The Rectal Gland of the Shark: The Road to Understanding the Mechanism and Regulation of Transepithelial Chloride Transport.

Pictured, described, and speculated on, for close to 400 years, the function of the rectal gland of elasmobranchs remained unknown. In the late 1950s, Burger discovered that the rectal gland of Squalus acanthias secreted an almost pure solution of sodium chloride, isosmotic with blood, which could be stimulated by volume expansion of the fish. Twenty five years later, Stoff discovered that the secretion of the gland was mediated by adenyl cyclase. Studies since then have shown that vasoactive intestinal peptide (VIP) is the neurotransmitter responsible for activating adenyl cyclase; however, the amount of circulating VIP does not change in response to volume expansion. The humoral factor involved in activating the secretion of the gland is C-type natriuretic peptide, secreted from the heart in response to volume expansion. C-type natriuretic peptide circulates to the gland where it stimulates the release of VIP from nerves within the gland, but it also has a direct effect, independent of VIP. Sodium, potassium, and chloride are required for the gland to secrete, and the secretion of the gland is inhibited by ouabain or furosemide. The current model for the secretion of chloride was developed from this information. Basolateral NaKATPase maintains a low intracellular concentration of sodium, which establishes the large electrochemical gradient for sodium directed into the cell. Sodium moves from the blood into the cell (together with potassium and chloride) down this electrochemical gradient, through a coupled sodium, potassium, and two chloride cotransporter (NKCC1). On activation, chloride moves from the cell into the gland lumen, down its electrical gradient through apical cystic fibrosis transmembrane regulator. The fall in intracellular chloride leads to the phosphorylation and activation of NKCC1 that allows more chloride into the cell. Transepithelial sodium secretion into the lumen is driven by an electrical gradient through a paracellular pathway. The aim of this review was to examine the history of the origin of this model for the transport of chloride and suggest that it is applicable to many epithelia that transport chloride, both in resorptive and secretory directions.

The RING zinc finger protein LbRZF1 promotes salt gland development and salt tolerance in Limonium bicolor.

The recretohalophyte Limonium bicolor thrives in high-salinity environments because salt glands on the above-ground parts of the plant help to expel excess salt. Here, we characterize a nucleus-localized C3HC4 (RING-HC)-type zinc finger protein of L. bicolor named  RING  ZINC  FINGER PROTEIN  1 (LbRZF1). LbRZF1 was expressed in salt glands and in response to NaCl treatment. LbRZF1 showed no E3 ubiquitin ligase activity. The phenotypes of overexpression and knockout lines for LbRZF1 indicated that LbRZF1 positively regulated salt gland development and salt tolerance in L. bicolor. lbrzf1 mutants had fewer salt glands and secreted less salt than did the wild-type, whereas LbRZF1-overexpressing lines had opposite phenotypes, in keeping with the overall salt tolerance of these plants. A yeast two-hybrid screen revealed that LbRZF1 interacted with LbCATALASE2 (LbCAT2) and the transcription factor LbMYB113, leading to their stabilization. Silencing of LbCAT2 or LbMYB113 decreased salt gland density and salt tolerance. The heterologous expression of LbRZF1 in Arabidopsis thaliana conferred salt tolerance to this non-halophyte. We also identified the transcription factor LbMYB48 as an upstream regulator of LbRZF1 transcription. The study of LbRZF1 in the regulation network of salt gland development also provides a good foundation for transforming crops and improving their salt resistance.

Leaf sodium homeostasis controlled by salt gland is associated with salt tolerance in mangrove plant Avicennia marina.

