Gross and microscopic pathology of West Indian sea eggs (Tripneustes ventricosus).

In this study, we performed comprehensive pathology examinations on 83 Tripneustes ventricosus from 11 locations on St. Kitts to build baseline data necessary for disease diagnosis in this species. Gross abnormalities were observed in 23/83 (28%) urchins and included spine loss, visceral hyperpigmentation, test discoloration, and test ulceration. Ciliates were the only protists identified in this study via examination of tissue wet mounts and histology, documented in 50/83 (60%) urchins. Microscopic observations associated with visibly abnormal status included muscle necrosis, test and appendage inflammation, appendage (tube feet, spines, and pedicellariae) degeneration, severe coelomocytosis, and generalized hypermelanosis. Enterocyte intranuclear inclusion bodies, microbial aggregates, nerve pigmentation, enteric pigmentation, integument-associated crustaceans, and encysted metazoan parasites were of uncertain pathological significance. The etiology for any lesion was not microscopically apparent, contrasting literature implicating common marine bacteria in urchin diseases. This study highlights the importance of histopathology in urchin disease investigations and facilitates the recognition of disease in T. ventricosus.

Effect of dopamine on early larvae of sea urchins, Mesocentrotus nudus and Strongylocentrotus intermedius.

Larvae of many echinoids are known to be phenotypically plastic and capable of changing the growth rate of their post-oral arms depending on the microalgae concentration in their habitat. As literature data show, developing larvae use chemosensation to detect algae in the environment and "adjust" the rate of growth of their post-oral arms through dopamine signaling. According to our results, dopamine has a significant effect on the post-oral arm growth in early larvae of two sea urchin species, Mesocentrotus nudus and Strongylocentrotus intermedius. The dopamine effect depends on concentration: the higher the dopamine concentration in the water, the shorter the post-oral arms. We suggest that the pattern of response to variation in dopamine concentration, manifested by early larvae of both species, is similar to that observed at different concentrations of microalgae.

A new ophiocistioid with soft-tissue preservation from the Silurian Herefordshire Lagerstätte, and the evolution of the holothurian body plan.

Reconstructing the evolutionary assembly of animal body plans is challenging when there are large morphological gaps between extant sister taxa, as in the case of echinozoans (echinoids and holothurians). However, the inclusion of extinct taxa can help bridge these gaps. Here we describe a new species of echinozoan, Sollasina cthulhu, from the Silurian Herefordshire Lagerstätte, UK. Sollasina cthulhu belongs to the ophiocistioids, an extinct group that shares characters with both echinoids and holothurians. Using physical-optical tomography and computer reconstruction, we visualize the internal anatomy of S. cthulhu in three dimensions, revealing inner soft tissues that we interpret as the ring canal, a key part of the water vascular system that was previously unknown in fossil echinozoans. Phylogenetic analyses strongly suggest that Sollasina and other ophiocistioids represent a paraphyletic group of stem holothurians, as previously hypothesized. This allows us to reconstruct the stepwise reduction of the skeleton during the assembly of the holothurian body plan, which may have been controlled by changes in the expression of biomineralization genes.

Comparative studies on the skeletogenic mesenchyme of echinoids.

Skeletogenic mesenchyme cells in echinoids are suitable for studying developmental mechanisms, and have been used extensively. Most of these studies have been performed on species in the order Camarodonta, which are modern echinoids (subclass Euechinoidea) and are considered "model" echinoid species. In contrast, species belonging to other orders are studied less frequently, especially investigations of their molecular developmental biology such as gene regulatory networks. Recent studies on mesenchyme development in non-camarodont species suggest that these species are potential sources of comparative information to elucidate the mechanisms underlying skeletogenic mesenchyme development. In this review, the importance of using comparative data to understand development and evolution is discussed.

A phylogenomic resolution of the sea urchin tree of life.

