Onset of feeding in juvenile sea urchins and its relation to nutrient signalling

The purple sea urchin, Strongylocentrotus purpuratus, has been the focus of extensive ecological and developmental research over the years. Strongylocentrotus purpuratus larvae transition into the juvenile stage after an extensive planktonic period. The metamorphic transition is characterized by dramatic changes in morphology and physiology of the juvenile compared to the larval form and mechanisms underlying this process, especially the early days post-settlement, remain poorly understood. We used SEM and phalloidin stain analysis as well as whole mount in situ hybridization to gain a detailed understanding of juvenile development in relation to nutrient signalling [insulin-like growth factor (IIS), FoxO (forkhead box, sub-group ‘O’) and TOR (target of rampamycin), also known as IIS/TOR/FoxO signalling]. Our results show that the majority of juvenile feeding features are fully developed only after 8-days of juvenile development, leaving an extensive period of nutritional stress. We found that FoxO gene expression increases during that time period and is localized in juvenile tube feet, potentially associated with sensory structures involved in nutrient signalling. Our data complement existing work on sea urchin juvenile development and shed new light on the perimetamorphic period of Strongylocentrotus purpuratus, with respect to nutrient signalling and the potential stressful pre-feeding period of juvenile sea urchins.     

Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

Background

A metamorphic life-history is present in the majority of animal phyla. This developmental mode is particularly prominent among marine invertebrates with a bentho-planktonic life cycle, where a pelagic larval form transforms into a benthic adult. Metamorphic competence (the stage at which a larva is capable to undergo the metamorphic transformation and settlement) is an important adaptation both ecologically and physiologically. The competence period maintains the larval state until suitable settlement sites are encountered, at which point the larvae settle in response to settlement cues. The mechanistic basis for metamorphosis (the morphogenetic transition from a larva to a juvenile including settlement), i.e. the molecular and cellular processes underlying metamorphosis in marine invertebrate species, is poorly understood. Histamine (HA), a neurotransmitter used for various physiological and developmental functions among animals, has a critical role in sea urchin fertilization and in the induction of metamorphosis. Here we test the premise that HA functions as a developmental modulator of metamorphic competence in the sea urchin Strongylocentrotus purpuratus.

Results

Our results provide strong evidence that HA leads to the acquisition of metamorphic competence in S. purpuratus larvae. Pharmacological analysis of several HA receptor antagonists and an inhibitor of HA synthesis indicates a function of HA in metamorphic competence as well as programmed cell death (PCD) during arm retraction. Furthermore we identified an extensive network of histaminergic neurons in pre-metamorphic and metamorphically competent larvae. Analysis of this network throughout larval development indicates that the maturation of specific neuronal clusters correlates with the acquisition of metamorphic competence. Moreover, histamine receptor antagonist treatment leads to the induction of caspase mediated apoptosis in competent larvae.

Conclusions

We conclude that HA is a modulator of metamorphic competence in S. purpuratus development and hypothesize that HA may have played an important role in the evolution of settlement strategies in echinoids. Our findings provide novel insights into the evolution of HA signalling and its function in one of the most important and widespread life history transitions in the animal kingdom - metamorphosis.     

Evolutionary changes in sites and timing of actin gene expression in embryos of the direct- and indirect-developing sea urchins, Heliocidaris erythrogramma and H. tuberculata

 We describe an evolutionary comparison of expression of the actin gene families of two congeneric sea urchins. Heliocidaris tuberculata develops indirectly via a planktonic feeding pluteus that forms a juvenile rudiment after a long period of larval development. H. erythrogramma is a direct developer that initiates formation of a juvenile rudiment immediately following gastrulation. The developmental expression of each actin isoform of both species was determined by in situ hybridization. The observed expression patterns are compared with known expression patterns in a related indirect-developing sea urchin, Strongylocentrotus purpuratus. Comparisons reveal unexpected patterns of conserved and divergent expression. Cytoplasmic actin, CyIII, is expressed in the aboral ectoderm cells of the indirect developers, but is an unexpressed pseudogene in H. erythrogramma, which lacks aboral ectoderm. This change is correlated with developmental mode. Two CyII actins are expressed in S. purpuratus, and one in H. erythrogramma, but no CyII is expressed in H. tuberculata despite its great developmental similarity to S. purpuratus. CyI expression differs slightly between Heliocidaris and Strongylocentrotus with more ectodermal expression in Heliocidaris. Evolutionary changes in actin gene expression reflect both evolution of developmental mode as well as a surprising flexibility in gene expression within a developmental mode. Received: 27 July 1997 / Accepted: 30 December 1997     

