Galaxea, Journal of Studies 11: 1-11（2009）

Original paper

Coral symbiotic complex: Hypothesis through

vitamin B12 for a new evaluation

Sylvain AGOSTINI1, *, Yoshimi SUZUKI1, Beatriz E. CASARETO1, 2, Yoshikatsu NAKANO3, Michio HIDAKA3, and Nesa BADRUN3

1 Shizuoka University, 836 Oya, Suruga-ku, Shizuoka 422-8529, Japan 2 Laboratory of Aquatic Science and Consultant Co. Ltd., 1-14-1 Kamiikedai, Meishin, Ota-ku, Tokyo 145-0064, Japan 3 University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan

* Corresponding author: S. Agostini E-mail: [email protected]

Communicated by Hiroya Yamano (Environment and Conservation Editor)

Abstract Historically, hermatypic corals are defined as a prokaryotes are the drivers of internal processes, such as symbiotic system composed of an host, corals vitamin B12 production, occurring directly inside the coral, belonging to the taxa Cnidarian, and a photosynthetic forming a semi-closed system. Furthermore, most main­ organism, the dinoflagellate Symbiodinium spp., also tenance of the symbiotic complex is due to internal pro­ known as zooxanthellae. The high gross productivity and cesses rather than the supply from outside the coral. stability of coral reefs have been explained by the effi­ ciency of the coral-algal symbiotic system in using the Keywords corals, coelenteric , symbiosis, vitamin low nutrient concentrations found in the surrounding B12, semi-closed system water and their rapid recycling in the water. Although several studies have reported the presence of bacteria closely associated with corals, the mechanisms of the relationships among them, the host and the zooxanthellae, Introduction remain to be shown. In this study, evidence for the im­ portance of coelenteric bacteria in corals as a component The symbiotic relationship between a coral host and its of the coral symbiotic complex was shown by using a new zooxanthellae consists of the production of organic matter, approach. Vitamin B12, which is produced only by pro­ mainly carbohydrates, by the zooxanthellae and their karyotes, was chosen as a chemical tool to clarify the translocation to the coral at rates of 60-90% (Leletkin symbiotic relationships among bacteria, coral, and zoo­ 2000). The coral uses these carbohydrates to grow, and its xanthellae, which require vitamin B12. High vitamin B12 catabolites, especially CO2 and nutrients, are then used by concentrations (up to 700 pmol l−1 compared with max. the zooxanthellae. This relationship can be defined as 20 pmol l−1 in the surrounding water) and high bacteria mutualism or symbiosis in the strict sense of the term abundances (100 times higher than surrounding water) because there are reciprocal benefits for both organisms. were found in the coelenteron of live corals using a new From the 1970’s through the 80’s, bacteria in coral reefs sampling method. The results led to the hypothesis that were mainly considered as responsible for the regeneration 2 Agostini et al.: Coral symbiotic complex of nutrients through the decomposition of organic matter hypothesis that coral forms a semi-closed system was in the water column and sediment, and as a food source used, meaning that there is only limited exchange between for and benthic organisms. Recently, bacteria corals and the surrounding water, and internal processes have been shown to cause coral bleaching and other involving bacteria occur and create a micro-environment diseases (Rosenberg et al. 2007 and references therein), suitable for the zooxanthellae and coral. To investigate the and therefore the scientific community has taken an linkage between the coral and bacteria we chose vitamin interest in coral-associated bacteria. Qualitative studies B12 because it is known to be produced only by prokaryotes have described the associated bacteria, especially the (Warren et al. 2002). Moreover, vitamin B12 is known to bacteria living in the mucus on the surface of corals be required by all and by a majority of phyto­ (Ducklow and Mitchell 1979), and some concluded the plankton (Croft et al. 2005). However, the requirement of specificity of these communities (Rohwer et al. 2001; zooxanthellae for vitamin B12 has not been studied until Bourne and Munn 2005). Archae were also reported to now. live in association with corals (Kellogg 2004; Wegley et Porter (1978) described a method for sampling the al. 2004). One study, based on microscopic investigation, coelenteric fluid of corals to investigate the feeding of reported the discovery of a symbiotic cyanobacteria with­ corals. The gastral contents of giant anemone were sam­ in the coral tissue (Lesser et al. 2004). Inoculation with pled using Porter's methods (Herndl et al. 1985). In both bacteria to provoke disease in corals led to the discovery cases, the sizes of the polyps allowed manual sampling of several coral pathogenic bacteria (Bourne 2005; with a syringe. Concentrations of bacteria ranging from Rosenberg et al. 2007). Vibrio shiloi successfully provoked 105 up to 5×106 cells ml−1 were found in the anemone bleaching of the coral Oculina patagonica, but the coral coelenteron. These results led us to hypothesize that the showed resistance to this pathogen after several inocu­ coelenteron of corals contains high abundances of bacteria lations, thereby suggesting an adaptation of the sym- and so could be the location of vitamin B12 production. To biotic complex. The probiotic hypothesis (Reshef et al. verify this hypothesis, bacteria abundances and vitamin

