Utilization of Dissolved Organic Matter (DOM), from Living Macrophytes, by Pulmonate Snails: Implications to the “Food Web” and “Module” Concepts

Comp. Biochem. Physiol. Vol. 117A, No. 1, pp. 105–119, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00377-5

Utilization of Dissolved Organic Matter (DOM), from Living Macrophytes, by Pulmonate Snails: Implications to the ‘‘Food Web’’ and ‘‘Module’’ Concepts J. D. Thomas and C. Kowalczyk School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9OG, U.K. ABSTRACT. The rates at which freshwater macrophytes (axenic and nonaxenic Lemna minor L. and nonaxenic Ceratophyllum demersum L.) accumulated inorganic carbon (IC) into plant organic carbon (PLOC) and then released it as dissolved organic carbon (DOC) and fine particulate organic carbon (FPOC) were measured by incubating the plants in media containing 14C-labelled inorganic carbon (IC 5 CO2 1 HCO23 ). Both the rate of fixation of 14C and the rate of loss of PLO14C were higher in the case of the faster growing Lemna than for Ceratophyllum. The fraction of the 14C fixed by nonaxenic Lemna and Ceratophyllum and then released as DO14C increased significantly with time to give values of 4.2% and 4.6%, respectively, after 9 days’ incubation. There were no significant differences between the rates of release of DO14C by axenic and nonaxenic Lemna but nonaxenic Lemna released significantly more of the fixed 14C as FPO14C (1%) than axenic Lemna (0.1%). Nonaxenic Ceratophyllum released significantly more FPO14C (8.2%) than Lemna (0.9%) after 9 days of conditioning. Some of the 14C in the DO14C accumulated as carboxylic acids (C6, C7, C16, C18) in the surface film. Living adult snails incubated for 38 hr in DO14C, from nonaxenic Lemna, accumulated significantly more of the 14C in their body tissues, haemolymph and shell (9.5%, 6.3%, 0.4% of the total carbon, respectively; concentration factor 10–15) than control snails. Snails incubated in media with DO14C from axenic Lemna accumulated proportionately less of the 14C label and produced more respiratory CO2 than snails incubated in media from nonaxenic Lemna. The results are discussed with particular reference to the food web and modular concepts. comp biochem physiol 117A;1:105–119, 1997.  1997 Elsevier Science Inc. KEY WORDS. Aquatic macrophytes, dissolved and particulate organic matter, freshwater pulmonate snails, Biomphalaria glabrata, uptake of DOC, food web, modular systems

INTRODUCTION Freshwater pulmonate snails tend to live in close association with macrophytes (9,10,54,55,63,84–86,95). As a result, many ecologists have considered them to be simple herbivores that obtain their energy by consuming and digesting the living green tissues of macrophytes or their epiphytic algae (26–28,62,68,73). By implication these authors, therefore, consider that snails regulate the population densities of macrophytes and their epiphyton. However, the following considerations suggest that this simplistic view may be incorrect. First, there is now accumulating evidence that pulmonate snails tend to exploit very little, if any, of the living macrophyte tissues and that they are primarily detriAddress reprint requests to: Dr. J. D. Thomas, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K. Tel. 01273606755; Fax 01273-678433. Received 26 June 1996; accepted 16 September 1996.

tivorous (8,70,72,90,91,95,97,98). Furthermore, although epiphytic algae are ingested, many of them remain viable after gut passage (99). Second, it has been demonstrated that the growth of aquatic macrophytes such as Ceratophyllum may be enhanced by the presence of pulmonate snails (9,10,85,86,97,98). These latter findings led Thomas (85,90) to postulate that pulmonate snails, the macrophytes and the associated epiphytic algae and bacteria should be regarded as a four component modular, mutualistic system, with six interacting subsets. Analysis of these subsets produced evidence of mutualistic interactions including the utilization of metabolic end products exchanged between living organisms as nutrients (85). In previous articles, attention has been focussed on the release of short-chain carboxylic acids (C2–C 5) by living bacteria involved in the decomposition of dead organic matter (15,16,64,78,79,85) and their detection and utilization as nutrients by pulmonate snails (78,83,93). The aim

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of the present work was to further examine the snail macrophyte subset and specifically to determine the rate of release of photoassimilates, in the form of DO14C (dissolved organic carbon) and FPO14 C (fine particulate organic carbon), by macrophyte species, their distribution in the water column and the extent to which they might be accumulated and metabolized by the snails. MATERIALS AND METHODS Pretreatment Axenic cultures of Lemna minor L., prepared according to the method of Baker and Farr (4), were obtained from Dr J. H. Baker, IFE, River Laboratory, Wareham, and maintained by serial culturing in sterile Jacob’s medium (pH 6) (6). Nonaxenic L. minor and Ceratophyllum demersum L., collected from the Lewes Brooks, were similarly maintained in Jacob’s medium for 7 days prior to experimentation. The albino, sexually mature (400–600 mg wet weight) Biomphalaria glabrata used in the experiments were obtained from stock cultures of known ages (91). The methods for culturing, selecting, and pretreating the snails in standard snail water (SSW2) are given in Thomas (83,88,94). Measurement of Compartmentation of 14 C-Labelled Organic Carbon within the Macrophyte Culture Systems AXENIC AND NONAXENIC LEMNA. Twenty microCurie (0.2 ml) of NaH14CO3 was initially injected through rubber seals into 9.8 ml aliquots of Jacob’s medium (pH 6.0) containing 40 axenic or nonaxenic Lemna fronds. Each treatment was replicated six times. Tests for sterility on the axenic Lemna were carried out at intervals by placing the fronds on CPS agar plates (4). Lemna cultures were incubated for 1, 4 and 6 days at 22°C (12 hr L/D cycle) in plant growth chambers and swirled gently once daily. Controls without plants were also run in parallel. At the end of each incubation period the Lemna cultures were dried at 180°C for 3 hr and then weighed prior to solubilization in the tissue solubilizer/scintillation fluid (Fluorosol; National Diagnostics). Aliquots (0.1 and 0.2 ml) of the solubilized plant material and the incubation medium were then added to scintillation vials containing 10 and 15 ml Fluorosol, respectively. After adjusting the pH of the remaining incubation medium from 6 to 2–3 by the addition of HCl, it was then bubbled with N2 gas for 10 min to remove all the inorganic carbon as CO2. This method is known to be efficacious, as in a preliminary test bubbling a solution of NaH14CO3 with nitrogen for 5 min at a pH of 3.0 removed 99.99% of the inorganic carbon. After removal of the inorganic carbon, a 0.2 ml aliquot from each replicate was placed in 15 ml Fluorosol in a scintillation vial. Finally, after the remaining incubation medium had been filtered through a 0.22 µm Millipore GSWF filter, 0.2 ml of the filtrate from each replicate was added to 15 ml Fluorosol in

a scintillation vial. The 14C activity was then counted in each sample in a Beckman LS5800 liquid scintillation counter. NONAXENIC LEMNA AND CERATOPHYLLUM. Forty microCurie (0.1 ml) of NaH14CO3 was added to 9.9 ml of nonsterile Jacob’s medium (pH 6.0) in 25 ml flasks containing either 40 fronds of Lemna or short stems of Ceratophyllum (2– 3 cm lengths, with two nodes). Controls without plants were also set up. Cultures were incubated for 1, 2, 5, 7 and 9 days (three replicates in each case) in plant growth chambers at 22°C (12 hr L/D regimen) and swirled daily. At the end of each incubation period the various compartments were dealt with as described above.