BACKGROUND: Echinoidea is a clade of marine animals including sea urchins, heart urchins, sand dollars and sea biscuits. Found in benthic habitats across all latitudes, echinoids are key components of marine communities such as coral reefs and kelp forests. A little over 1000 species inhabit the oceans today, a diversity that traces its roots back at least to the Permian. Although much effort has been devoted to elucidating the echinoid tree of life using a variety of morphological data, molecular attempts have relied on only a handful of genes. Both of these approaches have had limited success at resolving the deepest nodes of the tree, and their disagreement over the positions of a number of clades remains unresolved. RESULTS: We performed de novo sequencing and assembly of 17 transcriptomes to complement available genomic resources of sea urchins and produce the first phylogenomic analysis of the clade. Multiple methods of probabilistic inference recovered identical topologies, with virtually all nodes showing maximum support. In contrast, the coalescent-based method ASTRAL-II resolved one node differently, a result apparently driven by gene tree error induced by evolutionary rate heterogeneity. Regardless of the method employed, our phylogenetic structure deviates from the currently accepted classification of echinoids, with neither Acroechinoidea (all euechinoids except echinothurioids), nor Clypeasteroida (sand dollars and sea biscuits) being monophyletic as currently defined. We show that phylogenetic signal for novel resolutions of these lineages is strong and distributed throughout the genome, and fail to recover systematic biases as drivers of our results. CONCLUSIONS: Our investigation substantially augments the molecular resources available for sea urchins, providing the first transcriptomes for many of its main lineages. Using this expanded genomic dataset, we resolve the position of several clades in agreement with early molecular analyses but in disagreement with morphological data. Our efforts settle multiple phylogenetic uncertainties, including the position of the enigmatic deep-sea echinothurioids and the identity of the sister clade to sand dollars. We offer a detailed assessment of evolutionary scenarios that could reconcile our findings with morphological evidence, opening up new lines of research into the development and evolutionary history of this ancient clade.

Synthesis of Zinc Oxide Nanorods via the Formation of Sea Urchin Structures and Their Photoluminescence after Heat Treatment.

A protocol for the aqueous synthesis of ca. 1-µm-long zinc oxide (ZnO) nanorods and their growth at intermediate reaction progression is presented, together with photoluminescence (PL) characteristics after heat treatment at temperatures of up to 1000 °C. The existence of solitary rods after the complete reaction (60 min) was traced back to the development of sea urchin structures during the first 5 s of the precipitation. The rods primarily formed in later stages during the reaction due to fracture, which was supported by the frequently observed broken rod ends with sharp edges in the final material, in addition to tapered uniform rod ends consistent with their natural growth direction. The more dominant rod growth in the c direction (extending the length of the rods), together with the appearance of faceted surfaces on the sides of the rods, occurred at longer reaction times (>5 min) and generated zinc-terminated particles that were more resistant to alkaline dissolution. A heat treatment for 1 h at 600 or 800 °C resulted in a smoothing of the rod surfaces, and PL measurements displayed a decreased defect emission at ca. 600 nm, which was related to the disappearance of lattice imperfections formed during the synthesis. A heat treatment at 1000 °C resulted in significant crystal growth reflected as an increase in luminescence at shorter wavelengths (ca. 510 nm). Electron microscopy revealed that the faceted rod structure was lost for ZnO rods exposed to temperatures above 600 °C, whereas even higher temperatures resulted in particle sintering and/or mass redistribution along the initially long and slender ZnO rods. The synthesized ZnO rods were a more stable Wurtzite crystal structure than previously reported ball-shaped ZnO consisting of merging sheets, which was supported by the shifts in PL spectra occurring at ca. 200 °C higher annealing temperature, in combination with a smaller thermogravimetric mass loss occurring upon heating the rods to 800 °C.

The conserved genetic background for pluteus arm development in brittle stars and sea urchin.

