Characterisation of the physical and chemical properties influencing bacterial epibiont communities on benthic gelatinous egg masses of the pulmonate Siphonaria diemenensis

Journal of Experimental Marine Biology and Ecology 432–433 (2012) 138–147

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Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe

Characterisation of the physical and chemical properties influencing bacterial epibiont communities on benthic gelatinous egg masses of the pulmonate Siphonaria diemenensis Casey Peters a, Geoffrey M. Collins b, Kirsten Benkendorff a, c,⁎ a b c

School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia National Marine Science Centre, Southern Cross University, PO Box 4321, Coffs Harbour, NSW 2450, Australia Marine Ecology Research Centre, School of Environment, Science and Engineering, Southern Cross University, PO Box 157, Lismore, NSW 2480, Australia

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Article history: Received 8 December 2011 Received in revised form 20 July 2012 Accepted 21 July 2012 Available online 10 August 2012 Keywords: Molluscan egg masses Epibionts Microbial fouling Microscopy Antibacterial activity Bacillus psychrodurans

a b s t r a c t The ability of sessile benthic egg masses to deter or prevent epibiosis is essential to the success of species that employ this life-history strategy. This study characterised the physical structure and bacterial communities on the surface of egg masses from the Siphonariid mollusc Siphonaria diemenensis (Quoy & Gaimard, 1833). Egg masses at the veliger stage of development were collected from two intertidal sites in the Gulf St. Vincent, South Australia. Physical structure was assessed using a combination of light microscopy and scanning electron microscopy. Egg mass surfaces were characterised by wave-like elevations 1–3 μm apart, fouled only by cocci, and longitudinal ridges (5–20 μm) fouled by a diversity of microorganisms and dense exopolymeric substance. Bacteria from the surface of egg masses and adjacent rock substratum were then isolated using standard culture procedures. The biochemical profiles of the isolates were used, along with Gram stain and visual morphological observations, to identify the bacteria. Eight species of bacteria were isolated and the composition of culturable epibiont communities from the egg mass was found to be significantly different from those found on the adjacent substrata. One species of bacterium on egg masses exhibited antibacterial activity in mixed culture and was identified as Bacillus psychrodurans using PCR of the partial 16S rRNA gene and sequence alignment on the GenBank database. Chemical extraction was performed on ‘clean’ and ‘fouled’ eggs and antibacterial activity was assessed against the marine pathogen Vibrio harveyi using the disc diffusion assay. Extracts from the cleaned egg masses were found to inhibit the growth of V. harveyi, whilst the fouled egg masses and extracts from the epibionts showed no antibacterial activity. However, extracts from the supernatant and cell pellet from the cultured B. psychrodurans exhibited antibacterial activity against V. harveyi and two human pathogens, Staphylococcus aureus and Escherichia coli. The results obtained in this study suggest that the surfaces of S. diemenensis egg masses are selective towards coccoid bacteria, which may result from a combination of physical structure and chemical antimicrobial properties, with further competitive interactions possibly occurring between the epibionts post settlement. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The ability of marine macro-organisms to prevent or deter epibiosis is necessary for health and survival. The marine environment contains numerous micro-fouling organisms such as bacteria, viruses, fungi, diatoms and protozoans and the interactions that occur between these organisms initially dictate the formation of the resultant mature biofouling community (Davis et al., 1989; Harder, 2009). Bacterial biofilms can influence the settlement of algal spores (Holmström and Kjelleberg, 1999), fungi (Egan et al., 2000) and the larvae of a wide range of invertebrates (Davis et al., 1989; Dobretsov, ⁎ Corresponding author at: School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia. Tel.: +61 2 66203755; fax: +61 2 66212669. E-mail address: [email protected] (K. Benkendorff). 0022-0981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jembe.2012.07.018

2010; Holmström and Kjelleberg, 1994; Wahl, 1989). Even in low densities, some epiphytic bacteria are effective in preventing settlement in a diversity of fouling organisms through the production of inhibitory compounds (Rao et al., 2007). Surface fouling can act as an environmental stressor for marine macro-organisms by decreasing survivorship, particularly at the juvenile stages (Dobretsov, 2010). Mortality in the tropical rock lobster Panulirus ornatus coincides with the formation of filamentous bacterial biofilms (Payne et al., 2007). Biofouling also prevents water from circulating through the ostia of the marine sponge Ianthella basta causing disease and mortality (Cervino et al., 2006). Higher incidences of surface fouling have also been associated with significantly higher incidences of embryonic mortality in gastropod egg masses (Biermann et al., 1992; Przeslawski and Benkendorff, 2005). Fouling on the surface of nudibranch and polychaete egg masses has been shown to affect the oxygen

