Temperature-Dependent Inhibition of Opportunistic Vibrio Pathogens by Native Coral Commensal Bacteria

Microb Ecol (2014) 67:392–401 DOI 10.1007/s00248-013-0334-9

ENVIRONMENTAL MICROBIOLOGY

Temperature-Dependent Inhibition of Opportunistic Vibrio Pathogens by Native Coral Commensal Bacteria Beck R. Frydenborg & Cory J. Krediet & Max Teplitski & Kim B. Ritchie

Received: 1 February 2013 / Accepted: 18 November 2013 / Published online: 27 December 2013 # Springer Science+Business Media New York 2013

Abstract Bacteria living within the surface mucus layer of corals compete for nutrients and space. A number of stresses affect the outcome of this competition. The interactions between native microorganisms and opportunistic pathogens largely determine the coral holobiont's overall health and fitness. In this study, we tested the hypothesis that commensal bacteria isolated from the mucus layer of a healthy elkhorn coral, Acropora palmata, are capable of inhibition of opportunistic pathogens, Vibrio shiloi AK1 and Vibrio coralliilyticus. These vibrios are known to cause disease in corals and their virulence is temperature dependent. Elevated temperature (30 °C) increased the cell numbers of one commensal and both Vibrio pathogens in monocultures. We further tested the hypothesis that elevated temperature favors pathogenic organisms by simultaneously increasing the fitness of vibrios and decreasing the fitness of commensals by measuring growth of each species within a co-culture over the course of 1 week. In competition experiments between vibrios and commensals, the proportion of Vibrio spp. increased significantly under elevated temperature. We finished by Electronic supplementary material The online version of this article (doi:10.1007/s00248-013-0334-9) contains supplementary material, which is available to authorized users. B. R. Frydenborg Microbiology and Cell Science Department, University of Florida-IFAS, Gainesville, FL 32611, USA C. J. Krediet : M. Teplitski Interdisciplinary Ecology Graduate Program, University of Florida-IFAS, Gainesville, FL 32611, USA M. Teplitski : K. B. Ritchie Soil and Water Science Department, University of Florida-IFAS, Gainesville, FL 32611, USA B. R. Frydenborg : K. B. Ritchie (*) Mote Marine Laboratory, Sarasota, FL 34236, USA e-mail: [email protected]

investigating several temperature–dependent mechanisms that could influence co-culture differences via changes in competitive fitness. The ability of Vibrio spp. to utilize glycoproteins found in A. palmata mucus increased or remained stable when exposed to elevated temperature, while commensals' tended to decrease utilization. In both vibrios and commensals, protease activity increased at 30 °C, while chiA expression increased under elevated temperatures for Vibrio spp. These results provide insight into potential mechanisms through which elevated temperature may select for pathogenic bacterial dominance and lead to disease or a decrease in coral fitness.

Introduction Known as the “coral holobiont”, corals consist of an invertebrate host, endosymbiotic dinoflagellates (Symbiodinium spp.), and microorganisms including bacteria, archaea, fungi, and viruses associated with both the polyp and the dinoflagellates [1]. Evidence points to co-evolution of these groups, as different coral species exhibit unique microbial communities and host specific clades of Symbiodinium [1, 2]. Corals secrete mucus, which contains a mixture of glycoproteins (carbohydrate moieties which are from photosynthate via dinoflagellate photosynthesis). This surface mucus layer serves to protect the coral from desiccation, sedimentation, and UV damage [3, 4]. The glycoproteins that compose the mucus provide habitat and nutrients to resident microbiota [5, 6]. Under optimal environmental conditions, coral commensal bacteria are capable of producing antimicrobials, cell-to-cell communication inhibitors, and other compounds that interfere with swarming, biofilm formation, and ability of pathogens to utilize specific carbon sources present in mucus [7–11]. Mucus acts as a medium in which secreted allelochemicals (produced by the members of the holobiont) are thought to accumulate. Previous studies have shown that up to 70 % of culturable

