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Home , Assimilation (biology), Mulinia, Mulinia lateralis

l MARINE ECOLOGY PROGRESS SERIES Vol. 71: 27-35. 1991 Published March 28 Mar. Ecol. Prog. Ser. l

Assimilation of by the dwarf surf clam lateralis (: )

Kashane Chalermwat, Timothy R. Jacobsen, Richard A. Lutz

Rutgers University. Institute of Marine and Coastal Sciences. ShellfishResearch Laboratory, PO Box 678, Port Norris, New Jersey 08349, USA

ABSTRACT: Estimation of assimilation efficiency of bivalves, fed with radiolabeled bacteria, differs substantially depending on choice of tracer. We have investigated assimilation of bactena by the dwarf surf clam (Say) using bacteria labeled wth either ~-[~~S]-methionineor [methyl-3~]- thymidme. Labeled bacteria were fed to M. lateralis 6.18 f 0.45 mm (mean +- SD) in shell length in static pulse-chase experiments. After feeding and 4 consecutive 1 h chases in 0.2 pm flltered seawater, M. lateralis retained 93.34 f 6.45 % (n = 20) of incorporated 35S-methionine radioactivity. Altemahvely, when 3H-thymidine was used, only 51.40 f 16.54 % (n = 13) was retained. During our chase procedure, patterns also differed conspicuously For excreted 35~activity, 49.05 13.30 % was recovered m the particulate phase (> 0.2 ~m)and 50.94 f 13 30 % was dissolved. The corresponding values for excreted 3~ were 11.43 -t 4.70 '10 and 88.57 + 4.70 %. Assidation efficiency, estlrnated using 35S- methionine, is considerably hlgher than previously reported values using I4C, 3H and 15N tracers. We can explain assimilation and excretion differences between tracers in terms of metabolic pathways associated wth the labeled moiebes. The assimilation of methlonine and high retention of 35S tracer demonstrates the role of bacteria as a source of proteins and for M. lateralis.

INTRODUCTION et al. (1982), Lucas et al. (1987), and Langdon & Newell (1990). Kemp et al. (1990) have shown that small- (shell The dwarf surf clam Mulinia lateralis (Say) is a com- height = 16 mm) to large- (40 mm) sized individuals of the mon bivalve that inhabits soft substrata and exhibits ribbed mussel Geukensia dernissa (Dillwyn) can filter hgh growth and reproduction rates. Opporturustic bacteria-sized particles with differing efficiency. The such as M. lateralis increase dramatically in assimilation of both free-living and particle-bound bac- numbers under favorable conditions and can become teria (not necessarily natural bacterioplankton) has been dominant in benthic communities (Levinton 1970, San- demonstrated by Birkbeck & McHenery (1982),Harvey tos & Simon 1980). Over short time periods, M. laterahs & Luoma (1984), Amouroux (1986), and Crosby et al. may attain local average densities of up to 21 000 ind. (1990). In addition to clearance and assimilation studies, m-2 (Santos & Simon 1980). Elevated filtration and the presence of bacteriolytic has been con- metabolic rates suggest that M. lateralis is adapted to firmed in bivalves by McHenery et al. (1979), Seiderer et exploit high concentrations (Shumway 1983, al. (1984), and Jamieson & Wardlaw (1989). Shumway & Newel1 1984). The possibility that food Many researchers studying feeding and assimilation resources may seasonally fall below the energy of bacteria by suspension-feeding bivalves have requirements of M. lateralis, and the inabhty to utilized isotopic tracers to label bacteria. These studies catabolize reserves during starvation, may have included the use of 3~-thymidine(Holhbaugh et account for the mass mortalities that characterize thls al. 1980, Birkbeck & McHenery 1982), '4C-glutamic species (Shumway & Newel1 1984). acid (Amouroux 1986), 14C-[~]-Dglucose (Harvey & It is conceivable that suspended bacteria may consti- Luoma 1984, Crosby et al. 1990) and [15N]-ammonium tute a food source for Mulinia lateralis in its natural sulfate (Crosby et al. 1990). In this study, we investi- habitat. The ability of bivalves to uhlize bacteria has been gated the abllity of Muhnia lateralis to assimilate a verified by many authors (reviewed by Langdon & mixed assemblage of bacterioplankton using 35S-meth- Newel1 1990). Effective clearance of free-living and ionine in comparison with 3~-thymidineas tracers. natural-sized bacteria has been demonstrated by Wright The goal of our study was 2-fold: (1) to investigate

