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Southern Cross University ePublications@SCU School of Environment, Science and Engineering Papers School of Environment, Science and Engineering 2012 Characterisation of the physical and chemical properties influencing bacterial epibiont communities on benthic gelatinous egg masses of the pulmonate Siphonaria diemenensis Casey Peters Flinders University Geoffrey M. Collins Southern Cross University Kirsten Benkendorff Southern Cross University Publication details Postprint of: Peters, C, Collins, GM & Benkendorff, K 2012, '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, vol. 432-433, pp. 138-147. Published version available from: http://dx.doi.org/10.1016/j.jembe.2012.07.018 ePublications@SCU is an electronic repository administered by Southern Cross University Library. Its goal is to capture and preserve the intellectual output of Southern Cross University authors and researchers, and to increase visibility and impact through open access to researchers around the world. For further information please contact [email protected]. Characterisation of the physical and chemical properties influencing bacterial epibiont communities on benthic gelatinous egg masses of the pulmonate Siphonaria diemenensis. Casey Peters1# Geoffrey M. Collins2, Kirsten Benkendorff 1,3 * 1 School of Biological Sciences Flinders University, GPO Box 2100, Adelaide, SA, 5001 2 National Marine Science Centre, Southern Cross University, PO Box 4321, Coffs Harbour, NSW, Australia, 2450 3 Marine Ecology Research Centre, School of Environment, Science and Engineering, Southern Cross University, PO Box 157, Lismore, NSW, Australia, 2480 *Corresponding author Phone: +61 2 66203755 Fax: +61 2 66212669 E-mail: [email protected] 1 1 ABSTRACT 2 The ability of sessile benthic egg masses to deter or prevent epibiosis is essential to the 3 success of species that employ this life-history strategy. This study characterised the physical 4 structure and bacterial communities on the surface of egg masses from the Siphonariid 5 mollusc Siphonaria diemenensis Quoy & Gaimard, 1833. Egg masses at the veliger stage of 6 development were collected from two intertidal sites in the Gulf St. Vincent, South Australia. 7 Physical structure was assessed using a combination of light microscopy and scanning 8 electron microscopy. Egg mass surfaces were characterised by wave-like elevations 1 to 3 9 μm apart, fouled only by cocci, and longitudinal ridges (5-20 μm) fouled by a diversity of 10 microorganisms and dense exopolymeric substance. Bacteria from the surface of egg masses 11 and adjacent rock substratum were then isolated using standard culture procedures. The 12 biochemical profiles of the isolates were used, along with Gram stain and visual 13 morphological observations, to identify the bacteria. Eight species of bacteria were isolated 14 and the composition of culturable epibiont communities from the egg mass was found to be 15 significantly different from those found on the adjacent substrata. One species of bacterium 16 on egg masses exhibited antibacterial activity in mixed culture and was identified as Bacillus 17 psychrodurans using PCR of the partial16S rRNA gene and sequence alignment on the 18 GenBank database. Chemical extraction was performed on ‘clean’ and ‘fouled’ eggs and 19 antibacterial activity was assessed against the marine pathogen Vibrio harveyi using the disc 20 diffusion assay. Extracts from the cleaned egg masses were found to inhibit the growth of V. 21 harveyi, whilst the fouled egg masses and extracts from the epibionts showed no antibacterial 22 activity. However, extracts from the supernatant and cell pellet from the cultured B. 23 psychrodurans exhibited antibacterial activity against V. harveyi and two human pathogens, 24 Staphylococcus aureus and Escherichia coli. The results obtained in this study suggest that 25 the surfaces of S. diemenensis egg masses are selective towards coccoid bacteria, which may 2 26 result from a combination of physical structure and chemical antimicrobial properties, with 27 further competitive interactions possibly occurring between the epibionts post settlement. 28 29 Keywords 30 Molluscan egg masses, epibionts, microbial fouling, microscopy, antibacterial activity, 31 Bacillus psychrodurans. 32 33 1. Introduction 34 35 The ability of marine macro-organisms to prevent or deter epibiosis is necessary for 36 health and survival. The marine environment contains numerous micro- fouling organisms 37 such as bacteria, viruses, fungi, diatoms and protozoans and the interactions that occur 38 between these organisms initially dictates the formation of the resultant mature biofouling 39 community (Davis et al., 1989; Harder, 2009). Bacterial biofilms can influence the settlement 40 of algal spores (Holmström and Kjelleberg, 1999), fungi (Egan et al., 2000) and the larvae of 41 a wide range of invertebrates (Wahl, 1989; Davis et al., 1989; Holmström and Kjelleberg, 42 1994; Dobretsov, 2010). Even in low densities, some epiphytic bacteria are effective in 43 preventing settlement in a diversity of fouling organisms through the production of inhibitory 44 compounds (Roa et al., 2007). 45 Surface fouling can act as an environmental stressor for marine macro-organisms by 46 decreasing survivorship, particularly at the juvenile stages (Dobretsov, 2010). Mortality in the 47 tropical rock lobster Panulirus ornatus coincides with the formation of filamentous bacterial 48 biofilms (Payne et al., 2007). Biofouling also prevents water from circulating through the 49 ostia of the marine sponge Ianthella basta causing disease and mortality (Cervino et al., 50 2006). Higher incidences of surface fouling have also been associated with significantly 3 51 higher incidences of embryonic mortality in gastropod egg masses (Biermann et. al. 1992, 52 Przeslawski and Benkendorff, 2005). Fouling on the surface of nudibranch and polychaete 53 egg masses has been shown to affect the oxygen supply to internal embryos (Cohen and 54 Strathmann, 1996). These studies suggest that biofouling often has detrimental impacts and 55 highlight the potential for the evolution of defensive strategies for protection. Nevertheless, 56 there are some potential benefits from surface fouling on marine invertebrate egg masses. 57 Symbiotic bacteria on the surface crustacean eggs have been found to prevent infection by 58 pathogenic fungi (Gil-Turnes et al, 1989, Gil-Turnes and Fenical, 1992). Fouling by 59 photosynthetic microphytes can increase embryonic development rates in some species 60 (Przeslawski and Benkendorff, 2005, Fernandes and Podolsky, 2011). A coating of fouling 61 organisms can also provide an effective camouflage (Davis et al, 1989) or reduce the 62 palatability. Surface fouling could also provide protection from solar (UV) radiation, which 63 has been shown to have detrimental effects on encapsulated molluscan embryos (Biermann et 64 al 1992, Rawlings, 1996, Przeslawski et al. 2004, 2005). 65 Submerged surfaces are not equally colonised by marine microbes and many organisms 66 respond to epibiosis by the production of antifouling defences (Davis et al, 1989; Harder, 67 2010). Wahl (1989) describes three common mechanisms of defence: mechanical (shedding, 68 moulting and cleaning of body surfaces), physical (surface free energy, roughness, and 69 surface microtexture) and chemical (the production of bioactive secondary metabolites). 70 Evidence for this combination of antifouling mechanisms has been reported for the surface of 71 egg capsules of the muricid whelk Dicathais orbita (Lim et al., 2007). These mechanisms of 72 defence are often complementary to inhibit or prevent fouling (Camps et al., 2011). The 73 ability of surface microtexture to act as an antifouling defence mechanism is dependent on 74 the scale of the topographical features and the target organisms for repulsion (de Nys et al., 75 2010). Circular elevations 200 μm in diameter are distributed evenly across the carapace of 4 76 the crab Cancer pagurus and deter settlement by the barnacle Balanus improvises (Bers and 77 Wahl, 2004). The brittle sea star Ophiura texturata has 10 μm diameter knob-like structures 78 on its surface, which repel microfouling ciliates, including Zoothamnium commune and 79 Vorticella sp. (Bers and Wahl, 2004). These examples illustrate that surface microtopography 80 alone can be an effective inhibitor of fouling against a range of organisms, depending on the 81 size and structure of the features. On the other hand, some surface topographies may facilitate 82 microfouling, such as the waves and troughs observed on the surface of Sepioteuthis australis 83 (Cephalopoda) egg capsules (Lim et al., 2007). 