Osmotic stress and muscle tissue volume response of a freshwater bivalve

The freshwater bivalve, Corbicula fluminea, when submitted to hyperosmotic solutions, behaves as a hyperosmoconformer; we have observed an increase in osmolality and ions in its extracellular fluid. Osmotic and ionic changes in its watery environment represent a challenge for the tissues of this mollusk. Thus we evaluated, in vitro, muscle tissue volume variations (based on wet weight change) under anisosmotic salines, as well the possible regulatory mechanisms involved in the processes. This tissue did not exhibit complete volume regulation under anisosmotic saline solutions, but showed less variation than would be predicted by Van't Hoff's law, and tissue volume remained essentially stable throughout 90 min of exposure. To minimize tissue swelling in hyposmotic situations, C. fluminea muscle mobilizes organic osmolytes (ninhydrin positive substances) and inorganic ions (K(+) and Cl(-)). While under hyperosmotic stimulus, apparently only inorganic osmolytes (Na(+) and Cl(-)) are mobilized by the tissue. Our results indicate ionic accumulation by the Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)/H(+) coupled to Cl(-)/HCO(3)(-) exchangers. Exposure of the muscle tissue to Ca(2+)-free anisosmotic saline did not result in a detectable inhibition of the mechanisms described above. The Ca(2+) gradient that derives from the absence of this ion, even apparently enhances the regulatory mechanisms. These responses of this freshwater mollusk in hyperosmotic solutions, and the muscle tissue under anisosmotic (hypo and hyperosmotic) saline solutions, have not been previously characterized in the manner and approach as reported here. Specifically, we analyze both organic and inorganic osmolytes mobilized under hyposmotic stress, and can infer the participation of Na(+) and Cl(-) pathways stimulated by hyperosmotic stress. From the perspective gained in this study, tissue volume responses may be used as models for toxicological investigations.     

Heart action of the freshwater bivalve Anodonta anatina during activity.

1. Heart action of Anodonta anatina (L.) was investigated by recording the electrocardiogram (ECG), heart impedance, and ventricular and pericardial cavity pressure during different aspects of the normal behaviour. The contribution of mechanical and nervous mechanisms in controlling changes in heart action is discussed. 2. Pressure recordings were generally more reliable than the other methods and it is suggested that pericardial pressure pulses indicate the stroke volume output of the ventricle. 3. During spontaneous periods of prolonged shell closure there was an initial small increase in heart activity followed by a large reduction in both heart rate and systolic pressure, indicating that total heart output was considerably reduced. When the shell reopened, heart rate increased very rapidly with an initial overshoot of the normal level; systolic pressure increased more slowly with no overshoot. 4. These major changes in heart activity appear to be associated with respiratory changes and are controlled largely by the nervous regulatory system, but some minor rhythmic variations in the amplitude of heart beat are probably caused by mechanical factors. 5. Characteristic patterns of change in heart action were recorded during burrowing. These appear to result from haemodynamic changes associated with the muscular movements of the digging cycle. Control of the heart by the nervous regulatory system is apparently of much greater importance in relation to respiratory control than in relation to the haemodynamic functioning of the fluid-muscle system in locomotion.     

Polarized site of sperm entrance in the egg of a freshwater bivalve, Unio elongatulus

We studied the organization of the egg of a freshwater bivalve, Unio elongatulus. This egg is markedly polarized. At the vegetal pole there is a crater which constitutes the point of attachment of the growing oocyte to the ovarian wall. This has previously been interpreted as a micropyle. We show that the sperm does not enter the egg through the crater but in a differentiated region around it, mostly at its base. This region is characterized by a wrinkled surface and is the only site of the vitelline coat which specifically binds the lectin from Lotus tetragonolobus. The egg reacts explosively upon fertilization, ejecting vacuolar material from the crater. The role of this "egg reaction" in relation to the prevention of polyspermy is discussed.     

