ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis.

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates' biomineralizing cells, yet, little is known on ROCK's role in invertebrates' biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

Riqueza y densidad de equinodermos en los arrecifes rocosos de Punta Amapala, El Salvador / Richness and density of echinoderms in the rocky reefs of Punta Amapala, El Salvador

Resumen Introducción: Los equinodermos son animales estrictamente acuáticos y de los grupos de invertebrados marinos cuya importancia ecológica es crucial para los ambientes en donde habitan. La característica más destacada es su simetría pentarradial. Estos organismos pueden distribuirse en una amplia variedad de ambientes oceánicos, y no ser exclusivamente habitantes de las formaciones arrecifales. Se han registrado el total de 1 539 especies, de las cuales 717 pertenecen a Pacífico y 60 de ellas se han registrado en El Salvador. Objetivo: El objetivo del trabajo fue registrar la riqueza y densidad de especies de equinodermos en arrecifes rocosos de Punta Amapala (La Unión). Métodos: El muestreo fue llevado a cabo en cinco sitios, dos a 3 m de profundidad y tres a 6 m. Para esto, se utilizó snorkeling y buceo autónomo, recorriendo dos transectos de banda de 30 x 1 m. Dentro de los transectos, se realizó la identificación (riqueza) y contabilización (abundancia) de los organismos. Resultados: La especie que presentó mayor densidad en los arrecifes de 3 m fue Echinometra vanbrunti (0.83 indv/m2), seguido por el ofiuroideo Ophiocomella alexandri (0.16 indv/m2) y Ophiocoma aethiops (0.16 indv/m2). Y en los arrecifes a 6 m fueron Ophiothela mirabilis (0.83 indv/m2), Ophionereis annulata (0.21 indv/m2), Ophiothrix rudis (0.08 indv/m2) y Ophiocomella alexandri (0.01 indv/m2). Conclusiones: Estos organismos, cumplen con diversos roles ecológicos, como ser bioindicadores de calidad de agua al tolerar ciertos cambios en las condiciones físico-químicas, así como cumplir el papel de hospederos de macrofauna por las cavidades que forman algunos erizos. Es por ello que los esfuerzos de muestreo deben incrementarse en estas zonas para monitorear la calidad y salud de los arrecifes rocosos, cuyo papel es clave, por ser junto con Los Cóbanos, los de mayor extensión en el país.

Abstract Introduction: Echinoderms are strictly aquatic animals and one of the groups of marine invertebrates whose ecological importance is crucial for the environments where they inhabit. The most outstanding characteristic is their pentaradial symmetry. These organisms can be distributed in a wide variety of oceanic environments, and are not exclusive inhabitants of reef formations. A total of 1 539 species have been registered, of which 717 belong to the Pacific and 60 of them have been registered for El Salvador. Objective: The objective of the work was to record the richness and density of echinoderm species in rocky reefs of Punta Amapala (La Unión). Methods: The sampling was conducted in five sites, two of them at 3 m of depth and three at 6 m. For this, snorkeling and autonomous diving were used, covering two band transects of 30 x 1 m. Within the transects, the identification (richness) and counting (abundance) of the organisms was carried out. Results: The species that presented the highest density at 3 m reefs was Echinometra vanbrunti (0.83 indv/m2), followed by the ophiuroid Ophiocomella alexandri (0.16 indv/m2) and Ophiocoma aethiops (0.16 indv/m2). And at the 6 m reefs were Ophiothela mirabilis (0.83 indv/m2), Ophionereis annulata (0.21 indv/m2), Ophiothrix rudis (0.08 indv/m2) and Ophiocomella alexandri (0.01 indv/m2). Conclusions: These organisms fulfill various ecological roles, such as being bioindicators of water quality by tolerating certain changes in physicochemical conditions and fulfilling the role of macrofauna hosts due to the cavities formed by some sea urchins. That is why sampling efforts must increase in these areas to monitor the quality and health of the rocky reefs, whose role is key, as they are, together with Los Cóbanos, the largest in the country.

Recent molecular techniques to strengthen ecological studies in echinoderms / Técnicas moleculares recientes que fortalecen los estudios ecológicos de equinodermos

Abstract Introduction: Echinoderms, an integral component of marine ecosystems worldwide, have captivated scientific interest for centuries. Despite this longstanding attention, comprehending key facets such as trophic relationships, diet composition, and host-microbiota relationships still represents a challenge using traditional techniques. Recent years, however, have witnessed a transformative shift, thanks to the emergence of advanced molecular techniques, offering new approaches to strengthen ecological studies in echinoderms. Objective: Explore how recent advancements in molecular tools have impacted ecological research on echinoderms. Specifically, we aim to investigate the potential of these tools to shed light on trophic interactions, diet composition, and the characterization of gut microbial communities in these organisms. Methods: Available literature was used to clarify how novel molecular techniques can improve ecological studies. The focus is diet, trophic relationships, and gut microbiota. Results: Traditionally, studies of stomach contents using compound microscopy have provided an idea of ingested material; nevertheless, sometimes a simple magnified visualization of dietary content does not allow exhaustive identification of the entire food spectrum, as it is limited due to the rapid digestion and maceration of food items within the echinoderm's digestive tract. The use of DNA-metabarcoding, targeting specific DNA regions, such as the mitochondrial COI gene, has allowed us to enhance the accuracy and precision of diet characterization by enabling the identification of prey items down to the species or even genetic variant level, providing valuable insights into specific dietary preferences. Another approach is the use of stable isotopes, particularly carbon and nitrogen, which provide a powerful tool to trace the origin and flow of nutrients through food webs. By analyzing the isotopic signatures in muscular tissues and food items, we can discern the sources of their primary food items and gain insights into their trophic position within the ecosystem. Lastly, a third new technique used to elucidate the characterization of the prokaryotic community is 16S rRNA sequencing. This method allows us to explore the composition and dynamics of the digestive tract microbial communities. Conclusions: This is a promising era for ecological research on echinoderms, where advances of molecular tools have enabled an unprecedented level of detail, resolving longstanding challenges in comprehending their trophic interactions, diet composition, and host-microbiota relationships, and opening new avenues of investigation in ecological studies.