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supply to internal embryos (Cohen and Strathmann, 1996). These studies suggest that biofouling often has detrimental impacts and highlight the potential for the evolution of defensive strategies for protection. Nevertheless, there are some potential benefits from surface fouling on marine invertebrate egg masses. Symbiotic bacteria on the surface of crustacean eggs have been found to prevent infection by pathogenic fungi (Gil-Turnes and Fenical, 1992; Gil-Turnes et al., 1989). Fouling by photosynthetic microphytes can increase embryonic development rates in some species (Fernandes and Podolsky, 2011; Przeslawski and Benkendorff, 2005). A coating of fouling organisms can also provide an effective camouflage (Davis et al., 1989) or reduce the palatability. Surface fouling could also provide protection from solar (UV) radiation, which has been shown to have detrimental effects on encapsulated molluscan embryos (Biermann et al., 1992; Przeslawski et al., 2004, 2005; Rawlings, 1996). Submerged surfaces are not equally colonised by marine microbes and many organisms respond to epibiosis by the production of antifouling defences (Davis et al., 1989; Harder, 2009). Wahl (1989) describes three common mechanisms of defence: mechanical (shedding, moulting and cleaning of body surfaces), physical (surface free energy, roughness, and surface microtexture) and chemical (the production of bioactive secondary metabolites). Evidence for this combination of antifouling mechanisms has been reported for the surface of egg capsules of the muricid whelk Dicathais orbita (Lim et al., 2007). These mechanisms of defence are often complementary to inhibit or prevent fouling (Camps et al., 2011). The ability of surface microtexture to act as an antifouling defence mechanism is dependent on the scale of the topographical features and the target organisms for repulsion (de Nys et al., 2010). Circular elevations 200 μm in diameter are distributed evenly across the carapace of the crab Cancer pagurus and deter settlement by the barnacle Balanus improvises (Bers and Wahl, 2004). The brittle sea star Ophiura texturata has 10 μm diameter knob-like structures on its surface, which repel microfouling ciliates, including Zoothamnium commune and Vorticella sp. (Bers and Wahl, 2004). These examples illustrate that surface microtopography alone can be an effective inhibitor of fouling against a range of organisms, depending on the size and structure of the features. On the other hand, some surface topographies may facilitate microfouling, such as the waves and troughs observed on the surface of Sepioteuthis australis (Cephalopoda) egg capsules (Lim et al., 2007). Among sessile benthic organisms, chemical antifouling defence is particularly common. Recently discovered compounds with broad-spectrum antimicrobial activity are continually isolated from marine microorganisms and invertebrates (reviewed by Blunt et al., 2007, 2008, 2010, 2011; Liu et al., 2010; Smith et al., 2010). Chemical compounds with antimicrobial activity have been isolated from the egg masses of many marine molluscs. Benkendorff et al. (2001b) conducted a comprehensive investigation into antimicrobial activity of molluscan egg masses from 23 families, 18 of which were found to exhibit antimicrobial properties. Polyunsaturated fatty acids were found to contribute to the antibacterial activity in some gelatinous gastropod egg masses (Benkendorff et al., 2005), whereas brominated indoles were identified as the bioactive agents in egg capsules from the Muricidae family (Benkendorff et al., 2000a, 2001a). Ramasamy and Murugan (2005) assessed the activity of macerated molluscan egg masses from the Aplysiidae, Buccinidae, Cypraeidae, Conidae, Cassidae and Muricidae against 40 biofilm bacteria, all of which displayed antimicrobial activity against a portion of these. This high level of activity suggests that chemical defence is widespread in molluscan egg masses; however, it is mostly unknown whether the active compounds are contained within the egg masses to prevent invasion, or if they diffuse to the surface to prevent fouling. The biosynthetic origin of the antimicrobial compounds in gelatinous egg masses is also unclear. Marine microorganisms have been found to produce a wide range of antimicrobial metabolites (Rahman et al., 2010) and symbiotic bacteria associated with marine invertebrates are increasingly being identified as the source of bioactive

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compounds (Thomas et al., 2010; Zheng et al., 2005). In recent years, significant advances have been made in recognizing the key role of microbial symbionts in natural products originally thought to be produced by marine invertebrates (Lane and Moore, 2011). Marine molluscs of the genus Siphonaria are marine intertidal, herbivorous pulmonates commonly referred to as false limpets. Siphonariids are hermaphrodites with internal fertilisation (Hodgson, 1999; Smith et al., 1989) and, following copulation, most species lay gelatinous egg masses as ribbons on rocky substratum. The egg masses are composed largely of mucopolysaccharides (Pal and Hodgson, 2003; Przeslawski, 2004) and each egg capsule is surrounded by a mucous strand and an inner mucous layer (Mapstone, 1978). Siphonariids are characterised by their ability to synthesise polypropionate secondary metabolites (Darias et al., 2006). These compounds, such as the denticulatins, have demonstrated antibacterial activity (Darias et al., 2006; Hochlowski et al., 1983). Lipophylic extracts from egg masses of Siphonaria denticulata and S. zelandica have also been shown to possess antibacterial activity (Benkendorff et al., 2000b, 2001b); however, preliminary chemical analysis did not reveal the presence of polyproprionates (unpublished data, Benkendorff, 1999). Interestingly, antimicrobial activity detected in the freshly laid Siphonaria egg masses diminished as the embryos matured into shelled veligers (Benkendorff et al., 2001b), and coincidently, visual fouling by algae and protists is also higher at this later stage of development (Przeslawski and Benkendorff, 2005). Consequently, these egg masses may contain defense mechanisms that initially modulate the microbial fouling communities when the embryos are most vulnerable but then degrade to permit fouling prior to hatching. Siphonaria diemensis are commonly found in South Australia where they deposit their egg masses on intertidal rocky shores. These gelatinous benthic egg masses are present for 7–10 days, after which veliger larvae are released. Little is known about the bacterial epibiont communities that settle on the egg masses prior to hatching, or the physical and chemical properties that influence their settlement. The aims of this study were, firstly, to characterise the bacterial epibionts on the surface of S. diemenensis egg masses (Fig. 1) using microscopy and biochemical tests. We tested the hypothesis that egg-specific microbial communities exist in comparison with the adjacent rock substratum. We also aimed to determine whether the epibiont community was influenced by chemical defence by testing whether the surface of the egg masses or associated epibionts exhibit antibacterial activity. 2. Materials and methods 2.1. Sample collection Egg masses (n = 25) of Siphonaria diemenensis at the veliger stage of development (Fig. 1) were collected from August to November 2007 at low tide (0.2 m) from two rocky intertidal reefs in the Gulf St. Vincent, South Australia: Marino Rocks and South Port. All samples were collected from rocks exposed by the low tide. All specimens were transported in fresh seawater collected from the same site to Flinders University where they were either processed immediately for microbiological analysis or maintained overnight in glass aquaria containing sterile seawater for subsequent microscopic and chemical analysis. 2.2. Microscopy Fouling communities on egg masses were examined using an optical microscope (Olympus BH-2). Samples were washed for 15 s with sterile sea water to remove transient microorganisms and salt crystals, and then dissected into 5 mm2 sections using a sterile blade. Specimens were mounted on glass slides and viewed under cover slips and also examined after Gram stain. Egg masses (n = 3) were fixed in a solution containing 0.5% glutaraldehyde, 4% sucrose and 4% paraformaldehyde with either phosphate

