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a Department ofBiology, University of California, Santa Cruz 1156 High Street, SantaCruz, CA 95064
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: eubacteria • galls • Halymeniaceae • insitu hybridization • Prionitislanceolata • redalgae • symbiosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Galls on P. lanceolata are conspicuous in nature, appearingas small light-pink "domes" or "protuberances"and are typically restricted to thalli of the low intertidal andsubtidal zones (Apt and Gibor, 1989).Thalli exposed to high wave or surge action are usually found bearinggalls, the number of which varies per individual. Affected P.lanceolata appear to fall into two groups, with lightly affectedthalli bearing ~1–10 galls each and heavily affectedindividuals bearing less than ~100 each.
Galls on Prionitis were originally described from P.decipiens (Peru) as the parasitic red alga Lobocolaxdeformans Howe (Howe, 1914).Ultrastructural investigation of Lobocolax on P.lanceolata in central California revealed this "parasiticalga" to be a bacterial gall composed of hypertrophied algal cellscontaining aggregations of intercellular bacteria (1 x 2 µm insize) (McBride, Kugrens, and West,1974).
Gall induction and development on P. lanceolata were firstinvestigated by Apt and Gibor (1989), whoused size-fractionated homogenates of galls collected in nature todemonstrate the required presence of a "live infectiousparticle" of bacterial size (0.2–10 µm) for successfullaboratory induction (Apt and Gibor,1989). Wounding of the algal thallus to the medulla, followedby incubation with this homogenate allowed successful gall induction,but the specific microorganism responsible was not isolated or shown tobe the causative agent.
Bacterial gall formation has also been described from a number ofother red algal species, although there is little direct evidence forthe causative roles of any microorganism in gall formation (Andrews, 1977; Apt,1988) This is probably due to the generally recognizeddifficulties encountered when attempting to cultivate symbiotic bacteriaand isolate them in pure culture (Breznak,1984; Smith and Douglas, 1987;Bermudes, Chase, and Margulis, 1988;Apt and Gibor, 1989; Vetter, 1991). To date, only Cantcauzene (1930) has reported thedemonstration of the causative role of an isolated bacterium in algalgall formation.
Recently, the bacterial symbiont associated with gall formation onP. lanceolata, was identified by whole-cell in situhybridization using a fluorescently labeled, species-specific ribosomalRNA-targeted oligodeoxynucleotide probe (S-S-P.l.sym-0949-a-A-25)(Ashen and Goff, 1996). Phylogeneticinference based on 16S rDNA sequence comparisons suggests that thiseubacterium is most closely related to other gall-forming bacterialsymbionts from the genus Prionitis and the marine algalepiphyte isolated from Japan, Roseobacter denitrificans (alphasubclass of the Proteobacteria) (Shiba,1992; Ashen and Goff,1996)
The current investigation of P. lanceolata gall inductionemployed whole-cell in situ hybridization to investigate the causativerole of the gall symbiont in tumor formation. In situ hybridization isparticularly suited to investigations of symbiotic associations wherethe cultivation and isolation of the microbial partner has not beenachieved (Amann et al., 1991; Hahn, Amann, and Zeyer, 1993; Amann, Ludwig, and Schleifer, 1995). The algalhost response during gall development was also investigated using insitu hybridization in section and in standard preparations for lightmicroscopy. The possibility that the bacterial symbiont is notrestricted intercellularly but undergoes an intracellular phase incertain regions of gall proliferation or at particular times during gallformation was also re-investigated in preparations for transmissionelectron microscopy (TEM) (McBride, Kugrens, andWest, 1974).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Laboratory gallinduction from field-collected material
Galls used for laboratory induction trials were prepared as follows.Galls were excised from thalli collected at Lover's Pt. and washedten times in sterile seawater (0.22 µm filtered). Galls (~5 gfresh mass) were homogenized in a sterile mortar and pestle andresuspended in 45 mL sterile seawater in 50-mL Falcon tubes on ice.Algal thalli were prepared as follows. Blade tips (5–10 cm) wereexcised from thalli that had been kept in indoor flow tanks for 2 mo;these thalli showed no evidence of gall formation. Thalli were wipedclean of surface epiphytes and wounded to the medulla by injection ofgall homogenate. Negative controls were prepared simultaneously usingblade tips taken from the same individual thalli. Sterile seawater wassubstituted as an inoculum. Inoculated blades were incubated in 50-mLFalcon tubes containing gall homogenate at 12°C for 2–3 h.After incubation, blade tips were immobilized in short lengths of tygontubing epoxyed to the flat face of a cement brick. This assemblage wasincubated in an indoor seawater tank with high ambient flow andaeration. Induction trials were performed six times, totaling ~600inoculation events. Successful induction was scored as a percentage ofinoculated sites that formed pigmented cell masses associated withintercellular bacteria.