O Inter-ResearcWPrinted in Germany 28 Mar. Ecol. Prog. Ser. 71: 27-35, 1991

the ability of M. lateralis to assimilate bacterioplank- filter were enumerated under oil immersion. Bacterio- ton, and (2) to compare assimilation efficiency estima- present in 50 to 60 m1 of the bacterial sus- tions obtained by using 2 isotopic tracers to label differ- pension were caught on 47 mm diameter, 0.2 pm pore ent macromolecules. The amino acid 35~-methionine size polycarbonate filters. They were then washed labels bacterial protein and has been used in trophic (10 ml, 0.45 ym filtered seawater) and resuspended by studies by Wikner et al. (1986). The sulfur in shaking (1 min) in 50 m1 polypropylene centrifuge methionine plays an important structural role in tubes with 5 m1 of 0.45 p filtered seawater (salinity intracellular proteins and functions as part of important 22 ppt). Additional seawater was added to the initial coenzymes in reactions (Linder 1985). The volume. Bacteria were counted after resuspension by tracer 3H-thymidine labels bacterial DNA and was AODC to determine percentage recovery. Resus- oriqnally proposed for trophlc studes by Hollibaugh et pended bacteria were subsequently incubated in al. (1980). Under certain circumstances, however, 3H- 50 m1 sterile plastic bags (Whirl-Pak) in subdued light thymidlne may label other bacterial cellular compo- with either L-[35~]-methionine(specific activity = 1134 nents (see Hollibaugh 1988). Few tracer studies have Ci mmol-l, final conc. = 0.002 nmol ml-l; NEN been addressed towards differential utilization of bac- Research Products) or [methyl-35HJ-thymidine (specific terial macromolecules. We hypothesized that assimila- activity = 2.0 Ci mmol-l, final conc. = 11.25 nmol tion efficiency estimates would vary depending on the mlp'). Bacteria were incubated in 35~-methionine for metabolic pathways associated with the different 60 min; incubation time for 3~-thymidinewas either tracers. 30, 60 or 120 min depending on rate of 3H-thymidine uptake. We used 35S-methionine to label bacterial pro- tein (Wikner et al. 1986) and 3H-thymidine to label MATERIALS AND METHODS bacterial DNA (Hollibaugh et al. 1980; see also Hol- libaugh 1988). The specimens of Mulinia lateralis used in our experimental procedures were raised at the Rutgers Shellfish Research Laboratory, New Jersey, USA. PULSE-FEEDING Specimens were maintained in flow-through contain- (1) Labeledbacteria caughton fiter were ers and fed daily with algae batch-cultured in Kal- resuqxndcd. Bacteriawere enumeratedby AODC. wallTM tubes. Experimental organisms averaged 6.18 Volumebrought back ro 60 rnl. + 0.45 mm in shell length (mean + SD, n = 33). * Experiments were conducted at room temperature (2) Labeledbaclerial suspension divided inro (24.0 0.5"C) in sterile culture wells (Falcon, 3.6 two equalaliquols. m1 capacity). Bivalves were maintained in 0.45 pm * filtered seawater for 6 to 8 h before being used in (3) 30 rnl labeledbacterial 30 ml labeledbacterial experiments. During that period, if bivalves defecated, suspension with associated suspensionidtend through DOM. 25 mm dia.0.2 pm pore siu: the was changed. A Tracor Analytic Mark 111, PC filter to removebactena. Model 6881 LSC, was used for scintillation counting. W C) Radioactivity measurements in disintegrations per (4) 30 m1 bacterialsuspension. 30 m1 DOM solution. minute (dpm) were corrected for quenching and count- ing efficiency using external standards to establish a t C) (5)2 rnl aliquotsadded to each 2 m1 aliquouadded to each quench curve. feeding-chamber. feedingthamber. Our experimental protocol consisted of 3 steps: (1) isolation of bacteria and radioisotope labeling; (2) live 0 0 0 pulse-feeding; and (3) chase, which includes purging of residual radioactivity and excretion. Bacteria isolation and labeling. Bacterial assem- ...d-.. . . 000 blages used in thls study were obtained from Tahitian Isochrysis aff. galbana Green (strain T-ISO) cultures .no-..O 0 0 raised in ~alwall~'*'tubes. Bacteria were separated from algae by filtration through 47 mm diameter, 1 pm .. pore size polycarbonate filters (vacuum < 20 kPa). After separation, bacteria were counted by the Fig. 1. Protocol for pulse-feeding Mulinia lateralis with Orange direct count Inethod (AODC) de- labeled bacteria in tissue culture wells. AODC: acridine "eloped by Hobbie et al. (1977). Subsamples of 100 oranoe dlrect count method; DOM: dissolved oraanic matter: were taken from the stock and 10 random fields per PC: polycarbonate Chalermwat et al.: Bacteria assimilation by a clam 29

