Plasma homeostasis ad cloacal urie compositio i Crocodylus porosus caught alog a saliity gradiet Gordo C. Grigg Zoology, A08, Uiversity of Sydey, Sydey, N.S.W. 2006, Australia Accepted April l, 1981 Summary. Juveiles of the Estuarie or Saltwater Crocodile, Crocodylus porosus, maitai both osmotic pressure ad plasma electrolyte homeostasis alog a saliity gradiet from fresh water to the sea. I fresh water (FW) the cloacal urie is a clear solutio rich i ammoium ad bicarboate ad cotaiig small amouts of white precipitated solids with high cocetratios of calcium ad magesium. I salt water (SW) the cloacal urie has a much higher proportio of solids, cream rather tha white i colour, which are the major route for ecretio of potassium i additio to calcium ad magesium. Neither liquid or solid fractios of the cloacal urie represet a major route for ecretio of sodium chloride. The solids are urates ad uric acid, ad their productio probably costitutes a importat strategy for water coservatio by C. porosus i SW. These data, coupled with atural history observatios ad the recet idetificatio of ligual salt glads, cotribute to the coclusio that C. porosus is able to live ad breed i either fresh or salt water ad may be as euryhalie as ay reptile. Itroductio Util recetly it was thought that the marie ad/or estuarie Crocodilia, C. porosus ad C. acutus, lacked fuctioal etra-real salt glads (Duso 1969, 1970; Duso ad Moll 1980). This apparet lack led to doubt about the ability of these aimals to maitai electrolyte balace i salt water, so they came to be regarded as havig estuarie rather tha marie capabilities (Schmidt-Nielse 1975). Geographic distributio ad various observatios of large crocodiles at sea (Neill 1971 ; Alle 1974) were eplaied i terms of a relatively impermeable ski, a small surface area/mass ratio ad a fish diet (Duso 1976). However. the occurrece of juveile ad hatchlig C. porosus as residets i hypertoic estuarie coditios i orther Australia (persoal observatio) suggested physiological competece rather tha shortcomigs. Accordigly a study was made i 1975 to determie the etet to which juveile C. porosus are homeostatic alog a atural saliity gradiet. As will be described i this paper, the aimals maitai a very high degree of homeostasis, but whether or ot this homeostasis depeds o fresh water for drikig was ot kow. I a subsequet field study, i July 1979, Grigg et al. (1980) foud that hatchlig C. porosus survive ad grow i sea water, without access to fresh water. All these data implied a active salt glad ad the eistece of such glads was cofirmed recetly i both C. porosus (Tapli ad Grigg 1981) ad C. acutus (Tapli et al., i preparatio). A more complete uderstadig of the water ad electrolyte physiology of C. porosus, ad the related problem of itroge ecretio, requires iformatio o typical levels of electrolytes ad metabolites i both plasma ad cloacal urie from crocodiles captured over a wide rage of saliity. The preset paper reports these data, eamies the etet to which plasma homeostasis is maitaied by juveiles, ad describes cloacal electrolyte ad itroge ecretio over a wide atural saliity gradiet. Materials ad Methods The study was carried out i the Liverpool ad Tomkiso Rivers ear Maigrida, orther Australia, durig July 1975. Blood ad cloacal urie were take from as may o-hatchlig captives as possible. Stomach cotets also were take from may of the same aimals (Taylor 1977, 1979). Some supplemetary data are icluded from a field trip to the same area i July 1979. Study Area, Saliity Profiles ad Crocodiles. Detailed descriptios of the Liverpool-Tomkiso river system have appeared previously (Webb ad Messel 1978; Maguso et al. 1975; Messel et al. 1979) ad a brief descriptio oly eed be give here. The rivers meader across a etesive shared flood plai before they joi ad eter the Arafura Sea via a commo estuary (Fig. 1). Three major (Mugardobolo, Maragulidba ad Gudjerama) ad several mior creeks form part of the system.
Fig. 1. Map of the study area To uderstad the saliity eviromet of C. porosus ad to assig a likely saliity history to crocodiles caught, oe must uderstad the seasoal ad tidal cycles which occur i these rivers ad creeks. All are subject to a tidal rise ad fall of 2-3 m approimately twice daily, eve as far as 70-80 km upstream from the mouth. The water is mostly turbid with suspeded material ad its saliity varies i a distict seasoal cycle, overlai by the tidal cycle. Etesive raifall durig the mosoo seaso (November-April) flushes the rivers, ad potable water may be foud as far dowstream as Bat Islad. With the cessatio of the mosoo ad reductio of flow at the headwaters, the marie ifluece moves progressively up river by miig associated with the tidal flows util, by the ed of the dry seaso, water may be too salty for humas to drik eve as far upstream as A ad B i Fig. 1. I additio to the seasoal cycle of saliity chage, substatial chages ofte accompay each tidal cycle. These are most marked i the dry seaso whe saliity alog much of each river oscillates betwee high ad low values with the tidal flow (Fig. 2). Our samplig was carried out from 18-26 July, 1975, i mid dryseaso. High ad low tide saliitydistace profiles were determied i each river several times durig the study, usig a coductivity meter (Autolab). Seasoal chages i the ature of the saliity gradiet durig the samplig period were small eough to be igored, but tidal chages were quite large (Fig. 2a, b). The differet shape of the profile i each river reflects the lesser fresh water iflow to the Tomkiso river. Fortuately there was o top-bottom gradiet i saliity ad o 'salt-wedge' i either river, as occurs sometimes. This simplified the ature of the gradiet cosiderably. Crocodiles were caught o the ight low tides, as described by Webb ad Messel (1977) ad the capture site of each was recorded. It was assumed that each aimal had bee eposed to the saliity rage typical of its capture site ad was acclimatised to that saliity regime. It is kow that short term mobility of juveile crocodiles is low (Webb ad Messel 1978; Grigg et al. 1980; Messel et al. 1981). The supplemetary data collected i 1979 were gaied at the same time of year (18 July-2 August) ad high ad low tide saliity-distace profiles determied as before. Thus, the saliity history of the 1979 aimals was assessed i the same way as i 1975, allowig data to be pooled.