Resumen Introducción: Los equinodermos, un componente integral de los ecosistemas marinos en todo el mundo, han captado el interés científico durante siglos. A pesar de esta prolongada atención, el comprender facetas clave como las relaciones tróficas, la composición de la dieta y las relaciones huésped-microbiota todavía representa un desafío utilizando técnicas tradicionales. Sin embargo, los últimos años han sido testigos de un cambio transformador, gracias a la aparición de técnicas moleculares avanzadas, que ofrecen nuevos enfoques para fortalecer los estudios ecológicos en equinodermos. Objetivo: Explorar cómo los avances recientes en herramientas moleculares han impactado la investigación ecológica sobre equinodermos. Específicamente, nuestro objetivo es investigar el potencial de estas herramientas para arrojar luz sobre las interacciones tróficas, la composición de la dieta y la caracterización de las comunidades microbianas intestinales en estos organismos. Métodos: Se utilizó la literatura disponible para aclarar cómo las nuevas técnicas moleculares pueden mejorar los estudios ecológicos. La atención se centra en la dieta, las relaciones tróficas y la microbiota intestinal. Resultados: Tradicionalmente, los estudios del contenido estomacal mediante microscopía compuesta han proporcionado una idea del material ingerido; Sin embargo, a veces una simple visualización ampliada del contenido dietético no permite una identificación exhaustiva de todo el espectro alimentario, ya que está limitado debido a la rápida digestión y maceración de los alimentos dentro del tracto digestivo del equinodermo. El uso de metabarcoding de ADN, dirigidos a regiones específicas del ADN, como el gen COI mitocondrial, nos ha permitido mejorar la exactitud y precisión de la caracterización de la dieta al permitir la identificación de presas hasta el nivel de especie o incluso de variante genética, lo que proporciona valiosos resultados sobre preferencias dietéticas específicas. Otro enfoque es el uso de isótopos estables, en particular carbono y nitrógeno, que proporcionan una poderosa herramienta para rastrear el origen y el flujo de nutrientes a través de las redes alimentarias. Al analizar las firmas isotópicas en los tejidos musculares y los alimentos, podemos discernir las fuentes de sus alimentos primarios y obtener información sobre su posición trófica dentro del ecosistema. Por último, una tercera técnica nueva utilizada para dilucidar la caracterización de la comunidad procariótica es la secuenciación del ARNr 16S. Este método nos permite explorar la composición y dinámica de las comunidades microbianas del tracto digestivo. Conclusiones: Esta es una era prometedora para la investigación ecológica sobre equinodermos, donde los avances de las herramientas moleculares han permitido un nivel de detalle sin precedentes, resolviendo desafíos de larga data en la comprensión de sus interacciones tróficas, composición de la dieta y relaciones huésped-microbiota, y abriendo nuevas vías de investigación. en estudios ecológicos.

Equinodermos de la colección biológica del Laboratorio de Ecología Molecular, Microbiología y Taxonomía (LEMITAX) de la Universidad de Guadalajara, México / Echinoderms of the biological collection of the Laboratorio de Ecología Molecular, Microbiología y Taxonomía (LEMITAX) of the Universidad de Guadalajara, Mexico

