J. Phycol. 43, 235241 (2007) r 2007 by the Phycological Society of America DOI: 10.1111/j.1529-8817.2007.00329.

SPORE RELEASE IN ACROCHAETIUM SP. (RHODOPHYTA) IS BACTERIALLY CONTROLLED1

Florian Weinberger2,3
Pierre et Marie Curie-Paris, 6) Marine Plants and Biomolecules and LIA-DIAMS, Station Biologique, UMR 7139 (CNRS, Universite Place Georges Teissier, F-29682 Roscoff Cedex, France

Jessica Beltran, Juan A. Correa

a, Center for Advanced Studies in Ecology & Biodiversity and LIA-DIAMS, Departamento de Ecolog gicas, Ponticia Universidad Cato lica de Chile, Casilla 114-D, Santiago, Chile Facultad de Ciencias Biolo

Ulrich Lion, Georg Pohnert4

kologie, Hans-Kno Max-Planck-Institut fu ll-Str. 8, D-07745 Jena, Germany r Chemische O

Naresh Kumar
School of Chemistry, University of New-South-Wales, Sydney NSW 2052, Australia

Peter Steinberg
School of Biological, Earth and Environmental Sciences, University of New-South-Wales, Sydney NSW 2052, Australia

Bernard Kloareg and Philippe Potin

Pierre et Marie Curie-Paris, 6) Marine Plants and Biomolecules and LIA-DIAMS, UMR 7139 (CNRS, Universite Station Biologique, Place Georges Teissier, F-29682 Roscoff Cedex, France

The facultative red algal epiphyte Acrochaetium sp. liberated spores preferentially and recruited more successfully in laboratory cultures when its host Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira was present. The same effect was also induced by cell-free medium from G. chilensis, suggesting it contained a molecular signal. Antibiotics prevented spore release in Acrochaetium sp., even when G. chilensis was present, suggesting a prokaryotic origin of the signal. Simultaneous application of N-butyl-homoserine-lactone (BHL) restored the spore-release capacity, which demonstrated that spore release was not directly inhibited by the antibiotics and indicated that bacterially generated Nacyl-homoserine-lactones (AHLs) regulate spore release. An involvement of AHL was further indicated by the fact that two different halofuranone inhibitors of AHL receptors also inhibited spore release when they were applied at relatively low concentrations. Of seven different AHLs tested, only BHL induced the effect. However, BHL was only active at relatively high concentrations (100 lM), and it was not detected in spore-release-inducing medium of
Received 28 April 2006. Accepted 2 January 2007. Author for correspondence: e-mail fweinberger@ifm-geomar.de. Current address: IFM-GEOMAR, Du sternbrooker Weg 20, D24105 Kiel, Germany. 4 cole Polytechnique Fe de rale de Lausanne Present address: E (EPFL), Institute of Chemical Sciences and Engeneering, Laboratory for Chemical Ecology, Batochime, CH-1015 Lausanne, Switzerland.
2 3 1

G. chilensis. Another water-soluble AHL or an AHL structure analog is therefore probably the active compound in G. chilensis cultures. The data presented demonstrate that life cycle completion in Acrochaetium sp. strongly depends on bacteria, which are not always present in sufcient numbers on the alga itself. Exogenous bacteria that are associated with G. chilensis or with other potential substrates may therefore trigger timely spore liberation in Acrochaetium sp., provided that the necessary concentration of AHL is reached. This rst nding of AHL perception in a red alga conrms that AHL signalling is more widespread among eukaryotes than was thought until recently. However, spore release of a second red alga, Sahlingia subintegra (Rosenv.) Kornmann, was unaffected by AHL, and the reaction observed is therefore not universal. Key index words: Acrochaetium; epiphytism; Gracilaria; halofuranone; N-acyl-homoserine-lactone; Sahlingia; sporulation Abbreviations: AHL, N-acyl-homoserine-lactone; BHL, N-butyl-homoserine-lactone

