Mantle protein excretion and calcification in the hardshell clam Mercenaria mercenaria. II. Protein synthesis and excretion by the isolated mantle

Sheets of mantle tissue from above the mantle line of Mercenaria mercenaria were incubated for 2, 6 and 20 h at 20°C in 50 Ci ³H-glycine in 50 ml artificial seawater. Incorporation of tritium into soluble proteins excreted by the mantle and into tissue proteins was followed. The excretion of soluble protein continued throughout the experiment; the proportion of incorporated label excreted reached 17% by 20 h. The initiation of excretion of labelled protein seemed to lag 60 to 70 min behind the initiation of protein synthesis. Protein synthesis by the mantle contributed to proteins of the extrapallium and mantle chambers and, thus, may be involved in synthesis and regulation of proteins involved in the shell-formation process.     

Nucleic acid synthesis in oceanic microplankton from the drake passage,antarctica: Evaluation of steady-state growth

Differential uptake of [³H]adenine and [³H]thymidine, and incorporation of tritium label into proteins, RNA and DNA as a function of depth, indicate that surface microplankton incorporated most of the assimilated radioisotopes into nucleic acids. Growth processes for deep-sea microplankton were shifted towards higher rates of [³H]-adenine incorporation into RNA compared to rates of thymidine incorporation into DNA. Deep-sea microplankton also diverted a larger portion (up to 80%) of the assimilated tritium into biosynthetic pathways for amino acid and eventual incorporation into proteins. These results imply that protein synthesis is vital for deep-sea microplankton where populations may be stressed by low levels of available nutrients. The rates at which microplankton incorporated [³H]adenine into RNA and DNA, and [³H]thymidine into DNA, suggest that oceanic microplankton are in a transient state of balanced growth, i.e. between two steady-state growth conditions, irrespective of potential growth rates. Our results support the hypothesis that oceanic microplankton are in various growth states.     

Mantle-to-shell CaCO<Subscript>3</Subscript> transfer during shell repair at different hydrostatic pressures in the deep-sea vent mussel <Emphasis Type="Italic">Bathymodiolus azoricus</Emphasis> (Bivalvia: Mytilidae)

Calcium carbonate transfer was experimentally examined in hydrothermal mussels Bathymodiolus azoricus, which were collected from 850 m depth at Menez Gwen hydrothermal vent site (31°31′W, 37°50′N) on the Mid Atlantic Ridge in May 2007. In each of four 10-day experiments, groups of mussels were maintained at atmospheric pressure or re-pressurised to depths relevant to their site of occurrence, i.e. 850 m depth at Menez Gwen, 1,750 m at Lucky Strike (31°31′W, 37°18′N) and 2,300 m at Rainbow (31°31′W, 36°13′N). The shells of experimental mussels were perforated and mantle tissue was fixed for light and TEM studies at days 7 and 10 following the injury. Simultaneously, haemocytes from the extrapallial fluid (EPF) at the site of induction were studied. At day 7 the response was most intense in the middle fold of the mantle margin and possibly proportional to hydrostatic pressure. At day 10 the epithelial cells on the mantle surface facing the body cavity produced copious organic secretions that avidly bound calcium. Haemocyte migration was noted within the mantle tissue, and the haemocytes at the mantle surface facing the shell had a Ca-positive granular content. Large haemocytes were detected in the EPF at the injury site, and some showed evidence of an immune reaction while others showed Ca-positive granular content. These results suggest that haemocytes are involved in shell repair in these deep-sea mussels just as in some freshwater and shallow marine molluscs.     

