bioactivity of echinoderm ethanolic body-wall extracts: an assessment of marine bacterial attachment...

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ELSEVIER Journal of Experimental Marine Biology and Ecology 196 (1996) 79-96 JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY Bioactivity of echinoderm ethanolic body-wall extracts: an assessment of marine bacterial attachment and macroinvertebrate larval settlement Patrick J. Bryan”‘*, Dan Rittschofb, James B. McClintock” “1300 University Blvd. Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA hDuke University Marine Laboratory, Beaufort, NC 28516, USA Received 2 February 1995; revised 16 April 1995; accepted 27 June 1995 Abstract The ethanolic body-wall extracts of 16 species of echinoderms from 16 genera were screened for their ability to affect the attachment of the marine bacteria Dekya marina (Baumann) and Alteromonas luteo-violucea (Gauthier). Body-wall extracts were tested at concentrations which mimic mean natural tissue concentration, 3.0 mglml seawater, and four half-log dilutions of this initial concentration. The extracts of three echinoderm species caused significant inhibition of bacterial attachment, while extracts of eight species caused significant enhancement of attachment. The body-wall extract of the asteroid Goniaster tesselutus (Lamarck) displayed the most potent antimicrobial activity, completely inhibiting attachment of both bacterial species at a concentration of 3.0 mg/ml seawater. The ethanolic extracts of 20 echinoderm species were also tested at a similar range of concentrations for their ability to affect the settlement of cyprid larvae of the barnacle Balanus amphitrite (Darwin), and coronate larvae of the bryozoan Bug&a neritina (Lind). All echinoderm extracts inhibited settlement of both barnacle and bryozoan larvae at the highest concentration tested (3.0 mg/ml seawater). Eleven of the 20 echinoderms tested (13 asteroids, 3 holothuroids, 3 ophiuroids and a crinoid) had body-wall extracts that inhibited settlement of competent barnacle and bryozoan larvae at concentrations as low as 0.12 mg/ml seawater. These extracted echinoderm compounds may function as non-toxic or toxic antifoulants, and to promote bacterial surface colonization, which could be valuable to the organisms disease resistance. Keywords: Antifoulant; Bacterial attachment; Echinoderm extracts; Larval settlement * Corresponding author. Fax: (1) (205) 975-6097 0022.0981/96/$15.00 @ 1996 Elsevier Science B.V. All rights reserved SSDI 0022.0981(95)00124-7

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ELSEVIER Journal of Experimental Marine Biology and Ecology

196 (1996) 79-96

JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY

Bioactivity of echinoderm ethanolic body-wall extracts: an assessment of marine bacterial attachment and

macroinvertebrate larval settlement

Patrick J. Bryan”‘*, Dan Rittschofb, James B. McClintock”

“1300 University Blvd. Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA

hDuke University Marine Laboratory, Beaufort, NC 28516, USA

Received 2 February 1995; revised 16 April 1995; accepted 27 June 1995

Abstract

The ethanolic body-wall extracts of 16 species of echinoderms from 16 genera were screened for their ability to affect the attachment of the marine bacteria Dekya marina (Baumann) and Alteromonas luteo-violucea (Gauthier). Body-wall extracts were tested at concentrations which mimic mean natural tissue concentration, 3.0 mglml seawater, and four half-log dilutions of this initial concentration. The extracts of three echinoderm species caused significant inhibition of bacterial attachment, while extracts of eight species caused significant enhancement of attachment. The body-wall extract of the asteroid Goniaster tesselutus (Lamarck) displayed the most potent antimicrobial activity, completely inhibiting attachment of both bacterial species at a concentration of 3.0 mg/ml seawater. The ethanolic extracts of 20 echinoderm species were also tested at a similar range of concentrations for their ability to affect the settlement of cyprid larvae of the barnacle Balanus amphitrite (Darwin), and coronate larvae of the bryozoan Bug&a neritina (Lind). All echinoderm extracts inhibited settlement of both barnacle and bryozoan larvae at the highest concentration tested (3.0 mg/ml seawater). Eleven of the 20 echinoderms tested (13 asteroids, 3 holothuroids, 3 ophiuroids and a crinoid) had body-wall extracts that inhibited settlement of competent barnacle and bryozoan larvae at concentrations as low as 0.12 mg/ml seawater. These extracted echinoderm compounds may function as non-toxic or toxic antifoulants, and to promote bacterial surface colonization, which could be valuable to the organisms disease resistance.

