Lingual Salt Glands Research Articles

Salt-water acclimation of the estuarine crocodile Crocodylus porosus involves enhanced ion transport properties of the urodaeum and rectum.

Estuarine crocodiles, Crocodylus porosus, inhabit freshwater, estuarine and marine environments. Despite being known to undertake extensive movements throughout and between hypo-osmotic and hyperosmotic environments, little is known about the role of the cloaca in coping with changes in salinity. We report here that, in addition to the well-documented functional plasticity of the lingual salt glands, the middle of the three cloacal segments (i.e. the urodaeum) responds to increased ambient salinity to enhance solute-coupled water absorption. This post-renal modification of urine serves to conserve water when exposed to hyperosmotic environments and, in conjunction with lingual salt gland secretions, enables C. porosus to maintain salt and water balance and thereby thrive in hyperosmotic environments. Isolated epithelia from the urodaeum of 70% seawater-acclimated C. porosus had a strongly enhanced short-circuit current (an indicator of active ion transport) compared with freshwater-acclimated crocodiles. This enhanced active ion absorption was driven by increased Na+/K+-ATPase activity, and possibly enhanced proton pump activity, and was facilitated by the apical epithelial Na+ channel (ENaC) and/or the apical Na+/H+ exchanger (NHE2), both of which are expressed in the urodaeum. NHE3 was expressed at very low levels in the urodaeum and probably does not contribute to solute-coupled water absorption in this cloacal segment. As C. porosus does not appear to drink water of salinities above 18ppt, observations of elevated short-circuit current in the rectum as well as a trend for increased NHE2 expression in the oesophagus, the anterior intestine and the rectum suggest that dietary salt intake may stimulate salt and possibly water absorption by the gastrointestinal tract of C. porosus living in hyperosmotic environments.

Activity, abundance, distribution and expression of Na+/K+-ATPase in the salt glands ofCrocodylus porosusfollowing chronic saltwater acclimation

Saltwater crocodiles, Crocodylus porosus, possess lingual salt glands which function to remove excess Na(+) and Cl(-) accumulated as a consequence of living in salt water. Little is known about the nature of ion transport systems in C. porosus salt glands and how these systems respond to an osmotic challenge. In the present study, we examined the distribution and regulation of the Na(+)/K(+)-ATPase (NKA) pump, specifically the alpha-(catalytic) subunit in the salt glands of C. porosus chronically acclimated (6 months) to freshwater (FW) or 70% seawater (SW). We hypothesised that in the SW-acclimated C. porosus there would be an up-regulation of the abundance, activity and gene expression of the NKA transporter. NKA was immunolocalised to the lateral and basal membrane of secretory cells. As predicted, the NKA alpha-subunit was 2-fold more abundant in SW-acclimated C. porosus salt glands. NKA gene expression was also elevated in the salt glands of SW- vs FW-acclimated crocodiles. There was no increase in the specific activity of NKA in SW-acclimated animals and the in vitro rate of oxygen consumption by salt gland slices from SW-acclimated animals was not significantly different from that of FW-acclimated animals. The proportion of tissue oxygen consumption rate attributable to NKA activity was not different between SW- and FW-acclimated animals (approximately 50%). These data suggest that either chronic SW acclimation does not affect NKA in crocodile salt glands in the same manner as seen in other models or crocodiles possess the capacity to moderate NKA activity following prolonged exposure to SW.

Hormone-dependent dissociation of blood flow and secretion rate in the lingual salt glands of the estuarine crocodile, Crocodylus porosus

