A Metamorphic Inducer in the Opisthobranch Haminaea callidegenita: Partial Purification and Biological Activity

10 Pages·1994·4.6 MB·English

by  G D Gibson

Reference: Bio/. Bull. 187: 133-142. (October, 1994)
A
Metamorphic Inducer in the Opisthobranch
Haminaea Partial Purification
callidegenita:
and
Biological Activity
GLENYS D. GIBSON1 AND FU-SHIANG CHIA2
ZoologyDepartment, University ofAlberta. Edmonton, Alberta, Canada T6G 2E9
Abstract. LarvaeofHaminaeacallidegenita (Mollusca: physical, that identifies a specific habitat as suitable for
Cephalaspidea) were induced to metamorphose by a juvenile or adult existence (reviewed by Pawlik, 1992).
compound found in the gelatinous matrix composing This cue, or metamorphic inducer, triggers a radical
most ofthe egg mass. A functionally similar compound transformation of the morphology, physiology, ecology,
isolated from adult tissue also induced metamorphosis in and behavior ofthe larva to thejuvenile stage. Chemical
H. callidegenitalarvae. Opisthobranchsare frequently in- inducers are water-borne or tactile, and originate from
duced to metamorphose by a specific prey item ora sub- conspecifics, prey species, or a characteristic micro- or
strate characteristic of the adult habitat, but this is the macrobiotic substrate (bacterial film or an algal, inver-
first known instance of metamorphosis occurring in re- tebrate, or vertebrate species; see Pawlik, 1992). For ex-
sponse to a compound produced by adult conspecifics. ample, most progress in the isolation ofmetamorphic in-
The inducer was purified from egg mass jelly (EMJ) by ducers in opisthobranchs has involved two nudibranchs,
high pressure liquid chromatography (HPLC) and was both ofwhich are induced to metamorphose by a highly
found to be smaller than 1000 Da. polar, non-protein- specific adult prey species. Larvae of Phestilla sibogae
aceous, and very stable. We isolated acompound ofiden- metamorphose in response to a small, water-soluble mol-
tical activity from egg masses produced by four otherop- ecule derived from the coral Ponies (Hadfield, 1977;
isthobranchspecies, suggestingthatthesameorchemically Hadfield and Scheuer. 1985). and those of Eiibranchus
similar compounds are intrinsic to opisthobranch egg doriaerespondtoasmall, polarcompoundwithglucosidic
masses. However, only H. callidegenita larvae metamor- residues originating from the hydroid Kirchenpaueria
phosed in responsetoEMJ. Competent larvaeoffiveother (Bahamondes-Rojas, 1988; Bahamondes-Rojas and
mollusc species did not respond to the partially purified Dherbomez, 1990). Despite many attempts to purify
EMJ inducer but did respond to a specific substrate as- metamorphic inducers in marine invertebrates in general,
sociated with each species. The presence ofthe inducer such inducers have been identified for only a few species
within the egg mass causes an unusual developmental and include a variety ofchemical groups (see review by
pattern in H. callidegenita, a poecilogonous species that Pawlik, 1992; also Kitamura et a!.. 1993).
produces both swimming veliger and crawling juvenile TheopisthobranchHaminaeacallidegenita(Haminaea
offspring. = Haminoea [cf. Giannuzzi-Savelli andGentry, 1990]) is
induced to metamorphose by a compound found in the
Introduction
gelatinous layerscomposing most oftheegg mass(Gibson
Larvae ofmost benthic marine invertebrate speciesare andChia. 1989a). An unusual lifehistory resultsin which
induced to metamorphose by a cue. either chemical or halfthe siblings per egg mass metamorphose in response
toeggmassjelly(EMJ; the metamorphic inducer)to hatch
Received 31 May 1994;accepted 4August 1994. as crawlingjuveniles. The other halfhatch as swimming
Res1eParrecshenGtrAodudpr,es9s5:00ScOriiplpmsaInnsDtriitvuet.ionLaofJoOlclae.anCoAgr9a2p0h9y3,-0M2a1r8i.ne Life veligers and, during a two-week planktonic period, grad-
2Present Address: Hong Kong Institute ofScience and Technology, uallybecome competent to metamorphose in responseto
ClearWater Bay. Kowloon. HongKong. EMJ as well as to other substrates indicative ofajuvenile
133
134 G. D. GIBSON AND F.-S. CHIA
food source (Gibson, 1993). Only EMJ induces intracap- inducer (defined below) at 17C. About 15 to 20 larvae
sular metamorphosis; veligersdo notbecome sensitive to were placed in each well, depending on the size oftheegg
theothersubstrates until alV; latching(Gibson and Chia, mass usedand the numberofwellsrequired fortheassay.
