The Origins, Fate, and Ecological Significance of Free Amino Compounds Released by Freshwater Pulmonate Snails

Comp. Biochem. Physiol. Vol. 119A, No. 1, pp. 341–349, 1998 Copyright  1998 Elsevier Science Inc. All rights reserved.

ISSN 1095-6433/98/$19.00 PII S1095-6433(97)00433-9

The Origins, Fate, and Ecological Significance of Free Amino Compounds Released by Freshwater Pulmonate Snails J. D. Thomas and P. Eaton School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, U.K. ABSTRACT. The mass-specific accumulation rates (MSAR) of both total (TFAC) and individual free amino compounds (FAC) in conditioned media were measured by HPLC, using the orthophthaldialdehyde (OPA) methods, in the following cases: (a) laboratory-reared freshwater snails (B. glabrata) with chemosterilized shells; (b) Biomphalaria glabrata with non-chemosterilized shells; (c) B. glabrata faeces; (d) isolated shells of B. glabrata; and (e) 10 other species of freshwater gastropods from the Lewes Brooks, East Sussex, U.K. The MSAR values for B. glabrata show that 95% of the TFAC’s (predominantly ethanolamine, phosphoserine, and the amino acids leucine, isoleucine, valine, aspartic acid, and glycine/threonine) originated from the snails themselves as the faeces and shells contributed only 5.0 and 0%, respectively. In contrast, epizootic organisms on the shells of all 10 snail species from the Lewes Brooks released significant amounts of FAC with the two smallest species (Planorbis vortex and Planorbis contortus) having the highest MSAR values. The MSAR for isolated B. glabrata mucus was 42.45 µmol⋅g21h21. As 500 mg snails can release 16.67 mg of mucus daily, this could potentially result in the daily loss of 707.5 µmol of FAC. The cost/benefits of mucus secretion and the various anatomical, physiological, biochemical, and ecological mechanisms which allow freshwater snails to recover FAC’s lost as a result of a high rate of urine production in their hypotonic environment, are discussed. comp biochem physiol 119A;1: 341–349, 1998.  1998 Elsevier Science Inc. KEY WORDS. Ammonia, Biomphalaria glabrata, free-amino-acids, free-amino-compounds, mucus, pulmonate snails

INTRODUCTION Free amino compounds (FAC), which are ubiquitous components of dissolved organic matter (DOM) in freshwater ecosystems, are important as sources of information and nutrient to aquatic organisms (12). It is important, therefore, to ascertain the sources of the FAC in the external pool. These may include radiation-energized chemosynthesis from CO2, water and ammonia, UV, or chemically-mediated degradation of biopolymers (e.g., humic substances or mucopolysaccharides) released from living organisms or by dying or ‘‘dead’’ organisms as a result of sloppy feeding and microbially-mediated decomposition (11,14). In freshwater ecosystems dominated by macrophytes and molluscs, the latter may often be major contributors due to their high densities (. 2000 m3) and high FAC release rate (13). Thus, in experimental modular ecosystems, the mass specific accumulation rate of total FAC in media conditioned by snails (Biomphalaria glabrata (Say)) was 89.4 nmol g21 (wet Address reprint requests to: School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K. Tel. 01273 606755; Fax 01273 678433; E-mail: [email protected] Received 5 May 1997; revised 24 June 1997; accepted 3 July 1997.

mass of body without shell) day21, which is approximately 10 times greater than that for plants (Ceratophyllum demersum L.), which was 8.4 nmol g21 (wet mass) day21 (13). The purpose of the present investigation was to ascertain the possible sources and fate of FAC released by freshwater pulmonate snails. These sources include the shell, the epizootic mirco-organisms on its surface, the epidermis, faecal matter, urine, and mucus released by the snails. MATERIALS AND METHODS The following species of freshwater gastropod snails were used in the experiments: B. glabrata (Say), Planorbis (Planorbarius) corneus (L.), Planorbis (Tropodiscus) planorbis L., Planorbis (Tropodiscus) carinatus Mull., Planorbis (Anisus) vortex L., Planorbis (Bathymorphalus) contortus (L.), Lymnaea (Lymnaea) stagnalis (L.), Lymnaea (Radix) peregra (Mu¨ll.), Lymnaea (Stagnicola) palustris (Mu¨ll.), Physa fontinalis (L.), and Bithynia tentaculata L. The cultures of B. glabrata, a snail host of human schistosomes from South America, were maintained in flow-through (7.0 ml h21) aquaria (32.5 1) fed with chalk aquifer water at a temperature of 25 6 1°C (17). Their food consisted of epiphytes growing on the