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Fig. 1. Siphonaria diemenensis (A) adults and (B) egg masses. Light microscopy of S diemenensis egg mass showing (C) encapsulated veliger larvae and (D) compartment-like organisation of gelatinous matrix contain egg capsules, inner mucous layer and mucous strand that connects adjacent embryos (indicated by the arrow).

buffered saline (PBS) or Tris buffer for 24 h and subsequently washed twice in their respective buffers. Each egg mass was sliced into 5 mm2 sections using a sterile blade and fixed in 1% osmium tetroxide (OsO4), containing 300 μl OsO4 and 300 μl of the respective buffer, for 90 min. Following fixation, samples were dehydrated in 70% ethanol for 15 min, 90% ethanol for 15 min, 95% ethanol for 15 min, and then twice in 100% ethanol for 15 min. Following dehydration, samples were washed twice in Milli-Q water for 10 min, and then for a further 15 min. Specimens were then maintained in 100% ethanol to prevent artefacts and dried using a critical point drier (Emscope CPD750). Dried specimens were fixed to metal stubs using carbon adhesive and sputter coated with gold (Emscope SC500A Sputter Coater) for viewing under a scanning electron microscope (Siemens Autoscan). Images were captured using a digital camera (Ilford FD4 120).

further observed for an additional 5 days to allow for the isolation of slow growing bacteria. Bacterial growth was recorded as colony forming units per ml (CFU ml −1 equivalent to 10 mm 2 of swabbed substrate), and mean CFU counts were calculated for each morphologically different colony by combining counts from triplicate plates yielding 30–300 colonies for each replicate. Biochemical characterisation of cultivated bacterial isolates differentiated by colony and cell morphology was performed using a biochemical characterisation kit (API20E, bioMerieux). Test strips from the biochemical characterisation kit were prepared following the manufacturers’ instructions. Incubation parameters were modified following the method described by Popovic et al. (2007) for the isolation of fish pathogens, by incubating strips at 25 °C for 72 h. 2.4. Chemical extraction

2.3. Isolation and characterisation of bacteria Small portions (10 mm 2) from independent egg masses (n = 4 per site) were washed with sterile seawater for 5 s to remove loosely attached microorganisms, then semi-quantitatively sampled by swabbing with sterile cotton tips to collect epibiotic bacteria. Each swab was individually suspended in 1 ml sterile seawater in 1.5 ml tubes (Eppendorf) and transported on ice to Flinders University. This procedure was repeated for rocky substratum surface areas adjacent to the egg masses at each site. Bacterial homogenates were suspended in 9 ml sterile seawater and 10-fold serial dilutions were prepared to 10 −3. Sub-samples of each dilution (100 μl) were spread-plated in triplicate on nutrient agar (Oxoid No. 2) in seawater and incubated at 25 °C for 72 h. Bacterial growth was recorded and plates were

An investigation was undertaken to assess antimicrobial activity of egg masses with epibiotic communities removed compared to those that were left intact. S. diemenensis egg masses (n = 12) from South Port were pooled and 2 g of egg material was swabbed using sterile cotton tips to collect epibiotic bacteria, vortexed briefly in sterile seawater. The swabbed egg masses were designated ‘clean’. Egg masses with intact epibiotic communities were designated as ‘fouled’. Rapid surface extraction was used to assess the antimicrobial activity of compounds more likely to be present on the surface of egg masses. Samples were submerged in approximately 8 ml dichloromethane (DCM, HPLC grade; Sigma), and vortexed for 20 s. The solvent was then decanted and the sample was transferred into fresh vials for overnight extraction to assess the antimicrobial activity of any

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additional compounds associated with the internal egg mass matrix. Following extraction the solvent was concentrated using a rotary evaporator (Bucci) at 37 °C under 337 mbar pressure. Extracts were reconstituted with 3 ml of DCM and transferred to pre-weighed vials, then completely dried under a stream of high purity nitrogen gas. Crude extract yields from the rapid extraction were 0.81 and 0.89 mg ml −1 for fouled and clean egg mass samples, respectively, whereas overnight extraction yielded 3.81 and 2.34 mg ml −1 of extract respectively. Swabs containing epibionts were processed in 9 ml DCM using the rapid (20 s) extraction technique described above. Sterile swabs were included as a negative control. A bacterial isolate from the surface of egg masses collected from Marino Rocks was also found to inhibit the growth of other epibionts when initially plated onto agar. This isolate was subcultured for antibacterial testing in 50 ml nutrient broth (Oxoid No. 2) overnight at 25 °C on an orbital shaker (Ratec). Cells in the culture were separated by centrifugation at 6000 ×g for 10 min and the resulting bacterial pellet and culture supernatant were both assessed for antibacterial activity. The bacterial pellet was processed in 9 ml DCM using the rapid extraction technique as described above. The culture supernatant (cell‐free extract) was concentrated by ion exchange chromatography on Supelco Diaion™ HP-20 (Sigma-Aldrich) resin. Resin (10 g) was added to a glass column and washed twice with two volumes of 100% methanol. The resin was then washed with two volumes of Milli‐Q water then left for 10 min before washing for a third time. Then 20 ml cell-free extract (culture supernatant) was added to the column and the mixture was left for 2 h to allow maximum adsorption. The resin was then washed sequentially with 100% methanol and the eluate collected. This extract was concentrated under rotary evaporation (Bucci, 37 °C; 337 mbar pressure), reconstituted with approximately 3 ml of methanol and dried to completion under a stream of high purity nitrogen gas.