1989a). Theecological < < > .,uence ofthisdevelopmental At 24-h intervals, new hatchlings were counted and re-
pattern isthat each r ;it simultaneously produces both moved under a Wild M-5 microscope. Hatchlings were
dispersive (lecithotrophic veliger) and nondispersive (ju- removed daily so that estimates would not include post-
venile) offspring. Haichlingtypedepends on thepresence hatching metamorphosis (either spontaneous or in re-
ofEMJ before hatching and the time at which metamor- sponse to the tested inducer), which begins within 3 days
phic competence occurs (before or after hatching). ofhatching(Gibson, 1993). Hatchlingswereimmediately
In this paper, we characterize the metamorphic inducer scored as veligers orjuveniles. Veligers were identified by
found in EMJ and describe purification techniques. The the presence ofan intact, ciliated velum and were either
inducer was smaller than 1000 Da in molecular weight, swimming or, less frequently, crawling. Individuals iden-
polar,andstabletotemperatureandacid. Wealsodescribe tified as metamorphosedjuveniles had lost the velarcilia
the ecological distribution ofthe EMJ inducer that influ- and the velar lobes had been resorbed (in many cephal-
ences its activity. A functionally similar compound was aspideans, the shell is retained through the adult stage).
extracted from adult//. callidegenitatissueaswell asfrom Additional morphological changes (shell growth, elon-
the egg masses ofotheropisthobranch species. Metamor- gation of the foot, acquisition offeeding structures and
phic inducing activity ofEMJ was specific to H. callide- onset of feeding) occur within 2-3 days after hatching
genita larvae. We have focused on intracapsular meta- (Gibson and Chia, 1989a). The percentage ofjuveniles
morphosis only, because this is the basis for poecilogony hatching in each well was determined for each day ofthe
in this species; substrates that induce metamorphosis after hatchingperiod and isgenerally presented asacumulative
hatching are described elsewhere (Gibson. 1993). percent foreach clutch by theend ofthe hatching period.
Data are presented as the mean ( standard error) per-
Materials and Methods centage of intracapsular metamorphosis occurring per
treatment; data were arcsin transformed before further
Culture andbioassayprocedures statistical analysis. Sample size refers to the number of
Haminaea callidegenita were collected at Padilla Bay egg masses (= number of clutches) used. Subgroups of
or Spencer Spit and maintained after collection at the larvae from each masswere simultaneously tested foreach
University of Washington, Friday Harbor Laboratories, treatment per assay to allow an inter-clutch comparison
all in Washington State. Egg masses used in these assays ofmetamorphic rates. Thiscomparison wasnecessary be-
werecollected either in the field orwithin 24 h ofnatural cause larvae from different clutches are known to show
spawning by animals maintained in the laboratory. Cul- variable rates of intracapsular metamorphosis (ranging
turetechniquesforadultsaredescribedelsewhere(Gibson, from 4-100% in intact egg masses) arising from a genet-
1993). Gastrulae were separated from the sausage-shaped ically determined time of metamorphic competence
egg mass by removing thejelly layers composing most of (Gibson, 1993).
the mass because thisjelly is known to induce metamor- All assays contained two standard controls: (1) sepa-
phosis within the egg mass (Gibson and Chia, 1989a). rated embryos cultured in fSW to determine the per-
EMJ was removed from developing embryos with fine centage of siblings per clutch undergoing spontaneous
forceps, then the embryos were rinsed in 1-^m filtered metamorphosis in absence of inducer; and (2) embryos
seawater (fSW). Embryos were still contained by an em- cultured with untreated EMJ (about 1 mm3 per well) to
bryonic capsule. Unlike the egg masses of other opis- determine the proportion ofembryos per masscompetent
thobranchs, H. callidegenita egg masses fall apart easily to metamorphose during the assay period. Antibiotics
and all traces of EMJ can be removed. Embryos were were added to culture water to prevent degradation of
cultured in 100 ml of fSW at 17C, and cultures were EMJ orjellyextract(40 mg/1each ofstreptomycin sulfate
cleaned twice weekly. Hatching, defined as the escape of and penicillin G).
