Biol. Bull. 211: 66-75. (August 2006)
© 2006 Marine Biological Laboratory
Ionic and Acid-Base Consequences of Exposure to Increased Salinity in the Zebra Mussel, Dreissena polymorpha
R. A. Byrne1,* and
T. H. Dietz2
1 Department of Biology, SUNY Fredonia, New York 14063
2 Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70808
* To whom correspondence should be addressed. E-mail:Roger.Byrne{at}fredonia.edu
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Abstract
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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.
Abbreviations: ASW, artificial seawater • PW, artificial pondwater • SID, strong ion difference
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Introduction
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The zebra mussel, Dreissena polymorpha (Pallas, 1771), is a relatively recent invader of freshwater, probably evolving from an estuarine ancestor during the Pleistocene (Mackie et al., 1989; McMahon, 1991). The first record of D. polymorpha outside of its more brackish Ponto-Caspian range was in 1824 in the Volga River (de Martonne, 1927; Lee and Bell, 1999). The species retains some characteristics of its estuarine ancestry—for example, retention of a byssal attachment and a free-swimming veliger stage; the latter is regarded as nonadaptive to freshwater environments (McMahon, 1991).
Physiologically, D. polymorpha displays some extreme responses to living in a dilute environment. Its hemolymph osmolality is among the lowest ever recorded for any metazoan (Krogh, 1939; Horohov et al., 1992); individuals are relatively intolerant of ion-deficient media (Dietz et al., 1994) and cannot withstand moderate salinities unless sufficient K+ is available for cellular homeostasis (Dietz et al., 1997). In addition, this species has a leaky external epithelium that makes it very permeable to solutes and water (Dietz et al., 1995), and its mechanisms for ion transport and kidney ion reabsorption seem to be working at close to the physiological limit to maintain ionic homeostasis in freshwaters (Wilcox and Dietz, 1995; Dietz and Byrne, 1997, 1999). Thus D. polymorpha appears to be "stenohaline" within its freshwater environment, displaying only a moderate ability to withstand higher salinities. It has, however, established populations both in Eurasia and North America in brackish water and estuaries at salinities approaching 12
(= 34% seawater) (Klimowicz, 1958; Aladin and Potts, 1992; Strayer and Smith, 1993).
In this study we examine the response of D. polymorpha to the challenges of acute exposure to a modest salinity stress (10% seawater), long-term acclimation to this salinity, and the return to freshwater. We selected 10% seawater on the basis of prior work showing that D. polymorpha tolerated this challenge well (Wilcox and Dietz, 1995; Dietz and Byrne, 1997, 1999) and also because we could ensure 100% survival during the experiments and maintenance of body fluids that would have sufficient volume to enable successful sampling of hemolymph yet provide sufficiently large gradients to elevate the passive flux of solute and water. We monitored the effects on osmotic and ionic regulation and changes in hemolymph acid-base balance during the transitions. We were particularly interested in examining the interactions between the several ionic solutes and any resultant acid-base changes in the hemolymph as the animals were transferred into and out of the elevated salinity. We found that diffusional movement of solutes is a predominant factor, and that differential solubility may affect compositional changes in hemolymph ions, while acid-base changes seem to be the result of passive processes.
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Materials and Methods
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Animals and media
Specimens of Dreissena polymorpha were collected from the Huron River in Michigan and transported overnight in insulated containers to the laboratory in Baton Rouge, Louisiana. The animals were maintained unfed in aerated aquaria containing artificial pondwater (PW) at 22 ± 2 °C (PW composition in mmol · l–1: 0.5 NaCl, 0.4 CaCl2, 0.2 NaHCO3, 0.2 MgSO4, 0.05 KCl; 4 mOsm · kg–1; Dietz et al., 1994) for 10–24 days prior to experimentation. Specimens held under these conditions showed no mortality and no significant change in hemolymph ion composition over the course of the experimental period (Dietz et al., 1994). Stock artificial seawater (composition in mmol · l–1: 449.1 NaCl, 27.5 MgSO4, 24.4 MgCl2, 9.9 CaCl2, 6.6 KCl, 2.4 KHCO3, 0.8 KBr, 0.4 H3BO3, 1076 mOsm · kg–1, 35 salinity; Chambers and de Armendi, 1979; Dietz and Byrne, 1999) was diluted with artificial pondwater to be nominally 10% ASW (105 mOsm/kg, approximately). All acclimation media were changed twice a week, and all containers and water were treated with chlorine bleach before being discarded.