84 Among sessile benthic organisms, chemical antifouling defence is particularly common. 85 Recently discovered compounds with broad-spectrum antimicrobial activity are continually 86 isolated from marine microorganisms and invertebrates (reviewed by Blunt et al., 2007; 2008; 87 2010, 2011; Lui et al, 2010; Smith et al. 2010). Chemical compounds with antimicrobial 88 activity have been isolated from the egg masses of many marine molluscs. Benkendorff et al. 89 (2001a) conducted a comprehensive investigation into antimicrobial activity of molluscan 90 egg masses from 23 families, 18 of which were found to exhibit antimicrobial properties. 91 Polyunsaturated fatty acids were found to contribute to the antibacterial activity in some 92 gelatinous gastropod egg masses (Benkendorff et al. 2005), whereas brominated indoles were 93 identified as the bioactive agents in egg capsules from the Muricidae family (Benkendorff et 94 al., 2000, 2001b). Ramasamy and Murugan (2005) assessed the activity of macerated 95 molluscan egg masses from the Aplysiidae, Buccinidae, Cypraeidae, Conidae, Cassidae and 96 Muricidae against 40 biofilm bacteria, all of which displayed antimicrobial activity against a 97 portion of these. This high level of activity suggests that chemical defence is widespread in 98 molluscan egg masses, however it is mostly unknown whether the active compounds are 99 contained within the egg masses to prevent invasion, or if they diffuse to the surface to 100 prevent fouling. The biosynthetic origin of the antimicrobial compounds in gelatinous egg 5 101 masses is also unclear. Marine microorganisms have been found to produce a wide range of 102 antimicrobial metabolites (Rahman et al, 2010) and symbiotic bacteria associated with marine 103 invertebrates are increasingly being identified as the source of bioactive compounds (Zheng 104 et al., 2005; Thomas et al., 2010). In recent years, significant advances have been made in 105 recognizing the key role of microbial symbionts in natural products originally thought to be 106 produced by marine invertebrates (Lane and Moore 2011). 107 Marine molluscs of the genus Siphonaria are marine intertidal, herbivorous pulmonates 108 commonly referred to as false limpets. Siphonariids are hermaphrodites with internal 109 fertilisation (Smith et al., 1989; Hodgson 1999) and, following copulation, most species lay 110 gelatinous egg masses as ribbons on rocky substratum. The egg masses are composed largely 111 of mucopolysaccharides (Pal and Hodgson, 2003; Przeslawski, 2004) and each egg capsule is 112 surrounded by a mucous strand and an inner mucous layer (Mapstone, 1978). Siphonariids 113 are characterised by their ability to synthesise polypropionate secondary metabolites (Darias 114 et al., 2006). These compounds, such as the denticulatins, have demonstrated antibacterial 115 activity (Hochlowski et al., 1983; Darias et al., 2006). Lipophylic extracts from egg masses of 116 Siphonaria denticulata and S. zelandica have also been shown to possess antibacterial 117 activity (Benkendorff et al., 2001a), however, preliminary chemical analysis did not reveal 118 the presence of polyproprionates (unpublished data, Benkendorff, 1999). Interestingly, 119 antimicrobial activity detected in the freshly laid Siphonaria egg masses diminished as the 120 embryos matured into shelled veligers (Benkendorff et al., 2001a) and coincidently, visual 121 fouling by algae and protists is also higher at this later stage of development (Przeslawski and 122 Benkendorff, 2005). Consequently, these egg masses may contain defense mechanisms that 123 initially modulate the microbial fouling communities when the embryos are most vulnerable 124 but then degrade to permit fouling prior to hatching. 6 125 Siphonaria diemensis are commonly found in South Australia where they deposit their 126 egg masses on intertidal rocky shores. These gelatinous benthic egg masses are present for 7- 127 10 days, after which veliger larvae are released. Little is known about the bacterial epibiont 128 communities that settle on the egg masses prior to hatching, or the physical and chemical 129 properties that influence their settlement. The aims of this study were, firstly, to characterise 130 the bacterial epibionts on the surface of S. diemenensis egg masses (Fig. 1) using microscopy 131 and biochemical tests. We tested the hypothesis that egg-specific microbial communities exist 132 in comparison with the adjacent rock substratum. We also aimed to determine whether the 133 epibiont community was influenced by chemical defence by testing whether the surface of 134 the egg-masses or associated epibionts exhibit antibacterial activity. 135 136 2. Materials and Methods 137 138 2.1 Sample Collection 139 140 Egg masses (n = 25) of Siphonaria diemenensis at the veliger stage of development 141 (Fig. 1) were collected from August to November 2007 at low tide (0.2 m) from two 142 rocky intertidal reefs in the Gulf St. Vincent, South Australia: Marino Rocks and South 143 Port. All samples were collected from rocks exposed by the low tide. All specimens were 144 transported in fresh seawater collected from the same site to Flinders University where 145 they were either processed immediately for microbiological analysis or maintained 146 overnight in glass aquaria containing sterile seawater for subsequent microscopic and 147 chemical analysis. 148 149 2.2 Microscopy 7 150 151 Fouling communities on egg masses were examined using an optical microscope 152 (Olympus BH-2). Samples were washed for 15 s with sterile sea water to remove 153 transient microorganisms and salt crystals, and then dissected into 5 mm2 sections using a 154 sterile blade. Specimens were mounted on glass slides and viewed under cover slips and 155 also examined after Gram stain. 156 Egg masses (n = 3) were fixed in a solution containing 0.5 % glutaraldehyde, 4 % 157 sucrose and 4 % paraformaldehyde with either phosphate buffered saline (PBS) or TRIS 158 buffer for 24 h and subsequently washed twice in their respective buffers. Each egg mass 159 was sliced into 5 mm2 sections using a sterile blade and fixed in 1 % osmium tetroxide 160 (OsO4), containing 300 μl OsO4 and 300 μl of the respective buffer, for 90 min. 161 Following fixation, samples were dehydrated in 70 % ethanol for 15 min, 90 % ethanol 162 for 15 min, 95 % ethanol for 15 min, and then twice in 100 % ethanol for 15 min. 163 Following dehydration, samples were washed twice in Milli-Q water for 10 mins, and 164 then for a further 15 mins. Specimens were then maintained in 100 % ethanol to prevent 165 artefacts and dried using a critical point drier (Emscope CPD750). Dried specimens were 166 fixed to metal stubs using carbon adhesive and sputter coated with gold (Emscope 167 SC500A Sputter Coater) for viewing under a scanning electron microscope (Siemens 168 Autoscan). Images were captured using a digital camera (Ilford FD4 120). 169 170 2.3 Isolation and Characterisation of Bacteria 171 172 Small portions (10 mm2) from independent egg masses (n = 4 per site) were washed 173 with sterile seawater for 5 s to remove loosely attached microorganisms, then semi- 174 quantitatively sampled by swabbing with sterile cotton tips to collect epibiotic bacteria. 8 175 Each swab was individually suspended in 1 ml sterile seawater in 1.5 ml tubes 176 (Eppendorf) and transported on ice to Flinders University. This procedure was repeated 177 for rocky substratum surface areas adjacent to the egg masses at each site. Bacterial 178 homogenates were suspended in 9 ml sterile seawater and 10-fold serial dilutions were 179 prepared to 10-3. Sub-samples of each dilution (100 μl) were spread-plated in triplicate on 180 nutrient agar (Oxoid No. 2) in seawater and incubated at 25 oC for 72 h. Bacterial growth 181 was recorded and plates were further observed for an additional 5 days to allow for the 182 isolation of slow growing bacteria. Bacterial growth was recorded as colony forming 183 units per ml (CFU ml-1 equivalent to 10mm2 of swabbed substrate), and mean CFU counts 184 were calculated for each morphologically different colony by combining counts from 185 triplicate plates yielding 30-300 colonies for each replicate. Biochemical characterisation 186 of cultivated bacterial isolates differentiated by colonial and cell morphology was 187 performed using a biochemical characterisation kit (API20E, bioMerieux). Test strips 188 from the biochemical characterisation kit were prepared following the manufacturers’ 189 instructions. Incubation parameters were modified following the method described by 190 Popovic et al. (2007) for the isolation of fish pathogens, by incubating strips at 25 oC for 191 72 h. 192 193 2.4 Chemical Extraction 194 195 An investigation was undertaken to assess antimicrobial activity of egg masses with 196 epibiotic communities removed compared to those that were left intact. S. diemenensis 197 egg masses (n = 12) from South Port were pooled and 2 g of egg material was swabbed 198 using sterile cotton tips to collect epibiotic bacteria, vortexed briefly in sterile seawater. 