Ionic and acid-base consequences of exposure to increased salinity in the zebra mussel, Dreissena polymorpha.

Dreissena polymorpha, an invasive freshwater bivalve, displays physiological characteristics that reflect its ancestry in brackish water, yet it has limited ability to withstand modest increases in salinity. We examined changes in hemolymph ion concentrations and acid-base variables in mussels transferred to and incubated in 10% artificial seawater (ASW) for 7 days and then returned to pondwater (PW) for a further 7 days. Hemolymph was sampled (10 animals per sample period) every 4 h for the first 24-h incubation and at 72 h and 168 h for both the transfer to 10% ASW and the transfer back to PW. The initial response to transfer to 10% ASW was a rapid attainment of an apparent isoosmotic steady state, with most hemolymph ion concentrations rising and attaining steady state within 12 h. Hemolymph magnesium rose more slowly, and hemolymph calcium declined despite an increase in its concentration in the bathing medium. Hemolymph pH rose significantly during the first 24 h, from 7.96 to 8.25, as a result of increases in bicarbonate; pH subsequently returned to normal through increases in PCO2. When animals were returned to PW after 7 days' incubation in ASW, the response of the major hemolymph ions was largely the reverse of that effected by the transfer to ASW. Hemolymph pH was not altered significantly until after 72 h in PW, when declines in bicarbonate lowered the pH to 7.73. Strong ion difference (SID) was related significantly to hemolymph pH. Hemolymph calcium and magnesium showed a reciprocal relationship throughout both transfer and incubation. Solubility interactions between sulfate and calcium and magnesium may be important in determining calcium availability in solution. The Na/K ratio in hemolymph was maintained within relatively narrow bounds throughout the procedure and may contribute to the mussels' ability to volume-regulate during an osmotic challenge. Overall, the responses of D. polymorpha to modest changes in salinity were largely the result of passive processes.

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.

Paracellular Solute Uptake in the Freshwater Bivalves Corbicula fluminea and Toxolasma texasensis.

Two species of freshwater bivalve were exposed to hyperosmotic solutions of various nonelectrolytes to compare the paracellular permeability of their gill epithelia. In Corbicula fluminea, exposure resulted in an elevation of blood solutes that was primarily due to dehydration. After 36 h of exposure, the concentration of Na in the blood decreased precipitously, and the nonelectrolyte accumulated. When lanthanum was added to the solution as a diffusion tracer, its electron-dense precipitate was rarely observed to penetrate the paracellular spaces of the gill epithelial cells in the absence of hyperosmotic stress. In contrast, precipitated lanthanum was commonly observed in the paracellular junctional complexes of the gill in animals that were subjected to hyperosmotic conditions. When the second species, Toxolasma texasensis, was exposed to hyperosmotic solutions of nonelectrolyte, dehydration appeared to be minimal and a seemingly normal concentration of ions was maintained in the blood. This, however, was because of the simultaneous loss of ions and water and a small gain in nonelectrolytes. Longer exposure (12 h or more) produced a precipitous decrease in most blood solutes and an extensive accumulation of nonelectrolyte. More lanthanum precipitate was seen in the paracellular spaces of both control and hyperosmotically stressed T. texasensis than in identically treated C. fluminea. We conclude that the epithelial junctions found in C. fluminea are relatively tight, which probably contributes to the ability of this species to maintain the solute in its body fluid at concentrations higher than are possible in T. texasensis.

Ion Transport in the Freshwater Bivalve Corbicula fluminea.

In freshwater bivalves such as the mussel Corbicula fluminea, uptake of chloride depends on the external concentration of the chloride ion. In C. fluminea, Cl- uptake displayed saturation kinetics both in animals acclimated to pondwater and in those subjected to salt depletion by storage in deionized water. The transport capacity (Jmax) was 7.00 +/- 0.51 {mu}eq g-1 dry tissue h-1 and the transport affinity (Km) was 0.21 +/- 0.08 mM in animals acclimated to pondwater. Animals subjected to salt depletion had a higher rate of Cl- uptake than did animals acclimated to pondwater. After 4 weeks in deionized water, the longer the animals were salt-depleted, the higher their rate of Cl- uptake. Na+ and Cl- transport were independent in pondwater-acclimated C. fluminea. For salt-depleted animals, Cl- transport was Na+-independent, but Na+ transport depended partially on external Cl-. Serotonin stimulated Cl- and Na+ transport in pond-water-acclimated animals by increasing influx while having little influence on efflux. Acetazolamide increased the Cl- and Na+ efflux of salt-depleted animals. Both serotonin and acetazolamide elevated the net loss of titratable base.

