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The specificity of the relationship between cnidarian hosts and symbiotic dinoflagellates (zooxanthellae) differs among host species. Some cnidarian hosts can establish symbiotic relationship with various types of zooxanthellae, while others exhibit high fidelity to specific symbiont type. It is not known how compatibility or specificity of the relationship is determined. We hypothesized that some cnidarian hosts select symbiont type that leads to highest fitness when the host is flexible with symbiont type and more than one types of symbionts are available. As a first step to study this possibility, compatibility of clonal polyps of Cassiopea sp. with six strains of cultured zooxanthellae and the fitness of the host associated with different types of symbionts were studied. Polyp diameter was measured and the number of asexual buds were calculated as a measure of host fitness. The number of zooxanthellae in host and in asexual buds was also measured as a measure of symbiont fitness. Three strains KB8 (clade A), Y106 (clade A), and K100 (clade B) were compatible with the Cassiopea polyps, while other three strains, Y103 (clade C), K111 (clade D), and K102 (clade F) were incompatible. No clear difference in the fitness was found among the polyps inoculated with compatible and incompatible symbiont strains. In one experiment, a compatible strain Y106 seemed to decrease host fitness, but this should be checked by further studies. This study suggests that feeding regimes and long observation period might be important when fitness of hosts associated with different types of symbionts is investigated.

Due to their pluripotent nature and unlimited cell renewal, stem cells have been proposed as an ideal material for establishing long-term cnidarian cell cultures. However, the lack of unifying principles associated with “stemness” across the phylum complicates stem cells’ identification and isolation. Here, we for the first time report gene expression profiles for cultured coral cells, focusing on regulatory gene networks underlying pluripotency and differentiation. Cultures were initiated from Acropora digitifera tip fragments, the fastest growing tissue in Acropora. Overall, in vitro transcription resembled early larvae, overexpressing orthologs of premetazoan and Hydra stem cell markers, and transcripts with roles in cell division, migration, and differentiation. Our results suggest the presence of pluripotent cell types in cultures and indicate the existence of ancestral genome regulatory modules underlying pluripotency and cell differentiation in cnidaria. Cultured cells appear to be synthesizing protein, differentiating, and proliferating.

Various allogeneic responses have been characterized in stony corals. The responses were thought to be genetically control. However, very little is known about the genetic mechanism of allorecognition in corals. Therefore, the contact reaction between parent-offspring and between siblings of the coral Pocillopora damicornis has been studied. Three types of contact reactions were observed: between young colonies, between adult and young colonies, and between adult colonies. Siblings, which were primary polyps or young colonies derived from the same broodparent, invariably fused. All but one pair of parent-offspring grafts exhibited fusion. However, some pairs of young colonies derived from the same two broodparents showed a different contact response than did the broodparents when their branch tips were paired. When the same pairs of the young colonies were repeated to contact at different age, most pairs yielded the same results. Together with the findings that young colonies were able to recognize their partner less than 1 month after the initial contact, we suggest that contact reaction in coral and time needed for the stable reaction are most probably under genetic control. Keywords: coral, young colonies, reproduction, parent-offspring relationship, histocompatibility

Theory predicts that passing symbionts directly from parent to offspring should benefit the host by reducing conflict between diverse symbionts and benefit the symbiont by guaranteeing transmission. However, in some symbiotic associations, such as those between corals and algae of the family Symbiodiniaceae, vertical transmission is rare, suggesting a substantial cost. Here, we show that coral larvae which contained symbionts had lower survivorship than larvae of the same species that lacked symbionts when exposed to natural sunlight. Higher activity of antioxidant defenses and higher levels of oxidative cellular damage observed in symbiotic larvae suggest that reactive oxygen species, produced by stress on the photosynthetic apparatus of the symbionts, are responsible for these higher rates of mortality under solar radiation. We hypothesize that symbionts are a burden in the early life history of broadcast spawning corals under conditions typically experienced on the ocean surface during dispersal. This is one possible reason why species with an obligate pre-motile period of between 12 and 36 h very rarely have vertical transmission of symbionts, in contrast to species whose larvae are small or swim on release that often have vertical transmission of symbionts.

The increasing tourist industry along Java seashore might cause pollution and harm the coral reef ecosystems. The present study was carried out to quantify indicators of coral disease prevalence at sites inside and outside in the snorkeling tourism hotspot of Pasir Putih coastal waters, East Java, Indonesia. Coral disease rapid assessment was conducted in June, 2015. The prevalence of coral disease on the reef building corals was calculated as the mean percentage of coral colonies affected by disease per 50 m2 transect quadrats. Results of the present study showed that total of 78 coral colonies observed, 33 colonies were affected by diseases. Mean prevalence for all diseases observed was 42.30±4.9% SE. The disease prevalence at site inside the snorkeling tourism hotspot (50.00%) was higher than that of outside (38.88 %). Six disease states detected were white syndrome (13.89±1.31% SE), white band (7.41±2.62% SE), yellow blotch (3.70±2.62% SE), dark spot syndrome (3.70%), white plague (1.85%) and growth anomaly (1.85%). The prevalences of disease in the coral genus were found to be 17.95% in Acropora sp., 12.82% in Favia sp., 1.28% in Diploastrea sp., 3.85±2.71% SE in Porites sp., 2.56% in Favites sp., 1.28% in Goniastrea sp. and 1.28% in Montipora sp. To our knowledge, this is the first study of quantitatively coral disease on Pasir Putih coastal waters, Java Sea. This early study revealed that tourism activity might be resulting in the decrease of the coral health in the coral tourism areas.

