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Effect of Salinity on Osmoregulatory Patch Epithelia in Gills of the Blue Crab Callinectes sapidus

¹ Department of Biology, The College of New Jersey, Ewing, New Jersey 08628
² Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672

^* To whom correspondence should be addressed. E-mail: lovett{at}tcnj.edu

	Abstract

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In euryhaline crabs, ion-transporting cells are clustered into osmoregulatory patches on the lamellae of the posterior gills. To examine changes in the branchial osmoregulatory patch in the blue crab Callinectes sapidus in response to change in salinity and to correlate these changes with other osmoregulatory responses, crabs were acclimated to a range of salinities between 10 and 35 ppt. When crabs that had been acclimated to 35 ppt were subsequently transferred to 10 ppt, both the size of the osmoregulatory patch on individual gill lamellae and the specific activity of Na⁺,K⁺-ATPase in whole-gill homogenates increased only after the first 24 h of exposure to dilute seawater. Enzyme activity and size of patch area increased gradually and reached their maxima (increasing by 200% and 60%, respectively) 6 days following transfer to 10 ppt seawater and then remained at these levels. Patch size at acclimation varied inversely with the salinity for seawater dilutions below 26 ppt (the isosmotic point of the crab), although it did not vary in salinities at or above 26 ppt. Thus, the size of the patch clearly is modulated with acclimation salinity, but it increases only in those salinities in which the crab hyperosmoregulates. An increase in the total RNA/DNA ratio in gill homogenates, the lack of mitotic figures in the lamellae, and the lack of incorporation of bromodeoxyuridine into nuclei of lamellar epithelial cells during acclimation to dilute seawater were interpreted as evidence that no cell proliferation had occurred and that increases in the size of the osmoregulatory patch occurred through differentiation of existing gas exchange cells or of undifferentiated epithelial cells into ion-transporting cells.

Abbreviations: BrdU, 5-bromo-2'-deoxyuridine • DAPI, 4',6-diamidino-2-phenylindole dihydrochloride

	Introduction

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Estuarine crabs, such as the blue crab Callinectes sapidus Rathbun, 1896, hyperosmoregulate in dilute seawater. This regulation is facilitated by the enzyme Na⁺,K⁺-ATPase located in epithelial cells of the gills, the primary osmoregulatory organ in these crabs (reviewed by Mantel and Farmer, 1983; Péqueux, 1995; Lucu and Towle, 2003). The phyllobranchiate gills of crabs are composed of many parallel lamellae that extend out from a central core (or raphe) and decrease in size distally along the gill. When posterior crab gills (gill pairs 5–8 in C. sapidus) are treated with either AgNO₃ or OsO₄, a distinct dark region within a more lightly staining periphery can be visualized in each gill lamella (Copeland, 1968; Copeland and Fitzjarrell, 1968; Aldridge and Cameron, 1982; Barra et al., 1983; Compère et al., 1989; Genovese et al., 2000; Luquet et al., 2000). This dark region adjacent to the afferent branchial vessel represents the "osmoregulatory patch" and is composed of thick ion-transporting cells, which are osmoregulatory in function, while the remainder of each lamella is composed of thinner cells, which function primarily in gas exchange (Copeland and Fitzjarrell, 1968; Aldridge and Cameron, 1982; Barra et al., 1983; Martelo and Zanders, 1986; Taylor and Taylor, 1986; Compère et al., 1989; Goodman and Cavey, 1990; Lawson et al., 1994). The gas exchange cells are short, squamous cells (about 0.5 µm thick), with relatively few mitochondria and with little elaboration of either the apical or basolateral membranes. In contrast, the ion-transporting cells are tall, cuboidal to columnar cells (about 10 µm thick), with large numbers of mitochondria interposed among the extensively folded basolateral membrane; the apical surface is elaborated into extensive microvillar plates (Copeland and Fitzjarrell, 1968; Johnson, 1980; Barra et al., 1983; Martelo and Zanders, 1986; Towle and Kays, 1986; Compère et al., 1989; Lawson et al., 1994).