Spatial Distribution of Total Organic Carbon in Media Conditioned by Plants Cultures of both axenic and nonaxenic Lemna were incubated in sealed glass dishes (8 cm diameter 3 4 cm high) containing 50 ml Jacob’s medium (pH 6.0) and 20–60 µCi NaH14CO3. The cultures were incubated at 22°C (12 hr L/D cycle) for 5–33 days. A sampling device was constructed to collect a visible microlayer in media conditioned by the plants. This consisted of a circular, 25 mm diameter, perspex frame supporting fine tygan mesh (diameter 15 mm, mesh size 20 3 21, aperture size 1 mm) mounted on a perspex frame with a handle. When the tygan mesh was allowed to make contact with the surface it, was found that it collected 213 µ1 (SD 5 16 µ1, n 5 15) to a depth of 2.00 mm. This sample was then washed off the mesh into a collecting vessel with 2 ml distilled water followed by 2 ml n-hexane. The 14C activity of the total organic carbon (TOC) (FPO14C 1 DO14C) in the aqueous and hexane fractions was determined as described above. Samples of the medium below the surface layer were also assayed. Analysis of Long-Chain Carboxylic Acids in the Surface Films of Lemna-Conditioned Media These were analysed by gas chromatography using a Philips PU4500 GC with an FID detector and a 10 m, 530 µm diameter FFAP column with a 1 µm film of OV351 (Phase Separations Ltd). Filtered nitrogen was used as the carrier gas at a flow rate of 15 ml min21. The injector and detector temperatures were 250°C. After injecting the sample onto the column at 40°C, the temperature was then raised from 40°C–100°C within 1 min and then again from 100°C– 235°C at 4°C min21. Measurement of Accumulation of 14C from DO14C in the Various Snail ‘‘Compartments’’ 14

Both nonaxenic and axenic Lemna were used for producing DO14 C. The method involved incubating varying numbers of either axePRODUCTION OF DO C OF PLANT ORIGIN.

Utilization of Dissolved Organic Matter

nic or nonaxenic Lemna fronds in 125 ml SSW2 (88) (pH 7.80 6 0.05, conductivity 550 µmhos/cm2), containing 300 µCi NaH14 CO3 at 22°C in a 12 hr L/D regimen for 4 days. The media containing only DO14C were prepared as described above and the pH adjusted to 7.8 6 0.5 by the addition of NaOH. When necessary they were stored frozen (220°C) prior to use. Media without Lemna, kept under the same conditions as those used for producing DO14 C, were used as controls. After the conditioned media had been placed in the experimental vials, 0.2 ml samples were taken for the initial DO14C counting. 14

The adult (400–600 mg) B. glabrata used in the experiments were acclimated individually with excess of lettuce as food in 100 ml SSW2 for 2 days and then deprived of food for 1 day prior to experimentation. After their shells had been dried with tissue paper, the snails were placed individually in 10 ml of the test media in 50 ml boiling tubes. The snails were prevented from leaving the medium by fixing another slightly smaller test tube inside the boiling tube. This arrangement allowed the snails access to the air–water interface. The treatments were as follows: INCUBATION OF SNAILS IN DO C.

1. Media conditioned by nonaxenic Lemna containing: (a) snails with unswabbed shells (seven replicates); (b) snails whose shells had been swabbed with 70% ethanol then rinsed in SSW2 (seven replicates); and (c) unswabbed, heat-killed (immersion for 5 sec in water at 70°C) snails in media containing 10% sodium hypochlorite (9 ml medium 1 1 ml sodium hypochlorite) (three replicates); 2. Control media subjected to the same treatments as above but not conditioned by Lemna, with unswabbed snails (four replicates). The snails were incubated in the various media in the environmental unit at 26°C 6 1°C (12 hr L/D regimen) for 38 hr. At the end of this period the snails were removed and, after rinsing in SSW2, placed in scintillation vials and stored at 220°C. The 14C activity in the following compartments was then measured. Medium. The medium from each tube was transferred to scintillation vials. After the faeces and particulate organic matter (if any) had settled, 5 ml of the supernatant was transferred to new vials and frozen at 220°C. After removing an additional 4 ml of the supernatant from the vials containing the faecal matter, the remaining contents were acidified (by the addition of 1 drop of HCl) to remove any 14 CO2. The vials were dried at 180°C for 10 min. Then 0.1 ml of distilled water and 15 ml of Fluorosol were added to solubilize the material prior to counting. After the supernatants from the original vials had thawed, 15 ml of Fluorosol was added, and the 14C activity was then measured in 0.2 ml aliquots. The pH of the remainder was then adjusted to 2–3 by the addition of HCl before bubbling with N2 for 15 min to remove 14CO2. The 14C activity in 0.2 ml aliquots

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to which 15 ml Fluorosol had been added was then counted to determine the total 14C activity. The difference between the readings for total 14C and total organic carbon (TO14C) provides a measure of dissolved inorganic carbon (DIC 5 14 CO2 1 H14 CO32). To determine the levels of 14C activity in mucus on the walls of the incubation tubes, these were first rinsed with SSW2. Then 0.2 ml of 5M NaOH and 15 ml of Fluorosol were added. After vigorous mixing the solutions were transferred to scintillation vials prior to counting. 14 C activity was also measured in control treatments, containing either plants or snails to which no 14C label had been added. Snail Compartments. (a) Haemolymph. After the snails had been thawed, 20 µl of haemolymph was extracted from near the heart region. Then 0.1 ml of distilled water and 15 ml of Fluorosol were added to this prior to counting. The total 14 C activity in the haemolymph was then calculated by using the following equation: haemolymph volume (µl) 5 0.3752 3 total snail wet weight (mg) 1 43.87. This relationship between snail weight and haemolymph volume was determined in a separate experiment (n 5 35, r 5 0.94). (b) Snail body. The body was extracted from the shell by means of forceps, washed twice in 150 ml of distilled water and then placed in a vial containing 15 ml of Fluorosol. Counting was repeated until constant readings were obtained. (c) Shell. After thawing, the shell was washed thoroughly in 5 ml SSW2 and then transferred to a special 14CO2 measuring apparatus (94). This consisted of five replicated series of two 65 ml cylindrical glass vessels connected by plastic tubing via a peristaltic pump. Both vessels contained sintered glass inlet tubes. The snail shell was placed in the first chamber containing 40 ml of 20%(v/v) HCl to dissolve the shell and release the CO2, while the second chamber contained 2.0 ml of ethanolamine-methyl cellosolve (1:2 v/v) to trap the released CO2. Previous tests had shown that the trapping efficiency of the apparatus was 99.4%. The process was allowed to continue for 60 min with occasional swirling. This is known to be more than enough time to release all the 14C from the shell. Aliquots (0.2 ml) were taken from the trapping solutions and, after the addition of 15 ml of Fluorosol, the 14 C activity was counted. Controls without snail shells and shells from snails kept in media without DO14C were also run for comparison. RESULTS Rate of Accumulation and Release of 14C-Labelled Compounds by Plants NONAXENIC LEMNA MINOR AND CERATOPHYLLUM DE-

Both plant species converted large proportions of dissolved inorganic carbon (DI14C as 14 CO2 and H14CO32) into plant organic matter (PLO14C), MERSUM IN EXPERIMENT 1.

