Characterization of a Latent Virus-Like Infection of Symbiotic Zooxanthellae

A latent virus-like agent, which we designated zooxanthellafilamentous virus 1 (ZFV1), was isolated from Symbiodinium sp.strain CCMP 2465 and characterized. Transmission electron microscopyand analytical flow cytometry revealed the presence of a newgroup of distinctive filamentous virus-like particles afterexposure of the zooxanthellae to UV light. Examination of thinsections of the zooxanthellae revealed the formation and proliferationof filamentous virus-like particles in the UV-induced cells.Assessment of Symbiodinium sp. cultures was used here as a modelto show the effects of UV irradiance and induction of potentiallatent viruses. The unique host-virus system described hereprovides insight into the role of latent infections in zooxanthellaethrough environmentally regulated viral induction mechanisms.

INTRODUCTION

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INTRODUCTION

The frequency and intensity of coral bleaching have increasedin the last two decades, leading to mass mortality of coralsand a resulting reduction in the biodiversity of reefs (7, 33).Bleaching has been observed in more than 50 countries and inthe three major oceans (41). Many environmental factors havebeen linked to coral bleaching; these factors include elevatedand reduced temperatures (13), exposure to UV radiation (17),and bacterial infections (5, 19). However, the underlying causesof bleaching and the mechanisms involved remain largely unknown.

Reef-building corals associate with a diverse array of eukaryoticand prokaryotic microbes. The coral colony has been modeledas a holobiont comprising multispecies mutualisms consistingof the coral animal, endosymbiotic dinoflagellates (zooxanthellae),bacteria, fungi, protozoans, endolithic algae, and other unknowncomponents (27). Disruption of any of these components may causephysiological changes that result in coral disease or death.

Zooxanthellae (genus Symbiodinium) are now known to comprisenumerous clades (2). Zooxanthellae form a symbiotic relationshipwith many Cnidaria species and are by far the best-understoodmicrobial associates of corals, and they have a clearly beneficialrelationship with the coral. They release photosynthetic productsto their hosts, providing an important source of organic carbonand nitrogen for host metabolism, growth, and reproduction (12,24, 40). The symbiosis with zooxanthellae is believed to explainthe success of reef-building corals in nutrient-poor tropicalseas (15). Disruption of the zooxanthella-host symbiosis (bleaching)can lead to expulsion of zooxanthellae from the host and/ora decrease in the amount of the zooxanthella photosyntheticpigment (15, 18). Zooxanthellae have been shown to undergo necrosis,apoptosis, and lysis during bleaching (4, 31). The expulsionof Symbiodinium cells may help corals adapt to changing environmentalconditions by allowing symbiont populations to redistributethemselves (3, 10 ). If the symbiont-host relationship is notrecovered, the coral does not survive (33). It has been arguedthat the random mosaic patterns of bleaching are difficult toattribute to the effect of temperature stress alone as neighboringregions of a colony must be exposed to the same conditions (16).One explanation for the patchy spatial distribution of coralbleaching involves localized infections. Bacterial infectionby Vibrio sp. has been shown to be responsible for some typesof coral bleaching (5, 28, 37). Given the great abundance ofviruses in marine systems, in which concentrations reach >10⁸particles ml^–1 (6, 44), it is likely that viruses arealso involved as agents of coral disease (11), and involvementof viruses in the coral bleaching process must be considered.It has been established that the majority of marine virusesinfect bacteria, but viruses that infect eukaryotic algae havealso been shown to be abundant in the marine environment (8).Virus-like particles (VLPs) have been found in cells of about50 different algal species representing nearly all major algalclasses (26, 38 ), and evidence suggests that viruses play asignificant role in the population dynamics and community compositionof marine phytoplankton (8, 22, 29, 32). Despite this, therehave been relatively few reports on viruses of dinoflagellates(14, 30, 35) and fewer still on the presence of viruses in zooxanthellae.Previous studies in our laboratory have shown that zooxanthellaeand corals produce VLPs when they are exposed to stress andelevated temperatures (11, 42, 43), suggesting that zooxanthellaeharbor latent viruses. In order to determine whether VLPs areinduced by exposure of the zooxanthellae to UV light, we utilizedSymbiodinium sp. cultured in vitro to simplify the complexityfound in whole organisms containing symbionts.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Zooxanthella cultures.
During this study, 16 strains of Symbiodinium species isolatedfrom a variety of cnidarian hosts were maintained in culture(nonaxenic). Strains 12, 61, 104,133, 135, 141, 152, 154, 203,292, 368, 379, 383, and 385 were obtained from a collectionat the Marine Biological Association and have been describedin a previous study (20). Strains 200X and 200Y were isolatedfrom Acropora formosa. The zooxanthellae were grown in ASP-8Amedium with antibiotics (25) and were subcultured monthly. Algalcultures were grown by using a cycle consisting of 16 of lightand 8 h of darkness at 26°C. The light intensity used was40 to 50 mol quanta m^–2 s^–1. Zooxanthella isolate292 used in this study has been deposited in the Center forCulture of Marine Phytoplankton (http://ccmp.bigelow.org) asstrain CCMP 2465.