Fig. 2. Saliity profiles i the study area at high ad low tide i July 1975. Mea plasma osmotic pressure was used to defie four physiologically realistic saliity categories, SAL 1 to SAL 4 (see tet) Blood ad Cloacal Samplig ad Aalytical Procedures. Blood was sampled from 0 idividuals i 1975, of which 65 gave a sample of cloacal urie. Eightee samples of cloacal urie were collected i 1979. Blood was draw from the caudal sius (Grigg ad Caircross 1980) ad lithium hepari was used to prevet clottig. Sub-samples of whole blood were stored froze for later aalysis of blood glucose, lactate, hemoglobi ad 2,3-diphosphoglycerate, reported elsewhere (Grigg ad Gruca 1979; Gruca ad Grigg 1980; Grigg ad Caircross 1980). Plasma samples were prepared by cetrifugatio at 3,000 r.p.m. for 5 mi. Cloacal urie samples were cetrifuged at the time of collectio ad the superatat sub-sampled for osmotic pressure determiatio. Osmotic pressures of both plasma ad cloacal urie were determied withi two hours of samplig, usig a Kauer Model M Semi-micro Osmometer. The remaiders of plasma ad cloacal urie samples (liquid plus solid) ad all other samples were trasported froze to Sydey. Sodium, potassium, chloride, bicarboate, iorgaic phosphate, ammoia, urea, uric acid ad creatiie were aalysed at St. Vicet's Hospital, Sydey, o a Autolab 12/60 auto-aalyser. Calcium ad magesium were aalysed by atomic absorptio spectrophotometry (Perki-Elmer) at Royal Price Alfred Hospital, Sydey. Aalysis of cloacal urie precipitates was effected followig their digestio by acid. Groupig of the Aimals by Size, Se ad Saliity History. The crocodiles (69 males, 41 females) were all ohatchligs i their secod dry-seaso or older. Although they represeted a fairly cotiuous size distributio from 0.89 (sout-vet legth= 34.5 cm) to 46.4 kg, five weight classes were defied (Table 1), eablig some aalyses usig weight as a discotiuous rather tha a cotiuous variable.
Based o kowledge of saliity profiles of the river at high ad low tide ad the mea osmotic cocetratio of C. porosus plasma (304 mosm: 1-1 ). four 'physiologically reasoable' categories of saliity history were decided upo (Fig. 2). These were as follows: SAL 1 - fresh water above the limit of ay measurable marie ifluece. SAL 2 - some marie ifluece, but throughout the tidal cycle water saliity ever eceeds the osmotic cocetratio of the plasma. SAL 3 - tidal saliity fluctuatios are such that osmotic cocetratio of the water fluctuates alterately above ad below that of the plasma. SAL 4 - water saliity at a higher osmoticity tha the plasma at all stages of the tidal cycle. Table 1. Breakdow by weight, se ad saliity category of 0 juveile C. porosus Saliity category a SAL 1 SAL 2 SAL 3 SAL 4 Totals 0.8-2.4 kg Male 5 8 4 3 20 Female 1 6 s 2 12 2.41-5.0 kg Male 5 0 3 4 12 Female 6 3 0 0 9 5.01-8.0 kg Male 4 1 4 3 12 Female 4 1 0 7 8.01-10.2kg kg Male 1 2 1 6 10 Female 2 0 0 2 4 Above10.2kg Male 8 2 3 2 15 Female 7 1 0 I 9 Totals 43 24 20 23 0 a See tet (Methods) ad Fig. 2 for defiitio Statistical Aalysis ad Data Maagemet. Data maagemet ad aalysis were udertake usig the SPSS system (Nie et al. 1975) ad 5% levels of probability were adopted as sigificat. The mai thrust of the aalysis was to determie the effect of saliity o plasma ad urie electrolytes ad itrogeous ecretory products. Saliity was treated sometimes as a cotiuous variable (e.g. i correlatio matries) ad sometimes as a discotiuous variable (as i categorisatio of aimals by saliity history for aalysis of variace ad for tabulatio of results). Despite a reasoably eve spread of samples from both sees over a wide rage of saliity history ad weights (Table 1) there was the possibility of cofusio betwee effects of weight ad/or se ad those due to saliity. Accordigly, the possibility of weight ad/or se effects was eamied first, usig aalysis of variace techiques. Results Plasma Electrolytes ad Osmotic Pressure; Effect of' Saliity (i) Effects of Size ad Se. Complete (or early so) sets of data were available for plasma from 99 idividuals, ad for urie from 58 crocodiles. I order to determie whether or ot plasma electrolyte values deped upo size ad/or se, a two-factor aalysis of variace was carried out usig both sees ad five weight classes (Table 1) as factors for each electrolyte species ad plasma osmotic pressure. Four replicates for each weight-se cell i the aalysis were selected at radom (four beig the smallest umber of sets of observatios i a sigle cell; females 8.01-10.2 kg). No differeces due to se were apparet. A sigificat depedece o weight was