Settlement-competency period of planulae and genetic differentiation of the scleractinian coral Acropora digitifera

Broadcast-spawning corals expel eggs and sperm, and the fertilized eggs develop into planulae in the water column. As these sessile corals generally disperse during the planktonic larval stage, their larval characteristics (e.g., survival and settlement rates) are thought to be important for their dispersal. Although some studies of coral larval dispersal have focused on the maximum time that larvae can remain viable and settle, the relevance of this maximum settlement competency period for long distance dispersal remains unclear. To examine the relationship between competency periods and genetic differentiation, we performed laboratory experiments to investigate settlement rates of planulae and determine the degree of genetic differentiation in Acropora digitifera in the Ryukyu Archipelago, southern Japan. In addition, we compared our findings to published data for A. tenuis, which was studied using our methods. Our results indicated that the maximum settlement competency period was lower in A. digitifera planulae (54 days) than in A. tenuis (69 days) planulae. The mean survival rates at 45 days and 59 days after spawning were less than 10%. Furthermore, percentages of planulae that remained viable and settle at 30 days after spawning (survival rate x settlement rates at 30 days) were approximately 18% and 25% in A. digitifera and A. tenuis, respectively. By contrast, gene flow (N(e)m: number of migrations per generation) was significantly higher in A. digitifera (7.8 to 41.4) than in A. tenuis (3.1 to 22.5). These results indicate that the settlement competency period and survival rates are unlikely to be robust predictors of gene flow. Overall, we detected significant genetic differentiation between Kerama and Okinawa in A. digitifera. As direct observation of planula dispersal between Kerama and Okinawa has been reported, we concluded that genetic mixing is not complete, but that some localized planulae may disperse from Kerama to Okinawa via a specific current depending on reef or locality.     

Seasonal calcification of the coral Acropora digitifera from a subtropical marginal Okinawa reef under ocean acidification

Coral Reefs - Coral calcification is affected by the decrease in aragonite saturation state (Ωarag) caused by ocean acidification (OA). However, OA effects are modulated by other environmental...     

Ocean acidification does not affect the physiology of the tropical coral Acropora digitifera during a 5-week experiment

The increase in atmospheric CO₂ concentration, which has resulted from the burning of fossil fuels, is being absorbed by the oceans and is causing ocean acidification. Ocean acidification involves the decrease of both the pH and the calcium carbonate saturation state. Ocean acidification is predicted to impact the physiology of marine organisms and reduce the calcification rates of corals. In the present study, we measured the rates of calcification, respiration, photosynthesis, and zooxanthellae density of the tropical coral Acropora digitifera under near-natural summertime temperature and sunlight for a 5-week period. We found that these key physiological parameters were not affected by both mid-CO₂ (pCO₂ = 744 ± 38, pH = 7.97 ± 0.02, Ω_arag = 2.6 ± 0.1) and high-CO₂ conditions (pCO₂ = 2,142 ± 205, pH = 7.56 ± 0.04, Ω_arag = 1.1 ± 0.2) throughout the 35 days experimental period. Additionally, there was no significant correlation between calcification rate and seawater aragonite saturation (Ω_arag). These results suggest that the impacts of ocean acidification on corals physiology may be more complex than have been previously proposed.     

Spectral and structural analysis of a red fluorescent protein from Acropora digitifera

Fluorescent proteins (FPs) possess a wide variety of spectral properties that make them of widespread interest as optical markers. These proteins can be applied as pH indicators or metal biosensors. The discovery and characterization of new fluorescent proteins is expected to further extend their application. Here, we report the spectral and structural analysis of a red fluorescent protein from Acropora digitifera (designated AdRed). This protein shows a tetrameric state and is red emitting, with excitation and emission maxima at 567 and 612 nm, respectively. Its crystal structure shows the tetrameric interface stabilized by hydrogen bonding and salt bridges. The electron density map of the chromophore, consisting of Asp66–Tyr67–Gly68, shows the decarboxylated side chain of Asp66. Ser223, located near the chromophore, has the role of bridging His202 and Glu221, and is part of the hydrogen bond network. Mutated AdRed with Cys148Ser reveals a blue shift in fluorescence excitation and emission. Our results provide insights into understanding the molecular function of AdRed and other FPs.     

