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The nitrogenous products of degradation—Ammonia, urea and uric acid—In the hemolymph of the snail Biomphalaria glabrata
Comp. Biochem. Physiol., 1975, VoL 51A,pp. 407 to 411. PergamonPress. Printed in Great Britain
THE NITROGENOUS PRODUCTS OF DEGRADATION-AMMONIA, UREA AND URIC ACID--IN THE HEMOLYMPH OF THE SNAIL BIOMPHALARIA GLABRATA W. BECKER AND H. SCHMALE Zoologisches Institut und Zoologisches Museum der Universit~it Hamburg, Hamburg, Germany (Received 18 March 1974) Absa-act--1. The concentrations of ammonia, urea and uric acid in the hemolymph of Biomphalaria glabrata were studied. 2. In normally fed snails, the concentration of NHs-N varied between 0"031 and 0.145 mg/100 ml with a mean of 0.079 rag/100 ml. After a starvation period of 5 days, the concentration of NHa-N rose to an average value of 0.111 mg/100 ml. The individual values lay between 0"056 and 0.212 mg/ 100 ml. 3. In normally fed snails, the urea concentration fluctuated between 0 and 1-05mg/100 ml with a mean of 0.160 mg/100 ml. Within a starvation period of 5 days the urea concentration increased on average to 5.12 mg/100 ml. The individual values varied between 0-053 and 16.5 mg/100 ml. 4. Uric acid could neither be detected in the hemolymph of normally fed nor of starved snails. 5. The possible causes of the large individual variation of NHa-N and urea concentration, as well as of the concentration increase of those compounds containing N during the starvation periods, are discussed.
INTRODUCTION
MATERIALS AND METHODS
THE NITROGENOUSdegradation products, ammonia, urea and uric acid, are generally transported with the body fluids to places where they are excreted or further processed. Up to now research into the occurrence of these substances in the hemolymph of molluscs has been very scanty (Myers, 1920; Delaunay, 1931; Florkin & Houet, 1938; Florkin & Renwart, 1939; Friedl, 1961; Vasu & Giese, 1966; Potts, 1965; Little, 1968; de Jorge et al., 1965). The amounts of nitrogenous degradation products produced are indicative of the activity of the protein and nucleic acid metabolism of an animal so that any unusual stress imposed on the latter must make itself apparent in a change in the amounts of ammonia, urea and uric acid produced. Such stress can be brought about by phases of strong reproductive activity or by parasitic invasion. The latter is an aspect which must be considered, especially in the case of Biomphalaria glabrata, the snail we examined, as the larval stages of Schistosoma mansoni developing in this snail show a quick rate of reproduction (Becker, 1968). Heightened activity of the protein metabolism must, however, also appear when, as a result of starvation, the snails must use the materials of their own bodies. In this account the amounts of nitrogenous degradation products in normally fed animals and in snails starved for 5 days are determined.
Snails of the species Biomphalaria glabrata to be examined were kept at a temperature of 26 + 2°C. Their only food consisted of fresh lettuce leaves, fed as required. Fasting snails were kept in aquaria and these were cleaned every second day to prevent any growth of algae which might have served the animals as food. For the examination snails were chosen whose shell measured 14-20 mm dia. After thorough cleaning and drying the shell of the last but one whorl from the inside was carefully lifted and, after a quick puncture of the body wall by a glass capillary tube, the hemolymph was extracted. According to the size of the snail approximately 50-100 ~1 of hemolymph could be obtained. The hemolymph was stored in a closed container in an icebath until subsequent use. NH3-N analysis NHa-N analysis was carded out according to the method of Miiller-Beissenhirtz (1965), with several modifications made necessary by the small volume of the specimens. The phenol and hypochlorite reagents required (0-106 M phenol; 0.17 mM sodium nitroprussate and 11 mM NaOC1; 0"125 N NaOH) were taken from the Biochemica test combination "Urea" (Boehringer, Mannheim). Urea analysis For urea analysis the method of Fawcett & Scott (1960) and Bernt & Bergmeyer (1970) was used. The protein was removed from the hemolymph with 10%
407
408
W. BECKERAND H. S C r ~ . E
trichioracetic acid. Neutralization was carried out with potassium hydrogenous carbonate solution. Phenol and hypochlorite reagent, urease (2 mg/#l; 0.05 M phosphate buffer, pH---6.5) and a standard urea solution were taken from the Biochemica test combination "Urea" (Boehringer, Mannheim).
Uric acid analysis Uric acid analysis was carried out according to the method of Praetorius & Poulsen (1953). It was possible to use the reagents of the Biochemica test combination "Uric acid" (Boehringer, Mannheim) (borate buffer 0.2 M, pH = 9.5; uricase 2 mg/ml in 50% glycerine). ~S~TS
F o r the analysis of NH3-N, urea and uric acid in the hemolymph of B. glabrata, animals were used which had been differently fed as follows: Test Series A contains animals which were fed normally and were taken off lettuce leaves directly before the hemolymph was extracted. The snails of Test Series B had been starved for 5 days before the hemolymph was extracted.
Results of NHa-N analysis The results of NH3-N analysis are summarized in Table 1. Comparison of both test series with the t-test gave: m = 39,
t = 2.85,
Comparison of both test series with the F-test showed: quotient of variance F = 297.3,
P<0.001.