Resumen Introducción: Las colecciones biológicas representan los cimientos para el conocimiento y manejo de la biodiversidad de una región. No obstante, en México, y en particular en el Pacífico central mexicano (PCM), las colecciones regionales enfocadas en equinodermos, son escasas. La colección biológica del Laboratorio de Ecología Molecular, Microbiología y Taxonomía (LEMITAX) pertenece al Centro Universitario de Ciencias Biológicas y Agropecuarias de la Universidad de Guadalajara, Jalisco, México y sirve como referencia de la biodiversidad marina de la región. Objetivo: Dar a conocer la riqueza de equinodermos resguardada en la colección del LEMITAX. Métodos: Los organismos depositados en LEMITAX se han recolectado mediante buceo libre, SCUBA, y arrastres de fondo con dragas biológicas en diversas áreas del Pacífico mexicano. Los especímenes están preservados en húmedo (alcohol al 70 %; Asteroidea, Echinoidea, Holothuroidea y Ophiuroidea) o en seco (Asteroidea y Echinoidea). Resultados: Los especímenes provienen de los estados de Sinaloa, Nayarit, Jalisco y Colima, incluyendo dos áreas naturales protegidas, el Parque Nacional Isla Isabel y el Santuario de las islas e islotes de Bahía de Chamela. La colección dispone de 20 761 ejemplares de equinodermos distribuidos en 75 especies (10 asteroideos, 17 ofiuroideos, 17 equinoideos y 31 holoturoideos). El estado mejor representado es Jalisco (64 especies) seguido de Nayarit (31), Colima (20) y Sinaloa (11). Bahía de Chamela es la mejor representada (60), seguido de Isla Isabel (22). Se aportan 34 registros nuevos, la mayor contribución es para Bahía de Chamela con 14 registros nuevos, seguido del estado de Jalisco (siete), Nayarit (cuatro), Colima (tres) e Isla Isabel (tres). Para el PCM, se reporta por primera vez la presencia de Astropecten ornatissimus, Luidia phragma, Cucumaria crax y Holothuria (Cystipus) casoae, lo que actualiza su riqueza de equinodermos a 197 especies. Se amplía el intervalo de distribución batimétrica de Ophiactis simplex, Ophiocomella alexandri y Holothuria (Cystipus) casoae, así como el intervalo de distribución geográfica de Cucumaria crax. Conclusiones: Las colecciones biológicas de las universidades contribuyen de manera sustancial al conocimiento de la biodiversidad, como se refleja en la colección LEMITAX, cuya revisión resultó en la actualización de la riqueza de equinodermos de la región.

Abstract Introduction: The biological collections represent the foundation for the knowledge and management of the biodiversity of a region. However, regional collections focused on echinoderms are scarce in Mexico, particularly in the Central Mexican Pacific (CMP). The biological collection of the Laboratorio de Ecología Molecular, Microbiología y Taxonomía (LEMITAX) belongs to the Centro Universitario de Ciencias Biológicas y Agropecuarias of the Universidad de Guadalajara, Jalisco, Mexico and it aims to serve as a reference for the region's marine biodiversity. Objective: To state the richness of echinoderms in the LEMITAX collection. Methods: The organisms deposited at the LEMITAX have been collected by SCUBA, free-diving, and bottom trawls with biological dredges in different areas of the Mexican Pacific. The specimens are wet-preserved (70 % ethanol; Asteroidea, Echinoidea, Holothuroidea, and Ophiuroidea) or dried (Asteroidea and Echinoidea). Results: The specimens are from the states of Sinaloa, Nayarit, Jalisco, and Colima, including two natural protected areas (NPAs), the Isabel Island National Park and the sanctuary of the Islands and Islets of Bahía de Chamela. The collection has 20 761 specimens of echinoderms distributed in 75 species (10 asteroids, 17 ophiuroids, 17 echinoids, and 31 holothuroids). The best-represented state is Jalisco (64 species), followed by Nayarit (31), Colima (20), and Sinaloa (11). Concerning the NPAs, Chamela is the best represented (60), followed by Isabel Island (22). Thirty-four new records are added; the largest contribution is for Chamela, with 14 new records, followed by the state of Jalisco (seven), Nayarit (four), Colima (three), and Isabel Island (three). For the CMP, the presence of Astropecten ornatissimus, Luidia phragma, Cucumaria crax, and Holothuria (Cystipus) casoae, is reported for the first time, updating the echinoderm richness to 197 species. The bathymetric distribution range of Ophiactis simplex, Ophiocomella alexandri, and Holothuria (Cystipus) casoae is extended, as well as the geographic range of Cucumaria crax. Conclusions: The biological collections deposited in the universities contribute substantially to the knowledge of biodiversity, as reflected in the LEMITAX collection, whose revision resulted in the updating of the echinoderm richness in the region.

Crinoidea y Asteroidea (Echinodermata) del talud de la península de Yucatán, de la Colección Nacional de Equinodermos de México / Crinoidea and Asteroidea (Echinodermata) from the slope of the Yucatan Peninsula, Colección Nacional de Equinodermos, Mexico

Resumen Introducción: Las especies de las clases Crinoidea y Asteroidea se distribuyen en gran variedad de hábitats en todos los océanos desde la zona intermareal hasta grandes profundidades, existen pocos registros en aguas mexicanas, a profundidades superiores a los 200 m, los escasos registros existentes datan de documentos históricos de la literatura especializada y los ejemplares se resguardan en colecciones científicas extranjeras. Objetivo: Contribuir a la recopilación de información de los ejemplares resguardados en la Colección Nacional de Equinodermos (CNE) del Instituto de Ciencias del Mar y Limnología (ICML), UNAM y proveer un inventario de las especies de crinoideos y asteroideos que habitan en zonas profundas del talud de la Península de Yucatán, México. Métodos: Del año 2005 al 2014 se recolectaron ejemplares de crinoideos y asteriodeos en cuatro cruceros en el B/O "Justo Sierra" BIOREPES 1 y 2 y COBERPES 2 y 6. Se realizaron arrastres con red camaronera en 80 estaciones de muestreo. Resultados: El listado taxonómico comprende 1146 ejemplares, de los cuales 204 son crinoideos del orden Comatulida, distribuidos en tres familias tres géneros y tres especies y 942 asteroideos distribuidos en seis órdenes, 11 familias, 21 géneros y 28 especies. Se obtuvieron los registros nuevos para la CNE: Democrinus rawsonii y Atelecrinus balanoides en crinoideos. Astropecten alligator, Hymenaster modestus, Calyptraster personatus, Pteraster militarioides militarioides, Sclerasterias contorta en asteroideos. Con esta información se incrementan los registros de crinoideos y asteroideos (de profundidad mayor a 200 m) para los estados de Yucatán (16) y Quintana Roo (16).

