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Adaptation of the axolotl (Ambystoma mexicanum) to a hyperosmotic medium
ADAPTATION MEXKANUM)
OF THE AXOLOTL (AMBYSTOMA TO A HYPEROSMOTIC MEDIUM
M. P. IRELAND AND I.M. SIMONS Department of Zoology. University College of Wales. Aberystwyth, Wales. U.K. (Rrcriced 22 il4ay 1976) Abstrart---I. .4daptation of the axolotl to a 30”/ sea water medium for 5 and 35 days resulted in an increase in blood electrolytes and urea. 2. After both periods of sea water adaptation the blood osmotic concentration did not exceed that of the external environment and the major ions contributing to the ionic concentration were Na and Cl. 3. After 5 days in 30”; sea water there was a reduction in water uptake and urine flow. 4. A reduction in the size of the cells of the pars intermedia of the pituitary occurred after both periods of sea water adaptation. 5. The results are compared with the aquatic anuran Xe~zo~~s luetLs subjected to similar stimuli.
INTRODUCTION The tolerance of the aquatic anuran Xenopus laeuis to hyperosmotic solutions has been shown to result
in an increase in blood electrolytes and urea (MeBean & Goldstein, 196’7;Janssens & Cohen, 1967) together with a decrease in urine output and water uptake (Ireland. 1973; Schlisio et al., 1973). In response to hyperosmotic stimuli anurans show changes in the cells of the pars intermedia (Legait & Legait, 1962; Ireland, 1973). There has been no published reports of similar experiments carried out on urodele amphibians The axolotl is the neotenous form of the urodele A~~h~~stornu mesicurzurn and it is confined to an aquatic habitat. The purpose of the present investigation was to study the response of a urodele to a hyperosmotic environment and to compare it with an anuran naturally living in a similar environment, namely a ~rm~~nently aquatic habit.
Table 1. Composition of the serum of Axolotls in different media Site of adaptation Fresh water 30% sea water (5 days) 30% sea water (35 days)
Na (m-equiv~l)
Cl (m-equiv/l)
Urea (mM!f)
97.6 + 0.9 177.2 + 1.5
69.5 + 2.9 133.7 $- 2.1
0.78 k 0.08 8.86 + 0.51
161.6 f 1.4
132.8 + 4.8 10.57 + 1.40
Values are means k S.E.M. Number of animaIs in experiments = 8. 2OOml of either fresh water or 307; sea water which had been allowed to stand for 24 hr. After 3 hr the animals were removed from the test medium and weighed twice; before and after removal of the finger stall which contained
MATERIAL AND h1ETHODS
Axolotis weighing 2%40g were maintained in fresh water at IS-17’C before experimen~tion. They were fed daily on raw liver.
Axolotls were adapted to 30% sea water (100’,;, sea water. salinity, 35”,,3 by direct transfer from fresh water. The animals were kept in 3O”!isea water for 5 or 35 days and during the last 5 days food was withdrawn from the animals.
After 5 days of adaptation, axolotls were anaesthetised by immersion in a 1: 1000 solution of MS 222 (Sandoz) made up in the adaptation medium and a polyethylene cannula was inserted into the cloaca and secured with a purse-string suture. Twenty-four hours later the animals were gently squeezed to void any urine in the cannula. Fresh 30% sea water water dried with a cloth and weighed on an automatic balance to within 10mg. A thin-walled finger stall was connected Fig. 1. Water uptake (cross-hatched bars) and urine fiow to the cloaca1 cannual and the animals reweighed. Axolotls (open bars) in axolotls after adaptation to different media. were transferred individually to glass containers holding Vertical lines represent the S.E.M., number of animals = 5, 415
416
M. P.
IRLLAND
urine. Water uptake and urine volume were calculated using the formulae (1) and (3a) of Bentley & Hellcr (1964). Blood collection. chemical determinattons. histological techniques and estimation of nuclear size of pars intermedia cells were essentially the methods described by Ireland (1973). RESULTS
The results are summarized in Table I. After 5 days in 30”; sea water the concentration of the serum. calculated from the chemical analysis, showed an osmotic concentration of 320mOsm/l which indicated an increase of 152 mOsm/l from fresh water controls. Sodium and chloride ions constituted about 979; of the total difference. Compared with fresh water
Fig. 2. Axolotls Axolotls
adapted
adapted
AMI
I.