Interpretation of the results shows that the normally fed animals of Series A belong, with regard to their urea concentration, to a different group than snails of Test Series B, which had been starved for 5 days. A comparison of the mean values using the t-test was not possible, as the characteristic examined in both test series showed a different variability. A tendency already revealed in the NHs-N analysis, namely a higher NH3-N concentration in starved snails in comparison to normally fed ones, is found to a much greater extent in the urea analysis: the urea concentration in the hemolymph of snails starved for 5 days is approximately thirty times higher than in the case of normally fed animals.
Results of uric acid analysis F o r uric acid analysis thirty-three snails were examined. Seventeen of them had been fed normally and sixteen had been starved for 5 days. In no case could uric acid be found by the method used, which suggested a concentration of uric acid below 30/zg/100 ml. DISCUSSION
P<0.01.
As statistical interpretation of the results of the examination shows, the NI-Ia-N concentration in the hemolymph of normally fed snails is significantly lower than that of snails starved for 5 days.
Results of urea analysis The results of urea analysis for Test Series A and B are summarized in Table 2.
A comparison of the NH3-N concentration in the body fluid of various molluscs shows that the NH3-N concentration in the hemolymph o f B. glabrata is very low, lying at approximately 0-1 mg/ 100 ml. Only Florkin & Houet (1938) found an even lower NH3 value of 0.51-0.071mg/100ml in Anodonta cygnea. The highest N H s - N concentrations have been found in the terrestrial pulmonates, e.g. in Helix pomatia with 1.2 and in Arion rufus
Table 1. Results of NH3-N analysis
Test Series
Unit
No. of animals examined
A B
/~g/100 ml #.g/100 ml
19 22
Peak values
Mean value
31-145 56-212
79.2 111.5
Standard deviation of the mean value 7.5 8.3
Standard deviation 32.9 38.9
Table 2. Results of urea analysis
Test Series
Unit
No. of animals examined
A B
e.g/100 ml ~.g/100 ml
19 22
Peak values 0-1050 53-16,500
Mean value 160.3 5121
Standard deviation of the mean value 63.5 1017
Standard deviation 276"8 4773
Nitrogenous degradation products in snail hemolymph with 1.4 mg/100 ml NH3-N (Delaunay, 1951) and in the carnivorous cephalopods which are considered ammoniotelic. Thus Delaunay (1927) found 2.44.8 rag/100 ml NHa-N in Sepia officinalis. There are a few findings about the concentration of urea in the hemolymph. De Jorge et al. (1965) state a high value of 30.5 mg/100 ml of urea for Strophocheilus oblongus. On the other hand, Tramell & Campell (1970) were unable to identify any urea in S. oblongus. Vasu & Giese (1966) found approximately 9mg/100ml of urea in Cryptochiton stelleri during the winter. In two individuals of Lymnea stagnalis, a lung snail, which is systematically close to the planorbides, Friedl (1961) determined, chromatographically, 0.46 and 0.61 rag/100 ml of urea, respectively. Little (1968) was able to establish a degree of variation in urea concentration in the hemolymph of the amphibian prosobranchia, Pomacea lineata, with a mean value of 0-15 mg/100 ml of urea. This value closely approaches the 0-16 mg/100 ml of urea found in normally fed B. glabrata. Uric acid, being a typical storage excretory product, is often determined in molluscs in homogenates of the whole body or in certain organs such as the hepatopancreas, kidney or foot. De Jorge et aL (1965) determined the uric acid concentration in the hemolymph of S. oblongus at 0.34 mg/100 ml. In Pomacea depressa, Little (1968) found 0.11 mg/100ml, whereas in P. lineata there was 0.27 rag/100 ml of uric acid in the body fluid. The fact that no uric acid was found in the hemolymph of B. glabrata does not mean that no uric acid synthesis takes place. As an explanation the following possibilities should be considered: (a) the transportation of uric acid is not carried out by the hemolymph but by excretophorous cells (Florey, 1970), (b) uric acid synthesis takes place in the storage tissue itself, (c) a purinolytic enzyme system (Florkin & Bricteaux-Gr6goire, 1972) breaks uric acid down and thus, for example, uricase was identified in Planorbis (Florkin & Duchfiteau, 1943) and (d) the uric acid concentration lies below the detection sensitivity of the method used. Needham (1935) points to a uric acid concentration in the kidney of planorbides which is low, in comparison, to other pulmonates. Since the freshwater pulmonates, as is generally assumed, only changed over to life in the water secondarily, they usually have uric acid concentrations similar to those of their terrestrial relations. Needham sees an obvious, if hypothetical, explanation for the low uric acid concentrations of the planorbides in the fact that some fresh-water species are still uricotelic, whereas others no longer are, and that there is a connection between this difference and the length of time they have been living in water. Urea concentration in the hemolymph of B. glabrata shows a high degree of fluctuation from one
409
individual to another. Thus peak values of 0 and 1.05 mg/100 ml of urea were established in normally fed snails; after a starvation period of 5 days, the values varied between 0.64 and 16.5 mg/100 ml of urea. Campbell et al. (1972) also observed a significant degree of fluctuation in the hemolymph of different individuals of S. oblongus. Becker (1972) was able to determine similarly large fluctuations in the concentration of a metabolite in B. glabrata. Glucose concentration varied in extreme cases between 0-8 and 17.6 rag/100 ml. The metabolism of molluscs must also be subject to control, although next to none equivalent mechanism could be identified in gastropods. With regards to the degree of variation in the urea and glucose concentration it seems that the potential for regulating the metabolism of molluscs is less effective in comparison to that of vertebrates, with the result that fluctuations of internal and external factors cannot be counterbalanced. Findings of morphological changes of certain cells in the hepatopancreas could explain the great variability of certain metabolite concentrations from individual to