Biofilms:page1of8 C 2006 Cambridge University Press doi:10.1017/S1479050506002018

Temporal variation of an initial marine biofilm community and its effects on larval settlement and metamorphosis of the tubeworm elegans

* Corresponding author: ∗ Kewalo Marine Laboratory N. J. Shikuma and M. G. Hadfield University of Hawaii 41 Ahui Street Honolulu HI 96813 USA ABSTRACT T 1 808 539 7319 F 1 808 599 4817 E hadfi[email protected] Planktonic larvae of many sessile marine invertebrates settle and metamor- phose preferentially on surfaces covered by bacterial biofilms. The Kewalo Marine Laboratory, University of tubeworm is induced to settle by biofilms and is the primary Hawaii, 41 Ahui Street, Honolulu colonizer of newly submerged surfaces in the succession of macrofouling HI 96813, USA invertebrates in Pearl Harbor, Hawaii, USA. This study examines the succes- sion of bacterial community composition and cell density of marine biofilms, and how temporal changes in biofilms affect settlement of H. elegans. Settlement assays of H. elegans were conducted on naturally formed biofilms of increasing age from Pearl Harbor. Denaturing gradient gel electrophoresis and epifluoresence microscopy were used to identify community composition and densities of bacterial cells in biofilms. Results of this study suggest that increasing densities of a stable community, rather than a shift in dominant species composition of biofilm bacteria, are probably responsible for the primary colonization of submerged surfaces by H. elegans in Pearl Harbor. However, the current data leave open the question of why larvae of other biofouling invertebrate species known to settle in response to biofilms recruit to surfaces only at later times.

INTRODUCTION harbors and estuaries around the world (Hadfield et al., 1994). The larvae of H. elegans must settle on to newly The larvae of many sessile marine invertebrates are submerged surfaces and reproduce within a 2–4 week known to possess the ability to find suitable substrates on period before they are overgrown and out-competed for which to settle and metamorphose (Crisp, 1974). Patterns space by bryozoans, ascidians and sponges (Holm et al., of settlement, such as succession in marine biofoul- 2000). Unlike some polychaete species (Jensen & Morse, ing communities, indicate that there may be underly- 1984; Toonen & Pawlik, 1996; Keough, 1998), H. elegans ing mechanisms for this ordered recruitment (Chalmer, is not induced to settle by conspecific (Walters 1982). Settlement cues, usually biological in origin, often et al., 1997) but does settle in response to bacterial direct settlement of these larvae (Crisp, 1974; Hadfield & biofilms (Hadfield et al., 1994). Possible advantages of Paul, 2001; Steinberg et al., 2002). Bacterial biofilms have using bacterial biofilms as settlement cues include an been shown to induce settlement of larvae from many indication of food, preferable habitat, and likelihood that invertebrate phyla (Hofmann & Brand, 1987; Pawlik, 1992; the surface will be submerged long enough for the Hadfield et al., 1994; Unabia & Hadfield, 1999; Hamer to mature and reproduce (Keough & Raimondi, 1995). et al., 2001; Negri et al., 2001; Webster et al., 2004). Specific settlement cues for H. elegans have yet to be However, still there is little known about how microbial identified, but studies have shown that the cue is either succession may influence macrofouling succession and bound to the biofilm surface in the exopolysaccharides what qualities of a bacterial biofilm are responsible for or on the bacterial cells themselves (Huang & Hadfield, the induction of settlement. 2003). Hydroides elegans is an appropriate model organism for Natural biofilms are composed of many microbial studying the induction of settlement and metamorphosis species and their extracellular matrices attached to a by bacterial biofilms. It has separate sexes and starts surface (Decho, 1990; Costerton & Stewart, 2001). Rather to produce gametes at about 3 weeks in laboratory than conglomerations of cells and slime, biofilms are culture. This serpulid polychaete is an early colonizer instead organized communities complete with func- of newly submerged surfaces in subtropical and tropical tional microcolonies and channels to perform complex