foud oly for plasma iorgaic phosphate which was foud to be sigificatly higher i larger crocodiles. The questio of correlates with weight ad/or se was pursued also usig all data i a Pearso correlatio matri (a copy of which is available from the author, if desired). Agai, a sigificat icrease i iorgaic phosphate was idicated i larger crocodiles (P<0.001), though a scattergram showed that this result depeds heavily upo data from the few idividuals i the study which were approachig maturity. Plasma sodium, chloride, bicarboate, magesium, calcium, ad lactate were agai show to be idepedet of both se ad weight, but plasma potassium, ammoia ad osmotic pressure showed a sigificat icrease i larger aimals. Scattergrams of these latter relatioships suggested that such a result depeded upo the cotributio of oly a few poits from the largest aimals i the study. Whe the Pearso correlatio was
repeated without the eight crocodiles greater tha 15 kg i body weight, iorgaic phosphate ad ammoia were still sigificatly weight-depedet, with calcium i additio. I summary, the results from the aalysis of variace ad the Pearso correlatio showed that plasma electrolyte cocetratio ad osmotic pressure are idepedet of se, while size has little or o effect ecept that larger aimals ted to have higher levels of plasma iorgaic phosphate ad ammoia. More data are eeded from large aimals to cofirm whether levels of plasma potassium ad osmotic pressure icrease with icreasig body size. (ii) Effects of Saliity. There is a very high degree of plasma homeostasis alog the saliity gradiet. I the Pearso correlatio ot a sigle plasma electrolyte species was saliity-correlated ad oly plasma chloride came close (P=0.080). By aalysis of variace, plasma chloride is just (P=0.04) sigificatly saliity depedet, with a tred to higher chloride i fresh water (Table 2). Plasma sodium shows a similar (though ot sigificat) tred. Accordig to the correlatio matri, plasma osmotic pressure is idepedet of saliity, though the aalysis of variace idicated higher values i fresh water by a slight but statistically sigificat margi. Slightly higher values of plasma bicarboate seem typical of crocodiles from salt water (Table 2) ad may be a artifact of time betwee capture ad samplig (see later) though the relatioship did ot show up i the correlatio matri. I either method of aalysis was there ay suggestio of a sigificat saliity-related tred i the values of plasma sodium, potassium, iorgaic phosphate, calcium, magesium, ammoia or lactate. Table 2. Plasma electrolytes ad other data from C. porosus at differet levels of saliity. Values are mmol/1 uless stated otherwise. values (χ) are give, followed by the umber of observatios (). 95% cofidece limits are give i brackets χ SAL 1 χ SAL 2 χ SAL 3 χ SAL 4 Probability (1-way aalovar) Overall mea χ Osmotic pressure 307 37 305 19 299 19 302 24 < 0.001 304 99 (mosm) (305-309) (302-308) (295-302) (298-305) (302-305) Sodium 136 40 135 19 132 19 130 21 N. S. 134 99 (133-139) (130-139) (129-135) (126-135) (132-136) Potassium 4.1 35 3.7 3.5 18 3.8 21 N.S. 3.8 91 (3.7-4.4) (3.4-4.0) (3.3-3.7) (3.6-4.0) (3.6-4.0) Calcium 2.8 20 3.0 10 2.7 2.8 10 N. S. 2.8 51 (2.7-3.0) (2.9-3.1) (2.5-2.9) (2.6-3.0) (2.7-2.9) Magesium 1.4 21 1.6 9 1.4 6 1.5 9 N. S. 1.5 45 (1.3-1.5) (1.4-1.8) (1:2-1.6) (1.2-1.8) (1.4-1.6) Ammoia 0.8 33 0.6 15 0.45 0.8 20 N. S. 0.7 85 (0.5-1.1) (0.3-0.9) (0.3-0.6) (0.4-1.1) (0.5-0.8) Chloride 121 40 6 19 7 19 4 21 <0.05 8 99 (7-135) (4-8) (4-120) (1-7) (6-120) Bicarboate 14.9 40 16.7 19 18.9 19.8 20 <0.01 16.6 98 (13.7-16.1) (14.5-19.0) (16.7-21.2) (ib.l-19.5) (15.7-.5) Iorgaic 2.06 40 1.81 19 1.88 19 1.94 21 N.S. 1.95 99 phosphate (1.92-2.21) (1.53=?.10 (1.68-2.08) (1.72-2.15) (1.86-2.05) ) 4.6 16 4.4 9 2.9 10 4.0 10 N.S. 4.1 45 Lactate (3.4-5.9) (2.1-6.8) (1.013.7) (1.9fi.1) (3.3-4.9) (iii) Capture Artifact. The strugglig of a crocodile at the time of capture ca be epected to have a ifluece o plasma metabolites ad electrolytes. Accordigly the time betwee capture ad blood samplig was itroduced as a variable i the correlatio matri aalysis. Cogizace of ay variables related to elapsed time is importat i terms of reportig `ormal' values for C. porosus ad may shed light o the physiological correlates of recovery from a struggle. Followig capture, sigificat treds towards lower plasma lactate, osmotic pressure ad plasma calcium were foud, while bicarboate levels rose. I all cases, however, the variability was very great ad correlatio coefficiets were too low for the relatioship to have ay predictive value.
