2797.full

2797The Journal of Experimental Biology 200, 2797–2805 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JEB1132

,

THE EFFECTS OF EXPOSURE TO AMMONIA ON AMMONIA AND TAURINE POOLSOF THE SYMBIOTIC CLAM SOLEMYA REIDI

RAYMOND W. LEE*, JAMES J. CHILDRESS AND NICOLE T. DESAULNIERSMarine Science Institute and Department of Biological Sciences, University of California, Santa Barbara

CA 93106, USA

Accepted 18 August 1997

The nutrition of the gutless clam Solemya reidi issupported by the activity of intracellularchemoautotrophic bacteria housed in its gill filaments.Ammonia (the sum of NH3 and NH4+) is utilized as anitrogen source by the association and is abundant in theclam’s environment. In the present study, clams wereexposed to 0.01–1.3 mmol l−1 ammonia for 22–23 h in thepresence of thiosulfate as a sulfur substrate. Ammoniaexposure increased the ammonia concentration in thetissue pools of the gill, foot and visceral mass from 0.5 to2µmol g−1 wet mass, without added ammonia, to as muchas 12µmol g−1 wet mass in the presence of 0.7 and1.3 mmol l−1 external ammonia. Gill tissue ammoniaconcentrations were consistently higher than those in thefoot and visceral mass. The elevation of tissue ammoniaconcentration compared with the medium may be due inpart to an ammonia trapping mechanism resulting froma lower intracellular pH compared with sea water andgreater permeability to NH3 compared with NH4+. Rates

of ammonia incorporation into organic matter(assimilation) were determined using 15N as a tracer. 15N-labeled ammonia assimilation was higher in gill than infoot and increased as a function of 15N-labeled ammoniaconcentration in the medium. The size of the free aminoacid (FAA) pool in the gill also increased as a function ofammonia concentration in the medium. This entireincrease was accounted for by a single amino acid,taurine, which was the predominant FAA in both gill andfoot tissue. Aspartate, glutamate, arginine and alaninewere also abundant but their levels were not influencedby external ammonia concentration. Ammoniaassimilation appeared to occur at rates sufficient toaccount for the observed increase in taurine level. Thesefindings suggest that taurine is a major product ofammonia assimilation.

Key words: ammonia, taurine, clam, symbiosis, Solemya reidi,nitrogen metabolism.

Summary

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Solemya reidiis a gutless protobranch clam that inhabiburrows in sulfidic sediments on the Pacific coast of the UnitStates and Canada (Bernard, 1980). Reduction or absencstructures associated with particulate feeding is characteriof clams of the genus Solemya(Reid and Bernard, 1980, andreferences cited therein). The discovery of chemoautotropbacteria–invertebrate symbiosis at deep-sea hydrothervents in the Pacific led to the subsequent discovery of this tof symbiosis in S. reidi(Cavanaugh et al.1981; Felbeck et al.1981). High densities of chemoautotrophic intracellulasymbiotic bacteria are found in the gills of this species as was in two other species, S. velumand S. borealis(Cavanaugh,1983; Conway et al.1992; Felbeck, 1983; Felbeck et al.1981).Chemosynthetic bacterial symbionts have been documenteover 100 invertebrate species from at least five phy(Cavanaugh, 1994; Polz and Cavanaugh, 1995). In genethese associations are found in areas where reduced chem

Introduction

*Present address: Department of Organismic and Evolutionary B02138, USA (e-mail: [email protected]).

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species are abundant. Sulfide, and in some cases methanoxidized as an energy source by the intracellular bacterwhich are then believed to provide organic compounds for hocatabolism and biosynthesis.