Corals belong to the most basal class of the Phylum Cnidaria, which is considered the sister group of bilaterian animals, and thus have become an emerging model to study the evolution of developmental mechanisms. Although cell renewal, differentiation, and maintenance of pluripotency are cellular events shared by multicellular animals, the cellular basis of these fundamental biological processes are still poorly understood. To understand how changes in gene expression regulate morphogenetic transitions at the base of the eumetazoa, we performed quantitative RNA-seq analysis during Acropora digitifera’s development. We collected embryonic, larval, and adult samples to characterize stage-specific transcription profiles, as well as broad expression patterns. Transcription profiles reconstructed development revealing two main expression clusters. The first cluster grouped blastula and gastrula and the second grouped subsequent developmental time points. Consistently, we observed clear differences in gene expression between early and late developmental transitions, with higher numbers of differentially expressed genes and fold changes around gastrulation. Furthermore, we identified three coexpression clusters that represented discrete gene expression patterns. During early transitions, transcriptional networks seemed to regulate cellular fate and morphogenesis of the larval body. In late transitions, these networks seemed to play important roles preparing planulae for switch in lifestyle and regulation of adult processes. Although developmental progression in A. digitifera is regulated to some extent by differential coexpression of well-defined gene networks, stage-specific transcription profiles appear to be independent entities. While negative regulation of transcription is predominant in early development, cell differentiation was upregulated in larval and adult stages.

Background The marine dinoflagellate, Symbiodinium, is a well-known photosynthetic partner for coral and other diverse, non-photosynthetic hosts in subtropical and tropical shallows, where it comprises an essential component of marine ecosystems. Using molecular phylogenetics, the genus Symbiodinium has been classified into nine major clades, A-I, and one of the reported differences among phenotypes is their capacity to synthesize mycosporine-like amino acids (MAAs), which absorb UV radiation. However, the genetic basis for this difference in synthetic capacity is unknown. To understand genetics underlying Symbiodinium diversity, we report two draft genomes, one from clade A, presumed to have been the earliest branching clade, and the other from clade C, in the terminal branch. Results The nuclear genome of Symbiodinium clade A (SymA) has more gene families than that of clade C, with larger numbers of organelle-related genes, including mitochondrial transcription terminal factor (mTERF) and Rubisco. While clade C (SymC) has fewer gene families, it displays specific expansions of repeat domain-containing genes, such as leucine-rich repeats (LRRs) and retrovirus-related dUTPases. Interestingly, the SymA genome encodes a gene cluster for MAA biosynthesis, potentially transferred from an endosymbiotic red alga (probably of bacterial origin), while SymC has completely lost these genes. Conclusions Our analysis demonstrates that SymC appears to have evolved by losing gene families, such as the MAA biosynthesis gene cluster. In contrast to the conservation of genes related to photosynthetic ability, the terminal clade has suffered more gene family losses than other clades, suggesting a possible adaptation to symbiosis. Overall, this study implies that Symbiodinium ecology drives acquisition and loss of gene families. Electronic supplementary material The online version of this article (10.1186/s12864-018-4857-9) contains supplementary material, which is available to authorized users.

To examine the possible variation in responses of corals to ocean acidification (OA) among populations, we compared the sensitivity of two Okinawan populations (Sesoko and Bise) of the scleractinian coral Acropora digitifera to high pCO2. We found that both light and dark calcification rates of Sesoko corals did not change with an increase in seawater pCO2, while the calcification rates of Bise corals significantly decreased. Additionally, calcification rate of Sesoko corals was significantly lower than Bise corals at control conditions. Expressions of two putative calcification-related genes (BAT: bicarbonate transporter and galaxin) were up-regulated at high CO2 compared to the control and expression of the BAT gene was significantly higher in Sesoko compared to Bise corals. Consequently, differences in the calcification rate between populations and differences in the expression of genes related to inorganic carbon transport regulation could be reasons that explain the difference in the response to OA between the two populations. Furthermore, taking into account that Sesoko corals were located in relatively nearshore areas where the environmental conditions are more variable, while Bise corals were located in the forereef which shows more stable conditions, plasticity for coral calcification in response to different environmental conditions and/or acclimation response to changes such as seawater pCO2 may lead to differences in sensitivity between the two populations to high seawater pCO2. Studies considering the potential variability in corals sensitivity to OA among local populations from different habitats are important to predict the potential effects of climate change on reef ecosystems.

Crustaceans generally act as oxy-regulators, maintaining constant oxygen uptake as oxygen partial pressures decrease, but when a critical low level is reached, ventilation and aerobic metabolism shut down. Cave-adapted animals, including crustaceans, often show a reduced metabolic rate possibly owing in part to the hypoxic nature of such environments. However, metabolic rates have not been thoroughly explored in crustaceans from anchialine habitats (coastal ponds and caves), which can experience variable oxygenic regimes. Here, an atypical oxy-conforming pattern of oxygen uptake is reported in the Hawaiian anchialine atyid Halocaridina rubra , along with other unusual metabolic characteristics. Ventilatory rates are near-maximal in normoxia and did not increase appreciably as PO 2 declined, resulting in a decline in VO 2 during progressive hypoxia. Halocaridina rubra maintained in anoxic waters survived for seven days (the duration of the experiment) with no measureable oxygen uptake, suggesting a reliance on anaerobic metabolism. Supporting this, lactate dehydrogenase activity was high, even in normoxia, and oxygen debts were quickly repaid by an unusually extreme increase in oxygen uptake upon exposure to normoxia. In contrast, four related anchialine shrimp species from the Ryukyu Islands, Japan, exhibited physiological properties consistent with previously studied crustaceans. The unusual respiratory patterns found in H. rubra are discussed in the context of a trade-off in gill morphology for osmoregulatory ion transport vs. diffusion of respiratory gasses. Future focus on anchialine species may offer novel insight into the diversity of metabolic responses to hypoxia and other physiological challenges experienced by crustaceans.