Biofilm succession and effects on settlement 1 2 N. J. Shikuma and M. G. Hadfield

metabolic processes (Costerton et al., 1999). Bacterial hung from plastic frames with nylon monofilament fishing succession on newly submerged surfaces is known to line and plastic cable ties. Slides were hung horizontally occur in multiple habitats (Jackson et al., 2001; Kelly & along each frame and spaced approximately 10 cm apart. Chistoserdov, 2001; Rao, 2003), but how these natural Nylon rope was used to suspend the frames from a pier microbial assemblages influence macrofouling settlement on Ford Island, Pearl Harbor, Hawaii, between about has been little studied. 0.5 m and 2 m below the water surface, depending on Single-species biofilms composed of bacteria previously the tide. Preliminary experiments to examine variance isolated from natural multi-species biofilms were shown in community composition of 1 week old biofilms along to induce varying rates of settlement of H. elegans (Unabia the 50 m pier length using DGGE revealed no differences & Hadfield, 1999; Lau & Qian, 2002; Huang & Hadfield, in band patterns. Six frames were built, each holding 2003). Monospecific biofilms of Cytophaga lytica induced 18 slides. One frame was submerged initially, and an about 40% metamorphosis of H. elegans, while biofilms additional frame was submerged every 2 days after the of Pseudoalteromonas luteoviolacea induced about 60–80% previousframe untilallsixframeswere inthe water.Twelve metamorphosis (Huang & Hadfield, 2003). However, few days after the first frame was submerged, all six frames single species of bacterium cause rates of metamorphosis were removed from the water at the same time in order as high as natural multi-species biofilms, although mono- to decrease variance in subsequent experiments. Biofilms specific biofilms of P. luteoviolacea may approach those therefore ranged in age from 2 to 12 days in intervals of levels (Huang & Hadfield, 2003). Percentage metamorph- 2days. osis of H. elegans and cell density in a monospecific biofilm of P. luteoviolacea were also shown to be positively correlated (Huang & Hadfield, 2003). These results suggest Larval culture of Hydroides elegans that there may be an increase in the amount of cue to settle Hydroides elegans settles naturally on to newly submerged when more bacterial cells are present (Huang & Hadfield, biofilmed surfaces near Ford Island, Pearl Harbor, Hawaii, 2003). Until the present study, it has not been shown throughout the year (Carpizo-Ituarte & Hadfield, 1998). whether variations in the species composition of natural  Plastic screens (Vexar R ) were submerged in the water at biofilms over time play a role in the increasing settlement Ford Island for about 4 weeks to recruit H. elegans. Adults rates of larvae of H. elegans. were transported to the Kewalo Marine Laboratory of Until recently, knowledge of microbe-induced settle- the University of Hawaii at Manoa, Honolulu, Hawaii, ment of H. elegans larvae has been limited to biofilms andkeptinseatablesforupto14daysincontinuously of species cultured with artificial media and natural flowing unfiltered sea water. Calcareous tubes of about 15 multi-species biofilms of unknown species composition adult worms were gently broken to induce spawning in a (Hadfield et al., 1994; Unabia & Hadfield, 1999; Lau & common dish filled with about 10 ml of 0.22 µmfiltered Qian, 2002; Huang & Hadfield, 2003). Artificial media sea water (FSW). To reduce polyspermy of eggs, several can bias estimates of species composition of natural multi- males were removed before completely spawning. Zygotes species communities due to variations in metabolic and were transferred into 2liter beakers filled with FSW growth-condition requirements among bacterial species shortly after the two-cell stage. Larvae were maintained (Amann et al., 1995). Unculturable species present in − ◦ at a density of 5–10 ml 1 and a temperature of 22–24 C. natural biofilms may also have an important role in the Isochrysis galbana wasprovidedasfoodthroughoutlarval induction of settlement of larvae of H. elegans. culture at a density of 105 cells/ml. On days 2 through The present study focused on three aspects of marine 4ofculture,larvaeweresievedfromthewaterwith biofilms and how they may affect settlement preferences a41µmmeshandtransferredintofreshFSW,algae of H. elegans. (1) Denaturing gradient gel electrophoresis and clean beakers to decrease exposure to biofilms and (DGGE) was used to determine culture-independent thus settlement prior to the experiments. Larvae became species compositions of bacteria on newly submerged competent to settle and metamorphose on day 5 of surfaces in Pearl Harbor, Hawaii, USA, over a 2 week laboratory culture. interval. (2) Temporal changes in bacterial density and community composition of biofilms were examined and correlated with settlement rates of H. elegans.(3)Natural Settlement assays with larvae of H. elegans biofilms from Pearl Harbor were examined to determine whether the bacterial species P. luteoviolacea and C. lytica, Sixreplicateslidesofeachbiofilmagewereexposedto known to induce settlement of H. elegans in monospecific about 100 larvae of H. elegans in 95 mm diameter sterile biofilms, were present. Petri dishes and 6 ml of FSW.A negative control treatment, which consisted of larvae exposed to clean glass slides, was included. The percentage of larvae in each dish that MATERIALS AND METHODS settled on to biofilmed surfaces was determined 24 h after initial exposure. Settlement was defined as adherence of the larvae to the substratum, formation of a primary Biofilm site and collection proteinacious tube and initial formation of branchial Glass microscope slides were cleaned with 99% (v/v) rudiments. Animals that recruited to the slides in the field ethanol, sterilized with ultraviolet (UV) radiation, and could be distinguished from newly settled larvae in the Biofilm succession and effects on settlement 3

