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Nitrogen excretion in intra- and extrauterine larvae of the ovoviviparous salamander, Salamandra salamandra (L.) (amphibia, urodela)
(‘ornp. B,oclwm PIII.PIOI. Vol. 70A. PP. 563 lo 565. 1981 Pnnted I” Great Br~ra~n. All nghts reserved
Copjrqhf
030%Y629,Xl.l20563-03802.00,‘O 0 1981 Pergamon Press Ltd
NITROGEN EXCRETION IN INTRA- AND EXTRAUTERINE LARVAE OF THE OVOVIVIPAROUS SALAMANDER, SALAMANDRA SALAMANDRA (L.) (AMPHIBIA, URODELA) JOCHEN SCHINDELMEISER’ and HARTMUT GREVEN’ ‘Anatomisches
und ‘Zoologisches
Institut
der Universitgt
(Receicrd
Miinster,
D-4400 Miinster,
West-Germany
27 April 1981)
Abstract-l. Uterine fluid of pregnant females of the ovoviviparous Sulamandra sulamandru contains rather high amounts of urea (576 mmol/l) as compared to non pregnant females (150 mmol/l). 2. The same applies to the urea content of the plasma (pregnant female 19.4mmol/l; non pregnant female 7.8 mmol/l). 3. These data indicate ureotelism in intrauterine larvae. 4. During extrauterine larval life ammonia excretion was found to be slightly higher than urea excretion. 5. During metamorphosis ureotelism again becomes predominant. 6. The capacity for urea synthesis in intrauterine larvae can be regarded as an adaptation to the prolonged stay in utero, where availability of fluid is greatly limited.
MATERIAL
INTRODUCTION
The larvae of amphibians are generally regarded as being ammonotelic; this ammonotelism changes to ureotelism in the course of metamorphosis, particularly in terrestrial forms (e.g. Munro, 1953; Cohen, 1966; Balinsky, 1970). Besides this transition to a terrestric way of life being dependent on development, environmental conditions such as water shortage or osmotic stress have been studied. They lead to ureotelism in amphibia normally known as ammonotelic (e.g. Balinsky et al., 1967a,b; Seiter ef al., 1978). Furthermore it has been suggested that ureotelism seems to be widespread in the larvae of such species which spent their development at least partly in a restricted volume of water (e.g. eggs and larvae of land-nesting Leptodactylids, Candelas & Gomez, 1963; Martin & Cooper, 1972; Shoemaker & McClanahan, 1973) or in ephemeral ponds (e.g. the larvae of &q&opus-species, Jones, 1980). An extreme water shortage during development is also found in some viviparous and ovoviviparous amphibians, where the offspring develops in brood pouches or specialized parts of the female genital tract. Osmoregulatory capacities or nitrogen excretion in those larvae have not been investigated so far to our knowledge (see also Deyrup, 1964). In the terrestrial ovoviviparous European fire salamander, Salamundra salamandra, the offspring has to develop during an up to 14 months lasting gestation period (Joly, 1968) in a rather small amount of fluid present in the uterus, a fact which involves problems regarding the disposal of nitrogenous wastes. Studying some metabolic and morphological adaptations to the intrauterine environment of the larvae of S. salumandra, the present paper deals with the nitrogen excretion of intra- and extrauterine larvae.