Algal spore release is known to be controlled by numerous factors, including light and temperature (Kaliaperumal and Rao 1987, Kaliaperumal 1990, Narasimha Rao and Rangaiah 1991, Ganesan et al. 1999, Guzman-Uriostegui and Robledo 1999, West
235

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FLORIAN WEINBERGER ET AL.

and McBride 1999, Sukumaran and Kaliaperumal 2001, Han et al. 2003), nutrients (Agrawal 1983, Agrawal and Sharma 1994), desiccation (Suto 1952, Infante and Candia 1988), water movement (Gordon and Brawley 2001), or mechanic tissue disruption by grazers (Buschmann and Santelices 1987). Once released, the spore needs to complete a succession of processesinitiated by attachment, followed by adhesion, and ended by germinationbefore achieving a successful settlement (Maggs and Callow 2002). Attachment of Ulva spores has been shown to be mediated by bacteria. Spores of this genus settle preferentially on surfaces that contain N-acyl-homoserinelactones (AHLs) or are covered by bacterial biolms that have the capacity to liberate AHL (Joint et al. 2002, Tait et al. 2005). Naturally occurring AHLs consist of a ve-membered homoserine lactone ring with an amide-linked acyl side chain of variable length (four to 18 carbons), saturated or unsaturated, and with or without a substitute on the C3 carbon (Chhabra et al. 2005). The AHLs are quorum-sensing signals in certain genera of marine and nonmarine gram-negative bacteria (e.g., Pseudomonas, Vibrio) and control the activation of genes involved in biolm development, virulence, and a wide range of other responses (Swift et al. 2001, Withers et al. 2001). Some algae have the capacity to interfere with AHLsignaling (Givskov et al. 1996, Kjelleberg et al. 1997, Borchardt et al. 2001, Teplitski et al. 2004). A particularly well-described model is the red alga Delisea pulchra (Grev.) Mont., which excretes halofuranones (Steinberg et al. 1997, Steinberg and De Nys 2002). These are structural homologs of AHL, bind in a competitive manner to the AHL-receptor-protein complex LuxR (Maneeld et al. 1999), and consequently cause its destabilization and denaturation (Maneeld et al. 2002). A main limiting factor for algal spore settlement is the availability of suitable substrata. Even though epiphytism allows for settlement on substrata that are not accessible to most benthic organisms, epiphytes need to adapt to overcome the defense mechanisms of their hosts. Aquaculture of Gracilaria chilensis is often marred by epiphytism, which decreases both yield and the quality of the extracted agar (Kuschel and Buschmann 1991, Buschmann and Gomez 1993, Westermeier et al. 1993, Buschmann et al. 1997, Leonardi et al. 2006). In this context, and as a common research effort, it was the aim of the EPIFIGHT (Control of epiphytism in Gracilaria chilensis mariculture) consortium of ve partners in Latin America and Europe to identify the mechanisms that control epiphytism in G. chilensis. In order to allow for comparable results, standardized bioassays based on the infection of G. chilensis with Sahlingia subintegra and Acrochaetium sp. were developed in Chile and transferred to the participating laboratories in Europe. Both model epiphytes were chosen because of their suitability for cultivation under laboratory conditions, where large numbers of spores were easily obtained from

stock cultures. Surprisingly, however, only S. subintegra proved to be a continuously reliable ephiphyte under laboratory conditions, while Acrochaetium sp. ceased after some weeks to liberate spores once it had been transferred from Chile to France and Germany. As a result, the need to identify the factors that induced spore release in the latter species became apparent. As will be shown here, spore release in Acrochaetium sp., but not in S. subintegra, is induced by AHL. Moreover, bacteria that were associated with G. chilensis significantly stimulated spore release in Acrochaetium sp. We conclude that this effect was apparently due to release of AHL or structurally similar compounds.
MATERIALS AND METHODS

