Mar Biotechnol (2009) 11:188–198 DOI 10.1007/s10126-008-9132-7
ORIGINAL ARTICLE
Convergent Antifouling Activities of Structurally Distinct Bioactive Compounds Synthesized Within Two Sympatric Haliclona Demosponges K. E. Roper & H. Beamish & M. J. Garson & G. A. Skilleter & B. M. Degnan
Received: 6 November 2007 / Accepted: 15 July 2008 / Published online: 9 August 2008 # Springer Science + Business Media, LLC 2008
Abstract A wide range of sessile and sedentary marine invertebrates synthesize secondary metabolites that have potential as industrial antifoulants. These antifoulants tend to differ in structure, even between closely related species. Here, we determine if structurally divergent secondary metabolites produced within two sympatric haliclonid demosponges have similar effects on the larvae of a wide range of benthic competitors and potential fouling metazoans (ascidians, molluscs, bryozoans, polychaetes, and sponges). The sponges Haliclona sp. 628 and sp. 1031 synthesize the tetracyclic alkaloid, haliclonacyclamine A (HA), and the long chain alkyl amino alcohol, halaminol A (LA), respectively. Despite structural differences, HA and LA have identical effects on phylogenetically disparate ascidian larvae, inducing rapid larval settlement but preventing subsequent metamorphosis at precisely the same stage. HA and LA also have similar effects on sponge, polychaete, gastropod and bryozoan larvae, inhibiting both settlement and metamorphosis. Despite having identical roles in preventing fouling and colonisation, HA and LA differentially affect the physiology of cultured HeLa human cells, indicating they have different molecular targets. From these data, we infer that the secondary metabolites within K. E. Roper : G. A. Skilleter : B. M. Degnan (*) School of Integrative Biology, University of Queensland, Brisbane 4072 Queensland, Australia e-mail:
[email protected] H. Beamish Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Brisbane 4102 Queensland, Australia M. J. Garson School of Molecular and Microbial Science, University of Queensland, Brisbane 4072 Queensland, Australia
marine sponges may emerge by varying evolutionary and biosynthetic trajectories that converge on specific ecological roles. Keywords Allelochemicals . Biofouling . Invertebrate larvae . Settlement ecology
Introduction Marine environments, such as coral reefs, maintain high levels of biodiversity in intensely competitive communities. Sessile and sedentary marine invertebrates have evolved extensive chemical repertoires to mediate many ecological interactions (Paul 1992; Faulkner 2002; Fusetani 2004). The synthesis of these compounds is considered to be derived from, but independent of, the constitutive pathways that underlie primary cellular metabolism and may occur in either the host invertebrate or within the microbial populations associated with the host (e.g., Piel 2006; Konig et al. 2006). As these secondary metabolites are not involved in normal growth or development, they are often considered as being dispensable to the producer (Haslam 1986; Luckner 1990). However, they serve important roles in many ecological interactions and, hence, may confer an evolutionary advantage. The occurrence and diversity of secondary metabolites in disparate marine invertebrates is well documented (Faulkner 2002; Fusetani 2004; Paul et al. 2006), and in a number of cases, their biological and ecological roles have been demonstrated (reviewed in Williams et al. 1989). The widespread existence of these compounds in particular taxa (e.g., sponges, bryozoans, and ascidians) is compatible with the supposition that secondary metabolites are a conserved and ancient feature of these bauplans. Irrespective of the origin of secondary metabolites (i.e., poriferan, microbial, or a combination of
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both), individuals and species that present allelochemicals appear to have enhanced defenses against biofouling and overgrowth, and greater reproductive success (Paul et al. 2006). Most marine surfaces, both biotic and abiotic, are subject to colonisation by microbes, algae, fungi, and animals (Railkin 2004). In the case of established benthic residents, incoming settlers (biofoulers) potentially present ecological challenges (e.g., niche competitors) and opportunities (e.g., conspecific reproductive partners). The differential and often specific responses of a wide range of larvae to signals emanating from different biotic and abiotic substrata reflect selection upon larval sensory systems and behaviour (Crisp 1974; Pawlik 1992; Rodriguez et al. 1993; Fusetani 1997). Conversely, benthic marine organisms in danger of being overgrown by newly arrived recruits of potential competitors produce bioactive compounds that can prevent successful larvae settlement and thus function as antifouling agents (Crisp 1974; Bakus et al. 1986; Pawlik 1992; Fusetani 1997; Zimmer and Butman 2000; Steinberg et al. 2002). This suite of complex interactions is often played out on built structures, leading to the establishment of fouling communities. Understanding the ecological and chemical basis of these interactions informs efforts to understand the establishment and maintenance of biofouling communities and to discover natural antifoulants (Yebra et al. 2004). Members of the phylum Porifera have been a rich source of bioactive compounds, suggesting that secondary metabolite synthesis has played a central role in their ecology and evolution. This is supported by their success in a wide range of marine habitats, despite an apparent lack of morphological and structural defense (Porter and Targett 1988; Munro et al. 1999; Faulkner 2002; Fusetani 2004). At first glance, the immense structural diversity of compounds produced within different sponges appears counter-intuitive. If the production of ecologically important secondary metabolites is a core feature of sponge bauplan, why do closely related sponge species (i.e., congeners) produce markedly different compounds that may be derived from unrelated metabolic pathways? Conversely, if similar selective forces are at play, why is there not repeated evidence of evolutionary convergence in secondary metabolite chemistries in phylogenetically distant species? Based on chemical diversity, it can be inferred that sponges and their symbionts have evolved the capacity to change the operational secondary metabolic pathways within a short evolutionary time frame. Selective forces do not appear to act upon single genes encoding components of specific pathways but instead on the overall capacity to generate a diversity of compounds, which can impact on conspecifics, and both general and specific competitors. Thus, closely related, sympatric species will often produce markedly different suites of compounds that have essentially the same life history roles.
