Some euryhalinity may be more common than expected in ... - Core

Comparative Biochemistry and Physiology, Part A 166 (2013) 36–43

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Some euryhalinity may be more common than expected in marine elasmobranchs: The example of the South American skate Zapteryx brevirostris (Elasmobranchii, Rajiformes, Rhinobatidae) Natascha Wosnick, Carolina A. Freire ⁎ Departamento de Fisiologia, Setor de Ciências Biológicas, Centro Politécnico, Universidade Federal do Paraná, Curitiba, Paraná, CEP 81531-990, Brazil

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Article history: Received 17 January 2013 Received in revised form 30 April 2013 Accepted 2 May 2013 Available online 7 May 2013 Keywords: Carbonic anhydrase Gills Kidney Na+,K+-ATPase Rectal gland

a b s t r a c t Elasmobranchs are essentially marine, but ~ 15% of the species occur in brackish or freshwater. The Brazilian marine coastal skate Zapteryx brevirostris, non-reported in nearby estuaries, was submitted to 35, 25, 15, and 5 psu, for 6 or 12 h (n = 6). Plasma was assayed for osmolality, urea, and ions (Na+, Cl −, K +, Mg 2+). Muscle water content was determined, and the rectal gland, kidney and gills were removed for carbonic anhydrase (CA) and Na +,K+-ATPase (NKA) activities. The skate survived to all treatments. Plasma osmolality and urea levels decreased respectively by 27% and 38% after 12 h in 5 psu (with respect to levels when in seawater), but plasma Na+, Cl −, and Mg2+ were well regulated. Plasma K+ showed some conformation after 12 h. Muscle hydration was maintained. Branchial CA and NKA did not respond to salinity. Rectal gland NKA decreased upon seawater dilution, while renal NKA increased. This skate was shown to be partially euryhaline. The analysis of plasma urea of elasmobranchs in brackish and freshwater versus salinity and time—allied to the widespread occurrence of some euryhalinity in the group—led us to revisit the hypothesis of a brackish water habitat for elasmobranch ancestors. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Elasmobranchs are essentially marine, and in general considered as relatively stenohaline, with the euryhaline and freshwater being considered as the exceptions. Out of the ~ 1100 known species, ~ 170 (belonging to 34 families) are reported to occur in brackish estuarine waters, or in freshwater (Martin, 2005). Their osmoregulatory strategy in seawater is characterized by hyporegulation of salt with the maintenance of high plasma levels of urea and the protective trimethyl-amine oxide (TMAO). These organic osmolytes raise plasma osmolality to values commonly slightly above seawater, thus driving osmotic water uptake, with a distinct reduction in the role of sodium and chloride as main extracellular osmolytes. Elasmobranchs are thus described as typically isosmotic or slightly hyperosmotic to seawater (Smith, 1936; Cohen et al., 1958; Boylan, 1972; Evans et al., 2004, 2005; Hammerschlag, 2006); salt is secreted by their characteristic rectal gland. While hyporegulating NaCl, urea levels are maintained through a complex renal countercurrent structure (Boylan, 1972; Shuttleworth, 1988; Janech et al., 2006). The rectal gland is the main site of NaCl secretion (Epstein and Silva, 2005), and the kidneys the site of urea and TMAO retention (Cohen et al., 1958; Janech et al., 2006). The gastrointestinal ⁎ Corresponding author at: Departamento de Fisiologia, Setor de Ciências Biológicas, UFPR – Centro Politécnico, Curitiba, PR 81531-990, Brazil. Tel.: +55 41 3361 1712; fax: +55 41 3361 1714. E-mail addresses: [email protected], [email protected] (C.A. Freire). 1095-6433/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2013.05.002

tract rejects magnesium and sulphate (Wood et al., 2007), and finally the gills of elasmobranchs are considered the site of acid–base regulation, and are more relevant in salt uptake in very dilute seawater or freshwater (Smith, 1936; Hammerschlag, 2006; Evans and Claiborne, 2009; Ballantyne and Robinson, 2010; Reilly et al., 2011). Despite being essentially an ancient and very successful marine group, it has been increasingly recognized that many supposedly stenohaline marine elasmobranchs may display a certain capacity to deal with salinity reduction. Many researchers have recently acknowledged that most studies on elasmobranch osmoregulation deal with a few very well studied species (Anderson et al., 2002, 2007; Hazon et al., 2007; Evans and Claiborne, 2009). Two species are actually viewed as models of elasmobranch euryhalinity: Dasyatis sabina and Carcharhinus leucas. But these two species are unquestionably fully euryhaline, as they are found living in freshwater, in brackish water, and in seawater (Holmes and Donaldson, 1969; de Vlaming and Sage, 1973; Piermarini and Evans, 1998; Pillans and Franklin, 2004; Pillans et al., 2006). Quite interestingly, mechanisms of branchial salt absorption typical of fully euryhaline elasmobranchs were also detected in the marine stenohaline Squalus acanthias (Choe et al., 2007). Upon salinity decrease, euryhaline sharks and rays display reduced plasma concentrations of urea and TMAO, resulting in reduced plasma osmolalities (Hammerschlag, 2006; Evans and Claiborne, 2009). In the osmoregulatory organs, a distinct reduction in the action of the rectal gland is observed (Pillans et al., 2008; Evans and Claiborne, 2009; Ballantyne and Robinson, 2010); an increase in urinary output with increased