199 The swabbed egg masses were designated ‘clean’. Egg masses with intact epibiotic 9 200 communities were designated as ‘fouled’. Rapid surface extraction was used to assess the 201 antimicrobial activity of compounds more likely to be present on the surface of egg 202 masses. Samples were submerged in approximately 8 ml dichloromethane (DCM, HPLC 203 grade; Sigma), and vortexed for 20 s. The solvent was then decanted and the sample was 204 transferred into fresh vials for overnight extraction to assess the antimicrobial activity of 205 any additional compounds associated with the internal egg mass matrix. Following 206 extraction the solvent was concentrated using a rotary evaporator (Bucci) at 37 oC under 207 337 mbar pressure. Extracts were reconstituted with 3 ml of DCM and transferred to pre- 208 weighed vials, then completely dried under a stream of high purity nitrogen gas. Crude 209 extract yields from the rapid extraction were 0.81 and 0.89 mg mL-1 for fouled and clean 210 egg mass samples respectively, whereas overnight extraction yielded 3.81 and 2.34 mg 211 mL-1 of extract respectively. 212 213 Swabs containing epibionts were processed in 9 ml DCM using the rapid (20 s) 214 extraction technique described above. Sterile swabs were included as a negative control. 215 A bacterial isolate from the surface of egg masses collected from Marino Rocks was also 216 found to inhibit the growth of other epibionts when initially plated onto agar. This isolate 217 was subcultured for antibacterial testing in 50 ml nutrient broth (Oxoid No. 2) overnight 218 at 25 oC on an orbital shaker (Ratec). Cells in the culture were separated by centrifugation 219 at 6000x g for 10 min and the resulting bacterial pellet and culture supernatant were both 220 assessed for antibacterial activity. 221 The bacterial pellet was processed in 9 ml DCM using the rapid extraction technique 222 as described above. The culture supernatant (cell free extract) was concentrated by ion 223 exchange chromatography on Supelco DiaionTM HP-20 (Sigma-Aldrich) resin. Ten g resin 224 was added to a glass column and washed twice with two volumes of 100 % methanol. The 10 225 resin was then washed with two volumes of MilliQ water then left for 10 min before 226 washing for a third time. Then 20 ml cell-free extract (culture supernatant) was added to 227 the column and the mixture was left for 2 h to allow maximum adsorption. The resin was 228 then washed sequentially with 100 % methanol and the eluate collected. This extract was 229 concentrated under rotary evaporation (Bucci, 37 oC; 337 mbar pressure), reconstituted 230 with approximately 3 ml of methanol and dried to completion under a stream of high 231 purity nitrogen gas. 232 233 2.5 Antimicrobial Assays 234 235 Antibacterial activity was tested against a known mollusc pathogen; Vibrio harveyi. 236 Cultures were obtained from stock held at -80 oC at Flinders University (provided by the 237 Department of Primary Industries and Fisheries: Launceston, Tasmania, Australia). 238 Cultures were inoculated onto nutrient agar and incubated overnight at 25 oC, then a 239 single, isolated colony was used to inoculate a 25 ml culture in nutrient broth (Oxoid No. 240 2), which was then incubated overnight at 25 oC on an orbital shaker. Cultures were 241 diluted to an absorbance of 0.1 (600 nm, Metertech, UV/VIS SP8001 Spectrophotometer) 242 and grown to an absorbance of 0.2 to reach exponential growth. 243 A standard disc diffusion assay was used to assess the antimicrobial activity of all the 244 egg mass and epibiont extracts against V. harveyi, following the methodology described 245 by Becerro et al. (1994). A final amount of 50 μg of extract was loaded onto each disk (50 246 μl of a 10 mg ml -1 solution) and all solvent was evaporated in a fume hood prior to 247 placement on the bacterial lawn. The zone of inhibition assay only provides a qualitative 248 indication of antibacterial activity and should not be used for quantitative estimates of the 249 minimum inhibitory concentration. Consequently, we have tested at ~10 x higher than 11 250 natural concentration to maximise the chance of detecting activity accounting for lack of 251 migration of lipophylic active compounds on the agar and potential degradation of some 252 active compounds. Following incubation at 25 oC for 24 h, the zone of inhibition (mm) 253 was measured from the edge of the paper disc to the unaffected bacterial growth, to 254 provide a preliminary assessment of antibacterial activity. Extracts from the bacterial 255 isolate displaying antimicrobial properties from Marino Rocks, were further tested using 256 the zone of inhibition assay against human pathogens Escherichia coli (ACM845), S. 257 aureus (ACM844) (obtained from the Queensland Culture Collection and maintained at - 258 78°C in 15 % glycerol), Candida albicans (Queensland Culture Collection, maintained in 259 saline) and the marine pathogens V. harveyi, V. alginolyticus and V. tubiashi (Department 260 of Primary Industries and Fisheries: Launceston, Tasmania) using culture conditions 261 according to Benkendorff et al. (2001). 262 263 2.6 Molecular Identification of the Antibacterial Isolate 264 265 The epibiont isolated from Marino Rocks displaying antimicrobial activity was 266 identified using PCR of the partial 16 sRNA gene. Cells were grown overnight at 25 oC 267 on nutrient agar (Oxoid No. 2), then an isolated colony was suspended in sterile water in a 268 microcentrifuge tube and pelleted by centrifugation at 5000 x g for 10 min. The 269 supernatant was discarded and the pellet resuspended in 180 μl of enzymatic lysis buffer 270 (20 mM TRIS.Cl pH 8.0; 2 mM sodium EDTA; 1.2 % Triton x-100), with the addition of 271 3.6 mg lysozyme (Sigma) to a final concentration of 20 mg ml-1. DNA extraction was 272 performed using a DNeasy blood and tissue kit (Qiagen) following the manufacturer’s 273 instructions. Extracted DNA was quantified using a GeneQuant II spectrophotometer 274 (620 nm, Pharmacia Biotech). Samples were read in triplicate at 1:20 dilution and DNA 12 275 integrity was assessed via 1 % agarose gel electrophoresis (Bio-Rad Mini Sub-Cell GT 276 gel apparatus) using standard procedures, then stained in a 0.5 μg ml-1 aqueous solution 277 of ethidium bromide for 5 min and visualised under UV. PCR of the extracted DNA was 278 performed on a ThermoHyabid thermocycler using bacterial 16S rDNA gene specific 279 primers: 341F (5’-GCCTACGGGAGGCAGCAG-3’) and 907F (5’- 280 CCGTCAATTCMTTTGAGTTT-3’) (Romero and Navarrete, 2006). Cycling conditions 281 were: initial denaturation at 94 oC for 2 min then 35 cycles of 94oC for 45 s, 54oC for 45 s 282 and 72oC for 90 s. The PCR product was purified using a Wizard DNA clean up kit 283 (Promega) and sequencing was performed by the Australian Genome Research Facility 284 (AGRF). Sequence alignment was performed using GenBank and BLAST through 285 BioManager (Cattley and Arthur, 2007). 286 287 2.7 Statistics 288 289 All statistical analyses were conducted using Primer V5 (Plymouth Marine Lab). The 290 number of culturable bacterial epibionts was calculated using mean CFU ml-1, log 291 transformed, and used to generate a Bray-Curtis similarity matrix between samples. A 292 non parametric multidimensional scaling (nMDS) ordination plot was generated to 293 illustrate the relative similarity between bacterial communities on the rock substratum and 294 those found on the egg masses from the two locations. A two-way analysis of similarity 295 (ANOSIM) was conducted to test the null hypothesis that within group community profile 296 similarity was greater than between groups (Clarke, 1993). Similarity percentages 297 (SIMPER) analysis was used to establish the contribution of each bacterial species to the 298 mean dissimilarity between significantly different groups. 