Movements of tiger sharks (Galeocerdo cuvier) in coastal Hawaiian waters

The giant clam Tridacna crocea harbors in the mantle tissue symbiotic microalgae commonly called zooxanthellae. Isolated zooxanthellae release glycerol into the medium in the presence of mantle tissue homogenate (MH), but it is not clear whether the cells do so in situ. In order to determine the photosynthetic products released by zooxanthellae in the mantle of the giant clam we traced photosynthetic fixation products from ¹³C- and ¹⁴C-bicarbonate both in the clam and in isolated zooxanthellae (IZ) in the presence or absence of MH. After 15 min incubation in the absence of MH the IZ released less than 0.6% of the fixed labeled carbon, mainly as glucose. The major intracellular photosynthates were neutral lipids, which constituted 20 to 40% of the total extractable ¹⁴C. In the presence of MH, the IZ released up to 5.6% of the total fixed ¹⁴C, mostly as glycerol, and the major intracellular photosynthate was glucose. In an intact clam incubated in sea water containing ¹⁴C-bicarbonate, 46 to 80% of the fixed ¹⁴C was translocated from the zooxanthellae to the host tissues. Most of the ¹⁴C in the hemolymph, in the isolated zooxanthellae and in intact mantle tissue (containing zooxanthellae) was recovered as glucose. No ¹⁴C-glycerol was detected in the mantle after 1 to 30 min incubation, and, even after 60 min, far less ¹⁴C-glycerol was synthesized than by IZ in the presence of MH. The possibility that in clam tissue glycerol is converted to glucose was examined by tracing the labeled carbon from ¹⁴C-glycerol injected into the adductor muscle. After 5 min incubation, no labeled glucose was found in the hemolymph, but after 60 min, some 20% was found as glucose. Thin slices containing zooxanthellae, cut from the surface of the mantle, fixed inorganic carbon supplied as NaH¹⁴CO₃ in the medium and mainly released ¹⁴C-glucose. The addition of MH to the surrounding medium did not affect the release rate or form of release product. When the slices were cut into smaller pieces, however, the ratio of glycerol to glucose in the release product increased. These results indicate that in the presence of MH the metabolism of isolated zooxan- thellae was different from that of zooxanthellae in the mantle. In the presence of MH, isolated zooxanthellae release mostly glycerol, whereas in the mantle they release glucose. Received: 18 February 1998 / Accepted: 4 December 1998     

The uptake and distribution of 65Zn in oysters

Over a period of 6 weeks in aquaria, Portuguese oysters Crassostrea angulata, accumulate ⁶⁵Zn to a greater extent than do native Ostrea edulis, although intake rates for any particular organ in either species are quite similar. The general distribution pattern of radioactivity in the tissues is similar to that observed for stable zine and ⁶⁵Zn in other oyster species, concentration occurring to the greatest extent in gills and mantle, and least in muscle. The observed equilibrium concentrations and biological half-lives are considerably less than those measured in the natural environment, and the significance of this and its bearing on the mechanism of uptake is discussed. Cobalt and iron depress the rate of ⁶⁵Zn uptake by both oyster soft tissues and the shell. The limiting effect in soft tissues is probably due to competition for sites at the actual point of uptake. The distribution of ⁶⁵Zn in tissue subcellular fractions separated by centrifugation shows the greatest concentration of the radioisotope in the insoluble tissue components of gills, mantle and heart. Appreciable amounts of ⁶⁵Zn are associated with tissue proteins.     

Chemische tarnung. Die stoffliche grundlage der anpassung von anemonenfischen an riffanemonen

Dissolved amino acids (³H-phenylalanine, ³H-proline) were accumulated and incorporated into proteins and glycoproteins by sea anemones. On objects (glass rods, pipe cleaners or filter paper) which had touched labelled anemones, tritiated substances could be detected. The mucus of labelled anemones was analysed by disc-electrophoresis. Tritium activity was found in the bands (stained by amino black). Anemone fishes adapted to labelled anemones had 4 times more tritium activity on their surface than control fishes. Most radioactivity was found on those regions which made closest contact with the anemones. The mucus of anemone fishes adapted to labelled anemones was also analysed by disc-electrophoresis. The analysis revealed protein-containing fractions in which ³H-amino acids were incorporated. The pattern of tritium activity in disc-electropherograms from labelled anemones corresponds to that of fishes adapted to those anemones. Anemones produce specific substances which influence the discharge of their nematocytes. These substances have special functions in the normal behaviour of the anemones. The substances provide protection against self-nettling and prevent the discharge of nematocytes into nearby objects which the tentacles continuously contact. The production of these substances by anemones is completely separate from the association with anemone fishes. The fishes thus simply exploit a mechanism existing independently in the anemones. Therefore, it is possible to form, in an aquarium, unnatural associations between anemone fishes and anemone species which never live in association with fishes in their natural biotope. On the basis of previous information, as well as on these new data, it is possible to develop a model which explains the control of nematocyte discharge: Substances with inhibitory qualities (protecting substances) are produced by the anemones themselves, and de-sensitize the sensory inputs of the anemones (nematocytes and sensory cells). Sensitization takes place as soon as the anemones come into contact with “stimulating substances”. This happens if anemones are touched by food objects or by anemone fishes which have been previously isolated from anemones. The surfaces of these fishes are not impregnated with protecting substances. Adapted anemone fishes, neighbouring anemones of the same species and other “adapted” objects are coated with the inhibitory substances and thus do not induce nematocyte discharge.     