Mechanisms of Temperature-Dependent Coral Diseases

bacteria isolated from healthy coral colonies display some type of antimicrobial or inhibitory activity [9, 12–16]. Widespread loss of corals caused by diseases following abnormal sea surface temperature events is now a well documented phenomenon [17]. Multiple stressors, including temperature, affect the composition of coral microbial communities [18]. Perturbations to environmental conditions shift the resident population in coral mucus from commensal to pathogendominated [16–18]. Prolonged stressful conditions result in an increase in opportunistic bacterial infections of corals [19]. Recent metagenomic studies found that elevated temperatures increase the prevalence of the virulence genes within microbial communities similar to stress from changes in dissolved organic carbon, pH, and nutrient levels [18]. Elevated temperatures are associated with an increase in Vibrio abundance not only in corals but also in other environments [18]. Examples include temperature-dependent increases of V. cholerae outbreaks in Africa and India and encroachments of V. parahaemolyticus and V. vulnificus into previously cooler regions [20–22]. It is likely that elevated temperatures expand the habitat of these pathogens, which may act as one of many factors to increase the incidence of infection. The particular mechanisms of the temperature-mediated shifts in microbial communities preceding disease are not well understood. It is reasonable to hypothesize that to cause disease in corals, pathogens first establish within or on the coral and then dominate the microbial community. Virulence genes found in association with temperature-stressed corals include chitinases, proteases, and those involved in numerous secretion and iron sequestration pathways [18]. The recently sequenced genomes of Vibrio coralliilyticus VC450 and VCP1 contain an array of virulence factors that are temperature dependent [23]. In this study, we tested the influence of elevated temperature on the interactions between commensal bacteria from Acropora palmata and two model coral opportunistic pathogens, and examined potential mechanisms of temperaturedependent inhibition of pathogens mediated by commensals. Two well characterized isolates from mucus of apparently asymptomatic A. palmata were chosen: Planococcus sp. 34D8 and Photobacterium mandapamensis 34E11 [16]. Because many coral diseases are not well characterized and lack a known causative agent, we used known coral pathogens V. coralliilyticus and Vibrio shiloi AK1. V. shiloi AK1 has long been associated with bleaching of Mediterranean corals and V. coralliilyticus was originally associated with white syndrome of Indo-Pacific corals [24, 25]. Both Vibrio spp. have been found in disease lesions of A. palmata as well as healthy areas on diseased A. palmata and surrounding sediment [26]. Vibrio spp. are often associated with bleaching and other coral diseases and syndromes, and V. coralliilyticus has a worldwide distribution and has demonstrated temperature-dependent pathogenicity [16, 27–29].

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A variety of models have been advanced to explain the mechanisms behind temperature-dependent microbial community shifts, including overgrowth by opportunistic pathogens, loss of the ability of commensals to block opportunistic pathogens, or a combination of the two [30–32]. A recent model proposes a tight competition between commensal bacteria and opportunistic pathogens, with warmer temperatures tipping the balance to pathogen dominance [33]. Mechanisms were investigated by quantifying each bacterial strain's temperature-dependent substrate usage, protease production, and chinitanse A (chiA) regulation under each experimental condition. The substrates chosen are representative of the most common glycoside bonds in the Acropora mucus glycoprotein [34]. Protease and chiA expression were specifically investigated due to their known importance in V. coralliilyticus virulence, and because one of the common substrates in A. palmata mucus, N-acetyl-D -glucosaminidase, is the monomer of chitin [35].

Materials and Methods Collection of Bacterial Strains and Coral Mucus Bacteria were originally isolated in 2006 from A. palmata mucus taken from 12 visually asymptomatic colonies in Looe Key Reef (24° 32.76′ N, 81° 24.21′ W, and 24° 32.75′ N, 81° 24.35′ W). Coral isolates from glycerol stocks were identified by amplifying partial 16S rDNA fragments with primers 8 F (pA) 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1489R 5′TACCTTGTTACGACTTCA-3′ [36, 37]. Fragments were cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA, USA), sequenced at the University of Florida Biotechnology Core Facility, and identified with the BLAST algorithm against the NCBI (GenBank) non-redundant database. Two isolates identified as Planococcus sp. 34D8 and P. mandapamensis 34E11 were used as representative coral commensals. V. shiloi AK1 was obtained from ATCC, and V. coralliilyticus was kindly provided by James Cervino (Pace University). All strains matched previously submitted data (accession numbers JF346760, JF346761, and HM771346). A. palmata mucus was collected at Looe Key Reef from asymptomatic A. palmata colonies using a 60-ml syringe as previously described [16]. Mucus was transferred to 50-ml Falcon tubes and frozen for transportation to the lab and stored at −20 °C. Thawed mucus was filtered through a glass fiber filter to remove large particles before filtration through a 0.22-μm MCE filter (Fisher Scientific, Pittsburgh, PA, USA). Mucus was stored at 4 °C after filtration. High molecular weight (HMW) mucus was prepared by fractionation in a VivaSpin 15 (5000 MWCO) spin dialysis column (Sartorius, Goettingen, Germany) for 1 h at 3,500×g as previously described [38]. The HMW fraction was brought up to the

