36–40 Hz, and possibly to 21–25 Hz. In contrast, NANA shifts maximal discharge of basitrichous isorhiza nematocysts downwards to 1–20 Hz. Thus, discharge of cnidae (‘stinging capsules’) is differentially regulated according to the type of cnida. Furthermore, it appears that chemodetection of N-acetylated sugars is not a prerequisite to capturing prey because, in seawater alone, maximal discharge of cnidae occurs at frequencies overlapping movements of calmly swimming prey. Nevertheless, chemodetection of N-acetylated sugars broadens the range of frequencies stimulating maximal discharge of cnidae and, therefore, likely enhances prey capture.     

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.     

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.     

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     

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.     

210Po content in sea water and its accumulation in southern Baltic plankton

This paper presents the results of an investigation from 1980 to 1985 on the content of ²¹⁰Po in sea water and its accumulation in southern Baltic plankton. The mean value of the ²¹⁰Po concentration in sea water has been determined to be about 0.5 mBq dm^-3, with the proportion of the dissolved forms being ca 80%. It has been proved that ²¹⁰Po is accumulated by plankton organisms. The mean ²¹⁰Po concentration factors calculated on the basis of its content in plankton fall within the range from 5×10³ to 4.2×10⁴ and increase as follows: phytoplankton210Po concentration in mesozooplankton collected at the Slupsk Trough and the Gdask Basin have been observed. The above differences have been related to intense blue-green alga blooming in the Gdask Basin.     

Effect of light on the total lipid content and storage lipids of the symbiotic sea anemoneAnemonia viridis

In order to examine the effect of light level on the storage lipids of the symbiotic sea anemoneAnemonia virudis (Forskäl), anemones were exposed to three experimental light regimes of 10, 100 and 300 E m^-2s^-1. Anemones were fed once a week. After 30 d there were no significant differences in the total lipid levels between anemones at any of the light intensities. However, after 60 d lipids had increased in proportion to light level in both the animal-tissue and zooxanthellae compartments. The higher levels of total lipid were in part due to increases in storage lipid (wax esters and triglycerides). Wax ester levels increased in the animal tissues but remained constant in the zooxanthellae, whereas triglycerides increased in both compartments. In contrast to fed anemones, starved anemones which were maintained at 300 E m^-2s^-1 for 30 or 60 d did not show a statistically significant change in lipid levels at 60 d, although a slight increase in the lipid level was observed. However, there was a significant increase in the storage lipids, which suggested that the non-storage phospholipids and structural lipids had declined as a result of cellular catabolism. The composition of the wax esters and triglycerides of both fed and starved anemones was analysed and compositional changes were observed at higher light intensities.     

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.     

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.     

A kinetic and autoradiographic study of the direct assimilation of amino acids and glucose by organs of the musselMytilus edulis

The uptake of amino acids and glucose dissolved in sea water by different parts of common mussels (Mytilus edulis, L.) is studied from the first minutes up to 1 week. Autoradiography and donble countings on¹⁴C and²H labelled samples show that, in the first hour, the gills and mantle can concentrate several hundred times the dissolved nutritive molecules from very dilute solutions, whereas the digestive tract is not involved significantly during the first hours. An important -amylasic activity has been detected in the gills. The gill epithelium shows a strong positive reaction with mixtures used for the histochemical detection of chymotrypsin. This suggests that the digestion of small particles as well as the absorption of dissolved food might be initiated on the surface of the palleal-gill areas, and completed later in the hepatic caeca.     

The musselsMytilus galloprovincialis andM. trossulus on the Pacific coast of North America

Most recent authors have called the bay mussels of the Pacific coast of North AmericaMytilus edulis Linnaeus, 1758. Thirteen samples ofedulis-like mussels were collected from California, Oregon, and Alaska, USA, in 1985, 1986 and 1987. Electrophoretic evidence from wight loci indicates that these samples consist of two genetically distinct groups, neither of which is similar toM. edulis from the Atlantic Ocean. Mussels in southern California are very similar toM. galloprovincialis Lamarck, 1819 from the Mediterranean Sea; it is probable thatM. galloprovincialis was introduced accidentally to southern California. Mussels in Oregon and Alaska are similar to those from the Baltic Sea and parts of eastern Canada; the nameM. trossulus Gould, 1850 has priority for this taxon. In central and nothern California,M. galloprovincialis, M. trossulus and their hybrids co-occur. Despite the presence of hybrids betweenM. galloprovincialis andM. trossulus, the genetic integrity which they maintain across large areas of the world warrants their recognition as two distinct species.     

Normalisation models for accounting for fat content in stable isotope measurements in salmonid muscle tissue

Stable isotope analysis is increasingly used in ecological studies. Because lipid content influences δ¹³C, lipids should be removed from lipid-rich samples before δ¹³C analysis. To account for differences in δ¹³C arising from differences in lipid content, relationships between lipid content, C:N ratio and Δδ¹³C with lipid removal can be used to normalise lipid content to uniform levels. We investigate these relationships for salmonid muscle and evaluate the suitability of previously published normalisation equations for these fish. Salmonids with a wide range of condition (muscle lipid content = 3–35% of dry weight) were considered. There were no consistent relationships between lipid content or C:N ratio and Δδ¹⁵N. There were linear relationships between C:N ratio and lipid content (L = −16.53 + 6.27 × C:N); C:N ratio and Δδ¹³C (Δδ¹³C = −1.87 + 0.65 × C:N); and lipid content and Δδ¹³C (Δδ¹³C = 0.01 + 0.10 × L), which should be used on salmonid stable isotope studies.     

Ultrastructure of the byssal apparatus of Mytilus galloprovincialis. I. Associated fungal hyphae

Electron-microscope observations on ultrathin sections of Mytilus galloprovincialis byssus have revealed the existence of fungal hyphae. The ultrastructural features of these hyphae identify them as ascomycetes. A saprophytic nature is suggested for this fungus, and its probable role in the degradation of the byssus is discussed.     

Antimicrobial activity of tissue and associated bacteria from benthic sea anemone Stichodactyla haddoni against microbial pathogens

Associated bacteria from Stichodactyla haddoni are found maximum in tentacle tissues than the body tissue. There are eight associated bacterial species viz., Alcaligenes sp, Corynebacterium sp, Aeromonas sp, Sporosarcina sp, Renibacterium sp, Camobacterium sp1, Camobacterium sp2 and Salinococcus sp were recorded. The culture extracts from the associated bacterial species showed sensitivity against human bacterial and fungalpathogens. However, the hexane tissue extract of sea anemone showed maximum sensitivity (24 mm dia.) against the fish bacterial pathogen Aeromonas hydrophila than the other chosen pathogens. Comparatively the tissue extracts showed promising antimicrobial sensitivity than the cell free extracts of associated bacteria, and hence, the tissue samples from the sea anemone Stichodactyla haddoni is recommended for further exploration of novel antimicrobial drugs than the associated bacteria.