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Settlement inducers for larvae of the tropical fouling serpulid, Spirobranchus kraussii (Baird, 1865) (Polychaeta: Annelida)
International Biodeterioration & Biodegradation 94 (2014) 192e199
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Settlement inducers for larvae of the tropical fouling serpulid, Spirobranchus kraussii (Baird, 1865) (Polychaeta: Annelida) Janlin Y-H. Chan a, Serina S-C. Lee a, *, Siti Zarina Zainul Rahim b, Serena L-M. Teo a a b
Tropical Marine Science Institute, National University of Singapore, Singapore 119227, Singapore Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 January 2014 Received in revised form 29 July 2014 Accepted 29 July 2014 Available online
Calcareous tubeworms are one of the most abundant fouling organisms found on substrates submerged in marine environments. Research has clearly shown that tubeworms select surfaces with a biofilm, and attachment and adhesion strength is enhanced in the presence of a biofilm. Studies have also shown that abiotic materials in the environment are strongly linked to proliferation of aquatic benthic microbes, which in turn may influence larval recruitment. Soil-derived nutrients are capable of supporting large, diverse bacterial communities and have been used to encourage growth of heterotrophic microbes. In this study we investigate the effect of organic nutrient-rich extract derived from sediments on larval settlement of the fouling calcareous polychaete, Spirobranchus kraussii (Baird, 1865). Application of a soil extract, as used in the preparation of GSe microalgal culture media, induced larval settlement within 24 h in laboratory assays. Our observations suggest that soil extract promoted the growth of an inductive biofilm from microbiota present on the larvae during the incubation period. However, the presence of a chemical inducer could not be conclusively discounted. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Biofouling Larval settlement Serpulidae Biofilms Spirobranchus (syn. Pomatoleios) kraussii
1. Introduction The understanding of larval behaviour and inducers of settlement of common marine fouling organisms provides valuable clues for the development of methods to control macrofouling of underwater structures. Much of the earlier work has focused on the biology of barnacles, in particular that of Amphibalanus amphitrite (Holm, 2012). Interest in tubeworm fouling on man-made materials has grown in recent years as the adhesion strength of these organisms on antifouling foul-release coatings appears to be stronger than that of barnacles (Holm et al., 2006). Subsequently, Zardus et al. (2008) reported that adhesion strength increases in the presence of a biofilm. Biofilms have long been recognized as a natural stimulus for marine polychaete larvae settlement (Kirchman et al., 1981; Unabia and Hadfield, 1999), and larvae appear to be highly discriminant between biofilms originating from different bacterial species (Huang and Hadfield, 2003; Lau et al., 2003). For example, Janua brasiliensis only settles in the presence of a bacterial species associated with its natural substratum, the green macroalga Ulva lobata * Corresponding author. 18 Kent Ridge Road, Tropical Marine Science Institute, National University of Singapore, Singapore 119227, Singapore. Tel.: þ65 98223849. E-mail address: [email protected] (S.S.-C. Lee). http://dx.doi.org/10.1016/j.ibiod.2014.07.017 0964-8305/© 2014 Elsevier Ltd. All rights reserved.
(Kirchman et al., 1981). The larvae of Hydroides elegans have been shown to metamorphose in the presence of phage tail-like structures produced by the bacterium Pseudoalteromonas luteoviolacea (Shikuma et al., 2014). Recently, Tait and Havenhand (2013) suggested that N-acylhomoserine lactones involved in bacterial quorum sensing may also provide cues for settling barnacle larvae. These observations provide new insights into the ubiquitous way bacteria and biofilms are shaping sessile invertebrate communities. Perspectives of bacterialeinvertebrate interactions have evolved with the increasing acceptance of bacteria as key contributors in the energy transfer, stability of the natural environment, and its diversification (Bonar et al., 1986; Battin et al., 2003; McFall-Ngai et al., 2013). It is known that bacterial diversity and abundance in microbial films fluctuate in response to availability of nutrients in the environment (Barranguet et al., 2002; Fischer et al., 2002). The density and identity of aquatic benthic microbes may exhibit strong dependency and sensitivity to variations in abiotic factors such as particulate organic matter, amino acids and peptide concentrations in the environment (Fischer et al., 2002; Pringault et al., 2008; Hochbaum et al., 2011; Sawall et al., 2012). Experiments conducted by numerous authors (Keough and Raimondi, 1996; Hung et al., 2009; Chung et al., 2010) have established a strong interdependency between settlement in marine invertebrates and bacterial colonization.
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In this study we investigate the effect of organic nutrient-rich extract derived from sediments (henceforth referred to as soil extract) on larval settlement of the fouling calcareous polychaete, Spirobranchus kraussii (Baird, 1865) (Pomatoleios kraussii of authors; see Pillai, 2009). S. kraussii is a common intertidal serpulid found across the Indo-Pacific (Straughan, 1969). On natural substrates, these tubeworms appear to be gregarious and occur as reef builders. In Singapore mangroves, S. kraussii occurs in dense aggregates on Avicennia and Rhizophora mangrove roots. It is also common in fouling communities on hard structures in coastal areas, on ship hulls, wooden boardwalks and fish farms (Straughan, 1969; Cinar, 2006). Soil, as a medium rich in abiotic nutrients, is capable of supporting a high magnitude of bacterial communities and species diversity (Torsvik et al., 1990; Gans et al., 2005), inspiring the use of soil-derived nutrients as an enhancer of heterotrophic microbial growth (Fischer, 2003; Liebeke et al., 2009; Tikhonov et al., 2010). As S. kraussii is an estuarine species, it was hypothesized that soil nutrients may act as a cue for settlement. For our experiments, we considered soil sourced from pristine, uncultured land to minimise variables such as contamination by inductive adult conspecifics, toxic substances and chemical residues from coastal pollution that may have an adverse effect on larval mortality and settlement (Bayen et al., 2005; Cuong et al., 2005). The use of pristine soil also reduces fluctuations in organic levels as wastewater and runoff can affect the microbial population and composition of mangrove soils (Tam, 1998). 2. Materials and methods 2.1. Larval culture S. kraussii adults were collected from mangrove roots in Lim Chu Kang, Singapore. Fertilization and culturing were modified from methods first described by Hadfield et al. (1994) and Bryan et al. (1997). Larvae were reared on 1:1 mixture of Isochrysis galbana (CS-177) and Nannochloropsis oculata (CS-179) at an approximate concentration of 4.6 105 cells per ml suspended in 27 ppt 0.22 mm-filtered seawater (FSW). Cultures were maintained at 27 C with a 12:12 h light: dark cycle and gentle aeration. The culture medium was renewed every two days. After seven to nine days, competent larvae were visually assessed before harvest for experimentation use. Metamorphically competent larvae acquired elongated tails with visible straightening of the abdominal mass with appearance of two slight indentations on the anterior end (Crisp, 1977; Nedved and Hadfield, 2009). Behavioural changes in mature larvae were also evident with larvae actively crawling and exploring the surfaces of culture beakers. 2.2. General design of settlement experiments Settlement experiments were performed in FSW using seven to nine day old competent larvae. Larvae were harvested by filtering through a 50 mm mesh and rinsed thrice in FSW prior to use. Every experiment consisted of five replicates, each containing 20e30 larvae in 2 ml FSW. All treatments were conducted twice with different batches of larvae for consistency. Settlement dishes were incubated in the dark for 24 h at 27 C, and scored under a dissecting microscope for settled, swimming and dead larvae. Individuals with secondary or primary tubes were classified as settled. Secondary tubes were identified by the appearance of an opaque calcified tube, and well-differentiated branchial radioles on the worm. Primary tubes were discernible by transparent colouring and developing branchial lobes of the attached juveniles (Nedved and Hadfield, 2009).
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Biofilm density was determined in all settlement dishes by staining with BacLight bacterial viability kit (Invitrogen) according to manufacturer's instructions (Roth et al., 1997). Bacterial abundance was visualized under an epifluorescence microscope. Three photographs of approximate 3.80 mm2 each were taken for every dish under 100 magnification. Five random spots per photo were selected for assessment of microbial biomass with an estimated area of 2.17 mm2 quantified for each dish. The only two exceptions to this procedure were when IBMX was used (See Section 2.3.2) and when biofilm was harvested for fingerprinting via terminal restriction fragment length polymorphism, TRFLP (See Section 2.4). 2.3. Settlement of tubeworms in response to soil extract Sterile soil extract was prepared following protocols provided by the CSIRO Australian National Algae Culture Collection for the preparation of CMAR soil extract (http://www.marine.csiro.au/ microalgae/methods/). Soil collected from uncultivated land on St John's Island, Singapore was dried and sieved through a 2 and 1 mm mesh to obtain fine powder. Distilled water was added at a ratio of 1 kg soil to 2 L water, and the mixture was sterilized in an autoclave. The suspension was left to cool and settle over night at room temperature before collection. The aqueous portion was twice filtered through absorbent cotton until a clear dark brown solution was produced. This solution was again sterilized, then sealed and stored at 4 C prior to use. As the soil extract was sterile, and tubeworm larvae were rinsed in sterile FSW before use, the main source of bacteria present in the assays would be those adhered to the larvae. 2.3.1 Liquid versus dried soil extract To determine if the method of applying soil extract affects settlement, aqueous soil extract was added to FSW with larvae to give final concentrations of 100, 200 and 300 ml ml1. Addition of liquid soil extract resulted in reduced salinity of approximately 25, 23 and 20 ppt at 100, 200 and 300 ul/ml respectively. However, salinity in mangrove swamps in the Johor Straits, where S. kraussii were collected, fluctuate between 20 and 33 ppt due to runoffs and tidal flow (Chou and Lee, 1997) and S. kraussii has even been shown to thrive in 16e20 ppt (Perthuisot et al., 1990; Shalla and Holt, 1999) environments. Another set was prepared by air-drying an equivalent amount of soil extract on sterile 35 mm petri dishes before topping up with FSW and larvae. Negative controls consisted of petri dishes with FSW and larvae only. Dried soil extract was selected for use as it resulted in higher settlement rates than liquid (Fig 1). As there was only a marginal increase in settlement even at a three-fold increase of soil extract (Fig 2A), 100 ml1 was selected as the final working concentration for all assays. 2.3.2 Soil extract versus IBMX To compare the effect of soil extract with a known chemical inducer of larval settlement, settlement assays were set up as in Section 2.2 using 10e4 M of IBMX and dried soil extract at a final concentration of 100 ml ml1. 2.3.3 Antibiotic assays Antibiotics were applied to inhibit the growth of bacteria on soil extract-coated dishes to determine if a reduction in biofilm affects the settlement-inducing properties of soil extract. Stock solutions of tetracycline hydrochloride, ampicillin sodium and streptomycin sulfate (SigmaeAldrich) at 10 mg ml1 were prepared in distilled water. Final testing concentrations of 1, 10, 50 and 100 mg ml1 were obtained by diluting antibiotic stock solutions in FSW dispensed
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Fig. 1. Presence of dried soil extract increases (A) metamorphosis of S. kraussii larvae and (B) biofilm density after 24 h incubation compared to equal volumes of liquid soil extract. Asterisks (*) indicate significant difference between the two bars under the line.
into petri dishes with larvae. Negative controls consisted of Petri dishes with only FSW and larvae. Dried soil extract at 100 ml ml1 was used as a positive control.