Predation of the sea urchin Heliocidaris erythrogramma by rock lobsters (Jasus edwardsii) in no-take marine reserves

The formation of sea urchin ‘barrens’ on shallow temperate rocky reefs is well documented. However there has been much conjecture about the underlying mechanisms leading to sea urchin barrens, and relatively little experimentation to test these ideas critically. We conducted a series of manipulative experiments to determine whether predation mortality is an important mechanism structuring populations of the sea urchin Heliocidaris erythrogramma in Tasmania. Tethered juvenile and adult sea urchins experienced much higher rates of mortality inside no-take marine reserves where sea urchin predators were abundant compared to adjacent fished areas where predators were fewer. Mortality of tagged (but not tethered) sea urchins was also notably higher in marine reserves than in adjacent areas open to fishing. When a range of sizes of sea urchins was exposed to three sizes of rock lobsters in a caging experiment, juvenile sea urchins were eaten more frequently than larger sea urchins by all sizes of rock lobster, but only the largest rock lobsters (> 120 mm CL) were able to consume large adult sea urchins. Tagging (but not tethering) juvenile and adult sea urchins in two separate marine reserves indicated that adult sea urchins experience higher predation mortality than juveniles, probably because juveniles can shelter in cryptic microhabitat more effectively. In a field experiment in which exposure of sea urchins to rock lobster (Jasus edwardsii) and demersal reef fish predators was manipulated, rock lobsters were shown to be more important than fish as predators of adult sea urchins in a marine reserve. We conclude that predators, and particularly rock lobsters, exert significant predation mortality on H. erythrogramma in Tasmanian marine reserves, and that adult sea urchins are more vulnerable than smaller cryptic individuals. Fishing of rock lobsters is likely to reduce an important component of mortality in H. erythrogramma populations.     

Complete regulation of development throughout metamorphosis of sea urchin embryos devoid of macromeres

The developmental potential of the animal cap (consisting of eight mesomeres) recombined with micromeres or of micromere progeny was examined in sea urchin embryos. The embryos derived from the animal cap recombined with a quartet of micromeres or their descendants developed into four-armed plutei. After feeding, the larvae developed into eight-armed plutei. The left-right polarity of the larvae, recognized by the location of the echinus rudiment, was essentially normal, regardless of the orientation of animal-vegetal polarity in micromeres combining with the animal cap. The larvae had sufficient potential to metamorphose into complete juvenile sea urchins with five-fold radial symmetry. Cell lineage tracing experiments showed that: (i) macromere progeny were not required for formation of the typical pattern of primary mesenchyme cells derived exclusively from large micromeres; (ii) the progeny of large micromeres did not contribute to cells in the endodermal gut with three compartments of normal function; (iii) the presumptive ectoderm had the potential to differentiate into endodermal gut and mesodermal secondary mesenchyme cells, from which pigment cells likely differentiated; and (iv) behavior of the progeny of small micromeres was the same as that in normal embryos through the gastrula stage. These results indicate that the mesomeres respecify their fate under the inductive influence of micromeres so perfectly that complete juvenile sea urchins are produced.     

Utilization of purple and red sea urchins (Strongylocentrotus purpuratus Stimpson and S. franciscanus Agassiz) as food by the white sea urchin (Lytechinus anamesus Clark) in the field and laboratory

White sea urchins (Lytechinus anamesus Clark) attacked purple (Strongylocentrotus purpuratus Stimpson) and red (S. franciscanus Agassiz) sea urchins at Anacapa Island, California. Densities of white urchins were highest in the deep algal crust-dominated community where up to 6% of purple and 25% of red urchins were being attacked by white urchins. Up to 9% of Lytechinus anamesus in an area were actively eating stronglylocentrotids and usually, more than one white urchin was involved in the attack. In areas with low densities of white urchins, no strongylocentrotids were being attacked.After 36 h in the laboratory, there was no difference in the number of white urchins attacking injured or healthy purple urchins in each of the three experimental densities of white urchins. However, both injured and healthy urchins were attacked by more white urchins in high density. When given a choice between injured purple urchins or fresh kelp, white urchins overwhelmingly chose kelp. Data suggest that white urchins utilize other urchin species as an alternative source of food when more preferred food is absent, but will switch to preferred food should it become available.     