Laboratory gall induction from experimentally inducedgalls
Bacterial galls (60) induced in the laboratory were harvested after 8wk, rinsed, and homogenized (6–8 each) with sterile tissuegrinders (Kontes, Vineyard, New Jersey) in sterile 1.5-mL Eppendorftubes containing 200 µL of sterile seawater. The volume of gallhomogenate in each tube was increased to 1.5 mL with sterile seawaterand the samples pooled, yielding a final volume of 12 mL. Subsamples (2x 1 mL) were fixed for whole-cell in situ hybridization and theremainder brought to 45 mL in sterile seawater. This suspension was usedto initiate a second round of gall induction on P. lanceolatablade tips. Inoculated blades were incubated as above and scored after 8wk as a percentage of galls successfully formed in inoculationsites.
Fluorescent in situhybridization of symbiotic bacteria
Galls from P. lanceolata collected from the state beach inCarmel and Lighthouse Point in Santa Cruz were excised from host thalliand cored by sterile dissection. Cores were washed five times in sterileseawater and homogenized in Eppendorf tubes as above. Specimens werefixed, dehydrated, and attached to 10-well Teflon-coated hybridizationslides as described previously (Ashen and Goff,1996). Identification of the gall symbiont was then carriedout by dual hybridization using a 5' TAMARA-conjugated universaleubacterial probe (S-D-Bact-0338-a-A-18) and a 5'fluorescein-conjugated symbiont-specific probe (S-S-P.l.sym-0949-a-A-25)(DeLong, 1993; Ashen and Goff, 1996). Hybridizations and washeswere performed at 43°C in 0.2 x SET (30 mmol/L NaCl, 2mmol/L Na2EDTA, 4 mmol/L Tris base) as describedpreviously, mounted in 3:1 Citifluor:DAPI (0.5 µg/mL4,6-Diamidino-2-phenylindole), sealed with nail polish, and imaged on anOlympus IMT2 inverted photoscope using a Biorad 600 laser confocalimaging system and an Olympus S-Plan-apo 60x, oil objective(Ashen and Goff, 1996). Excitationwavelengths were 510 nm, corresponding to the maximal excitationwavelength of fluorescein and 560 nm, corresponding to that of TAMRA(the excitation wavelength of this proprietary chromophore is equivalentto that of rhodamine) (Applied Biosystems, Foster City, California).Slides were stored dark at 4°C for up to 2 mo with little to nonoticeable loss of fluorescence. Agrobacterium tumefaciens andRoseobacter denitrificans cells were included on the sameslides to control for oligonucleotide probe specificity.
Homogenates used to investigate the bacterial role in gall inductionwere prepared, fixed, hybridized, and imaged as above. Images of labeledcells were captured from single transects across individual wells of thehybridization slides (ten random fields of view, 2x zoom). Therelative percentage of symbiotic bacteria among the total eubacteriahybridized was determined in each induction homogenate by merging of thered and green fluorescence signals produced by each excitationwavelength and subtraction of cells hybridized only by the universaleubacterial probe from the total hybridizedeubacteria.
