A Review of Asteroid Biology in the Context of Sea Star Wasting: Possible Causes and Consequences.

AbstractSea star wasting-marked in a variety of sea star species as varying degrees of skin lesions followed by disintegration-recently caused one of the largest marine die-offs ever recorded on the west coast of North America, killing billions of sea stars. Despite the important ramifications this mortality had for coastal benthic ecosystems, such as increased abundance of prey, little is known about the causes of the disease or the mechanisms of its progression. Although there have been studies indicating a range of causal mechanisms, including viruses and environmental effects, the broad spatial and depth range of affected populations leaves many questions remaining about either infectious or non-infectious mechanisms. Wasting appears to start with degradation of mutable connective tissue in the body wall, leading to disintegration of the epidermis. Here, we briefly review basic sea star biology in the context of sea star wasting and present our current knowledge and hypotheses related to the symptoms, the microbiome, the viruses, and the associated environmental stressors. We also highlight throughout the article knowledge gaps and the data needed to better understand sea star wasting mechanistically, its causes, and potential management.

Culturing echinoderm larvae through metamorphosis.

Echinoderms are favored study organisms not only in cell and developmental biology, but also physiology, larval biology, benthic ecology, population biology and paleontology, among other fields. However, many echinoderm embryology labs are not well-equipped to continue to rear the post-embryonic stages that result. This is unfortunate, as such labs are thus unable to address many intriguing biological phenomena, related to their own cell and developmental biology studies, that emerge during larval and juvenile stages. To facilitate broader studies of post-embryonic echinoderms, we provide here our collective experience rearing these organisms, with suggestions to try and pitfalls to avoid. Furthermore, we present information on rearing larvae from small laboratory to large aquaculture scales. Finally, we review taxon-specific approaches to larval rearing through metamorphosis in each of the four most commonly-studied echinoderm classes-asteroids, echinoids, holothuroids and ophiuroids.

Brief exposure to intense turbulence induces a sustained life-history shift in echinoids.

In coastal ecosystems, attributes of fluid motion can prompt animal larvae to rise or sink in the water column and to select microhabitats within which they attach and commit to a benthic existence. In echinoid (sea urchin and sand dollar) larvae living along wave-exposed shorelines, intense turbulence characteristic of surf zones can cause individuals to undergo an abrupt life-history shift characterized by precocious entry into competence - the stage at which larvae will settle and complete metamorphosis in response to local cues. However, the mechanistic details of this turbulence-triggered onset of competence remain poorly defined. Here, we evaluate in a series of laboratory experiments the time course of this turbulence effect, both the rapidity with which it initiates and whether it perdures. We found that larvae become competent with turbulence exposures as brief as 30 s, with longer exposures inducing a greater proportion of larvae to become competent. Intriguingly, larvae can remember such exposures for a protracted period (at least 24 h), a pattern reminiscent of long-term potentiation. Turbulence also induces short-term behavioral responses that last less than 30 min, including cessation of swimming, that facilitate sinking and thus contact of echinoid larvae with the substratum. Together, these results yield a novel perspective on how larvae find their way to suitable adult habitat at the critical settlement transition, and also open new experimental opportunities to elucidate the mechanisms by which planktonic animals respond to fluid motion.

Sand Dollar Larvae Show Within-Population Variation in Their Settlement Induction by Turbulence.

Settlement-the generally irreversible transition from a planktonic phase to a benthic phase-is a critical stage in the life history of many shoreline organisms. It is reasonable to expect that larvae are under intense selection pressure to identify appropriate settlement habitat. Several decades of studies have focused mainly on local indicators that larvae use to identify suitable habitat, such as olfactory cues that indicate the presence of conspecifics or a favored food source. Our recent work has shown that the larvae of seashore-dwelling echinoids (sea urchins, sand dollars, and kin) can be primed to settle following a brief exposure to a broader-scale indicator of their approach to shore: an increase in fluid turbulence. Here we demonstrate that this priming shows within-population variation: the offspring of certain Pacific sand dollar (Dendraster excentricus) parents-both specific fathers and specific mothers, regardless of the other parent-are more responsive to turbulence than others. In particular, the observation of the effect correlating, in some cases, with specific fathers leads us to conclude that these behavioral differences are likely genetic and thus heritable. We also report that turbulence exposure causes larvae to temporarily sink to the bottom of a container of seawater and that larvae that respond in this way are also more likely to subsequently settle. We hypothesize a two-step scenario for the evolution of turbulence responsiveness at settlement and suggest that the evolutionary origin of these behaviors could be a driving force for population differentiation and speciation.

A newly identified left-right asymmetry in larval sea urchins.

