High-throughput segmentation, data visualization, and analysis of sea star skeletal networks.

The remarkably complex skeletal systems of the sea stars (Echinodermata, Asteroidea), consisting of hundreds to thousands of individual elements (ossicles), have intrigued investigators for more than 150 years. While the general features and structural diversity of isolated asteroid ossicles have been well documented in the literature, the task of mapping the spatial organization of these constituent skeletal elements in a whole-animal context represents an incredibly laborious process, and as such, has remained largely unexplored. To address this unmet need, particularly in the context of understanding structure-function relationships in these complex skeletal systems, we present an integrated approach that combines micro-computed tomography, automated ossicle segmentation, data visualization tools, and the production of additively manufactured tangible models to reveal biologically relevant structural data that can be rapidly analyzed in an intuitive manner. In the present study, we demonstrate this high-throughput workflow by segmenting and analyzing entire skeletal systems of the giant knobby star, Pisaster giganteus, at four different stages of growth. The in-depth analysis, presented herein, provides a fundamental understanding of the three-dimensional skeletal architecture of the sea star body wall, the process of skeletal maturation during growth, and the relationship between skeletal organization and morphological characteristics of individual ossicles. The widespread implementation of this approach for investigating other species, subspecies, and growth series has the potential to fundamentally improve our understanding of asteroid skeletal architecture and biodiversity in relation to mobility, feeding habits, and environmental specialization in this fascinating group of echinoderms.

The crown-of-thorns starfish genome as a guide for biocontrol of this coral reef pest.

The crown-of-thorns starfish (COTS, the Acanthaster planci species group) is a highly fecund predator of reef-building corals throughout the Indo-Pacific region. COTS population outbreaks cause substantial loss of coral cover, diminishing the integrity and resilience of reef ecosystems. Here we sequenced genomes of COTS from the Great Barrier Reef, Australia and Okinawa, Japan to identify gene products that underlie species-specific communication and could potentially be used in biocontrol strategies. We focused on water-borne chemical plumes released from aggregating COTS, which make the normally sedentary starfish become highly active. Peptide sequences detected in these plumes by mass spectrometry are encoded in the COTS genome and expressed in external tissues. The exoproteome released by aggregating COTS consists largely of signalling factors and hydrolytic enzymes, and includes an expanded and rapidly evolving set of starfish-specific ependymin-related proteins. These secreted proteins may be detected by members of a large family of olfactory-receptor-like G-protein-coupled receptors that are expressed externally, sometimes in a sex-specific manner. This study provides insights into COTS-specific communication that may guide the generation of peptide mimetics for use on reefs with COTS outbreaks.

Deep-sea starfish from the Arctic have well-developed eyes in the dark.

Asteroids, starfish, are important members of the macro-benthos in almost all marine environments including the deep sea. Starfish are in general assumed to be largely olfactory guided, but recent studies have shown that two tropical shallow water species rely on vision alone to find their habitat at short distances. Their compound eyes are found at the tip of each arm and they vary little between examined species. Still, nothing is known about vision in the species found in the aphotic zone of the deep sea or whether they even have eyes. Here, 13 species of starfish from Greenland waters, covering a depth range from shallow waters to the deep sea below 1000 m, were examined for the presence of eyes and optical and morphological examinations were used to estimate the quality of vision. Further, species found in the aphotic zone below 320 m were checked for bioluminescence. All species, except the infaunal Ctenodiscus crispatus, had eyes, and two were found to be bioluminescent. Interestingly, one of the species found in the aphotic zone, Novodinia americana, had close to the highest spatial resolution known for starfish eyes along with being bioluminescent. Accordingly, we hypothesize that this species communicates visually using bioluminescent flashes putatively for reproductive purposes. Other species have greatly enhanced sensitivity with few large ommatidia but at the sacrifice of spatial resolution. The discovery of eyes in deep-sea starfish with a huge variation in optical quality and sensitivity indicates that their visual ecology also differs greatly.