Salt and water balance in the estuarine crocodile, Crocodylus porosus, involves the coordinated action of both renal and extra-renal tissues. The highly vascularised, lingual salt glands of C. porosus excrete a concentrated sodium chloride solution. In the present study, we examined the in vivo actions of vasoactive intestinal peptide (VIP), B-type natriuretic peptide (BNP) and angiotensin II (ANG II) on the secretion rate and blood perfusion of the lingual salt glands. These peptides were selected for their vasoactive properties in addition to their reported actions on salt gland activity in birds and turtles and rectal gland activity in elasmobranchs. The femoral artery was cannulated in seven juvenile crocodiles for delivery of peptides and measurement of mean blood pressure and heart rate. In addition, secretion rate of, and blood flow to, the salt glands were recorded simultaneously using laser Doppler flowmetry. VIP stimulated salt secretion was coupled to an increase in blood flow and vascular conductance of the lingual salt glands. BNP was a potent stimulant of salt gland secretion, resulting in a maximal secretion rate of more than 15-fold higher than baseline; however, this was not coupled to an increase in perfusion rate, which remained unchanged. ANG II failed to stimulate salt gland secretion and there was a transient decrease in salt gland blood flow and vascular conductance. It is evident from this study that blood flow to, and secretion rate from, the lingual salt glands of C. porosus are regulated independently; indeed, it is apparent that maximal secretion from the salt glands may not require maximal blood flow.

The effects of saltwater acclimation on Na–K–2Cl and Na–K-ATPase distribution, activity and gene expression in salt glands of the estuarine crocodile

The estuarine crocodile, Crocodylus porosus utilises lingual salt glands to excrete excess NaCl accumulated whilst living in saline environments. In avian salt gland models, Na–K-ATPase (NKA) is essential for the maintenance of the electrochemical gradients which facilitate the secretory activity of other ion pumps, while Na–K–2Cl (NKCC) functions to pump Na+ and K+ into the paracellular space, in exchange for Cl− which is transported into the cell. We have investigated the effects of chronic saltwater acclimation on the distribution, activity and gene expression patterns of both NKA and NKCC in the crocodilian salt gland. Using immunohistochemistry, both NKA and NKCC were localized to the basolateral membranes of salt gland secretory cells in both freshwater (fw) and saltwater (sw) acclimated salt gland tissues. The intensity of staining was slightly, but significantly, higher in tissues from SW-acclimated animals. Western blotting indicated that there was an increase in NKA and NKCC abundances in SW-acclimated tissues. The relative metabolic costs associated with these two pumps were investigated in vitro. While there were no differences between fw- and sw-acclimated tissues in the relative costs associated with NKA activity, NKCC activity was almost 50% higher in SW-acclimated tissues. We are currently exploring patterns of NKA and NKCC gene expression in sw- and fw-acclimated animals. This study has demonstrated the presence of NKA and NKCC in the salt glands of C. porosus and has shown that the activity and abundance of these transporters can be influenced by prolonged saltwater acclimation. Copyright © 2008 Published by Elsevier Inc.

Functional and morphological plasticity of crocodile (Crocodylus porosus) salt glands

The estuarine crocodile, Crocodylus porosus, inhabits both freshwater and hypersaline waterways and maintains ionic homeostasis by excreting excess sodium and chloride ions via lingual salt glands. In the present study, we sought to investigate the phenotypic plasticity, both morphological and functional, in the lingual salt glands of the estuarine crocodile associated with chronic exposure to freshwater (FW) and saltwater (SW) environments. Examination of haematological parameters indicated that there were no long-term disruptions to ionic homeostasis with prolonged exposure to SW. Maximal secretory rates from the salt glands of SW-acclimated animals (100.8+/-14.7 micromol 100 g(-0.7) body mass h(-1)) were almost three times greater than those of FW-acclimated animals (31.6+/-6.2 micromol 100 g(-0.7) body mass h(-1)). There were no differences in the mass-specific metabolic rate of salt gland tissue slices from FW- and SW-acclimated animals (558.9+/-49.6 and 527.3+/-142.8 microl O(2) g(-1) h(-1), respectively). Stimulation of the tissue slices from SW-acclimated animals by methacholine resulted in a 33% increase in oxygen consumption rate. There was no significant increase in the metabolic rate of tissues from FW-acclimated animals in response to methacholine. Morphologically, the secretory cells from the salt glands of SW-acclimated animals were larger than those of FW-acclimated animals. In addition, there were significantly more mitochondria per unit volume in secretory tissue from SW-acclimated animals. The results from this study demonstrate that the salt glands of C. porosus are phenotypically plastic, both morphologically and functionally and acclimate to changes in environmental salinity.