Directional asymmetry (DA) in body form is a widespread phenomenon in animals and plants alike, and a functional understanding of such asymmetries can offer insights into the ways in which ecology and development interface to drive evolution. Echinoids (sea urchins, sand dollars and their kin) with planktotrophic development have a bilaterally symmetrical feeding pluteus larva that undergoes a dramatic metamorphosis into a pentameral juvenile that enters the benthos at settlement. The earliest stage of this transformation involves a DA: a left-side invagination in mid-stage larvae leads to the formation of the oral field of the juvenile via a directionally asymmetric structure called the echinus rudiment. Here, we show for the first time in two echinoid species that there is a corresponding DA in the overall shape of the larva: late-stage plutei have consistently shorter arms specifically on the rudiment (left) side. We then demonstrate a mechanistic connection between the rudiment and arm length asymmetries by examining rare, anomalous purple urchin larvae that have rudiments on both the left and the right side. Our data suggest that this asymmetry is probably a broadly shared feature characterizing ontogeny in the class Echinoidea. We propose several functional hypotheses-including developmental constraints and water column stability-to account for this newly identified asymmetry.

Rethinking competence in marine life cycles: ontogenetic changes in the settlement response of sand dollar larvae exposed to turbulence.

Complex life cycles have evolved independently numerous times in marine animals as well as in disparate algae. Such life histories typically involve a dispersive immature stage followed by settlement and metamorphosis to an adult stage on the sea floor. One commonality among animals exhibiting transitions of this type is that their larvae pass through a 'precompetent' period in which they do not respond to localized settlement cues, before entering a 'competent' period, during which cues can induce settlement. Despite the widespread existence of these two phases, relatively little is known about how larvae transition between them. Moreover, recent studies have blurred the distinction between the phases by demonstrating that fluid turbulence can spark precocious activation of competence. Here, we further investigate this phenomenon by exploring how larval interactions with turbulence change across ontogeny, focusing on offspring of the sand dollar Dendraster excentricus (Eschscholtz). Our data indicate that larvae exhibit increased responsiveness to turbulence as they get older. We also demonstrate a likely cost to precocious competence: the resulting juveniles are smaller. Based upon these findings, we outline a new, testable conception of competence that has the potential to reshape our understanding of larval dispersal and connectivity among marine populations.

Manipulation of developing juvenile structures in purple sea urchins (Strongylocentrotus purpuratus) by morpholino injection into late stage larvae.

Sea urchins have been used as experimental organisms for developmental biology for over a century. Yet, as is the case for many other marine invertebrates, understanding the development of the juveniles and adults has lagged far behind that of their embryos and larvae. The reasons for this are, in large part, due to the difficulty of experimentally manipulating juvenile development. Here we develop and validate a technique for injecting compounds into juvenile rudiments of the purple sea urchin, Strongylocentrotus purpuratus. We first document the distribution of rhodaminated dextran injected into different compartments of the juvenile rudiment of sea urchin larvae. Then, to test the potential of this technique to manipulate development, we injected Vivo-Morpholinos (vMOs) designed to knock down p58b and p16, two proteins involved in the elongation of S. purpuratus larval skeleton. Rudiments injected with these vMOs showed a delay in the growth of some juvenile skeletal elements relative to controls. These data provide the first evidence that vMOs, which are designed to cross cell membranes, can be used to transiently manipulate gene function in later developmental stages in sea urchins. We therefore propose that injection of vMOs into juvenile rudiments, as shown here, is a viable approach to testing hypotheses about gene function during development, including metamorphosis.

A detailed staging scheme for late larval development in Strongylocentrotus purpuratus focused on readily-visible juvenile structures within the rudiment.

BACKGROUND: The purple sea urchin, Strongylocentrotus purpuratus, has long been the focus of developmental and ecological studies, and its recently-sequenced genome has spawned a diversity of functional genomics approaches. S. purpuratus has an indirect developmental mode with a pluteus larva that transforms after 1-3 months in the plankton into a juvenile urchin. Compared to insects and frogs, mechanisms underlying the correspondingly dramatic metamorphosis in sea urchins remain poorly understood. In order to take advantage of modern techniques to further our understanding of juvenile morphogenesis, organ formation, metamorphosis and the evolution of the pentameral sea urchin body plan, it is critical to assess developmental progression and rate during the late larval phase. This requires a staging scheme that describes developmental landmarks that can quickly and consistently be used to identify the stage of individual living larvae, and can be tracked during the final two weeks of larval development, as the juvenile is forming. RESULTS: Notable structures that are easily observable in developing urchin larvae are the developing spines, test and tube feet within the juvenile rudiment that constitute much of the oral portion of the adult body plan. Here we present a detailed staging scheme of rudiment development in the purple urchin using soft structures of the rudiment and the primordia of these juvenile skeletal elements. We provide evidence that this scheme is robust and applicable across a range of temperature and feeding regimes. CONCLUSIONS: Our proposed staging scheme provides both a useful method to study late larval development in the purple urchin, and a framework for developing similar staging schemes across echinoderms. Such efforts will have a high impact on evolutionary developmental studies and larval ecology, and facilitate research on this important deuterostome group.