Abstract Introduction: The species of the Crinoidea and Asteroidea classes are distributed in a wide variety of habitats in all oceans from the intertidal zone to great depths, there are few Mexican records in depths greater than 200 m, the few existing records date from historical documents in the specialized literature and the specimens are kept in foreign scientific collections. Objective: Contribute to the collection of information on the specimens kept in the Colección Nacional de Equinodermos (CNE) of Instituto de Ciencias del Mar y Limnología (ICML), UNAM and provide an inventory of the species of crinoids and asteroids that inhabit deep areas of the slope of the Yucatan Peninsula, Mexico. Methods: From 2005 to 2014, specimens of crinoids and asterioids were collected through four cruises in the B/O "Justo Sierra" BIOREPES 1 and 2 and COBERPES 2 and 6, shrimp net trawls were carried out at 80 sampling stations. Results: The taxonomic list includes 1146 specimens, of which 204 crinoids are of the order Comatulida, distributed in three families, three genera and three species and 942 asteroids distributed in six orders, 11 families, 21 genera and 28 species. New records were obtained for CNE: Democrinus rawsonii and Atelecrinus balanoides in crinoids. Astropecten alligator, Hymenaster modestus, Calyptraster personatus, Pteraster militarioides militarioides, Sclerasterias contorta in asteroids. With this information, the records of crinoids and asteroids (deeper than 200 m) increase for the states of Yucatan (16) and Quintana Roo (16).

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.

Evaluating the reliability of DNA Barcoding for Central American Pacific shallow water echinoderms identification: a molecular taxonomy and database accuracy analysis / Evaluando la fiabilidad del Código de Barras de ADN para la identificación de equinodermos en aguas poco profundas del Pacífico de América Central: un análisis de taxonomía molecular y precisión de bases de datos

Abstract Introduction: Molecular divergence thresholds have been proposed to distinguish recently separated evolutive units, often displaying more accurate putative species assignments in taxonomic research compared to traditional morphological approaches. This makes DNA barcoding an attractive identification tool for a variety of marine invertebrates, especially for cryptic species complexes. Although GenBank and the Barcode of Life Data System (BOLD) are the major sequence repositories worldwide, very few have tested their performance in the identification of echinoderm sequences. Objective: We use COI echinoderm sequences from local samples and the molecular identification platforms from GenBank and BOLD, in order to test their accuracy and reliability in the DNA barcoding identification for Central American shallow water echinoderms, at genus and species level. Methods: We conducted sampling, tissue extraction, COI amplification, sequencing, and taxonomic identification for 475 specimens. The 348 obtained sequences were individually enquired with BLAST in GenBank as well as using the Identification System (IDS) in BOLD. Query sequences were classified depending on the best match result. McNemar's chi-squared, Kruskal-Wallis's and Mann-Whitney's U tests were performed to prove differences between the results from both databases. Additionally, we recorded an updated list of species reported for the shallow waters of the Central American Pacific. Results: We found 324 echinoderm species reported for Central American Pacific shallow waters. Only 118 and 110 were present in GenBank and BOLD databases respectively. We proposed 325 solved morphology-based identities and 21 provisional identifications in 50 putative taxa. GenBank retrieved 348 molecular-based identifications in 58 species, including twelve provisional identifications in tree taxa. BOLD recovered 170 COI identifications in 23 species with one provisional identification. Nevertheless, 178 sequences retrieved unmatched terms (in 34 morphology-based taxa). Only 86 sequences (25 %) were retrieved as correct identifications and 128 (37 %) as identification errors in both platforms. We include 84 sequences for eleven species not represented in GenBank and 65 sequences for ten species in BOLD Echinoderm COI databases. The identification accuracy using BLAST (175 correct and 152 incorrect identifications) was greater than with IDS engine (110 correct and 218 identification errors), therefore GenBank outperforms BOLD (Kruskal-Wallis = 41.625, df = 1, p < 0.001). Conclusions: Additional echinoderm sample references are needed to improve the utility of the evaluated DNA barcoding identification tools. Identification discordances in both databases may obey specific parameters used in each search algorithm engine and the available sequences. We recommend the use of barcoding as a complementary identification source for Central American Pacific shallow water echinoderm species.