M.
SIMONS
adapted animals the axolotls adapted for 35 days in 30”,, sea water showed an elevated serum osmotic concentration of I37 mOsm/l and the level of serum sodium was significantly lower than 5 day adapted sea water animals (P < 0.001). Despite this, the sum electrolytes were the major contributing factor to the elevated osmotic concentration. Serum urea concentration was significantly higher in the sea water adapted axolotls compared with fresh water controls (P < 0.001) and it-was signiticantly the highest in animals adapted to se;~ water for 35 days (P < 0.01). but urea only contributed 3 4”,, to the total osmotic concentration of the strum. After both periods of sea water adaptation the blood osmotic concentration was not significantly different from the external medium.
to fresh water.
Normal
to 30Y0 sea water for 5 days. Note shrinkage of pars intermedia
pars intermcdia.
reduced cells.
nuclear
size and
cytc Jplasmic
Adaptation of the axolotl to a hyperosmotic medium
The results are shown in Fig. I of water uptake and urine volume in axolotls after adaptation to sea water for 5 days. There was no significant difference between water uptake and urine volume in animals adapted to either sea water or fresh water. In the sea water adapted axolotls there was a significant decrease in urine volume (P < 0.05) and water uptake (P < 0.0 I ) compared with fresh water controls of 694: and 66”,, respectively. Pir&Iq
&lIid
Changes occurred in the pars intermedia cells but no obvious changes could be observed in the pars distalis cells. As shown in Figs 2 and 3. the sea water adapted axolotls showed a reduction in cell cytoplasm and a significant reduction in the cell nuclei from X0.6 + 1.4pm’ in the controls to 58.8 i 1.5pm” in 5 days adapted animals and 59.2 k 2.3 in 35 days adapted axolotfs (P < O.OOl). The difference between the sea water adapted axolotls was not significant.
amount of blood urea increase was considerably less in axolotls. Urine flow rate and water uptake in fresh water adapted animals were lower than in X. h~~is in fresh water and the decrease in these parameters after adaptation to sea water for 5 days was less in extent than in X. luecis (Ireland, 1973). The cells of the pars intermedia of axoloti are larger than Xer~~p~4s but they give the same tinctorial afhnity for lead haematoxylin. The overall response to hyperosmotic stimuli in axolotls was similar to Xrr1opu.s in that the cells of the pars intermedia showed cytoplasmic shrinkage and a redllcti~~ll in the cell nuclei of about 27”;,. A decrease in the size of the pars intermedia cells in the lower vertebrates. in response to hyperosmotic stress. has been reported in fish (Olivereau & Ball, 1970) and in X. lawis (Legait & L.egait. 1962; Ireland. 1973). It might be concluded from the present results that axolotls respond to a hy~rosmotic stress in a similar way to X. Lrr~is but the magnitude of these responses is less in axolotls. REFERENCES
DISCUSSION
The tolerance of axolotls to 300, sea water was probably close to the limit of endurance since the blood osmotic concentration did not exceed the concentration of the external medium and under these conditions the animals would be steadily losing water to the environment. X. Iurcis under similar conditions could remain hyperosmotic in 33”;, sea water (Ireland, 1973). Under both sea water adaptation conditions, Na and Cl ions co~ltribllted substantially to the blood osmotic concentrations. Axolotls adapted to Xd water for 35 days appeared to show a reduction in the blood Na levels but the calculated blood osmotic concentration was not significantly different from 5 days adapted animals due to an elevation in the blood urea. These results are in good agreement with similar findings in X. lut)Gs (Ireland, 1973). Elevated blood urea concentrations as a result of hyperosmotic stress appears to he a general reaction of anurans but apart from Rnrw cmc’riwru it does not contribute. to any significant amount, to the osmotic concentration of the blood (Dicker & Elliot, 1973). The rise in the blood urea in sea water adapted axolotls was over IO times compdrcd with a 4 fold increase in X. lurcis under similar conditions (Ireland, 1973). Elevation of the rate limiting enzyme in urea biosynthesis in X. /trer?.s. carbamyl phosphate synthetase, is increased in saline solutions (Janssens & Cohn. 1967). This enzyme has also been identified in A/nhJ~ottru (Mora et ul. 1965). Despite the m~~gnitude of increased response in axolotls compared with X. lu&~. the absolute
417
B~~NTLEY P. J. & HELLI:KH. (1964) The action of neurohy-
pophysial hormones on the water and sodium metabolism of mod& amphibians. J, P~z~si~~~.. Land. 171, 434453.