(New England Biolabs) before capillary electrophoresis and analysis using GeneMapper software (Applied Biosystems). 2.5 Statistical analysis
2.3.4 Biofilm formation in marine nutrient broth compared to soil extract To determine if the inductive effect of the biofilm was due to bacterial numbers or composition, biofilms were grown with commercially available nutrient-rich broth. Marine broth (Conda™), prepared as per manufacturer's instructions, was applied to sterile 35 mm petri dishes and allowed to dry over night before topping up with FSW and larvae resulting in final test concentrations of 0.4, 1 and 2 mg ml1. Negative controls consisted of Petri dishes with only FSW and larvae. Dried soil extract at 100 ml ml1 was used as positive control. 2.4 Biofilm fingerprinting (terminal restriction fragment length polymorphism, TRFLP)
Untransformed settlement data were tested for parametric assumptions based on similar studies (Wilson et al., 2010; Warton and Hui 2011; Huang et al., 2012; Sokal and Rolf 2012), then analyzed by use of the KruskaleWallis non-parametric ANOVA, where p < 0.05 considered significant, and presented as the mean ± standard error. TRFLP data were exported and transformed into an absence-presence matrix, then a non-metric multidimensional scaling (NDMS) was conducted using BrayeCurtis distance measures and k ¼ 2 in conjunction with the function “ordiellipse” to delimit the 95% confidence interval around the clusters. Data were analyzed by use of R software package (R Development Core Team, 2010). 3. Results
In order to harvest sufficient biofilm for terminal restriction fragment length polymorphism (TRFLP) analysis, the exact experimental set up described in Section 2.3.4 was scaled up using 90 mm petri dishes with a final volume of 15 ml. Only petri dishes with settlement were used to harvest biofilm. Bacteria were removed from the surface using 1% 3M enzymatic cleaner (Burke et al., 2009). DNA was extracted using phenol:chloroform as previously described (Chen et al., 2013) and amplified using universal bacterial primers 926F (Matsuki et al., 2002) and 1392R (Lane et al., 1985) modified with 6-FAM and ATTO 565 dyes respectively. Amplicons were digested with AluI and Bsu361
3.1 Liquid versus dried soil extract Addition of soil extract increased settlement in S. kraussii larvae and biofilm growth. This effect was more pronounced when the extract was dried onto the dish surface compared to the equivalent amount inoculated into solution (Fig. 1A and B). As concentrations of liquid soil extract increased, the settlement rate presented a downward trend, opposite to that in dried soil extract treatments. Mortality (not presented) was not significantly different between liquid and dried soil extract at the various concentrations tested.
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larvae in the presence of antibiotics were low across all concentrations and not significantly different from the positive control containing only soil extract and larvae. 3.4 Biofilm formation in marine nutrient broth compared to soil extract Nutrient rich marine broth promotes bacterial biofilm growth but denser biofilm, obtained from applying higher concentrations of broth, did not correlate to increased settlement. It was notable that even though the lowest concentration of marine broth resulted in a comparable bacterial biofilm density to that of soil extract (Fig. 4B), the larval settlement in biofilms from broth was significantly lower (Fig. 4A). Mortality of S. kraussii larvae was significantly higher in marine broth treatments compared to positive control of soil extract, but we did not observe a doseeresponse curve when higher concentrations of marine broth were applied. 3.5 Biofilm fingerprinting (TRFLP) TRFLP analysis of the biofilms that developed when tubeworms were allowed to settle on Petri dishes with soil extract and marine broth indicate that these were significantly different communities from biofilms that developed with no treatment (Fig. 5). The bacterial community of soil extract-associated biofilms appears to be a subset of the marine broth associated community, showing that soil extract selects for a more specific set of bacteria within the biofilm. 4. Discussion Fig. 2. (A) Settlement induction of soil extract (SE) does not display a doseeresponse curve over the range of treatment levels that were used. (B) Dried soil extract is as effective as 104 M IBMX at inducing settlement, but with less mortality. Asterisks (*) indicate significant difference from (A) negative control and (B) IBMX control.
There was no significant difference in settlement rates of S. kraussii larvae across 3 concentrations of dried soil extract (Fig. 2A). 3.2 Soil extract versus IBMX The presence of soil extract induced settlement in S. kraussii larvae at levels comparable to IBMX but with significantly less mortality (Fig. 2B). Secondary tubes were obtained with soil extract, whereas in presence of IBMX most of the worms had primary tubes after 24 h. Where IBMX was used, some of the larvae that did metamorphose appeared bloated and laid outside of their tubes. Instances of mass settlement in clumps and on water surfaces have also been observed. Developmental abnormalities also increased with appearance of developing branchial lobes seen in swimming larvae. These were scored as swimming. 3.3 Antibiotic assays Bacterial density appeared to influence larval settlement as the application of antibiotics resulted in a significant reduction of settlement even in the presence of soil extract. For all three antibiotics, a dose-dependent significant decrease in bacterial density was observed with increase in antibiotic concentration (Fig. 3B, D, F), which generally corresponded to an equivalent reduction in settlement (Fig. 3A, C, E). Ampicillin produced the strongest inhibitory effect followed by streptomycin (Fig. 3A, C). Although there was no statistical significance between settlement in different concentrations of tetracycline (Fig. 3E), there appeared to be a reduction in numbers as concentration increased. Mortality rates of S. kraussii
In this study we considered the possible effects of soil associated nutrients on the recruitment of S. kraussii, and the effect of soil extract cultured biofilm on larvae settlement. Three hypotheses can be inferred from the results of the experiments. Firstly, the chemical cues in soil extract may stimulate S. kraussii metamorphosis, promoting settlement. Secondly, as soil associated nutrients have been found to promote proliferation of specific bacterial communities soil extract may indirectly promote settlement by enhancing growth of this inductive bacterial consortium. Thirdly, the settlement inducing effect of soil extract may be a result of a combination of both factors mentioned above. The complex mixture of compounds present in soil extract may be acting directly on larval receptors, or increasing settlement rates indirectly by enhancing the growth of suitable biofilms. Microbial presence is ubiquitous in all invertebrate settlement assays (Jin and Qian, 2004) and antibiotics are often used to minimize bacteria. Ampicillin, streptomycin and tetracycline are commonly used to eradicate bacteria; however, complete elimination was difficult due to the high antibiotic resistance of marine bacteria (Grabow et al., 1974; Cooke, 1976; Baya et al., 1986) in a biofilm (Gander, 1996; Costerton et al., 1999; Brooun et al., 2000). Lower settlement of larvae was observed in the treatments with fewer bacteria as a result of antibiotic treatment, indicating an association between settlement and bacterial abundance. These findings are consistent with conclusions of Keough and Raimondi (1996) and Jin and Qian (2004): surfaces with higher microbial density are more attractive to invertebrate larvae. The only condition when settlement did not occur was in the negative control where soil abstract was absent (Fig. 3). However, the possibility that soil extract may contain bioactive substances that induce settlement could not be discounted as we were unable to kill all the bacteria present, and we still observed settlement even after biofilm density had been substantially reduced (Fig. 3B and D). In the literature, artificial surfaces pre-treated with a biofilm are commonly used for laboratory experiments involving tubeworm
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Fig. 3. The presence of antibiotics reduces tubeworm larval settlement and biofilm growth. Mean percentage of S. kraussii settlement in four concentrations of (A) ampicillin (C) streptomycin, and (E) tetracycline in the presence of soil extract. Microbial density in four concentrations of (B) ampicillin (D) streptomycin, and (F) tetracycline in the presence of soil extract after 24 h incubation. Asterisks (*) indicate significant difference from the positive control of dried soil extract with no antibiotics.
larvae. Examples include Hadfield et al. (1994), Lau and Qian (1997), Qian et al. (2007), Hung et al. (2009), Hadfield (2011) and Huang et al. (2012). Alternatively, settlement can also be stimulated by addition of chemical inducers, such as neuroactive agents 3isobutyl-1-methylxanthine (IBMX) and P-3,4-dihydroxypheny lalanine (L-DOPA) (Bryan et al., 1997; Okamoto et al., 1998; Qian and Pechenik, 1998) or monovalent cations Kþ and Csþ (Yool, 1986; Carpizo-Ituarte and Hadfield, 1998). Pure chemicals such as IBMX have been used in bioassay systems to hasten the settlement of H. elegans, but these often result in abnormal development and metamorphosis of S. kraussii without attachment (unpublished data). Soil extract contains numerous organic components such as humic acids and ammonia (Forward et al., 1997), which are utilized for microbe growth (Poeton et al., 1999; Coates et al., 2002; et al., 2005; Tikhonov et al., 2010). These had most Feificova likely stimulated formation of an inductive biofilm that in turn promoted settlement of S. kraussii larvae. Differences in settlement rates of S. kraussii larvae in liquid and air dried soil extract suggested that the inductivity and increased density of soil extract associated biofilms may be due to a concentration of nutrients. Alternatively, adhesion of soil extract components or attachment of selected bacterial species could also have increased the attractiveness of the substrate. Soil retained on substrates may facilitate attachment of microorganisms and provide nutrients for growth (Whitehead and Verran, 2009). Furthermore, biofilms can alter the physical
characteristics of the substratum, including texture and wettability, both of which are important to recruiting larvae (Faimali et al., 2004; Zardus et al., 2008; Huggett et al., 2009). It was also likely that reduced settlement in liquid soil extract treatments might be due to lower salinities (see Section 2.3.1) although the latter did not affect the biofilm density as much. It is not known if the lower salinities impacted S. kraussii larvae directly or indirectly through modification of the bacterial community in the biofilm. Lau et al. (2005) documented differing settlement of H. elegans among biofilms developed in different salinities but did not find any correlations with bacterial community composition of the biofilms. TRFLP analysis of the bacterial biofilm community indicates an overlap between marine broth and soil extract associated communities, with soil extract species being a subset of marine broth species. Marine broth is a commercial media containing nutrients for microbial growth and is a non-specific promoter of all marine bacterial species, whereas soil extract may only act to promote the growth of heterotrophic bacteria. LaMontagne et al. (2004), Mathur zaro et al. (2011) showed that selection of et al. (2007) and Sanz-La bacterial communities can be influenced by soil and various organic nutrients. This is consistent with observations by Liebeke et al. (2009) of increased yield and culture of previously unculturable, novel bacteria in media enriched with soil nutrients. Chiu et al. (2008) also demonstrated that soils can influence larval metamorphosis in barnacle (A. amphitrite) and slipper limpet (Crepidula
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Fig. 4. Settlement of larvae in soil extract compared to marine broth shows decreased settlement is not correlated to bacterial density. (A) Larval settlement in petri dishes coated with 0.4 mg ml1, 1 mg ml1 and 2 mg ml1 marine broth. (B) Microbial abundance of assays treated with dried soil extract or dried marine broth after 24 h incubation with S. kraussii larvae. Asterisks (*) indicate significant difference from the positive control (SE 100 ul ml1).
onyx) by acceleration of microbial film formation due to availability of chemical nutrients. Thus, S. kraussii settlement may have been influenced by presence, density, and specific species of bacteria in the soil extract associated biofilm.
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Our results indicate that soil extract promotes growth of a specific biofilm community. The most plausible source of bacteria in this community would be the microbiota found on the S. kraussii larvae. Research has shown that inductive environmental bacterial species usually belong to the same consortia present in the organism's natural habitat (Lau et al., 2005). Huggett et al. (2006) demonstrated that larvae of sea urchin Heliocidaris erythrogramma settle only on bacterial communities associated with coralline algae, a host for the sea urchin adults. Although transmission of endosymbionts have been documented across some marine phyla such as sponges (Usher et al., 2001; Sharp et al., 2006; Lee et al., 2009), bryozoans (Lim and Haygood, 2004), ascidians (Hirose, 2000; Hirose and Fukuda, 2006), bivalves (Cary and Giovannoni, 1993; Krueger et al., 1996) and deep sea clams (Vrijenhoek, 2010), it is not known when and how bacteria came to be associated with S. kraussii larvae. The possibility remains that the larvae picked up bacteria from the water column during culture. Whichever may be the case, if we consider the perspective of animals as host-microbe ecosystems (McFall-Ngai et al., 2013), it then seems probable that a biofilm resulting from a subset of bacteria associated with the organism would have a positive effect. Soil forms the basic substrate in terrestrial ecosystems and plays an important role in modulating species diversity (Nielsen et al., 2011; Wall and Nielsen, 2012). Preliminary estimates of the value of ecosystem services provided by soil biota and soils globally range from 1.5 to 13 trillion US dollars annually (van der Putten et al., 2004; Wall and Nielsen, 2012). The influence of soil on aquatic biota is less documented. Research in the chemistry of soil nutrients suggest that soil associated molecules may act as recruitment or location specific cues for marine invertebrate larval settlement. Metamorphosis in blue crab (Callinectes sapidus) larvae was visibly accelerated in the presence of high humic acids levels in estuarine waters (Forward et al., 1997). The recruitment of benthic polychaete, Capitella sp. is correlated with soils of high organic content (Dubilier, 1988; Tsutsumi et al., 1990), and the Pacific oyster (Crassostrea gigas) has also been documented to undergo metamorphosis in the presence of ammonia (Pawlik, 1992). The results from this study suggest that soil nutrients may modulate the settlement of sessile invertebrates by promoting the formation of a specific biofilm. Soil nutrients would be present in significant amounts in estuarine intertidal waters, and play a significant role in shaping the biofilms found on substrates in these environments. In such a scenario, rising levels of eutrophication in aquatic systems from coastal pollution may be expected to have significant impact on prokaryotic communities, resulting in consequences for inverzaro et al., 2011) and tebrate recruitment (Chiu et al., 2008; Sanz-La increasing fouling of coastal structures. 5. Conclusion
Fig. 5. NMDS plot of tubeworm assay biofilm community fingerprints using TRFLP data indicate that the soil extract-associated bacteria is a subset of the marine broth associated bacteria, and both are significantly different from bacteria grown in no treatment. Ellipses delimit community clusters to 95% confidence interval, stress ¼ 0.066.