Mucociliary Transport in Living Tissue: The Two-Layer Model Confirmed in The Mussel Mytilus edulis L.

The present study combined video confocal laser microscopy (1) and tissue reflectance and autofluorescence to visualize mucus position and mucociliary transport in excised living gill tissue from the blue mussel Mytilus edulis. Rafts of mucus and embedded particles were transported atop a periciliary space traversed by frontal cilia, which engaged the mucus layer and moved it during the effective stroke, disengaging and completing the cycle during the recovery stroke. These results confirm the two-layer model for mucociliary transport in the mussel gill. Given the conservative nature of ciliated epithelial structure and function (2, 3), and the structural similarity of mucociliary surfaces as diverse as terrestrial vertebrate respiratory epithelium and molluscan gill, the two-layer mechanism of mucociliary transport may be a general feature of Metazoan biology.

Particle Capture by the Gills of Dreissena polymorpha: Structure and Function of Latero-frontal Cirri.

Microscopic techniques were used to examine the role of gill cirri in particle capture by Dreissena polymorpha. The latero-frontal cirri, formed from two fused ciliary plates, consist of about 40 pairs of cilia. Each cilium in the plate contains a typical 9 + 2 axoneme in the fused region of the cirrus, but the structure of the axoneme in the long, free ciliary tips is reduced. The cilia in a cirrus are graded in length, with the shortest cilia positioned frontally. The cirral cilia move in unison, allowing the cirrus to move from a flexed position with its tip arched over the front of the gill filament, to an extended position with the cirrus projected in the plane of the latero-frontal cell and extending across the interfilament space. In the latter position, the free ciliary tips of opposing and neighboring cirri form a "trap" (net) with a spacing of about 0.5 µm. Observations with laser confocal microscopy indicated that these structures can physically trap particles <1 µm in diameter. Particles captured by the extended cirri are moved to the frontal surface of the gill, where the cirri are swept by the lateralmost frontal cilia. During cirral movement the shift from extended to flexed position is, in part, achieved by the base of the cirrus pivoting at a hinge region. Morphologically, the hinge region shows axonemal specializations that consist of electron-dense plates and other structures of undefined function that may be important in the overall movement of the cirrus. In addition to trapping by cirri, we also observed particles moving in the water currents, particularly in the frontal current located over the apical surface of the filament, suggesting that some particles are captured in these water currents without being physically trapped. Probably, therefore, trapping by the cirri and establishment of water currents by the filaments both participate in the interception of particles by Dreissena polymorpha.

Paracellular solute uptake by the freshwater zebra mussel Dreissena polymorpha.

A hyperosmotic solution of mannitol or glucose (100 mM) in pond water caused an increase in paracellular solute movement between the bathing medium and body fluids of Dreissena polymorpha. Small molecules (< 5,000 Da) in the bath entered the mussel, and 80-85% of the sodium and chloride in the blood was lost within 12 h. Blood total solute was elevated within 4 h of exposure to hyperosmotic conditions, but the rise was attributed to the gain of glucose or mannitol from the bath and not to an elevation of ion concentration as a result of the osmotic loss of water. Lanthanum in the bathing solution was able to penetrate the paracellular junctional complex between gill epithelial cells in mussels exposed to hyperosmotic conditions but was rarely observed in pond water-acclimated animals. Colloidal gold (6 nm diam) was unable to penetrate the paracellular space but was accumulated in endocytotic vesicles in many epithelial cells. The "leakiness" of the epithelial tissue may be a critical factor in the low blood solute concentrations in freshwater mussels despite high rates of ion transport in these animals.

Filtration and Utilization of Laboratory-Cultured Bacteria by Dreissena polymorpha, Corbicula fluminea, and Carunculina texasensis.