Avicennia marina, a mangrove plant growing in coastal wetland habitats, is frequently affected by tidal salinity. To understand its salinity tolerance, the seedlings of A. marina were treated with 0, 200, 400 and 600 mM NaCl. We found the whole-plant dry weight and photosynthetic parameters increased at 200 mM NaCl but decreased over 400 mM NaCl. The maximum quantum yield of primary photochemistry (Fv/Fm) significantly decreased at 600 mM NaCl. Transmission electron microscopy observations showed high salinity caused the reduction in starch grain size, swelling of the thylakoids and separation of the granal stacks, and even destruction of the envelope. In addition, the dense protoplasm and abundant mitochondria in the secretory and stalk cells, and abundant plasmodesmata between salt gland cells were observed in the salt glands of the adaxial epidermis. At all salinities, Na+ content was higher in leaves than in stems and roots; however, Na+ content increased in the roots while it remained at a constant level in the leaves over 400 mM NaCl treatment, due to salt secretion from the salt glands. As a result, salt crystals on the leaf adaxial surface increased with salinity. On the other hand, salt treatment increased Na+ and K+ efflux and decreased H+ efflux from the salt glands by the non-invasive micro-test technology, although Na+ efflux reached the maximum at 400 mM NaCl. Further real-time quantitative PCR analysis indicated that the expression of Na+/H+ antiporter (SOS1 and NHX1), H+-ATPase (AHA1 and VHA-c1) and K+ channel (AKT1, HAK5 and GORK) were up-regulated, and only the only Na+ inward transporter (HKT1) was down-regulated in the salt glands enriched adaxial epidermis of the leaves under 400 mM NaCl treatment. In conclusion, salinity below 200 mM NaCl was beneficial to the growth of A. marina, and below 400 mM, the salt glands could excrete Na+ effectively, thus improving its salt tolerance.

Proteomics of Salt Gland-Secreted Sap Indicates a Pivotal Role for Vesicle Transport and Energy Metabolism in Plant Salt Secretion.

Soil salinization is one of the major factors restricting crop growth and agricultural production worldwide. Recretohalophytes have developed unique epidermal structures in their aboveground tissues, such as salt glands or salt bladders, to secrete excess salt out of the plant body as a protective mechanism from ion damage. Three hypotheses were proposed to explain how salt glands secrete salts: the osmotic hypothesis, a hypothesis similar to animal fluid transport, and vesicle-mediated exocytosis. However, there is no direct evidence to show whether the salt gland-secreted liquid contains landmark proteins or peptides which would elucidate the salt secretion mechanism. In this study, we collected the secreted liquid of salt glands from Limonium bicolor, followed by extraction and identification of its constituent proteins and peptides by SDS-PAGE and mass spectrometry. We detected 214 proteins and 440 polypeptides in the salt gland-secreted droplets of plants grown under control conditions. Unexpectedly, the proportion of energy metabolism-related proteins increased significantly though only 16 proteins and 35 polypeptides in the droplets of salt-treated plants were detected. In addition, vesicle transport proteins such as the Golgi marker enzyme glycosyltransferase were present in the secreted sap of salt glands from both control and salt-treated plants. These results suggest that trans-Golgi network-mediated vesicular transport and energy production contributes to salt secretion in salt glands.

Exogenous hydrogen sulfide mediates Na+ and K+ fluxes of salt gland in salt-secreting mangrove plant Avicennia marina.

Hydrogen sulfide (H2S), is a crucial biological player in plants. Here, we primarily explored the interaction between sodium hydrosulfide (NaHS, a H2S donor) and the fluxes of Na+ and K+ from the salt glands of mangrove species Avicennia marina (Forsk.) Vierh. with non-invasive micro-test technology (NMT) and quantitative real-time PCR (qRT-PCR) approaches under salinity treatments. The results showed that under 400-mM NaCl treatment, the addition of 200-µM NaHS markedly increased the quantity of salt crystals in the adaxial epidermis of A. marina leaves, accompanied by an increase in the K+/Na+ ratio. Meanwhile, the endogenous content of H2S was dramatically elevated in this process. The NMT result revealed that the Na+ efflux was increased from salt glands, whereas K+ efflux was decreased with NaHS application. On the contrary, the effects of NaHS were reversed by H2S scavenger hypotaurine (HT), and DL-propargylglycine (PAG), an inhibitor of cystathionine-γ-lyase (CES, a H2S synthase). Moreover, enzymic assay revealed that NaHS increased the activities of plasma membrane and tonoplast H+-ATPase. qRT-PCR analysis revealed that NaHS significantly increased the genes transcript levels of tonoplast Na+/H+ antiporter (NHX1), plasma membrane Na+/H+ antiporter (SOS1), plasma membrane H+-ATPase (AHA1) and tonoplast H+-ATPase subunit c (VHA-c1), while suppressed above-mentioned gene expressions by the application of HT and PAG. Overall, H2S promotes Na+ secretion from the salt glands of A. marina by up-regulating the plasma membrane and tonoplast Na+/H+ antiporter and H+-ATPase.

