Defense Against Epibiosis in the Sponge Mycale Adhaerens: Modulating the Bacterial Community Associated with Its Surface

AQUATIC MICROBIAL ECOLOGY Vol. 43: 55–65, 2006 Published May 3 Aquat Microb Ecol Defense against epibiosis in the sponge Mycale adhaerens: modulating the bacterial community associated with its surface On On Lee, Stanley C. K. Lau, Pei-Yuan Qian* Department of Biology/Coastal Marine Laboratory, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, SAR ABSTRACT: It has long been hypothesized that sponges can produce bioactive compounds to modulate bacterial communities on their body surfaces and that the modulated bacterial communities can protect the sponge host from being colonized by other invertebrates. Conventionally, testing of this hypothesis has relied on bioassays involving bacteria isolated from ecologically relevant sites. How- ever, this approach generally suffers from the fact that only <1% of the total bacterial population in the natural environment is culturable using existing techniques. In this study, a bacterial culture- independent method was employed to investigate if the metabolites of the sponge Mycale adhaerens could render surface-colonizing bacterial communities inhibitive to larval settlement of Hydroides elegans, a polychaete worm that co-exists with M. adhaerens in Hong Kong waters. Organic extracts of the sponge tissue were immobilized in hydrogels and then exposed to a continuous flow of natural seawater for the development of microbial films. Epifluorescence microscopy and DNA fingerprint- ing analysis of bacterial community in the microbial films revealed that the extracts drastically altered bacterial community composition on the hydrogel surfaces. Subsequently, these microbial films demonstrated either an anti-settlement or toxic effect on the larvae of H. elegans. These results suggested that sponge extracts can potentially prevent epibiosis via the modulation of sponge- associated bacterial communities. KEY WORDS: Mycale adhaerens · Hydroides elegans · Surface bacterial community · Bioactive compounds · Bacterial density · Culture-independent method · Hydrogels Resale or republication not permitted without written consent of the publisher INTRODUCTION hosts for nutrients and resources (Witman & Suchanek 1984, Davis et al. 1989, Wahl 1989, Lesser et al. 1992). The life cycle of many marine sessile organisms Furthermore, the hosts may suffer from tissue damage includes a free-living planktonic larval stage. Larvae or reduced growth due to an increase in weight, rigid- may spend hours to months in the water column before ity and drag (Dixon et al. 1981, Keough 1984, Wahl & settling on a substratum and eventually developing Mark 1999). into adults. Larvae may settle on virtually any surface It is a widely accepted ecological concept that soft- deemed suitable for their subsequent growth and sur- bodied invertebrates have evolved defensive mecha- vival, including the body surfaces of other organisms. nisms to prevent epibiosis. These mechanisms are For soft-bodied invertebrates, such as corals, sponges believed to involve the production of bioactive com- and ascidians, the consequences of allowing larvae of pounds which inhibit larval settlement of epibiotic other organisms to settle on their body surfaces (i.e. organisms. A number of low-molecular weight, anti- epibiosis) can be detrimental. For instance, epibiotic larval settlement substances have been discovered organisms may obstruct feeding, gas exchange and from sponges (Keifer et al. 1991, Hirota et al. 1996, waste excretion in the hosts, and compete with the Okino et al. 1996), gorgonian corals (Standing et al. *Corresponding author. Email: [email protected] © Inter-Research 2006 · www.int-res.com 56 Aquat Microb Ecol 43:55–65, 2006 1984, Gerhart et al. 1988) and ascidians (Davis & of sponge metabolites against epibiosis (Lee & Qian Wright 1990). There is growing evidence for the hy- 2003). The aim of this study was to investigate a possi- pothesis that soft-bodied invertebrates may produce ble indirect protective role of sponge metabolites bioactive compounds to modulate the bacterial com- against epibiosis via the modulation of bacterial communities associated with their body surfaces so as to munities associated with the surface of M. adhaerens. render their body surfaces unsuitable for larval settle- To overcome the limitations and biases due to utiliza- ment of epibiotic organisms (Lee & Qian 2003, tion of bacterial-culture-dependent assay techniques, Dobretsov & Qian 2004, Dobretsov et al. 2005). This we employed a recently developed culture-indepen- hypothesis is largely based on the following 3 argu- dent technique, in which organic extracts of sponge ments. Firstly, all surfaces submerged in the marine tissue were embedded into hydrogel