Resumen Introducción: Se han propuesto los umbrales de divergencia molecular para distinguir unidades evolutivas recientemente separadas, que a menudo muestran asignaciones de especies putativas más precisas en la investigación taxonómica en comparación con los enfoques morfológicos tradicionales. Esto hace que los Códigos de Barras de ADN sean una herramienta de identificación atractiva para una variedad de invertebrados marinos, especialmente para complejos de especies crípticas. Aunque GenBank y Barcode of Life Data System (BOLD) son los principales repositorios de secuencias en todo el mundo, muy pocos han probado su desempeño en la identificación de secuencias de equinodermos. Objetivo: Utilizamos secuencias de equinodermos COI de muestras locales y las plataformas de identificación molecular de GenBank y BOLD, para probar su precisión y confiabilidad en la implementación de códigos de barras de ADN para equinodermos de aguas someras de Centroamérica, a nivel de género y especie. Métodos: Realizamos muestreo, extracción de tejido, amplificación de COI, secuenciación e identificación taxonómica de 475 especímenes. Las 348 secuencias obtenidas fueron consultadas individualmente con BLAST en GenBank así como utilizando el Sistema de Identificación (IDS) en BOLD. Las secuencias consultadas se clasificaron según el mejor resultado de coincidencia. Se realizaron las pruebas chi-cuadrado de McNemar, Kruskal-Wallis y U de Mann-Whitney para comprobar diferencias entre los resultados de ambas bases de datos. Además, registramos una lista actualizada de especies reportadas para las aguas someras del Pacífico Centroamericano. Resultados: Encontramos 324 especies de equinodermos reportadas para aguas someras (< 200 m) del Pacífico centroamericano. Sólo 118 y 110 estaban presentes en las bases de datos GenBank y BOLD respectivamente. Propusimos 325 identidades resueltas basadas en morfología y 21 identificaciones provisionales en 50 taxones putativos. GenBank recuperó 348 identificaciones de base molecular en 58 especies, incluidas doce identificaciones provisionales en tres taxones. BOLD recuperó 170 identificaciones de COI en 23 especies con una identificación provisional. Sin embargo, 178 secuencias recuperaron términos no coincidentes (en 34 taxones basados en morfología). Sólo 86 secuencias (25 %) se recuperaron como identificaciones correctas y 128 (37 %) como errores de identificación en ambas plataformas. Incluimos 84 secuencias para once especies no representadas en GenBank y 65 secuencias para diez especies ausentes en las bases de datos BOLD Echinoderm COI. La precisión de la identificación usando BLAST (175 identificaciones correctas y 152 incorrectas) fue mayor que con el motor IDS (110 correctas y 218 errores de identificación), por lo tanto, GenBank supera a BOLD (Kruskal-Wallis = 41.625, df = 1, p < 0.001). Conclusiones: Se necesitan muestras adicionales de equinodermos de referencia para mejorar la utilidad de las herramientas de identificación de códigos de barras de ADN evaluadas. Las discordancias de identificación en ambas bases de datos pueden obedecer a parámetros específicos utilizados en cada algoritmo de búsqueda y a las secuencias disponibles. Recomendamos el uso de códigos de barras como fuente de identificación complementaria para las especies de equinodermos de aguas someras del Pacífico centroamericano.

Rare Ophiuroid-Type Steroid 3ß,21-, 3ß,22-, and 3α,22-Disulfates from the Slime Sea Star Pteraster marsippus and Their Colony-Inhibiting Effects against Human Breast Cancer Cells.

Two new steroid 3ß,21-disulfates (1, 2) and two new steroid 3ß,22- and 3α,22-disulfates (3, 4), along with the previously known monoamine alkaloid tryptamine (5) were found in the ethanolic extract of the Far Eastern slime sea star Pteraster marsippus. Their structures were determined on the basis of detailed analysis of one-dimensional and two-dimensional NMR, HRESIMS, and HRESIMS/MS data. Compounds 1 and 2 have a Δ22-21-sulfoxy-24-norcholestane side chain. Compounds 3 and 4 contain a Δ24(28)-22-sulfoxy-24-methylcholestane side chain, which was first discovered in the polar steroids of starfish and brittle stars. The influence of substances 1-4 on cell viability, colony formation, and growth of human breast cancer T-47D, MCF-7, and MDA-MB-231 cells was investigated. It was shown that compounds 1 and 2 possess significant colony-inhibiting activity against T-47D cells, while compounds 3 and 4 were more effective against MDA-MB-231 cells.

Marine Antimicrobial Peptides: An Emerging Nightmare to the Life-Threatening Pathogens.

The emergence of multidrug-resistant pathogens due to improper usage of conventional antibiotics has created a global health crisis. Alternatives to antibiotics being an urgent need, the scientific community is forced to search for new antimicrobials. This exploration has led to the discovery of antimicrobial peptides, a group of small peptides occurring in different phyla such as Porifera, Cnidaria, Annelida, Arthropoda, Mollusca, Echinodermata, and Chordata, as a component of their innate immune system. The marine environment, possessing immense diversity of organisms, is undoubtedly one of the richest sources of unique potential antimicrobial peptides. The distinctiveness of marine antimicrobial peptides lies in their broad-spectrum activity, mechanism of action, less cytotoxicity, and high stability, which form the benchmark for developing a potential therapeutic. This review aims to (1) synthesise the available information on the distinctive antimicrobial peptides discovered from marine organisms, particularly over the last decade, and (2) discuss the distinctiveness of marine antimicrobial peptides and their prospects.

Systematics of deep-sea starfish order Brisingida (Echinodermata: Asteroidea), with a revised classification and assessments of morphological characters.