Sampling and analysis
Hemolymph samples were obtained from specimens by pericardiac puncture (Fyhn and Costlow, 1975). Despite the small size of these animals, the uniformity of sample ionic composition gives us confidence that we consistently sampled pericardial fluid. Although the pericardial fluid is a filtrate of hemolymph (McMahon, 1991), it is similar in ionic and protein composition to extracellular fluid (Fyhn and Costlow, 1975). Animals chosen for experiments were a minimum of 20 mm in shell length (about 1 g total animal weight [tissue and shell]) to ensure collection of an adequate hemolymph sample. A total of 200 µl was obtained from each specimen. A 50-µl sample was obtained anaerobically for pH and CO2 analysis. The remaining 150 µl was centrifuged for 3 min at 8000 x g to remove cell debris before solute analysis. Osmolality was determined in undiluted samples by freezing point depression. Osmolality measurements were an average of 6% lower than the total solute estimates based on the algebraic sum of individual ion concentrations. This discrepancy would be expected considering that the activities of the ions are less than their total concentrations, suggesting that there was no significant underestimation of any dissolved ion species that might have become adsorbed to any precipitate. Sodium and potassium concentrations were determined on diluted samples by flame photometry. Calcium and magnesium concentrations were determined by atomic absorption spectrophotometry on samples diluted with LaO3-HCl. Chloride concentration was determined by electrometric titration. Sulfate was determined using a modified turbidometric technique (Dietz and Byrne, 1999). Total CO2 was measured by gas chromatography, using a 10-µl anaerobic sample (Byrne and Dietz, 1997). Hemolymph pH (±0.002 units) was determined immediately on 40-µl samples, using a Cameron Instruments blood-gas cell maintained at 23 °C and fitted with a Microelectrodes Inc. micro-pH electrode (MI-710) connected to a Corning model 10 pH meter with expanded scale. Hemolymph Pco2 (mm Hg; 1 mm Hg = 133.3 Pa) and bicarbonate concentration ([HCO3–]) were calculated by rearrangement of the Henderson-Hasselbalch equation:
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where pKapp (apparent pK) at 23 °C for freshwater bivalve hemolymph is 6.324 (Byrne et al., 1991). Cco2 is total CO2, and 
co2 is the solubility of CO2 in a salt solution similar to mussel blood at the experimental temperature (0.046 mmol · l–1 · mm Hg–1; Cameron, 1986). No correction for changes in hemolymph osmolality was necessary because the solubility coefficient was not significantly affected over the range of osmolality encountered. Hemolymph bicarbonate (mmol · l–1) was calculated as
Strong ion difference (SID) is the algebraic sum of the equivalents of strong mineral ions with consideration of valence sign. An approximation of SID was derived from measured values for Na, K, Cl, Ca, Mg, SO4 as follows: SID (mEq · l–1) = 1Na + 1K + 2Ca + 2Mg – 1Cl – 2SO4 (ion values in mmol · l–1).