Expression of maternal and paternal histone genes during early cleavage stages of the echinoderm hybrid Strongylocentrotus purpuratus × Lytechinus pictus

Interspecific hybrids of the sea urchins Strongylocentrotus purpuratus (♀) and Lytechinus pictus (♂) were used to estimate the contributions of the maternal and paternal genomes to histone mRNA synthesis during early development. Radiolabeled histone mRNAs from the two sea urchin species were identified by hybridization to cloned histone genes from both S. purpuratus and L. pictus and shown to be electrophoretically distinguishable. The synthesis of maternal and paternal histone mRNA in these hybrid embryos is evident as early as the two-cell stage. By at least the 16-cell stage, both maternal and paternal histone mRNAs are associated with polysomes. The relative amounts of the maternal and paternal histone mRNAs synthesized by the zygote appear to be similar.     

Evolution of direct-developing larvae: selection vs loss

Observations of a sea urchin larvae show that most species adopt one of two life history strategies. One strategy is to make numerous small eggs, which develop into a larva with a required feeding period in the water column before metamorphosis. In contrast, the second strategy is to make fewer large eggs with a larva that does not feed, which reduces the time to metamorphosis and thus the time spent in the water column. The larvae associated with each strategy have distinct morphologies and developmental processes that reflect their feeding requirements, so that those that feed exhibit indirect development with a complex larva, and those that do not feed form a morphologically simplified larva and exhibit direct development. Phylogenetic studies show that, in sea urchins, a feeding larva, the pluteus, is the ancestral form and the morphologically simplified direct-developing larva is derived. The current hypothesis for evolution of the direct-developing larval form in sea urchins suggests that major developmental changes occur by neutral loss of larval features after the crucial transition to a nonfeeding life history strategy. We present evidence from Clypeaster rosaceus, a sea urchin with a life history intermediate to the two strategies, which indicates that major developmental changes for accelerated development have been selected for in a larva that can still feed and maintains an outward, pluteus morphology. We suggest that transformation of larval form has resulted from strong selection on early initiation and acceleration of adult development.     

Early development and neurogenesis of Temnopleurus reevesii

Sea urchins are model non‐chordate deuterostomes, and studying the nervous system of their embryos can aid in the understanding of the universal mechanisms of neurogenesis. However, despite the long history of sea urchin embryology research, the molecular mechanisms of their neurogenesis have not been well investigated, in part because neurons appear relatively late during embryogenesis. In this study, we used the species Temnopleurus reevesii as a new sea urchin model and investigated the detail of its development and neurogenesis during early embryogenesis. We found that the embryos of T. reevesii were tolerant of high temperatures and could be cultured successfully at 15–30°C during early embryogenesis. At 30°C, the embryos developed rapidly enough that the neurons appeared at just after 24 h. This is faster than the development of other model urchins, such as Hemicentrotus pulcherrimus or Strongylocentrotus purpuratus. In addition, the body of the embryo was highly transparent, allowing the details of the neural network to be easily captured by ordinary epifluorescent and confocal microscopy without any additional treatments. Because of its rapid development and high transparency during embryogenesis, T. reevesii may be a suitable sea urchin model for studying neurogenesis. Moreover, the males and females are easily distinguishable, and the style of early cleavages is intriguingly unusual, suggesting that this sea urchin might be a good candidate for addressing not only neurology but also cell and developmental biology.     

Developmental plasticity in larval development in the echinometrid sea urchin Evechinus chloroticus with varying food ration

