Área de conservación Guanacaste Echinoderms, North Pacific of Costa Rica / Equinodermos del Área de Conservación Guanacaste, Pacífico Norte de Costa Rica

Introduction: The study of the marine diversity of the North Pacific of Costa Rica began with isolated foreign expeditions in the 1930s and was systematically developed in the mid-1990s by the Center for Research in Marine Sciences and Limnology, Universidad de Costa Rica, as consequence there are now a total of 1 479 reported species in this region. Objective: Present an update to the echinoderm richness of the Guanacaste Conservation Area. Methods: We sampled 25 localities exhaustively and estimated similarity between sites based on the family richness and environmental heterogeneity. Results: We found 61 taxa, which represent 26 % of the echinoderm reported species for the country's Pacific coast. Of these, 43 species are new records for the Guanacaste Conservation Area, and seven for Costa Rica and Central American Pacific coasts. We found three morpho-species that do not match to available descriptions of the Eastern Tropical Pacific echinoderm species. We also found the holothuroid Epitomapta tabogae, and the ophiuroid Ophioplocus hancocki, previously thought endemic to Panama and the Galapagos Islands, respectively. The proximity of the sampled sites and the redundancy of certain families may explain why we did not find important differences among localities. Conclusions: The echinoderm richness of this conservation area is at least 20 % higher than previously reported, reaching similar levels to those in other high diversity sites of the Eastern Tropical Pacific.

Introducción: El estudio de la diversidad marina del Pacífico Norte de Costa Rica inició con expediciones extranjeras aisladas en la década de 1930, y fue desarrollado sistemáticamente a mediados de la década de 1990 por el Centro de Investigaciones en Ciencias del Mar y Limnología de la Universidad de Costa Rica, como consecuencia ahora se reporta un total de 1 479 especies en esta región. Objetivo: Presentar una actualización de la riqueza de equinodermos del Área de Conservación Guanacaste. Métodos: Realizamos muestreos exhaustivos en 25 localidades y estimamos la similitud entre sitios con base en la riqueaza de familias y la heterogeneidad ambiental. Resultados: Encontramos 61 taxa, que representan el 26% de las especies reportadas para la costa pacífica del país. De estas, 43 especies son nuevos registros para el Área de Conservación Guanacaste y siete para las costas de Costa Rica y el Pacífico centroamericano. Tres morfoespecies no coinciden con las descripciones disponibles para las especies del Pacífico Tropical Oriental. Por último, hallamos un ejemplar del holoturoideo Epitomapta tabogae y otro del ofiuroideo Ophioplocus hancocki, considerados endémicos para Panamá y las Islas Galápagos respectivamente. La proximidad entre los sitios muestreados y la redundancia de ciertas familias pueden explicar por qué no se encontraron diferencias entre las localidades. Conclusiones: La riqueza de equinodermos de esta área de conservación es al menos 20% mayor que la reportada anteriormente, alcanzando niveles similares a los de otros sitios de alta diversidad del Pacífico Tropical Oriental.

Gene expression analysis of three homeobox genes throughout early and late development of a feather star Anneissia japonica.

Crinoids are considered as the most basal extant echinoderms. They retain aboral nervous system with a nerve center, which has been degraded in the eleutherozoan echinoderms. To investigate the evolution of patterning of the nervous systems in crinoids, we examined temporal and spatial expression patterns of three neural patterning-related homeobox genes, six3, pax6, and otx, throughout the development of a feather star Anneissia japonica. These genes were involved in the patterning of endomesodermal tissues instead of the ectodermal neural tissues in the early planktonic stages. In the stages after larval attachment, the expression of these genes was mainly observed in the podia and the oral nervous systems instead of the aboral nerve center. Our results indicate the involvement of these three genes in the formation of oral nervous system in the common ancestor of the echinoderms and suggest that the aboral nerve center is not evolutionally related to the brain of other bilaterians.

Genomic insights of body plan transitions from bilateral to pentameral symmetry in Echinoderms.