2006), followed by the hologenome theory of evolution B12 levels were measured in the gastral cavity of the polyps (Rosenberg et al. 2007), that a dynamic relationship be­ from coral colonies of fascicularis. In order to tween symbiotic microorganisms and environmental con­ take samples of the coelenteric fluid of coral, Porter’s ditions brings about the selection of the most advantage­ method was modified mainly by miniaturizing it to fit the ous coral holobiont, were then proposed. However, the polyp mouth, which is less than 1 mm. With our method, mechanisms of the relationship remained unknown. Ex­ the sample volume is limited to less than 1 ml. Therefore, cept for the study of Rosenberg et al. (2007), the role of the usual methods for measuring bacteria abundance using coral-associated bacteria is still poorly documented. In epifluorescence microscopy and vitamin B12 using HPLC order to study the relationships among the bacteria, coral and bioassay are not applicable; thus, new methods were host, and zooxanthellae, a biological approach alone is not applied. sufficient—the biochemical processes involved must also be described. The only notable quantitative study of the relationship between coral and bacteria highlighted the Materials and methods importance of endolithic bacteria in the coral skeleton as a nitrogen fixer, thus providing assimilable fixed nitrogen Study sites to the coral (Shashar et al. 1994). Water samples to measure vitamin B12 levels in surface Until now, coral metabolism has been studied by mea­ water were taken from the reef flat at Sesoko Beach, suring parameters outside the coral, i.e., in the surrounding Okinawa, Japan. Two fixed stations were chosen: station water, supposing that nutrients and any other chemical A (26°38′57.4″N, 127°51′13.9″E), characterized by higher compounds were mainly supplied by exchange of water coral coverage (1.7±5.1%, average±SD), was dominated from outside (Falter et al. 2004). In this report, the by Psamocora contigua, Goniastrea aspera, Montipora Agostini et al.: Coral symbiotic complex 3 digitata, Millepora exaesa, and station B (26°38′44.6″N, sampling tube that was kept in the dark. The volume 127°51′15.8″E), with lower coral cover­age (0.5±1.5%, collected under anesthesia by this method was 0.5 to 1 ml average±SD), was dominated by Goniastrea aspera, and by sampling up to 50 polyps of the same colony. Millepora exaesa, Favia spp., and Porites massive spp (as of 2007). Colonies of Galaxea fascicularis were taken Determination of bacteria abundance from the reef in front of Sesoko Station, University of the Flow cytometry was used to determine the abundances Ryukyus, Okinawa, Japan, and kept at the station in an of bacteria in the coelenteric fluid. Bacteria were stained aquarium with a continuous flow of seawater. with SYBR-Green and then counted using a Beckman Coulter flow cytometer. Bacteria abundances in surround­

Vitamin B12 requirement of zooxanthellae ing water were determined by epifluorescence using DAPI Zooxanthellae of clade A, isolated from Cassiopea, staining and a 0.2 µ black membrane filter (Nucleopore) were cultured in different conditions. Because the ino­ (Porter and Feig 1980). culum was not axenic, antibiotics (polymixin B sulfate −1 −1 100 µg l and ampicillin 100 µg l ) were added to limit Vitamin B12 concentration the in situ production of vitamin B12 by bacteria. Finally, The only methods available to determine the concent­ four conditions were tested with four replicates each: ration of vitamin B12 in sea water are HPLC, which requires modified f/2 medium with or without vitamin B12 (1.5 several liters (Okbamichael and Sañudo-Wilhelmy 2004), nmol l−1), and each with or without antibiotics, in a 2×2 and bioassay, which requires several milliliters (Carlucci factorial design. First, cells were cultured, and in vivo and Bowes 1972). These methods are not applicable to chlorophyll fluorescence was measured every day and samples with a very limited volume, so a new radioassay converted to cells ml−1 using a standard made from the method was developed. HPLC methods was used for inoculum. Then the cultures were transferred to new determination of the vitamin B12 concentration in surface medium with the same conditions. water. The original method (Okbamichael and Sañudo- Wilhelmy 2004) was slightly modified. Coelenteric fluid sampling Radioassay. Radioassay was commonly used for deter­