Removal of glycine from solution by the sea urchin Strongylocentrotus purpuratus

Radioautography and liquid-scintillation counting of juvenile sea urchins — Strongylocentrotus purpuratus (Stimpson) — exposed to ³H- and ¹⁴C-labeled glycine showed that the ³H is rapidly translocated throughout the animal and lost, while the ¹⁴C is slowly translocated into the interior of the urchin and incorporated into insoluble material.     

Incorporation of pesticides by soil micro-organisms as a way of bound residues formation

We studied the ¹⁴C-tagged biochemical compartments such as proteins, lipids and polysaccharides from bacterial and fungal cultures supplied with ¹⁴C-pesticides and ¹⁴C-glucose. We showed that ¹⁴C incorporation depends both on the substrate structure and on the nature of the micro-organism. Both bacterial and fungal cells incorporated glucose ¹⁴C mainly into cell wall proteins. By contrast, glyphosate ¹⁴C was mainly incorporated into cytoplasm carbohydrates by fungi. On the other hand, 2,4-dichlorophenoxyacetic acid ¹⁴C was mainly found in the carbohydrate fractions of the bacteria walls.     

The essential amino-acid requirements of the prawnPalaemon serratus. The growth of prawns on diets containing proteins of different amino-acid compositions

Two specimens ofPalaemon serratus Pennant (of about 4 g wet weight) were each given intrahaemocoelic injections of [U-¹⁴C] acetate. They were maintained under controlled conditions, and the release of¹⁴CO₂ and other labelled metabolites was followed for 6 days. The prawns were then sacrificed and fractionated into trichloroacetic acid soluble compounds, lipids, nucleic acids and a protein/chitin residue. Little or no¹⁴C had been incorporated into arginine, methionine, valine, threonine, isoleucine, leucine, lysine, histidine, phenylalanine and tryptophan. It was inferred thatP. serratus cannot synthesize these amino acids from ordinarily available materials, and that the prawns have an absolute dietary requirement for them. The glucosamine isolated from the protein/chitin residue contained more radioactivity than any of the other compounds examined. This implies a rapid turnover of glucosamine and chitin inP. serratus. The gain in weight of prawns fed on compound diets containing freeze-dried cod muscle was about 70% that of prawns fed on fresh mussel mantle. Predigestion of freeze-dried cod muscle with proteolytic enzymes did not significantly enhance its nutritional valve as assessed by increases in weight. Prawns fed two other compounded diets containing proteins of low nutritional value (gelatin and zein, respectively) grew at about 1/5 the rate of prawns fed fresh mussel mantle. Supplementation of the gelatin and zein in these diets with tryptophan, and with lysine plus tryptophan, respectively, failed to augment their nutritional value significantly.     

Uptake,binding and clearance of divalent cadmium in Glycera dibranchiata (Annelida: Polychaeta)

The bloodworm Glycera dibranchiata Ehlers, 1968 accumulates cadmium through the general body surface and the intestine. Absorption through the gut accounts for cadmium which rapidly binds to coelomic proteins. Intracoelomic injection of ¹⁰⁹Cd demonstrates that cadmium binds readily to hemoglobin and other proteins. The degree of cadmium binding is pH-dependent. The apparent pK of binding sites in body wall and musculature homogenates is 5.39. Cadmium ions injected into the coelom at 7 g g^-1 tissue increase proline incorporation rates into the positively charged hemoglobin (cathode fraction) by 15-fold in 3 days. A 3-fold increase of proline incorporation was observed in the anode hemoglobin fraction over the same time period. Radioactivity in various protein fractions decreases at different rates after injection of ¹⁰⁹Cd. Comparisons between the function of mammalian metallothionein and the coelomic fluid proteins of G. dibranchiata as a detoxification mechanism are discussed.     