Turbulent shear spurs settlement in larval sea urchins.

Marine invertebrates commonly produce larvae that disperse in ocean waters before settling into adult shoreline habitat. Chemical and other seafloor-associated cues often facilitate this latter transition. However, the range of effectiveness of such cues is limited to small spatial scales, creating challenges for larvae in finding suitable sites at which to settle, especially given that they may be carried many kilometers by currents during their planktonic phase. One possible solution is for larvae to use additional, broader-scale environmental signposts to first narrow their search to the general vicinity of a candidate settlement location. Here we demonstrate strong effects of just such a habitat-scale cue, one with the potential to signal larvae that they have arrived in appropriate coastal areas. Larvae of the purple sea urchin (Strongylocentrotus purpuratus) exhibit dramatic enhancement in settlement following stimulation by turbulent shear typical of wave-swept shores where adults of this species live. This response manifests in an unprecedented fashion relative to previously identified cues. Turbulent shear does not boost settlement by itself. Instead, it drives a marked developmental acceleration that causes "precompetent" larvae refractory to chemical settlement inducers to immediately become "competent" and thereby reactive to such inducers. These findings reveal an unrecognized ability of larval invertebrates to shift the trajectory of a major life history event in response to fluid-dynamic attributes of a target environment. Such an ability may improve performance and survival in marine organisms by encouraging completion of their life cycle in advantageous locations.

Interspecific variation in metamorphic competence in marine invertebrates: the significance for comparative investigations into the timing of metamorphosis.

Metamorphosis in marine invertebrate larvae is a dynamic, environmentally dependent process that integrates ontogeny with habitat selection. The capacity of many marine invertebrate larvae to survive and maintain metamorphic competence in the absence of environmental cues has been hypothesized to be an adaptive convergence (Hadfield and others 2001). A survey of the literature reveals that a single generalized hypothesis about metamorphic competence as an adaptive convergence is not sufficient to account for interspecific variation in this character. In an attempt to capture this variation, we discuss the "desperate larva hypothesis" and propose two additional hypotheses called the "variable retention hypothesis" and the "death before dishonor hypothesis." To validate these additional hypotheses we collected data on taxa from the published literature and performed a contingency analysis to detect correlations between spontaneous metamorphosis, habitat specificity and/or larval life-history mode, three characters relevant to environmentally induced settlement and metamorphosis. In order to account for phylogenetic bias in these correlations, we also constructed a phylogeny of these taxa and again performed a character-correlation analysis. Both these tests suggest that juvenile habitat specificity is correlated to the capacity of individuals to retain the competent larval state in the absence of substrate cues and therefore validate the existence of more than one hypothesis about metamorphic competence. We provide new data from the sea urchin Lytechinus pictus that suggest that nitric oxide (NO) and thyroxine hormone signaling interact to determine the probability of settlement in response to a settlement cue. Similarly, we provide evidence that thyroxine signaling in the sand dollar Dendraster excentricus increases spontaneous metamorphosis in the absence of cues from adult conspecifics in a manner that is independent of larval age.

Expanding networks: Signaling components in and a hypothesis for the evolution of metamorphosis.

Metamorphosis is a substantial morphological transition between 2 multicellular phases in an organism's life cycle, often marking the passage from a prereproductive to a reproductive life stage. It generally involves major physiological changes and a shift in habitat and feeding mode, and can be subdivided into an extended phase of substantial morphological change and/or remodeling, and a shorter-term phase (for example, marine invertebrate "settlement," insect "adult eclosion," mushroom fruiting body emergence) where the actual habitat shift occurs. Disparate metamorphic taxa differ substantially with respect to when the habitat shift occurs relative to the timing of the major events of morphogenetic change. I will present comparative evidence across a broad taxonomic scope suggesting that longer-term processes (morphogenetic changes) are generally hormonally regulated, whereas nitric oxide (NO) repressive signaling often controls the habitat shift itself. Furthermore, new evidence from echinoids (sea urchins, sand dollars) indicates a direct connection between hormonal and NO signaling during metamorphosis. I incorporate 2 hypotheses for the evolution of metamorphosis-one involving heterochrony, the other involving phenotypic integration and evolutionarily stable configurations (ESCs)-into a network model for metamorphosis in echinoderms (sea urchins, starfish, and their kin). Early indications are that this core regulatory network can be acted upon by natural selection to suit the diverse ecological needs of disparate metamorphic organisms, resulting in evolutionary expansions and contractions in the core network. I briefly speculate on the ways that exposure to xenobiotic pollutants and other compounds might influence successful settlement of juveniles in the wild. Indeed, environmentally regulated life history transitions-such as settlement, metamorphosis, and reproductive maturation-may be developmental periods that are especially sensitive to such pollutants.