individual and also in the same organism within a short period of time (Becker, 1972). According to Thiele (1953), two sorts of cells can be distinguished in the hepatopancreas of Helix pomatia, the secretory-resorption cells (s-r cells) and the calcium cells. The s-r cells work at the same rhythm in the whole gland. Since one finds all the cells at the same phase in a microtome section the whole period of activity can only be demonstrated by means of a step-by-step examination. The hepatopancreas works rhythmically even in a state of starvation. After feeding the rhythm accelerates. Parallel to the rhythmic activity of the hepatopancreas there might be a rise and fall of the concentration of those metabolites which are resorbed or secreted by the s-r cells. As the extraction of the hemolymph takes place at different phases of the process, in each snail a certain degree of fluctuation of the metabolite concentrations concerned can be expected. In the hemolymph of B. glabrata fasting for 5 days a rise of 38% in the NH3-N concentration and of approximately 3000yo in the urea concentration in comparison to normally fed animals could be observed. Several explanations for this speedy accumulation, and above all that of urea, are possible. If one assumes that there is a constant production of urea and a continuous excretion with normal feeding, then there might be an increased concentration of urea during a starvation period, due to kidney insufficiency, active retention or generally reduced excretion. In the terrestial lung snail, Bulimulus dealbatus, H o m e (1971) was able to demonstrate a more or less
410
W. BECKERAND H. SCHMALE
linear rise of urea concentration in the snail's body during the first 7 months of aestivation. Since the activity of the urea cycle enzyme is the same in active and aestivating animals, B. dealbatus appears to produce urea in similar quantities in both periods. The osmotic pressure resulting from the reduced excretion of urea could, according to Home, contribute to a reduction in the loss of water during a longer period of drought. De Jorge & Petersen (1970) were able to determine a similar rise in the already high urea concentration in the hepatopancreas, lung and kidney of S. oblongus during hibernation and aestivation. Now, in the case of B. dealbatus and S. oblongus, one is dealing with terrestrial gastropods which are threatened by loss of water in the course of their aestivation, whereas with B. glabrata, which is a water snail, urea accumulation under this physiological aspect is out of the question. There is the possibility, however, that a decrease in the activity of the excretion processes, caused by starvation, brings about an increased concentration of urea. Another hypothesis to explain the rise in the NH3-N and urea concentrations is based on the assumption that the body decomposes its own protein during a period of starvation. As protein is significantly more nitrogenous than the lettuce which usually serves as food [protein approximately 16~o N, lettuce approximately 0"2~o N (de Jorge et al., 1969)] there is an increased supply of nitrogen which leads to a greater production of degradation products of the protein metabolism. Apparently, B. glabrata is not in a position to adapt its rate of excretion to these altered conditions and this results in an increased concentration of NHz-N and urea in the hemolymph. The fact that the protein concentration of the hemolymph of B. glabrata is, after several days of fasting, significantly lower than that of normally fed animals also supports this hypothesis (Lee & Cheng, 1972; Hirtbach, 1973). Vasu & Giese (1966) give a similar correlation in Cryptochiton stelleri. In an examination of the body fluid of C. stelleri for protein and residual nitrogen concentration during a complete yearly reproductive cycle and under starvation it could be determined that during a 53-day starvation period the nonprotein nitrogen level increased sharply. Since this increase was not coupled with a drop in the protein concentration in the hemolymph Vasu inferred a mobilization of extravascular protein as a source of energy. While the gonads were developing in the course of the reproductive cycle the residual nitrogen level, especially the urea concentration, rose too. In examination of the urea cycle enzymes in molluscs extensive conformity between the biochemical processes of the formation of urea in vertebrates and molluscs can be determined
(Campbell & Bishop, 1963; Campbell & Speeg, 1968; H o m e & Boonkoom, 1970; Andrews & Reid, 1972). However, whereas the urea cycle and the urea produced in the cycle have been proved, in the case of ureotelic vertebrates, to serve for detoxification of NH3 and excretion of nitrogen these hypotheses are not necessarily applicable in the case of the molluscs hitherto examined for two reasons: (a) Urea can be produced in other ways without playing a role in the detoxification of NH3 (enzymatic effect of arginase on exogenous arginine; uricolysis). (b) In no case as yet was it possible to prove that the urea cycle found in a mollusc mainly serves for detoxification of NH3. H o m e (1971) considers it possible that it might be the physiological task of the urea cycle to reduce the loss of water by increased osmotic pressure resulting from urea accumulation during aestivation. Another possible function of urea could have a connection with the formation of the shell. According to Speeg & Campbell (1969) the ammonia set free by the controlled enzymatic effect of urease on urea is responsible for an alkaline environment which promotes the deposition of calcium carbonate. F o r the reasons given, the increase in urea concentration in the hemolymph observed in B. glabrata in a state of starvation cannot be valued unequivocally as an indication of the mobilization of the protein of the body itself for the production of energy, as in the case of an animal proved to be ureotelic. To clarify the significance of the urea cycle of molluscs in connection with the detoxification of NHa and the excretion of nitrogen more precisely it is necessary to carry out activity analyses under various physiological conditions as well as demonstrating the presence of the urea cycle enzymes.