299 13 300 3. Results 301 302 3.1 Microscopy 303 304 The gelatinous matrix was easily distinguishable from egg capsules containing 305 embryos. Light microscopy revealed that each egg capsule within the egg mass was 306 surrounded by a mucous strand, which was connected to adjacent embryos (Fig. 1D). 307 Siphonaria diemenensis development was synchronous, as the egg masses studied 308 contained embryos that were all at the veliger stage of development (e.g. Fig. 1C). 309 Bacteria, protozoa and diatoms were observed in the gelatinous matrix, but never within 310 the egg capsules. No macrofouling organisms were observed by light microscopy, either 311 on or within the egg masses. Embryonic mortality was calculated at 16 % ± 8.9 of the 312 veligers within the egg masses. 313 Scanning electron microscopy revealed a diverse assemblage of fouling organisms on 314 the surface of S. diemenensis egg masses. The surface of the gelatinous matrix was 315 heavily fouled by bacterial rods and cocci (Fig. 2A, B), which formed a biofilm consisting 316 of dense exopolymeric substances (Fig. 2G). Cocci (0.1 μm diameter) were also attached 317 to the surface of the egg capsules within the gelatinous matrix (Fig. 2C). The surface was 318 also fouled by several macrofouling species including filamentous algae (Fig. 2D), 319 dinoflagellates (Fig. 2E) and nematodes (Fig. 2F). 320 Two microtopographies were identified on the surface of the egg masses: 321 longitudinal ridges (Fig. 2H) and wave-like elevations (Fig. 2B, I). The wave-like 322 topography was marked by irregular peaks and troughs, 1 to 3 μm apart and was fouled 323 only by cocci shaped bacteria 0.5 to 1 μm in diameter. Longitudinal ridges (5-20 μm) 324 dominated the surface and were heavily fouled with a dense exopolymeric layer (Fig. 2G). 14 325 In some areas of the egg masses, short lateral valleys were enclosed in longitudinal ridges. 326 Less exopolymeric substance was observed within the valleys and side walls, and these 327 areas also appeared to be preferentially fouled by cocci approximately 0.5 μm in diameter. 328 No bacilli were observed fouling the valleys and side walls. In comparison, bacilli were 329 observed fouling the elevated regions of the ridges found across the surface of the egg 330 masses. Both surface microtopographies were consistent across TRIS and PBS fixed 331 samples. 332 333 3.2 Characterisation of Culturable Bacteria 334 335 Seven morphologically distinct isolates were identified from S. diemenensis eggs and 336 rock substratum samples collected from South Port and Marino (Table 1). The swab and 337 culture techniques used here do not provide a quantitative assessment of absolute 338 abundance of all epibiotic bacteria, but nevertheless they provide a semi-quantitative or at 339 least a qualitative indication of relative abundance. Samples from different surface types 340 displayed different bacterial profiles and not all isolates were present on each substratum 341 at each site (indicated by 0 CFU). Morphologically, the most common isolate exhibited 342 opaque colonies that were rhizoid and flat. This isolate was cultured at densities ranging 343 from 360-810 and 140-270 CFU ml-1, equivalent to 10mm-2 of swabbed substrate, on 344 substratum and egg surfaces respectively, but resisted subculture. As this isolate could not 345 be cultivated in purity and identified, it is referred to as an unknown Gram positive cocci. 346 The second most common isolate consisted of colonies that were circular, smooth, 347 slightly convex, glistening and pale yellow-brown. Based on morphological and 348 biochemical traits, it was identified as Mesophilobacter marinus (Table 1). This bacterial 349 isolate ranged in abundance from 150-26 CFU mm-2 on rock substrata to 2.5-7 CFU mm-2 15 350 on egg surfaces. Mesophilobacter marinus is the only reported Gram negative cocco- 351 bacillus that is oxidase positive, catalase positive, reduces nitrate, produces acid from 352 glucose and mannitol, but not from sucrose and sorbitol. 353 An orange isolate was cultured from some replicate samples at both sites at a density 354 of 0-6 CFU mm-2 for rock substratum and 2-17 CFU mm-2 for egg surfaces. Colonies 355 were smooth, round, glistening and punctiform, and were distinguished from other Gram 356 negative bacilli based on pigmentation, oxidase activity, acid from glucose and indole 357 production from tryptophan (Table 1). Based on this biochemical profile, the isolate was 358 identified to the genus Pseudoalteromonas. The species P. aurantia produces an orange 359 pigment, and does not reduce nitrate or utilise melibiose or D-mannitol carbon sources, 360 which is consistent with the biochemical results for this isolate (Table 1). 361 Yellow colonies were cultivated from rock substratum and egg samples from South 362 Port only (Table 1). Abundance ranged from 5-14 CFU mm-2 on eggs to 2-9 CFU ml-2 on 363 substratum. These colonies were also smooth, round, glistening and punctiform. This 364 isolate was distinguished from other oxidase negative, Gram negative rods bacilli based 365 on pigmentation, acetoin production, and acid production from glucose, arabinose, 366 inositol, mannitol, melibiose and sorbitol (Table 1). Based on this profile the organism 367 was identified to the genus Erwinia. Colonies were further identified as E. uredovora, as 368 this is the only species to produce a yellow pigment, hydrolyse gelatin, and to produce 369 indole from tryptophan and acid from rhamnose. 370 Pink colonies were isolated sporadically from rock substratum (density range from 371 not present (0) to2 CFU mm-2) and egg samples (0-9 CFU mm-2). Colonies were 372 distinguished from other oxidase negative, Gram positive cocci, and identified as 373 Micrococcus, based on their catalase activity and the production of non-diffusible 374 pigment (Table 1). Micrococcus roseus is the only pink-pigmented species that is 16 375 gelatinase negative, reduces nitrate, and produces acid only from glucose, which is 376 consistent with the biochemical profile from this isolate (Table 1). 377 White colonies were isolated consistently across rock substratum (5-15 CFU mm-2) 378 and egg samples (5-20 CFU mm-2) collected from both sites. Differentiation of this isolate 379 from other Gram negative bacilli was based on oxidase activity, the presence of 380 gelatinase, acid from glucose and the lack of pigment (Table 1). Based on this profile, the 381 isolate was identified as another Pseudoalteromonas sp. Psudoalteromonas espejiana is 382 the only un-pigmented species to produce acid from glucose, mannitol and melibiose. 383 Fluorescent colonies were only isolated from substratum samples collected from 384 South Port, at a density of 1-4 CFU mm-2. These colonies were smooth, round and 385 glistening, with yellow-green water soluble fluorescent pigments. This isolate was 386 differentiated from other Gram negative bacilli based on their biochemical profile (Table 387 1) and characteristic yellow-green fluorescence, which is consistent with Azotobacter. 388 Other luminescent strains such as Vibrio and Pseudomonas are oxidase positive, which is 389 inconsistent with this isolate (Table 1). Based on the ability to utilise rhamnose, inositol 390 and mannitol as carbon sources, this isolate was further identified as A. vinelandii. 391 White, butyroid colonies were cultured exclusively from egg samples collected from 392 Marino (1-2.5 CFU mm-2). This isolate was identified as a Gram positive, spore-forming 393 bacillus, which is consistent with the genus Bacillus. However, members of this genus 394 share a large number of morphological and biochemical similarities, therefore genomic 395 analysis was required to identify the isolate further (see Section 3.3 below). On agar 396 plates containing these Bacillus colonies, a zone of inhibition was observed extending 2-4 397 mm out from the colonies, where other isolates were unable to grow, hence this isolate 398 was further tested for antimicrobial activity (see Section 3.5 below). 