Proposal for a simple hydromorphological habitat survey method for freshwater bivalve (Unionidae) inventories

Numerous species of Unionidae are presently threatened by anthropogenic impacts on freshwater ecosystems. Field inventories, based on reliable presence/absence observations, are urgently needed to improve their protection. Such observations should be comparable between sites and collected with minimal surveying effort. Here, we demonstrate a strategy that minimises sampling effort while maximising inventory efficiency, formulated using hydromorphological data from 26 river sites sampled in north-eastern France between 2009 and 2010. Our findings indicate that a comprehensive visual survey of seven x river width with a bathyscope confirmed unionid presence in 96 % of cases, and presence of all unionid species in 88 % of cases. A further seven x width search increased this latter figure to 96 %, while a third (=21 × width) ensured that all species were registered in all rivers surveyed. Based on these results, we recommend that surveyors first undertake an initial seven x river width visual survey to confirm unionid presence. If no Unionidae are observed over this distance, sampling ceases and the site is marked negative. If at least one Unionidae is observed over this distance, an additional upstream stretch of the same length is surveyed with identical sampling effort. If at least one new species is observed within this second stretch, then a third and final stretch can be surveyed. This method is discussed in the light of representativeness of hydromorphological habitats (e.g. pool spacing and meander wavelength).     

Sphaeriid and corbiculid clams represent separate heterodont bivalve radiations into freshwater environments

Nine families of bivalve molluscs have undergone successful radiations in freshwater habitats, including three heterodont taxa: the Sphaeriidae, Corbiculidae, and Dreissenidae. Although the phylogenetic relationships of these freshwater heterodont families are controversial, most workers place the first two in the superfamily Corbiculoidea and assume that they represent a monophyletic grouping. We have tested competing phylogenetic hypotheses for the Corbiculoidea by constructing a representative molecular phylogeny, based on domains D1-D3 of the nuclear large subunit 28S rDNA, for 18 heterodont bivalves and for two oyster outgroup taxa. Our results do not support the monophyly of the Corbiculoidea and are consistent with the hypothesis that all three families of freshwater heterodonts represent independent colonization events by marine ancestors. Similarities in developmental mode specializations exhibited by some sphaeriids and corbiculids, such as sequential direct-developing broods, represent convergent adaptations to the freshwater environment. The corbiculid taxa form a clade with venerid and mactrid outgroups but we were not able to identify a putative marine outgroup for the sphaeriids.     

Absence of sterols constrains food quality of cyanobacteria for an invasive freshwater bivalve

The accumulation of cyanobacterial biomass may severely affect the performance of aquatic consumers. Here, we investigated the role of sterols in determining the food quality of cyanobacteria for the invasive clam Corbicula fluminea, which has become a common benthic invertebrate in many freshwater ecosystems throughout the world. In standardized growth experiments, juvenile clams were fed mixtures of different cyanobacteria (Anabaena variabilis, Aphanothece clathrata, Synechococcus elongatus) or sterol-containing eukaryotic algae (Cryptomonas sp., Nannochloropsis limnetica, Scenedesmus obliquus). In addition, the cyanobacterial food was supplemented with different sterols. We provide evidence that somatic growth of C. fluminea on cyanobacterial diets is constrained by the absence of sterols, as indicated by a growth-enhancing effect of sterol supplementation. Thus, our findings contribute to our understanding of the consequences of cyanobacterial mass developments for benthic consumers and highlight the importance of considering sterols as potentially limiting nutrients in aquatic food webs.     

Kidney function and sulfate uptake and loss in the freshwater bivalve Toxolasma texasensis

Toxolasma texasensis acclimated to an artificial pondwater (PW) maintained a concentration of SO4 in the blood of about 1-2 mmol l(-1) . The anion transport inhibitor DIDS (5,5'-diisothiocyanatostilbene 2, 2'-disulfonic acid) reduced the uptake of 35SO4 from the bathing medium by 54%. The clearance of polyethylene glycol (PEG) injected into the blood of T. texasensis ranged between 0.8 and 1.3 ml g(-1) dry tissue h(-1), and provided an estimate of renal filtration in PW-acclimated animals. The clearance of radioactive 35SO4 simultaneously injected into the same animal was about 16% of the PEG clearance, suggesting that sulfate was being reabsorbed by the kidney. Para-aminohippuric acid was cleared about 4.6 times faster than PEG, indicating that this organic acid was subjected to secretion in addition to filtration. When the normal osmotic gradient was abolished by acclimating T. texasensis to 10% seawater (SW), the PEG clearance decreased to 0.17 ml g(-1) dry tissue h(-1). Sulfate clearance in animals acclimated to PW or 10% SW was the same. However, in mussels acclimated to 10% SW, the calculated amount of SO4 reabsorbed was significantly reduced relative to mussels acclimated to PW. T. texasensis conserved SO4 when acclimated to PW, and reduced reabsorption when acclimated to the sulfate-rich 10% SW. When mussels acclimated to 10% SW were returned to PW, there was a transient increase in sulfate clearance during the first 8 h because filtration exceeded reabsorption.     

