Effect of gamete aging on fertilization success of the sea urchin Arbacia dufresnii (Arbacioida: Arbaciidae) / Efecto del envejecimiento de los gametos sobre el éxito en la fecundación del erizo de mar Arbacia dufresnii (Arbacioida: Arbaciidae)

Abstract Introduction: Short-term gametes storage is an inexpensive and simple technique that allows the use of the same batch of eggs or sperm at different times, maximizing the application of research protocols and the use of gametes in production. Arbacia dufresnii is a sea urchin species with proven aquaculture potential and already used in the nutraceutical industry. Aging of its gametes is unknown and is a needed information to scale up the production. Objective: Determine the effect of male and female gamete aging on the fertilization success of Arbacia dufresnii. This will allow optimizing the use of gametes after collection decoupling spawning from fertilization. Methods: A. dufresnii individuals were induced to spawn and gametes were kept at 12 ± 1 °C throughout each bioassay. Sperm was separated into two treatments: activated sperm in seawater (AS), and dry sperm (DS). Two bioassays were made: Bioassay 1 evaluated the effect of time on fertility by performing fertilization tests at 0 h, 24 h, 48 h, 72 h, and 96 h after spawning. Bioassay 2 evaluated the contribution of each type of aged gamete on fertility, combining aged gametes (96 h) with fresh gametes (0 h). Results: Bioassay 1: the fertilization success obtained by combining eggs (E) with AS or DS presented important differences. While the fertilization success remained acceptable (greater than 50 %) for up to 72 h using ExDS, it only remained acceptable for up to 48 h using ExAS. Bioassay 2: acceptable fertilization success was found by combining aged E (96 h) with fresh sperm, or aged DS (96 h) with fresh E, but not using aged AS with fresh E. Conclusions: The findings of this work show that fertilization success in A. dufresnii gametes remains relatively unchanged for up to 48 h after spawning when combining ExAS, and for up to 72 h when combining ExDS. However, when combining aged E or aged DS with a fresh gamete, post-collection fertilization can be extended up to 96 h. In this work, the first steps have been taken to understand the conservation time of A. dufresnii gametes with minimum intervention.

Resumen Introducción: El almacenamiento de gametos a corto plazo es una técnica económica y sencilla que permite utilizar el mismo lote de óvulos o espermatozoides en diferentes momentos, maximizando la aplicación de protocolos de investigación y el uso de gametos en la producción. Arbacia dufresnii es una especie con probado potencial acuícola como fuente de gametos para la industria nutracéutica. Sin embargo, se desconoce el envejecimiento de sus gametos y es una información necesaria para escalar la producción. Objetivo: Determinar el efecto del envejecimiento de los gametos masculinos y femeninos en el éxito de la fecundación de Arbacia dufresnii con el fin de optimizar el aprovechamiento de los gametos después de la recolecta desincronizando el desove de la fecundación. Métodos: Se indujo el desove de individuos de A. dufresnii y los gametos se mantuvieron a 12 ± 1 °C durante cada bioensayo. El esperma se separó en dos tratamientos: esperma activado en agua de mar (AS) y esperma seco (DS). Se realizaron dos bioensayos: El Bioensayo 1 evaluó el efecto del tiempo sobre la fertilidad realizando pruebas de fecundación a las 0 h, 24 h, 48 h, 72 h y 96 h después del desove. El bioensayo 2 evaluó la contribución de cada tipo de gameta envejecida (96 h) sobre la fertilidad, combinando gametos envejecidas (96 h) con gametos frescas (0 h). Resultados: Bioensayo 1: el éxito de fecundación obtenido combinando huevos (E) con AS o DS presentó diferencias importantes. Si bien el éxito de la fecundación se mantuvo aceptable (más del 50 %) durante un máximo de 72 h con ExDS, solo permaneció aceptable hasta 48 h con ExAS. Bioensayo 2: se encontró un éxito de fecundación aceptable combinando E envejecidos (96 h) con esperma fresco, o DS envejecido (96 h) con E fresco (0 h), pero no usando AS envejecido con E fresco (0 h). Conclusiones: Los hallazgos de este trabajo muestran que el éxito de la fecundación en los gametos de A. dufresnii permanece relativamente sin cambios hasta 48 h después del desove cuando se combina ExAS, y hasta 72 h cuando se combina ExDS. Sin embargo, cuando se combina E envejecido o DS envejecido con un gameto fresco, el tiempo entre la recolección y la fecundación puede extenderse hasta 96 h. En este trabajo se han dado los primeros pasos para entender el tiempo de conservación de los gametos de A. dufresnii con mínima intervención.

