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Nitrogen excretion by the fresh water pulmonate snail, Lymnaea stagnalis jugularis say
Comp. Biochem. Physiol., 1974, Vol. 49A, pp. 617 to 622. Pergamon Press. Printed in Great Britain.
NITROGEN EXCRETION BY THE FRESH WATER PULMONATE SNAIL, L YMNAEA S T A G N A L I S JUGULARIS SAY* F. E. F R I E D L Department of Biology, University of South Florida, Tampa, Florida 33620, U.S.A.
(Received 26 September 1973) Abstract--1. The average per cent partition of excretory nitrogen produced into an ambient medium by Lymnaea stagnalis was found to be approximately: ammonia, 50; urea, 30; unidentified residual N, 20. Of the residual, about one-quarter appears to be volatile. 2. The endogenous nature of the ammonia and urea produced is supported by their appearance in the presence of both penicillin and streptomycin as bacteriostatic agents. INTRODUCTION IT HAS long been appreciated that organisms contribute nitrogenous end products of metabolism to their environment. The characterization of their excretory patterns by ammonotelic, ureotelic or uricotelic has tended to oversimplify the situation in many cases. Although a primary metabolic end product may be much in evidence, a total pattern of nitrogen excretion can involve many substances disposed of in various ways. In gastropod molluscs a number of such products have been categorized, and a recent summary of work on the group is reported by Campbell & Bishop (1970). The pulmonate gastropod Lymnaea stagnalis jugularis Say was studied by Bayne & Friedl (1968), who related the rates of accumulation of ammonia and urea in an ambient medium to size groups. The following work, partially reported in abstract by Friedl (1967), was undertaken to substantiate the endogenous origins of ammonia and urea in Lymnaea and to establish their proportions relative to a total amount of nitrogenous material found. MATERIALS AND METHODS The snails used in this work were laboratory-reared Lymnaea stagnalis jugularis Say, originally obtained from Minnesota. They were placed singly, or in groups, in a salt solution consisting of: KH,PO, 136rag/1.; Na2HPO, 331 mg/l.; KCI 25mg/l.; MgSO,.7H,O 50 mg/l. and CaC12 50 mg/1. in distilled water. * This investigation was assisted by Research Grant No. GB 3158 from the National Science Foundation. 617 22
618
F . E . FRIEDL
After a period of incubation at 25°C, the ambient fluids were removed, centrifuged and aliquots were analyzed in duplicate for total nitrogen, ammonia, urea and additional volatile and non-volatile nitrogenous residues. Total nitrogen was determined by a Kjeldahl-type digestion followed by direct nesslerization. In general, ammonia was determined by direct nesslerization of samples, and urea by difference, using a similar direct nesslerization procedure after incubation of samples with buffered urease. Microdiffusion (adapted from Seligson & Seligson, 1951) was used to estimate ammonia and urea in the presence of antibiotics. The formulation of the Nessler reagent and the general procedure for its use was as described in Umbreit et al. (1957). Nitrogenous bases were volatilized by the addition of NaOH to samples, followed by evaporation to dryness under reduced pressure over concentrated H2SO4. After total nitrogen had been determined, the residual volatile and non-volatile nitrogenous components could be estimated by difference using data obtained from the analysis of undesiccated samples. Weights reported are those from whole animals, including shells, after removing excess water by draining or blotting. Other experimental details are explained in the text or with figures. RESULTS AND D I S C U S S I O N Five experimental groups are characterized in T a b l e 1 and data obtained f r o m these groups are displayed in Tables 2-4. As a supplement to the study of Bayne & Friedl (1968), these data show that a small, variable a m o u n t of nitrogenous material in addition to a m m o n i a and urea accumulates in media surrounding L. stagnalis. T h e concomitant production of ammonia and urea (0.13 and 0.03 /~mole/g per hr of each respectively) falls within the ranges found in the m o r e extensive former study. Figure 1 summarizes the partition of nitrogenous material and portrays the additional volatile and non-volatile substances as an average of 17 per cent of the total nitrogen found. Spitzer (1937) noted somewhat larger TABLE 1--TOTAL
NITROGEN
PRODUCTION
OF FIVE EXPERIMENTAL
GROUPS OF
L. stagnalis
Group
Total N found Ozg) Rate of accumulation Oxg N/g whole weight per hr)* Ammonia found (% of total N) Number of snails in pool Average whole weight (g) Hours of incubation at 25°C
I
II
III
IV
V
680 2-82
360 1"18
1340 3"74
950 3'95
925 5"41
70"6
39"0
40'0
58-0
54.1
9 1 "66 24
5 1 "81 19
5 2-54 19
5 2"03 30
5 2"58 19
* Average rate of accumulation, 3-4/zg N/g whole weight per hr; range, 1"25"4/zg N. Snail weights are whole, wet weights including shell. Average whole weight of twenty-nine experimental snails, 2-06 g. Snails were isolated in sealed 500-ml flasks usually with 10 ml of ambient fluid present per snail.