In many euryhaline crabs the relative size of the osmoregulatory patch varies among the posterior gills and also varies within a gill, depending on the acclimation salinity (Copeland, 1968; Copeland and Fitzjarrell, 1968; Aldridge and Cameron, 1982; Compère et al., 1989; Genovese et al., 2000; Luquet et al., 2000). When crabs acclimated to full-strength seawater are subsequently transferred to low salinity, both the size of the osmoregulatory patch and the specific activity of Na⁺,K⁺-ATPase increase in the posterior gills (reviewed in Mantel and Farmer, 1983; Péqueux, 1995; Lucu and Towle, 2003).

Time-course studies of acclimation by C. sapidus to dilute seawater have demonstrated that hemolymph osmotic concentration reaches a new equilibrium value within 24 h following transfer from seawater to dilute environments (Neufeld et al., 1980; Lovett et al., 2003a,b). However, Na⁺,K⁺-ATPase activity in gill homogenates does not increase significantly within the first 24 h after crabs have been transferred. In fact, specific activity of Na⁺,K⁺-ATPase increases only 2–4 days after transfer to low salinity, and maximum activity is attained 6–8 days after transfer (Neufeld et al., 1980; Lovett et al., 2003a,b). It is not known whether changes in the histology of the gill epithelium occur at that same time that Na⁺,K⁺-ATPase activity increases (King and Schoffeniels, 1969; Towle et al., 1976; Mantel and Landesman, 1977; Péqueux and Gilles, 1977; Neufeld et al., 1980; Siebers et al., 1982, 1985; Martelo and Zanders, 1986).

This report presents the results of a study of several aspects of the acclimation change in the gill lamellae of C. sapidus. First, to determine how rapidly the osmoregulatory patch size increases after a crab has been exposed to a lower salinity, this study examined the time course of the change in the size of the patch and whether this change occurred immediately or gradually (i.e., whether the osmoregulatory patch occupied an increasingly greater proportion of the lamellae as the crab became acclimated to the lower salinity). A second goal was to determine whether the size of the osmoregulatory patch in acclimated crabs was graded with respect to the salinity of the seawater. A final goal of this study was to determine whether the increase in the size of the osmoregulatory patch was due to differentiation of existing gas exchange cells into ion-transporting cells, or rather was due to proliferation of existing ion-transporting cells within the lamella.

	Materials and Methods

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Specimen preparation
Intermolt male blue crabs (Callinectes sapidus Rathbun) (13.1 ± 0.2 cm average carapace width) were maintained in recirculating tanks with biological filters and were acclimated to 35 ppt (1088 ± 4 mOsm/kg) artificial seawater (Instant Ocean, Aquarium Systems) for 3 weeks. Crabs were fed squid mantle ad libitum on alternate days. Following acclimation to 35 ppt, crabs were transferred to 10 ppt (336 ± 5 mOsm/kg) seawater and then were harvested at regular intervals over 18 days. To determine hemolymph osmolality, samples were collected from the base of the fifth pereiopod and frozen. Gill pairs 3 and 4 were chosen to represent the anterior gas exchange gills, while gill pairs 6 and 7 were selected to represent the posterior ion-transporting gills, as has been done in most studies of osmoregulation in C. sapidus (e.g., Neufeld et al., 1980; Savage and Robinson, 1983; Burnett and Towle, 1990; Henry and Watts, 2001; Henry, 2005). Because gill pairs 1 and 2 are small and provide only limited amounts of tissue with which to work, they were not utilized in this study. The anterior lamellae of gill pair 5 are histologically and functionally similar to lamellae of posterior ion-transporting gills, but the posterior lamellae of gill 5 are similar to lamellae of anterior gas exchange gills (Aldrich and Cameron, 1982). Furthermore, the activity of Na⁺,K⁺-ATPase in gill pairs 5 and 8 is relatively low, suggesting that these gills are intermediate in function between the anterior gas exchange gills and the posterior osmoregulatory gills (Neufeld et al., 1980). Therefore, these two gill pairs were not examined in the present study. Na⁺,K⁺-ATPase activity was measured in gills 3, 4, 6, and 7 from one side of the crab. Each gill was blotted dry, weighed, and stored at –70 °C until assayed. Gills 3 and 6 from the contralateral side were used to determine the size of the osmoregulatory patch area. Each gill was fixed in 4% glutaraldehyde and postfixed in 1% osmium tetroxide. RNA and DNA content was measured in gills 4 and 7 from the contralateral side. Each of these gills was stored in ethanol until assayed.