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reabsorption of ions in the kidneys is reported (Anderson et al., 2007), along with a higher role of the gills in ion uptake (Ballantyne and Robinson, 2010; Reilly et al., 2011). Drinking is not traditionally ascribed to marine elasmobranchs but has been demonstrated in euryhaline species returning to more concentrated media, seawater (Anderson et al., 2007; Evans and Claiborne, 2009). A broader knowledge about elasmobranch euryhalinity can help us understand their past evolutionary history and predict their future in an unstable environment (Martin, 2005), given that many elasmobranch species are low-depth coastal species. Their osmoregulatory strategy, with reduced plasma salt levels, has led to the proposal, not free from debate, that the group had a common ancestry developing in brackish/freshwater habitats (Smith, 1936, 1961; Lutz, 1975; Griffith, 1985; Vize and Smith, 2004; Ditrich, 2007). Euryhalinity is one relevant issue for the occupation of new habitats (Heithaus et al., 2009), including putative situations of climate change and global warming. Data are here presented for Zapteryx brevirostris, a marine coastal skate, found from Southeastern Brazil to the coast of Argentina and Uruguay. The species is informally reported to occasionally occur in estuaries on the Southern Brazilian coast. However, it does not appear in fish surveys performed along these large estuarine Bays, such as Paranaguá Bay and Guaratuba Bay, where other elasmobranchs have been found in very low numbers (Chaves and Corrêa, 1998; Queiroz et al., 2006). The goal of this study was to examine how this small coastal skate would respond to short-range seawater dilution experiments, simulating short sojourns in estuarine waters. Its physiological capacity to resist for up to 12 h in water of 5 psu raised the interest in echoing previous voices that questioned the general stenohaline view of marine elasmobranchs. Data on urea have been reviewed to strengthen the patterns, and records of at least partial euryhalinity have been displayed in an elasmobranch cladogram, all to revisit the hypothesis of an ancestral brackish water habitat for the group.

After the experimental period, skates were anesthetized using clove oil (diluted 1:10 in 90% ethanol), 6 mL of anesthetic solution per liter of aquarium water. After approximately 8 min, they displayed absence of spiracle movement and lack of response to tail grabbing/touching. The fish were then removed from the water, and had their spinal cord quickly sectioned. This procedure is in agreement with the standard protocols of fish laboratory experimentation. Clove oil is considered a good anesthetic for fish, leading to deep surgical sedation in doses such as the one employed here (Neiffer and Stamper, 2009). Skates were then weighed, measured for their total length, and evaluated for state of sexual maturity. The ventral side of the animal was carefully opened using scissors, to allow blood sampling from the heart. The blood sample was then immediately centrifuged (2000 g for 5 min, room temperature) and the plasma pipetted to another tube and frozen at − 20 °C. Tissues were dissected right after blood sampling: a muscle slice, the rectal gland, the kidney, and all gills, and were immediately placed in liquid nitrogen for storage. After 3–4 days, tissues were transferred to an ultra-freezer (− 80 °C) until assayed for muscle hydration or enzyme activities.

2. Materials and methods

Muscle samples (~400 mg) were initially weighed (wet weight, Ww) and were then dried for 24 h at 60 °C. Samples were then weighed again (dry weight, Wd), and muscle water content (MWC) was expressed as: MWC (%) = [(Ww-Wd)/Ww]x100.

2.1. Animals Skates (Zapteryx brevirostris) captured with gillnets were purchased from fishermen of the fish market of Shangrilá (25° 41' 41" S, 48° 28' 7" W), on the coast of the Southern State of Paraná. Upon arrival at the beach, specimens were carefully and quickly transferred from the boat to plastic boxes (56.4 cm × 38.5 cm × 37.1 cm) containing 30 L of seawater, 4 rays in each box. Water was aerated using battery aerators, and boxes with skates were transported to the laboratory (9 km, ~ 10 min ride). Three collecting sessions were conducted between May and July 2011, yielding a total of 48 animals: 23 males and 25 females; all were adults and in good physical conditions (mean ± SD; total length 52.8 ± 4.5 cm; disk width 20.3 ± 3.2 cm; mass 640 ± 200 g. Authors had a permit from the Brazilian Environmental Agency (IBAMA/ICMBio-SISBIO #20030) to obtain and use these animals for research purposes. Fish were transported to the laboratory, where they were immediately transferred to the stock tanks (500 L), filled with seawater and a thin layer of local beach sand as substrate, under constant aeration and biological filtration, at a density of 2 skates/100 L of seawater. Salinity was kept at 35 psu and temperature at 20 °C during the acclimation period of 24 h, and animals were not fed. After 24 h of acclimation, fish were individually transferred to boxes (same boxes used for transporting fish from the collection site) containing 30 L of control (full-strength seawater of salinity 35 psu) or experimental (dilute seawater of salinities 25, 15, or 5 psu). Skates were exposed to these conditions for 6 or 12 h. Each experiment (4 salinities × 2 times of exposure = 8 different experimental conditions) was repeated 6 times (n = 6 for each group).