Treatments
We measured hemolymph ion and acid-base variables in mussels incubated in 10% ASW for 7 days and transferred acutely—that is, abruptly, with no transition—back to PW for a further 7 days. Animals (about 130 for each replicate) were rinsed in PW prior to use, and any byssus attachments were carefully removed. Animals were subdivided into two groups. One (the ASW-acclimated group) was acutely transferred to 10% ASW and incubated in daily changes of this medium for 168 h (7 days). Animals from the other group (PW-acclimated) were placed in groups of 8–10 in polypropylene containers with 1 liter of 10% ASW. Animals were sampled as described above (n = 5 per time-slot) at 0 (= PW), 4, 8, 12, 16, 20, and 24 h, and at 72- and 168-h after transfer to 10% ASW. For each sampling time, animals were removed from one of the containers with care taken to dislodge any byssus attachment without damaging the animal. Medium was changed daily for the 72- and 168-h animals.
To determine the response to returning to PW after 168 h in 10% ASW, animals from the ASW-acclimated group were placed in containers with 1 liter of PW. Animals from this transfer were sampled (also n = 5 per time slot) at 168 (= ASW), 172, 176, 180, 184, 188, and 192 h, and at 240 and 336 h from the initial incubation in 10% ASW. Thus we generated a time series of sampling over a 2-week period that combined sampling every 4 h over the first 24 h of acute transfer either from PW to 10% ASW or from 10% ASW back to PW with further samples taken at the approximate midpoint (72 h) and at the end (168 h) of the incubation periods. Experiments were performed twice. Two-way analysis of variance indicated no significant difference (P > 0.05) between experimental runs, so data were pooled (resulting in n = 10 animals per sampling interval). All experiments were completed within 4 weeks.
Statistical analysis
All data are expressed as means ± standard error of the mean (SE) to indicate variability; note that because pH is a logarithmic variable, its SE is not an accurate depiction of dispersion, but is retained for uniformity; significance was accepted at P < 0.05. Overall differences between mean values were determined by a single-factor analysis of variance followed by Tukey-Kramer post hoc tests to determine significant differences between individual means. Simple regression analysis was used to examine relationships between individual variables. All statistical analysis used SuperAnova ver. 1.11 (Abacus Concepts) or Statgraphics Plus ver. 5 (Statistical Graphics Corp.) procedures.
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Results
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Dreissena polymorpha individuals acclimated to pondwater (PW) are hyperregulators, but maintain their hemolymph osmolality at a very low level (Table 1a). The ionic composition consists primarily of Na and Cl in about equal concentrations. Calcium concentration is approximately 4 mmol · l–1, and is about 4 times the magnesium concentration; bicarbonate and sulfate are roughly equal in concentration at about 3 mmol · l–1 (Tables 1a and 2). Blood pH is midrange for freshwater bivalves, at about 8, and derived blood Pco2 values of around 1.5 mm Hg suggest that animals are ventilating normally (Table 2) (Byrne and Dietz, 1997).
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Table 1 Changes in hemolymph solute concentrations (mmol · l–1), strong ion difference (SID, meq · l–1), and osmolality (mosm · kg–1) in Dreissena polymorpha
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Table 2 Changes in pH, and calculated Pco2 (mm Hg) and HCO3 (mmol · l–1) in the hemolymph of Dreissena polymorpha transferred from pondwater into 10% artificial seawater (0–168 h) then back to pondwater (168–336 h)
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Transfer to 10% seawater
On transfer to 10% artificial seawater (ASW), the mussels initially act as osmoconformers and ionoconformers. Details of the change in hemolymph ion concentrations on this transfer are given in Table 1a. Hemolymph osmolality rose to within the 10% ASW value (105 mOsm · kg–1) within 12 h, but continued to increase slowly by a further 10% throughout the 168-h immersion. Hemolymph chloride and sulfate also were close to steady state within the first 12 h: chloride was approximately isoionic with the bathing medium, whereas sulfate was maintained at somewhat higher concentrations than the bathing medium. Magnesium values rose at a slower pace, reaching steady state by 20 h post-immersion. The pattern for calcium was opposite that for magnesium. Calcium values declined by almost a half by the first 12 h of immersion and remained at a very steady value of about 2.2–2.8 mmol · l–1 throughout the period in 10% ASW. Hemolymph total CO2, although variable, also increased 2-fold during the first 20 h of immersion and remained elevated throughout the initial immersion period, though it was not significantly different from that of PW-acclimated animals by the end of the 7-day treatment. Derived bicarbonate values showed a similar trend (Table 2).