In situhybridization: paraffin sections using a digoxigenin-labeledoligonucleotide
Field-collected and laboratory-induced galls were excised from algalthalli, dissected, and fixed for 3–16 h in a 1:1 mixture ofbuffered paraformaldehyde (8%) and seawater at 4°C (Ashen and Goff, 1996). Laboratory-inducedspecimens were fixed at 2, 3, 4, 6, and 8 wk after inoculation.Specimens were washed twice in 1x phosphate-buffered saline (PBS)for 10 min each and dehydrated in a graded ethanol series (Sambrook, Fritsch, and Maniatis, 1989). Theethanol was replaced by three washes in xylenes (20 min each) and thespecimens gradually infiltrated with Periplast plus (Oxford Scientific,St. Louis, Missouri). Infiltration was completed through three changesof Periplast over 48 h and embedded blocs stored for up to 4 mo(4°C) before sectioning.
Sections (5 µm) were adhered overnight at 45°C to acid-washed,3-aminopropyltriethoxysilane (Sigma, St. Louis, Missouri) coated glassslides and stored at -20°C for no longer than 1 mo. Forhybridization a number of previously used protocols available in themicrobial and botanical literature were combined and modified (Cary et al., 1993; Hahn,Amann, and Zeyer, 1993; Polz et al.,1994; Dubilier et al.1995). Specimens were brought to 25°C, deparaffinized inthree changes of xylenes (30 min total) and rehydrated through ethanolto deionized water. Sections were treated with freshly prepared HCl(0.2mol/L for 20 min at 25°C), rinsed in 1x PBS andneutralized in 2x SSC (0.3 mol/L NaCl, 30 mmol/L sodiumcitrate) for 20 min at 25°C. This was followed by two rinses ofdeionized water and incubation in TE-100 (100 mmol/L Tris-HCl, 5mmol/L Na2EDTA [pH 7.5]) for 5 min at25°C.
Following equilibration, sections were treated with lysozyme (1mg/mLin TE-100 for 30 min at 25°C) in a humidchamber under parafilm coverslips. Slides were rinsed twice with TE-100and prehybridized in 150 µL prehybridization buffer (6x SSC,0.2% SDS, 5x Denhart's, 100 µg/mL shearedsalmon sperm DNA, and 25 µg/mL yeast tRNA) at 43°C underparafilm coverslips. The prehybridization buffer was replaced after 2 hby 100 µL hybridization buffer, identical in composition butcontaining either S-S-P.l.sym-0949-a-A-25 or an Alvinellapompjiana specific probe 5' labeled with digoxigenin-11-dUTP(final concentration = 75 ng/section) (Cary et al., 1993). Sections were then hybridizedovernight at 43°C under parafilm coverslips. Sections oflaboratory-induced galls to which no oligonucleotide probe was added andsections of P. lanceolata wounded with sterile seawater andhybridized with S-S-P.l.sym-0949-a-A-25 were included as negativecontrols.
After hybridization, specimens were washed at 43°C as follows:2x SSC, once for 30 min and once for 1 h; 0.2x SSC, twicefor 1.5 h each. Digoxigenin moieties conjugated to hybridizedoligonucleotides were detected using an antidigoxigenin antibody (FABfragment) conjugated to alkaline phosphatase (Boehringer Mannheim,Indianapolis, Indiana). The activity of alkaline phosphatase isvisualized colorimetrically as a dark precipitate following reactionwith 5-bromo-4-chloro-3-indolyl-phosphate-4-toluidine salt/4-Nitroblue tetrazolium chloride (BCIP/NBT) as per the manufacturersrecommendations (Boehringer Mannheim). Specimens were dehydrated throughethanol and xylenes, mounted in Permount (Fisher), and photographedusing a Leitz Diaplan microscope equipped with PL-fluotar objectives andKodachrome 160T color slide film.
Light and epifluorescence microscopy
P. lanceolata galls (laboratory-induced andfield-collected), uninfected vegetative thalli, and seawater inoculatedthalli were embedded in JB4 plus resin (Ted Pella, Redwood City,California) and prepared for light and epifluorescence microscopy usingstandard techniques (McCully, Goff, and Adshead,1980; Goff and Zuccarello,1994). Galls induced in the laboratory were also prepared forlight microscopy by squashing following the methodology of Goff and Zuccarello (1994).
