Table Of ContentReference: Biol. Bull. 197: .132-340. (December IsW)
Inhibition of Settlement and Metamorphosis of the
Ascidian Herdmania curvata by Non-geniculate
Coralline Algae
BERNARD M. DEGNAN1-* AND CRAIG R. JOHNSON2
1 Department ofZoology and Entomology, University ofQueensland, Brisbane, Qkl 4072. Australia: and
'Zoology Department, University ofTasmania, GPO Box 252-05, Sandy Bay, Tasmania 7001, Australia
Abstract. The surfaces of non-geniculate coralline algae not exposed to NCA metamorphosed at high rates in KCl-
(NCA) are known to induce the settlement and metamor- elevated seawater.
phosis ofdisparate marine taxa. In this study we investigate
the responsiveness of larvae of Herdmania curvata (As-
Introduction
NCA
cidiacea: Stolidobranchia) to three species of (Neo-
goniolithon brassica-florida, Hydrolithon onkodes, and Marine invertebrate larvae traverse a complex chemical
Lithothamnium prolifer) that cohabit the slope and crest of seascape and can encounter factors that promote or deter
Heron Reef, Great Barrier Reef. H. curvata larvae were first settlement and metamorphosis. The unique and sometimes
exposed to these NCA at or within 2 h ofhatching, which is opposite responses ofdifferent larvae to chemicals emanat-
1 to 2 h prior to attaining competence, and then cultured ing from or on the surface of a particular substratum has
continuously with the NCA for 12 to 14 h. Rates of settle- been attributed to differences in larval chemosensory capa-
ment and metamorphosis of H. curvata cultured in labora- bilities (reviewed in Hadfield. 1986; Hadfield and Penning-
tory chambers in the presence of the different NCA were ton. 1990; Morse. 1990. 1993; Pawlik. 1992; Leitz. 1997)
significantly lower than spontaneous rates in seawater. The and developmental states (Trapido-Rosenthal and Morse,
limited settlement in treatments containing NCA were con- 1986; Degnan and Morse. 1995). At different ages, compe-
fined entirely to the chamber periphery, and settlement tent larvae ofa given species can respond differentially to a
never occurred on the surface of the NCA. The inhibitory particular inductive cue. either by settling or metamorphos-
effect was dose-dependent and was stronger in N. brassica- ing at different rates (e.g.. Coon et al., 1990; Degnan et al.,
1995)..
f/orida and H. onkodes than in L. prolifer. Larvae that did
not settle in treatments with NCA had rounded anterior Despite the apparent diversity of larval chemosensory
systems, disparate marine invertebrate taxaoften respond in
trunks and. in extreme cases, kinked tails with rounded and
dissociated tail muscle cells. In some individuals, we ob- a similar manner to particular substrata. Bacterial films on
both biotic and abiotic substrata induce settlement in a wide
served the anterior chemosensory papillae being sloughed range of marine invertebrates (reviewed in Pawlik, 1992;
off the larval body. Morphological analysis of trunk ecto- Johnson et al., 1997), although several species are inhibited
dermal and meseNncChAymal nuclei of larvae cultured in the from settling by bacteria or their products (e.g.. Maki etal.,
presence of the revealed that general necrotic cell 1988. 1992) or show no response to bacteria (e.g., Keough
death was occurring. Importantly, H. cumita larvae that and Ramondi, 1995). In contrast, in most cases the surfaces
were exposed to NCA could not subsequently be induced to of sessile marine invertebrates such as sponges, bryozoans.
metamorphose in KCl-elevated seawater, whereas larvae and ascidians either contain inhibitors or do not provide
morphogenic cues to induce the settlement of larvae (re-
Received 24 February 1999: accepted 22 September IW9. viewed in Davis etal.. 1989; Pawlik, 1992). A finerscale of
Authortowhomcorrespondenceshouldheaddressed- E-mail:bdegnan<" analysis reveals greater complexity in larval/substratum in-
zoology.uq.edu.au teractions. For example, in the case of microbial films.
