Ammonium Metabolism in the Green Hydra Symbiosis

9 Pages·1995·3.8 MB·English

by  P J Mcauley

Reference: Bail. Hull 188: 210-21X. (April. 1445)
Ammonium Metabolism in the Green Hydra Symbiosis
McAULEY
P. J.
School of Biological ami Medical Sciences. Sir Harold Mitchell Building. University ofSt. Andrews.
St. Andrews. Fife. KYI6 9TH. L'nited Kingdom
Abstract. Inhibitors ofenzymes ofammonium assimi- to take up and assimilate ammonium from the environ-
lation were used to test ifassimilation ofammonium in ment (Kawaguti, 1953: Gates and McLaughlin, 1976;
the green hydra-Chlorella symbiosis was due to host or Szmant-Froelich and Pilson, 1977; Muscatine and D'Elia,
symbionts. Both methionine sulphoximine (MSX, an in- 1978; Muscatine el at.. 1979; Wilkerson and Muscatine,
hibitor ofglutamine synthetase, found in both host and 1984; Wilkerson and Trench. 1986; Rees, 1986; McAuley,
symbionts) and azaserine (AZS, an inhibitor of2-oxoglu- 1990). However, there is some controversy over whether
tarate amido transferase, not found in the host) inhibited ammonium assimilation isdue to the symbiotic algae, or
ammonium uptake by the intact symbiosis. MSX was to the animal host, or to the combined activities ofboth
taken up and caused predictable changes in pools ofglu- (Millerand Vellowlees, 1989). Thepossibilitythatanimal
tamate and glutamine in both freshly isolated symbionts rather than algal enzymes may be responsible, in part or
and cultured ex-symbiotic C/ilorel/u. However, after in whole, for assimilation ofammonium has important
treatment of the intact symbiosis with MSX. no MSX implications for the mechanisms by which animal hosts
was found in the symbiotic Chlorella. and glutamine and regulate cell division in populations ofsymbiotic algae,
glutamate pools ofboth host and symbionts were unaf- since a numberofworkershavesuggested that thegrowth
fected. Although both MSX and AZS inhibited ammo- ofpopulations ofsymbiotic algae may be nitrogen-limited
nium uptake by Chlorella. MSX caused seven times as (Rees. 1986; Cook and D'Elia. 1987; McAuley. 1987a).
much ammonium release from the intact symbiosis as Millerand Yellowlees(1989) pointed out that in corals
did AZS. AZS treatment of the intact symbiosis caused symbiotic with dnioflagellates (zooxanthellae), levels of
an increase inglutamine poolsin both hostandsymbionts, the ammonium assimilatory enzyme NADPH-glutamate
and AZS also competitively inhibited glutamine uptake dehydrogenase (NADPH-GDH) are higher in host tissue
byChlorella. Further, ammonium treatment ofintact hy- than in symbionts, although the catabolic production of
dradid notaffectthenitrogen statusofthealgal symbionts, ammonium probably exceeds the capacity of host
although it did cause a small increase in the number of NADPH-GDH (Rahav etui.. 1989; Falkowski el al.. 1993;
algae in each digestive cell ofthe host. It is suggested that Spencer-Davies, 1992), which has a low specificity for
primaryammonium assimilation in thegreen hydrasym- ammonium (Catmull el al.. 1987). More recently, glu-
biosis occurs by means ofanimal glutamine synthetase, tamine synthetase (GS)activitywas reported in giant clam
and that the resulting glutamine may be taken up and (Tridacnagigas) and coral (Pocillopora damicornis) host
furtherprocessed by the symbiotic algae. Freshly isolated tissues (Fitt et a/.. 1993; Yellowlees et al.. 1994). Because
symbionts were able to processglutamine into glutamate thereare relatively high levelsofammonium-assimilating
even when incubated at low pH, which causes them to enzyme in host tissues, it is possible that much of the
releaseasubstantial proportion offixedcarbonasmaltose. ammonium present in natural seawaterconcentrations is
Introduction assimilated before it reaches the symbiotic zooxanthellae
(Yellowlees et al., 1994).
Invertebrate-microalgal symbioses are able not only to However, several lines ofevidence suggest that some if
reassimilate catabolically produced ammonium but also not all ammonium assimilation is due to the zooxan-
thellae. Assimilation islight dependentanddoes not occur
Received 10 March 1444;accepted 12 January 1495. in aposymbiotic animals (for review, see Rees, 1986).
210
AMMONIUM METABOLISM IN GREEN HYDRA
Ratesofuptake by intact associationsare similarto those (McAuley. 1986a), but unlike assays forenzyme activity,
of freshly isolated symbionts (D'Elia et al.. 1983: Wil- measurement offreeamino acid pools by HPLC requires
kerson and Trench, 1986), and intactassociationsexhibit only small numbers ofalgae (the equivalent of I04 cells
MSX
uptake kinetics which suggest that symbionts assimilate or fewer per sample injection). Uptake of by algae
ammonium from seawater by depleting ammonium in ;/; vitro and in the intact symbiosis was also measured
hosttissueandcausingadiffusion gradient toform(D'Elia using HPLC.