Echinoderm pluteus larvae are considered a classical example of convergent evolution that occurred in sea urchins and brittle stars. Several genes are known to be involved in the development of pluteus arms in sea urchins, including fgfA, pax2/5/8, pea3, otp, wnt5, and tet. To determine whether the convergent evolution of larval arms also involves these genes in brittle stars, their expression patterns were determined in brittle star. We found that all genes showed similar expression in the arms of ophiopluteus to that seen in echinopluteus, suggesting that convergent evolution of pluteus arms occurred by recruitment of a similar set of genes. This may be explained by our observation that some of these genes are also expressed in the spine rudiment of direct-type development sea urchins. We propose an evolutionary scenario wherein the pluteus arms of both echinopluteus and ophiopluteus were acquired by independent co-options of the genetic module responsible for the projection of the adult skeleton.

Morphological and genetic analyses reveal a cryptic species complex in the echinoid Echinocardium cordatum and rule out a stabilizing selection explanation.

Preliminary analyses revealed the presence of at least five mitochondrial clades within the widespread sea urchin Echinocardium cordatum (Spatangoida). In this study, we analyzed the genetic (two mitochondrial and two nuclear sequence loci) and morphological characteristics (20 indices) from worldwide samples of this taxon to establish the species limits, morphological diversity and differentiation. Co-occurring spatangoid species were also analyzed with mitochondrial DNA. The nuclear sequences confirm that mitochondrial lineages correspond to true genetic entities and reveal that two clades (named A and B1) hybridize in their sympatry area, although a more closely related pair of clades (B1 and B2), whose distributions widely overlap, does not display hybridization. The morphology of all E. cordatum clade pairs was significantly differentiated, but no morphological diagnostic character was evidenced. By contrast, other spatangoid species pairs that diverged more recently than the E. cordatum clades display clear diagnostic characters. Morphological diversity thus appears responsible for the absence of diagnostic characters, ruling out stabilizing selection, a classical explanation for cryptic species. Alternative classical explanations are (i) environmental plasticity or (ii) a high diversity of genes determining morphology, maintained by varying environmental conditions. We suggest a new hypothesis that the observed morphological diversity is selectively neutral and reflects high effective population sizes in the E. cordatum complex. It is supported by the higher abundance of this taxon compared with other taxa, a trend for the genetic and morphological diversity to be correlated in Europe, and the higher genetic and morphological diversities found in clades of E cordatum (except B1) than in other spatangoid samples in Europe. However, the Pacific clades do not confirm these trends.

Size-dependent mortality rate profiles.

Knowledge of mortality rates is crucial to the understanding of population dynamics in populations of free-living fish and invertebrates in marine and freshwater environments, and consequently to sustainable resource management. There is a well developed theory of population dynamics based on age distributions that allow direct estimation of mortality rates. However, for most cases the aging of individuals is difficult or age distributions are not available for other reasons. The body size distribution is a widely available alternative although the theory underlying the formation of its shape is more complicated than in the case of age distributions. A solid theory of the time evolution of a population structured by any physiological variable has been developed in 1960s and 1970s by adapting the Hamilton-Jacobi formulation of classical mechanics, and equations to estimate the body size-distributed mortality profile have been derived for simple cases. Here I extend those results with regards to the size-distributed mortality profile to complex cases of non-stationary populations, individuals growing according to a generalised growth model and seasonally patterned recruitment pulses. I apply resulting methods to two cases in the marine environment, a benthic crustacean population that was growing during the period of observation and whose individuals grow with negative acceleration, and a sea urchin coastal population that is undergoing a stable cycle of two equilibrium points in population size whose individuals grow with varying acceleration that switches sign along the size range. The extension is very general and substantially widens the applicability of the theory.

Ancient animal microRNAs and the evolution of tissue identity.