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Collection, maintained in saline) and the marine pathogens V. harveyi, V. alginolyticus and V. tubiashi (Department of Primary Industries and Fisheries, Launceston, Tasmania) using culture conditions according to Benkendorff et al. (2000b, 2001b). 2.6. Molecular identification of the antibacterial isolate The epibiont isolated from Marino Rocks displaying antimicrobial activity was identified using PCR of the partial 16 sRNA gene. Cells were grown overnight at 25 °C on nutrient agar (Oxoid No. 2), then an isolated colony was suspended in sterile water in a microcentrifuge tube and pelleted by centrifugation at 5000 ×g for 10 min. The supernatant was discarded and the pellet resuspended in 180 μl of enzymatic lysis buffer (20 mM Tris–Cl pH 8.0; 2 mM sodium EDTA; 1.2% Triton x-100), with the addition of 3.6 mg lysozyme (Sigma) to a final concentration of 20 mg ml−1. DNA extraction was performed using a DNeasy blood and tissue kit (Qiagen) following the manufacturer's instructions. Extracted DNA was quantified using a GeneQuant II spectrophotometer (620 nm, Pharmacia Biotech). Samples were read in triplicate at 1:20 dilution and DNA integrity was assessed via 1% agarose gel electrophoresis (Bio-Rad Mini Sub-Cell GT gel apparatus) using standard procedures, then stained in a 0.5 μg ml−1 aqueous solution of ethidium bromide for 5 min and visualised under UV. PCR of the extracted DNA was performed on a ThermoHyabid thermocycler using bacterial 16S rDNA gene specific primers: 341F (5′-GCCTACGGGAGGCAGCAG-3′) and 907F (5′-CCGTCAATTCMTTTGAGTTT-3′) (Romero and Navarrete, 2006). Cycling conditions were as follows: initial denaturation at 94 °C for 2 min then 35 cycles of 94 °C for 45 s, 54 °C for 45 s and 72 °C for 90 s. The PCR product was purified using a Wizard DNA clean up kit (Promega) and sequencing was performed by the Australian Genome Research Facility (AGRF). Sequence alignment was performed using GenBank and BLAST through BioManager (Cattley and Arthur, 2007). 2.7. Statistics

2.5. Antimicrobial assays Antibacterial activity was tested against a known mollusc pathogen, Vibrio harveyi. Cultures were obtained from stock held at −80 °C at Flinders University (provided by the Department of Primary Industries and Fisheries, Launceston, Tasmania, Australia). Cultures were inoculated onto nutrient agar and incubated overnight at 25 °C, then a single, isolated colony was used to inoculate a 25 ml culture in nutrient broth (Oxoid No. 2), which was then incubated overnight at 25 °C on an orbital shaker. Cultures were diluted to an absorbance of 0.1 (600 nm, Metertech, UV/VIS SP8001 Spectrophotometer) and grown to an absorbance of 0.2 to reach exponential growth. A standard disc diffusion assay was used to assess the antimicrobial activity of all the egg mass and epibiont extracts against V. harveyi, following the methodology described by Becerro et al. (1994). A final amount of 50 μg of extract was loaded onto each disk (50 μl of a 10 mg ml−1 solution) and all solvent was evaporated in a fume hood prior to placement on the bacterial lawn. The zone of inhibition assay only provides a qualitative indication of antibacterial activity and should not be used for quantitative estimates of the minimum inhibitory concentration. Consequently, we have tested at ~10× higher than natural concentration to maximise the chance of detecting activity accounting for lack of migration of lipophylic active compounds on the agar and potential degradation of some active compounds. Following incubation at 25 °C for 24 h, the zone of inhibition (mm) was measured from the edge of the paper disc to the unaffected bacterial growth, to provide a preliminary assessment of antibacterial activity. Extracts from the bacterial isolate displaying antimicrobial properties from Marino Rocks were further tested using the zone of inhibition assay against human pathogens Escherichia coli (ACM845), S. aureus (ACM844) (obtained from the Queensland Culture Collection and maintained at −78 °C in 15% glycerol), Candida albicans (Queensland Culture

All statistical analyses were conducted using Primer V5 (Plymouth Marine Lab). The number of culturable bacterial epibionts was calculated using mean CFU ml −1, log transformed, and used to generate a Bray–Curtis similarity matrix between samples. A non‐parametric multidimensional scaling (nMDS) ordination plot was generated to illustrate the relative similarity between bacterial communities on the rock substratum and those found on the egg masses from the two locations. A two-way analysis of similarity (ANOSIM) was conducted to test the null hypothesis that within group community profile similarity was greater than between groups (Clarke, 1993). Similarity percentages (SIMPER) analysis was used to establish the contribution of each bacterial species to the mean dissimilarity between significantly different groups. 3. Results 3.1. Microscopy The gelatinous matrix was easily distinguishable from egg capsules containing embryos. Light microscopy revealed that each egg capsule within the egg mass was surrounded by a mucous strand, which was connected to adjacent embryos (Fig. 1D). Siphonaria diemenensis development was synchronous, as the egg masses studied contained embryos that were all at the veliger stage of development (e.g. Fig. 1C). Bacteria, protozoa and diatoms were observed in the gelatinous matrix, but never within the egg capsules. No macrofouling organisms were observed by light microscopy, either on or within the egg masses. Embryonic mortality was calculated at 16%± 8.9 of the veligers within the egg masses. Scanning electron microscopy revealed a diverse assemblage of fouling organisms on the surface of S. diemenensis egg masses. The

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surface of the gelatinous matrix was heavily fouled by bacterial rods and cocci (Fig. 2A and B), which formed a biofilm consisting of dense exopolymeric substances (Fig. 2G). Cocci (0.1 μm diameter) were also attached to the surface of the egg capsules within the gelatinous matrix (Fig. 2C). The surface was also fouled by several macrofouling species including filamentous algae (Fig. 2D), dinoflagellates (Fig. 2E) and nematodes (Fig. 2F). Two microtopographies were identified on the surface of the egg masses: longitudinal ridges (Fig. 2H) and wave-like elevations (Fig. 2B

and I). The wave-like topography was marked by irregular peaks and troughs, 1–3 μm apart and was fouled only by cocci shaped bacteria 0.5–1 μm in diameter. Longitudinal ridges (5–20 μm) dominated the surface and were heavily fouled with a dense exopolymeric layer (Fig. 2G). In some areas of the egg masses, short lateral valleys were enclosed in longitudinal ridges. Less exopolymeric substance was observed within the valleys and side walls, and these areas also appeared to be preferentially fouled by cocci approximately 0.5 μm in diameter. No bacilli were observed fouling the valleys and side walls. In comparison,

A

B

C

D

E

F

G

H

I

Fig. 2. Scanning electron microscopy of Siphonaria diemensis egg masses. The surface of the eggs is fouled by (A) rods and (B) cocci. Egg capsules within the gelatinous matrix are fouled by (C) cocci. Macrofouling species include (D) filamentous algae, (E) dinoflagellates and (F) nematodes. The two topographies were (G) under extensive exopolymeric fouling and were characterised by (H) longitudinal ridges and (I) wave-like elevations.