A nonolfactory shark adenosine receptor activates CFTR with unique pharmacology and structural features.

Adenosine receptors (ADORs) are G protein-coupled purinoceptors that have several functions including regulation of chloride secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in human airway and kidney. We cloned an ADOR from Squalus acanthias (shark) that likely regulates CFTR in the rectal gland. Phylogenic and expression analyses indicate that elasmobranch ADORs are nonolfactory and appear to represent extant predecessors of mammalian ADORs. We therefore designate the shark ADOR as the A0 receptor. We coexpressed A0 with CFTR in Xenopus laevis oocytes and characterized the coupling of A0 to the chloride channel. Two-electrode voltage clamping was performed, and current-voltage (I-V) responses were recorded to monitor CFTR status. Only in A0- and CFTR-coinjected oocytes did adenosine analogs produce a significant concentration-dependent activation of CFTR consistent with its electrophysiological signature. A pharmacological profile for A0 was obtained for ADOR agonists and antagonists that differed markedly from all mammalian ADOR subtypes [agonists: R-phenyl-isopropyl adenosine (R-PIA) > S-phenyl-isopropyl adenosine (S-PIA) > CGS21680 > N6-cyclopentyladenosine (CPA) > 2-chloroadenosine (2ClAdo) > CV1808 = N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA) > N-ethyl-carboxyl adenosine (NECA); and antagonists: 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > PD115199 > 1,3-dimethyl-8-phenylxanthine (8PT) > CGS15943]. Structures of human ADORs permitted a high-confidence homology model of the shark A0 core that revealed unique structural features of ancestral receptors. We conclude that 1) A0 is a novel and unique adenosine receptor ancestor by functional and structural criteria; 2) A0 likely activates CFTR in vivo, and this receptor activates CFTR in oocytes, indicating an evolutionary coupling between ADORs and chloride secretion; and 3) A0 appears to be a nonolfactory evolutionary ancestor of all four mammalian ADOR subtypes.

Sugar uptake, metabolism, and chloride secretion in the rectal gland of the spiny dogfish Squalus acanthias.

The rectal gland of the spiny dogfish Squalus acanthias secretes a salt solution isosmotic with plasma that maintains the salt homeostasis of the fish. It secretes salt against an electrochemical gradient that requires the expenditure of energy. Isolated rectal glands perfused without glucose secrete salt, albeit at a rate about 30% of glands perfused with 5 mM glucose. Gradually reducing the glucose concentration is associated with a progressive decrease in the secretion of chloride. The apparent Km for the exogenous glucose-dependent chloride secretion is around 2 mM. Phloretin and cytochalasin B, agents that inhibit facilitated glucose carriers of the solute carrier 2 (Slc2) family such as glucose transporter 2 (GLUT2), do not inhibit the secretion of chloride by the perfused rectal glands. Phloridzin, which inhibits Slc5 family of glucose symporters, or α-methyl-d-glucoside, which competitively inhibits the uptake of glucose through Slc5 symporters, inhibit the secretion of chloride. Thus the movement of glucose into the rectal gland cells appears to be mediated by a sodium-glucose symporter. Sodium-glucose cotransporter 1 (SGLT1), the first member of the Slc5 family of sodium-linked glucose symporters, was cloned from the rectal gland. No evidence of GLUT2 was found. The persistence of secretion of chloride in the absence of glucose in the perfusate suggests that there is an additional source of energy within the cells. The use of 2-mercapto-acetate did not result in any change in the secretion of chloride, suggesting that the oxidation of fatty acids is not the source of energy for the secretion of chloride. Perfusion of isolated glands with KCN in the absence of glucose further reduces the secretion of chloride but does not abolish it, again suggesting that there is another source of energy within the cells. Glucose was measured in the rectal gland cells and found to be at concentrations in the range of that in the perfusate. Glycogen measurements indicated that there are significant stores of glucose in the rectal gland. Moreover, glycogen synthase was partially cloned from rectal gland cells. The open reading frame of glycogen phosphorylase was also cloned from rectal gland cells. Measurements of glycogen phosphorylase showed that the enzyme is mostly in its active form in the cells. The cells of the rectal gland of the spiny dogfish require exogenous glucose to fully support the active secretion of salt. They have the means to transport glucose into the cells in the form of SGLT1. The cells also have an endogenous supply of glucose as glycogen and have the necessary elements to synthesize, store, and hydrolyze it.