AND
METHODS
Pregnant (3) and non pregnant (1) females of S. sahmundra were sacrificed in December and January. Fully developed larvae (for definition see Joly, 1968) were removed from the uterus and placed into tap water. Samples of uterine fluid were drawn from the uterus of pregnant and non pregnant females with calibrated capillary micropipettes; the blood of the same specimens was collected in heparinized micropipettes from the truncus arteriosus and the plasma was separated by centrifugation. Uterine fluid and blood plasma were appropriately diluted and analyzed for urea and ammonia. The total excretion of nitrogen and the relative amounts of urea and ammonia produced by larval S. salammdra were measured at three different age groups: (1) larvae taken directly from the uterus, (2) larvae which spent 16 days and (3) larvae which spent 51 days in tap water (they were shortly before metamorphosis) after the artificial birth. Before analysis the larvae were kept for 24 hr in 10ml tap water at 15°C without feeding during this time. Aliquots (200~1) were taken from the water, filtered to remove faecal material and then analyzed for urea and ammonia. Urea was determined calorimetrically with the commercial test kit of Boehringer (Mannheim).according to Fawcett & Scott (1960) with some slight modifications. For the determination of ammonia-nitrogen, urease was replaced by distilled water; the concentration of urea-nitrogen was calculated by subtracting ammonia-nitrogen from total waste nitrogen. Urea and ammonium chloride solutions were used as standards. Preliminary tests on urea and ammonia tolerance were performed using two groups of 10 larvae each immediately after having been removed from the uterus. The first group was placed in 100 ml of tap water containing 30 mmol/l of urea. This concentration was doubled daily to 60, 120 and 240 mmol/l. The second group was placed in 100 ml of tap water (buffered to pH 7.0 with Na,HPO,) containing 5 mmol/l NH,CI. This concentration was raised to 10 mmol/l after 24 hr. 563
JOCHEN SCHINDELMEISERand HARTMUT GREVEN
564
Table 1. Mean values of molar concentrations of urea nitrogen, ammonia nitrogen and total nitrogen in the uterine fluid of pregnant and
non pregnant females of S. salamandra Urea nitrogen mmol/l (percentage of total nitrogen)
Total waste nitrogen mmol/l
Pregnant
51.6 (93YA)
4.3 (77,))
61.9
Non pregnant
15.0 (71:,,)
6.1 (29”,,)
21.2
RESULTS
Nitroyenous wustes in the uterine jluid The molar concentration of urea nitrogen was nearly 4 times higher in the uterine fluid of the pregnant females compared to that of the non pregnant females (Table 1). The amount of ammonia nitrogen of the pregnant as well as of the non pregnant females, was found to be at about a 10% level of the nitrogen in the uterine fluid of the pregnant females excreted, as urea. Regarding the total nitrogen content in the uterine fluid, a nearly three times higher amount could be demonstrated for the pregnant animals: urea nitrogen formed 937; and ammonia nitrogen 7”,, of the nitrogen excreted. Nitrogenous wustes in the blood plusma A concentration of 19.4mmol/l urea nitrogen was measured in the serum of the pregnant females, a concentration that is lowered by a factor three compared to the uterine fluid (see Table 1). A similar relation was found for the non pregnant females, in which the concentration of urea nitrogen in the serum only amounts to 7.8 mmol/l. Any ammonia nitrogen present in the blood plasma could not be determined because of an interference of the test with the coloured serum. Nitroyen excretion
Ammonia nitrogen mmol/l (percentage of total nitrogen)
of extrauterine
same larvae showed a reduced excretion of total waste nitrogen within 24 hr with a percentage of 46% urea nitrogen and 54% ammonia nitrogen. On the 51st day of extrauterine life (larvae were just before climax of metamorphosis) there was again a relatively high output of total waste nitrogen, slightly higher than at the beginning of the experiments. Urea nitrogen again constituted the majority of nitrogen excretion (Table 2). The absolute amount of ammonia nitrogen continuously increased from 84 to 147 mmol/l tapwater, whereas the relative percentage is the highest after 16 days. Regarding urea nitrogen, the absolute and relative amounts are the highest at 0 and 51 days. Tolerunce of the larvae to urea and ammonia Larvae exposed to a concentration of 5 mmol/l NH&l in tap water-a similar concentration was found in the uterine fluid-showed no mortality within 24 hr. An increase of this concentration up to 10 mmol/l NH4C1 resulted in a mortality of 100% within 26 hr. All larvae placed for 24 hr into 30mmol/l urea, which corresponds to the approx. 60mmol/l urea nitrogen measured in the uterine fluid of pregnant females, survived. Increasing this concentration to 120,240 and 480 mmol/l urea nitrogen, the larvae survived for at least three days.