In conclusion, the present study demonstrates the potent effect of environmental soil extract to induce S. kraussii larval settlement. Our findings indicate that this effect is predominantly a result of the way the soil nutrients have accelerated the formation of an inductive biofilm. The same effect however could not be achieved with commercially available marine broth even when higher biofilm density was obtained. The possibility remains that bioactive substances in the extract may have enhanced the effect but this effect is likely to be small compared to the effect of the biofilm. This study offers an alternative method for bioassays where a biofilm is required for settlement of the test organisms. Biofilms obtained by soaking in “aquarium seawater” for a few days do not consistently produce the same settlement-inducing effect (Olivier et al., 2000; Lau et al., 2005; Khandeparker et al., 2006). This simple approach of adding nutrients into a bioassay may be a
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useful means for promoting the growth of an inductive biofilm from microbiota present on the larvae during the incubation period. Acknowledgements The authors acknowledge support from the Agency for Science, Technology and Research (A*STAR) “Innovative Marine Antifouling Solutions” Program Grant number 102-166-0102, and the US Office for Naval Research Global Grant Award No. N62909-13-1-N056. The authors also wish to thank colleagues from NUS Tropical Marine Science Institute and Department of Biological Sciences, NUS Environmental Research Institute and the US ONR Coatings Program for their assistance and guidance in the study. References Baird, W., 1865. Description of several new species and varieties of tubicolous annelids (¼tribe Limivora of Grube) in the collection of the British Museum. J. Linn. Soc. Lond. (Zool.) 8, 10e22. Barranguet, C., Plans, M., Van Der Grinten, E., Sinke, J.J., Admiraal, W., 2002. Development of photosynthetic biofilms affected by dissolved and sorbed copper in a eutrophic river. Environ. Toxicol. Chem. 21, 1955e1965. Battin, T.J., Kaplan, L.A., Denis Newbold, J., Hansen, C.M., 2003. Contributions of microbial biofilms to ecosystem processes in stream mesocosms. Nature 426, 439e442. Baya, A.M., Brayton, P.R., Brown, V.L., Grimes, D.J., Russek-Cohen, E., Colwell, R.R., 1986. Coincident plasmids and antimicrobial resistance in marine bacteria isolated from polluted and unpolluted Atlantic Ocean samples. Appl. Environ. Microbiol. 51, 1285e1292. Bayen, S., Wurl, O., Karuppiah, S., Sivasothi, N., Lee, H.K., Obbard, J.P., 2005. Persistent organic pollutants in mangrove food webs in Singapore. Chemosphere 61, 303e313. Bonar, D.B., Weiner, R.M., Colwell, R.R., 1986. Microbial-invertebrate interactions and potential for biotechnology. Microb. Ecol. 12, 101e110. Brooun, A., Liu, S.H., Lewis, K., 2000. A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 44, 640e646. Bryan, P.J., Qian, P.Y., Kreider, J.L., Chia, F.S., 1997. Induction of larval settlement and metamorphosis by pharmacological and conspecific associated compounds in the serpulid polychaete Hydroides elegans. Mar. Ecol. Prog. Ser. 146, 81e90. Burke, C., Kjelleberg, S., Thomas, T., 2009. Selective extraction of bacterial DNA from the surfaces of macroalgae. Appl. Environ. Microbiol. 75, 252e256. Carpizo-Ituarte, E., Hadfield, M.G., 1998. Stimulation of metamorphosis in the polychaete Hydroides elegans Haswell (Serpulidae). Biol. Bull. 194, 14e24. Cary, S.C., Giovannoni, S.J., 1993. Transovarial inheritance of endosymbiotic bacteria in clams inhabiting deep-sea hydrothermal vents and cold seeps. Proc. Natl. Acad. Sci. 90, 5695e5699. Chen, C.L., Maki, J.S., Rittschof, D., Teo, S.L.M., 2013. Early marine bacterial biofilm on a copper-based antifouling paint. Int. Biodeterior. Biodegrad. 83, 71e76. Chiu, J.M.Y., Zhang, R., Wang, H., Thiyagarajan, V., Qian, P.Y., 2008. Nutrient effects on intertidal community: from bacteria to invertebrates. Mar. Ecol. Prog. Ser. 358, 41e50. Chou, R., Lee, H.B., 1997. Commercial marine fish farming in Singapore. Aquac. Res. 28, 767e776. Chung, H.C., Lee, O.O., Huang, Y.L., Mok, S.Y., Kolter, R., Qian, P.Y., 2010. Bacterial community succession and chemical profiles of subtidal biofilms in relation to larval settlement of the polychaete Hydroides elegans. ISME J. 4, 817e828. Cinar, M.E., 2006. Serpulid species (Polychaeta: Serpulidae) from the Levantine coast of Turkey (eastern Mediterranean), with special emphasis on alien species. Aquat. Invasions 1, 223e240. Coates, J.D., Cole, K.A., Chakraborty, R., O'Connor, S.M., Achenbach, L.A., 2002. Diversity and ubiquity of bacteria capable of utilizing humic substances as electron donors for anaerobic respiration. Appl. Environ. Microbiol. 68, 2445e2452. Cooke, M.D., 1976. Antibiotic resistance among coliform and fecal coliform bacteria isolated from sewage, seawater, and marine shellfish. Antimicrob. Agents Chemother. 9, 879e884. Costerton, J.W., Stewart, P.S., Greenberg, E.P., 1999. Bacterial biofilms: a common cause of persistent infections. Science 284, 1318e1322. Crisp, M., 1977. The development of the serpulid Pomatoleios kraussii (Annelida, Polychaeta). J. Zool. 183, 147e160. Cuong, D.T., Bayen, S., Wurl, O., Subramanian, K., Wong, K.K., Sivasothi, N., Obbard, J.P., 2005. Heavy metal contamination in mangrove habitats of Singapore. Mar. Pollut. Bull. 50, 1732e1738. Dubilier, N., 1988. H2S e a settlement cue or a toxic substance for capitella sp. I larvae? Biol. Bull. 174, 30e38. Faimali, M., Garaventa, F., Terlizzi, A., Chiantore, M., Cattaneo-Vietti, R., 2004. The interplay of substrate nature and biofilm formation in regulating Balanus amphitrite Darwin, 1854 larval settlement. J. Exp. Mar. Biol. Ecol. 306, 37e50.
, M., Cejkov , A., Mas Feifi cov a, D., Snajdr, J., Siglova a ak, J., Jirk u, V., 2005. Influence of humic acids on the growth of the microorganisms utilizing toxic compounds (comparison between yeast and bacteria). CHIMIA Int. J. Chem. 59, 749e752. Fischer, H., 2003. The role of biofilms in the uptake and transformation of dissolved organic matter. In: Findlay, S., Sinsabaugh, R.L. (Eds.), Aquatic Ecosystems: Interactivity of Dissolved Organic Matter. Academic Press, USA, pp. 285e313. Fischer, H., Wanner, S.C., Pusch, M., 2002. Bacterial abundance and production in river sediments as related to the biochemical composition of particulate organic matter (POM). Biogeochemistry 61, 37e55. Forward, R.B., Tankersley, R.A., Blondel, D., Rittschof, D., 1997. Metamorphosis of the blue crab Callinectes sapidus: effects of humic acids and ammonium. Mar. Ecol. Prog. Ser. 157, 277e286. Gander, S., 1996. Bacterial biofilms: resistance to antimicrobial agents. J. Antimicrob. Chemother. 37, 1047e1050. Gans, J., Wolinsky, M., Dunbar, J., 2005. Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science 309, 1387e1390. Grabow, W.O.K., Prozesky, O.W., Smith, L.S., 1974. Drug resistant coliforms call for review of water quality standards. Water Res. 8, 1e9. Hadfield, M.G., 2011. Biofilms and Marine invertebrate larvae: what bacteria produce that larvae use to choose settlement sites. Annu. Rev. Mar. Sci. 3, 453e470. Hadfield, M.G., Unabia, C.C., Smith, C.M., Michael, T.M., 1994. Settlement preferences of the ubiquitous fouler Hydroides elegans. Recent Dev. Biofouling Control, 65e75. Hirose, E., 2000. Plant rake and algal pouch of the larvae in the tropical ascidian Diplosoma similis: an adaptation for vertical transmission of photosynthetic symbionts Prochloron sp. Zool. Sci. 17, 233e240. Hirose, E., Fukuda, T., 2006. Vertical transmission of photosymbionts in the colonial ascidian Didemnum molle: the larval tunic prevents symbionts from attaching to the anterior part of larvae. Zool. Sci. 23, 669e674. Hochbaum, A.I., Kolodkin-Gal, I., Foulston, L., Kolter, R., Aizenberg, J., Losick, R., 2011. Inhibitory effects of D-amino acids on Staphylococcus aureus biofilm development. J. Bacteriol. 193, 5616e5622. Holm, E.R., 2012. Barnacles and biofouling. Integr. Comp. Biol. 52, 348e355. Holm, E.R., Kavanagh, C.J., Meyer, A.E., Wiebe, D., Nedved, B.T., Wendt, D., Smith, C.M., Hadfield, M.G., Swain, G., Wood, C.D., Truby, K., Stein, J., Montemarano, J., 2006. Interspecific variation in patterns of adhesion of marine fouling to silicone surfaces. Biofouling 22, 233e243. Huang, S., Hadfield, M.G., 2003. Composition and density of bacterial biofilms determine larval settlement of the polychaete Hydroides elegans. Mar. Ecol. Prog. Ser. 260, 161e172. Huang, Y., Callahan, S., Hadfield, M.G., 2012. Recruitment in the sea: bacterial genes required for inducing larval settlement in a polychaete worm. Sci. Reports 2, 228. Huggett, M.J., Williamson, J.E., de Nys, R., Kjelleberg, S., Steinberg, P.D., 2006. Larval settlement of the common Australian sea urchin Heliocidaris erythrogramma in response to bacteria from the surface of coralline algae. Oecologia 149, 604e619. Huggett, M.J., Nedved, B.T., Hadfield, M.G., 2009. Effects of initial surface wettability on biofilm formation and subsequent settlement of Hydroides elegans. Biofouling 25, 387e399. Hung, O.S., Lee, O.O., Thiyagarajan, V., He, H.P., Xu, Y., Chung, H.C., Qiu, J.W., Qian, P.Y., 2009. Characterization of cues from natural multi-species biofilms that induce larval attachment of the polychaete Hydroides elegans. Aquat. Biol. 4, 253e262. Jin, T., Qian, P.Y., 2004. Effect of amino acids on larval metamorphosis of the polychaete Hydroides elegans. Mar. Ecol. Prog. Ser. 267, 209e218. Keough, M.J., Raimondi, P.T., 1996. Responses of settling invertebrate larvae to bioorganic films: effects of large-scale variation in films. J. Exp. Mar. Biol. Ecol. 207, 59e78. Khandeparker, L., Chandrashekar Anil, A., Raghukumar, S., 2006. Relevance of biofilm bacteria in modulating the larval metamorphosis of Balanus amphitrite. FEMS Microbiol. Ecol. 58, 425e438. Kirchman, D., Graham, S., Reish, D., Mitchell, R., 1981. Bacteria induce settlement and metamorphosis of Janua (Dexiospira) brasiliensis Grube (Polychaeta, Spirorbidae). J. Exp. Mar. Biol. Ecol. 56, 153e163. Krueger, D.M., Gustafson, R.G., Cavanaugh, C.M., 1996. Vertical transmission of chemoautotrophic symbionts in the bivalve Solemya velum (Bivalvia: Protobranchia). Biol. Bull. 190, 195. LaMontagne, M.G., Leifer, I., Bergmann, S., Van De Werfhorst, L.C., Holden, P.A., 2004. Bacterial diversity in marine hydrocarbon seep sediments. Environ. Microbiol. 6, 799e808. Lane, D.J., Pace, B., Olsen, G.J., Stahl, D.A., Sogin, M.L., Pace, N.R., 1985. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. U. S. A 82, 6955e6959. Lau, S.C.K., Qian, P.Y., 1997. Phlorotannins and related compounds as larval settlement inhibitors of a tube-building polychaete Hydroides elegans (Haswell). Mar. Ecol. Prog. Ser. 159, 219e227. Lau, S.C.K., Thiyagarajan, V., Qian, P.Y., 2003. The bioactivity of bacterial isolates in Hongkong water for the inhibition of barnacle (Balanus amphitrite Darwin) settlement. J. Exp. Mar. Biol. Ecol. 282, 43e60. Lau, S.C.K., Thiyagarajan, V., Cheung, S.C.K., Qian, P.Y., 2005. Roles of bacterial community composition in biofilms as mediator for larval settlement of three marine invertebrates. Aquat. Microb. Ecol. 38, 41e51.