DICKEKS. E. & ELLIOTTA. B. (1973) Neurohypophysial hormones and homeostasis in the crab-eating frog, Rutu cuncricoro. Horrn. Rvs. 4, X4-260. IR~ILAW M. P. (1973) Studies on the adaptation of Xetropus kwis to hyperosmotic media. Conrp. Biocltc~rz. P~,~.s~~~. 46A. 4694%. JANSSF:NSP. A. & COHLX P. P. (1968) Biosvnthesis of urea , in the estivating African lungfish and in X~topus icxris under conditions of water-shortage. Cornp. Biochm Physiol. 24, 887-X98. LISAIT H. & L~.GAITE. (1962) Relationships between the hypothalamus and pars intermedia in some mammals and amphibians. In ~~~~rf~s~~~~rj~~t~ (Edited by HIILUR H. & CLAW R. B.). pp. 165-171. Academic Press, London. MCBEAN R. L. & GOLDSTLINL. (1967) Omithine-urea cycle activity in Xrrropus lurris: adaptation in saline. Sciotc,c. 1V.Y 157, 931. 932. MORA J.. MARTUSCELLIJ.. ORTIT-PINEDA J. & SOBERON
G. (1965) The regulation of urea-biosynthesis enzymes in vertebrates. ~~~~r~~,~?~. d. 96. Z--35. OLIVI-K~ACI M. & BALL J. N. (1970) Pituitary influences on osmoregulation in teleosts. in Hormones uhd rite Encirorrrtrcrtt (Edited by BENSENG. K. & PHILLIPSJ. G.) pp. 57785. Cambridge University Press, Cambridge. SCHLISIO W.. JCIRSS K. & SPAWHOP L. (1973) Osmo- und Ionenrcgulation von Xrrropus Irreris Daud. nach Adaptation in serschiedenen osmotisch wirksamen Losungen I. Toleranz und ~~asserh~~ushalt. Zool. Jh. Plrtsioi. 77, 235-290
OF THE AXOLOTL (AMBYSTOMA TO A HYPEROSMOTIC MEDIUM
M. P. IRELAND AND I.M. SIMONS Department of Zoology. University College of Wales. Aberystwyth, Wales. U.K. (Rrcriced 22 il4ay 1976) Abstrart---I. .4daptation of the axolotl to a 30”/ sea water medium for 5 and 35 days resulted in an increase in blood electrolytes and urea. 2. After both periods of sea water adaptation the blood osmotic concentration did not exceed that of the external environment and the major ions contributing to the ionic concentration were Na and Cl. 3. After 5 days in 30”; sea water there was a reduction in water uptake and urine flow. 4. A reduction in the size of the cells of the pars intermedia of the pituitary occurred after both periods of sea water adaptation. 5. The results are compared with the aquatic anuran Xe~zo~~s luetLs subjected to similar stimuli.
INTRODUCTION The tolerance of the aquatic anuran Xenopus laeuis to hyperosmotic solutions has been shown to result
in an increase in blood electrolytes and urea (MeBean & Goldstein, 196’7;Janssens & Cohen, 1967) together with a decrease in urine output and water uptake (Ireland. 1973; Schlisio et al., 1973). In response to hyperosmotic stimuli anurans show changes in the cells of the pars intermedia (Legait & Legait, 1962; Ireland, 1973). There has been no published reports of similar experiments carried out on urodele amphibians The axolotl is the neotenous form of the urodele A~~h~~stornu mesicurzurn and it is confined to an aquatic habitat. The purpose of the present investigation was to study the response of a urodele to a hyperosmotic environment and to compare it with an anuran naturally living in a similar environment, namely a ~rm~~nently aquatic habit.