Galaxea fascicularis was chosen for this experiment mination of the level of vitamin B12 in human blood (Houts because of the size and structure of its polyps. To sample and Carney 1981), and attempts were made to use it for the coelenteric fluid, a specimen was transferred to a sea water by using a different binder to decrease the smaller aquarium. To avoid retraction of the polyps during detection limit (Sahni et al. 2001). The method used in sampling, the coral was anesthetized by scattering ground this study was based on competitive binding radioassay crystals of menthol (Moore 1989) on the surface of the using the binder and tracer from the commercial kit water and putting the aquarium in the dark for 45 min. Simultrac-SNB (MP Biomedicals). It was optimized for Coral could be kept insensitive for 3 h, until the water was seawater and required a sample of less than 0.5 ml (mea­ changed. After the water change, the coral recovered surement in duplicate). The standards were done in vita­ quickly and could be anesthetized several times in one min B12-free seawater or artificial sea water. Some samples day without any harm. Coelenteric fluid was sampled were boiled 30 min to verify that no endogenic binder using a glass capillary mounted on a micromanipulator interfered with the assay. The samples (200 µl) were (Sutter Instrument model MM-33) under a zoom stereo­ incubated with 50 µl of [57Co]-labeled cyanocobalamin­ microscope. The capillary used for sampling was made and 750 µl of the binder (purified porcine intrinsic factor from glass tubing and its outer diameter was less than bound on a solid support) for 2 h at constant temperature 1 mm. The diameter can be adjusted to the polyp size. and shaken slightly. After centrifugation at 1000 g for After introduction of the capillary into the mouth, the 10 min, the supernatant was gently discarded, and the gastral content was gently extracted using a vacuum pump radioactivity of the pellet was counted for 5 min using a (maximum vacuum 0.09 Bar). The fluid was collected in a gamma counter (Aloca, ARC-380, Tokyo Japan) set up 4 Agostini et al.: Coral symbiotic complex for [57Co] with a window of 100-180 KeV. Con­centrations were read against standards made with pure cyanocobala­ min (Sigma-Aldrich) in vitamin B12-free seawater plotted Results with a logit-log scale.

Modifications of HPLC methods. Five liters of seawater Vitamin B12 requirement of zooxanthellae was filtered using a 0.2 µm cartridge filter (Advantec, type Zooxanthellae grew in all four types of medium and TCYE-NS-S1FE). Strata X (Phenomenex) solid phase replicates during the first culture. The growth rates were extraction column was used to extract the vitamin B12. determined by measuring in vivo fluorescence every day, Elution was done using 5 ml of methanol. The eluate was and the growth curves are shown in Figure 1. The growth evaporated and the volume adjusted to 0.5 ml with MilliQ rates with and without vitamin B12 in the exponential water. The final concentration factor was of 10,000. phase during the first culture showed no significant dif­ Cyanocobalamin concentrations were determined using ference (ANOVA p＞0.05) . However, the abundances in an HPLC equipped with a C18 column (Phenomenex) the final stage of this experiment showed a significant and a UV-Visible detector. difference between cultures with and without vitamin B12 Comparison HPLC radioassay. Seawater was collected in the presence of antibiotics (Tukey’s HSD, α＝ 0.05, p＝ at a depth of 2 m in Suruga Bay, Shizuoka, Japan, and 0.0001). Also no significant difference was found between brought immediately to the laboratory, where it was fil­ with and without vitamin when the bacteria production tered through a 0.2 µm cartridge filter (Advantec, type was not limited by antibiotics (Tuckey HSD, α＝ 0.05, TCYE-NS-S1FE). Cyanocobalamin was dissolved in the p＝0.66). During the second culture, an inoculum from seawater at different concentrations to obtain approximate the first culture was transferred to new medium. The zoo­ concentrations that were higher than the radioassay and xanthellae grew in all cases except for the case without

HPLC detection limits after concentrating 1 l to 1 ml. vitamin B12 but with antibiotics. These results show that

The radioassay used in this study had a detection limit vitamin B12 is an essential micronutritient for zooxanthellae. −1 of 35 pmol l (when defined as 3 times the standard The vitamin B12 requirement was shown only in the deviation of the trace binding). The concentrations of cyanocobalamin detected by HPLC and radioassay in the prepared solutions are shown in Table 1. No significant differences were found between the two methods. Dif­ ferences observed may be due to the extraction of bound vitamin B12 (as opposed to free vitamin B12) during solid phase extraction. Bound vitamin B12 was not measured by radioassay.