To test whether telomere length can be used in estimating the age of colonial corals, we used terminal restriction fragment (TRF) length analysis to compare the telomere lengths of the coral Acropora digitifera at three developmental stages: sperm, planula larvae, and polyps of adult colonies. We also compared the mean TRF lengths between branches at the center and periphery of tabular colonies of A. digitifera. A significant difference was observed in the mean TRF lengths in sperm, planulae, and polyps. The mean TRF length was longest in sperm and shortest in polyps from adult colonies. These results suggest that telomere length decreases during coral development and may be useful for estimating coral age. However, the mean TRF length of branches at the center of a table-form colony tended to be longer than that of peripheral branches, although this difference was not statistically significant. This suggests that both the chronological age of polyps and cell proliferation rate influence telomere length in polyps, and that estimating coral age based on telomere length is not a simple endeavor.

The ability to estimate coral age using soft tissue would be useful for population biology or aging studies on corals. In this study, we investigated whether telomere length can be used to estimate coral age. We applied single telomere length analysis to a colonial coral, Galaxea fascicularis, and estimated telomere lengths of specific coral chromosomes at different developmental stages. If the telomere shortened at each cell division, the telomere length of the coral would be longest in sperm and shortest in adult colonies. However, the mean telomere length of sperm, planula larvae, and polyps was approximately 4 kb, with no significant differences among the developmental stages. The telomerase restriction fragment (TRF) analysis also showed no significant difference in the mean TRF length among the developmental stages. Our results suggested that telomere length is maintained during developmental stages and that estimating the age of colonial coral based on telomere length may not be possible. However, our findings can be used to examine avoidance of aging and rejuvenation during regeneration and asexual reproduction in colonial corals.

Growth anomalies (GAs) in corals are characterized by morphological abnormalities of the skeleton as well as polyps and coenosarcs. GAs commonly appear as protuberances with fewer polyps and are paler in color due to decreased zooxanthellae density. To test the hypothesis that morphological anomalies in GAs may be caused by unregulated cellular kinetics, the relative abundances of apoptotic cells and proliferating cells were compared between GAs and apparently healthy regions in 2 corals, Porites australiensis and Montipora informis. Apoptotic cells and pro- liferating cells were detected using TUNEL assays and BrdU incorporation assays, respectively. The labeling indices for apoptotic nuclei and BrdU-labeled nuclei were measured in the epider- mis, oral gastrodermis, aboral gastrodermis, and calicodermis. The labeling index for apoptotic nuclei in the oral gastrodermis and the calicodermis was significantly lower in GAs than in healthy regions in both coral species. The index for BrdU-labeled cells in the calicodermis was signifi- cantly higher in GAs than in healthy regions in both coral species. When GA regions partially died, the GA tissues directly adjacent to the dead areas exhibited signs of necrosis, although some apoptotic cells were also present. Healthy oral gastrodermis adjacent to the border between the healthy and GA regions exhibited higher frequencies of apoptotic cells. These results suggest that apoptotic pathways were suppressed and cell proliferation was promoted in GA regions, although cells in GAs may die through both necrosis and apoptosis.

We describe a strategy to identify stress-responsive genes in corals by means of high-coverage cDNA expression profiling (HiCEP). In this study, the effects of thermal and chemical (DCMU and TBT-Cl) stresses on Acropora tenuis juvenile polyps were examined, and 19 genes were identified as stress-responsive genes. Of these, seven had sequences significantly similar to sequences of known proteins. We quantified the expression levels of these seven using real-time PCR. In corals, the genes encoding heat shock protein 90, calreticulin, methionine adenosyltransferase, and aquaglyceroporin showed differential stress-related expression patterns. In the endosymbiotic algae associated with the corals, genes coding for DnaJ-like protein and chloroplast light-harvesting protein were identified as stress-responsive genes. The only gene showing increased expression in response to all three stresses (thermal and both chemical stresses) was the coral gene coding for the oxidative stress-responsive protein, suggesting that oxidative stress generally occurs in each stress-exposed polyp.

Symbiosis with zooxanthellae (symbiotic dinoflagellates) and high capacities for clonal reproduction and regeneration are important life history traits of reef-building corals. This chapter reviews the life history of reef-building corals and the symbiotic relationships between corals and their algal symbionts and discusses possible collaborative defense systems against environmental stresses as well as mechanisms of adaptation to environmental changes in coral–zooxanthella symbiotic systems. Most corals associate with one main type of symbiont, although minor or cryptic symbiont types are often detected within a colony. The association between the coral host and algal symbiont appears to be stable, and the original symbiont often returns when corals recover from bleaching. Some corals, such as the massive Porites, exhibit high fidelity to certain types of algal symbionts and still have high stress tolerance as well as long life spans suggesting the possibility that stem cells and algal symbiont cells in such long-lived colonies accumulate mutations; in turn, if cells with a higher fitness for a new environment proliferate within a colony, the colony may be able to adapt to the new environment.