and Cook, 1988). Finally, addition ofammonium to sea-
watercauses increases in zooxanthellar mitosis, biomass, Materials and Methods
and amino acid levels (Cook ct a/., 1988; Muscatine el Maintenance oforganisms
a/.. 1989; Hoegh-Guldberg and Smith, 1989; Dubinsky
el al.. 1990; Fitt and Cook, 1990; Stambler el ai. 1991; Green hydra of the European strain (EE hydra) were
McAuley, 1994; Muller-Parkerel al.. 1994; McAuleyand grown in unbuffered 'M'solution (Muscatineand Lenhofi,
Cook, 1994), and reduces ammonium uptake rates (Yel- 1965)at 15Cin constant light(60 jumol photonsm 2 s '
lowleest1/al.. 1994). Ammonium assimilation isassumed PAR). Cultures were fed each Monday, Wednesday, and
toinvolvethecoupled glutamine synthetase/2-oxoglutar- Friday with freshly hatched brine shrimp. All hydra used
ate amido transferase (GS/GOGAT) pathway. At present. in experiments had not been fed for the previous 3 days
GS but not GOGAT has been detected in zooxathellae to allow complete digestion of food. Cultures of the
(SummonsandOsmond, 1981;Wilkerson and Muscatine, 3N813A strain of maltose-releasing Chlorella, grown in
1984), although azaserine, a potent inhibitorofGOGAT, Kessler's medium (Kessler et ai. 1963), pH 6.3, were
prevents ammonium uptake in intact corals (Rahav et maintained in a shaking, illuminated incubatorin growth
al.. 1989), and the presence of GS/GOGAT activity is conditions similar to those ofhydra cultures.
supported by the data ofSummons et al. (1986).
In contrast to marine symbioscs. in the symbiosis be- Isolation ofsymbiotic algaefrom hydra
tween the freshwater cnidarian Hydra viridissima (green The SDS-washing technique (McAuley, 1986a) was
hydra) and Chlorella algae, the host rather than the algal used to isolate symbiotic Chlorella algae from hydra.
symbionts may be responsible for assimilation of am-
monium. Ammonium assimilation by maltose-releasing Determination ofnumbers ofalgaeper digestive cell
ChlorellaisinhibitedatthelowpH thatstimulatesmaltose
release (Rees, 1989). Recent evidence suggests that in Five gastric regions ofhydra were isolated in a drop of
symbiosis the algae are carbon- ratherthan nitrogen-lim- macerating fluid (David. 1973). After 10 min, pieces of
ited, in that in vivo release ofmaltose consumes consid- hydrawereteasedapart into individual cellsandexamined
erable amounts of photosynthetically fixed carbon that using phase contrast microscopy. Numbers ofalgae were
would otherwise be used to assimilate ammonium into determined in 100 randomly selected digestive cells in
amino acids (McAuley. 1992). Treatment of the intact each preparation.
DCMU
symbiosis with the photosynthetic inhibitor does
not cause ammonium excretion into the medium, and Measurement ofammonium uptake
rates ofammonium uptake by freshly isolated symbionts Ammonium uptake was measured as depletion from
are only40% ofthose ofthe intact symbiosis (Rees, 1986; the medium ofeither hydra or algae. Duplicate samples
oMfcAGuSleayct,iv1i9t9y0a)r.eFhiingalhleyr,itnhesyhmobsitostpiocsstehsasnGaSp,osaynmdbiloevteilcs (m2e0n0ts^,1)anwderaemotuaknetnsaotfathmemboengiinunminwgeraenddeetnedrmoifneedxpweirtih-
animals (Rees, 1986).
Although green hydra do not normally excrete am- a(Rseceasl,ed1-9d8o6)w.n hAyfptoecrhlroeraigteen-tnsitwreorpreusasdiddeedc,olsoammeptlreiscawsesraey
monium into the medium, release can be induced by incubated for 20 min in darkness, then absorbence was
etifnrfheeiacbttimtoesfnGtMSwSiXitnhoimnnettaahcltigaolhnyiGdnrSeash(auRslepenhsoo,txi1bm9e8ie6nn)e.t(eHsMotSewXde,)v,eberwc,haiutcshhee Cprehhaaedtmeiactsa6tl3a0nCdonamrmpdasanny(d)re.caogmenptasreadndtostthaatndoafradsmmfornomiuSmigsumla-
of the difficulty in isolating sufficient numbers of sym-
bionts uncontaminated by host material. This paper de-
Inhibitors
scribestheeffectsofinhibitors ofenzymesofammonium
assimilation on perturbation ofanimal and algal internal Methionine sulphoximine (MSX), an inhibitor ofGS
pools of glutamate and glutamine, amino acids closely (Ranziorfa/.. 1969; Meister, 1974), and azaserine(AZS),
associated with ammonium assimilation (Miflin and Lea, an inhibitor of 2-oxoglutarate amido transferase (GO-
1976). Recovery of symbiotic algae after SDS washing, GAT) (Wallsgrove et al.. 1977; Elrifi and Turpin, 1986),
which is necessary to remove host contamination, is low were purchased from the Sigma Chemical Company.
McAULEY
212 P. J.
Stock solutions were stored at 4C and routinely tested GOGAT activity by determining the rate ofoxidation of
by measuringtheireffectivenessin inhibitingammonium NAD(P)H at 340 nm by the method ofBhandari and Ni-
uptakebycultured 3N813Acells. Inallexperiments, MSX cholas (1981). Blanks were run without addition of n-
was used at a final concentration of200 fiMand AZS at ketoglutarate.
mM.