larvaeorjuveniles from the capsule, began about 14 days
afterspawning. In intact egg masses, hatchlings must also Characterization andpurification ofthe metamorphic
crawl through thejelly matrix; thus hatching takes slightly inducer in egg massjelly
longer in intact masses (4-5 days; Gibson, 1993) than in EMJ wascollected, drainedofseawater, and stored fro-
mm
separated capsules (maximum of2-3 days). zen at either -9 or -60C. About 1 3 ofuntreated or
About 10 days after oviposition, encapsulated larvae experimentally treated EMJ (wet volume) was added to
were placed in culturescontaining potential metamorphic the indicated assay. In all cases, EMJ was thawed and
inducers. Cultureswere maintained in tissueculture plates thoroughly rinsed, first in distilledwaterand then in fSW,
(Falcon Wells) containing 1 to 2 ml offSW and a single to remove any residual contaminants ofeach treatment.
METAMORPHOSIS IN HAM1NAEA 135
The general stability of the metamorphic inducer in to 100% water over 30 min. Fractions were collected at
EMJ was determined by boiling ( 100C. 20 min) or by 1-min intervals. All fractions were tested for inductive
lyophilizing( 46C, 24 h) manually homogenized EMJ. activity regardless ofthe presence ofa visible peak (UV-
Stability ofinducerin the presence ofacid wasdetermined vis detector set at 254 nm). Solvent was evaporated, the
by acidifying an aqueous EMJ homogenate with acetic residue was resuspended in fSW. and the resuspension
acid (0.1 M. 2 h). EMJ was also treated with one oftwo wasadded to Falcon wellsand assayed as outlined above.
nonspecificproteasestodetermine ifthe inducerwaspro- Two fractions were found to contain activity (min 9-10,
teinaceous. Proteinase K (final concentration 0.1 mg/ml) 17-18). To determine if both peaks represent the same
was added to 5 ml EMJ for90 min at 18-20C. Protease orsimilarcompounds, fractions containing the first peak
XIV from Streptomycesgriseus(Sigma P-5147;finalcon- ofactivity (min 9-10)werecollected forthree runs(300 /ul
centration 1 mg/ml) was added to 20 ml EMJ in 20 ml original crude extract), concentrated in absolute MeOH.
fSW at 17-20C for 12 h. with continual mixing. and repartitioned at the same gradient conditions. Frac-
The molecularweight oftheinducerwasestimated with tions were collected and assayed as described above. In
Amicon diaflo ultrafilters. We used a methanol (MeOH) the final step ofthe purification procedure, thetwo active
EMJ extract, rather than EMJ pieces, in this bioassay to peaks (min 9-10. 17-18) were collected for three runs
prevent clogging ofthe ultrafilters with the large muco- (300jul crude extract), pooled, dissolved in MeOH, and
proteins composing much ofthe gelatinous matrix. Ly- subsequently reinjected through the same column under
ophilized EMJ (15 ml wet volume) was twice extracted isocratic buffer conditions (absolute MeOH). Fractions
(1 h each) with 25 ml MeOH. The combined extract was were collected at intervals corresponding to visible peaks
centrifuged (3000 rpm. 5 min.), the supernatant lyophi- (at 254 nm) on the chromatograph, and both peaks and
lized, resuspended in distilled water, then filtered through inter-peak intervals were assayed as outlined above.
both 5000 Da (Amicon YM5) and 1000 Da (Amicon
YM2) ultrafilters (flow rates of 1 ml/2.5 min.). All solu- Biological distribution ofthe metamorphic inducer
tions were lyophilized and resuspended in 30 ml fSW.