Fluxes of dissolved and particulate calcium in selected tissues of Crassostrea virginica

Rates of calcium incorporation by selected tissues of Crassostrea virginica increased in a step-wise fashion from lowest values among organisms exposed to ambient total calcium concentrations of 45 mg l^-1, intermediate values among oysters exposed to 135, 225, and 315 mg l^-1, to highest rates for oysters exposed to 360 mg l^-1. Although excised visceral mass tissue had highest rates of calcium incorporation relative to mantle, muscle, and both organic and inorganic portions of the shell, mantle tissue appeared to have the most dynamic response to changes in ambient calcium concentrations. Rates of dissolved calcium incorporation from ambient water were approximately two to three orders of magnitude higher than comparable rates from ingested algal food. Behavioral response to concentrations of selected inoic, species in the ambient environment may have been responsible for observed differences in rates of calcium incorporation.     

Hypoxic life of intertidal acorn barnacles

Oxygen levels were monitored within the mantle cavities of three barnacle species ( Chthamalus stellatus, Semibalanus balanoides, Elminius modestus), using optode microsensors. Conditions were always hypoxic, even when barnacles were actively using the prosoma and cirri to pump aerated seawater into the mantle cavity. Mantle fluid oxygen concentrations were extremely variable; behaviour and oxygen concentrations were not closely coupled. Ventilation of the mantle cavity depended partially on external water flow, with higher and more stable mantle fluid oxygen concentrations being sustained when the water around barnacles was agitated. During emersion, barnacles initially pumped seawater between the mantle cavity and the cone above the opercular plates to achieve ventilation. As water was lost it was replaced by air bubbles, eventually resulting in an air-filled mantle cavity. In S. balanoides and E. modestus, once the mantle cavity was filled with air, the barnacle usually used up the oxygen within the bubble within 2–3 h and did not regain oxic conditions until the barnacle was reimmersed in seawater. In C. stellatus, the air bubble was repeatedly refreshed for many hours by pneumostome formation. In response to low environmental salinity, all three species closed the opercular plates firmly and rapidly used up oxygen within the mantle fluid.     

Energy maximization by selective feeding on tissues of the venerid clam <Emphasis Type="Italic">Marcia hiantina</Emphasis> in the marine scavenger <Emphasis Type="Italic">Nassarius festivus</Emphasis> (Gastropoda: Nassariidae)

Feeding selectivity of the intertidal scavenging gastropod Nassarius festivus on four types of tissue (soft tissue, adductor muscle, foot, mantle margin) of the venerid clam Marcia hiantina was studied by field observations and laboratory experiments. Both approaches showed similar results with the soft tissue and adductor muscle being much preferred. As the energy content of the four types of tissue was similar and individuals of N. festivus rearing in different types of tissue obtained similar growth rates both in shell length and in body wet weight, differential consumption was most likely determined by the palatability of the tissues. Energy intake in a meal for N. festivus was estimated at 10.92, 9.17, 3.86 and 2.02 cal meal⁻¹ ind⁻¹ for soft tissue, adductor muscle, mantle margin, and foot, respectively. In view of the small size and sporadic distribution of the carrion, and intense competition among the conspecifics, selective feeding on more palatable tissues maximizes energy intake for scavengers like N. festivus.     

Effect of Cd on accumulation and loss of 210Po in the mussel Mytilus edulis

As a result of ²¹⁰Po's previously identified association with sulphur-rich proteins, metallothioneins could have a significant effect on the behaviour and fate of ²¹⁰Po in molluscs. Starved control and cadmium-exposed mussels, Mytilus edulis, were fed ²¹⁰Po-labelled algae (Isochrysis galbana) for 5 d and then allowed to depurate in clean sea water. Cadmium-exposed M. edulis accumulated less ²¹⁰Po in the digestive gland and the remainder of the tissue than control mussels, although this was due to a decrease in tissue weight. More than 40% of ²¹⁰Po was identified as being associated with high molecular weight and heat-treated cytosol proteins in M. edulis. Mussels in a starved state are known to recycle as much as 90% of their amino acids. It is proposed that ²¹⁰Po associated with these and other proteins is recycled, explaining why no significant loss of ²¹⁰Po was observed from the remainder of the tissue in either control or Cd-exposed mussels. Cadmium-induced metallothioneins had no effect on the distribution of ²¹⁰Po in M. edulis; <5% associated with the cytosolic fraction was considered to principally contain metallothioneins. It is suggested that ²¹⁰Po's apparent relationship with metallothioneins is coincidental rather than connected with its role in the regulation of metals. Received: 30 March 1998 / Accepted: 3 August 1998     

