APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1992, p. 2111-21150099-2240/92/072111-05$02.00/0Copyright C) 1992, American Society for Microbiology

Inhibition of Settlement by Larvae of Balanus amphitrite andCiona intestinalis by a Surface-Colonizing Marine Bacterium

CAROLA HOLMSTROM,' RITYLSCHOF,2 KJELLEBERGl*

Department of General and Marine Microbiology, University of Goteborg, Carl Skottbergs Gata 22, 5-413 19Goteberg, Sweden, 1 and Duke University Marine Laboratory, Beaufort, North Carolina 285162

Received 12 February 1992/Accepted 22 April 1992

In an attempt to isolate bacteria with inhibitory effects against settlement by larvae of sessile invertebrates, 40marine bacterial isolates were screened for effects against laboratory-reared barnacle larvae (Balanus amphitrite)and ascidian larvae (Ciona intestinalis). Five isolates displayed non-pH-dependent inhibitory effects against thelarvae. The initial characterization of a toxic component released from an isolate, designated D2 (CCUG 26757),and its effect on laboratory-reared barnacle and ascidian larvae were studied. D2 is a facultative, anaerobic,gram-negative bacterium isolated from the surface of C. intestinalis from waters off the Swedish west coast at a

depth of 10 m. Results suggest that the toxic component is released by D2 during the stationary phase. Agedbiofilms were more toxic to the larvae than unaged films. The biologically active compound was in the supernatantofD2 and was heat stable and <500 Da in molecular mass. No evidence of protein or peptide moieties was found.On the basis of two phase and chromatography separations, the component is polar and neutral and contains or

binds to carbohydrate moieties. Metaperiodate treatment increased toxicity; undiluted supernatant from a 24-hgrowth culture of D2 killed barnacle and ascidian larvae within a few hours of exposure, whereas aftermetaperiodate treatment, the larvae were killed in approximately 30 min.

Barnacle and tunicate larvae in the settling stage of theirlife cycle respond to stimulatory (13, 23, 24) and inhibitory(12) external signals. Biochemical cues adsorbed to surfacesas well as those free in solution serve to transmit specificinformation in marine environments. Such cues are impor-tant in the settlement, attachment, and metamorphosis ofsessile marine invertebrates (3, 17, 24). Chemical com-pounds that affect the settling have been isolated both fromwithin the species (9, 10) and from other organisms (1, 22).Pheromones released from living adult Balanus spp. havebeen reported to mediate aggregation by settling larvae (18).Adult barnacle peptides affect the behavior and inducemetamorphosis of larvae in the settling stage (26).Of settlement-inhibitory compounds, those from octocor-

als are among the best understood. All known such com-

pounds effective against barnacle settlement that have beenpurified are diterpenoids. At least six unique diterpenoidsthat prevent barnacle settlement have been characterized.These include renillafoulins a and b (8); pukalide, epoxy-pukalide, and 20-OH pukalide from Leptogorgia virgulata(6); and several juncellins. These compounds are effective atnanogram-per-milliliter or lower concentrations (21).

Surfaces colonized by bacteria can serve as a source ofregulatory signals for larval settlement (2, 27). Whether thelarvae require contact with the bacterial film or may sense

soluble cues released from the film has not been determinedfor most bacterial species identified as producers of regula-tory signals. A study by Fitt et al. (4) found that supernatantsfrom Alteromonas colwelliana and Vibrio cholerae affectedoyster larvae (Crassostrea gigas) in the same way as whenthe larvae were exposed to the neurotransmitter precursorL-3,4-dihydroxyphenylalanine (L-DOPA).

In this study, we show that the supernatant from themarine isolate D2 contains a substance that inhibits settle-ment and kills the larvae of Balanus amphitrite and Ciona

* Corresponding author.

intestinalis. These organisms are frequent colonizers ofman-made surfaces immersed in seawater. The inhibitorybacterium was isolated from the surface of the attachmentorgan of an adult C. intestinalis. The mode of action,formation, and initial characterization of the inhibitory com-ponent are presented here.