2.2. Plasma assays Osmolality and urea, and ionic concentrations (Na +, Cl −, K +, Mg 2+) were assayed in plasma samples. Osmolality was determined using a vapor pressure osmometer (VAPRO 5520, Wescor, USA), in undiluted samples. Urea, chloride, and magnesium levels were quantified colorimetrically—Labtest kits, Brazil, and Ultrospec 2100 PRO Amersham Pharmacia biotech, Sweden. Plasma samples were appropriately diluted in ultrapure water: 1:50 for urea, 1:2 for chloride, and 1:15 for magnesium. Sodium (Na +) and potassium (K +) were quantified using flame photometry (CELM FC-180, Brazil) in samples diluted (1:200) in ultrapure water. 2.3. Muscle hydration

2.4. Enzyme assays Carbonic anhydrase (CA; EC 4.2.1.1) activity was determined through the detection of pH reduction upon the addition of ice-cold CO2-saturated water (Vitale et al., 1999, based on Henry, 1991). Kidneys (173.7 ± 3.7 mg, mean ± SD) and gills (132.1 ± 2.2 mg) were thawed and homogenized at 10% (mass/volume in g/mL) in 10 mM phosphate buffer, pH 7.4. Homogenates were then centrifuged (~ 2000 g for 5 min, room temperature), and the supernatant was aliquoted for the assays of total protein and specific activity of the enzyme. CA activity was quantified in buffer containing mannitol (225 mM), sucrose (75 mM), and tris-phosphate (10 mM), pH 7.4. The homogenate (50 μL) was added to 7.5 mL of the buffer. Ice-cold (2.5 °C) distilled and CO2-saturated water (1 mL) was then added to the mixture. Immediately after the addition of carbon dioxidesaturated cold water, pH reduction was monitored (inoLAB pH meter Level 1, WTW, Germany) for 20 s, with pH readings every 4 s. The slope of the regression line (pH × time) represents the catalyzed rate (CR); r2 of the regression lines was always above 0.98. The non-CR (NCR) was the slope of the regression line of pH drop upon the addition of plain buffer, without any tissue homogenate. NCR was always determined in quadruplicates, in every session of sample assays. CA specific activity was calculated as CA activity = [(CR/NCR) – 1)]/mg protein (Burnett et al., 1981; Vitale et al., 1999). The Na +,K +-ATPase (NKA) (EC 3.6.3.9) specific activity was quantified through the rate of NADH oxidation in a coupled assay of

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ouabain-sensitive ATPase activity in the presence of Na +, K +, and Mg 2 +, and lactate dehydrogenase and pyruvate kinase activities (McCormick, 1993). Rectal glands (25.0 ± 7 mg), kidneys (35 ± 8 mg), and gills (35 ± 5 mg) were thawed and homogenized in 400 μL of buffer (150 mM sucrose, 10 mM EDTA, 50 mM imidazole, pH 7.3), containing 0.1% sodium deoxycholate. The homogenate was centrifuged at 5000 g for 30 s at 4 °C. ATPase activity was determined in the presence and absence of ouabain (0.5 mM), and with final salt concentrations of 47 mM NaCl, 21 mM KCl, and 2.6 mM MgCl2. All samples were analysed in triplicates in microplates (Elisa Sunrise, Germany). Specific activity was calculated as the difference between the slope of absorbance decrease without ouabain and the slope of absorbance decrease in the presence of ouabain, divided by the slope of absorbance decrease of the ATP standard. The result is divided by the protein concentration and multiplied by 60, to be expressed in mmol ADP/mg protein/h. Total protein content of the homogenates was determined using the Bradford (1976) method, and BSA as standard.

