Metabolism of Gemmules From the Freshwater Sponge Eunapius fragilis During Diapause and Post-Diapause States

8 Pages·1996·3.1 MB·English

by  S H Loomis

Reference:Biol. Bull 191:385-392.(December, 1996)
Metabolism of Gemmules From the Freshwater
Sponge Eunapiusfragilis During Diapause
and Post-Diapause States
STEPHEN H. LOOMIS1 STEVEN C. HAND: AND PAUL E. FELL1
, ,
1DepartmentofZoology, Connecticut College, NewLondon, Connecticut 06320;
and2DepartmentofEPOBiology, UniversityofColorado, Boulder. Colorado80309-0334
Abstract. Post-diapause gemmules of the freshwater ture, it isunlikelythat levelswereseverely compromised
sponge Eunapius fragilis remained quiescent when duringthediapausecondition.
maintained at 5C. Germination occurred within 48 to
72 h following warming to 20-23C, culminating with Introduction
theemergenceofanewspongefromthecollagenouscap- Gemmules ofthe sponge Eunapiusfragilisare asexu-
sule. Both heat dissipation and oxygen consumption ally produced reproductive bodies composed of un-
climbed steadily during germination and eventually differentiatedcellssurroundedbyacomplexcollagenous
reached 600% ofthe startingvalues. By comparison, en- capsule. Such bodies serve as the overwintering stage in
ergyflowwasmuchloweroverthesameperiodoftimein the life cycle of this sponge. The possession of resting
diapausing gemmules, clearly demonstrating metabolic stages in the life cycles of invertebrates is common for
depression during diapause. The calorimetricrespiro- organismsinhabitinginconsistentorephemeralenviron-
mmeotlriOc2(tCoR)-5r4a1tiokJi/ncmroelasOed2 bseigtnwiefeicnanhtoluyrfsro3.m5-a3n5d456k.J5/ mNeenwtlsyafnodrmisedwigdeesmpmruelaedsphayrleoginenaentiocballilgyat(eHasntdat,e 1o9f9d1e)-.
ofgermination, with an average value across this period velopmental (and potentially metabolic) arrest that is
ofabout -495 kJ/mo!O2. ThelowCR ratioathour 12.5 termed diapause. Vernalization in the cold for 2-
(-374 21; 1 SE, H = 3) was statistically below the 3 months releases the gemmules from diapause and al-
oxycaloric equivalent, which suggests that gemmules lows them to resume development when warmed to
may have experienced hypoxia during the more than 20-23C.
3 monthsofstorage at 5Cpriortoexperiments. The in- The molecular mechanisms ofthis diapause breakage
crease in metabolism during germination could be areunexplained. E.fragilisiswell-suited forusein inves-
blocked by perfusing the gemmules with nitrogen-satu- tigating these mechanisms because diapause in its gem-
rated medium (nominally oxygen free). Developing mules is easily manipulated and is quite distinct from
gemmuleswereabletosurviveoxygen limitationforsev- quiescence (Fell, 1987), a metabolically arrested condi-
eral hours at least; during that time energy flow was de- tion promoteddirectlybyenvironmental insult. In other
pressed to 6% ofnormoxic values. Duringgermination, sponges,thesetwostatesareapparentlyintertwined(e.g.,
the range ofvalues was 3.5 to 4.0 nmol/mg protein for Spongilla lacustris, Simpson and Rodan, 1976), and it
ATP, 0.2 to 0.4 nmol/mg protein for ADP, and 0.5 to can be quite difficult to distinguish whether or not gem-
0.8 nmol/mg protein for AMP. Because ATP was high mules are in diapause orwhetherthe state has been bro-
even before gemmules were warmed to room tempera- ken. Other species apparently do not enter diapause at
all and exhibit only quiescence (Ephydatia fhiviati/is.
Received 10October 1995;accepted27September 1996. Rasmont, 1963, 1965).