Algal material. Two strains, CS1 and CR14, of G. chilensis isolated from a commercial sea-based farm at Caldera (northern Chile, 27104 0 S, 70150 0 W) were cultivated at 151C and under a photon ux density of 40 mmol m 2 s 1 in Erlenmeyer asks containing sterile ltrated SFC-enriched seawater medium (Correa and McLachlan 1991). Acrochaetium sp. strain A1-C and S. subintegra strain S1-C were isolated from G. chilensis and maintained in petri dishes under the same conditions. Bioassays. Two different bioassays were conducted in order to quantify the potential of Acrochaetium sp. for settlement and germination on G. chilensis (infection trials) and for spore release (spore release trials). For infection trials, beakers containing 60 mL of seawater medium and glass slides as substrata were inoculated with sporogenic individuals of Acrochaetium sp. (20 40 in different experiments) or with sporogenic tissue of S. subintegra. One centimeter long tips of G. chilensis were added, which had been cut, cultivated for 1 week, carefully brushed, and sonicated for 20 s before being transferred into the beakers with the infective epiphytes. Four days later, the numbers of settled spores along 1 cm long transects of tips and glass slides were counted under a Nikon SMZ-10A stereomicroscope (Nikon Corp., Tokyo, Japan). The fertile epiphytes were discarded, and G. chilensis and the glass slides were incubated for four more days in a clean beaker containing fresh medium. After this second incubation period, spores that had successfully germinated were counted as before. The exact positions of transects counted on G. chilensis could not be marked but were determined by thallus incurvations or other irregularities, which allowed for only few stable tip positions under the microscope. Nonetheless, the transects evaluated during the rst and second counting were not always identical. As a consequence, more germinated than settled spores were detected in some cases, which resulted in percentages of germination 4 100%. In spore release trials, Costar wells (Corning Inc., Corning, NY, USA) containing 5 mL of medium were inoculated with three sporogenic individuals of Acrochaetium sp. or with sporogenic tissue of S. subintegra. After 1 d of incubation on a shaker, the agitation was stopped until unattached spores sunk to the bottom of the well (Carrier 1984), where the number of spores present per area was counted under an inverted microscope (Nikon Corp., Tokyo, Japan). For a pharmacological analysis of spore release, AHLs, halofuranones, and other antibiotics were applied in some spore-release trials. N-butyl-homoserine-lactone (BHL), N-hexyl-homoserine-lactone, N-heptyl-homoserine-lactone, N-octyl-homoserine-lactone, N-decyl-homoserine-lactone, N-dodecyl-homoserine-lactone, and N-tetradecyl-homoserine-lactone, as well as other chemicals used, were from Sigma Ltd. (St Louis, MO, USA). Vancomycin and cefotaxim were added from stock solutions in water, and AHLs from stock solutions in DMSO. Halofuranones C2 [4-bro-

BACTERIAL CONTROL OF SPORE RELEASE

237

mo-5-(bromomethylene)-3-butyl-2(5H)-furanone] and C30 [4-bromo-5-(bromomethylene)- 2(5H)-furanone] were isolated from Delisea pulchra and synthesized, respectively, as described previously (Maneeld et al. 2002, Hentzer et al. 2003). N-butyl-homoserine-lactone analysis. Detection of BHL in seawater samples was performed after solid phase extraction on RP18ec cartridges (Chromabond, Macherey-Nagel GmbH, Du ren, Germany), using liquid chromatography mass spectrometry (LCMS). Separation was carried out on an Agilent HP1100 HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a Grom Sil C18 Column (Alltech-Grom GmbH, Rottenburg-Hailngen, Germany) using a linear gradient of acetonitrile and water 0.5% acetic acid. Identication of AHL was done on the basis of retention times in mass spectra [electrospray ionization (ESI), Finnigan LCQ, Thermo Fisher Scientific Inc., Waltham, MA, USA] compared with the above-mentioned standard.
RESULTS

FIG. 2. Effect of increasing concentrations of N-butylhomoserine-lactone (BHL) on spore release in Acrochaetium sp. Spore settlement on glass substrata was quantied after an exposure of 24 h. Bars indicate minima and maxima; n 5 5. Different letters indicate data that were significantly different in Tukeys test (P<0.05) after Box-Cox transformation.