332
INHIBITION OF ASCID1AN SETTLEMENT 333
larvae of some species respond to a variety of bacterial metamorphosis facilitates the analysis of inhibition ofthese
genera (e.g., Fitt et ul., 1989), whereas others apparently processes.
require particular strains or communities (e.g., Wilson, Here wedemonstrate that all three speciesofNCA inhibit
1955: Kirchman el al.. 1982: see also Johnson and Sutton, settlement and metamorphosis of H. citn'ata in the labora-
1994). Similarly, a particular strain may induce settlement tory. In the presence of the algae, settlement rates were
in some species but not others (Tritar et ai, 1992) or even much less than rates of spontaneous settlement in the ab-
inhibit settlement in some species but promote settlement in senceofthe NCA, andthe limited settlement inthe presence
others (cf. Kirchman et ai. 1982; Maki et ul.. 1990, 1992). of NCA was confined to the periphery ofthe culture cham-
The range ofinteractions between invertebrate larvae and ber and never occurred on the surface of the plant. Mor-
the surface ofnon-geniculate coralline algae (NCA) is sim- phological analysis oflarvae cultured in the presence ofthe
ilar to that exhibited between larvae and microbial films. NCA revealed that the NCA were toxic to the larvae,
NCA induce settlement, metamorphosis, orboth in a variety resulting in necrotic cell death and sloughing ofthe anterior
ofechinoderms (e.g.. Rowley, 1989; Johnson et ul., 1991), chemosensory papillae. This toxic inhibition prevents these
molluscs (e.g., Barnes and Gonor, 1973: Heslinga, 1981; larvae from subseqmueMntly responding to a strong artificial
Rurnrill and Cameron, 1983; Morse and Morse, 1984; Moss inductive cue (40 KCl-elevated seawater; Degnan et
and long. 1992), annelids (e.g.. Gee and Knight-Jones, ul.. 1997). This result contrasts with that observed for some
1962; Gee, 1965), and coelenterates (e.g., Harrigan. 1972; echinoderm larvae, which are readily induced to settle and
Sebens, 1983a, b; Morse et al., 1988), but do not provide a metamorphose on contact with some of these NCA species
morphogenic signal for several species of tubicolous anne- (Johnson et al.. 1991; Johnson and Sutton. 1994).
lids (e.g., De Silva. 1962; Gee and Knight-Jones. 1962;
Jensen and Morse, 1984). Among species whose larvae are Materials and Methods
induced by NCA. the specificity ofthe interaction covers a
spectrum from species that manifest specificity for a partic- Collection, niaintentinee, am! cultivation ofHerdmania
ular species of NCA (Gee and Knight-Jones, 1962; Gee, curvata and coralline algae
1965; Johnson et al., 1991), to those requiring contact with Gravid Herdmania curvata and pieces of the non-genic-
NCA
any of a variety of (Morse et al., 1988), to those ulate coralline algae (NCA) Neogoniolithon brassicu-
requiring a cue from NCA or any of a variety of other fiorida. Hvdrolitlion onkodes. and Lithothamnium prolifer
substrata (Harrigan. 1972; Heslinga. 1981; Harm, 1989). were collected from the reefcrest and slope ofHeron Reef.
In this study we investigate the responsiveness of larvae Great Barrier Reef. Australia (23 27' S; 151 55' E), and
of the tropical solitary ascidian Herdmania curvata (previ- maintained separately in flowing, ambient seawater on site.
ously known as H. mounts "Heron Reef or H. mounts NCAwere collected by chiselling the algae from the surface
forma curvata: see Degnan and Lavin, 1995) to three spe- of coral rubble.
cies of NCA. viz. Neogoniolithon brassica-florida (previ- H. curvata eggs were fertilized by pooling the gametes
ously known as N.foslei), Hydrolitlwn onkodes (previously from at least three individuals into 0.2-jum-filtered seawater
known as Pololithon onkodes). and Lithothamniumprolifer. (FSW), and embryos were cultured in FSW as described in
H. citi-vata and these NCA inhabit the slope and crest of Degnan et al. (1996). Thin ( 1-2 mm) shards of NCA were
reefs along the Great Barrier Reef. Since larval ontogeny prepared by chipping away most of the coral rubble from
and the cellular basis of morphological change are under- underneath the algae. Only shards free ofmacroscopic foul-
stood in solitary ascidians to a greater extent than in most ing organisms were used in the experiments.