Determination of osmoregulatory ability
The salinity of the seawater in each tank was established using a refractometer, and its osmotic concentration was measured with a vapor pressure osmometer (Wescor, Inc.). The osmolality of each hemolymph sample also was measured with a vapor pressure osmometer. To measure Na⁺,K⁺-ATPase activity, gills were thawed and homogenized in buffer (2 mM di-sodium EDTA, 50 mM imidazole pH 7.2, 250 mM sucrose) with 0.15% deoxycholate; crude homogenates were partially purified by centrifugation at 10,000 x g. Supernatants were assayed spectrophotometrically for ouabain-sensitive Na⁺,K⁺-ATPase with a linked pyruvate kinase/lactate dehydrogenase-NADH assay (Saintsing and Towle, 1978). Total protein concentration in homogenates was measured using the Bradford (1976) assay.

Measurement of osmoregulatory patch size
After treatment with osmium tetroxide, the region of the gill lamella that contained the thick ion-transporting epithelial cells (i.e., the osmoregulatory patch) could be distinguished from other regions of the lamella composed of thin cells because it appeared distinctly darker. Individual lamellae from each of the gills were removed after treatment with osmium tetroxide, and the area of the entire lamella and its osmoregulatory patch were measured using camera lucida and an MOP Videoplan digitizing pad with image analysis software (Kontron Image Analysis, ver. 6.20; Kontron Electronik). Measurement of lamellar area in each gill commenced with lamella 6 (lamellae 1–5, the most proximal lamellae of each gill, were substantially reduced in size in comparison with adjacent lamellae) and continued with every fifth lamella until either the size of the entire lamella was less than 7 mm² or the size of the osmoregulatory patch was less than 2 mm². Typically, measurement was terminated at lamella 150–200 (of the 290–350 total lamellae in each gill). Initial examination of specimens confirmed previous reports (Copeland and Fitzjarrell, 1968; Aldrich and Cameron, 1982) that lamellae from gill 3 lack an osmoregulatory patch, and so no patch size data were reported for gill 3 in this study.

Evaluation of mechanism of patch growth
To measure RNA and DNA concentration, gills were homogenized and processed following the method of Dagg and Littlepage (1972). Briefly, after nucleic acids were extracted and RNA was isolated from DNA with perchloric acid, RNA was quantified at 260 nm, while DNA was reacted with indole, extracted with chloroform, and quantified at 490 nm. Total protein concentration in the original homogenate was measured with the Bradford assay and was used to normalize concentrations of RNA and DNA in each gill homogenate. To determine whether epithelial cells of posterior gills underwent hyperplasia during acclimation to dilute seawater, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to detect mitotic activity and 5-bromo-2'-deoxyuridine (BrdU) was used to detect DNA replication. Mitotic figures were identified and enumerated by incubating isolated gill lamellae with DAPI (Schnedl et al., 1981). BrdU was dissolved in Pantin’s (1934) crab saline and injected (60 mg/100 g wet weight) into the hemal sinus near the fifth pereiopod. After 6 h, gill tissue and hepatopancreatic (control) tissue were fixed in paraformaldehyde, embedded in paraffin, and sectioned. The incorporation of BrdU, a thymidine analog (Morstyn et al., 1986; Gould et al., 1998), into cells during DNA replication was detected immunohistochemically with anti-BrdU mouse primary antibody and anti-mouse secondary antibody conjugated with horseradish peroxidase, and visualized with 3,3'-diaminobenzidine.