Feeding habits of Holothuria (Stauropora) fuscocinerea (Echinodermata: Holothuroidea) in a Mexican Pacific reef / Hábitos alimenticios de Holothuria (Stauropora) fuscocinerea (Echinodermata: Holothuroidea) en un arrecife del Pacífico Mexicano

Introduction: The importance of holothuroids in marine ecosystems is related to their feeding activities, which have been little studied in the eastern Pacific. Objective: To describe the feeding habits of the sea cucumber Holothuria (Stauropora) fuscocinerea and their bioturbation potential in La Entrega reef, México. Methods: The population size of H. (Stauropora) fuscocinerea was determined by means of four 20 x 4 m (320 m2) band transects, while the available sediment for the sea cucumbers and their feces were characterized in three stations (A-C) via granulometry and quantification of organic carbon and organic matter. Excretion rate was determined every 4 hr over a 24 hr period and the relationship between sea cucumber length and biomass and the excretion rate evaluated. Results: According to non-parametric permutation, ordination and percentage similarity analyses, sea cucumber feces mostly comprised a large proportion of small grains compared to the available sediment, indicating that H. (Stauropora) fuscocinerea is selective on grain size. Preliminary bioturbation potential of the species may reach up to 8.71 ± 0.17 kg day of sediment in the study area. Conclusion: The spatial distribution of H. (Stauropora) fuscocinerea in the study area is a product of its selective feeding activity, evidencing the preliminary importance of the species in the trophic dynamics of La Entrega reef.

Introducción: La relevancia de los holoturoideos en los ecosistemas marinos está relacionada con sus actividades alimenticias, las cuales han sido escasamente estudiadas en el Pacífico oriental. Objetivo: Describir los hábitos alimenticios del pepino de mar Holothuria (Stauropora) fuscocinerea y su potencial de bioturbación en el arrecife La Entrega, México. Métodos: Se determinó el tamaño poblacional de H. (Stauropora) fuscocinerea mediante cuatro transectos de banda de 20 x 4 m (320 m2), mientras que la caracterización de los sedimentos disponibles y las heces de los pepinos se realizó en tres estaciones (A-C) mediante granulometría y cuantificación de carbono orgánico y materia orgánica. Se determinó la tasa de excreción cada 4 hr durante un periodo de 24 hr y se evaluó la relación entre la talla y la biomasa con la tasa de excreción. Resultados: De acuerdo con un análisis no paramétrico mediante permutaciones, ordenación y similitud, las heces de los pepinos estuvieron compuestas en su mayoría por una gran cantidad de granos pequeños en comparación con el sedimento disponible, lo que sugiere que H. (Stauropora) fuscocinerea es una especie selectiva respecto al tamaño de grano. El potencial de bioturbación preliminar de la especie puede alcanzar 8.71 ± 0.17 kg día en el área de estudio. Conclusiones: La distribución espacial de H. (Stauropora) fuscocinerea en el área de estudio es producto de su selectividad alimenticia, evidenciando la importancia de la especie en la dinámica trófica del arrecife La Entrega.

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.

¿Hacia dónde va la acuicultura de equinodermos en América Latina? Potencial, retos y oportunidades / Where is echinoderm aquaculture heading in Latin America? Potential, challenges and opportunities

Introducción: La acuicultura de equinodermos en América Latina es incipiente, pero representa una nueva frontera para la producción de alimentos marinos altamente nutritivos, con especies nativas de alto valor comercial que ofrecen un gran potencial en comparación con las debilitadas pesquerías bentónicas artesanales. Metodología: Se revisó la literatura sobre pepinos de mar, erizos de mar y estrellas de mar hasta el año 2020. Resultados: Para el 2020, había 17 especies nativas con importantes avances en reproducción, larvicultura, crecimiento, tratamiento e identificación de enfermedades y nutrición en México, Belice, Panamá, Colombia, Ecuador, Perú, Brasil, Chile y Argentina. Conclusiones: La acuicultura de equinodermos en América Latina está avanzando y se beneficiaría de los sistemas multitróficos para expandirse y diversificarse, reduciendo la presión sobre las pesquerías agotadas.