Developmental plasticity in the morphology of planktotrophic echinoid larvae has been studied primarily in temperate sea urchins from the Northern Hemisphere. These studies have shown that echinoplutei with reduced food ration generally respond by increasing the length of the larval arms, and thus the length of the ciliated band, and delaying formation of the rudiment. Using the endemic New Zealand sea urchin Evechinus chloroticus we tested for the presence of developmental plasticity in larvae fed a high or low algal food ration (6000 or 600 Dunaliella cells/ml), or with no algal food. Our results show that developmental plasticity in larval form is seen in a Southern Hemisphere representative of the Family Echinometridae, but the general pattern of longer arms with low food ration was only seen in the earliest part of development. Larvae in the High food treatment were largest in all dimensions and formed rudiments within 23 days of fertilization. Larvae fed a low algal ration or no algal food stalled at the four-arm echinopluteus stage, and were significantly smaller in size, and differed in shape, when compared in a multivariate analysis to the High food treatment. We suggest that the response of echinoplutei to low levels of particulate food is a species-specific trait, depending in part on the level of dependence of the larvae on exogenous food. Previous studies in Lytechinus variegatus, and the present study with E. chloroticus, suggest that in species where development stalls at the four-arm stage, the pattern of longer larval arms with low food ration will only be seen during the initial early period of larval growth. Additionally our results show that there can be significant variation in larval morphology between replicate jars in the same feeding treatment, suggesting that future research on developmental plasticity should also consider differences in larval morphology between culture containers.     

Extraordinary Diversity of Immune Response Proteins among Sea Urchins: Nickel-Isolated Sp185/333 Proteins Show Broad Variations in Size and Charge

Effective protection against pathogens requires the host to produce a wide range of immune effector proteins. The Sp185/333 gene family, which is expressed by the California purple sea urchin Strongylocentrotus purpuratus in response to bacterial infection, encodes a highly diverse repertoire of anti-pathogen proteins. A subset of these proteins can be isolated by affinity to metal ions based on multiple histidines, resulting in one to four bands of unique molecular weight on standard Western blots, which vary depending on the individual sea urchin. Two dimensional gel electrophoresis (2DE) of nickel-isolated protein samples followed by Western blot was employed to detect nickel-isolated Sp185/333 (Ni-Sp185/333) proteins and to evaluate protein diversity in animals before and after immune challenge with marine bacteria. Ni-Sp185/333 proteins of the same molecular weight on standard Western blots appear as a broad complex of variants that differ in pI on 2DE Western blots. The Ni-Sp185/333 protein repertoire is variable among animals, and shows a variety of changes among individual sea urchins in response to immune challenges with both the same and different species of bacteria. The extraordinary diversity of the Ni-Sp185/333 proteins may provide significant anti-pathogen capabilities for sea urchins that survive solely on innate immunity.     

Larval feeding structure plasticity during pre-feeding stages of echinoids: Not all species respond to the same cues

Much of the work on phenotypic plasticity has focused on inducible defenses. As a result, little is known about induced phenotypes that improve the acquisition of resources (i.e. inducible offenses). Feeding larvae of several marine invertebrate species, gastropods and echinoderms, have inducible offenses, and produce larger feeding structures when given less food. To better understand inducible offenses, I investigated when in development sea urchin and sand dollar larvae can first alter their feeding morphology in response to different concentrations of food. Food induced feeding structure changes in both sea urchin and sand dollar larvae before larvae were able to ingest food. This suggests that the nervous system and a regulator gene, orthopedia, play a mechanistic role. In addition, larvae of the two species, Strongylocentrotus purpuratus and Dendraster excentricus, responded to different cues. Pre-feeding larvae of both species developed relatively shorter arms when given algal cells (i.e. chemical and physical stimuli), whereas only pre-feeding larvae of D. excentricus developed shorter arms when exposed to algal exudates (i.e. chemical stimuli). Larvae of neither species responded morphologically to the presence of polystyrene beads (i.e. physical stimuli).     

Evolutionary convergence in<Emphasis Type="Italic"> Otx</Emphasis> expression in the pentameral adult rudiment in direct-developing sea urchins