Seasonal stable isotope variations of the modern Amazonian freshwater bivalve Anodontites trapesialis

In a floodplain lake of the Amazon River near the city of Iquitos, northeastern Peru, a one-year monitoring experiment was conducted during which water samples and living bivalves (Anodontites trapesialis) were collected with the aim to investigate seasonal δ¹⁸O variation in and fractionation between bivalve aragonite and host water. Both host water and molluscan growth increments show more than 8‰ seasonal variation in δ¹⁸O. In the floodplain lake under study the δ¹⁸O variation of the water is controlled by contrasting dry and wet season evaporation-precipitation regimes. Molluscan δ¹⁸O appears to be in equilibrium with the host water. Although an approximately 4.0‰ offset occurs, δ¹³C records of water and bivalves are in good agreement, suggesting that both δ¹⁸O and δ¹³C of the shells of freshwater bivalve A. trapesialis are good recorders of (palaeo-)environmental conditions. The δ¹³C of Dissolved Inorganic Carbon (DIC) is governed by plant growth and/or by changes in aquatic chemistry, affecting the DIC pool.     

Evaluation of a nonlethal technique for hemolymph collection in Elliptio complanata, a freshwater bivalve (Mollusca: Unionidae)

Hemolymph, the circulatory fluid of bivalves, transports nutrients, respiratory gases, enzymes, metabolic wastes, and toxicants throughout the body. Hemolymph can provide information pertinent to health assessment of animals or populations, but is not commonly used in freshwater bivalves partly because of the lack of tested, practical techniques for its nonlethal collection. The objective of this study was to evaluate the effect of hemolymph collection on the growth and survival of Elliptio complanata, a freshwater bivalve (Unionidae). We describe a simple technique for the collection of hemolymph from the anterior adductor muscle sinus of E. complanata. To evaluate the effect of hemolymph sampling on mussel survival and growth, 30 mussels sampled using the technique and 30 unsampled controls were followed for 3 mo post collection. Nine animals were sampled 3 times over 7 mo to monitor effects of repeated sampling. No negative impacts on survival or growth were observed in either the singly or repeatedly sampled animals. We also compared the composition of hemolymph collected from the adductor muscle sinus with that collected from the ventricle of the heart. Calcium levels and cell count of hemolymph obtained from the adductor sinus and ventricle were significantly different. There was no significant difference between collection sites for magnesium, phosphorus, ammonia, protein, sodium, potassium, or chloride. We conclude that collection of hemolymph from the adductor sinus is safe for sampled E. complanata and should be explored as a relatively non-invasive, and potentially useful, approach to the evaluation of freshwater mussel health.     

Role of the cytoskeleton in sperm entry during fertilization in the freshwater bivalve Dreissena polymorpha

The present study examined the role of the cytoskeleton in sperm entry and migration through the egg cytoplasm during fertilization in the zebra mussel, Dreissena polymorpha (Bivalvia: Veneroida: Dreissenidae). Fertilization in this freshwater bivalve occurs outside the mantle cavity, permitting detailed observations of fertilization. After its initial binding to the egg surface, the sperm is incorporated in two stages: (1) a gradual incorporation of the sperm nucleus into the egg cortex, followed by (2) a more rapid incorporation of the sperm axoneme, and translocation of the sperm head through the egg cytoplasm. Initial incorporation into the egg cortex was shown to be microfilament dependent. Microfilaments were found in the sperm's preformed acrosomal filament, the microvilli on the egg surface, and in an actin-filled insemination cone surrounding the incorporating sperm. Treatment of eggs with cytochalasin B inhibited sperm entry in a dose- and time-dependent manner. Microtubule polymerization was not necessary for initial sperm entry. Following incorporation of the sperm head, the flagellar axoneme entered the egg cytoplasm and remained active for several minutes. Associated with the incorporated axoneme was a flow of cytoplasmic particles originating near the proximal end of the flagella. Inhibition of microtubule polymerization prevented entry of the sperm axoneme, and the subsequent cytoplasmic current was not observed. After sperm incorporation into the egg cortex, no appreciable microfilaments were associated with the sperm nucleus. A diminutive sperm aster was associated with the sperm nucleus during its decondensation, but no obvious extension toward the female pronucleus was observed. The sperm aster was significantly smaller than the spindle associated with the female pronucleus, suggesting a reduced role for the sperm aster in amphimixis.     