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hornwort, C. demersum L., detritus, and lettuce, as a supplementary food. All the other species of snails, which were collected during the summer (August) from the Lewes Brooks, near Southease, East Sussex (50° 50°N; 0° 01′E) immediately prior to experimentation, were maintained under similar conditions at a temperature of 20 6 1°C. Prior to experimentation, snails were carefully selected on the basis of health, and then acclimated individually in 50 ml of a defined, standard snail water (SSW2) (16) in environmentally controlled units at 25 6 1°C in the case of B. glabrata, and 20 6 1°C in the case of the other species under a 12 hr light:12 hr dark regimen for 7 days. During this period they were fed on a daily ration of 2, 2.9-cm diameter lettuce discs. The water was changed daily and any uneaten lettuce removed. All snails were deprived of food for 24 hr prior to experimentation. The treatments, each replicated five times unless otherwise stated, were as follows: a. 500 6 50 mg (wet mass) adult B. glabrata with shells chemosterilized using 70% ethanol swabs. During the swabbing process the snails were encouraged to retract into their shells and care was taken not to touch the snail bodies with the swab. After the chemosterilization, the snails were transferred to fresh SSW2 and their behaviour was checked for normality. The snails were then incubated at 25 6 1°C for 24 hr in 2.5 ml of aerated SSW2 contained in a large test-tube containing a smaller inner test-tube (Fig. 1). The latter prevented the snail from leaving the medium while allowing it access to the air-water interface for gaseous exchange. b. 500 mg 6 50 mg adult B. glabrata with non-chemosterilized shells, incubated as described in ‘‘a’’. c. Snails with shell apertures closed with plasticine and then sealed with previously warmed wax to prevent the snails emerging from their shells. It was shown that these materials do not release amino compounds in measurable amounts. All the snail species listed above were subjected to this treatment. As it was demonstrated by using B. glabrata with chemosterilized shells that this method prevents the release of FAC by the snails themselves, it can be concluded that the FAC accumulating in the incubation medium had originated from epizootic organisms inhabiting the shell surface. The snail species used in this treatment were incubated under the conditions described in ‘‘a’’, except that those from the Lewes Brooks were maintained at a temperature of 20 6 1°C. d. Snail faeces collected from 500 mg B. glabrata over a 24hr period. The faeces, which are deposited in the form of compact cylinders (approximately 20-mm long per day), consists of intertwined hepatopancreas, gizzard and caecal strings (Fig. 2) and are easily handled. Faecal samples collected from individual snails were incubated in 2.5 ml SSW2 over a 24 hr-period. e. Snail mucus collected from 500-mg adult B. glabrata. The mucus was collected with a glass rod from the feet of

FIG. 1. Diagram of snail incubation apparatus.

snails held in an inverted position with the aid of a split plastic tube located at the air-water interface of 50-ml SSW2 contained in a beaker. Each sample of mucus, which weighed an averagae 200 6 18 mg (SE) as wet weight, was placed at the bottom of a small glass test tube to which 5-ml SSW2 was then added slowly so as not to disturb the mucus. 100-µl samples were then taken at intervals of 5, 15, 30, and 45 min from the mid-point. f. Topical samples (50 µl, 10 replicates) from various regions on the surface of 500-mg adult B. glabrata. These were taken from the tip of the tentacle, the oral region, the mantle inlet, the mantle outlet, the anterior region of the foot, and the posterior region of the foot (Fig. 2) with the aid of a 10-µl pipette to which a 2-cm length of fine wire tubing had been added, while the snail was held in an inverted position, as described in ‘‘e’’. Prior to analysis, the samples collected (usually 200 µl) were first filtered through a 0.22-µm filter and then centrifuged (average 10,000 g) to ensure removal of particulate matter. Individual amino acids and other amino compounds were analyzed using the ortho-phthaldialdehyde (OPA)