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original volume with filter sterilized artificial seawater (FSW). Total mucus was used for growth assays, and HMW mucus was used for substrate utilization assays. Bacterial Growth and Competition We first examined the effect of temperature on the growth of individual bacteria, followed by co-culture experiments containing commensal and Vibrio strains. Experiments were performed using both a standard marine bacterial media (marine broth) and an environmentally relevant coral-derived medium (A. palmata mucus). Unless otherwise noted, strains were grown overnight in 5 ml of Marine Broth (BD Biosciences, Franklin Lakes, NJ, USA). An overnight culture of each individual isolate was grown at 25 and 30 °C for 14 to 16 h to log phase. The OD600 was measured and cultures were standardized using sterile marine broth. Serial dilutions to 104 CFU ml−1 were made from the overnight culture in FSW. Three biological replicate cultures were inoculated at 102 CFU ml−1 for each strain, which were grown at 25 and 30 °C. Colony counts were performed at the start of the assay and then at 1, 3, 5, and 7 days postinoculation. Serial dilutions of the cultures were performed and 50 μl of the appropriate dilution was spread onto marine agar plates in triplicate and incubated at 30 °C to calculate colony forming units per milliliter. A series of co-inoculation experiments between coral commensal bacteria and Vibrio pathogens were performed in both marine broth and filter-sterilized A. palmata mucus at 25 and 30 °C. Dilutions to 104 CFU ml−1 were made from an overnight culture in FSW and commensal and Vibrio strains were mixed 1:1. Time points were taken in the same manner as monoculture experiments. Vibrios were distinguished from coral commensals by patching onto thiosulfate-citrate-bile salts-sucrose agar (TCBS) plates (Fisher Scientific, Pittsburgh, PA, USA). In co-culture experiments, a difference of more than three orders of magnitude between a commensal and Vibrio could not be detected due to limitations with the dilution and spread plating protocol. We conducted six biological replicates for each time point. Glycosidase Assays Two overnight cultures of each isolate were grown in 5 ml of marine broth for approximately 16 h at both 25 and 30 °C. Cells were then transferred to microcentrifuge tubes, pelleted, and washed three times in filter-sterilized FSW before being resuspended in 5 ml of FSW. Bacteria were starved for 3 days in FSW while shaking at 25 or 30 °C. To test carbon substrate usage, 1 ml of starved cells was combined with 2 ml of filtersterilized A. palmata HMW mucus. As a negative control, FSW replaced the cell suspension. Cells were incubated in mucus for 2 and 18 h at the appropriate temperature and population densities were measured (OD590). Following

B.R. Frydenborg et al.

previously established protocols [39], chromogenic p -nitrophenyl substrates were mixed with lysed cells, and the reactions were incubated for exactly 24 h. Cellular debris, unused substrate, and salts were pelleted by centrifugation. Reactions were stopped with 1 M Na2CO3 before supernatant was transferred to a clear polystyrene 96-well plate and the absorbance at 405 nm (A405) was read using a VICTOR3 plate reader (PerkinElmer, Waltham, MA, USA). Activity was calculated using modified Miller Units (A405/OD590). Protease Assays In order to measure virulence-associated activities, a quantitative protease assay was adopted [40]. Briefly, three replicates of an overnight culture of each strain were grown in marine broth at 25 and 30 °C. The OD600 of each culture was measured and serial dilutions plated in order to determine cell number (colony forming units per milliliter). The culture was pelleted at 12,000×g for 20 min, and the resulting supernatant was filter sterilized through 0.22-μm syringe filters. The remaining pellet was washed with FSW, followed by resuspension in 10 ml sterile deionized (DI) water to lyse cells. Supernatant was concentrated in VivaSpin 15 columns with a 5,000 molecular weight cutoff. Columns were placed in 50-ml Falcon tubes and centrifuged at 2,500×g for 30 min. The concentrated supernatant was brought up to 1 ml in FSW. One hundred microliters of concentrated cell free supernatant or lysed pellet was combined with 100 μl of azocasein solution (5 mg ml−1 in 50 mM tris–HCl, pH 8.0 with 0.4 % sodium azide) and incubated in microcentrifuge tubes at 37 °C for 60 minutes. Sterile media was used as a negative control, and 1 mg ml−1 trypsin in 1× PBS solution was used as a positive control. The reaction was stopped by adding 500 μl of 8 % (wt/ vol) trichloracetic acid and was incubated at room temperature for 2 minutes before centrifugation at 12,000×g for 4 minutes. The resulting supernatant was added to a new tube containing 700 μl of 525 mM NaOH to intensify color, and 200 μl of the mixture was transferred to a 96-well clear, polystyrene, flat bottom plate. Absorbance was read at 450 nm with a VICTOR3 plate reader (PerkinElmer, Waltham, MA, USA). Protease activity units were calculated with the formula: 1 protease activity unit=[1,000*A450 /CFU]*109. Quantitative PCR Accumulation of the chitinase A chiA transcript (a known virulence factor) was characterized in the two Vibrio spp. at 25 and 30 °C using known housekeeping genes 16S rRNA, recA, and rpoA genes as a reference [41]. Primer pairs were designed to amplify 180 to 240 base pairs, with a melting temperature of 60 °C (Table S1). RNA isolation was performed from at least two independent overnight cultures per strain with the Ambion RNAqueous Total RNA Isolation Kit