1
Results
Extraction amimeasurement offree cimino acidpools Effect ojammonium on symhionts in intact hydra
Samplesofalgae, orofanimal homogenatescentrifuged Incubation ofEE hydra for 7 days in M solution sup-
at 1000 X g for 5 min to remove algae, were added to plementedwith 50 nAfammonium chloride had noeffect
absolute ethanol to give 80% ethanol (v:v) and extracted on the size ofthe glutamate and glutamine pools oftheir
dfIonertse2or4mmeihnceaatdse4uss,Ci.anlggPatrlhoestaeBimrnpaldcefosonrtwdeenrmteestfhoilfotdehroe(dmBroaagtdelfnooarwtd,evsa1c9wu7e6ur)me. aeallxggpaaeleris(myoemnnbeti-sow)natywseAr(NeTaObpVlreAes.eI)n.tPHi<onwe0d.vi0eg5re,,stnisvi=egni6cefliilcnsadnteolpfyenhmdyoedrnreta
throughWhatman GF/Cfilters,whichwerethenextracted maintained in ammonium-supplemented medium (24.94
trweiscuespiennd8e0d%ienthaannola.ppErxotprraicattsewveroeludmreiedof//;12v.a5c/nuoA/anad- 1.71 SD) than in controls (22.58 1.48).
astmainndoa-rbdu.tAyrmiicnoaciadcid(AcAonBtAe)n,tswohfiaclhiqaucottesdofasexatnracitnstewrenrael Effect ofMSX andA/.S on ammonium uptake
determined by ophthaldialdehyde pretreatment and re- As previously observed (Rees, 1986; McAuley, 1990).
verse-phase HPLC as previously described (McAuley, MSX not only inhibited ammonium assimilation but also
1992). MSX but not AZSwasdetectable usingthissystem; caused release ofammonium from both intact hydraand
the MSX peak occurred 0.8 min after that ofserine. cultured 3N813A algae (Table II). AZS inhibited am-
monium assimilation butdid not cause release in 3N813A
Glutamine and methylamine uptake algae;ammonium releasebygreen hydratreatedwith AZS
MSX
was seven times lower than that observed with
Glutamine uptake was determined from cultured
3N813A cells resuspended in 10 mM MES buffer pH 7 treatment.
acttuabmaaitdnieeonnsc,iotntythaeoifnai5snsgXay3.1w70a7^s/cBesqltlas[rUtm-elMdCb'].y-Agfaltduedtrianm3gi0n2em-i(2An0moepfrMpsrhegilanum-- EafnfdecKt/ioiftMamSinXcaimni3NASZ1S3Aon internalpools ofglutamate
International; specific activity9.25 GBq mmol ')with or 3N813A algaewereincubated witheitherMSX orAZS
without additional 20 \iM AZS. At intervals of 1. 2, and in the absence or presence of a nitrogen source (either
3 minafteraddition ofradioactivity, 200-^1sampleswere glutamine or ammonium), and amino acid pools were
filtered onto Whatman GF/C filters at low vacuum and compared tothoseofuntreatedcontrols(Table III). MSX
washed with 20 ml distilled water. Filters were dried in and AZS had distinct effects on internal glutamine and
scintillation vials, 10 ml ofscintillation fluid was added glutamate pools irrespective ofthe nitrogen supply, and
(McAuley, 1988), and samples were counted on an LKB
1214 Rackbeta scintillation counter. Vmax and KM were
determined from Lineweaver Burke double reciprocal Table I
plots ofuptake rates against substrate concentration. Effect <>tinciihalion /inlucl hydra inammonium onglittamaicanil
Uptake of [l4C]-methylamine was determinedM by the i>o<il\ olalgal vrw/i;uv
same method as glutamine uptake, except that [ C]-me-
Ammonium
thylamine (Amersham International; specific activity
2.22 GBq mmor')wasaddedtogiveafinalconcentration
of 5 juA/. At intervals. 100-jul samples were filtered onto
Whatman GF/C niters and radioactivity was determined
as described above.
Enzyme assay
About 100 hydra, previously starved for 3 days, were
homogenized in extraction buffer as described by Rees
(1986). The homogenate was centrifuged at 13000 X g
for 5 min at 4C, and the supernatant was assayed for
AMMONIUM METABOLISM IN GREEN HYDRA 213
Table II Table III
EffectofMSXandAZSon uptake(+) orrelease(-)ofammonium hy EffectofMSXanil.1ZSon treeglulamalcandglutaminepools
intactgreenhydraand3NSI3.4 algae ol3X813A algae
Hydra 3N813A
(nmol hydra" h ') (fmol ceir1 h*')
Control +0.192 +0.045 +1.51 0.35
MSX -0.220 0.036 -0.86 0.25
AZS -0.031 +0.006 +0.05 + 0.03
Hydrawerepreincubated in MSX orAZS for24 handwashedtwice
inlargevolumesofgrowthmediumbeforebeingplacedinnewmedium;
3N813A algae(5 X 107cells ml"1) were preincubated for 30 min in 10
nMMES pH 7. Uptakeofammonium wasmeasured bythedifference
in ammonium content ofaliquotstaken immediately afteraddition of
ammonium chloride to give a concentration of50 nM(hydra) or 100
liM(algae)and ofaliquotstaken after 1 h. Figuresarethe means+ SD
ofthree(hydra)orfour(algae)independentexperiments. All treatment
values are significantly different from controls (one-way ANOVA, P
<
0.001).
theseeffectswereconsistentwith inhibition ofGSorGO-
GAT MSX
respectively. significantly decreased glutamine
and AZS significantly decreased glutamate in all treat-
ments. In algaewithouta nitrogen sourceorsupplied with
MSX
glutamine, also significantly decreased glutamate
pools; in algaewithout a nitrogen sourceorsupplied with
ammonium, AZS significantly increased glutamine pools.