Pulse-feeding. After incubation, labeled bacteria After termination of the chase procedure, bivalves were collected on 47 mm diameter, 0.2 pm polycarbo- were blotted dry, placed in scintillation vials, and nate filters and rinsed twice with 5 m1 each of 0.45 pm digested in Scintigest tissue solubilizer for 24 h at 60 'C. filtered seawater to remove unincorporated label. Bac- After , the shells were removed with fine teria were then resuspended as described above. Bac- forceps and 35 p1 glacial acetic acid was added to the teria were counted again (AODC) to estimate percent- digestion mixture to decrease chemiluminescence. Tis- age, recovery and degree of clumping. sue dpm values were corrected for variable quench and The resulting resuspended bacteria were used for for self-absorption. The pulse-chase experiments were feeding and assimilation studies. Bacterial concen- repeated 6 times for 35S-methionine and 3 times for 3H- trations added to feeding chambers ranged from 4.0 to thymidine, using different batches of bacteria. 8.5 X 106 cells ml-'. Radioactivity in the bacterial sus- Added radioactivity. Radioactivity added in particu- pension was determined for particles caught on 0.2 pm late phase (R,) was calculated by subtracting dissolved pore size filters. (filtrate) radioactivity (Rd) from total radioactivity in Preliminary experiments indicated that some label stock bacterial suspension from bacteria was released during our resuspension procedure. Therefore, prior to feeding experiments, the labeled bacterial suspension was divided into 2 equal Incorporated radioactivity. Incorporated particulate portions (Fig. 1). One portion was filtered through a radioactivity (IR,) was calculated by subtracting 25 mm diameter, 0.2 pm pore size polycarbonate filter radioactivity remaining in dissolved set (R%) from total to remove bacteria. The filtrate was collected in acid- remaining radioactivity in wells (RR(p+d))and subtract- washed and combusted glass scintillation vials. This ing this remaining dpm value from added particulate filtrate was used for dissolved-phase uptake controls. radioactivity: Each experimental run consisted of 2 replicate sets of feeding chambers. One set was used for bacteria plus associated dissolved organic matter (DOM), the other for Isotope excretion. For each experimental set, DOM only. Each set consisted of at least 12 chambers: 6 excreted radioactivity was obtained by subtracting wells with bivalves (usually 3 live and 3 formalin-killed) excreted radioactivity from treatment controls (dis- and 6 wells without. For each chamber, 2 m1 of suspen- solved) from corresponding total excretion values. sion (bacteria + DOM or DOM only) was added. Suspen- Particulate excretion: For calculation of particulate sions were added to empty wells as controls for label phase excretion we used the following formula: disappearance resulting from feeding-chamber wall adsorption. Radioactivity in 3 wells without bivalves EPR, = EPR(,+*) - EPRd were sampled at the beginning of the experiment. The 3 where EPR, = excreted particulate radioactivity from remaining no-bivalve wells were sampled after 1 h. The particulate uptake; EPR(p+d, = excreted particulate difference in activity was used to correct uptake values. radioactivity from total uptake; and EPRd = excreted Feeding was carried out in a laminar flow hood under particulate radioactivity from dissolved uptake. continuous lighting. Bivalves were allowed to feed for Dissolved excretion: For calculation of dissolved l h, after which they were removed and placed in new phase excretion resulting from particulate uptake we stenle wells. Remaining radioactivity was assayed by used the formula: taking 1 m1 samples from each well. Samples were assayed in 7 m1 plastic Minivials with 5 m1 ScintiVerse LC cocktail. Chase. In the chase procedure, live bivalves incu- where EDRp = excreted dissolved radioactivity from bated with both bacteria and DOM, and with DOM particulate uptake; EDR(p+d) = excreted dissolved only, were blotted dry and placed in new culture wells radioactivity from total uptake; and EDRd = excreted with 2 m1 of 0.2 pm filtered seawater. After each 1 h dissolved radioactivity from dissolved uptake. incubation (total 4 h), bivalves were removed from the Total excretion: Cumulative excretion (particulate wells, blotted dry, and transferred to new wells. phase and dissolved phase) after 4 h was calculated as: Radioactivity excreted in wells was assayed by filtering C ER, = C EPR, + C EDR, (5) each well's total volume through 25 mm diameter, 0.2 pm polycarbonate filters. Particles caught on each filter where C ER, = total excreted radioactivity from par- were washed with a total of 3 m1 of 0.45 pm filtered ticulate uptake after 4 chases, CEPR, = particulate seawater and assayed (particulate excretion). Filtrate excretion from particulate uptake after 4 chases, and from each well (1 ml) was assayed separately for dis- ZEDR, = dissolved excretion from particulate uptake solved excretion. after 4 chases. 30 Mar. Ecol. Prog. Ser. 71: 27-35, 1991

