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. Accepted: 25 February 1999     

Osmoregulation of the Nile crocodile, Crocodylus niloticus, in Lake St. Lucia, Kwazulu/Natal, South Africa

Nile crocodiles of three age classes, hatched in captivity and reared in fresh water, when exposed acutely to water of 17 and 35 ppt NaCl, suffered marked dehydration, were lethargic, ceased to feed and lost mass. When exposed to gradually increasing salinities (3-35 ppt), with a short acclimation period at each salinity, crocodiles survived, continued to feed and increased in mass and size. All age classes had a relatively constant plasma osmolality across the salinity spectrum. Cloacal urine osmolality varied throughout the acclimation experiment, but did not increase with increasing salinity. No significant increase was found in plasma concentrations of any of the osmolytes. There was a trend of decreasing cloacal urine [Na(+)] and [Cl(-)] and increasing cloacal urine [K(+)] with increased salinity, indicating that urine was not an important route for Na(+) and Cl(-) excretion. Crocodiles exposed to saline conditions maintained relatively constant plasma uric acid concentrations, but urinary uric acid concentrations increased markedly with increasing salinities. This suggests that uric acid is the main constituent of nitrogenous waste excretion in saline exposed Nile crocodiles. As in Crocodylus porosus, C.niloticus has the physiological ability to survive and thrive in periodically hyper-osmotic environments. However, its euryhalinity is restricted, in that acute exposure to sea water leads to dehydration, but with an acclimation period at lower salinities, it survives and thrives in sea water.     

Effects of recombinant human growth hormone in juvenile Nile crocodiles (Crocodylus niloticus)

1. Recombinant human growth hormone (hGH) showed somatotropic activity in juvenile Nile crocodiles (Crocodylus niloticus). 2. Body weight of crocodiles receiving 3.25 micrograms hGH/g body weight twice a week was increased by 49% after five weeks of treatment, compared to 31% increase in controls. 3. Total length was increased by 15 and 5%, respectively, in the two groups. 4. Food conversion efficiency increased from 28% in the controls to 36% in the hormone injected animals. 5. Cessation of hormone treatment was followed by reduced appetite and decreasing body growth.     

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.     

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.     

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.

     

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. Accepted: 7 September 1996     

Biotelemetry monitoring of translocated Crocodylus niloticus in Lake Ngezi, Zimbabwe

Radiotelemetry was used from June 1986 through February 1987 to observe the movements of four Nile crocodiles, Crocodylus niloticus Laurenti, introduced into a resident population at Lake Ngezi, Zimbabwe. The use of surgically-implanted transmitters was the first application of this technique on wild Nile crocodiles. A subadult female (194 cm TL, 35 kg) and two males (256 cm, 81 kg; 348 cm, 260 kg) wandered throughout the area. A large female (297 cm, 149 kg) moved > 12 km from her point of release to Ngezi River and nested; she guarded the nest site through 21 January 1987 when she moved the hatchlings to a nearby creche. A juvenile crocodile (120 cm TL, 8 kg) captured in Lake Ngezi was also monitored from June 1986 to February 1987. It traversed some 15 km of the shoreline of Lake Ngezi and moved as much as 10 km overnight. Evidence is presented that the translocated crocodiles, excluding the nesting female, exhibited exploratory behaviour throughout the nine-month observation period, and did not establish home ranges per se.     

Nest opening response of the Nile crocodile Crocodylus niloticus

Observations on captive Nile crocodiles, Crocodylus niloticus confirm earlier reports on parental care of nests and of young. Male and female appear to form a pair bond after courtship, the male defending nesting territory with the female rarely leaving the nest until the sounds of the young hatching stimulate her to open the nest to release them. The female transports unhatched eggs and live young in her buccal pouch to water and establishes a nursery where the young are defended for several weeks. Experiments involving playback of distress calls by tape recorder, offering of live young and eggs to the adults, the parent opening an artificial nest and vocalisations and resultant interactions between adults and young are described.     