Origins of Free Amino Compounds

FIG. 2. Diagrammatic representation of ventral surface of

adult B. glabrata showing ciliary pathways, faeces, and mucus sheath.

method (8,9) with a Shimadzu HPLC system and a 15 3 0.04-cm i.d., 5 µm OD3 column (Jones Chromatography, Hengoed, U.K.) protected with a ‘‘guard-pak’’ column (Waters Associates, MA, U.S.A.). OPA amino compounds were detected with a fluorimeter (Fluoromonitor III, model 1311 LDC Milton Roy, Stone, U.K.) equipped with a 360-nm excitation filter, a 470 to 700-nm emission filter, and a 30µl cell column. Data were collected and concentrations calculated by means of a Spectra-Physics SP4270 integrator and a PC with a Microsoft Excel, respectively. Milli-Q water (Millipore Ltd.) was used to prepare buffers and solutions, including aqueous eluent (10 mM phosphate buffer at pH 7.3 prepared from NaH2PO4 and Na2PO4) and organic eluent (80% methanol HPLC grade, BDH, Poole, U.K., with 20% of the aqueous eluent). A flow rate of 1 ml min21 was used throughout. The OPA reagent solution (Sigma) buffered at pH 10.4 contained boric acid, potassium hydroxide, the surfactant Brij 35, 1 mg ml21 OPA, methanol, and mercaptomethanol. A mixture of the amino acids, l-asparagine, l-citrulline, Ophospho-l-serine, and l-tryptophan was added as an lamino-acid calibration standard (Sigma). Individual amino

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acids and ammonium chloride (as ammonia standard) were used as peak markers. Either homo-l-serine or homo-l-arginine was used as internal standards as it was verified that they were absent from the samples to be analyzed. OPA derivatization and injection were undertaken by an autosampler attached to the chromatograph. A 10-µl vol of internal standard and 50 µl of the sample were added together and mixed twice with 50 µl of the OPA reagent solution. After 1.5 min, incubation of the 50 µl of the derivatized sample were injected. As glycine and threonine are not easily separated by the method used, they were measured together. When measuring total free amino compounds (TFAC), the 200-µl samples were first treated as described above for the FAC analysis. They were then treated with one drop of 10 M NaOH, followed by bubbling with N2 gas for 2 hr to remove the ammonia. The latter procedure was necessary because although OPA derivatives of ammonia are only weakly fluorescent, with a quantum yield of approximately 1/50 of an equimolar OPA amino acid solution, the relatively high ammonia concentrations in snail conditioned media were found to interfere with the TFAC measurements. The latter were undertaken with the aid of fluorescence spectroscopy after derivatization with OPA (19). A volume of 250 µl of Sigma OPA reagent was added to each of the 750-µl samples. After shaking several times over a 1.5 min period, fluorescence intensity was measured at 340 nm excitation and 455 nm emission (1 cm path length) with a Perkin-Elmer fluorimeter. Total FAC concentrations were determined using a linear calibration line prepared by using 750 µl of a leucine standard, at different concentrations, and 250 µl of Sigma OPA reagent solution treated in the same way as the samples. Unless otherwise stated, each result is based on five replicates and is given as the mean 6 the standard error. Statistical differences were verified by the application of t-tests for small samples (3). RESULTS Excretory ammonia was the dominant amino compound in media conditioned by snails as it constituted 98.4% of the total amino compounds measured in media conditioned by both snails with chemosterilized shells, as well as those with non-chemosterilized shells. In contrast, ethanolamine, phosphoserine, and total amino acids formed only 0.35, 0.10, and 1.13%, respectively, of the total amino compounds that had accumulated in media conditioned by snails with chemosterilized shells. The comparable values for snails with non-chemosterilized shells were very similar, being 0.22, 0.12, and 1.30%, respectively. When the results were expressed in terms of mass-specific accumulation rates (MSAR; the mass being the total wet mass of snail), the mean values for snails with chemosterilized shells, nonchemosterilized shells, and isolated faeces that had been deposited by the snails over a 24-hr period were 35.96