larvae
When larvae were placed into a defined small volume of tap water immediately after excision from the uterus, a relatively high excretion of nitrogen with about 80% of urea nitrogen and 20% ammonia nitrogen could be observed (Table 2). Sixteen days later the
DISCUSSION
Considering the long period of gestation (12-14 months) in S. salamandra as well as the limited availability of water in the female genital tract and the
Table 2. Mean values of molar concentrations of urea nitrogen, ammonia nitrogen and total waste nitrogen expressed as the excretion of one larva placed in 10 ml of tap water during 24 hr, at 0, 16, and 51 days after artificial birth Urea nitrogen ~mol/l (percentage of total nitrogen) 0
days
329 (80%)
16 days (4:;) 51 days
304 (67%)
Ammonia nitrogen pmol/l (percentage of total nitrogen)
Total waste nitrogen pmol/l
84 (20%)
413
105 (54%) 147 (33%)
194 451
Nitrogen excretion in larval Salamandra
high toxicity of ammonia, ammoniotelism in intrauterine larvae appears a priori to be improbable. The high amount of urea nitrogen (57.6 mmol~) and the smal1 amount of ammonia nitrogen (4.3 mmol/l) measured in the uterine fluid of pregnant females, as well as the comparatively small amount of both substances in the uterine fluid of non pregnant females, indicate larval ureotelism. This suggestion is also confirmed by the sensitivity of the larvae to ammonia. Unfed extrauterine larvae could tolerate a concentration of 5 mmol/i ammonia (as NH,Cl) for at least 24 hr. the con~ntration of which corresponds well to that of the uterine fluid in pregnant females. A double concentration already results in 100% mortality. Thus, survival of intrauterine larvae is only guaranteed by excreting less toxic nitrogenous wastes in form of urea to offset excessive ammonia concentrations. Urea can be tolerated by extrauterine fed larvae up to at least 480 mmol/l urea nitrogen (ea. 205 mGsm), the amount of which exceeds the urea content in the uterine fluid by a factor of about 8. This tolerance may also be interpreted as an adaptation to the intrauterine environment, in which urea content (e.g. in the time of augmented vitellary protein metabolism during the first period of development) may be higher than the levels measured in December and January. Such changes, however, are not known at the present time. The ability of the intrauterine larvae to a~umulate urea in the serum or in tissues remains to be investigated, too. Such an ability has been demonstrated for the larvae of leptodactylid frogs (Candelas & Gomez, 1963) and some other amphibians (Xenopus lueuis, Balinsky et al., 1967b; Scaphiopus couchi and S. multiplicatus, Jones, 1980). The increased concentration of urea nitrogen in the blood plasma of the pregnant females can easily be interpreted as a consequence of the considerable amount in the uterine fluid. Obviously urea reaches the circulatory system via the tiny one-layered uterine epithelium and the numerous blood vessels underlying it (see @even, 1980). Urea excretion has also been found in larvae investigated immediately after (artificial) birth. Within 24 hr there was a relative high output of nitrogen (where urea constituted about 80% of the urea plus ammonia nitrogen), a fact that may be correlated with a metabolism of own as well as of still available vitellary proteins. Similar conditions have been described in Leptodnctylus bufonius larvae by Shoemaker & McClanahan (1973). The total amount of nitrogen excreted was diminished, when 16 days old fed larvae were investigated. Urea and ammonia were excreted in about equal proportions. Predominant ureotelism is not obvious. This may be regarded as a consequence of feeding the lar-
565
