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1. Changing horses in midstream: Secondary zooxanthella uptake by the Caribbean octocoral Briareum asbestinum following an experimentally induced bleaching event 10 μm LEWIS, C L and COFFROTH, M A Department of Biological Sciences; University at Buffalo, NY Abstract: Coral bleaching events are typically characterized by the breakdown of the coral-algal symbiosis and the loss/expulsion of the algal symbionts (i.e., zooxanthellae). After a bleaching event, corals often re-establish their symbiotic zooxanthella population. Reconstituted zooxanthella populations are generally believed to derive from some combination of zooxanthella cells that remained in the host and newly acquired cells. However, the actual source of these zooxanthella symbionts has yet to be established. In this study we examined whether adult colonies of the octocoral Briareum asbestinum were capable of secondarily acquiring zooxanthella from the environment. Zooxanthellae populations within healthy colonies were genetically characterized based on their chloroplast 23S r-DNA (cp-genotype). These colonies were then induced to bleach by maintaining them in a darkened tank for 12 weeks. Zooxanthella genotype was reassessed at the end of the bleaching period. The bleached colonies were then placed in aquaria subject to natural lighting and inoculated with cultured zooxanthellae that had a rare cp-genotype. Subsequent chloroplast genotyping of the zooxanthellae within the B. asbestinum colonies revealed the presence of zooxanthellae identical to the specific zooxanthellae cultures used for inoculations. This is the first demonstration that octocorals are capable of secondarily acquiring zooxanthellae as adults. Introduction:Many coral species produce asymbiotic planulae that establish their symbiosis upon metamorphosis to the juvenile polyp stage. Juvenile clade specificity is indiscriminate and a sorting out of optimal symbionts usually occurs within the first year (Coffroth et al, 2001). Each coral species appears to preferentially establish and maintain a symbiosis with a specific clade of zooxanthellae, which may represent an optimization of its adaptation to its particular environment (Rowan & Powers, 1991). During a “coral bleaching event”, zooxanthellae leave or are expelled and the symbiosis is dissolved in response to various environmental changes (e.g., variations in temperature, light levels, salinity). Most corals must re-establish their symbiosis or perish. After a bleaching event, the coral animal is often able to reestablish a symbiosis, provided the coral has not been irreparably damaged (Toller et al, 2001; Baker, 2001). The source of symbionts is unknown but is presumably from either residual symbionts within the host tissue or from an environmental pool. Identification of the Symbiodinium spp.within a host can be determined to the clade level using Restriction Fragment Length Polymorphism (RFLP) of the 18S small subunit nuclear rDNA (Rowan & Power 1991). Clades of Symbiodinium can further be characterized by Polymerize Chain Reaction (PCR) amplification of the 23S rDNA region of the symbiont’s chloroplast, followed by electrophoresis through a polyacrylamide gel. Chloroplast genotype is designated according to base pair number and can be determined by fragment size relative to a DNA reference ladder (Santos et al, 2002a,b). The purpose of this study was to determine whether Briareum asbestinum could acquire new zooxanthellae from the environment after a bleaching event. The symbiont chloroplast genotype was tracked over time: before bleaching, after bleaching, and after inoculation with zooxanthellae cultures of a novel genotype (marker allele cultures). Figure 7: A typical polyacrylamide electrophoresis gel illustrating the uptake of zooxanthellae with the marker alleles by adult Briareum asbestinum. Representative colonies are shown from two treatment groups. Zooxanthellae, sampled from each individual colony pre-bleaching, post-bleaching, and post inoculation, are shown in lanes labeled 1, 2, and 3, respectively. The first three colonies were inoculated with marker allele culture 224bp (MA-224), the second three with marker allele culture 211bp (MA-211). “L” indicates the DNA ladder used for size reference. Arrows indicate acquisition of the zooxanthellae containing the marker allele in five out of the six colonies shown. Figure 6: Treatment tanks for the inoculation of the B. asbestinum colonies. Colonies were maintained in 20 gallon aquaria with circulating, aerated FSW (six colonies per tank). Aquaria were partially submerged in a water bath of flow-through sea water to maintain water temperature. The water was changed every three days for six weeks. Tanks were inoculated with appropriate Marker Allele (MA) cultures after each water change, at a density of 1000 cells per ml (Tank 1 & 2 MA-224; Tank 3 & 4 MA-211; Tank 5 MA-223; Tank 6 Control). Crypt colonies were situated between Tank 6 and the crypt wall, near the intake port for the sea water. Figure 5: Bleached colonies were moved to aquaria containing circulating, filtered (0.2 μm) sea water (FSW) and treated with antibiotics (“antibiotic cocktail” Polne-Fuller, 1991) for three days. Antibiotic treatment was designed to reduce stress-related bacterial infection after bleaching. Colonies were then divided among the six treatment tanks. Conclusions: Adult Briareum asbestinum are capable of acquiring new algal symbionts from their environment following an experimentally induced