Reef-building corals harbor symbiotic dinoflagellates, Symbiodinium spp., which are currently divided into several clades. The responses of corals associated with different Symbiodinium clades to thermal stress are not well understood, especially at a gene expression level. Juveniles of the coral Acropora tenuis inoculated with different algal types (clade A or D) were exposed to thermal stress and the expression levels of four putative stress-responsive genes, including genes coding green and red fluorescent proteins, an oxidative stress-responsive protein, and an ascorbic acid transporter, were analyzed by quantitative real-time PCR. The expression levels of the four genes decreased at high temperatures if juveniles were associated with clade A symbionts but increased if the symbionts were in clade D. The intensity of green fluorescence increased with temperature in clade D symbionts harboring juveniles, but not in juveniles associated with clade A symbionts. The present results suggest that genotypes of endosymbiotic algae affect the thermal stress responses of the coral juveniles.

One of the most threatened ecosystems on many islands may be anchialine habitats, or coastal land-locked water bodies with no surface connection to the sea yet containing brackish water that fluctuates with the tides. To better manage these habitats, it is important to develop a broader understanding of the biodiversity within them since such knowledge plays critical roles in establishing conservation strategies. In this study, the genetic variation and population structure of an anchialine atyid shrimp, Caridina rubella Fujino and Shokita, 1975 was investigated in the Southern Ryukyu Archipelago, Japan. Given a planktotrophic larval stage and its potential amphidromous life cycle, populations of C. rubella on the island of Miyako-jima (to which it is apparently restricted) were hypothesized to have little to no structure across the island. To test this, 61 individuals were collected from four anchialine caves and sequence variation examined at the cytochrome c oxidase subunit I (COI) region of the mitochondrial DNA (mtDNA). Surprisingly, significant genetic structure was exhibited across distances ranging from 10 km. Additionally, deep (∼17% p-distance) genetic divergence correlating with distinct variation in rostrum lengths was found between closely situated, but more-or-less completely isolated, populations. This implies “C. rubella ”m ay actually represent two distinct species on Miyako-jima. Given that this atyid is already listed as a threatened species by the Japanese government, the results presented here are useful in the formulation and implementation of future conservation plans for populations of C. rubella on Miyako-jima.

This ten-year study examined the morphological, physiological, and ecological characteristics of coral growth anomalies on Acropora cytherea on Amuro Island, Okinawa, Japan. The objectives of the study were to assess whether the growth anomalies, identified as diffuse disruptions on the skeleton: (i) were more prevalent on large colonies than on small colonies, (ii) were more common near the center of the colonies than peripherally, (iii) affected colony growth and mortality, and (iv) affected coral-colony fecundity and photosynthetic capacity. We hypothesized that the growth anomalies were signs of the onset of aging. The growth anomalies were more prevalent on colonies >2 m diameter, and were concentrated near the central (older) portions of the colonies. The growth anomalies were also associated with reduced productivity and dysfunctional gametogenesis. Still, the growth anomalies did not appear to affect colony survival. The contact experiments showed that the growth anomalies were not contagious, and were most likely a sign of aging that was exacerbated by thermal stress.

Mutualisms are often viewed as reciprocal exploitations that nonetheless provide net benefits to each partner. While the benefits of symbiosis with dinoflagellates of the genus Symbio- dinium (zooxanthellae) for corals are well understood, the costs of the association, particularly when under stress, remain a focus of much research. One of the primary impediments to exploring the costs of symbiosis in zooxanthellate corals is that it is impossible to examine the animal host in isolation. Evidence for a cost of symbiosis with zooxanthellae includes the fact that direct transmission of zoox- anthellae between generations is rare, particularly in broadcast spawning corals. Fortuitously, the absence of zooxanthellae in oocytes of many species and the ability to readily infect larvae with zoox- anthellae provide an opportunity to compare individuals with and without these symbionts. Here, we use this larval model to show that individuals with zooxanthellae have lower survival than those lack- ing zooxanthellae when exposed to high temperature. Higher activity of anti-oxidant defenses and higher levels of oxidative cellular damage in larvae with zooxanthellae suggest that oxidative stress originating in the symbionts is a cause of tissue damage in the host under heat stress. We hypothesize that this may be one reason for the absence of direct transmission of zooxanthellae in most broadcast spawning corals whose propagules must spend at least 1 d on the ocean surface.

The establishment of symbiosis in early developmental stages is important for reef-building corals because of the need for photosynthetically derived nutrition. Corals spawn eggs and sperm, or brood planula larvae and shed them into the water. Some coral eggs or planulae directly inherit symbiotic dinoflagellates (Symbiodinium spp.) from their parents, while others acquire them at each generation. In most species examined to date, the larvae without dinoflagellates (aposymbiotic larvae) can acquire symbionts during the larval stage, but little is known regarding the timing and detailed process of the onset of symbiosis. We examined larval uptake of symbiotic dinoflagellates in nine species of scleractinian corals, the onset of symbiosis through the early larval stages, and the distribution pattern of symbionts within the larval host, while living and with histology, of two acroporid corals under laboratory conditions. The larvae acquired symbiotic dinoflagellates during the planktonic phase in all corals examined which included Acropora digitifera, A. florida, A. intermedia, A. tenuis, Isopora palifera, Favia pallida, F. lizardensis, Pseudosiderastrea tayamai, and Ctenactis echinata. The larvae of A. digitifera and A. tenuis first acquired symbionts 6 and 5 days after fertilization, respectively. In A. digitifera larvae, this coincided with the formation of an oral pore and coelenteron. The number of symbiotic dinoflagellates increased over the experimental periods in both species. To test the hypothesis that nutrients promotes symbiotic uptake, the number of incorporated dinoflagellates was compared in the presence and absence of homogenized Artemia sp. A likelihood ratio test assuming a log-linear model indicated that Artemia sp. had a significantly positive effect on symbiont acquisition. These results suggest that the acquisition of symbiotic dinoflagellates during larval stages is in common with many coral species, and that the development of both a mouth and coelenteron play important roles in symbiont acquisition.

