Na+/K+-ATPase α1 mRNA expression in the gill and rectal gland of the Atlantic stingray, Dasyatis sabina, following acclimation to increased salinity
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The salt-secreting rectal gland plays a major role in elasmobranch osmoregulation, facilitating ion balance in hyperosmotic environments in a manner analogous to the teleost gill. Several studies have examined the central role of the sodium pump Na+/K+-ATPase in osmoregulatory tissues of euryhaline elasmobranch species, including regulation of Na+/K+-ATPase activity and abundance in response to salinity acclimation. However, while the transcriptional regulation of Na+/K+-ATPase in the teleost gill has been well documented the potential for mRNA regulation to facilitate rectal gland plasticity during salinity acclimation in elasmobranchs has not been examined. Therefore, in this study we acclimated Atlantic stingrays, Dasyatis sabina (Lesueur) from 11 to 34 ppt salinity over 3 days, and examined changes in plasma components as well as gill and rectal gland Na+/K+-ATPase α1 (atp1a1) mRNA expression.
Acclimation to increased salinity did not affect hematocrit but resulted in significant increases in plasma osmolality, chloride and urea. Rectal gland atp1a1 mRNA expression was higher in 34 ppt-acclimated D. sabina vs. controls. There was no significant change in gill atp1a1 mRNA expression, however mRNA expression of this gene in the gill and rectal gland were negatively correlated.
This study demonstrates regulation of atp1a1 in the elasmobranch salt-secreting gland in response to salinity acclimation and a negative relationship between rectal gland and gill atp1a1 expression. These results support the hypothesis that the gill and rectal gland play opposing roles in ion balance with the gill potentially facilitating ion uptake in hypoosmotic environments. Future studies should further examine this possibility as well as potential differences in the regulation of Na+/K+-ATPase gene expression between euryhaline and stenohaline elasmobranch species.
KeywordsNa+/K+-ATPase Rectal gland Osmoregulation Elasmobranch Euryhaline
Na+/K+-ATPase α1 subunit mRNA
18S ribosomal RNA
quantitative reverse transcription-polymerase chain reaction
parts per thousand
Significant aspects of elasmobranch osmoregulatory physiology include a heavy reliance on nitrogenous compounds for urea-based osmoregulation and the presence of the salt-secreting rectal gland, an organ that is unique to these taxa. The rectal gland is the primary site for sodium and chloride secretion in euryhaline and marine elasmobranchs, an osmoregulatory role analogous to that of the teleost gill. Therefore, in contrast to the teleost fishes the elasmobranch gill is thought to play a comparatively lesser role in salt balance. However, a series of studies in the Atlantic stingray (Dasyatis sabina) demonstrated changes in gill ion exchange proteins in response to changes in environmental salinity and suggested a role for the elasmobranch gill in ion uptake in addition to acid–base regulation [1, 2, 3]. Therefore the elasmobranch gill may play a more significant role in ion balance than previously accepted, particularly in euryhaline species that may experience frequent and/or rapid changes in salinity.
The Atlantic stingray is an excellent model for studies regarding the osmoregulatory physiology of elasmobranchs, as this euryhaline species is a common inhabitant of coastal bays and estuaries ranging from the east coast of the United States to Central America . D. sabina therefore experience daily and seasonal fluctuations in environmental salinity and have been collected from coastal areas with salinities ranging from 2.2 to 36.7 ppt [5, 6, 7] as well as in hypersaline habitats such as the Laguna Madre in south Texas . Furthermore, it has been reported that some populations of Atlantic stingrays migrate into freshwater rivers [9, 10], and a permanent freshwater population is established in the St. John’s River of Florida .
The enzyme Na+/K+-ATPase plays a central role in ion transport and is highly abundant in secretory cells of both the gill (ionocytes) and rectal gland, driving the secondary active transport of chloride involving ion channels and symport proteins such as the Na–K–Cl cotransporter [12, 13]. Multiple studies have specifically examined the osmoregulatory role of Na+/K+-ATPase in the gill and rectal gland of euryhaline elasmobranch fishes challenged with changes in salinity. In the bull shark (Carcharhinus leucas), seawater acclimation increases Na+/K+-ATPase activity in rectal gland with no corresponding change in gill Na+/K+-ATPase activity . Rectal gland Na+/K+-ATPase abundance and activity also increase in D. sabina following seawater acclimation, with a corresponding decrease in gill Na+/K+-ATPase activity along with changes in gill protein localization . Finally, it has been demonstrated that gill Na+/K+-ATPase alpha subunit (atp1a1) mRNA expression in both D. sabina and C. leucas is higher in freshwater vs. seawater individuals, supporting the hypothesis that the elasmobranch gill plays a role in ion uptake and also suggesting that transcriptional regulation is one mechanism by which euryhaline taxa respond to salinity challenges [15, 16].