MATERIALS AND METHODS

Larvae. C. intestinalis larvae were prepared as describedpreviously (25). Adult Ciona specimens were collected in theGullmarsfjorden on the Swedish west coast at a depth of 25to 30 m. They were then stored in a 500-liter aquarium withseawater at 5°C. All seawater used in this study was col-lected at the Kristineberg Marine Biological Laboratory atthe Gullmarsfjorden on the Swedish west coast or at DukeUniversity Marine Laboratory and was passed through a

100,000-Da filter. B. amphitrite larvae were reared in mass

culture from spawnings of laboratory brood stock (19) at theDuke University Marine Laboratory. Both nauplius (feedingstage) and cypris (settling stage) larvae were used in the testassays, which were performed at both Duke UniversityMarine Laboratory and the University of Goteborg. The dayof transformation from the sixth nauplius stage to the cypridwas considered day 0. Cyprids were stored at 4°C until theday of use. Toxicity and settlement assays followed theprotocols described by Rittschof et al. (20).

Bacteria. Marine bacteria were used in the larval attach-ment experiments. They were isolated from rock surfaces atvarious depths and from adult C. intestinalis after colonyformation of the marine medium VNSS agar (15). Bacteriawere grown in VNSS and stored in 50% glycerol ampoules at-70°C. The isolate D2 (CCUG 26757), which was used indetailed larva-inhibitory experiments, is a dark-green-pig-mented bacterium. D2 was isolated from an adult C. intes-tinalis collected from waters off the Swedish west coast. It isa facultative, anaerobic, gram-negative rod and is catalasenegative.

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Growing biofilms for larval attachment assays. Biofilmswere produced by adding 25 ml of the growth medium(VNSS) to petri dishes, which were inoculated with 0.1 ml ofovernight cultures of the individual strains. After 24 h, thesupernatant was poured off and the biofilm on the dish waswashed three times with 20 ml of artificial seawater (NSS)prior to the larval attachment assay. The number of attachedbacteria on surfaces was determined microscopically. Forassessing the effect of exposure of the larvae in the bacterialsupernatant assays, the bacterial cultures were grown for 24h in petri dishes and the supernatant was poured off andcentrifuged at 15,000 x g for 30 min to remove the bacterialcells. The supernatant was then filter sterilized.Attachment assay with larvae. Aliquots (20 ml) of larval

suspensions were transferred to each of the biofilm-coatedpetri dishes, which were incubated in the dark at 25°C for 24h. The number of attached larvae was determined by micros-copy (x 10) and calculated as follows: the degree of attach-ment = [(number of adhering larvae)/(total number of larvaeadded to the system)] x 100. Values are presented as themeans ± standard deviations. For experiments with thebacterial supernatant, the supernatants were mixed withlarvae (15 to 20 larvae per ml) and incubated at 25°C. Thenumber of viable larvae was determined at timed intervalsuntil all larvae were dead.

Pressure dialysis experiments. The supernatant from 24-hgrowth in VNSS of isolate D2 was passed through mem-branes with cutoff sizes of 10,000 (Millipore), 1,000 (Milli-pore) and 500 (Amicon, Ycos) Da. The membranes wereprepared by following the manufacturers' instructions. Non-fractionated supernatants as well as the filtered supernatant,after passage through a given membrane, were used in larvalbioassay studies. Further, dilution series of the supernatantand filtered seawater, as presented in Results, were in-cluded in the barnacle larval bioassays. The residue at the500-Da cutoff filter was redissolved in 6 ml of NSS and testedfor its effect on the settlement and viability of C. intestinalislarvae. This was done to analyze whether the bacterialpigment, which accumulated on the filter, would decreaselarval settlement and viability. It was observed that thepigment was produced and released into the supernatant atthe same time as the larva-inhibitory component.Heat treatment. D2 supernatant was exposed to 80°C heat

for 10 min and filter sterilized before exposure of both Cionaand Balanus larvae.

Ion-exchange chromatography. Separation of the size-frac-tionated and nonfractionated supernatants, using the ionexchangers Amberlite IR 120 P and Dowex 1 (chloride form),was included to determine whether the inhibitory componentcarries charged moieties. One milliliter of filter-sterilized D2supernatant was applied to these packed columns. NSS wasused for elution and as a control.