Functional characterization of olfactory receptors in three Dacini fruit flies (Diptera: Tephritidae) that respond to 1-nonanol analogs as components in the rectal glands.

Dacini fruit flies (Tephritidae: Diptera), including destructive pest species, are strongly affected in their reproductive behaviors by semiochemicals. Notably, male lures have been developed for pest management e.g., aromatic compounds for the Oriental fruit fly Bactrocera dorsalis and the melon fruit fly Zeugodacus cucurbitae; terpenic α-ionone analogs for the solanaceous fruit fly, B. latifrons. Other than those specific male attractants, 1-nonanol analogs have been noticed as major aliphatic components in the male rectal gland, which is considered as a secretory organ of male sex pheromones. Although multiple semiochemicals associated with the life cycle of Dacini fruit flies have been identified, their behavioral role(s) and chemosensory mechanisms by which the perception occurs have not been fully elucidated. In this study, we conducted RNA sequencing analysis of the chemosensory organs of B. latifrons and Z. cucurbitae to identify the genes coding for chemosensory receptors. Because the skeletons of male attractants are different among Dacini fruit fly species, we analyzed phylogenetic relationships of candidate olfactory receptors (ORs) among the three species. We found that the OR phylogeny reflects the taxonomic relationships of the three species. We further characterized functional properties of OR74a in the three Dacini species to the 1-nonanol analogs related to components in the rectal glands. The three OR74a homologs responded to 1-nonanol, but their sensitivities differed from each other. The OR74a homologs identified from B. dorsalis and Z. cucurbitae responded significantly to 6-oxo-1-nonanol, but not to 1,3-nonanediol and nonyl acetate, indicating similar binding properties of the homologous ORs.

Evidence of accumulation and elimination of inorganic contaminants from the lachrymal salt glands of leatherback sea turtles (Dermochelys coriacea).

Plasma osmolalities of marine vertebrates are generally lower than the surrounding medium; therefore, marine organisms must cope with the osmoregulatory challenges of life in a salty environment. The salt glands serve to maintain osmotic and ionic homeostasis in a number of lower marine vertebrates. One marine reptile, the leatherback sea turtle (Dermochelys coriacea), ingests excessive amounts of salts due to their diet of gelatinous zooplankton. Outside of the normal osmoregulatory function of the salt gland, little research has been conducted on contaminant accumulation and excretion in this organ. Here, we established arsenic, cadmium, lead, mercury, and selenium concentrations in red blood cells (RBCs) and salt gland secretions (SGSs) of nesting leatherbacks. We also collected salt glands from different life stage classes of dead stranded leatherbacks from the western Atlantic Ocean to determine if inorganic contaminants accumulate in this organ. Using non-metric multidimensional scaling and regression analyses, we determined that RBC and SGS inorganic contaminant concentrations were not correlated. Additionally, RBCs showed significantly higher concentrations of these contaminants in comparison to SGSs, likely due to the affinity of inorganic contaminants for the heme group of RBCs. Lastly, we found that salt gland cadmium and mercury concentrations tended to increase with increasing curved carapace length (CCL) in stranded leatherbacks. Our results indicate that different physiological mechanisms determine the distribution of inorganic contaminants in blood and SGSs. Increases in salt gland contaminant concentrations with increasing CCL suggest this organ as a potential target for accumulation.