vae, which, thus, do not use own proteins as energy source (see also Shoemaker & McClanahan, 1973). The increased amount of excreted toxic ammonia can be removed by the water now sufficiently available. Larvae just before the climax of metamorphosis (51 days out of the uterus) again show a clear shift to ureotelism, as it can be expected for this terrestrial urodelan species. Here, catabolism of endogenous proteins, especially present in anurans during metamorphosis but also existent in urodels-fire salamander larvae stop feeding during metamorphosisgives rise to the higher excretion rate of nitrogen observed. REFERENCES
E. L., LOE C. G. L. & VAN DER SCHAANSG. S. (1967a) Urea cycle enzymes and urea excretion during the development and metamorphosis of Xenopus laevis. Comp. Biochem. Physiol. 22, 53-57. BALINSKYJ. B., CHORITZE. L., LOE C. C. L. & VAN DER Sc~arjs G. S. (1967b) Amino acid metabolism and urea synthesis in naturally aestivating Xenorus /aeris. Camp. Biochem. Physiol. 22, 59-68. BALINSKY J. B. (1970) Nitrogen metabolism in amphibians. In Comparative Biochemistry qf Nitrogen Metabolism (Edited by CAMPBELJ_ J. W.) Vol. II, pp. 519-638. Academic Press, New York. CANDELAS G. C. & GOMEZM. (1963) Nitrogen excretion in tadpoles of Leptodactylus alhilahris and Ranu catesbiana. BALINSKY J. B., CHORITZ
Am. 2001. 3, 521.-522. P. P. (1966) Biochemical aspects of metamorphosis:
COHEN
transition from ammonote~ism to ureotelism. Hurse) Lect. Ser. 60, 119-164. DEYRUPI. (1964) Water balance and kidney. In Physioiog? of the Amphibia (Edited by MOOREJ. A.), pp. 251-328. Academic Press, New York. FAWCETTJ. K. & SCOTT J. E. (1960) A rapid and precise method for the determination of urea. J. clin. Parh. 13, 156-159. GREVEN H. (1980) Ultrahistochemical and autoradiographic evidence for epitheiiaf transport in the uterus of the ovoviviparous salamander, Sula#zandru .sa~~~ma~dru (L.) (Amphibia, Urodela). Cell. 7%~. Res. 212, 147-162. JOLY J. (1968) Dorm&es ecologiques sur la salamandra tachetee Salamandra salamandra (L.) Ann/s Sci. nat. Zoo\. Paris 10, 301-366. JONESR. M. (1980) Nitrogen excretion by Swphious tadpoles in ephemeral ponds. Physiol. Zoo/. 53, 26-3 1. MARTINA. A. & C~IIPERA. K. (1972) The ecology of terrestrial anuran eggs, genus Crinicr (Leptodactylidae). Cop& 1972, 163-168. MUNRO A. F. (19.53)The ammonia and urea excretion of different species of Amphibia during their development and metamorphosis. Biochem. J. 54, 29-36, SEITERP., SCHULTHEISS H. & HANKE W. (1978) Osmotic stress and excretion of ammonia and urea in Xenopns laevis. Comp. Biochem. Physiol. 6lA, 571.-576.
SHOEMAKER V. H. & MCCLANAHANL. L. (1973) Nitrogen excretion in the larvae of a land-nesting frog (Lrptodartyks bufbnius). Camp. Biochem. Physiol. 44A, 1149-l156.
Copjrqhf
030%Y629,Xl.l20563-03802.00,‘O 0 1981 Pergamon Press Ltd
NITROGEN EXCRETION IN INTRA- AND EXTRAUTERINE LARVAE OF THE OVOVIVIPAROUS SALAMANDER, SALAMANDRA SALAMANDRA (L.) (AMPHIBIA, URODELA) JOCHEN SCHINDELMEISER’ and HARTMUT GREVEN’ ‘Anatomisches
und ‘Zoologisches
Institut
der Universitgt
(Receicrd
Miinster,
D-4400 Miinster,
West-Germany
27 April 1981)
Abstract-l. Uterine fluid of pregnant females of the ovoviviparous Sulamandra sulamandru contains rather high amounts of urea (576 mmol/l) as compared to non pregnant females (150 mmol/l). 2. The same applies to the urea content of the plasma (pregnant female 19.4mmol/l; non pregnant female 7.8 mmol/l). 3. These data indicate ureotelism in intrauterine larvae. 4. During extrauterine larval life ammonia excretion was found to be slightly higher than urea excretion. 5. During metamorphosis ureotelism again becomes predominant. 6. The capacity for urea synthesis in intrauterine larvae can be regarded as an adaptation to the prolonged stay in utero, where availability of fluid is greatly limited.