Twenty-four hours after mussels were transferred to 10% ASW, hemolymph pH rose from an initial value of 7.957 ± 0.012 to 8.250 ± 0.044, a significant elevation above the control value (Table 2). Hemolymph pH trended downward after this peak and was not significantly different from the PW control by 72 h of immersion. Upon transfer to 10% ASW, mussels showed an immediate significant increase—from about 1.5 mm Hg to about 3.2 mm Hg—in their derived values for hemolymph Pco2 (Table 2). After this initial spike, hemolymph Pco2 trended back toward PW values and was not significantly elevated for the remainder of the immersion period.
Transfer to pondwater
When mussels were transferred back to artificial PW after 7 days’ incubation in 10% ASW, their hemolymph osmolality declined exponentially and was not significantly different from that of PW-acclimated animals by 16 h after return (Table 1b). Hemolymph sodium and chloride had returned to values not significantly different from those of PW-acclimated animals by 8 h, and they continued to decline over the course of the incubation in PW (Table 1b). Other ionic components of hemolymph that returned to values not significantly different from PW-acclimated animals by 8 h included K, Ca, and SO4 (Table 1b). Hemolymph Mg, which had increased over 6-fold during 10% ASW incubation, was slower to decline upon return to PW, and it had not achieved PW-acclimated values until after the full 7 days’ incubation (recovery) period. Although CO2 levels declined on return to PW, there was no significant change in total CO2 from the end of ASW incubation up to 72 h after return to PW. Animals that had returned to PW for 7 days did have a total CO2 concentration that was significantly below that of ASW-acclimated animals (Table 1b).
Hemolymph pH remained remarkably unperturbed by the return to PW, despite the dramatic changes in measured solutes (Table 2). Within the first 24 h of reincubation in PW, mean hemolymph pH varied only within a narrow range (0.1 pH unit), but trended downwards in a slow decline. Subsequently, hemolymph pH was significantly reduced by 168 h (time since transfer to PW), or 336 h (168 h plus time spent in ASW), when compared to the 24-h (192-h) sampling, but it was still not significantly different from that of PW-acclimated animals (Table 2). Derived hemolymph Pco2 did not change significantly at any time during the return to PW: it remained in the range of 1.4–2.6 mm Hg, which was not different from PW control values. Derived bicarbonate concentrations mirrored total CO2 trends, with only the 168-h (336-h) sample having bicarbonate values significantly below those of ASW-acclimated animals (Table 2).
Calcium-magnesium interaction
Hemolymph calcium declined during incubation in 10% ASW despite the 2-fold increase in calcium concentration in the external medium (Table 1a), and rose to normal levels upon return to PW (Table 1b). In contrast, hemolymph magnesium increased 7-fold when animals were in 10% ASW, reaching values that were higher than in the external medium (Table 1a). Magnesium concentration returned to normal values slowly upon return to PW (Table 1b). This relationship is well illustrated in Figure 1, where a reciprocal relationship between hemolymph calcium and magnesium that depends on the incubation medium is evident. A regression analysis of the relationship between hemolymph calcium and hemolymph magnesium (Fig. 2) reveals a highly significant inverse relationship: [Ca] = (4.64 ± 0.12) – (0.358 ± 0.029)*[Mg]; (± SE; R2 = 0.470; F1,168 = 148.9; P << 0.001). Although both [Ca] and [Mg] vary across a wide range, each cation seems to be have a minimum value, as illustrated in Figure 2 (Camin = 1.3 mmol · l–1; Mgmin = 0.63 mmol · l–1).
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Figure 1. Changes in hemolymph Mg (closed symbol) and Ca (open symbol) concentration in Dreissena polymorpha transferred from pondwater to 10% artificial seawater (ASW) (0–168 h) and returned to pondwater (168–336 h). Error bars are ± SE.