Cholinergic and adrenergic innervation of lingual salt glands of the estuarine crocodile, Crocodylus porosus

Many marine reptiles and birds possess extrarenal salt glands that facilitate the excretion of excess sodium and chloride ions accumulated as a consequence of living in saline environments. Control of the secretory activity of avian salt glands is under neural control, but little information is available on the control of reptilian salt glands. Innervation of the lingual salt glands of the salt water crocodile, Crocodylus porosus, was examined in salt water-acclimated animals using histological methods. Extensive networks of both cholinergic and adrenergic nerve fibres were identified close to salt-secreting lobules and vasculature. The identification of both catecholamine-containing and cholinergic neurons in the salt gland epithelium and close to major blood vessels in the tissue suggests the action of the neurotransmitters on the salt-secreting epithelium itself and the rich vascular network of the lingual salt glands.

Osmoregulatory mechanisms of the Australian freshwater crocodile, Crocodylus johnstoni , in freshwater and estuarine habitats

The estuary of the Limmen Bight River in Australia's Northern Territory is home to an unusual salt water-adapted population of the Australian `freshwater' crocodile, Crocodylus johnstoni. Crocodiles were captured from tidal reaches of the estuary ranging in salinity from 0.5–24‰ and from several permanent fresh water reaches more or less remote from saline waters. C. johnstoni is an effective osmoregulator in moderately saline waters and has osmoregulatory mechanisms very similar to its more marine-adapted relative, the estuarine crocodile Crocodylus porosus. Fasted C. johnstoni in brackish water appear to lose little sodium in cloacal urine, relying on their lingual salt glands for excretion of excess sodium chloride. The lingual glands show clear evidence of short-term and long-term acclimation to salt water. Like estuarine crocodiles, C. johnstoni drinks fresh water and will not drink sea water. Gross sodium and water fluxes in brackish water are very similar to those in other crocodilians, suggesting differences in integumental permeability are not a major influence on osmoregulatory differences between crocodilians. The data reinforce the hypothesis that crocodylids differ fundamentally from alligatorids in the structure and function of the renal-cloacal-salt gland complex and are of interest in current debate over the evolutionary and zoogeographical history of the eusuchian crocodilians.

Osmoregulation by the broad-snouted caiman, Caiman latirostris , in estuarine habitat in southern Brazil

The broad-snouted caiman Caiman latirostris, of South America mostly frequents freshwater but occurs also in estuaries. Nothing of substance is known of its osmoregulatory physiology but, in the light of accumulating evidence that alligatorids lack specialised adaptations for life in hyperosmotic waters, we anticipated its physiology would be more similar to that of Alligator mississippiensis than the euryhaline Crocodylus porosus, which has both lingual salt glands and a more complex renal:cloacal system. This proved to be the case. Caiman captured in estuaries of the Ilha do Cardoso in southern Brazil were effective hypo-osmotic osmoregulators in salinities of 0–24 ppt (seawater = 35 ppt). Plasma osmolarity, sodium and chloride were similar to those in other crocodilians and not influenced by salinity. Plasma urea was low and did not vary with salinity. We found no evidence of lingual or other salt glands. Urinary electrolyte concentrations varied considerably with salinity and in ways reminiscent of A. mississippiensis but very different from C. porosus.

Kidney and Cloaca Function in the Estuarine Crocodile ( Crocodylus porosus) at Different Salinities: Evidence for Solute-linked Water Uptake

Kidney function and the role of the cloacal complex in osmoregulation was investigated in estuarine crocodile ( Crocodylus porosus) exposed to three environmental salinities: hypo-, iso- and hyperosmotic to the plasma. Plasma homeostasis was maintained over the range of salinities. Antidiuresis occurred with increased salinity. Although urine from the kidneys retained an osmotic pressure between 77% and 82% of the plasma, over 93% and 98% of plasma chloride filtered at the glomeruli was reabsorbed during passage through the kidneys under hypo- and hyperosmotic conditions, respectively, and only 64% in iso-osmotic water. The kidneys were the primary site of sodium reabsorption under hypo- and hyperosmotic conditions. Secondary processing of urine during storage in the cloaca varied with salinity. During post-renal storage of urine, the difference in urine osmotic pressure increased from −26.1 ± 15.5 to 35.66 ± 9.29 mOsM with increased salinity, and potassium concentration of urine increased over 3-fold in C. porosus from freshwater. The almost complete reabsorption of both sodium and chloride under hyperosmotic conditions indicates the necessity for secretory activity by the lingual salt glands. The osmoregulatory response of the kidneys and cloacal complex to environmental salinity is both plastic and complementary.