Immunohistochemical colocalization of G protein alpha subunits and 5-HT in the rectal gland of the cartilaginous fish Scyliorhinus canicula.

Serotonin [5-hydroxytryptamine (5-HT)] is an important neuromodulator involved in a wide range of physiological functions. The effects of serotonin are mediated by an extended family of receptors coupled to multiple heterotrimeric G-proteins, associated with cellular membrane. G proteins connect receptors to effectors and thus trigger intracellular signaling pathways. These cellular processes several regulate systemic functions such as embryonic development, gonadal development, learning and memory, and organismal homeostasis. Generally, elasmobranch fish dwell a hypersaline environment and utilize a specialized extrarenal salt secreting organ, the rectal gland, to face ionic homeostasis. In this study in addition to the morphological, histochemical and immunohistochemical description of the Scyliorhinus canicula rectal gland, for the first time, the presence of serotonin (5-HT), and distribution of different types of G protein alpha subunits (Gα o, Gα q/11, and Gα s/olf) has been investigated in the rectal gland epithelium by confocal immunofluorescence techniques. Colocalization G proteins and 5-HT in the secretory epithelium of the gland suggests serotonin acts as a hormone and involves G proteins in an autocrine-paracrine control of rectal gland homeostasis.

Crying a river: how much salt-laden jelly can a leatherback turtle really eat?

Leatherback turtles (Dermochelys coriacea) are capital breeders that accumulate blubber (33 kJ g-1 wet mass) by hyperphagia on a gelatinous diet at high latitudes; they breed in the tropics. A jellyfish diet is energy poor (0.1-0.2 kJ g-1 wet mass) so leatherbacks must ingest large quantities. Two published estimates of feeding rate [50% body mass day-1 (on Rhizostoma pulmo) and 73% body mass day-1 (on Cyanea capillata)] have been criticised as too high. Jellyfish have high salt and water contents that must be removed to access organic material and energy. Most salt is removed (as NaCl) by paired lachrymal salt glands. Divalent ions are lost via the gut. In this study, the size of adult salt glands (0.622 kg for a 450 kg turtle; relatively three times the size of salt glands in cheloniid turtles) was measured for the first time by computed tomography scanning. Various published values for leatherback field metabolic rate, body fluid composition and likely blubber accumulation rates are combined with known jellyfish salt, water and organic compositions to calculate feasible salt gland secretion rates and feeding rates. The results indicate that leatherbacks can produce about 10-15 ml secretion g-1 salt gland mass h-1 (tear osmolality 1800 mOsm kg-1). This will permit consumption of 80% body mass day-1 of Ccapillata Calculations suggest that leatherbacks will find it difficult/impossible to accumulate sufficient blubber for reproduction in a single feeding season. Rapid jellyfish digestion and short gut transit times are essential.

Functional and molecular identification of a TASK-1 potassium channel regulating chloride secretion through CFTR channels in the shark rectal gland: implications for cystic fibrosis.

In the shark rectal gland (SRG), apical chloride secretion through CFTR channels is electrically coupled to a basolateral K+ conductance whose type and molecular identity are unknown. We performed studies in the perfused SRG with 17 K+ channel inhibitors to begin this search. Maximal chloride secretion was markedly inhibited by low-perfusate pH, bupivicaine, anandamide, zinc, quinidine, and quinine, consistent with the properties of an acid-sensitive, four-transmembrane, two-pore-domain K+ channel (4TM-K2P). Using PCR with degenerate primers to this family, we identified a TASK-1 fragment in shark rectal gland, brain, gill, and kidney. Using 5' and 3' rapid amplification of cDNA ends PCR and genomic walking, we cloned the full-length shark gene (1,282 bp), whose open reading frame encodes a protein of 375 amino acids that was 80% identical to the human TASK-1 protein. We expressed shark and human TASK-1 cRNA in Xenopus oocytes and characterized these channels using two-electrode voltage clamping. Both channels had identical current-voltage relationships (outward rectifying) and a reversal potential of -90 mV. Both were inhibited by quinine, bupivicaine, and acidic pH. The pKa for current inhibition was 7.75 for shark TASK-1 vs. 7.37 for human TASK-1, values similar to the arterial pH for each species. We identified this protein in SRG by Western blot and confocal immunofluorescent microscopy and detected the protein in SRG and human airway cells. Shark TASK-1 is the major K+ channel coupled to chloride secretion in the SRG, is the oldest 4TM 2P family member identified, and is the first TASK-1 channel identified to play a role in setting the driving force for chloride secretion in epithelia. The detection of this potassium channel in mammalian lung tissue has implications for human biology and disease.