The spectacular escalation in complexity in early bilaterian evolution correlates with a strong increase in the number of microRNAs. To explore the link between the birth of ancient microRNAs and body plan evolution, we set out to determine the ancient sites of activity of conserved bilaterian microRNA families in a comparative approach. We reason that any specific localization shared between protostomes and deuterostomes (the two major superphyla of bilaterian animals) should probably reflect an ancient specificity of that microRNA in their last common ancestor. Here, we investigate the expression of conserved bilaterian microRNAs in Platynereis dumerilii, a protostome retaining ancestral bilaterian features, in Capitella, another marine annelid, in the sea urchin Strongylocentrotus, a deuterostome, and in sea anemone Nematostella, representing an outgroup to the bilaterians. Our comparative data indicate that the oldest known animal microRNA, miR-100, and the related miR-125 and let-7 were initially active in neurosecretory cells located around the mouth. Other sets of ancient microRNAs were first present in locomotor ciliated cells, specific brain centres, or, more broadly, one of four major organ systems: central nervous system, sensory tissue, musculature and gut. These findings reveal that microRNA evolution and the establishment of tissue identities were closely coupled in bilaterian evolution. Also, they outline a minimum set of cell types and tissues that existed in the protostome-deuterostome ancestor.

Coordination between catch connective tissue and muscles through nerves in the spine joint of the sea urchin Diadema setosum.

Echinoderms have catch connective tissues that change their stiffness as a result of nervous control. The coordination between catch connective tissue and muscles was studied in the spine joint of the sea urchin Diadema setosum. Spine joints are equipped with two kinds of effector: spine muscles and a kind of catch connective tissue, which is called the catch apparatus (CA). The former is responsible for spine movements and the latter for maintenance of spine posture. Diadema show a shadow reaction in which they wave spines when a shadow falls on them, which is a reflex involving the radial nerves. Dynamic mechanical tests were performed on the CA in a joint at which the muscles were severed so as not to interfere with the mechanical measurements. The joint was on a piece of the test that contained other spines and a radial nerve. Darkening of the preparation invoked softening of the CA and spine waving (the shadow reaction). Electrical stimulation of the radial nerve invoked a similar response. These responses were abolished after the nerve pathways from the radial nerve to spines had been cut. A touch applied to the CA stiffened it and the adjacent spines inclined toward the touched CA. A touch to the base of the adjacent spine softened the CA and the spines around the touched spine inclined towards it. The softening of the CA can be interpreted as a response that reduces the resistance of the ligaments to spine movements. Our results clearly show coordination between catch connective tissue and muscles through nerves.

Branching out: origins of the sea urchin larval skeleton in development and evolution.

It is a challenge to understand how the information encoded in DNA is used to build a three-dimensional structure. To explore how this works the assembly of a relatively simple skeleton has been examined at multiple control levels. The skeleton of the sea urchin embryo consists of a number of calcite rods produced by 64 skeletogenic cells. The ectoderm supplies spatial cues for patterning, essentially telling the skeletogenic cells where to position themselves and providing the factors for skeletal growth. Here, we describe the information known about how this works. First the ectoderm must be patterned so that the signaling cues are released from precise positions. The skeletogenic cells respond by initiating skeletogenesis immediately beneath two regions (one on the right and the other on the left side). Growth of the skeletal rods requires additional signaling from defined ectodermal locations, and the skeletogenic cells respond to produce a membrane-bound template in which the calcite crystal grows. Important in this process are three signals, fibroblast growth factor, vascular endothelial growth factor, and Wnt5. Each is necessary for explicit tasks in skeleton production.

Wnt6 activates endoderm in the sea urchin gene regulatory network.

In the sea urchin, entry of ß-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of ß-catenin, of a dominant-negative variant of GSK-3ß or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.

Annual reversible plasticity of feeding structures: cyclical changes of jaw allometry in a sea urchin.