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14 C activity expressed as mean DPM (3 105) 6 SE per mg dry weight of nonaxenic Lemna and Ceratophyllum in various compartments including the plant material (PLO14 C), fine particulate organic matter (FPO14 C) and dissolved organic matter (DO14C) in the media conditioned by plants for various time intervals, and the mean percentages of the total initial 14C activity (6 SE) in various compartments, including dissolved inorganic carbon (DI14C) and 14CO2 , lost to the aerial compartment

TABLE 1. Compartmentation of

Conditioning time (days) Nonaxenic Lemna 1 2 5 7 9 Nonaxenic Ceratophyllum 1 2 5 7 9

PLO14C X 6 SE

FPO14cC

% 6 SE

X 6 SE

DO14C

% 6 SE

X 6 SE

DI14C

% 6 SE

% 6 SE

14

CO2

% 6 SE

44.47 6 1.61 32.55 6 1.88 26.50 6 1.76 22.92 6 0.91 21.48 6 0.91

74.1 78.5 72.8 65.8 68.5

6 6 6 6 6

0.5 0.7 2.2 1.9 2.3

0.16 0.14 0.12 0.30 0.20

6 6 6 6 6

0.06 0.01 0.03 0.06 0.13

0.3 0.3 0.3 0.9 0.7

6 6 6 6 6

0.1 0.0 0.1 0.2 0.4

0.27 0.19 0.46 0.69 1.13

6 0.01 6 0.00 6 0.04 6 0.13 6 0.31

0.4 0.5 1.3 2.0 3.6

6 6 6 6 6

0.0 0.0 0.1 0.4 1.0

1.1 1.1 1.4 2.4 4.5

6 6 6 6 6

0.2 0.2 0.2 1.1 1.8

24.1 19.6 24.2 28.9 22.7

6 0.5 6 0.5 6 2.4 6 0.7 6 0.7

4.41 6 2.55 4.44 6 1.07 3.62 6 0.87 3.64 6 1.50 4.70 6 0.95

41.6 50.2 47.0 41.1 40.6

6 6 6 6 6

9.3 6.9 4.1 5.4 5.7

0.04 0.06 0.10 0.10 0.37

6 6 6 6 6

0.01 0.01 0.04 0.04 0.11

0.5 0.7 1.4 1.1 3.1

6 6 6 6 6

0.1 0.1 0.8 0.2 0.7

0.02 0.04 0.13 0.12 0.20

6 0.01 6 0.01 6 0.00 6 0.05 6 0.03

0.2 0.5 1.8 1.4 1.8

6 6 6 6 6

0.1 0.2 0.3 0.3 0.3

1.6 0.7 3.1 2.1 2.3

6 6 6 6 6

0.4 0.2 0.9 0.0 1.0

56.1 47.9 46.7 54.3 52.2

6 9.5 6 7.1 6 4.1 6 5.8 6 7.1

expressed as DPM per unit biomass (dry weight) within 24 hr (Table 1). However, the nonaxenic Lemna converted approximately 10 times more of the inorganic carbon into PLO14C than was the case with nonaxenic Ceratophyllum over the same period. This difference is reflected in the higher (P , 0.001) proportion of PLO14C occurring in Lemna (74.1%) than in Ceratophyllum (41.6%) after a 1day incubation. As might be expected, the accumulation of the label in the plant was accompanied by a steep decline in the DI14C in the medium and a loss of 14CO2 to the atmospheric component. This loss to the atmosphere was less in the case of Lemna (24.1%) than for Ceratophyllum (56.1%), partly because the floating Lemna fronds acquired CO2 directly from the atmosphere via the stomata on their dorsal surface. By the end of the experiment, the PLO14C values for the nonaxenic Lemna had declined to less than half of those at the end of the first day (Table 1). In contrast, the corresponding PLO14C values for nonaxenic Ceratophyllum changed very little over time. Similar trends are also apparent in the case of percentage values for PLO14C, except that those for Lemna increased significantly (P , 0.05) by the second day before declining. Despite the fact that the PLO14C values for Lemna declined at a greater rate than those for Ceratophyllum, they remained consistently higher than the values for the latter plant. Both the FPO14C and DO14 C values expressed in terms of 14C activity or as percentages of the total 14C activity tended to increase with time in media conditioned by the two nonaxenic plant species (Table 1). With one exception the DO14C values were invariably higher in the media conditioned by Lemna. In contrast, the FPO14C values expressed as percentages of the total 14C activity tended to be higher in the case of Ceratophyllum than Lemna. Nevertheless, only a small amount of

labelled organic carbon was released as FPO14C and DO14C, as these constituted only 0.7% and 3.6%, respectively of the total 14C at the end of the experiment in Lemna-conditioned media. The corresponding values for Ceratophyllum were 3.1% and 1.8%, respectively. The amount of DO14C released, expressed as a percentage of total fixed carbon (PLO14C), increased with time (P , 0.05 and , 0.01) (Table 2) for both species of nonaxenic plants. However, although there were also tendencies for the amount of FPO14C released, expressed as percentages of PLO14C, to increase with time, these trends were not significant (P . 0.05) (Table 3). By the end of the 9-day incubation period, the nonaxenic Lemna and Ceratophyllum had released 5.24% and 4.4%, respectively, of the total fixed carbon as DO14C. The nonaxenic Ceratophyllum also released more FPO14C (8.2%) by the end of the experiment than nonaxenic Lemna (0.9%) (Table 3). AXENIC AND NONAXENIC LEMNA IN EXPERIMENT 2. The axenic Lemna accumulated much more of the 14C label as PLO14 C on a biomass basis by the end of the first day of incubation than nonaxenic Lemna (Table 4). Despite this the proportion of the label in the plant tissue, expressed as a percentage of total carbon, was the same for the two Lemna systems (approximately 78%), suggesting that there were differences between the metabolic and growth rates of the two plant systems. On subsequent days the PLO14C values did not change a great deal, implying that the plants were taking up 14C (as 14CO2 or H14CO32 ) at a significant, albeit at a slower rate, as measurable amounts of the label were being released as DO14C, FPO14C and respiratory 14CO2. It is likely that the decline in the rate of accumulation of 14 CO2 by the plants from the first day onward was due to resource depletion.