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snails with chemosterilized and non-chemosterilized shells. In both cases, ethanolamine, phosphoserine, leucine, isoleucine, valine, aspartic acid, and glycine1threonine were the dominant amino compounds with MSAR’s . 2 nmol⋅ g21day21. Other amino acids that were present in both the snail treatments but with MSAR values less than 1.0 nmol⋅g21day21 were citrulline, phenylalanine, methionine, histidine, arginine, glutamic acid, tyrosine, and serine. In contrast, tryptophan, glutamine, and asparagine could not be detected in the media containing snails, although both tryptophan and asparagine were present in measurable amounts in media conditioned by faeces isolated from the snails. The MSAR values for the individual amino compounds in faeces-conditioned media were all much lower (, 0.25 nmol⋅g21day21) than those conditioned by the snails, when the MSAR values are determined on the basis of the wet weight of the snails. It can be concluded, therefore, that most (. 95% in the case of snails with chemosterilized shells) of the amino compounds such as FAA, phosphoserine, and ethanolamine in snail-conditioned media originate from the snails themselves rather than from their

FIG. 3. Mean mass specific accumulation rates (MSAR) (6SE) in nmol g21 (wet weight of snail) day1 for exogenous amino acids and amino compounds (other than ammonia) in media conditioned by adult B. glabrata kept under standard conditions (with chemosterilized shells) (Mean 1); adult B. glabrata with non-chemosterilized shells (Mean 2); and also for faeces released by snails over 24 hr and incubated separately for the same period (Mean 3). The MSAR for the incubated faeces was also expressed in nmol g21 (wet weight of snail) day21 in this case. g and t: Glycine and threonine; ser.: serine; tyr.: tyrosine; asn.: asparagine; gln.: glutamine; asp.: aspartic acid; glu.: glutamic acid; lys.: lysine; arg.: arginine; his.: histidine; ala.: alanine; met.: methionine; val.: valine; trp.: tryptophan; phe.: phenylalanine; isl.: isoleucine; leu.: leucine; cit.: citrulline; orn.: ornithine; pser.: phosphoserine; etha.: ethanolamine.

nmol⋅g21day21, 29.68 nmol⋅g21day21, and 1.48 nmol⋅g21, day21, respectively. Figure 3 shows that snails with chemosterilized shells have significantly higher MSAR values for ethanolamine, ornithine, and serine (P , 0.01 in all cases) on a mass-specific basis than was the case with snails with non-chemosterilized shells, as a result, it was also found that snails with chemosterilized shells have significantly higher (P , 0.05) MSAR values for the total non-ammonia amino compounds than snails with non-chemosterilized shells. Otherwise, there are no other significant differences between the MSAR values for amino compounds released by

FIG. 4. Mean mass specific accumulations rates (MSAR)

(6SE) in nmol g21 (wet weight of faeces) day1 for exogenous amino acids and amino compounds (other than ammonia) in media conditioned by the amount of faeces released by the snail over 24 hr.

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(TFAC) in mM in topical samples taken from various locations on the ventral surface of tethered adult, B. glabrata.

