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Stable Symbiodinium composition in the sea fan Gorgonia ventalina during temperature and disease stress.

Introduction

Gorgonian corals, including the common sea fan Gorgonia ventalina Linnaeus, 1758, are prevalent on reefs across the Caribbean Sea (Kinzie, 1973; Opresko, 1973; Sanchez et al., 1998; Jordan-Dahlgren, 2002). Like many other cnidarians, octocorals harbor endosymbiotic dinoflagellates of the genus Symbiodinium. These endosymbionts, known collectively as zooxanthellae, can be phylogenetically sorted into groups, or clades, based on restriction fragment length polymorphism (RFLP) analysis of the 18S small subunit rDNA (Rowan and Powers, 1991a; Rowan and Powers, 1991b). Gorgonia ventalina adults, like most Caribbean octocorals, harbor Symbiodinium clade B zooxanthellae (Goulet, 1999; Coffroth et al., 2001; LaJeunesse, 2002; Santos et al., 2003a; Goulet and Coffroth, 2004). However, many cnidarians harbor multiple types of algal symbionts, and the composition can vary in response to environmental factors (Rowan and Knowlton, 1995; Rowan et al., 1997; Baker, 2001; Toller et al., 2001; van Oppen et al., 2001; Knowlton and Rohwer, 2003; Douglas, 2003). If an environmental change is extreme, the host may lose algal endosymbionts, a phenomenon termed "coral bleaching." High and low temperature, light, salinity, and other stresses have been implicated in coral bleaching (Steen and Muscatine, 1987; Glynn and D'Croz, 1990; Buddemeier and Fautin, 1993; Glynn, 1996; Hoegh-Guldberg, 1999; Downs et al., 2002; Douglas, 2003; McClanahan, 2004; Strychar et al., 2004).

Disease and parasitism can also lead to a change or loss of algal symbionts. For example, Toller et al. (2001) reported variation in Symbiodinium type in diseased versus healthy areas of colonies of Montastraea spp. affected by yellow blotch disease. Bacteria of the genus Vibrio infect corals and induce bleaching (Kushmaro et al., 1996, 2001; Banin et al., 2001; Ben-Haim et al., 2003a, b). These bacteria infect only corals harboring endosymbiotic dinoflagellates, and they cause death and eventual expulsion of the dinoflagellates (Banin et al., 2001; Kushmaro et al., 2001; Ben-Haim et al., 2003a, b).

Bleached octocorals (and possibly other cnidarians) can acquire Symbiodinium from the water column and from cryptic populations within the colony (Rodriguez-Lanetty et al., 2003; Lewis and Coffroth, 2004). These can be a source for repopulation after a bleaching event, opening the way for changes in symbionts (Buddemeier and Fautin, 1993; Rowan et al., 1997; Rowan, 1998; Kinzie et al., 2001; Toller et al., 2001; Douglas, 2003; Baker, 2004; Buddemeier et al., 2004; Lewis and Coffroth, 2004). Especially under stressful conditions, this may lead to recombination between host and endosymbiont.

Over the past 15 years, Gorgonia ventalina and Gorgonia flabellum have suffered widespread mortality associated with an infection from Aspergillus sydowii (Bainier & Sartory 1913) Thom & Church 1926, a common soil saprobe (Smith et al., 1996; Nagelkerken et al., 1997a, b; Geiser et al., 1998; Harvell et al., 1999; Kim et al., 2000; Alker et al., 2001). The fungus may be introduced to the marine environment through soil runoff (Smith et al., 1996; Nagelkerken et al., 1997a; Jolles et al., 2002) or air-borne dust (Shinn et al., 2000). Once in the water column, it can be spread to other sea fans via water currents (Nagelkerken et al., 1997a; Jolles et al., 2002). Aspergillus causes lesions and tissue loss in the host (Nagelkerken et al., 1997b; Alker et al., 2001; Petes et al., 2003), which triggers a nonspecific innate immune response that includes production of antifungal and antibacterial compounds (Kim et al., 2000; Petes et al., 2003). Furthermore, severity of Aspergillus infections is highest in areas of poor water quality (i.e., high nitrogen concentrations and low water clarity) (Kim and Harvell, 2002) and those with experimentally elevated nutrient levels (Bruno et al., 2003).