619
NITROGEN EXCRETION BY L YMNAEA TABLE 2--RELATIVE QUANTITIES OF NITROGENOUS MATERIAL PRODUCED BY Z. (PER CENT OF TOTAL N )
stagnalis
Group*
Ammonia Urea Unidentified remaining N
I
II
III
IV
V
Average
40
54 27 19
58 26 16
39 32 29
71 25 4
52 28 17
-
-
--
* As in Table 1. TABLE 3--RELATIVE QUANTITIES OF VOLATILE AND NON-VOLATILE NITROGENOUS MATERIAL (EXCLUDING AMMONIA AND UREA) PRODUCED BY L . stag~aali$ (PER CENT OF UNIDENTIFIED REMAINING N )
Group*
Non-volatile Volatile
I
II
III
IV
V
Average
---
91 9
50 50
57 43
100 0
75 26
* As in Table 1. RATESOF PRODUCTIONOF AMMONIAAND UREABY L. stagnalis (~m/g whole weight per hr)
TABLE 4
Group *
Ammonia Urea
I
II
III
IV
V
Average
0"107 --
0"209 0-052
0-163 0"037
0"033 0-013
0"142 0"025
0"131 0-032
* As in Table 1. amounts of additional material f r o m s u m m e r snails kept at r o o m t e m p e r a t u r e ; he found 47-3% a m m o n i a N, 13-8% urea N, 5.2% uric acid N plus a remaining 38.5 per cent of unidentified nitrogen. It is not entirely clear what the volatile and non-volatile additional constituents of the ambient fluids m a y be, although Spitzer (1937) reported uric acid, purine nitrogen and amino nitrogen in some of his excretion studies. Volatile substances, in addition to ammonia, could be represented b y amines; non-volatile substances, in addition to urea, could include soluble proteins, peptides, amino acids and purines.
620
F. E. FRIEDL Nitrogen partition in Lymnaea stagna//$
FIG. 1. Pie diagram summarizing the partition of nitrogenous material produced by L. stagnalis. Data from Tables 1-3. Relative average amounts of ammonia N, urea N and unidentified residual N are shown. Undetermined volatile and non-volatile residual material represents about 17 per cent of the total nitrogen. An ultraviolet absorption spectrum recorded from one sample of ambient fluid (Table 1, Group I) shows inflections approaching 260 and 280 nm (As60 = 0.1249; A2a 0 = 0.1051; 280/260 = 0.842; dual beam recording; sample vs fresh ambient fluid) suggesting the possible presence of purines and phenolic groups. The Folin phenol reagent (applied as the Lowry protein method, Lowry et al., 1951) reacts with ambient fluid samples, and ninhydrin-positive material, over and above the amount expected from urea on a color basis, is found in ammoniafreed samples. Efforts were made to standardize as much of the sample collection procedure as possible. All snails were laboratory-reared, temperatures were controlled and a uniformly formulated ambient fluid, derived from one developed in previous studies on axenic Lymnaea (Friedl, 1964), was employed. Unfortunately, there was no feasible way to evaluate the nutritional state or overall metabolic condition of the animals. It has been suggested by Duerr (1966a, 1967, 1968) that ammonia and urea production by Lymnaea and other snails, when measured by analysis of ambient fluids, is apparent as a result of microbial decomposition of fecal masses. He found low levels of nitrogenous material in the presence of 1000 units/ml of penicillin (less than 6 Fg of nitrogen in any form over 24 hr), and considered L. stagnalis to produce little, if any, endogenous ammonia or urea.