Mechanisms of Temperature-Dependent Coral Diseases

(Ambion, Carlsbad, CA, USA) and eluted with DNA grade water (Fisher Scientific, Pittsburgh, PA, USA). An Ambion DNA-free kit was used to treat samples immediately following RNA isolation, and the isolation yield and purity were determined using a N1000 NanoDrop (Thermo Fisher Scientific, Wilmington, DE, USA). Uniform concentrations of cDNA were created by modifying the amount of RNA added to the iScript Select cDNA Synthesis Kit reaction (Bio-Rad, Hercules, CA, USA), which used random primers. A 20 μl reaction consisting of 10 μliQ SYBR Green Supermix (BioRad, Hercules, CA, USA), 8 μl of primer probes (producing a final concentration of 30 nM), and 2 μl of cDNA (for a final concentration of 30 ng μl−1) was added to each well of a twin.tec 96-well PCR plate (Eppendorf, Hauppauge, NY, USA). Samples were run on an iCycler IQ™ Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA) with cycle consisting of 50 °C for 2 minutes, 95 °C for 30 s, and 95 °C for 10 minutes, followed by 45 repeats of 95 °C for 15 s and 60 °C for 1 minute. C T values were used to calculate a range of fold change using the comparative C T method [42].

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Results Monoculture Growth Characterization Under Elevated Temperature in A. palmata Mucus For monocultures grown in A. palmata mucus, the overall time–temperature interaction was significant for V. shiloi, V. coralliilyticus , and P. mandapamensis 34E11 (Table 1). Monocultures were compared for the effect size of elevated temperature (Fig. 1), which was calculated from the growth curves of monocultures (Fig. S4). For V. shiloi, growth at 30 °C resulted in significantly more bacteria on days 1, 3, 5, and 7 as compared to the strain grown at 25 °C (Fig. 1a). V. coralliilyticus growth was significantly greater on days 1 and 3, but not significantly different than growth at 25 °C by days 5 and 7 (Fig. 1b). For P. mandapamensis 34E11, growth at elevated temperature resulted in significantly and increasingly greater cell densities on days 3, 5, and 7 (Fig. 1c). Growth of Planococcus sp. 34D8 was not significantly affected by elevated temperature (Fig. 1d). Co-culture Growth Characterization Under Elevated Temperature in A. palmata Mucus

Statistical Analysis All statistical analyses were performed with SAS 9.3 (Cary, NC, USA). Colony forming unit counts over the course of 1 week were used to compare the temperature-dependent outcomes of monoculture and co-culture experiments. Data were tested for normality and homoscedacity, and were log transformed (colony forming units per milliliter data) or arcsin-square root transformed (percentage data). For monocultures, colony forming units per milliliter was used as the response, and for co-cultures, the percent of Vibrio spp. was used. Effect size was calculated as the difference between elevated temperature (30 °C) and baseline temperature (25 °C) means, and the 95 % confidence interval determined using the standard error for all time points specific to the inoculums. Effect size estimates the magnitude of effect by quantifying the difference between the control group (25 °C) and treatment group (30 °C). A confidence interval is associated with each difference, which together conveys the same information as a statistical significance test, but with emphasis placed on the significance of the effect rather than on sample size. Time series ANOVAs were initially constructed with media type included as a block effect and in a factorial design with temperature and media type (Tables S2 and S3). Due to significant influence of media type, final time series ANOVAs were separated by strain and media type, and temperature was the only effect included. A profile analysis was used to examine the influence of temperature between time points (Table S4). Results of marine broth cultures are discussed in the supplementary material (Figs. S1, S2, and S3).

