Experimental observation of microplastics invading the endoderm of anthozoan polyps.

Coral reefs are being degraded worldwide by land reclamation and environmental factors, such as high seawater temperature, resulting in mass bleaching events. In addition, microplastics disturb the formation of coral-algae symbiotic relationships in primary polyps. In our experiments, we observed this effect in the bleached primary polyp Seriatopora caliendrum that lost its symbiont Symbiodiniaceae as a result of high water temperature. There was a higher incorporation of microspheres into bleached corals than in healthy ones. To understand the interference in symbiosis, we used the sea anemone Exaiptasia (as an anthozoan model organism) and fed it with microspheres. TEM results suggested the incorporation of microspheres and symbionts from the same phagocytosis zones in the mesenterial filament and endocytosis by the cells. In the tentacles, microspheres were in the same cell layer as the symbionts. These results suggest that microplastics occupy the spaces inhabited by Symbiodiniaceae, thereby hindering their symbiotic association.

Microplastics disturb the anthozoan-algae symbiotic relationship.

World production of plastic has dramatically increased from the 1950's and now it reaches approximately 311 million tons per year. The resulting accumulation of small plastic detritus less than 5 mm in size, termed "microplastics", has started threatening the life cycles of marine organisms. Here we show the first evidence that microplastics disturb the initiation of symbiotic relationships in anthozoan-algae symbiosis. We found in both the aposymbiotic sea-anemone Aiptasia sp. and the coral Favites chinensis that the infectivity of symbiotic algae into the host is severely suppressed by microspheres fed either directly or indirectly through microsphere-fed Artemia sp. Similar trends were seen when microplastics collected from commercial facewash were used instead of microspheres. Therefore, ongoing accumulation of microplastics in the ocean might disturb the healthy anthozoan-algae symbiotic relationships, which are cornerstones of the biologically enriched coral reef ecosystem.

Coral individuality - confluence of change physical splitting and developmental ability of embryos.

Previous studies have suggested that blastomeres from the 2-, 4-, or 8-cell stage of corals have the ability to develop into normal primary polyps. However, it is still not known which developmental stage's blastomere produces which juvenile. In this study, we demonstrated that only the blastomeres with animal hemispheres have the capacity to develop into normal primary polyps. Individuality was evaluated using blastomeres isolated from the corals Acropora digitifera, A. intermedia, Dipsastraea lizardensis, and Favites chinensis. On commencement of embryo cleavage, the animal pole was marked using Neutral red staining, and at the 2-, 4-, and 8-cell stages, embryos were divided into individual blastomeres using glass needles. We found that the survival rate and percentage metamorphosis were higher in the larger-sized blastomeres with animal hemispheres. The vegetal hemisphere alone is incapable of developing into a normal primary polyp; however, a ball-shaped embryo with incomplete mesenteries and no pharynx developed in some cases. These results indicate that the animal hemisphere is needed for corals to develop into normal primary polyps, and that the individuality of corals is possibly determined by a combination of the chance physical splitting of embryos by waves and their innate developmental ability.

Acceptable symbiont cell size differs among cnidarian species and may limit symbiont diversity.

Reef-building corals form symbiotic relationships with dinoflagellates of the genus Symbiodinium. Symbiodinium are genetically and physiologically diverse, and corals may be able to adapt to different environments by altering their dominant Symbiodinium phylotype. Notably, each coral species associates only with specific Symbiodinium phylotypes, and consequently the diversity of symbionts available to the host is limited by the species specificity. Currently, it is widely presumed that species specificity is determined by the combination of cell-surface molecules on the host and symbiont. Here we show experimental evidence supporting a new model to explain at least part of the specificity in coral-Symbiodinium symbiosis. Using the laboratory model Aiptasia-Symbiodinium system, we found that symbiont infectivity is related to cell size; larger Symbiodinium phylotypes are less likely to establish a symbiotic relationship with the host Aiptasia. This size dependency is further supported by experiments where symbionts were replaced by artificial fluorescent microspheres. Finally, experiments using two different coral species demonstrate that our size-dependent-infection model can be expanded to coral-Symbiodinium symbiosis, with the acceptability of large-sized Symbiodinium phylotypes differing between two coral species. Thus the selectivity of the host for symbiont cell size can affect the diversity of symbionts in corals.

A comparative view of early development in the corals Favia lizardensis, Ctenactis echinata, and Acropora millepora - morphology, transcriptome, and developmental gene expression.