Introduction: Echinoderm aquaculture in Latin America is incipient, but it represents a new frontier for the highly nutritious marine food production, with native species of high commercial value offering a vast potential when compared to the weakened artisanal benthic fisheries. Methodology: We reviewed the literature on sea cucumbers, sea urchins, and starfish up to the year 2020. Results: By 2020, there were 17 native species with important advances in reproduction, larviculture, growth, treatment and identification of diseases, and nutrition, in Mexico, Belize, Panamá, Colombia, Ecuador, Peru, Brazil, Chile and Argentina. Conclusions: Echinoderm aquaculture in Latin America is advancing and would benefit from multitrophic systems to expand and diversify, reducing the pressure on depleted fisheries.

The influence of density on survival and larval development in the sea urchin Arbacia dufresnii (Echinodermata: Echinoidea)

Introduction: Density is one of the critical factors in echinoderm larvae for aquaculture purposes. Echinoplutei larvae are very sensitive to overcrowding. High culture density can lead to problems with bacteria or protozoa, decreasing survival and generating abnormal morphotypes. Objective: To evaluate the effect of culture density on survival and larval growth in the sea urchin Arbacia dufresnii. Methods: Two days after fertilization of A. dufresnii we we kept treatments at 1, 3, 5 and 10 larvae.ml-1, with three replicates each. We recorded survival and abnormal morphotypes periodically, as well as growth:somatic rod length, total length, and length of the post oral arms,. we applied generalized linear models. Results: Survival is dependent on density, time and replicates, and their interactions. Larval growth depended on density and time, also with interaction between the variables. The treatment of 5 larvae.ml-1 had the highest survival and larval condition. Conclusions: Larval culture of A. dufresnii had the best results at 5 larvae.ml-1.

Introducción: La densidad es uno de los factores críticos para las larvas de equinodermo para fines de acuicultura. Las larvas de Echinoplutei son muy sensibles al hacinamiento. Una alta densidad de cultivo puede provocar problemas con bacterias o protozoos, disminuyendo la supervivencia y generando morfotipos anormales. Objetivo: Evaluar el efecto de la densidad de cultivo sobre la supervivencia y el crecimiento larvario del erizo de mar Arbacia dufresnii. Métodos: Dos días después de la fertilización de A. dufresnii se mantuvieron los tratamientos a 1, 3, 5 y 10 larvas.ml-1, con tres repeticiones cada uno. Registramos la supervivencia y los morfotipos anormales periódicamente, así como el crecimiento: longitud de la varilla somática, longitud total y longitud de los brazos posorales. aplicamos modelos lineales generalizados. Resultados: La supervivencia depende de la densidad, el tiempo y las réplicas y sus interacciones. El crecimiento larvario dependió de la densidad y el tiempo, también de la interacción entre las variables. El tratamiento de 5 larvas.ml-1 tuvo la mayor supervivencia y condición larvaria. Conclusiones: El cultivo larvario de A. dufresnii tuvo los mejores resultados con 5 larvas.ml-1.

Variabilidad en el tamaño y alimentación de Acanthaster planci (Echinodermata: Asteroidea) en el sur del Golfo de California, México / Variability of size and food type of Acanthaster planci (Echinodermata: Asteroidea) in the southern Gulf of California, Mexico