matrix. The environment, including those of soft-bodied inverte- hydrogels were then exposed to the natural bacterial brates, are covered with microbial films, which are population in flow-through seawater for the develop- mainly composed of bacteria, fungi, algae and proto- ment of microbial films. Hydrogels coated with micro- zoa (Wahl 1989). Secondly, the body tissues of many bial films were subsequently subject to larval set- soft-bodied invertebrates (e.g. soft-corals and sponges) tlement bioassay. The effect of sponge extract on are rich in antibacterial compounds (Amade et al. modulation of bacterial community was analyzed 1987, Aceret et al. 1998). Thirdly, it is believed that the using epifluorescence microscopy and a DNA finger- composition and physiology of microbial films, espe- printing technique. cially the bacterial population, can have drastic effects on larval settlement of many marine invertebrates (Wieczorek & Todd 1997, Maki 1999, Harder et al. MATERIALS AND METHODS 2002, Qian et al. 2003). Previous investigations of this hypothesis have Collection and extraction of sponge tissue. Mycale mainly relied on the standard agar disc-diffusion assay adhaerens was collected at a fish farm in Long Har- to demonstrate antibacterial effects of bioactive com- bour, Hong Kong (22° 27.33’ N, 114° 21.00’ E). Col- pounds produced by soft-bodied invertebrates (Acar lected sponge colonies were flushed with autoclaved 1980). However, the agar disc diffusion assay, which 0.22 µm filtered seawater (AFSW) to remove loosely was originally developed for clinical testing of antibi- attached bacteria and sealed in sterile plastic bags for otics on pure bacterial cultures, has limited applicabil- transportation to the laboratory. Approximately 550 ml ity for ecological studies. The limitation is largely due of sponge tissue were blot-dried, cut into small pieces to the fact that only an insignificant proportion (<1%) and extracted twice in 550 ml of 1:1 mixture of of bacteria living in natural habitats is culturable using methanol/chloroform (MeOH/CHCl3) for 12 h each existing techniques (Pace 1996). Therefore, investiga- with gentle agitation. This 1:1 mixture of methanol/ tions relying on culturable bacteria to reveal the effects chloroform solvent system could extract compounds of of bioactive substances on natural bacterial community a wide range of polarity, including both polar and non- usually result in rather fragmented and more specula- polar metabolites from the sponge tissue. After extrac- tive conclusions (Harder et al. 2004). Moreover, in tion, sponge tissue was removed from the solvents by almost all previous studies, efforts have been made to centrifugation. Then, the organic extract was sepa- examine either the antimicrobial activities of bioactive rated into 2 layers (MeOH and CHCl3) in a separation compounds from soft-bodied invertebrates or the anti- funnel and each layer was dried by rotary evaporation. larval settlement activities of the associated microbes. In total, 10.1 and 14.3 g dry weight of MeOH and The correlation between antimicrobial activities with CHCl3 extracts were obtained, respectively. reduced larval settlement has rarely been examined in Gel immobilization of tissue extracts and prepara- detail. tion of gel discs. The dry MeOH and CHCl3 extracts The sponge Mycale adhaerens usually occurs in were re-dissolved in 1/20 of the original sponge tissue well-established benthic communities where open (i.e. 27.5 ml) of double-distilled water (ddH2O) and substratum space is limited; its body surface is con- CHCl3, respectively. This resulted in 2 concentrated ex- stantly under the pressure of colonization by other tracts, both at a concentration equivalent to 20× tissue organisms, such as the larvae of many benthic inverte- level concentration (TLC). Extracts at 2 and 10× TLC brates, but is rarely fouled. By demonstrating an in- were also prepared by dilution with corresponding sol- hibitive effect of the waterborne metabolites from vents. Gel immobilization of the organic extracts was M. adhaerens on larval settlement of Hydroides ele- adopted from Henrikson & Pawlik (1995) and Harder et gans (a polychaete that is usually found in the same al. (2004) with slight modifications. Briefly, 1 ml of habitat of M. adhaerens but not on its surface), our pre- aliquots of each extract at 2, 10 and 20× TLC were vious investigation suggested a direct protective role transferred to individual 15 ml sterile polypropylene Lee et al.: Anti-epibiosis mechanisms in sponges 57 tubes (Falcon 2096), each of which con- Sponge organic extracts (MeOH or CHCl ) tained 9 ml of 4% (w/v) hydrogel (Phy- 3 TM tagel , Sigma Chemical) solution pre- Immobilization of extracts pared in hot ddH2O (ca. 70°C). The tubes and preparation of gel were closed and turned

Load more