Effects of short-term hyper- and hypo-osmotic exposure on the osmoregulatory strategy of unfed North Pacific spiny dogfish (Squalus suckleyi).

The North Pacific spiny dogfish (Squalus suckleyi) is a partially euryhaline species of elasmobranch that often enter estuaries where they experience relatively large fluctuations in environmental salinity that can affect plasma osmolality. Previous studies have investigated the effects of altered salinity on elasmobranchs over the long term, but fewer studies have conducted time courses to investigate how rapidly they can adapt to such changes. In this study, we exposed unfed (no exogenous source of nitrogen or TMAO) spiny dogfish to hyper- and hypo-osmotic conditions and measured plasma and tissue osmolytes, nitrogen excretion, and changes in enzyme activity and mRNA levels in the rectal gland over 24h. It was shown that plasma osmolality changes to approximately match the ambient seawater within 18-24h. In the hypersaline environment, significant increases in urea, sodium, and chloride were observed, whereas in the hyposaline environment, only significant decreases in TMAO and sodium were observed. Both urea and ammonia excretion increased at low salinities suggesting a reduction in urea retention and possibly urea production. qPCR and enzyme activity data for Na(+)/K(+)-ATPase did not support the idea of rectal gland activation following exposure to increased salinities. Therefore, we suggest that the rectal gland may not be a quantitatively important aspect of the dogfish osmoregulatory strategy during changes in environmental salinity, or it may be active only in the very early stages (i.e., less than 6h) of responses to altered salinity.

Osmoregulation by juvenile brown-banded bamboo sharks, Chiloscyllium punctatum, in hypo- and hyper-saline waters.

While there is a considerable body of work describing osmoregulation by elasmobranchs in brackish and saltwater, far fewer studies have investigated osmoregulation in hypersaline waters. We examined osmo- and ionoregulatory function and plasticity in juvenile brown-banded bamboo sharks, Chiloscyllium punctatum, exposed to three experimental salinities (25, 34 and 40‰) for two weeks. C. punctatum inhabits sheltered coastal areas and bays which can naturally become hypersaline as a consequence of evaporation of water but can also become hyposaline during flood events. We hypothesised that C. punctatum would demonstrate a phenotypically plastic osmoregulatory physiology. Plasma osmolality, urea, Na(+) and Cl(-) levels increased significantly with increasing environmental salinity. Rectal gland and branchial sodium-potassium ATPase (NKA) activities were unaffected by salinity. Using immunohistochemistry and Western Blotting we found evidence for the presence of the key ion-regulatory proteins vacuolar H(+)-ATPase (VHA), pendrin (Cl(-)/HCO3(-) co-transporter) and the Na(+)-H(+) exchanger isoform 3 (NHE3) in discrete cells within the branchial epithelia. These results indicate that C. punctatum is a partially euryhaline elasmobranch able to maintain osmo- and ionoregulatory function between environmental salinities of 25‰ and 40‰. As suggested for other elasmobranchs, the gills of C. punctatum likely play a limited role in maintaining Na(+) homeostasis over the salinity range studied, but may play an important role in acid-base balance.

Gastric inhibitory peptide, serotonin, and glucagon are unexpected chloride secretagogues in the rectal gland of the skate (Leucoraja erinacea).