experiment by the large size of the animals and length of initial denaturing step at 94 ◦C for 2 min, followed by 35 their calcareous tubes. Field-recruited animals were not cycles of a 45 s denaturing step at 94 ◦C, a 45 s annealing included in subsequent analyses. Few other macrofouling step at 55 ◦C,anda1minextensionstepat72◦C. A final invertebrates settled onto the experimental slides, and extension step of 6 min at 72 ◦C was carried out. The 16 S those that did were too small to consume the larvae of ribosomal gene fragments from the previously isolated H. elegans or affect subsequent settlement assays. bacteria, P. luteoviolacea and C. lytica (Huang & Hadfield, 2003), were amplified using PCR with the protocols described above. Each PCR reaction was carried out with Bacterial biofilm density estimates a negative control where the volume of template DNA Bacteria on six replicate slides of each biofilm age were was replaced with an equal volume of sterile water. stored at 4 ◦C for up to 1 month in 3.7% (v/v) formalin Each PCR reaction product was run on a 1% (w/v) in FSW. Bacteria on these slides were stained using the agarose gel to verify the DNA fragment length. A Kodak nuclear stain propidium iodide (5 µg/ml) in FSW and Gel Logic 100 Imaging System and Kodak 1D Image counted using a confocal laser scanning microscope (Zeiss Analysis Software (Kodak) were used to take and analyze LSM510). On each slide, bacteria were viewed at 630× digital images of the agarose gels stained with ethidium magnification and 10 square fields measuring 145 µmper bromide (0.5 µg/ml). side. Areas were chosen using a grid on the stage of the microscope and randomly generated numbers. Bacteria were counted in each digital image, averaged for each slide Denaturing gradient gel electrophoresis and averaged over all replicate slides for each biofilm age Gradient 8% (w/v) polyacrylamide gels (acrylamide- to provide bacterial density estimates. N,N-methylenebisacrylamide) containing 40–100% de- naturant, from top to bottom, were poured. One hundred per cent denaturant is defined as a 7 M urea and 40% (v/v) DNA extraction formamide solution. Two gels were poured simultaneously Six replicate slides for each biofilm age were used for using a multi-gel caster, a 40 ml gradient maker, and mini- biofilm DNA extraction. Biofilms from two slides were peristaltic pump (CBS Scientific). Approximately half of pooled into a 1.5 ml centrifuge tube by scraping them from each of the 25 µl PCR reactions was loaded into each the surface using a sterile razor, creating three replicates of the gradient acrylamide gels. The gels were loaded per age group. The tubes were stored at 4 ◦C for 1–2 h into a denaturing gradient gel electrophoresis system before DNA extraction. Genomic DNA from biofilms was (CBS Scientific) containing 1 × TAE buffer (20 mm Tris, ◦ extracted using an UltraClean Microbial DNA Kit (Mo 10 mm acetate, 0.5 mm EDTA, pH 7.4) at 60 Cand Bio Laboratories Inc.) according to the manufacturer’s run at 30V for 14h. Gels were stained in 1× Cybr instructions. Gold solution (Molecular Probes) in 1 × TAE buffer for The bacteria P. luteoviolacea and C. lytica were 30 min. Digital images of the gels were taken using a previously isolated and employed to induce settlement Kodak Gel Logic 100 Imaging System (Kodak) under UV of H. elegans in this laboratory (Huang & Hadfield, 2003). illumination. The PCR–DGGE process was repeated for Single colonies from each species were grown to log each of three replicate biofilm samples of each age to phase in half-strength sea water–tryptone (SWT) broth determine reproducibility of community fingerprints. (Boettcher & Ruby, 1990), and DNA was extracted using Bands were excised from PCR–DGGE gels, placed in the UltraClean Microbial DNA Kit, as described above. 20 µl of molecular biology grade water (Eppendorf) and eluted overnight. Reamplified products of bands labeled P1, P2 and C2 were cloned using a pGEM-T Easy Vector PCR amplification System (Promega) and three clones of each band were sequenced. Reamplified PCR products of bands A, B, The polymerase chain reaction (PCR) was used to amplify C and D were sequenced directly. Sequence homologies an approximately 419 base-pair (bp) fragment of the 16 S were determined using the BLAST search in GenBank. ribosomal gene. Universal bacterial forward and reverse Sequence data were submitted to the GenBank databases primers were complementary to positions 341 to 357 with  under accession numbers DQ226055–DQ226067. a 40 bp GC clamp (underlined) (5 -CGCCCGCCGCGCG- CGGCGGGCGGGGCGGGGGCACGGGGGGCCTACG- GGAGGCAGCAG-3)(Muyzeret al., 1993), and position Statistical analyses 758 to 776 (5-CTACCAGGGTATCTAATCC-3)(Juck et al., 2000) (Escherichia coli numbering). Reactions A one-way analysis of variance (ANOVA) and Tukey’s (25 µl) were carried out with final concentrations of pairwise comparison were performed to determine × 1.5 mM MgCl2,1 reaction buffer (MgCl2 free), 800 µM significant differences between log-transformed bacterial PCR Nucleotide Mix, 0.025 U/µl Taq DNA polymerase density counts of biofilms of sequential age. Although the (Promega, Madison, WI, USA), molecular biology grade homogeneity of variance criterion was not met, ANOVA water (Eppendorf, Westbury, NY, USA), and 1–5 µl is considered robust and was performed to determine template in 200 µl thin-walled PCR tubes (USA Scientific, significant differences between settlement of H. elegans Ocala, FL, USA). PCR reactions were carried out with an larvae on biofilms of sequential age. Tukey’s pairwise 4 N. J. Shikuma and M. G. Hadfield