Brisingida Fisher 1928 is one of the seven currently recognised starfish orders, and one of the least known taxa as being exclusive deep-sea inhabitants. Modern deep-sea expeditions revealed their common occurrences in various deep-sea settings including seamounts, basins and hydrothermal vent peripheral, underlining the necessity of clarifying their global diversity and phylogeny. In this study, we present a comprehensive molecular phylogeny of Brisingida which encompasses the highest taxonomic diversity to date. DNA sequences (COI, 16S, 12S and 28S) were obtained from 225 specimens collected in the global ocean, identified as 58 species spanning 15 of the 17 extant genera. Phylogenetic relationship was inferred using both maximum likelihood and Bayesian inference methods, revealing polyphyletic families and genera and indicating nonnegligible bias in prior morphology-based systematics. Based on the new phylogeny, a novel classification of the order, consisting of 5 families and 17 genera, is proposed. Families Odinellidae, Brisingasteridae and Novodiniidae (sensu Clark and Mah, 2001) were resurrected to encompass the genera Odinella, Brisingaster and Novodinia. Brisingidae and Freyellidae were revised to include 11 and 3 genera, respectively. A new genus and species, two new subgenera and seven new combinations are described and a key to each genus and family is provided. Transformations of morphological traits were evaluated under the present phylogenetic hypothesis. A series of paedomorphic characters were found in many genera and species, which led to a high degree of homoplasy across phylogenetically distant groups. Our results provide new insights in the phylogeny and ontogeny of the order, and highlight the necessity to evaluate character convergence under sound phylogenetic hypothesis.

Microplastic contamination in the digestive tract of sea urchins (Echinodermata: Echinoidea) in Kepulauan Seribu, Indonesia.

The accumulation of microplastics on sediment surfaces contributed to the digestive tract of sea urchins contamination during foraging. Therefore, the aim of this study was to investigate the potential relationship between the accumulation of microplastics on sediment surfaces and the contamination of sea urchins' digestive tracts during their foraging activities. Sediment and sea urchins' samples were collected from Pari and Harapan Islands, in Kepulauan Seribu, DKI Jakarta, Indonesia. Microplastics were extracted and observed in sediment and the digestive tract of sea urchins' samples. Fourier transform infrared (FTIR) spectroscopy identified microplastic polymers. The average microplastic concentration on Pari Island was 160 ± 158.75 particles/kg dry weight sediment and 3.93 ± 2.25 particles/g dry weight in the digestive tract of sea urchins. Correspondingly, on Harapan Island, the values were 113 ± 41.63 particles/kg dry weight and 0.27 ± 0.28 particles/g dry weight. Fragment-type microplastics (75%) were predominantly detected in the digestive tract of sea urchins, while fiber-type microplastics (59%) were more common in sediments on Pari Island. Conversely, on Harapan Island fragment types were more prevalent (53%). Microplastics larger than 1000 µm were identified in both sediment and the digestive tract of sea urchins. The observed plastic polymers, such as polyethylene, polyester, and polypropylene were dominant at both study sites. This study postulated that microplastics in sediments may be ingested during sea urchins digestion, supported by a significant correlation of 0.016. Consequently, the presence of microplastics in sea urchins from Pari Island and Harapan Island in Kepulauan Seribu was confirmed. Future investigations should explore the toxic effects of absorbed microplastics on sea urchins' physiology, requiring further analysis.

Extracellular Matrix of Echinoderms.

This review considers available data on the composition of the extracellular matrix (ECM) in echinoderms. The connective tissue in these animals has a rather complex organization. It includes a wide range of structural ECM proteins, as well as various proteases and their inhibitors. Members of almost all major groups of collagens, various glycoproteins, and proteoglycans have been found in echinoderms. There are enzymes for the synthesis of structural proteins and their modification by polysaccharides. However, the ECM of echinoderms substantially differs from that of vertebrates by the lack of elastin, fibronectins, tenascins, and some other glycoproteins and proteoglycans. Echinoderms have a wide variety of proteinases, with serine, cysteine, aspartic, and metal peptidases identified among them. Their active centers have a typical structure and can break down various ECM molecules. Echinoderms are also distinguished by a wide range of proteinase inhibitors. The complex ECM structure and the variety of intermolecular interactions evidently explain the complexity of the mechanisms responsible for variations in the mechanical properties of connective tissue in echinoderms. These mechanisms probably depend not only on the number of cross-links between the molecules, but also on the composition of ECM and the properties of its proteins.

Structure and proteomic analysis of the crown-of-thorns starfish (Acanthaster sp.) radial nerve cord.

The nervous system of the Asteroidea (starfish or seastar) consists of radial nerve cords (RNCs) that interconnect with a ring nerve. Despite its relative simplicity, it facilitates the movement of multiple arms and numerous tube feet, as well as regeneration of damaged limbs. Here, we investigated the RNC ultrastructure and its molecular components within the of Pacific crown-of-thorns starfish (COTS; Acanthaster sp.), a well-known coral predator that in high-density outbreaks has major ecological impacts on coral reefs. We describe the presence of an array of unique small bulbous bulbs (40-100 µm diameter) that project from the ectoneural region of the adult RNC. Each comprise large secretory-like cells and prominent cilia. In contrast, juvenile COTS and its congener Acanthaster brevispinus lack these features, both of which are non-corallivorous. Proteomic analysis of the RNC (and isolated neural bulbs) provides the first comprehensive echinoderm protein database for neural tissue, including numerous secreted proteins associated with signalling, transport and defence. The neural bulbs contained several neuropeptides (e.g., bombyxin-type, starfish myorelaxant peptide, secretogranin 7B2-like, Ap15a-like, and ApNp35) and Deleted in Malignant Brain Tumor 1-like proteins. In summary, this study provides a new insight into the novel traits of COTS, a major pest on coral reefs, and a proteomics resource that can be used to develop (bio)control strategies and understand molecular mechanisms of regeneration.