Post-metamorphic ontogeny of Zoroaster fulgens Thomson, 1873 (Asteroidea, Forcipulatacea).

The complete ontogenetic development of an asteroid skeleton has never been described formally for any species. Here, we describe in detail the post-metamorphic ontogeny of Zoroaster fulgens Thomson, 1873. The major novelty of our work is the description of patterns of plate addition, the ontogeny of the internal ossicles, as well as the variability of ossicles according to their position along series. Seven specimens collected in the Rockall Basin (North Atlantic) were dissected with bleach and their anatomy was documented using a scanning electron microscope. The external anatomy was additionally observed on more than 30 specimens. We found that the overall structure of the skeleton does not change much between juveniles and adults, but the shape of individual ossicle changes during growth. Allometric scaling was particularly visible on the orals, ambulacrals and adambulacrals. The shape of an ossicle is more dependent of its position along the arm series than of its individual size. Many morphological features differentiate progressively during ontogeny, while others are expressed consistently among specimens. The study of this ontogenetic series allows discussing the homology between the structures present on the ossicles of Z. fulgens in particular and other forcipulatacean sea stars in general (i.e. muscles insertions and articulation areas). The new data obtained in this study provide a comprehensive framework of the anatomy and ontogeny of Z. fulgens that will help resolve taxonomic and phylogenetic controversies in the future.

An integrated view of asteroid regeneration: tissues, cells and molecules.

The potential for repairing and replacing cells, tissues, organs and body parts is considered a primitive attribute of life shared by all the organisms, even though it may be expressed to a different extent and which is essential for the survival of both individual and whole species. The ability to regenerate is particularly evident and widespread within invertebrates. In spite of the wide availability of experimental models, regeneration has been comprehensively explored in only a few animal systems (i.e., hydrozoans, planarians, urodeles) leaving many other animal groups unexplored. The regenerative potential finds its maximum expression in echinoderms. Among echinoderm classes, asteroids offer an impressive range of experimental models in which to study arm regeneration at different levels. Many studies have been recently carried out in order to understand the regenerative mechanisms in asteroids and the overall morphological processes have been well documented in different starfish species, such as Asterias rubens, Leptasterias hexactis and Echinaster sepositus. In contrast, very little is known about the molecular mechanisms that control regeneration development and patterning in these models. The origin and the fate of cells involved in the regenerative process remain a matter of debate and clear insights will require the use of complementary molecular and proteomic approaches to study this problem. Here, we review the current knowledge regarding the cellular, proteomic and molecular aspects of asteroid regeneration.

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.

Visual navigation in starfish: first evidence for the use of vision and eyes in starfish.

Most known starfish species possess a compound eye at the tip of each arm, which, except for the lack of true optics, resembles an arthropod compound eye. Although these compound eyes have been known for about two centuries, no visually guided behaviour has ever been directly associated with their presence. There are indications that they are involved in negative phototaxis but this may also be governed by extraocular photoreceptors. Here, we show that the eyes of the coral-reef-associated starfish Linckia laevigata are slow and colour blind. The eyes are capable of true image formation although with low spatial resolution. Further, our behavioural experiments reveal that only specimens with intact eyes can navigate back to their reef habitat when displaced, demonstrating that this is a visually guided behaviour. This is, to our knowledge, the first report of a function of starfish compound eyes. We also show that the spectral sensitivity optimizes the contrast between the reef and the open ocean. Our results provide an example of an eye supporting only low-resolution vision, which is believed to be an essential stage in eye evolution, preceding the high-resolution vision required for detecting prey, predators and conspecifics.

New taxa and taxonomic revisions to the Poraniidae (Valvatacea; Asteroidea) with comments on feeding biology.