Data analysis
Data are reported as means ± standard errors. Results were analyzed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keul’s multiple comparisons test (InStat, ver. 2.04a, GraphPad Software).

	Results

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Osmoregulatory ability
To correlate the time course of change in the relative size of the osmoregulatory patch with other osmoregulatory responses in the crab during acclimation, hemolymph osmolality and specific activity of Na⁺,K⁺-ATPase in gills were measured. Hemolymph osmolality had dropped from 1082 ± 3 mOsm/kg to 782 ± 17 mOsm/kg within one day after transfer to 10 ppt seawater, and had reached acclimation levels of about 725 mOsm/kg by day 4, after which hemolymph osmolality did not change (P > 0.05) (Fig. 1A). During this period, specific activity of Na⁺,K⁺-ATPase in posterior gills (gills 6 and 7) had increased up to threefold (from 12 ± 2 µmol P_i/h/mg protein to about 30–40 µmol P_i/h/mg protein), while specific activity in anterior gills (gills 3 and 4) had increased only slightly. Acclimation levels of Na⁺,K⁺-ATPase activity were attained by day 6 after transfer to dilute seawater (Fig. 1B). Although the ability to osmoregulate was immediate (i.e., animals maintained a hemolymph concentration higher than that of the seawater after transfer), osmoregulatory changes in Na⁺,K⁺-ATPase activity took several days to become established.

View larger version (14K): [in this window] [in a new window]   Figure 1. Blue crabs Callinectes sapidus acclimated to 35 ppt salinity and then transferred to 10 ppt for up to 18 days. (A) Hemolymph osmolality. (B) Specific activity of Na+, K+-ATPase in gill homogenates. (C) Relative size of osmoregulatory patch on gill lamellae. Asterisks indicate means that were significantly different from the mean at t = 0 (*P < 0.05; **P < 0.01; ***P < 0.001). Mean ± SE indicated; n = 6.

View larger version (16K): [in this window] [in a new window]   Figure 2. Size of osmoregulatory patch on individual lamellae along the length of gill 6 in the blue crab Callinectes sapidus acclimated to either 35 ppt or 10 ppt salinity. (A) Relative size of osmoregulatory patch on each lamella. Asterisks indicate means for the same lamellae that are significantly different between the two acclimation salinities (*P < 0.05; ***P < 0.001). (B) Ratio of size of patch on lamellae at 10 ppt to size of same lamellae at 35 ppt. Asterisks indicate means that are significantly different from the mean ratio for lamella 10. (*P < 0.05, **P < 0.01). Mean ± SE indicated; n = 6.

View larger version (21K): [in this window] [in a new window]   Figure 3. Osmoregulatory responses of blue crabs Callinectes sapidus acclimated to various seawater salinities. (A) Osmolality of seawater and crab hemolymph. Means with the same letter are not significantly different (P > 0.05). (B) Ratio of osmoregulatory patch size to total area of lamella for lamellae 5–200 of gill 6. Asterisks indicate means significantly different from the mean at 35 ppt (**P < 0.01, ***P < 0.001). Mean ± SE indicated; n = 6.

View larger version (13K): [in this window] [in a new window]   Figure 4. Ratio of RNA concentration to DNA concentration in gills 4 and 7 of the blue crab Callinectes sapidus acclimated to 35 ppt salinity seawater and then transferred to 10 ppt salinity. Asterisks indicate means significantly different from mean at t = 0 (**P < 0.01). Mean ± SE indicated; n = 6.