BACKGROUND: Research into various aspects of coral biology has greatly increased in recent years due to anthropogenic threats to coral health including pollution, ocean warming and acidification. However, knowledge of coral early development has lagged. The present paper describes the embryonic development of two previously uncharacterized robust corals, Favia lizardensis (a massive brain coral) and Ctenactis echinata (a solitary coral) and compares it to that of the previously characterized complex coral, Acropora millepora, both morphologically and in terms of the expression of a set of key developmental genes. RESULTS: Illumina sequencing of mixed age embryos was carried out, resulting in embryonic transcriptomes consisting of 40605 contigs for C.echinata (N50 = 1080 bp) and 48536 contigs for F.lizardensis (N50 = 1496 bp). The transcriptomes have been annotated against Swiss-Prot and were sufficiently complete to enable the identification of orthologs of many key genes controlling development in bilaterians. Developmental series of images of whole mounts and sections reveal that the early stages of both species contain a blastocoel, consistent with their membership of the robust clade. In situ hybridization was used to examine the expression of the developmentally important genes brachyury, chordin and forkhead. The expression of brachyury and forkhead was consistent with that previously reported for Acropora and allowed us to confirm that the pseudo-blastopore sometimes seen in robust corals such as Favia spp. is not directly associated with gastrulation. C.echinata chordin expression, however, differed from that seen in the other two corals. CONCLUSIONS: Embryonic transcriptomes were assembled for the brain coral Favia lizardensis and the solitary coral Ctenactis echinata. Both species have a blastocoel in their early developmental stages, consistent with their phylogenetic position as members of the robust clade. Expression of the key developmental genes brachyury, chordin and forkhead was investigated, allowing comparison to that of their orthologs in Acropora, Nematostella and bilaterians and demonstrating that even within the Anthozoa there are significant differences in expression patterns.

Restructuring the Traditional Suborders in the Order Scleractinia Based on Embryogenetic Morphological Characteristics.

The order Scleractinia includes two distinct groups, which are termed "complex" and "robust" as indicated by the molecular phylogeny of mitochondrial 16S ribosomal gene sequences. Since this discovery, coral taxonomists have been seeking morphological characters for grouping this deep division in the order Scleractinia. Recently, morphological characteristics during embryogenesis that facilitate grouping the two clades as "complex" and "robust" were reported, thus clarifying a deep division in the Scleractinia. In the present report, I establish two new suborders, Refertina and Vacatina, on the basis of the embryogenetic morphological characteristics, molecular data, and new observations of Tubastraea coccinea and Cyphastrea serailia embryogenesis. In particular, the embryo of T. coccinea has a possible fertilization membrane that was first observed in the phylum Cnidaria. The new suborder Refertina consists of the families that belong to the "complex" clade and have no or little blastocoel. The new suborder Vacatina is composed of the families that fall into the "robust" clade and have an apparent blastocoel.

Comparative embryology of eleven species of stony corals (Scleractinia).

A comprehensive understanding of coral reproduction and development is needed because corals are threatened in many ways by human activity. Major threats include the loss of their photosynthetic symbionts (Symbiodinium) caused by rising temperatures (bleaching), reduced ability to calcify caused by ocean acidification, increased storm severity associated with global climate change and an increase in predators caused by runoff from human agricultural activity. In spite of these threats, detailed descriptions of embryonic development are not available for many coral species. The current consensus is that there are two major groups of stony corals, the "complex" and the "robust". In this paper we describe the embryonic development of four "complex" species, Pseudosiderastrea tayamai, Galaxea fascicularis, Montipora hispida, and Pavona Decussata, and seven "robust" species, Oulastrea crispata, Platygyra contorta, Favites abdita, Echinophyllia aspera, Goniastrea favulus, Dipsastraea speciosa (previously Favia speciosa), and Phymastrea valenciennesi (previously Montastrea valenciennesi). Data from both histologically sectioned embryos and whole mounts are presented. One apparent difference between these two major groups is that before gastrulation the cells of the complex corals thus far described (mainly Acropora species) spread and flatten to produce the so-called prawn chip, which lacks a blastocoel. Our present broad survey of robust and complex corals reveals that prawn chip formation is not a synapomorphy of complex corals, as Pavona Decussata does not form a prawn chip and has a well-developed blastocoel. Although prawn chip formation cannot be used to separate the two clades, none of the robust corals which we surveyed has such a stage. Many robust coral embryos pass through two periods of invagination, separated by a return to a spherical shape. However, only the second of these periods is associated with endoderm formation. We have therefore termed the first invagination a pseudo-blastopore.

Survival dynamics of reef coral larvae with special consideration of larval size and the genus Acropora.

Temporal dynamics of larval survival were examined in vitro in four broadcast-spawning reef coral species, Acropora hyacinthus, A. japonica, A. solitaryensis, and Goniastrea pectinata. Larval size was treated as an important characteristic that may relate to larval lifespan. Two patterns were observed in larval survival dynamics between the three Acropora species (mean initial larval size; 0.05-0.08 mm(3)) and G. pectinata (0.02 mm(3)), based on the timing of a sharp drop in larval survival rates (ca. > 50% reduction over a 1-2 week period). Consequently, the majority of larvae of the three Acropora species had a lifespan of less than 2-3 weeks, whereas those of G. pectinata were extended a further 2-3 weeks despite the smaller larval size. No significant relationship was detected between the initial larval size and larval lifespan in any of the four reef coral species. These results suggest that (1) larval dispersal of spawning Acropora species may be on a more local scale than that of G. pectinata and most other reef coral species previously reported, and (2) larval size is not a good estimator of larval lifespan in reef coral species.

