Morphological evolution of newly metamorphosed sea urchins--a phylogenetic and functional analysis

Newly metamorphosed juvenile sea urchins are highly variable across taxa. This contribution documents and illustrates structural, functional, and phylogenetic variation among newly metamorphosed juvenile sea urchins for 31 species from 12 ordinal or familial lineages. The classic juvenile with five primary podia, 20 interambulacral spines, and variable numbers of juvenile spines is found commonly among new metamorphs across lineages, but there are many examples, which depart from this pattern and most likely reflect adaptation to settlement habitats. At metamorphosis juveniles can have 5-25 functional podia. They can have 0-65 spines, 0 or 5 sphaeridia (balance organs). They may have zero or up to eight pedicellariae. While competent larvae that delay metamorphosis may continue to develop juvenile structures, variation across species is much greater than within species and there are strong phylogenetic and functional differences among juveniles. Heterochronic changes in expression of these structures can account for differences among taxa. Based on this sample, juvenile characters such as spines, podia, and larval pedicellariae are expressed in ways that suggest they are developmental modules whose expression can be readily changed relative to one another and to the time of metamorphosis.     

The gastropod nervous system in metamorphosis

Many gastropods, including the sea hare Aplysia californica, undergo metamorphosis in passing from the larval to the juvenile phases of their life cycle. During metamorphosis, the gastropod nervous system is affected by both progressive and regressive neuronal events. In addition to this metamorphic reorganization, the nervous system appears to be centrally involved in initiating metamorphosis. We propose that gastropods not only possess temporally distinct neuronal adaptations for the specific needs of the larval and juvenile phases, but also another transient neuronal adaptation specialized to subserve the metamorphic episode.     

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.     

How do changes in parental investment influence development in echinoid echinoderms?

SUMMARY Understanding the relationship between egg size, development time, and juvenile size is critical to explaining patterns of life-history evolution in marine invertebrates. Currently there is conflicting information about the effects of changes in egg size on the life histories of echinoid echinoderms. We sought to resolve this conflict by manipulating egg size and food level during the development of two planktotrophic echinoid echinoderms: the green sea urchin, Strongylocentrotus droebachiensis and the sand dollar, Echinarachnius parma . Based on comparative datasets, we predicted that decreasing food availability and egg size would increase development time and reduce juvenile size. To test our prediction, blastomere separations were performed in both species at the two-cell stage to reduce egg volume by 50%, producing whole- and half-size larvae that were reared to metamorphosis under high or low food levels. Upon settlement, age at metamorphosis, juvenile size, spine number, and spine length were measured. As predicted, reducing egg size and food availability significantly increased age at metamorphosis and reduced juvenile quality. Along with previous egg size manipulations in other echinoids, this study suggests that the relationship between egg size, development time, and juvenile size is strongly dependent upon the initial size of the egg.     

Soldier determination in Pheidole bicarinata: Effect of methoprene on caste and size within castes

Topical application of methoprene to final-instar larvae of the ant Pheidole bicarinata can induce soldier development. Soldier induction takes place if methoprene levels are above a soldier-determining threshold during a critical period of juvenile hormone-sensitivity that occurs during about days 4–6 of the final instar. Furthermore, the amount of exogenous methoprene applied affects the timing of metamorphosis and the adult size in both the minor worker and soldier castes. When larvae that receive methoprene treatment become minor workers these are always larger than acetone-treated controls. In larvae that become soldiers, growth and timing of metamorphosis vary with the dose of methoprene, but in a more complex way. A high dose of methoprene produces a metamorphic delay and large soldiers. However, the lowest effective dose for soldier induction produces early metamorphosis and small adults. On the basis of these results, we have expanded our model of a mechanism by which juvenile hormone could control determination of worker castes in Pheidole bicarinata.     

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called “CLARITY” was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.     