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bacilli were observed fouling the elevated regions of the ridges found across the surface of the egg masses. Both surface microtopographies were consistent across Tris and PBS fixed samples. 3.2. Characterisation of culturable bacteria Seven morphologically distinct isolates were identified from S. diemenensis eggs and rock substratum samples collected from South Port and Marino (Table 1). The swab and culture techniques used here do not provide a quantitative assessment of absolute abundance of all epibiotic bacteria, but nevertheless they provide a semi-quantitative or at least a qualitative indication of relative abundance. Samples from different surface types displayed different bacterial profiles and not all isolates were present on each substratum at each site (indicated by 0 CFU). Morphologically, the most common isolate exhibited opaque colonies that were rhizoid and flat. This isolate was cultured at densities ranging from 360 to 810 and 140–270 CFU ml−1, equivalent to 10 mm−2 of swabbed substrate, on substratum and egg surfaces respectively, but resisted subculture. As this isolate could not be cultivated in purity and identified, it is referred to as an unknown Gram positive cocci. The second most common isolate consisted of colonies that were circular, smooth, slightly convex, glistening and pale yellow-brown. Based on morphological and biochemical traits, it was identified as Mesophilobacter marinus (Table 1). This bacterial isolate ranged in abundance from 150 to 26 CFU mm −2 on rock substrata to 2.5– 7 CFU mm −2 on egg surfaces. Mesophilobacter marinus is the only reported Gram negative cocco-bacillus that is oxidase positive, catalase positive, reduces nitrate, produces acid from glucose and mannitol, but not from sucrose and sorbitol. An orange isolate was cultured from some replicate samples at both sites at a density of 0–6 CFU mm −2 for rock substratum and

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2–17 CFU mm −2 for egg surfaces. Colonies were smooth, round, glistening and punctiform, and were distinguished from other Gram negative bacilli based on pigmentation, oxidase activity, acid from glucose and indole production from tryptophan (Table 1). Based on this biochemical profile, the isolate was identified to the genus Pseudoalteromonas. The species P. aurantia produces an orange pigment, and does not reduce nitrate or utilise melibiose or D-mannitol carbon sources, which is consistent with the biochemical results for this isolate (Table 1). Yellow colonies were cultivated from rock substratum and egg samples from South Port only (Table 1). Abundance ranged from 5 to 14 CFU mm−2 on eggs to 2–9 CFU ml-2 on substratum. These colonies were also smooth, round, glistening and punctiform. This isolate was distinguished from other oxidase negative, Gram negative rods bacilli based on pigmentation, acetoin production, and acid production from glucose, arabinose, inositol, mannitol, melibiose and sorbitol (Table 1). Based on this profile the organism was identified to the genus Erwinia. Colonies were further identified as E. uredovora, as this is the only species to produce a yellow pigment, hydrolyse gelatin, and to produce indole from tryptophan and acid from rhamnose. Pink colonies were isolated sporadically from rock substratum (density range from not present (0) to2 CFU mm −2) and egg samples (0–9 CFU mm −2). Colonies were distinguished from other oxidase negative, Gram positive cocci, and identified as Micrococcus, based on their catalase activity and the production of non-diffusible pigment (Table 1). Micrococcus roseus is the only pink-pigmented species that is gelatinase negative, reduces nitrate, and produces acid only from glucose, which is consistent with the biochemical profile from this isolate (Table 1). White colonies were isolated consistently across rock substratum (5–15 CFU mm−2) and egg samples (5–20 CFU mm−2) collected from both sites. Differentiation of this isolate from other Gram negative bacilli

Table 1 Biochemical characteristics of bacterial isolates cultured from the surfaces of Siphonaria diemensis egg masses (EM) and rocky substratum (R) at South Port (SP) and Marino Rocks (M), Gulf St. Vincent, South Australia. Trait

Pau

Eur

Pes

Mro

Mma

Avi

Bps

Source Location Pigmentation Gram reaction Cell shape Spore

EM, R SP, M Orange − Bacillus −

EM, R SP Yellow − Bacillus −

EM, R SP, M White − Bacillus −

EM, R SP, M Pink + Coccus −

EM, R SP, M Yellow- brown + Cocco-Bacillus −

R SP Yellow-green − Bacillus −

EM M White + Bacillus −

+ + − − − − − − − − − − +

+ − − − − − + − − − + + +

− + − − − − − − − − − + +

+ − − − − − + − − − − − −

+ + − − − − − − − − + − +

+ − − − − − − − − − − + −

+ + − − − − − − − − − − +

+ − + + + − − + + −

+ + + + + − + − + +

+ + + + + + + + + −

+ − − − − − − − − +

+ + − − − − − − − +

+ + + + + − + − + +

+ − − + + − + − + +

Biochemistry Catalase Oxidase Ortho-nitro phenyl β-d-galactopyranoside (ONPG) Arginine dihydrolase (ADH) Lysine decarboxylase (LDC) Ornithine decarboxylase (ODC) Citrate utilisation (CIT) H2S production Urease (URE) Tryptophan deaminase (TDA) Indole production (IND) Acetoin production (VP) Gelatinase gel Acid from D-glucose (GLU) D-mannitol (MAN) Inositol (INO) D-sorbitol (SOR) L-rhamnose (RHA) D-sucrose (SAC) D-melibiose (MEL) Amygladin (AMY) L-arabinose (ARA) Reduction of NO3− to NO2

Pau = Pseudoalteromonas aurantia; Eur = Erwinia uredovora; Pes = Pseudoalteromonas espejiana; Mro = Micrococcus roseus; Mma = Mesophilobacter marinus; Avi = Azotobacter vinelandii; Bps = Bacillus psychrodurans.