Rates of ammonium turnover and the role of amino-acid deamination in seagrass (Zostera capricorni) beds of Moreton Bay,Australia

Samples of sediments from Australian seagrass (Zostera capricorni Aschers.) beds were taken in June to August 1983 (for¹⁵N experiments) and November 1982 to January 1983 (¹⁴N experiments). The ammonium pool turned-over every 0.4 to 0.8 d, as determined with a¹⁵N isotope-dilution technique. The ammonium pool in subtidal bare areas turned-over two to three times more slowly than in adjacent seagrass beds. Gross rates ofin situ ammonium regeneration equalled those of utilization, and ranged from 0.04 to 0.35 mol cm^-3 d^-1, or from 50 to 490 mg N m^-2 d^-1 over the upper 10 cm of the sediment. The potential rate of glycine utilization, measured with a large excess of glycine added to anaerobic incubations, ranged from 0.21 to 0.39mol cm^-3 d^-1, butin situ rates were probably much lower. Between 35 and 65% of added¹⁵N-glycine was deaminated over 12 h, and the remainder was most likely assimilated by microbes. Evidence for the seagrasses taking up glycine was equivocal, owing to the rapid deamination of the amino acid and the likelihood that they assimilated the labelled ammonium produced from the glycine.     

Assimilation of 210Po by the mussel Mytilus edulis from the alga Isochrysis galbana

Marine invertebrates are thought to accumulate ²¹⁰Po primarily from their food. In this study, a pulse-chase methodology was used to examine the assimilation and depuration of ²¹⁰Po by Mytilus edulis from the common marine alga Isochrysis galbana. The digestion of ²¹⁰Po from I. galbana occurred via a biphasic process, characteristic of a rapid (extracellular) and slow (intracellular) digestion typical of marine bivalves. The mantle/gill and foot have no known digestive role, yet their ²¹⁰Po specific activities increased after 24 h. It is proposed that this increase in ²¹⁰Po specific activity was related to ²¹⁰Po being incorporated into these tissues from ²¹⁰Po assimilated from I. galbana during extracellular digestion. It is proposed that the linear loss of ²¹⁰Po previously accumulated by control mussels was related to the continual state of renewal and replacement of cellular proteins, with ²¹⁰Po turnover and metabolism governed by protein turnover and metabolism. M. edulis' assimilation efficiency of ²¹⁰Po from the ²¹⁰Po-labelled alga was calculated to be 17.2 ± 2.1%, and thus similar to that of Ag, Cd, Co, Se and Zn by bivalves from other marine algae species. It is proposed that the assimilation efficiency of ²¹⁰Po is a function of protein assimilation. Received: 27 August 1998 / Accepted: 3 September 1999     

Uptake of L-valine and other amino acids by the polychaete Nereis virens

For valine uptake by the polychaete Nereis virens Sars, the kinetic constants were: V _max=355 nmol g^-1 fresh weight h^-1, K _m=20 M. Leucine and some other amino acids acted as partial inhibitors of valine uptake. Valine uptake rate was 78% higher at 21.5 S than at 14 S. The major portion of valine absorbed by the polychaete could be extracted as free valine, with 6.5 to 15.6% being respired, and 3.6 to 9.5% incorporated into proteins. Calculations indicate that 7 to 12% of the metabolism of N. virens may be sustained by uptake of glycine and aspartic acid from natural concentrations. It is suggested that uptake of amino acids by this worm is important in the nitrogen cycling of marine sediments.     