Table 1 Comparison of radioassay and HPLC for de­ termination of cyanocobalamin concentration in natural seawater

Fig. 1 Growth curves of zooxanthellae under different

conditions: B12＋AB＋(▲): cyanocobalamin and antibiotics

were added ; B12＋AB-(▼): only cyanocobalamin was added;

B12−AB＋(■): only antibiotics were added, B12−AB-(◆): neither cyanocobalamin nor antibiotics were added. Error bar represent the standard error (n＝4) Agostini et al.: Coral symbiotic complex 5

presence of antibiotics, that is, when bacteria production Vitamin B12 concentration in surface water and coe­ is limited; when no vitamin B12 and no antibiotics were lenteric fluid added, the zooxanthellae grew as much as with the Vitamin B12 concentrations in samples are shown in addition of vitamin. Table 2. Vitamin B12 concentrations in surface reef waters were measured by HPLC after pre-concentration by solid Bacteria abundances in surface water and coelenteric phase extraction. Concentrations were found to vary be­ fluid tween 5 and 20 pmol l−1 with an average of 10.2±7.0 Bacteria were stained with SYBR-Green and counted pmol l−1 (mean±SD). Variations in concentration were by flow cytometry. Thus, only living bacteria were count­ observed between days of sampling, but no difference was ed. Abundances were always higher in the coelenteric observed for samples taken at both stations at the same fluid at 7.5×106 to 4.6×107 cell ml−1 (Fig. 2) than in the time. 5 −1 surrounding aquarium water at 3.54×10 cell ml and In the coelenteric fluid, the vitamin B12 concentrations 5 −1 −1 −1 natural surface water at an average of 3.9×10 cell ml . ranged from 108 pmol l to 704 pmol l . Vitamin B12 in Variations in abundance were observed, but no trend, such surface water and aquarium water was not detectable by as diurnal variation, was observed. Bacteria abundances radioassay. The difference between vitamin B12 concent­ in the aquarium water remained quite constant during the rations in the coelenteric fluid and surrounding water was sampling period and did not seem to have a remarkable 1-2 orders of magnitude. The correlation between the affect on the coelenteric abundances. Picocyanobacteria concentration of vitamin B12 and the abundance of bacteria (Synechococcus spp. and Prochlorococcus spp.) abun­ could not be investigated because the glutaraldehyde used dances were also quantified once, and concentrations for preservation of the bacteria seemed to interfere with similar to that of aquarium water: Synechococcus spp. the radioassay. As shown in Table 2 and Figure 3, the 2 −1 2 −1 at 8.9×10 cell ml (aquarium) and 6.1×10 cell ml concentrations of vitamin B12 were low in the evening (coelenteric) and Prochlorococcus spp. at 4.8×102 cell and night at 108-160 pmol l−1, except once at midnight ml−1 (aquarium) and 6.1×102 cell ml−1 (coelenteric), were (647 pmol l−1), and were high in the daytime at 293- −1 observed. 708 pmol l . These could mean that vitamin B12 is taken up by the zooxanthellae through during

Fig. 2 Coelenteric bacteria abundances May 2007 gray zone Fig. 3 Concentrations of vitamin B12 observed in coelenteric represents night, the dotted line (May 2007) represents the cavity. Error bars represent the standard deviation; if no error bar variation range found in natural sea water, and the striated zone is present only one measurement could be done. ＋× is not represents the standard deviation (n＝16) (average 3.9×105± included in the exponential regression, and the standard deviation 1.4×105) for this point is 0 (See Table 2 for details) 6 Agostini et al.: Coral symbiotic complex

Table 2 Vitamin B12 concentrations at different locations. Samples for determination of the

vitamin B12 concentration in aquarium water at the time of each coelenteric sampling were taken but were not detectable by radioassay. a Galaxea fascicularis colony A, b Galaxea fascicularis colony B, c Galaxea fascicularis colony C, d Galaxea fascicularis colony D, e Sesoko Beach Station A, f Sesoko Beach Station B, ±standard deviation of n replicates

the day, and is produced by the bacteria, not only at night, possible only if there is a reliable external supply in the but also during the day. More samples collected just before environment. Zooxanthellae, as symbionts, fit the con­ sunrise are required to confirm this interpretation. ditions described by Croft well, the reliable external supply being possibly a symbiotic relationship with other components of the coral symbiotic complex. Therefore, Discussion zooxanthellae may have lost the capacity to synthesize

vitamin B12 or became B12 auxotrophs due to changes in

Vitamin B12 requirement of zooxanthellae their metabolic enzymes, i.e., from vitamin B12-independ­