Assimilation efficiency. Assimilation efficiency (AE) was calculated as: Fig. 2. Mulinia lat- eralis. Difference in IR, - X ER, AE = X 100 radiolabel excretion IRP rate over hme by bi- valves expressed as percentage of incorpo- RESULTS rated radoactivity. Re- tenhon of 35S was Preliminary clearance studies showed that within 1 h higher than that of 3H. $ After 4 h bivalves re- Mulinia % 3 lateralis could clear 33.48 k 5.50 of bacterial tained 93.34 + 6.45 % cr - 0 cells from suspension. Incubation of bacteria with (n = 20) of 35~activity i 5 j 4 isotopes did not noticeably alter size or appearance of and 51.40 2 16.54 % z cells. We observed no clumping of bacteria after resus- (n = 13) of 3~ activity TIME (HOURS) pension. The recovery of bacteria from filters ranged from 50 to 100 % (mean 77 %). Uptake of particulate radioactivity by clams was vari- is shown in Fig. 3A, B. Particulate radioactivity de- able for both isotopes and was apparently related to creased rapidly, with a proportional increase in dis- activity of the organisms during feeding. An average of solved excretion. A comparison between isotopes indi- 74.92 f 18.54 % of methionine was taken up (range = cates that 3H activity was excreted primarily in dis- 19.05 to 94.82), whereas the corresponding value for solved form from the beginning of the chase. thymidine was 49.51 f 15.28% (range = 19.88 to Cumulative excretion in both fractions for 35S- 69.60). methionine and 3H-thymidine after 4 chases is shown Average tracer radioactivity present in bivalve tissue in Fig. 3C, D, respectively. When 35S-methionine was (assimilation) after digestion and scintillation counting used, 49.05 + 13.30% of excreted radioactivity was was within 5 % of values obtained by subtraction associated with particles ('feces') and 50.94 f 13.30 O/O (Table 1). Assimilation estimates reported in this study was dissolved. The corresponding values associated were obtained by subtraction. with 3H-thymidine-labeled macromolecules were 11.43 The discrepancy in tracer retention is shown graphi- + 4.71 O/O particulate and 88.57 f 4.70 O/O dissolved. cally in Fig. 2. Assimilated radioactivity values, ex- Resuspension of labeled bacteria caught on filters pressed as percentage of incorporated radioactivity, consistently caused the release of DOM. An average of after 4 chases were 93.34 k 6.45 (n = 20) for 35S- 7.43 f 4.45 % (n = 6) of 35S label and 21.08 f 15.78 O/O methionine and 51.40 f 16.54 (n = 13) for 3~-thy- (n = 3) of 3~ label was released in solution during midine. Retention of incorporated 35S was higher than resuspension of bacteria. Preliminary experiments that of 3H from the beginning of the chase period. showed that after 2 washes (5 m1 each), less than The difference between particulate and dissolved 0.01 % of initial L-[35S]-methionine and [methyl-3~]- fractions of excreted radioactivity, in relation to time, thymidine activity remained bound to the filter. We attribute the release of labeled DOM (< 1 % of initially Table 1. Mulinialateralis. Label activity remaining in tissue added activity) to bacterial damage. We did not from digestion and scintillation counting, compared to values attempt to characterize these dissolved substances, and obtained by subtraction of excreted radioactivity. Experiments their chemical affinity remains unknown. The percent- with 35S-methionine were run 6 times. Experiments with 3~- thymidine were run 3 times. Average M, lateralis 35~back- age uptake of DOM associated with both isotopes was ground dpm = 49 t 7 (n = 18); 3~ background dprn = 176 f lower than that of particulate uptake (compare Fig. 42 (n = 9); Subtr.: subtraction; Scint.: scinhllation 4A, B with Fig. 4C, D).