Effect of salinity fluctuation on the osmotic pressure and Na+, Ca2+ and Mg2+ ion concentrations in the hemolymph of bivalve molluscs

Specimens of Chlamys opercularis, Modiolus modiolus, Mytilus edulis, Crassostrea gigas, Scrobicularia plana and Mya arenaria were exposed to both gradual (sinusoidal) and abrupt (square-wave) salinity fluctuations and measurements made of osmotic, Na⁺, Mg²⁺ and Ca²⁺ concentrations in the hemolymph and where applicable in the mantle fluid. In both sinusoidal and square-wave regimes fluctuating between 100 and 50% seawater (100%=ca. 32 S), the hemolymph Na⁺, Mg²⁺, Ca²⁺ and osmotic concentrations followed the concentrations of the external medium in Chlamys opercularis. The hemolymph and mantle fluid osmotic Na⁺, Mg²⁺ and Ca²⁺ concentrations of Modiolus modiolus, Mytilus edulis, Crassostrea gigas and S. plana followed those of the external medium as long as the molluscs' shell valves remained open. There were no changes in the ionic or osmotic concentrations of the hemolymph or mantle fluid of any of these species during periods of shell-valve closure. The hemolymph osmotic, Na⁺ and Mg²⁺ concentrations of wedged-open Modiolus modiolus, Mytilus edulis, C. gigas and S. plana followed those of the external medium. Hemolymph Ca²⁺ concentrations showed a damped response in C. gigas and Mytilus edulis. The hemolymph osmotic, Na⁺, Ca²⁺ and Mg²⁺ concentrations of Mya arenaria fluctuated in a similar manner to the external medium, but were damped. Wedged-open Mytilus edulis exposed to fluctuating salinity and supplied with a constant supply of 10 mM Ca²⁺ showed greater changes in hemolymph ionic and osmotic concentrations than M. edulis exposed to the same salinity fluctuation without a constant Ca²⁺ supply. Chlamys opercularis and Modiolus modiolus survived in a 50% seawater minimum sinusoidal salinity fluctuation for 10 days; wedged-open M. modiolus survived only 3 days. Burrowing had no effect on the osmotic, Na⁺, Mg²⁺ or Ca²⁺ concentrations of the hemolymph of Mya arenaria or S. plana exposed to fluctuating salinities. All of the species studied were shown to be osmoconformers.     

Uptake of dissolved organics by marine bacteria as a function of fluid motion

A mass transfer analysis predicts that fluid motion can increase the assimilation of dissolved organics by attached compared to free-living microorganisms under certain conditions. To test this we examined the effect of advective flow and fluid shear on the uptake of model compounds (leucine and glucose) by natural assemblages of heterotrophic bacteria, collected from Roosevelt Inlet, Delaware Bay (USA), in 1989. We found that [³H]leucine uptake by cells held in fluid moving at 20 to 70 m d^–1 was eight times larger than uptake by cells at a velocity of 3 m d^–1. This effect was only observed at low leucine concentrations (ca. 1 nM), when uptake was likely not saturated. When we added leucine at concentrations expected to saturate leucine uptake (ca. 11 nM), fluid motion past cells did not affect uptake. Fluid flow past bacteria did not increase [³H]glucose uptake, and laminar shear rates of 0.5 to 2.1 s^–1 did not increase either glucose or leucine uptake by suspended bacteria. These results indicate that fluid motion increases bacterial uptake of certain lowmolecular-weight dissolved organics only when the microorganism exists in an advective flow field. As predicted from a mass transfer model, fluid shear rates in natural systems are too low to affect bacterial uptake of such compounds.     

Role of carbonic anhydrase in the supply of inorganic carbon to the giant clam—zooxanthellate symbiosis

The mechanism whereby inorganic carbon (C_i) is acquired by the symbiotic association between the giant clam (Tridacna derasa) and zooxanthellae (Symbiodinium sp.) has been investigated. C_i in the haemolymph of the clam is in equilibrium with the surrounding sea water. The photosynthesis rate exhibited by the intact clam varies as a function of the C_i concentration in the clam haemolymph. The gill tissue contains high carbonic anhydrase activity which may be important in adjusting the C_i equilibrium between haemolymph and sea water. Zooxanthellae (Symbiodinium sp.) isolated from the clam mantle prefer CO₂ to HCO ₃ ^- as a source of inorganic carbon. The zooxanthellae have low levels of carbonic anhydrase on the external surface of the cell; however, mantle extracts display high carbonic anhydrase activity. Carbonic anhydrase is absent from the mantle of aposymbiotic clams (T. gigas), indicating that this enzyme may be essential to the symbiosis. The enzyme is probably associated with the zooxanthellae tubes in the mantle. The results indicate that carbonic anhydrase plays an important role in the supply of carbon dioxide within the clam symbiosis.