Echinoderms are an exceptional group of bilaterians that develop pentameral adult symmetry from a bilaterally symmetric larva. However, the genetic basis in evolution and development of this unique transformation remains to be clarified. Here we report newly sequenced genomes, developmental transcriptomes, and proteomes of diverse echinoderms including the green sea urchin (L. variegatus), a sea cucumber (A. japonicus), and with particular emphasis on a sister group of the earliest-diverged echinoderms, the feather star (A. japonica). We learned that the last common ancestor of echinoderms retained a well-organized Hox cluster reminiscent of the hemichordate, and had gene sets involved in endoskeleton development. Further, unlike in other animal groups, the most conserved developmental stages were not at the body plan establishing phase, and genes normally involved in bilaterality appear to function in pentameric axis development. These results enhance our understanding of the divergence of protostomes and deuterostomes almost 500 Mya.

Retinoic Acid Signaling Regulates the Metamorphosis of Feather Stars (Crinoidea, Echinodermata): Insight into the Evolution of the Animal Life Cycle.

Many marine invertebrates have a life cycle with planktonic larvae, although the evolution of this type of life cycle remains enigmatic. We recently proposed that the regulatory mechanism of life cycle transition is conserved between jellyfish (Cnidaria) and starfish (Echinoderm); retinoic acid (RA) signaling regulates strobilation and metamorphosis, respectively. However, the function of RA signaling in other animal groups is poorly understood in this context. Here, to determine the ancestral function of RA signaling in echinoderms, we investigated the role of RA signaling during the metamorphosis of the feather star, Antedon serrata (Crinoidea, Echinodermata). Although feather stars have different larval forms from starfish, we found that exogenous RA treatment on doliolaria larvae induced metamorphosis, like in starfish. Furthermore, blocking RA synthesis or binding to the RA receptor suppressed metamorphosis. These results suggested that RA signaling functions as a regulator of metamorphosis in the ancestor of echinoderms. Our data provides insight into the evolution of the animal life cycle from the viewpoint of RA signaling.

Using ATAC-seq and RNA-seq to increase resolution in GRN connectivity.

Echinoderms have some of the most complete reconstructed developmental gene regulatory networks (GRN) of any embryo, accounting for the formation of most embryo tissues and organs. Yet, many nodes (genes and regulators) and their regulatory interactions are still to be uncovered. Traditionally, knockdown/knockout experiments are performed to determine regulator-gene interactions, which are individually validated by cis-regulatory analysis. Differential RNA-seq, combined with perturbation analysis, allows for genome-wide reconstruction of a GRN around given regulators; however, this level of resolution cannot determine direct interactions. ChiP-chip or ChIP-seq is better equipped for determining, genome-wide, whether binding of a given transcription factor (TF) to cis-regulatory elements occurs. Antibodies for the TFs of interest must be available, and if not, this presents a limiting factor. ATAC-seq identifies regions of open chromatin, that are typically trimethylated at H3K4, H3K36 and H3K79 (Kouzarides, 2007), for a given time point, condition, or tissue. This technology combined with RNA-seq and perturbation analysis provides high resolution of the possible functional interactions occurring during development. Additionally, ATAC-seq is less expensive than ChIP-seq, requires less starting material, and provides a global view of regulatory regions. This chapter provides detailed steps to identify potential regulatory relationships between the nodes of a GRN, given a well assembled genome, annotated with gene models, and ATAC-seq data combined with RNA-seq and knockdown experiments.

Identifying gene expression from single cells to single genes.

Gene regulatory networks reveal how transcription factors contribute to a dynamic cascade of cellular information processing. Recent advances in technologies have enhanced the toolkit for testing GRN mechanisms and connections. Here we emphasize three approaches that we have found important for interrogating transcriptional mechanisms in echinoderms: single cell mRNA sequencing (drop-seq), nascent RNA detection and identification, and chromatin immunoprecipitation (ChIP). We present these applications in order since it is a logical experimental protocol. With preliminary information from bulk mRNA transcriptome analysis and differential gene expression studies (DE-seq), one may need to test in what specific cells important genes may be expressed and to use single cell sequencing to define such links. Nascent RNA analysis with the Click-iT chemistry allows the investigator to deduce when the RNA was transcribed, not just identify its presence, and ChIP allows the investigator to study direct interactions of putative transcriptional regulators with the gene promoter of interest. This flow of thinking, and the technologies to support it, is presented here for echinoderms. While many of the procedures are general and applicable to many organisms and cell types, we emphasize unique aspects of the protocols for consideration in using echinoderm embryos, larvae, and adult tissues.