Historically, hermatypic corals are defined as a symbiotic system composed of an animal host, corals belonging to the taxa Cnidarian, and a photosynthetic organism, the dinoflagellate Symbiodinium spp., also known as zooxanthellae. The high gross productivity and stability of coral reefs have been explained by the efficiency of the coral-algal symbiotic system in using the low nutrient concentrations found in the surrounding 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, remain to be shown. In this study, evidence for the importance of coelenteric bacteria in corals as a component of the coral symbiotic complex was shown by using a new approach. Vitamin B12, which is produced only by prokaryotes, was chosen as a chemical tool to clarify the symbiotic relationships among bacteria, coral, and zooxanthellae, which require vitamin B12. High vitamin B12 concentrations (up to 700pmol l-1 compared with max. 20pmol l-1 in the surrounding water) and high bacteria abundances (100 times higher than surrounding water) were found in the coelenteron of live corals using a new sampling method. The results led to the hypothesis that prokaryotes are the drivers of internal processes, such as vitamin B12 production, occurring directly inside the coral, forming a semi-closed system. Furthermore, most maintenance of the symbiotic complex is due to internal processes rather than the supply from outside the coral.

The coral Galaxea fascicularis exhibits marked variation in polyp color and several color morphs have been described (e.g. brown polyp with green oral disc, BG; brown with green septal tentacles, Gs; brown with white fluorescent tentacles, Wt). Color morphs of sessile marine inver tebrate species may exhibit considerable differences in their capacity to absorb UV radiation, al­ though only a few studies have examined intra­species variation in the UV­absorbing com pounds, mycosporine­ like amino acids (MAAs). The objective of this study was to investigate possible differences in the composi­ tion and quantity of MAAs in the coral tissue and symbio­ tic algae of several G. fascicularis color morphs. Marked differences in the composition and quantity of MAAs were found between the BG and Gs/Wt morphs of G. fascicularis, both in the coral tissue and freshly isolated symbiotic algae. Future studies, especially genetic analy­ ses of corals and their symbionts, are necessary to un­ derstand whether these observed differences in the com­ position and quantity of MAAs reflect phylogenetic or bio geographical differences among the corals.

To investigate bleaching mechanisms in coral–zooxanthella symbiotic systems, it is important to study the cellular- or tissue-level responses of corals to stress. We established an experimental system to study the stress responses of coral cells using coral cell aggregates. Dissociated coral cells aggregate to form spherical bodies, which rotate by ciliary movement. These spherical bodies (tissue balls) stop rotating and disintegrate when exposed to a thermal stress. Tissue balls prepared from dissociated cells of Fungia sp. and Pavona divaricata were exposed to either elevated temperature (31 °C, with 25 °C as the control) or elevated temperature in the presence of exogenous antioxidants (ascorbic acid and catalase, or mannitol). The survival curves of tissue balls were markedly different between 31 and 25 °C. At 31 °C, most tissue balls disintegrated within 24 h, whereas at 25 °C, most tissue balls survived for more than 24 h. There was a negative correlation between survival time and the zooxanthella density of tissue balls at 31 °C, but no significant relationship was found at 25 °C. Antioxidants extended the survival time of tissue balls at high temperature, suggesting that zooxanthellae produce reactive oxygen species under stress. These results indicate that zooxanthellae produce harmful substances and damage coral cells under high-temperature stress. Tissue balls provide a good experimental system with which to study the effects of stress and various chemical reagents on corals cells.

The existence of “free-living” Symbiodinium that can form symbioses with hosts is implied by the presence of hosts that produce Symbiodinium-free gametes and expulsion and/or expelled symbiotic algae from host. However, it is still unclear if potentially symbiotic Symbiodinium are found “free-living” in the coral reef environment. Sixteen Symbiodinium strains were established from samples taken from three sampling locations of coral reef sand in Okinawa, Japan. Phylogenetic analyses of the partial large subunit ribosomal DNA (28S-rDNA) and the internal transcribed spacer of ribosomal DNA (ITS-rDNA) conclusively showed that all 16 isolates belonged to Symbiodinium clade A sensu Rowan and Powers (1991). The lack of other Symbiodinium clades besides clade A in this study may be due to other clades not being readily culturable under culture conditions used here. The new isolates could be phylogenetically divided into four groups, though no sequences were identical to previously reported Symbiodinium. Two of the four groups were closely related to symbiotic Symbiodinium clade A isolated from a variety of host species. One isolate group formed a highly supported monophyly with Symbiodinium types that have previously been characterized as “free-living”. The remaining isolate group, although within clade A, was quite divergent from other clade A Symbiodinium. These results indicate that novel diversity of free-living Symbiodinium exists in coral sand.