399 17 400 3.3 Molecular Analysis of Bacillus sp. 401 402 Genetic analysis based on partial 16S rDNA sequencing identified the isolate with 403 antimicrobial activity collected from Marino as Bacillus psychrodurans. Genome analysis 404 showed the amplified 16S rDNA fragment shared 100% sequence similarity with that of 405 B. psychrodurans (Fig. 3), based on the 536 bp sequence. The biochemical profile 406 obtained for this isolate was identical to that described for B. psychrodurans (Fig. 3 407 GenBank accession no. EU 2495666.1 ); however the type strain produces acid from 408 mannitol (Abd El-Rahman et al., 2002). 409 410 3.4 Comparison of Bacterial Communities 411 412 nMDS ordination indicates distinct culturable bacterial community profiles occur on 413 the egg masses as opposed to rock substratum and further, that samples within each 414 location form distinct groups (Fig. 4, stress = 0.08). Two-way ANOSIM revealed 415 significant differences in the community profiles between the two locations (R = 0.85; p = 416 0.001). A significant difference between community profiles was also found between 417 substratum and egg surface samples (R = 0.46, p = 0.003). 418 SIMPER analysis revealed that most of the bacterial species contributed to the 419 differences between substratum and locations (Table 2) with dissimilarity ratios close to, 420 or exceeding 1. P. aurantia, E. uredovora, M. roseus and the unknown Gram positive 421 cocci were all more abundant on the egg masses compared to the rock substratum, 422 whereas M. marinus was more abundant on the rock substratum (Table 2A). Azotobacter 423 vinelandii was the only bacteria found on the rock substratum but not on the egg masses, 424 whereas B. psychrodurans was exclusively cultured form the egg masses (Table 2A). 18 425 Comparison between the two locations revealed that E. uredovora abundance contributed 426 to 31.2 % of the differences between locations, being abundant at South Port and absent 427 from Marino (Table 2B). Azotobacter vinelandii was also absent from Marino and P. 428 aurantia was more abundant at South Port. Bacillus psychrodurans was only cultured 429 from egg masses at Marino, whereas M. roseus and the unknown Gram positive cocci 430 were consistently present at both locations (Table 2B). 431 432 433 3.5 Antimicrobial Activity Using the disc diffusion assay, antibacterial activity against V. harveyi was only 434 detected in extracts of the clean S. diemenensis egg masses. The mean (± standard 435 deviation) width of the zone of inhibition was greater for overnight extracts (4.67 ± 0.6 436 mm) compared to the rapid surface extract (2.00 ± 0.0 mm). No zones of inhibition were 437 observed around the crude extracts collected from fouled egg masses. Crude extracts 438 collected from the swabs of epibiotic communities taken from the egg mass surface were 439 also unable to inhibit the growth of V. harveyi. 440 When tested against a panel of Gram positive and Gram negative bacteria, and fungi, 441 culture supernatant and cell extracts from B. psychrodurans exhibited antibacterial 442 activity against two human pathogens and one marine pathogen. Bacteria consistently 443 sensitive to extracts were E. coli, S. aureus, and V. harveyi, whereas the extracts had no 444 effect on V. alginolyticus or V. tubiashi. No antifungal activity was detected against C. 445 albicans. 446 447 4. Discussion 448 19 449 This study provides a novel insight into the fouling organisms and anti-fouling 450 defence strategies employed by the egg masses of the Siphonariid mollusc S. diemenensis. 451 Combining cultivation techniques with direct observation we provide a detailed view of 452 the bacterial landscape of these molluscan egg mass surfaces. Although only a small 453 proportion of epibiotic bacteria are likely to have been cultured using our techniques, the 454 dominance of coccoid bacteria in culture was confirmed by their abundance on the egg 455 mass surface using scanning electron microscopy. Multivariate SIMPER confirmed that 456 the unknown Gram positive cocci and the coccoid M. roseus were more abundant on the 457 egg masses than the surrounding rock substratum, whereas several bacilli bacterial 458 species were under-represented on the egg masses. Valleys between elevations on the 459 surface of the egg masses appear to provide a favourable attachment site for cocci. This 460 physical structure may facilitate selection of coccoid bacteria and their colonisation of the 461 surface may subsequently allow the attachment of other epibionts (Whitehead and Verran, 462 2006). Antimicrobial defence of the egg masses may also influence the settlement of 463 bacteria on the surface. The antimicrobial activity in DCM extracts prepared from several 464 pooled egg masses was associated with the egg mass matrix, rather than the culturable 465 epibiont communities, with the exception of one antibacterial isolate identified as Bacillus 466 psychrodurans. This species was only found on egg masses from one location and 467 assuming it secretes antibacterial compounds in situ, its presence may have influenced the 468 differences in epibiont community composition detected between sites. Notably no 469 spirochetes were cultured from the egg mass or observed using scanning electron 470 microscopy. Spirochetes are a phylum of morphologically unique prokaryotes that are 471 widespread in aquatic environments, but often resist cultivation (Madigan et al. 2003), as 472 do most other marine strains. As standard culturing techniques are very selective, 473 spirochetes and other uncultivable bacteria are likely to be represented and thus this 20 474 study should only be considered a preliminary assessment of the diversity of bacteria 475 found on the egg masses of S. deimenensis. 476 Using light microscopy, the egg masses of S. diemenensis were found to have a 477 similar physical structure to that reported previously for S. serrata, which also undergoes 478 benthic development (Pal and Hodgson, 2003). Pal and Hodgson (2003) did not observe 479 microorganisms within the egg masses of S. serrata, although they were found in the egg 480 masses of a Siphonariid with planktonic larval development (S. capensis). The degree of 481 bacterial penetration into the gelatinous matrix of molluscan egg masses may be 482 influenced by stage of embryonic development and length of exposure in the 483 environment. The inner mucous layer of some gastropods (Cephalaspidea; Nudibranchia) 484 dissolves during intracapsular development (Klussmann-Kolb and Wägele, 2001) and 485 microbial degradation of the gelatinous matrix around the time of hatching is thought to 486 facilitate the release of juveniles into the water column or onto the substratum. The 487 Siphonaria egg masses examined in this study were at the veliger stage of development 488 and would have been at least a week old. Observations show that the gelatinous matrix 489 degrades in Siphonaria egg masses containing late stage veligers (Smith et al., 1989) and 490 this could be facilitated by specific microbial symbionts. 491 Egg masses exist for only a short period of time in the environment compared to 492 surrounding non-living substrata. Although we can only speculate about the role of 493 specific bacterial epibionts at this stage, it remains possible that some of those that are 494 abundant on the surface of S. diemenensis egg masses may assist with degradation of the 495 gelatinous matrix and facilitate the escape of juveniles. Symbiotic cocci, localised for the 496 purpose of polysaccharide degradation, are associated with eukaryotic hosts. 497 Pseudoalteromonas espejiana was abundant on the egg masses and this bacterium is able 498 to secrete a range of hydrolytic enzymes (Andreev et al., 2007). Bacteria of the genus 21 499 Erwinia also contain a complex arsenal of degradative enzymes (Pirhonen et al., 1993; 500 Venturi et al., 2004). Erwinia uredovora was more abundant on the egg masses than the 501 substratum, but was only recorded on samples from South Port, suggesting this may be an 502 opportunistic facultative epibiont. 503 Multivariate analysis revealed significant differences in the proportion of cuturable 504 bacterial communities occurring on the egg masses of S. diemenensis compared to the 505 rock substratum, however significant differences were also found between locations. 