Differences in renal-cloacal function between Crocodylus porosus and Alligator mississippiensis have implications for crocodilian evolution

Major electrolytes and nitrogenous excretory products were analysed in the blood plasma, ureteral urine and cloacal urine of juvenile Alligator mississippiensis and Crocodylus porosus in fresh and hypoosmotic salt water (206 mosmol · l−1). Both species coped well with saline water, showing little (Alligator) or no (Crocodylus) change in plasma composition. Comparisons of renal-cloacal function point to major differences in their osmoregulatory physiology. The cloaca of C. porosus is a very active osmoregulatory organ in salt and fresh water, contributing to water conservation and NaCl excretion through the lingual salt glands. In contrast, the cloaca of Alligator has little impact on the composition of excreted urine. It seems likely that A.␣mississippiensis is largely constrained to a renal response to osmotic and ionic stress while C. porosus is able to call on a more complex mix of renal response, post-renal modification of urine in the cloaca, and excretion of excess NaCl through the salt glands. The results support the idea that there are deep-seated differences in the osmoregulatory physiology of alligatorids and crocodylids (Eusuchia), an understanding of which should provide valuable insights into their evolution and zoogeography.

A Preliminary Investigation of the Effects of Vasoactive Intestinal Peptide on Secretion from the Lingual Salt Glands ofCrocodylus porosus

Vasoactive intestinal peptide (VIP)-like immunoreactivity was demonstrated in the lingual salt glands of the estuarine crocodile,Crocodylus porosus.Varicose fibers showing VIP-like immunoreactivity ramified the salt glands, forming a dense network around the basal region of the exocrine cells. Secretions from the lingual salt glands were monitored in hatchlingC. porosus.Spontaneous secretory activity was variable, ranging from 0.3 to 5.3 μmol Na 100 g−0.7bm hr−1(bm, body mass). Administration of a 100-μl bolus of 0.9% NaCl, via a cannulated femoral vein, did not affect the spontaneous secretory rate. However, injection of 100 pmol porcine VIP resulted in a massive increase in secretory activity, reaching a maximum of 58.2 μmol of Na 100 g−0.7bm hr−1. The presence of VIP-like immunoreactivity and the positive secretory effect of administered VIP indicate a potential action of VIP on salt gland activity.

Salt loading stimulates secretion by the lingual salt glands in unrestrained Crocodylus porosus

AbstractIn previous studies, the lingual salt glands of crocodiles have failed to secrete in response to a salt load. This is unlike salt glands in other reptiles and in birds, and it has led to some doubt about the extent to which the salt glands in crocodiles are of functional importance.Believing that the failure to secrete may be associated with the artificial restraint associated with measurement, or with the removal of study animals from the water, we designed and built an electronic sensor which would monitor secretions from the salt glands of crocodiles while they were in water and undisturbed.Under these conditions, the salt glands were observed to secrete spontaneously. Under salt loading, delivered via a femoral vein cannula, there was a rapid production of secretion from the glands. Secretion was stimulated by methacholine and acetylcholine chlorides also. The results support the view that the lingual salt glands in crocodilians are stimulated by a salt load and that previous attempts to demonstrate this may have been influenced by the experimental conditions. © 1995 Wiley‐Liss, Inc.

Increased vascularity of the lingual salt glands of the estuarine crocodile, Crocodylus porosus, kept in hyperosmotic salinity.