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109

TABLE 2. Relationships between time and quantity of DO14C released, expressed as the mean percentage (6 SE), of DO14C/

PLO14 by nonaxenic Lemna and Ceratophyllum and axenic Lemna Time (days) 1 2 4 5 6 7 9 P value Sig. level

Nonaxenic Lemna

Nonaxenic Ceratophyllum

Nonaxenic Lemna

Axenic Lemna

0.60 (6 0.04) 0.60 (6 0.03)

0.57 (6 0.12) 0.95 (6 0.23)

0.38 (6 0.05)

0.20 (6 0.02)

0.69 (6 0.04)

0.65 (6 0.04)

1.73 (6 0.05)

3.96 (6 0.92) 0.78 (6 0.05)

0.85 (6 0.06)

3.01 (6 0.51) 5.37 (6 1.62) 0.01 P , 0.01

3.44 (6 0.74) 4.51 (6 0.55) 0.01 P , 0.01

0.22 NS

0.08 NS

TABLE 3. Relationships between time and quantity of FPO14C released, expressed as the mean percentage (6 SE), of FPO14C/

PLO14 by nonaxenic Lemna and Ceratophyllum and axenic Lemna

Time (days) 1 2 4 5 6 7 9 P value Sig. level

Nonaxenic Lemna

Nonaxenic Ceratophyllum

0.37 (6 0.15) 0.44 (6 0.03)

1.33 (6 0.54) 1.40 (6 0.23)

0.46 (6 0.11)

3.30 (6 2.09)

1.28 (6 0.51) 0.93 (6 0.63) 0.12 NS

2.56 (6 0.23) 7.59 (6 1.01) 0.06 NS

Nonaxenic Lemna (Exp. 2)

Axenic Lemna (Exp. 3)

0.30 (6 0.05)

0.00 (6 0.01)

1.19 (6 0.65)

0.05 (6 0.02)

1.02 (6 0.25)

0.07 (6 0.06)

0.45 NS

0.41 NS

14 C activity expressed as mean DPM (3 105 ) 6 SE per mg dry weight of nonaxenic Lemna and axenic Lemna in various compartments including the plant material (PLO14 C), fine particulate organic material (FPO14C) and dissolved organic matter (DO14C) in the media conditioned by plants for various time intervals, and the mean percentages of the total initial 14C activity (6 SE) in the various compartments, including dissolved inorganic carbon (DI14C) and 14 CO2 , lost to the aerial compartment

TABLE 4. Compartmentation of

Conditioning time (days)

PLO14 C

FPO14C

DO14C

DI14C

14

CO2

X 6 SE

%

X 6 SE

%

X 6 SE

%

%

%

Nonaxenic Lemna 1 4 5

57.50 6 3.40 48.89 6 0.79 61.12 6 6.31

78.3 6 2.8 77.4 6 3.8 80.4 6 1.5

0.17 6 0.03 0.61 6 0.31 0.59 6 0.10

0.2 6 0.0 0.1 6 0.5 0.8 6 0.2

0.21 6 0.02 0.37 6 0.04 0.48 6 0.07

0.3 6 0.0 0.5 6 0.0 0.6 6 0.0

2.1 6 0.5 0.5 6 0.7 0.5 6 0.2

19.1 6 2.3 20.6 6 3.8 17.6 6 1.5

Axenic Lemna 1 4 6

93.27 6 7.40 93.29 6 5.53 99.91 6 9.33

77.8 6 2.9 83.7 6 5.3 83.1 6 2.2

0.00 6 0.01 0.05 6 0.02 0.09 6 0.06

0.0 6 0.0 0.0 6 0.0 0.1 6 0.0

0.19 6 0.02 0.61 6 0.05 0.83 6 0.08

0.2 6 0.0 0.5 6 0.0 0.7 6 0.0

3.8 6 1.3 0.7 6 0.2 0.4 6 0.1

18.3 6 1.7 15.1 6 5.1 15.7 6 2.3

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

37.7 6 9.8 29.8 6 5.7 45.7 6 2.2

62.3 6 9.8 70.2 6 5.7 54.3 6 2.2

Control 1 4 6

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14 C activity (in DPM 3 1023) per ml (6 SD, n 5 10) in the surface and subsurface layers of media conditioned by axenic and nonaxenic Lemna minor

TABLE 5. Mean

Condition of Lemna Incubation period (days) } surface layer } subsurface } surface layer } subsurface

Aqueous extract Hexane extract

Nonaxenic 7

Axenic 7

Axenic 25

13.5 6 9.3* 5.9 6 4.6 4.7 6 1.9** 1.7 6 0.8

11.6 6 1.2 10.6 6 1.7 1.1 6 1.0* 0.3 6 0.2

15.4 6 3.2 13.1 6 3.6 2.3 6 1.5* 0.2 6 0.01

*, **Indicate that 14C activity is significantly higher in the surface layer than in the subsurface layer at P , 0.05 and P , 0.01, respectively.

DO14C appeared to accumulate at a faster rate over the first 4 days than subsequently in both the Lemna cultures. Nevertheless, the DO14C values continued to increase and by the sixth day the DO14C constituted 0.6% and 0.7%, respectively, of the total 14C in the nonaxenic and axenic Lemna systems, respectively (Table 4). When the DOC values were expressed as a percentage of photosynthetically fixed carbon, the tendency for the values to increase with time was not significant (P . 0.04) (Table 4). At the end of the sixth day of incubation, the percentage of photosynthetically fixed carbon was approximately 0.8% for both axenic and nonaxenic Lemna. There were no significant differences between the values of DO14C in media conditioned by nonaxenic and axenic Lemna at any time. In the case of the nonaxenic Lemna the pattern of release of FPO14C was similar to that of DO14C, and it constituted approximately 0.8% of the total 14 C label or 1.0% of total fixed carbon by the end of the experiment. There was no significant tendency for the FPO 14C released by nonaxenic Lemna, expressed as a percentage of total fixed carbon, to increase with time (Table 4). In contrast, the quantity of PO14C released by axenic Lemna was barely detectable, and by the end of the experiment it formed only 0.1% of the total 14 C. In the case of the control treatment without plants, most of the 14C label entered the gaseous phase as 14CO2, and no 14 C-labelled organic material could be detected. COMPARTMENTATION OF

14

C LABEL IN LEMNA CULTURES.

After 7-day conditioning by nonaxenic Lemna the aqueous extract of the surface film contained significantly higher levels of 14C activity per unit volume than the underlying water column (Table 5). In contrast, the 14C activity levels in aqueous extracts of surface films conditioned by axenic Lemna for 7 and 25 days were found not to be significantly different from those in similar volumes of the underlying water column. However, the levels of 14C activity in hexane extracts of surface films in media conditioned by both nonaxenic and axenic Lemna were significantly higher than those found in the underlying water column in all three cases (Table 5). Longer term conditioning of the medium by nonaxenic Lemna over a period of 33 days resulted in the formation of a solid, white, translucent surface film among senescing Lemna fronds. This was found to contain 2.6% of the total

TABLE 6. Mean 14C activity (6 SE) expressed as percentages

of total 14C activity in various compartments including nonaxenic, senescing Lemna, the surface film and remainder of water column in Jacob’s medium conditioned by plants for 33 days Compartments Plant organic 14C Surface film 14C Dissolved inorganic 14C Dissolved organic 14C Particulate organic 14C Gaseous 14CO2

X 6 SE 19.4 6 0.2 2.6 6 0.3 0.1 6 0.0 1.4 6 0.1 0.2 6 0.0 76.3 6 0.3

In media without plants, all the 14C was lost to the atmosphere as 14CO2.