Since the discovery of the rectal gland of the dogfish shark 50 years ago, experiments with this tissue have greatly aided our understanding of secondary active chloride secretion and the secretagogues responsible for this function. In contrast, very little is known about the rectal gland of skates. In the present experiments, we performed the first studies in the perfused rectal gland of the little skate (Leucoraja erinacea), an organ weighing less than one-tenth of the shark rectal gland. Our results indicate that the skate gland can be studied by modified perfusion techniques and in primary culture monolayers, and that secretion is blocked by the inhibitors of membrane proteins required for secondary active chloride secretion. Our major finding is that three G protein-coupled receptor agonists, the incretin gastric inhibitory polypeptide (GIP), also known as glucose-dependent insulinotropic peptide, as well as glucagon and serotonin, are unexpected potent chloride secretagogues in the skate but not the shark. Glucagon stimulated chloride secretion to a mean value of 1,661 ± 587 µeq·h(-1)·g(-1) and serotonin stimulated to 2,893 ± 699 µeq·h(-1)·g(-1). GIP stimulated chloride secretion to 3,733 ± 679 µeq·h(-1)·g(-1) and significantly increased tissue cAMP content compared with basal conditions. This is the first report of GIP functioning as a chloride secretagogue in any species or tissue.

Transcriptome responses in the rectal gland of fed and fasted spiny dogfish shark (Squalus acanthias) determined by suppression subtractive hybridization.

Prior studies of the elasmobranch rectal gland have demonstrated that feeding induces profound and rapid up regulation of the gland's ability to secrete concentrated NaCl solutions and the metabolic capacity to support this highly ATP consuming process. We undertook the current study to attempt to determine the degree to which up regulation of mRNA transcription was involved in the gland's activation. cDNA libraries were created from mRNA isolated from rectal glands of fasted (7days post-feeding) and fed (6h and 22h post-feeding) spiny dogfish sharks (Squalus acanthias), and the libraries were subjected to suppression subtractive hybridization (SSH) analysis. Quantitative real time PCR (qPCR) was also used to ascertain the mRNA expression of several genes revealed by the SSH analysis. In total the treatments changed the abundance of 170 transcripts, with 103 up regulated by feeding, and 67 up regulated by fasting. While many of the changes took place in 'expected' Gene Ontology (GO) categories (e.g., metabolism, transport, structural proteins, DNA and RNA turnover, etc.), KEGG analysis revealed a number of categories which identify oxidative stress as a topic of interest for the gland. GO analysis also revealed that branched chain essential amino acids (e.g., valine, leucine, isoleucine) are potential metabolic fuels for the rectal gland. In addition, up regulation of transcripts for many genes in the anticipated GO categories did not agree (i.e., fasting down regulated in feeding treatments) with previously observed increases in their respective proteins/enzyme activities. These results suggest an 'anticipatory' storage of selected mRNAs which presumably supports the rapid translation of proteins upon feeding activation of the gland.

Osmoregulatory and metabolic costs of salt excretion in the Rufous-collared sparrow Zonotrichia capensis.

Recent experiments on shorebirds have demonstrated that maintaining an active osmoregulatory machinery is energetically expensive. This may, in part, explain diet and habitat selection in birds with salt glands. However little is known about the osmoregulatory costs in birds lacking functional salt glands. In these birds, osmotic work is done almost exclusively by the kidneys. We investigated the osmoregulatory cost in a bird species lacking functional salt glands, the passerine Zonotrichia capensis. After 20 days of acclimation to fresh water (FW) and salt water (200 mM NaCl, SW), SW birds tended to be heavier than FW birds. However, this difference was not statistically significant. Total basal metabolic rate was higher in SW birds as compared with FW birds. Renal and heart masses were also higher in the SW group. We also found greater medullary development and an increase in urine osmolality in the SW group. In spite of Z. capensis' ability to tolerate a moderate salt load in the laboratory, we hypothesize that increased cost of maintenance produced by salt consumption may significantly affect energy budget, dietary, and habitat choices in the field.

Morphological and biochemical evidence for the evolution of salt glands in snakes.