Acknowledgement--The authors are grateful for the assistance of the Deutsche Forschungsgemeinschaft. REFERENCES ANDREWST. R. &REtD R. G. B. (1972) Ornithine cycle and uricolytic enzymes in four bivalve molluscs. Comp. Biochem. Physiol. 42B, 475-491. BECrd/R W. (1968) Untersuchungen fiber die aus der Muttersporocyste auswandernden Tochtersporocysten yon Schistosoma mansoni--I. Beitr~ige zum Kohlenhydratstoffwechsel dieser Stadien. Z. Parasit. 30, 233-251. BECKERW. (1972) The glucose content in haemolymph of Australorbis glabratus. Comp. Biochem. Physiol. 43A, 809-814. BERNT E. & BERGMEYER H. U. (1970) Harnstoff. In Methoden der enzymatischen Analyse (Edited by BERGM~YER H. U.), 2. Auflage, Bd. II, pp. 1738-1741. Verlag Chemie, Weinheim/Bergstr. CAMPBELLJ. W. • BISHOP S, H. (1963) Urea biosynthesis in invertebrates: [a4C] urea formation in the land snail Otala lactea and earthworm. Biochim. biophys. Acta 77, 149-152.
Nitrogenous degradation products in snail hemolymph CAMPBELL J. W., DROTMAN R. B., McDONALD J. A. & TRAMELL P. R. (1972) Nitrogen metabolism in terrestrial invertebrates. In Nitrogen Metabolism and the Environment (Edited by CAMPBELLJ. W. & GOLDSTEIN L.). Academic Press, New York. CAMPBELL J. W. & SPEEG K. V. (1968) Arginine biosynthesis and metabolism in terrestrial snails. Comp. Biochem. Physiol. 25, 3-32. DELAUNAY H. (1927) Recherches biocbimiques sur l'excr6tion azot6e des invert6br6s. Bull. biol. Sm. Arcachon 24, 95-214. DELAUNAYH. (1931) L'excr6tion azot6e des invert6br6s. Biol. Rev. 6, 265-301. FAWCETTJ. K. & SCOTTJ. E. (1960) A rapid and precise method for the determination of urea. J. clin. Path. 13, 156-159. FLOREY E. (1970) Lehrbuch der Tierphysiologie. Georg Thieme Verlag, Stuttgart. FLORKIN M. & BRICTEUX-GRI~GOIRES. (1972) Nitrogen metabolism in mollusks. In Chemical Zoology (Edited by FLORKIN M. & SCHEERB. T.), Vol. VII, pp. 301-348. Academic Press, New York. FLORKIN M. & DUCH.~TEAUG. (1943) Les formes du systeme enzymatique de l'uricolyse et r6volution du catabolisme purique chez les animaux. Archs. int. Physiol. Biochem. 53, 267-307. FLORKIN M. d~. HOUET R. (1938) Concentration de rammoniaque in vivo et in vitro dans le milieu int6rieur des invert6br6s--I. L'Anodonte. Archs. int. Physiol. Biochem. 47, 125-131. FLORKIN M. & RENWARI" H. (1939) Concentration de l'ammoniaque in vivo et in vitro dans le milieu int6rieur des invert6br6s--II. Escargot et Homard. Arch. int. Physiol. Biochem. 49, 127-128. FRIEDL F. E. (1961) Studies on larval Fascioloides magna --IV. Chromatographic analyses of free amino acids in the hemolymph of a host snail. J. Paras#. 47, 773-776. HIRTBACH E. (1973) Protein- und H~'noglobingehalt bei Biomphalaria glabrata. Diplomarbeit. Zoologisches Institut und Museum, Hamburg. HORNE F. (1971) Accumulation of urea by a pulmonate snail during aestivation. Comp. Biochem. PhysioL 38A, 565-570. HORNE F. & BOONKOOMV. (1970) The distribution of the ornithine cycle enzymes in twelve gastropods. Comp. Biochem. Physiol. 32, 141-153. DE JORGE F. B. & PETERSENJ. A. (1970) Urea and uric acid contents in the hepatopancreas, kidney and lung of active and dormant snails, Strophocheilus and Thaumastus (Pulmonata; Mollusca). Comp. Biochem. Physiol. 35, 211-219.