Supply ofnitrogen also affected glutamine and gluta-
mate pools. Glutamine increased in algae supplied with
ammonium and glutamine, and glutamate increased in
algae supplied with glutamine (one-way ANOVA. P <
0.05). Tukey post-hoc tests showed that the size of the
glutaminepoolswassimilarin all MSX andall AZStreat-
ments (P > 0.05). The size of the glutamate pools was
also similar in all AZS treatments (P > 0.05), but gluta-
MSX
mate levels were higher in algae treated with in the
presenceofglutaminethan in thepresenceofammonium
or the absence ofa nitrogen source.
Effect ofMSXandAZS on internalpools ofglutamate
andglutamine infreshly isolatedsymbionts and in the
intact symbiosis
The effect of maltose release on the changes the two
inhibitors produce in glutamate and glutamine pools was
tested on freshly isolated symbionts treated at pH 5 and
at pH 7. Maltose release is pH dependent (Cernichiari et
a!.. 1969) and occurs at the former but not the latter pH.
When freshly isolated symbionts were treated with
MSX and AZS at pH 7, the effects were similar to those
seen when 3N813A algaeweretreated without a nitrogen
MSX
source: caused a significant decrease in glutamine
and glutamate; AZS caused a significant decrease in glu-
tamate and an increase in glutamine (Table IV). At pH
214 P. J. McAULEY
Treatment offreshly isolatedsymbiontsatpH 5 caused 0.06 -
a significant reduction in both glutamate and glutamine
pools compared to those at pH 7 (one-way ANOVA, P
<0.05).
Incubation of intact green hydra in MSX caused no
significant change in the glutamate orglutamine pools of
eitherthesymbioticalgae orthe host(Table V). Only 16.2
30.2 SD amol cell ' MSX was detected in algal pools
after 24-h treatment of intact hydra, compared to high
levels in freshly isolated symbionts after only 90 min of
treatment (3126.0 227.6 amol cell ' in freshly isolated
symbionts treated at pH 5; 495.3 175.7 amol cell ' in
freshly isolated symbionts treated at pH 7). Treatment of
intact hydra with AZS increased glutamine pools in both
hydraand algae, although the increase wassignificant only
in hydra. AZS treatment did not cause a reduction in
either host or algal glutamate pools, in contrast to the
consistent effect ofAZS on glutamate pools measured in
both freshly isolated symbionts and 3N813A irrespective
ofpH or nitrogen supply.
Effect ofMSXandAZS on glutamine ami methylamine
uptake by 3N813A algae
u
[ C]-glutamine uptakeby 3N813A algaewascompeti-
tively inhibited by a low concentration ofAZS (Fig. 1).
KM was 17.3 ^M in the absence ofAZS and 28.6 /uA/ in
the presence of AZS. In both cases, Vmax was 250 amol
glutamine cell ' h '.
Therewaslinearuptakeoftheammonium analog I4C]-
[
methylamine in the presence ofboth MSX and AZS, al-
though uptake rates were reduced by 25.1% and 33.4%
respectively, in comparison to controls (Fig. 2). In con-
trast, ammonium chloride inhibited methylamine uptake
Table V
Effect ofinciihatmxhyilra in MSXorA7Sonfrees-lulamateand
glutaminepooh <>thvclra (pmolngprotein'1) andsymbioticalgae
(amolcell'1)
Control
AMMONIUM METABOLISM IN GREEN HYDRA 215
lOO-i 1200-]
a)
1000-
80-
<ou
800-
<u 60-
600-
w
s
"o
E
o
T3
'0
Time (minutes) O
bmueftFfhiegyrulpraeHmi2n.7efboEryff33e0cNt8mo1ifn3MAbSeaXflog,areeA.ZaAdSld,giataeinowdneraoefmpm5ropeniMnicuu['bm4aCto]en-dmueipntth1ay0klemamoMifn[eM14ECt]oS-- 03
gether with either 10pM MSX ( ). 10nM AZS (O O), "o
10nM ammonium chloride (A A), or an equivalent volume of
bufferonlyascontrol(D D)togiveafinaldensity of5 > 107cells
ml'1. At intervals, 100-^1 samples were filtered and radioactivity was
determined.
noeffectonglutamineandglutamatepoolsorontheGin:
Gluratioofthealgae(Table I),thelatterbeingan indicator
ofnitrogen sufficiency in microalgae (Flynn ci til.. 1989).
Inmarinesymbioses. inwhich itisbelievedthatsymbionts
are able to assimilate ammonium from seawater, am-
monium supplementation notonlycauseslarge increases
in the population density ofsymbionts (Muscatine etai,
1989;Hoegh-GuldbergandSmith. 1989; Dubinsky eta!..