Keywords: Antifoulant; Bacterial attachment; Echinoderm extracts; Larval settlement

* Corresponding author. Fax: (1) (205) 975-6097

0022.0981/96/$15.00 @ 1996 Elsevier Science B.V. All rights reserved

SSDI 0022.0981(95)00124-7

80 P. Bryan et al. I .I. Exp. Mar. Bid. Ecol. 196 (1996) 7%96

1. Introduction

Fouling, the colonization of an organisms surface by bacteria (Lappin-Scott and Costerton, 1989), diatoms, algae, and sessile marine invertebrates (reviewed by Wahl, 1989) is a threat to organisms in the marine environment. Fouling of the body surfaces of marine invertebrates causes such problems as increased weight, reduced elasticity (hindering motion and flexibility), increased surface friction, and damage to soft body-wall surfaces (reviewed by Davis et al., 1989; Wahl, 1989). Some marine organisms tolerate such fouling (Sand-Jensen, 1977), while others have adaptations to prevent fouling through avoidance (e.g. habitat selection) or defense. Defensive mechanisms that marine invertebrates may employ to prevent their tissues from being fouled include sloughing of the epidermis (Filion-Myklebust and Norton, 1981; Barthel and Wolfrath, 1989). copious mucous production (Targett et al., 1983; Krupp, 1985), scraping or cleaning of the body wall tissues (Dyrynda, 1986), and the production of noxious organic compounds or sequestration of heavy metals which inhibit other organ- isms from attaching to or living on their tissues (reviewed by Pawlik, 1993).

Bacterial attachment is one of the primary stages in the ontogeny of the fouling process (ZoBell and Allen, 1935; Clare et al.. 1992). Once in contact with a substratum, microbes explore their surroundings to detect how favorable the conditions are for settlement (Chet and Mitchell, 1976; Little et al., 1988). After sensing the nutrient and environmental conditions, bacteria may anchor and permanently attach to the surface, or they may migrate to a more suitable substrate before attaching (Costerton et al., 1978; Little et al., 1988). The formation of a biofilm consisting of bacteria, protozoans, and microalgae immedi- ately follows initial bacterial attachment (Characklis and Cooksey, 1983). Bacteri- al biofilms can influence subsequent stages of fouling (Mihm et al., 1981; Maki et al., 1988; Clare et al., 1992).

The larvae of fouling marine invertebrates face intense competition for substratum (Jackson and Buss, 1975; Sutherland and Karlson, 1977). Many factors influence selection of settlement substrata by invertebrate larvae, including chemical induction (Clare et al., 1992; Rittschof, 1993), available nutrients (Crisp, 1974; Hudon et al., 1983), surface energy (Vrolijk et al., 1990; Roberts et al.. 1991; Maki et al., 1992), and currents and surface texture (Hudon et al., 1983; Rittschof et al., 1984). The presence of a biofilm on a substrate may have an effect on both the induction (Brancato and Woollacott, 1982) and inhibition (Maki et al., 1988. 1990) of larval attachment.

Echinoderms often occur in high abundance, offering a potentially valuable source of substratum for the attachment and growth of bacteria (Jangoux, 1987), diatoms, algae, and larvae of fouling marine invertebrates (Davis et al., 1989; Levin, 1989). Although most echinoderms are free from fouling organisms there are several reports of echinoderms fouled by sponges (Mortensen, 1936; Fell. 1961), cnidarians (Clark, 1921; Smirnov and Stepanyants, 1980), bryozoans (Gautier, 1959; Moyano and Wendt, 1981), and barnacles (Boolootian, 1964; Strachan, 1970). Campbell (1983) suggested that echinoderms may clean their body-wall surfaces of fouling organisms using their pincer-like pedicellariae.

P. Bryan et al. I J. Exp. Mar. Bid. Ecol. 196 (1996) 79-96 81

However, only echinoids and asteroids possess pedicellariae, and of these groups, pedicellariae are present in a low percentage of species (Burke, 1983). It is evident that echinoderms must utilize defensive strategies other than simply the use of pedicellariae to protect themselves from fouling organisms.