Vertebrate salt glands have evolved independently multiple times, yet there are few hypotheses about the processes underlying the convergent evolution of salt glands across taxa. Here, we compare the morphology and molecular biology of specialized salt-secreting glands from a marine snake (Laticauda semifasciata) with the cephalic glands from semi-marine (Nerodia clarkii clarkii) and freshwater (N. fasciata) watersnakes to look for evidence of a salt gland in the former and to develop hypotheses about the evolution of snake salt glands. Like the salt gland of L. semifasciata, the nasal and anterior/posterior sublingual glands in both species of Nerodia exhibit a compound tubular shape, and express basolateral Na(+)/K(+)-ATPase (NKA) and Na(+)/K(+)/2Cl(-)cotransporter (NKCC); however, the abundance of NKA and NKCC in N. fasciata appears lower than in N. c. clarkii. Aquaporin 3 (AQP3) is also basolateral in the sublingual glands of both species of Nerodia, as is abundant neutral mucin; both AQP3 and mucin are absent from the salt gland in L. semifasciata. Thus, we propose that the evolution of the snake salt gland by co-option of an existing gland involved at least two steps: (i) an increase in the abundance of NKA and NKCC in the basolateral membranes of the secretory epithelia, and (ii) loss of AQP3/mucus secretion from these epithelia.

Renal responses to salinity change in snakes with and without salt glands.

To understand renal responses to salinity change in aquatic reptiles, we examined the structure and function of the kidney in three species of snake: a marine species with a salt gland (Laticauda semifasciata), a marine species without a salt gland (Nerodia clarkii clarkii) and a freshwater species without a salt gland (Nerodia fasciata). Both marine species maintained relatively constant plasma ions, even after acclimation to saltwater. By contrast, both plasma Cl(-) and mortality increased with salinity in the freshwater species. To investigate putative renal ion regulatory mechanisms, we examined the distribution and abundance of Na(+)/K(+)-ATPase (NKA) and the Na(+)/K(+)/2Cl(-) cotransporter (NKCC2). In all species, NKA localized to the basolateral membranes of the distal tubule and the connecting segments and collecting ducts only; there was no effect of salinity on the distribution of NKA or on the abundance of NKA mRNA in any species. NKCC2 protein was undetectable in the kidney of any of the species and there was no effect of salinity on NKCC2 mRNA abundance. We also examined the distribution and abundance of aquaporin 3 (AQP3) in the kidney of these species; although putative AQP3 localized to the basolateral membranes of the connecting segments and collecting ducts of all three species, there was no effect of salinity on the localization of the protein or the abundance of the transcript. Interestingly, we found very few differences across species, suggesting that the snake kidney may play a trivial role in limiting habitat use.

Secretion by the nasal salt glands of two insectivorous lizard species is initiated by an ecologically relevant dietary ion, chloride.

Salt glands are used by some vertebrates to excrete hyperosmotic NaCl or KCl solutions in response to dietary salt loads. Control of secretion varies across taxa; some secrete in response to osmotic challenges while others secrete in response to specific dietary ions. We hypothesized that differences in control could be related to different diet-related selective pressures on herbivorous, marine, and insectivorous species. We studied control of secretion and flexibility of cation (sodium or potassium) and anion (chloride or bicarbonate) secretion in two insectivorous lizard species, Schneider's skinks (Eumeces schneideri, Scincidae) and green anoles (Anolis carolinensis, Polychrotidae). Lizards were injected daily for four days with combinations of cations (potassium, sodium, and histidine control) and anions (chloride and acetate control), isoosmotic saline, or sham injection. Secretions were collected daily and analyzed for sodium, potassium, and chloride. Both species secreted only in response to chloride; sodium appeared to have a slight inhibitory effect. Regardless of cation load, skinks secreted a combination of potassium and sodium, while anoles secreted solely potassium. In both species, total cation secretion was matched closely by chloride; very little bicarbonate was secreted. As predicted, secretion in insectivorous lizards was initiated by the dietary ion ecologically most important for these species, chloride, which otherwise cannot be excreted without significant water loss (unlike the cations, which may be excreted as insoluble urate salts). This gives further support to the hypothesis that ecological factors drive the evolution of control mechanisms in lizard salt glands.