Methyl methacrylate corrosion casts were made of the blood-vascular system of the lingual salt glands of the estuarine crocodile, Crocodylus porosus, and examined with light and scanning electron microscopy. The 28-40 individual salt glands, each opening separately via a single pore onto the dorsal surface of the tongue, are supplied by a pair of lingual arteries. Each gland is richly vascularized and is composed of 14-20 lobular sub-units, each having a dense network of capillaries. The blood flow in each gland is from the centre to its periphery, opposite to the direction of the flow of secretions in the ducts of the gland. The main collecting duct leading from the gland to the external pore was well vascularized. The blood supply to the glands of juvenile crocodiles raised in 20‰ salt water was more dense than in freshwater and, from cast masses, had a three-fold greater vascular volume. This study provides the first evidence which shows that the salt glands of crocodiles are morphologically labile and can adapt to the environmental salinity. © 1993 Wiley-Liss, Inc.

Osmoregulation in Crocodilians

Recent crocodilians live primarily in freshwater habitats. However two species ( Crocodylus acutus and C. porosus ) are estuarine specialists; two others ( C. niloticus and C. johnstoni ) that are primarily found in fresh water, have estuarine populations. Routes of uptake of water and sodium include drinking, feeding and associated incidental drinking, and integumental and buccal diffusion. Routes of loss include faeces-cloacal fluid, lingual salt glands, integumental and buccal diffusion, and respiratory loss. The least understood route of salt and water exchange is that of the oral and buccal epithelia, which are much more permeable to water and sodium than the general integument. The freshwater alligator ( Alligator mississippiensis ) osmoregulates in a manner typical for an amphibious reptile. Body sodium turnover is low and the general integument is quite low in permeability to sodium. Water turnover is more rapid (in terms of molar exchange) but still relatively low for an aquatic reptile. Most water exchange occurs across the integument and buccal epithelia. The presence of lingual salt glands in freshwater crocodilians remains enigmatic, as does the failure of these exocrine glands in estuarine species to respond to saline loading. Secretion does occur after injection of the parasympathetic stimulant methacholine chloride. The “salt water crocodile” ( C. porosus ) possesses a suite of osmoregulatory adaptations similar to those found in other estuarine reptiles. Water and sodium balance are maintained primarily by an extremely low general permeability to sodium, by economies in water loss, and by excretion of excess sodium by the lingual salt glands. Further work is needed to examine newly hatched C. porosus , and the possibility of ontogenetic change in lingual gland function in C. acutus. The importance of incidental drinking of sea water during feeding (recently discovered in turtles) needs to be evaluated in crocodilians. The use of osmoregulatory data in interpretation of the evolutionary history of the genus Crocodylus needs to be viewed with caution. The hypothesis that all species of Crocodylus originated from the transoceanic migration of a saline-tolerant form may not be the most parsimonious explanation.

Nile crocodiles, Crocodylus niloticus, and estuarine crocodiles, Crocodylus porosus, show similar osmoregulatory responses on exposure to seawater

1. 1. Nile crocodiles, reared in fresh water and exposed acutely to sea water, suffer marked dehydration and hypernatraemia. Cloacal urine osmolarity and potassium concentration increase markedly but urine sodium remains low. 2. 2. Hypernatraemia is increased when secretion from the lingual salt glands is prevented. 3. 3. C. niloticus appears not to drink seawater. 4. 4. Similarities in osmoregulatory response between estuarine and Nile crocodiles suggest that the lingual salt glands of C. niloticus are functional in salt water, playing an important role in sodium balance. 5. 5. Significant differences in the function of the renal/cloacal complex of Alligator and Crocodylus emphasize further the differences between these two groups of crocodilian and provide support for the postulated marine ancestry of many or all of the Crocodylidae.

Sodium and water budgets of the fasted estuarine crocodile,Crocodylus porosus, in sea water

1. Quantitative sodium and water budgets have been constructed for the estuarine crocodile,Crocodylus porosus, unfed in sea water in the laboratory.C. porosus is homeostatic for exchangeable Na in sea water but is unable to achieve homeostasis of plasma Na concentration or total body water when fasted. 2. The lingual salt glands account for 55% of the total Na efflux compared with only 2% excreted by the renal/cloacal complex. The urinary system appears to be the principal route for excretion of K. 3. The integument is essentially impermeable to Na but permeable to water, as in other estuarine/marine reptiles. Much of the Na and water exchange across the skin appears to take place across cephalic epithelia, particularly the buccal mocosa. 4. The permeable buccal mucosa is costly in terms of direct water loss and indirect losses tied to excretion of excess Na. Lowering of buccal permeability may be limited by the necessity to retain some functional glandular tissue and sensory organs in the mouth. 5. There is no evidence to suggest thatC. porosus feeds selectively on invertebrate prey of low Na or high water content despite the apparent selective advantage of minimizing water losses associated with extra-renal Na excretion.