J.Y.-H. Chan et al. / International Biodeterioration & Biodegradation 94 (2014) 192e199 Lee, O.O., Chui, P.Y., Wong, Y.H., Pawlik, J.Y., Qian, P.Y., 2009. Evidence for vertical transmission of bacterial symbionts from adult to embryo in the Caribbean sponge Svenzea zeai. Appl. Environ. Microbiol. 75, 6147e6156. Liebeke, M., Brozel, V.S., Hecker, M., Lalk, M., 2009. Chemical characterization of soil extract as growth media for the ecophysiological study of bacteria. Appl. Microbiol. Biotechnol. 83, 161e173. Lim, G.E., Haygood, M.G., 2004. “Candidatus Endobugula glebosa,” a specific bacterial symbiont of the marine bryozoan Bugula simplex. Appl. Environ. Microbiol. 70, 4921e4929. Mathur, J., Bizzoco, R.W., Ellis, D.G., Lipson, D.A., Poole, A.W., Levine, R., Kelley, S.T., 2007. Effects of abiotic factors on the phylogenetic diversity of bacterial communities in acidic thermal springs. Appl. Environ. Microbiol. 73, 2612e2623. Matsuki, T., Watanabe, K., Fukimoto, J., Miyamoto, Y., Takada, T., Matsumoto, K., Oyaizu, H., Tanaka, R., 2002. Development of 16S rRNA-gene-targeted groupspecific primers for the detection and identification of predominant bacteria in human feces. Appl. Environ. Microbiol. 68, 5445e5451. McFall-Ngai, M., Hadfield, M.G., Bosch, T.C., Carey, H.V., Domazet-Loso, T., Douglas, A.E., Dubilier, N., Eberl, G., Fukami, T., Gilbert, S.F., Hentschel, U., King, N., Kjelleberg, S., Knoll, A.H., Kremer, N., Mazmanian, S.K., Metcalf, J.L., Nealson, K., Pierce, N.E., Rawls, J.F., Reid, A., Ruby, E.G., Rumpho, M., Sanders, J.G., Tautz, D., Wernegreen, J.J., 2013. Animals in a bacterial world, a new imperative for the life sciences. Proc. Natl. Acad. Sci. U. S. A 110, 3229e3236. Nedved, B.T., Hadfield, M.G., 2009. Hydroides elegans (Annelida: Polychaeta): a model for biofouling research. In: Flemming, H.C., Venkatesan, R., Murthy, S.P., Cooksey, K. (Eds.), Marine and Industrial Biofouling, Springer Series on Biofilms. Springer-Vergla, Berlin, pp. 203e217. Nielsen, U.N., Ayres, E., Wall, D.H., Bardgett, R.D., 2011. Soil biodiversity and carbon cycling: a review and synthesis of studies examining diversity-function relationships. Eur. J. Soil Sci. 62, 105e116. Okamoto, K., Watanabe, A., Sakata, K., Watanabe, N., 1998. Chemical signals involved in larval metamorphosis in Hydroides ezoensis (Serpulidae; Polychaeta). Part I: induction of larval metamorphosis by extract of adult tube clumps. J. Mar. Biotechnol. 6, 7e10. Olivier, F., Tremblay, R., Bourget, E., Ritschoff, D., 2000. Barnacle settlement: field experiments on the influence of larval supply, tidal level, biofilm quality and age on Balanus amphitrite cyprids. Mar. Ecol. Prog. Ser. 199, 185e204. Pawlik, J.R., 1992. Chemical ecology of the settlement of benthic marine invertebrates. Oceanogr. Mar. Biol. 30, 273e335. Perthuisot, J.P., Guelorget, O., Ibrahim, A., Jusserand, C., Margerel, J.P., Maurin, A., 1990. L'organisation biogeologique du lac. Temsah (lsmailia, Egypte). Rev. d'Hydrobiologie Trop. 23, 77e90. Pillai, T.G., 2009. Descriptions of new serpulid polychaetes from the Kimberleys of Australia and discussion of Australian and Indo-West Pacific species of Spirobranchus and superficially similar taxa. Rec. Aust. Mus. 61, 93e199. Poeton, T.S., Stensel, H.D., Strand, S.E., 1999. Biodegradation of polyaromatic hydrocarbons by marine bacteria: effect of solid phase on degradation kinetics. Water Res. 33, 868e880. Pringault, O., Duran, R., Jacquet, S., Torreton, J.P., 2008. Temporal variations of microbial activity and diversity in marine tropical sediments (New Caledonia lagoon). Microb. Ecol. 55, 247e258. Qian, P.Y., Pechenik, J.A., 1998. Effects of larval starvation and delayed metamorphosis on juvenile survival and growth of the tube-dwelling polychaete Hydroides elegans (Haswell). J. Exp. Mar. Biol. Ecol. 227, 169e185. Qian, P.Y., Lau, S.C.K., Dahms, H.U., Dobretsov, S., Harder, T., 2007. Marine biofilms as mediators of colonization by marine macroorganisms: implications for antifouling and aquaculture. Mar. Biotechnol. 9, 399e410. R Development Core Team, 2010. R: a Language and Environment for Statistical Computing, Vienna, Austria. Retrieved from: http://www.R-project.org. Roth, B.L., Poot, M., Yue, S.T., Millard, P.J., 1997. Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl. Environ. Microbiol. 63, 2421e2431.
199
Sanz-L azaro, C., Navarrete-Mier, F., Marin, A., 2011. Biofilm responses to marine fish farm wastes. Environ. Pollut. 159, 825e832. Sawall, Y., Richter, C., Ramette, A., 2012. Effects of eutrophication, seasonality and macrofouling on the diversity of bacterial biofilms in equatorial coral reefs. Plos One 7, e39951. Shalla, S.H., Holt, T.J., 1999. The Lessepsian migrant Pomatoleios kraussii (Annelida, Polychaeta) e recent formation of dense aggregations in Lake Timsah and the Bitter Lakes (Suez Canal, Egypt). Egypt. J. Biol. 1, 133e137. Sharp, K.H., Eam, B., Faulkner, D.J., Haygood, M.G., 2006. Vertical transmission of diverse microbes in the tropical sponge Corticium sp. Appl. Environ. Microbiol. 73, 622e629. Shikuma, N.J., Pilhofer, M., Weiss, G.L., Hadfield, M.G., Jensen, G.J., Newman, D.K., 2014. Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures. Science 343, 529e533. Sokal, R.R., Rohlf, F.J., 2012. Biometry: the principles and practice of statistics in biological research. W. H. Freeman and Co, New York, p. 937. Straughan, D., 1969. Intertidal zone-formation in Pomatoleios kraussii (Annelida e Polychaeta). Biol. Bull. 136, 469e482. Tait, K., Havenhand, J., 2013. Investigating a possible role for the bacterial signal molecules N-acylhomoserine lactones in Balanus improvisus cyprid settlement. Mol. Ecol. 22, 2588e2602. Tam, N.F.Y., 1998. Effects of wastewater discharge on microbial populations and enzyme activities in mangrove soils. Environ. Pollut. 102, 233e242. Tikhonov, V.V., Yakushev, A.V., Zavgorodnyaya, Y.A., Byzov, B.A., Demin, V.V., 2010. Effects of humic acids on the growth of bacteria. Eurasian Soil Sci. 43, 305e313. Torsvik, V., Goksoyr, J., Daae, F.L., 1990. High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56, 782e787. Tsutsumi, H., Fukunaga, S., Fujita, N., Sumida, M., 1990. Relationship between growth of Capitella sp. and organic enrichment of the sediment. Mar. Ecol. Prog. Ser. 63, 157e162. Unabia, C.R.C., Hadfield, M.G., 1999. Role of bacteria in larval settlement and metamorphosis of the polychaete Hydroides elegans. Mar. Biol. 133, 55e64. Usher, K.M., Kuo, J., Fromont, J., Sutton, D.C., 2001. Vertical transmission of cyanobacterial symbionts in the marine sponge Chondrilla australiensis (Demospongiae). Hydrobiologia 461, 15e23. van der Putten, W.H., Anderson, J.M., Bardgett, R.D., Behan-Pelletier, V., Bignell, D.E., Brown, G.G., Brown, V.K., Brussaard, L., Hunt, H.W., Ineson, P., Jones, T.H., Lavelle, P., Paul, E.A., John, M.S., Wardle, D.A., Wojtowicz, T., Wall, D.H., 2004. The sustainable delivery of goods and services provided by soil biota. In: Wall, D. (Ed.), Sustaining Biodiversity and Ecosystem Services in Soils and Sediments. Island Press, Washington, pp. 15e44. Vrijenhoek, R.C., 2010. Genetics and evolution of deep-sea chemosynthetic bacteria and their invertebrate hosts. In: Kiel, S. (Ed.), The Vent and Seep Biota, Topics in Geobiology, vol. 33. Springer, Dordrecht Heidelberg London New York, pp. 15e49. Wall, D.H., Nielsen, U.N., 2012. Biodiversity and ecosystem services: is it the same below ground? Nat. Educ. Knowl. 3, 8. Warton, D.I., Hui, F.K.C., 2011. The arcsine is asinine: the analysis of proportions in ecology. Ecology 92, 3e10. Whitehead, K.A., Verran, J., 2009. The effect of substratum properties on the survival of attached microorganisms on insert surfaces. In: Flemming, H.C., Murthy, P.S., Venkatesan, R., Cooksey, K.E. (Eds.), Marine and Industrial Biofouling. Springer Series on Biofilms. Springer-Vergla, Berlin, pp. 13e34. Wilson, E., Underwood, M., Puckrin, O., Letto, K., Doyle, R., Caravan, H., Camus, S., Bassett, K., 2010. The Arcsine Transformation: has the Time Come for Retirement? Memorial University of Newfoundland, Newfoundland and Labrador, Canada. http://www.mun.ca/biology/dschneider/b7932/B7932Final10Dec2010.pdf. Yool, A.J., Grau, S.M., Hadfield, M.G., Jensen, R.A., Markell, D.A., Morse, D.E., 1986. Excess potassium induces larval metamorphosis in 4 marine invertebrate species. Biol. Bull. 170, 255e266. Zardus, J.D., Nedved, B.T., Huang, Y., Tran, C., Hadfield, M.G., 2008. Microbial biofilms facilitate adhesion in biofouling invertebrates. Biol. Bull. 214, 91e98.