The importance of sulfur-based autotrophy in Solemya reidiis well documented. Sulfide concentration ranges from 0.41.9 mmol l−1 in the porewater of the sediment from whichclams are collected (Lee et al. 1992a) and it can be oxidizedas an energy source by both the symbionts and hmitochondria (Powell and Somero, 1986). Symbiont sulfuoxidation results in net assimilation of CO2 by theCalvin–Benson cycle (Anderson et al. 1987). Organiccompounds are then translocated to the host tissues (FisherChildress, 1986). Utilization of dissolved organic compoundmay also be important since free amino acids (FAAs) can taken up and are present in sediments where S. reidiare found(Felbeck, 1983; Lee et al. 1992a). However, assimilation of

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inorganic compounds (autotrophy) is probably the main souof organic carbon and nitrogen in S. reidi. The overallcontribution of autotrophy can be inferred from naturabundance stable carbon and nitrogen isotope studies oS.velumand S. borealis(Conway et al.1989, 1992). The tissuesof these associations are highly depleted of 13C and 15N(δ13C=−31 to −35 ‰; δ15N=+4 to −10 ‰) compared withbivalves that rely on particulate feeding. The δ13C and δ15Nvalues of purified symbionts do not differ from those of thhost tissues, and this is evidence that the carbon and nitroused in biosynthesis are primarily derived from autotrop(Conway et al.1989).

Assimilation of nitrogen is an important and potentialcomplex physiological capability of marine symbioseNitrogen is often a limiting nutrient for marine autotrophorganisms. The ability to assimilate inorganic nitrogecompounds facilitates the recycling of waste ammonia froamino acid catabolism and the utilization of inorgannitrogen from the environment in autotrophic marinsymbioses such as algal–invertebrate associations (Musca1980). The metabolism of ammonia and nitrate in symbioassociations is complicated by the possibility that both hand symbiont are involved in assimilation. Ammoniumassimilation by algal–invertebrate associations, which wonce thought to involve primarily the algal symbiont, apparently facilitated in part by the invertebrate ho(McAuley, 1995; Rees, 1987; Rees et al. 1994). Since the‘essential’ amino acids (those that cannot be synthesized must be obtained from the diet) of invertebrates are probathe same as those of other metazoans (Bishop et al. 1983),even if the host can assimilate ammonia into amino acids,symbiont may be required for synthesis of essential amacids. This is particularly applicable to S. reidi, since theseclams cannot obtain amino acids from particulate food.

In earlier studies, we documented that Solemya reidicantake up and assimilate ammonia as well as nitrate (Lee Childress, 1994; Lee et al. 1992a). Ammonia is the mostabundant dissolved nitrogen source in the sewage sluoutfall environment where clams were collected. Porewaammonia concentrations are around 50–60µmol l−1

compared with 1–11µmol l−1 nitrate and 3–15µmol l−1 totalFAA (Lee and Childress, 1994; Lee et al.1992a). Ammoniaassimilation appears to be dependent on conditions favorsulfide-based chemoautotrophy. Sulfide stimulates ammouptake, and detectable ammonia excretion is only obserafter prolonged maintenance in the laboratory maintenance in sulfide-free sea water (Lee et al. 1992a).Ammonia is incorporated into organic compounds, and thighest rates of incorporation are in the gills (Lee aChildress, 1994). In the present study, we investigated fate of ammonia within the symbiotic association bmeasuring tissue ammonia pools and 15N-labeled ammoniaassimilation. Biosynthesis of amino acids was investigatby measuring changes in tissue FAA pools in responseincreased ammonia availability.

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Materials and methodsClam collection and maintenance

Solemya reidiBernard were collected by Van Veen grabfrom depths of approximately 100 m in Santa Monica BaCalifornia, near the Hyperion sewage sludge outfall anmaintained in laboratory mudtanks at 5–9 °C as describpreviously (Lee et al.1992a).