A wide variety of organisms show morphologically plastic responses to environmental stressors but in general these changes are not reversible. Though less common, reversible morphological structures are shown by a range of species in response to changes in predators, competitors or food. Theoretical analysis indicates that reversible plasticity increases fitness if organisms are long-lived relative to the frequency of changes in the stressor and morphological changes are rapid. Many sea urchin species show differences in the sizes of jaws (demi-pyramids) of the feeding apparatus, Aristotle's lantern, relative to overall body size, and these differences have been correlated with available food. The question addressed here is whether reversible changes of relative jaw size occur in the field as available food changes with season. Monthly samples of the North American Pacific coast sea urchin Strongylocentrotus purpuratus were collected from Gregory Point on the Oregon (USA) coast and showed an annual cycle of relative jaw size together with a linear trend from 2007 to 2009. Strongylocentrotus purpuratus is a long-lived species and under field conditions individuals experience multiple episodes of changes in food resources both seasonally and from year to year. Their rapid and reversible jaw plasticity fits well with theoretical expectations.

Unique system of photoreceptors in sea urchin tube feet.

Different sea urchin species show a vast variety of responses to variations in light intensity; however, despite this behavioral evidence for photosensitivity, light sensing in these animals has remained an enigma. Genome information of the recently sequenced purple sea urchin (Strongylocentrotus purpuratus) allowed us to address this question from a previously unexplored molecular perspective by localizing expression of the rhabdomeric opsin Sp-opsin4 and Sp-pax6, two genes essential for photoreceptor function and development, respectively. Using a specifically designed antibody against Sp-Opsin4 and in situ hybridization for both genes, we detected expression in two distinct groups of photoreceptor cells (PRCs) located in the animal's numerous tube feet. Specific reactivity of the Sp-Opsin4 antibody with sea star optic cushions, which regulate phototaxis, suggests a similar visual function in sea urchins. Ultrastructural characterization of the sea urchin PRCs revealed them to be of a microvillar receptor type. Our data suggest that echinoderms, in contrast to chordates, deploy a microvillar, r-opsin-expressing PRC type for vision, a feature that has been so far documented only in protostome animals. Surprisingly, sea urchin PRCs lack any associated screening pigment. Indeed, one of the tube foot PRC clusters may account for directional vision by being shaded through the opaque calcite skeleton. The PRC axons connect to the animal internal nervous system, suggesting an integrative function beyond local short circuits. Because juveniles display no phototaxis until skeleton completion, we suggest a model in which the entire sea urchin, deploying its skeleton as PRC screening device, functions as a huge compound eye.

Encoding anatomy: developmental gene regulatory networks and morphogenesis.

A central challenge of developmental and evolutionary biology is to explain how anatomy is encoded in the genome. Anatomy emerges progressively during embryonic development, as a consequence of morphogenetic processes. The specialized properties of embryonic cells and tissues that drive morphogenesis, like other specialized properties of cells, arise as a consequence of differential gene expression. Recently, gene regulatory networks (GRNs) have proven to be powerful conceptual and experimental tools for analyzing the genetic control and evolution of developmental processes. A major current goal is to link these transcriptional networks directly to morphogenetic processes. This review highlights three experimental models (sea urchin skeletogenesis, ascidian notochord morphogenesis, and the formation of somatic muscles in Drosophila) that are currently being used to analyze the genetic control of anatomy by integrating information of several important kinds: (1) morphogenetic mechanisms at the molecular, cellular and tissue levels that are responsible for shaping a specific anatomical feature, (2) the underlying GRN circuitry deployed in the relevant cells, and (3) modifications to gene regulatory circuitry that have accompanied evolutionary changes in the anatomical feature.

SM30 protein function during sea urchin larval spicule formation.

A central issue in better understanding the process of biomineralization is to elucidate the function of occluded matrix proteins present in mineralized tissues. A potent approach to addressing this issue utilizes specific inhibitors of expression of known genes. Application of antisense oligonucleotides that specifically suppress translation of a given mRNA are capable of causing aberrant biomineralization, thereby revealing, at least in part, a likely function of the protein and gene under investigation. We have applied this approach to study the possible function(s) of the SM30 family of proteins, which are found in spicules, teeth, spines, and tests of Strongylocentrotus purpuratus as well as other euechinoid sea urchins. It is possible using the anti-SM30 morpholino-oligonucleotides (MO's) to reduce the level of these proteins to very low levels, yet the development of skeletal spicules in the embryo shows little or no aberration. This surprising result requires re-thinking about the role of these, and possibly other occluded matrix proteins.