marine invertebrates (reviewed in Satoh, 1994), and the
developmental and morphogenetic processes that regulate Settlement experiments
the attainment ofcompetence, settlement, and metamorpho-
sis have been documented in H. citn'ata and other ascidians Three experimental designs were used to examine the
(Cloney, 1982; Torrence and Cloney, 1983; Degnan et ul.. settlement response of H. citn'ata to the NCA: (1) larvae
NCA
1996, 1997; Eri et al., 1999), this tropical ascidian is a were presented with all species of simultaneously (the
useful model for investigating the inductive and inhibitory "choice" experiment); (2) larvae had access to only a single
activities ofvarious substrata. Settlement and metamorpho- species ofNCA (the "nochoice" experiment); and (3) a "no
sis can be induced rapidly in H. citn'ata by a range of choice" design in which a dose-dependent response was
artificial and natural cues, with most competent larvae ini- examined.
tiating metamorphosis within 1 h ofcontact with the induc- Choice experiment. A variable numberofsmall shards of
tive signal (Degnan et al.. 1997). In addition. H. curvata each species ofNCA (total epithallial area ofca. 1.5-3 cnr
larvae spontaneously settle and metamorphose in axenic for each species) were placed in the center of 100-mm-
cultures at a slower rate, such that up to 80% will be settled diameter polycarbonate chambers containing 200 ml of
within 24 h ofhatching. This "spontaneous" settlement and seawater. Shards of each species were arranged so that the
334 B. M. DEGNAN AND C. R. JOHNSON
epithallial surfaces faced both upwards and downwards. the rubble surface, settled on the sides of the chamber,
H. citn-ata embryos were mass cultured in FSW at 24C metamorphosed but not settled, and neither metamorphosed
until hatching, which is about 10 h after fertilization (Deg- nor settled. Larvae that had not yet metamorphosed or
nan et at., 1996); newly hatched larvae require another 3-4 settled in chambers containing large shards of L. prolifer
h of development to attain competence (Degnan et al.. and the smallest shards ofN. brassica-florida and H. onkode
1997). Pre-competent H. cun'ata larvae ( 10-12 h old) were were transferred to 40 mA/ KCl-elevated FSW (see below).
transferred from the mass culture to chambers (mean num-
NCA
ber of larvae cu. 130 per chamber) containing and Artificial induction ofmetamorphosis
incubated at the ambient water temperature (22-24C) for
12-14 h. For controls, larvae were added to chambers To determine whether H. cun-ata larvae were competent
containing seawater only. The experiment was repeated on to metamorphose afterexposure to the NCA, we transferred
consecutive days with different batches of larvae. Since the 20 larvae that had been in the presence of large shards of
results of the two experiments were similar, data were L. prolifer. small shards of H. onkodes or N. hrassica-
combined for presentation, yielding a total of 15 replicate florida, or seawater to new sterile 35-mm polycarbonate
chambers of each of the NCA and control treatments. chambers containing 5 ml of 40 mA/ KCl-elevated FSW;
After incubation, the NCA were removedfrom the cham- this was performed in triplicate for each treatment (i.e., 20
bienrs,pecptleadcedmicirnosacosphiaclallolwy dfoirshsectotnltedainHi.ngcusne'aawtaatero,n atnhde slaarmveaefeXrti3lirzeaptliiocna.teKsC)l,-aenldevaaltleldarFvaSeWweirsenodremriavleldyfaropomtetnhte
epithallial and rubble surfaces. The seawaterin the chamber artificial inducer of metamorphosis (Degnan et al., 1997).
was filtered through a 60-/u.m mesh, and the chamber was Because most larvae that were previously exposed to sea-
gently washed with an additional 100 ml ofseawater, which water had metamorphosed by the time of the transfer, ad-
was also filtered. The chamberand the mesh were inspected ditional cultures were established as described in the above
alanrdvaseco(rie.ed.,folrarlvaarevaet,hastetutnledderpwosetnltarmvaeet.amaonrdpuhnosseitstlebdutponsot-t csoencttriooln tloarevnaseurweertehatal6s0o ltarravnasefewrreerdettoraFnsSfWerrteod.coUnnttrroelatfeodr
settlement). the transfer process (an additional 60 larvae).