621
NITROGEN EXCRETION BY L YMNAEA
Microbial action is indeed a most likely component of any investigation in which axenic animals are not used; however, when antibiotics were employed as bacteriostatic agents by the author, ammonia and urea production continued. Table 5 shows the results of an experiment wherein various concentrations and TABLE 5 - - T H E
PRODUCTION OF NITROGENOUS EXCRETORY MATERIALS BY L . $tagnali$ IN THE PRESENCE OF ANTIBIOTICS
Rate of accumulation (/zmoles/g per hr) Sample 1 (30 hr) Snail
Antibiotics
1 2 3 4 5 6 7 8 9
None P+S 500 P+S 500 P + S 1000 P + S 1000 P 500 P 500 P 1000 P 1000
Ammonia 0.040 0-055 0"064 0"105 0"067 0'065 0"089 0"035 0"095
Sample 2 (18 hr)
Urea
Ammonia
Urea
0"041 0"035 0"049 0"033 0"020 -0"071 0"047 0"072
0"000 0.129 0"087 0"112 0"073 0"070 0"155 0"000 0"071
0.282 0-064 0"053 0"072 0"040 0"040 0"064 0"082 0-091
Snail weights (whole) were between 0.54 and 2"05 g. Snails were placed individually in 250-ml sealed flasks with 10 ml of the salt solution described in text and kept at 25°C for the sample times indicated. Penicillin G, 500 or 1000 units/ml and streptomycin sulfate, 500 or 1000/zg/ml were included in the solutions for sample 1, as indicated by P or S. For sample 2, the same snails were washed, drained and then placed in new salt solution without antibiotics. An additional control snail (no antibiotic exposure) died before final weighing; it produced 0"860 and 1"07/~moles of ammonia and 1-90 and 1"61/zmoles of urea in samples 1 and 2, respectively. combinations of penicillin and streptomycin were present with single snails in 10 ml fluid. Although considerable variation is seen, ammonia and urea are both produced during and subsequent to antibiotic exposure. Up to 117/zg of ammonia and urea N were found for a 1.6-g snail kept 30 hr in the presence of 1000 units and/zgs respectively of both penicillin and streptomycin. In this test, the rate of ammonia production, although somewhat low, was still generally within ranges found in other experiments. Since there is evidence from studies on axenic snails that streptomycin inhibits growth (Chernin, 1959, on Australorbis) the author has preferred not to use antibiotics. Obviously, axenic animals, although abnormal in their own right, would be much desired for such work. In a general sense, as exemplified by the earlier investigations of Delaunay (1931), Spitzer (1937) and others, excretion in many invertebrates, unlike that in more rigidly compartmentalized and insulated forms, is difficult to evaluate in its entirety. Renal organs, digestive glands, whole animals and ambient fluids have all been studied. Kidney function, intestinal activity, diffusion, leakage
622
F . E . FRIEDL
and precipitation, all of which at various times may contribute in a major sense, were undoubtedly considered. Of these aspects, the precipitation of materials in tissues or organs (storage excretion) has commanded much interest. Spitzer (1937) noted uric acid and other purine material in digestive gland and nephridia of his snails (Limnea stagnalis). Duerr (1966b) also reports uric acid in L. stagnalis tissues. These findings tend to complicate the assignment of such animals to ammonotelism, ureotelism or uricotelism. The relationship between alternate modes of excretion and the ability of organisms to utilize them preferentially is not within the scope of this paper; although such plasticity can be a perplexing problem for the investigator, it remains one of the most fascinating aspects of the study of nitrogen catabolism in invertebrates. REFERENCES BAVNE R. & FRmDL F. (1968) The production of extemaUy measurable ammonia and urea in the snail, Lymnaea stagnalisjugularis Say. Comp. Biochem. Physiol. 