Aspergillus sydowii infection, a stressful condition, could lead to the loss of some Symbiodinium strains and replacement with others more tolerant to stress. We characterized the symbiont genetic diversity within Gorgonia ventalina, examined Symbiodinium endosymbionts present in healthy and naturally infected G. ventalina sea fans, and experimentally tested the effects of temperature stress and bleaching on symbiont diversity.

Materials and Methods

In situ comparison of Symbiodinium within healthy and diseased hosts

To compare the symbiont taxa in healthy and diseased sea fans, Gorgonia ventalina was sampled at Pickles Reef (24[degrees] 59.079' N, 80[degrees] 24.978' W) and Eastern Sambo Reef (24[degrees] 30.329' N, 81[degrees] 39.260' W) in the Florida Keys. At each site, 2 [cm.sup.2] of tissue was cut from healthy and diseased colonies. From each diseased fan, a second 2 [cm.sup.2] of tissue was removed from a healthy area about 10 cm from the infection. Forty colonies (20 healthy, 20 diseased) were sampled at Pickles Reef, and 25 healthy and 15 diseased colonies were sampled at Eastern Sambo. Half of each sample (1 [cm.sup.2]) was preserved in 95% ethanol for molecular analysis and half in 5% formalin for quantification of zooxanthella-densities.

Laboratory experiments

Elevated temperature. Five pieces (each 36 [cm.sup.2]) were collected from each of 36 Gorgonia ventalina colonies on Eastern Sambo Reef and transported to the Mote Tropical Research Laboratory, Summerland Key, Florida. One fragment from each colony was processed immediately as an initial control, and three pieces were blocked across temperature treatments: 27[degrees] (control), 30[degrees] and 31.5 [degrees]C in temperature-controlled aquaria. The fifth fragment served as an in situ control (n = 36). In situ controls were mounted on 5-cm-tall polyvinyl chloride (PVC) racks attached to the substrate with nails and two-part marine epoxy. The racks were situated within 5 m of the collection area. Each fragment was held on the rack with a hose clamp at the fragment base. After 24 h of acclimation, all pieces from a particular colony received either control or fungal inoculations using sterile cotton wicks embedded with agar only (control) or agar and a GFP-modified strain of Aspergillus sydowii (treatment). The fragments were sampled 6 days post-inoculation and preserved for Symbiodinium density counts (5% formalin) and Symbiodinium identification (95% ethanol). In situ controls all received control inoculations to prevent spread of the pathogen on the reef.

Temperature duration. The effects of duration of temperature stress were tested with a second experiment with the following temperature treatments: 27 [degrees]C for 12 days (no temperature stress), 30.5 [degrees]C for 12 days (long-duration temperature stress), 30.5 [degrees]C for 6 days followed by 27 [degrees]C for 6 days (short-duration temperature stress). All collections and methods were the same as those in the elevated temperature experiment. Treatments were sampled 6 and 12 days post-inoculation. An additional 36-[cm.sup.2] fragment was collected from each sea fan colony sampled for the experiment to serve as an in situ control (n = 36).

Molecular analyses

Molecular determination of Symbiodinium type. Symbiodinium clade was determined using restriction fragment length polymorphisms (RFLP) of the 18S small subunit ribosomal DNA (18S ssrDNA). Symbiodinium DNA was extracted from the 1-[cm.sup.2] samples collected during surveys or from experimentally manipulated fragments. Standard methods were used (Coffroth et al., 1992), with the following modifications. Due to high levels of contaminating compounds, half of the samples were extracted twice with phenol/chloroform/isoamyl alcohol (25:24:1). Other samples were extracted one time with phenol/chloroform/isoamyl alcohol and then further purified using a polyethylene glycol precipitation (Glenn et al., 1999). Some samples were transferred to a sodium-chloride-saturated, 20% dimethyl sulfoxide solution 2 weeks prior to extraction (Seutin et al., 1991) to aid in the removal of contaminating compounds.

