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CHEMICAL COMPOSITION and RELEASE in SITU DUE to INJURY of the INVASIVE CORAL Tubastraea (CNIDARIA, SCLERACTINIA)*

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CHEMICAL COMPOSITION and RELEASE in SITU DUE to INJURY of the INVASIVE CORAL Tubastraea (CNIDARIA, SCLERACTINIA)* BRAZILIAN JOURNAL OF OCEANOGRAPHY, 58(special issue IICBBM):47-56, 2010 CHEMICAL COMPOSITION AND RELEASE IN SITU DUE TO INJURY OF THE INVASIVE CORAL Tubastraea (CNIDARIA, SCLERACTINIA)* Bruno G. Lages 1**, Beatriz G. Fleury 2, Cláudia M. Rezende 3, Angelo C. Pinto 3 and Joel C.Creed 2 1Universidade do Estado do Rio de Janeiro Programa de Pós-graduação em Ecologia e Evolução, IBRAG (PHLC Sala 524, Rua São Francisco Xavier 524, 20559-900 Rio de Janeiro, RJ, Brasil) 2Universidade do Estado do Rio de Janeiro Departamento de Ecologia, IBRAG (PHLC Sala 220, Rua São Francisco Xavier 524, 20559-900 Rio de Janeiro, RJ, Brasil) 3Universidade Federal do Rio de Janeiro (Instituto de Química, 21.945-970, Rio de Janeiro, RJ, Brasil) **Corresponding author: [email protected] A B S T R A C T Defensive chemistry may be used against consumers and competitors by invasive species as a strategy for colonization and perpetuation in a new area. There are relatively few studies of negative chemical interactions between scleratinian corals. This study characterizes the secondary metabolites in the invasive corals Tubastraea tagusensis and T. coccinea and relates these to an in situ experiment using a submersible apparatus with Sep-Paks ® cartridges to trap substances released by T. tagusensis directly from the sea-water. Colonies of Tubastraea spp were collected in Ilha Grande Bay, RJ, extracted with methanol (MeOH), and the extracts washed with hexane, dichloromethane (DCM) and methanol, and analyzed by GC/MS. Methyl stearate and methyl palmitate were the major components of the hexane and hexane:MeOH fractions, while cholesterol was the most abundant in the DCM and DCM:MeOH fractions from Tubastraea spp . The organic material retained in Sep- Paks ® cartridges was tentatively identified as hydrocarbons. There was a significant difference between treatments and controls for 1-hexadecene, n-hexadecane and n-eicosane contents. The production of defensive substances by the invasive corals may be a threat to the benthic communities of the region, which include endemic species. R E S U M O Substâncias químicas de defesa contra consumidores e competidores podem ser usadas por espécies invasoras marinhas como estratégia de colonização e perpetuação em novo ambiente. Entretanto, há poucos estudos experimentais que demonstrem as possíveis interações negativas entre corais escleractínios. Este trabalho tem como objetivo caracterizar os metabólitos secundários dos corais invasores Tubastraea tagusensis e T. coccinea ; avaliar através da técnica de amostragem in situ quais são as substâncias de T. tagusensis liberadas na água do mar, com o auxílio de aparelho subaquático com colunas Sep-Paks ®. Colônias dos corais invasores Tubastraea spp foram coletadas na Baía de Ilha Grande, RJ, e extraídas com MeOH. Os extratos foram submetidos à eluições com hexano, DCM e MeOH, e analisados por CG-EM. Estearato de metila e palmitato de metila foram as substâncias majoritárias das frações hexânicas e hexano: DCM, enquanto o colesterol foi a substância mais abundante das frações DCM e DCM:MeOH de Tubastraea spp. O material orgânico retido nas colunas Sep-Paks ® foi identificado como hidrocarbonetos. Diferenças significativas entre controle e tratamento foram relacionadas a diferentes quantidades de 1-hexadeceno, n-hexadecano e n-eicosano. A produção de substâncias de defesas em Tubastraea spp permite especular sobre a ameaça que estes corais invasores representam para as comunidades bentônicas da Ilha Grande. Descriptors: Injury, Secondary metabolites, Tubastraea coccinea , Tubastraea tagusensis , Submersible apparatus. Descritores: Lesão artificial, Metabólitos secundários, Tubastraea coccinea , Tubastraea tagusensis , Aparelho submersível. __________ (*) Paper presented at the 2 nd Brazilian Congress of Marine Biology, on 24-28 May. Búzios, RJ, Brazil. 2009. 48 BRAZILIAN JOURNAL OF OCEANOGRAPHY, 58(special issue IICBBM), 2010 INTRODUCTION use of large quantities of solvents for extraction (COLL et al., 1982; SCHULTE et al., 1991). The ahermatypic coral T. tagusensis Wells, Chemical defense against consumers and 1982 is a scleractinian coral non-indigenous to the competitors have been studied in great detail for South Atlantic and was probably introduced into sessile invertebrates and plants and the detailed Brazil in the late 1980's (CASTRO; PIRES, 2001) mechanisms, in several instances, have been jointly with T. coccinea Lesson, 1829. It is well- elucidated by manipulative field experiments (HAY; established on the rocky shores of Ilha Grande Bay, FENICAL, 1988; DE NYS et al., 1991; HAY; STEIN southeastern Brazil (PAULA; CREED, 2004). These BERG, 1992; PAWLIK, 1993; SCHMITT et al., 1995; species are non-zooxanthellate corals which mainly THACKER et al., 1998; IRFANULLAH; MOSS, inhabit overhangs and vertical surfaces (PAULA; 2005). In the marine environment, the success of CREED, 2005). Studies have demonstrated that this defense used