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Settlement inducers for larvae of the tropical fouling serpulid, Spirobranchus kraussii (Baird, 1865) (Polychaeta: Annelida) Janlin Y-H. Chan a, Serina S-C. Lee a, *, Siti Zarina Zainul Rahim b, Serena L-M. Teo a a b
Tropical Marine Science Institute, National University of Singapore, Singapore 119227, Singapore Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 January 2014 Received in revised form 29 July 2014 Accepted 29 July 2014 Available online
Calcareous tubeworms are one of the most abundant fouling organisms found on substrates submerged in marine environments. Research has clearly shown that tubeworms select surfaces with a biofilm, and attachment and adhesion strength is enhanced in the presence of a biofilm. Studies have also shown that abiotic materials in the environment are strongly linked to proliferation of aquatic benthic microbes, which in turn may influence larval recruitment. Soil-derived nutrients are capable of supporting large, diverse bacterial communities and have been used to encourage growth of heterotrophic microbes. In this study we investigate the effect of organic nutrient-rich extract derived from sediments on larval settlement of the fouling calcareous polychaete, Spirobranchus kraussii (Baird, 1865). Application of a soil extract, as used in the preparation of GSe microalgal culture media, induced larval settlement within 24 h in laboratory assays. Our observations suggest that soil extract promoted the growth of an inductive biofilm from microbiota present on the larvae during the incubation period. However, the presence of a chemical inducer could not be conclusively discounted. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Biofouling Larval settlement Serpulidae Biofilms Spirobranchus (syn. Pomatoleios) kraussii
1. Introduction The understanding of larval behaviour and inducers of settlement of common marine fouling organisms provides valuable clues for the development of methods to control macrofouling of underwater structures. Much of the earlier work has focused on the biology of barnacles, in particular that of Amphibalanus amphitrite (Holm, 2012). Interest in tubeworm fouling on man-made materials has grown in recent years as the adhesion strength of these organisms on antifouling foul-release coatings appears to be stronger than that of barnacles (Holm et al., 2006). Subsequently, Zardus et al. (2008) reported that adhesion strength increases in the presence of a biofilm. Biofilms have long been recognized as a natural stimulus for marine polychaete larvae settlement (Kirchman et al., 1981; Unabia and Hadfield, 1999), and larvae appear to be highly discriminant between biofilms originating from different bacterial species (Huang and Hadfield, 2003; Lau et al., 2003). For example, Janua brasiliensis only settles in the presence of a bacterial species associated with its natural substratum, the green macroalga Ulva lobata * Corresponding author. 18 Kent Ridge Road, Tropical Marine Science Institute, National University of Singapore, Singapore 119227, Singapore. Tel.: þ65 98223849. E-mail address: [email protected] (S.S.-C. Lee). http://dx.doi.org/10.1016/j.ibiod.2014.07.017 0964-8305/© 2014 Elsevier Ltd. All rights reserved.
(Kirchman et al., 1981). The larvae of Hydroides elegans have been shown to metamorphose in the presence of phage tail-like structures produced by the bacterium Pseudoalteromonas luteoviolacea (Shikuma et al., 2014). Recently, Tait and Havenhand (2013) suggested that N-acylhomoserine lactones involved in bacterial quorum sensing may also provide cues for settling barnacle larvae. These observations provide new insights into the ubiquitous way bacteria and biofilms are shaping sessile invertebrate communities. Perspectives of bacterialeinvertebrate interactions have evolved with the increasing acceptance of bacteria as key contributors in the energy transfer, stability of the natural environment, and its diversification (Bonar et al., 1986; Battin et al., 2003; McFall-Ngai et al., 2013). It is known that bacterial diversity and abundance in microbial films fluctuate in response to availability of nutrients in the environment (Barranguet et al., 2002; Fischer et al., 2002). The density and identity of aquatic benthic microbes may exhibit strong dependency and sensitivity to variations in abiotic factors such as particulate organic matter, amino acids and peptide concentrations in the environment (Fischer et al., 2002; Pringault et al., 2008; Hochbaum et al., 2011; Sawall et al., 2012). Experiments conducted by numerous authors (Keough and Raimondi, 1996; Hung et al., 2009; Chung et al., 2010) have established a strong interdependency between settlement in marine invertebrates and bacterial colonization.
J.Y.-H. Chan et al. / International Biodeterioration & Biodegradation 94 (2014) 192e199
In this study we investigate the effect of organic nutrient-rich extract derived from sediments (henceforth referred to as soil extract) on larval settlement of the fouling calcareous polychaete, Spirobranchus kraussii (Baird, 1865) (Pomatoleios kraussii of authors; see Pillai, 2009). S. kraussii is a common intertidal serpulid found across the Indo-Pacific (Straughan, 1969). On natural substrates, these tubeworms appear to be gregarious and occur as reef builders. In Singapore mangroves, S. kraussii occurs in dense aggregates on Avicennia and Rhizophora mangrove roots. It is also common in fouling communities on hard structures in coastal areas, on ship hulls, wooden boardwalks and fish farms (Straughan, 1969; Cinar, 2006). Soil, as a medium rich in abiotic nutrients, is capable of supporting a high magnitude of bacterial communities and species diversity (Torsvik et al., 1990; Gans et al., 2005), inspiring the use of soil-derived nutrients as an enhancer of heterotrophic microbial growth (Fischer, 2003; Liebeke et al., 2009; Tikhonov et al., 2010). As S. kraussii is an estuarine species, it was hypothesized that soil nutrients may act as a cue for settlement. For our experiments, we considered soil sourced from pristine, uncultured land to minimise variables such as contamination by inductive adult conspecifics, toxic substances and chemical residues from coastal pollution that may have an adverse effect on larval mortality and settlement (Bayen et al., 2005; Cuong et al., 2005). The use of pristine soil also reduces fluctuations in organic levels as wastewater and runoff can affect the microbial population and composition of mangrove soils (Tam, 1998). 2. Materials and methods 2.1. Larval culture S. kraussii adults were collected from mangrove roots in Lim Chu Kang, Singapore. Fertilization and culturing were modified from methods first described by Hadfield et al. (1994) and Bryan et al. (1997). Larvae were reared on 1:1 mixture of Isochrysis galbana (CS-177) and Nannochloropsis oculata (CS-179) at an approximate concentration of 4.6 105 cells per ml suspended in 27 ppt 0.22 mm-filtered seawater (FSW). Cultures were maintained at 27 C with a 12:12 h light: dark cycle and gentle aeration. The culture medium was renewed every two days. After seven to nine days, competent larvae were visually assessed before harvest for experimentation use. Metamorphically competent larvae acquired elongated tails with visible straightening of the abdominal mass with appearance of two slight indentations on the anterior end (Crisp, 1977; Nedved and Hadfield, 2009). Behavioural changes in mature larvae were also evident with larvae actively crawling and exploring the surfaces of culture beakers. 2.2. General design of settlement experiments Settlement experiments were performed in FSW using seven to nine day old competent larvae. Larvae were harvested by filtering through a 50 mm mesh and rinsed thrice in FSW prior to use. Every experiment consisted of five replicates, each containing 20e30 larvae in 2 ml FSW. All treatments were conducted twice with different batches of larvae for consistency. Settlement dishes were incubated in the dark for 24 h at 27 C, and scored under a dissecting microscope for settled, swimming and dead larvae. Individuals with secondary or primary tubes were classified as settled. Secondary tubes were identified by the appearance of an opaque calcified tube, and well-differentiated branchial radioles on the worm. Primary tubes were discernible by transparent colouring and developing branchial lobes of the attached juveniles (Nedved and Hadfield, 2009).
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Biofilm density was determined in all settlement dishes by staining with BacLight bacterial viability kit (Invitrogen) according to manufacturer's instructions (Roth et al., 1997). Bacterial abundance was visualized under an epifluorescence microscope. Three photographs of approximate 3.80 mm2 each were taken for every dish under 100 magnification. Five random spots per photo were selected for assessment of microbial biomass with an estimated area of 2.17 mm2 quantified for each dish. The only two exceptions to this procedure were when IBMX was used (See Section 2.3.2) and when biofilm was harvested for fingerprinting via terminal restriction fragment length polymorphism, TRFLP (See Section 2.4). 2.3. Settlement of tubeworms in response to soil extract Sterile soil extract was prepared following protocols provided by the CSIRO Australian National Algae Culture Collection for the preparation of CMAR soil extract (http://www.marine.csiro.au/ microalgae/methods/). Soil collected from uncultivated land on St John's Island, Singapore was dried and sieved through a 2 and 1 mm mesh to obtain fine powder. Distilled water was added at a ratio of 1 kg soil to 2 L water, and the mixture was sterilized in an autoclave. The suspension was left to cool and settle over night at room temperature before collection. The aqueous portion was twice filtered through absorbent cotton until a clear dark brown solution was produced. This solution was again sterilized, then sealed and stored at 4 C prior to use. As the soil extract was sterile, and tubeworm larvae were rinsed in sterile FSW before use, the main source of bacteria present in the assays would be those adhered to the larvae. 2.3.1 Liquid versus dried soil extract To determine if the method of applying soil extract affects settlement, aqueous soil extract was added to FSW with larvae to give final concentrations of 100, 200 and 300 ml ml1. Addition of liquid soil extract resulted in reduced salinity of approximately 25, 23 and 20 ppt at 100, 200 and 300 ul/ml respectively. However, salinity in mangrove swamps in the Johor Straits, where S. kraussii were collected, fluctuate between 20 and 33 ppt due to runoffs and tidal flow (Chou and Lee, 1997) and S. kraussii has even been shown to thrive in 16e20 ppt (Perthuisot et al., 1990; Shalla and Holt, 1999) environments. Another set was prepared by air-drying an equivalent amount of soil extract on sterile 35 mm petri dishes before topping up with FSW and larvae. Negative controls consisted of petri dishes with FSW and larvae only. Dried soil extract was selected for use as it resulted in higher settlement rates than liquid (Fig 1). As there was only a marginal increase in settlement even at a three-fold increase of soil extract (Fig 2A), 100 ml1 was selected as the final working concentration for all assays. 2.3.2 Soil extract versus IBMX To compare the effect of soil extract with a known chemical inducer of larval settlement, settlement assays were set up as in Section 2.2 using 10e4 M of IBMX and dried soil extract at a final concentration of 100 ml ml1. 2.3.3 Antibiotic assays Antibiotics were applied to inhibit the growth of bacteria on soil extract-coated dishes to determine if a reduction in biofilm affects the settlement-inducing properties of soil extract. Stock solutions of tetracycline hydrochloride, ampicillin sodium and streptomycin sulfate (SigmaeAldrich) at 10 mg ml1 were prepared in distilled water. Final testing concentrations of 1, 10, 50 and 100 mg ml1 were obtained by diluting antibiotic stock solutions in FSW dispensed
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Fig. 1. Presence of dried soil extract increases (A) metamorphosis of S. kraussii larvae and (B) biofilm density after 24 h incubation compared to equal volumes of liquid soil extract. Asterisks (*) indicate significant difference between the two bars under the line.
into petri dishes with larvae. Negative controls consisted of Petri dishes with only FSW and larvae. Dried soil extract at 100 ml ml1 was used as a positive control.