The 18S ssrDNA region was amplified with a universal primer, SS5, and a dinoflagellate-biased primer, SS3Z (Rowan and Powers, 1991a). PCR products were digested with Taq I restriction enzyme and compared with standards to identify Symbiodinium clade. Some samples did not amplify with SS5 and SS3Z primers, and for these the PCR was repeated using the universal primers SS5-SS3 (Rowan and Powers, 1991a) to verify the presence of amplifiable DNA. If a product was obtained with these primers, then the PCR was repeated using the dinoflagellate-biased primer set SS5Za+b and the universal primer SS3 (Rowan and Powers, 1991a).

To further resolve Symbiodinium taxa, the hypervariable region of domain V in the chloroplast 23S large subunit of ribosomal DNA (cp-23S rDNA type) was examined from a subset of the samples. These included all of the in situ comparison samples from each of the treatments from Pickles Reef and Eastern Sambo (healthy, diseased, and healthy portions of diseased colonies) and from most of the samples from the elevated temperature study (n = 114). Reaction conditions followed those of Santos et al. (2003a) using the primer set 23SHYPERUP and 23SHYPERDNM13, and fragments were separated on a 6.5% Long Ranger (FMC Bioproducts, Rockland, ME) gel on a LI-COR Gene ReadIR 4200 automatic DNA sequencer (LI-COR Biotechnology Divison, Lincoln, NE) (Santos et al., 2003a). Base pair length of the fragments was determined using ladders and standards of known size.

A single microsatellite locus (CA 6.38) was examined to increase taxon resolution. Primers were developed using zooxanthellae isolated from the octocoral Pseudopterogorgia elisabethae (Santos and Coffroth, 2003). The locus was amplified and analyzed on a 6.5% Long Ranger gel on a LI-COR Gene ReadIR 4200 automatic DNA Sequencer (Santos and Coffroth, 2003). The base pair size of the microsatellite alleles was determined by comparing the samples to a ladder and standards of known size.

Sequencing aberrant 18S ribosomal DNA amplicons. To identify an aberrant Symbiodinium PCR product amplified by the dinoflagellate-specific 18S rDNA primer set SS5 and SS3z, four samples from the East Sambo field survey and laboratory experiment were gel-purified and sequenced bidirectionally using IRD-800 M13 primers (LI-COR Biotechnology Division, Lincoln, NE) and the SequiTherm EXCEL II DNA sequencing kit-LC (Epicentre Technologies, Madison, WI) following manufacturer's specifications (Santos et al., 2003b). The sequencing products were visualized on a LI-COR Gene ReadIR 4200 automatic DNA sequencer using a 5.5% Long Ranger gel. The forward and reverse sequences were compared to remove ambiguities using AlignIR version 1.2 (LI-COR Biotechnology Division, Lincoln, NE). Consensus sequences of all four samples were identical, and one was deposited into Genbank under accession number DQ191042.

Symbiodinium cell counts

Symbiodinium densities were determined by homogenizing 1 [cm.sup.2] of tissue in 1 ml filtered seawater, washing twice, and resuspending in 1 ml of 5% formalin. Four independent aliquots from each sample were stained with Lugol's solution and counted on a hemacytometer at 40X. All data were checked for normality and homogeneity of variances and subsequently analyzed with ANOVA. Symbiodinium densities from field survey data were analyzed with a one-way ANOVA. To analyze the change in Symbiodinium density from initial controls to experimental pieces used in laboratory experiments a three-way nested ANOVA (fan nested within fungal treatment and temperature) was used (SAS ver. 8.0).

Results

In situ comparison of Symbiodinium within healthy and diseased hosts

Healthy and diseased sea fans from Pickles and Eastern Sambo harbored only Symbiodinium clade B zooxanthellae. Analysis of a hypervariable region within the large subunit of the chloroplast ribosomal gene of algal symbionts isolated from healthy colonies and from both healthy and diseased areas of diseased colonies at both sites yielded a PCR product of 184 bp (Cp-23S-rDNA type, n = 110). A single sample contained the 184-bp allele and a 206-bp allele. Therefore, almost all colonies harbor the same type of Symbiodinium within clade B, namely B184 (B1) (sensu Santos et al., 2003a; LaJeunesse, 2002).