The requirement for vitamin B12 by the zooxanthellae ent methionine synthase (metE) to vitamin B12-dependent studied could be shown only when bacterial production methionine synthase (metH), during their evolution. Such was not limited by antibiotics. This result suggests the changes are common in symbionts, who often evolve possibility of a direct translocation of the vitamin between towards a simplification of their metabolism when it can the bacteria present in the culture and the zooxanthellae. be compensated by a supply by a symbiont partner. Thus,

Similar results and conclusions were found for phyto­ the requirement for vitamin B12 by zooxanthellae can be plankton by Croft et al. (2005). The vitamin B12 require­ accepted, while the source of the vitamin for the coral- ment was shown for a wide range of phytoplankton and algal symbiotic system remains to be investigated. algae. In his review, Croft et al. (2006) concluded that auxotrophy of vitamins may have arisen because vita- Vitamin B12 in coral reef surface water mins are difficult to synthesize and required in only trace No other data are available for coral reefs water. Con­ amounts, and suggested that there may be a selective centrations in vitamin B12 in coastal surface water ranging advantage in dispensing with their production, which is from 2-446 pmol l−1 were measured by the HPLC method Agostini et al.: Coral symbiotic complex 7

(Okbamichael and Sañudo-Wilhelmy 2004), and the derstand the dynamics of vitamin B12 in reef waters. lowest concentrations in surface water were found at However, the concentrations found here, 5-20 pmol l−1, Gardiners Bay and Shelter Island Sound and the highest may not be sufficient to satisfy the requirements of the ones in brackish water overlying sediments of Flax Pond coral host and the zooxanthellae. These results imply the and in the headwaters of Stony Brook Harbor, NY. existence of another source in order to support the growth Considering the oligotrophic nature of reef water, con­ of the coral-algal symbiotic system. A possible source of centrations in the lower range of the previous study vitamin B12 may be internal production inside the semi- (Okbamichael and Sañudo-Wilhelmy 2004) may be rea­ closed system formed by the coral symbiotic complex. sonable. However, there is no consensus on the critical concentration required by marine organisms. Some au­ Coelenteric fluid: chemical and biological characteristics thors reported minimum required concentrations as low Concentrations of vitamin B12 as high as those found in as 0.1-0.3 pmol l−1 (Droop 2007) for diatoms and as high coral coelenterons have not been reported in most seawater as 62-97 pmol l−1 (Hoffmann 1990) for the chlorophyte environments, with the exception of water in areas with Cladophora glomerata, with an intermediate value of 7 high bacterial activity such as the brackish water overlying pmol l−1 (Croft et al. 2005) for Dinophytes, Rhodophytes, sediments in Stony Brook Harbor (Okbamichael and and a Euglenophyte. However, several studies reported Sañudo-Wilhelmy 2004). Finding high abundances of limitations (Bertrand and Saito 2007) or at least changes bacteria and high concentrations of vitamin B12 together in the composition of phytoplankton communities and supports the idea that high bacterial activity results in high its dynamics in natural water at vitamin B12 levels rang- production of vitamin B12 in the coelenteric fluid. Probably −1 ing from 5-87 pmol l (Sanudo-Wilhelmy et al. 2006). the concentrations in vitamin B12, as standing stock, could Because we analyzed only a few samples, variations be determined by the balance between production by bac­ observed between the days of sampling cannot be linked teria and consumption by the coral host and zooxanthellae. to biological (i.e., photosynthesis activity) or physical Thus, the abundance of bacteria and the concentration of changes (i.e., tidal cycle). More data are required to un­ vitamin B12 may not directly reflect the dynamic of the

Fig. 4 Schema of symbiotic relationship of symbionts of the coral system. a) Current scheme: only relationships between zooxanthellae and host and strong exchange between the inside of the coral and the surrounding water are