Na+/K+-ATPase activity of the elasmobranch rectal gland is more than tenfold higher than that of gill [1, 14], and this organ is critical for ion balance in euryhaline and marine elasmobranch species. It is therefore important to understand potential mechanisms for regulation of the activity of this gland, including transcriptional, translational and post-translational processes. Changes in rectal gland atp1a1 mRNA expression following feeding in the stenohaline European dogfish (Scyliorhinus canicula) and spiny dogfish (Squalus acanthias) have been reported, supporting the hypothesis that transcriptional mechanisms play a role in the plasticity of this organ in response to salt-loading [17, 18]. However, while changes in Na+/K+-ATPase activity in response to altered salinity has been demonstrated as described above, the potential role for mRNA regulation in the rectal gland to facilitate salinity acclimation has not been examined. Therefore, for the laboratory component of the 2014 Summer Field Program undergraduate course Stingray Physiology at the University of Southern Mississippi Gulf Coast Research Laboratory, we examined the regulation of plasma components as well as atp1a1 mRNA expression in the gill and rectal gland of D. sabina acclimated to increased environmental salinity.
Atlantic stingrays were captured by otter trawl in coastal waters near Ocean Springs, Mississippi (salinity ~11 ppt). A total of six mature (>22 cm disk width; ) animals were used for this study (two males, four females; disk width: 23–29 cm; mass: 0.5–1.0 kg). Stingrays were transferred to the laboratory and maintained at 11 ppt and ambient temperature (water temperature 26–27°C) in recirculating 1,700 L tanks. Animals were fed chopped shrimp ad libitum every other day and were acclimated to captivity for at least 2 weeks prior to experimentation. Following completion of the experiment animals were sacrificed by immersion in 1 g L−1 buffered tricaine methanesulfonate (MS-222) and subsequent severing of the spinal column posterior to the gill arches, as approved by the University of Southern Mississippi Animal Care and Use Committee (IACUC protocol #13031403).
Stingrays (two tanks, n = 3 per tank) were maintained at 11 ppt prior to the start of the salinity acclimation experiment. Before salinity acclimation, individual stingrays were removed from the tank by net and 0.5 mL blood was drawn from the wing of each animal using a 22-gauge needle on a 1 mL syringe. Blood was immediately transferred to lithium heparin vacutainers and placed on ice. After hematocrit determination using whole blood as described below, samples were centrifuged for 5 min at 5,000×g, with plasma transferred to a new microcentrifuge tube and stored at −80°C. Following a recovery period of 4 h, salinity acclimation was initiated by adding concentrated seawater brine (60–90 ppt) to the experimental tank’s filter box to facilitate a step-wise increase in salinity from 11 to 34 ppt over the course of 3 days (15–25–34 ppt). This protocol is similar to that used in previous SW-acclimation studies using the Atlantic stingray as a model species, in which animals were acclimated from brackish salinities (15–16 ppt) to seawater (30–32 ppt) over a period of 2–3 days [1, 20]. Stingrays were not fed during this time, and animals from both tanks were sampled 1 day after the salinity increase was completed (day four). Following blood collection as described above, individuals were sacrificed and rectal glands were then rapidly removed, coarsely minced and placed in microcentrifuge tubes containing RNAlater (Ambion). To collect gill tissue, a single gill arch was removed from each individual and scraped using a sterile scalpel blade; all tissue was then rapidly transferred to a microcentrifuge tube containing RNAlater.
All plasma analyses were completed in duplicate. Plasma osmolality was determined using a Vapro 5520 vapor pressure osmometer (Wescor), and chloride was quantified using a digital chloridometer (Labconco). For determining urea concentrations, plasma was diluted 1:50 and analyzed using a commercial colorimetric assay (QuantChrom Urea Assay, Bioassay Systems). Hematocrit was measured in approximately 50 µL of whole blood per replicate using 75 mm ammonium-heparin hematocrit tubes and an IEC Micro MB microhematocrit centrifuge (Thermo), with values determined using a microhematocrit capillary tube reader disk.
RNA isolation, cDNA synthesis and quantitative PCR
qRT-PCR primer sets and reaction efficiencies
Paired Student’s t test comparisons were used for statistical analysis of pre- and post-acclimation plasma component values within experimental groups. Rectal gland and gill atp1a1 mRNA levels were compared using ANOVA followed by a Student–Newman–Keuls post hoc test. In all cases, p-values less than 0.05 were considered significant.
Results and discussion
Euryhaline elasmobranch species such as D. sabina can acclimate to a wide range of environmental salinities, and may be frequently challenged with significant salinity gradients over a short period of time. The ability of these taxa to maintain enantiostasis regardless of salinity relies upon the rapid and efficient balance of plasma osmolytes including sodium and chloride ions via tissues such as the gill, kidney, gut and rectal gland. Therefore elucidating the cellular and molecular mechanisms that facilitate ion balance provides critical insight into the physiology of euryhalinity, including the potential to understand why some taxa are successful across a wide range of salinity whereas others are not. This study represents a significant step in this direction, demonstrating gene regulation in the primary salt-secreting organ of a euryhaline elasmobranch following acclimation to a hyperosmotic environment. Future studies should further examine atp1a1 gene transcription in the rectal gland and gill of D. sabina with regards to cellular mechanism e.g. promoter regulation, and also use D. sabina as a comparative model to examine the role and regulation of Na+/K+-ATPase in osmoregulatory tissues of euryhaline vs. stenohaline elasmobranch species.
Both AE and FL designed the experiments and collected tissue samples. AE completed statistical analyses and prepared the manuscript, figures and tables. FL performed the experimental work including qPCR assays and revised the manuscript. All authors read and approved the final manuscript.
We would like to thank all the participants of the 2014 Summer Field Program undergraduate course Stingray Physiology, who conducted this study as a laboratory exercise examining the osmoregulatory capabilities of the Atlantic stingray.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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