1990; Fittand Cook, 1990; Stambler et /.. 1991; Muller-
Parker et ai. 1994). but. in a coral and a hydroid, also
increasesthesize ofinternal glutamine poolsofsymbionts
and hencetheirGln:Glu ratio (McAuley, 1994; McAuley
and Cook, 1994). Time
Further, although MSX inhibitsammonium uptakeby (minutes)
intact hydra (Rees, 1986; McAuley. 1990; Table II. this
paper), almost no MSX was detected in symbiotic algae
MSX
isolated from hydra that had been incubated in for
24 h, and there was no change in algal internal pools of Figure3. Effectofglutamine uptakeon internalpoolsofglutamine
glutamate and glutamine. In contrast, freshly isolated (O O) and glutamate (D D) in freshly isolated symbionts.
asymmobuinotnstsoftrtehaetedinwhiibtihtoMr,SXandrapMidSlXy atcrceuamtumleanttedcaluasregde aiFfnrtee1srhltmyheMissotaMlracttlelodvfastiyhnmeeb'eisxopbneutrfsifem(re1n0att7,ceeigltllhsuetmrlap"m'Hi)ne5we(wara)esoprardpediHendc7utb(oba)tg.eiTdveefnaormc3io0nnucmteienns-
predictable changes in glutamate and glutamine pools of tration of 100/i.U. Samples (200nM) taken at each time point were
freshly isolated symbionts and cultured 3N813A algae in filteredontoGF/Cdisksunderlow vacuum,washedwith20mlice-cold
a variety ofconditions (Tables III and IV). distilledwater, and extracted foraminoacid analysis.
216 P. J. McAULEY
In addition to inhibiting GS, MSX has been shown to differentiallycontrol entryofsubstancesintotheperialgal
be a noncompetitive inhibitor of active ammonium vacuole.
transport in Anahaenajlos-aquae (Turpin et a/.. 1984). Supply ofnitrogen to algal symbionts as glutamine or
In 3N813A, both MSX and AZS caused a degree ofin- otheramino acids rather than asammonium may be im-
hibition of uptake of methylamine (Fig. 2). a structural portant because a supply ofcarbon is required forassimi-
analog ofammonium that istaken up by the same trans- lation of ammonium into amino acids (Turpin, 1991),
port system (Hackette et a/.. 1970; Pelley and Bannister, and symbionts release a high proportion offixed carbon
1979; Smith, 1982; Wright and Syrett, 1983). However, to their host in the form of maltose (Mews, 1980). In
ifboth MSX and AZS caused noncompetitive inhibition cultured 3N813A, maltose synthesis and release inhibits
of entry of ammonium into symbionts, release of am- ammonium uptake because it diverts photosynthetically
monium from the intact symbiosis would presumably be fixedcarbon from assimilation ofammonium intoamino
the same in both treatments. Instead, release was seven acids (McAuley, 1992). Maltose release is stimulated by
times higher in hydra treated with MSX than in hydra low pH, and ammonium uptake by freshly isolated sym-
treated with AZS (Table II). As shown in 3N813A cells, bionts and cultured maltose-releasing algae falls as pH is
MSX blocks assimilation ofammonium into glutamine reduced and maltose release increases (Rees, 1989). Below
via GS (Ranzio et al., 1969; Meister, 1974), leading to acritical pH value, thealgaeactually begin to releaseam-
lbloowckgslustuabmsieqnueenptoomlestaabnodlirsemleoasfeglouftaammimneontoiuglmu.tamAaZtSe tmhoenTiuCmA,cyncolteotnolymabletcoasueserelfeiaxseed,cbaurtbaolnsoisbdeicvaeursteedamfirnomo
via GOGAT (Wallsgrove et al.. 1977; Elrifi and Turpin, acids may be deaminated to provide carbon skeletons for
mm1a9o8tr6ee),paolmoelmsa,odinannigdutmnooawachcmeumnmoutlnraeitautiemodnrweoilftehagsleMu.StTaXhmaittnheha,yndlwroiawtrhgellAueZtaasS-e amsaslGitimoviseleantsityohnantthahersieigshin(rcaRoteemespsa,otfi1mb9al9le0t,)o.saemirenloeasaeciadnsdmaamympornoiviudme
isconsistentwith inhibition ofGSactivitypathways rather an alternative source of nitrogen for algae in symbiosis
than with noncompetitive inhibition of ammonium with green hydra. Freshly isolated symbionts have active
transport into the algae. Because very little MSX was de- ainmithneoianctiadcttrsaynmsbpoirotsissysctaenmsas(sMicmAiulalteey,a 1v9a8ri6ebt)y,oafndamalignaoe
tected in symbionts after treatment of intact hydra, the
ammonium release observed during MSX treatment of acids supplied by host feeding (McAuley, 1987b, 1988,
1991). Further, the rate ofglutamine uptake by freshly
the intact symbiosis must have been due to inhibition of isolated symbionts peaks between pH 4 and 5, the same
hosNtoGSG.OGAT was detected in crude, alga-free extracts range over which release of maltose is maximum (Mc-
Auley, 1991). Maltose-releasing symbionts using gluta-
ohfydhroamowgietnhiAzZeSd,graeneninhhyidbriat.orHoofweGvOerG,AtTr,eatpmreenvtenotfeidntuapc-t mininteheasfoarnmitorfog2e-noxsoogulructearwaotueldtorperquoidruecceatrwboonmsokleelceutloenss
take ofammonium from the medium by the intact sym- ofglutamate from each molecule ofglutamine. But even
biosis and caused a significant increase in host glutamine ifone molecule ofglutamate wasdeaminated, thecarbon
pools (Tables II and V). This may be explained ifgluta- recycled to 2-oxoglutarate, and the ammonium released
mine resulting from activity ofhost GS was taken up by to be reassimilated by the host, the algae would still gain
thealgae and converted into glutamate viaGOGAT.MUSpX- a new molecule of glutamate for each molecule of glu-
take ofglutamine by algae would also explain why tamine taken up. Short-term experiments showed that
treatment did not cause an increase in host glutamine uptake ofglutamine by freshly isolated symbiontsat both
pools. pH 5 (with maltose release) and pH 7 (without maltose
AZS treatment may have affected this coupling in two release)causedan increaseinthesizeofinternal glutamate
ways. First, AZS may have specifically inhibited algal pools (Fig. 3). Although the increase in glutamate pools
GOGAT, although AZStreatmentoftheintactsymbiosis was similar, it should be noted that the rate ofglutamine
did not cause the reduction in glutamate and increase in uptake by freshly isolated symbiontsis more than 5 times
glutamine consistently observed in 3N813A or in freshly higher at pH 5 than at pH 7 (McAuley. 1991 ).