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3.2. Plasma ions Six hours of exposure to diluted seawater did not result in reduction of plasma sodium. However, after 12 h in dilute seawater plasma sodium decreased from 227 mM in 35 psu to 195 mM in 25 psu, and then to 168 mM in 5 psu (Fig. 2A). Plasma chloride was kept stable upon seawater dilution, in both times of exposure (Fig. 2B). Sodium and chloride were hypo-regulated in 35 and 25 psu, were approximately iso-ionic in 15 psu, and were hyper-regulated in the lowest salinity tested, 5 psu (Fig. 2A). Plasma potassium was regulated upon 6 h of exposure to dilute seawater, but was reduced in skates exposed for 12 h: from 8–10 mM in salinities 35–25, to ~ 4 mM in salinities 15–5 (Fig. 2C). After 6 h

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Plasma osmolality of Z. brevirostris was of 980–985 mOsm/kgH2O in full-strength seawater (salinity 35 psu), and has shown progressive reduction upon seawater dilution, both after 6 and 12 h of exposure (Fig. 1A). When salinity reached 15 psu, plasma osmolality after 12 h of exposure (795 mOsm/kgH2O) was lower than after 6 h of exposure (880 mOsm/kgH2O). The effect of time was intensified at salinity 5 psu: 825 mOsm/kgH2O after 6 h, and 713 mOsm/kgH2O after 12 h (Fig. 1A). These skates are hyposmotic when in full-strength seawater, as the 95% confidence intervals (980 ± 23.8 and 985 ± 23.4) did not include seawater osmolality of 1050 mOsm/kgH2O. Upon seawater dilution, they turn hyperosmotic (Fig. 1A). Plasma urea was of ~ 406–458 mM in 35 psu, and reached 356–335 mM after 6 h of exposure to 25–5 psu. Given the large individual variability, this reduction was not significant (Fig. 1B). After 12 h of exposure to seawater dilution, plasma urea was kept stable (387–406 mM), and was only reduced in 5 psu: 266 mM (Fig. 1B). Muscle hydration was kept stable at all salinities and times of exposure: between 77.2 and 78.9% (Fig. 1C).

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3. Results 3.1. Plasma osmolality and urea, and muscle hydration

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2.5. Statistical analyses The effects of salinity reduction and time on the measured parameters have been evaluated through two-way ANOVAs, and post hoc test of Holm–Sidak. Shapiro–Wilk test was employed to verify normality and homogeneity of variances. Paired Student's t-tests or Wilcoxon Signed-Rank tests (when data failed Shapiro–Wilk test) were used to verify differences in the specific activities of the enzymes between tissues, pairwise. All tests were performed with limit of significance of 0.05, using Sigma Plot software version 11.0.

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Water Salinity (psu) Fig. 1. Plasma osmolality (A) and urea (B), and muscle water content (C) of Zapteryx brevirostris after 6 (black circles) and 12 (white circles) h of exposure to seawater of salinities 35, 25, 15, and 5. Water osmolality was calculated based on the relationship: 1 salinity unit (psu) equals approximately 30 mOsm/kg H2O. Black symbols that do not share a common lower case letter are significantly different (6 h). White symbols that do not share a common upper case letter are significantly different (12 h). # indicates that the value after 12 h is different from the value after 6 h at a same salinity. No differences were detected in muscle water content values.

of exposure, plasma potassium was hyporegulated in 35 psu, approximately isoionic in 15 and 25 psu, and hyper-regulated in 5 psu. This regulation turned into ionic conformation after 12 h of exposure (Fig. 2C). Plasma magnesium was strongly hyporegulated at all salinities and both times; values in 35 psu (~ 3.4 mM) were higher than values in 5 psu (~ 1.4 mM), but the ANOVA and the post hoc test employed did not localize for which time of exposure (Fig. 2D). 3.3. CA specific activity Renal activity of the CA decreased in skates in 5 psu when compared to rays in 15 psu, considering both times of exposure (Fig. 3A). There

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Fig. 2. Plasma sodium (A), chloride (B), potassium (C), and magnesium (D) of Zapteryx brevirostris after 6 (black circles) and 12 (white circles) h of exposure to seawater of salinities 35, 25, 15, and 5. Water ionic levels on the X axes of the graphs have been calculated based on standard seawater ion levels provided in Prosser (1973). Black symbols that do not share a common lower case letter are significantly different (6 h). White symbols that do not share a common upper case letter are significantly different (12 h). # indicates that the value after 12 h is different from the value after 6 h at a same salinity. * indicates that groups submitted to the different salinities are different, but considering both exposure times. No differences were detected in plasma chloride levels.

was no effect of salinity or time of exposure on branchial CA activity (Fig. 3B). Renal CA activity was higher than branchial CA activity.

4. Discussion

3.4. NKA specific activity

4.1. Hyposmotic in seawater, plasma dilution in brackish water, and regulation of MWC

Rectal gland NKA activity displayed a 63% decrease from control values in 35 psu (2.49 mmol ADP mg protein −1 h −1) to 0.93 mmol ADP.mg protein −1 h −1 after 6 h in 5 psu (Fig. 4A). Upon 12 h of exposure, NKA activity was lower in rectal glands of skates exposed to the lower salinities, when compared to those kept in higher salinities. In an opposite pattern, renal NKA activity increased by 30-fold when compared to renal activity in skates kept in 35 psu, considering both times of exposure together (Fig. 4B). In the gills, no change in NKA activity was observed upon exposure of the skates to seawater dilution (Fig. 4C). Average rectal gland NKA activities were higher than renal activities, which were on their turn higher than branchial activities.