25, 711-717. CAMeBELL J. & BISHOP S. (1970) Nitrogen metabolism in molluscs. In Comparative Biochemistry of Nitrogen Metabolism (Edited by CAMPBELL J.), Vol. 1, The Invertebrates, Chap. 5, pp. 103-206. Academic Press, New York. CrmRmN E. (1959) Cultivation of the snail, Australorbis glabratus, under axenic conditions. Ann. N. Y. Acad. Sci. 77, 237-245. DELAUNAYH. (1931) L'excr6tion azot6e des invert6br6s. Biol. Rev. 6, 265-301. DUERR F. (1966a) Nitrogen excretion in the fresh water pulmonate snail Lymnaea stagnalis appressa Say. The Physiologist 9, 172. DUERR F. (1966b) Qualitative analysis of the uric acid, xanthine, and guanine content of several snails. American Malacological Union Annual Reports, pp. 69-70. DUEaR F. (1967) The uric acid content of several species of prosobranch and pulmonate snails as related to nitrogen excretion. Comp. Biochem. Physiol. 22, 333-340. DUERa F. (1968) Excretion of ammonia and urea in seven species of marine prosobranch snails. Comp. Biochem. Physiol. 26, 1051-1059. FRIEDL F. (1964) A method for securing the snail, Lymnaea stagnalis jugularis Say, free from bacteria. Expl. Parasit. 15, 7-13. FRIEDL F. (1967) The partition of externally detectable nitrogenous material from the aquatic pulmonate snail Lymnaea stagnalis jugularis Say. Am. Zool. 7, 202. LOWRY O. H., ROSEBROUGHN. J., FARRA. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. )t. Biol. Chem. 193, 265-275. SELmSON D. & SELIGSON H. (1951) A microdiffusion method for the determination of nitrogen liberated as ammonia, ft. Lab. clin. Med. 38, 324-330. SPITZER J. (1937) Physiologisch-6kologische Untersuchungen fiber den Exkretstoffwechsel der Mollusken. Zool. ffahrb. Abt. Allg. Zool. Physiol. $7, 457-496. UMBREIT W., BUaRIS R. & STAUFFERJ. (1957) Manometric Techniques, p. 238. Burgess, Minneapolis.
Key Word Index--Ammonia; excretion; Lymnaea stagnalis; nitrogen catabolism; nitrogen partition; urea.
NITROGEN EXCRETION BY THE FRESH WATER PULMONATE SNAIL, L YMNAEA S T A G N A L I S JUGULARIS SAY* F. E. F R I E D L Department of Biology, University of South Florida, Tampa, Florida 33620, U.S.A.
(Received 26 September 1973) Abstract--1. The average per cent partition of excretory nitrogen produced into an ambient medium by Lymnaea stagnalis was found to be approximately: ammonia, 50; urea, 30; unidentified residual N, 20. Of the residual, about one-quarter appears to be volatile. 2. The endogenous nature of the ammonia and urea produced is supported by their appearance in the presence of both penicillin and streptomycin as bacteriostatic agents. INTRODUCTION IT HAS long been appreciated that organisms contribute nitrogenous end products of metabolism to their environment. The characterization of their excretory patterns by ammonotelic, ureotelic or uricotelic has tended to oversimplify the situation in many cases. Although a primary metabolic end product may be much in evidence, a total pattern of nitrogen excretion can involve many substances disposed of in various ways. In gastropod molluscs a number of such products have been categorized, and a recent summary of work on the group is reported by Campbell & Bishop (1970). The pulmonate gastropod Lymnaea stagnalis jugularis Say was studied by Bayne & Friedl (1968), who related the rates of accumulation of ammonia and urea in an ambient medium to size groups. The following work, partially reported in abstract by Friedl (1967), was undertaken to substantiate the endogenous origins of ammonia and urea in Lymnaea and to establish their proportions relative to a total amount of nitrogenous material found. MATERIALS AND METHODS The snails used in this work were laboratory-reared Lymnaea stagnalis jugularis Say, originally obtained from Minnesota. They were placed singly, or in groups, in a salt solution consisting of: KH,PO, 136rag/1.; Na2HPO, 331 mg/l.; KCI 25mg/l.; MgSO,.7H,O 50 mg/l. and CaC12 50 mg/1. in distilled water. * This investigation was assisted by Research Grant No. GB 3158 from the National Science Foundation. 617 22
618
F . E . FRIEDL