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DE JORGE F. B., PE~aSEN J. A. & DITADI A. S. F. (1969) Variations in nitrogenous compounds in the urine of Strophocheilus (Pulmonata; Mollusca) with different diets. Experientia 25, 614-615. DE JORGEE. B., ULHOACINTRAA. B., HAESERP. E. (S.J.) SAWAYAP. (1965) Biochemical studies on the snail Strophoeheilus oblongus musculus (Bequaert). Comp. Biochem. PhysioL 14, 35-42. LEE F. O. & CHENG T. C. (1972) Sehistosoma mansoni: alterations in total protein and hemoglobin in the hemolymph of infected Biomphalaria glabrata. Expt. Paras#. 31, 203-216. LITTLE C. (1968) Aestivation and ionic regulation in two species of Pomacea (Gastropoda; Prosobranchia). J. exp. Biol. 48, 569-585. M/3LLER-BEISSENmRTZW. (1965) Eine einfache Methode zur Bestimmung des Blutammoniaks. Z. anal. Chem. 212, 145-155. MYERS R. G. (1920) A chemical study of the blood of several invertebrate animals. J. biol. Chem. 41, 119-147. NEEOHAM J. (1935) Problems of nitrogen catabolism in invertebrates--II. Correlation between uricotelic metabolism and habitat in the phylum Mollusca. Biochem. J. 29, 238-251. POTTS W. T. W. (1965) Ammonia excretion in Octopus dofleini. Comp. Biochem. PhysioL 14, 339-355. PRAETORIUS E. & POULSEN H. (1953) Enzymatic determination of uric acid. Scand. J. clin. Lab. Invest. 5, 273-280. SPEEG K. V., JR. t~ CAMPBELLJ. W. (1969) Arginine and urea metabolism in terrestrial snails. Am. J. Physiol. 216, 1003-1012. TmELE G. (1953) Vergleichende Untersuchungen fiber den Feinbau und die Funktion der Mitteldarmdriise einheimischer Gastropoden. Z. Zellforsch. mikrosk. Anat. 38, 87-138. TRAMELL P. R. & CAMPBELLJ. W. (1970) Nitrogenous excretory products of the giant South American land snail, Strophocheilus oblongus. Comp. Biochem. Physiol. 32, 569-571. VASU B. S. & GIESE A. C. (1966) Variations in body fluid nitrogenous constituents of Cryptochiton stelleri (Mollusca) in relation to nutrition and reproduction. Comp. Biochem. Physiol. 19, 737-744.
Key Word Index--Urea; ammonia; uric acid; Biomphalaria glabrata.
starvation;
THE NITROGENOUS PRODUCTS OF DEGRADATION-AMMONIA, UREA AND URIC ACID--IN THE HEMOLYMPH OF THE SNAIL BIOMPHALARIA GLABRATA W. BECKER AND H. SCHMALE Zoologisches Institut und Zoologisches Museum der Universit~it Hamburg, Hamburg, Germany (Received 18 March 1974) Absa-act--1. The concentrations of ammonia, urea and uric acid in the hemolymph of Biomphalaria glabrata were studied. 2. In normally fed snails, the concentration of NHs-N varied between 0"031 and 0.145 mg/100 ml with a mean of 0.079 rag/100 ml. After a starvation period of 5 days, the concentration of NHa-N rose to an average value of 0.111 mg/100 ml. The individual values lay between 0"056 and 0.212 mg/ 100 ml. 3. In normally fed snails, the urea concentration fluctuated between 0 and 1-05mg/100 ml with a mean of 0.160 mg/100 ml. Within a starvation period of 5 days the urea concentration increased on average to 5.12 mg/100 ml. The individual values varied between 0-053 and 16.5 mg/100 ml. 4. Uric acid could neither be detected in the hemolymph of normally fed nor of starved snails. 5. The possible causes of the large individual variation of NHa-N and urea concentration, as well as of the concentration increase of those compounds containing N during the starvation periods, are discussed.
INTRODUCTION
MATERIALS AND METHODS
THE NITROGENOUSdegradation products, ammonia, urea and uric acid, are generally transported with the body fluids to places where they are excreted or further processed. Up to now research into the occurrence of these substances in the hemolymph of molluscs has been very scanty (Myers, 1920; Delaunay, 1931; Florkin & Houet, 1938; Florkin & Renwart, 1939; Friedl, 1961; Vasu & Giese, 1966; Potts, 1965; Little, 1968; de Jorge et al., 1965). The amounts of nitrogenous degradation products produced are indicative of the activity of the protein and nucleic acid metabolism of an animal so that any unusual stress imposed on the latter must make itself apparent in a change in the amounts of ammonia, urea and uric acid produced. Such stress can be brought about by phases of strong reproductive activity or by parasitic invasion. The latter is an aspect which must be considered, especially in the case of Biomphalaria glabrata, the snail we examined, as the larval stages of Schistosoma mansoni developing in this snail show a quick rate of reproduction (Becker, 1968). Heightened activity of the protein metabolism must, however, also appear when, as a result of starvation, the snails must use the materials of their own bodies. In this account the amounts of nitrogenous degradation products in normally fed animals and in snails starved for 5 days are determined.
