Chemical Antifouling Defences of Sea Stars: Effects of the Natural Products Hexadecanoic Acid, Cholesterol, Lathosterol and Sitosterol

Vol. 385: 137–149, 2009 MARINE ECOLOGY PROGRESS SERIES Published June 18 doi: 10.3354/meps08034 Mar Ecol Prog Ser Chemical antifouling defences of sea stars: effects of the natural products hexadecanoic acid, cholesterol, lathosterol and sitosterol Jana Guenther1, 2, 4,*, Anthony D. Wright2, 3, 5, Kathryn Burns3, Rocky de Nys1, 2 1School of Marine and Tropical Biology, and 2AIMS@JCU, James Cook University, Townsville, Queensland 4811, Australia 3Australian Institute of Marine Science, Townsville, Queensland 4810, Australia 4Present address: Centre for Research-based Innovation in Aquaculture Technology, SINTEF Fisheries and Aquaculture, 7465 Trondheim, Norway 5Present address: College of Pharmacy, University of Hawaii, Hilo, Hawaii 96720, USA ABSTRACT: The role of natural products in keeping the surfaces of the sea stars Linckia laevigata, Fromia indica, Cryptasterina pentagona and Archaster typicus free of fouling organisms was investigated. Conditioned seawater of these sea stars did not have any effects on the settlement of the ecologically relevant diatoms Amphora sp. and Nitzschia closterium and the bryozoan Bugula neritina. However, dichloromethane, methanol and aqueous extracts of whole sea stars at 100, 10, 1 and 0.1 µg cm–2 had concentration-dependent effects on the settlement of these fouling species and the polychaete Hydroides elegans. Based on bioassay-guided fractionation and analysis with proton nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry (GC-MS), the most bioactive fractions contained several fatty acids and sterols. To determine whether the compounds responsible for the observed antifouling effects were present on the surface of all 4 sea star species, surface-associated compounds were tested against Amphora sp., N. closterium, B. neritina and H. elegans. These compounds reduced the settlement of at least 2 of the 4 fouling species for each sea star species. Using surface extractions and GC-MS analysis, the most abundant surface-associated fatty acids and sterols of each sea star species were identified and quantified. Hexadecanoic acid, cholesterol, lathosterol and sitosterol were tested at surface-associated concentrations, ranging from 1000 to 1 ng cm–2, in settlement assays. Whereas the settlement of B. neritina was not affected by any of the compounds, hexadecanoic acid, cholesterol and lathosterol significantly reduced the settlement of N. closterium at surface-associated concentrations. KEY WORDS: Biofouling · Sea star · Fatty acid · Sterol · Diatom Resale or republication not permitted without written consent of the publisher INTRODUCTION rate and survival of bivalves (Lodeiros & Himmelman 1996, Taylor et al. 1997, Kaehler & McQuaid 1999, Marine biofouling, which is the formation of a com- Thieltges & Buschbaum 2007) and affect the condition plex layer of organisms on submerged surfaces, can and function of the appendages of crabs (Botton 1981, have negative ecological impacts on living surfaces Cadee 1991). Many marine plants and animals have (reviewed by Wahl 1989, de Nys & Steinberg 1999). evolved behavioural, mechanical, physical and/or For example, biofouling may reduce the photosynthe- chemical defences to reduce these disadvantages and sis and growth of marine algae (Dixon et al. 1981, ecological costs (reviewed by Wahl 1989). In common Drake et al. 2003), affect the shell condition, growth with many organisms investigated for natural antifoul- *Email: [email protected] © Inter-Research 2009 · www.int-res.com 138 Mar Ecol Prog Ser 385: 137–149, 2009 ing defences, sea stars generally have surfaces that are predict any antifouling role of compounds, because remarkably free of fouling organisms (McKenzie & natural products, which are only found within cells and Grigolava 1996, Guenther et al. 2007a). However, me- would never be found on exposed surfaces under nat- chanical antifouling defences associated with the pedi- ural conditions, may also be extracted and have an cellariae of the crown-of-thorns sea star Acanthaster inhibitory effect on fouling organisms (Nylund & Pavia planci (Guenther et al. 2007b) and physical antifouling 2003, Nylund et al. 2007). To determine whether ma- defences associated with the wettabilities and surface rine organisms use natural products to keep their sur- microtopographies of the sea stars Linckia laevigata, faces free of fouling organisms, several criteria need to Fromia indica, Cryptasterina pentagona and Archaster be fulfilled (Schmitt et al. 1995, Hay 1996, Nylund & typicus (Guenther & de Nys 2007) are inefficient and Pavia 2003, Dworjanyn et al. 2006, Nylund et al. 2007). do not explain how these sea stars keep their surfaces First, the marine organisms need to be generally