Phylogenetic characterization of culturable actinomycetes associated with the mucus of the coral Acropora digitifera from Gulf of Mannar

The marine environment is a virtually untapped source of novel actinomycete diversity and its metabolites. Investigating the diversity of actinomycetes in other marine macroorganisms, like seaweeds and sponges, have resulted in isolation of novel bioactive metabolites. Actinomycetes diversity associated with corals and their produced metabolites have not yet been explored. Hence, in this study we attempted to characterize the culturable actinomycetes population associated with the coral Acropora digitifera. Actinomycetes were isolated from the mucus of the coral wherein the actinomycetes count was much higher when compared with the surrounding seawater and sediment. Actinobacteria-specific 16S rRNA gene primers were used for identifying the isolates at the molecular level in addition to biochemical tests. Amplified ribosomal DNA restriction analysis using three restriction enzymes revealed several polymorphic groups within the isolates. Sequencing and blast analysis of the isolates revealed that some isolates had only 96.7% similarity with its nearest match in GenBank indicating that they may be novel isolates at the species level. The isolated actinomycetes exhibited good antibacterial activity against various human pathogens. This study offers for the first time a prelude about the unexplored culturable actinomycetes diversity associated with a scleractinian coral and their bioactive capabilities.     

Identification of fast-evolving genes in the scleractinian coral Acropora using comparative EST analysis

To identify fast-evolving genes in reef-building corals, we performed direct comparative sequence analysis with expressed sequence tag (EST) datasets from two acroporid species: Acropora palmata from the Caribbean Sea and A. millepora from the Great Barrier Reef in Australia. Comparison of 589 independent sequences from 1,421 A. palmata contigs, with 10,247 A. millepora contigs resulted in the identification of 196 putative homologues. Most of the homologous pairs demonstrated high amino acid similarities (over 90%). Comparisons of putative homologues showing low amino acid similarities (under 90%) among the Acropora species to the near complete datasets from two other cnidarians (Hydra magnipapillata and Nematostella vectensis) implied that some were non-orthologous. Within 86 homologous pairs, 39 exhibited dN/dS ratios significantly less than 1, suggesting that these genes are under purifying selection associated with functional constraints. Eight independent genes showed dN/dS ratios exceeding 1, while three deviated significantly from 1, suggesting that these genes may play important roles in the adaptive evolution of Acropora. Our results also indicated that CEL-III lectin was under positive selection, consistent with a possible role in immunity or symbiont recognition. Further studies are needed to clarify the possible functions of the genes under positive selection to provide insight into the evolutionary process of corals.     

Phylogenetic characterization of culturable bacterial diversity associated with the mucus and tissue of the coral Acropora digitifera from the Gulf of Mannar

Corals, considered the rainforests of the oceans, harbour an abundance of different bacterial populations throughout the coral structure. In the present study we attempted to characterize the cultivable bacterial population associated within the mucus and tissue of the coral Acropora digitifera from the Gulf of Mannar. 16S rRNA gene was amplified from the cultured mucus and tissue isolates. Amplified ribosomal DNA restriction analysis, performed with a combination of restriction enzymes to determine the polymorphic groups of bacteria, generated 19 distinct groups in the coral mucus and 17 distinct groups in the coral tissue. Phylogenetic analyses based on the full-length sequences of 16S rRNA gene sequences showed that the majority of bacterial isolates belonged to the group Firmicutes , followed by Gammaproteobacteria and Actinobacteria . On investigating their antimicrobial activity, mucus isolates showed about 25% activity and tissue isolates showed 48% activity. This study revealed the presence of actinomycetes in both the coral mucus and the coral tissue, which had high activity against pathogens. This study, for the first time, demonstrates that actinomycetes existing within corals also have potential antibacterial activity. This has been overlooked so far, and indicates that, in addition to mucus, bacteria within the tissue of corals might defend the coral host against pathogens.     

Embryogenesis in the reef-building coral Acropora spp

Embryogenesis in the reef building corals Acropora intermedia, A. solitaryensis, A. hyacinthus, A. digitifera, and A. tenuis was studied in detail at the morphological level, and the relationships among the animal pole, blastopore, and mouth were investigated for the first time in corals. These species showed essentially the same sequence of development. The embryo undergoes spiral-like holoblastic cleavage despite the presence of a dense isolecithal yolk. After the morula stage, the embryo enters the "prawn-chip" stage, which consists of an irregularly shaped cellular bilayer. The embryo begins to roll inward to form the bowl stage; the round shape observed during this stage suggests that it may be the beginning of gastrulation. However, the blastopore closes and the stomodeum (mouth and pharynx) is formed via invagination at a site near the closed blastopore. During the planula stage, a concavity forms in the aboral region in conjunction with numerous spirocysts, suggesting that spirocysts are used to attach to the substrate before the onset of metamorphosis.     