treatments. Mean percentage metamorphosis increased incrementally to 99% between 6 and 12 day old biofilm treatments.

Density of bacteria in biofilms Bacterial densities in the biofilms were estimated by

Percentage metamorphosis Percentage staining the cells with a fluorescent DNA stain, counting them using confocal microscopy and averaging the mean number of cells per field of view. Mean bacterial densities increased from 1053 to 31 795 cells/mm2 between 2 and

) 12 day old biofilms (Fig. 1b). Mean bacterial densities 2 were significantly different between biofilms that were 4 and 6, 6 and 8, and 8 and 10 days old (ANOVA, Tukey’s pairwise comparison, P < 0.01). As bacterial densities increased incrementally within biofilms that were 4 and 12 days old, settlement rates of the negative control, 2 and 4 day old biofilms were all below 26% and increased to above 85% between biofilms that were 4 and 6 days old. Mean percentage metamorphosis of H. elegans and bacterial densities were not significantly correlated

Biofilm density (thousands of cells/mm (one-way ANOVA, R2 = 0.58, P > 0.05). However, mean percentage metamorphosis and natural-log-transformed bacterial densities were significantly correlated (one-way Fig. 1: (a) Mean percentage metamorphosis of larvae of H. elegans ANOVA, R2 = 0.87, P < 0.01). exposed to 2 to12 day old biofilms. A negative control was included where larvae were exposed to clean glass surfaces. Bars indicate mean percentage metamorphosis in 24 h + 1 standard deviation (SD; n = 6 replicates per treatment). (b) Bacterial cell density in 2 to 12 day old Bacterial community analysis using DGGE biofilms enumerated by fluorescent staining and confocal microscopy. Bars indicate mean cell density per mm2 +1SD(n = 6 replicates per Bacterial fingerprints of biofilm communities between 2 treatment). Letters above bars indicate significant differences between and 12 days old were obtained using the DGGE technique means (one-way ANOVA and Tukey’s pairwise comparison; (a) P < 0.001, (Fig. 2). Replicate samples of community fingerprints (b) P < 0.01). of biofilms were consistently reproduced on separate DGGE gels, and seven clearly distinguishable bands were shared between all biofilms between the ages of 2 and comparison was employed to determine significant dif- 12 days. DGGE fingerprints of biofilm communities of ferences between individual treatments. The above tests increasing age were not completely identical, as a number were completed using the statistical program SPSS of bands occurred only in one or a subset of the biofilm (Student Version 10.0). A one-way ANOVA was used to communities. For example, several bands in the 2 day determine whether H. elegans percentage metamorphosis old biofilm fingerprint did not appear in other biofilm and bacterial density, as well as percentage metamorphosis community fingerprints, such as band A (Fig. 2, Table 1). and natural-log-transformed bacterial density, were Furthermore, biofilms that were similar in age tended to correlated in Microsoft Excel. have similar community fingerprints. To further charac- terize the biofilm communities, four bands of interest were excised, reamplified, and sequenced directly, to determine RESULTS band identities within the biofilm community (bands A–D in Fig. 2 and Table 1). Amplified 16 S ribosomal gene DNA from the bacte- Induction of metamorphosis of H. elegans rial species P. luteoviolacea and C. lytica,showntoinduce Settlement assays of H. elegans larvae were conducted on metamorphosis of H. elegans in monospecific biofilms biofilms