Convergence is a significant evolutionary phenomenon. Arrival at similar morphologies from different starting points indicates a strong role for natural selection in shaping morphological phenotypes. There is no evidence yet of convergence in the developmental mechanisms that underlie the evolution of convergent developmental phenotypes. Here we report the expression domains in sea urchins of two important developmental regulatory genes ( Orthodenticle and Runt), and show evidence of molecular convergence in the evolution of direct-developing sea urchins. Indirect development is ancestral in sea urchins. Evolutionary loss of the feeding pluteus stage and precocious formation of the radially symmetric juvenile has evolved independently in numerous sea urchin lineages, thus direct development is an evolutionary convergence. Indirect-developing species do not express Otx during the formation of their five primordial tube feet, the ancestral condition. However, each direct-developing urchin examined does express Otx in the tube feet. Otx expression in the radial arms of direct-developing sea urchins is thus convergent, and may indicate a specific need for Otx use in direct development, a constraint that would make direct development less able to evolve than if there were multiple molecular means for it to evolve. In contrast, Runt is expressed in tube feet in both direct- and indirect-developing species. Because echinoderms are closely related to chordates and postdate the protostome/deuterostome divergence, they must have evolved from bilaterally symmetrical ancestors. Arthropods and chordates use Otx in patterning their anterior axis, and Runt has multiple roles including embryonic patterning in arthropods, and blood and bone cell differentiation in vertebrates. Runt has apparently been co-opted in echinoderms for patterning of pentamery, and Otx in pentameral patterning among direct-developing echinoids. The surprisingly dynamic nature of Otx evolution reinvigorates debate on the role of natural selection vs shared ancestry in the evolution of novel features.     

The dopamine effect on sea urchin larvae depends on their age

Activation of the dopamine type-D₂ receptor in late gastrula of sea urchins is known to decrease the growth rate of post-oral arms of larvae, and, as a result, the phenotype of these larvae mimics that of larvae developing in the abundance of food. Our data indicate that the effect of dopamine on sea urchin larvae is stage-dependent. In our experiment, the early four-armed plutei (96 hours post fertilization, hpf) of Strongylocentrotus intermedius had substantially shorter post-oral arms if they developed from the larvae treated with dopamine at the early pluteus stage (48 hpf), when they had already formed the first dopaminergic neurons, as compared to the plutei from the larvae treated with dopamine at the mid to late gastrula stage (24 hpf), when they did not have any neurons yet. The pre-treatment of larvae in 6-hydroxydopamine, a neurotoxic analog of dopamine that specifically disrupts activity of dopaminergic neurons, prevented the development of the short post-oral arms phenotype in larvae. These results confirm the assumption that dopaminergic neurons play an important role in the development of the short post-oral arms phenotype in sea urchin larvae. Another finding of our study is that the dopamine treatment also affects the growth of the body rods and the overall larval body growth. Based on these observations, we suggest researchers to carefully select the developmental stage, pharmacological agents, and incubation time for experimental manipulation of sea urchin larvae phenotypes through dopaminergic nervous system.     

Predator‐induced selection on urchin activity level depends on urchin body size

Temporally consistent individual differences in behavior impact many ecological processes. We simultaneously examined the effects of individual variation in prey activity level, covering behavior, and body size on prey survival with predators using an urchin–lobster system. Specifically, we tested the hypothesis that slow‐moving purple sea urchins (Strongylocentrotus purpuratus) and urchins who deploy extensive substrate (pebbles and stones) covering behavior will out‐survive active urchins that deploy little to no covering behavior when pitted against a predator, the California spiny lobster (Panulirus interruptus). We evaluated this hypothesis by first confirming whether individual urchins exhibit temporally consistent differences in activity level and covering behavior, which they did. Next, we placed groups of four urchins in mesocosms with single lobster and monitored urchin survival for 108 hr. High activity level was negatively associated with survival, whereas urchin size and covering behavior independently did not influence survival. The negative effect of urchin activity level on urchin survival was strong for smaller urchins and weaker for large urchins. Taken together, these results suggest that purple urchin activity level and size jointly determine their susceptibility to predation by lobsters. This is potentially of great interest, because predation by recovering lobster populations could alter the stability of kelp forests by culling specific phenotypes, like foraging phenotypes, from urchin populations.     

Constraint, flexibility, and phylogenetic history in the evolution of direct development in sea urchins

Development in sea urchins typically involves the production of an elaborate feeding larva, the pluteus, within which the juvenile sea urchin grows. However, a significant fraction of sea urchins have completely or partially eliminated the pluteus, and instead undergo direct development from a large egg. Direct development is achieved primarily by heterochrony, that is, by the abbreviation or elimination of larval developmental processes and the acceleration of processes involved in development of adult features. Direct development has evolved independently several times, and in several ways. These radically altered ontogenies offer remarkable opportunities for the study of the mechanisms by which early development undergoes evolutionary modification. The recent availability of monoclonal antibody and cDNA probes that recognize homologous cells in embryos of closely related typical and direct developing species makes possible an experimental analysis of the cellular and molecular bases for heterochronic changes in development.