Hormone signaling in evolution and development: a non-model system approach.

Cooption and modularity are informative concepts in evolutionary developmental biology. Genes function within complex networks that act as modules in development. These modules can then be coopted in various functional and evolutionary contexts. Hormonal signaling, the main focus of this review, has a modular character. By regulating the activities of genes, proteins and other cellular molecules, a hormonal signal can have major effects on physiological and ontogenetic processes within and across tissues over a wide spatial and temporal scale. Because of this property, we argue that hormones are frequently involved in the coordination of life history transitions (LHTs) and their evolution (LHE). Finally, we promote the usefulness of a comparative, non-model system approach towards understanding how hormones function and guide development and evolution, highlighting thyroid hormone function in echinoids as an example.

Thyroid hormones determine developmental mode in sand dollars (Echinodermata: Echinoidea).

Evolutionary transitions in larval nutritional mode have occurred on numerous occasions independently in many marine invertebrate phyla. Although the evolutionary transition from feeding to nonfeeding development has received considerable attention through both experimental and theoretical studies, mechanisms underlying the change in life history remain poorly understood. Facultative feeding larvae (larvae that can feed but will complete metamorphosis without food) presumably represent an intermediate developmental mode between obligate feeding and nonfeeding. Here we show that an obligatorily feeding larva can be transformed into a facultative feeding larva when exposed to the thyroid hormone thyroxine. We report that larvae of the subtropical sand dollar Leodia sexiesperforata (Echinodermata: Echinoidea) completed metamorphosis without exogenous food when treated with thyroxine, whereas the starved controls (no thyroxine added) did not. Leodia sexiesperforata juveniles from the thyroxine treatment were viable after metamorphosis but were significantly smaller and contained less energy than sibling juveniles reared with exogenous food. In a second starvation experiment, using an L. sexiesperforata female whose eggs were substantially larger than in the first experiment (202+/-5 vs. 187+/-5 microm), a small percentage of starved L. sexiesperforata larvae completed metamorphosis in the absence of food. Still, thyroxine-treated larvae in this experiment completed metamorphosis faster and in much higher numbers than in the starved controls. Furthermore, starved larvae of the sand dollar Mellita tenuis, which developed from much smaller eggs (100+/-2 microm), did not complete metamorphosis either with or without excess thyroxine. Based on these data, and from recent experiments with other echinoids, we hypothesize that thyroxine plays a major role in echinoderm metamorphosis and the evolution of life history transitions in this group. We discuss our results in the context of current life history models for marine invertebrates, emphasizing the role of egg size, juvenile size, and endogenous hormone production for the evolution of nonfeeding larval development.

Heterochronic developmental shift caused by thyroid hormone in larval sand dollars and its implications for phenotypic plasticity and the evolution of nonfeeding development.

Recent work on a diverse array of echinoderm species has demonstrated, as is true in amphibians, that thyroid hormone (TH) accelerates development to metamorphosis. Interestingly, the feeding larvae of several species of sea urchins seem to obtain TH through their diet of planktonic algae (exogenous source), whereas nonfeeding larvae of the sand dollar Peronella japonica produce TH themselves (endogenous source). Here we examine the effects of TH (thyroxine) and a TH synthesis inhibitor (thiourea) on the development of Dendraster excentricus, a sand dollar with a feeding larva. We report reduced larval skeleton lengths and more rapid development of the juvenile rudiment in the exogenous TH treatments when compared to controls. Also, larvae treated with exogenous TH reached metamorphic competence faster at a significantly reduced juvenile size, representing the greatest reduction in juvenile size ever reported for an echinoid species with feeding larvae. These effects of TH on D. excentricus larval development are strikingly similar to the phenotypically plastic response of D. excentricus larvae reared under high food conditions. We hypothesize that exogenous (algae-derived) TH is the plasticity cue in echinoid larvae, and that the larvae use ingested TH levels as an indicator for larval nutrition, ultimately signaling the attainment of metamorphic competence. Furthermore, our experiments with the TH synthesis inhibitor thiourea indicate that D. excentricus larvae can produce some TH endogenously. Endogenous TH production might, therefore, be a shared feature among sand dollars, facilitating the evolution of nonfeeding larval development in that group. Mounting evidence on the effects of thyroid hormones in echinoderm development suggests life-history models need to incorporate metamorphic hormone effects and the evolution of metamorphic hormone production.