that had developed in the field from 2 to 12 days (Huang & Hadfield, 2003), was loaded into lanes adja- in intervals of 2 days. Mean percentage metamorphosis cent to the amplified biofilm DNA in order to determine of H. elegans was significantly different between biofilms whether bands within multi-species community finger- of sequential age and increased as biofilm age increased prints could be identified (lanes P and C in Fig. 2). from 2 to 12 days (ANOVA, Tukey’s pairwise comparison, These single species were previously isolated from the P < 0.001) (Fig. 1a). Metamorphosis in negative control sea tables of Kewalo Marine Laboratory, Kewalo Basin, assays, where larvae were exposed to clean surfaces, approximately 15 km away from the experimental field averaged 5%. Twenty-six per cent of larvae were induced site in Pearl Harbor. Migration rates of ribosomal gene to metamorphose by 4 day old biofilms, and rates of DNA from the isolated bacterial species closely matched metamorphosis increased to 86% in 6 day old biofilm bands within the bacterial biofilm community. Bands P1, Biofilm succession and effects on settlement 5

Fig. 2: DGGE profiles of PCR-amplified 16 S ribosomal gene DNA fragments from replicate bacterial biofilms from Pearl Harbor, Hawaii, USA. Numbers above lanes indicate age of biofilms in days at time of DNA extraction. Ribosomal gene DNA from the bacterial species P. luteoviolacea (P) and C. lytica (C) was extracted, amplified and loaded alongside biofilm DNA. Labeled bands were excised and sequenced (see Table 1).

P2 and C1, with migration rates similar to those of DISCUSSION P. luteoviolacea and C. lytica (indicated in Fig. 2), were excised, reamplified and cloned using the pGEM-T Easy The community composition and cell density of newly Vector System (Promega). DNA inserts from three clones formed bacterial biofilms were studied with DGGE and from each band were sequenced to determine band epifluorescence microscopy to determine how bacterial identities, and none was identified as P. luteoviolacea or succession of marine biofilms might affect preferences C. lytica (Table 1). of H. elegans. It was found that bacterial community 6 N. J. Shikuma and M. G. Hadfield

Table 1: Homologous 16 S rDNA sequences searched on the GenBank BLAST search tool. Bands were excised from a DGGE gel, reamplifed, and directly sequenced (bands A, B, C and D), or cloned into plasmids (bands P1, P2 and C1) and sequenced

DGGE band Clone Closest sequence (accession no.) Phylogenetic group % identity

P1 1 Uncult. bacterium from alkaline district heating system biofilm (AY753398) β-Proteobacteria 92.5 2 Uncult. symbiont in squid accessory nidamental glands (AJ633953) α-Proteobacteria 92.3 3 Uncult. bacterium from intertidal flat, Ganghwa Island (AY568804) α-Proteobacteria 98.4 P2 1 Uncult. bacterium from alkaline district heating system biofilm (AY753398) β-Proteobacteria 92.5 2 Uncult. bacterium from grey whale carcass, Santa Cruz Basin (AY922228) α-Proteobacteria 98.0 3 Uncult. bacterium from intertidal flat, Ganghwa Island (AY568928) α-Proteobacteria 98.4 C1 1 Uncult. Phyllobacteriaceae bacterium (AY711355) α-Proteobacteria 99.3 2 Flavobacterium spp. (AY536567) Bacteroidetes 95.3 3 Uncult. bacterium from Nankai Trough sediments (AY191341) Bacteroidetes 99.1 A Saprospira grandis (AY527408) Bacteroidetes 94.2 B Jannaschia rubra (AJ748747) α-Proteobacteria 96.9 C Uncult. from bioactive biofilms for H. elegans settlement (AY600952) α-Proteobacteria 97.6 D Uncult. bacterium from fluidized-bed reactor (AY263729) α-Proteobacteria 92.5