Assessing physiological tipping point of sea urchin larvae exposed to a broad range of pH.

Our ability to project the impact of global change on marine ecosystem is limited by our poor understanding on how to predict species sensitivity. For example, the impact of ocean acidification is highly species-specific, even in closely related taxa. The aim of this study was to test the hypothesis that the tolerance range of a given species to decreased pH corresponds to their natural range of exposure. Larvae of the green sea urchin Strongylocentrotus droebachiensis were cultured from fertilization to metamorphic competence (29 days) under a wide range of pH (from pHT  = 8.0/pCO2  ≈ 480 µatm to pHT  = 6.5/pCO2  ≈ 20 000 µatm) covering present (from pHT 8.7 to 7.6), projected near-future variability (from pHT 8.3 to 7.2) and beyond. Decreasing pH impacted all tested parameters (mortality, symmetry, growth, morphometry and respiration). Development of normal, although showing morphological plasticity, swimming larvae was possible as low as pHT  ≥ 7.0. Within that range, decreasing pH increased mortality and asymmetry and decreased body length (BL) growth rate. Larvae raised at lowered pH and with similar BL had shorter arms and a wider body. Relative to a given BL, respiration rates and stomach volume both increased with decreasing pH suggesting changes in energy budget. At the lowest pHs (pHT  ≤ 6.5), all the tested parameters were strongly negatively affected and no larva survived past 13 days post fertilization. In conclusion, sea urchin larvae appeared to be highly plastic when exposed to decreased pH until a physiological tipping point at pHT  = 7.0. However, this plasticity was associated with direct (increased mortality) and indirect (decreased growth) consequences for fitness.

A new late Cenozoic species of Abertella (Echinoidea: Clypeasteroida) from Patagonia.

A new species of abertellid sand dollar, Abertella miskellyi n. sp., is described from the Miocene Camarones Formation of Patagonia, southern Argentina. The new taxon corroborates the existence of the genus in South America, given that Abertella is most common in the southeastern USA and the eastern coast of Central America. It is characterized by a unique basicoronal circle, in which the interambulacral basicoronal plates are very heterogeneous in size (small in interambulacrum 5, largest in interambulacra 2 and 3). Additionally, it features disjunct oral interambulacra involving two ambulacral plates in some of the interambulacra rather than one, thus being the most disjunct of all known species of Abertella. A key to the species of the genus is provided.

A new species of Diadema (Echinodermata: Echinoidea: Diadematidae) from the eastern Atlantic Ocean and a neotype designation of Diadema antillarum (Philippi, 1845).

Diadenia africanum sp. nov. Rodríguez et al. 2013 occurs in the eastern Atlantic Ocean at depths of 1-80 meters off Ma- deira Islands, Salvage Islands, Canary Islands, Cape Verde Islands, Sâo Tome Islands and at the continental coast off Sen- egal and Ghana. This species was previously considered an eastern Atlantic population of D. antillarum. Genetic distances between the holotype of D. africanum and the neotype of D. antillarun herein designated, measured 3.34% in Cytochrome oxidase I, 3.80% in ATPase-8 and 2.31% in ATPase-6. Such divergence is similar to that already highlighted between other accepted species of Diadena. Morphometric analysis of test, spine and pedicellarial characters also separated D. africanum from D. antillartn and reveals that this new species is morphologically similar to D. antillarum ascensionis from the mid Atlantic. The tridentate pedicellariae, which have been shown to have diagnostic characters which discriminate among species of Diadema, occur as both broad and narrow valved forms in D. antillarumn from the western Atlantic. In D. africanum the tridentate pedicellariae occur only as a single form which is characterized by moderately broad and curved valves, with an expanded distal gripping region. This form of tridentate pedicellaria is very similar to that of D. antillarum ascensionis from the central Atlantic, with only slight variations in valve serration and valve curvature differ- entiating the two forms.