Urie Electrolytes ad Osmotic Pressure; Effects of Saliity I both fresh ad salt water, C. porosus ecretes urie with some isoluble precipitate. Although the precipitated fractio is particularly substatial i salt water, eve i freshwater the solids are a persistet ad characteristic compoet (Fig. 3). The results of aalysis of liquid ad precipitated urie fractios will be give separately. Fig. 3. Nitroge ecretio i liquid cloacal urie by C. porosus at differet levels of saliity, ad comparative percetages of solids i cloacal urie. Error bars are 95% cofidece limits (i) The Electrolyte Compositio ad Osmotic Pressure of the Liquid Urie at Differet Saliities. Whereas plasma electrolytes showed a high degree of homeostasis alog the saliity gradiet, a markedly differet patter emerged for electrolytes i cloacal urie (Table 3). Crocodiles from SAL 4 had sigificatly higher cloacal osmotic pressure, though always hypoosmotic to the plasma (Table 3). Large icreases i potassium, calcium ad magesium were observed ad i each of these cases the most.dramatic icrease was i SAL 4 with respect to SAL 1-3 (Table 3). Iorgaic phosphate levels were broadly similar i all four saliity categories. A very large declie i both ammoia ad bicarboate was observed ad agai i this case the biggest chage was i SAL 4 with respect to SAL 3 (Fig. 3). The most strikig result, however, is that sodium ad chloride are preset i oly very small amouts ad, furthermore, there is o suggestio of ay icrease i sodium ad chloride i the liquid fractio of the cloacal urie towards salt water at higher saliities (Table 3).
Table 3. Electrolyte compoets of liquid cloacal urie of C. porosus at differet levels of saliity. Values are mmol/1 uless stated otherwise. Mea values () are give, followed by the umber of observatios (). 95% cofidece limits are give i brackets SAL I SAL 2 SAL 3 SAL 4 Probability (1-way aalovar) Osmotic pressure (mosm) 252 (240-265) 15 256 (246-266) 19 257 (250-266) 15 277 16 P < 0.03 (259-296) Sodium a - Potassium 2.2 (1.6-2.9) Calcium 1.0 (0.6-1.4) Magesium Ammoia Chloride a - 8 7.9 (0-.5) 9-9.8 (2.4-.2) 9 N.S. 2.7 (1.5-3.9) 15 3.3 (1.5-5.2) 2.0 (0.6-3.5) 7 2.0 (0.6-3.3) 0.06 (0.03-0.08) 7 0.40 (0-0.87) 10 0.32 (0-0.86) 98 (85-1) 84 (66-101) 16 86 (69-104) 5 6 8.8 P < 0.0001 (5.5-12.0) 4.4 (0-9.0) 6 N.S. (P=0.08).69 (0-28.2) 5 P<0.01 43 (31-56) 13 P<0.0001 9.1 (4.4-13.9) 7-9.7 9 N. S. (5.0-14.3) Bicarboate 41.8 (33.7-50.0) 38.4 (23.3-53.4) 16 35.8 (18.6-53.1) 0.8 (0-1.84) Iorgaic phosphate 14 P < 0.0001 7.76 (4.66-10.86) 6.75 (2.73-10.77) 16 7.30 (3.93-10.67) 9.90 14 N. S. (2.96-.03) (ii) The Electrolyte Compositio of the Precipitated Urie at Differet Saliities. Substatial chages i catio compositio i urie solids are see betwee the four saliity categories (Table 4, Fig. 4). While the amout of potassium icreases more tha tefold, the proportioal cotributio of calcium ad magesium decreases betwee fresh water ad salt water. The low cocetratio of sodium i the liquid urie is mirrored i the compositio of the solids where sodium occurs i oly trace amouts. Table 4. Compositio of cloacal urie solids from C. porosus at differet saliities. Values are mg/g solid uless stated otherwise. The mea () is give ad umber of observatios (), with 95% cofidece limits i brackets SAL 1 SAL 2 SAL 3 SAL 4 Ca 21.5 (.3-31.7) 14 7.9 (1.7-14.0) 8 8.5 (2.3-14.6) 8 1.3 (0.6-2.0) M g 34.0 (23.8-44.1) 14 16.4 (8.4-24.4) 8 16.0 (9.7-22.3) 8 5.0 (1.2-8.8) Na 0.0 14 0.2 0.2 0.3 8 8 (0-0.7) (0-0.7) (0.1-0.5) K 0.2 2.7 0.8 12.7 14 8 8 (0-0.4) (0-7.9) (0.2-1.4) (4.1-21.3) % by volume 2.7 4.5 6.6 30.5 16 (1.7-3.7) (1.1-7.9) (3.9-9.3) (18.9-42.2) 12 12 12 14
Fig. 4. Comparative cotributio of liquid (ope sectios) ad solid (shaded sectios) urie to electrolyte ecretio i each of the four saliity categories (calculated accordig to method described i tet) (iii) Total Urie Electrolyte Compositio (Liquid+ Solid) at Differet Saliities. Combiig data o liquid (Table 3) ad solid (Table 4) urie ad allowig a specific gravity of 1.3 for the precipitate, oe ca arrive at a approimatio of electrolyte ecretio per litre of mied cloacal urie i each of the four saliity categories (Fig. 4). This has bee doe usig mea values oly, ot for each idividual aimal, ad o statistical comparisos were made. I fresh water the solids form a major vehicle for Ca ad Mg ecretio. I salt water, however, the additioal role of the solids i potassium ecretio is