Uncult., uncultured.

succession may not play as crucial a role in the settlement Although P. luteoviolacea and C. lytica were not found of the larvae of H. elegans as we initially hypothesized. in Pearl Harbor biofilms, it is not surprising that other Replicate DNA extractions, PCR amplifications and bacterial species within the biofilm were able to induce DGGE gels revealed a set of seven clearly distinguishable metamorphosis of H. elegans. Sequence identities of bands bands that appeared in all biofilms of all ages in all excisedfromDGGEgelswerefoundtobehomologousto three replicates. Although the seven clearly distinguishable several groups of distantly related bacteria isolated from bands that appeared consistently in 2 to 12 day old biofilms around the world. Monospecific biofilms of several species cannot be shown to represent the primary inducers of of bacteria from disparate groups were shown to induce settlement in H. elegans, the strong correlation between settlement of H. elegans (Unabia & Hadfield, 1999; Lau & natural-log-transformed bacterial density and percentage Qian, 2002; Huang & Hadfield, 2003). settlement is evidence that the increase in percentage Although the strong correlation between natural-log- settlement of H. elegans asbiofilmsageisduetoincreasing transformed bacterial densities and H. elegans settlement bacterial density of species that are present throughout suggests that existing bacterial cells or their specific the study period. This relationship was also shown in extracellular polymeric substances provide the cue(s) for previous experiments where increases in densities of short settlement, a shift in bacterial species composition over rod-shaped bacteria in natural biofilms and settlement of time may still play a role in settlement of H. elegans.It H. elegans was correlated (Hadfield et al., 1994). Rates of was shown that P. luteoviolacea induces higher rates of metamorphosis and densities of bacteria in monospecific settlement than C. lytica in monospecific biofilms, even biofilms were also shown to be directly related (Huang & when in dramatically lower densities (Huang & Hadfield, Hadfield, 2003). 2003). An inherent limitation of the DGGE technique is Similar conclusions about the effects of bacterial that only relative species compositions may be determined biofilm communities on the settlement preferences of (Vallaeys et al., 1997). Unculturable biofilm bacteria in low H. elegans were found in another study using DGGE. Lau densities, but with high settlement inductive qualities, may et al. (2005) created biofilms composed of different cell have a significant impact on H. elegans settlement and not densities and community compositions in the laboratory appear on DGGE gels. by submerging surfaces in unfiltered sea water in an Bacterial species in low densities may not amplify in the array of three salinities and three temperatures (Lau PCR process or may be overwhelmed by DNA from more et al., 2005). Biofilms in laboratory aquaria may develop prevalent bacterial species (Muyzer et al., 1993; Kisand & differently from those in the field owing to the fixed Wikner, 2003). The number of bands that are separated volume of unfiltered water and quantity of free-living from a single bacterial assemblage by DGGE can give bacteria available for attachment on to the experimental insight into the numbers of species present (Muyzer et al., surface. However, it is interesting to note that biofilms 1993), although 16 S ribosomal gene sequences are known developed in the environment over time (this study) to yield multiple bands from a single species (Dahllof and biofilms developed in the laboratory, where bacterial et al., 2000). Furthermore, species diversity may not be communities differed as a result of varied temperature and completely realized using DGGE (Vallaeys et al., 1997), salinity (Lau et al., 2005), appeared to have the same effect where multiple 16 S ribosomal gene sequences may appear on H. elegans settlement preferences. Factors influencing as one band on a DGGE gel, as observed in this study biofilm community composition, including salinity and (Fig. 2, Table 1). The 16 S ribosomal gene DNA fragment temperature (Lau et al., 2005) or succession over time (this amplified by PCR may also be too short to determine study), did not influence settlement whereas cell densities species level differences between sequences (Kisand & of biofilms and settlement were significantly correlated. Wikner, 2003). Biofilm succession and effects on settlement 7