FIG. 6. Mean accumulation rates (AR) in nmol⋅day1 (6SE) of total free amino acids and amino compounds released from the shell surface of 11 snail species incubated in 2.5 ml of SSW2.

faeces. However, when the MSAR values for the individual amino compounds released from the faeces are expressed in terms of the wet mass of the faeces, rather than the wet mass of snails from which they originated, they become appreciably higher (in some cases .1.5 nmol⋅g21day21). These values are also more equitable than those for amino compounds in snail-conditioned media (Fig. 4). Figure 5 shows that there were no significant differences between the concentrations of TFAC sampled at the various locations on the surface of the snails with the exception of the tentacle tips where the concentrations were significantly lower than at the other location (P , 0.05). Shells of all the species of the temperate zone pulmonate snails collected from Lewes Brooks released measurable amounts of amino compounds measured as TFAC, but this was not the case with the laboratory-reared tropical pulmonate, B. glabrata (Fig. 6). There were no statistically significant differences between the rates at which the TFAC had accumulated in media conditioned by the snail species from the Lewes Brooks, with the exception of L. stagnalis, which has a much higher accumulation rate than the other species. This is not unexpected since it, has a much higher biomass than any of the other species (Table 1). When the results are expressed in terms of mass-specific accumulation rates, it was found, however, that it was the two smallest spe-

cies (Table 1), namely P. vortex and P. contortus, which had the highest values of 13.4 nmol⋅g21day21 and 40.3 nmol⋅g21day21, respectively (Fig. 7). Figure 8 shows that the various amino compounds present in 200 µg (wet weight) of foot mucus collected from B. glabrata are released into the incubation medium immediately following introduction, and although their concentrations varied, these did not change to any significant extent during the course of the 65-min incubation period. Mucus is, therefore, a potentially rich source of amino compounds, and using the above data, it was calculated that 1 g of mucus from B. glabrata (wet weight) could release approximately 42.45 µmol of TFAC per hour and 0.95–3.81 µmol of individual FAC per hr. As the amount of TFACs present in a unit mass of mucus is known, it should be possible to calculate the quantity of amino compounds that potentially could be released by all the mucus secreted by 500 mg snails on a daily basis. As it is known that a 500 mg snail releases approximately 250 µg of protein per day (J. D. Thomas, unpublished data), it can be calculated that it will secrete approximately 16.67 mg of mucus per day if it is assumed that mucus contains, on average, 30% of protein in terms of dry weight and 95% water (5,6). The amount of mucus secreted per day is, therefore, approximately 3.3% of the wet mass of the snail. It must be

FIG. 5. Mean concentrations of total free amino acids

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TABLE 1. Mean weights in mg. (x 6 SD) of freshwater gastropods used in experiments

x SD Biomphalaria glabrata (Say) Planorbis (T.) planorbis L. Planorbis (T.) carinatus (Mu¨ll.) Planorbis (A.) vortex L. Planorbis (B.) contortus (L.) Planorbis (P.) corneus (L.) Physa fontinalis (L.) Lymnaea (R.) peregra (Mu¨ll.) Lymnaea (S.) palustris (Mu¨ll.) Lymnaea (L.) stagnalis (L.) Bithynia tentaculata L.

500 6 50 258 6 20 72 6 31 24 6 7 15 6 4 604 6 85 56 6 13 250 6 43 354 6 37 2052 6 229 172 6 23

noted, however, that most of the water in the mucus will have been sequestered from the medium after secretion. As 1 mg (wet weight) of mucus contains up to 42.45 nmol of TFAA and up to 0.95–3.81 nmol of individual amino compounds, the mucus secreted by the 500-mg snail on a daily basis could potentially release up to 707.5 nmol of TFAC and from 15.8–63.5 nmol of individual amino compounds. DISCUSSION Origins and Fate of Amino Compounds Unlike the temperate zone snails, the tropical snails, B. glabrata, did not release measurable amounts of amino compounds from their shells. This unique feature can be attributed to the absence of algae on their shell surfaces as a result of intense mutual grazing in the high density culture systems. These results are also in accord with those described for foraminiferous shells (10) buried in the sediments, as these also failed to release free amino acids. As the faeces of B. glabrata with chemosterilized shells, as well as those without chemosterilized shells, contribute only 4.1 and 5.0%, respectively, to the mass of amino compounds present in the conditioned medium on a weight-specific basis, it can be concluded that the remaining 95.9 and 95% of the amino compounds in the medium must have originated from the bodies of the snails. The question of why the MSARs of total amino compounds in general, and of ethanolamine, ornithine, and serine, in particular, are significantly lower in media conditioned by B. glabrata with non-chemosterilized shells than in media conditioned by snails which had their shells chemosterilized, needs to be addressed. The most plausible explanation is that it is due to the selective uptake of ethanolamine, ornithine, and serine by epizooic or heterotrophic algae bacteria present on the non-chemosterilized shells. This hypothesis is supported by scanning electron microscopy, which revealed that the non-chemosterilized shells were sparsely colonized with bacteria. It can be postulated that the bacteria, unlike the algae, had avoided grazing by the snails because of their very small size. However, except