Chemical studies of secondary metabolites isolated from echinoderms have revealed an abundance of saponin or saponin-like compounds in their tissues (reviews by Stonik and Elyakov, 1988; Habermehl and Krebs, 1990; Kalinin et al., 1996). Investigations of bioactive properties of crude echinoderm extracts and isolated saponins have led to discoveries of extracts and compounds with cytotoxic (Ruggieri and Nigrelli, 1960; Mackie et al., 1975) ichthyotoxic (Makie et al., 1975; McClintock, 1989), feeding deterrent (Lucas et al., 1979; McClintock and Vernon, 1990), antimicrobial (Rinehart et al., 1981; Bryan et al., 1994) and antifoulant (Iorizzi et al., 1996) activities. Bioactive compounds have been found in a high percentage of those echinoderms examined to date, especially among the classes Asteroidea and Holothuroidea (Stonik and Elyakov, 1988; Habermehl and Krebs, 1990; Kalinin et al., 1996). The functional significance of echinoderm bioactive compounds to predators and fouling organisms has received little attention. Nonetheless, one important role may be in the prevention of fouling.

One objective of this study was to determine if ethanolic extracts of echinoderm body-wall tissues can inhibit attachment of marine bacteria. The marine bacterium Deleya marina (Baumann) was selected to study this aspect because it has a broad oceanic distribution, is motile, and much is known of its method of attachment. Afteromonas luteo-violucea (Gauthier), another marine bacterium, was also utilized in this study because it was isolated from water samples sympatric with the echinoderms examined in the present study. A second objective of this study was to determine if ethanolic body-wall extracts from echinoderms can inhibit attachment of the larvae of common fouling marine invertebrates. Cyprid larvae of the acorn barnacle Balunus umphitrite (Darwin) and coronate larvae of the chelistome bryozoan Bugulu neritinu (Linne) were selected, because their larval development has been well studied (Mihm et al., 1981) and laboratory settlement assays with natural products have been established (Rittschof et al., 1988). Moreover, they represent a documented fouling threat to echinoderms (e.g. Boolootian, 1964; Moyano and Wendt, 1981).

2. Methods

2.1. Collections

Twenty species of echinoderms (Table 1) were collected from selected sites in the northern Gulf of Mexico (Fig. 1) using an otter trawl (Sites A and B) and SCUBA (Sites C and D). Collection sites A and B are described in detail by Hopkins et al. (1991). Site C is characterized by a sand substratum and occurs at a depth of ~20 m. Site D is located =l km off the coast of St. Petersburg, Florida and consists primarily of a sand substratum at a depth of =lO m . Echinoderms were immediately frozen and transported to the University of Alabama at

82

Table 1

P. Bryan et al. I J. Exp. Mar. Biol. Ecol. 196 (1996) 79-96

Echinoderm species and collection sites (see Fig. 1)

Class Asteroidea Family Astropectinidae

Astropecten articulatus (Say)

Tethyaster grandis (Verrill)

Family Echinasteridae

Echinaster sp

Henricia downeyae (Clark)

Family Goniasteridae

Anthenoides piercei (Perrier)

Goniaster tesselatus (Lamarck)

Tosia parva (Perrier)

Family Luidiidae

Luidia clathrata (Say)

Family Ophidasteridae

Chaetaster nodosus (Perrier)

Linkia nodosa (Perrier)

Narcissia trigonaria (Sladen)

Tamaria halperni (Downey)

Family Oreasteridae Oreaster reticulatus (Linnacus)

Class Ophiuroidea Family Gorgonocephalidae

Astrocyclus caecilia (Lutken)

Astroporpa annulata (O[umlaut]rsted and Lu[umlaut]tken)

Astrophyton muricatum (Lamarck)

Class Holothuroidea Family Holothuriidae

Holothuria lentigenosa (Marenzeller)

Holothuria thomasae (Pawson and Caycedo)

Family Stichopodidae

Isostichopus hadionotus (Selenka)

Class Crinoidea Family Comasteridae

C omactinia merdionalis (Agassizi)

Collection site

A

B

C B

B

B

B

A

B

B

B

B

D

B B

D

B

C

c

B

Birmingham. Water samples were collected from site B using a CTD instrument equiped with water samplers and transferred to sterile 200 ml glass jars for the culturing of marine bacteria.