Snails of the species Biomphalaria glabrata to be examined were kept at a temperature of 26 + 2°C. Their only food consisted of fresh lettuce leaves, fed as required. Fasting snails were kept in aquaria and these were cleaned every second day to prevent any growth of algae which might have served the animals as food. For the examination snails were chosen whose shell measured 14-20 mm dia. After thorough cleaning and drying the shell of the last but one whorl from the inside was carefully lifted and, after a quick puncture of the body wall by a glass capillary tube, the hemolymph was extracted. According to the size of the snail approximately 50-100 ~1 of hemolymph could be obtained. The hemolymph was stored in a closed container in an icebath until subsequent use. NH3-N analysis NHa-N analysis was carded out according to the method of Miiller-Beissenhirtz (1965), with several modifications made necessary by the small volume of the specimens. The phenol and hypochlorite reagents required (0-106 M phenol; 0.17 mM sodium nitroprussate and 11 mM NaOC1; 0"125 N NaOH) were taken from the Biochemica test combination "Urea" (Boehringer, Mannheim). Urea analysis For urea analysis the method of Fawcett & Scott (1960) and Bernt & Bergmeyer (1970) was used. The protein was removed from the hemolymph with 10%
407
408
W. BECKERAND H. S C r ~ . E
trichioracetic acid. Neutralization was carried out with potassium hydrogenous carbonate solution. Phenol and hypochlorite reagent, urease (2 mg/#l; 0.05 M phosphate buffer, pH---6.5) and a standard urea solution were taken from the Biochemica test combination "Urea" (Boehringer, Mannheim).
Uric acid analysis Uric acid analysis was carried out according to the method of Praetorius & Poulsen (1953). It was possible to use the reagents of the Biochemica test combination "Uric acid" (Boehringer, Mannheim) (borate buffer 0.2 M, pH = 9.5; uricase 2 mg/ml in 50% glycerine). ~S~TS
F o r the analysis of NH3-N, urea and uric acid in the hemolymph of B. glabrata, animals were used which had been differently fed as follows: Test Series A contains animals which were fed normally and were taken off lettuce leaves directly before the hemolymph was extracted. The snails of Test Series B had been starved for 5 days before the hemolymph was extracted.
Results of NHa-N analysis The results of NH3-N analysis are summarized in Table 1. Comparison of both test series with the t-test gave: m = 39,
t = 2.85,
Comparison of both test series with the F-test showed: quotient of variance F = 297.3,
P<0.001.
Interpretation of the results shows that the normally fed animals of Series A belong, with regard to their urea concentration, to a different group than snails of Test Series B, which had been starved for 5 days. A comparison of the mean values using the t-test was not possible, as the characteristic examined in both test series showed a different variability. A tendency already revealed in the NHs-N analysis, namely a higher NH3-N concentration in starved snails in comparison to normally fed ones, is found to a much greater extent in the urea analysis: the urea concentration in the hemolymph of snails starved for 5 days is approximately thirty times higher than in the case of normally fed animals.
Results of uric acid analysis F o r uric acid analysis thirty-three snails were examined. Seventeen of them had been fed normally and sixteen had been starved for 5 days. In no case could uric acid be found by the method used, which suggested a concentration of uric acid below 30/zg/100 ml. DISCUSSION
P<0.01.
As statistical interpretation of the results of the examination shows, the NI-Ia-N concentration in the hemolymph of normally fed snails is significantly lower than that of snails starved for 5 days.
Results of urea analysis The results of urea analysis for Test Series A and B are summarized in Table 2.
A comparison of the NH3-N concentration in the body fluid of various molluscs shows that the NH3-N concentration in the hemolymph o f B. glabrata is very low, lying at approximately 0-1 mg/ 100 ml. Only Florkin & Houet (1938) found an even lower NH3 value of 0.51-0.071mg/100ml in Anodonta cygnea. The highest N H s - N concentrations have been found in the terrestrial pulmonates, e.g. in Helix pomatia with 1.2 and in Arion rufus
Table 1. Results of NH3-N analysis
Test Series
Unit
No. of animals examined
A B
/~g/100 ml #.g/100 ml
19 22
Peak values
Mean value
31-145 56-212
79.2 111.5
Standard deviation of the mean value 7.5 8.3
Standard deviation 32.9 38.9
Table 2. Results of urea analysis
Test Series
Unit
No. of animals examined
A B
e.g/100 ml ~.g/100 ml
19 22
Peak values 0-1050 53-16,500
Mean value 160.3 5121
Standard deviation of the mean value 63.5 1017
Standard deviation 276"8 4773
Nitrogenous degradation products in snail hemolymph with 1.4 mg/100 ml NH3-N (Delaunay, 1951) and in the carnivorous cephalopods which are considered ammoniotelic. Thus Delaunay (1927) found 2.44.8 rag/100 ml NHa-N in Sepia officinalis. There are a few findings about the concentration of urea in the hemolymph. De Jorge et al. (1965) state a high value of 30.5 mg/100 ml of urea for Strophocheilus oblongus. On the other hand, Tramell & Campell (1970) were unable to identify any urea in S. oblongus. Vasu & Giese (1966) found approximately 9mg/100ml of urea in Cryptochiton stelleri during the winter. In two individuals of Lymnea stagnalis, a lung snail, which is systematically close to the planorbides, Friedl (1961) determined, chromatographically, 0.46 and 0.61 rag/100 ml of urea, respectively. Little (1968) was able to establish a degree of variation in urea concentration in the hemolymph of the amphibian prosobranchia, Pomacea lineata, with