506 Furthermore, bacterial epibionts cultivated from egg mass surfaces were identified on 507 surrounding substratum in seven out of eight cases, thus suggesting facultative 508 association. It is possible that more specific associations with the egg masses could be 509 found in the uncultivable bacteria, although in the marine environment, specific 510 associations between bacterial epibionts and macro-organisms appear to be rare. Wahl 511 and Mark (1999) investigated over 2000 epibiotic associations and found that within any 512 microhabitat, settlers prefer non-living surfaces over macro-organisms. Only one biofilm 513 bacterium, Azotobacter vinelandii, was recorded on the substratum but not the egg masses 514 in this study. However, this bacterium was relatively uncommon and only found at one 515 location. A. vinelandii is a nitrogen fixing bacterium found in plant rhizospheres in the 516 soil (Gorin and Spencer, 1966; Vermani et al., 1995). However, this species has also 517 been identified from mangrove habitats (Ravikumar et al., 2004) and Azotobacter spp. 518 have been found in marine cyanobacterial mats (Zehr et al., 1995). The distribution of A. 519 vinelandii at South Port may be explained by the presence of the sediment on the soft 520 limestone substrata, whereas Marino Rocks is composed of metamorphic conglomerates 521 not covered by sediment particles. An interesting characteristic of Azotobacter spp. is the 522 production of the copolymer alginate (Sabra et al., 2001), which is used by other biofilm 22 523 bacteria to enhance adhesion to surfaces (Rehm and Valla, 1997). The copolymer may be 524 utilised in this way by A. vinelandii to attach to sediment particles on substrata. 525 One bacterial species, which was identified as Bacillus psychrodurans, was isolated 526 exclusively from the surface of egg masses collected from Marino Rocks but not from the 527 rock substratum or any samples at South Port. In light of the low density, site specific 528 association (low density and site-specific), this more likely represents an obligate 529 association rather than an egg mass specific epibiosis. Previous work has identified B. 530 psychrodurans from a range of marine and terrestrial habitats including the surface of 531 brown algae (Lee et al., 2006) , the hindgut of a terrestrial arthropod (Kostanjšek et al., 532 2002) and deep sea Antarctic sponges (Xin et al., 2011). Despite a 100 % sequence match 533 in the 16S DNA with B. psychrodurans on EU 249566.1, our isolate was negative for the 534 production of acid from mannitol, whereas this is a biochemical attribute of the type 535 strain (Abd El-Rahman et al., 2002). 536 Antimicrobial activity has also not been reported for the type stain of B. 537 psychrodurans. However, Xin et al. (2011) reported antimicrobial activity in cultures of 538 B. psychrodurans isolated from Antarctic deep-sea sponges against several 539 microorganisms (Erwinia carotovora, Xanthomonas campestris, and X. oryzae). They 540 also detected the presence of polyketide synthase (PKS) genes in this B. psychrodurans 541 strain, which are responsible for the synthesis of a range of biologically active secondary 542 metabotites (Xin et al., 2011). The biosynthesis of antimicrobial compounds promoted by 543 microbial competition on surfaces can assist the producer in competing for nutrients and 544 space, while at the same time inhibiting pathogens on the host surface. Low cell densities 545 of Pseudoalteromonas tunicata (102-103 cell cm-2), an epibiont of the green algae Ulva 546 australis, effectively prevents the settlement of other fouling organisms, such as algal 547 spores and marine fungi (Rao et al., 2007). In this study, B. psychrodurans was found to 23 548 produce large inhibitory zones against other epibionts in primary mixed plate culture, as 549 well as exerting antimicrobial activity against a number of Gram negative and Gram 550 positive microorganisms, including both marine and human pathogens. Although our 551 DCM extracts were tested at higher than natural concentrations the qualitative assay used 552 typically underestimates the activity of lipophylic extracts (Benkendorff et al. 2000a, 553 2001a). Consequently it is possible that antibacterial production results in competitive 554 exclusion by B.psychrodurans on the surface of the Siphonaria egg masses, influencing 555 the bacterial community composition on the egg masses from Marino. 556 More generally however, antibacterial activity appears to be associated with the 557 internal matrix (including embryos) of S. diemenensis rather than the surface epibionts. In 558 fact, crude extracts from the egg masses of S. diemenensis from South Port only showed 559 antibacterial activity against V. harveyi when removed of their epibionts. The activity was 560 also greater in overnight extracts compared to rapid extraction, suggesting that the 561 antimicrobial compounds are not specifically associated with the surface where they 562 would be most effective at inhibiting biofilm formation. Antibacterial compounds are 563 most likely directed towards inhibiting infection of the egg capsules, which protect the 564 embryos within the gelatinous matrix. This is consistent with our observation that no 565 microorganisms were present with the egg capsules. The egg masses of S. denticulata 566 were also found to be more effective at inhibiting growth when placed on a lawn of 567 bacteria after crushing as opposed to intact (Benkendorff, 1999; Benkendorff et al., 2000). 568 Ramasamy and Murugan (2005) have also reported that activity is localised to the internal 569 matrix of egg masses in a number of other gastropods and it appears that this is the case 570 also for S. diemenensis. 571 572 This study provides an initial characterisation of the physical structure and associated fouling communities on the egg masses of the Siphonariid mollusc S. 24 573 diemenensis. The composition of culturable egg surface epibionts was significantly 574 different from adjacent substrata, suggesting that the surface chemistry and structure of 575 these gelatinous egg masses may favour the settlement of specific bacteria, and in 576 particular cocci. Overall, the bacterial ecology at the surface appears complex, and while 577 antibacterial activity and surface microtexture appear to be weak inhibitors of fouling per 578 se, they could combine along with competitive interactions in the biofilm to form a 579 selective antifouling strategy. To gain a better understanding of antimicrobial defensive 580 strategies in S. diemenensis, an investigation into the change in community composition 581 over time would be beneficial. Further studies to investigate the diversity of uncultivable 582 epibiotic communities on the surface of benthic mollusc egg masses would also be 583 beneficial, using molecular analysis to obtain comprehensive microbial community 584 profiles (e.g. Rudi et al., 2007). 585 586 587 Acknowledgement We are grateful to Kerry Gascoigne for the Flinders Medical Centre for assistance 588 with the Scanning electron microscopy. We thank members of the Molluscan Research lab, 589 Flinders University for useful discussions and assistance in the field. This project was 590 supported by Honours research funding from the School of Biological Sciences, Flinders 591 University and a research grant from the Marine Ecology Research Centre, Southern Cross 592 University. 593 594 References 595 596 597 598 Abd El-Rahman, H.A., Fritze, D., Sproer, C., Claus, D., 2002. Two novel psychrotolerant species, Bacillus psychrotolerans sp. nov. and Bacillus psychrodurans sp. nov., which contain ornithine in their cell walls. International Journal of Systematic and Evolutionary Microbiology. 52, 2127-2133. 25 599 600 601 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. 602 603 604 Becerro, M.A., Lopez, N.I., Turon, X., Uriz, M.J., 1994. Antimicrobial activity and surface bacterial film in marine sponges. Journal of Experimental Marine Biology and Ecology. 179, 195-205. 605 606 607 608 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/ 609 610 611 Benkendorff,K., Bremner, J.B., Davis, A.R. 2000b. Tyrian purple precursors in the egg masses of the Australian muricid, Dicathais orbita: A possible defensive role. Journal of Chemical Ecology. 26, 1037-1050. 612 613 Benkendorff, K. Bremner, J.B., Davis, A.R. 2001b. Indole derivatives from the egg masses of muricid molluscs. Molecules. 6, 70-78. 614 615 616 Benkendorff, K., Davis, A., Bremner, J., 2000a. Rapid screening for antimicrobial agents in the egg masses of marine muricid molluscs. Journal of Medicine and Applied Malacology. 10, 211-223. 617 618 619 Benkendorff, K., Davis, A.R., Bremner, J.B., 2001a. Chemical defense in the egg masses of benthic invertebrates: An assessment of antibacterial activity in 39 mollusks and 4 polychaetes. Journal of Invertebrate Pathology. 