Shifting patterns of nitrogen excretion and amino acid catabolism capacity during the life cycle of the sea lamprey (Petromyzon marinus)

The jawless fish, the sea lamprey (Petromyzon marinus), spends part of its life as a burrow-dwelling, suspension-feeding larva (ammocoete) before undergoing a metamorphosis into a free swimming, parasitic juvenile that feeds on the blood of fishes. We predicted that animals in this juvenile, parasitic stage have a great capacity for catabolizing amino acids when large quantities of protein-rich blood are ingested. The sixfold to 20-fold greater ammonia excretion rates (J(Amm)) in postmetamorphic (nonfeeding) and parasitic lampreys compared with ammocoetes suggested that basal rates of amino acid catabolism increased following metamorphosis. This was likely due to a greater basal amino acid catabolizing capacity in which there was a sixfold higher hepatic glutamate dehydrogenase (GDH) activity in parasitic lampreys compared with ammocoetes. Immunoblotting also revealed that GDH quantity was 10-fold and threefold greater in parasitic lampreys than in ammocoetes and upstream migrant lampreys, respectively. Higher hepatic alanine and aspartate aminotransferase activities in the parasitic lampreys also suggested an enhanced amino acid catabolizing capacity in this life stage. In contrast to parasitic lampreys, the twofold larger free amino acid pool in the muscle of upstream migrant lampreys confirmed that this period of natural starvation is accompanied by a prominent proteolysis. Carbamoyl phosphate synthetase III was detected at low levels in the liver of parasitic and upstream migrant lampreys, but there was no evidence of extrahepatic (muscle, intestine) urea production via the ornithine urea cycle. However, detection of arginase activity and high concentrations of arginine in the liver at all life stages examined infers that arginine hydrolysis is an important source of urea. We conclude that metamorphosis is accompanied by a metabolic reorganization that increases the capacity of parasitic sea lampreys to catabolize intermittently large amino acid loads arising from the ingestion of protein rich blood from their prey/hosts. The subsequent generation of energy-rich carbon skeletons can then be oxidized or retained for glycogen and fatty acid synthesis, which are essential fuels for the upstream migratory and spawning phases of the sea lamprey's life cycle.     

Gene expression during metamorphosis: An ideal model for post-embryonic development

The precocious induction in vivo and in culture of insect and amphibian metamorphosis by exogenous ecdysteroids and thyroid hormones, and its retardation or inhibition by juvenile hormone and prolactin, respectively, has allowed the analysis of such diverse processes of post-embryonic development as morphogenesis, tissue remodelling, functional reorganization, and programmed cell death. Metamorphosis in vertebrates also shares many similarities with mammalian development in the late foetal and perinatal period. This review describes the regulation of expression of some of the ‘adult’ gene products during metamorphosis in invertebrates and vertebrates. Recent studies on metamorphosis have revealed the important role played by auto-induction of hormone receptor genes, based on which a model will be presented to explain the activation of ‘downstream’ genes which give rise to the adult phenotype. It will also be argued that metamorphosis is an ideal model for analyzing some of the major mechanisms governing post-embryonic development.     

Induction of larval metamorphosis in the sea cucumber Stichopus japonicus by periphitic diatoms

The mass production of juvenile seeds of the sea cucumber,Stichopus japonicus has recently developed by the SeaFarming Center of Saga Prefecture. Methods for the culture ofperiphicic diatoms have been improved. There are three importantsteps in propagating the diatoms. The first step is theenrichment, with the addition of the nutrient salts, undercontrolled light intensity. The second step is washing withhigh pressure seawater and reversal of the plates. The laststep is elimination of copepods, which feed on diatoms, usinga pesticide. Small periphitic diatoms such as Navicula,Amphora, Achnanthes, and Nitzschia are easily culturedat a density of more than one million cells cm^–2, andthese diatoms are able to induce larval metamorphosis andserve as a food source for juvenile sea cucumbers.     

DNA SYNTHESIS AND CELL TURNOVER IN RANA PIPIENS TADPOLE LIVER DURING SPONTANEOUS METAMORPHOSIS1

The relationship of DNA synthesis and cellular turnover to biochemical differentiation during metamorphosis of R. pipiens liver was investigated. Average DNA/cell was constant at 11.6 pg/ nucleus through stage XXV; but increased during juvenile growth; during metamorphosis stages, changes in total DNA content must correspond to changes in cell number. Rates of DNA synthesis were estimated by rates of ³H-thymidine incorporated into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. DNA content increased steadily from premetamorphosis until late prometamorphosis; at preclimax stages XVIII and XX there were two successive decreases in DNA content of approximately 30%. Fluctuations in synthesis rates preceded corresponding fluctuations in content; DNA synthesis was maximal at stages XVI and XVIII, decreased nearly ten-fold at metamorphic climax, and then gradually rose again during late climax stages. The size of the endogenous thymine pool increased transitorily during spontaneous metamorphosis corresponding to a stage of maximal DNA synthesis. These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthesis rates and content during metamorphosis. Metamorphosis of the tadpole liver appears to be associated with both proliferation and cellular death, perhaps a replacement of “larval” by “adult” cells. Metamorphosis of the liver cannot be occuring in a “fixed population of cells” as is commonly assumed. An interpretation of the population dynamics of the metamorphic liver is presented.     