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was based on oxidase activity, the presence of gelatinase, acid from glucose and the lack of pigment (Table 1). Based on this profile, the isolate was identified as another Pseudoalteromonas sp. Psudoalteromonas espejiana is the only un-pigmented species to produce acid from glucose, mannitol and melibiose. Fluorescent colonies were only isolated from substratum samples collected from South Port, at a density of 1–4 CFU mm −2. These colonies were smooth, round and glistening, with yellow-green water soluble fluorescent pigments. This isolate was differentiated from other Gram negative bacilli based on their biochemical profile (Table 1) and characteristic yellow-green fluorescence, which is consistent with Azotobacter. Other luminescent strains such as Vibrio and Pseudomonas are oxidase positive, which is inconsistent with this isolate (Table 1). Based on the ability to utilise rhamnose, inositol and mannitol as carbon sources, this isolate was further identified as A. vinelandii. White, butyroid colonies were cultured exclusively from egg samples collected from Marino (1–2.5 CFU mm −2). This isolate was identified as a Gram positive, spore-forming bacillus, which is consistent with the genus Bacillus. However, members of this genus share a large number of morphological and biochemical similarities; therefore, genomic analysis was required to identify the isolate further (see Section 3.3 below). On agar plates containing these Bacillus colonies, a zone of inhibition was observed extending 2–4 mm out from the colonies, where other isolates were unable to grow; hence, this isolate was further tested for antimicrobial activity (see Section 3.5 below). 3.3. Molecular analysis of Bacillus sp. Genetic analysis based on partial 16S rDNA sequencing identified the isolate with antimicrobial activity collected from Marino as Bacillus psychrodurans. Genetic analysis showed the amplified 16S rDNA fragment shared 100% sequence similarity with that of B. psychrodurans (Fig. 3), based on the 536 bp sequence. The biochemical profile obtained for this isolate was identical to that described for B. psychrodurans

(Fig. 3; GenBank accession no. EU 2495666.1); however, the type strain produces acid from mannitol (Abd El-Rahman et al., 2002). 3.4. Comparison of bacterial communities nMDS ordination indicates distinct culturable bacterial community profiles occur on the egg masses as opposed to rock substratum and further, that samples within each location form distinct groups (Fig. 4, stress = 0.08). Two-way ANOSIM revealed significant differences in the community profiles between the two locations (R = 0.85; p = 0.001). A significant difference between community profiles was also found between substratum and egg surface samples (R = 0.46, p = 0.003). SIMPER analysis revealed that most of the bacterial species contributed to the differences between substratum and locations (Table 2) with dissimilarity ratios close to, or exceeding 1. P. aurantia, E. uredovora, M. roseus and the unknown Gram positive cocci were all more abundant on the egg masses compared to the rock substratum, whereas M. marinus was more abundant on the rock substratum (Table 2A). Azotobacter vinelandii was the only bacteria found on the rock substratum but not on the egg masses, whereas B. psychrodurans was exclusively cultured from the egg masses (Table 2A). Comparison between the two locations revealed that E. uredovora abundance contributed to 31.2% of the differences between locations, being abundant at South Port and absent from Marino (Table 2B). Azotobacter vinelandii was also absent from Marino and P. aurantia was more abundant at South Port. Bacillus psychrodurans was only cultured from egg masses at Marino, whereas M. roseus and the unknown Gram positive cocci were consistently present at both locations (Table 2B). 3.5. Antimicrobial activity Using the disc diffusion assay, antibacterial activity against V. harveyi was only detected in extracts of the clean S. diemenensis egg masses. The mean (± standard deviation) width of the zone of inhibition was greater

Fig. 3. BLASTn alignment between the sequences obtained from an antimicrobial isolate cultured from the surface of Siphonaria diemensis egg masses (labelled ‘Unknown isolate’) and Bacillus psychrodurans partial 16S rRNA gene (GenBank accession number EU 249566.1).

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4. Discussion

Substratum, South Port

Siphonaria diemenensis, Southport

Substratum, Marino

Siphonaria diemenensis, Marino

Fig. 4. Non‐parametric multidimensional scaling ordination showing the similarities in culturable bacterial epibiotic communities isolated from Siphonaria diemenensis egg masses (Δ) and substratum (□) from South Port (open symbols) and Marino (filled symbols). The two dimensional plot is generated from the log-transformed abundance data using a Bray–Curtis similarity matrix in PRIMER V.5. Stress = 0.08.

for overnight extracts (4.67 ±0.6 mm) compared to the rapid surface extract (2.00±0.0 mm). No zones of inhibition were observed around the crude extracts collected from fouled egg masses. Crude extracts collected from the swabs of epibiotic communities taken from the egg mass surface were also unable to inhibit the growth of V. harveyi. When tested against a panel of Gram positive and Gram negative bacteria, and fungi, culture supernatant and cell extracts from B. psychrodurans exhibited antibacterial activity against two human pathogens and one marine pathogen. Bacteria consistently sensitive to extracts were E. coli, S. aureus, and V. harveyi, whereas the extracts had no effect on V. alginolyticus or V. tubiashi. No antifungal activity was detected against C. albicans.