New molecular phylogenetic data and new specimens provide the basis for a revision of the family Poraniidae. We present molecular phylogenetic data for five out of 11 genera in the Poraniidae including a newly discovered taxon from the North Pacific. Bathyporania ascendens nov. gen., nov. sp., is described from Davidson Seamount (35º43'N, 122º43'W). Another newly discovered poraniid taxon, Clavaporania fitchorum nov. gen., nov. sp. is described from south of Macquarie Island (56º21'S, 158º 28'E) but was not included in the analysis. Revision of the Poraniidae has been undertaken. We present two new genera and reinstate the previously synonymized genus Glabraster and return Culcitopsis to genus level. The genus Porania sensu Clark (1993) and Clark and Downey (1992) is not monophyletic. Porania, Poraniomorpha and Poraniopsis are revised. In situ feeding observations of Bathyporania are described and compared with other poraniid feeding accounts.

Seasonal variation in body size and diet of the sea star Astropecten marginatus (Paxillosida, Astropectinidae) off coast of Paraná, Southern Brazil.

The sea star Astropecten marginatus has a neotropical distribution and is a highly abundant and frequent species in shrimp trawling by-catchin many places along the Brazilian coast. This has caused its threat to extinction and in addition, its bio-ecological aspects are poorly known. Thus, the main objective of this study was to analyze the seasonal variations of population length structure and feeding habits of the sea stars A. marginatus inhabiting off state of Paraná, Southern Brazil. The analyzed specimens were collected in February (summer), April (fall), June (winter) and October (spring) of 2008 from shrimp by-catch trawling. In the laboratory, each individual had its length measured and then weighed on an analytical scale. Afterwards, the stomach contents of 10 individuals of each of two most frequent length classes were seasonally analyzed. The relative frequency and abundance for each prey category was determined and, then combined into an index of alimentary importance. A total of 994 individuals of A. marginatus were collected with length ranging from 7.0 to 56.2mm, but most individuals were in the 20.1-25mm length classes. Individuals larger than 40mm were only collected in the spring while a few recruits (<10mm) were found in fall and winter. The total weight of individuals ranged from 0.1 to 15.3g and the weight-length relationships showed a negative allometric growth (b<2.54). Regarding its food consumption, this sea star explored eleven food items, with cumaceans and mollusks as the most frequent items. High frequency of empty stomach was recorded at fall. Seasonal differences in the amount explored preys and ingested items as well as in the prey composition were also observed. Higher amount of explored prey categories and ingested items were recorded at winter-spring than summer-fall periods. Predominance in prey category changed from gastropods (summer and fall) to cumaceans (winter and spring). The importance of gastropods as main prey category at summer and fall should be carefully considered since it was coincidently observed with high frequency of empty stomach and low amount of ingested items. The observed seasonal differences in feeding behavior pattern were mainly associated to low prey availability and to changes in the sea star feeding rates, and probably reflected in some biological traits such as small body size of the population inhabiting waters off the Paraná coast, Southern Brazil.

Intertidal sea stars (Pisaster ochraceus) alter body shape in response to wave action.

Sea stars are some of the largest mobile animals able to live in the harsh flow environment of wave-exposed, rocky intertidal shores. In addition, some species, such as the northeastern Pacific Pisaster ochraceus, are ecologically significant predators in a broad range of environments, from sheltered lagoons to the most wave-exposed shorelines. How they function and survive under such an extreme range of wave exposures remains a puzzle. Here we examine the ability of P. ochraceus to alter body form in response to variation in flow conditions. We found that sea stars in wave-exposed sites had narrower arms and were lighter per unit arm length than those from sheltered sites. Body form was tightly correlated with maximum velocity of breaking waves across four sites and also varied over time. In addition, field transplant experiments showed that these differences in shape were due primarily to phenotypic plasticity. Sea stars transplanted from a sheltered site to a more wave-exposed site became lighter per unit arm length, and developed narrower arms, after 3 months. The tight correlation between water flow and morphology suggests that wave force must be a significant selective factor acting on body shape. On exposed shores, narrower arms probably reduce both lift and drag in breaking waves. On protected shores, fatter arms may provide more thermal inertia to resist overheating, or more body volume for gametes. Such plastic changes in body shape represent a unique method by which sea stars adapt to spatial, seasonal and possibly short-term variation in flow conditions.