2.2. Extraction qf hioactive secondary metabolites

Echinoderms were dissected and body-wall tissues were weighed, lyophihzed, and reweighed. Saponins, which are the most predominant bioactive compounds

P. Bryan et al. I J. Exp. Mar. Biol. Ecol. 196 (1996) 79-96 83

89’ 88* 87” 86”

Gulf of Mexico

Fig. 1. Map of collection sites in the northern Gulf of Mexico. Echinoderms were collected from sites

A and B by trawl, while collections from sites C and D wer by SCUBA. Depth is in meters.

isolated from echinoderms, are characteristically polar. In an attempt to extract all saponin and saponin-like compounds, the lyophilized tissue was extracted in aqueous ethanol (ethanol:water, 80:20) (Yasumoto et al., 1966). Tissues were extracted by submerging body-wall tissues in the solvent mixture at a ratio of three to one (volume:wt) for 24 h at 21°C while being agitated on a shaker table. The solvent was decanted, filtered through Whatman #l filter paper, and dried in a rotary evaporator. Extracts were resolublized in ethanol:methanol (80:20) to precipitate any salt present from the initial extraction. This precipitate, assumed to be inorganic salts, was saved. Once desalted extracts were dried under nitrogen and weighed, the natural concentration (% dry wt) of extract recovered from the body-wall was determined for each species of echinoderm. Extracts were stored at -20°C and rehydrated to appropriate concentrations in sterile seawater (32 ppt) prior to use in bioassays.

2.3. Bacterial attachment assay

One species of bacteria was isolated from the water sample and identified by gas chromatography-fatty acid methyl ester (GC-FAME) analysis and a

84 P. Bryan et al. I J. Exp. Mar. Biol. Ecol. 196 (1996) 79-96

BIOLOGQsystems analysis (Biolog, Inc., Hayward, California). This Gram- negative bacterial species was determined to be A. luteo-violacea. A bacterial attachment assay was performed by a modification of the procedure of Shea and Williamson (1990). One hundred ~1 volumes of a stationary phase culture of D. marina (ATCC 25791) or A. luteo-violacea (ATCC 29580) grown in marine broth (Difco #2216), were pipetted into 88 wells of a 96 well microtiter plate (Falcon #3872). The remaining eight wells were filled with 100 ~1 of sodium desoxycho- late to serve as reference blanks. The bacterial cells were centrifuged at 1000 rpm for 10 min. The broth solution was removed and 100 ~1 of sterile seawater (control) or sterile seawater containing solublized body-wall extract (experimen- tal) was added to each well. This caused a re-suspension of the bacterial cells. Microtiter plates were incubated at 25°C for 2 h for D. marina and 4 h for A.

luteo-violacea. The seawater with bacterial cells was then removed from the wells by inverting the plates and 100 ,ul of a 0.1% crystal violet solution was added to each well for a 5 min staining period. The dye solution and any unattached cells were removed by rinsing the wells with deionized water. One hundred microliters of a 2% aqueous sodium desoxycholate solution was added to each well and the release of crystal violet encouraged by gently rocking the plates. The absorbances of the 100 ,ul dye solutions in the wells were measured by a Flow Laboratories Multiscan Microplate Reader equipped with a 595 nm filter.

Extracts were assayed at a concentration of 3.0 mg/ml seawater, which is within the range of that occurring naturally in the organism (from 2.1 to 3.5 mgigm of body-wall tissue for 20 species examined), and then at four half-log dilutions of that concentration (0.6, 0.12, 0.024, 0.0048 mglml seawater). A standard curve was generated so that absorbance values could be converted into numbers of bacterial cells. The number of attached bacterial cells from four replicate wells yielded mean values for each concentration of echinoderm body-wall extract tested.

The standard curve was determined by incubating polystyrene coverslips in separate broth cultures of D. marina and A. luteo-violacea for 1, 2, 4, 8, and 16 h (n = 3 per incubation period). The coverslips were removed from the cultures, rinsed, and stained with a 0.1% crystal violet solution. Cells were directly counted under a Zeiss compound microscope with a Petroff-Hausser counter. Ten l/400 mm* areas were counted for each slide and the total number of bacteria per coverslip were extrapolated. The coverslips were then extracted in 1 ml of sodium desoxycholate and 100 ,ul aliquots of each of the solutions were pipetted into each of four wells of a 96-well falcon plate. Absorbances were measured on a Flow Laboratories Multiscaner and values were plotted to generate a standard curve for each bacterial species. The r2 values for the D. marina and A. luteo-violacea standard curves were 0.975 and 0.982, respectively. These curves were utilized to transform the absorbance values into values representative of numbers of bacterial cells attached in a given well. The mean and standard deviation of the number of attached bacterial cells in each group of wells (n = 4 wells per extract concentration) were calculated. An Analysis of Variance was utilized with a post-hoc Scheffe’s F-test to determine if there was a statistically significant