After being scored, specimens ofH. cun'ata were either At 1-h intervals after transferral of larvae, each chamber
was scanned to record the number of larvae that were
inspected microscopically, fixed forhistological analysis, or
artificially induced to metamorphose (see below). Light undergoing metamorphosis. H. cun-ata larvae were consid-
microscopy was performed on living individuals with an ered to have initiated metamorphosis when the tail was at
Olympus BH-2 light microscope fitted with Nomarski least 50% resorbed (Degnan et al.. 1996. 1997).
optics; photomicrographs were obtained on an Olympus
C-35AD-2 camera attached to this microscope. Histology anil microscopy
No choice experiment. This experiment was conducted Larvae that had been cultured in seawater or in the
similarly to the choice experiment outlined above except presence of the NCA were fixed in 4% (w/v) paraformal-
tThhaet emaecahnchnaummbbeerrcoofntparien-ecdomonpleyteantsilnagrlveaespeacdideesdoftoNeCaAc.h dEeGhTydAefionr a1t00leamsAt/3Hhepaneds s(tpaHine6.d9)w,it2hm2Am/gM/mglSpOr4o.pi1dmiuMm
chamber was 1 10, and there were 10 replicate chambers of iodide for 1 h as described in Hinman and Degnan (1998).
each treatment containing NCA, and 9 replicate controls Larvae were examined on a BioRad 600 laser confocal
containing seawater but no NCA. scanning microscope, exciting samples with 488-nm laser
Dose-dependency (no choice): To determine the nature of light and monitoring emission at 515 nm.
any dose-dependency in the response to NCA, larvae were
added topolycarbonatetissueculture vessels (35-mmdiameter
Statistics
XlO-mm depth) containing 5 ml of seawater and either a
singlesmall,medium,orlarge(surfaceareaofbaseofchamber Since there was no settlement on the epithallial surface of
respectively 5%-15%, 25<7r-35<7c, and 50%-757r) shard of any NCA in the choice and no choice experiments, settle-
NCA. Each chamber contained a single species ofNCA, and ment rates on the chambersides only were compared among
about 30newly hatched larvae (i.e., 3 h beforedevelopmentof NCA and control treatments with one-way Model I
competence) wereaddedtoeach. Therewere6replicatecham- ANOVA. Similarly, in the dose-dependent experiment,
bers per treatment, to give a total of 3 species NCA X 3 size there was little (ifany) settlement on NCA surfaces; there-
classes X 6 replicates = 54 chambers containing NCA, and 6 fore, total settlement (on NCA and the plastic sides of
=
control chambers containing larvae but no NCA, to yield a chambers) was compared among the different "doses" (
total of60 chambers in the experiment. size classes, 3 levels) and species (3 levels) using Model I
After 12-14 h of incubation, the following categories of two-way ANOVA. For this experiment the proportion of
larvae were scored: settled on NCA (epithallus). settled on larvae neithersettled normetamorphosed and the proportion
INHIBITION OF ASCIDIAN SETTLEMENT 335
metamorphosed but not settled were analyzed in separate in the presence of Lithothamnium prolifer than either of
Model I two-way ANOVAs. All aposteriori multiple range the other species. Larvae showed greatest sensitivity to
tests used Tukey's HSD criteria (P = 0.05). Neogoniolithon brassica-florida, and the inhibitory effect
For all analyses, the relationship between group means increased dramatically when the percentage of the base of
and standard deviations was examined to determine the the settlement chamber that was covered with shards in-
appropriate transformation (if any) to stabilize variances creased from small (5%'-15% coverage) to medium (25%-
(Draperand Smith, 1991 ). Transformations are expressed in 35% coverage). Ofthese treatments, normal metamorphosis
terms of the untransformed variate Y. All analyses were occurred only in H. curvata cultured in the presence of
undertaken using the SAS/STAT software package (version small and medium shards of L. prolifer. Overall, the inhib-
6.12. SAS Institute Inc.. Gary. NC). itory effect on larval settlement was ranked N. brassica-
florida > Hydrolithon onkodes > L. prolifer.