Sections (3 µm) were stained directly with 1% aniline blue(aq), toluidine blue O (pH 4.4 in benzoate buffer), or the nucleic acidfluorochrome DAPI (0.1–0.5 µg/mL in filtered seawater)(McCully, Goff, and Adshead, 1980).Paraffin sections (5 µm) were deparaffinized in xylenes, rehydratedthrough a graded ethanol series, and stained as above. DAPI-treatedsections were mounted in Citifluor (Ted Pella), observed on a Leitzdiaplan microscope equipped with PL-fluotar objectives, and photographedusing Kodachrome color slide film (ASA 160T or elite 400) (Goff and Zuccarello,1994).
Transmissionelectron microscopy
Laboratory-induced and field-collected material were prepared fortransmission electron microscopy using chemical fixation protocolsdescribed previously and modified as described below (Bozzola and Russell, 1992; Goff and Zuccarello, 1994). Specimens weretransferred from 70% acetone to 2% uranyl acetate in70% methanol (w/v) and en bloc stained overnight at 4°Cin the dark. Dehydration in acetone was completed and gradualinfiltration with Spurr's resin completed over 1–2 wk. Silversections were cut on a Sorvall Porter-Blum Ultra MT-2, collected oncopper grids, stained for 5–10 min with Sato's lead stain,and observed and photographed on a JEOL JEM-100B electron microscope at80 kV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fluorescentin situ hybridization of symbiotic bacteria
Whole-cell in situ hybridization was successful in confirming theidentity of the bacteria associated with all galls tested in nature andinduced in the laboratory as well as in characterizing the relativepercentage of eubacterial gall symbionts from induction homogenates.Bacteria from galls collected at Carmel and Lighthouse Pt., Santa Cruz,were hybridized by both S-S-P.l.sym-0949-a-A-25 andS-D-Bact-0338-a-A-18r. Agrobacterium and Roseobactercells included as controls were hybridized by S-D-Bact-0338-a-A-18r butnever S-S-P.l.sym-0949-a-A-25. Assays of gall induction homogenates(galls collected in nature) using S-S-P.l.sym-0949-a-A-25 identified arange of 85–90% (±3%) of the hybridizableeubacteria as the P. lanceolata symbiont (Table 2) (Figs. 5–6). The relativepercentage of eubacterial symbionts in homogenates prepared fromlaboratory-induced galls was 97% (±3%) (Table 2).
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Light andepifluorescence microscopy
Several distinct cell layers were seen in cross sections ofunaffected P. lanceolata thallus (Fig. 7). The epidermal region oftightly packed cells was subdivided into epidermal cortex and corticallayers, which were underlain by a loosely woven, filamentous medulla.The epidermal cortex was composed of 3–4 layers of tightly packed,isodiametric cells (5–10 µm), each of which was derived fromthe cortex, a region composed of 3–5 cell layers of increasingdiameter (10–50 µm). Cells in this region formed extensivesecondary pit connections with each other. The central region of theunaffected P. lanceolata thallus was composed of a loose,filamentous medulla (Figs.7–8). Thallus expansion during growth resulted in a"network" of cells composed of stellate regions connected byfilamentous projections (Fig.8).
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Gall-induced medullary cells and bacterial symbionts were foundconsistently in close proximity to each other, suggesting that contactmay be involved in the induction response (<5 µm) (Fig. 17). Symbiotic bacteria were alsoassociated with the hypertrophy and hyperplasia of algal cortical andsubcortical cells, although specific cell-to-cell contact was notobserved in all cases (Figs.18–20). In regions adjacent to and surrounding gallproliferation (<20 µm) filamentous medullary cells that did notappear affected (these cells retained their normal elongate morphologyand were not induced to divide or produce bud initial cells) bybacterial infection were physically displaced by the expanding gall(Fig. 21).
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In situ hybridization: paraffin sections using adigoxigenin-labeled oligonucleotide
Hybridization of both field-collected and laboratory-induced gallswith S-S-P.l.sym-0949-a-A-25 localized a single intercellular bacterialmorphotype (Fig. 23).Hybridized cells were uniform and of the same size as whole cellshybridized in gall induction homogenates. The Alvinellaepibiont probe did not hybridize to any bacteria in sections of P.lanceolata galls (Fig.24). No bacteria were detected in sections not hybridized withan oligonucleotide. This confirmed the specificity of the colorimetricdetection method used (data not shown). Similarly, hybridization ofnegative control wound sites with S-S-P.l.sym-0949-a-A-25 did not detectthe gall bacterium. The gall symbiont was also localized in sections ofdeveloping, laboratory-induced galls (2–3 and 6 wk postinduction)(Figs. 25–28). Nobacteria in these sections were detected either by hybridization withthe Alvinella epibiont-specific probe or in the absence of anoligonucleotide.