isolated symbionts. Second, AZS, which competitively This scheme is qualitatively but not quantitatively dif-
inhibited uptake ofglutamine in 3N813A algae (Fig. 1), ferent from that in marine algal-invertebrate symbioses.
preventedentryofglutamine intoeitherthe perialgal vac- in which symbiotic zooxanthellae appear to assimilate
uoleorthe symbiotic algae themselves. In eithercase, the ammonium via coupled GS/GOGAT (for review, see
glutamine transport system must be specific forglutamine Spencer-Davies, 1992). In green hydra, although both GS
and AZS but not MSX. Given that symbionts accumu- and GOGAT are involved in ammonium assimilation,
lated MSX in vitro but not /// hox/iice. it is possible that they may be located in different compartments, and the
transport systemsin the host perialgal vacuolarmembrane necessity for glutamine to traverse the perialgal vacuole
AMMONIUM METABOLISM IN GREEN HYDRA 217
would produce a point at which nitrogen supply to the Hackette, S. L., G. E. Skye, C. Burton, and I. H. Segel.
algae could be regulated. m19e7n0t.ousChfaurnagcitewriitshatmieotnhoyflaanmmaomnmiounmi-uMmCtarsatnhseposrutbsstyrsattee.m/inBifiolla.-
Chcm 245:4241-4250.
Acknowledgments Hoegh-Guldberg,O.,andG.J.Smith. 1989. Influenceofthepopulation
density ofzooxanthellae and supply ofammonium on the biomass
This workwassupported bygrants from the Royal So- and metabolic characteristics ofthe reefcorals Serialopora hy.\lri.\
ciety and the Natural Environmental Research Council. andSly/opiumpistillata. Mar. Ecol. Prog. Ser. 57: 173-186.
My thanks to Mr. Harry Hodge for his help in HPLC Kawaguti, S. 1953. Ammonium metabolism ofthe reefcorals. Biol.
analysis ofamino acids, and toan anonymous referee for J. Okayama Univ. 1: 171-176.
helpful comments on a draft ofthis paper. Kessvloenr,SEe.k,uVnVd.aeLra-nCgaerro,tiI.noLiuddewnigb,eianSdtiHc.ks\to\fifemcahnmgaelnnu.n1d96H3y.drogBeinladsuen-g
ActivataetalstaxonomischeMerkmaleinderGattungChlorclla. Pp.
Literature Cited 7-20 in Studies <i Microalgae und Photosynthetic Bacteria, Jpn.
Soc. Plant Physiol., University ofTokyo Press.Tokyo.
Bhandari,B.,andD.J.D.Nicholas.1981. Somepropertiesofglutamine McAuley, P. J. 1986a. Isolation ofviable uncontaminated Chlorclla
synthetase from the nitrifying bacterium. Nitrasomnnas eurupai'u. from green hydra. Limnol. Oceanogr. 31: 222-224.
Aust. J. Biol Sci. 34: 527-539. McAuley, P.J. 1986b. Uptakeofaminoacidsbyculturedandfreshly
Bradford, M. M. 1976. A rapid sensitive method forthequantitation isolatedsymbiotic Chlorellu. AVirPhylol. 104: 415-427.
ofmicrogram quantitiesofprotein utilizingthe principle ofprotein McAuley, P.J. I987a. Nitrogenlimitationandamino-acidmetabolism
dyebinding.Analyl. Biochem. 72: 248-254. ofChlorcllasymbioticwithgreen hydra. Planta 171: 532-538.
Gates, N., and J. J. A. McLaughlin. 1976. Differences in ammonia McAuley, P. J. 1987b. Quantitative estimation ofmovement ofan
metabolism in symbiotic and aposymbiotic Ctindylactus and Cas- amino acid from host to Chlorclla symbionts in green hydra. Biol.
siopeaspp. J. Exp. Mar. Biol. Ecol. 21: 1-5. Bull 173: 504-512.