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Figure 2. Relationship between hemolymph Ca and Mg concentration in Dreissena polymorpha during incubation in 10% artificial seawater for 7 d and during return to pondwater for a further 7 d. Solid line is simple regression line; dotted lines indicate minimal concentrations for Mg and Ca in mussel hemolymph.
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Na/K ratio
The mean hemolymph Na/K ratio in PW-acclimated animals was 42.5 ± 2.4 (Fig. 3). Immediately upon transfer of mussels to 10% ASW, the ratio rose significantly—to 72.3 ± 4.4 within 4 h—and then declined as the incubation period progressed; after 8 h the value was not different from that of PW-acclimated animals (Fig. 3). The ratio continued to decline during the incubation period and stabilized at a value that was remarkably similar to that of PW-acclimated animals (40.9 ± 1.5 at 72 h; 42.4 ± 2.2 at 168 h).
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Figure 3. Changes in hemolymph Na/K ratio in Dreissena polymorpha transferred from pondwater to 10% artificial seawater (0–168 h) and returned to pondwater (168–336 h). Error bars are ± SE.
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When mussels were transferred back to PW after 7 days’ incubation in 10% ASW, the hemolymph Na/K ratio declined, but not significantly, and remained at a value of about 30 for the first 24 h. The ratio did decline further, to 23.3 ± 1.5, after 72 h in pondwater (240 h)—a value significantly lower than that of either PW-acclimated, or 10% ASW-acclimated mussels (Fig. 3)—which largely reflected a reduction in hemolymph sodium levels (Table 1b). By 7 days in PW (336 h), the Na/K ratio had recovered to 31.7 ± 2.8, which is not significantly different from values for PW-acclimated or 10% ASW-acclimated mussels (Fig 3).
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Discussion
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Acid-base balance
In freshwater bivalves, the analysis of hemolymph acid-base state is less complex than in other groups of organisms (e.g., vertebrates, crustaceans), largely because circulating hemolymph does not contain any gas-carrying pigment and protein-based buffering is not significant (Byrne and McMahon, 1994). The nonbicarbonate buffering capacity of freshwater bivalve hemolymph at this temperature is about 1 slyke (mmol HCO3– · l–1 · pH–1) (Byrne et al., 1991), which is comparable to the low values reported for marine bivalves such as Mytilus edulis (0.4 slykes) (Booth et al., 1984) and Crassostrea virginica (0.88 slykes) (Michaelidis et al., 2005), and among the lowest recorded for any group of organisms (Withers, 1992). As a consequence, hemolymph pH can vary dramatically (e.g., 6.9–8.3; Byrne et al., 1989, 1991), particularly in response to changes in Pco2, and regulation of extracellular pH seems to be within a broad range. Under stressful conditions of aerial exposure, exogenous buffering occurs through addition of bicarbonate with concomitant increases in hemolymph calcium, suggesting that the shell has a role in mediating severe acidosis resulting from accumulation of CO2 (Dugal, 1939; Byrne et al., 1989; Byrne and McMahon, 1991). Ion transport processes, particularly those for Na/H and Cl/HCO3, have also been implicated in affecting acid-base state in both corbiculids and unionids (Byrne and Dietz, 1997), through the addition or deletion of acid or base equivalents from either the external environment or mantle cavity fluid. Although ion transport processes have a role in acid-base balance in bivalves acclimated to freshwater media, any effect in higher ionic strength media may be masked by the large passive flux component.