(New England Biolabs) before capillary electrophoresis and analysis using GeneMapper software (Applied Biosystems). 2.5 Statistical analysis
2.3.4 Biofilm formation in marine nutrient broth compared to soil extract To determine if the inductive effect of the biofilm was due to bacterial numbers or composition, biofilms were grown with commercially available nutrient-rich broth. Marine broth (Conda™), prepared as per manufacturer's instructions, was applied to sterile 35 mm petri dishes and allowed to dry over night before topping up with FSW and larvae resulting in final test concentrations of 0.4, 1 and 2 mg ml1. Negative controls consisted of Petri dishes with only FSW and larvae. Dried soil extract at 100 ml ml1 was used as positive control. 2.4 Biofilm fingerprinting (terminal restriction fragment length polymorphism, TRFLP)
Untransformed settlement data were tested for parametric assumptions based on similar studies (Wilson et al., 2010; Warton and Hui 2011; Huang et al., 2012; Sokal and Rolf 2012), then analyzed by use of the KruskaleWallis non-parametric ANOVA, where p < 0.05 considered significant, and presented as the mean ± standard error. TRFLP data were exported and transformed into an absence-presence matrix, then a non-metric multidimensional scaling (NDMS) was conducted using BrayeCurtis distance measures and k ¼ 2 in conjunction with the function “ordiellipse” to delimit the 95% confidence interval around the clusters. Data were analyzed by use of R software package (R Development Core Team, 2010). 3. Results
In order to harvest sufficient biofilm for terminal restriction fragment length polymorphism (TRFLP) analysis, the exact experimental set up described in Section 2.3.4 was scaled up using 90 mm petri dishes with a final volume of 15 ml. Only petri dishes with settlement were used to harvest biofilm. Bacteria were removed from the surface using 1% 3M enzymatic cleaner (Burke et al., 2009). DNA was extracted using phenol:chloroform as previously described (Chen et al., 2013) and amplified using universal bacterial primers 926F (Matsuki et al., 2002) and 1392R (Lane et al., 1985) modified with 6-FAM and ATTO 565 dyes respectively. Amplicons were digested with AluI and Bsu361
3.1 Liquid versus dried soil extract Addition of soil extract increased settlement in S. kraussii larvae and biofilm growth. This effect was more pronounced when the extract was dried onto the dish surface compared to the equivalent amount inoculated into solution (Fig. 1A and B). As concentrations of liquid soil extract increased, the settlement rate presented a downward trend, opposite to that in dried soil extract treatments. Mortality (not presented) was not significantly different between liquid and dried soil extract at the various concentrations tested.
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larvae in the presence of antibiotics were low across all concentrations and not significantly different from the positive control containing only soil extract and larvae. 3.4 Biofilm formation in marine nutrient broth compared to soil extract Nutrient rich marine broth promotes bacterial biofilm growth but denser biofilm, obtained from applying higher concentrations of broth, did not correlate to increased settlement. It was notable that even though the lowest concentration of marine broth resulted in a comparable bacterial biofilm density to that of soil extract (Fig. 4B), the larval settlement in biofilms from broth was significantly lower (Fig. 4A). Mortality of S. kraussii larvae was significantly higher in marine broth treatments compared to positive control of soil extract, but we did not observe a doseeresponse curve when higher concentrations of marine broth were applied. 3.5 Biofilm fingerprinting (TRFLP) TRFLP analysis of the biofilms that developed when tubeworms were allowed to settle on Petri dishes with soil extract and marine broth indicate that these were significantly different communities from biofilms that developed with no treatment (Fig. 5). The bacterial community of soil extract-associated biofilms appears to be a subset of the marine broth associated community, showing that soil extract selects for a more specific set of bacteria within the biofilm. 4. Discussion Fig. 2. (A) Settlement induction of soil extract (SE) does not display a doseeresponse curve over the range of treatment levels that were used. (B) Dried soil extract is as effective as 104 M IBMX at inducing settlement, but with less mortality. Asterisks (*) indicate significant difference from (A) negative control and (B) IBMX control.
There was no significant difference in settlement rates of S. kraussii larvae across 3 concentrations of dried soil extract (Fig. 2A). 3.2 Soil extract versus IBMX The presence of soil extract induced settlement in S. kraussii larvae at levels comparable to IBMX but with significantly less mortality (Fig. 2B). Secondary tubes were obtained with soil extract, whereas in presence of IBMX most of the worms had primary tubes after 24 h. Where IBMX was used, some of the larvae that did metamorphose appeared bloated and laid outside of their tubes. Instances of mass settlement in clumps and on water surfaces have also been observed. Developmental abnormalities also increased with appearance of developing branchial lobes seen in swimming larvae. These were scored as swimming. 3.3 Antibiotic assays Bacterial density appeared to influence larval settlement as the application of antibiotics resulted in a significant reduction of settlement even in the presence of soil extract. For all three antibiotics, a dose-dependent significant decrease in bacterial density was observed with increase in antibiotic concentration (Fig. 3B, D, F), which generally corresponded to an equivalent reduction in settlement (Fig. 3A, C, E). Ampicillin produced the strongest inhibitory effect followed by streptomycin (Fig. 3A, C). Although there was no statistical significance between settlement in different concentrations of tetracycline (Fig. 3E), there appeared to be a reduction in numbers as concentration increased. Mortality rates of S. kraussii
In this study we considered the possible effects of soil associated nutrients on the recruitment of S. kraussii, and the effect of soil extract cultured biofilm on larvae settlement. Three hypotheses can be inferred from the results of the experiments. Firstly, the chemical cues in soil extract may stimulate S. kraussii metamorphosis, promoting settlement. Secondly, as soil associated nutrients have been found to promote proliferation of specific bacterial communities soil extract may indirectly promote settlement by enhancing growth of this inductive bacterial consortium. Thirdly, the settlement inducing effect of soil extract may be a result of a combination of both factors mentioned above. The complex mixture of compounds present in soil extract may be acting directly on larval receptors, or increasing settlement rates indirectly by enhancing the growth of suitable biofilms. Microbial presence is ubiquitous in all invertebrate settlement assays (Jin and Qian, 2004) and antibiotics are often used to minimize bacteria. Ampicillin, streptomycin and tetracycline are commonly used to eradicate bacteria; however, complete elimination was difficult due to the high antibiotic resistance of marine bacteria (Grabow et al., 1974; Cooke, 1976; Baya et al., 1986) in a biofilm (Gander, 1996; Costerton et al., 1999; Brooun et al., 2000). Lower settlement of larvae was observed in the treatments with fewer bacteria as a result of antibiotic treatment, indicating an association between settlement and bacterial abundance. These findings are consistent with conclusions of Keough and Raimondi (1996) and Jin and Qian (2004): surfaces with higher microbial density are more attractive to invertebrate larvae. The only condition when settlement did not occur was in the negative control where soil abstract was absent (Fig. 3). However, the possibility that soil extract may contain bioactive substances that induce settlement could not be discounted as we were unable to kill all the bacteria present, and we still observed settlement even after biofilm density had been substantially reduced (Fig. 3B and D). In the literature, artificial surfaces pre-treated with a biofilm are commonly used for laboratory experiments involving tubeworm
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Fig. 3. The presence of antibiotics reduces tubeworm larval settlement and biofilm growth. Mean percentage of S. kraussii settlement in four concentrations of (A) ampicillin (C) streptomycin, and (E) tetracycline in the presence of soil extract. Microbial density in four concentrations of (B) ampicillin (D) streptomycin, and (F) tetracycline in the presence of soil extract after 24 h incubation. Asterisks (*) indicate significant difference from the positive control of dried soil extract with no antibiotics.
larvae. Examples include Hadfield et al. (1994), Lau and Qian (1997), Qian et al. (2007), Hung et al. (2009), Hadfield (2011) and Huang et al. (2012). Alternatively, settlement can also be stimulated by addition of chemical inducers, such as neuroactive agents 3isobutyl-1-methylxanthine (IBMX) and P-3,4-dihydroxypheny lalanine (L-DOPA) (Bryan et al., 1997; Okamoto et al., 1998; Qian and Pechenik, 1998) or monovalent cations Kþ and Csþ (Yool, 1986; Carpizo-Ituarte and Hadfield, 1998). Pure chemicals such as IBMX have been used in bioassay systems to hasten the settlement of H. elegans, but these often result in abnormal development and metamorphosis of S. kraussii without attachment (unpublished data). Soil extract contains numerous organic components such as humic acids and ammonia (Forward et al., 1997), which are utilized for microbe growth (Poeton et al., 1999; Coates et al., 2002; et al., 2005; Tikhonov et al., 2010). These had most Feificova likely stimulated formation of an inductive biofilm that in turn promoted settlement of S. kraussii larvae. Differences in settlement rates of S. kraussii larvae in liquid and air dried soil extract suggested that the inductivity and increased density of soil extract associated biofilms may be due to a concentration of nutrients. Alternatively, adhesion of soil extract components or attachment of selected bacterial species could also have increased the attractiveness of the substrate. Soil retained on substrates may facilitate attachment of microorganisms and provide nutrients for growth (Whitehead and Verran, 2009). Furthermore, biofilms can alter the physical
characteristics of the substratum, including texture and wettability, both of which are important to recruiting larvae (Faimali et al., 2004; Zardus et al., 2008; Huggett et al., 2009). It was also likely that reduced settlement in liquid soil extract treatments might be due to lower salinities (see Section 2.3.1) although the latter did not affect the biofilm density as much. It is not known if the lower salinities impacted S. kraussii larvae directly or indirectly through modification of the bacterial community in the biofilm. Lau et al. (2005) documented differing settlement of H. elegans among biofilms developed in different salinities but did not find any correlations with bacterial community composition of the biofilms. TRFLP analysis of the bacterial biofilm community indicates an overlap between marine broth and soil extract associated communities, with soil extract species being a subset of marine broth species. Marine broth is a commercial media containing nutrients for microbial growth and is a non-specific promoter of all marine bacterial species, whereas soil extract may only act to promote the growth of heterotrophic bacteria. LaMontagne et al. (2004), Mathur zaro et al. (2011) showed that selection of et al. (2007) and Sanz-La bacterial communities can be influenced by soil and various organic nutrients. This is consistent with observations by Liebeke et al. (2009) of increased yield and culture of previously unculturable, novel bacteria in media enriched with soil nutrients. Chiu et al. (2008) also demonstrated that soils can influence larval metamorphosis in barnacle (A. amphitrite) and slipper limpet (Crepidula
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Fig. 4. Settlement of larvae in soil extract compared to marine broth shows decreased settlement is not correlated to bacterial density. (A) Larval settlement in petri dishes coated with 0.4 mg ml1, 1 mg ml1 and 2 mg ml1 marine broth. (B) Microbial abundance of assays treated with dried soil extract or dried marine broth after 24 h incubation with S. kraussii larvae. Asterisks (*) indicate significant difference from the positive control (SE 100 ul ml1).
onyx) by acceleration of microbial film formation due to availability of chemical nutrients. Thus, S. kraussii settlement may have been influenced by presence, density, and specific species of bacteria in the soil extract associated biofilm.
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Our results indicate that soil extract promotes growth of a specific biofilm community. The most plausible source of bacteria in this community would be the microbiota found on the S. kraussii larvae. Research has shown that inductive environmental bacterial species usually belong to the same consortia present in the organism's natural habitat (Lau et al., 2005). Huggett et al. (2006) demonstrated that larvae of sea urchin Heliocidaris erythrogramma settle only on bacterial communities associated with coralline algae, a host for the sea urchin adults. Although transmission of endosymbionts have been documented across some marine phyla such as sponges (Usher et al., 2001; Sharp et al., 2006; Lee et al., 2009), bryozoans (Lim and Haygood, 2004), ascidians (Hirose, 2000; Hirose and Fukuda, 2006), bivalves (Cary and Giovannoni, 1993; Krueger et al., 1996) and deep sea clams (Vrijenhoek, 2010), it is not known when and how bacteria came to be associated with S. kraussii larvae. The possibility remains that the larvae picked up bacteria from the water column during culture. Whichever may be the case, if we consider the perspective of animals as host-microbe ecosystems (McFall-Ngai et al., 2013), it then seems probable that a biofilm resulting from a subset of bacteria associated with the organism would have a positive effect. Soil forms the basic substrate in terrestrial ecosystems and plays an important role in modulating species diversity (Nielsen et al., 2011; Wall and Nielsen, 2012). Preliminary estimates of the value of ecosystem services provided by soil biota and soils globally range from 1.5 to 13 trillion US dollars annually (van der Putten et al., 2004; Wall and Nielsen, 2012). The influence of soil on aquatic biota is less documented. Research in the chemistry of soil nutrients suggest that soil associated molecules may act as recruitment or location specific cues for marine invertebrate larval settlement. Metamorphosis in blue crab (Callinectes sapidus) larvae was visibly accelerated in the presence of high humic acids levels in estuarine waters (Forward et al., 1997). The recruitment of benthic polychaete, Capitella sp. is correlated with soils of high organic content (Dubilier, 1988; Tsutsumi et al., 1990), and the Pacific oyster (Crassostrea gigas) has also been documented to undergo metamorphosis in the presence of ammonia (Pawlik, 1992). The results from this study suggest that soil nutrients may modulate the settlement of sessile invertebrates by promoting the formation of a specific biofilm. Soil nutrients would be present in significant amounts in estuarine intertidal waters, and play a significant role in shaping the biofilms found on substrates in these environments. In such a scenario, rising levels of eutrophication in aquatic systems from coastal pollution may be expected to have significant impact on prokaryotic communities, resulting in consequences for inverzaro et al., 2011) and tebrate recruitment (Chiu et al., 2008; Sanz-La increasing fouling of coastal structures. 5. Conclusion
Fig. 5. NMDS plot of tubeworm assay biofilm community fingerprints using TRFLP data indicate that the soil extract-associated bacteria is a subset of the marine broth associated bacteria, and both are significantly different from bacteria grown in no treatment. Ellipses delimit community clusters to 95% confidence interval, stress ¼ 0.066.