Table 2 SIMPER result for the determination of dissimilar bacterial epibionts, calculated as percentage abundance for (A) egg masses and surrounding substratum, and (B) location. A Average dissimilarity = 27.24 Substratum

Egg

Species

Av. abund

Av. abund

Diss/SD

Contrib. %

Cum. %

P. aurantia E. urevodora M. roseus Unknown A. vinelandii B. psychrodurans M. marinus

15.00 26.25 6.25 37.50 12.50 0.00 502.50

46.25 36.25 32.50 45.00 0.00 7.50 215.00

1.44 1.09 1.31 3.35 0.96 0.96 2.47

9.06 17.37 17.34 13.38 11.22 10.97 6.99

9.06 36.43 53.77 67.15 78.37 89.34 96.32

B Average dissimilarity = 28.12 South Port

Marino

Species

Av. abund

Av. abund

Diss/SD

Contrib. %

Cum. %

E. uredovora M. roseus P. aurantia A. vinelandii B. psychrodurans Unknown

62.50 18.75 45.00 12.50 0.00 130.00

0.00 20.00 16.25 0.00 7.50 138.75

6.86 1.43 1.13 0.96 0.97 1.31

31.20 15.18 14.80 11.67 9.78 7.66

31.20 46.38 61.18 72.85 82.63 90.29

This study provides a novel insight into the fouling organisms and antifouling defence strategies employed by the egg masses of the Siphonariid mollusc S. diemenensis. Combining cultivation techniques with direct observation we provide a detailed view of the bacterial landscape of these molluscan egg mass surfaces. Although only a small proportion of epibiotic bacteria are likely to have been cultured using our techniques, the dominance of coccoid bacteria in culture was confirmed by their abundance on the egg mass surface using scanning electron microscopy. Multivariate SIMPER confirmed that the unknown Gram positive cocci and the coccoid M. roseus were more abundant on the egg masses than the surrounding rock substratum, whereas several bacilli bacterial species were under-represented on the egg masses. Valleys between elevations on the surface of the egg masses appear to provide a favourable attachment site for cocci. This physical structure may facilitate selection of coccoid bacteria and their colonisation of the surface may subsequently allow the attachment of other epibionts (Whitehead and Verran, 2006). Antimicrobial defence of the egg masses may also influence the settlement of bacteria on the surface. The antimicrobial activity in DCM extracts prepared from several pooled egg masses was associated with the egg mass matrix, rather than the culturable epibiont communities, with the exception of one antibacterial isolate identified as Bacillus psychrodurans. This species was only found on egg masses from one location and assuming it secretes antibacterial compounds in situ, its presence may have influenced the differences in epibiont community composition detected between sites. Notably no spirochetes were cultured from the egg mass or observed using scanning electron microscopy. Spirochetes are a phylum of morphologically unique prokaryotes that are widespread in aquatic environments, but often resist cultivation (Madigan et al., 2003), as do most other marine strains. As standard culturing techniques are very selective, spirochetes and other uncultivable bacteria are likely to be under-represented and thus this study should only be considered a preliminary assessment of the diversity of bacteria found on the egg masses of S. deimenensis. Using light microscopy, the egg masses of S. diemenensis were found to have a similar physical structure to that reported previously for S. serrata, which also undergoes benthic development (Pal and Hodgson, 2003). Pal and Hodgson (2003) did not observe microorganisms within the egg masses of S. serrata, although they were found in the egg masses of a Siphonariid with planktonic larval development (S. capensis). The degree of bacterial penetration into the gelatinous matrix of molluscan egg masses may be influenced by stage of embryonic development and length of exposure in the environment. The inner mucous layer of some gastropods (Cephalaspidea; Nudibranchia) dissolves during intracapsular development (Klussmann-Kolb and Wägele, 2001) and microbial degradation of the gelatinous matrix around the time of hatching is thought to facilitate the release of juveniles into the water column or onto the substratum. The Siphonaria egg masses examined in this study were at the veliger stage of development and would have been at least a week old. Observations show that the gelatinous matrix degrades in Siphonaria egg masses containing late stage veligers (Smith et al., 1989) and this could be facilitated by specific microbial symbionts. Egg masses exist for only a short period of time in the environment compared to surrounding non-living substrata. Although we can only speculate about the role of specific bacterial epibionts at this stage, it remains possible that some of those that are abundant on the surface of S. diemenensis egg masses may assist with degradation of the gelatinous matrix and facilitate the escape of juveniles. Symbiotic cocci, localised for the purpose of polysaccharide degradation, are associated with eukaryotic hosts. Pseudoalteromonas espejiana was abundant on the egg masses and this bacterium is able to secrete a range of hydrolytic enzymes (Andreev et al., 2007). Bacteria of the genus Erwinia also contain a complex arsenal of degradative enzymes (Pirhonen et al.,