Identification of New Purpuroine Analogues from the Arctic Echinodermata Pteraster militaris That Inhibit FLT3-ITD+ AML Cell Lines.

Isolation of bioactive products from the marine environment is considered a very promising approach to identify new compounds that can be used for further drug development. In this work we have isolated three new compounds from the purpuroine family by mass-guided preparative HPLC; purpuroine K-M. These compounds where screened for antibacterial- and antifungal activity, antibiofilm formation and anti-cell proliferation activity. Additionally, apoptosis-, cell cycle-, kinase binding- and docking studies were performed to evaluate the mechanism-of-action. None of the compounds showed activity in antibacterial-, antibiofilm- or antifungal assays. However, one of the isolated compounds, purpuroine K, showed activity against two cell lines, MV-4-11 and MOLM-13, two AML cell lines both carrying the FTL3-ITD mutation. In MV-4-11 cells, purpuroine K was found to increase apoptosis and arrest cells cycle in G1/G0, which is a common feature of FLT3 inhibitors. Interactions between purpuroine K and the FLT3 wild type or FLT3 ITD mutant proteins could however not be elucidated in our kinase binding and docking studies. In conclusion, we have isolated three novel molecules, purpuroine K-M, one of which (purpuroine K) shows a potent activity against FLT3-ITD mutated AML cell lines, however, the molecular target(s) of purpuroine K still need to be further investigated.

More than a simple epithelial layer: multifunctional role of echinoderm coelomic epithelium.

In echinoderms, the coelomic epithelium (CE) is reportedly the source of new circulating cells (coelomocytes) as well as the provider of molecular factors such as immunity-related molecules. However, its overall functions have been scarcely studied in detail. In this work, we used an integrated approach based on both microscopy (light and electron) and proteomic analyses to investigate the arm CE in the starfish Marthasterias glacialis during different physiological conditions (i.e., non-regenerating and/or regenerating). Our results show that CE cells share both ultrastructural and proteomic features with circulating coelomocytes (echinoderm immune cells). Additionally, microscopy and proteomic analyses indicate that CE cells are actively involved in protein synthesis and processing, and membrane trafficking processes such as phagocytosis (particularly of myocytes) and massive secretion phenomena. The latter might provide molecules (e.g., immune factors) and fluids for proper arm growth/regrowth. No stem cell marker was identified and no pre-existing stem cell was observed within the CE. Rather, during regeneration, CE cells undergo dedifferentiation and epithelial-mesenchymal transition to deliver progenitor cells for tissue replacement. Overall, our work underlines that echinoderm CE is not a "simple epithelial lining" and that instead it plays multiple functions which span from immunity-related roles as well as being a source of regeneration-competent cells for arm growth/regrowth.

Receptor deorphanization in an echinoderm reveals kisspeptin evolution and relationship with SALMFamide neuropeptides.

BACKGROUND: Kisspeptins are neuropeptides that regulate reproductive maturation in mammals via G-protein-coupled receptor-mediated stimulation of gonadotropin-releasing hormone secretion from the hypothalamus. Phylogenetic analysis of kisspeptin-type receptors indicates that this neuropeptide signaling system originated in a common ancestor of the Bilateria, but little is known about kisspeptin signaling in invertebrates. RESULTS: Contrasting with the occurrence of a single kisspeptin receptor in mammalian species, here, we report the discovery of an expanded family of eleven kisspeptin-type receptors in a deuterostome invertebrate - the starfish Asterias rubens (phylum Echinodermata). Furthermore, neuropeptides derived from four precursor proteins were identified as ligands for six of these receptors. One or more kisspeptin-like neuropeptides derived from two precursor proteins (ArKPP1, ArKPP2) act as ligands for four A. rubens kisspeptin-type receptors (ArKPR1,3,8,9). Furthermore, a family of neuropeptides that act as muscle relaxants in echinoderms (SALMFamides) are ligands for two A. rubens kisspeptin-type receptors (ArKPR6,7). The SALMFamide neuropeptide S1 (or ArS1.4) and a 'cocktail' of the seven neuropeptides derived from the S1 precursor protein (ArS1.1-ArS1.7) act as ligands for ArKPR7. The SALMFamide neuropeptide S2 (or ArS2.3) and a 'cocktail' of the eight neuropeptides derived from the S2 precursor protein (ArS2.1-ArS2.8) act as ligands for ArKPR6. CONCLUSIONS: Our findings reveal a remarkable diversity of neuropeptides that act as ligands for kisspeptin-type receptors in starfish and provide important new insights into the evolution of kisspeptin signaling. Furthermore, the discovery of the hitherto unknown relationship of kisspeptins with SALMFamides, neuropeptides that were discovered in starfish prior to the identification of kisspeptins in mammals, presents a radical change in perspective for research on kisspeptin signaling.

Vibrio splendidus flagellin C binds tropomodulin to induce p38 MAPK-mediated p53-dependent coelomocyte apoptosis in Echinodermata.