An aberrant band appeared in the PCR product of the 18S small subunit rDNA in many of the Eastern Sambo samples (Fig. 1). The band, 565 bp in length, occurred either alone (Fig. 1a, Lane 3) or with the normal 1800-bp product (Fig. 1a, Lane 2). Samples (n = 122) containing only the aberrant band, and missing the normal 1800-bp PCR product, were amplified with the SS5Za+b-SS3 dinoflagellate-biased primers. For most of the samples, this resulted in a normal (1.8-kb) product in addition to the 565-bp band. These samples were identified as Symbiodinium clade B when compared to clade standards. Very little DNA was recovered from samples containing just the 565-bp band (n = 37), which may explain our inability to amplify the functional copy of the gene. No samples from Pickles Reef contained the abnormal PCR product.

Cp-23S-rDNA type was determined for 54 of the samples from Eastern Sambo. These were samples that had either the 565-bp fragment, the normal 1.8-kb PCR product for the 18S ssrDNA, or both. All of the samples yielded a PCR product of 184 bp, typical of Symbiodinium in clade B.

The CA 6.38 microsatellite locus was polymorphic, with four distinct alleles (Table 1). Significant differences in allele frequencies were found between the Pickles and Eastern Sambo sites ([chi square] = 25.064, df = 3, P < 0.05). Symbiodinium isolated from healthy (H:D) and diseased areas (D:D) of the same colony contained the same allele in all cases (n = 35), confirming that the symbiont taxon was ubiquitous throughout the diseased fan. At Eastern Sambo, four alleles were detected among the colonies sampled, and allele frequencies were not significantly different between healthy and diseased colonies ([chi square] = 6.975, df = 3, P > 0.05). In contrast, there was a significant difference in the frequency of the alleles in Symbiodinium isolated from healthy and diseased colonies at Pickles Reef: diseased colonies were more likely to harbor symbionts with the 108-bp allele ([chi square] = 8.179, df = 1, P < 0.05).

Laboratory experiments

In both laboratory experiments (elevated temperature and temperature duration) all Symbiodinium yielded an RFLP pattern characteristic of Symbiodinium clade B. The cp-23S rDNA type of a subset of these samples was determined and all were B184 (n = 114). Fifty-seven percent of the 70 colonies harbored Symbiodinium with the 565-bp fragment, but presence of the extra band was not correlated with treatment. Furthermore, in the laboratory experiment, no differences in allele frequency were detected at the CA 6.38 microsatellite locus in Symbiodinium isolated from the initial healthy tissue versus tissue blocked across all temperatures and treatments.

Symbiodinium cell counts

Symbiodinium densities were quantified for samples from Pickles Reef. Healthy areas of healthy fans (H:H) contained more zooxanthellae than both healthy (H:D) and diseased (D:D) areas of diseased fans (H:H vs. H:D--Student's t = -2.48, df = 57, P = 0.0418; H:H vs. D:D--t = -7.35, df = 57, P = < 0.0001). Healthy areas of diseased fans contained more zooxanthellae than diseased areas of the same fans (H:D vs. D:D--t = -4.87, df = 57, P = < 0.0001) (Table 2). Although the data were not normal (Shipiro-Wilk, W = 0.987, P = 0.0008; Kolmogorov-Smirnov, D = 0.047, P = 0.0213) the variances were homoscedastic (Levene's, P = 0.4193). A three-way nested ANOVA (fan nested within fungal treatment and temperature) showed a significant difference between Symbiodinium density, temperature treatments, and inoculations (F = 4.29, P = 0.0143).