P. Bryan et al. I J. Exp. Mar. Biol. Ecol. 196 (1996) 79-96 85

difference in the number of cells attached in the control wells from the experimental wells. The alpha value was set at 0.05.

2.4. Barnacle larval settlement assay

Larvae of the barnacle Balanus amphitrite were cultured at the Duke University Marine Laboratory in Beaufort, North Carolina. After nauplii molted to the cyprid stage, larvae were stored at 6°C for 3 days. These 3-day-old cyprid larvae were at an optimal state of competence for use in settlement assays (Rittschof et al., 1988). Approximately 35 cyprid larvae in a volume of 200-450 ,ul seawater were added to Falcon 9 X 50 mm (depth X width) covered polystyrene Petri dishes containing either 5 ml of lOO-kDa-filtered seawater (32 ppt) (control) or a similar amount of seawater containing echinoderm body-wall extract. The extracts were evaluated at five concentrations, beginning at a concentration within the range of that occurring naturally in echinoderm body-wall tissues (3.0 mg/ml seawater) and then at four half-log dilutions of that concentration (0.6, 0.12, 0.024, 0.0048 mg/ml seawater). Three replicates of each extract concentration were prepared, along with triplicate control Petri dishes containing seawater alone. The cyprid larvae were incubated at 28°C on a 15L: 9D photoperiod for 24 h. After incubation, several drops of 10% formalin solution were added to each dish to terminate the bioassay. Using a dissecting microscope, the numbers of attached and unattached larvae were determined. Larvae that were permanently attached or had metamorphosed on the polystyrene dish were considered settled. Differ- ences in the settlement frequencies of experimental and control treatments were tested for significance using a G-test for independence (Sokal and Rohlf, 1981).

2.5. Bryozoan larval settlement assay

The bryozoan settlement assay was performed with a combination of techniques designed for bryozoan (Mihm et al., 1981; Maki et al., 1989) and barnacle (Rittschof et al., 1988) larvae. Colonies of adult bryozoa, Bug&a neritina, were collected near the Duke University Marine Laboratory in November of 1992, and were held with aeration in the dark for ~24 h. Bug&a neritina colonies were subsequently shocked with light to trigger release of coronate larvae. The larvae released from their brood sacs were concentrated with a fiber optic light source and used immediately in settlement assays. Forty to 60 larvae were added to 9 X 50 mm circular polystyrene dishes, each containing 5 ml of filtered seawater (n = 3 controls) or seawater containing echinoderm body-wall extract (n = 3 per extract concentration). The dishes were incubated at 20 ? 2°C for 30 min. Assays were terminated with the addition of several drops of formalin to each dish. Dishes were viewed under a dissecting microscope, and larvae that were attached and deciliated were considered to be settled. Differences in the settlement of experimental and control treatments were tested for significance using a G-test for independence (Sokal and Rohlf, 1981).

86 P. Bryan et al. I J. Exp. Mar. Bid. Ecol. 196 (1996) 79-96

3. Results

There was a statistically significant reduction in attachment of D. marina in wells of a microtiter plate in which body-wall extracts of the asteroids Goniaster tesselatus, and the basketstar Astrophyton muricatum were added (Fig. 2). No bacterial cells attached in wells containing the body-wall extract of G. tesselatus at a concentration of 3.0 mg/ml seawater. Significant inhibition of attachment was observed in wells containing G. tessefutus body-wall extract at a concentration as low as 0.12 mg/ml seawater. A statistically significant reduction in attached cells occurred in wells containing the body-wall extract of Astrophyton muricatum at a concentration of 0.6 mg/ml seawater. In contrast, a significant increase in the numbers of attached cells of D. marina occurred in wells containing the body-wall

Concentration of Extract (mg/ml seawater)

Fig. 2. Mean number of attached cells from the marine bacteria D. nmrian (n = 4). The asterick (*)

indicates a statistically significant difference in the mean number of cells attached in the experimental

wells from the controls using ANOVA with a post-hoc Scheffe’s F-test (u = 0.0.5). Note that the y-axis

is different to account for variation between experiments.