385
386 S. H. LOOMIS ET AL.
4C
Theecological importance ofdormant states hasbeen ber of 1993 and maintained at until early October
emphasized by a number ofauthors (e.g.. Clutter, 1978; when calorimetry was performed (storage for 2 weeksat
4C
Crowe and Clegg, 1973; Danks, 1987; Elgmork, 1980; isinsufficienttimetobreakdiapause). Post-diapause
Hand, 1991;Handand Hardewig, 1996; Hairston, 1987; gemmules were collected from the same location in
King, 1980; Lees, 1955; Pourriot and Snell, 1983; February of 1993 and maintained for an additional
Tauber et a/., 1986). A recent example ofthe ecological 3 months in the laboratory at 4C priorto metabolic ex-
relevance ofresting stages comes from observations on periments. The hatching percentage of post-diapause
copepods and rotifers, where sediment cores from ma- gemmuleswasalwaysgreaterthan 95%.
rine environments have uncovered viable embryos that
have apparently withstood severe hypoxia or anoxia for Closed-system respirometry
10-40 years; these forms are now thought to serve as
"eggbanks" forfuturegenerations(Marcuselai, 1994). Experiments were initiated by filtering vernalized
A report on diapausing eggs from freshwater copepods gemmules from the 4C storage water and placing them
indicate that even longer survival times are possible into 20C stream water that was continuously bubbled
(Hairston eta/., 1995). with air. Respiratory measurementswere madeat the in-
Without metabolic energy conservation, survivorship dicated time intervals by transferring samples of gem-
during dormancy would be greatly reduced. General mules to a sealed respiration vessel containing air-satu-
principles governing metabolic arrest are beginning to rated stream water. Partial pressure ofoxygen was mea-
emerge(forreviews see Guppy et a/.. 1994;Hand, 1991, sured using a model 5300 Yellow Springs Instruments
1993a, b; Hand and Hardewig, 1996; Hochachka and biological oxygen monitor. Gemmules were continu-
Guppy, 1987; Storey and Storey, 1990). Recent physio- ously stirred through the 15-min measurement interval.
logical studieshave investigated cuesassociated with the Therateofoxygenconsumptionwaslineardown to40%
termination ofdiapause (Drinkwater and Clegg, 1991; saturation, and during these experiments the oxygen
Drinkwaterand Crowe, 1987, 1991; Lavens and Sorgel- level neverdropped below60% saturation.
oos, 1987; van der Linden el ai, 1988) and have docu-
mented the metabolic transitions associated with this Flow-throughcalorimetryandrespirometry
state (Clegg et ai, 1996). In this study, we wanted to es-
tablish a quantitative metabolic framework from which Heat dissipation and oxygen consumption of gem-
to ultimately investigate molecular mechanisms of mulesweremeasuredsimultaneouslywithan LKB2277
diapause breakage. We employed microcalorimetry thermal activity monitor coupled via stainless steel tub-
(Gnaiger, 1983a; Hand and Gnaiger, 1988) to assess ing to a twin-flow respirometer (Cyclobios, Innsbruck,
overall energy flow in diapausing and post-diapause Austria) (Gnaiger. 1983a). All experiments were per-
gemmules. Oxygen consumption was measured both si- formed at 21C. At time zero, gemmules were filtered
4C
multaneously and independently of the calorimetric from the stream water, transferred to the calorime-
measurements. Finally, we measured ATP, ADP, and ter, and perfused at 15ml/h with 21C air-saturated
AMP in post-diapausegemmulestogain insight intothe stream water (<10mOsm/kg H:O; 0.22 /um filtered).
adenylate statusduringdevelopment. The partial pressure ofoxygen wasabove60%airsatura-
In the accompanying paper (Loomis et ai, 1996), we tion on averagein theexcurrent flowafterpassageby the
address changes in carbohydrate levels during germina- gemmulesample. Onedatapointwassampledevery60 s
tion and potential enzymatic mechanisms for modulat- and recorded with Datgrafacquisition software (Cyclo-
ing these changes. Results of these studies could have bios; Innsbruck, Austria). Aftereach run, the gemmules
general implications for organisms or tissues in which were removed and dried at 60C to constant weight.