Presence of 100 mM BHL in the medium induced an increase in the number of spores that were released by Acrochaetium sp. and settled (Fig. 1A, C4), while the other six AHLs did not show any significant effect. Spore release in S. subintegra, on the other hand, was unaffected by any of the AHLs tested (Fig. 1B). Increasing concentrations of BHL resulted in an average increase in spore release, but a significant effect was only observed when BHL was present at the highest-tested concentration of 100 mM (Fig. 2). The

Spores settled [mm2]

100 75 50 25 0 50 40 30 20 10 0

A A A A

spore-release-inducing effect of 100 mM BHL was not significantly reduced when either the C2 or C30 halofuranones were present in the medium at 1 mM, while both of these compounds with structures similar to BHL completely prevented spore release at 10 mM (Fig. 3). Settlement of Acrochaetium sp. spores on glass slides was significantly higher when either of the two G. chilensis strains was present than in their absence (Table 1), with 7.517.5 times more spores released. No such effect was detected with S. subintegra, as spores of this species settled in similar quantities on glass, irrespective of the presence or absence of G. chilensis. Sahlingia subintegra demonstrated a significant settling preference for glass, while similar quantities of Acrochaetium sp. spores settled on the host and on glass. The germination success of both epiphytes on glass was not significantly affected by the presence or absence of G.

Spores settled [mm2]

No C4 C6 C7 C8 C10 C12 C14

AHL present
FIG. 1. Effect of different N-acyl-homoserine-lactones (AHLs; 100 mM) on the spore release in (a) Acrochaetium sp. and (b) Sahlingia subintegra. Labels indicate the length of N-acyl side chains increasing from C4 (N-butyl-homoserine-lactone) to C14 (Ntetradecayl-homoserine-lactone). The spore settlement on glass substrata was quantied after an exposure of 24 h. Bars indicate minima and maxima; n 5 3. Different letters indicate data that were significantly different in Tukeys test (P<0.05) after BoxCox transformation.

FIG. 3. Halofuranones inhibit the inducing effect of N-butylhomoserine-lactone (BHL) on the spore release in Acrochaetium sp. Spore settlement on glass substrata was quantied after 24 h of incubation in the presence of 100 mM BHL and increasing concentrations of halofuranones C2 or C30. Bars indicate minima and maxima; n 5 5. Different letters indicate data that were significantly different in a Nemenyi test (a<0.05).

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FLORIAN WEINBERGER ET AL.

TABLE 1. Settlement (average SD) and germination success (median; minima and maxima in brackets) of spores of Acrochaetium sp. and Sahlingia subintegra in the presence or absence of Gracilaria chilensis strains CS1 or CR14.
Settled spores (cm 1) on Epiphyte tested Gracilaria strain tested Glass Gracilaria Germination success (%) on Glass Gracilaria

Acrochaetium sp. Acrochaetium sp. Acrochaetium sp. S. subintegra S. subintegra S. subintegra

None CS1 CR14 None CS1 CR14

0.4 0.3 5.7 3.6 6.5 5.9 557.8 205.6 333.6 79.4 480.8 145.1

5.5 2.8 7.0 3.9 51 20.2 64.9 21.5

100.0 (50.0100.0) 50.0 (28.60.0) 87.5 (44.4145.5) 67.3 (48.471.4) 75.1 (64.8104.5) 67.2 (32.084.7)

133.3 (73.3210.5) 72.7 (37.592.3) 96.9 (80.0131.6) 81.3 (39.4143.3)

Spores that settled along transects, on glass or on G. chilensis, were counted after 34 d, and germinated spores were counted 3 d later. Bold numbers indicate values on glass in the presence of G. chilensis that were significantly different from values on glass in the absence of G. chilensis (U-test, a<0.05). Italics indicate values on G. chilensis that were significantly different from those on glass and in the presence of G. chilensis (U-test, a<0.05).