Discussion

Symbiodinium clade B is the predominant symbiont taxa in adult Gorgonia ventalina (LaJeunesse, 2002; Goulet and Coffroth, 2004). All Symbiodinium isolated from sea fans sampled at Pickles Reef and Eastern Sambo were within clade B, regardless of sea fan health or sample site. In the laboratory experiments, all the sea fans harbored Symbiodinium clade B throughout the experiment, regardless of temperature treatment or length of exposure, indicating that Symbiodinium clade does not change when the sea fan host is infected with the Aspergillus fungus. All samples except one harbored the B184 type of the hypervariable region of domain V of the chloroplast 23S large subunit rDNA. This is the most common cp-23S-rDNA type found in Caribbean gorgonians (Santos et al., 2003a) and is equivalent to ITS-type B1 (based on the internal transcribed spacer region of the nuclear rDNA), as described in LaJeunesse (2002).

In the laboratory studies and at Eastern Sambo, allele frequencies at the microsatellite locus did not correlate with infection status. However, in the samples collected at Pickles Reef, the 108-bp allele was associated with infected G. ventalina more frequently than expected. Given that this is a single microsatellite locus, which is a putative neutral marker, it is unclear whether this finding is significant to the etiology of the disease. This allele was found abundantly in healthy fans from East Sambo. Therefore, it seems unlikely that the correlation between algae with this allele and infection status has any biological relevance. Significant differences in allele frequencies were also detected between sites. Larger sample sizes using more loci are currently being analyzed to determine the population structure of the algal symbionts harbored by G. ventalina in the Florida Keys. Previous work by Santos et al. (2003) also detected population structure among the symbionts of another member of the Gorgoniidae, Pseudopterogorgia elisabethae, on Bahamian reefs, with each reef harboring a unique Symbiodinium genotype.

Fewer algal symbionts were present in diseased tissue than in healthy tissue, although these tissues always harbored the same symbiont type. The loss of algal symbionts is highly correlated with infection with Aspergillus sydowii. Symbiont density is higher in samples from tissue peripheral to the infection site than in samples from the diseased tissue itself, but lower than in tissue sampled from healthy fans. Loss of algal symbionts from diseased fans could be considered bleaching caused by infection with the fungal pathogen. However, it is also possible that reduced zooxanthellar density is due to a reduction in the number of polyps in diseased tissue (Kim and Harvell, 2002).

Among the Symbiodinium isolated from Eastern Sambo colonies in the field, 64.4% (n = 104) of the samples produced a 565-bp fragment when amplified with the dinoflagellate-biased 18S-rDNA primers. The presence of a smaller amplicon in addition to the expected full-length rDNA following PCR has been reported in a number of free-living and symbiotic dinoflagellates (Santos et al., 2003b). Blast searches of Genbank have found that the 565-bp fragments are most similar to clade B 18S small subunit rDNA (M88509 submitted to Genbank by Rowan and Powers, 1992; and AF238257 submitted to Genbank by Toller et al., 2001). In the case reported here, the truncated copy is created by a deletion of approximately 1200 base pairs, similar to that described in Santos et al. (2003b). Some samples, when amplified with the primers SS5-SS3Z, contained only the shortened length of DNA. If a deletion generated the short segment of DNA (i.e., a pseudogene), a larger, functional copy of the gene must also be present. When another dinoflagellate-biased primer set, SS5Za+b-SS3, was used, the normal-sized DNA fragment was recovered. This indicates that the normal, functional copy of the 18S rDNA gene is present in the zooxanthellae from these samples and, because the samples also amplified with the universal primers SS5-SS3 (Rowan and Powers, 1991a), it suggests that a mutation may be present in the flanking region of the 18S rDNA near the binding site of the SS3Z primer.

As in the case of free-living dinoflagellates and other symbiotic dinoflagellates, the presence or absence of the putative "pseudogene" can be used to differentiate Symbiodinium types present in different study sites and experimental treatments (Santos et al., 2003b). Only G. ventalina collected from Eastern Sambo harbored Symbiodinium containing the aberrant PCR product. No samples from Pickles Reef or samples from Gorgonia ventalina in previous studies contained this aberrant copy of the 18S rDNA (Goulet, 1999; Goulet and Coffroth, 2004). The aberrant PCR product occurred--regardless of treatment and temperature--in 57.1% of 70 samples used in the laboratory experiment, and in 64.4% of all 104 colonies collected in Eastern Sambo. The presence or absence of the putative pseudogene can be used to distinguish taxa of Symbiodinium among sites, but no correlation occurs between the pseudogene and the infection status of the sea fan.