Results
Settlement ant! metamorphosis ofHerdmania curvata are Non-geniculate coralline algae cause sloughing of larval
inhibited by non-geniculate coralline algae sensory papillae and necrotic cell death
Responses of Herdmania cun'ata larvae to NCA were Unlike normal 14-16 h posthatch larvae, which have
NCA
similar in experiments in which larvae had access to all extended papillae, larvae cultured in the presence of
three NCA species simultaneously (choice experiment; Fig. had rounded trunks and lacked papillae (Fig. 3). Normal
1A). and those in which larvae were presented with only a larvae were evident only in the treatment containing small
single species (no choice experiment; Fig. IB). In control and medium shards of L. prolifer (Fig. 2A). Microscopic
NCA
cultures containing seawater only, the majority of larvae inspection of larvae revealed that all three species
(60.8% SE = 4.4% and 51.1% SE = 4.6% in the choice appeared to induce this same effect on larval morphology,
and no choice trials respectively) had settled and metamor- although the amount of NCA required to affect larval mor-
phosed on the bottom of the plastic chamber 11-13 h after phology differed (Fig. 2A). For these reasons, only the
attaining competence ( 14-16 h after hatching; Figs. 1A, effects of L. prolifer, the least potent of the NCA, on
IB). These larvae were metamorphosing normally; i.e., tail H. cun'ata larvae were documented further.
muscle cells were in the process ofbeing degraded, ampul- Large shards of L. prolifer (Fig. 2A) induced a range of
lae and the postlarval tunic were forming, and the endoder- changes in H. curvata larval morphology that appearedtobe
mal primordia had turned and commenced morphogenesis. related but differed in severity. Slightly abnormal larvae had
Larvae that had not settled or metamorphosed were also normal axial structures (notochord. neural tube, and muscle)
normal and possessed extended sensory papillae. and trunk structures (sensory vesicle containing otolith and
In contrast, in treatments containing NCA the majority of ocellus, and endoderm rudiment) but lacked projecting pa-
larvae (ca. 80%) neithersettlednormetamorphosed (Fig. 1), pillae and had a slightly rounded trunk (Fig. 3C, D). Some
no postlarvae settled on the epithallial surface ofany ofthe of these larvae still had papillae associated with them;
NCA, and the low levels of settlement (ca. 20%) that did however, the papillae were no longerattachedtothe anterior
occur were confined wholly to the periphery of the plastic trunk and appeared to be in the process of being sloughed
chambers. Although we did not quantify larval behavior off (Fig. 3E). In the most severely altered larvae, the trunk
during the experiment, we did observe some larvae directly was small and rounded, the tail was kinked, and the muscle
contacting the NCA, and we noted that larvae exhibited cells had lost integrity and their usual columnar shape (Fig.
their usual weak and irregular swimming behavior. Al- 3F). Between these extremes of NCA-induced abnormali-
though early metamorphosis appeared normal for H. curvata ties was a continuum of morphological defects.
settled on the plastic in the presence ofNCA (i.e., larval tail To determine whether the inhibitory factor or factors
resorption. papillae retraction, and initiation of the pro- associated with L. prolifer or the other NCA were inducing
grammed degradation of muscle myofibrils appeared nor- generalcelldeath, we investigatedthe structureofthe nuclei
mal), metamorphosis was abnormal in that postlarvae did oflarvae that exhibited an intermediate abnormal morphol-
not develop ampullae or undergo the rotation of the ogy (i.e.. rounded trunk, straight tail with slightly rounded
endodermal primordium (see Degnan et ai. 1996. 1997). muscle cells). Nuclei were stained with propidium iodide
Most of the unsettled H. cun'ata larvae appeared abnormal and analyzed by laser scanning confocal microscopy. Opti-
(see below). cal sections were taken through the trunk epidermis and
By exposing larvae to different quantities of NCA, sig- mesenchyme ofnormal and abnormal larvae, andcompared.