Early and later studies on action potential and fertilization potential of echinoderm oocytes and Ca2+ response of mammalian oocytes.

This is a personal essay starting from the early study on fertilization signals in echinoderm and mammalian oocytes. It presents actual examples showing that a unexpected discovery leads to unimaginable development of the research in diverse directions in later years and yields a common concept after long years' effort and accumulation. Those outcomes are the happiest gift for researchers. We also learn many precepts in our own research life.

Techniques for analyzing gene expression using BAC-based reporter constructs.

To characterize the complex regulatory control of gene expression using fluorescent protein reporters, it is often necessary to analyze large genomic regions. Bacteria artificial chromosome (BAC) vectors, which are able to support DNA fragments of up to 300kb, provide stable platforms for experimental manipulation. Using phage-based systems of homologous recombination, BACs can be efficiently engineered for a variety of aims. These include expressing fluorescent proteins to delineate gene expression boundaries using high-resolution, in vivo microscopy, tracing cell lineages using stable fluorescent proteins, perturbing endogenous protein function by expressing dominant negative forms, interfering with development by mis-expressing transcription factors, and identifying regulatory regions through deletion analysis. Here, we present a series of protocols for identifying BAC clones that contain genes of interest, modifying BACs for use as reporter constructs, and preparing BAC DNA for microinjection into fertilized eggs. Although the protocols here are tailored for use in echinoderm embryonic and larval stages, these methods are easily adaptable for use in other transgenic systems. As fluorescent protein technology continues to expand, so do the potential applications for recombinant BACs.

Genome-wide analysis of chromatin accessibility using ATAC-seq.

Programs of gene transcription are controlled by cis-acting DNA elements, including enhancers, silencers, and promoters. Local accessibility of chromatin has proven to be a highly informative structural feature for identifying such regulatory elements, which tend to be relatively open due to their interactions with proteins. Recently, ATAC-seq (assay for transposase-accessible chromatin using sequencing) has emerged as one of the most powerful approaches for genome-wide chromatin accessibility profiling. This method assesses DNA accessibility using hyperactive Tn5 transposase, which simultaneously cuts DNA and inserts sequencing adaptors, preferentially in regions of open chromatin. ATAC-seq is a relatively simple procedure which can be applied to only a few thousand cells. It is well-suited to developing embryos of sea urchins and other echinoderms, which are a prominent experimental model for understanding the genomic control of animal development. In this chapter, we present a protocol for applying ATAC-seq to embryonic cells of sea urchins.

Echinoderm eggs as a model for discoveries in cell biology.

I happen to have been trained in cell and developmental biology in the early 1970s, which was near the beginning of the explosive growth of the field of cell biology. The American Society for Cell Biology had been founded in 1960 and so the field was in its early days. Cell biology research was dominated by the use of the electron microscope and by protein biochemistry. Molecular biology and the use of genetics were in their infancy. When we track the path of discoveries in cell biology contributed by research using echinoderm eggs, we follow the development of new technologies in genetics, molecular biology, biochemistry and biophysics, bioengineering, and imaging. The changes in approaches and methods have led to many key discoveries in cell biology through the use of sea urchin, sand dollar and sea star eggs. These include the discovery of cyclin, cytoplasmic dynein, rho activation for cytokinesis, new membrane addition as a late event in cytokinesis, multiple kinesins playing multiple roles, how flagella beat, the dynamics of microtubules in the mitotic apparatus, control over centrosomes and cell cycle checkpoints, the process of nuclear envelope breakdown for cell division, the discovery of 1-methyl adenine (hormones) as the trigger for meiotic maturation, Ca++ transients controlling cell activation and exocytosis among others. What I hope to provide in this perspective is to highlight some of those wonderful discoveries as my own career evolved to contribute to the field.

Genomic resources for the study of echinoderm development and evolution.