see. The data suggest that the urie is ot a major route for ecretio of ecess sodium. Nitrogeous Ecretory Products i the Plasma ad Urie; Effects of Saliity Levels of the four major itrogeous ecretory products i plasma ad liquid urie from crocodiles i each of the four saliity categories are show i Table 5. To eable easy comparisos, the data for ammoia are repeated from Tables 2 ad 3 i which it appears as a electrolyte. Homeostasis of plasma across the saliity categories is agai emphasised, with o sigificat effect of saliity o ammoia, creatiie or uric acid. Plasma urea is, however, sigificatly higher i SAL 4 tha i all the other categories. I the liquid urie, ammoia decreases progressively from fresh to salt water whereas urea, creatiie ad uric acid i solutio are all icreased sigificatly i SAL 4 by compariso with SAL 1-3. The large icrease i the percetage by volume of solid precipitate i salt compared with fresh water has bee referred to earlier. Fig. 3 summarises the levels of major itroge ecretio-related variables at differet saliities. Discussio Plasma Homeostasis It is clear that C. porosus is able to maitai plasma homeostasis over a wide rage of saliity (Table 2). No saliity-related chages were observed i plasma sodium, potassium, calcium, magesium, iorgaic phosphate or lactate. Very small, though statistically sigificat, treds dowwards with icreasig saliity were see i osmotic pressure ad chloride, parallelig a isigificat tred for sodium i the same directio. A icrease i plasma bicarboate was foud at higher saliities, but this is almost certaily the result of a greater time elapsig betwee capture ad blood samplig i the salt water areas (see later).
Though statistically sigificat, total chages i each of these variables are very small ad it is adequate to report average values (Table 2). Amog itroge compouds i the plasma, oly urea was sigificatly higher i SAL 4, though still preset i oly trivial amouts, while ammoia, uric acid ad creatiie are low ad idepedet of saliity (Table 3). The low level of plasma urea i salt water (12 mmol/1) is i sharp cotrast to the large icrease i urea see i the oly other euryhalie reptile studied i detail, Malaclemys terrapi, from 22 mmol/1 i fresh water to 5 mmol/1 i salt water (Gilles-Baillie 1970). The same species shows a rise i plasma osmotic pressure from 160 to 400 mosm/1 i salt water, while both Na ad Cl rise sigificatly. There is a marked similarity i the values of major aios ad catios i C. porosus with those reported i tabular form for other crocodilias by Betley (1976) ad Miich (1979). Capture Artifact Capture led to high levels of blood lactate, presumably associated with the aaerobic metabolism of strugglig, ad cocomitat fall i bicarboate. Blood bufferig has bee discussed by Grigg ad Caircross (1980). Coulso ad Heradez (1964) discussed the icrease i blood lactate followig hadlig of alligators. They reported ormal values ragig from trace to 1.0 mmol/1 (average 0.7) i alligators kept udisturbed ad i a soudproof bo prior to samplig. Thus our mea values of approimately 4 mmol/t are probably rather too high. The rage was from 0.7 to 10 mmol/1 with the mode at 3.0 mmol/1. Ufortuately, although the depedece of blood lactate o time elapsed betwee capture ad samplig, is very marked, the correlatio coefficiet is too low to eable ay cofidet predictio of mea levels either at capture or at, say, 24 h later. This eample highlights the difficulty of reportig ormal = levels. Appearace of Cloacal Urie from Crocodiles at Differet Saliities Eve to the casual observer the differeces betwee cloacal urie produced by C. porosus i salt ad fresh water are strikig. I fresh water a clear fluid is produced, which has oly a very small amout of white solid matter. I salt water however (Table 5) proportioally more solid is preset; a cream-coloured mucous-rich graular/crystallie material which may represet by far the bulk of the cloacal cotets. I some cases, almost o liquid is voided ad the product is a loosely compacted moist solid. It should be oted that the samples i this study were ot voided aturally but represet cloacal samples take at radom with respect to ay `secodary processig' which occurs i the cloaca. Hece, all reported values ca be epected to show a wider rage tha the values of ureteral urie or cloacal urie voided volutarily. Table 5. Plasma ad cloacal urie itroge compouds i C. porosus from differet saliities. Values