Z. brevirostris is hyposmotic to full-strength seawater (~980 mOsm/ kgH2O), such as reported for the coastal Australian Port Jackson shark Heterodontus portusjacksoni (987 mOsm/kgH2O, Cooper and Morris, 1998), or the dogfish Scyliorhinus canicula (~900–960 mOsm/kgH2O, Anderson et al., 2002), or else C. leucas acclimated to seawater (~940– 947 mOsm/kgH2O, Pillans et al., 2005, 2008). However, in another study, the same C. leucas captured in seawater displayed plasma osmolality of 1067 mOsm/kgH2O (Pillans and Franklin, 2004). In any case, it seems that elasmobranchs in seawater are not always slightly hyperosmotic to full-strength seawater. This hyposmotic condition results in an opposite direction for the osmotic gradient: instead of a steady water influx allowing for glomerular filtration and urine formation,

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Water Salinity (psu) Fig. 3. Specific activity of the enzyme carbonic anhydrase in the kidney (A) and gills (B) of Zapteryx brevirostris after 6 (spotted bars) and 12 (white bars) h of exposure to seawater of salinities 35, 25, 15, and 5. *indicates that groups submitted to the different salinities are different, but considering both exposure times. No differences were detected in branchial carbonic anhydrase activities.

the skate and other hyposmotic species may suffer osmotic water loss, and as a consequence lower urinary flow, when compared to other marine elasmobranchs. This difference in renal function would have to be demonstrated, in this and other elasmobranchs when hyposmotic in seawater. Exposure to seawater dilution for 6–12 h led to plasma osmotic dilution in the skate Z. brevirostris, as expected, with the skates remaining highly hyperosmotic to the dilute seawater. Extracellular dilution is also displayed by the euryhaline elasmobranchs D. sabina and C. leucas, when acclimated to dilute seawater or freshwater (de Vlaming and Sage, 1973; Piermarini and Evans, 1998; Pillans et. al., 2006). In freshwater plasma osmolality of these fully euryhaline species reaches ~600 mOsm/kgH2O (Piermarini and Evans, 1998; Pillans et al., 2006; Pillans et al., 2008). Teleosts, that are much more hyposmotic in seawater, display the strategy of seawater drinking, absorption through the gastrointestinal tract and salt excretion through the gills (Evans et al., 2005; Evans and Claiborne, 2009). Elasmobranchs are not commonly reported to display a drinking habit, actually not expected given their normally hyperosmotic status (Hammerschlag, 2006; Evans and Claiborne, 2009). However, recent evidence has demonstrated drinking habit in Scyliorhinus canicula and Triakis scyllia (Anderson et al., 2002) after transfer from 80% seawater to 100% seawater (reviewed in Evans and Claiborne, 2009). It is thus possible that Z. brevirostris and other species also display this habit, which may be a general feature of hyposmotic elasmobranchs in seawater, or in general of elasmobranchs returning from brackish water to full-strength seawater (Anderson et al., 2007; Evans and Claiborne, 2009). Despite some plasma dilution, muscle hydration was maintained unchanged, such as found and expected for euryhaline species (e.g., D. sabina in Anderson et al., 2007). Good maintenance of muscle

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Water Salinity (psu) Fig. 4. Specific activity of the enzyme Na+,K + -ATPase in the rectal gland (A), kidney (B) and gills (C) of Zapteryx brevirostris after 6 (bars with “scale” filling) and 12 (white bars) h of exposure to seawater of salinities 35, 25, 15, and 5. Scale-filled bars that do not share a common lower case letter are significantly different (6 h). White bars that do not share a common upper case letter are significantly different (12 h). * indicates that groups submitted to the different salinities are different, but considering both exposure times. No differences were detected in branchial Na+,K+-ATPase activities.

hydration has also been described for the euryhaline Dasyatis sabina in dilute seawater of 10 psu, after 3–12 days of transference from full-strength seawater (~ 79%, de Vlaming and Sage, 1973). In the partially euryhaline leopard shark Triakis semifasciata, significant increase in muscle moisture was noted upon exposure of 24 h to 3 weeks to 50–60% seawater (Dowd et al., 2010). In summary, the maintenance of muscle hydration by Z. brevirostris was indicative of its reasonable degree of euryhalinity, under the challenge presented here. 4.2. Plasma urea and ions Urea and sodium chloride are the main extracellular osmolytes in elasmobranchs, together with TMAO. Plasma urea of Zapteryx brevirostris was of ~ 430 mM in full-strength seawater. Similar values were reported for Dasyatis americana (444 mM, Cain et. al., 2004), and Squalus acanthias (438 mM, Wood et al., 2007). Somewhat lower plasma urea levels in full-strength seawater are normally found in fully euryhaline species such as Carcharhinus leucas (370 mM, Pillans and Franklin, 2004; 356 mM, Thorson et al., 1973) and Dasyatis sabina (394 mM, Piermarini and Evans, 1998; 383 mM, de Vlaming and Sage, 1973). Urea levels of Z. brevirostris are thus similar to those of other marine elasmobranchs. Exposure to seawater dilution leads to expected reduction in plasma urea. For example, D. sabina exposed for 6 days to salinity 19 psu