Effects of salinity on solute clearance from the freshwater bivalve, Dreissena polymorpha Pallas

Clearance of polyethylene glycol (PEG), inulin, or dextran that had been injected into the hemolymph of the mussel, Dreissena polymorpha, was measured in animals acclimated to pondwater (PW) or 10% seawater (SW). In addition, we measured the clearance of PEG from mussels acutely transferred into 10% SW and following return to PW after acclimation to 10% SW. Clearance values calculated for PW-acclimated mussels ranged from 2.0 to 3.3 ml (g dry tissue ċ h)^-1 and declined to 0.28 ml (g dry tissue ċ h)^-1 in 10% SW-acclimated animals. Transferring mussels into 10% SW resulted in a reduction in PEG clearance from the blood, coincident with the reduction of osmotic gradient. When 10% SW-acclimated mussels were returned to PW the clearance of PEG increased to rates observed in PW-acclimated animals within 1 h. The PEG clearance remained constant during the re-acclimation to PW even though the osmotic gradient declined from about 100 to 30 mosmol kg^-1. Clearance of the solutes used in this study was likely to be a measurement of renal filtration rate. The clearance values appeared to be maximal when the animals were in PW. The limited capacity to increase clearance in the face of an osmotic challenge may be a critical factor in restricting D. polymorpha to freshwater or lower salinity environments with small ranges in salinity.     

An electron microscope study of the formation of the periostracum in the freshwater bivalve, Anodonta cygnea

The periostracum and cells lining the periostracal groove of Anodonta cygnea L. have been studied at the electron microscope level. The cells lining the inner face of the outer fold differ in fine structural details, five cell types being recognized. Along the length of the outer surface of the middle fold, to which the periostracum is closely applied, only two cell types are evident. At the base of the periostracal groove the two epithelia are separated by a bulbous region containing a group of basal cells which initiate the periostracum. The periostracum, which is homogenously electron-lucid, originates in the intercellular space between a basal cell and the first cell of the middle fold. It increases in thickness in the periostracal groove due to the secretory activity of the different outer fold cells. The cells of the middle fold do not appear to be involved in periostracum formation.     

The effects of a harmful alga on bivalve larval lipid stores

Marine invertebrates often have complex life histories that include a swimming planktivorous larval stage, at which time they are vulnerable to a variety of stressors, including those associated with nutritional stress and harmful algal blooms. Lipid stores have been shown to be especially important for post-metamorphic survivorship and growth in a variety of marine invertebrates. We investigated the effects of the harmful brown tide alga Aureococcus anophagefferens on the lipid stores and growth of larvae of the hard clam (northern quahog, Mercenaria mercenaria), a dominant bivalve in many western Atlantic bays and estuaries. M. mercenaria was the dominant bivalve in Great South Bay, Long Island, until the mid-1970s, but very few larvae are presently found in these waters. Recent brown tide blooms have been hypothesized to pose a barrier to recovery of M. mercenaria populations and hinder recent restoration efforts by negatively affecting clam larvae. To test whether a diet of the brown tide alga affects the accumulation of beneficial lipid stores, we fed larvae one of three diets representing equal biovolumes of Isochrysis galbana, a nutritious control alga; A. anophagefferens, the brown tide alga for which nutritional quality is not presently known; or a mixture of the two. Larvae fed only brown tide had significantly less lipid stores than those in the other dietary treatments. In addition, brown tide negatively affected larval size. We also tested for evidence of tradeoffs between larval growth and lipid stores, predicting that when the diet was less nutritious as in the brown tide treatments, larval size and lipids would be negatively correlated. In contrast, we found that larvae fed a mixed algal diet or only A. anophagefferens showed a significant positive correlation between lipid stores and size, suggesting that some larvae were simply better at obtaining food and associated nutrients. Larval success likely depends on a complex interplay between genetic and environmental factors. Our study suggests that poor nutrition associated with a harmful alga can have negative effects on larval size and lipids stores, which in turn are mediated by the inter-individual variability in the ability to grow and accumulate necessary lipid stores. Phytoplankton quality is likely to be important for the sustainability of bivalve populations even when it primarily impacts the larval phase; and a diet of brown tide algae may have lasting legacies for juveniles and adults.     