78, 109-118. 620 621 622 Benkendorff, K., Davis, A.R., Rogers, C.N., Bremner, J.B. 2005. Free fatty acids and sterols in the benthic spawn of aquatic molluscs, and their associated antimicrobial properties. Journal of Experimental Marine Biology and Ecology. 316, 29-44. 623 624 Bers, A.V., Wahl, M., 2004. The influence of natural surface microtopographies on fouling. Biofouling. 20, 43-51. 625 626 627 Biermann, C.H., Schinner, G.O., Strathmann, R.R., 1992. Influence of solarradiation,microalgal fouling, and current on deposition site and survival of embryos of a dorid nudibranch gastropod. Mar. Ecol. Prog. Ser. 86, 205–215. 628 629 Blunt, J.W., Copp, B.R., Hu, W.-P., Munro, M.H., Northcote, P.T., Prinsep, M.R., 2007. Marine natural products. Natural Product Reports. 24, 31 - 86. 630 631 Blunt, J.W., Copp, B.R., Hu, W.-P., Munro, M.H., Northcote, P.T., Prinsep, M.R., 2008. Review: Marine natural products. Natural Product Reports. 25, 35-94. 26 632 633 Blunt, J.W., Copp, B.R., Munro, M.H., Northcote, P.T., Prinsep, M.R., 2010. Review: Marine natural products. Natural Product Reports. 27, 165-237. 634 635 Blunt, J.W., Copp, B.R., Munro, M.H., Northcote, P.T., Prinsep, M.R., 2011. Review: Marine natural products. Natural Product Reports. 28, 196-268. 636 637 638 639 Camps, M., Dombrowsky, L., Viano, Y., Blache, Y., Briand, J.-F., 2011. Chemical defense of marine organisms against biofouling explored with a bacterial adhesion bioassay, in: Ceccaldi, H.-J., Dekeyser, I., Girault, M., Stora, G. (Eds.). Global Change: MankindMarine Environment Interactions. Springer Netherlands, Part 6 pp. 341-345. 640 641 642 Cattley, S., Arthur, J.W., 2007. BioManager: the use of a bioinformatics web application as a teaching tool in undergraduate bioinformatics training. Briefings in Bioinformatics. 8, 457-465. 643 644 645 646 Cervino, J.M., Winiarski-Cervino, K., Polson, S.W., Goreau, T., Smith, G.W., 2006. Identification of bacteria associated with a disease affecting the marine sponge Ianthella basta in New Britain, Papua New Guinea. Marine Ecology Progress Series. 324, 139-150. 647 648 Clarke, K.R., 1993. Non-parametric multivariate analyses of changes in community structure. Australian Journal of Ecology. 18, 117-143. 649 650 651 Cohen, C.S., Strathmann, R.R., 1996. Embryos at the edge of tolerance: effects of environment and structure of egg masses on supply of oxygen to embryos. Biol. Bull. 190, 8–15. 652 653 Darias, J., Cueto, M., Diaz-Marrero, A.R., 2006. The chemistry of marine pulmonate gastropods. Progress in Molecular and Subcellular Biology. 43, 105-131. 654 655 656 657 Davis, A.R., Targett, N.M, McConnell, O.J. and Young, C.M. 1989. Epibiosis of marine algae and benthic invertebrates: Natural products chemistry and other mechanisms inhibiting settlement and overgrowth, in Scheuer, P.J. (Ed). Bioorganic Marine Chemistry Volume 3. Springer-Verlang, Heidelburg. pp 85-114. 658 659 de Nys, R., Guenther, J., Uriz, M.J., 2010. Natural control of fouling, in Dürr, S and Thomason, J.C. (Eds), Biofouling. Wiley-Blackwell, Oxford, UK. pp. 109-120. 660 661 Dobretsov, S., 2010. Marine Biofilms, in Dürr, S and Thomason, J.C. (Eds), Biofouling. Wiley-Blackwell, Oxford, UK pp. 123-136. 662 663 664 Egan, S., Thomas, T., Holmström, C., Kjelleberg, S., 2000. Phylogenetic relationship and antifouling activity of bacterial epiphytes from the marine alga Ulva lactuca. Environmental Microbiology. 2, 343-347. 27 665 666 667 668 669 670 Fernandes, D.A.O., Podolsky, R.D. 2011. Developmental consequences of association with a photosynthetic substrate for encapsulated embryos of an intertidal gastropod. Journal of Experimental Marine Biology and Ecology. 407, 370–376. 671 672 Gil-Turnes, M.S., Fenical, W., 1992. Embryos of Homarus americanus are protected by epibiotic bacteria. The Biological Bulletin. 182, 105-108. 673 674 Gorin, P.A.J., Spencer, J.F.T., 1966. Exocellular alginic acid from Azotobacter vinelandii. Canadian Journal of Chemistry. 44, 993-998. 675 676 Harder, T. 2009. Marine epibiosis: Concepts, ecological consequences and host defence. Marine and Industrial Biofouling. Springer Series on Biofilms, Volume 4, II, 219-231. 677 678 679 Hochlowski, J.E., Faulkner, D.J., Matsumoto, G.K., Clardy, J., 1983. The denticulatins, two polypropionate metabolites from the pulmonate Siphonaria denticulata. Journal of the American Chemical Society. 105, 7413-7415. 680 681 Hodgson, A.N., 1999. The biology of siphonariid limpets (Gastropoda : Pulmonata). Oceanography and Marine Biology. 37, 245-314. 682 683 Holmström, C., Kjelleberg, S., 1994. The effect of external biological factors on settlement of marine invertebrates and new antifouling technology. Biofouling. 8, 147-160. 684 685 686 Holmström, C., Kjelleberg, S., 1999. Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents. FEMS Microbiology Ecology. 30, 285-293. 687 688 689 Klussmann-Kolb, A., Wägele, H., 2001. On the fine structure of opisthobranch egg masses (Mollusca, Gastropoda). Zoologischer Anzeiger - A Journal of Comparative Zoology. 240, 101-118. 690 691 692 Kostanjšek, R., Štrus, J., Avguštin, G., 2002. Genetic diversity of bacteria associated with the hindgut of the terrestrial crustacean Porcellio scaber (Crustacea: Isopoda). FEMS Microbiology Ecology. 40, 171-179. 693 694 Lane, A.L., Moore, B.S. 2011. A sea of biosynthesis: marine natural products meet the molecular age. Natural Product Reports. 28, 411-428 695 696 Lee, Y.K., Jung, H.J., Lee, H.K., 2006. Marine bacteria associate with the Korean brown alga, Undaria pinnatifida. The Journal of Microbiology. 44, 694-698. 697 698 Lim, N.S.H., Everuss, K.J., Goodman, A.E., Benkendorff, K., 2007. Comparison of surface microfouling and bacterial attachment on the egg capsules of two molluscan species Gil-Turnes, M., Hay, M., Fenical, W., 1989. Symbiotic marine bacteria chemically defend crustacean embryos from a pathogenic fungus. Science. 246, 116-118. 28 699 700 representing Cephalopoda and Neogastropoda. Aquatic Microbial Ecology. 47, 275287. 701 702 703 Liu, X.Y., Ashforth, E., Ren, B.A., Song, F.H, Dai, H.Q. , Liu, M., Wang, J.A., Xie, Q.O., Zhang, L.X. 2010. Bioprospecting microbial natural product libraries from the marine environment for drug discovery. Journal of Antibiotics. 63, 415-422 704 705 Madigan, M.T., Martinko, J.M., Parker, J., 2003. Brock Biology of Microorganisms. Prentice Hall. Upper Saddle River NJ. 706 707 708 Mapstone, G., 1978. Egg capsules and early development in Siphonaria diemensis (Quoy and Gaimard, 1833) and Siphonaria baconi (Reeve, 1856). Journal of the Malacological Society. 4, 85-92. 709 710 711 Pal, P., Hodgson, A.N., 2003. The structure of the egg ribbons of a planktonic and intracapsular developing siphonariid limpet (Gastropoda: Pulmonata). Invertebrate Reproduction and Development. 43, 243-253. 712 713 714 Payne, M.S., Hall, M.R., Sly, L., Bourne, D.G., 2007. Microbial diversity within early-stage cultured Panulirus ornatus phyllosomas. Applied and Environmental Microbiology. 73, 1940-1951. 715 716 717 718 Pirhonen, M., Flego, D., Heikinheimo, R., Palva, E., 1993. A small diffusable signal molecule is responsible for the globabl control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora. The EMBO Journal. 12, 24672476. 719 720 Popovic, N., Coz-Rakovac, R., Strunjak-Perovic, L., 2007. Commercial phenotypic tests (API20E) in diagnosis of fish bacteria: a review. Veterinarni Medicina. 52, 167-173. 721 722 Przeslawski, R., 2004. A review of the effects of environmental stress on embryonic development within intertidal gastropod egg masses. Molluscan Research. 24, 43-63. 723 724 Przeslawski, R., Benkendorff, K., 2005. The role of surface fouling in the development of encapsulated gastropod embryos. Journal of Molluscan Studies. 71, 75-83. 725 726 Przeslawski, R., Benkendorff, K., Davis, AR. 2005. Synergies, climate change and the development of rocky shore invertebrates. Global Change Biology. 11, 515-522. 727 728 729 Przeslawski, R., Davis, A., Benkendorff, K. 2004. Effects of ultraviolet radiation and visible light on the development of encapsulated molluscan embryos. Marine Ecology Progress Series. 268,151-160. 730 731 732 Rahman, H., Austin, B., Mitchell, W.J., Morris, P.C., Jamieson, D.J., Adams, D.R., Mearns Spragg, A., Schweizer, M. 2010. Novel anti-infective compounds from marine bacteria. Marine Drugs. 8, 498–518. 29 733 734 735 Ramasamy, M.S., Murugan, A., 2005. Potential antimicrobial activity of marine molluscs from Tuticorin, southeast coast of India against 40 biofilm bacteria. Journal of Shellfish Research. 