Bioactivitywastested at several stages: ( 1) MeOH residue We examined specific tissues ofadult Haminaea cal-
resuspended in distilled water before centrifugation: (2) lidegenitaforinductiveactivity. Tissuesinvestigatedwere
distilled water suspension before filtration: (3) filtrates: parapodial lobes, anterior and posterior mucous glands
and (4) residue from both YM5 and YM2 filters. ofthe female reproductive system, gametolytic sac, and
The inducer was isolated from EMJ by sequentially digestive gland. Tissue samples from three adults were
extractingthejellywith organicsolventsofincreasingpo- combined, then extracted with MeOH and assayed as
larity (20 ml each), including hexane, diethyl ether. 1:1 outlined above.
diethyl ether: methanol. methanol. and distilled water EMJ offouradditional opisthobranch specieswastested
(Pawlik. 1986). Solvent was added to lyophilized EMJ for ability to induce metamorphosis in H. callidegenita
( 10 ml wet volume) and sonicated for 1 min, followed by veligers. EMJ assayed for inductive potential came from
extraction for 1 h with continual gentle agitation. Solvents the following opisthobranchs: H. callidegenita: H. vesi-
were filtered, dried, resuspended in fSW, and added to citla. a sympatric congener that is morphologically and
each Falconwell (2-ml aliquots). Individual solventswere ecologically similar: Melanochlamys diomedea. a cephal-
also tested (in absence of EMJ) for potential effects by aspidean found in the same habitat; Alderia modesta, an
evaporating an equivalent volume ofsolvent and resus- ascoglossan found in association with the zanthophyte
pending any possible residue in fSW (<.;/.' Pennington and I'aucheria sp. located in the high intertidal zone of the
Hadfield. 1989). samebaysas//. callidegenita:and Onchidorisbilamellata,
The metamorphic inducer was partitioned from the a nudibranch found in rocky intertidal zones in associa-
active fraction (MeOH extract) with HPLC (Spectra- tion with barnacles. We tested both piecesofEMJ aswell
Physics) usingtwobuffersystems(gradient and isocratic). as a MeOH jelly extract for inductive potential on H.
MeOH
Bioactivitywasdetermined forboth crude extract callidegenita larvae. Egg masses ofthe first three species
(50 n\ resuspended in 2 ml fSW) and HPLC fractions werecollected from False Bayand Onchidorisegg masses
pooled over 2-min intervals (corresponding to 50 ^1 crude were collected at Friday Harbor Laboratories, both on
extract/2 ml fSW). Lyophilized EMJ was extracted with San Juan Island. Washington State.
hexane to remove nonpolar compounds: eluant was dis- The metamorphic inducer found in //. callidegenita
carded and EMJ dried. Residual EMJ wasextracted with EMJ was tested on five other species ofmollusc: the four
absolute MeOH as described above. The centrifuged opisthobranchs mentioned above (H. vesicula, Melano-
MeOH
extract was injected (100/ul) onto an Alltech chlamys diomedea, Alderia modesta, Onchidoris bila-
RPC18 column (25 cm X 4.6 mm. 10 /jm particle size) mellata)andthe oysterCrassostreagigas. Planktotrophic
and partitioned on a buffergradient from 100% methanol veligers ofall five species were cultured as by Kempfand
136 G. D. GIBSON AND F.-S. CHIA
Willows(1977), except thatantibioticswere not used and Inducer activity ofEMJ was not destroyed by boiling
larvae were supplied with a 1:1:1 mixture of Isochrysis (49%> intracapsularmetamorphosiswasinduced;Table I),
galhana, Pavlova litiheti. ;;nd Rhodomonas sp. as food. lyophilizing (56%), or acidifying (46%) thejelly. Rates of
Larvae were identified as metamorphically competent intracapsular metamorphosis in these treatments were
when the propodium was well developed and the mantle similartoratesobservedin larvaeculturedwith untreated
was retracted from the shell (Bickell. 1978). These larvae EMJ in the same experiment (67% intracapsular meta-
were placed in 100-mljars (n = 10 larvae/jar) containing morphosis occurred; Table I). The metamorphic inducer
fSW and one ofthe following substrates: EMJ (pieces of passed through ultranlters ofboth 5000 (46% metamor-
jellyoraMeOH extract);a metamorphosis-inducing sub- phosis)and 1000 molecularweightcutoff(36%;Table II),
strateassociated with that species; or 19 mmol Kf in fSW. indicating that the inducer is less than 1000 Da in mo-
The lattertwo treatments were used to ensure that tested lecularweight. Activity wassomewhat lowerin treatments
veligerswere metamorphically competent. Metamorpho- containing ultrafiltrate than in those with untreated EMJ
sis-inducing substrates included diatoms and adult mucus (71%), but activity appeared to have been lost during ex-
for//, vesicula(Gibson and Chia, 1989b), sand containing traction before filtration took place (the resuspended ex-
nematodes for Melanochlamys (Gibson, pers. obs.), the tract induced only 31% metamorphosis; Table II). Induc-
alga I'anc/iena forAlderia (Seeleman, 1933), the barnacle tive potential was also unaffected by the action of two
Chthamalus dalli for Onchidoris (Chia and Koss, 1988), general proteases. EMJ treatedwith proteinase Kinduced
andadultshell forCrassostrea(Crisp, 1967). Allsubstrates 45% intracapsular metamorphosis, and 43% occurred in
were freshly collected from the adult habitat ofeach spe- response to untreated EMJ (in absence ofproteinase; Ta-
cies. Competence was also determined using seawater ble III). Results were similar when EMJ was treated with
containing excess potassium (19 mmol K+), which is pronase XIV, with 53% metamorphosis occurring in re-
known to induce metamorphosis in several phyla ofma- sponse to pronase-treated EMJ and 54% occurring in re-
rine invertebrate (Yool el ai, 1986; Todd el ai, 1991). sponse to untreated EMJ (Table III).