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Transmissionelectron microscopy
Despite an extensive survey of laboratory-induced galls (2 and 4 wkpostinoculation) no evidence of an intracellular stage of bacterialproliferation was observed. Gall-affected cells contained reducedvacuoles and enlarged plastids in comparison to nonaffected cells.Symbiotic bacteria were found consistently in intercellular spaces andwere always surrounded by an electron-transparent region. This regionformed an external layer similar in appearance to other bacterialexopolysaccharide layers, making complete infiltration of gall tissuesextremely problematic and resulting in the intermittent loss ofbacterial cells from sections.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The metabolic consequences of gall formation for either P.lanceolata or its bacterial symbiont remain unknown. Thehypertrophied growth of gall-induced algal cells apparently provides adesirable microhabitat for the growth and proliferation of the bacterialsymbiont. Observations of the host alga in nature, however, suggest thatthere is little to no ecological consequence to the gall-bearing thallus(Ashen, personal observations). The subtidal population at Pacific Grovecontains both gall and nongall-bearing individuals, which can be eithersmall, perennating thalli or large (>1 m) 1st-yr thalli regardless ofbacterial presence. The extent of gall formation on a particularindividual is extremely variable but ranges from several (5–10)galls to hundreds of galls per thallus, irrespective of size.Gall-affected thalli were not obviously different from nonaffectedindividuals except for the localized effects of gall formation (Ashen,personal observations).
Gall formation in nature appears to affect between 45 and 70%of the thalli from a given location (Apt and Gibor,1989). Extensive collection of P. lanceolatathroughout California suggests that gall formation is common on algalthalli (perhaps >80%) from low intertidal to subtidal zones,which experience moderate to high wave action (Ashen, personalobservations). Thalli in other littoral regimes were rarely observed tobear galls.
The genus Prionitis is highly pleiomorphic with the speciesP. lanceolata being an extreme example. Thallus morphology isapparently related to position within the intertidal, with lowerintertidal and subtidal thalli manifesting a more cylindrical and lessbranched habit (Abbott and Hollenberg,1976). Galls were consistently found on thalli of thismorphology and were never observed on mid-to-high intertidal individualscharacterized by flatter, wider blades and increasing proliferation ofmarginal bladelets.
The presence of a single, predominant eubacterial phylotype (16s rDNAsignature) was confirmed on all collected individuals of P.lanceolata from in and around Monterey Bay, California. Thespecies-specificity of particular regions of the 16S rDNA sequence hasbeen demonstrated and widely applied to the investigation of specificmicroorganisms in a wide range of symbiotic associations (Amann et al., 1991; Distel, DeLong, and Waterbury, 1991; Cary et al., 1993; DeLong,1993; Hahn, Amann, and Zeyer,1993; Amann, Ludwig, and Schleifer,1995; Dubilier et al.,1995; Fischer et al., 1995;Ashen and Goff, 1996; Bianciotto et al., 1996). Gall formation onP. lanceolata has not been found in the absence of theidentified gall symbiont. This supports the hypothesis that gallformation is a species-specific phenomenon requiring the presence of aparticular eubacterium and satisfies Koch's first postulate for thedemonstrated presence of the disease agent in all cases of manifestsymptomology.
The predominant eubacterium identified in induction homogenates fromgalls collected in nature (85–95%) was the P.lanceolata proteobacterial symbiont. The low percentage ofextraneous eubacteria detected in these homogenates were undoubtedlybacterial epiphytes, ubiquitous among intertidal algae (Provasoli and Pintner, 1980; Tatewaki, Provasoli, and Pintner, 1983;Shiba, 1992). Laboratory induction usingthe above homogenates showed a significant correlation between thepresence of the identified symbiotic eubacterium and gall formation.Wound sites that were inoculated with sterile seawater formed galls asmall percentage of the time (4%). This was likely due to theincubation of P. lanceolata blade tips in ambient seawater. Theassumption at present is that the gall symbiont is present in the watercolumn and was able to enter the wound sites of the control bladesbefore they had healed.