After a period of incubation at 25°C, the ambient fluids were removed, centrifuged and aliquots were analyzed in duplicate for total nitrogen, ammonia, urea and additional volatile and non-volatile nitrogenous residues. Total nitrogen was determined by a Kjeldahl-type digestion followed by direct nesslerization. In general, ammonia was determined by direct nesslerization of samples, and urea by difference, using a similar direct nesslerization procedure after incubation of samples with buffered urease. Microdiffusion (adapted from Seligson & Seligson, 1951) was used to estimate ammonia and urea in the presence of antibiotics. The formulation of the Nessler reagent and the general procedure for its use was as described in Umbreit et al. (1957). Nitrogenous bases were volatilized by the addition of NaOH to samples, followed by evaporation to dryness under reduced pressure over concentrated H2SO4. After total nitrogen had been determined, the residual volatile and non-volatile nitrogenous components could be estimated by difference using data obtained from the analysis of undesiccated samples. Weights reported are those from whole animals, including shells, after removing excess water by draining or blotting. Other experimental details are explained in the text or with figures. RESULTS AND D I S C U S S I O N Five experimental groups are characterized in T a b l e 1 and data obtained f r o m these groups are displayed in Tables 2-4. As a supplement to the study of Bayne & Friedl (1968), these data show that a small, variable a m o u n t of nitrogenous material in addition to a m m o n i a and urea accumulates in media surrounding L. stagnalis. T h e concomitant production of ammonia and urea (0.13 and 0.03 /~mole/g per hr of each respectively) falls within the ranges found in the m o r e extensive former study. Figure 1 summarizes the partition of nitrogenous material and portrays the additional volatile and non-volatile substances as an average of 17 per cent of the total nitrogen found. Spitzer (1937) noted somewhat larger TABLE 1--TOTAL
NITROGEN
PRODUCTION
OF FIVE EXPERIMENTAL
GROUPS OF
L. stagnalis
Group
Total N found Ozg) Rate of accumulation Oxg N/g whole weight per hr)* Ammonia found (% of total N) Number of snails in pool Average whole weight (g) Hours of incubation at 25°C
I
II
III
IV
V
680 2-82
360 1"18
1340 3"74
950 3'95
925 5"41
70"6
39"0
40'0
58-0
54.1
9 1 "66 24
5 1 "81 19
5 2-54 19
5 2"03 30
5 2"58 19
* Average rate of accumulation, 3-4/zg N/g whole weight per hr; range, 1"25"4/zg N. Snail weights are whole, wet weights including shell. Average whole weight of twenty-nine experimental snails, 2-06 g. Snails were isolated in sealed 500-ml flasks usually with 10 ml of ambient fluid present per snail.
619
NITROGEN EXCRETION BY L YMNAEA TABLE 2--RELATIVE QUANTITIES OF NITROGENOUS MATERIAL PRODUCED BY Z. (PER CENT OF TOTAL N )
stagnalis
Group*
Ammonia Urea Unidentified remaining N
I
II
III
IV
V
Average
40
54 27 19
58 26 16
39 32 29
71 25 4
52 28 17
-
-
--
* As in Table 1. TABLE 3--RELATIVE QUANTITIES OF VOLATILE AND NON-VOLATILE NITROGENOUS MATERIAL (EXCLUDING AMMONIA AND UREA) PRODUCED BY L . stag~aali$ (PER CENT OF UNIDENTIFIED REMAINING N )
Group*
Non-volatile Volatile
I
II
III
IV
V
Average
---
91 9
50 50
57 43
100 0
75 26
* As in Table 1. RATESOF PRODUCTIONOF AMMONIAAND UREABY L. stagnalis (~m/g whole weight per hr)
TABLE 4
Group *
Ammonia Urea
I
II
III
IV
V
Average
0"107 --
0"209 0-052
0-163 0"037
0"033 0-013
0"142 0"025
0"131 0-032
* As in Table 1. amounts of additional material f r o m s u m m e r snails kept at r o o m t e m p e r a t u r e ; he found 47-3% a m m o n i a N, 13-8% urea N, 5.2% uric acid N plus a remaining 38.5 per cent of unidentified nitrogen. It is not entirely clear what the volatile and non-volatile additional constituents of the ambient fluids m a y be, although Spitzer (1937) reported uric acid, purine nitrogen and amino nitrogen in some of his excretion studies. Volatile substances, in addition to ammonia, could be represented b y amines; non-volatile substances, in addition to urea, could include soluble proteins, peptides, amino acids and purines.