Time series ANOVAs for the co-inoculations of V. shiloi with Planococcus sp. 34D8, V. coralliilyticus with P. mandapamensis 34E11, and V. coralliilyticus with Planococcus sp. 34D8 were significant for an overall time–temperature interaction (Table 1). Graphs of colony forming units per milliliter from these coinoculations illustrate the temperature-dependent outcomes (Fig. 2). The effect of temperature on each co-culture increased the percentage of vibrios present, although it was not always significant (Fig. S5). For the co-culture of V. shiloi with P. mandapamensis 34E11, commensal density is lowered by an order of magnitude when temperature is elevated, while V. shiloi maintains a consistent density (Fig. 2a and b). There is an increase in vibrio proportion within the co-culture due to elevated temperature on days 3 to 7, but it is only significant on day 3 (Fig. S5 a). This same pattern repeats for the co-culture of V. shiloi with Planococcus sp. 34D8 (Fig. 2c and d). The effect of elevated temperature on vibrio growth is significant on days 3, 5, and 7, showing an increase in the percent vibrios present within the co-inoculation due to elevated temperature (Fig. S5 b). In the co-inoculation of V. coralliilyticus with P. mandapamensis 34E11 showed a decrease in commensal growth at the elevated temperature (Fig. 2e and f). Effect size was positive and significant at days 1 and 5 (Fig. S5 c). The co-culture of V. coralliilyticus with Planococcus sp. 34D8 showed a clear temperature dependence, with equal growth at 25 °C transitioning to a two orders of magnitude difference at 30 °C (Fig. 2g and h). For this co-culture, the effect of elevated temperature was significant for days 1, 3, 5, and 7 (Fig. S5 d).

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Table 1 Time series ANOVAs in mucus and marine broth with temperature as the main effect. The overall effect of the temperature and time interaction is reported as the univariate F ratio, and p values are corrected with the Greenhouse–Geisser correction factor. The degrees of freedom (df) are presented as (n −1, error) Acropora palmata total mucus

Marine broth

Strains

df

F ratio

p value

Strains

df

F ratio

p value

Vs Vc D8 E11 Vs/D8 Vs/E11 Vc/D8 Vc/E11

4,16 4,8 4,8 4,16 4,24 4,32 4,36 4,36

58.2 12.31 11.76 14.81 7.14 0.86 10.08 5.6

0.0005 0.0303 0.055 0.0087 0.0171 0.4159 0.0005 0.0219

Vs Vc D8 E11 Vs/D8 Vs/E11 Vc/D8 Vc/E11

4,16 4,16 4,8 4,16 4,24 4,36 4,28 4,32

5.4 19.27 110.64 1.18 1.83 5.96 3.28 13.1

0.0565 0.0004 0.0028 0.3575 0.1973 0.0169 0.1101 0.001

Vs Vibrio shiloi, Vc Vibrio coralliilyticus, D8 Planococcus sp., E11 Photobacterium mandapamensis

Temperature Affects Substrate Usage and Enzymatic Activity Enzymatic activities corresponding to 11 substrates abundant in A. palmata mucus were measured for each strain at 25 and 30 °C. Independent measurements were taken at 2 h (early) and 18 h (late) to capture changes in enzymatic activities (Table 2). Unless otherwise noted, reported changes in enzymatic activity due to elevated temperature were consistent (both increased or decreased from baseline) for early and late time points. Elevated temperature resulted in Planococcus sp. 34D8 enzyme activity decreasing by twofold or greater for Nacetyl-D-glucosaminidase, α-D-galactopyranosidase, α-DFig. 1 The effect size of temperature for strains grown as a monoculture in A. palmata mucus. Error bars are at associated 95 % confidence intervals. Graphs are of V. shiloi (a), V. coralliilyticus (b), P. mandapamensis 34E11 (c), and Plannococcus sp. 34D8 (d)

glucopyranosidase, ß-D-glucopyranosidase, α-Larabinopyranosidase, ß-L-arabinopyranosidase, α-Lfucopyranosidase, ß-D-fucopyranosidase, and ß-Dmannopyranosidase. Enzymatic activity of P. mandapamensis 34E11 decreased by twofold or greater for ß-Dgalactopyranosidase, α-D-glucopyranosidase, ß-Dglucopyranosidase, α-L-arabinopyranosidase, ß-Larabinopyranosidase, and ß-D-fucopyranosidase under elevated temperature. Elevated temperature also caused enzyme activity of α-D-galactopyranosidase and α-Lfucopyranosidase to increase for P. mandapamensis 34E11 (Table 2). When V. shiloi was subjected to elevated temperature, a twofold or greater decrease in enzymatic activity was observed for α-D-galactopyranosidase, ß-D-glucopyranosidase, ß-Larabinopyranosidase, α-D-mannopyranosidase, and ß-Dmannopyranosidase. Elevated temperature also caused a twofold or greater decrease in activity for α-L-arabinopyranosidase and ß-D-fucopyranosidase at 2 h. V. coralliilyticus enzyme activity decreased for α-D-galactopyranosidase and ß-Dgalactopyranosidase with elevated temperature. Activity of Nacetyl-D-glucosaminidase was decreased at 2 h only, while αL-fucopyranosidase experienced a twofold decrease only at 18 h under elevated temperature. Enzymatic activity of V. coralliilyticus increased for α-D-glucopyranosidase, ß-Dglucopyranosidase, and ß-L-arabinopyranosidase at 30 °C (Table 2). Overall, elevated temperature decreased glycosidase activity for commensals, and V. shiloi. V. shiloi , and Planococcus sp. 34D8 experienced no increased activity with elevated temperature. Though P. mandapamensis 34E11 experienced decreased activity for several substrates, it also displayed increased activity for two. V. coralliilyticus displayed