14

C activity (Table 6). Only 19.4% of the 14C activity remained in the plant by this time, the remainder being found in the gaseous phase as 14CO2 (74.4%) or in the medium as dissolved inorganic carbon, dissolved organic carbon and particulate organic carbon (0.1%, 1.4%, and 0.2%, respectively) in the water column (Table 6). Microscopic examination of the solid surface film revealed the presence of dense populations of coccoid, rod, spiral or filamentous shaped bacteria, cyanobacteria (including species of Ana baena, Oscillatoria), green algae (including species of Chlorella, Chlamydomonas, Carteria) and diatoms (Cocconeis placentula, Epithemia, Rhoicosphenia and Navicula). With the aid of the API 20E diagnostic system some of the bacteria present were identified as Klebsiella pneumoniae (Shroeter), Pseudomonas paucimobilis and Enterobacter agglomerans (Beijenck). The dry weight of the surface film produced over a period of 12 days was 91 µg cm 22 and appeared to consist mainly of lipid material as well as protein (9.1 µg cm22) and carbohydrate (0.02 µg cm22). Hexanoic, heptanoic, palmitic and stearic acids were identified as being present in the lipid material from day 7 onward (Table 7). Accumulation of the Pulmonate Snail, B. Glabrata, in Media Containing DO14C Produced by Nonaxenic Lemna and the Subsequent Accumulation of 14C in Various Compartments Including the Snail Tissues The experimental adult B. glabrata were incubated for 38 hr at 26°C 6 1°C (12 hr L/D regimen) in media containing

Utilization of Dissolved Organic Matter

111

TABLE 7. Concentrations (mM) of carboxylic acids in media

conditioned by axenic Lemna minor for up to 67 days Time (days)

C6 hexanoic acid C7 heptanoic acid C16 palmitic acid C18 stearic acid

7

16

67

— — 4 12

28 15 — 36

— — 14 31

only 14C-labelled DOC, released by healthy, rapidly growing, nonaxenic Lemna cultured for 4 days in SSW2. In the freshly conditioned medium the 14C in DO14C, FPO14C, plant organic carbon (PLO14 C) and dissolved inorganic carbon (DIC 5 14CO2 1 H14CO32) constituted 1.6, 4.2, 26.6 and 67.7% of the total 14C, respectively. However, before the snails were incubated in the medium the PLO14C and FPO14C were removed by filtration and the DO14C by bubbling with nitrogen for 15 min after adjusting the pH of the medium to 2–3. By using unconditioned controls it has been demonstrated that the latter method is efficient in removing at least 99.3% of the DI14C. Before placing the snails in the test media the pH of the SSW2 was restored to 7.8. When control snails were incubated in medium to which the DI14C had been added in the absence of Lemna, no measurable amounts of the 14C label became incorporated in their body tissues, faeces or mucus, and only trace amounts were found in their haemolymph and shell (Table 8). Likewise none of the 14C label could be detected in the tissues, shells or mucus of previously killed snails incubated in media containing DO14C of Lemna origin, although traces of 14 C were detected in their haemolymph and faeces. In con-

trast, significantly higher levels (P , 0.001) of 14C activity were found in the body tissues, haemolymph, shell, faeces and mucus of both normally treated and shell-sterilized experimental snails compared with the two groups of control snails. However, there were no significant differences between levels of 14C activity in the body tissues, haemolymph, shell, faeces and mucus of the normal snails and those that had chemosterilized shells. The highest levels of activity in both of these treatments were found to occur in the body tissues and haemolymph of the snails. By the end of the experiment much of the 14C activity in the treatments containing normal snails, snails with chemosterilized shells and dead snails was still either retained in the DOC compartment or had been released into the atmosphere as CO2. Approximately 9–10% of the 14C label also occurred as inorganic 14 C in the medium in each of the three treatments containing DO14C originating from the Lemna. In contrast only very low levels of 14C activity could be detected in the DOC, DIC and atmospheric CO2 compartments in the treatment containing control SSW2 that had not been previously conditioned by Lemna. Table 9 provides a measure of the extent to which the snails kept in various treatments had concentrated the 14 DOC from the medium. On the basis of the DO14C activity within the snails the concentration values range from 1.8–4.0, but if the respiratory 14CO2 or H14CO32 is included, then the concentration factors range from 10.0–15.1. There were differences in the distribution patterns of 14C activity after snails had been incubated in media containing 14 C-DOC derived from axenic and nonaxenic Lemna (Table 10). Thus, snails in media conditioned by axenic Lemna had lower percentages of 14 C in their body tissues and haemolymph and more in the 14C-DIC in the medium and shell (3.4, 4.3, 14.4 and 1.2%, respectively) than was the case with snails incubated in DOC produced by nonaxenic

TABLE 8. Mean 14C activity expressed in terms of DPM (3 104) and mean percentages of total 4C activity (6 SE) in parentheses

in various compartments after the adult snails (B. glabrata) had been incubated for 38 hr in media containing 14C-labelled dissolved organic carbon (DO14C) produced by nonaxenic Lemna over a 4-day period Treatments using DO14C from nonaxenic Lemna Normal snails Compartments Initial DO14C Snail body tissues Snail haemolymph Snail shell Snail faeces Snail mucus Total organic 14C in medium Dissolved inorganic 14 C in medium 14 CO2 in atmosphere

Shell sterilized snails

Control dead snails

Non-Lemna control normal snails DPM

%

6 6 6 6 6 6 6

(0.4 6 1.0) (17.5 6 10.6) (2.3 6 0.9) (0.9 6 0.5) (0.2 6 0.1) (23.7 6 15.5)

DPM

%

DPM

%

DPM

%

77.1 6 4.0 7.2 6 1.5 4.9 6 0.8 0.3 6 0.1 2.3 6 0.7 0.1 6 0.0 34.5 6 1.1

(9.5 6 2.0) (6.3 6 1.0) (0.4 6 0.1) (3.1 6 0.8) (0.2 6 0.1) (46.5 6 4.8)

81.1 6 1.0 8.2 6 1.5 4.7 6 0.8 0.3 6 0.1 2.7 6 1.0 0.1 6 0.0 36.9 6 1.7

(10.0 6 1.8) (5.7 6 1.0) (0.4 6 0.1) (3.4 6 1.2) (0.2 6 0.0) (45.5 6 2.1)

94.3 6 15.0 0.0 6 0.0 0.3 6 0.1 0.0 6 0.0 1.0 6 0.1 0.0 6 0.0 27.7 6 1.8

(0.0 6 0.1) (0.3 6 0.1) (0.0 6 0.0) (1.1 6 0.2) (0.0 6 0.0) (30.2 6 2.6)

6.8 6 0.4

(9.0 6 0.7)

8.1 6 1.6

(10.0 6 2.1)

8.1 6 0.2

(9.0 6 1.2)

0.7 6 0.2

(17.3 6 5.5)

20.8 6 4.8

(25.0 6 6.0)

20.1 6 1.8

(24.8 6 2.2)

57.1 6 12.9

(59.4 6 3.8)

1.4 6 0.5

(37.7 6 12.8)

3.8 0.0 0.7 0.1 0.0 0.0 0.9

0.0 0.0 0.4 0.0 0.0 0.0 0.5

J. D. Thomas and C. Kowalczyk

112

14 C within snails in the various treatments expressed as (A) the ratio of 14C per g wet weight of snails over the amount of 14C DOC per g of medium at the end of the incubation and (B) the ratio of 14C per g wet weight of snails over the amount of 14C DOC per g of medium at the end of incubation, assuming that 14C-CO2 or 14C-HCO32 had been metabolized by the snail