Catmull,J., D. Yellowlees,and D.J. Miller. 1987. NADP-dependent McAuley, P. J. 1988. Uptake ofleucine by CMorella symbionts of
glutamate dehydrogenase from Acroporaformosa: purification and green hydra. Proc. R Soc Lorn/ B234: 319-332.
properties. Mar Biol. 95: 559-563. McAuley, P.J. 1990. Uptakeofammonium bygreen hydra. Proc. R.
Cernichiari,E.,L.Muscatine,andD.C.Smith. 1969. Maltoseexcretion Soc. Lond. B242:45-50.
bythe symbioticalgae ofHydra viridis. Proc. R Soc Lund. B 173: McAuley, P.J. 1991. Aminoacidsasa nitrogen source forChlorella
557-576. symbiotic withgreen hydra. Hydrobiologica 216/217: 369-376.
Cook, C. B., and C. F. D'Elia. 1987. Are natural populations ofzoo- McAuley, P. J. 1992. The effect of maltose release on growth and
xanthellaeevernutrient-limited?Symhiosis4: 199-212. nitrogen metabolism ofsymbioticChlorella. Br. Phycol. J 27:417-
Cook,C. B.,C. F. D'Elia, and G. Muller-Parker. 1988. Host feeding 422.
andnutrientsufficiencyforzooxanthellaeintheseaanemoneAiptasia McAuley, P. J. 1994. Amino acid content ofzooxanthellae freshly
pallida. Mar. Biol. 98: 253-262. isolated from Pocillopora damicornis. Pac. Sci. 48: 247-253.
David, C. N. 1973. A quantitative method for maceration ofhydra McAuley, P. J., and C. B. Cook. 1994. Effects of host feeding and
tissue, Wilhelm RoiixArch. EnlwMech. Org 171: 259-268. dissolvedammoniumoncelldivisionandnitrogenstatusofzooxan-
D'Elia, C. F., and C. B. Cook. 1988. Methylamine uptake by zoo- thellaeinthehydroidMvrionemaamboinense. Mar. Biol. 121:343-
xanthellae-invertebratesymbioses:insightsintohostammoniumen- 348.
vironmentand nutrition. Limnol. Oceanogr 35: 1153-1165. Meister,A. 1974. Glutaminesynthetaseinmammals. Pp.699-754in
D'Elia,C. F.,S. L. Domotor, and K. L.Webb. 1983. Nutrient uptake The Eii:yiuc.s. Vol. 10. 3rd ed.. P. P. Boyer, ed. Academic Press.
kineticsoffreshly isolatedzooxanthellae. Mar. Biol 75: 157-169. NewYork.
Dubicranetssioknuyer,,ceaZsn.,donNP..thGSe.taoFmpabtllikceaorlw,spkMrio..peB1re9tni9-e0sZ.iaonnTd,hepL.heoftRf.oesctyMncotCfhleeotxsitkceerenyfa,flicLni.uetnMrciuyesno-tf Mewp3so7,r7t-L4o.0f11c.9a8r0b.ohyTdrhaetegrfereonmhayldgraatsoyamnbiimoasli.s.PrIIoIc..TRheSboci.otLroapnhdicBtr2a0n9s:-
Stylophorapistillata. Proc. R Soc Loud. B239: 231-246.
Elrifi, I. R.,and D. H.Turpin. 1986. Nitrateandammonium induced Miflin, B. J., and P. J. Lea. 1976. Review: the pathway ofnitrogen
photosyntheticsuppressionin N-limitedSelenastrummimttiim. Plan/ assimilation in plants. Phytochcmistry 15: 873-885.
Physio/. 81: 273-279. Miller, D.J., and D. Yellowlees. 1989. Inorganic nitrogen uptake by
Falkowski, P. G., Z. Dubinsky, L. Muscatine, and L. McCloskey. symbiotic marine cnidarians: a critical review. Prnc. R Soc. Lond.
1993. Populationcontrolinsymbioticcorals.BioScience43:606- 5237: 109-125.
611. Muscaline, L.,and H.M. LenhofT. 1965. Symbiosisofhydraandalgae.
Fill, W. K., and C. B. Cook. 1990. Some effects ofhost feeding on I. Effectsofsomeenvironmentalcationsonthegrowthofsymbiotic
growthofzooxanthellaeinthemarinehydroidMvnonemaambionese andaposymbiotic hydra. Biol. Bull. 128: 415-424.
inthelaboratoryandinnature.Pp.281-284inEndocvtohiologvII'. Muscatine, L., and C. F. D'Elia. 1978. The uptake, retention, and
P.Nardon, V. Gianinazzi-Pearson, A. M.Grenier. L. Margulis,and release ofammonium by reefcorals. Limnol. Oceanogr. 23: 725-
D. C. Smith,eds. 1NRA, Paris. 734.
Fitt,VV.K.,T.A.V.Rees,R.D.Braley,J.S.Lucas,and D.Yellowlees. Muscatine, L.,H.Masuda,and R. Burnap. 1979. Ammonium uptake
1993. Nitrogenfluxingiantclams:size-dependencyandrelationship bysymbioticand aposymbioticreefcorals. Bull. Mar. Sci. 29: 572-
tozooxanthellaedensityandclambiomassintheuptakeofdissolved 575.
nitrogen. Mar. Biol 117: 381-386. Muscatine, L., P. G. Falkowski, Z. Dubinsky, P. A. Cook, and L. R.