On nitric oxide signaling,metamorphosis, and the evolution of biphasic life cycles

Summary Complex life cycles are ancient and widely distributed, particularly so in the marine environment. Generally, the marine biphasic life cycle consists of pre‐reproductive stages that exist in the plankton for various periods of time before settling and transforming into a benthic reproductive stage. Pre‐reproductive stages are frequently phenotypically distinct from the reproductive stage, and the life cycle transition (metamorphosis) linking the larval and juvenile stages varies in extent of change but is usually rapid. Selection of suitable adult sites apparently involves the capacity to retain the larval state after metamorphic competence is reached. Thus two perennial and related questions arise: How are environmentally dependent rapid transitions between two differentiated functional life history stages regulated (a physiological issue) and how does biphasy arise (a developmental issue)? Two species of solitary ascidian, a sea urchin and a gastropod, share a nitric oxide (NO)‐dependent signaling pathway as a repressive regulator of metamorphosis. NO also regulates life history transitions among several simple eukaryotes. We review the unique properties of inhibitory NO signaling and propose that (a) NO is an ancient and widely used regulator of biphasic life histories, (b) the evolution of biphasy in the metazoa involved repression of juvenile development, (c) functional reasons why NO‐based signaling is well suited as an inhibitory regulator of metamorphosis after competence is reached, and (d) signaling pathways that regulate metamorphosis of extant marine animals may have participated in the evolution of larvae.     

Endogenous thyroid hormone synthesis in facultative planktotrophic larvae of the sand dollar Clypeaster rosaceus: implications for the evolutionary loss of larval feeding

Critical roles of hormones in metamorphic life history transitions are well documented in amphibians, lampreys, insects, and many plant species. Recent evidence suggests that thyroid hormones (TH) or TH-like compounds can regulate development to metamorphosis in echinoids (sea urchins, sand dollars, and their relatives). Moreover, previous research has provided evidence for endogenous hormone synthesis in both feeding and nonfeeding echinoderm larvae. However, the mechanisms for endogenous synthesis remain largely unknown. Here, we show that facultatively planktotrophic larvae (larvae that reach metamorphosis in the absence of food but have the ability to feed) from the subtropical sea biscuit Clypeaster rosaceus can synthesize thyroxine endogenously from incorporated iodine (I(125)). When treated with the goitrogen thiourea (a peroxidase inhibitor), iodine incorporation, thyroxine synthesis, and metamorphosis are all blocked in a dose-dependent manner. The inhibitory effect on metamorphosis can be rescued by administration of exogenous thyroxine. Finally, we demonstrate that thiourea induces morphological changes in feeding structures comparable to the phenotypic plastic response of larval structures to low food conditions, further supporting a signaling role of thyroxine in regulating larval morphogenesis and phenotypic plasticity. We conclude that upregulation of endogenous hormone synthesis might have been associated with the evolution of nonfeeding development, subsequently leading to morphological changes characteristic of nonfeeding development.     

Alternative Ways to Become a Juvenile or a Definitive Phenotype (and on Some Persisting Linguistic Offenses)

