Larval Transcriptomic Responses of a Stony Coral, Acropora Tenuis, During Initial Contact with the Native Symbiont, Symbiodinium Microadriaticum

Although numerous dinoflagellate species (Family Symbiodiniaceae) are present in coral reef environments, Acropora corals tend to select a single species, Symbiodinium microadriaticum, in early life stages, even though this species is rarely found in mature colonies. In order to identify molecular mechanisms involved in initial contact with native symbionts, we analyzed transcriptomic responses of Acropora tenuis larvae at 1, 3, 6, 12, and 24 h after their first contact, together with inoculation using non-native symbionts, including the non-symbiotic S. natans and the occasional symbiont, S. tridacnidorum. Some gene expression changes were detected in larvae inoculated with non-native symbionts 1 h post-inoculation (hpi)), but those returned to baseline levels afterward. In contrast, we found that the number of differentially expressed genes gradually increased in relation to inoculation time when larvae were exposed to native symbionts. As a specific response to native symbionts, upregulation of pattern recognition receptor-like and transporter genes, and suppression of cellular function genes related to immunity and apoptosis, were exclusively observed. These findings indicate that coral larvae recognize differences between symbionts, and when the appropriate symbionts infect, they coordinate gene expression to establish stable mutualism.

Genetic Variation in Released Gametes Produces Genetic Diversity in the Offspring of the Broadcast Spawning Coral Acropora Tenuis

All coral species in the genus Acropora are broadcast-spawning hermaphrodites. Fertilization in the ocean requires sufficient numbers of gametes from conspecifics and the contact time for fertilization is thought to be limited by the rapid diffusion of sperm. Many studies have reported a positive correlation between sperm concentration and fertilization success, but it is not clear how released gametes are fertilized in nature (in situ) and how genetic diversity arises at a fertilization event. To elucidate this, we analyzed the changes in sperm concentration in situ after spawning and genotyped sperm and fertilized eggs from seawater using seven microsatellite (MS) markers. This showed that the sperm concentration in situ was suboptimal (<106 sperm/mL), but most of the eggs were fertilized. MS genotyping showed that the alleles of released sperm were diverse and those alleles also appeared in the fertilized eggs. In addition, the MS fragment peak height in released sperm, which presumable reflects the allele frequency of the sperm, was positively correlated with the allele frequencies of the fertilized eggs. Collectively, the genetic diversity of colonies with high fecundity dictates the genetic diversity of their descendants.

In vitro Symbiosis of Reef-Building Coral Cells With Photosynthetic Dinoflagellates

Coral reefs are the biodiversity hot spots of the oceans, but they have suffered from increasing environmental stresses caused principally by anthropogenic global warming. The keystone species of coral reefs are scleractinian corals, which maintain obligatory symbiotic relationships with photosynthetic dinoflagellates. Understanding cellular and molecular mechanisms of symbiosis is therefore essential for future preservation of coral reefs. To date, however, almost no in vitro experimental systems have been devised to illuminate such mechanisms. To this end, our previous study established stable in vitro cell culture lines, including IVB5, originating from planula larvae of the scleractinian coral, Acropora tenuis. Here, we show that soon after mixture with the dinoflagellate, Breviolum minutum, flattened amorphous coral cells with endodermal properties exhibited elevated locomotor activity using filopodia and lamellipodia and interacted with dinoflagellates. Several minutes thereafter, coral cells began to incorporate B. minutum, and in vitro symbiosis appeared to have been accomplished within 30 min. Nearly a half of the coral cells had incorporated algal cells within 24 h in a reproducible manner. Coral cells that harbored algal cells gradually became round and less mobile, and the algal cells sometimes settled in vacuole-like structures in coral cell cytoplasm. This symbiosis state was maintained for at least a month. The IVB5 line of A. tenuis therefore provides an experimental system to explore cellular and molecular mechanisms involved in coral-dinoflagellate symbiosis at the single-cell level, results of which may be useful for future preservation of coral reefs.

Can Acropora tenuis larvae attract native Symbiodiniaceae cells by green fluorescence at the initial establishment of symbiosis?

Most corals acquire symbiodiniacean symbionts from the surrounding environment to initiate symbiosis. The cell densities of Symbiodiniaceae in the environment are usually low, and mechanisms may exist by which new coral generations attract suitable endosymbionts. Phototaxis of suitable symbiodiniacean cells toward green fluorescence in corals has been proposed as one such mechanism. In the present study, we observed the phototaxis action wavelength of various strains of Symbiodiniaceae and the fluorescence spectra of aposymbiotic Acropora tenuis larvae at the time of endosymbiont uptake. The phototaxis patterns varied among the Symbiodiniaceae species and “native” endosymbionts—commonly found in Acropora juveniles present in natural environments; that is, Symbiodinium microadriaticum was attracted to blue light rather than to green light. Another native endosymbiont, Durusdinium trenchii, showed no phototaxis specific to any wavelength. Although the larvae exhibited green and broad orange fluorescence under blue-violet excitation light, the maximum green fluorescence peak did not coincide with that of the phototaxis action spectrum of S. microadriaticum. Rather, around the peak wavelength of larval green fluorescence, this native endosymbiont showed slightly negative phototaxis, suggesting that the green fluorescence of A. tenuis larvae may not play a role in the initial attraction of native endosymbionts. Conversely, broad blue larval fluorescence under UV-A excitation covered the maximum phototaxis action wavelength of S. microadriaticum. We also conducted infection tests using native endosymbionts and aposymbiotic larvae under red LED light that does not excite visible larval fluorescence. Almost all larvae failed to acquire S. microadriaticum cells, whereas D. trenchii cells were acquired by larvae even under red illumination. Thus, attraction mechanisms other than visible fluorescence might exist, at least in the case of D. trenchii. Our results suggest that further investigation and discussion, not limited to green fluorescence, would be required to elucidate the initial attraction mechanisms.