Methylene chloride extraction. To determine whether thetoxic component is polar or nonpolar, D2 supernatants wereextracted with 1:1 volumes of methylene chloride (Burdickand Jackson). The aqueous and methylene chloride fractionswere evaporated to dryness, redissolved in filtered seawater,and mixed with a barnacle larval suspension to determine theeffect on viability.

Metaperiodate oxidation. The supernatant of isolate D2was treated with 0.001 M metaperiodate (Merck), pH 4.5, tooxidize carbohydrate moieties. Sodium iodate (0.001 M;Merck) was used as the control. The suspensions wereincubated at 25°C for at least 1 h. The pH was then raised to7.5, and the suspensions were filter sterilized before expo-sure of Ciona larvae.

TABLE 1. Number of bacterial isolates with inhibitory effects onlarval settlement from different substratum sampling sites in

marine waters off the Swedish west coast

Sampling Total no. No. of No. of non-pH-Sitelg Tofaisols inhibitory dependentisolates inhibitory isolates

Rock surface1-m depth 26 5 210-m depth 7 3 2

Adult C. intestinalis 7 2 laa Isolate D2.

Pronase treatment. A stock solution with 0.02 g of pronase(Calbiochem) per ml was made; 0.1 ml of this solution wasadded to 1 ml of D2 supernatant, and the mixture wasincubated at 37°C for 1 h. The sample was filter sterilized andtested against the larvae.

Peptidase treatment. Various concentrations of peptidase(Sigma) were suspended in 6-ml, filter-sterilized superna-tants of D2: 10 ,±g, 0.1 mg, 0.2 mg, and 0.3 mg. Thesuspensions were incubated at 37°C for 4 h at pH 7.1 prior toheat treatment for 10 min at 80°C in order to remove thepeptidase activity before the suspensions were mixed withthe larval suspensions.

RESULTS

Supernatant experiment. The initial screening for bacteriawith inhibitory effects on larval attachment and viability wasdone with 40 bacterial isolates from different marine surfaces(Table 1). Ten of these isolates showed a toxic effect on theC. intestinalis larvae in that a significant number or all oflarvae were dead after 24 h of exposure to the undilutednonfractionated supernatant. However, five of these inhibi-tory isolates had an antagonistic effect as a result of areduction in pH. The remaining five isolates with non-pH-dependent inhibitory effects against larval viability and at-tachment originated from all three surface sampling sites(Table 1).Growing biofilms. After 12 h, the bacterium D2 formed a

surface layer consisting of 1.75 x 107 attached bacteria percm on the polystyrene petri dishes. By 24 h, a confluentmonolayer was obtained. Three-day-old cyprids settled at afrequency of 1.1% to a 12-h-old biofilm, while no larvae (0%)were observed attached to 24- and 48-h-old biofilms. By 144h, however, the degree of attachment was the same as thaton unfilmed control surfaces on which 33% attached larvaewere observed (Table 2). The bacterial cell density varied

TABLE 2. Degree of attachment of B. amphitrite larvae tobacterium D2 biofilms of different ages

Age of biofilm Degree of attachment(h) (%)a

Control (no biofilm) .................................... 32.2 ± 3.4612.................................... 1.1 ± 0.00524.................................... 048 .................................... 0120 ....... ............................. 1.1 + 0.51144 .................................... 33.0 ± 8.93

a Values represent means + standard deviations of three parallel experi-ments.

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TABLE 3. Dilution assay of toxic activity of supernatants of 24-hgrown D2 cells against nauplius and cypris larvae of

B. amphitritea

% Dead B. amphitrite larvae

Nonfractionated Supernatant after size frac-Dilution series supernatant tionation (nauplius larvae)

Nauplius Cypris 10 kDa 1 kDa 500 Dalarvae larvae

Not diluted 100 100 100 100 1001:1 100 100 100 100 1001:3 100 100 NDb ND ND1:4 ND ND 86 98 261:9 40 48 69 46 171:19 ND ND 55 31 201:24 ND ND 54 29 141:99 4 19 11 21 8Control (seawater) 0 0 ND ND ND

a The percentage of dead larvae was determined after 24 h of incubation ofD2 supernatant and larvae. The experiment was repeated two times to confirmreproducibility.

ND, not determined.

between 1.75 x 107 and 2.54 x 107 per cm2 during the timecourse of this experiment.