A phylogenetic hypothesis for Crocodylus (Crocodylia) based on mitochondrial DNA: evidence for a trans-Atlantic voyage from Africa to the New World

The phylogenetic relationships among extant species of Crocodylus (Crocodylia) have been inconsistently resolved by previous systematic studies. Here we used nearly complete mitochondrial (mt) genomes (～16,200 base pairs) for all described Crocodylus species, eight of which are new to this study, to derive a generally well-supported phylogenetic hypothesis for the genus. Model-based analyses support monophyly of all Asian+Australian species and paraphyly of Crocodylus niloticus (Nile crocodile) with a monophyletic New World clade nested within this species. Wild-caught Nile crocodiles from eastern populations group robustly with the four New World species to the exclusion of Nile crocodiles from western populations, a result that is also favored by parsimony analyses and by various subpartitions of the overall mt dataset. The fossil record of Crocodylus extends back only to the Late Miocene, while the earliest fossils assigned to C. niloticus and to New World Crocodylus are Pliocene. Therefore, in combination with paleontological evidence, mt DNA trees imply a relatively recent migration of Crocodylus from Africa to the Americas, a voyage that would have covered hundreds of miles at sea.     

Incubation temperatures, sex ratios and sex determination in a population of Nile crocodiles (Crocodylus niloticus)

A demographic study of the Nile crocodile Crocodylus niloticus at Lake Ngezi, Zimbabwe, revealed that females predominated in all size classes and among embryos. The sex of C. niloticus was shown to be determined by the temperature of egg incubation in constant temperature laboratory experiments. At 31 °C and below only females were produced. The threshold temperature for maleness was between 31 ° and 34 °C, but appeared to vary between clutches. The duration of the incubation period varied with temperature and was 110 days at 28 °C, falling to 85 days at 34 °C. Incubation temperature affected hatchling length, but not mass. Hatchlings from incubation at 34 °C were shorter on average than those from incubation at 28 °C and 31 °C, but by three months had outgrown them. There was no sex-related difference in length in a random sample of 200 two-year-old C. niloticus on a crocodile farm. Mean temperatures in wild nests were consistently lower than 31 °C and therefore the male threshold as determined in the laboratory. Embryonic development was slow and hatching success poor. The shallowest eggs in a nest had higher mean temperatures and more advanced embryos than the deepest eggs. They also experienced daily temperature fluctuations of up to 10 °C during which the maximum occasionally rose to 35 °C. Constant temperature incubation was not a good model of field conditions, but the correlation between nest temperatures and embryonic sex is consistent with temperature-dependent sex determination in the wild.     

Thermal relations of large crocodiles,Crocodylus porosus,free-ranging in a naturalistic situation

We monitored behaviour and environmental and body temperatures (T_b) in summer and winter in 11 salt-water crocodiles (Crocodylus porosus), of body mass 32 to 1010kg, free-ranging in naturalistic captivity in northern Australia. We found pronounced daily cycles in air and water temperatures in both winter (16 to 33 degrees C and 20 to 31degrees C, respectively) and summer (21 to 45 degrees C and 24 to 36 degrees C, respectively). In winter, crocodiles exposed themselves to the sun during the day and stayed in the water at night. In summer, they remained in the water during the day and emerged onto land at night. Body temperature showed a daily cycle the amplitude of which decreased with increasing mass, from 3.5 degrees C (mass 32kg) to 1.0 degrees C (660kg) in summer, and from 3.5 degrees C (42kg) to 1.4 degrees C (1010kg) in winter. Underlying the daily cycles in T_b were intermediate (10 to 13 day, tidal?) and seasonal cycles. Overall, values of modal T_b ranged from 25.1 to 28.7 degrees C in winter and from 28.4 to 33.6 degrees C in summer, trending upwards with body size. This pattern of continuous oscillations in T_b, with no daily plateau, is conspicuously different from that seen in crocodilians of small sizes and from the pattern usually regarded as typical of reptiles in general.     

Hematologic and plasma biochemical reference intervals for Morelet's crocodiles (Crocodylus moreletii) in the northern wetlands of Campeche, Mexico

Health surveys and hematologic and plasma biochemical analyses were conducted in 52 free-ranging and 51 captive Morelet's crocodiles (Crocodylus moreletii) in Campeche, Mexico, March-September 2007. Blood samples from 92 crocodiles (45 free-ranging and 47 captive) were collected for hematologic and plasma biochemical analyses. Average values of erythrocytes of free-ranging crocodiles were 1,046,166 cells/μl, and total white cells were 1.03 × 10(4) cells/μl. Captive crocodiles had erythrocyte and leukocyte values of 1,100,416 cells/μl and 8.51 × 10(3) cells/μl, respectively. There were no significant differences in values of erythrocytes or in hematocrit between free-ranging and captive crocodiles, or between sexes, or among size classes. Counts of leukocytes in free-ranging crocodiles were significantly higher than in captive individuals. The mean values of plasma analytes were 69.55 mg/l (glucose), 250.14 mg/l (cholesterol), 3.04 mg/l (uric acid), 2.70 mg/l (creatinine), and 20.20 IU/l (alanine aminotransferase). There were significant differences in cholesterol between free-ranging and captive crocodiles and between sexes.     