P. Bryan et al. I J. Exp. Mar. Biol. Ecol. 196 (1996) 79-96 87

extracts of the asteroids Astropecten articulatus, Linckia nodosus and Tamaria halperni, the ophiuroid Asteroporpa annulata, and the crinoid Comactinia mer- dionalis. A statistically significant increase in bacterial attachment occurred in wells containing body-wall extracts of Comactinia merdionalis and Linckia nodosus both only at a concentration of 3.0 mg/ml seawater. Statistically significant increases in bacterial attachment occurred in wells in which body-wall extract of the ophiuroid Asteroporpa annuluta was present at concentrations of 3.0 and 0.6 mg/ml seawater. Attachment of D. marina was statistically greater in wells containing the extracts of both Astropecten articulatus and Tamaria halperni at a concentration of 0.024 mg/ml seawater. No significant effect on the attachment of D. marina in wells of Falcon microtiter plates was observed for the body-wall extracts of all other echinoderms tested.

A statistically significant effect on the attachment of A. luteo-violucea was observed for 7 of the 16 echinoderm body-wall extracts tested (Fig. 3). Bacterial attachment was significantly lower than controls in wells containing the body-wall extract of the asteroid G. tesselutus at 3.0 and 0.6 mg/ml seawater. In contrast, a significant increase in bacterial attachment was observed in wells containing the body-wall extract of the ophiuroid Astrocyclus caecilia, the holothuroid Holothuria lentigenosa, and the asteroids Astropecten articulatus, Luidia clathrata, Narcissia trigonaria, and Tethyaster grandis at a concentration of 3.0 mg/ml seawater.

Rates of settlement for barnacle cyprid and bryozoan coronate larvae in seawater alone (control) ranged from 50.4 to 78.3 and 62.1 to 98.9%, respectively (Fig. 4 and Fig. 5). Barnacle and bryozoan larvae settled significantly less frequent in petri dishes containing body-wall extracts of all echinoderms tested at a concentration of 3.0 mg/ml seawater. Moreover, 15 of 20 extracts tested sig- nificantly inhibited settlement of both types of larvae at 0.6 mg/ml seawater. Eight of the 20 echinoderm body-wall extracts significantly inhibited bryozoan settle- ment to the lowest extract concentration tested (0.0048 mg/ml seawater), while only one of 20 extracts inhibited barnacle settlement at a similar concentration. In contrast, body-wall extracts of the asteroids Tethyaster grandis and Echinaster sp. significantly increased the percent of barnacle larval settlement at a concentration of 0.6 mg/ml seawater. The body-wall extract of the asteroid G. tesselatus was the most potent of all extracts tested, with significant activity against both species of larvae at all concentrations tested.

4. Discussion

The effect of echinoderm compounds on bacterial and larval fouling of echinoderm body-wall surfaces was not tested in this study. Our experimental approach utilizes plastic surfaces as the attachment substratum in our laboratory attachment experiments. This design provides an evaluation of the general effects of polar compounds extracted from echinoderm body-wall tissues on bacterial and larval attachment. The data presented in this study may not reflect the true

88 P. Bryan et al. I J. Exp. Mar. Bid. Ed. 196 (1996) 79-96

23 AsmoplIytm 2 2

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d 2 Y

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u

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b a 0

Concentration of Extract (mg/ml seawater)

Fig. 3. Mean number of attached cells from the marine bacteria Alteromonas ltrteo-violacea (n = 4). The asterick (*) indicates a statistically significant diference in the mean number of cells attached in

the experimental wells from the controls using ANOVA with a post-hoc Scheffe’s F-test (a = 0.0s).

Note that the y-axis is different to account for variation between experiments.

activity of echinoderm compounds in nature or attachment of bacteria and larvae to living tissues. Nonetheless, these compounds are the polar/water-soluble components of the echinoderm body-wall tissues, and are likely to be detectable by larvae or bacteria within close proximity of echinoderm surfaces in nature. The activity of surface active steroidal glycosides of echinoderms has been well studied (Mackie et al., 1975). These compounds, which are extractable in aqueous ethanol, are detectable by many marine organisms over great distances. The design of the laboratory assays involves the extracts being solublized in filtered seawater at various concentrations prior to being added to plastic dishes. When the bacteria or larvae are suspended in this seawater, they are being constantly exposed to the compounds in the seawater at the experimental concentration.