Lack of knowledge of early and juvenile development often makes it difficult to decide when a fish becomes a juvenile or, for that matter, a definitive phenotype. According to the established life-history model, a fish develops naturally in a saltatory manner, its entire life consisting of a sequence of stabilized self-organizing steps, separated by distinct less stabilized thresholds. Changes are usually introduced during thresholds. In principle, there are two ways to reach the juvenile period: by indirect or by direct development. Indirectly developing fishes have a distinct larva period that ends in a cataclysmic or mild remodeling process, called metamorphosis, from which the fishes emerge as juveniles. During metamorphosis, most temporary organs and structures of the embryos and larvae are replaced by definitive organs and structures that are also possessed by the adults. In contrast, directly developing fishes have no larvae. Their embryos develop directly into juveniles and do not need major remodeling. Consequently, the beginning of their juvenile period is morphologically and functionally less distinct than in indirect development. The life-history model helps to find criteria that identify the natural boundaries between the different periods in the life of a fish, among them, the beginning of the juvenile period. Looking at it from a different angle, when ontogeny progresses from small eggs with little yolk, larvae are required as the necessary providers of additional nutrients (feeding entities similar to amphibian tadpoles or butterfly caterpillars) in order to accumulate materials for the metamorphosis into the definitive phenotypes. Directly developing fishes start with large demersal eggs provided with an adequate volume of high density yolk and so require no or little external nutrients to develop into the definitive phenotype. These large eggs are released and develop in concentrated clutches. It therefore becomes possible and highly effective to guard them in nests or bear them in external pouches, gill chambers or the buccal cavity. Viviparity is the next natural step. Now the maternal investment into large yolks can be supplemented or replaced by direct food supply to the developing embryos like, for example, the secretion of uterine histotrophe or nutrient transfer via placental analogues. When the young of guarders and bearers start exogenous feeding, they are much larger or better developed than larvae of nonguarders and the larva period in the former is reduced to a vestige or eliminated entirely. In the latter case, the juvenile period begins with the first exogenous feeding. Such precocial fishes are more specialized and able to survive better in competitive environments. In contrast, altricial forms retain or revert to a life-history style with indirect development and high fecundity when dispersal is advantageous or essential. Fishes become juveniles when the definitive phenotype is formed in most structures, either indirectly from a larva via metamorphosis or directly from the embryo.     

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.     

Sources of genetic and phenotypic variance in fertilization rates and larval traits in a sea urchin

In nonresource based mating systems females are thought to derive indirect genetic benefits by mating with high-quality males. Such benefits can be due either to the intrinsic genetic quality of sires or to beneficial interactions between maternal and paternal haplotypes. Animals with external fertilization and no parental care offer unrivaled opportunities to address these hypotheses. With these systems, cross-classified breeding designs and in vitro fertilization can be used to disentangle sources of genetic and environmental variance in offspring fitness. Here, we employ these approaches in the Australian sea urchin Heliocidaris erythrogramma and explore how sire-dam identities influence fertilization rates, embryo viability (survival to hatching), and metamorphosis, as well as the interrelationships between these potential fitness traits. We show that fertilization is influenced by a combination of strong maternal effects and intrinsic male effects. Our subsequent analysis of embryo viability, however, revealed a highly significant interaction between parental genotypes, indicating that partial incompatibilities can severely limit offspring survival at this life-history stage. Importantly, we detected no significant relationship between fertilization rates and embryo viability. This finding suggests that fertilization rates should not be inferred from hatching rates, which is commonly practiced in species in which it is not possible to estimate fertilization at conception. Finally, we detected significant additive genetic variance due to sires in rates of juvenile metamorphosis, and a positive correlation between fertilization rates and metamorphosis. This latter finding indicates that the performance of a male's ejaculate in noncompetitive IVF trials predicts heritable offspring traits, although the fitness implications of variance in rates of spontaneous juvenile metamorphosis have yet to be determined.     

Pedomorphosis revisited: thyroid hormone receptors are functional in Necturus maculosus

Heterochrony, a difference in developmental timing, is a central concept in modern evolutionary biology. An example is pedomorphosis, retention of juvenile characteristics in sexually mature adults, a phenomenon largely represented in salamanders. The mudpuppy (Necturus maculosus) is an obligate pedomorphic amphibian, never undergoing metamorphosis. Thyroid hormone induces tissue transformation in metamorphosing species and this action is mediated by nuclear thyroid hormone (TH) receptors (TRs). The absence of metamorphosis in Necturus has been attributed to a resistance to TH action as treatment with exogenous TH fails to induce transformation. The failure to metamorphose could be due to the lack of TR expression in target tissues, or to a loss of TR function. Toward understanding the molecular basis for the failure of Necturus tissues to respond to TH, and the ultimate cause for the expression of the obligate pedomorphic life history, we characterized the structure, function, and expression of TR genes in Necturus. Strikingly, we found that Necturus TRalpha and TRbeta genes encode fully functional TR proteins. These TRs bind both DNA and TH and can transactivate target genes in response to TH. Both TRalpha and TRbeta are expressed in various tissues. TH treatment in vivo induced expression in the gill of some but not all genes known to be activated by TH in anuran larvae, caused whole organism metabolic effects, but induced no external morphological changes in adults or larvae. Thus, Necturus possesses fully functional TRs and its tissues are not generally resistant to the actions of TH. Rather, the absence of metamorphosis may be due to the loss of TH-dependent control of key genes required for tissue transformation.