FIG. 7. Mean mass specific accumulation rates (MSAR) in

nmol g21 (wet weight of snail) day1 of exogenous total free amino acids and amino compounds (other than ammonia) released from the shell surfaces of eleven snail species incubated in 2.5 ml of SSW2.

for the above differences, the MSAR values for amino compounds released by snails with non-chemosterilized and chemosterilized shell are very similar. It seems probable that the amino compounds released into the medium by the experimental snails originate from the large volumes of dilute urine produced by them as a consequence of living in a hypotonic medium. In the case of freshwater pulmonate snails such as Lymnaea stagnalis, for example, it has been shown that the water turnover rate is approximately 500–750% of the body water per hr (18). Evidence in support of this hypothesis is provided by the fact that the amino acids in the urine are derived from the haemolymph by ultrafiltration and also by the significant correlation coefficient (0.53; P , 0.05) obtained when comparisons were made between the compositions of amino compounds in the haemolymph and in the medium (6). The dominant amino compounds in both cases were ethanolamine, phosphoserine, leucine, isoleucine, valine, aspartic acid, and glycine-threonine. Citrulline, phenylalanine, methionine, histidine, arginine, glutamic acid, tyrosine, and serine, on the other hand, were present at lower concentrations in both cases. Although tryptophan, glutamine, and asparagine were present in the haemolymph, they were ab-

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FIG. 8. Mean concentrations of free amino acids and other amino compounds (other than ammonia) in nM (6SE) at successive time intervals of 1, 5, 15, 30, 45, 55, and 65 min following the introduction of a 200 mg 6 18 (wet weight) sample of mucus into 5 ml of SSW2.

sent from the medium, suggesting that the transport mechanisms involved in their uptake are highly efficient. In contrast, the correlation coefficient obtained when comparisons were made between the amino acid compositions in the foot muscle cytosol and the medium were very low (0.13) and non-significant (P . 0.05), thus indicating that the epidermal cells were unlikely to be a major source of exogenous amino compounds (6). Furthermore, there is no evidence that the mucus itself degrades to produce more amino compounds, as the composition of the amino compounds and their concentrations remain constant at least over a shortterm (65 min) incubation period. It seems probable, therefore, that the shedding of the FAC by the mucus sample placed in the relatively large volume of incubation medium is attributable to visco-elastic transformation. As the ureter in pulmonates opens within the mantle cavity above the rectal ridge (Fig. 9), the amino compounds released in the urine will be recycled within the mantle cavity by the inhalant and exhalant currents. Further transintegumental uptake of the amino compounds may, therefore,

occur during the course of this passage within the mantle cavity as autoradiographic studies (W. Pan, unpublished) have shown that the entire wall of the mantle cavity has the capacity for accumulating amino acids. After leaving the mantle, via the exhalant current, the remaining amino compounds will tend to be distributed over the entire body surface by the ciliary currents (Fig. 2). Topical sampling revealed that although the concentrations of TFAA were uniform along the body surface, they had declined markedly at the tip of the tentacle, approximately 10 mm away from the pseudobranch, where the exhalant current originates. As a result of the ciliary currents along the body surface, further resorbtion of amino acids can take place via the mucus as it consists of glycoprotein and mucopolysaccaride molecules which form a gel-like network with a high affinity for both polarized and non-polarized amino compounds (1,2). This proposed model for recycling of organic solutes resembles that suggested for ‘‘epidermal-cuticular complexes’’ in the case of the marine polychaete worm Nereis (7). Calculations made on the basis of the amount of mucus released