Adaptations of Crocodylus acutus and Alligator for life in saline water

1. 1. American crocodiles ( C. acutus) weighing less than 200 g are unable to grow when kept in 35 ppt sea water in the laboratory. Yet paradoxically there are some highly saline areas in south Florida where rapid growth occurs. It is possible that these conflicting observations can be reconciled by behavioral osmoregulation of young crocodiles. 2. 2. Hatching occurs during the rainy season and small crocodiles may drink from the brackish “lens” available during and after rainfall. 3. 3. Using a weekly regime of alternating exposure to 35 ppt (6 days) and 4 ppt (12–24 hr), it has been demonstrated that growth of small crocodiles occurs. Feeding takes place primarily when brackish water is available. Salinities as high as 18 ppt were drunk when crocodiles were dehydrated by 15–20% of initial mass. 4. 4. C. acutus and Alligator have a rather low rate of water efflux in sea water (0.2ml/100g-hr). 5. 5. Sodium influx in sea water of C. acutus is low, but higher than efflux. Thus there is no evidence yet for a significant role of the lingual salt glands in sodium excretion. 6. 6. The major adaptations to saline water of hatchling C. acutus are a low intake of sodium, an ability to selectively drink water of lower salinities, and to grow very rapidly (within 3–4 months) to a size much more tolerant of immersion in 35 ppt sea water.

Lingual salt glands inCrocodylus acutus andC. johnstoni and their absence fromAlligator mississipiensis andCaiman crocodilus

1. Lingual salt glands, secreting hyperosmotic Na/K solutions in response to methacholine, are present inCrocodylus acutus andC. johnstoni but apparently absent from the alligatorids,Alligator mississipiensis andCaiman crocodilus. 2. Both secretory rates (6–20 μmol/100 g·h) and concentrations (450–600 mM Na) of glandular secretions are essentially identical in the marine/estuarineC. acutus andC. porosus and significantly higher than in the freshwaterC. johnstoni (1–2 μmol/100 g·h; 320–420 mM Na). 3. Lingual glands inAlligator secrete isosmotic Na/K at low rates (1–2 μmol/100 g·h) while those ofCaiman show no response to methacholine. 4. The physiological contrast between alligatorids and crocodylids is reflected in distinct differences in the superficial appearance of the tongue and lingual pores. 5. It is postulated that the alligatorid condition of low secretory capacity and isosmotic secretion reflects the primitive salivary function of lingual glands from which the salt-secreting capability in crocodylids was derived.

Plasma homeostasis and cloacal urine composition inCrocodylus porosus caught along a salinity gradient

Juveniles of the Estuarine or Saltwater Crocodile,Crocodylus porosus, maintain both osmotic pressure and plasma electrolyte homeostasis along a salinity gradient from fresh water to the sea. In fresh water (FW) the cloacal urine is a clear solution rich in ammonium and bicarbonate and containing small amounts of white precipitated solids with high concentrations of calcium and magnesium. In salt water (SW) the cloacal urine has a much higher proportion of solids, cream rather than white in colour, which are the major route for excretion of potassium in addition to calcium and magnesium. Neither liquid nor solid fractions of the cloacal urine represent a major route for excretion of sodium chloride. The solids are urates and uric acid, and their production probably constitutes an important strategy for water conservation byC. porosus in SW. These data, coupled with natural history observations and the recent identification of lingual salt glands, contribute to the conclusion thatC. porosus is able to live and breed in either fresh or salt water and may be as euryhaline as any reptile.

Electrolyte excretion by the salt gland of the Galapagos marine iguana

Electrolyte excretion by the salt gland of the Galapagos marine iguana