Echinoderms are important research models for a wide range of biological questions. In particular, echinoderm embryos are exemplary models for dissecting the molecular and cellular processes that drive development and testing how these processes can be modified through evolution to produce the extensive morphological diversity observed in the phylum. Modern attempts to characterize these processes depend on some level of genomic analysis; from querying annotated gene sets to functional genomics experiments to identify candidate cis-regulatory sequences. Given how essential these data have become, it is important that researchers using available datasets or performing their own genome-scale experiments understand the nature and limitations of echinoderm genomic analyses. In this chapter we highlight the current state of echinoderm genomic data and provide methodological considerations for common approaches, including analysis of transcriptome and functional genomics datasets.

Nervous system characterization during the development of a basal echinoderm, the feather star Antedon mediterranea.

Neural development of echinoderms has always been difficult to interpret, as larval neurons degenerate at metamorphosis and a tripartite nervous system differentiates in the adult. Despite their key phylogenetic position as basal echinoderms, crinoids have been scarcely studied in developmental research. However, since they are the only extant echinoderms retaining the ancestral body plan of the group, crinoids are extremely valuable models to clarify neural evolution in deuterostomes. Antedon mediterranea is a feather star, endemic to the Mediterranean Sea. Its development includes a swimming lecithotrophic larva, the doliolaria, with basiepithelial nerve plexus, and a sessile filter-feeding juvenile, the pentacrinoid, whose nervous system has never been described in detail. Thus, we characterized the nervous system of both these developmental stages by means of immunohistochemistry and, for the first time, in situ hybridization techniques. The results confirmed previous descriptions of doliolaria morphology and revealed that the larval apical organ contains two bilateral clusters of serotonergic cells while GABAergic neurons are localized under the adhesive pit. This suggested that different larval activities (e.g., attachment and metamorphosis) are under the control of different neural populations. In pentacrinoids, the analysis showed the presence of a cholinergic entoneural system while the ectoneural plexus appeared more composite, displaying different neural populations. The expression of three neural-related microRNAs was described for the first time, suggesting that these are evolutionarily conserved also in basal echinoderms. Overall, our results set the stage for future investigations that will reveal new information on echinoderm evo-devo neurobiology.

Two more Posterior Hox genes and Hox cluster dispersal in echinoderms.

BACKGROUND: Hox genes are key elements in patterning animal development. They are renowned for their, often, clustered organisation in the genome, with supposed mechanistic links between the organisation of the genes and their expression. The widespread distribution and comparable functions of Hox genes across the animals has led to them being a major study system for comparing the molecular bases for construction and divergence of animal morphologies. Echinoderms (including sea urchins, sea stars, sea cucumbers, feather stars and brittle stars) possess one of the most unusual body plans in the animal kingdom with pronounced pentameral symmetry in the adults. Consequently, much interest has focused on their development, evolution and the role of the Hox genes in these processes. In this context, the organisation of echinoderm Hox gene clusters is distinctive. Within the classificatory system of Duboule, echinoderms constitute one of the clearest examples of Disorganized (D) clusters (i.e. intact clusters but with a gene order or orientation rearranged relative to the ancestral state). RESULTS: Here we describe two Hox genes (Hox11/13d and e) that have been overlooked in most previous work and have not been considered in reconstructions of echinoderm Hox complements and cluster organisation. The two genes are related to Posterior Hox genes and are present in all classes of echinoderm. Importantly, they do not reside in the Hox cluster of any species for which genomic linkage data is available. CONCLUSION: Incorporating the two neglected Posterior Hox genes into assessments of echinoderm Hox gene complements and organisation shows that these animals in fact have Split (S) Hox clusters rather than simply Disorganized (D) clusters within the Duboule classification scheme. This then has implications for how these genes are likely regulated, with them no longer covered by any potential long-range Hox cluster-wide, or multigenic sub-cluster, regulatory mechanisms.

Collagen Fibril Assembly and Function.

Collagen fibrils are the major mechanical component in the extracellular matrix of a broad range of multicellular animals from echinoderms to vertebrates where they provide a stable framework for tissues. They form the key tension-resisting element of a complex fiber-composite system that has a tissue-specific hierarchical structure linked to mechanical demands. Remarkably, these tissues are self-maintaining and avoid fatigue failure over the lifetime of the animal. Collagen fibrils can assemble spontaneously from purified solutions of collagen molecules. In developing tissues, however, in addition to the intrinsic self-assembly properties, there is cellular machinery that regulates fibril nucleation, spatial orientation, and fibril size, according to the tissue and stage of development. The intricate mechanisms underlying the generation of a collagen fibril network of defined architecture and mechanical properties are now becoming apparent. Impairment of this system leads ultimately to mechanical failure or tissue fibrosis.