Introducción: Es poco lo que se conoce sobre aspectos biológicos y poblacionales de Acanthaster planci en México. Su tamaño se ha relacionado con la cantidad de alimento que consume, por lo que es relevante conocer esta variable. Objetivo: Describir la variabilidad en el diámetro del disco de la especie y de su alimentación en cuatro sitios al sur del Golfo de California. Métodos: De febrero 2008 a marzo 2009 se visitaron mensualmente los sitios. Se registró el diámetro del disco de un total de 389 individuos observados. El diámetro del disco se midió con una cinta métrica flexible colocada de lado a lado, en el borde donde comienzan los brazos. Para facilitar la descripción, el diámetro del disco se clasificó en tres grupos: pequeños ( 26 cm). Del total, 298 organismos se encontraron alimentándose. Se clasificaron los tipos de organismos consumidos en corales duros, otros invertebrados y algas. Para calcular la probabilidad de que individuos del mismo tamaño se alimenten de diferente variedad de organismos, se aplicó el índice de Diversidad de Simpson. Con el fin de detectar diferencias del diámetro del disco entre los meses de estudio y los sitios de muestreo, se aplicó un análisis permutacional de varianza. Resultados: Acanthaster planci tuvo un promedio de diámetro del disco de 18.23 ± 0.21 cm, con un mínimo de 7 cm y un máximo de 36 cm. La estructura de tallas fue unimodal y se caracterizó por la predominancia de individuos medianos, seguida por los pequeños y en baja proporción por los grandes. El diámetro del disco fue estadísticamente más grande en febrero y más pequeño en los últimos meses del año. En San Rafaelito se encontraron los ejemplares más grandes y en Ensenada de Muertos los más pequeños. Los individuos de tamaño mediano fueron más frecuentes a lo largo del año y en todos los sitios. Los individuos pequeños se alimentaron significativamente más de Porites panamensis, seguido de algas coralinas y tapetes. Los más grandes tuvieron los valores de diversidad más bajos, más del 90 % de los individuos en este grupo se alimentaron de Pocillopora spp. y algunos de macroalgas. Los medianos presentaron los valores más altos de diversidad y consumieron todos los tipos de alimento encontrados, sin embargo, como era de esperarse, muchos fueron selectivos hacia Pocillopora spp. Conclusiones: Acanthaster planci se considera un individuo de tamaño mediano en el área de estudio. Se detectó una evidente relación entre el diámetro del disco y la diversidad del tipo de alimento. Los tres grupos de tallas fueron positivamente selectivos hacia corales duros; los más pequeños se alimentaron preferentemente de Porites panamensis y en conjunto con los grandes, presentaron valores bajos de diversidad en el tipo de alimento, mientras que los medianos fueron los que consumieron una mayor diversidad de presas.

Introduction: Little is known about the biology and population dynamics of Acanthaster planci in Mexico. Its size is considered relevant because it is related to consumption rate. Objective: To describe the variability of the disk diameter and food type of the species in four sampling sites in the southern Gulf of California. Methods: Each site was visited on a monthly basis from February 2008 to March 2009. Disk diameter was registered from a total of 389 observed specimens. Measurements included only the main disk from side to side where the arms begin. A flexible tape was used to measure disk diameter. Three size groups were created for easier explanations: small ( 26 cm). From the total, only 298 asteroids were feeding. Preyed organisms were classified in hard corals, other invertebrates, and algae. The Simpson's Diversity Index was used to calculate the probability that any specimen would be feeding on different types of organisms. A permutational analysis of variance was used to detect differences of disk diameter amongst months and study sites. Results: This population of Acanthaster planci had an average disk diameter = 18.23 ± 0.21 cm, with a minimum of 7 cm and maximum of 36 cm. Size structure was unimodal and largely dominated by medium sized specimens, followed by small ones. The larger were less frequent. Disk diameter was statistically higher in February and lower in the last months of the year. San Rafaelito was the site with the largest and Ensenada de Muertos with the smallest organisms. Medium sized individuals were dominant throughout the year and sampling sites. The small sized fed significantly more on Porites panamensis and coralline algae and turf. Larger specimens had the lowest diversity values, more than 90 % of this size group fed on Pocillopora spp. and very few on macroalgae. Medium sized specimens showed the highest diversity, feeding on all the food types, although as expected, many of them were selective to Pocillopora spp. Conclusions: Acanthaster planci can be considered a medium sized species in the study area. There is an evident food selectivity regarding disk diameter size. The three size groups were positively selective to hard corals, but only the small favored Porites panamensis; and together with large individuals, they had the lowest diversity on food types, whereas medium sized asteroids fed on a high diversity of prey.

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.

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.

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.