MATERIAL
INTRODUCTION
The larvae of amphibians are generally regarded as being ammonotelic; this ammonotelism changes to ureotelism in the course of metamorphosis, particularly in terrestrial forms (e.g. Munro, 1953; Cohen, 1966; Balinsky, 1970). Besides this transition to a terrestric way of life being dependent on development, environmental conditions such as water shortage or osmotic stress have been studied. They lead to ureotelism in amphibia normally known as ammonotelic (e.g. Balinsky et al., 1967a,b; Seiter ef al., 1978). Furthermore it has been suggested that ureotelism seems to be widespread in the larvae of such species which spent their development at least partly in a restricted volume of water (e.g. eggs and larvae of land-nesting Leptodactylids, Candelas & Gomez, 1963; Martin & Cooper, 1972; Shoemaker & McClanahan, 1973) or in ephemeral ponds (e.g. the larvae of &q&opus-species, Jones, 1980). An extreme water shortage during development is also found in some viviparous and ovoviviparous amphibians, where the offspring develops in brood pouches or specialized parts of the female genital tract. Osmoregulatory capacities or nitrogen excretion in those larvae have not been investigated so far to our knowledge (see also Deyrup, 1964). In the terrestrial ovoviviparous European fire salamander, Salamundra salamandra, the offspring has to develop during an up to 14 months lasting gestation period (Joly, 1968) in a rather small amount of fluid present in the uterus, a fact which involves problems regarding the disposal of nitrogenous wastes. Studying some metabolic and morphological adaptations to the intrauterine environment of the larvae of S. salumandra, the present paper deals with the nitrogen excretion of intra- and extrauterine larvae.
AND
METHODS
Pregnant (3) and non pregnant (1) females of S. sahmundra were sacrificed in December and January. Fully developed larvae (for definition see Joly, 1968) were removed from the uterus and placed into tap water. Samples of uterine fluid were drawn from the uterus of pregnant and non pregnant females with calibrated capillary micropipettes; the blood of the same specimens was collected in heparinized micropipettes from the truncus arteriosus and the plasma was separated by centrifugation. Uterine fluid and blood plasma were appropriately diluted and analyzed for urea and ammonia. The total excretion of nitrogen and the relative amounts of urea and ammonia produced by larval S. salammdra were measured at three different age groups: (1) larvae taken directly from the uterus, (2) larvae which spent 16 days and (3) larvae which spent 51 days in tap water (they were shortly before metamorphosis) after the artificial birth. Before analysis the larvae were kept for 24 hr in 10ml tap water at 15°C without feeding during this time. Aliquots (200~1) were taken from the water, filtered to remove faecal material and then analyzed for urea and ammonia. Urea was determined calorimetrically with the commercial test kit of Boehringer (Mannheim).according to Fawcett & Scott (1960) with some slight modifications. For the determination of ammonia-nitrogen, urease was replaced by distilled water; the concentration of urea-nitrogen was calculated by subtracting ammonia-nitrogen from total waste nitrogen. Urea and ammonium chloride solutions were used as standards. Preliminary tests on urea and ammonia tolerance were performed using two groups of 10 larvae each immediately after having been removed from the uterus. The first group was placed in 100 ml of tap water containing 30 mmol/l of urea. This concentration was doubled daily to 60, 120 and 240 mmol/l. The second group was placed in 100 ml of tap water (buffered to pH 7.0 with Na,HPO,) containing 5 mmol/l NH,CI. This concentration was raised to 10 mmol/l after 24 hr. 563
JOCHEN SCHINDELMEISERand HARTMUT GREVEN
564
Table 1. Mean values of molar concentrations of urea nitrogen, ammonia nitrogen and total nitrogen in the uterine fluid of pregnant and
non pregnant females of S. salamandra Urea nitrogen mmol/l (percentage of total nitrogen)
Total waste nitrogen mmol/l
Pregnant
51.6 (93YA)
4.3 (77,))
61.9
Non pregnant
15.0 (71:,,)
6.1 (29”,,)
21.2
RESULTS