15N-labeled ammonia incubations

All ammonia incubations involved the addition of 100 %15N-labeled ammonia to facilitate measurement of assimilatirates. Solemya reidi, maintained for 3 days in laboratorymudtanks, were removed from their burrows, rinsed with swater, then placed in filtered sea water for 5 h. Clams weexposed to 100 % 15N-labeled ammonia (0.01–1.3 mmol l−1;five treatments) for 22–23 h. Incubations consisted of 2clams in 0.5–1.0 l of filtered sea water at 5 °C containin500µmol l−1 sodium thiosulfate. ΣNH3 (the sum of[NH3]+[NH4+] measured in our analyses) concentration wadetermined by flow-injection analysis (FIA; Willason andJohnson, 1986). Following exposure to ammonia, clams weremoved from the incubation medium and separated into gfoot and visceral mass. In our sampling, visceral mass refto the soft body parts remaining after the removal of the gand foot. Excised tissues were frozen in liquid nitrogen thstored at −80 °C until analyses of ΣNH3, FAAs and 15Nincorporation were made.

Tissue extracts and ΣNH3 and FAA determinations

Frozen tissue samples were homogenized in nine volumof 50 % ethanol using a ground-glass homogenizer and thcentrifuged (Millipore microfuge, 6400 revs min−1). Tissueextracts were analyzed for ΣNH3 by FIA. Free amino acidanalyses were performed by high-pressure liquchromatography (HPLC) and precolumn fluorimetriderivatization with o-phthalaldehyde (OPA; Lindroth andMopper, 1979; Mopper and Lindroth, 1982). Derivatizeamino acids were separated on a Beckman C-18 column usa methanol–acetate buffer gradient and detectfluorometrically as described previously (Lee et al. 1992a,b).These tissue values are probably below true intracellular ΣNH3

and FAA concentrations owing to dilution by hemolymph ithe tissues (discussed in more detail below in DiscussioResults are presented as µmol g−1wet mass.

15N determinations

Subsamples of frozen tissues were dried at 60 °C, thground to a fine powder. A portion of the ground sample wtreated with 2 mol l−1 NaOH to remove ammonia quantitatively(Lee and Childress, 1996). Treated and untreated samples wanalyzed for 15N/14N by continuous-flow isotope ratio massspectrometry (CF-IRMS) using a Europa Scientific RobopreCN/Tracermass instrument. Operating conditions were described previously (Lee and Childress, 1995). In the prespaper, ‘assimilation’ refers to incorporation observed

2799Ammonia metabolism of a symbiotic clam

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samples after treatment with NaOH. ‘Σ15NH3’ refers to theamount of 15N-labeled total ammonia (15NH3 and 15NH4+)present in samples determined from the difference in 15Ncontent between NaOH-treated and untreated samples.

ResultsTissue ΣNH3

Concentrations of ΣNH3 in Solemya reiditissues rangedfrom 0.5 to 12µmol g−1 (Fig. 1A–C). Tissue ΣNH3

concentrations correlated with external ΣNH3 concentration inall tissues tested (Fig. 1A–C). ΣNH3 concentration was clearlyelevated in the tissues compared with the medium (Fig. 1A–Foot and visceral mass ΣNH3 concentrations were generallylower than gill ΣNH3 concentration. Hemolymph ΣNH3

concentration was lower than that in tissues and was adependent on external ΣNH3 (Fig. 1D).

ΣNH3 concentrations measured from tissues frozen −80 °C may be slight overestimates, since ΣNH3 concentrationcan increase in frozen biological samples. Although there conflicting reports, one study shows an increase 5–7µmol l−1 in human blood samples stored at −70 °C(Howanitz et al.1984). Tissues of a deep-sea mussel symbio

Fig. 1. Tissue and hemolymph total ammonia (ΣNH3) concentration othiosulfate as sulfur substrate. ΣNH3 values are for tissue (A–C) angram wet mass. The solid line is the isoline for tissue [ΣNH3] = externfor estimated intracellular pH values of 7.3 and 6.5.