Pressure dialysis. The D2 supernatant that passed througha 500-Da-cutoff filter was still toxic against larvae, althougha decrease in its inhibitory effect was observed. The dilutionseries of the supematant prior to exposure to barnacle larvaeshowed that nonfractionated supernatant can be dilutedfourfold without a reduction in the rate at which nauplius andcypris larvae lose viability. Fractionated supernatants can bediluted twofold and still display 100% activity towards thebarnacle larvae. While further dilution of the supernatantsreduced the toxicity effect (Table 3), the highest dilutiontested, 1:99, still reduced larval viability to a significantextent. This was the case for both unfractionated andfractionated supernatants. The redissolved residue of the500-Da-cutoff filter was found not to affect the larvae interms of viability or any other visible phenotypic character-istic (Table 4).

Ion-exchange chromatography experiments. The results ofion-exchange chromatography experiments showed that thetoxic component did not bind to either the cation or theanion exchanger (Table 4). The samples (1 ml) applied toboth the anion- and cation-exchange absorption chromatog-raphy columns were fractionated. A full inhibitory effect

TABLE 4. Summary of the characterization of the larva-inhibitory component produced by bacterial isolate D2

Conditions or treatments of Loss of larva viabilitybacterial supernatant Barnacles Ascidians

Cation-exchange eluate Yes YesAnion-exchange eluate Yes YesMethyl chloride extraction No NDa

(nonpolar components)Heat treatment Yes YesPeptidase treatment ND YesPronase treatment Yes YesPigment ND NoBiofilm Yes Yes

a ND, not determined.

TABLE 5. Effect of metaperiodate and iodate (control) treatmentprior to exposure of C. intestinalis larvae to D2 supernatant

after 24 h of growtha

Treatment Time (min)

Nonfractionated Fractionated Treatment of 100% loss insupernatant supematanth supernatantc larval viability

JO4d 180 10Jo3e 180 150NTf 180 155

104 120 30103 120 210NT 120 >210

I04 195 30103 195 180NT 195 180

104 200 60I03 200 165NT 200 180

104 240 20103 240 150NT 240 180

a Results are expressed as the time of incubation necessary to obtain 100%loss in larval viability. Results of four different experiments with differentincubation times (nonfractionated supematants) and one experiment withfractionated supernatant are reported.

b D2 supematant passed through a 500-Da-cutoff filter.c Time of metaperiodate and iodate treatment of D2 supernatant at 37°C, pH

4.5.d Metaperiodate treatment of D2 supernatant.e Sodium iodate treatment of D2 supernatant.f NT, not treated.

against both Ciona and Balanus larvae was seen in theeluate, the main part of which was eluted in the first 1 ml.Methylene chloride extraction. The residues of the aqueous

and methylene chloride phases were redissolved in 10 ml offiltered seawater and transferred to petri dishes. The expo-sure of the methylene chloride resuspension to the barnaclelarvae had no effect on larval viability or attachment, and itis concluded that the methyl chloride extract did not containthe toxic component. A full inhibitory effect, as measured bythe percentage of both viable and attached larvae, wasobtained by exposing the larvae to the aqueous supernatantextract (Table 4). These results indicate that the toxiccomponent is polar.Heat treatment. The results obtained after exposure of the

larvae to heat-treated (80°C) supernatant were the same asfor the control, i.e., nontreated supernatant, experiment. Noreduction in larval viability or attachment was observed,suggesting that the active component is heat stable (Table 4).

Viability of C. intestinalis larvae after metaperiodate, pro-nase, and peptidase treatment. The D2 supernatant becamesignificantly more toxic against the larvae after metaperiod-ate treatment. This effect was observed in a series ofexperiments in which the time of metaperiodate incubationwas varied between 2 and 4 h. As can be seen in Table 5, thisresult was obtained for both the nonfractionated supernatantand the supernatant that passed through a 500-Da-cutofffilter. Table 5 also shows the results of the appropriatecontrol experiment, i.e., those with nontreated supernatantsand after the addition of sodium iodate to the supernatants torule out that the metaperiodate and not iodate would account

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for the observed effect. No change in the die-off rate oflarvae was seen after pronase and peptidase treatment.