chilensis. Spores that settled on strain CS1 germinated at a higher percentage than spores that settled on glass slides, incubated together with this strain. This trend was only significant in the case of Acrochaetium sp. and conrms the nding that G. chilensis strain CS1 is more susceptible to epiphytism than strain CR14 (Table 1). An increased spore release by Acrochaetium sp. was also observed when G. chilensis was absent, but 7-dayold culture medium of G. chilensis was used instead of fresh medium (Table 2). However, similar absolute numbers of spores germinated successfully, irrespective of the number initially released. Neither release nor germination of S. subintegra spores was affected by the use of culture medium of G. chilensis. The spore-release-inducing effect of G. chilensis on Acrochaetium sp. was largely inhibited when either cefotaxim or vancomycin was present in the medium at a concentration of 100 ppm, and the combination of both antibiotics completely prevented the release of spores by the epiphyte (Fig. 4). The presence of 100 mM BHL fully prevented the inhibitory effect of the antibiotics (Fig. 4).
DISCUSSION

Our results show that spore release in Acrochaetium sp. was stimulated by BHL in a dose-dependent manner. Two different AHL-receptor-blocking halofura-

nones fully inhibited the effect of BHL, which suggests that BHL acted through stimulation of such a receptor in Acrochaetium sp. Out of all tested forms of AHL, only BHL induced spore release, which may indicate specicity for BHL. However, BHL is more water soluble than all other tested AHLs, as a consequence of the relatively short N-acyl chain (Joint et al. 2002, Tait et al. 2005). Thus, the lack of effect of AHL may also result from an insufcient availability in the algal medium. Increased recruitment of Acrochaetium sp. was also observed when G. chilensis was present. This effect was not due to a better germination success of spores, as spore germination proved to be unaffected by the presence of G. chilensis. On the contrary, the release and subsequent settlement of spores strongly increased when G. chilensis was present. The same effect was also observed when only culture medium of G. chilensis was added to Acrochaetium sp., suggesting that a chemical cue is present in cultures of the host that triggers spore release in the epiphyte. This molecular signal is likely to be released by bacteria that are associated with G. chilensis, since the antibiotics vancomycin and cefotaxim prevented spore-release induction. These two antibiotics are specific inhibitors of prokaryotic cell wall biosynthesis and do not affect species of the genus Gracilaria (Weinberger et al. 1997). Furthermore, the spore-release-preventing effect of vancomycin and ce-

TABLE 2. Settlement and germination of spores of Acrochaetium sp. and Sahlingia subintegra in the presence or absence of Gracilaria chilensis medium.
Epiphyte tested Medium tested Settled spores (number cm 1) Germinated spores (number cm 1) Germination success (%)

Acrochaetium sp. S. subintegra

Fresh Used Fresh Used

30.1 247.5 159.6 99.6

8.5 64.4 35.7 28.9

23.4 6.9 23.1 12.2 85.2 10.2 68.2 14.3

89.4 (54.5106.9) 9.6 (5.113.0) 53.8 (36.280.0) 84.7 (33.6100.0)

Both epiphytes were incubated in fresh medium or in 1-week-old culture medium of Gracilaria chilensis (50% of strain CS1% and 50% of strain CR14) (denoted as used). Spores that settled along transects on glass were counted after 34 d, and germinated spores were recounted 3 d later. Bold numbers indicate values in used medium that were significantly different from those in fresh medium (U-test, a<0.05).

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239

FIG. 4. Effect of antibiotics on the induction of spore release in Acrochaetium sp. by Gracilaria chilensis. Spore settlement on glass substrates was quantied after 24 h of treatment. With the exception of treatment 1 (control without presence of G. chilensis), sporogenic individuals of the epiphyte were incubated in the presence of a tip of the host (2 mm long) and (2) no antibiotics, (3) 100 ppm cefotaxim, (4) 100 ppm vancomycin, (5) 100 ppm cefotaxim 100 ppm vancomycin, (6) 100 ppm cefotaxim 100 mM N-butyl-homoserine-lactone (BHL), (7) 100 ppm vancomycin 100 mM BHL, (8) 100 ppm cefotaxim 100 ppm vancomycin 100 mM BHL. Bars indicate minima and maxima; n 5 5. Different letters indicate data that were significantly different in Tukeys test (P<0.05) after BoxCox transformation.