We simultaneously examined seasonal fluctuations in maximum quantum yield (Fv/Fm), cell density and genotypic clade composition of zooxanthellae in the sympatric corals Pavona divari- cata and P. decussata at a high latitude site in Okinawa, Japan. The 2 species had different responses to seasonal changes in light and seawater temperature. P. decussata was associated only with clade C zooxanthellae (at all sampling times) while genotypic clade composition fluctuated in P. divaricata. Three colonies of P. divaricata harbored clade D throughout the year and the other 7 colonies changed clade type from C to D or vice versa, or contained a mixture of both clades on some sampling occasions. Though neither coral species suffered bleaching during the experimental period, there were Fv/Fm decreases in P. decussata and P. divaricata harboring clade C during cold and warm sea- sons; Fv/Fm of P. divaricata harboring clade D was stable. When exposed to low or high temperature under normal or high light, P. divaricata harboring clade D was less damaged and Photosystem II recovery was faster than in P. decussata harboring clade C. These results suggest that the sympatric congeneric corals have different symbiont switching flexibilities and this difference might account for the observed difference in stress susceptibility between the two species. In high-latitude reefs, both winter low temperatures and summer high temperatures may be potential stressors that cause coral bleaching, at least in corals with high clade C zooxanthellar fidelity.

Photochemical efficiency (Fv/Fm) was compared between a common symbiotic bioeroding sponge, Cliona cf. orientalis, and a common reef-builder, Acropora palifera using pulse amplitude modulated fluorescence (PAM). The study was conducted on Sesoko Island, Okinawa, where reefs were severely damaged during previous bleaching episodes. Sponge and coral dinoflagellate symbionts were treated with heat and light in a tank experiment, both in hospite (=still within their host) and isolated from their hosts. We found significant differences for photochemical efficiency of holobionts (=host and symbiont together) compared to the isolate symbionts and over time. All symbionts suffered in isolation and displayed stronger reactions to the treatments, and there was evidence for increasing damage despite returning to control conditions. However, because of large variability of the bi-symbiont coral samples and restrictions of the experimental design, our main results remained inconclusive, with no significant differences between sponge and coral samples and between the different stress treatments. Judging the results based on the uniform trends in the subsets of data, the G-clade sponge symbionts appeared to be more stress tolerant than the C- and D-clade coral symbionts, with no treatment effects in hospite and less damage in isolation compared to the coral symbionts, but this is an unconfirmed assumption. Isolated sponge symbionts were very resistant against heat stress, but may have suffered from light stress. In hospite, the latter risk can be countered by the sponge's 3-dimensional morphology, the endolithic life style that affords shading, and by behavioural adaptation, i.e. the ability to move symbionts away from the source of stress. Overall, C. cf. orientalis symbionts displayed a more stable photochemical efficiency during and after stress than those of A. palifera. Results of this study suggest that with climate change C. cf. orientalis might have a better survival potential than A. palifera, but further investigations are necessary.

Coral bleaching is caused by environmental stress and susceptibility to bleaching stress varies among types of coral. The physiological properties of the algal symbionts (Symbiodinium spp.), especially extent of damage to PSII and its repair capacity, contribute importantly to this variability in stress susceptibility. The objective of the present study was to investigate the relationship between the growth rates and photosynthetic activities of six cultured strains of Symbiodinium spp. (clades A, B, C, D, and F) at elevated temperature (33 °C). We also observed the recovery of photodamaged-PSII in the presence or absence of a chloroplast protein synthesis inhibitor (lincomycin). The growth rates and photochemical efficiencies of PSII (Fv/Fm) decreased in parallel at high temperature in thermally sensitive strains, B-K100 (clade B followed by culture name) and A-Y106, but not in thermally tolerant strains, F-K102 and D-K111. In strains A-KB8 and C-Y103, growth declined markedly at high temperature, but Fv/Fm decreased only slightly. These strains may reallocate energy from growth to the repair of damaged photosynthetic machineries or protection pathways. Alternatively, since recoveries of photo-damaged PSII at 33 °C were modest in strains A-KB8 and C-Y103, thermal stressing of other metabolic pathways may have reduced growth rates in these two strains. This possibility should be explored in future research efforts.

To examine whether damage to zooxanthellar photosystem II (PSII) is the primary step of thermally induced coral bleaching, we first assessed the relationship between the maximum quantum yield of PSII (Fv/Fm) or active PSII centers (Fv/Fo) and maximum electron transport rate (ETRmax), which represent the PSII activity and electron flow beyond the PSII, respectively, in the symbiotic algae of the coral Pachyseris rugosa. Next, the photo-physiological responses of the symbionts to stress treatments of 1h high temperature (33.5°C) and/or high light (1030μmol quanta m-2 s-1) stress and 6h recovery were investigated. The 1h high temperature treatment in darkness produced a significant, but reversible (6h recovery) decline in ETRmax without any change in Fv/Fm. The 1h high light treatment reduced Fv/Fm, but not ETRmax. High temperature in combination with high light resulted in a more pronounced reduction in Fv/Fm, along with a significant decline in ETRmax. Neither Fv/Fm nor ETRmax recovered fully within 6h. Reversible increases in non-photochemical quenching (NPQ), energy dissipation in PSII, were also recorded. Chloramphenicol (CAP), an inhibitor of synthesis of D1 protein of PSII reaction center, revealed that a minimum of -60% of the Fv/Fm or -30% of active PSII centers (Fv/Fo) is required to maintain ETRmax in the in hospite symbionts. These results suggest that the primary step of heat-induced damage in the symbiont's photosynthetic apparatus involves a component beyond the PSII, probably at the level of the dark reaction as indicated by reduced ETRmax and the PSII damage is secondary.