DISCUSSION

The cypris larvae of barnacles and the tadpole larvae of C.intestinalis respond to both stimulatory (11, 23, 25) andinhibitory (7) surface-associated signals during recruitmentfrom the plankton to solid substrata. Although many con-flicting results have been presented in the literature, it seemssafe to conclude that microbial films represent one source ofthese signals (2, 14, 25, 28). Most of the studies aimed atdefining the interactions between a bacterial film and larvaeof sessile invertebrates have described systems in which thebacterial cells stimulate larval settlement. Apart from theisolation of inhibitory components from other animals, thereis only one report on inhibitory effects by a bacterial film onlarval attachment. Maki et al. (14) reported possible reasonsfor the fact that a bacterial film of Deleya manina onhydrophobic substrata resulted in a lower number of at-tached cypris larvae. They reported furthermore that nineadditional marine bacterial isolates were found to lower thedegree of attachment of cypris larvae to hydrophobic sur-faces. No attempts to identify the mechanisms of theseinhibitory effects were made. On the other hand, preliminaryattempts to isolate and identify signals that affect larvalsettlement have been made for stimulatory bacteria (11). Inview of severe technical and economical problems related tobiofilm formation on man-made surfaces by macrofoulingorganisms such as barnacles and tunicates, it is surprisingthat additional studies on inhibitory bacterial biofilms havenot been pursued.

This study demonstrates that a significant fraction ofbacteria on surfaces in the natural environment may producecomponents that inhibit the attachment of barnacle andtunicate larvae. Of 40 bacterial strains isolated from differentmarine surfaces, 5 strains were found to produce extracel-lular inhibitory components that are released from biofilms.These components inhibit and/or kill the larvae in thesuspension above the biofilm in a non-pH-dependent man-ner. Interestingly, the relatively high frequency of suchinhibitory strains supports the notion that bacterium-larvainteractions may occur frequently in the natural environment(2, 11, 28). High frequencies of stimulatory bacterial isolateshave been reported previously (13, 14). It should also bepointed out that bacterial strains with inhibitory actionagainst invertebrate larvae could be isolated from the surfaceof an adult specimen of the invertebrate organism. It istempting to speculate that adult animals thus prevent settle-ment of larvae of the same and possibly other species ofsessile invertebrates.One of the five inhibitory bacterial strains was analyzed in

more detail with respect to formation and nature of thelarva-inhibitory component. Most of the experiments per-formed on this bacterium and its extracellular componentincluded assays of the response by larvae of both B. am-phitnte and C. intestinalis. It may be concluded that thesame component has very similar or the same effect on bothof these organisms. The generality of this effect has not beenfurther explored; i.e., we have not yet included additionalsessile invertebrates in these tests. It is clear, however, thatthe inhibitory component is not effective against other pro-karyotes. We exposed cells of 15 different marine bacterialstrains to the supernatant produced by the marine bacterialisolate D2. No reduction in viability or growth was ob-served. Attempts have been made to find a convenient

bioassay that allows experiments to be performed indepen-dently of the field and seasonal conditions. Preliminaryexperiments with a human cell line, AGS, indicate that thiscell line responds in a manner similar to that of invertebratelarvae.The marine bacterial isolate D2, isolated from the attach-

ment site of an adult C. intestinalis, produces a <500-Dacomponent which is toxic to larvae of both B. amphitnte andC. intestinalis. This component is released into the suspen-sion of batch-grown as well as biofilm-grown cells. Theformation of the inhibitory component coincides with thedevelopment of dark-green pigmentation, characteristic ofstationary-phase cells of isolate D2. However, size fraction-ation experiments clearly demonstrated that the pigment isnot the toxic component. The 500-Da filtrate, which stronglyinhibits and kills the larvae, is pigmentless. The retentate ofthe 500-Da filter contains the pigment but has no or only asmall effect on barnacle or tunicate larvae. On the basis ofinitial characterizations of this component described in thisreport (Table 4), the extracellular inhibitory molecule is heatstable, polar, and uncharged. The enzyme exposure experi-ments indicate that the component is not a protein or apeptide. It should also be reported that the low-molecular-weight component shows long-term stability. No loss inactivity was seen after storage at 4°C for a period of 6 days(data not shown). Similarly, no loss in activity was foundafter frozen storage at -70°C. On the basis of incubation ofthe suspension containing the active component withmetaperiodate, the inhibitory molecule either contains car-bohydrate moieties or is released by the cells in a form inwhich it is bound to or surrounded by carbohydrate-contain-ing molecules.That the retentate of the 500-Da filter occasionally exhib-