TABLE 9. Concentration factors for

Treatments Source of Medium Snail A B

Nonaxenic Lemna Unsterilized shell

Nonaxenic Lemna Sterilized shell

Axenic Lemna Unsterilized shell

Nonaxenic Lemna Dead snail

Unconditioned medium Unsterilized shell

3.15 10.17

3.17 9.98

1.78 14.17

0.08 —

3.99 15.11

Lemna (9.5, 6.3, 9.0 and 0.4% for the body tissues, haemolymph and DIC in the medium, respectively). DISCUSSION DOC Release by the Macrophytes The validity of the 14C method for measuring DOC or EOC (extracellular organic carbon) release rate by macrophytes depends on the time required for isotopic equilibrium to occur. As this is at least 24 hr for algae (45), it is likely to be somewhat longer for macrophytes. It follows, therefore, that 14C-DOC release rates for plants, based on 24-hr incubation periods, will tend to underestimate the true DOC release rate and that subsequent estimates based on incubation periods of 48 hr or more will be more accurate. Several of the present observations support the generalizations made by several workers (6,31,104) that the rate of secretion of DOC by macrophytes tends to be positively correlated with the photosynthetic and growth rates of the plants. Thus, the nonaxenic Lemna fronds, which had incorporated 74% of the 14C-DIC into 14C-PLOC over 24 hr compared with only 42% for Ceratophyllum, had also grown faster and released more 14C-DOC than Ceratophyllum on a weight specific basis. Although this relatively high level of efficiency in the uptake of inorganic carbon (DIC) by

Lemna may be partly due to its ability to use both atmospheric and aqueous sources of inorganic carbon (105), it is likely that the inherent capacity for faster growth exhibited by the small Lemna fronds was the major factor. The rapid growth of the Lemna fronds also helps to explain why the weight specific PLO14C values for the nonaxenic Lemna in the first experiment declined much more rapidly than was the case for Ceratophyllum. Finally, the weight specific DO14C accumulation rates for the rapidly growing nonaxenic Lemna in the first experiment were much higher than those for the nonaxenic Lemna used in the second experiment. Nevertheless, the rates of growth and photosynthesis are not the only factors involved in determining the DO14C release rate. Thus, it has been shown that the rate of accumulation of DO14C was much higher (4.3% of the carbon fixed) when the source of inorganic carbon was limiting than when this was not the case (<1.0% of the carbon fixed) (4). As the plants used in the present experiments were also incubated in closed systems, which would result in inorganic carbon becoming limiting, this helps to explain the close similarity between the percentages of 14C released as DOC from the carbon fixed by Lemna (5.2%) and Ceratophyllum (4.4%) with that found for axenic Lemna (4.3%) (4). The much lower values of 0.02–0.07% for axenic

C activity expressed in terms of DPM (3 104) and mean percentages of total initial 14C activity (6 SE) in various compartments of adult snails (B. glabrata) with untreated shells, which had been incubated for 38 hr in media previously conditioned with 14C-labelled dissolved organic carbon (DO14C) produced by axenic and nonaxenic Lemna over a 4day period

TABLE 10. Mean

14

Treatments 14

DO C from nonaxenic Lemna

DO14C from axenic Lemna

Compartments (initial DO14C levels)

77.1

% 6 SE

18.0

% 6 SE

Snail body tissue Snail haemolymph Snail shell Snail faeces Snail mucus Total organic 14C in medium Dissolved organic 14C in medium 14 CO2 in atmosphere

7.2 4.9 0.3 2.3 0.1 34.5 6.8 20.8

9.5 6 2.0 6.3 6 1.0 0.4 6 0.1 3.1 6 0.8 0.2 6 0.1 46.5 6 4.8 9.0 6 0.7 25.0 6 6.0

0.6 0.7 0.2 0.3 0.0 4.8 2.6 8.5

3.4 6 0.7 4.3 6 0.2 1.2 6 0.3 1.8 6 0.3 0.3 6 0.0 27.2 6 3.7 14.4 6 1.6 47.5 6 4.6

Utilization of Dissolved Organic Matter

Lemna perpusilla (104) are attributable to the short incubation period of a few hours. After 24 hr incubation the corresponding value for axenic Lemna in the present experiment was only 0.20%, but after 6–9 days’ incubation the present values were comparable to those cited by other workers for various macrophyte species: 1.3%–3.8% for Myriophyllum spicatum (31); 7% for Najas flexilis (1); and 1–3% for N. flexilis in Lawrence Lake (61). However, much wider ranges of DOC released (1%–100%) as percentages of fixed carbon have been quoted (11,42,49,75,104), although according to the latter authors most of the values were ,10%. When plants such as Zostera and Thalassia are growing rapidly under optimal conditions in nature, they may release up to 20% of their fixed carbon (11,65). Most of DOC released by aquatic macrophytes is considered to originate from leaves, but there is also accumulating evidence that the roots and rhizomes of both aquatic and terrestrial macrophytes also release substantial amounts of DOC (31,57,58). These estimates of DOC release by macrophytes are comparable to those of 12%–20% for photosynthetically produced organic carbon released as DOC by phytoplankton (35,36,80,81) or during sloppy feeding by zooplankton (52). As axenic Lemna fronds are devoid of surface living bacteria, which are regarded as the main consumers of DOC (71,81,96), it might be expected that they would release more DOC on a weight specific basis than nonaxenic fronds. However, this hypothesis receives no support from the present results as there was no difference between the weight specific DOC release rates for axenic and nonaxenic Lemna. In contrast, nonaxenic Myriophyllum releases more DOC on a weight specific basis than axenic plants (31). These authors attributed this finding to the slower growth rate of the axenic plants and to the absence of epiphytic algae on their surface. As both the axenic and nonaxenic plants were cultured in a similar medium, it is possible that the poorer growth and lower DOC release rate of the axenic macrophytes were due to the absence of growth factors that would normally be released by epiphytic bacteria or algae. In contrast to macrophytes it has been found that axenic cultures of algal species (103) release significantly more DOC than the nonaxenic forms on a weight specific basis. The present results indicate that some of the nonpolar, lipid DOC material released by macrophytes tends to become concentrated in the surface film within 7 days of conditioning. After 33 days of conditioning by nonaxenic Lemna, the surface film became a solid, waxy layer colonized by bacterial and algal members of the neuston community. Among the fatty acids identified as major components in the surface film produced by Lemna were hexanoic, heptanoic, palmitic and stearic acids. These experimentally produced surface films therefore resemble those found in nature where lipid materials in the form of stable saturated fatty acids (3,50), long-chain fatty acids (lauric, palmitic, stearic, myristic, palmitoleic and oleic) (60), fatty acids, esters, and