Nitroyenous wustes in the uterine jluid The molar concentration of urea nitrogen was nearly 4 times higher in the uterine fluid of the pregnant females compared to that of the non pregnant females (Table 1). The amount of ammonia nitrogen of the pregnant as well as of the non pregnant females, was found to be at about a 10% level of the nitrogen in the uterine fluid of the pregnant females excreted, as urea. Regarding the total nitrogen content in the uterine fluid, a nearly three times higher amount could be demonstrated for the pregnant animals: urea nitrogen formed 937; and ammonia nitrogen 7”,, of the nitrogen excreted. Nitrogenous wustes in the blood plusma A concentration of 19.4mmol/l urea nitrogen was measured in the serum of the pregnant females, a concentration that is lowered by a factor three compared to the uterine fluid (see Table 1). A similar relation was found for the non pregnant females, in which the concentration of urea nitrogen in the serum only amounts to 7.8 mmol/l. Any ammonia nitrogen present in the blood plasma could not be determined because of an interference of the test with the coloured serum. Nitroyen excretion
Ammonia nitrogen mmol/l (percentage of total nitrogen)
of extrauterine
same larvae showed a reduced excretion of total waste nitrogen within 24 hr with a percentage of 46% urea nitrogen and 54% ammonia nitrogen. On the 51st day of extrauterine life (larvae were just before climax of metamorphosis) there was again a relatively high output of total waste nitrogen, slightly higher than at the beginning of the experiments. Urea nitrogen again constituted the majority of nitrogen excretion (Table 2). The absolute amount of ammonia nitrogen continuously increased from 84 to 147 mmol/l tapwater, whereas the relative percentage is the highest after 16 days. Regarding urea nitrogen, the absolute and relative amounts are the highest at 0 and 51 days. Tolerunce of the larvae to urea and ammonia Larvae exposed to a concentration of 5 mmol/l NH&l in tap water-a similar concentration was found in the uterine fluid-showed no mortality within 24 hr. An increase of this concentration up to 10 mmol/l NH4C1 resulted in a mortality of 100% within 26 hr. All larvae placed for 24 hr into 30mmol/l urea, which corresponds to the approx. 60mmol/l urea nitrogen measured in the uterine fluid of pregnant females, survived. Increasing this concentration to 120,240 and 480 mmol/l urea nitrogen, the larvae survived for at least three days.
larvae
When larvae were placed into a defined small volume of tap water immediately after excision from the uterus, a relatively high excretion of nitrogen with about 80% of urea nitrogen and 20% ammonia nitrogen could be observed (Table 2). Sixteen days later the
DISCUSSION
Considering the long period of gestation (12-14 months) in S. salamandra as well as the limited availability of water in the female genital tract and the
Table 2. Mean values of molar concentrations of urea nitrogen, ammonia nitrogen and total waste nitrogen expressed as the excretion of one larva placed in 10 ml of tap water during 24 hr, at 0, 16, and 51 days after artificial birth Urea nitrogen ~mol/l (percentage of total nitrogen) 0
days
329 (80%)
16 days (4:;) 51 days
304 (67%)
Ammonia nitrogen pmol/l (percentage of total nitrogen)
Total waste nitrogen pmol/l
84 (20%)
413
105 (54%) 147 (33%)
194 451
Nitrogen excretion in larval Salamandra
high toxicity of ammonia, ammoniotelism in intrauterine larvae appears a priori to be improbable. The high amount of urea nitrogen (57.6 mmol~) and the smal1 amount of ammonia nitrogen (4.3 mmol/l) measured in the uterine fluid of pregnant females, as well as the comparatively small amount of both substances in the uterine fluid of non pregnant females, indicate larval ureotelism. This suggestion is also confirmed by the sensitivity of the larvae to ammonia. Unfed extrauterine larvae could tolerate a concentration of 5 mmol/i ammonia (as NH,Cl) for at least 24 hr. the con~ntration of which corresponds well to that of the uterine fluid in pregnant females. A double concentration already results in 100% mortality. Thus, survival of intrauterine larvae is only guaranteed by excreting less toxic nitrogenous wastes in form of urea to offset excessive ammonia concentrations. Urea can be tolerated by extrauterine fed larvae up to at least 480 mmol/l urea nitrogen (ea. 205 mGsm), the amount of which exceeds the urea content in the uterine fluid by a factor of about 8. This tolerance may also be interpreted as an adaptation to the intrauterine