The acid-base response of Dreissena polymorpha to transfer to 10% artificial seawater (ASW) for 7 days and subsequent return to pondwater (PW) for a further 7 days is summarized in a pH-bicarbonate diagram (Fig. 4, using data from Table 2). The initial response upon transfer to 10% ASW was an accumulation of bicarbonate and an increase in Pco2 in the first 8 h, both of which combined to maintain hemolymph pH at PW values. The increase in Pco2 was likely due to a reduction in mantle cavity ventilation or reduced efficiency of gas offloading, or both, as a result of the transfer, similar to the results of valve closure seen when other species (e.g., Modiolus spp., Pierce, 1970) are transferred to higher or lower salinities. The accumulation of bicarbonate was maintained throughout the incubation period. After the initial shock, hemolymph Pco2 declined, causing hemolymph pH to increase gradually, reaching its highest value at 24 h. Thereafter, there was a form of respiratory "compensation" in which hemolymph Pco2 values rose gradually through the 72- and 168-h incubation, with the result that hemolymph pH returned to PW-acclimated values while bicarbonate remained elevated (Fig. 4). Thus changes in hemolymph pH were due primarily to changes in Pco2, while hemolymph bicarbonate remained elevated throughout the incubation period in 10% ASW.
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Figure 4. pH-Bicarbonate diagram illustrating the effects of transfer and incubation of Dreissena polymorpha from pondwater (PW) to 10% artificial seawater (ASW) (filled squares) and from 10% ASW back to PW (open squares). Data are as presented in Table 2; error bars are omitted for clarity. Numbers adjacent to squares indicate the time (hours) of incubation throughout the experiment (0–168: PW–10% ASW; 168–366: 10% ASW–PW, italics; the symbol for 168 h is grayed, indicating both the end of the incubation in 10% ASW and the beginning of the transfer to PW). The straight line is the nonbicarbonate buffer line for freshwater bivalve blood at the experimental temperature (from Byrne et al., 1991) and was fitted through the 0-h value. The isopleths are for Pco2 values in equilibrium with clam blood; values are in mm Hg (1 mm Hg = 133.3 Pa).
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As noted earlier, hemolymph pH remained remarkably stable during the first 24 h after mussels were returned to PW after 7 days’ incubation in 10% ASW (Fig. 4). During this time, mussels tended to become bloated with excess water as a result of osmotic uptake. Swelling of the tissue interfered with the mussel’s ability to move its valves and probably compromised ventilation efficiency, while at the same time providing an extensive surface for passive gas exchange. After the first 24 h, hemolymph pH declined steadily, following the Pco2 isopleth, as a result of a reduction in hemolymph bicarbonate. This "metabolic" acidosis coincided with a similar decrease in measurements of strong ion difference (SID).
SID is the algebraic difference in hemolymph mineral cations and anions, and strong organic ions, particularly lactate. It is regarded as an independent variable that, together with Pco2 and the total concentration of weak electrolytes, will largely determine pH (Stewart, 1978, 1981). Previous research has not demonstrated that freshwater bivalves accumulate much in the way of organic ion metabolites in hemolymph during hypoxia (Byrne and McMahon, 1991, 1994; Byrne et al., 1991), though Corbicula fluminea has been shown to produce succinate as an end product of anaerobic metabolism (Ortmann and Grieshaber, 2003). Moreover, derived Pco2 values for D. polymorpha in this study suggest that the mussels were aerobic during the incubation periods. Thus, SID and Pco2 may be important determinants of pH in mussel hemolymph, particularly as nonbicarbonate buffering capacity is very low (Byrne and McMahon, 1991; Byrne et al., 1991). Because our measurement of Pco2 was derived from Cco2 and pH measurements, we cannot use our values of Pco2 in directly testing the relationship of Pco2 to pH.
Our estimate of SID was derived from all of the measured cations and anions in the hemolymph (Na+, K+, Cl–, Ca2+, Mg2+, and SO42–). The relationship between hemolymph pH and SID (Fig. 5) is significant: pH = (7.822 ± 0.033) + (0.0242 ± 0.0039) * SID (±SE; R2 = 0.188; F1, 168 = 38.8; P << 0.0001); it fails to be predictive only at very low pH, where hemolymph Pco2 is likely to be a significant determinant of pH. For D. polymorpha, SID is largely the result of the difference between the major cation (Na+) and the major anion (Cl–), particularly as Ca2+ and Mg2+ have a reciprocal relationship. However, a similar analysis simply looking at the relationship between the Na+-Cl– difference and pH yields a less robust result (R2 = 0.078; F1,168 = 14.1), suggesting that other ionic components within SID are also important in determining hemolymph pH.