35~-methlorune 3~-thymidine Mean dpm n Mean dpm n DISCUSSION Subtr. Scint. Subtr. Scint.

89 150 85 531 Assimilation in this study is defined as incorporated 205 659 172 284 radioactivity minus radioactivity lost as excreta (par- 407 586 400 417 ticulate and dissolved; see also Bayne 1983 p. 312). The 224 767 219 100 use of radiolabeled food to measure absorption and 479 746 464 990 assimilation poses considerable problems of interpreta- 132 512 137 809 tion (Conover & Francis 1973). The focus of our study Mean 256570 246 689 Diff. (O/O) 3.9 was the assimilation of bacterial (particulate) radio- activity. However, we have utilized a 2-compartment Chalernlwat et al.: Bacteria assimilation by a clam 3 1

A particulate 35s B particulate H - KSl dissolved ES3I dissolved I &-

TIME (HOURS)

Fig. 3. Mulinia lateralis. Difference in ex- creted radioactivity in particulate and dis- solved fractions over time for (A) ^S and (B) Excreted radioactivity for ^S was close to background levels for the 4 h period. Also shown are cumulative radioactivity excretion after 4 h for (C) "S and (D)3H - particulate dissolved particulate dissolved

particulate particulate - - 3u

total I'rve formolin no anlmal total live forrnolin no particulota onimol killed particulote animal killed