620
F. E. FRIEDL Nitrogen partition in Lymnaea stagna//$
FIG. 1. Pie diagram summarizing the partition of nitrogenous material produced by L. stagnalis. Data from Tables 1-3. Relative average amounts of ammonia N, urea N and unidentified residual N are shown. Undetermined volatile and non-volatile residual material represents about 17 per cent of the total nitrogen. An ultraviolet absorption spectrum recorded from one sample of ambient fluid (Table 1, Group I) shows inflections approaching 260 and 280 nm (As60 = 0.1249; A2a 0 = 0.1051; 280/260 = 0.842; dual beam recording; sample vs fresh ambient fluid) suggesting the possible presence of purines and phenolic groups. The Folin phenol reagent (applied as the Lowry protein method, Lowry et al., 1951) reacts with ambient fluid samples, and ninhydrin-positive material, over and above the amount expected from urea on a color basis, is found in ammoniafreed samples. Efforts were made to standardize as much of the sample collection procedure as possible. All snails were laboratory-reared, temperatures were controlled and a uniformly formulated ambient fluid, derived from one developed in previous studies on axenic Lymnaea (Friedl, 1964), was employed. Unfortunately, there was no feasible way to evaluate the nutritional state or overall metabolic condition of the animals. It has been suggested by Duerr (1966a, 1967, 1968) that ammonia and urea production by Lymnaea and other snails, when measured by analysis of ambient fluids, is apparent as a result of microbial decomposition of fecal masses. He found low levels of nitrogenous material in the presence of 1000 units/ml of penicillin (less than 6 Fg of nitrogen in any form over 24 hr), and considered L. stagnalis to produce little, if any, endogenous ammonia or urea.
621
NITROGEN EXCRETION BY L YMNAEA
Microbial action is indeed a most likely component of any investigation in which axenic animals are not used; however, when antibiotics were employed as bacteriostatic agents by the author, ammonia and urea production continued. Table 5 shows the results of an experiment wherein various concentrations and TABLE 5 - - T H E
PRODUCTION OF NITROGENOUS EXCRETORY MATERIALS BY L . $tagnali$ IN THE PRESENCE OF ANTIBIOTICS
Rate of accumulation (/zmoles/g per hr) Sample 1 (30 hr) Snail
Antibiotics
1 2 3 4 5 6 7 8 9
None P+S 500 P+S 500 P + S 1000 P + S 1000 P 500 P 500 P 1000 P 1000
Ammonia 0.040 0-055 0"064 0"105 0"067 0'065 0"089 0"035 0"095
Sample 2 (18 hr)
Urea
Ammonia
Urea
0"041 0"035 0"049 0"033 0"020 -0"071 0"047 0"072
0"000 0.129 0"087 0"112 0"073 0"070 0"155 0"000 0"071
0.282 0-064 0"053 0"072 0"040 0"040 0"064 0"082 0-091
Snail weights (whole) were between 0.54 and 2"05 g. Snails were placed individually in 250-ml sealed flasks with 10 ml of the salt solution described in text and kept at 25°C for the sample times indicated. Penicillin G, 500 or 1000 units/ml and streptomycin sulfate, 500 or 1000/zg/ml were included in the solutions for sample 1, as indicated by P or S. For sample 2, the same snails were washed, drained and then placed in new salt solution without antibiotics. An additional control snail (no antibiotic exposure) died before final weighing; it produced 0"860 and 1"07/~moles of ammonia and 1-90 and 1"61/zmoles of urea in samples 1 and 2, respectively. combinations of penicillin and streptomycin were present with single snails in 10 ml fluid. Although considerable variation is seen, ammonia and urea are both produced during and subsequent to antibiotic exposure. Up to 117/zg of ammonia and urea N were found for a 1.6-g snail kept 30 hr in the presence of 1000 units and/zgs respectively of both penicillin and streptomycin. In this test, the rate of ammonia