Expression of putative immune response genes during early ontogeny in the coral Acropora millepora

Background

Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals.

Methodology/Principal Findings

Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A.millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned.

Conclusions/Significance

Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are needed to further clarify emerging evidence of a complex innate immunity system in corals.     

Differential expression of three galaxin-related genes during settlement and metamorphosis in the scleractinian coral Acropora millepora

Background  

The coral skeleton consists of CaCO₃ deposited upon an organic matrix primarily as aragonite. Currently galaxin, from Galaxea fascicularis, is the only soluble protein component of the organic matrix that has been characterized from a coral. Three genes related to galaxin were identified in the coral Acropora millepora.     

Construction of a high-resolution genetic linkage map and comparative genome analysis for the reef-building coral Acropora millepora

Background  

Worldwide, coral reefs are in decline due to a range of anthropogenic disturbances, and are now also under threat from global climate change. Virtually nothing is currently known about the genetic factors that might determine whether corals adapt to the changing climate or continue to decline. Quantitative genetics studies aiming to identify the adaptively important genomic loci will require a high-resolution genetic linkage map. The phylogenetic position of corals also suggests important applications for a coral genetic map in studies of ancestral metazoan genome architecture.     

Cryopreservation of sperm from the coral Acropora humilis

Corals are sensitive to minute changes in their environments, and their continued existence is substantially threatened by the increasing number of destructive anthropogenic activities and unprecedented rates of climate change. Although cryopreservation has been successfully to preserve mammalian gametes for decades, coral cryopreservation was attempted for the first time less than 15 years ago, and freezing protocols exist for only a handful of coral species. The present study developed a cryopreservation protocol for the sperm of the common Indo-Pacific reef-builder Acropora humilis. Colonies of reefs of Sattahip Bay, Chonburi Province, Thailand were collected from 3 m depth with a mesh net during a spawning event. Immediately after collection, the sperm were isolated and subjected to a two-step freezing method featuring DMSO, polyethylene glycol, or methanol as the cryoprotectant. Viability and motility were assessed via a bioluminescence technique and a “computer-assisted semen analysis, and it was found that a 15-min equilibration with 2 M DMSO followed by cooling at 41.7 °C was the optimum cryopreservation protocol for A. humilis sperm. The post-thaw sperm achieved 45% fertilization success, and 35% of the fertilized eggs developed into blastopore larvae. The present optimized protocol can therefore facilitate the preservation of sperm for future propagation efforts of this species and provide an experimental platform for optimizing cryopreservation protocols for gametes of other scleractinian coral species.     

Localization of two L-glutamate dehydrogenases in the coral Acropora latistella

The coral Acropora latistella has been shown to contain two distinct L-glutamate dehydrogenases. An NADPH-specific GDH, closely resembling the enzyme from other coelenterates, is located in the cytosol; a distinct NADH-specific GDH is localized within the mitochondria. This is in contrast to earlier reports, where the NADPH-specific enzyme was thought to be the only GDH present and was assumed to be mitochondrial. Preliminary kinetic and stability data are presented for the two GDHs.     

Sympatric populations of the highly cross-fertile coral species Acropora hyacinthus and Acropora cytherea are genetically distinct

High cross-fertilization rates in vitro and non-monophyletic patterns in molecular phylogenies challenge the taxonomic status of species in the coral genus Acropora. We present data from eight polymorphic allozyme loci that indicate small, but significant, differentiation between sympatric populations of Acropora cytherea and Acropora hyacinthus (F(ST) = 0.025-0.068, p < 0.05), a pair of acroporid corals with very high interspecific fertilization rates in vitro. Although no fixed allelic differences were found between these species, the absence of genetic differentiation between widely allopatric populations suggests that allele frequency differences between A. cytherea and A. hyacinthus in sympatry are biologically significant. By contrast, populations of Acropora tenuis, a species which spawns 2-3 hours earlier and shows low cross-fertilization rates with congeners in vitro, were clearly distinct from A. cytherea and A. hyacinthus (F(ST) = 0.427-0.465, p < 0.05). Moreover, allopatric populations of A. tenuis differed significantly, possibly as a consequence of its relatively short period of larval competency. Our results effectively rule out the possibility that A. hyacinthus and A. cytherea are morphotypes within a single species, and indicate that hybridization occurs relatively infrequently between these taxa in nature.