113

alcohols (30), fatty acids, waxy esters and tripalmitic and palmitic acids (3,37,53) have been reported. These lipids dominate the physical characteristics of the surface of water (3,32,34,37) even though their mass may be considerably less than the underlying film of macromolecules variously described as polysaccharides, glycoproteins, proteoglycans and humic acids (37). The fatty acids found in the surface film are included in the low molecular weight fraction of DOM (,50–700 Da), which is generally considered to be the dominant fraction of DOM (.60%) (4,14,44,71). However, the esters formed from these acids, together with the glycoproteins, plysaccharides and humic acids, form part of the high molecular weight fraction. Other low molecular weight components in the water column that have been described include sugars, amino, hydroxy- and short-chain carboxylic acids (102,103,105). Some of the compounds, such as short-chain carboxylic acids, may be metabolic end products of bacteria involved in the decomposition of plant material (16). Fine Particulate Organic Carbon Release by Macrophytes The FPOC constituted only a small fraction of the total organic carbon in the experimental systems (, 1.0 and 3.1% in the case of nonaxenic Lemna and Ceratophyllum, respectively) or up to 1.3% and 8.3% as fractions of total fixed carbon for Lemna and Ceratophyllum, respectively. Although there was an apparent tendency for the amount of FPOC, as a fraction of fixed carbon, to increase with time, for both Lemna and Ceratophyllum cultures these trends were nonsignificant. The fact that Ceratophyllum releases more FPOC than Lemna can be attributed to Ceratophyllum having a larger biomass of epiphytes and more senescing decaying tissues as indicated by its loss of weight. The virtual absence of FPOC in the cultures of axenic Lemna is correlated with the absence of epiphytic microorganisms. It is likely that the traces of FPOC in the axenic Lemna cultures were produced as a result of autolysis of tissues following death. The present results, therefore, support the generalization made that particle size reduction is accomplished primarily through microbial maceration of the tissues and subsequent sloughing off or fragmentation in response to physical disturbance (101). Uptake of DOC by Pulmonate Snails The present results provide incontrovertible evidence that the snails can achieve a net uptake of DOC produced by macrophytes and also metabolize it. Thus, normal snails with untreated shells were found to have accumulated 9.5, 6.3 and 0.4% of the total labelled carbon into their body tissues, haemolymph and shell, respectively, during an incubation period of 38 hr. The presence of 3.1, 0.2 and 33.9% of the 14C label in faeces, mucus and respiratory CO2, respec-

114

tively, also indicates that the carbon compounds that had been accumulated had also been metabolized. By comparison, no traces of 14C were found in either the bodies or shells of killed snails incubated in the same medium, and these snails only had 4.1% of the amount of 14C label found in the haemolymph of the normal untreated snails. Control snails incubated in media to which 14C-labelled HCO 32 had been introduced in the absence of Lemna and subsequently treated to get rid of inorganic carbon were also devoid of 14 C label in either their body tissue or shell. The traces of 14 C found in the haemolymph of the control snails may have originated from either small residues of 14C-labelled HCO32 left in the medium or to a small amount of labelled organic contaminant. It must be concluded, therefore, that only healthy, living snails had accumulated and metabolized DOC of Lemna origin in significant amounts. The close similarities between the percentage of the 4C accumulated in the bodies, shell and haemolymph for normal snails (9.5, 0.4 and 6.3%, respectively) and those with chemosterilized shells (10.0, 0.4 and 5.7%, respectively) indicate that any microorganisms that might have been present on the external shell surface of normal snails accumulate very little, if any, 14 C label. This result was not unexpected, as it is known that the shells of laboratory reared B. glabrata are characterized by a dearth of periphyton (18) (Akinluyi, unpublished), possibly due to mutual grazing by the crowded snails. As the integuments of healthy, softbodied aquatic animals, such as pulmonate snails, are generally free of bacteria, possibly due to the release of antibiotics (43), it can be concluded that uptake of DOC is not mediated by surface living bacteria. This consensus is strengthened by the findings that there were no significant differences between the rates of amino acid uptake by axenic and nonaxenic sea urchin larvae (17). It is of interest that snails incubated in media conditioned by axenic Lemna had smaller percentages of 14C in their body tissues and haemolymph and more 14C label in their shell and 14C-DIC in their media than was the case with snails incubated in media produced by nonaxenic Lemna. It is likely that the 14 C in the shell was derived from respiratory CO2, thus allowing the snail to use its excretory products in a cost-effective manner. It can be concluded, therefore, that the snails in media produced by axenic Lemna had been metabolically more active and produced more respiratory CO2 than the snails in media produced by nonaxenic Lemna. It is likely that these differences are attributable to the DOC produced by axenic Lemna having a larger proportion of low molecular weight, readily metabolizable compounds than the DOC produced by nonaxenic Lemna. This hypothesis is supported by the well-documented tendency for bacteria in nonaxenic systems to sequester the low molecular weight fraction of the DOC preferentially (47,48,71, 76). It has been shown that B. glabrata can concentrate DOC in the medium by a factor of up to 10.0–15.1 over a period

J. D. Thomas and C. Kowalczyk

of 38 hr. Other molluscs such as the Japanese oyster, Crassostrea gigas, the mussel, Mytilus californicus, and the rock scallop, Hinnites multivagosus, have also been shown to concentrate DOC of macrophyte origin (20–22,66). As these bivalves rely almost entirely on their efficient suction and filtration system for their food supply, it is not surprising to find that intermediate sized and young rock scallops have higher concentration factors of 90 and 150, respectively, than B. glabrata (66). However, these scallops had similar percentages of the total 14C in their biochemical fractions (24.6% and 5–17% in the case of small and intermediate sized scallops, respectively) to B. glabrata (66). The bivalves sequester the DOC by uptake through the inhalent siphons, but in the case of pulmonate snails the uptake sites have yet to be identified with certainty. One possibility is that they ingest the surface film, which has been shown to have a high concentration of fatty acids. Several authors (13,46,59,82) have noted the ability of freshwater snails and other freshwater organisms, such as flatworms and tadpoles, to feed on surface films. In fact, Physa can consume a surface film containing 14C-labelled palmitic acid at a rate of 0.35 mg hr21 (; 12 cm2 hr21 of the surface film) (13). However, calculations based on the empirically determined normal drinking rate of the 500 mg (wet weight) snails used in the present experiments, which was 12.5 µl g21 hr21 (87,93), would appear to indicate that they could only have sequestered a small proportion of the DOC in the medium by drinking. The arguments are as follows: first, if the snails were drinking at the normal rate they could only have consumed 240 µl or 2.4% of the incubation medium, which had a volume of 10 ml, during the incubation period of 38 hr. Second, as the snails had, in fact, sequestered a minimum of 15.8% of the 14C label from the medium (this being the sum of the percentage of the label in the body tissue and haemolymph) or a maximum of 53.5% (assuming that the 14C in the shell, mucus, faeces and inorganic carbon in the medium and lost to the atmosphere were all by-products of the snail’s metabolism) it can be concluded that the snails could only have acquired from 4.5–15% by drinking at the normal rate. On the other hand, if it is assumed that the snails were drinking at the maximum rate of 75 µl g21 hr21, which they are capable of doing in the presence of a strong phagostimulant like maltose (87,93) throughout the incubation period, then they could possibly have acquired from 27%–91% of the DOC by the oral route. As this scenario can be ruled out, it is, therefore, necessary to conclude that the snails probably accumulate most of the DOC by a route other than the oral one. One possible alternative route for the DOC uptake is the epithelium on the floor of the mantle cavity. This has a large, well-vascularized surface area that is ventilated by inhalent and exhalent ciliary-driven currents (Fig. 1) (94). Recent autoradiographic evidence, based on the use of a nonmetabolizable sugar (2-deoxy-glucose) supports this hypothesis (Pan, unpublished report). It may be concluded,