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Fig. 2 Growth curves of coinoculations in A. palmata mucus. Vibrio–commensal competition was quantified in sextuplicate by total and TCBS CFU ml−1 counts at 25 and 30 °C over 1 week. Figure abbreviations for strains: Vs Vibrio shiloi AK1, Vc Vibrio coralliilyticus, D8 Planococcus sp. 34D8, Photobacterium mandapamensis 34E11 (E11). Co-inoculations are Vs/E11 at 25 °C (a) and 30 °C (b), Vs/D8 at 25 °C (c), and 30 °C (d), Vc/ E11 at 25 °C (e) and 30 °C (f), and Vc/D8 at 25 °C (g) and 30 °C (h). This plating method allowed for a difference of no more than three orders of magnitude in growth difference to be distinguished. This occurred in b, day 3, when no commensal was detected

an overall high glycosidase activity, with increased activity for several substrates at 30 °C, though there was also a decrease in activity for others (Table 2). Elevated Temperature Increases Protease Activity and Upregulates Chitinase Protease activity was measured for both a suspension of lysed cells (Fig. S6a) and for cell-free supernatant (Fig. S6b) for each strain. The A. palmata commensal isolates showed little protease activity at 25 °C, with only extracellular protease

activity for Planococcus sp. 34D8 (12.12±4.94, mean ± S.D.) detected. Growth at elevated temperature increased extracellular and cell-associated supernatant protease activity for both commensal isolates. Planococcus sp. 34D8 cell-free supernatant increased by 9.9-fold to 119.85 ± 9.66, while P. mandapamensis 34E11 had a fold change increase of 64.9 for cell free supernatant and 52.2 for lysed cell suspension. At 25 °C, V. coralliilyticus had consistently high protease activity for both cell-free supernatant (166.84±7.02, mean ± S.D.) and lysed cell suspension (130.54±8.82). This activity increased with temperature by 1.8-fold for cell-free

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Table 2 Utilization of carbon substrates. A twofold decrease in usage is indicated by bold, while italics indicate a twofold increase in usage. All data are Miller Units ± SD 25 °C

30 °C

2h

18 h

2h

18 h

N-Acetyl-β-D -glucosaminidase α-D -Galactopyranosidase β-D -Galactopyranosidase α-D -Glucopyranosidase β-D -Glucopyranosidase α-L -Arabinopyranosidase β-L -Arabinopyranosidase α-L-fucopyranosidase β-D-fucopyranosidase α-D-mannopyranosidase β-D-mannopyranosidase Vibrio corallylliticus N-Acetyl-β-D -glucosaminidase α-D -Galactopyranosidase β-D -Galactopyranosidase α-D -Glucopyranosidase β-D -Glucopyranosidase α-L -Arabinopyranosidase

98.37±4.86 7.40±0.90 50.23±3.52 112.68±8.95 12.73±1.37 6.08±0.63 4.48±0.40 1.54±0.20 6.97±0.73 5.23±0.68 1.95±0.10

138.64±29.98 14.70±1.740 70.05±10.22 149.82±21.52 20.60±1.95 5.92±0.70 10.98±1.40 1.00±0.18 5.05±0.85 0.02±0.02 7.56±1.10

162.20±2.95 0.65 ±0.05 49.97±0.19 159.64±2.41 5.16 ±0.08 2.29 ±0.11 0.62 ±0.22 0.36 ±0.07 2.92 ±0.09 0.49 ±0.038 0.70 ±0.07

147.05±7.30 0.86 ±0.07 45.46±6.43 157.72±7.89 5.03 ±1.03 3.29±0.67 0.45 ±0.04 0.31 ±0.02 4.06±1.66 0.53 ±0.08 0.99 ±0.30