Mono- and Dimeric Naphthalenones from the Marine-Derived Fungus Leptosphaerulina chartarum 3608.

Five new naphthalenones, two enantiomers (−)-1 and (+)-1 leptothalenone A, (−)-4,8-dihydroxy-7-(2-hydroxy-ethyl)-6-methoxy-3,4-dihydro-2H-naphthalen-1-one ((−)-2), (4S, 10R, 4’S)-leptotha-lenone B (5), (4R, 10S, 4’S)-leptothalenone B (6), and a new isocoumarine, 6-hydroxy-5,8-dimethoxy-3-methyl-1H-isochromen-1-one (4), along with two known compounds (+)-4,8-dihydroxy-7-(2-hydroxy-ethyl)-6-methoxy-3,4-dihydro-2H-naphthalen-1-one ((+)-2) and (+)-10-norparvulenone (3) were isolated from the marine-derived fungus Leptosphaerulina chartarum 3608. The structures of new compounds were elucidated by HR-ESIMS, NMR, and ECD analysis. All compounds were evaluated for cytotoxicity and anti-inflammatory activity. Compound 6 showed moderate anti-inflammatory activity by inhibiting the production of nitric oxide (NO) in lipopolysaccharide-stimulated RAW264.7 cells, with an IC50 value of 44.5 μM.

Developmental transcriptomics of the brittle star Amphiura filiformis reveals gene regulatory network rewiring in echinoderm larval skeleton evolution.

BACKGROUND: Amongst the echinoderms the class Ophiuroidea is of particular interest for its phylogenetic position, ecological importance and developmental and regenerative biology. However, compared to other echinoderms, notably echinoids (sea urchins), relatively little is known about developmental changes in gene expression in ophiuroids. To address this issue, we have generated and assembled a large RNAseq data set of four key stages of development in the brittle star Amphiura filiformis and a de novo reference transcriptome of comparable quality to that of a model echinoderm-the sea urchin Strongylocentrotus purpuratus. Furthermore, we provide access to the new data via a web interface: http://www.echinonet.eu/shiny/Amphiura_filiformis/ . RESULTS: We have identified highly conserved genes associated with the development of a biomineralised skeleton. We also identify important class-specific characters, including the independent duplication of the msp130 class of genes in different echinoderm classes and the unique occurrence of spicule matrix (sm) genes in echinoids. Using a new quantification pipeline for our de novo transcriptome, validated with other methodologies, we find major differences between brittle stars and sea urchins in the temporal expression of many transcription factor genes. This divergence in developmental regulatory states is more evident in early stages of development when cell specification begins, rather than when cells initiate differentiation. CONCLUSIONS: Our findings indicate that there has been a high degree of gene regulatory network rewiring and clade-specific gene duplication, supporting the hypothesis of a convergent evolution of larval skeleton development in echinoderms.

Chemical Ecology of Chemosensation in Asteroidea: Insights Towards Management Strategies of Pest Species.

Within the Phylum Echinodermata, the class Asteroidea, commonly known as starfish and sea stars, encompasses a large number of benthos inhabiting genera and species with various feeding modalities including herbivores, carnivores, omnivores and detritivores. The Asteroidea rely on chemosensation throughout their life histories including hunting prey, avoiding or deterring predators, in the formation of spawning aggregations, synchronizing gamete release and targeting appropriate locations for larval settlement. The identities of many of the chemical stimuli that mediate these physiological and behavioural processes remain unresolved even though evidence indicates they play pivotal roles in the functionality of benthic communities. Aspects of chemosensation, as well as putative chemically-mediated behaviours and the molecular mechanisms of chemoreception, within the Asteroidea are reviewed here, with particular reference to the coral reef pest the Crown-of-Thorns starfish Acanthaster planci species complex, in the context of mitigation of population outbreaks.

Variability in larval gut pH regulation defines sensitivity to ocean acidification in six species of the Ambulacraria superphylum.