The invasive freshwater bivalve Limnoperna fortunei in South America: multiannual changes in its predation and effects on associated benthic invertebrates

Hydrobiologia - The invasive golden mussel Limnoperna fortunei is known to strongly affect benthic communities in South American freshwaters, but the evolution of these effects after the early...     

Evaluation of a catalase inhibitor in the developmental regulation of maize catalase

A protein which has been shown to inhibit catalase in vitro appears to vary inversely with catalase activity in the maize scutellum during early sporophytic development when assayed using a catalase inhibition assay. This result suggested that the inhibitor protein may play a direct role in regulating catalase activity during this time period. Four experimental approaches were used to evaluate this putative regulatory role, including immunological quantitation of individual catalase isozymes during germination using rocket immunoelectrophoresis, perturbation of normal catalase expression with hydrogen peroxide or allylisopropylacetamide (AIA), examination of a mutant line with an altered catalase developmental program, and direct radioimmunoassay of the inhibitor protein during germination. The results of these experiments indicate that the quantitative changes in catalase activity during development are not mainly due to changes in the expression of the catalase inhibitor. Other possible roles of this protein in catalase regulation are discussed.     

The SEM and TEM study on the laminated structure of individual aragonitic nacre tablet in freshwater bivalve H. cumingii Lea shell

Nacre (mother-of-pearl), one of the natural composite materials, is renowned for its excellent mechanical properties and becomes a model for study on the biominerals. In the present study on bivalve H. cumingii Lea, the forming nacre tablet was observed with SEM to show laminated character on the lateral growing surfaces. Correspondingly, HRTEM showed dense crystal defects on (0 0 1) plane of the aragonite nacre tablet which might be caused by the adsorption of organic macromolecules on the plane. The correlation of the laminated growth mode and crystal defects on (0 0 1) plane was discussed. These findings could enhance our understanding to the formation mechanism of the nacre tablet as well as the superior mechanical properties of the nacre.     

The catalase activity of ferrihaems

1. The variation of the specific stoicheiometric catalatic activity of proto- and deuteroferrihaem with total ferrihaem concentration has been studied at 25 degrees C over a wide range of pH. For deuteroferrihaem the results imply that only monomeric ferrihaem species contribute significantly to the catalatic activity. Protoferrihaem is more highly dimerized in solution and, in this system, contributions to the catalatic activity from both monomeric and dimeric ferrihaem species were observed. The ratio of the specific activity of protoferrihaem monomer to that of dimer varied from approximately 20 at pH7 to 5x10(4) at pH12.2. 2. The specific activity of protoferrihaem monomer closely resembles that of deuteroferrihaem monomer, both in magnitude and pH-dependence. In both cases the activity is inversely proportional to [H(+)]. In contrast, the activity of catalase is independent of pH in the range 5-10. At pH13 the activity of ferrihaem monomer becomes equal to the maximal activity of catalase. The results are in good agreement with those reported by Brown et al. (1970b) and provide support for the assumptions upon which this previous analysis relied. 3. Information from the literature concerning the catalatic activity and dimerization of the iron(III) complex of 4,4',4',4'-tetrasulphophthalocyanine (Waldmeier & Sigel, 1971; Sigel et al., 1971) have been re-analysed. The results imply that both the monomeric and dimeric complexes contribute to catalatic activity and these activities closely resemble those of the corresponding protoferrihaem species.