Dreissena polymorpha consumed about 6 x 108 Escherichia coli from 20 ml of artificial pondwater (APW) in 30 min under laboratory conditions. The clearance rate per mussel was 143 +/- 25 ml g-1 dry tissue min-1. The E. coli used in these studies ranged from about 1.7 to 2.9 {mu}m in length. 35S-labeled E. coli were used to demonstrate that bacteria-derived nutrients were incorporated into mussel tissue. Electrophoretic analysis of mussel and bacterial proteins on 12% polyacrylamide gels allowed the visual determination of incorporation of labeled amino acids into bivalve proteins and demonstrated that intact bacteria were not simply trapped in mussel tissues. The conversion of bacterial-labeled amino acids into mussel protein was about 26%. Similarly, we demonstrated that D. polymorpha can use other bacterial species ranging in size from about 1.3 to 4.1 µm, including Citrobacter freundii, Enterobacter aerogenes, Serratia marcescens, Bacillus megaterium, and B. subtilus. The ability of D. polymorpha to take up E. coli was compared with that of two other freshwater mussels, Corbicula fluminea and Carunculina texasensis. On a mussel-dry-weight basis, D. polymorpha cleared bacteria 30 to 100 times faster than Corbicula fluminea and Carunculina texasensis, respectively. The ability to filter E. coli appears to be related to the architecture of the cirri on the latero-frontal cells of the gill. Cirri from Corbicula and Dreissena are similar in size, but Dreissena has a larger gill compared to the tissue dry-weight, and has 102 times more cirri than found in Corbicula. Carunculina, the unionid representative, has smaller and fewer cirri, and has relatively limited ability to capture E. coli.

Osmoregulation in Dreissena polymorpha: the Importance of Na, Cl, K, and Particularly Mg.

Zebra mussels (Dreissena polymorpha) are unusual in that they cannot survive in Mg-deficient water. Analysis of blood samples from mussels obtained in the field indicated a Mg concentration of 1.5-2.0 mM immediately after animal collection. However, Mg concentration in the blood decreased rapidly when the mussels were transferred to Mg-free artificial pondwater (PW); the t1/2 was 24 h. Blood Mg decreased to the limits of detection within 2 weeks, and the time to 50% mortality was about 17 days in Mg-free PW. When Mg-depleted specimens of D. polymorpha were returned to PW containing Mg, the net flux was 3 µmol Mg (g dry tissue.h)-1, and blood Mg concentration was restored within a day to 0.4-0.6 mM. Mussels depleted of Mg did not survive beyond 51 days. When mussels were acclimated to K-free pondwater (containing Mg), their osmoregulatory ability was impaired, and the total solute of the blood dropped from 30-36 to 21-24 mosm, with blood Na and Cl concentrations declining 30-50%. This ion-depleted condition was reversed within 45 h upon return of K to the pondwater bathing medium. D. polymorpha individuals were unable to survive beyond 5 days in deionized water and required minimal concentrations of Na, Cl, K, and Mg for prolonged storage (>51 days) under laboratory conditions. Mussels survived Ca-deficient solutions for more than 51 days, presumably because they were able to mobilize Ca from internal stores (shell) to maintain blood calcium at 1 mM.

Changes in monoamine transmitter concentration in freshwater mussel tissues.

Freshwater mussels were analyzed for biogenic amine transmitter substances in gill tissue, suprabranchial nerve and blood. Gill tissue from normal pondwater-acclimated mussels contained significant amounts of monoamine neurotransmitter substances. In comparison with the suprabranchial nerve the gill tissue contained 42% of the dopamine, 7% of the serotonin and 490% of norepinephrine. Exposing the animals to deionized water (salt-depleted) resulted in a loss of transmitter substances from gill tissue, but serotonin reduction was modest. The mussel gill tissue content of serotonin and the precursor tryptophan was regulated at nearly constant levels. Serotonin is an important transmitter substance in mussels and the many functions it controls, including sodium transport regulation, would depend on its continued presence.

Ion Transport in the Freshwater Zebra Mussel, Dreissena polymorpha.