Experimental treatments.
Triplicate exponentially growing cultures (50 ml; approximately10⁵ cells ml^–1) were exposed for 2 min in open petri dishesto UV light (254 nm) from a Chromato-Vue transilluminator (modelTM-20) which was placed upside down 12 cm above the petri dishesto allow direct radiation. After exposure, the cultures weretransferred back into 50-ml flasks and maintained at 26°Cwith illumination using a cycle consisting of 16 of light and8 h of darkness. Samples used for enumeration of zooxanthellaeand VLPs were collected daily for 1 week. Zooxanthellae wereenumerated immediately, and 1-ml aliquots were fixed in 0.5%glutaraldehyde, stored at 4°C for 30 min, snap frozen inliquid nitrogen, and stored at –80°C until they wereprocessed for enumeration of VLPs and bacteria.

Enumeration of zooxanthellae, VLPs, and bacteria by analytical flow cytometry (AFC).
All analyses were performed with a Becton Dickinson FACScanflow cytometer using the Cellquest software. For enumerationof live zooxanthellae, undiluted samples were examined for 2min using a high flow rate (ca. 50 to 70 µl min^–1)with the discriminator set on red fluorescence. For enumerationof VLPs and bacteria, counting was performed using fixed samplesdiluted 1:10 to 1:1,000 in TE buffer (10 mM Tris, 1 mM EDTA;pH 8.0), filtered with a 50-kDa filter using tangential flowfiltration (Vivaflow flip flow), and stained for 10 to 15 minat 80°C with a 10^–5 dilution of SYBR green I stain(Molecular Probes). The samples were analyzed for 2 min at alow flow rate (ca. 30 µl min^–1) with the discriminatorset on green fluorescence (23). VLPs and bacteria were enumeratedusing plots of side scatter (SSC) versus green fluorescence.All 16 zooxanthella strains were screened and exhibited differentialsensitivity to UV induction. Strain CCMP 2465 exhibited thestrongest response and was therefore utilized for further assessment.

TEM.
Samples potentially containing VLPs were filtered through 0.45-µmfilter units (Whatman) with a BD Plastipak sterile 10-ml syringeto remove debris and large cells. Aliquots were fixed in 0.5%glutaraldehyde, stored at 4°C for 30 min, and snap frozenin liquid nitrogen. "Spot" grids were prepared by placing gridsonto 15 to 30 µl of a fixed suspension for 30 min. Theexcess liquid was removed with filter paper, and each preparationwas negatively stained with uranyl acetate (2% [wt/vol] in water).For preparation of thin sections, UV-induced zooxanthellae werepelleted by centrifugation at 14,000 rpm in 1.5-ml Eppendorftubes for 1 min. The pellets were resuspended, washed, and repelletedtwice in filtered seawater (pore size, 0.2 µm). Moltenagar (2% Fischer agar in distilled H₂O; 0.5 ml) was added toeach washed pellet and allowed to set; the excess agar was trimmedfrom the agar plugs. A 4% osmium tetroxide solution was mixed1:1 with filtered seawater and added to cover the agar plugs.The preparations were left for 1 h at room temperature, andthis was followed by two washes in filtered seawater (pore size,0.2 µm), dehydration with a graded ethanol series, andinfiltration with Spurr's resin in preshaped molds. Dried (48h, 35°C) blocks were mounted and cut into sections thatwere 70 to 80 nm thick with a Reichert-Jung Ultracut microtome;the sections were floated onto transmission electron microscopy(TEM) grids, stained with 2% uranyl acetate in 70% ethanol (15min), washed in double-distilled H₂O, stained with Reynoldslead citrate for 15 min, and then rinsed in double-distilledH₂O. Prepared grids and thin sections were examined with a JEOL200 CX TEM (magnification, 80 to 600,000) at 160 kV. Photographswere taken at magnifications between x5,000 and x50,000.