0

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8

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Gill

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pHi=6.5

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CVisceral mass

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External

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with methanotrophic bacteria (seep mytilid Ia) exhibited gΣNH3 concentrations in samples stored at −80 °C of1.46±0.14µmol g−1 (mean ± S.D., N=5) compared with1.22±0.17µmol g−1 (mean ±S.D, N=5) in samples that wereextracted and analyzed immediately. The ΣNH3 concentrationsof these fresh and frozen samples were not significandifferent (analysis of variance, P>0.05; R. W. Lee, unpublishedobservations). Thus, storage at −80 °C may have resulted inincreased ΣNH3 concentrations in S. reidi tissue samples, butthese changes are probably negligible compared with absolute concentrations and large increases observed function of external ΣNH3 concentration (Fig. 1A–D).

Effect of external ammonia concentration on 15Nassimilation and isotope dilution of 15NH3

Assimilation of 15N was greatest in gill tissue although labewas also detected in the visceral mass (Fig. 2). The rate of 15Nassimilation increased as a function of external ΣNH3

concentration (Fig. 2).Although clams were exposed to 100 % 15N-labeled

ammonia, only part of the gill ΣNH3 was 15N-labeled (Fig. 3).The amount of 15N-labeled ammonia (Σ15NH3) in some gilltissue samples was determined by quantifying 15N lost

f Solemya reidiexposed for 22–23 h to ammonia-enriched sea water withd hemolymph (D) samples from single individuals and are expressed peral [ΣNH3]. Broken lines are isolines for tissue [NH3] = external [NH3]

0

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Visceral mass

External ∑NH3 (mmol l )

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Fig. 2. Incorporation of 15N label into Solemya reidifrom ammoniaexposure experiments. Ammonia added to the medium was 100 %15N-labeled ammonia. Data points represent determinations made ontissue samples from a single individual following treatment withNaOH to remove label present as 15NH3. Filled circles, gill; opencircles, visceral mass.

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Fig. 3. 15N-labeled and total ammonia concentration in Solemya reidigills. Open circles, labeled and unlabeled ammonia as measured byflow-injection analysis (see Fig. 1A). Filled circles, 15N-labeledammonia determined by quantifying 15N lost by treatment of tissuesamples with NaOH. Each data point represents a determination fromthe gill of a single individual.

following treatment with 2 mol l−1 NaOH. Σ15NH3

concentration was as high as 2µmol g−1 in gill exposed to 0.7and 1.3 mmol l−1 external ammonia. The percentage of ΣNH3

present as Σ15NH3 (%Σ15NH3) was variable, with a mean of19.4±8.8 % (S.D., N=12). At higher external Σ15NH3

concentrations, unlabeled ΣNH3 concentration as well asΣ15NH3 concentration increased in gills.

Tissue free amino acid composition and response toammonia

FAA compositions of gill tissue from individual clams

Table 1.Free amino acid concentrations in gill tissue oconcen

External ∑

Amino acid 0.005 0.005 0.012 0.012 0.012 0.

Aspartate 24.2 30.5 20.7 16.2 13.6 2Glutamate 11.2 14 6.7 10 15.9 13Glutamine 2.3 4.3 1.6 1.2 3.3 1Glycine 2.9 4 2.4 2.3 − −Threonine 2 2.8 1.6 1.6 1.9 1Arginine 4.7 3.6 4.6 2.2 5.9 2Taurine 58.4 84.4 51.1 83.8 106.8 9Alanine 5.5 8.1 4.3 3.7 7.7 4Tyrosine 0.9 1.5 − 0.7 − −Methionine − − 0.5 − − −Valine 1.5 2.6 4.2 0.6 − 0.8Isoleucine 0.9 1.3 0.5 0.5 − −Leucine 1.2 2.3 1 0.8 0.6 −

Total 115.6 159.3 99.2 123.6 155.7 15

Free amino acid concentrations are presented as µmol g−1wet mass.Dashes denote concentrations too low to quantify reliably (less

exposed to varying ammonia concentrations are given in Ta1. The most abundant FAAs were taurine, aspartate, glutamalanine and arginine. Taurine concentration was conspicuouelevated compared with the concentrations of all other FAmeasured. Ammonia exposure resulted in an increase in tauand total FAA concentrations (Fig. 4A,B). No increase in totnon-taurine FAA concentration was observed (Fig. 4B).