The Gorgonia ventalina symbiosis does not respond to stresses such as increased temperatures or fungal infection through symbiont shuffling or switching (sensu Baker, 2003). Although changes in symbiont type are correlated with increased temperature and some coral diseases (i.e., Rowan et al., 1997; Toller et al., 2001), symbiont change is not universal and appears to depend on the host species. Further studies will be necessary to understand the interaction of the host and symbiont under these stresses. This work further supports a growing number of studies that have demonstrated that many symbioses (perhaps the majority, see references in Goulet and Coffroth, 2003b) are extraordinarily stable and do not change seasonally or under different environmental conditions (Billinghurst et al., 1997; Diekmann et al., 2002; Goulet and Coffroth, 2003a, b; Rodriguez-Lanetty et al., 2003; Thornhill et al., in press.). Future work must focus not only on how symbioses might change, but on how less flexible symbioses survive in the face of disease and environmental change.

Acknowledgments

We thank S.R. Santos, T.L. Shearer, A.R. Hannes, J.L. Weaver, C.L. Lewis, D.M. Poland, K. Kim, J. Stamos, C.D. Harvell, K. Rypien, and A. Shaw for all their technical support and help. We thank S.R. Santos and H.R. Lasker for comments on the manuscript. The research was supported by NSF OCE-99-07319 (MAC), NSF OCE-9818830 (D. Harvell and K. Kim), NSF Graduate Research Fellowship (JRW), Edna Bailey Sussman Fund (JRW), an ISRS/OC fellowship (JRW), the Mellon Foundation (JRW), and the Cornell University Field of Zoology (JRW). We also thank the staff at the National Undersea Research Center and the Mote Marine Tropical Research Laboratory for their assistance. Collections were done under a special activity permit (Florida Fish and Wildlife Conservation Commission 02IMP-334) and FKNMS-2002-018.

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NATHAN L. KIRK (1), JESSICA R. WARD (2), AND MARY ALICE COFFROTH (1,*)

(1) Department of Biological Sciences, 109 Cooke Hall, University at Buffalo (State University of New York), Buffalo, New York 14260; and (2) Department of Ecology & Evolutionary Biology, Corson Hall, Cornell University, Ithaca, New York 14853

Received 16 November 2004; accepted 20 September 2005.

* To whom correspondence should be addressed. E-mail: coffroth@buffalo.edu
Table 1 Allelic variation at microsatellite locus CA6.38 among
Symbiodinium isolated from Gorgonia ventalina collected at Pickies and
Eastern Sambo Reefs

                        Allele size (bp)
Reef        Fan type  104  106  108  110

East Sambo
            H         0     8   15   0
            D         1     1   12   1
Pickles
            H         0    18    0   0
            D         0    12    7   0

The number of samples with each allele size for samples from healthy (H)
fans (not visibly infected with the Aspergillus fungi) and samples from
diseased (D) fans (those inoculated with the fungi) are shown.

Table 2 Density of zooxanthellae (Symbiodinium) in samples from healthy
and diseased (infected with Aspergillus fungi) specimens of Gorgonia
ventalina

             Mean X [10.sup.5] cells [cm.sup.-2]
Sample type  ([+ or -] std. dev.)

H:H          5.27 (1.46)
H:D          4.45 (1.64)
D:D          2.83 (2.42)

Cell counts are given as the average of 20 samples with the standard
deviations. (H:H) are healthy fans. (D:D) are infected areas of diseased
fans, and (H:D) samples are taken from diseased fans peripheral to the
infection site.
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Author:Kirk, Nathan L.; Ward, Jessica R.; Coffroth, Mary Alice
Publication:The Biological Bulletin
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Date:Dec 1, 2005
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