a mean value of 0-15 mg/100 ml of urea. This value closely approaches the 0-16 mg/100 ml of urea found in normally fed B. glabrata. Uric acid, being a typical storage excretory product, is often determined in molluscs in homogenates of the whole body or in certain organs such as the hepatopancreas, kidney or foot. De Jorge et aL (1965) determined the uric acid concentration in the hemolymph of S. oblongus at 0.34 mg/100 ml. In Pomacea depressa, Little (1968) found 0.11 mg/100ml, whereas in P. lineata there was 0.27 rag/100 ml of uric acid in the body fluid. The fact that no uric acid was found in the hemolymph of B. glabrata does not mean that no uric acid synthesis takes place. As an explanation the following possibilities should be considered: (a) the transportation of uric acid is not carried out by the hemolymph but by excretophorous cells (Florey, 1970), (b) uric acid synthesis takes place in the storage tissue itself, (c) a purinolytic enzyme system (Florkin & Bricteaux-Gr6goire, 1972) breaks uric acid down and thus, for example, uricase was identified in Planorbis (Florkin & Duchfiteau, 1943) and (d) the uric acid concentration lies below the detection sensitivity of the method used. Needham (1935) points to a uric acid concentration in the kidney of planorbides which is low, in comparison, to other pulmonates. Since the freshwater pulmonates, as is generally assumed, only changed over to life in the water secondarily, they usually have uric acid concentrations similar to those of their terrestrial relations. Needham sees an obvious, if hypothetical, explanation for the low uric acid concentrations of the planorbides in the fact that some fresh-water species are still uricotelic, whereas others no longer are, and that there is a connection between this difference and the length of time they have been living in water. Urea concentration in the hemolymph of B. glabrata shows a high degree of fluctuation from one
409
individual to another. Thus peak values of 0 and 1.05 mg/100 ml of urea were established in normally fed snails; after a starvation period of 5 days, the values varied between 0.64 and 16.5 mg/100 ml of urea. Campbell et al. (1972) also observed a significant degree of fluctuation in the hemolymph of different individuals of S. oblongus. Becker (1972) was able to determine similarly large fluctuations in the concentration of a metabolite in B. glabrata. Glucose concentration varied in extreme cases between 0-8 and 17.6 rag/100 ml. The metabolism of molluscs must also be subject to control, although next to none equivalent mechanism could be identified in gastropods. With regards to the degree of variation in the urea and glucose concentration it seems that the potential for regulating the metabolism of molluscs is less effective in comparison to that of vertebrates, with the result that fluctuations of internal and external factors cannot be counterbalanced. Findings of morphological changes of certain cells in the hepatopancreas could explain the great variability of certain metabolite concentrations from individual to individual and also in the same organism within a short period of time (Becker, 1972). According to Thiele (1953), two sorts of cells can be distinguished in the hepatopancreas of Helix pomatia, the secretory-resorption cells (s-r cells) and the calcium cells. The s-r cells work at the same rhythm in the whole gland. Since one finds all the cells at the same phase in a microtome section the whole period of activity can only be demonstrated by means of a step-by-step examination. The hepatopancreas works rhythmically even in a state of starvation. After feeding the rhythm accelerates. Parallel to the rhythmic activity of the hepatopancreas there might be a rise and fall of the concentration of those metabolites which are resorbed or secreted by the s-r cells. As the extraction of the hemolymph takes place at different phases of the process, in each snail a certain degree of fluctuation of the metabolite concentrations concerned can be expected. In the hemolymph of B. glabrata fasting for 5 days a rise of 38% in the NH3-N concentration and of approximately 3000yo in the urea concentration in comparison to normally fed animals could be observed. Several explanations for this speedy accumulation, and above all that of urea, are possible. If one assumes that there is a constant production of urea and a continuous excretion with normal feeding, then there might be an increased concentration of urea during a starvation period, due to kidney insufficiency, active retention or generally reduced excretion. In the terrestial lung snail, Bulimulus dealbatus, H o m e (1971) was able to demonstrate a more or less
410
W. BECKERAND H. SCHMALE
linear rise of urea concentration in the snail's body during the first 7 months of aestivation. Since the activity of the urea cycle enzyme is the same in active and aestivating animals, B. dealbatus appears to produce urea in similar quantities in both periods. The osmotic pressure resulting from the reduced excretion of urea could, according to Home, contribute to a reduction in the loss of water during a longer period of drought. De Jorge & Petersen (1970) were able to determine a similar rise in the already high urea concentration in the hepatopancreas, lung and kidney of S. oblongus during hibernation and aestivation. Now, in the case of B. dealbatus and S. oblongus, one is dealing with terrestrial gastropods which are threatened by loss of water in the course of their aestivation, whereas with B. glabrata, which is a water snail, urea accumulation under this physiological aspect is out of the question. There is the possibility, however, that a decrease