acute modulation ofdevelopment and energy metabo- Baselineswere measuredbeforeandaftereach run, with-
lismareknowntooccur. From anecological perspective, outgemmules in the perfusion chamber. Forthe experi-
theongoingwork mayeventuallyprovideabetterunder- ment involving a bout of oxygen limitation, post-dia-
standingofhow environmental stimuli, ones promoting pause gemmules were given aerobic perfusion for 21 h,
entryintoandexit from dormancy,arelinkedtothereq- and then the perfusion was switched to nitrogen-satu-
uisite physiological responsesofinvertebrates. rated stream water for 7.5 h. After the bout of oxygen
limitation,gemmuleswerereturnedtoaerobicperfusion
Materialsand Methods to initiate a recovery period. One advantage of flow-
throughcalorimetry forsuchexperimentsistheeaseand
Diapausing gemmules were collected from Stony speed with which oxygen availability can be manipu-
Brook in South Hadley, Massachusetts, in late Septem- lated. Although considerable effort is devoted to main-
METABOLISM OF SPONGE GEMMULES 387
taining a nominally oxygen-free status in the flow- -8.0
through system (for methodological precautions em-
ployed, see Gnaiger 1983a; Hand and Gnaiger, 1988;
Hand, 1990), evidence indicates that a sealed ampoule Post-Diapause(germinating)Gemmules
system, while experimentally cumbersome in terms of
reversibility, may be slightly more effective in providing
an anoxicenvironment (Hand, 1995).
Theinfluenceofantibiotics(50 mg/1 eachofpenicillin
and streptomycin) on the heat dissipation wasevaluated
by exposing gemmules for 5 h to perfusion with nitro-
gen-saturated medium and then switching to the same
nitrogen-saturated medium containing antibiotics. The
slope ofthe iiW-versus-time curve before the switch to
antibiotics was compared to the slope 4 h after the
switch. The slopes were not significantly different (P =
0.343, n = 3, paired, two-tailed / test). Similarly, the ab-
solute jiW values before and after the switch were not
= =
significantly different (P 0.258, n 3, paired, two- 10 20 30 40 50 60
tailed t test). Thus, antibiotics were not included in the Hours at 21 C
waterused forcalorespirometryexperiments.
Figure 1. Calorimetnc measurementsofenergy flow in germinat-
ing and diapausing gemmules ofEunapiusfragilisat 21C. Data for
Adenylatemeasurements germinating gemmules are expressed as means ofthree independent
experiments;barsrepresentonestandarderrorofthemeanatthetime
Post-diapause gemmules were incubated aerobically pointsindicated. Valuesfordiapausingembryosarepresentedfortwo
in stream water at 20-23C. At the indicated time independentexperimentsatthetimeintervalsshown.
points, triplicate samples were extracted with perchloric
acid(Reesand Hand, 1991). Perchloricacid precipitates
were resuspended in 0.5 TV NaOH and assayed for total
protein with a modified Lowry assay (Peterson, 1977). mules at the same temperature. Thus, breakage ofdia-
The neutralized supernatants were analyzed for adenyl- pause apparently results in an increased heat flow. Sec-
ates by high-performance liquid chromatography using ond, the large increase in heat dissipation observed over
weak anion exchange as previously described (Rees and time for the post-diapause condition is absent with the
Hand, 1991). Briefly, the separation column ofamino diapausing embryos. Even though the diapausing em-
propyl silica (250 X 4.6 mm, 5 ^m particle size; Pheno- bryosarepresentedwith optimalconditionsfordevelop-
menex, Torrance, CA)waseluted isocraticallywitha2:1 ment and germination, they remain dormant (no mor-
mixtureofacetonitrileand60 mA/potassium phosphate phological signs of germination were observed). These
buffer (pH 6.5). The potassium phosphate replaced the quantitativedata illustratethedevelopmental and meta-
ammonium bicarbonate used by Reesand Hand (1991) bolic differences between these two states of diapause
in the mobile phase, which avoided the changes in com- and post-diapause.
position caused byslowoutgassingofCO2 from the mix- A similar overall trend is observed when respiration
ture. Adenylates were monitored at 254 nm and identi- rate is plotted as a function ofdevelopmental time (Fig.