production, although somewhat low, was still generally within ranges found in other experiments. Since there is evidence from studies on axenic snails that streptomycin inhibits growth (Chernin, 1959, on Australorbis) the author has preferred not to use antibiotics. Obviously, axenic animals, although abnormal in their own right, would be much desired for such work. In a general sense, as exemplified by the earlier investigations of Delaunay (1931), Spitzer (1937) and others, excretion in many invertebrates, unlike that in more rigidly compartmentalized and insulated forms, is difficult to evaluate in its entirety. Renal organs, digestive glands, whole animals and ambient fluids have all been studied. Kidney function, intestinal activity, diffusion, leakage
622
F . E . FRIEDL
and precipitation, all of which at various times may contribute in a major sense, were undoubtedly considered. Of these aspects, the precipitation of materials in tissues or organs (storage excretion) has commanded much interest. Spitzer (1937) noted uric acid and other purine material in digestive gland and nephridia of his snails (Limnea stagnalis). Duerr (1966b) also reports uric acid in L. stagnalis tissues. These findings tend to complicate the assignment of such animals to ammonotelism, ureotelism or uricotelism. The relationship between alternate modes of excretion and the ability of organisms to utilize them preferentially is not within the scope of this paper; although such plasticity can be a perplexing problem for the investigator, it remains one of the most fascinating aspects of the study of nitrogen catabolism in invertebrates. REFERENCES BAVNE R. & FRmDL F. (1968) The production of extemaUy measurable ammonia and urea in the snail, Lymnaea stagnalisjugularis Say. Comp. Biochem. Physiol. 25, 711-717. CAMeBELL J. & BISHOP S. (1970) Nitrogen metabolism in molluscs. In Comparative Biochemistry of Nitrogen Metabolism (Edited by CAMPBELL J.), Vol. 1, The Invertebrates, Chap. 5, pp. 103-206. Academic Press, New York. CrmRmN E. (1959) Cultivation of the snail, Australorbis glabratus, under axenic conditions. Ann. N. Y. Acad. Sci. 77, 237-245. DELAUNAYH. (1931) L'excr6tion azot6e des invert6br6s. Biol. Rev. 6, 265-301. DUERR F. (1966a) Nitrogen excretion in the fresh water pulmonate snail Lymnaea stagnalis appressa Say. The Physiologist 9, 172. DUERR F. (1966b) Qualitative analysis of the uric acid, xanthine, and guanine content of several snails. American Malacological Union Annual Reports, pp. 69-70. DUEaR F. (1967) The uric acid content of several species of prosobranch and pulmonate snails as related to nitrogen excretion. Comp. Biochem. Physiol. 22, 333-340. DUERa F. (1968) Excretion of ammonia and urea in seven species of marine prosobranch snails. Comp. Biochem. Physiol. 26, 1051-1059. FRIEDL F. (1964) A method for securing the snail, Lymnaea stagnalis jugularis Say, free from bacteria. Expl. Parasit. 15, 7-13. FRIEDL F. (1967) The partition of externally detectable nitrogenous material from the aquatic pulmonate snail Lymnaea stagnalis jugularis Say. Am. Zool. 7, 202. LOWRY O. H., ROSEBROUGHN. J., FARRA. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. )t. Biol. Chem. 193, 265-275. SELmSON D. & SELIGSON H. (1951) A microdiffusion method for the determination of nitrogen liberated as ammonia, ft. Lab. clin. Med. 38, 324-330. SPITZER J. (1937) Physiologisch-6kologische Untersuchungen fiber den Exkretstoffwechsel der Mollusken. Zool. ffahrb. Abt. Allg. Zool. Physiol. $7, 457-496. UMBREIT W., BUaRIS R. & STAUFFERJ. (1957) Manometric Techniques, p. 238. Burgess, Minneapolis.
Key Word Index--Ammonia; excretion; Lymnaea stagnalis; nitrogen catabolism; nitrogen partition; urea.