In conclusion, the present study demonstrates the potent effect of environmental soil extract to induce S. kraussii larval settlement. Our findings indicate that this effect is predominantly a result of the way the soil nutrients have accelerated the formation of an inductive biofilm. The same effect however could not be achieved with commercially available marine broth even when higher biofilm density was obtained. The possibility remains that bioactive substances in the extract may have enhanced the effect but this effect is likely to be small compared to the effect of the biofilm. This study offers an alternative method for bioassays where a biofilm is required for settlement of the test organisms. Biofilms obtained by soaking in “aquarium seawater” for a few days do not consistently produce the same settlement-inducing effect (Olivier et al., 2000; Lau et al., 2005; Khandeparker et al., 2006). This simple approach of adding nutrients into a bioassay may be a
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useful means for promoting the growth of an inductive biofilm from microbiota present on the larvae during the incubation period. Acknowledgements The authors acknowledge support from the Agency for Science, Technology and Research (A*STAR) “Innovative Marine Antifouling Solutions” Program Grant number 102-166-0102, and the US Office for Naval Research Global Grant Award No. N62909-13-1-N056. The authors also wish to thank colleagues from NUS Tropical Marine Science Institute and Department of Biological Sciences, NUS Environmental Research Institute and the US ONR Coatings Program for their assistance and guidance in the study. References Baird, W., 1865. Description of several new species and varieties of tubicolous annelids (¼tribe Limivora of Grube) in the collection of the British Museum. J. Linn. Soc. Lond. (Zool.) 8, 10e22. Barranguet, C., Plans, M., Van Der Grinten, E., Sinke, J.J., Admiraal, W., 2002. Development of photosynthetic biofilms affected by dissolved and sorbed copper in a eutrophic river. Environ. Toxicol. Chem. 21, 1955e1965. Battin, T.J., Kaplan, L.A., Denis Newbold, J., Hansen, C.M., 2003. Contributions of microbial biofilms to ecosystem processes in stream mesocosms. Nature 426, 439e442. Baya, A.M., Brayton, P.R., Brown, V.L., Grimes, D.J., Russek-Cohen, E., Colwell, R.R., 1986. Coincident plasmids and antimicrobial resistance in marine bacteria isolated from polluted and unpolluted Atlantic Ocean samples. Appl. Environ. Microbiol. 51, 1285e1292. Bayen, S., Wurl, O., Karuppiah, S., Sivasothi, N., Lee, H.K., Obbard, J.P., 2005. Persistent organic pollutants in mangrove food webs in Singapore. Chemosphere 61, 303e313. Bonar, D.B., Weiner, R.M., Colwell, R.R., 1986. Microbial-invertebrate interactions and potential for biotechnology. Microb. Ecol. 12, 101e110. Brooun, A., Liu, S.H., Lewis, K., 2000. A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 44, 640e646. Bryan, P.J., Qian, P.Y., Kreider, J.L., Chia, F.S., 1997. Induction of larval settlement and metamorphosis by pharmacological and conspecific associated compounds in the serpulid polychaete Hydroides elegans. Mar. Ecol. Prog. Ser. 146, 81e90. Burke, C., Kjelleberg, S., Thomas, T., 2009. Selective extraction of bacterial DNA from the surfaces of macroalgae. Appl. Environ. Microbiol. 75, 252e256. Carpizo-Ituarte, E., Hadfield, M.G., 1998. Stimulation of metamorphosis in the polychaete Hydroides elegans Haswell (Serpulidae). Biol. Bull. 194, 14e24. Cary, S.C., Giovannoni, S.J., 1993. Transovarial inheritance of endosymbiotic bacteria in clams inhabiting deep-sea hydrothermal vents and cold seeps. Proc. Natl. Acad. Sci. 90, 5695e5699. Chen, C.L., Maki, J.S., Rittschof, D., Teo, S.L.M., 2013. Early marine bacterial biofilm on a copper-based antifouling paint. Int. Biodeterior. Biodegrad. 83, 71e76. Chiu, J.M.Y., Zhang, R., Wang, H., Thiyagarajan, V., Qian, P.Y., 2008. Nutrient effects on intertidal community: from bacteria to invertebrates. Mar. Ecol. Prog. Ser. 358, 41e50. Chou, R., Lee, H.B., 1997. Commercial marine fish farming in Singapore. Aquac. Res. 28, 767e776. Chung, H.C., Lee, O.O., Huang, Y.L., Mok, S.Y., Kolter, R., Qian, P.Y., 2010. Bacterial community succession and chemical profiles of subtidal biofilms in relation to larval settlement of the polychaete Hydroides elegans. ISME J. 4, 817e828. Cinar, M.E., 2006. Serpulid species (Polychaeta: Serpulidae) from the Levantine coast of Turkey (eastern Mediterranean), with special emphasis on alien species. Aquat. Invasions 1, 223e240. Coates, J.D., Cole, K.A., Chakraborty, R., O'Connor, S.M., Achenbach, L.A., 2002. Diversity and ubiquity of bacteria capable of utilizing humic substances as electron donors for anaerobic respiration. Appl. Environ. Microbiol. 68, 2445e2452. Cooke, M.D., 1976. Antibiotic resistance among coliform and fecal coliform bacteria isolated from sewage, seawater, and marine shellfish. Antimicrob. Agents Chemother. 9, 879e884. Costerton, J.W., Stewart, P.S., Greenberg, E.P., 1999. Bacterial biofilms: a common cause of persistent infections. Science 284, 1318e1322. Crisp, M., 1977. The development of the serpulid Pomatoleios kraussii (Annelida, Polychaeta). J. Zool. 183, 147e160. Cuong, D.T., Bayen, S., Wurl, O., Subramanian, K., Wong, K.K., Sivasothi, N., Obbard, J.P., 2005. Heavy metal contamination in mangrove habitats of Singapore. Mar. Pollut. Bull. 50, 1732e1738. Dubilier, N., 1988. H2S e a settlement cue or a toxic substance for capitella sp. I larvae? Biol. Bull. 174, 30e38. Faimali, M., Garaventa, F., Terlizzi, A., Chiantore, M., Cattaneo-Vietti, R., 2004. The interplay of substrate nature and biofilm formation in regulating Balanus amphitrite Darwin, 1854 larval settlement. J. Exp. Mar. Biol. Ecol. 306, 37e50.
, M., Cejkov , A., Mas Feifi cov a, D., Snajdr, J., Siglova a ak, J., Jirk u, V., 2005. Influence of humic acids on the growth of the microorganisms utilizing toxic compounds (comparison between yeast and bacteria). CHIMIA Int. J. Chem. 59, 749e752. Fischer, H., 2003. The role of biofilms in the uptake and transformation of dissolved organic matter. In: Findlay, S., Sinsabaugh, R.L. (Eds.), Aquatic Ecosystems: Interactivity of Dissolved Organic Matter. Academic Press, USA, pp. 285e313. Fischer, H., Wanner, S.C., Pusch, M., 2002. Bacterial abundance and production in river sediments as related to the biochemical composition of particulate organic matter (POM). Biogeochemistry 61, 37e55. Forward, R.B., Tankersley, R.A., Blondel, D., Rittschof, D., 1997. Metamorphosis of the blue crab Callinectes sapidus: effects of humic acids and ammonium. Mar. Ecol. Prog. Ser. 157, 277e286. Gander, S., 1996. Bacterial biofilms: resistance to antimicrobial agents. J. Antimicrob. Chemother. 37, 1047e1050. Gans, J., Wolinsky, M., Dunbar, J., 2005. Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science 309, 1387e1390. Grabow, W.O.K., Prozesky, O.W., Smith, L.S., 1974. Drug resistant coliforms call for review of water quality standards. Water Res. 8, 1e9. Hadfield, M.G., 2011. Biofilms and Marine invertebrate larvae: what bacteria produce that larvae use to choose settlement sites. Annu. Rev. Mar. Sci. 3, 453e470. Hadfield, M.G., Unabia, C.C., Smith, C.M., Michael, T.M., 1994. Settlement preferences of the ubiquitous fouler Hydroides elegans. Recent Dev. Biofouling Control, 65e75. Hirose, E., 2000. Plant rake and algal pouch of the larvae in the tropical ascidian Diplosoma similis: an adaptation for vertical transmission of photosynthetic symbionts Prochloron sp. Zool. Sci. 17, 233e240. Hirose, E., Fukuda, T., 2006. Vertical transmission of photosymbionts in the colonial ascidian Didemnum molle: the larval tunic prevents symbionts from attaching to the anterior part of larvae. Zool. Sci. 23, 669e674. Hochbaum, A.I., Kolodkin-Gal, I., Foulston, L., Kolter, R., Aizenberg, J., Losick, R., 2011. Inhibitory effects of D-amino acids on Staphylococcus aureus biofilm development. J. Bacteriol. 193, 5616e5622. Holm, E.R., 2012. Barnacles and biofouling. Integr. Comp. Biol. 52, 348e355. Holm, E.R., Kavanagh, C.J., Meyer, A.E., Wiebe, D., Nedved, B.T., Wendt, D., Smith, C.M., Hadfield, M.G., Swain, G., Wood, C.D., Truby, K., Stein, J., Montemarano, J., 2006. Interspecific variation in patterns of adhesion of marine fouling to silicone surfaces. Biofouling 22, 233e243. Huang, S., Hadfield, M.G., 2003. Composition and density of bacterial biofilms determine larval settlement of the polychaete Hydroides elegans. Mar. Ecol. Prog. Ser. 260, 161e172. Huang, Y., Callahan, S., Hadfield, M.G., 2012. Recruitment in the sea: bacterial genes required for inducing larval settlement in a polychaete worm. Sci. Reports 2, 228. Huggett, M.J., Williamson, J.E., de Nys, R., Kjelleberg, S., Steinberg, P.D., 2006. Larval settlement of the common Australian sea urchin Heliocidaris erythrogramma in response to bacteria from the surface of coralline algae. Oecologia 149, 604e619. Huggett, M.J., Nedved, B.T., Hadfield, M.G., 2009. Effects of initial surface wettability on biofilm formation and subsequent settlement of Hydroides elegans. Biofouling 25, 387e399. Hung, O.S., Lee, O.O., Thiyagarajan, V., He, H.P., Xu, Y., Chung, H.C., Qiu, J.W., Qian, P.Y., 2009. Characterization of cues from natural multi-species biofilms that induce larval attachment of the polychaete Hydroides elegans. Aquat. Biol. 4, 253e262. Jin, T., Qian, P.Y., 2004. Effect of amino acids on larval metamorphosis of the polychaete Hydroides elegans. Mar. Ecol. Prog. Ser. 267, 209e218. Keough, M.J., Raimondi, P.T., 1996. Responses of settling invertebrate larvae to bioorganic films: effects of large-scale variation in films. J. Exp. Mar. Biol. Ecol. 207, 59e78. Khandeparker, L., Chandrashekar Anil, A., Raghukumar, S., 2006. Relevance of biofilm bacteria in modulating the larval metamorphosis of Balanus amphitrite. FEMS Microbiol. Ecol. 58, 425e438. Kirchman, D., Graham, S., Reish, D., Mitchell, R., 1981. Bacteria induce settlement and metamorphosis of Janua (Dexiospira) brasiliensis Grube (Polychaeta, Spirorbidae). J. Exp. Mar. Biol. Ecol. 56, 153e163. Krueger, D.M., Gustafson, R.G., Cavanaugh, C.M., 1996. Vertical transmission of chemoautotrophic symbionts in the bivalve Solemya velum (Bivalvia: Protobranchia). Biol. Bull. 190, 195. LaMontagne, M.G., Leifer, I., Bergmann, S., Van De Werfhorst, L.C., Holden, P.A., 2004. Bacterial diversity in marine hydrocarbon seep sediments. Environ. Microbiol. 6, 799e808. Lane, D.J., Pace, B., Olsen, G.J., Stahl, D.A., Sogin, M.L., Pace, N.R., 1985. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. U. S. A 82, 6955e6959. Lau, S.C.K., Qian, P.Y., 1997. Phlorotannins and related compounds as larval settlement inhibitors of a tube-building polychaete Hydroides elegans (Haswell). Mar. Ecol. Prog. Ser. 159, 219e227. Lau, S.C.K., Thiyagarajan, V., Qian, P.Y., 2003. The bioactivity of bacterial isolates in Hongkong water for the inhibition of barnacle (Balanus amphitrite Darwin) settlement. J. Exp. Mar. Biol. Ecol. 282, 43e60. Lau, S.C.K., Thiyagarajan, V., Cheung, S.C.K., Qian, P.Y., 2005. Roles of bacterial community composition in biofilms as mediator for larval settlement of three marine invertebrates. Aquat. Microb. Ecol. 38, 41e51.