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displays 316 mM of plasma urea (de Vlaming and Sage, 1973). Further reduction in salinity and/or increase in time of exposure to low salinity leads to further dilution in plasma urea: plasma urea of D. sabina decreases by 59%, from 352 mM (in seawater) to 208 mM after 12 days in salinity 0 psu (de Vlaming and Sage, 1973); D. sabina living in fresh water shows 195 mM of plasma urea (Piermarini and Evans, 1998). C. leucas (Pillans and Franklin, 2004) displayed a reduction of 51% in plasma urea, from 370 mM (living in seawater) to 192 mM (living in freshwater). The longer the time and the lower the salinity, the lower the plasma urea levels, as a rule. The pattern is clear when plasma urea of our current Z. brevirostris data and data from the literature on elasmobranchs are plotted in a 3-D mesh plot across salinity and time of exposure (Fig. 5). Higher plasma urea levels are measured in elasmobranchs when in high salinities (i.e., seawater), or also in low salinities for short times (few h) of exposure. Z. brevirostris displayed remarkable regulation of plasma sodium chloride, such as typical of euryhaline elasmobranchs (Pillans and Franklin, 2004; Hammerschlag, 2006; Pillans et al., 2006), and also found in the considered partially euryhaline leopard shark Triakis semifasciata (Dowd et al., 2010). It may well be that longer times of exposure would result in weakening of the ionic regulatory capacity, but in any case the regulation exhibited was surprising for a marine species with no reported presence in nearby estuaries (Chaves and Corrêa, 1998; Queiroz et al., 2006). Again, as indicated for urea, of course, the longer the time of exposure/acclimation to changing salinities, the widest the range of variation in plasma ionic levels, yet with these fish showing remarkable regulation and tolerance (Pillans and Franklin, 2004; Pillans et al., 2006). Plasma osmolytes were rather consistent here. For ~1000 mOsm/kg H2O of plasma osmolality of the skate in seawater, some 400 mM is ascribed to urea, also ~400 mM to

NaCl, and the remaining ~150–200 mOsm/kg H2O are probably due to TMAO and the other minor organic and inorganic components. Plasma potassium was regulated after 6 h of exposure to dilute seawater but showed some conformation after 12 h, different for example from the fully euryhaline shark C. leucas, which shows distinct regulation of potassium even in freshwater, with values around 4 mM (Pillans and Franklin, 2004; Pillans et al., 2005), or ranging between 4 and 6 mM, between freshwater and seawater animals (Pillans et al., 2008). However, even the euryhaline D. sabina also shows some plasma potassium variation between seawater and freshwater: ~ 6–2 mM (de Vlaming and Sage, 1973). Plasma magnesium was distinctly hypo-regulated by the skate Z. brevirostris under all experimental conditions, despite some reduction observed in the lowest salinity of exposure. Overall values of potassium and magnesium were similar to the ones previously provided in the literature for sharks, rays and skates (de Vlaming and Sage, 1973; Thorson et al., 1973; MandrupPoulsen, 1981; Piermarini and Evans, 1998; Pillans and Franklin, 2004; Pillans et al., 2005). 4.3. CA activity CA activity increased in the kidney upon mild seawater dilution, with the value in 15 psu being higher than the value in 5 psu. The reduction in the lowest salinity would not be expected; the literature reports increased renal activity upon salinity reduction (Evans, 1984; Ballantyne and Robinson, 2010). Increase in CA activity in the gills, related to ion absorption in dilute seawater, would also be expected (Evans, 1984; Evans et al., 2005), but was not observed. It may well be that a longer exposure to dilute brackish waters would cause increased activity of the CA in the gills of Z. brevirostris. In any case this enzyme deserves additional studies in this group, especially concerning its role in acid–base regulation and salt absorption in freshwater. 4.4. NKA activity

500

M)

400

300

200 35 30 25 20 15 10

100

ity

Plasma Urea (m

41

250

200

Time

Sa lin

0 150

(h)