P. Bryan et al. I J. Exp. Mar. Biol. Ecol. 196 (1996) 79-96 89

Concentration of Extract (mg / ml seawater)

Fig. 4. Percent setlement of barnacle cyprid larvae after exposure to ethanolic echinoderm body-wall

extracts. Astericks indicate statistically significant differences from controls (p < 0.05; G-test of

Independence) (n = 3 for each treatment).

This situation attempts to mimic conditions in the seawater near an echinoderms surface. The activity of these compounds on the larvae is likely a toxic effect. If the effect is toxic, the modification of plastic surface properties by interaction with extracted compounds becomes irrelevant, as the larvae and bacteria are being affected by the compounds while suspended in the water.

Bacterial attachment is one of the primary stages of colonization of surfaces in the marine environment (ZoBell and Allen, 1935). Bacterial films which colonize surfaces can potentially induce, inhibit, or have no effect on the succesion of fouling organisms (Mimh et al., 1981; Maki et al., 1988). Our results indicate that while the body-wall extracts of some echinoderms, such as the asteroids G. tesselatus and Luidia clathrata and the basket star Astrophyton muricatum inhibit

90 P. Bryan rt al. I J. Exp. Mar. Bid. Ed. 196 (1996) 79-96

Fig. 5. Percent settlement of coronate hryozoan larvae after exposure to ethanolic echinoderm

body-wall extracts. Astericks indicate statistically significant differences from controls ( ,D < 0.05; G-test

of Independence) (n = 3 for each treatment).

the attachment of marine bacteria in a microtiter plate assay, body-wall extracts from other species of echinoderms can enhance bacterial attachment (e.g. the asteroids Astropecten articulatus, Linckia nodosus, and the crinoid Comactiniu merdionalis). The adaptive significance of enhancing the attachment of marine bacteria to body-wall tissues in echinoderms is unknown.

Epithelial bacteria have been demonstrated to aid in nutrient transport across the epidermis of marine organisms (Goering and Parker, 1972; Lynch et al., 1979; Giere and Langheld, 1987). It has been suggested that epidermal cells of seastar larvae can readily phagocytize bacterial symbionts and derive energy from them (Bosch, 1992). The induction of bacterial attachment by compounds extracted

P. Bryan et al. I J. Exp. Mar. Biol. Ecol. 196 (1996) 79-96 91

from an echinoderms body-wall tissue could be explained as an adaptation to increase nutrient uptake through facilitating bacterial colonization. However, it is unknown if the cuticle of healthy echinoderms becomes colonized by bacteria and if bacteria that colonize the cuticle could be transported into the sub-cuticular space. One of the primary difficulties in studying marine bacteria is the inability of most to be cultured in the laboratory. Many questions concerning the actual colonization of living surfaces and origin of subcuticular bacteria are in need of further study (McKenzie and Kelly, 1994). As the normal flora of many organisms aid in protection from infection (e.g. Gil-Turnes et al., 1989) it is also possible that these echinoderms tolerate colonization of their surfaces by bacteria which aid in their immune defenses. Moreover, Maki et al. (1988) reported that a surface film of D. marina inhibits the attachment of cyprid larvae of BaZunus amphitrite. Therefore, bacterial epithelial growth could benefit echinoderms by providing a refuge from macroinvertebrate fouling.

Other investigators who have addressed bacterial fouling, have designed experiments to measure the inhibition of bacterial growth (Uriz et al., 1992; Walls et al., 1993). Growth inhibition would take place after bacteria had already attached to a surface. Some attempts have been made to correlate antimicrobial activity of extracts from marine invertebrates with their degree of surface fouling (Thompson et al., 1985; Walls et al., 1993). The inhibition of bacterial growth and bacterial attachment may or may not be related processes. If the inhibition of bacterial growth and attachment are unique processes, they may be governed by different types of chemical and/or physical defenses. Bryan et al. (1994) demon- strated that ethanolic extracts from G. tesselutus had no effect on the growth of D. marina, while the same extract significantly inhibited the attachment of this bacterial species. Additionally, extracts of the asteroids G. tessefutus and Tamaria halperni inhibited growth of A. luteo-violacea, but only the extract of G. tesselatus affected its attachment. There was an increase in the percent attachment of D. marina in wells containing the extracts of Astropecten articulatus and Tamaria halperni at only a concentration of 0.024 mg/ml seawater. This indicates that there is likely to be a mixture of inducing and inhibiting compounds in the extract (e.g. Standing et al., 1982). At this concentration, 0.024 mg/ml seawater, we can speculate that the inhibitor is no longer effective while the inducing agent retains its activity. These types of observations indicate that organisms may possess compounds which affect bacterial attachment, growth, or both, and that different compounds may be responsible for each specific activity.