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To test this supposition, this study has assessed the impact of purified bioactive compounds derived from two Haliclona species on the settlement and metamorphosis of ecologically relevant larvae. These two species are found in the same habitats on Heron Island Reef, often living adjacent to each other (Skilleter et al. 2005). The predominant bioactive compound from Haliclona sp. 628 (Demospongiae: Haplosclerida, Chalinidae: Queensland Museum Voucher G304086) is the tetracyclic alkaloid, haliclonacyclamine A (HA; Charan et al. 1996; Clark et al. 1998). The primary secondary metabolite of the second species, Haliclona sp. 1031 (Queensland Museum Voucher G312726) is a long-chain alkyl amino alcohol, halaminol A (LA; Clark et al. 2001; Fig. 1). Although both HA and compounds structurally related to LA are known to be associated with sponge cell tissue (Garson et al. 1998; Richelle-Maurer et al. 2001), a microbial role in their production cannot be excluded. These compounds have previously been shown to display cytotoxic activities in laboratory-based assays (Clark et al. 1998, 2001). In this study, we test how these compounds affect the colonization of potential benthic competitors—the ascidians Herdmania momus and Ciona intestinalis, the gastropod Haliotis asinina, the polychaete Filograna implexa, the bryozoan Triphyllozoon mucronatum, and the demosponge Amphimedon queenslandica—by assessing their impact on these well-characterized invertebrate larvae (e.g., Degnan et al. 1996; 2005; Nakayama et al. 2001, 2002; Satou et al. 2001; Jackson et al. 2002, 2005; Leys and Degnan 2002; Wanninger et al. 2005). To gain insight into the molecular and cellular mechanisms underlying the impact of these two allelochemicals, we assessed how these compounds influenced human cells in culture (e.g., Carté 1996; Haefner 2003; Livett et al. 2004).
Materials and Methods Extraction of Sponge Allelochemicals HA (C32H56N2, MW 468.8), the major haliclonacyclamine from extracts of Haliclona sp. 628, was isolated from crude organic extracts as previously described (Charan et al. 1996;
a
b NH2
H H N H
N
OH
H
Fig. 1 Structures of sponge allelochemicals. a Haliclonacyclamine A (HA), from Haliclona sp. 628 (Charan et al. 1996; Clark et al. 1998). b Halaminol A (LA), from Haliclona sp. 1031 (Clark et al. 2001)
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Clark et al. 1998). The long-chain alkyl amino alcohol LA (C14H21NO, MW 227.39), which dominates organic extracts of Haliclona sp.1031, was purified according to Clark et al. (2001). Larval Settlement Assays Whatman GFC filter discs (5-cm diameter) were prepared according to Green et al. (2001). Sponge compounds were dissolved in appropriate solvents (DCM for HA and MeOH for LA) to working concentrations, and a 10-μl aliquot was transferred onto filter discs. All experiments were run with controls for the addition of filter discs containing solvent only and for larvae grown in only 0.2-μm filtered seawater (FSW). For assays, larvae were placed in sterile six-well plates in 5 ml of FSW, except for abalone larvae that were assayed in 10 ml of FSW. At competency (Table 1), treatments were added to larvae in each well of a six-well plate, and this was deemed t=0 h. Each treatment was performed in triplicate in each experiment. Between 25 and 40 larvae were used for each replicate, depending on the species. A one-way analysis of variance was used to compare the effects of each treatment at 1 h post-treatment using R Software (http://www.R-project.org). Where significant differences between treatments were detected, a Tukey honestly significant difference (HSD) test was performed for a posteriori comparison of treatment means. Treatments were considered significantly different if p value50% tail resorption) was recorded over time. a, b Allelochemicals tested on Herdmania momus larvae. c, d Allelochemicals tested on Ciona intestinalis larvae. a, c Treatment with purified haliclonacyclamine A (HA) from Haliclona sp.
628. b, d Treatment with purified halaminol A (LA) from Haliclona sp. 1031. Open triangles filtered seawater (FSW) controls, full triangles 40 mM potassium chloride (KCl) positive control for H. momus, open circles 5 μg ml−1 (11 μM) HA, full circles 10 μg ml−1 (22 μM) HA, asterisks 5 μg ml−1 (22 μM) LA, crosses 10 μg ml−1 (44 μM) LA, and bars standard error
death was seen in H. asinina (Fig. 4d). One hundred percent of larvae in corresponding FSW controls were alive. By 4 h post addition of HA or LA, 100% of T. mucronatum, A. queenslandica, and F. implexa larvae were dead; contrasting to FSW controls, which in all cases, showed no larval death. Only a marginal (5%) increase was seen from 1 to 4 h posttreatment in the percentage of necrotic/dead H. asinina larvae in allelochemical treatments. However, many of the abalone larvae in these treatments were seen lying on the side of their shells at the bottom of the culture dish. Tissue blebbing and other morphological signs of necrosis in H. asinina were not evident until 24 h post-treatment. By 24 h, 100% of larvae from all four species were dead in HA or LA treatments. While sponge, polychaete, and gastropod larvae reared in control FSW for 24 h appeared morphologically and behaviorally (i.e., rapidly swimming) healthy, the few T. mucronatum larvae that had not spontaneously settled began to die.
showed an increase in the percentage of cells with a DNA content of less than 2n (sub-G10 population) from