Two extracts of foot tissue were also analyzed (from 0.and 0.19 mmol l−1 ΣNH3 treatments). The dominant FAAswere similar to those observed in gill tissue, with taurinpresent at a higher concentration than all other FAA

f individual Solemya reidiexposed to various external ∑NH3

trations

NH3 concentration (mmol l−1)

061 0.061 0.061 0.187 0.687 0.687 1.299 1.299 1.299

6.3 20.9 7.7 22.9 19.9 24.2 25.9 18.4 44.5.3 15.1 25.9 11.3 12.1 5.6 20 12.2 14.2.3 0.8 1.1 1.4 0.4 0.9 − − 2.3

− 2.8 0.8 − 1.3 − 1.4 2.3.5 1.1 1.2 1.5 1.7 0.1 3.4 1.7 2.2.9 1.2 2.6 1.9 3.8 4.7 2.7 3.5 5.29.7 80.5 44.3 128.4 126.2 153.1 129.4 155.3 182.2.6 6.2 10.6 3.1 5.7 1.7 9.4 3.2 3.4

0.1 0.8 0.7 − − 0.7 0.5 0.2− − − − − − − −1 1.3 1.1 1.3 2 1.7 1.4 1.5− 0.9 0.6 − 4.3 0.5 0.3 0.9− 1.4 1 0.4 0.8 0.9 0.5 1.5

0.5 127 100.4 174.6 171.5 198.8 194.7 198.4 260.4

than approximately 0.3µmol g−1) in 1:10 diluted ethanol extracts.


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Fig. 4. Free amino acid (FAA) concentrations in Solemya reidigill.Single determinations (expressed per gram wet mass) from indiviclams exposed for 22–23 h to ammonia-enriched sea water thiosulfate as sulfur substrate. Slopes of regression equationsgiven ±95 % confidence intervals. (A) Total identified FAAs (sTable 1) measured by OPA derivatization and HPLC. (B) FAseparated into taurine (d) and total non-taurine (s) FAAs.

Aspartate, glutamate, arginine and taurine concentrations whigher in the 0.19 mmol l−1 external ΣNH3 treatment. Taurineconcentration was 73.5µmol g−1 in the 0.06 mmol l−1 ΣNH3

treatment and 111.9µmol g−1 in the 0.19 mmol l−1 ΣNH3

treatment.Four additional OPA reactive compounds, that did n

correspond to standards used in our analyses, were consistdetected in gill. The concentrations of these compounds didchange as a function ammonia concentration.

DiscussionBecause whole tissues were used in determinations of ΣNH3

and FAA concentrations in Solemya reidi, our values reflectintracellular as well as extracellular concentrationExtracellular FAA concentrations are generally lo(0.2–5 mmol l−1; Bishop et al. 1983) compared withintracellular FAA concentrations, and hemolymph ΣNH3

concentrations were lower than concentrations measuredtissues (Fig. 1). Thus, variation in the amount of extracellufluid present in these samples is a source of variability in oresults. By dissecting and treating samples as consistentlpossible, differences in the proportion of extracellular flu

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were probably kept to a minimum between samples of the satissue type. Differences in hemolymph content could explawhy gill ΣNH3 concentration was consistently higher than thain the foot or visceral mass.

In S. reidi, internal ΣNH3 concentrations were elevatedcompared with levels in the medium across a wide rangeexternal ΣNH3 concentrations (Fig. 1A–C). This elevation maybe accounted for in part by the acidic intracellular pH (pHcompared with that of the medium (pHe) and by the greapermeability to NH3 compared with NH4+. If only NH3 ispermeable, and if NH3 is in equilibrium between the internaland external compartments, the relationship between interand external ΣNH3 concentration is (Roos and Boron, 1981):

It follows that, since intracellular pH is generally lower thathat of sea water (pH 8), internal ΣNH3 concentration will begreater than external ΣNH3 concentration.