considered, and b) proposed scheme: coelenteric bacteria produce vitamin B12 and translocations of compounds among the bacteria, zooxanthellae, and coral are added. Coral is considered to be a semi-closed system, meaning that water exchanges between the inside of the coral and the surrounding water are limited 8 Agostini et al.: Coral symbiotic complex processes. The diurnal variations found suggest that there may be due to a higher grazing of zooplankton during the is an interactive relationship between zooxanthellae and night, because higher abundances of these in the sur­ bacteria through vitamin B12 within the coelenteric cavity. rounding water were reported after sunset (Sorokin 1990; Furthermore, according to Starr et al. (1957), different Heidelberg et al. 2004). bacteria produce different amounts of vitamin B12. Higher abundances of bacteria and vitamin B12 con­ Therefore, simply to establish a relationship between centrations in the coelenteric fluid than in the surrounding abundances of bacteria and vitamin B12 concentrations water could be explained with the assumption that corals may not be relevant. The vitamin B12 concentrations found form a semi-closed system. The biological structure of in the coelenteron may also include some vitamin B12 coral also supports this hypothesis because corals have present in the coral tissues because during sampling (as­ narrow paths (mouths) for exchange of water between the piration of coelenteric fluid), mesenteric filaments were surrounding environment and the internal fluid. also aspirated, resulting in lesions of the host tissue. The Our results suggest that zooxanthellae use the vitamin vitamin B12 requirement of zooxanthellae may be around B12 produced by coelenteric bacteria. Therefore, the coe­ several pmol l−1, considering the high concentration found lenteric bacteria may play one of the main roles in the in the coelenteron, vitamin B12 should not limit their maintenance of the coral symbiotic complex as producers growth. of vitamin B12, which is then used by the coral host and Heterotrophic nutrition should also be considered to the zooxanthellae (Fig. 4). Feeding on organic matter by explain the high concentrations of vitamin B12 in the corals (Sorokin 1973) seems to provide a protected and coelenteron. The corals are known to feed on zooplankton, enriched organic environment, so that bacteria are able to phytoplankton (Porter 1978), bacteria (Ducklow 1990), multiply rapidly. As shown in Figure 4b, there are strong cyanobacteria (Houlbrèque et al. 2006), and particulate linkages among coelenteric bacteria, zooxanthellae, and organic matter (Sorokin 1973). B12-active substances were coral through vitamin B12 and organic matter. Therefore, it detected in marine bacteria cell pellets (Starr et al. 1957). is reasonable to consider the coelenteric microbes as a Prochlorococcus spp. were reported to synthesize vitamin third symbiont in the coral symbiotic complex.