dissolved dissolved 55s ^H

Fig. 4. Mulinia laterals. Uptake of par- ticulate-phase (bacteria) and dissolved- phase radioactivity by experimental bivalves compared to formalin-killed con- trols, and disappearance of radioactivity due to feeding-chamber adsorption, for total lii formalin no animal live formalin no animal (A, C) "S and (B, D) 'H dissolved animal killed animal killed system consisting of bacteria + DOM and DOM only. From our experiments, we have shown that Mulinia Appropriate corrections for DOM were necessary (see lateralis can clear and assimilate bacteria smaller than Amouroux & Amouroux 1988) to provide an adequate 1 pm. Assimilation efficiency estimated by using ^S- index of the ability of Mulinia laterals to assimilate methionine in this study (93.34 O/O) is higher than most bacterioplankton. The duration of our experiments was previously reported values. Birkbeck & McHenery kept short (1 h), to minimize recycling of radioisotopes (1982) utilized several cultured bacteria as food and H in feeding chambers while allowing sufficient radioac- and 14C as tracers in their studies with Mytilus edulis L. tivity to be incorporated for liquid scintillation count- Assimilation estimations based on '^H tracers used in ing. We adopted a closed system for our study and their study differed depending on the labeled bacterial make no inferences as to the efficiency of bacterial macromolecule. When ^-thymidine was used to label clearance by M. lateralis in natural systems. DNA in bacteria, AE ranged from 7 to 36 %. When '^H- 32 Mar. Ecol. Prog. Ser. 71: 27-35, 1991

diamiopimelic acid was used to label bacterial cell wall, less specific than originally proposed. Hollibaugh an AE of 81 O/O was reported. An efficiency of 84 O/O was (1988) has demonstrated that some microbial com- reported for 14C-labeled bacterial macromolecules. munities incorporate up to 53 % of [methyl-3H]-thy- Birkbeck & McHenery (1982) were the first authors to midine into protein. [Methyl-3H]-thymidine can be demonstrate differential utilization of bacterial mac- degraded rapidly within bacterial cells. The 3H on the romolecules in bivalves. methyl group may subsequently be transferred to other The tracer 14C-glucose was used by Harvey & Luoma compounds and incorporated by de novo pathways into (1984) in their studies on Macoma balthica (L.).Assimi- RNA, DNA and proteins (Moriarty 1986). In our studies, lation efficiency reported as 14C activity remaining in as much as 47.75 k 5.32 % of radioactivity associated the bivalve's foot after 14 d was ca 20%. Amouroux with 3H was found in hot TCA precipitated mac- (1986) studied Venus verrucosa L. and reported 14C romolecules (non-DNA) after l h incubation. Because assimilation values between 39.9 and 51 % during a 3H-methyl can enter a general CH3 pool, interpretation 40h period. Studying Crassostrea virginica (Gmelin) of its production and excretion pathways is difficult. and using I4C and 15N as tracers, Crosby et al. (1990) CH3 has many functions in cellular reported AE values of 52.5 and 57.2 % for bacterial (Zubay 1988), making 3~ a non-specific label. The carbon and respectively. large portion of dissolved 3~ may have included a Hollibaugh et al. (1980) were the first researchers to variety of substances, in particular 3H20, a product of propose the use of 3H-thymidine as a tracer in bacteria- cellular . That this dissolved 3~ radioactivity feeding studies. Using Dons gouldii Dall and Mytilus was not taken up by bacteria in earlier pulse-chase edulis as study organisms, they reported extremely experiments suggests that it is not in a utilizable form high uptake values for both species after 1 h feeding. for bacteria. Release of dissolved 3H from digestion of Average clearance rates for the 2 species used in their labeled bacteria was first noted by Birkbeck & McHen- study were 4.6 m1 h-' for D. gouldii and 6.5 m1 h-' for ery (1982). These authors suggested that Mytilus edulis M. edulis. Based on these clearance values D, gouldii selectively utilized different bacterial polymers, reject- incorporated 99.69 O/O of cleared particulate radioactiv- ing DNA. ity after 1 h. During the same time period, M. edulis Methionine is classified as an essential amino acid in incorporated ca 100 O/O of cleared particulate radioactiv- mammalian studies (Zubay 1988). The min- ity. These results suggest 100 % retention of natural- eral sulfur can be absorbed either as part of sized bacteria. Later studies have, however, shown that amino acids or as inorganic sulfate with equal effi- M. edulis and related Donax spp. are not efficient at ciency (Linder 1985). Sulfur is the site for attachment clearing natural-sized bacteria (Wright et al. 1982, and transfer of l-C methyl groups, via S-adenosyl- Matthews et al. 1989). We attribute the high 'assimila- methionine. It is also part of the important reducing tion' values of Hollibaugh et al. (1980) to a possible agent, glutathone, and various important coenzymes underestimation of clearance rate. Methods for estima- and , including coenzyme A (Linder 1985). tion of clam and mussel clearance rates were not given The 35S isotope in methionine is utilized by bivalves, in this pioneering study. specifically, in several metabolic pathways (Florkin & Direct comparison of assimilation efficiencies Bricteux-Gregoire 1972). The few studies on intercon- reported in different studies is difficult. Generally, versions of sulfur amino acids in molluscs have focused, information is not given concerning the specific bac- primarily, on taurine and the methyltransferase en- terial components labeled. Apart from the discrepancy zymes (Bishop et al. 1983). in choice of tracers, assimilation values are reported Allen & Awapara (1960) and Allen & Garrett (1972) after variable time periods ranging from 1 h to 14 d. An report labeling of taurine with 35~-methioninein tissues appropriate time period of several hours to allow for of Mya arenaria L. and Mytilus edulis. The amino acid purging of unabsorbed food is necessary. Extremely derivative taurine is conserved to different degrees in long 'chase' periods, however, increase the chances of these bivalves and functions in intracellular osmoregula- isotope recycling within the system (Conover & Francis tion. In M. arenana subjected to osmotic shock, taurine 1973), especially in static systems. was rapidly degraded but 35S was retained within the The discrepancy in assimilation and excretion of bivalve. The extent to which taurine is synthesized and radiolabel between isotopes in this study can be degraded in Mulinia lateralis is unknown. explained by the difference in labeled moieties. Triti- For both 35S and 3H the initially excreted radioactiv- ated thymidine used in this study is labeled at its ity in particulate phase may be attributed to gut pas- methyl group at the C5 position of thymine. The tracers sage of unassimilated bacterial fragments. Bacterial 35S-methionine and 3H-thymidine were chosen to fragments, demonstrating digestion of bacteria, have selectively label bacterial proteins and DNA. Tritiated been observed in the alimentary canal of Mytilus edulis thymidine, however, has recently been shown to be larvae by Prieur (1983). Table 2. Studies investigating clearance and/or assimilation of attached or unattached planktonic bacteria and cultured bacteria by suspension-leeding bivalves. NR: not reported; NS: not studied; (+) positive result for clearance or assimilation; (-) negative result for clearance