are i mmol/1, with 95% cofidece limits i brackets. =umber of observatios z SAL 1 SAL 2 SAL 3 SAL 4 Probability z z z (1-way aalovar) Plasma Ammoia 0.8 0.6 0.4 0.8 N.S. 33 15 20 (0.5-1.1) (0.31-0.9) (0.3-0.6) - (0.4-I.l) Urea 1.2 1.1 1.1 2.2 P<0.0001 39 33 19 21 (l.1-1.3) (0.8-1.3) (0.6-1.5) (1.6-2.8) Uric acid 0.62 0.69 0.67 0.73 N. S. 40 19 19 21 (0.53-0.70) (0.60-0.77) (0.56-0.77) (0.62-0.84) 0.064 0.065 0.064 0.069 N. S. Creatiie (0.057-40 (0.052-19 (0.51-19 (0.061-21 0.071) 0.077) 0.076) 0.076) Urie Ammoia Urea 98 (85-1) 4.54 (3.77-5.31) Uric acid 0.28 (0.24-0.33) 0.102 Creatiie (0.042-0.162) Solids i 2.7 urie (1.7-3.7) 84 (66-101) 6.52 (4.08-8.97) 0.35 (0.22-0.49) 0.108 (0.087-0.130) 4.5 (1.1-7.9) 16 16 16 16 16 86 (69-104) 7.07 (5.30-8.85) 0.51 (0.-0.85) 0.4 (0.072-0.157) 6.6 (3.9-9.3) 43 (31-56) 20.96 (14.05-27.87) 3.12 (1.80-4.44) 0.4 (0.188 -- 0 633) 30.5 (18.9-13 14 14 13 14 P<0.0001 P<0.0001 P<0.0001 P<0.001 P<0.0001
Electrolytes i Cloacal Urie The observed stability of plasma osmotic pressure, electrolytes ad other costituets throughout the saliity gradiet ca result oly from active hyperosmotic ad hypoosmotic regulatio. I fresh water, crocodiles must retai salts, whereas i sea water they are likely to have a ecess of salts, otably sodium ad chloride. The problems of osmoregulatio by reptiles i various eviromets, ad the mechaisms by which regulatio is effected, have bee reviewed by Duso (1976, 1979) Betley (1976), Datzler (1976) ad Miich (1979). Etesive review here would, therefore, be superfluous. The preset results emphasise the overwhelmig importace of the precipitated urie for ecretio of K, Ca ad Mg (Fig. 4). Sodium Chloride. The results show that either the liquid or the solid fractio of the cloacal urie is a major vehicle for ecretio of ecess NaCl i salt water (Tables 3, 4). I the liquid cloacal urie, mea values of 8-10 mmol/1 Na ad Cl were foud i both SAL 2 ad SAL 4, with o sigificat differeces betwee them (Table 3). These values compare favourably with Na values of mmol/1 i Caima crocodilus (Betley ad Schmidt-Nielse 1965) ad 6 mmol/1 i Crocodylus acutus (Schmidt-Nielse ad Skadhauge 1967) i fresh water. Oly traces of sodium were foud i acid digests of the precipitated urie (Table 4) ad this has bee cofirmed by X-ray diffractio ad electro probe aalyses (Grigg et al., i progress). As the diet of C. porosus i salt water (Taylor 1979) probably provides a ecess of sodium chloride, fuctio of a etra-real salt glad is implied ad, as reported i the itroductio, Tapli ad Grigg (1981) have described ligual salt glads capable of producig a secretio of NaCl 3-4 times as cocetrated as the plasma. Further studies are ow i progress to elucidate the role of these glads i the electrolyte regulatio of C. porosus. Potassium. Tapli ad Grigg (1981) foud oly small quatities of K ecreted by the salt glad. There ca be little doubt that the major route for ecretio of ecess potassium is the isoluble precipitate i cloacal urie (Fig. 4) though cofirmatio of this awaits data o volumes of cloacal urie productio. Prelimiary results idicate that this occurs i the form of potassium urate (Grigg et al., i progress). As see i Fig. 4, the liquid urie cotais little K i either fresh or salt water. Similarly low values were reported by Schmidt-Nielse ad Skadhauge (1967) for C. acutus i fresh water. Calcium ad Magesium. Although the levels of both Ca ad Mg are higher i the liquid urie of crocodiles from SAL 4, by far the major part of uriary loss of Ca ad Mg occurs i the isoluble fractio (Fig. 4). This is true i both fresh ad salt water. Prelimiary results show that Ca ad Mg phosphates make up the bulk of the isoluble fractio of the urie i fresh water, whereas i salt water Ca ad Mg are ecreted maily as urates (Grigg et al., upublished). These results differ from those of Coulso ad Heradez (1964) who showed that alligators i fresh water lose Ca through the faeces, ot the urie. Phosphate was, however, foud i the urie. Datzler (1976), reviewig this ad other data o Ca i the urie of reptiles, cocluded that Ca is ecreted i the faeces, ad P i the urie; otherwise isoluble calcium phosphate would ted to precipitate i the real tubules. This seems to be the case eactly i C. porosus, with uriary ecretio of precipitated calcium phosphate. Ammoium Bicarboate. As foud for crocodilias i fresh water by other workers (Coulso ad Heradez 1964; Schmidt-Nielse ad