Table 1. Composition of the serum of Axolotls in different media Site of adaptation Fresh water 30% sea water (5 days) 30% sea water (35 days)
Na (m-equiv~l)
Cl (m-equiv/l)
Urea (mM!f)
97.6 + 0.9 177.2 + 1.5
69.5 + 2.9 133.7 $- 2.1
0.78 k 0.08 8.86 + 0.51
161.6 f 1.4
132.8 + 4.8 10.57 + 1.40
Values are means k S.E.M. Number of animaIs in experiments = 8. 2OOml of either fresh water or 307; sea water which had been allowed to stand for 24 hr. After 3 hr the animals were removed from the test medium and weighed twice; before and after removal of the finger stall which contained
MATERIAL AND h1ETHODS
Axolotis weighing 2%40g were maintained in fresh water at IS-17’C before experimen~tion. They were fed daily on raw liver.
Axolotls were adapted to 30% sea water (100’,;, sea water. salinity, 35”,,3 by direct transfer from fresh water. The animals were kept in 3O”!isea water for 5 or 35 days and during the last 5 days food was withdrawn from the animals.
After 5 days of adaptation, axolotls were anaesthetised by immersion in a 1: 1000 solution of MS 222 (Sandoz) made up in the adaptation medium and a polyethylene cannula was inserted into the cloaca and secured with a purse-string suture. Twenty-four hours later the animals were gently squeezed to void any urine in the cannula. Fresh 30% sea water water dried with a cloth and weighed on an automatic balance to within 10mg. A thin-walled finger stall was connected Fig. 1. Water uptake (cross-hatched bars) and urine fiow to the cloaca1 cannual and the animals reweighed. Axolotls (open bars) in axolotls after adaptation to different media. were transferred individually to glass containers holding Vertical lines represent the S.E.M., number of animals = 5, 415
416
M. P.
IRLLAND
urine. Water uptake and urine volume were calculated using the formulae (1) and (3a) of Bentley & Hellcr (1964). Blood collection. chemical determinattons. histological techniques and estimation of nuclear size of pars intermedia cells were essentially the methods described by Ireland (1973). RESULTS
The results are summarized in Table I. After 5 days in 30”; sea water the concentration of the serum. calculated from the chemical analysis, showed an osmotic concentration of 320mOsm/l which indicated an increase of 152 mOsm/l from fresh water controls. Sodium and chloride ions constituted about 979; of the total difference. Compared with fresh water
Fig. 2. Axolotls Axolotls
adapted
adapted
AMI
I.
M.
SIMONS
adapted animals the axolotls adapted for 35 days in 30”,, sea water showed an elevated serum osmotic concentration of I37 mOsm/l and the level of serum sodium was significantly lower than 5 day adapted sea water animals (P < 0.001). Despite this, the sum electrolytes were the major contributing factor to the elevated osmotic concentration. Serum urea concentration was significantly higher in the sea water adapted axolotls compared with fresh water controls (P < 0.001) and it-was signiticantly the highest in animals adapted to se;~ water for 35 days (P < 0.01). but urea only contributed 3 4”,, to the total osmotic concentration of the strum. After both periods of sea water adaptation the blood osmotic concentration was not significantly different from the external medium.
to fresh water.
Normal
to 30Y0 sea water for 5 days. Note shrinkage of pars intermedia
pars intermcdia.
reduced cells.
nuclear
size and
cytc Jplasmic
Adaptation of the axolotl to a hyperosmotic medium
The results are shown in Fig. I of water uptake and urine volume in axolotls after adaptation to sea water for 5 days. There was no significant difference between water uptake and urine volume in animals adapted to either sea water or fresh water. In the sea water adapted axolotls there was a significant decrease in urine volume (P < 0.05) and water uptake (P < 0.0 I ) compared with fresh water controls of 694: and 66”,, respectively. Pir&Iq
&lIid
Changes occurred in the pars intermedia cells but no obvious changes could be observed in the pars distalis cells. As shown in Figs 2 and 3. the sea water adapted axolotls showed a reduction in cell cytoplasm and a significant reduction in the cell nuclei from X0.6 + 1.4pm’ in the controls to 58.8 i 1.5pm” in 5 days adapted animals and 59.2 k 2.3 in 35 days adapted axolotfs (P < O.OOl). The difference between the sea water adapted axolotls was not significant.