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Figure 5. Relationship between hemolymph pH and strong ion difference (SID) in Dreissena polymorpha during incubation in 10% artificial seawater for 7 d and during return to pondwater for a further 7 d. The straight line is a simple regression line.
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Hemolymph ionic composition and changes
In 10% ASW, D. polymorpha is an osmotic and ionic conformer, as has been demonstrated in other investigations (Dietz et al., 1994, 1997; Wilcox and Dietz, 1998). Mussels can volume-regulate in 10% ASW because Na/K ratios in the bathing medium, and thus in the hemolymph or extracellular fluid (ECF), allow animals to maintain an ECF to intracellular fluid (ICF) potassium gradient and redistribute water between these two compartments (Dietz et al., 1994, 1997). Salinity tolerance in D. polymorpha has been attributed to this limited ability to volume-regulate under hyperosmotic challenge (Dietz et al., 1997). Our study extends this analysis to a more lengthy incubation period in which we observe that ECF Na/K ratios are maintained during incubation in 10% ASW. We do note that, although the ECF Na/K ratio remains at about normal values during return to PW, ECF sodium declines during the weeklong reacclimation period.
The increase in hemolymph Mg upon incubation in 10% ASW is likely the result of diffusion, as was noted under shorter term incubations of D. polymorpha in various salinities (Dietz et al., 1997; Wilcox and Dietz, 1998). Magnesium concentration in hemolymph is most strongly correlated with sodium and chloride concentrations. Because both of these ions are entering the mussel largely as the result of diffusion, this also suggests that the increase in magnesium is due to a similar process. Our study confirms the inverse relationship between hemolymph Mg and Ca that was suggested by earlier studies. This relationship was noted by Wilcox and Dietz (1998), though hemolymph Ca had returned to PW-acclimated values by 24 h. Dietz et al. (1997) demonstrated an inverse relationship for Ca and Mg in hemolymph after a 26-h incubation in 10% ASW; however, no alteration in Ca-Mg dynamics was noted after a 12-h incubation in PW containing 45 mmol · l–1 NaCl, suggesting that a component of dilute seawater was inducing a reduction in hemolymph Ca, rather than the response being simply the result of a hyperosmotic challenge.
In our study, hemolymph Ca remained depressed throughout the 7-day period, and returned slowly to PW-acclimated values when the mussels were returned to PW. We demonstrated that D. polymorpha has elevated hemolymph sulfate concentrations upon incubation in 10% ASW and can maintain a concentration higher than bathing medium (Dietz and Byrne, 1999; this study). Of all the inorganic ions measured, the ion pair calcium and sulfate have the lowest solubility; thus the presence of additional sulfate may affect the amount of calcium that can be maintained in solution. The solubility of CaSO4 in water is given by the solubility product constant (Ksp: 4.93 x 10–5 M2; Lide, 2004; though there is some small variation in published values). The solubility product of Ca and SO4 is determined by multiplying the activity of both ions thus:
where the activity coefficients are determined by the ionic strength of the medium and temperature. Deriving solubility products for Ca and SO4 yields values that are within an order of magnitude of the solubility product constant (Table 3), which—considering the variability in measuring ionic strength and the fact that biological fluids may not act as a perfect solvent—suggests that CaSO4 solubility may have an effect on calcium dynamics in hemolymph. CaSO4 may precipitate within the hemolymph (ECF) space, producing the observed decline in hemolymph calcium levels. As our hemolymph sampling is by pericardiac puncture, we are essentially sampling an ultrafiltrate of circulating hemolymph; thus it is unlikely that we would bring precipitates of calcium sulfate into our samples.