Skadhauge 1967), the major ios i cloacal urie of C. porosus i SAL 1, SAL 2 ad SAL 3 are ammoium ad bicarboate. I SAL 4, however, oly traces of bicarboate are preset ad ammoium is much reduced (Table 3). This represets a strikig differece betwee SAL 4 ad the other categories. Grigg (1978) cofirmed, i fresh water at least, suggestios by Coulso ad Heradez (1964) that gasometric RQ of crocodilias would be low because of the uriary ecretio of much CO 2 as bicarboate, bufferig NH 3. Values of RQ=0.49 (rage 0.32-0.74) were foud for C. porosus i fresh water. I fresh water, some ammoium is lost also as ammoium urate (Grigg et al., upublished). Ecretio of' Nitroge Whereas i SAL 1-3 most itrogeous waste is i solutio as ammoium bicarboate (Table 5), i SAL 4 the domiat itroge ecretio is i the form of isoluble uriary precipitates, uric acid ad its salts (Grigg et al., upublished). There is also a sigificat amout of urea i the liquid urie i SAL 4. The presece of urea was ot recorded i Alligator urie (Coulso ad Heradez 1964) but Khalil ad Haggag (1958) report its presece i C. iloticus urie collected uder uspecified coditios. Whether or ot alligators would ecrete urea i salt water is ukow. The chage-over i ecretory products from ammoia to uric acid ad its salts is udoubtedly a major strategy for water coservatio i a hyperosmotic eviromet. Some prelimiary data are available which allow the effectiveess of this to be gauged. Tapli et al. (upublished), usig radioisotope techiques, have measured water ad sodium flues i 30 C. porosus (hatchligs ad juveiles) uder hypoosmotic
(SAL 2) ad hyperosmotic (SAL 4) field coditios i the Tomkiso River. Water flu i SAL 4 was approimately half that i SAL 2, whereas sodium flu early doubled. Comparable data are uavailable from other free-ragig euryhalie reptiles. Osmoregulatory Strategy i SAL 3 Aimals uder SAL 3 coditios are eposed to ambiet saliities which cycle with the tide betwee hypoad hyperosmotic. Overall, the urie from aimals caught i SAL 3 is more similar to urie from SAL 1 ad SAL 2 tha to SAL 4 urie. This suggests that strategies for hypoosmotic regulatio are beig employed more i SAL 4 tha 3, although i SAL 3 the ambiet water is hyperosmotic to plasma for part of every tidal cycle. Thus, crocodiles may show behavioural correlates with saliity as it chages durig a tidal cycle. The most likely behavioural strategies would be for aimals to leave the water at high tide i SAL 3 areas ad/or drikig whe hypoosmotic water is available at low tide. Messel et al. (1981) have aalysed thousads of sightigs of C. porosus but their data led o support to the idea that crocodiles leave the water at high tide or to escape higher saliities. Saliity Tolerace C. porosus emerges from this ad parallel studies (Grigg et al. 1980; Tapli ad Grigg 1981) as a reptile which is far more euryhalie tha has bee thought previously. It is able to maitai the homeostasis of the body fluids over a saliity rage from fresh water to salt water, has salt glads, ad hatchligs ca survive ad grow i salt water without drikig fresh water. May ests are located where fresh water is ot available i the dry seaso, although there is some preferece for sites with fresh water (Grigg et al., upublished). Hece, C. porosus seems to have the capability to live ad breed i either fresh or sea water. The maimum tolerace of C. porosus for high saliities is ukow, but 1-2 m aimals have bee see at saliities up to 70 0 / 00, are see commoly at 45 0 / 00 (Messel et al. 1980; Tapli, persoal commuicatio) ad hatchligs have bee see i Mugardobolo Creek, a brach of the Tomkiso River, at 64 0 / 00 (Tapli, persoal commuicatio). I am grateful to Professor Harry Messel, Director of the Sciece Foudatio for Physics, Uiversity of Sydey, for makig it possible for me to udertake the field work for this study. Michael Caircross gave valuable assistace both i the field ad i the laboratory. The study could ot have bee carried out without the willig help ad participatio from so may people i the field, particularly Harry Messel, Grahame Webb, Jacky Agaral, Graeme Wells, Bill Magusso, Jaet Taylor ad Bill Gree. Staff i the Biochemistry Departmets of both St. Vicets ad Royal Price Alfred Hospitals gave ustitigly of their advice ad assistace with aalyses ad I am particularly grateful to Val Lacaster from St. Vicets. Margie Gruca carried out lactate aalyses. Ro Siclair ad Do Scott-Kemmis helped with