free free of fouling organisms. In contrast, crude extracts of fouling organisms in their natural environment. Sec- and isolated compounds from sea star tissues have ond, the presence of putative compounds on or near commonly been tested against selected fouling organ- the surface of the organism needs to be verified and isms using disc diffusion assays (Bryan et al. 1994, Ior- their natural concentrations quantified. Third, surface- izzi et al. 1995), motion analysis assays (Iken et al. associated concentrations of these compounds need to 2001, 2003, Greer et al. 2006) and settlement assays be tested in bioassays with ecologically relevant foul- (Iorizzi et al. 1995, Bryan et al. 1996, De Marino et al. ing species to determine whether these compounds 2000, Greer et al. 2003). Iorizzi et al. (1995) demon- have any antifouling effects. strated that the ethanol whole body extracts of the sea The aim of the present study was to identify surface- star Luidia clathrata inhibited the growth of the bacte- associated chemical defences against fouling in the ria Bacillus subtilis and Staphylococcus aureus. Fur- tropical sea stars Linckia laevigata, Fromia indica, thermore, spores of the brown alga Hincksia irregu- Cryptasterina pentagona and Archaster typicus by laris exposed to low concentrations (<91 µg ml–1) of investigating the effects of conditioned seawater, aqueous and organic body wall extracts of the sea stars whole body extracts and selected surface-associated Astropecten articulatus and L. clathrata changed their compounds of these sea stars on the settlement of the normally fast and straight swimming movements to ecologically relevant diatoms Amphora sp. and Nitz- helical and erratic (Iken et al. 2003). At the highest schia closterium, the serpulid polychaete Hydroides concentration (909 µg ml–1), spores were severely dam- elegans and the bryozoan Bugula neritina, which are aged or completely immobilized (Iken et al. 2003). commonly found in tropical and subtropical waters Finally, isolated asterosaponins of the sea star Gonio- (Hall 1984, Hallegraeff & Jeffrey 1984, Lewis et al. pecten demonstrans reduced the settlement of the 2006, Gottschalk et al. 2007). brown alga H. irregularis (De Marino et al. 2000), whereas water and acetone body wall extracts of the sea stars Astropecten articulatus and L. clathrata had MATERIALS AND METHODS significant effects on the settlement and germination of H. irregularis (Greer et al. 2003). Ethanol body wall Collection of specimens. Live specimens of the sea extracts of 13 sea star species reduced the settlement of stars Linckia laevigata and Fromia indica were col- the barnacle Balanus amphitrite and the bryozoan lected from John Brewer Reef (18° 38’ S, 147° 03’ E), Bugula neritina (Bryan et al. 1996). Of these, the Cryptasterina pentagona from Kissing Point in extract of the sea star Goniaster tesselatus was the Townsville (19° 14’ S, 146° 48’ E) and Archaster typicus most active, reducing the settlement of Balanus am- from Picnic Bay on Magnetic Island (19° 11’ S, phitrite and Bugula neritina at concentrations of 146° 51’ E), Queensland, Australia, in February 2006. ≥4.8 µg ml–1 (Bryan et al. 1996). Similarly, the ethanol Specimens were transported in ambient seawater and whole body extracts of the sea star L. clathrata inhib- kept temporarily in an outdoor recirculating tank ited the settlement of Balanus amphitrite and Bugula (26°C, 35 ppt) at the Marine and Aquaculture Research neritina at concentrations of ≥120 and ≥4.8 µg ml–1, Facilities Unit at James Cook University. respectively (Iorizzi et al. 1995). Conditioned seawater. To test the antifouling activ- Testing whole-cell crude extracts of unfouled marine ity of waterborne compounds of Linckia laevigata, Fro- species against the settlement of ecologically relevant mia indica, Cryptasterina pentagona and Archaster fouling organisms screens the widest possible range of typicus using conditioned seawater, specimens of each compounds present within tissues, where synergism species (n = 5) were blot-dried, weighed and then kept between compounds of an extract may be important individually in plastic containers (L. laevigata) or glass (Teo & Ryland 1995). Results from settlement assays beakers (F. indica, C. pentagona and A. typicus) with with whole-cell extracts, however, are insufficient to 0.45 µm filtered seawater (FSW) (75 ml FSW per 1 g of Guenther et al.: Chemical antifouling defences of sea stars 139 sea star) and aerated for 24 h. Seawater controls were Dry extracts were kept individually and stored at prepared by keeping FSW in plastic containers and –18°C until they were redissolved for use in settlement glass beakers for the same amount of time. assays. Natural concentrations (mg g–1) of dichloro- Bioassays with conditioned

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