	Discussion

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Results from DAPI-stained lamellae also indicated that it is unlikely that additional new cells were formed within the lamellae during the increase of the patch size, because no mitotic figures were observed and because the distance between nuclei did not decrease. The size of each lamella cannot increase without ecdysis, so if the number of cells had increased, the distance between nuclei would have decreased. Furthermore, the increase in patch size was not due to the hypertrophy of existing ion-transporting cells and the atrophy of existing gas exchange cells, because the distance between nuclei (and hence the size of individual cells as measured from lateral membrane to lateral membrane) in existing ion-transporting cells did not increase and because the distance between nuclei in gas exchange cells did not decrease. The lack of BrdU incorporation into DNA in cells in the gill lamellae also indicates that no DNA synthesis occurred in the lamellae. Therefore, we conclude that the increase in the osmoregulatory patch size was due to differentiation of existing gas exchange cells into new ion-transporting cells. This conclusion was based on the increase in the RNA/DNA ratio, the absence of any evidence of cell proliferation, and the absence of evidence of hypertrophy of existing ion-transporting cells. Genovese et al. (2000) also found no evidence of mitotic activity during the first 48 h after transfer of Chasmagnathus granulatus, an estuarine crab, to low-salinity seawater, but could not rule out the possibility that cell proliferation might occur later in the acclimation process.

When salinity is decreased to levels at which Callinectes sapidus hyperosmoregulates (i.e., below 26 ppt), the response by gill lamellae appears to be graded because the size of the osmoregulatory patch was inversely proportional to the acclimation salinity. Not all gas exchange cells or undifferentiated epithelial cells that potentially could differentiate into ion-transporting cells actually did so. A discrete population of these precursor cells apparently differentiates into ion-transporting cells at each particular salinity. At lower salinities many cells are stimulated to differentiate, but at intermediate dilute salinities only some of these cells are stimulated. There also appears to be a basal size below which the patch area does not decrease, even when salinity is well above the isosmotic point. Thus, it appears that there may be some ion-transporting cells (near the afferent branchial vessel) that never differentiate into gas exchange cells and some gas exchange cells (near the efferent branchial vessel) that never differentiate into ion-transporting cells. Furthermore, not all cells differentiate simultaneously, since the size of the osmoregulatory patch increased gradually (over several days) during acclimation to dilute seawater. Only precursor cells located immediately adjacent to existing ion-transporting cells differentiated. Lawson et al. (1994) report that the region of the lamella nearest the afferent vessel in Carcinus maenas, the green crab, acclimated to 35 ppt is composed almost exclusively of taller ion-transporting cells, while the region of the lamella near the edge of the osmoregulatory patch is composed of a mixture of both shorter ion-transporting cells and gas exchange cells. Our results suggest that the stimulation or modulation of the differentiation process is complex.

An increase in the size of the osmoregulatory patch is not directly responsible for the initial ability of the crab to maintain the osmotic concentration of its hemolymph above that of the surrounding dilute seawater. Although hemolymph osmolality decreased within 4 h after transfer of C. sapidus to dilute (10 ppt) seawater and continued to drop throughout the first 24 h (Lovett, unpubl. data), it did not decrease substantially after 24 h. The hemolymph osmolality decreased steadily to the acclimation level (substantially above the osmotic concentration of the seawater) and never dropped below this level. The size of the osmoregulatory patch did not increase to its acclimation size until after the osmotic concentration of the hemolymph had already stabilized, and it did not attain maximum size until day 6 after transfer. Therefore, an increase in the size of the osmoregulatory patch does not appear to be necessary to prevent decreases in hemolymph osmolality below the acclimation level. The crab apparently dampens initial decreases in hemolymph osmolality after transfer to dilute seawater by reducing the permeability of its body surface to minimize osmotic influx of water and efflux of Na⁺ (Robinson, 1982, 1994). Furthermore, during the period of patch growth (days 4–6), there was no change in the osmotic concentration of the hemolymph. Thus, growth in the size of the patch later in the acclimation process does not alter the level at which the hemolymph osmotic concentration is maintained.