J.Y.-H. Chan et al. / International Biodeterioration & Biodegradation 94 (2014) 192e199 Lee, O.O., Chui, P.Y., Wong, Y.H., Pawlik, J.Y., Qian, P.Y., 2009. Evidence for vertical transmission of bacterial symbionts from adult to embryo in the Caribbean sponge Svenzea zeai. Appl. Environ. Microbiol. 75, 6147e6156. Liebeke, M., Brozel, V.S., Hecker, M., Lalk, M., 2009. Chemical characterization of soil extract as growth media for the ecophysiological study of bacteria. Appl. Microbiol. Biotechnol. 83, 161e173. Lim, G.E., Haygood, M.G., 2004. “Candidatus Endobugula glebosa,” a specific bacterial symbiont of the marine bryozoan Bugula simplex. Appl. Environ. Microbiol. 70, 4921e4929. Mathur, J., Bizzoco, R.W., Ellis, D.G., Lipson, D.A., Poole, A.W., Levine, R., Kelley, S.T., 2007. Effects of abiotic factors on the phylogenetic diversity of bacterial communities in acidic thermal springs. Appl. Environ. Microbiol. 73, 2612e2623. Matsuki, T., Watanabe, K., Fukimoto, J., Miyamoto, Y., Takada, T., Matsumoto, K., Oyaizu, H., Tanaka, R., 2002. Development of 16S rRNA-gene-targeted groupspecific primers for the detection and identification of predominant bacteria in human feces. Appl. Environ. Microbiol. 68, 5445e5451. McFall-Ngai, M., Hadfield, M.G., Bosch, T.C., Carey, H.V., Domazet-Loso, T., Douglas, A.E., Dubilier, N., Eberl, G., Fukami, T., Gilbert, S.F., Hentschel, U., King, N., Kjelleberg, S., Knoll, A.H., Kremer, N., Mazmanian, S.K., Metcalf, J.L., Nealson, K., Pierce, N.E., Rawls, J.F., Reid, A., Ruby, E.G., Rumpho, M., Sanders, J.G., Tautz, D., Wernegreen, J.J., 2013. Animals in a bacterial world, a new imperative for the life sciences. Proc. Natl. Acad. Sci. U. S. A 110, 3229e3236. Nedved, B.T., Hadfield, M.G., 2009. Hydroides elegans (Annelida: Polychaeta): a model for biofouling research. In: Flemming, H.C., Venkatesan, R., Murthy, S.P., Cooksey, K. (Eds.), Marine and Industrial Biofouling, Springer Series on Biofilms. Springer-Vergla, Berlin, pp. 203e217. Nielsen, U.N., Ayres, E., Wall, D.H., Bardgett, R.D., 2011. Soil biodiversity and carbon cycling: a review and synthesis of studies examining diversity-function relationships. Eur. J. Soil Sci. 62, 105e116. Okamoto, K., Watanabe, A., Sakata, K., Watanabe, N., 1998. Chemical signals involved in larval metamorphosis in Hydroides ezoensis (Serpulidae; Polychaeta). Part I: induction of larval metamorphosis by extract of adult tube clumps. J. Mar. Biotechnol. 6, 7e10. Olivier, F., Tremblay, R., Bourget, E., Ritschoff, D., 2000. Barnacle settlement: field experiments on the influence of larval supply, tidal level, biofilm quality and age on Balanus amphitrite cyprids. Mar. Ecol. Prog. Ser. 199, 185e204. Pawlik, J.R., 1992. Chemical ecology of the settlement of benthic marine invertebrates. Oceanogr. Mar. Biol. 30, 273e335. Perthuisot, J.P., Guelorget, O., Ibrahim, A., Jusserand, C., Margerel, J.P., Maurin, A., 1990. L'organisation biogeologique du lac. Temsah (lsmailia, Egypte). Rev. d'Hydrobiologie Trop. 23, 77e90. Pillai, T.G., 2009. Descriptions of new serpulid polychaetes from the Kimberleys of Australia and discussion of Australian and Indo-West Pacific species of Spirobranchus and superficially similar taxa. Rec. Aust. Mus. 61, 93e199. Poeton, T.S., Stensel, H.D., Strand, S.E., 1999. Biodegradation of polyaromatic hydrocarbons by marine bacteria: effect of solid phase on degradation kinetics. Water Res. 33, 868e880. Pringault, O., Duran, R., Jacquet, S., Torreton, J.P., 2008. Temporal variations of microbial activity and diversity in marine tropical sediments (New Caledonia lagoon). Microb. Ecol. 55, 247e258. Qian, P.Y., Pechenik, J.A., 1998. Effects of larval starvation and delayed metamorphosis on juvenile survival and growth of the tube-dwelling polychaete Hydroides elegans (Haswell). J. Exp. Mar. Biol. Ecol. 227, 169e185. Qian, P.Y., Lau, S.C.K., Dahms, H.U., Dobretsov, S., Harder, T., 2007. Marine biofilms as mediators of colonization by marine macroorganisms: implications for antifouling and aquaculture. Mar. Biotechnol. 9, 399e410. R Development Core Team, 2010. R: a Language and Environment for Statistical Computing, Vienna, Austria. Retrieved from: http://www.R-project.org. Roth, B.L., Poot, M., Yue, S.T., Millard, P.J., 1997. Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl. Environ. Microbiol. 63, 2421e2431.
199
Sanz-L azaro, C., Navarrete-Mier, F., Marin, A., 2011. Biofilm responses to marine fish farm wastes. Environ. Pollut. 159, 825e832. Sawall, Y., Richter, C., Ramette, A., 2012. Effects of eutrophication, seasonality and macrofouling on the diversity of bacterial biofilms in equatorial coral reefs. Plos One 7, e39951. Shalla, S.H., Holt, T.J., 1999. The Lessepsian migrant Pomatoleios kraussii (Annelida, Polychaeta) e recent formation of dense aggregations in Lake Timsah and the Bitter Lakes (Suez Canal, Egypt). Egypt. J. Biol. 1, 133e137. Sharp, K.H., Eam, B., Faulkner, D.J., Haygood, M.G., 2006. Vertical transmission of diverse microbes in the tropical sponge Corticium sp. Appl. Environ. Microbiol. 73, 622e629. Shikuma, N.J., Pilhofer, M., Weiss, G.L., Hadfield, M.G., Jensen, G.J., Newman, D.K., 2014. Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures. Science 343, 529e533. Sokal, R.R., Rohlf, F.J., 2012. Biometry: the principles and practice of statistics in biological research. W. H. Freeman and Co, New York, p. 937. Straughan, D., 1969. Intertidal zone-formation in Pomatoleios kraussii (Annelida e Polychaeta). Biol. Bull. 136, 469e482. Tait, K., Havenhand, J., 2013. Investigating a possible role for the bacterial signal molecules N-acylhomoserine lactones in Balanus improvisus cyprid settlement. Mol. Ecol. 22, 2588e2602. Tam, N.F.Y., 1998. Effects of wastewater discharge on microbial populations and enzyme activities in mangrove soils. Environ. Pollut. 102, 233e242. Tikhonov, V.V., Yakushev, A.V., Zavgorodnyaya, Y.A., Byzov, B.A., Demin, V.V., 2010. Effects of humic acids on the growth of bacteria. Eurasian Soil Sci. 43, 305e313. Torsvik, V., Goksoyr, J., Daae, F.L., 1990. High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56, 782e787. Tsutsumi, H., Fukunaga, S., Fujita, N., Sumida, M., 1990. Relationship between growth of Capitella sp. and organic enrichment of the sediment. Mar. Ecol. Prog. Ser. 63, 157e162. Unabia, C.R.C., Hadfield, M.G., 1999. Role of bacteria in larval settlement and metamorphosis of the polychaete Hydroides elegans. Mar. Biol. 133, 55e64. Usher, K.M., Kuo, J., Fromont, J., Sutton, D.C., 2001. Vertical transmission of cyanobacterial symbionts in the marine sponge Chondrilla australiensis (Demospongiae). Hydrobiologia 461, 15e23. van der Putten, W.H., Anderson, J.M., Bardgett, R.D., Behan-Pelletier, V., Bignell, D.E., Brown, G.G., Brown, V.K., Brussaard, L., Hunt, H.W., Ineson, P., Jones, T.H., Lavelle, P., Paul, E.A., John, M.S., Wardle, D.A., Wojtowicz, T., Wall, D.H., 2004. The sustainable delivery of goods and services provided by soil biota. In: Wall, D. (Ed.), Sustaining Biodiversity and Ecosystem Services in Soils and Sediments. Island Press, Washington, pp. 15e44. Vrijenhoek, R.C., 2010. Genetics and evolution of deep-sea chemosynthetic bacteria and their invertebrate hosts. In: Kiel, S. (Ed.), The Vent and Seep Biota, Topics in Geobiology, vol. 33. Springer, Dordrecht Heidelberg London New York, pp. 15e49. Wall, D.H., Nielsen, U.N., 2012. Biodiversity and ecosystem services: is it the same below ground? Nat. Educ. Knowl. 3, 8. Warton, D.I., Hui, F.K.C., 2011. The arcsine is asinine: the analysis of proportions in ecology. Ecology 92, 3e10. Whitehead, K.A., Verran, J., 2009. The effect of substratum properties on the survival of attached microorganisms on insert surfaces. In: Flemming, H.C., Murthy, P.S., Venkatesan, R., Cooksey, K.E. (Eds.), Marine and Industrial Biofouling. Springer Series on Biofilms. Springer-Vergla, Berlin, pp. 13e34. Wilson, E., Underwood, M., Puckrin, O., Letto, K., Doyle, R., Caravan, H., Camus, S., Bassett, K., 2010. The Arcsine Transformation: has the Time Come for Retirement? Memorial University of Newfoundland, Newfoundland and Labrador, Canada. http://www.mun.ca/biology/dschneider/b7932/B7932Final10Dec2010.pdf. Yool, A.J., Grau, S.M., Hadfield, M.G., Jensen, R.A., Markell, D.A., Morse, D.E., 1986. Excess potassium induces larval metamorphosis in 4 marine invertebrate species. Biol. Bull. 170, 255e266. Zardus, J.D., Nedved, B.T., Huang, Y., Tran, C., Hadfield, M.G., 2008. Microbial biofilms facilitate adhesion in biofouling invertebrates. Biol. Bull. 214, 91e98.