bleaching event. The Adaptive Bleaching Hypothesis (Rowan and Powers 1991, Buddemeier and Fautin 1993) postulates that after a bleaching event, different zooxanthellae, better suited to the change in the host’s habitat, may repopulate corals. Many studies have shown that asymbiotic juvenile anthozoans (Coffroth et al 2001 and references therein) and scyphozoans (Fitt & Trench 1983) can take up zooxanthellae from the water column. It has also been demonstrated that adult actiniarians (anemones) can secondarily acquire zooxanthellae from the water column after a bleaching event (Kinzie et al 2001 and references therein, Belda-Baillie et al 2002 and references therein). However, to date, this has not been documented in adult hard corals or gorgonians (the predominant members of coral reefs). This study is the first to demonstration that adult Octocorals (Alcyonacea) are also capable of acquiring new zooxanthellae from their environment. Figure 2: Fragment sizes (bp) of Symbiodinium cp23S-rDNA domain V alleles and their relationship to the Symbiodinium cp23S-rDNA phylogeny (Santos, et al in press). Throughout the Caribbean, adult B. asbestinum commonly form a symbiosis with clade B zooxanthellae of chloroplast genotypes 178 bp and/or 184 bp, in blue. The rare “marker allele” cultures used, also clade B zooxanthellae, are chloroplast genotypes 211 bp, 223 bp, and 224 bp, in red. Results: Table 2:Briareum asbestinum acquisition of zooxanthellae with marker allele after six weeks of inoculations. *One colony sampled in Control Tank 6 contained 223bp genotype, after six weeks of inoculation. It is likely that this was contamination due to the close proximity of MA-223 Treatment Tank 5 to Control Tank 6 (see figure 6).No marker allele genotypes were found in the Crypt Colonies (maintained in the flow-through sea water outside of the aquaria). Methods: Figure 3: Flow through sea water crypt where 40 adult B. asbestinum colonies were maintained for 12 weeks (March 5th to May 27th) to experimentally induce bleaching by limiting ambient light. Acknowledgements:We would like to thank the entire staff at the Florida Keys Marine Lab, Long Key, FL for the use of their facilities and their gracious assistance during the field season. We would also like to thank K. Benz, P. Bouwma, P. Bowerman, M. Dorner, A. Hannes and C. Lewis for their part in helping to maintain and sample the Briareum colonies. This research was supported by NFS Grant OCE-95-07319 (MAC). References cited: Belda-Baillie, C A; Baillie, B K; Maruyama, T; (2002) Specificity of a Model Cnidarian-Dinoflagellate Symbiosis; Biol. Bull. 202: 74-85 Buddemeier, RW; Fautin, DC; (1993) Coral Bleaching as an adaptive mechanism Bioscience 43(5): 322-326 Coffroth, MA; Santos, SR; Goulet, T L; (2001) Early ontogenetic expression of specificity in a cnidarian-algal symbiosis; Marine Ecology Progress Series; 222: 85-96 Fitt, W K; Trench, R K; (1983) Infection of coelenterate hosts with the symbiotic dinoflagellate Symbiodinium microadriaticum: Endocytobiology Vol. II: Intracellualar Space as Oligogenetic Ecosytem; Proceedings 2nd International Colloquium on Endocytobiology; Tubingen, Germany Kinzie, R A; Takayama, M; Santos, S R; Coffroth, M A; (February 2001) The Adaptive Bleaching Hypothesis: Experimental Tests of Critical Assumptions; Biol. Bull. 200: 51-58 Polne-Fuller, M. (1991) A novel technique for preparation of axenic cultures of Symbiodinium (Pyrrophyta) through selective digestion by amoebae J. of Phycology 27:552-554 Rowan, R; Powers, D A; (1991) A molecular genetic classification of zooxanthellae and the evolution of animal-algal symbiosis; Science; 251:1348-1351 Santos, S; Gutierrez-Rodfiguez, C; Coffroth, M A; (in press) Phlyogenetic identification of symbiotic dinoflagellates via length heteroplasmy in domain V of the chloroplast large subunit )cp23S)-rDNA sequences Marine Biotechnology 7/02 Santos, S R; Taylor, D J; Coffroth, M A; (2001) Genetic comparisons of freshly isolated vs. cultured symbiotic dinoflagellates: implications for extrapolating to the intact symbiosis Journal of Phycology 37: 900-912 Santos, S R; Taylor, D J; Kinzie, R A; Sakai, K; Coffroth, M A; (2002a) Evolution of length variation and heteroplasmy in the chloroplast rDNA of symbiotic dinoflagellates (Symbiodinium, Dinophyta) and a novel insertion in the universal core region of the large subunit rDNA; Phycologia; 41(4): 311-318 Santos, S R; Taylor, D J; Kinzie, R A, Hidaka M, Sakai, K; Coffroth, M A; (2002b) Molecular phylogeny of symbiotic dinoflagellates inferred from partial chloroplast large subunit (23S)-rDNA sequences Molecular Phylogenetics and Evolution 23: 97-111 Toller, W W; Rowan, R; Knowlton, N; (December 2001) Repopulation of Zooxanthellae in the Caribbean Corals Montastrea annularis and M. faveolata following Experimental and Disease-Associated Bleaching; Biol. Bull. 201: 360-373 Figure 1:Symbiodinium spp. (cultured from Plexaura kuna), photosynthetic dinoflagellate endosymbionts (zooxanthellae) found in a wide variety of Cnidarians. (photo by A. Siegel) Table 1: Single lines of algal symbionts were cultured and maintained under laboratory conditions (Santos et al, 2001). Chloroplast genotypes of these selected cultures were rare marker alleles, not commonly found in adult Briareum asbestinum around the Caribbean. A B Figure 4: Visual effects of experimentally induced bleaching on Briareum asbestinum. Normal B. asbestinum colony (A), with polyp pigmentation due to algal symbiont. Bleached B. asbestinum colonies(B), lacking pigmentation.