fotaxim could be reversed by additional application of BHL, strongly suggesting that the antibiotics acted through prevention of bacterial AHL production. The sensitivity of Acrochaetium sp. toward BHL was, however, relatively low, as about 10100 mM of BHL was necessary for induction of spore release. Interestingly, halofuranones inhibited the effect of BHL at concentrations 10 times lower. Moreover, BHL was not detected in the medium of G. chilensis. We suggest that another water soluble AHL or an AHL analog could be the active principle and that this signal could possibly act at lower concentrations than BHL. The results presented here demonstrate that bacterial production of AHL was, in monocultures of Acrochaetium sp., insufcient for induction of spore release. This lack of AHL was not due to an inhibition of AHL biosynthesis, because sufcient amounts of the signal were obviously generated when a small thallus fragment of G. chilensis was also present. A possible explanation is that only few, if any, AHL-generating bacteria were associated with Acrochaetium sp., while larger quantities were associated with G. chilensis. Epiphytic bacterial communities from seaweeds are not homogenous, and each algal species has its own characteristic ora (Bolinches et al. 1988, Weidner et al. 1996, Ashen and Goff 2000, Meusnier et al. 2001, Robertson et al. 2002). It may well be that such differences exist as well among G. chilensis and Acrochaetium sp. and account for the presence or absence of the specific AHL-type signal. Furthermore, the composition of associated bacterial communities is affected by the age and physiological condition of the algal host as well as by environmental parameters (Cundell et al. 1978, Bolinches et al. 1988, Robertson et al. 2002). Enriched

seawater media can differ in their constituents, as the water used originates from different water bodies. Differences in the readiness of Acrochaetium sp. to liberate spores, as observed among laboratories in Europe and Latin America (see the introduction) may therefore be the result of different abundances of AHL-generating bacteria. Suppression of bacterial epiphytes with antibiotics prevented spore release of Acrochaetium sp. completely, which demonstrates that this alga strongly depends on bacterial AHL. Manipulation of abiotic parameters, such as temperature, light, inorganic nutrients, and desiccation could not activate spore release (data not shown). The microorganisms providing AHL are obviously not always present on Acrochaetium sp., and this may possibly constitute an advantage: exogenous bacteria that are associated with G. chilensis, with other basiphytes, or with nonliving surfaces may trigger spore liberation in Acrochaetium sp., provided that the necessary concentration of AHL accumulates. Sensing of AHL may therefore indicate the availability of suitable substrata to Acrochaetium sp. sporangia in small closed water bodies, such as rock pools, and trigger a timely release of spores when wave action is minor or absent and the danger of drift into less suitable environments is low (Gaylord et al. 2002). The interaction revealed here points to the importance of associated microbial communities in seaweedseaweed interactions. A sustainable management of epiphytes, such as Acrochaetium sp., would probably require selective tools for the specific components of seaweed-associated microbial communities. However, the general understanding of these associations is still very poor. The evidence of response to AHL by a red alga demonstrates that this type of communication between prokaryotes and eukaryotes is widely distributed and not restricted to Ulva sp. Agrawal and Sharma (1994) reported that incubation in culture medium of the green algae Spirogyra decimina (O. F. Mu ll.) Ku tz. and Chaetophora attenuata Hazen induced spore release in Westiellopsis prolifica M. Janet, leaving open the possibility that the effect was also due to AHL. On the other hand, S. subintegra in our study proved to be insensitive to AHL, and the mechanisms observed with Acrochaetium sp. are therefore not universal. The AHLs are nonetheless a promising new tool for phycologists interested in successful completion of algal life cycles under axenic conditions or in instantaneous liberation of large amounts of algal spores.
This study was supported by the European Commission INCO-DEV Programme (contribution number VII of the joint research effort INCO-EPIFIGHT ICA4-CT-200110021). We are grateful to Dr. Sylvain Faugeron (Santiago, Chile) who provided us with G. chilensis.

Agrawal, S. C. 1983. Effects of nutrients present in Bolds basal medium on sporulation of the green alga Pithophora oedogonia (Mont.) Wittrock. Microb. Lett. 93:279.

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