Survival and arm abscission are linked to regional heterothermy in an intertidal sea star.

Body temperature is a more pertinent variable to physiological stress than ambient air temperature. Modeling and empirical studies on the impacts of climate change on ectotherms usually assume that body temperature within organisms is uniform. However, many ectotherms show significant within-body temperature heterogeneity. The relationship between regional heterothermy and the response of ectotherms to sublethal and lethal conditions remains underexplored. We quantified within-body thermal heterogeneity in an intertidal sea star (Pisaster ochraceus) during aerial exposure at low tide to examine the lethal and sublethal effects of temperatures of different body regions. In manipulative experiments, we measured the temperature of the arms and central disc, as well as survival and arm abscission under extreme aerial conditions. Survival was related strongly to central disc temperature. Arms were generally warmer than the central disc in individuals that survived aerial heating, but we found the reverse in those that died. When the central disc reached sublethal temperatures of 31-35°C, arms reached temperatures of 33-39°C, inducing arm abscission. The absolute temperature of individual arms was a poor predictor of arm abscission, but the arms lost were consistently the hottest at the within-individual scale. Therefore, the vital region of this sea star may remain below the lethal threshold under extreme conditions, possibly through water movement from the arms to the central disc and/or evaporative cooling, but at the cost of increased risk of arm abscission. Initiation of arm abscission seems to reflect a whole-organism response while death occurs as a result of stress acting directly on central disc tissues.

Asterinid seastars from the Mozambique Channel (Echinodermata: Asteroidea: Asterinidae).

A new fissiparous asterinid seastar Aquilonastra chantalae sp. nov. is described from Europa Island, one of the Scattered Islands (Les îles Éparses) in the Mozambique Channel. It is compared with the fissiparous asterinid Aquilonastra conandae O'Loughlin & Rowe from the Mascarene Islands. A table of distinguishing diagnostic characters is provided. The asterinid Aquilonastra richmondi O'Loughlin & Rowe is reported for Europa Island.

Eyes, Vision, and Bioluminescence in Deep-Sea Brisingid Sea Stars.

AbstractSea stars are a major component of the megabenthos in most marine habitats, including those within the deep sea. Being radially symmetric, sea stars have sensory structures that are evenly distributed along the arms, with a compound eye located on each arm tip of most examined species. Surprisingly, eyes with a spatial resolution that rivals the highest acuity known among sea stars so far were recently found in Novodinia americana, a member of the deep-sea sea star order Brisingida. Here, we examined 21 species across 11 brisingid genera for the presence of eyes; where eyes were present, we used morphological characteristics to evaluate spatial resolution and sensitivity. This study found that eyes were present within 43% of the examined species. These brisingid eyes were relatively large compared to those of other deep-sea sea stars, with a high number of densely packed ommatidia. One of the examined species, Brisingaster robillardi, had more than 600 ommatidia per eye, which is the highest number of ommatidia found in any sea star eye so far. Combined, the results indicate that brisingid eyes are adapted for spatial resolution over sensitivity. Together with results showing that many brisingids are bioluminescent, this relatively high spatial resolution suggests that the group may use their eyes to support visually guided intraspecific communication based on bioluminescent signals. Phylogenetic analysis indicated that the common ancestor of brisingids had eyes (P = 0.72) and that eyes were lost once within the clade.

Echinoderm phylogeny including Xyloplax, a progenetic asteroid.