Utilization of Dissolved Organic Matter

115

FIG. 1. Diagrammatic representation of B. glabrata to illustrate the flow of the mantle cavity where transintegumentary uptake

of dissolved organic matter is likely to take place. The broken line indicates where cuts have been made to expose the rectal ridge. The arrows indicate the direction of the inhalent and exhalent water currents.

therefore, that freshwater pulmonate snails appear to possess a mechanism that is analogous to that in bivalve molluscs, albeit less powerful, for sequestering DOC and also possibly FPOC. Finally, it is necessary to ask how much of the energy requirements of the snail can be satisfied by the uptake of DOC at naturally occurring concentrations. As a first step to resolve this question, it would be necessary to identify and then quantify the organic constituents of the DOC. After measuring the basal metabolism of the snail and the uptake characteristics Ks and Vmax for the particular organic chemicals involved, it would be possible to determine the contributions made by each one to the basal metabolism of the snail when the chemicals were being taken up at naturally occurring concentrations. Such calculations have been made in the case of B. glabrata when it takes up short-chain carboxylic acids and sugars at naturally occurring concentrations (85,87,92,93). For example, when acetate, butanoate and glucose occur at naturally occurring concentrations of 945, 75 and 550 µM, respectively (38–41,100), it has been calculated, on the basis of empirically determined K m and Vmax values, that these would contribute 22.4%, 33.5% and 14.5%, respectively, of the basal metabolism of B. glabrata (87,93). It is possible, therefore, that pulmonate snails moving on the surface of plants or sediments where the concentration of DOC is maximal (16) could satisfy all

their basal metabolism requirements without ingesting any living organisms. Relevance of the Present Findings to the Food Web and Modular Concepts The dominance of the concepts of the food web and energy flow in present-day ecological theory is encapsulated in the statements that ‘‘the food web is the centrepiece of the biotic community part of ecosystem models’’ (67) and that food chain dynamics is the central theory of ecology (24). Implicit in these concepts is the belief that higher eukaryotic organisms obtain all their energy by ingesting and digesting other organisms. As a result, a great deal of emphasis is placed on 1/2 or 2/2 interactions (predation [1/ 2], herbivory [1/2], parasitism [1/2] and competition [2/ 2]). The currently accepted conventional view is summarized in Fig. 2. In essence this model envisages that carbon compounds, manufactured by primary producers, are conveyed along the food chain in the form of living tissues and that the DOC released during excretion by living organisms, ‘‘sloppy feeding’’ or cell death is utilized almost entirely by bacteria or fungi. These microorganisms are then ingested and digested by protozoan predators, macrodetrivores or grazer-scrapers forming what has been described as the microbial loop (2,69).

116

J. D. Thomas and C. Kowalczyk

FIG. 2. Diagrammatic representation of the flow of carbon in an aquatic ecosystem. → 5 Flow of dissolved organic carbon (DOC) into the external pool of DOC; - - → 5 Uptake of DOM from the external pool by bacteria and fungi, - ⋅ ⋅ - ⋅ ⋅ - ⋅ ⋅ 5 Uptake of DOM from the external pool by organisms other than bacteria and fungi; ⇒ 5 Uptake of organic carbon by ingestion of living organisms.

However, the following recent findings, including those described in the present article, have made it necessary to re-examine this model critically. First, there is now accumulating evidence that other classes of organisms, as well as bacteria and fungi, can sequester appreciable quantities of DOM from the water without killing other organisms. These organisms include algae (7,33,56,80,81), protozoa (74), macrophytes (25), zooplanktonic organisms (29), both freshwater (12,19,38–41,88,93,94) and marine (77,108), invertebrates (85,93) and even fish (23). In the case of freshwater and marine aquatic invertebrates there is evidence that they can satisfy either appreciable proportions or even more than their entire basal metabolism requirements from the integumental uptake of organic compounds such as short-chain carboxylic acids, amino acids and sugars at naturally occurring concentrations (38–41,77,85,92,93, 107,108). Furthermore, the labile low molecular weight compounds present in the detritus could also be assimilated in the gut of detritivorous invertebrates and fish following ingestion of detritus material (85). As a result, several workers have concluded that DOM present in detrital food material is more important as a food source than the bacteria as such (5,51,70,106). Second, it is to be expected, on evolutionary grounds, that vulnerable organisms such as bacteria and plants will have evolved defensive strategies to prevent overexploitation by potential consumers. These predation pressures will be further reduced by the fact that these vulnerable elements release metabolic end products that can provide potential consumers such as detritivore-herbivores with sources of information and nutrients. Examples are provided by pulmonate snails utilizing metabolic end products of bacteria such as C 2–C4 carboxylic acids (83,85,89,91,92) and photoassimilates from plants (83,87,93,94) as sources of information and nutrients. It must be concluded, therefore,

FIG. 3. Snails as components of modular systems where ex-

change of organic and inorganic metabolites take place between living components. The evolutionary primitive module (1) consists of epilithic algae, epilithic microorganisms such as bacteria or fungi and snails. It has three subsets and is associated with inorganic sediments such as sand. In contrast, the evolutionarily more advanced module (2) has four major components (aquatic macrophytes, epiphytic bacteria and fungi, epiphytic algae and snails) and has six subsets.

that it will be necessary to quantify the extent to which higher eukaryotic organisms, as well as bacteria, utilize DOM as a source of information and nutrients before we can hope to develop a complete understanding of the flow of carbon and the role of organic compounds in aquatic ecosystems. These findings raise another very important question. As aquatic ecosystems are extremely complex, which levels of organization should be used to investigate the biochemical role of DOM? Thomas (85) suggested that investigations should be focussed on co-evolved resource sets or ‘‘modules’’ within complex ecosystems. Two examples of such modules involving molluscs are shown in Fig. 3. The simplest module (Fig. 3, panel 1) has three components and three subsets and the more complex module has four components and six subsets. Although the linkage strengths between the components of various modules may vary, all modules are characterized by the following features (85): (a) the components of a particular module will have co-evolved; (b) the more vulnerable elements in the module will have evolved effective mechanisms to reduce exploitation by the more aggres-

Utilization of Dissolved Organic Matter

sive heterotrophs; (c) the components of the module derive some mutual benefits and, as a result, the biological fitness of the components will be enhanced by the presence of others; (d) as components of the module derive benefit from co-existence it will be advantageous for the more sedentary components to produce specific kairomones to serve as attractants and developmental inducers; (e) the remainder of the module will disappear following the removal of strongly interactive ‘‘keystone’’ organisms; and (f) living components of the module will exchange inorganic chemicals and also organic chemicals that provide information and energy. Evidence in support of this hypothesis has also been given previously (85,86), but much more work remains to be done in the field of freshwater biochemical ecology to test it further.

117

16. 17. 18. 19. 20. 21.

The authors wish to thank the NERC and the UNDP/World Bank/ WHO Special Programme for financial help. Thanks are also due to Professor M. Wallis for providing facilities and to Mrs. P. Chatfield for typing the manuscript.

22.

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