Regulation of salt gland, gut and kidney interactions

Marine birds can drink seawater because their cephalic 'salt' glands secrete a sodium chloride (NaCl) solution more concentrated than seawater. Salt gland secretion generates osmotically free water that sustains their other physiological processes. Acclimation to saline induces interstitial water and Na move into cells. When the bird drinks seawater, Na enters the plasma from the gut and plasma osmolality (Osm(pl)) increases. This induces water to move out cells expanding the extracellular fluid volume (ECFV). Both increases in Osm(pl) and ECFV stimulate salt gland secretion. The augmented intracellular fluid content should allow more rapid expansion of ECFV in response to elevated Osm(pl) and facilitate activation of salt gland secretion. To fully utilize the potential of the salt glands, intestinally absorbed NaCl must be reabsorbed by the kidneys. Thus, Na uptake at gut and renal levels may constrain extrarenal NaCl secretion. High NaCl intake elevates plasma aldosterone concentration of Pekin ducks and aldosterone stimulates intestinal and renal water and sodium uptake. High NaCl intake induces lengthening of the small intestine of adult Mallards, especially males. High NaCl intake has little effect on glomerular filtration rate or tubular sodium Na uptake of birds with competent salt glands. Relative to body mass, kidney mass and glomerular filtration rate (GFR) are greater in birds with salt glands than in birds that do not have them. Birds with salt glands do not change GFR, when they drink saline. Thus, their renal filtrate contains excess Na that is, in some species, almost completely renally reabsorbed and excreted in a more concentrated salt gland secretion. Na reabsorption by kidneys of other species, like mallards is less complete and their salt glands make less concentrated secretion. Such species may reflux urine into the hindgut, where additional Na may also be reabsorbed for extrarenal secretion. During exposure to saline, marine birds maintain elevated aldosterone levels despite high Na intake. Marine birds are excellent examples of physiological plasticity.     

Exercise training enhances aerobic capacity in juvenile estuarine crocodiles (Crocodylus porosus)

Aerobic capacity (VO2max) of endothermic vertebrates is known to increase with exercise training, but this effect has not been found to-date in non-avian reptiles. We exercised juvenile estuarine crocodiles (Crocodylus porosus) to walk at 0.75-0.88 km/h on a treadmill for up to 20 min a day over 16 weeks, and compared their aerobic performance with that of unexercised crocodiles. In the exercised group, VO2max increased from 6.9 to 8.5 mLO2/kg/min (+28%), and locomotor endurance increased from 3.8 to 6.9 min (+82%). Neither VO2max nor endurance changed significantly in the sedentary group. This finding extends the exercise training effect onto another vertebrate clade, and demonstrates that ectothermic amniotes are capable of elevating their aerobic capacity in response to exercise training. We propose that differences in cardiopulmonary structure and function in non-avian reptiles may be responsible for the absence (in squamates) or presence (in crocodilians) of a strong training effect on aerobic capacity.     

Evolutionary history of Cuban crocodiles Crocodylus rhombifer and Crocodylus acutus inferred from multilocus markers

Among crocodilians, Crocodylus rhombifer is one of the world's most endangered species with the smallest natural distribution. In Cuba, this endemic species coexists with the American crocodile (Crocodylus acutus). Hybridization between these two species is well known in captivity and might occur in the wild, but has never been demonstrated genetically. Here, we combined molecular data with environmental, geographic, and fossil data to infer the evolutionary history of Crocodylus in the Cuban Archipelago, and to evaluate genealogical support for species boundaries. We analyzed seven microsatellite loci plus DNA sequence data from nuclear (RAG-1) and mitochondrial (cytochrome b and cytochrome oxidase I) genes from 89 wild-caught individuals in Cuba, Grand Cayman Island, Jamaica, and Central America, and two samples from zoo collections. Microsatellites showed evidence of introgression, suggesting potential hybridization among Cuban groups. In Cuba, C. acutus contained one mitochondrial DNA (mtDNA) haplotype, whereas C. rhombifer contained two haplotypes. MtDNA data showed that C. acutus is paraphyletic with respect to C. rhombifer, revealing 1% sequence divergence between species within Cuba vs. 8% divergence between Cuban forms and mainland C. acutus. We suggest that hybridization has been a historical as well as a current phenomenon between C. acutus and C. rhombifer. These findings suggest that long-term conservation of crocodiles in Cuba will require identification of genetically pure and hybrid individuals, and a decrease in anthropogenic activities. We also recommend more extensive morphological and genetic analyses of Cuban population to establish clear boundaries of the hybrid zone between C. acutus and C. rhombifer.     