Reconstruction of the phylogeny of the five extant classes of the phylum Echinodermata has proven difficult. Results concerning higher-level taxonomic relationships among echinoderms are sensitive to the choice of analytical parameters and methods. Moreover, the proposal of a putative sixth class based on a small enigmatic disc-shaped echinoderm, Xyloplax, from the deep seas of the Bahamas and New Zealand in the 1980s further complicated the problem. Although clearly an echinoderm, Xyloplax did not have clear affinity among known groups. Using molecular sequence and developmental data from recently collected Xyloplax adults and embryos, we show that rather than representing an ancient distinct lineage as implied by its status as a class, Xyloplax is simply a starfish that is closely related to the asteroid family Pterasteridae. Many members of the Pterasteridae and all Xyloplax inhabit deep or polar seas and brood young. Brooding pterasterids and Xyloplax hold their young in specialized adult chambers until the young reach an advanced juvenile stage after which they are released as free-living individuals. We hypothesize that the unique morphology of Xyloplax evolved via progenesis--the truncation of somatic growth at a juvenile body plan but with gonadal growth to maturity. Although the overall phylogeny of extant echinoderms remains sensitive to the choice of analytical methods, the placement of Xyloplax as sister to pterasterid asteroids is unequivocal. Based on this, we argue that the proposed class and infraclass status of Xyloplax should be suppressed.

[Distribution, density and size structure of Oreaster reticulatus and Luidia senegalensis (Echinodermata: Asteroidea) in Cubagua Island, Venezuela]. / Distribución, densidad y estructura de talla de Oreaster reticulatus y Luidia senegalensis (Echinodermata: Asteroidea) en isla de Cubagua, Venezuela.

There is limited biological information about the starfish Oreaster reticulatus and the nine-armed starfish Luidia senegalensis in Venezuelan waters. These species are currently considered threatened in many localities of the Caribbean, Brazil and Colombia. Therefore, the aim of this study was to describe their population density, size distribution and population structure in Cubagua Island. During 2008, 52 stations located around the island were evaluated using band transects of 50m2. Each organism was counted and measured (maximum radius), and its size was compared to the maturity length reported in the literature. The results for O. reticulatus include: 167.3ind./ha; 33% juveniles and 67% adults; average size of 10.7 +/- 5cm (range: 2.2-21cm); a wide distribution around the island, with higher densities in the Eastern and Southwest areas. The 50% of the specimens were found in seagrass beds, 25% in sand, 16% in areas covered by decomposed seaweeds, 9% in oyster beds and 1% coralline patches. The densities of O. reticulatus were higher than those reported in the Caribbean, Panama and Puerto Rico, but lower than Venezuelan National Parks: Mochima and Morrocoy; as well as in the Virgin Islands and Belize. For L. senegalensis we found an average density of 40ind./ha; 95% exceeded the reported size at maturity, with mean length of 12cm +/- 3.5cm (range: 3.5-22.3cm); they were found only in 15% of the stations of which 92.5% were sandy bottoms and the other 7.5% oyster beds. The degree of aggregation of L. senegalensis was greater than O. reticulatus, with an estimated k of 0.06. However, it was not possible to compare the densities of L. senegalensis with any other study. For both species is recommended to carry out reproductive studies and to monitor their population densities to infer temporal variations.

A taxonomic review of the genus Astrocharis Koehler (Echinodermata: Ophiuroidea: Asteroschematidae), with a description of a new species.

A revision of the genus Astrocharis Koehler (Echinodermata: Ophiuroidea: Asteroschematidae) is based on 41 specimens, including three syntypes of Astrocharis virgo Koehler, one syntype of Astrocharis ijimai Matsumoto and the holotype of Astrocharis gracilis Mortensen. Astrocharis gracilis is a junior subjective synonym of A. ijimai. A. virgo and A. ijimai are redescribed. A new species, Astrocharis monospinosa is described from southwestern Japan. A tabular key to the three species of the genus Astrocharis is provided.

Sea stars generate downforce to stay attached to surfaces.