The blood solute concentration (36 mosm) of pondwater acclimated zebra mussels is among the lowest found in freshwater bivalves. Blood ion concentrations were Na (11-14 mM) and Cl (12-15 mM), with lesser amounts of Ca (4-5 mM), HCO3 (about 2-4 mM), and K (0.5 mM). Sodium, Ca and Cl transport rates were 20-30 µeq (g dry tissue · h)-1 for pondwater acclimated mussels. The influx of both Na and Cl was stimulated by exogenous serotonin (0.1 mM). Sodium transport in zebra mussels was not inhibited by amiloride. Zebra mussels became isosmotic in 30 mM NaCl solutions and did not survive beyond a week in 45 mM NaCl. Zebra mussels are well adapted to their dilute freshwater habitat, but are more stenohaline than other freshwater bivalves as reflected by their intolerance of elevated ion concentrations in the bathing solution.

Long-Term Culture of Freshwater Mussel Gill Strips: Use of Serotonin to Affect Aseptic Conditions.

Serotonin relaxes the musculature and increases epithelial ciliary activity in freshwater mussel gills. This results in greater than normal water flow through the labyrinth of water canals and channels of the gill. These water spaces harbor significant microbial populations that make aseptic culture of freshwater mussel gills difficult. High concentrations of antibiotics can maintain short-term cultures, but are toxic to the tissue and reduce the lifespan of the culture. Moderate levels of antibiotics used in combination with 0.1 mM serotonin during a single, short pretreatment produces aseptic cultures. These cultures can now be established routinely and are viable for over a month as assayed by gill structural integrity, trypan blue exclusion, leucine incorporation into TCA precipitable protein, and normal physiological responsiveness to serotonin re-exposure.

Musculature Associated with the Water Canals in Freshwater Mussels and Response to Monoamines In Vitro.

The gills of freshwater mussels perform many functions that depend on water flow through the water canals and channels. Regulation of water flow depends in part on ciliary activity and in part on the contraction of musculature underlying the gill filament and water channel epithelium. Obliquely striated muscles control water canal openings (ostia) at the base of the filaments and also at the entry into the water channel (internal ostia, IO). The muscles adjacent to the ostia are oriented in an anterior-posterior direction (perpendicular to gill filaments), and those controlling the internal ostia are oriented in a dorso-ventral direction (parallel to gill filaments). Small bundles of fibers radiate from the major dorso-ventral IO muscle bands and appear to insert at the base of the water canal epithelial cells at the canal-channel junction. Both muscular bands are closely associated with the branchial nerves in the gill. When gills are exposed to 10-5 M serotonin in vitro, both ostial openings dilate and gill ciliary activity increases. The net result of serotonin treatment is an increase in ciliary activity, a maximal opening of the water canal ostia, and, presumably, an increase in water flow through the gill.

Behavioral and Metabolic Responses to Emersion and Subsequent Reimmersion in the Freshwater Bivalve, Corbicula fluminea.

When exposed to air, the freshwater bivalve, Corbicula fluminea, displayed valve movement behaviors, such as mantle edge exposure, wider gaping "ventilatory" response, and an escape or "burrowing" response. The proportion of the emersion period spent in these behaviors, relative to valve closure, increased with decreasing temperature. Emersion at 35°C inhibited valve movement behaviors, whereas emersion in a nitrogen atmosphere stimulated ventilatory activity. High rates of aerial oxygen uptake (Mo2) were associated with initial valve opening and ventilatory behaviors, and lower Mo2 occurred during bouts of mantle edge exposure. Heart rate was affected by temperature, but not by mantle edge exposure. Heart rate increased during burrowing and ventilatory behaviors suggesting a hydraulic function for hemolymph. Emersed C. fluminea had short bursts of heat production followed by longer periods of lower heat flux when measured by direct calorimetry. The mean heat production rate was 1.11 mW (g dry tissue)-1, significantly higher than the mean value for clams exposed in a nitrogen atmosphere, 0.50 mW (g dry tissue)-1. On reimmersion, C. fluminea showed no significant "oxygen debt" until after three days aerial exposure. The bursts of activity, while emersed, may be the result of periodic renewal of oxygen stores followed by immediate oxygen use.