statistical aalysis, Peter Harlow gave techical help ad Laurie Tapli ad Joh Miich read the mauscript. Jue Jeffery drew the figures ad Tess Maalag typed the mauscript. To all these colleagues ad frieds I am very grateful. Refereces Alle GR (1974) The marie crocodile, Crocodylus porosus, from Poape, Easter Carolie Islads with otes o food habits of crocodiles from the Palau Archipelago. Copeia 1974:553 Betley PJ (1976) Osmoregulatio i reptiles. I: Gas C, Dawso WR (eds) Biology of the reptilia. Physiology A, vol 5. Academic Press, New York, pp 365-412 Betley PJ, Schmidt-Nietse K (1965) Permeability to water ad sodium of the crocodilia Caima sclerops. J Cell Comp Physiol 66:303-309 Coulso RA, Heradez T (1964) Biochemistry of the alligator. Louisiaa State Uiversity Press, Bato Rouge Datzler WH (1976) Real fuctio (with special emphasis o itroge ecretio). I: Gas C, Dawso WR (eds) Biology of the reptilia. Physiology A, vol 5. Academic Press, New York, pp 447-503 Duso WA (1969) Reptilia salt glads. I: Botelho SY, Brooks FP, Shelley WB (eds) Eocrie glads. Uiv of Pesylvaia Press, Philadelphia, pp 83-103 Duso WA (1970) Some aspects of electrolyte ad water balace i three estuarie reptiles, the diamodback terrapi, America ad "salt-water" crocodiles. Comp Biochem Physiol 32:1674 Duso WA (1976) Salt glads i reptiles. I: Gas C, Dawso W R (eds) Biology of the reptilia. Physiology A, vol 5. Academic Press, New York, pp 413-445 Duso WA (1979) Cotrol mechaics i reptiles, chap 7. I: Gilles R (ed) Mechaisms of osmoregulatio i aimals: Maiteace of cell volume. Wiley-Itersciece, New York, pp 273322 Duso WA, Moll EO (1980) Osmoregulatio i sea water of hatchlig emydid turtles Callagur boreoeis from a Malaysia sea beach. J Herpetol 14:31-36 Gilles-Baillie M (1970) Urea ad osmoregulatio i the diamodback terrapi Malaclemys cetrata cetrata (Latreille). J Ep Biol 52:691-697 Grigg GC (1975) Metabolic rate, Q 10 ad respiratory quotiet (RQ) i Crocodylus porosus, ad some geeralizatios about low RQ i reptiles. Physiol Zool 51:354-360
Grigg GC. Caircross M (1980) Respiratory properties of the blood of Crocodylus porosus. Respir Physiol 41:367-380 Grigg GC. Gruca M (1979) Possible adaptive sigificace of low red cell orgaic phosphates i crocodiles. J Ep Zool 209:1667 Grigg GC, Tapli LE, Harlow P, Wright J (1981) Survival ad growth of hatchlig Crocodylus porosus i saltwater without access to fresh drikig water. Oecologia (i press) Gruca M. Grigg GC (1980) Methemoglobi reductio i crocodile blood: are high levels of met-hb typical of healthy reptiles? J Ep Zoo1213:305-308 Khalil F, Haggag G (1958) Nitrogeous ecretio i crocodiles. J Ep Biol 35:552-555 Magusso WE, Grigg GC, Taylor JA (1978) A aerial survey of potetial estig areas of the Saltwater Crocodile, Crocodylus porosus Scheider, o the North Coast of Arhem Lad, Norther Australia. Aust Wildl Res 5:401-415 Messel H, Wells AG, Gree WJ (1979) Surveys of tidal river systems i the Norther Territory of Australia ad their crocodile populatios. Moograph 7. Pergamo Press, Sydey Messel H, Vorlicek GC, Wells AG, Gree WJ (1981) Surveys of tidal river systems i the Norther Territory of Australia ad their crocodile populatios. Moograph 1. Pergamo Press Sydey (i press) Miich JE, (1979) Reptiles. I: Maloiy GMO (ed) Comparative physiology of osmoregulatio i aimals. Academic Press, New York, pp 391-641 Neill WT (1971) The last of the rulig reptiles. Columbia Uiversity Press, New York Nie NH, Hadlai Hull C, Jekis JG, Steibreer K, Bet DH (1975) Statistical package for the social scieces. McGraw-Hill, New York Schmidt-Nielse K (1975) Aimal physiology; adaptatio ad eviromet. Cambridge Uiversity Press, Lodo Schmidt-Nielse B, Skadhauge E (1967) Fuctio of the ecretory system of the crocodile (Crocodylus acutus). Am J Physiol 212:973-980 Tapli LE, Grigg GC Salt glads i the togue of the estuarie crocodile Crocodylus porosus. Sciece (i press) Taylor JA (1977) The foods ad feedig habits of sub-adult Crocodylus porosus Scheider, i Norther Australia (Crocodilia: Reptilia). MSc thesis, Uiversity of Sydey Taylor JA (1979) The foods ad feedig habits of subadult Crocodylus porosus Scheider i North Australia. Aust Wildl Res 6:347-359 Webb GJW, Messel H (1977) Crocodile capture techiques. J Wildl Maage 41:572-575 Webb GJW, Messel H (1978) Movemet ad dispersal patters of Crocodylus porosus i some rivers of Arhem Lad, Norther Australia. Aust Wildl Res 5:363-283