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1993; Venturi et al., 2004). Erwinia uredovora was more abundant on the egg masses than the substratum, but was only recorded on samples from South Port, suggesting this may be an opportunistic facultative epibiont. Multivariate analysis revealed significant differences in the proportion of culturable bacterial communities occurring on the egg masses of S. diemenensis compared to the rock substratum; however, significant differences were also found between locations. Furthermore, bacterial epibionts cultivated from egg mass surfaces were identified on surrounding substratum in seven out of eight cases, thus suggesting facultative association. It is possible that more specific associations with the egg masses could be found in the uncultivable bacteria, although in the marine environment, specific associations between bacterial epibionts and macro-organisms appear to be rare. Wahl and Mark (1999) investigated over 2000 epibiotic associations and found that within any microhabitat, settlers prefer non-living surfaces over macro-organisms. Only one biofilm bacterium, Azotobacter vinelandii, was recorded on the substratum but not the egg masses in this study. However, this bacterium was relatively uncommon and only found at one location. A. vinelandii is a nitrogen fixing bacterium found in plant rhizospheres in the soil (Gorin and Spencer, 1966; Vermani et al., 1995). However, this species has also been identified from mangrove habitats (Ravikumar et al., 2004) and Azotobacter spp. have been found in marine cyanobacterial mats (Zehr et al., 1995). The distribution of A. vinelandii at South Port may be explained by the presence of the sediment on the soft limestone substrata, whereas Marino Rocks is composed of metamorphic conglomerates not covered by sediment particles. An interesting characteristic of Azotobacter spp. is the production of the copolymer alginate (Sabra et al., 2001), which is used by other biofilm bacteria to enhance adhesion to surfaces (Rehm and Valla, 1997). The copolymer may be utilised in this way by A. vinelandii to attach to sediment particles on substrata. One bacterial species, which was identified as Bacillus psychrodurans, was isolated exclusively from the surface of egg masses collected from Marino Rocks but not from the rock substratum or any samples at South Port. In light of the low density, site‐specific association (low density and site-specific), this more likely represents an obligate association rather than an egg mass specific epibiosis. Previous work has identified B. psychrodurans from a range of marine and terrestrial habitats including the surface of brown algae (Lee et al., 2006), the hindgut of a terrestrial arthropod (Kostanjšek et al., 2002) and deep‐sea Antarctic sponges (Xin et al., 2011). Despite a 100% sequence match in the 16S DNA with B. psychrodurans on EU 249566.1, our isolate was negative for the production of acid from mannitol, whereas this is a biochemical attribute of the type strain (Abd El-Rahman et al., 2002). Antimicrobial activity has also not been reported for the type stain of B. psychrodurans. However, Xin et al. (2011) reported antimicrobial activity in cultures of B. psychrodurans isolated from Antarctic deepsea sponges against several microorganisms (Erwinia carotovora, Xanthomonas campestris, and X. oryzae). They also detected the presence of polyketide synthase (PKS) genes in this B. psychrodurans strain, which are responsible for the synthesis of a range of biologically active secondary metabolites (Xin et al., 2011). The biosynthesis of antimicrobial compounds promoted by microbial competition on surfaces can assist the producer in competing for nutrients and space, while at the same time inhibiting pathogens on the host surface. Low cell densities of Pseudoalteromonas tunicata (10 2–103 cells cm−2), an epibiont of the green algae Ulva australis, effectively prevents the settlement of other fouling organisms, such as algal spores and marine fungi (Rao et al., 2007). In this study, B. psychrodurans was found to produce large inhibitory zones against other epibionts in primary mixed plate culture, as well as exerting antimicrobial activity against a number of Gram negative and Gram positive microorganisms, including both marine and human pathogens. Although our DCM extracts were tested at higher than natural concentrations the qualitative assay used typically

underestimates the activity of lipophylic extracts (Benkendorff et al., 2000b, 2001b). Consequently it is possible that antibacterial production results in competitive exclusion by B. psychrodurans on the surface of the Siphonaria egg masses, influencing the bacterial community composition on the egg masses from Marino. More generally however, antibacterial activity appears to be associated with the internal matrix (including embryos) of S. diemenensis rather than the surface epibionts. In fact, crude extracts from the egg masses of S. diemenensis from South Port only showed antibacterial activity against V. harveyi when removed of their epibionts. The activity was also greater in overnight extracts compared to rapid extraction, suggesting that the antimicrobial compounds are not specifically associated with the surface where they would be most effective at inhibiting biofilm formation. Antibacterial compounds are most likely directed towards inhibiting infection of the egg capsules, which protect the embryos within the gelatinous matrix. This is consistent with our observation that no microorganisms were present with the egg capsules. The egg masses of S. denticulata were also found to be more effective at inhibiting growth when placed on a lawn of bacteria after crushing as opposed to intact (Benkendorff, 1999; Benkendorff et al., 2000b). Ramasamy and Murugan (2005) have also reported that activity is localised to the internal matrix of egg masses in a number of other gastropods and it appears that this is the case also for S. diemenensis. This study provides an initial characterisation of the physical structure and associated fouling communities on the egg masses of the Siphonariid mollusc S. diemenensis. The composition of culturable egg surface epibionts was significantly different from adjacent substrata, suggesting that the surface chemistry and structure of these gelatinous egg masses may favour the settlement of specific bacteria, and in particular cocci. Overall, the bacterial ecology at the surface appears complex, and while antibacterial activity and surface microtexture appear to be weak inhibitors of fouling per se, they could combine along with competitive interactions in the biofilm to form a selective antifouling strategy. To gain a better understanding of antimicrobial defensive strategies in S. diemenensis, an investigation into the change in community composition over time would be beneficial. Further studies to investigate the diversity of uncultivable epibiotic communities on the surface of benthic mollusc egg masses would also be beneficial, using molecular analysis to obtain comprehensive microbial community profiles (e.g. Rudi et al., 2007). Acknowledgement We are grateful to Kerry Gascoigne for the Flinders Medical Centre for assistance with the Scanning electron microscopy. We thank members of the Molluscan Research lab, Flinders University for useful discussions and assistance in the field. This project was supported by Honours research funding from the School of Biological Sciences, Flinders University and a research grant from the Marine Ecology Research Centre, Southern Cross University. [SS] References Abd El-Rahman, H.A., Fritze, D., Sproer, C., Claus, D., 2002. Two novel psychotolerant species, Bacillus psychrotolerans sp. nov. and Bacillus psychrodurans sp. nov., which contain ornithine in their cell walls. Int. J. Syst. Evol. Microbiol. 52, 2127–2133. Andreev, V., Gonikberg, E., Kuznetsova, N., 2007. Application of the complex of DNA with the congo red anionic diazo dye for detection of nuclease-producing colonies of marine bacteria. Microbiology 76, 585–589. Becerro, M.A., Lopez, N.I., Turon, X., Uriz, M.J., 1994. Antimicrobial activity and surface bacterial film in marine sponges. J. Exp. Mar. Biol. Ecol. 179, 195–205. Benkendorff, K., 1999. Bioactive molluscan resources and their conservation: chemical and biological studies on the egg masses of marine molluscs. Ph.D. Thesis Department of Biological Sciences, Department of Chemistry. University of Wollongong, Wollongong, pp. 563. http://ro.uow.edu.au/theses/278/. Benkendorff, K., Bremner, J.B., Davis, A.R., 2000a. Tyrian purple precursors in the egg masses of the Australian muricid, Dicathais orbita: a possible defensive role. J. Chem. Ecol. 26, 1037–1050. Benkendorff, K., Davis, A., Bremner, J., 2000b. Rapid screening for antimicrobial agents in the egg masses of marine muricid molluscs. J. Med. Appl. Malacol. 10, 211–223.

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