Percent metamorphosis was determined after 48 h. Theonly organicsolventtoextractsignificantamounts
EMJ from masses containing embryos ofdifferent de- of inducer from EMJ was methanol (46% intracapsular
velopmental stages was assayed for inductive activity to metamorphosis; Table IV). Some inducer was retained
test for effects ofegg mass age. EMJ was separated and by the EMJ, as evidenced by subsequent extraction of
pooled from fiveeggmassesateach ofthe followingstages: inducer with distilled water and the small amount ofac-
gastrula; early veliger (showing cephalopedal rudiment); tivity remaining in the EMJ after extraction (Table IV).
mid-veliger(statocysts visible, growth oflarval shell com- The MeOH extract showed peak absorbance at 254 and
plete); late veliger (eyes and heart well developed); and asmallerpeak at 303 nm, aswell asapeak corresponding
posthatch (all hatchingwascompleted). EMJ wasassayed to the absorbance maximum for pure MeOH at 205 nm.
as described above. Data were analyzed with a one-way Metamorphosiswasnot inducedbypotential residuesleft
ANOVA usingthe Scheffeprocedure forunplannedcom- after evaporation ofany ofthe organic solvents used in
parisons among treatments (Day and Quinn, 1989). theseextractions(Table IV). Spontaneous metamorphosis
(in seawateronly, and in absence ofknown cue) occurred
in 2% ofveligerstested, whereas 68% intracapsular meta-
Results
Characterization and isolation ofthe metamorphic Table I
inducerfrom egg massjelly
Characteristicsofthemetamorphicinducerfoundin Haminaea
Haminaea callidegenita larvae were induced to meta- callidegenitaeggmassjelly
morphose within the embryonic capsule byegg massjelly. Treatment Metamorphosis
EmbryosculturedinthepresenceofEMJ metamorphosed
within the embryonic capsule (X SE 52.68 4.49% Seawater
juveniles were released at hatching, /; = 24 cultures) at
rates expected ofintact egg masses (61.32 1.41% juve-
niles, // = 288 egg masses). Most embryos cultured in
the absence ofEMJ hatched as swimming veligers (2.12
1.22% juveniles, n = 24 culturesof 15-20 larvae each).
In assays involving piecesofEMJ,jellywasin the culture
water but not necessarily in contact with developing em-
bryos, indicating that the inducer is a water-soluble com-
pound.
METAMORPHOSIS IN ll.l.\/l\AEA 137
Table II likely because the crude extract retains high levels ofac-
Molecular weight estimatesforthemetamnrphicinduccr/unmlin tivity for at least several weeks (9C).
Hannnaeacallidegenitaeggmassjelly Thecombined active fractions(min 9-10, 17-18)were
re-eluted under isocratic buffer conditions (absolute
Treatment Metamorphosis MeOH). This resulted in a single peak ofinducer eluting
Seawater 6.95 3.27 8 min after injection (peak E in Fig. IB), as well as the
EMJ 71.34 + 1.85 partitioning of several contaminants. Peak E was accu-
Resuspendedextract 31.02 5.87 mulated overseveral runsand thesolvent wasevaporated.