Flynn,K.J.,D.M.J.Dickson,andO.A.AI-Amoudi. 1989. Theratio McCloskey. 1989. Theeffectofexternal nutrientresourceson the
ofglutamine:glutamateinmicroalgae:abiomarkerforN-statussuit- population dynamicsofzooxanthellae in a reefcoral. Proc. R Soc.
able for useat natural celldensities. J. Plankton Res. 11: 165-170. Lond 236: 311-324.
218 P. J. McAULEY
Muller-Parkcr, G., L. R. McCloskey, O. Hoegh-Guldberg, and P. J. analysis of I5N incorporation by GC-MS methods. Phytochemistry
McAuley. 1994. Responseofanimal-algalbiomassofthecoralPo- 20: 575-578.
cilliipura diiinii<>rni\ l>> seawaterammonium enrichment. Pac. Sci Summons, R. K., T. S. Boag, and K. R. S. Osmond. 1986. The effect
48: 273-283. ofammonium on photosynthesis and the pathway ofammonium
Pelley, J. L., and I. I. Bannister. 1979. Methylamine uptake in the assimilation in Gymnodinium microadriaticum in n/ioand in sym-
greenalgaChlorclla[>yreni>idn\a. .1 Pliycul 15: 110-112. biosis with tridacnid clams and corals. Prot.: R Soc Lontl. Ser. B
Raha, O., L. Dubinsky, V. Achituv, and P. G. Falkowski. 227: 147-159.
1989. Ammonium metabolism in the /ooxanthellatecoral. Sty/o- S/mant-Froclich, A., and M. K. Q. Pilson. 1977. Nitrogen excretion
phorapiMillatu. Proc. R Soc. Loud. B236: 325-337. bycoloniesofthetemperatecoralAstrungiadanacwithandwithout
Ranzio, A. A., VV. B. Rowe, and A. Meister. 1969. Studies on the zooxanthellae. Pnn:. 3rdInt. CoralReefSymp. 1:417-423.
mechanismofinhibitionofglutaminesynthetasebymethioninesul- Turpin, D. 1991. EffectsofinorganicNavailabilityonalgalphotosyn-
foximine. BinclicniistryS: 1066-1075. thesisandcarbon metabolism. / Phycol. 27: 14-20.
ReesT,heT.roAl.eVo.ft1h9e86h.ostTinheamgmreoennihuymdraasssiymmibliaotsiionsaannddiatsmpmoossniibluemreg1-. Turprieng.ulDa.tiIoIn.,bSy.aA.mmF.odine,iaunmdaDn.dT.meCtahnvyilna.mi1n9e84a.ndItnheuwef>fencittroofgMenSasXe
ulatory significance. Pruc. R Sue I.ond B229: 299-314. onammonium transport inAnabaenaflos-aguae. PlumPln'.iiol 74:
Rees, T. A. V. 1989. Thegreen hydra symbiosisand ammonium. II. 701-704.
Ammonium assimilation and release by freshly isolated symbionts \\allsgrove,R.M.,H.Harel,P.J.Lea,andB.J.Millin. 1977. Studies
and cultured algae. Pruc R Sue l.ond B 235: 365-382. onglutamatesynthase from leavesofhigherplants. ,/ A'.v/i Bui 28:
588-546.
Rees,T. A. V. 1990. Thegreen hydrasymbiosisand ammonium. III.
sChyamrbaicotteircissttircasinofofaCmlimloorncliluamPrroecleaRseSboycaLohiugdh Bmal2-t1o2s:e-2r4e9l-e2a5s3i.ng \\ildSkioescrssoolLnvo,euddF..iPn#.o,2rg2aan1nd:icI7..1n-iM8tu6rso.cguelninbey.a19s8y4m.biotUipctaskeea aanndemaosnseim.ilPartoico.nRof
Smith, F. A. 1982. Transport ofmethylammonium and ammonium
\\ilkerson,F.P.,and R.K.Trench. 1986. Uptakeofdissolvedinorganic
ionsby Llodcndcn\a J L.\p But 33:221-232. nitrogenbythesymbioticclam TridacnagigusandthecoralAcropora
Spencer-Daues, P. 1992. Endosymbiosis in marine cnidanans. Pp. sp. Mar. Biol 93: 237-246.
51 1-540in Plant-AnimalInteraction^ inilicMarineBenthos, D. M. XYright,S.A.,and P.J.Syrett. 1983. Theuptakeofmethylammonium
John. S. J. Hawkins, and J. H. Price, eds, Systematics Association anddimethylammonium bythediatom.PhaeodaclylumInconnttiiin.
Special Volume No. 46. Clarendon Press, Oxford. NewPhytol. 95: 189-202.
Stambler, IN., N. Popper, Z. Dubinsky, and J. Stimson. 1991. Effects \ellowlees, D., T. A. V. Rees, and \V. K. Fitt. 1994. Effect ofam-
ofnutrient enrichment and water motion on the coral Pocilloporu monium-supplemented seawater on glutamine synthetase and glu-
damn-onus I'm Sci 45: 299-307. tamate dehydrogenase activities in host tissue and zooxanthellae of
Summons, R. K., and C. B. Osmond. 1981. Nitrogen assimilation in Pocillopura<.Uinucrni\andonammonium uptakeratesofthezoox-
the symbiotic marine alga Gymnodinium microadrialicwn direct anthellae. Pai: Sci 48: 291-295.