Two values of intracellular pH were used to calculate threlationship between internal and external ΣNH3 concentration(see Fig. 1): the pHi reported for S. reidi in the literature anda low estimate based on hemolymph measurements. Tintracellular pH of excised gill filaments of S. reidi is 7.3(Kraus et al.1996), which is in the expected range for a marinmollusc at 5–9 °C (Hochachka and Somero, 1984). Howevthe intracellular pH of tissues from intact clams under somconditions may be lower than 7.3. Hemolymph draining fromincisions in the mantle and visceral mass was taken up intsyringe and analyzed immediately using a water-jacket(10 °C) microvolume cell and double-junction pH electrodeThe hemolymph pH of sulfide-incubated clams ([sulfide] up 150µmol l−1; [O2] between 160 and 211µmol l−1; 9 °C)averaged 6.8 (range 6.6–7.2), and that of clams incubatedsea water averaged 7.1 (range 6.9–7.3; Lee et al.1992a). Thehemolymph pH of three S. reidi measured immediatelyfollowing collection ranged from 7.2 to 7.5 (R. W. Leeunpublished data). Assuming that intracellular pH is 0.4 unlower than hemolymph pH (Hochachka and Somero, 198and a low hemolymph pH value of 6.9, intracellular pH mabe as low as 6.5. Using a pK value (5 °C; 35 ‰ salinity) of 9.9(Whitfield, 1974), it is predicted that intracellular ΣNH3

concentration would be five times greater than external ΣNH3

concentration for a pHi value of 7.3 and 31 times greater fopHi value of 6.5. Such predicted values are as high as thobserved for whole gill tissue (Fig. 1A–C). Although wecannot distinguish between intracellular and extracellulammonia in our measurements and values of pHi are estimathese findings are consistent with a mechanism whereinternal ammonia concentration is elevated compared with thin the medium owing to the relatively acidic intracellular pHand the higher permeability of NH3.

Because ammonia is potentially an important nitrogesource for autotrophic symbionts, the finding of millimolaconcentrations of ammonia in Solemya reidi gill tissue

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suggests that the symbionts encounter high nitrogavailability. In contrast, low ammonia concentration(µmol l−1) are present in symbioses between cnidarians aalgae (Crossland and Barnes, 1977; Falkowski et al. 1993;Wilkerson and Muscatine, 1984). The concentrations ammonia in S. reidi appear to be typical of other bivalvesAmmonia concentrations of 12µmol g−1wet mass are reportedfor mantle tissue of Crassostrea virginica(Heavers andHammen, 1985) and 21µmol g−1dry mass for Mytilus edulis(Livingstone et al. 1979). Similarly, S. reidi hemolymphammonia concentrations (0.06–0.21 mmol l−1) from lowexternal ammonia (0.05–0.06 mmol l−1) treatments are withinthe range of hemolymph ammonia concentrations observethe clam Rangia cuneata(Henry and Mangum, 1980).

High ammonia concentrations in clam tissues may enhaautotrophy by the chemoautotrophic symbionts. Sourcesnitrogen are often limiting to marine autotrophs, and ammoconcentrations in sea water are generally in the low micromorange. Therefore, compared with free-living bacteria living the water column, the symbionts encounter abundant ammoHigh nitrogen availability may have effects on symbioassimilation pathways and their regulation. In bacterammonia assimilation is catalyzed by either glutamisynthetase (GS), which has a high affinity for ammonia, glutamate dehydrogenase (GDH), which has a low affinity ammonia (Reitzer and Magasanik, 1987). In free-livinbacteria, GS is believed to be the primary enzyme involvedammonia assimilation (Merrick, 1988; Reitzer and Magasan1987), but in the symbiotic bacteria GDH may function assimilation since ammonia concentrations are potentiahigh. High ammonia concentrations, which would act promote symbiont growth, may exacerbate the problemsmaintaining stable symbiont populations. The host mpossibly regulate symbiont nitrogen assimilation or restrsymbiont access to ammonia.