L
nificant differences were observed in larval responses to the In both tissues, the nuclei of larvae exposed to prolifer
different coralline species, and to different quantities ofany were larger Ihan those ofnormal larvae (Fig. 4). Mesenchy-
given species (Fig. 2). For a given amount of NCA. larval mal nuclei of normal larvae were circular (diameters be-
settlement was consistently (and often significantly) greater tween 3.5 and 4.3 jam) and appeared granular when stained
336 B. M. DEGNAN AND C. R. JOHNSON
80
i.8
T
60
INHIBITION OF ASCIDIAN SETTLEMENT 337
A
338 B. M. DEGNAN AND C. R. JOHNSON
B
1 2
\
h post-treatment
E
Figure 5. Percentage of Herdmania cun-ata larvae metamorphosing
Figure 3. The effect of Liilintlmiiiniiini I'mlifer on normal develop- when treated with 40 m/W KCl-elevated FSW. Prior to treatment with
ment and larval structures ofHcn/iminui curnitu. Larval anterioris to the KCl-elevatedFSW, larvae wereculturedeitherin FSW. orin the presence
right in all micrographs. (A) A normal postlarva approximately 12 h after of Nei>Kniolitlmn brassica-florida. Hydrolithon onkodes. or Litlinllhiiii-
initiating metamorphosis; degenerating larval muscle cells (Imc) and pro- niiini/uvi/j/c; for 12-14h. Filledtriangles, larvaeculturedin seawaterand
jectingampullae (amp)areevident. (B)Normaltadpolelarvawithsensory then transferred to KCl-elevated FSW; open circles, larvae cultured in
papillae(arrow). (C-F)Larvaeculturedwith Lfirnlifcr. (C, D)Larvawith seawater and then transferred to FSW; open squares, larvae exposed to
arounded trunkand nopapillae;arrowspoint toregionwherepapillaeare Neogoniolithon brassica-florida and then transferred to KCl-elevated
normally located. (E) Larva in the process ofshedding a papilla (arrow). FSW; diamonds (hidden behind open squares), larvae exposed to Hydro-
(F) Larva with rounded trunk, no papillae, kinked tail, and necrosing liihuii (inkoiU's and then transferred to KCl-elevated FSW; half-filled
muscle cells (arrow). Scale bars: A, B, C, F, 100 mm); D. E, 50 /urn. squares (hidden behind open squares), larvae exposed to iithothamnium
proliferandthentransferredtoKCl-elevatedFSW. Dataaremeans( SE).
cultured with NCA and had not settled lacked papillae and
had rounded anterior trunks. In some of the least morpho- being sloughed from the trunk of the larva, suggesting that
logically disturbed individuals, the palps were observed the toxic effect of the NCA upon the larva first disables the
chemosensory and primary signaling system. Hemps is ex-
pressed in the papillae and the papillae-associated tissue
(PAT) which is located in the anterior epidermis between
the three papillae and consists of about 5 cells (Eri ct <//.,
1999). In the vicinity ofthe sloughed papillae are individual
cells that may have been part ofPAT (Fig. 3E). Secretion of
the Hemps protein is essential for the induction and pro-
gression of metamorphosis (Eri el /., 1999). It is possible
that PATcells are exuded precociously in larvae exposed to
NCA, disrupting the Hemps signaling system. H. cun'iitu
larvae do not have the ability to regenerate these papillae
nor regain the ability to respond to artificial (Fig. 5) or
natural cues (data not shown). The global toxic effect ot the
NCA upon H. ciirvutu larvae is demonstrated by the high
percentage of death within 24 h of larvae being cultured
Figure 4. Degeneration of the nuclei of Herdmania cun-aia larvae with NCA. Experimental inhibition ofpapillae morphogen-
cultured in thepresence ofLillinilitiiiiiiiiini/>////</. Confocal micrographs esis by ecotopic application ofretinoic acid results in a very
of nuclei stained with propidium iodide. (A) Normal larval trunk mesen- similar phenotype to larvae cultured with NCA: however,
ecxhpyomseed(trtounkL.vepnmtlriaflerc.ells(;C)SatNoohr.ma1l996l)a.rv(aBl)ITrruunknkempiedseernmicsh.ym(eD)ofTlraurnvka metamorphosis is not inhibited (Hinman and Degnan,
epidermis oflarva exposed to L. pmlifcr. Scale bar, 10 /j.m. 1998). Toxicity is not limitedtothe larvae individuals that
INHIBITION OF ASCIDIAN SETTLEMENT 339
did settle and initiate metamorphosis in these assays did not data), indicating that the inhibitors can be released from the
complete metamorphosis and died within 24 h (data not surface, it is likely that the inhibitors would operate on a
shown). microscopic scale because of dilution to noneffective con-
The inhibitory signals produced by these NCA appear to centrations a short distance from the NCA. The inability of
be taxa specific. Importantly, under similar laboratory con- NCA to induce settlement and metamorphosis in H. cumita
NCA
ditions, we have induced normal settlement and metamor- is consistent with a general pattern that induce settle-
phosis ofan asteroid (with L. prolifer; Johnson ettil., 1991; ment and metamorphosis in motile species whose feeding
Johnson and Sutton, 1994) and observed that coral and activities reduce fouling of the plant surface, and in sessile
mollusc larvae are not affected by these algae under iden- species that provide preferred habitat for NCA, but not in
tical assay conditions (unpub. data). Given that the effect of sessile species that potentially grow to smother and kill
each of the NCA species on H. citwata larval morphology NCA (C. R. Johnson, unpub. manuscript).