Although DGGE has limitations, data gathered in this gene intraspecies heterogeneity. Applied and Environmental study are evidence that succession of the bacterial biofilm Microbiology 66, 3376–3380 communities may not be an influential factor in de- Decho, A. W. (1990) Microbial exopolymer secretions in ocean termining primary invertebrate succession of biofouling environments: their role(s) in food webs and marine processes. communities over the period examined. As a ubiquitous Oceanography Marine Biology Annual Review 28, 73–153 Hadfield, M. G. & Paul, V. J. (2001) Natural chemical cues for marine fouling invertebrate found in harbors and estuaries settlement and metamorphosis of marine-invertebrate larvae. around the world, H. elegans may be induced to settle In Marine Chemical Ecology, pp. 431–461. Edited by J. B. by numerous bacterial species, increasing its chances of McClintock&B.J.Baker.BocaRaton,FL:CRCPress finding a suitable habitat. Moreover, the bacterial species Hadfield, M. G., Unabia, C. R. C., Smith, C. M. & Michael, T. M. that induce settlement of H. elegans may be the more (1994) Settlement preferences of the ubiquitous fouler Hydroides common or abundant colonizers of submerged surfaces, elegans.InRecent Developments in Biofouling Control, pp. 65–74. thus increasing the probability that larvae of H. elegans Edited by M. F. Thompson, R. Nagabhushanam, R. Sarojini & will find a suitable habitat. Further research into the M. Fingerman. New Delhi: Oxford University Press and IBH genetics of settlement-inducing bacteria may reveal the Hamer, J. P., Walker, G. & Latchford, J. W. (2001) Settlement of specific qualities of marine biofilms responsible for Pomatoceros lamarkii () larvae on biofilmed surfaces and the effect of aerial drying. Journal of Experimental Marine larval settlement. It would also be most useful to com- Biology and Ecology 260, 113–131 pare biofilm compositions among a group of harbors Hofmann, D. K. & Brand, U. (1987) Induction of metamorphosis in where fouling invertebrates such as H. elegans differ in the symbiotic scyphozoan Cassiopea andromeda:rollofmarine occurrence. bacteria and biochemicals. Symbiosis 4, 99–116 Holm, E. R., Nedved, B. T., Phillips, N., Deangelis, K. L., Hadfield, M. G. & Smith, C. M. (2000) Temporal and spatial variation in ACKNOWLEDGEMENTS the fouling of silicone coatings in Pearl Harbor, Hawaii. Biofouling 15, 95–107 The authors thank A. Alvarez and E. Seaver for their Huang, S. & Hadfield, M. G. (2003) Composition and density of baterial biofilms determine larval settlement of the polychaete support and constructive criticism of the manuscript. We Hydroides elegans. Marine Ecology Progress Series 260, also thank B. Vine for his expertise in DGGE and the rest of 161–172 the Alvarez group for their support and technical help. We Jackson, C. R., Churchill, P. F. & Roden, E. E. (2001) Successional thank B. Nedved for help with microscopy and T. Du Buc changes in bacterial assemblage structure during epilithic for help in the laboratory. This research was supported in biofilm development. Ecology 82,555–566 part by grants to M.G.H. from the Office of Naval Research Jensen, R. B. & Morse, D. E. (1984) Intraspecific facilitation of larval (ONR) (N00014-03-1-0078) and a grant from the Pacific recruitment: gregarious settlement of the polychaete Research Center for Marine Biomedicine (PRCMB), a Phragmatopoma californica (Fewkes). Journal of Experimental Center for Oceans and Human Health (COHH), funded Marine Biology and Ecology 83, 107–126 by the National Institute of Environmental Health Sciences Juck, D., Charles, T., Whyte, L. G. & Greer, C. W. (2000) Polyphasic microbial community analysis of petroleum (NIEHS) and the National Science Foundation (NSF). hydrocarbon-contaminated soils from two northern Canadian communities. FEMS Microbiology Ecology 33, 241–249 Kelly, K. M. & Chistoserdov, A. Y. (2001) Phylogenetic analysis of REFERENCES the succession of bacterial communities in the Great South Bay (Long Island). FEMS Microbiology Ecology 35, 85–95 Amann, R. I., Ludwig, W. & Schleifer, K.-H. (1995) Phylogenetic Keough, M. J. (1998) Responses of settling invertebrate larvae to the identification and in situ detection of individual microbial cells presence of established recruits. Journal of Experimental Marine without cultivation. Microbiological Reviews 59, 143–169 Biology and Ecology 231,1–19 Boettcher, K. J. & Ruby, E. G. (1990) Depressed light emission by Keough, M. J. & Raimondi, P. T. (1995) Responses of settling symbiotic Vibrio fisheri of the sepiolid Euprymna scolopes. invertebrate larvae to bioorganic films: effects of different types Journal of Bacteriology 172, 3701–3706 of films. Journal of Experimental Marine Biology and Ecology Carpizo-Ituarte, E. & Hadfield, M. G. (1998) Stimulation of 185, 235–253 metamorphosis in the polychaete Hydroides elegans Haswell Kisand, V. & Wikner, J. (2003) Limited resolution of 16S rDNA (Serpulidae). Biological Bulletin 194, 12–24 DGGE caused by melting properties and closely related DNA Chalmer, P. N. (1982) Settlement patterns of species in a marine sequences. Journal of Microbiological Methods 54, 183–191 fouling community and some mechanisms of succession. Lau, S. C. K. & Qian, P. Y. (2002) Bioactivity of bacterial strains Journal of Experimental Marine Biology and Ecology 8,73–85 isolated from marine biofilms in Hong Kong waters for the Costerton, J. W. & Stewart, P. S. (2001) Battling biofilms. Scientific induction of larval settlement in the marine polychaete American 285,74–81 Hydroides elegans. Marine Ecology Progress Series 226, 301–310 Costerton, J. W., Stewart, P. S. & Greenberg, E. P. (1999) Bacterial Lau, S. C. K., Thiyagarajan, V., Cheung, S. C. K. & Qian, P. Y. (2005) biofilms: a common cause of persistent infection. Science 284, Roles of bacterial community composition in biofilms as a 1318–1322 mediator for larval settlement of three marine invertebrates. Crisp, D. J. (1974) Factors influencing settlement of marine Aquatic Microbial Ecology 38, 41–51 invertebrate larvae. In Chemoreception in Marine Organisms, Muyzer,G.,Wall,E.C.D.&Uitterlinden,A.G.(1993)Profilingof pp. 177–265. Edited by P. T. Grant & A. M. Mackie. New York: complex microbial populations by denaturing gradient gel Academic Press electrophoresis analysis of polymerase chain reaction-amplified Dahllof, I., Baillie, H. & Kjelleberg, S. (2000) rpoB-based microbial genes coding for 16S rRNA. Applied and Environmental community analysis avoids limitations inherent in 16S rRNA Microbiology 59, 695–700 8 N. J. Shikuma and M. G. Hadfield