Enhancing Coral Survival on Deployment Devices With Microrefugia

Surviving after settlement through the first year of life is a recognised bottleneck in up-scaling reef coral restoration. Incorporating spatial refugia in settlement devices has the potential to alleviate some hazards experienced by young recruits, such as predation and accidental grazing, and can increase the likelihood of survival to size-escape thresholds. Yet optimising the design of microrefugia is challenging due to the complexity of physical and biological processes that occur at fine spatial scales around a recruit. Here, we investigated the effects of microhabitat features on the survival of Acropora tenuis spat in a year-long experimental field deployment of two types of artificial settlement devices—grooved-tiles and lattice-grids—onto three replicate racks on a shallow, central mid-shelf reef of the Great Barrier Reef. Spat survival across device types averaged between 2 and 39% and about half of all devices had at least one surviving coral after a year. While the larvae settled across all micro-habitats available on the devices, there was strong post-settlement selection for corals on the lower edges, lower surfaces, and in the grooves, with 100% mortality of recruits on upper surfaces, nearly all within the first 6 months of deployment. The device type that conferred the highest average survival (39%) was a tile with wide grooves (4 mm) cut all the way through, which significantly improved survival success over flat and comparatively featureless control tiles (13%). We hypothesise that the wide grooves provided protection from accidental grazing while also minimising sediment accumulation and allowing higher levels of light and water flow to reach the recruits than featureless control devices. We conclude that incorporating design features into deployment devices such as wide slits has the potential to substantially increase post-deployment survival success of restored corals.

Establishing Sustainable Cell Lines of a Coral, Acropora tenuis

Planula larvae of the scleractinian coral,Acropora tenuis, consist of elongated ectodermal cells and developing inner endodermal cells. To establish in vitro cell lines for future studies of cellular and developmental potential of coral cells, larvae were successfully dissociated into single cells by treating them with a tissue dissociation solution consisting of trypsin, EDTA, and collagenase. Brown-colored cells, translucent cells, and pale blue cells were the major components of dissociated larvae. Brown-colored cells began to proliferate transiently in the culture medium that was devised for the coral, while translucent cells and pale blue cells decreased in number about 1 week after cell dissociation. In addition, when a modular protease, plasmin, was added to the cell culture medium, brown-colored cells extended pseudopodia and assumed amorphous shapes. They then continued to proliferate in clumps for more than 6 months with a doubling time of approximately 4–5 days. From 3 weeks of cell culture onward, brown-colored cells often aggregated and exhibited morphogenesis-like behavior to form flat sheets, and blastula-like clusters or gastrula-like spheres. Single cells or cell-clusters of the cell lines were analyzed by RNA-seq. This analysis showed that genes expressed in these cells in vitro wereA. tenuisgenes. Furthermore, each cell line expressed a specific set of genes, suggesting that their properties include gastroderm, secretory cells, undifferentiated cells, neuronal cells, and epidermis. All cell properties were maintained stably throughout successive cell cultures. These results confirm the successful establishment of a coral in vitro cell line.

N-Acetyl-d-Glucosamine-Binding Lectin in Acropora tenuis Attracts Specific Symbiodiniaceae Cell Culture Strains

Many corals establish symbiosis with Symbiodiniaceae cells from surrounding environments, but very few Symbiodiniaceae cells exist in the water column. Given that the N-acetyl-d-glucosamine-binding lectin ActL attracts Symbiodiniaceae cells, we hypothesized that corals must attract Symbiodiniaceae cells using ActL to acquire them. Anti-ActL antibody inhibited acquisition of Symbiodiniaceae cells, and rearing seawater for juvenile Acropora tenuis contained ActL, suggesting that juvenile A. tenuis discharge ActL to attract these cells. Among eight Symbiodiniaceae cultured strains, ActL attracted NBRC102920 (Symbiodinium tridacnidorum) most strongly followed by CS-161 (Symbiodinium tridacnidorum), CCMP2556 (Durusdinium trenchii), and CCMP1633 (Breviolum sp.); however, it did not attract GTP-A6-Sy (Symbiodinium natans), CCMP421 (Effrenium voratum), FKM0207 (Fugacium sp.), and CS-156 (Fugacium sp.). Juvenile polyps of A. tenuis acquired limited Symbiodiniaceae cell strains, and the number of acquired Symbiodiniaceae cells in a polyp also differed from each other. The number of Symbiodiniaceae cells acquired by juvenile polyps of A. tenuis was correlated with the ActL chemotactic activity. Thus, ActL could be used to attract select Symbiodiniaceae cells and help Symbiodiniaceae cell acquisition in juvenile polyps of A. tenuis, facilitating establishment of symbiosis between A. tenuis and Symbiodiniaceae cells.