An analysis of the solubility product of calcium and sulfate also reveals that there is no significant difference between solubility products throughout the transfer to ASW and return to PW, except at the 12-h sampling and 72 h after return to PW (Table 3). Thus the effect of transfer to 10% ASW and back to PW is not reflected very strongly in the calculated solubility product, despite distinctive changes in the concentrations of these ions in the hemolymph. If calcium and sulfate dynamics in both PW-incubated and 10% ASW-incubated mussels involved a significant solubility component, then we would expect little change in the CaSO4 solubility product during the course of the transfers. Increases in sulfate would result in declines in calcium while mussels were incubated in 10% ASW, and more calcium could be maintained in solution as the sulfate concentration in pondwater declined, with the result that the solubility product remained relatively constant over time. Using data reported in Dietz et al. (1997) for D. polymorpha incubated in 10% ASW for 12 h, we calculate that the precipitation of approximately 3 µmol CaSO4, representing half the total ECF calcium for PW-acclimated mussels, would be required to account for the reduction in ECF Ca when animals were transferred to 10% ASW, if all of the loss was due to precipitation. The location of any putative precipitates within the ECF space is uncertain, as is any possible effect on circulation. Possible sources for the rise of calcium in the hemolymph on return to PW include shell (hemolymph calcium derived from shell can rise quickly under stress conditions of aerial exposure in other species [Dugal, 1939; Byrne et al., 1991]) and other extracellular stores (Silverman et al., 1983). It should be noted that our analysis is approximate and does not rule out solubility as a component of hemolymph calcium dynamics in this animal. We have not considered other aspects of CaSO4 solubility such as neutral salt effects, or common ion effects, or the effects of CaSO4 ion pair solubility (see Martin, 1986).
Acclimation to a well-tolerated increase in salinity and back again to PW in Dreissena polymorpha, as measured by changes in extracellular parameters, seems to be largely the result of passive processes. Hemolymph acid-base disturbances are well within the range of tolerance compared to those seen under the more extreme conditions of aerial exposure (Byrne et al., 1989; Byrne and McMahon, 1991). Maintenance of cell volume under osmotic stress requires an appropriate Na/K ratio in the ECF, and such a ratio was observed in dilute seawater bathing medium. The reciprocal relationship between magnesium and calcium may be due to passive solubility effects related to changes in sulfate concentration, and not to any active regulation. Thus the very limited ability of D. polymorpha to withstand modest increases in salinity seems to suggest that the process of adaptation to a freshwater environment from an ancestral brackish water existence may result in a decrease in "euryhalinity" rather than an extension of an ability to withstand a range of salinities. This decrease in ability to tolerate a range of salinities in freshwater-adapted forms has also been noted in comparisons between anadromous populations of arctic char, Salvelinus alpinus, and those raised totally in freshwater (Staurnes et al., 1992). However, other bivalve Ponto-Caspian invaders (e.g., Corbicula fluminea, the Asian clam, which is in the same order [Veneroida] as D. polymorpha and is also a recent inhabitant of freshwaters) apparently retain abilities to withstand significant salinity stress (Morton and Tong, 1985). Indeed, a euryhaline capacity is regarded as one of the major characteristics that enabled recent Ponto-Caspian invaders of North American Great Lakes (Ricciardi and MacIsaac, 2000) to survive trans-oceanic transport in saline ballast tanks. However, the short-term capacity of D. polymorpha to withstand more saline environments is seemingly not a result of adaptive physiological mechanisms, but more a consequence of passive processes, and those processes confer only a modest and somewhat restrictive ability to live in higher salinity environments.
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Acknowledgments
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We thank Julie Cherry for technical assistance, and Drs. Tom Janik and Jim Bowser (SUNY Fredonia, Chemistry Dept.) for helpful discussions on chemical solubility. This study was partially supported by Louisiana Sea Grant College grant NOAA NA46RG0096 Project R/ZM-18.
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Footnotes
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Received 2 August 2005; accepted 1 February 2006.
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Abstract
Introduction