Most colonial corals vary intraspecifically in growth forms, and the diversity in branching morphology is especially striking. While the effects of environmental factors on growth forms have been studied, the genetic control of coral branching patterns has received little attention. The discovery of ontogenetic changes in the capacity to originate branching would set the stage for studies of how branch formation is genetically controlled. During experiments investigating contact reactions in the coral Pocillopora damicornis, we observed that young colonies derived from settled planulae and colonies regenerated from adult branch tips assumed different growth forms. Young colonies formed at least one branch from the central region of the colony, while colonies regenerated from adult branch tips (3-5 mm long) did not form branches during the 9-month observation period. This pattern was invariable, regardless of the types and outcomes of the contact experiments or the orientation of the branch tips. However, some fragments taken from 1- or 2-year-old colonies formed branches. This suggests that the rate of branch formation in P. damicornis colonies decreases with age. These findings will facilitate investigations of the mechanism of coral branch formation at the molecular level.

This study examined the importance of mycosporine-glycine (Myc-Gly) as a functional antioxidant in the thermal-stress susceptibility of two scleractinian corals, Platygyra ryukyuensis and Stylophora pistillata . Photochemical efficiency of PSII ( F v / F m ), activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), and composition and abundance of mycosporine-like amino acids (MAAs) in the coral tissue and in symbiotic zooxanthellae were analyzed during 12-h exposure to high temperature (33 °C). After 6- and 12-h exposures at 33 °C, S. pistillata showed a significantly more pronounced decline in F v / F m compared to P. ryukyuensis . A 6-h exposure at 33 °C induced a significant increase in the activities of SOD and CAT in both host and zooxanthellae components of S. pistillata while in P. ryukyuensis a significant increase was observed only in the CAT activity of zooxanthellae. After 12-h exposure, the SOD activity of P. ryukyuensis was unaffected in the coral tissue but slightly increased in zooxanthellae, whereas the CAT activity in the coral tissue showed a 2.5-fold increase. The total activity of antioxidant enzymes was significantly higher in S. pistillata than in P. ryukyuensis , suggesting that P. ryukyuensis is less sensitive to oxidative stress than S. pistillata . This differential susceptibility of the corals is consistent with a 20-fold higher initial concentration of Myc-Gly in P. ryukyuensis compared to S. pistillata . In the coral tissue and zooxanthellae of both species investigated, the first 6 h of exposure to thermal stress induced a pronounced reduction in the abundance of Myc-Gly but not in other MAAs. When exposure was prolonged to 12 h, the Myc-Gly pool continued to decrease in P. ryukyuensis and was completely depleted in S. pistillata . The delay in the onset of oxidative stress in P. ryukyuensis and the dramatic increase in the activities of the antioxidant enzymes in S. pistillata , which contains low concentrations of Myc-Gly suggest that Myc-Gly provides rapid protection against oxidative stress before the antioxidant enzymes are induced. These findings strongly suggest that Myc-Gly is functioning as a biological antioxidant in the coral tissue and zooxanthellae and demonstrate its importance in the survival of reef-building corals under thermal stress.

Reports of bathymetric decrease in the total mycosporine-like amino acid (MAA) concentration of benthic invertebrates suggest that light gradients may be important determinants of MAA content. With the pronounced diel light changes, distinct temporal variations in MAA concentrations might also be expected. We examined the changes in the abundance of MAA in three shallow-water scleractinian corals, Pavona divaricata, Galaxea fascicularis and Montipora digitata from Okinawa, Japan, in relation to daily cycles in solar radiation and tested whether the species have different capabilities for protection against UVR depending on their MAA composition. The results show that symbiotic algae freshly isolated from the investigated coral species do not contain MAAs and that distribution of these compounds resided only within the animal tissue. Total MAA content in the tissue of P. divaricata, G. fascicularis and M. digitata rose rapidly at midday and significantly dropped at night. The observed variations were by a factor of two and, thus, very dramatic. For all the investigated coral species, total MAA concentrations were significantly correlated with the diurnal cycle in solar radiation, during both winter and summer seasons. Seawater temperature was significantly correlated with MAA levels only in the June experiment, but represented no more than 20% of the MAA variation in all three coral species, whereas solar radiation explained 60–70% of the MAA fluctuations. This suggests that MAAs are an integral component of the hard coral’s biochemical defense system against high solar irradiance stress. The diurnal increase in total MAA concentrations was due to an increase in the concentration of imino-MAA species of up to 2–2.5-fold of their pre-dawn values. In contrast, the oxocarbonyl-MAA mycosporine-glycine (Myc-Gly) showed the lowest (Tukey–Kramer HSD test: P