ited inhibitory effects (data not shown) can be explained byeither (i) the existence of a larger precursor molecule or (ii)complexing of the small molecule with other molecules (16).Both the non-size-fractionated and the fractionated cell-freesuspensions became significantly more toxic to the larvaeafter metaperiodate treatment (Table 5). Other studies havepresented evidence of the complex arrangement of surface-localized molecules that affect the settlement and metamor-phosis of invertebrate larvae (11). Szewzyk et al. (25)demonstrated that the exopolysaccharide of marine Pseudo-monas sp. strain S9 influences settlement of tadpole larvaeof C. intestinalis. The attachment of larvae of the polychaeteJanua brasiliensis has been suggested to be mediated bylarva-derived lectinlike substances that bind to receptors inthe exopolysaccharide layer of the bacterial biofilm of D.marina (11). Furthermore, Maki et al. (13) proposed thatalthough bacterial polysaccharides have been suggested tobe important cues in the settlement and metamorphosis theymay also act as binding components for other settlementregulatory components. Preliminary studies on the attach-ment of fertilized eggs of C. intestinalis suggest that ahigh-molecular-weight proteinaceous component is trappedby the exopolysaccharide layer that surrounds the cells ofPseudomonas sp. strain S9 (unpublished data). The com-plexity of the signals produced by bacteria is evidenced alsoby recent studies by Maki et al. (13). D. manina appears toproduce both inhibitory and stimulatory components forattachment of B. amphitrite larvae.

All experiments in this study were performed with immo-bilized bacteria. Exposure of larvae to the suspension ofbacterial extracellular products was made on the supernatantderived from growth of bacteria in the biofilm on the bottomof plastic petri dishes. Even though this study differs from

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experiments in which larval regulatory components aresurface associated, it is of value to relate this study to larvalattachment studies of similar experimental design. Bacterialimmobilization to surfaces was approximately 107 cells percm2, in agreement with previous reports on bacterial attach-ment on polystyrene (5, 13). Maki et al. (13) concluded thatbacterial density is an important factor and that no inhibitoryeffects by the bacteria on larval attachment were observed atcell densities of <107/cm2. They observed that aging of thebacterial films resulted in a decrease in the percentage ofattached larvae. These experiments were performed with D.manna films on polystyrene surfaces. The biofilm used inthis study secretes the inhibitory component into the me-dium. Stationary-phase biofilms were more inhibitory tolarval attachment than 12-h-old biofilms of approximatelythe same number of bacterial cells. This suggests that theinhibitory component is synthesized and/or released as thebacterial cells enter the stationary phase.

It is noteworthy that the inhibitory low-molecular-weightcomponent is highly toxic to larvae. A relatively low numberof cells grown for 24 h, i.e., 2.54 x 107 bacteria per cm2, issufficient to produce a bacterial supernatant that kills asmany as 50 larvae in a 4-ml suspension in <3 h. Unfraction-ated and fractionated samples can be diluted 100-fold andstill kill all B. amphitnte larvae within 22 h. This test wasonly performed with larvae of B. amphitnte.Our present experimental efforts are directed at the iden-

tification and structural analysis of the inhibitory compo-nent. We have also begun exploring the possibility of immo-bilizing cells of the marine bacterial isolate D2 in polymersthat are used for coating surface test panels immersed in situin marine waters. The use of biological rather than chemicalmeans to reduce macrofouling of man-made surfaces inmarine systems is an attractive possibility.

ACKNOWLEDGMENTS

We gratefully acknowledge valuable discussions with James S.Maki and Ulrich Szewzyk. We thank Anders Henriksson for helpduring the diving and in the sampling of tunicate specimens.

This work was supported by grants from the Swedish NationalBoard of Technical Development, BioVast Foundation for Biotech-nology Goteborg, and the Gullmarn Foundation.

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