Free amino acid pools

Free amino acid levels in the gill tissue of Solemya reidiarein the low range reported for marine mollusc(50–400µmol g−1wet mass; reviewed in Bishop et al. 1983).The total concentration of amino acids measured w99–159µmol g−1wet mass in clams exposed to externammonia concentrations of 0.005–0.06 mmol l−1. Theseconcentrations are similar to total FAA concentrations reporfor Crassostrea virginicaof 146µmol g−1wet mass (Heaversand Hammen, 1985) and Mytilus edulis of70–200µmol g−1wet mass (Zurburg and De Zwaan, 1981).

It is well documented that FAAs are important in marininvertebrates as intracellular osmolytes (Pierce, 1982; Somand Bowlus, 1983). In the present study, environmental salinwas not altered, and the expansion of the total FAA pool oS.reidi gills from 100–150µmol g−1 to up to 260µmol g−1 inresponse to an increased external ΣNH3 concentration couldpotentially result in cellular swelling. This may have beeavoided by a compensatory loss of an amino acid osmolytemeasured in the present study or by the loss of other org

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osmolytes such as methylammonium compounds, e.g. glycbetaine, which are important for osmoregulation in othmarine invertebrates (Pierce et al. 1992; Schoffeniels, 1976;Somero and Bowlus, 1983). The effects of increased taurlevels on intracellular osmolarity and the role of other aminacids, related compounds and ions as osmolytes are cleareas for further investigation.

Taurine is the dominant FAA in all clams of the genuSolemyathat have been investigated. In S. velumand S.borealis, which also have chemoautotrophic symbionts, taurinconstitutes 63–74 % of the FAA pool. As in S. reidi, otherabundant FAAs are glutamate, alanine and (in S. velum)aspartate. The total concentration of non-taurine FAAs in gtissue is similar between species: 83µmol g−1 in S. velum(Conway, 1990; Conway and McDowell Capuzzo, 199260µmol g−1 in S. borealis (Conway et al. 1992) and53±12µmol g−1 (mean ±S.D., N=14; from all treatments) in S.reidi. However, taurine levels differ greatly between specie235µmol g−1 in S. velumgill and 100µmol g−1 in S. borealisgill. The taurine levels of S. velumexceed the concentrationsobserved in S. reidi even at high external ammoniaconcentrations, whereas S. borealislevels are comparable tolevels in S. reidiexposed to low to moderate external ammonconcentrations (0.005–0.06 mmol l−1; Table 1).

Taurine is a common FAA in other chemoautotrophsymbioses as well as in some (but not all) non-symbiomarine invertebrates (reviewed in Conway and McDoweCapuzzo, 1992). In the symbiotic seep mytilid Ia from the Guof Mexico, taurine and glycine were the dominant FAAs, wittaurine levels of approximately 40–50µmol g−1 (Lee et al.1992a). Taurine, glycine and alanine were the dominant FAAin symbiotic deep-sea mussels of the genus Bathymodioluscollected in the South Pacific (Pranal et al.1995).

Taurine synthesis

The increase in taurine concentration observed in respoto ammonia in S. reidi is not easily explained by host orsymbiont metabolism alone. Although biosynthesis of taurinis well documented in animals, there do not appear to reports of taurine biosynthesis by bacteria. Like vertebratmarine molluscs can apparently synthesize taurine frocysteine and methionine (Bishop et al.1983). Recently, a highcapacity for taurine synthesis has been demonstrated in bivalarvae (Welborn and Manahan, 1995). However, methioninwhich is the precursor for cysteine, is an essential amino athat cannot be synthesized by animals. Methioninconcentrations were low to undetectable in the FAA pool of S.reidi, and cysteine concentrations (not tested in S. reidi) arelow in the FAA pool of other Solemyaclams (Conway et al.1992; Conway and McDowell Capuzzo, 1992). Therefore,source of cysteine or methionine is needed for the hostsynthesize taurine. Cysteine may be provided by the symbiobacteria since it is well documented that cysteine synthesisthe predominant way in which bacteria incorporate inorgansulfur, such as sulfide and thiosulfate, into organic compoun(Kredich, 1996). If sulfide is the sulfur source, cystein