<5000 Da Filtrate 46.53 7.97 A very small amount ofdry residue remained as a fine,
<1000 Da Filtrate 36.45 6.12 white powder that reacted strongly with vanillin, indicat-
Data are the mean ( standard error) percentage of intracapsular ing the presence of steroids, phenols, or fatty acids as
metamorphosis that occurred in response to EMJ extract and extract functionalgroups:alternatively,thestrengthofthevanillin
filtrates,asindicated. Datafromtwocontrolsarealsolistedandinclude reaction may indicate the presence ofa residual contam-
EraMtJes.ofnm=etnaummorbpehrosoifsegogccmuarsrsinegsitnessteeadw.aterandinresponsetountreated pinraenste,ncseucohfaesggaspionlatrheliopriidgitnhaaltemgagymabsesdeerxtirvaecdt.frTohmetihne-
ducer residue also had a slight reaction with ninhydrin,
indicative ofthe presence of primary amines. To detect
morphosis occurred in response to untreated EMJ (Ta- possible contaminants, the pure fraction was re-eluted
ble IV). with HPLC and each fraction scanned with a refractive-
The inducer was partitioned from an EMJ extract in index detector to reveal the presence ofall compounds,
methanol with HPLC over a gradient from absolute regardlessofspecific ultravioletorvisual absorbance. The
MeOH toabsolutewater. Theinducerelutedat 9-1 min resultwasasingle sharppeak, suggestingthat this fraction
(at a buffer concentration ofabout 70% MeOH) and at represents pure inducer, although the presence ofa con-
17-18 min (about 40%> MeOH) after injection (Fig. 1A), taminant eluting precisely with the inducer can not be
with some spreading of activity around the first active eliminated.
peak. Fraction 9-10 induced slightly less intracapsular
metamorphosis (60%) than did EMJ (70%) or the crude Biological distribution
MeOH extract (93%). Fraction 17-18 induced metamor- A metamorphic inducer was present in methanol ex-
phosis at a much lower rate (32%; Fig. 1A). Rates ofin- tractsofseveraladulttissues, with mostactivityoccurring
tracapsular metamorphosis in cultures containing most in extracts of the posterior mucous gland ofthe female
other HPLC fractions were similar to rates in embryos reproductivetract (61% intracapsular metamorphosis oc-
cultured in seawater only. Fraction 9-10 was collected curred), digestive gland (49%), and parapodial lobes (56%-;
and pooled overthree runs, then re-eluted underthe same Fig. 2). All three extracts induced intracapsular meta-
conditionsto determine ifthe inducerwould separate into morphosis at rates comparable to that induced by un-
two fractions. Again, the inducer eluted at two intervals, treated EMJ (48%-). Less activity was evident in extracts
thefirstappearing 7-8 min afterinjection (59.19 3.04% ofthe anterior mucous gland ofthe female reproductive
intracapsular metamorphosis) and the second at 15- tract. Extracts ofthe gametolytic sac showed no activity
18 min (27.48 4.23%). This suggests that the inducer (Fig. 2).
eitherracemizesunderpolarconditionsordegradesduring EMJ produced by other opisthobranch species also in-
the elution procedure. Degradation ofthe inducer is less duced high rates of intracapsular metamorphosis in
Table III
Theeffectsoftwogeneralproteaseson themetamorphicinducerfoundinHaminaeacallidegenitaeggmassjelly
Proteinase K
138 G. D. GIBSON AND F.-S. CHIA
TableIV
Extractionofthemetamorphic loundin Haminaeacallidegenitaeggmassjellyusingsequentialsolventsofincreasingpolarity
A. EXTRACTS
Treatment
METAMORPHOSIS IN HAMl.\.IHA 139
100-1
Source of Metamorphic Indueer
Figure 2. Activity ofadult tissue as a metamorphic indueer. Data
are the percentage ofHaminaea callidegenita veligers that underwent
intracapsular metamorphosis (mean standard error) in response to
seawater(SW), untreated egg massjelly (EMJ; both in hatched bars)as
well as to a methanol extract ofthe indicated adult tissues (solid bars).
n = numberofegg massesassayed.
piiinata (Bahamondes-Rojas. 1988). In this case, the in-
dueer is water soluble and contains galactosidic residues.