fied byco-elution with bona fide standards. 2). As seen in the inset to Figure 2, closed-system respi-
rometry provides results comparable to those obtained
with the open-flow system. Figure 3 depicts the changes
Results CR
in the ratio during development of post-diapause
Heatdissipation andoxygen consumption ofgemmules embryos. The value increases significantly (P = 0.023,
unpaired, two-tailed / test) from -354 46 kJ/mol
A pronounced increase in heat dissipation of5.8-fold ( 1 SE, n = 3) to -541 36 kJ/mol ( 1 SE; n = 4) be-
is observed upon incubating post-diapause gemmules at tween hours 3.5 and 56.5 ofgermination, with an aver-
21 C(Fig. 1). In comparison, twodistinctdifferencesare age value ofabout -495 kJ/mol across this period. For
seen with diapausing gemmules (Fig. 1). First, the abso- comparison, the oxycaloric equivalent for carbohydrate
2lu1teCleisve5l0o%fohfeatthadtismseiapsautiroendaffotrerth3e.5pohsotf-diinacpuabuasteigonema-t c(Gantaaibgoelri,sm19u8n3dbe,rc)a.erTohbeicCcRonvdailtuieonosf-is3-744782k1J/(m1olSOE;:
388 S. H. LOOMIS ET AL
brine shrimp Anemiafranciscana (Hand and Gnaiger,
140 1988; Hand. 1990). Even the biphasic profile seen with
CO thegemmulesduringrecovery (hours 28-30) is virtually
12.0 identical tothat observedwithA.franciscanaembryos.
O)
^ 100
Adenylatelevelsduringgermination
o
80 Considerable variance was noted in the ATP levels of
co gemmulesduringtheearlystagesofthegerminationpro-
'o. cess, as manifested in the large error bars over the first
E 6.0 20 h (Fig. 5). Overall, however, the mean valuesare rela-
O tivelyconsistentacrosstheentireperiod. There isasmall
O 4.0 decrease in ADP at hour42 (Fig. 5) that results in a sig-
c nificant change (P = 0.008) in the ATP:ADP ratiowhen
O0OX>)), 2.0 Diapausing Gemmules tcwoemepnar0e.5datnodho0.u8rnm(oTla/blmegIp).roAteMiPn (lFeivge.ls5)f.lTuchteuactaelcbeu--
0.0 lated adenylate energy charge was statistically un-
10 20 30 40 50 60 changed over the 54 h of aerobic incubation (Table I).
Hours at 21 C Even at the hour time point (i.e., immediately before
Figure 2. Oxygen consumption rales at 2IC. measured usingthe transferring the post-diapause gemmules to room tem-
flow-through respiration system, for post-diapause and diapausmg perature),theadenylatepool isalreadywellcharged. The
gemmules ofEunapiusfragilis during germination. Values for post-
diapause gemmules are means for 3-h intervals (except for a 1.25-h
intervalatthefirsttimepoint),withthesymbolplacedatthemid-point
ofeachtimeinterval. Errorbarsrepresent 1 SEforthreeindependent
experiments, except at hours 50.5 and 56.5, where n = 4. Values for -650
diapausingembryosarepresented fortwoindependent batchesofem-
bryosat the time intervals shown. Inset: For comparison, respiration
rfoartepsomset-adsiuarpeadusuesignegmmcluolseesd;-snys=te1m. respirometryat20Carepresented Eoi -550
im3
8 -450
c
CD
n = 3) determined at hour 12.5 is statistically different Ox) -350
from the oxycaloric equivalent (t, = 4.95). ThislowerC:
R ratio near the beginning of the incubation could be "o
attributed to gemmules that are undergoing recovery -250
from hypoxia or anoxia. Because they were stored for o
monthsat4Cwithoutaeration,itislikelythatthesequi- I -150
escent gemmulescould have experienced oxygen limita-
cr:
tion. d
-50
Toleranceofgemmules to oxygen limitation
10 20 30 40 50 60
Figure 4 illustrates the effect ofinterrupting the aero- Hours at 21 C
bic germination ofpost-diapausegemmules with a 7.5-h
bout ofoxygen limitation. Heat flow is depressed to 6% Figure3. Calonmetncrespirometric(CR)ratiosforpost-diapause
gemmulesofEunapiusfragilisduringgerminationat21 C. Valuesare
ofthe aerobic rate. Upon reperfusion with air-saturated means for 3-h intervals (except for a 1.25-h interval at the first time
medium (at hour 28), heat flow increases, and in due point), with thesymbol placedat the mid-pointofeach time interval.