The body-wall extracts of all species of echinoderms investigated in this study significantly inhibited the settlement of both barnacle and bryozoan larvae at concentrations at or below natural tissue levels. This suggests that regardless of taxonomic class, all echinoderms studied possess compounds which may deter fouling by barnacles and bryozoans. Extracts from sponges (Sears et al., 1990), ascidians (Davis, 1991), and octocorals (Vitalina et al., 1991) are active at similar concentrations. It is likely that organisms from many phyla produce compounds which aid in the prevention of fouling. Once more is known of the compound structures and specific activity, comparisons can be made between the evolution of

92 P. Bryan et al. I J. Exp. Mar. Rid. Ed. 196 (1996) 79-96

chemical defenses among phyla. Bryozoan larvae were more sensitive to ech- inoderm body-wall extracts than barnacle larvae. Fourteen of 20 (70%) ech- inoderm body-wall extracts inhibited settlement in coronate bryozoan larvae at concentrations as low as 0.12 mg/ml seawater, whereas 9 of 20 (45%) inhibited barnacle cyprid settlement at a similar concentration.

Clark (1921) reported several species of hydroids fouling the stalk and cirri of comatulid crinoids, while Gravier (1918) reported the anemone Sicyopus com-

mensafis attached to the body-wall surface of the holothuroid Pseudostichopus villosus. The asteroid Leptometru phalangium was fouled by several species of bryozoans including Microporella ciliata, Smittina landsborovii, Tubilopora

liliacea, and Lichenoporu harmeri (Gautier, 1959). Cidaroid urchins are unique among the echinoderms in having their spine surface devoid of an epithelium. The cidaroid Heterucentrotus trigonaris has been reported with its spines heavily fouled by the corraline algae Fosliella furinosu (Lawrence and Dawes. 1969). Clearly, body-surfaces lacking an epidermis from which organic compounds can be synthesized and released are particularly susceptible to fouling.

The true function of the compounds present in the tissues of these echinoderms remains unknown. Future studies which assay echinoderm compounds under natural field conditions will provide more information on their role in echinoderm defenses. Once techniques for the culture of marine bacteria become further developed, studies addressing the bacterial colonization of tissues and origin of bacteria in the subcuticular space can be pursued. Observations of fouled echinoderm tissues are rare. Further descriptions of fouled echinoderms may also provide more insight of the defenses of those echinoderms which are never fouled. The physical properties of echinoderm body-wall surfaces and sloughing of tissues are also possible antifouling defenses that require further investigation in echinoderms.

Acknowledgments

We thank Dr. T.S. Hopkins for the identification of the echinoderms used in this study. We would also like to thank Dr. C. Shea and Dr. J.J. Gauthier for advice concerning the bacterial attachment assay. Kim Cook identified the species of marine bacterium. Dr. A. Clare and M. McCleary aided in the rearing of competent barnacle cyprid larvae. Dr. G. Poirier kindly donated the use of his centrifuge and Flow Laboratories Multiscan Microplate Reader during bacterial attachment assays. We would also like to thank members of P.J. Bryan’s graduate committee; Dr. J.M. Lawrence, Dr. T.S. Hopkins, Dr. J.J. Gauthier, and Dr. K. Marion for helpful comments concerning this manuscript. We would additionally like to thank Dr. J.D. McKenzie and one anonymous reviewer for their comments which improved this manuscript. This work was funded by NSF EPSCoR grant # EHR9108761 to J.B.M.. T.S.H. and K.M. and S.A. Watts.

P. Bryan et al. I J. Exp. Mar. Biol. Ecol. 196 (1996) 79-96 93

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