B12 (Sullivan et al. 2005), which indicates that picocyano­ Although our research focused on vitamin B12, asso­ bacteria cells may also contain vitamin B12. Phytoplankton ciated prokaryotes may be the drivers of internal processes were reported to require a minimum cellular concentration for other chemical compounds. Herndl et al. (1985) −1 of vitamin B12 of about 1.4 pg µg C for Pavlova lutheri studied the abundance of coelenteric bacteria of giant and 0.8-1.3 pg µg C−1 for Cladophora in order to have anemones in two different states, fed and starved, and their growth not limited (Hoffmann 1990). Intracellular concluded that the anemone farmed bacteria in its coe­ vitamin B12 levels of zooplankton have not been reported lenteron in order to use them as a source of organic matter. in the literature but may be high considering that they feed However, coelenteric bacteria were evaluated as a minor on bacteria through the microbial loop. Therefore, diges­ source of organic matter under natural conditions. More­ tion of ingested living and non-living organic matter in the over, even when starved, the anemone did not digest all its coelenteron would release the vitamin B12 contained in bacteria and seemed to maintain a minimum abundance. this matter. The bacteria quantified in this report were They concluded that the coelenteric bacteria might be living bacteria, indicating that balance between digestion essential for anthozoans. The role of associated bacteria in and growth of bacteria is achieved in the coelenteron. This the production of different compounds including mono- shows that internal production of vitamin B12 by coelenteric and disaccharides, alcohols, sugar acids, and amino acids, bacteria should also be considered as well as its release removal of waste, contribution in the structural rigidity, from digested organic matter. Higher concentrations in and uptake of dissolved organic matter was also shown in vitamin B12 in the morning were interpreted as the result of sponges by Wilkinson (1978a, b, c). Others studies also a lower uptake by the coral-algal symbiosis system during suggested different roles for coelenteric bacteria, such as the night, but a significant contribution to this increase providing trace elements, vitamins (Sorokin 1973), or Agostini et al.: Coral symbiotic complex 9 antibiotic substances (Burkholder 1973). pecially related to the fact that corals thrive in oligotrophic Until now, the high gross productivity of corals was seas. Further studies using the method described in this explained by rapid recycling of nutrients in the water or in report, i.e., sampling the internal fluid of living coral with the sediment driven by bacteria (Ducklow 1990; D'Elia a minimum of perturbations, are required to explore the and Wiebe 1990) and/or exchange of reef water by tidal processes occurring inside the coral. Variations of the cycles. Direct evidence suggests that like the production internal chemical and biological compounds due to of vitamin B12 occurring inside the coral itself, the high changes in the environment, such as a temperature rise, gross productivity of the zooxanthellae may also be due to must be investigated in order to understand coral phys­ internal recycling of nutrients and micronutrients rather iology and, in consequence, the mechanisms of bacteria- than uptake from the surrounding reef water. A similar provoked disease, coral bleaching, the stability of coral conclusion concerning endolithic organisms as part of the reefs over time, and their future. symbiotic complex throughout nitrogen fixation was reported (Shashar et al. 1994). Previously, prokaryotes have not been included in Acknowledgments theories of coral symbiosis by showing the mechanisms of their relationships, with the exception of the previously We thank K. Takagaki, Shizuoka University, Japan, for cited papers of Reshef et al. (2006), with prokaryotes HPLC measurement of vitamin B12 in surface water and contributing to coral defense, and Shashar et al. (1994), assistance during the development of the radioassay. This with prokaryotes contributing a supply of fixed nitrogen, work was supported by grants from Mitsubishi Corpo­ as summarized in the review of Rosenberg et al. (2007). ration, the Ministry of Education, Culture, Sports, Science Furthermore, corals were thought to form an open system and Technology, and a scholarship for foreign students having a robust water exchange with the surrounding from Honda Benjiro. water. Our results lead to a fundamental reevaluation of the principles of the coral symbiotic complex which consists of (1) essential compounds such as vitamin B12 References and nutrients for the symbiotic complex being produced through internal processes driven by bacteria, (2) coral Bertrand E, Saito M (2007) Vitamin B12 and iron colimitation of forming a semi-closed system that allows maintenance of phytoplankton growth in the Ross sea. Limnol Oceanogr high concentrations of essential chemical compounds, 52: 1079-1093 thus sustaining productivity, and (3) the coral symbiotic Bourne D (2005) Microbiological assessment of a disease complex including several components such as the host, outbreak on corals from Magnetic Island (Great Barrier Reef, Australia). Coral Reefs 24: 304-312 zooxanthellae, coelenteric prokaryotes (as shown in this Bourne D, Munn C (2005) Diversity of bacteria associated with report), and possibly other associated prokaryotes. the coral Pocillopora damicornis. Environ Microbiol 7: Bacteria are often considered to exert negative effects 1162-1174 on corals such as causing disease and bleaching, and a Burkholder P (1973) The ecology of marine antibiotics and coral methodology to examine the microscale processes inside reefs. In: Jones OA, Endean R (eds) Biology and geology coral had not yet been confirmed. In addition, previous of coral reefs, vol. 2, Biol. 1. Academic Press, New. York, investigations had assumed that the entire coral and coral pp 117-182 reef system could be understood completely only by ob­ Carlucci A, Bowes PM (1972) Determination of vitamin B12, servation and measurement of the physical, chemical, and thiamine, and biotin in lake Tahoe waters using modified biological parameters of the surrounding water based on marine bioassay techniques. Limnol Oceanogr 17: 774- the assumption that coral was an open system. However, 777 our results show that internal processes cannot be described Croft M, Lawrence A, Raux-Deery E, Warren M, Smith A (2005) by such a methodology resulting paradoxical results, es­ Algae acquire vitamin B12 through a symbiotic relationship 10 Agostini et al.: Coral symbiotic complex

with bacteria. Nature 438: 90-93 UK 69: 803-811 Croft MT, Warren MJ, Smith AG (2006) Algae need their Okbamichael M, Sañudo-Wilhelmy S (2004) A new method for

vitamins. Eukaryot Cell 5: 1175-1183 the determination of vitamin B12 in seawater. Anal Chim D’Elia CF, Wiebe WJ (1990) Biogeochemical nutrient cycles in Acta 517: 33-38 coral reef ecosystems. In: Dubinsky Z (ed) Ecosystem of Porter JW (1978) Coral feeding on zooplankton. In: Stoddart the world, vol 25 Coral reefs. Elsevier Science Publisher, DR, Johannes RE (eds) Coral reefs: research methods. New York, pp 49-74 UNESCO, Paris, pp 515-521 Droop M (2007) Vitamins, phytoplankton and bacteria: sym­ Porter K, Feig Y (1980) The use of DAPI for identifying and biosis or scavenging? J Plankton Res 29: 107-113 counting aquatic microflora. Limnol Oceanogr 25: 943- Ducklow H, Mitchell R (1979) Bacterial populations and ad­ 948 aptations in the mucus layer of living corals. Limnol Reshef L, Koren O, Loya Y, Zilber-Rosenberg I, Rosenberg E Oceanogr 24: 715-725 (2006) The coral probiotic hypothesis. Environ Microbiol Ducklow HW (1990) The biomass, production and fate of 8: 2068-2073 bacteria in coral reefs. In: Dubinsky Z (ed) Ecosystem of Rohwer F, Breitbart M, Jara J, Azam F, Knowlton N (2001) the world, vol 25 Coral reefs. Elsevier Science Publisher, Diversity of bacteria associated with the Caribbean coral New York, pp 265-289 Montastrea fransk. Coral Reefs 20: 85-91 Falter JL, Atkinson M, Merrifield M (2004) Mass-transfer lim­ Rosenberg E, Koren O, Reshef L, Efrony R, Zilber-Rosenberg I itation of nutrient uptake by a wave-dominated reef flat (2007) The role of microorganisms in coral health, disease community. Limnol Oceanogr 49: 1820-1831 and evolution. Nature Rev: Microbiol 5: 355-362 Heidelberg K, Sebens K, Purcell J (2004) Composition and Sahni MK, Spanos S, Wahrman MZ, Sharma GM (2001) Marine sources of near reef zooplankton on a Jamaican forereef corrinoid-binding proteins for the direct determination of