In situ identification of the P. lanceolata symbiont fromgalls collected in nature satisfies Koch's second postulate byidentifying the suspected disease agent. The need for pure cultureisolation has been required traditionally to ensure that only a singleorganism is involved in reinfection trials. In this case moleculartechniques were used to identify an uncultivable microorganism andmonitor that organism in re-infection trials. While not the onlyeubacterium present in "natural" induction homogenates, theidentified gall symbiont was the numerically predominant microbe and wassubsequently identified in situ, in proliferating stages of thereforming association.
Whole-cell in situ hybridization of the gall symbiont withS-S-P.l.sym-0949-a-A-25 confirmed the presence of this specificeubacterium in laboratory-induced galls Symbionts in homogenates ofinduced galls comprised a higher percentage of the hybridizableeubacteria than in homogenates of galls collected in nature(97%). This was probably due to removal of these galls fromaffected thalli shortly after eruption through the thallus surface,affording epiphytes only a short time for colonization. Localization ofthe bacterial symbiont was performed in section throughout the course ofthe induction trials. The in situ monitoring, during consecutive roundsof laboratory induction, satisfies Koch's third and fourthpostulates by demonstration of the disease symptomology in the reformedassociation in response to the presence of a specific, suspected diseaseagent.
The overall timing of cellular events involved in gall developmentand formation may be characterized as follows. The production ofepidermal cell layers from the cortex adjacent to the inoculation siteis generally complete within 2–4 wk, sealing the thallus interiorfrom the external environment. In situ hybridization confirms that theeubacteria lying within the wound site and observed to proliferateintercellularly from the inoculation site are the gall symbiont.Bacterial induction of algal hypertrophy does not appear to be localizedin one region of the wound site but is related to symbiont proximity.Expansion of gall-induced tissues continues within the thallus throughthe 4th wk, with increasing numbers of cells of the algal cortex,subcortex, and medulla induced to hypertrophy and hyperplasia by contactwith the proliferating gall. The finding of cortical cell induction hasnot been reported elsewhere where red algal gall induction anddevelopment have been examined.
Eruption of the gall mass through the algal epidermis occurs~8–12 wk after inoculation. Release from the thallus exterioris followed by a marked change in the orientation of gall-induced cells,with growth expanding externally in anticlinal, albeit irregular filesto form the gall mass observable in nature. This is an apparentlyphysical response resulting from the release of pressure on theexpanding gall mass once the surface of the algal thallus has beenbreached.
The evidence presented here demonstrates the causative role of aspecific eubacterial phylotype in gall induction and formation on P.lanceolata. This microorganism is associated with all cases of gallformation on P. lanceolata. It can be specifically identifiedand comprises the vast majority of metabolically active bacterial cellsin homogenates from field-collected and laboratory-induced galls. Thepresence of this eubacterium is significant for the reformation of thegall symbiosis, and this microbe has been identified in situ, as theonly eubacterium present in consecutive rounds of gall induction andformation.
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FOOTNOTES |
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2 Author for correspondence, current address: Mail Stop
239-12, NASA/Ames Research Center, Moffett Field, CA
94035-1000.
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  M. J. Pujalte, M. C. Macian, D. R. Arahal, W. Ludwig, K. H. Schleifer, and E. Garay Nereida ignava gen. nov., sp. nov., a novel aerobic marine {alpha}-proteobacterium that is closely related to uncultured Prionitis (alga) gall symbionts Int J Syst Evol Microbiol, March 1, 2005; 55(2): 631 - 636. [Abstract] [Full Text] [PDF]
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 J. B. Ashen and L. J. Goff Molecular and Ecological Evidence for Species Specificity and Coevolution in a Group of Marine Algal-Bacterial Symbioses Appl. Envir. Microbiol., July 1, 2000; 66(7): 3024 - 3030. [Abstract] [Full Text]
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