by soft corals and gorgonians against genus produces mainly steroids and alkaloids and that consumers and competitors has been attributed to their some are toxic to cells and bioactive (FUSETANI et production of secondary metabolites, many of which al., 1986; GUELLA et al., 1988; RASHID et al. 1995; show predator deterrence and allelopathic activities IWAGAWA et al., 2008; MEYER et al., 2009). They (COLL et al., 1982; PAWLIK et al., 1987; DE NYS et also may function against competitors (KOH, 1997). al., 1991; VAN ALSTYNE et al., 1994; KELMAN et Therefore, the chemical study of these alien species is al., 1999; KOH et al., 2000; LAGES et al., 2006; important to understand the behavior in colonization CHANGYUN et al., 2008). and range expansion. The compounds produced by Despite the fact that hard corals produce a Tubastraea may be aiding these species to establish protective external skeleton some experiments have and persist among the endemic flora and fauna and so showed that these also produce bioactive compounds change community structure. (TARRANT et al., 2003; KONTIZA et al., 2006) This study described the secondary some of which are deterrent to fish (LAGES et al., metabolites in the invasive corals T. tagusensis and T. 2010) and may function as allelochemicals (KOH; coccinea , and evaluated, in a field assay, whether T. SWEATMAN, 2000). tagusensis releases secondary metabolites in seawater The compounds involved in allelopathic and whether the method applied here may collect, in interactions can act in different concentrations situ , these compounds. (repellent or toxic) and distances and some of these are soluble or volatile so as to diffuse away from the source (HADFIELD; SCHEUR, 1985; SLATTERY et MATERIAL AND METHODS al., 1997, VAN ALSTYNE et al., 2001). Others may be deposited on the surface of the organism and act by Coral material and extraction: T. coccinea and T. direct contact with potential competitors. The tagusensis were collected at a depth of production of secondary metabolites likely requires approximately 3 m from tropical rocky shores at considerable cost to the organism (COLL, 1992) Ilha Itacoatiba (23°4'00"S and 44°15'00"W), because the resources allocated to production will not, located on the southeastern coast of Brazil in the therefore, be available for other processes such as state of Rio de Janeiro. After collection, the growth and reproduction (VAN ALSTYNE et al., colonies were immediately frozen at -25°C. Pieces 2001). of different colonies of T. coccinea (total weight The secondary metabolites used against 665 g) and of T. tagusensis (total weight 666 g) consumers and competitors may affect the behavior were extracted 3 times consecutively using and distribution of organisms in marine systems methanol (MeOH) as solvent and an ultrasound (LUBCHENCO; GAINES, 1981; ESTES; machine was used to increase the efficiency of STEINBERG, 1988; KVITEK et al., 1991; COLL, extraction. Evaporation of this solvent under 1992; STACHOWICZ, 2001). However, compounds reduced pressure yielded two corresponding brown responsible for the competitive effects have rarely residues (11.0 g and 9.6g, respectively), that were been isolated and identified (COLL et. al., 1982; submitted to an open silica gel column SULLIVAN et al. 1983, DE NYS et al. 1991). The use chromatography with hexane, dichloromethane of the submersible sampling apparatus described by (DCM) and MeOH elution that produced different Schulte and collaborators (1991) is a method available fractions: hexane (19.1 mg and 6.4 mg), to collect, in situ , trace quantities of organic hexane:DCM (1:1) (8.8 mg and 4.5 mg), DCM compounds from seawater. This method provides a (17.8 mg and 6.2 mg) and DCM:MeOH (1:1) simple means of sampling the water around aquatic (278.9 mg and 0.4 mg), respectively. Their organisms for bioactive substances thus avoiding the secondary metabolites were compared after being analyzed by GC-MS. LAGES ET AL.: CHEMICAL COMPOSITION OF INVASIVE CORAL Tubastraea 49 Chemical analysis : The gas chromatography mass SCUBA tank (Fig. 2). Previous studies have suggested spectrometry (GC/MS) analysis for substances that sampling procedures which involve plastic hoses from MeOH fractions were carried out in a HP or other similar material should be avoided since the Model 5973 with electron impact ionization at 70 hose has a strongly lipophilic surface capable of eV. A HP5 MS capillary column (30 m × 0.25 adsorbing the allelopathic compounds to be examined, mm; coating thickness 0.25 µm / J&W Scientific) while, at the same time, releasing considerable was used. Sample volumes of 1 µl (DCM solution) quantities of plasticizers (FAULKNER et al., 1980). were injected in a split mode ratio (1:20) using a We used an apparatus constructed using silicon hoses manual syringe. Helium was employed as carrier to avoid adsorption of compounds by the apparatus. gas. Oven temperature was programmed from Before the field experiment, the apparatus 60°C to 290°C at 15°C min -1, where it remained were previously tested in a pool by using ink to verify constant for 15 minutes. Injector and detector the rate of flow of water moving through Sep-Paks ® temperature were kept constant at 280 and 290°C, columns.
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