24, 243(249). 736 737 738 739 Rao, D., Webb, J.S., Holmström, C., Case, R., Low, A., Steinberg, P., Kjelleberg, S., 2007. Low densities of epiphytic bacteria from the marine alga Ulva australis inhibit settlement of fouling organisms. Applied and Environmental Microbiology. 73, 78447852. 740 741 742 Ravikumar, S., Kathiresan, K., Ignatiammal, S.T.M., Babu Selvam, M., Shanthy, S., 2004. Nitrogen-fixing azotobacters from mangrove habitat and their utility as marine biofertilizers. Journal of Experimental Marine Biology and Ecology. 312, 5-17. 743 744 Rawlings, T.A. 1996. Sheilds against ultraviolet radiation: an additional protective role for the egg capsules of benthic marine gastropods. Mar. Ecol. Prog. Ser. 136, 81-95. 745 746 Rehm, B.H.A., Valla, S., 1997. Bacterial alginates: biosynthesis and applications. Applied Microbiology and Biotechnology. 48, 281-288. 747 748 749 Romero, J., Navarrete, P., 2006. 16S rDNA-Based analysis of dominant bacterial populations associated with early life stages of Coho salmon (Oncorhynchus kisutch). Microbial Ecology. 51, 422-430. 750 751 752 Rudi, K., Zimonja, M., Trosvik, P., Naes, T. 2007. Use of multivariate statistics for 16s rRNA gene analysis of microbial communities. International Journal of Food Microbiology. 120, 95-99. 753 754 Sabra, W., Zeng, A.P., Deckwer, W.D., 2001. Bacterial alginate: physiology, product quality and process aspects. Applied Microbiology and Biotechnology. 56, 315-325. 755 756 757 Smith, B., Black, J., Shepherd, S., 1989. Molluscan egg masses and capsules. in: Shepherd, S., Thomas, I. (Eds.), Marine Invertebrates of Southern Australia: Part II. South Australia Government Printing Division, Adelaide, pp. 786. 758 759 760 Smith, V.J., Desbois, A.P., Dyrynda, E.A. 2010. Conventional and unconventional antimicrobials from fish, marine invertebrates and micro-algae. Marine Drugs. 8, 1213-1262 761 762 Thomas, T.R.A., Kavlekar, D.P., LokaBharathi, P.A., 2010. Marine drugs from spongemicrobe association—A Review. Marine Drugs. 8, 1417-1468. 763 764 765 766 Venturi, V., Venuti, C., Devescovi, G., Lucchese, C., Friscina, A., Degrassi, G., Aguilar, C., Mazzucchi, U., 2004. The plant pathogen Erwinia amylovora produces acylhomoserine lactone signal molecules in vitro and in planta. FEMS Microbiology Letters. 241, 179-183. 30 767 768 769 Vermani, M.V., Kelkar, S.M., Kamat, M.Y., 1995. Novel polysaccharide produced by Azotobacter vinelandii isolated from plant rhizosphere. Biotechnology Letters. 17, 917-920. 770 771 Wahl, M., 1989. Marine Epibiosis. I. Fouling and Antifouling: Some basic aspects. Marine Ecology Progress Series. 58, 175-189. 772 773 Wahl, M., Mark, O., 1999. The predominantly facultative nature of epibiosis: experimental and observational evidence. Marine Ecology Progress Series. 187, 59-66. 774 775 Whitehead, K.A., Verran, J., 2006. The effect of surface topography on the retention of microorganisms. Food and Bioproducts Processing. 84, 253-259. 776 777 778 Xin, Y., Kanagasabhapathy, M., Janussen, D., Xue, S., Zhang, W., 2011. Phylogenetic diversity of Gram-positive bacteria cultured from Antarctic deep-sea sponges. Polar Biology. 34, 1501-1512. 779 780 781 Zehr, J.P., Mellon, M., Braun, S., Litaker, W., Steppe, T., Paerl, H.W., 1995. Diversity of heterotrophic nitrogen fixation genes in a marine cyanobacterial mat. Applied and Environmental Microbiology. 61, 2527-2532. 782 783 784 Zheng, L., Han, X., Chen, H., Lin, W., Yan, X., 2005. Marine bacteria associated with marine macroorganisms: the potential antimicrobial resources. Annals of Microbiology. 55, 119-124. 785 786 787 31 788 Table 1: Biochemical characteristics of bacterial isolates cultured from the surfaces of 789 Siphonaria diemensis egg masses (EM) and rocky substratum (R) at South Port (SP) and 790 Marino Rocks (M), Gulf St. Vincent, South Australia Trait Source Location Pigmentation Gram Reaction Cell Shape Pau EM, R SP, M Orange Eur EM, R SP Yellow Pes EM, R SP, M White Mro EM, R SP, M Pink Mma Avi Bps EM, R R EM SP, M SP M Yellow- Yellow- White Brown Green + + + Bacillus Bacillus Bacillus Coccus Cocco- Bacillus Bacillus Bacillus - Spore Biochemistry Catalase + + + + + + Oxidase + + + + ortho-nitro phenyl β-dgalactopyranoside 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 32 791 792 Table 2 793 SIMPER result for the determination of dissimilar bacterial epibionts, calculated as 794 percentage abundance for (A) egg masses and surrounding substratum, and (B) location 795 A Average Dissimilarity = 27.24 Substratum Egg Species P. aurantia E. urevodora M. roseus Unknown A. vinelandii B. psychrodurans M. marinus Av. Abund 15.00 26.25 6.25 37.50 12.50 Av. Abund 46.25 36.25 32.50 45.00 0.00 Diss/SD 1.44 1.09 1.31 3.35 0.96 Contrib. % 9.06 17.37 17.34 13.38 11.22 Cum. % 9.06 36.43 53.77 67.15 78.37 0.00 502.50 7.50 215.00 0.96 2.47 10.97 6.99 89.34 96.32 B Average Dissimilarity = 28.12 Species E. uredovora M. roseus P. aurantia A. vinelandii B. psychrodurans Unknown South Port Marino Av. Abund 62.50 18.75 45.00 12.50 Av. Abund 0.00 20.00 16.25 0.00 Diss/SD 6.86 1.43 1.13 0.96 Contrib. % 31.20 15.18 14.80 11.67 Cum. % 31.20 46.38 61.18 72.85 0.00 130.00 7.50 138.75 0.97 1.31 9.78 7.66 82.63 90.29 796 797 798 33 799 Captions 800 801 Figure 1: Siphonaria diemenensis A) adults and B) egg masses. Light microscopy of S 802 diemenensis egg mass showing C) encapsulated veliger larvae and D) compartment-like 803 organisation of gelatinous matrix contain egg capsules, inner mucous layer and mucous 804 strand that connects adjacent embryos (indicated by the arrow). 805 806 Figure 2: Scanning electron microscopy of Siphonaria diemensis egg masses. The surface of 807 the eggs are fouled by; A) rods and B) cocci. Egg capsules within the gelatinous matrix are 808 fouled by C) cocci. Macrofouling species include D) filamentous algae, E) dinoflagellates 809 and F) nematodes. The two topographies were G) under extensive exoploymeric fouling and 810 were characterised by H) longitudinal ridges and I) wave-like elevations. 811 812 Figure 3: BLASTn alignment between the sequences obtained from an antimicrobial isolate 813 cultured from the surface of Siphonaria diemensis egg masses (labelled ‘Unknown isolate’) 814 and Bacillus psychrodurans partial 16S rRNA gene (GenBank accession number EU 815 249566.1). 816 817 Figure 4: Non parametric multi-dimensional scaling ordination showing the similarities in 818 culturable bacterial epibiotic communities isolated from Siphonaria diemenensis egg masses 819 () and substratum () from South Port (open symbols) and Marino (filled symbols). The two 820 dimensional plot is generated from the log-transformed abundance data using a Bray-Curtis 821 similarity matrix in PRIMER V.5. Stress = 0.08 822 34 823 824 825 Fig. 1 826 35 827 828 Fig. 2 829 830 831 36 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 Unknown isolate AAGTCTGATGGAGCAATGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTG B. psychrodurans AAGTCTGATGGAGCAATGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTG 859 Fig. 3 Unknown isolate TGAGGGAAGAACAAGTACGAGTAACTGCGCTCGTACCTTGACGGTACCTCATTAGAAAGC B. psychrodurans TGAGGGAAGAACAAGTACGAGTAACTGCGCTCGTACCTTGACGGTACCTCATTAGAAAGC Unknown isolate CACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAAT B. psychrodurans CACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAAT Unknown isolate TATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGATGTGAAATCCCACGGCTCA B. psychrodurans TATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGATGTGAAATCCCACGGCTCA Unknown isolate ACCGTGGAAGGTCATTGGAAACTGGGGGACTTGAGTACAGAAGAGGAAAGCGGAATTCC B. psychrodurans ACCGTGGAAGGTCATTGGAAACTGGGGGACTTGAGTACAGAAGAGGAAAGCGGAATTCC Unknown isolate AAGTGTAGCGGTGAAATGCGTAGAGATTTGGAGGAACACCAGTGGCGAAGGCGGCTTTC B. psychrodurans ACCGTGGAAGGTCATTGGAAACTGGGGGACTTGAGTACAGAAGAGGAAAGCGGAATTCC Unknown isolate TGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTG B. psychrodurans TGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTG Unknown isolate TGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCT B. psychrodurans TGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCT Unknown isolate GCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAG B. psychrodurans GCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAG 860 37 Substratum, South Port Siphonaria diemenensis, Southport Substratum, Marino Siphonaria diemenensis, Marino 861 862 Fig. 4 863 38