Species Clearance Assimilation Bacterial source Bacterial dimensions (pm) Cells ml-' Source

Mytilus californianus + + Cultured 0.8 X 1.0 2.0 x 10' ZoBell & Landon (1937) Growth 0.8 to 1.8-7.0 X 9.2 ZoBell & Feltham (1938) Donax gouldii + + Natural 0.3 (cocci) 0.5-5.0 X 106 Hollibaugh et al. (1980) Mytdus edulis + [Methyl-%] lhymidine 0.5 X 2.0 (rods) Mytilus edulis + + Cultured NR 0.5-1.0 X 10' Birkbeck & McHenery (1982) Lysozyme-sensitive sp. [MethylL3H] thymidine '4C-glucose %I-diam~nopimelicacld Geukensia demlssa NS Natural Wright et al. (1982) Myllus edulis Mya arenaria Macoma balthicad + Cultured 8.0-15.0 x 106 Harvey & Luoma (1984) I4C-[u]~glucose Venus verrucosa + Cultured Amouroux (1986) I4C-L glutamic acia

MyUus edulis + NS Natural 0.513-0.524 mean spherical diameter 1.38-3.43 X 106 Lucas et al. (1987)

Donax serra NS Natural 0.25-1.15 mean spherical diameter 1.17-6.14 X 106 Matthews et al. (1989) Mactra lilacea Crassostrea virginica + + Cultured 1.12-3.14 X 10' Crosby et al. (1990) [I4c1glucose 115~1ammonium sulfate Crassostrea virginica + NS Natural 5.58 x 106 Langdon & Newell (1990) Geukensia demissa + Mulinia lateralis + + Algal culture 4.0-8.5 X lob Present studv IMethyl-3~]thyrnidine ~-~~~-methionine

a Macoma baltica is a facultative deposit feeder (J. Lin pers. comm.) 34 Mar. Ecol. Prog. Ser. 71: 27-35, 1991