Concentration of VLPs.
Zooxanthellae were grown to a density of ca. 10⁵ cells ml^–1in 3 liters of ASP-8A medium and exposed to UV light (254 nm)for 2 min in open petri dishes. VLPs were isolated from thegrowth media 96 h after exposure to UV. Debris, bacteria, andalgal cells were removed by filtration though 0.45-µm-poresize, 47-mm-diameter filters (PALL Corp.). VLPs in the supernatantwere concentrated by tangential flow filtration (Vivaflow flipflow) with a 50-kDa cutoff to obtain ca. 50 ml. Further concentrationto ca. 3 ml was accomplished by ultracentrifugation with a BeckmanL8-M at 100,000 x g for 2 h. VLPs were further purified andconcentrated on a CsCl gradient prepared with CsCl in TE bufferat densities of 1.0, 1.2, 1.4, and 1.6 g cm^–3, and thepelleted VLPs were added to the 1.0-g cm^–3 layer. Thispreparation was then centrifuged at 100,000 x g for 2 h at 20°C,with the deceleration set at 8. A translucent white band wasremoved by piercing the ultracentrifuge tube just below theband, which allowed aliquots to be separated. Aliquots weredialyzed (Fisher; diameter, 0.32 mm; 12- to 14-kDa molecular-masscutoff) overnight against 1 liter of TE buffer at 4°C, andthe TE buffer was changed twice in this time. The dialyzed VLPconcentrate was stored at 4°C until it was analyzed.

RESULTS AND DISCUSSION

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RESULTS AND DISCUSSION

Induction of VLPs by UV irradiation of zooxanthellae.
AFC analysis of UV-induced zooxanthella cultures revealed thepresence of a separate group of high-SSC, low-green-fluorescence(GFL) particles which were visible ca. 24 h after UV induction.By 96 h, this group was clearly distinguishable (Fig. 1B). Itwas likely that this group contained VLPs, despite the factthat it had a unique SSC-versus-GFL signal with the SSC higherthan that of previously described bacteriophages and viruses(9). The higher SSC suggested that the particles might be filamentous,since SSC is influenced by internal structure and its refractiveindex (9). Of the zooxanthella isolates screened for UV inductionof potential latent viruses, 38% showed proliferation of a groupof VLPs similar to that shown in Fig. 1B. VLPs in this high-SSCgroup were concentrated from a 3-liter culture of zooxanthellastrain CCMP 2465. CsCl gradient centrifugation produced a discretewhite band at a density of 1.25 g cm^–3 (Fig. 1C). AFCanalysis of this band revealed that it was dominated by thesame high-SSC group of VLPs (Fig. 1D).

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FIG. 1. AFC analysis of VLPs induced following UV treatment of zooxanthella cultures. (A) AFC analysis of control culture of zooxanthellae, showing bacteria (cultures were nonaxenic). (B) AFC analysis of UV-treated culture (after 96 h), showing a new VLP group with high SSC (indicated by arrow). (C) CsCl gradient of the VLPs with a translucent white band at ca. 1.25 g cm^–3. (D) AFC analysis of concentrated VLPs taken from the CsCl gradient, showing the presence of the high-SSC VLP group.

In UV induction experiments, a decline in the zooxanthella concentrationwas observed starting 24 h after UV induction, which correlatedwith the appearance and rapid increase in the concentrationof the high-SSC VLP group (Fig. 2A). The concentration of theVLPs continued to increase to just over 2 x 10⁶ particles ml^–1at 96 h after UV treatment, at which point the UV-induced zooxanthellaculture had lysed.

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FIG. 2. (A) Growth curves showing numbers of cells of zooxanthella strain CCMP 2465 ml^–1 in control cultures ( ${circ}$ ) and UV-treated cultures (•), together with the numbers of the high-SSC VLPs ml^–1 in the unirradiated control cultures ( ${triangleup}$ ) and the UV-treated cultures ( ${blacktriangleup}$ ). (B) Concentrations of bacteria in the control cultures ( ${square}$ ) and the UV-treated cultures ( ${blacksquare}$ ), together with the numbers of bacteriophage particles ml^–1 (10⁶) in the control cultures ( ${diamond}$ ) and the UV-treated cultures ( ${blacklozenge}$ ). The error bars indicate the standard errors for measurements from triplicate cultures.

Since the zooxanthella cultures were nonaxenic, a group witha signal characteristic of bacteria was present in the control(Fig. 1A), as well as in the UV-treated zooxanthella cultures(Fig. 1B). To rule out the possibility that lysogenic bacteriawere the source of the high-SSC VLPs, bacterial concentrationswere also monitored in the induction experiments. The temporalchanges in bacterial concentrations were similar in the controland UV-treated cultures, although there was a slight decreasein the UV-treated cultures between 24 and 48 h, before theyagain exhibited the same growth rate as the control (Fig. 2B).This initial decrease in bacterial concentrations could havebeen caused by several factors, such as lysis of UV-sensitivebacteria and/or induction of lysogenic bacteriophage, althoughthere was no apparent increase in the number of bacteriophageparticles compared to the control during the induction experiment(Fig. 2B). Bacteriophages usually group in the area above theinstrument noise in the bottom left corner of a GFL-versus-SSCplot (Fig. 1A) (9). This group was analyzed, and the numberof events remained consistent for the controls and the experimentalsamples throughout the experiment. While the experimental treatmentappeared to have an effect on the numbers of bacteria presentin the nonaxenic zooxanthella culture, the clear correlationbetween the decrease in the size of the zooxanthella populationand the concurrent increase in the VLP concentration suggeststhat bacterial contaminants were not the source of the new VLPgroup observed by AFC.