71.32±7.31 7.66±1.46 13.46±5.06 1.68±0.14 0±0 0.49±0.10

49.13±5.14 5.66±0.38 2.34±0.91 0±0 0±0 0.43±0.01

18.55 ±1.95 1.37 ±0.01 0.25 ±0.14 20.10 ±2.00 1.33 ±0.06 0.75±0.08

36.843±4.08 1.18 ±0.24 0 ±0 43.70 ±5.86 0.73 ±0.04 1.33±0.19

β-L -Arabinopyranosidase α-L-fucopyranosidase β-D-fucopyranosidase α-D-mannopyranosidase β-D-mannopyranosidase Planococcus sp. 34D8 N-Acetyl-β-D -glucosaminidase α-D -Galactopyranosidase β-D -Galactopyranosidase α-D -Glucopyranosidase β-D -Glucopyranosidase α-L -Arabinopyranosidase β-L -Arabinopyranosidase α-L-fucopyranosidase β-D-fucopyranosidase α-D-mannopyranosidase β-D-mannopyranosidase Photobacterium mandapamensis 34E11

0.25±0.042 1.15±0.30 0.34±0.04 1.44±0.39 0.53±0.10

0.09±0.04 1.62±0.18 0.30±0.15 0.61±0.12 0.46±0.05

1.39 ±0.05 0.61±0.12 0.58±0.02 0.81±0.21 0.60±0.02

1.70 ±0.32 0.13 ±0.09 1.72±0.73 1.00±0.55 0.55±0.07

3.35±1.20 7.87±0.99 0.26±0.11 19.32±1.79 5.51±0.72 4.48±0.70 3.26±0.37 3.45±1.90 3.05±1.66 0.39±0.060 3.42±1.80

3.46±0.20 6.97±0.08 0.87±0.04 4.00±0.05 4.07±0.14 1.83±0.27 1.67±0.41 0.46±0.15 0.84±0.15 0.79±0.08 0.95±0.02

0.91 ±0.08 0.08 ±0.05 0±0 0.74 ±0.13 0.23 ±0.04 0.67 ±0.07 0.05 ±0.03 0 ±0 0.15 ±0.08 0.29±0.22 0.18 ±0.06

1.45 ±0.16 0.17 ±0.03 0.08±0.03 1.69 ±0.07 0.88 ±0.11 0.28 ±0.04 0.44 ±0.13 0.11 ±0.02 0.10 ±0.01 0.62±0.46 0.16 ±0.03

202.54±9.31 5.13±0.45 5.09±1.01 198.11±8.70 3.64±0.47 3.70±0.22

222.34±3.75 4.90±0.22 8.14±0.66 220.61±5.30 4.37±0.42 5.61±0.38

330.88±152.80 17.75 ±3.71 1.07 ±0.34 22.52 ±8.07 1.15 ±0.72 1.58 ±0.35

288.72±116.81 0±0 0.60 ±0.09 0 ±0 0 ±0 0.84 ±0.09

Vibrio shiloi AK1

N-Acetyl-β-D -glucosaminidase α-D -Galactopyranosidase β-D -Galactopyranosidase α-D -Glucopyranosidase β-d-Glucopyranosidase α-L -Arabinopyranosidase

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Table 2 (continued) 25 °C

β-L -Arabinopyranosidase α-L-fucopyranosidase β-D-fucopyranosidase α-D-mannopyranosidase β-D-mannopyranosidase

30 °C

2h

18 h

2h

18 h

5.76±0.34 0.82±0.08 2.67±0.35 3.29±1.72 2.70±0.16

8.81±0.09 1.24±0.07 3.47±0.08 0+0 3.77±0.07

5.53±2.02 2.72 ±0.47 1.22 ±0.34 1.74±0.30 2.79±0.93

1.05 ±0.10 3.77 ±1.27 1.15 ±0.24 1.33±0.47 1.74±0.59

Enzymatic activities that increased (italics) or decreased (bold) at least twofold after incubation (2 or 18 h) with the high molecular weight fraction of mucus from A. palmata are indicated

supernatant and 1.9–fold for lysed cells. V. shiloi did not demonstrate a high level of protease activity at either temperature. At 25 °C, cell-free supernatant was 3.14±1.26 and lysed cell suspension showed no activity. At 30 °C, V. shiloi protease activity increased 9.1-fold for cell-free supernatant, and the activity associated with lysed cells remained below detection. When transcript accumulation of chiA was compared between baseline conditions (25 °C) and elevated temperature (30 °C), V. shiloi showed an increased fold change of 0.74. These same conditions caused V. coralliilyticus to increase chiA expression by 1.65-fold (Fig. S7).