2 Institute of Marine Biology, Far East Branch of Russian Academy of Sciences, Vladivostok 690041, Russia ABSTRACT: Differences in the time course of recovery of the photochemical efficiency (Fv /Fm) of PSII and the photosynthetic capacity (relative maximum electron transport rate; rETRmax), of symbiotic dinoflagellates from damage due to thermal and light stresses might have profound effects on the subsequent photodamage of photosynthetic apparatus. The reduction and recovery of Fv /Fm and rETRmax in symbiotic dinoflagellates were investigated at different temperatures (25, 28 and 31°C) after 3 h exposure to high light (1100 µmol photons m -2 s -1 of photosynthetically active radiation (PAR)) in 2 corals with different bleaching susceptibility, Pavona divaricata and Montipora digitata. There was a marked difference in both the reduction in rETRmax after high light exposure and the time course of its recovery between P. divaricata and M. digitata. In P. divaricata, the reduction in Fv /Fm and rETRmax during high light exposure was independent of temperature, and their recovery was almost complete within 24 h after stress treatment at all temperatures examined. In M. digitata, while the reduction and recovery of Fv /Fm did not show temperature dependency, rETRmax exhibited a greater decrease and lower recovery at 31°C than at 25 and 28°C. The recovery of rETRmax was slower than that of Fv /Fm in symbiotic dinoflagellates of M. digitata, and this delay of rETRmax recov- ery increased with increasing temperature during the recovery period. Significant decreases in dinoflagellate density and chlorophyll content were evident only in the 31°C-treated fragments of M. digitata kept at 31°C during the recovery period. These results indicate that it is the difference in the rate of recovery between the PSII function and the dark reaction of symbiotic dinoflagellates that determines whether the photosynthetic ability of algae recovers from the combined effect of thermal and irradiance stresses. The delayed recovery of rETRmax at high temperature made fragments of M. digitata susceptible to photodamage even under moderate light conditions. P. divaricata showed a high ability to recover its rETRmax and consequently was resistant to high light and thermal stresses. It is likely that high temperature affects the rate of recovery of the carbon-fixation cycle and that the ability of symbiotic dinoflagellates to repair damage determines susceptibility of corals to bleaching.

The diversity and community structures of symbiotic dinoflagellates are described from reef invertebrates in southern and central provinces of the Great Barrier Reef (GBR), Australia, and Zamami Island, Okinawa, Japan. The symbiont assemblages from region to region were dominated by Clade C Symbiodinium spp. and consisted of numerous host-specific and/or rare types (specialists), and several types common to many hosts (generalists). Prevalence in the host community among certain host-generalist symbionts differed between inshore and offshore environments, across latitudinal (central versus southern GBR) gradients, and over wide geographic ranges (GBR versus Okinawa). One particular symbiont (C3h) from the GBR had a dramatic shift in dominance. Its prevalence ranged from being extremely rare, or absent on high-latitude reefs to dominating the scleractinian diversity on a mid-latitude inshore reef. These changes occurred among coral fauna whose larvae must acquire symbionts from environmental sources (horizontal symbiont acquisition). Such differences did not occur among 'vertical transmitters' such as Porites spp., Montipora spp. and pocilloporids (corals that directly transmit symbionts to their offspring) or among those hosts displaying 'horizontal acquisition', but that associate with specific symbionts. Most host-specialized types were found to be characteristic of a particular geographic region (i.e. Okinawa versus Central GBR versus Southern GBR). The mode of symbiont acquisition may play an important role in how symbiont composition may shift in west Pacific host communities in response to climate change. There is no indication that recent episodes of mass bleaching have provoked changes in host-symbiont combinations from the central GBR.

Coral species in a similar habitat often show different bleaching susceptibilities. It is not understood which partner of coral–zooxanthellae complexes is responsible for differential stress susceptibility. Stress susceptibilities of in hospite and isolated zooxanthellae from five species of corals collected from shallow water in Okinawa were compared. To estimate stress susceptibility, we measured the maximum quantum yields (Fv/Fm) of in hospite and isolated zooxanthellae after 3-h exposure to either 28 or 34 °C at various light intensities and their recovery after 12 h under dim light at 26 °C. Significant reduction in photochemical efficiency (Fv/Fm) of photosystem II (PSII) was observed in in hospite zooxanthellae exposed to high light intensity (1000 μmol quanta m−2 s−1), while PSII activity of isolated zooxanthellae decreased significantly even at a lower light intensity (70 μmol quanta m−2 s−1). The recovery of the PSII activity after 12 h was incomplete in both in hospite and isolated zooxanthellae, indicating the presence of chronic photoinhibition. The stress susceptibility of isolated zooxanthellae was more variable among species than in hospite zooxanthellae. The order of stress susceptibility among the five coral species was different between in hospite and isolated zooxanthellae. The present results suggest that the host plays a significant role in determining bleaching susceptibility of corals, though zooxanthellae from different host have different stress susceptibilities.

To study morphological changes in zooxanthellae in bleached corals, we observed zooxanthellae in colonies of Acropora nasuta that suffered various degrees of bleaching. Colonies were collected from a shallow reef at Bise, Okinawa, from July to September in 2001. Zooxanthellae within the tissue of A. nasuta were classified into 3 types based on their coloration and shape: healthy-looking zooxanthellae (Hz.), pale zooxanthellae (Pz.) and transparent zooxanthellae (Tz.). The Pz. were pale yellow with more or less disorganized cellular content, while Tz. were swelled and vacuolated with no pigmentation. Tz. were the dominant zooxanthellae in colonies that suffered severe bleaching, while non-bleached colonies and those recovered from bleaching had mostly Hz. Some Tz, were ruptured with vacuoles spreading out of the cell. The density of zooxanthellae in severely bleached colonies was about 105 cells cm-2. The present observation suggests that, during natural bleaching due to chronic stress, zooxanthellae remained in host tissue lose pigmentation and become swelled and vacuolated suggesting necrotic death of the zooxanthellae.