Identification of VLPs.
After induction free VLPs in the UV-treated supernatants andconcentrates were observed by TEM as long flexible filamentsthat were 2 to 3 µm long and approximately 30 nm wide(Fig. 3). In VLPs purified by CsCl gradient centrifugation therewas an increase in the density of the filamentous VLPs afterconcentration (3 x 10⁷ VLPs ml^–1) (Fig. 3B and E). Thinsections of UV-treated zooxanthellae revealed pockets of filamentousVLPs with similar morphology in the cytoplasm on the peripheryof zooxanthella cells (Fig. 4); filamentous VLPs were not seenin controls. The incidence of VLPs observed in the thin sectionsincreased markedly between 39 and 46 h postinduction comparedto the incidence in noninduced controls (in which no filamentswere observed). The presence of intracellular VLPs providesfurther evidence that the particles observed were actually infectingthe zooxanthellae and not the bacteria present in the cultures.Therefore, we designated the new group of VLPs "zooxanthellafilamentous virus 1" (ZFV1).

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FIG. 3. TEM images showing the morphology of the new VLP, ZFV1. The filamentous VLP is ca. 2 to 3 µm long. (A, C, D, and F) Images from spot grids containing unconcentrated lysate. Scale bars = 1 µm. (B and E) Images of VLPs concentrated on a CsCl gradient (shown in Fig. 1C). Scale bars = 200 nm.

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FIG. 4. TEM images showing the presence of filamentous VLPs in thin sections of zooxanthellae prepared 39 h (A and B) and 46 h (C and D) after induction with UV light. Scale bars = 3 µm (A), 200 nm (B), 2 µm (C), and 300 nm (D). The box in panel A indicates the area shown in panel B, and the box in panel C indicates the area shown in panel D. Chl, chloroplast; Nu, nucleus; Li, lipid vacuole.

The majority of algal viruses described to date are membersof the Phycodnaviridae, which is a family of large double-strandedDNA viruses that have an icosahedral shape (38, 44), althoughmore recently a number of algal RNA viruses have also been isolatedand characterized (21, 34, 36). ZFV1 is morphologically similarto RNA viruses which are known to infect plants, in particularviruses belonging to the family Closteroviridae. Members ofthis family are filamentous and flexuous, and they are 1,500to 2,200 nm long (1, 39). However, determination of the taxonomicaffiliation of ZFV1 will require further molecular characterization,and this certainly warrants further investigation.

Implications for the mechanism of coral bleaching.
Wilson et al. (43) first suggested that zooxanthellae may harbora latent infection, after showing that VLPs were present inzooxanthellae of thermally stressed anemones and that isolatedVLPs could reinfect zooxanthellae. Further studies have shownthat VLPs are present in three species of tropical coral (11).While the hosts of the numerous VLPs remain unknown, the abundanceof VLPs and their close association with corals and the symbioticzooxanthellae is evident. A variety of VLP forms have been observed,and while most of VLPs the are hexagonal, it is notable thata filamentous VLP (up to 3 µm long) whose morphology issimilar to that of ZFV1 has been observed in seawater followingexposure of the coral Acropora formosa to heat stress (11).Although in our experiments we utilized nonaxenic cultures,our results clearly indicate that zooxanthellae contain a latentvirus that is induced by UV treatment, leading to the lysisof the zooxanthellae. Extrapolation from this virus-host interactionand its effects on zooxanthella viability provides a novel linkto the impact of latent infection on symbiotic dinoflagellatesand the subsequent disruption of reef ecosystems. If stress-inducedviral induction in zooxanthellae occurs in the natural reefenvironment, the presence of this new group of filamentous VLPsis clearly important.

ACKNOWLEDGMENTS

We acknowledge help and advice provided by Roy Moate, PeterBond, and Glen Harper from the Plymouth Electron MicroscopyCentre, University of Plymouth.

W.H.W. was supported through the NERC-funded core strategicresearch program of the Plymouth Marine Laboratory.

FOOTNOTES

* Corresponding author. Mailing address: Bigelow Laboratory for Ocean Sciences, 180 McKown Point Road, P.O. Box 475, W. Boothbay Harbor, ME 04575-0475. Phone: (207) 633-9666. Fax: (207) 633-9641. E-mail: wwilson{at}bigelow.org

Published ahead of print on 9 March 2007.

REFERENCES

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