Download Characterisation of the physical and chemical properties influencing

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