environment, in which urea content (e.g. in the time of augmented vitellary protein metabolism during the first period of development) may be higher than the levels measured in December and January. Such changes, however, are not known at the present time. The ability of the intrauterine larvae to a~umulate urea in the serum or in tissues remains to be investigated, too. Such an ability has been demonstrated for the larvae of leptodactylid frogs (Candelas & Gomez, 1963) and some other amphibians (Xenopus lueuis, Balinsky et al., 1967b; Scaphiopus couchi and S. multiplicatus, Jones, 1980). The increased concentration of urea nitrogen in the blood plasma of the pregnant females can easily be interpreted as a consequence of the considerable amount in the uterine fluid. Obviously urea reaches the circulatory system via the tiny one-layered uterine epithelium and the numerous blood vessels underlying it (see @even, 1980). Urea excretion has also been found in larvae investigated immediately after (artificial) birth. Within 24 hr there was a relative high output of nitrogen (where urea constituted about 80% of the urea plus ammonia nitrogen), a fact that may be correlated with a metabolism of own as well as of still available vitellary proteins. Similar conditions have been described in Leptodnctylus bufonius larvae by Shoemaker & McClanahan (1973). The total amount of nitrogen excreted was diminished, when 16 days old fed larvae were investigated. Urea and ammonia were excreted in about equal proportions. Predominant ureotelism is not obvious. This may be regarded as a consequence of feeding the lar-
565
vae, which, thus, do not use own proteins as energy source (see also Shoemaker & McClanahan, 1973). The increased amount of excreted toxic ammonia can be removed by the water now sufficiently available. Larvae just before the climax of metamorphosis (51 days out of the uterus) again show a clear shift to ureotelism, as it can be expected for this terrestrial urodelan species. Here, catabolism of endogenous proteins, especially present in anurans during metamorphosis but also existent in urodels-fire salamander larvae stop feeding during metamorphosisgives rise to the higher excretion rate of nitrogen observed. REFERENCES
E. L., LOE C. G. L. & VAN DER SCHAANSG. S. (1967a) Urea cycle enzymes and urea excretion during the development and metamorphosis of Xenopus laevis. Comp. Biochem. Physiol. 22, 53-57. BALINSKYJ. B., CHORITZE. L., LOE C. C. L. & VAN DER Sc~arjs G. S. (1967b) Amino acid metabolism and urea synthesis in naturally aestivating Xenorus /aeris. Camp. Biochem. Physiol. 22, 59-68. BALINSKY J. B. (1970) Nitrogen metabolism in amphibians. In Comparative Biochemistry qf Nitrogen Metabolism (Edited by CAMPBELJ_ J. W.) Vol. II, pp. 519-638. Academic Press, New York. CANDELAS G. C. & GOMEZM. (1963) Nitrogen excretion in tadpoles of Leptodactylus alhilahris and Ranu catesbiana. BALINSKY J. B., CHORITZ
Am. 2001. 3, 521.-522. P. P. (1966) Biochemical aspects of metamorphosis:
COHEN
transition from ammonote~ism to ureotelism. Hurse) Lect. Ser. 60, 119-164. DEYRUPI. (1964) Water balance and kidney. In Physioiog? of the Amphibia (Edited by MOOREJ. A.), pp. 251-328. Academic Press, New York. FAWCETTJ. K. & SCOTT J. E. (1960) A rapid and precise method for the determination of urea. J. clin. Parh. 13, 156-159. GREVEN H. (1980) Ultrahistochemical and autoradiographic evidence for epitheiiaf transport in the uterus of the ovoviviparous salamander, Sula#zandru .sa~~~ma~dru (L.) (Amphibia, Urodela). Cell. 7%~. Res. 212, 147-162. JOLY J. (1968) Dorm&es ecologiques sur la salamandra tachetee Salamandra salamandra (L.) Ann/s Sci. nat. Zoo\. Paris 10, 301-366. JONESR. M. (1980) Nitrogen excretion by Swphious tadpoles in ephemeral ponds. Physiol. Zoo/. 53, 26-3 1. MARTINA. A. & C~IIPERA. K. (1972) The ecology of terrestrial anuran eggs, genus Crinicr (Leptodactylidae). Cop& 1972, 163-168. MUNRO A. F. (19.53)The ammonia and urea excretion of different species of Amphibia during their development and metamorphosis. Biochem. J. 54, 29-36, SEITERP., SCHULTHEISS H. & HANKE W. (1978) Osmotic stress and excretion of ammonia and urea in Xenopns laevis. Comp. Biochem. Physiol. 6lA, 571.-576.
SHOEMAKER V. H. & MCCLANAHANL. L. (1973) Nitrogen excretion in the larvae of a land-nesting frog (Lrptodartyks bufbnius). Camp. Biochem. Physiol. 44A, 1149-l156.