Intertidal sea stars often function in environments with extreme hydrodynamic loads that can compromise their ability to remain attached to surfaces. While behavioral responses such as burrowing into sand or sheltering in rock crevices can help minimize hydrodynamic loads, previous work shows that sea stars also alter body shape in response to flow conditions. This morphological plasticity suggests that sea star body shape may play an important hydrodynamic role. In this study, we measured the fluid forces acting on surface-mounted sea star and spherical dome models in water channel tests. All sea star models created downforce, i.e., the fluid pushed the body towards the surface. In contrast, the spherical dome generated lift. We also used Particle Image Velocimetry (PIV) to measure the midplane flow field around the models. Control volume analyses based on the PIV data show that downforce arises because the sea star bodies serve as ramps that divert fluid away from the surface. These observations are further rationalized using force predictions and flow visualizations from numerical simulations. The discovery of downforce generation could explain why sea stars are shaped as they are: the pentaradial geometry aids attachment to surfaces in the presence of high hydrodynamic loads.

Preliminary study of the population and reproductive dynamics of Echinaster sepositus (Spinulosida: Echinasteridae) in Cala del Racó / Estudio preliminar de la población y dinámica reproductiva de Echinaster sepositus (Spinulosida: Echinasteridae) en Cala del Racó

Abstract Introduction: The red starfish (Echinaster sepositus) is one of the most common asteroid species in the Mediterranean Sea. However, information about their biology or their role in benthic communities is scarce. Objective: This study aims to provide new information on the ecology of this species through the temporal characterization of the population of E. sepositus in Cala del Racó (Alicante, Spain) and the in situ monitoring of its reproductive cycle. Methods: For this purpose, three study areas were established at different depths. For each of the recorded starfish, data about the size, the substrate on which it was found, the area, the depth and the sex in the case of observing the reproduction were collected. Results: A total of 19 samplings have been carried out throughout a year of study. In this way, it has been possible to observe that the density of individuals increases in the shallower zone during autumn and winter, when the temperature drops to 14.13 ºC, while it decreases in spring and summer when the temperature rises to 27.17 ºC. Those results are reversed in the deepest part of the study. The highest density of individuals (0.51 ind/m2) occurred in October. Arborescent photophilic algae and crustose coralline algae were the substrates with the highest number of E. sepositus recorded. Medium to large specimens are located preferably on crustose coralline algae or arborescent photophilic algae, while smaller individuals were mostly located on Posidonia oceanica. No specimens of E. sepositus were observed spawning. Conclusions: Data leads to assume that there is a migration of starfishes towards more superficial areas when the water is at colder temperature and towards deeper areas when the temperature increases. It is valued the possibility that there is a change in the nutritional needs of E. sepositus throughout its development. According to our observations, the future reproduction studies should be concentrated between late-summer and early-autumn.

Resumen Introducción: La estrella de mar roja (Echinaster sepositus) es una de las especies de asteroideos más comunes del mar Mediterráneo. Sin embargo, la información sobre su biología o su papel en las comunidades bentónicas es escasa. Objetivo: Este estudio pretende aportar nueva información sobre la ecología de esta especie mediante la caracterización temporal de la población de E. sepositus en la Cala del Racó (Alicante, España) y el monitoreo in situ de su ciclo reproductivo. Métodos: Con este fin se establecieron tres zonas de estudio a distintas profundidades. Para cada una de las estrellas registradas se tomaron datos de tamaño, el sustrato sobre el que se encuentra, la zona, la profundidad y el sexo en caso de observar la reproducción. Resultados: A lo largo de un año de estudio se han realizado un total de 19 muestreos. De esta forma se ha podido observar que la densidad de individuos aumenta en la zona menos profunda durante otoño e invierno, cuando la temperatura del agua baja hasta los 14.13 ºC, mientras que se reduce en primavera y verano, cuando la temperatura se eleva hasta los 27.17 ºC. Este resultado se invierte en la zona más profunda del estudio. La mayor densidad de individuos ha sido observada en octubre (0.51 ind/m2). Las algas fotófilas arborescentes y las algas coralinales costrosas han sido los sustratos con un mayor número de E. sepositus registrados. Los ejemplares de tamaños medianos a grandes se localizan preferentemente sobre algas coralinales costrosas o algas fotófilas arborescentes, mientras que los individuos de menor tamaño se sitúan mayormente sobre Posidonia oceanica. No se observaron ejemplares de E. sepositus reproduciéndose. Conclusiones: Los datos permiten presuponer que existe una migración entre las zonas más superficiales, cuando la temperatura del agua es menor, y zonas más profundas cuando la temperatura aumenta. Se valora la posibilidad de la existencia de un cambio en los requerimientos nutricionales de E. sepositus a lo largo de su desarrollo. De acuerdo con nuestras observaciones, los estudios futuros sobre la reproducción de esta especie deben concentrarse entre finales de verano y principios de otoño.