Results from our study show that approximately half (Stuart et al. 1982, Newell & Field 1983, Seiderer et al. of the excreted 35S label was recovered in dissolved 1984, Benner et al. 1988, Crosby et al. 1990). Compared form. Dissolved 35S-labeled compounds were also with other planktonic components, natural planktonic found by Allen & Awapara (1960) and Allen & Garrett bacteria have a lower C:N ratio (Nagata 1986, Lee & (1972) in their studies using 35~-methionine.Using 14C- Fuhrman 1987). Nitrogen and crude protein content in glutamic acid as a tracer, Amouroux (1986) detected bacteria can be as high as 12.5 and 78 % respectively radioactivity associated with unidentified DOM in his (Young & Scrimshaw 1975). studies on Venus verrucosa. Based on previous studies, Although the contnbution of bacterial carbon and the dissolved 35S could have resulted from bacterial nitrogen to bivalve nutrition has been well degradation of fecal material, as suggested by documented, little information exists on the contribu- Amouroux & Amouroux (1988), or from excretion of tion of specific bacterial macromolecules. The degrada- dissolved SO4-' (Linder 1985, Allen & Garrett 1972). tion of methionine and high retention of 35S found in Activity from 35Scaught on filters ('particulate') during this study suggest a potentially important role of bac- later parts of the chase may have been that incorpo- teria as a source of mineral nutrients, as well as pro- rated into sulfonated mucopolysaccarides, a major teins, for Mulinia lateralis. Caution in isotope selection component of molluscan mucus (Allen & Garrett 1972). for use in bacteria-assimilation studies is recom- The ability of bivalves to effectively clear and assimi- mended. The importance of bacteria as a food source is late bacteria-sized particles seems to be species- dependent upon both bacterial quantity and quality. specific (Table 2). Comparative particle-retention studies in bivalves indicate that particles smaller than Acknowledgements. We thank Drs S. K. Allen and J. Scarpa 2 to 4 ym are not cleared efficiently (M~hlenberg& for supplying specimens of Mulinia lateralis. We also thank RiisgArd 1978, RisgBrd 1988). These studies are, how- Drs S. R. Fegley, R. G. Gustafson, J. N. Kraeuter, B. A. MacDonald and S. C. Wainright, and Mr D. Bushek and Mr W. ever, based on particle counts using electronic particle J. Canzonier, for critically reading the manuscript. Dr S. E. counters which cannot routinely detect particles Ford and MS H. L. Motto assisted In figure preparation. Com- < 2 km. Although clearance of larger particles may be ments from 2 anonymous reviewers helped improve the high, clearance of nano- and picoplankton-sized parti- manuscript. K. Chalermwat is under scholarship from the Royal Thai Government. This is New Jersey Agricultural cles may have been underesbmated. Experiment Station Publication no. D-32406-2-90 and Con- In this study, we have utilized 'natural'-sized bac- tribution no. 90-41 of the Institute of Marine and Coastal teria to estimate AEin Mulinia lateralis. Many previous Sciences. Rutgers University, supported by New Jersey State researchers investigating assimilation of marine bac- funds and the Fisheries and Aquaculture Technology Exten- teria by bivalves have relied on radiolabeled bacteria sion Center. grown on enriched organic media (Birkbeck & McHen- ery 1982, Harvey & Luoma 1984, Amouroux 1986). Cultured marine bacteria are known to be much larger LITERATURE CITED than natural forms (Lee & Fuhrman 1987). Therefore, Allen, J. A., Garrett, M R. (1972). Studles on taurine in the use of large bacteria may overestimate the contribution euryhaline bivalve, Mya arenaria. Comp. Biochem. 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This article was presented by S. Y. Newell, Sapelo Island, Manuscript first received: November 5, 1990 Georgia, USA Revised version accepted: January 16, 1991