amount of blood urea increase was considerably less in axolotls. Urine flow rate and water uptake in fresh water adapted animals were lower than in X. h~~is in fresh water and the decrease in these parameters after adaptation to sea water for 5 days was less in extent than in X. luecis (Ireland, 1973). The cells of the pars intermedia of axoloti are larger than Xer~~p~4s but they give the same tinctorial afhnity for lead haematoxylin. The overall response to hyperosmotic stimuli in axolotls was similar to Xrr1opu.s in that the cells of the pars intermedia showed cytoplasmic shrinkage and a redllcti~~ll in the cell nuclei of about 27”;,. A decrease in the size of the pars intermedia cells in the lower vertebrates. in response to hyperosmotic stress. has been reported in fish (Olivereau & Ball, 1970) and in X. lawis (Legait & L.egait. 1962; Ireland. 1973). It might be concluded from the present results that axolotls respond to a hy~rosmotic stress in a similar way to X. Lrr~is but the magnitude of these responses is less in axolotls. REFERENCES
DISCUSSION
The tolerance of axolotls to 300, sea water was probably close to the limit of endurance since the blood osmotic concentration did not exceed the concentration of the external medium and under these conditions the animals would be steadily losing water to the environment. X. Iurcis under similar conditions could remain hyperosmotic in 33”;, sea water (Ireland, 1973). Under both sea water adaptation conditions, Na and Cl ions co~ltribllted substantially to the blood osmotic concentrations. Axolotls adapted to Xd water for 35 days appeared to show a reduction in the blood Na levels but the calculated blood osmotic concentration was not significantly different from 5 days adapted animals due to an elevation in the blood urea. These results are in good agreement with similar findings in X. lut)Gs (Ireland, 1973). Elevated blood urea concentrations as a result of hyperosmotic stress appears to he a general reaction of anurans but apart from Rnrw cmc’riwru it does not contribute. to any significant amount, to the osmotic concentration of the blood (Dicker & Elliot, 1973). The rise in the blood urea in sea water adapted axolotls was over IO times compdrcd with a 4 fold increase in X. lurcis under similar conditions (Ireland, 1973). Elevation of the rate limiting enzyme in urea biosynthesis in X. /trer?.s. carbamyl phosphate synthetase, is increased in saline solutions (Janssens & Cohn. 1967). This enzyme has also been identified in A/nhJ~ottru (Mora et ul. 1965). Despite the m~~gnitude of increased response in axolotls compared with X. lu&~. the absolute
417
B~~NTLEY P. J. & HELLI:KH. (1964) The action of neurohy-
pophysial hormones on the water and sodium metabolism of mod& amphibians. J, P~z~si~~~.. Land. 171, 434453.
DICKEKS. E. & ELLIOTTA. B. (1973) Neurohypophysial hormones and homeostasis in the crab-eating frog, Rutu cuncricoro. Horrn. Rvs. 4, X4-260. IR~ILAW M. P. (1973) Studies on the adaptation of Xetropus kwis to hyperosmotic media. Conrp. Biocltc~rz. P~,~.s~~~. 46A. 4694%. JANSSF:NSP. A. & COHLX P. P. (1968) Biosvnthesis of urea , in the estivating African lungfish and in X~topus icxris under conditions of water-shortage. Cornp. Biochm Physiol. 24, 887-X98. LISAIT H. & L~.GAITE. (1962) Relationships between the hypothalamus and pars intermedia in some mammals and amphibians. In ~~~~rf~s~~~~rj~~t~ (Edited by HIILUR H. & CLAW R. B.). pp. 165-171. Academic Press, London. MCBEAN R. L. & GOLDSTLINL. (1967) Omithine-urea cycle activity in Xrrropus lurris: adaptation in saline. Sciotc,c. 1V.Y 157, 931. 932. MORA J.. MARTUSCELLIJ.. ORTIT-PINEDA J. & SOBERON
G. (1965) The regulation of urea-biosynthesis enzymes in vertebrates. ~~~~r~~,~?~. d. 96. Z--35. OLIVI-K~ACI M. & BALL J. N. (1970) Pituitary influences on osmoregulation in teleosts. in Hormones uhd rite Encirorrrtrcrtt (Edited by BENSENG. K. & PHILLIPSJ. G.) pp. 57785. Cambridge University Press, Cambridge. SCHLISIO W.. JCIRSS K. & SPAWHOP L. (1973) Osmo- und Ionenrcgulation von Xrrropus Irreris Daud. nach Adaptation in serschiedenen osmotisch wirksamen Losungen I. Toleranz und ~~asserh~~ushalt. Zool. Jh. Plrtsioi. 77, 235-290