course, new sponge tissue emerges from the collagenous Errorbarsrepresent 1 SE forthreeindependentexperiments,except
ceaqpusaullet.otEhmeebroguetnocfeniistdroegleanyepderbfyusainoni(ndcarteamneonttsohfowtni)m.e 0ant.if0hi2oc3au:nrtusni5pn0ac.ir5reaeasdne,ditnw5o6C-.Rt5.airwlahetdieor1ebteensttw)=.eeT4n.hetAhneaaslftyiersrsiitssakondfdevnlaaorsttieatsnictmeheesphooonilwnytssva(asPliug=e-
Thus, oxygen limitation appears to halt development in significantlydifferent(I, = 4.95)fromthetheoreticaloxycaloricequiv-
addition to depressing heat dissipation. These observa- alentforcarbohydrate(-478 kJ/molO:).basedoncomparisonofthe
tions are similar to those reported for embryos of the experimental meansandassociatedvariancestotheparametricvalue.
METABOLISM OF SPONGE GEMMULES 389
-6.0
g -5.0
n
a -4.0 NitrogenPerfusion
75h
|.30
g
to
t
-2-0
-1.0
0.0
10 20 30 40 60
Hours at21 C
Figure4. InterruptionofheatdissipationduringgerminationofEunapiusfragilisgemmulesbyperfu-
sion with nitrogen-saturated medium (nominally oxygen free). The periodofoxygen deprivation isindi-
catedonthefigure
observation impliesthat adenylate statusisprobably not and post-diapause (germination). Diapausinggemmules
compromisedbythediapauseperiodpriortoreinitiation exhibit an energy flow only 50% that of post-diapause
ofdifferentiation. Otherwise, one would expect to see a gemmules at the beginningofgermination, and this rate
substantial rise in ATP levels during the early stages of ofheat flow is only 1 1% ofthat seen in post-diapausing
germination. gemmulesatthe pointofcellemergence from thecollag-
enouscapsules. Thus, diapauseischaracterizedbyasub-
Discussion stantial metabolicanddevelopmental suppression. Nev-
ertheless, there is still energy metabolism in this state of
Energy flows in gemmules of the freshwater sponge obligate (constitutive) dormancy when measured at
Eunapius fragilis differ between the states of diapause 2\C. Presumably, the energy flow during diapause
would be depressed furtherby the lowtemperatures nat-
urally experienced during the overwintering state. Gas-
TableI
ATPADPratiosandadenylateenergychargeingemmules
ofEunapiusfragilisduringgermination
Timeatroom
01
E temperature(h)
Hours at Room Temperature
Figure 5. Chemical measurements ofATP, ADP. and AMP ex-
tracted from gemmules ofEunapiusfragilisat various points dunng
germination. Valuesareexpressedasmeans 1 SE(n = 3).
390 S. H. LOOMIS ET AL.
trula-stageembryosofthe brine shrimpArtemiafrancis- Adenylate poolsappeartobewellcharged in post-dia-
canaexhibitan exponential decline in respiration rateas pausegemmuleseven beforewarmingto room tempera-
a function oftime in diapause; after 5 days ofdiapause, ture (hour0). Preliminary data (not shown) suggest that
respiration reached "barely detectable" levels (Clegg el ATP levels are also quite high in diapausing gemmules,
a!.. 1996). Considering that gemmules in this study had andconsequently, severedepletion ofATPis unlikely in
been in diapause for 2 weeks before the metabolic mea- either ofthese two metabolic states. Data are currently
surements, it seems that gemmules ofEunapiusfragilis not available for anoxic gemmules, but our prediction
do not undergo as severe a metabolic depression as that is that ATP levels could be compromised under anoxia,
similar to the situation observed for Artemia embryos,
reported forbrine shrimpembryos.