Production of prostaglandins E2 and F2 alpha in the freshwater mussel Ligumia subrostrata: relation to sodium transport.

Pharmacological experiments indicate that prostaglandins (PGs) have a role in the control of sodium regulation in freshwater mussels and the mechanism may be linked to cyclic AMP and serotonin. To test this hypothesis we used radioimmunoassay to investigate the ability of freshwater mussels to synthesize PGs. The levels of precursor fatty acids were determined in a gas-liquid chromatograph. Arachidonic acid (precursor to the diene PGs) was the major fatty acid component of total lipids in the gill and accounted for 14% of the total. In addition, gill homogenates synthesize PG-like material from [3H]arachidonic acid. Material corresponding to PGE2 and PGF2 alpha were identified on thin-layer radiochromatograms. These data indicate that gills (the primary site of Na transport) can produce PGs. The presence of PGs in freshwater mussels was verified by radioimmunoassay of blood. Both PGE2 and PGF2 alpha were identified using highly specific antisera. The concentrations of both PGs was significantly reduced when the mussels were injected with inhibitors of phospholipase A2 or cyclooxygenase before sampling blood. Stimulation of Na transport by serotonin and cyclic AMP results in a depression of blood PGE2 with no effect on circulating PGF2 alpha. PGE2 levels are inversely correlated with net Na flux. These data indicate endogenous PGE2 negatively modulated Na transport and PGE2 levels are regulated by a serotonin-cyclic AMP mediated system.

Modification of sodium transport in freshwater mussels by prostaglandins, cyclic AMP and 5-hydroxytryptamine: effects of inhibitors of prostaglandin synthesis.

Pondwater acclimated unionid mussels, Ligumia subrostrata, experienced an increased Na influx, compared to controls, when injected with the phospholipase A2 inhibitor dexamethasone, or the cyclooxygenase inhibitors N-(2,6-dichloro-m-tolyl) anthranilic acid (meclofenamate) and indomethacin. Prostaglandin E2 (PGE2) or PGF2 alpha injections inhibited Na transport by depressing Na influx with no change in Na efflux. Prostaglandin E2 injections inhibited the indomethacin and dexamethasone dependent increase in Na transport. The PGE2 inhibition of Na influx was reversed by the administration of dibutyryl cAMP. Injections of serotonin (5-HT) elevated Na influx in mussels and the stimulation of Na transport by 5-HT could be potentiated by the injection of meclofenamate.

Sodium transport in the freshwater mussel, Carunculina texasensis (Lea).

Freshwater bivalves maintain a Na steady state in artificial pondwater: JiNa = 1.2 +/- 0.1 mumol/g dry tissue per h. Na uptake is Cl independent. The affinity (KS) of the Na transport system is 0.15-0.23 mmol Na/1. Sodium influx is coupled to H and/or NH4 exchange. Salt depletion stimulates JiNa 300% relative to nondepleted animals with no change in Ks. Injected ammonium ion stimulates JiNa. Sodium transport is inhibited 84% by 0.5 mM amiloride but is not affected by 4 mM NH4 or 1 mM furosemide in the bathing solution or injection of acetazolamide (0.26 mumol/ml blood).

Chloride transport across isolated skin of Rana pipiens.

The influx of Cl- across isolated frog skin bathed on the outside by 0.8 mM NaCl is about 100 nmol cm-2 h-1, which is approximately twice the Cl- influx in intact animals. The influx consists of diffusion (1%), exchange diffusion (38%), and active transport (60%). About 80% of the influx is independent of Na+ in the outer bath and is also independent of concomitant inward movement of cations. Chloride is exchanged for anions, probably HCO-3. The Cl- transport system is saturable; Vmax is about 200 nmol cm-2 h-1, and Ks is about 0.7 mM Cl-. High external concentrations of NaCl increase unidirectional fluxes of Cl- and urea, indicating a change in paracellular pathways. Active transport of Cl- is temperature sensitive (Q10 equals 2.68) and is inhibited by cyanide, dinitrophenol, iodoacetic acid, iodide, thiocyanate, and acetazolamide. The Na-independent component of JClin was unaffected by amiloride, ouabain, or eserine, all of which inhibit Na+ transport.