was very similar (i.e., all induced rounded trunks and some-
times kinked tails and dissociated muscle cells), a similar
Acknowledgments
inhibitory factor may be being produced by all three species
ofNCA. We did not determine whether the inhibitors were We thank the Vision, Touch and Hearing Centre for use
produced by the NCA or by surface-associated bacteria or of their confocal microscope facilities, Colin McQueen for
micArlotahloguaeg.h it is well established that NCA provide mor- khiisndhleylpprwoivtihdicnognfsoocamle moifcrtohsecocpoyn.foVcearlonmiiccarogHrianpmhas,n tfhoer
phogenic cues for larvae of a variety of marine inverte- staff of the Heron Island Research Station for their assis-
brates, but do not induce settlement or metamorphosis in tance in maintenance of animals, and two anonymous re-
others (see Introduction), this report demonstrates toxic viewers for their valuable comments. This work was sup-
inhibition of settlement and metamorphosis of an inverte- ported by Australian Research Council grants to BMD and
brate by NCA. The distinction between the simple absence CRJ.
ofa morphogenic cue and inhibition is important. Although
larvae will not settle and metamorphose in eithercase, ifthe
initial contact is with a substratum that simply lacks an Literature Cited
inductive cue, the larva can continue to search and may Arnold,J. M., R. Eri, B. M. Degnan,and M. F. Lavin. 1997. A novel
subsequently receive the appropriate stimulus to settle and gene containing multiple EGF-like motifs transiently expressed in the
metamorphose. These NCA are apparently not toxic toother papillae ofthe ascidian tadpole larva. De\: Dyn. 210: 264-273.
larvae, since some coral and mollusc larvae swim in the Barnes,J.R.,andJ.J.Conor. 1973. Thelarvalsettlingresponseofthe
assay chambers and behave normally for several days with- Clonleinye,dRc.hiAt.on19T8o2n.icrlAlsucildinieaanhil.arMvaare.anBidole.ve2n0t:s2o5f9m-e2t6a4m.orphosis.Am.
out settling (unpub. data). Importantly, the ability of Zoo/. 22: 817-826.
H. cnn-ata larvae to undergo high rates of "spontaneous" Coon,S.L.,W.K.Fitt.andD.B.Bonar. 1990. Competenceanddelay
settlement and metamorphosis in seawater (Degnan et ul., ofmetamorphosis in the Pacific oyster Crassostrea gigas. Mar. Biol.
1997) or when exposed to a potent inducer (KC1) allows us 106: 379-387.
to determine whether a substratum is actually inhibiting DaviEsp,ibAi.osRi.s,oNf.mMa.riTnaergaeltgta.eOa.ndJ.hMecntChoincneilnvle,rtaebnrdatCe.s:Mn.atYuoraulngp.rod1u9c8t9s.
settlement and metamorphosis or is merely not providing a chemistryandothermechanismsinhibitingsettlementandovergrowth.
morphogeniccue. Ifthe ascidian larvacontacts an inhibitory Bioorg. Mar. Chem. 3: 85-1 14.
substratum in this case three species ofNCA then lower Degnan, B. M.. and M. F. Lavin. 1995. Highly repetitive DNA se-
(or zero) rates of settlement or metamorphosis will be quencesprovideevidenceforalackofgene flow between morpholog-
detected, even when the larvae are subsequently exposed to Degnicaanl,foBr.msM.o,faHnedn/Dm.anEi.aMmoorusnet.s.1M9a9r5.. BiDoel.ve1l2o4p:me2n9t3a-l29a9n.d morpho-
a potent inducer (KC1). In contrast, because many larvae do genetic gene regulation in Huliotis rufescenslarvaeat metamorphosis.
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