The unusual rate and extent of environmental changes due to human activities may exceed the capacity of marine organisms to deal with this phenomenon. The identification of physiological systems that set the tolerance limits and their potential for phenotypic buffering in the most vulnerable ontogenetic stages become increasingly important to make large-scale projections. Here, we demonstrate that the differential sensitivity of non-calcifying Ambulacraria (echinoderms and hemichordates) larvae towards simulated ocean acidification is dictated by the physiology of their digestive systems. Gastric pH regulation upon experimental ocean acidification was compared in six species of the superphylum Ambulacraria. We observed a strong correlation between sensitivity to ocean acidification and the ability to regulate gut pH. Surprisingly, species with tightly regulated gastric pH were more sensitive to ocean acidification. This study provides evidence that strict maintenance of highly alkaline conditions in the larval gut of Ambulacraria early life stages may dictate their sensitivity to decreases in seawater pH. These findings highlight the importance of identifying and understanding pH regulatory systems in marine larval stages that may contribute to substantial energetic challenges under near-future ocean acidification scenarios.

Genome-wide use of high- and low-affinity Tbrain transcription factor binding sites during echinoderm development.

Sea stars and sea urchins are model systems for interrogating the types of deep evolutionary changes that have restructured developmental gene regulatory networks (GRNs). Although cis-regulatory DNA evolution is likely the predominant mechanism of change, it was recently shown that Tbrain, a Tbox transcription factor protein, has evolved a changed preference for a low-affinity, secondary binding motif. The primary, high-affinity motif is conserved. To date, however, no genome-wide comparisons have been performed to provide an unbiased assessment of the evolution of GRNs between these taxa, and no study has attempted to determine the interplay between transcription factor binding motif evolution and GRN topology. The study here measures genome-wide binding of Tbrain orthologs by using ChIP-sequencing and associates these orthologs with putative target genes to assess global function. Targets of both factors are enriched for other regulatory genes, although nonoverlapping sets of functional enrichments in the two datasets suggest a much diverged function. The number of low-affinity binding motifs is significantly depressed in sea urchins compared with sea star, but both motif types are associated with genes from a range of functional categories. Only a small fraction (∼10%) of genes are predicted to be orthologous targets. Collectively, these data indicate that Tbr has evolved significantly different developmental roles in these echinoderms and that the targets and the binding motifs in associated cis-regulatory sequences are dispersed throughout the hierarchy of the GRN, rather than being biased toward terminal process or discrete functional blocks, which suggests extensive evolutionary tinkering.

A potential role for chondroitin sulfate/dermatan sulfate in arm regeneration in Amphiura filiformis.

Glycosaminoglycans (GAGs), such as chondroitin sulfate (CS) and dermatan sulfate (DS) from various vertebrate and invertebrate sources are known to be involved in diverse cellular mechanisms during repair and regenerative processes. Recently, we have identified CS/DS as the major GAG in the brittlestar Amphiura filiformis, with high proportions of di- and tri-O-sulfated disaccharide units. As this echinoderm is known for its exceptional regeneration capacity, we aimed to explore the role of these GAG chains during A. filiformis arm regeneration. Analysis of CS/DS chains during the regeneration process revealed an increase in the proportion of the tri-O-sulfated disaccharides. Conversely, treatment of A. filiformis with sodium chlorate, a potent inhibitor of sulfation reactions in GAG biosynthesis, resulted in a significant reduction in arm growth rates with total inhibition at concentrations higher than 5 mM. Differentiation was less impacted by sodium chlorate exposure or even slightly increased at 1-2 mM. Based on the structural changes observed during arm regeneration we identified chondroitin synthase, chondroitin-4-O-sulfotransferase 2 and dermatan-4-O-sulfotransferase as candidate genes and sought to correlate their expression with the expression of the A. filiformis orthologue of bone morphogenetic factors, AfBMP2/4. Quantitative amplification by real-time PCR indicated increased expression of chondroitin synthase and chondroitin-4-O-sulfotransferase 2, with a corresponding increase in AfBMP2/4 during regeneration relative to nonregenerating controls. Our findings suggest that proper sulfation of GAGs is important for A. filiformis arm regeneration and that these molecules may participate in mechanisms controlling cell proliferation.