100

5 50

0

0

100 mM

200 mM

300 mM

400 mM

500 mM

Fig. 5. Combined effects of time and salinity on plasma urea levels in several elasmobranchs. Data used to build the 3-D mesh plot came from: Carcharhinus leucas (Thorson et al., 1973; Pillans and Franklin, 2004; Pillans et al., 2005; Reilly et al., 2011), Pristis microdon (Holmes and Donaldson, 1969), Dasyatis sabina (de Vlaming and Sage,1973; Piermarini and Evans, 1998; Janech and Piermarini, 2002), Dasyatis americana (Cain et al., 2004), Himantura signifer (Tam et al., 2003; Chew et al., 2006), Triakis semifasciata (Dowd et al., 2010), Squalus acanthias (Robertson, 1975; Wood et al., 2007), Heterodontus portusjacksoni (Cooper and Morris, 1998), Chiloscyllium plagiosum (Anderson et al., 2010), Leucoraja erinacea (Anderson et al., 2010), Raja eglanteria (Anderson et al., 2010), Raja erinacea (Treberg et al., 2006), Sphyrna tiburo (Mandrup-Poulsen, 1981), Zapteryx brevirostris (current study). Strictly freshwater stingrays of the family Potamotrygonidae were not included in the plot. For the sake of simplicity, and to avoid compressing the data of shorter times of exposure, 300 h was considered as the time for urea data from studies in which: (1) animals were obtained directly from their habitat, (2) were fully acclimated to that salinity, that is, exposed/acclimated for more than 12 days.

NKA activity in the rectal gland displayed a distinct reduction upon seawater dilution, in total agreement with the literature, for euryhaline elasmobranchs such as C. leucas (Pillans et al., 2005) and D. sabina (Piermarini and Evans, 2000), compatible with the role of the rectal gland in salt secretion in higher salinities. Conversely, renal NKA activity increased remarkably (30-fold between salinities 35 and 5) upon seawater dilution, as expected from its increased role in salt retention in dilute media (Piermarini and Evans, 2000; Pillans et al., 2005). Data on the skate Z. brevirostris show fast regulation of NKA activity in the osmoregulatory organs, as a function of changing salinities. Also as expected, branchial NKA activity was much lower than that of the other two tissues (Pillans et al., 2005; Ballantyne and Robinson, 2010), but it did not increase upon seawater dilution, perhaps given the short time of exposure to salinity reduction (Piermarini and Evans, 2000). In conclusion, Z. brevirostris is physiologically capable of surviving short-term exposure to low salinity which is at odds with its known distribution that does not include areas subjected to reduced salinity. 4.5. Why is euryhalinity so common in marine elasmobranchs? When families containing partially or fully euryhaline species or even fully freshwater species are represented by symbols beside the names of their orders in an elasmobranch cladogram, it is clear that euryhalinity (even more considering that most species have not been investigated so far) is very widespread in the group (Fig. 6). This widespread euryhalinity (to varying degrees) or tolerance of dilute waters may be explained by a potential previous history of the group in waters more dilute than the ocean water. It could be a return to a habitat of the past. Their common osmoregulatory strategy also

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Carcharhiniformes Orectolobiformes Lamniformes Heterodontiformes

Galeomorphi

Squaliformes Squatiniformes Pristiophoriformes Echinorhiniformes Hexanchiformes Chamydoselachiformes

Squalimorphi

Myliobatiformes Rajiformes Torpediniformes Rhiniformes Rhynchobatiformes Pristiformes

Batoidea

Chimaeriformes Fig. 6. Cladogram of extant elasmobranchs, from Martin (2013). The stars depict the widespread occurrence of euryhalinity within the group, according to Martin (2005): the grey stars represent brackish marginal species (or partially euryhaline in laboratory studies, such as Z. brevirostris); the black stars represents true/fully euryhaline species (Carcharhinidae in Carcharhiniformes, Dasyatidae in Rajiformes, and Pristidae in Pristiformes), and the white star represents obligate freshwater species (Carcharhinidae in Carcharhiniformes, Potamotrygonidae in Myliobatiformes, and Dasyatidae in Rajiformes). The families represented by the grey stars were not named so that this legend would not be too long. Please see Martin (2005) for their names.

points to the same direction. The extracellular accumulation of urea is an ancient trait acquired early in the elasmobranch lineage, present in all members of the subclass (Smith, 1936). Actually, enzymes to produce urea were demonstrated to have been present in the earliest vertebrates (Mommsen and Walsh, 1989). Its role in the osmoregulatory strategy of the group is viewed as secondary, built on the branchial and rectal gland regulation of salt (Smith, 1936, 1961; Lutz, 1975). A complex kidney with a countercurrent system to avoid urinary loss of urea was selected in tandem. Reproductive adaptations are also seen: cleidoic eggs and viviparity and internal fertilization are selected, in order to protect embryos which should develop in the urea-rich environments (Smith, 1961; Dodd and Dodd, 1985; Griffith, 1985; Vize and Smith, 2004). Interestingly, viviparity is proposed to have been present already in palaeozoic chondrichthyans (Dodd and Dodd, 1985). Urea retention (along with TMAO and other protective organic molecules) is thus very adequate for the reinvasion of seawater (Lutz, 1975). Urea retention leading to an isosmotic/ hyperosmotic state results in elimination of the osmotic problem, allowing for constant supply of fluid to the glomeruli-rich kidneys, even in seawater (Smith, 1936, 1961; Griffith, 1985). It is hard to conceive that urea retention, coupled to sodium chloride hyporegulation, and its physiological and morphological consequences would be selected for by evolution within the context of a purely and entire seawater existence. Rather, early development of the group in brackish water seems the more likely explanation for the osmoregulatory characteristics of elasmobranchs (Smith, 1936, 1961; Lutz, 1975; Griffith, 1985; Ditrich, 2007). Plasma urea is extremely labile in current marine species when they enter brackish water or even freshwater (Fig. 5). So, it seems that a variable degree of urea accumulation is adaptive in areas of variable salinity, allied to some salt secretion (Lutz, 1975). Plasma salt content is claimed to be related to the habitat where animals evolved (Smith, 1961; Lutz, 1975). In addition, elasmobranchs ended up being so successful in the oceans, not only given this ureo-osmotic strategy, but also given their size, predatory capacity, swimming ability in large open spaces (Ilves and Randall, 2007). Zapteryx brevirostris is reported along the coast of Southeastern– Southern Brazil; specimens used here were obtained 20 km away