Discussion Models of the microbial dynamics within the holobiont are limited by a lack of mechanistic understanding. Multiple studies demonstrate the existence of inhibitory bacteria found in corals [9, 10, 16, 43]. When known coral pathogens are examined for their susceptibility to inhibition by a coral commensal, V. shiloi is inhibited by 6–12 %, V. coralliilyticus is inhibited by 3 %, and 8 % show an antagonistic response towards Serratia marcescens [7–9]. While antibiotic production may be a mechanism for opportunistic pathogen suppression, these low suppression rates in coral commensal bacteria suggest that other inhibitory mechanisms are involved. Vibrios are known to display increased growth rates under elevated temperatures, and communitywide population shifts to Vibrio spp. during times of coral thermal stress are well documented [16, 18]. In agreement with these observations, monoculture experiments with V. shiloi and V. coralliilyticus had significantly greater growth at 30 °C than 25 °C. We initially hypothesized that commensals would either respond negatively to elevated temperature or maintain similar growth to that at 25 °C. This hypothesis was confirmed for Planococcus sp. 34D8, which in monoculture was not significantly affected by elevated temperature. In co-culture experiments with the vibrios, abundance of Planococcus sp. 34D8 was lowered under elevated temperature while vibrios increased. This observation supports the hypothesis that greater vibrio growth at higher

temperatures leads to vibrio dominance [44]. In addition to this temperature effect on growth, we found lowered substrate utilization by Planococcus sp. 34D8 along with increases in both protease activity and chiA expression in vibrios under elevated temperature. Protease activity of Planococcus sp. 34D8 did increase under elevated temperature, though the conferred competitive advantage was not able to overcome the vibrio growth. These findings show that temperature influences the competition between the commensal and vibrios in favor of the vibrio through a variety of mechanisms. In contrast to the growth of Planococcus sp. 34D8, P. mandapamensis 34E11 monocultures displayed a significant increase in growth under elevated temperature. The ability of P. mandapamensis 34E11 to increase growth similar to V. shiloi, and V. coralliilyticus indicates that in co-culture, there should be no difference during elevated temperature due to differences in growth. Conversely, temperatures of 30 °C resulted in a decrease of P. mandapamensis 34E11 abundance. Mechanistic investigations examining usage of the 11 high molecular weight compounds most common to A. palmata mucus found that a key difference in substrate utilization at elevated temperatures was the drastic loss in activity for α-D-glucopyranosidase by P. mandapamensis 34E11, whereas vibrios increased (V. coralliilyticus) or maintained (V. shiloi) strong metabolism of this substrate. Although P. mandapamensis 34E11 produces extracellular and cell-bound proteases at elevated temperature, this does not appear sufficient to overcome the the vibrios at elevated temperature. It should be noted that these experiments were conducted in an artificial environment in order to test specific hypotheses. Metagenomic surveys of coral find the operational taxonomic units to number at least several hundred [45, 46]. With so many different species co-occurring, opportunities for more complex interactions and relationships exist, which would not be determined in a study design such as this. Our microcosms, by necessity, required us to remove mucus from a coral. However, many of the environmental Vibrio strains within the mucus of Oculina patagonica are in a viable but non-culturable state when the mucus is attached to a coral and that detachment of mucus leads to loss of the nonviable state independent of

400

temperature [47]. This mechanism of Vibrio control was, therefore, not present in our microcosms. Bacteriophages also exert influence in naturally occurring populations through “kill the winner” dynamics and can influence competitive advantage through the horizontal transfer of genes. In co-culture experiments, elevated temperature resulted in a significant increase in the abundance of Vibrio compared to our baseline temperature of 25 °C. Correlated with this trend, we found that V. coralliilyticus increased enzymatic activity for multiple coral mucus substrates, increased protease activity, and upregulated chiA with elevated temperature. V. shiloi maintained high enzymatic activity for several substrates and also had an increase in chiA expression with elevated temperature. These results demonstrate increased competitive advantage for these vibrios mediated by elevated temperature. Commensals showed not only decreases in several enzymatic substrates with elevated temperature but also increased protease expression under elevated temperature. Elevated temperature appears capable of increasing some virulence factors of at least these two commensal species, perhaps as they adopt a more pathogenic profile in order to compete with vibrios for resources in a stressed environment [48]. Together, these observations offer insight into additional mechanisms that explain the observed shifts to Vibrio -dominated microbial communities and genetically pathogenic profiles of thermally stressed corals. Acknowledgments We thank the three anonymous reviewers for their helpful criticism and feedback. This research was supported by the Dart Foundation funding to KBR and the “Protect Our Reefs” Grants Program to MT, KBR, and CJK, a Mote Marine Laboratory program which uses funds from statewide sales of specialty license plates to fund peer reviewed research. BF was funded by the American Society for Microbiology Undergraduate Research Fellowship (ASM URF) and by the National Science Foundation Research Experience for Undergraduates (NSF REU0453955). Coral mucus was collected under permit FKNMS-2008–075.

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