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Total ammonia assimilation ( mol g )

Fig. 5. Gill tissue taurine concentration and calculated total ammoniaassimilation based on 15N tracer incorporation and %Σ15NH3 in thegill tissue ammonia pool (see text) in Solemya reidi. Singledeterminations (expressed per gram wet mass) from individual clamsexposed for 22–23 h to ammonia-enriched sea water with thiosulfateas sulfur substrate.

biosynthesis involves a two-step process in which serineconverted to O-acetylserine which then reacts with sulfide form cysteine (Kredich, 1996).

Role of taurine

Increased taurine concentration in response to an increaammonia supply is unprecedented in symbiotic invertebraIt is not clear what the functional significance of this observincrease is since no functions for taurine, other than asosmolyte, have been identified in marine invertebrates. Tdramatic increase observed in the present study is suggeof hitherto unrecognized roles for taurine in symbiotinvertebrates.

Since there is a relationship between taurine levels ammonia availability, taurine may be involved in nitrogestorage and transport. Taurine is rich in N (C:N=2) and canmaintained at a high concentration in the cytosol. Not all amacids can be present at high concentration without affecprotein function (Somero and Bowlus, 1983). Amino acisuch as taurine, glycine, alanine and proline do not affenzyme Km or Vmax and have favorable effects on proteistructural stability (Somero and Bowlus, 1983). In additionbeing inert with regard to protein function, taurine is highsoluble and zwitterionic over the physiological pH range. a zwitterionic compound, taurine can be accumulated withperturbing membrane potential and has low lipophilicity, sois not readily lost by diffusion (Huxtable, 1992).

The finding that rates of taurine production were comparato rates of total ammonia assimilation is consistent with incorporation of ammonia into taurine. Total ammonassimilation (15N-labeled and unlabeled) is greater than 15Nassimilation when isotope dilution is taken into accouΣ15NH3 averaged 19.4 % in gill tissue and, assuming that ammonia is a single pool, total ammonia assimilation is times greater than 15N assimilation. The regression coefficienfor the relationship between taurine concentration (µmol g−1)and total ammonia assimilation (µmol g−1) was 1.17±0.60(95 % confidence interval; Fig. 5). Therefore, ammonassimilation can account for the increase in tauriconcentration. Since the lower 95 % confidence interval limwas 0.58, at least 58 % of the increase in taurine concentramay be due to ammonia assimilation. Further 15NH3 tracerstudies, in which 15N can be detected in individual aminamino acids, are needed to gain direct evidence that nitrofrom ammonia is incorporated into taurine.

To determine whether taurine can function as a nitrogstorage compound, further studies are needed to documwhether taurine can be catabolized by either host or symbiThe ability to use taurine as a nitrogen source is not univerMammals cannot catabolize taurine (Huxtable, 1992), aalthough marine molluscs appear to have a modest capacittaurine catabolism, the products and potential involvemenassociated bacteria are not known (Bishop et al. 1983). Themetabolism of taurine as a source of energy, carbon nitrogen, and the possible metabolic pathways, have only bdocumented in bacteria (reviewed in Huxtable, 1992). T

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taurine catabolism capabilities of Solemya reidiremain an openquestion that merits further investigation. If a high capacity ftaurine catabolism can be demonstrated, then the role of tauas a nitrogen (and carbon) storage compound will supported.

We thank the captain and crew of the R.V. Robert GordonSproul for assistance in animal collection, A. Seitz fodiscussions of bacterial sulfur metabolism, T. Garcia fassistance in sample preparation, and D. Manahan suggestions on the interpretation of some of the results. Twork was supported by NSF grants OCE-9301374, OC9632861 and DIR-901674 and an Office of Naval Researgrant NOOO14-92-J-11290 to J.J.C.

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