Changes in the size of the osmoregulatory patch correspond with other aspects of the osmoregulatory response in crabs. During the first 24 h after transfer to dilute seawater, there was no significant increase in the specific activity of Na⁺,K⁺-ATPase in the gills of C. sapidus. Enzyme activity did not increase significantly until day 4, and maximum Na⁺,K⁺-ATPase activity was not attained until day 6 (the same time that the osmoregulatory patch attained its maximum size). A similar time course for the response has been reported previously (Neufeld et al., 1980; Lucu and Flik, 1999; Lovett et al., 2003a,b). The increase in both the activity of Na⁺,K⁺-ATPase and the amount of enzyme is directly proportional to the increase in the relative size of the osmoregulatory patch, although there may not necessarily be a direct causality between the two. However, when comparing Na⁺,K⁺-ATPase activity in subdivided gill lamellae of C. sapidus acclimated to high and low salinity, Neufeld et al. (1980) reported a substantial increase in activity in the portion of the lamellae composed of thin cells in low-salinity crabs, but no significant change in activity in the portion of the lamella composed of thick, ion-transporting cells. As gas exchange cells (or undifferentiated precursor cells) differentiate into ion-transporting cells during acclimation to dilute seawater, they develop extensive basolateral folds in the plasma membrane. The Na⁺,K⁺-ATPase protein is localized in the basolateral membrane of the ion-transporting cells (Towle and Kays, 1986). Thus, the concomitant increase in the amount of basolateral membrane that occurs when gas exchange or precursor cells differentiate into ion-transporting cells would provide an increased surface area for the insertion of additional, newly synthesized Na⁺,K⁺-ATPase protein molecules into the membrane, linking growth in the size of the osmoregulatory patch area with increased Na⁺,K⁺-ATPase specific activity.

The osmoregulatory ability of a species is reflected in the size of the osmoregulatory patch in crabs acclimated to extremes of salinity. C. sapidus hyperosmoregulates in seawater concentrations below its isosmotic point (about 26 ppt), but osmoconforms at salinities greater than 26 ppt (Fig. 3A and Mangum and Towle, 1977). In salinities at which C. sapidus osmoconformed (26–35 ppt), the size of the osmoregulatory patch did not change significantly; the size increased only in salinities at which the crab osmoregulated. In contrast, the crab Chasmagnathus granulatus both hyperosmoregulates in dilute seawater and hypo-osmoregulates in concentrated seawater. The size of the osmoregulatory patch in C. granulatus increases when it has acclimated to either dilute or hyper-saline seawater (Genovese et al., 2000). Thus, patch size generally appears to increase in those salinities where a crab osmoregulates (i.e., actively maintains a hemolymph concentration significantly different from that of the environment).

This report describes the time-course changes during acclimation that lead to an increase in the size of the osmoregulatory patch of gill lamellae and the underlying histologic changes in the epithelial cells of the gill. However, long-term (acclimation) increases in either osmoregulatory patch size or Na⁺,K⁺-ATPase specific activity do not occur during the short-term osmoregulatory response to an acute decrease in salinity. Therefore, neither of these mechanisms would contribute significantly to regulation of the osmotic concentration of hemolymph during tidal flux or during the first 24–48 h after transfer of a crab to dilute seawater.

	Acknowledgments

 
Preliminary data that supported this research project were collected by M. McDonald, O. Fasiuddin, J. Turner, E. Terrill, R. Ott, and B. Smith. This study was supported in part by a New Jersey Marine Sciences Consortium Development Grant, New Jersey Sea Grant College Program, to DLL and an NSF grant DCB-8711427 and DCB-9024293 to DWT.

	Footnotes

 
Received 17 October 2005; accepted 9 January 2006.

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