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FIG. 9. Diagrammatic representation of B. glabrata to illustrate the direction of water flow within the mantle cavity where

the ureter opens. The broken line indicates where cuts were made to expose the rectal ridge. The arrows indicate the direction of inhalent and exhalent currents.

by a 500-mg snail per day and the mass of total amino compounds that the mucus can adsorb reveal that approximately 707.5 nmol of TFAC could be trapped and subsequently released by the mucus on a daily basis. However, as the 500mg snails only release approximately 13.3 nmol of TFAA and 18.0 nmol of TFAC per day, it can be postulated that most of the TFAA and TFAC adsorbed in the mucus (approximately 98.2 and 97.4%, respectively) is taken up by the epidermal cells. The hypothesis that mucus traps FAA, thus facilitating recovery by epidermal cells, requires further verification, but should it prove to be correct, this benefit can be added to those already listed for mucus (4–6). These include the provision of an unstirred layer which acts as a diffusion barrier thus decreasing water influx, facilitation of locomotion and adhesion to substrates, storage of pheromones and antibiotics for informational and defensive purposes, respectively, and provision of substrate and nutrient for micro-organisms from which the snails can derive mutual benefits subsequently. In addition, it has been suggested recently that the mucus may also act as a precursor of humic substances from which the snails may derive several other advantages (11,14,15). These benefits help to offset the high costs which molluscs incur when producing mucus. In the case of Patella vulgata, the costs may amount to 23% of ingested energy (4,5). It may be postulated, therefore, that in addition to the

kidney, pulmonate snails, such as B. glabrata, have evolved other efficient anatomical, physiological, biochemical, and ecological adaptations to recover amino compounds, including osmolytes such as alanine, glutamic acids, phenylalanine, serine, and phosphoserine that are lost during excretion (Fig. 10a and b). Despite living in a hypotonic environment, B. glabrata is able to reduce the ambient concentrations of amino acids to levels which are lower than those achieved by marine mussels. This is achieved by using efficient transporters, with Km values ranging from 1–200 µM (6) located in the integument. The absence of asparagine, glutamic acid, and tryptophan in conditioned media, despite their presence in the haemolymph, indicate that they are accumulated by highly efficient transporters. There is now accumulating evidence that other freshwater invertebrates are also capable of transintegumental uptake of other low molecular compounds (11). Ecological Significance of the Amino Compounds Released by Epizootic Organisms on the Shells and by the Faeces The concentrations of TFAA in media conditioned by epizootic organisms on snail shells, which varied from 32.4– 338.0 nM are comparable to those found in cultures of nonaxenic Lemna (approximately 100 nM) (15) but somewhat

Origins of Free Amino Compounds

FIG. 10. A summary of the various short term (a) and longer term processes (b) which allow snails to recover some of the exogenous organic compounds released by excretory and secretory mechanisms.

lower than those found in the water column of natural water bodies such as the Lewes Brooks and the Isle of Thorns (643–1330 nM and 411–691 nM, respectively) (14). Nevertheless, the epizootic organisms could benefit the snails by contributing amino compounds to their nutrient pool, as well as providing camouflage and nutrient to grazing conspecifics. Although only approximately 4–5% of the amino compounds released by snails originate from the faeces when they accumulate in the sediments, they could provide the snails and other detritivorous organisms with an important food source as their TFAA concentration may be as high as 293.8 µM compared with average values of 26.1– 29.5 and 30.4–58.4 µM in the sediments from the Isle of Thorns and the Lewes Brooks, respectively (14). This observation helps to explain why the coprophogous habit is so widespread amongst aquatic detritivores. The authors wish to thank the NERC and the UNDP/World Bank/ WHO Special Programme for financial help, Mr. C. Kowalczyk for technical help and useful discussions, Professor A. L. Moore for providing facilities, and Mrs. P. Chatfield for typing the manuscript.

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