Metamorphosis ofE. doriaeisalso induced bym-isomers
Figure 1. Isolation ofmetamorphic indueerwith high performance of various sugars (Bahamondes-Rojas and Dherbomez,
liquidchromatography(HPLC). Dataaretheresultsofpartitioningthe 1990). The EMJ indueer that was active on Haminaea
indueerfrom a crude methanol extract with a buffergradient from ab-
solute methanol toabsolutewater(A)and partitioningthepartially pu-
nned active fractions from A with an isocratic buffer system (absolute
methanol)(B). InbothAandB,thechromatograph indicatesabsorbance
ofeachfractionaselutedfroman RP-C18column,detectedat254nm, 100-,
andthehistogramgivesresultsofthebioassay usedtotesttheinductive
potentialofeachfraction. InA,bioassayresultsarethepercentage(mean
standard error) ofHaminaea callidegenita veligers that underwent
intracapsularmetamorphosisinresponsetoseawater(SW),withuntreated
indueer(EMJ), with a crude EMJ extract (all in hatched bars), as well
as with the indicated HPLC fractions (solid bars) pooled over 2-min
intervalsthroughouta30-minelutionperiod.Peaksofactivityarecross-
referencedtothechromatograph. In B,resultsincludethepercentageof
intracapsularmetamorphosisoccurringinresponsetostandardcontrols
(hatched bars) as well as in response to the fractions indicated in the
chromatograph (solid bars), n = numberofeggmassesassayed.
Isolation ofthe metamorphic indueer has been exten-
sivelystudiedintwospeciesofopisthobranch, but inboth
cases the indueer has yet to be identified. Veligers ofthe
nudibranch Pht'stilla silwgae are induced to metamor- Source of Egg MassJelly
phose by a water-borne compound that is released from Figure 3. Metamorphic inducing activity ofegg massjelly (EMJ)
its prey, the hard coral Porites (Hadfield, 1977). This producedbyotheropisthobranchspecies.Dataarethepercentage(mean
compound is small (200-500 Da) and stable to temper- standarderror)ofHaminaeacallidegenita veligersundergoingintra-
ature and pH (Hadfield and Scheuer, 1985). The nudi- c(saoplsiudlbaarrsm),etoarmtooraphmoestihsanionlrEeMspJonesxetrtaocts(ehaawtactheerd(bSaWrs)),.StoouErcMeJofpiEeMceJs
branch Eitbranchus doriae also metamorphoses in re- is indicated on the horizontal axis, n = numberofH callidegenita egg
sponse to its prey species, the hydroid Kirchenpaiicria massesassayed.
140 G. D. GIBSON AND F.-S. CHIA
LLJ
METAMORPHOSIS IN UAMINAEA 141
suggesting that this compound or group ofsimilar com- to a maternally derived egg mass, as well as the first de-
pounds isintrinsic tothe structure ofthesegelatinousegg scription of an opisthobranch metamorphosing in re-
masses. As such, production of the inducer, unlike the sponse to conspecifics.
release ofa more specific pheromone, would not require
anadditionalexpenditureofenergybytheadult. Response Acknowledgments
of larvae to the inducer may be a consequence of opis- We thank Dennis Willows, Director, Friday Harbor
thobranch veligers being sensitive to cues denoting aju- Laboratories, for kindly providing us with laboratory fa-
venile or adult food source. After hatching, juvenile H. cilities. We also thank the library at Friday Harbor Lab-
callidegenita often graze diatoms and detritus from the oratories. R. Strathmann provided us with Crassostrea
surface ofthe egg mass and appear to ingest EMJ as well, larvae. Doug Bulthius, Research Coordinator, Padilla Bay
before movingto nearby Zostera orChaetomorpha (Gib- National Estuarine Research Reserve and Washington
son, pers. obs.) State Parks, SpencerSpit State Park, generously permitted
Despite the fact that the inducing compound is not us access to collection sites. This paper benefited from
uniquely derived in //. callidegenita, itseffectsasa meta- the comments ofL. Barlow. P. Fankboner. J. Holmes, A.
morphic inducerare restricted tothis species. Competent Kohn, A. Spencer, and two reviewers. Much thanks goes
veligers of five other molluscan species did not meta- to R. Andersen for his advice on isolation protocols for
morphosein responsetothe metamorphic inducerin EMJ marine natural products. This research was funded by an
in a partially purifiedcondition. This is not surprising for NSERC grant to FSC.
stenophagous species (in this study, Oncludoris and Al-
deriahaverestrictedtrophicrequirements)astheselarvae Literature Cited
would be expected to metamorphose only on a highly
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