As a typical pathogen-associated molecular pattern, bacterial flagellin can bind Toll-like receptor 5 and the intracellular NAIP5 receptor component of the NLRC4 inflammasome to induce immune responses in mammals. However, these flagellin receptors are generally poorly understood in lower animal species. In this study, we found that the isolated flagellum of Vibrio splendidus AJ01 destroyed the integrity of the tissue structure of coelomocytes and promoted apoptosis in the sea cucumber Apostichopus japonicus. To further investigate the molecular mechanism, the novel intracellular LRR domain-containing protein tropomodulin (AjTmod) was identified as a protein that interacts with flagellin C (FliC) with a dissociation constant (Kd) of 0.0086 ± 0.33 µM by microscale thermophoresis assay. We show that knockdown of AjTmod also depressed FliC-induced apoptosis of coelomocytes. Further functional analysis with different inhibitor treatments revealed that the interaction between AjTmod and FliC could specifically activate p38 MAPK, but not JNK or ERK MAP kinases. We demonstrate that the transcription factor p38 is then translocated into the nucleus, where it mediates the expression of p53 to induce coelomocyte apoptosis. Our findings provide the first evidence that intracellular AjTmod serves as a novel receptor of FliC and mediates p53-dependent coelomocyte apoptosis by activating the p38 MAPK signaling pathway in Echinodermata.

Cytotoxic and antimicrobial activities of two new sesquiterpenoids from red sea brittle star Ophiocoma dentata.

Bioactive compounds were extracted from a locally available brittle star; Ophiocoma dentata, collected from the Red Sea, Egypt. Two new sesquiterpenoids; 8, 11-epoxy-9(15)-himachaladiene-4-ol (O8-ophiocomane) and, 11-epoxy-9(15)-himachaladiene-4-ol (O7-ophiocomane) were isolated and characterized using appropriate techniques. Structure elucidation was estimated via 1D NMR, 2D NMR, FT-IR and mass spectroscopy analyses. The isolated compounds were tested for cytotoxic, antibacterial and antifungal activities. Pure compounds showed a dose dependent reduction in MCF-7 cells viability with LC50 of 103.5 and 59.5 µg/ml for compounds 1 and 2 respectively compared to the chemotherapeutic drug cisplatin (47.4 µg/ml). In vivo experiments showed that O. dentate extract significantly reduced tumor progression and improved hematological parameters and liver functions of tumor-bearing mice when administered either before or after tumor cells' injection. The most remarkable antimicrobial effects of O. dentate crude extract were against Staphylococcus aureus, Vibrio damsela and Pseudomonas aeruginosa while the pure compounds showed activity against P. aeruginosa alone. Neither the crude extract nor the pure compounds have shown activity against Aeromonas hydrophila. These results indicates that O. dentata extract and newly isolated compounds have shown a promising cytotoxic, antiproliferative and antimicrobial activities that might be useful for pharmaceutical applications.

Sulfated Naphthopyrones and Anthraquinones from the Vietnamese Crinoid Comanthus delicata.

Three new sulfated naphthopyrone derivatives namely delicapyrons F-H (1-3) and two new sulfated anthraquinone derivatives namely delicaquinons A (4) and B (5), together with 6-methoxycomaparvin-5-methylether-8-O-sodium sulfate (6), 6-methoxycomaparvin-8-O-sodium sulfate (7), comaparvin-8-O-sodium sulfate (8), and 3-propyl-1,6,8-trihydroxy-9,10-anthraquinone-6-O-sodium sulfate (9) were isolated from the Vietnamese crinoid Comanthus delicata. Their chemical structures were elucidated by extensive analysis of the one dimensional (1D) and 2D-NMR, high resolution electrospray ionization quadrupole time-of-flight (HR-ESI-QTOF) mass spectra as well as calculation of optical rotation. In addition, significant cytotoxicity was observed for 6 against LNCaP (prostate cancer) cell line with IC50 value of 20.29 ± 2.43 µM, whereas moderate or weak cytotoxic effects were observed for 1-3 and 5-8 on SK-Mel-2 (melanoma) cell line and 7 and 8 against LNCaP cell line, with IC50 values ranging from 49.96 ± 1.74 to 76.92 ± 5.85 µM.

Contrasting Early Ordovician assembly patterns highlight the complex initial stages of the Ordovician Radiation.

The Early Ordovician is a key interval for our understanding of the evolution of life on Earth as it lays at the transition between the Cambrian Explosion and the Ordovician Radiation and because the fossil record of the late Cambrian is scarce. In this study, assembly processes of Early Ordovician trilobite and echinoderm communities from the Central Anti-Atlas (Morocco), the Montagne Noire (France), and the Cordillera Oriental (Argentina) are explored. The results show that dispersal increased diachronically in trilobite communities during the Early Ordovician. Dispersal did not increase for echinoderms. Dispersal was most probably proximally triggered by the planktic revolution, the fall in seawater temperatures, changes in oceanic circulation, with an overall control by tectonic frameworks and phylogenetic constraints. The diachronous increase in dispersal within trilobite communities in the Early Ordovician highlights the complexity of ecosystem structuring during the early stages of the Ordovician Radiation. As Early Ordovician regional dispersal was followed by well-documented continental dispersal in the Middle/Late Ordovician, it is possible to consider that alongside a global increase in taxonomic richness, the Ordovician Radiation is also characterized by a gradual increase in dispersal.