Respiration rates of diapausing and post-diapause where ATPdrops rapidly duringoxygen limitation (An-
gemmules were compared by Rasmont (1962), who chordoguy and Hand, 1994, 1995; Carpenterand Hand.
found that oxygen consumption increased threefold 1986; HandandGnaiger, 1988; Reese/al.. 1989;Stocco
upon breakage ofdiapause(promoted by coldtreatment etal.. 1972).
Post-diapause gemmules are tolerant of severe hyp-
ofgemmules). These studies were conducted with gem-
mules ofEphydatia muelleri, which apparently do not oxia or anoxia, at least for a period of7.5 h. During ni-
eExuhniabpitiuassfdriasgtiilnicst. aThdeiapcaururseentstaetveidaesndcoe gsuegmgmesutlsesthaotf ttsioronon,geiansftrpeeerrdfu5uc0seihdonotof(n6oox%myigonefanlthldeyepaorexiryvoagbteiinconvf,arelehu)ee,.athIendaitcssodimippsasatiripioa-n-
tshuemrpetiisoanmeotfadbeovleicloapcmtievnattiaofntetrhadticaopianucsiedebsrewiatkhagtehearned- dfrroopmsaptosleta-sdtiaanpaoursdeereomfbmraygonsitoufdeAratnedmiisastifllradneccilsicnainnag
subsequentexposure toelevated temperatureCs.R atthat point (Hand, 1990, 1995;comparewith Hontoria
gOe;Om,vmaeuvrlaetlhsueeoeinfntdiEirusentpiaenprgiiuuiossdhoaffbralggeeirlfimrsionamavtteihroeang,oextdyhcea-l4o9r5irckaetJiq/oumifovol-r desttiastaslei.,,paat1si9o9dn3e)f.iinnIetAdrisbtypeomtsihsaieblaeebmsbterhnayctoessoufrcehpflrleococtwesavsnaelsuaaemssestooacfbiohaletiaectd
a-l4e7nt8 kfJo/rmoalerOo:b.icAsmneottaedboplriesvmiousolfy,chaorwbeovheyrd,ratthee=CoRf wpeirtfhuAsiTonP,tuhrenaotvedri.ssUippaotniornetculrinmobfsgerampmiudllye,satondaergoebmi-c
ratio was significantly lower (-374 21 1 SE; n 3) mules resume development and emerge. Exploration of
at hour 12.5 ofthegermination process(similarly low at biochemical and metabolic features underlying this tol-
hour3.5),an observation thatleads ustosuspectthat the erance to oxygen limitation and the capacity for long-
gemmules were hypoxic prior to the aerobic incubation term survivorship under anoxia is biologically relevant
periodofgermination. Valuessignificantlybelowtheox- because the natural habitat of these gemmules during
ycaloric equivalent have been reported before fororgan- wintercan include burial in pond sedimentsoflow oxy-
Miystmislurseceodvuelriisn,gvaflruoems aasnolxoiaw.asFo-r21th0ekmJa/rmionleOm2uhsasevle gvaetniotnesn)s.ion (S. H. Loomis and P. E. Fell, unpubl. obser-
been observed duringthe first hourofrecovery from an- The results from thisstudy, in conjunction with those
A
oxia (Shick et al, 1986; Shick el nl.. 1988). small por- ofthe companion paper, provide a quantitative frame-
tion ofthe excess oxygen consumed (relative to heat lib- work from which to explore molecular bases for dia-
etrhaetperd)edcoamnibneanatttcraiubusteeodfttohreelooxwygCeRnartaitoinosoifstaipspsuaerse,ntbluyt pmaeutsaeb.olTihcetdraatnsairteiponrsesienntsptohnegfeirsgtecmhmarualcetsertihzaattiaorneocfaptah-e
the occurrence ofanabolic, heat-conserving metabolism ble ofenteringand exitingdiapause.
(Shick et al., 1986; Shick et a!., 1988). The theoretical
oxycaloric equivalent forgluconeogenic succinate clear-
ance is estimated to be about -200 kJ/mol O2, and the Acknowledgments
oxycaloric equivalent for the partial oxidation ofsucci-
nateto malateoraspartateisprojected tobequitelowas This work was supported by NSF grants DCB-
well (reviewedby Shick etal., 1988). Furthersupportfor 9018579 and IBN-9306652 to SCH.
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