Negri, A. P., Webster, N. S., Hill, R. T. & Heyward, A. J. (2001) Unabia, C. R. C. & Hadfield, M. G. (1999) Role of bacteria in larval Metamorphosis of broadcast spawning corals in response to settlement and metamorphosis of the polychaete Hydroides bacteria isolated from crustose algae. Marine Ecology Progress elegans. Marine Biology 133, 55–64 Series 223, 121–131 Vallaeys, T., Topp, E., Muyzer, G., Macheret, V., Laguerre, G., Pawlik, J. R. (1992) Chemical ecology of the settlement of benthic Rigaud, A. & Soulas, G. (1997) Evaluation of denaturing marine invertebrates. Oceanography Marine Biology Annual gradient gel electrophoresis in the detection of 16S rDNA Review 30, 273–335 sequencevariationinrhizobiaandmethanotrophs.FEMS Rao, T. S. (2003) Temporal variations in an estuarine biofilm: with Microbiology Ecology 24, 279–285 emphasis on nitrate reduction. Estuarine Coastal and Self Science Walters, L. J., Hadfield, M. G. & del Carmen, K. A. (1997) The 58, 67–75 importance of larval choice and hydrodynamics in creating Steinberg, P. D., De Nys, R. & Kjelleberg, S. (2002) Chemical cues aggregations of Hydroides elegans (Polychaeta: Serpulidae). for surface colonization. Journal of Chemical Ecology 28, Invertebrate Biology 116, 102–114 1935–1951 Webster,N.S.,Smith,L.D.,Heyward,A.J.,Watts,J.E.M.,Webb, Toonen, R. J. & Pawlik, J. R. (1996) Settlement of the tube worm R. I., Blackall, L. L. & Negri, A. P. (2004) Metamorphosis of a Hydroides dianthus (Polychaeta: Serpulidae): cues for gregarious scleractinian coral in response to microbial biofilms. Applied settlement. Marine Biology 126, 725–733 and Environmental Microbiology 70, 1213–1221