from the shoreline, but is not found in surveys of the fish fauna of nearby estuarine Bays. It is remarkably resistant, surviving to 2 h of air exposure during transport by the fishermen. Moreover, it survived dilution of up to 12 h to 5 psu. This physiological capacity allows the conclusion that the species could enter and explore estuaries, at least for some h. Of course the occupation of estuarine areas by a marine species is not solely dependent on its tolerance to dilute seawater. In fact, elasmobranchs are not ideally suited for estuaries and freshwater, in general, due to their limited maneuverability, and common large body size and top predatory feeding habit, making it harder for them to move quickly, changing direction fast, as necessary for prey capture, in shallower waters. The need to be constantly swimming for gill ventilation and respiration also makes them especially suited for marine habitats, with characteristic large volumes of water (Martin, 2005; Ilves and Randall, 2007). However, these constraints would not be applicable to small-sized elasmobranchs such as the skate Z. brevirostris, which is also consistent with the success of the freshwater stingrays of the family Potamotrygonidae in freshwater. 5. Conclusions Thus, Z. brevirostris can be considered a partially euryhaline species, a marine species that could potentially use estuarine brackish areas. In this study the species was not tested to the limits of its tolerance. It was not tested for survival in freshwater, or for longer times of exposure (days or weeks) in dilute seawater. Finally, as seen in our case study here with this skate, and reported in the literature by many authors (Choe et al., 2007; Hazon et al., 2007; Evans and Claiborne, 2009), euryhalinity (to varying degrees) seems to be more common than generally expected in elasmobranchs, and allied to the reasoning presented above—supported by several knowledgeable past researchers— also point to at least a brackish water environment for the early evolution of the group. Acknowledgments Authors wish to gratefully acknowledge the financial support by CNPq as a Masters's fellowship (#557061/2010-5) awarded to NW, and to CAF (PQ #306630/2011-7). Dr Érica Vidal from UFPR (Marine Studies Center) gently allowed laboratory space in Praia de Leste, for the maintenance and experiments with the skates. Authors also wish to thank MSc Hugo Bornatowski for all the pertinent information about the fishes, and for providing the contact with the fishermen. The crucial help of Dr Luciana R. Souza-Bastos and Dr Enelise M. Amado in the Na +,K +-ATPase assays must also be fondly acknowledged here. References Anderson, W.G., Good, J.P., Hazon, N., 2002. Changes in secretion rate and vascular perfusion in the rectal gland of the European lesser spotted dogfish (Scyliorhinus canicula) in response to environmental and hormonal stimuli. J. Fish Biol. 60, 1580–1590. Anderson, W.G., Taylor, J.R., Good, J.P., Hazon, N., Grosell, M., 2007. Body fluid volume regulation in elasmobranch fish. Comp. Biochem. Physiol. A 148, 3–13. Anderson, W.G., Dasiewicz, P.J., Liban, S., Ryan, C., Taylor, J.R., Grosell, M., Weihrauch, D., 2010. Gastro-intestinal handling of water and solutes in three species of elasmobranch fish, the white-spotted bamboo shark, Chiloscyllium plagiosum, little skate, Leucoraja erinacea and the clear nose skate Raja eglanteria. Comp. Biochem. Physiol. A 155, 493–502. Ballantyne, J.S., Robinson, J.W., 2010. Freshwater elasmobranchs: a review of their physiology and biochemistry. J. Comp. Physiol. B 180, 475–493. Boylan, J.W., 1972. A model for passive urea reabsorption in the elasmobranch kidney. Comp. Biochem. Physiol. A42, 27–30. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Bioanal. Biochem. 72, 248–254. Burnett, L.E., Woodson, P.B.J., Rietow, M.G., Vilicich, V.C., 1981. Crab gill intra-epithelial carbonic anhydrase plays a major role in haemolymph CO2 and chloride ion regulation. J. Exp. Biol. 92, 243–254.

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