along with implications for coral feeding. Coral Reefs 23: vitamin B12 by radioassay. Anal Biochem 289: 68-76 263-276 Sanudo-Wilhelmy S, Gobler C, Okbamichael M, Taylor G Herndl G, Velimirov B, Krauss R (1985) Heterotrophic nutrition (2006) Regulation of phytoplankton dynamics by vitamin

and control of bacteria density in the coelenteron of giant B12. Geophys Res Lett 33: L04604 sea anemone Stoichactis giganteum. Mar Ecol Prog Ser Shashar N, Cohen Y, Loya Y, Sar N (1994) Nitrogen fixation 22: 101-105 (acetylene reduction) in stony corals: evidence for coral- Hoffmann JP (1990) Dependence of photosynthesis and vitamin bacteria interactions. Mar Ecol Prog Ser 111: 259-264

B12 uptake on cellular vitamin B12 concentration in the Sorokin Y (1973) On the feeding of some sceratinians corals multicellular alga Cadophora glomerata (Chlorophyta). with bacteria and dissolved organic matter. Limnol Ocea­ Limnol Oceanogr 35: 100-108 nogr 18: 380-385 Houlbrèque F, Delesalle B, Blanchot J, Montel Y, Ferrier-Pagès Sorokin YI (1990) Plankton in the reef ecosystems. In: Dubinsky C (2006) Picoplankton removal by the coral reef communi­ Z (ed) Ecosystem of the world, vol 25 Coral reefs. Elsevier ty of La Révoyante, Mayotte Island. Aquat Microbial Ecol Science Publisher, New York, pp 291-328 44: 59-70 Starr TJ, Jones ME, Martinez D (1957) The production of

Houts TM, Carney JA (1981) Radioassay for cobalamin (vitamin vitamin B12- active substances by marine bacteria. Limnol

B12) requiring no pretreatment of serum. Clin Chem 27: Oceanogr 2: 114-119 263-267 Sullivan MB, Coleman ML, Weigele P, Rohwer F, Chisholm SW Kellogg CA (2004) Tropical archaea: diversity associated with (2005) Three prochlorococcus cyanophage genomes: the surface microlayer of corals. Mar Ecol Prog Ser 273: signature features and ecological interpretations. PLoS 81-88 Biol 3: e144 Leletkin V (2000) The energy budget of coral polyps. Russ J Warren MJ, Raux E, Schubert HL, Escalante-Semerena JC Mar Biol 26: 389-398 (2002) The biosynthesis of adenosylcobalamin (vitamin

Lesser M, Mazel C, Gorbunov M, Falkowski P (2004) Discovery B12). Nat Product Rep 19: 390-412 of symbiotic nitrogen fixing cyanobacteria in corals. Wegley L, Yu Y, Breitbart M, Casas V, Kline DI, Rohwer F Science 305: 997-1000 (2004) Coral-associated archaea. Mar Ecol Prog Ser 273: Moore S (1989) Narcotising sea anemones. J Mar Biol Assoc 89-96 Agostini et al.: Coral symbiotic complex 11

Wilkinson C (1978a) Microbial associations in sponges. i Ultrastructure of the in situ associations in coral reef Ecology, physiology and microbial populations of coral sponges. Mar Biol 49: 177-185 reef sponges. Mar Biol 49: 161-167 Wilkinson C (1978b) Microbial associations in sponges. ii Received: 2 December 2008 Numerical analysis of sponges and water bacterial popu­ Accepted: 11 December 2008 lations. Mar Biol 49: 169-176 Wilkinson C (1978c) Microbial associations in sponges. iii Ⓒ Japanese Coral Reef Society