Abbreviated Development of the Brooding Brittle Star Ophioplocus esmarki.

The bilaterally symmetrical, feeding larval stage is an ancestral condition in echinoderms. However, many echinoderms have evolved abbreviated development and form a pentamerous juvenile without a feeding larva. Abbreviated development with a non-feeding vitellaria larva is found in five families of brittle stars, but very little is known about this type of development. In this study, the external anatomy, ciliary bands, neurons, and muscles were examined in the development of the brooded vitellaria larva of Ophioplocus esmarki. The external morphology throughout development shows typical vitellaria features, including morphogenetic movements to set up the vitellaria body plan, an anterior preoral lobe, a posterior lobe, transverse ciliary bands, and development of juvenile structures on the mid-ventral side. An early population of neurons forms at the base of the preoral lobe at the pre-vitellaria stage after the initial formation of the coelomic cavities. These early neurons may be homologous to the apical neurons that develop in echinoderms with feeding larval forms. Neurons form close to the ciliary bands, but the vitellaria larva lacks the tracts of neurons associated with the ciliary bands found in echinoderms with feeding larvae. Additional neurons form in association with the axial complex and persist into the juvenile stage. Juvenile nerves and muscles form with pentamerous symmetry in the late vitellaria stage in a manner similar to their development within the late ophiopluteus larva. Even though O. esmarki is a brooding brittle star, its developmental sequence retains the general vitellaria shape and structure; however, the vitellaria larvae are unable to swim in the water column.

High-Throughput Segmentation of Tiled Biological Structures using Random-Walk Distance Transforms.

Various 3D imaging techniques are routinely used to examine biological materials, the results of which are usually a stack of grayscale images. In order to quantify structural aspects of the biological materials, however, they must first be extracted from the dataset in a process called segmentation. If the individual structures to be extracted are in contact or very close to each other, distance-based segmentation methods utilizing the Euclidean distance transform are commonly employed. Major disadvantages of the Euclidean distance transform, however, are its susceptibility to noise (very common in biological data), which often leads to incorrect segmentations (i.e., poor separation of objects of interest), and its limitation of being only effective for roundish objects. In the present work, we propose an alternative distance transform method, the random-walk distance transform, and demonstrate its effectiveness in high-throughput segmentation of three microCT datasets of biological tilings (i.e., structures composed of a large number of similar repeating units). In contrast to the Euclidean distance transform, the random-walk approach represents the global, rather than the local, geometric character of the objects to be segmented and, thus, is less susceptible to noise. In addition, it is directly applicable to structures with anisotropic shape characteristics. Using three case studies-tessellated cartilage from a stingray, the dermal endoskeleton of a starfish, and the prismatic layer of a bivalve mollusc shell-we provide a typical workflow for the segmentation of tiled structures, describe core image processing concepts that are underused in biological research, and show that for each study system, large amounts of biologically-relevant data can be rapidly segmented, visualized, and analyzed.