in the activity of the excretion processes, caused by starvation, brings about an increased concentration of urea. Another hypothesis to explain the rise in the NH3-N and urea concentrations is based on the assumption that the body decomposes its own protein during a period of starvation. As protein is significantly more nitrogenous than the lettuce which usually serves as food [protein approximately 16~o N, lettuce approximately 0"2~o N (de Jorge et al., 1969)] there is an increased supply of nitrogen which leads to a greater production of degradation products of the protein metabolism. Apparently, B. glabrata is not in a position to adapt its rate of excretion to these altered conditions and this results in an increased concentration of NHz-N and urea in the hemolymph. The fact that the protein concentration of the hemolymph of B. glabrata is, after several days of fasting, significantly lower than that of normally fed animals also supports this hypothesis (Lee & Cheng, 1972; Hirtbach, 1973). Vasu & Giese (1966) give a similar correlation in Cryptochiton stelleri. In an examination of the body fluid of C. stelleri for protein and residual nitrogen concentration during a complete yearly reproductive cycle and under starvation it could be determined that during a 53-day starvation period the nonprotein nitrogen level increased sharply. Since this increase was not coupled with a drop in the protein concentration in the hemolymph Vasu inferred a mobilization of extravascular protein as a source of energy. While the gonads were developing in the course of the reproductive cycle the residual nitrogen level, especially the urea concentration, rose too. In examination of the urea cycle enzymes in molluscs extensive conformity between the biochemical processes of the formation of urea in vertebrates and molluscs can be determined
(Campbell & Bishop, 1963; Campbell & Speeg, 1968; H o m e & Boonkoom, 1970; Andrews & Reid, 1972). However, whereas the urea cycle and the urea produced in the cycle have been proved, in the case of ureotelic vertebrates, to serve for detoxification of NH3 and excretion of nitrogen these hypotheses are not necessarily applicable in the case of the molluscs hitherto examined for two reasons: (a) Urea can be produced in other ways without playing a role in the detoxification of NH3 (enzymatic effect of arginase on exogenous arginine; uricolysis). (b) In no case as yet was it possible to prove that the urea cycle found in a mollusc mainly serves for detoxification of NH3. H o m e (1971) considers it possible that it might be the physiological task of the urea cycle to reduce the loss of water by increased osmotic pressure resulting from urea accumulation during aestivation. Another possible function of urea could have a connection with the formation of the shell. According to Speeg & Campbell (1969) the ammonia set free by the controlled enzymatic effect of urease on urea is responsible for an alkaline environment which promotes the deposition of calcium carbonate. F o r the reasons given, the increase in urea concentration in the hemolymph observed in B. glabrata in a state of starvation cannot be valued unequivocally as an indication of the mobilization of the protein of the body itself for the production of energy, as in the case of an animal proved to be ureotelic. To clarify the significance of the urea cycle of molluscs in connection with the detoxification of NHa and the excretion of nitrogen more precisely it is necessary to carry out activity analyses under various physiological conditions as well as demonstrating the presence of the urea cycle enzymes.
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DE JORGE F. B., PE~aSEN J. A. & DITADI A. S. F. (1969) Variations in nitrogenous compounds in the urine of Strophocheilus (Pulmonata; Mollusca) with different diets. Experientia 25, 614-615. DE JORGEE. B., ULHOACINTRAA. B., HAESERP. E. (S.J.) SAWAYAP. (1965) Biochemical studies on the snail Strophoeheilus oblongus musculus (Bequaert). Comp. Biochem. PhysioL 14, 35-42. LEE F. O. & CHENG T. C. (1972) Sehistosoma mansoni: alterations in total protein and hemoglobin in the hemolymph of infected Biomphalaria glabrata. Expt. Paras#. 31, 203-216. LITTLE C. (1968) Aestivation and ionic regulation in two species of Pomacea (Gastropoda; Prosobranchia). J. exp. Biol. 48, 569-585. M/3LLER-BEISSENmRTZW. (1965) Eine einfache Methode zur Bestimmung des Blutammoniaks. Z. anal. Chem. 212, 145-155. MYERS R. G. (1920) A chemical study of the blood of several invertebrate animals. J. biol. Chem. 41, 119-147. NEEOHAM J. (1935) Problems of nitrogen catabolism in invertebrates--II. Correlation between uricotelic metabolism and habitat in the phylum Mollusca. Biochem. J. 29, 238-251. POTTS W. T. W. (1965) Ammonia excretion in Octopus dofleini. Comp. Biochem. PhysioL 14, 339-355. PRAETORIUS E. & POULSEN H. (1953) Enzymatic determination of uric acid. Scand. J. clin. Lab. Invest. 5, 273-280. SPEEG K. V., JR. t~ CAMPBELLJ. W. (1969) Arginine and urea metabolism in terrestrial snails. Am. J. Physiol. 216, 1003-1012. TmELE G. (1953) Vergleichende Untersuchungen fiber den Feinbau und die Funktion der Mitteldarmdriise einheimischer Gastropoden. Z. Zellforsch. mikrosk. Anat. 38, 87-138. TRAMELL P. R. & CAMPBELLJ. W. (1970) Nitrogenous excretory products of the giant South American land snail, Strophocheilus oblongus. Comp. Biochem. Physiol. 32, 569-571. VASU B. S. & GIESE A. C. (1966) Variations in body fluid nitrogenous constituents of Cryptochiton stelleri (Mollusca) in relation to nutrition and reproduction. Comp. Biochem. Physiol. 19, 737-744.
Key Word Index--Urea; ammonia; uric acid; Biomphalaria glabrata.
starvation;