Ocular disease in American crocodiles (Crocodylus acutus) in Costa Rica

Beginning in early 2006, an ocular disease of unknown etiology was routinely observed in American crocodiles (Crocodylus acutus) inhabiting the highly polluted Tarcoles River in west-central Costa Rica. We examined the nature and incidence of ocular disease in Tarcoles crocodiles and assessed the possible association between the disease and accumulation of chemical pollutants in diseased individuals. During 12-15 September and 12-13 December 2007, crocodiles were captured and examined for ocular disease and sampled to determine environmental contaminant accumulation. Three of 11 (27.3%) crocodiles captured (all males) exhibited unilateral ocular disease, primarily characterized by corneal opacity and scarring, anterior synechia, and phthisis bulbi. Multiple pollutants were detected in crocodile caudal scutes (organochlorine pesticides [OCPs] and metals), crocodile blood (OCPs), and sediments (OCPs and metals) from the Tarcoles, but no associations were found between contaminant accumulation and the incidence of eye disease. On the basis of the limited number of diseased animals examined and the potential exposure of crocodiles to pathogens and other pollutants not targeted in this study, we cannot rule out infection or chemical toxicosis as causes of the eye lesions. However, circumstantial evidence suggests that the observed ocular disease is likely the result of injury-induced trauma (and possibly secondary infection) inflicted during aggressive encounters (e.g., territorial combat) among large adult crocodiles living at relatively high densities.     

Characterization of serum complement activity of saltwater (Crocodylus porosus) and freshwater (Crocodylus johnstoni) crocodiles

We employed a spectroscopic assay, based on the hemolysis of sheep red blood cells (SRBCs), to assess the innate immune function of saltwater and freshwater crocodiles in vitro. Incubation of serum from freshwater and saltwater crocodiles with SRBCs resulted in concentration-dependent increases in SRBC hemolysis. The hemolytic activity occurred rapidly, with detectable activity within 2 min and maximum activity at 20 min. These activities, in both crocodilian species, were heat sensitive, unaffected by 20 mM methylamine, and completely inhibited by low concentrations of EDTA, suggesting that the alternative serum complement cascade is responsible for the observed effects. The hemolytic activities of the sera were inhibited by other chelators of divalent metal ions, such as phosphate and citrate. The inhibition of SRBC hemolysis by EDTA could be completely restored by the addition of 10 mM Ca2+ or Mg2+, but not Ba2+, Cu2+ or Fe2+, indicating specificity for these metal ions. The serum complement activities of both crocodilians were temperature-dependent, with peak activities occurring at 25-30 degrees C and reduced activities below 25 degrees C and above 35 degrees C.     

Satellite tracking reveals long distance coastal travel and homing by translocated estuarine crocodiles, Crocodylus porosus

Crocodilians have a wide distribution, often in remote areas, are cryptic, secretive and are easily disturbed by human presence. Their capacity for large scale movements is poorly known. Here, we report the first study of post-release movement patterns in translocated adult crocodiles, and the first application of satellite telemetry to a crocodilian. Three large male Crocodylus porosus (3.1-4.5 m) were captured in northern Australia and translocated by helicopter for 56, 99 and 411 km of coastline, the last across Cape York Peninsula from the west coast to the east coast. All crocodiles spent time around their release site before returning rapidly and apparently purposefully to their capture locations. The animal that circumnavigated Cape York Peninsula to return to its capture site, travelled more than 400 km in 20 days, which is the longest homeward travel yet reported for a crocodilian. Such impressive homing ability is significant because translocation has sometimes been used to manage potentially dangerous C. porosus close to human settlement. It is clear that large male estuarine crocodiles can exhibit strong site fidelity, have remarkable navigational skills, and may move long distances following a coastline. These long journeys included impressive daily movements of 10-30 km, often consecutively.