Development and application of a simple LC-MS method for the determination of plasma maraviroc concentrations.

Maraviroc is an orally available antagonist of the CCR5 chemokine receptor, which acts as a human immunodeficiency virus type 1 (HIV-1) coreceptor. Binding of maraviroc to this receptor blocks HIV-1 attachment to the coreceptor and prevents HIV-1 from entering host cells. Maraviroc does not require intracellular processing to exert this activity. Drug interaction studies have shown changes in maraviroc exposure when given with other anti-HIV medications, and thus quantification of maraviroc in human plasma is important to manage drug interactions and to evaluate the relationship between plasma concentrations and treatment response. We developed a conventional LC-MS method for determining plasma maraviroc concentrations, validated by estimating precision and accuracy for inter- and intraday analysis in the concentration range of 0.011-2.188 µg/ml. The calibration curve was linear within this range. The average accuracy ranged from 92.7% to 99.7%, while the relative standard deviations of both inter- and intraday assays were less than 7.1%. Recovery of maraviroc exceeded 86.7%. Our LC-MS method provides a conventional, accurate and precise way to determine the maraviroc concentration in human plasma. This method enables dose adjustment based on monitoring plasma maraviroc concentrations and permits management of drug interactions and toxicity.

Reproduction in cultured versus wild coral colonies: fertilization, larval oxygen consumption, and survival.

In the late 1990s, the once prolific populations of the coral Acropora intermedia surrounding Okinawa, Japan, dramatically declined because of thermal stress, bleaching caused by heat stress, and consequent mortality. Before the bleaching event, 72 fragments (about 15 cm in length) were collected and transferred to the Okinawa Churami Aquarium. Through growth and repeated fragmentation, these original fragments developed into about 100 colonies that spawned from 1999 to 2009. In this study, we compared gametogenesis, fertilization, survival, and O(2) consumption in cultured and wild colonies of A. intermedia and their offspring. Cultured A. intermedia had larger oocytes and higher fertilization and survival rates than samples from wild colonies. O(2) consumption of cultured embryos was similar to that of wild embryos. These results suggest that cultured A. intermedia and their offspring are as viable as wild colonies. Aquaria can play a role in the conservation of endangered corals, and their cultured colonies could be used to re-establish devastated species on the Okinawa reefs.

Crystallization and preliminary crystallographic studies of putative RNA 3'-terminal phosphate cyclase from the crenarchaeon Sulfolobus tokodaii.

RNA 3'-terminal phosphate cyclase (Rtc) is an enzyme involved in RNA splicing that converts the 3'-terminal hydroxyl group of truncated RNA to 2',3'-cyclic phosphate, which is required just before its ligation. This reaction may occur in the following two steps: (i) Rtc + ATP --> Rtc-AMP + PP(i) and (ii) RNA-N3'p + Rtc-AMP --> RNA-N>p + Rtc + AMP. In order to reveal the reaction mechanism, Rtc of Sulfolobus tokodaii (St-Rtc) overexpressed in Escherichia coli was purified and crystallized in the following states: St-Rtc, St-Rtc+Mn, St-Rtc+ATP, St-Rtc+AMP and St-Rtc-AMP. The crystals diffracted to 2.25-3.00 A resolution and preliminary solutions of their structures have been obtained by molecular replacement using the structure of a selenomethionine-labelled St-Rtc crystal which was solved in advance using the MAD method as a model. These crystals grew in two different space groups (P3(1) and P4(2)), with the former space group displaying two distinct packing modes.

Possible self-fertilization in the brooding coral Acropora (Isopora) brueggemanni.

We examined gametogenesis and the periodicity of planula release in the brooding coral Acropora (Isopora) brueggemanni (Brook, 1893) on Akajima Island, Okinawa, Japan. We captured the moment when A. brueggemanni would be in the process of self-fertilization. Whole colonies of this species were cultured separately or together with other colonies in plastic containers. We observed no apparent periodicity of planula release in the collected colonies. A few planulae were released intermittently during the observation period, regardless of whether the colonies were cultured individually or with other colonies. Serial paraffin sections of A. brueggemanni showed follicle-like cells surrounding the oocyte during vitellogenesis. In September and October, some spermaries looked half-broken and some ova were surrounded by sperm or spermaries instead of follicle-like cells. Such ova were heart-shaped and may have been cells at the first cleavage stage. These observations suggest that the migration of spermaries and/or oocytes resulted in the close proximity of oocytes and sperm, which would allow self-fertilization. This possibility, together with the production of planula larvae by isolated colonies, suggests that this brooding coral engages in self-fertilization.

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.